- Formed w/in the macrophages & reduced into
bilirubin
2 TYPES OF BILIRUBIN
1. Conjugated
2. Unconjugated
- What comes out of bilirubin
- Non-polar water insoluble & when numerous in
plasma it causes jaundice
a. Unconjugated bilirubin will be released in the
plasma & for it to be normally excreted it will be
brought by albumin (transport protein) into the
liver for conjugation
b. In the liver w/ UDP-glucorynyl transferase which
will catalyze the addition of 2 glucoronic acids to
bilirubin forming now the conjugated
bilirubin/bilirubin diglucuronide which is water
soluble
c. Bilirubin diglucuronide will be stored in the bile.
COMPOSITION OF BILE: bile salts, bile acids, bile
pigments (conjugated bilirubin)
FUNCTION OF BILE: digestion of fats
d. During digestion, bile is released into the small
intestines (duodenum) via the common bile
duct.
e. Bilirubin will be acted by bacterial intestinal
enzymes converting it into urobilinogen. ½ of
urobilinogen will be reabsorb to the liver for
reprocessing while some will be filtered by the
kidneys and be excreted into the urine
f. Urine urobilinogen will give off the dark yellow
color of urine (normal)
g. The other half will be excreted into the feces
giving a color of light yellow, light brown to dark
brown due to urobilin and stercobilin coming
from urobilinogen
INTRAVASCULAR HUMAN HEMOGLOBIN
- Same w/ extravascular but w/out the
macrophages.
- Lysis occurs in the circulation
NORMAL HUMAN HEMOGLOBINS:
1. EMBRYONIC HEMOGLOBIN
- Produced in the mesoblastic stage
- First 3 months after conception
- zeta chain = analogue of a-chain
- epsilon chain = counterpart of the gamma, beta
and delta chains
3 EMBRYONIC HEMOGLIBIN:
- DIFFERENCE: Composition of globin structure
a. Hb Gower I (2 zeta & 2 epsilon)
b. Hb Gower II (2 alpha & 2 epsilon)
c. Hb Portland (2 zeta & 2 gamma)
2. Hb F (2 alpha & 2 gamma)
- Major Hb of the fetus and the newborn
- Has high affinity to O2 thus it will not easily
release the O2
- Resistant to both acid elution and alkali
denaturation
- After birth, smaller amounts of HbF are
produced
- Diseases associated: HPFH & B. Thalassemia
- Increased would lead to hypoxia
- In fetus, anaerobic respiration occurs, for it to
conserve its O2, it contains Hb F, but for new
born, aerobic respiration, Hb F is still increased
thus decreased O2, this decrease is
compensated by the increased Hb F
AFTER BIRTH, SMALLER AMOUNTS OF HbF ARE
PRODUCED:
o 6 months = <8%
o After age 2 years = <2%
o Adults = < 1-2%
GLOBIN SYNTHESIS:
- Hb F, Gower 1 & 2 & Portland are Hb that are
synthesized separately & produced separately
- Primitive RBC are the very first blood cell
produced during the mesoblastic stage which
will oxygenate the embryo’s tissue. This
primitive RBC contains the embryonic Hb
a. Zeta & Epsilon are synthesized in the
mesoblastic stage forming the Gower 1
Note: synthesis of zeta & epsilon is high at the
start but declines thereafter, thus decreasing
embryonic Hb.
b. After few days, alpha chains are synthesized
wherein a small amount of it combines w/
epsilon forming the Gower 2
c. A small amount of Gamma is synthesized &
combines w/ zeta forming the Portland
d. No synthesis of Epsilon and Zeta would lead to
dissolution of embryonic hb wherein it only
 persists for at least 3 months. As a  Methemoglobin is not a compound but only
counterpart, the A & Gamma increases. an Hb containing an oxidized Fe (Ferric)
e. Combination of these 2 forms the Hb F during  Sulfhemoglobin is a Hb w/ sulfide radical
the hepatic stage  Oxy & DeoxyHb are normal Hb formed during
f. Synthesis of A chain persist until after birth the normal transport of gases. These are
while gamma starts to decrease after birth & normal both in presence & function.
as a counterpart B chain gradually increases.  Carboxy, Met & SulfHb are abnormal in
g. Combination of A & B chain forms the Hb A1. function but normal in presence. These are
h. Delta chain, does not peak & combination of present in small amount in circulation.
this to A chain will form the Hb A2  Affinity of carboxyhemoglobin to Hb is >200x
thus instead that O2 binds,
carboxyhemoglobin binds w/ Hb thus it has an
abnormal function & is present in very minute
3. Hb A1 (2A & 2B): Major normal adult hemoglobin
concentration.
(97%)
 Everyday 3.5% of Hb is converted into
4. Hb A2 (2A & 2D): methemoglobin but since RBC have reducing
o Accounts for 1.5 % - 3.5 % of normal capacity, it is reduced back into a normal Hb.
adult Hb Deficiency in reducing capacity of RBC, RBC
o Increased in some – B Thalassemia’s, can no longer reverse the reaction thus
hyperthyroidism and in some cases of increase in methemoglobin causing
megaloblastic anemia methemoglobinemia.
 Affinity of Hb to O2 is dependent in the state
HEMOGLOBIN MOLECULAR STAGE OF LIFE of Fe – ferrous
STRUCTURE  Oxidation reduction is reversible, meaning it is
oxidized but it is also reduced.
Gower 1 2Z&2E EMBRYONIC
 Sulfhemoglobins presence & function is both
Gower 2 2A&2E EMBRYONIC abnormal thus it must not be present in
circulation. It is the only Hb that is not
Portland 2Z&2G EMBRYONIC measured by cyanmethemoglobin procedure.
 Conversion of Hb to Sulf is irreversible.
Fetal (F) 2A&G FETAL

A1 2A&2B ADULT
FORMS OF HEMOGLOBIN
A2 2A&2D ADULT NORMAL

1. OXYHEMOGLOBIN (HbO2)
- Circulates in the arterial circulation
HEMOGLOBIN COMPOUNDS - Formed when the Hb passes through the
OXYHEMOGLOBIN O2 + Hb Hb F, A1 & pulmonary capillaries of the lungs
- The O2 is loosely bound and unstable for easier
A2
release into the tissues
DEOXYHEMOGLOBIN CO2 + Hb Hb F, A1 & - Gives a scarlet red/bright red color
A2
NOTE:
CARBOCYHEMOGLOBIN CO + Hb Hb A2 Basilic is not appropriate for blood collection due to
possibility that brachial artery might be hit
METHEMOGLOBIN Ferric Hb Hb A1 & A2

SULFHEMOGLOBIN S + hb Hb A1 & A2 2. DEOXYHEMOGLOBIN

- Reduced form however increased CO2
- Hemoglobin with CO2 (carbaminohemoglobin)
NOTE: wherein CO2 binds to the free amino group of
the Hb to form carbaminohemoglobin
 - Gives a dark red color due to presence in venous
circulation
- Formed when Hb passes through the tissues
ABNORMAL
1. CARBOXYHEMOGLOBIN (HbCO2)
- Hemoglobin with CO (affinity of CO to Hb is 200-
210x greater than the affinity of O2 thus it binds
more in Hb)
- Normally present in body due to endogenous CO
source (<1%) but have an abnormal function
because it cannot bind with O2
- From the breakdown of protoporphyrin into
biliverdin & CO which is the endogenous source
which is only minimal (<1%). At the same time,
for people who are exposed to normal
atmospheric O2, the half-life of CO is only 4
hours & 50% is lost by exhalation & this is not
enough to cause CO poisoning
- Chief Sources of CO (exogenous): automobile
exhaust, industrial wastes, tobacco smoking
(10% of Hb of people expose to exogenous
source)
- Imparts a cherry red in color in blood & skin
- Absorption wavelength: 576 nm
CRITICAL VALUE:
5g /100 ml which may cause irreversible tissue
changes since COHb cannot bind w/ O2, thus it cannot
transport O2 thus it cannot oxygenate the tissues &
lungs. There are body tissues that are very dependent
on O2 (brain cells). When brain cells are depleted w/
O2 this will result to cell damage & brain cell damage
is irreversible. If values are >50 – 70% this may lead to
asphyxiation which is experienced by a person being
strangled, it is a condition wherein the patient is
depleted w/ O2 thus the patient will lose
consciousness & may lead to death, however
treatment may be employed w/ hyperbaric O2
Treatment: administration of hyperbaric O2 – involves
MEDICAL IMPORTANCE:
high amount of conc. O2 w/ pressure in a pressurize
room in a pressurize tube. Although the affinity of
COHb is greater than O2, w/ higher amount of
pressurize O2, the O2 exposure can displace CO from
attachment to Hb thus bringing the CO to its normal
Hb range
MANAGEMENT OF CYANIDE POISONING
NOTE: RAPID TESTS FOR HbCO
a. NaOH test
- Composition: 40% NaOh + EDTA – blood
- Process: warm gently
- Result: HbCO: red color HbO2: Black-Brown
b. Dilution test (addition of water)
- Compostion: 1 mL blood + 50 mL water
- Result:
o HbCO: cherry red – pink or bluish red
o HbO2: Yellowish Red
c. Tannic Acid test
- Composition: 1% Tannic acid which will tend
to acid digest the blood resulting to black-
brown color
- Result:
o HbCO: red precipitate
o HbO2: Black-brown discoloration
2. METHEMOGLOBIN/ HEMIGLOBIN (Hi)
- Iron (Fe++) is oxidized into its Fe+++ form
- Not a compound but an oxidized Hb
- Absorption wavelength: 630 – 635 nm
- Screening tests:
o Aeration: blood retains its color
o Addition of 1% MB: blood turns red
- Have an abnormal function but normally present
in blood w/ conc. of 1.5% because every day
almost 0.5 – 3% Hb is converted to hemoglobin
that does not accumulate even if it is oxidized it
is reduced back to normal Hb as oxidation
reduction is reversible. But if amount of
oxidation is >1.5-3.5% g/dL (10%) it will cause
cellular damage causing cyanosis wherein the
patient will suffer from bluish discoloration of
the skin as well as difficulty of breathing due to
depletion of O2.
- If present in blood at high conc. >1.5-3.5% g/dL
(10%) this will result to a chocolate brown color
- With strong affinity w/ cyanide forming
hemoglobincyanide (HiCN) which may take
place inside the body. It is important in the
treatment of cyanide poisoning. Free cyanide
in the circulation is very toxic.
- NO2 is given which will cause the oxidation.
NO2 will convert Hb into hemiglobin causing
oxidation of the patients Hb thus once
 hemiglobin is formed, it can bind to the
produced cyanide forming into HiCN. The free
poisonous cyanide is converted into less
poisonous cyanide.
 Maintenance of Hemoglobin in the Normal
Reduced state (Fe++): Reducing properties of
RBC
PRODUCTION OF:
 Reduced Diphosphopyridine dinucleotide
(DPNH) in the presence of methemoglobin
reductase (diaphorase)
- Reaction in Embden-Meyerhof Pathway that
Generates NADH:
o NAD is reduced into NADH which has a
reducing potential thus deficiency of it
will not make it to reduce the ferric into
ferrous.
 Reduced Triphosphopyridine nucleotide (TPNH)
in the presence of Glucose-6-PO4
dehydrogenase
- produced in Embden-Meyerhof Pathway in the
Hexose Monophosphate shunt
- Process: from phosphorylation of glucose to
glucose 6 phosphate it enters the hexose
monophosphate shunt & converted into 6-P
gluconate catalyze by G-6-PD with a coenzyme
NADP wherein in this reaction it is reduced which
is similar to TPNH. With the reduced NADP it can
caused the production or reduction of more
glutathione which is also a reducing potential.
The major reducing capacity of RBC is found in
the hexose monophosphate shunt because in
this shunt, it can be able to produce 1.) NADPH
& 2.) GSH
 Glutathione and its associated enzymes
o Glutathione is the major anti-oxidant
since it has a reducing property w/
NADPH
o Reduced glutathione is important in
protecting the Hb from being oxidized
NOTE:
Reduced NAD can bring back methemoglobin into
normal Hb, ferric to ferrous
OTHER REDUCING AGENTS:
a. METHYLENE BLUE
- Often given to patients suffering from
methemoglobinemia
- It will cause the reaction of glucose 6-P to be
faster wherein the H of NADH will be
transferred to the Hb thus reducing Hb
however the transfer of H is slow wherein
methylene blue will enhance this reaction
b. ASCORBIC ACID: a potent reducing agent &
also an anti-oxidant
METHEMOLOBINEMIA
- increase in the methemoglobin conc. in blood
CONDITIONS OF METHEMOGLOBINEMIA
a. Inherited (enzyme deficiency): commonly
attributed to NADH-Methemoglobin reductase
deficiency/Diaphorase deficiency which is
important in reducing ferric to ferrous
b. Inherited M (methemoglobin)
- results of various amino acid substitutions in the
globin chain that directly affect the heme group
- due to increase methemoglobin, RBC cannot
keep up thus it cannot reduced back to
hemoglobin
METHEMOGLOBIN VARIANTS
- Different structural abnormality but are not
protected from being oxidized meaning they are
easily oxidized
1. M-saskatoon
2. M-boston
3. M-iwate
4. M-hydePark
5. M-milwaekee
c. Acquired:
- effects of chemical or therapeutic agents (aniline
dyes in food, nitrate & nitrite-rich water and
foodstuffs ; anti-malarial drugs & sulfonamides)
- Therapy: Ascorbic acid & Methylthionimium Cl
NOTE:
 Methylene blue is not a reducing agent but
only accelerates the reaction.
 Carboxyhemoglobin and methemoglobin are
present in small quantities in the serum
however its function is abnormal.
SCREENING TEST FOR METHEMOGLOBIN 123 – 153  Higher in the morning & lower in
Composition: 1% Methylene Blue g/L the evening
Result: red or pink color of blood
AT BIRTH:  Lower if lying down

3. SULFHEMOGLOBIN (SHb) 150 – 200  Increased in smokers

- Formed when organic sulfides combine with Hb
- Observed in patients taking in oxidant drugs
(phenacetin & acetanilid, sulfonamides)
- Once Hb is converted into sulfhemoglobin it
remains as it is thus it is Irreversible for 120 days
however it can combine w/ CO forming
carboxysulfhemoglobin
- It is the irreversible formation which prevents it
to be measured by drabkins reagent thus it
cannot be converted in cyanmethemoglobin
- NOT normally present in the blood thus its
presence at 0.5 g/100 mL is a critical level
causing damage to tissues
- Imparts a greenish color
- Unstable Hb that can easily precipitate into a
Heinz bodies, an inclusion bodies, which when
present to RBC, they tend to attach themselves
on RBC membrane making RBC membrane rigid
thus cells w/ Heinz bodies are prone to hemolysis
leading to hemolytic anemia. Even normal Hb
can form few Heinz bodies when denatured
- Absorption wavelength: 600 - 620 nm
g/L
 Increased in high altitude
because the atmosphere is very
thin thus O2 is thin
 Estrogen tends to inhibit
erythropoietin while androgen
will tend to stimulate more
OTHER FORMS OF HEMOGLOBIN
a. CYANMETHEMOGLOBIN
- Feriicyanide + Fe++ of Hb
- An in vitro test that may also occur inside the
body (in vivo)
- Its presence can also be induced in vivo (inside
the body) especially during cyanide poisoning.
- The most stable among the Hb pigments thus
when performing cyanmethemoglobin
procedure the Hb result can be delayed because
the color produced does not fade
- Absorption wavelength: 540 nm

RFERENCE INCREASED Hb DECREASED Hb b. GLYCOSYLATED HEMOGLOBINS

VALUES - Also called “Glycated Hemoglobin” or simple
FOR  Polycythemi  All “Glycohemoglobin”.
HEMOGLO a - increase anemia - Described as “fast hemoglobins” because they
BIN RBC migrate fast in electrophoresis.
 Leukemi
production - Irreversibly glycosylated at 1 or both N-terminal
a
valines (or lysine) of the β-chains
 Dehydration
 Oligochr - Refers to the addition of a glucose molecule to
– due to
omia the hemoglobin molecule. Such addition is non-
hemoconcen
enzymatically catalyzed. The only basis of when
tration w/
the glucose binds in hemoglobin is the level of
decrease
plasma glucose that when the levels of plasma
plasma
glucose is elevated (like in hyperglycemia, DM
volume due
Type I and II), the excessive glucose in the plasma
to burns &
can bind with the hemoglobin called glycation or
diarrhea
glycosylation which is irreversible wherein once
MALE: PHYSIOLOGIC VARIATIONS the glucose is added to the Hgb molecule, this
Hgb will remain to be glycated so that the RBC,
140 – 175  After 50 years of age – slight once its Hgb is glycosylated, it will remain
g/L decrease glycolyated during the entire life span of the
FEMALE: RBC. This will now lead to the formation of:
o Hb A1a
 o Hb A1b days. It is important index in diabetes control,
o Hb A1c: the FBS & RBS are more accurate as an index of
 Major fraction which serves as metabolic control for the past 2-4 months,
an index of the metabolic meaning the glycosylated Hb is the same for the
control over that last 2-4 next 4 months.
months. This is more preferred
OXYGEN DISSOCIATION CURVE
in monitoring glycemic control.
Oxyhemoglobin
 Elevated 2-3 folds in patients
dissociation curve
with Diabetes Mellitus (DM).
(ODC), is a curve that
 Preferred than FBS and RBS.
plots the proportion of
NOTE: HBA1c hemoglobin in its
saturated form on the
 Patients with DM are being monitored
Y axis against the
for their glycemic control and if their
prevailing oxygen
medications can control the increase
tension on the X axis. It
of their glucose. The monitoring
is slightly S shape.
should be a form of review on their
Left shift indicates increase O2 saturation
conditions for the past 2-4 months.
Right shift indicates that O2 is released in the tissues.
 The present HbA1c levels of the
This relates oxygen saturation and partial pressure of
patient reflects on his/her HbA1c
O2 in the blood (PO2), and is determined by
levels for the past 2-4 months.
hemoglobin affinity for oxygen wherein the higher the
 HbA1c is just for monitoring and not
affinity, the curved shift towards the left & vice versa.
for diagnosis for DM.
Affinity is the main factor that influences the shifting.

- Necessary for diabetic individual

FACTOR SHIFT CAUSED BY
- Fast Hb
- Hb w/ attached glucose which occurs when FACTOR FACTOR
plasma glucose is high. Glycation is not INCREASE DECREASE
enzymatically catalyzed. When glucose attached
to Hb, the only factor which will promote this is pH Left shift due to Right shift due
the increased in plasma glucose or increase affinity to decrease pH –
hyperglycemia. – alkalosis acidosis

NOTE: 2,3 DPH/BPG Right shift due do Left shift due to

 To which part of the Hgb will the glucose decrease affinity increase affinity
attach? due to increase due to decrease
o Glucose attaches at 1 or both N- DPG DPG
terminal valines (or lysine) or the β-
CO2 (haldane Right shift due to Right shift due
chains.
effect), H+ & increase of these to decrease of
 Only HbA1 are glycosylated Hgb because the
glycosylation occurs in the β-chain. Therefore, Cl- - all factors thus salt these factors
only Hgb has the β-chain. maintains the bridge is thus salt bridge
salt bridge stabilized thus is not stabilized
decreasing the thus increasing
2 REACTIONS ARE INVOLVED: affinity the affinity
1. REVERSIBLE Blood (Body) Right shift due to Left shift due to
2. AMADORI: irreversible meaning the glucose Temperature increase decreased
attached to the terminal valine or lysine can no Temperature temperature
longer be detached. & once the Hb is Rule: the
glycosylated it will remain glycosylated until 120 higher the
temperature,
the faster is DISORDERS RELATED WITH ABNORMAL
the HEMOGLOBINS
metabolism 1. THALASSEMIA
- Synthesis defect
Hb F Left shift due to Right shift due - Decreased or non-existent production of one or
increase Hb F to decreased Hb more globin chain type
-Has innate
thus it will not F thus it will - For both α & β, there is a deletion in the gene
increased
easily release O2 easily release resulting to incomplete synthesis of A & B chain.
affinity which
O2 - Diverse group of genetic disorders characterized
is needed by
fetus in an by a primary, quantitative reduction in globin
anaerobic chain synthesis for hemoglobin.
environment CLASSIFICATIONS:
in the uterus
thus if A. ALPHA THALASSEMIA
increased in - Indication of Deletion:
adults, this 1. Silent carrier - (_A/AA) (AA/_A) = ¼ thus no
may lead to signs & symptoms of anemia
deoxygenatio 2. Thalassemia trait/A-Thal minor – (_ _/AA)
n. (_A/A_) = 2/4 w/ mild signs & symptoms
3. Hb H disease – (_ _ /A _) = ¾ thus the
Abnormal Hbs production of Alpha chain becomes too
(depends on insufficient & B chain will be used in excess
characteristic resulting to Hb having all B chains (4 B)
of 4. Bart’s Hydrops fetalis – (_ _/ _ _) = 4/4 thus
abnormality) no synthesis of A chain, as early as fetal life,
Hb F which consist of 2 A & 2 G, there would
be no Hb F synthesis & what will be
SEVERAL ABNORMALITIES OF HEMOGLOBIN synthesize is the G chain (4 G).
1. HEME IS ABNORMAL
- Iron is deficient thus affecting Hb synthesis B. BETA THALASSEMIA
leading to IDA - Indication of Deletion: 0 superscript
- Deficient Protoporphyrin IX because of enzyme 1. Beta Thalassemia Major/Cooley’s anemia –
defect along the synthesis leading to excess iron B0/B
& iron will not be utilized in the absence of 2. Beta Thalassemia Intermedia – B+/B+,
protoporphyrin 9. Unused iron will precipitate present but cannot effect the synthesis of B
into siderotic granules leading to sideroblastic chain because of partial suppression.
anemia 3. Beta Thalassemia Minor/Cooley’s trait -
B0/B0, the patient is devoid of Hb A1
NOTE:
because the patient cannot synthesize B
Sideroblastic Anemia – problem in the utilization of
chain & what is elevated is Hb F.
iron due to deficiency in protoporphyrin 9

2. HEMOGLOBINOPATHIES
2. GLOBIN IS ABNORMAL - Disorder in structure
- Ex.: Hb A1 – 2 A w/ 4 genes & 141 amino acids & - Results from the alteration of the DNA genetic
2 B chains w/ 2 genes & 146 amino acids code for the chains
- 2 FACTORS: - Conditions characterized by qualitative
o Alpha Thalassemia structural abnormalities of the globin
o B Thalassemia polypeptide chains that result from alteration of
 the DNA genetic code for those chains. Structural Hb S (SICKING HB)
abnormalities may involve amino acid. - The most well-known hb variant characterized
by substitution of glutamic acid by tRNA to valine
STRUCTURAL DEFECTS ON A OR B CHAIN RESULTING TO
on the 6th amino acid position of the β-chain.
Hb VARIANT:
Glutamic: CAG Hb S: β 6(A3) Glu → val
Valine: GUG
1. SUBSTITUTION
- Ex.: Hb Hb S: β 6(A3) Glu → val
- Common to black population
- The effect of alteration from glutamic acid to
2. DELETION
valine is a negative charge wherein glutamic is
- some amino acid is deleted
highly negatively charge compared to valine thus
- reason of thalassemia
leading to loss of negative charges & Hb
Ex.: Hb Gun Hill: (β91 to β 95) → 0 becomes less negative
- In electrophoresis in cellulose acetate, S
3. FUSION migrates slowly than A & F due to substitution of
- Hb Lepore-Baltimore: (G(1-50) B(86-146)) – caused by amino acid 6
cross over during mitosis resulting to fusion - Inheritance:
o Homozygous (SS) – sickle cell anemia
4. ELONGATION/ ADDITION o Heterozygous (AS) – sickle cell trait
5. Hb CONSTANT SPRING (a+31c)
EFFECTS OF HB S:
REASON FOR DELETION & ELONGATION: mutation in  Blacks who are suffering from this are
genetic code protected from being infected from
developing severe falciparum malaria
REASON OF HEMOGLOBINOPATHIES: mutation of gene (Resistance). Hb C, E, B-Thalassemia & G-6PD
NOMENCLATURE deficiency also confer protection in terms of
resistance but not immunity meaning these
1. COMMON NAME
patients may still be infected but may not
a. Morphology- sickling Hb (HbS)
proceed to chronic stage.
b. Place where they were discovered- (Hb
o Falciparum are intracellular species &
Boston) when present in RBC & when RBC is
2. SCIENTIFIC NAME sickle the parasite inside also dies
- Gives structural defect of Hb does no complication arise
- Example: Hb Hb S: β 6(A3) Glu → val o Falciparum metabolizes Hb, when Hb
B: affected polypeptide chain crystalizes it forms into a crystal &
6: sequential amino acid number(s) affected falciparum may no longer metabolize
(primary structure) the Hb
A3: Helix number involved (secondary structure: o RBC w/ falciparum will be
A-H); not necessary as long as primary structure phagocytosis before it causes severity
is indicated
Glu: nature of the abnormality (substitution, HOMOZYGOUS (SS) SICKLE CELL ANEMIA
deletion, addition or globin chain fusion) - Severe anemia because the patient does not
PRIMARY STRUCTURE synthesize the normal Hb A

- sequence or arrangement of amino acid NOTE:

On electrophoresis:
NOTE: o Hb S – 80 -90%
Hemoglobinopathies commonly affects the B chain. o Hb F – 10 -20 %
o Hb A2 – Normal

- Increased RDW
 - Retarded growth & sexual maturation & patient
suffers sickle cell crises
- Main cause of Sickle Cell Crises: sickling
- Normally when Hb S is fully oxygenated, Hb S is
fully soluble
- But when Hb S is w/ reduced O2, Hb S becomes
insoluble wherein it polymerizes (change in
molecular arrangement) & forms into tactoid
crystals/fluid crystals (elongated & thin, pointed
at both ends) leading to sickling & rigid cell
membrane thus it is no longer adjustable leading
to vassoocclusion thus it cannot easily pass
through the small circulations
- Sickling is reversible but repeated sickling leads
to permanent sickling due to permanent damage
to RBC membrane
- Causes vasso-obstruction
- Vasoocclusive crises occurs commonly on small
circulations like in the fingers & toes thus hand-
foot syndrome / dactylitis (inflamed or swollen,
cyanotic due to absence of circulation) occurs
- Vassoocclusion also occurs in bone & joint
causing pain
- Splenic circulation is also minute, since spleen
also serves as a filter of blood thus its circulation
is too small, that only deformable red cell may
pass through. Due to increase activity of spleen
& vassoocclusion, spleen also enlarges
(splenomegaly) causing sequestration of more
blood leading to hypovolemic shock. When
spleen is enlarged & bone marrow is affected
thus both are abnormal in function leading to
decrease blood production to the marrow
(aplastic crises) thus the immune system is
defective & the patient becomes prone to
infection (infectious crises) as a result of all of
this organ damage occur (marrow lungs)
- The no. 1 cause of death is infection & in children
the most common is S. pneumoniae.
NOTE:
SICKLE CELL TRAIT (AS)
• Predominant Hb is A, only 30-45% is Hb S
• Patient usually have no symptoms & sickling is
uncommon to occur unless in cases of extreme tissue
hypoxia because S is minimal in concentration
NOTE:
Hb S – Thalassemia:
o Hb S-A
o Hb S-B – more severe
NOTE:
OTHER SICKLING HBS:
o Abnormality: Hb S: β 6(A3) Glu → val + unique B-
substitution
o Hb C-Harlem, C-Ziguinchor, S-Travis
HEMOGLOBIN C
-
Composition: B 6 (A3) Glu → Lys
- A beta structural defect, where glutamic acid is
substituted w/ lysine
HOMOZYGOUS: Hb C DISEASE (CC)
- N/N in size & shape anemia with numerous
target cells (bull’s eye)
- Hb CC crystals formed when deoxygenated &
dehydrated & the crystals are hexagonal or rod-
shaped crystals or bar of gold shape & blunt ends
& does not deform the cell meaning it retains its
shape but becomes rigid
HETEROZYGOUS: Hb C TRAIT (AC)
- RBCs – slightly hypochromic
- Less abnormalities
HEMOGLOBIN SC DISEASE
- Signs & symptoms are milder than SS but more
severe than sickle cell
- Electrophoresis: C = S = 50 – 50%
- Cytoplasm appears folded like a pocketbook cells
(RBC w/ cytoplasm folded)
- Hb SC easily crystalize forming a fingerlike
projection w/ more than 1 protrusions where
one is long & protrude away. It also in the form
of hand in gloves crystals or pointing – finger or
Washington monument crystal
OTHER ABNORMAL HEMOGLOBINS
a. Hb METHEMOGLOBIN
- M-Saskatoon, Boston, Iwate, HydePark,
Milwaukee = all have different amino acid
substitution but the effect is the same & not
protected from oxidation
- Methemoglobinemia w/ congenital cyanosis due
to overwhelming oxidation even at baby stage &
the blood appears chocolate brown
- Easily precipitate & forms into Heinz bodies
-
b. HbS W/ ABNORMAL AFFINITY TO OXYGEN
1. Hb w/ Increase Affinity
- Example: Hb Chesapeake (a 93 Arg → Leu) – affects
affinity of Hb to O2 & O2 will shift to left

2. Hb with Decrease Affinity
- Example: Hb Kansas (B 120 Asp → Thr) – shift to the
right
NOTE:
Abnormal Hb are connected by weak bonds thus they
easily precipitate & flocculate
TEST FOR UNSTABLE HB
1. ISOPROPANOL PRECIPITATION TEST
- When heated, 17% Isopropanol at 37 C normal
Hb do not precipitate but few will do & unstable
Hb will easily precipitate.
- Purpose of Isopropanol: weaken the bonds
2. HEAT DENATURATION/INSTABILITY TEST
- At 50 C for 3 hours, normal Hb will not flocculate
but unstable Hb will
3. HEINZ BODY STAINING
- If ppt & flocculation happens in vivo forming
Heinz bodies which are not easily seen in normal
wrights blood smear
- Supravital staining is employed (brilliant cresyl
blue & crystal violet)
NOTE:
RED BLOOD CELLS
CHARACTERISTIC OF A NORMAL RBC:
 Shape: Biconcave disk
o Purpose:
 Promotes gas exchange
between CO2 & O2
 For RBC to be flexible &
deformable in order to squeeze
through the microcirculations or
else it will be prone to lysis.
 Diameter: 7 – 8 um
o <7: microcyte
o >8: macrocyte
 Thickness (MCAT): 1.5 – 2.5 um
 Average volume (MCV): 90 fL
 Average Surface Area: 160 um2
 Shape & Deformity: Flexible & deformable
o Responsibility of: shape, integrity of cell
membrane & biconcavity.
 Mature: anucleated w/ a dense outer rim
 Central pallor: 1/3 of its diameter, it is pale due
to lack of Hb
o >1/3: RBC is hypochromic
 Size: similar to small lymphocyte
o Rule: Slight variation in size & shape is
acceptable but increase in variation will
be abnormal
 Cytoplasm: uniformly pink without inclusions
 Average lifespan: 120 days
o <120 days: RBC undergoes pre-mature
hemolysis
 Chromium 51 (Cr51): used to measure the
effective survival of RBCs in vitro
o Half survival time range: 25-32 days
o <25 days: RBC undergoes hemolysis
o Process: Collect blood from the patient.
Radioactive Cr is added on the blood.
Chromated RBC are infuse back
intravenously. The disappearance of
chromated RBC will be measured by
performing blood cell count in a gamma
ray for every 1 – 2 days for 10 – 14 days.
Cr is eluted from circulation 1% a day, its
disappearance is shorter
o Purpose: label RBC in order to be
differentiated from unlabeled RBC
 Half – life: no. of days wherein at least 50% of
radioactivity of Cr or gamma ray disappear
from circulation
 Eliptocytes are usually utilize for thinner &
deformed cells due to acquired condition
while ovalocytes are egg shape & not thin,
which are often seen in hereditary type.
RBC MEMBRANE
- underlie the lipid layer & regulate shape &
deformability because if the membrane is rigid,
the cells cannot reshape itself & not flexible
- surrounds the cellular cytoplasm or internal
environment
- selective transport membrane: separates the
internal environment to external
o does not easily allow diffusion of
substances
o It selectively allows the entry of water &
electrolytes, inlet & exit
- consist of a phospholipid bilayer
 RBC MEMBRANE COMPOSITION:
1. 50% PROTEINS
a. INTEGRAL PROTEINS
- crosses phospholipid bilayer
- high amount of sialic acid for imparting negative
charge to maintain zeta potential
NOTE:
Zeta potential
- RBC repel each other due to negative cloud
surrounding them caused by sialic acid. When
zeta potential becomes altered this result to
rouleau formation.
- Component: Glycophorin A & component a
(band 3)
- Function: serves as membrane protein
mechanism in order to allow passage of
molecules either inward or outward
b. PERIPHERAL PROTEINS
- Component (cytoskeleton): Spectrin & Actin
- found facing the inner or cytoplasmic
environment or facing outwards found on the
outer or inner surface
- adds up to the adherence of lipids
MEMBRANE ENZYME SYSTEM FOUND OF THE SURFACE
OF RBC:
- Form the Active Transport System
a. Na/K ATPase
- Regulates entry of Na & K in & out of the cell in
order to maintain Na as a major extracellular
cation while K is the major intracellular cation
thus when it is impaired there would be
excessive inlet & outlet of cations. If excessive
cation entered, this will result to hypotonicity of
RBC causing now the entry of water, swelling &
lysis of RBC
b. Ca/Mg ATPase
2. 40% LIPIDS
a. PHOSPOLIPIDS
- Serves as a liquid sealer, because it is liquid
stabilized by proteins where they are connected
- Arrangement serves as liquid sealer wherein
there is 2 phospholipid layers containing a
phosphate head which is polar thus it reacts with
liquid environment with hydrocarbon tail which
is hydrophobic meaning it does not interact with
watery environment. The polar heads are
exposed to the watery environment while the
hydrocarbon tail is facing inwards because if
outwards it does not interact with watery
environment.
KINDS:
1. EXTERNAL SURFACE: phosphatidyl choline,
glycolipids & sphingomyelin
2. INTERNAL SURFACE: Cephalin,
phosphatidylinositol & phosphatidylserine
b. CHOLESTEROL
- Amount depends upon the concentration of
plasma cholesterol, bile acids & activity of the
enzyme LCAT
- Maintains regulation of membrane fluidity &
permeability to electrolytes. It also maintains the
surface area volume of RBC
- Water content of RBC depends mainly on its
ionic gradient wherein the greater the ionic
gradient, RBC becomes hypertonic thus water
enters RBC & too much entry of water causes
swelling of RBC & eventually burst & decrease
surface area volume
AMOUNT DEPENDS UPON THE CONCENTRATION OF:
1. PLASMA CHOLESTEROL: if plasma cholesterol
is high, patient has hypercholesterolemia
(liver diseases) macrocytes & target cells are
present, because of loading of some
cholesterol into the cell membrane causing
the cell to widen
2. BILE ACIDS – for solubilization
3. Activity of the enzyme LCAT
3. 10% CARBOHYDRATES
- Some serve as red cell antigens (e.g. ABH antigen
on surface of RBC)
- Often in combination w/ lipids (glycolipid) or
protein (glycoprotein)
- Ex.: Glycophorin A, B , C/D
ABH – blood antigen ABO – blood
group Ab
NOTE:
 A-spectrin, Ankyrin, Band 3, B-Spectrin,
Protein 4.2 & 4.1 & Glycoprotein are
 important in maintaining the shape & size of
RBC & abnormality of these will lead to
deformation of cells.
 Defective A-spectrin, cell will be spherocytes
or pyropoikilocytes
METABOLIC ACTIVITIES IN THE RBCS
- RBC is metabolically active when it is maturing
because when it becomes mature it does not
have nucleus & mitochodria
RULE:
The more immature the RBC, the more metabolically
active it is which is related to the amount of the RNA
which indicates the cellular immaturity & metabolic
activity.
1. EMBDEN MEYERHOF PATHWAY/GLYCOLYTIC
PATHWAY
- Since RBC are already devoid of mitochondria,
glycolytic pathway goes anaerobic.
- Main Function of Anaerobic Glycolysis:
production of the cells energy which is ATP
- Process: for every glucose metabolize, it will
produce 2 ATPs for the glyceraldehyde-3-
phosphate & for 2 ATPs for Dihydroxyacetone
Phosphate thus a total of 4 but the net energy
utilized is a net of 2
- Major source of red cell’s ATP (90-95%)
2. HEXOSE MONOPHOSPHATE SHUNT/PENTOSE
PHOSPHATE SHUNT
- Arises from Embden Meyerhof Pathway but it
undergoes oxidative glycolysis providing 5 –
10% ATP
- Contains the major anti-oxidant of cells
- Main function: generates NADPH and reduced
glutathione (GSH) in the presence of Glucose 6-
PO4 dehydrogenase (G6PD). GSH protects the
hemoglobin from oxidation by peroxides.
- Process: If G6P converts into 6-
phosphogluconate, it produces NADPH & the
enzyme used is G6PD & the substrate is NADP
which is reduced into NADPH which may
become H donor wherein it transfers the H to
ferric iron & ferric becomes ferrous. It also
maintains the glutathione in the reduced form
which also serves as a reducing agent wherein it
detoxifies the toxic radicals (H2O2 – strong
oxidizing agent causing oxidation of lipids & Hb
& this is detoxify into H2O & O2). Glutathione
also brings back the ferric form of iron into
ferrous.
3. RAPOPORT-LUEBERING SHUNT
- Generates 2,3-DPG /2,3-BPG which regulates
the affinity of Hb to O2 by lowering the affinity
promoting release of O2 causing a shift to the
right
4. METHEMOGLOBIN REDUCTASE PATHWAY
- Reduction of methemoglobin by NADPH is more
efficient in the presence of methemoglobin
reductase (cytochrome b5 reductase) which
serves as an intermediate electron carrier. It also
prevents oxidation of heme by reducing ferric to
ferrous with the use of NAD.
CONNECTIONS OF THE 4 PATHWAYS
- EMBDEN MEYERHOF PATHWAY is an anaerobic
& goes as anaerobic glycolytic pathway, mainly
for the production of 90-95% of RBC ATPs. The
HEXOSE MONOPHOSPHATE SHUNT is important
for the production of NADPH & GSH for the
maintenance of Hb iron in the reduced form
because it can reverse the ferric into ferrous.
GSH can also detoxify H2O2 converting it into
H2O & O2. RAPOPORT-LUEBERING SHUNT is
necessary for production of 2,3 DPG which
maintains affinity of Hb to O2.
METHEMOGLOBIN REDUCTASE PATHWAY
produces NADH which serves as reducing
property & may also donate its hydrogen to
cause reduction of ferric into ferrous.
 BREAKDOWN OF RBCs - Process: Hemoglobin is released into the plasma
- As RBC ages, there is a decrease in its enzymes and can be filtered by the kidneys. Plasma
causing slow reaction, thus causing a decrease in haptoglobin and hemopexin salvage the
ATP production & metabolic activity decreases & released hemoglobin so that its iron is not lost in
everything also decreases causing now the urine
distortion thus if more ions enter the RBC, more - Ex.: when RBC is not deformable they undergo
water will enter causing now swelling and size intravascular hemolysis (spherocytes)
also decrease and an increase in density.
- This natural deterioration leads to its
MECHANISM TO CONSERVE THE HB:
phagocytosis. When cells are deformed it will be
1. HAPTOGLOBIN
recognized by splenic macrophages as a damage
- synthesize by the liver & released into the
or senescent RBC wherein spleen filters blood to
plasma
removed old & damage cells (splenicullin), this
- Function: combine w/ hemoglobin preventing
process happens extravascularly. Approximately
Hb from being filtered by the kidneys &
1% of RBC leave the circulation each day & are
hemoglobin-haptoglobin will be brought into the
broken down by mononuclear phagocytic
liver for metabolism. Hb will undergo breakdown
system
by separating heme & globin. Globin will be
recycled as amino acids, & goes back to amino
a. 90% EXTRAVASCULAR (MACROPHAGE-
acid pool. Heme is recycled as iron is
MEDIATED)
immediately utilized or stored as ferritin but
- destruction of senescent red cells by splenic
haptoglobin cannot repeat the function, it will
macrophages
carry Hb to the liver but it cannot repeat its
- release of Hb occurs inside the macrophages &
function, it cannot go back. Thus, in increase
unconjugated Hb is released in the circulation
hemolysis haptoglobin is also depleted.
- Process: Hemoglobin undergoes degradation
- In short: haptoglobin binds w/ free Hb thus
within the macrophage where iron is stored as
forming hemoglobin-haptoglobin & delivered to
ferritin, amino acids of globin are returned to
the liver
the metabolic amino acid pool and
protoporphyrin is converted to bilirubin which
2. ALBUMIN
is released into the plasma and excreted by the
- Process: Hb in plasma becomes oxidized into
liver in bile.
methemoglobin which combines with albumin
forming methemealbumin, this will be brought
b. 10% INTRAVASCULAR DESTRUCTION
into the liver for processing.
(MECHANICAL HEMOLYSIS)
- In short: albumin bind to methemoglobin
- may be caused by the turbulent environment in
forming methemealbumin, & increases in
the circulation
hemolysis due to increase formation
- lysis occurs in the circulation thus release of Hb
is directly in the plasma & increase in
intravascular hemolysis can cause the RBC to
appear pink. Hb is a low molecular weight
protein that can be easily filtered by the kidneys
& be excreted into the urine causing
hemoglobinuria making urine red or pink &
when Hb stayed in room temp. for prolonged
time it will be oxidized into methemoglobin.
When Hb is excreted, it will lose the components
(60% Fe, Protoporphyrin 9, globin & amino acids)
but the body has a compensatory mechanism to
conserve the Hb.
 LABORATORY EVALUATION OF RBC
 RBC count
 Hemoglobin
 Hematocrit
 RBC Indices
 Peripheral Blood Examination
o Blood Smear for differential counting &
morphology examination of RBC (PBS)
 Reticulocyte measurement
 Osmotic Fragility test
 Erythrocyte Sedimentation Rate

CRITERIA FOR PATHOLOGIC RBC

 Size (7-8 um)
 Variation in size (80-100 fL)
 Area of Central Pallor (1/3 & clear)
3. HEMOPEXIN
 Cytoplasm (clear w/out inclusion)
- Process: Hemopexin combines w/ heme. When
heme is separated from methemoglobin it can
be still conserve by binding of hemopexin to
heme forming the hemopexin complex & will be
brought to the liver
- In short: heme bind with hemopexin forming
heme-hemopexin complex

NOTE:
Whether intravascular or extravascular, iron is
recycled.

ANISOCYTOSIS
 increase in variation in size thus increasing RDW a measure of variation in size
 Refers to variation in color after staining which is related to the hemoglobin content of the cell, since it is the
hemoglobin that takes up the stain wherein the appearance of the cell after staining is directly proportional to
the hemoglobin content
CELL TYPE MORPHOLOGIC APPEARANCE DEFECT OR CHANGE ASSOCIATED CONDITIONS
NORMOCYTE Biconcave disc  Normal
 Not all cells are normal there is a classification
known as normocytic anemia which are normal
in size (MCV: 80-100) but there is anemia which
is related to the decrease in production
MICROCYTE  Smaller RBCs  Main cause: Any defect in heme or globin that
 Diameter: <7 µm results in IMPAIRED HB SYNTHESIS thus cells
 MCV= <80 fL undergo extra division & the cells hardly
reached its optimum MCHC
Note: It is efficient to use MCV  Develops from:
because diameter depends on the o ineffective iron utilization, absorption,
smear prepared. or release
o decreased or defective globin synthesis
 Diseases caused:

MACROCYTE  Larger than 10 µm
diameter due to failure of
mitosis
 Diameter: >9 µm
 MCV= >100 fL
o IRON DEFICIENCY ANEMIA: affects
heme
o SIDEROBLASTIC ANEMIA (heme) – iron
precipitate due to protoporphyrin 9
deficiency leading to siderotic granules
& its occurrence in RBC appears as
sideroblast
o THALASSEMIA (globin)
o ANEMIA OF CHRONIC
INFLAMMATION/DISEASE:
inflammation activates macrophages &
T lymphocytes causing increased
production of cytokines that will inhibit
normal iron metabolism thus affecting
heme synthesis
o LEAD POISONING – lead inhibits
enzymes (heme synthase) in heme
synthesis
CAUSES:
a. ACCELERATED ERYTHROPOIESIS – fast RBC
prod. Until faster release of reticulocyte in
circulation as a compensation to blood loss or
lysis. If patient suffers from blood loss or lysis,
blood cell count decreases causing hypoxia &
the kidneys will produce increase
erythropoietin causing acceleration of
erythropoiesis thus the bone marrow will
release even immature reticulocytes
(stress/shift reticulocytes) thus it is larger in
size
b. DEFECTIVE DNA SYNTHESIS – affects nuclear
maturation resulting to nuclear arrest which is
prolonged mitosis due to immaturity of nucleus
which is the central control of the cell due to
deficiency in DNA synthesis thus it undergoes
slow maturation & mitosis leading to large
cells; failure of division
c. IN CONDITIONS WHERE MEMBRANE
CHOLESTEROL AND LECITHIN LEVELS ARE
INCREASED – cell membrane is loaded w/
cholesterol thus increasing cell membrane &
the cell due to hypercholesterolemia this is due
to disturbance in the lipid conc. of plasma &
may be seen in obstructive liver disease
wherein liver is the main organ that
metabolizes fats and where VLDL came from.
Obstructive liver disease leads to incomplete
metabolism of fats thus patients suffer from
hypercholesterolemia.
 HEMOLYTIC ANEMIA & ACUTE BLOOD LOSS –
bone marrow compensates for the blood loss
 or lysis by producing immature RBC thus high
reticulocyte count
 MEGALOBLASTIC PROCESS – affected by
impaired DNA synthesis because cells fail to
divide or suffer from long mitosis resulting to
oval macrocytes
 CHEMOTHERAPY – the chemical infused to
destroy the malignant cells often target the
DNA of cell thus affecting DNA
 LIVER DISEASE - increase cholesterol lipids
causing loading to phospholipid bilayer
 HYPOTHYROIDISM – thyroid is necessary for
metabolism, T3 & T4, for regulating other
hormone
 POST-SPLENECTOMY – not only macrocytes
persist in circulation but also all cell
abnormalities. The spleen filters 5 – 10% of
blood allowing inclusions & abnormal cells to
be removed thus removing the spleen will lead
to no filtration of blood causing abnormality in
circulation.
 ALCOHOLISM
DINMORPHIC RBCs  Presence of 2 distinct populations of red cells that may differ in size, shape or
hemoglobin content thus it may be a mixture of hypochromic cell & normochromic
cell or a microcytic cell or macro
 SEEN IN:
o ANEMIA AFTER TRANSFUSION – after transfusion, on the examination of
patient’s blood smear, there is 2 population of cell, the patient RBC &
transfused RBC, the cells are pale (hypochromic) & microcytic, mixed w/ the
transfused blood (normocytic & normochromic)
o IRON DEFICIENCY DURING THERAPY – cells are microcytic & hypochromic
o IRON DEFICIENCY & VIT. B12 – Iron stored in bone marrow, but storage of
iron is not proportional where the other parts are proportional while the
others are not, thus the developing cells near w/ iron develops as normocytes
while cells w/ developing iron develops as microcytes thus it is a mixture of
microcytic & normochromic cells
o COMBINED VIT. B12 /FOLATE – macrocytosis, 2 most common causes of
megaloblastic anemia DEFICIENCY AN IN SIDEROBLASTIC ANEMIA –
microcytosis, normocytic cell ; a group of anemias that show different
morphology

ANISOCYTOSIS & POIKILOCYTOSIS MACROCYTOSIS GRADING (no

GRADING variation)
Percentage of RBC differ in size or shape from normal RBCs.
Normal 5% 1+ (slight) 25%
Slight 5-10%
1+ 10-25% 2+ to 3+ (moderate) 25-50%
2+ 25=50%
3+ 50-75% 4+ (marked) >50%
4+ >75%
Used if cells vary in size and shape
ANISOCHROMASIA
 variation in the hemoglobin concentration
 absence of uniformity of color
 directly relates to Hb
 Color of cells when stained depends on the Hb
CELL TYPE MORPHOLOGIC APPEARANCE DEFECT OR CHANGEASSOCIATED CONDITIONS
NORMOCHROMIC Homogenous with central Normal
pallor (1/3)
Measure: MCHC
HYPOCHROMIC  RBCs show central DEFECT IN HB SYNTHESIS (HEME/GLOBIN) DUE TO:
pallor (exceeds 1/3 of  decrease in heme synthesis
the diameter of the red  defect in globin chain synthesis
cell)  Iron Deficiency Anemia (IDA)
 Thalassemia
ANULOCYTE/ GHOST  Thin & poorly
CELLS hemoglobinized cells RULE: Not all hypochromic cells are microcytic but all
- very hypochromic (anulocyte) microcytic cell are hypochromic
cells
ANISOCHROMASIA & POIKILOCYTES
SPHEROCYTIC Cells that lack central pallor  Defects in the cytoskeletal proteins
and with reduced diameter  Hereditary and acquired spherocytosis
TARGET CELLS  Central pale area is
filled with hemoglobin
resulting to bulls eye &
further surrounded by
thin rim of Hb
 On side view, there is
protrusion on the
center
POLYCHROMASIA PROBLEM: difference in RNA & Hb among RNA & Hb
 Refers to the display of different shades of color which is Diffused bluish-gray tint of
RBCs & is not directly related to Hb content
 Already fully hemoglobinized but the display of different colors is caused by the
differences in the affinity of the cellular component (Hb & rRNA) wherein Hb takes
up the acidic dye & the rRNA takes up the basic dye
 Exemplified by the fifth stage of erythropoiesis: reticulocytes which is the first non-
nucleated stage but it has remnants of RNA & is fully hemoglobinized

RULE: The more immature, the more RNA it has thus the more chromatophilic it is.
 Not directly related to Hb content but on the RNA content
 The reddish or orange color caused by eosin of Hb & the bluish color stained by
methylene blue of rRNA will be mixed in the cell & the colors produced is mixed
resulting to diffuse coloration of cells
 Increase polychromatic cells indicates immature cell (reticulocyte) which is
synonymous to reticulocyte

APPEARANCE: diffuse coloration, no central pale area

RETICULOCYTE IDENTIFICATION:
 Supravital stains (new methylene blue & brilliant cresyl blue) – appropriate staining
of living reticulocyte & the cells will appear as larger cells w/ granules (RNA).
  Supravital stains precipitate the RNA thus they appear as dark blue precipitate
against pale blue cytoplasm. The granules appear in a network or reticulum thus
called reticulocytes

HYPOCHROMIA GRADING POLYCHROMASIA GRADING

1+ Area of central pallor is ½ of cell diameter Slight 1%
2+ Area of pallor id 2/3 of cell diameter 1+ 3%
3+ Are of pallor is three-quarters 2+ 5%
4+ Thin rim of hgb 3+ 10%
4+ >11%

1+ (slight) Central pallor: 1/3 ro 2/3 of cell’s diameter 1+ 1-3 polychromatophilic cells/ field
2+ to 3+ More than 2/3 2+ 3-5 PcC/f
(moderate)
4+ (marked) Thin rim on the periphery 3+ >5 PcC/f

VARIATION IN SHAPE OF RBC

POIKILOCYTOSIS- increase variation in shape ; named after their shape
PROTEINS – maintain shape & size of the cell, preventing it from collapsing
LIPIDS – main fluid barrier

 Abnormality in proteins, lipids & carbohydrates affects the shape of the cell
CELL TYPE MORPHOLOGIC DEFECT OR CHANGE DEFECT OR CHANGE ASSOCIATED
APPEARANCE ASSOCIATED CONDITIONS
CONDITIONS
OVALOCYTES  Range from egg- HEREDITARY OVALOCYTES:
(ELLIPTOCYTES) shape, slightly oval ELLIPTOCYTOSIS: (25-  Myelodysplastic syndrome
to sausage, rod, or 90%)  Thalassemic syndrome
pencil forms  decrease in  Megaloblastic syndrome –
 Despite of their skeletal megaloblastic
shape they function membrane ELLIPTOCTE:
normally depending CHON band 4.1  Hereditary elliptocytosis
on their hemoglobin  increase heat  Idiopathic myelofibrosis
content, flexibility & sensitivity of  Small numbers may be
deformability spectrin acquired in:
 Some appear o iron deficiency,
normochromic or ACQUIRED: 10% o megaloblastic
hypochromic  No problem or anemia,
meaning they are deficiency o myelophthisic
abnormal in shape affecting the anemia,
but normal Hb protein o thalassemias &
content thus membrane but sickle cell anemia
function normally the cell
 Some are became
normocytic but distorted
elongated sized but because of
the MCV is the different
same. Some are oval condition
& macrocytic
(macroovalocyte)
which is a significant
 observation related
to megaloblastic
anemia
 Hb appears to be
concentrated at the
two ends of the cell,
leaving a normal
central pallor area.

NOTE: Membrane
abnormality for both is the
same
NOTES:
Peripheral proteins can be found on the surface or inner surface underlying the phospholipid layer, these proteins are
connected to the transmembrane proteins & phospholipid bilayer which adds on the integrity thus deficiency in
spectrins, protein 4.1, 4.2 will alter the membrane thus deformed.
SPHEROCYTE  sphere & not  indicates a TWO MOST COMMON CAUSES:
biconcave hemolytic  HEREDITARY
 Smaller in diameter process SPHEROCYTOSIS/INTRINSI
than normal RBC & (hemolysis C MEMBRANE DEFECT –
w/ smaller volume results from a protein or spectrin
because it is fully membrane abnormality, deficiency in
distended due to abnormality) ankyrin or protein band 3.
increase hb thus it cannot Spectrin dimer is
 On side view it has a enter onto the connected to the integral
small biconcavity microcirculatio protein, ankyrin underlies
but very small not n & as they the phospholipid bilayer,
enough to make enter they these add to the integrity
them flexible or immediately of phospholipid bilayer
deformable burst or lyse thus a deficiency in these
 With concentrated protein will make the
hb content phospholipid bilayer,
 No visible or weaker, & some of portion
decrease central of phospholipid will be
pallor easily detached into the
form of microvesicles.
IN SHORT:
A deficiency in spectrin, ankyrin &
protein band 3 leads uncoupling of
phospholipid layer & some of lipids
will be easily detached into the
form of microvesicles. Repeated
detachment leads to a smaller cell
having no central pallor,
spherocyte.

OTHER EXPLANATION:
Weak membrane causing weak
dissociation of deficiency in
cytoskeletal protein

 AUTOIMMUNE
HEMOLYTIC ANEMIA: Ab
 to the red cell will
attached to the red cell w/
their FC portion exposed
which will be recognized
by macrophages that have
Fc receptors on their
receptors & they pit off.
Macrophages pull off Ab
attached on RBC but as it
pulls off, it removes
portion of RBC. The
remaining fragment of RBC
will reseal to form a
smaller cell w/ a filled
volume. Repeated pitting
may occur resulting to
microspherocyte.

SPLENIC CULLING – removal of old

& damage red cell
PITTING – process of removing
inclusions & Ab attached to RBC

OTHER MECHANISMS (LOSS OF

MEMBRANE):
 NORMAL AGING PROCESS:
lose enzymatic properties
& will lack ATP caused by
decreased in enzymatic
activity of enzymes involve
in Embden Meyerhof
pathway resulting to lack
of ability to maintain their
shape & size. Na/K ATPase
needs ATP for activity thus
when cell lacks ATP, this
can no longer be
maintained & transport of
Na/K is compromised & as
an effect the cell can be
crenate or distorted.
 STORAGE PHENOMENON
– after transfusion of
stored blood, patient
blood smear shows lots of
spherocytes but no
abnormality in the
membrane but the cells
undergo change in their
shape due to storage
lesion. During the storage
of blood in blood bag, it
will be consumed, the cell
 will lack glucose to
produce ATP & the main
source is glycolysis, but as
the cell lack glucose this
will lead to lack of ATP
production.
 SEVERE BURNS:
Since phospholipid is a fat,
it is easily dissolve by heat
thus will fragment &
vesicle out of phospholipid
bilayer resulting to
spherocyte & repeated
fragmentation leads to
microspherocyte.
BURR CELL  Microscopically burr  Depletion of BURR CELL
& echinocyte ATP 1. Occur in situations that
appear similar.  Exposure to cause a change in tonicity
ECHINOCYTE hypertonic (hypertonic) of the
 Sea urchin solution intravascular fluid plasma
 Crenation occurs in seen in dehydration &
vitro azotemia related to uremia
 An artefact &
insignificant which 2. ASSOCIATED W/ RENAL
may be seen on INSUFFICIENCY - wherein
thinner part of the kidneys important in
cell removing metabolic waste
 With evenly in the blood, it also filters
distributed, blood but do not remove
uniformly sized 10- abnormally shaped cells &
30 blunt spicules or inclusions. It functions to
bumps remove metabolic
products, dissolve
RULE: Both reactions are chemicals & substances
reversible (salts, acids, & excessive
water). In renal
BURR CELL insufficiency the kidneys
 Very clinically cannot function, resulting
significant which to non-removal of
indicates changed in metabolic products such as
the plasma tonicity the non-protein
 Microscopically the nitrogenous substances,
appearance of the blood urea nitrogen &
spicules is the same creatinine, these
w/ echinocytes thus substances builds up in the
relate in conditions. blood altering the tonicity
 Crenation occurs in thus cells will crenate
vivo while still in the
 Reversible in intravascular fluid.
formation, when
patient underwent RULE: The no. of burr cell indicates
dialysis thus NPN is the severity of renal damage.
cleared out from the
 plasma maintaining ECHONOCYTES
the normal tonicity 1. Several hours old
of plasma anticoagulant blood stored
 With irregularly at room temperature
sized & unevenly causing an artefactual
spaced 10-30 blunt effect due to prolonged
spicules storage at prolonged
temperature
2. Stored blood causes
crenation of cell due to
depletion of ATP &
biochemical activities

RULE: Both reactions are reversible

ACANTHOCYTES  Irreversible  May be caused  ASSOCIATED W/ END
formation by changes in STAGE LIVER DISEASE –
 No longer a disc the ratio of liver is main organ which
shaped, with few (3- plasma lipids. metabolizes & stores fats
12) irregularly Since cell such as cholesterol &
spaced, pointed membrane is lipoproteins but when it is
spicules/thornlike made up of 2 diseased it will not be able
projections of layers of to metabolize the different
various lengths and phospholipids phospholipids &
widths + cholesterol cholesterol resulting to
 Indicates that maintain abnormal ratio in plasma.
permanent the When there is
membrane damage, permeability of hypercholesterolemia due
some are long & membrane to to liver disease this may
short thus it the passage of lead to abnormal ratio of
indicates that the ions. plasma lipids leading to
cell can no longer  Changes in distortion of RBC into
regain its normal cholesterol, acanthocyte.
shape lecithin &
 sphingomyelin NOTE: The conc. of cholesterol
depends on the conc. of plasma
cholesterol, activity of LCAT & bile
acids.

 ALCOHOL INTOXICATION –
affects liver leading to
hepatic cirrhosis, the
alcohol has a direct effect
on RBC
 ALCOHOLIC CIRRHOSIS
 PK DEFICIENCY – pyruvate
kinase is involved in EMP,
it catalyzes the conversion
of phosphoenolpyruvate
to pyruvate wherein ATP is
produced thus deficiency
in PK leads to deficiency in
ATP which is important in
 maintaining cell
membrane
 CONGENITAL
ABETALIPOPROTEINEMIA
– absence of
betalipoprotein which is
related to the inability of
the liver to metabolize it.
LDL is necessary but must
not be in excess thus the
absence of betalipoprotein
leads to red cell distortion
 VITAMIN E DEFICIENCY –
Vit. E is needed for
preventing peroxidase
oxidation of the cell
membrane
POST-SPLENECTOMY – removal of
spleen, all abnormalities will be
seen in the circulation
STOMATOCYTES  Normal sized cell Membrane defect that COMMON CAUSES: HEREDITARY
(MOUTH CELLS) with an elongated results in high cellular  HEREDITARY
HYDROCYTE or a slit like mouth sodium and low STOMATOCYTOSIS – main
or stoma of central potassium content problem in the transport
pallor of Na/K. Due to defect in
 cup-shaped on SEM the transport of Na/K,
 fully hydrated cell there is high cellular Na
due to defect in the thus water enters into the
transport of Na/K cell & the cell becomes
ATPase hydrated & low K
 swollen cell due to  RH NULL DISEASE – second
increase water major blood group (rhesus
content blood group), the Ag in this
blood group are the capital
D indication of positive or
negative & also C & E. Rh
null means no D, C & E Rh
Ag located on the cell
membrane. Rh Ag are
integral part of cell
membrane for the
transport of molecules
thus absence affects the
maintenance of RBC thus
transport of Na/K is
altered leading to
stomatocytosis

ACQUIRED CASES: MOST

COMMON CAUSE:
 Obstructive liver disease
 Alcohol excess or
intoxication
 LESS COMMON:
 myelodysplastic
syndromes
 hydroxyurea therapy –
therapy for leukemia
 Hereditary stomatocytosis
TARGET CELLS  Has a bulls eye, w/ a  Increase in HB DEFICIENCY:
(CODOCYTES)/ button or cholesterol &  Hemoglobinopathies
PLATYCTES/LEPTOCYT concentrate Hb thus phospholipids  Thalassemia
E a dark center  Excess surface  Hemoglobin C Disease or
followed by clear Hb area to volume Trait
area & followed by ratio  Iron deficiency anemia
thin rim of Hb  Any hemoglobin
 May have or have abnormality
not clinical
significance (Hb OTHERS:
deficiency)  Liver diseases – affects
 Large cell w/ small level of plasma cholesterol
Hb as compared to  LCAT deficiency –
its volume or size hypercholesterolemia,
causing the some of cholesterol will
redistribution of Hb load causing the cell to be
into a button of Hb larger in size & Hb content
at the center (Iron  Post-splenectomy
deficiency anemia,
thalassemia,
hemoglobinopathies
)
 With a central area
or hb surrounded by
a colorless ring and
a peripheral ring of
hb
 bell-shaped (codon)
or tall hat shaped on
SEM
 resembles a tall
Mexican hat cell on
SEM
 non-specific finding,
seen on several
findings & are
artIfactual in nature:

1. Drying Artefact –
slow drying due to
humid environment
causing Hb to
crystallize at the
center & dissolve
forming a
concentrated Hb at
 the center resulting
to a target cell
2. Decrease Hb

PLATICYTES - On front view,

it appears as a flat cell
LEPTOCYTE – remains
connected w/ the peripheral
Hb but clinical significance is
the same
NOTES:
ACANTHOCYTE: abetalipoproteinemia TEARDROP CELL: myelofibrosis
BURR CELL: renal disorder TARGET CELL: non-specific

POIKILOCYTES SECONDARY TO TRAUMA

EXCESSIVE PHYSICAL TRAUMA IN THE CVS DUE TO:
1. Presence of fibrin strands causing physical trauma to the red cells passing through leading to fragmentation
2. Blood vessels that are abnormally too small for RBC to circulate

TWO PATHWAYS OF FRAGMENTATION ARE RECOGNIZED

a. Alteration of normal fluid circulation (DIC, TTP, MAHA)
b. Intrinsic defects of the red cell (spherocytes, antibody altered red cells, inclusions
CELL TYPE MORPHOLOGIC DEFECT/S OR CHANGE/S
APPEARANCE ASSOCIATED CONDITIONS
SCHISTOCYTES  Very significant FRAGMENTATION DUE TO:
(SCHIZOCYTES) finding indicating a 1. Exposure of cells to
hemolytic anemia abnormal conditions like
heat (affects RBC in
General term for fragments circulation) or mechanical
of RBCs varying in shapes: trauma
 Helmet cells 2. Contact with fibrin
 Triangular cells strands or damaged
 Keratocytes/Horn endothelium – fibrin
cell deposits & fibrin strands
present slicing of RBC
during their passage. If
the current of blood flow
is strong this will push the
cell to the fibrin strand
causing now
fragmentation seen in
microangiopatic anemia &
disseminated
intravascular coagulation
3. Intrinsic defects of RBCs –
RBC has the problem
 Spherocytes (no more
adjustment in shape
& volume), Ab altered
red cells (recognized
by macrophages &
hemolyze them), Red
ASSOCIATED
CONDITIONS
Hall mark of Hemolytic
anemias
CAUSES:
PROSTHETIC HEART
VALVE – damaged on
artificial heart valve will
exposed artificial
components such as
rubber, causing damage
to RBC & RBC suffer from
physical trauma leading
to hemolysis
MICROANGIOPATHIC
HEMOLYTIC ANEMIA &
DISSEMINATED
INTRAVASCULAR
COAGULATION
SIMILARITIES:
coagulation mechanism
is activated causing now
the formation of blood
clots due to fibrin
threads deposited on the
circulation causing now
obstruction & limit the
normal circulation

KERATOCYTES (HORN KERATOCYTE – pair of
CELL), BITE CELLS, spicule surrounded by a
DEGMACYTES gap
BITE CELL – bite
appearance
 Erythrocytes with a
pair of spicules or
horns surrounding
a gap in the cell
outline
cells containing
inclusions (pitting of
cell causing formation
of bite cell), etc.
BITE CELL: Results from
removal of a Heinz body from
a cell by splenic macrophages
(splenic pitting) or when an
erythrocyte is caught on a
fibrin strand or during pitting.
 When a cell has an
inclusion it will pit off
or pull off by
macrophages thus
removing a part of
RBC & the cells will
assume a bite cell
1. Heinz inclusion on a cell
will pass through a limited
basement membrane
leading to physical
trauma. If clots are
bigger the more
obstruction which will be
difficult for RBC to pass
thru.
DIFFERENCE:
MAHA – abnormal
formation & deposition
of blood clots occurs in
blood vessels
DIC – affects all vessels
since it is disseminated
CLOSTRIDIAL
INFECTIONS – toxin
produced damages RBC
& activate coagulation to
cause abnormal clot
formation
THROMBOTIC
THROMBOCYTOPENIC
PURPURA & HEMOLYTIC
UREMIA SYNDROME
SIMILARITIES: platelet
aggregates causes
obstruction of circulation
due to abnormal
activation of platelets
wherein when platelets
are aggregated they form
together to form a
thrombus
Hemolytic anemias
G6PD deficiency
 2. Rigid portion will be left &
SEMILUNAR BODIES  Half-moon / crescent
cells
 Large pale pink
staining ghost of the
RBC
 Complete round w/ a
visible crescent shape
of Hb or complete
crescent
TEARDROP CELLS  Pear-shaped or
(DACROCYTES) teardrop-shaped
w/ a single
elongated point
 One end is
rounded the other
end is pointed
resulting to a pear,
drop or rocket
shape
 Hall mark of
myelofibrosis
if able to pass through it
will become a teardrop
cell but if removed RBC
will reseal & adjust the
volume & shape
 Frequently observed Overt Hemolysis
in malaria which is a
hemolytic disorder &
in other conditions
causing overt
hemolysis.
 As RBC is hemolyze it
will release its Hb
content & only a small
portion will be left
that is remain
attached on the
membrane indicating
an overt hemolysis
 Caused by excessive MOST COMMON:
squeezing & results to
fragmentation during MYELOFIBROSIS W/
splenic passage MYELOID METAPLASIA –
 Vessels constricted bone marrow is
causing RBC to infiltrated by fibrous
squeeze itself which materials, thus there is
causes too much overgrowth of fibrous
squeezing resulting to cells thus if bone marrow
permanent damage & is unable to produce
may no longer revert blood cells resulting to
to its shape thus metaplasia & spleen is
loosing capacity to involve in the production
regain normal shape of RBC which may suffer
also of fibrosis causing a
limitation of its
circulation. Blood vessels
in the spleen are
normally small or minute
but in myelofibrosis the
more they become
limited thus increase
microcirculation causing
teardrop cell which is the
hall mark of
myelofibrosis.
HYPERSPLENISM –
spleen enlarge causing
circulation to be limited
PRESENCE OF INCLUSION
BODIES – makes RBC

MICROSPHEROCYTES &  Are red cell
PYROPOIKILOCYTE
 MCV 60 fL
 Smaller than
 Also lack central
OTHER APPEARANCES RELATED W/ HEMOLYSIS:
Results from repeated
fragments fragmentation of RBC & from
thermal damage to cell
membrane
spherocytes
KNIZOCYTES – appear like
pinched bottle having
rigid like Heinz bodies
which commonly
attached on the
membrane of RBC
making the membrane
rigid & when it passes
through the splenic
circulation which is too
limited, it has to squeeze
itself leading to its:
1. Detached & cell
becomes a bite cell
2. Pulled & be pointed
because it is the last to
exceed the membrane
causing a tear drop shape
MYELOPHTHISIC
ANEMIA – bone marrow
is infiltrated w/ abnormal
cell like in leukemia thus
circulation becomes
limited
UNCOMMON:
Pernicious anemia
B thalassemia
MICROSPHEROCYTE:
Severe burns – thermal
injury on RBC membrane
wherein fats are easily
damage causing the
membrane to vesicle, if
detachment is mild it will
result to spherocyte but
if there is continuous
fragmentation, it will
become the largest
fragment, the
microspherocyte
PYROPOIKILOCYTE:
Hereditary
pyropoikilocytosis –
there is a membrane
damage of red cell due to
sensitivity to heat. A
normal RBC fragments at
49 C but cells with HPP
will fragment at low T
(45-46)
PYKNOCYTES – distorted
or irregularly contracted
BLISTER CELL – with thinned portion due to cell caught constriction on one of its part red cells, seen in
on fibrin strands wherein it might have encountered indicating that it might blister hemolytic anemia
along the circulation such the portion of the membrane or bud off wherein the bud
is pull apart. may detached & become
defragment. The remaining
fragment, the larger one may
become a spherocyte or the
smaller being
microspherocyte, seen in
hemolytic anemia normally in
infants but w/ lesser number

POIKILOCYTES & CRYSTALS SECONDARY TO ABNORMAL HEMOGLOBIN CONTENT

CELL TYPE MORPHOLOGIC DEFECT/ CHANGES ASSOCIATED CONDITIONS
APPEARANCE
DREPANOCYTES/SICKLE  Most insoluble of  Polymerization of Sickle cell anemia
CELLS the Hb variants hemoglobin Indicates Sickling Hb
 Fully soluble Hb  Sickling is
when exposed to reversible when
normal O2 but exposed to normal
when O2 but repeated
deoxygenated it sickling causes
becomes insoluble permanent
& crystallizes at damage on the
low O2 tension. membrane &
The crystals are assumes
thin & elongated
with pointed ends
altering the shape
of the cell having a
curved, straight L,
V, & S shape

