Midterms

HEMATOLOGY I MTAP II Lecture

COMPLETE BLOOD COUNT
REVIEW OF CONCEPTS, PRINCIPLES, AND CORRELATION: COMPLETE BLOOD COUNT
 A quantitative determination yielding qualitative results.
Outline
i. Complete Blood Count RESULT FORM
a. Result form
s

b. Parameters of CBC  List of numbers where reference ranges vary according to

- Manual Method of CBC sex, age, and population group.
ii. Review of Hematopoiesis o Population group: e.g., Caucasians vs. Asians, Black
- Conditions that affect bone marrow Americans vs. Asians
- Three Major Phases of Hematopoiesis  In this case, there is a discrepancy; hence, we
- Hemoglobin Compounds cannot make use of the same values for the
- Cellularity
- Collection of Bone Marrow\
current populations.
- Normal Marrow Cells  Medical technologists compare values derived from
- Cytokines in Hematopoiesis patients and reference range.
iii. Erythrocytes  Contains quantitative results  align different parameters
a. Production
b. Metabolic pathways
c. Rbc hemolysis
iv. Laboratory methods:
a. Hematocrit
b. Red cell indices
v. GRANULOCYTES
a. Neutrophils
b. Eosinophils
c. Basophils
vi. BASOPHIL VS. MAST CELL
vii. AGRANULOCYTES: Monocytes and Macrophages
a. Characteristics
b. Function
viii. AGRANULOCYTES: Lymphocytes
ix. B LYMPHOCYTE DEVELOPMENT
x. T LYMPHOCYTE DEVELOPMENT
a. Summary of T Cell Maturation
b. T Cell Blastogenesis
c. NK Cells PARAMETERS OF CBC
d. Lymphocyte Function
s

xi. Lymphocyte Function

xii. Cell Counts
 Fuchs-Rosenthal Hemocytometer
 Speirs-Levy Hemocytometer
 Improved Neubauer Counting Chamber
xiii. General Principles
xiv. Outline
xv. The Inverted L Rule
xvi. Poisson’s Law of Distribution
xvii. Routine Computations
xviii. Leukocyte Count Normal Reference Values
xix. Differential Count
xx. The Schilling Hemogram
xxi. Arneth Count All of these properties
xxii. Corrected WBC Count are numerically given Normal cells seen in
xxiii. Absolute Leukocyte Fraction Count to describe red cells. peripheral circulation.
xxiv. Eosinophil Count
a) Thorn Test  RBC Morphology – Input of medical technologist
xxv. Basophil Count
because it is not given by analyzers.
a) Cooper and Cruickshank Method
xxvi. Megakaryopoiesis and thrombopoiesis  WBC Count – In cases of blastic crisis, expect the
xxvii. Platelet Quantitation presence of immature forms. In more specificized
a) Direct: Rees and Ecker Method analyzer, include metamyelocyte, myelocyte, and
b) PBS: Platelet Evaluation blasts.
c) Automation: MPV (Coulter)  These parameters of CBC are only possible when one is
xxviii. Peripheral Blood Smear using an automated analyzer.
a) Preparation
b) Evaluation
c) Reporting

Page 1 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 MANUAL METHOD OF CBC
Reports will include:
 Hematocrit THREE (3) MAJOR PHASES OF HEMATOPOIESIS
s

 WBC count 1. Primitive

 Differential count o Red cells, blood cells, etc. cannot be morphologically
 Platelet estimate identical with the cells.
o In short, there is difficulty in the identification of the
NOTE: Laboratories should not be allowed to report
hemoglobin and RBC count in the manual method IF they morphology of the cell.
did not perform: o Begins with: Mesoblastic phase
1. Cyanmethemoglobin
2. No actual counting of red cells 2. Definitive
o Blood cells show morphologic characteristics that are
RULE OF THREE unique to each cell group.
s

o Able to identify them.

 It is not an advantageous source of report for RBC and
Hemoglobin.
 When using this technique, initially one must:
MESOBLASTIC PHASE
1. Have an automated analyzer  May also be referred as the “yolk-sac” phase.
2. The cells are normocytic, normochromic  There is production of primitive erythroblasts and
angioblasts.
REVIEW OF HEMATOPOIESIS o Primitive erythroblasts
 Different from the erythroblast that comes out
Mesoblastic Hepatic Phase Medullary from the bone marrow (the earliest recognizable
Phase Phase precursor of red cell: pronormoblast)
Period of 18th-20th day 5th-7th week End of 5th  They are unable to extrude their nucleus. The
occurrence AOG AOG month AOG nucleus stays.
Site of Blood islands Liver, thymus, Bone o Angioblasts
occurrence of the yolk spleen, and marrow  Cells that are bound to become blood vessel
sac (notable) kidneys cells in the future.
Cells Primitive - Red blood ALL
produced erythroblasts, cells, HEPATIC PHASE
Angioblasts - Granulocytes  Overlaps with the Mesoblastic Phase.
and  Marks the beginning of definitive hematopoiesis.
Megakaryocytes o There is a recognition of other cell types.
(3rd month
AOG), NOTE: When asked in exam about the sequence of
production in blood cells, just follow the cells on the table.
- Lymphocytes
(4th month
AOG), RBCs  Granulocytes & Megakaryocytes  Lymphocytes
- Monocytes (5th  Monocytes
month AOG)
Hemoglobi Embryonic - Fetal - Adult MEDULLARY PHASE
n detected forms Hemoglobin Hemoglobin  What is special about the 5th month AOG?
- Adult hemoglobin - Fetal o There is a completion of the different organ
Hemoglobin systems of the body (kaya nadedetect na ang sex ng
NOTE: baby).
Blood cells of adults will come solely from the bone o Stem cells migrate in the medulla of the long bones
marrow. so that there is a continuation of blood cell production.
So, in any circumstance that the bone marrow condition is  Thrombocytopenia with absent radius – also detectable in
affected, then we get to see the impact in CBC result form. this age because by the 5th month, the bones of a
developing baby should be complete; hence, incomplete
WHAT ARE THE CONDITIONS THAT AFFECT THE BONE development can be seen in this condition.
MARROW THAT COULD TREMENDOUSLY AFFECT CBC  Traces of Fetal hemoglobin are bound to disappear as the
RESULT?
s
baby grows.
 Aplastic anemia – absence of cellular production 
decreased CBC result -- INTENTIONALLY LEFT BLANK --
 Leukemia
o Increased WBC count HEMOGLOBIN COMPOUNDS
o Abnormality in diff count s

