FUNDAMENTAL HEMATOLOGY PRINCIPLES

BLOOD COMPOSITION
1. Whole blood includes:
A. erythrocytes
B. leukocytes
C. platelets
D. plasma.
2. When a specimen is centrifuged, leukocytes and platelets make up the buffy coat (small white
layer of cells lying between the packed red blood cells and the plasma).

Plasma/serum
Buffy coat
Packed RBC

3. Plasma is the liquid portion of one clotted blood. Serum is the fluid that remains after
coagulation has occurred and a clot has formed.
A. Plasma is composed of 90% water and contains proteins, enzymes, hormones, lipids and
salts.
B. Plasma normally appears hazy and pale yellow (contains all coagulation proteins) and serum
normally appears clear and straw colored (lacks fibrinogen group coagulation proteins).

BASIC HEMATOLOGY TERMINOLOGY

a- Without micro- Small
-blast Youngest/nucleated myelo- Marrow
-chromic Colored normo- Normal
-cyte Cell -oid Like
dys- Abnormal -osis Increased
-emia In the blood pan- All
ferro- Iron -penia Decreased
hyper- Increased -plasia Formation
hypo- Decreased -poiesis Cell production
iso- Equal poly- Many
macro- Large pro- Before
mega- Very large/huge thrombo- Clot

FORMED ELEMENTS AND SIZES

FORMED ELEMENTS SIZE
Thrombocytes (platelets) 2-4um
Erythrocytes (RBCs) 6-8um
Normal lymphocytes 6-9um
Reactive lymphocytes 10-22um
Basophils 10-15um
Segmented neutrophils 10-15um
Band neutrophils 10-15um
Eosinophils 12-16um
Monocytes 12-20 um

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BASIC HOMEOSTASIS
1. Homeostasis is the bodys tendency to move toward physiological stability. In vitro testing of
blood and other body fluids must replicate exact environmental body conditions. These
conditions should include the following:

A. Osmotic concentration is the body/cellular water concentration composed of 0.85%

sodium chloride. This normal osmotic concentration relationship to lesser of amount of
solutes), water enters the cell; the cell swells and may lyse. In a hypertonic solution
(lesser amount of H2O in relationship to greater amount of solutes), water leaves the
cell; the cell may crenate.

Osmosis is the basic principle underlying the previously popular erythrocyte

fragility test.

Osmotic Fragility Test

- A measurement of the resistance of erythrocytes to hemolysis in hypotonic saline
solutions (usually ranging from 0.85-0.10% sodium chloride)
- Results:
a. Normal erythrocytes show initial hemolysis at concentrations of 0.45-0.39%
and complete hemolysis at 0.33-0.30
b. Hereditary spherocytosis, the fragility of the erythrocytes is markedly
increased
c. Thalassemia, sickle cell anemia, and obstructive jaundice the fragility of the
erythrocytes is usually reduced

B. pH reference range: venous blood range 7.36-7.41; arterial blood range 7.38 7.44.

C. Temperature: Normal body temperature is 37.0C. Blood specimens should be analyzed

as soon as possible to prevent cellular breakdown (refer to individual tests for specimen
collection requirements, stability times and storage temperature).

RBC INDICES
-The erythrocyte indices are used to mathematically define cell size and the concentration of
hemoglobin within the cell.
They are:
1. MCV
2. MCH
3. MCHC

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 1. MCV (mean corpuscular volume)
Reference range (SI/conventional units) is 80-100 femtoliters (fL)
It is an indicator of the average/mean volume of erythrocytes (RBCs). Calculate using the
hematocrit (Hct) and RBC count:
Hct (%) x 10
MCV in fL RBC count (x 1012 /L)

Example: If the patients hematocrit is 35%, or 0.35L/L, and the erythrocyte count is 4.0 x 1012/L, the MCV
is determined thus:
Answer:

a. increased in the following conditions:

1. megaloblastic anemia
2. hemolytic anemia with reticulocytosis
3. liver disease
4. normal newborn

b. decreased in the following conditions:

