AMLDT
Hemoglobin is converted to stable
– Cyanmethemoglobin
Chapter 1: An Overview of Clinical Laboratory
 5 Liters – average human blood
 Plasma – liquid portion of blood; provides
coagulation enzymes that protect vessels from
trauma and maintain the circulation; transports
and nourishes blood cells
 Hematology – study of blood cells
Three Categories of Blood Cells:
 Red Blood Cells (Erythrocytes)
 White Blood Cells (Leukocytes)
 Platelets (Thrombocytes)
HISTORY
 Athanasius Kircher – described worms in the blood
 Anton van Leeuwenhoek – gave an account of
RBCs
 Giulio Bizzozero – described platelets as “petites
plaques”
 James Homer Wright – discovered Wright’s stain;
opened a new world of visual blood film
examination through the microscope
 Wright’s Romanowsky-type stain – polychromatic;
mixture of acidic and basic dyes; remains the
foundation of blood cell identification
 Wright or Wright-Geimsa stain – present-day
hematology stain (appearance analyzed: 500x to
1000x)
 Morphology – cell appearance which
encompasses color, size, shape, cytoplasmic
inclusions and nuclear condensation
RED BLOOD CELLS
 Anucleated, biconcave, discoid cells filled with
reddish protein (Hemoglobin)
 Appears pink to red
 Measures 6-8 µm in diameter
 Pallor occupies 1/3 of the center (reflects
biconcavity)
 Hemoglobin – transports oxygen and carbon
dioxide
 Anemia – loss of oxygen-carrying capacity; often
reflected in a reduced RBC count or decreased
RBC hemoglobin concentration
 Polycythemia – increased in RBC count reflecting
increased circulating RBC mass (leads to
Hyperviscosity)
 Normal Saline Solution – 0.85%; matches the
osmolality of blood, retained intrinsic morphology
(no swelling/shrinking)
 1:200 dilution – typical for RBC counts with the use
of Thoma Pipettes (before automation)
 Hemacytometer – glass counting chamber; reports
RBC count in µL, mcL (sometimes mm3, mL or cc, L)
 Coulter Counters – by Joseph and Wallace Coulter
of Chicago; first electronic counter
HEMOGLOBIN, HEMATOCRIT, AND RED BLOOD CELL INDICES
 Drabkin Reagent – a weak solution of potassium
cyanide and potassium ferricyanide
 Hemoglobin Measurement: an aliquot amount of
blood is mixed with Drabkin Reagent ->
absorbance or
(Hemoglobincyanide)
color is measured
->
in
spectrophotometer with a wavelength of 540 nm
 Sodium Lauryl Sulfate (ionic surfactant, detergent) –
reduce environmental cyanide
 Hematocrit – ratio of the volume of packed RBCs to
volume of whole blood; also called Packed Cell
Volume (PCV); manually determined by transferring
blood to a graduated plastic tube with a uniform
bore, centrifuging, measuring the column of RBCs
plus plasma.
 Buffy Coat - light-colored layer between plasma
and packed RBCs; contains WBC and platelets;
excluded from hematocrit determination
In computing RBC Indices: used to detect/diagnose assess
the severity of, and monitor treatment of anemia,
polycythemia, and the numerous systemic conditions that
affect the RBC
 Mean Cell Volume (MCV); fL (femtoliter)
 reflects RBC diameter
 Mean Cell Hemoglobin (MCH); pg (picogram)
 mass of hemoglobin and parallels the MCHC
 Mean Cell Hemoglobin Concentration (MCHC);
g/dL
 reflects RBC staining intensity and amount of
central pallor
 Red Cell Distribution Width (RDW)
 degree of variation in RBC volume
 Anisocytosis – extreme RBC volume variability
RETICULOCYTE
 6-8 µm average diameter
 Stain slightly blue-gray
 Polychromatic/polychromatophilic erythrocytes
 Newly released from the bone marrow (RBC
production site)
 Indicate the ability of the bone marrow to increase
RBC production in anemia due to blood loss or
excessive RBC destruction
 Contains RNA (visualized by vital stains)
 Determine: Reticulocyte Count, Immature
Reticulocyte Fraction/Immature Reticulocyte Count
 Methylene Blue Dye – nucleic acid stains or vital
stains (supravital); used to differentiate and count
young RBCs; absorbed by live cells
WHITE BLOOD CELLS (Leukocytes)
 Dedicated to protecting their host from infections
and injury
 Transported in the blood from bone marrow or
lymphoid tissue
 Nearly colorless in an unstained cell suspensions
(that’s why named as white blood cells)
 Dilution is 1:20 (Diluent is dilute acid solution – to lyse
RBCs)
 Ranges from 4,500 to 11,500/µL
 Uses hemacytometer for visual counting
 Uses Wright stain
 Leukopenia – decreased WBC count
 Leukocytosis – increased WBC count
 Leukemia – uncontrolled proliferation of WBCs;
identified through Wright-stained bone marrow

