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
 (totipotent stem cell)
Rubriblast prorubricyte rubricyte metarubricyte reticulocyte ERYTHROCYTE
Megakaryoblast promegakaryocyte megakaryocyte THROMBOCYTE
also known as: hemocytoblasts (Maximow & Bloom), hemohistioblasts (Ferrata & Di
Guglielmo), lymphoidocytes (Pappenheim) and hemopoietic reticulum cells.
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 hemopoiesis

Myeloblast promyelocyte myelocyte metamyelocyte stab (band or staff cell) I. Mesoblastic Stage
Segmenters (NEUTROPHIL, EOSINOPHIL, BASOPHIL) - The chief site of hemopoiesis is in the yolk sac.
- The fetus is 2.25 to 5'mm in size.
Monoblast promonocyte MONOCYTE
0n the second week of fetal life there is the formation of blood islands wherein the primitive
Lymphoblast prolymphocyte LYMPHOCYTE 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
Plasmoblast proplasmocyte PLASMOCYTE transformation of peripherally located mesenchymal cells into endothelial cells. Thus, the
primitive blood cells are now enclosed in endothelial-lined spaces. Upon further differentia-
2. Polyphyletic or Dualistic Theory tion into cells .known as erythroblasts, and with the secretion of plasma, blood is established
- this theory, believes that there is a separate and distinct stem cell compartment for as a definitive somatic component. Leucocytes and megakaryocytes are seldom found during
each of the blood cells. This was suggested by Sabin and co-workers and was concurred the earliest phase of the mesoblastic stage.
by Naegeli, Schilling & Downey. On the 9th wek of fetal life, the predominant cell.is the Primitive Erythroblast (PE), a large
cell measuring from 15-25 u in diameter with coarse, clumped chromatin in the nucleus,
RETICULOENDOTHELIAL CELL several nucleoli and homogenous basophilic cytoplasm. The PE elaborates hemoglobin to
Hemohistioblast take care of the oxygen needs of the fetus, after which PE dies cut_and is' replaced by
definitive normoblastic cells that do differentiate into adult erythrocytes.
Rubriblast prorubricyte rubricyte metarubricyte reticulocyte
ERYTHROCYTE II. Hepatic Stage
- This starts on the second month; embryo is 5 to 7 mm in size.
Myeloblast promyelocyte myelocyte metamyelocyte stab segmenters - On the 3rd month of fetal life, the liver becomes the chief site of hemopoiesis. It
(NEUTROPHIL, EOSINOPHIL, BASOPHIL) reaches peak activity during the 3rd or 4th month and remains active until a few
months before birth.
Megakaryoblast promegakaryocyte megakaryocyte THROMBOCYTE
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,
Tissue hemohistioblast 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
Monoblast promonocyte MONOCYTE continues until the end of normal gestation and lymphopoiesis continues throughout life.,

Lymphoblast prolymphocyte LYMPHOCYTE
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
primitive cells. The primitive cells have large nucleus with spongy chromatin, one or two
nucleolar chromatin condensations, and a deeply basophilic cytoplasm. The primitive cells are
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
- This is the final phase wherein the red bone-marrow assumes the chief role in
hemopoiesis.
- It starts on the 5th month of fetal life and increases during the last trimester and at
birth the marrow is and then remains, the chief site of normal hemopoiesis.
Extramedullary hemopoiesis is negligible except for lymphopoiesis in the spleen, lymph - nuclear chromatin rich in DNA: young cell
nodes and thymus. **nucleoli with RNA: Feulgen negative
NATURE OF CHROMATIN- most important criterion to det age of rbc
The first appearance of each cell type in the peripheral blood corresponds to maximal - chromatin strands coarse and clumped: mature cell
hemopoietic activity in the parent tissue. Early in fetal life many nucleated RBC are present. - decreased number of nucleoli: mature cell
The number gradually decreases until at birth a normal infant never shows more than 10 per - changes in shape
100 leucocytes. Non-nucleated rbcs actually increase after which granulocytes, then - more lobulations: more mature cell
lymphocytes and finally monocytes can be recognized in the peripheral blood. 3. Reduction in cell size
- smaller: more mature cell
In normal infant and in adult, the bone marrow is the only site of erythropoiesis, myelopoiesis
and thrombopoiesis. In general, newborn infant has little marrow reserve. Increased BLOOD VOLUME
production, if needed, must take place at extramedullary sites (liver, lymph nodes, spleen, Blood volume determinations are important in the detection and treatment of fluid and
thymus). electrolyte imbalances. Direct measurements of total blood volume have shown that blood
makes up about 7 to 8% of the total body weight. Since direct measurements depend on
In the adult, the bone marrow represents a weight of tissue at least equal to the weight of the complete exsanguination, most of available data refer to animal experiments. Laboratory
liver. Normally, only about one-half of the total volume is active but even so an estimated 900 methods, therefore, for determining blood volume, plasma volume and total RBC mass
billion rbcs are produced daily. Under physiologic conditions, hemopoietic needs are met by are necessarily indirect.
mitotic division of the young cells of the marrow (homoplastic hemopoiesis). In case of a) For Plasma Volume determination
increased requirements, there is mitotic division as well as - IV administration of a foreign substance which dilution in the plasma allows
multiplication of younger precursors (heteroplastic hemopoiesis). measurement of fluid volume:
1. Evan's blue dye
Dyspoiesis - profound defect in the maturation of rbc, wbc and platelets. 2. Congo red dye
3. I131 labeled human serum albumin (RISA)
HOW CELLS ARE RELEASED FROM BONE NARROW INTO THE b) For Packed Cell Volume
CIRCULATION: - administration of a substance that attaches to the erythrocytes and gives a
1. RBC measurement of erythrocyte mass:
- hypoxia and erythropoietin are the factors that regulate the rate of production 1. Cr51
of new erythrocytes in the bone marrow as well as the release of these cells into 2. P32
the circulation. 3. radioactive Iron
2. WBC
- presence of chemotoxins in the blood will chemically tract WBC to go out into the Normal Values Male Female
circulation by the process known chemotaxis. Total Blood Volume (ml/kg) 76 (+ or - 8) 68 (+ or 6)
- Chemotaxis is a directional locomotion in response to a chemical substance nearby Plasma Volume (ml/kg) 42 (+ or - 5) 40 (+ or 4)
3. Platelets Packed Cell Volume (ml/kg) 35 (+ or - 4) 28 (+ or 3)
- produced and released by a shedding of megakaryocytic cytoplasm
Decreased Blood Volume Increased Blood Volume
PRINCIPLES OF NORMAL CELL MATURATION 1. loss of whole blood 1. during excessive fluid intake
1. Cytoplasmic differentiation 2. loss of rbc 2. during blood transfusion
- loss of basophilia (more basophilia due to cytoplasmic RNA means less mature cell) 3. loss of plasma / water 3. during IV injection of fluids
- cytoplasmic granules (more granules means mature cell) Terms
- elaboration of hemoglobin for RBC (no hemoglobin means younger cell) 1. Normovolemia - normal blood volume 3. Hypervolemia - increased blood volume
2. Hypovolemia - decreased blood volume 4. Oligemia - total reduction of blood volume
2. Nuclear maturation
- structure and cytochemistry BLOOD COLLECTION
- round or oval nucleus: young cell The basis of hematologic techniques is correct collection of blood sample and attention to
- large nucleus/cytoplasm ratio: young cell. precise methodology. lf the blood sample is not collected with proper attention to detail, the
whole hematologic examination is put into question. puncture site.
5. The values for red blood cell count, hematocrit, hemoglobin and platelets are lower in
Blood examination should be performed in accordance with the following general guidelines: capillary blood, but higher white blood cell count by as much as 1,000/mm as compared
As much as possible, blood examinations should be done in the morning, in the fasting to venousblood.
patient, before the usual breakfast time, particularly so if values to be determined are subject
to diurnal variations such as all chemical determinations, notably serum iron and blood sugar. VENIPUNCTURE
Any repeat examinations should be done in the morning of the following day. Heavy meals as - easiest and most convenient method of obtaining enough volume of venous blood
well as prolonged fasting can lead to appreciable leucocytosis. suitable for a variety of tests.
- three factors are involved in a good venipuncture:
The 2 general methods of collecting blood for haematological studies are: a) the venipuncturist
a) Skin puncture b) Venipuncture b) the patient and his veins
c) the equipment
SKIN PUNCTURE
- used when only small quantities of blood are required Two methods of collecting blood by venipuncture:
- collection of blood from puncture made on skin 1. Syringe Method
- blood obtained is known as: capillary blood 2. Vacuum Tube Method (Evacuated tube method)
peripheral blood Advantages over the Syringe method:
arteriolar blood a) requires no prior preparation as it is a prepackaged sterile unit.
b) offers a wider range of tube size and contained anticoagulants.
Sites of puncture: Sites to avoid: c) safer method of blood collection as samples are taken directly into
1. margin of earlobe 1. inflammed and pallor areas labeled tube
2. palmar surface of the finger 2. cold and cyanotic areas d) avoidance of syringe breakage.
3. plantar surface of heel and big toe 3. congested and edematous areas e) disposable
4. scarred and heavily calloused areas
Anticoagulants in vacuum tubes:
Advantages of skin puncture - finger: Advantages of skin puncture - earlobe 1. Pink stopper - no anticoagulant; used for tissue or blood culture.
1. easily accessible to the operator 1. Less pain (less nerve endings) 2. Red stopper - no anticoagulant; used for tests which require serum such as chemistry and
2. easy to manipulate. 2. More free flow of blood (thin skin) .serological exams.
3. ideal for peripheral blood smears 3. Less tissue fluid contamination (less muscle) 3. Amber stopper - no anticoagulant; used for blood lead determination.
4. less intimidating. 4. Ideal when searching for abnormal cells 4. Yellow stopper - no anticoagulant; used for bacterial culture and unknowns; can be
(histiocytes in bacterial endocarditis) Incubated or autoclaved.
5. Black stopper - 0.5 ml of 0.1M sodium citrate and collects 4.5 ml of blood for
Disadvantages of skin puncture: prothrombin time determination.
1. Less amount of blood can be obtained 6. Black stopper - 1 ml of 3.8% sodium citrate solution and collects 9ml of blood for
2. Additional and repeated tests cannot be done. coagulation tests requiring plasma.
3. Blood obtained by skin puncture lyses easily. 7. Blue stopper - 1 ml of 3.8% sodium citrate and collects 4 ml blood for sedimentation rate
determination, Westergren method.
Things to remember in doing skin puncture: . 8. Blue stopper - dry mixture of potassium (4 mg) & ammonium oxalate (6mg) and collects
1. Puncture should be 2.5 to 3 mm deep so as to hit the capillary bed, thus, ensuring free 5ml for blood cell counts
flow of blood. 9. Gray stopper - lithium oxalate; for blood chemistry determinations
2. Pressure and squeezing should be avoided to minimize a mixture of blood with tissue fluid 10. Green stopper - heparin 286 U.S.P. sodium and collects 15 ml of blood for determination
which will affect the accuracy of the tests. of serum iron concentration and total iron-binding capacity. Also for
3. The first drop of blood is usually discarded since it contains tissue fluids and other foreign special blood tests like arterial blood gas and research studies.
materials like dead epidermal cells. 11. Lavender stopper - 0.06 ml of l5% ethylenediaminetetraacetic acid (EDTA)and collects
4. When collecting blood for hematologic tests, the punctured finger must be wiped dry 7 ml of blood for blood cell counts and hematologic
after each test since platelets will begin to clump immediately in the blood at the examinations.
