HEMATOLOGY - Haima = blood ; logos = study ; pathos = suffering

Blood Collection 5%

Hematology Test 30%

Hematopoiesis 40%

Coagulation 20%

Quality Assurance 5%
Total: 100%

PHLEBOTOMY
● Types of blood vessels
○ Veins: 5 mm in diameter (longest)
○ Arteries: 4 mm in diameter
○ Capillaries: 8 um (smallest)
I. Skin/Capillary/Microsampling Puncture Site
A. Infant
● Site: Medial or lateral plantar surface of the heel
● Depth: <2.0 mm
B. Children/Adult
● Site: Lateral to the fleshy pad of the last phalanx on the palmar surface of the 3rd or 4th
finger of the hand that is less used
● Puncture: Perpendicular to the fingerprint
● Depth: 2.0-2.5 mm Fingerprint whorls
C. Notes:
● Avoid applying pressure/squeezing/milking the site.
○ Possible effects
■ Hemolysis
■ Excess interstitial fluid or tissue
● Disinfectant: 70% isopropyl alcohol then air dry
○ Iodine can falsely decrease the Potassium
● Discard first drop of blood
○ Tissue fluid
○ Dead epidermal cells
○ Will facilitate free flow of blood
● Order of draw: BSEOS
○ Blood gas tubes
○ Slide
○ EDTA
○ Other anticoagulated tube
○ Serum collection tube is last to avoid clotting
● Warming the site can increase blood flow up to 7x
○ Warm washcloth (40-42 C) for 3-5 mins.

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II. Venipuncture
A. Most common site:
➢ Superficial veins of the antecubital fossa
B. Two anatomical pattern: H or M pattern
● H pattern : 70% - Median, Cephalic, Basilic
● Preferred site: Median (First choice lagi si median, second si cephalic tas pang third si
Basilic) Kahit di nakikita si median, practice lang doon kasi mas okay talaga don saka
masakit kumuha at magalaw sa cephalic saka basilic.
● Median vein is largest, well anchored, stable and least painful.
C. Skin Antiseptic:
➢ 70% Isopropyl alcohol ; 30% H2O2
D. Gauge:
● Adults: 21 or 23 ; Length of needle. 1 inch
● Children: 23
● The higher the gauge the smaller the bore
E. Notes:
● Angle between skin and needle: < 30 degree
● Tourniquet application
○ Less than 1 minute or 60 seconds
○ Effects of prolonged tourniquet application
■ Hemoconcentration
■ Hemolysis
■ Shortened coagulation time due to stasis
● Local accumulation of factors VIII and VWF
● False shortening of the coagulation time
○ Distance of the tourniquet: 3 to 4 inches
● Standard Precautions:
○ “Universal Precautions”
○ To treat all kinds of blood, body fluids and unfixed tissues as if they are potentially
infectious
○ Handwashing: Rub hands vigorously for at least 15 seconds. Rinse in a downward
flow. Dry using a paper towel. Turn the faucet off using a paper towel.
● Phlebotomist must never puncture the same patient more than twice
● Avoid pumping fist - Falsely increase of Potassium
○ Veins in inner wrist
○ Veins in feet : Dx: Diabetes mellitus
○ Fistula - seen in dialysis patient
○ Arteries - Kumukuha lang ay ang Physicians and Nurses lang.
○ Inflamed sites
○ Edematous area → There’s accumulation of fluids. Pag tumusok ka sa edema di
blood makukuha mo kundi yung contaminated tissue fluids.
○ Mastectomy location → collect on opposite sides, pag wala lahat at di alam san
kukuha, ask the physician.

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 ○ Scarred/burned areas → Risk of infection, tanggal na superficial dermis nila.
● Cause of hemolysis
○ Prolonged tourniquet application
○ Moisture/Contamination → basa pa ng alcohol tumusok agad bida bida amp.
○ Use of two small bore → cause of hemolyzation of blood sample
○ Excessive agitation –. Sobra sa invert tapos baka shinake ng malala
○ Frotting of blood sample

III. Anticoagulants
A. EDTA
● Color: lavender/purple. May pink pero intended sa blood banking
● Inversion: 8x
● Uses: Hematology tests
● Optimal concentration: 15mg/mL of blood
● Action: chelation of Ca → important component of blood clotting
● Viability
○ CBC → RT up to 4 hrs
○ WBC count/ HCT / Plt count → refrigerator or 4°C up to 24 hours
○ ESR → under RT up to 2 hours; 4-8°C up to 6 hours
○ Blood smear → it should be made within 2 hrs of collection
● Insufficient:
○ Cause: Overfield
○ Effect: Clotting “microclot”
■ insufficient ratio between the blood and anticoagulant
● Excessive
○ Cause: due to underfield
○ Effects: Falsely decrease of Hematocrit, ESR; Falsely increase MCHC and pH
count, Degenerative change in WBC
B. Heparin
● Color: Green
● Inversion: 8x
● Uses: Flow cytometry, Plasma chemistry, blood gas, OFT, Ammonia
● Optimal concentration: 15-20 IU
● Action: activates antithrombin III which will inactivate thrombin
● Notes:
○ Three formulation
■ Ammonium heparin
■ Sodium Heparin
■ Lithium Heparin → lithium determination
○ Causes Cellular clumping → false results in Hema analyzer
■ Pseudoleukocytosis, pseudothrombocytopenia
○ Causes morphologic distortion of leukocytes and platelets
■ Not to be used in blood smear
○ Causes bluish discoloration on background using Romanowsky stain due to pH

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 ○ Not for coagulation studies due to thrombin
C. 3.2 % Sodium Citrate
● Color: Light blue top
● Inversions: 3 to 4x
● Uses: Coagulation test → PT, PTT
● Action: the same with EDTA which is chelation of calcium
● Ratio: Blood to anticoagulant - 9:1, 9 parts blood, 1 part coagulant
● Note: Forceful mixing and excessive inversions can activate platelets → shorten
clotting time

IV. Specimen Preparation

A. Peripheral Blood Smear
● Source: EDTA
● Should be made within 2 hrs of collection
● Advantages:
○ Multiple blood smears can be made
○ Can be prepared at a later time
○ EDTA prevents platelet clumping
● Disadvantages
Disadvantages Effects Correction

Platelet Satellitosis Pseudothrombocytopenia Recollect using 3.2 % Na Citrate

Platelet clumping Pseudoleukocytosis Use correction factor of 1.1 (to

compensate for dilution brought
about by blue top)

NOTE: Satellitosis & clumping have the same remedy/correction; Line is visible in correction

CASE:
The patient demonstrates platelet clumping in his blood collected in a lavender top tube. CBC results
showed low platelet count at 90,000/mm3 and high leukocyte count at 15,000/mm3. Phlebotomist
recollected using light blue top and CBC result changed to platelet count at 107,800/mm3 and WBC ct at
11,000/mm3. What set of values should appear on Px’s blood?
ANSWER:
It is only used in platelet count & WBC count in cases of platelet satellitosis and platelet clumping
Plt Count: (107, 800) (1.1) = 118, 580
WBC Count: (11,000) (1.1) = 12,100
REFERENCE RANGE (PLATELETS):
- 150,000 - 450,000

● Source: anticoagulant free blood

○ Best for blood cell morphology
○ Advantages :
■ Made beside the patient

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 ■ Artifacts can be prevented
○ Disadvantages
■ Platelet clumping
■ Few films can be made

B. Smear Preparation
A. Methods of Manual Method
1. COVER GLASS METHOD
- Preferred to be used during bone marrow smears
2. WEDGE METHOD
● Specimen of choice for thin blood smears
● Procedure
○ Stationary Slide
○ Spreader/Pusher Slide
■ Amount of blood placed in the stationary slide should be formed 2-3 mm
diameter.
■ Angles should be between 30-45° to produce smear thick and thin in the end
with feathery transition must be gradual. ( Affect the thickness and thinness
of smear)
● Advantages:
○ Large examination area
○ Easy to prepare, label, stain and transport
○ Allows immediate storage without mounting
○ Easier to find abnormal cells that accumulates at the feathery edge
● Disadvantages:
○ Uneven distribution of cells
○ Greater trauma to the cells because of technique
■ Smudge cells can be seen in granulocytes and are not pathogenic in origin,
only due to the trauma of smear.
Notes: If there are no abnormal cells, there will be no uneven distribution for large cells to be brought to the
feathery edge. (Abnormal cells are usually larger than normal cells)

○ Large cells accumulate on the tail

○ The angle of the smear affects the thinness/thickness of the smear
○ Blood should run along the junction
○ Preferred:thick at one end and thin and feathery at the other edge
● Characteristics of a Well-prepared Smear:
○ About two-thirds to three-fourths of the length of the slide is covered by the
smear.
○ It is slightly rounded at the feather edge (thin portion), not bullet shaped.
○ Lateral edges of the smear should be visible. The use of slides with chamfered
(beveled) corners may facilitate this appearance.
○ It is smooth without irregularities, holes, or streaks.
○ When the slide is held up to light, the feather edge of the smear should have a
“rainbow” appearance.

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 ○ The whole drop is picked up and spread

● Characteristics of an Unsatisfactory Smear:

○ Too short, doesn’t cover two-thirds/three-fourth of the slide
○ Thick , then thin areas if you didn’t immediately place the stationary glass at the
table
● Difficulties/Irregularities
○ Extremely thick smears
■ Due to excess plasma which can cause the cells to shrink
■ If RBCs form a rouleaux formation
■ Causes: too large drop of blood used; increased angle of the spreader slide;
too light pressure on the stationary slide; too fast spreading motion.
■ Factors: Volume of blood; angle of spreader, speed of smear, pressure
exerted in stationary slide.
■ Disadvantages:
● Can easily be peeled during staining
● What makes it unacceptable? RBCs may obscure leukocytes.
○ Extremely thin smears
■ Able to see smudge and basket cells with protruding nuclei
■ RBCs tend to look spheroid/abnormal in shape
■ Causes: too small drop of blood uses; decrease angle of the spreader slide;
too heavy pressure on the stationary slide; too slow spreading motion.
○ Streaks on the Feathery edge
■ High leukocyte count with blast, dirt or dust particles on the slide, picking up
only part of the drop of blood.
■ Large amount of time elapses after the blood has been placed.
■ Causes: due to inability to prepare smear right after blood is dropped
○ Bubbles or holes
■ Excess fat or blood samples
■ Dirt or dust particles on the slide.
○ Waves
■ Jerky spreading motion (Not stable smearing)

Notes: General Evaluation of RBC Morphology and Distribution is part of evaluation while doing leukocyte
differential count.

3. COVERGLASS METHOD (Not for automated staining methods)

● Procedure:

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 ○ Should be mounted on a slide first with a mounting medium before microscopic
examination.
● Advantages:
○ Preferred for bone marrow smear preparation.
○ Even distribution of cells.
● Disadvantages:
○ More difficult to prepare, label, stain and transport.
○ Require mounting
○ Time-consuming and hard to dry

C. Scanning methods:
Longitudinal Battlement
Pattern: Tail to Head Back and Forth (Serpentine)

Note: Always read on transition from thick to thin, where RBCs do not overlap

HEMATOPOIESIS
A. Hematopoiesis
1. Production and differentiation of blood cells.
2. Blood cell production, maturation, and death occur in organs of the reticuloendothelial system
(RES).
● Bone marrow
● Spleen
● Liver
● Thymus
● Lymph Nodes
● Roles of RES:
○ Hematopoiesis
○ Phagocytosis
○ Immune Defense

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3. Intrauterine hematopoiesis/Prenatal Hematopoiesis includes three phases:
○ Mesoblastic (yolk sac) phase - goal is to begin the primitive hematopoiesis to promote
primitive erythroblasts which brings oxygen
■ Begins at 19 days gestation.
■ The first cell to be produced is a primitive nucleated erythroblast.
■ This cell produces embryonic hemoglobins:
● Hb Gower I - 2 zeta, 2 epsilon
● Hb Gower II - 2 alpha, 2 epsilon
● Hb Portland - 2 gamma, 2 epsilon
■ Alpha-globin chain production begins at this phase and continues throughout life.
■ At the 2nd month liver assumes the primary role of cell production
○ Hepatic (liver) phase
■ Begins at 6 weeks gestation with production of mainly red blood cells
■ But granulocytes, monocytes, and megakaryocytes appear.
■ Alpha- and gamma-globin chain production predominates forming: the Hb F (Fetal
hemoglobin)
● 2 alpha, 2 gamma
■ Adult hemoglobin appears at a SMALL PERCENTAGE
● 2 alpha, 2 beta
■ Hb A2
● 2 alpha, 2 delta
■ Extrahepatic secondary hematopoiesis occurs: Thymus, spleen, lymph nodes,
kidney (Secondary organs)
● Thymus - T cell production
● Kidney/Spleen - B cell production
○ Myeloid/Medullary Phase:
■ Begins around the 5th month of gestation and around 6th to 7th month BONE
MARROW assumes the primary role of cell production (hematopoiesis)
■ The M:E (myeloid {produced WBCs} : erythroid{Produced RBCs}) ratio approaches the
adult level of 3:1. MORE WBC, LESS RBC
■ Alpha- and gamma-globin chain production predominates at birth, forming Hgb F;
(Persists after 1 to 2 weeks after birth)
■ Hgb A andA2 are also present.

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 ■ Hgb A will not predominate until 6 months of age when the gamma-beta globin chain
switch is complete.
4. At birth, the bone marrow is very cellular with mainly red marrow, indicating very active blood cell
production. Red marrow is gradually replaced by inactive yellow marrow composed of fat. Under
physiological stress, yellow marrow may revert to active red marrow.
Note: Process of replacing red marrow with yellow marrow during development is known as RETROGRESSION

● At 4 yrs of Age: Adipocytes (fat cells) replace the hematopoietic stem cells in your red
marrow
● At 5 yrs of Age: Amount of yellow marrow dominates the amount of red marrow
● At 18 yrs of Age: Transition will be complete from red marrow to yellow marrow

B. Pediatric and Adult Hematopoiesis

1. Bone Marrow
a. Newborn: 80-90% of bone marrow is an ACTIVE RED BONE MARROW
b. Young adult (age 20): Hematopoiesis is confined to the proximal ends of large flat bones,
pelvis, and sternum.
c. Older adult (age 55): 40% of bone marrow is active, while 60% is fat
d. Cellularity is the ratio of marrow cells to fat (red marrow/yellow marrow) and is described in
adults as:
i. Normocellular - Marrow has 30-70% of hematopoietic stem cells
ii. Hypercellular/hyperplastic - Marrow has >70% of HSC
iii. Hypocellular/hypoplastic- Marrow has <30% of HSC
iv. Aplastic—Marrow has few or no hematopoietic cells.
e. M:E (myeloid:erythroid) ratio is the ratio of granulocytes and their precursors to nucleated
erythroid precursors.
i. Normal M:E ratio is 3:1 and 4:1
ii. Granulocytes are more numerous because of their short survival (1-2 days) as
compared to erythrocytes with a 120-day life span.
iii. Lymphocytes and monocytes are excluded from the M:E ratio.
f. Stem cell theory
i. Hematopoiesis involves the production of pluripotent stem cells that develop into
committed progenitor cells (lymphoid or myeloid) and finally mature blood cells,
ii. Progenitor cells:
● Hematopoietic Stem Cell is capable of:
a. Multipotential
b. Totipotential
c. Self-renewal
i. Common Lymphoid Progenitor:
- T Cells, B Cells, Nk Cells
ii. Myeloid:
- Granulocytes: Neutrophils, Eosinophils, Basophils, Monocyte
- Erythrocytes: RBCs
C. Lymphoid Tissue
1. Primary Lymphoid Tissue

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 a. Antigen Independent Lymphopoiesis
● develop into B cell or T cell without antigen to stimulate their production
b. Bone Marrow - site of B cell differentiation
c. Thymus - site of T cell differentiation
2. Secondary Lymphoid Tissue
1) B and T lymphocytes enter the blood and population secondary lymphoid tissue, where
antigen contact occurs.
2) Includes lymph nodes, spleen, gut-associated tissue (Peyer's patches)
3) Antigen-dependent lymphopoiesis depends on antigenic stimulation of T and B
lymphocytes.
- When there is a contact between T cell and B cell with an antigen, they become
specific lymphocytes into that antigen which is referred to as antigen-dependent
lymphopoiesis.
- Example: Plasma cells → Production of immunoglobulins → Ab-Ag
D. Medullary versus Extramedullary Hematopoiesis
1. Medullary hematopoiesis (Bone marrow)
● Blood cell production within the bone marrow
a) Begins in the fifth month of gestation and continues throughout life
b) Instances where bone marrow cannot produce enough cells:
(1) BM cannot meet body reqs
(2) BM cannot meet demand

Example:
- Cases of hemolytic anemia (there is an acute blood loss but BM can’t meet the demand (huge amount of
oxygen & RBC) and BM can’t immediately compensate for the loss due to sudden blood loss.

