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HSC- 128 PARASITOLOGY AND BLOOD CELL DISORDERS

LIST OF PRACTICALS

Sr.No. Topic Page No. 1. Routine examination of feces. 2

2. Gross examination and Physical examination of stool. 4

3. Concentration method of microscopic stool examination. 5

4. Microscopic examination of stool specimen. 6

5. Detection of Malarial parasite. 8

6. Detection of Trypanozomes (The casual agent of sleeping sickness).

9

7. Laboratory diagnosis of Kala Azar. 10

8. Laboratory diagnosis of Microfilaria (Wuchereria bancrofti). 11

9. Quantitative determination of Serum (or plasma) IgG class antibodies to Toxoplasma gondii by Elisa.

13

10. Determination of IgM class antibodies to Toxoplasma gondii by Elisa.

15

11. Preparation of staining of blood smear. 16

12. Study of morphology of blood cells. 19

13. Blood cells disorder in leukemia. 22

14. Screening for sickle cell anemia. 29

15. Determination of Osmotic fragility of red blood cells. 31

16. Determination of fetal Hemoglobin. 34

17. Preparation of Lupu8s Erythromatosus (LE) cell. 37

18. Preparation of Heinz bodies. 38

19. Microscopic examination of bone marrow smear. 39

20. Detection of presence of Iron in the bone marrow smear. 41

21. Laboratory tests for diagnosis of Aplastic anemia. 42

22. Investigations of Megaloblastic anemia. 44

23. Laboratory tests in Iron deficiency anemia. 46

24. Laboratory tests for diagnosis of Hemolytic disorders. 48

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Practical No.: -1

ROUTINE EXAMINATION OF FECES Collection of stool specimen: - The various precautions taken are as follows: (1)- Collection of sufficient quantity: Morning specimen (at least about 5-6 ml. Capacity) is collected in a 50 ml. Clean and dry container. (2)- Dry containers (50 ml. Capacity) The various types of containers are used are: (A)- Disposable wide mouth plastic bottle or glass bottle. (B)- Waxed carbon box (C)- Glass jar with fitting lid. Precautions taken after collection :- (1)- The specimen container should be labeled properly. (2)- Stool specimen should not be left uncovered. It is necessary to prevent drying effect. (3)- Stool specimen should be examined within 1 hour of collection. (4)- Specimen should be disposed properly after the examination. Preservation of specimen :- The preservative preserves protozoan morphology and further development of certain helminth eggs. For the preservation of the specimen following formalin saline reagent can be used. = 10 % (v/v) formalin saline

-(commercial 40 %) formaldehyde: 25 ml. -Normal saline: 75 ml. Mix and store in a clean and dry container. Use: - Mix 3 parts of 10 % formal saline with one part of stool.

Laboratory Investigations: - The various aspects studied for stool examination are as follows: (1)- Gross and Physical examination by visual observation: (A)- Consistency (B)- Color (C)- Mucus (D)- Blood (E)- Parts of parasite (F)- Adult parasite

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(2)- Chemical examination: (A)- Reaction (B)- pH (C)- Occult blood (3)- Microscopic examination: (A)- Protozoan (B)- Nematohelminths (C)- Platyhelminths (D)- Plant cells and fibers (E)- Meat fibers (F)- Crystals (G)- Fat globules (H)- Yeasts cells (I)- Bacteria (J)- Erythrocytes (K)- Pus cells Use of special technique for collection of Pineworm Eggs: - The eggs pf Enterobius vermicularis (Pinworm), rarely appear in the stools. These are usually collected in the folds of skin around the anus. These pinworm eggs are collected by using following special technique. (1)- Use a cotton wool swab. (2)- Wipe round the anus with swab. (3)- Dip the swab into a test-tube containing about 0.5 ml. Of normal saline. (4)- Observe under microscope by making a cover slip preparation.

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Practical No.: - 2

GROSS EXAMINATIO N AND PHYSICAL EXAMINATION OF STOOL Gross examination: - Observe the stool specimen for the following: (1)- Consistency and form: - Normal consistency: Well formed.

Abnormal consistency Expected reasons Pale, bulky, frothy Steatorrhea (poor fat digestion)

Hard Constipation Flattened and ribbon like Obstruction in the lumen of the bowel

Semi solid Mild diarrhea, after taking laxative

digestive upsets Watery Bacterial infection, Purgative

Rice water stools (copious thin with white flakes)

Cholera

(2)- Presence of adult worms or their parts (mature segments).

- Large round worms - Pinworms - Whipworms - Hookworms or - Tapeworms

(3)- Color Normal color: Light to dark brown (due to presence of bile pigment)

Abnormal color Possible reasons Black Bleeding in the upper gastrointestinal tract, Iron

administration (in iron deficiency, anemia) Bright red Bleeding at the lower level of gastrointestinal tract, Bleeding

piles, contamination with menstrual blood. Fresh blood, mucus Amoebic dysentery Clay colored Post-hepatic jaundice, obstruction to the flow of bile to

intestine White After barium meal

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PRACTICAL NO: 3

CONCENTRATION METHOD OF MICROSCOPIC STOOL EXAMINATION

If the presence of ova is small in number, they are not detected by direct examination. The concentration method is useful to detect the ova. Principle: - The ova whose specific gravity is less than that of the saturated saline will float to the top, when a coverslip is placed on the top of the container, the ova cling to the cover slip and by placing the cover slip on a glass slide the ova can be observed under microscope. Requirements: - (1)- Glass slides (2)- Cover slips (3)- Saturated saline solution (4)- Small bottle or bulbs or test tubes Procedure: - (1)- Fill half of the small bottle or test tube with saturated saline solution. (2)- Place small amount of fecal material in it by applicator stick. (3)- Mix well. (4)- Fill the bottle or test tube completely with saturated saline. (5)- Place a cover slip on it (air bubbles should not be produced). (6)- Leave for 15-20 minutes. (7)- Lift the cover slip gently (by taking care that no spillage of the clinging fluid taken place). (8)- Place it on a glass slide and observe under microscope. Result: - - Ova of A.lumbricoid seen. Or

- Ova not seen.

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Practical No.: - 4

MICROSCOPIC EXAMINATION OF STOOL SPECIMEN

Requirements: - (1)- Glass slides (2)- Cover slips (3)- Normal saline (4)- Lugol’s iodine solution Procedure: - (1)- Saline – specimen preparation: - (A)- Place a drop of Normal saline on a glass slide. (B)- Take a little fecal material by using an applicator stick and mix with a drop of normal saline. (C)- Place a cover slip over it. Avoid formation of air bubbles below the cover slip. (2)- Iodine-specimen preparation: - (A)- Place a drop of Lugol’s iodine on the other side of the slide. (B)- Mix little fecal material with a drop of iodine solution. (C)- Place a cover slip on it. (3)- Examine the slide under microscope. Observation: - Observe following things and record the result. (1)- Cells (A)- Pus cells (B)- Epithelial cells (C)- Macrophages (D)- Erythrocytes. (2)- Crystals (A)- Triple phosphate and calcium oxalate (B)- Charcot-Lynden crystals (C)- Hematoidin crystals (3)- Vegetable matter (4)- Animal matter (5)- Undigested ingredients (A)- Starch (B)- Fat (6)- Other findings (A)- Yeasts cells (B)- Bacteria (7)- Cysts and vegetative forms (A)- E.histolitica (B)- E.hartmanni (C)- E.coli (D)- I.butschlii (E)-E.nana(F)- G.lamblia (8)- Eggs that can be found in feces.