Sites of Venipuncture:
In newborn infants up to 18 months old:
1. external jugular vein
2. temporal vein (scalp vein)
3. superior longitudinal sinus
In children 3 years old up to adult life:
1. wrist vein
2. veins on dorsal of hand and fingers
3. veins on the antecubital fossa
III. General delayed complications:
1. Serum hepatitis - viral infection characterized by yellow coloration of the skin and eyes as
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
the use of contaminated lancets/needles.

In older children 18 months to 3 years old: 2. AIDS (Acquired Immunodeficiency Syndrome) caused by HIV virus.
1. femoral vein - Two ways by which it is acquired:
2. long saphenous vein 1. through blood and its by-products
3. popliteal vein 2. sexual contact with infected individual
4. ankle vein - incubation period may be from 5 to I5 years.
- no known cure yet
Advantages of Venipuncture:
1. Large amount of blood can be obtained for a variety of tests. Sample can be divided and ANTICOAGULANTS
treated as the prescribed test demands prescribed investigations demand. Anticoagulants are usually chemical preparations added to the blood to prevent clotting. The
2. Additional and repeated tests can be done. correct choice and amount of anticoagulant are very important in making sure that the correct
3. Fastest method of collecting samples from a large number of patients. sample is prepared for a test. An insufficient amount may lead to partial clotting while too
4. Blood can be transported to the laboratory and stored for future use. much liquid anticoagulant dilutes the blood sample. The incorrect choice of anticoagulant
5. Blood collected is ideal for blood chemistry determinations. may lead to distortion of cells.

Disadvantages of Venipuncture: I. OXALATES

1. Requires more time and skill on the part of the operator. - prevent coagulation by combining with calcium to form an insoluble calcium oxalate salt.
2. Requires more equipment. 1. dried Potassium Oxalate
3. More complications that may arise. - distorts wbc and shrinks rbc
4. Hard to do on infants, children and obese individuals. - not ideal for ESR, hematocrit, blood smears and blood K determination
- used at a concentration of 1-2 mg per ml of blood
Complications in Venipuncture: - dried sodium or lithium oxalate may be substituted for potassium oxalate
I. Local immediate complications -
application of tourniquet. 2. dried Ammonium & Potassium Oxalate
2. Failure of blood to enter syringe as in: (balanced oxalate, double oxalate, Winthrobe's solution, Paul-Heller's solution)
a. collapsed vein which may be due to
- nervousness Stock solution - dried Ammonium & Potassium Oxalate
- excessive pull of plunger of the syringe 1.2 gm of ammonium oxalate (3 parts)
b. hitting the vein through and through 0.8 gm of potassium oxalate (2 parts)
c. hitting just the wall of the vein (as in sclerotic and movable veins) 100.0 ml distilled water A
3. Circulatory failure - sudden stop or decrease of blood flow due to nervousness or shock.
4. Fainting or syncope - due to sudden decrease of blood supply to the brain brought about - Ammonium oxalate swells rbc, Potassium oxalate shrinks rbc. However, when used
by nervousness or shock. together in a proportion of 3:2. they form a balanced action on rbc (no significant
shrinkage or enlargement)
II. Local delayed complications: - ideal for ESR, hematocrit and blood cell counts
1. Hematoma - inflammation and discoloration of surrounding tissues due to extravasation - not ideal for blood chem. determinations such as BUN, uric acid and K determination
of blood brought about by trauma. since it will increase the values.
2. Thrombosis of the vein - formation of clot at the site of puncture due to trauma, - not ideal for blood smears because the anticoagulant causes:
3. Thrombophlebitis - inflammation of vein at the site of puncture wherein a thrombus is a) nuclear degeneration of leucocytes;
present. b) cytoplasmic vacuolation of granulocytes;
 c) pseudolobulation and clumping of agranulocytes Distilled Water - 100 ml
d) artifact formation in nuclei of lymphocytes and`monocytes;
e)phagocytosis of oxalate crystals Advantages of Sequestrene
1. Dipotassium salt is preferred due to its solubility
- used at 5ml (dried-to powder form) per 6 ml blood 2. Used in many hematologic tests even if blood has stood for many hours
- decomposes at high temperature (>8O oC) from oxalates into carbonates 3. Blood smears are well done, leucocytes and erythrocytes are morphologically well
preserved even after 3 or 4 hours
II. CITRATES 4. Prevents surface adhesion and clumping of RBC, WBC and platelets
- prevent coagulation by combining with calcium in a non ionized form; 5. Prevents formation of artifacts even upon long standing
- 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. IV. HEPARIN
- prevents coagulation by acting as anti-thromboplastin and anti-thrombin
1. 3.8% Na citrate - complex acidic mucopolysaccharide
- used in forms of disodium and trisodium citrates for clotting mechanism and ESR. - best natural anticoagulant
- Formula: - best anticoagulant for the minimal hemolysis of red blood cells
Trisodium citrate - 1.32 gm - ideal for ESR, hematocrit, RBC, osmotic fragility test and hemolytic disease of the
Citric acid - 0.48 gm newborn (HHN) cases
Dextrose - 1.40 gm - also used for special blood chemistry determinations such as pH determination and
Distilled water - 100.0 ml blood gas analysis
- 1 part.of 3.8% Na citrate added to 4 parts of blood can be used for ESR determination, - less toxic than Na citrate therefore it is often used for open heart surgery and exchange
Westergren method transfusion wherein large quantities of blood are given rapidly
- 1 part of- 8.8% Na citrate added to 9 parts of blood can be used for coagulation - available as sodium, calcium or ammonium salts
Studies using plasma. - preferred for electrolyte determination is ammonium salt
- used at a concentration of 0.1 - 0.2 mg/ml of blood or 0.1 ml of liquid heparin (1,000
2. Acid Citrate Dextrose (ACD) Solution units) per 10 ml blood.
- preserves erythrocytes better than trisodium citrate alone and is therefore preferred
For blood transfusions and for the study of the hemolytic process
- dextrose serves as food for the red blood cells in vitro leading to longer survival time
- Formula: Disadvantages:
Trisodium citrate - 22.0 gm Dextrose - 24.5 gm 1. Very expensive
Citric_acid - 8.0 gm Distilled water - 1,000.0 ml 2. Cannot be used for morphological studies of cells
- 15 ml ACD is used for every 100 ml blood 3. Blood preserved with heparin is not ideal for blood smear preparation using Wrights or
Leishmans stain due to the blue coloration of the background
3. Citrate- Phosphate- Dextrose (CPD) Solution
V. FLUORIDES
III. EDTA (Ethylenediaminetetraacetic acid) - prevent coagulation by forming a weakly dissociated calcium component.
- prevents coagulation by chelation (preventing Ca from ionizing) - anticoagulant with preservative action
- may be used as dipotassium salt (Sequestrene) or as disodium salt (Versene) - interfere with the enzyme system having to do with glycolysis
- used for blood cell counts including platelet count and also for preparing peripheral - ideal for CSF and blood dsugar determinations especially when there is likely one-
blood smears even after 3-4 hours half to one hour delay between specimen collection and actual analysis
- used at 1-2 mg/ml blood - used either as potassium fluoride or sodium fluoride
- if in liquid form,it is used at 0.1 ml of 10% aqueous solution of dipotassium EDTA per 5 - potassium fluoride is considered the simplest preparation because just 2 drops of
ml of blood, it should, however, be evaporated to dryness 40% aqueous solution of it is enough to collect 5 ml blood
- sodium fluoride is used in conjunction with potassium oxalate to prevent glycolysis
Preparation of Sequestrene Solution: for 24 hours or longer
Sequestrene (dry powder) - 10gm
 - usually, 4 gm potassium oxalate and 5 gm sodium fluoride are dissolved in 100 ml
distilled water then 0.5 ml of the mixture is placed in a tube or vial and Hemocytometry
evaporated to dryness. - numerical evaluation of formed elements of blood
- after evaporation, there should be 20 mg potassium oxalate and 25 mg sodium - estimation of the number of blood cells in a known volume of blood
fluoride left in the tube or vial to prevent coagulation of 10 ml blood or 10 mg
sodium fluoride per ml blood. Methods:
- Thymol (1 mg) may be added if bacterial action is to be controlled as in specimens I. Turbidimetric Method
that are sent to the laboratory by mail. II. Microscopic Method
a. Pipet Method b. Test Tube Dilution Method
VI. DEFIBRINATION III. Automated Method (Impedance)
- prevents coagulation by removing fibrins as they are formed a. Optical b. Electrical
- to defibrinate: fresh venous blood is placed in an Erlenmeyer flask with glass beads,
paper clips or marbles or glass rods. The flask is rotated in a figure of eight for at TURBIDIMETRIC METHOD
least 5-10 minutes by which time all the fibrin would have adhered to the beads or - based on the assumption that the more turbid the solution, the more cells are present
- paper clips or hedgehog end of the rod in that solution"
- ideal for: a) obtaining high serum yield from sample - obsolete and very erroneous.
b) preserving the morphology of rbc & wbc.
c) preparing smears from buffy coat for LE cell demonstration. MICROSCOPIC METHOD
- counting cells under the microscope with the use of the following materials:
VII. SILICONIZED GLASSWARES 1. counting chamber
- use of siliconized glasswares prevents coagulation by preventing the adhesion of 2. pipets
platelets to wetable surfaces, thereby preventing breakage of platelets andsubsequent 3. diluting fluids
release of platelet factor 3 (PF-3)
- the glasswares are immersed in a dilution of water-soluble silicone concentration
(like Clay-Adams Siliclad) for 5 seconds or longer and then rinsed with distilled
water and dried at room temperature for 24 hours or in a hot air oven at 100 C for 10 1. Counting chamber or haemocytometer
minutes. A. According to type:
1. Open type (Spencer, Burker, Levy, Levy-Hausser)
EXAMINATION OF BLOOD AND ITS COMPONENTS 2. Closed type (Thoma-Zeiss)
The laboratory study of blood is divided into 2 groups: 3. Addis
1) Tests for the detection of diseases of the blood 4. Exton
2) Tests for the detection of diseases of other organs 5. Petroff

The laboratory tests for the detection of diseases of blood: B. According to rulings:
1. Chemical tests (haemoglobin determination and its abnormalities). 1. Thoma 4. Neubauer
2. Morphologic tests 2. Tuerk 5. Improved Neubauer
Qualitative tests - determination of the structure and appearances of the cells as 3. Fuchs-Rosenthal 6. Bass-Jones
analyzed and classified.
Quantitative tests - determination of the number of cells such as RBC count WBC The most commonly used counting chamber is the Open Type Spencer
count platelet count, reticulocyte count, differential count etc. and Improved Neubauer

THE COMPLETE BLOOD COUNT (CBC):
- consists of 6 determinations, namely:
1. Red cell count
2. White cell count
3. Hemoglobin determination
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
4. Hematocrit determination central large square, which is used for RBC count, is subdivided into 25 intermediate squares,
5. Differential count each with an area of 0.04 sq; mm. Each intermediate square is further subdivided into 16
6. Stained red cell examination 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 opaqe
into 16 smaller squares and are used for WBC ct. Depth of the counting chamber is 0.1 mm.
