HEMATOLOGY

Hematology from the Greek word “haima” meaning blood and “logy” meaning study.
Hematology, the discipline that studies the development and diseases of blood, is an essential medical science.
BLOOD
3 LAYERS OF BUFFY COAT
Total volume: (Ave. of 5 to 6 L for male, 4 to 5L for female)
-Upper most: Platelets
Solid portion: 20g / 100 ml of blood
-Middle layer: Agranulocytes
45 % Formed elements
-Lower layer: Granulocytes, nRBC
55 % Fluid portion
- 90% water
- 10% proteins, carbohydrates, Salts, hormones and other substances

GENERAL CHARACTERSITICS OF BLOOD

[Link] vivo, blood is in fluid form; in vitro, it coagulates 5-10 minutes
2. Thick and viscous; 3.5-4.5 times thicker than water
3. Approximately 20 grams solid per 100ml blood
4. Blood pH: 7.35-7.45 (average of 7.40)
PLASMA SERUM
5. Blood specific gravity
Whole blood: 1.045-1.066 Fluid portion of anticoagulated Fluid portion of non-
blood anticoagulated blood
Serum: 1.024-1.028
Plasma: 1.025 – 1.029 Slightly hazy appearance Clear appearance

[Link] Contains all coagulation factor Lacks fibrinogen group

Arterial (oxygenated) blood: Bright red
Venous (deoxygenated) blood: Dark Purplish Red
In the pulmonary area the color is reverse. The color of the blood in the pulmonary vein is bright red while the color of the blood in
the pulmonary artery is dark-purple red

7. Osmolality- concentration of solutes dissolved in the blood

Uses OSMOMETER for measurement
Reference range: 281-297 milliosmoles/ kg
8. Makes up 75 to 85 ml blood per kilogram body weight or 7 to 8% of the total Body weight
FUNCTIONS OF BLOOD
[Link] – most important
2. Nutritional
[Link]
4. Buffering Action
5. Maintenance of constant body temperature
[Link] of hormones and other endocrine secretions that regulate cell function
7. Body defense mechanism

ANTICOAGULANTS

ETHYLENEDIAMINE TETRA ACETIC ACID (EDTA)

EDTA is used in concentrations of 1.5 mg/1 mL of whole blood. The mode of action of this anticoagulant is that it removes
ionized calcium (Ca2+) through a process referred to as chelation. This process forms an insoluble calcium salt that prevents
blood coagulation
EDTA is available into 3 forms: Dry form (Na2 EDTA, and K2 EDTA), liquid form (K3 EDTA)
It is the anticoagulant of choice in HEMATOLOGY
Disodium salt also known as VERSENE
Tri-potassium salt also known as SEQUESTRENE
Action is to chelates calcium
K2 EDTA are recommended by the International Council for Standardization in Hematology (ICSH) and CLSI as the
anticoagulant of choice for blood cell counting and sizing because they produce less shrinkage of RBCs and less of an
increase in cell volume on standing.
For routine cell count and blood smear preparation
Preferred anticoagulant for platelet count
If concentration of EDTA exceeds 2mg/ml of Whole Blood, platelets may SWELL and FRAGMENT, causing False
Higher Count

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 Platelet satellitosis
-The phenomenon of “platelet satellitosis” may occur when using EDTA anticoagulant. Platelets adhere around
neutrophils, forming a ring of satellite effect. Using sodium citrate as an anticoagulant should correct this problem. Because
of the dilution in the citrate tubes, it is necessary to multiply the obtained platelet by 1.1(Rodak’s 5th Ed.)
Increase/excess EDTA = ______________________________
Not for coagulation tests
Factor V is not stable
It Inhibits fibrinogen –thrombin reaction

SODIUM CITRATE
Action is to binds calcium and form soluble complex
Anticoagulant of choice in coagulation studies
o Ratio of blood to anticoagulant is _________
o Should be buffered and use in concentration of 3.2 % or 0.109 M
o It preserves factor V and VIII

Anticoagulant for Standard Westergren (black top)

o Ratio of blood to anticoagulant is 4:1
o Use in Concentration of 3.8%
o Not recommended in coagulation studies because it can cause falsely prolonged coagulation results

REMINDERS
Underfilling/excess anticoagulant/short draw of the tube can cause prolonged PT and PTT due to excess
citrate in plasma

In Polycythemic patient, where there is an increase in hematocrit (≥ 55%) and less plasma present, there would be excess
in citrate and can cause prolong coagulation test results.
• The remedy is to reduce the volume of citrate
Formula #1: 100-Hct x ml of Whole blood
595-Hct

Formula #2: Volume of citrate (ml) = (1.85 x10-3) (100- Hematocrit) x Whole blood volume

OXALATE
Used in concentration of 1 to 2 mg/ml
Binds calcium to form insoluble calcium oxalate
Different forms: Lithium oxalate, sodium oxalate, potassium oxalate, and Double oxalate
Potassium oxalate is the most widely used
Double balanced oxalate (potassium oxalate + ammonium oxalate)
o (2parts) Ammonium oxalate (Winthrobe’s) = can cause cell swelling
o (3 parts) Potassium oxalate (Paul Heller’s) = can cause cell shrinkage

HEPARIN
Natural anticoagulant
Heparin is used as an in vitro and in vivo anticoagulant
Anticoagulant of choice on most chemistry test (lithium heparin)
Optimum concentration: 15 to 20 U/ml of blood or 15 to 30 U/ml of blood
Action: an acid mucopolysaccharide that inhibits coagulation by inactivation of thrombin
Anticoagulant for Osmotic Fragility Test, and LAP test
Not for blood film preparation
▪ Heparin destroys WBC and platelets
▪ Heparin can produce a bluish background on Romanowsky stained smear
Not for coagulation studies
▪ Heparin inhibit all stages of coagulation cascade specially thrombin
Heparin anticoagulation is used during percutaneous transluminal coronary angioplasty (PTCA) and cardiopulmonary
bypass (CPB) to prevent clot formation

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 SODIUM FLUORIDE
❖ Used for preserving glucose
❖ Inhibitor of glycolysis
❖ Can also be used in determination of lactic and blood alcohol

VENIPUNCTURE ORDER OF DRAW: ETS AND SYRINGE SKIN PUNCTURE ORDER OF DRAW
[Link] cultures- SPS (Yellow stopper) 8-10x inversion [Link] gases
[Link]- Sodium citrate (Blue stopper) 3-4x inversion [Link] unless made from EDTA microcollection tube
[Link] tubes with or without clot activator or gel separator [Link] microcollection tube
[Link] tube with or without gel- (Green stopper) 8-10x inversion [Link] anticoagulated microcollection tube
[Link] acetic acid- (Lavender stopper) 8-10x inversion [Link] anticoagulated microcollection tube
6. K2 EDTA with gel (White stopper) 8-10x inversion
[Link] fluoride Glycolytic inhibitor tubes or Oxalate- (Gray stopper)8-10
inversion

NOTE ABOUT YELLOW TUBE/STOPPER 😊

Sodium polyanethol sulfonate (SPS) -for blood culture specimen collections in microbiology. First tube in the sequence
of order of draw
Acid Citrate Dextrose (ACD) - used in blood bank studies, Lymphocytotoxicity testing, HLA phenotyping, and DNA and
paternity testing. Last tube in the sequence of order of draw

PHLEBOTOMY / BLOOD COLLECTION

☺A phlebotomist is frequently the only laboratory staff member that a patient sees.
☺Pediatric patient - When working with children, it is important to be gentle and treat them with compassion, empathy, and
kindness.
☺When obtaining a blood specimen from an adolescent, it is important to be relaxed and perceptive about any anxiety that he or
she may have.
☺Geatric patient - It is extremely important to treat geriatric patients with dignity and respect. Do not demean the patient. It is best
to address the patient with a more formal title such as Mrs., Ms., or Mr. rather than by his or her first name.

3 MAJOR WAYS TO COLLEC BLOOD: Skin puncture, Veni puncture, Winged Blood Collection Set (Butterfly)

SKIN PUNCTURE/CAPILLARY PUNCTURE

Skin puncture is the technique of choice to obtain a blood specimen from newborns and pediatric patients.
In adults skin puncture may be used in patients who are:
▪ Extreme obese
▪ Burned patient
▪ Vein with therapeutic
▪ Extremely fragile veins

Blood obtained from skin puncture is a mixture of blood from venules, arterioles, capillaries, and interstitial and
intracellular fluids.
Depth of puncture: NOT DEEPER THAN 2mm because of the risk of bone injury and osteomyelitis

SITES
1. Infants under 1 year of age is the lateral (outside) or medial (inside) plantar (bottom) surface of the heel
or big toe
2. Distal portion of the third (middle/long) or fourth (ring) finger on the nondominant hand may be used
3. Earlobe – less pain, fewer never endings, and less tissue juices.

CONSIDERATIONS
a. The phlebotomist should warm the site with a commercial heel warmer or a warm washcloth to a temperature no
greater than 42° C and for no longer than 3 to 5 minutes
b. Phlebotomist should carry RED, puncture resistant containers in their collection trays.
c. In finger puncture, puncture is made perpendicular to the fingerprint lines
d. False increase: WBC count
e. False decrease: RBC count, Hematocrit, and Hemoglobin, and platelet counts

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 VENIPUNCTURE (SYRINGE AND ETS METHOD)

EVACUATED TUBE SYSTEM

▪ Double pointed needle
▪ Most widely used for collecting venous blood sample
▪ Affected by environmental factors such as Ambient
temperature, altitude, humidity, and sunlight
▪ If evacuated tubes are stored at low temperature, the
pressure of the gas inside the tube will decrease. This would
lead to an increase in draw volume for the evacuated tube.
(Vice versa)
▪ In situations where blood is drawn at high altitudes
(>5,000ft), the resulting draw volume will be lower
▪ Storage temperature: should not exceed 25 Ç or 77’F
▪ Shelf-life: at least 1 year
▪ Shelf life of an evacuated tube is defined by the
stability of the additive and vacuum retention.
▪ Sterilization: through gamma irradiation

SITES
A. Veins in the arms- superficial veins of the antecubital fossa (bend in the elbow)
are the most common sites for venipuncture.
▪ Three major veins in the arm
• __________________________
• __________________________
• __________________________
B. Alternate vein sites including the ventral forearm, wrist area, back of the hand,
ankle or foot.

VENIPUNCTURE PROCEDURE
PATIENT INTERACTION Identify the patient, note patient isolation restrictions, note patient dietary restrictions,
Reassure patient, Verify paperwork, Position patient
ASSEMBLE OF SUPPLIES AND
EQUIPMENT
VENIPUNCTURE Select general venipuncture location
Apply tourniquet
Select exact venipuncture site
Cleanse area
Inspect needle
Perform venipuncture
Release tourniquet
Position gauze over puncture site
Remove needle and apply pressure
SPECIMEN PREPARATION If syringe used, fill tubes
Discard needle
Label specimens
Transport specimens promptly and properly

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 ADDENDUM IN VENIPUNCTURE
Color coded needle (18gauge: Pink), (21gauge: green), (22gauge: black), (23gauge: blue/torquise)
The larger the gauge the smaller the internal bore diameter (vice versa)
Routinely used gauge of 19,20, 21 gauge
needles 20,21,22 gauge (Brown)
IN INSERTING OF NEEDLE, THE BEVEL SHOULD BE UPWARD
THE COMMONLY/STANDARD USED NEEDLE: 21g (Rodak’s and Turgeon)
Length of needle Standard = 1inch
Range = 1 to 1.5inches
Venipuncture angle Standard:15-degree angle
Range of 15 to 30-degree angle
Torniquet application -Should not exceed 1 minute/60 seconds, 2mins (if walang 1minute -Turgeon)
-3 to 4 inches (7.5 cm to 10cm) above venipuncture site
Blood pressure cuff as Standard:60mmHg
tourniquet Range: 40 to 60mmHg
Number of attempts Twice then call another Medtech
Position of the patient Lying down – hemodilution: ↓ Packed cell Volume by 8 %, ↓ WBC
Standing/Up- hemoconcentration: ↑ Packed Cell Volume 8%, ↑ WBC
Waste disposal (DOH) Yellow bag = Infectious waste
Orange bag = Radioactive waste
Red bag = Sharp waste
Yellow with black band = Chemical waste
Green bag = Non-infectious Wet waste or Biodegradable waste
Black bag = Non-infectious Dry waste or Non-biodegradable waste
Special considerations Blood should never be drawn from a vein in an arm with a cannula
(temporary dialysis access device) or fistula (a permanent surgical fusion of a
vein and an artery).
Venipuncture should not be performed on the same side as the mastectomy.
Venipuncture should not be performed on areas with scars or burns
Venipuncture should not be performed in edematous areas because the extra fluid
can make it difficult to palpate the veins, and the specimen may be contaminated
with the fluid and produce erroneous test results.

Patient with IV line Mastectomy Patient

1st: Use the opposite arm 1st: draw blood from the opposite arm
2nd: in case of both arm with IV line, ask the nurse to stop the IV for 2nd: In case of double mastectomy,
2 mins, discard 5 ml draw blood from the back of the hand
3rd: then collect sample below the IV line (1 to 2inches below) or perform skin puncture.
do not use tourniquet

PHLEBOTOMY PROBLEMS
Problem Remedy
Refusal by the patient to have blood drawn The response to this problem is to politely excuse yourself from the
patient’s room, note the refusal on the requisition, and notify the
hematology supervisor.
Difficulty in obtaining a specimen because the Slightly pulling back on the needle may solve this problem.
bore of the needle is against the wall of the vein
Movement of the vein To guard against this problem, always have firm pressure on the arm
below the intended venipuncture site.
Sudden movement by the patient or phlebotomist Immediately remove the tourniquet, place a gauze pad on the
that causes the needle to come out of the arm venipuncture site, and apply pressure until bleeding has stopped to prevent
prematurely. the formation of a hematoma
Fainting or illness subsequent to venipuncture. The first aid procedures of the laboratory should be practiced in this event
Blood clot formation in anticoagulated tubes Promptly after termination of the venipuncture procedure, any tubes
containing an anticoagulant should be gently inverted several times to mix
the specimen

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 PHLEBOTOMY COMPLICATIONS
Vascular complications Bleeding, hematoma, bruising, psuedoaneurysm, thrombosis, reflex arteriospasm, and arteriovenous
(MOST COMMON) fistula formation
Infectious complications Cellulitis (inflammation of tissue), phlebitis (inflammation of blood vessel), sepsis, septic arthritis, and
osteomyelitis
Cardiovascular Orthostatic hypotension, syncope, shock, and cardiac arrest
Anemia Iatrogenic, nosocomial, physician induced, anemia resulting from blood loss
Neurological Diaphoresis, seizure, pain, and nerve damage
Dermatological Allergic reaction to iodine, necrosis, basal cell carcinoma, and scarring

VASCULAR COMPLICATIONS OF PHLEBOTOMY

Pseudoaneurysm Fibrous capsule around encapsulated blood caused by a break in the blood vessel
Thrombosis The patient usually has a coagulation disorder. Thrombosis in a vein produces edema and swelling. If
thrombosis is in an arterial blood vessel, a decreased oxygen supply caused by impaired circulation can
occur beyond the thrombosis.
Reflex arteriospasm Occurs when a needle sticks an artery. Prevents blood from moving through the vessel
Arteriovenous fistula Abnormal connection between a vein and an artery can occur after repeated venipuncture
Hematoma results when leakage of a large amount of blood around the puncture site causes the area to
rapidly swell.
Bruising(ecchymosis) The most common complication encountered in obtaining a blood specimen
-It is caused by leakage of a small amount of blood in the tissue around the puncture site.

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 BLOOD SMEAR PREPARATION AND STAINING

METHODS OF BLOOD FILM PREPARATION

1. Cover glass smear (Ehrlich) – 2 cover glass smears

2. Cover glass and slide (Beacom)

3. Wedge smear = uses 2 slides; angle between the 2 slides → 25 / 30-40 / 30-450 degree angle (PREFERRED)
Easiest to master
Most convenient, and most commonly used method, recommend by CLSI for WBC differential counting
Slide: 3 x 1 inch or 75mm x 25mm, 1-1.2 mm thick

4. Spun smear
-Buffy coat smear → for platelet and WBC count (<1.0x109/L), For demonstration of LE cells
-Thick blood smear → blood parasites (malaria)

5. Automated methods
Miniprep – semi automatic, portable instrument, simulates the manual wedge technique
Cenrifugal (Spinner) type – uses approximately 0.2ml of well mixed anticoagulated blood. It produces a uniform blood film

Considerations in Preparation of blood film

Size (drop of blood) = _________________________
Speed of spreader = rapid and smooth
Hematocrit of patient
▪ Polyctemia vera (increased Hct) = angle of the spreader must be lower to 250
▪ Anemia (decreased Hct) = angle of the spread must be higher

Too Thin smears Too Thick smears

Pressure
Angle
Speed
Size of blood

CHARACTERISTIC OF AN IDEAL BLOOD SMEAR (Wedge method)

1. Gradual transition from thick to thin area
2. 2/3 to ¾ the length of the film slide
3. Finger-shaped
4. Visible lateral edges
5. Appears smooth without irregularities, holes, streaks, waves or gaps
6. Feather edge has rainbow appearance
7. It does not touch the outer borders of the slide or run off the sides or ends of the slide.

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 SCANNING / COUNTING METHODS OF CELLS
1. Cross sectional / Crenellation: WBCs are counted in consecutive fields as the blood film is moved from side to side

2. Longitudinal: WBCs are counted in consecutive fields from tail toward the head of the smear

3. Battlement/Track pattern/ Back and forth serpentine: uses a pattern of consecutive fields beginning near the tail on a
horizontal edge: count three consecutive horizontal edge field, count two fields towards the center of the smear, count two
fields horizontally, count two fields vertically to the edge. Most preferred counting method

BLOOD FILM STAINING

Romanowsky stains - A polychrome stain that is defined as any stain containing methylene blue and/or its products of
oxidation and a halogenated fluorescein dye, usually eosin B or Y.
Examples of Romanowsky stain are Wright’s stain, Giemsa stain, Leishman stain, Jenner stain and May Grunwald
The oxidation of methylene blue results in a solution containing primarily methylene blue; azures A, B, and C; methylene violet;
and thionin symdimethylthionin
The characteristic colors are purple in the cell nucleus, blue and pink in the cytoplasm, and various colors in specific granules.
Films stained well with Wright’s stain have a pink color when viewed with the naked eye.

Fixative Methanol
Stain: Wright’s stain- methylene blue + eosin Y
Buffer Aged distilled water or 0.05M sodium phosphate (pH 6.4-6.8)

For best results, blood smears should be stained 2 to 3 hours of specimen collection
High-quality blood films can be made from the blood in the EDTA tube, provided that they are made within 2 to 3 hours of
drawing the specimen (Rodak’s 5th edition)
High-quality blood films can be made from the blood in the EDTA tube, provided that they are made within 4 hours of drawing
the specimen (Rodak’s 6th edition)
For blood and Bone marrow staining: pH 6.8
For Malarial parasites: pH of 7.2

WELL STAINED PERIPHERAL BLOOD FILM

Macroscopically Microscopically
a well-stained blood film should be RBCs should appear orange to salmon pink
pink to purple. WBC nuclei should be purple to blue.
Cytoplasm of neutrophils should be pink to tan with violet or lilac granules.
Eosinophils should have bright orange refractile granules.

STAINING PROBLEMS
EXCESSIVELY BLUE STAIN EXCESSIVELY PINK STAIN
1. Thick films 1. Thin smears
2. Prolonged staining time 2. Insufficient staining
3. Inadequate washing 3. Prolonged washing
4. Too high alkalinity in the stain or buffer 4. Mounting the coverslips before they are dry
5. Too high acidity of the stain or the buffer

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 ADDITIONAL INFORMATION
1. film that is bluer overall than normal may indicate that the patient has increased blood proteins, as in plasma cell
myeloma, and that rouleaux may be seen on the film
2. A grainy appearance to the film may indicate RBC agglutination, as found in cold hemagglutinin diseases.
3. Holes all over the film could mean that the patient has increased lipid levels.
4. Slides stained after 1 week or longer turn out too blue.

Other staining problems:

1. Inadequately stained red cells, nuclei, or eosinophilic granules may be due to understaining or excessive washing.
2. Precipitate on the film may be due to unclean slides
3. Drying during the period of staining
4. Inadequate washing of the slide at the end of the staining period, especially failure to hold the slide horizontally during
initial washing
5. Inadequate filtration of the stain
6. Permitting dust to settle on the slide or smear

DRYING TECHNIQUES FOR BLOOD FILM

➢ Blood films and bone marrow smears should be dried as quickly as possible to avoid drying artifact
➢ A small fan can be used to facilitate drying
➢ Blowing breath on a slide causes RBC to become echinocyte or to develop water artifact (Drying artifacts)

Water Artifacts or Drying artifacts after blowing breath on SLIDE

1. moth-eaten look to the RBCs, or it may appear as a heavily demarcated central pallor
2. appear as refractive (shiny) blotches on the RBCs
3. manifests simply as echinocytes (crenation)

HEMATOPOIESIS
Hematopoiesis is a continuous, regulated process of blood cell production that includes cell renewal, proliferation,
differentiation, and maturation. These processes result in the formation, development, and specialization of all of the
functional blood cells that are released from the bone marrow to the circulation.

HUMAN STEM CELL

❖ Hematopoietic stem cells (HSCs) are the foundation of the adult hematopoietic system.
❖ Functionally, three types of human stem cell exist:

1. TOTIPOTENTIAL STEM CELL-These cells are present in the first few hours after an ovum is fertilized.
Totipotential stem cells, the most versatile type of stem cell, can develop into any human cell type, including
development from embryo into fetus.
2. PLURIPOTENTIAL STEM CELL- These cells are present several days after fertilization. Pluripotent stem cells can
develop into any cell type, except they cannot develop into a fetus.
3. MULTIPOTENTIAL STEM CELL-. These cells are derived from pluripotent stem cells. They can be found in adults, but
they are limited to specific types of cells to form tissues. For example, bone marrow stem cells can produce all
types of blood cells, bone cartilage, and adipose (fat) cells.

STEM CELL THEORY

A. Monophyletic theory- suggests that all blood cells are derived from a single progenitor stem cell called a pluripotent
hematopoietic stem cell. The monophyletic theory is the most widely accepted theory among experimental hematologists
today
B. Polyphyletic theory- suggests that each of the blood cell lineages is derived from its own unique stem cell.

HEMATOPOEITIC MICROENVIRONMENT
It is an ideal environment of HSC is the allowance for: Self renewal, Proliferation and Differentiation,
Apoptosis
Stromal cells: General term for specialized cells within the BM that provide protective and nourishing environment
to the HSCs. All these cells secrete substances that make up the extracellular matrix which are essential for cell growth
and support of the HSC’s.

Example of Stromal cells Reticular adventitial cells or Fibroblast, Adipocytes, Lymphocytes, Endothelial cells,
Monocyte/Macrophages, Osteoclast and Osteoblast, Glial cells, Perivascular

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 THREE STAGES OF HEMATOPOIETIC DEVELOPMENT
Mesoblastic Begins around 19th day of embryonic development after fertilization.
or Early in embryonic development, cells from the mesoderm migrate to the yolk sac.
Formation of primitive erythroblast in the central cavity of the yolk sac.
Yolk Sac
These primitive but transient yolk sac erythroblasts are important in early embryogenesis to produce
phase
hemoglobin (Gower-1, Gower-2, and Portland)
Stage for primitive hematopoiesis

EMBRYONIC HEMOGLOBIN GLOBIN CHAIN COMBINATION

Gower I
Gower II
Portland
Hepatic The hepatic phase of hematopoiesis begins at 5th to 7th week of gestation
Phase Characterized by recognizable clusters of developing erythroblasts, granulocytes, and monocytes colonizing
the fetal liver, thymus, spleen, placenta, and ultimately the bone marrow space in the final medullary phase.
Production of megakaryocytes also begins during the hepatic phase
Hematopoiesis during this phase occurs extravascularly, with the liver remaining the major site of
hematopoiesis during the second trimester of fetal life
During the hepatic phase, fetal hemoglobin (Hb F) is the predominant hemoglobin, but detectable
levels of adult hemoglobin (Hb A) may be present
Stage for the beginning of definitive hematopoiesis with a decline primitive hematopoiesis in
the yolk sac

FETAL HEMOGLOBIN GLOBIN CHAIN COMBINATION

Hb F
Medullary Prior to the fifth month of fetal development, hematopoiesis
(Myeloid) begins in the bone marrow cavity
Phase By the end of 24 weeks’ gestation, the bone marrow becomes the primary site of hematopoiesis.
Measurable levels of erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocyte-
macrophage colony stimulating factor (GM-CSF), and hemoglobins F and A can be detected
In adult hematopoiesis, the red marrow is found only in the:
R-Ribs
S-Sternum, Shoulder blade, skull(children)
V-Vertebrae
P-Pelvic bone, Proximal end of long bones
Note ☺ Sternum and other flat bones are the principal source of production in adult

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 ADULT HEMOGLOBINS GLOBIN CHAIN COMBINATION
Hb A (predominant)
Hb A2

Site of adult hematopoietic tissue Bone marrow (major site), lymph nodes, spleen, liver, and thymus

Reference range for normal adult Hgb is 95- 97 %Hb A, 2 to 3%Hb A2, and ≤1% Hb F

BONE MARROW
Bone marrow, one of the largest organs in the body, is the tissue located within the cavities of the cortical bones

Red/Active marrow Hematopoietically active marrow consisting of the developing blood cells and their progenitors
Yellow /Inactive Hematopoietically inactive marrow composed primarily of adipocytes (fat cells), with undifferentiated
marrow mesenchymal cells and macrophages

During infancy and early childhood, all the bones in the body contain primarily red (active) marrow.
The process of replacing the active marrow by adipocytes (yellow marrow) during development is called retrogression
NORMAL MYELOID TO ERYTHROID RATIO 2:1, 3:1, 4:1 (Monocytes and Lymphocytes are excluded from the M:E ratio)

Scenario M:E ratio Interpretation (M:E ratio)

Infection 6:1 Increase
Leukemia 25:1 Increase
Myeloid hyperplasia 20:1 Increase
Myeloid hypoplasia 3:20 Decrease
Erythroid hyperplasia 1:20 Decrease
Erythroid hypoplasia 5:1 Increase

[Link] Cellularity- ratio of marrow cells to fat

Normocellular Marrow has 30 to 70% HSCs

Hypercellular/Hyperplastic Marrow has >70% HSCs
Hypocellular/Hypoplastic Marrow has <30% HSCs
Aplastic Marrow has few or no HSCs

[Link] Marrow Cells

a. Macrophages
b. Mast cells – Tissue basophils
c. Osteoblasts
▪ Bone forming cells
▪ Waterbug or comet appearance.
▪ They are Mistaken as PLASMA CELLS
d. Osteoclasts
▪ Bone resorption or destroying cells.
▪ Mistaken as MEGAKARYOCYTE

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 III. Bone Marrow smear collection and Preparation
a. Posterior iliac crest – most commonly used site
b. Sternum- occasionally used site
c. Anterior iliac crest and spinal/vertebral bodies- very rarely used site
d. Upper end of tibial bone– for newborns and infants

IV. Bone Marrow Specimen and Instruments

a. Trephine (Core) biopsy – trephine biopsy needle / Jamshidi needle / Westerman-Jensen needle
b. Bone marrow Aspirate – aspiration needle / University of Illinois sternal needle

[Link] of Bone marrow Specimen

a. Direct aspirate smears
b. Anticoagulated aspirate smears
c. Crush smears
d. Imprints/ Touch preparation
e. Concentrate/ Buffy coat smear
f. Histologic/ Cell block

VI. Bone marrow smears – should be retained for 10 years

VI. Extramedullary hematopoiesis

Blood cell production outside the bone marrow
Occurs when the bone marrow cannot meet body requirements
Occurs mainly in the liver and spleen; hepatomegaly and/ or splenomegaly often accompany this.
Can also occur the in lymph nodes
Found in cases of:
- When the bone marrow becomes dysfunctional in cases such as aplastic anemia, infiltration by malignant cells, or
overproliferation of a cell line (e.g., leukemia, myeloproliferative disorder).
- When the bone marrow is unable to meet the demands placed on it, as in the hemolytic anemias

VII. Bone Marrow Differential - a procedure which requires counting at least 500, and preferably 1000 cells be counted for a
marrow differential
NORMAL RANGES OF BONE MARROW
CELLS IN ADULT
CELL PERCENTAGE
Myeloblast 0.3- 4.0

Promyelocyte 1.0-5.0

Myelocytes
a. Neutrophil 5.0-19.0
b. Eosinophil 0.5-3.0
c. Basophil 0.0-0.5

Metamyelocyte/ Juvenile 13.0-32.0

Neutrophils 7.0-25.0

Eosinophils 0.5-4.0

Basophils 0.0-0.7

Lymphocytes 3.0-20.0

Monocytes 0.5-3.0

Megakaryocytes 0.1-3.0

Plasma cells 0.1-3.0

Reticulum cells 0.1-.0

Pronormoblast 1.0-5.0

Basophilic normoblast 7.0-32.0

Polychromatophilic normoblast
Orthochromic normoblast

Mitotic cells 0.0-2.0

Note😊 Polychromatophilic and orthochromic normoblast = two most common erythrocytic maturation stages = fried egg app.

