INTRODUCTION AND GENERAL CONSIDERATIONS 2. It is red in color due to hemoglobin.
3. It is slightly alkaline with an average pH of 7.4.
HEMATOLOGY
- branch of physiology that deals with the study of the structure, functions and
diseases of the blood; as well as of the body tissues and organs that produce the
constituents of the blood.
BLOOD
- the red liquid that is circulated by the heart and flows in the veins, arteries and
capillaries. Although blood normally looks red, the blood of humans and most other
vertebrates actually consists of a pale-yellow plasma and cellular elements, the red
and white blood cells and platelets.
FUNCTIONS OF BLOOD
By virtue of its circulation through every organ, the blood participates in every major
functional activity in.the body. Its primary roles are:
1. Respiratory - carries oxygen from lungs to tissues and carbon dioxide from body tissues to
the lungs.
2. Nutritional - supplies tissues throughout the body with food materials and substances
absorbed from gastrointestinal tract.
3. Excretory - carries waste products of catabolism of the tissues to the main excretory
organs, the lungs and kidneys, for elimination.
4. Buffering action - assists in the preservation of an almost neutral reaction in the tissues by
its selective excretion of soluble substances and its buffering power. The
maintenance of a normal water balance and fluid distribution
throughout the body depends on the mobilityof the water contained in
the blood.
5. Maintains body temperature - maintains organs of the body within closely restricted limits
of temperature. The metabolic processes which occur
during cell activity produce heat and blood tends to
minimize even minor variations in local temperature as it
passes through capillaries of the different body organs.
6. Transportation of hormones and other endocrine secretions that regulate cell function.
7. Maintenance of a degree of irritability of the tissue cells so that functional activity may be
carried on satisfactorily.
8. Body defense mechanism - helps protect the body against infection by the phagocytic
activity of certain white blood cells and by the production of
proteolytic enzymes and antibodies in the blood stream.
PHYSICAL CHARACTERISTICS OF BLOOD
1. In vivo, it is in fluid form because of naturally circulating anticoagulants but in vitro, it
coagulates within 5-10 minutes
4. It has an average specific gravity of 1.055.
5. It is thick and viscous; 3.5 - 4.5 times thicker than water.
6. It makes up 7-8% of the total body component or 75-85 ml blood per kg body weight.
7. There are approximately 20 gm solid per 100 ml blood.
COMPOSITION OF BLOOD
I. Liquid portion
1. Plasma - liquid portion of unclotted blood with the protein fibrinogen. It is obtained when
fresh blood is mixed with an anticoagulant and then centrifuged or allowed to
stand until the cells have settled.
2. Serum - liquid portion of clotted blood without fibrinogen since clotting is due to the
polymerization of the plasma protein fibrinogen into fibrin. When whole blood
coagulates, the cellular elements are trapped in the fibrin mesh. Upon standing,
the clotted blood undergoes retraction, separating from the wall of the container
and shrinking in. volume, thereby squeezing out a straw colored fluid known as
serum. Serum has essentially the same composition as plasma except that its
fibrinogen and clotting factors II, V & VIII have been removed, and it has high
serotonin content due to breakdown of platelets during clotting.
II. Solid portion (cellular elements or hemocytes)
1. Red blood cells ( erythrocytes, normocytes, akaryocytes and erythroplastids)
2. White blood cells (leucocytes, leucoplastids)
A. Granular WBC
- Neutrophils, Eosinophils, Basophils
B. Agranular WBC
- Lymphocytes, Monocytes t
3. Platelets (thrombocytes, thromboplastids)
III. Gaseous portion
- Usually, there is an exchange between oxygen and carbon
dioxide.
BLOOD CELL FORMATION
1. Monophyletic or Unitarian Theory
- this theory believes that there is only one stem cell (parent cell) - the hemocytoblast-
REC - which is capable of giving rise to all types of blood cells. This was advocated by
Maximow and Pappenheim and was supported by Bloom & Barthelmez
 RETICULOENDOTHELIAL CELL primitive cells. The primitive cells have large nucleus with spongy chromatin, one or two
(totipotent stem cell) nucleolar chromatin condensations, and a deeply basophilic cytoplasm. The primitive cells
are also known as: hemocytoblasts (Maximow & Bloom), hemohistioblasts (Ferrata »& Di
Rubriblast prorubricyte rubricyte metarubricyte reticulocyte ERYTHROCYTE Guglielmo), lymphoidocytes (Pappenheim) and hemopoietic reticulum cells.

Megakaryoblast promegakaryocyte megakaryocyte THROMBOCYTE ‘ By definition, the primitive cells are the transition forms from an undifferentiated
mesenchymal cell to one that can produce only blood cells. There are 3 stages of
Myeloblast promyelocyte myelocyte metamyelocyte stab (band or staff cell) hemopoiesis
Segmenters (NEUTROPHIL, EOSINOPHIL, BASOPHIL)
I. Mesoblastic Stage
Monoblast promonocyte MONOCYTE - The chief site of hemopoiesis is in the yolk sac.
- The fetus is 2.25 to 5'mm in size.
Lymphoblast prolymphocyte LYMPHOCYTE
0n the second week of fetal life there is the formation of blood islands wherein the primitive
Plasmoblast proplasmocyte PLASMOCYTE cells aggregate. These cells fulfill their function of fetal erythropoiesis. Later on the blood
islands are connected to one another by primitive endothelial tubes which are formed by the
2. Polyphyletic or Dualistic Theory transformation of peripherally located mesenchymal cells into endothelial cells. Thus, the
- this theory, believes that there is a separate and distinct stem cell compartment for primitive ‘blood cells are now enclosed in endothelial-lined spaces. Upon further differentia-
each of the blood cells. This was suggested by Sabin and co-workers and was concurred tion into cells .known as erythroblasts, and with the secretion of plasma, blood is established
by Naegeli, Schilling & Downey. as a definitive somatic component. Leucocytes and megakaryocytes are seldom found during
the earliest phase of the mesoblastic stage.
RETICULOENDOTHELIAL CELL On the 9th weék of fetal life, the predominant cell.is the Primitive Erythroblast (PE), a large
Hemohistioblast cell measuring from 15-25 u in diameter with coarse, clumped chromatin in the nucleus,
several nucleoli and homogenous basophilic cytoplasm. The PE elaborates hemoglobin to
Rubriblast prorubricyte rubricyte metarubricyte reticulocyte take care of the oxygen needs of the fetus, after which PE dies cut_and is' replaced by
ERYTHROCYTE definitive normoblastic cells that do differentiate into adult erythrocytes.

Myeloblast promyelocyte myelocyte metamyelocyte stab segmenters II. Hepatic Stage

(NEUTROPHIL, EOSINOPHIL, BASOPHIL) - This starts on the second month; embryo is 5 to 7 mm in size.
- On the 3rd month of fetal life, the liver becomes the chief site of hemopoiesis. It
Megakaryoblast promegakaryocyte megakaryocyte THROMBOCYTE reaches peak activity during the 3rd or 4th month and remains active until a few
months before birth.

Tissue hemohistioblast Although hepatic hemopoiesis is the chief mechanism for production of blood cells during
the middle third of fetal development ,there are significant contributions by the spleen,
Monoblast promonocyte MONOCYTE thymus and lymph nodes. The spleen is at first active in erythropoiesis, myelopoiesis and
lymphopoiesis but by the 5th month myelopoiesis becomes minimal. Splenic erythropoiesis
Lymphoblast prolymphocyte LYMPHOCYTE continues until the end of normal gestation and lymphopoiesis continues throughout life.,

Plasmoblast proplasmocyte PLASMOCYTE
HEMATOPOIESIS
Hemopoiesis deals with the processes of blood cell derivation and maturation. In the embryo
the mesenchymal cells of the yolk sac differentiate into groups of cells known as the
The lymph nodes also contribute to hemopoiesis by manufacturing lymphocytes
(lymphopoiesis) during the 4th and 5th months of fetal life and on throughout life.
The 2nd stage (hepatic) in fetus has an important counterpart in adult since normal
hemopoiesis in adult is in the bone marrow (medullary hemopoiesis).
III. Medullary Stage - elaboration of hemoglobin for RBC (no hemoglobin means younger cell)
- This is the final phase wherein the red bone-marrow assumes the chief role in
hemopoiesis. 2. Nuclear maturation
- It starts on the 5th month of fetal life and increases during the last trimester and at - structure and cytochemistry
birth the marrow is and then remains, the chief site of normal hemopoiesis. - round or oval nucleus: young cell
Extramedullary hemopoiesis is negligible except for lymphopoiesis in the spleen, lymph - large nucleus/cytoplasm ratio: young cell.
nodes and thymus. - nuclear chromatin rich in DNA: young cell
**nucleoli with RNA: Feulgen negative
The first appearance of each cell type in the peripheral blood corresponds to maximal •NATURE OF CHROMATIN- most important criterion to det age of rbc
hemopoietic activity in the parent tissue. Early in fetal life many nucleated RBC are present. - chromatin strands coarse and clumped: mature cell
The number gradually decreases until at birth a normal infant never shows more than 10 per - decreased number of nucleoli: mature cell
100 leucocytes. Non-nucleated rbcs actually increase after which granulocytes, then - changes in shape
lymphocytes and finally monocytes can be recognized in the peripheral blood. - more lobulations: more mature cell
3. Reduction in cell size
In normal infant and in adult, the bone marrow is the only site of erythropoiesis, - smaller: more mature cell
myelopoiesis and thrombopoiesis. In general, newborn infant has little marrow reserve.
Increased production, if needed, must take place at extramedullary sites (liver, lymph nodes, BLOOD VOLUME
spleen, thymus). Blood volume determinations are important in the detection and treatment of fluid and
electrolyte imbalances. Direct measurements of total blood volume have shown that blood
In the adult, the bone marrow represents a weight of tissue at least equal to the weight of makes up about 7 to 8% of the total body weight. Since direct measurements depend on
the liver. Normally, only about one-half of the total volume is active but even so an complete exsanguination, most of available data refer to animal experiments. Laboratory
estimated 900 billion rbcs are produced daily. Under physiologic conditions, hemopoietic methods, therefore, for determining blood volume, plasma volume and total RBC mass
needs are met by mitotic division of the young cells of the marrow (homoplastic are necessarily indirect.
hemopoiesis). In case of increased requirements, there is mitotic division as well as a) For Plasma Volume determination
multiplication of younger precursors (heteroplastic hemopoiesis). - IV administration of a foreign substance which dilution in the plasma allows
measurement of fluid volume:
Dyspoiesis - profound defect in the maturation of rbc, wbc and platelets. 1. Evan's blue dye
2. Congo red dye
HOW CELLS ARE RELEASED FROM BONE NARROW INTO THE CIRCULATION: 3. I131 labeled human serum albumin (RISA)
1. RBC b) For Packed Cell Volume
- hypoxia and erythropoietin are the factors that regulate the rate of production - administration of a substance that attaches to the erythrocytes and gives a
of new erythrocytes in the bone marrow as well as the release of these cells into measurement of erythrocyte mass:
the circulation. 1. Cr51
2. WBC 2. P32
- presence of chemotoxins in the blood will chemically tract WBC to go out into the 3. radioactive Iron
circulation by the process known chemotaxis.
- Chemotaxis is a directional locomotion in response to a chemical substance nearby Normal Values Male Female
3. Platelets Total Blood Volume (ml/kg) 76 (+ or - 8) 68 (+ or – 6)
- produced and released by a shedding of megakaryocytic cytoplasm Plasma Volume (ml/kg) 42 (+ or - 5) 40 (+ or – 4)
Packed Cell Volume (ml/kg) 35 (+ or - 4) 28 (+ or – 3)
PRINCIPLES OF NORMAL CELL MATURATION
1. Cytoplasmic differentiation Decreased Blood Volume Increased Blood Volume
- loss of basophilia (more basophilia due to cytoplasmic RNA means less mature cell) 1. loss of whole blood 1. during excessive fluid intake
- cytoplasmic granules (more granules means mature cell) 2. loss of rbc 2. during blood transfusion
3. loss of plasma / water 3. during IV injection of fluids
Terms Things to remember in doing skin puncture: .
1. Normovolemia - normal blood volume 3. Hypervolemia - increased blood volume 1. Puncture should be 2.5 to 3 mm deep so as to hit the capillary bed, thus, ensuring free
2. Hypovolemia - decreased blood volume 4. Oligemia - total reduction of blood volume flow of blood.
2. Pressure and squeezing should be avoided to minimize a mixture of blood with tissue fluid
BLOOD COLLECTION which will affect the accuracy of the tests.
The basis of hematologic techniques is correct collection of blood sample and attention to 3. The first drop of blood is usually discarded since it contains tissue fluids and other foreign
precise methodology. lf the blood sample is not collected with proper attention to detail, the materials like dead epidermal cells.
whole hematologic examination is put into question. 4. When collecting blood for hematologic tests, the punctured finger must be wiped dry
after each test since platelets will begin to clump immediately in the blood at the
Blood examination should be performed in accordance with the following general guidelines: puncture site.
As much as possible, blood examinations should be done in the morning, in the fasting 5. The values for red blood cell count, hematocrit, hemoglobin and platelets are lower in
patient, before the usual breakfast time, particularly so if values to be determined are capillary blood, but higher white blood cell count by as much as 1,000/mm as compared
subject to diurnal variations such as all chemical determinations, notably serum iron and to venousblood.
blood sugar. Any repeat examinations should be done in the morning of the following day.
Heavy meals as well as prolonged fasting can lead to appreciable leucocytosis. VENIPUNCTURE
- easiest and most convenient method of obtaining enough volume of venous blood
The 2 general methods of collecting blood for haematological studies are: suitable for a variety of tests.
a) Skin puncture b) Venipuncture - three factors are involved in a good venipuncture:
a) the venipuncturist
SKIN PUNCTURE b) the patient and his veins
- used when only small quantities of blood are required c) the equipment
- collection of blood from puncture made on skin
- blood obtained is known as: capillary blood Two methods of collecting blood by venipuncture:
peripheral blood 1. Syringe Method
arteriolar blood 2. Vacuum Tube Method (Evacuated tube method)
Advantages over the Syringe method:
Sites of puncture: Sites to avoid: a) requires no prior preparation as it is a prepackaged sterile unit.
1. margin of earlobe 1. inflammed and pallor areas b) offers a wider range of tube size and contained anticoagulants.
2. palmar surface of the finger 2. cold and cyanotic areas c) safer method of blood collection as samples are taken directly into
3. plantar surface of heel and big toe 3. congested and edematous areas labeled tube
4. scarred and heavily calloused areas d) avoidance of syringe breakage.
e) disposable
Advantages of skin puncture - finger: Advantages of skin puncture - earlobe
1. easily accessible to the operator 1. Less pain (less nerve endings) Anticoagulants in vacuum tubes:
2. easy to manipulate. 2. More free flow of blood (thin skin) 1. Pink stopper - no anticoagulant; used for tissue or blood culture.
3. ideal for peripheral blood smears 3. Less tissue fluid contamination (less muscle) 2. Red stopper - no anticoagulant; used for tests which require serum such as chemistry and
4. less intimidating. 4. Ideal when searching for abnormal cells .serological exams.
(histiocytes in bacterial endocarditis) 3. Amber stopper - no anticoagulant; used for blood lead determination.
4. Yellow stopper - no anticoagulant; used for bacterial culture and unknowns; can be
Disadvantages of skin puncture: Incubated or autoclaved.
1. Less amount of blood can be obtained 5. Black stopper - 0.5 ml of 0.1M sodium citrate and collects 4.5 ml of blood for
2. Additional and repeated tests cannot be done. prothrombin time determination.
3. Blood obtained by skin puncture lyses easily. 6. Black stopper - 1 ml of 3.8% sodium citrate solution and collects 9ml of blood for
 coagulation tests requiring plasma. - excessive pull of plunger of the syringe
7. Blue stopper - 1 ml of 3.8% sodium citrate and collects 4 ml blood for sedimentation rate b. hitting the vein through and through
determination, Westergren method. c. hitting just the wall of the vein (as in sclerotic and movable veins)
8. Blue stopper - dry mixture of potassium (4 mg) & ammonium oxalate (6mg) and collects 3. Circulatory failure - sudden stop or decrease of blood flow due to nervousness or shock.
5ml for blood cell counts 4. Fainting or syncope - due to sudden decrease of blood supply to the brain brought about
9. Gray stopper - lithium oxalate; for blood chemistry determinations by nervousness or shock.
10. Green stopper - heparin 286 U.S.P. sodium and collects 15 ml of blood for determination
of serum iron concentration and total iron-binding capacity. Also for II. Local delayed complications:
special blood tests like arterial blood gas and research studies. 1. Hematoma - inflammation and discoloration of surrounding tissues due to extravasation
11. Lavender stopper - 0.06 ml of l5% ethylenediaminetetraacetic acid (EDTA)and collects of blood brought about by trauma.
7 ml of blood for blood cell counts and hematologic examinations. 2. Thrombosis of the vein - formation of clot at the site of puncture due to trauma,
3. Thrombophlebitis - inflammation of vein at the site of puncture wherein a thrombus is
Sites of Venipuncture: present.
In newborn infants up to 18 months old: 3. veins on the antecubital fossa
1. external jugular vein 2. veins on dorsal of hand and fingers III. General delayed complications:
2. temporal vein (scalp vein) 1. wrist vein 1. Serum hepatitis - viral infection characterized by yellow coloration of the skin and eyes as
3. superior longitudinal sinus In children 3 years old up to adult life: well as presence of bile in the urine. There is inflammation of the liver
and we may transmit this infection from one patient to the next with
In older children 18 months to 3 years old: the use of contaminated lancets/needles.
1. femoral vein
2. long saphenous vein 2. AIDS (Acquired Immunodeficiency Syndrome) caused by HIV virus.
3. popliteal vein - Two ways by which it is acquired:
4. ankle vein 1. through blood and its by-products
2. sexual contact with infected individual
Advantages of Venipuncture: - incubation period may be from 5 to I5 years.
1. Large amount of blood can be obtained for a variety of tests. Sample can be divided and - no known cure yet
treated as the prescribed test demands prescribed investigations demand.
2. Additional and repeated tests can be done. ANTICOAGULANTS
3. Fastest method of collecting samples from a large number of patients. Anticoagulants are usually chemical preparations added to the blood to prevent clotting. The
4. Blood can be transported to the laboratory and stored for future use. correct choice and amount of anticoagulant are very important in making sure that the
5. Blood collected is ideal for blood chemistry determinations. correct sample is prepared for a test. An insufficient amount may lead to partial clotting
while too much liquid anticoagulant dilutes the blood sample. The incorrect choice of
Disadvantages of Venipuncture: anticoagulant may lead to distortion of cells.
1. Requires more time and skill on the part of the operator.
2. Requires more equipment. I. OXALATES
3. More complications that may arise. - prevent coagulation by combining with calcium to form an insoluble calcium oxalate salt.
4. Hard to do on infants, children and obese individuals. 1. dried Potassium Oxalate
- distorts wbc and shrinks rbc
Complications in Venipuncture: - not ideal for ESR, hematocrit, blood smears and blood K determination
I. Local immediate complications - - used at a concentration of 1-2 mg per ml of blood
application of tourniquet. - dried sodium or lithium oxalate may be substituted for potassium oxalate
2. Failure of blood to enter syringe as in:
a. collapsed vein which may be due to 2. dried Ammonium & Potassium Oxalate
- nervousness (balanced oxalate, double oxalate, Winthrobe's solution, Paul-Heller's solution)
 Trisodium citrate - 22.0 gm Dextrose - 24.5 gm
Stock solution - dried Ammonium & Potassium Oxalate Citric_acid - 8.0 gm Distilled water - 1,000.0 ml
1.2 gm of ammonium oxalate (3 parts) - 15 ml ACD is used for every 100 ml blood
0.8 gm of potassium oxalate (2 parts)
100.0 ml distilled water A 3. Citrate- Phosphate- Dextrose (CPD) Solution