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(A)- Taenia saginata (B)- Taenia solium (Tape worms)(C)- H.nana (D)- E.vermicularis (E)- Trichuris trichiura (whip worm) (F)- Ascaris lumbricoides (round worm) (G)- Ancylostoma duodenale (hook worm) (H)- P.westermani (I)- Schistosoma hematobium (J)- Schistosoma mansoni (K)- F.hepatica (L)- F.buski. (M)- Necator Americanus (N)- Strongyloides stercoralis (O)- Schistosoma japonicum

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Practical No.: - 5

DETECTION OF MALARIAL PARASITE Principle: - Two blood smears are made (1)- thin and (2)- thick. (1)- The thin smear is stained by using Leishman’s stains (or Wright’s stain). The stained blood, gives an idea of the morphology of red cells and also helps to identify the stage of malarial parasite in its life cycle. (2)- The thick smear is stained by Field’s stain, without fixing in methanol. This removes the red cells and leaves behind the imprints and pink parasite dots and other structures. Requirements: - (1)- Glass slides (2)- Spreader slide (3)- Microscope Specimen: - EDTA anticoagulated or capillary blood. Procedure: - (1)- Prepare thin and thick blood smear. (2)- Stain thin smear in Leishmana’s stain (or Wright’s stain). (3)- Stain thick smear (without fixing in methanol) in Field stain A and B. (4)- Examine under microscope. Observations: - (1)- The various stages of parasite seen (A)- Immature and mature trophozoites (B)- Schizont and (C)- Gamatocytes (2)- The various types of the parasite seen. (A)- Plasmodium vivax (B)- P.malariae (C)- P.falciparum (D)- P. ovale

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Practical No.: - 6

DETECTION OF TRYPANOZOMES (THE CAUSAL AGENT OF SLEEPING SICKNESS) Introduction: - Trypanozomes are found in lymph gland at early stage of the infection. When the infection is about 4 months old, the parasites disappear from the lymph gland and are found in the blood. Requirements: - (1)- Glass slides (2)- Cover slips (3)- Syringes and needles (4)- Microscope Specimen: - Lymph node aspirate. Specimen Collection: - (1)- Disinfect the lymph node site with tincture iodine and wash off later with 70 % alcohol. (2)- First pierce the skin by the needle and then penetrate the lymph gland. (3)- Collect the aspirate. Apply iodine at the collected site. Preparation of slide: - (1)- Put one drop each on three glass slides. (2)- Put cover slips and examine under microscope. Observation: - (1)- The motile flagellar trypanosomes appear 2-3 times larger than re3d blood cells (length wise). (2)- They form zigzag course among the cellular elements by flagellar movements.

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Practical No. :- 7

LABORATORY DIAGNOSIS OF KALA AZAR Introduction :- Leishmania species (flagellate protozoa) cause Kala-Azar. In India Lesishmania donovani is the most common cause of Kala Azar. The disease is characterized by remittent type of fever, massive enlargement of the spleen and generalized weakness. The disease is fatal if untreated. Specimen :- Serum Name of the method :- Chemical : Formal gel test Principle :- Gammaglobin increases in Kala Azar. It forms a solid gel with formalin. Reagent :- Formalin 40 % (v/v) Procedure :- (1)- Add 0.5 ml. Of serum in a test tube (100x10 mm) (2)- Add 0.5 ml. Of 40 % (v/v) formalin (3)- Observe the reaction after 10 minutes. (4)- Perform the test by also using normal serum (Control). Observation :- (1)- A white colored gel develops within 20 minutes in the case of a person suffering from Kala Azar. (2)- A positive reaction is not observed in the case of control tube (Normal serum). Additional Information :- (1)- The bone marrow smear stained with Giemsa stain indicates the round non-flagellar form (2-4 microns) of the Leishmania species within the reticuloendothelial cells. These are called Donovan bodies. (2)- Serum protein electrophoretic fractions indicate elevated gamma globulin fraction.

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Practical No.: - 8

LABORATORY DIAGNOSIS OF MICROFILARIA (Wuchereria banchrofti) Introduction :- The filarial worms are long, and are nematodes that inhabit parts of the lymphatic system and subcutaneous and deep connective tissues. The parasites do not produce eggs or ova and reproduce through microfilariae. A mosquito takes the blood from an infected human among with the microfilariae. The infective stages develops in the mosquito and returns to a healthy human through the bite of the mosquito. The worms then mature in the circulatory system of the human body. The adult worm live in the lymph nodes. The end result is Elephantiasis (enlargement of the affected parts). And chyluria or escape of chyle or fat particles through urine, resulting in a milky appearance of the urine. Blood is usually drawn at night because the organism exhibits nocturnal periodically. Requirements :-

(1)- Glass slides (2)- Cover slips (3)- Test tubes (4)- Giemsa stain (5)- 2% Formaldehyde solution. Procedure :- (1)- Collect blood between 10 PM and 4 AM by the finger prick method or by venipuncture. (2)- Put a drop of blood directly on a clean slide and add a drop of saline solution. With the help of corner of cover slip mix the blood specimen with the saline and then make a wet mount. (3)- Examine the wet mount under reduced illumination. The first sign of the presence of microfilariae is the rapid movement of the red cells (4)- In addition to the wet mount, prepare a thick smear of blood. To prepare the thick smear place three drops of blood in the center of the slide. Spread the blood out in a circular manner with the help of an inoculating loop. (5)- Stain the blood smear with Giemsa stain. Examine the stained slide under microscope. Look the size (length and diameter) , presence of sheath , and the tail (tapered, rounded or hooked).

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Observation :- Presence of microfilariae is easily recognized from the wet mount but it also be identified on stained smear. Microfilariae are thicker (almost the diameter of a red cell, 7 microns) and longer 250 microns. It posses a pink sheath , they are rather straight and have tapered tail. Blood staining procedure :- (1)- Prepare a thick smear of blood taken from finger or anticoagulated venous blood. (2)- Air dry. (3)- Place the slide vertically in the beaker filled with clean water. Wait for 10 minutes for complete haemolysis. 1) Take slide out, drain them and air dry. 2) Stain for 30 minutes, using a 1 : 10 dilution of geimsa. 3) Wash the stain off and air dry. 4) Examine under microscope. Concentration Technique :- 1) Take 1 ml of citrated blood in a bottle and mix with 2 % formaldehyde solution in a conical centrifuge tube. 2) After waiting for 5 minutes to haemolysis the red cells, centrifuge for 5 minutes. 3) Save the sediment and pour off the supernatant. 4) Take a drop of well-mixed sediment on a slide. Spread the drop to form a thin smear, air dry, fix with alcohol and stain immediately with geimsa stain. 5) Examine the slide under microscope.

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Practical No.: - 9

QUANTITATIVE DETERMINATION OF SERUM (OR PLASMA) IgG CLASS ANTIBODIES TO TOXOPLASMA GONDII BY ELISA

Introduction :- Toxoplasma gondii is an obligate intracellular protozoa parasite that is probably capable of infecting all species of mammals, including man where infection may cause a variety of manifestations. While acute infection in healthy individual may resemble infectious mononucleosis,congenital infection acquired in the uterus from infected mothers, may cause a devastating syndrome with severe abnormalities. The detection of IgG/IgM specific antibodies to T.gondii is particularly helpful for the diagnosis of acute/primary infections in ‘risk’ individuals in association with AIDS, organ transplantation and pregnancy. Principle :- In the first incubation period, microplate coated with a purified prepatation of T.gondii (antigen) is incubated with the specimen. If specific antiviral antibodies are present in the specimen, formation of antigen-antibody complexes take place. After washing out the other components of the sample,, specific anti-T.gondii antibodies are detected with a goat antihuman IgG antibody conjugated with peroxidase. The intensity of the color generated by the enzyme on the substrate/cromogen mixture (in the last incubation), is proportional to the content of anti-T.gondii antibodies in the sample. Results are calculated by means of a standard curve . Main Requirements :- (1)- Serum or plasma (heparin, EDTA,citrate) (2)- Diagnostic kit : It contains the following materials :

- Microtitre plates : Coated with Toxoplasma gondii specific antigen - Enzyme conjugate : Human anti – IgG polyclonal antibodies conjugated with

peroxidase. - Buffered substrate: Peroxidase in phosphate/citrate buffer. - Chromogen : Tetramethylbenzidine (TMB) with activators and stabilizers. - Stop solution : IM hydrochloric and phosphoric acids. - Washing solution : Phosphate buffer and detergent.. - Standards : Calibrate set containing following IgG standards

0,50,100,200,250,500,1000 IU/ml. - Diluents for reagents and specimen .

(3)- Microplate – ELISA reader.