2. Extramedullary hematopoiesis
● Blood cell production outside the bone marrow
- In such cases, Extramedullary Hematopoiesis occurs where your spleen & liver revert
back the cell production in order to compensate for the blood loss. If this action where
the two organs have added function, this will be evident in physical examination
(splenomegaly: abnormally enlarged spleen and hepatomegaly: abnormally enlarged
liver).
- Lumalaki ‘tong mga organs na ito kapag merong added function which is cell
production (because they’re functioning abnormally: hindi dapat sila ang gumagawa ng
function na ito)

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E. Marrow hematopoiesis is divided into three major compartments or cell types:
1) Hematopoietic Stem Cells (HSC)
- also known as CFU-S or Colony Forming Unit Spleen (has the capability to self renew)
2) Multipotent Progenitors
Two multipotential pathways:

● Multipotential Stem cells (CMP)

○ which proliferates and differentiates into individual granulocytic, erythrocytic,
monocytic, and megakaryocytic lineages
● Multipotential Stem Cells (CLP)
○ Proliferates and differentiates into T, B, and natural killer lymphocyte and dendritic
lineages
3) Lineage
● Morphologically recognizable, lineage- specific precursor cells
● Committed and Restricted precursor
● CD34
○ use serum containing CD, recognized due to additional expression of antigens
○ (cell expression due to antigen) where upon development, “antigenic expression”
occur that serves as receptor in the cell membrane
○ not specific in Lineage precursors cell; it can also be found in progenitor cells and
hematopoietic stem cell
ADDITIONAL EXPRESSIONS

CD38 known as precursor page

CD71 responsible for erythroid differentiation

CD33 for myeloid differentiation

CD10 for myeloid differentiation

CD7/CD5 for T cell differentiation

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 Early-Acting Multilineage Growth Factors
➢ KIT ligand & FLT3 ligand
○ stimulates cell to proliferate
○ work synergistically with IL-3, GM-CSF, and others cytokines to promote early
HSC proliferation and differentiation
➢ IL-3 (Interleukin 3)
○ regulates blood cell production by controlling the production, differentiation, and
function of granulocytes and macrophages
➢ GM-CSF (Granulocyte Macrophage-Colony Stimulating Factor)
○ induces expression of specific genes that stimulate HSC differentiation to the
common myeloid progenitor
QUESTION: So paano malalaman ng mga cell na mag m-mature sila? Ewan ko tangina
● due to the early-acting multilineage growth factors (CFU-GM will differentiate into CFU-G & CFU-M) this differentiation is
enhanced by these growth factors
● KTI & LFT3 stimulate the cell to proliferate and work synergistically with IL3 and GM-CSF and other cytokines to promote
early HSC proliferation and differentiation.

F. Basic Cell Morphology

1) Nucleus
● Chromatin: contains DNA & proteins
● Contains nucleoli rich in RNA
2) Cytoplasm
● Golgi complex: form lysosomes (found in phagocytic cells kaya they can kill pathogenic
microorganism)
○ Lysosome contains hydrolytic enzymes (phagocytosis) which can kill foreign
microorganism
● Ribosome: assembles amino acid into protein
● Mitochondria: produce necessary ATP for basic cellular processes
○ In order to convert amino acid into proteins, it needs ATP (source of energy)

General Characteristics of Blasts

1) Size
● Generally blast cell are larger with high NC (Nuclear Cytoplasmic) ratio
○ NC - nucleus occupies most spaces compared to cytoplasm
2) Cytoplasm
● basophilic in color (deeply purple) due to the presence of RNA
3) Nucleus
● large, round to oval in shape
4) Capable of Mitosis
● DNA - chromatin (euchromatin): very fine, smooth, reddish-purple in color, no
clumping because it still undergoing mitosis
● NUCLEOLI: pale light blue
QUESTION: Kelan nagiging mature cell?
● SIZE: smaller than blast with low NC ratio
● CYTOPLASM: less basophilic due to loss of RNA (pale blue/purple
● CHROMATIN: coarse and clump (no longer capable of mitosis or division)

Three Erythroid Precursor Nomenclature Systems

Normoblastic Rubriblastic Erythroblastic

Pronormoblast Rubriblast Proerythroblast

Basophilic normoblast Prorubricyte Basophilic erythroblast

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 Polychromatic (polychromatophilic) Rubricyte Polychromatic (polychromatophilic) erythroblast
normoblast

Orthochromic normoblast Metarubricyte Orthochromic erythroblast

Polychromatic (polychromatophilic) Polychromatic (polychromatophilic) Polychromatic (polychromatophilic) erythrocyte*

erythrocyte* erythrocyte*

Erythrocyte Erythrocyte Erythrocyte

A) As the cell matures, cell

diameter decreases and
cytoplasm becomes less
basophilic
● Ribosomes: responsible for
the conversion of amino acid to
protein
● Hemoglobin: protein component of RBCs
● An exception to the diameter decreasing is that in the granulocytic series, the
promyelocyte may be larger than its precursor, the myeloblast (see Chapter 5).
● In the erythroid series, hemoglobin development in the cytoplasm imparts a
pink/salmon color.
B) N:C ratio decreases; Nucleus changes from purplish red(initial color of the cell) to
dark blue (mature cells)
C) Nuclear chromatin becomes coarser, clumped, and condensed.
● Nucleoli disappear
● In the granulocytic series, the nuclear shape changes and the nucleus becomes
segmented.
○ Nuclear chromatin becomes coarser, clumped, and condensed.
● Granules appear in cytoplasm
● In the erythroid series, the nucleus becomes fully condensed and is ejected

ERYTHROCYTES
A. General Characteristics
1. Oxygen transport, removal of metabolic waste

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 a. Primary function: deliver oxygen all throughout the body through hemoglobin (metabolic
waste: CO2)
2. Loss of nucleus is required for function
3. Normal life span is 120 days
B. Erythropoietin
1) Produced mainly by the kidneys.
2) Growth factor that stimulates erythrocyte production from myeloid progenitor cell;
influences colony-forming unit-erythrocytes (CFU-Es) to differentiate into
erythroblasts
C. Erythrocyte Maturation

ERYTHROCYTE SIZE N:C NUCLEOLI CHROMATIN NUCLEUS CYTOPLASM Reference

RATIO Range

Pronormoblast 20µm 8:1 1-3 nucleoli fine & dark areas of Deep blue; no
(rubriblast) uniform; DNA granules
- Earliest RBC stains
intensely

Basophilic normoblast 16µm 6:1 0-1 nucleoli coarsening Centrally less blue;
(prorubricyte) located nucleus intensely
- RNA: in preparation basophilic (RNA)
for Hgb synthesis

Polychromatophilic 12µm 4:1 no nucleoli Shows Eccentric gray-blue

normoblasts significant nucleus cytoplasm;
(rubricyte) clumping Begins to
produce
- Last stage capable of
hemoglobin
mitosis

Orthochromic 10µm 0.5:1 no nucleoli Eccentric with Pale blue to

normoblast small, fully salmon;
(metarubricyte) condensed Hemoglobin
(pyknotic) synthesis
- Youngest cell not
nucleus decreases
capable of mitosis
- Last nucleated stage

Adult: 0.5-1.5%
Reticulocyte NB: 2.5-6.5%
- Last stage to synthesize no nucleus; has
hemoglobin 10µm mitochondria & - w/ slightly
- Last stage to synthesize ribosomes increased
hemoglobin
ranges at higher
altitudes

Mature erythrocyte 6-8µm Salmon with F: 4.0-5.4x1012/L

- round biconcave, central pallor F: 4.0-5.4x106/µL
discocyte (clearing in the
center) when a M:4.6-6.0x1012/L
- Donut shaped
blood smear is M:4.6-6.0x106/µL
Wright's stained
*NB: Newborns; F: Female; M: Male
● Reticulocyte

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 ○ A supravital stain is used to enumerate reticulocytes
■ Methylene blue/New methylene blue (NMB) - stains basophilic reticulum of RNA
○ Reticulocyte count is one of the best indicators of bone marrow function
○ Stress reticulocytes are young cells released from bone marrow after older reticulocytes
have been released. This is a response to increase
■ Seen in peripheral blood
■ Larger in size
○ Hemoglobin continues to be produced by reticulocytes for approximately 24 hours after
exiting the bone marrow.
● Mature erythrocyte
○ Central pallor: normal size is 1/3 of its diameter
■ Decreased central pallor: seen spherocytic cells also known as spherocyte
■ Increased central pallor: seen in microcytic anemia and hypochromic cell
○ Erythropoiesis: regulated by erythropoietin produced in the kidney. Additional regulation
includes:
■ Tissue Hypoxia- low oxygen tissue; trigger of erythropoietin
● Heart, Lung dysfunction, anemia
● In order for the organs to survive, continuous supply of oxygen is needed
○ Too much production of erythrocytes, erythrocytosis
❖ Polycythemia vera
○ Decrease in RBC production
❖ Aplastic anemia
■ Androgens (male hormones that appear to enhance the activity of erythropoietin) and
hemolytic anemias (increased erythrocyte destruction
RED BLOOD CELL MATURATION

Cell or Stage Diameter Nucleus-to- Nucleoli % In Bone Bone Marrow

Cytoplasm Marrow Transit Time (hr)
Ratio

Pronormoblast 12 - 20µm 8:1 1-2 1% 24 hrs

Basophilic normoblast 10 - 15µm 6:1 0-1 1-4% 24 hrs

Polychromatic Normoblast 10 - 12µm 4:1 0 10-20% 30 hrs

Orthochromic normoblast 8 - 10µm 1:2 0 5-10% 48 hrs

Shift (stress) reticulocyte 8 - 10µm No nucleus 0 1% 48-72 hrs

(polychromatic erythrocyte)

Polychromatic erythrocyte 8 - 8.5 µm No nucleus 0 1% 24-48 hrs

NOTE:
● Up to 16 RBCs are produced from single Polychromatophilic normoblast/rubricyte

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 ● RBC lifespan: 120 days
● Start of hemoglobin synthesis: Polychromatophilic normoblast/rubricyte
● End of hemoglobin synthesis: Reticulocyte/Polychromatophilic erythrocyte

D. Erythrocyte Physiology
1) Early RBCs get energy from oxidative phosphorylation. During maturation, the
mitochondria are lost, and energy is derived from glycolysis.
2) Erythrocytes need proper volume ratio for exchange of blood gasses and flexibility to
travel through capillaries. This is accomplished by the cation pump, a mechanism that
keeps sodium outside and potassium inside the cell.
3) Erythrocyte membrane is 50-60% lipid (phospholipids, cholesterol, and glycolipids)
and 40-50% protein.
a) Erythrocyte Membrane
i) Protein (50%)0
(1) Integral protein - Glycophorin A
➢ Contains SALICYLIC ACID
○ Negative charge (Zeta potential) - repel each other
(a) Ex. LISS- “enhancer” for agglutination
● Reduce the zeta potential
(2) Peripheral protein
➢ Spectrin - maintains the RBC biconcavity
➢ Actin
ii) Lipid (40%)
(1) Increase cholesterol and phospholipid
● codocyte or target mexican hot cell
(2) External surface:
● phospholipids phosphatidylcholine,
● glycolipid and sphingomyelin
(3) Internal surface:
● phosphatidylethanolamine
● phosphatidylinositol
● phosphatidylserine
(4) Cholesterol content
● plasma cholesterol
● bile acids
● the activity of the enzyme lecithin: cholesterol acyltransferase (LCAT)
which maintains the cholesterol content of RBC.
iii) Carbohydrate (10%)

METABOLISM OF RED BLOOD CELLS

1. EMBDEN-MEYERHOF PATHWAY
● MAjor source of red cell energy
● 90% glycolysis
● Every glucose broken down to lactate yields 2 ATP

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 ○ End product
● ATP controls Na* and K* and prevent oxidation of membrane lipid
2. HEXOSE MONOPHOSPHATE SHUNT/PENTOSE PHOSPHATE PATHWAY
● 10% glycolysis
● Provides REDUCED GLUTATHIONE to prevent denaturation of hemoglobin
● G6PD- absence; Heinz bodies
● End result- reduced glutathione
● He, He, He
○ HEinz bodies is by product of denatured HEmoglobin found in a defect in the
HExose monophosphate shunt
3. Rapoport-Luebering Pathway
● Generation of 2,3-BPG which regulates hemoglobin affinity for 02
● 2,3 BPG Increase, O Decrease- Shift to the right
● 2,3 BPG Decrease, O Increase- Shift to the right
○ Inverted in affinity
4. Methemoglobin Reductase Pathway
● Maintains hemoglobin in Fe2+(Ferrous) state to be functional

BREAKDOWN OF RED BLOOD CELL

● Culling- destruction of senescent red blood cells
● As RBC ages- Decrease enzyme, ATP and size; Increase density
● Approximately 1% of the red blood cells leave the circulation each day and are broken down
by the mononuclear phagocytic system (MPS)
○ Found in RES specifically the spleen
● Recycled component- iron

1. EXTRAVASCULAR (90% AGED RED CELL DESTRUCTION)

● Major
➢ Within RES
● When complement is not activated or incompletely activated
● Rh incompatibility
2. INTRAVASCULAR (10% AGED RED CELL DESTRUCTION) Win AN
● Within blood vessels
● Complement is activated
● Hemoglobinuria
● Hemopexin is decreased
● Haptoglobin is decreased
➢ Carrier protein of iron
● ABO incompatibility

E. Substances Needed for Erythropoiesis

1. Iron:
2. Amino acids:
3. Folic acid/vitamin B12:

17
 4. Others:

F. Erythrocytic Morphology and Associated Disease (Size and Shape)

1. Anisocytosis
● Variation in RBC size, indicating a heterogeneous RBC population (dimorphism)
● Correlates with RDW (red blood cell distribution width)
→ Post-transfusion
→ post-treatment for a deficiency (e.g., iron),
→ presence of two concurrent deficiencies (e.g., iron and vitamin B12),
→ idiopathic sideroblastic anemia

2. Normocytes (discocytes) are normal erythrocytes that are approximately the same size
as the nucleus of a small lymphocyte
● Normal MCV (80-100fL)
○ Acute hemorrhagic anemia
○ Hemolytic anemia
○ Aplastic anemia
3. Macrocytes
a. RBCs greater than 8um in diameter; MCV greater than 100 fL
● Chemotherapy
○ Affect the progenitor stem cell in bone marrow
● Liver disease
● Alcoholism
● Megaloblast Anemia
○ Folic acid deficiency
■ Pregnancy
■ Dietary deficiency (vegetables-source of folic acid)
■ Sprue/Steatorrhea
○ Vit. B12 (Cobalamin) deficiency- Pernicious anemia; decrease in intrinsic
factor
■ Vegetarian diet
■ D. latum
■ Malabsorption syndrome
4. Microcytes