HB CRYSTAL HB CC CRYSTAL
 With one or more  Hexagonal
fingerlike, blunt-pointed  Both ends are blunt &
projections that protrude rectangular
from the cell membrane  Parallel sides
leaving a pale area at the  Formed within the membrane (RBC) but does not
opposite end deform RBC causing a normal shape but it makes
 Has more than one protrusion & the longest one RBC rigid thus the RBC is not deformable &
protrude away flexible thus prone to hemolysis
 Washington monument, Pointing finger, Hand  Bar of gold
glove  Crystalizes when Hb is O2 or dehydrated
 INCLUSIONS: ABNORMAL HEMOGLOBIN PRECIPITATION
REASON:
1. Precipitation of abnormal component in the cell
2. Disturbance or abnormality in the developmental or maturation of RBC (developmental organelles)
INCLUSION MORPHOLOGIC DEFECT/S OR CHANGE/S ASSOCIATED CONDITIONS
APPEARANCE
HEINZ BODIES  Large (1 to 3 Results from  G-6-PDH DEFICIENCY
um), single or precipitation of ANEMIA – most common
multiple small, hemoglobin that has disorder associated w/ Heinz
round, oval or been denatured body. G-6-PDH is an involve
serrated, in hexose monophosphate
purplish shunt. When G6P is
inclusions on RBC converted into 6PG catalyze
periphery by G6PD & the cofactor is
(distorts the NADP which becomes
membrane) NADPH a reducing property
depending on of RBC which in turn
the oxidation maintains GSH into reduced
form, which serves as the
STAINS (SUPRAVITAL major antioxidant property
STAINS): Crystal violet & of RBC which can breakdown
Brilliant Cresyl Blue H2O2 into H2O & O2
preventing oxidation of Hb.
The NADPH formed will
reduced the ferric back to
ferrous thus protecting RBC
to oxidation. Deficiency will
result to deficiency in NADPH
& GSH
 RED CELL INJURY DUE TO
CHEMICAL INSULT OR
UNDER OXIDANT STRESS
(sulfonamides) – causes
accumulation of oxidized Hb
 HEMOGLOBINOPATHIES
AND THALASSEMIA MAJOR
 UNSTABLE HEMOGLOBIN
SYNDROMES
 ZURICH & KOLN HB –
abnormal Hb prone to
precipitation & oxidation,
unstable Hb
 POST-SPLENECTOMY
HB H INCLUSIONS  Small greenish- Precipitate of Hb H, an Seen in Hb H disease
blue inclusion abnormal Hb which has 4 Alpha Thalassemia
bodies polypeptide chains as B
 Seen in occurs when A chain is
unstained cells not completely
 May also be seen synthesize
w/ supravital
stain
 Golf ball w/ pits
when numerous
in RBC
HOWELL-JOLLY BODY  Small, coarse  Nuclear CAUSES:
round granules remnants  SPLENECTOMY
 deep blue or red-  with Feulgen &  FUNCTIONAL
purple, appears Methyl Green HYPOSPLENIA - spleen
singly & is off the Pyronin stains w/ decreased function
center but not at (stains for DNA) thus unable to remove
the periphery  seen when there inclusion
 DNA containing is increase
inclusion karyorrhexis DISORDERS ASSOCIATED W/
meaning it is a (fragmentation KARYORHEXIS:
nuclear fragment of nucleus) •ACCELERATED OR ABNORMAL
 Very tiny ERYTHROPOIESIS
inclusion w/ <1  hemolytic anemias – too
um much RBC are
hemolyzed thus bone
marrow compensate by
accelerating RBC
production thus nuclear
dissolution is also fast &
more nuclear fragments
will be seen
 megaloblastic anemia –
caused by abnormal
nuclear dissolution (Vit.
B12 & Folate deficiency)
PAPPENHEIMER BODIES  small (2-3 um)  Unused Iron  SIDEROBLASTIC
SIDEROTIC GRANULES faint blue deposits ANEMIA – red cell
coccoid bodies precipitate & cannot make use of the
that aggregate in become siderotic iron due to deficiency in
small clusters at granules protoporphyrin 9 thus
the periphery of  Sometimes may iron precipitate &
the cell. appear as Ringed becomes siderotic
 Smaller than H-J sideroblast granules
bodies, deep  THALASSEMIA – affects
blue & are more globin
likely to be  HEMOGLOBINOPATHIES
multiple - affects globin
 Iron containing  ALCOHOLISM – affects
granules positive enzymes in heme
when stained w/ synthesis
Perls Prussian  POST-SPLENECTOMY
 blue & named as  CONDITIONS LEADING
siderotic TO
granules & if HEMOCHROMATOSIS or
positive when HEMOSIDEROSIS (iron)
stained w/ – both increase iron or
wrights/giemsa it iron overload
is named as
pappenheimer
bodies
BASOPHILIC STIPPLING  Dark blue Aggregates of ribosomes DEFECTIVE OR ACCELERATED
granules, and mitochondrial HEME SYNTHESIS
multiple, uniform remnants thus acidic in  Lead or other heavy
and evenly component because they metal poisoning –
distributed as took up the basic dye affects hb synthesis
fine dots or large due to rRNA  Thalassemia - globin
granules  Hemoglobinopathies -
 Often confused globin
w/  Megaloblastic anemia -
pappenheimer rarely
bodies (+
Prussian Blue) PYRAMIDINE-5-NUCLEOTIDASE
but basophilic DEFICIENCY
stippling do not Explanation:
stain w/ Prussian When RNA degrades, they
blue thus it is not release their purine &
seen due to lack pyrimidine bases. For the
of iron but if complete degredation of
present in wright pyramidine bases, it needs
& Prussian then Pyramidine-5-nucleotidase thus
it is siderotic deficiency of Pyramidine-5-
granules nucleotidase, RNA degradation
will also be defective or
APPEARANCE: incomplete
COARSE: granules are
much more outlined and
easily distinguished &
larger
PUNCTATE: coalescing
into smaller forms
DIFFUSE: Granules are
fine blue dusting
CABOT RINGS Reddish-violet thread- Remnants of Dyserythropoiesis
like that assumes a loop, microtubules of mitotic megaloblastic anemias
or an incomplete ring or spindle & can reshape Homozygous
a figure of 8 into a loop or a fragment Thalassemias
of the nuclear  Lead poisoning
membrane  Other severe anemia
 Post splenectomy
STRUCTURES NOT SEEN IN WRIGHTS: STRUCTURES NOT SEEN IN WRIGHTS:
1. Granules of reticulocyte 1. Polychromatophilic cell
2. Siderotic granules 2. Pappenheimer bodies
3. Heinz bodies
 ABNORMAL RED CELL DISTRIBUTION
AGGLUTINATION Clumping of RBCs Presence of antibodies on  Cold agglutinin
Indication of Ag-Ab the RBC surface produced  Autoimmune
reaction by patient (autoAb) or from Hemolytic Anemia
different person (isoAb)

ROULEAUX  Linear alignment of Increased concentration of  HYPERPROTEINEMIA’S

RBCs appearing as globulin – plasma protein
stack of coins component is altered
 RBC due to resulting to alteration of
destruction of zeta the negative charges
potential surrounding the RBC
 Side by side flat which is seen in
surfaces Multiple myeloma &
 biconcave surfaces Waldenstrom’s
in apposition macroglobulinemia
appearing as a which are plasma cell
stack of coins or discrasias which are
flat plates leukemias affecting
plasma cell causing
increase proliferation of
plasma cells

 MULTIPLE MYELOMA –
most of the affected
plasma cell will
produce excessive IgM

 WALDENSTROM’S

PROTOZOAN INCLUSIONS
 RBC is normal but infected by hematozoic parasite
a. PLASMODIUM SPECIES
• Seen in different stages of smear mostly ringed forms

1) MALARIAL INCLUSION
• Obligate intraerythrocytic parasites
• w/ hepatic stage (liver) where they rapture the liver & proceed to
rupture the RBC & metabolize the Hb but not completely & leave behind a
pigment (hematin or hemozoin pigment)

PIGMENTS INSIDE THE INFECTED RBC:

a. P. vivax - Schuffners dots
b. P. falcifarum – Maurer’s dots
c. P. malariae – ziemann’s dots
d. P. ovale – james dots

2) BABESIA PARASITE
• Parasite of cattle’s causing red fever on cattle’s
• Discovered by babes & may also affect men
• w/ ring forms in RBC
 ARTIFACTS
a. FIXATION b. HEAT ARTEFACTS
• When fixative is contaminated w/ water which EFFECTS:
must be water free methanol 1. Red cell bud off into vesicles which might be reported
EFFECTS: microspherocytes
1. Refractile rings in red cells 2. WBC disintegrate
2. Mouth eaten cells – caused by blowing leading to 3. Proteins coagulate producing small weak basophilic
spicules particles which are similar to platelets

WHITE BLOOD CELLS

CLASSIFICATION OF WBC
a. Granulocytes (neutrophil, eosinophil, basophil) & Non-granulocytes (lymphocytes & monocytes)
 Non-granulocytes have granules however too small to be seen
 Cytoplasm of monocytes appear like a ground glass due to the tiny granules present

b. Polymorphonuclears (those w/ segmented or lobulated nuclei – neutrophil, eosinophil & basophil) &
Mononuclears (unsegmented nucleus – monocytes & lymphocytes)
c. Phagocytes (non -specific function – neutrophil (most active phagocyte), monocyte & macrophages,
eosinophil & basophil (both are less efficient phagocytes) & Immunocytes (involve in production of Ab – B
cells)

Formation: Bone marrow & lymphoid tissues (thymus (T-cells), spleen & lymph nodes)
General Function: Defense
NON-MALIGNANT/REACTIVE CHANGES OF WBCS
CYTOPLASMIC MORPHOLOGIC DEFECT OR CHANGE ASSOCIATED CONDITION/S
CHANGES APPEARANCE
TOXIC  Large purple to Altered primary  SEVERE INFECTION – a reaction of the
GRANULATION black azurophilic granules infection
granules especially EXPLANATION: during infection since
in the neutrophils  Neutrophils have neutrophil is involve in this mechanism, it
which contains primary granules undergoes several changes:
primary granules. but when it is 1. EXEMPLIFY & ENHANCE ENZYME
 Larger than 2- altered, it PRODUCTION as they need to
degree granules & appears as a toxic destroy the invading organism by
stain dark blue-black granulation which phagocytosis. The phagocytized
are larger than material must be destroyed thus the
the usual primary cell produces lots of enzymes to
granules perform respiratory burst & must
also alter its granulation by
increasing the function of its
granules. Enzymes such as the
myeloperoxidase is contained in the
primary granules. As the cell
increase its function w/ the
granules, the granules also function
to increase their content resulting to
alteration & toxic granulation.
2. SHIFT TO THE LEFT – even immature
WBC will be released as a
 compensation for the severe
infection
3. INCREASED PHAGOCYTIC ACTIVITY
– in infection, the neutrophils have
to phagocytized the invading
organism & the phagocytized cell
will be contained inside the vacuoles
& it will appear as many toxic
vacuolation & granulations. The
cytoplasm also appears as Dohle
bodies as larger inclusion.
 CHEMICAL POISONING
 TOXIC STATES

 Toxic granulation is a normal reaction in

these conditions
DOHLE  Blue, round, or Aggregates of free INFECTION – due to increase activity of the
BODIES/AMATO mostly elongated ribosomes cell
bodies
 Represents rRNA CELLS INCREASE METABOLIC ACTIVITY AS A
 Not permanently RESPONSE IN:
seen in cytoplasm  Severe infections, burns,
except for may- Chemotherapy, surgery
hegglin anomaly  If these conditions disappear,
 Single or multiple Amato bodies will also
arranged in parallel disappear
rows
MAY-HEGGLIN ANOMALY – an inherited
condition, thus appearance of amato is
permanent

 Basophilic stippling: If RNA

precipitates & seen as granules in
RBC
 Amato bodies: if RNA precipitates &
seen as granules seen inside of WBC
 RNA is important in metabolism, as
the cell increases its production of
enzyme, this will increase metabolic
activity thus increased RNA function.
CYTOPLASMIC  Many presence Represents end stage SEPTICEMIA, SEVERE INFECTIONS, TOXIC
VACUOLATION indicate phagocytosis of phagocytosis STATES
particularly the end
stage  During phagocytosis the phagocytize
 Presence of many material will be contained in a vacuole
toxic vacuoles thus increasing the vacuole inside the
containing the neutrophil. Once in the vacuole the
phagocytized phagosome will fused w/ lysosome
material w/ the forming the phagolysosome. Once
contents of lysosome destroyed the debris will be contained in
or debris of the a vacuole
phagocytized
material
  Large empty areas
within the cell
ATYPICAL/  Cells show foamy & Seen in INFECTIOUS
REACTIVE abundant cytoplasm MONONUCLEOSIS/KISSING DISEASE –
LYMPHOCYTE/  ≥40% associated w/ caused by Epstein barr virus
DOWNEY CELLS infectious
mononucleosis

INHERITED DISORDERS
CYTOPLASMIC CHANGES MORPHOLOGIC APPEARANCE ASSOCIATED CONDITION/S
ALDER-REILLY  Large, coarse blue – black granules Associated w/
GRANULATION in all WBCs MUCOPOLYSACCHARIDOSIS/G
 Abnormal polysaccharide granules ARGOYLISM – abnormalities in
which are not completely mucopolysaccharide
metabolize due to an enzyme metabolism
deficiency (congenital disorder) which leads to non-  Hurler’s
metabolism of mucopolysaccharide & prevents the  Hunters
formation of secondary granules

MAY-HEGGLIN  Resembles Dohle bodies but are permanently LEUCOPENIA, VARIABLE

ANOMALY present THROMBOCYTOPENIA, GIANT
 Presence of gray – blue spindle shaped inclusions in PLATELETS
the cytoplasm of Neutrophils and Monocytes

FUNCTIONS OF NEUTROPHIL:
1. DIAPEDESIS – squeezing out through the circulation via the endothelial lining & proceeds via:
Random motility – no particular direction
Direct motility/Chemotaxis – w/ particular direction
2. PHAGOCYTOSIS – killing of phagocytized agent
INHERITED ABNORMAL GRANULOCYTE FUNCTION
CYTOPLASMIC DESCRIPTION
CHANGES
JOB’S SYNDROME  Impaired directional motility
 Random movement of phagocytes is normal thus neutrophil may still move
elsewhere but directional motility is impaired because cells respond slowly to
chemotactic agents thus do not directly proceed to the chemical attractant.
EFFECT: it gives more time for the bacteria to proliferate or cross the infection. If bacteria
are dislodged in the tissue, & immediately phagocytized thus it will not be able to cause
infection but due to slow movement of cells, the bacteria will be able to lodge in the site of
infection causing infection.
LAZY LEUKOCYTE  Both random and directional movement of the cells are defective thus neutrophil
SYNDROME will only stay but the organism will approach the neutrophil thus phagocytosing it
  Cells fail to respond to inflammatory stimuli but have normal phagocytic and
bactericidal activity.

NON-MALIGNANT: DEFECTIVE KILLING OF MICROORGANISMS

CYTOPLASMIC DESCRIPTION
CHANGES
CHEDIAK – HIGASHI 
Characterized by large, blue to grayish round granules or inclusions in Neutrophils
ANOMALY that vary in size and color
 Stain positive with peroxidase, Sudan black B (SBB) and Acid phosphatase (ACP)
stains.
 Represents the fused primary granules. Primary granules contain the usual
composition such as peroxidase, sudan black b & ACP but these are abnormally
package, meaning they are normal in contents but abnormally package, thus
appears as chediak-higashi anomaly.
IN SHORT: granules are normal in content but abnormally packaged thus it cannot perform
its normal function
CHRONIC  Phagocytes ingest all but cannot kill catalase (+) organism because of lack of
GRANULOMATOUS appropriate respiratory burst
DISEASE  Neutrophils have normal direct & random motility & phagocytosis

RESPIRATORY BURST – reaction inside the neutrophil needed for the killing of organism.

In respiratory burst the neutrophil is able to produce potent oxygen radicals like H2O2 &
O2 for killing organisms. In chronic respiratory disease, the respiratory burst function of
neutrophil is decreased or inappropriate thus inadequate production of H2O2 & O2 which
are important bactericidal agent.

Catalase + Organisms: Staph aureus – produce enzyme catalase which will breakdown
H2O2 into H2O & O2

OTHER APPEARANCES
TART CELL  A monocyte (phagocyte) that engulf a lymphocyte (engulf material)
 Formation is no known significance

FERRATA CELL  a stimulated or atypical lymphocyte with denser and more opaque cytoplasm
 associated with SBE
REIDER CELL  a lymphocyte with notched, lobulated, segmented, clover – leaf like nucleus
 associated with Chronic Lymphocytic Leukemia
L.E. CELL (LUPUS  Neutrophil engulf the lysed nucleus of another neutrophil containing a large purple
ERYTHEMATOSUS) homogenous round inclusion (phagocytized cell) with nucleus wrapped around the
phagocytized material which occupies the entire cytoplasm of neutrophil thus
neutrophils nucleus is pushed around the side appearing as a wrapping itself into the
phagocytized cell
 For it to occur there has to be a neutrophil, ANA or L.E. factor & nucleus of another
neutrophil
 Seen in SLE & other autoimmune disorders because it is related to the presence of
anti-nuclear Ab (ANA)
HAIRY CELL  A lymphocyte affected by hairy cell leukemia
  with abundant hair-like cytoplasmic projections

SEZARY CELL  Round lymph cell with nucleus that is grooved or convoluted
 Seen in Sezary syndrome which is an advanced case of mycosis fungoides. Both are T
cell disorder but they represent different stage of the disorder:
Mycosis fungoides – only skin is affected by T cells ; initial stage
Sezary Syndrome – when affected T cell migrate deeper into the dermal layer, lymph
nodes & visceral organs
 Mononuclear cell w/ a brain-like nucleus or cerebriform nucleus & a narrow rim of
cytoplasm
FLAME CELL  plasma cell with red to pink cytoplasm that appears like a flickering candle
 Associated with increase in IgA associated w/ multiple myeloma which is a malignancy
of plasma cell which is responsible for the production of Ig.

GRAPE CELL/ MOTT  plasma cell that contains small colorless (or blue or pink) vacuoles or globules
CELL/ MORULA CELL  large protein globules giving the appearance of grapes
 blue or pink protein that are excessively produced in multiple myeloma

RUSSEL BODIES  accumulation of IgG found in the cytoplasm & also seen in plasma cell

DUTCHER BODIES  intracellular crystalline structures of abnormal IgG

 inclusions of nucleus

NON-MALIGNANT/REACTIVE CHANGES
UCLEAR CHANGES MORPHOLOGIC APPEARANCE DEFECT OR CHANGE ASSOCIATED
CONDITION/S
HYPOLOBULATION 
Neutrophils have single or Decreased segmentation PELGER-HUET ANOMALY:
bilobed nuclei TRUE – inherited
 The 2 lobes appear as a PSEUDO – acquired
peanut or dumbbell shape or
a pince-nez spectacles (pair of
eye glasses)
HYPERSEGMENTATION  Neutrophils show more than 4 Abnormal DNA synthesis Megaloblastic anemia
lobes  Cells are abnormally
 Abnormal in size & lobulation large
& large as a macropolycyte
2 TYPES:
Normal size of neutrophil: 9-15 a. B 12 deficiency
um anemia
b. Folate deficiency
anemia
 OTHER NUCLEAR AND CYTOPLASMIC CHANGES
PYKNOTIC CELL  shrunken and dehydrated nucleus of cells that are about to die

PYKNOSIS – cell dissolution

SMUDGE CELL  Nuclear remnant of lymphocyte, seen after smear preparation

 Thumbprint appearance
 Associated w/ Chronic Lymphocytic Leukemia wherein cells are fragile thus they are easily
smudge.

BASKET CELL  Nuclear remnants of granulocytic cell

 Seen in Chronic Granulocytic Anemia
 Basket appearance

BARR BODY  Drumstick like body protrudes away from some of segments of neutrophils & attached on
one of the lobes of neutrophil nucleus
 Indicate an extra X chromosome & seen in females

AUER RODS  Pink or red rod-shaped structures

 Fused primary granules seen in immature neutrophils
 If seen in bundles this are called faggot cells
 Seen in Acute Myeloid Leukemia

INHERITED DISORDERS OF MONOCYTES & MACROPHAGES

Monocytes & macrophages are also involved in phagocytosis & also dispose of. They help in metabolism of lipids &
carbohydrates where they need enzymes & deficiency of these enzymes leads incomplete metabolism thus
incomplete disposal. Non-metabolized substances are contained in the cytoplasm causing macrophage overload.
DISORDER DESCRIPTION ENZYME DEFICIENCIES
MUCOPOLYSACCHARIDOSES  Deficiencies of specific enzymes involved I Hurler’s (most common)
/GARGOYLISM in the degradation of IV Morquio – Ullrich
mucopolysaccharides II Hunter’s
 Non-metabolized mucopolysaccharide will V Scheie
be contained in the cytoplasm of the cell III Sanfilippo
as granules VI Maroteaux – Lamy
 Alder-Reilly granules represents the non-
metabolized mucopolysaccharide. It is
associated w/ the enzyme deficiencies.
LIPIDOSES (abnormal lipid metabolism)
DISORDER DESCRIPTION & ENZYME DEFICIENT
GAUCHER’S DISEASE (MOST  lack of  - glucosidase resulting inability to metabolized glucocerebroside
COMMON) which will accumulate of in the spleen, liver and bone marrow

Gaucher cell – cytoplasm is distended with many glucocerebroside; sponge-like

appearance, crinkled or crumpled tissue paper
NIEMANN – PICK DISEASE
SEA –BLUE HISTROCYTES
 deficiency of sphingomyelinase resulting to accumulation of sphingomyelin
and cholesterol in the macrophages
 Foamy appearance
 w/ non-metabolized lipid-rich granules that stain blue green with polychrome
stains
 accumulation in the spleen and BM of histocytes giving an appearance of a
blue sea
 s/s: hepatomegaly, thrombocytopenia

FABRY’S DISEASE  lack of a-galactosidase resulting to accumulated glycolipid trihexosyl ceramide

but will not give an unusual appearance of macrophage
WOLMAN DISEASE  deficiency of acid esterase resulting to accumulation of TG and cholesterol
TANGIER’S DISEASE  Patient is unable to produce HDL (good cholesterol) which responsible for the
metabolism of the cholesterol by bringing it into the liver to prevent
hypercholesterolemia, as a result, cholesterol esters accumulate

TESTS FOR NEUTROPHIL FUNCTION: EVALUATION OF RESPIRATORY BURST

NTR (NITROBLUE  screening test for neutrophil function (ability of neutrophils to attack bacteria)
TETRAZOLIUM  test for the ability of normal neutrophil to reduce normal yellow water-soluble dye
REDUCTION) TEST into an insoluble blue formazan
 abnormal neutrophils are unable to reduce the yellow water-soluble dye into blue
formazan
CHEMILUMINESCENCE  Based on the ability of the cell for emission of low-level light pulses by
stimulated cells.
 Normal neutrophils when not stimulated or in resting state do not emit light
but when stimulated they emit a low level of light.
 Abnormal neutrophils w/ impaired respiratory burst cannot emit light whether
stimulated or resting
BOYDEN MICROPORE  Utilizes a micropore filter where neutrophils are counted. This is placed on an
FILTER abrasion to determine the ability of neutrophils to migrate
 Tests migration of neutrophil (fastest to migrate) in response to a chemotactic
factor.
REBUCK SKIN WINDOW  Requires performance of superficial abrasion on the skin to evaluate the
speed, type and number of phagocytes that respond to a skin abrasion.
 On top of the abrasion is the slide. After, perform a count on the slide &
observe.
 PLATELET
 Cytoplasmic fragments of megakaryocyte
 Small packages of cytoplasm that are nipped off from
the cytoplasm of megakaryocyte
 Are complex and metabolically active. They function
in hemostasis by interacting with their environment
to initiate and control hemostasis
GENERAL CHARACTERISTICS OF THROMBOCYTES
Composition: 60% proteins, 30% lipids, 8% CHO,
minerals, water, nucleotide, glycogen & glucose (main
ATP source)
Shape & Origin: Thin disc cytoplasmic fragment
Diameter: 2-4 um
Volume: 5 – 7 fL
Reference Platelet Count: 150,000 – 450, 000/ uL
Daily Turnover: 35 x 109/L (+/- 4.3) & then destroyed by
the spleen & produced by the bone marrow
2 pools: 1/3 spleen & 2/3 circulation
Lifespan: 8-11 days in vivo
Function: Hemostasis or stoppage of bleeding by
forming into plugs for the maintenance of vascular
integrity & continuity & blood coagulation
HEMOSTASIS
During vessel injury the blood contained in the
vasculature may leave the circulation (extravasation).
The blood vessels will constrict to limit the entry of
blood into the vessel causing limited blood loss. The
constriction of vessels is aided by thromboxane A2 &
serotonin from the platelets. Platelets immediately
form into plugs to prevent further bleeding. The plug is
an initial plug, w/ the current of flowing plug, the plug
may be removed or dislodge thus bleeding continues.
When secondary hemostatic mechanism is activated
when fibrin is form of fibrinogen, the fibrin thread will
be measured around the platelet plug, stabilizing now
the platelet plug thus further enhancing the hemostatic
effect of platelets.
NOTE:
In the 4 stages of megakaryopoiesis, only the
metamegakaryocyte (MK 4) with a 4 nucleus or more,
will be able to shed off as platelet.
 THROMBOPOIETIN – growth factor for
megakaryopoiesis produced mainly by the
liver & some in kidneys
 ENDOMITOSIS – division of nuclear
components but no cellular division thus as
cell mature it increases in size
 PLATELET BITS/STRING/RIBBON – hydrolytic enzymes
interconnected platelets indicating newly
released form BM which may be increased in
number in cases of hemolytic anemia &
hemorrhagic anemia
PLATELET STRUCTURE: FUNCTIONAL PLATELET
ZONES
A. PERIPHERAL ZONE: outermost zone
 Glycocalyx bears the different glycoproteins (Gp
1a, 1b, 1c, 2a, 2b, 3, 4, 5) which are not present
in other cell membrane. These glycoproteins
serves as surface for the adherence of other
coagulation factor (CF 1, 5, 7, 11, 12, 13)
Ex.: fibrinogen converted into fibrin on the
surface of platelets
Platelet can only aggregate to another
platelet if it has the important glycoprotein
for aggregation
NOTE:
SUB-MEMBRANOUS AREA: Underlies the plasma
membrane, this contains the communication w/ the
external environment of cell via receives message
from the outside
B. SOL-GEL ZONE:
 Consists of a stable gel that regulates
arrangement of internal organelles.
 Contains communication of organelles &
maintains location of the different organelles
 Where important protein serving as
cytoskeleton of the cell is located
MICROTUBULES (tubulin) & MICROFILAMENTS
(actin & myosin) – provide the cytoskeleton thus
maintaining the discoid shape of platelets
 When actin & myosin interacts, they form
into actomyosin where they form into
thrombosthenin
NOTE:
THROMBOSTHENIN – important contractile acting
force, provides contractile force after activation of
platelets.
C. ORGANELLE ZONE:
 Where the different organelles, alpha granules
(most abundant), dense bodies, mitochondria,
lysosomes and peroxisomes are located.
 Alpha & dense granules are for storage of
important contents while mitochondria for
ATP production, lysosomes containing
 ALPHA GRANULES CONTENT ROLE IN SUBSTANCE SOURCE
a. High molecular weight kininogen – cofactor in HEMOSTASIS
coagulation system Promote High Molecular Alpha granules
b. Factor 5 – cofactor for coagulation system coagulation Weight
c. Fibrinogen – same as coagulation factor 1, when Kininogen
acted by thrombin it is converted into fibrin thus it Fibrinogen;
is the precursor of fibrin Factor V von
d. Von Willebrand Factor – for platelet adhesion Willebrand
e. Thrombospondin – platelet function Factor
f. Platelet Factor 4 – platelet function Promote ADP; Calcium Dense bodies
g. Beta thromboglobulin – blood vessel repair aggregation Platelet factor 4 Alpha granules
h. Platelet Derived Growth Factor – blood vessel repair Thrombospondin Alpha granules
 When a vessel is injured, it will undergo healing Promote Serotonin Dense bodies
where smooth muscles & fibrous tissues must vasoconstriction Thromboxane A2 Membrane
regenerate. Beta thromboglobulin & PDGF both precursors phospholipids
stimulate smooth muscle repair & chemotactic Promote Platelet derived Alpha granules
to fibroblast vascular repair growth factor
i. Plasminogen – when acted on by its activator, Beta
converts into plasmin ; important for fibrinolysis thromboglobulin
Fibrinolysis – dissolution of the clot by plasminogen Other system Plasminogen Alpha granules
affected Alpha 2-
j. A-2 antiplasmin – Inhibits plasminogen to prevent antiplasmin C1
excessive clot dissolution esterase
k. C1 esterase inhibitor – found in complement system inhibitor

DENSE GRANULES: CAMAS

a. ADP – for platelet aggregation & adhesion
b. ATP – for platelet aggregation & adhesion
c. Calcium – for platelet aggregation & adhesion
d. Magnesium – for platelet aggregation & adhesion
e. Serotonin – for vasoconstriction

PLASMA COAGULATION FACTORS – roman numerals

PLATELET FACTORS – ARABIC NUMBERS
PLATELET FACTORS
a. PF 3 – platelet phospholipids found on surface of
platelet
b. PF 4 – inhibitor of heparin thus anti-heparin factor
MEMBRANE SYSTEM
 2 membrane systems
a. OPEN CANALICULAR SYSTEM – delivery routes thus
it is where nutrients enter & waste products leave
the cell
b. DENSE TUBULAR SYSTEM – remnant of endoplasmic
reticulum ; site of arachidonic acid synthesis and
functions as a calcium sequestering pump
MICROSCOPY FOR PLATELET COUNT
 light & phase contrast microscopy
PLATELET ESTIMATION
IMPORTANCE: for correlation for performed platelet
count
FORMULA: (total no. of platelets per field) / 10 x 20, 000
REPORTING: qualitative/descriptive reporting
Ex.: (70 / 10) x 20,000 = 140, 000 ; slight decreased
 Correlate w/ the chart
TYPE OF BLOOD SAMPLE PERFORMED
EDTA
 because it prevents platelets from adhering &
aggregating together thus appear separated
under the microscope but if examined via fresh
blood, platelet appears clump or aggregated
  DISADVATAGE OF EDTA: platelet sateletism for
patients who develop Ab against EDTA
DIRECT PLATELET COUNT REPORTING: 150 – 450 x 109/L
PROPERTIES AND FUNCTIONS OF PLATELETS
1. ADHESION: platelet to non-platelet
 Adherence to injured blood vessel
 Ability of platelets to attach/stick to non-
platelet surface. This allows platelets to adhere
to damaged endothelium.
 requires plasma von willebrand factor and gp
ib-ix
NOTE: MODE OF ACTION
When activated or discoid, they change their
morphology where their cytoplasm will have
expansions for adherence. When an injury occurs,
some of the connective tissue & smooth muscles
components enters the circulation & be exposed to
the circulation such as the collagen. Collagen is
outside the blood vessel which maintains the integrity
of blood vessels, but during injury, some of the
collagen are exposed to the plasma & platelets. The
platelets adhere on the exposed collagen via the GP
1B9 complex but GP 1B9 complex will not adhere
directly to the collagen but needs a receptor the, von
willebrand factor thus allowing adhesion to occur.
2. PLATELET RELEASE REACTION
 Alpha & dense granules release substances that
will contribute to platelet aggregation and
activation of the coagulation system
Ex.: Serotonin for vasoconstriction – when
vessels are injured vessels constrict
Thromboxane A2 form the cytoplasm of the
membrane phospholipids
ADP from the dense granules promotes
aggregation for efficient plug strength
3. PLATELET AGGREGATION: platelet to platelet
 Platelets stick to one another platelet to form
an initial platelet plug.
 Induced by stimuli such as ADP, thrombin, TxA2,
collagen & epinephrine.
 Requires Gp IIb-IIIa and plasma
fibrinogen/FACTOR 1.
 FACTOR 1 serves as the adhesive glycan or
glue of the 2 platelets together
4. VASOCONSTRICTION & CLOT FORMATION
QUALITATIVE PLATELET DISORDERS
ADHESION DEFECTS:
1. BERNARD-SOULIER: DEFICIENCY/DEFECT IN GP IB-IX
 Characterized by large platelets,
thrombocytopenioa, prolonged and Bleeding
Time.
 Platelets show normal aggregation with
epinephrine, ADP and collagen but not with
ristocetin.
2. VON WILLEBRAND’S DISEASE
Aggregation test: same with Bernard-Soulier
PLATELET AGGREGATION STUDY TEST
 Platelets + agonist (aggregating agents) =
stimulate aggregation
RESULT: BOTH AGGREGATES TO: ADP, collagen &
epinephrine
BOTH DO NOT AGGREGATES TO: Ristocetin
AGGREGATION DEFECTS
1. GLANZMANN’S THROMBASTHENIA:
DEFICIENCY/DEFECT IN GP IIB-IIIA
 Platelets show normal aggregation with
ristocetin but not with epinephrine, ADP nor
collagen.
2. AFIBRINOGENEMIA (severe/absent),
HYPOFIBRINOGENEMIA (decrease) OR
DYSFIBRINOGENEMIA (defective)
RELEASE DISORDER/GRANULE DEFECTS/STORAGE
GRANULE DEFECT
1. Gray platelet syndrome – deficiency in Alpha
granules where platelets appear large pale or gray
or pale blue wherein platelet must be blue or purple
2. Wiskott-aldrich syndrome – deficiency in dense
granules that appears as smallest platelets
3. Chediak-higashi – deficiency in dense granules
4. Hermansky-pudlak syndrome – deficiency in dense
granules
QUANTITATIVE PLATELET DISORDERS
THROMBOCYTOPENIA: (<150, 000)
1. Decreased production – BM is ineffective in
producing platelets due to:
a. Megakaryocyte Hypoproliferation
b. Ineffective thrombopoiesis
 c. Marrow replacement/myelopthesis – BM space
is replaced by abnormal cells (cancer, leukemic
or myeloma cells)
2. Dilutional – related to massive blood transfusion
which is the transfusion of >1.5 L blood per 24 hr.
The available circulating platelet of the patient will
be diluted by transfused blood. If transfused blood
is not fresh do not have viable platelets.
 Outside the body platelets lifespan is only 3
days
3. Increased destruction (in vivo)
a. Immune – presence of Ab mostly IgG such as in
immune thrombocytopenic purpura
ITP – caused by autoimmune Ab destroying the
platelets
b. non-immune – no Ab implicated such as in
abnormal activation of platelets (thrombotic
thrombocytopenic purpura)
TTP – platelets are consumed as they are
converted into platelet aggregates or thrombus
leading to decreased in platelet count
4. Splenic sequestration – in cases of splenomegaly,
platelets will be sequestered more in spleen than in
circulation thus decreased platelet count
5. Acquired
a. Uremia – increase in NPN thus damaging cells
b. Paraproteinemias - increase in abnormal
protein
THROMBOCYTOSIS/THROMBOCYTHEMIA
1. Primary Thrombocytosis – excessive production is
the main defect
 Myeloproliferative diseases – disorder of the
bone marrow that results to excessive
production of blood cells such as in cases of
polycythemia vera which is excessive
production of all blood cells ; uncontrolled
production caused by the permanent damage of
bone marrow
 Essential Thrombocytosis – excessive
production of abnormal platelet
2. Secondary/Reactive thrombocytosis – reaction to
another condition ; irreversible
 hemolytic anemias
 hemorrhagic blood loss anemia
BOTH: body will compensate for the loss of
blood by causing splenic mobilization wherein
the 1/3 in the spleen will move into the
circulation as a compensation thus transiently
increasing platelets but returns to normal after
a few hours
 splenectomy
 SPECIAL HEMATOLOGY PROCEDURES
TEST FOR SICKLING HEMOGLOBINS
 SICKLE CELLS are distorted cells appearing as
thin, dense and elongated cells with both ends
pointed. SICKLING is caused by the
PRECIPITATION of an ATYPICAL HEMOGLOBIN
(commonly hemoglobin S) which DISTORTS the
RED BLOOD CELLS into a SICKLE or CRESCENT
SHAPE. HEMOGLOBIN S is fully soluble when
oxygenated but becomes insoluble when the
oxygen level is decreased. It first POLYMERIZES
then forms into CRYSTALS which cause the red
cell to become RIGID.
SCREENING TESTS
 PRINCIPLE: HbS forms an insoluble tactoid
crystals when oxygen supply to the red cell is
decreased. When Hb S is deoxygenized outside
the cell into the plasma it will cause the solution
to be TURBID.
 Positive result:
o Sickle RBC
o Turbidity
 Degree of sicking or turbidity depends on the
concentrstion of HbS in the red cell is decreased.
HB S in the RBC
1. SEALED WHOLE BLOOD METHOD (SCRIVER &
WAUGH)
 PRINCIPLE: RBC take on a SICKLE-LIKE SHAPE
when oxygen supply to the red cell is decreased.
HbS forms INSOLUBLE TACTOID CRYSTALS when
exposed to LOW OXYGEN TENSION.
 PROCESS: Perform IN VIVO. Using a RUBBER
BAND, the rubber band is TIED at the BASE of the
FINGER or at the BALL of the finger intended to
be prick & maintain it for at least 5-10 minutes
to cause obstruction in order to obstruct the
circulation thus decreasing the oxygen & Hb S
will form into crystals. Prick the deoxygenated
finger & drop into the center of the slide & cover
w/ cover slip. Seal the sides w/ petroleum jelly or
paraffin to prevent entry of atmospheric O2
which contains abundant O2 which may cause
the sickled cell to revert to normal shape.
Incubate for 1 hour at room temperature &
examine microscopically if still none, incubate
for 2 hours until 3rd hour. Perform interval
microscopic examination.
 POSITIVE RESULT: 1. Sickle RBC (Hb S) but does
determine if heterozygous AS or homozygous SS
 REPORTING: positive or negative
NOTE:
 Degree of sickling depends on the
concentration of the Hb S in the RBC
 Readings are made at an hourly interval for 2-
3 hours
 POSITIVE RESULT is diagnostic but does not
distinguish hb s trait & hb s anemia
2. SODIUM METABISULFATE METHODS (DALAND
& CASTLE)
 REAGENT: 2% Sodium metabisulfide or sodium
bisulfate which are reducing agent which bind &
remove O2 from the blood
 PRINCIPLE: a drop of blood is mixed w/ a drop
of 2% sodium metabisulfite (a reducing agent)
on a slide, & the mixture is sealed under a
coverslip. The hemoglobin inside the RBCs
becomes deoxygenated causing polymerization
& the resultant sickle cell formation
 PROCESS: add a drops of blood & the reagent
into the center of the slide. Cover the slide w/
coverslip & seal the sides. Observe
microscopically.
 POSITIVE RESULT: sickled RBC deoxygenated by
reducing agent
3. DITHIONITE SOLUBILITY TUBE TEST
 REAGENT: sodium hydrosulfite (dithionite)
removes O2
 SPECIMEN: whole blood w/ EDTA heparin or
sodium citrate in order to prevent clotting
 PRINCIPLE: Red cells are lysed by saponin
allowing Hb to escape. Sodium dithionine binds
& removes oxygen from the test environment.
Hb S polymerizes in the deoxygenated state &
forms a precipitate in a high-molarity
phosphate buffer solution. The tactoid refract
or deflect light & make the solution turbid.
 PROCESS: anticoagulated whole blood + sodium
dithionite. Saponin lyses the RBC releasing now
Hb. Hb binds w/ sodium dithionite which
removes O2 from solution causing Hb to
crystalizes but polymerization happens outside
 the RBC thus it will not cause sickling but
turbidity of solution & is determined by
refraction & deflection of the solution in the
presence of light. Observe specimen for
turbidity by holding the tube 2.5 cm in front of a
newsprint or a card reader w/ thin black line
 RULE: if the solution is observed in the solution
then the solution is not turbid thus negative
result but if lines are no longer visible then it is
positive for tactoid crystals & sickling.
 POSITIVE RESULT: turbidity
NOTE:
 Turbidity indicates presence of sickling Hb
regardless of any genotype
 Screening method of choice
4. HEMOCARD Hb A & S
 PRINCIPLE: Hb A & S contains monoclonal Ab
(IgG), which specifically bind to the amino acids
at or near the 6th position of the B-chain of the
Hb S & A
NOTE:
 Hb S monoclonal Ab will react w/ the B-chain
of Hb S but not w/ the Hb A
 Hb A monoclonal Ab will react w/ the B-chain
of the Hb A but not w/ Hb S
 Abs are adsorbed to suspended metal sol
particles giving the reagent raspberry-like
color
NOTE:
 After electrophoresis, densitometry is
performed to determine the density of the
bands created by the migrating proteins
converted into concentration.
 In cellulose Acetate, at an alkaline pH, Hbs are
negatively charge thus they migrate towards
the anode. Hb S together w/ D & G upon
migration appears as a band of protein thus
densitometry is performed to determine the
concentration of Hb S.
 On Citrate Agar, at an acidic pH, Hb S migrates
towards the anode thus citrate agar is more
confirmatory.
RETICULOCYTE COUNT AND ITS ASSOCIATED
CORRECTIONS
RETICULOCYTES
 Are young erythrocytes that are in a discrete,
penultimate phase of maturation where the
nucleus has been removed, however, some of
the extranuclear RNA remains in the cytoplasm.
 The term “reticulocyte” is derived from the fact
that the cell contains a small network of
basophilic materials called reticulum which is
demonstrable only by supravital stain.
Reticulocytes is the 5th stage of erythropoiesis
having no nucleus as well as contains remnants
of RNA. The presence of RNA is used to
determine presence of reticulocyte which is
stained w/ supravital stain which will precipitate
the RNA into granulofilamentous particles.
 In the peripheral blood using Romanowsky stain,
they are called as polychromatophilic
erythrocytes. Reticulocyte count and its
associated corrections can be used to assess
bone marrow erythrophoietic activity. Wherein
BM is the main organ that produces
erythrocytes.
 PRINCIPLE: The ribosomal RNA is stained
supravitally. Any non-nucleated RBC that
contains 2 or more blue-stained
particles/granulofilamentous materials, is
counted as reticulocyte.
 PROCEDURE: In a test tube, add an equal
number of blood with supravital stain (new
methylene blue or brilliant cresyl blue or janus
green for granulofilamentous staining). Incubate
for 10-20 minutes inside of an incubator to
enhance the staining because staining of living
cell is longer because they resist staining due to
their intact membrane. After incubation, remix
the sample because the reticulocyte has a low
specific gravity thus they tend to float in the
solution. After, transfer a drop into a slide &
prepare smear.
 METHODS OF STAINING: Wet method and Dry
Method (schilling’s method)
METHOD OF COUNTING:
A. LIGHT MICROSCOPE: DIRECT COUNT
 Reticulocytes are enumerated among 1000
RBCs in areas where RBCs are close but not
 overlapping and reticulocytes appear to be well
stained.
 Formula:
RETIC % = (No. of Retics / 1000 RBCs observed) x 100
B. MILLER DISC METHOD
 Miller Disc is a small disc, connected to one of
the ocular
 The calibrated Miller disc appears in the field
with 2 squares: a large square and a small square
inside the large square which is 1 /9 the size of
the larger square which can be located anywhere
 Reticulocytes are counted in the large square
while RBCs are counted in the small square in
successive fields on the film until a total of 500
RBCs have been counted
 RULE: include in the count the reticulocyte
present in small squares
FORMULA:
𝒕𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝒓𝒆𝒕𝒊𝒄𝒔 𝒊𝒏 𝒕𝒉𝒆 𝒍𝒂𝒓𝒈𝒆 𝒔𝒒𝒖𝒂𝒓𝒆
Retic (%)= × 𝟏𝟎𝟎
𝒕𝒐𝒕𝒂𝒍 𝒏𝒐.𝒐𝒇 𝑹𝑩𝑪𝒔 𝒊𝒏 𝒕𝒉𝒆 𝒔𝒎𝒂𝒍𝒍 𝒔𝒒𝒖𝒂𝒓𝒆 ×𝟗
REFERENCE VALUE:
ADULTS: 0.5 - 1.5 %
NEWBORN: 2.5 - 6.5 %
NOTE:
 Counterstain (wrights or giemsa) is not
advisable because it may destroy the image
 Reticulocyte have lower specific gravity
 Increased glucose inhibits staining thus
granules may not be seen when observed.
INCLUSIONS CONFUSED WITH RETICULOCYTES
 Pappenheimer bodies – iron containing
granules ; stain w/ Romanowsky or Prussian
blue stain (siderotic granules)
 Howell-Jolly bodies – appears singly ; stain
smear w/ Romanowsky to differentiate
 Heinz bodies – appears pitted golf ball ; stain
w/ supravital stain
 Hb H – appears at the periphery attached to
the membrane of RBC ; stain w/ supravital
stain
 Artifact – if stained smear is stored longer it
may create into precipitate which may
deposit in the surface of RBC & be mistaken
as the granules thus filter the stain & move
the fine adjustment. Artefacts when you are
out of focus appear as refractile, while
granulofilamentous particles will disappear.
ASSOCIATED CORRECTION
ABSOLUTE RETICULOCYTE COUNT
 Actual no. of reticulocytes in 1 L of whole blood
o ARC = Retic% / 100 x RBC count =
(_x1012/L) x 1000
o EXAMPLE: (1.5% / 100) x 4 000 000 = 60,
000
o REFERENCE VALUE: 25, 000 – 75, 000
CORRECTED RETIC COUNT / RETICULOCYTE INDEX
 This corrects the reticulocyte count to a normal
Hct to allow correction for the degree of patient
anemia. The percentage of reticulocytes may
appear increased because of early release into
the circulation or because of a decrease in the
no. of mature RBC in the circulation
o CRC: Retic % x (Hct (L/L) / 0.45 L/L)
0.45 L/L = normal mean Hct
o REFERENCE VALUE: 1% (depends on the
degree of anemia)
RETICULOCYTE PRODUCTION INDEX/SHIFT
CORRECTION (RPI)
 More appropriate test for determining efficiency
of BM compensation for anemia
 General indicator of the rate of erythrocyte
production increase above normal in anemias
meaning the previous production rate increases
for compensation for blood loss
 Index calculated to correct for the presence of
shift reticulocytes that otherwise may falsely
elevate the visual reticulocyte count.
 Done if shift cells/stress reticulocyte are present
& computing it
 RULE: the more severe the anemia, the higher
the production must be to normalize the
condition
o RPI = Corrected Reticulocyte count /
Maturation time (days)
Maturation Time: depends on the
patient’s hct
o REFERENCE VALUE: 1 maturation time if
hct is 0.45
 RETICULOCYTE MATURATION
HCT TIME (DAYS)
40-45% 1.0
35-39% 1.5
25-34% 2.0
15-24% 2.5
<15% 3.0
NOTE:
 Reticulocyte mature in circulation after 1 day
but in the presence of anemia the bone
marrow has to release even the immature
reticulocyte. Immature reticulocyte stay
longer in the circulation
 RULE: the shorter the immature reticulocyte
spent in the bone marrow, the longer they are
in the circulation
 RULE: the lower the Hct, the longer the
maturation time because of the severity of
anemia
CLINICAL SIGNIFICANCE OF RETICULOCYTES
METHODS:
 Increased in hemolytic & hemorrhagic anemias
OSMOTIC FRAGILITY TEST (OFT)
 OFT measures the ability of the RBCs to take in
fluid without lysing. As the cells take in water
(hypotonic solution), they become bloated or
swollen. If the cell is normal or biconcave disk, it
can take in more water w/out lysis because the
biconcavity can be distended w/out lysing but
the cell is already a spherocyte, its volume is
occupied thus as it takes more water, it will
immediately hemolyzed. It Reflects RBC shape
and size (surface area to-volume ratio). This is
used to detect spherocytosis, because
spherocytes rupture in saline concentrations
near the normal level. It may also detect target
cells, which, owing to their reduced hemoglobin
content, are able to withstand osmotic stress
and rupture only at very dilute saline
concentrations.
 IMPORTANCE: Diagnosis of Hereditary
Spherocytosis (HS) and other hemolytic anemias
associated with spherocytes.
CLINICAL SIGNIFICANCE OF OFT
 ABILITY OF CELL W/OUT LYSING IS AFFECTED
BY: shape & size of the RBC, which in turns
depends on the volume, surface area wherein a
hypochromic cell has a wider area than
spherocytic cell and functional state of the cell
membrane.
 SPECIMEN: Heparinized blood or defibrinated
blood because other anticoagulants alter the pH
of the blood
o Blood sample should be obtained with
minimum trauma and stasis thus upon
collection of the blood there must be no
hemolysis
o Ensure uniform size of blood drops
NORMAL RESULT:
a. Incomplete hemolysis: 0.45 % saline, solution
w/ pinkish tinge & an intact button of RBC at
the bottom upon centrifugation
b. Complete hemolysis: 0.35 % saline, red
solution w/out an intact button at the bottom
Note: Spherocytes show hemolysis as early as 0.60%
saline indicating prone to lysis or fragility
1. SANFORD METHOD
 Whole blood is pipetted to each of a series of
hypotonic saline solutions of graduated
concentration.
 Visual observation
 (+) result: Hemolysis
2. DACIE’S METHOD
 Uses hypotonic saline buffered with PO4 at ph
7.4 Hemolysis is determined
spectrophotometrically at 540 nm
 If the degree of spherocytosis is mild the degree
of hemolysis is not detected at first thus incubate
for hours to determine presence of hemolysis
 (+) result: Hemolysis
NOTE:
 INCUBATED OFT: sterile incubation at 37C for
18 – 24 hrs to detect mild forms of hereditary
spherocytosis.
a. INCREASED OFT (cells are fragile): Hereditary
spherocytosis; red cells with abnormal
membrane; severe G-6-PDH deficiency;
Pyruvate kinase deficiency; hemolytic anemias
 characterized by abnormal membrane or
enzyme deficiency
b. DECREASED OFT (cells are resistant to lysis due
to enough surface area to volume ratio thus
these are hypochromic cells & target cell): Sickle
cell anemia, severe Iron deficiency anemia,
thalassemia
o Sickling is reversible, if a sickle cell
intakes water it reverses back to its
normal shape, thus ability of sickle cell
to react in a hypotonic solution will
depend on the degree of sickling
ERYTHROCYTE SEDIMENTATION RATE
 Measures the degree or rate of settling of
erythrocytes at the bottom of the tube in
plasma in an anticoagulated whole-blood during
a specified period of time (1 hour)
IMPORTANCE:
1. Non-specific measure of inflammation
o The main factor affecting the rate of
settling is the presence of varied plasma
protein such as acute phase proteins
produced during inflammation
2. It is a good index for the presence of a hidden but
active disease like in the case of inflammation &
tuberculosis
3. A hematology test to indicate inflammatory
response to tissue injury however it does not
determine the severity of inflammation
4. Follow the progress of certain disease but not
identify the severity of the condition
5. It measures the suspension ability of the RBCs
6. It measures the abnormal concentration of
fibrinogen and globulin
STAGES OF ESR:
1. LAG PHASE/INITIAL ROULEAUX
 occurs during the first 10 minutes & the settling
is slower but after the first 10 minutes where
they form in rouleau, RBC sediment faster
 ROULEAU – cells in apposition to one another w/
their flat surface
2. DECANTATION PHASE/ PERIOD
RAPID/CONSTANT SETTLING
 occurs within 40 minutes.
3. FINAL SETTLING/PACKING
 occurs during the last 10 minutes settling slower
at the bottom of the tube
FACTORS AFFECTING SETTLING OF RBC
1. ERYTHROCYTE FACTORS
 RBC SIZE OR MASS: directly proportional to ESR
or rate of settling
 RULE: the larger, the faster is the settling & the
smaller RBC is, the slower they settle
 NUMBER OF RBC: inversely proportional
o In anemia where there are only few
cells present thus more space to settle.
In anemia, lesser cells, faster settling. In
polycythemia vera, cells are crowded
thus settling is slower.
 PRESENCE OF ANISOCYTES & POIKILOCYTES:
slower settling due to no rouleaux formation
wherein these cells are abnormal in shape &
size
2. PLASMA FACTORS
 PLASMA VISCOSITY: inversely proportional to
ESR meaning the more viscous the plasma is, the
slower is the rate of settling
o The most important factor which affects
the rate of settling is the plasma
composition
 PLASMA COMPOSITION: increase fibrinogen, α-
1 globulin, α-2 globulin: increase ESR thus
altering the zeta potential of cells causing
formation rouleaux
o increase albumin & lecitihin: decreases
ESR due to rouleaux formation inhibition
3. MECHANICAL/ TECHNICAL FACTORS
 POSITION OF THE TUBE: should be exactly
straight vertical because a slight tilting of the
tube 3 degree causes 30% error thus faster
settling
 INCUBATION ENVIRONMENT: should be free
from any movement or vibration at room
temperature however fluctuation caused
vibration causes a significant error because
vibration causes more cells settle at the bottom
OF
 INCUBATION TEMPERATURE: should be at room
temperature
 LENGTH AND DIAMETER OF THE TUBE: the
longer & wider the tube, the faster is the ESR due
 to increase pressure & wider space for cells to ESR: WESTERGREN METHOD
settle down AGE MALES FEMALES
 ANTICOAGULANT: should not be over- <50 y/o 0-15 mm/hr 0-20 mm/hr
anticoagulated (EDTA or citrate) of causes slower >50 y/0 0-20 mm/hr 0-30 mm/hr
ESR because cells are shrunken which may not >85 y/o 0-30 mm/hr 0-42 mm/hr
form into rouleau Children: 0-10 mm/hr
 TIME OF SETTING UP: first 2 hours or 6th hour CLINICAL SIGNIFICANCE OF ESR
from collection because beyond that cells will a. INCREASED ESR IN DUE TO INFLAMMATION OF
swell thus no rouleaux formation THE TISSUES
 Inflammations; acute & chronic infections,
ESR METHODS tuberculosis, multiple myeloma
1. WINTROBE & LANDSBERG METHOD  Rheumatic fever, rheumatoid arthritis,
(WINTROBE TUBE) myocardial infarction, nephrosis
 WINTROBE TUBE: glass tube w/ thick walls  SBE, Waldenstrom’s macroglobulinemia,
where one end is open & the other is close & hepatitis, menstruation, pregnancy
feeding of blood is done manually, w/ 2
graduation 10 on top & 0 at the bottom (left) b. DECREASED OR NORMAL ESR IN
for microhematocrit reading while for ESR it is 0  Polycythemia due to many blood cells,
on top & 10 on the bottom (right). Internal spherocytosis, conditions associated with Hb S
diameter is 3 mm & total height is 11 cm but & Hb C disease
column of blood is 10 cm  TO DETERMINE DECREASED ESR: if cells do not
 MATERIAL: wintrobe tube, Pasteur pipet w/ settle at all
thin needle pipet
 ANTICOAGULANT: Ammonium-Potassium OTHER METHODS OF ESR
Oxalate or wintrobe mixture or double oxalate 1. GRAPHIC-CUTLER METHOD: uses 3.8% sodium
citrate and Cutler tube
2. WESTERGREN METHOD (WESTERGREN TUBE) 2. LINZENMEIER METHOD: uses 3.8% sodium
 WESTERGREN TUBE: plastic or glass tube, both citrate and Linzenmeier tube
ends are open. Length is 200 while the blood 3. MICRO-LANDAU METHOD: uses 5 % sodium
column is 200 mm. The internal diameter is 2.65 citrate
(+- .15) 4. SMITH MICRO METHOD: uses 5 % sodium citrate
 MATERIALS: Westergren tube w/ stopper, 5. CRISTA OR HELLIGE-VOLLMER METHOD
commercial tube w/ anticoagulant, Westergren 6. ROARKE-ERNSTENE METHOD: uses heparin
rack 7. BRAY’S METHOD: 1.3% sodium oxalate
 ANTICOGULANT: Original Westergren = 3.8% NEW ESR METHOD
sodium citrate 1. Automated Erythrocyte Sedimentation Rate
 Modified Method = 2 ml of EDTA- a. Ves-Matic system – based on optoelectronic
anticoagulated blood with 0.5 ml NSS or 3.8% sensor wherein it measures the change in
sodium citra as diluents in order to adjust hct opacity of a column of blood as
 REFERENCE: adjust hct 0.35 sedimentation occurs
 PROCESS: Westergren tube is pushed into a b. Sedimat 15 – based on infrared
bottle of blood, the blood will move up via measurement wherein tubes are slightly
capillary action making sure that the blood is inclined thus the normal values are also
properly mixed via inversion & incubate for 1 adjusted
hour in ESR rack by letting it stand on a c. ESR STAT PLUS system - based on how fast
Westergren rack cells settle upon centrifugation for pediatric
 REPORTING: mm/hr or mm patients because it requires smaller samples
o More accurate if hct is adjusted to 0.35
to limit effect of anemia on RBC
 ZETA SEDIMENTATION RATIO (ZSR)
- Uses zetafuge which applies controlled
centrifugation of blood, producing alternating
compaction and dispersion of erythrocytes to
measure how closely the erythrocytes approach
one another under a specific standardized
gravitational force
- Also uses small amount of blood due to a special
capillary tube, 75 mm in length with an internal
diameter of 2 mm
- After centrifugation, Zetacrit% is determined at
the “knee” of curve to determine zeta
sedimentation ratio
FORMULA:
𝒉𝒄𝒕 %
ZSR (%) = × 𝟏𝟎𝟎
𝒁𝒆𝒕𝒂𝒄𝒓𝒊𝒕 %
REFERENCE VALUE:
(ZSR): 41 – 54%
ZETA SEDIMENTATION RATIO
51-54% Borderline normal
55-59% Mildly elevated
60-64% Moderately elevated
>65% Markedly elevated
ADVANTAGES OF ZSR
 It requires a smaller amount of blood
 It is not affected by anemia and is faster than
other methods
 Reference range is the same for both sexes
 Has only 1 reference value
 Faster due to involvement of centrifugation
ABSOLUTE EOSINOPHIL and BASOPHIL COUNTS
 DILUTION FOR EOSINOPHIL & BASOPHIL: 1:10
to increase observation because the higher the
dilution the fewer the cells seen
 DILUTING FLUID FOR EOSINOPHIL GRANULES:
o Phloxine – stains eosinophil red
o Propylene glycol – lyses RBC
o Heparin – inhibits leukocyte clumping
o Na Carbonate – enhances eosinophil
granule staining & hemolyze other WBC
o ALTERNATIVE STAINS: Pilot’s solution &
Randolph’s stain
NOTE:
ABSOLUTE EOSINOPHIL COUNT:
 FORMULA: Absolute count x WBC count
WBC COUNT: 9000/ uL
DIFFERENTIAL COUNT:
 NETROPHIL: 0.60
 LEUKOCYTE: 0.30
 MONOCYTE: 0.08
 EOSINOPHIL: 0.02
NOTE:
 There are some cells that needs to be counted
directly such as the eosinophil & basophil
 We rarely see eosinophil & very rare for
basophil thus performing count directly &
manually is necessary
THORN TEST (Test for adrenocortical functions)
- Does not determine bone marrow function but
adrenal cortical function
- Perform direct absolute eosinophil count w/
fasting sample w/ ACTH injection. After,
introduce ACTH in order to stimulate the adrenal
gland to produce corticosteroid hormones which
will cause the circulation to leave the circulation
& proceed towards the tissues. After 4 hrs., draw
second specimen for absolute eosinophil count.
After 4-8 hours ACTH injection, when the adrenal
gland reacts it will produce corticosteroid
hormones causing now eosinophil to proceed to
the tissues thus giving a lower count. If the
adrenal gland function is normal it will be able to
cause increase production of adrenal hormones.
- NORMAL: 2nd count should at least be 50%
lower than initial count
o If 1st & 2nd count is the same meaning
adrenal gland is not functioning causing
hypoadrenalism
ABSOLUTE BASOPHIL COUNT
- uses Cooper and Cruickshank stain (neutral red)
- COUNTING CHAMBER: Speirs – Levy or Fuchs-
Rosenthal
- DILUTION: 1:10
LUPUS ERYTHEMATOSUS (L.E.) CELL PREPARATION
- An LE cell is a neutrophil or macrophage that has
phagocytized (engulfed) the denatured nuclear
material of another cell that has been lysed by
anti-nuclear Ab. Its formation is characterized by
 the production of a number of autoantibodies.
The most important of these are the antinuclear
antibodies which occur in the serum of patients
with some autoimmune disorders including
systemic lupus erythematosus (SLE).
Demonstration of LE cells therefore suggests the
presence of these antinuclear antibodies also
termed as the LE factor
TO PERFORM LE:
 The LE factor or antinuclear antibodies causes
nuclear lysis (lysed nucleus becomes a
homogenous amorphous mass) and this material
is then phagocytized by a neutrophil.
 Extruded nuclei is acted by antinuclear
antibodies
 The phagocytic neutrophil which has
phagocytized the lysed nucleus is now called an
L.E. cell. (seen on a buffy coat smear)
 Rosette formation a flower like formation which
has an extruded nucleus surrounded by smaller
neutrophils is NOT considered positive
PROCEDURE:
a. Collect blood w/out an anticoagulant, 10-15 mL
WB & allowed to completely clot for 1 hr at 37 C
or 2 hr at RT
b. Discard the serum to demonstrate the LE cell by
obtaining the buffy coat layer
c. Get the blood clot & macerate to extrude some
of nucleus thus if antinuclear antibodies is
present it will act on the extruded nucleus thus
lysing the nucleus
d. Macerated blood will be transferred into 3-4
wintrobe tube & centrifuge at 2550 rpm for 30
mins.
e. After centrifugation, there is a higher volume of
buffy coat
f. Remove serum to obtain buffy coat & perform
buffy coat smear
g. Stain w/ wirghts & giemsa & observed for LE cell
which is a neutrophil that has engulf a large mass
h. Phagocytic neutrophil appears to wrap into the
lysed nucleus
OTHER ANTI-NUCLEAR ANTIBODY TEST
a. FLUORESCENT TEST (has replaced the LE test)
- more accurate because it identify what kind of
nuclear Ab is produced by patient
- serological test
- Uses fluorescein-conjugated antihuman IgG
BONE MARROW STUDY
COLLECTION OF SPECIMEN
 ADULTS: posterior iliac crest (posterior
preferred or anterior) – more common because
the tissue is more thinner at the hips & sternum
 CHILDREN: tibia
PROCESS: the aspirating needle must be able to reach
the medulla
 Examination of BM requires examination of
blood film which must be collected first because
bone marrow collection requires boring
because the outer layer of bone is calcified thus
it is very painful & stressful for the patient
which causes falsely increase WBC
1. TREPHINE BIOPSY/CORE
- Utilizes the jamshidi needle for obtaining the
core to get a rounded portion of the BM tissue
thus it will not disturb the nearby tissue
- This is taken before aspiration biopsy to avoid
any disruption of marrow architecture.
- Requires small sample of BM
- Imprint biopsy is prepared by touching the
specimen on a slide.
- FIXATIVE: 5% Zenker fluid
2. ASPIRATION
- This follows immediately trephine biopsy
- Disturb the architectural pattern of cell
- 1-3 mL of marrow in a 30-35 mL syringe w/ EDTa
to prevent cellular distortion & processed within
1hr.
- NEEDLE: university of Illinois sternal needle
BONE MARROW SMEAR PREPARATION:
1. WEDGE OR COVERSLIP METHOD – 2 coverslip
method giving excellent distribution of blood
cells
2. PARTICLE SMEAR/SQUASH METHOD – on slide
place the core collected & cover w/ another slide
& squash
3. BUFFY COAT (CONCENTRATE) SMEAR – collect
in a wintrobe tube & prepare buffy coat smear
same w/ wedge smear & centrifuged for 8-10
mins. at 2800 rpm this will compensates for
 hypocellular marrow and allows for examination o HYPOCELLULAR/HYPOPLASIA:
of large numbers of nucleated cells without incomplete development in one or more
interference from fat or RBCs. lines thus it contains more fats thus less
4. HISTOLOGIC SECTION (CELL BLOCK) blood production like in aplastic anemia
o FIXATIVE: 10% formalin, Zenker glacial
MARROW DIFFERENTIAL
acetic acid, or
- include all nucleated hematopoietic cells EXCEPT
o B5 FIXATIVE STAINS: Romanowsky; iron
megakaryocytes & macrophages
stain; H and E
- at least 500 cells are counted preferably 1000
NORMAL MARROW CELLS cells, 500/slide (2 slides)

 Hematopoietic cells – stain w/ romanowsky Guidelines for adult bone marrow differential in
stain concentrated smears, 1000 cell counts
 Lymphoblasts CELL TYPE RANGE (%)
 Monocytic cells Eryhroblast 18-24
 Myelocytic cell Myeloblasts, Type I 0-1
 Erythrocytic precursors Myeloblasts, Type II 0-2
Promyelocytes 1-4
 Macrophages/ Histiocyte or osteoclast
Neutrophils & Precursors 53-63
o with storage iron – stain w/ iron stain
Monocytes 0-2
(Prussian blue stain)
Eosinophi;s & Precursors 1-3
o with lipids (Gaucher cells)– monocyte or
Basophils & Precursors 0-1
macrophage w/ cytoplasm appearing a
Lymphocytes 8-12
crinkled tissue paper because of Plasma Cells 0-2
leucocerebrocytes)
 Mast Cells / Tissue Basophil
 Osteoblasts – immature bone cells, waterbug or MYELOID ERYTHROID RATIO
comet appearance - ratio between the granulocytic precursors &
 Osteoclasts/macrophages – for bone marrow erythroid precursors but only the nucleated
resorption precursors
- on smear, count all myeloid cells & erythroid
precursor which has 3 pronormoblast &
basophilic normoblast & 4 orthochromic
normoblast & for granulocyte only the
neutrophil, eosinophil & basophil
o M:E RATIO = 2 granulocyte:1 RBC – 4:1
o AVERAGE: 3:1

SMEAR PREPARATION FOR MALARIA

MARROW CELLULARITY
- Percentage of marrow space occupied by
hematopoietic cells compared w/ fat (yellow or
red marrow)
 MARROW FAT: hematopoietic cell = 1 : 2 (adults)
 MYELOID: Erythroid (M:E) ratio = 2 : 1 - 4 : 1
 RED MARROW CELLULARITY
o HYPERCELLULAR/HYPERPLASIA:
increased in one or more cell lines w/
few fats thus increase marrow
production like in polycythemia vera
EXAMINATION: SPECIMEN
- EDTA-anticoagulated blood or fresh capillary
blood collected before the initiation of
treatment.
- At least two thick and two thin peripheral blood
films should be made.
a. THIN BLOOD SMEAR
- For morphologic examination and species
identification and determination of Percent
arasitemia
- PREPARATION: same as wedge method
 o A drop of blood w/ 2-3 mm & 1 cm away
from the end & smear via wedge method
o First fixed in methanol then add distilled
water to let RBC to hemolyze to
removed Hb then stained w/ pH of 7.2
pH due to hemozoin pH & examine
o Cells are still intact & not overlapping
thus observing morphology
b. THICK BLOOD SMEAR
- For screening purposes because cells are
distorted
- PREPARATION: place 3 small drops or large drop
of blood close together near one end of the slide.
Spread in a 25 centavo coin & dry. With one
corner of a clean slide, stir the blood for about
30 seconds to mix the three drops over an area
approximately 1 to 2 cm in diameter
o First dehemoglobinized; air-dried then
stained
c. STAINS FOR MALARIAL BLOOD SMEAR
- Wright’s; Giemsa
CYTOCHEMICAL STAINS
CYTOCHEMISTRY
- Defined as the microscopic study and
identification of chemical constituents within
individual cells. It is useful in the identification of
malignant cell types on the basis of cytoplasmic
or nuclear chemistry; cellular constituents that
are present in abnormal form or amount; lack of
cellular constituent; and cells exhibiting
functional abnormalities
LEUKEMIA
- refers to group of disorders
- In the category of cell line, leukemia can be
differentiated to either Myeloid or Lymphoid
Leukemia
- In the basis of Blast cell
o Pre-dominance of lymphoblast is called
Acute Lymphocytic Leukemia
o Pre-dominance of mature with few
lymphoblast is called Chronic
Lymphocytic Leukemia
o Acute leukemia refers to a type of
leukemia with poorer prognosis with an
increase predominance of blast cells or
immature cells
o The decrease of blast cells and increase
of mature cells is called chronic
leukemia
o Predominance of myeloblast is
associated with Acute Myeloid
Leukemia
o Predominance of mature myelocyte is
associated with Chronic Myeloid
Leukemia
- Blast cells are not easily identified because of
their common characteristics (larger in size, large
nucleus, smaller and deeply basophilic
cytoplasm), cytochemical stains are used to
differentiate in the absence of CD marker
- Acute Myeloid Leukemia can be further classified
knowing it is a stem cell
- French American British System classified
leukemia based on the morphology of the cells
after staining with Romanowski stain and their
reaction to cytochemical stain
- FAB further classified ALL as ALL1, ALL2, ALL3.
While AML is classified into seven categories
(AML1-AML7)
- In AML:
o AML1-AML3 predominance cell is
Myeloblast, promyeolocyte myelocyte
(granulocytic series)
o AML4 and AML5 uses alpha naphthyl
Chloroacetate esterase
o AML4 stain positive in both Specific and
non- specific esterase because it
contains both granulocytic and
monocytic cells
o AML5- is the acute monocytic leukemia
o AML6 is called Erythroid Leukemia,
leukemic cells are precursors of
erythrocyte. Positive in Periodic acid
Schiff
o AML7 leukemia of megakaryocytes
called acute megakaryoblastic leukemia
- One example for Chronic Lymphocytic Leukemia
is the Hairy Cell Leukemia which affects B-cells.
Hairy cells contain ACP.
o ACP isoenzyme is present in all blood
cells and can be inhibited by tartaric
acid except the isoenzyme number 5
 which is the one present in hairy cell
leukemia
- In Chronic Myeloid Leukemia the predominant
cells are the mature myeloid cells especially the
granulocytes. CML is also called Chronic
Granulocytic Cells. Granulocytes can range from
50,00-300,000
- Having a very high WBC count can also be
observed in Leukemoid Reaction which is due to
underlying condition
- Both leukemoid reaction and chronic myeloid
leukemia are characterized by a very high WBC
count. However, Leukemoid Reaction is
reversible that when the underlying condition is
resolved the WBC will return to normal count.
CLM is a permanent condition. To differentiate
Leukemoid reaction from CML, alkaline
phosphatase is used.

