MORPHOLOGIC CHANGES DUE TO AREA OF SMEAR

1. MORPHOLOGIC CHANGES DUE TO AREA OF SMEAR • Thin area- Spherocytes which are really "spheroidocytes" or flattened red cells. True spherocytes will be found in other (Good) areas of smear. • Thick area - Rouleaux, which is normal in such areas. Confirm by examining thin areas. If true rouleaux, two-three RBC's will stick together in a "stack of coins" fashion..

2. Common causes of a poor blood smear • Drop of blood too large or too small. • Spreader slide pushed across the slide in a jerky manner. • Failure to keep the entire edge of the spreader slide against the slide while making the smear. • Failure to keep the spreader slide at a 30° angle with the slide. • Failure to push the spreader slide completely across the slide. • Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide • Holes in film: Slide contaminated with fat or grease • Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water.

3. Biologic causes of a poor smear • Cold agglutinin - RBCs will clump together. Warm the blood at 37° C for 5 minutes, and then remake the smear. • Lipemia - holes will appear in the smear. There is nothing you can do to correct this. • Rouleaux - RBC’s will form into stacks resembling coins. There is nothing you can do to correct this

4. Principle of Romanowsky Stain Giemsa stain : polychromatic stain • Methanol : fixes cells to slide also use as solvent • methylene blue stains RNA,DNA blue-grey color • Eosin stains hemoglobin orange-red color • parasites cytoplasm: blue • nuclear material: purple-red • pigment : depending on type& species • pH value of phosphate buffer is very important

5. Staining Procedure • Thin smear are air dried after fixation with absolute alcohol. • Dilute Giemsa 10% • Flood the smear with stain. • Leave the stain on the slide for 10 min. • Wash off by running water directly to the centre of the slide to prevent a residue of precipitated stain. • Stand slide on end, and let dry in air.

6. too acidic suitable too basicStaining result

7. Causes and correction • Too Acid Stain: • insufficient staining time • prolonged buffering or washing • old stain • Correction: • lengthen staining time • check stain and buffer pH • shorten buffering or wash time • Too Alkaline Stain: • thick blood smear • prolonged staining • insufficient washing • alkaline pH of stain components •  Correction : • check pH • shorten stain time • prolong buffering time

8. Life cycle of Plasmodium

9. Components of the Malaria Parasite

10. Malaria in Thin Blood Film

11. Morphology of erythrocytic stages of Plasmoduimfalciparum • Trophozoite • size: small to medium • number: often numerous • shape: ring and comma forms common • chromatin: often 2 dots • cytoplasm: regular, fine to fleshy

12. Morphology of erythrocytic stages of Plasmodium falciparum • Schizont • size: small • number: few • shape: compact • uncommon, usually seen in severe malaria • mature forms: 12-30 or more merozoits • pigment: single dark mass

13. Morphology of erythrocytic stages of Plasmodium falciparum • Gametocyte • shape: • -mature, banana-shaped or round • -immature, pointed-end (uncommon) • chromatin: single, well defined • pigment: scattered, coarse, rice-grain-like

14. Malaria in Thin Blood Film

15. Plasmodium malariae • Morphology of erythrocytic stages of P. malariae • Trophozoite • size:small • number:usually few • shape:ring to rounded, compact forms • chromatin:single, large • cytoplasm:regular, dense • pigment:scattered, abundant, with yellow tinge • in older forms

16. Morphology of erythrocytic stages of Plasmodium malariae • Schizont • size:small, compact • number:usually few • mature forms:6-12 merozoits, usually 8, in • loose cluster, some apparently • without cytoplasm • pigment:concentrated

17. Morphology of erythrocytic stages of Plasmodium malariae • Gametocyte shape: • -immature:difficult to distinguish from mature • trophozoit • -mature:round, compact • chromatin:single, well defined • pigment:scattered, coarse, may be peripherally • distributed • Eroded formswith only chromatin and pigment • present