18
 a. RBCs less than 6um in diameter; MCV less than 80 fL
● Iron deficiency
● Thalassemia
● Sideroblastic anemia
● Anemia of chronic disease
5. Poikilocytosis
● Variation of size
a. Poikilocytosis secondary to developmental defect
● Megalocytes/Macroovalocytes/Macrocytes
● Megaloblastic anemia
■ Asynchronous development
Type Cause Pathology Cells Disorder

Megaloblastic Cobalamin or ● Nuclear maturation lags behind Macrocytes Pernicious

folate cytoplasmic maturation anemia
● Cells grow larger without dividing

Iron deficiency Iron deficiency ● Cytoplasmic maturation lags behind Microcytic IDA
nuclear maturation due to deficiency Hypochromic
of Iron needed for hemoglobin
synthesis

b. Poikilocytosis secondary to membrane defect

6. Echinocytes
● Include created and burr cells
● Have evenly spaced round projections; central pallor area present
○ Liver disease uremia
○ Uremia; anemia associated with renal insufficiency
○ Heparin therapy
○ Pyruvate kinase deficiency
○ Artifact/effect of surreal drying
● Caused by changes in osmotic pressure
7. Acanthocytes (spur cells)
● Have unevenly spaced pointed projections; lack a central pallor area
○ Alcoholic liver disease
○ Post-splenectomy,
○ Abretalipoproteinemia
● Caused by excessive cholesterol in the membrane
8. Target cells (codocytes or Mexican hat cells)
● Show a central area of hemoglobin surrounded by a colorless ring and a
peripheral ring of hemoglobin; cells have an increased surface-to-volume ratio
○ Liver disease
○ Hemoglobinopathies
○ Thalassemia
○ IDA

19
 ● Caused by excessive cholesterol in the membrane or a hemoglobin distribution
imbalance
9. Spherocytes
● Disk-shaped cell with a smaller volume than a normal erythrocyte;
● Cells have a decreased surface-to-volume ratio
● Lack of central pallor area; due to abnormal spectrin
● Associated with defects of the red cell membrane proteins
● MCHC >37% ; Increase in Osmotic Fragility
● Damaged RBC; seen in:
○ Hereditary spherocytosis; Increase OFT, MCHC, (-) DAT
○ G6PD deficiency
○ Immune hemolytic anemias; Increase OFT, MCHC, (+) DAT
● Banked blood for a long time
● Microspherocytes
○ (<4 um) are frequently seen in severe thermal injury (burns)
10. Stomatocytes (mouth cells)
● Characterized by an elongated or slit-like area of central pallor
■ Liver disease
■ Hereditary stomatocytosis
■ Rh null disease
● Caused by osmotic changes due to cation imbalance (Na+/K+)
11. Elliptocytes (ovalocytes)
● Cigar to egg-shaped erythrocytes
● Abnormal aggregation of hemoglobin; settle in bipolar ends
● Associated with defects of the red cell membrane protein
○ Hereditary elliptocytosis
■ Membrane protein band 4.1
○ IDA
○ Megaloblastic anemia (macro-ovalocytes)
○ Thalassemia major

c. Poikilocytosis secondary to trauma

12. Teardrops (dacryocytes)

● Pear-shaped cell with one blunt projection
● Squeezing and fragmentation due to splenic passage
■ Hypersplenism - enlarged spleen
■ Thalassemia
13. Helmet cells (horn cells or keratocytes)
● Interior portion of cell is hollow, resembling a horn or helmet
● Clothesline effect
● PITTING - removal of inclusions resulting to BITE CELL
■ Howell jolly bodies
■ Heinz bodies

20
 ● degradation of hemoglobin found in defect in the HExose
monophosphate shunt
● Seen in:
■ MAHA- Microangiopathic Hemolytic Anemia
● DIC- Disseminated Intravascular Coagulation
● Throbocytophilic purpura
● Hemolytic Urinic Syndrome
■ Burns
extramedullary hematopoiesis (myelofibrosis, myelophthisic anemia)
14. Schistocytes (RBC fragments)
● Damaged RBC; fragments of various sizes and shapes are present, often with
pointed projections
■ Microangiopathic hemolytic anemias (e.g., DIC, HUS, TTP)
■ Thermal injury
■ Renal transplant rejection
■ 6 GPD deficiency
■ Prosthetic heart valve
● Damage due to pumping
d. Poikilocytosis secondary to hemoglobin content
15. Sickle cells (drepanocytes)
● Shapes vary but show thin, elongated, pointed ends and will appear crescent
shaped; usually lack a central pallor area
○ Contains polymer of abnormal hemoglobin S
○ HemoglobinopathiesSickle
● Cell shape is caused by cell membrane alterations due to an amino acid
substitution

G. Erythrocyte Inclusions and Associated Diseases

1. Nucleated RECS (MRBCS, nUORBCS)
● Orthochromatic/Orthochromic normoblast
➢ metarubricyte
● Indicate bone marrow stimulation or increased erythropoiesis
● Associated with:
i. thalassemia major
ii. sickle cell anemia
iii. hemolytic anemias
iv. erythroleukemia
v. myeloproliferative disorders- defect in bone marrow production
● Sunny side up appearance
■ Normal (small amount); newborns but in adults associated with disorder
2. Howell-Jolly bodies
● Small, round DNA fragments (0.5-1.0um in diameter) usually one per cell, but can be
multiple
● Dark purple to black
■ Wright's stain
● Not seen in normal erythrocytes; normally pitted by splenic macrophages

21
 ● (+) Fuelgen reaction
● Seen in:
i. sickle cell anemia
ii. beta-thalassemia
iii. major megaloblastic anemia
iv. Post-splenectomy
■ Wala nang mag f-filter sa cells (splenic pitting)
3. Basophilic stippling
● Multiple, tiny, fine, or coarse inclusions (ribosomal RNA remnants) evenly dispersed
throughout the cell; "blueberry bagel" appearance
● Well distributed
● Dark blue
i. Wright's stain
● Precipitation of ribosomes/RNA
● Seen in:
i. Thalassemias
ii. Megaloblastic anemias,
iii. Sideroblastic anemia
iv. Pyrimidine 5-nucleotidase deficiency
v. Lead poisoning, and alcoholism
■ Heavy metal poisoning; inhibits
a. Ferrochelatase
➢ Covernts Protoporphyrin IX to Heme
b. ALA dehydratase
➢ Enzyme that responsible converting ALA to PBG
4. Pappenheimer bodies
● Small, irregular, dark-staining iron granules usually clumped together at
periphery of the cell
■ Concentrated in the periphery
● (+) Perls prussian blue
■ For iron granules
● Dark violet
■ Wright’s stain
● Caused by an accumulation of ribosomes, mitochondria, and iron fragments
● Seen in:
i. Sideroblastic anemia
ii. Hemoglobinopathies & thalassemia
iii. Megaloblastic anemia
5. Cabot rings
● Thin, red-violet, single to multiple ringlike structures that may appear in loop or
figure-eight shapes
● Seen in:
○ Megaloblastic anemia,
○ Myelodysplastic syndromes,

22
 ○ Lead poisoning
● Composed of fragments of nuclear material
● Remnants of microtubules and mitotic spindle

6. Hemoglobin C crystals
● Condensed, intracellular, rod-shaped crystal
● Seen in:
○ Hemoglobin C or SC disease, but not in trait
➢ Dapat homozygous ang genetic condition bago siya mag appear
7. Hemoglobin SC crystals (Washington monument)
● 1-2 blunt, finger like projections extending from the cell membrane
● Seen in:
i. Hemoglobin SC disease
8. Heinz bodies
● Multiple inclusions ranging in size from 0.3 to 2.0um; deep purple irregularly shaped
inclusions
● Supravital stain to visualize;
○ Crystal violet- Heinz bodies
○ NMB(new methylene blue)- Reticulocyte
● Invisible with Wright's stain;
● Seen in:
○ G6PD deficiency
○ beta-thalassemia major
○ HbH disease- unstable hemoglobin
○ Hereditary defect in hexose monophosphate shunt
○ Favism; “Fava beans”
○ Represent denatured hemoglobin
9. Malarial parasites--includes;
● P. vivax, P-falciparum,
● P. malariae
● P.ovale
10. Babesia - protozoa inclusion (B.microti) transmitted from deer to humans by tick bite

H. Erythrocyte Hemoglobin Content and Associated Diseases

1. Normochromic
a. Cells have the normal one third clear, central pallor area
b. MCHC- 31-36%
2. Hypochromic
a. Central pallor area is greater than one-third the diameter of the celli
b. MCH and MCHC usually decreased (<31%)
c. Often associated with microcytosis
d. Seen in:

23
 i. IDA
1. Increase TIBC- measures the available transferrin
ii. Thalassemias
iii. Anemia of chronic disease
iv. Sideroblastic anemia,
v. Myelodysplastic syndromes
3. Hyperchromic
a. Current terminology is spherocyte; lacks a central pallor area
b. Seen in:
i. Increase MCHC (>36%
4. Polychromasia
● Variation in hemoglobin content showing a slight blue tinge when stained with
Wright's stain; residual RNA
■ Blue gray coloration/Pink
● Indicates young red blood cells or presence of reticulocytes
● Usually slightly macrocytic

I. Abnormal Erythrocyte Distributions and Associated Diseases

1. Rouleaux
● Stacking or "coining" pattern of erythrocytes due to abnormal or increased plasma
proteins
● Seen in:
○ hyperproteinemia
○ Multiple myeloma
■ Proliferation of immunoglobulin (produced by plasma cells)
○ Walden Strom macroglobulinemia
○ increased fibrinogen (chronic inflammation)
■ Acute phase reactant
○ Increased ESR
● May be an artifact; considered normal in the thicker area of the peripheral smear
● True rouleaux formation is determined in the thin area of the peripheral smear.
2. Agglutination
● Characterized by clumping of erythrocytes with no pattern
● Occurs when erythrocytes are coated with IgM antibodies and complement
● Seen in:
○ Cold autoimmune hemolytic anemia (cold agglutinin disease) due to presence of of
Anti-I
● Warm blood to 37C to correct a falsely low/decreased RBC or hematocrit and also cause
falsely increase in MCHC (in hemodialyzers)

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HEMOGLOBIN
A. Introduction
a. Hemoglobin is an oxygen-transporting protein contained within erythrocytes.
b. The heme portion of hemoglobin gives erythrocytes their characteristic red color.
B. Hemoglobin Structure
1. Four identical heme groups, each consisting of a protoporphyrin ring and ferrous (Fe2+) iron
2. Four globin (polypeptide) chains
a. Alpha chains have 141 amino acids
b. Beta, gamma, and delta chains have 146 amino acids.
3. The amino acid sequence of the globin chain determines the type of hemoglobin, normal
adult hemoglobin consists of two alpha and two non-alpha (beta) chains in pairs.
a. 1 heme can carry 1 mole of O2
b. 1 hemoglobin can carry 4 moles of O2
C. Hemoglobin Synthesis
1. 65% hemoglobin synthesis occurs in immature nRBCs.
2. 35% hemoglobin synthesis occurs in reticulocytes.
3. Heme synthesis occurs in the mitochondria of normoblast and is dependent on glycine,
succinyl coenzyme A, aminolevulinic acid synthetase, and vitamin B6 (pyridoxine).
4. Globin synthesis occurs in the ribosomes, and it is controlled on:
a. Chromosome 16 - responsible for encoding alpha and zeta chains
b. Chromosome 11 - responsible for encoding beta, delta, epsilon gamma
5. Each globin chain binds to a heme molecule in the cytoplasm of the immature RBC.
D. Hemoglobin Synthesis
1. Intravascular hemolysis (10%)
● Occurs when hemoglobin breaks down in the blood and free hemoglobin is released into
plasma
● Free hemoglobin binds to haptoglobin, hemopexin, and albumin, and it is phagocytized by
liver macrophages.
● Laboratory:
○ Increased plasma hemoglobin
○ serum bilirubin
○ urine urobilinogen
○ Hemoglobinuria
■ decreased serum haptoglobin (haptoglobin is the carrier protein of free hemoglobin)
● Consumed or used up due to the increased free hemoglobin in the circulation
(maraming kailangan i-transport kaya nauubos siya)
2. Extravascular hemolysis (90%)
● Occurs when senescent/old RBCs are phagocytized by macrophages in the liver or
spleen
● Protoporphyrin ring metabolized to bilirubin and urobilinogen; excreted in urine and
feces
● Globin chains are recycled into the amino acid pool for protein synthesis.

25
 ● Iron binds to transferrin and is transported to bone marrow for new RBC production, or it
is stored for future use in the form of ferritin or hemosiderin
E. Hemoglobin and Iron
● Most iron in the body is in hemoglobin and must be in the ferrous state (Fe2+) to be used.
Fe2+ binds to oxygen for transport to lungs and body tissues. Ferric iron (Fe3+) is not
able to bind to hemoglobin, but does bind to transferrin. Iron is an essential mineral and is
not produced by the body.
○ Serum iron - lab test measures the amount of Fe3+ bound to transferrin
○ Total iron-binding capacity (TIBC) measures the total amount of iron that transferrin can
bind when fully saturated.
○ Serum ferritin - indirect measurement of storage iron in tissues and bone marrow
F. Types of Hemoglobin
1. Hgb F (Fetal Hemoglobin) (Compensatory Hemoglobin)
● two alpha and two gamma-globin chains.
● Has higher affinity to oxygen compared to adult hemoglobin
● functions in a reduced oxygen environment.
● Hgb F predominates at birth (80%)
● Laboratory:
○ Alkali denaturation test and Kleihauer-Betke acid elution stain
■ Hgb F is resistant to denaturation
○ column chromatography
○ radial immunodiffusion
■ Hgb F is a compensatory hemoglobin and can be increased in homozygous
hemoglobinopathies and beta-thalassemia major.
2. Adult
● Hgb A contains:
○ two alpha- and two beta-globin chains
○ subdivided into glycosylated fractions.
■ A1c (HbA1c): fraction/variant that reflects glucose levels in the blood and is used to
monitor individuals with diabetes mellitus.
● Hgb A2 contains:
○ two alpha- and two delta-globin chains.