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Note :- Bring all reagents and samples to room temperature at least 60 minutes before the analysis. Procedure :- (1)- Add 0.1 ml. Sample and standards in the respective wells of the micro strip. (2)- Incubate at 37-degree centrigrate. (3)- Aspirate reaction solution from the well, wash 4-5 times with diluted wash solution, aspirate off the remaining liquid. (4)- Add 0.1 ml. Conjugate. (5)- Incubate ate 37 degree centigrade. (6)- Aspirate reaction mixture, wash 4-5 times with diluted wash solution, aspirate off the remaining liquid. (7)- Add 50 micro liter each of buffered substrate and chromogen and incubate at room temperature for 20 minutes. (8)- Read absorbance against blank well solution at 450 nm (and bichromatically at 620-630 nm). (9)- Calculate ‘test’ value by using a standard graph prepared by using the various IgG standards.

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Practical No. :- 10

DETERMINATION OF IgM CLASS ANTIBODIES TO TOXOPLASMA GONDII BY ELISA

Principle :- Microtiter strip wells as a solid phase are coated with purified Toxoplasma gondii membrane antigens (Toxo-Ag). If corresponding specific antibodies are present in a patient sample they are bound to the antigens at the solid phase. The sample dilution buffer contains antihuman IgG to prevent rheumatoid factor(RF) interference and competition from specific IgG present in the specimen. After washing step to remove unbound material, peroxidase- labeled antihuman IgM antibody conjugate is added which binds specifically to IgM class antibodies resulting in the formation of sandwich complexes : Toxo-Ag + Anti IgM + Anti IgM.Ab-peroxidase. After a second washing step(to remove unbound material), the enzyme- linked complexes are detected by incubation with a TMB/Substrate solution and the formation of color is directly proportional to the amount of anti-Toxoplasma gondii IgM antibodies in the specimen. Absorbance at 450 nm is noted and the test results are calculated by means of a cutoff value able to discriminate between the anti T.gondii IgM negative and positive controls. Main requirements and procedure :- Main requirements and procedure are the same, which is mentioned, in the practical ‘Quantitative determination of serum (or plasma) IgG class antibodies to T.gondii by Elisa’ Note :- Calculate the cutoff value as follows : Negative control mean (at 405 nm) + 0.25 = Cut-off.

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Practical No.:- 11

PREPARATION AND STAINING OF BLOOD SMEAR

Introduction :- A good smeared and stained slide is used to count different leukocytic cells (differential count),morphology of RBCs and WBCs, approximate quantity of platelets and parasite study. Specimen :- The blood smears should be preferably prepared immediately after skin puncture or venipuncture before mixing with anticoagulant. If EDTA blood is used the smears should be prepared within 1 to 2 hours after blood drawing. Other anticoagulants do not give satisfactory results. The blood smears should be immediately fixed in methanol. Requirements :- (1)- Glass slides (2)- Glass spreader (3)- Cedar wood oil. Reagents :- (1)- Leishman’s stain :- It is prepared by dissolving 0.15 Gms. Of powdered stain in 100 ml. Of (acetone free) methyl Alcohol. (2)- Buffer (pH 7.0) :- It is prepared as follows : (A)- Sodium hydrogen phosphate 3.76 gms. (B)- Potassium hydrogen phosphate 2.1gms. (C)- Distilled water 1000 ml. Principle :- The polychromic staining solutions (Wright, Leishman, Gimsa) contain methelene blue and eosin. These basic and acidic dyes induce multiple colors when applied to cells. Methanol acts as fixative and also as a solvent. The fixative does not allow any further change in the cells and makes them adhere to the glass slide. The basic component of cells (i.e. cytoplasm) is stained by acidic dye and they are described as eosinophilic or acidophylic. The acidic components (e.g. nucleus with nucleic acid) take blue to purple shades by the basic dye and they are called basophilic.

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Procedure:- A thin smear is prepared by spreading a small drop of blood evenly on a slide. (1)- Making a film- (A)- Take a clean, dry (grease free) slide. (B)- Transfer a small drop of blood near the edge of the slide. (C)- Place the spreader slide at an angle of 30 degree , pull back the spreader until it touches the drop of blood. Let the blood run along the edge of the spreader. (D)- Push the spreader forward to the end of the slide with a smooth movement. (E)- Dry the blood smear at room temperature. Adequate drying is essential to preserve the quality of the film. (F)- Precautions-

- Dirty slides do not give and even smear. - Use an appropriate size of blood drop. - After putting the drop on the slide, make the smear immediately for even

distribution of blood cells on the slide. - The thickness of the smear depends on the angle of the spreader. If the angle

is less than 30 degree , a thinner smear is obtained and if the angle is more than 30 degree , a thicker smear is obtained.

- The film must be smooth at the end. There should be no lines extending across or down through the film and it should not contain holes.

(2)- Identification marking- By using a lead pencil write the identification number on the slide. (3)- Fixing the smear- The slide should be stained after making the smear. Methanol present in the stain fixes the smear. If the staining is to be done later, the blood smear must be fixed with methanol for 2-3 minutes to prevent distortion of cells. (4)- Staining the film- (A)- Cover the smear with the staining solution by adding 10-15 drops on the smear. Wait exactly for one minute. (B)- Add equal number of the drops of buffer solution. Mix the reaction mixture adequately by blowing on it through a pipette. Wait for 10 minutes. (C)- Wash the smear by using tap water. (D)- Stand the slide in a draining rack to dry. (E)- Precautions-

- Avoid formation of deposits of stain. They appear on the film as masses of little black spots. In that case rinse the slide twice with methanol. Dry and restain using filtered stain.

- Poor staining makes the film blue, pink or too dark. - Use neutral water for washing.

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(5)- Examination of film- (A)- First examine the stained smear under the low power. In an ideal smear three zones will appear (i)- Thick area (head) (ii)- body and (iii)- thin end of the smear (tail). (B)- Choose the portion slightly before the tail end where the red cells are beginning to overlap. (C)- Place a drop of immersion oil on the smear. Switch to the oil immersion objective and increase the light by opening the iris diaphram. (D)- Examine the film by moving from one field to the next systematically. (E)- Record yours observation. Observation and Result :- Study the morphology of cells and record the result. Examine following things on smear.

- Abnormality of leukocytes. - Abnormality of erythrocytes. - Approximate platelete count. - Parasite seen

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Practical No. :- 12

STUDY OF MORPHOLOGY OF BLOOD CELLS

Introduction :- The stained smear is observed under oil immersion objective to study red blood cells morphology, abnormalities of white blood cells and also the approximate quantity of platelets. Clinical Significance :- In various anemia’s and in other diseases such as thalassemia, malaria etc.,the red blood cells of peripheral blood may show certain significant changes. Procedure :- (1)- Choose the portion of the smear where the red cells are beginning to overlap. (2)- Place a drop of immersion oil on the smear. Switch to the oil immersion objective and examine the film by moving from one field to the next , systematically. The various observations are as follows. Observation :- Various erythrocytes observed under oil immersion objective. (1)- Normal red blood cells :-

- Round cells - They have a small area of central pallor - Only slight variation in size

(2)- Microcytes :- - Decrease in the size

(3)- Macrocytes :- - Increase in the size

(4)- Hypochromia :- - Red cells with a large area of central pallor (Decreased concentration of

hemoglobin) (5)- Spherocyte :-

- Cells are not biconcave - No central area of pallor - Smaller surface area for the cell size

(6)- Target cells :- - These cells have a centrally stained area

(7)- Stomatocytes :- - These cells show an oval or rectangular area of central pallor

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(8)- Anisocytosis :- - A variation in the size of the red blood cell is observed

(9)- Poikilocytosis :- - A variation in the shape of red blood cell is observed

(10)- Sickle cells :- - Red cells in the shape of a sickle or crescent. - They come to a point at one end

(11)- Ovalocyte :- - Ovalshaped red cells

(12)- Elliptocyte :- - More oval than ovalocyte (cigar shaped)

(13)- Acanthocytes :- - Red cells with sharp, irregular, thorn like projections on the outer edge

(14)- Burr cells :- - Red blood cells with pointed projections on their outer edge

(15)- Basophylic stippling :- - Presence of fine or course purple staining granules in the red cells.

(16)- Siderocyte (Pappenheimer bodies) :- - Purple colored granules of iron in red cell

(17)- Howell- Jolly body :- - Round purple-staining nuclear fragment in the red blood cell

(18)- Cabot ring :- - Purple staining, thread like filaments in the shape of a ring in the red cell

(19)- Schistocyte - Red blood cell fragments

(20)- Crescent bodies :- - Ruptured red cells in the shape of a quarter moon.