- Ammonium oxalate swells rbc, Potassium oxalate shrinks rbc. However, when used III. EDTA (Ethylenediaminetetraacetic acid)
together in a proportion of 3:2. they form a balanced action on rbc (no significant - prevents coagulation by chelation (preventing Ca from ionizing)
shrinkage or enlargement) - may be used as dipotassium salt (Sequestrene) or as disodium salt (Versene)
- ideal for ESR, hematocrit and blood cell counts - used for blood cell counts including platelet count and also for preparing peripheral
- not ideal for blood chem. determinations such as BUN, uric acid and K determination blood smears even after 3-4 hours
since it will increase the values. - used at 1-2 mg/ml blood
- not ideal for blood smears because the anticoagulant causes: - if in liquid form,it is used at 0.1 ml of 10% aqueous solution of dipotassium EDTA per 5
a) nuclear degeneration of leucocytes; ml of blood, it should, however, be evaporated to dryness
b) cytoplasmic vacuolation of granulocytes;
c) pseudolobulation and clumping of agranulocytes Preparation of Sequestrene Solution:
d) artifact formation in nuclei of lymphocytes and`monocytes; Sequestrene (dry powder) - 10gm
e)phagocytosis of oxalate crystals Distilled Water - 100 ml

- used at 5ml (dried-to powder form) per 6 ml blood Advantages of Sequestrene

- decomposes at high temperature (>8O oC) from oxalates into carbonates
II. CITRATES
- prevent coagulation by combining with calcium in a non ionized form;
- widely used for blood collection for transfusion since it is a relatively non-toxic salt that is
rapidly used up by the body or excreted by the kidneys.
1. Dipotassium salt is preferred due to its solubility
2. Used in many hematologic tests even if blood has stood for many hours
3. Blood smears are well done, leucocytes and erythrocytes are morphologically well
preserved even after 3 or 4 hours
4. Prevents surface adhesion and clumping of RBC, WBC and platelets
5. Prevents formation of artifacts even upon long standing

1. 3.8% Na citrate IV. HEPARIN

- used in forms of disodium and trisodium citrates for clotting mechanism and ESR. - prevents coagulation by acting as anti-thromboplastin and anti-thrombin
- Formula: - complex acidic mucopolysaccharide
Trisodium citrate - 1.32 gm - best natural anticoagulant
Citric acid - 0.48 gm - best anticoagulant for the minimal hemolysis of red blood cells
Dextrose - 1.40 gm - ideal for ESR, hematocrit, RBC, osmotic fragility test and hemolytic disease of the
Distilled water - 100.0 ml newborn (HHN) cases
- 1 part.of 3.8% Na citrate added to 4 parts of blood can be used for ESR determination, - also used for special blood chemistry determinations such as pH determination and
Westergren method blood gas analysis
- 1 part of- 8.8% Na citrate added to 9 parts of blood can be used for coagulation - less toxic than Na citrate therefore it is often used for open heart surgery and exchange
Studies using plasma. transfusion wherein large quantities of blood are given rapidly
- available as sodium, calcium or ammonium salts
2. Acid Citrate Dextrose (ACD) Solution - preferred for electrolyte determination is ammonium salt
- preserves erythrocytes better than trisodium citrate alone and is therefore preferred - used at a concentration of 0.1 - 0.2 mg/ml of blood or 0.1 ml of liquid heparin (1,000
For blood transfusions and for the study of the hemolytic process units) per 10 ml blood.
- dextrose serves as food for the red blood cells in vitro leading to longer survival time
- Formula:
 water and dried at room temperature for 24 hours or in a hot air oven at 100 C for
Disadvantages: 10 minutes.
1. Very expensive
2. Cannot be used for morphological studies of cells EXAMINATION OF BLOOD AND ITS COMPONENTS
3. Blood preserved with heparin is not ideal for blood smear preparation using Wright’s or The laboratory study of blood is divided into 2 groups:
Leishman’s stain due to the blue coloration of the background 1) Tests for the detection of diseases of the blood
2) Tests for the detection of diseases of other organs
V. FLUORIDES
- prevent coagulation by forming a weakly dissociated calcium component. The laboratory tests for the detection of diseases of blood:
- anticoagulant with preservative action 1. Chemical tests (haemoglobin determination and its abnormalities).
- interfere with the enzyme system having to do with glycolysis 2. Morphologic tests
- ideal for CSF and blood dsugar determinations especially when there is likely one-  Qualitative tests - determination of the structure and appearances of the cells as
half to one hour delay between specimen collection and actual analysis analyzed and classified.
- used either as potassium fluoride or sodium fluoride  Quantitative tests - determination of the number of cells such as RBC count WBC
- potassium fluoride is considered the simplest preparation because just 2 drops of count platelet count, reticulocyte count, differential count etc.
40% aqueous solution of it is enough to collect 5 ml blood
- sodium fluoride is used in conjunction with potassium oxalate to prevent glycolysis THE COMPLETE BLOOD COUNT (CBC):
for 24 hours or longer - consists of 6 determinations, namely:
- usually, 4 gm potassium oxalate and 5 gm sodium fluoride are dissolved in 100 ml 1. Red cell count 4. Hematocrit determination
distilled water then 0.5 ml of the mixture is placed in a tube or vial and 2. White cell count 5. Differential count
evaporated to dryness. 3. Hemoglobin determination 6. Stained red cell examination
- after evaporation, there should be 20 mg potassium oxalate and 25 mg sodium
fluoride left in the tube or vial to prevent coagulation of 10 ml blood or 10 mg Hemocytometry
sodium fluoride per ml blood. - numerical evaluation of formed elements of blood
- Thymol (1 mg) may be added if bacterial action is to be controlled as in specimens - estimation of the number of blood cells in a known volume of blood
that are sent to the laboratory by mail.
Methods:
VI. DEFIBRINATION I. Turbidimetric Method
- prevents coagulation by removing fibrins as they are formed II. Microscopic Method
- to defibrinate: fresh venous blood is placed in an Erlenmeyer flask with glass beads, a. Pipet Method b. Test Tube Dilution Method
paper clips or marbles or glass rods. The flask is rotated in a figure of eight for at III. Automated Method (Impedance)
least 5-10 minutes by which time all the fibrin would have adhered to the beads or a. Optical b. Electrical
- paper clips or hedgehog end of the rod
- ideal for: a) obtaining high serum yield from sample TURBIDIMETRIC METHOD
b) preserving the morphology of rbc & wbc. - based on the assumption that the more turbid the solution, the more cells are
c) preparing smears from buffy coat for LE cell demonstration. present in that solution"
- obsolete and very erroneous.
VII. SILICONIZED GLASSWARES
- use of siliconized glasswares prevents coagulation by preventing the adhesion of MICROSCOPIC METHOD
platelets to wetable surfaces, thereby preventing breakage of platelets - counting cells under the microscope with the use of the following materials:
andsubsequent release of platelet factor 3 (PF-3) 1. counting chamber
- the glasswares are immersed in a dilution of water-soluble silicone concentration 2. pipets
(like Clay-Adams “Siliclad”) for 5 seconds or longer and then rinsed with distilled 3. diluting fluids
 Comparison of the 2 pipets RBC WBC
1. size of bulb larger smaller
1. Counting chamber or haemocytometer 2. color of bead red white
A. According to type: 3. volume in bulb 100 10
1. Open type (Spencer, Burker, Levy, Levy-Hausser) 4. size of bore smaller larger
2. Closed type (Thoma-Zeiss) 5. upper calibration 101 11
3. Addis
4. Exton RBC STUDIES
5. Petroff NORMAL MATURATION SERIES
1. RUBRIBLAST OR NORMOBLAST
B. According to rulings: - makes up 5-10% of total nucleated cells in bone
1. Thoma 4. Neubauer - Size : 14-19 u
2. Tuerk 5. Improved Neubauer - Nucleus: Round or slightly oval; thin nuclear membrane; may be central or slightly
3. Fuchs-Rosenthal 6. Bass-Jones eccentric; chromatin varies from finely reticular to coarsely reticular with
a tendency to clumping; parachromatin is indistinct and scant.
 The most commonly used counting chamber is the Open Type – Spencer - Nucleoli: 1 to 2; usually very faint and pale blue
and Improved Neubauer - Cytoplasm: small in amount; moderately basophilic, homogeneous and opaque.