ADULT NEONATE

HbA1 ≥ 95% HbF 60-90%

HbA2 ≤ 3.5% HbA 10-40%

HbF < 2% (normal)

● HFPF (Hereditary Persistent Fetal Hemoglobin): If HbF is increased in adulthood

G. Different Forms of Normal Hemoglobin

1. Oxyhemoglobin
● Hemoglobin with Fe2+ + O2; seen in arterial circulation of oxygenated hemoglobin

26
 2. Deoxyhemoglobin
● Hemoglobin with Fe2+ but no O2; seen in venous circulation
3. Carboxyhemoglobin:
● Hemoglobin with Fe2+ and carbon monoxide (CO) [delikado raw]
○ If CO is present, hemoglobin will attach more in CO than O2
○ CO has 200x affinity to hemoglobin compared to oxygen C
○ an result to death → fatal
○ Reversible if given pure O2
4. Sulfhemoglobin:
● Hemoglobin with Sulfur
○ cannot transport O2
○ seldom reaches fatal levels
● caused by drugs and chemicals
● Irreversible
● Cannot be measured by cyanmethemoglobin method
5. Methemoglobin:
● Hemoglobin with Fe3+
● Cannot transport O2
● Increased level can cause cyanosis or anemia
○ Cyanosis: bluish discoloration of skin due to decreased O2 in tissues

H. Oxygen Dissociation Curve

a. Oxygen affinity is the ability of hemoglobin to bind or release oxygen.
b. Expressed in terms of the oxygen tension at which hemoglobin is 50% saturated with oxygen.
● Right shift
○ decreased oxygen affinity
■ More O2 is released to tissues
○ Increased or high 2,3-bisphosphoglycerate level
■ O2 affinity is inversely proportional to 2-3 BPG
○ increased body temperature
○ decreased body pH
● Left shift
○ increased oxygen affinity
■ Less O2 release to tissue
○ Decreased or low 2,3-bisphosphoglycerate (2,3-BPG) level
○ decreased body temperature
○ increased body pH

HEMOGLOBINOPATHIES
A. Introduction:
● group of inherited disorders
● structurally abnormal globin chain synthesis due to amino acid substitutions

27
 ● Qualitative defects in hemoglobin
● changes in RBC deformability and electrophoretic mobility can occur.
● Homozygous/disease conditions (both globin chains affected) are more serious than
heterozygous/trait conditions (only one globin chain affected).
● Target cells are commonly associated with the hemoglobinopathies.
● Hemoglobin electrophoresis, isoelectric focusing and/or DNA (PCR) analysis may be used to
confirm the diagnosis.
● Incidence: Hgb S > Hgb C > Hgb E
○ Sickle hemoglobin has higher incidence compared with Hgb C and Hgb E Hgb E - variant
seen in Southeast Asia countries

B. Sickle Cell Disease (Hgb SS) (α2β2^6Val)

● Sickle cell disease is caused when valine replaces glutamic acid at position 6 on both beta
chains.
○ Disease inherited from both parents
○ No adult hgb is produced (absent)
○ 80% Hgb S and 20% Hgb F Hgb A2 is variable.
○ Can be present or absent Homozygous condition
● Hemoglobin insolubility results when deoxyhemoglobin is formed.
○ Hemoglobin crystallizes in erythrocytes.
○ It is characterized by the classic sickled shape of erythrocytes.
● Clinical findings
○ Erythrocytes become rigid and trapped in capillaries; blood flow restriction causes lack of
oxygen to the tissues, resulting in tissue necrosis.
○ All organs are affected, with kidney failure being a common outcome; hyposplenism (lack
of splenic function) and joint swelling also occur.
○ Apparent immunity to Plasmodium falciparum
● Laboratory
○ Severe normochromic/normocytic hemolytic anemia with polychromasia resulting from
premature release of reticulocytes; bone marrow erythroid hyperplasia (M:E ratio
decreases)
■ M:E - myelo erythroid
■ Standard M:E - Adult (3:1)
○ Sickle cells, target cells, nucleated RBCs, Pappenheimer bodies, and Howell-Jolly bodies
are seen.
○ Increased bilirubin and decreased haptoglobin are characteristic due to hemolysis.
○ (+) hemoglobin solubility screening test
○ Hgb S migrates with hemoglobins D and G on alkaline hemoglobin electrophoresis; can
differentiate using acid electrophoresis

C. Sickle Cell Trait (Hgb AS)

● Sickle cell trait
○ caused when valine replaces glutamic acid at position 6 on one beta chain.
○ Defect is inherited from one parent ONLY

28
 ○ One normal beta chain can produce some Hgb A.
■ 60% Hgb A
■ 40% Hgb S are produced
■ with normal amounts of Hgbs A2 and F.
● Sickle cell trait generally produces no clinical symptoms.
● (+) hemoglobin solubility screening test
● Apparent immunity to Plasmodium falciparum
○ Plasmodium - ayaw nila mag multiply sa may defect na RBC, maarte sila vebs

D. Hgb C Disease/Hgb CC (α2β2^6Lys)

● Hgb C disease is caused when lysine replaces glutamic acid at position 6 on both beta
chains.
○ No Hgb A is produced
○ 90% Hgb C
○ 2% Hgb A2
○ 7% Hgb F
○ Mild anemia may be present.
● Laboratory:
○ Normochromic/normocytic anemia with target cells;
○ characterized by intracellular rodlike C crystals
● The heterozygous Hgb C trait patient is asymptomatic, with no anemia; the one normal beta
chain is able to produce approximately 60% Hgb A and 40% Hgb C, with normal amounts of
Hgb A2 and Hgb F.
○ Kapag trait, asymptomatic

E. Other Hemoglobinopathies
1. Hemoglobin E (α2β2^26Lys)
a. Caused when lysine replaces glutamic acid at position 26 on the beta chain
b. Found more commonly in Southeast Asian, African, and African-American populations
c. Homozygous condition results in mild anemia with microcytes and target cells;
heterozygotes are asymptomatic.
2. Hemoglobin D (α2β2^121Gln)
a. Caused when glycine replaces glutamic acid at position 121 on the beta chain
b. Both homozygous and heterozygous conditions are asymptomatic.

THALASSEMIAS
A. Introduction
● Group of inherited disorders
● decreased rate of synthesis of a structurally normal globin chain (quantitative defect)
● microcytic/hypochromic RBCs and target cells
○ Classified according to the globin chain affected
○ Thalassemia major
■ Severe anemia because of the absence of alpha or beta chains
○ Thalassemia minor/trait:

29
 ■ Mild anemia; sufficient alpha and beta chains produced to make normal
hemoglobins A, A2, and F, but may be in abnormal amounts
B. Beta-Thalassemia
● Major/homozygous (Cooley anemia)
○ Decreased or absent beta chains
○ no Hgb A; compensate with up to 90% Hgb F.
○ Excess alpha chains precipitate on the RBC membrane - Heinz bodies (causes cell
rigidity)
■ Easily destroyed in the BM
■ Easily removed in the spleen
○ Laboratory Findings:
■ Severe microcytic/hypochromic anemia
■ target cells; teardrops cells
■ many nRBCS
● indicative if increased rate of RBC production due to increased O2 demand
■ basophilic stippling
■ Howell-Jolly bodies,
■ Pappenheimer bodies
■ Heinz bodies
■ increased serum iron and increased bilirubin reflect the hemolysis
C. Alpha-Thalassemia
● Major (hydrops fetalis) (hemoglobin Bart synthesis)
○ All four alpha genes are deleted; no normal hemoglobins are produced. 80%
hemoglobin Bart's (4 gamma) produced
● Hgb H disease
○ Three alpha genes are deleted.
○ Decrease in alpha chains leads to beta chain excess.
○ Hemoglobin H (4 Beta),an unstable hemoglobin, is produced.
■ If there is unstable hemoglobin, Heinz bodies are present.

ANEMIAS
A. Introduction
● decrease in erythrocytes and hemoglobin - decreased oxygen delivery to the tissues.
● classified morphologically using RBC indices (MCV, MCH, and MCHC).
● Suspected when the hemoglobin levels in:
○ male: <12g/dL
○ female: <11g/dL

1. Relative (pseudo) anemia

● RBC mass is normal but plasma volume is DECREASED
● Secondary to an unrelated condition and can be transient in nature

30
 ○ Side effect or additional effect due to primary disorder
● Causes include conditions that result in hemodilution, such as pregnancy and
volume overload.
● Reticulocyte count normal; normocytic/normochromic anemia
2. Absolute anemia (True Anemia)
● RBC mass is decreased, but plasma volume is NORMAL
● This is indicative of a true decrease in erythrocytes and hemoglobin.
● Mechanisms involved include:
○ Decreased delivery of red cells into circulation
■ Caused by impaired or defective production
■ Bone marrow fails to respond; reticulocytopenia
○ Increased loss of red cells from the circulation
■ Caused by acute bleeding or accelerated destruction (hemolytic)
■ Bone marrow can respond; reticulocytosis

B. Impaired or Defective Production Anemias

1. Iron-deficiency anemia (IDA)
● Prevalent in infants and children, pregnancy, excessive menstrual flow, elderly with poor
diets, malabsorption syndromes, chronic blood loss (GI blood loss, hookworm infection:
Necator americanus, new world hookworm and old world hookworm)
D. latum Competes with vitamin B12 absorption

Hookworm Competes with Iron absorption

● Laboratory:
○ Microcytic/hypochromic anemia;
○ Levels of the following are DECREASED:
■ serum iron
■ ferritin hemoglobin/hematocrit
■ RBC indices
■ reticulocyte count
○ Increased RDW and total iron-binding capacity (TIBC)
○ smear shows ovalocytes/pencil forms

2. Anemia of chronic disease (ACD)

● Due to an inability to use available iron for hemoglobin production
● Impaired release of storage iron associated with increased hepcidin levels
○ Hepcidin
■ Liver hormone → responsible for release of storage iron from macrophages
■ Acts as a positive acute phase reactant (infection or inflammation)
■ Influences intestinal Iron absorption
○ Inflammation and infection cause hepcidin levels to increase; this decreases release
of iron from stores.
● Laboratory:

31
 ○ Normocytic/ normochromic anemia, or slightly microcytic/hypochromic
anemia
○ increased ESR
○ normal to increased ferritin
○ low serum iron and TIBC
○ Associated with persistent infections, chronic inflammatory disorders
■ SLE
■ rheumatoid arthritis
■ Hodgkin lymphoma cancer

3. Sideroblastic anemia
● Caused by blocks in the protoporphyrin pathway resulting in defective hemoglobin
synthesis and iron overload
● Excess iron accumulates in the mitochondrial region of the immature erythrocyte
○ Ringed sideroblasts
● Excess iron accumulates in the mitochondrial region of the mature erythrocyte
○ Ringed siderocyte
● Inclusions - Pappenheimer bodies on Wright's stained smears.
● Siderocytes are best demonstrated using Perl's Prussian blue stain.
● Laboratory:
○ Microcytic/hypochromic anemia
○ Increased ferritin and serum iron
○ TIBC is decreased

4. Lead poisoning
● Multiple blocks in the protoporphyrin pathway affect heme synthesis.
● Laboratory:
○ Normocytic/normochromic
○ Basophilic stippling

5. Porphyrias
● There is a blockage in the protoporphyrin pathway
● Heme precursors before the block accumulate in the tissues, and large amounts are
excreted in urine and/or feces.

6. Megaloblastic anemias
● Defective DNA synthesis - abnormal nuclear maturation;
● Caused by either a vitamin B12 or folic acid deficiency
● Laboratory:
○ Pancytopenia
■ RBC and granulocytic lineage are affected
○ Size and shape:
■ macrocytic/normochromic anemia with oval macrocytes and teardrop cells,
○ WBC: hypersegmented neutrophils

32
 ○ Inclusions:
■ Howell-Jolly bodies
■ nucleated RBCs
■ basophilic stippling
■ Pappenheimer bodies
■ Cabot rings
● Vitamin B12 deficiency (cobalamin)
○ Intrinsic factor is secreted by parietal cells and is needed to bind vitamin B12
for absorption into the intestine.
■ Pernicious anemia: Caused by deficiency of intrinsic factor
■ Characterized by achlorhydria and atrophy of gastric parietal cells
○ Other causes of vitamin B12 deficiency include malabsorption syndromes
(steatorrhea) . Diphyllobothrium latum tapeworm, total gastrectomy

7. Aplastic Anemia
● Bone marrow failure causes pancytopenia → all cell lines are affected (wbc or rbc)
● Laboratory:
○ Low hemoglobin/hematocrit and reticulocytes;
○ normocytic/normochromic anemia
● Patients have a poor prognosis with complications that include bleeding, infection and
iron overload due to frequent transfusion needs.
● Can be genetic, acquired or idiopathic
1) Genetic aplastic anemia (Fanconi anemia)
a) Autosomal recessive trait
2) Acquired aplastic anemia (secondary) caused by:
a) Antibiotics: Chloramphenicol and sulfonamides
b) Chemicals: Benzene and herbicides
c) About 30% of acquired aplastic anemias are due to drug exposure
d) Viruses: B19 Parvovirus secondary to hepatitis, measles, CMV, and
Epstein-Barr Virus
e) Radiation or chemotherapy
f) Myelodysplastic syndromes, leukemia, solid tumors, paroxysmal nocturnal
hemoglobinuria.
3) Idiopathic (primary): 50-70% of aplastic anemias have no known cause.

8. Diamond-Blackfan Anemia
a. True red cell aplasia → RBC lines are affected wherein wbc and platelet are
NORMAL. RBC LANG KAYA NGA DAW RED CELL EH. GE PO

9. Myelophthisic (marrow replacement) anemia

a. Hypoproliferative anemia - caused by replacement of bone marrow hematopoiesis cells
by malignant or fibrotic tissue.
b. Associated with cancers (breast, prostate, lung, melanoma) with bone metastasis.
c. Laboratory: Normocytic/normochromic anemia; leukoerythroblastic blood picture

33
C. Blood Loss Anemia
1. Acute blood loss anemia
● sudden loss of blood resulting from trauma or other severe forms of injury
● Laboratory:
○ Normocytic/normochromic anemia;
○ initially normal reticulocyte count, hemoglobin/hematocrit;
○ in a few hours, increase in platelet count and leukocytosis with a left shift, drop in
hemoglobin/hematocrit and RBC; reticulocytosis in 3-5 days.
2. Chronic blood loss anemia
● gradual, long-term loss of blood; often caused by gastrointestinal bleeding
● Laboratory:
○ Initially normocytic/normochromic anemia that over time causes a decrease in
hemoglobin/hematocrit;
○ gradual loss of iron causes microcytic/hypochromic anemia

D. Hemolytic Anemias Due to Intrinsic Defects

1. Paroxysmal nocturnal hemoglobinuria (PNH)
a. An acquired membrane defect in which the red cell membrane has an increased
sensitivity for complement binding as compared to normal erythrocytes
b. All cells are abnormally sensitive to lysis by complement.
c. Characterized by:
i. Pancytopenia
ii. chronic intravascular hemolysis causes hemoglobinuria and hemosiderinuria at an
acid pH at night
iii. PNH noted for low leukocyte alkaline phosphatase (LAP) score
iv. Ham's and sugar water tests used in diagnosis;
d. Although Ham's and sugar water tests have been traditionally used in diagnosis of PNH,
the standard now used is flow cytometry to detect deficiencies for surface expression of
glycosyl phosphatidylinositol (GPI)- linked proteins such as CD55 and CD59.

E. Hemolytic Anemias Due to Extrinsic/Immune Defect

1. All cause normocytic/normochromic anemia due to defects extrinsic to the RBC. All
are acquired disorders that cause accelerated destruction with reticulocytosis.
2. Warm autoimmune hemolytic anemia (WAIHA)
a. RBCs are coated with IgG and/or complement. Macrophages may phagocytize
these RBCs, or they may remove the antibody or complement from the RBC's
surface → membrane loss and spherocyte
3. Cold autoimmune hemolytic anemia (CAIHA or cold hemagglutinin disease)
a. RBCs are coated with IgM and complement at temperatures below 37°C.
RBCs are lysed by complement or phagocytized by macrophages. Antibody
is usually anti-I but can be anti-i.
i. Associated with M. pneumoniae infections and also infectious
mononucleosis.