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 The bone marrow is estimated to be capable of producing approximately 2.5 billion erythrocytes, 2.5 billion platelets, and
1 billion granulocytes per kilogram of body weight daily

HSCs exist in the marrow in the ratio of 1 per 1000 nucleated blood cells

The largest cell in the Bone Marrow

The most predominant cell in the Bone Marrow

Mitotic index Can be calculated to establish the percentage of cells in mitosis in relation to the total number of cells. Factors
affecting the mitotic index include the duration of mitosis and the length of the resting state. Normally the mitotic
index is approximately 1% to 2%. An increased mitotic index implies increased proliferation. An exception to this
rule is in the case of megaloblastic anemia, in which mitosis is prolonged

Flow cytometry The identification and origin of HSCs can be determined by immunophenotypic analysis using

Culture assays Functional characterization of HSCs can be accomplished through in vitro techniques using____. These involve
the enumeration of colony-forming units (e.g., CFU-GEMM) on semisolid media, such as methylcellulose.
In vivo functional assays also are available and require transplantation of cells into syngeneic, lethally irradiated
animals, followed by transference of the engrafted bone marrow cells into a secondary recipient

TGF-Beta, TNF-Alpha, and Interferons Cytokines that exert a negative influence on hematopoiesis
KIT ligand, FLT3 ligand, GM-CSF, IL-1, IL-3, Cytokines or Hematopoietic growth factors that exert a positive influence on HSCs
IL-6, and IL-11 and progenitor cells

SPLEEN
Largest lymphoid organ in the body
Site of extramedullary hematopoiesis
Stores 30 % of platelets (1/3 of total platelet)

WHITE PULP= lymphoid tissue contains Lymphocytes, macrophages and dendritic cells
a. PALS (periarteriolar lymphoid sheaths) – where T cells reside
b. Primary follicles – where B cells reside
c. Germinal center or Secondary follicle – where activated B cells reside

The marginal zone surrounds the white pulp and forms a reticular meshwork containing blood vessels, macrophages, memory B cells,
and CD4 T cells

RED PULP= Cord of billroth, Contains specialized macrophages for removal of the senescent RBC’s:
The red pulp makes up more than one half of the total volume, and its function is to destroy senescent RBCs (Culling) or remove
RBC inclusion (pitting)
Culling – removal of mature or senescent RBC through phagocytosis that leads to eventual degradation of cell organelles
Pitting- removal of cell inclusions.

HYPERSPLENISM/SPLENOMEGALY – Leads to pancytopenia

ASPLENIA /SPLENECTOMY -Leads to pancytosis
AUTOSPLENECTOMY – Necrosis of spleen due to entrapment of sickled RBC such as in case of sickle cell anemia.

ASPLENIA (the absence of splenic function) can be caused by either surgical removal (e.g., splenectomy) or radiation
overexposure. This condition can also occur in association with malabsorption syndromes. The hematologic results of asplenia
include:
▪ Increased susceptibility to infection
▪ Acute granulocytosis
▪ Acute thrombocytosis, with occasional giant platelets
▪ Chronic and absolute lymphocytosis and monocytosis
▪ Increased appearance of immature RBCs in the circulation
▪ Increased amount of circulating RBCs with cytoplasmic inclusions or abnormal forms (e.g., Howell-Jolly bodies, target cells,
and burr cells)

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 COMMONLY USED MARKERS
CD 2, CD 3 Lymphoid pan T cells
CD 4 Helper / Inducer T cells
CD 8 Suppressor / Cytotoxic T cells
CD 13 Pan Myeloid
CD 11c, CD 14 Monocytes
CD 19, CD 20 Lymphoid, pan B cells
CD 33 Pan myeloid cells
CD 34 Hematopoieitic stem cell marker
CD 16, CD 56 NK Cells
CD 10 Pre-CALLA (Common acute
lymphoblastic leukemia) marker

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 ERYTHROCYTE PRODUCTION AND DESTRUCTION

[Link]
A process by which erythroid precursor cells differentiate to become mature RBC. The primary regulator of this process is
ERYTHROPOIETIN (produced mainly by the peritubular cells of the Kidney, and to a lesser extent by the liver). It
normally takes around 3 to 5 days for the production of reticulocytes from pronormoblasts.
The reticulocytes remain in the bone marrow for 1 to 2 days before being released in the circulation. In the peripheral circulation,
continues to mature for one more day.
The morphologically identifiable erythrocyte precursors develop from two functionally identifiable progenitors, burst-forming
unit–erythroid (BFU-E) and colony-forming unit–erythroid (CFU-E), both committed to the erythroid cell line. Estimates of time
spent at each stage suggest that it takes about one week for the BFU-E to mature to the CFU-E and another week for the
CFU-E to become a pronormoblast, which is the first morphologically identifiable RBC precursor
It takes approximately 18 to 21 days for the BFU-E to mature to an RBC, of which about 6 days are spent as identifiable
precursors in the bone marrow.
The basic substances needed for normal erythrocyte and hemoglobin production are amino acids (proteins), iron, vitamin B12,
vitamin B6, folic acid (a member of the vitamin B2 complex), and the trace minerals cobalt and nickel

Major Stimulus Hypoxia

Other Stimulus Testosterone = directly stimulates erythropoiesis
Pituitary and Thyroid hormone = indirect effect to erythropoiesis

NOMENCLATURE OF ERYTHROID PRECURSORS

RUBRI NORMOBLAST ERYTHROBLAST
Rubriblast Pronormoblast Proerythroblast
Prorubricyte Basophilic normoblast Basophilic erythroblast
Rubricyte Polychromatophilic normoblast Polychromatophilic erythroblast
Metarubricyte Orthochromic normoblast Orthochromic erythroblast
Reticulocyte Reticulocyte Reticulocyte
Mature Eryhtrocyte Mature Erythrocyte Mature erythrocyte
Note: Erythron – General term for RBC Maturation series

Summary of General Maturational Characteristics

Erythroid Progenitors Morphological Feature Usual Development
General size Decreases with maturity
Burst Forming Unit (BFU-E)
Nuclear cytoplasmic: ratio Decreases with maturity
(resembles a cluster of grapes)
NUCLES

↓ Chromatin pattern Becomes more condensed

Colony Forming Unit (CFU-E) Presence of Nucleoli Not visible in mature cells
(they have many receptors for EPO) Nuclear sized Decreases with maturity
↓
CYTOPLASM
Color Progresses from darker blue to lighter
Precursors
blue, blue gray or pink
(loss of basophilia)
Granulation Progresses from no granules to
nonspecific to specific granules
Vacuoles Increases with age

BFU-E The CFU-GEMM gives rise to the earliest identifiable colony of RBCs, called the____ that contains only a
few receptors for EPO

Chromatin pattern In differentiating mature from immature RBCs, the emphasis should be in ___________

Raspberry-like The nuclear chromatin of erythroid precursors is inherently coarser than that of myeloid precursors. It
becomes even coarser and more clumped as the cell matures, developing a ______ appearance, in which
the dark staining of the chromatin is distinct from the almost white appearance of the parachromatin

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 ERYTHROPOIETIN
EPO is a thermostable, nondialyzable, glycoprotein hormone with a molecular weight of 34 kiloDalton
EPO is a true hormone, being produced at one location (kidney) and acting at a distant location (bone marrow). It is a growth
factor (or cytokine) that initiates an intracellular message to the developing erythroid cells; this process is called signal
transduction.
The EPO receptor is a transmembrane protein homodimer with extracellular and cytoplasmic [Link] binding of EPO to
the extracellular domain of the EPO receptor (on erythrocyte progenitors and early precursors) results in a change in the
conformation of the receptor.2 This activates Janus activated tyrosine kinase 2 (JAK2) signal transducers that are associated
with the cytoplasmic domains of the EPO receptor.JAK2 then activates downstream signal transduction pathways (such as
the signal transduction and activator of transcription 5 or STAT5 pathway) that ultimately promotes transcription of specific
genes in the RBC nucleus

Functions of EPO (Turgeon, 6th edition)

1. Accelerates the rate of mRNA and protein (hemoglobin) synthesis
2. Decreases the time for the maturation of metarubricyte
3. Stimulates the premature release of immature RBC, Reticulocytes from the bone marrow
4. Increases the rate of extrusion of a red blood cell nucleus (enucleation)

Three Major Effects of EPO (Rodak’s 6th edition)

(1) allowing early release of reticulocytes from the bone marrow,
(2) preventing apoptotic cell death,
(3) reducing the transit time or time needed for cells to mature in the bone marrow.

Measurement
Quantitative measurements of EPO are performed on plasma and other body fluids. EPO can be measured by chemiluminescence level
of 10 to 30 U/L is sufficient to maintain steady-state erythropoiesis in a healthy adult.

RED BLOOD CELL MATURATION SERIES (PRECURSORS)

Rubriblast 12 to 19 um diameter
(1st stage) Deeply basophilic cytoplasm
Non granular
N/C ratio is about 8:1
Fine chromatin
Usually 1-2 nucleoli
Earliest morphologically recognizable precursor
The nucleus is round or oval
This stage lasts slightly more than 24hrs
Most of the iron destined for hemoglobin synthesis is taken into the cell at
this stage.

Prorubricyte 12-17 um diameter

(2nd stage) Intensely basophilic cytoplasm
N/C ratio is about 6:1
Chromatin slightly coarse
Nucleoli are usually not visible
This stage lasts slightly more than 24hrs

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 Rubricyte 11-15 um diameter
(3rd stage) Blue-gray to pink-gray cytoplasm or muddy/murky, light gray cytoplasm
N/C ratio is about 4:1
More condensed; coarse and clumped chromatin
Distinct parachromatin
Last stage capable of mitosis
First stage of hemoglobin synthesis
Hemoglobin appears for the first time
This stage lasts approximately 30 hours

Metarubricyte 8-12 um diameter

(4th stage) Salmon Pink cytoplasm
N/C ratio is about 1:2
Small pyknotic nucleus (homogenous blue-black mass)
The nucleus moves to the cell membrane and into a pseudopod-like projection
The enveloped extruded nucleus, called a pyrenocyte, is then engulfed by bone marrow
macrophages. The macrophages recognize phosphatidylserine on the pyrenocyte surface as an “eat
me” flag
Last nucleated stage
This stage last approximately 48 hours
The first stage that is INCAPABLE OF MITOSIS
Three mitoses are believed to occur in the 2- to 3-day interval between the rubriblast and the end
of the metarubricyte stage

Reticulocyte 7-10 um diameter

Pink to slightly pinkish gray cytoplasm
Polychromatic Contains fine basophilic reticulum of RNA, which is only visible with supravital stain
erythrocyte or Last stage capable for the production of hemoglobin
macrocyte
In a Wright-stained blood smear, young reticulocytes with a high amount of RNA residual have a
blue appearance, which is referred to as polychromatophilia or polychromasia
Diffusely basophilic By the end of the polychromatic erythrocyte stage, the cell is the same color as a mature RBC,
erythrocyte salmon pink.
The shape of the cell is irregular in electron micrographs

STRESS OR SHIFT RETICULOCYTES

Stress reticulocytes are recognizable on Wright-stained blood smears by their larger size
and increased blue tint
Stress reticulocytes are prematurely released earlier than normal retics from the
BM such as in cases of hemolytic anemia. They compensate anemia.
Stress or shift reticulocytes are larger than normal reticulocytes
Instead of losing their reticulum in 1 day, as do most normal circulating reticulocytes, these stress
reticulocytes take 2 to 3 days to lose their reticula.

Note: Wright-stained polychromatic erythrocytes are also called diffusely basophilic erythrocytes for their regular bluish
tinge. This term distinguishes polychromatic erythrocytes from red blood cells with punctate basophilia (basophilic
stipplings), in which the blue appears in distinct dots throughout the cytoplasm. In a Wright-stained blood smear, young
reticulocytes with a high amount of RNA residual have a blue appearance, which is referred to as polychromatophilia

Mature Erythrocyte ❖ 6-8 um diameter

/ Discocytes ❖ Salmon-Pink in color
❖ Non- nucleated, round, biconcave disc with a central pallor that occupies the middle 1/3 of the cell.
❖ With an average of 120 ±20 days lifespan

NOTE! ☺
Up to 16 erythrocytes produced from a single rubriblast
8-32 erythrocytes are produced from a single rubriblast (Rodak’s 5th, Ed.)

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 The transition of a reticulocyte into a mature erythrocyte is accompanied by extensive changes in the structure and
properties of the plasma membrane. These changes include:
1. Increase in shear resistance
2. Loss of surface area (about 20%) due to loss of membrane lipid
3. Acquisition of a biconcave shape
4. Others: loss of organelles, and will not undergo exocytosis or endocytosis

ASYCHRONOUS ERYTHROPOIESIS
TYPE CAUSE EXPLANATION CHARACTERISTIC EXAMPLE
Iron deficiency Iron deficiency Cytoplasm lags behind nucleus Microcytic, hypochromic IDA
in maturation due to inadequate RBC
iron for hemoglobin synthesis
Megaloblastic Vit.B12 or folic Nucleus lags behind cytoplasm Oval macrocytes Pernicious anemia
acid deficiency in maturation. Cells grow larger
without dividing

ROLE OF MACROPHAGES IN ERYTHROPOIESIS

Erythropoiesis typically occurs in what are called erythroid islands that is surrounded by macrophages
Suckling pig phenomenon- macrophages provides iron directly to the normoblast for hemoglobin synthesis
Serves as the major cellular anchor for the RBC

ADDENDUM
Inversely Blood levels of erythropoietin are ________ related to tissue oxygenation
10 TO 15% ______ percent of erythropoietin production occurs in the liver, which is the primary source of erythropoietin
in the unborn
BFU-E Are not actively proliferating. Most of these cells are in the GO/G1 phase of the cell cycle.
Reticulocytes The earliest Erythrocyte precursor that is normally found in the peripheral blood/circulation
New methylene blue Supravital stain to demonstrate the reticulum of RNA of reticulocytes
1 to 2 days The reticulocyte resides in the bone marrow for __ day/s and then moves into the peripheral blood for about
___ day before reaching maturity
RBC mass The erythron is the entirety of erythroid cells in the body, whereas the ___ refers only to the cells in circulation
20% EPO also can reduce the time it takes for cells to mature in the bone marrow by reducing individual cell cycle
time, specifically the length of time that cells spend between mitoses. This effect is only about a ______
reduction
Present only in the BM in healthy individuals Rubriblast, Prorubricyte, Rubricyte, Metarubricyte
Peritubular fibroblasts of the kidney The primary oxygen-sensing system of the body is located in ____
Reduced In case of hypertransfusion, the quantity of EPO is_______
Decrease PMCA and HbA1c = increase RBC Plasma membrane Ca2+ (PMCA) and glycated hemoglobin, HbA1c, are reliable age
age markers for normal RBCs.

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 [Link] MEMBRANE PHYSIOLOGY

The biconcave disc shape of a mature RBC is crucial to its function, allowing for close to maximum surface- to –volume
ratio and optimal gaseous exchange. The ability of the circulating erythrocyte to perform function of gaseous exchange relies
almost exclusively on the integral and structural composition and functional capabilities of the membrane components and
sufficient generated ATP.
RBCs are biconcave and average 90fl in volume, with an average surface area of 140 um
A normal RBC = Increase deformability
The RBC membrane is impermeable to cations Na, K, and Ca. It is permeable to water and the anions bicarbonate (HCO3) and
chloride (Cl), which freely exchange between plasma and RBC cytoplasm.

Main physiologic functions of the RBC membrane:

▪ Maintain cell and deformability
▪ Maintain osmotic balance between plasma and the cell cytoplasm
▪ Act as supporting skeletal system for surface antigens and receptors
▪ Aid in the transportation of essential cellular ions and gases

RBC MEMBRANE COMPONENTS

PROTEIN (50%) LIPID (40%) CARBOHYDRATES
(10%)
A. Integral/Transmembrane proteins A. External surface- phospholipids,
• Glycophorin A- for MN phosphatidylcholine, glycolipid, and
sphingomyelin
• Glycophorin B- for Ss
B. Internal surface- phosphatidyl ethanolamine
• Glycophorin C/D- for Gerbich
Phospatidyl inositol, Phosphatidyl serine
• Band 3
• Aquaporin 1
Cholesterol content of the membrane depends upon
• RhAg
the concentration of plasma cholesterols, bile acids,
B. Peripheral/ Cytoskeletal proteins-
and the activity of the enzyme Lecithin:Cholesterol
• Spectrin acyltransferase (LCAT)
• Ankyrin
• Actin
• Myosin
• Band 4.1
• Adducin and dematin
• Tropomodulin

Note ☺ ! OTHER BOOKS: 52% PROTEIN, 40% LIPID, 8% CARBOHYDRATES

RBC transmembrane/Integral proteins -They serve as transport and adhesion sites and signaling receptors
-They support carbohydrate-defined blood group antigens
Cytoskeletal protein/Peripheral proteins Provides the shape and flexibility of the RBC, which are essential to its function

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 [Link] Blood Cell Energy Metabolism
Emden-Meyerhof _____________________
Pathway Major energy provider
Glucose enters the RBC without energy expenditure via the transmembrane protein GLUT-1
EMP - Converts glucose to lactic acid and generate a net ATP gain of 2 ATP molecules
_______________________- most common enzyme deficiency

Hexose __________________________
monophosphate The HMP detoxifies peroxide (H2O2), which arises from O2 reduction in the cell’s aqueous environment,
or Pentose where it oxidizes and destroys heme iron, proteins, and lipids, especially lipids containing thiol groups. By
phosphate detoxifying peroxide, the HMP extends the functional life span of the RBC.
Pathway Reduction of NADP (nicotinamide-adenine dinucleotide phosphate) to NADPH (the reduced form of NADP),
which is subsequently required to reduce glutathione
G6PD provides the only means of generating NADPH for glutathione reduction, and in its absence,
erythrocytes are particularly vulnerable to oxidative damage
Provides reduced glutathione (GSH) to prevent oxidative denaturation of hemoglobin which makes it a
protective mechanism
______________-most common inherited enzyme deficiency and is associated with Heinz bodies

Methemoglobin Heme iron is constantly exposed to oxygen and peroxides. Peroxide oxidizes heme iron from the ferrous
Reductase (2+) to the ferric (3+) state. When the iron state is ferric, the affected hemoglobin molecule is called
Pathway methemoglobin.
This pathway Converts methemoglobin back to normal hemoglobin using the methemoglobin reductase
enzyme which makes it a corrective mechanism
Cytochrome B5 reductase / methemoglobin reductase is involved for reducing ferric iron into ferrous state

Rapaport- Generates 2,3 Diphosphoglycerate (2,3-DPG) that decreases hemoglobin affinity to oxygen
Luebering This pathway is important in the oxygen-carrying capability of erythrocytes. Because of this pathway,
Pathway the erythrocyte has a built-in mechanism that is low in energy expenditure and is capable of regulating
oxygen transport during conditions of hypoxia and disorders of acid-base balance.

↑2,3DPG = ↓Hb affinity to Oxygen (Shift to the Right)

↓2,3DPG = ↑ Hb affinity to Oxygen (Shift to the Left)

IV. OXYGEN DISSOCIATION CURVE

The curve produced when the two variables (partial pressure of oxygen and affinity of hemoglobin for oxygen) are plotted on
a graph (oxygen saturation of hemoglobin versus the partial pressure of oxygen)
Sigmoid in shape
P50 values: defined in terms of the amount of oxygen needed to saturate 50% of hemoglobin
Arterial blood: 95% - 95 mmHg
Venous blood: 70% - 40 mmHg
p50: 26.6 mmHg or 27mmHg

If there is a shift of the curve to the left, 50% oxygen saturation of hemoglobin occurs at a PO2 of less than 27 mm Hg.
If there is a shift of the curve to the right, 50% oxygen saturation of hemoglobin occurs at a PO2 higher than 27 mm Hg.

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 PARAMETER SHIFT TO THE LEFT SHIFT TO THE RIGHT
Ph Increase Decrease
PCO2 Decrease Increase
2, 3- DPG Decease Increase
Temperature Decrease Increase
Hb affinity for O2 Increase Decrease
*Presence of increase myoglobin and fetal hemoglobin will lead to shift to the left

Bohr effect
Demonstration the relationship between blood pH and oxygen affinity of Hb. A shift in the curve due to a change in pH or
hydrogen ion concentration

V. RED BLOOD CELL BREAKDOWN OR DESTRUCTION

As red blood cell ages, there is a decrease in its enzyme, a decrease in ATP, a decrease in size, and an increase in density
Approximately 1 % of the red blood cells leave the circulation each day and broken down by the mononuclear phagocytic system
CULLING is a splenic function wherein old/aged/senescent red blood cells are filtered and destroyed through the
phagocytosis of splenic macrophages.

As an erythrocyte ages, the following processes occur:

1. The membrane becomes less flexible.
2. The concentration of cellular hemoglobin increases.
3. Enzyme activity, particularly glycolysis, diminishes.

Types of Destruction

Extravascular Hemolysis-90% of RBC destruction

Also known as the Macrophage mediated hemolysis

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 Intravascular/Fragmentation Hemolysis-
≤10% of RBC destruction (Turgeon), 10 to 20% (Rodak’s 5th edition)
Associated with Increase Unconjugated bilirubin
Hemoglobinuria, hemoglobinemia, and Hemosiderinuria (confirmed by Perl’s Prussian blue)
Decrease Haptoglobin values, Increase LD enzymes

HEMOGLOBIN
Hemoglobin is red globular protein, which have a molecular weight of about 64,000 and compromise almost one third of the
weight of a red cell. Each red cell contains approximately 640 million Hb molecules
Hemoglobin’s main function is to transport oxygen from the lungs to tissues and transport carbon dioxide from the
tissues to the lungs for exhalation
Hemoglobin is composed of four subunits. Each subunit with heme and globin
▪ 1 Heme:1 moles of Oxygen
▪ 1 Hb: 4 moles of Oxygen
Hemoglobin is an oxygen transporting protein contained within erythrocytes
The heme portion of hemoglobin gives erythrocytes their characteristic red color.
A third function of hemoglobin involves the binding, inactivation, and transport of nitric oxide.1,10 Nitric oxide is secreted by vascular
endothelial cells and causes relaxation of vascular wall smooth muscle and vasodilation.1 When released, free nitric oxide has a very
short half-life, but some enters RBCs and can bind to cysteine in the b chain of hemoglobin, forming S-nitrosohemoglobin

Reference range
AGE GROUP CONVENTIONAL S.I UNITS
Children (8 to 13 y.o) 12 to 15 g/dl 120 to 150 g/L
Adult (male) 14 to 18 g/dl 140 to 180 g/L
Adult (female) 12 to 15 g/dl 120 to 150 g/L

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 One gram of hemoglobin can carry 1.34ml of Oxygen
One gram of hemoglobin can carry a constant 3.47mg of Iron

[Link] STRUCTURE
1. Four identical heme groups, each consisting of a protoporphyrin ring and ferrous (Fe 2+) iron
2. Four globin (polypeptide) chains
3. a tetramer of four globin polypeptide chains, with a heme molecule attached to each chain
4. The hemoglobin molecule can be described by its primary, secondary, tertiary, and quaternary protein structures.

Primary structure of hemoglobin Refers to the amino acid sequence of the polypeptide chains
Secondary structure of Refers to chain arrangements in helices and nonhelices.
hemoglobin
Tertiary structure of hemoglobin Refers to the arrangement of the helices into a pretzel-like configuration
Quarternary structure The complete hemoglobin molecule structure

5. The quaternary structure of hemoglobin, also called a tetramer, describes the complete hemoglobin molecule

Globins
GREEK GREEK # OF
DESIGNATION NAME AMINO
ACIDS
Α Alpha 141
Β Beta 146
Δ Delta 146
Γ Gamma 146
Ε Epsilon 146
Ζ Zeta 141

Heme Heme consists of a ring of carbon, hydrogen, and nitrogen atoms called protoporphyrin IX, with a central atom of
divalent ferrous iron (Fe2+)
Must ✓ Hemoglobin = HEME + TWO DIMERS OF GLOBIN CHAINS
know ✓ 1 RBC = consists of 4 HEME
✓ 1 RBC = CAN CARRY 4 MOLECULES OF O2
✓ 1 HEME MOLECULE= 4 PYRROLE RINGS
✓ 1 Heme molecule = 1 ferrous iron
What is the ratio of pyrrole ring to ferrous iron in a single heme molecule? 4:1

II. Hemoglobin Synthesis / Production

65 % hemoglobin synthesis occur in immature nRBCs
35 % hemoglobin synthesis occur in reticulocytes
Heme synthesis occurs in the mitochondria of normoblasts and is dependent on glycine, succinyl coenzyme A, Aminolevulinic
acid synthase, and vitamin B6 (pyridoxine)
Globin synthesis occurs in the ribosome, and it is controlled on chromosome 16 for alpha and zeta chains and
chromosome 11 for all other chains
Rubricyte = first stage of hemoglobin synthesis
Reticulocytes = last stage capable of hemoglobin synthesis

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 Porphyria is defined as a disease of heme metabolism in which
a primary abnormality in porphyrin biosynthesis leads to
excessive accumulation and excretion of porphyrins
or their precursors by the biliary and/or renal route.

Porphyrias can be classified based on various characteristics:

■ Clinical presentation (acute versus chronic)
■ Source of enzyme deficiency
■ Site of enzyme deficiency in the heme biosynthetic pathway

Clinically, patients with porphyria have either neurological

complications or skin problems

Porphyria is derived from the Greek word, porphyra, which

means purple. The purple-red pigment (porphyrins)
is responsible for the wine-red or portwine color characteristic
of porphyric urine.

