Haemoparasites in blood smear

most common hemoparasites in peripheral blood smear

1. HEMOPARASITES IN PERIPHERAL SMEAR 1

2. HEMOPARASITES 1. Malaria 2. Filaria 3. Leishmania 4. B abesia 5. Trypanosoma Cruzi 2

3. MALARIA Thin smear Thick smear Peripheral smear 3

4. BLOOD EXAMINATION BLOOD FILMS Thin Thick Bld drop spread Air dry methyl alcohol Geimsa Air dry Geimsa Circular motion Malaria, Babesia, Filaria , Tryp . 4

5. Malaria - Blood Smear 5 should be examined about 20-40 minutes from an experienced observer Thin films: Dry Fix Stain

6. Thick films: Dry Dehemoglobinate with water Stain Stains : Giemsa stain Leishman's stain Field’s stain / JSB stain Malaria Blood Smear 6

7. Interpreting Thick and Thin Films 7 THICK FILM lysed RBCs larger volume 0.25 μl blood/100 fields more difficult to diagnose species good screening test THIN FILM fixed RBCs, single layer smaller volume 0.005 μl blood/100 fields good species differentiation requires more time to read low density infections can be missed

8. Thick smear-Mixed infection- numerous small rings of P.f and pigmented forms of P.v 8

9. Malaria Life Cycle 9

10. RING TROPHOZOITE SCHIZONT GAMETOCYTE Blue Cytoplasm Red Chromatin Brown Pigment Recognizing Erythrocytic Stages: Schematic Morphology 10

11. Appearance of P.falciparum in the blood films Ring and trophozoite Many cells infected – same with more than one parasite Red cell size unaltered Parasite is often attatch to the margin of the host cell: called as accole form (arrow) Schizont Very rarely seen except in cerebral malaria A single brown pigment dot along with 18-32 merozoites Gamatocyte Sickle shape “ cresent ” Matuer gametocyte is about 1.5 times larger than RBC harbouring it Microgamatocyte : Broader, shorter, blunt ends. Cytoplasm light blue Macrogamatocytes : Longer, narrower, pointed ends. Cytoplasm deep blue 11

12. P. facliparum- rings and gametocytes in thin smear 12

13. Appearance of P. vivax in film Ring and trophozoit e Unoccupied portion by parasite shows a dotted or stripped appearance “ Schuffner’s dot” Schizont Represent the full grown trophozoite Contain 12-24 merozoites Arranged in the form of rosette with yellow brown pigment at the center Gametocyte Microgametocyte: Spherical. Cytoplasm light blue Macrogamatocytes : spherical. Cytoplasm deep blue 13

14. Plasmodium vivax Trophozoites: ameboid; deforms the erythrocyte Gametocytes: round-oval Schizonts : 12-24 merozoites Rings Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots) 14

15. Plasmodium ovale Rings Trophozoites: compact Schizonts: 6-14 merozoites; dark pigment; (“rosettes”) Gametocytes: round-oval 15

16. P. malariae – rings and trophozoites (band forms) P. malariae - gametocytes P. malariae – schizonts, few chromatin divisions 16

17. 17

18. Malaria Parasite Mimics in thick blood smear Looks like rings but No bluish parasite cytoplasm Looks like Schizont But No pigments Looks like Falci gametocyte But No pigment No pink chromatin 18

19. Staining variations due to pH Alkaline buffer Chromatin also looks bluish Optimum pH Chromatin is pink and parasite cytoplasm blue Acidic buffer Parasite cytoplasm also looks pink 19

20. Calculating Parasite Density 20 Count the number of parasitized and nonparasitized RBCs in the same fields on thin smear Count 500-2000 RBCs % Parasitemia = no. of parasitized RBCs total no. of RBCs X 100 If ≥10 parasites are counted on thick film parasites/ l = parasites counted WBC counted X WBC count/ l

21. If 200 WBC ‘s are counted No. of parasites / µl = No. of parasites counted X 40 21

22. Estimating Parasite Density Alternate Method 22 Count the number of asexual parasites per high-power field (HPF) on a thick blood film 1-10 parasites per 100 HPF + 11-100 parasites per 100 HPF ++ 1-10 parasites per each HPF +++ > 10 parasites per each HPF ++++

23. Fluorescent Microscopy 23 Fluorescent dyes detect RNA and DNA that is contained in parasites Nucleic material not normally seen in mature RBCs Kawamoto technique Stain thin film with acridine orange (AO) Requires special equipment – fluorescent microscope Nuclei of malaria parasites florescence bright green