34
 4. Paroxysmal cold hemoglobinuria (PCH) → (+)
a. An IgG biphasic Donath-Landsteiner antibody with P specificity fixes complement
to RBCs in the cold (less than 20°C); the complement-coated RBCs lyse when
warmed to 37°C.
i. DAT (+); Donath-Landsteiner test (+)

F. Hemolytic Anemias Due to Extrinsic/Non-immune Defects

1. Microangiopathic hemolytic anemias (MAHAs)
a. Disseminated intravascular coagulation (DIC)
1) Systemic clotting is initiated by activation of the coagulation cascade due to toxins or
conditions that trigger release of procoagulants (tissue factor). Multiple organ failure
can occur due to clotting. (Di na reregulate yung clotting)
2) Fibrin is deposited in small vessels, causing RBC fragmentation.
b. Hemolytic uremic syndrome (HUS)
1) Occurs most often in children following a gastrointestinal infection (e.g., E.
coli)
2) Clots form, causing renal damage.
3) Neutrophilic Pools
a) Bone Marrow → divided into two
b) Mitotic Pool → Hematopoietic stem cell - CMP - GMP - Myeloblast -
promyelocyte - myelocyte
4) Circulation
a. Circulating Neutrophilic Pool → 50%
b. Margining Neutrophilic pool → 50%

LEUKOCYTE
❖ Overview
1. Classified as:
● Phagocytes - granulocytes, monocyte
● Immunocytes → lymphocytes, plasma cell and monocyte (Antigen presenting cell)
2. WBC reference range
● (SI units) is 4.0-11.0 x 10 9/L
● (Conventional units) 4.0-11.0 x 10 3/uL
3. Neutrophils
● First cells to reach the tissue; phagocytic bacteria
● Cell death → apoptosis “end stage” cell
4. Monocytes
● Differentiate into macrophages
● Phagocytize foreign bodies in tissues
● They arrive at the site of inflammation after neutrophils and do not die in the process.
5. T Lymphocytes
● Provide cellular immunity
● 80% of lymphocytes
6. B Lymphocytes
● Develop into plasma cells → Production of antibodies - Ig

35
 ● 20% of lymphocyte
7. NK (natural killer) lymphocytes
● Destroy tumor cells and cells infected with viruses.
● Known as large granular lymphocytes (LGLs)
8. Eosinophils
● Modulate the allergic response caused by basophil degranulation which releases
histamine.
9. Basophils
● Mediate immediate hypersensitivity reactions (type I, anaphylactic)

GRANULOCYTES
A. Maturation and Morphology of Immature Granulocytes
1. Myeloblast: → earliest recognizable granulocyte precursor
a. 14-20 um
b. N:C ratio 7:1-4:1
c. Round/oval nucleus with fine reddish-purple staining chromatin
d. 2-5 nucleoli
e. Dark blue cytoplasm
f. No cytoplasmic granules

2. Promyelocyte → appearance of prominent primary granules

a. 15-21 um
b. N:C ratio 3:1
c. Round/oval nucleus with slightly coarsening chromatin
d. 1-3 nucleoli
e. Dark blue cytoplasm
f. Cytoplasm has large, nonspecific/ primary granules containing myeloperoxidase.
3. Myelocyte:
a. 12-18 um
b. N:C ratio 2:1
c. Round nucleus with coarse chromatin
d. Early myelocytes may have visible nucleoli.
e. Light blue to light pink cytoplasm
f. Prominent golgi apparatus—clear area located in the cytoplasm next to the nucleus
g. Cytoplasm has specific/ secondary granules that contain hydrolytic enzymes,
including alkaline phosphatase and lysozyme.
h. Nonspecific/primary granules are present and may still stain.
4. Metamyelocyte
a. 10-18 um
b. N:C ratio 1.5:1
c. Nucleus is indented in a kidney bean shape and has coarse, clumped chromatin.
d. Nuclear indent is less than half the width of a hypothetical round nucleus.
e. Cytoplasm is pink and filled with pale blue to pink specific/secondary granules.

36
 f. Nonspecific/primary granules are present but usually do not stain.
5. Band neutrophil
a. 10-15 um
b. N:C ratio 1:2
c. Nucleus is "C" or "S"-shaped with coarse, clumped chromatin lacking
segmentation.
d. Cytoplasm is pink and filled with pale blue to pink specific/secondary granules.
e. Stored in the bone marrow and released when there is an increased demand for
neutrophils
NOTE: Band Neutrophil VS Segmented neutrophil → as long as there is no filament — thread
like segments

B. Morphology of Mature Granulocytes

1. Segmented neutrophil
a. 10-15um
b. N:C ratio 1:3
c. Nucleus has coarse, clumped chromatin with 3-5 lobes connected by thin filaments.
d. Cytoplasm is pink and filled with small pale blue to pink specific/ secondary
granules.
● Half life in blood: 7 hrs
● Defense against bacteria and infection
2. Eosinophil
a. Eosinophils are 12-16 um
b. Nucleus is usually bilobed.
c. Cytoplasm contains large, bright red-orange, secondary granules that contain enzymes
and proteins.
d. Survival in tissues: 2-5 days
e. Defense against some helminthic parasites; express Fc receptors for IgE, which is a
response to parasitic infections
3. Basophil
a. Basophils are 10-15 um
b. Cytoplasm contains large, purple-black, secondary granules that contain heparin
and histamine.
c. Granules may be numerous and obscure the nucleus, or they may "wash out" in
staining (because the granules are water soluble) and leave empty areas in the
cytoplasm.
d. Life span: 60 hrs

C. Visible response to infection by neutrophils (toxic changes)

a. Toxic changes → Associated with bacterial infection or growth factor therapy.
● Toxic granules, vacuolation, dohle bodies
b. Shift to the left
● Increased number of myelocytes, metamyelocytes, and/or bands in the peripheral blood

37
 ● Regenerative shift to the left is an appropriate bone marrow response to increased
demand for neutrophils. It is seen in infection or in other physiological or pathological
conditions requiring neutrophils
a. WBC count above the reference range
b. Most common type of left shift
● Degenerative shift to the left is seen after an overwhelming infection in which bone
marrow production cannot keep up with increased need for neutrophils.
a. Associated with a poor prognosis
b. WBC count below the reference range.

D. Nonmalignant Granulocytic Disorders

1. Shift/Physiologic/Pseudo Neutrophilia
a. Short-term increase in the total WBC count and in the absolute number of neutrophils in
the circulating granulocyte pool.
b. Caused by exercise, stress, pain, pregnancy
2. Pathologic neutrophilia
a. Neutrophils leave the circulating pool, enter the marginating pool, and then move to the
tissues in response to tissue damage.
b. Bone marrow reserves are released into the blood to replenish the circulating pool. The
WBC count can increase up to 50.0 X 109 / L
c. Shift to the left with toxic changes to the neutrophils
d. Bone marrow increases production of neutrophils to replenish reserves.
e. Occurs in response to bacterial and other infections, tissue destruction, drugs or toxins,
growth factor etc. Many fine azurophilic granules give the appearance of “ground glass”.
3. Neutrophilic leukemoid reaction (NLR)
a. Blood picture mimics that seen in chronic myelogenous leukemia.
b. Benign, extreme response to a specific agent or stimulus
c. The WBC count can increase to between 50.0 and 100.0 X 109/L
4. Leukoerythroblastic reaction
a. Presence of immature leukocytes and immature (nucleated) erythrocytes in the blood
b. Occurs in marrow replacement disorders, such as myelofibrosis

MONOCYTES AND MACROPHAGES

A. Basic review
1. The myeloid progenitor cell gives rise to a committed progenitor cell, CFU-GM
(colony-forming-unit-granulocyte-macrophage), that is acted on by growth factors (GM-CSF)
and interleukins (ILs) to form monocyte. Monocytes form in the bone marrow, pass through
the peripheral blood, and then migrate into the tissues (macrophages), where they fight
infection. Macrophages are named according to their location in the body.
a. Monocytes → Peripheral blood smear
b. Kupffer cells → Liver
c. Microglial cells → CNS
d. Osteoclasts → bone

38
 e. Langerhans cells → skin
f. Alveolar cells → Lungs
B. Maturation and Morphology of Monocytes
1. Monoblast:
a. 12-18 um; N:C ratio 4:1
b. Round/oval eccentric nucleus with fine chromatin; 1-2 nucleoli
c. Dark blue cytoplasm; may have a gray tint; no cytoplasmic granules
2. Promonocyte
a. 12-20 um; N:C ratio 3:1
b. Irregularly shaped, indented nucleus with fine chromatin; 0-1 nucleoli
c. Blue to gray cytoplasm; fine azurophilic granules
3. Monocyte
a. 12-20 um
b. Horseshoe or kidney-bean shaped nucleus, often with “brainlike” convolutions
c. Fine, lacy chromatin
d. Blue-gray cytoplasm; may have pseudopods and vacuoles
4. Macrophage
a. 15-80 um
b. Indented, elongated or egg-shaped nucleus with fine chromatin
c. Blue-gray cytoplasm with many vacuoles and coarse azurophilic granules; may contain
ingested material
C. Monocyte Characteristics
1. Granules are lysosomes that contain hydrolytic enzymes, including peroxidase and acid
phosphatase.
2. Highly motile cell that marginates against vessel walls and into the tissues
3. Reference range is 2-10% in peripheral blood
D. Monocyte/Macrophage Function
1. Play a major role in initiating and regulating the immune response
2. They process ingested material and also process antigenic information, which is relayed to
the T-helper lymphocyte to coordinate the immune response to foreign antigens.
3. They arrive at the site of inflammation after neutrophils. Unlike neutrophils, the phagocytic
process does not kill the monocyte.
4. Very efficient phagocytic cells with receptors for IgG or complement-coated organisms.
5. Known as “scavenger cells” because of their ability to ingest foreign material
a. Blood monocytes ingest antigen-antibody complexes and activated clotting factors,
limiting the coagulation response.
b. Splenic macrophages remove old/damaged RBCs and conserve iron for recycling.
c. Liver macrophages remove fibrin degradation products
d. Bone marrow macrophages remove abnormal RBCs, ingest bare megakaryocyte nuclei
or extruded RBC nuclei, and store and supply iron for hemoglobin synthesis.
E. Nonmalignant monocytic Disorders
1. Monocytosis
a. Increase in the absolute number of monocytes associated with:

39
 1. Recovery stage from acute bacterial infections and recovery following marrow
suppression drugs
2. Tuberculosis, syphilis, subacute bacterial endocarditis
3. Autoimmune disorders
2. Monocytopenia
a. Decrease in the absolute number.

LYMPHOCYTES AND PLASMA CELLS

A. Basic Review
a. Bone marrow and thymus are primary lymphoid tissues.
i. Pre-B lymphocytes
● Found in bone marrow which will eventually become B Cell
● Development in bone marrow is antigen-independent lymphopoiesis (Will mature
w/o the stimulation of an antigen)
ii. Pre-T lymphocytes
● Found in thymus, which will eventually become T Cell
● Development in thymus is antigen-independent lymphopoiesis (Will mature w/o the
stimulation of an antigen)
b. B and T cells enter the blood and populate the secondary lymphoid tissues
i. (lymph nodes, spleen, and Peyer's patches in the intestine)
ii. Antigen-dependent lymphopoiesis
● Needing stimulation of antigen, the B and T Cells’ development in the secondary
lymphoid organs are targeted to kill a specific microorganism, especially viruses.

B. Maturation and Morphology of Lymphocytes

a. Lymphoblast: Earliest recognizable lymphocyte precursor
i. 10-18um; N:C ratio 4:1
ii. Round/oval eccentric nucleus with fine chromatin; 1 or more nucleoli
iii. Dark blue cytoplasm; no cytoplasmic granules
b. Prolymphocyte:
i. 9-18um; N:C ratio 3:1
ii. Round or indented nucleus with coarsening chromatin; 0-1 nucleoli
iii. Basophilic cytoplasm; no cytoplasmic granules
c. Lymphocyte:
i. 7-18 um
ii. Round, oval ,or slightly indented nucleus; condensed chromatin
iii. Scant to moderate amount of blue cytoplasm; few azurophilic granules

40
 iv. May occur in 3 sizes
1. Small - 7 - 10 um
2. Medium - 10 - 12 um
3. Large - 11 - 25 um
v. Are NOT obligate end cells
d. Reactive lymphocytes
● Have become activated as part of the immune response (and is seen only as an immune
response). Associated with lymphocytosis and can show the following characteristics:
● Also known as Variant Lymphocyte (Downey Cell, Atypical lymphocyte, Stressed
lymphocyte, Virocyte)
● Usually seen in response to VIRAL infections
1. Generally, larger cell with increased amount of dark blue cytoplasm (RNA)
2. Fine chromatin pattern with nucleoli
3. Irregular shape to the nucleus
4. Irregular shape to the cytoplasm (tags, sharp ridges); indented by red cells (Meaning
NAHUHULMA siya ng RBC as per the lovely drawing provided)
● Types of Reactive/Variant/Atypical Lymphocytes
○ Type 1 (Turks Cell) - Seen in German Measles
○ Type 2 (Infectious Mononucleosis Cell) - Fried egg/Flamed skirt in
appearance
○ Type 3 - Swiss cheese/Moth-eaten cell in appearance

C. T Lymphocytes (T cells)
a. T lymphocyte function
i. Cellular immunity
ii. Graft rejections and lysis of neoplastic cells
iii. Attack/destroy viral and fungal organisms
iv. Obtain antigenic information from monocytes then passed onto other T cells and B cells
v. Regulate humoral response by helping antigens activate B cells
vi. The END PRODUCT of the activation of T lymphocyte is to release cytokine, lymphokines,
and interleukines

D. B Lymphocytes (B cells)
a. Are identified by surface membrane markers CD19, CD 20 (Cluster of differentiation)
b. B lymphocyte function
i. Contact with foreign antigens stimulates B lymphocytes to become reactive
lymphocytes, with the characteristic morphology associated with reactivity.
ii. Reactive lymphocytes transform into immunoblasts
iii. Plasma cells
1. End stage of B lymphocyte; dominant in lymph nodes; not normally seen in circulation
2. 10-20um
3. Abundant blue cytoplasm with prominent perinuclear (golgi) zone
4. Eccentric nucleus with a very coarse, clumped chromatin pattern
5. Primary function is to produce antibodies

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 6. Short lived: 3-4 days

E. Natural Killer (NK)/Large Granular Lymphocytes (LGLs)

a. Large cells with low N:C ratio, large cytoplasmic granules, and pale blue cytoplasm
b. Lack B cell or T cell membrane markers; are CD16 and CD56 positive
c. Surveillance of cells for surface alterations such as tumor cells/cells infected with viruses
i. Primary cells responsible for tumor cells are NK Cells
d. Attack antigens with attached IgG; called antibody-dependent cytotoxic cells

F. Nonmalignant (Non cancerous in origin) Lymphocytosis Associated with Viral Infections

a. Infectious mononucleosis
i. Epstein-Barr virus (EBV) infects B lymphocytes.
b. Cytomegalovirus (CMV)
i. Transmission is by blood transfusions and saliva exchange.
c. Infectious lymphocytosis
i. Associated with adenovirus and coxsackie A virus
G. Other Conditions Associated with Lymphocytosis
a. Viral — hepatitis, influenza, mumps, measles, rubella, and varicella
b. Non Viral — Bordetella pertussis (whooping cough), brucellosis, toxoplasmosis

WBC DISORDERS
A. Nuclear Abnormalities of Neutrophils
1. Hypersegmentation characterized by 5 or more lobes in the neutrophil (Normal: 2-5 lobes);
associated with megaloblastic anemia due to vitamin B12 or folic acid deficiencies
2. Hyposegmentation refers to a tendency in neutrophils to have 1 or 2 lobes; may indicate an
anomaly or a shift to the left.
a. Pelger-Huet anomaly
i. Autosomal dominant inheritance
ii. Nucleus dumbbell- or peanut shaped; referred to as "pince-nez"
iii. Morphologically abnormal, but functionally normal
b. Pseudo Pelger-Huet
i. Acquired abnormality associated with myeloproliferative disorders and
myelodysplastic syndromes

Note: There is NO Hypo- and Hypersegmentation in Lymphocytes and Monocytes

B. Abnormalities in Granules
Quick Recap:
● Primary Granules - cannot be stained by Wright stain, appears purple-black
○ Myeloperoxidase (MPO)
○ Bacterial Cationic Proteins
○ Proteases