Porphyrias are diseases in which porphyrin production is

impaired. They can be acquired, such as lead poisoning, or
inherited with mutations affecting enzymes in the heme
synthetic pathway

Porphyria Enzyme Deficiency Elevated Compound(s) Clinical Symptoms

Acute intermittent porphyria Porphobilinogen deaminase ALA Porphobilinogen Neurologic/psychiatric
Porphyria Cutanea Tarda Uroporphyrinogen decarboxylase Uroporphyrin Photosensitivity
Congenital erythropoietic Uroporphyrinogen cosynthase Uroporphyrin and Photosensitivity
porphyria Coproporphyrin
Variegate porphyria Protoporphyrinogen oxidase Coproporphyrin Photosensitivity/neurologic
Erythropoietic protoporphyria Ferrochelatase Protoporphyrin Photosensitivity
Lead poisoning D-ALA synthase and ferrochelatase ALA and Protoporphyrin Neurologic

[Link]
Iron is the most abundant transition metal in the body
Most iron in the body is in Hemoglobin and must be in the ferrous state to be used. Ferrous iron binds to oxygen for
transport to lungs and body tissues. Ferric iron (Fe 3+) is not able to bind to hemoglobin, but does bind to transferrin
Iron is an essential mineral and is not produced in the body
Normal daily diet contains about 15mg iron and only 1-2mg of iron is absorbed in the duodenum and upper jejunum
In the duodenum, dietary free iron is reduced to ferrous iron and taken up from the intestinal lumen into the enterocytes
by the iron transport protein Divalent monotransporter 1 (DMT)
Once absorbed, iron may be stored as ferritin in the enterocytes or exported into the circulation by another iron transport
protein, FERROPORTIN (Fpn1)
In the plasma, ferric iron binds to transferrin which is delivered into cells by binding to transmembrane glycoprotein the
transferrin receptors (TfR)
Hepcidin, a liver-produced peptide hormone, it is the master regulatory hormone of systemic iron metabolism. The
interaction of hepcidin with the plasma iron transporter, ferroportin, coordinates iron acquisition with iron utilization and storage.
Hepcidin deficiency causes common iron overload syndromes but overexpression of hepcidin is responsible for microcytic anemia
(anemia of chronic inflammation).
Hepicidin = It regulates the transport of iron from enterocyte into the circulation by binding through ferroportin.

TWO STORAGE FORMS OF IRON

a. Ferritin = Major storage form, Water soluble, considered as an acute phase reactants.
b. Hemosiderin = Second storage form of iron, Water insoluble

[Link] OF HEMOGLOBIN AND DERIVATIVES

Hemoglobin A1 2 alpha + 2 beta globin chains
Predominate hemoglobin in an adult
Subdivided into glycosylated fractions A1C which reflects glucose level in the blood

Reference range for normal adult Hgb is 95- 97 %Hb A, 2 to 3%Hb A2, and ≤1% Hb F

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 Hemoglobin F Composed of 2alpha + 2 gamma
Predominates at birth (80%)
Hgb F is a compensatory hemoglobin
Increased in hemoglobinopathies and B-thalassemia Major
Oxyhemoglobin Hemoglobin with Ferrous + oxygen; seen in arterial circulation (RELAXED STATE)
Deoxyhemoglobin Hemoglobin with Ferrous but no Oxygen; seen in venous circulation (TENSED STATE)
Carboxyhemoglobin Hemoglobin with carbon monoxide (CO); hemoglobin has 200x/240x more affinity for CO than O2
Carbon monoxide will bind with Hb even if its concentration in the air is extremely low (eg 0.02–0.04 %)
Cannot bind and carry oxygen
Increasing concentration of HbCO shift the ODC (Oxygen dissociation curve) to the left, thus adding to
anoxia
Light sensitive and imparts a typical brilliant CHERRY RED COLOR to the blood
Chief sources of the gas are gasoline motors, illuminating gas, gas heaters, defective stoves, and
smoking of tobacco
Quantitated by differential spectrophotometry or by gas chromatography

Methemoglobin / Hemoglobin with Fe 3+ (ferric); cannot transport oxygen

Hemiglobin Cause chocolate brown discoloration of blood
Causes Cyanosis and functional anemia if present in high enough concentration
Sources: Chemical or drugs such as chlorate, nitrate, and nitrite
Quantitated by spectrophotometry
Methemoglobin is assayed by spectral absorption analysis instruments such as the CO-oximeter.
Methemoglobin shows an absorption peak at 630 nm

Level Signs and Symptoms

Less than 25% Asymptomatic
More than 30% Cyanosis (bluish discoloration of skin and mucous membranes) and symptoms of
hypoxia (dyspnea, headache, vertigo, change in mental status) occur
More than 50% Coma and Death

Methylene blue If the level of methemoglobin increases to 30% or more of total hemoglobin, intravenous
____ is administered. It reduces methemoglobin ferric iron to the ferrous state through
NADPH-methemoglobin reductase and NADPH produced by glucose-6-phosphate
dehydrogenase in the hexose monophosphate shunt

An abnormal hemoglobin (Hb M) may also be responsible for methemoglobinemia noted at birth or first
months

Sulfhemoglobin ▪ Hemoglobin with Sulfur; cannot transport Oxygen

▪ IRREVERSIBLE
▪ During oxidation of hemoglobin, sulfur (from some source, which may vary) is incorporated into
heme rings of hemoglobin, resulting in a green hemochrome
▪ Blood is mauve-lavender in sulfhemoglobinemia
▪ Usually reported in following situations:
a. Patients under prolonged treatment with sulfonamide or aromatic amine compounds such as
Phenacetin, sulfanilamides, nitrites, and phenylhydrazine
b. Patients with severe constipation
c. In cases of bacteremia caused by Clostridium perfringens
d. In condition known as enterogenous cyanosis
▪ It can also combine to Carbon monoxide to form carboxysulfhemoglobin
▪ CANNOT BE REDUCED BACK TO HEMOGLOBIN and it remains in the cell until break down
▪ Quantitated by spectrophotometry
▪ Sulfhemoglobin has a similar peak to methemoglobin on a spectral absorption instrument. The
sulfhemoglobin spectral curve, however, does not shift when cyanide is added, a feature that
distinguishes it from methemoglobin

Dyshemoglobins / Abnormal hemoglobin variants Carboxyhemoglobin, Sulfhemoglobin, Methemoglobin, Carboxysulfhemoglobin

I.K Aytona Page 26

 [Link] DETERMINATION
Colorimetric method: I. Acid hematin (Sahli’s method) = uses HCL
Direct/Visual Principle: When blood is mixed with an acid solution, the hemoglobin converts into the brown-colored
acid hematin. The acid hematin is then diluted with distilled water till the color of the acid hematin
matches that of the yellowish-brown glass standard. The hemoglobin is estimated by reading the value
directly from the scale.

[Link] hematin = uses NaOH

Colorimetric method: CYANMETHEMOGLOBIN / HEMIGLOBINCYANIDE (HiCN) METHOD
Indirect/Photoelectric ➢ Cyanmethemoglobin rgt contains: Modified DRABKIN’S reagents
➢ It is sensitive to light, it should be stored in a brown bottle or dark place
➢ Color intensity is measured at 540nm
➢ All types of hemoglobin may be measured except SULFHEMOGLOBIN
➢ Overanticoagulation of blood does not affect results
➢ Turbidity in the mixture will cause falsely elevated result

NOTE 😊
a. Abnormal globulins: remedy- add 0.1g of potassium carbonate to the drabkin’s reagent
b. Carboxyhemoglobin takes 1 hour to convert to cyanmethemoglobin and can cause
erroneous results in heavy smokers

Composition of Reagent
Potassium ferricyanide – converts Hemoglobin (w/ferrous iron) to methemoglobin
Potassium cyanide – converts methemoglobin to cyanmethemoglobin
Non-ionic detergent –improves lysis of RBC and decrease turbidity
Dihydrogen potassium phosphate-allows the solution to be read after 3 minutes

Causes Of Specimen Turbidity

Cause Remedy
High WBC count(>20x10 /L)
9
Centrifuge then read the supernatant
High platelet count (>700 x10 /L)
9

Presence of Hb S or Hb C Make a 1:1 or 1:2 dilution with distilled water then multiply
result by 2
Lipemic sample Use patient blank /reagent blank (0.01ml patient plasma +
5ml Drabkin’s reagent)

PROCEDURE
1. Create a standard curve, using a commercially available cyanmethemoglobin standard
A. When a standard containing 80 mg/dL of hemoglobin is used, the following dilutions
should be made:

B. Transfer the dilutions to cuvettes. Set the wavelength on the spectrophotometer to

540 nm and use the blank to set to 100% transmittance.
C. Using semilogarithmic paper, plot percent transmittance on the y-axis and the
hemoglobin concentration on the x-axis. The hemoglobin concentrations of the control
and patient samples can be read from this standard curve. A standard curve should be
set up with each new lot of reagents. It also should be checked when alterations are
made to the spectrophotometer (e.g., bulb change).
2. Controls should be run with each batch of samples. Commercial controls are available.
3. Using the patient’s whole blood anticoagulated with EDTA or heparin or blood from a
capillary puncture, make a 1:251 dilution by adding 0.02 mL (20 uL) of blood to 5
mL of cyanmethemoglobin reagent. The pipette should be rinsed thoroughly with the
reagent to ensure that no blood remains. Follow the same procedure for the control
samples.
4. Cover and mix well by inversion or use a vortex mixer. Let stand for 10 minutes at room
temperature to allow full conversion of hemoglobin to cyanmethemoglobin
5. Transfer all of the solutions to cuvettes. Set the spectrophotometer to 100% transmittance
at the wavelength of 540 nm, using cyanmethemoglobin reagent as a blank.
6. Using a matched cuvette, continue reading the % transmittance of the patient samples and
record the values
7. Determine the hemoglobin concentration of the control samples and the patient samples
from the standard curve.

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 STANDARD HEMOGLOBIN CURVE
X-axis / Abscissa Hemoglobin value (g/dl)
Y-axis / Ordinate Optical density (absorbance)
Copper sulfate method / 30 ml container
Specific Gravity method CuSO4 specific gravity should be _______
Solution should be changed daily
Distance between drop of blood and solution is 1cm
Acceptable drop of blood will sink in solution within 15 seconds if
Hb concentration is ≥ 12.5 g/dl
Gasometric method VAN SLYKE OXYGEN CAPACITY METHOD
▪ 1 gram of hb = 1.34ml Oxygen

Chemical method KENNEDY’S, WONG’S

▪ 1 gram of Hb = 3.47mg Iron

RED BLOOD CELL ABNORMALITIES

CELL SIZE

Anisocytosis Variation of cell size

RDW Assess anisocytosis. Increase in heterogenous population of cells. It is the coefficient of
(Red cell distribution width) variation of RBC volume expressed as percentage
Computed by dividing the SD by the mean of the RBC size distribution
Formula = (SD / Mean) x 100

R.V = 11.5% to 14.5%

Increased in Post Transfusion, idiopathic or primary sideroblastic anemia, and combine
anemia

Normocytic RBC Normal RBC size (6-8um)

Normal MCV (80-100fl)
Microcytic RBC Small size (<6 um in size)
MCV= less than 80 femtoliter
Macrocytic RBC Larger size (>9 um in size)
MCV = greater than 100 femtoliter

HEMOGLOBIN CONTENT

Anisochromia Variation in hemoglobin content

Normochromic Normal amount of hemoglobin, normal color
Normal RBCs have central pallor of approximately 1/3 in diameter
MCHC = 32-36%
Hypochromic Central pallor area exceeds 1/3 of the diameter of the cell
Decrease MCHC (<32%)
Hyperchromic/ Spherocyte No central pallor
Increased MCHC (>36%)
SPHEROCYTOSIS (Bronze cells)
Polychromasia / Indicates immature/young red blood cell
Reticulocytosis/Polychromatophilia Blue green discoloration (residual of RNA)
Increase erythropoietic activity

GRADE Percentage of RBCs that are HYPOCHROMIA GRADING

Polychromatophilic 1+ Area of central pallor is one half of cell diameter
Slight 1% 2+ Area of pallor is two-thirds of cell diameter
1+ 3% 3+ Area of pallor is three-quarters of cell diameter
2+ 5% 4+ Thin rim of hemoglobin
3+ 10%
4+ >11%

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 CELL SHAPE

Poikilocytosis variation in RBC cell shape

POIKILOCYTOSIS SECONDARY TO MEMBRANE ABNORMALITIES

RED CELL DESCRIPTION ASSOCIATED CONDITIONS
Acanthocyte/Spur Spheroid with 3-12 irregular spikes/ spicules Alcoholic cirrhosis with hemolytic anemia
Cells/Thorn cells Malabsorption state
Abnormal ratios of membrane lecithin and Post-splenectomy
sphingomyelins Hepatitis of the newborn
Pyruvate kinase deficiency
Severe hemolytic anemia associated with cirrhosis and
in metastatic liver disease (spur cells)
Abetalipoproteinemia
McLeod Syndrome
Echinocyte/Crenated Regular 10-30 scalloped short projections Depletion of ATP
cell/Sea urchin evenly distributed. Exposure to hypertonic solution
cell/Burr cells -RBC with blunt or pointed, short projections Artifact in air drying
that are usually evenly spaced over the
Anemia associated with renal insufficiency
surface of cell; present in all fields of blood
(Uremia)
film but in variable numbers per field
Pyruvate kinase deficiency
Target -Peripheral rim of hb surrounded by clear area Hemoglobinopathies
cell/Codocyte/Mexican and central hemoglobinized area (bull’s eye) Thalassemia
hat cell -RBC with hemoglobin concentrated in the Obstructive liver disease
center and around periphery resembling a
Post splenectomy states
target
Iron deficiency anemia
-Bell or tall hat shape on scanning electron
microscopy (Gr. Kodon: bell)
-Inrease in cholesterol and phospholipid
-Increase of surface membrane to volume
ratio
Spherocyte or Low surface area to volume ratio Hereditary spherocytosis
Bronze cells Lack of central pallor Isoimmune and autoimmune hemolytic anemia
Defect of loss of membrane Severe burns (microspherocyte)
Blood banked stored for a long time
Note: After splenectomy in a patient with HS,
spherocytes persist, indicating that the
abnormality involves red blood cell membrane
rather than splenic damage to the cells
Stomatocytes Characterized by an elongated or slit-like area Hereditary stomatocytosis
of central pallor Rh null disease
Alcoholism
Increased permeability of the membrane to Idiopathic sideroblastic anemia
sodium
Obstructive liver disease
Artifact
Elliptocyte Elliptical or Rod or cigar shaped, narrower Hereditary elliptocytosis
than ovalocytes Gerbich null disease (Ge: -2, -3, -4)
Ovalocyte Egg like or oval shaped, wider than Megaloblastic bone marrow
elliptocytes Myelodysplasia
Bipolar arrangement in hemoglobin Hereditary ovalocytosis
Reduction in membrane cholesterol

POIKILOCYTES SECONDARY TO TRAUMA

RED CELL DESCRIPTION ASSOCIATED CONDITION
Schistocyte/Schizocyte Fragmentation produced by damage of RBC by DIC
fibrin; altered vessel; prosthetic heart valves Microangiopathic hemolytic anemia
Thrombocytopenic purpura (TTP)
Keratocytes – Schistocyte with hornlike Extensive Burns
projections
Dacryocyte Tear drop or pear shaped with one blunt Myelopthisic anemia
projection Myeloid fibrosis with myeloid metaplasia
Squeezing and fragmentation during Spleenic Hypersplenism
passage
Megaloblastic anemia and Thlassemia

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 Pyropoikilocytes RBCs that fragment at 45 0C (Normal RBC 490 C) Severe burns
Hereditary pyropoikilocytosis
Semilunar bodies/Half Large pale pink -staining ghost of the red cell Malaria
moon

POIKILOCYTES SECONDARY TO ABNORMAL HEMOGLOBIN

Drepanocytes/ Crescent shaped cell, thin, dense, elongated RBC pointed Sickle cell anemia
Sickle cell at each end; may be curbed SC disease – “Holly leaf” formation
Menisocytes
-Due to polymerization of Deoxygenated Hb, thin, dense,
elongated RBC pointed at each end; may be curved
Folded cells RBC with membrane folded over Hb SC disease
Hb C disease

RBC INCLUSIONS

1. Howell Jolly bodies

Small, round fragments of the nucleus resulting from karyorrhexis or nuclear disintegration
Dark-blue purple dense, round granule; usually one per cell; occasionally multiple
Nuclear remnants of DNA
Stain dark purple to black with Wright’s stain; positive in Fuelgen reaction
o Megaloblastic anemia
o Physiologic atrophy of the spleen
o Thalassemia
o Accelerated Erythropoiesis / Hemolytic anemia
o Post splenectomy
o Myelodysplastic syndrome
o Hyposplenism

2. Diffuse basophilia
Dark blue granules and filaments in cytoplasm (supravital stain)
Bluish tinge throughout cytoplasm; also called polychromasia
Inclusion is composed of RNA
o Hemolytic anemia
o After treatment for iron, vitamin 12, or folate deficiency

3. Basophilic Stippling (punctuate basophilia)

Multiple, tiny, fine, or coarse inclusion, evenly dispersed throughout the cell; “blue berry bagel” appearance
Dark blue-purple granules, fine or coarse punctuate granules distributed throughout the cytoplasm
Precipitation of ribosomes and RNA
o Lead poisoning
o Pyrimidine-5-nucleotidase deficiency
o Heavy metal poisoning
o Thalassemia
o Hemoglobinopathies
o Megaloblastic anemia
o Myelodysplastic syndrome

4. Cabot rings
Rings, looplike figure of eight, red to purple color
Remnants of Microtubules of mitotic spindle
o Megaloblastic anemia
o Myelodysplastic syndrome
o Lead poisoning

5. Heinz bodies
Deep purple irregularly shaped inclusions
Round, dark-blue purple granule attached to inner RBC membrane
Precipitated, denatured hemoglobin due to oxidative injury
Cannot be seen in Wright’s stain
Multiple Heinz bodies may give a cell appearance of a pitted golf ball
Requires Supravital stain (Brilliant cresyl blue or crystal violet) for demonstration

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 o Hereditary defects in HMP shunt
o G6PD deficiency
o Unstable hemoglobins
o Splenectomized patients
o Thalassemia
o Oxidant drugs/chemicals
o Favism –inherited condition resulting from sensitivity to the fava beans

6. Pappenheimer bodies / siderotic granules

Irregular cluster of small, light to dark blue granules, often near periphery of cell
Unused iron deposits
Stains with perl’s Prussian blue
o Sideroblastic anemia
o Myelodysplastic syndrome such as RARS
o Thalassemia and hemoglobinopathies
o Megaloblastic anemia
o Post splenectomy and hyposplenism

7. Hemoglobin H
Inclusion represented by precipitated Hb H / precipitate of B-globin chains of hemolglobin
Fine, evenly dispersed, dark blue granules; imparts golf ball appearance to RBCs
Demonstrated with a supravital stain: Brilliant cresyl blue
o Hb H disease; an alpha thalassemia

8. Hb C or CC crystals
Hexagonal with blunt ends and stain darkly
Barr of Gold/ Clam shell appearance
o Homozygous C (Hb CC) disease

9. Hb SC crystals
Dark hued crystal of condensed Hb distort to red cell membrane
Crystalline fingkerlike/ quartzlike crystal projection is often straight with parallel sides and one blunt, pointed,
protruding end
Washington monument crystal
o Hb SC disease

10. Ringed sideroblast

Nucleate RBC that contains non heme iron particles
Excessive iron overload in mitochondria of normoblast
Due to defective heme synthesis
Stains with Perl’s Prussian blue fordemonstration of Hemosiderin
o Sideroblastic anemia
o MDS
o Alcoholism

11. Malarial inclusions

Protozoan transmitted by bite of female Anopheles mosquito
Maturation stages (from least to most mature): rings, trophozoite, schizonts and gametocytes
Plasmodium spp. Infected RBC size Name of the Inclusions
[Link] Normal Ziemann’s dot
[Link] Enlarged James dot
P. vivax Enlarged Schuffner’s dot
[Link] Normal Maurer’s dot

12. Babesia spp.

Protozoan inclusion transmitted from deer to human by tick bite
RBC with “Maltese cross appearance” – tiny rings or occasionally as tetrads inside the RBCs
Babesia microti – causative agent of Nantucket fever

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 ABNORMAL ERYTHROCYTE DISTRIBUTION
1. Rouleaux
➢ Stack of Coin pattern of RBC due to abnormal or increased plasma proteins
➢ True rouleaux formation is determined in the thin area of the peripheral smear
➢ To confirm true rouleaux formation, saline replacement is employed. A drop of NSS will disperse false rouleaux while
true rouleaux remains INTACT
➢ May be due to an artifact as a result of delay in smearing the blood once the drop has been placed on the slide
o Multiple Myeloma
o Waldenstrom macroglobulinemia
o Hyperproteinemia
2. Agglutination
➢ Characterized by clumping of erythrocytes with no pattern
➢ Autoagglutination occurs when an individual red cell agglutinates in his own plasma or serum that contains no
specific known agglutinins
o Cold agglutinin disease
o Certain hemolytic anemias
o Primary Atypical pneumonia / Walking pneumonia

ERYTHROCYTE MORPHOLOGY AND INCLUSION

1. Macrocytosis Is the result of a defect in either nuclear maturation or stimulated erythropoiesis. True macrocytes
represent a nuclear maturation defect associated with a deficiency of either vit. B12 or folate
2. Microcytosis Is associated with a decrease in hemoglobin synthesis. This decrease in hemoglobin content may
be produced by a deficiency of iron, an impaired globulin synthesis, or a mitochondrial abnormality affecting
the synthesis of the heme unit of the hemoglobin molecule
3. Acanthocytes Have multiple thorny, spike-like projections that are irregularly distributed around the cellular membrane
and may vary in size. They have few spicules.
4. Blister cells Are erythrocytes containing one or more vacuoles that resemble a blister on the skin. This cell
has a significantly thinned area at the periphery or outer border of the cell membrane
-The vacuoles may rupture. If rupturing does occur, distorted cells (keratocytes)
and cell fragments (schistocytes) are produced
-Blister cells result from the traumatic interaction of blood vessels and circulating blood such as fibrin
deposits. Clinically, increased numbers can be seen as the result of pulmonary emboli in sickle cell
anemia and microangiopathic hemolytic anemia
5. Burr cells Are erythrocytes having one or more spiny projections of cellular membrane. Pointed projections
on the outer edge are uniformly shaped.
6. Echinocytes Have short, scalloped, or spike-like projections that are regularly distributed around the cell membrane.
/Crenated cells The projections can vary in number and appearance. Crenation can occur as the result of the physical loss
of intracorpuscular water
7. Elliptocytes Are generally narrower and more elongated than megalocytes.
-Associated clinical disorders include hereditary elliptocytosis, anemias associated with malignancy,
hemoglobin (Hb) C disease, hemolytic anemias (occasionally), iron deficiency anemia, pernicious anemia,
sickle cell trait, and thalassemia.
8. Helmet cells Are usually the larger scooped out part of the cell that remains after the rupturing of a blister cell and are
formed as a result of the physical process of fragmentation
9. Keratocytes Are erythrocytes that are partially deformed but not cut. The spicules, resembling two horns, result from a
ruptured vacuole. Usually the cell appears like a half-moon or spindle. These cells are seen in conditions
such as disseminated (diffuse) intravascular coagulation (DIC).
10. Knizocyte Resemble a pinched bottle. This abnormality is associated with hemolytic anemias, including hereditary
spherocytosis.
11. Leptocytes Resemble target cells (codocytes) but the inner, central portion is not completely detached from the outer
membrane. This variation of the target cell is clinically associated with hepatic disorders, iron deficiency
anemia, and thalassemia.
12. Oval macrocytes Oval macrocytes (megalocytes) have an oval or egg-like appearance . Although these cells are similar in
appearance to elliptocytes, megalocytes are macrocytic and have a fuller and rounder appearance.
Increases in this abnormality are seen in vitamin B12 and folate deficiencies and may be observed in
erythrocytes that are in the reticulocyte stage.
13. Pyknocytes are distorted, contracted erythrocytes that are similar to burr cells. These cells are seen in acute, severe
hemolytic anemia; glucose-6-phosphate dehydrogenase (G6PD) defi ciency; and hereditary lipoprotein defi
ciency and may be seen in small numbers during the first 2 to 3 months of life as infantile pyknocytes.
14. Schistocytes are fragments of erythrocytes that are small and irregularly shaped. Because these cells are produced as
the result of the breaking apart of an erythrocyte, the schistocyte is about half the size of a normal
erythrocyte and may have a deeper red appearance
15. Sickle cells Sickle cells (drepanocytes) resemble a crescent. At least one of the ends of the cell must be pointed.
Generally, the membrane is smooth and the cell stains uniformly throughout. Sickle cells result from the
gelation of polymerized deoxygenated Hb S.
16. spherocytes Spherocytes are erythrocytes that have lost their normal biconcave shape . This type of cell has an
extremely compact, round shape. It is usually smaller than 6 mm and has an intense orange-red color when
stained. Spherocytelike erythrocytes may appear as artifacts if a slide is examined at the thin end of a
normal blood smear.
-Clinical disorders associated with spherocytes include acquired hemolytic anemia, blood transfusion
reactions, congenital spherocytosis, and DIC. Microspherocytes are associated with ABO hemolytic disease
of the fetus and newborn and a storage phenomenon that produces microspherocytes in the recipient of a
blood transfusion.
17. Spiculated Spiculated erythrocytes are irregularly contracted erythrocytes. Spiculated erythrocytes may also be
erythrocytes referred to as burr cells, crenated cells, pyknocytes, spur cells, acanthocytes, and echinocytes.

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 18. Stomatocytes Stomatocytes have a slitlike opening that resembles a mouth. The slitlike opening is on one side of the
cell. Stomatocytes result from increased sodium (Na+) ion and decreased potassium (K+) ion
concentrations within the cytoplasm of the erythrocyte
-Clinical conditions associated with an increase in stomatocytes include acute alcoholism, alcoholic cirrhosis,
glutathione deficiency, hereditary spherocytosis, infectious mononucleosis, lead poisoning, malignancies,
thalassemia minor, and transiently accompanying hemolytic anemia. These cells can also be seen in
hereditary stomatocytosis and Rh null disease
19. Target cells Target cells (codocytes) are erythrocytes that resemble a shooting target . A central red bull’s-eye is
surrounded by a clear ring and then an outer red ring. The cells are thinner than normal, which may be
because of an excessive ratio of membrane lipid to cell volume

Clinically, target cells are seen in the hemoglobinopathies (Hb C disease, S-C and S-S disease, sickle cell
thalassemia, and thalassemia), hemolytic anemias, hepatic disease with or without jaundice, and iron
deficiency anemia as well as after a splenectomy.
20. Tear drop cells Teardrop cells (dacryocytes) are usually smaller than normal erythrocytes .As the term implies, teardrop
cells resemble tears. This cellular abnormality is associated with homozygous beta-thalassemia,
myeloproliferative syndromes, pernicious anemia, and severe anemias
21. anisochromia The general term for a variation in the normal coloration or RBC
22. Hypochromia Term used when the central pallor exceeds one third of the cell’s diameter
23. Basophilic Fine stippling appears as tiny, round, solid staining, dark-blue granules. The granules are usually evenly
stippling distributed throughout the cell and often require careful examination to detect them. Coarse basophilic
stippling is sometimes referred to as punctate stippling. These granules are larger than in the fi ne form
and are considered to be more serious in terms of pathological significance. Stippling represents granules
composed of ribosomes and RNA that are precipitated
-Stippling is associated clinically with disturbed erythropoiesis (defective or accelerated heme synthesis),
lead poisoning, and severe anemias.
24. Cabot rings are ring-shaped, figure-eight, or loop-shaped structures. Occasionally, the inclusions may be formed of
either double or multiple rings They may represent remnants of microtubules from the mitotic spindle.
However, recent research suggests that these inclusions represent nuclear remnants or abnormal histone
biosynthesis
25. Heinz bodies are inclusions, 0.2 to 2.0 mm in size, that can be seen with a stain such as crystal violet or brilliant cresyl
blue. They represent precipitated, denatured hemoglobin and are clinically associated with congenital
hemolytic anemia, G6PD deficiency, hemolytic anemias secondary to drugs such as
phenacetin, and some hemoglobinopathies.
26. Howell-jolly are round, solid staining, dark-blue to purple inclusions, 1 to 2 mm in size. If present, cells contain
bodies only one or two Howell-Jolly bodies. Although these inclusions are most frequently seen in mature
erythrocytes that lack a nucleus, they may be seen in immature, nucleated erythrocytes.
Howell-Jolly bodies are not seen in normal erythrocytes. They are nuclear remnants predominantly
composed of DNA.