o Decreased in both red cells and platelets due to Hemoglobin Compounds Globin Chain Composition
overcrowding of precursors as seen in the bone in Early Life Development
marrow. Embryonic Forms
Gower I 2 Zeta and 2 Epsilon
Portland 2 Zeta and 2 Gamma
Gower II 2 Alpha and 2 Epsilon
Fetal Hemoglobin 2 Alpha and 2 Gamma

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LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 Adult Hemoglobin  Delta chain can be temporarily acting as a
Hb A1 2 Alpha and 2 Beta substitute for the missing globin chain, most
Hb A2 2 Alpha and 2 Delta esp. for adults.
Produced before birth  Consistently
produced but undetectable

CELLULARITY
s

X-Axis – Age of Gestation (before birth and post-partum); Y-

Axis – Percent of Globin Chain Synthesis

 Epsilon and Zeta Chains  All cells will be concentrated in the blood islands of the
o Have the same phenomenon yolk sac for the first month AOG, which will be decreasing
o They are synthesized in the highest level at zero subsequently before the 3rd month AOG.
month, and subsequently decline on the 3rd month  Cellularity peaks on the hepatic period before on the
AOG. second AOG (care of the liver).
 Liver
Synthesized at zero month  declines at 3rd month
o It can produce red cells even after 2 weeks after birth.
o It takes over blood cell production once yolk sac
 Alpha Chain
declines.
o Starts initially to reproduce during the mesoblastic
 Lymph Nodes
phase at zero month.
 Bone Marrow
o Consistently increases, plateaus, but never declines.
o Before the 5th month AOG
 Reason why it is detectable in adults.
Increases  Plateaus  Never declines  Different Bones
o They are capable of storing red cells after months.
o The capacity of Tibia and Femur will be diminished
 Gamma Chain
o Produced before 3rd month AOG. before age 25.
o Consistently increases, peaks, plateaus, and
declines.
o Lowest level during 6th months post-partum.
 Hence, the fetal hemoglobin shouldn’t be
detected when the baby grows.
Increases  Peaks  Plateaus  Declines

 Beta Chain
o Has a dramatic presentation.
o Produced before 3rd month AOG, but not detectable.
o Has a slow progression.
o Peaks before birth, consistently increasing joining
alpha chain in its plateau; hence, in adult hgb ang
dominant is alpha 2 and beta 2.
Peaks before birth  Consistently increasing 
Joins alpha chain in its plateau
 Delta Chain
o Produced before birth
o It is consistently produced but not in detectable levels.  Before Age 1, all of the bones of the body are
haematopoietically active.
o It is there but not significant.
o However, because of retrogression at age 4 to 7,
o In Thalassemia, we see the disease according to the
there is the beginning of fat deposition in the bone
globin chain that is not present.
marrow.
Page 3 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 Ideal site for bone marrow collection
o Adult: Superior Ileac Crest (most ideal site)
o In the medulla of long bones, the majority is yellow
marrow.

