INTRODUCTION AND GENERAL CONSIDERATIONS 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

2. It is red in color due to hemoglobin. 3. It is slightly alkaline with an average pH of 7.4. 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 hemocytoblastREC - 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 Myeloblast promyelocyte myelocyte metamyelocyte stab (band or staff cell) Segmenters (NEUTROPHIL, EOSINOPHIL, BASOPHIL) Monoblast promonocyte MONOCYTE Lymphoblast prolymphocyte LYMPHOCYTE Plasmoblast proplasmocyte PLASMOCYTE 2. Polyphyletic or Dualistic Theory - this theory, believes that there is a separate and distinct stem cell compartment for each of the blood cells. This was suggested by Sabin and co-workers and was concurred by Naegeli, Schilling & Downey. RETICULOENDOTHELIAL CELL Hemohistioblast Rubriblast prorubricyte rubricyte metarubricyte reticulocyte ERYTHROCYTE Myeloblast promyelocyte myelocyte metamyelocyte stab segmenters (NEUTROPHIL, EOSINOPHIL, BASOPHIL) 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 I. Mesoblastic Stage - The chief site of hemopoiesis is in the yolk sac. - The fetus is 2.25 to 5'mm in size. 0n the second week of fetal life there is the formation of blood islands wherein the primitive 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 transformation of peripherally located mesenchymal cells into endothelial cells. Thus, the primitive blood cells are now enclosed in endothelial-lined spaces. Upon further differentiation into cells .known as erythroblasts, and with the secretion of plasma, blood is established as a definitive somatic component. Leucocytes and megakaryocytes are seldom found during the earliest phase of the mesoblastic stage. 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, several nucleoli and homogenous basophilic cytoplasm. The PE elaborates hemoglobin to 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. II. Hepatic Stage - This starts on the second month; embryo is 5 to 7 mm in size. - On the 3rd month of fetal life, the liver becomes the chief site of hemopoiesis. It reaches peak activity during the 3rd or 4th month and remains active until a few months before birth. 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, 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 continues until the end of normal gestation and lymphopoiesis continues throughout life., 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.

Tissue hemohistioblast Monoblast promonocyte MONOCYTE 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

Extramedullary hemopoiesis is negligible except for lymphopoiesis in the spleen, lymph nodes and thymus. The first appearance of each cell type in the peripheral blood corresponds to maximal hemopoietic activity in the parent tissue. Early in fetal life many nucleated RBC are present. The number gradually decreases until at birth a normal infant never shows more than 10 per 100 leucocytes. Non-nucleated rbcs actually increase after which granulocytes, then lymphocytes and finally monocytes can be recognized in the peripheral blood. 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 production, if needed, must take place at extramedullary sites (liver, lymph nodes, spleen, thymus). In the adult, the bone marrow represents a weight of tissue at least equal to the weight of the liver. Normally, only about one-half of the total volume is active but even so an estimated 900 billion rbcs are produced daily. Under physiologic conditions, hemopoietic needs are met by mitotic division of the young cells of the marrow (homoplastic hemopoiesis). In case of increased requirements, there is mitotic division as well as multiplication of younger precursors (heteroplastic hemopoiesis). Dyspoiesis - profound defect in the maturation of rbc, wbc and platelets. HOW CELLS ARE RELEASED FROM BONE NARROW INTO THE CIRCULATION: 1. RBC - hypoxia and erythropoietin are the factors that regulate the rate of production of new erythrocytes in the bone marrow as well as the release of these cells into the circulation. 2. WBC - presence of chemotoxins in the blood will chemically tract WBC to go out into the circulation by the process known chemotaxis. - Chemotaxis is a directional locomotion in response to a chemical substance nearby 3. Platelets - produced and released by a shedding of megakaryocytic cytoplasm PRINCIPLES OF NORMAL CELL MATURATION 1. Cytoplasmic differentiation - loss of basophilia (more basophilia due to cytoplasmic RNA means less mature cell) - cytoplasmic granules (more granules means mature cell) - elaboration of hemoglobin for RBC (no hemoglobin means younger cell) 2. Nuclear maturation - structure and cytochemistry - round or oval nucleus: young cell - large nucleus/cytoplasm ratio: young cell.

- nuclear chromatin rich in DNA: young cell **nucleoli with RNA: Feulgen negative NATURE OF CHROMATIN- most important criterion to det age of rbc - chromatin strands coarse and clumped: mature cell - decreased number of nucleoli: mature cell - changes in shape - more lobulations: more mature cell 3. Reduction in cell size - smaller: more mature cell BLOOD VOLUME Blood volume determinations are important in the detection and treatment of fluid and electrolyte imbalances. Direct measurements of total blood volume have shown that blood makes up about 7 to 8% of the total body weight. Since direct measurements depend on complete exsanguination, most of available data refer to animal experiments. Laboratory methods, therefore, for determining blood volume, plasma volume and total RBC mass are necessarily indirect. a) For Plasma Volume determination - IV administration of a foreign substance which dilution in the plasma allows measurement of fluid volume: 1. Evan's blue dye 2. Congo red dye 3. I131 labeled human serum albumin (RISA) b) For Packed Cell Volume - administration of a substance that attaches to the erythrocytes and gives a measurement of erythrocyte mass: 1. Cr51 2. P32 3. radioactive Iron Normal Values Total Blood Volume (ml/kg) Plasma Volume (ml/kg) Packed Cell Volume (ml/kg) Male 76 (+ or - 8) 42 (+ or - 5) 35 (+ or - 4) Female 68 (+ or 6) 40 (+ or 4) 28 (+ or 3)

Decreased Blood Volume 1. loss of whole blood 2. loss of rbc 3. loss of plasma / water Terms 1. Normovolemia - normal blood volume 2. Hypovolemia - decreased blood volume

Increased Blood Volume 1. during excessive fluid intake 2. during blood transfusion 3. during IV injection of fluids 3. Hypervolemia - increased blood volume 4. Oligemia - total reduction of blood volume

BLOOD COLLECTION The basis of hematologic techniques is correct collection of blood sample and attention to precise methodology. lf the blood sample is not collected with proper attention to detail, the

whole hematologic examination is put into question. Blood examination should be performed in accordance with the following general guidelines: As much as possible, blood examinations should be done in the morning, in the fasting 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. Any repeat examinations should be done in the morning of the following day. Heavy meals as well as prolonged fasting can lead to appreciable leucocytosis. The 2 general methods of collecting blood for haematological studies are: a) Skin puncture b) Venipuncture SKIN PUNCTURE used when only small quantities of blood are required collection of blood from puncture made on skin blood obtained is known as: capillary blood peripheral blood arteriolar blood Sites of puncture: 1. margin of earlobe 2. palmar surface of the finger 3. plantar surface of heel and big toe Sites to avoid: 1. inflammed and pallor areas 2. cold and cyanotic areas 3. congested and edematous areas 4. scarred and heavily calloused areas

puncture site. 5. The values for red blood cell count, hematocrit, hemoglobin and platelets are lower in capillary blood, but higher white blood cell count by as much as 1,000/mm as compared to venousblood. VENIPUNCTURE easiest and most convenient method of obtaining enough volume of venous blood suitable for a variety of tests. - three factors are involved in a good venipuncture: a) the venipuncturist b) the patient and his veins c) the equipment Two methods of collecting blood by venipuncture: 1. Syringe Method 2. Vacuum Tube Method (Evacuated tube method) Advantages over the Syringe method: a) requires no prior preparation as it is a prepackaged sterile unit. b) offers a wider range of tube size and contained anticoagulants. c) safer method of blood collection as samples are taken directly into labeled tube d) avoidance of syringe breakage. e) disposable Anticoagulants in vacuum tubes: 1. Pink stopper - no anticoagulant; used for tissue or blood culture. 2. Red stopper - no anticoagulant; used for tests which require serum such as chemistry and .serological exams. 3. Amber stopper - no anticoagulant; used for blood lead determination. 4. Yellow stopper - no anticoagulant; used for bacterial culture and unknowns; can be Incubated or autoclaved. 5. Black stopper - 0.5 ml of 0.1M sodium citrate and collects 4.5 ml of blood for prothrombin time determination. 6. Black stopper - 1 ml of 3.8% sodium citrate solution and collects 9ml of blood for coagulation tests requiring plasma. 7. Blue stopper - 1 ml of 3.8% sodium citrate and collects 4 ml blood for sedimentation rate determination, Westergren method. 8. Blue stopper - dry mixture of potassium (4 mg) & ammonium oxalate (6mg) and collects 5ml for blood cell counts 9. Gray stopper - lithium oxalate; for blood chemistry determinations 10. Green stopper - heparin 286 U.S.P. sodium and collects 15 ml of blood for determination of serum iron concentration and total iron-binding capacity. Also for special blood tests like arterial blood gas and research studies. 11. Lavender stopper - 0.06 ml of l5% ethylenediaminetetraacetic acid (EDTA)and collects 7 ml of blood for blood cell counts and hematologic examinations.

