Thick and Thin Blood Smear for Malaria Diagnosis

By Acharya Tankeshwar •  Updated: 06/06/21 •  4 min read

The direct microscopic visualization of the malarial parasite on the thick and/or thin blood smears has been the “gold standard” for malaria diagnosis.

Preparation of Thick and Thin Blood Smear
Preparation of Thick and Thin Blood Smear

Thick Blood smear 

Thick blood film samples a relatively large volume of blood thus allowing more efficient detection of parasites (increased sensitivity).

Thick smears consist of a thick layer of dehemoglobinized (lysed) red blood cells (RBCs) which provides better opportunity to detect parasitic forms against a more transparent background. However, they do not permit an optimal review of parasite morphology.  

Making Thick Blood Smear

  1. Using the corner of a clean slide, spread the drop of blood in a  circle the size of a dime (diameter 1-2 cm).
    • Do not make the smear too thick or it will fall off the slide. (you should be able to read newsprint through it.)
  2. Allow the smear to dry thoroughly. Insufficiently dried smears (and/or smears that are too thick) can detach from the slides during staining. You can accelerate the drying by using a fan or hairdryer.
    • #Do not fix thick smears with methanol or heat.
  3. If there will be a delay in staining smears, dip the thick smear briefly in water to hemolyse the RBCs.

Quality Control

Visually, the smear should appear as a round to oval smear of blood about 2 cm in diameter. It should be of such thickness that newsprint can barely be seen through the wet or dry smear.

Limitation of Thick Smear

Thin Blood Smear

Prepared smear
Prepared smear

Thin smears consist of blood spread in a layer such that the thickness decreases progressively toward monolayer. It allows optimal assessment of the morphology of any parasitic forms that may be present. Thin blood film is prepared similarly to that of the differential white-cell count.

Making Thin Blood Smear

  1. Bring a clean spreader slide, held at a 45° angle, toward the drop of blood on the specimen slide.
  2. Wait until the blood spreads along the entire width of the spreader slide.
  3. While holding the spreader slide at the same angle, push it forward rapidly and smoothly.
  4. Wait until the thin films are completely dry before staining.
  5. Fix the thin film with methanol (100% or absolute) for 15-30 second and let it dry completely before staining.
    • Note: fixation time for ethanol is 20 minutes

Limitation of a thin smear

Staining of the thick/thin smear with Giemsa Stain

Note: As alternates to this 45-60 minutes in 2.5% Giemsa stain, the smears could be stained for shorter times
in more concentrated stains. One alternate is 10 minutes in 10% Giemsa; the shorter stains yield faster results,
but use more stain and might be of less predictable quality

Microscopic examination

First screen the thick/thin smear at low magnification (10× or 20× objective lens), to detect large parasites(microfilaria) then examine the smear using oil immersion objective. NCCLS recommends the examination of at least 300 oil immersion fields for the determination of “No Parasite Seen”.

P. falciparum trophozoite stage in thick (right) and thin (left) smear.
P. falciparum trophozoite stage in thick (right) and thin (left) smear.

Diagnostic Points for Plasmodium falciparum

  1. Red cells are not enlarged.
  2. Rings (trophozoite ring stage) appear fine and delicate and there may be several in one cell.
  3. Some rings may have two chromatin dots.
  4. Presence of marginal or applique/accole forms.
  5. It is unusual to see developing forms in peripheral blood films.
  6. Gametocytes have a characteristic crescent shape appearance. However, they do not usually appear in the blood for the first four weeks of infection.
  7. Maurer’s dots may be present.

Diagnostic Points for P. vivax

  1. Red cells containing parasites are usually enlarged.
  2. Schuffner’s dots are frequently present in the red cells as shown above.
  3. The mature ring forms tend to be large and coarse.
  4. Developing forms are frequently present.

Further Resources:

Staining for Malarial Parasites; a guideline by DPDx

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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One response to “RDTs for Malaria Diagnosis: Principle, Results, Advantages”

  1. DUFATANYE says:

    thank you for your initiative

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