Thin Smear Diagnosis of Malaria

 

Blood Samples

Blood can be collected at any time, but the best way is midway between episodes of fever and chills, when the greatest number of malaria parasites are present. It may be necessary to collect several specimens at intervals of 4-6 hours.

Draw blood from the patient's vein according to the following steps:

 

The blood sample should be stored like this.... and tested within x time.

 

 

 

Blood Smears

Microscopic evaluation of thin and thick films is an important method for confirming clinical diagnosis of malaria and identifying the specific species. Thick film is a concentration method useful for detecting the presence of parasites, and, with training, the species. Thin film is most useful for determining species identification.

 

Preparing the Thin Smear

 

 

 

 

 

 

 

 

 

 

 

 

 

Slide Staining

The slides should be stained with Giemsa, which stains the nucleus of malarial parasites red and the cytoplasm blue.

 

Procedure

1) Fix blood film in acetone-free absolute methanol for 30 seconds. Allow slides to air dry.

 

2) Immerse slide insolution of 1 part Giemsa stock to 10 to 50 parts of trotin-buffered water (pH 7.0-7.2) Fresh working stain should be prepared from stock solution each day. Stain for 10-60 minutes. A good general rule is if dilution is 1:20, stain for 20 minutes; if it is 1:30, stain for 30 minutes, etc.

 

3) Dip slides briefly in Triton X-100 buffered water. Drain thoroughly in vertical position and allow to air dry.

 

For thick films, the same protocol is followed withouth the fixation and airdrying.

 

Reagents

 

 

Slide Evaluation

Examining properly prepared slides for malarial parasites is perhaps the most challenging aspect of malarial diagnosis, and success is directly proportional to time spent and skill. Parasites are often confused with platelets. Start at 40x (x10 ocular).

Look for intracellular organaisms, as well as gametes.

Erythrocytes stain pale grey-blue. White blood cells have purple nuclei and pale purple cytoplasm. Paraites are purple-blue, with reddish nuclei.

 

Mixed infections often occur, requiring thorough examinations of blood films. There are differences among the different malaria species. With P. vivax, typical oval host cells can be seen, with Schuffner's dots and a ragged cell wall. P. malariae infections often are characterized by bar and band forms, with rosette schizonts. P. falciparum infections result in characteristic rings, frequently in multiples within a single cells as well as in the accole position. Distinctive crescent gametocytes are also present.

 

Parasitemia is measured by grade, or the percentage of RBCs infected. A P. falciparum with a grade of over 5% carries a very high risk of cerebral malaria and renal complications.

 

Never rule out malaria based on a negative smear. Carry out at least 3 before strongly considering something else. Different infections can have different periodicities.

 

Here is how to view the slides, and what to look for:

 

Here are some examples...