(21)- Crenated cells :- - Red cells having irregular outer edges. This is often due to faulty drying of

blood smears. Abnormalities in white blood cells :- Most of the abnormalities are seen in neutrophils. (1)- Toxic granulation - Neutrophils contain prominent granules

- It may be due to severe bacterial infection and also in some hereditary disorders.

(2)- Vacuoles - Occurrence of vacuoles in the cytoplasm and nucleus

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- It is observed in conditions such as severe infections, burns, chemical poisoning and in malignancy.

(3)- Dohle bodies - Appearance of small round or oval light blue stained bodies in the cytoplasm. - Appearance in severe infections, burns and on exposure to toxic agents.

(4)- Hypersegmentation - In segmented neutrophils more than 4 lobes are seen. - May be due to inherited disorder. Also seen in macrocytic anemia.

(5)- Hyposegmentation - Failure of normal lobe development (less than 3) - It occurs in acute myelocytic leukemia, severe infections, toxic states and in

hereditary disorders (6)- Auer bodies

- Needle like red purple - Found in myeloblasts with Romanowsky stains, in case of acute myelogenous

leukemia (7)- Smudge cells

- Basket like cells with no cellular wall. These are degenerated lymphocytes - Presence of few smudge cells is probably due to faulty technique and it is

considered normal. However, appearance of large number is associated with chronic lymphocytic Leukemia’s

Approximate Platelet count Determine the ratio of platelets to red cells on the blood smear. If the average number of platelets is 25 in 10 fields it is reported as adequate. If it is 0-5 it is reported as inadequate.

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Practical No. :- 13

BLOOD CELLS DISORDER IN LEUKEMIA It is a disease of the blood forming tissues and the bone marrow is always involved. The different terms which are commonly used according to the number of cells present in the peripheral blood are as follows :

Type No. of white blood cells Leukemic leukemia More than 15,000 cells/cu.mm. Subleukemic leukemia Less than 15,000 cells/cu.mm.

Presence of immature (abnormal) forms of white cells in the peripheral blood

Aleukemic leukemia Less than 15,000 cells/cu.mm..No immature or abnormal white cells present in peripheral blood smears.

Following different terms which are commonly used according to type of cells involved – Condition Type of cells involved Myelogenous (Granulocytic) leukemia Neutrophils and their precursors Eosinophilic leukemia (a rare type) Eosinophils and their precursors Basophilic leukemia (a rare type) Basophiles and their precursors Lymphocytic leukemia Lymphocytes and their precursors Monocytic leukemia Monocytes and their precursors Myelomonocytic leukemia Two or more types of cells and their

precursors (usually megakaryocytes and red cell precursors along with monocytic and granulocytic cells)

Erythroleukemia ( Di Gugliemo’s disease)

Red cells and granulocytic precursors

Lymphosarcoma cell leukemia Lymphosarcoma cells Stem cell leukemia Primitive blast cells Erythremia myelosis (Di gugliemo’s disease)

Red cell precursors

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Laboratory features of :- Acute myelogenous leukemia (AML) :-

- The total leukocyte count is usually elevated but may be normal or low. - Anemia is present - Thrombocytopenia is common and patients may present with hematuria , or

gastrointestinal bleeding. - Examination of peripheral blood smear may reveal dysplastic changes in the

red cells, granulocytes and platelets. In peripheral blood typical blast cells are seen.

- The bone marrow is usually hypercellular and is often replaced with leukemic blast cells.

- The rapid turnover of leukemic blasts produces a hypermetabolic state with increased levels of lactate dehydrogenase and uric acid. Pseudohypoglycemia may occur as result of glucose utilization by blast cells.

Acute lymphoblastic leukemia (ALL) :-

- Anemia is present if clinical manifestations are fully developed . It is normocytic red blood cells are occasionally present.

- Moderate to marked thrombocytopenia is present. - The leukocyte count is occasionally very high (over 1,00,000 cells / cu.mm.) It

may be moderately elevated, normal or decreased. The predominant cell is lymphoblast or immature lymphocyte. Pancytopenia is common finding in acute leukemia.

- Bone marrow examination : By the time the patient is symptomatic the hematopoietic cells and fat are usually replaced by diffuse infiltration of lymphoblasts.

- The diaganosis of ALL cannot be made with certainty until the Sudan black B or peroxidase reaction has been performed to show that the blasts are negative by cytochemistry techniques.

- The acid phosphatase reaction is moderately or strongly positive in the blasts in about 20 % cases of ALL

Chronic lymphocytic leukemia (CLL) :-

- Anemia or thrombocytopenia may be present at the time of diagnosis (mainly in the advanced stages of CLL)

- Lymphocytosis is of a variable degree and is the most consistent finding. The peripheral white blood is typically in the range of 1,00,000 – 2,00,000 cells/cu.mm.,but may be much higher , with more than 90 % mature appearing lymphocytes.

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- The bone marrow in patients with CLL may have a nodular pattern or a diffuse infiltration of small and medium sized lymphocytes with mature morphologic features. Myeloid elements of the marrow are usually normal with respect to their morphology and maturation.

- Hypogammaglobulinemia occurs in approximately two thirds of patients with CLL

Other chronic lymphoid leukemia’s :- A variety of lymphoproliferative disorders distinct from CLL that are distinguished by unique clinical features and cellular morphology and phenotype have been described. (1)- Polymphocyte leukemia :-

- patients usually have extremely high blast counts and splenomegaly but often lack significant lymphadenopathy.

(2)- Hairy cell leukemia :- - Characterized by lymphocytes having cytoplasmic projections from the

periphery of the cell. These cells contain tartarate-resistant acid phosphatase in their cytoplasm.

- Pancytopenia and splenomegaly are common findings, and lymphadenopathy is distinctly uncommon.

Chronic Myelogenous Leukemia (CML) :- CML is amyeloproliferatve disease characterized by a proliferation of myeloid cells without loss of their capacity to differentiate. (1)- Peripheral blood :-

- A moderate to marked increase in the white blood cell (WBC) count . (50,000 – 3,00,000 cells / cu.mm.)

- An occasional blast cell may be present. - Platelet count may be decreased in early stage. - Mild to moderate anemia is usually present . - No evidence of hemolysis, Reticulocyte is normal. - Mild increase in the percentage of basophiles and eosinophils.

(2)- The bone marrow studies :- - Hypercellular with marked proliferation of all granulocytic elements. - Megakaryocytes are frequently prominent. - Increased number of eosinophils and basophils are observed.

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Erythroleukemia :- It is an abnormal proliferation of both erythroid precursors and granulocytic precursors.

- Myeloblasts are increased in erythroleukemia, and Auer rods may be them or in pormyelocyte. Abnormalities in neutrophils and eosinophils may be seen. Monocytic proliferation may be a part of the process.

- Abnormal megakaryocytes are often prominent and include giant forms with bizarre nuclear fragmentation and small fragmented megakaryocytes with one or two apparently diploid nuclei.

- Atypical platelets may be found in the blood. - There is an apparent arrest of maturation and fewer polychromatic and

orthochromatic forms are present. - In chronic forms ,later normoblast are present in large numbers.

Pernicious Anemia :- This disease is caused by Vitamin B12 deficiency. - Pancytopenia

- Macrocytic – normochromic red cells - Moderate to marked anisocytosis and poikilocytosis. - Few microcytes and tear drop shaped cells. - Basophylic stippling, Howell-Jolly bodies and nucleated red blood cells. - Bone marrow contains increased number of erythroid cells, which are

characteristically megaloblastic. - Serum iron level may be normal or slightly elevated. Serum bilirubin may be

increased. Sideroblastic Anemia :-

- Hypochromic and microcytic red cells. - Moderate anisocytosis and poikilocytosis. - Presence of target cells and basophilic stippling - Marked increased in storage iron of the bone marrow. - Increase in serum iron. - Bone marrow shows erythroid hyperplasia and presence of ringed

sideroblasts. Anemia of Chronic Renal Insufficiency :- This type of anemia develops due to failure of the kidneys to produce erythropoiein.

- Normocytic and normochromic red cells. - Presence of burr cells.

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- Reticulocyte count is generally normal or sometimes increased. - Macrocytosis (if patient is on dialysis since folic acid is lost in dialysis.) - Platelet count is normal or slightly increased. - Bone marrow may show erythroid hyperplasia in acute renal failure.