3. RUBRICYTE OR POLYCHROMATOPHILIC NORMOBLAST .
The area of one large square (1 sq.mm) and the depth of the counting chamber (0.1 mm). C - characterized by the first appearance of hemoglobin,
Compute for the volume per large square: - usually perinuclear, so that cytoplasm stains pink to basophilic.
- Size : 8-12 u
Volume = Area X Depth = 1 sq.mm X 0.1 mm= 0.1 cu.mm - Nucleus: round and smaller than in prorubricyte; usually eccentric; thick' nuclear
membrane; coarse and clumped chromatin so that nucleus stains very
This volume of 0.1 cu.mm is always multiplied by 10 to give the contents of 1 cu. mm blood; dark; distinct parachromatin.
thus 10 here is considered as the depth correction factor. - Nucleoli: none
- Cytoplasm: more abundant than in precursors; varies from basophilic to diffusely
2. Pipets lilac, depending upon the amount of hemoglobin.
A. Automatic pipets ( Ex: Trenner, Unopette)
- microglass capillary pipets that automatically suck in just the right amount of 4. METARUBRICYTE OR ORTHOCHROMIC OR ACIDOPHILIC NORMOBLAST
sample. - fully hemoglobinated_cell; constitutes 50% of nucleated red cells in normal marrow
- connected to a plastic container containing just the right amount of diluting fluid - Size : 7-10 u
- Nucleus: small and/shrunken; dense and dark staining because of marked
B. Non-automatic pipets (Ex: Thoma pipet) condensation of chromatin. Parachromatin no longer distinguishable, may be round,
a. RBC Thoma pipet b. WBC pipet oval or have various bizarre forms and is usually eccentric
- Nucleoli: none
Comparison of the 2 pipets RBC WBC - Cytoplasm: orange-red, as in adult erythrocyte
1. size of bulb larger smaller
2. color of bead red white 5. RETICULOCYTE
3. volume in bulb 100 10 - constitutes 0.5 to 1.5% of circulating red blood cells.
4. size of bore smaller larger - slightly larger than mature erythrocyte.
5. upper calibration 101 11 - after expulsion of the nucleus in metarubricyte, a large somewhat basophilic
anuclclear cell remains, which, when stained with new methylene blue, a vital stain,
RBC STUDIES is seen to contain a network of bluish granules or what is known as reticulum
NORMAL MATURATION SERIES network. As cell matures the network becomes smaller, finer, thinner and finally
1. RUBRIBLAST OR NORMOBLAST disappears within 2-4 days.
- makes up 5-10% of total nucleated cells in bone - cytoplasm is pink and reddish brown.
- Size : 14-19 u - Size: 8-10 u, Nucleus : absent
- Nucleus: Round or slightly oval; thin nuclear membrane; may be central or slightly - Cytoplasm: cell outline may be irregular because of shallow indentations; faintly
eccentric; chromatin varies from finely reticular to coarsely reticular with polychromatophilic (basophilic)
a tendency to clumping; parachromatin is indistinct and scant.
- Nucleoli: 1 to 2; usually very faint and pale blue 6. ERYTHROCYTE
- Cytoplasm: small in amount; moderately basophilic, homogeneous and opaque. - Size : 6.2 - 8.2 u in diameter (Ave.= 7.2 u), Nucleus : none
- Cytoplasm: biconcave orange-pink cytoplasm has a paler staining center occupying
2. PRORUBRICYTE OR BASOPHILIC NORMOBLAST one-third of the cell area.
- Size - 10-15 u
- Nucleus: smaller than in normoblast; generally round and slightly eccentric; thin NOTE:
nuclear membrane; chromatin is coarse and irregular so that nucleus - no hemoglobin yet in cells 1 & 2.
stains dark; parachromatin is sparse but distinct - cells 1 to 5 are normally seen only in the bone marrow except for cell 5 (retic)
- Nucleoli: 0 - 1 wherein 0.5 to 1.5% is seen in the circulation.
- Cytoplasm: appears more abundant than in normoblast because of smaller nucleus; - cell 6 (erythrocyte) is definitely seen only in the circulation and it is
varies from intense to moderately basophilic and is royal blue and eosinophilic/acidophilic in staining reaction due to hemoglobin.
 - lifespan is around 100 days (+ or - 20 days)
GENERAL RULES IN THE NORMAL MATURATIONl SERIES: - erythrocytes are uniform in size and have a small area of central pallor.
- the younger the cell is, the bigger in size and the bigger in nucleus - in every 100-red blood cells, 3-8 platelets may be seen; in every 1000 red blood
- the older the cell is, the smaller in size and the smaller the nucleus until eventually in cells, there is 1 white blood cell
erythrocyte no more nucleus is seen.
:Normal Values:
RETICULOCYTE Conventional SI
- mature red blood cell; matures into erythrocyte in 2-4 days with reticulum network Male - 5.5 - 6.5 million/ mm3 5.5 - 6.5 X 1012/L
which are RNA and protoporphyrin remnants, the latter being responsible for the Female: 4.5 - 5.5 million/ mm3 4.5 5.5 X 1012/L
fluorescence of some erythrocytes when viewed by ultraviolet light
- reticulum network is demonstrable with the use of supra-vital stains only like Erythrocyte is composed of a protein-lipid stroma that contains hemoglobin in solution and is
brilliant cresyl blue and methylene blue. condensed into a lipid-rich peripheral cell membrane. The cell membrane is non-elastic,
- normally about 0.5 - 1.5 % (20,000 - 60,000/cu. mm) of all erythrocytes in adults are although the cell can become distorted in order to pass through narrow capillaries.
reticulocytes
- normal values at birth range from 2.5 - 6.5%, falling to the normal adult level by the In order for normal erythrocytes to form, certain requirements must be met:
end of the second week. 1. There must be an adequate supply of the hemopoietic factors (erythropoietin)
responsible for normal and orderly maturation;
2. The maturing erythrocyte must be supplied with an adequate amount of iron in a
RETICULOCYTE COUNT usable form;
- Degreeof reticulocytosis is proportional to erythropoietic activity. Retic count above 3. Adequate amounts of protoporphyrinIll must be supplied;
normal indicate that erythropoiesis increased. The discovery of reticulocytosis 4. There must be available sufficient globin.
lead to the recognition of an otherwise occult disease such hidden hemorrhage for
unrecognized hemolysis. Persistently low reticulocyte counts particularly in the LIFE HISTORY OF THE ERYTHROCYTE:
presence of anemia, suggest markedly defective erythropoiesis. If the normal blood volume is taken to be 5L and the RBC count as 5,000,000 per mm blood,
RETICULOCYTE COUNT then a total of 25 x 1012 red blood cells are in active circulation at any one time. Their speed
Physiologic increase: Low Count or Absent in: of travel varies in different parts of the vascular system, the whole circuit taking 45 seconds,
1. at birth 1. Idiopathic aplastic anemia on the average. lt has been computed that each rbc travels about 700 miles before
2. menstruation 2. Acute benzol poisoning disintegration
3. pregnancy 3. In anaplastic crisis of hemolytic anemias
NORMAL MECHANISM OF RBC DESTRUCTION:
Increased Count: While the red blood cell is in the circulation its membrane is mechanically injured by constant
1. Hemolytic anemias 7. Polyoythemia vera buffeting in the blood stream and by alterations in its tension which is dependent on the ionic
2. Kala-azar 8. Relapsing fever changes associated with oxygen and carbon dioxide exchange. Furthermore, as the red cell
3. Lead poisoning 9. Sickle cell anemia matures it becomes more spherical and this facilitates rupture of the cell membrane.
4. Leukemia 10. Splenic tumor Normally, the end stages of cell destruction takes place in the reticulo-endothelial system,
5. Malaria 11. Blood intoxication particularly in the bone marrow, liver and spleen. The spleen has been long regarded as the
6. Erythroblastic anemias 12. Parasitic infections main graveyard of rbc.

Factors that determine number of reticulocytes in the circulation: ABNORMAL MECHANISM OF RBC DESTRUCTlON
1. Speed of release of reticulocytes from bone marrowi into the circulation - include various types of hemolysis, hemoglobin alterations and parasitic infections.
2. Degree of immaturity of released reticulocytes; and
3. Speed of disappearance of reticulum network from the reticulocytes. RBC COUNT
Six factors have statistically significant effect on the RBC
ERYTHROCYTE 1. posture 4. age
- mature red blood cell, non-nucleated, biconcave disc-like cell. 2. extreme physical exercise 5. gender
- 6.2 - 8.2 u in diameter and around 2.8 u in thickness 3. severe dehydration 6. High altitude
 CATABOLISM OF HEMOGLOBIN:
Increased RBC Count When old red blood cells are destroyed in the RES, the globin portion of the hemoglobin
1. polycythemia vera 4. dehydration molecule is split off, and the heme is converted into biliverdin. Most of the biliverdin formed
2. chronic heart disease 5. acute poisoning from heme is converted to bilirubin which-is excreted in the bile. Subsequent degradation of
3. lung diseases (TB, fibrosis) 6. residing in'a place of high altitude bilirubin will give rise to urobilinogen and stercobilinogen, oxidation of which will result
to urobilin and stercobilin. The iron from the heme is reused for hemoglobin synthesis.
Decreased RBC Count
1. Anemia Hemoglobin Increased in: Decreased in:
2. hemorrhage 1 1. hyperchromia 1. all anemias
3. oligocythemia 2. high altitude 2. leukemia
3. polycythemia 3. oligochromia
HEMOGLOBINOMETRY 4. dehydration in burns & diarrhea
- determination of the amount of hemoglobin in a known volume of blood screening 5. heart diseases .
test for the presence of diseases associated with anemia and for following the 6. erythremia .
response of these diseases to treatment in as much as the oxygen-carrying capacity of
the blood is directly proportional to hemoglobin and not to RBC ct. TYPES OF HEMOGLOBIN
A. NORMAL or FUNCTIONAL HB
HEMOGLOBIN (Ib) A 1. Oxyhemoglobin (HbO2)
- main component of rbc; it makes up 35 % Hb + Oxygen = 0xyhemoglobin (in arterial blood; bright red)
- molecular weight of more than 64,400. 2. Deoxygenated or Reduced Hb (HbCO2)
- gives red color to the blood so that it is also known as respiratory pigment. ` Hb + Carbon dioxide = Deoxygenated or Reduced Hb (in venous blood; dark
- responsible for the oxygen and carbon dioxide-carrying capacity of rbc. red)
- 1 gm of Hb can carry 1.34 ml of oxygen and 3.47 mg iron
- adult RBC mass with 600.gm Hb can carry around 800 ml oxygen. B. ABNORMAL OR NON-FUNCTIONAL HB - unable to act as an oxygen carrier.
- conjugated protein; made up of 1 globin molecule and 4 heme groups. 1. Carboxyhemoglobin (HbCO2)
- globin molecule: 4 polypeptide chains and each chain is attached to 1 heme group-a Hb + Carbon monoxide.: Carboxyhemoglobin
ferroprotoporphyrin responsible for the red color of the compound, and consist of 4 - the combination is irreversible. The affinity of Hb for C0 is 218x greater
protoporphyrinrings, each ring complexed with single bivalent iron atom. than for Oxygen at 37 C and is not affected if helium is substituted for
- globin molecule has 2 pairs of polypeptide chains: nitrogen as the inert gas (as in prolonged underwater diving). Since
alpha chain (in pairs) = 141 amino acids/chain = 282/pair carboxyhemoglobin is not capable of transporting oxygen, hypoxia
beta chain (in pairs)' = 146 amino acids/chain = 292/pair
results. Death may be due to anoxia, accompanied with irreversible
Normal Values: Conventional S.I. tissue changes.
