MEDICAL PARASITOLOGY LAB .

Detection of Blood Parasites

Thick & Thin

Blood Smear

BLOOD EXAMINATION The most commonly used technique for blood

examination is stained blood films. Geimsa stain is usually used to stain the films. Delafild’s haematoxylin stain is used for microfilariae. Either thick or thin films may be used depending on the

circumstances. The thick film is more sensitive in detecting parasite and

also saves time in examination. The thin film technique cause very little distortion of the

parasite, and permits species identification when it may not be possible in thick films, but many fields must be examined to detect parasite when they are few in number.

CONTINUE……… Therefore, both thick and thin films must always be prepared

when searching for plasmodia and trypanosomes. If a precise identification can not be made from thick film,

the thin film will be available. Thick films should be used when searching for microfilariae. The most economical use of slides is achieved by making a

combination thick and thin slide. However, combination films must dry thoroughly 8-10 hrs.

to overnight before they can be satisfactorily stained. Slides for malaria should be stained in the same day.

CONTINUE……… The thin films will dry quickly and can be stained as soon as

they are dry, and examine for parasites. If parasites are not seen in the thin film, stain the thick film

using Field’s stain, and examine for parasites. Direct wet mounts of fresh whole blood (or centrifuged blood)

are usually used for detection of microfilariae and trypanosomes, this only gives evidence of infection and stained films are necessary for confirmation of species present.

In areas where malaria, trypanosomes, and/or microfilariae may all present, both wet and stained films should be prepared and examined.

If neither trypanosomes nor microfilariae occur in region, only stained smears need to be made for detection of plasmodia.

EXAMINATION OF THICK & THIN BLOOD SMEAR For optimum staining, the thick and thin films should be made

on separate slides and different concentrations used for staining.

When it’s done good quality staining of thick film is of primary importance, best results are obtained if the blood smear have dried overnight.

Fixation of thin blood film done by adding 3 drops of methanol, or dipping it in a container of methanol for few seconds, with prolonged fixation it may be difficult to demonstrate Schuffner’s dots and Maurer’s dots.

To permit dehemoglobinization, thick film should not be fixed; therefore avoid exposure to methanol or methanol vapor

READING OF THICK FILM Focus on film with 10x objective and search for microfilariae.

They are easily detected with 10x objective. If microfilariae are present, switch to oil- immersion objective

and identify the species. Also, look for malaria parasites with oil- immersion objective, at

least 100 fields should be examined. Microscopy of thick film should reveal the following

features: The background should be clean, free from debris, with a pale

mottled- gray color derived from the lysed erythrocytes. Leukocyte nuclei are stained a deep, rich purple. Malaria parasite are well defined with deep- red chromatin and

pale purplish blue cytoplasm.

READING OF THIN FILM Microscopy of thin film should reveal the following

features: The background should be clean and free from debris;

erythrocytes are stained a pale greyish pink. Neutrophil leukocytes have deep purple nuclei and well defined

granules. Malaria parasite are well defined with deep- red chromatin and

pale purplish blue cytoplasm. Like plasmodia, the cytoplasm of trypanosomes stain blue, the

nucleus and kinetoplast stain red or purple.

IDENTIFICATION OF MALARIAL PARASITES

In thin films, look at : The appearance of the parasites The appearance of the RBC containing the parasites: Size: Is the parasitized cell the same size as the blood cell

without parasite or Is it enlarged? Stippling: Is the RBC filled with pink or red staining dots? Schuffner’s stippling in the “ghost” of the erythrocyte can some

times be seen at the edges of the film and indicate infection with Plasmodium vivax or P. ovale,.

Maurer’s dots show as stippling in erythrocytes containing the larger ring forms of Plasmodium falciparum.

COMPARISON Thick smear Thin smear

Lysed RBCs, many layer Fixed RBCs, single layer

0.25 μl blood/100 fields ( large volume ) 0.005 μl blood/100 fields ( small volume )

Good screening test ( positive or negative )

Good species differentiation

Save time Requires more time to read

Low density infection can be detected as blood elements more concentrate ( more

sensitive )

Low density infections can be missed

More difficult to diagnose species Good species differentiation

Blood Parasites

BLOOD PROTOZOA Blood ParasiteMicrofilariae

Trypanosoma

Leishmania

PlasmodiumPlasmodium falciparum

Plasmodium vivax

Plasmodium ovale

Plasmodium malariae

Trypanosoma spp.

Blood Parasites

TRYPANOSOMA SPP. Trypanosoma cruci (Americans) cause Chaga’s disease. Trypanosoma bruci (Africans) cause sleeping sickness disease. Trypanosoma have many stages: Amastigote, Promastigote, Epimastigote and Trypomastigote. Reservoir host: mammalian animal. Intermediate host: Tse tse fly (Glossina spp.) Definitive host: Human. Infective stage: Metacyclic trypomastigote. Diagnostic stage: Trypomastigote.

CONTINUE…… • Diagnosis:

o Detection of Trypanosoma chancer after biteo Blood smear within 21 days from the bite, it will show the

parasites.o Lymph node aspiration (most reliable).o Lumber puncture if brain affected.

Undulating membrane

Flagellum Nucleus

TRYPANOSOMA TRYPOMASTIGOTES

Leishmania spp .

Blood Parasites

LEISHMANIA SPP. There is many species affect man: Leishmania tropica : cause skin lesion ( cutaneous ) Leishmania braziliense : cause muco-cutaneous lesion. Leishmania donovani : cause visceral lesion. Leishmania have two stages: Amastigote (Leishman form), in man (reticuloendothelial

cell). Promastigote (Leptomonas stage), the infective stage and

present in the lumen gut of the sand fly. Reservoir host: dogs and rodents. Intermediate host: Sand fly (Phlebotomus). Definitive host: Human.

CONTINUE…… Diagnosis:

Thick and thin blood filmSkin scrapingBlood culture on N.N.N media*Serological tests

Nucleus

Flagellum

LEISHMANIA PROMASTIGOTES

Plasmodium spp.

Blood Parasites

PLASMODIUM SPP. Four species of Plasmodium are the causative agent

of malaria, these are: P. vivax, P. malariae, P. falciparum, and P. ovale. Intermediate host: Human. Definitive host: Anopheles mosquitoes. Plasmodium spp. have 4 stages: Ring form (young trophozoite.) Late ( old ) trophozoite Schizonts Gametocyte. Infective stage: Sporozoites. Diagnosis: Thick and stained thin blood film to detect parasites.

RING FORM

P. vivax P. ovale

P. malariae P. falciparum

TROPHOZOITE FORM

P. vivax P. ovale

P. malariae P. falciparum

SCHIZONTS FORM

P. vivax P. ovale

P. malariae P. falciparum

GAMETOCYTE FORM

P. vivax P. ovale

P. malariae P. falciparum

 

Species Differentiation On Thin Films

Feature P. falciparum P. vivax P. ovale P. malariae

Enlarged infected RBC

- + + -

Infected RBC shape round round,

distortedoval,

fimbriated round

Stippling infected RBC

Maurer’s clefts

Schuffner's spots

Schuffner's dots none

Trophozoite shape

small ring, applique

large ring, amoeboid

large ring, compact

small ring, compact

Chromatin dot often double single large single

Mature schizont rare, 12-30 merozoites

12-24 merozoites

4-12 merozoites( scattered

)

6-12 merozoites( rosette )

Gametocyte crescent shape

large, round

large, round

compact, round