Hereditary Spherocytosis :- (Congenital hemolytic anemia and congenital hemolytic jaundice ) This disorder is caused by a defect in the red blood cells membrane.

- The red cells show increased rate of destruction. - The red cells are hyperpermeable to sodium ions. - Presence of spherocyte (vary from few to many) - Enlarged liver and splenomegaly. - Jaundice is commonly present (elevated serum bilirubin) - Patient exhibits pallor. - Presence of nucleated red cells - Increase in osmotic fragility of red cells. - Hypercellular bone marrow with increase in erythroid cells.

Hereditary Elliptocytosis :- Characterized by the presence of variable numbers of elliptical, or oval shaped, mature red blood cells. Approximately 90 % of the individuals showing elliptocytosis have no clinical symptoms, the remaining individuals, however, display hemolytic anemia, similar to hereditary spherocytosis. Sickle cell anemia :- In this disorder the person is homozygous fro HbS. The red cells contain 90 –100 % hemoglobin S, with the remainder being hemoglobin F. This disease is usually fatal by the age of 30. Under decreased oxygen tension, hemoglobin S is less soluble than hemoglobin A, which forces the red cells into a rigid sickle shaped cells.

- Normocytic , normochromic red cells. - Presence of sickle cells, Howell-Jolly bodies and nucleated red blood cells. - Increased reticulocytes - Increased platelets - Decrease in osmotic fragility of red cells - Hemoglobin electrophoresis show a single ‘S’ band (which migrates more

slowly than HbA) - Positive sickle cell test using sodium metabisulfite.

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Sickle cell Trait :- The patient is heterozygous for HbS. The red cells of the patient contain 20 –40 % of HbS and 60-80 % HbA.

- Normal red cell morphology with occasional target cells. - Normal reticulocytes - Positive sickle cell test and presence of HbS by hemoglobin electrophoresis.

Acquired Autoimmune Hemolytic Anemia :- As a result of antibodies, red blood cell destruction occurs. These antibodies are not formed in response to a specific antigen. The antibodies are active against all red cells. Most of the cases are secondary to other conditions such as (1)- lymphoma (2)- leukemia (3)- renal carcinoma (4)- lupus erythematosus and various infections such as infective hepatitis, syphilis, etc.

- Massive red cell hemolysis. - Pallor, hepatoshplenomegaly, increased bilirubin - Appearance of large spherocytes - Elevated reticulocyte count - Hemoglobin and hematocrit varies from normal to extremely low levels - The bone marrow is hypercellular - Positive direct antiglobulin (Coomb’s ) test.

Hemolytic Disease of the Newborn :- This disease is usually found in cases of Rh incompatibility where mother is Rh negative, and the newborn Rh positive.

- Large increased in nucleated red cells - Polychromatophilia - Normochromic, macrocytic red cells - Increased reticulocyte count. - Platelets will be normal or decreased - Low hemoglobin at birth. It decreases rapidly as the disease progresses. - Umbilical cord blood bilirubin is about 3 mg/dl.and rises very rapidly after birth

and may reach 40-50 mg/dl. (increase in indirect bilirubin ).Bilirubin more than 20 mg/dl. Cause severe brain damage, called kernicterus.

- Direct antiglobulin (Coomb’s) test on the baby’s red cells is positive in all cases

- Hepatosplenomegaly Infectious Mononucleosis :- A viral disease of children.

- Normal red cell and platelet counts

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- White cell count normal to slightly elevated - Increase in atypical lymphocytes (from 20 –90 %) - The lymphocytes vary in size, irregular shaped, larger than normal

lymphocytes. - Nucleus of lymphocytes may be oval or irregular, with increase in basophilic

cytoplasm. - Positive Paul-Bunnel heterophil antibody test (Positive in dilutions 1:110 and

above) - Elevations in serum alkaline phosphatase, LDH,and GOT.

Multiple Myeloma :-

- Increase in plasma proteins. Electrophoretic pattern (serum proteins) shows additional M band near gamma globulin

- Urine test for Bence Jone’s proteins is positive - Normocytic and normochromic anemia. - Rouleaux formation of red cells - Polychromatophilia and reticulocytosis may be present. - Myeloma cells or abnormal plasma cells are present in the bone marrow and

in tumor deposits. - Increase in serum calcium

Glucose - 6 – Phosphate Dehydrogenase (G-6-PD) Deficiency :-

- Destruction of 30-50 % of red cells - Presence of Heinz bodies, basophilic stippling and polychromatophilia - Increase in reticulocyte count - Elevated serum bilirubin level.

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Practical No.: 14

SCREENING FOR SICKLE CELL ANEMIA Specimen :- EDTA or heparinized capillary blood .(Fasting not necessary.) Principle:- A strong reducing agent, Sodium metabisulfite is mixed with the blood specimen. If the red cells contain HbS, they become sickle shaped. Requirements :- (1)- Microscope (2)- Glass slides and cover slips (3)- Pasteur pipettes (4)- Filter papers (5)- Petri- Dish Reagents :- 2 g./dl. Sodium metabisulfite in distilled water. Prepare fresh (about 25 ml.) before use. Procedure :- (1)- Place a drop of blood on the glass slide. (2)- Add equal drop of Sodium metabisulfite. (3)- Mix carefully with the corner of a cover slip, seal edges, and place in a petri dish that has wet filter paper. Incubate the petri dish for 15 minutes at 37 degree centigrade. (4)- Examine the slide under microscope. (5)- If the result is negative, re-examine the slide after 2 hours and after 24 hours. Result :- - Sickle cells seen. or - Sickle cells not seen.

Solubility Test For Sickle Cell Screening Specimen: - 0.02 ml. Of blood. Principle: - Dithionate in phosphate buffer with saponin reduces HbS, which is less soluble than HbA (normal) in reduced state. It is indicated by formation of Turbidity. Saponin lyses the red cells.

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Requirements: - (1)- Dithionate (2)- Phosphate buffer (A)- K2HPo4 (B)- KH2Po4 (3)- Saponin (4)- Distilled water. Preparation of reagents: - Dissolve Dithionate 0.5 g/dl. In phosphate buffer (21.6 gms. K2HPo4 and 16.9 gms. Of KH2Po4) with 0.1 gms. Saponin. Procedure: - (1)- Take 2.0 ml. Of dithionate reagent in a test tube. (2)- Add 0.02 ml. Of blood. (3)- Mix the tube and observe the turbidity after 10 minutes. Observation :-

- If turbidity present then it indicates presence of HbS. - If turbidity absent then it indicates absence of HbS.

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Practical No.:- 15

DETERMINATION OF OSMOTIC FRAGILITY OF RED BLOOD CELLS Introduction :- Red blood cell membrane restricts the solutes and allows only water to pass through them (Osmosis). If the cells are placed in hypertonic sodium chloride (Solution concentrated than normal saline e.g. 2 % w/v), they shrink due to osmosis . The red cells absorb water when kept in hypotonic solution (Solution concentration less than even 0.55 % w/v saline). Due to endomosis, hemolysis of the cells take place due to the excessive Swelling. In isotonic solution (normal saline) however the red cells stay intact. Clinical significance :- In condition such as congenital spherocytosis the red cells are highly fragile and start to hemolyze at 0.55 % (w/v) sodium chloride while normal cells start to hemolyze at 0.45 % (w/v) of sodium chloride. Thalassemic target cells hemolyze below 0.3 % sodium chloride. Specimen :- (1)- Heparinized blood or (2)- Defibrinated blood. For this type of specimen, about 10 ml. Of blood is collected in a conical flask containing 10-15 glass beads. After rotating the blood gently for 5 minutes it is centrifuged to gel red blood. Principle :- The red cells are suspended in decreasing concentrations of sodium chloride, ranging from 0.9 (w/v) to 0.0 % (water). Degree of hemolysis is measured in each tube by using, a photometer. Requirements :- (1)- Test tubes (15x125 mm.) (2)- 10 ml. And 5 ml. Pipette , 1.0 ml. volumetric pipette. (3)- Photometer (4)- Graph-paper

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Reagents :- (1)- 10 g/dl. Stock buffered sodium chloride. - Sodium chloride : 90 gms. - Na2HPo4 (anhydrous) : 13.65 gms. - NaH2Po4 (anhydrous) : 2.43 gms. - Distilled water : 1000 ml. Note :- Due to the presence of phosphate ions the solution is osmotically equivalent to 10 g/dl. (2)- Prepare 1 g/dl. Of buffered solution by mixing 10 ml. Of solution (1) and 90 ml. Of distilled water. (3)- Now prepare working solutions in centrifuge tube tubes (17x120 ml.) as follows.: Working Solution

No. 1g/dl.buffered

saline (ml.) Distilled water

(ml.) Conc. Of working

soln. (g/dl.) 1. 0.9 8.1 0.1 2. 1.8 7.2 0.2 3. 2.7 6.3 0.3 4. 3.15 5.85 0.35 5. 3.6 5.4 0.4 6. 4.05 4.95 0.45 7. 4.5 4.5 0.5 8. 4.95 4.05 0.55 9. 5.4 3.6 0.6 10. 6.3 2.7 0.7 11.