The presence of Howell-Jolly bodies is associated with accelerated or abnormal erythropoiesis,

hemolytic anemias, pernicious anemia, and particularly postsplenectomy, physiological
atrophy of the spleen,
27. Pappenheimer Observed in Wright-stained smears as purple dots. These inclusions are infrequently seen in peripheral
bodies blood smears. Siderotic granules are dark-staining particles of iron in the erythrocyte that are visible with
(siderotic a special iron stain—Prussian blue. They appear as blue dots and represent ferric (Fe3+) ions.
granules) Pappenheimer bodies are aggregates of mitochondria, ribosomes, and iron particles. Clinically, they are
associated with iron-loading anemias, hyposplenism, and hemolytic anemias.
28. Alteration in -Agglutination and rouleaux formation are two alterations in erythrocytic distribution that may be observed
RBC on a stained peripheral smear
distribution -Agglutination, or the clumping of erythrocytes, may be observed. Rouleaux formation the
arrangement of erythrocytes in groups that resemble stacks of coins, is usually present in the thick portions
of normal blood smears

HEMATOLOGY LABORATORY TESTS

Hemacytometer
The most commonly used is the Levy chamber
Composed of 3mm x 3mm square counting area or grid (total area 9mm2), separated by an H-shaped moat
the total volume of one entire grid or counting area on one side of the hemacytometer is 0.9 mm3

I.K Aytona Page 33

 General formula/ calculation for cell counting in neubauer counting chamber:

A. Cell counted x Area correction factor x Dilution Factor x Dilution (1/200)

B. Total cells counted X Dilution

Total area counted (mm2) X Depth (0.1)

The difference between the total cells counted on each side should be <10%. A greater variation could
indicate an uneven distribution, which requires the procedure to be repeated

MANUAL CELL COUNTS WITH MOST COMMON DILUTIONS, AND COUNTING AREA
Cells counted Diluting fluid Dilution Objective Area Counted
WBC 1% ammonium oxalate 1:20 (standard) 10x (LPO) 4mm2
3% acetic acid 1:100 10x (LPO) 9mm2
1%HCL
RBC Isotonic saline / NSS 1:100 40x(HPO) 0.2mm2 (5small squares of
center square)
Platelets 1% ammonium oxalate 1:100 40x (HPO) 1mm2

[Link] RBC COUNT

REFERENCE VALUE
Male 4.2-6.0 x1012/L
Female 3.6-5.6 x1012/L
At birth 5.0-6.5 x1012/L

RBC Diluting Fluids

NSS, Dacies or Formol citrate (BEST), 3.8 % Sodium citrate, Hayem’s, Toisson’s, Gower’s, Betthell’s

RBC Pipette Calibration Marks = 0.5, 1, and 101

Example question
1. 100 cells were counted in the 5 RBC center squares of the counting chamber using a 1:100 dilution. Calculate for the
RBC count

[Link] WBC COUNT

PROCEDURE
1. Clean the hemacytometer and coverslip with alcohol and dry thoroughly with a lint-free tissue. Place the coverslip on the
hemacytometer.
2. 2. Make a 1:20 dilution by placing 25 uL of well-mixed blood into 475 uL of WBC diluting fluid in a small test tube.
3. Cover the tube and mix by inversion.
4. Allow the dilution to sit for 10 minutes to ensure that the red blood cells have lysed. The solution will be clear
once lysis has occurred. WBC counts should be performed within 3 hours of dilution.
5. Mix again by inversion and fill a plain microhematocrit tube.
6. Charge both sides of the hemacytometer by holding the microhematocrit tube at a 45-degree angle and
touching the tip to the coverslip edge where it meets the chamber floor.
7. After charging the hemacytometer, place it in a moist chamber for 10 minutes before counting the cells to give
them time to settle. Care should be taken not to disturb the coverslip.
8. While keeping the hemacytometer in a horizontal position, place it on the microscope stage.
9. Lower the condenser on the microscope and focus by using the low-power objective lens. The cells should be distributed
evenly in all of the squares.
10. For a 1:20 dilution, count all of the cells in the four corner squares, starting with the square in the upper left-hand corner.
Cells that touch the top and left lines should be counted; cells that touch the bottom and right lines should be ignored for
the appearance of WBCs in the hemacytometer using the low-power objective lens of a microscope.

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 11. Repeat the count on the other side of the counting chamber. The difference between the total cells counted on each side
should be less than 10%. A greater variation could indicate an uneven distribution, which requires that the procedure be
repeated.
12. Average the number of WBCs counted on the two sides. Using the average, calculate the WBC count

Reference Value:
Adult 4.0-11.0 x109/L
At birth 10.0-30.0 x109/L

WBC Diluting Fluids

1-3% Acetic acid
1% HCL
Turk’s diluting fluid
(Glacial acetic acid + Crystal violet)

WBC Pipette Calibration

Marks= 0.5, 1, 11

NOTE! ☺
For WBC count <3.0 x109 /L 1:10 or 1:11 Dilution used
For WBC count >30.0 x109 /L 1:100 or 1:101 Dilution used
For WBC count 100-300.0 x109 / L 1: 200 or 1:201 Dilution used

Example question
1. 200 cells were counted in the 4 WBC large squares of the counting chamber using a 1:100 dilution. Calculate for the
WBC count

EOSINOPHIL COUNT
Eosinopenia is found in hyperadrenalism (Cushing’s disease), shock, and following administration of ACTH

DIRECT METHOD FOR EOSINOPHIL COUNT (REAGENTS)

Diluting fluid: Pilot’s fluid, Randolph’s fluid, Manner’s fluid, Tannen’s fluid, Hinkelman’s’fluid

Diluting fluid composition:

A. Phloxine, Eosin, and Neutral red iodide- stains eosinophil red
B. Propylene glycol – Lyses Rbc
C. Sodium carbonate (Na2CO3) – Lysis WBCs except eosinophil
D. Heparin- prevents clumping of cells

-Dilution Factor = 1:32 under LPO

-Area counted =9mm2

[Link] WBC COUNT

Any nucleated red blood cells (NRBCs) present in the sample are not lysed by the diluting fluid. The NRBCs are counted as WBCs
because they are indistinguishable when seen on the hemacytometer.

CRITERIA BEFORE PERFORMING THE CORRECTION Example: (Compute for the CWC)
Check for the Presence of N-RBC -10 nRBCs present,
Adult = ____________ -WBC count is 5x109 / L
Newborn = _________

Formula: Corrected WBC Count= WBC count X 100

100 + nRBCs

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 IV. WHITE BLOOD CELL DIFFERENTIAL COUNT

FROM BARBARA BROWN

100 Differential count 50 Differential Count 200 Differential Count
Routinely used If patient WBC count is <1.0 x 109 / L If there are:
✓ Over 10 % eosinophil
-multiply results by 2 ✓ Over 2 % basophils
✓ Over 11% monocytes
✓ More lymphocytes than neutrophils (except
in children)

FROM RODAK’S (6th Edition)

100 Differential Count 200 Differential Count 300-400 Differential Count
Routinely used When WBC count is higher than 40x109/L. When WBC count is ≥100x109 /L.

Cell Relative Count Absolute Count

Segmenters 44- 67 % 1.8 to 7.8 x109 / L
Band cells 0-7 % 0 to 0.7 x109 / L ABSOLUTE WBC COUNT FORMULA
Eosinophil 0-6 % 0 to 0.6 x109 / L
Basophils 0 to 1 % 0 to 0.2 x109 / L Relative WBC count x Total WBC Count
Monocytes 2 to 10 % 0.01 to 0.8 x109 / L
Lymphocytes 20 to 40 % 1 to 4.8 x109 / L

TOTAL WHITE CELL COUNT ESTIMATION

2-5 WBC/ Hpf = 4000-7000 WBC/ul
4-6 WBC/ Hpf = 7000-10000 WBC/ul
6-10 WBC/ Hpf = 10000-13000 WBC/ul
10-20 WBC/ Hpf = 13000-18000 WBC/ul

WBC Estimation factor in Blood 1. Average number of WBC per field x 2000 (if using 40x HPO)
film counting 2. Average number of WBC per field x 3000 (if using 50x OIO)

LIST OF SOME CONDITIONS ASSOCIATED WITH SPEFIC INCREASE OF WBC

Neutrophilia Lymphocytosis Monocytosis Eosinophilia Basophilia
Viral infections TB Allergies Allergic reaction
Whooping cough Brucellosis Parasitic infection Type 1 hypersensitivity
Appendicitis Infectious Monocytic leukemia Eosinophilic leukemia
Bacterial infections mononucleosis SBE Note: glucocorticoids
Myelogenous leukemia Lymphocytic leukemia Typhoid fever decreases basophil
count
Pyogenic bacteria Ricketssial
Pancreatitis infections
Gaucher’s disease

Condition Absolute WBC count

Neutropenia <2.0 x 109/ L
Neutrophilia >7.0 x 109/ L (adults), >8.0 x 109/ L(children)
Eosinophilia >0.4 x 109/ L
Eosinopenia <0.09 x 109/ L
Basophilia <0.15 x 109/ L, commonly associated in CML
Monocytosis >1.0 x 109/ L (adults), >3.5 x 109/ L(neonates)
Frequently the first sign of recovery from acute infection or severe neutropenia
Monocytopenia <0.2 x 109/ L
Lymphocytosis >4.5 x 109/ L in adults, >10 x 109/ L in children
Lymphocytopenia <1.0 x 109/ L in adults, <2.0 x 109/ L in children

I.K Aytona Page 36

 [Link] INDICES /WINTHROBE’S INDICES
MCV ▪ Indicator of the average/ mean volume of RBCs.
(Mean cell volume) ▪ A RBC index that reflects RBC diameter in blood
▪ Reference range is 80-100 femtoliter

𝐻𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡(%)
MCV = 𝑥10
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡

MCH ▪ Indicator of the average weight of hemoglobin and RBC count

(Mean corpuscular hemoglobin) ▪ A RBC index that express the mass of hemoglobin
▪ Reference range is 26-32picograms

𝐻𝐸𝑀𝑂𝐺𝐿𝑂𝐵𝐼𝑁(𝑔/𝑑𝑙)
MCH= 𝑥10
𝑅𝐵𝐶 𝐶𝑂𝑈𝑁𝑇

MCHC ▪ It is a measure of the average concentration of hemoglobin and hematocrit values

(Mean cell hemoglobin ▪ Is the average concentration of hemoglobin in each individual cell
concentration) ▪ It reflects RBC staining intensity and amount of central pallor
▪ Reference range is 32-36 g/dl
▪ MCHC above 38% - presence Cold agglutinin
▪ MCHC below 22% - due to lipemia, or presence of Hb S or Hb C
▪ Cold agglutinin – causes markedly increase MCHC

𝐻𝐸𝑀𝑂𝐺𝐿𝑂𝐵𝐼𝑁(𝑔/𝑑𝑙)
MCHC = 𝑥100
𝐻𝐸𝑀𝐴𝑇𝑂𝐶𝑅𝐼𝑇 (%)

RDW ▪ It is the fourth RBC index

▪ It asses the degree of anisocytosis
▪ It is used in conjunction with MCV to classify anemia

[Link] COUNTS
Reticulocytes are used to assess bone marrow functionality
They are demonstrated primarily by a supravital stain (new methylene blue)

Supravital stain The new methylene blue stain is used to demonstrate reticulum in reticulocytes. Any
nonnucleated red blood cell that contains two or more particles of blue-stained
granulofilamentous material after new methylene blue staining is defined as a reticulocyte
High blood glucose High blood glucose level will cause reticulocyte to stain poorly

PROCEDURE
1. Mix equal amounts of blood and new methylene blue stain (2 to 3 drops, or approximately 50 uL each), and allow to
incubate at room temperature for 3 to 10 minutes.
2. Remix the preparation.
3. Prepare two wedge films
4. In an area in which cells are close together but not touching, count 1000 RBCs under the oil immersion objective lens (1000x
total magnification). Reticulocytes are included in the total RBC count (i.e., a reticulocyte counts as both an RBC and a
reticulocyte).
5. To improve accuracy, have another laboratorian count the other film; counts should agree within 20%.
6. Calculate the % reticulocyte count

SOURCES OF ERROR and COMMENTS

If a patient is very anemic or polycythemic, the proportion of dye to blood should be adjusted accordingly
An error may occur if the blood and stain are not mixed before the films are made. The specific gravity of the
reticulocytes is lower than that of mature RBCs, and reticulocytes settle at the top of the mixture during incubation
Moisture in the air, poor drying of the slide, or both may cause areas of the slide to appear refractile, and these areas
could be confused with reticulocytes. The RNA remnants in a reticulocyte are not refractile
Other RBC inclusions that stain supravitally include Heinz, Howell-Jolly, and Pappenheimer bodies may be mistaken as
reticulocytes
If a Miller disc is used, it is important to heed the “edge rule” as described in the WBC count procedure

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 Retic Count formula Relative count:
Reference range is 0.5-1.5 % in Adult, 0.5% to 2.5% (Rodak’s 5th ed)

Retics (%) = __number of Retics x 100_

1000 RBCs observed

Absolute retic count:

It is the actual number of reticulocytes in 1 Liter or microliter of blood
Reference range is 25-75 x109 / L, 20 to 115x109 / L (Rodak’s 5th ed)

Absolute retic = Retics (%) x RBC count x1000

100
(CRC) Calculated to account for the degree of anemia by using standard normal hct of 45%
Corrected reticulocyte count Calculation performed to correct the visual reticulocyte count for specimens with a
hematocrit below 45% to the equivalent reticulocyte count at a hematocrit of 45%. In
anemia, the visual reticulocyte percentage is misleadingly elevated because whole blood
contains fewer red blood cells relative to reticulocytes

Patient Hematocrit (%)

CRC = Retics (%) x 45

Reticulocyte production General indicator of the rate of erythrocyte production increase above normal in
Index / Shift correction anemias
Index calculated to correct for a low hematocrit and the presence of shift reticulocytes
that otherwise may falsely elevate the visual reticulocyte count

RPI = CRC value_________

Maturation time of retics (in blood)

HEMATOCRIT MATURATION OF RETICS IN BLOOD

36-45 % 1.0 Days
26-35 % 1.5 Days
16-25 % 2.0 Days
<15 % 2.5 Days

Interpretation:
RPI is 1 when Hematocrit is equal to 0.45 L/L

RPI when >3 Adequate bone marrow response to anemia

→Chronic hemolysis, recent hemorrhage, and response to therapy

RPI when <2 Inadequate response of the BM to anemia

→BM failure, aplastic anemia, ineffective erythropoiesis like vitamin B12 deficiency

Reticulocyte count using The disc is composed of two squares,

miller ocular disk the area of smaller square(B)
measuring 1/9 of the larger
square.
RBCs are counted of smaller square,
Reticulocytes are counted on larger
square
A minimum of 112 cells should be
counted in the small square

Formula % Retic = number of retics in square (A) x 100

Number of RBC in square(B) x 9

Immature Reticulocyte Done in automated instrument

Fraction It reflects the proportion of the more immature reticulocytes in the specimen useful in
detecting early erythropoietic activity after chemotherapy or hematopoietic stem cell
transplantation.
The immature reticulocyte fraction and the immature platelet fraction provide an early
indication of engraftment success after hematopoietic stem cell transplant

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 [Link] DETERMINATION
Hematocrit is the volume of packed red blood cells that occupies a given volume of whole blood
It is also referred as Packed cell volume
The packed cell volume (PCV) is a measurement of the ratio of the volume occupied by the RBCs to the volume of whole
blood in a sample of capillary or venous blood. Following centrifugation, this ratio is measured and expressed as a
percentage or decimal fraction (Turgeon)
The PCV is used for detecting anemia, polycythemia, hemodilution, or hemoconcentration.

Population CONVENTIONAL UNITS S.I UNITS

Adult males 40 to 55 % 0.40 to 0.54 L/L
Adult females 36 to 48 % 0.35 to 049 L/L
Newborn 45 to 60 % 0.53 to 0.65 L/L

1. MACROHEMATOCRIT METHOD
• Winthrobe and Landsberg Double oxalate
• Van allen Sodium oxalate
• Haden Sodium oxalate
• Sanford- Magath Sodium oxalate
• Bray Heparin

Speed of centrifugation = 2000 To 2300g for 30minutes

2. ADAM’S MICROHEMATOCRIT METHOD

Capillary tube:
• Length: 7-7.5cm or 70-75 mm
• Bore: 1-1.2 mm in diameter
• Can hold up to 0.05mL of blood
• Plug: 4 to 6 mm long (seal the capillary tubes at the end of the tube with the colored ring)
• 2 type: With red band (with anticoagulant heparin)
With blue band (without anticoagulant)

LAYERS OF SPUN HEMATOCRIT

Top layer Fatty layer Normally, layer is barely visible
In the presence of lipidemia, layer several mm thick
Second layer Plasma Normally pale yellow and fairly clear
Excessive hemolysis results in cherry red color
Jaundice produces a deep yellow color
Third layer Buffy coat Normally less than 1 mm thick
Thick layer when white cell count exceeds 10,000 cu/mm
Packed platelets are found in upper part layer
Fourth layer (bottom) Packed Red Cell Volume (packed) is read as hematocrit

PROCEDURE
Perform skin puncture
Wipe off the first drop of blood
Fill two heparinized capillary tubes two third with blood (air bubbles denote poor skills but does not affect
test result)
In a vertical position, carefully seal the dry end of the heparinized capillary tubes with the sealing clay and the plug
should be 4 to 6 mm long.
Place two heparinized capillary tubes in the radial grooves of the microcentrifuge with their heads exactly opposite
each other. The sealed end should be away from the center of the centrifuge
Spin for 5minutes at 10000-15000 RPM (RPM Should be checked periodically with a Tachometer)
After centrifugation, read the hematocrit within 10 minutes (The buffy coat layer should not be included)

REMINDERS
Trapped plasma (microhematocrit) may cause the Hct to be falsely increased by as much 1% to 3% (0.01 to
0.03 L/L) higher than the value obtained using automated instruments
When determined by fully automated methods, the Hematocrit may be 0.01 to 0.03 L/L lower than the
microhematocrit method because it is electronically calculated and therefore in unaffected by trapped plasma
Automated hematocrit – a calculated value from RBC and MCV

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 Trapped plasma- small amount of plasma that remains in the RBC portion of the spun Hematocrit even when properly
centrifuged.
Trapped plasma increased in conditions such as Sickle cell anemia, Hypochromic anemia, Spherocytosis,
Macrocytosis and Thalassemia
Certain abnormal RBC shapes like spherocytes and sickle cells inhibit complete packing
Excess anticoagulant / Short draw/underfilled tubes = decrease hematocrit results
The values of duplicate hematocrits should agree within 1%
Specimens for microhematocrit determination should be centrifuged within 6 hours of collecting.

Errors in Microhematocrit Results

False Increase Hematocrit False Decrease Hematocrit
Hemoconcentration Hemolysis and overcentrifugation
Dehydration Introduction of interstitial fluid
Insufficient centrifugation time Increase anticoagulant
Buffy coat is read Improper sealing of capillary tube
Allowing the tubes to stand longer than several Improper flushing of an intravenous catheter
minutes

3. RULE OF THREE
Used for checking the validity of test results
Works only on Normocytic, Normochromic specimens
3 x RBC Count = Hb
3 x Hb = Hct ± 3 (%)

[Link] SEDIMENTATION RATE

▪ A non-specific measurement used to detect and monitor an inflammatory response to tissue injury
▪ The erythrocyte sedimentation rate (ESR) is ordered with other tests to detect and monitor the course of inflammatory
conditions such as, rheumatoid arthritis, infections, or certain malignancies. It is also useful in the diagnosis of temporal
arteritis and polymyalgia rheumatic
▪ The ESR value is determined by measuring the distance from the surface meniscus to the top of the erythrocyte
sedimented in a special tube that is placed perpendicular in a rack for 1 hour.
▪ Elevated ESR: Pregnancy, acute and chronic infections, rheumatic fever, rheumatic arthritis, MI, nephrosis, acute
hepatitis, menstruation tuberculosis, macroglobulinemia, cryoglobulinemia, hypothyroidism, and hyperthyroidism
▪ Decreased ESR: polycythemia, congestive heart failure, hypofibrinogenemia and in the presence of RBC abnormalities
such as poikilocytosis, spherocytosis and sickle cells
▪ The ESR is directly proportional to the red blood cell mass and inversely proportional to plasma viscosity.
▪ The ESR is directly proportional to the weight of the cell aggregate and inversely proportional to the surface area

STAGES
Initial rouleaux formation 10 minutes
Rapid settling of RBCs/Decantation 40 minutes
Final sedimentation of RBCs 10 minutes

Setting the rack of sedimentation rate tubes on top of the refrigerator could lead to the following (Steinenger)
a. False decrease ESR because of lower temperature from air rushing out on opening the refrigerator or freezer
b. A falsely increased ESR attributable to vibrations from opening and closing the refrigerator and freezer doors
c. A falsely increased ESR because of heat

METHODS FOR DETERMINATION OF ESR

Wintrobe and Landsberg ▪ Anticoagulant: Double oxalate or EDTA
▪ Length of 11.5 cm/ 115 mm
▪ Internal Bore of 3.0 mm

Standard/ Original ▪ Anticoagulant: 3.8 % Citrate (black)

Westergren ▪ Length of tube is 30 cm/ 300 mm
▪ Internal bore of 2.5mm

Modified Westergren ▪ Most commonly used method

▪ Anticoagulant: EDTA + 0.05 ml NSS/ Citrate
▪ Length tube: 200mm
▪ Internal bore: 2.55 mm

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 ▪ Westergren method is the most sensitive of the methods, because of the longer tube it requires more blood
▪ Wintrobe method requires a smaller amount of blood and involves no dilution
▪ Blood specimens must be analyzed within 4 hours of collection if kept at room temperature (18° to 25° C). If the
specimen is allowed to sit at room temperature for more than 4 hours, the RBCs start to become spherical, which may inhibit
the formation of rouleaux. Blood specimens may be stored at 4° C up to 24 hours before testing, but must be rewarmed by
holding the specimen at ambient room temperature for at least 15 minutes before testing. (Rodak’s 6th edition)
▪ Blood should be at room temperature for testing and should be no more than 2 hours old. If anticoagulated blood is
refrigerated, the test must be set up within 6 hours. Hemolyzed specimens cannot be used. (Turgeon)

Reference Range
Westergren Male 0–15mm/hr (0–50 years old)
0–20 mm/hr (>50 years old)
Female 0–20 mm/hr (0–50 years old)
0–30 mm/hr (>50 y)
Children 0-10mm/hr
Wintrobe Male 0-9mm/hr
Female 0-20mm/hr
Children 0-13mm/hr

For every 1 to 3 degree angle tilt will lead to _________error

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 ESR MARKEDLY INCREASE (100 MM OR MORE PER HOUR) ESR MODERATELY INCREASED
Multiple myeloma Acute and chronic infectious diseases
Waldenstrom macroglobulinemia Acute localized infections
Malignant lymphoma Reactivation of a chronic infection
Leukemia Rheumatic fever
Acute and severe bacterial infection Rheumatoid arthritis
Severe anemia Myocardial infarction
Carcinoma Malignant tumors with necrosis
Collagen diseases Hyperthyroidism and hypothyroidism
Sarcoma Lead and arsenic intoxication
Portal or biliary cirrhosis Nephrosis
Ulcerative Colitis Internal hemorrhage
Severe renal diseases Acute hepatitis
Unruptured ectopic pregnancy after third month
Ruptured ectopic pregnancy
Menstruation
Normal pregnancy after third month
Tuberculosis
Ingestion of oral contraceptives
Intravenous dextran

ESR USUALLY NORMAL ESR DECREASED

Early acute appendicitis Spherocytosis
Early unruptured ectopic pregnancy Poikilocytosis
Malarial paroxysm Sickle cell anemia
Cirrhosis of liver Hemolytic jaundice
Degenerative arthritis Severe iron deficiency
IM Thalassemia
Acute allergies
Uncomplicated virus diseases
Peptic ulcer
Typhoid fever and Undulant fever
Rheumatic carditis with cardiac failure
Pertussis

VIII. LABORATORY TEST FOR IRON METABOLISM

TESTS REFERENCE RANGE COMMENTS
Serum Iron 50 to 150 ug/dl ▪ Indicator of available transport iron
▪ Used for the differential diagnosis of disorders of iron metabolism
▪ Specimen: non hemolyzed serum and should be obtained in the
morning because serum iron levels may be approximately 25 % lower
in the evening
▪ 12 hours fasting required
Total iron binding 250 to 400 ug/dl ▪ Indirect indicator of iron stores
capacity (TIBC) ▪ Indirectly measures the concentration of transferrin by
measuring its ability to bind iron
▪ Specimen: non hemolyzed serum
▪ 12 hours fasting required
▪ TIBC Values are independent on the time of day the sample is drawn
Serum Ferritin 15 to 200 ug/ L (men) ▪ Reveals the body’s tissue on iron stores
12 to 150 ug/L (women) ▪ Good indicator of iron storage status
▪ Useful in diagnosis of iron deficiency
40 to 400 ng/ml ▪ Generally, the first laboratory test to become abnormal when iron
(Rodak’s) stores become to decline
▪ Measured using radioimmunoassay
Free Erythrocye Depends on the method ▪ RBCs produce slightly more protoporphyrin than is necessary, but in
Protoporphyrin being used but an iron deficiency, protoporphyrin levels build up in RBCs to several times
approximate reference the normal
value is <50 ug/dl of RBC ▪ May be measured directly by a hemato fluorometer or by an extraction
and fluorescence method
▪ Differentiating thalassemia, IDA, & anemia of chronic infection

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 IRON COMPARTMENTS IN NORMAL HUMAN
COMPARTMENTS FORM AND ANATOMICAL SITE
Functional Hemoglobin iron in the blood
Myoglobin iron in muscles
Peroxidase, catalase, cytochromes, riboflavin enzymes in all cells
Storage Ferritin and hemosiderin mostly in macrophages and hepatocytes; small amounts in all cells except
mature RBC
Transport Transferrin in plasma

IX. ZETA SEDIMENTATION RATIO

Performed using a zetafuge and special capillary tubes
Result is dependent on the concentration of fibrinogen and gamma globulins
Requires a small amount of specimen, is not affected by anemia
Same reference range for male and female: 40 to 51 %

ZSR = Hct(%) x 100

Zetacrit (%)

X. OSMOTIC FRAGILITY TEST

Measures the ability of the RBC to take up fluid without lysing
Primary factor affecting OFT is shape of RBC, which, in turn, depends on the volume, surface and functional state of RBC
membrane
Principle: Red blood cells are diluted in 0 to 0.85 % hypotonic saline solutions and the amount of hemolysis at each
concentration is determined by measuring the absorbance of the supernatant at 540 nm
Spherocytes already have a decreased surface area–to–volume ratio, they lyse in less hypotonic solutions than normal-
shaped, biconcave RBCs and thus have increased osmotic fragility.
Anticoagulant used: Heparin
Specimen: stable for 2 hours at room temp or 6 hours if refrigerated
Lack of sensitivity, and is replaced by Eosin-5’-maleimide (EMA) binding test for detecting hereditary
spherocytosis

Griffin and Sanford Method

TUBE 25 24 23 22 21 20 19 18 17 16 15 14
0.5% NaCl 25 24 23 22 21 20 19 18 17 16 15 14
Water 0 1 2 3 4 5 6 7 8 9 10 11
%NaCl 0.50 0.48 0.46 0.44 0.42 0.40 0.38 0.36 0.34 0.32 0.30 0.28

Normal values: Initial hemolysis is in TUBE 21 or 22 (0.42-0.44%)

Complete hemolysis is in Tube 16 or 17 (0.32- 0.34%)

I.K Aytona Page 43

 Increased OFT Decreased OFT
(Decreased resistance in hypotonic solution) (Increased resistance in hypotonic solution)
Hereditary spherocytosis Splenectomy and Liver diseases
Acquired hemolytic anemias with spherocyte Conditions in which target cells are present
Conditions where spherocyte is found Sickle cell anemia
Old cells IDA
Thalassemia
Reticulocytes shows decrease OFT

XI. SICKLE CELL TESTS

SODIUM METABISULFITE TEST

-Whole blood is mixed with sodium metabisulfite, a strong reducing agent which deoxygenated the hemoglobin. Under these
conditions, Hemoglobin S present in the red blood cells causes the formation of sickle shaped red cells

Positive result: Formation of sickle cell shaped red cells (Hb S)

SOLUBILITY/DITHIONITE TEST
The most common screening test for Hb S, called the hemoglobin solubility test, capitalizes on the decreased
solubility of deoxygenated Hb S in solution, producing turbidity. Blood is added to a buffered salt solution containing a
reducing agent, such as sodium hydrosulfite (dithionite), and a detergent-based lysing agent (saponin). The saponin
dissolves membrane lipids, causing the release of hemoglobin from the RBCs, and the dithionite reduces the iron from
the ferrous to the ferric oxidation state. Ferric iron is unable to bind oxygen, converting the hemoglobin to the
deoxygenated form. Deoxygenated HbS polymerizes in solution, which renders it turbid, whereas solutions containing
non-sickling hemoglobins remain clear.
This is a biphasic system consisting of an upper organic phase of toluene and a lower, aqueous phase containing
phosphate buffer, saponin, and reducing agents. Erythrocytes are lysed by toluene and saponin, with the released
hemoglobin being reduced by sodium hydrosulfite. The resulting colors of the aqueous phase and the interface phase
allow for the differentiation of hemoglobin types AA, AS, and SS (Turgeon)

When red blood cells are added to the working solution, the red cells immediately lyse due to the saponin (detergent
based lysing agent) present. Hemoglobin S and other sickling hemoglobins, in the reduced state, in a concentrated buffer
solution, forms liquid crystals and yields a turbid appearance

Hemoglobin S is insoluble to reducing agent such as the sodium dithionite / sodium hydrosulfite, producing turbidity.
It is done by assessing the turbidity of tubes in a background with black lines
This test cannot differentiate sickle cell trait and sickle cell disease
This test is also positive for Hemoglobin C disease

Interpretation
Negative for Hb S absence of turbidity and lines are visible
Positive for Hb S presence of turbidity and lines are not visible

False positive result False negative result

a. Hyperlipidemia a. infants less than 6 months of age
b. few rare hemoglobinopathies b. low hematocrit values
c. too much blood is added to the test
solution

Confirmatory test for Sickle cell disease Hemoglobin electrophoresis, HPLC, or capillary electrophoresis

XII. QUANTITATION OF FETAL HEMOGLOBIN OR HEMOGLOBIN F

A. Betke method of alkali denaturation
▪ A red cell hemolysate is prepared to lyse the red blood cells and free the hemoglobin. This test utilizes the
characteristics of fetal hemoglobin to resist denaturation in an alkaline solution. The hemolysate is added to
cyanmethemoglobin reagent and then exposed to an alkaline reagent sodium hydroxide, for a specified period.
During this time, normal hemoglobin is denatured or destroyed, but the fetal hemoglobin remains intact;
ammonium sulfate is added to halt the denaturation process and to precipitate the denatured hemoglobin. The
solution is filtered, measured spectrophotometrically, and compared with the spectrophotometric readings of the
original cyanmethemoglobin solution to determine the percent of hemoglobin F.