1. iron deficiency anemia
2. thalassemia
3. sideroblastic anemia
4. lead poisoning

2. MCH (mean corpuscular hemoglobin)

Reference range (SI/conventional units) is 26-34 picograms (pg)
It is directly proportional to the amount of hemoglobin of the erythrocyte.
It is an indicator of the average weight of hemoglobin in individual RBCs. Calculate using the
hemoglobin (Hgb) and RBC count:

Hgb (g/dL) x 10
MCH in pg RBC count (x 1012 /L)

Example: If the patients hemoglobin is 14 g/dL and the erythrocyte count is 4 x 1012, the MCH would
equal to:
Answer:

a. Increased in microcytic anemia

b. Decreased in microcytic, hypochromic anemia

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 3. MCHC (mean corpuscular hemoglobin concentration)
Reference range (conventional is 32-37g/dl, (SI units 320-370g/L)
It is a measure of the average concentration of hemoglobin in grams per deciliter
Calculate using the hemoglobin in grams per deciliter.
Calculate using the hemoglobin and hematocrit values:

MCHC Hemoglobin (g/dL) x 100

Hematocrit

Example: If the patients hemoglobin is 14 g/dL and the hematocrit is 0.45L/L, the MCHC would equal to:
Answer:

a. 32-37g/dL MCHC indicates normochromic RBCs

b. Lesser than (<) 32g/dL MCHC indicates hypochromic RBCs, which is seen in iron
deficiency and thalassemia
c. Greater than (>) 37g/dL MCHC indicates a possible error in RBC or hemoglobin
measurement or the presence of Spherocytes.

OTHER RBC PARAMETERS

1. RDW (RBC distribution width):
reference range (conventional units) is 11.5 14.5%
Determined from the RBC histogram
Increased proportional to the degree of anisocytosis (variation in size); coefficient of variation
of the mean corpuscular volume
High RDW is seen in the following condition:
1. post-transfusion
2. post-treatment (e.g. iron, B12 or folic acid therapy);
3. idiopathic sideroblastic anemia
4. in the presence of two concurrent deficiencies (iron and folic acid deficiencies)

2. Hct (hematocrit)
Reference range for males (conventional units) is 41-53% (SI units 0.41- 0.53L/L)
Reference range for females (conventional units) is 36-46% (SI units 0.36-0.46L/L)
Reference range for hematocrit is age and sex dependent
Hematocrit is the percentage of RBCs in a given volume of whole blood
Note!
a. Spun microhematocrit is the reference manual method
b. The buffy coat layer of leukocytes and platelets is not included in the measurement, can
be seen, between plasma (upper) and RBC (lower) layers
c. Hematocrit is calculated by many automated cell counters using the MCV and RBC
count:

MCV (fL) x RBC count (x 1012/L)

Hct (%) 10

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 3. Hgb (Hemoglobin)
Reference range for males (conventional units) is 13.5-17.5 g/dL (SI units 135-175g/L).
Reference range for females (conventional units) is 12.0-16.0g/dL (SI units 120- 160 g/L).
Reference range for hemoglobin is age and sex dependent.

PLATELETS
1. PLT (Platelets)
Reference (SI units) is 150-450 x 109 (conventional units 150,000 - 450,000/uL).
2. MPV (mean platelet volume)
Reference range (SI/conventional units) is 6.8-10.2 fL
MPV is analogous to the MCV for erythrocytes

RELATIVE AND ABSOLUTE BLOOD CELL COUNTS

Relative count
The amount of a cell type in relation to other blood components.
relative lymphocytosis is an increase in the percentage of lymphocytes
this is frequently associated with neutropenia
In relative polycythemia, RBCs appear increased due to a decreased plasma volume

Absolute count
The number of each cell type without respect to other blood components.
Absolute lymphocytosis is a true increase in the number of lymphocytes.
Absolute polycythemia is a true increase in red cell mass.