(Blank) 7.2 1.8 0.8

12. 0.0 9.0 0.0 Note :- (1)- Tube No. 11 (blank) gives 0 % hymolysis. (2)- Tube No. 12 gives 100 % hymolysis. (4)- Preparation of 50 % suspension of patient’s red cells : (A)- Take about 5.0 ml. Of blood in a graduated centrifuge tube. (B)- Centrifuge at 1,500 RPM for 10 minutes (C)- Discard the supernatant and add 2 to 3 ml. Of 1g/dl. Buffered saline, mix and centrifuge at 1,500 RPM for 5 minutes.

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(D)- Discard the supernatant and repeat step © at least 3 times. Discard the supernatant. (E)- Measure the volume of packed cells. (F)- Add equal quantity of 1g/dl. Buffered saline and mix well. Procedure :- (1)- Add 1 ml. Of 50 % cell suspension to each of the 12 working solutions of buffered saline (prepared at step 3) (2)- Mix well and keep all the tubes at room temperature for 30 minutes. (3)- Mix again and centrifuge at 1,500 RPM for 10 minutes. (4)- Read absorbance’s of the supernatant of each tube at 540 nm.(green filter) by setting blank (tube No.11) to 100 % T. (5)- Use following formula to calculate percent hymolysis- O.D. of individual tube X 100 Percent hemolysis = ------------------------------------------

*Absorbance with 100 % hymolysis *Absorbance with 100 % hymolysis is obtained in tube No.12. (6)- Draw the fragility curve by plotting percent hemolysis on Y-axis and concentration of buffered saline on X-axis. (7)- Determine median corpuscular fragility (MCF) which causes 50 % lysis. Normal value :- Osmotic fragility in healthy person-

- Initial lysis - 5.0 g/l. NaCl. - Complete lysis - 3.0 g/l. NaCl. - MCF (50 % lysis) - 4.0 – 4.45 %

Information :- - In case of a normal person the MCF is close to 0.45 % or slightly lower. - MCF is about 0.5 % in hereditary sherocytosis. - MCF decreases in sickle cell anemia and in thalassemia (0.35 to 4.0 %)

Result :- Osmotic fragility = g/l. NaCl.

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Practical No.: - 16

DETERMINATION OF FETAL HEMOGLOBIN Clinical significance :- Marked increased in the levels of HbF are found in Beta- thalassemia major. In Beta-thalassemia trait, sickle cell disease and in other congenital or acquired hematological conditions, mild but significant increase is observed in HbF. Specimen :- EDTA anticoagulated blood (5to 6 ml.) Principle :- All the hemoglobin’s except HbF are easily denatured by an alkali. Total hemoglobin is determined of one aliquot of hemolysalte by cyanmethemoglobin method without alkali treatment. The second aliquot is treated with alkali and followed by precipitation of denatured hemoglobin’s by ammonium sulfate. When filtered, the filtrate contains the alkali resistant HbF. Requirement :- (1)- Graduated pipettes (1,2,5 and 10 ml.) (2)- Centrifuge tubes. (3)- Pasteur pipettes. (4)- Watman filter papers No.44 (5)- Timer (6)- Photometer Reagents :- (1)- Drabkin’s reagent (2)- 1.2 N Sodium hydroxide. (3)- Saturated ammonium sulfate solution (4)- Carbon tetrachloride. Procedure :- (A)- Preparation of hemolysate – (1)- Centrifuge 5-6 ml. Of blood at 1,500 RPM for 10 minutes. (2)- Aspirate and plasma and buffy coat. (3)- Wash the packed cells by normal saline ( at least four times).

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(4)- Add ¼ part of distilled water, to the packed cells and ¼ part of carbon tetrachloride. (5)- Shake vigorously and centrifuge at 3000 RPM for 20 minutes. (6)- Aspirate the hemolysate and determine its hemoglobin concentration (by cynmethemoglobin method.) (7)- By using normal saline adjust hemoglobin concentration of hemolysate to 10 g/dl. Example

- If (a)- Hb Conc. Of the hemolysate = 14.7 g/dl. And (b)- Quantity = 3.0 ml. - Use formula C1V1=C2V2 i.e. 14.7 x 3 = 10 x V2 There fore 14.7 x 3 = V2 10 - V2 = 4.41 i.e. 4.41 – 3.0 = 1.41 ml. Of Saline to be added in the hemolysate to make the concentration 10 g/dl. Hemoglobin.

(B)- Estimation of fetal hemoglobin – Prepare cyanmethemoglobin solution as follows : (8)- Pipette 9.5 ml. Of Drabkin’s reagent in a test tube and add 0.5 ml. Of the hemolysate (from step 7). Mix well. (9)- Now in a test tube labeled as ‘F’ , pipette the following :

- Cyanmethemoglobin solution (from step 8) 2.8 ml. - 1.2 N Sodium hydroxide 0.2 ml. - Mix well and keep at room temperature for 5 minutes. - Filter through double layers of Whatman No.42 filter paper. - Read absorbance of the filtrate (fetal hemoglobin) against Drabkin’s reagent

at 415 nm. (C)- Estimation of total hemoglobin In a test tube labeled as ‘T’ pipette as following : - Cynmethemoglobin solution 1.4 ml. - Distilled water 1.6 ml. - Saturated ammonium sulfate 2.0 ml. - Mix well and filter through watman No.42 filter paper. - Pipette 0.5 ml. Of filtrate in a test tube (labeled as ‘T’)and add 4.5 ml. Of

Drabkin’s reagent. Mix well. - Read the absorbance at 415 nm. against Drabkin’s reagent.

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(D)- Calculations : O.D. of HbF x 100 % HbF = ---------------------------- O.D. of Hb (total) x 20 O.D. of HbF = -------------------- X 5

O.D. of Hb (total) (E)- Result (1)- Less than 1 % (adult) : Normal (2)- 10-98 % : Beta thalasemia major (3)- Reduced : Alfa thalasemia (4)- Normal : Sickle cell trait (5)- 1-20 % : Sickle cell anemia.

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Practical No.:- 17

PREPARATION OF LUPUS ERYTHEMATOSUS (LE) CELL Principle :- The leukocytes are traumatized by rotating anticoagulated blood with glass beads.)Since LE factor does not attack healthy living leukocytes). The patient’s serum containing LE factor i.e. anti nuclear antibody (gamma globulin function) is then directed towards the nuclear material of leukocytes. The nuclear material is then transformed into an LE body, which attracts the neutrophils and then ingested by one of them. A smear of buffy coat is stained by using Leishman stain and examined under microscope. Specimen :- EDTA anticoagulated or clotted blood. Requirments :- (1)- Centrifuge tubes, glass beads, rubber bungs (2)- Glass slides, test tubes (10x100 mm.) (3)- Leishman’s stain (4)- Centrifuge, microscope, blood mixing rotor. Procedure :- (1)- Take about 5 ml. Of EDTA blood in a test tube. (2)- Add 5 glass beads and stopper the tube. (3)- Rotate on a blood mixing rotar at 50 RPM for 30 minutes. (4)- Place the tube at 370 C for 10 minutes. (5)- Transfer the blood to a wintrobe tube. (6)- Centrifuge wintrobe tube at 3000 RPM for 30 minutes. (7)- Remove hemolyzed serum and then remove carefully the buffy coat. (8)- Make smears of the buffy coat on clean and dry glass slides. (9)- Fix the air-dried smears in methanol. (10)- Stain by using Leishman’s staining solution. (11)- Examine the stained smear under microscope. Result :- (1) LE cells appear as a neutrophil containing a large spherical body in its cytoplasm. (2) LE body does not show nuclear structure and stains as a pale purple homogenous mass. (3) The ‘Tart’ cells retain their nuclear structure and should not be confused with LE cells.