Advantages of skin puncture - finger: Advantages of skin puncture - earlobe 1. easily accessible to the operator 1. Less pain (less nerve endings) 2. easy to manipulate. 2. More free flow of blood (thin skin) 3. ideal for peripheral blood smears 3. Less tissue fluid contamination (less muscle) 4. less intimidating. 4. Ideal when searching for abnormal cells (histiocytes in bacterial endocarditis) Disadvantages of skin puncture: 1. Less amount of blood can be obtained 2. Additional and repeated tests cannot be done. 3. Blood obtained by skin puncture lyses easily. Things to remember in doing skin puncture: . 1. Puncture should be 2.5 to 3 mm deep so as to hit the capillary bed, thus, ensuring free flow of blood. 2. Pressure and squeezing should be avoided to minimize a mixture of blood with tissue fluid which will affect the accuracy of the tests. 3. The first drop of blood is usually discarded since it contains tissue fluids and other foreign materials like dead epidermal cells. 4. When collecting blood for hematologic tests, the punctured finger must be wiped dry after each test since platelets will begin to clump immediately in the blood at the

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 older children 18 months to 3 years old: 1. femoral vein 2. long saphenous vein 3. popliteal vein 4. ankle vein

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. 2. AIDS (Acquired Immunodeficiency Syndrome) caused by HIV virus. - Two ways by which it is acquired: 1. through blood and its by-products 2. sexual contact with infected individual - incubation period may be from 5 to I5 years. - no known cure yet ANTICOAGULANTS Anticoagulants are usually chemical preparations added to the blood to prevent clotting. The correct choice and amount of anticoagulant are very important in making sure that the correct sample is prepared for a test. An insufficient amount may lead to partial clotting while too much liquid anticoagulant dilutes the blood sample. The incorrect choice of anticoagulant may lead to distortion of cells. I. OXALATES - prevent coagulation by combining with calcium to form an insoluble calcium oxalate salt. 1. dried Potassium Oxalate - distorts wbc and shrinks rbc - not ideal for ESR, hematocrit, blood smears and blood K determination - used at a concentration of 1-2 mg per ml of blood - dried sodium or lithium oxalate may be substituted for potassium oxalate 2. dried Ammonium & Potassium Oxalate (balanced oxalate, double oxalate, Winthrobe's solution, Paul-Heller's solution) Stock solution - dried Ammonium & Potassium Oxalate 1.2 gm of ammonium oxalate (3 parts) 0.8 gm of potassium oxalate (2 parts) 100.0 ml distilled water A - Ammonium oxalate swells rbc, Potassium oxalate shrinks rbc. However, when used together in a proportion of 3:2. they form a balanced action on rbc (no significant shrinkage or enlargement) - ideal for ESR, hematocrit and blood cell counts - not ideal for blood chem. determinations such as BUN, uric acid and K determination since it will increase the values. - not ideal for blood smears because the anticoagulant causes: a) nuclear degeneration of leucocytes; b) cytoplasmic vacuolation of granulocytes;

Advantages of Venipuncture: 1. Large amount of blood can be obtained for a variety of tests. Sample can be divided and treated as the prescribed test demands prescribed investigations demand. 2. Additional and repeated tests can be done. 3. Fastest method of collecting samples from a large number of patients. 4. Blood can be transported to the laboratory and stored for future use. 5. Blood collected is ideal for blood chemistry determinations. Disadvantages of Venipuncture: 1. Requires more time and skill on the part of the operator. 2. Requires more equipment. 3. More complications that may arise. 4. Hard to do on infants, children and obese individuals. Complications in Venipuncture: I. Local immediate complications application of tourniquet. 2. Failure of blood to enter syringe as in: a. collapsed vein which may be due to - nervousness - excessive pull of plunger of the syringe b. hitting the vein through and through c. hitting just the wall of the vein (as in sclerotic and movable veins) 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 by nervousness or shock. II. Local delayed complications: 1. Hematoma - inflammation and discoloration of surrounding tissues due to extravasation of blood brought about by trauma. 2. Thrombosis of the vein - formation of clot at the site of puncture due to trauma, 3. Thrombophlebitis - inflammation of vein at the site of puncture wherein a thrombus is present.

c) pseudolobulation and clumping of agranulocytes d) artifact formation in nuclei of lymphocytes and`monocytes; e)phagocytosis of oxalate crystals - used at 5ml (dried-to powder form) per 6 ml blood - decomposes at high temperature (>8O oC) from oxalates into carbonates II. CITRATES - prevent coagulation by combining with calcium in a non ionized form; - widely used for blood collection for transfusion since it is a relatively non-toxic salt that is rapidly used up by the body or excreted by the kidneys. 1. 3.8% Na citrate - used in forms of disodium and trisodium citrates for clotting mechanism and ESR. - Formula: Trisodium citrate - 1.32 gm Citric acid - 0.48 gm Dextrose - 1.40 gm Distilled water - 100.0 ml - 1 part.of 3.8% Na citrate added to 4 parts of blood can be used for ESR determination, Westergren method - 1 part of- 8.8% Na citrate added to 9 parts of blood can be used for coagulation Studies using plasma. 2. Acid Citrate Dextrose (ACD) Solution - 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: Trisodium citrate - 22.0 gm Dextrose - 24.5 gm Citric_acid - 8.0 gm Distilled water - 1,000.0 ml - 15 ml ACD is used for every 100 ml blood 3. Citrate- Phosphate- Dextrose (CPD) Solution III. EDTA (Ethylenediaminetetraacetic acid) - prevents coagulation by chelation (preventing Ca from ionizing) - may be used as dipotassium salt (Sequestrene) or as disodium salt (Versene) - used for blood cell counts including platelet count and also for preparing peripheral blood smears even after 3-4 hours - used at 1-2 mg/ml blood - if in liquid form,it is used at 0.1 ml of 10% aqueous solution of dipotassium EDTA per 5 ml of blood, it should, however, be evaporated to dryness Preparation of Sequestrene Solution: Sequestrene (dry powder) - 10gm

Distilled Water

- 100 ml

Advantages of Sequestrene 1. Dipotassium salt is preferred due to its solubility 2. Used in many hematologic tests even if blood has stood for many hours 3. Blood smears are well done, leucocytes and erythrocytes are morphologically well preserved even after 3 or 4 hours 4. Prevents surface adhesion and clumping of RBC, WBC and platelets 5. Prevents formation of artifacts even upon long standing IV. HEPARIN - prevents coagulation by acting as anti-thromboplastin and anti-thrombin - complex acidic mucopolysaccharide - best natural anticoagulant - best anticoagulant for the minimal hemolysis of red blood cells - ideal for ESR, hematocrit, RBC, osmotic fragility test and hemolytic disease of the newborn (HHN) cases - also used for special blood chemistry determinations such as pH determination and blood gas analysis - less toxic than Na citrate therefore it is often used for open heart surgery and exchange transfusion wherein large quantities of blood are given rapidly - available as sodium, calcium or ammonium salts - 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 units) per 10 ml blood.

Disadvantages: 1. Very expensive 2. Cannot be used for morphological studies of cells 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 V. FLUORIDES prevent coagulation by forming a weakly dissociated calcium component. anticoagulant with preservative action interfere with the enzyme system having to do with glycolysis ideal for CSF and blood dsugar determinations especially when there is likely onehalf to one hour delay between specimen collection and actual analysis used either as potassium fluoride or sodium fluoride potassium fluoride is considered the simplest preparation because just 2 drops of 40% aqueous solution of it is enough to collect 5 ml blood sodium fluoride is used in conjunction with potassium oxalate to prevent glycolysis for 24 hours or longer

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 evaporated to dryness. after evaporation, there should be 20 mg potassium oxalate and 25 mg sodium fluoride left in the tube or vial to prevent coagulation of 10 ml blood or 10 mg sodium fluoride per ml blood. Thymol (1 mg) may be added if bacterial action is to be controlled as in specimens that are sent to the laboratory by mail.

Hemocytometry numerical evaluation of formed elements of blood estimation of the number of blood cells in a known volume of blood Methods: I. Turbidimetric Method II. Microscopic Method a. Pipet Method b. Test Tube Dilution Method III. Automated Method (Impedance) a. Optical b. Electrical TURBIDIMETRIC METHOD based on the assumption that the more turbid the solution, the more cells are present in that solution" obsolete and very erroneous. MICROSCOPIC METHOD counting cells under the microscope with the use of the following materials: 1. counting chamber 2. pipets 3. diluting fluids

VI. DEFIBRINATION prevents coagulation by removing fibrins as they are formed 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 least 5-10 minutes by which time all the fibrin would have adhered to the beads or paper clips or hedgehog end of the rod ideal for: a) obtaining high serum yield from sample b) preserving the morphology of rbc & wbc. c) preparing smears from buffy coat for LE cell demonstration. VII. SILICONIZED GLASSWARES use of siliconized glasswares prevents coagulation by preventing the adhesion of platelets to wetable surfaces, thereby preventing breakage of platelets andsubsequent 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 minutes. EXAMINATION OF BLOOD AND ITS COMPONENTS The laboratory study of blood is divided into 2 groups: 1) Tests for the detection of diseases of the blood 2) Tests for the detection of diseases of other organs The laboratory tests for the detection of diseases of blood: 1. Chemical tests (haemoglobin determination and its abnormalities). 2. Morphologic tests Qualitative tests - determination of the structure and appearances of the cells as analyzed and classified. Quantitative tests - determination of the number of cells such as RBC count WBC count platelet count, reticulocyte count, differential count etc. THE COMPLETE BLOOD COUNT (CBC): - consists of 6 determinations, namely: 1. Red cell count 4. Hematocrit determination 2. White cell count 5. Differential count 3. Hemoglobin determination 6. Stained red cell examination

1. Counting chamber or haemocytometer A. According to type: 1. Open type (Spencer, Burker, Levy, Levy-Hausser) 2. Closed type (Thoma-Zeiss) 3. Addis 4. Exton 5. Petroff B. According to rulings: 1. Thoma 2. Tuerk 3. Fuchs-Rosenthal