ENZYMATIC STAINS
Peroxidase/Myeloperoxi - The stain itself does not contain the peroxidase enzyme. The peroxidase enzyme
dase (MPO) is inside the cell, the composition of the stain is the substrate for peroxidase
enzyme
- Myeloperoxidase is a normal constituent of the primary granules of the
myelocytic cells which is observe as early as promyeolocyte
- Stain marker for primary granules & auer rods (fused primary granules)
- Stain positive in AML but negative in ALL
- Differentiates AML from ALL
- Stain marker for immature myeloid cells
- Stain positive in granulocyte but not in lymphocytes
- Positive Activity: reddish-brown deposits (in cytoplasm of granulocytes and
monocytes)
- Note: Myeloperoxidase enzyme deteriorates; Stain should be done only on fresh
specimens
Alpha-Naphthyl Acetate - Use to detect the granulocyte of monocytic origin
Esterase & Alpha- - Non specific because it can be seen in other cells
Naphthyl Butyrate - The stain does not contain esterase but rather substrate for the enzyme
Esterase (Non-Specific - If the cell contains the enzyme it will catalyze the hydrolysis of the butyrate or
Esterases/NSE) acetate in the stain
- Unfixed sample can be used so long in the dark for as long as 2 weeks
- Marker for cells of Monocytic origin
- Other cells (+) : Megakaryocytes, Platelets, Histiocytes, Plasmacytes, some T-
lymphocytes
- Positive Activity: red-brown/dark red
Leukocyte/Neutrophil - Stains NEUTROPHILS (the only leukocytes that contain this activity)
Alkaline Phosphatase - Neutrophils contain various amount of ALP
(LAP/NAP) - Principle: Differential count involving neutrophils
- Differentiates Leukemoid reaction (LR) from Chronic myeloid leukemia (CML)
- Reference Value: 30 –185 LAP score
- Increase LAP score is observed in the following:
o during the last trimester of pregnancy - Hodgkin disease
o Polycythemia vera - Multiple myeloma
 o Aplastic anemia - Obstructive jaundice
- KAPLOW’s Scoring (Count 100 cells and grade them as follows)
o 0 = no staining
o 1+ = faint & diffuse staining
o 2+ = pale moderate amount of blue staining
o 3+ = strong blue precipitate
o 4+ = deep blue or brilliant
- To compute multiply the counted neutrophils by their grade
- Normal to High indicates Leukemoid reaction
- Below than normal value to zero indicates CML
Tartaric Acid Resistant - For the diagnosis of Hairy cell leukemia (HCL)
Acid Phosphatase (TRAP) - General Principle: ACP is Detected in almost all blood cells but when treated with
tartaric acid it is inhibited except isoenzyme number 5
- Labile if unpreserved
- Sample should first be fixed and be stored at -20 C and can be used atleast 2
weeks
- Other Tartrate Resistant cells: Sezary cells; Histiocyte; T-cell of acute lymphocytic
leukemia
- Activity is indicated by purple to dark red granules in cytoplasm
Cyanide-Resistant - For identification of Eosinophilic components
Peroxidase - Positive Activity: brown

Naphthol ASD - Marker for mature & immature Neutrophil and mast cells
Chloroacetate Esterase - Substrate is the chloroacetate
(Specific Esterase) - Use to identify immature or primitive granulocyte ALM1-ALM3
- Positive Activity: bright red granules in cytoplasm
- This enzyme is stable and may last for months
Terminal - Stains DNA polymerase immunoperoxidase
Deoxyribonucleotidyl - Marker for immature Lymphoid cell
Transferase (TdT) - Positive in almost 90% cases of ALL
- Also useful in the detection of the “lymphoblastic transformation” of chronic
myeloid leukemia (CML)
- Detects blastic transformation of CML
o CML can transform into an acute leukemia
o Acute leukemia more than or equal to 30% blast cells
o CML can transform either AML or ALL. If the blast cells presence stain
positive in MPO stain and SBB it is AML while if it stain positive in TdT it
means the it transforms into ALL
Acid Phosphatase - Present in all hematopoietic cells and found in lysosomes
- Activity is indicated by purple to red granules
- Unstable, cannot be stored

NON-ENZYMATIC STAINS
Prussian Blue stain - Stains siderotic granules (for the diagnosis of sideroblastic anemia)
- Used to detect hemosiderin in urine
- Use to identify iron in the ferric state (Fe+3)
Periodic Acid Schiff (PAS) - Stain reacts w/ aldehyde groups in glycogen, mucoprotein, glycoproteins, and
high molecular weight carbohydrates
- Does not detect stains instead it combines with the aldehyde group of glycogen
- Positive to all blood cells except normal erythroblast or pronormoblast
 - Positive erythroblast indicates that it is affected by AML6
- Positive in erythroblasts in Di Guglielmo disease / Erythroleukemia
- L1 and L2 also stain positive in PAS
- The positivity of L1 to Pas is described to be chunky or block-like positivity
- L2 is not that large as L1
- Negative Activity: bright fuchsia pink (Pattern of staining varies with each cell
type)
- Also differentiates Acute Lymphoblastic leukemias
- Peroxidase of eosinophil is resistance to cyanide.
- Cyanide resistance peroxidase is used to identify peroxidase of eosinophils
Sudan Black B (SBB) - Marker for phospholipids and lipids
- Has the same staining reaction as myeloperoxidase
- Gives the same information as Peroxidase in the interpretation
- Positive in AML
- Advantage over myeloperoxidase is that it is more stable, because
myeloperoxidase detects enzyme which is somewhat labile and may disappear in
prolong storage. Sample stained in SBB can give reliable result for months
- Differentiates AML from ALL
- (+) Result: Dark purple-black granules
- Notes:
 Can be done on stored specimens.
 More sensitive than chloroacetate in the demonstration of mature &
immature neutrophils and mast cells
Toluidine Blue O - A metachromatic stain
- Binds with mucopolysaccharides in blood cells
- For the recognition of basophils and mast cells
- (+) Result: Reddish-Violet
 HEMATOLOGY 2
GLOSSARY
HEMOSTASIS
- Stoppage of bleeding
- To maintain normal fluidity of blood in vivo.
There must be a balance in hemostasis. If not
needed, the clot should not be formed, but when
needed, the clot should be formed.
- Imbalance in hemostasis system will lead to
excessive bleeding/hemorrhage or excessive
clotting or abnormal thrombosis.
- Bleeding can be external or internal, severe or
mild which is indicated by red purple or blue
black which occur in the vessels particularly in
the capillaries thus blood deposit on the skin and
causes discoloration of the skin
PETECHIAE
- purplish red, pinpoint hemorrhagic spots in the
skin caused by loss of capillary ability to
withstand normal blood pressure and trauma
- diameter of <3mm
PURPURA
- produced by hemorrhage of blood into small
areas of skin, mucous membranes, and other
tissues
- Red-purple-brownish yellow
- diameter of >3 mm but <1 cm
- larger than petechiae or a combination of
petechiae
- simultaneous rupture of capillaries
ECCHYMOSIS/ BRUISE
- a form of purpura in which blood escapes into
large areas of skin or mucous membranes, but
not into deep tissue
- Black/blue – greenish brown or yellow
- diameter of >3 cm
- due increase rupture of capillaries but not on the
visceral organs but directly on the skin
NOTE:
All of these are caused by the breaks of small vessels
particularly the capillaries.
CAPILLARIES
- smallest blood vessels w/ the thinnest lining or
walls thus easily ruptured which may be caused
by increase in blood pressure/hypertension
leading to discoloration of the skin depending on
the amount of blood that deposited on the skin
NOTE:
Discoloration of the skin depends on the volume of
the blood.
COLOR TRANSITION
- bluish black if blood volume is greater or bluish
reddish/purplish to greenish to dark brown or
greenish brown to yellow
NOTE:
 Changes in the color of skin is associated with
the degradation of Hb wherein Hb is degraded
into globin & heme wherein heme is further
broken down into iron & protoporphyrin &
protoporphyrin is oxidized into biliverdin
(green) & further converted into bilirubin
(yellow)
 Petechiae, purpura & ecchymosis are
indication of primary hemostasis
POSSIBLE CAUSES OF RUPTURE OF CAPILLARIES:
1. Increase in blood pressure or hydrostatic
pressure
2. Pressure exerted outside
DISORDERS AFFECTING THE INTEGRITY OF CAPILLARIES:
1. Platelet abnormalities
2. Blood vessel wall abnormalities
NOTE:
 Even during normal situations, there are gaps
formed in between the endothelial lining
which is made up of connected endothelial
cells
 Whenever gaps formed, platelet functions to
aggregate to plug the gaps in order to
maintain continuity of blood vessel thus
limiting blood loss. Upon aggregation,
platelets are consumed leading to increase
appearance of petechiae or platelet may not
 HEMATOLOGY 2
properly perform its function due to platelet
abnormalities
 Petechiae and Purpura may occur without
platelet & blood vessel wall abnormalities
EPITAXIS- nosebleed
HEMARUTHROSIS
- leakage of blood into a joint cavity
- bleeding in the joints resulting to clot formation
causing obstruction in the movement causing
damage to the affected joint leading to crippling
which occur in severe hemocoagulation
deficiencies such as hemophilia
HEMATEMESIS
- vomiting of blood
- content of esophagus or stomach which is acidic
(HCl)
- brown color blood due to reaction of blood to
HCl
- pH of <7.35
HEMOPTYSIS
- expectoration of blood secondary to
hemorrhage in the larynx, trachea, bronchi, or
lungs
- coughs out blood
- red color w/ pH of 7.35 – 7.45
SPUTUM- product of the goblet cells of the lungs
HEMATOMA
- a swelling or tumor in the tissues or a body cavity
that contains clotted blood
- clotted blood with swelling
TRANSFIXATION- through & through of needle causing
hematoma
THROMBUS/CLOT
- In vivo blood clot causing vascular occlusion and
tissue ischemia
- composed of mostly platelets occurring in the
arteries causing arterial thrombosis or fibrin & a
small amount of platelet occurring in vein
causing deep vein thrombosis wherein both
causes obstruction.
DEEP VEIN THROMBOSIS
- vaso-obstruction causing the amount of blood
flow to be limited & the tissue will suffer from
ischemia due to decrease O2
EMBOLUS/EMBOLISM
- some parts of the clot detach & there is increase
in thrombus causing increase in pressure causing
the lumen to be smaller & the heart
compensates causing increase blood output &
pressure leading to rupture & detachment of clot
& the detach clot may obstruct smaller vessel
THROMBOSIS- formation, presence of a clot in a blood
vessel
 2 TYPES:
o PHYSIOLOGIC – if activation of
coagulation system to form a thrombus
is normal
o PATHOLOGIC – inadvertent formation of
blood clot
NOTE:
CAUSES OF PATHOLOGIC THROMBOSIS:
 TTP
 Cholesterol Plaque formation seen in DM
which thickens causing the vessel to be
sclerotic resulting to less capacity of the vessel
to dilate & constrict thus tissue reaction occur
causing obstruction leading to slower
circulation & this increases platelet activation
& will form into thrombus
 Varicose veins / superficial thrombosis
 Deep veins thrombosis
 Arterial thrombosis
HEMATURIA
- presence of intact red cells in the urine or simply
blood in urine
- red with intact RBC thus presence of turbidity or
smoky red
HEMOGLOBINURIA- presence of hb in the urine with
clear red color.
 HEMATOLOGY 2
METHEMOGLOBINURIA – methemoglobin in urine or
oxidized Hb in urine due to storage at room temperature
giving a color of chocolate brown urine
MENORRHAGIA – excessive menstrual bleeding
MELENA
- Stool containing dark red or black blood due to
oxidized Hb.
- Bleeding in the upper GIT such as in stomach or
upper part of small intestine.
HEMATOCHEZIA
- The passage of blood in feces.
- Red color bleeding in lower GIT such as in rectum
or colon.
OTHER CAUSES OF RED COLOR IN STOOL
 Tomato, red beets, dragon fruit, diet
 NOTE:
o the passage of blood in feces
o red color bleeding in lower GIT such as in
rectum or colon
HEMOSTASIS IS A COMPLEX MECHANISM THAT:
1. Retains the blood within the vascular system
during periods of injury.
- vessels constrict/ vasoconstriction/vasospasm
thus limiting blood passage leading to limited
blood loss
2. Localizes the reaction involved to the site of
injury.
- Platelet plug formation by first adhering of
platelet & will release their content & will
aggregate causing plug formation thus stoppage
of bleeding initially.
3. Repairs and re-establishes blood flow through
the injured vessels
- Fibrin clot formation by converting fibrinogen
into fibrin causing fibrin clot formation which will
stabilized the plug formation by forming a
meshwork.
- Promoted by thrombospondin & platelet derived
factor for smooth muscle & connected tissue
muscle repair thus dissolving fibrin.
2 STAGES OF HEMOSTASIS
a. Primary – consist of vasoconstriction & plug
formation
 Components: blood vessels (endothelial lining),
platelets
b. Secondary – consist of fibrin clot formation
 Component: Coagulation factors
(Fibrinogen/CF1 converted by thrombin from
prothrombin wherein the conversion of
prothrombin to thrombin is caused by CF10a, 5a,
Ca & PL to fibrin), Inhibitors (deactivates further
action) & Fibrinolytic proteins
PLASMIN/FIBRONOLYSIN – dissolves/lysis clot
FIBRINOLYSIS – process of dissolution of clot
PROCOAGULANTS- Promoters of coagulation
 Components: clotting factor for fibrin,
phospholipids, platelets for plug formation
ANTICOAGULANTS- Prevents excessive formation of
platelet plug
 Components: Natural inhibitors (ATIII, proteins-
C & S), Fibrinolysis
RESULT OF BOTH:
a. Thrombosis – increase procoagulant, decrease
anticoagulant
b. Hemorrhage – decrease procoagulant, increase
anticoagulant
CAPILLARY FRAGILITY TEST
- This test measures the ability of small capillaries
to retain blood when subjected to increased
hydrostatic pressure and anoxia.
- It is a non-specific evaluation to measure
capillary weakness and deficiencies in platelet
number and function meaning when there is
abnormal platelet number, there is abnormal
CFT this is due to thrombocytopenia or when
there is abnormal plt count but normal plt
function thus abnormal CFT
- Decreased capillary resistance causes the
capillaries to rupture which leads to bleeding
and formation of petechiae.
 HEMATOLOGY 2
- Requires exposure to increase hydrostatic
pressure & anoxia by applying a positive
pressure via BP apparatus or Tourniquet using
Rumpel-Leede Tourniquet Test or Negative
pressure from the outside using suction cup
- Measures fragility or stability of capillaries as
well as a non-specific evaluation of platelet
number & function
RESULT: petechiae
RUMPEL-LEEDE TOURNIQUET TEST
1. Examine the forearm, hand, and fingers to make
certain that no petechiae are present.
2. With a blood pressure cuff, apply 100 mmHg
pressure to upper arm. To those who do not
have a blood pressure cuff, use a tourniquet or
rubber/cloth strip instead. Apply the tourniquet
not too tight, not too loose to employ just
enough pressure.
3. Maintain pressure for 5 minutes.
4. Release cuff and wait for 5 – 10 minutes before
making a final reading.
5. Examine the forearm, hands and fingers for
petechiae.
NOTE:
Disregard any petechiae within ½ inch of the
blood pressure cuff (tourniquet) because this
may be due to pinching of the skin by the cuff.
6. Count the number of petechiae and roughly
interpret as follows:
1+ A few petechial on the anterior part of the
forearm
2+ Many petechial on the anterior part of the
forearm
3+ Multiple petechial over the whole arm and back
of the hand
4+ Confluent petechial on the arm and back of the
hand
HEMOSTASIS
VASCULAR SPASM
- very first reaction to injury
- function of blood vessels
NOTE:
 Vascular spasm & platelet plug formation are
under the primary hemostasis while the clot
formation is under the secondary hemostasis
 The primary hemostatic response towards
injury is immediate but since the platelet plug
is unstable the effect is short term
 Secondary hemostatic response is more
complex but the response is quite delayed but
once the clot is formed & is stabilized it will be
efficient in effect which is long term
 In primary hemostasis, the platelets clump
together & in secondary hemostasis, fibrin
monomers are formed & connected together
or polymerized thus forming a meshwork
around the platelet aggregate thus stabilizing
the platelet aggregate
 In the presence of meshwork, some of the
blood cell in the circulation (RBC) are trapped
in the fibrin meshwork & add up to the bulk &
the larger the size of the clot, the more
efficient it is as a plug
THREE HEMOSTATIC COMPONENTS
EXTRAVASCULAR - Play a part in hemostasis by
COMPONENTS providing back pressure on the
injured vessel causing some to
flow outside & the tissues will
absorbed & the blood causing
swelling which will exert a back
pressure preventing the further
release or extravasation of the
blood
DEPENDS ON:
a) BULK or amount of surrounding
tissue.
b) TYPE of tissue (skeletal muscles
will be absorbed more as
compared to lose connective
tissues).
c) TONE of the surrounding tissue
(tissues that lack tonicity will not
 HEMATOLOGY 2

absorbed that much such as PRIMARY HEMOSTASIS SECONDARY

tissues of elderly) HEMOSTASIS
VASCULAR - Involves the blood vessels INVOLVES: INVOLVES: activation of a
COMPONENTS Vasoconstriction, Platelet series of plasma proteins
DEPENDS ON: plug, Platelet adhesion, in the coagulation system
a) SIZE of the blood vessels release reaction & until fibrin clot formation
b) AMOUNT of smooth muscle aggregation (final product)
within their wall ACTIVATED BY: ACTIVATED BY: large
c) INTEGRITY of the endothelial desquamation and small injuries to blood vessels
cell lining injuries to blood vessels and surrounding tissues
Procoagulant substances Tissue factor exposed on
ENDOTHELIAL LINING are exposed or released cell membranes or
- composed of endothelial cells by damage or activated circulation
INTRAVASCULAR - Includes cells, plasma proteins & endothelial cells in order
COMPONENTS fibrinolytic agents such as the to activate the secondary Ex.:
plasminogen, plasmin & tissues hemostasis 1. Collagen exposed to
plasminogen activator and the surrounding tissue,
inhibitor causes platelets to
Platelets and biochemical in the aggregate & adhere &
plasma. activate CF 12. COLLAGEN
is the chief component of
 Clots are actually the the connective tissues.
plasma not the RBC 2. Tissue Factor
 There are inhibitors for introduced to the
coagulation and circulation because of
fibrinolysis large injury to the tissues,
 PECAM & ICAM contains it will activate factor 7
adhesion molecules causing the in vivo
 Platelets are also coagulation
involved in 2nd COMPONENTS: vascular COMPONENTS: platelet
hemostasis by exposing intima & platelets & coagulation system
their surface for the Rapid, short-lived Delayed, long term
clotting factors to be response response
activated & react Ends with platelet plug Naturally occurring
formation inhibitors in blood will
Primary Hemostatic block activated
ARTERIES VEINS CAPILLARY Disorders: Blood vessel & coagulation factors so
SIZE Large Smallest; platelet disorder that widespread
easily coagulation does not
rupture occur.
LININGS 3 layers of 3 layers of Endothelium FIBRINOLYSIS – slow breakdown & removal of fibrin
endothelial endothelial (inner) and clot as healing of the injured vessel occurs
lining, lining, Epithelial
smooth smooth cells
muscle and muscle and SPECIAL FUNCTIONS OF VASCULAR ENDOTHELIUM
connective connective
1. MECHANICAL ABILITY
tissues tissues
WALL Thicker Thinnest a. Vasodilation
o Prostacyclin – induces
vasodilation ; produce by
endothelial cell

b. Vasoconstriction
- 2 Substances that Promotes
Vasoconstriction:
o Serotonin/5 hydroxytriptamine
– from dense granules
o Thromboxane A2
NOTE:
Both serotonin & thromboxane A2 activates the
surrounding tissues for it to constrict causing now
limited amount of blood to enter
2. SYNTHESIS
- Aids in anticoagulation & procoagulation
 ANTICOAGULANT: during intact skin ; by smooth
surface allowing blood cells to circulate
smoothly
o Prostacyclin & nitrous oxide – secreted
by intact endothelial cell; induces
vasodilation & inhibit platelet
aggregation
o Heparan sulfate – Increases the activity
of antithrombin 3 (major inhibitor of
thrombin
o Tissue Factor Pathway Inhibitor – major
inhibitor of tissue factor/extrinsic
pathway which is initiated by factor 12
o Anti-thrombin 3 – from the liver; major
inhibitor of the entire coagulation
system
o Thrombomodulin – Inhibitor of Factor
2a; binds w/ thrombin thus inhibiting
thrombin & cause deactivation of
protein C which is the cofactor of protein
S. Protein C comes from the liver
produced as an inactive protein which is
inactivated by thrombin
thrombomodulin. Protein C becomes an
inhibitor. Protein C, thrombomodulin &
thrombin combines to form a
trimolecular complex & with endothelial
cell as a receptor, Protein C becomes
activated
HEMATOLOGY 2
 PROCOAGULANT: during vascular damage
o Serotonin & thromboxane A2 –
vasoconstriction
o Von Willebrand Factor – from alpha
granules which is involve in platelet
adhesion & in the endothelial cell they
are stored in the weibel palate
o ADAMTS-13 – cleaves ultra large von
Willebrand factor which may adhere
even in the absence of damage which
may lead to TTP
o P-selectin – produce by other WBC &
produce by endothelial cell ; enhances
adhesion to damage
o Collagen & Tissue Factor exposed –
enters the circulation during damage &
be exposed wherein collagen will cause
platelets to adhere & aggregate &
activate coagulation factors such as
Factor 7 & 7a causing clotting factors to
occur for tissue factor
o Fibrinolysis – produces tissue
plasminogen activator which converts
plasminogen into plasmin however
excessive TPA causes excessive plasmin
causing excessive fibrinolysis thus
PLASMINOGEN ACTIVATOR INHIBITOR is
activated to inhibit activation of
plasminogen in order to decrease the
synthesis of plasmin & THROMBIN
ACTIVATABLE FIBRINOLYSIS INHIBITOR
inactivates fibrinolysis
&
 HEMATOLOGY 2

PRIMARY HEMOSTASIS: Components, Functions, ADHESION •P-selectin; Procoagulant

Disorders MOLECULES Intracellular
BLOOD VESSELS (Vascular Intima) Adhesion
Molecules
ANTITHROMBOTIC, FIBRINOLYTIC AND COAGULANT •Platelet
SUBSTANCES RELEASED FROM OR FOUND ON THE endothelial
SURFACE OF ENDOTHELIAL CELLS cell adhesion
molecules
SUBSTANCE ACTION HEMOSTATIC
(PECAMs)
ROLE
•Help cells
PROSTACYCLIN •Inhibits Anticoagulan stick to each
(PGI2) platelet t and to their
activation surroundings
•Stimulates
vasodilation
HEPARAN SULFATE Coats the Anticoagulan NOTE:
endothelial t  Vascular disorders affect hemostasis
cell surface  Bleeding occurs if the connection of the
and weakly endothelial lining is loose.
enhances  Connective tissues are disseminated in every
activity of part of the body. Thus, all tissues of the body
antithrombin with defective connective tissue will also be
-3 affected.
THROMBOMODULI •Endothelial Anticoagulan
N (ENDOTHELIAL surface t
PROTEIN C receptor for Fibrinolytic BLEEDING DISORDER CAUSED BY VASCULAR
RECEPTOR) thrombin DEFECTS
(binds & HEREDITARY CONNECTIVE TISSUE DEFECTS
inactivates
thrombin) 1. EHLERS-DANLOS SYNDROME
•Enhances - Sex-linked ; caused by a defect in the gene
anticoagulant responsible for peptidase enzyme production
and which converts procollagen to collagen (chief
fibrinolytic component of the connective tissues of blood
action of vessels)
protein C - Characterized by: Hyper-extensive joints and
TISSUE FACTOR Controls Anticoagulan hyperplastic skin
PATHWAY Activation of t - LABORATORY FINDINGS: Normal Coagulation
INHIBITOR the extrinsic Test & Platelet function studies
pathway
TISSUE Converts Fibrinolytic
2. MARFAN SYNDROME
PLASMINOGEN plasminogen
- Defect in fibrillin
ACTIVATOR to plasmin
ADENOSINE Stimulates Reduces - Characterized by: Elongated upper & lower
vasodilation blood flow digits.
rate
VON WILLEBRAND Required for Procoagulant 3. PSEUDOXANTHOMA ELASTICUM
FACTOR (VWF) platelet - Autosomal recessive ; defect in elastic tissue
WEIBEL-PALADE adhesion to - Characterized by: calcified & formed into
BODIES site of injury plaques thus affecting the ability of the vessel to
 HEMATOLOGY 2
constrict & dilate and structurally abnormal
Elastic fibers
- Yellowish plaque, observed on skin where the
skin is abnormally loose (neck)
ACQUIRED CONNECTIVE TISSUE DEFECT
1. VITAMIN C DEFICIENCY (SCURVY/SCORBUTUS)
- Vitamin C deficiency common among children
thus prone to petechial formation
- IMPORTANCE OF VITAMIN C: for the formation
of the intact structure of the vascular basement
membrane & in synthesis of collagen for
hydroxylation of Vitamin C’s proline & lysine
- Characterized by: gingival bleeding; hemorrhage
into subcutaneous tissues and muscles; large
hemorrhagic areas may develop just below the
eyes; splinter-like hemorrhage may also appear
in the fingernail beds
- LABORATORY FINDINGS: CFT = usually positive
2. SENILE PURPURA
- Related with normal aging process;
discoloration of the skin which may not
disappear & will remain as age spot because
even the function of the macrophages which
supposed to remove it has also declined
- LABORATORY FINDINGS: CFT = positive ;
Bleeding time = normal or only slightly prolonged
HEREDITARY ALTERATION OF VESSEL WALL
STRUCTURE
1. HEREDITARY HEMORRHAGIC TELAGIECTASIA
(RENDU-OSLER-WEBER SYNDROME)
- Common among Scandinavian population ;
discoloration of the skin where the skin is thin ;
blood vessels are abnormally thin thus they
dilate & will be seen as spots
- Autosomal dominant trait characterized by thin-
walled, focally disorganized and dilated blood
vessels with a discontinuous endothelium
appearing as red pinpoint lesions most
commonly on the face, lips, tongue, mucous
membranes of the mouth and nose, ears,
conjunctiva, palms and soles
- LABORATORY FINDINGS: bleeding time, platelet
function tests, capillary fragility test and
coagulation test are all normal
2. CONGENITAL HEMAGIOMATA (KASABACH-
MERRITT SYNDROME)
- Disorder associated with tumors composed of
many large vessels ; benign & can be surgically
remove
- Formation of fibrin clots, platelets consumption
and red cell destruction secondary to vascular
obstruction occur at the site of tumor in the
mucous membrane
ACQUIRED ALTERATIONS OF VESSEL WALL
STRUCTURE
1. DIABETES MELLITUS
- Deposition of glycosylated proteins leading to
thickening of capillary basement membrane
plaques affecting often the capillaries of the
renal glomeruli and retina
2. AMYLOIDOSIS
- Excessive amyloid or cholesterol deposit in
small vessels thus forming plaques causing now
inflammatory process leading to cellular
inflammation and activation of cellular
inflammatory process leading to injury & the
normal vessel flow will contain thrombus
formation
- Causes vasoobstruction thus less oxygenation &
may also lead to rupture resulting to stroke &
other condition
ENDOTHELIAL DAMAGE
1. AUTOIMMUNE VASCULAR PURPURA
- Problem with antibodies
A. DRUG INDUCED PURPURA
- Common drugs that induce purpura:
sulfonamides & iodides quinine, procaine,
penicillin, aspirin, sedatives, coumarin
- Endothelial cell might no longer be recognized as
self-cells & the immune system will induce Ab
against the endothelial cell & iodide or
sulfonamide
- May be due to development of antibodies to
vessel walls components, development of
immune complexes and change in vessel wall
permeability
 HEMATOLOGY 2

B. ALLERGIC PURPURA/ANAPHYLACTOID NOTE:

PURPURA
- Associated with certain food and drugs, cold
temperature, insect bites and vaccinations
- HENOCHS PURPURA – associated with
abdominal pain secondary to GIT hemorrhaging
- SCHONLEINS PURPURA – associated with joint,
especially in the knees, ankles and wrist
 Only metamegakaryocyte has the ability to
produce platelets
 The more nuclear lobes, the more platelets it
will produce
4 FUNCTIONAL ZONES OF PLATELETS

2. INFECTIOUS PURPURA a. PERIPHERAL ZONE

- Associated with bacterial ; viral, rickettsial & - Contains glycocalyx which contains
protozoal infections or substance it produces glycoproteins (4, 6, 9)
- Contains glycoprotein 1b/9 for platelet adhesion
PLATELETS & GP 2b/3a for platelet aggregation
CHARACTERISTICS:

- Cytoplasmic fragments from megakaryocytes b. ZOL GEL ZONE

- Contains gel components that maintains
Diameter 2-4 um & 1/8 the diameter of different organelles at their position & allows
RBC them to communicate with one another
Site of production Bone marrow - Contains the cytoskeleton of the cell which
2 Pools 1/3 in spleen maintains the size & shape of platelet for it to not
2/3 in circulation
collapse which is made up of microtubules and
Growth Factor Thrombopoietin
microfilaments, the actin & myosin forms the
Manner of Division Endomitosis – increase
contractile protein, thrombosthenin.
nuclear component while
cytoplasmic component is
absent c. ORGANELLE ZONE
Rule As it matures, the cell size - Contains the dense granules, alpha granules
increases (most abundant), mitochondria & lysosomes
Mean Platelet 8-10 fL
Volume (MPV) d. MEMBRANE SYSTEM
Reference Platelet 150,000-450,000/ uL o Open Canalicular System – Membrane
count that allows communication to the
Daily Turnover 35 X 109/L (+/- 4.3) outside environment & route of entry &
Average production 2,000 – 4,000/megakaryocyte exit of substances
Lifespan 8-11 days o Dense Tubular System – for calcium
Function Maintenance of vascular pump & arachidonic acid production
integrity and blood
coagulation

DEMARCATING MEMBRANE SYSTEM

- Demarcation lines in cytoplasm ; where platelet

fragmentation occurs
- Appears as early as megakaryoblast & prominent
in promegakaryocyte & absent in megakaryocyte
& metamegakaryocyte.
 HEMATOLOGY 2

PLATELET FACTORS

PF1 Plasma coagulation factor V

PF2 A globulin that inhibits antithrombin III;
increases platelet aggregation and
accelerates interaction of thrombin and
fibrinogen (fibrinoplastic platelet factor)
PF3 A lipoprotein (platelet phospholipid) found
in platelet granules & membrane and
required in 2 steps of the coagulation
process ; functions for coagulation factors to
assemble in order to cause fibrin formation
; component of prothrombinase together
w/ CF10, 5a, Ca for conversion of
prothrombin into thrombin for secondary
hemostasis ; assembly molecule
PF4 A glycoprotein stored in the alpha granules
and is extruded during the platelet release
reaction; aids in ADP-induced platelet
aggregation and inhibits effect of heparin

NOTE:
 In heparin therapy, patients might
develop autoantibodies against PF4
in Heparin Induce
Thrombocytopenia disease
 Heparin Induce Thrombocytopenia
– Acquired condition caused by
autoimmune disorder caused by
heparin therapy
PF5 Platelet fibrinogen
PF6 A plasma inhibitor associated with platelets
PF7 Cothromboplastin
PF8 Antithromboplastin factor
PF9 Accelator globulin stabilizing factor
PF10 Serotonin found in the dense granules
 HEMATOLOGY 2

ROLE IN SUBSTANCE SOURCE PRINCIPAL FUNCTION/COMMENTS

HOMEOSTASIS
Promote HMWK (high molecular Alpha Granules  Contact Activation of intrinsic coagulation
coagulation weight kininogen) pathway
 Cofactor of 12a in converting prokalycrine into
kalycrine
Fibrinogen  Converted to fibrin for clot formation
Factor V  Cofactor to Factor 10 to convert prothrombin
into thrombin in fibrin clot formation
Von Willebrand Factor  Assists platelet adhesion to subendothelium
to provide coagulation surface
Promote ADP (strong) Dense bodies
Aggregation Calcium  Promote platelet aggregation
Platelet factor 4 Alpha granules  Calcium is involved in coagulation
Thrombospondin  Thrombospondin & platelet factor 4 is for
platelet function
Promote Serotonin Dense bodies
Vasoconstriction Thromboaxane A2 Membrane  Promotes vasoconstriction at injury site
precursors (inhibited phospholipids  Thromboxane A2 is a very strong aggregator
by aspirin therapy) of platelet
Promote Platelet derived Alpha granules  Promotes smooth muscle growth
vascular repair growth factor  For vessel repair
Beta-thromboglobulin  Chemotactic for fibroblasts
 For vessel repair
Other systems Plasminogen (from Alpha granules  Precursor to plasmin, which induces clot lysis
affected liver converted into
plasmin by TPA &
inhibited by A2-
antiplasmin)
A2 – antiplasmin  Plasmin inhibitor, inhibits clot lysis
C1 esterase inhibitor  Complement system inhibitor

PHYSICAL PROPERTIES AND FUNCTION OF - Von Willebrand Factor is necessary for areas
PLATELETS where circulation is with high shear (arteries &
1. ADHESION (platelet-to-injury) arterioles) such that if the platelets will simply
- Platelets during injury first cling & roll on the adhere, It will be easily dislodge but in veins &
damage endothelium & adhere on damage capillaries, since the pressure is not that strong,
endothelium the platelets can directly adhere on the exposed
- Sticking of platelets to a non-platelet structure; surface such as GP 6
platelets adhere only on detached or injured - Reversible.
endothelium
- Requires the GP Ib/IX in complex with V for 2. PLATELET RELEASE REACTION
adhesion which serves as a receptor for Von - Platelet undergoes shape or morphological
Willebrand Factor which is readily available in change. Alpha & dense granules release
plasma substances that will contribute to platelet
- Plasma Von Willebrand Factor is used for aggregation and activation of the coagulation
platelet attachment to the exposed system.
subendothelial matrix such as the collagen
 HEMATOLOGY 2
- Releases ADP from dense for aggregation,
serotonin from dense for vasoconstriction &
thromboxane A2 from membrane phospholipid
for vasoconstriction & aggregation
- Irreversible
3. PLATELET AGGREGATION (platelet-to-platelet)
- Requires Gp IIb-IIIa as a receptor for fibrinogen
and plasma fibrinogen/Factor 1 which serves as
glue or adhesive for platelet to platelet adhesion
- Viscous metamorphosis
- INITIAL AGGREGATION: reversible
- SECONDARY AGGREGATION: irreversible
- AGGREGATING AGENTS: ADP, Thrombin,
Collagen, Thromboxane A2, Epinephrine,
Arachidonic Acid, Ristocetin, Thrombospondin,
Reptilase (from reptile)
OTHER FUCNTIONS:
4. VASOCONSTRICTION
- Enhanced by serotonin released from platelets &
thromboxane A2 from membrane phospholipids
- Reaction in primary hemostasis
5. CLOT FORMATION
- Platelet factor 3 or the assembly molecule is
needed for the formation of active plasma
thromboplastin
- Reaction in secondary reaction
QUALITATIVE PLATELETS (Platelet-vessel wall
interaction)
A. ADHESION DEFECTS:
1. BERNARD-SOULIER SYNDROME
- Features:
o Deficiency in Gp 1b/IX thus an intrinsic
defect
o Platelets are abnormally low & abnormal
morphology
o Abnormally large platelet/giant platelet
w/ thrombocytopenia & prolonged
bleeding time
o AGGREGATION STUDIES: platelet
 Abnormal: Ristocetin
 Normal: ADP, collagen &
epinephrine
2. VON WILLEBRAND DISEASE
- Most common coagulopathy
- Autosomal inheritance & function at
chromosome 12 synthesized by the endothelial
cell & platelets; stable.
- Autosomal inheritance in Chromosome 12;
deficiency in plasma VIIIc: VWF.
- Characterized by prolonged bleeding time &
same aggregation test result with Bernard-
Soulier; prolonged APTT due to deficiency or
decrease in CF 8c
- FACTOR 8C/ANTIHEMOPHILIC FACTOR –
cofactor in the intrinsic pathway
- ACTIVATED PARTIAL THROMBOPLASTIN TIME –
test for intrinsic pathway
- Multimeric molecule w/ a receptor for GP
Ib/IX/V complex & collagen which is used to
bind w/ platelets causing platelet adhesion
- Contains Ag epitope & has a receptor for & a
protein carrier for FACTOR
8C/ANTIHAEMOPHILIC 8C which is a sex-linked
inheritance in the X chromosome synthesized by
the liver & is labile thus it is prone to proteolysis
thus causing secondary deficiency causing
prolongation of APTT but becomes relatively
stable w/ Von Willebrand Factor.
ANTIHEMOPHILIC FACTORS:
 A – Factor VIII (2 components: 8:VWF for
adhesion & 8C for coagulation intrinsic pathway)
 B – Factor IX
 C – Factor XI
LABORATORY: PLATELET COUNT AND MORPHOLOGY:
generally NORMAL
COMMON SIGN: mucocutaneous bleeding
BLEEDING TIME (severe) & APTT TEST (prolonged) –
screening test
DIAGNOSIS: STANDARD VMD TEST PANEL TESTS:
1. Quantitative VWF test (VWF Ag assay) – to
determine if VWF is quantitative or qualitative.
2. VWF activity test/ VWF RCo assay – qualitative
test to determine ability of vWF to bind to
platelets.
 HEMATOLOGY 2

3. Factor VIII activity assay – to determine the

extent of VWF disease which may lead to
NOTE:
secondary deficiency of Factor 8
ACQUIRED VWF DEFICIENCY:
TREATMENT: - hypothyroidism, autoimmune,
lymphoproliperative & myeloproliferative,
 Cryoprecipitate – contains the complete disorders; benign monoclonal gammopathies;
component of factor VIII & VWF wherein this Wilms tumor ; intestinal angiodysplasoa;
control bleeding by supplying the deficient congenital heart disease ; pesticide exposure;
Factor VIII & VWF but it is no longer & hemolytic uremic syndrome
recommended because it does not undergo viral
inactivation wherein the specimen source is the
B. AGGREGATION DEFECTS (Platelet-platelet
blood donor.
interaction)
 Desmopressin acetate (1-desamino-8-D
arginine vasopressin/ DDAVP) – standard
1. GLANSMANN’S THROMBASTHENIA
treatment which cause release of VWF & Factor
- Features:
8 from endogenous sources in order to prevent
o Deficiency/ Gp IIb-IIIa; Inherited as an
bleeding.
autosomal recessive trait
 A plasma-derived factor VIII/VWF concentrate
o Bleeding time is prolonged, platelets are
CLASSIFICATIONS OF VON WILLEBRAND DISEASE morphologically normal but isolated w/
one another
TYPES DESCRIPTION o AGGREGATION STUDIES: Platelets
1  Partial quantitative deficiency of von aggregate normally with Ristocetin but
Willebrand factor (vWF) not with Epinephrine, ADP, Collagen
 Most common to occur about 75 -80% ;
a quantitative disorder
2. (CONGENITAL) AFRIBRINOGENEMIA OR
2  Qualitative (Function) deficiency of vWF
HYPOFIBRINOGENEMIA
 Level of vWF is normal but function is
o AFIBRINOGENEMIA: Acquired/Inherited
abnormal
total absence or severe deficiency in
2A  Decreased platelet-dependent vWF
fibrinogen.
function with selective deficiency of high-
molecular-weight multimers (the vWF is o HYPOFIBRINOGENEMIA:
more susceptible to proteolysis by Acquired/Inherited slight decrease in
ADAMTS-13 which is secreted by fibrinogen.
endothelial cells which destroys low o DYSFIBRINOGENEMIA: Inherited lack of
molecular weight vWF) fibrinogenemia
2B  Increased affinity for platelet
glycoprotein lb/IX/V DISORDERS OF PLATELET SECRETION & SIGNAL
TRANSDUCTION DEFICIENCY OF GRANULES
2M  Decreased platelet receptor binding
(STORAGE POOL DISORDERS)
(glycoprotein lb/IX/V)
ALPHA GRANULES DEFICIENCY
2N  (Normandy Variant) Impaired factor VIII
binding site leading to deficiency to APTT 1. GRAY PLATELET SYNDROME
thus APTT is prolonged - Deficiency of alpha granules
 Autosomal Hemophilia (royal disease) - Platelets aggregate abnormally & are abnormally
3  VWF is absent or nearly absent from large
plasma - Characterized by larger platelets colored gray to
 Rarest vWF & severe or complete blue-gray, and thrombocytopenia
deficiency
- LABORATORY: Prolonged Bleeding time;
 Autosomal Recessive
decreased aggregation with all agents.
 HEMATOLOGY 2
2. QUEBEC PLATELET DISORDER
- Autosomal dominant disorder characterized by
abnormal proteolysis (destruction of proteins) &
deficiency of alpha granules
- Abnormal increase in urokinase-type
Plasminogen Activator
- Platelet count is normal or decrease
- Characterized by delayed bleeding when
exposed to trauma (12-24 hrs from the time of
exposure) from mucocutaneous bleeding.
DENSE GRANULES DEFICIENCIES
1. WISKOTT-ALDRICH SYNDROME
- (Sex-linked) deficiency of dense granules, with a
defect in cytoskeletal assembly.
- Characterized by predominance of small
platelets
- TRIAD OF SYMPTOMS: thrombocytopenia,
recurrent infection & eczema
2. HERMANSKY-PUDLAK SYNDROME
(AUTOSOMAL)
- TRIAD OF SYMPTOM: oculocutaneous albinism,
accumulation of ceroid-like pigment in
macrophages & bleeding tendencies
3. CHEDIAK-HIGASHI (AUTOSOMAL)
- TRIAD OF SYMPTOM: albinism (no pigment),
recurrent infection (immune deficiency) & giant
lysosomes
4. THROMBOCYTOPENIA WITH ABSENT RADII
SYNDROME (TAR)
- Reduced radial bone or totally absent ;
appearing like a t-rex syndrome
- Structural defect in beta granules with
NOTE:
corresponding abnormal aggregation responses
and thrombocytopenia
- Characterized by severe neonatal
thrombocytopenia and congenital absence or
extreme hypoplasia of the radial bones (most
pronounced skeletal abnormality), cardiac and
other skeletal abnormalities
- Abnormal aggregation
- Observed as early as birth
DISORDERS OF PLATELET PROCOAGULANT
ACTIVITIES
- In resting platelets, phosphatidylserine (PS) &
Phosphatidylethanolamine (PE) are located
predominantly on the inner leaflet while
phosphatidylcholine has the opposite
distribution & this arrangement is maintained
by Aminophospholipid Translocase Enzyme.
During platelet activation, it undergoes a
morphological change such that its cytoplasm
will be extruded with extensions & the
distribution of phospholipids will be scrambled
wherein phosphatidyl serine &
phosphatidylethanolamine will be exposed &
acted by the enzyme, phosphatidyl
scramblase/PF 3 & coagulation factors will be
activated & be assembled
1. SCOTT SYNDROME
- Surface expression of phosphatidylserine is
decreased
- Disorder of calcium-induced membrane
phospholipid scrambling that when a platelet is
activated it can assume a resting phase of
platelet & may also be activated wherein the
exposure of phosphatidyl serine is deficient thus
scrambling occurs & affects the secondary
hemostasis
2. STORMORKEN SYNDROME
- Platelets are always in an activated state without
prior activation
- Defect in aminophospholipid translocase
- Phosphatidyl choline is always on the outside
causing abnormal platelet activation
 Phosphatidyl Choline – major component of
the outer layer
 Phosphatidyl serine &
Phosphatidylethanolamine – internal later
 HEMATOLOGY 2
ACQUIRED DEFECTS OF PLATELET FUNCTION
1. DRUG-RELATED
 ASPIRIN – permanent cyclooxygenase inhibition
which is needed in the synthesis of thromboxane
A2 thus affecting platelet aggregation
- Phospholipid A2 will convert the membrane
phospholipid of platelets into cyclooxygenase
- Prevents conversion of arachidonic acid into
prostaglandin by cyclooxygenase leading to
deficiency in thromboxane A2
 NSAIDs and other COX-2 inhibitors (naproxen
and ibuprofen)
 Other Antiplatelet agents: TICLOPIDINE AND
CLOPIDROGEL – blood thinning drugs making
blood viscous ; affects fibrinogen & Gp2b/3a
 Dextran – coating of platelets thus platelet
Causes:
cannot adhere & aggregate
2. PARAPROTEINEMIAS ((MULTIPLE MYELOMA
AND WALDENSTROM MACROGLOBULINEMIA)
- Dysproteneimias – Excessive plasma proteins to
lead to hyper-viscosity syndrome making the
circulation slow leading to thrombosis & the
pressure is increase
- Disorder of the bone marrow thus production of
intrinsically abnormal platelets
- Increase in bens jones protein &
immunoglobulins wherein for MM it is increase
IgG while for WM it is increase IgM
3. RENAL DISEASE
- Uremia: increase in non-protein nitrogenous
substances ; leads to decreased thromboxane
synthesis causing platelet destruction ;
Decrease: adhesion, platelet release, and
aggregation ; Increase: non-protein nitrogenous
substances
QUANTITATIVE PLATELET DISORDERS
- Platelets function is normal but platelets are
few or increased in number
SIGNIFICANT PLATELET LEVELS (platelet count and
clinical manifestation):
 150 – 450 x 109/L – REFERENCE PLATELET
COUNT
o <150, 000: thrombocytopenia
o >450, 000: thrombocytosis
 < 100,000/ uL – ABNORMALLY LOW; significant
decrease ; bleeding is not apparent
 30,000 – 50,000/ uL – BLEEDING IS POSSIBLE
especially when exposed to trauma or bleeding
secondary to surgical procedures
 <30,000 – SPONTANEOUS BLEEDING, meaning
even without trauma or surgical procedure,
bleeding may occur in the internal organs
 <5,000/ uL – SEVERE SPONTANEOUS BLEEDING,
may cause intracranial bleeding and bleeding in
visceral organ due to inability of platelets to
maintain the continuity of blood vessels ;
considered as a life-threatening condition
THROMBOCYTOPENIA
1. DECREASED PRODUCTION
- Bone marrow defect, where platelet production
remains abnormal
 MEGAKARYOCYTE HYPO-PROLIFERATION in:
o APLASTIC ANEMIA – affects the bone
marrow; bone marrow is infiltrated with
fats which are haematopoietically
inactive; all blood cells are decrease.
o MAY-HEGGLIN – inherited condition;
abnormally large megakaryocyte but are
decrease in number; WBC w/ dohle
bodies.
o TOXIC CONDITIONS – when the toxic
agents destroy megakaryocyte
 INFECTIVE THROMBOPOIESIS – Infective
thrombopoiesis due to decrease in
thrombopoietin which is mainly produced by the
liver and a few in kidneys ; associated among
chronic alcoholics among ethanol abused thus
damaging platelet production
 NEONATAL THROMBOCYTOPENIA – among
newborns who are exposed to infection inside
the uterus ; associated with infections with
toxoplasma, rubella, cytomegalovirus, and
herpes and HIV (TORCH); and in-utero exposure
to certain drugs (particularly chlorothiazide
diuretics and the oral hypoglycemic
tolbutamide)
 HEMATOLOGY 2
 MARROW REPLACEMENT/MYELOPTHISIS –
replacement of entire bone marrow with
abnormal cells such as cancerous cells
2. BLOOD TRANSFUSION
- Transfused platelets will add on the patients
platelets improving now the patients platelet
count
- Occurs after transfusion of platelet-containing
blood products (recipient’s plasma is found to
contain alloantibodies to antigens on the
platelets of the transfused blood product)
- Normal lifespan of platelets in blood banks is
only 5 days
 DILUTIONAL LOSS – blood product transfused
does not contain enough viable platelets thus it’s
effect is to dilute only the patients’ blood and
improve the blood volume ; Massive transfusion
where patient receives 4 or more units of blood
for 24 hours may also lead to dilution of patient’s
platelets ; transfusion of intravenous fluids and
pure plasma
 POST TRANSFUSION PURPURA – occurs a week
after ; related to antibody if the patient is already
been sensitized or received blood component
before causing now antibody production against
the platelets ; severe type of thrombocytopenia
3. INCREASES DESTRUCTION OR CONSUMPTION
a. NON-IMMUNE
- premature activation of platelets in secondary
aggregation causing platelets to be consumed
- associated with activation and consumption
1. THROMBOTIC THROMBOCYTOPENIC
PURPURA (TTP): caused by a deficiency of a
ADAMTS-13 ; common in young adults ;
persistence of ultra large VWF which may
bind prematurely to platelets causing
platelets to aggregate which is irreversible &
the clot may detach & may cause
vasooclussion; platelets are consumed when
formed into thrombus ; deficiency in
disintegrin and metalloproteinase with a
thrombospondin type 1 motif, member 13
(ADAMTS-13)
2. DISSEMINATED INTRAVASCULAR
COAGULATION (DIC) – patient produces a
substance that will activate both coagulation
and fibrinolysis leading to imbalance in
coagulation as clotting occurs there is a
corresponding fibrinolysis, when clot occurs
some coagulation factors are consumed
such as the CF 1, 5,8 and 13 and platelets ;
occurs elsewhere the body ; involves the
activation of both coagulation and
fibrinolysis due to liberation of
thromboplastic substance by damaged or
abnormal cells
3. HEMOLYTIC URENIC SYNDROME (HUS) –
associated with diarrheal or non-diarrheal
a. DIARRHEAL among children after
children has been infected with GIT
problem with any organism that can
produce a shiga-like organism the
verotoxin (E. coli H7:0157, Strep.
Pneumoniae, shigella) where it will it
bind to glomerular capillaries on the
endothelial cells causing them to be
activated and become thrombotic
promoting thrombosis; often occurs in
the kidneys ; It is hemolytic when
platelets become thrombus they cause
lysis ; urine appears as pinkish ;
Associated with mild febrile illnesses,
certain immunizations, and
gastrointestinal disturbances (E.coli
O157:H7 or other Shiga/Vero toxin-
producing bacteria)
b. NON-DIARRHEAL among children
associated with vaccination
4. PLATELET LOSS ON ARTIFICIAL SURFACE – in
cases of artificial heart valve, though it is
match, when platelets are in contact to
them, platelets are destroyed
5. INFECTIONS (eg. Dengue) – leads to platelet
consumption, because dengue infects some
of the macrophages and monocytes which
produces cytokines & the effect make the
endothelial lining hyper permeable and
platelets will plug the area until platelet
count is consumed
 HEMATOLOGY 2
b. IMMUNE DESTRUCTION
- Associated with antibody (IgG)
1. IDIOPATHIC/IMMUNE
THROMBOCYTOPENIC PURPURA (TTP) –
cause is unknown for ITP; common among
young children ; due to presence of anti-
platelet antibodies of the IgG type
associated with previous infection; most
common in young females <15 y/o ; if occurs
in children, the prognosis improves but in
adults it is life threatening
2. DRUG-INDUCED: HEPARIN-INDUCED
THROMBOCYTOPENIA (HIT)- patient
develops IgG antibody specific for heparin-
platelet factor 4 complexes; A. common side
effect of unfractionated heparin
administration
4. SPLENIC SEQUESTRATION
- Seen in hypersplenism and splenomegaly where
there is enlargement of spleen thus sequestering
>1/3 of platelets & keeping 50-90% of platelets,
leaving 10% of platelets in the circulation thus
leading to thrombocytopenia.
5. UREMIA
- Increase of NPN in the blood due to inability of
the kidney to remove these substances due to
renal insufficiency; also causes platelet damage.
THROMBOCYTOSIS/ THROMBOCYTHEMIA
- Increases platelet count (>450,000/uL)
1. PRIMARY THROMBOCYTOSIS
- main defect ; abnormal production ; seen in
myeloproliferative disorders where there is
abnormal proliferation of blood
a. POLYCYTHEMIA VERA – chronic
myeloproliferative disorder ; all blood cells
are increase
b. ESSENTIAL THROMBOCYTOSIS – increase
platelets >1, 000, 000/uL w/ abnormal
function
c. CHRONIC GRANULOCYTIC LEUKEMIA –
leads to abnormal marrow cell production
2. SECONDARY/ REACTIVE THROMBOCYTOSIS
- due to underlying condition ; Seen in splenic
mobilization and in hemolytic anemias
a. SPLENIC MOBILIZATION – occurs in
hemorrhagic conditions ; due to acute
hemorrhage, platelets moves into systemic
circulation causing transient increase in the
platelet circulation ; response to stress such
as in acute blood loss anemias ; platelets
formed and mature from the spleen
b. RAPID REGENERATION – involves the bone
marrow but production is normal ; during
hemolytic anemia where there is a need to
loss of blood cells thus the bone marrow will
immediately try to compensate and increase
production of blood cells ; platelets newly
released from the bone marrow indicated by
reticulated platelets in circulation in order to
respond to underlying condition
LABORATORY EVALUATION OF PRIMARY
HEMEOSTASIS
- Evaluates platelet and blood vessels.
A. DIRECT METHOD OF PLATELET COUNT
- Can be performed directly or indirectly and can
also be done manually or with the use of
automated machines.
- Manual Procedures for platelet count can be
done with the use of Light Microscopy Principle
or the Phase-Contrast Microscopy Principle.
- EDTA: Anticoagulant of choice whether
automated or manual procedure. It prevents
platelets from adhering and as well as
aggregating together.
- When aggregation and adhesion occurs, this will
result to a false decrease in platelet count.
However, even if the blood is EDTA
anticoagulated, platelet count should be
performed as early as possible. Preferable within
the first 3-5 hours from collection.
o 5 Hours: if blood is left in a room
temperature (20°C). Otherwise, on
prolonged standing, platelets will start
to swell and break into smaller
fragment resulting to a false increase in
platelet count.
 HEMATOLOGY 2
o 24 hours at 4°C: cell count (Platelet, RBC
count, WBC count) are still valid if
performed within the first 24 hours.
AUTOMATED PRINCIPLE:
- Counted within the volume range of 2-20 fL. All
cellular factors that falls within this value are
considered by the machine as platelets.
a. Electric impedance – platelets are counted in
triplicate (3x)
b. Light scattering
DILUENT:
1. LIGHT MICROSCOPY METHOD (Tocantin
SOURCES OF ERRORS:
Method)
- Manual counting and requires additional stain
- Principle: Hemocytometry
- Counting chamber: improved Neubauer
 Rees & Ecker Diluent: contains formalin as a
fixative which preserves platelets and RBCs. It
also contains Sodium Citrate as an additional
anticoagulant and a supravital stain which is the
Brillian Cresyl Blue (BCB).
2. PHASE – CONTRAST MICROSCOPY METHOD
- Identify cells based on their index of refraction.
- Reference method for manual platelet counting.
- Platelets are counted in 5R squares & appear as
refractile or glistening bodies
- Counting Chamber: Spencer-Bright line (S-B)
1475
 Brecker Cronkite Method
o 1% NH4 Oxalate diluent: no fixative and
stains. It removes RBCs from the
background wherein RBCs are
hemolyzed and platelets will remain in
the field.
PROCEDURE FOR ALL:
PHYSIOLOGIC VARIATION:
a. First dispense 2-3 drops of the mixture before
loading the counting chamber
b. Dispense the mixture on both side of the
counting chamber
c. Place the loaded counting chamber on the wet
house counting chamber which contains a
moistened cotton/filter paper w/ distilled water
to provide moisture and prevent evaporation for
15 minutes to allow the cells to settle
d. After 15 minutes, place on the microscope &
start counting on the 5 small square or 25 central
square or 10 small squares
e. Compute:
FORMULA:
𝑇𝑉−1
DF=
𝐵𝑙𝑜𝑜𝑑 𝑣𝑜𝑙𝑢𝑚𝑒
1 𝑚𝑚3
VCF=
𝑛𝑜.𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 ×𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠
Plt. Count= no. of platelet counted × 𝑉𝐶𝐹 × DF
1. PLATELET CLUMPS – occurs if blood is not
immediately mix w/ anticoagulant during
collection causing false decrease platelet count.
2. PLATELET SATELLITISM
- Surrounding of platelets in a neutrophil.
- Common among individuals who have develop
Ab (IgG) against EDTA.
- Platelets attaches on WBC, band cells &
monocytes due to cell receptors on their cell
surface.
- Causes false decrease platelet count.
- Remedy: collect using Na citrate & perform
manual platelet count. However, platelet must
be multiplied by 1.1 to correct the dillutional
effect of Na citrate to blood.
3. EXCESS EDTA (1/2 full)
- Platelets will swell & fragmentize causing false
increase in platelet count.
- Fill EDTA half full (2.5 mL)
4. 3 HOURS AT RT – platelet will swell &
disintegrate causing false decrease platelet
count thus, refrigeration of the sample at 4°C for
24 hours.
1. Decreased at birth
2. Increased after the 1st week of birth until adult
normal values.
3. Decreased during menstruation.
 HEMATOLOGY 2

B. INDIRECT METHOD release cuff & wait for 5-10 minutes before
reading.
1. FONO’S METHOD - The forearm, hands, and fingers are then
- DILUENT: 14% of MgSO4 – prevents platelets examined for petechiae 5 inches away from
from adhering & aggregating. blood pressure cuff.
- If there is a need to re-perform, perform on the
PLATELET ESTIMATION
other arm. It can only be repeated on the same
- The average number of platelets observed in 10 arm after a week.
fields & divided by 10 & multiplied by 20,000 in - NORMAL: 0 to occasional petechiae.
a blood smear anticoagulated with EDTA thus - INTERPRETATION: +, ++, +++, ++++
appearing clear & separated at the neck region
counted via cross sectional (side by side) or b. HESS/SUCTION TEST (Negative pressure)
battlefield method (serpentine) - A suction cup (2cm diameter) is placed in close
- In capillary puncture platelet appears as clump. contact with the skin at midpoint of upper arm
Thus, resulting to a false decrease of platelet for 1 min (pressure of 200-250 torr). An area
count. within a circle of 1 cm diameter is observed for
- Platelet shows 8-20 platelets per oil immersion petechiae 5 minutes after removal of suction
field of 200 RBC & 2-3 platelet clumps only and cup.
reported as follows: - CLINICAL SIGNIFICANCE: Positive in
thrombocytopenia, hypofibrinogenemia, and in
0-49,000 Marked decreased vascular purpura.
50,000- 99,000 Moderate decreased
100,000-149,000 Slight decreased TESTS FOR PLATELET FUNCTION & VASCULAR
150,000-199,000 Low normal FUNCTION
200,000-400,000 Normal 1. BLEEDING TIME
401,000-599,000 Slight increase - Measures the ability of the small blood vessels
600,000-800,000 Moderate increase to control bleeding after injury.
Above 800,000 Marked increased - Time it takes for a standard wound to stop
bleeding at a standard pressure (40mmHg)
- Standardized both incision & pressure applied
PLATELET & VASCULAR FUNCTION
- FACTORS AFFECTING BT:
1. TOURNIQUET TEST/CAPILLARY FRAGILITY TEST
a. Number of platelets and the ability of
- Tests the ability of small capillaries to retain
platelets to form plugs.
blood when subjected to increased hydrostatic
b. Thickness and vascularity of the skin.
pressure and anoxia.
c. Ability of the blood vessels to constrict.
- Affected by capillary integrity & platelet function
d. Severe coagulation factor deficiency causing
and number.
prolonged bleeding time.
- Becomes normal in presence of
- PROLONGED BT:
thrombocytopenia, hypofibrinogenemia &
o When platelet count is lower than 30-
vascular purpura
50,000/uL, or when platelets are
- Requires application of pressure which is applied
dysfunctional in VWD.
as a positive or negative pressure.
o After ingestion of aspirin/aspirin-
containing compounds, anti-
a. RUMPLE-LEEDE METHOD (positive pressure)
inflammatory, anticoagulants, and
- PRINCIPLE: an inflated blood pressure cuff on
some antibiotics.
the upper arm is used to apply pressure (100
mmHg) to the capillaries for 5 minutes. After,

2. PLATELET ADHESIVENESS/RETENTION TEST
- Reference Values: 26-60%
a. GLASS BEAD METHOD/SALZMAN METHOD
- Collect 2 whole blood samples, 1 using EDTA and
the other using glass bead collecting system.
Perform platelet counts separately. (higher in
EDTA)
- COMPUTATION:
𝑝𝑙𝑡 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑔𝑙𝑎𝑠𝑠 𝑏𝑒𝑎𝑑𝑠−𝑝𝑙𝑡 𝑤𝑖𝑡ℎ 𝑔𝑙𝑎𝑠𝑠 𝑏𝑒𝑎𝑑𝑠
%= × 100
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑔𝑙𝑎𝑠𝑠 𝑏𝑒𝑎𝑑𝑠
b. BORSHGERVINCT METHOD (in vivo test)
- Perform separate platelet count on venous
blood and capillary blood samples
- Formula:
𝑝𝑙𝑡 𝑐𝑜𝑢𝑛𝑡 𝑣𝑒𝑛𝑜𝑢𝑠 𝑏𝑙𝑜𝑜𝑑−𝑝𝑙𝑡 𝑐𝑜𝑢𝑛𝑡 𝑐𝑎𝑝𝑖𝑙𝑙𝑎𝑟𝑦 𝑏𝑙𝑜𝑜𝑑
%=
𝑝𝑙𝑎𝑡𝑒𝑙𝑒𝑡 𝑐𝑜𝑢𝑛𝑡 𝑖𝑛 𝑣𝑒𝑛𝑜𝑢𝑠 𝑏𝑙𝑜𝑜𝑑
3. PLATELET AGGREGATION TEST
- Aggregating agents; thrombin, arachidonic acid,
ADP, collagen, epinephrine and ristocetin.
- Currently, this test is considered the gold
standard for evaluation of aspirin resistance.
- PRINCIPLE: Aggregating agents added to a
stirred suspension of PRP induce a shape change
and aggregation of platelets. As a result, the PRP
changes from a turbid suspension to tone that
transmits more light (clear) as the aggregates
are formed. The aggregometer records changes
in optical density in the form of a graph.
READING OF AGGREGATION RESPONSE:
1. PLATELET-RICH PLASMA AGGREGOMETRY: light
transmittance aggregometer
2. WHOLE-BLOOD PLATELET AGGREGOMETRY:
electrical impedance
3. OPTICAL LUMI-AGGREGOMETER: for
simultaneous measurement of platelet
aggregation and the secretion of ATP.
HEMATOLOGY 2
OTHER TESTS
CLOT RETRACTION TIME
- PRINCIPLE: When blood coagulation is complete,
clot normally undergoes retraction where clot
becomes denser and serum is expressed.
Normally, clot retraction begins within 30
minutes after the blood has clotted &
completed within 24 hours.
- Normal clot retraction requires normal number
of functioning platelets. Ca, ATP, fibrinogen,
normal interaction of the platelets with
fibrinogen.
- Thrombosthenin; ratio of the plasma volume
and red cell mass; activity of a retraction-
promoting principle in serum & nature of the
surface on which CRT is being measured.
CLINICAL SIGNIFICANCE:
× 100
- CRT is poor when platelet count is less than
100,000/uL
- in dysfibrinogenemia or hypofibrinogenemia,
paraprotinemias
a. HIRSCHBOECK METHOD (Castor oil method)
- Qualitative: test for the presence or absence of
retraction
- Formation of dimpling/droplet like serum on the
surface of blood drop.
- Normal values equals to 15-45 minutes.
b. STEFANINI METHOD (test tube)
- Normal: clot retraction begins within 1 hour,
complete within 18 to 24 hours.
c. MAC FARLANE METHOD
- Provides quantitative estimate of the degree of
retraction.
- Normal values equals to 44 - 67%.
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑒𝑟𝑢𝑚
- %CRT= 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 × 100
𝑏𝑙𝑜𝑜𝑑
 HEMATOLOGY 2
SECONDARY HEMOSTASIS: COMPONENTS,
PATHWAYS, TESTS; DISORDERS
SECONDARY HEMOSTASIS
- involves coagulation factor deficiency
(hemophilia A/B/C)
- Depends on availability and functionality of
different plasma coagulation factors.
PLASMA FACTORS
- These are soluble plasma coagulation proteins
that have crucial functions in the formation of a
clot. These proteins circulate as inactive
zymogens (proenzymes) that become activated
during the process of coagulation and, in turn,
react with other factors in the system to
ultimately generate thrombin which converts
fibrinogen to a localized fibrin clot.
CLASSIFICATION OF COAGULATION FACTORS:
 Substrate: are the substance upon which
enzymes act.
 Cofactors: accelerate the activities of the
enzymes that are involved in the cascade. (TF,V,
VIII, HMWK)
 Enzymes: serine proteases and transaminase
NOMENCLATURE:
- Except for Prekallikrein (PK) and HMWK which
are referred to by their name only, each factor
was assigned a Roman numeral number in order
of its discovery. The use of “a” that follows as
Roman numeral denotes the activated form. A
“f” refers to fragmented factor (e.g. XIIf)
GENERAL CHARACTERISTICS: (SUMMARY)
 All are synthesized by the Liver except factors
VWF, III, and IV
 Deficiency in any factor may produce abnormal
hemostats; except of factors XII, PK and HMWK
 Labile factors: V and VIII (short shelf life)
 Factors prematurely activated by refrigerator
temperature.
QUESTIONS TO REMEMBER:
 Composition of Prothrombinase: Xa-Va-Ca++-
PL
 Composition of the intrinsic tenase: IXa-VIIIa-
Ca++-PL
 Factors present in fresh plasma: all are
present: no deterioration
 Factors present in aged serum: II, VII, IX, X
(prothrombin); XI, XII, HMWK, PK (contact)
 Clotting → fresh serum: (-) I, II, V, VIII, XIII
 Storage: plasma: (-) V and VIII (labile factors)
 Adsorption → absent in plasma(-): II, VII, IX,
X
 Present in adsorbed plasma: I, V, VIII, XIII
(fibrinogen): XI, XII, HMWK, PK (contact
group)
 Most abundant: fibrinogen and prothrombin
→ less likely to be deficient.
 Factor II → 80% convert to thrombin and only
20% remain in circulation
 Factors in the Common Pathway: I, II, V, X
 Factors tested by both PT and APTT: I, V, II, X
 Fibrinogen Factors: I, V, VIII, XIII
 Coagulation factor deficiencies not related to
bleeding: XII, PK, HMWK.
GENERAL RULE:
All coagulation factors are produced by the LIVER.
EXCEPT factors: VWF, III, and IV
VON WILLEBRAND FACTOR (VWF)
- Produced by endothelial cells or endothelial
lining (Weibel palate bodies), platelets and
megakaryocytes (alpha granules).
FACTOR III: THROMBOPLASTIN
- Abundant: produced by all tissues in the body
except blood cells.
- When abnormally produced can trigger DIC
patients release thromboplastin-like substance
that will activate both coagulation and
fibrinolysis.
- Crude extract from the tissue
- Component: tissue factor and phospholipid
- COMPLETE: Tissue factor and phospholipid
 HEMATOLOGY 2
- PARTIAL/INCOMPLETE: phospholipid
component only
- PTT: reagent used is complete thromboplastin
- APTT: reagent used is phospholipid from partial
thromboplastin
FACTOR IV (CALCIUM)
- Abundant in the plasma in ionized form.
- Can interact in coagulation mechanism.
- Sources: diet, bone, reabsorbed in the filtrate
ZYMOGENS: II, VII, IX, X, XI, XII, AND PK
 Zymogens – proenzymes: inactive enzymes; no
enzymatic function unless activated.
 II (Prothrombin): inactivated → IIa (thrombin) →
fibrinogen → fibrin; and activate other factors.
 VII (stable factor: no fxn) → VIIa → activate
factor X and IX
 PK → kallikrein → enzymatic ability: serine
proteases
SERINE PROTEASES
- When zymogen is activated and functions as
serine proteases → which when activated,
functions to activate the next enzyme or factor
in the pathway.
- e.g. XIIa → activate XI → XIa → IX → IXa → X →
Xa → prothrombin → thrombin
NOTE:
EXCEPT: I and XIII
- Factor I (fibrinogen): never activated (Note: never
write it as Ia) → write it as fibrin or fibrin monomer
(activated)
e.g. thrombin once formed will activate fibrinogen to
become fibrin (wrong) → use the word cleaves
- Last to occur/be activated: cleavage of fibrinogen to
fibrin.
FIBRINOGEN: ultimate substrate in the coagulation
mechanism (acted by thrombin).
Cofactors: V, VIII, III, HMWK
FACTOR XIII: FIBRIN STABILIZING FACTOR/LAKI-
LORAND FACTOR
- (activated by thrombin) → transaminase or
transglutaminase enzyme → create covalent
bonds between and among fibrin monomers →
stabilization (cross-linkages) → fibrin as
insoluble clot
- Fibrin polymer: first clot formed (soluble and
unstable) unless factor XIII acted on it to stabilize
it.
o The one stabilizes the unstable and
soluble clot by forming covalent linkages
or cross-linking the fibrin monomers
together.
NOTE:
EXCEPT: Co-factors: V, VIII (need to be activated); III,
HMWK (need not to be activated)
Co-factors: increase activity of another coagulation
factor
Activated by thrombin: V → Va and VIII → VIIIa →
function as cofactor.
Va cofactor of Xa → Prothrombinase (Xa-Va-Ca++- PL)
Xa → directly converts prothrombin to thrombin;
need Va to increase its function
VIIIa cofactor of IXa → Intrinsic tenase (IXa-VIIIa-Ca++-
PL) needed to activate factor X to Xa
VIIIa: increases the activity of IXa in activating X
Factor III (Tissue factor) – cofactor of VIII- when
combined to factor III, will be able to generate X and
Xa
Extrinsic tenase: TF-VIIa → activate Factor X
HMWK: cofactor of XIIa; enhances XIIa activity
Function of XIIa: 1.) XIIa → PK → Kal; 2) activate XI
→ XIa
NOTE:
XII, PK, and HMWK deficiency – NO HEMOSTATIC
(BLEEDING) ABNORMALITY (IN VIVO STATEMENT)
IN VITRO: Prolonged APTT
In some, PK and XII deficiency  thrombosis
(abnormal clot formation)
NOTE:
FACTOR I (FIBRINOGEN)
- First to be discovered and most abundant
- Fibrinogen deficiency is unlikely to occur.
- PLASMA PROTEIN (absent in serum due to
consumption → fibrin) → NOT a serum
protein)
LABILE FACTORS: V and VIII (AHF-A)
- Immediately disappear in stored plasma at
room or ref temperature (in vitro).
 HEMATOLOGY 2

- Factor V (labile factor/proaccelerin) – most

labile.
- Factor VIII: labile in vitro and in vivo if it is not
being complexed with VWF.
- APTT (intrinsic pathway test): prolonged in
factor VIII deficiency and liver diseases.

FACTOR VII (STABLE FACTOR)

- IN VITRO: most stable → present in plasma
even if it is stored for several hours.
- IN VIVO: has shortest circulating life (3 hours)
and it is the first to disappear in the
circulation.
- In liver disease: first to be deficient
- FXN: in extrinsic pathway, it is the only
coagulation factor involved in extrinsic
pathway
- Prothrombin Time Test (extrinsic): prolonged
in VII deficiency and liver diseases.
- Prothrombin time: test that is more sensitive
in evaluating liver diseases, vitamin K
deficiency or antagonist.

PLASMA COAGULATION FACTORS (13)

- 12 coagulation factors assigned with Roman

numeral number (RNN) and named according to
order of their discovery.
- Two assigned with RNN but often referred by
their name: Factor III (Tissue factor or Tissue
thromboplastin) and IV (Ionized calcium).
- Factor VI: assigned to a discovered coagulation
factor (removed)  same as activated factor V
(Va): did not move/rearranged assignment of
coagulation number.