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Practical No. :- 18

PREPARATION OF HEINZ BODIES Introduction :- Heinz bodies are hemoglobin precipitates that appear in red blood cells due to metabolic defect caused by deficiency of G-6-PD or glutathione. During therapy with drugs such as antimalarials and sulfonamides, Heinz body formation takes place due to the denaturation of hemoglobin. Heinz bodies appear as single or multiple, round or oval, refractile granules in the red cells. Principle :- Heinz bodies are seen in wet preparation, when supravital stains like crystal violet are used. The stain enters the red cells when they are in functional condition. The stained red blood cells are detected by preparing a wet mount or a smear. Specimen :- EDTA anticoagulated or double oxalated blood. Requirments :- (1)- Test tubes (10x100 mm) (2)- Glass slides, cover slips, and Pasteur pipettes. (3)- Microscope Reagents :- (1)- Normal saline (Sodium chloride 0.85 g/dl.) (2)- 1 g/dl. Crystal violet in normal saline. Procedure :- (1)- Add 5 drops of well-mixed blood in a test tube. (2)- Add 10 drops of 1g/dl. Crystal violet reagent. (3)- Keep at room temperature for 15 minutes. (4)- Mix and take a drop on a glass slide and cover it with a cover slip. (5)- Examine under microscope. Result :- (1)- Heinz bodies stain deep purple, vary in diameter from 1-4 microns and are attached to red cell membrane. (2)- The percentage of Heinz bodies can be calculated by counting 100 red cells.

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Practical No. :- 19

MICR0SCOPIC EXAMINATION OF BONE MARROW SMEAR Clinical Significance :- In various types of anemia’s such as aplastic, megaloblastic, sideroblastic and in various diseases such as acute leukemia’s, multiple myeloma and disease of reticuloendothelial system, bone marrow microscopic examination is useful. Performing bone marrow biopsy :- There are three methods. (1)- Needle aspiration biopsy :- It is a simple, sage and relatively painless method and performed by using a specially designed needle such as Salah or Klema. The disadvantages by this technique are - (A)- Aspiration of fibrotic marrow cannot be performed. (B)- Special relationship of marrow cells may be destroyed. (2)- Microtrephine method :- This method is not very simple but can be performed in O.P.D. by using needles such as Jasshidi-Swaim or Islam trephine needles. (3)- Surgical biopsy :- This method is carried out under aseptic condition in an operation theatre. Site of Biopsy :- (A)- Sternum (B)- Iliac crest (C)- Anterior and posterior Iliac spines (D)- Upper end of the tibia in children below 2 years of age. Note :- The aspirated material tends to clot rapidly, hence the smears should be made immediately after the aspiration. When the contents are transferred on the slide, blood is sucked with a needle and yellowish irregular shaped fragments are used to prepare smears. Procedure :- (1)- Place a drop of bone marrow aspirate on a microscope slide (if blood is present in the aspirate it is removed before the smear preparations). (2)- Dry the smears at room temperature. (3)- Fix it in methanol. (4)- Stain by using Giemsa’s , Leishman’s or Wright’s staining method. (5)- Observe the smear under microscope.

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Observations :- (1)- The cells appear as dark blue masses and fats as round vacuoles. (2)- A normocellular fragment contains about 40 % of fat cells and 60 % of marrow cells. (3)- In hyper cellular fragments fat is reduced. (4)- In hypocellular fragments the amount of fat is increased. (5)- In aplastic fragments cells are rarely detected. (6)- It is necessary to note the following- (A)- Erythropoiesis, normoblastic or megaloblastic. (B)- Activity of erythroid cells from number of erythroid cells and the ratio of myeloid/erythroid cells. (C)- Number of maturity of developing granulocyte cells. (D)- Abnormal cells and toxic granules. (E)- Megakaryocytes (F)- Any abnormality in plasma cells and lymphocytes. (G)- Parasite and bacteria.

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Practical No. :- 20

DETECTION OF PRESENCE OF IRON IN THE BONE MARROW SMEAR

Method :- Perl’s staining method (Prussian blue reaction). Clinical Significance :- In iron deficiency anemia the iron stores are completely depleted while in sideroblastic anemia prominent increase in the number of iron granules is observed. Principle :- The iron containing compounds hemosiderin or ferritin reacts with Potassium ferrocyanide in acid medium to produce Prussian blue color. Reagents :- (1)- 4 % (v/v) hydrochloric acid (2)- 4 g/dl. Potassium ferrocyanide. (3)- Stock basic fuchsin solution. It is prepared by mixing the following : (A)- Basic fuchsin 1.0 gms. (B)- Absolute Alcohol 10 ml. (C)- 5 % (w/v) Phenol 90 ml. (4)- Working basic fuchsin solution : Add 3.0 ml. Of stock basic fuchsin to 100 ml. Of Distilled water. (5)- Formaldehyde reagent (6)- Absolute alcohol. Procedure :- (1)- Mix equal volume of reagent 1 and 2 in a staining jar. (2)- Dip the smear in the freshly prepared solution for 10 minutes. (3)- Rinse in tap water. Immerse in working basic fuchsin solution fro 5 minutes. Wash with tap water. (4)- Rinse with absolute alcohol and wash again with tap water. (5)- Air-dry and observe under microscope. Observation :- Iron containing compounds produce Prussian blue color.

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Practical No.: -21

LABORATORY TESTS FOR DIAGNOSIS OF APLASTIC ANEMIA

Introduction :- Aplastic anemia is a hematologic disorder characterized by the presence of anemia, leukopenia and thrombocytopenia. Classification :- (1)- Primary or idiopathic aplastic anemia :- (A)- Acquired (B)- Familial or constitutional : Fanconi’s anemia, Pancreatic deficiency in children. (2)- Secondary aplastic anemia :- (A)- Due to effects of chemicals (B)- Pharmacological chemicals have bone marrow depression effects (probably idiosyncratic) (C)- Due to effects of physical agents :- X-ray, gamma ray. (D)- Infections :- viral hepatitis,miliary tuberculosis (E)- Metabolic reasons :- due to pancreatitis, pregnancy (F)- Paroxysmal norcturnal hemoglobinuria. Laboratory tests and expected results :- (1)- Complete hemogram Hemoglobin : decreased may be less than 3g/dl. (2)- Total erythrocyte count and PCV : reduced, usually proportionate to decrease in hemoglobin. (3)- MCV : Usually within normal range, sometimes more than 94 fl.,slightly macrocytic. (4)- MCH : Within normal range : Normochromic (5)- MCHC : within normal range : Normochromic (6)- Reticulocyte count : Low count, may be slightly increased when aplasia is patchy. (7)- Morphology of red blood cells (A)- Anisocytosis : May be slight or moderate (B)- Poikilocytosis : May be occasionally present. (8)- Total leukocyte count : Considerably reduced, may be less than 200 cells / cu.mm.

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(9)- Differential leukocyte count : (A)- Lymphocytes may constitute 80 to 90 % of peripheral blood leukocytes. (B)- Toxic granules in neutrophils may be present in excess. Especially in secondary aplastic anemia. (10)- Thrombocytes : Marked decrease is observed may be less than 10,000 /cu.mm. (11)- ESR : Increase in ESR (A normal ESR excludes diagnosis of aplastic anemia) (12)- Bleeding time : Prolonged (13)- Coagulation time :- Normal (14)- Clot retraction time :- Prolonged (15)- Bone marrow examination : (A)- Marrow may be completely aplastic but more often the changes are partial and patchy. (B)- Reduction in erythropoietic elements. (C)- Reduction in myeloid elements (D)- Reduction oar absence of megakaryocytes (E)- Increase in lymphocytes.