4. Neubauer 5. Improved Neubauer 6. Bass-Jones

The most commonly used counting chamber is the Open Type Spencer and Improved Neubauer The counting chamber (hemocytometer) has 2 ruled areas etched on its surface, each consisting of a 3 mm square divided into 9 large squares, each with an area of 1 sq. mm. The central large square, which is used for RBC count, is subdivided into 25 intermediate squares, each with an area of 0.04 sq; mm. Each intermediate square is further subdivided into 16 small squares. Red blood cells are usually counted in the central and four corner intermediate

squares (R). The 4 corner large squares (W), each with an area of 1 sq. mm, are subdivided into 16 smaller squares and are used for WBC ct. Depth of the counting chamber is 0.1 mm. The area of one large square (1 sq.mm) and the depth of the counting chamber (0.1 mm). C Compute for the volume per large square: Volume = Area X Depth = 1 sq.mm X 0.1 mm= 0.1 cu.mm This volume of 0.1 cu.mm is always multiplied by 10 to give the contents of 1 cu. mm blood; thus 10 here is considered as the depth correction factor. 2. Pipets A. Automatic pipets ( Ex: Trenner, Unopette) microglass capillary pipets that automatically suck in just the right amount of sample. connected to a plastic container containing just the right amount of diluting fluid B. Non-automatic pipets (Ex: Thoma pipet) a. RBC Thoma pipet b. WBC pipet Comparison of the 2 pipets 1. size of bulb 2. color of bead 3. volume in bulb 4. size of bore 5. upper calibration RBC larger red 100 smaller 101 WBC smaller white 10 larger 11

opaqe 3. RUBRICYTE OR POLYCHROMATOPHILIC NORMOBLAST . characterized by the first appearance of hemoglobin, usually perinuclear, so that cytoplasm stains pink to basophilic. Size : 8-12 u Nucleus: round and smaller than in prorubricyte; usually eccentric; thick' nuclear membrane; coarse and clumped chromatin so that nucleus stains very dark; distinct parachromatin. Nucleoli: none Cytoplasm: more abundant than in precursors; varies from basophilic to diffusely lilac, depending upon the amount of hemoglobin. 4. METARUBRICYTE OR ORTHOCHROMIC OR ACIDOPHILIC NORMOBLAST fully hemoglobinated_cell; constitutes 50% of nucleated red cells in normal marrow Size : 7-10 u Nucleus: small and/shrunken; dense and dark staining because of marked condensation of chromatin. Parachromatin no longer distinguishable, may be round, oval or have various bizarre forms and is usually eccentric Nucleoli: none Cytoplasm: orange-red, as in adult erythrocyte 5. RETICULOCYTE constitutes 0.5 to 1.5% of circulating red blood cells. slightly larger than mature erythrocyte. after expulsion of the nucleus in metarubricyte, a large somewhat basophilic anuclclear cell remains, which, when stained with new methylene blue, a vital stain, is seen to contain a network of bluish granules or what is known as reticulum network. As cell matures the network becomes smaller, finer, thinner and finally disappears within 2-4 days. cytoplasm is pink and reddish brown. Size: 8-10 u, Nucleus : absent Cytoplasm: cell outline may be irregular because of shallow indentations; faintly polychromatophilic (basophilic) 6. ERYTHROCYTE 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 one-third of the cell area. NOTE: no hemoglobin yet in cells 1 & 2. cells 1 to 5 are normally seen only in the bone marrow except for cell 5 (retic) wherein 0.5 to 1.5% is seen in the circulation. cell 6 (erythrocyte) is definitely seen only in the circulation and it is eosinophilic/acidophilic in staining reaction due to hemoglobin.

RBC STUDIES NORMAL MATURATION SERIES 1. RUBRIBLAST OR NORMOBLAST makes up 5-10% of total nucleated cells in bone Size : 14-19 u Nucleus: Round or slightly oval; thin nuclear membrane; may be central or slightly eccentric; chromatin varies from finely reticular to coarsely reticular with a tendency to clumping; parachromatin is indistinct and scant. Nucleoli: 1 to 2; usually very faint and pale blue Cytoplasm: small in amount; moderately basophilic, homogeneous and opaque. 2. PRORUBRICYTE OR BASOPHILIC NORMOBLAST Size - 10-15 u Nucleus: smaller than in normoblast; generally round and slightly eccentric; thin nuclear membrane; chromatin is coarse and irregular so that nucleus stains dark; parachromatin is sparse but distinct Nucleoli: 0 - 1 Cytoplasm: appears more abundant than in normoblast because of smaller nucleus; varies from intense to moderately basophilic and is royal blue and

GENERAL RULES IN THE NORMAL MATURATIONl SERIES: the younger the cell is, the bigger in size and the bigger in nucleus the older the cell is, the smaller in size and the smaller the nucleus until eventually in erythrocyte no more nucleus is seen. RETICULOCYTE mature red blood cell; matures into erythrocyte in 2-4 days with reticulum network which are RNA and protoporphyrin remnants, the latter being responsible for the fluorescence of some erythrocytes when viewed by ultraviolet light reticulum network is demonstrable with the use of supra-vital stains only like brilliant cresyl blue and methylene blue. normally about 0.5 - 1.5 % (20,000 - 60,000/cu. mm) of all erythrocytes in adults are reticulocytes normal values at birth range from 2.5 - 6.5%, falling to the normal adult level by the end of the second week.

lifespan is around 100 days (+ or - 20 days) erythrocytes are uniform in size and have a small area of central pallor. in every 100-red blood cells, 3-8 platelets may be seen; in every 1000 red blood cells, there is 1 white blood cell

:Normal Values: Male Female: Conventional 5.5 - 6.5 million/ mm3 4.5 - 5.5 million/ mm3 SI 5.5 - 6.5 X 1012/L 4.5 5.5 X 1012/L

Erythrocyte is composed of a protein-lipid stroma that contains hemoglobin in solution and is condensed into a lipid-rich peripheral cell membrane. The cell membrane is non-elastic, although the cell can become distorted in order to pass through narrow capillaries. In order for normal erythrocytes to form, certain requirements must be met: 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 usable form; 3. Adequate amounts of protoporphyrinIll must be supplied; 4. There must be available sufficient globin. LIFE HISTORY OF THE ERYTHROCYTE: If the normal blood volume is taken to be 5L and the RBC count as 5,000,000 per mm blood, then a total of 25 x 1012 red blood cells are in active circulation at any one time. Their speed of travel varies in different parts of the vascular system, the whole circuit taking 45 seconds, on the average. lt has been computed that each rbc travels about 700 miles before disintegration NORMAL MECHANISM OF RBC DESTRUCTION: While the red blood cell is in the circulation its membrane is mechanically injured by constant buffeting in the blood stream and by alterations in its tension which is dependent on the ionic changes associated with oxygen and carbon dioxide exchange. Furthermore, as the red cell matures it becomes more spherical and this facilitates rupture of the cell membrane. Normally, the end stages of cell destruction takes place in the reticulo-endothelial system, particularly in the bone marrow, liver and spleen. The spleen has been long regarded as the main graveyard of rbc. ABNORMAL MECHANISM OF RBC DESTRUCTlON include various types of hemolysis, hemoglobin alterations and parasitic infections. RBC COUNT Six factors have statistically significant effect on the RBC 1. posture 4. age 2. extreme physical exercise 5. gender 3. severe dehydration 6. High altitude

RETICULOCYTE COUNT Degreeof reticulocytosis is proportional to erythropoietic activity. Retic count above normal indicate that erythropoiesis increased. The discovery of reticulocytosis lead to the recognition of an otherwise occult disease such hidden hemorrhage for unrecognized hemolysis. Persistently low reticulocyte counts particularly in the presence of anemia, suggest markedly defective erythropoiesis. RETICULOCYTE COUNT Physiologic increase: Low Count or Absent in: 1. at birth 1. Idiopathic aplastic anemia 2. menstruation 2. Acute benzol poisoning 3. pregnancy 3. In anaplastic crisis of hemolytic anemias Increased Count: 1. Hemolytic anemias 2. Kala-azar 3. Lead poisoning 4. Leukemia 5. Malaria 6. Erythroblastic anemias

7. Polyoythemia vera 8. Relapsing fever 9. Sickle cell anemia 10. Splenic tumor 11. Blood intoxication 12. Parasitic infections

Factors that determine number of reticulocytes in the circulation: 1. Speed of release of reticulocytes from bone marrowi into the circulation 2. Degree of immaturity of released reticulocytes; and 3. Speed of disappearance of reticulum network from the reticulocytes. ERYTHROCYTE mature red blood cell, non-nucleated, biconcave disc-like cell. 6.2 - 8.2 u in diameter and around 2.8 u in thickness

Increased RBC Count 1. polycythemia vera 2. chronic heart disease 3. lung diseases (TB, fibrosis) Decreased RBC Count 1. Anemia 2. hemorrhage 1 3. oligocythemia

4. dehydration 5. acute poisoning 6. residing in'a place of high altitude

CATABOLISM OF HEMOGLOBIN: When old red blood cells are destroyed in the RES, the globin portion of the hemoglobin molecule is split off, and the heme is converted into biliverdin. Most of the biliverdin formed from heme is converted to bilirubin which-is excreted in the bile. Subsequent degradation of 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. Hemoglobin Increased in: 1. hyperchromia 2. high altitude 3. polycythemia 4. dehydration in burns & diarrhea 5. heart diseases . 6. erythremia . Decreased in: 1. all anemias 2. leukemia 3. oligochromia