COAGULATION FACTOR NOT ASSIGNED WITH ROMAN

NUMERAL NUMBER:

1. HMWK (Kinin system)

2. PK (Kallikrein system)
- Belong and functions to other system.
- Also have a role in coagulation (minor).
3. Platelet factor 3: Platelet phospholipids →
exposed when platelets are activated:
phosphatidyl serine (major) and phosphatidyl
ethanolamine → assembly molecule →
formation of enzyme complex.
 HEMATOLOGY 2

NUMERAL NO. PREFERRED NAME & FUNCTION BIOLOGIC HALF- MEAN

SYNONYM/S LIFE; REMARKS CONCENTRATION
I Fibrinogen Thrombin 2-4 days 200-400 mg/dl
substrate
II Prothrombin Serine protease 3-4 days 10 mg/dl
 Prethrombin
III Tissue Factor Cofactor Lipoprotein found in None
 Tissue Thromboplastin most of the body
tissues; insoluble
IV Calcium Mineral Ionized state 8-10 mg/dl
V Proaccelerin Cofactor 36 hrs 1 mg/dl
 Labile factor Most unstable &
 Accelerator globulin deteriorates
(AcG) Rapidly at RT
VII Proconvertin Serine protease 3-6 hrs; disappears 0.05 mg/dl
 Stable factor rapidly from the
 Serum Prothrombin blood when
Conversion Accelerator production is halted
 Autoprothrombin I (e.g. coumarin)
VIII:C Antihemophilic factor (AHF) Cofactor 10-14 hrs 0.01 mg/dl
 Antihemophilic globulin Bound to vWF
(AHG) (Factor VIII complex)
 Antihemophilic factor A
(AHF A)
 Platelet cofactor I
IX Plasma thromboplastin Serine protease 18-24 hrs 0.3 mg/dl
component (PTC)
 Christmas factor
 Antihemophilic factor B
(AHF B)
 Platelet cofactor 2
X Stuart-Prower factor Serine protease 40-60 hrs 1 mg/dl
 Stuart factor May be stored up to
 Prower factor 2 mons at 40C
 Autoprothrombin III
XI Plasma thromboplastin Serine protease 40-70 hrs 0.5 mg/dl
antecedent (PTA) Circulates in
 Antihemophilic factor X complex with
(AHF C) HMWK
XII Hageman factor Serine protease 50-70 hrs 3 mg/dl
 Glass factor Heat stable; can be
 Contact factor stored at 40C for 3
mons
XIII Fibrin stabilizing factor (FSF) Function? 11-14 days 2 mg/dl
 Laki-Lorand factor
 Fibrinase; Fibrinoligase
 Plasma
transglutaminase
Prekallikrein (PK) Serine Protease 35 hrs 35-50 mcg/ml
 HEMATOLOGY 2

 Fletcher Factor
High Molecular Weight Kininogen (HMWK/HK) cofactor 9-10 hrs 5 mg/dl
 Fitzgerald factor; Contact activation
cofactor
 William factor; Flaujeac factor
Other Procoagulants:
VWF von Willebrand factor Factor VIII carrier; 24 hrs 1 mg/dl
Functions in platelet
adhesion
Platelet factor 3 Phospholipids- Assembly molecule Released by
primarily the platelets
Phosphatidylserine,
PF3

NOMENCLATURE FOR FACTOR VIII OF THE Test: Bleeding time test

INTERNATIONAL COMMITTEE ON THROMBOSIS AND Portion of molecule responsible for
HEMOSTASIS binding to endothelium and supporting
- In circulation, FACTOR VIII circulates in complex normal platelet adhesion and function.
with vWF. Tested by bleeding time
VIIIC: Ag Test: Immunologic monoclonal Ab
Antigenic property of procoagulant
2 MAJOR COMPOSITIONS portion as measured by immunologic
monoclonal antibody techniques
a. High Molecular Weight VWF (autosomal/
vWF: Ag Formerly known: VIIIR: Ag
chromosome 12)
Test: Laurell rocket or
- stable and serve as protein carrier for VIII and immunoradiometric factor assay
low molecular weight VIII:C (sex-linked in X Factor VIII-related antigen, which is a
chromosome) property of the large vWF portion of the
- labile if not carried by vWF  in plasma circulate molecules and measured by immunologic
in complex techniques of Laurell rocket or
b. Low Molecular Weight VIIIC (sex-linked in X immunoradiometric assay
chromosome) The antigen portion of the protein
- labile if not carried by vWF)  in plasma portion of vWF; now vWF:Ag
circulate in complex VIIIR:RCo Ristocetin cofactor activity
Allows platelets to interact or aggregate
VIII/vWF VIII:C and VWF with ristocetin
The molecule as it circulates in the plasma Ristocetin cofactor activity, which is
Composed of VIII:C and VIII:vWF portions factor VIII-related activity required for
non-covalently bound aggregation of human platelets with
VIII:C Function as a cofactor in intrinsic ristocetin in in vitro aggregation studies.
pathway coagulation (antibiotic no longer used
Test: Activated Partial Thromboplastin therapeutically)
Time (APTT/ PTT)
Portion of molecule acting in intrinsic
system as cofactor to IXa (with calcium) in NOTE:
the conversion of factor X to Xa. Tested by FACTOR DEFICIENCY- Leads to bleeding
partial thromboplastin time XII, PK & HMWK Deficiency- No bleeding abnormality
The pro-coagulant portion
VIII: vWF Function in primary hemostasis:
adhesion (platelet function)
 HEMATOLOGY 2

THE 3 GROUPS OF COAGULATION FACTORS (12)

- Based on their common characteristics or
properties

1. FIBRINOGEN GROUP
- Factors: I, V, VIII, XIII
- They are large molecules
- Act as Substrate for Plasmin
- Consumed during the process of coagulation
- They are susceptible to denaturation (most
labile).
- Increase in concentration during an
inflammatory response.
- Increased in women using contraceptive pills
and in pregnancy

2. PROTHROMBIN GROUP (VIT.K DEPENDENT

GROUP)
- Factors II,VII,IX,X
- They are dependent on vitamin K for their
synthesis in the liver
- Oral anticoagulants: cause a functional decrease
in these factors by producing abnormal proteins
that cannot bind calcium
- They are not consumed during coagulation
- They are stable compounds that are well
preserved in stored plasma.
- They require calcium as a cofactor for binding to
phospholipids surfaces.
- Are readily adsorbed from the plasma by BaSO4,
or Al(OH)3.
- They are decreased in patients with problems in
gastrointestinal absorption

3. CONTACT GROUP
- Factors: XI,XII, Prekallikrein, HMWK
- They are fairly stable and not consumed during
coagulation.
- They require contact with negatively charged
surface for activation.
- They are closely related to fibrinolytic, kinin, and
complement systems.
 HEMATOLOGY 2

Characteristics FIBRINOGEN GROUP PROTHROMBIN GROUP CONTACT GROUP

Members I, V, VIII, XIII II, VII, IX, X XI, XII, HMWK, PK
Consumed during CONSUMED NOT CONSUMED NOT CONSUMED
Coagulation/ Exception: Factor II-
clotting partially consumed

Factors in serum ABSENT in Serum PRESENT PRESENT

PRESENT in Plasma
Adsorbed by NOT ABSORBED ADSORBED NOT
BaSO4/ Al(OH)3

Factors present in PRESENT ABSENT PRESENT

BaSO4 adsorbed
plasma
Vit. K dependent NOT REQUIRED
Require Ca++ for NOT
binding to PL
surface
HIGHEST Molecular Weight
MOST LABILE
(V and VIII)
Absent in stored plasma: V
and VIII
Substrate to plasmin (main
CHON for fibrinolysis)
Thrombin cleaves factor I, V,
VIII, XIII
Found in platelets:
I and V: alpha granules
XIII – general cytoplasm of
the platelets (not stored in
granules)
EXCEPT: VIII:C  plasma
bound to VWF (protein
carrier)
Increase in inflammation
(APP) and pregnancy; in
women using contraceptive
(V and VIII)
DEPENDENT NOT REQUIRED
LEVELS NOT AFFECTED
(For functional ability of the
factors)  binding on surface
of platelet or any phospholipid
REQUIRES NOT
Needs to be carboxylated in
order to interact with Ca++ in
the presence of vit. K
STABLE STABLE
Vitamin ADEK fat soluble
vitamins  should be
properly digested bile for
fat solubilisation PRESENT IN ALL SAMPLES
If there’s an obstructive
jaundice  bile can’t be
drained in duodenum
Undigested fats and vit K.
will not be reabsorbed Closely related to fibrinolytic,
kinin and complement systems
Decrease GIT absorption
problems: (e.g. Obstructive 1. XII  XIIa = coagulation
jaundice, excessive bacterial 2. XIIa  PK Kal  Kininogen
overgrowth) = Kinins
3. XIIa-Kal: plasminogen
Affected by oral activator  plasmin =
anticoagulants: immediate fibrinolysis
action: coumarin, 4. Plasmin  complement
coumadin, warfarin - inhibit activation
synthesis of vit. K; vit. K
antagonist
 HEMATOLOGY 2

NOTE: THEORIES ON COAGULATION

ADSORPTION: process of removal of different
coagulation factor; adherence of smaller molecule to 1. MORAWITZ/CLASSICAL THEORY (1905)
larger molecule or surface. - Believes that there are 2 stages coagulation:
BaSO4/ Al(OH)3: large molecule unto which small a. Thrombin formation: by the combination of
molecules adhere prothrombin plus calcium ions plus tissue
Prothrombin Group: needs Vit. K for them to interact thrombokinase.
with Ca++ → bind with platelet PL
Excessive fibrinolysis: plasmin destroys intact factor V
and VIII

b. Fibrin formation: the formed thrombin acts

on the fibrinogen in the plasma.

2. HOWELL’S THEORY (1910)

- This theory believed that there are 5
coagulation factors involved: (Fibrinogen,
FACTORS II, VII, IX, X: Terminal AA (Glutamic acid): Calcium, Prothrombin, Anti-prothrombin,
requires additional –COOH (carboxyl radicals)  Thromboplastin)
gamma carboxylation (addition of another –COOH)
 to make it an anion (net negative charge)
interact with cation (Ca++) adhere or assembly on
the surface of platelet phospholipid (negatively
charge).
Gamma carboxylation: Vitamin K is required as a
cofactor for Factor IX to gain a negative charge for it
to attach with calcium.

Sequence of reactions:
NOTE:
1. Clotting (Serum): (-) I, V. VIII, XIII a. Prothrombin is inactivated by anti-
2. Adsorption: (-) II, VII, IX, X prothrombin/ heparin
3. Storage Age: (-) V and VIII b. When prothrombin needs to be activated
(vessel injury) → Thromboplastin neutralizes
anti-prothrombin and releases prothrombin
COAGUALATION MECHANISM c. Prothrombin → (Ca++ and thromboplastin) 
- Coagulation is a complex series of cascading Thrombin
reactions involving development of enzymes d. Thrombin + Fibrinogen → Fibrin
from their precursor. As one enzyme formed, it
converts the next zymogen to its activated 3. MODERN THEORIES: “WATERFALL” &
enzyme. This process continues until a fibrin “CASCADE” (1964)
meshwork clot has formed. Fibrin forms a - By Breckenridge and Ratnoff
meshwork in and around the primary hemostatic - 3 distinct phases:
platelet plug-like spider web, producing a stable
physical barrier.
 HEMATOLOGY 2

 Activation of factor X via the intrinsic or extrinsic

pathway (start of COMMON PATHWAY).
 Conversion of prothrombin to thrombin
 Conversion of fibrinogen to fibrin
 Advocated the phases of coagulation

PHASES OF COAGULATION: Coagulation is divided

into four phases
1. Contact Activation Phase (XII – IX)
- Starts with the activation XII (comes in contact
with negative surface), then activation of
prekallikrein to kallikrein; and factor XI.
2. Activation of Factor X (via intrinsic Pathway or
via Extrinsic Pathway)
- Formation of Thrombin: Conversion of
Prothrombin to Thrombin
3. Formation of Fibrin
COAGULATION CASCADE
IN VITRO COAGULATION
1. INTRINSIC SYSTEM
- All necessary factors are IN the blood
(circulation) VIII, IX, XI, XII, HMWK, PK
- All components are found in the circulating
blood. To be activated, the blood must have
direct contact with a foreign object such as a
damaged blood vessel or glass. This results to
absorption of XII of the negatively-charged
collagen exposed by vessel wall damage.

Sequence of reactions:

a. Factor XII is activated by the foreign material

into XIIa;
b. XIIa reacts weakly with XI, prekallikrein (also by
XIIf), and plasminogen, thus converting them
into their forms
 HEMATOLOGY 2
- Formed kallikrein feeds back to XII and causes
enzymatic cleavage producing more XIIa.
c. XIa in the presence of Ca activates IX
Note: IX can also be activated by VIIa-Ca-TF complex:
(Current concept of Coagualation)
d. IXa in the presence of Ca, platelet phospholipids,
and VIIIa activates X.
NOTE:
APTT: deficiency in any factors  PROLONGED APTT
Activators: Exposed collagen (major component of
connective tissue that surrounds and support BV)
and chemical  XII (contact factor)  XIIa 
perform several functions:
XII (glass factor/contact factor)  XIIa
1. Coagulation cascade: 1. (CONTACT ACTIVATION
PHASE: Activate XI  IXa); (IXa-VIIIa –Ca++-PL:
Intrinsic tenase); convert PK to Kal
2. Initiate fibrinolysis: XIIa  plasminogen to
plasmin
e.g. Which of the pathway occurs when the blood is
placed on a tube or glass slide and allowed to clot?
INTRINSIC PATHWAY  coagulation XII (glass factor)
 XIIa
2. EXTRINSIC SYSTEM/ TISSUE FACTOR PATHWAY
- All factors needed are in the tissue NOT in the
circulation. Produce by all tissues but not by
blood cells. ONLY FACTOR VII is involved.
- Does not occur outside the body unless you add
a reagent
- Tissue Factor/Tissue thromboplastin
introduced in the circulation when there’s
vessel/tissue injury.
- In classical concept: TF activates VII  VIIa and
combines with VIIa
- In current concept: VIIa (inert material) is
already available in the circulation in small
amount (1-2%)  combine with TF/Factor III
(cofactor)
 EXTRINSIC TENASE: TF + VIIa = TF/VIIa 
activates X in common pathway
 Tissue factor is necessary for the
activation of this system. All cells with
the possible exception of those in the
blood, contain tissue factor. When
injury occurs, the tissue factor is
released and acts as a cofactor in
initiating coagulation. The released
tissue factor bind to VII and activates it
to VIIa. Then, VIIa, together with
calcium, activates X to Xa of the
common pathway.
 VII  prothrombin time: deficiency of
VII  prolonged PT
3. COMMON PATHWAY
- Where both intrinsic and extrinsic pathway
usually meet or merge. The application is in in-
vitro coagulation. Superseded by the current
concept of the coagulation  REVISED
COAGULATION MECHANISM (In vitro:
laboratory tests)
- Begins with activation of factor X via the
Intrinsic Pathway (IXa-VIIIa-Ca-PL) or via the
Extrinsic Pathway (VIIa-III/TF) to form
Prothrombinase/Xa-Va-Ca-PL. This
prothombinase complex converts prothrombin
to thrombin which enzymatically cleaves
fibrinogen into fibrin.
- Thrombin activates XIII  XIIIa
(transaminase/transglutaminase)
- 4 FACTORS INVOLVED: X, V, II, I  tested by
PT/APTT.
 HEMATOLOGY 2
NOTE:
In performing a laboratory test on coagulation, follow
and interpret the test using the CLASSICAL CONCEPT
OF COAGULATION
Routine Screening Test for coagulation factor
deficiency: COAGULATION TESTS: measure how long
does the plasma take to form a clot; end result 
clot formation
Intrinsic: APTT
Extrinsic: PTT (Prothrombin Time)
PT: prolonged; APTT: Normal  Extrinsic factor
deficiency (VII)
PT: Normal APTT: prolonged  Intrinsic factor
deficiency (XII, XI, IX, VIII, HMWK, PK)
Both prolonged  Coagulation factor deficiency of
the common pathway (X, V, II, I)
In APTT/PTT (in vitro testing): XIII is not included 
not detected
XIII not needed for the formation of clot  stabilize
the clot
NOTE:
COAGULATION ROLES OF THROMBIN: activates V,
VIII, XIII, cleaves fibrinogen + activates XI.
FIBRIN FORMATION
FIBRINOGEN STRUCTURE:
- D-terminal domains and a central-E domain
- 3 types of polypeptide chain (pairs):
o + Fibrinopeptides (2A)
o Alpha (2B) beta
o Gamma (no fibrinopeptide)
- Fibrinopeptide: removed by thrombin
- Fibrinogen: soluble protein; not seen in plasma,
but once fibrinopeptide A and B is removed 
insoluble (clot is seen).
 alpha, beta and gamma chains terminates in D-
domain and meet at the center coil E-domain
 exposing alpha and beta fibrinopeptides 
thrombin (proteolytic enzyme) cleaves
fibrinopeptides A and B  removed  fibrin
monomer (insoluble)  several formed 
polymerization: end to end and side to side
connection  fibrin polymer (clot) soluble and
unstable clot  fibrin cross-linking by factor
XIII  covalent linkages in D-terminal 
stabilized fibrin
STEPS IN CONVERTING FIBRINOGEN TO FIBRIN
1. Thrombin cleaves the alpha and beta chains
only (releasing fibrinopeptides A and B) 
soluble fibrin monomer/unstable gel.
2. Fibrin monomers aggregate spontaneously end
to end, side to side to form soluble fibrin
polymers thus vulnerable to enzyme plasmin
(also soluble in 5M urea).
3. Factor XIII: XIIIa + Ca crosslinks adjacent fibrin
monomers by forming covalent bonds to form
stabilized fibrin.
OTHER RELATED SYSTEMS:
1. Kinin System: this system is involved in
chemotaxis, pain sensation, and mediate
inflammatory responses, increased vascular
permeability, and vasoconstriction and induce
smooth muscle contraction.
2. Fibrinolytic system: The Kallikrein, XIIa, &
HMWK act as plasminogen activator.
3. Complement System: this mediate immune and
allergic responses.
 HEMATOLOGY 2
CLASSICAL (IN VITRO)
- Does not explain how physiologic hemostasis
occur in vivo.
- Interdependent pathways
IN VIVO CONCEPT OF COAGULATION/ CURRENT
CONCEPT/ REVISED CONCEPT OF COAGULATION
- Current Concept of the Coagulation Cascade (In
vivo coagulation)
- INITIATING step: exposure of TF and its reaction
with VII.
2 FACTORS ACTIVATED BY TF/VIIa: X and XI 
production of thrombin
a. INITIATION: Extrinsic tenase  3%- 5% lIa
- Small effect of activation of IX and X + Tissue
Factor Pathway Inhibitor (naturally occurring
inhibitor) inhibits VIIa and Xa  less thrombin
production.
b. PROPAGATION (platelets)  95%
lIa/Thrombin:
- Self-perpetuating /autocatalytic.
- When complexes are increased in phospholipids,
the more thrombin production.
NOTE:
The key initiating step is the exposure of TF to the
circulation and reaction of TF with factor VII. The TF-
factor/VIIa complex can enzymatically activate
factors X and IX. The initial activation of factor X to Xa
may be important in getting the coagulation cascade
started, however, tissue factor pathway inhibitor
(TFPI) rapidly inactivates the TF-VIIa-Xa complex. The
major action of the TF-VIIa complex in vivo is the
activation of factor IX to IXa, which then activates
factor X to Xa. Consequently, prothrombin is
converted to thrombin, then fibrinogen is converted
to fibrin. Factors Xa and IIa have positive feedback
activity on earlier steps of the cascade.
NOTE:
FACTOR XII: In vivo coagulation; their role is not
necessary; can be by passed; XI can be activated by
thrombin; deficiency will not cause bleeding
conditions.
Key initiating step: cause to start the reaction:
activation of XII and VII (more important to occur);
VIIa/TF  series of reaction
Xa and IIa (positive feedback activity: once formed go
back to their initial reaction and proceed to cause
more activation)
 Xa  VII  VIIa (2 chain) more effective in
function
 VIIa (single chain) produce by other factors or in
plasma; less efficient.
 Thrombin: once formed, it can cause increase in
production.
THROMBIN FEEDBACK MECHANISM
ACTIVATION OF COAGULATION
1. Autocatalytic or self-perpetuating: many more
thrombin from small amount present
2. LOW level of thrombin activates V and VIII
3. Activates XIII  XIIIa; calcium dependent
4. Induces platelet aggregation; agonist
5. Also activates factor XI; current concept
6. Procoagulant: Activates TAFI (thrombin
activatable fibrinolysis inhibitor) – inhibits
fibrinolysis; produced by endothelial cell
INHIBITOR TO COAGULATION
1. Increased thrombin: destroys Va and VIIIa  prevents
clotting/continuous clotting Disseminated coagulation
2. Activate Protein C (w/ Ca++ and thrombomodulin) 
inhibitor of coagulation (CHON S)  plasmin generation
 inactivation of Va Vi and VIIIa  VIIIi
3. Promote plasmin generation (fibrinolysis)
 Controls excessive coagulation
 Increase concentration of thrombin (HIGH level);
destroys V and VII; activated protein C
 Protein C and S increase plasminogen activation
 HEMATOLOGY 2
QUESTIONS TO REMEMBER:
1. What may activate II: PROTHROMBINASE: II  (Xa-
Va-Ca-PL)  IIa
2. What may activate XII: in vivo: collagen or any
other negative surface or chemicals; in vitro: glass
contact
3. What may activate X: X  (IXa-VIIIa-Ca-PL)  Xa
4. What may activate V and VIII: THROMBIN (IIa): V
(thrombin)  Va ; VIII  (thrombin)  VIIIa
5. What may activate XI: XI  (XIIa, IIa)  XIa for
propagation
6. What may activate IX: Extrinsic tenase and XIa: IX
 (VIIa/TF) or (XIa)  IXa
7. What activation reactions can be by-passed: XII 
XIIa (even without, coagulation can still occur)
FIBRINOLYSIS
- Last phase of Coagulation
- Re-establishment of blood flow → lysis of clot.
- It is defined as the dissolution of clot or lysis of
clot by plasmin or fibrinolysin.
- Plasmin: not normally present in plasma and it is
derived from plasminogen, which is produced by
the liver.
- Plasminogen: normally present in plasma;
precursor synthesized by liver; it will convert into
plasmin by plasminogen activators (3 activation
phases).
EXCESSIVE/ UNREGULATED FIBRINOLYSIS:
- May lead to disorders: inability to clot/excessive
clotting.
REGULATE FIBRINOLYSIS: REGULATORY PROTEINS
 PLASMINOGEN ACTIVATOR INHIBITOR (PAI)
- It inhibits the activators of plasminogen to
prevent excessive production of plasmin.
 ALPHA-2-ANTIPLASMIN
- Inhibit plasmin; prevent excessive function of
plasmin
FIBRIN DEGRADATION PRODUCTS (FDP) OR FIBRIN
SPLIT PRODUCTS (FSP)
- released after the fibrin clot is broken down
ACTIVATION OF PLASMINOGEN → PLASMIN
WAYS OR PHASES: PLASMINOGEN ACTIVATORS
1. INTRINSIC ACTIVATION: XIIa AND KALLIKREIN
- by XIIa-Kallikrein-HMWK
- Contact factors are closely related to the
fibrinolytic system: → XIIa-Kal → plasminogen
 plasmin
 HEMATOLOGY 2
2. EXTRINSIC ACTIVATION: TPA (tissue type)
- Tissue Plasminogen Activator(TPA): produced by
endothelial cells
3. EXOGENOUS ACTIVATION
- introduced into patient: Urokinase and
Streptokinase Therapeutic and fibrinolytic agent:
prevent occurrence of abnormal clot
o Urokinase: present in the urine
o Streptokinase: exogenous; comes from
strep organisms
INHIBITORS
 Plasminogen: Histidine-rich glycoprotein
 Activators (TPA): Thrombin Activatable
Fibrinolysis Inhibitor(TAFI) & Plasminogen
Activator Inhibitor (PAI)
 Plasmin: plasmin inhibitor (alpha-2-antiplasmin;
alpha-2-macroglobulin)
FIBRINOGEN LYSIS
FIBRINOGEN
- Made up of three polypeptide chains: alpha,
beta, and gamma.
ALPHA CHAIN
- first site that plasmin will be acted on:
1. Plasmin will cleave alpha chains  Fragment X
(D-E-D)
- Fragment X: first fragment to be formed;
largest.
2. Fragment X will be acted on by plasmin
asymmetrically (one side) to separate one of
the D’s  Fragment Y (E-D combination)
3. Plasmin will act on Fragment Y and separate E
from the D.
FIBRINOGEN DEGRADATION PRODUCTS (FDP)
 X and Y: early degradation products
 D and E: late degradation products
NOTE:
Fibrinogen Degradation Product = XYDE
FIBRINOLYSIS
FIBRIN
- Fibrin monomers (connect end-to-end (D-D
connection) and side-by-side (D-E-D connection)
 fibrin polymerization XIIa (stabilizes clot;
crosslinks by forming covalent linkages between
D-D).
FIBRIN DEGRADATION PRODUCTS X, Y, D-D DIMER, E
- Acted on by Plasmin
D-DIMER/ D-D PRODUCT
- indication (specific) of fibrin lysis (clot formed)
- main difference between the products
YY COMPLEX/ YY FRAGMENT: E-D-D-E
DXD COMPLEX: D-D-E-D-D
 HEMATOLOGY 2

DIMER: clot Fibrinolysis is activated within 24-48 hours.

MONOMER (FIBRINOGEN): no clot formed COAGULATION MECHANISM

Coagulation and Fibrinolysis should have balance
Excessive coagulation → thrombosis
Excessive fibrinolysis → bleeding

NOTE:
 Fibrin Degradation Product: X Y D (D=D; D-
dimer) E
 In normal coagulation/hemostasis/fibrinolysis
→ X, Y, D (D-D Dimer), E will be cleared by the
liver but not in excessive fibrinolysis and liver
diseases; or if increase production of FDP → NATURALLY OCCURRING INHIBITORS OF
bleeding. COAGULATION AND FIBRINOLYSIS
SERPIN FAMILY (Serine Protease Inhibitors)

EFFECTS OF FDP: INHIBIT OR INTERFERE IN NORMAL SERINE PROTEASES

COAGULATION - Those when activated there function is to
 X and Y: with anticoagulant effects activate another enzyme in the cascade.
 Y and D: inhibit fibrin polymerization - e.g. VII → VIIa → IXa
 Fragment E: inhibits thrombin
NAME FUNCTION
NOTE: Tissue factor pathway With Xa, binds TF:VIIa
Primary Hemostasis: rapid in response; platelet plug: inhbitor
short lived and unstable Thrombomodulin Endothelial cell surface
Secondary Hemostasis: slower response; fibrin clot: receptor from thrombin
long live and stable Protein C Serine protease
Protein S Cofactor
SUMMARY OF STEPS IN HEMOSTASIS Antithrombin Serpin
1. Vessel injury Heparin Cofactor II Serpin
2. Vasospasm (TxA2; serotonin) Z-dependent protease Serpin
3. Platelet plug → PRIMARY HEMOSTASIS inhibitor
4. Insoluble fibrin clot
Alpha 1 -protease Serpin
5. Clot retraction
inhibitor (alpha1 –
6. Clot dissolution or fibrinolysis
antitrypsin)
Alpha2-macroglobulin Serpin
Clot formation (in vivo)= 6 minutes
Clot formation (in vitro) = Red tube (15-20 minutes →
1 hour completely clotted)
 HEMATOLOGY 2

INHIBITOR COAGULATION FIBRINOLYSIS NOTES

Component(s) Component(s)
inhibited inhibited
Antithrombin III Thrombin, VIIa, IXa, Plasmin  First inhibitor discovered and tested
Xa, XIa and XIIa and  Main inhibitor of the entire coagulation
Kallikrein mechanism; major inhibitor of thrombin
 Enhanced by a co-factor Heparin
 It is a serpin.

THROMBIN FUNCTIONS:
1. Induces platelet aggregation
2. Activates factors XIII, V, VIII, XI
HEPARIN: does not remove Ca++

ROLE of HEPARIN: Cofactor of AT-III  inhibit

thrombin; serves as a bridge. Heparin when
bound to AT-III causes a morphological change to
be more specific to the size of thrombin.
Thrombomodulin Thrombin  It binds to thrombin and inhibits
thrombin (functions to activate protein
C).
 Produced by endothelial lining and it is a
membrane bound.
 Thrombin when bound to
thrombomodulin can’t activate factor VIII
and V instead it will activate protein C to
become Activated Protein C (APC).
 Trimolecular complex: Thrombomodulin
+ thrombin + Protein C + (receptor:
EPCR)  APC.
Thrombin INHIBIT
Activatable FIBRINOLYSIS
Fibrinolysis
Inhibitor(TAFI)

Plasminogen
Activator Inhibitor
(PAI)
Tissue Factor Xa & TF:VIIa  Kunitz-type serine protease inhibitor
Pathway Inhibitor complex that has a double inhibitory effect
(TFPI)  Major inhibitor of extrinsic pathway
3 Kunitz type:
Lipoprotein Kunitz 1: binds with VIIa; inactivating therefore
Associated VIIa-TF complex
Coagulation Kunitz 2: binds with Xa; inactivating Xa
Inhibitor (LACI) Kunitz 3: unknown fxn

Extrinsic Pathway X (intrinsic tenase: IXa-VIIIa-Ca-PL) or (extrinsic

Inhibitor (EPI) tenase: TF-VIIa) Inhibited by TFPI  Xa Inhibited
 HEMATOLOGY 2

by TFPI  NO: CAN’T HAPPEN: II 

(prothrombinase)  IIa

Alpha-2 Thrombin & Plasmin  Inhibits plasmin when alpha-2

macroglobulin Kallikrein antiplasmin is depleted.
C’1 inactivator/ XIIa, XIa,& Plasmin  Inhibitor in the complement pathway
C’1 esterase Kallikrein
inhibitor
Protein C and S Va and VIIIa Enhances plasmin  Cofactor: Protein S
system formation   Vit. K dependent inhibitor
ENHANCES  Inactivates inhibitors of plasminogen
FIBRINOLYSIS activators
 Produce as zymogen by the liver
 Activated by thrombin
 APC will inactivate VIIIa and Va  VIIIi
and Vi
Protein S: exists in 2 forms in plasma
Bound to C4bBP(complement pathway;
inflammation): 60%
Free Protein S: 40%  can ONLY serve as
cofactor
In inflammation (excessive coagulation): 85-90%
bound and 10-15% is free: can’t inactivate VIII
and V.
Alpha-1- XIa Plasmin  Inhibits plasmin only when a-2
antitryspin/ A-1 antiplasmin and a-2 macroglobulin are
protease inhibitor depleted.
Alpha-2- Plasmin  Major inhibitor of plasmin
antiplasmin  First to inhibit plasmin
Z-dependent Xa, XIa  Enhanced by its co-factor protein Z
protease inhibitor (CHON –Z) additional protein that is vit. K
(ZPI) – dependent like II, VII, IX, X
 Serpin – inhibits serine protease

Protein C inhibitor Thrombin, APC, TPA, urokinase  Anticoagulant

thrombin-  Pro-coagulant
thrombomodulin  Fibrinolytic inhibitor
complex

QUESTIONS ASKED:
1. Factors in the intrinsic pathway: VII, IX, XI, XII, HK, PK (deficiency in any prolongs APTT)
2. What are the cofactors: TF-VIIa; Va-Xa; VIIIa-IXa; HK-XIIa
3. The only transaminase/transglutaminase among the coagulation factor: XIII
4. What stabilizes fibrin: XIIIa
5. Vitamin K-dependent: II, VII, IX, X, C, S, Z
V- (labile factor) Pro-accelarin / acceleratorgobulin
VII- (stable factor) Pro-convertin / plasma conversion
IX- Plasma thromboplastin component = anti hemolytic factor B
XI- Plasma thromboplastin antecedent = anti hemolytic factor C
 HEMATOLOGY 2

Coagulation Group Pathway Test - One reagent in APTT/PT is calcium, therefore the
factor involved prolonged excessive citrate in blood sample will bind with
If factor is the reagent calcium  prolonged test result;
deficient calcium is inactivated by excessive citrate.
(PT/APPT/
BOTH) 2. Clots in specimen
Fletcher Contact Intrinsic APTT - Clotting happens due to consumption
factor (PK) group
coagulation factors (I, V, VII, XIII).
Accelerator Fibrinogen Common Both
- REJECT SAMPLE even the clots are micro clots
Globulin (V) group
- In coagulation studies, presence of even micro
Antihemophili Fibrinogen Intrinsic APTT
c factor A clots would cause the rejection of sample.
(VIII:C)
Proconvertin Prothrom Extrinsic PTT 3. Hemolysis and Traumatic phlebotomy
(VII) bin - Tissue Factor introduction
Plasma Contact Intrinsic APTT - Hemolyzed RBC: exerts thromboplastin-like
thromboplasti activity
n antecedent - Excessive probing  damage tissue 
(XI) thromboplastin: TF:VIIa  coagulation
Plasma Prothrom Intrinsic APTT mechanism.
thromboplasti bin
n component; 4. Icterus; Lipemia
Christmas - Affects spectrophotometry: more light
factor (IX)
absorbed  concentrated.
Stuart-Prower Prothrom Common BOTH
factor (X) bin COLLECTION OF SAMPLE
1. NEEDLE GAUGE
 Adult (<25ml): G20 - 21; 1- 1.25 in.
LABORATORY EVALUATION OF COAGULATION AND
FIBRINOLYSIS  Adult (>25 mL): G19; 1 or 1.25 in.
- All coagulation testing critically depends on the  G23: Child or adult with friable, or hardened
quality of the specimen. Minimum tissue veins.
trauma and the avoidance of hemolysis are  G20, 21, or 23: Syringe with winged-needle set
essential. It is therefore important to adhere to o The smaller the bore, the slower the
principles of proper specimen collection, blood will flow into the needle
handling and preparation. o The larger the bore, the easier will blood
will flow  prevent HEMOLYSIS.
GENERAL INSTRUCTION FOR COAGULATION TESTS o For G23(smaller): Avoid hemolysis by
- Time is important: Coagulation test like in XIII gently pulling of the plunger/ gentle
assay; any factors that may affect time should be aspiration
avoided. 2. SYRINGE
- Plastic syringe or silicone-coated
SOURCES OF ERRORS AFFECTING COAGULATION TESTS: - AVOID using GLASS in all procedure: use plastic
1. Blood volume is inadequate causing excess test tube, pipettes and syringes
anticoagulant - Glass will activate XII (glass factor)
- Recommended ratio: 1:9 using Na citrate
- In routine hematology: half full is accepted 3. ANTICOAGULANT
- 3.2% sodium citrate
- In coagulation studies: tubes should be at least
90% full.
 HEMATOLOGY 2
- If patient’s Hct is more than or equal to 55%,
adjust the volume of citrate since excess citrate
will bind with reagent calcium.
- ADJUSTING THE VOLUME OF CITRATE:
o C= (0.00185) (V)(100-H)
o Where:
 V = blood volume in mL
 H = patient’s Hct
 C= volume (mL) of anticoagulant
needed.
4. TEMPERATURE
- Most of the tests are performed at 37°C
- Perform: STAT
- But if there is delay  Freeze the PPP only; DO
NOT FREEZE WHOLE BLOOD/ BLOOD WITH RBC.
- Storage:
o PT (extrinsic pathway: VII (stable
factor)): uncentrifuge (with Na citrate)
and well capped at room temperature
for 24 hours
o Never refrigerate samples for PT: cold
temp  cold activation: VII and XI
o APTT/PTT (intrinsic pathway factors:
VIII, IX, XI, XII, HK, PK): uncentrifuge for
4 hours at 4°C.
o Factor VIII (labile at room and ref. temp.)
note also V (labile factor; common
pathway).
o Never leave at RT: VIII will deteriorate
o Test to monitor unfractionated heparin
therapy is APTT: heparin+antiheparin
factor (PF4)
o Unfractionated heparin (PTT) 
separate/centrifuge in 1 hour  Platelet
poor plasma (PPP) to prevent the action
of PF4 against the heparin.
o PPP: at 20°C for 2 weeks; at -70°C for 6
months.
SAMPLE PREPARATION
- Sample Used:
o Whole Blood
o Plasma
 Anticoagulant used: 3.2%
Sodium Citrate
 Aspirate always using plastic
pipette.
 Platelets are found in the buffy
coat layer.
NOTE:
Platelet Poor Plasma (PPP) preparation (<10,000/uL)
 Centrifugation: 2000 xg for 10 minutes;
Rodak’s: 1500 g for 15 minutes
 Collect only the upper 3⁄4 of the plasma
layer.
 Test immediately because exposure to air
changes the pH due to loss of carbon dioxide
(↑pH).
 Store plasma at 4°C but not to exceed 2
hours or rapid freezing to -20°C.
Platelet Rich Plasma (PRP) Preparation (200,000 –
300,000/uL)
 Centrifugation: 60 – 100 xg for 10 minutes at
RT; Rodak’s 50 g for 30 mins
 Separate upper portion of the sample
NOTE:
Coagulation factors are in plasma.
2 concentration of sodium citrate:
3.2% SODIUM CITRATE (recommended by NCLSI)
 V and VII (preserve well)
 Less affected by polycythemia vera
 Normal Ratio: 1 (citrate): 9 (blood)
In px with high hct: adjust volume of citrate to prevent
excessive citrate which will bind to reagent calcium.
3.8% SODIUM CITRATE
 easily affected by high hct samples or polycytemia
vera.
DO NOT use EDTA (labile factors are not well preserve,
inhibit thrombin-fibrinogen interaction, affects calcium)
and HEPARIN (inhibits thrombin).
 HEMATOLOGY 2
NOTE:
ROUTINE SCREENING TEST (PT/APPT): Light blue first
SPECIFIC FACTOR ASSAY (like VIII, VII assay): use a
prime/dummy tube, red top, light blue
FOR OPEN SYSTEM: Two-syringe method: after
collecting blood using the first syringe, detach
syringe without withdrawing the needle and replace
with another syringe. The blood in the first syringe
will be discarded and the blood in second syringe is
the one be used.
RULE:
1st: DISCARD
2nd: USE
NOTE:
What factors will deteriorate at room temperature? V
and VIII
Refrigerator temperature affects what factors? VII
and XI
PT plasma samples capped, are stable for 24 hours at
what temperature? Room temperature
APTT samples are stable for 4 hours if stored at 4°C.
COAGULATION TESTS
- All based on the time it takes for the blood
samples to form into a clot whether plasma or
whole blood.
- Accuracy in measuring time is necessary because
little excess may be reported as falsely
prolonged instead of normal.
TEST FOR INTRINSIC AND COMMON PATHWAYS
FACTORS TO DETECT: VII, IX, XI, XII, HMWK, PK
CONTACT FACTORS: I, II, V, X
TISSUE FACTOR: where activation starts in vivo.
1. COAGULATION TIME (Whole Blood)
- All of the components needed for clotting are
present in the blood.
- When blood is placed in a glass test tube or glass
slide, the blood will be activated.
- Factor XII is activated first by contact with glass
slide (PK to Kalikrein; XIIa activate XI to XIa; Xia
activates IX to IXa).
- Activation starts with contact activation phase
- Measures the time required for whole blood to
clot after it has been removed from the body
- Affected by amount of blood used.
A. SLIDE METHOD
 REFERENCE VALUE: 2-4 minutes
 REQUIREMENT: glass slide method
B. CAPILLARY METHOD (Date and Laidlaws
Method)
 REFERENCE VALUE: 2-4 minutes
 REQUIREMENT: Non-heparinized
capillary tube (blue ring) or else no
clotting will occur.
PROCEDURE FOR BOTH (SLIDE AND CAPILLARY
METHOD):
a. Fill the non-anticoagulated capillary tube & let it
stand for 2 minutes
- NOTE: do not wipe off the first drop of blood but
include it.
b. Start time as soon as blood starts to appear from
the site of puncture
c. For the slide method place 2-3 drops of blood in
a slide while for capillary tube, fill it with whole
blood
d. Wait for 2 minutes initial time before checking
for fibrin clot formation
e. CHECKING:
o SLIDE METHOD: check by using the same
lancet from collection by pricking into
the blood sample and raising the blood
 HEMATOLOGY 2
sample until a thin fibrin thread is
observed as thin as the hair & record the
time when the thin fibrin thread is
observed as the clotting time
o CAPILLARY METHOD: Break the glass
tube on one side without pulling them
apart and slowly pull them apart and
observed for a thin fibrin thread
connection, connecting both fragments.
f. If no fibrin thread is observed wait for another
30 seconds and check again and if it is not yet
visible again continue waiting for another 30
seconds and check again.
NOTE:
BOTH NORMAL VALUE: 2-4 minutes at RT
AFFECTED BY: amount of blood used
Micromethod requires fewer blood sample while for
macromethod, the longer is the clotting time.
C. LEE AND WHITE METHOD (Macromethod)
o REFERENCE VALUE: 7-15 minutes
o DISADVANTAGE: Longer clotting
time due to usage of larger blood
volume.
PROCEDURE (LEE AND WHITE METHOD):
a. Place 1 mL of each into 3 tubes with equal size
and dimension (12 x 75)
b. Label the tube as 1, 2 and 3
c. Extract 4 mL of whole blood
d. Start time as soon as blood appears at the
puncture site.
e. Place 1 mL of blood in each tube starting from
the 3rd tube up to the first
f. Discard the last remaining 1 mL
g. Stoppered the three tubes and incubate at 37°C
in a water bath for 5 minutes undisturbed
h. After 5 minutes, check for clot formation by
tilting the tubes at 45° angle
i. observed first the tube 1 up to tube 3
j. If blood flows to the tube, it is not yet fully
clotted thus incubate further for 30 sec.
k. After 30 secs, check tube 1 again and when
tube 1 does not spill its content upon total
inversion, clotting time is complete and record
- NOTE: do not report clotting time in tube 1
l. After 30 secs. Check for clotting for tube 2 and
same procedure will be employed as with tube
1
m. Once clotting is complete in tube 2, wait for
another 30 secs. Before reading tube 3
n. Once 30 secs is reach, check for clotting with
tube employing the same procedure with tube
and report the clotting time
NOTE:
TIME DIFFERENCE BETWEEN TUBES: 30 SECONDS
REPORTING: from time the blood starts to appear until
complete clotting time in tube 3.
REFERENCE VALUE: 7- 15 MINUTES
2. ACTIVATING CLOTTING TIME (reliable)
- PRINCIPLE: Whole blood contains all the
components necessary to produce a clot when
put into a glass tube. By adding an activator (e.g.
Diatomite) and keeping blood at constant 37°C.
- The test becomes more reliable and rapid.
- REFERENCE VALUE: 75-120 seconds
- ACTIVATOR: Diatomite – shortens clotting time
and enhances the coagulation factors
NOTE:
PLASMA CLOTTING TIME
- Coagulation factors are in the plasma
- Plasma is the one which clots
- Plasma clotting is translucent
1. No clot
2. Clotted
3. Clotted
4. Clotted
3. PLASMA RECALCIFICATION TIME
- Plasma was prepared using Na Citrate thus Ca is
removed.
- Plasma is calcium free thus add calcium (reagent)
- Upon addition of calcium take the time it takes
for plasma to clot
 HEMATOLOGY 2
- Reference value depends on the plasma used
(platelet-rich plasma or platelet poor plasma)
- No longer performed
- PRINCIPLE: except for Ca, normal PRP/PPP
contains all the components necessary for
generating fibrin.
- REFERENCE VALUE:
o PRP: 100-150 seconds
o PPP: 130-240 seconds
4. ACTIVATED PARTIAL THROMBOPLASTIN TIME
(APTT)
- Routine screening test
- Thromboplastin/Tissue Factor is involved in the
extrinsic pathway which activates VII to VIIa
and binds to it
- Test for Heparin therapy
- REAGENT: partial thromboplastin: cephalin
(phospholipid only) & 0.025 M Ca (reaction
initiation
- SAMPLE: Platelet Poor Plasma (PPP) (Hct <55%)
to avoid inappropriate with citrate and plasma
volume.
- ACTIVATORS: Kaolin, Celite, Ellagic acid
- PURPOSE: for determination of deficient in
intrinsic pathway
- PROCEDURE: Starts with the activation of FXII
upon contact with a negative surface. In vivo, it
may be collagen while for in vitro it is the glass
test tube. Coagulation cascade will not proceed
if intrinsic tenase and prothrombinase will not
be formed thus no fibrin formation. Both
intrinsic tenase and prothrombinase requires
Ca & phospholipid. The source of phospholipid
source is the platelets but since the sample is
platelet poor plasma, there would be no source
of phospholipid. While, Ca is removed by
citrate. Thus, cephalin and Ca is added in order
to observe clotting. Upon addition of cephalin
and Ca, observed the time it takes to form fibrin
clot
- Most useful procedure for routine screening of
coagulation disorders in the intrinsic system, for
detecting the presence of circulating anti-
coagulant and for monitoring heparin therapy.
Also measures factors present in the common
pathway, except for platelet and factor XIII.
- PRINCIPLE: Expect for Ca and Platelet
phospholipid (PPL), PPP contains all the
coagulation factors needed for the generation
of intrinsic prothrombinase. When Ca is added
with “incomplete” thromboplastin (serves as
platelet substitute), intrinsic prothrombinase is
generated.
- Difference:
o APTT: w/ activator
o PTT: w/out activator
NOTE: APTT
FACTORS THAT MAY PROLONG THE TEST:
A. Any factor deficiency (Intrinsic (VIII, IX, XI, XII,
HMWK, PK) and common pathway (I, II, V, X))
B. Presence of inhibitor – Factors VIII-I and FIX-I
C. Heparin (immediately process the sample and
prepare the plasma within 1 hour to prevent PF4 from
inhibiting the heparin)
D. <60 mg/dL fibrinogen – Factor 1 belongs to
common pathway.
E. Presence of increased Fibrin Split Products (FSP) –
all clot-based test will be prolonged in the presence of
FSP.
NORMAL VALUE: 25 – 35 SECONDS
NOTE:
 All of clot-based test cannot measure Factor
XIII deficiency & FXIII will be normal because it
is not needed for formation of clot.
 When thrombin acts on XIII, it will now
stabilize the clot.
 XIII is involved in common pathway.
TEST FOR EXTRINSIC AND COMMON PATHWAYS
EXTRINSIC: VII
COMMON PATHWAY: I, II, V, X
1. PROTHROMBIN TIME (PT) / QUICK’S TEST
- Starts with the action of VII.
- Dominant pathway that occurs in vivo, once
thromboplastin is introduced in the circulation,
it immediately combines with VIIa already
present in circulation in vivo but in vitro, VII
must be activated by thromboplastin. In vitro,
there is no source of thromboplastin when
 HEMATOLOGY 2

placed in a test tube. The reagent  Oral anticoagulants (warfarin, coumarin,

thromboplastin will provide the thromboplastin
which comes from tissues. Complete
thromboplastin is needed because it has to
supply the other missing factors such as
phospholipid and thromboplastin. The
composition of complete thromboplastin is
tissue factor and phospholipid. The tissue factor
in the reagent will activate VII and the
phospholipid in the reagent will supply the
missing phospholipid. Since citrate is utilized, Ca
is also removed thus 0.025 M CaCl2 is utilized
as a substitute. Upon addition of
Thromboplastin and CaCl2, record the time it
takes for plasma to form into clots
- Useful screening procedure for the extrinsic
coagulation mechanism including the common
pathway, also monitors oral anticoagulant
therapy with coumadin.
NOTE: PT
 PRINCIPLE: when tissue extract or
thromboplastin is added to PPP along with Ca,
it reacts with factor VII leading to subsequent
activation of factor X.
 NORMAL VALUE: 10 – 12 SECONDS
 THROMBOPLASTIN SOURCE: rabbit brain
extract or similar
 SPECIMEN: Citrated PPP to prevent excessive
citrate in plasma
 REAGENTS: Complete Thromboplastin (TF &
phospholipid) and 0.025 M CaCL2
 PURPOSE: in monitoring extrinsic and
common pathway deficiency, more sensitive
for monitoring oral anticoagulant therapy,
vitamin K deficiency and liver diseases
coumadin).
SOURCES OF ERRORS:
 Prolonged in the presence of heparin
contamination; can be shortened in traumatic
venipuncture.
REPORTING:
 INR to monitor effect of therapy and dose of
oral anticoagulant
ADDITIONAL REMINDER:
 Prolonged PT causes VII to disappear
immediately since it has a shortest lifespan in
vivo.
INTERNATIONAL NORMALIZED RATIO (INR)
- A standardized way of reporting PT in
monitoring oral anticoagulant therapy,
calculated as a ratio of the patient’s PT to a
control PT standardized for the potency of the
thromboplastin reagent developed by the
World Health Organization (WHO).
- Reference Values for INR:
o 2.0-3.0
 In prevention and treatment of
venous thrombosis
 Treatment of pulmonary
embolism
 Prevention of stroke in MI
 Peripheral arterial disease
 Prevention of systemic
embolism in atrial fibrillation
 Cardiac value replacement
(tissue valves).

o 2.5-3.5
NOTE:  In prevention of recurrent MI
PROLONGED PT IS SEEN IN:  Reduction of mortality in MI
 Any factor deficiency under the extrinsic (VII,  Mechanical prosthetic heart
I, II, V, X) and common pathway. valve (high risk).
 Fibrinogen concentration <100mg/dl
 Dysfibrinogenemia – abnormal fibrin FORMULA:
𝑝𝑎𝑡𝑖𝑒𝑛𝑡 𝑃𝑇
structure affecting PT. INR= [ ]𝐼𝑆𝐼
𝑃𝑇 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑚𝑒𝑎𝑛)
 Vitamin K deficiency (II, VII, IX, X) and certain
liver disease (affecting all factors except, VWF,
Ca & TF synthesis) thus prolong PT and APTT
 HEMATOLOGY 2

SURFACE ACTIVATION – initiates intrinsic pathway Perform

when factor XII is in contact with any surface; mixing
monitored by APTT. studies

TISSUE THROMBOPLASTIN
2. STYPVEN TIME/ RUSSEL VIPER VENOM TEST
- for extrinsic and monitored by PT - Uses a venom (from (Viperarusselli/Daboia
INTRINSIC PATHWAY – PTT russelli) with a “thromboplastin like” action
- Principle: Addition of the venom bypasses the
VII – PT activation of VII and directly activates factor X
INTRINSIC PATHWAY AND FACTOR VII – common - Reagent: Russel Viper Venom 
pathway; APTT & PT Thromboplastin-like (“VIIa”): VIIa like activity 
immediately activate X.
- Activation of VII is bypassed in the reaction
because reagent have VIIa-like activity
- Initial utilization: Differentiate X and VII
deficiency
o X deficiency: ST: Prolonged
o VII deficiency: ST: Normal
- Clinical utility now: for identification or detect
the presence of Lupus anticoagulant (LA) or
Anti-phospholipid Antibody (APA) – antibody
against phospholipid; inhibits PF3 and
phospholipid (interferes normal coagulation)
prolonged test.
- Screening test performed using diluted plasma:
Dilute Russel Viper Venom Test (DRVVT) or
Dilute Stypven Time (DST) for Lupus
Anticoagulant: factor activity INCREASES with
increasing dilutions of patient plasma used in the
assay (Corresponding dilutions of LA/APA).
- Performed in combination with APTT: ST and
APTT  identify LA/APA.
TEST INTRINSIC EXTRINSIC COMMON - Interpretation:
PT- None Prolonged None o CF deficiency (dilution) : more
prolonged Factor VII prolonged
APTT- deficiency o LA-causing (diluted): improved in the
normal test/ shorten
APTT- Prolonged None None  Rationale: while the plasma is
prolonged VIII, IX, XI, being diluted, the LA is also
PT- Normal XII, HMWK, diluted  increase CF activity
PK - Circulating anticoagulant: abnormal
deficiency
anticoagulants that inhibit certain coagulation
Perform
proteins.
additional
testing - Specific inhibitor: inhibitor/anticoagulant that
BOTH PT & None None Prolonged inhibits specific coagulation protein like factor
APTT- I, II, V, X VIII or IX
prolonged
 HEMATOLOGY 2
- Non-specific inhibitor/ non-specific circulating
anticoagulant: inhibitor/anticoagulant that
inhibits no specific coagulation protein like LA.
INDIRECT TESTS:
FOR FIBRINOGEN DEFICIENCY ONLY
THROMBIN TIME (FIBRINOGEN DEFICIENCY TEST)
- Modification of Thrombin Clotting Time (TCT) by
Clauss.
- Principle: Addition of thrombin bypasses all
coagulation pathways except the polymerization
of Fibrinogen.
- Specimen: citrated PPP
- Reagent: standardized thrombin solution and
calcium.
o Fibrinogen --- Standard Thrombin + Ca++
 Fibrin
- Will not detect deficiency in the CF VII, V, X, VIII,
IX, XI, XII, HK, PK  reactions bypassed
- Cannot determine deficiency or availability of
thrombin.
- DETECT FIBRINOGEN DEFICIENCY ONLY
- Reactions involved conversion of fibrinogen to
fibrin.
- Measures the availability of adequate and
functional fibrinogen
- Used to monitor heparin therapy and is sensitive
to presence of increased levels of FSP.
- Presence of FSP: XYDE; inhibits fibrin
polymerization and inhibits conversion of
fibrinogen to fibrin by thrombin  inhibits
clotting/prevent clot formation  increased/
prolonged TCT (more sensitive).
- Monitors Heparin Therapy
o Heparin: anti-thrombin; only
anticoagulant (in vivo and in vitro) that
does not remove Ca++; functions to
inhibit thrombin by increasing the
activity of AT-III.
o APTT and TT: monitors heparin therapy
- Reference value: 17-25 secs. (may depend on
the thrombin concentration)
- Prolonged Thrombin time:
o Fibrinogen level is below 75-100 mg/dL;
functional disorder of fibrinogen
o Presence of Heparin, fibrin degradation
products (FDP) and thrombolytic agents
o Normally prolonged in the newborn and
in multiple myeloma.
APTT: intravenous anticoagulant
PT: oral anticoagulant  INR
TT: fibrinogen deficiency and heparin therapy
TT together with APTT: Heparin therapy
DST together with APTT: LA
REPTILASE TIME
- Uses the reagent reptilase enzyme (collected
from Bothropsatrox) which has a thrombin-like
effect.
- Functionality same as thrombin time: IDENTIFY
PRESENCE, AVAILABILITY AND FUNCTIONALITY
OF FIBRINOGEN.
- REAGENT: Reptilase (“IIa” – like): toxic and
potent: very unsafe to handle (should not be in
contact in open skin  blood will clot)
- Fibrinogen --- Reptilase  Fibrin ; immediate
clotting will occur
- PRINCIPLE: Reptilase bypasses all coagulation
pathways except the polymerization of
fibrinogen.
- FIBRINOGEN: 3 polypeptide chains (alpha, beta
and gamma) ends with D-terminals and a central
E domain where alpha, beta and gamma
polypeptide chains meet and coil extended at E
central domain is fibrinopeptides A and B.
- If thrombin cleaves fibrinogen: thrombin
cleaves both fibrinopeptides A and B.
- If reptilase cleaves fibrinogen: reptilase cleaves
only fibrinopeptides A.
- Effect are similar: Fibrinogen  fibrin monomer
 polymerization
 HEMATOLOGY 2

- Reptilase cleaves I  monomers polymerize F XIII Deficiency (soluble Dissolved Not

(end-to-end)  formation of clot clot) dissolved
o Thrombin: end to end and side to side Excessive fibrinolysis Dissolved Dissolved
connection (excessive plasmin)
o Reptilase: end to end only
- Prolonged reptilase time is observed in OTHER TEST:
Fibrinogen deficiency and other functional
disorder of fibrinogen; presence of FSP/FDP 1. BETHESDA ASSAY: determine and measure the
- REFERENCE TIME: 18-20 secs titer of specific factor inhibitor of VIII.
- More sensitive to fibrinogen deficiency 2. ECARIN CLOTTING TIME

NOTE: HISTORICAL TESTS:

 All coagulation assay is affected by the Basis of coagulation tests
presence of FSP  prolonged PROTHROMBIN CONSUMPTION TEST
 BOTH (Fibrinogen Assay) are used to identify
- Deficiency in factors V, VIII, IX, X, XII or platelets
availability and functionality of fibrinogen and
will slow the rate of prothrombin conversion
both sensitive to fibrinogen deficiency or
dysfunctional  prolonged test leaving significant amount of prothrombin in the
serum.
- Prothrombin Time (PT) is performed on SERUM
THROMBIN REPTILASE - Deficiency in some factors will SLOW the rate of
TIME (standard TIME prothrombin conversion leaving significant
thrombin Rgt) (thrombin-like amount (60%) of prothrombin in serum
rgt.) o Slow pathway: Prothrombin (60%) 
Heparin – both Prolonged Normal or
Thrombin (40%)
APTT and TT Slightly
- Can’t identify which coagulation factor is
Prolonged
deficient  needs mixing studies.
(overdosed)
(+) FSP – all Prolonged Prolonged - GENERAL RULE: Only consumable factor are
clotting test FIBRINOGEN GROUP (I, V, VIII and XIII) and
Decreased Prolonged Prolonged factor II.
Fibrinogen - During clotting (serum): II (20%; residual
prothrombin)  IIa (Thrombin: 80%)
- SERUM: product of clotting of blood
DUKERT TEST (5M UREA SOLUBILITY TEST)

- Test for Factor XIII

- PRINCIPLE: clot formed in normal plasma is
insoluble in 5M urea during a 24-hr incubation.
- If factor XIII is deficient in the patient’s plasma,
the clot is dissolved in less than 24 hours by the
urea.

1% monochloroacetic acid Incubate for 24 hours

at RT
Clot + 5M Clot+
THROMBOPLASTIN GENERATION TEST (TGT)
Urea NaCl
(NSS) - By Biggs & Douglas
Normal (insoluble clot) Not Not - Uses BaSO4/Al(OH)3- adsorbed plasma or fresh
dissolved dissolved serum
 HEMATOLOGY 2
- Basis of mixing studies AUTOMATED INSTRUMENTS:
- No longer performed
- Mixing agent + Patient’s plasma  clotting
- Mixing agents: BaSO4/Al (OH)3 – Adsorbed
plasma; Fresh serum; stored serum.
HICKS-PITNEY TEST
- Modification of Thromboplastin Generation
Time (TGT) and requires re-calcification of
diluted plasma.
AUTOMATION IN COAGULATION
- Detects formation of FIBRIN
FIBRINOMETER
- A semi-automated mechanical instrument that RESULT AND INTERPRETATION:
detects fibrin strand formation using a wire loop
or hook.
- Only machine in hematology that uses electro-
mechanical (manual) principle.
- Fibrinometer has wells where samples are
placed, and has a wire loop or needle which dips
in the blood sample in the wells and keeps
dipping in 0.5 seconds interval (up and down)
until fibrin thread or clot formation is observed.
When fibrin threads from the sample connects
with the wire loop, stop the time and record the
clotting time
- Semi-automated.
- Identify fibrin formation FACTOR ASSAYS:
PHOTO-OPTICAL
- Depends on the increase in light scattering
associated with the conversion of soluble
fibrinogen molecules to the insoluble
polymerized fibrin clot.
- Machines measures: increase in light scattering
- Identify fibrin formation from fibrinogen
- In plasma, fibrinogen is dissolved thus it is not
able to scatter the light into different direction
- Fibrinogen  Fibrin (insoluble) fibrin particles
will block and scatter the light in different
direction.
SEMI-AUTOMATED INSTRUMENTS:
 Electra 750 and 750A, Fibrin timer series; and FP
910 coagulation analyzer
 Ortho Koagulab 16S and 40A
 Coag-A-Mate X2 and XCm
 MLA Electra 700 and 800
MIXING STUDY OR SUBSTITUTION STUDIES
- Performed when PTT & PT are prolonged to
determine whether it is caused by a nonspecific
inhibitor, specific factor inhibitor, or coagulation
factor deficiency, and to determine the deficient
coagulation factor.
- Procedure: Mix equal parts of patient’s plasma
plus Fresh normal plasma then repeat the
prolonged test.
 Presence of a pathological circulating
anticoagulant: if test is not corrected (remains
prolonged) when mixed with mixing normal
plasma.
 Factor deficiency: if repeated test is corrected
when mixed with normal plasma. The next thing
to do is to identify the deficient factor by
performing additional mixing study or factor
assay.
 Identification of deficient factor: mix equal parts
of patient’s plasma plus an appropriate mixing
sample.
1. Fibrinogen assay: Clauss modification of
Thrombin time.
 SINGLE FACTOR ASSAY:
o Quantification of remaining deficient
factor; determine the factor level in
relation to
o Applicable only in A and B: sex linked
o Not for: C (autosomal)
o Do dilution: 1:10; 1:20; 1:40; 1:80  CT
interval (Reference Curve).
Hemophilia: Factor Level vs. Severity
Severity Factor Level Manifestation
Severe <1% Spontaneous
bleeding; bleeding
with minor surgery or
trauma
 HEMATOLOGY 2

Moderate 1-5% Spontaneous bleeding Factor Deficiency Mixed Reagent Result

uncommon; may (Patient’s
bleed with surgery or sample)
trauma. VII Factor-VII No correction
Mild 5-20% No spontaneous Deficient Plasma
bleeding; may bleed
with major trauma or
surgery 4. Factor XIII assay: Chromogenic assay
Example: - Transglutaminase/Fibrinase/Transaminase
APTT: prolonged PT: Normal reaction  Release of ammonia
APTT: not corrected by Factor XI- deficient plasma but is - Directly proportional: More ammonia release:
corrected by Factor IX-deficient plasma more XIII deficiency; Less ammonia release: less
 What type of hemophilia? XIII deficiency
o Hemophilia C - (Glu DH Rxn): NADPH consumption is measured
by decrease of A at 340 nm
2. Single factor assays using PTT - Absorbance is inversely proportional to factor
- For factors VIII (A-most common), IX (B- second XIII activity.
common), and XI (C-rarest) deficiencies - Inversely proportional: High absorbance:
(hemophilias). Increase XIII deficiency; Lower absorbance:
- Uses a factor-depleted platelet-poor plasma (like Normal or Increase
factor VIII-depleted PPP which provides normal - Factor XIII: Assay cannot be detected by PT and
activity of all procoagulant except factor VIII). APTT
- Normal plasma  normal result
e.g. Px plasma + factor deficient plasma NOTE:
Prolonged Test (PT, APTT/Both coagulation test) 
APTT reagent
CONSIDER/POSSIBLE REASONS: px is suffering in
PT reagent
coagulation factor deficiency or abnormal component
FACTOR MIXED REAGENT RESULT (inhibitor/circulating anticoagulant) that keeps
DEFICIENCY inhibiting the factors  perform mixing study
(Patient’s Reagent: 1:1 mixture
- Equal parts: Patient’s plasma + correction
Sample)
reagent or mixing reagent
VIII Factor-VIII Deficient No correction
PRINCIPLE:
Plasma - If patient is deficient and we add a mixing
Factor-IX Deficient Corrected reagent that contains all necessary
Plasma components  corrected: factor deficiency.
IX Factor-VIII Deficient Corrected - If patient is deficient and we add a mixing
Plasma reagent that contains all necessary
Factor-IX Deficient No correction components but still not corrected: presence
Plasma of abnormal component.

e.g. repeat prolonged test but: (1:1) Px plasma+

3. Single factor assays using PT normal fresh plasma(contains all coagulation factor)
- For factors II, V, VII, and X deficiencies
 HEMATOLOGY 2

PROLONGED TEST
INTERPRETATION NORMAL FRESH PLASMA
Factor Deficiency Normal: Corrected test
Inhibitor; C Remains Prolonged: not
anticoagulant corrected test

NOTE:
After Coagulation test (prolonged)  perform mixing
study with Fresh Normal Plasma (FNP): identify
whether it is coagulation deficiency or presence of
abnormal component  then identify which factor is
deficient or abnormal component
PROLONGED TEST: Deficiency  continue mixing
studies
PT: VII
APTT: VIII, IX, XI, XII, HK, PK
PT and APTT: I, II, V, X

MIXING STUDIES
MIXING/CORRECTION REAGENT CF PRESENT CF ABSENT
FRESH NORMAL PLASMA ALL NONE
ADSORBED PLASMA I,V, VIII, XIII, XI, XII, HK,PK II, VII, IX, X
AGED PLASMA I, XIII, II, VII, IX, X, XI,XIII, HK, PK V and VIII
FRESH SERUM II, VII, IX,X,XI,XII,HK,PK I, V, VIII, XIII
AGED SERUM VII,IX,X,XI,XII,HK,PK I, V, VIII,XIII, II (residual II
deteriorates)
ADSORBED SERUM XI, XII, HK, PK I, V, VIII, XIII, II, VII, IX, X
FACTOR DEFICIENT PLASMA Deficient of particular CF; removed through immunoadsorption
Commercially available plasma (named)
e.g. Factor VIII deficient plasma  deficient of VIII

3 WAYS COAGULATION FACTORS ARE REMOVED: EXAMPLES

PROBLEM 1: PT: Prolonged & APTT: Prolonged
1. Clotting/Coagulation: Consumable factors (I, V,
XIII) and partially II. 1. Establish whether it is coagulation deficiency or
2. Adsorption: Adsorbable factors presence of abnormal component
3. Storage/Aging: Labile factors (V and VIII) Perform test with FNP  if corrected: CF
- Aged Serum: residual prothrombin deficiency
 HEMATOLOGY 2

2. Identify which pathway is affected: Common  Elimination:

pathway. Adsorbed plasma (VIII and XI present) (IX absent)
3. Coagulation Factor Deficiency (CFD) (I, II, V, X)  Fresh serum (IX and XI present) (VIII absent)
Mixing study.
ANSWER: Factor VIII deficiency
4. CFD: Pxt Plasma + Mixing Rgt.  Repeat PT and
APTT.

PROBLEM 2: COMMON PATHWAY PROBLEM 3: PT and APTT – prolonged

 Coagulation Factor Deficiency: I, II, V, X  Both are corrected by fresh serum but not with
1. Pxt Plasma + Mixing Rgt: Fresh Serum (I, V stored serum. Identify factor/s deficient.
absent; II, X present)  Repeat PT and Aptt.  Common pathway deficiency: I, II,V, X  factor II
Deficient: deficiency
I = not corrected
II = corrected A. VIII B. I C. V D. II E. X
V = not corrected  Elimination:
X = corrected o Fresh serum (II and X present) (I, V
2. Pxt Plasma + Mixing Rgt: Aged Plasma (V absent)
absent; I, II, X present)  Repeat PT and APTT o Stored serum (X present) (I, V, II absent)
Deficient:
I = corrected NOTE:
II = corrected WAYS OF ANALYZATION
V = not corrected: prolonged  ELIMINATION
X = corrected  WRITING
 RECORDING
PROBLEM 3: PT and APTT are prolonged and corrected
by addition adsorbed plasma. Which coagulation is/are
deficient? PROBLEM 4: PT and APTT – prolonged

 Pxt Plasma + Mixing Rgt: Adsorbed Plasma ( II  Both are corrected with fresh serum but not with
and X absent; I and V present)  Repeat PT and adsorbed plasma.
APTT o Fresh serum: II, VII, IX, X, XI, XII, HK, PK
 Deficient: (present)
I = corrected o Adsorbed plasma: II, VII, IX, X (absent)
II = not corrected  What may be mixed to identify the factor
V = corrected deficient?
X = not corrected o Stored serum: II and X

ANSWER: Factor I and V deficiency PROBLEM 5: PT and APTT – prolonged

PROBLEM 4: PT: Normal APTT: Prolonged  COMMON PATHWAY: I, II, V, X

 Corrected (it contains the CF) by adsorbed
plasma but NOT(absent) by fresh serum Factor
VIII deficiency
 Factor deficiency in INTRINSIC PATHWAY: VIII, IX,
XI (common deficiencies to occur); (XII, PK, HK –
deficiency will not cause bleeding disorder but
may cause prolongation of the test and are very
rare to occur)
 Both are NOT corrected with fresh serum but
corrected with adsorbed plasma.
o Fresh serum: I, V,VIII,XIII (absent)
o Adsorbed plasma: I,V, VIII, XIII (present)
 What may be mixed to identify the factor
deficient?
o Aged Plasma: I
 HEMATOLOGY 2

PROBLEM 6: PT and APTT – prolonged

 COMMON PATHWAY: I, II, V, X

 Both are NOT corrected with fresh serum but
corrected with adsorbed plasma and aged
plasma.

FACTOR PROLONGED TEST/S CORRECTED BY NOT CORRECTED BY

DEFICIENCY
I PT and APTT  Adsorbed plasma  Fresh Serum
 Aged Plasma  Aged Serum
 Adsorbed Serum
II PT and APTT  Aged plasma  Adsorbed plasma
 Fresh serum  Aged serum
 Adsorbed serum
V PT and APTT Adsorbed plasma  Aged plasma
 Fresh serum
 Aged serum
 Adsorbed serum
VII PT  Aged plasma  Adsorbed plasma
 Fresh serum  Adsorbed serum
 Aged serum
VIII APTT Adsorbed plasma  Aged plasma
 Fresh serum
 Aged serum
 Adsorbed serum
IX APTT  Aged plasma  Adsorbed plasma
 Fresh serum  Adsorbed serum
 Aged serum
X PT and APTT  Aged plasma  Adsorbed plasma
Stypven Time  Fresh serum  Adsorbed serum
 Aged serum
XI APTT ALL NONE
Presence: APTT and Stypven Time NONE ALL
AntiPL-
Antibody
NOTE: Factor XIII Assay cannot be detected by PT and APTT
 HEMATOLOGY 2

MIDTERMS - The effect of coagulation is consumption of

DISORDERS OF FIBRINOLYSIS coagulation factors (I, V, VIII and XIII) and
PLASMIN platelets. The effects of activation of fibrinolysis
is production of FSP. Main result: continuous
- Main protein involved that dissolves the clot
bleeding
and may cause excessive fibrinolysis.
- Regulatory proteins: alpha 2 antiplasmin: main
- Comes from plasminogen in the presence of
inhibitor of plasmin; and other inhibitors are
plasminogen activators like tissue plasminogen
consumed because of too much production of
activator and activated factor XII (XIIa) when
plasminogen activators.
produced can dissolve fibrin and if excess
fibrinogen. TESTS FOR FIBRINOLYSIS
EUGLOBULIN CLOT LYSIS TIME
FUNCTIONS OF PLASMIN:
- Principle: Plasma euglobulins are precipitated
 Plasmin  fibrinogen  Fragment X, Y and
with 1% HAC. Precipitates are redissolved and
Fragment D, E
clotted with thrombin. The clot is incubated, and
 Cross-linked fibrin  X-oligomers and D-dimer
the time for complete lysis at 37°C is measured.
NOTE: - Procedure: Precipitation of plasma euglobin
In both degradation and split products of fibrin or with 1% Acetic acid  re-dissolved ppt  add
fibrinogen are produced. thrombin: fibrinogen immediately converted
into fibrin  plasminogen present in fraction
now present in fibrin will be slowly converted
1. PRIMARY FIBRINOLYSIS (FIBRINOGEN LYSIS)
into plasmin by activators of plasminogen 
- This is due to the release of excessive activators
fibrin clot with plasmin  check the
of plasminogen resulting to the conversion of
ability/activity of plasmin to dissolve the clot 
plasminogen to plasmin in the absence of fibrin
at 37°C  CLOT LYSIS TIME
formation
- TEST FOR PRIMARY FIBRINOLYSIS
- Fibrinolytic system: activated in the absence of
- Detects excess production of plasmin
clot formation.
- Separation through PRECIPITATION: inhibitors
- Main problem: Increase activators of
left in the supernatant
plasminogen that leads to increase production of
- Plasma Euglobulin Fraction contains
plasmin.
Plasminogen, Activators of plasminogen and
- Since the function of plasmin is not specific to
fibrinogen.
the clot, it also destroys intact fibrinogen
- Normal clot lysis time: within 2-4 hours.
together with factors V and VIII.
- Clot lysis in less than 2 hours is indicative of
- Effect of plasmin on plasminogen: will cause the
abnormal fibrinolytic activity
release of FSP which interferes in normal
- Increase fibrinolysis is seen in: circulatory
clotting; unable to clot  BLEEDING.
collapse; adrenalin injections; sudden death;
- To inhibit the role of plasmin: treatment
pulmonary surgery; pyrogen reaction; obstetric
involves introduction of anti-fibrinolytic
complications
drugs/antiplasmin.
TESTS FOR SECONDARY FIBRINOLYSIS
2. SECONDARY FIBRINOLYSIS (FIBRIN LYSIS) - Detects fibrin monomers
- This clot dissolution results to increase FSP/FDP
ETHANOL GELATION TEST (BREEN AND TULLIS) AND
that interfere with coagulation and platelet
PROTAMINE SULFATE TEST
function.
- In DIC where there is a simultaneous occurrence - Principle: 50% ethanol (and Protamine sulfate)
of both coagulation and fibrinolysis. will cause the soluble fibrin monomers
 HEMATOLOGY 2

complexes to dissociate resulting in the product came from primary or secondary

polymerization of the monomers and fibrinolysis.
subsequent gel like clot formation or - POSITIVE: presence of FSP  agglutination.
paracoagulum (paracoagulation)
PRIMARY SECONDARY
- Both tests are designed to detect the presence
FIBRINOLYSIS FIBRINOLYSIS
of fibrin monomers in the plasma. A screening
Platelet Normal Decreased
procedure utilized as an aid in the diagnosis of
count  Platelet  Platelets
DIC does not are
- Normally, there is NO gel formation. Because undergo consumed
there should be no fibrin monomers. aggregati during clot
- Presence of paracoagulation: indicates on thus is formation
presence of fibrin monomers and secondary not
fibrinolysis. consume
- Differentiate Primary and Secondary Fibrinolysis: d
o Negative: Primary fibrinolysis RBC Normal RBC
o Positive: Secondary fibrinolysis morpholog  No clot fragmentation:
y formatio Schistocytes
D-DIMER TEST/ D-D ASSAY n thus  Caused by
there is trauma
- Specific to SECONDARY FIBRINOLYSIS only no
- Measures fibrin formation (presence of D-dimer trauma
indicates: fibrin clot and fibrin lysis) caused in
- D-D fragment arises from the degradation of RBCs in
cross-linked fibrin. Its presence is specific of the
fibrin formation. circulatio
- The test uses latex beads with monoclonal Ab n
that will react only with D-dimer (Turbidimetry PT and Prolonged Prolonged
or Nephelometry) APTT  Excessive  Consumpti
- Normal: <200ng (D-dimer)/ (blood) mL : action of on of I,V,
<200ng/mL. plasmin VIII and XIII
on I, V during clot
- Increased results may be seen in DIC,
and VIII formation
thrombosis, phlebitis or other condition

characterized by fibrin clot formation. excessive
THROMBO-WELLCOTEST (TEST FOR FSP) fibrinolysi
s
- PROCEDURE: Whole blood + thrombin (for FDP’s or POSITIVE POSITIVE
complete clotting of whole blood)  serum + FSP’s
soya bean enzyme (as inhibitors)  diluted D-dimer NEGATIVE POSITIVE
serum + latex particles (coated with anti-FSP)  Euglobin POSITIVE NEGATIVE
presence of FSP  agglutination Clot Lysis
- Whole blood is added to thrombin to ensure Protamine NEGATIVE POSITIVE
complete clotting and soya bean enzyme Sulfate(SO
4) Test
inhibitor after incubation, the patient’s serum is
Ethanol NEGATIVE POSITIVE
diluted and mixed with latex particles that have
Gelation
been coated with anti-fibrin split products.
- Determines presence of split or degradation
products but will not identify whether this split
 HEMATOLOGY 2

THROMBOTIC DISORDERS
THROMBOSIS: platelet or fibrin clots

PHYSIOLOGIC THROMBOSIS: normal response due to

injury.