The counting chamber (hemocytometer) has 2 ruled areas etched on its surface, each
consisting of a 3 mm square divided into 9 large squares, each with an area of 1 sq. mm. The
central large square, which is used for RBC count, is subdivided into 25 intermediate squares,
each with an area of 0.04 sq; mm. Each intermediate square is further subdivided into 16
small squares. Red blood cells are usually counted in the central and four corner
intermediate squares (R). The 4 corner large squares (W), each with an area of 1 sq. mm, are
subdivided into 16 smaller squares and are used for WBC ct. Depth of the counting chamber
is 0.1 mm.
The area of one large square (1 sq.mm) and the depth of the counting chamber (0.1 mm). C
Compute for the volume per large square:
Volume = Area X Depth = 1 sq.mm X 0.1 mm= 0.1 cu.mm
This volume of 0.1 cu.mm is always multiplied by 10 to give the contents of 1 cu. mm blood;
thus 10 here is considered as the depth correction factor.
2. Pipets
A. Automatic pipets ( Ex: Trenner, Unopette)
- microglass capillary pipets that automatically suck in just the right amount of
sample.
- connected to a plastic container containing just the right amount of diluting fluid
B. Non-automatic pipets (Ex: Thoma pipet)
a. RBC Thoma pipet b. WBC pipet
2. PRORUBRICYTE OR BASOPHILIC NORMOBLAST
- Size - 10-15 u
- Nucleus: smaller than in normoblast; generally round and slightly eccentric; thin
nuclear membrane; chromatin is coarse and irregular so that nucleus
stains dark; parachromatin is sparse but distinct
- Nucleoli: 0 - 1
- Cytoplasm: appears more abundant than in normoblast because of smaller nucleus;
varies from intense to moderately basophilic and is royal blue and
opaqe
3. RUBRICYTE OR POLYCHROMATOPHILIC NORMOBLAST .
- characterized by the first appearance of hemoglobin,
- usually perinuclear, so that cytoplasm stains pink to basophilic.
- Size : 8-12 u
- Nucleus: round and smaller than in prorubricyte; usually eccentric; thick' nuclear
membrane; coarse and clumped chromatin so that nucleus stains very
dark; distinct parachromatin.
- Nucleoli: none
- Cytoplasm: more abundant than in precursors; varies from basophilic to diffusely
lilac, depending upon the amount of hemoglobin.
4. METARUBRICYTE OR ORTHOCHROMIC OR ACIDOPHILIC NORMOBLAST
- fully hemoglobinated_cell; constitutes 50% of nucleated red cells in normal marrow
- Size : 7-10 u
 - Nucleus: small and/shrunken; dense and dark staining because of marked - normal values at birth range from 2.5 - 6.5%, falling to the normal adult level by the
condensation of chromatin. Parachromatin no longer distinguishable, may be end of the second week.
round, oval or have various bizarre forms and is usually eccentric
- Nucleoli: none
- Cytoplasm: orange-red, as in adult erythrocyte RETICULOCYTE COUNT
- Degreeof reticulocytosis is proportional to erythropoietic activity. Retic count above
5. RETICULOCYTE normal indicate that erythropoiesis increased. The discovery of reticulocytosis
- constitutes 0.5 to 1.5% of circulating red blood cells. lead to the recognition of an otherwise occult disease such hidden hemorrhage for
- slightly larger than mature erythrocyte. unrecognized hemolysis. Persistently low reticulocyte counts particularly in the
- after expulsion of the nucleus in metarubricyte, a large somewhat basophilic presence of anemia, suggest markedly defective erythropoiesis.
anuclclear cell remains, which, when stained with new methylene blue, a vital stain, RETICULOCYTE COUNT
is seen to contain a network of bluish granules or what is known as reticulum Physiologic increase: Low Count or Absent in:
network. As cell matures the network becomes smaller, finer, thinner and finally 1. at birth 1. Idiopathic aplastic anemia
disappears within 2-4 days. 2. menstruation 2. Acute benzol poisoning
- cytoplasm is pink and reddish brown. 3. pregnancy 3. In anaplastic crisis of hemolytic anemias
- Size: 8-10 u, Nucleus : absent
- Cytoplasm: cell outline may be irregular because of shallow indentations; faintly Increased Count:
polychromatophilic (basophilic) 1. Hemolytic anemias 7. Polyoythemia vera
2. Kala-azar 8. Relapsing fever
6. ERYTHROCYTE 3. Lead poisoning 9. Sickle cell anemia
- Size : 6.2 - 8.2 u in diameter (Ave.= 7.2 u), Nucleus : none 4. Leukemia 10. Splenic tumor
- Cytoplasm: biconcave orange-pink cytoplasm has a paler staining center occupying 5. Malaria 11. Blood intoxication
one-third of the cell area. 6. Erythroblastic anemias 12. Parasitic infections

NOTE: Factors that determine number of reticulocytes in the circulation:

- no hemoglobin yet in cells 1 & 2. 1. Speed of release of reticulocytes from bone marrowi into the circulation
- cells 1 to 5 are normally seen only in the bone marrow except for cell 5 (retic) 2. Degree of immaturity of released reticulocytes; and
wherein 0.5 to 1.5% is seen in the circulation. 3. Speed of disappearance of reticulum network from the reticulocytes.
- cell 6 (erythrocyte) is definitely seen only in the circulation and it is
eosinophilic/acidophilic in staining reaction due to hemoglobin. ERYTHROCYTE
- mature red blood cell, non-nucleated, biconcave disc-like cell.
GENERAL RULES IN THE NORMAL MATURATIONl SERIES: - 6.2 - 8.2 u in diameter and around 2.8 u in thickness
- the younger the cell is, the bigger in size and the bigger in nucleus - lifespan is around 100 days (+ or - 20 days)
- the older the cell is, the smaller in size and the smaller the nucleus until eventually - erythrocytes are uniform in size and have a small area of central pallor.
in erythrocyte no more nucleus is seen. - in every 100-red blood cells, 3-8 platelets may be seen; in every 1000 red blood
cells, there is 1 white blood cell
RETICULOCYTE
- mature red blood cell; matures into erythrocyte in 2-4 days with reticulum network :Normal Values:
which are RNA and protoporphyrin remnants, the latter being responsible for the Conventional SI
fluorescence of some erythrocytes when viewed by ultraviolet light Male - 5.5 - 6.5 million/ mm 3 5.5 - 6.5 X 1012/L
- reticulum network is demonstrable with the use of supra-vital stains only like Female: 4.5 - 5.5 million/ mm 3 4.5 – 5.5 X 1012/L
brilliant cresyl blue and methylene blue.
- normally about 0.5 - 1.5 % (20,000 - 60,000/cu. mm) of all erythrocytes in adults are Erythrocyte is composed of a protein-lipid stroma that contains hemoglobin in solution and
reticulocytes is condensed into a lipid-rich peripheral cell membrane. The cell membrane is non-elastic,
although the cell can become distorted in order to pass through narrow capillaries.
HEMOGLOBINOMETRY
In order for normal erythrocytes to form, certain requirements must be met: - determination of the amount of hemoglobin in a known volume of blood screening
1. There must be an adequate supply of the hemopoietic factors (erythropoietin) test for the presence of diseases associated with anemia and for following the
responsible for normal and orderly maturation; response of these diseases to treatment in as much as the oxygen-carrying capacity
2. The maturing erythrocyte must be supplied with an adequate amount of iron in a of the blood is directly proportional to hemoglobin and not to RBC ct.
usable form;
3. Adequate amounts of protoporphyrinIll must be supplied; HEMOGLOBIN (Ib) A
4. There must be available sufficient globin. - main component of rbc; it makes up 35 %
- molecular weight of more than 64,400.
LIFE HISTORY OF THE ERYTHROCYTE: - gives red color to the blood so that it is also known as respiratory pigment. `
If the normal blood volume is taken to be 5L and the RBC count as 5,000,000 per mm blood, - responsible for the oxygen and carbon dioxide-carrying capacity of rbc.
then a total of 25 x 1012 red blood cells are in active circulation at any one time. Their speed - 1 gm of Hb can carry 1.34 ml of oxygen and 3.47 mg iron
of travel varies in different parts of the vascular system, the whole circuit taking 45 seconds, - adult RBC mass with 600.gm Hb can carry around 800 ml oxygen.
on the average. lt has been computed that each rbc travels about 700 miles before - conjugated protein; made up of 1 globin molecule and 4 heme groups.
disintegration - globin molecule: 4 polypeptide chains and each chain is attached to 1 heme group-a
ferroprotoporphyrin responsible for the red color of the compound, and consist of 4
NORMAL MECHANISM OF RBC DESTRUCTION: protoporphyrinrings, each ring complexed with single bivalent iron atom.
While the red blood cell is in the circulation its membrane is mechanically injured by - globin molecule has 2 pairs of polypeptide chains:
constant buffeting in the blood stream and by alterations in its tension which is dependent alpha chain (in pairs) = 141 amino acids/chain = 282/pair
on the ionic changes associated with oxygen and carbon dioxide exchange. Furthermore, as beta chain (in pairs)' = 146 amino acids/chain = 292/pair
the red cell matures it becomes more spherical and this facilitates rupture of the cell
membrane. Normally, the end stages of cell destruction takes place in the reticulo- Normal Values: Conventional S.I.
endothelial system, particularly in the bone marrow, liver and spleen. The spleen has been Men - 14 - 18 gm/100 ml blood 2.17 - 2.79 mmoles/L
long regarded as the main graveyard of rbc. Women - 12 - 16 gm/100 ml blood 1.86 - 2.48 mmoles/L
Children- 14 - 26 gm/100 ml blood 2.17 - 4.03¢mmoles/L
ABNORMAL MECHANISM OF RBC DESTRUCTlON
- include various types of hemolysis, hemoglobin alterations and parasitic infections. Hemoglobin in Normal Adult Blood
1. Hb A - 94.5% with 2 alpha and 2 beta polypeptide chains
RBC COUNT 2. Hb A2 - 3.5% with 2 alpha and 2 delta chains
Six factors have statistically significant effect on the RBC 3. Hb F- 2.0% with 2 alpha and 2 gamma chains
1. posture 4. age
2. extreme physical exercise 5. gender  Hb F is measured by:
3. severe dehydration 6. High altitude a) alkali denaturation test by Singer
b) acid elution technic by Kleihauer-Betke.
Increased RBC Count
1. polycythemia vera 4. dehydration CATABOLISM OF HEMOGLOBIN:
2. chronic heart disease 5. acute poisoning When old red blood cells are destroyed in the RES, the globin portion of the hemoglobin
3. lung diseases (TB, fibrosis) 6. residing in'a place of high altitude molecule is split off, and the heme is converted into biliverdin. Most of the biliverdin formed
from heme is converted to bilirubin which-is excreted in the bile. Subsequent degradation of
Decreased RBC Count bilirubin will give rise to urobilinogen and stercobilinogen, oxidation of which will result
1. Anemia to urobilin and stercobilin. The iron from the heme is reused for hemoglobin synthesis.
2. hemorrhage 1
3. oligocythemia Hemoglobin Increased in: Decreased in:
1. hyperchromia 1. all anemias 2.2% in premature infants
2. high altitude 2. leukemia 1 - 1.5% in infants up to 1-year old
3. polycythemia 3. oligochromia 1% in older children and adults.
4. dehydration in burns & diarrhea - critical level is 1.5 gm/100 ml blood and it gives a characteristic chocolate
5. heart diseases . brown color to the blood
6. erythremia . 3. Sulfhemoglobin (SHb)
Hb + sulfides = Sulfhemoglobin
TYPES OF HEMOGLOBIN - reaction is irreversible since SHb is so stable that once formed, it.
A. NORMAL or FUNCTIONAL HB disappears from the circulation only after the rbcs containing it are
1. Oxyhemoglobin (HbO2) naturally destroyed. .Erythrooytes containing SHb have normal survival
Hb + Oxygen = 0xyhemoglobin (in arterial blood; bright red) and osmotic fragility.
2. Deoxygenated or Reduced Hb (HbCO2) - normal concentration of SHb in the blood is 0 - 2.2% of total
Hb + Carbon dioxide = Deoxygenated or Reduced Hb (in venous blood; dark red) hemoglobin.
- critical level is .5 gm/100 ml blood
B. ABNORMAL OR NON-FUNCTIONAL HB - unable to act as an oxygen carrier.
1. Carboxyhemoglobin (HbCO2) C. HEMOGLOBIN VARIANTS:
Hb + Carbon monoxide.: Carboxyhemoglobin - variants result due to differences in the .arrangement of amino acids in the
- the combination is irreversible. The affinity of Hb for C0 is 218x greater polypeptide chain.
than for Oxygen at 37 C and is not affected if helium is substituted for - example: Hb A becomes Hb S when glutamic acid (negative charge) on the
nitrogen as the inert gas (as in prolonged underwater diving). Since 6th position of the beta chain is replaced by valine (neutral
carboxyhemoglobin is not capable of transporting oxygen, hypoxia charge). Hb A becomes Hb C when glutamic acid on the the 6th
chain is replaced by lysine.
results. Death may be due to anoxia, accompanied with irreversible - alphabetically A designated. There are over 200 hemoglobin variants listed but only
tissue changes. a few (like Hb S,Hb C, Hb D, Hb E) are important agents in the production of
- Carbokyhemoglobin produces a typical cherry red color of patient's hemolytic anemia.
blood and skin. - differentiated from one another by:
- Carboxyhemoglobin concentration in normal non-smokers is 0 - 2.3% of a. solubility
total hemoglobin and in smokers, 2.1 - 4.2%. Critical level is 5 g/100 ml b. mobility in an electrophoretic field at pH 8.6
blood. fastest = Hb H and Hb I
- measured by: slowest = Hb C
a) Palmer's Carboxyhemoglobin Methods
b) Sunderman Dithionite Spectroscopic Test (Na2S2O4 -Dithionite) METHODS OF HEMOGLOBIN DETERMINATION
2. Methemoglobin (Hi) Principle: Hemoglobin is measured as oxyhemoglobin. It is first converted into one of several
- also known as ferrihemoglobin, oxidized Hb and hemiglobin compounds, such as acid hematin, alkaline hematin, cyanmethemoglobin, and other less
Hb + oxidizing agent = Methemoglobin widelyl employed., The measurement is done by comparing the unknown with a standard.
- the reaction is reversible since methemoglobin is unstable. The method of comparison may be visual or photoelectric. The latter is ,considerably more
- differs from oxyhemoglobin in that it contains ferric, rather than ferrous accurate.
iron; the oxygen being replaced by -OH. It is, therefore, oxidized Hb or
ferriHb and results when Hb is exposed to oxidizing agents such as I. COLORIMETRIC METHOD
laniline dyes, potassium ferricyanide or potassium permanganate. A. Direct Visual Colorimetric Method
- formation of methemoglobin is a normalprocess, kept within bounds by 1. Tallquist Method
a normal intraerythrocytic system capable of reducing methemoglobin 2. Dare's Hemoglobinometer
to hemoglobin again 3. Acid Hematin Methods
- Kravitz et al. give the ff. values for methemoglobin in normal subjects: Methods:
 a. Sahli-Adams d. Hayden-Hausser M Hematocrit after 30 minutes of centrifugation at 3,500 rpm.
b. Sahli-Hellige e. Newcomer - each calibration has 10 mm so there is a total of 100 mm in the tube.
c. Sahli-Hayden f. Osgood-Haskin - the bore is 3 mm in diameter.
4. Alkaline Hematin Method Normal Values:
B. Photoelectric Colorimetric Method Male Adult : 0 - 9 mm/hr
1. Oxyhemoglobin Method Female Adult: 0 -20 mm/hr
2. Cyanmethemoglobin Method (HiCN) (Drabkin’s Reagent) Children : 0 -13 mm/hr
Layers in Wintrobe tube after l hour
II. SPECIFIC GRAVITY METHOD a. Plasma layer - uppermost layer
A. Copper Sulfate Method b. Buffy coat layer - middle layer which contains the platelets, WBC, and other
cells such as L.E. cells and other malignant cells. 1mm of buffy
III. GASOMETRIC METHOD coat contains about 10,000 WBC. ,It is whitish to cream in color.
- oxygen content of blood is directly measured c. Packed red blood cells layer – lowermost layer which contains nucleated young
red cells on top and non-nucleated mature red
IV. CHEMICAL METHOD cells at the lower layer.
- iron content of blood is measured Correct ESR for Anemia:
- when ESR is high and hematocrit is low.
ERYTHROCYTE SEDIMENTATION RATE Winthrobe method can be used to do other tests like studies on the plasma, preparing
- rate of settling of red blood cells from their plasma after the smears from the buffy coat and hematocrit determination
addition of anticoagulant
- settling of red blood cells is due to changes in surface charge. B: WESTERGREN METHOD
- red blood cells are normally negatively charged but sometimes - most sensitive ESR method 'for serial study of chronic diseases
some changes in the plasma will cause_some red blood cells to become positively - 2 readings are done: after 1 hour and after 2 hours
charged and this leads to rouleaux formation since unlike chargesl attract.
- Rouleaux formation leads to settling of red blood cells. Anticoagulant -3.8% Na citrate
Westergren tube
IMPORTANCE OF ESR: - Long tube with both ends open; calibration is up to 200 mm.
- index for the presence of hidden but active diseases such as TB and carcinoma. - bore is 2.5 mm in diameter
- measures the suspension stability of red blood cells Normal Values:
- measures the abnormal concentration of fibrinogen and serum globulin. Male Adult = 3 - 5 mm/1 hr 7 -15 mm/2 hrs
Female Adult = 4 - 7 mm/1 hr 12 -17 mm/2 hrs
There is a significant difference between the sedimentation rate of normal men and women
regardless of age, women showing a higher rate than men. This is due to the concentration C. GRAPHIC – CUTLER METHOD
of androgenic steroids in males. Castration of the male produces a lower ESR to normal male - 3.8% Na Citrate and Cutler tube
levels. D. LINZENMEIER METHOD
- 3.8% Na Citrate and Cutler tube
METHODS OF ESR DETERMINATION: E. MICROMETHODS
A. WINTHROBE AND LANDSBERG METHOD 1. Micro Landau – 5% Na Citrate and Micro Landau tube
Anticoagulant - Ammonium-Potassium oxalate (also known as: double oxalate, balanced 2. Smith Micro Method
oxalate, Paul-Heller's solution, Winthrobe's solution) 3. Crista or Hellige-Vollmer Method
Winthrobe tube F. ROARKE-ERNSTENE
- flat-bottomed with two calibrated sides: G. BRAY’S METHOD
Left side - colored red; calibrated 0 on top and 10 cm at bottom; used for ESR
Right side - colored white; calibrated 10 cm on top and 0 at the bottom; for STAGES OF ESR
1. Initial rouleaux formation (during'the first 10 minutes). 2. Low temperature - this tends to slow ESR since there is increased viscosity of the blood.
2. Period of rapid settling (during the next 40 minutes when settling is constant). 3. Excess of dry anticoagulant - slows down ESR because rouleaux formation is inhibited. '
3. Period of-final settling or packing (during the last 10 minutes). 4. Diameter of the tube - tubes with less than 2 mm in diameter will slow down ESR.