HEMOGLOBINOMETRY determination of the amount of hemoglobin in a known volume of blood screening test for the presence of diseases associated with anemia and for following the 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. HEMOGLOBIN (Ib) A main component of rbc; it makes up 35 % molecular weight of more than 64,400. gives red color to the blood so that it is also known as respiratory pigment. ` responsible for the oxygen and carbon dioxide-carrying capacity of rbc. 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. conjugated protein; made up of 1 globin molecule and 4 heme groups. globin molecule: 4 polypeptide chains and each chain is attached to 1 heme group-a ferroprotoporphyrin responsible for the red color of the compound, and consist of 4 protoporphyrinrings, each ring complexed with single bivalent iron atom. globin molecule has 2 pairs of polypeptide chains: alpha chain (in pairs) = 141 amino acids/chain = 282/pair beta chain (in pairs)' = 146 amino acids/chain = 292/pair Normal Values: Men Women ChildrenConventional 14 - 18 gm/100 ml blood 12 - 16 gm/100 ml blood 14 - 26 gm/100 ml blood S.I. 2.17 - 2.79 mmoles/L 1.86 - 2.48 mmoles/L 2.17 - 4.03mmoles/L

TYPES OF HEMOGLOBIN A. NORMAL or FUNCTIONAL HB 1. Oxyhemoglobin (HbO2) Hb + Oxygen = 0xyhemoglobin (in arterial blood; bright red) 2. Deoxygenated or Reduced Hb (HbCO2) Hb + Carbon dioxide = Deoxygenated or Reduced Hb (in venous blood; dark red) B. ABNORMAL OR NON-FUNCTIONAL HB - unable to act as an oxygen carrier. 1. Carboxyhemoglobin (HbCO2) Hb + Carbon monoxide.: Carboxyhemoglobin - the combination is irreversible. The affinity of Hb for C0 is 218x greater than for Oxygen at 37 C and is not affected if helium is substituted for nitrogen as the inert gas (as in prolonged underwater diving). Since carboxyhemoglobin is not capable of transporting oxygen, hypoxia results. Death may be due to anoxia, accompanied with irreversible tissue changes. - Carbokyhemoglobin produces a typical cherry red color of patient's blood and skin. - 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 blood. - measured by: a) Palmer's Carboxyhemoglobin Methods b) Sunderman Dithionite Spectroscopic Test (Na2S2O4 -Dithionite) 2. Methemoglobin (Hi) - also known as ferrihemoglobin, oxidized Hb and hemiglobin Hb + oxidizing agent = Methemoglobin - the reaction is reversible since methemoglobin is unstable. - differs from oxyhemoglobin in that it contains ferric, rather than ferrous

Hemoglobin in Normal Adult Blood 1. Hb A - 94.5% with 2 alpha and 2 beta polypeptide chains 2. Hb A2 - 3.5% with 2 alpha and 2 delta chains 3. Hb F- 2.0% with 2 alpha and 2 gamma chains Hb F is measured by: a) alkali denaturation test by Singer b) acid elution technic by Kleihauer-Betke.

iron; the oxygen being replaced by -OH. It is, therefore, oxidized Hb or ferriHb and results when Hb is exposed to oxidizing agents such as laniline dyes, potassium ferricyanide or potassium permanganate. - formation of methemoglobin is a normalprocess, kept within bounds by a normal intraerythrocytic system capable of reducing methemoglobin to hemoglobin again - Kravitz et al. give the ff. values for methemoglobin in normal subjects: 2.2% in premature infants 1 - 1.5% in infants up to 1-year old 1% in older children and adults. - critical level is 1.5 gm/100 ml blood and it gives a characteristic chocolate brown color to the blood 3. Sulfhemoglobin (SHb) Hb + sulfides = Sulfhemoglobin - reaction is irreversible since SHb is so stable that once formed, it. disappears from the circulation only after the rbcs containing it are naturally destroyed. .Erythrooytes containing SHb have normal survival and osmotic fragility. - normal concentration of SHb in the blood is 0 - 2.2% of total hemoglobin. - critical level is .5 gm/100 ml blood C. HEMOGLOBIN VARIANTS: 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

I. COLORIMETRIC METHOD A. Direct Visual Colorimetric Method 1. Tallquist Method 2. Dare's Hemoglobinometer 3. Acid Hematin Methods Methods: a. Sahli-Adams d. Hayden-Hausser M b. Sahli-Hellige e. Newcomer c. Sahli-Hayden f. Osgood-Haskin 4. Alkaline Hematin Method B. Photoelectric Colorimetric Method 1. Oxyhemoglobin Method 2. Cyanmethemoglobin Method (HiCN) (Drabkins Reagent) II. SPECIFIC GRAVITY METHOD A. Copper Sulfate Method III. GASOMETRIC METHOD oxygen content of blood is directly measured IV. CHEMICAL METHOD iron content of blood is measured ERYTHROCYTE SEDIMENTATION RATE 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)

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.

Winthrobe tube flat-bottomed with two calibrated sides: 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 Hematocrit after 30 minutes of centrifugation at 3,500 rpm. each calibration has 10 mm so there is a total of 100 mm in the tube. the bore is 3 mm in diameter. Normal Values: Male Adult : 0 - 9 mm/hr Female Adult: 0 -20 mm/hr Children : 0 -13 mm/hr Layers in Wintrobe tube after l hour a. Plasma layer - uppermost layer b. Buffy coat layer - middle layer which contains the platelets, WBC, and other cells such as L.E. cells and other malignant cells. 1mm of buffy coat contains about 10,000 WBC. ,It is whitish to cream in color. c. Packed red blood cells layer lowermost layer which contains nucleated young red cells on top and non-nucleated mature red cells at the lower layer. Correct ESR for Anemia: - 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 B: WESTERGREN METHOD most sensitive ESR method 'for serial study of chronic diseases 2 readings are done: after 1 hour and after 2 hours Anticoagulant -3.8% Na citrate Westergren tube Long tube with both ends open; calibration is up to 200 mm. bore is 2.5 mm in diameter Normal Values: Male Adult = 3 - 5 mm/1 hr 7 -15 mm/2 hrs Female Adult = 4 - 7 mm/1 hr 12 -17 mm/2 hrs C. GRAPHIC CUTLER METHOD 3.8% Na Citrate and Cutler tube D. LINZENMEIER METHOD 3.8% Na Citrate and Cutler tube E. MICROMETHODS 1. Micro Landau 5% Na Citrate and Micro Landau tube 2. Smith Micro Method 3. Crista or Hellige-Vollmer Method

F. ROARKE-ERNSTENE G. BRAYS METHOD STAGES OF ESR 1. Initial rouleaux formation (during'the first 10 minutes). 2. Period of rapid settling (during the next 40 minutes when settling is constant). 3. Period of-final settling or packing (during the last 10 minutes). FACTORS IN ESR: I. INTRINSlC FACTORS (inherent factors in rbc and plasma) 1. No. of RBC/mm3 (inversely proportional to_ESR) polycythemia = slower ESR anemia = faster ESR 2. Size of RBC (directly proportional to ESR) macrocytes = faster ESR microcytes = slower ESR 3.Viscosity of the pla na (inversely proportional) more viscous = slower ESR less viscous = faster ESR 4. Plasma protein contents All proteins (alpha-, beta- and gamma-globulin. and fibrinogen) except albumin will increase ESR.

II. EXTRINSIC FACTORS 1. Length of tube longer tube = faster ESR (because more space at the bottom wherein cells can settle down) 2. Diameter of tube: wider tube = faster ESR 3. Position of the tube slightly inclined = faster ESR (since the' RBC's have a shorter distance through which to settle down and the up-current of the plasma is along the slope, thus by-passing the sedimenting RBC's.) 4. Temperature (ESR increases with temperature) most ideal temp.= 2 - 27C (Room Temp.) 5. Pipeting - if with bubbles= faster ESR (since less blood and less rbc in tube) 6. Volume of blood more lood in tube = slower ESR less blood in tube = faster ESR 7. Anticoagulant: excess = slower ESR (since rouleaux formation is inhibited) 8. Time of setting up the test after 2 hours from blood collection = slower ESR (since red blood cells become spherical upon standing for a longer time) 9. Time of reading results before 1 hour = lower reading (since no final settling yet of red blood cells)

FACTORS FAVORING SLOW ESR: 1. Defibrination - removal of fibrin either by artificial or natural means. 2. Low temperature - this tends to slow ESR since there is increased viscosity of the blood. 3. Excess of dry anticoagulant - slows down ESR because rouleaux formation is inhibited. ' 4. Diameter of the tube - tubes with less than 2 mm in diameter will slow down ESR. HEMATOCRIT volume occupied by erythrocytes in a given volume of blood and usually expressed as volume of erythrocytes per 100 ml blood. venous hematocrit is the hematocrit of venous blood which represents the hematocrit of peripheral blood and does not indicate the proportion of erythrocytes to plasma in the entire circulation. hematocrit is the ratio of total erythrocyte mass to total blood volume and is calculated by a different method. used in determining erythrocyte indices, calculating blood volume and total erythrocyte mass, and establishing whether or not a patient is anemic, in as much as a low hematocrit indicates that the concentration of erythrocytes is reduced. hematocrit cannot completely replace RBC counts since the latter are needed to calculate some of the erythrocyte indices. for normal blood, hemoglobin and RBC counts may be estimated from the microhematocrit reading METHODS OF HEMATOCRITDETERMINATl0N: A. WINTHROBE METHOD Anticoagulant S double oxalate Winthrobe tube (same as tube used in ESR) Normal Values: Conventional: S.I. Adult Male 47 vol% (+ or - 5) 0. 47 (+ or - .05) Adult Female 42 vol% (+ or - 5) 0.42 (+ or - .05) B. HADEN'S MODIFICATION METHOD Anticoagulant = 1.1% Sodium oxalate in distilled C. VAN ALLENS METHOD Anticoagulant: 1.6% sodium oxalate in distilled water Tube: with bulb and calibrated 1-10 or 10-100 mm. D. SANFORD-MAGATH METHOD Anticoagulant = 1.3% Sodium oxalate tube = calibrated to 6 ml at 1 mm per division; tube is short (about 5 inches long) and with a funnel-like mouth. It has a-6 ml capacity. OSMOTIC FRAGILITY TEST semi-permeable characteristics of the surface membrane of an erythrocyte make it vulnerable to changes in the osmotic pressure of the external environment. -