smears, cytogenetics, flow cytometric
immunophenotyping, cytochemical staining
 Acute Myeloid Leukemia – acute
 Chronic Myelogenous Leukemia – 
granulocytic
TYPES OF WBC
1. Neutrophil
 phagocytic cells
 engulf and destroy microorganisms and
foreign material
 Segmented – multi-lobed nuclei
 Neutrophilia – increased neutrophils;
signals bacterial infection
 Neutropenia – decreased neutrophils;
often caused by certain medications or
viral infections
2. Bands
 less differentiated or less mature neutrophils
 contains submicroscopic, pink or lavender-
staining granules filled with bactericidal
secretions in the cytoplasm
 Left Shift – increase in bands; bacterial
infection
3. Eosinophils
 cells with bright orange-red, regular
cytoplasmic granules filled with proteins
involved in immune system regulation
 Eosinophilia – increased eosinophil count;
response to allergy of parasitic infection
 Eosinopenia – theoretical
4. Basophils
 cells with dark purple, irregular cytoplasmic
granules that obscure the nucleus
 granules contain histamines and other various
proteins
 Basophilia – increased basophil count; rare
and often signals hematologic disease
 Basopenia – theoretical
5. Lymphocytes
 comprise a complex system of cells that
provide for host immunity
 recognize foreign antigens and mount humoral
(antibodies) and cell-mediated antagonistic
responses
 Nearly round, slightly larger than RBCs, have
round featureless nuclei and a thin rim of non-
granular cytoplasm
 Lymphocytosis – increase in lymphocyte
count; associated with viral infections
 Lymphopenia/Lymphocytopenia –  “wedge prep” blood film on a glass slide, then
decrease in lymphocyte count; drug
deficiency or immunodeficiency
 Chronic Lymphocytic Leukemia – more
prevalent in 65 years old or older
 Acute Lymphoblastic Leukemia – most
common form of childhood leukemia
6. Monocytes
 Immature macrophage
 slightly larger diameter than other WBCs, blue-
gray cytoplasm with fine azure granules, and a
nucleus that is usually indented or folded
 Macrophage – most abundant cell type in
the body; phagocytose foreign particles
and assist
AMLDT
 Monocytosis – increased monocytes;
certain infections, collagen-vascular
disease; acute and chronic leukemia
Monocytopenia – theoretical
PLATELETS
 True blood cells that maintain blood vessel
integrity by initiating vessel wall repairs
 Rapidly adhere to the surfaces of damaged blood
vessels, form aggregates and secrete proteins and
small molecules that trigger thrombosis (clot
formation)
 Control hemostasis (series of cellular and plasma-
based mechanisms that seal wounds, repair vessel
walls and maintain vascular patency
 2-4 µm in diameter, round or oval, anucleated,
slightly granular
 Uncontrolled platelet and hemostatic activation –
causes deep vein thrombosis, pulmonary emboli,
acute myocardial infarctions (heart attacks),
cerebrovascular accidents, peripheral artery
disease, and repeated spontaneous abortions
(miscarriages)
 Thrombocytosis – increased platelet count;
inflammation or trauma but convey modest
intrinsic significance;
 Essential Thrombocythemia – rare malignant
condition; extremely high platelet counts and
uncontrolled platelet production; life-
threatening hematologic disorder
 Thrombocytopenia – common consequence
of drug treatment and may be life
threatening; accompanied by easy bruising
and uncontrolled hemorrhage
COMPLETE BLOOD COUNT
 Accession – may be automated, relying on bar
code or radio frequency identification
technology; reduces instances of identification
error
 Flag – indication that one of the results from the
profiling instrument is abnormal
BLOOD FILM EXAMINATION
 After “flag” is seen, the scientist performs a reflex
blood film examination
 A specialized, demanding and fundamental CBC
activity
 A physician can request this on the basis of clinical
suspicion even when the profiling instrument results
are within normal
allows it to dry and fixes and stains it using Wright or
Wright-Giemsa stain
 Checking for abnormalities of the cell using 50x or
100x in OIO
 Scientist reviews, identifies, and tabulates 100 WBC
to determine their percent distribution
ENDOTHELIAL CELLS
 Structural, do not flow in bloodstream
 Endodermal cells that form the inner surface of
blood vessels
 Important in maintaining the normal blood flow, in
snaring platelets during times of injury

COAGULATION
 Plasma coagulation: second important
component of hemostasis
 Employs a complex sequence of plasma proteins,
some enzymes, and some enzyme cofactors to
produce clot formation after blood vessel injury
 Fibrinolysis – digestion of clots to restore vessel
patency
 Prothrombin Time and Partial Thromboplastin Time,
Fibrinogen Assay, D-dimer Assay - high volume
assays used in screening profiles (PT, PTT); assess
each portion of the coagulation pathway for
deficiencies and are used to monitor
anticoagulant therapy
ADVANCED HEMATOLOGY PROCEDURES
 Bone Marrow Examination, Flow Cytometry
Immunophenotyping, Cytogenetic Analysis,
Molecular Diagnosis Assays
 Bone Marrow and Biopsy Specimens – collected
and stained to analyze nucleated cells that are
precursors to RBCs
 Myeloid Series – mature to form bands and
segmented neutrophils, eosinophils, and basophils
 Megakaryocyte – produce platelets
 Biopsy Specimen – enhanced by Hematoxylin and
Eosin Stain (H&E); abnormalities may include
leukemia, aplastic anemia, one of the host of other
hematologic disorders
 Cytochemical Stains – may be employed to
differentiate abnormal myeloid, erythroid and
lymphoid cells
 Stains:
 Myeloperoxidase
 Sudan Black B
 Nonspecific and Specific Esterase
 Periodic Acid-Schiff
 Tartrate-Resistant Acid Phosphatase
 Alkaline Phosphatase
 Immunostaining Methods – used to identify cell
lines, particularly lymphocyte precursors with
certainty
 Qualitative Laser-Based Flow Cytometers – simple
but more demanding
 Qualitative and Quantitative Cytometers –
employed to analyze cell populations by
measuring the effects of individual cells on laser
light such as forward-angle fluorescent light scatter
and right-angle fluorescent light scatter
 Immunophenotyping – for cell membrane epitopes
ADDITIONAL HEMATOLOGY PROCEDURES
 Osmotic Fragility Test – uses graduated
concentrations of saline solutions to detect
spherocytes (RBCs with proportionally reduced
surface membrane areas) in hereditary
spherocytosis or warm autoimmune leukemia
 Glucose-6-Phosphate Dehydrogenase Assay –
phenotypically detects an inherited RBC enzyme
deficiency causing severe episodic hemolytic
anemia
 Sickle Cell Solubility Screening Assay and High
Performance Liquid Chromatography – detects
sickle cell anemia and other inherited qualitative
hemoglobin abnormalities and thalassemia
AMLDT
 Erythrocyte Sedimentation Rate – detects
inflammation and roughly estimates its intensity
 Non-blood body fluid is always performed with a
rapid turn- around because cells in these hostile
environment rapidly lose their integrity.
HEMATOLOGY QUALITY ASSURANCE AND QUALITY CONTROL
 Moving Average – internal standard method that
supports hematology laboratory applications
Chapter 2: Safety in the Hematology Laboratory
 A well-defined safety program is the key to
prevention of accidents and laboratory-acquired
infections.
 One of the greater risks associated with the
hematology laboratory is the exposure to blood
and bodily fluids.
 Occupational Safety and Health Administration
(OSHA) issued the final rule for the Occupational
Exposure to Bloodborne Pathogens Standard. That
protects workers and other health care professionals
(From Universal precautions to Standard
precautions).
 Bloodborne pathogens: pathogenic
microorganisms that when present in blood can
cause disease. (EG. hepatitis B virus (HBV), hepatitis
C virus (HCV), and human immunodeficiency virus
(HIV).
APPLICABLE SAFETY PRACTICES REQUIRED BY THE OSHA
STANDARD
1. Hand washing is one of the most important safety
practices. Hands must be washed with soap and
water. If water is not readily available, alcohol hand
gels (minimum 62% alcohol) may be used
2. Eating, drinking, smoking, and applying cosmetics
or lip balm must be prohibited in the laboratory
work area.
3. Hands, pens, and other fomites must be kept away
from the mouth and all mucous membranes.
4. Food and drink, including oral medications and
tolerance testing beverages, must not be kept in
the same refrigerator as laboratory specimens.
5-8. *More examples*
9. Personal protective clothing and equipment must be
provided to the laboratory staff.
Such as Outer covering (Lab gowns and sleeve
protectors, Gloves, Eyewear, Phlebotomy trays,
Pneumatic Tube System - used to transport
specimens, the specimens should be transported in
the appropriate tube (primary containment), and
placed into a special self-sealing leak- proof bag
appropriately labeled with the biohazard symbol
(secondary containment).
HOUSEKEEPING
 All work surfaces should be cleaned when
procedures are completed and whenever the
bench area of floor becomes visibly contaminated.
An appropriate disinfectant solution is household
bleach, used in a 1:10 volume/volume dilution
(10%), which can be made by adding 10 mL of
bleach to 90 mL of water or 1½ cups of bleach to 1
gallon of water to achieve the recommended
concentration of chlorine (5500 ppm).