Men - 14 - 18 gm/100 ml blood 2.17 - 2.79 mmoles/L - Carbokyhemoglobin produces a typical cherry red color of patient's
Women - 12 - 16 gm/100 ml blood 1.86 - 2.48 mmoles/L blood and skin.
Children- 14 - 26 gm/100 ml blood 2.17 - 4.03mmoles/L - Carboxyhemoglobin concentration in normal non-smokers is 0 - 2.3% of
total hemoglobin and in smokers, 2.1 - 4.2%. Critical level is 5 g/100 ml
Hemoglobin in Normal Adult Blood blood.
1. Hb A - 94.5% with 2 alpha and 2 beta polypeptide chains - measured by:
2. Hb A2 - 3.5% with 2 alpha and 2 delta chains a) Palmer's Carboxyhemoglobin Methods
3. Hb F- 2.0% with 2 alpha and 2 gamma chains b) Sunderman Dithionite Spectroscopic Test (Na2S2O4 -Dithionite)
2. Methemoglobin (Hi)
Hb F is measured by: - also known as ferrihemoglobin, oxidized Hb and hemiglobin
a) alkali denaturation test by Singer Hb + oxidizing agent = Methemoglobin
b) acid elution technic by Kleihauer-Betke. - the reaction is reversible since methemoglobin is unstable.
- differs from oxyhemoglobin in that it contains ferric, rather than ferrous
 iron; the oxygen being replaced by -OH. It is, therefore, oxidized Hb or I. COLORIMETRIC METHOD
ferriHb and results when Hb is exposed to oxidizing agents such as A. Direct Visual Colorimetric Method
laniline dyes, potassium ferricyanide or potassium permanganate. 1. Tallquist Method
- formation of methemoglobin is a normalprocess, kept within bounds by 2. Dare's Hemoglobinometer
a normal intraerythrocytic system capable of reducing methemoglobin 3. Acid Hematin Methods
to hemoglobin again Methods:
- Kravitz et al. give the ff. values for methemoglobin in normal subjects: a. Sahli-Adams d. Hayden-Hausser M
2.2% in premature infants b. Sahli-Hellige e. Newcomer
1 - 1.5% in infants up to 1-year old c. Sahli-Hayden f. Osgood-Haskin
1% in older children and adults. 4. Alkaline Hematin Method
- critical level is 1.5 gm/100 ml blood and it gives a characteristic B. Photoelectric Colorimetric Method
chocolate 1. Oxyhemoglobin Method
brown color to the blood 2. Cyanmethemoglobin Method (HiCN) (Drabkins Reagent)
3. Sulfhemoglobin (SHb)
Hb + sulfides = Sulfhemoglobin II. SPECIFIC GRAVITY METHOD
- reaction is irreversible since SHb is so stable that once formed, it. A. Copper Sulfate Method
disappears from the circulation only after the rbcs containing it are
naturally destroyed. .Erythrooytes containing SHb have normal survival III. GASOMETRIC METHOD
and osmotic fragility. - oxygen content of blood is directly measured
- normal concentration of SHb in the blood is 0 - 2.2% of total
hemoglobin. IV. CHEMICAL METHOD
- critical level is .5 gm/100 ml blood - iron content of blood is measured

C. HEMOGLOBIN VARIANTS: ERYTHROCYTE SEDIMENTATION RATE

- variants result due to differences in the .arrangement of amino acids in the
polypeptide chain.
- example: Hb A becomes Hb S when glutamic acid (negative charge) on the
6th position of the beta chain is replaced by valine (neutral
charge). Hb A becomes Hb C when glutamic acid on the the 6th
chain is replaced by lysine.
- alphabetically A designated. There are over 200 hemoglobin variants listed but only
a few (like Hb S,Hb C, Hb D, Hb E) are important agents in the production of
hemolytic anemia.
- differentiated from one another by:
a. solubility
b. mobility in an electrophoretic field at pH 8.6
fastest = Hb H and Hb I
slowest = Hb C
METHODS OF HEMOGLOBIN DETERMINATION
Principle: Hemoglobin is measured as oxyhemoglobin. It is first converted into one of several
compounds, such as acid hematin, alkaline hematin, cyanmethemoglobin, and other less
widelyl employed., The measurement is done by comparing the unknown with a standard.
The method of comparison may be visual or photoelectric. The latter is ,considerably more
accurate.
- rate of settling of red blood cells from their plasma after the
addition of anticoagulant
- settling of red blood cells is due to changes in surface charge.
- red blood cells are normally negatively charged but sometimes
some changes in the plasma will cause_some red blood cells to become positively
charged and this leads to rouleaux formation since unlike chargesl attract.
- Rouleaux formation leads to settling of red blood cells.
IMPORTANCE OF ESR:
- index for the presence of hidden but active diseases such as TB and carcinoma.
- measures the suspension stability of red blood cells
- measures the abnormal concentration of fibrinogen and serum globulin.
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 of
androgenic steroids in males. Castration of the male produces a lower ESR to normal male
levels.
METHODS OF ESR DETERMINATION:
A. WINTHROBE AND LANDSBERG METHOD
Anticoagulant - Ammonium-Potassium oxalate (also known as: double oxalate, balanced
oxalate, Paul-Heller's solution, Winthrobe's solution)
 Winthrobe tube F. ROARKE-ERNSTENE
- flat-bottomed with two calibrated sides: G. BRAYS 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
Hematocrit after 30 minutes of centrifugation at 3,500 rpm. 1. Initial rouleaux formation (during'the first 10 minutes).
- each calibration has 10 mm so there is a total of 100 mm in the tube. 2. Period of rapid settling (during the next 40 minutes when settling is constant).
- the bore is 3 mm in diameter. 3. Period of-final settling or packing (during the last 10 minutes).
Normal Values:
Male Adult : 0 - 9 mm/hr FACTORS IN ESR:
Female Adult: 0 -20 mm/hr I. INTRINSlC FACTORS (inherent factors in rbc and plasma)
Children : 0 -13 mm/hr 1. No. of RBC/mm3 (inversely proportional to_ESR)
Layers in Wintrobe tube after l hour polycythemia = slower ESR
a. Plasma layer - uppermost layer anemia = faster ESR
b. Buffy coat layer - middle layer which contains the platelets, WBC, and other 2. Size of RBC (directly proportional to ESR)
cells such as L.E. cells and other malignant cells. 1mm of macrocytes = faster ESR
buffy microcytes = slower ESR
coat contains about 10,000 WBC. ,It is whitish to cream in 3.Viscosity of the pla na (inversely proportional)
color. more viscous = slower ESR
c. Packed red blood cells layer lowermost layer which contains nucleated young less viscous = faster ESR
red cells on top and non-nucleated mature red 4. Plasma protein contents
cells at the lower layer. All proteins (alpha-, beta- and gamma-globulin.
Correct ESR for Anemia: and fibrinogen) except albumin will increase ESR.
- when ESR is high and hematocrit is low.
Winthrobe method can be used to do other tests like studies on the plasma, preparing
smears from the buffy coat and hematocrit determination II. EXTRINSIC FACTORS
1. Length of tube
B: WESTERGREN METHOD longer tube = faster ESR (because more space at the bottom wherein
- most sensitive ESR method 'for serial study of chronic diseases cells can settle down)
- 2 readings are done: after 1 hour and after 2 hours 2. Diameter of tube: wider tube = faster ESR
3. Position of the tube
Anticoagulant -3.8% Na citrate slightly inclined = faster ESR (since the' RBC's have a shorter distance through which
Westergren tube to
- Long tube with both ends open; calibration is up to 200 mm. settle down and the up-current of the plasma is along the slope, thus
- bore is 2.5 mm in diameter by-passing the sedimenting RBC's.)
Normal Values: 4. Temperature (ESR increases with temperature)
Male Adult = 3 - 5 mm/1 hr 7 -15 mm/2 hrs most ideal temp.= 2 - 27C (Room Temp.)
Female Adult = 4 - 7 mm/1 hr 12 -17 mm/2 hrs 5. Pipeting - if with bubbles= faster ESR (since less blood and less rbc in tube)
6. Volume of blood
C. GRAPHIC CUTLER METHOD more lood in tube = slower ESR
- 3.8% Na Citrate and Cutler tube less blood in tube = faster ESR
D. LINZENMEIER METHOD 7. Anticoagulant: excess = slower ESR (since rouleaux formation is inhibited)
- 3.8% Na Citrate and Cutler tube 8. Time of setting up the test
E. MICROMETHODS after 2 hours from blood collection = slower ESR (since red blood cells become
1. Micro Landau 5% Na Citrate and Micro Landau tube spherical upon standing for a longer time)
2. Smith Micro Method 9. Time of reading results
3. Crista or Hellige-Vollmer Method before 1 hour = lower reading (since no final settling yet of red blood cells)
 - 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 swelling
1. Defibrination - removal of fibrin either by artificial or natural means. of rbc and rbc + hypertonic solution will result to shrinking of rbc.
2. Low temperature - this tends to slow ESR since there is increased viscosity of the blood. - compared to a normal biconcave erythrocyte, a spherocyte can imbibe in less water
3. Excess of dry anticoagulant - slows down ESR because rouleaux formation is inhibited. ' before it lyses because its cell membrane is already fully distended so we refer to it
4. Diameter of the tube - tubes with less than 2 mm in diameter will slow down ESR. as a fragile cell.
- on the other hand, a sickle cell can take in more water before it lyses because its cell
HEMATOCRIT membrane can be distended more, so we refer to it as a resistant cell
- volume occupied by erythrocytes in a given volume of blood and usually expressed
as volume of erythrocytes per 100 ml blood. METHODS OF 0SMOTIC FRAGILITY TESTS:
- venous hematocrit is the hematocrit of venous blood which represents the hematocrit 1. SANFORD METHOD. '
of peripheral blood and does not indicate the proportion of erythrocytes to plasma in Principle: It tests the stability of red blood cells under different concentrations of
the entire circulation. hypotonic NaCl solutions.