PATHOLOGIC THROMBOSIS

- Abnormal that may lead to obstruction of

circulation.
- Can be acquired or inherited.
- Risk factors:
o Acquired conditions are related with life
events such as age, smoking and other
diseases.
o Inherited conditions are related with
congenital deficiencies in the regulatory
proteins (inhibitors) and defective
factors.

TYPES OF PATHOLOGIC THROMBOSIS

a. ARTERIAL THROMBOSIS
- Composed of many platelets with small amounts
of fibrin, RE and white cells – “white clot”.
- CAUSES: Hypertension, Hyperviscosity,
qualitative platelet abnormalities and
atherosclerosis.

b. VENOUS THROMBOSIS
- Few platelets and blood cells, and MANY FIBRIN
- Composed of large amounts of fibrin and red
cells.
- CAUSES: Abnormalities: associated with slow
blood flow (hyper viscosity of blood), activation
of coagulation system, platelet dysfunction,
leukocyte activation: produces a lot of adhesive
molecules, anatomical abnormalities of blood
vessel wall, impairment of the fibrinolytic
system, and deficiency of physiologic inhibitors
- RISK FACTORS: Life events: age, smoking, DM,
hypercholesterolemia.
 HEMATOLOGY 2

INHERITED THROMBOTIC NOTES ASSOCIATED ACQUIRED

DISORDERS CONDITIONS
Anti-thrombin (AT) Deficiency Recurrent venous thrombosis DIC, liver disease, nephrotic
AT-III: main inhibitor of thrombin syndrome, oral contraceptives and
(continuous activation of V, VIII, XI, pregnancy
XIII)
Heparin Cofactor II Deficiency Not associated with thrombosis
Regulatory Proteins important in inactivation of V and VIII
Protein C Deficiency Thrombopoietin Vitamin K deficiency, liver disease,
malnutrition, DIC and warfarin
therapy
Protein S Deficiency Vitamin K deficiency, liver disease
and DIC
Activated Protein C Resistance Factor V Leiden is not inactivated  excessive clot formation
(Factor V Leiden) Mutated factor V  Resistant to inactivation of activated Protein C  Va remains
to be activated
Prothrombin Mutations Increase in the concentration of plasma prothrombin
Other Inherited Thrombotic Disorders:
 Elevated activity levels of Factor VIII are associated with venous thrombosis embolism
 Factor XII deficiency: XIIa serves as plasminogen activator  deficiency: decrease production of plasmin and
persistence of clot in the circulation
 Dysfribrinogenemia
 Hyperhomocysteinemia
 Tissue Factor Pathway Inhibitor Deficiency: TFPI main inhibitor of tissue factor pathway
THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP)
 Deficiency of ADAMTS-13 (metalloproteinase enzyme important for the cleavage of ultra large vWF)
 activation of platelets  platelet plug  obstruction

HEMOLYTIC UREMIC SYNDROME

 Associated with diarrhea
 Caused by verotoxin or shigatoxin producing bacteria: E.coli 0157:H7 or Shigella strain
 This toxin will cause damage to glomerular capillaries and activation of platelets  hemolytic anemia;
fragmentation of RBC
COMMON SIGNS: TTP and HUS are fever, petechiae, neurologic signs (more often occur in TTP) and renal diseases
(more often occur in HUS); this happens when platelet aggregates is present in small circulations.
 HEMATOLOGY 2

ACQUIRED THROMBOTIC DISORDERS - It inhibits coagulation by combining with

1. LUPUS ANTICOAGULANT / ANTIPHOSPHOLIPID
SYNDROME
- Lupus anticoagulant does not inhibit in vivo
coagulation but may cause prolonged in vitro
tests.
2. HEPARIN-INDUCED THROMBOCYTOPENIA
- Development of antibodies against heparin-
platelet factor 4 complex.
- This immune complex causes platelet activation,
platelet microparticles, thrombocytopenia, and
hypercoagulable state.
NOTE:
ANTICOAGULANT THERAPY
Prevention of thrombosis: can be anticoagulant
drugs, antiplatelet or thrombolytic drugs
ANTIPLATELET DRUGS – prevent activation and
aggregation and are most effective in the treatment of
the arterial diseases; platelets are more often
associated.
1. ASPIRIN – MOST COMMONLY USED: Inhibits
Thromboxane A2 synthesis by irreversibly actetylating
the enzyme cyclooxygenase; acetylsalicylic acid;
irreversibly affects platelet function by inhibiting the
cyclooxygenase (COX) enzyme and thereby the
formation of thromboxane A2 (TXA2).
2. OTHER ANTIPLATELET DRUGS: dipyridamole,
thienopyridines, ticlopidine and clopidogrel.
ANTICOAGULANT DRUGS
- Inhibit thrombin and fibrin formation.
- Prevention of venous thrombosis.
antithrombin-III in inhibiting thrombin and Xa.
2. ORAL ANTICOAGULANTS: COUMADIN,
WARFARIN, DICUMAROL
- These inhibit vitamin K-dependent factors and
other vitamin K-dependent proteins
- They alter hepatic synthesis resulting to inability
of the liver to carboxylate the glutamyl residue
of the factor leading to their functional
deficiency. These factors / proteins formed are
referred to as PIVKA/des – gamma-carboxy
proteins.
- Coumadin (warfarin): crosses the placenta and
is present in human milk.
- Therapy is monitored by PT / international
normalized ratio (INR).
- When overdosed, high levels vitamin K reverse
the action.
- For long term management.
THROMBOLYTIC DRUGS
- Used to break down fibrin clots to restore
vascular function and to prevent loss of tissues
and organs.
- Also used in acute arterial thrombosis for
immediate thrombolysis.
SERVE AS PLASMINOGEN ACTIVATORS
1. UROKINASE – this is not a fibrin specific; it is used in
the treatment of venous thromboembolism, MI and
thrombolysis of clotted catheters.
2. STREPTOKINASE – not fibrin specific.

1. INTRAVENOUS ANTICOAGULANT: HEPARIN

- The anticoagulant activity of heparin is enhanced
by binding to AT.
- Heparin-AT complex inactivates thrombin and
Factor Xa.
- Heparin dosage is monitored by APTT &
activated clotting time.
- When overdosed, its action is reversed by
protamine sulfate.
- Effect is immediate because it enters directly
into the vessel.
 HEMATOLOGY 2

RECAP: PRIMARY AND SECONDARY HEMOSTASIS

PATTERNS OF CLINICAL BLEEDING IN DISORDERS OF HEMOSTASIS

PRIMARY HEMOSTASIS SECONDARY HEMOSTASIS
Characteristics platelet/vascular problem coagulation factor problems or presence of
inhibitors and systemic disease
Onset Spontaneous bleeding, immediate after trauma Delayed after trauma
Sites Skin, mucous membranes Deep tissues
Form Petechiae, ecchymosis Hematomas (bluing of skin with tumor/swelling
 clot within tissues
Mucous Common (nasal, oral, gastrointestinal, Less common
membrane genitourinary)
Other sites Rare in deep tissues Joint, muscle, central nervous system,
retroperitoneal spaces
Clinical examples Quantitative Platelet Defects: Factor deficiency, liver disease, acquired
 Thrombocytopenia inhibitors
Qualitative Platelet Defects: (adhesion,
aggregation or release reaction, vascular defects)
 platelet defects, vWD, scurvy

DISORDER OF COAGULATION, FIBRINOLYSIS AND THROMBOSIS

INHERITED DISORDERS OF COAGULATION
COAGULATION FACTORS DEFICIENCY

All CF are produced by the liver except PF3, CF IV(Ca++) and vWF
FACTOR INHERITED COAGULOPATHIES ACQUIRED NOTES
INHERITANCE COAGULOPATHY COAGULOPATHY
PATTERN
PROTHROMBIN GROUP
II Autosomal Prothrombin Liver disease Least to occur
recessive deficiency Vitamin K deficiency
VII Autosomal F VII deficiency (II,VII,IX,X)  functional Most profound
recessive deficiency; no gamma disorder/deficiency;
carboxylation shortest circulating life 
first to disappear.
Oral anticoagulant PT: immediately prolonged;
therapy: inhibits vit. K  more sensitive to liver
functional deficiency diseases and vit. K.
X Autosomal F X deficiency Bile duct obstruction (gall
recessive stone)  incomplete
IX Sex -linked digestion and absorption Hemophilia B; Christmas
of vit. K  functional Disease
deficiency
FIBRINOGEN GROUP
I Autosomal Afibrinogenemia Severe liver disease Rarest to occur
recessive DIC
 HEMATOLOGY 2

Autosomal Dysfibrinogenemia Fibrinolysis Consumable factors during

dominant Normal levels of I but clotting
has abnormal Sensitive to high levels of
structure plasmin
V Autosomal Owren’s disease
recessive Labile Factor
Deficiency
Parahemophilia
XIII Autosomal F XIII deficiency
recessive
VIII Sex linked Inflammatory liver
(hepatic) diseases
 factor VIII increases
CONTACT GROUP
XI Autosomal Most common among
recessive contact factors: Hemophilia
C but least likely to occur in
hemophilia
XII Autosomal F XII deficiency; 2nd most common; not
recessive Hageman deficiency related to bleeding but
related to
thrombosis/abnormal clot
formation
PK Autosomal Fletcher trait
recessive
HK Autosomal Fitzgerald trait
recessive

HEMOPHILIAS
HEMOPHILIA

- Love of hemorrhage
- Inherited disorders characterized by a deficiency of the anti-hemophilic factors.

HEMOPHILIA A B C
FACTOR DEFICIENT VIII (VIII:C) IX XI
OTHER NAME Royal disease and Classic Christmas disease Rosenthal syndrome
hemophilia
INHERITANCE Sex-linked Sex-linked Autosomal
OCCURENCE 1 in 5,000-10,000 males 1 in 25,000 -30,000 in males Ashkenazi Jews
REGARDED AS Most severe congenital 2nd most severe congenital Least and rarest to occur
bleeding anomaly bleeding anomaly same S/s
with A but milder
PROLONGED TEST APTT APTT APTT
TREATMENT Commercial Factor VIII Factor IX concentrate Fresh frozen plasma (FFP) or
concentrate (prevent (prevent bleeding) whole blood: Replenish
Does not correct bleeding) Recombinant F-IX ( in deficient factor
hemophilias but only Recombinant F-VIII (in developed countries: No single blood component
ARREST BLEEDING or developed countries: standard standard treatment and
treatment and management) management)
 HEMATOLOGY 2

PREVENT POSSIBLE DDAVP (1-desamino-B-

BLEEDING arginine-vasopressin)

Contraindication: ASPIRIN,
ANALGESICS (anti-platelets)

Notes: Royal disease: first observed in Named after Stephen Rosenthal: means “rose-
royal family of England (Queen Christmas; case published valley”: German compound
Victoria: Carrier) (December 22, 2011) in The
New England Journal of
Medicine

1. HEMOPHILIA A/CLASSIC HEMOPHILIA with major trauma or

- A sex-linked disorder characterized by a surgery
deficiency of Factor VIII:C
- Most common hemophilia
- Clinical signs and symptoms depend on NOTE:
remaining factor VIII:C level  Generally applies to both Factor VIII and factor
IX deficiencies; may not apply to deficiency of
- Therapy:
other factors.
o Commercial Factor VIII concentrate
 To determine the remaining factor level 
o DDAVP (1-desamino-B-arginine-
Factor Assay (+ Factor VIII –deficient plasma
vasopressin) or + Factor IX-deficient plasma)
 Bleeding manifestation: Hematoma,
2. HEMOPHILIA B (CHRISTMAS DISEASE) hemarthrosis  severe hemophilias
- Sex-linked disorder characterized by a deficiency
of Factor IX
- Second most common hemophilia with clinical
signs and symptoms similar to hemophilia A HOW IS SEX-LINKED HEMOPHILIA (A AND B)
- Therapy: Factor IX concentrate TRANSMITTED?
- Applies PUNNET SQUARE
SEVERITY OF HEMOPHILA A AND B - 50:50 Rule of inheritance of genes including
the sex chromosomes( XX:Female; XY: Male)
- Severity of bleeding depends on factor level For females:
which classify hemophilias as severe, moderate, XXH: Only one chromosome is affected  CARRIER
or mild; applicable only to sex-linked hemophilia:  Carrier (does not manifest s/s of hemophilias
A and B but can transmit abnormal gene to either son
or daughter)
CLASSIFICATION FACTOR COMMON XHXH: When both X chromosome have the abnormal
OF BLEEDING LEVEL MANIFESTATION gene  HEMOPHILIAC
Severe <1% Spontaneous bleeding;  Happens only if father is hemophiliac and
bleeding with minor mother is either hemophiliac or carrier
surgery or trauma XHO: One X chromosome is inactivated; Female is
Moderate 1–5% Spontaneous bleeding suffering from Turner’s syndrome  HEMOPHILIAC
uncommon; may bleed
with surgery or trauma For males:
Mild 5- 20% No spontaneous XHY: Once X is affected  HEMOPHILIAC (one’s that
bleeding; may bleed have the disease and shows s/s)
 HEMATOLOGY 2
 HEMOPHILIAC (one’s that have the disease
and shows s/s)
IMAGE 1: Union of Carrier Mother and Normal Father
will produce sons: (50% normal and 50%
haemophiliac) and daughters: (50% normal and 50%
carrier)
IMAGE 2: Union of Normal Mother and Hemophiliac
Father will produce sons: (100% normal) and
daughters: (100% carriers)
NOTE:
HOW TO DETECT HEMOPHILIA
 Signs and symptoms
 Presence of FAMILY HISTORY of bleeding
among male relatives in the mother’s side
 Laboratory tests: APTT and Clotting Factor
Assays
EARLY CLUES FOR THE DETECTION OF HEMOPHILIA
 Prolonged bleeding after cutting the umbilical
cord
 Hematomas and joint swelling in babies
beginning to creep, crawl and walk
 Limited range of motion of certain joint
 Refusal to use limb (young child)
 Prolonged bleeding after circumcision
 Prolonged bleeding after tooth extraction
 Swelling and hematomas after blood
extraction or IM injection
 Muscle tightness
3. HEMOPHILIA C (ROSENTHAL SYNDROME)
- An autosomal recessive disorder characterized
by a deficiency of Factor XI
- Has an increased incidence among Ashkenazi
Jews
- Bleeding is mild unless stressed by trauma or
surgery
- Considered as silent type
- Not associated with bleeding disorder or just
mild bleeding disorder: explain by current
coagulation pathway
- Therapy: Fresh frozen plasma or whole blood
4. PREKALLIKREIN ; HMWK ; FACTOR XII
DEFICIENCY
- Autosomal recessive disorders with no
associated bleeding tendencies.
- Patients are more vulnerable to excessive
clotting (thrombosis).
ACQUIRED DISORDERS OF COAGULATION
- No genetic abnormality or anomaly has been
associated with these condition. Gene is
something that underwent mutation due to
some mutagens. Main causes are the other
underlying conditions or diseases.
1. INHIBITORS
A. NON-SPECIFIC FACTOR INHIBITORS
- Directed not against coagulation factor but
other molecules and proteins involved in
coagulation.
- Example: Lupus Anticoagulant (LA)
NOTE:
LUPUS ANTICOAGULANT (LA)/ ANTIPHOSPHOLIPID
ANTIBODIES (APA)/ ANTICARDIOLIPIN ANTIBODIES
 A non-specific inhibitor directed against
platelet phospholipid and phospholipid-
protein complexes.
 Its presence will prolong
 Directed to epitopes of proteins bound to
phospholipids  THROMBOSIS
 HEMATOLOGY 2
 Prolonged: APTT (effect is less common) and
dilute Rusell Viper Venom Test (dRVVT)
o The combination of these two tests
serves as a screening test for the
presence of Lupus Anticoagulant.
 Immuno assays (ELISA): for confirmatory test
as well as for titer.
 Not at risk from suffering bleeding conditions.
Instead, they are more at risk to suffer from
THROMBOSIS.
B. SPECIFIC FACTOR INHIBITORS
o These are IgG autoantibodies
directed against specific
coagulation factors (e.g. Factor
VIII:C, IX, X and Factor XI inhibitors)
NOTE:
FACTOR VIII:C INHIBITOR
 Most likely to occur
 Commonly developed from patients suffering
from B-cell malignancies, autoimmune
diseases (Systemic Lupus Erythematosus), and
elderly.
 Production may also be induced in patients
who have received the Factor VIII
concentrate.
 Present bleeding conditions similar with
Hemophilia A (Acquired hemophilia).
 Prolonged APTT and will serve as a screening
test to detect VIII:C Inhibitor.
 Bethesda Assay-titer: determine the titer of
antibody.
 High-dose Factor VIII: given to prevent from
suffering bleeding conditions.
 Immunosuppressant drugs (Cytoxan,
Rituximab): given for longer management of
the patient. It is used to inhibit or prevent the
immune system from continuous production
of antibody against factor VIII.
2. VITAMIN K DEFICIENCY
- More on functional deficiency and less on
synthesis deficiency
- Will result to functional decrease of Factor II, VII,
IX, and X, protein C, S and Z
- Causes of Vitamin K Deficiency:
 Decrease intake of vit. K.: Malnutrition;
source of Vit. K is diet (green leafy
vegetables)
 Use of oral anticoagulant (warfarin,
coumarin, dicoumarol and other
derivatives) or presence of Vitamin K
antagonist: inhibits vitamin K 
inhibiting normal synthesis of vit. K
dependent coagulation factors 
referred as PROTEINS INDUCED BY
VITAMIN K ANTAGONIST (PIVKA) 
abnormal in function
 Decreased normal flora in the GIT
(bacteroides fragilis, E. coli); prolonged
use of strong antibiotics: broad
spectrum antibiotics  damage normal
flora  growth of abnormal flora
 Decreased intestinal absorption intake
 Intestinal infection and Biliary duct
obstruction (obstructive jaundice).
 Vitamin K is fat soluble vitamin it
requires bile for its digestion thus
obstruction along the normal flow of bile
which can be caused by the presence of
tumor or gallstone that constricts this
bile duct and cause obstruction to bile
flow  inadequate digestion and
absorption.
NOTE:
 Infants: given vitamin K at birth to
improve the baby’s synthesis of vitamin
K dependent factors; intestine is sterile.
Bacteria (from maternal skin) grows
only when the baby start to breastfeed.
 Importance of Vitamin K: gamma
carboxylation of II, VII, IX, X; the addition
of this carboxyl radicals to their terminal
glutamic acid will cause them to gain a
negative charge thus will allow them to
interact with Calcium which is positively
charged and on bind to platelet
phospholipid which is negatively
charged.
 HEMATOLOGY 2
3. LIVER DISEASE
- Both clearance and synthesis function will be
affected  Bleeding disorders.
- Leads to synthesis deficiencies (FVII deficiency is
the most profound; first to decrease due to its
short circulating lifespan)
- Decrease clearance activity of activated
components including plasminogen activators
- Increase in FSP due to increase fibrinolysis
- DECREASES: VII (first), PK, I
 Factor VII: most profound to decrease.
When the liver is unable to produce
coagulation factors, Factor VII is the first
to decrease due to its short circulating
life span.
 PK: one of the first to decrease
 Factor I (Fibrinogen): last to decrease
- INCREASE: VIII; if the liver disease is
inflammatory in nature, Factor VIII increases.
NOTE:
Liver functions in the synthesis of all coagulation
factors except calcium and tissue factor and in
clearance, it removes activated coagulation
components to prevent further activation and
excessive coagulation. It also removes
plasminogen activators to prevent excessive
fibrinolysis or excessive production of plasmin.
It removes products of fibrinolysis: Fibrin split
products.
4. MASSIVE TRANSFUSION
- Replacement of more than 1.5 L blood volume
or more than 3 units of blood bags in 24 hours
resulting to dilution of coagulation factor;
DILUTIONAL COAGULOPATHY (in transfusion of
pRBC and NSS) and increased introduction of
anticoagulants (each unit contains 63 mL of
anticoagulant; 10% or more enough to interfere
to normal coagulation).
5. DISSEMINATED INTRAVASCULAR
COAGULATION (DIC)
- Disseminated: clot formation occurs elsewhere
in the body
- Often described as CONSUMPTION
COAGULOPATHY
- CAN OCCUR IN ANYONE, NO AGE GROUP NOR
GENDER PREFERENCE.
- Occurs as complication of another disorder 
causes release of thromboplastic substance 
initiates both Coagulation and Fibrinolysis: both
system simultaneously occurs and may either
predominate the other.
 Result from the liberation of thromboplastic
substance that activates coagulation
 Release of thromboplastic-like substance may
be secondary to trauma, sepsis or toxin that
tissues suffering from this may cause the release
of inflammatory substances that may then exerts
thromboplastic-like activity
 Complication of childbirth obstetrics as
placental tissue are separated, they release
tissue fluids that can exert thromboplastin-like
activity, so the substance will activate both
coagulation and fibrinolysis.
 Plasminogen is also activated through the
contact phase (TPa)
NOTE:
Thus, coagulation and fibrinolysis occur
simultaneously and either may dominate, resulting to:
 Consumption of coagulation factors (I,V, VIII
and XIII) and platelets as thrombi are formed
and deposited locally and widely in the
circulation
o As COAGULATION is activated,
platelet aggregates  they are
consumed resulting to
thrombocytopenia and bleeding.
The platelet aggregate will also
obstruct normal circulation 
thrombosis  vasoocclusion
because of thrombus within the
circulation. Because of the clot
deposited in the tissues, the
 HEMATOLOGY 2

normal flow of oxygen and blood Hematom II, VIII, IX Umbilical X, XIII
will be obstructed resulting to as cord
ischemia or decrease in tissue bleeding
oxygenation  tissues die Mucosal II, VIII, IX, XI Miscarriag I, XIII
(necrosis): kidneys, lungs, liver bleeding e
and brain. Hemarthr VII, IX, X Thrombosi Abnormal
 RBC fragmentation  SCHISTOCYTES osis s fibrinogens;
o Clot deposits cause trauma to red Postsurgic I, II, V, VII, LA;
cells; some are caught or slice/cut al VIII, IX, X, inhibitor
off by fibrin strands  bleeding XI, XIII deficiencies
fragmentation or lysis  Intracrani VII, VIII, IX, Asymptom FXII, PK, HK
hemolysis lead to presence of RBC al XIII atic
fragments called schistocytes on bleeding -They do
circulation and blood smear. not play a
 Increased in FDP and D-dimer major role
o FIBRINOLYSIS ACTIVATION: in in vivo
production of fibrin coagulatio
split/degradation products: XYDE n
 interferes in normal platelet NOTE:
function as well as in the Factors VIII and IX is the most severe coagulation
polymerization of fibrin factor deficiency.
monomers  affects normal
coagulation  more bleeding will
occur. CYTOCHEMICAL STAINS
CYTOCHEMISTRY

NOTE: DIC - Microscopic study and identification of chemical

LAB FINDINGS: all laboratory test will appear constituents within individual cells. It is useful in
abnormal like platelet count (decrease: the identification of malignant cell types on the
thrombocytopenia) and prolonged bleeding time and basis of cytoplasmic or nuclear chemistry;
clotting time(PT, APTT, TT, ST) cellular constituents that are present in
abnormal form or amount; lack of cellular
TREATMENT: underlying cause of DIC should be constituent; and cells exhibiting functional
considered abnormalities.
 Plasma and platelet transfusions – restore
consumed BRIEF INTRODUCTION TO LEUKEMIA
 Heparin –prevent further activation of
clotting mechanism - Leukemia refers to group of disorders
- In the category of cell line, leukemia can be
differentiated to either Myeloid or Lymphoid
Leukemia
CLINICAL MANIFESTATIONS OF COAGULATION - In the basis of Blast cell
FACTOR DEFICIENCIES o Acute Lymphocytic Leukemia: Pre-
dominance of lymphoblast.
TYPE OF COAGULATI TYPE OF COAGULATI o Chronic Lymphocytic Leukemia:
BLEEDING ON FACTOR BLEEDING ON FACTOR Pre-dominance of mature with few
DEFICIENCY DEFICIENCY lymphoblast.
Easy II, VIII, IX Delayed I, XIII o Acute leukemia: refers to a type of
bruising wound leukemia with poorer prognosis with
healing
 HEMATOLOGY 2
an increase predominance of blast
cells or immature cells
o Chronic Leukemia: the decrease of
blast cells and increase of mature
cells.
o Predominance of myeloblast is
associated with Acute Myeloid
Leukemia
o Predominance of mature myelocyte
is associated with Chronic Myeloid
Leukemia
- Blast cells are not easily identified because of
their common characteristics (larger in size, large
nucleus, smaller and deeply basophilic
cytoplasm), cytochemical stains are used to
differentiate in the absence of CD marker
- Acute Myeloid Leukemia can be further
classified knowing it is a stem cell.
- French American British System classified
leukemia based on the morphology of the cells
after staining with Romanowski stain and their
reaction to cytochemical stain.
- FAB further classified ALL as ALL1, ALL2, ALL3.
While AML is classified into seven categories
(AML1-AML7).
- In AML
o AML1-AML3 predominance cell is
Myeloblast, promyeolocyte myelocyte
(granulocytic series)
o AML4 and AML5 uses alpha naphthyl
Chloroacetate esterase
o AML4 stain positive in both Specific and
non- specific esterase because it
contains both granulocytic and
monocytic cells
o AML5- is the acute monocytic leukemia
o AML6 is called Erythroid Leukemia,
leukemic cells are precursors of
erythrocyte. Positive in Periodic Acid
Schiff.
o AML7 leukemia of megakaryocytes
called acute megakaryoblastic leukemia.
- One example for Chronic Lymphocytic Leukemia
is the Hairy Cell Leukemia which affects B-cells.
Hairy cells contain ACP.
o ACP isoenzyme is present in all blood
cells and can be inhibited by tartaric
acid except the isoenzyme number 5
which is the one present in hairy cell
leukemia.
- In Chronic Myeloid Leukemia the predominant
cells are the mature myeloid cells especially the
granulocytes. CML is also called Chronic
Granulocytic Cells. Granulocytes can range from
50,00-300,000
- Having a very high WBC count can also be
observed in Leukemoid Reaction which is due to
underlying condition.
- Both leukemoid reaction and chronic myeloid
leukemia are characterized by a very high WBC
count. However, Leukemoid Reaction is
reversible that when the underlying condition is
resolved, the WBC will return to normal count.
CLM is a permanent condition. To differentiate
Leukemoid reaction from CML, alkaline
phosphatase is used.
ENZYMATIC STAINS
PEROXIDASE/MYELOPEROXIDASE (MPO)
- The stain itself does not contain the peroxidase
enzyme. The peroxidase enzyme is inside the
cell, the composition of the stain is the substrate
for peroxidase enzyme
- Myeloperoxidase is a normal constituent of the
primary granules of the myelocytic cells which is
observed as early as promyeolocyte.
- Stain marker for primary granules & auer rods
(fused primary granules)
- Stain positive in AML but negative in ALL
- Differentiates AML from ALL
- Stain marker for immature myeloid cells
- Stain positive in granulocyte but not in
lymphocytes
- Positive Activity: reddish-brown deposits (in
cytoplasm of granulocytes and monocytes)
- Note: Myeloperoxidase enzyme deteriorates;
Stain should be done only on fresh specimens
ALPHA-NAPHTHYL ACETATE ESTERASE & ALPHA-
NAPHTHYL BUTYRATE ESTERASE (NON-SPECIFIC
ESTERASES/NSE)
- Used to detect the granulocyte of monocytic
origin
- Nonspecific because it can be seen in other cells
 HEMATOLOGY 2
- The stain does not contain esterase but rather
substrate for the enzyme
- If the cell contains the enzyme it will catalyze the
hydrolysis of the butyrate or acetate in the stain
- Unfixed sample can be used so long in the dark
for as long as 2 weeks
- Marker for cells of Monocytic origin
- Other cells (+): Megakaryocytes, Platelets,
Histiocytes, Plasmacytes, some T-lymphocytes
- Positive Activity: red-brown/dark red
LEUKOCYTE/NEUTROPHIL ALKALINE PHOSPHATASE
(LAP/NAP)
- Stains NEUTROPHILS (the only leukocytes that
contain this activity)
- Neutrophils contain various amount of ALP
- Principle: Differential count involving
neutrophils
- Differentiates Leukemoid reaction (LR) from
Chronic myeloid leukemia (CML)
- Reference Value: 30 –185 LAP score
- Increase LAP score is observed in the following:
o during the last trimester of pregnancy -
Hodgkin disease
o Polycythemia vera - Multiple myeloma
o Aplastic anemia - Obstructive jaundice
- To compute, multiply the counted neutrophils by
their grade.
- Normal to High: Leukemoid reaction
- Below than normal value to zero: CML
TARTARIC ACID RESISTANT ACID PHOSPHATASE (TRAP)
- For the diagnosis of Hairy cell leukemia (HCL)
- General Principle: ACP is Detected in almost all
blood cells but when treated with tartaric acid it
is inhibited except isoenzyme number 5
- Labile if unpreserved
- Sample should first be fixed and be stored at -
20°C and can be used atleast 2 weeks
- Other Tartrate Resistant cells: Sezary cells;
Histiocyte; T-cell of acute lymphocytic leukemia
- Activity is indicated by purple to dark red
granules in cytoplasm
CYANIDE-RESISTANT PEROXIDASE
- For identification of Eosinophilic components
- Positive Activity: brown
NAPHTHOL ASD CHLOROACETATE ESTERASE (SPECIFIC
ESTERASE)
 Marker for mature & immature Neutrophil and
mast cells.
 Substrate is the chloroacetate
 Use to identify immature or primitive
granulocyte ALM1-ALM3
 Positive Activity: bright red granules in
cytoplasm.
 This enzyme is stable and may last for months.
TERMINAL DEOXYRIBONUCLEOTIDYL TRANSFERASE
(TDT)
 Stains DNA polymerase immunoperoxidase
 Marker for immature Lymphoid cell
 Positive in almost 90% cases of ALL
 Also useful in the detection of the
“lymphoblastic transformation” of chronic
myeloid leukemia (CML)
 Detects blastic transformation of CML
o CML can transform into an acute
leukemia
o Acute leukemia more than or equal to
30% blast cells
o CML can transform either AML or ALL.
 AML: (+) MPO stain and SBB
 ALL: (+) TdT, it means that it
transforms into ALL.
ACID PHOSPHATASE
 Present in all hematopoietic cells and found in
lysosomes
 Activity is indicated by purple to red granules
 Unstable, cannot be stored.
NON-ENZYMATIC STAINS
PRUSSIAN BLUE STAIN
 Stains siderotic granules (for the diagnosis of
sideroblastic anemia).
 Used to detect hemosiderin in urine
 Used to identify iron in the ferric state (Fe+3)
 HEMATOLOGY 2
PERIODIC ACID SCHIFF (PAS)
 Stain reacts w/ aldehyde groups in glycogen,
mucoprotein, glycoproteins, and high molecular
weight carbohydrates.
 Does not detect stains instead it combines with
the aldehyde group of glycogen.
 Positive to all blood cells except normal
erythroblast or pronormoblast
 Positive erythroblast indicates that it is affected
by AML6.
 Positive in erythroblasts in Di Guglielmo disease
/ Erythroleukemia.
 L1 and L2 also stain positive in PAS
 The positivity of L1 to Pas is described to be
chunky or block-like positivity
 L2 is not that large as L1
 Negative Activity: bright fuchsia pink (Pattern of
staining varies with each cell type)
 Also differentiates Acute Lymphoblastic
Leukemia.
 Peroxidase of eosinophil is resistance to cyanide
 Cyanide resistance peroxidase is used to identify
peroxidase of eosinophils
SUDAN BLACK B (SBB)
 Marker for phospholipids and lipids
 Has the same staining reaction as
myeloperoxidase
 Gives the same information as Peroxidase in the
interpretation.
 Positive in AML.
 Advantage over myeloperoxidase is that it is
more stable, because myeloperoxidase detects
enzyme which is somewhat labile and may
disappear in prolonged storage. Sample stained
in SBB can give reliable result for months.
 Differentiates AML from ALL
 (+) Result: Dark purple-black granules
NOTE:
 Can be done on stored specimens.
 More sensitive than chloroacetate in the
demonstration of mature & immature
neutrophils and mast cells.
TOLUIDINE BLUE O
 A metachromatic stain
 Binds with mucopolysaccharides in blood cells
 For the recognition of basophils and mast cells
 (+) Result: Reddish-Violet
ADDITIONAL NOTES:
Primary fibrinolysis/fibrinogenolysis: Increase
activators of plasminogen  increased plasmin: not
normally present
Secondary fibrinolysis: Release of thromboplastin-like
substance  activates both coagulation and
fibrinolysis  increased plasmin.
Naturally occurring inhibitors: naturally present in the
circulation and their purpose is for normal regulation
of coagulation mechanism. They maintain
homeostasis. Examples: AT-III; TFPI, Protein C, Protein
S, alpha-2 macroglobulin, alpha-2 anti-plasmin, etc.
Abnormally, we could also develop inhibitors or
autoantibodies. Under ACQUIRED COAGULATION
DISORDER; not normally present in the system or
circulation but we produce them when our system is
abnormally activated.
INHIBITORS: autoantibodies that cause prolongation
of certain test because they interfere in normal
coagulation.
2 CATEGORIES:
1. SPECIFIC
 inhibitor directed against specific coagulation
factor like FVIII, FIX or FVII inhibitor FACTOR
VIII: C INHIBITOR
 Most common and severe coagulation factor
inhibitor in VIII:C portion not the complex
 Inhibit Factor of VIII: C only: does not have an
effect on vWF.
 Prevents VIII:C in participating as cofactor in
the formation of intrinsic tenase.
 Normally present in elderly with B-cell
anomalies
 Patients are high risk for suffering bleeding
 Prolonged: APTT
 Bethesda assay – titer: antibody
measurement
 Acute management  high-dose F VIII  not
all will be inhibit  coagulation proceeds (for
short term only).
 HEMATOLOGY 2
 Long term management  MYELODYSPLASTIC, MYELOPROLIFERATIVE and
Immunosuppression  patient is given
cytosan  inhibit production of all antibodies:
should be well-planned.
2. NON-SPECIFIC
 directed against factor or protein involved in
coagulation but is not specific to a certain
coagulation factors; targets anything.
LUPUS ANTICOAGULANT/APA/ ANTICARDIOLIPIN
ANTIBODY
 Inhibits phospholipids and proteins bound to
phospholipids but it has no anticoagulant
effect in vivo  does not lead to bleeding.
 Directed to epitopes of proteins bound to
phospholipids  Thrombosis; abnormal
clotting
 Seen in immunocompromised patients:
higher in patients suffering from SLE, HIV, and
other autoimmune disorders that affects B-
cells production
 Effect is different in vivo and in vitro
 In vitro: has anticoagulant effect and causes
prolongation of clotting time tests like APTT
and dRVVT; less likely in PT
 Immunoassay: for diagnosis  ELISA
LYMPHOPROLIFERATIVE DISEASES
MYELODYSPLASTIC SYNDROME
 myelo – bone marrow; dys – abnormality; plastic
– development  abnormality in development
of cells arising from bone marrow.
MYELOPROLIFERATIVE DISEASE
 Abnormal proliferation of cells in the bone
marrow.
MYELODYSPLASTIC-MYELOPROLIFERATIVE
 Abnormal proliferation and development of cells
in the bone marrow.
NOTE:
NORMAL LIFE: cell production = cell destruction 
NORMAL cell count
Main parent cell: PPSC  Stem cell: LSC (T and B
lymphocytes and non-A non-B population) and MSC
(RBCs, Platelets, Monocytes and granulocytes)
UNILINEAGE DYSPLASIA: only one cell line is affected
MULTILINEAGE DYSPLASIA: one or more cell line is
affected
 HEMATOLOGY 2

MYELODYSPLASTIC MYELOPROLIFERATIVE MYELODYSPLASTIC-

Myelodysplastic Syndrome (MDS) MYELOPROLIFERATIVE
or Dysmyelopoetic Syndrome
(DMS)
abnormality in development of abnormal proliferation of cells in the bone abnormal proliferation and
bone marrow cells marrow development of cells in the bone
marrow
MAIN ABNORMALITY: MAIN ABNORMALITY: Uncontrolled Can’t classify as MDS or MPD alone
Hypercellular maturation  cells proliferation of one or more of the BM  MDS/MPD  with variable
are abnormally large of cells Dysplasia might be present (little) increases in cells as well as
myelo-cell line (RBCs and WBCs but not common cytopenia and morphologic
but NOT LYMPHOCYTES) dysplasia
Abnormal morphology: RBC and Abnormal number of cell: RBC and WBC Show mixed characteristics of MDS
WBC and MPD.
MAIN DIFFERENCE: Many MAIN DIFFERENCE: Little or no dysplasia
dysplasia
Apoptosis (programmed cell Proliferation outspaces apoptosis
death) predominates  easily
perish/destroyed
BLOOD PICTURE: Cytopenia  BLOOD PICTURE: Overproliferation 
decrease in blood cells increase in blood cells
Pre-leukemia DISORDERS: DISORDERS:
• Not leukemia but can 1. Leukemia: genetic involvement; 1.Chronic Myelomonocytic
progress to leukemia  complicated classification Leukemia (CMML)
px are at risk of suffering A. ACUTE LEUKEMIA 2. Atypical Chronic Myeloid
leukemia (AML) B. CHRONIC LEUKEMIA Leukemia (aCML)
• Common in elderly 3. Juvenile Myelo-Monocytic
2. Chronic Myeloproliferative Disorders
patient: Age 50 and above Leukemia (JMML)
• Acquired condition 4. MDS/MPD-U: unclassified
CLINICAL COURSE/PROGNOSIS:
survival
Shorter than CMPD longer than
Acute Leukemia  AML: short
clinical course
DISORDERS: Refractory 
unresponsiveness
1. Refractory Cytopenia Unilineage
Dysplasia (RCUD)
Refractory Anemia (RA): only red
cells are affected
Refractory Neutropenia (RN): only
neutrophils are affected
Refractory Thrombocytopenia
(RT): only thrombocytes or
platelets are affected
2. Refractory Anemia with Ringed
Sideroblast (RARS): immature
RBCs with siderotic granules
 HEMATOLOGY 2

3. Refractory Anemia with

Multilineage
Dysplasia (RAMD)
Refractory Anemia with Excess
blast (RAEB)
4. 5q- SYNDROME OR
ISOLATED 5q DELETION
5. MDS-U: unclassified

TWO SYSTEMS EMPLOYED IN CLASSIFICATION OF LEUKEMIAS AND MYELOID DISORDERS

The FAB Classification The WHO Classification

French-American British System
Group of scientist and doctors
Basis: Morphologic appearance 
examination of blood smear stained with
Wright’s or Giemsa; cellular level  not
accurate and precise
FIRST BASIS
RA Refractory anemia
RARS Refractory anemia with ringed sideroblasts
RAEB Refractory anemia with excess blasts
CMML Chronic Myelomonocytic Leukemia
RAEBIT Refractory anemia with excess blasts in
transformation
World Health Organization
Basis includes more criteria: morphology,
chromosomal study (genetic abnormality) and
immunologic probe (CD markers); more
genetic level of disease
CURRENT BASIS
Refractory anemia RCUD
Refractory anemia with ringed sideroblasts
Refractory cytopenia with multilineage dysplasia
Refractory anemia with excess blast
Type I
Type II: characteristics are similar with RAEBIT
Myelodysplastic syndrome unclassifiable
5(q) chromosome abnormality

MYELODYSPLASTIC SYNDROME (MDS)

 MDS are group of disorders that result from clonal abnormalities of hematopoietic pluripotential stem cells. These
are characterized by hypercellular maturation of the erythroid cells, granulocytes and megakaryocytes.
 They are often described as “Pre-Leukemias” FEATURES
 Occur primarily in persons over age 50
 Characterized by peripheral Cytopenias
 Also characterized by presence of dysplasia of the myeloid cell lines
 Clinical course is shorter than CMPD and longer than acute leukemias.
 HEMATOLOGY 2

MAJOR CLASSIFICATION OF MDS/DMS (WHO Classification, 2008)

MDS PERIPHERAL BLOOD FINDINGS BONE MARROW NOTES

FINDINGS
Refractory Cytopenia Unicytopenia Unilineage dysplasia: Refractory:
with Unilineage Dysplasia No or rare blasts (<1% blast) only one cell is affected; Unresponsive to either
(RCUD) >10% of cells in one treatment or stimulation.
• Refractory anemia (RA): myeloid lineage Dysplastic: should be
RBCs (most common) more than or equal to
• Refractory neutropenia 10% of cells.
(RN) Main abnormality:
•Refractory hypercellular maturation
thrombocytopenia: Prognosis: >5 years; good
Platelets prognosis Risk of
acquiring
leukemia  AML (6%) 
Refractory Anemia Dyserythropoiesis: BM: <5% blasts AML: poor prognosis
megaloblastoid maturation (RA): none or <15%
(ovalocytes); ringed sideroblasts
macroovalocytes ( more than 8
micra)
PB: <1% blasts; reticulocytopenia
 increased apoptosis

Refractory anemia with Features are similar with RA. •Erythroid dysplasia only Reasons for the presence
ring sideroblasts (RARS) Dyserythropoiesis: •15% or more ringed of ringed sideroblast:
megaloblastoid maturation sideroblasts (ONLY Abnormal development
(ovalocytes); DIFFERENCE with RA) of cells and impaired RBC
macroovalocytes ( more than 8 •<5% blasts hemoglobinization: Iron
micra) ppt  siderotic granules
PB: <1% blasts; reticulocytopenia Stain to confirm presence Heme synthesis:
 increased apoptosis of iron: mitochondria; near
Anemia, no blast PRUSSIAN BLUE nucleus: iron not utilized
 siderotic granules
around nucleus forming
a ring
Prognosis: >5 years
 AML (2 years)

Refractory Cytopenia Cytopenia(s) Dysplasia in >10% of cells Multilineage: more than

With Multilineage PB: more than or equals to 10% in two or more myeloid one lineage in myelo cell
Dysplasia (RMCD) dysplasia in more than one cell lineages (neutrophil &/or line is affected; any
lines: (> 2 cell line) erythroid precursors combination
No or rare blasts (<1%) &/or Prognosis: 33 months 
<1 x 109/L monocytes; megakaryocytes <5% AML (11%)
monocytopenia is observed blasts in marrow + (less
No Auer rods than 15%) or - ringed
 HEMATOLOGY 2

sideroblasts; No Auer
rods

Refractory Anemia With Cytopenia(s) Unilineage or PROGNOSIS: less than

Excess Blasts 1 (RAEB-1) <5% blasts multilineage dysplasia 2 years Incidence to
<1 x 109/L monocytes Hypercellularity progressing as AML
NO AUER RODS dyspoiesis: 5-9% blasts (25%)
without Auer rods

Refractory Anemia With Cytopenia(s) Unilineage or Incidence to progressing

Excess Blasts 2 (RAEB-2) 5-19% blasts multilineage dysplasia as AML (33%)
<1 x 109/L monocytes Hypercellularity
+ or – ; with or without: Auer rods dyspoiesis: 10-19% blasts
(fused primary granules) with Auer rods

Myelodysplastic Cytopenia(s) •Unequivocal dysplasia

Syndrome Unclassified <1% blasts in <10% of cells in one or
(MDS-U) more myeloid cell lines
PB: Rare blast when accompanied by a
cytogenic abnormality
considered as
presumptive evidence for
a diagnosis of MDS
•<5% blasts
MDS Associated With Anemia •Normal to increased 5q: long arm of
Isolated Del (5q) Usually normal or increased megakaryocytes with chromosome; there’s
Also known as: platelet count increased hypolobulated deletion of gene 
5-q Syndrome or Isolated No or rare blasts (<1%) nuclei affecting megakaryocytes
5q Deletion •<5% blasts p-arm: short arm
•No Auer rods Prognosis is good
 HEMATOLOGY 2
MYELODYSPLASTIC/MYELOPROLIFERATIVE
DISORDERS
These are disorders that show features of both
myelodysplastic and myeloproliferative disorders. They
are characterized by variable increases in cells as well as
cytopenia and morphologic dysplasia.
TYPES:
1. CHRONIC MYELOMONOCYTIC LEUKEMIA
(CMML)
 Predominant feature is persistent monocytosis
of >1 X 109/L for more than 3 months duration;
with <20% blasts (monoblasts) and few
promonocytes in the peripheral blood and bone
marrow with dysplasia in one or more myeloid
cell line.
 Cytochemical stain: (+) with alpha naphthyl
acetate and alpha naphthyl butyrate esterase
stains  nonspecific esterase stains
 Used to be under MDS in FAB classification.
 In WHO, it is under MDS-MPD  used currently
 Chronic means long/persistent or prolonged;
more than 3 months
NOTE:
TYPES:
 CMLL-1: <5% blasts and promonocytes in the
PB, <10% in the BM.
 CMML-2: 5-19% blasts and promonocytes in
the PB, or 10-19% in the BM  more severe
o Whenever the blast is increased, the
disease is more severe and the
poorer the prognosis
ACUTE LEUKEMIA: poor prognosis; ↑ blasts
CHRONIC LEUKEMIA: good prognosis; ↓ blasts
ESTERASE STAINS:
 SPECIFIC: for immature granulocytes like
myeloblast  alpha naphthyl chloroacetate
esterase (combined substrate)
 NON-SPECIFIC: for monocytic cells  alpha
naphthyl acetate and alpha naphthyl
butyrate esterase stains
2. ATYPICAL CHRONIC MYELOID LEUKEMIA, BCR-
ABL1 NEGATIVE
 Characterized by leukocytosis with prominent
dysgranulopoiesis  (mature and immature
granulocytes)
3. JUVENILE MYELOMONOCYTIC LEUKEMIA
(JMML)
 This is a clonal disorder of predominantly
granulocytic and monocytic lineages.
 MDS/MPD: no Ph1 chromosome
 Juvenile - immature or young patients
 Previously referred to as Ph1 negative CML or
Monosomy 7.
o Normal chromosome: 23 pairs  22
pairs autosomes and 1 pair of sex
chromosomes; but in monosomy 7, no
pair.
 Ph : Philadelphia chromosomes; abnormal
1
translocated chromosome seen in patient
suffering CML ( leukemia and stem cell disorder
wherein most patients but not all have Ph1 )
o Ph1 (+): mostly adults; when Ph1 is
present, it will fall under CML (MPD).
o Ph1 (-): infants or newborns and young
children.
4. MYELODYSPLASTIC/MYELOPROLIFERATIVE
DISEASE, UNCLASSIFIABLE
 These include those cases that meet the criteria
for MDS/MPN but do not fit into one of the
specified subcategories.
 HEMATOLOGY 2

MYELOPROLIFERATIVE DISORDERS (MPD) ENVIRONMENTAL:

LEUKEMIAS
 Group of disorders: Leukemia is a disease of
the blood-forming tissues, predominantly the
bone marrow. It is a malignant neoplasm
characterized by disorderly, purposeless, and
uncontrolled proliferation of one or more of the
hematopoietic cells in the bone marrow.
HEMATOPOIETIC CELLS
 Medullary: bone marrow: disorder  leukemia
(cancer of bone marrow of the blood forming
cells)
 Extra medullary: thymus, lymph nodes 
lymphoma
ETIOLOGY: genetic mutation and/or oncogene activation
3. CHEMICAL AGENTS: carcinogen or leukomogens
 Through inhalation, ingestion or simply exposure
4. IONIZING RADIATION
 Strong dosage
5. VIRUSES: EBV and HTLV (I and II Retrovirus)
 Because of their nature (viral replication)
 EBV and HTLV (I and II Retrovirus): Burkitt
lymphoma  ALL –L3 (leukemic phase)
 Bacteria: can’t cause leukemia: reproduce or
divide by binary fission
 Virus: enters host cell  dissolve its capsid to
release its viral genes or genetic component then
combine with host’s cell gene  form a new
gene  new viral elements  replicate inside
cell using their own and host’s gene  mutation.

 Genetic mutation: cancerous gene  over 6. IMMUNOLOGICAL DEFECTS

proliferation  Patient’s immune system is suppressed 
 Oncogene activation: a gene when activated genetic mutations/oncogene activation  Prone
converts normal gene/cell into cancerous one  to suffer cancer: *Kaposis sarcoma: HIV 
Onco means cancer Breakdown of the immunosurveillance that
normally keeps neoplastic growths in check.
IMPLICATED/ASSOCIATED CONDITIONS: Leukemia  Especially seen in lymphocytic leukemia and
occur as a combination of genetic and environmental lymphoma
factors. 7. NEOPLASIA
GENETIC:

1. HEREDITY/ GENETIC
 Familial incidence: cancer that runs in the
family.
2. CHROMOSOMAL ABNORMALITY/CONGENITAL
FACTORS
 Ph1 chromosome (CML)
 Trisomy 21 (Down syndrome)  predisposed
to suffer AML
 Translocation of a part of Chromosome 8 -14 
or written as t (8:14)  Burkitt lymphoma (ALL-
L3)
o Leukemia and lymphoma are two
different terms but lymphoma can
progress to leukemia when the
cancerous cells in the lymph nodes
infiltrate the bone marrow  leukemic
phase.
 HEMATOLOGY 2

NOTE:
CLINICAL FEATURES: Cancerous cell clone will
grow rapidly and the expense of normal
hematopoietic cells
Leukemic cells (LC) are cancer cells that grows
rapidly or replicate easily thus spread rapidly; found
in bone marrow  space is limited by hard bone 
LC grow rapidly  occupy the bone marrow at the
expense of the normal ones  overcrowd and
infiltrated and take up the nutrients fast
(competition) for normal cells  decreased normal
hematopoietic cells and increased abnormal cells or
LC.

CONSEQUENCES MANIFESTATIONS TREATMENT FOR LEUKEMIA

Decrease RBC production
Decrease normal WBC production
Decrease platelet production
1.Systemic Symptoms 1. Chemotherapy: destroy all cells
- Decreased RBC: including normal  monitor CBC
 Anemia: Fatigue, headaches, 2. Radiotherapy: radiation destroys
dizziness, paleness and others also abnormal cell.
seen in leukemia with anemia 3. Bone Marrow Transplant
- Decreased WBC: multitude of infection  - Graft vs. Host (GVHD)
fever, increase sweating 4.Differentiation Treatment:
- Decreased Platelets: Bleeding (more manipulation of growth or
important to address) and Clotting replication of cancerous cell into a
problems less or non-cancerous one  turn
into mature cell
2.Hypermetabolism - All Trans Retinoic Acid (ATRA)
5. Supportive Management:
Transfusion of blood for anemia and
Antibiotics for infections
 HEMATOLOGY 2
NOTE:
2 MAJOR CLASSIFICATION OF LEUKEMIA BASED ON
CELL LINE:
1. Myeloid/ Non-Lymphoid/ Myeloblastic/
Myelocytic/ Myelogenous Leukemia (AML or ANLL)
2. Lymphoid/ Lymphocytic/ Lymphoblastic/
Lymphogenous Leukemia
BASED ON THE NUMBER OF BLAST CELLS:
 Upper: immature/blasts  increased in blast
cells  ACUTE LEUKEMIA (AML or ALL)
 Acute: more blast (30 (FAB) or 20 (WHO) more
percent)
 Lower: Mature cells increased  CHRONIC
LEUKEMIA (CML or CLL)
 Chronic: less blast (20 (WHO) or 30 (FAB) less
percent); more mature cells
e.g. Lymphocytic/Myeloid cell line
Acute/Chronic  number of blast or prognosis/clinical
course
NUMBER OF BLAST (> or < 20/30):
 Acute: more
 Chronic: less
PROGNOSIS:
 Acute: Days to months (poor; short life span)
 Chronic: 1-2 years or more (good, longer life
span)
4 MAJOR TYPES OF LEUKEMIA
1. Acute Myeloid Leukemia (AML)
 increased in blast: myeloblast (>30%); stem
cell disorder; elderly and newborns
2. Chronic Myeloid Leukemia (CML)
 decreased blast(<30%); increase mature cell:
myelocytes
3. Acute Lymphoblastic Leukemia (ALL)
 increased in blast: lymphoblast (>30%);
common in children
4. Chronic Lymphoblastic Leukemia (CLL)
 decreased blast(<30%); increase mature cell:
lymphocytes
 HEMATOLOGY 2

CLASSIFICATIONS OF LEUKEMIAS
No. of WBC (PB)
(15 x 109/L)
CHARACTERIZATION:

 LEUKEMIC: based on the number of WBC in

peripheral blood (PB)  if WBC count is higher
or more than 15x 109/L
o In leukemia, WBC/RBC count is high
however these cells are abnormal in
function because it produced by
abnormal bone marrow.
 ALEUKEMIC: leukemia if WBC count less than
15x 109/L
o Absence of leukemic or blast cells in the
circulation, leukemic cells are confined
ONLY in bone marrow
 SUBLEUKEMIC: leukemia if WBC count less than
15x 109/L found in the bone marrow and in the
circulation.

There are presence of blast and leukemic cells  both in bone marrow and peripheral blood

CRITERIA CLASSIFICATION; CHARACTERISTICS

I. Cell line 1. Myeloid/Myelocytic/Myelogenous Leukemia/Non-Lymphocytic Leukemia
2. Lymphoid/Lymphocytic/Lymphogenous Leukemia/Lymphoblastic
II. % of Blasts in 1. ACUTE LEUKEMIA:
the Peripheral  With increased blast cells in the bone marrow and peripheral blood
blood and  FAB critertia: >30% bone marrow blasts
bone marrow  WHO criteria: >20% bone marrow blasts
 Prognosis: Rapidly progressive, with a shorter prognosis of days to 6 month
2. CHRONIC LEUKEMIA
 Less than 10% blasts in PB
 With better and longer prognosis
3. SUB-ACUTE LEUKEMIA
 10-30% blast in the PB plus other symptoms
 Prognosis: at least 2-6 months
III. Number of 1. LEUKEMIC LEUKEMIA: WBC count is more than 15000/uL
WBCs in the 2. SUB-LEUKEMIC LEUKEMIA: WBC count is less than 15,000 uL, and
Peripheral  Presence of immature or abnormal cells in the PB
blood 3. ALEUKEMIC LEUKEMIA: WBC count is less than 15,000/Ul
 No immature nor abnormal cells in the PB
TWO SYSTEMS FOR CLASSIFYING LEUKEMIA
French-American British 1976; group of scientist
(FAB) Criteria: Blood smear with Romanowsky stain (Wright’s or Giemsa)  observe
Classification morphology (M1-M7; L1-L3) Cytochemical staining  identify cell line affected
>30% Acute Leukemia
World Health Improvements in 1997, 2001, 2008, 2014  classification is subject to changes
 HEMATOLOGY 2

Organization (WHO) Criteria: Cellular morphology, cytochemical stains, immunologic probes of cell
classification markers (CD markers), cytogenetic or chromosomal abnormalities and clinical
syndrome
Current basis: Standard system of classifying leukemia >20%  Acute Leukemia

NOTE: ACUTE LEUKEMIA  Key myeloid antigens: Myeloperoxidase,

 Acute Myelocytic Leukemia (AML or ANLL)
 AML is a stem cell disorder with
predominance of Blast Cells (>20%) in the
blood or marrow and may resemble acute
infection at presentation. It affects all ages,
but increases with older age (>60 years). This
is also the most common form of acute
leukemia during the first few months of life.
 Stem cell disorder/leukemia  affect all cell
line that arise from it CFU- GEMM
 Ionizing Radiation, Leukomogens;
Congenital/Genetic factors (Down’s
syndrome); Viruses, Neoplasia (cancer that
transform into different or poorer type of
cancer; transformation of one type of
malignancy into another form of malignancy)
Clinical and Laboratory features of AML
 Affects all ages, but increases with older age
 Two peaks: newborns or babies (almost
always AML) and elderly (>60 years)  In
children, ALL is more common
 May resemble acute infection at
presentation.
 Requires 20% blasts in blood or marrow for
diagnosis.
CD13, CD33, CD117, CD14/CD64
 Signs and symptoms: Anemia, infection;
bleeding (decrease in plts.); coagulation
mechanism abnormalities (disturbance in CF)
(AML –M3)
 FAB: Classified into seven; requires 30% or
more blast in the myeloid series in the
marrow or circulation.
 WHO: requires 20% or more blast in the
myeloid series in the marrow or circulation
 Key myeloid antigens: Myeloperoxidase,
CD13, CD33, CD117, CD14/CD64
 Classification made by morphology,
cytogenetics, flow cytometry and
cytochemistry  Recurrent
cytogenetic abnormalities that characterize
defined subtypes

AML
(M1, M2, M3: MPO (+); SBB (+); aNCAE(+)); TdT
( -)
Myeloid M4 and M5: aNAE and aNBE (+)
M6: PAS (+)
Leukemia
CML
LAP (+)
Leukemia
ALL
MPO (-); SBB(-); Alpha NCAE( -); (TdT (+) L1-L3)
Lymphocytic
Leukemia L1 (intense) and L2: PAS (+)

CLL
 HEMATOLOGY 2

French-American-British (FAB) Classification of the Acute Myeloid Leukemias (stem cell disorder)
CRITERIA: MORPHOLOGY AND REACTION TO CYTOCHEMICAL STAINS
M0 (AML with minimal differentiation/ Undifferentiated leukemia)
 Cells are not well differentiated
 Cytochemical staining: Negative with SBB and peroxidase  stains used to identify cells in myeloid series
(granulocytes).
MYELOCYTIC or GRANULOCYTIC SERIES
M1 (Acute Myeloblastic L. without Maturation) Myelocytic origin
No maturation: >30% myeloblast
Auer rods present (fused primary granules with pencil or
rod like shaped; spindle-shaped, red-purple)

Primary granules: promyelocyte

Secondary granules: myelocyte
M2 (Acute Myeloblastic L. with Maturation) Myelocyctic origin
With maturation: >30% myeloblast with >10%
granulocytic component (promye- to neutrophil) 
mature forms
Auer rods present
Similar to WHO: t (8:21)

M3 (Acute Promyelocytic Leukemia) With heavy granulation or Hypergranulation 

Also called Hypergranular Promyelocytic Leukemia abundant promyelocyte: primary granules of
granulocytes
Many Auer rods in bundles called “faggot cells”

WHO: t (15:17)

Associated with DIC: normal tissue (damage)  release

of thromboplastic substances  Secondary fibrinolysis:
ACTIVATION of:
Coagulation  Fibrinolysis promyelocyte lysis 
lysosomal contents  activate coagulation and fibrinolytic
cascade
MONOCYTIC SERIES [(+) with a-naphthyl acetate esterase & a-naphthyl butyrate e.]
M4 (Acute Myelomonocytic Leukemia) Myelo represents granulocytic cells
Also called “Naegeli” monocytic Leukemia Monocytic represents monocytic cells
>20% of PB WBCs are monocytes or monocytic
precursors the rest are mixture of the granulocytic cells
Predominance of both myelocytic and monocytic cells at
80:20 ratio WHO: inv (16)

M5 (Acute Monocytic Leukemia) >80% of BM elements are monocytic series; mono-, pro-
Also known as Schilling’s Leukemia and monocytes

WHO: t (9:11)
 HEMATOLOGY 2

TYPES:
M5a: poorly differentiated monocytic Leukemia;
predominant cell is promonocyte (increased in BLASTS;
>80% are mostly monoblasts)
M5b: well differentiated; more of promonocytes and
monocytes (more MATURE)
ERYHTRPID SERIES
M6 (Pure Erythroid Leukemia) With neoplastic Myeloblasts & Erythroblasts
Cancerous cells in BM represents: >50% are erythroid cells
Other names: in all stages of maturation
Erythroleukemia
Erythremic Myelosis Cytochemical Stains (+) w/ Periodic Acid Schiff (PAS)
DiGuglielmo Disease PAS: stain for aldehyde; stains glycogen in the cells;
combined with aldehyde portion of the glycogen therefore
it is positive in ALL BLOOD CELLS except the normal
erythroblast or precursor or immature RBCs Normally,
erythroblast should be negative in PAS however in AML-
M6; erythroblast are abnormal  Positive in PAS.
MEGAKARYOCYTIC SERIES
M7 (Acute Megakaryocytic Leukemia) Predominantly Megakaryoblasts (>30%) and
micromegakaryoblast Cytochemical stain: Negative (-) w/
SBB and peroxidase

NOTE:
 HEMATOLOGY 2

World Health Organization (WHO) CLASSIFICATION: AML

In order to classify the cell under AML they should be

positive with the following: Key Myeloid Antigens:
CD13, CD33, CD117, and CD14/CD64

Primitive Myeloblasts: (+) Myeloid associated antigens:

MPO (peroxidase enzyme in primary granules), CD13,
CD33, CD117

More mature myeblasts: produces enzymes  (+)

Cytochemical stains: MPO (peroxidase), SBB (lipids),
CAE (enzymes; esterase)

WHO Classification - Main Categories of AML;

current basis
1. AML with recurrent cytogenetic abnormalities
 Translocation (t) and inversion (inv)
 M3

2. AML with multilineage dysplasia

 There is a combination of cell line affected

3. AML, Therapy related: has mutagenic effects 

cause mutation
 Alkylating agents: chlorambucil,
cyclophosphamide, thiotepa, and busulfan
 Exposure to radiation
 Appears 5 years after exposure to both
alkylating agents and/or radiation: MDS  AML

4. AML, Not otherwise specific/classified (sub-

classified by morphology and
immunophenotype)
 FAB: M1-M7 (except M3) retained as sub-
classification

With stars: AML with recurrent cytogenetic

abnormalities (translocation and inversion) 
Myeloid associated Ag: MPO, CD13, CD33, CD117.
Second table: AML, Not otherwise specific/classified;
retained FAB
M3: under AML with recurrent cytogenetic abnormalities
 APL with t (15:17).
 HEMATOLOGY 2

KEY FEATURES OF THE MAJOR ACUTE MYELOID LEUKEMIAS (AML)

Category Cell Morphology Cell surface markers Cytogenetics Prognosis

AML with t (8;21) >20% blasts, >10% CD 13, 33, 117, t(8;21)(q22;q22) More favorable
maturing granulocytes, CD19, CD34 AML1/ETO
Auer rods, dysplasia
AML with inv (16) Blasts with both CD 13,33,14,4,64 inv(16)(p13;q22) or More favorable
monocytic & t(16;16)(p13;q22)
neutrophilic
differentiation,
increased
eosinophils/immature
eosinophils
APL with t (15;17) Promyelocytes with CD 13,33 t(15;17)(q22;q12) More favorable if
azurophilic granules, CD2, +CD117 PML/RARa Variants responsive to ATRA
Auer rods all involve 17q12
AML with (t9;11) Monoblasts and CD 33,65,4, HLA-DR t(9;11)(p22;q23), Intermediate
promonocytes MLLT3-MLL
predominate
AML, therapy Multilineage dysplasia, CD 13, 33, 34, +CD 11q23 Less favorable;
related RS, increased basophils 56, 57 abnormality Median survival < 3
seen with years
topoisomerase
II
inhibitor-associated
AML
AML with t(6;9) Any morphology may be CD 13, 33, 38, DEK-NUP214; t(6;9) Less favorable
seen but HLADR, CD117 (p23;q35)
AML with inv(3) or myelomonocytic is most CD13, CD33, CD38, Inv(3)(121; q26.2) or Less favorable
t(3;3) common Any HLA-DR, CD117 t(3;3) (q21;q26.2)
morphology may be RPN1-EV11
seen except APL
AML with t(1;22) Megakaryoblastic CD41, CD61, square} t(1;22)(p13;q13) Less favorable
morphology CD13, CD33 RBM15-MKL1
with small and large
megakaryocytes
AML with FLT3 Any Any t(6;9)(p23;q34), Less favorable
mutation/duplication t(15;17)(q22;q12) or
normal
AML with NPM1 Myelomonocytic and CD13,CD33, CD34 is NPM1 mutation, More favorable in
mutation monocytic features negative cytoplasmic the absence of FLT3
expression
AML with CEBPA Variable, generally CD13, CD33, CD65, CEBPA mutation More favorable
mutation similar to less CD11b, CD15
differentiated AMLs in
FAB scheme (M1,2)
 HEMATOLOGY 2

CYTOCHEMICAL STAINS: Cytochemical Reactions in Acute Leukemia

CYTOCHEMICAL CELLULAR ELEMENT BLASTS IDENTIFIED NOTES

REACTIONS STAINED
ENZYMATIC STAINS
 Enzymatic staining: the stain is not the enzyme. (e.g. CAE stain  enzyme is what we want to detect which is
inside in the cell) Purpose/Composition of stain: serve as a substrate to cause a reaction
Myeloperoxidase (MPO) Neutrophil primary Myeloblasts strong positive Differentiates ALL from
granules and auer rods Monoblast faint positive ANLL
For AML identification
(+) Activity: reddish brown
deposits (in cytoplasm of
granulocytes and
monocytes)

MPO enzyme deteriorates;

stain should be done only
on fresh specimens
Luekocyte Alkaline Present in tertiary granules Identify CML (leukemia; uncontrolled proliferation) from
Phosphatase of neutrophils leukemoid reaction (LR): transient
Reference Value: 30-185 LAP score

CML: WBC: >50,000/uL  LAP score: <0

LR: WBC: >50,000/uL  LAP score: Normal to High

Smear with LAP stain  observe microscopically  count

100 neutrophils  Grade

Kaplow’s LAP SCORE:

0 = no staining
+ = faint and diffuse staining
++ = pale and moderate amount of blue staining
+++ = strong blue ppt
++++ = deep blue or brilliant

Computation: Multiply the number of cell

by their grade e.g.
0 = 30  0
+ = 50  50
++ = 20  40
= 90 LAP score

Increased LAP Score:

(Controls: During the last trimester of pregnancy),
Polycythemia vera, Aplastic anemia, Hodgkin disease,
multiple myeloma, obstructive jaundice
Specific esterase Cellular enzyme Myeloblasts strong positive For AML identification
 HEMATOLOGY 2

alpha-Naphthyl Marker for mature and

chloroacetate esterase immature neutrophil and
mast cells
(+) Activity: bright red
granules in cytoplasm
This enzyme is stable and
may last for months
Nonspecific esterase Cellular enzyme Monoblast strong positive For M4 and M5
alpha-naphthyl Acetate Marker for cells of
Esterase and alpha- monocytic origin
Naphthyl Butyrate Other cells: (+)
Esterase Megakaryocytes,
Platelets, Histiocytes,
Plasmacytes, some T-
lymphocytes
(+) Activity: red-brown/
dark red
Terminal deoxynucleotidyl Intranuclear enzyme Lymphoblast positive Present in the nucleus of
transferase (TdT) immature lymphocytes
primarily lymphoblast
Stain for ALL
Important in identifying
blastic transformation of
CML
e.g. CML  TdT  ALL CML
 MPO  AML
Tartaric Acid Resistant Acid For hairy cell leukemia(HCL) Other tartrate resistant Activity is indicated by
Phosphatase (TRAP) cells: sezary cells, purple to dark-red granules
histiocytes, T cell of acute in cytoplasm
lymphocytic leukemia
ACP: 5 isoenzymes
1-4: inhibited by tartaric
acid  NEGATIVE
5: resistant to tartaric acid;
inside lymphocytes of HCL
 positive to TRAP
Cyanide- Resistant For identification of Eosinophils (+) Activity: brown
Peroxidase Eosinophilic components
Acid Phosphatase Present in all hematopoietic Activity is indicated by
cells and found in purple to red granules
lysosomes Unstable and cannot be
stored
NON-ENZYMATIC STAINS
Periodic acid-Schiff (PAS) Glycogen Variable, coarse, block-like It stains glycogen,
positivity often seen in mucoprotein,
lymphoblast and glycoproteins, and high
pronormoblasts, molecular weight
myeloblasts usually carbohydrates by
negative although faint combining with the
 HEMATOLOGY 2

diffuse reaction may aldehyde portion of the

occasionally be seen glycogen.