HEMATOLOGY STAINS
1. Non vital (dead cells) polychrome stain (Romanowsky)
a. Most commonly used routine-peripheral blood smear stain is Wright's stain.
b. Wrights stain contain methylene blue a basic dye, which stains acidic cellular components
(DNA and RNA) blue and eosin an acidic dye, which stains basic components
(hemoglobin and eosinophilic cytoplasmic granules) red orange.
c. Methanol fixative is used in the staining process to fix the cells to the slide.
d. Staining does not begin until a phosphate buffer (pH between 6.4 and 6.8) is added.

Note!
Causes of RBCs too red and WBC nuclei poorly stained:
1. Buffer or stain below pH 6.4
2. Excess buffer
3. Decreased staining time
4. Increased washing time
5. Thin smear
6. Expired stains

Causes of RBCs and WBC nuclei too blue:

1. Buffer or stain above pH 6.8
2. Too little buffer
3. Increased staining time
4. Poor washing
5. Thick smear
6. Increased protein
7. Heparinized blood sample

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 e. Examples of polychrome stains include:
Wrights stain Jenner Stain
Giemsas stain May-Grunwald Stain
Leishman stain

2. Non vital monochrome stain

a. Stains specific cellular components
b. Prussian blue stain is an example
Contains potassium ferrocyanide, HCl and a safranin counterstain
Used to visualize the following:
1. Iron granules in RBCs (siderotic iron granules)
2. Histiocytes
3. Urine epithelial cells

3. Supravital (living cell) monochrome stain

a. Used to stain specific cellular components
b. No fixatives are used in the staining process
c. Includes:
1. New methylene blue
used to precipitate RNA reticulocytes; measure of bone marrow erythropoiesis

2. Neutral red with brilliant cresyl green

as a counterstain is used to visualize Heinz bodies
Note! Clinical disorders associated with Heinz bodies include:
1. G6PD deficiency
2. and other unstable hemoglobin disorders

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 HEMATOPOIESIS
HEMATOPOIESIS
Production and differentiation of blood cells
Blood cell production, maturation and death occur in organs of the reticulo-endothelial system
(RES)

a. RES includes:
1. bone marrow
2. spleen
3. liver
4. thymus
5. lymph nodes

b. RES functions :
1. hematopoiesis
2. phagocytosis
3. immune defense

INTRAUTERINE HEMATOPOIESIS INCLUDES THREE PHASES:

1. Mesoblastic (yolk sac) phase

begins at 19 days gestation
The yolk sac is located outside the developing embryo.
The first cell to be produced is a primitive nucleated erythroblast. This cell produces
embryonic hemoglobins:
Portland
Gower I
Gower II
Alpha-globin chain production begins at this phase and continues throughout life.

Note!
NORMAL HEMOGLOBINS

Hemoglobiin Composition
Embryonic
Gower I 2 Zeta + 2 Epsilon
Gower II 2 Alpha + 2 Epsilon
Portland 2 Zeta + 2 Gamma
Birth
Hb F 2 Alpha + 2 Gamma (60%-90%)
Hb A 2 Alpha + 2 Beta (10%-40%)
Adult
HbF 2 Alpha + 2 Gamma (1%-2%)
Hb A2 2 Alpha + 2 Delta (<3.5%)
Hb A 2 Alpha + 2 Beta (95%)

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2. Hepatic (liver) phase
begins at 6 weeks gestation with production of mainly red blood cells, but also granulocytes,
monocytes and megakaryocytes.
Alpha and gamma globin chain production dominates forming Hgb F; detectable Hgb A and A2 are
also present.

3. Myeloid/medullary phase
begins around the fifth month of gestation, with the bone marrow producing mainly granulocytes.
The M:E (Myeloid:Erythroid) ratio approaches the adult level 3:1.
Alpha- and gamma-globin chain production predominates at birth, forming Hgb F; Hgb A and A2,
are also present. Hgb A will not predominate until 6 months of age when the gamma- beta globin
chain switch is complete.

Note!
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.