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Practical no. :- 22

INVESTIGATIONS OF MEGALOBLASTIC ANEMIA

Introduction :- Presence in a blood film of macrocytes, oval red cells, pear shaped poikilocyte and neutrophils with hypersegmented (more than 5 lobes) nuclei indicate Megaloblastic anemia. The diagnosis may be confirmed by the presence of megaloblasts and giant metamyelocytes in the marrow. Megaloblastic anemia is caused mainly by deficiency of Vitamin B12 or folic acid or both. Laboratory Investigations of Megaloblastic anemia :- (1)-Complete hemogram

- Reduction in total erythrocyte count is much more than reduction in hemoglobin

- MCV :- more than 94 fl. - MCH :- more than 32 pg. - MCHC :- More than 32 % - RDW :- Increased - Reticulocyte count :- usually within normal limits or less.

(2)- Morphology of red blood cells - Presence of large number of macrocytes (marked macrocytosis) - Presence of anisocytes and poikilocytes - Presence of polychromasia - Presence of megalogblasts - Moderate leukopenia - Marked neutropenia - Mild thrombocytopenia

(3)- Bone marrow examination - Hypercellular bone marrow with megaloblastic erythropoiesis - Increase in retuculum cells - Large atypical granulocytes in all stages of development and presence of

‘giant stab’ form of metamyelocytes. - Macropolycytes are present - Myeloid to erythroid ratio: reduced - Megakaryocytes : Usually normal or slightly increased.

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(4)- Biochemical tests - Serum total bilirubin and indirect bilirubin are moderately elevated. - Urine urobilinogen : Increased - Serum levels of vitamin B12 (normal 72-512 ng/L.): levels are decreased.

(5)- Other investigations required – - Determination of serum and blood cell folate. - Determination of intrinsic factor in gastric juice - Determination of intrinsic factor antibodies.

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Practical No.: - 23

LABORATORY TESTS IN IRON DEFICIENCY ANEMIA

The normal adult body contains 4000 mg of iron. About 10% of this iron is present in the circulating blood. The remaining is stored in the liver and reticuloendothelial cells of the bone marrow. When the iron stores become exhausted, iron deficiency anemia results. Iron deficiency is the commonest cause of hypochromic microcytic anemia. Expected test results in iron deficiency anemia :- (1)- Hemoglobin estimation :- Less than 10 g/dl. (2)- Examination of peripheral blood film: - Microcytic, elliptocytosis, hypochromia, anisochromia. (3)- Erythrocyte count :- It varies with the severity of anemia. (4)- MCH :- 15-26 pg. (5)- MCV :- 60-80 fl. (6)- MCHC :- 20-30 % (7)- RWD :- More than 17.5 (8)- Reticulocytes :- May be over 2 % after hemorrhage. (9)- ESR :- Rarely elevated (10)- Total leukocyte count :- Mild to moderate leukopenia in severe anemia. After hemorrhage, Leukocytosis may be observed. Bone marrow examination :- (1)- Erythroid hyperplasia which is polychromatic macronormoblastic in type. (2)- Myeloid series is usually normal though myeloid to erythroid ratio is reduced. (3)- Iron staining of bone marrow shows absence of stainable iron in reticulum cells and less than 10 % of normoblast stain as sideroblasts. Examination of feces :- Iron deficiency may be associated with intestinal blood loss hence occult blood test should be performed. This test should be repeated at least 3 times on different days to confirm the test result. In microscopic examination, presence of cysts, ova, parasites, should be examined carefully. Hookworm infection is a potent cause of severe anemia.

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Routine examination of urine :- Presence of hematuria and kidney disease may operate as etiological agents for iron deficiency. Gastric juice analysis :- Absolute achlorhydria is frequently detected in iron deficiency anemia. It may help to detect gastric carcinoma, which may lead to iron deficiency anemia. Determination of serum bilirubin and SGPT :- To rule out cirrhosis of liver, which may lead to iron deficiency anemia. Additional tests in iron deficiency anemia :- (1)- Detection of serum iron and iron-binding capacity. (2)- Determination of serum transferring (3)- Determination of serum ferritin

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Practical No :- 24

LABORATORY TESTS FOR DIAGNOSIS OF HEMOLYTIC DISORDERS Introduction :- Hemolytic disorders are conditions in which the rate of destruction of red blood cells is increased, and the bone marrow response to anemia is not compromised. (The life span of the red cells is short termed and the red cell production is accelerated).When shortening of red cell life span is beyond the regenerative capacity of bone marrow, hemolytic anemia occurs. Hemolytic anemia is characterized by excessive destruction as well as formation of erythrocytes and anemia developing despite increased erythropoisis. Mechanism of hemolysis are broadly of two types : (1)- Intravascular lysis :- Occurs due to – (A)- Trauma (B)- Complement :- Incompatible transfusion , (C) - Toxins :- Snake venom, spider poison,etc (D)- Parasitization :- Plasmodium, Brtonella, etc. (2)- Extravascular hemolysis is due to :- (A)- Abnormality in cell shape :- Spherocytes, elliptocytosis (B)- Abnormality in membrane composition : Spur cells (C) - Abnormality in erythrocytic contents :- HbS,HbC,due to thalassemia, unstable hemoglobins, etc. Expected laboratory test results :- (1)- Hemoglobin and red blood cell count :- Reduction in hemoglobin is often proportional to fall in RBC count. (2)- PCV is proportional to RBC count. (3)- MCV : Normocytic : Macrocytic when regeneration is rapid : Microcytic in chronic cases (4)- MCHC: Normochromic : Hypochromic in cronic cases (5)- RDW: Increased (6)- Reticulocytes: Increased usually 5-20 % (may be as high as 70-90 %. Normal range is : 0.2 – 2.0 %) Note :- Low Hb values and high reticulocyte count are important evidence of Hemolytic anemia.

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(7) Nucleated red blood cells : Normally they are absent but commonly found in hemolytic anemia. (8)- Normoblasts increase in hemolytic anemia. Bone marrow examination (Microscopic) : (1)- Erythroid hyperplasia is macronormoblastic. (2)- Myeloid-erythroid ratio is reduced (may be 1:1, Normally it is 3 to4:1) Morphological changes in erythrocytes : (1)- Presence of spherocytes (may be due to congenital or acquired spsherocytosis) (2)- Acanthocytes are present in Spur- cell anemia. (3)- Target cells are seen in Thalassemia (4)- Elliptocytes are seen in hereditary elliptocytosis. (5)- Sickle cells are seen in sickle cell anemia and in anemia’s where HbS is associated with other abnormal hemoglobin’s. Osmotic fragility test : Normal values : Partial hemolysis begins at 40 % and complete hemolysis at 30 % saline. These results are also observed after 24 hrs. incubation at 37 degree centigrade. (1)- Osmotic fragility is increased in spherocytosis. (2)- Osmotic fragility is decreased in thalassemia, sickle cell anemia and hemolytic anemia associated with target cells. Autohemolysis test : Spontaneous hemolysis of sterile defibrinated blood is observed on incubation ate 37 degree centigrade for 48 hours. (1)- Increased rate of hemolysis is observed in hereditary spherocytosis and in pyruvate kinase deficiency. (2)- Hemolysis is also increased in some cases of auto-immune hemolytic anemia and anemias caused by oxidant drugs. Determination of G-6-PD , pyruvate kinase and estimation of reduced glutathione (GSH) : (1)- Positive finding reveal the cause of hemolytic anemia (i.e. due to enzyme deficiency) Electrophoresis of hemoglobin : (1)- Increase in HbA2 is observed in Beta-thalassemia and Beta- unstable hemoglobinopathy. (2)- Increase in HbF is observed in Beta-thelassemia major trait and also in sickle cell disease. Sickling test : It is positive in HbS trait, homozygous Hb-S disease, Hb-S/C disease, Hb-S/Beta thalassemia.

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Coomb’s test :- (1)- Direct coomb’s test :- Positive test indicates immunohematologic anemia. (2)- Indirect Coomb’s test :- Positive reaction indicates presence of Rh antibodies in patient’s serum. Biochemical tests : These tests gives evidence of increased hemolysis. (1)- Serum bilirubin total and indirect both types are increased. (2)- SGOT and LDH: Increased (3)- Serum heptoglobin : Reduced Note: - Serum heptoglobin is reduced in both intra and extravascular hemolysis but it is more pronounced in intravascular hemolysis. (4)- Increased urobilinogen and presence of hemoglobin and hemosiderin in urine indicates hemolytic condition.

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