COLLECTION OF THE BONE MARROW

 s
CYTOKINES IN HEMATOPOIESIS
 s

Core Biopsy Trephine Jamshidi needle Cytokine Source Target Cell Effect
(a small fraction of the biopsy needle EPO Peritubular BFU-E and Stimulates
bone is obtained) Interstitial cells CFU-E proliferation of
Aspiration Aspiration
University of erythroid
(extracting red marrow needle
Illinois sternal progenitors and
from the patient) needle prevents apoptosis
 BM collection is one of the most bothersome and of CFU-E.
aggressive collection. G-CSF Endothelial Neutrophil Stimulates
 BM studies are done to: cells, precursors, granulocyte
1. Check the M:E ratio. placenta, fibroblasts, colonies,
2. Check for cellularity. monocytes, leukemic differentiation of
macrophages myeloblasts progenitors toward
RATIONALE – M:E Ratio Observation neutrophil lineage
Normal Infection Leukemia and maturation
2:1 to 4:1 6:1 25:1 GM-CSF T cells, Bone marrow Promotes antigen
(Average: 3:1) macrophages, progenitor presentation, T cell
 M is higher than E (even if they come from same endothelial cells, dendritic homeostasis,
common progenitor), WHY? cells, cells, hematopoietic cell
o Remember: These cells come from the common fibroblasts, macrophages, growth factor
mast cells NKT cells
myeloid progenitor (CFU-GEMM) - source.
IL-2 CD4+ T cells, T cells, NK Cell
o Mas mabilis ang turnover ng other blood cells
NK cells, B cells, B cells, growth/activation of
compared to the red cell.
cells Monocytes CD4+ and CD8+ T
o The red cell has BFU-E (Burst-forming Unit)
cells, suppress
 Hence, it is guaranteed that even if you have Treg responses,
individual cell precursors, they can undergo mediator of
multiple mitotic divisions to give out the need for immune tolerance.
red cells.
IL-3 Activated T Hematopoietic Proliferation of
 In infections, the M cells increases because the body cells, NK cells stem cell and hematopoietic
needs it. After the infection, the ratio will return back to progenitors progenitors
normal values. IL-6 T cells, T cells Costimulation with
 In Leukemia, there is a high M ratio due to unregulated macrophages, B cells other cytokines,
production. fibroblasts Liver cell
growth/activation of
NORMAL MARROW CELLS
 s
T cells and B cells,
All developing hematopoietic cells megakaryocyte
Macrophages maturation, neural
Mast cells differentiation,
Osteoblasts acute phase
(commonly confused as plasma cells) reactant
Osteoclasts IL-10 CD4+, Th2 T T cells Inhibits cytokine
(commonly confused as megakaryocytes) cells, CD8+ T Macrophages production, inhibits
 Mast Cells cells, macrophages
o Cells in the bone marrow, they are not blood cells. monocytes,
macrophages
o They only make use of the blood for transport medium
IL-12 Macrophages T cells T cell, Th1
to go to the tissues.
differentiation
IL-15 Activated CD4+ T cells CD4+/CD8+ T cell
CD4+ T cells CD8+ T cells proliferation
NK Cells CD8+/NK cell
cytotoxicity
IFN-α Dendritic cells, Macrophages, Antiviral, Enhances
NK cells, T NK cells MHC expression
and B cells,
-- INTENTIONALLY LEFT BLANK -- macrophages,
fibroblasts,
endothelial
cells,
osteoblasts
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LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
REMEMBER:
 The specific growth factor that can induce growth for such cell.
 IL-3, IL-5, IL-6, IL-11, and IFN-α – board exam essentials
 G-CSF – specific for associated term (granulocyte)
 IL-3 – affect almost all cells
 IL-15 – specific for affecting T cell maturation and development.

ERYTHROCYTES

PRODUCTION

TOTIPOTENT HEMATOPOIETIC STEM CELL

 youngest progenitor cell for all type of red cells

PLURIPOTENT HEMATOPOIETIC STEM CELL

 nakababa na from totipotent stem cell

PRECURSOR VS PROGENITOR

PRECUROSOR PROGENITOR
 from CFU-GEMM,  non-committed
pronormoblast is  it could develop to any
developed and is now cell
the precursor cell ex. Common myeloid
committed to become a progenitor- can give out
red cell granulocyte, erythrocyte,
megakaryocyte or monocyte

CFU-GEMM
 progenitor cell for all types of blood cell except for
 (1st column) general rule of maturation:
lymphocyte  As cells mature- decrease in size
CFU-L  As the cells mature- loss of basophilia
 will give out/ source of lymphocyte
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LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 o Cell will initially start with a blue color. Because of  Composed of DNA
maturation the blue color will change into PINK  Seen in normal red cells but they are
removed by the spleen.
 (2nd column) As cells mature the N:C ratio decreases  So, when there are Howell-jolly bodies
 Size of the nucleus decreases. in the initial cells produced, they will
 There will be disappearance of the nucleus that will be circulate in the spleen and after they will
applicable for red cells only because for other cells there is be eaten by the spleen.
imposition of the unique nuclear characteristics  There will be a lot of Howell-jolly bodies
seen in patient with removed spleen.
 (3rd column) As the cell matures the chromatin pattern  Stays in the BM for 24 hrs. before it goes to the peripheral
becomes more coarse, dense, and clumped. circulation for another day and will fully become a mature
 Open/ loose chromatin appearance- euchromatin cell
 Euchromatin will become heterochromatin as it goes down

 (4th column) Representation of how cells change in

morphology

Nomenclatures: (naming of different cells according to maturation)

 Normoblastic nomenclature
 Rubriblastic nomenclature
 Erythroblastic nomenclature (almost the same as
normoblastic nomenclature)