LAUNDRY
 If non-disposable laboratory coats are used, they
must be placed in appropriate containers for
transport to the laundry at the facility or to a
contract service and not taken to the employee’s
home.
HEPATITIS B VIRUS VACCINATION
 Lab employees should receive the HIV vaccination
series at no cost before or within the 10 days after
beginning work in the laboratory.
TRAINING AND DOCUMENTATION
 Hematology staff should be properly educated in
epidemiology and symptoms of bloodborne
diseases.
REGULATED MEDICAL WASTE MANAGEMENT
 Specimens from the hematology laboratory are
identified as regulated waste.
 Occupational Exposure to Bloodborne Pathogens
Standards: provides information on the handling of
regulated medical waste.
OCCUPATIONAL HAZARDS
FIRE HAZARD
 Fire Response Plan: written fire prevention and
response procedures
Common Sources of Fire in the Laboratory:
 Electrical overloading
 Poor electric maintenance
 Excessive long gas tubing and electricity leads
 Equipment left switched on unnecessarily
 Naked flames
 Deteriorated gas tubing
 Misuse of matches
 Carelessness with flammable materials
 Flammable and explosive chemicals stored in
ordinary refrigerators.
Safety/Prevention Plan:
 Enforcement of a no smoking policy.
 Placement of fire extinguishers every 75 ft.
 Placement of fire detection systems which should
be tested every 3 months.
 Written fire prevention and response procedures,
commonly referred to as the fire response plan.
 Conducting quarterly fire drills
 A well-organized fire safety training program.
Good Practices:
 Checking fire extinguishers at least once a year.
 Turn off the flame before leaving the laboratory
 Keep the portable fire extinguisher within reach.
 In case of fire accident in the laboratory, close all
doors and windows and prevent draft.
 For a small blaze, use sand or water and a fire
blanket while fire extinguishers are used for a larger
blaze.
 Do not use water to put out an electric fire or fire
caused by grease, oil or gasoline.
 While escaping, it is safest to crawl and stay close
to the floor. Cover the mouth and nose to reduce
the danger of inhaling flame.
Four Classes of Fire: A, B, C, D
 Class AL ordinary combustibles such as wood,
paper and cloth
Use: pressurized-water and dry-chemical
extinguishers
AMLDT
 Class B: flammable liquids, gases or grease
 Class C: electrical equipment, motors and switches
Use: dry-chemical and carbon dioxide extinguishers
 Class D: flammable metals such as magnesium
Extinguishment is left to trained firefighters using
special dry-chemical extinguishers
Operating Fire Extinguisher:
1. Pull the pin
2. Aim nozzle at base fire
3. Squeeze the handle
4. Sweep nozzle side to side
CHEMICAL HAZARDS
 Safety Data Sheets/Material Safety Data Sheets
(MSDS): written by the manufacturers of chemicals
to provide information on the chemicals that
cannot be put on label
 All chemicals in the laboratory must be considered
as poison.
 Label all chemicals properly.
 Follow all handling and storage requirements for the
chemical. Refer to MSDS when you have problems
regarding a chemical.
 The wearing of contact lens should not be
permitted when an employee is working with
xylene, acetone, alcohols, formaldehyde and other
solvents.
 Exposure to fumes must be kept within permissible
limits.
 Do not store inflammable chemicals in refrigerators.
 Strong acids and alkalis are corrosive compounds.
Always store them near the floor with a warning sign
on the bottle.
ELECTRICAL HAZARDS
Most Common Causes of Electrical Hazards by WHO:
 Wet or moist surface near electrical equipment
 Long flexible electrical connecting cables
 Poor and perished insulation on cables
 Overloading of circuits by use of adapters
 Sparking equipment near flammable substances
and vapors
 Electrical equipment left switched on and
unattended
How to Avoid:
 Electric wirings inside the laboratory should be
inspected regularly.
 Use of extension cords should be avoided.
 Use of “cheater adapters” and gang plugs should
be prohibited.
 All new instruments should be thoroughly inspected
first before being used out for service.
 Electrical equipment should not be placed in areas
where ignitable vapors might accumulate.
 If electrical equipment fails to function properly,
disconnect the apparatus.
 In case of electric fire, use only carbon dioxide
extinguisher. Never throw water.
CHAPTER 3: Blood Collection
SKIN PUNCTURE
 Blood sample collected is called peripheral blood
instead of capillary blood
 Mixture of capillary, venous, & arterial blood
with interstitial and intracellular fluid
 AMLDT

 Different from venous blood because of  3 years to adult life

admixing of tissue juice which leads to the  Wrist vein
following: ↓ Hct, Hgb, RBC ct., Plt & ↑ WBC ct.  Dorsal veins of the hands
 Less amount can be obtained  Dorsal veins of the ankle or foot
 Additional & repeated test cannot be done  Veins of the antecubital region ( Dorsal, Median
 Hemolyses easily Cubital, Basilic )