- hematocrit is the ratio of total erythrocyte mass to total blood volume and is
calculated by a different method. PRECAUTIONS IN ERYTHROCYTE OSMOTIC FRAGILITY TEST
- used in determining erythrocyte indices, calculating blood volume and total - the blood sample should be obtained with a minimum of stasis and trauma
erythrocyte mass, and establishing whether or not a patient is anemic, in as much as a - test procedure should be set up as soon as possible
low hematocrit indicates that the concentration of erythrocytes is reduced. - the capillary pipet must be held in approximately the same angle so as to ensure
- hematocrit cannot completely replace RBC counts since the latter are needed to uniform size of drops
calculate some of the erythrocyte indices. - blood should fall directly on the saline solution and not on the dry sldes of the tubes
- for normal blood, hemoglobin and RBC counts may be estimated from the
microhematocrit reading ERYTHROCYTE INDICES
- important in assessing border line types of anemia
METHODS OF HEMATOCRITDETERMINATl0N: - values should be interpreted only in the light of other findings such as the appearance
A. WINTHROBE METHOD of erythrocytes on fixed smears
- Anticoagulant S double oxalate - computed using 3 determinations RBC count, Hemoglobin & hematocrit
- Winthrobe tube (same as tube used in ESR)
Normal Values: Conventional: S.I. 1. MCV (MEAN CORPUSCULAR VOLUME)
Adult Male 47 vol% (+ or - 5) 0. 47 (+ or - .05) - average volume of an individual red blood cell
Adult Female 42 vol% (+ or - 5) 0.42 (+ or - .05) - computed using hematocrit & RBC count x 10
2. MCH (MEAN CORPUSCULAR HEMOGLOBIN)
B. HADEN'S MODIFICATION METHOD - ratio of Hb to RBC count x 10
- Anticoagulant = 1.1% Sodium oxalate in distilled - average weight or amount of Hb in an individual RBC
C. VAN ALLENS METHOD 3. MCHC (MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION
- Anticoagulant: 1.6% sodium oxalate in distilled water - concentration of hemoglobin in a given volume of packed red blood cells
- Tube: with bulb and calibrated 1-10 or 10-100 mm. - computed using Hb values over Hct x 100
D. SANFORD-MAGATH METHOD
- Anticoagulant = 1.3% Sodium oxalate USE OF ERYTHROCYTE INDICES:
- tube = calibrated to 6 ml at 1 mm per division; tube is short (about 5 inches long) and The MCV, MCH & MCHC are sometimes collectively referred to as red cell indices. These
with a funnel-like mouth. It has a-6 ml capacity. 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.
OSMOTIC FRAGILITY TEST
- semi-permeable characteristics of the surface membrane of an erythrocyte make it In summary, the determination of the MCV, MCH, and MCHC as valuable information 'that
vulnerable to changes in the osmotic pressure of the external environment. 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 2. MICROCYTIC NORMOCHROMIC
per erythrocyte. - blood picture shows small red cells with normal Hb contents.
ANEMIAS - anemia is a disorder characterized by a reduction in the oxygen-carrying - computation of Erythrocyte Indices shows:
substance of a certain volume of blood due to a reduction below normal of RBC ct., Hb & Decreased MCV & MCH but Normal MCHC
Hct. - seen in chronic inflammations.
3. MICROCYTlC HYPOCHROMIC
Factors that determine the point at which the decreased oxygen-carrying capacity of the blood - blood picture shows small red cells that are pale in color due to decreased Hb
may produce symptoms of hypoxia are: - computation of Erythrocyte Indices shows: Decreased MCV, MCH & MCHC
1. rapidity with which anemia develops - seen in Thalassemia and severe iron deficiency anemia
2. degree of physiologic adjustment to the anemia 4. MACROCYTIC NORHOCHROMIC
3. effect of physical activity on oxygen demand. - blood picture shows red cells that are larger than normal. Although they contain a
larger than normal weight of Hb, the MCHC is normal so that the cells
Hypoxia symptoms as well as their severity depend on: therefore, are normochromic.
1. how great an oxygen-carrying deficiency exists - computation of Erythrocyte Indices shows: inc. MCV & MCH but Normal MCHC
2. how rapidly the anemia develops - seen in Pernicious anemia
3. the degree of physiologic compensation
4. level of physical activity
5. MACROCYTIC HYPOCHROMIC
- when anemia develops rapidly, as in massive hemorrhage, the severity of the - blood picture shows red cells that are larger than normal but are hypochromic due to
symptoms is proportional to the haemoglobin concentration. When anemia develops decreased MCHC.
slowly, the patient can adjust to progressive hypoxia that symptoms will - computation of Erythrocyte Indices shows: inc MCV but Decreased MCH and
be minimal in spite of the very low Hb and RBC count. Symptoms are also MCHC.
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 CLASSIFICATION OF ANEMIAS ACCORDING TO CAUSE
tachycardia. I. Decreased production of red blood IV. Hemolytic anemia
- tachycardia occurs when the heart attempts to improve oxygenation of the tissues by cells due to: A. Acute Stage
increasing the heartbeat and cardiac output per minute. A. Marrow damage - Hemorrhage
- because these factors may vary, the diagnosis of anemia is only partially based on 1. Leukemias B. Chronic Stage
laboratory measurements of erythrocyte and Hb concentrations.. Laboratory data, 2. Leukoerythroblastosis 1. Congenital
however, must be interpreted with reference to the clinical picture. 3. Aplastic anemia a. Red cell membrane defect
B. Decreased erythropoietin - Hereditary spherocytosis
An adult person is said to be suffering from anemia if: 1. Inflammatory process b. Hemoglobinopathies
- For males: 2. Renal disease - Hemoglobin C
RBC ct. = < 4.2 million/mm3 3. Hypothyroidism - Hemoglobin S
Hb = < 12 gm/100 ml C. Iron-deficiency c. Enzyme defects
- For females: II. Nuclear maturation abnormality - Glucose-6-phosphate
RBC ct. = < 3.6 million/mm3 A. Vitamin B12 deficiency dehydrogenase deficiency
Hb = < 10 gm/100 ml 1. Pernieious anemia 2. Acquired
B. Folic acid deficiency a. Overactivity of the RES
MORPHOLOGIC CLASSIFICATIONS OF ANEMIA C. Refractory macrocytic anemia - Heinz Body anemia
1. NORMOCYTIC NORMOCHROMIC 1. Di Guglielmo's anemia b. Auto-immune hemolytic anemias
- blood picture shows-red cells that are normal in size and normal in Hb contents. III. Cytoplasmic m turation abnormality 3. Hereditary elliptocytosis
- computation of Erythrocyte Indices shows: Normal MCV, MCH & MCHC A. Severe iron-deficiency
- seen in: hemodilution, hemorrhage, hemolytics anemia and aplastic anemia. B. Defect in globin production
- Thalassemia
C. Defect in heme synthesis
 - Sideroblastic anemia TYPES OF HEMOLYTIC ANEMIA
1. Paroxysmal Cold Hemoglobinuria cold hemolysin Donald-Landsteiner
COMMON ANEMIAS 2. Paroxysmal Nocturnal Hemoglobinuria Marchiafava- Micheli Syndrome
1. APLASTIC ANEMIA 3. Sickle Cell Anemia
- "aplastic anemia" due to the functional inability of the bone marrow to replace lost
red blood cells with proportionate decrease of RBC ot., Hb and Hct. 3. SICKLE CELL ANEMIA
- Blood picture: normocytic normochromic red cells, with normal MCV, MCH & - due to the presence of Hb S variant which causes sickling of red cells under reduced
MCHC. Reticulocytes are very few or none at all. Aside from the low RBC ct., there oxygen tension. Under normal oxygen tension, red cells appear normocytic
is leucopenia as well as thrombocytopenia. This decrease of all cell elements is normochromic so that all the three erythrocyte indices (MCV, MCH & MCHC) are
known as "pancytopenia. normal
Classifications of Aplastic Anemia According to Cause - under reduced oxygen tension, the normocytes will become sickle cells
1. Bone marrow injury 6. Metabolic inhibition of bone marrow - Sickle cells are crescent-shaped and they are also known as drepanocytes or
2. Congenital aplastic anemia 7. Erythroid hypoplasia of bone marrow in meniscocytes. This sickling phenomenon is an irreversible reaction
3. Familial aplastic anemia hemolytic-disease - Vaso-occlusive crisis is observed since sickle cells will clog the bleed vessels
4. Chronic erythrocytic hypoplasia 8. Idiopathic aplastic anemia especiallyiat the sites of the joints.
5. Aplastic anemia assoc. with thymoma 9. Aplasia in myeloproliferative disorders - swelling and too much pain in the occluded blood vessels and this may lead to
patient's death
2. HEMOLYTIC ANEMIA - patient's red cells also exhibit chronic hemolytic tendency.
- due to the excessive destruction and shortened life span of red blood cells brought - blood picture will also further show the following:
about by: a..intrinsie or corpuscular defects anisocytosis, poikilocytosis (cigar-shaped, crescent-like, ovalocytes,
b. extrinsic or extracorpuscular abnormalities acanthocytes, target cells), neutrophilia, thrombocytosis, decreased osmotic
and mechanical fragility, presence of nucleated red cells and red cells with
Etiologic Classifications of Hemolytic Anemia: basophilic stipplings as well as abnormal inclusion bodies such as Howell-
I. Due to intrinsic (corpusoular) defects Jolly
A. Defect of erythrocytic membrane bodies, Pappenheimer bodies and Cabot's ring.
1. Hereditary spherocytosis 4. Zieve's syndrome - commonly seen among the black race
2. Elliptocytosis or ovalocytosis 5. Paroxysmal Nocturnal Hemoglobinuria - presence of homologous Hb S and S in the patient indicates sickle cell anemia
3. Acanthocytosis whereas presence of heterologous Hb S and Hb A indicates sickle cell trait
B. Defect of intracellular enzyme (non-spherocytic type of Hemolytie anemia)
1. Enzymes involved in anaerobic glycolysis - Hexokinase, Aldolase def. Methods for demonstrating sickling of cells in the laboratory:
2. Enzymes involved in hexose monophosphate shunt G6PD def. 1. Scriver and Waugh
3. Enzymes involved in methemoglobin formation glutathione synthase def. 2. Daland and Da Silva
II. Due to extrinsic (extracorpuscular) defects 3. Sherman's Test (also known as Test Tube Method)
A. Acquired autoimmune haemolytic anemia
B. Acquired isoimmune haemolytic anemia Principle Involved:
C. Paroxysmal Cold Hemoglobinuria The reduced form of Hb S which is not loaded. With oxygen is less soluble than normal
D. infectious agents hemoglobin and solidifies within 15-30 seconds if there is an absolute .lack of oxygen. . In the
subsequent crystallization of Hb S the shape of the erythrocyte changes from that of a disc to
Blood picture in hemolytic anemia: a sickle form which serves as a diagnostic criterion for the sickle cell anomaly and can be
- proportionate decrease of RBC, Hb and Hematocrit. observed in blood preparation under the microscope.
- the blood picture shows normocytic normochromic red blood cells
- computation of erythrocyte indices shows normal MCV, MCH & LCHC. 4. THALASSEMIA
- increased nucleated red cells high reticulocyte count (10 - 2O%), increased osmotic - due to the abnormal production rate of one of the polypeptide chains of hemoglobin
fragility index, poikilocytosis and presence of'abnormal inclusion molecule.
bodies such as Howell-Jolly bodies. - also known as: a. Cooley's anemia
b. Mediterranean anemia
 c. Hereditary leptocytosis 4. Low excretlon of radioactive Vit B12 in the urine (Schilllng s test)
- blood picture: microcytic hypochromic cells with a predominance of target cells. 5. Low concentration of Vit B12 in the serum
- computation of erythrocyte indices shows decreased MCV, MCH & MCHC
RBC ANOMALIES
Thalassemia major 1. ANISOCYTOSIS - variation in size of red blood cells ( sign of regeneration)
- usually associated with increased Hb F, most severe expression of the thalassemia Normocyte - 6.2 to 8.2 micra in diameter. Macrocyte - > 9 micra in diameter.
abnormality and is usually detected in childhood. Affected infants fail to thrive. Microcyte - < 6 micra in diameter. Megalocyte 12 to 21 micra in diameter
Thalassemia minor 2. POIKILOCYTOSIS - variation in shape of red blood cells; seen in all forms of severe
- associated with increased Hb A2, less serious type and is usually detected in adults anemias, nephritis and bleeding peptic ulcers. May be graded 1+ to 4+
Beta Thalassemia a. Acanthocyte
- deficit of beta chain production relative to that of normal alpha production - (thorn cell, spur cell) - thorny, rounded rbc with_irregularly arranged
Alpha-thalassemia - 5-10 spicules of various lengths, some of which are bent at their tips.