PEDIATRIC AND ADULT HEMATOPOIESIS

1. Bone marrow
a. Newborn
80-90% of bone marrow is active red marrow
b. Young adult (age 20):
60% of bone marrow is active
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; 60% is fat
d. Cellularity
is the ratio of marrow cells to fat (red marrow/yellow marrow) and is described in
adults as:
1) Normocellular - marrow has 30-70% hematopoietic cells.
2) Hypercellular/ hyperplastic - marrow has >70% hematopoietic cells
3) Hypocellular/ hypoplastic - marrow has <30% hematopoietic cells
4) 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.
A normal ratio is between 3:1 and 4:1.
Granulocytes are more numerous because of their short survival (1-2 days) as
compared to erythrocytes with a 120 days life span. Lymphocytes and monocytes are
excluded from the M:E ratio.

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STEM CELL THEORY
Hematopoiesis involves the production of pluripotential stem cells that develop into committed
progenitor cells (lymphoid or myeloid) and finally mature blood cells.

Progenitor cells
1. Lymphoid: differentiate into either B or T lymphocytes in response to cytokines
lymphokines/CSFs/ growth factors.

2. Myeloid: gives rise to the multipotential progenitor CFU-GEMM (colony forming-unit-

granulocyte-erythrocyte-macrophage-megakaryocyte), which will
differentiate into committed progenitor cells and finally mature blood cells
in response to cytokines/interleukins/colony stimulating factors/growth
factors:

TYPES OF HUMAN STEM CELLS

1. Totipotential Stem Cells
Present in the first few hours after an ovum is fertilized.
Most versatile of stem cell, can develop into any human cell type.
May develop into fetus.
2. Pluripotential Stem Cell
Present several days after fertilization.
Can develop into any type of cell except fetus.
3. Multipotential Stem Cell
Derived from pluripotent stem cells .
Found in adults and limited to specific types of cells
Example:
a. Bone Marrow Stem Cell All types of bloods cells, adipose cells and bone cartilage

CELLULAR ELEMENTS OF BONE MARROW

1. Progenitor Blood Cells
Multipotential Hematopoietic Stem Cell Progenitor of all blood cells

3 PHASES OF CELL MATURITY

1. Premitive, multipotential cells
most immature group
capable of self-renewal and differentiation into all blood cell lines
2. Intermediate cells
consist of committed progenitor cells destined to develop into distinct cell lines
3. Mature cells
the most developed group with specific functions

HEMATOPOIETIC GROWTH FACTORS

Regulating the proliferation and differentiation of Hematopoietic Progenitor Cells (HPC).
Regulates the survival and function of mature blood cells.
Mediated through specific binding receptors on the surface of target cells.

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 Committed Progenitor Cell Growth factors or Interleukins Mature cell
CFU-MEG Thrombopoietin, GM-CSF Thrombocytes
CFU-GM CFU-M GM-CSF, M-CSF, IL-3 Monocytes
CFU-GM CFU-G GM-CSF, G-CSF, IL-3 Neutrophils
BFU-E CFU-E Erythropoietin, GM-CSF, IL-3 Erythrocyte
CFU-Eo GM-CSF, IL-3,IL-5 Eosinophils
CFU-Ba IL-3, IL-4 Basophils

LYMPHOID TISSUE
a. Primary lymphoid tissue
1) Bone marrow: site of pre-B cell differentiation
2) Thymus: site of pre-T cell differentiation
3) This is antigen-independent lymphopoiesis
b. Secondary lymphoid tissue
1) B and T lymphocytes enter the blood and populate secondary lymphoid tissue, where
antigen contact occurs
2) Includes lymph nodes, spleen, gut-associated tissue (Peyers patches)
3) Antigen-dependent lymphopoiesis depends on antigenic stimulation of T and B
lymphocytes
CD markers are surface proteins expressed by specific cell lines at different maturation stages. As a cell
matures, some markers vanish and new ones appear. More than 200 CD markers have been identified.
Commonly used markers include the following:

CD2, CD3 Lymphoid, pan T cells

CD4 Helper/inducer T cells
CD 8 Suppressor/cytotoxic T cells
CD13 Pan myeloid
CD11c, CD14 Monocytes
CD19, CD20 Lymphoid, pan B cells
CD33 Pan myeloid cells
CD34 Stem cell marker/lymphoid and myeloid
precursor)
CD16, CD56 NK cells