FACTORS IN ESR: HEMATOCRIT

I. INTRINSlC FACTORS (inherent factors in rbc and plasma) - volume occupied by erythrocytes in a given volume of blood and usually expressed
1. No. of RBC/mm3 (inversely proportional to_ESR) as volume of erythrocytes per 100 ml blood.
polycythemia = slower ESR - venous hematocrit is the hematocrit of venous blood which represents the
anemia = faster ESR hematocrit of peripheral blood and does not indicate the proportion of erythrocytes
2. Size of RBC (directly proportional to ESR) to plasma in the entire circulation.
macrocytes = faster ESR - hematocrit is the ratio of total erythrocyte mass to total blood volume and is
microcytes = slower ESR calculated by a different method.
3.Viscosity of the pla na (inversely proportional) - used in determining erythrocyte indices, calculating blood volume and total
more viscous = slower ESR erythrocyte mass, and establishing whether or not a patient is anemic, in as much
less viscous = faster ESR as a low hematocrit indicates that the concentration of erythrocytes is reduced.
4. Plasma protein contents - hematocrit cannot completely replace RBC counts since the latter are needed to
All proteins (alpha-, beta- and gamma-globulin. calculate some of the erythrocyte indices.
and fibrinogen) except albumin will increase ESR. - for normal blood, hemoglobin and RBC counts may be estimated from the
microhematocrit reading

II. EXTRINSIC FACTORS METHODS OF HEMATOCRIT»DETERMINATl0N: »

1. Length of tube A. WINTHROBE METHOD
longer tube = faster ESR (because more space at the bottom wherein - Anticoagulant S double oxalate
cells can settle down) - Winthrobe tube (same as tube used in ESR)
2. Diameter of tube: wider tube = faster ESR Normal Values: Conventional: S.I.
3. Position of the tube Adult Male 47 vol% (+ or - 5) 0. 47 (+ or - .05)
slightly inclined = faster ESR (since the' RBC's have a shorter distance through which to Adult Female 42 vol% (+ or - 5) 0.42 (+ or - .05)
settle down and the up-current of the plasma is along the slope, thus
by-passing the sedimenting RBC's.) B. HADEN'S MODIFICATION METHOD
4. Temperature (ESR increases with temperature) - Anticoagulant = 1.1% Sodium oxalate in distilled
most ideal temp.= 2 - 27C (Room Temp.) C. VAN ALLEN‘S METHOD
5. Pipeting - if with bubbles= faster ESR (since less blood and less rbc in tube) - Anticoagulant: 1.6% sodium oxalate in distilled water
6. Volume of blood - Tube: with bulb and calibrated 1-10 or 10-100 mm.
more lood in tube = slower ESR D. SANFORD-MAGATH METHOD
less blood in tube = faster ESR - Anticoagulant = 1.3% Sodium oxalate
7. Anticoagulant: excess = slower ESR (since rouleaux formation is inhibited) - tube = calibrated to 6 ml at 1 mm per division; tube is short (about 5 inches long)
8. Time of setting up the test and with a funnel-like mouth. It has a-6 ml capacity.
after 2 hours from blood collection = slower ESR (since red blood cells become
spherical upon standing for a longer time) OSMOTIC FRAGILITY TEST
9. Time of reading results - semi-permeable characteristics of the surface membrane of an erythrocyte make it
before 1 hour = lower reading (since no final settling yet of red blood cells) vulnerable to changes in the osmotic pressure of the external environment.
- Follows the Lawof Osmosis which states that there is a transfer of solution from
FACTORS FAVORING SLOW ESR: lower to higher concentration so that: rbc + hypotonic solution will result to
1. Defibrination - removal of fibrin either by artificial or natural means. swelling of rbc and rbc + hypertonic solution will result to shrinking of rbc.
 - compared to a normal biconcave erythrocyte, a spherocyte can imbibe in less water
before it lyses because its cell membrane is already fully distended so we refer to it
as a fragile cell.
- on the other hand, a sickle cell can take in more water before it lyses because its
cell membrane can be distended more, so we refer to it as a resistant cell
METHODS OF 0SMOTIC FRAGILITY TESTS:
1. SANFORD METHOD. '
Principle: It tests the stability of red blood cells under different concentrations of
hypotonic NaCl solutions.
PRECAUTIONS IN ERYTHROCYTE OSMOTIC FRAGILITY TEST
- the blood sample should be obtained with a minimum of stasis and trauma
- test procedure should be set up as soon as possible
- the capillary pipet must be held in approximately the same angle so as to ensure
uniform size of drops
- blood should fall directly on the saline solution and not on the dry sldes of the tubes
ERYTHROCYTE INDICES
- important in assessing border line types of anemia
- values should be interpreted only in the light of other findings such as the
appearance of erythrocytes on fixed smears
- computed using 3 determinations RBC count, Hemoglobin & hematocrit
1. MCV (MEAN CORPUSCULAR VOLUME)
- average volume of an individual red blood cell
- computed using hematocrit & RBC count x 10
2. MCH (MEAN CORPUSCULAR HEMOGLOBIN)
- ratio of Hb to RBC count x 10
- average weight or amount of Hb in an individual RBC
3. MCHC (MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION
- concentration of hemoglobin in a given volume of packed red blood cells
- computed using Hb values over Hct x 100
USE OF ERYTHROCYTE INDICES:
The MCV, MCH & MCHC are sometimes collectively referred to as red cell indices. These
indices, in conjunction with the appearance of red blood cells in fixed smears, give an
accurate picture of the morphology of the red blood cell.
In summary, the determination of the MCV, MCH, and MCHC as valuable information 'that
helps to characterize erythrocytes. According to the MCV, erythrocytes may be classified as
normocytic, microcytic or macrocyticl. According to the MCHC, erythrocytes may be
classified as normochromic or hypochromic. A higher than normal MCHC does not occur
except in cases of hereditary spherocytosis. MCH expresses only the weight of hemoglobin
per erythrocyte.
ANEMIAS - anemia is a disorder characterized by a reduction in the oxygen-carrying
substance of a certain volume of blood due to a reduction below normal of RBC ct., Hb &
Hct.
Factors that determine the point at which the decreased oxygen-carrying capacity of the
blood may produce symptoms of hypoxia are:
1. rapidity with which anemia develops
2. degree of physiologic adjustment to the anemia
3. effect of physical activity on oxygen demand.
Hypoxia symptoms as well as their severity depend on:
1. how great an oxygen-carrying deficiency exists
2. how rapidly the anemia develops
3. the degree of physiologic compensation
4. level of physical activity
- when anemia develops rapidly, as in massive hemorrhage, the severity of the
symptoms is proportional to the haemoglobin concentration. When anemia
develops slowly, the patient can adjust to progressive hypoxia that symptoms will
be minimal in spite of the very low Hb and RBC count. Symptoms are also
proportional to physicalv activity. A patient at rest may not feel the symptoms even
though markedly anemic, but on exertion may have weakness, dizziness and
tachycardia.
- tachycardia occurs when the heart attempts to improve oxygenation of the tissues
by increasing the heartbeat and cardiac output per minute.
- because these factors may vary, the diagnosis of anemia is only partially based on
laboratory measurements of erythrocyte and Hb concentrations.. Laboratory data,
however, must be interpreted with reference to the clinical picture.
An adult person is said to be suffering from anemia if:
- For males:
RBC ct. = < 4.2 million/mm3
Hb = < 12 gm/100 ml
- For females:
RBC ct. = < 3.6 million/mm3
Hb = < 10 gm/100 ml
MORPHOLOGIC CLASSIFICATIONS OF ANEMIA
1. NORMOCYTIC NORMOCHROMIC
- blood picture shows-red cells that are normal in size and normal in Hb contents.
- computation of Erythrocyte Indices shows: Normal MCV, MCH & MCHC
- seen in: hemodilution, hemorrhage, hemolytics anemia and aplastic anemia.
 B. Defect in globin production
- Thalassemia
2. MICROCYTIC NORMOCHROMIC C. Defect in heme synthesis
- blood picture shows small red cells with normal Hb contents. - Sideroblastic anemia
- computation of Erythrocyte Indices shows:
Decreased MCV & MCH but Normal MCHC COMMON ANEMIAS
- seen in chronic inflammations. 1. APLASTIC ANEMIA
3. MICROCYTlC HYPOCHROMIC - "aplastic anemia" due to the functional inability of the bone marrow to replace lost
- blood picture shows small red cells that are pale in color due to decreased Hb red blood cells with proportionate decrease of RBC ot., Hb and Hct.
- computation of Erythrocyte Indices shows: Decreased MCV, MCH & MCHC - Blood picture: normocytic normochromic red cells, with normal MCV, MCH &
- seen in Thalassemia and severe iron deficiency anemia MCHC. Reticulocytes are very few or none at all. Aside from the low RBC ct., there is
4. MACROCYTIC NORHOCHROMIC leucopenia as well as thrombocytopenia. This decrease of all cell elements is known
- blood picture shows red cells that are larger than normal. Although they contain a as "pancytopenia“.
larger than normal weight of Hb, the MCHC is normal so that the cells Classifications of Aplastic Anemia According to Cause
9. Aplasia in myeloproliferative disorders
therefore, are normochromic. 1. Bone marrow injury
8. Idiopathic aplastic anemia
- computation of Erythrocyte Indices shows: inc. MCV & MCH but Normal MCHC 2. Congenital aplastic anemia
hemolytic-disease
- seen in Pernicious anemia 3. Familial aplastic anemia
7. Erythroid hypoplasia of bone marrow in
4. Chronic erythrocytic hypoplasia
6. Metabolic inhibition of bone marrow
5. Aplastic anemia assoc. with thymoma
5. MACROCYTIC HYPOCHROMIC
- blood picture shows red cells that are larger than normal but are hypochromic due 2. HEMOLYTIC ANEMIA
to decreased MCHC. - due to the excessive destruction and shortened life span of red blood cells brought
- computation of Erythrocyte Indices shows: inc MCV but Decreased MCH and MCHC. about by: a..intrinsie or corpuscular defects
b. extrinsic or extracorpuscular abnormalities
CLASSIFICATION OF ANEMIAS ACCORDING TO CAUSE
I. Decreased production of red blood Etiologic Classifications of Hemolytic Anemia:
3. Hereditary elliptocytosis
cells due to: I. Due to intrinsic (corpusoular) defects
b. Auto-immune hemolytic anemias
A. Marrow damage A. Defect of erythrocytic membrane
- Heinz Body anemia
1. Leukemias 1. Hereditary spherocytosis 4. Zieve's syndrome
a. Overactivity of the RES
2. Leukoerythroblastosis 2. Elliptocytosis or ovalocytosis 5. Paroxysmal Nocturnal Hemoglobinuria
2. Acquired
3. Aplastic anemia 3. Acanthocytosis
dehydrogenase deficiency
B. Decreased erythropoietin B. Defect of intracellular enzyme (non-spherocytic type of Hemolytie anemia)
- Glucose-6-phosphate
1. Inflammatory process 1. Enzymes involved in anaerobic glycolysis - Hexokinase, Aldolase def.
c. Enzyme defects
2. Renal disease 2. Enzymes involved in hexose monophosphate shunt – G6PD def.
- Hemoglobin S
3. Hypothyroidism 3. Enzymes involved in methemoglobin formation – glutathione synthase def.
- Hemoglobin C
C. Iron-deficiency II. Due to extrinsic (extracorpuscular) defects
b. Hemoglobinopathies
II. Nuclear maturation abnormality A. Acquired autoimmune haemolytic anemia
- Hereditary spherocytosis
A. Vitamin B12 deficiency B. Acquired isoimmune haemolytic anemia
a. Red cell membrane defect
1. Pernieious anemia C. Paroxysmal Cold Hemoglobinuria
1. Congenital
B. Folic acid deficiency D. infectious agents
B. Chronic Stage
C. Refractory macrocytic anemia
- Hemorrhage
1. Di Guglielmo's anemia Blood picture in hemolytic anemia:
A. Acute Stage
III. Cytoplasmic m turation abnormality - proportionate decrease of RBC, Hb and Hematocrit.
IV. Hemolytic anemia
A. Severe iron-deficiency - the blood picture shows normocytic normochromic red blood cells
 - computation of erythrocyte indices shows normal MCV, MCH & LCHC.
- increased nucleated red cells high reticulocyte count (10 - 2O%), increased osmotic 4. THALASSEMIA
fragility index, poikilocytosis and presence of'abnormal inclusion - due to the abnormal production rate of one of the polypeptide chains of
bodies such as Howell-Jolly bodies. hemoglobin molecule.
- also known as: a. Cooley's anemia
TYPES OF HEMOLYTIC ANEMIA b. Mediterranean anemia
1. Paroxysmal Cold Hemoglobinuria – cold hemolysin Donald-Landsteiner c. Hereditary leptocytosis
2. Paroxysmal Nocturnal Hemoglobinuria – Marchiafava- Micheli Syndrome - blood picture: microcytic hypochromic cells with a predominance of target cells.
3. Sickle Cell Anemia - computation of erythrocyte indices shows decreased MCV, MCH & MCHC