24. malaria parasites fluorescent microscope 24

25. Quantitative Buffy Coat(QBC ) 25 Useful for screening large numbers of samples Quick, saves time Requires centrifuge M ain disadvantages High cost of capillaries and equipment Can’t store capillaries for later reference

26. Principle of QBC System 26

27. QBC (Fluorescent Test) 27

28. Comparison between peripheral smear and QBC test for detecting malaria Peripheral smear QBC Method Cumbersome Easy Time Longer, 60 - 120 minutes Faster, 15 - 30 minutes Sensitivity 5 parasites/µl in thick film and 200 / µl in thin film Claimed to be more sensitive, at least as good as a thick film Specificity Gold standard ? False positives, artifacts may be reported as positive by not-so-well-trained technicians Species identification Accurate, gold standard Difficult to impossible sometimes Cost Inexpensive Costly equipment and consumables Acceptability 100% Not so Availability Everywhere Limited Other   -- Accidentally can detect filarial worms 28

29. Malaria Serology 29 Antibody detection Immunologic assays to detect host response antibodies to asexual parasites appear some days after invasion of RBCs and may persist for months Positive test indicates past infection Useful for Identifying infective donor in transfusion-transmitted malaria Investigating congenital malaria ,

30. Malaria Antigen Detection 30 Target antigens for malaria (rapid detection test) RDT Card / cassette / dipstick HRP2 HRP2 & aldolase pLDH Pf & pan pLDH Pf & Pv HRP2, pLDH pan HRP2, pLDH pan & pLDH Pv aldolase "COMBO" tests A: HRP-2 (histidine-rich protein 2) (ICT) B: pLDH (parasite lactate dehydrogenase)(Flow) C: HRP-2 (histidine-rich protein 2) (PATH)

31. INDIRECT FLUORESCENT ANTIBODY(IFA) The fluorescence indicates that the patient serum being tested contains antibodies that are reacting with the antigen preparation. 31

32. ELISA Valuable epidemiologic tool Useful for Identifying infective donor in transfusion-transmitted malaria Retrospective confirmation of empirically-treated non-immunes 32

33. Polymerase Chain Reaction (PCR) 33 Molecular technique to identify parasite genetic material Threshold of detection 5 parasites/ µl Definitive species-specific diagnosis Can identify mutations – try to correlate to drug resistance

34. Brugia malayi 34

35. Brugia Malayi Common name : Malayan Filaria Geographic Distribution : Tropical; freshwater (but limited in Asia) Habitat : lymphatics and Blood Periodicity : Nocturnal/Sub-periodic( present at all hours but density increases during night 10pm – 2am ) 35

36. Brugia Malayi Infective Stage : L3 Larva MOT : Bite from infected mosquito(anopheles, mansonia Aedes ) Diagnosis : Giemsa stained smear(collected at night)/ Knotts’s Technique Pathogenesis : Malayan filariasis 36

37. Life cycle 37

38. Life cycle 38

39. BLOOD EXAMINATION KNOTT ’ S CONC. TECHNIQUE 10 ml 1 ml Air dry fix Geimsa anticoagulated blood Formalin 2 % sediment 2 min centrifuge 39

40. Morphology- Microfilariae : Size : 177 to 230 um; smaller than W. bancrofti Shape/appearance : curved/kink/stiff Terminal nuclei : Two Sheathed Nocturnal periodicity – 10pm to 2 am Locomotion : S-shaped motion 40

41. Microfilariae 41

42. Transmitted by : female sand fly Parasite are intracellular amastigote form. Leishmaniasis 42

43. BLOOD EXAMINATION Buffy coat film centrifuge RBC WBC (BC) plasma Citrated bld 30 min Air dry Fix spread Giemsa Tryp., L. donovani 43

44. Peripheral blood examination Amastigote ( leishman - Donovan bodies ) form are seen in monocytes, less commonly in neutrophils. small,round bodies 2-4µm in diameter with indistinct cytoplasm , a nucleus and a small rod – shaped kinetoplast . 44

45. Babesia Infect mice. Transmitted to host by ticks Infected humans may be asymptomatic, but in asplenic host fever, myalgia, hemolysis can be seen PBS – tiny multiple rings in blood in the red cells 45

46. Trypanosoma Cruzi 2 types – 1. African 2. American ( chaga’s disease ) PBS – long slender curvy body with a long flagella 46

47. Amastigote in striated muscles 47

48. Referrences 1.Dacie and lewis textbook of haematology 2. Dr.Tejindar singh , Text of haematology 3. Dennis et al . Estimating malaria parasite density ,malaria journal 2012: 11;238. 4.Textbook of microbiology 5 .Internet sources 48