Vol. 19 • Issue 6 • Page 60
Lab Limelight
For most routine analytes, serum and plasma measurement results are equivalent, but due to platelet clotting, platelet substances (e.g., potassium, lactate dehydrogenase, calcium, magnesium, etc.) can be higher in serum than plasma. And depending on how plasma is prepared (e.g., plastic versus glass collection, storage temperature, etc.), anticoagulants can interfere with the measurement of certain analytes.
For example, when measuring lithium in lithium heparin plasma for therapeutic drug monitoring, lithium interferes with the anticoagulant. In a lithium heparin tube filled with blood, plasma tests would reveal about 1 millimole per liter of lithium. Since the therapeutic range for lithium is between .5 and 1.5 millimole per liter, a patient would appear normal even if no lithium was circulating in the blood and the patient were not responding to medication.
“The patient may develop psychiatric symptoms as a result of non-compliance, and it won’t be until those symptoms are manifest that the physician would have any inclination the patient is non-compliant with this medication,” says William L. Roberts, MD, PhD, professor of Pathology, University of Utah, medical director Automated Core Laboratory, ARUP Laboratories. “So the potential [consequences] are pretty clinically significant.”
In a 2004 study published in Clinical Chemistry, Dr. Roberts and colleagues reported differences between serum and heparinized plasma specimen results for several chemistry analytes, although past results have often been considered equivalent for many assays. The reason for the discrepancy is because past results were compared to the wrong reference interval and due to misinterpretation of reference values at upper/lower limits.
For example, potassium and lactate dehydrogenase values can be higher in serum than in heparinized plasma. A patient may appear potassium deficient if results obtained on a plasma specimen are compared to a serum reference interval. Similarly, a patient with hyperkalemia (high blood potassium levels), which can result in cardiac problems, might be missed if the result from a plasma sample was compared to a serum reference interval.
“The difference is about a half a millimole per liter so it’s unlikely the patient’s going to have excessive toxicity but he could be borderline on the high end and it not be detected,” he says.
Measurement Accuracy
Ensuring the right sample type-serum or blood-is collected for a given test is essential to ensuring measurement accuracy. Phlebotomists need to be properly educated and have handy information on correct reference interval information when drawing blood. They would need to know that the B-type natriuretic peptide (BNP) analyte, a marker of heart failure, returns more accurate results with ethylenediaminetetraacetic acid (EDTA) plasma than serum because of activated clotting factors. Labs should establish standard procedures for sample collection.
“It’s increasingly important to have a very defined protocol for the collection of plasma or serum samples,” says Roger L. Lundblad, PhD, consultant and adjunct professor, Department of Pathology, University of North Carolina, Chapel Hill, NC. “And for the storage, the same.” Dr. Lundblad compares sample collection protocols to successful culinary pursuits: “If you don’t have a recipe, you can’t reduce the batch of cookies. Without an established protocol, you cannot get reproducible data.”
Most labs have test directories listing acceptable sample types. Dr. Roberts’ reference lab refers to data it receives from hospital labs around the country along with an online test directory and package inserts on FDA-cleared assays for acceptable/normal reference values. Labs can also defer to standards such as Clinical and Laboratory Standards Institute document C28-A3, which outlines how to establish reference intervals.1 “It’s recommended that each laboratory verify those reference intervals before the assay is placed in service,” Dr. Roberts says.
At times, a lab needs to adjust intervals to align with its patient population. Factors such as ethnic background and even geography can play a role. In Dr. Roberts’ lab in Salt Lake City, some blood gas parameters are different than they would be if measured at sea-level locations. The lab will do a validation experiment of additional sample types if needed.
“We’ll draw 10-20 individuals with the approved sample type in parallel with the type we’d like to validate,” he says. “Ideally, we’d have not only normal patients but also those with high or low values consistent with the disease for which that test is used. For lab developed tests, we do a more extensive reference interval validation and a specimen type and stability check.”
Jill Hoffman is a senior associate editor.
Reference
Horowitz GL, Altaie S, Boyd JC, et al. (2008). Clinical and Lab Standards Institute (C28-A3): Defining, Establishing and Verifying Reference Intervals in the Clinical Lab; Approved Guideline-3rd Edition; Vol. 28(30). At: www.clsi.org/source/orders/free/c28-a3.pdf (accessed April 12, 2010).
Proper Specimen Collection
By Michelle McLean
In reviewing analytical differences in serum versus plasma, it is important to emphasize the use of appropriate equipment and proper phlebotomy technique when collecting specimens. This can be more difficult than it appears when considering issues such as employee turnover, decentralized phlebotomy and ongoing training in laboratories.
Though choosing the correct tube type for the test ordered may seem the most obvious answer in avoiding analytical errors when considering specimen collection, the impact of pre-analytic errors such as hemolysis, fibrin and incorrect additive to blood ratio can further exacerbate the inaccuracies of randomly switching between serum and plasma. For example, potassium values can be falsely elevated by hemolysis, further elevating levels caused by excess platelets in a plasma sample.
The phlebotomist must practice good collection technique to avoid pre-analytic variables that can impact sample testing. Choosing the correct equipment to avoid traumatic collection, following the correct order of draw, adequately filling the tube to ensure proper additive to blood values and mixing the tube according to the manufacturer’s instructions after collection all help to ensure that a quality sample arrives in the laboratory for testing. Even serum tubes contain an additive to initiate coagulation now that plastic tubes are more prevalent than glass due to safety concerns.
Prior to centrifugation, serum samples must be allowed to sit for at least 30 minutes to ensure complete clot formation. If a serum sample is centrifuged prior to clot retraction, fibrin strands can be present in the serum portion of the sample, creating potential for instrument error and inaccurate test results. Plasma samples, on the other hand, do not require wait time and are often utilized to meet the tight turnaround times required of laboratories.
Finally, samples should be properly centrifuged to achieve good sample separation and, in the case of gel tubes, adequate gel barrier formation. Most analytical tests require platelet-poor plasma. Ideally, the manufacturer’s recommendations for centrifugation should be followed to obtain optimal sample separation. If changes are made as a result of equipment limitations or turnaround time requirements, appropriate validation studies should be performed to ensure that platelets are adequately removed and test values are not adversely affected.
Ensuring accuracy of test results depends on establishing appropriate reference ranges and utilizing proper test samples whether they are whole blood, serum or plasma. The process of obtaining accurate results begins, however, with proper collection technique.
Michelle McLean is technical marketing manager at Greiner Bio-One North America Inc., Monroe, NC.