- deficit of aprolpha chain duction relative to that of normal beta production - indicate permanent cell damage found in abetalipoproteinemia
b. Burr cell
In thalassemia, there is decreased HbA but increased HbF and Hb A2 - reversibly spiculated elongated cells that are not synonymous with the
non-reversible acanthocyte. Spicules usually are shorter than that seen in
5. SEVERE IRON DEFICIENCY ANEMIA acanthocytes.
- due to the depletion of iron storage in the body needed for normal hemoglobin c. Crenated RBC (echinocyte)
produition. - 10-30 short and blunt spicules that are evenly distributed over the surface of
the red cell.
Etiologic factors in iron deficiency - produced when blood is allowed to stand, when rbcs are exposed to lytic agents
A. Negative iron balance or when placed in hypertonic solutions. Not a clinically important phenomenon,
1. Decreased iron intake except that it has to be differentiated from other similar but significant changes.
2. Increased iron loss d. Elliptocyte (ovalocyte)
a. Gastrointestinal bleeding e. self-induced bleeding - elliptical or oval red cell, usually seen in pernicious anemia and in anemias
b. excessive menstrual flow f. Pulmonary hemosiderosis Associated with malignant lesions. It may also be due to a congenital
c. blood donation g. Hemorrhagic telangiectasia anomaly of rbc, which is Mendelian dominant and is not sex-linked.
d. hemoglobinuria h. Disorders of hemostasis e. Blister cell
B. Increased Requirements - red cell with single or multiple vacuoles or markedly thinned areas at the
1. pregnancy 3. lactation periphery and comes as a result of trauma to red cells during their passage
2. infancy through partially obstructed blood, vessels. Indicative of microangiopathic
hemolytic anemias.
PERNICIOUS ANEMIA f. Helmet cell (keratocyte of Bessis, triangle cell)
- lack of intrinsic factor needed for the normal absorption of Vit. B12 and folic acid. - irregularly contracted, triangular cell; usually half-moon shaped, the central
- also known as: a. Macrocytic anemia depression flanked by two horns and results after the rupture of blister cell.
b. Megalocytic anemia g. Pyknocyte
c. Addisonian's disease - distorted,`contracted, spiculated rbc similar to burr cell; normally present in
- blood picture shows macrocytes and megalocytes that are normochromic so that small numbers in the first 2 to 3 of months of life and may be increased up to
MCV & MCH are increased but MCHC isa normal. There is a three-fold increase in 50% in a condition known as infantile pyknocytosis.
erythropoiesis in the bone marrow. However, the rate of release of young cells from h. Sickle cell (drepanocyte, meniscocyte)
bone marrow into the circulation remains the same as during normal erythropoiesis. - crescent-shaped rbc, elongated,. Drawn-out, slightly curved cell with pointed
ends that resemble a sickle blade
Laboratory diagnosis of pernicious anemia - sickling usually occurs in red cells containin Hb S which are exposed to
1. peripheral blood smear reduced
2. Achlorhydria after histamine stimulation pH or oxygen tension.
3. Megaloblastic dysplasia of bone marrow 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
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)
- intraerythrocytic iron not yet incorporated into hemoglobin
- 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
Maurers 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
 22,800/mms) and declines gradually until the adult level is reached at about 21 years
TERMS DESCRIBING RBC INVOLVEMENT: of age (average of 7,500/mmg). There tis no significant change after this point
1. Po1ycythemia
- general term for the increase) of red cell concentration in the peripheral blood to 1. Leucocytosis - increase above normal in the number of leucocytes in the peripheral blood
above normal (>6.5 mil/mm3 of blood) a) Physiologic leucocytosis
- may be primary (unknown etiology) or secondary (accompanies a variety of - caused by conditions that are physiologic such as exercise.
conditions), may be a temporary or permanent condition. b) Pathologic leucocytosis
2. Polycythemia vera - caused by disease; always accompanied by an absolute increase in one or another type
- also known as primary polycythemia, polycythemia rubra, erythremia, Vaques of WBC making the terms: b1) neutrophilic leucocytosls (neutrophilia)
disease, Osler's disease, Vaquez-Osler disease, megalosplenica and crytogenic b2) lymphocytic leucocytosis (lymphocytosis),
polycythemi b3) monocytosis
- etiology not known but it is essentially a myeloproliferative disorder. b4) eosinophilia
- characterized by abnormal proliferation of erythroids, myeloids and b5) basophilia
megakaryocytes in the bone marrow (condition is known 2. Leucopenia - a decrease below normal in the number of leucocytes in peripheral blood.
as panmyelosis) as well as increased erythrocytes, leucocytes and thrombocytes 3. Leukemia - malignant increase in the number of leucocytes in the peripheral blood as well
in the circulation (condition is known as pancytosis) as in leucocyte producing organs.
3. Erythremia
- increase in -red blood dell count above 6.5 mil/mm3 of blood due to Increased WBC (leucocytosis) Decreased WBC (leucopenia)
polycythemia vera. 1. infections 1. in the morning
4. Erythrocytosis 2. inflammations 2. in older people above 40
- increase 'in red blood cell count above 6.5 mil/mm3 of blood. due to chronic 3. in the afternoon 3. in measles
heart diseases, lung diseases and high altitude. 4. after strenuous exercise 4. leucopenia of viral origin
5. Oligocythemia 5. after meals E. prolonged use of sulfa drugs
- general decrease of red blood cells in the circulation as well as in all rbc- S. during menstruation 6. replacement of marrow with malignant disease
forming 7. in newborns 7. hypersplenism
organs in the body.
NORMAL MATURATION SERIES:
WHITE BLOOD CELL STUDIES I. GRANULOCYTIC SERIES
LEUCOCYTE 1. MYELOBLAST
- larger than red blood cell, measures 9-1 u in diameter, nucleated lifespan of 5-8 days - Size : 10 - 20u; round or oval
- provides with natural immune defense mechanisms by means of phagocytosis and - Nucleus: Round or oval, thin nuclear membrane; abundant chromatin (light purple);
production of antibodies (Ab) and proteolytjc enzymes sparse parachromatin (pale-blue or pink)
- Nucleoli : 2 to 5;well-outlined;round or oval, pale blue
2 Types of WB - Cytoplasm : sparse; nucleus-to-cytoplasm ratio = 5:1 to 7:1; deeply basophilic; no
1. Granular WBC 2. Agranular WBC granules
- Neutrophil - Lymphocytes
- Eosinophil - Monocytes 2. PROMYELOCYTE (PROGRANULOCYTE}
- Basophil - Size : 14 to 20 u; round or oval
- Nucleus : large, round or oval; thin nuclear membrane; slightly coarse and network-
like chromatin
Normal Values: - Nucleoli : 1 to 3; less prominent; round or oval; pale blue
For both male and female adults = 5,000-10,000/mm3 - Cytoplasm : sparse, nuc-to-cytop ratio = 5:1; basophilic but higher than myeloblast;
in Sl = 5~lO X lflg/L few purplish granules that may be large and round or fine
3. MYELOCYTE
- Leucocyte count is normally higher in the newborn infant (average of 18,100/mm3) - Size: 10-18 u; round or oval
than in the adult. It rises to an even higher level 12 hours after birth (average of
 - Nucleus: indistinct thin nuclear membrane, coarse chromatin network with irregular
patches of pink staining parachromatin
- Nucleoli: absent or invisible; rarely more than one
- Cytoplasm: moderate in amount; nucleus-to-cytoplasm ratio = 2:1; large granules
which are purplish-blue in the early stage of development
4. METAMYELOCYTE (JUVENILE CELL)
- Size : 10~18u
- Nucleus : kidney-shaped; heavy nuclear membrane; coarse chromatin staining deep
purple; scanty parachromatin;
- Nucleolus: absent or invisible
- Cytoplasm : fairly abundant; nucleus-to-cytoplasm ratio = 1.5:1; pink cytoplasm;
granules are eosinophilic, basophilic or neutrophilic and are smaller and less
- uniform in size than in the myelocyte
5. BAND GRANULOCYTE (STAB; STAFF CELL
- Size: 10-15u
- Nucleus: sausage-shaped or band shaped or horse-shoe shaped with some areas of
constrictions; coarse deep purple-blue chromatin; scanty parachromatin.
- Nucleolus: absent or invisible
- Cytoplasm: abundant; pale blue or pink; nucleus-to-cytoplasm ratio = 1:2; with fine
lilac granules (neutrophil), large blue-black granules (basophil), or brick red granules
(eosinophil) which have the appearance of small hollow spheres
6. SEGMENTED GRANULOCYTE
- Size: 10-15 u
- Nucleus: segmented or lobulated nucleus connected by thin filaments; coarse and
dense deep purple-blue chromatin; scanty parachromatin.
- Cytoplasm: abundant; light pink or blue; nucleus-to- cytoplasm ratio = 1:3; with
specific granules
II. AGRANULOCYTIC SERIES
LYMPHOCYTIC SERIES
1. LYMPHOBLAST either absent in normal bone marrow or present in small numbers
- Size: 10 - 18u
- Nucleus: round or oval, no indentation and centrally located; definite nuclear
membrane; chromatin -in thin strands or stippled light red-purple; moderately
abundant, sharply demarcated and light blue parachromatin.
- Nucleoli: 1-2; small and pale blue
- Cytoplasm: homogeneous and moderately to heavily basophilic; sparse with
nucleus-to- cytoplasm ratio= 5:1-7:1; bftentimes shows a lighter perinuclear zone; no
granules. -
2. PROMYELOCYTE
- Size: 10-18 u; average smaller than lymphoblast
- Nucleus: round or oval, may be slightly indented; chromatin p more clumped than in
the lymphoblast but is still relatively fine and dark red-purple; parachromatin not as
well defined as in the lymphoblast; nor as smudged as in the adult lymphocyte.
- Nucleoli: usually one; round, blue and sharply outline
- Cytoplasm: more abundant; nucleus-to-cytoplasm ratio closer to 5:1; light blue to
medium dark blue; occasionally with azurophilic granules.
3. LYMPHOCYTE
- Size: Small = 6 - 8 u, Medium = 8 - 14 u, Large = 8 - 18 u
- Nucleus : round or oval; slightly or deeply indented; somewhat eccentric; heavy
nuclear membrane large coarse clumps of chromatin, blending into sparse pale blue
to pink "smudged parachromatin
- Nucleoli: one may be seen in the larger cells; generally none
- Cytoplasm: typically sky-blue, but may be clear and homogeneous with occasional
azuruphilic granules in large and medium lymphocytes
PLASMOCYTIC SERIES
- this is included here since plasma cells are known to arise from stimulated small
lymphocytes. Plasma cells are not normally found in the peripheral blood; although
1-2% of plasma cell-like transformed lymphocytes may be seen under physiologic
conditions. Plasma cells produce immunoglobulins.
1. PLASMABLAST
- Size: 8-20u
- Nucleus: round or oval and eccentric, bluish purple fine chromatin network with
some clumping; nucleoli difficult to discern.
- Cytoplasm: non-granular, blue, mottled and moderate in amount
2. PROPLASMACYTE
- Size: 15-25u
- Nucleus: round or oval and eccentric with coarse chromatin network; nucleoli may
be visible.