18. Malaria in Thin Blood Film

19. Morphology of erythrocytic stages of Plasmodium vivax • Trophozoite • size: small to large • number: few to moderate • shape: broken ring to irregular forms common • chromatin: single, occasionally 2 • cytoplasm: irregular or fragmented (amoeboid) • pigment: scattered, fine

20. Morphology of erythrocytic stages of Plasmodium vivax • Schizont • size: large • number: few to moderate • shape: compact • mature forms: 12-24 merozoits (usually 16) • pigment: loose mass

21. Morphology of erythrocytic stages of Plasmodium vivax • Gametocyte • shape: • -immature: difficult to distinguish from mature trophozoit • -mature: round, large • chromatin: single, well defined • pigment: scattered, fine • Eroded forms with scanty or no cytoplasm and only chromatin and pigment present

22. Malaria in Thin Blood Film

23. Morphology of erythrocytic stages of Plasmodium ovale • Trophozoite • size:may be smaller than P. vivax • number:usually few • shape:ring to rounded, compact forms • chromatin:single, prominent • cytoplasm:fairly regular, fleshy • pigment:scattered, coarse

24. Morphology of erythrocytic stages of Plasmodium ovale • Schizont • size:rather like P. malariae • number:few • mature forms:6-16 merozoits, usually 8, in loose cluster • pigment:concentrated mass

25. Morphology of erythrocytic stages of Plasmodium ovale • Gametocyte • shape: • -immature: difficult to distinguish from mature trophozoit • -mature:round may be smaller than P. vivax • chromatin: single, well defined • pigment: scattered, fine • Eroded forms with only chromatin and pigment present

26. Morphologic forms of haemoflagellate There are 4 morphologic forms seen in hemoflagellates: • Amastigote • Promastigote • Epimastigote • Trypomastigote - They can exist in two or more of the 4 morphologic forms  depending on the species.

27. Amastigotes of Leishmania species Structurally the amastigotes of Leishmaniaspecies that causing visceral, cutaneous and mucocutaneous are similar. • With variations in size between species. • Small, round to oval measuring 2–4 um. • Can be seen in groups inside blood monocytes ,less commonly in neutrophils, in macrophages in aspirates or skin smears, or lying free between cells. • The nucleus and kinetoplast stain dark reddish-mauve. • The cytoplasm stains palely and is difficult to see when the amastigotes are ingroups.

28. Promastigote of Leishmania species • Size: 9-15µm • Shape: long and slender. • Nucleus: one, central. • Kinetoplast: Anterior end of the organism, no undulating membrane. • Flagellum: Single, anterior free flagellum. • Is the infective stage • Also result from culture (NNN)media.

29. Cutaneousleishmaniasis • Infection is often referred to as wet or dry oriental • sore. The early papule is often inflamed and resembles • a boil of 5–10 mm in diameter which rapidly develops • into a large uneven ulcer which is self-healing • in as little as 3–6 months. Multiple lesions may occur

30. Mucocutaneousleishmanisis • is the most severe and destructive form of • cutaneousleishmaniasis in South America. • Lesions are similar in development to those of oriental sore and the resulting ulcers may become very large and • long-lasting.

31. Post kalazar dermal leishmaniasis • occur about 2 years after treatment and recovery from visceral leishmaniasis. • Affects about 20% of patients in India. • Hypopigmented and raised erythematous patches can be found on the face, trunk of the body, and limbs. • These may develop into nodules and resemble those of lepromatous leprosy, fungal infections or other skin disorders. • Amastigotes are present in the papules and nodules.

