HEMATOLOGY 1 -PRELIMS

b.He believed that disease occur from the disruption

Overview of Hematology of the balance between these humors

1.Safety in hematology 2.ATHANASIUS KIRCHER (1667)

2.Specimen collection and consideration a.Described “worms” is the blood
3.Basic componnents of blood Rouleaux formation
4.Hematopoiesis
5.Laboratory calculations of normal and
abnormal blood
6.Erythrocyte disorders
7.Leukocyte Disorders Blood stock of coin appearance due to
8.Automation and its principles utilize in the INCREASE protein concentration
hematological analyses
3.ANTON VAN LEEUWENHOEK
a. Microscopy
HEMATOLOGY AS A DISCIPLINE b. Morphology of RBC
1.Study of blood and it’s component in normal and
pathologic condtions and devlopment of disease 4.GIULO BIZZOZERO
relating to blood a.Described platelet as “petite plaques” aka little dirt

2.Entities involved in hema lab 5.JAMES HOMER WRIGHT

a. 1 MLS a.Develop the Right Stain (Romanowsky type stain)
b. 1 MLT open a new world of visual blood film examination
c. 1 Lab assistant through microscope
d. 1 Phlebotomist
e. 1 Physician Note:Romanowsky stain is not just a single stain but a
group of stains
3. Your role as a future MLS working in hematology • Ex:Wright Stain, Giemsa Stain, Wright-Giemsa
laboratory Stain, Leishman and Jenner
a. Establish or rule out a diagnosis
b. Confirm a physician clinical impression of 6.JOSEPH AND WALLACE COULTER
possible hematological disorder a.Patented the irst electronic counter used today on
c. Detect an unsuspected disorder automatic cell counts called cell counter
d. Monitor the effects off theraphy
e. Detect minimal residual disease following Coulter Principle- detection and measurement of
theraphy changes in electronic impedance

BRIEF HISTORYY OF HEMATOLOGY BASIC COMPONENTS OF BLOOD

1.HIPPOCRATES
1.Red blood Cells (erythrocyte)
a. Laid the foundation of hematology with the ffour
2.White blood cells(leukocytes)
humors
a. Basophil
i. Blood(haima) - 25%
b. Eosinophil
ii. Phlegm (phlegma) -25%
c.Neutrophil
iii. Black bile(melaniakhole) -25%
d.Lymphocyyte
iv. Yellow Bile (Xanthekole ) -25%
e.Monocyte

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 HEMATOLOGY 1 -PRELIMS
3.Platelts ii. Reflects the size/ volume on wright-stained
4.Plasma blood smear
iii. Uses hematocrit and hemoglobin values
PLASMA VS. SEERUM e.Mean Corposcular/Cell Hemoglobin
PLASMA SERUM Concentration
Straw yellow Strong yellow i. Expressed as g/dL or %
1.022 – 1.025 >1.025 ii. Reflects RBC staining intensit and amount of
With anticoagulat Without anticoagulant hemoglobin
FIBRINOGEN -helps in coagulation/ blood clotting iii. Uses RBC count and hemoglobin
f.Mean Corposcular Hemoglobin
RED BLOOD CELLS i. Expressed as picograms(pg)
1.Aka Erythrocyte ii. Reflect hemoglobin mass abd parallel with
2.Structure MCV
o Anucleate, biconcae, discoid with a reddish iii. Uses RBC count and hematocrit
protein known as hemoglobin
o Appear pink-red(under normal staining
MCH is directly proportional to MCV
procedures ; Recalls; salmon pink
-if they vary it is due to
o Diameter: 6-8 micro meter (7.2 micro meter)
✓ Lipemia
o Occupies 1/3 of the central structure
✓ Icteric
✓ Machine error
3. Measurement
a. Mixing aliquot of whole blood and 0.85%NSS g.Red Cell Distribution width
b. NSS is used because it matches the osmolality i. Degree o variation – anisocyytosis (variation of
of blood(isotonic solution) red cell size
c. Counting equipment- glass counting chamber Cyanmethemoglobin method
aka Hemacytometer or Hemocytometer 540nm-Hemoglobin
(Imrpoved Neubauer Counting Chamber) 450-Bilirubin
i. Unit for reporting (cells per___)
ii. Microliter NORMAL VALUES
iii. Millimeter ✓ MCH- 27-32pg
iv. Liter ✓ MCV= 80-100fl
4.Increased:Polycytemia vera/ Pancytosis = ✓ MCHC= 32-36%
increased of all blood cells ✓ RDW= 11.5-14.5%
Decreased:Aplastic Anemia/ Pancytopenia :Tissue
Hypoxia (Hallmark of anemia) = a.In Romanowsky stain (using Wright stain )
decreased of all blood cells ➢ Name: Polychromatic erthrocyte
➢ Size: 7-8um
5.RBC Indices ➢ Color: salmon pink
a.Internal quality control b.In supravital stain
b. There are four indices ➢ Name; Reticulocyte
i. Aids in diagnosis of anemia(categorizes ➢ Identifying characteristics:presence of RNA
anemia) based on morphology networks inside cell
ii. Internal qc ➢ Color:Blue
c.described morphology of RBC in blood
d.Mean Corposcular /Cell Value(MCV)
i. Expressed as fL femtoliters