3. SICKLE CELL ANEMIA Thalassemia major

- due to the presence of Hb S variant which causes sickling of red cells under reduced - usually associated with increased Hb F, most severe expression of the thalassemia
oxygen tension. Under normal oxygen tension, red cells appear normocytic abnormality and is usually detected in childhood. Affected infants fail to thrive.
normochromic so that all the three erythrocyte indices (MCV, MCH & MCHC) are Thalassemia minor
normal - associated with increased Hb A2, less serious type and is usually detected in adults
- under reduced oxygen tension, the normocytes will become sickle cells Beta Thalassemia
- Sickle cells are crescent-shaped and they are also known as drepanocytes or - deficit of beta chain production relative to that of normal alpha production
meniscocytes. This sickling phenomenon is an irreversible reaction Alpha-thalassemia
- Vaso-occlusive crisis is observed since sickle cells will clog the bleed vessels - deficit of aprolpha chain duction relative to that of normal beta production
especiallyiat the sites of the joints.
- swelling and too much pain in the occluded blood vessels and this may lead to In thalassemia, there is decreased HbA but increased HbF and Hb A2
patient's death
- patient's red cells also exhibit chronic hemolytic tendency. 5. SEVERE IRON DEFICIENCY ANEMIA
- blood picture will also further show the following: - due to the depletion of iron storage in the body needed for normal hemoglobin
anisocytosis, poikilocytosis (cigar-shaped, crescent-like, ovalocytes, produition.
acanthocytes, target cells), neutrophilia, thrombocytosis, decreased osmotic
and mechanical fragility, presence of nucleated red cells and red cells with Etiologic factors in iron deficiency
basophilic stipplings as well as abnormal inclusion bodies such as Howell-Jolly A. Negative iron balance
bodies, Pappenheimer bodies and Cabot's ring. 1. Decreased iron intake
- commonly seen among the black race 2. Increased iron loss
- presence of homologous Hb S and S in the patient indicates sickle cell anemia a. Gastrointestinal bleeding e. self-induced bleeding
whereas presence of heterologous Hb S and Hb A indicates sickle cell trait b. excessive menstrual flow f. Pulmonary hemosiderosis
c. blood donation g. Hemorrhagic telangiectasia
Methods for demonstrating sickling of cells in the laboratory: d. hemoglobinuria h. Disorders of hemostasis
1. Scriver and Waugh B. Increased Requirements
2. Daland and Da Silva 1. pregnancy 3. lactation
3. Sherman's Test (also known as Test Tube Method) 2. infancy