SITES METHODS
1. FINGER (middle or ring)  Syringe Methods
 Lateral palmar surface perpendicular to the  Evacuated Tube Method
fingerprints  Butterfly Infusion Set
 Accessible & easy to manipulate
 Ideal for peripheral smears PROCEDURE
 Less intimidating 1. PATIENT INTERACTION
2. EARLOBE  Identify patient
 Less free nerve ending, hence less pain & less tissue  Note patient isolation restrictions Reassure
juice patient
 More free flow of blood  Verify paperwork Position patient
 Ideal when searching for abnormal cells 2. ASSEMBLE SUPPLIES & EQUIPMENT Select general
(histiocytes in bacterial endocarditis) venipuncture location Apply tourniquet
 can be arteriolized by: heat (44OC), slight flicking  Select exact venipuncture site Cleanse area
with index finger until definite flushing, & chemical (70% Isopropyl Alcohol) Inspect needle
means (Trefuril paste)  Perform venipuncture Release tourniquet
3. HEEL or BIG TOE  Position gauze over the puncture site Remove
 For less than 1 you needle & apply pressure
 Lateral portion of the plantar surface of the heel 3. SPECIMEN PREPARATION
 If syringe is used, fill tubes Discard needle
SITES TO AVOID  Label specimens
 Inflamed & pallor areas  Transport specimens promptly
 Cold & cyanotic areas
 Congested & edematous areas KEY NOTES
 Scarred & heavily calloused areas  NEEDLE INSERTION : Bevel Up, 15 degrees
 NEEDLE GAUGE : 19, 20, 21
PUNCTURING DEVICES  TOURNIQUET
 NEEDLES  3-4 inches or 7.5-10cm above the site
 BLADES  Should NOT EXCEED 1-2 mins -> 1 min
 LANCETS  Prolonged application may lead to
 Should be more than 2.0mm deep in order that hemoconcentration
the lancet passes through the dermal-  Maximum of 2 attempts
subcutaneous junction  Reassure patient; crying may result to increased cell
 For newborns, must not exceed 2.4mm in count
length  Patient with IV lines: use the opposite arm without IV
line; if both arms have IV line, ask the nurse to stop
VENIPUNCTURE IV for 2 mins, then collect below the IV line with IV
 Blood sample collected is VENOUS blood line with the exception of Glucose & Phosphorus
 Manner of inserting a needle attached to a
syringe to a palpable vein to collect blood for ORDER OF DRAW
laboratory testing Anticoagulant - A chemical substance which interferes in
 venous blood == most widely used blood blood coagulation through various mechanisms.
sample in all laboratory tests not only in 1. EDTA - VERSENE/SEQUESTRENE
hematology  Most common used in hematology
 Mechanism of action: binds the non-ionized
THREE FACTORS INVOLVED IN GOOD COLLECTION: Calcium then chelates Calcium molecule in a
 The phlebotomist complex
 The patient & his/her veins  COLOR: Lavender/purple
 The equipment needed  Recommended amount: 1.2 mg/ml of blood
 USES: RBC, WBC Hgb, Hct, ESR, Plt, Peripheral
SITES Smear
 In newborns infants up to 18 months old  Should be prepared 2 hours after collection
 External jugular vein since EDTA may have adverse effect to RBCs
 Temporal vein (scalp vein)  Not for recommended for coagulation studies
 Superior longitudinal sinus (interferes with fibrinogen-thrombin reaction
 In older children 18 months to 3 years old  Platelet may adhere to neutrophils in old EDTA
 Femoral vein blood, a phenomenon called as PLATELET
 Long saphenous vein SATELLITISM
 Popliteal vein  Changes in old EDTA blood: vacuolization of
 Ankle vein leukocyte cytoplasm, artifact /crystal formation,
 AMLDT

phagocytosis of crystals by WBCs, clover  Addt’l & repeated tests can be done
leafing of WBC nucleus, RBC crenation,  Fastest method of collecting sample which requires
platelet disintegration various anticoagulation
 THREE FORMS:  Ideal for clinical chemistry & other serological tests
 Dipotassium EDTA – most soluble, hence DISADVANTAGES
preferred  Requires more time & skill on the part of the
 Disodium EDTA phlebotomist
 Tripotassium EDTA  Requires more equipment
2. CITRATES  More complications may arise
 Most common & preferred for coagulation  Difficult to do in infants, children & obese individuals
studies
 Mechanism of action: binds Calcium VENIPUNCTURE COMPLICATIONS
 COLOR: Light blue 1. LOCAL IMMEDIATE
 Blood-Anticoagulant Ratio: a. Hemoconcentration
 3.2% or 0.109M NaCitrate (9:1) <light b. Failure of blood to enter the syringe
blue> 2. LOCAL DELAYED
 0.105M NaCitrate (4:1) <black> for a. Hematoma
standard Westergren method for ESR b. Thrombosis of the vein
2. HEPARIN c. Thrombophlebitis
 Most common used for OFT and 3. GENERAL DELAYED COMPLICATIONS
immunophenotyping a. Infections
 Mechanism of action: inhibits thrombin
 COLOR: Green PHLEBOTOMY COMPLICATIONS
 10-20 units/ml of blood  Vascular: Infection, Cardiovascular, Anemia,
 TWO FORMS: Neurological, Dermatological
 Lithium heparin
 Sodium heparin Chapter 7: Hematopoiesis
 Not recommended for coagulation studies
because it affects all stages of blood HEMATOPOIESIS
coagulation  continuous regulated process of blood cell
 Not recommended for blood smear production
preparation because it cause BLUE  cell renewal, proliferation, differentiation,
BACKGROUND when stained with maturation
Romanowsky stains  result in the formation, development and
 Not for WBC ct, causes agglutination of specialization of all the functional blood cells
WBCs  Mature blood cells have limited lifespan (ex. 120
 Not for Plt ct, enhances platelet aggregation days for RBC)
 Most expensive  Hematopoietic stem cell is capable of cell renewal
3. OXALATES (replenishment) and directed differentiation into all
 Mechanism of action: binds Calcium required cell lineages.
 COLOR: Gray  select distribution of embryonic cells
 1-2 mg/ml of blood  In adults: bone marrow
 For RBC ct., Hgb, Hct, ESR (all RBC evaluation  In fetal development: yolk sac > aorta- gonad
tests since there is no effect on RBCs) mesonephrons > liver > bone marrow
 THREE FORMS:
 Double oxalate – most common MESOBLASTIC PHASE
 Lithium oxalate – collecting bloody body  begins at 19th day of embryonic development after
fluids fertilization in the yolk sac of the embryo
 Sodium oxalate – coagulation studies  Cells from the mesoderm migrate to the yolk sac.
4. DOUBLE OXALATE: Some of these cells form primitive erythroblasts in
 Salts of Ammonium & Potassium (NH4K) in 3:2 the central cavity of the yolk sac. Others
ratio (angioblasts) surround the cavity of the yolk sac
 Ammonium oxalate only – RBC swelling and form blood vessels.
 Potassium oxalate only – shrinkage of RBC  Primitive erythropoiesis. Only erythrocytes are made
 Sodium oxalate – coagulation studies (nucleated primitive erythroblasts)
 Known as Balance Oxalate, Wintrobe fluid  Intravascular
,Paul-Heller’s fluid  The RBCs contain fetal hemoglobin (Hgb F) [Gower
 Not for blood transfusion because it’s toxic I= zeta-2, epsilon-2; Gower II= alpha-2, epsilon-2;
 Causes agglutination or clumping of WBC & Portland= zeta-2, gamma-2]
platelets hence causing erroneous counting  About 6 weeks of gestation, yolk sac production of
 No recommended for peripheral blood smear erythrocytes DECREASES and production of RBCs in
because it has same ill effects as EDTA when the human embryo itself begins
use for more than 2 hours  Mesodermal cells also migrate to aorta- gonad
mesonephrons region to give rise to primitive
EVACUATED TUBE METHOD ADVANTAGES erythroblast
 Large amount can be obtained  Mesenchymal cells > aorta-gonad mesonephrons >
 Can be transported & stored for future use hematopoietic stem cells