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 B. Singer method of alkali denaturation
▪ The red cell hemolysate is incubated with potassium hydroxide, which destroys(denatures) normal and nonfatal
hemoglobin. Addition of the precipitating solution stops this reaction and precipitates the denatured hemoglobin.
The non-altered hemoglobin (F) is then measured spectrophotometrically.

C. KLIEHAUER BETKE TEST (ACID ELUTION TEST)

▪ Used to quantitate the number of fetal Rh-positive cells in the mother’s circulation as a result of a fetomaternal
hemorrhage.
▪ In the Kleihauer-Betke test, a maternal blood smear is treated with acid and then stained with counterstain
▪ Stain: Shepard’s stain
Interpretation
Positive Fetal RBC (with Hb F)
Result =RBCs are deeply stained red and resist acid denaturation
Negative Maternal RBC (Hb A)
result =RBC appear as pale pink ghost cells and susceptible to acid denaturation

XIII. TESTS FOR UNSTABLE HEMOGLOBIN

a. Heat precipitation test
▪ A red blood cell hemolysate is mixed with tris buffer pH 7.4 and incubated at 50 oC for 2 hours
▪ Most unstable hemoglobins will precipitate out more rapidly than normal hemoglobins at this elevated temperature

b. Isopropanol Precipitation test

▪ A red blood cell hemolysate is prepared, added to 17% solution of isopropanol, and incubated at 37 oC . The
specimens are examined at 5 minutes and 20 minutes for precipitation of unstable hemoglobin

XIV. ASCORBATE CYANIDE SCREENING TEST

▪ Non-specific procedure for detecting deficiencies in the pentose phosphate pathway; G6PD deficiency, Glutathione
peroxidase deficiency and glutathione reductase
▪ Reagent: sodium cyanide and sodium ascorbate
▪ Normal color: RED
▪ Abnormality / Enzyme deficient: Brown

XV. G6PD FLUORESCENT SCREENING TEST

▪ Normal result: Maximum fluorescence at 10 minutes
▪ G6PD deficiency: Little or no Fluorescence

XVI. TEST FOR PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Sugar water screening test Citrated WB: 1 part of 0.109 M sodium citrate to 9 parts whole blood. Obtain blood specimen
for the normal control at the same time the patient’s blood is collected.
-a simple screening procedure for PNH
-TEST SHOULD BE PERFORMED WITHIN 2 HOURS
-Use of defibrinated blood may cause false positive result due to hemolysis of the traumatized
RBCs
Sucrose hemolysis test Citrated WB: 1 part of 0.109 M sodium citrate to 9 parts whole blood. Obtain blood specimen
for the normal control at the same time the patient’s blood is collected
-used as a confirmatory test for PNH when the sugar water test is positive
- Erythrocytes in PNH lyse when exposed to serum solutions of low ionic strength containing
complement. This test demonstrates the sensitivity of erythrocytes to the protein, complement.
Normal erythrocytes under similar circumstances do not lyse.
Ham’s Acid serum test Whole blood, 10ml, to be defibrinated – CONFIRMATORY & DEFINITIVE TEST FOR
PNH
-reliably test for PNH
- Erythrocytes are incubated with fresh and heated serum to test for hemolysis. Weak acid is
used in specific serum cell mixtures to maximize hemolytic activity. The presence of hemolysis,
depending on the test conditions, may be observed in cases of antibody-sensitized coated
erythrocytes, spherocytes, or paroxysmal nocturnal hemoglobinuria (PNH).
Flow cytometric analysis of New approach to confirm PNH
GPI anchored proteins, CD 55
and CD 59

I.K Aytona Page 45

 XVII. AUTOHEMOLYSIS TEST
▪ Test for G6PD, PK deficiency, and hereditary spherocytosis
▪ Uses defibrinated blood, incubated at 48 hours at 37’C
a. G6PD deficiency = corrected upon addition of glucose
b. PK deficiency = corrected upon addition of ADP
c. HS= corrected upon addition of glucose and ADP

Principle: one portion of sterile, defibrinated blood is incubated at 48hours at 37Ç. A second sample of the defibrinated blood is
incubated with a specific amount of glucose. The percentage of hemolysis in each specimen is determined spectrophotometrically.
Normally, and in certain disorders such as hereditary spherocytosis, the amount of autohemolysis is reduced in the presence of glucose.
In other pathologic states, the addition of glucose does not effectively decrease the autohemolysis. The degree of hemolysis that takes
place in this procedure (with and without glucose) is a function of the metabolism and membrane properties of the RBC.

XVIII. EOSIN 5’MALEIMIDE

▪ A sensitive test for detecting Hereditary spherocytosis
▪ It uses EMA Fluorescent dye that binds to transmembrane protein band 3, Rh, RhAg, and CD47 in the RBC
membrane.
▪ Measured by Flow cytometry
▪ Hereditary Spherocytosis- has a lower Mean fluorescence intensity that is reported in % decrease MFI

[Link] LANDSTEINER TEST FOR PCH

TUBE 1 PX TEST SAMPLE RESULT

INCUBATED AT 4’C FOR 30 MINS HEMOLYSIS
INCUBATED AT 37’C FOR 30 MINS HEMOLYSIS

TUBE 2 PX CONTROL SAMPLE RESULT

INCUBATED AT 37’C FOR 30 MINS NO HEMOLYSIS
INCUBATED ANOTHER AT 37’C FOR 30 MINS NO HEMOLYSIS

INTERPRETATION 😊
*INCUBATE→ CENTRIFUGE→ CHECK SUPERNATANT FOR HEMOLYSIS
(+) PCH = If Patient sample shows hemolysis at 4’c and 37’c but the control sample shows no hemolysis on both 37’C

NOTE 😊
Initial test results may be falsely negative due to low complement and/or anti-P levels in the patient sample because of the brisk
hemolysis in vivo. Incubating patient serum with complement and papain-treated compatible group O RBCs
increases the sensitivity of the test in detecting anti-P. The enzyme treatment provides greater exposure of the P antigen
on the RBC surface for antibody binding

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 GRADING CHARTS IN HEMATOLOGY
MORPHOLOGY GRADED AS
Polychromatophilia 1+ = 1 to 5/field
Helmet cells 2+ = 6 to 10/ field
Teardrop RBC 3+ = >10/field
Acanthocytes
Schistocytes
Spherocytes
Poikilocytes 1+ = 3 to 10/ field
Ovalocytes 2+ = 11 to 20 / field
Elliptocyte 3+ = >20 / field
Burr cells
Bizzare- shaped RBC
Target cells
Stomatocytes
Rouleaux 1+ = aggregates of 3 to 4 RBC
2+ = aggregates of 5 to 10 RBC
3+ = numerous aggregates with only few free RBC
Sickle cells Grade as positive only
Basophilic stippling
Pappenheimer bodies
Howell- Jolly bodies

MORPHOLOGY GRADE AS
Rouleaux 1+ = aggregates of 3 to 4 RBC
2+ = aggregates of 5 to 10 RBC
3+ = numerous aggregates with only few free RBC
Sickle cell Grade as positive only
Basophilic stippling
Pappenheimer bodies
Howell-Jolly bodies

Sample Criteria For Erythrocytic Morphology Evaluation

Morphologic Characteristics WNL 1+ 2+ 3+ 4+
Macrocytes 0-5 5-10 10-20 20-50 >50
Microcytes 0-5 5-10 10-20 20-50 >50
Hypochromia 0-2 3-10 10-50 50-75 >75
Poikilocytosis (generalized variations in shape) 0-2 3-10 10-20 20-50 >50
Burr cells 0-2 3-10 10-20 20-50 >50
Acanthocytes <1 2-5 5-10 10-20 >20
Schistocytes <1 2-5 5-10 10-20 >20
Dacryocytes 0-2 2-5 5-10 10-20 >20
Codocytes 0-2 2-10 10-20 20-50 >50
Spherocytes 0-2 2-10 10-20 20-50 >50
Ovalocytes 0-2 2-10 10-20 20-50 >50
Stomatocytes 0-2 2-10 10-20 20-50 >50
Sickle cells Absent Report as to indicate presence, do Not
+1 quantitate
Polychromaatophilia <1 2-5 5-10 10-20 >20
Adult 1-6 7-15 15-20 20-50 >50
Newborn
Basophilic stippling 0-1 1-5 5-10 10-20 >20
Howell- Jolly bodies abs 1-2 3-5 5-10 >10
Siderocytes (Pappenheimer bodies) abs 1-2 3-5 5-10 >10

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 Polychromasia:
1+ if 1–3 polychromatic cells are found per microscopic field
2+ if 3–5 polychromatic cells are found per microscopic field
3+ if more than five polychromatic cells are found per microscopic field

Basophilic Stippling:
Slight if one stippled RBC is noted in every other microscopic field
Moderate if 1–2 stippled RBCs are noted in every microscopic field
Marked if three or more stippled RBCs are noted in every microscopic field

Macrocytosis:
1+ (i.e., slight) if there are approximately 25% macrocytic RBCs present per high-power field
2+–3+ (i.e., moderate) if there are 25% to 50% macrocytic RBCs present per high-power field
4+ (i.e., marked) if there is >50% macrocytic RBCs per high-power field

Rouleaux Formation:
Slight, if one to two RBC chains are found per thin microscopic field
Moderate, if three to four RBC chains are found per thin microscopic field
Marked, if five or more RBC chains are found per thin microscopic field

GRADING SCALE FOR RED CELL MORPHOLOGY

ANISOCYTOSIS / POIKILOCYTOSIS
Percentage of Red Cells that Differ in Size or Shape from Normal RBCs
Normal: 5%
Slight: 5-10%
1+ 10-25%
2+ 25-50%
3+ 50-75%
4+ >75%

Numerical Description
Scale
0 Normal appearance or slight variation in erythrocytes.
1+ Only a small population of erythrocytes displays a particular abnormality; the terms slightly increased or few
would be comparable.
2+ More than occasional numbers of abnormal erythrocytes can be seen in a microscopic field; an equivalent
descriptive term is moderately increased.
3+ Severe increase in abnormal erythrocytes in each microscopic field; an equivalent descriptive term is many.
4+ The most severe state of erythrocytic abnormality, with the abnormality prevalent throughout each microscopic
field; comparable terms are marked or marked increase

RED BLOOD CELL DISORDERS

ANEMIA

Anemia is defined as a decrease in RBC, Hb, and hematocrit resulting in decreased oxygen delivery to the tissues. The
anemias can be classified morphologically using RBC indices (MCV, MCH, MCHC). They can also be classified based on
etiology/cause.
Anemia is derived from the Greek word anaimia, meaning “without blood”
Anemia should not be thought of as a disease but rather as a manifestation of an underlying disease or deficiency
The usual complaints of an anemic patient are easy fatigability and dyspnea on exertion. Other general manifestations can
include vertigo, faintness, headache, and heart palpitations.
The most common physical expressions of anemia are pallor, low blood pressure, a slight fever, and some edema.
The causes of anemia fall into three major pathophysiological categories:
1. Blood loss
2. Impaired red cell production
3. Accelerated red cell destruction (hemolysis in excess of the ability of the marrow to replace these losses)

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 Ineffective erythropoiesis Refers to the production of erythroid precursor cells that are defective. These precursor cells
undergo apoptosis.
A. Megaloblastic anemia
B. Thalassemia
C. Sideroblastic anemia
D. Myelodysplastic syndrome
Insufficient erythropoiesis Refers to a decrease in the number of erythroid precursors in the bone marrow, resulting in
decreased RBC production and anemia

A. IDA
B. Anemia due to Renal diseases
C. Aplastic anemia
D. Sarcoidosis
E. Acute leukemia

Laboratory Test Used to Diagnose Anemia

• CBC
• RBC indices
• Reticulocyte count
• Peripheral blood film
• Patient history
• Physical examination
• BM examination
• Other lab test such as urinalysis and chemistry test

Initial LAB test for diagnosis of anemia = CBC (Hemogram), Reticulocyte count, and Peripheral Blood Smear

PATHOPHYSIOLOGIC CLASSIFICATION OF ANEMIAS

Anemia Caused by Decreased Production of Red Blood Cells

Bone marrow acquired and congenital aplastic anemia, pure red cell aplasia, myelopthisic anemia
failure
Impairment of Disorders of DNA synthesis Megaloblastic anemia
erythroid Disorders of hemoglobin Iron deficiency anemia, thalassemia, sideroblastic anemia, anemia of
development synthesis chronic inflammation
Decreased production of Anemia of renal disease
erythropoietin

Anemia Caused by Increased Red Blood Cell Destruction or Loss

Intrinsic RBC Membrane defect:
abnormality Hereditary spherocytosis, hereditary elliptocytosis or pyropoikilocytosis, paroxysmal nocturnal
hemoglobinuria

Enzyme deficiencies:
glucose-6-phosphate dehydrogenase deficiency, pyruvate kinase deficiency Globin abnormalities: sickle cell
anemia, other hemoglobinopathies

Extrinsic RBC Immune causes:

abnormality warm-type autoimmune hemolytic anemia, cold agglutinin disease, paroxysmal cold hemoglobinuria,
hemolytic transfusion reaction,
hemolytic disease of the fetus and newborn

Nonimmune causes:
microangiopathic hemolytic anemia (thrombotic
thrombocytopenic purpura, hemolytic uremic syndrome, HELLP syndrome, disseminated intravascular
coagulation), macroangiopathic hemolytic anemia (traumatic cardiac hemolysis), infectious agents (malaria,
babesiosis, bartonellosis, clostridial sepsis), other injury (chemicals, drugs, venoms, extensive burns)
Blood loss Acute and chronic blood loss anemia

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 MORPHOLOGIC CLASSIFICATIONS OF ANEMIA (BROWN)
MACROCYTIC - NORMOCHROMIC NORMOCYTIC, NORMOCHROMIC MICROCYTIC - HYPOCHROMIC
A. Vitamin B12 deficiency – Folic A. Defective formation of the red o Iron deficiency anemia
acid deficiency blood cells or the presence of o Thalassemia
o Pernicious anemia tumor cells in the bone marrow
o Sideroblastic anemia
o Sprue o Aplastic anemia
o Chronic blood loss
o Following gastrectomy o Leukemia
o Anemia of chronic
o Dietary o Hodgkin’s disease inflammation
o Abnormal intrinsic factor o Multiple myeloma o Lead poisoning
o Tapeworm o Leukoerythroblastosis o Hemoglobin E disease
infection([Link]) o Metastatic cancer
B. Disease of the liver o Anemia associated with “TAILS CHe”
C. Hypothyroidism renal and endocrine disease
B. Abnormal hemoglobin, increased
destruction of red blood cells
o Certain acquired hemolytic
anemia
o PNH
o Sickle cell anemia
o HDN
o Anemia of chronic renal
insufficiency

The RDW can help determine the cause of an anemia when used in conjunction with the MCV.

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 MICROCYTIC / HYPOCHROMIC ANEMIA
Iron deficiency ❖ Most common form of anemia
anemia ❖ Prevalent in infants and children, pregnancy, excessive menstrual flow, elderly with poor diets,
malabsorption syndromes, chronic blood loss

ETIOLOGY
a. Inadequate intake of iron
b. Increase demand (ex. Pregnancy, infancy, and childhood)
c. Impaired iron absorption (ex. Celiac disease and decrease stomach acidity)
d. Chronic blood loss (ex. Chronic GI bleeding, prolong menorrhagia, fibroid tumors, or
hemorrhoids)

Clinical signs and symptoms

Fatigue, weakness, and shortness of breath, especially with exertion, sore tongue (glossitis), inflamed
cracks at the corners of the mouth (angular cheilosis), Koilonychia (spooning of the fingernails) may
be seen if the deficiency is long-standing. Patients also may experience cravings for nonfood items,
called pica.
PICA = The cravings may be for things such as dirt, clay, laundry starch.

STAGES OF IDA
STAGE DESCRIPTION HEMOGLOBIN SERUM IRON TIBC FERRITIN
1 Storage depletion Normal Normal Normal Decreased
2 Transport Normal Decreased Increased Decreased
depletion
3 Functional Decreased Decreased Increased Decreased
depletion

Parasites Hookworms, Trichuris trichiura, Schistosoma mansoni, and

associated with IDA Schistosoma haematobium
Anemia of Chronic a. Second most common type of anemia
disease/Inflammation b. Commonly associated with systemic diseases, including chronic inflammatory
conditions such as rheumatoid arthritis, chronic infections such as tuberculosis or human
immunodeficiency virus infection, and malignancies

ETIOLOGY
c. Due to inability to use available iron for hemoglobin production
d. Impaired release of storage iron associated with increased Acute phase reactants such as
Hepcidin (decreases release of iron from stores) , Ferrtin, and Lactoferrin
Sideroblastic ❖ Caused by blocks in the protoporphyrin pathway resulting in defective hemoglobin synthesis
anemia and iron overload
❖ In this type of anemia, the body has adequate iron but is unable to incorporate it into
hemoglobin synthesis. The iron enters the developing erythrocyte but then accumulates in the
perinuclear mitochondria of metarubricytes
❖ Excess iron accumulates in the mitochondrial region of the immature RBC in the BM and
encircles the nucleus; cells are called ringed sideroblasts
❖ A ring sideroblast is an erythroid precursor containing at least five iron granules per cell, and
these iron-containing mitochondria must circle at least one-third of the nucleus
❖ Excess iron accumulates in the mitochondrial region of the mature RBC in circulation; cells are
called ringed siderocytes; inclusions are siderotic granules (Pappenheimer bodies on Wright’s
stained smears)
❖ Sideroblasts and Siderocytes are best demonstrated using Perl’s Prussian blue stain

TYPES
1. Primary – irreversible; causes of block is unknown
a. Two RBC populations (Dimorphic)
b. One of myelodysplastic syndromes- Refractory anemia with ringed
sideroblasts (RARS)
2 Secondary- reversible; causes include alcohol, anti-TB drugs, Chloramphenicol
Anemia due to lead Anemia, when present in lead poisoning, is most often normocytic and normochromic; however, with
poisoning chronic exposure to lead, a microcytic, hypochromic clinical picture may be seen
❖ Multiple blocks in the protoporphyrin pathway that affect heme synthesis
❖ Presence of many coarse Basophilic Stipplings
❖ lead poisoning will lead to acquired sideroblastic anemia and acquired porphyria
❖ It inhibits ferrochelatase and D-ala synthase enzyme in Heme/Protoporphyrin pathway

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 LABORATORY FEATURES IN MICROCYTIC / HYPOCHROMIC ANEMIAS
MARROW MARROW
TYPE SERUM SERUM % % IRON SERUM ZPP Hb Hb
IRON TIBC SATURATION SIDEROBLASTS STORES FERRITIN A2 F
Iron ↓ ↑ ↓ ↓ ↓ ↓ ↑ N-↓ N
deficiency
Anemia
B- N (↑) N N N N-↑ N-↑ N ↑ N-↑
thalassemia
Anemia of ↓ N-↓ ↓ ↓ N-↑ N-↑ ↑ N N
chronic
disease
Sideroblastic ↑ ↓ ↑ ↑ ↑ ↑ ↑ N N-↑
anemia
TIBC= Total iron binding capacity; ZPP=zinc protoporhyrin ; ↓=decreased ↑=increased N=normal

MACROCYTIC / NORMOCHROMIC ANEMIA

1. Megaloblastic anemias
❖ Defective DNA synthesis causes abnormal nuclear maturation; RNA synthesis is normal, so the cytoplasm is not
affected. The nucleus matures slower than the cytoplasm (Asynchronism)
❖ It is an example of ineffective erythropoiesis
❖ Caused by either vitamin B12 or folic acid deficiency
❖ Laboratory picture of Pancytopenia, Oval macrocyte, Howell- jolly bodies, Hypersegmented neutrophil.
Screening test used to Diagnose Megaloblastic anemia
Five tests used to screen for megaloblastic anemia are the
1. complete blood count (CBC)- check for oval macrocytes and pancytopenia
2. reticulocyte count- decrease retic count
3. white blood cell (WBC) manual differential- check for Hypersegmented cells
4. serum bilirubin- increase indirect bilirubin and total bilirubin
5. lactate dehydrogenase- elevated LD (flipped LD)

*NOTE = Megaloblastic anemia is associated with hemolytic anemia because many RBC precursors dies during division in the BM,
many RBCs never enter the circulation (Ineffective erythropoiesis)

a. Vitamin B12 (Cobalamin) deficiency

• Pernicious anemia – caused by deficiency of intrinsic factor, Antibody to intrinsic factor or antibodies to parietal
cells
• Other causes: Malabsorption, total gastrectomy, total vegetarian diet, [Link] infection, Intestinal blind loops,
and sprue
• Vit. B12 deficiency takes 3-6 years to develop because of high body stores
• Clinical symptoms: Jaundice, weakness, glossitis, GI bleeding, numbness and CNS PROBLEMS
b. Folic acid deficiency
• Associated with poor diet, pregnancy or chemotherapeutic anti folic drugs such as METHOTREXATE.
• Folic acid has low body stores
• Clinical symptoms: same with vitamin B12 deficiency, except NO CNS involvement

2. Non- Megaloblastic anemia

❖ Include Alcoholism, Liver disease, and conditions causes accelerated erythropoiesis
❖ RBC are round and not oval as seen in the megaloblastic anemias
❖ Nonmegaloblastic forms of macrocytic anemias are also characterized by large RBCs, but in contrast to megaloblastic
anemias, they are typically related to membrane changes caused by disruption of the cholesterol-to-
phospholipid ratio. These macrocytic cells are mostly round, and the erythroid precursors in the bone marrow do
not display megaloblastic changes. Macrocytic anemias are often seen in patients with chronic liver disease, alcohol
abuse, and bone marrow failure. It is rare for the MCV to be greater than 115 fL in nonmegaloblastic anemias

EXAMPLES ASSOCIATED WITH NON-MEGALOBLASTIC MACROCYTIC ANEMIA (RODAK’S 5th edition)

-BM failure, Chronic liver disease, Alcoholism, reticulocytosis, liver disease, and normal newborns

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 NORMOCYTIC / NORMOCHROMIC ANEMIA

APLASTIC ANEMIA
Bone marrow failure that causes pancytopenia, NO response to EPO
The bone marrow is hypocellular/hypoplastic with increase fat
Aplastic anemia may be a result of immune mediated destruction of hematopoietic stem cells causing pancytopenia and
characterized by an empty bone marrow
There is generally neutropenia with relative lymphocytosis
Patients have poor prognosis with complications that include bleeding
Can be genetic, acquired or idiopathic
Treatment includes bone marrow or stem cell transplant and immunosuppression
Elevated serum EPO, TPO, CSF-GEMM

TYPES OF APLASTIC ANEMIA

1. Genetic aplastic anemia / Fanconi’s anemia (familial aplastic anemia)

• Autosomal recessive trait or X-linked pattern
• Characterized by Dwarfism, renal disease, mental retardation, skeletal abnormalities,
hypo/hyperpigmentation of skin, café-au-lait lesions, hypogonadism, and low birth weight.
• Lab findings:
1. Pancytopenia, reticulocytopenia, and a hypocellular bone marrow.
2. Macrocytic RBCs
3. Elevated Hb F and Alpha feto-protein

OTHER INHERITED FORMS OF APLASTIC ANEMIA (Rodak’s 5th edition)

I. Dyskeratosis Congenita
❖ Characterized by mucocutaneous abnormalities, BM failure, MACROYCTIC RBC and pancytopenia
❖ Clinical triad manifestation: abnormal skin pigment, dystrophic nails, and oral leukoplakia
❖ DKC chromosomes have very short telomeres, and inherited defects in the telomerase complex are implicated in
the pathophysiology
II. Shwachman-Bodian-Diamond syndrome
❖ An autosomal recessive disorder with multisystem disorder characterized by pancreatic insufficiency, cytopenia,
skeletal abnormalities, and a pre-deposition of hematological malignancies

2. Acquired Aplastic anemia (secondary)

• Caused by radiation, or antibiotics such as Chloramphenicol and sulfonamides
• Chemicals: Benzene and herbicides
• Viruses: Parvo Virus B19, viral hepatitis, measles, HIV, CMV, and EBV
• Miscellaneous condition: PNH, pregnancy, and Autoimmune diseases (Rodak’s)
• Accounts 10 to 15%

3. Idiopathic (primary)
• Accounts about 50-70 percent of aplastic anemia with no known causes.

PURE RED CELL APLASIA

DIAMOND BLACK FAN ANEMIA

Autosomal recessive
The bone marrow is generally normal except for a marked decrease in erythroid cells
True red cell aplasia (leukocytes and platelets are normal in number)
TYPES OF PURE RED CELL APLASIA (Rodak’s 5th edition)
a. The acquired form of PRCA in young children is also known as transient erythroblastopenia of childhood – commonly
associated with viral infection
b. Primary PRCA – may be idiopathic or auto immune related
c. Secondary PRCA may occur in association with an underlying thymoma, hematologic malignancy, solid tumor, infection,
chronic hemolytic anemia, collagen vascular disease, or exposure to drugs or chemicals
d. Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplastic disorder of early infancy

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 MYELOPHTHISIC (MARROW REPLACEMENT) ANEMIA
Hypoproliferative anemia caused by replacement of bone marrow hematopoietic cells by malignant cells or fibrotic tissue
Associated with cancers with bone metastasis
Due to the infiltration of abnormal cells into the bone marrow and subsequent destruction and replacement of normal
hematopoietic cells. Metastatic solid tumor cells (particularly from lung, breast, and prostate), leukemic cells, fibroblasts, and
inflammatory cells (found in miliary tuberculosis and fungal infections) have been implicated.
Picture of leukoerythroblastic blood = Peripheral blood findings include teardrop erythrocytes and nucleated RBCs,
as well as immature myeloid cells/WBC and megakaryocyte fragments

BLOOD LOSS ANEMIA

Acute Blood Loss

The acute loss of blood is usually associated with traumatic conditions such as an accident or severe injury. Occasionally,
acute blood loss may occur during or after surgery.
An acute blood loss does not produce an immediate anemia. A severe hemorrhage or rapid blood loss amounting to
more than 20% of the circulating blood volume reduces an individual’s total blood volume and produces a condition of
shock and related cardiovascular problems
In acute blood loss, the body itself adjusts to the situation by expanding the circulatory volume, which produces the
subsequent anemia. Fluid from the extravascular spaces enters the blood circulation and has a diluting effect on the
remaining cells.