1. PRONORMOBLAST/ PROERYTHROBLAST
 Rubriblast- equivalent to the rubriblastic nomenclature
 Earliest recognizable precursor of the red cell
 Intensely blue in color
BFU-E
 In basic microscopy: they appear are very dark circular cells
o The light can no longer penetrate it burst forming unit erythroid
 Capable of mitotic cell division stays for 1 week
 Stays in the bone marrow in that stage for approx. 24 hours.
o Pronormoblast will gather materials for globin synthesis CFU-E
1 week
2. BASOPHILIC NORMOBLAST/ PRORUBRICYTE
 Another cell exclusively seen in the bone marrow for about
24 hrs.- undergoes mitotic division Pronormoblast
 They are needed to be highlighted because they are the 1st 7 days
stage to begin hemoglobin synthesis
 There will be a decrease in the size of the nucleus but a
Mature red cells
deeper staining/ intensity of the blue color in the cytoplasm
o What causes the very blue color? 1 pronormoblast= 8-32 red cells depending on the #
 the organelles are highly active in protein of mitotic divisions
synthesis= stain more blue
Is it possible to reduce 18-21 days? Yes, especially in the
case of hypoxia
3. POLYCHROMATOPHILIC NORMOBLAST HYPOXIA
 Equivalent to rubricyte  Low levels of oxygen in the tissue and the body needs to act on
 Polychromatic because there is an increase in hemoglobin this
synthesis that will affect the blue color of cytoplasm 3 basic methods in which the body will address the lack of
o Polychromia= increases hgb synthesis oxygen delivery:
 Seen in the bone marrow for about 24 hrs. 1. Chemical response:
 Last stage capable of mitotic cell division  Increase in 2,3-DPG
o Shift to the right of the oxyhemoglobin
4. ORTHOCHROMIC NORMOBLAST/ METARUBRICYTE
 Not capable of mitosis dissociation curve
 Possesses a single color because hemoglobin synthesis is  Release of oxygen content
peaking 2. Physical response
 Very high hemoglobin= pink in color  Selective redistribution of blood
 Stage wherein it extrudes the nucleus o Priority of blood supply: vital organs (brain)
 Nucleated red blood cell (ex. namumutla)
3. Hematologic response
5. POLYCHROMATOPHILIC ERYTHROCYTE
 Slow but sure that it will address the lack of oxygen
 Despite the pink color, there are remnants of RNA and
ribosomes that are seen in the cytoplasm
 Roles of RNA and ribosomes: For the completion of HYPOXIA AND EPO
hemoglobin synthesis
 Stain RNA and ribosomes supravitally= reticulocyte / a. Early release of reticulocytes
reticulum  Tries to affect the receptor that are found in the
 When the nucleus is extruded, will there be remaining DNA
endothelium of the trilaminar sinus of the bone
in the cell? YES
o they are known to be the HOWELL-JOLLY marrow so that they will allow the passage of
BODIES reticulocyte

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LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
  Reason why we see shift reticulocytes EXTRAVASCULAR HEMOLYSIS
o Shift reticulocyte- reticulocytes that did not stay in
the bone marrow for 1 week
o They should stay for 1 week in the bone marrow
before going to the peripheral circulation, but the
body needs red cell to work on oxygen delivery

b. Prevent apoptotic cell death

 ex. BFU-E gave a lot of CFU-E, EPO will tell CFU-E
to reproduce red cells  Death of the red cells in the reticuloendothelium.
 Splenomegaly, hepatomegaly
c. Reduces maturation time inside the BM  No:
 from the pronormoblast it will become a mature red o Hemoglobinuria
cell after 7 days, this can be shortened to 48-72 hrs. o Hemoglobinemia
only
o Methemoglobinuria
 red cells once they are in the circulation, they get to
INTRAVASCULAR HEMOLYSIS
live for 80-120 days.
o They need to sustain their membrane to
avoid hemolysis and do its functions

METABOLIC PATHWAYS

a. EMP (Embden-Meyerhof Pathway/ krebs cycle)

 anaerobic pathway that gives 90% of ATP in the body
b. HMP (hexose monophosphate shunt)
 Gives out about only 10% of energy source
c. Leubering-rapaport shunt
 Sidearm of EMP
 Synthesis 2,3-DPG
d. Methemoglobin reductase pathway  Death of the red cells that happens in the blood vessels.
 The functional form of iron is ferrous. Due to  There is:
subsequent oxidation in the body, it become ferric. o Hemoglobinuria
But this is reversible by means of cytochrome 5 o Hemoglobinemia
oxidase, glutathione and blood pH which are involved o Decreased haptoglobin and hemopexin
in this pathway. o Methemoglobinuria and methemoglobinemia
 Role: to reverse Methemoglobin which is the non-
functional/ ferric form into ferrous which is the
functional form.

RBC HEMOLYSIS

*Routine blood film: there polychromatophilia (↑in reticulocytes) b/c

there is cell death – needs to be replaced;
*Erythroid hyperplasia: ↑ red cell production.
*Bilirubin: plasma or serum; unconjugated

LABORATORY METHODS: TESTING RED CELLS

1. Hematocrit
2. Blood cell indices

MICROHEMATOCRIT METHOD
A small amount of whole blood is
centrifuged to determine maximum
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LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 Principle packing of erythrocytes, expressed as  Limitation: Only for normocytic and normochromic RBC.
PCV or Hct
 Anticoagulated Whole Blood (EDTA) LABORATORY METHODS: BLOOD CELL INDICES
 Capillary blood collected in
Specimen Heparinized Capillary Tubes MCV: Mean cell volume
Requirement  Specimens should be stored at room
s temperature and be processed within
6 hours after blood collection.
Note: more than 6 hours causes RC to be
macrocytic when exposed to anticoagulant for a
long period of time. = False ↑
 Capillary hematocrit tubes (with red
band or plain)  In automated determinations, MCV makes use of
Reagents and  Non-absorbent sealing clay electronic impedance / Coulter principle.
Equipment  Microhematocrit reader device  With range = Normocytic
 Microfuge with an RCF of 10,000 to  <80: Microcytic
15,000 RPM  >100: Macrocytic
 Fill at least two capillary tubes MCH: Mean corpuscular hemoglobin
approximately 2/3 full. (2 HCT prep)
 Seal the unfilled end.
Procedure  Centrifuge.
 Determine microhematocrit value
using the microhematocrit reader.
 Results should agree within 0.02L/L
for duplicates.  In manual determinations, Hgb is identified with the used
E.g.: 0.45 in the first HCT reading then 0.47 in of Cyanmethemoglobin principle.
the 2nd HCT reading, it is acceptable because
 Within range = Normochromic
0.02 is the difference.
 <26: Hypochromic
TECHNICAL ERRORS
 >32: ask yourself if it is a macrocyte or spherocyte =
 Excess anticoagulant (red cell shrinks) False ↓
cause increase MCH & MCHC
 Insufficient mixing of blood prior to obtaining Hct
MCHC: Mean corpuscular hemoglobin concentration
sample may increase or decrease Hct False ↑
 Improper sealing of capillary tube
 Inadequate centrifugation or allowing tubes to stand too
long after the centrifugation. False ↑
 Including a large buffy coat in the reading False ↑
 Improper use of the microhematocrit reader False ↑ / ↓ g/dL
 Heat sealing of capillary tubes may damage the blood
sample (bursting of RC) False ↓  Useful for the evaluation of iron therapy, anemia.
PHYSIOLOGIC ERRORS  However, the more significant indices for Red cell
 Trapped plasma may cause the Hct to be falsely evaluation are: MCV and MCH
increased by as much as 0.02 L/L. RDW: red cell distribution width
Note: trapped plasma causes: spherocytes, single cells, target
cells,
 Certain abnormal rbc shapes (spherocytes and sickle
cells interfere with rbc packing)
 During the first few hours of acute blood loss.
 Dehydration increases Hct  Elaborated with the use of histograms.
Note: Water comprises the majority of your plasma, wherein  Ratio that determines red cell population of homogeneity
dehydration would cause increase RCs. (uniformity in size)
 Specimen collection errors may alter the results. False ↑  High RDW: Anisocytosis
NORMAL VALUES
NEWBORN 0.50-0.58 OTHER INDICES
INFANTS 0.35-0.40
s

