Most laboratory testing for clinical purposes is done on samples obtained from blood. Whole blood contains the liquid fraction of blood (i.e., plasma) as well as the cellular elements that lead to clotting under certain circumstances. These include red blood cells (RBCs), white blood cells, and other components.
First, it is helpful to establish some baseline definitions:
- “Serum” is the fluid obtained when whole blood clots, as it will do spontaneously when it contacts a surface such as glass or plastic. Clotting is pre-programmed into the components of blood to prevent excessive blood loss from a minor wound. In a lab setting, it is common to centrifuge the clotted blood, including red cells, to the bottom of the collection tube, leaving a straw-colored liquid above the clot.
- “Plasma” is the fluid component of blood. It is obtained when a clotting-prevention agent is added to whole blood and then placed in a centrifuge to separate the cellular material from the lighter liquid layer. Common anti-coagulant agents are EDTA (ethylenediaminetetraacetic acid), heparin, and citrate.
Blood samples intended for laboratory analysis are usually collected in glass or plastic tubes that have been partially evacuated so that their internal air pressure is lower than atmospheric pressure. These tubes have color-coded polymer stoppers that indicate their contents. Plain tubes with no anticoagulants have red stoppers and are used in the preparation of serum after clotting and centrifugation. Purple or lavender top tubes contain EDTA, and green top tubes have heparin in them.
Blue top collection tubes containing citrate exist as well, but it far more common to encounter citrate containing plasma as reclaimed plasma from blood donations. When people donate blood for therapeutic purposes (e.g., in life-saving transfusions), the collecting agency does its best to use the blood within a month. Inevitably, due to a need to have enough blood on hand to meet unexpected demand, some of the blood cannot be transfused because it has been stored too long. When blood is collected, it fills a plastic bag that contains a solution of dextrose and citric acid. The dextrose is a nutrient for some of the cells in the blood, and the citric acid complexes with or chelates calcium which prevents the blood from clotting, Minutes after collection, the blood is centrifuged into packed RBCs and citrated plasma for better storage. Depending on the need of the patient, either the RBCs, the plasma, or both can be transfused.
Outdated citrate plasma becomes an industrial commodity and is processed to provide a number of components, including a synthetic normal human serum that is the starting point for many of the calibrators used in commercial immunoassay products. The segments of tubing used to transfer separated plasma from the red cells can sometimes be obtained from vendors of outdated plasma. These segments contain about 500 uL of plasma and are invaluable for informal normal-range studies as they come from ostensibly healthy normal donors, who can be identified by age and gender. The entire bag of plasma associated with the tubing segments may also be available for further studies, and it holds approx. 220 mL. This can be a valuable resource in the process of developing an assay.
Labs usually have time limits on how long serum or plasma can remain in touch with the red blood cells before being physically separated—usually by pouring off into another tube—while maintaining the identity of the patient from whom the blood was taken. This is necessary because RBCs can rupture over time, and the contents can interfere with a number of assays. RBCs contain high levels of potassium and the thyroid hormone thyroxine. Assays for either of these substances and others can be skewed by the contents of lysed red blood cells. Hemoglobin is the most abundant protein in blood. It can interfere in assays once released from RBCs.
One technical solution to the problem of RBCs remaining in contact with the serum or plasma is to use serum separator tubes (SSTs). These are evacuated blood drawing tubes that contain a silicone gel that has a density intermediate between serum and red blood cells. In a centrifuge, this silicone gel forms an impermeable layer between the red blood cells at the bottom of the tube and the serum above. Caution is advised in using these SSTs with hydrophobic analytes such as some drugs.[1] The silicone gel can act as an extracting organic solvent, and the drug can be removed from the plasma and end up in the gel, leading to misleading low drug levels in the serum.
Whole blood, serum, and various plasmas are not interchangeable sample matrices. While some analytes may give similar results, equivalence can only be ensured by testing matched samples. Whole blood contains RBCs that may occupy as much as 60% of the volume. Some analytes, such as Parathyroid Hormone (PTH), partition freely between the red blood cells and the plasma so that whole blood and plasma values are the same within experimental accuracy. Others, such as lipoproteins, are strongly influenced by red blood cell content.[2] Still others, such as some immunosuppressant drugs like cyclosporin, partition between RBCs and plasma in a temperature-dependent manner[3] that reflects the temperature history of the sample. To avoid this problem, whole blood is the preferred sample for measuring immunosuppressant drugs.
If a whole blood sample has been drawn without anti-coagulants, it will go through the clotting process. The resulting serum will therefore be depleted of various clotting factors and, after centrifugation, all cellular material. Citrated plasma drawn in a blue-topped tube is the sample preferred for coagulation factor testing. Complete blood count samples are drawn in a purple- or lavender-topped EDTA tube.
Plasmas are also not interchangeable. EDTA plasma has an excess of a powerful chelating agent that will sequester metal ions such as calcium and magnesium. EDTA plasma will also inactivate some enzymes that require a metal ion for their activity. This includes alkaline phosphatase used as a label in some immunoassays. To avoid this assay interference, EDTA plasma and the alkaline phosphatase containing label must never come in contact for any length of time.
Heparin can also interfere with some analytes. Troponin I values measured in heparinized plasma are often lower than when measured in EDTA plasma or serum from the same patient taken at the same time. Troponin I is a positively charged molecule, and there has been speculation that the highly negatively charged polymeric heparin effectively shields Troponin I from being recognized by antibodies.
If you are a user of an assay, you should consult the manufacturer’s instructions for use (IFU) to determine what sample types have been validated for use in the assays. If the sample type you wish to test is not listed in the IFU, consult the manufacture’s technical support staff as it is likely someone else has asked the same question before you.
If you are the developer of an assay, you should validate the use of the sample types you believe will be used in the assay. Assuming that a certain sample type will work in a given assay without proper validation is poor science and may elicit an unfavorable response from customers and regulatory agencies.
We work with a variety of sample types. For more information, click here.
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[1] Effect of serum separator blood collection tubes on drug concentrations. Quattrocchi F, Karnes HT, Robinson JD, Hendeles L.,Ther Drug Monit. 5:359-62. (1983)
[2] Influence of hematocrit on the measurement of lipoproteins demonstrated by the example of lipoprotein(a) Kronenberg F, Trenkwalder M, Kronenberg M, Koenig P, Utermann G and Dieplinger H,Kidney International, 54, 1385–1389 (1998)
[3] Cyclosporin Therapeutic Drug Monitoring – an Established Service Revisited, Morris, RG, Clin Biochem Rev 24 33-46 (2003)