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WHITE BLOOD CELLS EOSINOPHILS

1.Aka Leukocytes a) Bright orange red cytoplasmic granules
2. Dedicated on protecting their host from infection b) Incresed:EOSINOPHILIA- allergic response or
and injury parasitic infection (main function)
3.Presence of Different orientations of nucleous and Non-Eosinophilic Helminths
granules ▪ D.latum
4.Source: Bone marrow and Thymus ▪ Trichinella
▪ Taenia
Movement of the WBC ▪ T
1.Random Chemotaxis- just moving ▪ E.vermicularis
2.Diapidesis- from peripheral blood to tissues
BASOPHIL
-main purpose is for allergic infection
WBC- Leukocytosis a) Dark purple
b) Cytoplasmic contains
WBC-Leukocytopenia ▪ Histamin
▪ Heparin
NEUTROPHIL
-special cells because of its folding nucleus LYMPHOCYTES
1.Aka Segmenter and Polymorphonuclear cells 1.humoral and cellular immunity
2.Phagocytosis 2.can be either B-lmphocyytes or T-lymphocytes and
▪ .Directly- suicide bomber (usinng primitive NK cells
receptors) 3.round featureless nuclei with cyytplams
▪ Indirectly- after being labeled by immune
system(immunoglobulins) Lymphocyte is the only IMMUNOCYTES
Basophil, Eosinophil, Neutrophil and Monocyte are
Note: Antibodies has no capabilities in killing called PHAGOCYTES
bacteria .It only have the capability of tagging to
be defeated .
NK cells- target tumor cells and virally infected cells
B cells – Producing antibodies
3.Increased- indicator of bacterial infection Note:B cells need to be activated and become plasma
4.Decreased- due to drug infection or viral infection cells before becoming antibodies
5.Band Neutrophils T cells-
▪ Less mature neutrophil ❖ T cytotoxic cell (Tc)- tumoricidal and viricidal
▪ Good indicator of bacterial infection called left function
shift ❖ T supressor cell (Ts)- supressing all lmphatic
6.Cytoplasmic conditions action
▪ Enzymes: submicroscopic pink or lavander ❖ T regulatory cell(Tr)- regulate who activates
staining filled with bacterial secretions ❖ T helper cell(Th)- nagsasabi kung sino ang mag
1) Myeoloperoxidase aactivate
2) Gelatinase ❖ T delayed time hypersensitive cell (Tdth)-
3) Elastase allergic reaction
4) Lactoferrin