MEDULLARY VERSUS EXTRAMEDULLARY HEMATOPOIESIS

1. Medullary hematopoiesis
Blood cell production within the bone marrow
Begins in the fifth month of gestation and continues throughout life

2. Extramedullary hematopoiesis
Blood cell production outside the bone marrow
a. Occurs when the bone marrow cannot meet body requirements such as:
1. Hemolytic anemias
2. Aplastic anemia
3. Infiltration of malignant cells or over proliferation of a cell line (e.g. leukemia)

b. Occurs mainly in the-liver and spleen; hepatomegaly and/or splenomegaly often

accompany this

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GENERAL CELL MATURATION CHARACTERISTIC FOR ERYTHROCYTES
ERYTHROPOIESIS
Is a process by which erythroid precursor cells differentiate to become mature RBC.
ERYTHROPOIETIN is the primary regulator of this process.
It normally takes 3 to 5 days for the production of reticulocytes from pronormoblast.
The reticulocytes remain in the bone marrow for 1 to 2 days before being released in the circulation.
The reticulocyte continues to mature for one more day in the circulation

ERYTHROCYTES
A. General Characteristic
1. Oxygen transport, removal of metabolic waste
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 erythrocytes production from myeloid progenitor cells;
3. Influences colony- forming unit-erythrocytes (CPU-Es) to differentiate into erythroblasts.

C. Erythrocyte Maturation
1. Pronormoblast (rubriblast)
a. Earliest RBC
b. 14-20 um diameter
c. Deeply basophilic cytoplasm
d. Non-granular
e. N/C ratio is about 8:1
f. Fine chromatin
g. Usually 1-2 nucleoli

2. Basophilic normoblast(prorubricyte)
a. Size up to 16um diameter
b. N:C ratio of 6:1
c. Centrally located nucleus with 0-1 nucleoli
d. Chromatin slightly coarse
e. Cytoplasm, is less blue but intensely-basophilic (RNA)
f. Nucleoli are usually not visible

3. Polychromatophilic normoblast (rubricyte)

a. Size up to 10-15 um diameter (12um)
b. N:C ratio of 4:1
c. Eccentric nucleus with no nucleoli
d. Chromatin shows significant clumping
a. Begins to produce hemoglobin, resulting in
reddish-blue cytoplasm

4. Orthochromatic normoblast (metarubricyte)

a. Size up to 7-12 um diameter (10um)
b. N:C ratio of 0.5: 1
c. Pink cytoplasm
d. Eccentric nucleus
e. Fully condensed (pyknotic) nucleus; no nucleoli
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 f. Pale blue to salmon cytoplasm
g. Hemoglobin synthesis decreases

5. Reticulocyte
a. Size up to 7-10 um diameter
b. A reticulocyte contains no nucleus but has mitochondrial and
Ribosomes
c. Last stage to synthesize hemoglobin
d. Last stage in bone marrow before release to the blood
e. Reference ranges are 0.5-1.5% for adults and
2.5-6.5% for newborns, with slightly increased ranges at higher altitudes
f. A supravital stain is used to enumerate reticulocyte
g. Reticulocyte count is one of the best mediators of bone marrow function
h. Stress reticulocytes are young cells released from bone marrow after older reticulocytes
have been released. This is a response to increased need.
i. Hemoglobin continues to be produced by reticulocytes for approximately 24 hours after
exiting the bone marrow.