Principle Involved: PERNICIOUS ANEMIA

The reduced form of Hb S which is not loaded. With oxygen is less soluble than normal - lack of intrinsic factor needed for the normal absorption of Vit. B12 and folic acid.
hemoglobin and solidifies within 15-30 seconds if there is an absolute .lack of oxygen. . In - also known as: a. Macrocytic anemia
the subsequent crystallization of Hb S the shape of the erythrocyte changes from that of a b. Megalocytic anemia
disc to a sickle form which serves as a diagnostic criterion for the sickle cell anomaly and can c. Addisonian's disease
be observed in blood preparation under the microscope.
 - blood picture shows macrocytes and megalocytes that are normochromic so that
MCV & MCH are increased but MCHC isa normal. There is a three-fold increase in
erythropoiesis in the bone marrow. However, the rate of release of young cells from
bone marrow into the circulation remains the same as during normal erythropoiesis.
Laboratory diagnosis of pernicious anemia
1. peripheral blood smear
2. Achlorhydria after histamine stimulation
3. Megaloblastic dysplasia of bone marrow
4. Low excretlon of radioactive Vit B12 in the urine (Schilllng s test)
5. Low concentration of Vit B12 in the serum
RBC ANOMALIES
1. ANISOCYTOSIS - variation in size of red blood cells ( sign of regeneration)
Normocyte - 6.2 to 8.2 micra in diameter. Macrocyte - > 9 micra in diameter.
Microcyte - < 6 micra in diameter. Megalocyte – 12 to 21 micra in diameter
2. POIKILOCYTOSIS - variation in shape of red blood cells; seen in all forms of severe
anemias, nephritis and bleeding peptic ulcers. May be graded 1+ to 4+
a. Acanthocyte
- (thorn cell, spur cell) - thorny, rounded rbc with_irregularly arranged
- 5-10 spicules of various lengths, some of which are bent at their tips.
- indicate permanent cell damage found in abetalipoproteinemia
b. Burr cell
- reversibly spiculated elongated cells that are not synonymous with the
non-reversible acanthocyte. Spicules usually are shorter than that seen in
acanthocytes.
c. Crenated RBC (echinocyte)
- 10-30 short and blunt spicules that are evenly distributed over the surface of
the red cell.
- produced when blood is allowed to stand, when rbcs are exposed to lytic agents
or when placed in hypertonic solutions. Not a clinically important phenomenon,
except that it has to be differentiated from other similar but significant changes.
d. Elliptocyte (ovalocyte)
- elliptical or oval red cell, usually seen in pernicious anemia and in anemias
Associated with malignant lesions. It may also be due to a congenital
anomaly of rbc, which is Mendelian dominant and is not sex-linked.
e. Blister cell
- red cell with single or multiple vacuoles or markedly thinned areas at the
periphery and comes as a result of trauma to red cells during their passage
through partially obstructed blood, vessels. Indicative of microangiopathic
hemolytic anemias.
f. Helmet cell (keratocyte of Bessis, triangle cell)
- irregularly contracted, triangular cell; usually half-moon shaped, the central
depression flanked by two horns and results after the rupture of blister cell.
g. Pyknocyte
- distorted,`contracted, spiculated rbc similar to burr cell; normally present in
small numbers in the first 2 to 3 of months of life and may be increased up to
50% in a condition known as infantile pyknocytosis.
h. Sickle cell (drepanocyte, meniscocyte)
- crescent-shaped rbc, elongated,. Drawn-out, slightly curved cell with pointed
ends that resemble a sickle blade
- sickling usually occurs in red cells containin Hb S which are exposed to reduced
pH or oxygen tension.
i. Spherocyte
- spherical rbc with diminished diameter, sometimes reduced to 4 micra
(microspherocyte) and a central thickened portion instead of the normal pallor
' - cell with markedly reduced surface area
- occurs in large numbers in congenital spherocytic and acquired haemolytic
anemia: also in transfusion rea tions
j. Stomatocyte (mouth cell)
- morphologically abnormal rbc with a mouth like clear central area.
- exhibits abnormally' increased osmotic fragility and increased autohemolysis at
37 - 40 C.
k. Schistocyte
- fragmenting or disintegrating-rbc usually seen in haemolytic anemias, severe
burns and in DIC (diffuse intrayascular coagulation syndromes)
l. Poikilocyte
- shows marked variation not only in shape but also in size and Hb content
m. Stellar Cell (astrocyte)
- star-shaped red cell with cause similar to that of acanthocytes
n. Tear-drop Cell (dacryocyte)
- pear-shaped cell seen in severe anemias, myelofibrosis and homozygous beta-
thalassemia
o. Target cell (leptocyte, platycyte, codocyte, bull's eye cell, Mexican hat cell,
- abnormally thin cell that presents a bull's eye appearance due to a central and
peripheral condensation of haemoglobin with a clear zone in between.
- seen in hemolytic anemias, thalassemia, obstructive liver disease, HbC dis.
- unusually resistant to hypotonic solution of NaCl
p. Racket cell
- pear-shaped like the tear drop cell but with longer tail or projection so that it
now resembles the shape of a tennis racket.
3. ANISOCHROMASIA - variation in hemoglobin contents of red blood cells.
a . Normochromic (orthochromic)
- rbc with normal.hemog1obin content
b. Hypochromic
- rbc with decreased Hb contents as shown by a more pronounced pale central
area. Cells are usually microcytes and are see in all deficiency anemias.
 c. Hyperchromic
- rbc with seemingly increased Hb contents since the cell is thicker than normal but
the Hb contents is actually within normal limits. The entire cell stains deep pink
without the usual central pallor. Seen in spherocytes and megalocytes in
pernicious anemia and acute leukemia.
d. Polychromic
- rbc with patchy (uneven) distribution of Hb.
- cell appears mottled with orange (Hb) and bluish (basophilic substance) areas.
e. Anulocyte (pessary cell, ghost cell)
- rbc with just a thin rim of Hb and a large clear central area.
f. Target cell
- abnormally thin cell which tends to buckle.
- rbc with a central and peripheral condensation of Hb with a clear zone in between.
- present in Hb C and Hb S disease (lysine
4. ABNORMAL INCLUSION BODIES TERMS DESCRIBING RBC INVOLVEMENT:
a. Howell-Jolly bodies
- small (1u), spherical structures seen in rbc, may be nuclear remnants separated
during the normal process of karyorhexis or they may represent separated
chromosomes
- seen in hemolytic anemia. pernicious anemia and after splenectomy
b. Cabot s ring
- delicate, thread-like structures in figure-eight or loop-shaped found in
polychromatic or stippled red cells.
- formerly believed to be remnants of the nuclear membrane but they may be merely
artifacts produced by denatured proteins following cell degeneration; seen in
pernicious anemia and lead poisoning.
- distinguished from the ring forms of Plasmodium by their larger size and by absence
of a red chromatin
c. Basophilic stippling
- fine or coarse granules that stain blue or purple with Wright-Giemsa stain and are
scattered either around the edge or throughout the entire red cell
- characteristic of immature forms, seen in cases of disturbed erythropoiesis such as
in lead poisoning (also known as plumbism) and severe anemlas.
d. Heinz-Ehrlich bodies
- small, round inclusions that are denatured Hb caused by agents toxic to the rbc.
They are single or multiple, refractile, round, oval, or irregular bodies
and are never found in reticulocytes.
- seen after splenectomy, hemolytic anemias, hemoglobinopathies and
thalassemia major
e. Siderocytic granules (Pappenbeimer bodies) WHITE BLOOD CELL STUDIES
- intraerythrocytic iron not yet incorporated into hemoglobin LEUCOCYTE
- granules may be single or multiple and they appear as very faint bluish granules
in Wright-stained cells.
- seen in large numbers when hemoglobin synthesis is impaired and also after
splenectomy and in plumbism.
f. Maragliano bodies
- elliptical or round vacuole-like bodies seen at the
- center or periphery of the red blood cell
g. Malarial stipplings
- fine as well as coarse granules formed by malarial parasites in infected red cell.
Schuffner's dots: P. ovale and P. vivax
Zeiman's dots: P. Malariae
Maurer’s dots: P. falciparum
h. Crystals
- rodlike angular opaque reddish brown (in Wright Giemsa stained blood films) Hb C
crystals within some erythrocytes in Hb C dlsease. In Hb S-C disease the crystals are
often curved
1. Po1ycythemia
- general term for the increase) of red cell concentration in the peripheral blood to
above normal (>6.5 mil/mm3 of blood)
- may be primary (unknown etiology) or secondary (accompanies a variety of
conditions), may be a temporary or permanent condition.
2. Polycythemia vera
- also known as primary polycythemia, polycythemia rubra, erythremia, Vaques
disease, Osler's‘ disease, Vaquez-Osler disease, megalosplenica and crytogenic
polycythemi
- etiology not known but it is essentially a myeloproliferative disorder.
- characterized by abnormal proliferation of erythroids, myeloids and
megakaryocytes in the bone marrow (condition is known
as panmyelosis) as well as increased erythrocytes, leucocytes and thrombocytes
in the circulation (condition is known as pancytosis)
3. Erythremia
- increase in -red blood dell count above 6.5 mil/mm3 of blood due to
polycythemia vera.
4. Erythrocytosis
- increase 'in red blood cell count above 6.5 mil/mm3 of blood. due to chronic
heart diseases, lung diseases and high altitude.
5. Oligocythemia
- general decrease of red blood cells in the circulation as well as in all rbc-forming
organs in the body.
- larger than red blood cell, measures 9-1 u in diameter, nucleated lifespan of 5-8
days
 - provides with natural immune defense mechanisms by means of phagocytosis and 1. MYELOBLAST
production of antibodies (Ab) and proteolytjc enzymes - Size : 10 - 20u; round or oval
- Nucleus: Round or oval, thin nuclear membrane; abundant chromatin (light purple);
2 Types of WB sparse parachromatin (pale-blue or pink)
1. Granular WBC 2. Agranular WBC - Nucleoli : 2 to 5;well-outlined;round or oval, pale blue
- Neutrophil - Lymphocytes - Cytoplasm : sparse; nucleus-to-cytoplasm ratio = 5:1 to 7:1; deeply basophilic; no
- Eosinophil - Monocytes granules
- Basophil
2. PROMYELOCYTE (PROGRANULOCYTE}
- Size : 14 to 20 u; round or oval
Normal Values: - Nucleus : large, round or oval; thin nuclear membrane; slightly coarse and network-
For both male and female adults = 5,000-10,000/mm3 like chromatin
in Sl = 5~lO X lflg/L - Nucleoli : 1 to 3; less prominent; round or oval; pale blue
- Cytoplasm : sparse, nuc-to-cytop ratio = 5:1; basophilic but higher than myeloblast;
- Leucocyte count is normally higher in the newborn infant (average of 18,100/mm3) few purplish granules that may be large and round or fine
than in the adult. It rises to an even higher level 12 hours after birth (average of 3. MYELOCYTE
22,800/mms) and declines gradually until the adult level is reached at about 21 - Size: 10-18 u; round or oval
years of age (average of 7,500/mmg). There tis no significant change after this point - Nucleus: indistinct thin nuclear membrane, coarse chromatin network with irregular
patches of pink staining parachromatin
1. Leucocytosis - increase above normal in the number of leucocytes in the peripheral blood - Nucleoli: absent or invisible; rarely more than one
a) Physiologic leucocytosis - Cytoplasm: moderate in amount; nucleus-to-cytoplasm ratio = 2:1; large granules
- caused by conditions that are physiologic such as exercise. which are purplish-blue in the early stage of development
b) Pathologic leucocytosis 4. METAMYELOCYTE (JUVENILE CELL)
- caused by disease; always accompanied by an absolute increase in one or another type - Size : 10~18u
of WBC making the terms: b1) neutrophilic leucocytosls (neutrophilia) - Nucleus : kidney-shaped; heavy nuclear membrane; coarse chromatin staining deep
b2) lymphocytic leucocytosis (lymphocytosis), purple; scanty parachromatin;
b3) monocytosis - Nucleolus: absent or invisible
b4) eosinophilia - Cytoplasm : fairly abundant; nucleus-to-cytoplasm ratio = 1.5:1; pink cytoplasm;
b5) basophilia granules are eosinophilic, basophilic or neutrophilic and are smaller and less
2. Leucopenia - a decrease below normal in the number of leucocytes in peripheral blood. - uniform in size than in the myelocyte
3. Leukemia - malignant increase in the number of leucocytes in the peripheral blood as well 5. BAND GRANULOCYTE (STAB; STAFF CELL
as in leucocyte producing organs. - Size: 10-15u
- Nucleus: sausage-shaped or band shaped or horse-shoe shaped with some areas of
Increased WBC (leucocytosis) 7. hypersplenism constrictions; coarse deep purple-blue chromatin; scanty parachromatin.
1. infections 6. replacement of marrow with malignant disease - Nucleolus: absent or invisible
2. inflammations E. prolonged use of sulfa drugs - Cytoplasm: abundant; pale blue or pink; nucleus-to-cytoplasm ratio = 1:2; with fine
3. in the afternoon 4. leucopenia of viral origin lilac granules (neutrophil), large blue-black granules (basophil), or brick red granules
4. after strenuous exercise 3. in measles (eosinophil) which have the appearance of small hollow spheres
5. after meals 2. in older people above 40 6. SEGMENTED GRANULOCYTE
S. during menstruation 1. in the morning - Size: 10-15 u
7. in newborns Decreased WBC (leucopenia) - Nucleus: segmented or lobulated nucleus connected by thin filaments; coarse and
dense deep purple-blue chromatin; scanty parachromatin.
NORMAL MATURATION SERIES: - Cytoplasm: abundant; light pink or blue; nucleus-to- cytoplasm ratio = 1:3; with
I. GRANULOCYTIC SERIES specific granules
 - Nucleus: round or oval and eccentric with coarse chromatin network; nucleoli may
be visible.
II. AGRANULOCYTIC SERIES - Cytoplasm: abundant, deep blue, non-granular with clear peri-nuclear zone.
LYMPHOCYTIC SERIES 3. PLASMACYTE (PLASMA CELL)
1. LYMPHOBLAST – either absent in normal bone marrow or present in small numbers - Size: 14-20u
- Size: 10 - 18u - Nucleus: small, oval and eccentric; condensed chromatin forms large granular
- Nucleus: round or oval, no indentation and centrally located; definite nuclear clumps that may be concentrated in the periphery and center of the nucleus
membrane; chromatin -in thin strands or stippled light red-purple; moderately creating the so-called " cartwheel" pattern. Parachromatin is pale pink.
abundant, sharply demarcated and light blue parachromatin. - Cytoplasm: dark blue, ovoid and somewhat fibrillary; non-granular with Russell
- Nucleoli: 1-2; small and pale blue bodies (secretory globules that represent protein secretion). If cytoplasm is
- Cytoplasm: homogeneous and moderately to heavily basophilic; sparse with completely filled with secretory globoid structures, the cell is called
nucleus-to- cytoplasm ratio= 5:1-7:1; bftentimes shows a lighter perinuclear zone; grape cell or Mott cell).
no granules. -
2. PROMYELOCYTE MONOCYTIC SERIES
- Size: 10-18 u; average smaller than lymphoblast 1. MONOBLAST
- Nucleus: round or oval, may be slightly indented; chromatin p more clumped than - Size : 14-18u
in the lymphoblast but is still relatively fine and dark red-purple; parachromatin not - Nucleus : round or oval; thin nuclear membrane; chromatin structure similar to
as well defined as in the lymphoblast; nor as smudged as in the adult lymphocyte. myeloblast but stains lighter; abundant, sharply demarcated and pale pink' or blue
- Nucleoli: usually one; round, blue and sharply outline parachromatin.
- Cytoplasm: more abundant; nucleus-to-cytoplasm ratio closer to 5:1; light blue to - Nucleoli: 1 to 2
medium dark blue; occasionally with azurophilic granules. - Cytoplasm: moderate; basophilic; no granulations in the blast stage.
3. LYMPHOCYTE 2. PROMONOCYTE
- Size: Small = 6 - 8 u, Medium = 8 - 14 u, Large = 8 - 18 u - Size : 14-18u
- Nucleus : round or oval; slightly or deeply indented; somewhat eccentric; heavy - Nucleus : moderately indented; thin nuclear membrane; fine, threadlike chromatin;
nuclear membrane large coarse clumps of chromatin, blending into sparse pale blue abundant parachromatin
to pink "smudged“ parachromatin - Nucleoli: 0-1
- Nucleoli: one may be seen in the larger cells; generally none - Cytoplasm: gray-blue; opaque, with very fine lilac granules (azurophilic dust which
- Cytoplasm: typically sky-blue, but may be clear and homogeneous with occasional indicate cell maturation)
azuruphilic granules in large and medium lymphocytes 3. MONOCYTE
- Size : 12-18 u
PLASMOCYTIC SERIES - Nucleus : indented or folded-over or bean-shaped; delicate;d pale staining; fine
- this is included here since plasma cells are known to arise from stimulated small strands of reticulated or lacy in appearance chromatin; abundant and distinct
lymphocytes. Plasma cells are not normally found in the peripheral blood; although parachromatin.
1-2% of plasma cell-like transformed lymphocytes may be seen under physiologic - Nucleoli: usually none, occasionally one
conditions. Plasma cells produce immunoglobulins. - Cytoplasm: light gray or gray blue, opaque; characteristic numerous, fine, dust-like
lilac granules
1. PLASMABLAST
- Size: 8-20u BRIEF DESCRIPTION-OF WBCS NORMALLY SEEN IN PERIPHERAL BLOOD:
- Nucleus: round or oval and eccentric, bluish purple fine chromatin network with I. GRANULOCYTES (produced mainly in BM)
some clumping; nucleoli difficult to discern. 1. NEUTROPHIL
- Cytoplasm: non-granular, blue, mottled and moderate in amount - 9 -15u, nucleus usually trilobulated (2 - 5 lobes) with abundant fine lilac pink
2. PROPLASMACYTE granules and coarse and clumped chromatin pattern.
- Size: 15-25u - normally produced in the bone marrow and released in response to various stimuli
 - circulate in the peripheral blood and are sequestered in various organs for variable extensive burns
periods before they are destroyed. 2° to_bacterial invasion
- average lifespan is 10 - 14 days (4 days in the bone marrow in the course of benign and malignant neoplasms
maturation, 3 - 4 days represent the total time spent in the circulating_blood, for
periods of only 2 or 3 hours at a time, and the remaining hours spent in 4. Acute hemorrhage
sequestration in .various organs 5. Acute hemolysis
- actively phagocytic, ingesting bacteria and other foreign particles both in vivo hemolytic transfusion reactions
and in vitro. After ingestion, bacteria are destroyed by proteolytic enzymes in the acute hemolytic anemia
chronic hemolytic anemia
neutrophil after which the neutrophil dies and fragmentst into particles that are 2. EOSINOPHIL
removed by fresh phagocytes. - 9-15u, nucleus usually bilobulated with coarse, clumped
- show active ameboid locomotion. When this movement- is directional, in response chromatin pattern.
to a chemical substance nearby, it is called CHEMOTAXIS. - cytoplasm contains large reddish-orange granules function remains obscure
- Chemotaxis is governed by products released at the site of inflammation as well as - recent work has shown that eosinophils actually have antihistaminic activity as
directly by bacteria. opposed to the old belief that eosinophils contributed to allergic reactions
- Leucocytosis-promoting factors (LPF) such as leucotaxine, may also be active in as carriers of histamine.
chemotaxis. In fact, leucotaxine has been shown to attract neutrophils and - capable of phagocytosis, in the course of which there is degranulation as in the
monocytes in vitro. neutrophils.
- Pathologic neutrophiliai or pathologic neutrophilic leucocytosis (increase in - Eosinophilia is observed in :
neutrophils) may be due to the following: - allergies pernicious anemia, sickle cell anemia
- skin disorders (non-allergic) polycythemia vera, Hodgkin's disease,
1. Acute infections by pyogenic bacteria and other organisms: - parasitic infections - hemopoietic disorders such as:
empyema, etc.
A. Localized or limited such as: - after splenectomy
cholera
abscesses
peritonitis
osteomyelitis 3. BASOPHIL
septicemia
acute appendicitis , - 9 - 15 u; with most varied-shaped nucleus (may be indented,bi-lobulated or stab
acute rheumatic fever
tonsillitisf - form); usually indistinct as it is covered with large purple or purplish black
B. Generalized such as:
meningitis granules. Its cytoplasm contains large purple or purplish black granules.
- rarest leucocyte in the peripheral blood.
2. Intoxications bacterial vaccines - functional importance is probably minor
A. Metabolic E. Reaction to parenterally given - contains heparin and large amounts of histamine
uremia protein A - Basophilia may be observed in the ff:
acidosis D. Reaction to parenterally given foreign - chronic myelocytic leukemia - ulcerative colitis
diabetes, etc. black widow spider - polycythemia vera - urticarla plgmentosa