 Angioblasts - other parts of the yolk sac; blood
vessels
HEPATIC PERIOD
 begins at 5-7 (book) 4-5 (ppt) gestational weeks
 Developing erythroblasts, granulocytes, and
monocytes colonizing the fetal liver, thymus,
spleen, placenta and bone marrow
 Lymphoid cells begin to appear
 Extravascular
 Liver as the major site of hematopoiesis during the
second trimester of fetal life and reaches reaches
its peak by the third month of fetal development,
gradually declines after the 6th month, retaining
minimal activity until 1-2 weeks after birth
 Hematopoiesis in the AGMs region disappear
 Production of megakaryocytes begins
 Minor sites: sleep, kidney, thymus, lymph nodes
 Thymus - first fully developed organ in the fetus (T
cell production)
 Kidney and Spleen - produce B cells
 Hgb F (a-2, y-2)
 Hgb A (a-2, b-2)
 Hgb A2
MYELOID PHASE
 5th month of fetal development
 Medullary hematopoiesis (major site at 6th
month/24 gestational weeks of fetal life: medulla
of bone marrow; secondary site: liver and spleen)
 Extramedullary hematopoiesis (other areas other
than bone marrow)
 Detectable levels of EPO, G-CSF, GM-CSF
 mainly granulocytes
 M:E ratio = 3:1 (immature > mature)
 Red bone marrow: HSC (myeloid and erythroid)
 Yellow bone marrow: fats
 Hgb F (a-2, y-2)
 Hgb A2 (a-2, δ-2)
 Hgb A (a-2, b-2)
ADULT HEMATOPOIETIC TISSUE
 located in the bone marrow, lymph nodes, spleen,
liver, thymus, reticuloendothelial system, bursa
(birds), stomach, kidneys
 Bone marrow contains: erythroid, myeloid,
megakaryocytic, and lympoid cells
 Primary lymphoid tissue consists of: bone marrow
and thymus [where T and B lymphocytes are
derived]
 Secondary lymphoid tissue consists of: spleen,
lymph nodes, mucosa-associated lymphoid tissue
[where cells respond to foreign antigen]
RETICULOENDOTHELIAL SYSTEM
 cellular destruction
 Mononuclear phagocytic system
 Circulating monocytes, fixed macrophages, free
macrophages
 Free macrophages: engulfing
 Processing of antigens
 Removal of senescent (aged) cells
 Secretion of growth factor or interleukins
BONE MARROW
 tissue located between the cavities of cortical
bones
AMLDT
 Central space within the bone that results from the
resorption of cartilage and endosteal bone
 Trabeculae - projections of calcified bone; forms
honeycomb like 3D matrix; structural support got
developing blood cells
 Contains hematopoietic cells, stromal cells, blood
vessels
 Stromal cells - originate from mesenchymal cells;
includes endothelial cells, adipocytes,
macrophages and lymphocytes, osteoclasts,
osteoblasts, fibroblasts
 before birth - 100% red bone marrow
 after birth - 90% red, 10% yellow
 19th- 20th month - 60% red, 40% yellow
 Average adult - 50% red, 50% yellow (proximal ends
of large flat bones, pelvis, sternum)
 65 y/o above - 40% red, 60% yellow
 Retrogression: myeloid; replacement of red BM with
yellow BM
 Cellularity: ratio of marrow cells to fat
 Hypercellular/Hyperplastic: > 70% HSCs (yellow to
red)
 May be due to the following: acute blood loss,
severe chronic anemia, myeloma
 Hypocellular/Hypoplastic: <30% HSCs (red to yellow)
 May be due to the following: chemicals, genetics,
MPD
 Normocellular: 30-70% HSCs
 Aplastic: no HSCs
 Numerous granulocytes because of short survival (1-
2 days)
 Infection 6:1; leukemia 25:1; myeloid hyperplasia
20:1; myeloid hypoplasia 3:20)
MARROW CIRCULATION: NUTRIENT ARTERY
 supplies blood only to the marrow
 Divides into ascending and descending branches >
enters the endosteum of cortical bone > form
sinusoids > connect to the periosteal capillaries
PERIOSTEAL ARTERY
 provide nutrients to for the osseous bone and the
marrow
BONE MARROW COLLECTION
 Posterior Iliac Crest
 Trephine (CORE) biopsy = Jamshidi Needle
 Bone marrow aspirate = University of Illinois Sternal
Needle
 Staining of BM using Romanowsky's stain
LIVER
 major site of blood cells production during the 2nd
trimester of fetal development
FUNCTIONS IN THE FOLLOWING:
 protein synthesis and degradation
 Coagulation factor synthesis
 Carbohydrate and lipid metabolism
 Drug and toxin clearance
 Iron recycling and storage
 Hemoglobin degradation (bilirubin transported to
the small intestine)
 Contains phagocytic cells known as KUPFFER CELLS
that remove senescent cells and foreign debris and
regulate protein synthesis in the hepatocytes