6. Mature Erythrocyte
a. Size range to 6-8um
b. Round, biconcave discocyte
c. Salmon with central pallor (clearing in the center) when a
blood smear is Wright's stained
normal cells have a central pallor that is-one-third the
diameter of the cell
decreased central pallor is seen with:
1. spherocytic disorders, including
2. thermal injury
3. liver disease
d. Central pallor greater than one-third the diameter of the cell is seen:
1. microcytic anemias

e. RBC reference ranges in SI units:

Females: 4.0-5.4 x 1012/L (conventional units 4.0-5.4 x 106/uL)
Males: 4.6-6.0 x I012/L (conventional units 4.6-6.0 x 106/L)

Note!
Erythropoiesis is regulated by erythropoietin produced in the kidney. Additional regulation includes:
a. Hypoxia due:
1. high altitude
2. heart or lung dysfunction
3. anemia
b. Androgens (male hormones that appear to enhance the activity of erythropoietin)
c. Hemolytic anemias (increased erythrocyte destruction)

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ERYTHROCYTE PHYSIOLOGY
1. Early RBCs get energy from oxidative phosphorylation. During maturation, the mitochondria
are lost, and energy is derived from glycolysis.

Metabolism of Red Blood Cells

a. Embden Meyerhof Pathway
90% glycolysis
Anaerobe
For every glucose breakdown to lactic acid = 2 ATPs
ATP controls flow of Potassium and Sodium
Protects membrane Lipids

b. Hexose Monophosphate Shunt

Pentase Phosphate Pathways
10% glycolysis
Provide reduced glutathione to prevent denaturation of hemoglobin

c. Rapoport Leubering Pathway

Generates 2,3-DPG that regulates hemoglobin affinity to oxygen
Note!
1. Decreased 2,3 DPG = Increased Hb affinity for oxygen
2. Increased 2,3 DPG = Decreased Hb affinity for oxygen

d. Methemoglobin Reductase Pathway

Maintain iron in ferrous state

2. Erythrocytes need proper volume ratio for exchange of blood gases 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 composed of
a. 50-60% (phospholipids, cholesterol, and glycolipids)
b. 40-50% protein.

SUBSTANCES NEEDED FOR ERYTHROPOIESIS

1. Iron: must be in the ferrous state to transport oxygen
2. Amino acids: globin chain synthesis
3. Folic acid/vitamin B12: DNA replication/cell division
4. Others: erythropoietin, vitamin B6 (pyridoxine), trace minerals

GENERAL CELL MATURATION CHARACTERISTIC FOR LEUKOCYTES

INTRODUCTION TO LEUKOCYTES
1. Classified as phagocytes (granulocytes, monocytes) or immunocytes (lymphocytes, plasma
cells and monocytes)
2. WBC reference range:
a. 4.0-11.0 x 109/L (SI units)
b. 4.0-11.0 x 103/uL (conventional units)
3. Granulocytes include
a. Neutrophils
b. Eosinophils

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 c. Basophil
4. Neutrophils are the first to reach the tissues and phagocytize (destroy) bacteria. In the
process, they die.
5. Monocytes differentiate into macrophages, and such they work in the tissues to phagocytize
foreign bodies. They arrive at the site of inflammation after neutrophils and do not die in the
process.
6. T lymphocytes provide cellular immunity. They represent 80% of lymphocytes in the blood.
When activated, they proliferate and produce cytokines/interleukins
7. B lymphocytes develop into plasma cells in tissue and produce antibodies needed for humoral
immunity. B lymphocytes represent (20% of the lymphocytes in the blood).
8. NK (natural killer) lymphocytes destroy tumor cells and cells infected with viruses. They are
also known as large granular lymphocytes LGLs.
9. Eosinophils modulate the allergic response caused by basophil degranulation.
10. Basophils mediate immediate hypersensitivity reactions (type 1 anaphylactic)

MATURATION CHARACTERISTICS OF LEUKOCYTE

Immature Cells Mature Cells

Cell is large Cell becomes smaller
Nucleoli preset Nucleoli absent
Chromatin line and delicate Chromatin coarse and clumped
Nucleus round Nucleus round, lobulated or segmented
Cytoplasm dark blue (rich in RNA) Cytoplasm light blue (less RNA)
High N:C ratio Low N:C ratio

GRANULOCYTES
BASIC REVIEW
The myeloid progenitor cell gives rise to a committed progenitor cell that is acted on by growth
factors to form granulocytes.

