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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