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..

Common causes of a poor blood smear1. Drop of blood too large or too small.2. Spreader slide pushed across the slide in a

jerky manner.3. Failure to keep the entire edge of the

spreader slide against the slide while making the smear.

4. Failure to keep the spreader slide at a 30° angle with the slide.

5. Failure to push the spreader slide completely across the slide.

6. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide

7. Holes in film: Slide contaminated with fat or grease

8. Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water.

Biologic causes of a poor smear 1. Cold agglutinin - RBCs will

clump together. Warm the blood at 37° C for 5 minutes, and then remake the smear.

2. Lipemia - holes will appear in the smear. There is nothing you can do to correct this.

3. Rouleaux - RBC’s will form into stacks resembling coins. There is nothing you can do to correct this

Principle of Romanowsky Stain Giemsa stain : polychromatic stain

Methanol : fixes cells to slide also use as solventmethylene blue stains RNA,DNA blue-grey color Eosin stains hemoglobin orange-red color parasites cytoplasm: bluenuclear material: purple-redpigment : depending on type& speciespH value of phosphate buffer is very important

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.

too acidic suitable too basicStaining result

Causes and correctionToo Acid Stain:

1. insufficient staining time2. prolonged buffering or

washing3. old stain

Correction:1) lengthen staining time2) check stain and buffer pH3) shorten buffering or wash

time

Too Alkaline Stain:1. thick blood smear2. prolonged staining3. insufficient washing4. alkaline pH of stain

components Correction :

1) check pH2) shorten stain time3) prolong buffering time

Life cycle of Plasmodium

Components of the Malaria Parasite

Malaria in Thin Blood Film

Morphology of erythrocytic stages of Plasmoduim falciparum

Trophozoitesize: small to mediumnumber: often numerousshape: ring and comma forms commonchromatin: often 2 dotscytoplasm: regular, fine to fleshy

Morphology of erythrocytic stages of Plasmodium falciparum

Schizontsize: small number: fewshape: compactuncommon, usually seen in severe malariamature forms: 12-30 or more merozoitspigment: single dark mass

Morphology of erythrocytic stages of Plasmodium falciparum

Gametocyteshape: -mature, banana-shaped or round-immature, pointed-end (uncommon)chromatin: single, well definedpigment: scattered, coarse, rice-grain-like

Malaria in Thin Blood Film

Plasmodium malariaeMorphology of erythrocytic stages of P. malariaeTrophozoitesize:smallnumber:usually fewshape:ring to rounded, compact formschromatin:single, largecytoplasm:regular, densepigment:scattered, abundant, with yellow tinge in older forms

Morphology of erythrocytic stages of Plasmodium malariae

Schizontsize:small, compactnumber:usually fewmature forms:6-12 merozoits, usually 8, in loose cluster, some apparently without cytoplasmpigment:concentrated

Morphology of erythrocytic stages of Plasmodium malariae

Gametocyte shape:-immature:difficult to distinguish from mature trophozoit-mature:round, compactchromatin:single, well definedpigment:scattered, coarse, may be peripherally distributedEroded formswith only chromatin and pigment present

Malaria in Thin Blood Film

Morphology of erythrocytic stages of Plasmodium vivax

Trophozoitesize: small to largenumber: few to moderateshape: broken ring to irregular forms commonchromatin: single, occasionally 2cytoplasm: irregular or fragmented (amoeboid)pigment: scattered, fine

Morphology of erythrocytic stages of Plasmodium vivax

Schizontsize: large number: few to moderateshape: compactmature forms: 12-24 merozoits (usually 16)pigment: loose mass

Morphology of erythrocytic stages of Plasmodium vivax

Gametocyteshape: -immature: difficult to distinguish from mature trophozoit-mature: round, largechromatin: single, well definedpigment: scattered, fineEroded forms with scanty or no cytoplasm and only chromatin and pigment present

Malaria in Thin Blood Film

Morphology of erythrocytic stages of Plasmodium ovale

Trophozoitesize:may be smaller than P. vivaxnumber:usually fewshape:ring to rounded, compact formschromatin:single, prominentcytoplasm:fairly regular, fleshypigment:scattered, coarse

Morphology of erythrocytic stages of Plasmodium ovale

Schizontsize:rather like P. malariaenumber:fewmature forms:6-16 merozoits, usually 8, in loose clusterpigment:concentrated mass

Morphology of erythrocytic stages of Plasmodium ovale

Gametocyteshape:-immature: difficult to distinguish from mature trophozoit-mature:round may be smaller than P. vivaxchromatin: single, well definedpigment: scattered, fineEroded forms with only chromatin and pigment present

Morphologic forms of haemoflagellate

There are 4 morphologic forms seen in hemoflagellates:

1) Amastigote2) Promastigote3) Epimastigote4) Trypomastigote - They can exist in two or more of the 4

morphologic forms depending on the species.