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5.Mean Platelet Volume
4.Increased-can be due to viral infections
5.Decrease- drug theraph or immunodeficiencyy Platelet count and MPV is indirectly proportional
to each other
LEUKEMIA- cancer of blood
❖ CHILDREN- Acute lymphoblastic leukemia
(childhood leukemia ) more deadly HEMATOPOEISIS
❖ ADULT- Chronic lymphoblastic leukemia (>65
yrs old ) M:E RATIO (AKA MYELOID TO ERYTHROID RATIO)
-Pertains to differential counting in bone marrow spx
MONOCYTE → MACROPHAGE (if settled in organs or
localizes )
Normal 2:1 to 4:1
-most abundant cell type
-Immature macrophages present in the circulation Average 3:1
1) Tissue- Histiocytes Infection 6:1
2) Kidneys- Mesengeal cells Leukemia 10:1 or 25:1
3) CNS- Glial cells
4) Liver- Kupffer cells 1.Shows the quantitative relationship between
5) Spleen- Litoral cells granulocytic precursors (myeloid) and erythroid
6) Skeletal muscle- Histiocytes precursors (progenitors of RBC). Thus monocytic
7) Skin- Langerhans cells precursors are not included
8) Placenta- Hofbauer cells
9) Bone- Osteoclast MYELOID: will develop to granulocytes
10) Lungs- Alveolar macrophage or Dust cells • Megakaryocytes and Monocytes are also from
11) Joints- Synovicyte cells the Myeloid cell line
ERYTHROID: will develop to mature RBC
PLATELETS
1.Aka Megakaryocyte 2.In differential, the recommended number of cells to
2.thrombosis or clot formation or control of be examined is at least 500 cells (preferably, 1, 000
hemostatis cells on 2 slides if the cells are not aplastic)
3.Structure
a) Size: 1-3 um NORMAL MARROW CELLS
b) Standard structure 1.Developing hematopoietic cells
i. Hyalomer- light microscopy i. Blood cells undergo several stages of
ii. Granulomer- electron microscopy development
▪ Peripheral zone ii. Development from one stage to the next is
▪ Submembranous zone NOT SUDDEN, so commonly the cell being
▪ Organelle zone studied may be between stages (when this
▪ Membranous zone occurs, the cell is usually given the name of
c) Ultrastructures the more mature stage)
4.Measurement iii. When reading a bone marrow specimen, we
Increased:THROMBOCYTOSIS- inflammation or see the morphologic appearance between 2
trauma stages.
Decrease: THROMBOCYTOPENIA – drug treatment iv. We classify it by the stage where it is normally
related seen

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Examples
OSTEOBLAST OSTEOCLAST
MEGAKARYOCYTES (MK-III)
Synthesize new bone Large, multinucleated
• Largest bone marrow cell (when not matrix (using calcium)
cells; bone detroyers
fragmented) Comet/ water bug 100 um or greater
• Diameter: 30-169 um appearance (shorter than
• Develops and fragments into a platelet megakaryocyte)
• Its cytoplasm is fragmented later on becomes Confused with plasma Confused with
the platelet cells Megakaryocytes

METAMYELOCYTES BONE MARROW

MAIN SITES OF ADULT HEMATOPOIESIS
• Aka Juvenile Cells
a. R – ribs
• Most common cells in the bone marrow
b. S – sternum, skull, scapula
• Not seen in peripheral blood c. V – vertbrae
• Immediate stage of mature granulocyte = d. P – pelvis, proximal ends of the long bones
band cell (its precursor is the 1. Blood Islands of the Yolk Salk
metamyelocytes) 2. Hepatic Stage (STL)
• Changes in intravascular hematopoiesis to
1.MACROPHAGES extravascular hematopoiesis
• Diameter: more than 30 um • spleen, liver, thymus (primary sites of
• Cytochemical staining characteristics is hematopoiesis)
stained by a Non-specific esterase 3. Mesenchymal Cells then travel from the liver to
▪ Alpha naphtyl acetate the bone marrow
▪ Alphae naphtyl butyrate esterase • Transition from hepatic to Medullary phase
▪ Used to stain the cytoplasm of What specific bones is active hematopoietically?
macrophages (always smaller than Children – all bone are active hematopietically
megakaryocytes) Adult – reach the peak of growth (older = shrinkage of
• Smaller than megakaryocyte bones)

MK III- or Megakaryocytes= 30-160um SITES OF BONE MARROW COLLECTION SITES

2.MAST CELLS a. P – posterior superior iliac crest (best site

• Diameter:12 -25 um for adults together with “b”)
• Tissue cell that is also causative with b. A – Anterior superior iliac crest (best site for
Chloroacetate esterase adults together with “b”)
• High number of granules in the cytoplasm c. S – sternum
• In peripheral blood, it can be d. A- anterior medial surface of the Tibia –
mistaken/confused with basophils best for infant/children
e. S- spinous process of vertebrae, ribs, and red
marrow containing bones

