Malaria Site: Malaria Simplified

Peripheral smear examination for malarial parasite is the gold-standard in confirming the diagnosis of malaria. Thick and thin smears prepared from the peripheral blood are used for the purpose.

The peripheral blood smear provides comprehensive information on the species, the stages, and the density of parasitemia with a sensitivity of 5 to 10 parasites/無 of blood for an experienced laboratory professional. The efficiency of the test depends on the quality of the equipment and reagents, the type and quality of the smear, skill of the technician, the parasite density, and the time spent on reading the smear. The test takes about 20 to 60 minutes depending on the proximity of the laboratory and other factors mentioned above. It is estimated to cost about 12 to 40 US cents per slide in the endemic countries.

Problems: The exacting needs of the blood smear examination are often not met in certain remote and poor parts of the world.Detection of low levels of parasitemia, sequestered parasites of P. falciparum and past infections in aspiring blood donors; ascertaining viability of the detected parasites; difficulties in maintaining the required technical skills and resultant misdiagnosis due to poor familiarity and problems in accessing and activating the facility in emergencies are some of the deficiencies with the blood smear examination.

Alternative microscopic methods have been tried, including faster methods of preparation, dark-field microscopy, and stains like benzothiocarboxypurine, acridine orange and Rhodamine-123. Acridine orange has been tried as a direct staining technique, with concentration methods such as thick blood film or the centrifugal Quantitative Buffy Coat system and with excitation filter in the Kawamoto technique. Inability to easily differentiate the Plasmodium species, requirements of expensive equipment, supplies and special training as well as the high cost limit the use of these methods.

Preparation of the smear: Use universal precautions while preparing the smears for malarial parasites - use gloves; use only disposable needles/lancets; wash hands; handle and dispose the sharp instruments and other materials contaminated with blood carefully to avoid injury.

Step 1		Step 2

Hold the third finger of the left hand and wipe its tip with spirit/Savlon swab; allow to dry		Prick the finger with disposable needle/lancet; allow the blood to ooze out
Step 3		Step 4

Take a clean glass slide. Take 3 drops of blood 1 cm from the edge of the slide, take another drop of blood one cm from the first drop of blood		Take another clean slide with smooth edges and use it as a spreader...
Step 5		Step 6

...and make thick and thin smears. Allow it to dry		Prepared smear. (Slide number can be marked on the thin smear with a lead pencil.)

Thick smear: The thick smear of correct thickness is the one through which newsprint is barely visible. It is dried for 30 minutes and not fixed with methanol. This allows the red blood cells to be hemolyzed and leukocytes and any malaria parasites present will be the only detectable elements. However, due to the hemolysis and slow drying, the plasmodia morphology can get distorted, making differentiation of species difficult. Thick smears are therefore used to detect infection, and to estimate parasite concentration.

Thin smear: Air dry the thin smear for 10 minutes. After drying, the thin smear should be fixed in methanol. This can be done by either dipping the thin smear into methanol for 5 seconds or by dabbing the thin smear with a methanol-soaked cotton ball. While fixing the thin smear, all care should be taken to avoid exposure of the thick smear to methanol.

Staining: A number of Romanowsky stains like Field’s, Giemsa’s, Wright’s and Leishman’s are suitable for staining the smears. Thick films are ideally stained by the rapid Field’s technique or Giemsa’s stain for screening of parasites. The sensitivity of a thick blood film is 5-10 parasites/痞. Thin blood films stained by Giemsa’s or Leishman’s stain are useful for specification of parasites and for the stippling of infected red cells and have a sensitivity of 200 parasites/痞. The optimal pH of the stain is 7.2.

Thick films: The thick film is first de-hemoglobinised in water and then stained with Giemsa.

Rapid Giemsa: Prepare a 10% Giemsa in buffered water at pH 7.1. Immerse the slide in the stain for 5 minutes. Rinse gently for 1 or 2 seconds in a jar of tap water. Drain, dry and examine.

Standard Giemsa: Prepare a 4% Giemsa in buffered solution at pH 7.1. Immerse the slide (at least 12 hours old) in stain for 30 minutes. Rinse with fresh water, drain, dry and examine.

Thin films: Thin film examination is the gold standard in diagnosis of malarial infection.

Giemsa stain: Fix with 1-2 drops of methanol. Cover the film with 10% Giemsa stain and leave for 30 minutes, wash with distilled water, drain, dry and examine.

Leishman's stain: Add 7-8 drops of the stain and leave for 1-2 minutes. Then add 12-15 drops of buffered distilled water, mix thoroughly, leave for 4 - 8 minutes. Then wash off with clean water, drain, dry and examine.

Jaswant Singh Battacharya (JSB) Stain for thick and thin films: This is the standard method used by the laboratories under the National Malaria Eradication Programme in India.

JSB I stain: Medicinal methylene blue (0.5 g) is dissolved in 500 ml of distilled water and 3 ml of 1% sulphuric acid (H₂SO₄) is gradually added, followed by 0.5 g of potassium dichromate (K₂Cr₂O₇) when a purple precipitate forms. 3.5 g of disodium hydrogen phosphate dihydrate (Na₂HPO₄.2H₂O) is next added and when the precipitate has dissolved, the solution is boiled in a flask with a reflex condenser for 1 hour. The stain is ready for immediate use.

Buffered water: 0.22 g of disodium hydrogen phosphate dihydrate (Na₂HPO₄.2H₂O) and 0.74 g of potassium acid phosphate (KH₂PO₄) are added to 1000 ml of distilled water or filtered tap water.

After dehemoglobinisation, dip the thick smear in JSB II stain two to three times. Wash it by dipping in buffer water two to three times. Then keep the thick film dipped in JSB I stain for 40-60 seconds. Wash it with buffer water. Drain, dry and examine.

Thick Film Examination

	P. vivax - trophozoites and gametocytes	P. falciparum - trophozoites and gametocytes

Thin Film Examination

P. vivax - trophozoites		P. vivax - schizont		P. vivax - gametocyte


	P. falciparum - trophozoite		P. falciparum - gametocyte

Infected erythrocytes are counted in relation to a predetermined number of WBCs and an average of 8000/痞 is taken as standard. 200 leucocytes are counted in 100 fields (0.25 痞 of blood). All parasite species and forms including both sexual and asexual forms are counted together.

If the parasites are <9, then 500 WBCs should be counted and the formula will be -

In the Earle and Perez method, the number of asexual parasites per known volume of blood (usually 5痞) spread as a thick film are counted; this is used only in research studies.

Determining the percentage of parasitaemia will be essential for P. falciparum. The number of infected red cells (and not number of parasites) in 1000 RBCs is converted to percentage.

This method estimates the percentage of red blood cells infected with malarial parasites. The smear is scanned carefully, one 'row' at a time. The total number of red cells and the number of parasitised red cells are tabulated separately. If 1000 red cells are counted, then divide the number of parasitised red cells by 10 to get the percentage (i.e. if 30 out of 1000 cells are parasitised, then the parasitised red cell count is 3%). If lesser red cells are counted, then divide the number parasitized by the total number counted and multiply the result by 100 to obtain a percentage estimate of red blood cells parasitized. If occasional parasites are seen when scanning the smear, but none are identified during the process of counting 300-500 red blood cells, a percentage value of less than 1% of red blood cells parasitized is assigned. An estimate of less than 1% of red blood cells parasitized does not need to be refined, since no clinical predictive value is gained. It is values of 2-3% or above that are of clinical concern.

The "plus system" is less precise as variation in the thickness of the film results in false variation in parasite count.