Positive in erythroblasts in
Di Guglielmo disease/
Erythroleukemia

(-) Activity: bright

fuschia pink (pattern
of staining varies with
each cell type)

Principle: positive in all

blood cells except normal
erythroblast but the
abnormal erythroblast in
M6 is positive in PAS.
For AML- M6 Identification

Gives a faint reaction with

lymphocytes in ALL;
Differentiates
ALL-L1 from ALL-L2 of ALL
 Both positive
L1: chunky or block-like
positivity of granules
Sudan Black B (SBB) Lipids and phospholipids Myeloblasts strong positive Differentiates ALL from
Monoblast faint positive ANLL
For AML identification
Gives the same
information as peroxidase
in the interpretation (+)
Result: dark purple-black
granules
Can be done on stored
specimens More sensitive
than chloroacetate in the
demonstration of mature
and immature neutrophils
and mast cells
Perl’s Prussian Blue Stains siderotic granules Use to detect hemosiderin
( for diagnosis of in urine
sideroblastic anemia)
Toluidine Blue O Binds with For recognition of basophils A metachromatic stain:
mucopolysaccharides in and mast cells gives different colors
blood cells (+) Result: Reddish Violet
 HEMATOLOGY 2
ACUTE LYMPHOCYTIC LEUKEMIA
 All is characterized by an uncontrolled growth of
abnormal lymphoid cells. It is primarily a disease
of childhood (young children) and adolescence,
accounting 15% of childhood cancers and up to
75% of childhood leukemia.
 Peak: 2-5 years of age
 Clinical Manifestations: The same with other
acute leukemias but onset (s/s) is more sudden.
o In general, leukemia is characterized by
anemia (decreased RBC), recurrent
infection and bleeding disorders
 Immunocompromised patients:
2 MAJOR CLASSIFICATIONS OF LYMPHOCYTES:
o Humoral Immunity: B cell ALL: signs
include lymphadenopathy.
Splenomegaly and hepatomegaly.
Eventual infiltration of malignant cells
into the meninges (lymphoblasts appear
in the CSF), testes, or ovaries.
o Cell-mediated Immunity: T cell ALL:
there may be a large mass in the
mediastinum (near ribs) leading to
compromised/abnormality of regional
anatomic structures. More common in
teenage males.
 Prognosis: depends on age at the time of
diagnosis, lymphoblast load/tumor burden,
immunophenotype, and genetic abnormalities.
o The younger (common) the age the
better prognosis. In adults  worse
prognosis
o Immunophenotype: type of ALL: (L1, L2,
L3).
 L3: most severe and have worst
prognosis.
 L1: better prognosis among all
the types.
o T-ALL: bad prognosis
o Worse outcome if the count of
lymphoblast is too high (greater than 20
to 30x109/L)  poor prognosis:
hepatosplenomegaly, and
lymphadenopathy all are associated
with worse outcome (uncommon in
AML)
o Secondary lymphoid organs: spleen and
lymph nodes  needs antigenic
stimulation.
o Primary lymphoid organs: bone marrow
and thymus.
 Common causes of death are infection (1st) and
bleeding (2nd).
 In children, the common causes of infections are
S. aureus, P. aeruginosa, C. albicans, H. influenza,
and P. mirabilis  parasites are not that
pathogenic but causes severe outcome
especially in immunocompromised individuals.
1. B cells: humoral immunity  produces
antibodies
2. T cells: cell-mediated immunity
NOTE:
Unlike neutrophils, it involves on defense but the
activity is PHAGOCYTOSIS.
TOTAL NORMAL WBC COUNT: 4,500 -11,000/ 4.5-
11.0x109/L Peripheral blood lymphoblast counts
greater than 20 to 30 x 109/L
Laboratory Findings:
 WBC count vary from low (<5.0 x 109/uL) to
high (>100 X 109/L)  poor prognosis; most
are lymphocyte.
 Most abundant WBC: Neutrophil (60-80%); 
lymphocyte (20-40%);  monocyte 
eosinophils  basophils
 In ALL, predominant cell is immature
lymphoblasts.
 BM is hypercellular/hyperplastic (red marrow;
has more hematopoietic cells uncontrolled
production of lymphocytes) and heavily
infiltrated with lymphoid cells.
NOTE:
ALL- most are lymphoblasts indicated by immature
amount of basophilic cytoplasm
AML – most are myeloblasts
Hard to differentiate: Immature cells: larger in size,
large nucleus with basophilic cytoplasm  importance
of cytochemical staining and CD markers
differentiation.
 HEMATOLOGY 2

mature
cells are
seen in this
type; negative
in CD34
T-ALL CD2, CD3, CD4, CD5, Rare and more
CD7, CD8, TdT common on
teenage
males.

WHO: Immunophenotyping

- Regardless of the classification: from CFU-L 

lymphoid antigens: CD34, TdT (nucleus of
lymphoblasts).
Further classification of B-ALL: Based on recurrent genetic
ALL IMMUNOPHENOTYP NOTES abnormalities: 7 types T-ALL not further classified
SUBTYPE E
Early CD34, CD19,
5% (less NOTE:
(pro/pre- cytoplasmic CD22,
common in CALLA: monoclonal COMMON ACUTE
pre) B-ALL TdT LYMPHOBLASTIC LEUKEMIA ANTIGEN: present on
children);
the leukemia cells of 70% of all patients; not present
11% (most
on normal peripheral lymphocytes but not specific
common in
for leukemia because it is also present on normal BM
adults)
cells that are positive for TdT and HLA-DR antigen;
Intermediat CD34, CD19, CD10, Common B-
only aids in identification of type II: intermediate B-
e (common) cytoplasmic CD22, ALL;
ALL
B-ALL TdT Common ALL FAB Classification of ALL: based on difference in
antigen morphology and cytochemical staining
(CALLA) are
Bases of FAB: Occurrence of individual cytologic
found  CD10
features (cell size; chromatin; nuclear shape, nucleoli,
has
degree of basophilia in the cytoplasm & presence of
relationship
cytoplasmic vacuolation); Degree of heterogeneity in
with CALLA
distribution among the leukemic cell population of
Pre B-ALL -CD34, CD19, 15%
some or all of those cytologic features
cytoplasmic CD22, (child);
Hand-mirror cells: lymphoblast that has
cytoplasmic m, 10%
(adults) contracted cytoplasm
TdT (variable)
 OIF= RBC  Neutrophils  Lymphocytes 
commo Monocytes  Eosinophils  Basophils
n in
children
less in
adults;
most
 HEMATOLOGY 2

Criteria L1 L2 L3
(Burkitt type)
Cell size Small, homogenous Large, heterogeneous (large Large; homogenous
and small)
Nucleus Regular with occasional Irregular; clefting/indentation Regular, oval to round
clefting; Rare with occasional is common
clefting
Nucleoli Rare/small, inconspicuous Present (often large) 1-3 (vesicular)
Chromatin 1-3 (vesicular) Variable Finely stippled
Cytoplasm Scanty Moderate Moderately; vacuolation of the
cytoplasm
Basophilia Moderate Variable Intense
Incidence 15 y/o and below Older than 15 y/o Rarest to occur; Burkitt lymphoma
(ALL in t(8;14)
general is
more
common in
young
children (2-
5 y/o)
Most common and has the best Rarest and has the worst or
prognosis poorest prognosis;

NOTE: ALL resulting to gene fusion (BCR-ABL gene). Ph1 is

First stage of life: Infants and newborns: AML
Young Children: ALL
Adults: CML
Older adults: CLL
Late/Last stage of life: Elderly: AML
CHRONIC MYELOPROLIFERATIVE DISORDERS
(CMPD)
- CMPDs are a group of acquired, malignant
disorders that develop from the proliferation of
an abnormal pluripotential stem cell.
1. CHRONIC MYELOGENOUS LEUKEMIA
(CML/CGL)
 CML is a stem cell disorder commonly affecting
adult. Some patients show a chromosomal
abnormality called the Ph1 chromosome
(formed by the translocation of long arm of
Chromosome 22 to long arm of Chromosome 9
present in around 90% of CML adults, while
infants and children <2 years of age are often
Ph1 (-).
 Also known as Chronic Granulocytic Leukemia
(CGL): most abundant cells is granulocytes
(neutrophils)  Disease of adults
 Philadelphia chromosome: Ph1 or Ph’  BCR-
ABL gene  CML; truncated Chromosome 22.
o Chromosome 9 is longer chromosome;
chromosome 22 is shorter  both have
p arm (short) and q arm (long)
o Chromosome 9 (ABL gene is detached;
became longer) reciprocal translocation
 Chromosome 22 (part is truncated in
BCR; but not detached; became shorter
due to truncation)  truncated
Chromosome 22 (Ph’): where new gene
is formed: BCR-ABL gene  cause
chronic leukemia  more oncogenic
effect.
 HEMATOLOGY 2
o Truncated chromosome 22: Ph1 has
BCR-ABL gene expressed as mRNA (has
genetic code); template for CHON
synthesis  DNA transcription 
production of p210 protein: influence
 chronic phase of leukemia 
excessive production of mature cells
(granulocytic cells)
NOTE:
Truncated chromosome 22: Ph1 has BCR-ABL gene 
expressed as mRNA (has genetic code); template for
CHON synthesis  DNA transcription  production of
p210 protein: influence  chronic phase of leukemia
 excessive production of mature cells (granulocytic
cells)
Imatinib mesylate: drug for CML which targets p210
protein  produced as an effect of newly produced
BCR-ABL gene  Dominated by granulocytes 
neutrophils
Clinical Features: Splenomegaly; Hepatomegaly; Bone
pain
LAB FINDINGS:
•WBC count: 50-600 X 109/L; ↓/0 LAP score
•Basophilia; Eosinophilia; Monocytosis
•Bone marrow: hypercellular with predominant
granulocytic cells (different stages of maturation);
more red marrow  more hematopoietic cells)
• ↓/0 LAP score:
o Differentiated with leukemoid reaction (LR):
transient; cells with normal function 
normal to high LAP score.
o CML: bone marrow defect; cells with
abnormality  0 to low LAP score
o Neutrophils (Tertiary granules): with highest
and only cell with LAP activity  LAP is also
called Neutrophil Alkaline Phosphatase (NAP)
• Increased granulocytes: Basophilia; Eosinophilia;
and Monocytosis
o PPSC  CFU-GEMM  CFU GM  CFU-G
(granulocyte) and CFU-M (monocyte)
• RBC and Plt. count decreased: Anemia and bleeding
disorder
• M:E = 10:1 or higher
o M:E– relationship between nucleated
granulocytic cells and precursor versus
precursors of erythrocytes
o In bone marrow, granulocytic cells is higher in
number than erythrocytes
o Normal ratio: 2-4:1
 Every 1 RBC there is 2-4 granulocytic
precursors
o In ALL, hypercullar BM but M:E ratio is not
affected or increased because what increases
are lymphoid cells
PROGNOSIS: Not all CML patients have Ph’
• Ph1 (+) patients have better prognosis than Ph1 (-)
• Ph1 (+) : mostly Adults  CML; prognosis is better.
• Ph1 (-) : Infants and <2 y/o  JMML or Monosomy 7
under MSD/MPD
o If you have abnormal chromosome the better
is your prognosis.
o Effect of presence of Ph1 in CML  better
prognosis
o If you have abnormal chromosome the better
is your prognosis
• High risk of transforming from chronic to blast 
“blast transformation” or “acute exacerbation blast
crisis” is indicated by an increase of >20% blasts in the
PB or BM.
o If myeloblast fails to mature  acute
leukemia; >20% blasts  can change in cell
line due to release of abnormal chemicals
and substances like cytokines that will
influence the differentiation of cell.
CML/CGL  ALL: TdT (+) AML: MPO (+)
 HEMATOLOGY 2

Acute Leukemia Chronic Leukemia NOTE:

>20% are blasts/ <20% blast; more mature  Later stage  BM infiltrated with fibrous
immature cells  cells tissues  resembles and become
abnormal indistinguishable with BM myelofibrosis.
Fail to mature  More mature cells o Hallmark mark: teardrop cells in
prone to destruction myelofibrosis and later stage of
of blasts cell primary polycythemia vera.
Unlikely to become If blasts are destroyed  Hyperviscosity syndrome  increased cells
chronic  Can’t revert before maturing  can but few plasma volume  flow slower than
back to chronic progress to Acute Leukemia usual  increased pressure (to compensate)
leukemia o The slower flow  thrombotic
Poor prognosis Better prognosis condition.
NOTE:
Management: Repeated phlebotomy
2. POLYCYTHEMIA VERA  Prevent hyperviscosity of blood
 This is characterized by pancytosis/panmyelosis  Removes blood: Blood removed can’t be
due to clonal proliferation with an increase in all transfused  abnormal blood
cellular BM elements, specifically the red cell  Every blood volume removed; iron (60% in hb)
mass (RCM). is loss  possible develop Iron Deficiency
 Laboratory: Anemia (IDA).
o Generally lower EPO level
o High RBC count (>10M)
NORMAL POLYCYTHEMIA VERA
▪ Normal RBC count: 3.8-5.2
HCT = PCV x 100 2: Relative polycythemia
Millions TV vera
▪ Increased in all RBC 1: 50/100 x 100= 50% Decreased in plasma
measurement volume  changing the
o High Hct (>55% in males; >47% in total volume
females) 50/75 x100 = 66.67% 
o High Hb: >18.5 g/dL (M); >16.5 g/dL (F) increased hct
o High LAP, WBC count: 40-50,000/uL In here, bone marrow is
o High Plt count (>1000 x 109/L); more not defective but the hct
than 1 million is increased due to
reaction with underlying
▪ Normal plt. count: 150-
condition
450,000/uL
o High Basophils

WHO CRITERIA FOR THE DIAGNOSIS OF PV 3: True or Primary

polycythemia vera
 Elevated hb> 18.5 (M), 16.5 (F) g/dL, or other Plasma volume is the
evidence of increased Red cell volume. same however the PCV is
 Presence of JAK2 V617F or similar mutation such increased.
as AK2 exon 12 mutation. 75/100 x 100 = 75% 
increased hct
o Janus kinase gene  mutated gene 
dictates abnormal
Increased PCV  due to
production/proliferation and mutation increased production in
of cells. the bone marrow
(defective BM)
 HEMATOLOGY 2

presence of
abnormal
hemoglobin

Inappropriate
response
Associated with some
kidney diseases

3. ANGIOGENIC MYELOID METAPLASIA/

IDIOPATHIC MYELOFIBROSIS/ MYELOFIBROSIS
WITH MYELOID METAPLASIA/ PRIMARY
MYELOFIBROSIS
- AMM is characterized by fibrosis,
megakaryocytic and granulocytic hyperplasia in
the bone marrow (marrow fibroblast).
- This is caused by an increase in defective
platelets as a result of dysplastic
megakaryocytopoiesis. These dysplastic cells
Polycythemia/Erythrocytosis: Characterized by an are prematurely destroyed resulting in the
INCREASED in HEMATOCRIT which may be due to: release of their a-granules that contain platelet-
1. A true increase in Red Cell Mass: PV/Primary derived growth factor (PDGF) which stimulates
polycythemia and secondary polycythemia fibrosis.
2. Just a decreased in plasma volume (relative o Fibrosis (marrow fibroblast)  caused
polycythemia) by dysplastic megakaryopoiesis 
DIFFERENTIATION abnormal megakaryocytes and platelets
Primary Secondary Relative  easily destroyed and lysed 
Polycythemia Polycythemia Polycythe released the contents of their granules
With bone No bone marrow mia
(alpha  PDGF (normally, promote
marrow defect defect No bone
vessel repair; abnormally, cause fibrous
marrow
defect tissue proliferation  scar formation
This is the Appropriate/compens Associated and dense)
myeloprolifera atory with a - Blood film shows predominance of teardrop
tive disease  Characterized decrease cells. Marrow fibrosis often result to “dry tap”
Absolute type by an increase in the during bone marrow aspiration.
Belongs to in the RCM plasma - Bone marrow failure to produce blood 
CMPD &Hct due to volume increased in fibrous tissue and depleted blood
some with no cells; what will produce are liver and spleen that
identifiable increase in has residual function for hematopoiesis
cause RCM & however there would also be fibrosis in these
 High in RCM is EPO organs especially in the spleen.
secondary to
- With increased blood cells and fibrous tissues
high in EPO
- EXTRAMEDULLARY FIBROSIS: fibrosis in the
 Hypoxia: can
be seen in liver and spleen
high altitude, - Middle aged to older people; rare to occur in
high O2 children
affinity and - Bone marrow: 2 normal tissues
 HEMATOLOGY 2

o Yellow: Marrow fats - Spontaneous aggregation even in the absence

o Red: Hematopoietic marrow of vessel injury  Megakaryocyte proliferation
- Blood film shows predominance of teardrop with large and mature morphology; no or little
cells. granulocyte or erythroid proliferation.
o PBS: teardrop cells  hallmark finding; - Platelet aggregation studies show platelets have
indicates the disease decreased/abnormal aggregation with
o Formation of teardrop cell can be epinephrine.
medullary (BM) and extramedullary - In vivo aggregation  platelets tend to obstruct
(SPLEEN) circulation
o Due to narrow circulation in the fibrotic o Two types of thrombosis:
BM and spleen  RBCs squeeze ▪ Arterial: involves platelets 
themselves  deform in order to pass arterial vasoocclusion by
through or enter the limited platelets aggregates 
circulation severe damage in cell decreased circulation and
membrane  retained the shape  oxygenation  called ischemia
TEARDROP CELL ▪ Venous: involves fibrin
- During bone marrow aspiration/core Biopsy 
University of Illinois needle targeting marrow o Arterial or venous thrombosis: 
sinusoids where bone marrow sample is Transient Ischemic Attacks (e.g.
aspirated. priapism (penis); digital ischemia
o Marrow fibrosis often result to “dry (peripheral tissues (digits: toes and
tap” during bone marrow aspiration. finger))
o DRY TAP: difficulty in aspirating that is ▪ More on arterial  decreased
very painful to the patient oxygenation
o Bone marrow should be spongy but due
▪ Toe ischemia: bluish coloration
to fibrous tissues infiltrated it becomes
on the toes
scarred/hard  hard aspiration.
▪ Priapism is a prolonged
erection of the penis. The
4. ESSENTIAL/PRIMARY THROMBOCYTOSIS
persistent erection continues
- ET characterized by thrombocytosis with
hours beyond or isn't caused by
spontaneous aggregation of abnormal platelets.
sexual stimulation. Priapism is
Platelet aggregation studies show platelets have
usually painful. Although
decreased aggregation with epinephrine.
priapism is an uncommon
- 2008 WHO criteria: platelet count of
condition overall, it occurs
>450x109/L. Megakaryocyte proliferation with
commonly in certain groups,
large and mature morphology; no or little
such as people who have sickle
granulocyte or erythroid proliferation; does not
cell anemia.
meet WHO criteria for CML, PV, IMF, MDS, or
▪ Priapus: minor fertility god in
other myeloid neoplasm; demonstration of
Greek mythology, who was also
JAK2 (V817F) mutation or other clonal marker,
the protector of livestock, fruit
No evidence of reactive thrombocytosis.
plants, and male genitals. He
- PRIMARY: main defect is production  bone
was depicted as having an
marrow defect  cancer of BM  excessively
oversized and permanent
produce thrombocytes
erection.
- SECONDARY: in reaction, also known as
reactive thrombocytosis
 HEMATOLOGY 2

NORMAL SMEAR ABNORMAL SMEAR (Essential thrombocytosis)

Platelet count: 8-20/OIF that

contains 200 RBCs.

ET/ PRIMARY THROMBOCYTOSIS POLYCYTHEMIA VERA

Increased platelets (ONLY) with NO increased in RCM
and other blood cells
JK mutation gene
PRIMARY THROMBOCYTOSIS
Platelet count often exceeds 1M/uL or >1000 x109/L
Not reactive: Increased in the production by defective
bone marrow  JAK2(V617F) mutation
With permanent damage, unless treated with bone
marrow transplant
Increased in all blood cells with prominent increased RCM
JK mutation gene
SECONDARY/REACTIVE THROMBOCYTOSIS
Platelet count is high more than 450,000/uL but rarely
exceeds 1M/uL or >1000 x 109/L
Platelet pool: 1/3 spleen and 2/3 circulation
Reactive: When 1/3 platelet in the spleen is mobilized into
the circulation  reactive thrombocytosis (transient)

Abnormally produced due to defective marrow  Normally produced (from spleen; just mobilized) 
Abnormal platelet function normal platelet function
 HEMATOLOGY 2
CHRONIC LYMPHOPROLIFERATIVE DISORDERS
1. CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
 CLL is characterized by abnormal proliferation
of lymphocyte that are unresponsive to
antigenic stimuli. CLL is less likely to undergo
blastic transformation but CLL patients are
predisposed to develop autoimmune hemolytic
anemia (AIHA)
o Less likely to undergo blastic
transformation
▪ does not become ALL or AML
o Dormant B-cells: unresponsive to Ag
stimulation and treatment
▪ B cells are stimulated by
antigens  transform into
plasma cells  produce
antibodies.
▪ Decreased IgG production: IgG
(2nd; produce at chronic phase,
but once produce it continues
 confers long term or lifetime
protection but in here, it is
decreased thus patients prone
in reinfection).
▪ Affected B-cells does not
produce normal IgG but
produces abnormal ones that is
directed against the patient’s
RBC leading to AIHA.
▪ Warm Autoimmune Hemolytic
Anemia (WAIHA)  IgG –
related: warm type
▪ Recurrent skin infection:
Herpes zoster or Shingles 
pruritus
▪ Chickenpox: initially appears as
papules on the skin and when
reoccurs it is now called herpes
zoster
▪ IgM (1st; peaks immediately
but declines rapidly  does not
confers lifetime protection; cold
type AIHA).
 PROGNOSIS: 3-4 years
NOTE:
CLINICAL SIGNS AND SYMPTOMS INCLUDE:
 Enlarged lymph nodes (lymphadenopathy),
spleen (splenomegaly), and liver
(hepatomegaly)  CLL will more likely show
these S/s over CML.
 Pruritus due to recurrent skin infection (often
with Herpes zoster).
LAB FINDINGS:
Lymphocyte count: 10-150 X 109/L  very high and
mature
 More on lymphocytes: thin rim cytoplasm;
morphologically normal but is fragile 
during smearing  smudge cell  CLL
▪ Differentiate smudge cells from
basket cell  nuclear remnants of
granulocytes; seen in chronic
granulocytic leukemia (CGL/CML)
▪ Smudge cells: formed IN-VITRO 
caused by manner of smearing; does
not seen in wet preparation.
 Lymphoblasts: larger and basophilic
cytoplasm
Many smudge cells on the blood smear
Hypogammaglobulinemia.
TREATMENT/MANAGEMENT:
1. Leukapheresis
 Apheresis: separation technique for
blood components  Whole blood:
PRBC, Plasma (PPP and Fresh plasma,
cryoprecipitate and cryosupernate),
Platelets (plateletpheresis), and WBC
through leukapheresis  separates
into Granulocytes and/or
lymphocytes
2. Regular injection with gamma-globulins (IgG) 
manage infection.
2. HAIRY CELL LEUKEMIA/ LEUKEMIC
RETICULOENDOTHELIOSIS
 HCL shoes presence of small lymphocytes with
many fine cytoplasmic extensions.
o It affects B-cells with presence of ACP 5
o Microscopically, cells have cytoplasmic
hair-like protrusions
 Prognosis is quite good (benign), and can be
longer than 10 years.
o Benign and has a good prognosis
 HEMATOLOGY 2
 Both CLL and HCL  more common in older
adults (>50 y/o) and more on males
 Pancytopenia  unique finding and important
feature of HCL.
NOTE:
 Clinical sign and symptoms: splenomegaly is
most consistent (splenectomy is often
considered).
 Laboratory diagnosis: Cytochemical stain 
TRAP (+)
o Isoenzyme 5 of ACP  unique
because it is resistant and not
inhibited by Tartaric acid  when
employed with ACP stain  ACP 1-4
(negative); ACP 5 (positive)
3. PRO-LYMPHOCYTIC LEUKEMIA
 CLL is characterized by an increase in
prolymphocytes, with almost total replacement
of the bone marrow. This has a poorer
prognosis than HCL and CLL.
 RARE BUT MOST SEVERE
 PROGNOSIS: poor (<1 year)  life expectancy
if left untreated.
NOTE:
LAB FINDINGS:
 HIGH LYMPHOCYTES WITH PREDOMINANCE
OF PROLYMPHOCYTES  less mature
o STAGES of lymphocyte maturation:
LYMPHOBLAST  PROLYMPHOCYTE
 LYMPHOCYTE
OTHER LYMPHOCYTE and PLASMA CELL NEOPLASMS
DIFFERENT STAGES OF A SINGLE NEOPLASTIC
DISORDER
 Both affects T-cells
o T-helper cell (lymphatic tissue)  skin:
MYCOSIS FUNGOIDES
o SKIN: affected T-cells will cause damage
in the epidermal layer  parakeratosis
and pautrier’s microabscess. Skin
become scale that causes eczematoidal
or fungal lesions.
o Histological finding: accumulation of
lymphocytes called  Pautrier’s
microabscess in epidermal biopsy
 SEZARY SYNDROME: Flow through lymphatic
circulation and reach lymph
o Blood smear: Sezary cells  lymphoid
cells with convoluted or irregular
nucleus
NOTE:
MYCOSIS FUNGOIDES
 This affects the mature T cells. This is an
early stage of Sezary syndrome and is
characterized by pruritus, eczematoidal
psoriasis form non-specific exfoliative
dermatitis. Diagnostic evaluation of the skin
reveals Pautrier’smicroabscesses (clusters of
lymphocytes forming on the skin)
accompanied by parakeratosis.
 EARLIER STAGE
SEZARY SYNDROME
 This stage manifests infiltration of the lymph
nodes and viscera, characterized by band-like
infiltrates of lymphocytes with cerebriform
nuclei called Sezary cells.
 ADVANCE STAGE
PLASMA CELL DYSCRASIAS
Paraproteinemia: MM and WM  malignancies
involving plasma cells.
 Excessive proliferation of plasma cells even in
the absence of antigenic stimulation 
excessive production of abnormal
immunoglobulins.
1. MULTIPLE MYELOMA
 This is the most common clonal proliferative
malignancy of plasma cells.
 Monoclonal gammopathies  abnormal cells
produce a single type of immunoglobulins.
 Of the cases, approximately 50% produce only
IgG; 20% only IgA; and 15% only the L-chain
o Plasma cells: 50% - only IgG; or 20% -
only IgA and/ or 15% - only L-chain 
interfere in platelet function
 Marrow infiltration often leads to bone pain.
o Myeloma cells: cancerous and leukemic
plasma cells that cause increased
 HEMATOLOGY 2
production of antibodies  tumor
formation in bone marrow  lytic bone
lesions  bone pain
NOTE:
Clinical sign and symptoms:
 Lytic bone lesions
 Hyperviscosity: due to abnormal proteins in
the plasma
o Causes hypertension and
Cardiovascular Disease (CVD)
 Bleeding disorders
o Platelets and endothelial cells are
coated with the abnormal proteins
 preventing the expression of
glycoproteins and other receptors 
abnormal adhesion and aggregation
Laboratory findings reveal:
o Chemistry: Bence Jones Protein (BJP)
o Myeloma cells may produce BJP 
part of immunoglobulin but
represent only the L-chain  Low
MW  easily passes through
glomerulus  seen in urine 
urinary screening
o Urine screening: unique solubility
characteristic  when heated at
certain temperature it can
precipitate and be dissolved
 Not heated or <40°C = clear/
soluble
 40-60°C = urine becomes
turbid due to precipitation
of protein; BJP  insoluble
 100°C = clear; BJP  soluble
o Urinalysis: many hyaline casts and other
casts and positive BJP
o Hematology:
o Elevated ESR: Rouleaux formation
 Because of the abnormal
proteins produced, zeta
potential of the RBC is
altered  rouleaux
formation (normally in vivo
RBC does not form into
rouleaux  repel each other
through zeta potential; RBC
has sialic acid in their
phospholipid layer that
imparts a negative charge)
ESR: 3 stages
 Initial Rouleaux: first 10 mins
 rouleaux  becomes
heavier
 Rapid Settling: succeeding 40
mins  larger sediment will
settle faster
 Final Settling: last 10 mins
o Hypercalcemia: due to abnormal
metabolism of calcium  bone
lesions and pain
BLOOD FILM FINDINGS
 Flame cells: plasma cell with red to pink
cytoplasm associated with increase in IgA
o Plasma cells with cytoplasm filled
with IgA
o Appear like a flickering candle
 Dutcher bodies: intranuclear crystalline
structures of abnormal IgG.
o Contains precipitated IgG in the
nucleus of the plasma cells
 Grape cell/Mott cell, Morula cell: plasma cell
that contains small colorless or blue/pink
protein vacuoles that appear like grapes
o Plasma cells whose cytoplasm are
filled with several protein globules 
russel bodies: are globules seen in
the cytoplasm containing proteins
and IgG  made the cell look like a
bundle of grapes
o Russel bodies: accumulations of IgG

 Rouleaux formation
2. WALDENSTROM MACROGLOBULINEMIA
 This is the second most common type of plasma
cell dyscrasia.
 Almost similar with MM
 Myeloma plasma cells are more involve in the
production of IgM.
o Malignant plasma cells show increase
production of IgM (>3 g/dL)
o IgM: pentamer; big
macroglobulinemia
 Also called Lymphoplasmacytic lymphoma 
can affect lymphocytes that are not transformed
into plasma cells.
 Hyperviscosity: due to macroglobulins
NOTE:
LAB FINDINGS:
o Increased IgM (>3 g/dL)
o Rouleaux formation  Increased ESR
o BJP can be produced
DIFFERENCE BETWEEN WM AND MM
1. WM: predominantly IgM
MM: predominantly IgG/IgA/L-chain
2. MM: affects B-cells and plasma cells precursors
WM: only plasma cells are affected
RARE CONDITIONS:
3. PLASMA CELL LEUKEMIA
 >2x109/L plasma cell count
4. HEAVY CHAIN DISEASE (HCD)
 This shows an abnormal synthesis of heavy
chains (Gamma HCD; Alpha HCD)
HEMATOLOGY 2
LYMPHOMAS
- Lymphomas are cancers of the lymph nodes
characterized by uninhibited growth of cellular
elements normally found in lymphatic tissues
resulting to lymph node enlargement.
o Lymph nodes are well-organized
because they are involved in the
immunity.
- Proliferative disease affecting some of the blood
forming cells and tissues outside the bone
marrow  lymph tissues: lymph nodes and
vessels
- May affect either B-cells or T –cells
1. NON-HODGKIN LYMPHOMA (NHL)
 A more common type than Hodgkin lymphoma.
It is a B-cell lymphoma characterized by painless
lymph node enlargement (cervical nodes are
 most often involved)
 Cervical nodes  GIT lymph nodes 
enlargement.
 Represent 60% of lymphoma cases
 CAUSES: congenital immunodeficiency diseases
and viral agents
o Since it is a cancer, mechanism isn’t
clearly defined.
 CLASSIFICATION:
o Low grade: enlargement is slow; can
take years for the enlargement of lymph
nodes to be diagnosed as one
o Intermediate grade: Rapid enlargement
o High grade: More rapid enlargement
2. BURKITT HODGKIN LYMPHOMA
 This is a B-cell neoplasm associated with EBV
and HIV infections. It is endemic among African
children (observed as jaw mass). Cytogenic
abnormality involves a translocation of C-myc
gene on chromosome regions (Ch8:14)
 One type of non-hodgkin lymphoma which can
be B-type or T-type  can affects both B-cells
and T-cells
 Lymphoma that when it infiltrates the bone
marrow  it becomes leukemic  ALL-L3
 ALL-L3 and Burkitt  related to EBV and HIV
 Problem: translocation of a gene between 8 and
14 (more common; written as t (8;14)) or 8 and
 HEMATOLOGY 2
2 or 8 and 22; the gene translocated is is C-myc
gene
o C-myc gene is a normal gene that both
regulates proliferation and apoptosis
but its translocation will cause it to be
abnormal thus it will become
overexpressed  abnormal function
 Prominent feature: rapid proliferation of
affected cell  rapid enlargement of lymph
nodes
 DIAGNOSIS: Lymph node biopsy: diffused
lymphoid proliferation (many lymphoid cells;
monocyte and macrophage: clears cellular
debris)  STARRY SKY.
NOTE:
WHO Classification:
Endemic: more common in African children 
enlarged jaw or with jaw mass or upper lymph nodes
Sporadic: more common in Unites States 
American  begins at GIT  enlargement of GIT
lymph nodes
Immunodeficiency: EBV and HIV
3. HODGKIN LYMPHOMA
 This is characterized by painless enlarged lymph
nodes usually in the neck and/or in some, the
cervical nodes that spreads in an orderly fashion.
o If enlargement is rapid (severe cases) 
usually is painful.
o Orderly fashion enlargement  it
follows lymphatic circulation
 More on B cell lymphoma
 Signs and symptoms include:
o GENERAL: fever, night sweats, or 10%
unexplained weight loss in 6 months, or
a combination of both.
 Diagnosis: lymph node biopsy  SPECIFIC
IDENTIFICATION
 Hallmark finding: REED-STERNBERG (R-S) CELL
or LACUNAR HISTIOCYTES
 Giant lymph cell that is usually 4-8x larger than
lymphocytes.
 Giant multi/binucleated: more common and
each half is identical to each other
o Nuclei  shows distinct nucleolus with
very prominent nuclear halo and distinct
nuclear envelope  giving owl-eyes
image
 When cut in half; almost identical  MIRROR
IMAGE CELL.
 When seen is pathognomonic of the disease
 CBC is not the appropriate procedure because it
is not a cancer of the bone marrow thus the
disease have a normal CBC.
 But in cases wherein the lymphoma becomes
leukemic; CBC can be included.
 LEUKEMIC PHASE OF LYMPHOMA: CBC 
Leukocytosis and lymphocytopenia are observed
in advance cases  poor prognosis.
TWO CRITERIA:
A. ANN HARBOR CLASSIFICATION OF HIGKIN
LYMPHOMA
 Region of RN (I-IV):
o Suffix A: no s/s
o Suffix B: with s/s
o Subscript E: indicates involvement of
extra lymphatic tissue
o Subscript S: indicates involvement of
the spleen.
 Stages:
o Stage I: only one lymph node region
affected
o Stage II: two or more lymph node
region affected but all located in one
part of the diaphragm either upper or
lower.
 HEMATOLOGY 2

o Stage III: two or more lymph node

region affected but are disseminated in
both parts of the diaphragm
o Stage IV: involvement of an extra
lymphatic tissue (spleen, liver or any
other organs)

B. RYE CLASSIFICATION (WHO)

 based on extent of lymphocyte infiltration and
the abundance of R-S cells
a. Lymphocyte Predominant: Abundant
Lymphocytes with normal and abnormal
forms; no fibrosis; with few R-SCs
b. Nodular Sclerosis: Moderate Lymphocytes
with nodulating collagen bands; R-SCs in
clear zones
c. Mixed Cellularity: Moderate lymphocytes in
diffuse pattern
d. Lymphocyte Depleted: few lymphocytes
with diffused fibrosis and many R-SCs

Stage Rye Histological Prognosis

Classification Features
I Lymphocyte Lymphocytes BEST
predominant and R-SCs
II Nodular Nodules of GOOD
sclerosis lymphoreticu
lar cells and
lacunar RSCs
III Mixed Mixture of FAIR
cellularity lymphocytes,
eosinophils,
plasma cells
and RSCs
IV Lymphocyte Lymphocytes POOR
depleted and RSCs
 HEMATOLOGY 2

FINALS o Proper position of patient during

phlebotomy: in comfortable position,
whether upright/sitting or lying in bed.
ERYTHROCYTE DISORDERS
o Sudden change in position:
 ERYTHROCYTIC DISORDER: affects RBC
Hemodilution: upright to lying: shift of
functions  oxygen transport as conducted by
fluid from extravascular to
its hemoglobin content thus affecting the oxygen
intravascular/circulation  decreased
delivery function of RBCs called ANEMIA.
RCM.
 ANEMIAS: are group of clinical conditions o Hemoconcentration: lying to upright:
associated with either a decrease in the number shift of fluid from intravascular to
of total red blood cells, a decrease in the
extravascular increased RCM (PV) 
concentration of hemoglobin, and/or a decrease REMEDY: wait for at least 20 minutes to
in the hematocrit (in any or all of the RCM) in
elapse for the fluid to equilibrate
relation to the individual’s age, gender and o IV fluid: hemodilution
geographic location.
o Anemia conditions can be absolute or CLINICAL SIGNS:
relative.
General signs and symptoms due to lack of oxygenation
o Decrease in RCM:
in tissues
 Hemoglobin (single lab test
important in screening anemia)  Easy fatigability and dyspnea on exertion  lack
 RBC count of oxygenation
 Hematocrit  Vertigo and faintness  drop in circulation thus
 Decrease oxygen delivery in less circulation and oxygenation in the brain
tissue preventing normal o Nervousness/faint  REMEDY: patient
cellular respiration. must lay down improve circulation in
 POLYCYTHEMIA VERA: all findings are opposite the brain
of anemia  increase in all  increase in RCM  Palpitation, rapid bounding pulse, systolic
(absolute PV) or decrease murmurs and headache o Due to lack
plasma/hemoconcentration (relative PV)  oxygenation  systolic murmur  heart
hematocrit (single lab test important in compensates
screening PV) o Brain demands large amount of oxygen
therefore the heart compensates by
TYPES:
redistributing the circulation of blood
 ABSOLUTE: True decrease in RCM  Decreased through transporting more oxygen to
in production (bone marrow defect that does not the brain causing an increase in cardiac
release a normal RBCs); increased loss output.
(hemorrhagic); increased destruction o Compensation of heart: increase cardiac
(hemolytic) output  increase pressure
o Normal plasma volume but PRBCs or o Low Blood Pressure due to decrease
PCV is decreased blood output causing increase cardiac
 RELATIVE: related to volume of plasma: plasma output causing increase pressure
expansion  hemodilution: shift of fluid from leading to headache  Pallor (lacks
extravascular to intravascular; false hemoglobin  heme: gives the normal
interpretation: decreased in cell count, hb and red pigment of blood)
hct.  low BP (lack of blood)
o Specimen collection: Very important to  Slight fever (especially when coexistent with an
consider infection) and dependent edema
 HEMATOLOGY 2
SPECIFIC CLINCAL SIGNS
JAUNDICE: yellow discoloration of the LEG ULCERS: open wounds most
skin, eyes, nail bed due to commonly seen lower extremities
accumulation of unconjugated because it is affected by thrombosis
bilirubin (persist in circulation(yellow  in sickle cell anemia  rigid cells
plasma); water insoluble and bound  hyperviscosity of blood leading to
to albumin; not excreted in the urine) thrombosis
 specific sign seen in hemolytic
anemia In normal condition: RBCs are containing CHON (myoglobin and
discocyte, flexible and deformable  cytochrome) and tissues  heme
pass through small vessels.
In sickle cell anemia, rigid cells and color of blood and flesh.
not deformable therefore they
obstruct the circulation leading to
hypervicosity of blood  thrombosis
 vasoocclusion decrease in
oxygenation  delay wound healing
FRONTAL BOSSING: seen in NEUROLOGIC DEFICIT: seen in
Thalassemias: Betathalassemia major Vitamin B12 deficiency  decrease
(more common) and DNA production leading to
hemoglobinopathies megaloblastic anemia (affects blood
cell maturation) and destruction of
Cause: Skull (flat bone) that is active in CNS tissues due to inefficient myelin
hematopoiesis. In B-thalassemia sheath  severe cases: mood swings
major: no hemoglobin(A1) which is and change in behavior 
the major hemoglobin in adults so the Megaloblastic madness
blood production tries to compensate
 red marrow expansion inside the Vit. B12: important and serve as a
flat bone producing more blood cells cofactor in many metabolic reaction:
 bone structure also expands  DNA synthesis and maintenance of
skeletal deformity myelin sheath of the brain and spinal
B-THALASSEMIA: manifest mongoloid cord or CNS tissues
features due to skeletal deformity
KOILONYCHIAS: spooning of nail bed
 seen in Iron
Deficiency Anemia
IDA  hookworm infections
Iron is very much needed by the body:
hemoglobin and other heme
contains iron that give offs normal
CYANOSIS: seen in
methemoglobinemia  increase in
abnormal hemoglobin: hemoglobin
Hemoglobin “M”s  methemoglobin:
hb that is not protected from
oxidization  easily oxidized (ferrous
to ferric form)  Hb M Boston, Hb M
Iwate, Hb M Hyde park, Hb M
Saskatoon, Hb M Milwaukee, Hb M
Osaka and Hb M Fort Ripley.
Methemoglobin is normal in blood
but in small amounts: >3.5% but if
increased it becomes abnormal
because it cannot transport oxygen
especially in distal tissue like fingers.
 HEMATOLOGY 2
COMPENSATORY MECHANISMS
PHYSIOLOGIC RESPONSE
Immediately occur
. Shift to the right of the Oxygen Dissociation Curve
Y-axis = oxygen saturation; increase affinity of hb to O2
X-axis = partial pressure of oxygen; decrease affinity of
hb to O2
2.Increase 2,3 DPG shift to right in ODC and more
oxygen will be released towards the tissue
TWO FORMS OF HEMOGLOBIN BASED ON
OXYGENATION:
1. Oxyhemoglobin – “R” relax form; No 2,3 DPG;
fully oxygenated  increased affinity to
oxygen; but once in tissue there will be
decreased in the affinity 2,3 DPG inserts in the
hb
2. Deoxyhemoglobin – “T” tense form; with 2,3
DPG that lowers the hb affinity to oxygen
release oxygen in the tissue
3.Selective redistribution of blood flow to areas of
highest oxygen demand like in the brain
4.Increased cardiac output
RETICULOCYTE
 Immature cell
 Normal count: Adults: 0.5-1.5% (average: 1%
Retics)
HEMATOLOGIC RESPONSE
Take time to occur
Erythropoiesis: 3-5 days (from pronormoblast to
reticulocyte)
 Normally: 3.5 days will be spent in BM and 1 day
in the circulation mature RBC
 Anemia: reticulocyte will be released in the
circulation less than its normal (<3 days); if the
number of days in the BM is shorten, the longer
the number of days the reticulocyte
will take in the circulation
Increase in erythropoietin (EPO)
 Increase erythroid precursors
 Accelerates rate of proliferation and maturation
 Accelerate release from the BM to the PB
Increase shift cells/stress reticulocyte
SHIFT/ STRESS CELLS/ RETICS
 Prematurely released
 More immature than reticulocyte
 Increased RNA
 HEMATOLOGY 2
o Maintains 1% retics everyday
o In anemia, prolonged in the circulation
 increased retics count; and if they are
shift (more immature more RNA)
Romanowky stain – polychromatic
MAJOR CAUSES OF ANEMIA
1. GENETIC FACTORS
- Abnormalities in the structure of some
molecules present in the RBC.
a. Hemoglobin C, S: can’t function well in
delivering oxygen leading to anemia.
b. Cell membrane: abnormal spectrin and
ankyrin due to abnormal gene  RBC easily
deformed  spherocytes/elliptocytes)
c. Enzymes: affects metabolism of RBC (like
G6PD deficiency  needed in hexose
monophosphate shunt )
2. ENVIRONMENT FACTORS
- Factors that interfere with the normal
development and functioning of RBCs.
a. Lead exposure may lead to Sideroblastic
Anemia (SDA); lead interfere in heme
synthesis affecting the utilization of iron 
iron cannot be utilized therefore precipitate
as siderotic granules
b. BENZENE – laboratory reagent that exposure
may lead to developing aplastic anemia;
toxic and damages BM thus preventing
production of blood cells
c. Failure to acquire nutrients, vitamins,
minerals (e.g. from vegetables) 
Nutritional Deficiency Anemia: Iron
deficiency (IDA), Lacks dietary vegetable and
meat (poultry product)  B12 and Folate
Deficiency
3. PATHOLOGICAL FACTORS
- infectious organisms and parasites
a. PARASITES
o In anemia, prolonged in the circulation 
increased retics count; and if they are shift
(more immature more RNA)
Supravital stain – bluish granules More granules  shift cells
o D. latum - tapeworm that lives in ileum;
interferes in the absorption of Vit. B12
anemia leading to Vit. B12 deficiency
anemia or fish tapeworm anemia; often
referred as bothricephalus pernicious
anemia
o Hookworms – Ancylostoma duodenale
and Necator americanus: blood sucking
nematodes that attaches in the mucosal
of the small intestine through their
buccal cavity. As they move to one area
to another, the area of attachment will
be left behind as chronic/minute
bleeding area and mechanical sucking it
will cause blood loss (hb loss  iron
loss especially chronic infection with
heavy worm burden  IDA)
o Malaria/Plasmodium spp. and Babesia
– live inside the RBC  ruptures and
destroy RBCs hemolysis  hemolytic
anemia
4. IATROGENIC FACTORS
- These are results of side-effects of legitimate
medical treatment/procedures that may
interfere with the normal development and
function of RBC
a. Prolonged use of antibiotics:
Chlorampenicol – destroys BM leading to
development of aplastic anemia b. Surgery
– hemorrhagic/blood loss anemia
 HEMATOLOGY 2

LABORATORY DIAGNOSIS NOTE:

- To screen for the presence of anemia  Reference Value:
performs CBC  Normocytic: 80-100 fL
1. HEMOGLOBIN (ANEMIA) AND HEMATOCROT  Microcytic: <80 fL
(PCV)  Macrocytic: >100 fL
 Single test to screen anemia: Hemoglobin  if FORMULA:
low, proceed with stated test below. 𝑯𝒄𝒕 (𝑳/𝑳) × 𝟏𝟎𝟎 𝑯𝒄𝒕 (%) × 𝟏𝟎
𝒐𝒓
𝑹𝑩𝑪 (𝟏𝟎𝟏𝟐 /𝑳) 𝑹𝑩𝑪 (𝟏𝟎𝟏𝟐 /𝑳)
NOTE:
HEMOGLOBIN
 Reference Value:  MCHC: refers to average Hb concentration of
FORMULA: red cells in a given volume of blood
𝑨 𝒐𝒇 𝒕𝒆𝒔𝒕 (𝒖𝒏𝒌𝒏𝒐𝒘𝒏)
NOTE:
𝑨 𝒐𝒇 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅
Reference Value:
× 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅 𝒄𝒐𝒏𝒄. (𝟏𝟓𝒈𝒎/𝑳)
= 𝒈/𝑳 𝒐𝒓 𝒈/𝒅𝑳 (𝒘𝒊𝒕𝒉 𝒅𝒆𝒎𝒊𝒄𝒂𝒍)  Normochromic: 320-360 g/L or 32-36% or 32-
HEMATOCRIT 26 g/dL
 Reference Value:  Hypochromic: <32%
 Male: 140-175 g/L  Spherocytic  full of Hb: >36%
 Female: 123-153 g/L FORMULA:
𝑯𝒈𝑩 (𝒈/𝒅𝑳) 𝑯𝒈𝑩 (𝒈/𝒅𝑳) × 𝟏𝟎𝟎
 At birth: 150-200 g/L 𝒐𝒓
FORMULA 𝑯𝒄𝒕 (𝑳/𝑳) 𝑯𝒄𝒕 (%)
1. INDIRECT:
𝑯𝒄𝒕 = 𝑷𝑪𝑽 × 𝑹𝑩𝑪 𝑪𝒐𝒖𝒏𝒕
3. RETICULOCYTE COUNT; RETICULOCYTE
2. DIRECT
𝑯𝒕 𝒐𝒇 𝑷𝑪𝑽 × 𝟏𝟎𝟎 PRODUCTION INDEX (RPI) OR SHIFT
= 𝑽𝒐𝒍. 𝒐𝒇 %𝑯𝒄𝒕 CORRECTION
𝑯𝒕 𝒐𝒇 𝒘𝒉𝒐𝒍𝒆 𝒃𝒍𝒐𝒐𝒅 (𝑻𝑽)
𝒐𝒓  Compensatory mechanism of BM is to increase
𝑷𝑪𝑽 its reticulocyte production  increased
= 𝑳/𝑳 (𝒅𝒆𝒄𝒊𝒎𝒂𝒍)
𝑻𝑽 reticulocyte count.
 RPI: to measure whether the increase in
production is parallel or compensatory to the
2. BLOOD INDICES: MCV, MCHC, RDW
degree of anemia
 For morphological classification of anemia
o Index calculated to correct for the
 RDW: represents the ratio of the standard
presence of shift reticulocytes that
deviation to the MCV )Width of histogram)
otherwise may falsely elevate the visual
o Index of relative anisocytosis; may also
reticulocyte count
indicate poikilocytosis
o Increased in hemolytic and hemorrhagic
REFERENCE VALUE: anemia
 11.6- 14.6% o Reference value: 1 if hct is 0.45
 >14.6% indices variation in sizes
NOTE:
RETICULOCYTE COUNT:
 MCV: refers to the average volume of PCV of the  Reference value: 0.5 -1.5%  average: 1%
individual red cells in a given blood samples.
o It is used as an estimation of the average RETICULOCYTE PRODUCTION INDEX (RPI)
size of the RBC  Reference value: 1 if hct is 0.45
𝑪𝒐𝒓𝒓𝒆𝒄𝒕𝒆𝒅 𝒓𝒆𝒕𝒊𝒄𝒖𝒍𝒐𝒄𝒚𝒕𝒆 𝒄𝒐𝒖𝒏𝒕
o No dependable when RBC vary markedly  FORMULA:
𝑴𝒂𝒕𝒖𝒓𝒂𝒕𝒊𝒐𝒏 𝒕𝒊𝒎𝒆 (𝒅𝒂𝒚𝒔)
in size.
 HEMATOLOGY 2

4. MYELOID: ERYTHORID RATIO B. ANEMIA ASSOCIATED WITH DEFECTIVE HGB

 Ratio between granulocytes and erythrocytes SYNTHESIS
precursor inside the BM  Defect in globin or heme synthesis
 Normal ratio: 3:1  1 RBC precursor: 3  Heme: IDA, SDA
Granulocytic precursors  Globin: thalassemia
 CGL/CML ratio: >10:1  M:E ratio increases
 Anemia: 3:3  1:1  M:E ratio decreases C. ANEMIA WITH VITAMIN B12 OR FOLATE
DEFICIENCY
5. OTHER SPECIAL TEST DIAGNOSTIC OF THE  Nutritional deficiency anemia: IDA, B12 or
SPECIFIC TYPE OF ANEMIA folate deficiency anemia
 AIHA: serologic test: antibody testing to
indentify the specific autoantibody D. ANEMIA ASSOCIATED WITH IMPAIRED BONE
 Pernicious anemia: Schilling’s test MARROW OR STEM CELL FUNCTION
 Aplastic anemia; bone marrow is infiltrated with
CLASSIFICATION OF ANEMIA fats thus reduced or no red marrow that is the
- Some anemia have more than 1 pathologic
active marrow producing blood cells.
mechanism (e.g. B12 deficiency  tapeworm
infection + nutritional deficiency + antibody) and
E. ANEMIAS ASSOCIATED WUTH DECREASED RED
go through more than one morphologic stage
CELL SIRVIVAL AND INCREASED RED CELL
(IDA: microcytic-hypochromic; normocytic-
DESTRUCTION
hypochromic)
 Prone to damage: hemolytic anemia
1. MORPHOLOGIC CLASSIFICATION
2. ETIOLOGIC CLASSIFICATION
F. ANEMIA SECONDARY TO BLOOD LOSS
3. PHYSIOLOGIC CLASSIFICATION
 Hemorrhagic anemia:
 The ability of the bone marrow to respond
o Chronic blood loss
physiologically by producing erythrocytes is
o Acute blood loss iii. Post-acute
measured by RPI  Ineffective
hemorrhagic anemia  IDA
erythropoiesis (RPI <2.0)
 Effective erythropoiesis (RPI >3.0)  e.g. RPI= 4
means that the rate of production has increased
4x than its usual for compensation

NOTE:
Some anemia have more than 1 pathologic
mechanism & go through more than one morphologic
stage.

ETIOLOGIC CLASSIFICATION OF ANEMIAS: CAUSE OF

ANEMIA
A. RELATIVE ANEMIA
 Increase in plasma volume  hemodilution
 RBCs are NOT reduced but is diluted with
increase plasma volume
 TRUE/ABSOLUTE ANEMIA: reduced in number
of RBC due to defective production
 HEMATOLOGY 2

MORPHOLOGIC CLASSIFICATIONS OF ANEMIA

- Usually followed
- Basis: size and hemoglobin

MCV: according to size
<80 fL = microcytic
80-100 fL = normocytic
>100 fL = macrocytic
MCHC: hemoglobin concentration RDW: a measure of the degree of
MCH is not helpful in morphological anisocytosis and poikilocytosis  used
classification of anemia because it always because MCV and MCHC cannot be rely
follow the MCHC upon when the cell population is diverse.
<32% = hypochromic
32-36% = normochromic machine gives its value from histogram
>36% = spherocytic
According to MCV and MCHC: single
population
3 CLASSIFICATION OF ANEMIA
1. Normocytic – normochromic
2. Microcytic – hypochromic
3. Macrocytic – normochromic
4. Normocytic - hypochromic  gradual
changes seen in IDA

MCV: Single red cell index that can be used to CBC  anemia  retic count  morphologic classification
morphologically classify anemia; will suffice alone 
microcytic; macrocytic; normocytic

MICROCYTIC: the most common anemias included are

those
characterized by defect in hb: heme: SDA, IDA; globin: ACI,
Thalassemia  in general thalassemia fall under
microcytic anemia

GLOBIN DEFECTS: deficiency in alpha and beta chain

synthesis: thalassemia; defect in amino acid
structure/composition: hemoglobinopathies  fall under
normocytic anemia (Hb C and S) except Hb E disease or
trait.
 HEMATOLOGY 2

MACROCYTIC: include problems in DNA synthesis 

megaloblastic anemia

NORMOCYTIC: more on destruction (hemolytic) and

decrease in production (aplastic: BM failure)

MICROCYTIC HYPOCHROMIC MACROCYTIC HYPOCHROMIC NORMOCYTIC NORMOCHROMIC

Main cause: defective hb synthesis Commonly Macrocytic Commonly Normocytic
Heme synthesis: IDA, SDA, ACI Globin  Vitamin B12 deficiency  Hypoproliferative anemia
synthesis: alpha or beta thalassemia and anemia  Folic acid  Myelophthisic anemia
Hb E disease deficiency anemia  Hemolytic anemia
 Hemoglobinopathies
Commonly Microcytic Occasionally Macrocytic  Acute blood loss anemia
 Iron deficiency anemia  Hypoproliferative anemia  Anemia of chronic disease
 Sideroblastic anemia   Refractory anemia  Renal and endocrine
Thalassemia  Liver disease diseases
 Hemolytic anemia  Paroxysmal nocturnal
Occasionally Microcytic  Acute blood loss anemia hemoglobinuria
 Anemia of chronic
disease/inflammation Occasionally Normocytic
 Hemoglobinophaties (Hb E  Early iron deficiency
disease  Refractory anemia
The cells will keep on dividing until their optimum MCHC is
reached. In normal erythropoiesis, it takes only 4-5
divisions for mitosis  Pronormoblast will divide once
producing 2 basophilic normoblast (BN) and these BN will
divide twice and the last to go division is the rubicyte  4x
division = 16 ; 5x division = 32

Every time the cell divides the cell size becomes smaller

In microcytic-hypochromic, since due to decrease hb, it

will not reach its optimum MCHC therefore it will keeps on
dividing thus decrease in cell size Microcytic: extra division
Hypochromic: decrease Hb.
 HEMATOLOGY 2
MICROCYTIC- HYPOCHROMIC ANEMIAS
 ETIOLOGY: Microcytosis is caused by one or
more extra cell division due to the depression of
the MCHC. This conditions also exhibits
hypochromia which is cause by an impairment in
the hemoglobin synthesis due to abnormalities
in either the heme or globin synthesis or both.
1. IRON DEFICIENCY ANEMIA (IDA)
 Iron Metabolism: Iron is absorbed in the ferrous
form, later oxidized in the mucosal cells to the
ferric form. Absorbed iron passed on directly to
the blood stream by the mucosal cells, but most
of it is bound to Apoferritin, which then forms
Ferritin. Iron in the bloodstream is transported
by Transferrin which delivers it to the
mononuclear phagocytic cells (MPC) of the
bone marrow and other issues. During
erythropoiesis, the MPC of the bone marrow
incorporated iron into the developing red cell.
 NORMAL BODY IRON (FE): 4,000 mg
o 3 COMPARTMENTS: iron is distributed
among these compartments and sub-
compartments.
 STORAGE: in the form of ferritin
(major form in the liver and
tissue) and hemosiderin
 TRANSPORT: protein
transporter for iron
 FUNCTIONAL: hemoglobin,
myoglobin, iron-containing
enzyme. Most of the iron is in
the hemoglobin approximately
60%; thus deficiency in iron will
affect hemoglobin resulting in
anemia.
 IRON KINETICS:
o Fe++ & Fe+++ (diet)  Fe++ (stomach)
 Fe+++ (Mucosal cells)
 What the body can absorb is
only the ferrous form; the ferric
form can be reduced into
ferrous form in the stomach by
our gastric juices (very acidic
due to HCl) to promote
absorption, when the ferrous
form of iron reaches the small
intestine (particularly in
duodenum and jejunum) they
will be absorbed by mucosal
cells and immediately cause
oxidation of ferrous thus
converting back into ferric form.
The ferric form will be released
into the plasma circulation by
the mucosal cells.
o Fe+++  bloodstream
 Fe binds with apoferritin to form
FERRITIN (major storage form of
iron  stored in tissues and
liver).
 Fe-transferrin  transported to
Mononuclear Phagocytic Cell
(MPC) going into the bone
marrow and liver.
NOTE:
 In bone marrow, during erythropoiesis, when
the developing red cell start to synthesize hb,
mononuclear cell will transport and
incorporate iron into developing RBC 
incorporation is called ROPHEOCYTOSIS
 ROPHEOCYTOSIS: During hemoglobin
synthesis in immature red cell, mononuclear
cell will incorporate the iron thus iron (Fe+++)
will be released and immediately reduced into
ferrous (Fe++) and incorporate itself into
protoporphyrin IX to form heme.
ETIOLOGY/ RELATED CAUSES: IDA
1. CHRONIC BLOOD LOSS
 Most common cause of IDA in adults.
 HEMATOLOGY 2
 Small volume but chronic
 Male: GIT bleeding  unnoticed because it
occurs inside unless it develops into other
serious complications.
 Female: prolonged menstruation, bleeding
after childbirth or cesarean
 ACUTE BLOOD LOSS will NOT lead to IDA
STAGES IN THE DEVELOPMENT OF IDA
2. INCREASE DEMAND
 especially among pregnant women (30-
75mg), adolescents and infants
 Not pregnant female: 10-20 mg thus female
needs more iron due to monthly blood loss
 Pregnant: ferrous sulfate supplement to
prevent IDA
 Adolescent: fast growing  more iron needed
by the body
 Infants: breastmilk – poor in iron  pure
breastfed infants may develop IDA called
“milk anemia of infancy”  Male has less iron
demand because they do not have monthly
menstruation/blood loss
3. MALABSORPTION
 Absorption of iron happen in small intestine
(duodenum and jejunum).
 Regardless of the amount of iron take in, the
absorption is limited to 10% of dietary iron
 Celiac disease (sprue): disorder characterized
by antibody destruction of the small intestines
 destroyed absorption sites  total
gastrectomy: decreased absorption sites
 Intestinal Parasites:
o N. americanus: 0.03 ml/day  per
worm
o A. duodenale: 0.15 – 0.25 ml/day 
per worm
o T. trichiura: 0.005 ml/day  per
worm
4. POOR DIET
5. FREQUENT BLOOD DONATION (225 gm/unit)
 450 ml: blood donated
PROMOTE ABSORPTION REDUCE ABSORPTION
Ferrous form Ferric form
Inorganic form Organic form
Acids: HCl, Vitamin C Alkali: Antacids
Pancreatic secretions
Solubilizing agents: Precipitating agents:
sugars and amino acids phosphates
Iron deficiency Iron excess
Increased erythropoiesis Decreased erythropoiesis
infection
 STAGE I: Iron depletion stage: Iron is mobilized
from stores, storage iron decreases, plasma
ferritin decreases, iron absorption increases.
o First to decrease is iron storage
compartment which is measured by
transferrin13
o Serum ferritin first decreases and
reflects the iron storage or ferritin
o Heme is consisting of iron and
protoporphyrin IX. Deficiency in iron
would lead to increase in
protoporphyrin IX and will accumulate
inside the RBC which is measured as Free
Erythrocyte Porphyrin  FEP is increase
o Since functional iron is still normal then
hemoglobin and hematocrit are normal
thus, it is characterized as normocytic
and normochromic
o Normal serum iron and TIBC
 STAGE II: Iron deficient erythropoiesis: after
iron stores are depleted, the plasma iron
concentration falls, saturation of transferrin falls
below 15% and the percentage of sideroblasts
decreases in the marrow.
o Iron storage (ferritin) and transport iron
is depleted
o Decrease in serum iron with increase
FEP
o Increase TIBC: indirect measurement of
transferrin
o Transferrin: transport two molecules of
iron simultaneously
 When transferrin is bound into
iron because iron is normal, it is
fully saturated thus it will no
longer bind with additional iron.
In the absence of iron (IDA),
transferrin remains to be free.
 HEMATOLOGY 2
When added with the reagent
iron, it will bind with iron.
 Transferrin when it is not
saturated, its binding capacity
for iron is increased measured
by TIBC.
o Functional iron is still normal thus the
anemia is normocytic – normochromic
 When the cells are in the
circulation, they are fully
hemoglobinized and in the
presence of IDA, the mature RBC
will not be affected because the
mature RBC have fully
synthesized their hemoglobin.
 IDA affects the developing RBC
in the bone marrow wherein the
bone marrow will immediately
suffer in decreased in iron
because of the storage form of
iron.
 Aside from the liver and tissues
having the storage form, there is
also in the bone marrow which
will be first depleted.
 STAGE III: Iron deficiency anemia: anemia is now
detectable. The anemia is at first normochromic
acid normocytic, gradually becomes microcytic
IRON PROFILE TESTS FOR THE DIFFERENTIAL DIAGNOSIS
and finally microcytic & hypochromic.
OF IRON DEFICIENCY ANEMIA
o Iron deficient red cells from the bone
marrow will be released and will appear
as microcytic as reflected by the low
hemoglobin (hypochromic).
o Findings: decrease hemoglobin, serum
iron, ferritin ; increase TIBC
LABORATORY FINDINGS OF IDA
- In early IDA, RBCs are N/N; in later stages, RBCs
become Microcytic-Hypochromic. Severe cases
will exhibit poikilocytosis & anisocytosis 
Reticulocyte count is low; Platelet count is
slightly elevated
- Bone marrow smear is negative for hemosiderin
granules
- Serum iron is decreased, and serum ferritin may
be reduced to zero; increase Free Erythrocyte
Protoporphyrin (FEP); increase Total Iron-
Binding Capacity (TIBC)
- General blood picture: microcytic-hypochromic
with poikilocytosis and anisocytosis and elevated
RDW.
o RDW: measure in the variation of size
and shape
- Reticulocytopenia: Even though the red marrow
tries to compensate, the red cells are
ineffectively growing therefore
reticulocytopenia.
- Slight thrombocytosis: If it is associated with
bleeding because bleeding is also one of the
causes of IDA, chronic bleeding (most common
cause of IDA) especially in adults, it may also be
characterized by slight thrombosis.
- Prussian blue staining: hemosiderin granules are
negative.
 SERUM FERRITIN
o Reflects the body’s tissue iron stores
o Decrease in iron causes increase
protoporphyrin 9. Though normally, RBC
produces more protoporphyrin than
iron, but in the absence of iron, the more
the protoporphyrin will accumulate
causing increase FEP. It is free because it
is supposed to bind with iron but since
there is no iron, it remains to be free
erythrocyte protoporphyrin. Some
measured it as ZEP because the FEP can
combine with zinc and measured as zinc
erythrocyte protoporphyrin
o Reference value: 12-300 ug/dL
 SERUM IRON
 HEMATOLOGY 2
o A sensitive indicator or iron depletion
but has a marked diurnal variation by as
much as 30% with its highest values in
the morning and lowest values late in
the day.
o Reference value: 50-160 ug/dL
o Decreased in IDA
 FREE ERYTHROCYTE PROTOPORPHYRIN (FEP)
o Normally, red cells produce slightly more
protoporphyrin than what is needed, but
when iron is deficient, protoporphyrin
build up in the erythrocytes
o Reference value: 10-99 ug/dL
o Increased in IDA; decreased in most
SDAs.
 TOTAL IRON BINDING CAPACITY (TIBC)
o Binding capacity: measure the ability of
transferrin to bind with iron. Wherein, if
tranferrin is not bound to iron, it will
bind. But if the transferrin is already
saturated with iron, it can not further
bind with other iron (in the reagent).
o Wherein in normal iron status, iron is
plenty thus transferrin carry two iron
molecules.
o In IDA, iron is depleted thus they carry
no iron thus transferring is free. When
the reagent (iron) is added, the
transferrin can bind with it because TIBC
is increase.
o Reference value: 250-400 ug/dL
o Increased in IDA
 TRANFERRIN SATURATION/ PERCENT
SATURATION OF TIBC
o Measures how transferrin is saturated;
percent saturation
o Reference value: 20-55%
o Decreased in IDA
NOTE:
Since iron is not only for hemoglobin synthesis but also
important for the tissues, enzymes, myoglobin and
other proteins, these tissues and proteins will also be
damaged due to IDA.
CLINICAL FEATURES OF IDA
 TONGUE- ATROPHY: The tongue is red because
of iron in heme present in hemoglobin in blood
and myoglobin in tissues, but since it lacks iron,
it will atrophy (become smaller).
 ANGULAR STOMATITIS: the lips will get dry and
have cracks
 CHEILITIS: the drying and inflammation of the
lips; associated with IDA
 “Webs” of tissue or partial strictures at the
junction of the esophagus and hypopharynx:
there will be cracks and wounds inside 
resulting now to difficulty in swallowing; affects
entire GIT.
 CHRONIC GASTRITIS – decreased gastric fluid
secretion which is important because it is the
gastric juice that is acidic which helps in the
absorption of iron. The acidity of gastric juices
helps reduce the ferric into ferrous in order to
absorb the dietary iron even in the ferric form.
 NUMBNESS & TINGLING SENSATION: peripheral
tissues nerves will be affected especially the
distal parts of the body (toes, fingers, and feet).
 KOILONYCHIA: distortion of the nail bed, spoon
shaped of the nails due to lack of iron in the
tissues
 PICA: unusual and compulsive appetite for a
certain kind of food or non-food item; among
children with severe IDA wherein they even have
abnormal craving for unusual food.
 SPLENOMEGALY: spleen function to filter blood
and remove a lot of abnormal cells so it will be
overworked and abnormal cells will clog in the
splenic circulation resulting enlargement of
spleen.
NOTE:
Aside from the general manifestations of anemia,
other manifestations include paresthesia, pica,
koilonychia, atrophy of epithelium of the tongue with
burning or soreness, fissures/cracks or ulcers at the
corners of the mouth, difficulty swallowing owing to
webs of tissue or partial strictures at the junction of
the esophagus & hypopharynx. Chronic IDA also
manifests gastritis and splenomegaly.
GENERAL BLOOD PICTURE OF IDA
 Microcytic-hypochromic
 HEMATOLOGY 2
o Central pallor: >1/3 diameter
o Normal RBC has the same size as the
small lymphocyte
 So roughly, even with the absence of MCHC
computation; blood picture alone can suffice
that the cells are hypochromic  central
pallor is greater than 1/3 of the diameter.
TREATMENT OF IDA
 Dependent on the cause of IDA.
 Important to consider the etiology for the
treatment of anemia.
 Since iron is primarily deficient, iron supplement
is given.
 If the main cause of IDA is malnutrition, ferrous
sulfate supplement may be given.
 If the cause is chronic blood loss, blood loss must
be supervised and give iron supplement to
monitor the progression of disease.
 If it is caused by parasite, eradicate the parasite.
NOTE: RECAP
IRON DEFICIENCY ANEMIA
 Described as microcytic: hypochromic type of
anemia
 Morphologic changes: At first it is normocytic
and as it becomes more severe, it becomes
microcytic.
IRON DISTRIBUTION: 3 POOLS/ COMPARTMENTS
a. IRON STORAGE POOL: includes ferritin
measured using serum ferritin and
hemosiderin measured via Prussian blue
staining.
b. TRANSPORT COMPARTMENT: iron bound to
transferrin measured as serum iron or
transferrin saturation or total iron binding
capacity (TIBC).
c. FUNCTIONAL IRON COMPARTMENT: iron
present in functional proteins such as
hemoglobin, myoglobin and other enzymes.
2. SIDEROBLASTIC ANEMIA (SDA)
 It is a group of anemias characterized by
abnormalities in the synthesis or enzyme
involved in synthesis of heme.
 SDA are a group of heterogeneous disorders
characterized by hypochromic red cells, which
are microcytic in the hereditary forms and often
macrocytic in the acquired forms, as a result of
defective heme synthesis, the red cells may also
be mixed with normochromic cells (dimorphic
appearance).
 Associated with defective protoporphyrin
synthesis which leads to excessive accumulation
of iron. The unused iron is deposited in the
mitochondria of normoblasts as siderotic
granules.
NOTE:
In SDA, the first enzyme (D-ALA synthase) is commonly
affected or if not the last enzyme (ferrochelatase). If
the D-ALA synthetase is affected, defective or
deficient, it will slow down the very first reaction
which is the condensation between succinyl CoA and
glycine and deficiency in this reaction would lead to
deficient D-ALA and the following reactions will
become retarded or slow down thus protoporphyrin
IX will also be deficient. When iron is transported in
the RBC, it will not be utilized because of the
deficiency in protoporphyrin IX; wherein iron is
supposed to bind with protoporphyrin IX but in the
absence of protoporphyrin IX, the iron will be
unutilized and the non-utilized iron will precipitate
inside the RBC, inside the mitochondria of RBC
becoming now the siderotic granules. When siderotic
granules are found surrounding the nucleus of RBC, it
is called the ringed-sideroblast which are immature
RBC containing siderotic granules which are non-
utilized iron.
COMPARISON:
 In IDA, iron is deficient thus increase in
protoporphyrin IX
o Negative in prussian blue staining
 HEMATOLOGY 2
 In SDA, protoporphyrin IX is deficient thus iron
will not be utilized and precipitate as siderotic
granules
o Not in all types of SDA, the
protoporphyrin IX will be decrease.
There are some types wherein
protoporphyrin IX will be increased.
o Dimorphism is observed in
sideroblastic anemia.
o Some cases, protoporphyrin IX is
increase or decrease depending on
which stage in the heme synthesis is
inhibited.
o SDA is highly positive in Prussian blue
stain since iron is not utilized
CLASSIFICATION/ TYPES OF SDA
1. INHERITED
 Caused by an abnormal gene
 Inability to inherit the gene needed by the
enzyme
 Characterized by microcytic cells
2. ACQUIRED
OTHER RELATED DISORDER:
 Exposure to agent that will inhibit the function of
the enzyme like in lead poisoning wherein lead
interferes with some of the enzymes involved in
heme synthesis.
 Characterized by macrocytic cells.
 Example: lead poisoning  lead may interfere in
the synthesis by inhibiting pyridoxal phosphate
ALA synthetase, ALA dehydrase or
ferrochelatase. When ferrochelatase or heme
synthase (which catalyzes the addition of
protoporphyrin IX with iron) is inhibited by lead
 protoporphyrin IX may no longer bind with
iron thus there would be protoporphyrin IX and
iron build up
A. HEREDITARY SIDEROBLASTIC ANEMIA
 Sex-linked recessive trait, mostly caused by
decreased in D-ALA synthase activity
 Deficiency of D-ALA synthase causes the
retardation of heme synthesis pathway
 Associated with microcytosis
B. AQUIRED SIDEROBLASTIC ANEMIA
 Due to exposure
 Associated with macrocytosis
 Acquired Idiopathic Sideroblastic Anemia
(AISA)/ Refractory Anemia with Ringed
Sideroblast (RARS)
o Mostly encountered in elderly patients
(older than 50 y/o) affecting both sexes.
Actual etiology of the disease is still
unknown but consistent findings
included decreased activity of ALA-
synthetase or an abnormal high
requirement for the cofactor pyridoxial-
5PO4 to maintain ALA synthetase RBCs
are hypochromic, despite presence of
large quantities of iron.
 Secondary Sideroblastic Anemia: Secondary to
some agents that interfere with heme synthesis
(Lead Poisoning; Alcohol, Drug-induced)
o Known etiology
 PORPHYRIAS:
o Group of disorders characterized by the
accumulation of porphyrins.
o Diseases characterized by impaired
production of heme (can be hereditary
or acquired) causing porphyrins to not
be completely changed and metabolized
resulting them to accumulate inside the
RBC or liver cells.
o The causes of porphyria can be the same
with those of SDA and the more
prominent feature is the accumulation
of porphyrins.
 Lead poisoning can also lead to
porphyrias
o Acquired types are usually associated
with enzyme deficiency. The products
from earlier stages (porphyrins) in the
pathway accumulate in the cells that
actively produce heme proteins such as
RBC and hepatocytes. These porphyrins
 HEMATOLOGY 2
can also be excreted in the urine or feces
or be deposited in body tissues.
NOTE: PORPHYRIA
MAIN FEATURE: accumulation of porphyrins
 Porphyrins accumulate first in the RBC
because RBC is involved in heme synthesis and
in the liver because it is involved in the
synthesis of myoglobin.
o During damage of RBC, when it is
hemolyze, as it reaches its 120th day,
the RBC will hemolyze and all its
contents will diffuse out into the
plasma including the porphyrins.
o When liver cells are damaged, they
also release their contents like,
porphyrins into the plasma.
Porphyrins will be elevated in the
plasma and can be filtered by the
kidneys to appear in the urine
(porphyria) where the urine appears
as port wine red. Some are excreted
in the feces.
o It is more accurate and precise to
measure porphyrins in the feces than
in urine.
o Deposit in body tissues – when
porphyrins deposit in the skin, the
skin becomes photosensitive.
Porphyrins absorbs the zorret band
coming from the sun, so when
exposed to sun, the skin of the patient
suffers from sun burn, blindness
(eyes), brain damage or psychiatric
abnormality (brain).
o Some teeth and bones especially
among the growing children wherein
some of the substances will
fluorescence. Teeth and bones are
fluorescent because of the deposited
porphyrins.
PORPHYRIA: associated with vampire stories
 A common disease affecting the royal families
in Romania and Transylvania.
 Characterized by bleeding teeth due to lack of
hemoglobin; evasion from sunlight because
they are prone to suffer sunburns and
blindness; fluorescing teeth due to deposition
of fluorescing porphyrins in the bone
 Most common clinical features:
Photosensitivity and Psychiatric or neurologic
symptom due to deposition of porphyrins
 Hematologic manifestations: Acute
intermittent porphyria (abdominal pain),
congenital erythropoietic porphyria (port
wine red urine), variate porphyria (psychosis
and photosensitivity) & erythropoietic
protoporphyria (psychosis)
HEMOCHROMATOSIS
 Another disease related to Iron metabolism:
TRUE IRON OVERLOAD.
 TO COMPARE: SDA  Iron wasn’t used in the
phase of normal iron (NORMAL: Iron; Cannot be
used/metabolized = NO IRON OVERLOAD)
 NORMAL: RATE of IRON ACQUISITION (1%) =
RATE of IRON LOSS (at least 1% of Iron daily = Red
cell destruction 1% daily)
o Rate of Loss (1%) daily is replaced by
what is absorbed (1%) daily
 HEMOCHROMATOSIS: RATE of IRON
ACQUISITION > RATE of IRON LOSS
o Iron acquisition exceeds the rate of iron
loss  iron is accumulated in the body
 Results when the body’s state of iron acquisition
exceeds the rate of iron loss. It can be acquired
or hereditary (mutations affecting the proteins
of iron metabolism). The body’s first reaction is
to store excess iron in the form of ferritin, then
in the form of hemosiderin within cells.
CAUSES OF HEMOCHROMATOSIS
 ACQUIRED: Transfusion-related
o It cannot be due to DIETARY REASONS,
excessive iron in the diet is unlikely to
cause hemochromatosis because our
absorption for Iron is LIMITED
 HEMATOLOGY 2

o Commonly caused by TRANSFUSION- o Fe++ (+O2)  Superoxide and Free

RELATED radicals – strong oxidizing agents 
 IDA: one factor leading to this PEROXIDATION of membrane lipids
condition is Frequent Blood results to:
Donation that every time we  Cell respiration - (Destroying
donate blood we lose iron. Mitochondrial Membrane) –
 Hemochromatosis: Every time Impaired ATP production = No
the recipient receives blood Cellular Respiration
(transfusion) the recipient gains  Lysosomal enzymes - (Impairing
Iron. Lysosomal Membrane) -
• Example: Patients with Beta- Rupture = Release of Hydrolytic
Thalassemia would require Enzyme
frequent donation or  Lysosome = Suicide Bag of the
transfusion of blood (PRBC/WB) Cell because once it releases its
they gain Iron thus repeated hydrolytic enzymes it can lead to
transfusion will lead to Autolysis (Cell will digest itself)
Transfusion-Related cell damage it will be destroying
Hemochromatosis. the nucleus, nuclear membrane
and in general will cause an
 INHERITED: Mutations affecting the proteins of irreversible membrane damage
iron metabolism of the cell and the organelles.
o Results to Less Metabolism: More Iron  Irreversible membrane damage
Accumulation. (Cell Membrane)
o Excessive Iron (Iron Overload): most of o In the presence of Oxygen, free ferrous
the Iron will first be stored as ferritin, but iron initiates the generation of
when overwhelmed will be stored as superoxide and other free radicals,
hemosiderin. which results in the peroxidation of the
o 2 Storage form of iron: 1st - Ferritin  membrane lipids.
when overwhelmed  2nd - o Results to damage of cell membrane,
Hemosiderin nuclear membrane, mitochrondrial and
o Both storage forms have limits and when lysosomal mnembranes. These events
both get overwhelmed, the remaining ultimately affect cellular respiration,
excessive Iron will have nowhere to be enzyme digestion, cell death, and organ
stored thus the remaining Iron (Fe++) in damage.
the presence of free oxygen (O2) will o Hemosiderin also deposits into tissues
now cause the generation of and organs leading to further organ
SUPEROXIDES and FREE RADICAL which damage:
are TOXINS and STRONG OXIDIZING
AGENT that cause oxidation of proteins,
lipids and cells  causing
PEROXIDATION of Membrane Lipids,
Cell Membrane, Mitochondrial
Membrane, Lysosomal Membrane and o SKIN AND PANCREAS: “bronze diabetes” –
other organelles, even the Nuclear bronze skin plus diabetes meaning the
Membrane leading now to impaired ATP Hemochromatosis have already affected the skin
production. and pancreas.
 HEMATOLOGY 2
 SKIN: Bronze/ golden color due to
cellular damage and accumulation of
excessive iron. The skin will absorb the
excessive Iron and it will accumulate
resulting to a BRONZE SKIN COLOR
 PANCREAS: Pancreas will be destroyed
and its production of Hormones
(regulate Carbohydrate Digestion) like
NOTE:
INSULIN, GLUCAGON etc.  with that it
BOTH IDA and SDA  heme synthesis defect
affects the Glucose Metabolism and can
Thalassemia  globin synthesis defect
lead to Diabetic Mellitus
o LIVER: Cirrhosis-induced jaundice subsequent
Liver Cancer
o HEART: Congestive Heart Failure
o Other more organs can be passively destroyed
SCREENING TEST FOR HEMOCHROMATOSIS:
 Transferrin saturation (common test performed)
and Ferritin (excessively overwhelm).
 Measurement of Transferrin Saturation
o Transferrin is fully saturated due to
excess Iron/ Iron overload ELEVATED
Transferrin Saturation
 Transferrin and Iron-binding Capacity(TIBC) will
be DECREASED
o NOT THE APPROPRIATE TEST for
HEMACHOMATOSIS
TREATMENT:
 THERAPEUTIC/REPEATED PHLEBOTOMY
o Most practical treatment to prevent
excessive Iron and prevent the
successive chain reaction
o Some patients require 2-3 times removal
of blood/phlebotomy  removal of
excessive Iron
 IRON-CHELATING DRUGS to bind/chelate excess
iron for safe excretion.
o Desferrioxamine/Desferal
 Intake of Vit. E and C
o Slow down chain of reaction but effect
will be overwhelming  inefficient;
retard.
o Can somehow help because they are a
REDUCING SUBSTANCE (especially Vit. E)
resulting to the RETARDATION of the
Iron and
o Free Oxygen Reaction
o But itself can be overwhelmed and will
only be able to SLOW DOWN the CHAIN
of REACTIONS = Inefficient
o It should be alongside the
Therapeutic/repeated phlebotomy and
Iron-chelating drugs
 Hemoglobin are usually dimers, their globin
portion has 4 polypeptide chains  2 different
types of chain (alpha and beta).
o Example: A1 (heme + globin)  4 PPC
(2 alpha and 2 beta chains)
o To synthesize the chains, it need
genes (complete number) that is
inherited from parents (one per each
parent).
 Alpha chain: needs 4 alpha
genes
 Beta chain: 2 beta gene
o Each PPC has its amino acids
 Alpha chain: made up of 141
AA  per chain
 Beta chain: 146 AA  per
chain
 HEMOGLOBINOPATHIES: condition
characterized by qualitative structural
abnormalities of the globin polypeptide chains
that result from alteration of the DNA genetic
code for those chains.
o Structural defect due to defect in
amino acids; classified as normocytic
anemia except Hb E disease
 THALASSEMIA: diverse group of genetic
disorders characterized by a primary,
quantitative reduction in globin chain
synthesis for hemoglobin
o Synthesis defect (alpha or beta) 
disorder characterized by defective or
deficient number in chains due to
deletion of some gene thus proteins
that is encoded by the gene can’t be
synthesize.
 HEMATOLOGY 2