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2.DIFFERENTIATION
TECHNICALITIES OF BONE MARROW COLLECTION a.) Stochastic model
a.Bone marrow aspirate - Random differentiation and self-renewal (early
•Disturbs bone marrow architecture stages)
•Types of present and relative number b.)Instructive model
b.Bone marrow biopsy - Differentiation and self-renewal is influenced by
•Bone marrow is removed with intact structure the microenvironment of the bone marrow (later
and not disturbing the bone and architecture stages)
e.g. Cytokines/Growth factor influenced the cell
Additional notes to be a certain cell line
c.)Stochastic/Instructive model
1. Bone marrow allows smears should be retained in - The early decision for self-renewal and
10 yrs
differentiation is stochastic and the later stages
a. Extramedullary hematopoiesis: Blood cell of differentiation is instructive
production outside the bone marrow (occurs
when the bone maarow cannot meet he 3.MATURATION
requirements of the bodyto maintain
-progressive development of immature cells
homeosatis)
3 PHASES OF MATURITY
2. Occurs mainly in the liver and spleen
Stem Cells Most primitive; capable of
(characterized by hepatomegaly and
self-renewal
splenomegaly)
Precursor cells Immature but committed
HEMATOPOEITIC PROCESS progenitor cells
REGULATION OF HEMATOPOEISI
1) SELF-RENEWAL Mature Cells Most developed group
2) MATURATION with specific functions
3) DIFFERENTIATION
4) PROLIFERATION
5) APOPTOSIS General changes in the maturation is described in
1.SELF RENEWAL nuclear and cytoplasmic development
-ability to dublicate and replenish mitotic pool a.Changes in the Nuclear Stage
Mitotic pool: group of blood cells that undergo i. Loss of nucleoli
mitosis; immature because they primary function is to ii. Decrease in diameter of nucleus (leading to
proliferate pyknosis) Pyknotic nucleus = dead nucleus
i. Cells in mitotic pool preliferate , the first one iii. Changes in the structure of the nucleus
matures and second one goes back to being iv. Loss of nucleus (RBC)
HSC to replenish = ASYYMMETRIC b.Changes in the Cytoplasmic stage
ii. Cells divides and two of them go back to being i. Decrease in basophilia (loss of azurophilic
cell= SYMETIC capabilities, leading to acidic cytoplasm)
iii. Cell divides and one matures one goes *loss affinity to eosin
apoptosis= ASYMETRIC ii. Increase in the proportion of the cytoplasm
(due to nucleus shrinkage)
iii. Appearance of granules in the cytoplasm
(acidic due to granules)
iv. Fragmentation (Thrombocytes)

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TERMS
PLUROPOTENTIAL HEMATOPOIETIC STEM CELL: all
1. CFU = Colony Forming Unit
stem cell arises from here
2. BFU = Blast Forming Unit
• Pluripotential Hematopoietic Stem Cells can
3. CFU-GM (Granulocyte Monocyte)
be also called as Multipotential
4. CFU-MegE (Megakaryocyte Erythrocyte)
Hematopoietic Stem Cells
• Monophyletic theory: all cells come from
pluripotential hematopoietic stem cell. CYTOKINES AND GROWTH FACTORS
• If there is an influencer like erythropoietin or • Growth Factors are also called Colony-
thrombopoietin, PHSC will decide to go as a Stimulating Factors
Common Myeloid Progenitor. • Includes the different interleukins,
• If there is an influencer like (Colony- interferons, colony-stimulating factors,
Stimulating Factors) CSF-Agranulocyte, the hormones secreted by kidneys (erythropoietin
PHSC will pick the Common Lymphoid or thrombopoietin)
Progenitor
• Granulocytes have the same stages 1.KIT LIGAND OR FTL3 LIGAND

• In Precursor NK/T Cell, T-cells will go to a. Early acting multilineage growth factor – they
thymus first before it becomes fully matured. can have an interaction with multiple linkage
(can act with myeloid cell line and lymphoid
• Plasma cells are the ones who secrete cell line)
antibodies
b. Affects the earliest growth factors (colony
stimulating factors)