B. Chemical C. Insect venoms - myeloproliferatlve dlsorders - erythroderma
epinephrine
lead Il. AGRANULOCYTES
mercury 1. MONOCYTE
arsenic - largest cell in the circulation probably the circulating counterpart of the blood tlssue
kerosene, etc. macrophage with large semi-indented or bean shaped nucleus with superimposed
brainlike convolutlons; nuclear chromatin retlculated or lacy in appearance
3. Tissue necrosis from any cause 7. Myelocytic leukemia - cytoplasm is abundant grayish or muddy blue in color often described as having a
myocardial infarction 6. Myeloproliferative disorders ground glass appearance
gangrene
 - functions: the cell s phagocytic function is related to its immunologic activity which - Lymphocytogenesis is a function of the lymph nodes, bone marrow, spleen, and
consists of 2 phases: scattered .lymphoid tissues in the body. The bone marrow is one of the largest
a. durlng the inductlon of lmmunlty when antigenic informatlon is lymphocyte-containing tissues in the body.
transferred to lymphocytes
b. during expression of cellular responses and the development of delayed
hypersensltivity Pathologic Lymphocytosis:
6. Brucellosis
- contains large amounts of lipase thus specifically endowed to combat bacilli with 1. Acute viral and Non-Viral infections
5. Pertussis
lipoid capsule such as Mycobacterium tuberculosis and leprae - infectious mononucleosis
4. Congenital syphilis
- infectious lymphocytosis
3. infectious hepatitis
- mumps, chicken pox
2. lymphocytic leukemia
Monooytosis may be observed in the ff: - german measles
- Monocytic leukemia - Gaucher’s disease
- Subacute Bacterial Endocarditis - Pulmonary TB ABNORMAL WHITE BLOOD CELLS
- Brucellosis - Trypanosomiasis 1. SMUDGE CELL (BASKET CELL)
- Typhoid - Collagen diseases - ruptured WBC with bare nucleus, few may be seen in normal blood smear due to
- Rickettslal lnfections - Chronic Ulcerative Colitis improper and forceful smearing
- Kala-azar - large numbers may be seen in leukemia and their presence may indicate increased
fragility of the cells or abnormal destruction of cells, not counted
2. LYMPHOCYTE 2. HYPERSEGMENTED NEUTRDPHILS
- size: small = 6-8u, medium= 8-14u, large 8-18u - also known as macropolycytes and PA Polycell
- presence of small, medium and large lymphocytes in the blood of the adult and the - usually larger than a normal neutrophil and nucleus has 5-10 lobes instead of the
predominance of medium and large lymphocytes in the blood of infants are normal 3 lobes
common observations. - usually present in pernicious anemia
- Lymphocytes larger than polymorphonpclear cells are classified as large lympho - the number of hypersegmented neutrophils in the differential white cell count
cytes and the smaller cells as small lymphocytes, morphologic difference lies mainly should be reported under miscellaneous white cells
in the amount of cytoplasm 3. VACUOLATED CELL
- most small lymphocytes are T-cells and most large lymphocytes are B lymphocytes. - with holes or vacuoles in the cytoplasm which are signs of degeneration and If seen
- In Wright s stained cell, the nucleus is deep purplish blue and is round to oval to in smears made from fresh blood it should be counted and reported under
kidney- shaped or deeply indented usual eccentrically located and occupies nine miscellaneous white blood cell
tenths of the cell diameter so that the sky-blue cytoplasm is hardly seen. - may also be found in normal blood smears if the smear is made from oxalated
- Functionally lymphocytes can be divided into B-lymphocytes derived from the bone blood which is over 2 hours old.. It may be seen in severe infections, chemical
marrow or the bursa equivalent organ in humans T-lymphocytes derived from the poisoning and leukemia.
thymus. Morphologically, T and B lymphocytes cannot be distinguished in Wright- 4. TART CELL
Giemsa stained smear. - a phagocytic WBC, usually a monocyte with 'engulfed nucleus of another cell
- Lymphocytes that lack both T & B cell markers are called null cells. - usually, the ingested nucleus retains its characteristic nuclear structure such as
- Small lymphocytes that are short-lived (3-4 days), upon antigenic stimulation, chromatin clumps, nucleoli or nuclear membrane
become immunoglobulin-producing B lymphocytes which are the precursor - seen in drug sensitivity
of plasma cells. B lymphocytes carry surface markers not present on T cells. 5. L.E- CELL
- Small lymphocytes that are long-lived (200-300 days) are usually the T lymphocytes - LE (lupus erythematosus) cell is a phagocytic WBC (usually a neutrophil) that has
helper cells. ingested an altered, homogeneous globular nuclear mass of a destroyed cell.
- The following distribution of T and B cells is found in the peripheral blood of healthy - nucleus of the phagocyte is compressed to one side, its appearance is distorted. The
adults: noncommitted null cells = 0.5 - 1% ingested nuclear material is homogeneous and redder than the usual color
B cells = 7 - 23% of unaltered chromatin.
T cells = 70 - 90%
 - LE cell formation is observed in 80 % of cases with disseminated lupus
erythematosus.
6. HAIR CELL (MAC or Malignancy Associated Changes)
- seen in 88 % of patients with cancer.
- recognized in granulocytes ( and monocytes) as thin, threadlike, pointed
excrescences arising from the nucleus.
- recognized in monocytes as small( (1u) inclusions surrounded by halos within the
cytoplasm.
7. REIDER CELL
- lymphocyte with notched, lobulated or segmented or cloverleaf like nucleus in
chronic lymphocytic or lymphatic leukemia.
- Bizarre leukemic myeloblasts with pseudolobulations are also called Reider cells.
8. TURK IRRITATION CELL
- common morphologic variant of lymphocyte showing immature nucleus and a
basophilic cytoplasm similar to that of a plasma cell.
- seen in viral infections as in German measles
9. FERRATA CELL
- phagocytic WBC associated with subacute bacterial endocarditis
10. DOWNEY CELL
- transformed, stimulated' or atypical lymphocytes with denser and more opaque
cytoplasm and increased number of cytoplasmic granules.
- cytoplasm stains heavily red with pyronine, a red basic dye staining RNA so they are
also called pyroninophilic cells
- Cytoplasm at times is vacuolated and foamy usually associated with viral infections
such as acute infectious mononucleosis
11. PYKNOTIC CELL
- nucleus becomes smaller and denser and the chromatin bridges between the
nuclear segments disappear leaving several small balls of dense chromatin.
- may be seen in infections and in aging cells.
12. TWINNING DEFORMITY (Tetraploid neutrophils with diploid nuclei)
- segmented neutrophils are twice the size of normal neutrophils, usually round
- apparent hypersegmentation of the twinning deformity is due to the presence of 2
nuclei in 1 cell and occurs in pernicious anemia and in myeloproliferative states
ANOMALIES OF THE WHITE BLOOD CELLS
1. PELGER-HUET ANOMALY
- inherited as a non-sex linked dominant trait characterized by the failure of the
nucleus of neutrophils to segment or lobulate so that cells may appear bilobed or
band forms.
- may be congenital (true Pelger-Huet anomaly) or acquired (pseudo Pelger-Huet
anomaly).
- congenital form is asymptomatic in the heterozygous state and lethal in the
homozygous form.
- acquired form may be seen in blood diseases such as chronic myelocytic leukemia,
acute leukemia, agranulocytosis, infectious mononucleosis, etcdisease. ~ I »
2. DOHLE-AMATO BODIES
- found in neutrophils as irregular, round or oval, blue staining cytoplasmic inclusions
that vary from the size of cocci to about 2.u in diameter.
- identified by' electron microscopy to be lamellar aggregates of rough endoplasmic
reticulum.
- found in severe infections, severe burns, exposure to cytotoxic agents and in other
toxic states but they disappear as the infection subsides.
- similar to and must be differentiated from May – Hegglin bodies.
3. ALDER - REILLY BODIES
- inherited as a recessive trait. characterized by the presence of larger than normal
coarse, dark, azurophilic granules (Alder's bodies& Reilly bodies) in the cytoplasm
of granulocytes as well as of lymphocytes and monocytes and bone marrow
precursor cells that cover the nucleus. `
- granules are larger than toxic granulations seen in infections.
4. CZEDIAK-HIGASHI SYNDROHE
- usually familial, usually fatal and affects children, male and female
- part of hereditary autosomal recessive syndrome that includes albinism, abnormal
skin pigmentation, and repeated infections, ultimately ending up in anemia,
neutropenia, thrombocytopenia and death
- neutrophils show large Peroxidase (+), Sudan, Black B (+), acid phosphatase (+)
inclusions that vary in size and color (blue to red) and somewhat resemble Dohle
5. MAY-HEGGLIN ANOHALY
- multiple or single inclusions (similar to Dohle bodies) in polymorphonuclear cells,
monocytes and rarely in lymphocytes.
- not related to infections but with abnormal giant platelets and thrombocythemia
- clinically normal, but about 25% have a tendency to bleed abnormally, (epistaxis,
purpura or hematuria).
6..JORDAN'S ANOMALY
- characterizedy by presence of fat containing vacuoles in granulocytes and
monocytes. May be seen in muscular dystrophy and ichthyosis
7. TOXIC GRANULATION
- cytoplasm may contain large basophilic dark-staining granules (toxic granules)
different from abnormal granules of Alder's anomaly and Czediak-Higashi syndrome
and from artifacts produced by poor staining.
- seen in severe infections, in chemical poisoning and in toxic states
8. GRANULOCYTIC ANOMALY IN DOWN’S SYNDROME
- Down's syndrome (Trisomy 21) is characterized by a series of abnormalities
involving the heart, soft tissues and nervous system
9. DRUMSTICK
- sex chromatin of polymorphs.
- solid nuclear appendage shaped like drumstick (measuring 1 - 2 u in diameter)
attached by a narrow segment to one of the main lobes of the nucleus of 1 -8%
 neutrophils of females. May also be seen in eosinophils and basophils but granules
of these cells may obscure it
10. BARR BODIES
- sex chromatin of somatic cells in females
11. AUER BODIES
- rod-like bodies which stain reddish purple in the cytoplasm of monoblasts and
myeloblasts in acute monocytic or acute myelocytic leukemia
LEUKEMIA
- neoplastic disease characterized by the purposeless, malignant proliferation of
hemopoietic cells in the bone marrow and other organs. These cells can be
expected to appear in the peripheral blood.
- The two blood pictures that would highly characterize leukemia
are: high WBC count and increase of immature forms.
Etiology
- etiology of leukemia is not known but has 4 overlapping approaches:
1.Epidemiologic
- some groups/races are more susceptible than others.
2. Leukemogenic effect of ionizing radiation
- ionizing radiation induces leugbmiai by causing chromosomal.aberrations, bhiefly
aneuploidy (wherein chromosome number deviates from the normal complement
of 46) as well as structural abnormalities such as fragmentation, dicentric
chromosomes, and ring chromosomes.
3. Role of viruses
- Viral etiology of leukemia can be said to be more likely than the others not because
there is much evidence to implicate viruses in human leukemia, but because
viruses are so definitely implicated in leukemia of lower animals.
4.Genetic (chromosomal) determinants
- most constant and characteristic changes involve chromosome 21
- cells from patients with chronic myelocytic leukemia show an abnormal
chromosome called the Philadelphia or Phl chromosome. This is a small
chromosome formed by deletion or translocation of a portion of the normal
chromosome 21
- cells from patients with Down's syndrome (mongolism) show an extra chromosome
(trisomy 21).It has been shown that leukemia is about 17x more frequent in
mongoloids than it is in normal children and this is probably an underestimate
because many mongoloids die at a young age.
Miller identifies five classes of persons with an exceptionally high risk of leukemia:
1) If one identical twin has developed leukemia, there is a probability that the other will
also develop pleukemia. This is not so for non-identical twins.
2) Individuals with an inherited tendency for chromosomal breakage, as in-Bloom's
syndrome, aplastic anemia of the Fanconi itype, ataxia-telangiectasia, and possibly other
genetically induced diseases
3) Individuals with_acquired chromosomal damage due to exposure to ionizing radiation
and to other leukemogenic agents such as drugs.
4) Individuals with extra chromosome as in Down syndrome and Klinefelter s syndrome
5) Siblings of leukemia children incidence is 4x greater than the normal
CLASSIFICATIONS OF LEUKEMIA
I. ACCORDING T0 DURATION OF THE DISEASE
1. Acute leukemla
- blood picture shows large number of immature cells (predominantly blast cells)
- short life expectancy of 6 months or less
- encountered in all age groups and most frequent type in children
- onset is marked by pallor, fever, purpura, malaise, bone pain, splenomegaly,
lymphadenopathy, and central nervous systemilnvolvement.
- Rapidly developlng severe anemla and thrombocytopenia are characteristic.
- Anemla is usually normocytic normochronlc but may also show a tendency towards
macrocytosis.
- Thrombocytopenia is constant feature and that the diagnosis of acute leukemia
should not be made in its absence.
- Bone marrow is diagnostic showing massive infiltration with blast cells
2. Subacute Leukemia
- blood plcture shows slightly differentiated or older cells
- life expectancy is from 6 months to 1 year
3. Chronic Leukemla
- blood plcture shows high number of mature or old cells
- life expectancy is from 1 year to several years
- gradual onset with general body weakness and easy fatiguability due to anemia.
- bones are tender to pressure due leukemlc infiltration, splenomegaly
II. ACCORDING TO WBC/mm3 IN THE PERIPHERAL BLOOD
1. Leukemic leukemia
- blood picture shows hlgh percent of young/immature cells (blasts)
- WBC ct usually higher than 15,000/mm3
2. Subleukemic leukemla
- blood plcture shows sllghtly dlfferentlated cells.
- WBC count is less than 15,000/mm3
3. Aleukemic leukemia
- blood picture shows high percentage of mature and normal cells
- WBC count is less than 15,000/mm3
III. ACCORDING T0 TYPES OF CELLS INVOLVED
1. Blast cell leukemia or stem cell leukemia
 - predominant cell is a blast, cannot be identified at the time of diagnosis b) Leukemic reticuloendotheliosis
2. Lymphocytic leukemia c) Histiocytic.medullary reticulosis (Robb-Smith)
- acute lymphocytic leukemia - predominance of lymphoblasts
- chronic lymphocytic leukemia - predominance of mature lymphocytes LEUKEMOID REACTION
3. Promyelocytic leukemia - Leukemoid Reaction is not a disease or diagnosis but a descriptive term to indicate
- preponderance of atypical progranulocyte containing large irregular azurophilic a blood picture in which the peripheral blood findings resemble those found in
granulation. leukemia, with the important difference that while leukemia is a neoplastic
4. Chronic myelocytic leukemia (CML) proliferation and malignant in nature, leukemoid reactions are benign
- predominance of neutrophilic, eosinophilic and basophilic types that are variants proliferations.
and need not be classified separately. Philadelphia (Phl) chromosome is present in - LRs may be due to infections, intoxications, tumors and acute hemorrhage.
70-90% of patients with CML. - LR mimics leukemia and may show the same quantitative and
5. Neutrophilic leukemia qualitative changes
- preponderance of neutrophilic myelocytes. - differentiated from leukocytosis in the sense that its WBC count is usually above
6. Eosinophilic leukemia 50,000/mm3 and its blood smear shows more immature cells. It is the presence of
- preponderance of eosinophilic myelocytes. immature cells as much as the high WBC count that gives the leukemoid blood
7. Basophilic leukemia - picture the appearance of leukemia.
- preponderance of basophilic myelocytes - As a rule, LR is not accompanied by the anemia or thrombocytopenia that is
8. Monocytic leukemia (occurs in two forms that are basically not related) common in leukemia.
- Naegeli type (myelomonocytic leukemia) - variant of myelocytic leukemia and may
resemble granulocytic leukemia. Cytologically, myelomonocytes show the nuclear CONDITIONS THAT MAY CAUSE LEUKEHOID REACTIONS:
features of monocytes and the cytoplasmic features of myeloid cells. I. Infections
- Schilling's type (histiocytic leukemia) - true monocytic leukemia since it is A. Myelocytic reactions
characterized by primitive cells having the same general features as a true blood 1. Pneumonia 6. Bubonic plague
monocyte and its tissue counterpart, the histiocyte. 2. Empyema 7. Septicemia
9. Plasmacytic leukemia and multiple myeloma 3. Endocarditis 8. Tuberculosis
- myelomas are neoplasms of proliferating plasma cells. They may present as solitary 4. Meningitis 9. Leptospirosis
tumors involving a bone (solitary myeloma), or as neoplasms involving the BM and 5. Diphtheria .
other organs (multiple myeloma).
10. Plasma cell leukemia B. Lymphocytic reactions
7. Tuberculosis
- terminal leukemic phase of multiple myeloma. 1. Whooping cough
6. Congenital syphilis
11. Mast cell leukemia 2. Chicken pox
5. Infectious lymphocytosis
- rarest; leukemic cell is the,tissue mast cell. 3. Mumps
- Mast cells are essentially tissue basophils in the bone marrow. They arise from 4. Infectious mononucleosis
basophils by a process of differentiation and replication
12. Erythremic myelosis (Di Guglielmo's disease) PLATELET STUDIES
- Pure erythroblastic proliferation. - non-nucleated, irregular in size (1~4 u), irregular in shape, lifespan of,4 - 9 days
- may progress to a mixed erythroblastic myeloblastic phase and terminate as an - 3 main functions: hemostasis, blood coagulation, clot retraction
acute myeloblastic leukemia 6. Acute leukemia
13. Erythroleukemia (Di Guglielmo's syndrome) INCREASED PLATELET COUNT 5. Idiopathic -
- erythroblastic and myeloblastic proliferation. 1. Polycythemia vera ”. 4. Lesions involving the bone marrow
14. Chronic neutrophilic leukemia 2. Myeloproliferative syndrome' 3. Infectious diseases
- preponderance of adult segmented neutrophils and band neutrophils. 3. Splenic vein thrombosis 2. Aplastic anemia ' ,
15. Leukemias of uncertain type 4. Postsplenectomy states 1. Pernicious anemia
a) Reticulum cell leukemia 5. Acute blood loss DECREASED PLATELET COUNT
6. Carcinomatosis - Nucleus : large, indented and polylobulated; coarse, heavily stained strands of
7. Nephrotic syndrome without uremia chromatin.
8. Chronic myelocyticwleukemia - Cytoplasm: intensely basophilic; filled with increasing numbers of azurophilic
9. Acute alcoholic hepatitis granules, sparing a thin peripheral ring that remains blue in color.
10. Ulcerative colitis 3. MEGAKARYOCYTE
11. Cirrhosis of liver - Size: 30-100 u (largest cell in the bone marrow)
- Nucleus: plump, multilobulated, indented, and sometimes multinucleated. Nuclei
THROMBOCYTOPENIA are arranged in chains or rings and may partially cover each other; chromatin in
- refers to increase in platelet count accompanied by a disease heaxv clumps.
- platelet count is significantly higher than normal. - Cytoplasm: produces blunt, smooth, pseudopodia-like projections with aggregates
- term used for secondary cases (there is a coexistent of azurophilic granules surrounded by pale halos. These structures will give rise to
- disease that may be accompanied by an elevated platelet count platelets at the periphery of megakaryocyte. The line of cleavage goes thru the
- Examples: splenic vein thrombosis, postsplenectomy state, acute blood loss, hyaline cytoplasm of the halo, so that the granular mass becomes the platelet
various anemias, carcinomatosis, nephrotic syndrome without uremia, 4. PLATELET or THROMBOCYTE
- detached cytoplasmic fragmentations of, mature megakaryocytes
THROMBOCYTHEMIA - Size: 1-4u ( young platelets may be 2 - 3 times larger)
- platelet count is >1,000,000 or >1 million/mm3 - Nucleus : none
- term used for primary cases (no other cause for the increased platelet count exists - Cytoplasmr In Wright-Giemsa stained smears, platelets appear as small, bright,
azure, rounded or elongated bodies with a delicately granular structure. Each
THROMBOCYTOPENIA platelet consists of a central group of azurophilic granules (chromomere) and a
- subnormal number of platelets in the circulating blood and is the most common surrounding light blue hyalomere.
cause/of abnormal bleeding.
- It results from: Morphology of platelets varies greatly depending on:
1. deficient platelet production 1). the methods by which they are examined
2. accelerated platelet destruction 2). the anticoagulant
3. abnormal distribution or pooling of the platelets within the body. 3). and the temperature .