CHILDREN 0.38-0.44 VI
WOMEN 0.37-0.43  Same as MCV
MEN 0.40-0.50 Hct x 2.3
 Formula: M
LABORATORY METHODS: RULE OF THREE RBC ∈ x 20
mm 3
 Normal Values: 0.9 – 1.1

SI
 Same as MCHC

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LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 C.I . o Exiting blood vessels
 Formula: o Uses integrin and selectin
V .I o Perform phagocytosis
 Normal Values: 0.80 – 1.20

MCD
 Prince – Jones Method (Direct Micrometry)
 Normal Values: 6 – 9 u
 A result >9 is considered macrocytic

MCAT
 Hemoglobin content
MCV

( )
2
 Formula: MCD
π
2
 Normal Values: 1.7 – 2.5 u

GRANULOCYTES
NEUTROPHILS
STAGES OF NEUTROPHILS
1. Myeloblast
o Azurophilic/primary
o Quadrangle nucleus

2. Promyeloblast
o 15 – 20 azurophilic granules

3. Myelocyte
o Start of secondary/specific granule synthesis
o “Dawn of Neutrophilia” – 1st stage that shows
different color of granules
o D-shaped/circular nucleus
o Last stage capable of cell division

4. Metamyelocyte
o Juvenile cells
o Presence of indented nucleus
o Tertiary/gelatinase granules are produced

5. Band/Stab
o Increase constriction of nucleus (C-shaped)
o Synthesis of secretory vesicle
6. Segmenter/Neutrophil
o 3 - 5 lobes
o Hyposegmentation: Pelger-Huet Anomaly
NEUTROPHIL FUNCTION
o Hypersegmentation: Megaloblastic anemia  Attachment
o 1 neutrophil: 16 myeloblast  Includes killing and ingestion

NEUTROPHIL GRANULES
Primary Secondary Tertiary Secretory
(Azurophilic (Specific Granules Granules
Granules Granules) (Vesicles)
Myeloperoxidase B2- Gelatinase CD11b/CD18
Microglobulin
Acid B- Collagenase Lysozyme ALP
glycerophosphat
ase
Cathepsin Gelatinase Acetyltrans Vesicle-
ferase Associated
membrane 2
Defensin Lactoferrin B2- CD10, CD13,
 Diapedesis Microglobu CD14, CD16

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LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 lin  Colorless cytoplasm
Elastase Neutrophil - Cytochrome  Do NOT contain hydrolytic enzymes
Gelatinase- D558
Associated BASOPHIL FUNCTION
Protein  Responds to adrenal corticosteroids
Proteinase-3 Others - C1q receptor  Immediate hypersensitivity
Others - - Complement o IgE + basophil: degranulation
receptor 1  Delayed hypersensitivity reaction
o Cutaneous basophil hypersensitivity
 Other Functions:
o For IgE synthesis
o Angiogenesis: repair blood vessel due to production
of mitogenic factor
o Control helminth

EOSINOPHILS
 Myelocyte
o Stage first recognized
 Large granules
o Blue
o Has crystalloid “Charcot Leyden crystals” BASOPHIL VS. MAST CELL
o Major Basic Protein – basic > absorb red stain  Basophil is mistaken as Mast Cell because of its granular
o Color Development: Blue > olive green > deep orange appearance.
 MAST cells contain:
> red o Proteolytic Enzyme
 Most attractive granulocyte o Serotonin
 Parasitic infection
 Headphone appearance AGRANULOCYTES: Monocytes and Macrophages
 Promonocyte - earliest recognizable precursor of
EOSINOPHIL FUNCTION monocyte
 Degranulation
o Classical Exocytosis: ALL granules are released
o Compound Exocytosis: Granules COMBINE before
release
o Piecemeal: secretory vesicle release substance
 Regulation of Immune Response
 Indicator of Parasitic Infections
o ↑ eosinophils = ↑ parasitemia
 Hallmark of Allergic Disorders
MONOCYTE
s

 UNIQUE TO MONOCYTE MATURATION:

1. Presence of VACUOLES in both cytoplasm and
nucleus
2. Kidney-shaped appearance
3. Great Ground Glass Cytoplasm
4. Cytochemical staining - Specific for esterase
MONOCYTE FUNCTIONS
s

 Innate Immunity
 Adaptive Immunity
 Housekeeping Functions
BASOPHILS - act as scavengers
 Least in population -release growth factors affecting hematopoiesis
 ↑ = leukemia
AGRANULOCYTES: Lymphocytes
 Metamyelocyte – earliest recognizable stage
 Deep large basophilic
LYMPHOCYTE GROUPS
s

 T and B Cells
 “metachromatic” – accumulation of heparin, give color to
 Natural Killer Cells
basophil
 Have folded nucleus
 Bilobed, large granules that obscure nucleus

Page 10 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
LYMPHOCYTE INVOLVEMENT CATEGORIES
s

 Humoral Immunity - B cells are involved.

