LABORATORY 2 Staining and processing of blood parasites Differential counts of leukocytes (giemsa stains)

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1 LABORATORY 2 Staining and processing of blood parasites Differential counts of leukocytes (giemsa stains) SPECIMENS TO BE STAINED 1. Thin and thick blood smears from a patient returning from Africa - case history -- see pp 20 of your lab manual - reasons for examining these 2 preparations see pp 20 of your lab manual; -blood slide preparation methods described-pp 7-8 -Stained thick blood film provided 2. Peritonitis sample--cytocentrifuged mouse peritoneal leukocytes (identification of WBC & differential leukocyte count).

examining these 2 preparations see pp 20 of your lab manual; -blood slide preparation methods described-pp 7-8 -Stained thick blood

2 Staining technique with GIEMSA STAIN (PAGES 8&9) 1. GIEMSA STAIN (Gustav Giemsa, a German chemist and bateriologist): - a mixture of an acidic (eosin) and two basic dyes (methylene blue/azure A & azure B). - To use, dilute commercial stock 1:20 in distilled water). 2. Used for staining blood smear (differential leukocyte count), blood parasites (malaria, trypanosomes, microfilariae) and bone marrow, lymph node juice (Leishmania sp) list on pp # Collect blood from the patient with an anticoagulant or from finger, ear lobe or toe (of children). - for malaria - draw blood before or several hours after malarial paroxyms ( # infected RBCs) - for microfilarae- night blood (10pm- 2am) 4. THICK AND THIN BLOOD FILMS used for malaria diagnosis - thick film is parasite detection (low parasitemia) - thin film used for species identification

- To use, dilute commercial stock 1:20 in distilled water). 2.

THICK BLOOD SLIDE PREPARATION THIN Touch the blood drop with a clear slide or 2 drops of blood (30-40 ul) Using the corner of another slide, spread the blood into the shape of a circle of ~1 1.

3 THICK BLOOD SLIDE PREPARATION THIN Touch the blood drop with a clear slide or 2 drops of blood (30-40 ul) Using the corner of another slide, spread the blood into the shape of a circle of ~1 1.5 cm 2 Touch the blood drop with a clear slide or one drop of collected blood Hold the slide and with another slide at o angle touch the drop and wait until the blood spread along the edge of the second slide Rapidly and smoothly push the slide forward

5 cm 2 Touch the blood drop with a clear slide or one drop of collected blood Hold the slide and with another

4 THICK stained slide provided dry at RT 2-3 hrs lyse the blood film in dist-water, 1-2 min (longer if frozen) dry slide at RT, fix in methanol 3 min, let dry in a vertical position stain in giemsa min; wash in dist water dry the slide, examine under oil immersion (100x) objects seen: 1. Clear RBC ghosts and malaria parasites (difficult to identify species); 2. White Blood cells (WBC) 3. brownish hemozoin granules in schizonts (if present). THIN same technique (RBC not lysed) as used for white blood cells (WBC) differential count dry slide at RT, fix in methanol 3 min, let dry in a vertical position stain in Giemsa (1:20)- 30 min; wash in dist water dry the slide (5 min), examine under oil immersion (100x) objects seen: 1. intact RBC spread evenly on the slide; 2. scattered WBC; 3. different stages and species of malaria parasites.

brownish hemozoin granules in schizonts (if present).

5 THICK BLOOD FILM

6 THIN BLOOD SMEAR

7 Differential diagnosis of malaria P. vivax and P. falciparum Differential P. vivax P. falciparum Stage in Blood Multiple infection in RBC All stage (ring, trophozoite, early schizont, male and female gametocytes) rare Only 2 stages (early ring, male and female gametocytes) common Parasitemia low high Ring stage Relatively large usually 1 chromatin dot May have 2 chromatin dots Schizont stage ~18 merozoites Up to 36 merozoites (rare in blood smear) Gametocytes stage Round Sausage shaped

gametocytes) rare Only 2 stages (early ring, male and female gametocytes) common Parasitemia low high Ring stage

8 P. falciparum Gametocyte Multiple infection Ring with two chromatin dots

9 Late trophozoite P. vivax (with Schuffner`s dots Ring Early trophozoite Female gametocyte Male gametocyte Schizont plasmodium_vivax

gametocyte Male gametocyte Schizont http://www.trueknowledge.

10 REPORTING OF THE RESULTS 1. Based on thin blood film from the patient a. Stage/stages of malaria parasite that you have found on the blood slides b. Can you make a specific diagnosis (important for treatment)? 2. Based on thick blood film from the patient a. you will find ghost of RBCs, intact WBCs; distinguish platelets from stages of malaria (with or without hemozoin granules). b. now you are ready to determine and report the level of parasitemia described on pp BASED ON THE ASSUMPTION: 8000 leukocytes/ul of blood 500 WBCs should be counted and the formula to determine No. of parasites/µl will be: No. of parasites counted x 16 = No. of parasites/µl 8000/500= 16

you will find ghost of RBCs, intact WBCs; distinguish platelets from stages of malaria (with or without hemozoin granules). b.

11 3. Differential leukocyte count from the mouse peritonitis sample Use 100x oil immersion to identify and count at least 300 leukocytes See WBC diagrams on pp 18 of your lab manual % monocytes (kidney shaped nucleus) % neutrophils (multiple lobed nucleus) % macrophages (size ~20 um; one oval nucleus) % lymphocytes (size ~8 um; scanty cytoplasm) % eosinophils ( multi-lobed nucleus; red granules in cytoplasm) % mast cells

nucleus) % neutrophils (multiple lobed nucleus) % macrophages (size ~20 um; one oval nucleus) %

12 lymphocytes (size ~8 um; scanty cytoplasm) Google image

13 neutrophils (multiple lobed nucleus) Google image

14 Basophils bi or tri-lobed nucleus; large blue granules in cytoplasm) eosinophils eosinophils bi-lobed nucleus; pink-orange granules in cytoplasm Mast cells (uni nucleus; blue granules in cytoplasm) - large cells Google image

pink-orange granules in cytoplasm Mast cells (uni

15 Monocytes kidney shaped nucleus, Thin/light chromatin Macrophages size ~20 um; one oval nucleus) Large cytoplasm Google image

GIEMSA STAIN PREANALYTICAL CONSIDERATIONS

GIEMSA STAIN PREANALYTICAL CONSIDERATIONS I. Principle Giemsa stain is used to differentiate nuclear and/or cytoplasmic morphology of platelets, RBCs, WBCs, and parasites (1,2). The most dependable stain