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c. Works synergistically with other growth factors c. Produced by T cells, endothelial cells,
(i.e., IL-3 and GM-CSF) fibroblasts, macrophages, and mast cells.
d. Activates indirectly by stimulating other
2.GM-CSF
cytokines.
a. Granulocyte Monocyte Colony Stimulating
Factor 6.il-5
b. Increases of neutrophils, monocytes, and a.Interleukin-5
eosinophils, and in activation of phagocytic b.Common seen in lymphoid series
function c.Activates cytotoxic T-cells.
c. Can be used to combat neutropenia; d.Induces immunoglobulin (Ig) secretion (it has
Increased release and stimulation may cause a function towards B-cells) and stimulates
myeloid hyperplasia (most of the cell will eosinophils.
become myeloid cells) e. B-cells stimulated by IL-5, it becomes a
d. A pan-myeloid growth factor that is used plasma cells, then the plasma cells release
clinically to combat neutropenia. Immunoglobulins
e. It will mostly interact with CFUs, common
myeloid progenitors and sometimes to ERYTHROPOIETIN (production of RBC)
common lymphoid progenitors a. It has a dominant effect on erythroid
precursors primarily on CFU-E, rubriblast,
prorubricyte and rubricyte.
3.M-CSF
• Rubriblast, prorubricyte and rubricyte
a. Monocyte/macrophage colony-stimulating are dividing/mitotic cells acted by
factor erythropoietin
b. Aka. Colony-stimulating factor 1 b. Primarily produced in the kidney and is
c. Works with production of monocytes and induced by tissue hypoxia.
macrophages c. Recombinant EPO are used to treat anemia,
d. Induces the production of the IL-1 renal failure, chemotherapy or bone marrow
infiltration by cancer.

4.G-CSF THROMBOPOIETIN
a. Granulocyte Colony-Stimulating Factor a. Primary regulator of thrombopoeisis
b. Weaker version of GM-CSF b. Produced by the liver, kidneys and bone
c. Stimulates granulocyte production and marrow stroma.
functional activation. c. Stimulation of megakaryocytes and
d. Used to treat neutropenia with less toxicity fragmentation into platelets.
Used to mobilize CD34+ stem cells in the
peripheral blood PRIMITIVE NON-COMITTED CELLS
Totipotential
5.IL-3 1. The most versatile cell type that can develop
a. Interleukin-3 into any kind of human cells including the
b. Activity is mostly analogous with GM-CSF but development of embryo into fetus.
on an earlier stage – it is also the one who 2. Versatility means that it can transform into any
instructs PHSC to become common myeloid cell type
progenitors.

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Pluripotential
1. Also, can develop into other cell type but 2.Maturation Process
cannot transform into another fetus. a. erythroid progenitor
Multipotential i. Blast forming unit and colony forming unit
1. Pluripotential Stem Cell (Rodak/Henry); ii. Both are commited to the cell line
Multipotential (Turgeon) Progenitor cells- Immature and cannot be
distinguised morphologically
2. Symmetric division:
Precursor cells- immature cells and can be
• 2 daughter cells produced by the HSC will differentiated morphologically
continue with differentiation.
3. Asymmetric division: PBPOPE
P-Pronormoblast
• 1 daughter cell will undergo self-renewal
B-Basophilic
• Another daughter cell will undergo P-Polychromatophilic
differentiation or apoptosis O-Orthochromatophilic
4. Hematopoietic Stem Cell: CD34 P-Polychromatoplhilic
5. All cells that are hematopoietically active are E-Erthrocytes
marked by CD34.
6. Most of the stem cells are marked by CD34 but NOMENCLATURE FOR NAMING ERYTHROID
has no CD38 – they only acquire it when they PRECURSORS
mature. Normoblastic Erythroblastic Rubriblastic
Nomenclature Nomenclature Nomenclature
Pronormoblast Proerythroblast Rubriblast
Basophilic Basophilic Prorubricyte
Normoblast Erythroblast
ERYTHROPOEISIS Polychromatophilic Polychromatophilic Rubricyte
Normoblast Polychromatophilic
Erythroblast
HSC Orthochromatophilic Orthochromatophilic Metarubricyte
Normoblast Erythroblast
Polychromatophilic Polychromatophilic Polychromatophilic
Erythrocyte* Erythrocyte* Erythrocyte*
CMP Erythrocyte Erythrocyte Erythrocyte

b.Erythroid Precursors
I.Criteria used in Identification of precursors
BFU-E
I. Overall Diameter: As the cell matures the
diameter decreases
II. Nuclear Diameter :Decrease more rapidly
COMMITED CELL CFU-E Maturation than the cell leading to reduces N:C ratio
III. Nuclear Chromatin Pattern: Becomes
coarser, clumped and condensed. Ultimately
Differentiation restriction- loss of capacity of the nucleus becomes quite condensed, with
differentiating as it become commited to erythrocyyte no parachromati evident at all, and the
cell line nucleus is said to be pyknotic

