GC 3/12/2004
PROTOCOL FOR STAINING MALARIA BLOOD FILMS

Staining Blood Films for Malaria Parasites

Because some patients have low parasite densities, thick films, which increase the sensitivity of the diagnostic process 15 to 20 times, should always be made. However, in most cases there will be a sufficient number of parasites present in the blood for a diagnosis to be made using the thin film.

A significant cause of difficulty in the detection and identification of malaria parasites in thin films is the staining of slides at pH 6.8 rather than pH 7.2. At the acid pH neither Schuffner's stippling nor Maurer's clefts will be apparent. In addition, the overall staining will be far less intense, often making the parasites, especially early ring forms, difficult to detect.

Because of the detrimental effect of prolonged exposure of malaria parasites to anticoagulants such as EDTA, it is important that blood films should be made as soon as possible after venepuncture. In the low humidity of an air-conditioned building thin films can be fixed and stained immediately, but thick films usually require a period of drying. If the thick films are made on very clean slides they can be stained with Field's stain as soon as the blood has dried, provided extreme care is used. If this care is not taken the smear will wash off during staining. It is best to make several smears and if the first one or two fall off the slide, put the others aside for an hour or so before staining. The `Rapid Field's Stain' method for thin films described below provides a readable film within several minutes of receiving the blood in the laboratory. If no parasites have been detected by the time the thick films are sufficiently dry for staining, further examination can be on these slides with a consequent increase in sensitivity. The thick films should be examined for around 10 to 15 minutes before being declared`negative'. It is worth remembering that in order to examine an equivalent amount of blood on a thin film, it would have to be examined for several hours.

Field's stain can be used for both types of film in the routine laboratory.

Thick Smears:

If the blood has just dried, extreme care must be taken in staining. Thick films are stained UNFIXED.

Dip the slide in Field's A (blue stain) for about 5 seconds. Carefully dip the slide in water to wash off the excess stain. Dip the smear in Field's B (red stain) for around 1 second, no longer. Gently wash again and then leave the slide to drain vertically. The overall result should be a smear which is stained blue-green in the centre and pink at the edges. The best colour balance is in the region where the blue and pink grade into each other. The staining balance can be adjusted by further staining, though care must be taken to avoid washing the blood from the slide.

Thin Films:

After fixation with methanol, thin films are normally stained with a stain such as Giemsa (10% solution of Gurr's R 66 Giemsa in buffer at pH 7.2 for 30 minutes), and this method produces the best, most consistent staining. Staining at pH 7.2 is important because the stippling (Schuffner's for P. vivax and Maurer's clefts for P. falciparum) will not be apparent in films stained at pH 6.8 (standard haematological practice).

A quicker method which usually gives good results is to use Field's stain:

Dilute a stock of Field's B (red stain) 1:4 with pH 7.2 buffer. Place the fixed smear on a staining rack. Using a pipette, cover the surface of the smear with the diluted Field's B stain, then add an equal amount of undiluted Field's A (blue stain). Mix the stains. After one minute, wash the stain from the slide with running water. Do not prolong this step as the stain will wash out of the blood film. Drain the slide by standing it vertically.

This method should give good staining, with Schuffner's stippling of Plasmodium vivax being apparent. Maurer's clefts will not normally show.

A: Fields Stain

Solution A

Methylene Blue 0.8 g
Azure 1 (Azure A) 0.5 g
Anhydrous Na2HPO4 5.0 g
KH2PO4 6.25 g
Distilled Water 500 ml

Solution B

Eosin 1.0 g
Anhydrous Na2HPO4 5.0 g
KH2PO4 6.25 g
Distilled Water 500 ml

The stock solutions should be kept in tightly stoppered bottles to inhibit fungus growth but should be filtered approximately every two weeks. For routine staining of thick blood films three jars are required; Field's stain A, Field's stain B and water. For staining thin films a container of Field's stain B diluted 1:4 with pH 7.2 buffer is needed as well.

B: Buffer Solution - pH 7.2

A suitable pH 7.2 buffer for diluting stock stains is:

Anhydrous Na2HPO4 7.40 gm
KH2PO4 2.65 gm

Dissolve the salts in 50 ml of distilled water then dilute to 8 litres.


PARASITE DENSITY COUNTS

The object of counting malaria parasites in blood smears is to determine their concentration at a particular time. This is particularly relevant in ascertaining the success of therapy, especially for cases of P. falciparum.

Thick Blood Films

Probably the most useful method is to determine the parasite density relative to a predetermined number of white cells (or platelets) and then to calculate the number of parasites per microlitre or litre of blood. It is generally assumed that the white blood cell count is standard and 8,000 WBC per Šl is the count most frequently used. If an accurate white cell count is available, then parasite density can be determined with confidence. Usually at least 200 white cells are counted, but the number may be increased if the parasite density is low. In smears with high parasite densities it is easier to count the number of leucocytes against 500 parasites.

Thin Blood Films

Parasite counts done on thin films can be useful for assessment of individual cases, provided the density is high enough. The number of infected red cells per microscopic field is assessed and, after sufficient fields have been viewed, a percentage infection rate determined. This requires the estimation of the average number of erythrocytes per microscopic field with the microscope being used.

< Back             Next >


| Guy Cox Software | Tutorial | Life Cycle | Artefacts | Protocols |