 Natural Killer Cells -T Cells and Natural Killer Cells are
involved.

PHASES OF DEVELOPMENT
s

 When exposed to antigen, Immature B Cells undergo

BLASTOGENESIS in the secondary lymphoid organs. It will
become a plasma cell or a memory cell.
 Blastogenesis: The completion and incurring of new and
specific function (plasma cell or memory cell) of the
immature B lymphocyte.

T LYMPHOCYTE DEVELOPMENT

 same with B Lymphocyte Development

1. Antigen Independent Lymphopoiesis Development
-the NORMAL production of lymphocytes in the bone SUMMARY OF T CELL MATURATION
marrow
-IMMATURE LYMPHOCYTE - able to exit the bone marrow.

2. Antigen Dependent Lymphopoiesis

-occurs when lymphocytes reach secondary lymphoid
organs
-In secondary lymphoid organs, there is exposure to antigen
-When it is exposed to antigen = BLASTOGENESIS
happens

B LYMPHOCYTE DEVELOPMENT

 CFU-L
Markers:
1. HLA (Human Leukocyte Antigen)
2. TDT (Terminal deoxynucleotidyl transferase)

 Pro B
- becomes Pre B

 Pre B
-earliest recognizable pre-cursor of B lymphocyte
Markers:
1. CALLA (common acute lymphoblastic leukemia
antigen) – replaced HLA
2. TDT – still present T CELL BLASTOGENESIS
 The majority of lymphocytes that are circulating in the body
 Immature B are T lymphocytes.
-exits the bone marrow  In Differential Count, 85% of lymphocytes counted are T
-goes to the secondary lymphoid organs to perform its Lymphocytes.
function  T cell becomes Reactive or Atypical Lymphocyte once it
- also known as Naïve cell - never been exposed to an undergoes blastogenesis.
antigen
-also known as Hematogones
Marker:
1. sIg (Surface Immunoglobulin)
-No CALLA and TDT Lymphocyte
Development

B CELL BLASTOGENESIS
s

NK Cells
Page 11 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
  Large granular lymphocyte
 NK Cells comes from T Cell.
Development
LYMPHOCYTE FUNCTION
o Volume = 3.2 mm3
T cells are quite unique because of the clusters of differentiation that  Total volume of Fuchs-Rosenthal Hemocytometer: 3.2
are found on the surface. mm3 x 2 (Because it has 2 ruled areas)
 CD4+ T lymphocytes

Speirs-Levy Hemocytometer

 CD8+ T lymphocytes

LYMPHOCYTE FUNCTION

T Lymphocytes

CD8+ Killing target cells

NK Cells Killing of certain tumor cells and  Has 4 ruled areas and each is comprised of 10
smaller squares
virally infected cells  1 square = 1x1 mm2
o Depth is 0.2
o Volume 2 mm3
 Total volume: 8 mm3 (2 mm3 x 4 areas)

CELL COUNTS Improved Neubauer Counting Chamber

Fuchs-Rosenthal Hemocytometer  Used in the laboratory

s  Has 2 ruled areas (with 19 squares)
 Has 2 ruled areas (Neubauer Counting Chamber  1 square
look-alike) o Volume = 0.12 mm3
 Each of the root area contains 16 large squares and  Total volume of 1 ruled area = 0.9 mm3
each will be divided into 16 smaller squares  Considered unique because of the presence of 3
 Total area: 16 mm2. boundary lines
 1 ruled area has a measurement of 16 mm2 o Useful in the utilization of the Inverted “L”
o Depth = 0.2 mm Rule
Page 12 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 HgCl2 (0.25g)

Distilled water (to 100mL)

Toisson’s Sodium Sulfate (8g)

Solution
NaCl (1g)

Methyl Violet (0.025g)

Glycerin 30mL

Distilled water (180mL)

Dacie’s Solution 40% Formalin (10mL)

3% w/v trisodium citrate (to 990mL)

 Depth of the chamber: 0.1  The diluting fluid for WBC count should be able to
 Has 4 corner squares divided into 16 squares lyse the red cell, hence we use of 2% acetic acid.
 Central square divided into 25 intermediate squares  For the red cell, the intention is to preserve the
and each is further divided into 16 smaller squares morphology of the right cell.
o Ideal diluting fluid: NSS
GENERAL PRINCIPLES
WHY DO WE PERFORM MANUAL CELL COUNT?

