1HEMOPARASITES IN PERIPHERAL SMEAR

2HEMOPARASITES

1. Malaria 2. Filaria 3. Leishmania 4. Babesia 5. Trypanosoma Cruzi

3MALARIA

Thin smear Thick smear

A. Peripheral smear

4BLOOD EXAMINATIONBLOOD FILMS

Thin ThickBld drop

spread

Air dry

methyl alcohol

Geimsa

Air dry

Geimsa

Circular motion

Malaria, Babesia, Filaria, Tryp.

Malaria - Blood Smear 5

should be examined about 20-40 minutes from an experienced observer

Thin films:•Dry•Fix•Stain

Thick films:1. Dry2. Dehemoglobinate

with water 3. Stain Stains :

Giemsa stainLeishman's stainField’s stain / JSB stain

Malaria Blood Smear6

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

9Malaria Life Cycle

RING TROPHOZOITE

SCHIZONT GAMETOCYTE

BlueCytoplasm

RedChromatin

BrownPigment

Recognizing Erythrocytic Stages:Schematic Morphology

10

11Appearance 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

12

P. facliparum- rings and gametocytes in thin smear

13Appearance of P. vivax in film

Ring and trophozoite 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

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

Plasmodium ovale

RingsTrophozoites: compact

Schizonts: 6-14 merozoites; dark pigment; (“rosettes”) Gametocytes: round-oval

15

16

P. malariae – rings and trophozoites (band forms)

P. malariae - gametocytesP. malariae – schizonts, few chromatin divisions

17

18Malaria Parasite Mimicsin thick blood smear

Looks like rings

but No bluish parasite cytoplasm

Looks like Schizont

But No pigments

Looks like Falci gametocyte

But No pigmentNo pink chromatin

19Staining variations due to pHAlkaline bufferChromatin also looks bluish

Optimum pHChromatin is pink and parasite cytoplasm blue

Acidic bufferParasite cytoplasm also looks pink

Calculating Parasite Density 20

Count the number of parasitized and nonparasitized RBCs in the same fields on thin smearCount 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

Estimating Parasite DensityAlternate 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 ++++

Fluorescent Microscopy 23

• Fluorescent dyes detect RNA and DNA that is contained in parasites

• Nucleic material not normally seen in mature RBCsKawamoto technique

• Stain thin film with acridine orange (AO)• Requires special equipment – fluorescent

microscope• Nuclei of malaria parasites florescence bright

green

malaria parasites fluorescent microscope 24

Quantitative Buffy Coat(QBC)

25

• Useful for screening large numbers of samples• Quick, saves time• Requires centrifuge

• Main disadvantages• High cost of capillaries and equipment

• Can’t store capillaries for later reference

Principle of QBC System 26

27QBC (Fluorescent Test)

28

Comparison between peripheral smear and QBC test for detecting malaria

Peripheral smear QBC

Method Cumbersome EasyTime Longer, 60 - 120 minutes Faster, 15 - 30 minutes

Sensitivity5 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 techniciansSpecies identificat

ionAccurate, 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

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 malariaInvestigating congenital malaria,

Malaria Antigen Detection 30Target antigens for malaria(rapid detection test) RDTCard / cassette / dipstick

HRP2HRP2 & aldolasepLDH Pf & pan pLDH Pf & PvHRP2, pLDH panHRP2, pLDH pan & pLDH Pvaldolase

"COMBO" tests

A: HRP-2 (histidine-rich protein 2) (ICT) B: pLDH (parasite lactate dehydrogenase)(Flow)C: HRP-2 (histidine-rich protein 2) (PATH)

INDIRECT FLUORESCENT ANTIBODY(IFA)

The fluorescence indicates that the patient serum being tested contains antibodies that are reacting with the antigen preparation.

31

ELISA

• Valuable epidemiologic toolUseful for

• Identifying infective donor in transfusion-transmitted malaria

• Retrospective confirmation of empirically-treated non-immunes

32

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

35Brugia 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 )

36Brugia 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

37Life cycle

38Life cycle

39BLOOD EXAMINATIONKNOTT’S CONC. TECHNIQUE

10 ml

1 ml

Air dry fix Geimsa

anticoagulated blood

Formalin 2 % sediment

2 min

centrifuge

40Morphology- 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

41Microfilariae

42

Transmitted by : female sand fly Parasite are intracellular amastigote

form.

Leishmaniasis

43BLOOD EXAMINATIONBuffy coat film

centrifuge

RBC

WBC (BC)

plasma

Citrated bld

30 min

Air dry Fix

spread Giemsa

Tryp., L. donovani

44Peripheral 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.

45Babesia

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

46Trypanosoma Cruzi

2 types – 1. African 2. American ( chaga’s disease )

PBS – long slender curvy body with a long flagella

47

Amastigote in striated muscles

48Referrences

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