Lab Findings
The earliest hematological change in acute blood loss is a transient fall in the platelet count, which may
rise to elevated levels within 1 hour.
The next change is the development of neutrophilic leukocytosis (from 10 to 35 × 109/L) with a shift to the left. The
hemoglobin and hematocrit do not fall immediately but fall as tissue fluids move into the blood circulation.
It can be 48 or 72 hours after the hemorrhage until the full extent of the red cell loss is apparent.
the peripheral blood film at 24 hours should be essentially normochromic and normocytic with normal red
blood cell (RBC) indices (mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], and mean corpuscular
hemoglobin concentration [MCHC]).
It takes about 2 to 4 days after the blood loss for the total white blood cell (WBC) count to return to normal and about 2
weeks for the morphological changes to disappear

Chronic Blood loss

Chronic blood loss is frequently associated with disorders of the gastrointestinal (GI) tract, although chronic blood loss
may be related to heavy menstruation in women or urinary tract abnormalities

In chronic anemias, blood loss of small amounts occurs over an extended period, usually months.

The chronic and continual loss of small volumes of blood does not disrupt the blood volume

A noticeable anemia does not usually develop until after storage iron is depleted. At first, the anemia is normochromic
and normocytic. Gradually, the chronic bleeding results in an iron deficiency, and the newly formed cells are
morphologically hypochromic and microcytic

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 HEMOLYTIC ANEMIAS
Results when the rate of RBC destruction exceeds the increased rate of RBC production.
Can be classified as
a. Acute versus chronic
b. Inherited versus acquired
c. Intrinsic versus extrinsic
d. Fragmentation (INTRAVASCULAR) versus macrophage-mediated (Extravascular)

OVERVIEW CLASSIFICATION OF HEMOLYTIC ANEMIAS (Rodak’s 5th edition)

Intravascular hemolysis ACUTE OR EPISODIC
Intrinsic defect a. Enzyme defects such as G6PD deficiency
b. Paroxysmal nocturnal hemoglobinuria (acquired)

CHRONIC
a. Globin abnormalities such as hemoglobinopathies, sickle cell, and thalassemia
b. Spur cell anemia of severe liver disease (acquired)
Intravascular hemolysis ACUTE OR EPISODIC
Extrinsic defect a. Immune hemolysis: cold antibody (IgM)
b. Microangiopathic hemolysis
c. Infectious agents, as in malaria
d. Thermal injury
e. Chemicals/drugs
f. Venoms

CHRONIC
a. Prosthetic heart valve
Extravascular hemolysis CHRONIC
Intrinsic defect a. Hereditary membrane defect (Ex. Hereditary spherocytosis)
Extravascular hemolysis ACUTE
Extrinsic defect a. Immune hemolysis: warm antibody (IgG)
b. Drugs

Laboratory evidence of the hemolytic anemia includes a decreased hemoglobin level, increased reticulocyte count,
increased serum indirect (unconjugated) bilirubin, increased serum lactate dehydrogenase (LD) activity, decreased
serum haptoglobin level, and increased urine urobilinogen

In some cases, the fragmentation is so severe that intravascular hemolysis occurs with varying amounts of hemoglobinemia,
hemoglobinuria, and markedly decreased levels of serum haptoglobin

CLASSIFICATION OF HEMOLYTIC ANEMIA

[Link] ANEMIA DUE TO INTRINSIC DEFECT

Normocytic / Normochromic anemia usually hereditary with reticulocytosis due to accelerated destruction
a. Hereditary Spherocytosis
b. Hereditary Elliptocytosis
c. Hereditary Stomatocytosis
d. Hereditary Acanthocytosis
e. Hereditary enzyme defects (G6PD, hexokinase, glutathione reductase, and Pyruvate kinase)
f. Hereditary Hemoglobinopathies (Sickle cell, Hgb C disease, Unstable hemoglobin disease, and Hgb E disease)
g. Hereditary defective globin synthesis (Thalassemias)
h. RH null disease
i. ACQUIRED – Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal Hemoglobinuria

There is a mutation of the PIGA gene that codes for phosphatidylinositol glycan class for the expression of
CD55 AND CD59
Lacks CD55 and CD59, thus there is increase susceptibility to complement lysis
Associated with hemolytic anemia, thrombosis, and bone marrow failure
The most common thrombotic manifestation is hepatic vein thrombosis, which obstructs venous outflow from the liver
(Budd-Chiari syndrome)

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 [Link] ANEMIAS DUE TO EXTRINSIC , IMMUNE DEFECTS

All causes Normocytic / Normochromic anemia due to defects extrinsic to the RBC with antibody participation.
a. Warm autoimmune hemolytic anemia (WAIHA)
b. Cold autoimmune hemolytic anemia (CAIHA) or Cold hemagglutinin disease
c. Paroxysmal cold hemoglobinuria (PCH)- IgG biphasic Donath Landsteiner antibody with P specificity
d. Hemolytic transfusion reaction (HTR)
e. Hemolytic disease of newborn (HDN)

PAROXYSMAL COLD HEMOGLOBINURIA

an acute form of cold-reactive hemolytic anemia. PCH can be idiopathic or secondary. Historically, secondary PCH was
associated with late-stage syphilis, but now it is most commonly seen in young children after a viral respiratory infection
The anti-P autoantibody, also called the Donath-Landsteiner antibody, is a complement-binding IgG hemolysin with
specificity for the P antigen on RBCs
The anti-P autoantibody is biphasic in that at cold temperatures it binds to the P antigen on RBCs and partially activates
complement (C1 to C4), but full complement activation (C3 through C9) and hemolysis occur only upon warming to 37°
C.

III. HEMOLYTIC ANEMIAS DUE TO EXTRINSIC / NON-IMMUNE DEFECTS

All cause normocytic / normochromic anemia caused by trauma to RBC.

Disorders that cause intravascular hemolysis with schistocytes and thrombocytopenia
a. Disseminated Intravascular coagulation (DIC)
b. Hemolytic uremic syndrome (HUS)
c. Thrombotic thrombocytopenic purpura (TTP)
d. HELLP (Hemolysis, elevated liver enzymes, low platelet count syndrome)
e. March hemoglobinuria – e.g marathon runners,
f. Infections such as malaria, babesia and Clostridium perfringens
g. Chemicals, toxin and venoms
h. Physical trauma – Burns, Cardiac replacement valve (Waring Blender syndrome) and Microangiopathic hemolytic
anemia

MICROANGIOPATHIC HEMOLYTIC ANEMIA

Microangiopathic hemolytic anemias (MAHAs) are a group of potentially life-threatening disorders characterized by RBC
fragmentation and thrombocytopenia. The RBC fragmentation occurs intravascularly by the mechanical shearing of RBC
membranes as the cells rapidly pass-through turbulent areas of small blood vessels that are partially blocked by
microthrombi or damaged endothelium
Examples of microangiopathic hemolytic anemias are DIC, HUS, TTP, and HELLP syndrome

Findings on the Peripheral Blood Film

1. Schistocyte/schizocyte= characteristic feature
2. RBC shearing that may produce helmet cell, and occasionally, microspherocytes
3. Polychromasia
4. Nucleated RBCs

The presence of schistocytes on the peripheral blood film is a characteristic feature of microangiopathic hemolytic
anemia. RBC shearing may also produce helmet cells and, occasionally, microspherocytes. polychromasia and nucleated
RBCs may also be present

Triad of features characteristic of microangiopathic hemolytic anemias

[Link] decrease in platelets
[Link] polychromasia
[Link] fragmentation (microspherocytes, schistocytes, keratocytes)

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 Thrombotic Is a rare, life-threatening disorder characterized by the abrupt appearance of
Thrombocytopenic microangiopathic hemolytic anemia, severe thrombocytopenia, and markedly elevated
Purpura/Moschowitz serum lactate dehydrogenase activity
syndrome Caused by a deficiency of the von Willebrand factor cleaving protease known as a
disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13
(ADAMTS-13).

3 TYPES
a. Idiopathic – commonly associated with autoantibodies to ADAMTS-13
b. InheritedTTP / Upshaw-Schulman syndrome – mutation in ADAMTS-13 gene
c. Secondary TTP
-triggered by infections, pregnancy, surgery, trauma, inflammation, and malignancy

Lab Findings
Hematologic Decreased hemoglobin, Decreased platelets,
Increased reticulocyte count
Peripheral Blood Film Schistocytes, Polychromasia, Nucleated red blood cells
Biochemical Markedly increased lactate dehydrogenase activity
Increased serum total and indirect bilirubin
Decreased serum haptoglobin level
Hemoglobinemia
Hemoglobinuria
Proteinuria, hematuria, castsb
Hemolytic Uremic Is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute
Syndrome renal failure from damage to endothelial cells in the glomerular microvasculature

TWO TYPES

A. Typical HUS/ Shiga-toxin associated HUS/ stx-HUS

-most common cause is infection with Shiga toxin-producing Escherichia coli
(STEC), such as serotype O157:H7, but strains of toxin-producing Shigella have also
been implicated
-most commonly associated with young children

B. Atypical HUS
-10% cases of HUS and can first present in infancy, childhood, or adulthood.
-The characteristic feature is uncontrolled activation of the alternative complement system,
which causes endothelial cell injury, activation of platelets and coagulation factors, and
formation of platelet-fibrin thrombi that obstruct the microvasculature in the glomerulus and
other organs.
-it is categorized as hereditary (50 to 70% of aHUS), and acquired (5 to 10% of aHUS)
-An acquired form of aHUS is associated with autoantibodies to complement factor H

DIC Named as defibrination syndrome or consumption coagulopathy

(Disseminated Generalized uncontrolled activation of both hemostatic and fibrinolytic system
Intravascular In DIC, fibrin microthrombi partially occlude small vessels and consume platelets,
Coagulation) coagulation factors, coagulation control proteins, and fibrinolytic enzymes.
Acute DIC is seen in association with obstetric emergencies, intravascular hemolysis,
septicemia, viremia, burns, acute inflammation, crush injuries, dissecting aortic aneurysms,
and cardiac disorders.
Chronic DIC may be associated with vascular tumors, tissue necrosis, liver disease, renal
disease, chronic inflammation, use of prosthetic devices, and adenocarcinoma

*ADDITIONAL INFORMATIONS WILL BE DISCUSSED IN HEMATOLOGY 2 😊

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 HEMOGLOBINOPATHIES (STRUCTURAL DEFECTS IN HEMOGLOBIN)

These are group of inherited disorders causing structurally abnormal globin chain synthesis due to amino acid substitution
(QUALITATIVE DEFECT); changes in RBC deformability and electrophoretic mobility can occur
TARGET CELLS are associated with hemoglobinopathies
Homozygous / Disease conditions = both globin chains affected
Heterozygous / Trait conditions = only one globin chain affected

1. Sickle cell disease (Hgb SS)

❖ Caused when Valine replaces glutamic acid at position 6 on both beta chains.
❖ 80 % Hgb S, and 20 % Hgb F are seen. Hgb A2 is variable if present.
❖ Immunity to Plasmodium falciparum
❖ Hgb S migrates with Hgb G and D on alkaline hgb electrophoresis

Promotes Sickle cell formation a. hypoxic environment

b. lower pH
c. reduced blood flow
d. dehydration
Crisis Refers to episodes of recurring pain associated in sickle cell disease. It may occur as
vaso-occlusive or “painful,” aplastic, megaloblastic, sequestration, and chronic
hemolytic
Most common cause of death in sickle cell Bacterial or infectious crisis such as Acute infections are common causes of
disease hospitalization and have been the most frequent cause of death, especially in the
first 3 years of life.
Screening test -Sodium metabisulfite test- old method
-Dithionite solubility test- most commonly used
Confirmatory test for Sickle cell disease Hemoglobin electrophoresis, HPLC, or capillary electrophoresis

2. Sickle cell trait (Hgb AS)

❖ Caused when Valine replaces glutamic acid at position 6 on one beta chain.
❖ 60 % Hgb A, and 40 % Hgb S are produced, with normal amounts of Hgb F and Hgb A2

3. Hemoglobin C Disease / Hgb CC

❖ Caused when Lysine replaces glutamic acid at position 6 on both beta chains.
❖ 90 % Hgb C, 2% Hgb A2, and 7 % Hgb F
❖ Characterized by barr of gold crystals (Hb CC crystal)
❖ Hgb C migrates with A2, E, and O on alkaline hgb electrophoresis

4. Hemoglobin C trait
❖ Caused when Lysine replaces glutamic acid at position 6 on one beta chains.
❖ 60 % Hgb A, and 40 % Hgb C

5. Hemoglobin SC disease
❖ Hb SC is the most common compound heterozygous syndrome that results in a structural defect in the hemoglobin
molecule in which different amino acid substitutions are found on each of two b-globin chains.
❖ Hgb SC disease is a double heterozygous condition where an abnormal sickle gene from one parent and an
abnormal C gene from the other parent inherited.
❖ Symptoms are less severe than sickle anemia but more severe than Hgb C disease
❖ 50% Hgb S, and 50 % Hgb C

6. Hemoglobin E
❖ Caused when lysine replaces glutamic acid at position 26 on the beta chain
❖ Hgb E migrates with Hgb A2, C and O on alkaline hemoglobin electrophoresis

7. Hemoglobin D (Punjab)
❖ Caused when glycine replaces glutamic acid at position 121 on the beta chain
❖ Both homozygous and heterozygous conditions are asymptomatic
❖ Hgb D migrates with Hgb S and Hgb G on alkaline hemoglobin electrophoresis

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 HEMOGLOBIN ELECTROPHORESIS

SCD Caused when Valine replaces glutamic acid at position 6 on two beta chain
SC trait Caused when Valine replaces glutamic acid at position 6 on one beta chain.
Hb C disease Caused when Lysine replaces glutamic acid at position 6 on both beta chains
Hb C trait Caused when Lysine replaces glutamic acid at position 6 on one beta chains.
Hb SC Hgb SC disease is a double heterozygous condition where an abnormal sickle gene from one parent and an
abnormal C gene from the other parent inherited
Hb E Caused when lysine replaces glutamic acid at position 26 on the beta chains
Hb D Punjab Caused when glycine replaces glutamic acid at position 121 on the beta chain
Hb-O-Arab Caused when lysine replaces glutamic acid at position 121 on the beta chain
Hb G dis. Caused when lysine replaces asparagine at position 68 on alpha chain
Hb S-KorleBu Caused when aspartic acid replaces asparagine at position 73 on beta chain

HB S HB C Hb A1 Hb A2 Hb F
[Link] disease 80% - - variable 20%
[Link] trait 40% - 60% normal Normal
HB SC disease 50% 50% - - -
HB CC/disease - 90% 2% 7%
Hb C Trait - 40% 60% - -

THALASSEMIA
Group of inherited disorders causing decreased rate of synthesis of a structurally normal globin chain
(QUANTITATIVE DEFECT); characterized by microcytic / hypochromic RBCs and TARGET CELLS.
These are a diverse group of inherited disorders caused by genetic mutations that reduce or prevent the synthesis of one
or more of the globin chains of the hemoglobin (Hb) tetramer.
Thalassemia Major: Severe anemia; either no alpha or no beta chains produced
Thalassemia Minor: Mild anemia; sufficient alpha and beta chains produced to make normal hemoglobin A, A2, and F, but
maybe in abnormal amounts.

The clinical manifestations of thalassemia:

1. A reduced or absent production of a particular globin chain, which diminishes hemoglobin synthesis and produces microcytic,
hypochromic RBCs;
2. An unequal production of the a- or b-globin chains causing an imbalance in the a/b chain ratio; this leads to a markedly
decreased survival of RBCs and their precursors (Ineffective erythropoiesis
The paramount in the diagnosis of thalassemia = Individual and family histories

ALPHA THALASSEMIA
1. Alpha thalassemia Major ( Hydrops fetalis)
❖ All four alpha genes are deleted; no normal hemoglobins are produced.
❖ 80 % Hemoglobin Bart’s (4 Gamma) produced which cannot carry oxygen
❖ Patient die in utero or shortly after birth
2. Hemoglobin H disease
❖ Three alpha genes deleted. Decrease in alpha chains leads to BETA CHAIN EXCESS
❖ Hemoglobin H (4 Beta) , an unstable hemoglobin, is produced
❖ Heinz bodies are present
❖ 30 % Hgb H and the rest is Hgb A
3. Alpha thalassemia Minor/Trait
❖ Two alpha genes are deleted and are usually asymptomatic
❖ Up to 6 % Bart’s Hgb is present
4. Silent carrier
❖ One alpha gene is deleted.
❖ Diagnosed only by gene analysis

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 CLASSIFICATIONS OF ALPHA-THALASSEMIA (Rodak’s 5th edition)
Silent carrier -deletion of one a-globin gene, leaving three functional a-globin genes
- There is no reliable way to diagnose silent carrier state other than genetic analysis
-slight increase of BART’S hemoglobin (1 to 2%)
a-thalassemia minor -Deletion of two a-globin genes is the major cause of a-thalassemia minor.
-5 to 15% Bart’s Hb
Hb H disease -Deletion of three a-globin genes is the major cause of Hb H disease in which only one
a-globin gene remains to produce alpha chains
-common in Asians
- 1 to 40% Bart’s Hb
Hydrops fetalis/ a- -results in the absence of all alpha chain production and usually results in death in utero
thalassemia major or shortly after birth
-fetus is severely anemic, edema, with hepatomegaly, and cardiomegaly
-BM with marked erythroid hyperplasia
-predominant Bart’s Hb

BETA THALASSEMIA

1. Beta thalassemia Major (Cooley anemia)

❖ Markedly decreased rate of synthesis or absence of both beta chains results in an excess of alpha chains
❖ No hemoglobin A can be produced
❖ 90 % Hgb F (compensatory hemoglobin)
❖ RBC inclusions commonly found in B-thalassemia major are Basophilic stippling, Howell-Jolly bodies, and
Pappenheimer bodies

2. Beta thalassemia Minor/ Trait

❖ Decrease rate of synthesis of one of the beta chain
❖ Hgb A is slightly decreased, and Hgb A2 is slightly increased to compensate

TYPES OF B-THALASSEMIA (RODAK’S)

B-thalassemia major (homozygous or compound heterozygous state) with severe hemolytic anemia,
microcytic/hypochromic RBCs, severe clinical symptoms, and transfusion dependence
B-thalassemia intermedia With mild to moderate hemolytic anemia, microcytic/hypochromic RBCs, moderate clinical
symptoms, and transfusion dependence
B-thalassemia minor With mild hemolytic anemia, microcytic/ hypochromic RBCs, and no clinical symptoms
B-thalassemia silent carrier With no hematologic abnormalities or clinical symptoms

TYPES OF B-THALASSEMIA (TURGEON)

Thalassemia major Severe anemia caused by ineffective erythropoiesis, transfusion-dependent, organ damage (heart, liver,
(Cooley anemia) etc.) secondary to iron overload, extramedullary erythropoiesis, hepatosplenomegaly
Thalassemia Moderate anemia and ineffective erythropoiesis, microcytosis, abnormal erythrocyte morphology,
intermedia splenomegaly, iron overload, not transfusion-dependent
Thalassemia minor Mild anemia, microcytosis, abnormal erythrocyte morphology, splenomegaly
(Thalassemia trait)

WHITE BLOOD CELLS

Granulocytes
There are three types of mature granulocytes: neutrophil, eosinophil, and basophil
These three cells are distinguishable by their presence of specific granules that appear in myelocytic stage.
Individually, they include eosinophils, with granules containing basic proteins that stain with acid stains such as eosin;
basophils, with granules that are acidic and stain with basic stains such as methylene blue; and neutrophils, with granules
that react with both acid and basic stains, which gives them a pink to lavender color

SCHILLING’S CLASSIFICATION Neutrophil are classified according to their granulation

ARNETH’S CLASSIFICATION Neutrophil are classified according to their age, number of lobulation

Agranulocytes / Mononuclear cells

Categorized as monocyte and lymphocytes
These cells have a non-segmented nuclei but round, oval, indented, or folded

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 WBC with phagocytic function Macrophage/Monocyte, Neutrophil, Eosinophil, and Basophil
WBC Known as immune cells /immunocytes Lymphocytes (B and T cells)
Polymorphonuclear Neutrophils, Eosinophils, Basophils

Left Shift Presence of increase Immature WBC (myeloblast up to band cells) in the circulation
Right Shift Presence of increase Hypersegmented cells in the circulation

GRANULOPOIESIS
Orderly production of mature granulocytes. It takes 14 days from the blast stage to the release of mature
granulocytes into the peripheral blood.

General Cell Maturation Characteristics for Leukocytes

IMMATURE CELLS MATURE CELLS
Cell Is large Cell becomes smaller
Nucleoli is present Nucleoli absent
Chromatin fine and delicate Chromatin coarsed 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

THREE BONE MARROW POOLS OF GRANULOCYTE

Pool Stage/s involved Properties
Stem cell pool HSC (Hematopoietic stem cell) Self-renewal and differentiation
Proliferation or Mitotic pool Common myeloid progenitor (CMP), CFU-GEMM, Myeloblast, Dividing
Promyelocyte, Myelocyte
Maturation or Storage pool Metamyelocyte, Band cells, and Mature granulocytes Nuclear Maturation
Marrow reserve

GRANULOCYTE PRECURORS
STAGE INFORMATION /CHARACTERISTICS
MYELOBLAST 15-20 um in diameter
Basophilic cytoplasm
Round nucleus
NC ratio of 4:1
Fine chromatin
2-5 nucleoli
First recognizable stage in the bone marrow
Makes up 0 to 3 % of nucleated cells in the bone marrow
MYELOBLAST CANNOT BE DISTINGUISHED FROM MONOBLAST IN LIGHT MICROSCOPY
Type I myeloblast -N:C ratio of 8:1 to 4:1
-No visible granules, fine nuclear chromatin, 2-4 nucleoli
-HSCs, CMPs, and GMPs are not distinguishable with the light microscope
and Romanowsky staining and may resemble type 1 myeloblast and
lymphoblast
Type II myeloblast Shows dispersed primary (azurophilic) granules in cytoplasm
Type III myeloblast Darker chromatin and more purple cytoplasm
Rare in the BM and can be seen in certain types of AML
PROMYELOCYTE MYELOCYTE
15-21 um in diameter 12-18 um in diameter
Basophilic cytoplasm First synthesis/ Appearance of secondary or specific granules
Synthesis and Contains primary / non- N:C ratio is 1:1
specific azurophilic granules Generally shows no nucleoli
Coarse chromatin
N:C ratio of 3:1 to 2:1
Last stage capable of mitosis
2-3 nucleoli
Chromatin pattern slightly coarser
Normal promyelocyte has a paranuclear
halo or “hof”

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 METAMYELOCYTE 10-15 um in diameter
(JUVENILE CELLS) Decreased N:C ratio
Indented (≤1/2 indentation) or Kidney, peanut shaped nucleus
Chromatin pattern is coarse and clumped
Predominant cell in the Bone marrow
The First stage for the synthesis of tertiary/ gelatinase granules

BAND CELLS 9-15 um in diameter

(STAB CELLS) Elongated or Band shaped nucleus ( C or S shaped) / Curved or sausage shape
The nucleus is highly clumped, and the nuclear indentation that began in the metamyelocyte
stage now exceeds one half the diameter of the nucleus, but actual segmentation has not yet
occurred
Chromatin pattern is coarsed and clumped
Youngest cell in the series that is normally present in the Peripheral blood
Secretory granules/ secretory vesicles may begin to be formed during this phase

NOTE
The Clinical and Laboratory Standards Institute (CLSI) recommends that bands and segmented
neutrophils be counted together and placed in a single category rather than in separate categories
because it is difficult to reliably differentiate bands from segmented neutrophils

MATURE OR Segmented Neutrophil Eosinophil Basophil

SEGMENTED *9-15 um *9-15 um *9-15 um
GRANULOCYTES *Pink to rose violet specific granules *Reddish orange granules *Dark purple to blue black
*Normally with 2-5 lobes *Usually with 2 Lobes granules
*Coarse, clumped chromatin pattern *Coarse, clumped *Generally unsegemented, or
chromatin pattern bilobed, rarely 3-4 lobes

NEUTROPHIL GRANULES
Primary granules Secondary granules Tertiary granules Secretory granules
Formed during the Formed during myelocyte Formed during Formed during band and
promyelocyte stage and metamyelocyte stages metamyelocyte and band segmented neutrophil
stages stages
-Last to be released -Third to be released -First to be released (fuse
(exocytosis) -Second to be released to plasma membrane)
Contain (attached to
membrane)
• Myeloperoxidase • b2-Microglobulin Gelatinase • CD11b/CD18
• Acid Beta • Collagenase • Collagenase • Alkaline phosphatase
glycerophosphatase • Gelatinase • Lysozyme • Vesicle-associated
• Cathepsins • Lactoferrin • Acetyltransferase membrane-2
• Defensins • Neutrophil gelatinase- • b2-Microglobulin • CD10, CD13, CD14, CD16
• Elastase associated lipocalin • Cytochrome b558
• Proteinase-3 • Transcobalamin I • Complement 1q receptor
• Complement receptor-1

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 EOSINOPHIL GRANULES
Primary Secondary granules Small lysosomal Lipid bodies
granules granules
Formed during Formed throughout maturation series Formed during Formed during metamyelocyte
the promyelocyte metamyelocyte and band and band stages
stage stages

• Myeloperoxidase • Major basic protein (core) • Acid phosphatase • Cyclooxygenase

• Charcot • Eosinophil cationic protein (matrix) • Arylsulfatase B • 5-Lipoxygenase
leyden crystal • Eosinophil-derived neurotoxin (matrix) • Catalase • 15-Lipoxygenase
• Eosinophil peroxidase (matrix) • Cytochrome b558 • Leukotriene C4 synthase
• Lysozyme (matrix) • Elastase • Eosinophil peroxidase
• Catalase (core and matrix) • Eosinophil cationic protein • Esterase
• b-Glucuronidase (core and matrix)
• Cathepsin D (core and matrix)
• Interleukins 2, 4, and 5 (core)
• Interleukin-6 (matrix)
• Granulocyte-macrophage colony-
stimulating factor (core)

Basophil Secondary Histamine

Granules Platelet-activating factor
Leukotriene C4
Interleukin-4
Interleukin-13
Vascular endothelial growth factor A and B
Chondroitin sulfates (e.g., heparan)
Eosinophilic Chemotactic factor A

