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What are general guidelines for the production of blood serum and plasma for cytokine analysis using ELISA and ProcartaPlex assays? Product FAQ

Answer

Serum is the liquid fraction of whole blood that is collected after the blood has clotted. The clot is removed by centrifugation and the resulting supernatant is the serum. It is carefully removed and used right away or it can be stored at -20 degrees C or below. Plasma is produced when whole blood is collected in tubes that contain an anticoagulant. In this case, the blood does not clot and the red and white blood cells are removed by centrifugation. The supernatant, called plasma, is carefully removed from the cell pellet and can be used right away or stored frozen for testing later.

Here are some procedures for preparing serum and plasma:

For serum, collect whole blood in capped test tubes and allow it to clot. Typically, commercially available tubes such as Vacutainer tubes are used, and for serum, the researcher should use the ones with red tops (no anticoagulant added). Vacutainer tubes of various types and volumes are available from Fisher Scientific. After collecting the blood, leave it undisturbed at room temperature to clot, which usually takes 15-30 minutes. Remove the clot by centrifugation at 1,000-2,000 x g for 10 minutes in a refrigerated centrifuge. The resulting supernatant is the serum. Following centrifugation, it is important to immediately transfer the sera into clean polypropylene tubes. These samples should be kept on wet ice until they are used or frozen. If the sera are not analyzed immediately, they should be divided into 0.5 mL aliquots and stored frozen at -20 degrees C or lower. It is important to avoid freeze/thaw cycles because this is detrimental to many serum components. Also, serum derived from blood that has undergone hemolysis (erythrocyte lysis), is icteric (contains bilirubin) or is lipemic (contains lipids) can invalidate certain tests.

For plasma preparation, collect whole blood into commercially available anticoagulant-treated Vacutainer or equivalent tubes. Tubes that are EDTA-treated (lavender tops) or citrate-treated (light blue tops) are commonly used. Heparinized tubes (green tops) are indicated for some applications. However, heparin can be contaminated with endotoxin, which can stimulate white blood cells to release cytokines. Cells are removed from the non-coagulated blood by centrifugation for 10 min at 1,000-2,000 x g using a refrigerated centrifuge. Centrifugation for 15 min at 2,000 x g depletes platelets in the plasma sample, if desired. Either way, the resulting supernatant is the plasma. Following centrifugation, it is important to immediately transfer the plasma into clean polypropylene tubes. The samples should be maintained on wet ice during handling. If the plasma is not analyzed immediately, it should be divided into 0.5 mL aliquots and stored at –20 degrees C or lower. It is important to avoid freeze/thaw cycles with plasma as well. Like serum, plasma derived from hemolyzed, icteric, or lipemic blood can invalidate certain tests.

Besides Vacutainer tubes, there are other commercially available tubes for blood sample collection. However, we have not evaluated these tubes to see if sera and plasma samples derived with them are compatible with our ELISA and ProcartaPlex kits. Nevertheless, commercially available blood collection tubes for serum are designated as follows: red caps = no anticoagulant or red caps with black stripes = no anticoagulant and containing gel to help to separate the clot (not evaluated). Blood collection tubes for plasma are as designated as follows: lavender cap = treated with EDTA, blue cap = treated with citrate, green cap = treated with heparin, grey cap = treated with potassium oxalate/sodium fluoride (not evaluated), and yellow cap = treated with acid-citrate-dextrose (not evaluated).

Answer Id:: E5220

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How does one prepare serum and plasma samples? Product FAQ

Answer

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 anti-coagulant. 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.

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 Becton Dickinson Vacutainer tubes. 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 and 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.

Plasma preparation: Collect whole blood into commercially available anti-coagulant treated tube, such as EDTA treated (lavender tops) or citrate treated (light blue tops). Heparinized tubes (green tops) are indicted for some applications; however, heparin can often be contaminated with endotoxin and endotoxin 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 and 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. Thermo Fisher Scientific has not evaluated some of these tubes for compatibility with our ELISA kits. 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: Lavender: Treated with EDTA. Blue: Treated with citrate. Green: Treated with heparin. Grey: Treated with potassium oxalate/sodium fluoride (not evaluated). Yellow: Treated with ACD (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., S. Longhurst, S.P. Joel, M.L. Slevin, and F.R. Balkwill (1992) Measuring cytokine levels in blood. Importance of anticoagulants, processing, and storage conditions. J. Immunol. Methods 153:115-124.

Answer Id:: E5129

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Whole genome expression and biochemical correlates of extreme constitutional types defined in Ayurveda. Citations & References

  • Authors: Prasher B; Negi S; Aggarwal S; Mandal AK; Sethi TP; Deshmukh SR; Purohit SG; Sengupta S; Khanna S; Mohammad F; Garg G; Brahmachari SK; Indian Genome Variation Consortium; Mukerji M
  • Journal: Journal of Translational Medicine

Whole genome expression and biochemical correlates of extreme constitutional types defined in Ayurveda. Citations & References

  • Authors: Prasher B, Negi S, Aggarwal S, Mandal AK, Sethi TP, Deshmukh SR, Purohit SG, Sengupta S, Khanna S, Mohammad F, Garg G, Brahmachari SK, Mukerji M
  • Journal: J Transl Med
  • PubMed ID: 18782426
Catalog #

Variability in platelet procoagulant activity in healthy volunteers. Citations & References

  • Authors: Sumner WT, Monroe DM, Hoffman M
  • Journal: Thromb Res
  • PubMed ID: 8907312
Catalog # A13199

Dynabeads DNA DIRECT Blood Experiment Protocol

Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Citations & References

  • Authors: Kienast J, Schmitz G
  • Journal: Blood
  • PubMed ID: 1688494
Catalog # T1376

Dynabeads DNA DIRECT Universal Experiment Protocol

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