Follows the Lawof Osmosis which states that there is a transfer of solution from lower to higher concentration so that: rbc + hypotonic solution will result to swelling of rbc and rbc + hypertonic solution will result to shrinking of rbc. compared to a normal biconcave erythrocyte, a spherocyte can imbibe in less water before it lyses because its cell membrane is already fully distended so we refer to it as a fragile cell. on the other hand, a sickle cell can take in more water before it lyses because its cell membrane can be distended more, so we refer to it as a resistant cell

METHODS OF 0SMOTIC FRAGILITY TESTS: 1. SANFORD METHOD. ' Principle: It tests the stability of red blood cells under different concentrations of hypotonic NaCl solutions. PRECAUTIONS IN ERYTHROCYTE OSMOTIC FRAGILITY TEST the blood sample should be obtained with a minimum of stasis and trauma test procedure should be set up as soon as possible the capillary pipet must be held in approximately the same angle so as to ensure uniform size of drops blood should fall directly on the saline solution and not on the dry sldes of the tubes ERYTHROCYTE INDICES important in assessing border line types of anemia values should be interpreted only in the light of other findings such as the appearance of erythrocytes on fixed smears computed using 3 determinations RBC count, Hemoglobin & hematocrit 1. MCV (MEAN CORPUSCULAR VOLUME) average volume of an individual red blood cell computed using hematocrit & RBC count x 10 2. MCH (MEAN CORPUSCULAR HEMOGLOBIN) ratio of Hb to RBC count x 10 average weight or amount of Hb in an individual RBC 3. MCHC (MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION concentration of hemoglobin in a given volume of packed red blood cells computed using Hb values over Hct x 100 USE OF ERYTHROCYTE INDICES: The MCV, MCH & MCHC are sometimes collectively referred to as red cell indices. These indices, in conjunction with the appearance of red blood cells in fixed smears, give an accurate picture of the morphology of the red blood cell. In summary, the determination of the MCV, MCH, and MCHC as valuable information 'that helps to characterize erythrocytes. According to the MCV, erythrocytes may be classified as normocytic, microcytic or macrocyticl. According to the MCHC, erythrocytes may be classified as normochromic or hypochromic. A higher than normal MCHC does not occur

except in cases of hereditary spherocytosis. MCH expresses only the weight of hemoglobin per erythrocyte. ANEMIAS - anemia is a disorder characterized by a reduction in the oxygen-carrying substance of a certain volume of blood due to a reduction below normal of RBC ct., Hb & Hct. Factors that determine the point at which the decreased oxygen-carrying capacity of the blood may produce symptoms of hypoxia are: 1. rapidity with which anemia develops 2. degree of physiologic adjustment to the anemia 3. effect of physical activity on oxygen demand. Hypoxia symptoms as well as their severity depend on: 1. how great an oxygen-carrying deficiency exists 2. how rapidly the anemia develops 3. the degree of physiologic compensation 4. level of physical activity when anemia develops rapidly, as in massive hemorrhage, the severity of the symptoms is proportional to the haemoglobin concentration. When anemia develops slowly, the patient can adjust to progressive hypoxia that symptoms will be minimal in spite of the very low Hb and RBC count. Symptoms are also proportional to physicalv activity. A patient at rest may not feel the symptoms even though markedly anemic, but on exertion may have weakness, dizziness and tachycardia. tachycardia occurs when the heart attempts to improve oxygenation of the tissues by increasing the heartbeat and cardiac output per minute. because these factors may vary, the diagnosis of anemia is only partially based on laboratory measurements of erythrocyte and Hb concentrations.. Laboratory data, however, must be interpreted with reference to the clinical picture.

2. MICROCYTIC NORMOCHROMIC blood picture shows small red cells with normal Hb contents. computation of Erythrocyte Indices shows: Decreased MCV & MCH but Normal MCHC seen in chronic inflammations. 3. MICROCYTlC HYPOCHROMIC blood picture shows small red cells that are pale in color due to decreased Hb computation of Erythrocyte Indices shows: Decreased MCV, MCH & MCHC seen in Thalassemia and severe iron deficiency anemia 4. MACROCYTIC NORHOCHROMIC 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 therefore, are normochromic. computation of Erythrocyte Indices shows: inc. MCV & MCH but Normal MCHC seen in Pernicious anemia

5. MACROCYTIC HYPOCHROMIC blood picture shows red cells that are larger than normal but are hypochromic due to decreased MCHC. computation of Erythrocyte Indices shows: inc MCV but Decreased MCH and MCHC. CLASSIFICATION OF ANEMIAS ACCORDING TO CAUSE I. Decreased production of red blood IV. Hemolytic anemia cells due to: A. Acute Stage A. Marrow damage - Hemorrhage 1. Leukemias B. Chronic Stage 2. Leukoerythroblastosis 1. Congenital 3. Aplastic anemia a. Red cell membrane defect B. Decreased erythropoietin - Hereditary spherocytosis 1. Inflammatory process b. Hemoglobinopathies 2. Renal disease - Hemoglobin C 3. Hypothyroidism - Hemoglobin S C. Iron-deficiency c. Enzyme defects II. Nuclear maturation abnormality - Glucose-6-phosphate A. Vitamin B12 deficiency dehydrogenase deficiency 1. Pernieious anemia 2. Acquired B. Folic acid deficiency a. Overactivity of the RES C. Refractory macrocytic anemia - Heinz Body anemia 1. Di Guglielmo's anemia b. Auto-immune hemolytic anemias III. Cytoplasmic m turation abnormality 3. Hereditary elliptocytosis A. Severe iron-deficiency B. Defect in globin production - Thalassemia C. Defect in heme synthesis

An adult person is said to be suffering from anemia if: For males: RBC ct. = < 4.2 million/mm3 Hb = < 12 gm/100 ml For females: RBC ct. = < 3.6 million/mm3 Hb = < 10 gm/100 ml MORPHOLOGIC CLASSIFICATIONS OF ANEMIA 1. NORMOCYTIC NORMOCHROMIC blood picture shows-red cells that are normal in size and normal in Hb contents. computation of Erythrocyte Indices shows: Normal MCV, MCH & MCHC seen in: hemodilution, hemorrhage, hemolytics anemia and aplastic anemia.

- Sideroblastic anemia COMMON ANEMIAS 1. APLASTIC 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. Blood picture: normocytic normochromic red cells, with normal MCV, MCH & MCHC. Reticulocytes are very few or none at all. Aside from the low RBC ct., there is leucopenia as well as thrombocytopenia. This decrease of all cell elements is known as "pancytopenia. Classifications of Aplastic Anemia According to Cause 1. Bone marrow injury 6. Metabolic inhibition of bone marrow 2. Congenital aplastic anemia 7. Erythroid hypoplasia of bone marrow in 3. Familial aplastic anemia hemolytic-disease 4. Chronic erythrocytic hypoplasia 8. Idiopathic aplastic anemia 5. Aplastic anemia assoc. with thymoma 9. Aplasia in myeloproliferative disorders 2. HEMOLYTIC ANEMIA due to the excessive destruction and shortened life span of red blood cells brought about by: a..intrinsie or corpuscular defects b. extrinsic or extracorpuscular abnormalities Etiologic Classifications of Hemolytic Anemia: I. Due to intrinsic (corpusoular) defects A. Defect of erythrocytic membrane 1. Hereditary spherocytosis 4. Zieve's syndrome 2. Elliptocytosis or ovalocytosis 5. Paroxysmal Nocturnal Hemoglobinuria 3. Acanthocytosis B. Defect of intracellular enzyme (non-spherocytic type of Hemolytie anemia) 1. Enzymes involved in anaerobic glycolysis - Hexokinase, Aldolase def. 2. Enzymes involved in hexose monophosphate shunt G6PD def. 3. Enzymes involved in methemoglobin formation glutathione synthase def. II. Due to extrinsic (extracorpuscular) defects A. Acquired autoimmune haemolytic anemia B. Acquired isoimmune haemolytic anemia C. Paroxysmal Cold Hemoglobinuria D. infectious agents Blood picture in hemolytic anemia: proportionate decrease of RBC, Hb and Hematocrit. the blood picture shows normocytic normochromic red blood cells computation of erythrocyte indices shows normal MCV, MCH & LCHC. increased nucleated red cells high reticulocyte count (10 - 2O%), increased osmotic fragility index, poikilocytosis and presence of'abnormal inclusion bodies such as Howell-Jolly bodies.