32. Trypanosoma (cruzi,bruzei) trypomastigote • Extracellular Usually C-shaped. • measuring 12–30m in length • A narrow membrane and free flagellum. • large, round to oval, dark-red staining kinetoplast at the posterior end. • Nucleus is centrally placed and stains red mauve. • Disease: trypanosomiasis • Sleeping sickness in africa • Chagas disease in america • Sample: Blood or Cerebrospinal fluid

33. Life CycleAfrican Trypanosomiasis Life cycle of Trypanosoma brucei gambiense & T. b. rhodesiense

34. Cerebrospinal fluid • Lymph node aspiration • CATT test trypanosoma

35. Blood CoccidiaTachyzoites of Toxoplasmagondii • Parasites are frequently seen in neutrophils and mononuclear cells. • They are crescent shaped and small, • measuring about 37m. One end is rounded and the other end more pointed. • Nucleus is situated towards the roundedend and stains dark red. • Cytoplasm stains blue.

36. Diagnosis of Free-living Amoebae They are amoebae that normally inhabit: • Water (lakes, swimming pools, air-conditioning units) • Moist soil. • Decaying vegetations. Potentially Pathogenic Free-living amoebae Naegleria fowleri Acanthamoeba species In water In water or air Trophozoite Trophozoite form Amoeboid form Cyst form Cyst form Flagellate form

37. Diagnosis of Free living amoeba: Naegleriafowleri Disease Meningoencephalitis Sample: CSF • Geographical Distribution: some parts of the world. • Mode of infection: • Swimming and sniffing (inhalation) in contaminated water. • Sappinia sp cause nonlethal amoebic encephalitis إستنشاق

38. Free living amoeba: AcanthamoebaspeciesBalamuthiamandrillaris Granulomatous Amoebic Encephalitis Acanthamoeba Keratitis Mode of infection Nasal to Lower respiratory tract to Blood to Brain Through corneal trauma Exposure to contaminated water Wearing contaminated contact lenses Ulcerated skin and mucosa to Blood to Brain

40. Urinogenital protozoa Trichomonas vaginalis • vaginal, urethral and prostatic tissue • only in humans; no animal • Structuer contain Fg=flagella • Bb=basal body • Nu=nucleus • Ax=axostyle • um=undulating membrane • Cy=cytostomal groove • Cs=costa • No cyst stage. • Multiplies by binary fission • Sample: urine sample,vaginal or urethral swab

41. Class insectaAnopheles • Morphology and medical importance • Female are blood feeder • Spotted wings • Maxillary palps as long as proboscis • Egg: floated, layed single • Proboscis and body in same straight line • Malaria transmition • In some areas it can also transmit filariasis

42. Class insectaCulex • Morphology and medical importance • Female are blood feeder • Uniform wings • Blunt tip abdomen • Maxillary palps shorter than proboscis • Egg: not floated, layedin group • Proboscis and body at an angle • vectors of filariasis and some viral diseases

43. Class insectaAedes Morphology and medical importance • Female are blood feeder • Uniform wings • Maxillary palps shorter than proboscis • Pointed tip abdomen • Egg: not floated, layedsingly • Proboscis and body at an angle • vectors of dengue • yellow fever • and other viral diseases

44. Class insectaPhlebotomus morphology and medical importance • Also known as Sand fly • Vector of Leishmaniasis • Minute in size1.5–3 mm • yellowish in colour with black eyes • Hairy body, wings and legs. • The oval lanceolate wings are carried erect on the humped thorax

45. Class insectaSimulium(Black fly) morphology and medical importance • Transimitteonchocerciasis • Known as (river blindness) • small 1.5-5mm • humped thorax • short antennae • without hair

46. Class insectaCulicoides morphology and medical importance • Annoyance • Filarial disease • 1.5-5 mm • Pair antennae • small headcoered by black spott

47. Class insectaGlossina • Other name Tsetse fly • adults are relatively large flies • Measuring 6-15mm • have forward projecting proboscis • hatchet cell wing venation. • African trypanosoma

48. Order hemipteraFamily:Reduviidae • Triatomine bug • Measure 1-4 cm • Elongate snout-like • head with two eye • 4 segment antennae • 3 pairs of legs • Vector of Chagas disease