MATURATION AND MORPHOLOGY OF IMMATURE GRANULOCYTES

1. Myeloblast: earliest recognizable granulocyte precursor

b. 14-20 f um
c. NC ratio 4:1-7:1
d. Round/oval nucleus with fine reddish-purple
staining chromatin
e. 2-5 nucleoli
f. No cytoplasmic granules
g. Dark blue cytoplasm
h. 1% of the nucleated cells in the bone marrow

3. Promyelocyte
a. 15-21 um
b. NC ratio 3:1
c. Round/oval nucleus with slightly coarsening chromatin
d. 1-3 nucleoli
e. Dark blue cytoplasm
f. Cytoplasmhas large, non-specific/primary granules

14
 containing myeloperoxidase
g. 2-5% of the nucleated cells in the bone marrow

4. Myelocyte: first stage where granulocyte types can be

differentiated into eosinophils, basophils and neutrophils
a. 12-18 um in diameter
b. NC 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 loacated in the cytoplasm next to the nucleus
g. Cytoplasm has specific/secondary granules that contain hydrolytic enzymes, including
alkaline phosphatase and lysozyme
h. Non specific/primary granules are present and may still stain
i. Last stage capable of cell division
j. Neutrophilic myelocyte makes up 13% of the nucleated cells in the bone marrow.

5. Metamyelocyte
a. 10-18 um
b. NC 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.
f. Non specific/primary granules arc present but do not stain
g. Neutrophilic metamyelocyte make up 16% of nucleated cells in the bone marrow

6. Band
a. 10-15 um
b. NC ratio 1:2
c. Nucleus is C or S-shaped with coarse, clumped
chromatin lacking segmentation
d. Nuclear indent is greater than half the width of
a hypothetical round nucleus
e. Cytoplasm is pink and filled with pale blue to pink
specific/secondary granules
f. Non specific/primary granules are present but do not dually stain
g. Band neutrophils make up 12%of the nucleated cells in the bone marrow and 0-5% of the
peripheral white blood cells (WBCs).
h. Stored in the bone marrow and released when there is an increased demand for neutrophils
.

MORPHOLOGY OF MATURE GRANULOCYTES

7. A. Segmented neutrophill
(referred to as: seg, polymorphonuclear cell [PMN])
a. 10-15 um
b. NC 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
15
 specific/secondary granules
e. Non specific/primary granules are present but usually do not stain unless in response to
infection or growth factor
f. Segmented neutrophil makes up 12% of the nucleated cells in the bone marrow and 50-
80% of peripheral WBCs

7. B. Eosinophil
a. Recognizable maturation stages include the eosinophilic
myelocyte, eosinophilic metamyelocyte; eosinophilic
band, and eosinophil (segmented form)
b. Eosinophils are 12-16 um
c. Nucleus is usually bilobed
d. Cytoplasm contains large, bright red-orange, secondary
granules that contain enzymes and proteins
e. Eosinophils make up less than 1% of the nucleated cells in the bone marrow and 5% of
the peripheral WBCs

7. C. Basophil
a. Recognizable maturation stages include the basophilic
myelocyte, basophilic metamyelocyte, basophilic band
and basophil (segmented form)
b. Basophils are 10-15 um
c. Cytoplasm contains heparin and histamine
d. 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.
e. Basophils make up less than. 0.1% of the nucleated cells in the bone marrow and
peripheral blood

GRANULOCYTE FUNCTION
NEUTROPHILS
1. Blast, promyelocytes and myelocytes are in the bone marrow mitotic pool, 3-6 days and that is
where they divide
2. Metamyelocyte, bands and segmented neutrophils are in the bone marrow post-mitotic pool
about 6 days, and that is where they mature
3. Released into the circulation when matured or when needed
4. Total blood granulocyte pool
a. Contains 50%circulating granulocyte pool (mainly neutrophils) that is measured when WBC
count is performed
b. Contains 50% marginating pool (mainly neutrophils) that adheres to the vessel walls
c. There is a rapid and free exchange of neutrophils between the circulating and marginating
granulocyte pool
d. Neutrophils diapedese into the tissues from the marginating pool in response to antigenic
stimulation
e. Chemotactic factors attract the neutropil to the site of inflammation include complement,
bacterial products, injured tissue, hemotactic components
f. Opsonins such IgG and complement component C3b help neutrophils recognize a
substance as foreign
g. Phagocytosis involves neutrophil attachment to the foreign object formation of a vacuole
around it and neutrophilic degranulation to release lytic enzymes (respiratory burst) in
effort to kill the organism
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 h. Neutrophils are sensitive to oxidants they secrete and are destroyed in the process.