- Cytoplasm: abundant, deep blue, non-granular with clear peri-nuclear zone.
3. PLASMACYTE (PLASMA CELL)
- Size: 14-20u
- Nucleus: small, oval and eccentric; condensed chromatin forms large granular
clumps that may be concentrated in the periphery and center of the nucleus creating
the so-called " cartwheel" pattern. Parachromatin is pale pink.
- Cytoplasm: dark blue, ovoid and somewhat fibrillary; non-granular with Russell
bodies (secretory globules that represent protein secretion). If cytoplasm is
completely filled with secretory globoid structures, the cell is called
grape cell or Mott cell).
MONOCYTIC SERIES
1. MONOBLAST
- Size : 14-18u
- Nucleus : round or oval; thin nuclear membrane; chromatin structure similar to
myeloblast but stains lighter; abundant, sharply demarcated and pale pink' or blue
parachromatin.
- Nucleoli: 1 to 2
- Cytoplasm: moderate; basophilic; no granulations in the blast stage.
2. PROMONOCYTE
 - Size : 14-18u acute appendicitis ,
- Nucleus : moderately indented; thin nuclear membrane; fine, threadlike chromatin; tonsillitisf -
abundant parachromatin meningitis
- Nucleoli: 0-1
- Cytoplasm: gray-blue; opaque, with very fine lilac granules (azurophilic dust which 2. Intoxications
indicate cell maturation) A. Metabolic C. Insect venoms
3. MONOCYTE uremia black widow spider
- Size : 12-18 u acidosis D. Reaction to parenterally given foreign
- Nucleus : indented or folded-over or bean-shaped; delicate;d pale staining; fine diabetes, etc. protein A
strands of reticulated or lacy in appearance chromatin; abundant and distinct B. Chemical E. Reaction to parenterally given
parachromatin. epinephrine bacterial vaccines
- Nucleoli: usually none, occasionally one lead
- Cytoplasm: light gray or gray blue, opaque; characteristic numerous, fine, dust-like mercury
lilac granules arsenic
kerosene, etc.
BRIEF DESCRIPTION-OF WBCS NORMALLY SEEN IN PERIPHERAL BLOOD:
I. GRANULOCYTES (produced mainly in BM) 3. Tissue necrosis from any cause 6. Myeloproliferative disorders
1. NEUTROPHIL myocardial infarction 7. Myelocytic leukemia
- 9 -15u, nucleus usually trilobulated (2 - 5 lobes) with abundant fine lilac pink gangrene
granules and coarse and clumped chromatin pattern. extensive burns
- normally produced in the bone marrow and released in response to various stimuli 2 to_bacterial invasion
- circulate in the peripheral blood and are sequestered in various organs for variable benign and malignant neoplasms
periods before they are destroyed.
- average lifespan is 10 - 14 days (4 days in the bone marrow in the course of 4. Acute hemorrhage
maturation, 3 - 4 days represent the total time spent in the circulating_blood, for 5. Acute hemolysis
periods of only 2 or 3 hours at a time, and the remaining hours spent in hemolytic transfusion reactions
sequestration in .various organs acute hemolytic anemia
- actively phagocytic, ingesting bacteria and other foreign particles both in vivo chronic hemolytic anemia
and in vitro. After ingestion, bacteria are destroyed by proteolytic enzymes in the 2. EOSINOPHIL
- 9-15u, nucleus usually bilobulated with coarse, clumped
neutrophil after which the neutrophil dies and fragmentst into particles that are chromatin pattern.
removed by fresh phagocytes. - cytoplasm contains large reddish-orange granules function remains obscure
- show active ameboid locomotion. When this movement- is directional, in response to - recent work has shown that eosinophils actually have antihistaminic activity as
a chemical substance nearby, it is called CHEMOTAXIS. opposed to the old belief that eosinophils contributed to allergic reactions
- Chemotaxis is governed by products released at the site of inflammation as well as as carriers of histamine.
directly by bacteria. - capable of phagocytosis, in the course of which there is degranulation as in the
- Leucocytosis-promoting factors (LPF) such as leucotaxine, may also be active in neutrophils.
chemotaxis. In fact, leucotaxine has been shown to attract neutrophils and monocytes - Eosinophilia is observed in :
in vitro. - allergies - hemopoietic disorders such as:
- Pathologic neutrophiliai or pathologic neutrophilic leucocytosis (increase in - skin disorders (non-allergic) polycythemia vera, Hodgkin's disease,
neutrophils) may be due to the following: - parasitic infections pernicious anemia, sickle cell anemia
- after splenectomy
1. Acute infections by pyogenic bacteria and other organisms:
A. Localized or limited such as: B. Generalized such as: 3. BASOPHIL
abscesses acute rheumatic fever - 9 - 15 u; with most varied-shaped nucleus (may be indented,bi-lobulated or stab
osteomyelitis septicemia form); usually indistinct as it is covered with large purple or purplish black
peritonitis
cholera
empyema, etc.
 granules. Its cytoplasm contains large purple or purplish black granules. - Functionally lymphocytes can be divided into B-lymphocytes derived from the bone
- rarest leucocyte in the peripheral blood. marrow or the bursa equivalent organ in humans T-lymphocytes derived from the
- functional importance is probably minor thymus. Morphologically, T and B lymphocytes cannot be distinguished in Wright-
- contains heparin and large amounts of histamine Giemsa stained smear.
- Basophilia may be observed in the ff: - Lymphocytes that lack both T & B cell markers are called null cells.
- chronic myelocytic leukemia - erythroderma - Small lymphocytes that are short-lived (3-4 days), upon antigenic stimulation,
- polycythemia vera - urticarla plgmentosa become immunoglobulin-producing B lymphocytes which are the precursor
- myeloproliferatlve dlsorders - ulcerative colitis of plasma cells. B lymphocytes carry surface markers not present on T cells.
- Small lymphocytes that are long-lived (200-300 days) are usually the T lymphocytes
Il. AGRANULOCYTES helper cells.
1. MONOCYTE - The following distribution of T and B cells is found in the peripheral blood of
- largest cell in the circulation probably the circulating counterpart of the blood tlssue healthy adults: noncommitted null cells = 0.5 - 1%
macrophage with large semi-indented or bean shaped nucleus with superimposed B cells = 7 - 23%
brainlike convolutlons; nuclear chromatin retlculated or lacy in appearance T cells = 70 - 90%
- cytoplasm is abundant grayish or muddy blue in color often described as having a - Lymphocytogenesis is a function of the lymph nodes, bone marrow, spleen, and
ground glass appearance scattered .lymphoid tissues in the body. The bone marrow is one of the largest
- functions: the cell s phagocytic function is related to its immunologic activity which lymphocyte-containing tissues in the body.
consists of 2 phases:
a. durlng the inductlon of lmmunlty when antigenic informatlon is
transferred to lymphocytes Pathologic Lymphocytosis:
b. during expression of cellular responses and the development of delayed 1. Acute viral and Non-Viral infections 2. lymphocytic leukemia
hypersensltivity - infectious mononucleosis 3. infectious hepatitis
- contains large amounts of lipase thus specifically endowed to combat bacilli with - infectious lymphocytosis 4. Congenital syphilis
lipoid capsule such as Mycobacterium tuberculosis and leprae - mumps, chicken pox 5. Pertussis
- german measles 6. Brucellosis

Monooytosis may be observed in the ff: ABNORMAL WHITE BLOOD CELLS

- Monocytic leukemia - Chronic Ulcerative Colitis 1. SMUDGE CELL (BASKET CELL)
- Subacute Bacterial Endocarditis - Collagen diseases - ruptured WBC with bare nucleus, few may be seen in normal blood smear due to
- Brucellosis - Trypanosomiasis improper and forceful smearing
- Typhoid - Pulmonary TB - large numbers may be seen in leukemia and their presence may indicate increased
- Rickettslal lnfections - Gauchers disease fragility of the cells or abnormal destruction of cells, not counted
- Kala-azar 2. HYPERSEGMENTED NEUTRDPHILS
- also known as macropolycytes and PA Polycell
2. LYMPHOCYTE - usually larger than a normal neutrophil and nucleus has 5-10 lobes instead of the
- size: small = 6-8u, medium= 8-14u, large 8-18u normal 3 lobes
- presence of small, medium and large lymphocytes in the blood of the adult and the - usually present in pernicious anemia
predominance of medium and large lymphocytes in the blood of infants are common - the number of hypersegmented neutrophils in the differential white cell count should
observations. be reported under miscellaneous white cells
- Lymphocytes larger than polymorphonpclear cells are classified as large lympho 3. VACUOLATED CELL
cytes and the smaller cells as small lymphocytes, morphologic difference lies mainly - with holes or vacuoles in the cytoplasm which are signs of degeneration and If seen
in the amount of cytoplasm in smears made from fresh blood it should be counted and reported under
- most small lymphocytes are T-cells and most large lymphocytes are B lymphocytes. miscellaneous white blood cell
- In Wright s stained cell, the nucleus is deep purplish blue and is round to oval to - may also be found in normal blood smears if the smear is made from oxalated blood
kidney- shaped or deeply indented usual eccentrically located and occupies nine which is over 2 hours old.. It may be seen in severe infections, chemical poisoning
tenths of the cell diameter so that the sky-blue cytoplasm is hardly seen. and leukemia.
4. TART CELL
- a phagocytic WBC, usually a monocyte with 'engulfed nucleus of another cell
- usually, the ingested nucleus retains its characteristic nuclear structure such as
chromatin clumps, nucleoli or nuclear membrane
- seen in drug sensitivity
5. L.E- CELL
- LE (lupus erythematosus) cell is a phagocytic WBC (usually a neutrophil) that has
ingested an altered, homogeneous globular nuclear mass of a destroyed cell.
- nucleus of the phagocyte is compressed to one side, its appearance is distorted. The
ingested nuclear material is homogeneous and redder than the usual color
of unaltered chromatin.
- 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 DOWNS 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 c) Histiocytic.medullary reticulosis (Robb-Smith)
- predominant cell is a blast, cannot be identified at the time of diagnosis
2. Lymphocytic leukemia LEUKEMOID REACTION
- acute lymphocytic leukemia - predominance of lymphoblasts - Leukemoid Reaction is not a disease or diagnosis but a descriptive term to indicate a
- chronic lymphocytic leukemia - predominance of mature lymphocytes blood picture in which the peripheral blood findings resemble those found in
3. Promyelocytic leukemia leukemia, with the important difference that while leukemia is a neoplastic
- preponderance of atypical progranulocyte containing large irregular azurophilic proliferation and malignant in nature, leukemoid reactions are benign proliferations.
granulation. - LRs may be due to infections, intoxications, tumors and acute hemorrhage.
4. Chronic myelocytic leukemia (CML) - LR mimics leukemia and may show the same quantitative and
- predominance of neutrophilic, eosinophilic and basophilic types that are variants and qualitative changes
need not be classified separately. Philadelphia (Phl) chromosome is present in 70- - differentiated from leukocytosis in the sense that its WBC count is usually above
90% of patients with CML. 50,000/mm3 and its blood smear shows more immature cells. It is the presence of
5. Neutrophilic leukemia immature cells as much as the high WBC count that gives the leukemoid blood
- preponderance of neutrophilic myelocytes. - picture the appearance of leukemia.
6. Eosinophilic leukemia - As a rule, LR is not accompanied by the anemia or thrombocytopenia that is
- preponderance of eosinophilic myelocytes. common in leukemia.