NORMAL MATURATION SERIES PLATELETS IN HEMOSTASIS

The megakaryocytic series is characterized by some unusual features:
a). The youngest cell type (megakaryoblast) is generally much smaller than the adult
(megakaryocyte).
b). Repeated- nuclear division without cellular division takes place, so that instead of the
usual diploid nucleus in other cells the megakaryocyte has a polyploid nucleus.
c)., The functional end product is a cytoplasmic fragment of the mature megakaryocyte
1. MEGAKARYOBLAST
- Size : 10-30 u in diameter (smaller than its mature form, the megakaryocyte but
larger than all other blast cells).
- Nucleus : single, large, oval or indented with loose chromatin structure and delicate
nuclear membrane.
- Cytoplasm: scanty, bluish, patchy and irregular ring formed around the nucleus;
periphery with cytoplasmic projections and pseudopodia-like structures.
2. PROMEGAKARYOCYTE (Basophilic Megakaryocyte)
- Size : 20-50 u (larger than megakaryoblast)
There are at least 3 physical changes that can be observed in platelets which make platelets
very useful in hemostasis:
1. Adhesiveness which is the property of sticking to other surfaces to bacteria and to other
particles
2. Aggregation which is the clumping of platelets by sticking to each other
3. Viscous metamorphosis in which the individuality of the platelets is lost in the formation
of large amorphous hyaline-like clumps
PLATELETS IN BLOOD COAGULATION
Platelets function in blood coagulation because they contain various proteins or lipoprotein
substances designated as "platelet factors”. These factors are designated by Arabic
numerals differentiate them from coagulation factors which are designated as Roman
numerals.
The platelet factors are as follows.
1. Platelet factor 1 - Accelerates prothrombin conversion
2. Platelet factor 2 - Facilitates the interaction of thrombin and fibrinogen hence the
 fibronoplastic platelet factor.
3. Platelet factor 3 - thromboplastic factor necessary for the generation of plasma
thromplastin
4. Plat elet factor4 - Anti-heparin factor
PLATELETS IN CLOT RETRACTION
- Clot Lretraction. is largely dependent on the presence and action of platelets which
contain a specialized actomyosin-like contractile protein known as thrombosthenin.
- The contraction of thrombosthenin underlies the phenomenon of clot retraction. `
REASONS WHY PLATELETS ARE HARD T0 COUNT
1. Platelets adhere on foreign surfaces (like skin and dried walls of pipets).
2. Platelets easily disintegrate.
3. Hard to differentiate from debris.
4. Platelets are unevenly distributed in the blood because they tend to clump.
QUALITATTVE DISORDERS OF PLATELET FUNCTION
I. HEREDITARY-DISORDERS OF PLATELET FUNCTION
1. Primary Forms
A. Thrombasthenia (Glanzmann's disease, Glanzmann Naegeli's disease)
- Due to a general defect in the platelet membrane. dysfunction
- Thrombasthenic platelets are unable to adsorb various cationic proteins, including
factor XIIa, IgG & IgM immunoglobulins, and fibrinogen.
- Fibrinogen appears to be an important cofactor for ADP-induced platelet
aggregation and may be involved in, platelet adhesion to fibrin, a phenomenon
essential for clot retraction. "
B. Deficient release reaction (Storage pool disease, thrombopathia, Portsmouth synd.)
- inability of affected platelets to undergo a normal release reaction when
physiologically stimulated. In this disorder, platelets fail to release normal amounts
of endogenous ADP, not because of abnormalities in the pathways that supply
energy to the release mechanism but because of def. in the available stored ADP.
C. Thrombocytopathy (or Thrombopathy)
- familial defect due to deficient platelet factor 3 (PF 3) content or release.
2. Varieties with thrombocytopenia
A. Bernard-Soulier syndrome
- inherited as an autosomal recessive trait.
- rare disorder, morphologic abnormalities of the platelets are the most consistent
and striking feature of the disorder. Giant platelets with sizes up to 8 u
with relatively dense granulomere and described as "lymphocytoid" are observed.
B. Thrombopathic thrombocytopenia
- resembles Bernard-Soulier syndrome in most respects, but is inherited as an
autosomal dominant trait.
- Platelet counts of 20,000-80,000/mm3 with numerous "giant" platelets are
observed, common in certain populations of Mediterranean descent
- associated with other hereditary or congenital syndromes such as
monoclonal gammopathy, autosomal dominant nephritis and deafnes
C. Wiskott-Aldrich Syndrome
- inherited as a rare sex-linked recessive trait, characterized by qualitative
abnormalities of platelet function.
- Platelets from affected persons are smaller than normal and reveal deficiencies in
the number of alpha granules and other ultrastructural abnormalities.
- syndrome 'is icharacterized by a triad of clinical findings:
1. Recurrent infections with a variety of organisms, due to selective
deficiencies of cellular and humoral immunity.
2. Moderate to severe chronic thrombocytopenia.
3. Eczema
3. Miscellaneous Hereditary Forms
A. Hereditary afibrinogenemia
- since fibrinogen is required for ADP-induced platelet aggregation, severe
deficiencies of this factor produce a secondary abnormality in platelet
function manifested in vitro by deficient platelet aggregation with low
concentrations of ADP, markedly defective adhesiveness of platelets to
glass and variable abnormalities in PF-3 activity.
B. Heritable Disorders of Connective Tissue and Mucopolysaccharidoses
- abnormally giant platelets with abnormalities in ADP release have been described in
patients with various heritable disorders of connective tissue (e g Marfan syndrome,
ostsogenesis imperfecta and Ehlers Danlos syndrome) and in those with
mucopolysaccharidoses
ll. ACQUIRED DISORDERS OF PLATELET FUNCTION
1. Drug Induced Platelet Dysfunction
- Many chemically and biologically active substances in common use are shown to
inhibit platelet function when used in therapeutic concentration The abnormalities
produced by the drugs listed below are quite variable but resemble in most
respects those associated with hereditary deficiency of the platelet release
reaction.
- Examples of such drugs:
= Anti-inflammatory agents like aspirin, phenylbutazone sulfinpyrazone
and indomethicin
= Antidepressants like chlorpromavine. Promethazine, reserpine,
imiprimine, qamytryptilene and congeners
= Adrenergic blocking agents like .phentolamine, dihydroergotamine
= Miscellaneous drugs like ethanol, clofibrate, dextran and similar
 polymers, papaverine, carbenicilin d. Congenital, neonatal, or familial thrombocytopenic syndromes
2. Uremia - Wiskott-Aldrich syndrome - May-Hegglin anomaly
- Uremia was one of the first acquired thrombopathies to be described
- platelet dysfunction appears to be in the release reaction
- Bleeding time is variably prolonged and-prothrombin consumption and PF~3
activity are usually deficient
3. Disorders Involving the Hematopoietic System THROMBOCYTHEMIA
A. Paraproteinemias (macroglobulinemia multiple myeloma and others) A. Thrombocythemia hemorrhagica
B. Hemorrhagic (ldiopathic) Thrombocythemia, Myelofibrosis & Polycythemia vera - primary disease characterized by a persistent increase in platelets and frequently
C. Miscellaneous (acute and chronic leukemias, ITP, others) associated with bleeding tendency.
B. Secondary thrombocythemia
QUANTITATIVE PLATELET ABNORMALITIES - Seen in polycythemia and in chronic granulocytic leukemia.
A. Due to_excessive destruction or loss
1. Due to immunologic or hypersensitivity mechanisms STUDY OF BLOOD COAGULATION AND HEMOSTASIS
- Idiopathic Thrombocytopenic Purpura (ITP) – platelet destruction as_a result of an COAGULATION
immunologic process. - solidifying of fluid blood (whole blood or plasma) brought about by the different
2. Hypersensitivity to certain. drugs coagulation factors
- apronalide, guinine, quinidine. - formation of a visible coagulum, which is the physical manifestation of fibrin
3. Due splenomegaly and increased sequestration in the spleen formation, represents only the end result of an intricate series of reactions that
- Conditions above produce thrombocytopenia by sequestering normal and undamaged involve a number of coagulation factors.
platelets in the spleen. An enlarged spleen usually traps many normal cells and these - coagulation factors have an international standard nomenclature
cells are eventually destroyed due to normal lytic splenic mechanisms acting for a long
period of time on cells that are sequestered. NOMENCLATURE & SYNONYMS FOR COAGULATION FACTORS
4. Due to sequestration but not in spleen
- Sequestration of platelets in tumors and trapping of platelets in the fibrin network I Fibrinogen ---------------------------------
Deposited throughout the vascular system in the syndrome characterized by diffuse II Prothrombin ---------------------------------
Intravascular coagulopathy III Tissue factor Thromboplastin,Thrombokinase
5. Due to mechanical destruction IV Calcium ions ---------------------------------
- Extracorporeal circulation of blood produces moderate destruction of platelets. The VI Proaccelerin Labile factor, Thrombogen, Accelerator globulin (ACG)
cause is primarily mechanical (trauma, adhesion to tubing, artificial cardiac valves, etc). VII Proconvertin Stable factor, Serum Prothrombin Conversion
6. Due to miscellaneous factors Accelerator (SPCA), Autoprothrombin I,
- massive hemorrhage i cothromboplastin
- massive transfusion of platelet-poor blood VIII Antihemophilic Antihemophilic globulin (AHG), Anti- hemophilic
- chronic alcoholism factor (AHF) factor A, Platelet cofactor 1, Thromboplastinogen
IX Plasma Thromboplas- Christmas factor, Antihemophilic factor B, Platelet
B. Due to deficient production tin Component (PTC) cofactor 2, Autoprothrombin II
1. Bone marrow suppression of thrombocytopoiesis X Stuart-Prower factor Prower factor, Autoprothrombin III
a. Potentially myelotoxic drugs such as antifolates, nitrogen mustard, chloramphenicol, XI Plasma Thromboplas- Antihemophilic factor C
gold salts, DDT, organic chemicals, etc. ` tin Antecedent (PTA)
b. Physical and animal agents XII Hageman factor Glass factor, contact factor
- ionizing radiation - artificial fever XIII Fibrin stabilizing Fibrinase, Laki-Lorand factor
- heat stroke - burns factor-(FSF)
- insect bites
c. Bone marrow replacement of abnormal cells (metastatic tumor, lymphomas, etc.) GENERAL CHARACTERISTICS OF THE COAGULATION FACTORS
1. FIBRINOGEN
- protein clotted by thrombin in the formation of fibrin, plasma concentration is 250 -
400 mg/100 ml. formed in the liver and possibly also in the RES.
2. PROTHROHBIN ' `
- proenzyme, the precursor of thrombin and functions in thecommon pathway of
coagulation, plasma concentration is 10 - 15 mg/100 ml. '
3. THROMBOPLASTIN (TISSUE FACTOR) . '
- substance that, in the presence of. calcium ions, brings about the conversion of
prothrombin to thrombin. '
- a) tissue thromboplastin: from tissue extracts and it is the prothrombin activator in
the extrinsic system
- b) plasma thromboplastin: prothrombin activator in the intrinsic system
4. CALCIUM
- normally exists in the blood in an ionized form and combines with plasma proteins
and lipids, needed in prothrombin conversion and plasma thromboplastin
generation.
5. THROMBIN'
- active agent (enzyme) that clots fibrinogen. Its action is so powerful that it can clot
several hundred times its weight of fibrinogen. As.an enzyme, it splits the arginyl-
glycyl bands at the N terminal of the fibrinogen molecule to form fibrin.
6. FACTOR V
- called "accelerator" because it speeds up the conversion of prothrombin to
thrombin in the presence of Ca ions .and tissue thromboplastin in the common
pathway of coagulation, occurs in the plasma of all normal persons and is
synthesized in the liver
7. FACTOR VII
- needed in the conversion of prothrombin to thrombin but only in the extrinsic
system. Chief difference between factors V &'VII is that factor V can be obtained
from plasma, while VII is present in plasma but is more active in serum.
- plasma concentration is 3 mg/100 ml
8. FACTOR VIII
- the antihemophilic factorrequired for adequate evolution of plasma thromboplastin
(intrinsic pathway), usually absent or with molecular abnormality in hemophilia
and in von Willebrand's disease.
- trace protein with a plasma concentration of 1 ug/100 ml
9. FACTOR IX
- necessary for intrinsic thromboplastin generation.
- plasma concentration is trace amounts only
10. FACTOR X
- proenzyme that is important in the formation of prothrombinase in the common
pathway of coagulation. It is activated by the products of both the. intrinsic and
extrinsic pathways. Plasma concentration is 1.2 mg/100ml
11. FACTOR XI
- proenzyme that is essential in the intrinsic pathway of coagulation.. Little is known
regarding its biochemistry although it is stable in plasma and serum.
12. FACTOR XII
- activated by contact with foreign surfaces, and initiates the intrinsic pathway of
coagulation. It is also involved in the activation of fibrinolysis and in the plasma
kinin system, plasma levels of this factor usually normal even in severe
liver disease.
13. FACTOR XIII
- the enzymatic form of Factor XIII acts in the common pathway of coagulation where
it forms stabilizing covalent bonds with fibrin strands. It is also involved in wound
healing.
Vitamin K-dependent coagulation factors are the following:
4. Factor X
1. Factor II 3. Factor IX
2. Factor VII
HEMOSTASIS
- process by which spontaneous or induced hemorrhage is stopped.
- spontaneously arrests the flow of blood from vessels carrying blood under pressure
- entire mechanism by which bleeding from an injured blood vessel is spontaneously
controlled and stopped
Mechanical Hemostasis
- stops bleeding with the use of tourniquet; surgical or a first aid problem.
Physiologic hemostasis
- brought about by interaction of several factors, hemorrhagic disorders due to
deficient physiologic hemostasis are a laboratory concern.
Three Aspects of Hemostasis ` `
1. Extravascular
- includes physical constriction of injured skin and tissues resulting in the release of
tissue juice which contains tissue thromboplastin
2. Vascular Aspect
- includes, constriction of injured blood vessel brought about by reflex constriction
and by the serotonin released by disintegrated platelets
3. Intravascular Aspect _ A '
- physico-biochemical changes undergone by platelets and the interaction of the
different coagulation factors.