5. Blood and tissue cells in the body undergo cell death through necrosis or apoptosis:
a. Necrosis is induced by extracellular forces such as lethal chemical, biological or physical
events. The blood cell is killed

b. Apoptosis is programmed cell death due to extracellular or intracellular processes that

depend on a signal

EOSINOPHILS
1. Found in the blood, only a few hours before seeking a tissue site such as nasal passages, skin or
urinary tract
2. They can degranulate like neutrophils. They express Fc receptors for IgE, which is a response to
parasitic infection
3. They release substances that can neutralize products released by basophils and mast cells;
eosinophils modulate the allergic response.

BASOPHILS
1. Found in the blood only a few hours before migrating to the site of inflammation in the tissue
2. They express membrane receptors for IgE. Once activated, degranulation releases histamine.
This initiates the classic signs of immediate hypersensitivity reaction Type I
3. Basophils release a chemotactic factor that attracts eosinophil to the site

MONOCYTES
1. Monoblast
-12-20 um diameter
-Basophilic cytoplasm
-Non-granular
-N:C ratio is 4:1 to 3:1

2. Promyelocyte
-14-18 um diameter
-Blue-gray cytoplasm
-N:C ratio 3:1 to 2:1

3. Monocyte
-12-20 um diameter
-Blue-gray cytoplasm
-Many fine azurophilic granules
-Round kidney shaped nucleus, brain like convolutions
- No nucleoli are visible

LYMPHOCYTES
1. Lymphoblast
-10-18 um diameter
-Moderate to dark blue cytoplasm
-N:C ratio is 4:1
-1-2 nucleoli

2. Prolymphocyte
-May be the same size as the lymphoblast or smaller

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 3. Mature lymphocyte
-Small lymphocyte 8-10 um
-Medium lymphocyte 10-12 um
-Large lymphocyte 12-16 um

Note!
-T lymphocytes (60-80%) long lived (4-10 years)
-B lymphocytes (10-20%) short lived (3-4 days)
-Null lymphocyte (10% lymphoid population)

PLASMA CELLS
-Effector B cells are antibody producing cells
1. Plasmablast
2. Proplasmacyte
3. Plasmacyte/Plasma cell
-8-20 um diameter
-Cell shape may be oval
-Deeply basophilic cytoplasm
-Large well hof
-Eccentric nucleus
-Chromatin is condensed and coarse
-No nucleoli are visible

MATURATION OF PLATELETS
1. Maturation sequence of megakaryoblast takes about 5 days.
2. Platelets are produced directly from the megakaryocyte cytoplasm.
3. As the megakaryocyte matures, clusters of granules aggregate to form platelets

1. MEGAKARYOBLAST
a. 20-50 um diameter
b. Blue cytoplasm
c. N/C ratio is about 10:1
d. Multiple nucleoli
e. Fine chromatin

2. PROMEGAKARYOCYTE
a. 20-60 um diameter
b. Less basophilic cytoplasm
c. Chromatin becomes coarse
d. Irregularly shaped nucleus, may show slight lobulation
e. N/C ratio is 4:1 to 7:1

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3. MATURE MEGAKARYOCYTE
a. 40-120 UM diameter
b. Cytoplasm contains coarse clumps of granules aggregating
into little bundles which bud off from the periphery to
become platelets
c. Multiple nuclei are present
d. No nucleoli is visible
e. N/C ratio is less than 1:1

4. PLATELETS/THROMBOCYTE
a. 1-4 um diameter
b. Light blue to purple, very granular
*Chromomere granular and located centrally
*Hyalomere surrounds the chromomere, nongranular
and clear to light blue

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