TYPES OF HEMOLYTIC ANEMIA 1. Paroxysmal Cold Hemoglobinuria cold hemolysin Donald-Landsteiner 2. Paroxysmal Nocturnal Hemoglobinuria Marchiafava- Micheli Syndrome 3. Sickle Cell Anemia 3. SICKLE CELL ANEMIA due to the presence of Hb S variant which causes sickling of red cells under reduced oxygen tension. Under normal oxygen tension, red cells appear normocytic normochromic so that all the three erythrocyte indices (MCV, MCH & MCHC) are normal under reduced oxygen tension, the normocytes will become sickle cells Sickle cells are crescent-shaped and they are also known as drepanocytes or meniscocytes. This sickling phenomenon is an irreversible reaction Vaso-occlusive crisis is observed since sickle cells will clog the bleed vessels especiallyiat the sites of the joints. swelling and too much pain in the occluded blood vessels and this may lead to patient's death patient's red cells also exhibit chronic hemolytic tendency. blood picture will also further show the following: anisocytosis, poikilocytosis (cigar-shaped, crescent-like, ovalocytes, acanthocytes, target cells), neutrophilia, thrombocytosis, decreased osmotic and mechanical fragility, presence of nucleated red cells and red cells with basophilic stipplings as well as abnormal inclusion bodies such as HowellJolly bodies, Pappenheimer bodies and Cabot's ring. commonly seen among the black race presence of homologous Hb S and S in the patient indicates sickle cell anemia whereas presence of heterologous Hb S and Hb A indicates sickle cell trait Methods for demonstrating sickling of cells in the laboratory: 1. Scriver and Waugh 2. Daland and Da Silva 3. Sherman's Test (also known as Test Tube Method) Principle Involved: The reduced form of Hb S which is not loaded. With oxygen is less soluble than normal 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 a sickle form which serves as a diagnostic criterion for the sickle cell anomaly and can be observed in blood preparation under the microscope. 4. THALASSEMIA due to the abnormal production rate of one of the polypeptide chains of hemoglobin molecule. also known as: a. Cooley's anemia b. Mediterranean anemia

c. Hereditary leptocytosis blood picture: microcytic hypochromic cells with a predominance of target cells. computation of erythrocyte indices shows decreased MCV, MCH & MCHC

4. Low excretlon of radioactive Vit B12 in the urine (Schilllng s test) 5. Low concentration of Vit B12 in the serum RBC ANOMALIES 1. ANISOCYTOSIS - variation in size of red blood cells ( sign of regeneration) Normocyte - 6.2 to 8.2 micra in diameter. Macrocyte - > 9 micra in diameter. Microcyte - < 6 micra in diameter. Megalocyte 12 to 21 micra in diameter 2. POIKILOCYTOSIS - variation in shape of red blood cells; seen in all forms of severe anemias, nephritis and bleeding peptic ulcers. May be graded 1+ to 4+ a. Acanthocyte - (thorn cell, spur cell) - thorny, rounded rbc with_irregularly arranged - 5-10 spicules of various lengths, some of which are bent at their tips. - indicate permanent cell damage found in abetalipoproteinemia b. Burr cell - reversibly spiculated elongated cells that are not synonymous with the non-reversible acanthocyte. Spicules usually are shorter than that seen in acanthocytes. c. Crenated RBC (echinocyte) - 10-30 short and blunt spicules that are evenly distributed over the surface of the red cell. - produced when blood is allowed to stand, when rbcs are exposed to lytic agents or when placed in hypertonic solutions. Not a clinically important phenomenon, except that it has to be differentiated from other similar but significant changes. d. Elliptocyte (ovalocyte) - elliptical or oval red cell, usually seen in pernicious anemia and in anemias Associated with malignant lesions. It may also be due to a congenital anomaly of rbc, which is Mendelian dominant and is not sex-linked. e. Blister cell - red cell with single or multiple vacuoles or markedly thinned areas at the periphery and comes as a result of trauma to red cells during their passage through partially obstructed blood, vessels. Indicative of microangiopathic hemolytic anemias. f. Helmet cell (keratocyte of Bessis, triangle cell) - irregularly contracted, triangular cell; usually half-moon shaped, the central depression flanked by two horns and results after the rupture of blister cell. g. Pyknocyte - distorted,`contracted, spiculated rbc similar to burr cell; normally present in small numbers in the first 2 to 3 of months of life and may be increased up to 50% in a condition known as infantile pyknocytosis. h. Sickle cell (drepanocyte, meniscocyte) - crescent-shaped rbc, elongated,. Drawn-out, slightly curved cell with pointed ends that resemble a sickle blade - sickling usually occurs in red cells containin Hb S which are exposed to reduced pH or oxygen tension. i. Spherocyte

Thalassemia major usually associated with increased Hb F, most severe expression of the thalassemia abnormality and is usually detected in childhood. Affected infants fail to thrive. Thalassemia minor associated with increased Hb A2, less serious type and is usually detected in adults Beta Thalassemia deficit of beta chain production relative to that of normal alpha production Alpha-thalassemia deficit of aprolpha chain duction relative to that of normal beta production In thalassemia, there is decreased HbA but increased HbF and Hb A2 5. SEVERE IRON DEFICIENCY ANEMIA due to the depletion of iron storage in the body needed for normal hemoglobin produition. Etiologic factors in iron deficiency A. Negative iron balance 1. Decreased iron intake 2. Increased iron loss a. Gastrointestinal bleeding b. excessive menstrual flow c. blood donation d. hemoglobinuria B. Increased Requirements 1. pregnancy 3. lactation 2. infancy

e. self-induced bleeding f. Pulmonary hemosiderosis g. Hemorrhagic telangiectasia h. Disorders of hemostasis

PERNICIOUS ANEMIA lack of intrinsic factor needed for the normal absorption of Vit. B12 and folic acid. also known as: a. Macrocytic anemia b. Megalocytic anemia c. Addisonian's disease blood picture shows macrocytes and megalocytes that are normochromic so that MCV & MCH are increased but MCHC isa normal. There is a three-fold increase in erythropoiesis in the bone marrow. However, the rate of release of young cells from bone marrow into the circulation remains the same as during normal erythropoiesis. Laboratory diagnosis of pernicious anemia 1. peripheral blood smear 2. Achlorhydria after histamine stimulation 3. Megaloblastic dysplasia of bone marrow

- 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 betathalassemia 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

TERMS DESCRIBING RBC INVOLVEMENT: 1. Po1ycythemia general term for the increase) of red cell concentration in the peripheral blood to above normal (>6.5 mil/mm3 of blood) may be primary (unknown etiology) or secondary (accompanies a variety of conditions), may be a temporary or permanent condition. 2. Polycythemia vera also known as primary polycythemia, polycythemia rubra, erythremia, Vaques disease, Osler's disease, Vaquez-Osler disease, megalosplenica and crytogenic polycythemi etiology not known but it is essentially a myeloproliferative disorder. characterized by abnormal proliferation of erythroids, myeloids and megakaryocytes in the bone marrow (condition is known as panmyelosis) as well as increased erythrocytes, leucocytes and thrombocytes in the circulation (condition is known as pancytosis) 3. Erythremia - increase in -red blood dell count above 6.5 mil/mm3 of blood due to polycythemia vera. 4. Erythrocytosis - increase 'in red blood cell count above 6.5 mil/mm3 of blood. due to chronic heart diseases, lung diseases and high altitude. 5. Oligocythemia - general decrease of red blood cells in the circulation as well as in all rbcforming organs in the body. WHITE BLOOD CELL STUDIES LEUCOCYTE larger than red blood cell, measures 9-1 u in diameter, nucleated lifespan of 5-8 days provides with natural immune defense mechanisms by means of phagocytosis and production of antibodies (Ab) and proteolytjc enzymes 2 Types of WB 1. Granular WBC - Neutrophil - Eosinophil - Basophil

22,800/mms) and declines gradually until the adult level is reached at about 21 years of age (average of 7,500/mmg). There tis no significant change after this point 1. Leucocytosis - increase above normal in the number of leucocytes in the peripheral blood a) Physiologic leucocytosis - caused by conditions that are physiologic such as exercise. b) Pathologic leucocytosis - caused by disease; always accompanied by an absolute increase in one or another type of WBC making the terms: b1) neutrophilic leucocytosls (neutrophilia) b2) lymphocytic leucocytosis (lymphocytosis), b3) monocytosis b4) eosinophilia b5) basophilia 2. Leucopenia - a decrease below normal in the number of leucocytes in peripheral blood. 3. Leukemia - malignant increase in the number of leucocytes in the peripheral blood as well as in leucocyte producing organs. Increased WBC (leucocytosis) 1. infections 2. inflammations 3. in the afternoon 4. after strenuous exercise 5. after meals S. during menstruation 7. in newborns Decreased WBC (leucopenia) 1. in the morning 2. in older people above 40 3. in measles 4. leucopenia of viral origin E. prolonged use of sulfa drugs 6. replacement of marrow with malignant disease 7. hypersplenism

2. Agranular WBC - Lymphocytes - Monocytes

NORMAL MATURATION SERIES: I. GRANULOCYTIC SERIES 1. MYELOBLAST Size : 10 - 20u; round or oval Nucleus: Round or oval, thin nuclear membrane; abundant chromatin (light purple); sparse parachromatin (pale-blue or pink) Nucleoli : 2 to 5;well-outlined;round or oval, pale blue Cytoplasm : sparse; nucleus-to-cytoplasm ratio = 5:1 to 7:1; deeply basophilic; no granules 2. PROMYELOCYTE (PROGRANULOCYTE} Size : 14 to 20 u; round or oval Nucleus : large, round or oval; thin nuclear membrane; slightly coarse and networklike chromatin Nucleoli : 1 to 3; less prominent; round or oval; pale blue Cytoplasm : sparse, nuc-to-cytop ratio = 5:1; basophilic but higher than myeloblast; few purplish granules that may be large and round or fine 3. MYELOCYTE Size: 10-18 u; round or oval