42
 ○ Lysosome / Muramidase
● Secondary Granules
○ Lysozyme
○ Lactoferrin - competes with bacteria for iron absorption
● Tertiary Granules
○ Alkaline phosphatase (ALP)
○ Acetyl Transferases
○ Gelatinase
1. Chediak-Higashi syndrome
● large, gray-green, peroxidase positive granules; peroxidase negative in lymphocytes
○ To differentiate if granulocyte or lymphocyte in origin use peroxidase
(cytochemical stain)
■ Granulocyte: Peroxidase POSITIVE
■ Lymphocyte: Peroxidase NEGATIVE
● Abnormal fusion of primary and secondary neutrophilic granules – defect in Golgi
complex – responsible to granule assembly
● Both morphologically and functionally abnormal leukocytes;
● If granulocyte in origin it means that they are unable to degranulate and kill invading
bacteria
● Patients with chediak-higashi syndrome exhibit albinism and photosensitivity

2. May-Hegglin anomaly
● Large, crystalline, Dohle-like inclusions in the cytoplasm of neutrophils on Wright's
stain; gray-blue and spindle CIGAR shaped
● Morphologically abnormal, but functionally normal
● Giant platelets, thrombocytopenia, and clinical bleeding are also associated
with this anomaly.
Inclusions Size Shape PAS RNA

May-Hegglin Anomaly Large Spindle NEGATIVE (-) Inclusion is mRNA in origin

Dohle Bodies Small Round POSITIVE (+) Inclusion is rRNA in origin

*PAS - Periodic Acid Schiff

3. Alder-Reilly anomaly
● May resemble TOXIC GRANULES
● Large purple-black coarse cytoplasmic granules
● Accumulation of degraded-stored mucopolysaccharidoses
● Found in all leukocytes

4. Auer rods
● Pink or red rod shaped structures (spindle-shaped)
● Fused primary granules (peroxidase positive)
● Found in myeloid and monocytic series only

43
 ○ AML - Acute Myelogenous Leukemia
○ AMML - Acute Myelomonocytic Leukemia
● Faggot cells – cells with mass of auer rods
○ (AML) M3 - Acute Promyelocytic Leukemia
○ DIC - disseminated intravascular coagulation
● Origin of auer rods is the DEFECT in PRIMARY GRANULES
○ Prominent primary granules are found first in promyelocytes

5. Dohle bodies/ Dohle Amato bodies

● Single or multiple blue inclusions
● Aggregates of free ribosomes of rER
● Severe infections, burns, toxic states
6. Toxic granulation
● Prominent granulation due to persistent staining of primary granules.
● Infections, toxic states
● Lage purple-black granules
7. Toxic vacuolation:
● Colorless areas in the cytoplasm that indicate phagocytosis and degranulation
have occurred

C. Abnormalities in Functions
1. Chronic granulomatous disease (CGD)
● Morphologically normal, but functionally abnormal because of enzyme deficiency that
results in an inability to degranulate, which causes inhibited bactericidal function
● Inability of phagocytes to kill ingested microorganisms
● Deficiency is due to impaired NADPH Oxidase therefore hindering respiratory burst
● Test: Nitroblue Tetrazolium Dye Test (NBT Test)
2. Job’s syndrome
● Normal random activity
○ WITHOUT the presence of pathogen, normal movement of WBC
● Abnormal chemotactic activity
○ WITH presence of pathogen, abnormal movement of WBC
3. Lazy Leukocyte Syndrome
● BOTH random and chemotactic activity are ABNORMAL

D. Cell Exhibiting Phagocytosis

● LE (Lupus Erythematosus) Cell - In vitro phenomenon
○ Neutrophil with large purple homogenous round inclusion;
○ Buffy coat smear
○ LE
○ Found in SLE (Systemic Lupus Erythematosus): anti-DNA autoantibody
● Tart Cell
○ Monocyte with ingested lymphocyte

44
E. Abnormalities in Monocytes/Macrophage
● Lipid storage disorders
○ Gaucher disease (Glucocerebroside)
■ autosomal recessive inheritance pattern.
■ A deficiency in glucocerebrosidase causes glucocerebroside to accumulate in
macrophages called Gaucher cell
○ Niemann-Pick disease
■ autosomal recessive
■ A deficiency in sphingomyelinase causes sphingomyelin to accumulate in
macrophages which will then be called Niemann-pick cell
○ Sea-blue histiocytosis
■ unknown deficiency. - Sea-blue macrophages
○ Tay sachs
■ Deficiency in Hexosaminidase A
■ Accumulation of glycolipid and ganglioside
■ Vacuolated cytoplasm
○ Sandhoff
■ Deficiency in Hexosaminidase A and B
■ Accumulation of glycolipid and ganglioside
■ Vacuolated cytoplasm

F. Abnormal Lymphocytes
● Reactive lymphocytes
○ B-cells
○ EBV (Epstein-Barr virus), IM (Infectious Mononucleosis)
● Smudge cells - are nuclear remnants of lymphoid cell
○ Degenerated nucleus
○ Pressure in making smear
○ CLL (Chronic Lymphocytic Leukemia)
● Hairy cell
○ Small lymphocytes with hair-like cytoplasmic protrusions surrounding nucleus
○ Hairy cell leukemia is tested in TRAP+ (Tartrate Resistant Acid Phosphatase)
○ B - Cell in Origin
● Sezary cell
○ “Cerebri form” nuclei – brain-like convolutions
○ Round lymph cell with nucleus that is grooved or convoluted
○ T - Cell in Origin

G. Abnormality in Plasma Cells

● Flame cell
○ Plasma cell with red to pink cytoplasm
○ Assoc. with increased Ig (usually IgA)
○ Seen in cases of MM (Multiple Myeloma)
● Russell bodies

45
 ○ Individual globules of Ig
● Grape cell/Mott cell
○ Plasma cell with vacuoles
○ Accumulation of Russell bodies
○ Seen in cases of MM (Multiple Myeloma)
● Dutcher’s bodies
○ Intranuclear protein inclusions

LEUKEMIA
● Acute Leukemia
○ Is of sudden onset and rapid progression unless treated and is characterized by the
presence of blast cells in peripheral blood and bone marrow
● Chronic Leukemia
○ Is of gradual onset and slow progression and is characterized by the presence of more
mature cells in the peripheral blood and bone marrow.

A. Lymphocytic leukemia
FAB Classification:
● ALL (Acute Lymphocytic Leukemia)
○ L1
■ Small, homogenous
■ Childhood ALL
○ L2
■ Large and heterogenous
■ Adult ALL
○ L3
■ Large and homogenous
■ Burkitt’s Type
● Large lymphoblast with cytoplasmic vacuoles

Immunologic Markers PAS (Periodic Acid Schiff) Oil Red O Blast

L1 Calla, CD10 tdt, CD19, CD20 + - Many, Small

L2 tdt + - Few, Large

L3 CD19, CD20 CD22, CD24 - + Burkitt’s

● CLL (Chronic Lymphocytic Leukemia)

○ Presence/increase of smudge cells accompanied by cells with clover leaf-like
nucleus called rieder cells
○ CLL is due to a translocation between chromosome 11 and 14

46
 ○ Common Feature: Is the enlargement of lymph nodes known as
lymphadenopathy

B. Myelocytic/Non-lymphocytic Leukemia
● AML (Acute Myelogenous Leukemia)
○ FAB Classification:
Morphology MPO SBB Specific Nonspecif PAS (Periodic
(Myelopero (Sudan esterase ic Acid Schiff)
xidase) Black B) Esterase

M0 Acute myeloblastic >30% blasts (-) (-) (-) (-) (-)

leukemia: minimally No granules
differentiated

M1 Acute myeloblastic >30% blasts Present Present Present (-) (-)

leukemia with no Few granules
maturation +/- Auer rods

M2 Acute myeloblastic >30% blasts Present Present Present (-) (-)

leukemia with no Granules common +
maturation Auer rods

M3 Acute >30% blasts Present Present Present (-) (-)

promyelocytic Prominent granules
leukemia ++ Auer rods, very
distinct will cause
Faggot cells

M4 Acute >30% blasts Present Present Present Present (-)

myelomonocytic >20%monocytes
leukemia + Auer rods

M4 eo Acute >30% blasts Present Present Present Present (-)

myelomonocytic >20%monocytes
leukemia with >5% abn eos
eosinophilia + Auer rods

M5 Acute monoblastic >30% blasts Can be Can be Can be Present (-)

leukemia with or >80% monocytes Present Present Present
without maturation with/without
differentiation

M6 Acute >30% myeloblasts Present: Present: Present: Not (+) Present:

erythroleukemia >50% erythroblast + Myeloblasts Myeloblasts Myeloblasts Present Erythroblasts
Auer rods (Erythroblast
present would
be normoblast)

M7 Acute >30% (-) (-) (-) (-) (-)

megakaryocytic Megakaryoblasts
leukemia Cytoplasmic budding
Cytochemical Stains:
● MPO (Myeloperoxidase)

47
 ● SBB (Sudan Black B)
● Naphthol AS-D Chloroacetate
● Esterase Stain
○ Non-specific Esterase Stain - stain’s reaction is stronger in monocytes
because they only have primary granules and contain lysozyme and muramidase
whereas granulocytes have more prominent secondary granules
■ Acetate
■ Butyrate
○ Both can be found in ALL WBCs
○ Specific Esterase Stain
■ Chloroacetate - found in granulocytes only

Myeloproliferative Disorder - problem in the bone marrow specific to myeloid stem cell
● CML (Chronic Myelogenous Leukemia)
○ Translocation between chromosome 9 and 22 also known as the “Philadelphia
Chromosome”
○ Plt count:
■ >600 x 10⁹/L
■ <50 x 10⁹/L
○ To differentiate CML from Leukemoid Reaction test them against LAP
(Leukocyte Alkaline Phosphatase) score
■ CML - Decreased LAP score
■ Leukemoid Reaction - Increased LAP score
○ CMP Cell lines are affected (RBC, Granulocytes, Monocytes, Platelets)
● MMM (Myelofibrosis w/ Myeloid Metaplasia)
○ Problem occurring in granulocytic hyperplasia in the BM
○ Granulocytic and Megakaryocytic proliferation in liver and spleen
● (PV) Polycythemia Vera
○ All cell lines are affected
○ Increased RBC, WBC, Plt
● (ET) Essential Thrombocythemia
○ Increased platelet count (>1000 x 10⁹/L)
■ NV: 150 - 450
○ Platelet has abnormal function

Lymphoproliferative Disorder
● Hairy Cell Leukemia
○ B-cell in origin
○ Positive (+) in TRAP (Tartrate Resistant Acid Phosphatase)

● Mycosis Fungoides.Sezary Syndrome

○ T-cell in origin
○ Presence of neoplastic T-cells, these neoplastic T-cells are sezary cells with
cerebriform nucleus

48
 ● CLL (Chronic Lymphocytic Leukemia)
○ Smudge cells, Rieder cells
○ Translocation between chromosome 11 and 14

Staining blood smears

1. Type of stain
● Romanowsky stain
○ pH dependent 6.4-6.8 - buffer must always be incorporated
○ Polychrome stains consist of 2 types of aniline dye : methylene blue
(or oxidation product of methylene blue called azure) and eosin.
■ Methylene blue/ azure stains acidic components; blue or
purple
■ Eosin stains basic cellular components (acidophilic or
eosinophilic); orange to red
○ Contains Methanol - Acts as fixative
○ Acidic components are basophilic and takes up to basophilic dye like
WBC nuclei, RNA remnants and Platelets.
○ Basic components are acidophilic or eosinophilic
○ Neutrophilic takes up both dyes
○ Ex. Wrights, Giemsa
● Supravital stain
○ New Methylene blue, Janus green, Neutral Red, Brilliant Cresyl Blue
○ Stains the cell at living state and meant to demonstrate cytoplasmic
structures only like reticulocyte.
○ Buffer used Sorensen’s phosphate buffer consist of anhydrous
monobasic KH2PO4 and anhydrous dibasic Na2HPO4.
2. Criteria for a good stain
● Macroscopic
○ Reddish-brown or pinkish
● Microscopic
○ The red blood cells (RBCs) should be pink to salmon.
○ Nuclei are dark blue to purple.
○ Cytoplasmic granules of neutrophils are lavender to lilac.
○ Cytoplasmic granules of basophils are dark blue to black.
○ Cytoplasmic granules of eosinophils are red to orange.
○ The area between the cells should be colorless, clean, and free of
precipitated stain.
Staining Problems
(staining quality is dependent on pH 6.8)
a. Extremely Red or Acidic Smears
○ RBCs appear bright red or orange;
○ Nuclear chromatin of WBCs appear pale blue eosinophilic granules are
sparkling brilliant red

49
 ○ Causes: too acidic buffer/ stain ( <6.4), excess buffer in preparation to the
stain, excessive washing, very thin smears, contaminants in the wash water
(most common is chlorine), exposure of the buffer/ stain to acid fumes, old
stain were methanol has oxidized to formic acid.
b. Extremely blue or Alkaline smears
○ RBCs appear blue or green; nuclear chromatin is deep blue to black;
eosinophil granules are blue to gray.
○ Causes: too alkaline buffer/ stain (>6.4) , too little buffer for the stain, thick
smears. Inadequate washing, excessive washing time, short drying period,
heparinized blood sample, proteins abnormality, high WBC count with blasts,
low PCV, old samples.
○ Short Drying period should be 5 mins, (average shades of red may improve
during drying period).
○ Heparinized samples not acceptable for WBC count for it imparts blue green
background.
○ Oxalate samples not acceptable for WBC count as well for it imparts
cloverleafing of nuclei
c. Presence of Precipitation
○ Causes: Inadequate washing after the staining period, inadequate filtration of
the stain, dust particle on the slide or smear.
○ Precipitate: Purple/ Blue can be mistakenly recognized as lymphocytes or
leukocytes.

Peripheral Smear Examination

● 10x
○ Necessary to assess the overall quality of smear
○ Rapid detection of abnormal cells – blasts, reactive lymph and parasites
● 40x or 50x
○ Field wherein cells do not overlap
○ Determining the average number of WBC per field
○ WBC estimate:
■ 40x objective
● Find the average leukocytes in 10 fields
● Average no. of WBC x 2000 = estimated WBC/uL
■ 50x objective
● Average no. of WBC x 2500 = estimated WBC/uL
● 100x
○ WBC differential
○ WBC abnormalities, presence of NRBCs
○ RBC morphology based on RBC indices
○ Platelet estimate
■ Scan 10 OIF
■ Average no. of plt/OIFx20,000 = est. plt. Ct/uL

50
 Cells Found in a Normal White Blood Cell
Differential Count

Adult Reference
Cell Size Range
Cell Type (µm) Nucleus Chromatin Cytoplasm Granules
Peripheral Cells X
Blood (%) 10⁹/L

Segmented 10-15 2-5 Lobes Coarsely Pale pink, Primary: Rare 50-70 2.3-8.1
neutrophil connected clumped cream Secondary:
(Seg), by thin colored, or Abundant
Polymorphonuclear filaments colorless
neutrophil without
(Poly, PMN) visible
chromatin

Band Neutrophil 10-15 Constricte Coarsely Pale blue to Primary: Few 0-5 0.0-0.6
(Band) d but clumped pink Secondary:
chromatin Abundant
must be
visible
within the
thinnest
part

Lymphocyte 7-18* Round to Condensed Scant to 土 Few 20-40 0.8-4.8

(Lymph) oval may to deeply moderate; azurophilic
be slightly condensed sky blue
indented;
occasional
nucleoli

Monocyte 12-20 Variable; Moderately Blue-gray; Many fine 3-11 0.5-1.3

(Mono) may be clumped; may have granules,
round, lacy pseudopods; frequently
horseshoe, vacuoles giving the
or kidney may be appearance of
shaped absent or the ground
often has numerous glass
folds
producing
‘brain-like’
convolutio
ns

Eosinophil 12-17 2-3 Lobes Coarsely Cream to Primary: Rare 0-5 0.0-0.4

51
(Eos) connected clumped pink; may Secondary:
by thin have Abundant, red
filaments irregular to orange,
without borders round
visible
chromatin

Basophil 10-14 Usually Coarsely Lavender to Primary: Rare 0-1 0.0-0.1

(Baso) two lobes clumped colorless Secondary:
connected Lavender to
by thin dark purple;
filaments variable in
without number with
visible uneven
chromatin distribution;
may obscure
nucleus or
wash out during
staining, giving
the appearance
of empty areas
in cytoplasm

Evaluation of erythropoiesis
● Total erythropoiesis
○ Total production of RBC/Erythropoietin
● Effective erythropoiesis
○ Production of RBC that REACHED the circulation
● Ineffective erythropoiesis
○ Production of RBC that DIDN’T REACH the circulation
1. Measurement of the Total Erythropoiesis
○ Bone marrow examination/aspiration - cellularity and percentage of total
erythropoietic cells ; puncture sites would be posterior iliac crest & sternum;
○ Plasma iron turnover - calculated from the serum iron level and the rate of
removal of injected radioactive iron (iron 59/59Fe) from plasma; extent of iron
consumption from plasma
■ 70-75% of plasma iron is taken up or used in erythropoiesis
■ Can measure both effectiveness and ineffectiveness
○ EPO (Erythropoietin) assay - helpful in differentiating primary polycythemia
where there is an uncontrolled marrow production unregulated by EPO
from secondary erythropoiesis where there is a controlled excess of erythroid
production stimulated by tissue hypoxia and mediated by EPO.
2. Measurement of effective erythropoiesis
○ Reticulocyte count - important in assessing the status of erythrocyte
production in the bone marrow

52
○ Erythrocyte utilization of iron - measure of the amount of an injected dose
of iron that appears in the Hemoglobin of circulating erythrocytes; derived from
the plasma iron turnover and the percentage of radioactive iron that has been
injected and that appears in the Hb of circulating red cells
○ Siderocyte count - useful in differentiating anemia due to iron deficiency from
thalassemia or other disorders in which iron accumulates because of poor
utilization in Hb synthesis
○ Free erythrocyte protoporphyrin - provides a sensitive early indicator of iron
deficiency; also elevated in chronic disease states and lead poisoning.