Amastigotes of Leishmania species

Structurally the amastigotes of Leishmania species 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.

Promastigote of Leishmania species

Size: 9-15µmShape: long and slender.Nucleus: one, central.Kinetoplast: Anterior end of the organism, no undulating membrane.Flagellum: Single, anterior free flagellum.Is the infective stageAlso result from culture (NNN)media.

Cutaneous leishmaniasisInfection is often referred to as wet or dry orientalsore. The early papule is often inflamed and resemblesa boil of 5–10 mm in diameter which rapidly developsinto a large uneven ulcer which is self-healingin as little as 3–6 months. Multiple lesions may occur

Mucocutaneous leishmanisisis the most severe and destructive form ofcutaneous leishmaniasis in South America. Lesions are similar in development to those of oriental sore and the resulting ulcers may become very large andlong-lasting.

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.

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: trypanosomiasisSleeping sickness in africaChagas disease in americaSample: Blood or Cerebrospinal fluid

Life Cycle African Trypanosomiasis

Life cycle of Trypanosoma brucei gambiense & T. b. rhodesiense

Cerebrospinal fluid Lymph node aspiration

CATT test trypanosoma

Blood CoccidiaTachyzoites of Toxoplasma gondii

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.

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

Amoeboid form

Trophozoite form

Naegleria fowleri Acanthamoeba species

In water

Flagellate form

Cyst form Cyst form

Trophozoite

In water or air

Diagnosis of Free living amoeba: Naegleria fowleri

Geographical Distribution: some parts of the world.

Mode of infection:

- Swimming and sniffing (inhalation) in contaminated water.

- Sappinia sp cause nonlethal amoebic encephalitis

إستنشاق

Disease

Meningoencephalitis

Sample: CSF

Free living amoeba: Acanthamoeba species Balamuthia mandrillaris

Granulomatous Amoebic Encephalitis

Acanthamoeba Keratitis

Mode of infection

Nasal to Lower respiratory tract to Blood to BrainUlcerated skin and mucosa to Blood to Brain

Through corneal traumaExposure to contaminated waterWearing contaminated contact lenses

Morphology of free living amoebae

Naeglaria Acanthoamoeba Balamuthia

TROPHOZOITE

CYST

Urinogenital protozoa Trichomonas vaginalis

vaginal, urethral and prostatic tissue only in humans; no animal Structuer contain Fg=flagellaBb=basal bodyNu=nucleusAx=axostyleum=undulating membraneCy=cytostomal grooveCs=costaNo cyst stage. Multiplies by binary fission Sample: urine sample,vaginal or urethral swab

Class insecta Anopheles

Morphology and medical importanceFemale are blood feederSpotted wingsMaxillary palps as long as proboscisEgg: floated, layed singleProboscis and body in same straight lineMalaria transmitionIn some areas it can also transmit

filariasis

Class insectaCulex

Morphology and medical importanceFemale are blood feederUniform wingsBlunt tip abdomenMaxillary palps shorter than proboscisEgg: not floated, layed in groupProboscis and body at an anglevectors of filariasis and some viral

diseases

Class insectaAedes

Morphology and medical importanceFemale are blood feederUniform wingsMaxillary palps shorter than proboscisPointed tip abdomenEgg: not floated, layed singlyProboscis and body at an anglevectors of dengueyellow fever and other viral diseases

Class insectaPhlebotomus

morphology and medical importanceAlso known as Sand flyVector of Leishmaniasis Minute in size1.5–3 mmyellowish in colour with black eyes Hairy body, wings and legs.The oval lanceolate wings are carried

erect on the humped thorax

Class insectaSimulium(Black fly)

morphology and medical importance

Transimitte onchocerciasisKnown as (river blindness)small 1.5-5mmhumped thorax short antennae without hair

Class insectaCulicoides

morphology and medical importance

AnnoyanceFilarial disease1.5-5 mmPair antennaesmall headcoered by black

spott

Class insectaGlossina

Other name Tsetse fly adults are relatively large fliesMeasuring 6-15mmhave forward projecting proboscis hatchet cell wing venation.

African trypanosoma

Order hemipteraFamily:Reduviidae

Triatomine bugMeasure 1-4 cm Elongate snout-like head with two eye 4 segment antennae3 pairs of legsVector of Chagas disease