Quantitative Buffy Coat (QBC) Test

Two methods, thick and thin blood smear microscopy are regarded as the “gold standard” for the diagnosis of malaria. Quantitative Buffy Coat is another direct and rapid test for the diagnosis of malaria. It is based on acridine orange staining of centrifuged peripheral blood samples in a microhematocrit tube (QBC) and examination under UV light source (fluorescence microscopy).

The acridine orange stains all nucleic acid-containing cells and the associated fluorescence is observable under blue-violet light through a microscope.

According to the manufacturer, QBC Malaria Test is 5.5 to 7% more sensitive than Giemsa thick films. It can detect as little as 1 parasite per μL of blood and establish diagnosis earlier than thick film in 47% of low parasitemia (<10 parasites per μL) cases.

QBC is established as an effective tool for diagnosing blood parasites that cause malaria, filariasis, and visceral leishmaniasis.

Principle

Acridine orange binds deoxyribonucleic acids and ribonucleic acids. The malaria parasite binds acridine orange in the nucleus and the cytoplasm and emits green and red fluorescence when excited by blue light (at 460 nm) allowing the detection and examination of parasite morphology by fluorescent microscopy. The nuclei of the parasites emit yellowish-green fluorescence whereas the cytoplasm exhibits bright red fluorescence.

RBCs are not stained by the dye, hence remain inconspicuous under fluorescent light (dark background) while the brightly fluorescent parasites are easily seen. The outlines of stained parasites are well preserved and the general morphology is similar to that in specimens stained by the Giemsa stain.

Sample collection: Blood sample can be collected in either capillary finger-prick or phlebotomy in an ethylenediamine tetraacetate (EDTA) containing vials.

About the QBC tube

The QBC glass capillary tube (Becton Dickinson) is 75 mm in length and 1.677 mm in diameter. The tubes are internally coated with EDTA and heparin at the fill end and with acridine orange stain and potassium oxalate at the other end.

Procedure:

1. Draw samples of blood ( 55 µl) into the QBC tube by capillary action.
2. Rotate the tubes for 10 seconds to dissolve the contained residues in the blood.
   
3. Insert a close-fitting cylindrical insert or plastic float {having a specific gravity (1.055) i.e midway between that of plasma (1.028) and red blood cells (1.090)} inside acridine orange-coated capillary tube.
4. Centrifuge tubes at 12,000 g for 5 minutes.
   After gentrification blood components and malaria parasites separate based on density and concentrate in distinct layers.
   Note: The float by virtue of its density settles on top of the centrifuged packed red cells. It occupies 90% cross-sectional area of the tube which aids in the expansion of the centrifugally separated cell layers. It is surrounded by three discernible and now measurable layers of the buffy coat.
5. Insert the centrifuged QBC Malaria test into the Paraviewer. Position the tube so the closure end extends over the depressed area of the holder.
6. The area surrounding the float just beneath the buffy coat was examined under oil immersion. Individual cells within this layer were easily seen by microscopy; the malaria parasites staining green (DNA) and orange (RNA) under blue-violet light.
7. The entire circumference of the tube was examined systematically while moving away from the buffy coat through the erythrocyte layer.
8. Each tube was examined until parasites were detected or for a maximum of 5 minutes.

Note: You can download this QBC malaria test user guide to get step-by-step guidelines.  

Results/Analysis 

If a sample contains P. falciparum malaria parasites:

1.  Crescent-shaped gametocytes (1) will appear near the interface of the lymphocyte/monocyte and platelet layers.
2.  A small number of (2) schizonts and (3) mature trophozoites may appear in the granulocyte layer.
3. Ring-shaped (4) immature trophozoites will appear throughout the red blood cell layer, with a concentration near the interface with the granulocyte layer.

Other parasites species, including P. vivax, will also concentrate
during centrifugation, but exhibit different characteristics

For more information please visit www.qbcdiagnostics.com.