Normal Values: For both male and female adults = 5,000-10,000/mm3 in Sl = 5~lO X lflg/L Leucocyte count is normally higher in the newborn infant (average of 18,100/mm3) 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

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

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

Size : 14-18u Nucleus : moderately indented; thin nuclear membrane; fine, threadlike chromatin; abundant parachromatin Nucleoli: 0-1 Cytoplasm: gray-blue; opaque, with very fine lilac granules (azurophilic dust which indicate cell maturation) 3. MONOCYTE Size : 12-18 u Nucleus : indented or folded-over or bean-shaped; delicate;d pale staining; fine strands of reticulated or lacy in appearance chromatin; abundant and distinct parachromatin. Nucleoli: usually none, occasionally one Cytoplasm: light gray or gray blue, opaque; characteristic numerous, fine, dust-like lilac granules BRIEF DESCRIPTION-OF WBCS NORMALLY SEEN IN PERIPHERAL BLOOD: I. GRANULOCYTES (produced mainly in BM) 1. NEUTROPHIL 9 -15u, nucleus usually trilobulated (2 - 5 lobes) with abundant fine lilac pink granules and coarse and clumped chromatin pattern. normally produced in the bone marrow and released in response to various stimuli circulate in the peripheral blood and are sequestered in various organs for variable periods before they are destroyed. average lifespan is 10 - 14 days (4 days in the bone marrow in the course of maturation, 3 - 4 days represent the total time spent in the circulating_blood, for periods of only 2 or 3 hours at a time, and the remaining hours spent in sequestration in .various organs actively phagocytic, ingesting bacteria and other foreign particles both in vivo and in vitro. After ingestion, bacteria are destroyed by proteolytic enzymes in the neutrophil after which the neutrophil dies and fragmentst into particles that are removed by fresh phagocytes. show active ameboid locomotion. When this movement- is directional, in response to a chemical substance nearby, it is called CHEMOTAXIS. Chemotaxis is governed by products released at the site of inflammation as well as directly by bacteria. Leucocytosis-promoting factors (LPF) such as leucotaxine, may also be active in chemotaxis. In fact, leucotaxine has been shown to attract neutrophils and monocytes in vitro. Pathologic neutrophiliai or pathologic neutrophilic leucocytosis (increase in neutrophils) may be due to the following:

acute appendicitis , tonsillitisf meningitis 2. Intoxications A. Metabolic uremia acidosis diabetes, etc. B. Chemical epinephrine lead mercury arsenic kerosene, etc. 3. Tissue necrosis from any cause myocardial infarction gangrene extensive burns 2 to_bacterial invasion benign and malignant neoplasms

C. Insect venoms black widow spider D. Reaction to parenterally given foreign protein A E. Reaction to parenterally given bacterial vaccines

6. Myeloproliferative disorders 7. Myelocytic leukemia

4. Acute hemorrhage 5. Acute hemolysis hemolytic transfusion reactions acute hemolytic anemia chronic hemolytic anemia 2. EOSINOPHIL 9-15u, nucleus usually bilobulated with coarse, clumped chromatin pattern. cytoplasm contains large reddish-orange granules function remains obscure recent work has shown that eosinophils actually have antihistaminic activity as opposed to the old belief that eosinophils contributed to allergic reactions as carriers of histamine. capable of phagocytosis, in the course of which there is degranulation as in the neutrophils. Eosinophilia is observed in : - allergies - hemopoietic disorders such as: - skin disorders (non-allergic) polycythemia vera, Hodgkin's disease, - parasitic infections pernicious anemia, sickle cell anemia - after splenectomy 3. BASOPHIL 9 - 15 u; with most varied-shaped nucleus (may be indented,bi-lobulated or stab form); usually indistinct as it is covered with large purple or purplish black

1. Acute infections by pyogenic bacteria and other organisms: A. Localized or limited such as: B. Generalized such as: abscesses acute rheumatic fever osteomyelitis septicemia peritonitis cholera empyema, etc.

granules. Its cytoplasm contains large purple or purplish black granules. rarest leucocyte in the peripheral blood. functional importance is probably minor contains heparin and large amounts of histamine Basophilia may be observed in the ff: - chronic myelocytic leukemia - erythroderma - polycythemia vera - urticarla plgmentosa - myeloproliferatlve dlsorders - ulcerative colitis

Il. AGRANULOCYTES 1. MONOCYTE largest cell in the circulation probably the circulating counterpart of the blood tlssue macrophage with large semi-indented or bean shaped nucleus with superimposed brainlike convolutlons; nuclear chromatin retlculated or lacy in appearance cytoplasm is abundant grayish or muddy blue in color often described as having a ground glass appearance functions: the cell s phagocytic function is related to its immunologic activity which consists of 2 phases: a. durlng the inductlon of lmmunlty when antigenic informatlon is transferred to lymphocytes b. during expression of cellular responses and the development of delayed hypersensltivity contains large amounts of lipase thus specifically endowed to combat bacilli with lipoid capsule such as Mycobacterium tuberculosis and leprae

Functionally lymphocytes can be divided into B-lymphocytes derived from the bone marrow or the bursa equivalent organ in humans T-lymphocytes derived from the thymus. Morphologically, T and B lymphocytes cannot be distinguished in WrightGiemsa stained smear. Lymphocytes that lack both T & B cell markers are called null cells. Small lymphocytes that are short-lived (3-4 days), upon antigenic stimulation, become immunoglobulin-producing B lymphocytes which are the precursor 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 helper cells. The following distribution of T and B cells is found in the peripheral blood of healthy adults: noncommitted null cells = 0.5 - 1% B cells = 7 - 23% T cells = 70 - 90% Lymphocytogenesis is a function of the lymph nodes, bone marrow, spleen, and scattered .lymphoid tissues in the body. The bone marrow is one of the largest lymphocyte-containing tissues in the body.

Pathologic Lymphocytosis: 1. Acute viral and Non-Viral infections - infectious mononucleosis - infectious lymphocytosis - mumps, chicken pox - german measles

2. lymphocytic leukemia 3. infectious hepatitis 4. Congenital syphilis 5. Pertussis 6. Brucellosis

Monooytosis may be observed in the ff: - Chronic Ulcerative Colitis - Monocytic leukemia - Subacute Bacterial Endocarditis - Collagen diseases - Trypanosomiasis - Brucellosis - Pulmonary TB - Typhoid - Gauchers disease - Rickettslal lnfections - Kala-azar 2. LYMPHOCYTE size: small = 6-8u, medium= 8-14u, large 8-18u presence of small, medium and large lymphocytes in the blood of the adult and the predominance of medium and large lymphocytes in the blood of infants are common observations. Lymphocytes larger than polymorphonpclear cells are classified as large lympho cytes and the smaller cells as small lymphocytes, morphologic difference lies mainly in the amount of cytoplasm most small lymphocytes are T-cells and most large lymphocytes are B lymphocytes. In Wright s stained cell, the nucleus is deep purplish blue and is round to oval to kidney- shaped or deeply indented usual eccentrically located and occupies nine tenths of the cell diameter so that the sky-blue cytoplasm is hardly seen.

ABNORMAL WHITE BLOOD CELLS 1. SMUDGE CELL (BASKET CELL) ruptured WBC with bare nucleus, few may be seen in normal blood smear due to improper and forceful smearing - large numbers may be seen in leukemia and their presence may indicate increased fragility of the cells or abnormal destruction of cells, not counted 2. HYPERSEGMENTED NEUTRDPHILS also known as macropolycytes and PA Polycell usually larger than a normal neutrophil and nucleus has 5-10 lobes instead of the normal 3 lobes usually present in pernicious anemia the number of hypersegmented neutrophils in the differential white cell count should be reported under miscellaneous white cells 3. VACUOLATED CELL with holes or vacuoles in the cytoplasm which are signs of degeneration and If seen in smears made from fresh blood it should be counted and reported under miscellaneous white blood cell may also be found in normal blood smears if the smear is made from oxalated blood which is over 2 hours old.. It may be seen in severe infections, chemical poisoning 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 predominant cell is a blast, cannot be identified at the time of diagnosis 2. Lymphocytic leukemia acute lymphocytic leukemia - predominance of lymphoblasts chronic lymphocytic leukemia - predominance of mature lymphocytes 3. Promyelocytic leukemia preponderance of atypical progranulocyte containing large irregular azurophilic granulation. 4. Chronic myelocytic leukemia (CML) predominance of neutrophilic, eosinophilic and basophilic types that are variants and need not be classified separately. Philadelphia (Phl) chromosome is present in 7090% of patients with CML. 5. Neutrophilic leukemia preponderance of neutrophilic myelocytes. 6. Eosinophilic leukemia preponderance of eosinophilic myelocytes. 7. Basophilic leukemia preponderance of basophilic myelocytes 8. Monocytic leukemia (occurs in two forms that are basically not related) Naegeli type (myelomonocytic leukemia) - variant of myelocytic leukemia and may resemble granulocytic leukemia. Cytologically, myelomonocytes show the nuclear features of monocytes and the cytoplasmic features of myeloid cells. Schilling's type (histiocytic leukemia) - true monocytic leukemia since it is characterized by primitive cells having the same general features as a true blood monocyte and its tissue counterpart, the histiocyte. 9. Plasmacytic leukemia and multiple myeloma myelomas are neoplasms of proliferating plasma cells. They may present as solitary tumors involving a bone (solitary myeloma), or as neoplasms involving the BM and other organs (multiple myeloma). 10. Plasma cell leukemia terminal leukemic phase of multiple myeloma. 11. Mast cell leukemia rarest; leukemic cell is the,tissue mast cell. Mast cells are essentially tissue basophils in the bone marrow. They arise from basophils by a process of differentiation and replication 12. Erythremic myelosis (Di Guglielmo's disease) Pure erythroblastic proliferation. may progress to a mixed erythroblastic myeloblastic phase and terminate as an acute myeloblastic leukemia 13. Erythroleukemia (Di Guglielmo's syndrome) erythroblastic and myeloblastic proliferation. 14. Chronic neutrophilic leukemia preponderance of adult segmented neutrophils and band neutrophils. 15. Leukemias of uncertain type a) Reticulum cell leukemia b) Leukemic reticuloendotheliosis