3. THALASSEMIAS CLASSIFICATION CLINICAL HEMOGLOBI

 Thalassemias are the results of impaired MANIFESTATIO N
(deficient) globin chain synthesis. These may N
affect either the alpha or beta chain synthesis. Silent carrier Normal 3 functional
(one-gene hematologically, alpha gene
Greek No. of No. of Chromosome deletion) with few target A1= normal
Name genes Amino Location cells, elliptocyte (97%)
Acids
Alpha 4 (2 141 16 s/s:
mother; 2 asymptomatic
father)  normal
Beta 2 (1 146 11
mother; 1
father) Alpha- Mild anemia, 2 functional
Delta 2 146 11 Thalassemia trait: microcytic- alpha gene
Gamma 2 146 11 (two-gene hypochromic, remaining
Epsilon 2 146 11 deletion) elliptocytes, thus
Zeta 4 146 16 target cells synthesis of
(Rodak’s) Hb A1 is
slightly
decresed:
normal A1
levels atleast
97%.

Hb A (60%);
Hb Barts
(510%)
A. ALPHA THALASSEMIA HbH disease Microcytic, One
(Three-gene hypochromic, Hb functional
o A deficiency in the synthesis of Alpha-
deletion) H inclusions, gene
globin chains. Each individual has two
high retics remaining
sets of two alpha genes. Suppression of that
all four genes is needed to completely A1: 2 alpha & 2 is not enough
suppress alpha chain synthesis. The beta to cause
usual mechanism of suppression is by A2: 2 alpha and 2 normal
gene deletion. delta production of
o Main common cause: gene deletion or Hb F: 2 alpha and alpha
no inherited gene. 2 gamma polypeptide
o Deletion is indicated by blank: e.g. – Since alpha chain.
a/aa  one gene deletion; --/aa  two chain production
deletion; --/a  three deletion; --/--  is affected Hb H  seen
and these an pitted golf
four gene deletion.
hemoglobin ball in PBS.
needs the alpha
chain thus their
production will
also be
 HEMATOLOGY 2

retarded  RBC CLASSIFICATION OF BETA THALASSEMIA

will compensate Beta Beta Beta
by Thalassemia Thalassemi Thalassemi
increasing the Major/ a Major/ a Major/
synthesis of beta Cooley’s Cooley’s Cooley’s
chain  anemia anemia anemia
4 beta chain 0 0 + +
(B l B ) (B l B ) (B0l B)
producing Hb H
•Complete lack of •Caused by a •Sufficient
Bart’s Hydrops Stillborn, Hb Bart’s:
Beta globin partial quantities of
fetalis (four-gene hydrops tetramer Hb
production suppression of HbA1 is
deletion) (enlarged head) consisting of
•Most affected the Beta genes produced.
-Hb Bart’s has 4 gamma
patients exhibit •Symptoms •Hemoglobin
abnormal chains.
retarded growth are similar levels are
function
with mongoloid with that of slightly low,
wherein it can’t Since there is
facial features and Beta but blood
promote no
severe anemia Thalassemia smear shows
oxygenation of production of
•Marked by major, but similar
tissues. alpha chains
characteristics depend on the morphology as
due to total
changes in RBC extent of gene in Thalassemia
deletion of
morphology; suppression Major or
alpha gene,
microcytosis, • May produce Intermedia
the RBC will
hypochromia, varying •HbF is about
compensate
anisocytosis and amounts of A1 2-3%
by producing
poikilocytosis. depending on •HbA2 level is
gamma
Numerous target the degree of elevated 
chains thus
cells, Howell-Jolly suppression has delta chain
formation of
bodies and
Hb bart’s.
siderocytes. TYPES:
•HbF level is Homozygous
elevated (40-60%) (B+l B+): 2 beta
• Can synthesis A2 gene is
• Most severe of partially
all B-thalassemia suppressed
that requires Heterozygous
B. BETA THALASSEMIA frequent (B+IB): One
o Characterized by a deficiency in Beta- transfusion gene is
globin chain synthesis. Each individual partially
has one set of two beta genes. suppressed
Suppression or absence of these beta and the other
one is normal
genes results to deficient beta chain
synthesis.
o Gene deletion is indicated by 0
 HEMATOLOGY 2
NOTE:
Hereditary Persistence of Fetal Hemoglobin and B-
Thalassemia Major: highest concentration of Hb F can
be seen.
In thalassemias and other disorders characterized by
abnormal hemoglobin synthesis like IDA and SDA, it is
very common to see target cells  nonspecific finding
unlike schistocytes that is a hallmark finding in
hemolytic anemia indicating hemolysis and teardrop
cells in myelofibrosis
4. ANEMIA OF CHRONIC INFLAMMATION (ACI)
 These diseases are grouped into chronic
inflammatory diseases and malignant diseases.
It is the inflammation that may lead to anemia.
 Malignant disease/ cancer
 Chronic inflammatory disease
o Infectious TB
o Osteomyelitis
o Non-infectious RA
o SLE and Crohn’s disease
MECHANISMS OF ANEMIA OF CHRONIC DISEASES:
1. Altered Iron Metabolism  Microcytic
 Among the 4 pathophysiology, most common to
happen due to increase in acute-phase protein
and it is a direct effect of inflammation
2. Direct inhibition hematopoiesis, decreases
production of EPO. moderate destruction 
normocytic (do not involve iron)
 During acute/chronic inflammation, the
macrophages and lymphocytes are activated
thus produce cytokines of different types that
includes tumor necrosis factor-alpha (TNF-
alpha), Interferon-gamma and other
interleukins. Some of these cytokines will cause
inhibition of bone marrow function leading to
decrease hematopoiesis, inhibition of kidney
function thus decreased erythropoietin
production and some may cause direct damage
on RBCs
NOTE:
Anemias that develop in chronic diseases are caused
by the effects of the increased inflammatory
cytokines. (i.e. interleukins) produced by activated
cells during the chronic inflammation: These cytokines
may cause any of the following mechanisms:
1. ALTERED IRON METABOLISM OR IMPAIRED
FERROKINETICS
 Activated lymphocyte will produce IL-6 that
will act on the liver, it will activates the liver to
produce increase hepcidin (Positive Acute
Phase
 Protein: proteins that immediately increase
during inflammation) caused by an increase of
the following acute phase reactants that
interfere with iron metabolism:
o HEPCIDIN: this causes the
degradation of the transmembrane
protein ferroportin used by
macrophages and hepatocytes to
export iron into plasma.
 HEMATOLOGY 2
 Increase in hepcidin will result
to failure of iron to be
released in the plasma
because iron will be retain in
the cells
 Decrease iron absorption and
release  decrease plasma
iron
 FERROPORTIN: transport
protein found in the intestinal
enterocytes(cells of
duodenum), hepatocytes,
and macrophages
o LACTOFERRIN: An iron-binding
protein in the tertiary granules of
neutrophils. Its avidity for iron is
greater than that of transferrin.
During inflammation, it scavenges
available iron at the expense of
transferrin.
o FERRITIN: developing RBCs do not
have a ferritin receptor, thus, cannot
utilize iron for hemoglobin synthesis.
 Increase in the production of
apoferritin that binds with
iron to form ferritin, thus iron
will not be utilized because it
is trap in ferritin.
2. MODERATE INCREASE IN THE DESTRUCTION
OF RBC
3. DECREASED PRODUCTION OF
ERYTHOPOIETIN
 Production of inflammatory cytokines (such as
tissue necrosis factor-a and interleukin-1 from
activated macrophages and interferon-g from
activated T-cells) also impairs proliferation of
erythroid progenitor cells, diminishes their
response to erythropoietin, and decreases
production of erythropoietin by the kidney
5. HEMOGLOBIN E
 Only hemoglobinopathies wherein its
morphology is microcytic
 It a structural and synthesis defect
(hemoglobinopathy and thalassemia)
 Involves same mutation as Hb C(B6) wherein
Glu  Lysine but the point of
mutation(substitution) occurred in 26th amino
acid
o Hb C (B6 glu  lys) and Hb S(B6 glu 
val) has structural defect but Hb E has
both structural and synthesis defect
 Decrease in B-chain synthesis
 2nd most common hemoglobinopathies
MACROCYTIC ANEMIA
 MCV: >100 fL
 A macrocytic cell seems to have more
hemoglobin content compared to normocytic
but in relation to its size it is normal. 
Macrocytic  normochromic.
TWO TYPES OF MACROCYTIC ANEMIA:
 In cell maturation, the nuclear-cytoplasmic
maturation must be synchronous in order for the
cells to be normocytic when released. In
asynchronous maturation, the maturation of
nucleus and cytoplasm is not the same that can
lead to megaloblastic maturation.
1. NON-MEGALOBLASTIC
 Normal maturation therefore normal
development however when released in the
circulation they appear large
 PATHOPHYSIOLOGY: Aplastic anemia (but most
are in normocytic classification), chronic liver
disease, alcoholism and other disease or
condition that can lead to hypercholesterolemia
and reticulocytosis.
 LIVER DISEASES (chronic, obstructive jaundice),
LCAT deficiency:
o LIVER: main organ where cholesterol is
stored and metabolized thus when
damaged cholesterol will not be
completely metabolized leading to
hypercholesterolemia/ excess
cholesterol in plasma
o LCAT – enzyme involve in the
esterification of cholesterol thus when
 HEMATOLOGY 2
deficient the cholesterol(free) in the
plasma will accumulate leading now to
hypercholesterolemia
 Cholesterol in the plasma can
be in the form of free
cholesterol or cholesterol ester
 The RBC membrane consists of
phospholipid bilayer which
have some cholesterol,
proteins. CHO in the layer. The
cholesterol embedded in the
membrane depends on plasma
cholesterol that when
cholesterol plasma is increased,
there will be excessive loading
of cholesterol into the plasma
membrane thus enlarging the
size of the cell.
 RETICULOCYTOSIS: any condition resulting to
premature release of reticulocytes in the
circulation
o Reticulocyte (5th in erythropoiesis) are
immature cells seen as large cells but
they underwent maturation in the BM
o PBS: supravital stain: reticulocyte
(reticulocytosis)
o Wright’s Stain: polychromatophilic cell
(polychromatophilia)
o Shift/Stress cells: more larger
o Released as a compensatory
mechanism of bone marrow to the
following condition:
 Acute blood loss
 Hemolysis or hemolytic anemia
 Bone marrow infiltration: filled
with abnormal cells
 High level of EPO
 RBC should stay in the BM for 3.5 days and 1 day
in the peripheral circulation but due to the
stated condition above, the RBC transit time in
the BM will be shorter and prematurely released
in the circulation (prolonged stay) shift/stress
reticulocytes
2. MEGALOBLASTIC
 Asynchronous maturation
 Due to abnormal development  that during
their earliest developmental stage
(pronormoblast) their development is already
abnormal  promegaloblast  2nd stage:
basophilic megaloblast (b. normoblast should
undergo normal maturation)
 MAIN PROBLEM: Impaired DNA synthesis
 Defective nuclear maturation caused by
impaired DNA synthesis (prolonged intermitotic
resting phase; block early in mitosis)
 N-C asynchrony or “maturation arrest”
o Deficient DNA synthesis (nucleus)
resulting to retarded nuclear maturation
while the RNA synthesis in the cytoplasm
is less impeded thus cytoplasm
maturation proceed normally
o While the nucleus is not yet mature, it
will not cause division but the cytoplasm
keeps on maturing and enlarging
resulting to prolonged intermitotic
resting phase leading to block in early
mitosis or the cell will not divide at all
the cell become larger appearing now as
macrocytic.
 In contrast to microcytosis
wherein the cells are small
because they undergo
extradivision.
 RULE: as the cell divides, the cell
size decreases
o NUCLEUS: control center of the cell:
control all the process including the
division of the cell.
 Not only affect RBC but all rapidly proliferating
cells  enlargement of ALL rapidly proliferating
cells like neutrophils
 MCV: >100 fL; cannot be relied alone in
determining a megaloblastic maturation
 IMPORTANT FINDINGS INDICATING A
MEGALOBLASTIC MATURATION: MCV of >100 fL
+ macro-ovalocyte and macropolycyte
 RBC is often seen as macro-ovalocytes (large and
oval) and Neutrophils as macropolycyte (large in
size with hypersegmentation) indication of
megaloblastic anemia.
 HEMATOLOGY 2

o Neutrophil: has a normal 3-4 lobes but
when hypersegmented and stil has a
normal size (9-15 um) it indicates old
cell.
 Multiple Howell-Jolly bodies and many
nucleated RBCs with karyorrhexis.
o DNA containing inclusion indicating
nuclear remnants
o Problem in DNA synthesis which
affected the nuclear maturation thus
the nucleus is prone to fragmentation
 karyorrhexis
o Due to increase karyorrhexis
 Pancytopenia, leucopenia and
thrombocytopenia

ERYTHROKINETICS
 Total erythropoiesis is increased yet there is
reticulocytopenia indicating ineffective
erythropoiesis.
 Hyperplastic marrow: more red marrow in
order to compensate for decreased amount of
A. MACROCYTOSIS WITH NORMOBLASTIC RBCs.
MARROW o A marrow which has red
 Characterized by the presence of macrocytic hematopoietic cell (hematopoietically
cells that are not megaloblastic. This may be due active) over the marrow fats
to early release of immature erythrocytes from o Hypoplastic: more of marrow fats
the bone marrow in response to acute hemolysis o Normoplastic: normally, 50:50 ratio
of red cell and fats or 25:75 in long
and/or acute blood loss.
bones
 Not a complication but a compensation
 3x erythropoiesis
 Supravital stain: reticulocyte  Decrease M:E ratio = 1:1
 Wright’s stain: Polychromatophilic cells o Normal M:E ratio is 3:1
 Reticulocytopenia: Increased RBC in the
B. MEGALOBLASTIC ANEMIA marrow but not enough number of cells in
 Characterized by enlargement of all rapidly the circulation and due to premature lysis
proliferating cells of the body including marrow  Pancytopenia
cells. The major abnormality is the diminished
capacity for DNA synthesis, resulting to “nuclear- CHEMISTRY
cytoplasmic asynchrony” or “maturation arrest”  Related to hemolysis
RNA synthesis is less impeded than is DNA o Increase serum Lactate
synthesis, hence cytoplasmic maturation and Dehydrogenase (LD)  from RBC-
derived isoenzyme
growth continue.
o Increase bilirubin (both total and
 Most common causes: Vitamin B12 deficiency
unconjugated)
and folate deficiency o Increase endogenous carbon
NOTE: monoxide (CO)  while the
hemoglobin continuously degrade,
BLOOD PICTURE:
the heme and globin will separate
 Presence of macroovalocytes and giant
and heme will further separate into
hypersegmented neutrophils.
iron and protoporphyrin IX.
 Basophilic stippling: precipitated ribosomes
Protoporphyrin IX will further
indicating a disturbed erythropoiesis and
undergo degradation releasing
erythrocyte metabolism
biliverdin and CO. In normal
 HEMATOLOGY 2

hemolysis, carbon monoxide is

exhaled but if hemolysis is increased
CO is also increased.
o Common source of CO is exogenous:
factory and automobile exhaust
 Others: different mechanism
o Increased serum muramidase 
present in granules of neutrophils
 Disturbed granulopoiesis
thus enlarged and
hypersegmented neutrophils
and easily damaged thus
releasing their enzyme
content (muramidase).
o Increased homocysteine  due to
failure convert into methionine
 Homocysteine should receive
methyl from folate to
convert into methionine, but C. CYANOCOBALAMIN (VITAMIN B12) DEFICIENCY
due to folate deficiency it  Not common to occur because the body’s daily
fails to be converted. requirement of B12 is very minimal (around 2-5
DNA = double stranded helix consisting of sugar- ug/day)
phosphate backbone and nucleotides.  When intake is stopped, this type of anemia can
be acquired after 3-6 months or a year. Because
In the formation of thymidine structure to thymine, the body’s utilization of B12 is also minimal thus
both folate and B12 are needed. Therefore deficiency B12 deficiency takes gradually.
in folate and B12 will result to impaired DNA synthesis.
 Vitamin B12 is required in the Demethylation of
The bone marrow converts
folate during DNA synthesis
deoxyuridinemonophosphate into
deoxythymidinemonophosphate using the enzyme  Folate is absorbed through the ileum with the aid
thymidylate synthethase which needs a cofactor of the intrinsic factor (IF) or Castle’s factor
tetrahydrofolate (THF) which is from dietary folate. produced by the parietal cells, and is transported
Dietary folate enters the plasma in the form of 5- by transcobalamin II (TC II) in the plasma.
methyl THF and enters again the red cell in the form of o Intrinsic factor: a protein synthesized by
5- methyl THF but before it could participate in the gastric mucosa; without it, B12 can’t be
synthesis of DNA structure, it needs to undergo absorbed
demethylation wherein there should be removal of o In ileum, cells (enterocyte) with
the methyl group. The methyl removed will be receptors for B12 are available however
transferred to homocysteine thus converting it into it is specific only to B12-IF complex. Thus
methionine by the enzyme methionine synthase
when only B12 is pass through it, it will
needing the cofactor B12.
not be recognized therefore not
TWO MOST COMMON CAUSES OF MEGALOBLASTIC absorbed.
ANEMIA: o Transcoblamin II: transports B12 to sites
1. B12 deficiency anemia where it could be metabolized or stored
2. Folate deficiency anemia like in the liver, bone marrow and other
tissues.
 RDA: 2-5 ug/day
 TWO SOURCES OF B12:
 HEMATOLOGY 2
o DIET: primarily from food of animal
origin  main source of B12
o LARGE INTESTINE: synthesized by some
of the normal flora in the intestine
(lactobacilli spp. and bacteroides spp.).
However the absorption of B12 is in the
ileum but the production comes from
the large intestine. Thus, B12
synthesized in the large intestine is not
all/readily available for consumption of
the human body.
NOTE:
ACQUISITION OF METABOLISM OF B12
 B12 comes from the diet (meat origin) once
reaches the stomach, the gastric juice is very
acidic because of the HCl produced by the
parietal cells. Thus B12 can be damaged. In
order to protect B12 from acid destruction,
salivary glands produce haptocorrin or
Transcobalamin I (TC I). While masticating the
food, B12 combines with TC I. Since TC I
protects B12, it can bypass the acidity and
digestive action of the stomach in small
intestine (duodenum;1st portion) the pH of
the chyme is neutral to alkaline thereby
destroying haptocorrin and B12 will be
released. B12 combines with Intrinsic factor
(IF) coming from the parietal cells of the
stomach and thus forming the B12-IF
complex. The complex goes down until the
jejunum and terminal part – ileum where it
will be absorbed by the intestine. Once
absorbed, IF will be detached and B12 will be
released into the circulation. B12 will then
combine with Transcobalamin II and be
brought into the liver (for storage or
metabolism), bone marrow (for utilization and
DNA synthesis) and other tissues (for
utilization being a cofactor).
IMPORTANCE OF B12
 For metabolism
 Formation of RBCs
 Maintenance of CNS (brain and spinal cord)
CONSEQUENCES OF B12 DEFICIENCY
 Ineffective DNA synthesis: Megaloblastic
anemia
 Inadequate myelin synthesis: neurologic
damage
 Hyperhomocysteinemia: cardiovascular
disease.
NOTE:
RELATED CAUSES IF VITAMIN B12 DEFICIENCY:
1. INADEQUATE INTAKE
 RDA: 2-5 ug/day from meat diet
 Common in strict vegan people
2. MALABSORPTION SYNDROME
 Any condition affecting the ileum
(inflammation): Celiac disease, ileal disease
(Crohn’s disease/regional enteritis) tropical
sprue, resection of the small bowel
 Imerslund-Grasbeck syndrome – deficiency
or absence of IF-B12 receptors
 Zollinger-Ellison syndrome – hypersecretion
syndrome  gastric hypersecretion (acid and
juices) that can degrade B12 and prevent it
from binding with IF and therefore B12 is not
absorbed
3. LACK OF AVAILABILITY OF COBALAMIN
 Blind loop syndrome
o Refers to bacterial overgrowth
o The large intestine (colon) is rich in
normal flora that synthesis vitamins
but in this case, what will proliferate
in the LI are the abnormal flora which
will utilize the available B12 at the
expense of the host. And this
abnormal flora will also destroy the
normal flora in the expense of their
capacity of producing some
important vitamins (B12)
 Infection with Diphyllobothrium latum
o D. latum is the longest tapeworm
infecting human which resides in the
ileum
o Bothriocephalus anemia: long
tapeworm that competes with the
host with the absorption of B12 and
secretes substance that inhibit
binding of IF with B12.
4. PRESENCE OF AUTOANTIBODIES
 Can be either those who destroy the intrinsic
factor or those which destroys cells(parietal)
 HEMATOLOGY 2
that produce IF  Inability of the gastric
mucosa to secrete IF (Pernicious anemia)
5. DRUGS/ SUBSTABCE-RELATED
 e.g. Neomycin and ethanol  chronic
alcoholics
6. ABNORMAL PRODUCTION OF TC-II
D. PERNICIOUS ANEMIA
 Autoimmune type of vitamin B12 deficiency
anemia
 Types of Autoantibodies:
o Anti-parietal cell antibodies: destroys
parietal cells
 Intrinsic factor: complexes with
B12 in order for it to be
absorbed
 HCl: gives acidity of the
stomach  absence can lead to
achloridria
o Anti-intrinsic factor antibodies
 IF production is normal, but IF is
inhibited by the autoantibodies
thus it cannot bind with B12 
non-absorption of B12
 2 TYPES OF ANTI-INTRINSIC
FACTOR ANTIBODY:
NOTE: DIAGNOSIS OF VITAMIN B12 DEFICIENCY
Blocking antibody: blocks the
 Normal plasma vit. B12 levels: 180-914 ng/L
binding of cobalamin to IF
Binding antibody: binds to the
cobalamin-IF complex
becoming trimolecular complex
receptors will not recognize;
and prevents the complex from
binding to receptors in the
ileum
 Clinical signs & symptoms include:
o Atrophic glossitis: tongue will atrophy
and become inflamed due easy
desquamation of surface epithelial cells
(lack of integrity because of B12 def.) 
beefy red tongue
o Atrophic gastritis and achloridria:
leading to malabsorption; when
stomach become inflamed, parietal
cells become defective and can’t
produce HCl
 Achloridria: absence of HCl;
more specific of pernicious
anemia especially when the
autoantibodies destroy the
parietal cells.
 HCl: important in digestion in
the stomach  digestion of
proteins
o Episodic abdominal pain, constipation,
and diarrhea
o Symptoms of anemia with a
combination of skin pallor and lemon-
yellow skin
o Diffuse and irregular degeneration of
the white matter of the CNS
 Memory loss, numbness and
tingling in toes and fingers
damaged nerves
 Symmetric sensation of “pins &
needles” of the distal
extremities
 In advance cases, brain may be
affected resulting to irritability,
emotional instability, or a
change in personality
(megaloblastic madness)
1. SERUM COBALAMIN ASSAY
 A microbiological assay using an organism (E.
gracilis) that requires B12 for its growth.
 Organisms used: Euglena gracilis and
Lactobacillus leichmannii
 Patient’s plasma + organism incubated for
48-72 hours at 37 degree Celsius
 If the patient’s plasma has a normal B12, it will
support the growth of the organisms
organisms will grow and cause turbidity of the
medium
o If the patient’s plasma has an
abnormal B12  growth will be
retarded  organisms will not grow
and will not cause turbidity
o TURBIDITY WILL BE COMPARED WITH
STANDARD CONTROLS
 HEMATOLOGY 2
 Highly sensitive but it is lengthy to performed
but;
o Not sensitive to patients taking
antibiotics  antibiotics are in plasma
thus when incubated with the
organisms, it can destroy or kill these
thus this test should not be done with
patients undergoing an antibiotic
therapy
 Reference value: 200-900 mg/dL
2. METHYLMALONIC ACID & HOMOCYSTEINE
ASSAY
 B12 is a cofactor of methymalony CoA mutase
in the isomerization of methylmalonyl CoA to
Succinyl CoA. Deficiency of B12 will lead to
excretion of increased amount of
methylmalonate in the urine.
 Methyl malonyl CoA  MMCoA mutase with
cofactor Vit. B12  Succinyl CoA
 Measured by:
o Methyl malonic acid assay: GC-MS
o Homocysteine assay: GC-MS; HPLC;
Fluorescence Polarization
 In both B12 and folate deficiency,
homocysteine will increase.  homocysteine
assay
3. DEOXYURIDINE SUPPRESSION TEST
 Indirect test that measures the ability of the
bone marrow cells in vitro to utilize
deoxyuridine in DNA synthesis.
 It is abnormal in both cobalamin (B12) and
folate deficiency.
4. COMPETITIVE PROTEIN BINDING
RADIOASSAY
 Method of choice for both B12 and folate
deficiency quantitation
 Principle: patient’s B12 and folate compete
with Co-B12 and -folate in binding with hog IF
and B-lactoglobulin
 Patient’s B12  hog IF  Co-B12
o Competition between the patient’s
B12 against the cobalt-labeled B12 in
binding with hog IF
o Differentiation: using the radioassay
 Patient’s folate  B-lactoglobulin  I-folate
o Competition between patient’s folate
against the iodine-labeled folate in
binding with the reagent B-
lactoglobulin
o Differentiation: using the radioassay.
5. SCHILLING’S TEST
 Specific test for Pernicious Anemia
 Provides a measure of the body’s ability to
secrete viable IF & absorb orally administered
Co-labeled B12.
 PART ONE: patient is given a physiologic dose
of B12 labeled with radioactive cobalt (0.5 -2
ug) ingested orally to see whether the patient
can absorb the B12 and after 1-2 hours;
another flushing dose is given  injection of
non-radioactive vitamin B12 (1000 ug)
intramuscularly in order to saturate the
binding sites of B12 so that the minimal
amount of B12 absorbed will not bind in the
binding site instead it will be urinated. check
B12 levels in the patient’s urine
o Patients with IF or NO pernicious
anemia: the cobalt labeled B12 given
orally in the duodenum will combine
with IF available in the patient to now
allow B12 absorption. B12 absorbed
will proceed to the plasma and those
some will be stored in the liver,
plasma protein binding and urinary
excretion.
 Patients with normal IF: excrete >7% ingested
B12
 If the first part of the test is normal: excreted
>7% B12  report it as NORMAL. But if there
is abnormal result (<7% or none excreted) that
may be due to deficient or absent IF 
proceed to PART II
o If IF is absent, there will be no
intestinal absorption of B12 thus,
ingested B12 will be excreted through
defecation.
 PART TWO: Done after 24 hours of the
conduct of first part of the test. Patient is
given an oral cobalt-labeled B12 with IF that
will allow absorption of B12 in the ileum.
Result is therefore expected to be impoved.
o Urinary excretion: improved >7% or
15%  PERNICIOUS ANEMIA
o Low cobalt in urine  Other B12
deficiency anemia
 HEMATOLOGY 2
OTHER TESTS
1. TEST FOR AUTOANTIBODIES
 For pernicious anemia
 Identify serologically what specific antibody is
produced
 More specific than schilling’s
2. GASTRIC ANALYSIS (HCl) AND SERUM
GASTRIN
 For pernicious anemia
 HCl: Achloridria due to damage in parietal
cells
 Gastrin: peptide hormone secreted by the
gastric walls which functions to stimulate
stomach to secrete juice and acids. Since there
is decrease HCl, gastrin will be stimulated to
induce more HCl production. Although serum
gastrin is plenty, there will be still no HCl since
the parietal cells are destroyed.
 DECREASE HCl and INCREASE SERUM GASTRIN
3. SERUM HOLOTRANSCOBALAMIN ASSAY
 Complementary test
 Holotranscobalamin: metabolically active
form of cobalamin (B12)
4. FECALYSIS (FOR BOTHRICEOHALUY ANEMIA)
 Operculated eggs of D. latum
E. FOLIC ACID/ PTEROYLGLUTAMIC ACID
DEFICIENCY
 Features are similar to Vitamin B12 deficiency
but no neurologic symptoms; leukopenia and
thrombocytopenia are less constant
 RDA: 50 ug folic acid or 400 ug food folate
 The clinical s/s of it is very similar with B12
deficiency except NO NEUROLOGIC SYMPTOMS
 uncommon to occur even in severe cases
 Absorption: small intestine: duodenum and
jejunum (same with iron absorption)
o Small intestine parts: 1st duodenum 
2nd jejunum  3rd ileum (B12
absroption)
 Deficiency inhibits the thymidine
 SOURCE: vegetables and poultry products (eggs)
NOTE:
RELATED CAUSES IF FOLATE DEFICIENCY
 Inadequate intake/Dietary deficiency
 Liver disease
 Alcoholism: alcoholic is hepatotoxic
 Increased demand/requirement
o Prone to babies and pregnant women
 Defective absorption
 Inadequate utilization of folate
 Presence of folate antagonists (antimalarial
drugs that inhibits utilization of folate)
DIAGNOSIS OF FOLATE DEFICIANCY
1. Microbiologic assay using Lactobacillus casei
 Serum and Red Cell folate: usually < 3 ug/L
2. Deoxyuridine Suppression Test
 Ability of BM to utilize deoxyuridine into
deoxythymidine and finally into DNA
structure
3. Quantitation of Vitamin B12 and folate
through competitive protein binding
radioassay
 Method of choice
4. Homocysteine Assay
F. TROPICAL SPRUE & GLUTEN-SENSITIVE
ENTEROPATHY
 A condition that can cause both vitamin B12 and
folic acid malabsorption as a result of intestinal
atrophy.
 Intestinal atrophy (SI and proximal intestine)
Vit. B12 and folic acid malabsorption
o Tropical Sprue: intestinal atrophy of the
small intestine
o Gluten-sensitive enteropathy: intestinal
atrophy of the proximal intestine
NOTE: Tropical Sprue & Gluten-sensitive enteropathy
THERAPY: depends on the condition
1. ORAL COBALAMIN
 B12 deficiency: B12 supplement
2. PARENTERAL CYANOCOBALAMIN
 For pernicious anemia  through IV/
injection
3. ORAL FOLATE
 HEMATOLOGY 2

 Folate deficiency: Folate supplement o HYPOPLASTIC: preferred term meaning

severe depletion of blood cells
o APLASTIC: may be confused due to the
NOTE: RECAP prefix “a” meaning absence; but in
DEVELOPMENTAL DEFECT:
anemia it means the same as hypoplastic
MICROCYTIC: abnormal cytoplasm maturation (hb
 Clinical signs include the common signs of
synthesis)
MACROCYTIC: Megaloblastic: Asynchronous anemia plus infection and bleeding seen also in
maturation due to defective DNA synthesis leukemia
 Splenomegaly & lymphadenopathy are ABSENT
in aplastic anemia but present in leukemia
NORMOCYTIC-NORMOCHROMIC ANEMIAS o In leukemia, there are many leukemic
 Synchronous maturation: not a developmental cells that causes the spleen to be
defect but a survival or proliferation problem overworked (remove cells) 
 Anemia occurs due to: splenomegaly.
o Increased destruction  Hemolytic o For ALL, the lymph nodes are also
anemia: can be INTRINSIC or EXTRINSIC infiltrated  lymphadenopathy
 Problem in the
circulation/spleen wherein a
lysis occur
 Increased in reticulocyte count
due to compensatory
mechanism of a normal bone
marrow
NORMAL BM HYPOCELLULAR HYPERCELLUL
o Decreased in proliferation or
BM AR BM
hypoproliferation 50% of red Decrease in red Increase in red
 Bone marrow defect wherein marrow and marrow but marrow but
there is decrease production of 50% of marrow increase in white decrease in
cells fats marrow(fats) white marrow
 Decreased or normal (fats)
reticulocyte count due to bone Ratio depends Seen in
marrow failure on age: hypoplastic/aplas Seen in
 Differentiated through reticulocyte count Younger child: tic anemia polycythemia
100% red vera
I. ANEMIA OF BONE MARROW FAILURE marrow
Adults: 50:50;
 Could be due defect of bone marrow cells
25:75
(damaged)
 Abnormal or lack of stimulation of the hormone
(EPO) even with normal bone marrow PATHOGENESIS
1. Direct damage to the hematopoietic
A. APLASTIC/ HYPOPLASTIC ANEMIA stem & progenitor cells decreased in
 Characterized by a severe reduction of the blood cell count
amount of hematopoietic tissues that results to o Causes: ionizing radiation, toxic
deficient production of blood cells (hypocellular chemicals (benzene)
bone marrow) o Infiltration BM: myelophthistic
anemia
 BONE MARROW: hypocellular
o competition for nutrients by
 CIRCULATION: pancytopenia
normal and abnormal cells
cancerous cells take up the
 HEMATOLOGY 2
oxygen and nutrient supply
o PANCYTOPENIA
2. Immune-mediated
destruction of marrow cells
o Antibody destruction wherein
patient produce autoantibody
that damages its own cell.
o PANCYTOPENIA
3. Low effectiveness of EPO
(cannot activate erythroid
stem cell)
o Defective/inefficient hormone
that causes insufficient
stimulation of the bone marrow
o PURE RED CELL APLASIA
4. Low production of EPO like
in renal diseases
o Normal function of EPO but low
in number thus unable to
stimulate the bone marrow
o PURE RED CELL APLASIA
A.1. IDIOPATHIC APLASTIC ANEMIA
 The cause is of unknown etiology
 The development of sign and symptoms is
hideous and gradually occurring until it becomes
a full-blown anemia
A.2. ACQUIRED APLASTIC ANEMIA
 May be the effects of exposure to certain
physical and chemical agents some drugs like
antimicrobials, anticonvulsants analgesics
 SECONDARY TO:
o Ionizing radiation  high doses
damages bone marrow
o Benzene  chemical that has toxic
effect to BM  Benzene Hypersensitive
Aplastic Anemia
o Chloramphenicol  most common
antibiotic causing AML and Aplasic
anemia  toxic to BM
A.3. CONSTITUTIONAL APLASTIC ANEMIA
 Associated with other group of congenital
anomalies or with genetic predisposition to
chronic bone marrow failure. Predisposition to
cancer and chronic bone marrow failure.
 Patient is born without aplastic anemia but has
congenital genetic abnormality that made him
predispose to suffering other
malignancies/cancer.
FANCONI’S ANEMIA 9CONGENITAL APLASTIC ANEMIA)
 Congenital bone marrow defect that leads to
decrease production blood cells
 Hereditary Disorder: Autosomal recessive trait
or pattern of inheritance
 Chromosomal defect are found in lymphocytes
and BM cells.
 With features of developmental abnormalities
that include hyperpigmentation, short stature
(inability to grow), hypogonadism, mental
retardation, strabismus (cross-eyed) and
malformation of the extremities.
 Fanconi’s syndrome: different and has no
relationship from this kind of anemia
o Defect in PCT reabsorption thus it is a
renal tubular defect wherein the
important solute in the filtrate are not
reabsorbed.
FAMILIAL APLASTIC ANEMIA
 Subset of Fanconi’s anemia.
 SCHWACHMAN – Diamond syndrome prone to
develop familial aplastic anemia
 Patients may have pancytopenia and a
hypocellular marrow without major
developmental abnormalities.
A.4. PURE RED CELL PLASMA
 Only the red cell are decreased
NOTE:
1. Transitory Arrest of Erythropoiesis/
transient aplastic crises – may occur during
the course of a hemolytic anemia often
preceded by an infection with parvovirus B19
 HEMATOLOGY 2
infects CFU-E
2. Transient Erythroblastopenia of Childhood –
associated with a history of viral infection
within the last 3 months (humoral inhibition)
3. Acquired Pure Red Cell Aplasia – associated
with thymoma
4. Congenital Red Cell Aplasia/
Congenital Hypoplastic Anemia
(Diamond-Blackfan anemia)
 Defects in BFU-E and CFU-E with
accelerated apoptosis (programmed cell
ANEMIA OF RENAL INSUFFICIENCY
death) thus decrease production of RBCs
o Progenitor cells BFU-E CFU-E
that give rise to pronormoblast
mature erythrocytes
 Most common type caused by a defect in
the erythroid-committed progenitor cell
II. MYELOPLASTIC ANEMIA
 Associated with marrow replacement by
metastatic carcinoma, multiple myeloma,
leukemia or other conditions.
ANEMIA OF ENDOCRINE DISEASE
 This is characterized by the presence of varying
number of normoblasts and immature
neutrophils. (leukoerythroblastic-anemia)
o Leukoerythroblastic reaction: increase in
immature granulocytes or myeloblast
and immature erythrocyte.
o Occasionally macrocytic but more
ANEMIA IN LIVER DISEASE
commonly normocytic-normochromic
 Marrow replacement: space occupied by cancer
cells  overcrowding and consumption of
nutrients and oxygen supply at the expense of
normal cells by the cancer cells
 In leukemia, there is decrease RBC (anemia),
decrease WBC (infection) and decrease platelets
(bleeding tendencies). Anemia that develops in
leukemia is Normocytic-Normochromic.
III. ANEMIAS OF SYSTEMIC DISORDERS
(ASSOCIATED WITH CHRONIC DISEASES)
 These include anemia of renal insufficiency,
anemia in liver diseases, and anemia of
endocrine function, among others.
 RBCs are usually normocytic-normochromic,
occasionally microcytic-hypochromic.
 In chronic disorders/inflammation: WBC are
activated causing them to release cytokines.
Increased cytokines (interleukins, TNF,
interferons)  alter iron metabolism that affects
hb synthesis (microcytic – hypochromic) but if
the effect of it is on blood cell production like will
cause decreased EPO because it inhibits kidneys
or it causes decrease in RBC production as it
directly suppresses the erythroid progenitors
(normocytic - normochromic)
 Increased cytokines and Decreased EPO
 Normocytic anemia due to renal disease wherein
the kidney will not be able to produce enough
hormones (EPO) which is the main growth factor
for erythropoiesis
 Decreased EPO (inefficient stimulation)
decreased BM response thus decreased RBCs
 Patients on dialysis is often given commercially
produced EPO and undergo frequent transfusion
of packed red blood cells
 EPO has hormonal regulation and its function in
stimulating blood production is also affected by
hormones produced by the endocrine glands like
the following:
o Thyroid, pituitary, adrenal, gonads
 More characterized by macrocytic anemia but
occasionally may be normocytic
 Liver diseases are associated with abnormal
metabolism and storage of folate
megaloblastic anemia
IV. CONGENICAL DYSERYTHROPOIETIC
ANEMIA (CDA)
 Group of anemia: inherited refractory anemia
characterized by erythroid multinuclearity,
karyorrhexis and bizarre malformations.
o Refractory means unresponsiveness to
stimulation and medication
 HEMATOLOGY 2

NOTE: o Intrinsic defects (defects of the red cell

itself)
o Abnormal membrane of RBCs:
Hereditary spherocytosis, elliptocytosis,
South-east asian ovalocytosis
o Abnormal metabolism/ metabolic
disorder: embden-meyerhof pathway or
hexose-monophosphate shunt is not
occurring normally due to enzyme
deficiency.
TYPE I: Megaloblastic changes with some binuclearity o Hemoglobin defects: RBCs are
 has nuclear bridging. synthesizing abnormal hemoglobin that
 More on macrocytic. makes the cell prone to lysis.
 Defective gene (Ch 15) o Most are inherited defects
TYPE II: HEMPAS (Hereditary Erythroblastic  Extrinsic defects (factors outside the red cell)
Multinuclearity with a Positive Acid Serum Test) o Normal RBC but the environment of it is
abnormal.
TYPE III: Giant erythroid precursor with more o Damaged blood vessels/
pronounced multinuclearity,
microcirculation
 More on macrocytic
o Plasma has antibodies or toxic agents
 Defective gene (Ch 15)
that can destroy the cells
o Most are acquired defects
V. APLASTIC ANEMIA ASSOCIATED WITH
CHEMICAL OR PHYSICAL AGENTS
 Chemical Agents: Benzene  toxic to BM
causing Toxic Aplastic Anemia and
Chloramphenicol
 Physical Agent: Ionizing Radiation  RBC (most
sensitive)  WBC  Platelets (more resistant;
last to be affective)

HEMOLYTIC ANEMIAS
 Belong to normochromic-normocytic anemia
 Normally, 1% of our RBCs are destroyed daily by
extravascular hemolysis
 Hemolysis can occur:
o Extravascular hemolysis: outside the
blood vessels  spleen (splenic
macrophages)
o Intravascular hemolysis: within
the blood vessel or circulation
o In both cases, there will be the release of
hemoglobin that may cause the loss of it.
Hemoglobin contains most of the body’s
iron (60% of total iron)
 Increased red cell destruction can be cause by:

If the iron released from the
hemoglobin is not recycled, it will
be loss. The body’s mechanism in
order to recycle the iron: Our
iron source may come from the
diet and recycled iron, thus it is
important for our body is able to
recycle iron. When RBCs are
hemolyzed, it will release
hemoglobin and it will be
degraded to release the iron.
Iron can go directly to cells for
reuse, or can be bound to
transferrin for transport or
stored in the form of ferritin and
hemosiderin.
HEMATOLOGY 2
EXTRAVASCULAR HEMOLYSIS occuring in macrophages. When the RBCs are
abnormal, the spleen function in filtering the blood and removes any abnormal or
senescent/old red cells called splenic culling.When there are alot of abnormal cells,
splenic culling/ extravascular hemolysis will be increased resulting to the release of
hemoglobin due to hemolysis of RBCs. Hemoglobin undergoes degradation by
separating into heme and globin. Globin will be recycled as amino acid and goes
back to cytoplasm and plasma as part of of the amino acid pool for further protein
synthesis. Heme undergoes further degradation releasing iron and recycled.
Protoporphyrin IX will also be released from the heme and be broken down into
endogenous carbon monoxide (exhaled) and biliverdin. Biliverdin is reduced into
bilirubin (unconjugated) and released into the plasma. So what increases in the
plasma is the unconjugated type which is water-insoluble and is immediately bound
to albumin. Unconjugated bilirubin can’t be excreted in the urine because kidney is
not able to filter it due to the following reasons: it is water-insoluble and they are
bound to albumin (HMW). This albumin-unconjugated bilirubin complex is brought
into the liver for conjugation. It is conjugated with 2 molecules of glucuronic acid
catalyzed by the enzyme UDP-glucuronyl transferase to form the conjugated
bilirubin called bilirubin diglucuronide. Conjugated bilirubin is stored in the gall
bladder and when needed it forms part of the bile. When needed for digestion it
will be secreted along with the bile into the small intestine to be acted on by some
bacterial enzyme and intestinal enzymes thus it converts into urobilinogen. ½ of the
urobilinogen will reabsorbed going back to the circulation then into the liver. The
other half will be converted into urobilin and stercobilin to be excreted in the stool.
FINDINGS: Increased plasma bilirubin, increased urine urobilinogen, increased
endogenous carbon monoxide.
INTRAVASCULAR HEMOLYSIS: The red cell is not engulf but it lyses in the circulation
releasing now hemoglobin in the circulation resulting to free hemoglobin in the
plasma. Hemoglobin is a LMW protein thus it can be easily filtered (solutes with MW
of <70,000 daltons) by the kidney and excreted into the urine resulting to
unpreservation of the hemoglobin content like iron. To prevent haemoglobin loss,
haptoglobin from the liver will bind with it forming now the hemoglobin-
haptoglobin complex (HMW) and direct into liver for degradation. But due to this,
haptoglobin is consumed. When there is continuous hemolysis, haptoglobin will be
depleted. Thus, hemoglobin becomes free again and will be degraded into heme
and globin in the plasma. Some heme will be oxidized and binds with albumin
forming methemalbumin and some will also bind with hemopexin forming heme-
hemopexin complex and these two will be brought back in to the liver for
degradation.
FINDINGS: hemoglobinuria or methemoglobinuria; Decreased haptoglobin;
increased plasma methemalbumin; increased carbon monoxide
 HEMATOLOGY 2

TYPES OF HEMOLYSIS (intravascular hemolysis)

 Increased urine urobilinogen
 EXTRAVASCULAR (MACROPHAGE-MEDIATED):
(extravascular hemolysis)
destruction of senescent red cells by splenic
macrophages. RBC Survival Studies (Radioactive Chromium
 INTRAVASCULAR DESTRUCTION (MECHANICAL (51Cr)
HEMOLYSIS): may be caused by the turbulent o RBC half-life: 25-32 days
environment in the circulation o Cr-RBCs IV blood count every 1-2 days
for 10-14 days
3 MECHANISMS ON HOW THE BODY CONSERVES IRON o Residual activity: index of the
IN HGB DURING INTRAVASCULAR HEMOLYSIS intravascular lifespan
1. Haptoglobin binds with free Hb preventing it o Detects sites of red cell destruction
from being filtered by the kidneys
2. When haptoglobin is depleted, heme is liberated
from hb and binds with hemopexin.
3. Oxidized heme binds with albumin forming
Methemalbumin

LABORATORY FINDINGS: RBCs and HgB Destruction

HEMATOLOGY:
 Reticulocyte count measured by RPI
o Reticulocytosis/
polychromatophilia
 Decreased red cell hemoglobin, Hct, and
RBC count
 Normocytic and/or occasionally
Macrocytic anemia representing the
reticulocytosis that is prematurely
released in the circulation

BONE MARROW
 Erythroid hyperplasia (decreased M:E)
 Increased Plasma hemoglobin and
bilirubin (unconjugated)
o Intravascular: highly increased
o Extravascular: slightly increased
 Decreased Serum haptoglobin
o Intravascular: severely decreased
to 0
o Extravascular: slightly decreased
 Increased plasma methemalbumin 
formed when haptoglobin can’t no longer
support hemoglobin
 Increased LHD due to hemolysis 
increased in isoenzyme LD 1 and 2

URINALYSIS
 Hemoglobinuria and methemoglobinuria
 HEMATOLOGY 2

TEST PARAMETER INTRAVASCULAR HEMOLYSIS EXTRAVASCULAR

HEMOLYSIS
H Hb, Hct, RBC count Decreased Decreased
E
M Reticulocyte count Increased Increased
A
T Bone Erythroid hyperplasia Erythroid
O marrow (decreased M:E ratio) hyperplas
L examinatio ia
O n (decrease
G d M:E
Y ratio)
RBC morphology Normocytic some macrocytic Normocytic some
macrocytic

P Free hemoglobin Increased Slightly increased

L
A Bilirubin Increased Unconjugated Increased
S Unconjugated
M
Haptoglobin Decreased/Absent Slightly decreased
A
C
H Lactate dehydrogenase Increased Slightly increased
E
M
Hemopexin Decreased Slightly decreased
I
S Endogenous CO Increased Increased
T
R
Y

U Free hemoglobin Positive Negative

R
I Urobilinogen Increased Increased
N
E Hemosiderin Positive Negative

1. HEREDITARY SPHEROCYTOSIS  It may involve a defect or deficiency in spectrin

 A defect in the RBC membrane protein
composition leading to hyperpermeability of the
cell to sodium.
 A genetic problem that leads to production of
abnormal membrane protein or cause non-/ or
deficient production of red cell protein.
 Repeated removal of parts of the unsupported
membrane results to spherocyte formation.
 Loss of unsupported lipid membrane
or a defect in the binding of spectrin to protein
4.1, ankyrin, band 3, beta-spectrin, protein 4.2.
o Proteins involves are vertical proteins
that support the red cell membrane.
o Many proteins can be involve:
combination of which or it could also
mean the interaction of these proteins
to together like the connection of
spectrin to protein bands.
 HEMATOLOGY 2
 Uncoupling of unsupported
phospholipid bilayer due
defective protein. The detached
lipids will form vesicles and
remaining part becomes smaller
cell without a central pallor
spherocytes. (HEREDITARY)
 Spherocyte does not always mean a hereditary
condition, it could also be an acquired condition
(AUTOIMMUNE ANEMIA that can lead to
spherocytosis).
 It could also be ARTEFACTUAL: in an old stored
blood, most of the red blood cells are
spherocytes.
 Hence, it is needed to differentiate the true
hereditary membrane defect (HS) from other
causes of spherocytosis like in immune
hemolytic anemia.
o IMMUNE HEMOLYTIC ANEMIA:
antibody sensitized the red blood cell. So
when this sensitized RBCs passes
through the spleen, the macrophages
will recognize it as a foreign/abnormal
cell and remove the inclusion (attached
antibody) in the cell (splenic pitting). As
the macrophage pulls off the antibodies
from the red cell, it also knits-off some
parts of the red cell. The remaining RBC
fragments will reseal and form into
spherocytes.
 Since some of these proteins take part in ion-
exchange channel, deficiency will cause
alteration or have defect on the channel thus
ion transport is affected.
o Sodium-Potassium ATPase: instead of
normal ion-exchange (normal amount
of Na (major extracellular cation) and K
(major intracellular cation) is
maintained.  Defect in transport 
will result to accumulation of sodium
inside or the cell gains more sodium
causing also the cell to gain more water
hence the cell will be bloated.
LABORATORY FINDINGS:
FINDINGS IN BOTH HEREDITARY AND ACQUIRED
 Spherocytes, microspherocytes and many
reticulocytes
o Seen in both hereditary and acquired
condition
o Reticulocytes  as compensatory
mechanism of BM
 Osmotic Fragility Test – always increased
o Since the spherocytes are non-
deformable because they are already
fully distended, they are fragile and
easily lyse.
 MCV: normal/slightly decrease
o Normocyte is larger than spherocyte,
but when it comes to volume, they are
the same. The volume of spherocyte is
closely similar to normocyte despite
the difference in size because
although the spherocyte is smaller, its
entire volume is occupied by the
hemoglobin (no central pallor) fully
distended. Unlike the normocyte
wherein it may have a normal size but
its volume is not entirely occupied
(1/3 central pallor).  SPHEROCYTE
and NORMOCYTE have the SAME
VOLUME.
o DIFFERENCE: in SIZE  spherocyte
(smaller) vs. normocyte (larger)
 MCHC is increased  no central pallor due to
diminished volume thus the entire volume will
be occupied by the hemoglobin. When
hemoglobin is measured in relation to the cell
volume, it is high. Hence, MCHC is above 36%.
o Spherocytes are cells with highest
MCHC but the volume is normal
 Autohemolysis test – positive
o When the spherocytes are incubated
for 48 hours, the more they become
sphere therefore they hemolyse on
their own.
DIFFERENTIATION:
 Direct Antiglobulin/Coomb’s Test (DAT/DCT)
– negative
o Test done in blood banking to
determine occurrence of in vivo
sensitization of RBCs
 HEMATOLOGY 2

o Determine whether the RBCs were whether the spherocytosis is hereditary,

attached with antibodies in vivo immune or other causes.
o In HEREDITARY: no antibody-  For hereditary spherocytosis and other
associated whether in vivo or in vitro anemia characterized by spherocytosis
 NEGATIVE in DAT o Spherocytes are test using OFT
o In IMMUNE HEMOLYSIS that result to because they are the most fragile cell
SPHEROCYTOSIS: there are  able to detect the presence of
antibodies-associated  POSITIVE in spherocyte in the circulation.
DAT o RESULT IN HS: 0.60 - 0.65% saline
CLINICAL SIGNS AND SYMPTOMS solution, hemolysis is already
JAUNDICE AND SPLENOMEGALY observed: spherocyte (fragile) easily
 Since it is a type of hemolytic anemia, increase lyse
in bilirubin causing the jaundice.  PRINCIPLE: The cell is placed in a hypotonic
 With the predominance of spherocytes in the saline to cause osmosis (the shift of the fluid
circulation, the spleen becomes overworked from the hypotonic saline into the red blood
in the response of removing them.  cell). If the red cell could still contain its
SPLENOMEGALY: enlargement of the spleen volume, the RBC would be able to absorb
 When the spleen is enlarged, the more it water without lysing and if not, it will easily
sequesters blood and destroys more blood. hemolyse. POSITIVE RESULT: Hemolysis
 HEREDITARY SPHEROCYTOSIS (HS): the main  RESULTS:
cause of hemolysis is the spleen. It is the o Incomplete hemolysis: 0.45%
spleen that causes the hemolytic process  Faint pink tinge supernatant
because the function of the spleen is to filter with intact red cell button
the blood thus it removes abnormal cells. o Complete hemolysis: 0.35%
Since spherocytes are abnormal cells, they  Homogenous pink/red
remove it through hemolysis. solution with the absence of
 It is the hemolytic process that will lead to the red cell button.
complications  when the bilirubin levels  Fragility means increased OFT  cells are
increased and the hemoglobin is rearbsorbed easily lyse (spherocytes)
in the kidneys wherein the heme is toxic to the  INCREASED OFT:
kidney  HS. o HS, abnormal membrane, severe G-6-
TREATMENT PDH deficiency, PK deficiency,
SPLENECTOMY is considered to Hemolytic anemia
prevent/lessen/correct the extravascular hemolysis  DECREASED OFT:
however it will not correct the spherocytosis because o Resistant to red cell lysis even when
it is hereditary and therefore the abnormal gene will placed in hypotonic solution
always be there causing the production of  With increased surface area-
spherocytes. But the spherocytes will no longer to volume ratio
hemolyse because the spleen was removed.  Hypochromic cells, target
cells/leptocytes, sickle cell
o Severe IDA (microcytic-hypochromic
LABORATORY TESTS: cell)
1. OSMOTIC FRAGILITY TEST o Thalassemia (microcytic-
 A test to identify the ability of red cells to hypochromic and target cell)
absorb water without lysing. o Sickle cell anemia: If the sickling is still
 It reflects the shape and size of erythrocyte mild and is reversible, when the sickle
(specifically the surface area-to-volume ratio) cell absorbs water it goes back to
 Not a confirmatory test for HS; it will only being a normal cell. However, if its
identify the presence of fragile cells like membrane is totally damaged, the
spherocytes. But it will not determine
 HEMATOLOGY 2
sickling becomes irreversible
therefore becomes resistant.
 AFFECTED BY:
o Surface area-to-volume ratio
 A normocyte with normal
central pallor is resistant to
lysis because the space can
bloat to extend the capacity,
so the cell can absorb more
water without hemolyzing.
But a cell without a central
pallor will immediately lyse
upon absorption of water like
spherocyte.
 If the surface area-to-volume
ratio is still high, the cell is
resistant
 If the surface area-to-volume
ratio is decrease, the cell is
fragile  easily lyse
 Cells with decreased surface
are-to-volume ratio have a
limited capacity to expand in
hypotonic solution and
therefore lyse even at a less
hypotonic concentration of
saline than the normal
biconcave cells.
o Functional state of the cell
membrane
 If the red cell membrane is
defective, it is easily
hemolyse.
 Like in HS, there is membrane
problem wherein it lack
integrity due to loss of
protein.
o Sanford Method  macroscopic
 Reagent: 0.5% saline + add
corresponding drops of
DH2O
 SPECIMEN: Heparinized or
defibrinated blood
 PROCEDURE: Prepare
different concentration of
hypotonic saline using the
following procedure:
1. Set up 12 test tubes and
label no. 14 -25.
2. Place in each tube 0.5%
NaCl solution (The number of
the tube corresponds to the
drops of 0.5% NaCl)
3. Using the same dropper,
add distilled water into each
tube until a total of 25 drops
(NaCl & water) are in each
tube.
4. Dispense to each tube one
drop of heparinized or
defibrinated blood. Note:
Deliver at the same angle
directly to saline solution.
5. Mix tube and allow to
stand at room temperature
or centrifuge for 1 minute at
2,500 rpm.
6. Examine each tube for
initial/incomplete and
complete hemolysis,
 DETERMINE THE
CONCENTRATION OF NaCl
SOLUTION WHERE INITIAL
AND COMPLETE HEMOLYSIS
OCCURRED.
 FORMULA: %NaCl = test tube
number x 0.02
o Dacie Method  more accurate
 Hypotonic saline (pH 7.4)
 Buffered at pH 7.4 using PO4
in order to prevent
interference of inconsistent
pH
 RECAP: OSMOSIS: movement
of fluid in order to attain
homeostasis/equillibrium
 Outside is hypotonic, inside is
isotonic movement of
water from outside to inside
 Hemolysis: @540 nm
 INCUBATED OFT: incubate
the sample for at least 24
hours to detect mild forms of
HS
 It is done because if the
observation is done
immediate and the HS is mild
there will be no hemolysis
seen. false negative result
 Mild forms of HS: on
prolonged incubation they
 HEMATOLOGY 2
become fully spherocyte
therefore show hemolysis
 PURPOSE: detect mild forms
of HS
2. EOSIN-5’-MALEIMIDE (EMA) BINDING TEST
 Alternative confirmatory test  accurate
3. SODIUM DODECYL SULPHATE-
POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE)
4. OSMOTIC GRADIENT EKTACYTOMETRY
5. ACIDIFIED GLYCEROL LYSIS TEST
6. AUTOHEMOLYSIS TEST
7. HYPERTONIC CRYOHEMOLYSIS TEST
8. PCR
 single-stranded and conformational
polymorphism analysis
 alternative confirmatory test
NOTE:
Since HS is a genetic disorder, the confirmatory tests
are the genetic studies like chromosomal studies or
polymerase chain reaction (PCR).
2. HEREDITARY ELLIPTOCYTOSIS
 A defect in the skeletal protein interaction
(spectrin dimmers, ankyrin, protein 3 and 4.1)
anemia is normocytic normochromic with >25%
elliptocytes.
o Proteins involves are horizontal proteins
that support the red cell membrane.
 Clinical manifestions are mild, or none at all.
 Deficiency of glycophorin C
 ACQUIRED: <25% elliptocytes of the RBC
population
 HEREDITARY: >25% elliptocytes of the RBC
population
 Splenectomy is not considered
VARIANTS:
SOUTHEAST ASIAN OVALOCYTOSIS (SAO)
 In Southeast Asian Ovalocytosis/ (SAO), the
mutation in protein band 3 results to
increased membrane rigidity, making the red
cells resistant to Plasmodium invasion.
o Mutation: gene for band 3 increased
rigidity (not flexible) of the membrane
and resistance to invasion by malaria
o BAND 3: gives elasticity and flexibility
of the RBC membrane
 Common in southeast asian population
 Since the RBC membrane is rigid and instead
of showing a pale center area, a band is
formed at the center
 PINCER-LIKE CELLS: traverse ridge or
longitudinal slit
HERIDITARY PYROPOIKILOCYTOSIS (HPP)
 Affected red cells are heat sensitive and start
to bud & fragment at 45-46°C. Normal red
cells fragment starting at 49°C.
 Budding and fragmentation at 45-46 °C
 Normal RBC will start to fragment or bud at 49
degree Celsius  vesicle formation and
fragmentation
 But cells in HPP are heat-labile meaning that
even at lower temperature (45-46 C) they
easily bud off and fragment
 If they bud off they will form a small vesicle
which are called microspherocytes
buds/fragments from RBC
 PYROPOIKILOCYTES or MICROSPHEROCYTES:
cells that easily buds and fragment @45-46
degree Celsius
 Pyro: means heat/fire cells are resistant to
heat
SPHEROCYTIC HE
 Variant of both HS and HE
 HEMATOLOGY 2

 The patient has the abnormal gene resulting  Deficiency will lead to decreased membrane
to spherocytic and elliptocytic HEREDITARY transport mechanism hence it will lead to
 BLOOD SMEAR will show combination of dehydrated or overhydrated cell.
many spherocytes and elliptocytes  Normal cell should maintain more
concentration of sodium outside and
potassium inside but if the defect of the
3. HEREDITARY STOMATOCYTOSIS: transport protein results to:
HYDROCYTOSIS (OVERHYDRATED) & o A NET INCREASE in cellular cations (
HEREDITARY XEROCYTOSIS (DEHYDRATED)  Na and K inside)  cell becomes
 A mild hemolytic anemia characterized by too concentrated which will drive the
increased permeability and flux of both Na+& K+. water from the outside to shift inside
Intercellular water is influenced by ions,  cells gains more water appearing
primarily sodium, thus, a net increase or as STOMATOCYTE: a swollen cell/fully
decrease in cellular cations will influence the hydrated cell
shift of fluid shift into or out of the cell. o A NET DECREASE in cellular cation
almost all of the cations have left the
 ALTERED MEMBRANE TRANSPORT PROTEINS
RBC as the cations leaves the RBC,
 In phospholipid bilayer aside from cytoskeleton, even intracellular water will also shift
peripheral proteins at the inner leaflet or lining, outside the RBC  cells become
there are also proteins that traverse the bilayer dehydrated or dessicated 
which are called integral proteins, which serve as appearing as DESSICOCYTE or
ion channels or transport proteins. Integral XEROCYTE: a dehydrated cell
proteins can also be defective altering the
membrane transport mechanism which can
The blood antigens are
result into:
found on the red cell
o Overhydrated Hereditary
surface like the Rh
Stomatocytosis/Hydrocytosis antigens which add in
 Stomatocytes (hydrocytes) are the integrity of the RBC
formed due to the shift of fluid membrane. Hence their
into the cell (swollen cell) these absence (complete
cells have decreased surface-to- absence: Rh null disease)
volume ratio make the RBC
o Dehydrated Hereditary membrane incomplete
Stomatocytosis/Xerocytosis therefore it is
 Xerocytes (dessicocytes) are compromised.
formed due to the shift of fluid Net INCREASE in cellular
out of the cell (dehydrated cell). cations Decrease
surface-to-volume ratio
These cells have increased
OFT: Increase  prone
surface-to-volume ratio.
to lysis MCV: Decreased
CAUSES: Seen in Rh null
1. Mutation in the RHAG gene Net DECREASE in cellular
 RH Blood Group system: any of the antigen cations Increased
DCcEe surface-to-volume ratio
OFT: Decrease 
2. Absence of stomatin (band 7 region) resistant to lysis
3. Mutations in the PIEZO1 gene (Piezo-type MCHC: Elevated  cells
mechanosensitive ion channel component 1 hemoglobin appears
protein)
 HEMATOLOGY 2

concentrated due to 3. Rapoport-Luebering Pathway

intracellular water loss  Production of 2,3 DPG
Characterized by Mild  Regulates hemoglobin affinity to oxygen
hemolytic anemia  NOT involved in ATP production or RBC
hemoglobin protection
SUMMARY: MEMBRANE DAMAGE 4. Methemoglobin Reductase Pathway
HEREDITARY SPHEROCYTOSIS: most affected protein  Maintains hemoglobin in the reduced form
structure are the vertical ones like ankyrin and band 3

