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YLF1_Jan03.qxd 12/4/02 10:47 AM Page 49 your lab focus CE Update [coagulation and hematology | phlebotomy] Preanalytical Variables in the Coagulation Laboratory Jeffry B. Lawrence, MD Medical and Scientific Affairs, BD Clinical Laboratory Solutions, Franklin Lakes, NJ DOI: 10.1092/ER9P64EBMCFR47KY After reading this article, the reader should understand how errors occur in the preanalytical phase of testing, and how efforts can be made to correct these errors. Hematology exam 0301 questions and the corresponding answer form are located after the “Your Lab Focus” section on p. 69. 왘 Errors in coagulation laboratory 왘 왘 results, whether in assays performed for anticoagulant monitoring or for screening and diagnostic testing for hemorrhagic and thrombotic disorders, can lead to clinical mismanagement and significant risk for the patient. The majority of coagulation laboratory errors arise in the preanalytical phase; therefore, ensuring the quality of citrated blood samples is critical for accurate test results. Understanding the sources of preanalytical variability; educational and quality improvement programs; and standardization efforts directed at optimizing sample quality provide the most cost-effective opportunities for a laboratory to improve the quality of coagulation test data and enhance clinical care. Though cardiovascular disease remains the most common cause of death in Western countries, significant progress has been made in the management of it during the past 2 decades. Increased use of anticoagulant therapy has played an important role in the improved outlook for patients with occlusive arterial or venous thromboembolic disease. However, while anticoagulant drugs reduce a patient’s risk of heart attack and stroke by making the blood less clottable, this same effect increases the patient’s risk of hemorrhage. This necessitates laboratory monitoring of the patient’s response to these drugs on an ongoing basis. For instance, of the approximately 300 million coagulation tests performed annually in the United States, more than 40 million are prothrombin times (PT) performed for monitoring of warfarin therapy. While monitoring of anticoagulant therapy may have the most immediate impact on patient management and outcome, clinicians also rely heavily on the accuracy of screening and diagnostic PT and activated partial thromboplastin time (APTT) tests; platelet function studies; work-ups to identify inherited thrombotic predisposition; and measurement of coagulation factors and inhibitors, D-dimer, and other coagulation analytes. Because of the significantly improved instrumentation and highly sensitive reagents available in modern coagulation laboratories, preanalytical variability now represents the most important source of errors that can lead to inaccurate patient results, patient mismanagement, and preventable hemorrhagic or thrombotic complications. This article will review the importance of the quality and integrity of the coagulation © laboratorymedicine> january 2003> number 1> volume 34 sample for the accuracy and precision of patient test results, and discuss approaches to improve coagulation laboratory performance by controlling preanalytical variability. Laboratory Errors and Role of Preanalytical Variables Just as with all other laboratory testing, in the coagulation laboratory the ultimate goal is to reflect the patient’s actual state of hemostatic function in vivo. Whether testing is performed to diagnose a bleeding or thrombotic disorder, to screen a patient for surgery, or to monitor coagulation factor replacement or antithrombotic therapy, any factor that causes the test to misrepresent the actual state of coagulation function in the patient (whether in the preanalytical phase, during the analysis, or in postanalytical test reporting and interpretation given to the clinician) can lead to adverse outcomes for the patient. Anticoagulant drugs, whether intravenous heparin or oral compounds such as warfarin, interfere with several different coagulation enzymes, resulting in inhibition of the coagulation system. Both warfarin and heparin have a narrow therapeutic index. This means that there is a narrow range between inadequate doses that can lead to recurrent thrombosis and excessive 49 YLF1_Jan03.qxd 50 12/4/02 10:47 AM Page 50 doses which predispose to hemorrhage. Administration of these drugs results in prolongation of the PT or APTT, respectively, and the magnitude of the prolongation is used to determine the optimal dose of the drug for the patient. Thus, these tests, in essence, serve as therapeutic drug monitoring bioassays that directly determine drug dosage. Erroneously prolonged PT or APTT results would mislead the clinician to believe that the patient has a greater anticoagulant response to warfarin or heparin in vivo than is actually the case, leading to dose reductions and a corresponding risk of recurrent thrombosis. Alternatively, an artifactually shortened clotting time would typically cause the physician to inappropriately increase the anticoagulant dose, putting the patient at risk for hemorrhage. Similarly, inaccurate coagulation test results performed to diagnose or screen for potential hemorrhagic or bleeding disorders can either lead to the failure to detect a treatable life-threatening condition or erroneously suggest the presence of a disease when the patient is free from a coagulation disorder. The latter often results in cancellation of needed invasive procedures and/or unnecessary follow-up diagnostic testing. In an effort to better understand the source of laboratory errors across disciplines, Plebani and Carraro1 analyzed all 40,490 stat results reported over a 3month period in the University Hospital of Padua to the Intensive Care Unit and the inpatient Departments of Internal Medicine, Surgery, and Nephrology. They identified 189 errors (0.47% of all results) and determined that 68% of the errors originated in the preanalytical phase, compared with 13% of errors arising during analysis and 18% in the postanalytical phase. In over one-fourth of the cases, these laboratory errors resulted in unnecessary further investigations and/or inappropriate care or changes in therapy. In this paper, we define preanalytical variables as any influence before testing of the specimen that causes the coagulation test result to fail to reflect the patient’s in vivo hemostatic function. Susceptibility of Tests to Preanalytical Variability Clinical chemistry, immunochemistry, hematology, and other laboratory tests each show some dependence upon the quality of the sample. However, coagulation tests are exquisitely susceptible to error introduced by suboptimal specimen