GRANULOCYTE KINETICS
Neutrophil kinetics make up 7% to 30% of nucleated cells in the bone marrow
Neutrophil production has been calculated to be on the order of between 0.9 and1.0 x 109
cells/kg per day
The transit time from myeloblast through myelocyte has been estimated to be roughly 6 days
transit time through the maturation pool is approximately 4 to 6 days
Eosinophil Kinetics The time from the last myelocyte mitotic division to the emergence of mature eosinophils
from the marrow is about 3.5 days
The mean turnover of eosinophils is approximately 2.2x109 cells/kg per day
Circulating half-life of 18 hours
Basophil Kinetics Life span of 60 hours
Basophil are activated by IL-3

GRANULOCYTE KINETICS (Rodaks 5th edition, Turgeon 5th edition)

12 hours (Turgeon) Basophil remains in the maturation or storage phase/pool for _____
2.5 days (Turgeon) Eosinophil remains in the maturation or storage phase/pool for _____
4.3 days The stage from myelocyte to metamyelocyte lasts an average of
7 to 10 days (Turgeon) Neutrophil remains in the maturation or storage phase/pool for _____
3.5 days (Rodak’s) The time from the last myelocyte in BM to mature eosinophil is about____
7 hours (Rodak’s) The half-life of neutrophils in the blood is relatively short at approximately ____
18 hours (Rodak’s) The half-life of Eosinophils in the blood is relatively approximately ____
9 or 10 days (Steinenger) The lifespan of neutrophil is approximately __ from myeloblast to death
7 to 10 hours (Turgeon) The lifespan of neutrophils in the peripheral blood or circulation
8.5 hours (Turgeon) The lifespan of basophil in the peripheral blood or circulation
60 hours (Rodak’s)
6 days The transit time of neutrophil from myeloblast through myelocyte
15 hours Myeloblast stage lasts for approximately ___ hours
The stage from myeloblast to promyelocyte lasts and average of ____ hours
24 hours Promyelocyte stage lasts for approximately ___ hours
The stage from promyelocyte to myelocyte lasts an average of ______ hours

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 MONOCYTE MATURATION SERIES / MONOPOIESIS

MONOCYTE KINETICS
1. Monocytes remain in the circulation approximately 3 DAYS before migrating to tissues
2. Monocytes are the precursor of the macrophages (tissue monocytes)
3. Ratio of marginal pool to circulating pool of monocytes is 3.5:1

STAGES
MONOBLAST 12-20 um diameter
Basophilic cytoplasm
Non-granular
N:C ratio of 4:1 or 3:1
1-2 nucleoli
MONOBLAST CANNOT BE DISTINGUISHED FROM MYELOBLAST IN LIGHT MICROSCOPY

PROMONOCYTE 14-18 um diameter

Blue gray cytoplasm
N:C ratio of 3:1 to 2:1
The first recognizable cell in the marrow

MONOCYTE 14-20 um
Blue gray cytoplasm
Many fine azurophilic granules
Ground glass cytoplasm
Round, kidney shaped, or horse shoe shaped nucleus, may show slight lobulation; it
may be folded showing brain-like convultions
No nucleoli present
Largest cell in the peripheral blood

MACROPHAGES These are tissue monocytes

Cells that usually have an oval nucleus with a netlike (reticulated) chromatin pattern. Their
cytoplasm is pale, frequently vacuolated, and often filled with debris of phagocytized cells
or organisms

NOMENCLATURE OF DIFFERENT MACROPHAGES ON ITS SPECIFIED TISSUE LOCATION

Liver Kupffer cells
Kidney Mesanglial cells
Brain Microglial cells
Bone Osteoclast
Lungs Alveolar macrophage / Dust cells
Connective tissue Histiocytes
Skin Langerhan cells
Spleen Littoral cells
Placenta Hofbauer cells

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

2. PROLYMPHOCYTE
o May be the same size of the lymphoblast or smaller
o Moderate to dark blue cytoplasm
o Round or oval nucleus
o May contain 1-2 nucleoli

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 3. MATURE LYMPHOCYTE
o Small lymphocyte: 8 to 10 um in size
o Medium lymphocyte: 10 to 12 um in size
o Large lymphocyte: 12 to 16um in size
o Bluish cytoplasm sometimes referred as robin’s egg blue cytoplasm
o Compact nucleus
o They are not end cell, they are resting cells
o B cell differentiates into Effector B cells/ Plasma cells

PLASMA CELL MATURATION

1. PLASMABLAST
o 18 – 25 um in diameter
o Abundant basophilic cytoplasm
o Eccentric nucleus
o Perinuclear halo may be present

2. PROPLASMACYTE
o 15 to 25 um
o Intensely basophilic cytoplasm, usually bluer than the blast stage
o Eccentric nucleus

3. PLASMACYTE / PLASMA CELL

o 8 to 20 um
o Deeply basophilic cytoplasm (abundant antibodies found in the cytoplasm)
o With a Large, well defined hof (perinuclear halo) next to nucleus
o Chromatin is condensed and coarsed
o Nucleus is eccentric and Exhibits “Tortoise shell, “clock face”, “spoke’s wheel” or “CART WHEEL” like
pattern

DIS TI NG U IS HI NG L YMP H O CYT ES F R OM BL AS T

Check for Nuclear chromatin

Lymphocyte Blocks of heterochromatin stain dark purple, with sharp demarcation of unstained or lightly
stained
BLASTS Reveals delicate strands that have a stippled or sieve-like appearance that stains evenly and
lightly

DIS TI NG U IS HI NG L YMP H O CYT ES F R OM R UB R I CYT E

Check for Nuclear parachromatin, and cytoplasm

Nuclear parachromatin Cytoplasm

Lymphocyte Stains light purple with deep purple Clear blue / sky blue / Robbin’s egg
heterochromatin giving the appearance of blue
crushed velvet
Rubricyte More unstained, and the chromatin is small dense Muddy or gray appearance
spherical clumps, giving a CHECKERED BOARD
appearance

DISTINGUISHING LYMPHOCYTES FROM MONOCYTE

Check for Nuclear shape, and characteristic of the cytoplasm

Nuclear shape Characteristic of cytoplasm

Lymphocyte Round, ovoid Clear blue
Monocyte Often folded or U shaped Contains extremely small azure granules in blue gray
cytoplasm that gives it an opaque or ground glass
appearance

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 NON-MALIGNANT LEUKOCYTE DISORDERS
QUALITATIVE DISORDERS OF LEUKOCYOTE

NUCLEAR ABNORMALITIES

A. Hyposegmentation (Pelger Huet)

Decrease segmentation usually bilobed or unilobed
Function of the cell is considered normal despite morphologically abnormal
Characterized by coarse chromatin clumping pattern potentially affecting all leukocytes, although morphologic
changes are most obvious in mature neutrophils
The disorder is a result of a mutation in the lamin Beta-receptor gene
Nuclei appearance= Round, Oval, Dumbbell, spectacle like, pair of eyeglasses, peanut shape, “pince nez”
a. Pelger Huet anomaly / True or Congenital PHA
o Autosomal dominant, can be homozygous or heterozygous
o In true PHA, the number of affected cells is much higher than in pseudo-PHA
b. Acquired pseudo Pelger Huet anomaly
o Found on myeloproliferative disorders such as CML, Myelodsyplastic syndrome, HIV
infection, TB, Mycoplasma pneumoniae infection, and other bacterial infections.
o Drugs known to induce pseudo-PHA include mycophenolate mofetil, valproate, sulfisoxazole,
ganciclovir, ibuprofen, and chemotherapies such as paclitaxel and docetaxel.

B. Hypersegmentation
Presence of ≥6 segmentation and the cell is also larger than normal
Hereditary hypersegmentation- Undritz anomaly, Myelokathexis
Acquired Hypersegmentation- Megalobastic anemi
Pseudohypersegmentation may be seen on old segmented neutrophils

CYTOPLASMIC ABNORMALITIES

A. Alder-Reilly granules
Transmitted as a recessive trait and is characterized by granulocytes with large, darkly staining metachromatic
cytoplasmic granules composed primarily of partially digested mucopolysaccharides
Large purple black coarse cytoplasmic granules
Accumulation of degraded mucopolysaccharide
Can be found on all types leukocytes
Associated with Mucopolysaccharidosis disorder such as Hunter’s and Hurler’s disease
The granules morphology may resemble heavy toxic granulation
Leukocyte function is not affected in Alder-Reilly anomaly

B. Auer rods
Pink or red rod shaped structures
These are fused primary granules (peroxidase positive)
Found on myeloid and monocytic series only
Faggot cells = bundles of Auer rods that is mainly associated with M3(Acute Promyelocytic leukemia
Found in cases of AML and AMML

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 C. Chediak- Higashi granules
Autosomal recessive disorder causes large, gray-green, peroxidase positive granules in the cytoplasm of leukocytes;
abnormal fusion of primary and secondary neutrophilic granules.
This are large Lysosomal granules
Both morphologically and functionally abnormal leukocytes; WBC’s are unable to degranulate and kill invading
bacteria
Patients often have bleeding issues due to abnormal dense granules in platelets.
Chédiak-Higashi syndrome is associated with a mutation in the CHS1 LYST gene on chromosome 1q42.1-2 that
encodes for a protein involved in vesicle fusion or fission
Patient will present with photophobia and skin hypopigmentation (ALBINISM)

D. Dohle Bodies
Single or multiple blue inclusions
Aggregates of free ribosomes of rough E.R
These are cytoplasmic inclusions consisting of remnants of ribosomal ribonucleic acid (RNA) arranged in parallel
rows
Confused with May-Hegglin
Seen in: Severe infection and Toxic states

E. Toxic granules
Large to purple to black primary granules that are peroxidase positive
The granulation may represent the precipitation of ribosomal protein (RNA) caused by metabolic toxicity within the
cells
Prominent dark granulation, either fine or heavy, that can be observed in band cells, segmenters, and monocyte
Cannot be found in LYMPHOCYTES
Seen in: Infections, Toxic states, Burns, Malignant disorders

F. May Hegglin inclusions

May-Hegglin anomaly is a rare, autosomal dominant platelet disorder characterized by variable thrombocytopenia,
giant platelets, and large Döhle body–like inclusions in neutrophils, eosinophils, basophils, and monocytes
Large, crystalline, Dohle-like inclusions in the cytoplasm of neutrophils on Wright’s stain; gray blue and spindle
shaped
Morphologically abnormal but functionally normal
There is Mutations in the MYH9 gene for production of myosin heavy chain type IIA which affects megakaryocyte
maturation and platelet fragmentation
Presence of giant platelets, thrombocytopenia, and clinical bleeding are also associated with this anomaly

ABNORMAL FUNCTION

A. Job’s syndrome
Normal random activity but characterized by abnormal chemotactic activity

B. Lazy Leukocyte syndrome

Abnormal random activity and abnormal chemotactic activity

C. Chronic granulomatous disease (CGD)

Both sex-linked or autosomal recessive
Morphological normal, but functionally abnormal
Inability of a phagocyte to kill ingested microorganism because of enzyme deficiency (NADPH Oxidase)
Decreased ability of phagocytes to produce superoxide and reactive oxygen species.
NO RESPIRATORY BURST
Most patients experience bacterial and fungal infections of the lung, skin, lymph nodes, and liver. Macrophage-rich
granulomas can be found in the liver, spleen, and other organs.

TEST FOR CGD

1. Nitro blue tetrazolium reduction test
Normal WBC: able to reduced yellow water-soluble tetrazolium to a blue insoluble formazan
CGD: remains yellow/colorless

[Link] cytometry using dihydrorhodamine-123

Normal WBC: Presence of fluorescence when dihydrorhodamine is reduced

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 D. Myeloperoxidase deficiency /Alius-Grignaschi anomaly
Characterized by deficiency of MPO in the primary granules of neutrophil and lysosomes of monocyte
MPO normally stimulates the production of hypochlorite and hypochlorous acid, which are oxidant agents that
attack phagocytized microbes.
Defects originates in the mutation of MPO gene on chromosome 17
In the hematology laboratory, MPO deficiency can be easily detected by the Siemens Advia analyzer, which uses
myeloperoxidase to identify cells in the automated differential
Acquired myeloperoxidase deficiency can present in association with hematologic neoplasms and lead poisoning

E. Leukocyte Adhesion Disorders

Rare autosomal recessive inherited disorders that result in the inability of neutrophils and monocytes to adhere to
endothelial cells and to transmigrate from the blood to the tissues. The consequence is increased and potentially
lethal bacterial infections
Selectins and integrins are important adhesion molecules for Leukocytes

Leukocyte adhesion Mutation in gene(s) responsible for b2 integrin subunits, leads to decreased or truncated B2 integrin,
disorder – I needed for neutrophil adhesion to endothelial cells, recognition of bacteria, and outside-in signaling
Leukocyte adhesion Mutation in SLC35C1 which codes for a fucose transporter involved in synthesis of selectin ligands.
disorder – II Results in decreased amount or function of selectin ligands and defective leukocyte recruitment
Leukocyte adhesion Mutations in Kindlin-3 and defective protein product Kindlin-3, needed for B- integrin activation and
disorder – III leukocyte rolling. Failed response to external signals that would normally result in leukocyte activation

CELLS EXHIBITING ABNORMAL PHAGOCYTOSIS

A. LE cells
Neutrophil with homogenous round body
Smooth and evenly stained
Found on SLE
It is demonstrated in buffy coat preparation

B. Tart cells
Monocyte with ingested lymphocyte
Rough and unevenly distributed

CELL ABNORMALITIES INVOLVING LYMPHOCYTES

A. Atypical lymphocytes
Also referred as reactive/variant/stimulated lymphocytes or Downey cells
Reactive lymphocytes are the result of complex morphologic and biochemical events that occur as lymphocytes are
stimulated when interacting with antigens in peripheral lymphoid organs
A plasmacytoid lymphocyte is a type of reactive lymphocyte that has some of the morphologic features of
plasmacytes
Types of Downey cells
1. Type I –Turk’s irritation cell which is actually a plasmacytoid lymphocyte with large black chromatin
2. Type II – found on IM with round mass of chromatin (ballerina skirt appearance)
3. Type III – Vacuolated lymphocyte resembling a swiss cheese or moth-eaten appearance

B. Hairy cell
Originally B cells with hair like projection which are identified by being TRAP resistance

C. Basket Cell / Smudge cell

Destroyed lymphocyte can be found on
A. Pressured smear preparation (artifacts)
B. CLL – Chronic lymphocytic leukemia (thumbprint appearance)

D. Sezary cells
A T lymphocyte with cerebriform nucleus (Brain-like) usually seen in mycosis fungoides and Sezary syndrome

E. Reider cells
Cells are similar to normal lymphocytes except that the nucleus is notched, lobulated, and cloverleaf-like
Occurs in CLL (pathologic) or artificially through blood smear preparation

CELL ABNORMALITIES INVOLVING PLASMA CELLS

A. Flame cells
A plasma cell with red to pink cytoplasm; associated with increased IgA and usually seen in Multiple Myeloma

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 B. Russel bodies
Individual globules of immunoglobulins

C. Dutcher’s bodies
Intranuclear protein inclusions

D. Grape cell/ Berry/ Morula or MOTT cells

A plasma cell with vacuoles; with large protein globules called as “Russel bodies”

ABNORMALITIES INVOLVING MACROPHAGES / MONOCYTES

A. Gaucher disease
The most common lipid storage disorder and has an autosomal recessive inheritance pattern. A deficiency in
glucocerebrosidase / B-glucosidase causes glucocerebroside to accumulate in macrophages of the bone marrow,
spleen, and liver with Gaucher cells (Chicken scratch appearance) more commonly seen in the bone marrow.
The typical Gaucher cell is large, with one to three eccentric nuclei and characteristically wrinkled cytoplasm
The bone marrow contains Gaucher cells, distinctive macrophages occurring individually or in clusters, that have an
abundant fibrillar blue-gray cytoplasm with a striated or wrinkled appearance (sometimes described as onion skin–
like)
The clinical triad used in diagnosis is hepatomegaly, Gaucher cells in the bone marrow, and increase in serum
phosphatase
Hematologic features include anemia and thrombocytopenia as a result of hypersplenism that is common in these
patients.
Ashkenazi Jews are common carriers of this disease
Pseudo-Gaucher cells can be found in the bone marrow in some patients with thalassemia, chronic
myelogenous leukemia, and acute lymphoblastic leukemia. In these diseases, pseudo-Gaucher cells form as
a result of excessive cell turnover and overwhelming the glucocerebrosidase enzyme rather than a true decrease in
the enzyme

TESTS FOR GAUCHER’S DISEASE

I. Periodic acid Schiff (PAS) = Positive due to mucopolysaccharides in Gaucher cells
II. Chitotriosidase test for determining the level of glucocerebroside in storage. This biomarker can be
used in diagnosis and monitoring of the disease.
III. PCR and gene sequencing= screens for associated genetic mutations

B. Niemann- Pick disease

A lipid storage disease that has three subtypes: A, B, and C. Types A and B are characterized by recessive mutations
in the SMPD1 gene, which leads to a deficiency in the lysosomal hydrolase enzyme acid sphingomyelinase (ASM)
Deficiency of sphingomyelinase enzyme that leads to accumulation of sphingomyelin in macrophages and other
organs.
These are macrophages with a foamy cytoplasm packed with lipid-filled lysosomes that appear as vacuoles after
staining
Pick cell - Has a foamy appearance.

C. Sea blue histiocytosis

Caused by unknown deficiency. Macrophage appears as sea-blue in color

D. Tay-sachs disease
Characterized by deficiency in Hexosaminidase A, an enzyme which leads to the accumulation of glycolipids and
gangliosides exhibited by vacuolated cytoplasm.

E. Sand Hoff’s disease

Characterized by deficiency in Hexosaminidase A and B enzyme which leads to the accumulation of glycolipids and
gangliosides exhibited by vacuolated cytoplasm.

F. OTHERS
Fabry disease – deficiency of a-galactosidase
Farber’s disease- deficiency of ceramidase
Krabbe’s disease – deficiency of galactocerebrosidase / B-galactosidase
Metachromatic leukodystrophy- deficiency of arylsulfatase A

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 QUANTITATIVE ABNORMALITIES OF LEUKOCYTES

EOSINOPHILIA EOSINOPENIA
Nonmalignant causes of eosinophilia are generally a result of -Marrow hypoplasia
cytokine stimulation, especially from interleukin-3 and -Infection or inflammation that is accompanied by neutrophilia
interleukin-5
-Sepsis
-Parasitic infection
-Bone marrow failure
-Allergic reaction (asthma, hay fever, urticarial, atopic
-Steroid therapy
dermatitis)
-Cushing syndrome or disease
-Scarlet fever
-HIV
-Fungal infections
-Autoimmune disorders
-Malignancy (ALL)
-Hyper-eosinophilic syndrome (HES)
HES - eosinophilia (>1.5 x 109/L) lasting more than 6 months
without an identifiable cause. HES is considered to be a
myeloproliferative neoplasm

MONOCYTOSIS MONOCYTOPENIA
-TB -Aplastic anemia
-Brucellosis -Chemotherapy induced cytopenia
-Monocytic leukemia -Viral infections, especially those due to the Epstein-Barr virus (EBV)
-Sub acute bacterial endocarditis -Steroid therapy
-Typhoid fever -Hemodialysis
-Ricketssial infections -Sepsis
-Gaucher’s disease
-Malaria
-Leishmaniasis
-Syphilis
-SLE
-Myositis
-Wiskott Aldrich syndrome
-Acute, chronic, and cyclic neutropenia
-Alcoholic liver disease
-Lymphoma and Plasma cell dyscrasia

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 LYMPHOCYTOSIS LYMPHOCYTOPENIA
-Most viral infections -HIV
-Infectious mononucleosis -Congenital immunodeficiency
-Hyperthyroidism (Bruton’s agammaglobulinemia, Wiskott-aldrich
-Mycoplasma syndrome, ataxia telangiectasia, Di-George syndrome)
-Syphilis -Aplastic anemia
-Listeria -Hepatitis
-Typhoid fever -Influenza
-Toxoplasmosis -Herpes
-Sepsis, Measles, Tuberculosis

REACTIVE MORPHOLOGIC CHANGES IN NEUTROPHIL

Reactive change Morphology Association
Toxic granulation Dark, blue-black cytoplasmic granules Inflammation, infection, administration of granulocyte
colony stimulation factor (G-CSF)
Dohle bodies Intracytoplasmic pale blue round or Nonspecific finding, or associated with bacterial
elongated bodies between infections,
1 and 5 mm in diameter, usually adjacent to sepsis, and pregnancy
cellular membranes.
Vacuolization Small to large circular clear areas in -Septicemia or other infection;
cytoplasm, rarely may contain -autophagocytosis secondary to drug ingestion, acute
organism alcoholism, or storage artifact (stored in EDTA >2hrs)
Pyknotic nuclei nuclear water has been lost and the Imminent cell death
chromatin becomes very dense and dark;
however, filaments can still be seen
between segments

REACTIVE MORPHOLOGIC CHANGES IN MONOCYTES

Morphology Associated with
Thin and band-like, or segmentation of nucleus; increased Infection, recovery from bone marrow aplasia, and granulocyte
cytoplasmic volume and granulation, and/or evidence of monocyte colony stimulating factor (GM-CSF) administration
phagocytic activity (cytoplasmic vacuolation, intracellular
debris, and irregular cytoplasmic borders)

MORPHOLOGIC CHANGES IN REACTIVE LYMPHOCYTES

• Heterogeneous population of various shapes and sizes
• Cells exhibit increased amount of variably basophilic cytoplasm
• Lymphocyte population exhibits variation in nuclear/cytoplasmic ratio and/or nuclear shape.
• Chromatin is usually clumped however some cells may demonstrate less mature (less clumped) pattern.
• Nucleoli may be visible.
•The cytoplasm may be indented by surrounding RBCs

LEUKEMOID REACTION
▪ Refers to a reactive leukocytosis above 50 x 109/L with neutrophilia and a marked left shift (presence of immature
neutrophilic forms)
▪ Leukemoid reactions are mostly a result of acute and chronic infection, metabolic disease, inflammation, or response to
a malignancy
▪ It may be confused with CML
LAB FINDINGS:
a. Increase WBC count
b. Left shift of the WBC (Presence of increase IMMATURE WBC in blood)
c. Increase LAP scores
d. Presence of toxic granulations and Dohle bodies in WBC

LEUKEMOID REACTION CHRONIC MYELOGENOUS LEUKEMIA

✓ A response or reaction to infection or malignancy ✓ A form of malignancy in blood
✓ High WBC count ✓ High WBC count (malignant cells)
✓ Increase LAP ✓ DECREASE LAP
✓ Left shift ✓ Left shift
✓ Presence of Dohle bodies and toxic granules ✓ Presence of Auer rods
✓ ABSENCE OF AUER RODS ✓ PRESENCE OF PHILADELPHIA CHROMOSOME
✓ ABSENCE OF PHILADELPHIA CHROMOSOME

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 MALIGNANT LEUKOCYTE DISORDERS
LEUKEMIA
Virchow was the first to recognize leukemia as a distinct clinical disorder between 1839 and 1845. He named this disorder leukemia
because of the white appearance of the blood from patients with fever, weakness, and lymphadenopathy

Abnormal, uncontrolled proliferation and accumulation of one or more of the hematopoietic cells
Major symptoms of leukemia are fever, weight loss, and increased sweating. Enlargement of the liver, spleen and lymph nodes
may occur more predominantly in chronic leukemias. The basic metabolic rate is often elevated, and there may be hemorrhagic
tendencies. If marked thrombocytopenia is present. Bone pain from a large leukemia cell mass in the bone marrow is typical in
the acute leukemias.

Differentiating Luekemia From Other Malignant Leukocyte Disorders

Leukemia A disease, usually of leukocytes, in the blood and bone marrow. Overproduction of various types of immature or
mature leukocytes in the bone marrow and/ or peripheral blood
Lymphoma General term for malignancy that starts in the lymph system, mainly the lymph nodes. Two main types of
lymphomas are Hodgkin lymphoma and non-Hodgkin lymphoma
Myeloma A form of cancer of the plasma cells. In myeloma, the cells overgrow, forming a mass or tumor that is located in
the bone marrow.

Classification
Duration Acute leukemia = days to 6 mos.
Subacute leukemia = 2 to 6 mos.
Chronic leukemia = 1 or 2 years or more

Number of white blood Aleukemic leukemia = WBC ct. <15,000 cells/ul, no immature or abnormal WBC
cells present in PBS Sub leukemic leukemia = WBC ct. <15,000cells/ul, with immature or abnormal form
Leukemic leukemia = WBC ct. >15,000cells/ul, with immature and abnormal form

Type of WBC Involved Acute leukemia = predominance of immature (blast) WBC

Chronic Leukemia = predominance of mature/old WBC

Major Types of Leukemia

Acute lymphoblastic ▪ Primarily a disease of childhood and adolescence, accounting for 25% of childhood cancers and
leukemia (ALL) up to 75% of childhood leukemia
▪ Early pre-B cell or common ALL (60-70%)
▪ T cell ALL (10-20%) - associated with large mass in the mediastinum
▪ B cell ALL (rare)

Acute myeloid ▪ AML is the most common type of leukemia in adults, and the incidence increases with age.
leukemia ▪ Common abnormalities
(AML)
a. Uric acid - hyperuricemia
b. Electrolytes affected = Calcium, potassium, and phosphate
Chronic Lymphocytic ▪ Majority of cases of CLL appears to involve B lymphocytes, occurring more in men than in
leukemia (CLL) women
▪ Clinical signs are lymphadenopathy, fatigue, weight loss, splenomegaly, and hepatomegaly
▪ Lymphocytes are fragile
▪ Large number of SMUDGE cells are present
▪ In CLL, bone marrow and peripheral blood films show small lymphoid cells with a
characteristically coarse chromatin (“soccer-ball” pattern), absent or inconspicuous nucleoli, and
scant cytoplasm

Chronic Myelogenous ▪ A myeloproliferative disorder characterized by pancytosis

Leukemia (CML) ▪ 90% of cases are positive for Philadelphia chromosome t(9:22)

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 POTENTIAL PREDOPOSING FACTORS FOR DEVELOLPMENT OF LEUKEMIA AND LYMPHOMA
Chemicals Benzene, Hydrocarbons, hair dyes
Environmental Ionizing radiation, insecticides, herbicides, and fungicides
Drugs Alkylating agents, chloramphenicol
Viruses EBV, HIV, HTLV
Genetic syndrome Down syndrome, Fanconi anemia
Hematologic conditions Myelodysplastic syndromes

ORGANIZATIONS THAT CLASSIFY ANEMIA

FRENCH AMERICAN BRITISH (FAB) WORLD HEALTH ORGANIZATION
First system, still used by some but being replaced by Widely used to classify leukemia. It is now the standard
WHO classification in diagnosing leukemia
Classified acute leukemia as presence of ≥30 % WHO defines acute leukemia as ≥ 20% peripheral
blast in the peripheral blood and bone blood and bone marrow blasts.
marrow.
Subdivide leukemia according to:
Subdivide leukemia according to:
1. Cellular morphology
1. Cellular morphology 2. Cytochemical stains(cytochemistry)
2. Cytochemical staining results. 3. Immunophenotyping (Flow cytometry)
4. Cytogenetics abnormalities
5. Clinical syndrome.

