The direct microscopic visualization of the malarial parasite on the thick and/or thin blood smears has been the “gold standard” for malaria diagnosis.
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 a 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
- 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.)
- 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.
- If there will be a delay in staining smears, dip the thick smear briefly in water to hemolyse the RBCs.
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
- Making a species identification of malarial parasites may be impossible, even for experienced technicians.
- A thin film should always be examined if a definitive identification based on morphology is required.
- Smears must be prepared from anticoagulated blood within one hour after venipuncture. The morphology of parasitic forms and the erythrocytes become atypical after that time from the direct action of the anticoagulant.
Blood smears should be stained as soon as possible after they are prepared. Storage of unstained slides for a few days in the hot and humid atmosphere without staining will result in auto-fixation, and the thick film will be useless for microscopy.
Thin Blood Smear
Thin smears consist of blood spread in a layer such that the thickness decreases progressively toward a monolayer. It allows optimal assessment of the morphology of any parasitic forms that may be present. Thin blood film is prepared similarly to the differential white cell count.
Making Thin Blood Smear
- Bring a clean spreader slide, held at a 45° angle, toward the drop of blood on the specimen slide.
- Wait until the blood spreads along the entire width of the spreader slide.
- While holding the spreader slide at the same angle, push it forward rapidly and smoothly.
- Wait until the thin films are completely dry before staining.
- Fix the thin film with methanol (100% or absolute) for 15-30 seconds and let it dry completely before staining.
- Note: fixation time for ethanol is 20 minutes
Limitation of a thin smear
- Parasitic forms may be missed in light infections. In such instances, a thick film must be examined.
- Smears must be prepared from anticoagulated blood within 1 hour after venipuncture. The morphology of parasitic forms and the RBC become atypical after that time from the direct action of the anticoagulant.
Giemsa Staining of Thick and Think Blood Smear
Giemsa stain is the most reliable method for staining thick and thin blood films. Giemsa solution is composed of eosin and methylene blue (azure). The cytoplasm appears blue (stained by methylene blue), and the nucleus appears red (stained by eosin).
Counts the number of slides to be stained. Each slide requires approximately 3 mL of stain. If you have 16 slides to stain, you can prepare 50 mL Giemsa working solution. Two commonly used working solutions are;
- 10% Giemsa stain working solution: It is used in hospital/diagnostic laboratories for a quick diagnosis. It is slightly costly, as more stain is consumed.
- 3% working solution: This cost-effective slow method is mostly used to stain slides for teaching or epidemiological purposes.
- Fix the thin film by carefully dropping methanol onto the thin film using a Pasteur pipette.
Alternatively, you can dip the thin film for 2 seconds into a small container or beaker containing methanol. Avoid contact between the thick film and methanol. Methanol vapors quickly fix the thick film, thus interfering in its hemolysis.
- Let the blood film dry in the air or on a drying rack or tray.
Allow methanol-fixed thin smear to dry by placing the slides on a flat surface. It will approximately take 2 minutes to completely air dry the specimen. Never dry slide in a vertical position with the thin film down, as this may lead to fixing of the thick film by methanol vapor.
- Place the slides individually on the staining rack with blood films facing up.
Ensure that these slides are not touching each other.
- Gently pour the stain onto the top of the slides until the blood films are covered.
Each slide will require approximately 3 mL of stain. Avoid pouring the stain directly onto thick films.
- Set the timer for the staining. Leave the stain on the slides for 45-60 min (if you are using 3% Giemsa working solution) or 8-10 min (while using 10% Giemsa solution).
Optimum staining duration should be determined previously by testing the batch of stock staining solution used (i.e. your internal quality control will dictate the exact duration requirement for good staining).
- Gently flush all the stains from the slides by dropping buffered water (pH 7.2) over them.
Buffered water should be poured onto the slides from the thin film end to avoid undue disturbance and to wash off the thick films. The surface becomes covered with a metallic green scum during the staining process. You should avoid getting it onto blood films during rinsing, as it can impair examination.
- When the stain has been washed away, remove each slide individually and allow air drying.
Ensure that thick films are not scraped against the edge of the rack.
- Discard any remaining Giemsa working solution.
Examining the thick film
- Place the Giemsa-stained blood film to be examined on the microscope stage, with the label to the left. Position the thick film in line with the 10X objective lens.
- Switch on the microscope, adjust the light source optimally and find the focus by looking through the ocular and the 10x objective.
- Scan the blood film for parasites and blood elements. Select part of the film that is well stained and has evenly distributed white blood cells.
- Place a small drop of immersion oil on the thick film. To avoid cross-contamination, ensure that the immersion oil applicator never touches the slide. Do not allow the 40x objective to touch the oil.
- Switch the 100x oil immersion objective over the selected portion of the thick film. Use the fine focus adjustment to see the image. Raise the mechanical stage to avoid damaging the slide.
- Using the fine adjustment, focus on the cell elements, and confirm that the film is acceptable for routine examination; 15-20 white blood cells per thick film field will give a satisfactory film thickness. Films with fewer white blood cells per field will require more extensive examination.
- Examine the slide systematically. Start at the top left of the film (marked with a vertical green arrow) and begin at the periphery of the field, then move horizontally to the right, field by field.
- When the other end of the film is reached, move the slide slightly downwards, then to the left, field by field, and so forth. For efficient examination, continuously focus and refocus with the fine adjustment throughout the examination of each field.
- Examine the thick film under the oil immersion objective, field by field, horizontally or vertically. Use the fine adjustment to focus.
- A minimum of 100 high-power fields must be examined before a thick film can be declared as having “no malaria parasites seen.” If possible, the whole thick film should be scanned.
- If parasites are found, scan additional 100 fields to increase the chance of identifying mixed infections.
- Identify all species and stages observed, and record them.
Examining Thin Films
The thin blood film should always be examined to identify parasite species or mixed infections after examining the thick film. Unlike the thick film, the thin film allows visualization of parasite and red cell morphology. Perform an examination at the feathery end or edge of the thin film.
- Place a drop of immersion oil on the feathered edge of the thin film.
- Move from the 10x lens to the 100x oil immersion lens.
- Examine the feathery end of the edge of the thin film where red cells lay side by side, and there is minimal overlap. Follow the pattern of movement shown in Fig. 2. Move along the edge of the film, then move the slide outwards by one field, inwards, returning in a lateral movement, and so on.
- Continue examining the thin film until the presence and species of malaria parasites have been confirmed. Identify and record all species and stages observed in the malaria microscopy blood register.
- Staining for Malarial Parasites; a guideline by DPDx
- World Health Organization; Global Malaria Programme