 Can assume functions when bone marrow cannot
function
 Porphyrias: accumulation of intermediary
porphyrins that damage hepatocytes
 Severe hemolytic anemia = increase conjugation
of bilirubin and storage of iron
SPLEEN
 largest lymphoid organ in the body
 Beneath the diaphragm, behind the fundus, upper
left quadrant of abdomen
 Functions as an indiscriminate filter of the
circulating blood
 Contains 350mL of blood
 White pulp is composed of
llymphocytes, macrophages, and dendritic cells
 Red pulp is composed of vascular sinuses
separated by cords of Bill Roth containing
macrophages; serves as a filter
 Culling: cells are phagocytized with subsequent
degradation of cell organelles (filtering and
destruction)
 Pitting: removal of inclusions or damaged surface
membrane from the RBCs
 Also serve as a site for platelets (30% of platelet
found on the spleen)
 Slow-transit pathway
 through the red pulp
 RBC pass through the macrophage-lined cords
before reaching the sinuses
 Plasma freely enters the sinuses
 RBCs have more difficult time passing through the
inter-endothelial junctions
 Creates an acidic, hypoglycemic, and hypoxic
environment
 Rapid-transit pathway
 blood cells enter splenic artery and pass directly to
the sinuses in the red pulp and continue to the
venous system to exit the spleen
 Splenomegaly: speen becomes enlarged and
palpable
 Hypersplenism: enlargement of spleen resulting to
pancytopenia (leukopenia, thrombocytopenia,
anemia)
 Primary Hypersplenism: no underlying disease
 Secondary Hypersplenism: caused by underlying
disorder
LYMPH NODES
 located along lymphatic capillaries that parallel
but are not part or the circulatory system
 Composed of lymph nodes and lymphatic vessels
that drain into the left and right lymphatic duct
 Bean-shaped structures in groups or chains
 Lymph: fluid portion of blood
 Lymph nodes
 composed of lymphocytes, macrophages,
reticular networks
 Act as filters to remove foreign particles
 Afferent Lymphatic Vessel: carry circulating lymph
to the lymph nodes
 Efferent Lymphatic Vessel: where the lymph exits
 Cortex: outer region; consists of B lymphocytes
surrounded by T lymphocytes and macrophages
 Medulla: inner region; contains plasma cells
 Adenitis: infection of lymph node
AMLDT
THYMUS
 originates from endodermal and mesenchymal
tissue
 Located at the upper part of the anterior
mediastinum
 well-developed at birth and increases size at
puberty at which time it starts to decrease in size
 Serves as compartment for maturation of T
lymphocytes to immunocompetent T Cells
(hormone thymosin)
 Cortex: peripheral zone; densely packed with
lymphocytes and macrophages
 Medulla: central zone; less cellular with few
lymphocytes, macrophages and epithelial cells;
only 15% of mature T cells
 Mature T cells leave the thymus to populate spleen
and lymphoid tissues
 Also contains other cells such as B cells, eosinophils,
neutrophils and myeloid cells
STEM CELL THEORY
 Till and McCulloch: Stochastic Model of
Hematopoiesis
 Aplastic mice were given IV injection of marrow
cells and CFU-S were observed.
 Colony forming units – spleen
 Capable of self-renewal and production of
differentiated progeny
 refer to COMMITTED MYELOID PROGENITORS or CFU-
GEMM
 capable of giving rise to multiple lineages of blood
cells
 Monophyletic Theory: all blood cells are derived
from a single progenitor stem cell called
PLURIPOTENT HSC.
 Polyphyletic Theory: each blood cell lineages is
derived from its own unique stem cell
HEMATOPOIETIC STEM CELLS
 capable of self-renewal but limited ability only
 Pluripotent cells
 Single type of cell where all hematopoietic cells
arise
 Can differentiate into myeloid and lymphoid
lineages
 For self-renewal or differentiation or apoptosis
 Symmetric division: both daughter cells differentiate
 Asymmetric division: one daughter cell return to
stem cell pool and the other one differentiates or
undergoes apoptosis
 Stochastic Model: to self-renew or to differentiate
 Instructive Model: lineage differentiation
PROGENITOR CELLS
 more restricted
 Multipotent
 Do not self-renew
 Respond best to multiple cytokines
 Expand the number of cells dramatically
 CFU-GEMM
PRECURSOR CELLS ("committed")
 Blast cells committed to unilinear differentiation
 Develop into distinct cell lines
 Do not self-renew
 Respond best to 1 or 2 cytokines
 Still replicate until near terminal differentiation
 AMLDT

 CFU-G, CFU-M, CFU-E, CFU-B Chapter 8: Erythrocyte Production and Destruction

ERYTHROCYTE / RBC
HEMATOPOIETIC GROWTH FACTORS OR CYTOKINES  Carry oxygen from the lung to the tissues.
 soluble proteins that regulate the proliferation,  Attachment of the oxygen to hemoglobin
differentiation and maturation of hematopoietic  Major cytoplasmic component of mature RBCs
precursor cells  Returning carbon dioxide to the lungs
 Includes interleukins, lymphokines,  Buffering the pH of the blood
monokines, interferons, chemokines and CSFs.  Anemia – body’s response to diminished oxygen-
 Apoptosis: programmed cell death COLONY- carrying capacity of the blood.
STIMULATING FACTORS  Mammalian, Amphibians & Birds – possess RBCs
 have high specificity for their target cells having no nucleus in its mature, functional state
 Active at low concentrations
 Can influence other cell lineages ERYTHROBLASTS
 Nucleated precursors of RBCs in the bone marrow
EARLY-ACTING MULTILINEAGE GROWTH FACTORS  Also NORMOBLAST
 Multi-lineage  Developing nucleated cells with normal
 KIT ligand appearance
 stem cell factor (SCF)
 early acting growth factor a MEGALOBLAST
 KIT as the receptor (transmembrane protein;  Abnormal appearance of the developing
tyrosine-protein kinase) nucleated cells in megaloblastic anemia
 FLT3: another tyrosine-protein kinase  Erythroblast are large
 KIT and FLT3 work together with IL-3 and GM-CSF
 IL-3: regulates blood cell production by controlling MATURATION PROCESS ERYHTROID PROGENITORS
granulocytes and macrophages 1. Burst Forming Unit Erythroid (BFU-E)
 GM-CSF: induces expression of specific genes that  1 week to mature to CFU-E
stimulate HSB differentiation to the common 2. Colony Forming Unit Erythroid (CFU-E)
myeloid progenitor  1 week to become pronormoblast (first
identifiable RBC precursor)
INTERLEUKINS  3 -5 divisions before maturing further
 IL-1: lymphocyte activating factor  6-7 days to become mature cells ready to enter
 Proteins that exhibit multiple biologic activities (ex. the circulation
Regulation of autoimmune and inflammatory  18-21 days to produce a mature RBC from the
reactions, hematopoiesis) BFU-E ERYTHROID PRECURSORS
 Have synergistic interactions with other cytokines
 Effective at low concentrations NORMOBLASTIC PROLIFERATION
 Part of interacting systems with amplification  Process encompassing replication to increase cell
potential numbers and development from immature to
mature cell stages.
ERYTHROPOIESIS
 occurs in the bone marrow CRITERIA USED IN IDENTIFICATION OF THE ERYTHROID
 CFU-GEMM gives rise to BFU-E PRECURSORS
 BFU-E  Modified Romanowsky stain, such as Wright /
 resembles a cluster of grapes with bright Wright- Giemsa is commonly used.
hemoglobin  Stage of maturation is determined by nucleus and
 Has few EPO receptors cytoplasm
 CFU-E
 BFU-Es under the influence of IL-3, GM-CSF, TPO, KIT IMPORTANT QUALITIES IN RBC IDENTIFICATION
ligand 1. Nuclear chromatin pattern (texture, density,
 Has many EPO receptor homogeneity)
 EPO = lineage specific glycoprotein in the renal 2. Nuclear diameter
peritubular interstitial cells; in the kidneys 3. Nucleus:Cytoplasm (n:c) ratio
4. Presence or absence of nucleoli
LEUKOPOIESIS 5. Cytoplasmic color
 can be divided into myelopoiesis and
lymphopoiesis MATURE RBC TRENDS
 Includes GM-CSF, G-CSF, macrophage colony- 1. Overall diameter – decreases
stimulating factor (M-CSF), IL-3, IL-5, IL-11, and KIT 2. Nucleus:Cytoplasm ratio – decreases
ligand 3. Nuclear Chromatin – coarser, clumped and
 Eosinophils require: GM-CSF, IL-5, IL-3 condensed
 Basophils require: IL-3 and KIT ligand  develops raspberry-like appearance
 dark stained chromatin v. white appearance of
parachromatin
MEGAKARYOPOIESIS 4. Nucleoli disappear
 Includes GM-CSF, IL-3, IL-6, IL-11, KIT ligand, TPO  no more protein synthesis
 ribosomes are formed
5. Cytoplasm changes from blue to gray-blue to pink