HEREDITARY ELLIPTOCYTOSIS: the proteins involved

can be the same with HS but the position of the
protein is horizontal like actin

HEREDITARY STOMATOCYTOSIS: whether in

dehydrated or overhydrated, the problem are the
transport proteins

1. GLUCOSE-6-PHOSPHATE DEHYDROGENASE
DEFICIENCY ANEMIA
 This is the most common RBC enzymopathy
associated with hemolytic anemia, inherited as
a sex-linked disorder. Red cells need G6PD to
generate NADPH and reduced glutathione
HEMOLYTIC ANEMIA DUE TO METABOLIC
(GSH). These reducing potentials are important
ABNORMALITIES (ENZYMOPATHIES)
in protecting hemoglobin from oxidation.
 G-6-PD enzyme is found in hexose
Metabolism Pathway in RBCs: monophosphate shunt which functions to
provide the reducing potential of the RBC or to
1. Embden-meyerhoff Pathway: anaerobic provide the antioxidant property of RBCs.
glycolysis  In the absence of G6PD, hemoglobin is not
 Provides 90-95% ATP protected from oxidation by peroxides. It will be
 Mature RBCs lack mitochondria thus they easily oxidized becoming
produce ATP through EMP
methemoglobin/hemoglobin  iron is in the
ferric state
2. Hexose Monophosphate Shunt or Pentose
Phosphate Pathway:  Oxidized hemoglobin easily denatures and
 Oxidative pathway precipitates as Heinz bodies.  located at the
 Provides 5-10% ATP periphery
 Production of reduced glutathione  main o Heinz bodies are found at the periphery
function attach to red cell membrane inducing
 Glutathione is the main antioxidant property cell rigidity and fragility.
of RBC  detoxifying agent o When RBC with Heinz bodies enters a
microcirculation(splenic sinusoids), a
 HEMATOLOGY 2

part of the cell which is flexible can pass

through however the part where there
is inclusion (rigid) will not and will be
detached from the RBC  the
remaining cell will be knit off producing
now a pitted red cells called bite cell,
horned cells, thorned cell or
degmacyte.
 Increased oxidation may be associated with
exposure to oxidant drugs, diabetic acidosis &
during neonatal period.
o Included in newborn screening in the
Philippines.
 G6PD Mediterranean is occasionally associated
with severe hemolytics episodes ( CLASS ENZYMATIC ACTIVITY DEGREE OF
ASSOCIATED
hemoglobin) of following consumption of fava
HEMOLYSIS
beans (favism)
I Severely deficient  Chronic hemolysis
o G6PD Mediterranean - favisms most severe
 PBS: Heinz bodies, bite cells, polychromasia II Severely deficient Acute, episodic
MAIN REACTION: Glucose-6-Phosphate (<10% residual
Dehydrogenase (cofactor NADP; when functions it activity)
becomes reduced into NADPH  reducing potential) III Moderately to mildly Acute, episodic
catalyzes the conversion of Glucose-6-Phosphate into deficient (10% to 60%
6-phosphogluconate. residual activity)
IV Mildly deficient to Absent
Importance of reduced NADP  NADPH normal (60% to 150%)
1. Protects the hemoglobin from being oxidized; the V Increased (>150%) Absent
H of NADPH will be given to hemoglobin reverse the Not all G6PD deficiency is characterized by severe
oxidation and reduces methemoglobin (oxidized hgb) hemolysis, it depends on the type (5). With hemolysis
back to reduced hemoglobin. (1-3); without hemolysis (4-5)
2. Reduce another reducing potential: Reduced
Glutathione
DIAGNOSTIC LABORATORY TESTS FOR G6PD
 Good reducing potential if it is in reduced
DEFICIENCY:
form; it can reduce back methemoglobin into
1. FLUORESCENT SPOT TEST (REDUCTION)
normal form.
 Recommended screening test): hemolysate +
 Major antioxidant property: breaks down
reagent  NADH – NAD (NADH conversion to
hydrogen peroxide (toxic) into water and
NAD results to loss of fluorescence)
oxygen; detoxify peroxides and free radicals
 Blood + Reagent: G6P + NADP  NADPH + 6-
PG
Methemoglobin production: 3.5 himeglobin daily
 RESULTS:
If accumulated can suffer from methemoglobinemia
o With fluorescence: conversion of
 abnormal hgb that can’t transport oxygen
NADP into NADPH
o Without fluorescence: absence of
NADPH; conversion of NADPH into
NADP  reaction does not occur
normally

2. ASCORBATE CYANIDE TEST

 HEMATOLOGY 2
 Measures the ability of RBCs to detoxify
hydrogen peroxide when incubated with
ascorbate. A brown color develops
(methemoglobin) in the absence of adequate
RBC G6PD.
 If the patient RBC has a normal G6PD levels,
it is able to produce NADPH which will cause
the reduction of glutathione (major
antioxidant property of red cell that will
break down hydrogen peroxide)
 DETOXIFICATION OF HYDROGEN PEROXIDE
(H2O2)
 PROCEDURE:
o RBC (hemoglobin) + ascorbate
cyanide reagent –incubated-- 
produces H2O2 (hydrogen peroxide,
a toxic radical that destroys
hemoglobin)
o RBC (Normal – G6PD) + H2O2 
Hemoglobin (normal)  pink
solution: cell is maintained in normal
state
o RBC ( Decrease – G6PD) + H2O2 
Hemiglobin/Methemoglobin 
brown solution: G6PD deficient cell
3. METHEMOGLOBIN REDUCTASE TEST
 INDIRECT TEST that determines the ability of
red cell to reduce methemoglobin back to
normal hemoglobin.
 All hemoglobin compounds
(abnormal/normal) are reversible EXCEPT
sulfhemoglobin.
 Hemiglobin is reversible.
 Detects NADPH generation by G6PD. The
transfer of H+ from NADPH to
methemoglobin results to its reduction back
to normal hemoglobin.
o If patient RBC have normal levels of
G6PD, then it is able to generate
enough NADPH (reducing potential)
o Methylene Blue can be added to
make the transfer of hydrogen to
methemoglobin faster.
o It facilitates a fast transfer of
hydrogen
4. AUTOHEMOLYSIS TEST (SCREENING TEST
FOR PK AND G-6-PD DEFICIENCIES)

Measures the spontaneous lysis of RBCs
when incubated at 37°C for 48 hours.
Depletion of Glucose and ATP during
incubation results to membrane loss,
spherocyte formation, and then ultimately,
lysis. If positive, test is repeated with addition
of Glucose and ATP to protect against
autohemolysis.
INTERPRETATIONS:
 Normal sample: 0.2-2% hemolysis; 0%-0.8% /
0.9% if with ATP or glucose
 G-6-PD deficiency: moderate high
autohemolysis, partially corrected by glucose
 PK deficiency: greatly high autohemolysis,
corrected by ATP
 Hereditary spherocytosis: Greatly high
hemolysis corrected by glucose or ATP but not
to a normal value.
2. PYRUVATE KINASE DEFICIENCY ANEMIA
 Pyruvate kinase enzyme is found in EMPathway
(anaerobic glycolysis) which is important in the
synthesis of ATP. Hence, its deficiency will lead
as well to ATP deficiency.
 PK catalyzes phosphoenolpyruvate to pyruvate.
 ATP energizes the Na-K pump which is
necessary in the maintenance of RBC
membrane
 PK deficiency results to impaired Na+-K+ pump.
Water and K+ are lost resulting to shrinkage,
distortion, speculation and premature RBC
destruction.
 RBC morphology is generally normocytic-
normochromic, with presence of
polychromasia.
NOTE:
In embden meyerhoff pathway, there are two
reaction that are ATP producing:
(1) Conversion of 1,3 – biphosphoglycerate into 3-
phosphoglycerate
(2) Conversion of phosphoenolpyruvate (PEP)
into pyruvate 
catalyzed by pyruvate kinase
There is two (2) glyceraldehyde-3-phosphate so there
will be 4 ATP molecules produced. However, if there
is a deficient enzyme like PK, ATP production will also
be deficient.
 HEMATOLOGY 2

o Without fluorescence: Normal PK:

conversion of NADH into NAD+
(oxidized form)
 If patient RBC have PK, it will
cause the conversion of the
above reaction
 Loss of fluorescence means
normal pyruvate kinase
enzyme

2. AUTOHEMOLYSIS TEST (Screening Test for

IMPORTANCE OF ATP IN RBC:
1. Active Transport: Transport protein + ATP
 The transport of ion is mostly active thus
ATP is needed like in Calcium pump and
NaK ATPase pump
 Facilitated transport: only transport protein
is needed
Thus the effect of ATP deficiency is that the transport
of ions along cell membrane becomes abnormal thus
the cell loses more potassium and losses more water
 crenated/distorted cell that is prone to
lysis/destruction
DIAGNOSTIC LABORATORY TESTS FOR PK
DEFICIENCY
1. FLUOERSCENT SPOT TEST (OXIDATION)
 Blood RBC (Hemolysate) + reagent (PEP,
NADH, LD) –incubate--  NADH  NAD+
o PEP – PK  Pyruvate –Lactate
Dehydrogenase (with cofactor NADH)
 Lactate
 Pyruvate: aerobic product
 Lactate: anaerobic product
 RESULTS:
o With fluorescence:
Abnormal/Deficient PK: Presence of
NADH (reduced form)
PK and G-6-PD Deficiency)
 For diagnosis of PK, G6PD, HS
 Incubated in sterile condition at 37 degree
Celsius for 48 hours (2 days)
 Measures the spontaneous lysis of RBCs
when incubated at 37oC for 48 hours.
Depletion of Glucose and ATP during
incubation results to membrane loss,
spherocyte formation, then ultimately, lysis.
If positive, test is repeated with addition of
Glucose and ATP to protect against
autohemolysis.
 INTERPRETATION:
o POSITIVE RESULT: Hemolysis
o 1st test: positive for hemolysis 2nd
test: add Glucose or ATP repeat
autohemolysis test
 1st test: Greatly
High/Increase Hemolysis;
2nd test: hemolysis is
corrected by ATP  PK
deficiency
 1st test: Moderate Hemolysis
partially; 2nd test: hemolysis
is partially corrected by
glucose  G6PD deficiency
 3rd test: Greatly Hemolysis;
2nd test: hemolysis is
partially corrected by ATP
and Glucose  HS
INTERPRETATIONS:
 Normal sample: 0.2-2% hemolysis; 0%-0.8% /
0.9% if with ATP or glucose
 G-6-PD deficiency: moderate high
autohemolysis, partially corrected by glucose
 PK deficiency: greatly high autohemolysis,
corrected by ATP
 HEMATOLOGY 2
 Hereditary spherocytosis: Greatly high
hemolysis corrected by glucose or ATP but
not to a normal value.
ACQUIRED INTRACORPUSCULAR HEMOLYTIC
MECHANISM
1. PAROXYSMAL NOCTURNAL HEMOGLOBINURA
(PNH)
 Acquired intrinsic defect which arises in the
bone marrow stem cells affecting RBCs, WBCs
and platelets.
o The cause of mutation is not yet
established because many factors are
involved.
o Acquired but genetic in
nature/hereditary disease
o EFFECT OF MUTATION: the mutation
has lead to deficiency or absence of
complement defense protein like the
regulatory protein C3 convertase. The
complement system is part of the
body’s defense mechanism thus it
function normally if its activation occurs
normally. The complement protein
causes lysis but RBCs are resistant to it
because it is protected by C3
convertase however in this disease, the
RBC does not elaborate these defense
proteins thus prone to hemolysis.
Another cause is the deficiency or
absence of GPI which supposedly
should function to linked complement
proteins thus deficiency will lead to
hemolysis.
 ↓ / (-): complement defense
proteins (C3 convertase)
 ↓ / (-): Glycosylphosphatidyl
Inositol (GPI)  linked protein
 Affected cells show an abnormal sensitivity to
complement in acidified plasma or low ionic
strength environment, resulting to
complement-mediated lysis.
o Affected cells (acidified plasma/Low
Ionic Strength)  COMPLEMENT-
mediated lysis
o COMPLEMENT: a thermo-labile protein
coming from complement activation
that in the presence of antibodies may
mediate hemolysis
 ETIOLOGY:
o HEMOGLOBINURIA: presence of
hemoglobin in the urine due to
hemolytic anemia (intravascular
hemolysis)  hemoglobin is directly
freed into the plasma and plasma is
filtered by the kidney hence will appear
in the urine
 Hemolysis mainly is the cause
for hemoglobinuria
o NOCTURNAL: means NIGHT.
 The intravascular hemolysis and
hemoglobinuria occurs chiefly
at NIGHT  plasma is more
acidified thus the RBCs
hemolyse.
o PAROXYSMAL: mean INTERVAL
 The lysis does not occur
everytime or constantly
anytime but it occurs at an
INTERVAL  NIGHT
 Decrease or absence
 Most cases of PNH, the patient conditions are
related to aplastic anemia indicating that the
mutation is caused by weaken bone marrow.
o In aplastic anemia, bone marrow is
defective, it has decreased function due
to infiltration of marrow fats hence it is
prone to mutation.
NOTE:
The complement system has 3 pathway: classical,
alternative, and lectin pathway in which involve the
assembly of C3 convertase that will later cleave C3 into
C3a and C3b and other fragments. C3 convertase is
injurious to RBC, that C3 may bind with the RBC and
stress it. Sooner or later, the RBC bound with
complement hemolyse. However, if the RBC have on
their surface the expose CD55 and CD59 which are
protective proteins, the RBC will be protected against
complement. CD55 and CD59 inhihits the further
activation of complement.
In PNH, CD55 (also called DAF) and CD59 are
deficient/absent, thus on continued activation of the
complement pathway, there will be formation of
membrane-attack complex which can also assemble
 HEMATOLOGY 2

on surface of RBC together with the complement  PROCEDURE: incubate RBC with the
proteins that will induce further lysis. following:
o Weak acid: complement activation
MAIN MUTATION in PNH occurs in the gene and cause the complement to attach
responsible in the production of the normal GPI  on RBC surface causing stressful
mutation of GPI environment and later hemolysis
GPI: links regulatory protein like CD55 and CD59 on o RBC + N. serum + 0.1% HCl  weak
RBC surface DEFICIENCY OF EITHER/BOTH will lead to acid  POSITIVE
prone complement-mediated lysis. o RBC + Pxt. Serum
o RBC + Inactivated serum  NO
HEMOLYSIS: complement is there
but inactivated; complement
proteins are thermo-labile thus when
heated at 56 degree Celsius will
cause its inactivation.
 Affected RBCs are abnormally sensitive to
complement-mediated lysis in acidified
serum (use of weak acid as reagent)
 Positive screening + Positive confirmatory
test  PNH
 Also test CDA II (HEMPAS)

Currently, flow cytometry procedures are available

that can test white cells for the presence or absence
of GPI-linked proteins. White cells can be examined
DIAGNOSTIC LABORATORY TEST FOR PNH
for reactivity to anti-CD48, CD55, and CD59, all of
1. SUGAR WATER SCREENING TEST which are anchored proteins. A new diagnostic
 Whole blood and normal control are procedure, FLAER (fluorescent- labeled aerolysin) has
incubated at 37 degrees Celsius in a LISS. proved effective in detecting smaller populations of
Hemolysis in the test/sample may indicate abnormal leukocytes in PNH.
complement-mediated lysis. OTHER DIAGNOSTIC TESTS FOR PNH
o LISS promotes binding of
FLOW CYTOMETRY
complement to RBC  straining;
 Able to detect which specific GPI or
while the complement is on the RBC
defense/regulatory protein is deficient or
surface, it stresses the cell until such
defective in affected cells because it made
time the RBC can no longer bear the
use of specific anti- sera
stress and hemolyse
 ANTI-SERA for: CD48, CD55, CD59, WBC and
o WB in LISS at 37°C
other blood cells
o NC in LISS at 37°C
o When blood cell react to the anti-
 RESULTS:
sera means that they possess the
o WB: with hemolysis; NC: no
following markers have normal
hemolysis  valid but should not yet
regulatory proteins
reported  proceed to another test
 GPI – linked proteins; -WBCs reactivity to
 confirmatory test like sucrose
anti-CD48, CD55 and CD59
hemoysis test and Ham’s acidified
serum test
FLAER (FLUORESCENT-LABELED AEROLYSIN)
2. SUCROSE HEMOLYSIS TEST
 It uses fluorescent dyes and has an
 Confirmatory Test
advantage of being able to detect even the
small population of affected cells
3. HAM’S ACIDIFIED SERUM TEST
 More sensitive
 Confirmatory test for PNH
 HEMATOLOGY 2
ACQUIRED IMMUNE ANEMIC OF INCREASED
DESTRUCTION (Antibody Related)
1. ISOIMMUNE HEMOLYTIC ANEMIA
 Antibodies comes from another person:
o Hemolytic Disease of the Fetus and the
Newborn (HDFN)
 Rh negative mother; Rh positive
baby
o Hemolytic Transfusion Reactions (HTR)
2. AUTOIMMUNE HEMOLYTIC ANEMIA (AIHA)
 Antibodies produced by patient’s own body
 Happens when the patient’s immune system
becomes altered to cause the production of
antibodies that will attack the patient’s own
RBC causing lysis.
 Can be drug-induced, infections, unknown
 WARM AIHA: IgG production reacts @37
degree Celsius; more common
o Associated/Pre-exist with CLL, wherein
the affected lymphocytes are the
dormant B-cells but they start
producing abnormal antibodies that will
attack patient’s own red blood cell
causing WARM AIHA
 COLD AIHA: IgM production reacts @colder
temperature (0-10 degree Celsius)
o Bind with RBCs at cold temperature,
since IgM is pentamer, more RBC can
bind thus agglutinates more.
NOTE:
WARM-ANTIBODY AIHA
- Occurs when the patient’s own immune
system produces anti-RBC antibodies (IgG
antibodies) that react most effectively at
warm temperatures
COLD-ANTIBODY AIHA
- Caused by pathologic antibodies (IgM
antibodies against the I antigen) that react
most effectively at cold temperature (0-10
degrees Celsius). Often associated with M.
pneumoniae & Epstein-barr virus/EBV
infections. Patients exposed to these
infections may develop harmful Anti-I & Anti-
I antibodies, respectively.
IgM Ab  directed against I Ag: 0-10 degree Celsius
 Harmless cold antibodies: Anti-I, Anti-H and
Anti-IH
 Harmful antibodies: produced in infections 
M. pneumonia (Anti-I); EBV (Anti-i) 
hemolytic anemia
 Reynaud’s phenomenon/acrocyanosis:
Cyanotic appearance: red- blue-black
coloration plus painful sensation on the
exposed parts of the body due to
vasooclussion caused by agglutination of the
RBC by the abnormal antibody.
The exposed body parts like ears and fingertips which
are the coldest or easily cold part of the body. The
IgM which is the Anti-I will bind to I antigen causing
now agglutination. So the blood vessels supplying the
distal tissues will be obstruction resulting to non-
oxygenation  cyanotic  may even lead to
necrosis/death of tissue.
3. PAROXYSMAL COLD HEMOGLOBINURIA (PCH)
 The hemolysis does not occur anytime but
occurs in an interval  during cold temperature
o Technically, hemolysis occurs in warm
temperature but it is called cold because
antibodies bind in this temperature.
 Caused by the binding of the Donath-Landsteiner
antibody to the patient’s red cells following
exposure to cold. Donath-Landsteiner is an Ab
specific for Pp blood group system. It is biphasic
that reacts at cold and warm temperature. At
 HEMATOLOGY 2
cold temperature, it binds to the red cell
membrane without causing lysis. Intravascular
hemolysis and hemoglobinuria occurs as the
temperature returns to normal body temp.
 It is immune because of the presence of
antibody: Donath-Landsteiner (D-L) antibody is
directed for the Pp blood group (hence, also
called  anti-P). IgG antibody type but biphasic
which reacts in both warm and cold
temperature.
o In cold temperature, it does not cause
hemolysis, it only binds to surface of the
RBC. This binding will cause the partial
activation of complement mechanism
from C1 to C4. Nothing will happen if the
patient is maintained in the cold
temperature however when exposed to
warmer/normal (37 degree Celsius)
temperature, the D-L antibody will be
detached and the complement
mechanism will completely (C3-C9) be
activated thus hemolysis will occur.
o Complement – mediated lysis by D-L
antibody
 D-L TEST
o PROBLEM: presence of antibody which is
found in the serum
o TEST: Pxt serum + Reagent cells: O and
P+( for anti-P) normal cells incubate @
4 degree Celsius transfer to 37 degree
Celsius incubation
o RESULTS:
 HEMOLYSIS: Presence of D-L
antibody
 NO HEMOLYSIS: absence of D-L
antibody
ACQUIRED EXTRACORPUSCULAR HEMOLYTIC
ANEMIAS
 Vascular OBSTRUCTION caused by fibron clots or
threads (DIC and MAHA) and by abnormal
platelet aggregates (TTP and HUS)
1. MICROANGIOPATHIC HEMOLYTIC ANEMIA
(MAHA)
 SECONDARY HEMOSTATIC
DEFECTS/COAGULATION DISORDER
 Caused by abnormal deposition of fibrin or
atheroma, or anatomic defects of the small
blood vessels resulting to reduced blood vessel
lumen and increased blood pressure. The
combination of this abnormal increase in the
force of moving cells and the smaller vessel
cause injury and fragmentation of RBCs.
 When the cell passage is small and is obstructed,
the circulation becomes limited and to
compensate for the decreased blood supply,
there will be increase in cardiac output and the
effect is increased blood pressure.
o The combination of increased pressure
pushing the red cell and the limited
circulation where the red cell should
pass through  trauma to red cells
 PBS: presence of schistocytes  hallmark of
hemolytic anemia
 Affects or occurs in the small blood vessels
wherein it further narrows due to the:
o Presence of fibrin deposits causing
obstruction
o Presence of atheroma plaques like in
diabetes mellitus or
hypercholesterolemia  thickening and
hardening of blood vessels (rough)
o Anatomic defects: blood vessel is very
small  hemolysis
2. DISSEMINATED INTRAVASCULAR
COAGULATION (DIC)
 SECONDARY HEMOSTATIC
DEFECTS/COAGULATION DISORDER
 Caused by extensive damage to vessel
endothelium or exposure to compounds that
initiate clotting resulting to fibrin deposition
along and across the vessel lumen. RBCs are
fragmented as they are pushed through the
vessel by the action of rapid circulation.
 Affects all types of blood vessels: small,
medium, large and may also affects either
male/female
 Test: Coagulation & Fibrinolysis tests
 Due to release the release of thromboplastin-
like or tissue-factor like substance, it will cause
the activation of both coagulation and
fibrinolysis, the effect of which is because of the
 HEMATOLOGY 2

continous activation of coagulation decreased  Shiga toxin attach to glomerular

coagulation factors; Increased activation of capillaries(renal damage)
fibrinolysis will lead to increased fibrin-split causing the release and
product (FSP) thus will result to bleeding activation of vWF which causes
o Decreased coagulation and Increased the platelets to adhere with
FSP them and aggregate 
OBSTRUCTION
3. THROMBOTIC THROBOCYTOPENIC PURPURA o NON-DIARRHEAL: in infant and children
(TTP) / MOSCHKOVITZ SYNDROME following vaccination
 PRIMARY HEMOSTATIC DEFECTS
 Associated with defective ADAMTS-13 which is 5. HEMOLYSIS DUE TO MECHANICAL INJURY
an enzyme that cleaves the ultralong and  Damage to the heart of aorta – RBCs fragment
ultralarge von Willebrand factor so they as they come in contact with damaged heart
become a smaller or medium sized vWF that is valves.
more effective in hemostasis. However the o Patients with artificial or prosthetic
deficiency/absence in ADAMTS-13, the vWF will heart valves
persist as large which easily binds with  Stress or March hemoglobinuria – lysis is caused
platelets. Ultralarge vWF will bind with platelets by mechanical pressure during exercise or
causing them to aggregate on them thus during marching
blood circulation, the platelet aggregates can o Also called Cadet’s,Orthostatic
detached and flow along the circulation and can hemoglobinuria/proteinuria, Cyclic
be deposited elsewhere causing now hemoglobinuria/proteinuria occurs
obstruction.  thrombosis (thrombus) red cell during the day
trauma o Hemoglobinuria: presence of
 Caused by formation of platelet aggregates (due hemoglobin in the urine due to
to activation) resulting to vascular occlusions hemolysis which is caused by stress and
and RBC fragmentation. More common in force against renal vein
young adults and is characterized by fever,  Infections & Infestations
petechiae, neurologic signs and renal disease. o PLASMODIUM (hemolytic condition;
intraerythrocytic parasite (humans),
4. HEMOLYTIC UREMIC SYNDROME (HUS) Babesia (ring forms; intraerythrocytic
 PRIMARY HEMOSTATIC DEFECTS cattles  humans)), Bartonella
 Etiology is not clear, though some cases are bacilliformis (Oroya fever), Clostridium
associated with mild febrile illnesses, certain perfringens, Infectious Mononucleos
immunizations, and gastrointestinal (EBV).
disturbances. o INFESTATIONS: spider bites 
 This is common among infants and young Parasitism outside the body
children manifested with acute hemolysis, renal  Exposure to Chemicals and Toxins
failure, neurologic signs, abdominal pain,  Thermal Injury
vomiting, and diarrhea.
 Associated with either diarrheal and non-
diarrheal condition
o DIARRHEAL: seen commonly in young
individuals caused by shiga
(Shigella/Salmonella) or verotoxin
producing organism like E.coli O15H:7
 HEMATOLOGY 2

BLOOD LOSS ANEMIA (POST-HEMORRHAGIC

ANEMIA)

NOTE:
Post-Hemorrhagic because anemia occurs after the
blood loss.
TYPES:
1. ACUTE
 The amount loss is >20% of patient’s blood
volume and the occurrence is too short like in
overt bleeding (gunshot wounds, accident,
trauma)
2. CHRONIC
 The amount of blood being is loss quite low
but on prolonged duration like GIT bleeding,
Menstruation, Repeated Pregnancy. Gradual
loss of blood.

ACUTE POSTHEMORRHAGIC ANEMIA CHRONIC POSTHEMORRHAGIC ANEMIA

It is acute when the amount of blood loss is >20% of the
patient’s blood volume.
EXAMPLE: Normal blood volume is 5 Liter  20% is 1L
If a patient loss a 1L of blood in a single incident  ACUTE
BLOOD LOSS  most patient can adjust to a blood loss of
20% however more than 20%  patient may suffer in
HYPOVOLEMIA (decrease in blood volume)
Hematocrit: PRBC/Total Volume
Loss of small amount of blood but in long duration. In
chronic blood loss, there is no disruption in the blood
volume but hemoglobin is loss therefore even iron is lost
leading to iron deficiency anemia.
Morphology: before becoming a full-blown (Stage 1and 2)
IDA: NORMOCYTIC-NORMOCHROMIC  Stage 3:
MICROCYTIC

When a patient loses blood, it will not select RBC only but
it is a loss of proportionate amount plasma and RBC. Thus
both plasma and RBC will decrease and when we get the
hematocrit immediately, it will appear normal. But there
will be sudden decrease in platelets and after an hour will
rise. There will be decrease in platelets because they will
be needing in arresting the bleeding by forming clots and
increased due to splenic mobilization which is caused by
stress.

Since there is hypovolemia, there will be vasoconstriction

which will decrease the blood flow however it should not
be remain as that for too long thus the body will adjust to
bring back blood vessel into its normal size. So in order to
 HEMATOLOGY 2

have vasodilution, it will cause a shift of fluid from

extravascular and interstitial compartment into the
circulation in order to expand it thus causing an increased
plasma volume over the red blood cell  hemodilution
(will only correct blood volume)  decreased hct and
hgb. Starting 3-5 days after the blood loss, the BM will
compensate by producing more blood cells and releasing
even immature blood cells: red blood cells
(reticulocytes/shift cells) and leukocytes (immature
neutrophil: shilling’s hemogram: shift to the left:
increased in young forms).

BLOOD MORPHOLOGY
Initial: normocytic-normochromic/ unchanged
depending on px blood pic. Later (3rd -5th day): will
become transient macrocytic  immature cells 6th day:
no macrocytosis: immature forms start to mature.

1. ACUTE POSTHERMORRHAGIC ANEMIA o Reticulocyte count and its associated

 Blood loss over a short period of time in
amounts sufficient to cause anemia. Normal
individuals are able to compensate for rapid
blood loss of up to 20% of the circulating blood
volume with few symptoms.
 Major manifestations of anemia are due to
depletion of blood volume
 RBCs are Normocytic-Normochromic followed
by transient Macrocytosis and increased
polychromasia
2. CHRONIC POSTHEMORRHAGIC ANEMIA
 Blood loss in small amounts over an extended
period of time. This results to gradual iron loss
and slower regeneration of red cells.
 At first, the anemia is Normocytic-
Normochromic and gradually becomes
Microcytic-Hypochromic
NOTE:
RETICULOCYTE COUNT
 It measure the erythropoietic activity of the
bone marrow.
corrections can be used to assess
bone marrow erythropoietic activity.
 RETICULOCYTE: 5th stage in the erythopoiesis
 first non-nucleated stage but it has
remnant of RNA; fully hemoglobinized
o Young erythrocytes that are in a
discrete, penultimate phase of
maturation where the nucleus has
been removed, however some of the
extranuclear RNA remains in the
cytoplasm.
o The term reticulocyte is derived from
the fact that the cell contains a small
network of basophilic material called
reticulum which is demonstrable only
by supravital stain.
o In the peripheral blood they are called
polychromatophilic erythrocytes.
 PRINCIPLE: Supravital staining of RNA
o The ribosomal RNA is stained
supravitally. Any non-nucleated RBC
that contains stained
particles/granulofilamentous
materials are counted as reticulocyte.
 HEMATOLOGY 2

o Supravital staining: staining cells RETICULOCYTE POLYCHROMATOPHILI

while in their living state C CELLS
o STAINS: Most commonly used: New STAIN: Supravital stain Without granules 
Methylene Blue and Brilliant Cresyl The granules are arranged RNA is not precipitated
Blue; Others: Janus Green, Nile Blue in “reticulum pattern” Non- but the cell will be seen
sulfate nucleated; with 2 or more as polychromatophilic
 METHOD OF STAINING: Wet method and Dry blue-stained (polychromasia)
Method (schilling’s method) particles/granulofilamento showing a lot of colors
 SPECIMEN NEEDED: EDTA-anticoagulated us  precipitated RNA (grayish, bluish,
blood or capillary blood greenish) due to
 PROCEDURE: mixture of the cell.
a. Mix equal amounts of filtered stain COMPOSITION OF
and EDTA-anticoagulated blood or WRIGHT’S STAIN:
fresh capillary blood in a small test Methylene blue (basic
tube dye) and eosin(acidic
b. Incubate mixture at room dye)  reticulocyte
temperature/using incubator for 10 – have hemoglobin
15 minutes (eosin) and RNA(MB)
o Purpose of incubation: to colors are mixed
promote the staining of RBC.
Since the RBCs are alive, they
resist staining due to intact
membrane.
c. After incubation, mix thoroughly and
prepare a wedge smear.
d. Examine smear using 100x objective
(OIO) using the routine light
microscope method. Select an area NOTE:
where erythrocytes are close but not METHODS OF COUNTING
overlapping and reticulocytes appear 1. LIGHT MICROSCOPE
to be well stained.  Reticulocytes are enumerated among 1000
RBCs in areas where RBCs are close but not
APPEARANCE OF RBCs and RETICULOCYTES: overlapping and reticulocytes appear to be
 MATURE RBCs: light to medium green without well stained.
granules
 RETICULOCYTES: light green with granules/ 2. MILLER DISK METHOD
granulofilamentous that stain deep blue.  Still made use of light microscope but with
additional component attached to one of the
e. Count the number of reticulocytes in oculars (miller disk).
1000 red cells. Reticulocytes should  The calibrated Miller disc appears in the field
also be counted as erythrocytes. with 2 squares: a large square and a small
f. Calculate as follows: square inside the large square which is 1/9 of
𝑵𝒐. 𝒐𝒇 𝒓𝒆𝒕𝒊𝒄𝒖𝒍𝒐𝒄𝒚𝒕𝒆𝒔 × 𝟏𝟎𝟎 the size of the larger square.
% 𝑹𝒆𝒕𝒊𝒄𝒖𝒍𝒐𝒄𝒚𝒕𝒆𝒔 =
𝟏𝟎𝟎𝟎 𝑹𝑩𝑪  Reticulocytes are counted in the large square
while the RBCs are counted in the small
square in successive fields on the film until a
total of 500 RBCs have been counted.
 Counterstain is not advisable
 Reticulocytes have lower specific gravity 
after incubation, the reticulocyte appear to
 HEMATOLOGY 2
settle in the upper portion  always mix to
avoid false decrease count
 Patient with hyperglycemia: Increase glucose
 inhibit staining  false decrease
 TWO SQUARES:
o LARGER SQUARE: for reticulocyte
counting
o SMALLER SQUARE: for RBC counting
(mature and immature) Size: 1/9 of
the large square
 REFERENCE VALUES:
o Adults: 0.5 – 1.5%
o Newborn: 2.0-6.0%
𝑹𝒆𝒕𝒊𝒄 %
𝑻𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝒓𝒆𝒕𝒊𝒄𝒔 𝒊𝒏 𝒍𝒂𝒓𝒈𝒆 𝒔𝒒𝒖𝒂𝒓𝒆 × 𝟏𝟎𝟎
= artifacts will glisten.
𝑻𝒐𝒕𝒂𝒍 𝒏𝒐. 𝒐𝒇 𝑹𝑩𝑪𝒔 𝒊𝒏 𝒔𝒎𝒂𝒍𝒍 𝒔𝒒𝒖𝒂𝒓𝒆 × 𝟗
NOTE: INCLUSIONS & ARTIFACTS CONFUSED WITH
RETICS
 HOWELL-JOLLY BODIES
- Seen in Romanowky and methylene blue stain
but reticulocyte (RNA) will not be seen in
romanowsky stain.
 PAPPENHEIMER BODIES
- Pappenheimer bodies are present in
romanowsky, supravital stain and Prussian
blue stain (Siderotic granules) but reticulocyte
are negative in Prussian blue.
 HEMOGLOBIN H INCLUSIONS
- Hemoglobin H have no pattern; just
distributed in the red cell making the cell as
pitted golf ball whereas reticulocyte have
reticulum pattern
 HEINZ BODIES
- Most confusing because Heinz bodies are not
seen in romanowsky like the reticulocyte’s
RNA and are both present in supravital stain.
To differentiate, observe closely. Heinz bodies
are often few and found at the periphery
while reticulocyte RNA follows a reticulum
pattern
 ARTIFACTS
- Dust particles  move the the fine
adjustment and if the focus is lost. The
- OUT OF FOCUS: refractile  artifacts
disappearance  retics
 HEMATOLOGY 2

NOTE:
ASSOCIATED CORRECTION:
1. ABSOLUTE RETICULOCYE COUNT HEMOGLOBINOPATHIES
 Actual number of reticulocytes in 1 Liter of  Hemoglobinopathies are results of structural
whole blood defects in the globin component of hemoglobin.
 Formula: The structural defect result from the alteration
𝑅𝑒𝑡𝑖𝑐 % 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (× 1012 /𝐿) × 1000 of the DNA genetic code for the chains leading to
𝐴𝑅𝐶 =
100 the production of abnormal hemoglobins known
as hemoglobin variants.
 Reference value: 25,000 -75,000/uL  NOMENCLATURE:
o Denotes the affected polypeptide chain
2. CORRECTED RETIC COUNT / RETICULOCYTE
o The sequential amino acid number(s)
INDEX
affected (primary structure)
 This corrects the reticulocyte count to a
normal hematocrit to allow correction for the o The helix number involved (secondary
degree of patient anemia. The percentage of structure A-H).
the reticulocytes may appear increased o The nature of the abnormality (amino
because of early release in the circulation or acid substitution, deletion, addition or
because of a decrease in the number of globin chain fusion).
mature RBCs in the circulation.
 Also called Hematocrit Index or Hematocrit
correction
 Formula:  Disorder in structure
𝐻𝑐𝑡 (𝐿/𝐿)
𝐶𝑅𝐶 = 𝑅𝑒𝑡𝑖𝑐 % ×  Results from the alteration of the DNA genetic
0.45 𝐿/𝐿 code for the chains conditions characterized by
 Reference value: 1% (depends on the degree
qualitative structural abnormalities of the globin
of anemia)
polypeptide chains that result from alteration of
3. RETICULOCYTE PRODUCTION INDEX OR the DNA genetic code for those chains. Structural
SHIFT CORRECTION (RPI) abnormalities may involve amino acid
 Index calculated to correct for the presence of STRUCTURAL DEFECTS ON A OR B CHAIN RESULTING
shift reticulocytes that otherwise falsely TO HB VARIANT:
increased the visual reticulocyte count. 1. SUBSTITUTION
 General indicator of the rate of erythrocyte  Ex.: Hb Hb S: β 6(A3) Glu val
production INCREASE above normal in
anemias
2. DELETION
 Formula:
𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑅𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝐶𝑜𝑢𝑛𝑡  some amino acid is deleted
𝑅𝑃𝐼 =  reason of thalassemia
𝑀𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 (𝑑𝑎𝑦𝑠)
 Reference value: 1 if hct is 0.45  Ex.: Hb Gun Hill: (β91 to β 95)0
Patient’s Hct (%) Correction factor/
Maturation time 3. FUSION
(days)  Hb Lepore-Baltimore: (G(1-50) B(86-146)) caused
40-45 1.0 by cross over during mitosis resulting to fusion
35-39 1.5
25-34 2.0 4. ELONGATION
15-24 2.5 5. Hb CONSTANT SPRING (a+31c)
<15 3.0
Clinical Significance of Reticulocytes: Increased in
hemolytic and hemorrhagic anemias

NOTE:
REASON FOR DELETION & ELONGATION: mutation in
genetic code
REASON OF HEMOGLOBINOPATHIES: mutation of
gene
NOMENCLATURE:
 COMMON NAME
 Morphology: sickling Hb (Hb S)
 Place where they were discovered: (Hb
Boston)
 SCIENTIFIC NAME
 Gives structural defect of Hb
 Ex: Sickling Hb Hb S: β 6(A3) Glu val
 B: affected polypeptide chain
 6: sequential amino acid number(s) affected
(primary structure).
 A3: Helix number involved (secondary
structure: A-H); not necessary as long as
primary structure is indicated Glu:nature of
the abnormality (substitution, deletion,
addition or globin chain fusion)
 PRIMARY STRUCTURE: sequence or
arrangement of amino acid
o Hemoglobinopathies commonly
affected the B chain.
NOTE:
 HEMOGLOBIN ELECTROPHORESIS:
Laboratory test for variant identification and
separation.
o For variant identification
o Follows general principle
electrophoresis.
 PRINCIPLE: utilizes an electric chamber that
has both cathode & anode electrons.
o Hb like proteins are amphoteric.
o Proteins are somewhat amphoteric
wherein its negative charge is
neutralized by the + charge thus it can
show a net Negative & Positive
charges thus it can behave depending
on the pH of the medium.
HEMATOLOGY 2
CELLULOSE ACETATE ELECTROPHORESIS (PH 8.4-8.6)
 For the detection&preliminary identification
of both normal abnormal Hbs particularly, Hbs
A, F, S & C.
 PROCEDURE: a small quantity of red cell
hemolysate is placed on the cellulose acetate
membrane between the center & the
cathode. An electrical field is created in the
chamber through the use of a power supply&
the ones with greatest charges migrates
towards the anode.
 Hemoglobin molecules have a net NEGATIVE
charge at alkaline pH, & therefore migrates
towards the anode
 BASIS OF MIGRATION: net Negative charge
because all of them are negative but not all
have the same number of negative charges
Note: Hb that has the greatest no. of Net
charges migrates fastest towards the anode
farthest from the point of application but the
Hb with few Negative charges migrates slower
 Owing to the variations in amino acid content
of different Hbs the net charge of each varies&
this determines their rate of mobility in the
electrical field.
 MAJOR Hb: A (adult), F (fetal), S (most
common abnormal Hb that causes sickling of
RBC) &C (second most common)
 BANDS: indicates the migration
 FAST Hb: any other Hb that migrates faster
than A such as H & I
of
A – migrates fastest, followed by F
C – slowest followed by S
Arrangement: CSFA
 HEMATOLOGY 2

CITRATE AGAR ELECTROPHORESIS (PH 6-6.2)

 PURPOSE: For the separation of Hbs that
migrate together on cellulose acetate
 Also useful in detecting small amounts of
either HbA or F in the presence of large
amount of others& in revealing small amounts
of adult Hbs A & S present at birth in cord
blood
 PRINCIPLES:
o Samples are place at the center
between cathode & anode & apply
external electricity& migration will
occur
o Hbsare identified by their migration
toward the anode & cathode&
comparing the migration to that of
known control samples
 BASIS: Hbsare separated based on the
interactions among Hb variants, agar & citrate
buffer in addition to the altered electrical
charge of the various Hbs at acid Ph.

S & C migrates alone

F & A migrates towards the cathode
S & C migrates towards the anode
Arrangement: FASC

NOTE:
SUMMARY OF CHARACTERISTICS OF HBF:
 Resistant to both acid elution & alkaline
denaturation.
 In electrophoresis, it is slower than HbA

FUNCTION:
 Has high affinity to O2 thus when it goes to the
tissues, it will not readily release the O2.
 Decreased affinity to 2-3
Diphosphoglycerate: molecule that regulates
affinity of Hb to O2.
 HEMATOLOGY 2

CRITERIA CELLULOSE ACETATE ELECTROPHORESIS CITRATE AGAR ELECTROPHORESIS

pH pH 8.0 – 8.6 ( Turgeon) pH 6.0-6.2 (Bishop)
pH 8.4 - 8.6 (Bishop)
Principle A fresh hemolysate made from a packed RBC Citrate agar electrophoresis is performed at an
( Basis/bases of sample is applied to a cellulose acetate plate acid pH after abnormal haemoglobin is detected
hemoglobin using a buffer of alkaline Ph and electrophoresis on cellulose – acetate electrophoresis.
separation) is performed. After electrophoresis, the Hemoglobin F, with the fastest cathodial
membrane is stained and cleared. The patient’s mobility, is also the most soluble, probably
haemoglobin migration is compared with that because it is most resistant to denaturation at pH
of a control test interpretation. A rough 6. Adult hemoglobins with solubility similar to
estimate of proportions of different that of haemoglobin A, such as D, E, G, O, I and so
hemoglobins may be made using a forth, move with haemoglobin A. The relatively
densitometer. The order of electrophoretic insoluble haemoglobin S moves behind
mobility, from slowest to fastest, is haemoglobin A, and the even more insoluble
hemoglobins C, S, F and A. There are several haemoglobin C moves behind haemoglobin S.
mnemonic devices for remembering the
migration pattern, such as accelerated, fast,
slow, and Crawl. Hemoglobin that migrates
beyond haemoglobin A is termed fast
haemoglobin.
Hemoglobin Bart’s and haemoglobin H both
migrate here. Any abnormal haemoglobin or
any normal haemoglobin A2 and F, should be
confirmed. If there is abnormal haemoglobin,
confirm with citrate agar electrophoresis and
solubility test if haemoglobin S is suspected. If
an increased amount of A2 or F occurs, then
quantify the amount.
Hemoglobin D and G comigrate with
haemoglobin S in this method.
Importance/ Hemoglobin electrophoresis by cellulose An important factor in determining the mobility
purpose in acetate is useful in identifying and quantifying of hemoglobin
haemoglobin haemoglobin variants and abnormal quantities is solubility and provides ready separation of Hbs
separation of haemoglobin fractions. It is rapid and that migrate together on cellulose acetate: S
reproducible and separates the major Hb from D and G, and C from E and O.
variants S, D, G, C, and E from Hb A.
Quantification of the major bands is easily
accomplished.

SEVERAL ABNORMALITIES OF HEMOGLOBIN  SIDEROBLASTIC ANEMIA: problem in the

utilization of iron due to deficiency in
1. HEME IS ABNORMAL
protoporphyrin 9
 Iron is deficient thus affecting Hb synthesis
leading to IDA
 Deficient Protoporphyrin IX because of enzyme
2. GLOBIN IS ABNORMAL
defectalong the synthesis leading to excess
 Ex: Hb A1 - 2 A w/ 4 genes & 141 amino acids &
iron&iron will not be utilized in the absence of
2 B chains w/ 2 genes & 146 amino acids
protoporphyrin 9. Unused iron will precipitate
 2 FACTORS:
into siderotic granules leading to sideroblastic
o Alpha Thalassemia
anemia
 HEMATOLOGY 2
o B Thalassemia
VARIANTS CAUSED BY B-CHAIN SUBSTITUTION
- Most common cause of hemoglobinopathies
HEMOGLOBIN S (SICKLING HEMOGLOBIN)
 Results from the substitution of glutamic acid by
valine on the 6th position of the B-chain
 Inheritance may be homozygous (SS) known as
sickle cell anemia, or heterozygous (AS) known
as sickle cell trait.
 Hb S is fully soluble. Sickling occurs when oxygen
decreases at the tissue level because of the
formation of tactoid /fluid crystals.
 SICKLE CELL ANEMIA
ORGANS AFFECTED BY SICKLE CELL ANEMIA
- Anemia is usually severe;
(mainly related to vaso-occlusion)
characterized by normocytosis
(normocytic-normochromic)
- Blood Smear shows: Polychromasia;
Sickle cells, target cells, ovalocytes,
schitocytes; Howell-Jolly and
Pappenheimer bodies
- Growth and sexual maturation lag
behind that of normal adolescents
- Electrophoresis reveals HbS 80-90%
and Hb F 10-20%
NOTE:
 Sickle cell crises: Any situation that
produces excessive deoxygenation of
RBCs may cause a crisis. Every organ in the
body is affected and include pain of many
types.
 Vaso-occlusive crises
o Occur when rigid sickle cells
increase blood viscosity leading to
development of microthrombi &
microinfarctions in the joints,
extremities and in major organs.
o “Hand-foot syndrome” or
dactylitis refers to painful
swelling of the back of the
affected hands and feet due to
obstruction caused by many
sickle cells.
 Bone, Joint, and other crises: Pain occurs
in the joints where sickle cells accumulate.
Sickle cells may occlude the marrow
sinusoids, and the lungs, which are the
site of frequent infarcts in adults.
 Infectious crises: Due to abnormal splenic
function and depression of immune
function, patient becomes susceptible to
infections. In young patients,
Streptococcus penumoniae appears to be
the major infectious agent.
 Splenic sequestration crises: The spleen
enlarges and sequesters more blood and
hypovolemia. This may lead to shock and
death
 Aplastic crises: Infection and fever cause
temporary reduction in RBC count, Hb,
Hct and Reticulocytes
 Liver (hepatomegaly and liver
malfunction)
 Heart (Cardiomegaly & heart failure due
to iron deposition)
 Spleen (Splenomegaly, Abnormal splenic
function & Splenic infarction)
 Skin (ulcers/scars); Kidneys (infarcts &
hematuria); and the lungs
 SICKLE CELL TRAIT
- Hb A is present in higher percentage
and compensates for HbS. Patients
usually have no symptoms unless in
cases of extreme tissue hypoxia
- Electrophoresis: Hb= S 30-45%
 Hb S-THALASSEMIA
- Doubly heterozygous condition in
which the mutant genes for both
Hb S and Thalassemia are inherited
by a single individual. Hb S-a is
clinically insignificant while Hb S-B
has clinical features similar to,
although not as severe as sickle cell
anemia.
NOTE:
Other sickling Hbs: C Harlem; S Travis; C
Zinguinchor
 HEMATOLOGY 2

Hemoglobin C (B6 glulys): Second most
common hemoglobin variant
 Hb C DISEASE (CC)
- Characterized by a mild to moderate
normocytic-normochromic anemia
with numerous target cells. Under
low oxygen tension or when
dehydrated, Hb CC tend to
crystallize into hexagonal or rod-
shaped crystals with blunt ends. The
crystals does not alter the red cell’s
shape but maked the cells rigid.
 Hemoglobin O-Arab (B 121 Glu -> Lys) Rare,
found in Israel, Bulgaria, Egypt, Romania,
Jamaica, Kenya
 Hb G- Philadephia (a 68 Asn->Lys)
 Hb Gun Hill (B 91-95) Leu His Cys Asp Lys -> 0
 Hb Constant Spring (CS) a+31c (142 Gln) Due
to elongation of the a-chain (31 amino acids)
 Hb Lepore-Baltimore( (1-50) B (86-146)) Due
to fusion of B
 Hb C-Harlem/ Hb C: Georgetown 2 amino
acids substitution (B 6 Glu ->Val; Asp ->Asn)
 Hb Chesapeak: has increased affinity with
oxygen
 Hb Kansas: has decreased affinity with oxygen

 Hb TRAIT (AC)
- A heterogygous hemoglobinopathy.
Clinical symptoms are similar with
Hb SS, though milder and with fewer
complications. Often, the red cells
may appear with folded cytoplasm.
Hb SC crystals often appear
fingerlike or with projections.
Electrophoresis reveals equal
amounts of Hb S and C
LABORATORY TESTS FOR UNSTABLE HEMOGLOBIN
NOTE:
1. HEART DENATURATION/HEAT INSTABILITY
 IbM (M-Saskatoon; M-Boston; M-Iwate; M-  Blood sample is incubated at 50 degrees
HydePark; M-Mulwaukee): These are caused celcius for 3 hrs.
by structural defects (amino acid substitution)
in either the a or b chain. The structural defect 2. ISOPROPANOL PRECIPITATION
in the globin chain results to non-protection of
 Blood sample is incubated with 17%
iron from oxidation (methemoglobinemia).
isopropanol at 37°C
Patients appear cyanotic (with lavender-blue
 PRINCIPLE: Normal hemoglobins have intact
skin) due to tissue hypoxia. Babies may also
bonds and remain in the solution when
show clubbed fingers at birth. Blood appears
heated or incubated with Isopropanol.
brown with numerous Heinz bodies.
Unstable hemoglobins have weaker bonds
 Hb Barts (4 y-chains) and H (4-B chains):
and easily denature or precipitate under these
Found in patients with a-Thalassemia
conditions.
 Hb Barts disease: leads to stillbirth and
hypoxia because Hb Bart’s affinity to O2 is 3. HEINZ BODY STAINING
high
 requires supravital staining (e.g. crystal violet)
 Hb H diseases: leads to anemia

OTHER SIGNIFICANT ABNORMAL HEMOGLOBINS:

 Hemoglobin D Hb D-Punjab/Hb D: Los
Angeles (B121 glu->Gln)
 Hemoglobin E (B 26 Glu ->lys)
 HEMATOLOGY 2
NOTE:
 The HbS GENE, when present in homozygous
form, is an undesirable mutation, so a
selective advantage in the heterozygous form
must account for its high prevalence and
persistence. Malaria is possibly the selecting
agent because a concordance exists between
the prevalence of malaria and Hb S. Sickling
might protect a person from malaria by either
(1) accelerating sickling so that parasitized
cells are removed or (2) making it more
difficult for the parasite to metabolize or to
enter the sickled cell.
 SICKLE CELL TRAIT (AS) confers partial
protection against lethal Plasmodium
falciparum malaria. Multiple mechanisms for
this have been proposed, with a recent focus
on aberrant cytoadherence of parasite-
infected red blood cells (RBCs). Here we
investigate the mechanistic basis of AS
protection through detailed temporal
mapping. We find that parasites in AS RBCs
maintained at low oxygen concentrations stall
at a specific stage in the middle of intracellular
growth before DNA replication. We
demonstrate that polymerization of sickle
hemoglobin (HbS) is responsible for this
growth arrest of intraerythrocytic P.
falciparum parasites, with normal hemoglobin
digestion and growth restored in the presence
of carbon monoxide, a gaseous antisickling
agent. Modeling of growth inhibition and
sequestration revealed that HbS
polymerization-induced growth inhibition
following cytoadherence is the critical driver
of the reduced parasite densities observed in
malaria infections of individuals with AS. We
conclude that the protective effect of AS
derives largely from effective sequestration of
infected RBCs into the hypoxic
microcirculation.
ADDITIONAL INFORMATIONS:
Hb S (Sicking Hb)
 The most well-known hb variant
characterized by substitution of glutamic acid
by tRNA to valine on the 6th amino acid
position of the β- chain.
 Glutamic: CAG Hb S: β 6(A3) Glu  val
 Valine: GUG
 The effect of alteration from glutamic acid to
valine is a negative charge wherein glutamic is
highly negatively chargecompared to valine
 thus leading to loss of negative charges& Hb
becomes less negative
 In electrophoresis in cellulose acetate, S
migrates slowly than A & F due to substitution
of amino acid 6 Inheritance: Homozygous (SS)
– sickle cell anemia
 Heterozygous (AS): sickle cell trait
 EFFECTS OF HB S:
o Blacks who are suffering from this are
protected from being infected from
developing severe falciparum malaria
(Resistance). Hb C, E, B-Thalassemia
&G-6PD deficiency also confer
protection in terms of resistance but
not immunity meaning these patients
may still be infected but may not
proceed to chronic stage.
o Falciparum are intracellular species&
when present in RBC& when RBC is
sickle the parasite inside also dies
does no complication arise
o Falciparum metabolizes Hb, when Hb
crystalizes it forms into a crystal&
falciparum may no longer metabolize
the Hb
o RBC w/ falciparum will be
phagocytosis before it causes severity
HOMOZYGOUS (SS) SICKLE CELL ANEMIA
 Severe anemia because the patient does not
synthesize the normal Hb A
 On electrophoresis:
o Hb S: 80 -90%
o Hb F: 10 -20 %
o Hb A2: Normal
 Blood Smear: Polychromasia (Reticulocytes
are increased) ; Sickle cells, target cells,
ovalocytes, schistocytes ; Howell-Jolly &
Pappenheimer bodies
 HEMATOLOGY 2
 Increased RDW
 Retarded growth&sexual maturation&patient
suffers sickle cell crises Main cause of Sickle
Cell Crises: sickling
 Normally when Hb S is fully oxygenated, Hb S
is fully soluble
 But when Hb S is w/ reduced O2, Hb S
becomes insoluble wherein it polymerizes
(change in molecular arrangement) &forms
into tactoid crystals/fluid crystals (elongated
& thin, pointed at both ends) leading to
sickling &rigid cell membrane thus it is no
longer adjustable leading to vassoocclusion
thus it cannot easily pass through the small
circulations
 Sickling is reversible but repeated sickling
leads to permanent sickling due to permanent
damage to RBC membrane
 Causes vasso-obstruction
 VASOOCCLUSIVE crises occurs commonly on
small circulations like in the fingers &toes thus
hand-foot syndrome / dactylitis (inflamed or
swollen, cyanotic due to absence of
circulation) occurs
 VASSOOCCLUSION also occurs in bone&joint
causing pain
 SPLENIC CIRCULATION is also minute, since
spleen also serves as a filter of blood thus its
circulation is too small, that only deformable
red cell may pass through. Due to increase
activity of spleen&vassoocclusion, spleen also
enlarges (splenomegaly) causing
sequestration of more blood leading to
hypovolemic shock. When spleen is
enlarged&bone marrow is affected thus both
are abnormal in function leading to decrease
blood production to the marrow (aplastic
crises) thus the immune system is defective &
the patient becomes prone to infection
(infectious crises) as a result of all of this organ
damage occur (marrow lungs)
 The no. 1 cause of death is infection& in
children the most common is S. pneumoniae
SICKLE CELL TRAIT (AS)
 Predominant Hb is A, only 30-45% is Hb S
 Patient usually have no symptoms&sickling is
uncommon to occurunless in cases of extreme
tissue hypoxia because S is minimal in
 concentration
Hb S – THALASSEMIA
 Hb S-A
 Hb S-B – more severe
OTHER SICKLING HBS:
 Abnormality: Hb S: β 6(A3) Glu val
+ unique B-
substitution
 Hb C-Harlem, C-Ziguinchor, S-Travis
NOTE:
HEMOGLOBIN C
 A beta structural defect, where glutamic acid
is substituted w/ lysine
 Composition: B 6 (A3) Glu Lys
HETEROZYGOUS: Hb C Trait (AC)
 RBCs – slightly hypochromic
 Less abnormalities
HEMOGLOBIN SC DISEASE
 Signs & symptoms are milder than SS but
more severe than sickle cell
 Electrophoresis: C = S = 50 – 50%
 Cytoplasm appears folded like a pocketbook
cells (RBC w/ cytoplasm folded)
 Hb SC easily crystalize forming a fingerlike
projection w/ more than 1 protrusions where
one is long&protrude away. It also in the form
of hand in gloves crystals or pointing – finger
or Washington monument crystal
OTHER ABNORMAL HEMOGLOBINS
1. Hb METHEMOGLOBIN
 M-Saskatoon, Boston, Iwate, HydePark,
Milwaukee = all have different amino acid
substitution but the effect is the same &not
protected from oxidation
 Methemoglobinemia w/ congenital cyanosis
due to overwhelming oxidation even at baby
stage& the blood appears chocolate brown
 Easily precipitate&forms into Heinz bodies
2. Hbs WITH ABNORMAL AFFINITY TO OXYGEN
a. Hb w/ Increase Affinity
 Ex.: Hb Chesapeake (a 93 Arg Leu) –
affects affinity of Hb to O2 &O2
will shift to left
 HEMATOLOGY 2
b. Hb with Decrease Affinity
 Ex.: Hb Kansas (B 120 Asp Thr
)–
shift to the right
NOTE: Abnormal Hb are connected by weak bonds
thus they easily precipitate & flocculate.
TEST FOR UNSTABLE HB
1. ISOPROPANOL PRECIPITATION TEST
 When heated, 17% Isopropanol at 37 C
normal Hb do not precipitate but few will
do&unstable Hb will easily precipitate.
 Purpose of Isopropanol: weaken the bonds
2. HEAT DENATURATION/INSTABILITY TEST
 At 50°C for 3 hours, normal Hb will not
flocculate but unstable Hb will
3. HEINZ BODY STAINING
 If ppt & flocculation happens in vivo forming
Heinz bodies which are not easily seen in
normal wrights blood smear
 Supravital staining is employed (brilliant
cresyl blue & crystal violet).
TESTS FOR SICKLING HEMOGLOBINS
SICKLE CELLS are distorted cells appearing as thin,
dense and elongated cells with both ends pointed.
SICKLING is caused by the PRECIPITATION of an
ATYPICAL HEMOGLOBIN (commonly hemoglobin S)
which DISTORTS the RED BLOOD CELLS into a SICKLE
or CRESCENT SHAPE. HEMOGLOBIN S is fully soluble
when oxygenated but becomes insoluble when the
oxygen level is decreased. It first POLYMERIZES then
forms into CRYSTALS which cause the red cell to
become RIGID.
SCREENING TESTS:
 PRINCIPLE: HbS forms an insoluble tactoid
crystals when oxygen supply to the red cell is
decreased. When Hb S is deoxygenized
outside the cell into the plasma it will cause
the solution to be TURBID. POSITIVE RESULT:
1. Sickle RBC 2. Turbidity
 Degree of sickling or turbidity depends on the
concentration of Hb S in the RBC
1. SEALED WHOLE BLOOD METHOD (SCRIVER
& WAUGH)
 PRINCIPLE: RBC take on a SICKLE-LIKE SHAPE
when oxygen supply to the red cell is
decreased. HbSforms INSOLUBLE TACTOID
CRYSTALS when exposed to LOW OXYGEN
TENSION.
 PROCESS: Perform IN VIVO. Using a RUBBER
BAND, the rubber band is TIED at the BASE of
the FINGER or at the BALL of the finger
intended to be prick& maintain it for at least
5-10 minutes to cause obstruction in order to
obstruct the circulation thus decreasing the
oxygen& Hb S will form into crystals. Prick
the deoxygenated finger & drop into the
center of the slide &cover w/ cover slip. Seal
the sides w/ petroleum jelly or paraffin to
prevent entry of atmospheric O2 which
contains abundant O2 which may cause the
sickled cell to revert to normal shape.
Incubate for 1 hour at room temperature
&examine microscopically if still none,
incubate for 2 hours until 3rd hour. Perform
interval microscopic examination.
 POSITIVE RESULT: 1. Sickle RBC (Hb S) but
does determine if heterozygous AS or
homozygous SS REPORTING: positive or
negative
 DEGREE of sickling depends on the
CONCENTRATION of the Hb S in the RBC
 Readings are made at an HOURLY INTERVAL
for 2-3 HOURS
 POSITIVE RESULT is DIAGNOSTIC but DOES
NOT DISTINGUISH Hb S trait & Hb S anemia
2. SODIUM METABISULFATE METHODS
(DALAND & CASTLE)
 REAGENT: 2% Sodium metabisulfide or
sodium bisulfate which are reducing agent
which bind &remove O2 from the blood.
 PRINCIPLE: a drop of blood is mixed w/ a
drop of 2% sodium metabisulfite (a reducing
agent) on a slide, & the mixture is sealed
under a coverslip. The hemoglobin inside the
RBCs becomes deoxygenated causing
polymerization& the resultant sickle cell
formation
 PROCESS: add a drops of blood& the reagent
into the center of the slide.Cover the slide w/
 HEMATOLOGY 2
coverslip&seal the sides. Observe
microscopically.
 POSITIVE RESULT: sickled RBC deoxygenated
by reducing agent.
3. DITHIONITE SOLUBILITY TUBE TEST
 REAGENT: sodium hydrosulfite (dithionite)
removes O2
 SPECIMEN: whole blood w/ EDTA heparin or
sodium citrate in order to prevent clotting
 PRINCIPLE: Red cells are lysed by saponin
allowing Hb to escape. Sodium dithionine
binds&removes oxygen from the test
environment. Hb S polymerizes in the
deoxygenated state&forms a precipitate in a
high-molarity phosphate buffer solution. The
tactoid refract or deflect light& make the
solution turbid.
 PROCESS: anticoagulated whole blood +
sodium dithionite. Saponin lyses the RBC
releasing now Hb. Hb binds w/ sodium
dithionite which removes O2 from solution
causing Hb to crystalizes but polymerization
happens outside the RBC thus it will not
cause sickling but turbidity of solution& is
determined by refraction&deflection of the
solution in the presence of light. Observe
specimen for turbidity by holding the tube
2.5 cm in front of a newsprint or a card
reader w/ thin black line
 RULE: if the solution is observed in the
solution then the solution is not turbid thus
negative result but if lines are no longer
visible then it is positive for tactoid
crystals&sickling.
 POSITIVE RESULT: turbidity
 Turbidity indicates presence of sickling Hb
regardless of any genotype
 SCREENING METHOD OF CHOICE
4. HEMOCARD Hb A & S
 PRINCIPLE: Hb A& S contains monoclonal Ab
(IgG), which specifically bind to the amino
acids at or near the 6th position of the B-
chain of the Hb S&A
 Hb S monoclonal Ab will react w/ the B-chain
of Hb S but not w/ the Hb A
 Hb A monoclonal Ab will react w/ the B-chain
of the Hb A but not w/ Hb S
 Abs are adsorbed to suspended metal sol
particles giving the reagent raspberry-like
color
NOTE:
 After electrophoresis, densitometry is
performed to determine the density of the
bands created by the migrating proteins
converted into concentration.
 In cellulose Acetate, at an alkaline pH, Hbs are
negatively charge thus they migrate towards
the anode.Hb S together w/ D & G upon
migration appears as a band of protein thus
densitometry is performed to determine the
concentration of Hb S.
 On Citrate Agar, at an acidic pH, Hb S migrates
towards the anode thus citrate agar is more
confirmatory
EXPERIMENT MODULE:
Some hemoglobins that aggregate and have reduced
solubility are capable of polymerizing and crystallizing
within the red cell causing a distortion of cell shape
(sickle shape). Hb S (Sickling Hb), when fully
oxygenated is fully soluble. Polymerization and
formation into tactoid crystals occur only when
oxygen is decreased at tissue level.
 SPECIMEN NEEDED: Capillary blood
A. SCRIVER AND WAUGH METHOD
a. Place a rubber band around the base of
the middle finger and allow stayingin
place for 5 minutes.
b. Make a finger puncture on the ball of the
finger and place a drop of capillary blood
on a slide.
c. Immediately cover with a coverslip and
seal edges with petroleum jelly.
d. Incubate the preparation at room
temperature. Observe for red cell sickling
at hourly intervals for 2, 3 hours, or after
24 hours if desired.
e. Microscopic examination (400x): If more
than 10% of the cells are sickled, the result
is positive.
B. SICKLE CELL SLIDE TEST
 In the absence of the HbS solubility test, the
sickle cell slide test is useful in detecting
sickle cells in patients who have either sickle
cell disease or sickle cell trait.
 HEMATOLOGY 2
a. Weigh 0.1g of sodium metabisulphite and
transfer to a test tube capable of holding
15 ml of water.
b. Add 5 ml of distilled or deionized water,
stopper, and mix until the chemical is
fully dissolved. The chemical can only be
used within the day it was suspended
(within 8 hrs)
c. Deliver one drop of patient’s capillary
blood or well mix venous blood on a slide
and add an equal volume of freshly made
reagent, mix and cover with a cover glass.
Exclude any air bubbles.
d. Place the slide in a plastic box or Petri
dish with damp piece of blotting paper or
tissue at the bottom to prevent drying of
the preparation (moist chamber). Leave
at room temperature.
e. After 10-20 minutes, examine the
preparation microscopically for sickle
cells. Focus the cells first with the 10x
objective and examine for sickling using
the 40xobjective. Examine several fields.
Sickling usually takes place in one part of
the preparation than the other.
f. Sickle cells usually appear crescent shape
with pointed ends or holly leaf shape.
Report as “sickle cell test positive” when
crescent shape cells are seen, or ‘sickle
cell test negative” when cells appear
rounded or oval shape.
C. TUBE SOLUBILITY TEST
 The principle of solubility method was based
on turbidity created when Hb S is incubated
with sodium dithionate.
a. Add 20 uL of blood with 2 ml of the
reagent prepared in procedure B (2% w/v
sodium metabisulphite or sodium
dithionite)
b. Mix and stand at room temperature for 5
minutes.
c. Examine the tubes using a white board
with black lines as the background in
ambient light settings.
d. Interpret results as positive if the black
lines are not visible
OSMOTIC FRAGILITY TEST (OFT)
 A test to identify the ability of red cells to
absorb water without lysing.
 It reflects the shape and size of erythrocyte
(specifically the surface area-to-volume ratio)
 Not a confirmatory test for HS; it will only
identify the presence of fragile cells like
spherocytes. But it will not determine
whether the spherocytosis is hereditary,
immune or other causes.
 For hereditary spherocytosis and other
anemia characterized by spherocytosis
o Spherocytes are test using OFT
because they are the most fragile cell
 able to detect the presence of
spherocyte in the circulation
o RESULT IN HS: 0.60 - 0.65% saline
solution, hemolysis is already
observed: spherocyte (fragile) easily
lyse
 PRINCIPLE: The cell is placed in a hypotonic
saline to cause osmosis (the shift of the fluid
from the hypotonic saline into the red blood
cell). If the red cell could still contain its
volume, the RBC would be able to absorb
water without lysing and if not, it will easily
hemolyse. POSITIVE RESULT: Hemolysis
 RESULTS:
o Incomplete hemolysis: 0.45%
 Faint pink tinge supernatant with
intact red cell button
o Complete hemolysis: 0.35%
 Homogenous pink/red solution
with the absence of red cell
button
 Fragility means increased OFT cells are easily
lyse (spherocytes)
 INCREASED OFT:
o HS, abnormal membrane, severe G-6-
PDH deficiency, PK deficiency,
Hemolytic anemia
 DECREASED OFT:
o Resistant to red cell lysis even when
placed in hypotonic solution
 With increased surface area-to
volume ratio
 Hypochromic cells, target
cells/leptocytes, sickle cell
o Severe IDA (microcytic-hypochromic
cell)
 HEMATOLOGY 2
o Thalassemia (microcytic-hypochromic
and target cell)
o Sickle cell anemia: If the sickling is still
mild and is reversible, when the sickle
cell absorbs water it goes back to
being a normal cell. However, if its
membrane is totally damaged, the
sickling becomes irreversible
therefore becomes resistant.
 Affected by:
o Surface area-to-volume ratio
 A normocyte with normal
central pallor is resistant to
lysis because the space can
bloat to extend the capacity,
so the cell can absorb more
water without hemolyzing.
But a cell without a central
pallor will immediately lyse
upon absorption of water like
spherocyte.
 If the surface area-to-volume
ratio is still high, the cell is
resistant
 If the surface area-to-volume
ratio is decrease, the cell is
fragile  easily lyse
 Cells with decreased surface
are-to-volume ratio have a
limited capacity to expand in
hypotonic solution and
therefore lyse even at a less
hypotonic concentration of
saline than the normal
biconcave cells.
o Functional state of the cell
membrane
 If the red cell membrane is
defective, it is easily
hemolyse.
 Like in HS, there is membrane
problem wherein it lack
integrity due to loss of
protein.
 SANFORD METHOD  MACROSCOPIC
o REAGENT: 0.5% saline + add
corresponding drops of DH2O
o SPECIMEN: Heparinized or
defibrinated blood
o Determine the concentration of nacl
solution where initial and complete
hemolysis occurred.
o FORMULA: %NaCl = test tube
number x 0.02
o PROCEDURE: Prepare different
concentration of hypotonic saline
using the following procedure:
a. Set up 12 test tubes and label no.
14 -25.
b. Place in each tube 0.5% NaCl
solution (The number of the tube
corresponds to the drops of 0.5%
NaCl)
c. Using the same dropper, add
distilled water into each tube
until a total of 25 drops (NaCl &
water) are in each tube.
d. Dispense to each tube one drop
of heparinized or defibrinated
blood.
NOTE: Deliver at the same angle
directly to saline solution.
e. Mix tube and allow to stand at
room temperature or centrifuge
for 1 minute at 2,500 rpm.
f. Examine each tube for
initial/incomplete and complete
hemolysis,
 DACIE METHOD  MORE ACCURATE
o Hypotonic saline (pH 7.4)
 Buffered at pH 7.4 using PO4
in order to prevent
interference of inconsistent
Ph
 RECAP: OSMOSIS: movement
of fluid in order to attain
homeostasis/equilibrium
 Outside is hypotonic, inside is
isotonic  movement of
water from outside to inside
o HEMOLYSIS: @540 nm
o INCUBATED OFT: incubate the sample
for at least 24 hours to detect mild
forms of HS
 It is done because if the
observation is done
immediate and the HS is mild
there will be no hemolysis
seen  false negative result
 HEMATOLOGY 2

 Mild forms of HS: on

prolonged incubation they
become fully spherocyte
therefore show hemolysis
 PURPOSE: detect mild forms
of HS.