According to Henry’s (23rd edition)

 As a check on the validity of electronic methods

Sampling of diluted Counting for calibration purposes
Dilution suspension into of cells  As a check on the validity of electronic counts in
of blood measured volume patients with profound leukopenia or
in that
sample (CHARGING) thrombocytopenia
volume
 For blood specimens with platelet counting
interference (ie. Very microcytic RBCs)
o Severely microcytic red cell = platelet
count ↑
o Platelet clumps = platelet count ↓,
leukocyte count ↑ (because platelets are
DILUTING FLUIDS granulated and is now counted as
granulocytes)
 As a backup method
WBC Count
 It is also commonly used as a method for counting
2% Acetic Acid Glacial Acetic Acid cells in cerebrospinal fluid (CSF)

Distilled Water

Turk’s Solution 1% Aqueous gentian violet (1mL) -- INTENTIONALLY LEFT BLANK --

Glacial Acetic Acid (2mL)

Distilled water (to 100mL)

RBC Count
DILUTING PIPETTES
Gower’s Anhydrous sodium sulfate (12.5g)
Solution
Glacial Acetic Acid (33.3mL)

Distilled water (to 200mL)

Hayem’s Sodium Sulfate (2.5g)

Solution
NaCl (0.5g)

Page 13 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 ● Cells touching the innermost third line on the lower
and right grid SHOULD NOT BE INCLUDED (boxed
cells)

POISSON’S LAW OF DISTRIBUTION

● States that cells settle randomly.

● The allowable cell differences per square is 10 - 13 for
LPO and 15 - 20 for HPO.

ROUTINE COMPUTATION

WBC RBC Platelet

Count count Count

Dilution Factor 11-1 101-1 101-1

volume of fluid in bulb 0.5 0.5 0.5
 You may use any type of pipette to perform cell count. volume of sample used

ROUTINE DILUTION Volume Correction . 1 . . 1 . . 1 .

Factor (0.1)(4) (0.004)(5) (0.1)(4)
volume desired
WBC Count RBC Count Platelet Count volume used for counting

Blood up to 0.5 Blood up to 0.5 DF up to 0.5 mark

mark mark Blood up to 0.5 ● Note: REMEMBER THE FORMULA. There will be specific
mark problem sets for the exam instead of routine computations.
DF up to 11 mark DF up to 101 DF to 101 mark
mark LEUKOCYTE COUNT REFERENCE VALUES
 Platelet count is unique because of the need to
aspirate diluting fluid first (sodium citrate) because it
prevents the adhesion of platelet on the glass pipette Age Group Leukocyte Number
Concentration In SI Units
ROUTINE CELL COUNTS
Newborn 10.0 - 20.0
WBC Count RBC Count Platelet Count
Infants 4.0 - 15.0
Focus under LPO Focus under HPO Focus under HPO
Count in all Count in 5 Count in all Children 4.0 - 11.0
corner squares intermediate corner squares
squares
Adults 4.0 - 10.0

ABSOLUTE CELL COUNT = (cells counted) x VCF x DF

DIFFERENTIAL COUNT

THE INVERTED L RULE

● Prepare the peripheral blood smear, then, focus under

OIO, and lastly, count 100 WBCs (use the exaggerated
battlement method which is the ideal technique for
differential count).
○ Note on the Exaggerated Battlement Count: The
count starts at one edge of the smear and counting all
the cells, advancing inward to 1/3 of the width of the
smear, then on the line parallel to the edge, then out
of the edge, then along the edge.
● When performing dfferential count, choose the zone of
morphology. Pictographs may be useful in identifying the
cells counted

● Cells touching the outermost third line on the upper and

left grids should be INCLUDED (encircled cells)

Page 14 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 WBC is 30 x 10⁹ L (NV: 5-10
x 10⁹ L) and segmenter is
90% (NV: 65%).
Production of segmenter is
increased, thereby the total
WBC count also increased.
TECHNIQUES
s
Lymphocyte is 50 (NV: 45),
segmenter is 40, eosinophils
are 10, and WBC count is 6
x 10⁹ L.
Ratio is diferent from the
normal one because of an
increase in one or more
types of WBC but not
necessarily the whole WBC
count.

● Longitudinal Strip HW: What causes the differentiations in differential count?

● Crenellation Technique What leads to neutropenia, eosinophilia, etc.?
● Exaggerated Battlement Technique
● 2 or 3 Field Meander THE SCHILLING HEMOGRAM

● This arranging cells according to maturation sequence.

○ Most immature: Leftmost (myeloblast)
○ Most mature: Rightmost

SHIFT TO THE LEFT

s

● In differential count, a shift to the left means that there are

more band cells which is often ecountered in severe
infections or leukemia.
○ Regenerative: Cells are needed by the body hence the
left shift; advantageous
○ Degenerative: Low WBC count because of toxins
Regenerative Degenerative

High WBC count Low WBC count

REFERENCE RANGES
Rapid production of cells in Depression of leukopoietic
the BM centers
Age Cell Type
Group Acute need Irregularities in size and
shape;
Neu. Eo. Baso. Lym. Mono. decreased lymphoytes;
disappearance of
Newborn 55 - 65 2-4 0-1 30 - 25 3-6 eosinophils

Infants 40 - 48 2-5 0-1 40 - 48 5 - 10 Infection Toxins

Children 45 - 55 2-5 0-1 38 - 45 3-6

ARNETH COUNT
Adults 55 - 65 2-4 0-1 25 - 35 3-6 # of Segments Percentage

1 round, indented nucleus 5%

Absolute Increase Relative Increase
2 nuclear divisions 35%
True increase in the number Decline in the number of one
of cells cell type 3 nuclear divisions 41%

Example Example 4 nuclear divisions 17%

Page 15 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 5 or more nuclear divisions 2% Distilled Water (40 mL)