 Basophilia
 degree of the cytoplasmic basophilia
correlates with the amount of Ribosomal
RNA
 blue
 Eospinophilia
 correlates with the accumulation of
haemoglobin as the cell matures
 pink
THREE ERYTHROID PRECURSOR NOMENCLATURE SYSTEMS
 Erythroblast terminology - EUROPE
 Normoblastic Terminology - US
 Descriptive to the appearance of the cells
 Rubriblast
 Parallels the nomenclature used for WBC
development.
ERYTHROKINETICS
DYNAMICS OF RBC PRODUCTION AND DESTRUCTION
ERYTHRON
 Collection of all stages of erythrocytes throughout
the body
 Developing precursors in the bone marrow
 Circulating erythrocytes in the peripheral blood
and the vascular spaces within specific organs (i.e
spleen)
 UNIFIED FUNCTIONAL TISSUE
 Entirety of erythroid cells in the body
 not to be confused with RBC MASS
 Cells in the circulation
PERITUBULAR INTERSTITIAL CELLS OF THE KIDNEY
 primary oxygen-sensing system of the body
 produce Erythropoietin
 detects Hypoxia
 increase EPO production in the peritubular
cells
 Transcriptional regulation
HYPOXIA-SENSITIVE REGION
 3’ regulatory portion of the EPO gene
 Promotes gene transcription
HYPOXIA INDUCIBLE FACTOR -1
 transcription factor
 In cytoplasm when oxygen tension in the cells is
decreased then migrates to the nucleus
 interacts with the 3’ enhancer of the gene
 results: transcription of more EPO messenger RNA
molecules; production of more EPO
 With HYPOXIA = MORE messenger RNA molecules
are produced = more EPO production
ERYTHROPOIETIN STRUCTURE
 Thermostable, nondialyzable, glycoprotein
hormone
 MW = 34KD
 With carbohydrate unit that reacts specifically with
RBC receptors
 Terminal sialic acid unit
 Necessary for biologic activity in vivo
ACTION
 produced in kidneys ; acts in bone marrow
 Growth factor that initiates SIGNAL TRANSDUCTION
AMLDT
 an intracellular message to the developing
RBCs
 the EPO-responsive cells vary in their sensitivity
to EPO
 in healthy circumstances, CELLs only requires
LOW LEVEL of EPO respond
EPO(LIGAND) + RECEPTOR ON ERYTHROCYTE PROGENITORS
 Initiate a cascade of intracellular events that
ultimately leads to more RBCs entering the
circulation
 EPO’s effects are mediated by the intracellular
Janus tyrosine kinase signal transducers
 It affect gene activities in the RBC NUCLEUS
3 EFFECTS OF ERYTHROPOIETIN
1. Early release of reticulocytes from the bone marrow
2. Preventing apoptotic cell death
3. Reducing the time needed for cells to mature in the
bone marrow
 IMPORTANT: “EPO puts more RBCs into the
circulation at a faster rate than occurs without its
stimulation”
ACTION #1: EARLY RELEASE OF RETICULOCYTES
2 Mechanisms by EPO:
1. EPO induces changes in the adventitial cell layer of
the marrow/sinus barrier; increase the space for RBC
egress into sinus
 results to Shift Reticulocytes
 Reticulocytes that are still very basophilic
 Shifted from the marrow very early
 Even nucleated RBC can be release early
in cases of extreme anemia
 limited in their effectiveness
Action #2: INHIBITION OF APOPTOSIS
 increases the number of cells that will be able to
mature into circulating erythrocytes
APOPTOSIS – programmed cell death
a. it takes 18 days to produce an RBC
b. instead of storing cells (RBCs) for emergency cases,
the body produces more cells rapidly when needed
c. if not needed, the extra progenitors are allowed to
die
d. if needed, 8 -10 day head start in the production
process
e. the process of intentional wastage of cells occurs
PROCESS OF APOPTOSIS
 degradation of chromatin into large fragments of 5
to 300 kilobases further into 200 bases
 protein clustering
 activation of transglutamase
 NECROSIS
 Cell injury causes swelling and lysing with
release of cytoplasmic contents that stimulate
an inflammatory response
 APOPTOSIS
 Condensation of the nucleus
 Effect - increased basophilic staining of the
chromatin
 Nucleolar disintegration
 Shrinkage of cell volume width EFFECTS:
 increase in cell density
 compaction of cytoplasmic organelles

