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After centrifugation, one can distinguish a layer of clear fluid (the plasma), a layer of red fluid containing most of the red blood cells, and a thin layer in between. Making up less than 1% of the total volume of the blood sample, the buffy coat (so-called because it is usually buff in hue), contains most of the white blood cells and platelets. The buffy coat is used, for example, to extract DNA from the blood of mammals (since mammalian red blood cells are anucleate and do not contain DNA).
The buffy coat is usually whitish in color, but is sometimes green if the blood sample contains large amounts of neutrophils (which are high in green myeloperoxidase). The layer beneath the buffy coat contains granulocytes and red blood cells.
The following is a standard protocol for preparing a buffy coat from whole human blood. Biohazard precautions for working with potentially infectious material such as human body fluids should be strictly adhered to.
- Collect whole blood in tube containing an anticoagulant such as sodium heparin.
- Dilute fresh blood using an equal volume of calcium- and magnesium-free PBS with 2% v/v FBS.
- Centrifuge at 200 x g for 10 minutes at room temperature.
- Using a pipette, carefully remove the concentrated white blood cells at the plasma/erythrocyte interface. Higher numbers of cells can be collected by removing some of the serum and red blood cells, which can be lysed later if necessary.
Diagnostic uses of the buffy coat
- Quantitative buffy coat (QBC) is a laboratory test to detect infection with malaria or other blood parasites. The blood is taken in a QBC capillary tube which is coated with acridine orange (a fluorescent dye) and centrifuged; the fluorescing parasites can then be observed under ultraviolet light at the interface between red blood cells and buffy coat. This test is more sensitive than the conventional thick smear and in > 90% of cases the species of parasite can also be identified.
- In cases of extremely low white blood cell count, it may be difficult to perform a manual differential of the various types of white cells, and it may be virtually impossible to obtain an automated differential. In such cases the medical technologist may obtain a buffy coat, from which a blood smear is made. This smear contains a much higher number of white blood cells than whole blood.
- "Human Immunology Portal - Preparing a Buffy Coat from Whole Blood". Retrieved 1 July 2014.