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HEMATOLOGY 1 -PRELIMS
IV. Nucleoli Disappear: As the cells matures, UNDERGOING
indicating the cessation of active protein MITOSIS
synthesis Metarubricyte • Nucleus is lighly
V. Cyytoplasmic changes: Transition from blue -48 hours condensed and
to pink due to change in acidic and basic described as
components pyknotic
• In the later perio of
this stage, the
RBC MATURATION SERIES
nucleus will be
Pronormoblast • Fine and uniform
extruded from cell
-1 division chromatin pattern
• Last stage with
-2 daughter cells and stains intensely
nucleus
-more than 24 hrs • Earliest
• PYRENOCYTE- the
recognizable RBC
nuclear body that
precursor in light
was expelled byy the
microscopy
developing
Prorubricyte • Slightly smaller than
metarubricyyte
-2 division rubriblast
phagocytosed by
-4 daughter cells • Nuclear chromatin
macrophage
-more than 24hrs becomes more (osteoclast)
clumped
Polychromatophilic • Part of this phase
• Last stage with
erythrocyte occurs in the bone,
nucleolus
-2-3 Days marroew, and the
• Cyytoplasm is less
later part of the
but intensel
stage takes place in
basophilic due to
the circulating blood
RNA (DEEP BLUE)
• Residual ribosomes,
Rubricyte • Hemoglobin
mitochondria and
-1 division appears for the first
other organelles are
-2 daughter cells time (The stained
removed in the splee
-30 hours color reflects the or are intensely
accumulation of dissolved
hemoglobin • In Supravital stain:
pigmentation over New methylene blue
time and concurrent
• In Wright stain:
decreasing amounts
of RNA
• Last stage capable
• Muddy, light grayy of hemoglobin
appearance of cell syynthesis
due to variable
amounts of pink • 2 days are spent in
coloration mixed
bone marrow and 1
with basophilia
in peripheral blood
• Detectable
(1 day spent in
hemoglobin syntesizing hemoglobin,
synthesis occurs 1 day for mobilzation
• LAST STAGE IN after that it stays 1 day in
WHICH THE CELL IS peripheral blood)
CAPABLE OF

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• Same color with
mature RBC Earliest recognizable
Mature Erythrocyte • > On a stained blood Last stage capable of
film, it appears as a mitosis
salmon pink- First stage without
staining cell with a mitosis
central pale area Last stage with a
that corresponds to nucleolus
the concavity. Last stage with nucleus
• > central pallor is First stage without
about one third the nucleus
diameter of the cell. Last stage that can
• > average diameter snthesize hemoglobin
of 6 to 8 um; Dark-blue appearance
survivability of Deep blue/ Rich blue
erythrocytes can be appearance
determined by using Milky-gray appearance
radioactive First detactable levels of
chromium (51Cr) hemoglobin synthesis
First appearance of
SHIFT RETICULOCYTES- transitioning from bone hemoglobin
marrow to peripheral blooad more rapid than normal Difusely basophilic
due to anemia eryythrocytes
Punctuate basophilia
STRESS RETICULOCYTES- Basophilia
1) Severe blood loss Eosiniphilia
2) Hemolytic lysis
1.Accelerates erythropoetin process (shortens STAGE DIAMETER N:C NUCLEOLI TRANSIT
RATIO TIME
transitioning time between the precurssor
maturational cell
Pronormoblast
2.Inhibit apoptosis of excess cells
3.to stimulate early release of reticulocytes
Basophilic
16 daughter cells in total normoblast
8-if excess
32-if not enough Polychromatophilic
normoblast

Orthochromatic
normoblast

Polychromatophilic
eryythrocyte

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HEMATOLOGY 1 -PRELIMS

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