53
3. Measurement of the effective survival of erythrocytes in the blood
○ Radioactive chromium (51Cr)
■ Chromated red cells are injected intravenously and their
disappearance is measured by counting blood, which is sampled every
1-2 days for 10-14 days
■ Residual activity is an index of the intravascular life span of the labeled
red cells; external scanning (gamma ray emission) can detect sites of
red cell destruction
■ Half-life of 51Cr-labeled erythrocytes in normal individuals is 25-32
days
■ When rapid clearing is present there is an excessive intravascular
hemolysis
○ Flow cytometry
■ Uses ex vivo labeling of red cells by biotin followed by measurement of
the rate of cell disappearance using fluorochrome-conjugated avidin or
streptavidin
4. Measurement of total destruction of red cells or hemoglobin breakdown
○ Free hemoglobin
■ Normal values range from 0.5 - 5 mg/dL of plasma or 0.08 - 0.78
umol/L; INCREASED in hemolytic anemia, hemolytic transfusion
reaction, Paroxysmal Cold Hemoglobinuria, and Paroxysmal Nocturnal
Hemoglobinuria
○ Haptoglobin
■ Plasma protein which binds free Hb by forming a 1:1 complex with
alpha and beta dimers preventing their excretion through the kidneys;
normal binding capacity = 50 - 200 mg/dL of plasma
■ Carrier protein of FREE HEMOGLOBIN
○ Estimation of exhaled carbon monoxide
■ Measures heme catabolism immediately before and during the time of
the assay
■ Only available method to study short-term fluctuations in red cell
destruction
■ 0.5% Carboxyhemoglobin
■ Liberates one heme and undergoes conversion to biliverdin
○ Determination of fecal urobilinogen
■ Estimates the total excretion of bile pigments (breakdown products of
heme)
■ REDUCED in aplastic anemia and INCREASED in hemolytic anemia
and intramedullary destruction of hemoglobin characteristic of certain
hematologic diseases notably thalassemia, megaloblastic anemia,
refractory normoblastic anemia, and erythropoietic porphyria
■ While this is not reliable in cases of antibiotic therapy obstructive
jaundice or severe liver disease

54
Evaluation of erythrocyte structure and function
1. Measurement of the oxygen-carrying capacity of the blood
○ For diagnosis of anemia (↓ O2 carrying capacity)
○ RBC Count
■ Obtained using an automated method with the principle of Resistance
Pulse Counting/Impedance
■ Manual method is not used because of large inherent errors associated
○ Hemoglobin Determination
■ Reference method: Hi Cyanide method/Methemoglobin Method which
uses spectrophotometric determination
■ UV-visible spectrophotometer (quantitative procedure)
○ Hematocrit Determination
■ Adam’s Method or derived from the product of MCV(fL) and RBC
count(L)
2. Erythrocyte Indices
a. Mean Corpuscular Volume (MCV)
○ Indicates the average volume of red blood cells expressed in femtoliters (1 fL
= 10-15 L or 1 um3 )
○ ⬇ DECREASED MCV = Smaller Cells (Microcytic RBCs)
■ Common Cause: Iron Deficiency Anemia, Parasitism
○ ⬆INCREASED MCV = Larger Cells (Macrocytic RBCs)
■ Common Cause: Pernicious anemia, megaloblastic anemia, Vit. B12
def
𝐻𝐶𝑇 × 10 𝐻𝑐𝑡 × 1000
○ Formula: 𝑀𝐶𝑉 (𝑓𝐿) = or 𝑀𝐶𝑉 𝑓𝐿
𝑅𝐵𝐶/𝐿 𝑅𝐵𝐶/µ𝐿
○
○ Reference range: 80 - 96 fL
○ Interpretation:
■ < 80 fL = red cells are considered microcytic RBCs (IDA and
parasitism)
■ > 96 fL = red cells are macrocytic RBCs (pernicious anemia,
megaloblastic anemia, Vit. B12 def)

55
b. Mean Corpuscular Hemoglobin (MCH)
○ indicates the average weight of hemoglobin in the blood cell expressed in
picograms (1 pg = 10-12 g)
𝐻𝑏 (𝑔/𝑑𝐿) 𝑥 10 𝐻𝑏 (𝑔/𝑑𝐿)
○ Formula: MCH (pg) = or MCH (pg) =
𝑅𝐵𝐶/𝐿 𝑅𝐵𝐶/µ𝐿
○ Reference range: 27 - 33 pg
○ Interpretation:
■ MCH < 27 pg = microcytic, hypochromic red blood cells
■ MCH >33 pg = macrocytic anemias and in some cases of
spherocytosis (cells can be hyperchromic)
c. Mean Corpuscular Hemoglobin Concentration (MCHC)
○ Indicates the average content of Hb in the RBC

56
 ○ Ratio of weight in hemoglobin and volume of RBC
○ Detects interfering substances
○ Expression of the average concentration of hemoglobin in RBC
𝐻𝑏 𝑥 100
○ Formula: MCHC =
𝐻𝑐𝑡
○ Reference range: 33-36 g/dL
○ Interpretation:
■ MCHC < 33 g/dL indicates hypochromia
■ MCHC > 36 g/dL indicates hyperchromia
● Sources of erroneous hemoglobin value: (because
Spectrophotometery technique → Turbidity (Causes false
results)
○ Hyperlipidemia
○ Abnormal protein

HYPOCHROMASIA GRADING

Grade DESCRIPTION

1+ Central pallor is one half of cell diameter

2+ Central pallor is two thirds of cell diameter

3+ Central pallor is three fourth of cell diameter

4+ Thin rim of hemoglobin

1. Red cell Distribution Width (RDW)

● Expression of the coefficient of variation/ standard deviation of the distribution of individual
red cell volumes; estimates erythrocyte anisocytosis or size variation
● Affected by MCH
𝑆𝐷
● Formula: RDW (%)= 𝑋
× 100 [x refers to MCV]
● The more anemic the individual, the higher the RDW
● Reference range: 11.6-14.6%
○ Interpretation:
■ abnormally high results are seen in iron-deficiency anemia (microcytic and
hypochromic cells)
■ Vitamin B12 and folic acid deficiency
■ Megaloblastic anemia (macrocytic cells)

Morphologic Classification of Anemia Based on Red Blood Cell Mean Volume (MCV)
and Red Blood Cell Distribution Width (RDW)

MEAN CELL VOLUME

57
 DECREASED NORMAL INCREASED

RDW Normal ● α- or β-Thalassemia trait ● Anemia of chronic ● Aplastic anemia

● Anemia of chronic inflammation ● Chronic liver disease
inflammation ● Anemia of renal disease ● Alcoholism
● Hb E disease/trait ● Acute hemorrhage ● Chemotherapy
Hereditary spherocytosis

RDW Increased ● Iron deficiency ● Early iron, folate, or vitamin ● Folate or vitamin B12
● Sickle cell- β - B12 deficiency deficiency
thalassemia ● Mixed deficiency of iron + ● Myelodysplastic
vitamin B12 or folate syndromes
● Sickle cell anemia ● Cold agglutinin disease
● Hb SC disease ● Chronic liver disease
● Myelodysplastic ● Chemotherapy
syndromes
Hb: Hemoglobin*
2. Test for abnormal hemoglobins and derivatives
A. Shaking
a. Presumptive- OUTDATED
b. Whole blood is shaken for 15 minutes. The expected colors formed are as follows:
i. Bright red: HbO2 (normal) [oxygenated hemoglobin]
ii. Cherry red: HbCO [carboxyhemoglobin]
iii. Chocolate brown: Hi [methemoglobin/hemeglobin]
iv. Mauve lavender: SHb [sulfhemoglobin]
B. Spectrophotometry
a. based on the principle that hemoglobin pigments have characteristic absorbance
maxima:
i. HbCO - 555 nm (photosensitive)
ii. Hi - 630 nm
iii. SHb - 620 nm
C. Determination of fetal hemoglobin (Hb F)
a. In adults:
i. Detection of leukemia
ii. Hemoglobinopathies
iii. Hereditary Persistence of Fetal Hb (HPFH)
1) Alkali Denaturation Test (chemical test)
● Principle: Hb F resists alkali denaturation while HbA does not.
● A hemolysate is alkalized using NaOH, and then neutralized (sample is a mixture of
HbF and HbA), the denatured Hb A is precipitated by ammonium sulfate. The filtrate
which contains only alkali resistant Hb is measured and the result is expressed as a
% of the total.
● Only HbM resists denaturation after treatment with NaOH
● Normal values:
○ Adults - 0.2-1.0%
○ 1 year old - 1%
● Specimen: Hemolysate (lysed RBC)

58
 ● Increased HbF (HbF is compensatory Hgb): Beta Thalassemia - increased HbA2
[kapag walang HbA, expect that HbF is increased]
● Quantitate HbF using spectrophotometric method where superatant is subjected for
analysis
cathode A2 C E O L G D S F A K J Bart’s N I H Anode

*same color migrates →

2) Acid Elution Test (Kleihauer, Braun and Betke)

● cytochemical method of Hb F determination; uses a citric acid-phosphate buffer at pH
3.3
● Principle [cytochemical test: staining method]: Cells containing Hb F resist acid elution
to a greater extent than normal cells do appearing as isolated darkly staining cells
among a background of pale-staining ghost cells (normal cells containing HbA)
● Normal value: 1-5% of red cells contains residual Hb F
● Distinguishing factors for HPFH, B-thalassemia or hemoglobinopathy: all of these
show a high/large number of resistant cells but differ in their distribution of Hb
molecules.
● Clinical Significance: increase in cases of
○ HPFH - Hbf is evenly distributed
○ Beta thalassemia and hemoglobinopathies -HbF is heterogenous
D. Electrophoresis
● Principle: Hb molecules in an alkaline solution have a net negative charge and move
toward the anode (+).
● Components of electrophoretic system:
○ medium, electrodes (anodes and cathodes), power supply, sample, buffer
1. Cellulose acetate agar at an alkaline pH (8.4-8.6)
● screening method; rapid method- separates the Hb variant (S,D,G,C,E) from A
● Fast hemoglobins - include in order of increasing mobility, Hbs K, J, Bart's, N, I,
and H
● Slow hemoglobins: Hbs A2, C, E, and O Arab at the slow end are unresolved
from each other, as are S, D, G, and Lepore
NOTE: among all the abnormal hemoglobins, C is the slowest

2. Citrate agar at an acid pH (6.2)

● Used to separate hemoglobins that migrate together in cellulose acetate: S from
D and G and Lepore; C from E and O, sharp separation of Hb F and A2
● Separated S from D and G: We may able to identify sickle cell anemia
● C: sickle cell crystallization
3. Polyacrylamide gel (PAGE)
● Provides sharp separation of most hemoglobin variants/abnormal variants
3. Test to detect the presence of Hb S (Sickle hemoglobin)
1) Sickling Test/Metabisulfite Slide Test

59
 ● Principle: Addition of sodium metabisulfite to blood enhances deoxygenation of Hb
and sickling of Hb S
● Method of determination: microscopic because you determine the extent of citric
number of sickled cells after treatment with sodium metabisulfite which deoxygenated
and produces HbS causing the HbS containing cells to sickle
● Reduce/deoxygenated HbS
● To distinguish sickling test with dithionite solubility test:
○ The test does not distinguish sickle cell anemia from sickle trait or other Hb S
syndromes because all red cells sickle; however, sickling occurs more rapidly
with greater amounts of Hb S in the cells.
● False (+)
○ Presence of rare sickling disorders like HbS trans, HbC harlem
○ Hb Bart's
● False (-)
○ <10% HbS (infants)
○ Deterioration of Reagents
2) Sickle Solubility Test/Dithionite solubility Test
● Principle: Red cells are lysed with saponin, and Hb is reduced by dithionite (sodium
hydrosulfite). Reduced Hb S is insoluble in concentrated inorganic buffer (2.3 M
phosphate buffer) and the polymers of deoxy-Hb S obstruct light rays to produce opacity.
● Turbidimetric because polymers of HbS produce turbidity; reduced HbS is insoluble in
2.3M PO4 buffer
● (+) result is turbidity
● Dithionite reduced HbS
● 2.3M PO4 buffer precipitates the oxygenated Hbs
● useful in screening large numbers of people for the presence of Hb S or other sickling
Hbs
● false (+)
○ a large amount of Heinz bodies increased- in cases of G-6-P-D deficiency and
postsplenectomy, unstable Hb.
○ Precipitation of abnormal proteins
● False (-)
○ Low levels of HbS because of severe anemia
○ Deterioration of dithionite reagent

4. Hematocrit (HCT)/ Packed cell Volume (PCV)

● Represents the proportion of RBC to plasma in the peripheral blood
● Measured directly by centrifugation with macro methods and micromethods or indirectly as the
product of MCV and RBC count obtained from automated machines
● Packed Cell Volume (PCV)
● Procedure:
○ EDTA: blood : Capillary blood in heparinized tube
○ Capillary tubes (75mm long (length) with ID (internal diameter) of 1.155mm)
○ Blue band - used with EDTA-blood (non heparinized)

60
 ○ Red band - capillary blood
○ Fill the tube to about 3/4; no bubbles or spaces in between otherwise it can cause
elevation of result. Wipe side
○ Centrifuge for 5 mins in 10,000 - 15,000 rpm
a. Reference Range
● Male: 40-54% or 0.40-0.54
● Female: 37-47% or 0.37-0.47
● Newborn: 44-64% or 0.44-0.64
b. Clinical Significance
● Increased Hct - polycythemia
● Decreased Hct - anemia, blood loss
c. Sources of Error
● Speed and duration of centrifugation
○ Plasma trapping recounts for 1-3% of the RBC column (0.014 in a Hct of 0.47)
○ Slightly more in macrocytosis, spherocytosis and hypochromic anemias
○ And even greater in sickle cell anemia
● Type and amount of anticoagulants
○ Excess EDTA causes shrinkage of RBCs
○ Excess EDTA happen during unfilled tube (insufficient amount of blood
compared to anticoagulant)
● Improper collection and storage of samples
○ Falsely Increased: Hemoconcentration due to prolonged tourniquet
application increases the number of cell
○ False elevation: Storage at room temperature for about 6-24 hours causes
swelling of cells.
● Irregularity in the length and bore diameter of the tube used.
● Failure to mix blood adequately before sampling.
● Errors in reading or calculation of results.

