A buffy coat suspension is a concentrated leukocyte suspension. It contains most of the white blood cells and platelets from a whole blood sample. Blood stages can be concentrated using centrifugation of blood collected in EDTA anticoagulant, placed in a Wintrobe tube. The buffy coat is so-called because it is usually buff in hue.
Whole blood samples can be fractionated as a pretreatment to separate buffy coat, comprising white blood cells and platelets, from erythrocytes and plasma.
- Plasma: 55% of total Blood
- Buffy Coat: Leukocytes and Platelets (<1% of total blood)
- Erythrocytes: 45% of total blood
The buffy coat layer, containing most of the white blood cells and platelets, is situated between the plasma and erythrocytes.
Buffy coat uses
- The buffy coat is used to extract DNA from the blood of mammals (since mammalian red blood cells are anucleate and do not contain DNA). This offers an option for purifying large amounts of gDNA from relatively small sample sizes
- Quantitative buffy coat (QBC) is a laboratory test to detect infection with malaria or other blood parasites, such as Leishmania donovani, trypanosomes, microfilariae and a fungal pathogen Histoplasma capsulatum.
Method of preparing buffy coat preparation to concentrate malaria parasites:
- Using a narrow stem plastic bulb pipette or Pasteur pipette, fill two small narrow bore plastic or glass test tubes with EDTA (sequestrene) anticogulated blood.
- Centrifuge the blood at RCF 1000g for 15 minutes*.
- Note* To judge the best length of time to centrifuge, individual laboratories should try to centrifuging at different times, e.g. 10, 15, 20, 25 minutes to evaluate the times which give the best concentration of locally found Plasmodium species.
- Remove and discard (into disinfectant) the supernatant plasma above the buffy coat layer.
- Transfer the buffy coat layer and red cells immediately below it (to depth of about 1mm) to one end of a slide and mix with the end of the pipette. Using a smooth edged spreader, make a thin preparation.
- Note: Other parasites like trypanosomes and microfilariae are concentrated in the plasma immediately above the buffy coat layer. In practice, when withdrawing the buffy coat layer and red cells below it, a small amount of supernantant plasma is always withdrawn and this will contain any trypanosomes or microfilariae which may be in the specimen.
- Allow the preparation to air-dry. When dry, fix with absolute methanol or ethanol for 2 minutes.
- Stain using Field’s thin film staining technique or Giemsa staining method.
- Examine the preparation first with 40X objective and then with the 100X objective.
References and further reading