Red blood cell lysis method for parasite concentration

A sample of trypanosome infected blood after lysis of red cells and centrifugation. The sediment at the bottom contains both parasites and white blood cells

The parasitological tests that are commonly used in diagnosis of HAT have limited sensitivity, which can frequently cause misdiagnosis of patients with low numbers of parasites, especially T.b. gambiense infections.

One approach to enhance the sensitivity would be selective lysis of red blood cells (RBC), leaving behind intact trypanosomes that can be visualized more easily. A number of studies aimed at this were reported over 20 years ago, but were never pursued further. Researchers at Makerere University, Uganda, have revisited the approach using an ammonium chloride solution, to selectively lyse RBC. Blood samples containing trypanosomes were mixed with the lysis solution (or with a commercial RBC lysis solution) and allowed to stand for 5 minutes. The lysed mixture was then centrifuged, and the pellet obtained used to prepare a wet smear to detect parasites.

This parasite concentration procedure was very successful, with an increased sensitivity of up to 20 times when compared to untreated samples. Trypanosomes remained motile for several hours in lysis solutions, which is well over the time range during which diagnosis is normally done.

Parasite concentration by RBC lysis has several advantages over standard parasitological methods that are used to concentrate trypanosomes: it is a simple and fast technique, no cold chain is required and large volumes of blood (>5 ml) can be lysed to enhance the sensitivity of trypanosome detection. This method has great potential for inclusion into the HAT diagnostic algorithm, especially if it is used with advanced parasite visualization tools, such as the LED fluorescence microscope. It could also become an invaluable sample preparation method for other techniques that require concentration of parasites, such as the loop-mediated isothermal amplification (LAMP) of DNA.

• Revisiting red blood cell lysis as a critical step in demonstrating trypanosomes in patient blood
  Short report from Makerere University by Matovu E, Kazibwe A, Boobo A, Biéler S, Ndung'u J
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