Q. NCCLS is specific about time guidelines for coagulation testing on unopened tubes and stored plasma. It is not unusual for clinicians to request that tests be added to specimens already in the laboratory (e.g. PT, PTT, TT, fibrinogen, D-dimer). What are the guidelines for adding tests to specimens that have been centrifuged, opened, and held at room temperature?
A. The NCCLS guideline says coagulation testing should be completed within four hours of sample collection for PTTs because the levels of coagulation factors, such as factor V and factor VIII, decrease with time due to degradation by activated protein C. Thus, many coagulation assay results will become progressively more abnormal with prolonged storage at room temperature. For this reason, performing more coagulation tests on samples stored for more than four hours at room temperature is not advisable.
If additional assays need to be performed on routine coagulation specimens with frequency, it would be advisable to separate the plasma from the cells within four hours and freeze at -20°C or lower. It is important that the plasma is centrifuged to remove platelets (residual platelet count <10,000/µL) as residual platelets are a source of phospholipid, which can alter coagulation testing results and neutralize antiphospholipid antibodies, such as the lupus anticoagulant. The frozen samples can be defrosted and the additional coagulation tests performed at a later time.
In a large laboratory with a high volume of routine coagulation tests, it is not always feasible to freeze all coagulation samples because of the constraints on storage space or personnel resources. A potential solution is to freeze only coagulation samples that are likely to need additional coagulation evaluations—for example, samples with abnormal screening coagulation tests such as PT and PTT, specimens from certain hospital locations such as intensive care units, or tests ordered by certain physicians such as hematologists.
Kandice Kottke-Marchant, MD, PhD
Hemostasis and Thrombosis
Department of Clinical Pathology
Cleveland Clinic Foundation
CAP Coagulation Resource Committee
Q. Our laboratory has been asked to assess several varieties of plastic tubes. We use glass tubes now, but we plan to switch to plastic. What type of comparison tests should be performed and to what extent can past studies be used in our laboratory? What does the typical CAP inspector look for and expect to find in the laboratory?
A. The typical CAP inspector looks for a scientifically valid study, such as parallel testing or a collection of patient samples in both glass and plastic tubes. The data collected need to be statistically evaluated to demonstrate that results are consistent, regardless of whether glass or plastic collection tubes are used. The study conducted needs to assess not only the results obtained relatively soon after collection but also the results after storage to determine if precision and accuracy are affected.
Each laboratory should determine the parameters of its study, taking into account the laboratory’s instrumentation and methodologies as well as the stability of the analyte being tested, and challenge any known interferences or biases introduced by the use of plastic tubes as documented in the literature. At a minimum, the number of samples should be statistically valid (n=>30) and should cover the reportable range for each analyte. Precision studies require replicate testing (at least 10 repetitions) at one or more points. Several NCCLS documents would be helpful in developing such a study: EP5-A, “Evaluation of Precision Performance of Clinical Chemistry Devices”; EP7-P, “Interference Testing in Clinical Chemistry”; EP9-A, “Method Comparison and Bias Estimation Using Patient Samples”; and EP14-A, “Evaluation of Matrix Effects.” (These evaluation protocols can be ordered online at www.nccls.org or by calling 877-477-1888.)
The Laboratory Accreditation Program’s Laboratory General checklist contains one question, GEN.40942, that specifically relates to specimen containers. It says: “Has the laboratory evaluated its specimen containers to ensure that they do not contribute to analytic interference in the assays to be performed?” A note accompanying the question allows the combination of direct testing by the laboratory, review of clinical literature, and evaluation of information from manufacturers, but it does not require exhaustive testing by each laboratory. Performance data listed in the manufacturer’s information are calculated from samples collected in glass. Limited data are available for samples collected in plastic.
More extensive studies should be considered in the cases of tests with known problems when plastic tubes are used. Plastic is permeable by air, which, for coagulation testing, can cause a drop in pH and affect results. Plastic also has been shown to absorb drugs, such as cyclosporins, and heavy metals.
Glass activates clotting (plastic does not), forcing tube manufacturers to add a clot activator in red top tubes. The clot activator has been shown to cause positive interference in lithium results. DNA has been shown to denature if stored in plastic microtubes; similar results can be anticipated if polymerase chain reaction specimens are collected in plastic. Clot tubes have a silicone-coated interior; silicone-coated tubes have not been approved by the Food and Drug Administration for blood banking.
Previous studies can be used provided that no additional variables have been introduced, such as alterations to your testing/reagent systems or in the types of tubes themselves.
The use of glass has come into focus with the 1999 release of an FDA/NIOSH/OSHA joint safety advisory on the potential risks of using glass capillary tubes and the modification to the OSHA bloodborne pathogen standard through the Needlestick Safety and Prevention Act. Your institution’s planned response to the changes in safety requirements is laudable.
Dasgupta A, Blackwell W, Bard D. Stability of therapeutic drug measurement in specimens collected in Vacutainer plastic blood-collection tubes. Ther Drug Monit. 1996;18: 306-309.
Ernst DJ. Plastic collection tubes decrease risk of employee injury. Medical Laboratory Observer. 2001;33.
Gaillard C, Strauss F. Eliminating DNA loss and denaturation during storage in plastic microtubes. American Clinical Laboratory. 2001; 20(2):52-55.
Joint FDA/NIOSH/OSHA Advisory on Glass Capillary Tubes: Joint Safety Advisory about Potential Risks. Available at: www.cdc.gov/niosh/capssa9.html. Accessed Oct. 11, 2001.
NCCLS Electronic Document Delivery. Available at: www.nccls.org/howto.htm. Accessed Oct. 11, 2001.
Sampson M, Ruddel M, Albright S, et al. Positive interference in lithium determinations from clot activator in collection container. Clin Chem. 1997;43:
Jean L. Tenuta, MS, MBA, MT(ASCP)DLM, CLC(AMT)
Technical specialist, Laboratory Accreditation Programs
College of American Pathologists