Serum and Plasma Preparation

Introduction

Serum is the liquid fraction of whole blood that is collected after the blood is allowed to clot. The clot is removed by centrifugation and the resulting supernatant, designated serum, is carefully removed using a Pasteur pipette. Plasma is produced when whole blood is collected in tubes that are treated with an anticoagulant. The blood does not clot in the plasma tube. The cells are removed by centrifugation. The supernatant, designated plasma is carefully removed from the cell pellet using a Pasteur pipette.
 
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Serum Preparation

Collect whole blood in a covered test tube. If commercially available tubes are to be used, the researcher should use the red topped tubes. These are available from Becton Dickinson (BD). BD’s trade name for the blood handling tubes is Vacutainer. After collection of the whole blood, allow the blood to clot by leaving it undisturbed at room temperature. This usually takes 15-30 minutes. Remove the clot by centrifuging at 1,000-2,000 x g for 10 minutes in a refrigerated centrifuge.

The resulting supernatant is designated serum. Following centrifugation, it is important to immediately transfer the liquid component (serum) into a clean polypropylene tube using a Pasteur pipette. The samples should be maintained at 2-8°C while handling. If the serum is not analyzed immediately, the serum should be apportioned into 0.5 ml aliquots, stored, and transported at –20°C or lower. It is important to avoid freeze-thaw cycles because this is detrimental to many serum components. Samples which are hemolyzed, icteric or lipemic can invalidate certain tests.

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Plasma Preparation

Collect whole blood into commercially available anticoagulant-treated tubes e.g., EDTA-treated (lavender tops) or citrate-treated (light blue tops). Heparinized tubes (green tops) are indicated for some applications; however, heparin can often be contaminated with endotoxin, which can stimulate white blood cells to release cytokines. Cells are removed from plasma by centrifugation for 10 minutes at 1,000-2,000 x g using a refrigerated centrifuge. Centrifugation for 15 minutes at 2,000 x g depletes platelets in the plasma sample.

The resulting supernatant is designated plasma. Following centrifugation, it is important to immediately transfer the liquid component (plasma) into a clean polypropylene tube using a Pasteur pipette. The samples should be maintained at 2-8°C while handling. If the plasma is not analyzed immediately, the plasma should be apportioned into 0.5 ml aliquots, stored, and transported at –20°C or lower. It is important to avoid freeze-thaw cycles. Samples which are hemolyzed, icteric, or lipemic can invalidate certain tests. There are other commercially available tubes for blood sample collection. Invitrogen™ has not evaluated some of these tubes for compatibility with our ELISA kits.

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Table 2—Serum and Plasma Tubes

  The commercially available serum tubes are as follows:
Red
No anticoagulant.
Red with black
Treated with gel to help to separate the clot (not evaluated).


  The commercially available plasma tubes are as follows: 
Lavendar
Treated with EDTA.
Blue
Treated with citrate.
Green
Treated with heparin.
Grey
Treated with potassium oxalate/sodium fluoride (not evaluated).
Yellow
Treated with potassium oxalate/sodium fluoride (not evaluated).

References

  1. Henry, J.B. (1979) Clinical Diagnosis and Management by Laboratory Methods, Volume 1, W.B Saunders Company, Philadelphia, PA, p. 60.

  2. Thavasu, P.W., et al (1992) Measuring cytokine levels in blood. Importance of anticoagulants, processing, and storage conditions. J Immunol Methods 153:115-124.
BioSource   C-070276 1107      1-Jan-2007