c) Histiocytic.medullary reticulosis (Robb-Smith) LEUKEMOID REACTION Leukemoid Reaction is not a disease or diagnosis but a descriptive term to indicate a blood picture in which the peripheral blood findings resemble those found in leukemia, with the important difference that while leukemia is a neoplastic proliferation and malignant in nature, leukemoid reactions are benign proliferations. LRs may be due to infections, intoxications, tumors and acute hemorrhage. LR mimics leukemia and may show the same quantitative and qualitative changes differentiated from leukocytosis in the sense that its WBC count is usually above 50,000/mm3 and its blood smear shows more immature cells. It is the presence of immature cells as much as the high WBC count that gives the leukemoid blood picture the appearance of leukemia. As a rule, LR is not accompanied by the anemia or thrombocytopenia that is common in leukemia. CONDITIONS THAT MAY CAUSE LEUKEHOID REACTIONS: I. Infections A. Myelocytic reactions 1. Pneumonia 6. Bubonic plague 2. Empyema 7. Septicemia 3. Endocarditis 8. Tuberculosis 4. Meningitis 9. Leptospirosis 5. Diphtheria . B. Lymphocytic reactions 1. Whooping cough 2. Chicken pox 3. Mumps 4. Infectious mononucleosis

5. Infectious lymphocytosis 6. Congenital syphilis 7. Tuberculosis

PLATELET STUDIES non-nucleated, irregular in size (1~4 u), irregular in shape, lifespan of,4 - 9 days 3 main functions: hemostasis, blood coagulation, clot retraction INCREASED PLATELET COUNT 1. Polycythemia vera . 2. Myeloproliferative syndrome' 3. Splenic vein thrombosis 4. Postsplenectomy states 5. Acute blood loss 6. Carcinomatosis 7. Nephrotic syndrome without uremia 8. Chronic myelocyticwleukemia 9. Acute alcoholic hepatitis DECREASED PLATELET COUNT 1. Pernicious anemia 2. Aplastic anemia ' , 3. Infectious diseases 4. Lesions involving the bone marrow 5. Idiopathic 6. Acute leukemia

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

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

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.

1. Due to immunologic or hypersensitivity mechanisms - Idiopathic Thrombocytopenic Purpura (ITP) platelet destruction as_a result of an immunologic process. 2. Hypersensitivity to certain. drugs - apronalide, guinine, quinidine. 3. Due splenomegaly and increased sequestration in the spleen - Conditions above produce thrombocytopenia by sequestering normal and undamaged platelets in the spleen. An enlarged spleen usually traps many normal cells and these cells are eventually destroyed due to normal lytic splenic mechanisms acting for a long period of time on cells that are sequestered. 4. Due to sequestration but not in spleen - Sequestration of platelets in tumors and trapping of platelets in the fibrin network Deposited throughout the vascular system in the syndrome characterized by diffuse Intravascular coagulopathy 5. Due to mechanical destruction - Extracorporeal circulation of blood produces moderate destruction of platelets. The cause is primarily mechanical (trauma, adhesion to tubing, artificial cardiac valves, etc). 6. Due to miscellaneous factors - massive hemorrhage i - massive transfusion of platelet-poor blood - chronic alcoholism B. Due to deficient production 1. Bone marrow suppression of thrombocytopoiesis a. Potentially myelotoxic drugs such as antifolates, nitrogen mustard, chloramphenicol, gold salts, DDT, organic chemicals, etc. ` b. Physical and animal agents - ionizing radiation - artificial fever - heat stroke - burns - insect bites c. Bone marrow replacement of abnormal cells (metastatic tumor, lymphomas, etc.) d. Congenital, neonatal, or familial thrombocytopenic syndromes - Wiskott-Aldrich syndrome - May-Hegglin anomaly

solidifying of fluid blood (whole blood or plasma) brought about by the different coagulation factors formation of a visible coagulum, which is the physical manifestation of fibrin formation, represents only the end result of an intricate series of reactions that involve a number of coagulation factors. coagulation factors have an international standard nomenclature

NOMENCLATURE & SYNONYMS FOR COAGULATION FACTORS I II III IV VI (ACG) VII Fibrinogen Prothrombin Tissue factor Calcium ions Proaccelerin Proconvertin ----------------------------------------------------------------Thromboplastin,Thrombokinase --------------------------------Labile factor, Thrombogen, Accelerator globulin

VIII IX X XI XII XIII

Stable factor, Serum Prothrombin Conversion Accelerator (SPCA), Autoprothrombin I, cothromboplastin Antihemophilic Antihemophilic globulin (AHG), Anti- hemophilic factor (AHF) factor A, Platelet cofactor 1, Thromboplastinogen Plasma Thromboplas- Christmas factor, Antihemophilic factor B, Platelet tin Component (PTC) cofactor 2, Autoprothrombin II Stuart-Prower factor Prower factor, Autoprothrombin III Plasma Thromboplas- Antihemophilic factor C tin Antecedent (PTA) Hageman factor Glass factor, contact factor Fibrin stabilizing Fibrinase, Laki-Lorand factor factor-(FSF)

THROMBOCYTHEMIA A. Thrombocythemia hemorrhagica primary disease characterized by a persistent increase in platelets and frequently associated with bleeding tendency. B. Secondary thrombocythemia Seen in polycythemia and in chronic granulocytic leukemia. STUDY OF BLOOD COAGULATION AND HEMOSTASIS COAGULATION

GENERAL CHARACTERISTICS OF THE COAGULATION FACTORS 1. FIBRINOGEN protein clotted by thrombin in the formation of fibrin, plasma concentration is 250 400 mg/100 ml. formed in the liver and possibly also in the RES. 2. PROTHROHBIN ' ` proenzyme, the precursor of thrombin and functions in thecommon pathway of coagulation, plasma concentration is 10 - 15 mg/100 ml. ' 3. THROMBOPLASTIN (TISSUE FACTOR) . ' substance that, in the presence of. calcium ions, brings about the conversion of prothrombin to thrombin. ' a) tissue thromboplastin: from tissue extracts and it is the prothrombin activator in the extrinsic system b) plasma thromboplastin: prothrombin activator in the intrinsic system 4. CALCIUM normally exists in the blood in an ionized form and combines with plasma proteins and lipids, needed in prothrombin conversion and plasma thromboplastin generation. 5. THROMBIN'

active agent (enzyme) that clots fibrinogen. Its action is so powerful that it can clot several hundred times its weight of fibrinogen. As.an enzyme, it splits the arginylglycyl bands at the N terminal of the fibrinogen molecule to form fibrin. 6. FACTOR V called "accelerator" because it speeds up the conversion of prothrombin to thrombin in the presence of Ca ions .and tissue thromboplastin in the common pathway of coagulation, occurs in the plasma of all normal persons and is synthesized in the liver 7. FACTOR VII needed in the conversion of prothrombin to thrombin but only in the extrinsic system. Chief difference between factors V &'VII is that factor V can be obtained from plasma, while VII is present in plasma but is more active in serum. plasma concentration is 3 mg/100 ml 8. FACTOR VIII the antihemophilic factorrequired for adequate evolution of plasma thromboplastin (intrinsic pathway), usually absent or with molecular abnormality in hemophilia and in von Willebrand's disease. trace protein with a plasma concentration of 1 ug/100 ml 9. FACTOR IX necessary for intrinsic thromboplastin generation. plasma concentration is trace amounts only 10. FACTOR X proenzyme that is important in the formation of prothrombinase in the common pathway of coagulation. It is activated by the products of both the. intrinsic and extrinsic pathways. Plasma concentration is 1.2 mg/100ml 11. FACTOR XI proenzyme that is essential in the intrinsic pathway of coagulation.. Little is known regarding its biochemistry although it is stable in plasma and serum. 12. FACTOR XII activated by contact with foreign surfaces, and initiates the intrinsic pathway of coagulation. It is also involved in the activation of fibrinolysis and in the plasma kinin system, plasma levels of this factor usually normal even in severe liver disease. 13. FACTOR XIII the enzymatic form of Factor XIII acts in the common pathway of coagulation where it forms stabilizing covalent bonds with fibrin strands. It is also involved in wound healing. Vitamin K-dependent coagulation factors are the following: 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

entire mechanism by which bleeding from an injured blood vessel is spontaneously controlled and stopped

Mechanical Hemostasis stops bleeding with the use of tourniquet; surgical or a first aid problem. Physiologic hemostasis brought about by interaction of several factors, hemorrhagic disorders due to deficient physiologic hemostasis are a laboratory concern. Three Aspects of Hemostasis ` ` 1. Extravascular includes physical constriction of injured skin and tissues resulting in the release of tissue juice which contains tissue thromboplastin 2. Vascular Aspect includes, constriction of injured blood vessel brought about by reflex constriction and by the serotonin released by disintegrated platelets 3. Intravascular Aspect _ A ' physico-biochemical changes undergone by platelets and the interaction of the different coagulation factors.