 Partition of cytoplasm and nucleus into membrane-
bound apoptotic bodies
 LAST STAGE: degradation produces nuclear DNA
consisting of multimers 180 base pairs.
 Characteristic blebbing of the plasma membrane
 Apoptotic cell contents remain membrane-bound
and ingested by macrophages
 Prevents inflammation
EVASION OF APOPTOSIS by erythroid progenitors and
precursors
 Death receptor on the earliest RBCs precursors
 Crucial molecules in the messaging system
 Fas and FasL (ligand)
 Expressed by more mature RBCs
 EPO levels are LOW
 slow cell production
 Hypoxia not present
 Excess precursors should undergo apoptosis
 Occurs when older FasL-bearing erythroid
precursors (i.e polychromatic normoblasts)
cross-link with Fas marked immature erythroid
precursors (pronormoblast) which are then
stimulated to undergo apoptosis
 More mature cells with FasL present in the marrow,
erythropoiesis is subdued
 If FasL-bearing cells are depleted, as when EPO
stimulated early release occurs, the younger Fas-
positive precursors are allowed to develop.
 2nd mechanism : EPO RESCUE
 EPO is able to increase RBC production
 CFU-E
o Most EPO receptors and most sensitive to
EPO
o Without EPO, it will not survive
 EPO’s effect is mediated nu the transcription
factor GATA-1
 bcl-xL
o EPO stimulated cells develop this molecule
on their mitochondrial membranes
o Able to resist the FasL activation of Apoptosis
ACTION #3: REDUCED MARROW TRANSIT TIME
 APOPTOSIS RESCUE
 EPO increases RBC mass
 Increasing the number of erythroid cells that
survive and mature to enter the circulation
2 MEANS FOR EPO TO INCREASE THE RATE:
1. Increased rate of cellular processes
2. Decreased cell cycle times
 EPO actions:
 stimulates RBC RNA synthesis
 accelerates haemoglobin production and
 cessation of division
 increased EPO can reduce the time of
maturation in the marrow (reducing individual
cycle times) – 20% reduction – from 6 days
(normal) becomes 4 days
 STRESS RETICULOCYTES
 Large bluish cells lacking central pallor
 Exit the marrow early during condition of
hemoglobin accumulates slowly
 shift reticulocyte
MEASUREMENT OF ERYTHROPIETIN
 Quantitative measurements of EPO
AMLDT
 On plasma and other body fluids
 Measures by immunoassays
 Radioimmunoassay
 Chemiluminescence
 Reference range : 5 to 30mUnits/mL
 Increased EPO In urine are expected of most
patients with anemia (except anemia by renal
disease)
STIMULI TO ERYHTROPOIESIS
 Tissue Hypoxia
 Testosterone
 Pituitary hormones
 Thyroid hormones
MICROENVIRONMENT OF THE BONE MARROW
 Hematopoiesis occurs in marrow cords
 Occurs in ERYTHROID ISLANDS
o Macrophages surrounded by erythroid
precursors in the various stages of
development
 SUCKLING PIG phenomenon
 Provided iron directly to the normoblasts for the
synthesis of hemoglobin
 Developing RBCs probably obtain most iron via
transferrin
 Macrophages - major cellular anchor for the RBC
 fibronectin – anchors RBC to the extracellular matrix
of BM
3 COMPONENTS TO THE ANCHORING SYSTEM:
1. Stable matrix of stromal cells to which normoblasts
can attach
2. Bridging (adhesive) molecules for that attachment
3. Receptors on the erythrocyte membrane
ERYTHROCYTE DESTRUCTION
 natural catabolism – deterioration of enzymes
 mature erythrocyte is unable to generate new
proteins due to non-nucleatedness
 RBCs rely on Glycolysis for production of ATP,
because they don’t have any mitochondria
 SENESCENCE
 Cellular aging
 Loss of glycolytic enzymes
 Results in phagocytosis by macrophages
 NORMAL DEATH
RBC DESTRUCTION
1. Macrophage mediated (Extravascular Hemolysis)
 Substantial volume of blood is in the spleen
 sluggish movement through the red pulp
 Available glucose in the surrounding plasma is
depleted quickly as the cell flow stagnates, so
glycolysis slows
 low PH - iron oxidation
 In HOSTILE environment
 Deteriorating glycolytic processes lead to
reduced ATP production
o low levels of available glucose
 oxidation of the membrane lipids and
proteins
 sodium increases and potassium decreases
 Selective permeability is lost and water
entry
 Discoid shape is lost and cell becomes a
sphere
 AMLDT

 RBC must remain highly flexible to exit the

spleen by squeezing through the splenic sieve
 Spheric RBCs are rigid
 If they cannot pass through, they are readily
ingested by macrophages
 When RNC lyses within a macrophage, the
major components are catabolized
 Iron is removed from heme
 Globin is degraded and returned to the
metabolic amino acid pool
 MECHANICAL HEMOLYSIS (Intravascular
Hemolysis)
 Small portion of RBCs rupture intravascularly, in
the lumen of the blood vessels
 Small breaks in blood vessels and resulting clots
can trap and rupture cells

2. Mechanical Hemolysis (Fragmentation or

Intravascular Hemolysis)
 Small portion of RBCs rupture intravascularly, in
the lumen of the blood vessels
 Small breaks in blood vessels - clots
 Few cells lyse in the blood vessels from purely
mechanical or traumatic causes
 PLASMA PROTEINS
 Haptoglobin and Hemopexin
o Salvage the released hemoglobin so
that the iron is not lost
 AMLDT
Cellular Time in
Nucleus Cytoplasm N:C Ratio Division Location
Activity Stage
6 to 12 um Deeply Undergoes
Round to basophilic, Mitosis Accumulate
oval, reddish perinuclear More than components
Pronormoblast Bone
purple fine halo, 8:1 one division for 24 hours
(Rubliblast) marrow
chromatin irregular is possible hemogloin
patterns, 1-2 cytoplasm, before production
nucleoli non-granular maturation
12-17 um
Hemoglobin
Condensation
Basophilic Undergoes synthesis,
of chromatin Intensely Bone
Normoblast 6:1 Mitosis large 24 hours
(deep purple basophilic marrow
(Prorubricytes) 1 or more amount of
red), 0-1
mRNA
nucleoli
Increase
hemoglobin
10-12 um 4:1 or 1:1 synthesis
Polychromatophilic Mitosis (2
Condensation Blue-gray to by the end Bone decline in
Normoblast daughter 30 hours
of chromatin, pink-gray of the marrow DNA
(Rubricyte) cells)
no nucleoli stage transcription
Last mitotic
stage
Hemoglobin
Orthochromic 8-10 um production,
Bone
Normoblast Completely Salmon-pink 1:2 NONE ejection of 48 hours
marrow
(Metarubricyte) condensed nucleus (loss
of vimentin)
Completion
Polychromatophilic
BM, Spleen, of 3 days or
Erythrocyte 8-10 um Salmon-pink
PB hemoglobin more
(Reticuloycte)
production
Biconcave
disc, Delivers
Salmon-pink oxygen to
Mature RBC 6-8 um Circulation 120 days
staining cell tissues,
with central deformability
pallor