7. Basophilic leukemia
- preponderance of basophilic myelocytes CONDITIONS THAT MAY CAUSE LEUKEHOID REACTIONS:
8. Monocytic leukemia (occurs in two forms that are basically not related) I. Infections
- Naegeli type (myelomonocytic leukemia) - variant of myelocytic leukemia and may A. Myelocytic reactions
resemble granulocytic leukemia. Cytologically, myelomonocytes show the nuclear 1. Pneumonia 6. Bubonic plague
features of monocytes and the cytoplasmic features of myeloid cells. 2. Empyema 7. Septicemia
- Schilling's type (histiocytic leukemia) - true monocytic leukemia since it is 3. Endocarditis 8. Tuberculosis
characterized by primitive cells having the same general features as a true blood 4. Meningitis 9. Leptospirosis
monocyte and its tissue counterpart, the histiocyte. 5. Diphtheria .
9. Plasmacytic leukemia and multiple myeloma
- myelomas are neoplasms of proliferating plasma cells. They may present as solitary B. Lymphocytic reactions
tumors involving a bone (solitary myeloma), or as neoplasms involving the BM and 1. Whooping cough 5. Infectious lymphocytosis
other organs (multiple myeloma). 2. Chicken pox 6. Congenital syphilis
10. Plasma cell leukemia 3. Mumps 7. Tuberculosis
- terminal leukemic phase of multiple myeloma. 4. Infectious mononucleosis
11. Mast cell leukemia
- rarest; leukemic cell is the,tissue mast cell. PLATELET STUDIES
- Mast cells are essentially tissue basophils in the bone marrow. They arise from - non-nucleated, irregular in size (1~4 u), irregular in shape, lifespan of,4 - 9 days
basophils by a process of differentiation and replication - 3 main functions: hemostasis, blood coagulation, clot retraction
12. Erythremic myelosis (Di Guglielmo's disease)
- Pure erythroblastic proliferation. INCREASED PLATELET COUNT DECREASED PLATELET COUNT
- may progress to a mixed erythroblastic myeloblastic phase and terminate as an acute 1. Polycythemia vera . 1. Pernicious anemia
myeloblastic leukemia 2. Myeloproliferative syndrome' 2. Aplastic anemia ' ,
13. Erythroleukemia (Di Guglielmo's syndrome) 3. Splenic vein thrombosis 3. Infectious diseases
- erythroblastic and myeloblastic proliferation. 4. Postsplenectomy states 4. Lesions involving the bone marrow
14. Chronic neutrophilic leukemia 5. Acute blood loss 5. Idiopathic -
- preponderance of adult segmented neutrophils and band neutrophils. 6. Carcinomatosis 6. Acute leukemia
15. Leukemias of uncertain type 7. Nephrotic syndrome without uremia
a) Reticulum cell leukemia 8. Chronic myelocyticwleukemia
b) Leukemic reticuloendotheliosis 9. Acute alcoholic hepatitis
10. Ulcerative colitis
11. Cirrhosis of liver
THROMBOCYTOPENIA
- refers to increase in platelet count accompanied by a disease
- platelet count is significantly higher than normal.
- term used for secondary cases (there is a coexistent
- disease that may be accompanied by an elevated platelet count
- Examples: splenic vein thrombosis, postsplenectomy state, acute blood loss, various
anemias, carcinomatosis, nephrotic syndrome without uremia,
THROMBOCYTHEMIA
- platelet count is >1,000,000 or >1 million/mm3
- term used for primary cases (no other cause for the increased platelet count exists
THROMBOCYTOPENIA
- subnormal number of platelets in the circulating blood and is the most common
cause/of abnormal bleeding.
- It results from:
1. deficient platelet production
2. accelerated platelet destruction
3. abnormal distribution or pooling of the platelets within the body.
NORMAL MATURATION SERIES
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)
- Nucleus : large, indented and polylobulated; coarse, heavily stained strands of
chromatin.
- Cytoplasm: intensely basophilic; filled with increasing numbers of azurophilic
granules, sparing a thin peripheral ring that remains blue in color.
3. MEGAKARYOCYTE
- Size: 30-100 u (largest cell in the bone marrow)
- Nucleus: plump, multilobulated, indented, and sometimes multinucleated. Nuclei are
arranged in chains or rings and may partially cover each other; chromatin in heaxv
clumps.
- Cytoplasm: produces blunt, smooth, pseudopodia-like projections with aggregates of
azurophilic granules surrounded by pale halos. These structures will give rise to
platelets at the periphery of megakaryocyte. The line of cleavage goes thru the
hyaline cytoplasm of the halo, so that the granular mass becomes the platelet
4. PLATELET or THROMBOCYTE
- detached cytoplasmic fragmentations of, mature megakaryocytes
- Size: 1-4u ( young platelets may be 2 - 3 times larger)
- Nucleus : none
- Cytoplasmr In Wright-Giemsa stained smears, platelets appear as small, bright,
azure, rounded or elongated bodies with a delicately granular structure. Each platelet
consists of a central group of azurophilic granules (chromomere) and a
surrounding light blue hyalomere.
Morphology of platelets varies greatly depending on:
1). the methods by which they are examined
2). the anticoagulant
3). and the temperature .
PLATELETS IN HEMOSTASIS
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
2. Uremia
- 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
A. Paraproteinemias (macroglobulinemia multiple myeloma and others)
B. Hemorrhagic (ldiopathic) Thrombocythemia, Myelofibrosis & Polycythemia vera
C. Miscellaneous (acute and chronic leukemias, ITP, others)
QUANTITATIVE PLATELET ABNORMALITIES
A. Due to_excessive destruction or loss
1. Due to immunologic or hypersensitivity mechanisms - solidifying of fluid blood (whole blood or plasma) brought about by the different
- Idiopathic Thrombocytopenic Purpura (ITP) platelet destruction as_a result of an coagulation factors
immunologic process. - formation of a visible coagulum, which is the physical manifestation of fibrin
2. Hypersensitivity to certain. drugs formation, represents only the end result of an intricate series of reactions that
- apronalide, guinine, quinidine. involve a number of coagulation factors.
3. Due splenomegaly and increased sequestration in the spleen - coagulation factors have an international standard nomenclature
- Conditions above produce thrombocytopenia by sequestering normal and undamaged
platelets in the spleen. An enlarged spleen usually traps many normal cells and these NOMENCLATURE & SYNONYMS FOR COAGULATION FACTORS
cells are eventually destroyed due to normal lytic splenic mechanisms acting for a long
period of time on cells that are sequestered. I Fibrinogen ---------------------------------
4. Due to sequestration but not in spleen II Prothrombin ---------------------------------
- Sequestration of platelets in tumors and trapping of platelets in the fibrin network III Tissue factor Thromboplastin,Thrombokinase
Deposited throughout the vascular system in the syndrome characterized by diffuse IV Calcium ions ---------------------------------
Intravascular coagulopathy VI Proaccelerin Labile factor, Thrombogen, Accelerator globulin
5. Due to mechanical destruction (ACG)
- Extracorporeal circulation of blood produces moderate destruction of platelets. The VII Proconvertin Stable factor, Serum Prothrombin Conversion
cause is primarily mechanical (trauma, adhesion to tubing, artificial cardiac valves, etc). Accelerator (SPCA), Autoprothrombin I,
6. Due to miscellaneous factors cothromboplastin
- massive hemorrhage i VIII Antihemophilic Antihemophilic globulin (AHG), Anti- hemophilic
- massive transfusion of platelet-poor blood factor (AHF) factor A, Platelet cofactor 1, Thromboplastinogen
- chronic alcoholism IX Plasma Thromboplas- Christmas factor, Antihemophilic factor B, Platelet
tin Component (PTC) cofactor 2, Autoprothrombin II
B. Due to deficient production X Stuart-Prower factor Prower factor, Autoprothrombin III
1. Bone marrow suppression of thrombocytopoiesis XI Plasma Thromboplas- Antihemophilic factor C
a. Potentially myelotoxic drugs such as antifolates, nitrogen mustard, chloramphenicol, tin Antecedent (PTA)
gold salts, DDT, organic chemicals, etc. ` XII Hageman factor Glass factor, contact factor
b. Physical and animal agents XIII Fibrin stabilizing Fibrinase, Laki-Lorand factor
- ionizing radiation - artificial fever factor-(FSF)
- heat stroke - burns
- insect bites GENERAL CHARACTERISTICS OF THE COAGULATION FACTORS
c. Bone marrow replacement of abnormal cells (metastatic tumor, lymphomas, etc.) 1. FIBRINOGEN
d. Congenital, neonatal, or familial thrombocytopenic syndromes - protein clotted by thrombin in the formation of fibrin, plasma concentration is 250 -
- Wiskott-Aldrich syndrome - May-Hegglin anomaly 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) . '
THROMBOCYTHEMIA - substance that, in the presence of. calcium ions, brings about the conversion of
A. Thrombocythemia hemorrhagica prothrombin to thrombin. '
- primary disease characterized by a persistent increase in platelets and frequently - a) tissue thromboplastin: from tissue extracts and it is the prothrombin activator in
associated with bleeding tendency. the extrinsic system
B. Secondary thrombocythemia - b) plasma thromboplastin: prothrombin activator in the intrinsic system
- Seen in polycythemia and in chronic granulocytic leukemia. 4. CALCIUM
- normally exists in the blood in an ionized form and combines with plasma proteins
STUDY OF BLOOD COAGULATION AND HEMOSTASIS and lipids, needed in prothrombin conversion and plasma thromboplastin generation.
COAGULATION 5. THROMBIN'
 - active agent (enzyme) that clots fibrinogen. Its action is so powerful that it can clot - entire mechanism by which bleeding from an injured blood vessel is spontaneously
several hundred times its weight of fibrinogen. As.an enzyme, it splits the arginyl- controlled and stopped
glycyl bands at the N terminal of the fibrinogen molecule to form fibrin.
6. FACTOR V Mechanical Hemostasis
- called "accelerator" because it speeds up the conversion of prothrombin to thrombin - stops bleeding with the use of tourniquet; surgical or a first aid problem.
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 Physiologic hemostasis
synthesized in the liver - brought about by interaction of several factors, hemorrhagic disorders due to
7. FACTOR VII deficient physiologic hemostasis are a laboratory concern.
- 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 Three Aspects of Hemostasis ` `
from plasma, while VII is present in plasma but is more active in serum. 1. Extravascular
- plasma concentration is 3 mg/100 ml - includes physical constriction of injured skin and tissues resulting in the release of
8. FACTOR VIII tissue juice which contains tissue thromboplastin
- the antihemophilic factorrequired for adequate evolution of plasma thromboplastin 2. Vascular Aspect
(intrinsic pathway), usually absent or with molecular abnormality in hemophilia - includes, constriction of injured blood vessel brought about by reflex constriction
and in von Willebrand's disease. and by the serotonin released by disintegrated platelets
- trace protein with a plasma concentration of 1 ug/100 ml 3. Intravascular Aspect _ A '
9. FACTOR IX - physico-biochemical changes undergone by platelets and the interaction of the
- necessary for intrinsic thromboplastin generation. different coagulation factors.
- 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:

1. Factor II 3. Factor
2. Factor VII IX
4. Factor X
HEMOSTASIS
- process by which spontaneous or induced hemorrhage is stopped.
- spontaneously arrests the flow of blood from vessels carrying blood under pressure