5. Erythrocyte Sedimentation Rate (ESR)

➔ Rate of settling of erythrocytes from diluted plasma measured as:
◆ The distance from the surface meniscus to the top of sedimented column of RBC after a
given interval of time; or
◆ The amount of time required for erythrocytes to settle to a specified point

METHODS ANTICOAGULANT TUBE

Graduation Bore

Wintrobe Double Oxalate 0-100mm 3.0mm

Westergreen 3.8% Na Citrate 0-200mm 2.5mm

A. Phases of Sedimentation

61
 1. Agglomeration Phase- first 10 min; cells form agglomerates or rouleaux of
various sizes
2. Phase of fast settling- first 10 min; cells form agglomerates or rouleaux of
various sizes
3. Final phase of packing- last 10 min; rate of settling becomes slow due to
clogging of the agglomerates
- Overall time: 1 hour

B. Procedural Considerations
● ESR is most sensitive to changes in plasma of 0.3-0.4
● Heparin is not an acceptable anticoagulant
● Rate of settling depends on an interrelationship of variables

Increased ESR Decreased ESR

Physiologic Variation ● 3rd month of gestation to 1 ● Newborn

month of postpartum

Pathologic Variation ● Hemolytic Anemia ● Polycythemia

● Tissue Necrosis
● Myocardial Infarction
● Cardiac Thrombosis
● Multiple Myeloma
● Macroglobulinemia
● Hyperfibrinogenemia
● Chronic Infections
● Autoimmune Disorders
● Congestive Heart failure
● Liver disease
● Sickle Cell anemia
● Spherocytosis
● Hyperglycemia (inc FBS)
● Increase ACTH
● Hyperbilirubinemia

Intrinsic Factors

Plasma Factore ● Increase Fibrinogen ● Defrination

● Increased Globulins ● Increase in albumin
● Increased Cholesterol ● Increase lecithin

RBC Factors ● Large size (Macrocytes) ● Small Size (Microcytes)

● Increased Number ● Poikilocytosis
● Hemolysis

Extrinsic Factors ● Increased Temperature ● Decreased temperature

(>27°C) (<20°C)
● Longer tube or larger bore ● Dirty glasswares
diameter -Tilting (3° tilting ● Presence of clots
causes an error of about ● Old/stored blood
● 30%)
● Wet glasswares
● Air bubbles

RETICS COUNT
● New Methylene Blue (preferred)
○ Pwede rin si cresyl blue

62
● Principle: Supravital stains bind, neutralize and cross-link RNA causing the ribosome and
residual RNA to co precipitate with the few remaining mitochondria and ferritin masses in
lining reticulocytes microscopically as dark blue clusters or filaments (reticulum)
○ Specimen: EDTA or Heparinized blood
○ Procedure:
■ Mix 2 drops of blood with 2 drops of filtered new methylene blue
■ Stand for 10-15 minutes Prepare to wedge films and air dry
■ Examine each slide under OIO
● Retics
● Mature RBC
■ Methods of Counting
● Count a minimum of 1000 (hanap ng 10 fields na may 100 RBC) erythrocytes
both reticulin-containing and non-reticulated containing blood. (500 cells per
slide)- for QA purposes, reticulocytes reading should be done in 2 slides (dapat
match ‘tong 2 slides)
𝑇𝑜𝑡𝑎𝑙 # 𝑜𝑓 𝑟𝑒𝑡𝑖𝑐𝑠
○ Formula (Retics ct)= 1000
× 100
● Insert a Miller ocular disc into the eyepiece of the microscope. Count the retics
in the large square and RBCs in the small square in successive fields until 300
RBCs are counted.

CLINICAL SIGNIFICANCE
● Increased Retics: adequate bone marrow response
○ Indication of bone marrow compensatory mechanism
■ Blood Loss
■ Hemolytic anemia
○ Other causes:
■ Malaria
■ Blood intoxication
■ Lead poisoning
■ Splenic tumor
■ Response to therapy - Following treatment for pernicious anemia , IDA , Folic acid
deficiency (Megaloblastic Anemia)
○ Decrease Retics= inadequate BM response
■ Aplastic anemia
■ Aplastic crisis of sickle cell anemia
■ Chemotherapeutic or radiation induced hypo-proliferation
■ Pernicious anemia or megaloblastic anemia due to ineffective erythropoiesis
(asynchronous maturation where in precursor cells is more reliable to intramedullary
destruction or bone marrow destruction)
NOTE! Sundin yung table 16.1 for reference value!!! (Formulas for retic ct)

TABLE 16.1: Formulas for Reticulocyte Counts and Red Blood Cell Indices

63
 TEST FORMULA ADULT REFERENCE
INTERVAL

Absolute reticulocyte count (x10^9 /L) = [reticulocytes (%)/100] x RBC 20–115 x 10^9 /L
count (x10^12/L)

Corrected reticulocyte count (%) = 5 reticulocytes (%) x patient’s

HCT (%)/45

Reticulocyte production index (RPI) = corrected reticulocyte In anemic patients, RPI should
count/maturation time be >3

Mean cell volume (MCV) (fL) = HCT (%) x 10/RBC count 80–100 fL
(x10^12/L)

Mean cell hemoglobin (MCH) (pg) = HGB (g/dL) x10/RBC count 26–32 pg
(x10^12/L)

Mean cell hemoglobin concentration = HGB (g/dL) 3 100/HCT (%) 32–36 g/dL
(MCHC) (g/dL)
HGB, Hemoglobin; HCT, hematocrit; RBC, red blood cell.
(problem in BM- neoplastic cells replace normal hematopoietic stem cell)

Laboratory Features of Myeloproliferative Neoplasms

PARAMETER CML PV ET PMF

WBC Increased Normal or Normal or slightly Normal, increased,

increased increased or decreased

RBC Normal or Increased Normal or slightly Normal or

decreased decreased decreased

Platelets Normal or Normal or Increased Normal, increased,

increased increased or decreased

Molecular BCR-ABL1 JAK2 V617F or ±JAK2 ±JAK2

abnormalities other
JAK2 mutation

RBC Morphology Grading

Morphology Grade

Polychromatophilia 1+ = 1-5/field
Helmet cell, Dacrocyte 2+ = 6-10/field
Spherocyte, Acanthocyte 3+ = >10/field
Schistocyte

Poikilocytosis 1+ = 3-10/field
Codocyte, Burr cell 2+ = 11-20/field
Stomatocyte, Ovalocyte 3+ = >20/field
Elliptocyte

Rouleaux 1+ = 3-4 aggregates

64
 2+ = 5-10 aggregates
3+ = numerous aggregates

Sickle cells POSITIVE ONLY

Basophilic stippling
Pappenheimer bodies
Howell-Jolly

Polychromasia Grading
GRADE % POLYCHROMATIC RED
CELLS

Slight 1%

1+ 3%

2+ 5%

3+ 10 %

4+ > 11%

OFT (Osmotic Fragility Test)

● Whole blood is added to varying conc. of NSS
● Tests cell membrane integrity
● Isotonic solution = molecules will pass in and out of cells
● Hypotonic solution = water molecules will go accumulate inside the cell → hemolysis
○ 2-3 % Acetic acid → use in WBC count / Hemocytometry
○ Diluting fluid
○ Cell swelling → cell burst → lysis

CYTOCHEMISTRY

Main objective: Understand and establish the cell lineage in leukemia

Acceptable specimens:

65
 1. Peripheral film, bone marrow smears and smears from lymph nodes and spleen
2. Adequately and properly fixed well done smear

Enzymatic Techniques
A. PHOSPHATASES
● Group of isoenzyme that are capable of hydrolyzing monophosphate esters

● ACID PHOSPHATASE
○ Present in all hematopoietic cells and located on lysosomes
○ Used in preliminary identification of ACUTE T-CELL LYMPHOCYTIC
LEUKEMIA (ALL)
○ B-cell lymphocytes - NO ACID PHOSPHATASE
○ Reagents;
■ Fixative: Cold methanol-acetone mixture or RT citrate-acetone mixture
■ Acetate buffer
● Sodium acetate
● Acetic acid
● Distilled water
■ Staining substrate
● Naphthol AS-BI phosphate
● N,N-dimethylformamide
■ Mayer’s hematoxylin
■ Incubation mixture
○ Contains seven (7) non erythroid isoenzymes
(0,1,2,3b,4 and 5)

● Result: DARK-RED GRANULES

● interpretation :
○ Hematopoietic cells including blasts in T-cell ALL-POSITIVE
○ Blasts in non-T-cell ALL- NEGATIVE
○ Normal blood fil - (+) control

ISOENZYMES (+) CELLS

0 ● Gaucher cells
○ Dx: Gaucher’s disease
○ (Abundant cytoplasm: crumpled, tissue paper appearance

1, 2 AND 4 Neutrophils and Monocytes

3 Lymphocytes and Platelets

3B Primitive cells and Blasts

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 5 Hairy cells

TRAP (Tartrate Resistant Acid Phosphatase)

● Used in the diagnosis of HAIRY CELL LEUKEMIA
● Interpretation:
○ Hematopoietic cells - NEGATIVE
○ Hairy cell leukemia - POSITIVE

LEUKOCYTE ALKALINE PHOSPHATASE (LAP) - Present at cytoplasm of neutrophils and

band cells.
● AKA Neutrophil Alkaline Phosphatase (NAP) - 2 0 granules of mature granulocytes
(myelocytic stage)
● Used in differentiating Chronic Myelogenous Leukemia (CML) - lot of blast cells seen;
malignant myeloid cells - From leukemoid reaction / Reactive Neutrophilia - transient,
myeloproliferative disorder; mature neutrophils
● Interpretation:
○ Mature neutrophils and band cell - POSITIVE
○ All other leukocytes (malignant neutrophils - doesn’t have 20 granules) -
NEGATIVE or WEAKLY (+)
● Result: Blue-black (fast blue); red precipitate (fast red violet
● Scoring:
○ LAP activity is graded in 100 mature neutrophils and bands by scoring from
0-4
○ The number of cells seen in each particular grade is multiplied by that grade.
Products are then added to get the LAP score

Score Description

0 No granules in the cytoplasm

No staining

1+ Few small granules in the cytoplasm (could be counted)

Faint and diffuse staining

2+ Moderate staining granules in 50-80% of cytoplasm

Pale, with moderate amount of blue staining

3+ Strongly stained granules in 80-100% of cytoplasm and beginning to

coalesce
Strong blue precipitated staining

4+ Strongly stained granules packed in to the cytoplasm (nucleus is the

only visible component)
Deep blue or brilliant staining with no visible cytoplasm (obscured by

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 20 granules)

INCREASED SCORE DECREASED SCORE

Polycythemia Vera CML

Leukemoid reaction PNH
Infections Sideroblastic anemia
3rd trimester of pregnancy - blood from Marked eosinophilia
woman (+) control Sickle cell anemia

B. ESTERASES
● Are group of isoenzymes that are capable of hydrolyzing aliphatic and
aromatic esters at an acid or neutral pH
● Used to differentiate acute leukemias that are myeloid from monocytic in origin
● Consists of nine (9) isoenzymes with specific cellular location (found in all
hematopoietic cells except in erythroid series)

Esterase Isoenzyme # Cellular location

1, 2, 7, 8, 9 Neutrophils and mast cells

3, 4, 5, 6 Monocytes, megakaryocytes,
plasma cells

1, 2, 7, 8, 9 - referred to as “specific esterases”

3, 4, 5, 6 - referred to as nonspecific esterases

● NAPHTHOL AS-D CHLOROACETATE ESTERASE

○ Used to demonstrate “specific esterases”
○ To distinguish myeloid leukemias from monocytic leukemia
○ Result: Red cell granulation
○ Interpretation:
■ Myeloid cells - POSITIVE
● Differentiate M1, M2, M3 from M5
● M4 - mixture of myeloid and monocytic cells (+)
■ Monocytic cells - NEGATIVE or WEAKLY (+)

● ALPHA-NAPHTHYL ACETATE ESTERASE (ANAE)

○ Used to demonstrate “nonspecific esterases”
○ To distinguish monocytic leukemia from myeloid leukemia

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 ○ Result: Brown to dark brown
○ Interpretation:
■ Monocytic cells - POSITIVE - diffuse pattern
■ Myeloid and lymphoid cells - NEGATIVE
■ Erythroid cells - WEAKLY (+)

To differentiate MONOCYTIC CELLS (Na fluoride sensitive)

From ERYTHROID CELLS (Na fluoride resistant)

● ALPHA-NAPHTHYL BUTYRATE ESTERASE (ANBE)

○ Used to demonstrate “nonspecific esterases”
○ To distinguish monocytic leukemia from myeloid leukemias
○ Interpretation:
■ Monocytic cells - POSITIVE
■ Myeloid and lymphoid cells - NEGATIVE

C. PEROXIDASE (MYELOPEROXIDASE) - Stain of choice of Auer rods

● Enzymes that catalyzes the oxidation of substrates in the presence of
hydrogen peroxide
● Present in the primary granules of neutrophils, eosinophils and to lesser extent
monocytes while absent in lymphocytes in all erythroid cell lines
● Used in differentiating acute myelogenous leukemia and acute monocytic
leukemia form ALL
● Result: Gray-black granules in the cytoplasm while eosinophils stain
red-brown
● Interpretation:
○ Myeloid cells - POSITIVE (10 granules)
○ Monocytic cells - WEAKLY (+) or NEGATIVE
○ Lymphoid cells - NEGATIVE
○ Erythroid cell - ALL NEGATIVE

NONENZYMATIC TECHNIQUES

Periodic Acid Schiff (PAS)

● Aids in the diagnosis of ALL’s and M6
● Used to confirm the presence of glycogen in cells - found in all cells including
malignant erythroid precursors except for normal lymphocytes
● Involves 2 step reactions
1. Periodic acid is used to form aldehyde groups from glycogen
2. Aldehyde groups react with the Schiff reagent producing a bright reddish pink
● Interpretation:
○ Myeloid and monocytic cells - POSITIVE
○ ALL except L3 - POSITIVE

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 ○ M6 - POSITIVE

SUDAN BLACK B (SBB)

● Demonstrates lipid present in the cells (myeloid and monocytic cells)
● Useful in distinguishing AML from ALL (+) BLAST
● Result: Brown-black precipitate
● Interpretation:
○ Myeloid cells - POSITIVE
○ Monocytic cells - NEGATIVE or WEAKLY (+)
○ Lymphoid cells - NEGATIVE
● ADVANTAGE OF SBB
○ Reagent is not hazardous
○ Reagent is more stable

PRUSSIAN BLUE STAINS - screens defect in synthesis of Hgb; also stains Pappenheimer
bodies; stains iron present in mature and immature RBC
● Staining procedure for iron stores in the bone marrow
● Quantitated by using Perl’s reagent (potassium ferrocyanide)
● Indicated by a blue-green precipitate

IMMUNOCHEMISTRY
● Identification of the immunologic phenotype of a given cell population through the use
of specific monoclonal or polyclonal antibodies against selected cell antigens

FACTOR VIII ANTIBODIES

● Used for accurate diagnosis of M7 - acute megaloblastic anemia

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