● Count 100 neutrophils and categorize them according to STEPS

s

the number of segments they have. ● WBC pipette: blood to 1 mark, DF to 11 mark
● It is emphasizing that as neutrophils mature, their ● Charge hemocytometer, stand for 15 mins to allow lysis of
segments increase. cells
● If there are 5 or more segments, it is bound to undergo ● LPO; Count cells in all 9 squares
pyknosis (if it hasn’t encountered any bacteria).
EOSINOPHIL COUNT
CORRECTED WBC COUNT
THORN TEST
● Fasting patient at 8 am: Eo count is 180/cumm
● Diurnal Trend
● 20% midmorning decrease below the 8 AM level
● 30% increase at night compared to the 8AM level
BASOPHIL COUNT
COPPER AND CRUICKSHANK METHOD
● It ia performed when high levels of nucleated RBCs are ● Based on hemocytometry
observed to get WBC count in SI unit. ● Fuchs-Rosenthal or Speirs-Levy Hemocytometer
● Increased nRBCs are normally demonstrated by ● NV for adults: 0-200/cumm
newborns and geriatric patients but adult patients
should have no nRBCs.
MEGAKARYOPOIESIS AND THROMBOPOIESIS
● Corrected WBC is performed when:
○ ≥ 5 nRBCs in adults
○ > 10 nRBCs in newborns or geriatric patients

ABSOLUTE LEUKOCYTE FRACTION COUNT

● Platelets
○ comes from CFU-GEMM which will develop into CFU-
MEG-E
● Same formula is also used for eosinophil. ○ “Kapag nag-lyse yung red cell mo sa Rees and Ecker prep
● ANC (x 10⁹ L) = WBC count in SI units x total neutrophil mo, alam mo na na wala ka na ring platelet kasi pareho
in decimal
sila ng leading(?) susceptibility.”
● Total neutrophils would includeboth segmenters and band
○ BFU-MEG to CFU-MEG undergoes mitosis
cells
○ LD-CFU-MEG
▪ beginning of terminal differentiation
EOSINOPHIL COUNT
● It is NOT routinely done. ▪ no longer performs mitosis
● Diluting Fluids: Randolph’s Solution, Pilot’s Solution ▪ undergoes endomitosis
▪ Ex. CFU-MEG undergoes mitosis producing 16 LD-
Pilot’s Solution CFU-MEG (terminal number which will become
megakaryocytes, does not undergo division
Propylene Glycol (50 mL) lyses RBCs (act as stain anymore)
transporter due to its ▪ Undergoes endomitosis
viscosity and does not ⮚ Division of the nucleus of the cell instead of
evaporate quickly)
the entire cell itself
○ Megakaryocyte
10% Aqueous Solution lyses all WBCs, othrr than
▪ Largest most mature cell in the bone marrow
Na2CO3 (1 mL) the base- resistant
eosinophils PLATELET QUANTITATION

Phloxine stains eosinophils granules LABORATORY METHODS FOR PLT COUNT

red
DIRECT: REES AND ECKER METHOD
Heparine (100 units) prevents clumping of white
● Gold standard
cells
● Brilliant Cresyl Blue
● Formalin

Page 16 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 PBS: PLATELET ESTIMATION PERIPHERAL BLOOD SMEAR REPORTING

AUTOMATION: MPV (COULTER)

● MPV
○ Mean platelet volume
○ 1/9(/?) of the red cells
○ Normal MCV: 80-100 fL
○ Normal MPV: 7.5-11.5 fL
○ Tests:
▪ Flow cytometry
▪ Electric impedance

PERIPHERAL BLOOD SMEAR ● Ex. Smear shows normocytic normochromic (size and
color) erythrocytes. WBC count is the reference range
with a differential count of:
PERIPHERAL BLOOD SMEAR PREPARATION ○ Size and color: based on MCV and MCH
▪ Normocytic: size of red cell is the same with
the size of nucleus of the small lymphocyte
▪ Normochromic: only ⅓ of the cell exhibits
central pallor
○ Note for the presence of anisocytosis, variation in
size and staining
○ Presence of poikilocytes
▪ Large number: single population
⮚ Few: <5%
⮚ Moderate: 6-25%
⮚ Many: >25%
▪ Small number: more than one population [Ex.
SS (smear shows) moderately microcytic
hypochromic red cells with moderate
poikolicytosis.]
⮚ Slight: 6-10%
⮚ Moderate: 11-15%
⮚ Marked: >15%
● Ex. WBC count is above the reference range with a
differential count of:
○ Focus under HPO, obtain count in 10 fields, average
then multiply by two (Ex. WBC ct in 10 fields = 200,
200/10 (fields) = 20 x 2 = 40x109)
○ Reference range: 5-10x109
○ Differential count:
▪ Reported in percentage
● Ex. Platelets are above in number:
● More than ½ of the slide
○ Focus under OIO, obtain count in 10 fields, average
● NOT too thin or too thick
(Ex. PLT in 10 fields = 70, 70/10 (fields) = 7
● Staining should not have any irregularities
○ Decreased: <6
● Should have a feathery edge
○ Adequate: 8-20
○ When focused under OIO, locate the zone of
○ Increased: >25
morphology and write the report for PBS
● Ex. Atypical/immature cells are present/seen or
absent/not seen.
PERIPHERAL BLOOD SMEAR EVALUATION ○ Present: immature cells are included in the
differential count.
● Ex. Smear is positive/negative for malarial parasites.
o Positive: identify the genus, species, and form.

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LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB
 Page 18 of 18
LEJAT, M.L., DIAZ, D., IRA, S., SIORES, G., LACASTE, G., JUAREZ, J., LIBAO, R., LAPORGA, J. ~ SLU CLIN LAB