Cytogenetic Devoted to the laboratory study of visible chromosome abnormalities, such as deletions, translocations,
(Karyotyping)analysis and aneuploidy.
Cytochemistry Use of specialized stains to detect cellular enzymes and other chemicals in peripheral blood films and
bone marrow aspirate smears. Used to differentiate hematologic diseases, especially leukemias.
Immunophenotyping Used to identify cells on the basis of the types of markers or antigens present on the cell’s
(flow cytometry) surface, nucleus, or cytoplasm. This technique helps identify the lineage of cells using antibodies that
detect markers or antigens on the cells

Synthetic antibodies, often monoclonal antibodies produced by hybridoma technology, are used to
identify the antigens or CD markers by flow cytometry

ACUTE LYMPHOBLASTIC LEUKEMIA (FAB CLASSIFICATION)

ALL L1 →lymphoblasts are small and homogenous, varies little in size
→Scanty cytoplasm and inconspicuous nucleoli; nucleus is round and irregular/indistinct in shape; have high N:C ratio
→most common CHILDHOOD ALL with best prognosis
ALL L2 →Lymphoblasts are large and heterogenous, variable in size
→abundant, basophilic cytoplasm, and the nuclei are often clefted with nucleoli present
→Adult type ALL
ALL L3 → Burkitt-Type
→Lymphoblasts are large, homogenous and vacuolated
→rarest subclass, can be found in both children and adult
→poor prognosis

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 ACUTE MYELOID LEUKEMIA (FAB CLASSIFICATION)

M0 – ACUTE MYELOID LEUKEMIA, MINIMALLY DIFFERENTIATED

M1 – ACUTE MYELOID LEUKEMIA WITHOUT MATURATION
➢ >30 % blast
➢ <10% granulocytic cells

M2- ACUTE MYELOBLASTIC LEUKEMIA WITH MATURATION

➢ >30% blast
➢ >30% granulocytic cells

M3-ACUTE PROMYELOCYTIC LEUKEMIA

➢ >30% blast
➢ >10% granulocytic cells
➢ 30% or >50 % promyelocytes

M4- ACUTE MYELOMONOCYTIC LEUKEMIA

➢ 20 to <80% myelocytic cells
➢ <80% monoblast

M5a- ACUTE MONOBLASTIC LEUKEMIA WITHOUT MATURATION

➢ >80% monocytic cells
➢ >80% monoblast

M5b- ACUTE MONOBLASTIC LEUKEMIA WITH MATURATION

➢ >80% monocytic cells
➢ <80% monoblast

M6-Di GUGLIELMO’S SYNDROME (ERYTHROLEUKEMIA/ERYTHREMIC MYELOSIS)

➢ >30 % blast
➢ >50% erythrocytic precursors

M7-ACUTE MEGAKARYOCYTIC LEUKEMIA

➢ >30% blast
➢ >30% megakaryocytic cells

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 M1 FAB M1 is characterized by either a rapid or gradual onset that may resemble an acute infection. The patient may
have a history of fever, infections, fatigue, and bleeding episodes. Physical examination may reveal tenderness of
the bones, particularly the ribs and sternum; ulcerated mucous membranes; petechiae; and purpura. Additional
physical findings may include hepatomegaly, splenomegaly, and lymphadenopathy.
The outstanding feature of the peripheral blood smear and bone marrow is the predominance of myeloblasts.
These blasts usually have a regular cytoplasmic outline and may contain slender, red-staining Auer
rods in the cytoplasm. The nuclear chromatin is very fine and homogeneous
associated with CHLOROMA = localized tumor masses consisting of myeloblasts. In these tumors, the presence
of large quantities of the enzyme MPO produces a green appearance if the tissue is cut
M2 Hemorrhagic manifestations such as easy bruising, epistaxis, gingival bleeding, and petechiae are common initial
symptoms. Hepatomegaly, splenomegaly, and lymphadenopathy are seen infrequently
Myeloblasts predominate on peripheral blood smears. The nuclei are usually round or oval with one or more
prominent nucleoli and fi ne reticular chromatin. The cytoplasm is basophilic with a variable number of azurophilic
granules. Auer rods are commonly seen
M3 Fatigue and symptoms of bleeding such as bruising, hematuria, and petechiae are common. Hepatomegaly,
splenomegaly, and lymphadenopathy are seen infrequently.
Appears to be the most aggressive of acute leukemia with a severe bleeding tendency and a fatal course
Promyelocytes are the predominating cell type. The promyelocytes may be hypergranular, microgranular,
or hypogranular variations. Coarsely granular promyelocytes with dumbbell-shaped or bilobed nuclei may be
seen. The nuclear chromatin is finely reticular and the cells often lack nucleoli
M3 is characterized by a balanced reciprocal translocation between chromosomes 15 and 17, which results
in the fusion between PML gene and retinoic acid receptor a (RARA)
It is associated with DIC
M4 This form of leukemia may also be referred to as Naegeli type monocytic leukemia. Occurrence of this form of
leukemia is uncommon in children and young adults. The highest frequency of occurrence is in adults older than 50
years of age
Symptoms of this form of leukemia are similar to those of other forms of acute leukemia. Pharyngitis may be
observed. Gingival hyperplasia due to leukemic infiltration may be noted
Associated with leukostasis together with M5
Leukostasis refers to a pathological finding of slightly dilated, thin-walled vessels filled with leukemic cells. The brain
and lungs are the most commonly involved organs. Symptoms of leukostasis are headache, visual impairment, and
shortness of breath.
In FAB M4 proliferation of granulocytes and monocytes is characteristic.
M5 Also known as Schilling type
The onset of this form of leukemia is dramatic, with headaches and fevers being the chief complaints. Typical
symptoms of monocytic leukemia additionally include fatigue, weight loss, and bleeding from the mouth or nose.
Physical examination frequently reveals gingival (mouth and gums) hyperplasia, as in myelomonocytic leukemia;
pallor; and skin lesions
Monocytes and promonocytes constitute 25% to 75% of the nucleated cells. Blasts frequently have a muddy or
smoggy gray-blue cytoplasm containing tiny granules, and pseudopods are common. The nucleus has a
reticular granular chromatin pattern and may contain from one to five large nucleoli
M6 Acute erythroid leukemia. This form of leukemia, also referred to as erythemic myelosis or Di Guglielmo syndrome
represents a proliferation of both immature granulocytic and erythrocytic cells.
Erythroblasts on blood smears typically have an irregular outline with a high nuclear-cytoplasmic ratio
Blasts of myeloid origin may have Auer rods
Promyelocytes may also be present as well as monocytes and promonocytes.
M7 In this form of acute leukemia, 50% or more of the blasts are of megakaryocyte lineage.
Organomegaly is infrequent except in children. Radiographic evidence of bone lytic lesions has been observed in
children
Immunophenotyping reveals that megakaryoblasts express one or more of the platelet glycoprotein: CD41 or
CD61. Blasts are negative with anti-MPO antibody.

CYTOCHEMISTRY / CYTOCHEMICAL STAINS

Differentiating AML from ALL

STAIN AML ALL
MPO
SBB
TDT

MPO reactions and Sudan black B reactions frequently PARALLEL to each other

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 STAIN PURPOSE SPECIMEN
Leukocyte alkaline Stains ALP, present in the neutrophil and to a small degree, in Fresh capillary blood
phosphatase certain B cells. Alternatively, blood may be collected
Helpful in differentiating chronic myelogenous from a using heparin as anticoagulant.
leukemoid reaction or polycythemia vera
Peroxidase Stains peroxidases present in the granulocytes and monocytes Fresh blood smears made from capillary
Used to differentiate acute myelogenous and monocytic blood are recommended, or use of fresh
leukemia from acute lymphocytic leukemia whole blood anticoagulated with EDTA or
heparin
Sudan Black B Stains lipids present in the monocytes and granulocytes Air dried bone marrow smears
Used to differentiate acute myelogenous and myelomonocytic
leukemias from acute lymphocytic leukemia
Chloroacetate Stains esterases present in the granulocytes Air dried blood or bone marrow smears.
esterase Used to differentiate granulocytic cells from monocytic cells Blood anticoagulated with EDTA or
heparin may also be used
Non specific esterase Stains esterases present in the monocytic cells, macrophages, Air-dried blood or bone marrow smears.
megakaryocytes and platelets. Blood anticoagulated with EDTA or
Used to differentiate monocytic leukemias from granulocytic heparin may also be used
leukemias
Periodic acid shiff Stain mucoproteins, glycoproteins, and high molecular weight Air dried blood or bone marrow smears
carbohydrates normally present in almost all blood types
except pronormoblast
Used to help in the diagnosis of DiGugleilmo;s syndrome and
may be an aid, when used in conjunction with other stains, to
classify some acute leukemias
Acid phosphatase Stains ACP present in the myelogenous cell, lymphocytes, Air-dried blood or bone marrow smears,
plasma cells, monocytes and platelets or use blood anticoagulated with heparin
Using L (+) tartaric acid, the stain is helpful in diagnosing
hairy cell leukemia

NAME TYPE CONSTITUENTS STAINED INTERPRETATION IMPORTANT

INFORMATION
Myeloperoxidase Enzyme Marker for primary granules of Peroxidase activity produces MPO enzyme
auer rods dark brown granules in deteriorates
cytoplasm of granulocytes and Stain should be done
monocytes only on fresh specimens
Sudan Black B Nonenzyme Marker for phospholipids and Dark purple black granules in Can be done on stored
lipids neutrophil precursors specimens
Lymphoblasts are negative Parallel MPO For
interpretation
Terminal Enzyme DNA polymerase Is present in 90% cases of Used to differentiate
deoxyribonucleotidyl immunoperoxidase ALL AML to ALL
transferase
Periodic Acid Schiff Non-enzyme Marker for glycogen, Activity results in bright Lymphoblastic leukemia
glycoproteins, mucoproteins fuchsia pink or magenta red shows blocky or chunky
and high molecular weight Pattern of staining varies with pattern
carbohydrates each cell type L1 and L2 block pattern
Erythroblasts in M6
leukemia is positive
Naphthol AS-D Enzyme Marker for mature and Enzyme activity results in Known as specific
chloroacetate immature neutrophils and bright red granules in esterase
Esterase mast cells cytoplasm of neutrophils, Stable enzyme that
neutrophil precursors and lasts for months
mast cells
Alpha- napthyl Enzyme Marker for monocytes, Monocytic stain red-brown Known as nonspecific
acetate esterase megakaryocytes, and plasma esterase
cells
Alpha- naphthyl Enzyme Identifying monocytes, Enzyme activity results in dark Known as nonspecific
Butyrate esterase promonocytes, and red precipitates in cytoplasm esterase
monoblasts
Acid phosphatase Enzyme Present in all hematopoietic Activity is indicated by purple Cannot be stored
cells and found on lysosomes to red granules
Tartrate resistant acid Enzyme Marker for hairy cell leukemia Activity is indicated by purple Excellent marker for
phosphatase to dark red granules in hairy cell leukemia
cytoplasm

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 Leukocyte alkaline Enzyme Neutrophils is the only 100 cell count is done. Used to differentiate
Phosphatase (LAP) leukocyte that contain this Neutrophils are scored from CML from a leukamoid
activity with no activity to 4 with a reaction
large amount of activity
Toluidine Blue Non-enzyme Binds with acid Strongly metachromatic Useful for recognition of
mucopolysaccharides in blood mast cells and tissue
cells basophils

CELL RATING AMOUNT (%) SIZE OF GRANULES STAIN INTENSITY

0 none - None
1+ 50 small Faint to moderate
2+ 50 t0 80 small Moderate to strong
3+ 80 to 100 Medium to large Strong
4+ 100 Medium to large Brilliant
LAP STAIN SCORING (Turgeon)

OTHER LYMPHOPROLIFERATIVE DISORDERS:

LEUKEMIA, LYMPHOMA, NEOPLASM AND MYELOMA
Hairy Cell leukemia ✓ Hairy cell leukemia is characterized by small B lymphocytes with abundant cytoplasm and fine
(“hairy”) cytoplasmic projections. The postulated cell of origin is the peripheral B cell of post–
germinal center stage (memory B cell).
✓ More common in males than in women
✓ Clinical findings: fatigue, anemia, leukocytopenia, thrombocytopenia, splenomegaly, and marrow
fibrosis. Pancytopenia is common. Bleeding and infection can be present.
✓ The cytochemical features of HCL include a strong acid phosphatase reaction that is not
inhibited by tartaric acid or tartrate-resistant acid phosphatase (TRAP) stain.

Mantle cell ✓ Mantle cell lymphoma is a lymphoproliferative disorder characterized by medium-sized lymphoid
Lymphoma cells with irregular nuclear outlines derived from the follicular mantle zone
✓ lymph nodes show a replacement of normal nodal architecture with a diffuse proliferation of
monotonous, medium-sized lymphoid cells with irregular nuclear outlines

Follicular Lymphoma ✓ Follicular lymphoma originates from germinal center B cells and in most cases recapitulates
follicular architecture.
✓ Numerous closely spaced follicles replace the normal nodal architecture

Burkitt Lymphoma ✓ Burkitt lymphoma is characterized by medium- sized, highly proliferating lymphoid cells with
basophilic vacuolated cytoplasm
✓ The lymphoid proliferation is diffuse and at low magnification shows a prominent “starry sky”
pattern imparted by numerous tangible body macrophages
✓ The WHO classification lists three variants of this lymphoma: endemic (occurring predominantly
in Africa), sporadic, and immunodeficiency associated.

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 Mycosis fungoides & ✓ Mycosis fungoides is the most common cutaneous lymphoma
Sezary syndrome ✓ It affects T lymphocyte
✓ Sezary cells -composed of small to medium-sized lymphoid cells with irregular nuclear outlines
(cerebriform nuclei).
✓ Sézary syndrome is by definition a disseminated disease with leukemic presentation and skin and
lymph node involvement
Hodgkin’s lymphoma ✓ Malignant lymphoma but differs in that the cells reacting to the neoplasm predominant rather
than the neoplastic cells themselves
✓ Distinguished from other lymphomas by the presence of REED-STENBERG CELLS
✓ It is divided into 2 broad categories: CLASSICAL HODGKIN and NODULAR LYMPHOCYTE
PREDOMINANT HODGKIN
✓ NODULAR LYMPHOCYTE PREDOMINANT HODGKIN= a B cell neoplasm composed of relatively
rare neoplastic cells/ “Popcorn cells” that is scattered within the nodules of reactive lymphocytes

❖ Rye classification of Hodgkin = based to histologic appearance of the involved tissue from
lymph node biopsy
❖ Ann Arbor classification of Hodgkin = Most widely used staging scheme,depends on
histologic type and the extent of tissue involvement

MYELOPROLIFERATIVE DISORDERS/NEOPLASMS
❖ The MPNs are interrelated clonal hematopoietic stem cell disorders characterized by excessive proliferation of one or more
mature myeloid cell lines, for example, granulocytes, erythrocytes, megakaryocytes, or mast cells.
❖ Each MPN is characterized by the clonal expansion of one or more myeloid cell lines, but one cell line dominates. The MPNs
have the propensity to transform into other MPNs or progress into acute leukemias (ALs). Myeloproliferation largely is due to
hypersensitivity or independence of normal cytokine regulation resulting from genetic mutations that reduces cytokine levels
through negative feedback systems normally induced by mature cells
❖ All of the MPNs involve dysregulation at the multipotent hematopoietic stem cell (CD34), with one or more of the following
shared features: Cytogenetic abnormalities, Overproduction of one or more types of blood cells with, dominance of a
transformed clone, Hypercellular marrow or marrow fibrosis, Thrombotic and/or hemorrhagic bleeding, Extramedullary
hematopoiesis, Transformation to acute leukemia

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 TYPES OR CATEGORIES
Chronic Myelogenous Leukemia
Stem cell disorder affecting the granulocytic, monocytic, erythrocytic, and megakaryocytic cell lines
In 90% of the cases of this disease one arm of chromosome 22 is found to be translocated to chromosome 9(Philadelphia
chromosome)
Associated with BCR/ABL1 abnormality
Patients with this disorder who are negative for the Philadelphia chromosome usually have a poorer prognosis
Myelofibrosis with Myeloid Metaplasia/Primary myelofibrosis/Agnogenic myelofibrosis
Characterized by fibrosis and granulocytic hyperplasia of the bone marrow, with granulocytic and megakaryocytic
proliferation in the liver and spleen
Associated with splenomegaly, and ineffective hematopoiesis (marrow hypercellularity)
JAK2 V617F mutation is involved in the pathogenesis and is found in 65% of PMF patients
Presence of Dacryocytes
Essential thrombocythemia/ Primary thrombocytosis/ Hemorrhagic thrombocythaemia/ Idiopathic thrombocytosis
Chronic MPD characterized by thrombocytosis in excess of 1000x109/L, with spontaneous aggregation of functionally
abnormal platelets
The JAK2 V617F mutation is found in 50% to 60% of ET patients and supports the diagnosis of ET
The clinical manifestations of essential thrombocythemia are hemorrhage, platelet dysfunction, and thrombosis
Polycythemia vera
Characterized by an absolute increase in RBC, WBC and platelets
The specific JAK2 mutation, JAK2 V617F, is detected in 90% to 97% of patients with PV
Also known as the primary absolute polycythemia, polycythemia Rubra vera.
Patients exhibit a RUDDY skin coloration due to increase RBC concentration and viscosity of the blood
Increase RBC mass, Decrease EPO, increase RBC, WBC, and platelet count

PATHOPHYSIOLOGICAL CLASSIFICATION OF POLYCYTHEMIA

Relative polycythemia- Normal RBC mass, Increase hematocrit, Normal EPO
a. Diminished plasma volume; dehydration, diarrhea, burns and shock
b. Spurious polycythemia (Stress polycythemia: Gaisbock’s syndrome)

Absolute Polycythemia
a. Primary absolute- Polycythemia vera
b. Secondary polycythemia with appropriately increase EPO.
-Hypoxia, high altitudes, pulmonary disease, cyanotic heart disease, carboxyhemoglobinemia, high oxygen
hemoglobinopathy, 2-3DPG deficiency
c. Secondary polycythemia with inappropriately increase EPO
-Neoplasms, acute hepatitis, hepatoma, renal carcinoma, post renal transplant, wilm’s tumor
d. Genetic polycythemia / Congenital secondary Polycythemia
-primary familial congenital polycythemia, and Chuvash polycythemia

TYPE RBC MASS/COUNT HCT EPO

Relative polycythemia Normal Increase
Primary absolute (PV) Increase Increase
Secondary absolute Increase Increase
Genetic polycythemia Increase Increase

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 MYELODYSPLASTIC SYNDROMES(MDS)
Group of disorders that result from clonal abnormalities of hematopoietic pluripotential stem cells.
Characterized by a hypercellular bone marrow and abnormalities in the cellular maturation of the erythroid cells, granulocytes,
and megakaryocytes.
MDS are a group of acquired clonal hematologic disorders characterized by progressive cytopenias in the peripheral blood,
reflecting defects in erythroid, myeloid, and/or megakaryocytic maturation
Historically, this pattern of abnormalities was referred to as refractory anemia, smoldering leukemia, oligoblastic leukemia, or
preleukemia
The median age at diagnosis is 70

TRADITIONAL FAB COOPERATIVE GROUP CLASSIFICATION OF MDSs

MDS Blasts in Blasts in BM Ringed Peripheral blood
peripheral blood (%) sideroblasts Monocytes
(%) (%)
Refractory anemia (RA) <1% <5% <15 No
Refractory with ringed sideroblasts (RARS) <1% <5% >15 No
Refractory anemia with excess blast (RAEB) >5% 5-20% No No
Refractory anemia with excess blast in <5% 20-30% No No
transformation (RAEB-T)
Chronic myelomonocytic leukemia (CMML) <5% <20% No >1000

MORPHOLOGICAL ABNORMALITIES IN PERIPHERAL BLOOD AND BONE MARROW

Anemia, low platelet count, and low total leukocyte count, usually with an absolute neutropenia, are commonly present
Three major myeloid cell lines has dyspoietic morphologic features. These include Dyserythropoiesis, Dysmyelopoiesis, and
dysmegakaryopoiesis

Type of Dyspoietic feature Morphologic Evidence

Dyserythropoiesis Oval macrocytes
Hypochromic microcytes
Dimorphic red blood cell (RBC) population
RBC precursors with more than one nucleus
RBC precursors with abnormal nuclear shapes
RBC precursors with uneven cytoplasmic staining
Ring sideroblasts
Dysmyelopoiesis Persistent basophilic cytoplasm
Abnormal granulation
Abnormal nuclear shapes
Uneven cytoplasmic staining
Dysmegakaryopoiesis Giant platelets
Platelets with abnormal granulation
Circulating micromegakaryocytes
Large mononuclear megakaryocytes
Micromegakaryocytes or micromegakaryoblasts or both
Abnormal nuclear shapes in the megakaryocytes/megakaryoblasts

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 AUTOMATION IN HEMATOLOGY
Selection of a hematology analyzer for an individual laboratory requires careful evaluation of the laboratory’s needs and close
scrutiny of several important instrument issues, including instrument specifications and system requirements, methods used,
training requirements, maintenance needs, reagent usage, data management capabilities, staff response, and short-term and long-
term expenditures

Implementing automation in the hematology laboratory requires critical evaluation of the instrument’s methods and limitations, and
the performance goals for the individual laboratory. The Clinical and Laboratory Standards Institute (CLSI) has approved a standard
for validation, verification, and quality assurance of automated hematology analyzers. This standard provides guidelines for
instrument calibration and assessment of performance criteria, including accuracy, precision, linearity, sensitivity, and
specificity

BASIC COMPONENTS OF HEMA ANALYZERS

I. Hydraulics- aspirating unit, dispensers, dilutors, mixing chambers, aperture baths and /or flow cells, and hemoglobinometer
II. Pneumatics- vacuums and pressures for operating valves and moving the sample through the system
III. Electrical systems- electronic analyzers and computing circuitry for processing data

ELECTRICAL IMPEDANCE

A.K.A Low voltage Direct current (DC) resistance or “Coulter

Principle”

Cell counting and sizing is based on the detection and

measurement of changes in electrical impedances
(Resistance) produced by a particle as it passes through a
small aperture.

Particles such as blood cells are non-conductive but are

suspended in an electrically conductive diluent

Examples of machine uses this principle are SYSMEX and

Coulter

The number of pulses generated during a specific

period is proportional to the number of particles or
cells

The amplitude (magnitude) of the electrical pulses produced indicates the cell’s volume. Height or amplitude of
electrical pulse is directly proportional with the cell size or volume.

In Coulter, results are done in triplicate

Most common principle employed in Hema analyzer to measure hemoglobin concentration is Modified Cyanmethemoglobin.

DATAS GENERATED ARE PLOTTED IN BLOOD CELL HISTOGRAM

X-axis of Histogram Correlates with Cell Size or cell volume
Y-axis of Histogram Correlates with Cell Number

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 BLOOD CELL HISTOGRAMS
RBC HISTOGRAM
❖ RBCs volume size between 36 fL and 360 fL
❖ If the RBCs are larger than normal, the curve will shift toward the right
❖ if the RBCs are smaller than normal the curve will shift to the left
❖ if the histogram curve is bimodal then there is two population of red blood cells as might
be seen in patient received blood transfusion Or cold agglutinin disease.

WBC HISTOGRAM
❖ Provide a count and plot of cells in the WBC aperture bath larger than 35fL
❖ Normal WBC histograms have 3 distribution peaks
Peak Size Type of Cell
First 35 to 90 femtoliter, small
Second 90 to 160 femtoliter, medium
Third 160 to 450 femtoliter, large
PLATELET HISTOGRAM
❖ platelet derived histograms are obtained from volume sizes of 2 to 20fl
❖ The actual counting takes place in the RBC aperture.

RADIOFREQUENCY (ALTERNATING CURRENT / AC)

1. It is used in conjunction with the DC electronic impedance
2. The Radio Frequency or High voltage Alternating current resistance measures the cell interior density or complexity
[Link] pulse height of RF is directly proportional to the cell interior density

DILUTION FACTOR IN HEMA ANALYZERS

RBC Dilution factor
WBC dilution factor

OPTICAL LIGHT SCATTER (FLOW CYTOMETRY)

Optical scatter may be used as the primary methodology or in combination with other methods. In optical scatter systems (flow
cytometers), a hydrodynamically focused sample stream is directed through a quartz flow cell past a focused light source

LIGHT SOURCES
➢ Uses laser(helium-neon) and Non-Laser Light (E.g tungsten halogen lamp)

Three Independent processes in Optical scatter:

Light scatter results from the interaction between the processes of absorption, diffraction (bending around corners or the surface
of a cell), refraction (bending because of a change in speed), and reflection (backward scatter of rays caused by an obstruction).

a. Diffraction- bending of light around corners using small angles

b. Refraction- bending of light because of change in speed using intermediate angles
c. Reflection- light rays turned back by obstruction using large angles

The detection of scattered rays and their conversion into electrical signals is accomplished by photodetectors (photodiodes and
photomultiplier tubes) at specific angles.

Different Angles of light scatter that aid in cellular analysis

a. Forward light scatter (0°). This is diffracted light, which relates to the volume of the cell.

b. Forward low-angle light scatter (2 to 3°). This characteristic can relate to size or volume

c. Forward high angle (5° to 15°). This type of measurement allows for description of the refractive index of
cellular components.

d. Orthogonal or side angle light scatter (90°). The result of this application of light scatter is the production of
data based on reflection and refraction of internal components, which correlates with internal complexity.

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 Differential Scatter
Differential scatter is the combination of this low-angle and high-angle forward light scatter and is primarily used on
Siemens systems

Cytograms /Scatterplots
Scatter properties at different angles may be plotted against each other to generate two-dimensional cytograms or scatterplots

POTENTIAL EERORS FROM CELL COUNTING INSTRUMENT CAN BE CATEGORIZED IN TWO TYPES:
A. Instrumental errors
1. Aperture plugs are probably the most common problem in cell counting – POSITIVE ERROR
2. Extraneous electrical pulses from improperly grounded or shielded equipment may be picked up by the instrument
electrode – POSITIVE ERROR
3. Improper setting of aperture current or threshold – EITHER POSITIVEOR NEGATIVE ERROR
4. Bubbles in the sample caused by too vigorous mixing – POSITIVE ERROR
5. Excessive lysing of RBCs – NEGATIVE ERROR
B. Errors caused by the nature of the specimen
1. Giant platelets may be counted as RBCs or WBCs
2. Fragments of leukocyte cytoplasm, such as may be present during leukemia therapy, may be counted as platelets or
RBCs
3. Increased number of schistocytes may make accurate erythrocyte and platelet count impossible
4. Agglutination of RBC, WBC, or platelets will cause false negative results for each of the respective cell counts
5. Agglutinated red cells or platelets may also be causes false positive leukocyte counts
6. Platelet satellitism will result in falsely low platelet counts
7. Some abnormal RBCs tend to resist lysis, which may result in high WBC count. Examples sickle cells, extremely
hypochromic cells and target cells. The problem can be solved by delaying 2 to 3 minutes between the addition of
the lysing reagent and counting.

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 SOME RECALLS
1. Parameters that are directly obtained from histogram – WBC count, RBC count, Hemoglobin
2. Parameters that are derived from RBC Histogram – MCV and RDW
3. Parameters that are derived from platelet Histogram- MPV and PDW / MPV and platelet count
4. Parameters that are computed = Hematocrit, MCH, MCHC
5. THREE PART DIFFERENTIAL – Lymphocytes, Monocytes, and Granulocytes
6. FIVE PART DIFFERENTIAL – Neutrophils, Lymphocytes, Monocytes, Eosinophils, and Basophils
7. Ohm’s law: voltage = current x resistance

FLOW CYTOMETRY

Originally designed to measure physical properties of cells based on their ability to deflect light

For detection of fluorescent signals emitted by dyes bound directly to specific molecules or attached to proteins through
monoclonal antibodies

Main advantage: ability to rapidly and simultaneously analyze multiple parameters in a large number of cells.
A technique that can identify CD markers of a cell

In a flow cytometer, particles are suspended in fluid and pass one by one in front of a light source. As particles are
illuminated, they emit fluorescent signals registered by detectors. These results are later converted to digital output and
analyzed using flow cytometry software. The flow cytometer consists of fluidics, a light source (laser), a detection
system, and a computer.

THREE MAIN SYSTEM OR COMPONENTS:

FLUIDIC SYSTEM, OPTICAL SYSTEM, AND ELECTRONIC SYSTEM

Flow cytometric analysis is used for diagnosis and monitoring of immunodeficiencies, stem cell enumeration, detection of
fetal hemoglobin, tissue typing, diagnosing and classification of hematologic neoplasia by immunophenotyping, molecular
analysis, and drug testing.

“DREAM IT, WISH IT, AND DO IT”.

“IT’S GOING TO BE HARD, BUT HARD DOES NOT MEAN IMPOSSIBLE”
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