quality. This is due to the fact that the very act of obtaining a blood sample initiates the hemostatic response, the physiologic system that the testing is designed to assess. Coagulation assays are highly vulnerable to preanalytical variability because of a number of factors: (a) the complexity of the biochemical and cellular reactions measured in assays such as the PT and APTT; (b) the lability of several coagulation proteins; (c) the calcium dependence of many of these reactions; and (d) the highly excitable nature of blood platelets—the key cellular “player” in these assays. During the past 20 years, ever more sensitive and reliable PT and APTT reagent systems have been introduced into the laboratory, resulting from an explosion in understanding the basic biochemistry and physiology of the coagulation system. Furthermore, advances in laboratory instrumentation and automation have dramatically reduced analytical variability in coagulation analyzers. While these developments have substantially improved the accuracy and precision of coagulation laboratory data, paradoxically they have increased the dependence of the test results on the quality of the citrated blood sample. Consequently, preanalytical variability is more hazardous than ever, particularly given the recent dramatically increased use of anticoagulant therapy as well as the clinical importance of diagnostic testing for von Willebrand’s disease, prothrombotic disorders, disseminated intravascular coagulation, and other hemostatic abnormalities. It is impossible to completely reproduce physiologic conditions in the coagulation laboratory in vitro, since the act of removing the blood from the patient and putting it into a container causes initiation of clotting. However, calcium is required for many of the biochemical steps of the coagulation mechanism, and laboratorymedicine> january 2003> number 1> volume 34 © artificial removal of the calcium by an additive in the coagulation tube (ie, sodium citrate) prevents coagulation from occurring. The citrated blood sample is transported from the site of phlebotomy to the laboratory and centrifuged, resulting in the removal of the vast majority of blood cells and the remaining plasma is the specimen to be tested. The PT, APTT, or other coagulation reagent is incubated with the plasma, and the time it takes for a clot to form after the subsequent addition of calcium is inversely proportional to the patient’s coagulation response. Coagulation testing is optimized when these processes of phlebotomy, calcium chelation, sample transport, centrifugation, and recalcification proceed under maximally standardized and controlled conditions. Patient-Related Variables Strictly speaking, patient variables do not represent preanalytical variables as defined before, since they affect not only coagulation laboratory results but also hemostatic function in vivo. However, they are important to recognize for the determination of appropriate reference ranges and the evaluation of the significance of slight abnormalities detected in coagulation tests performed on patients suspected of having hemostatic disorders.2-19 [T1] For instance, use of age-specific reference ranges is critical for interpretation of coagulation data in newborns and infants,2 and we and others have demonstrated that age and gender influence platelet function as well as coagulation activation.3-6 Individuals with blood type O have significantly lower plasma von Willebrand factor and factor VIII activity than subjects with other blood types.7 It has been reported that platelet activation, and the incidence of myocardial infarction are highest in the morning,8 and increased coagulation activation has been described in cold weather, associated with an increased incidence of myocardial infarction and stroke.9 Diet and alcohol intake have been shown to affect coagulation activation in people with diabetes10 and in other populations. Smoking elevates plasma fibrinogen, von Willebrand factor, other YLF1_Jan03.qxd 12/4/02 10:47 AM Page 51 coagulation factors, thrombin generation, and platelet activation.11 Beyond the antioxidant and antithrombotic effect of red wine,10 moderate ethanol intake inhibits platelet reactivity,12 and this effect has been linked to the reduction in coronary heart disease associated with light to moderate alcohol consumption. In addition to the well-known platelet inhibitory effect of aspirin and non-steroidal antiinflammatory drugs, a number of other medications, including hormone replacement therapy and selective estrogen receptor modifiers, alter coagulation and platelet responsiveness.13 Oral contraceptives, pregnancy, menopause, and the different phases of the menstrual cycle are also associated with changes in coagulation.14 Vigorous physical exercise leads to coagulation activation,15 and psychological stress has been associated with changes in both the coagulation and fibrinolytic systems.16 Disease states which lead to anemia, polycythemia, or hemolysis can also interfere with coagulation tests since anemia results in an effectively lower plasma citrate concentration and shortened clotting times while polycythemia has the opposite effect.17 Because of this, the NCCLS coagulation guideline document H21-A3 provides recommendations for adjusting the amount of anticoagulant and blood for patients with hematocrit values >0.55.18 Hemolysis can both influence the biochemistry of coagulation reactions and interfere with clot detection in some analyzers. Furthermore, anemia itself can prolong the bleeding time because of the important rheologic role red cells play in platelet adhesion in primary hemostasis.19 Phlebotomy Practices Positive patient identification is critical for accurate test results in all disciplines within the laboratory. Identification errors can occur either at the time of phlebotomy or after centrifugation when specimens are aliquotted. Another source of error can be mistakes in transcribing the specific coagulation assays requested by the clinician and selection of inappropriate blood collection tubes (and needles). Because recent patient exercise can activate platelets and the coagulation Patient Variables That Can Influence Coagulation Test Results T1 · Age · Gender · Blood type · Intraindividual variability, circadian, and seasonal rhythms · Diet · Smoking · Alcohol intake · Medications · Menstrual cycle, pregnancy, menopausal status · Physical exercise · Emotional stress and psychiatric disorders · Disease states associated with anemia, polycythemia, or hemolysis system as noted,15 patients should rest comfortably for 15 to 30 minutes prior to phlebotomy. When patients move from the supine to the upright position, water moves from the intravascular compartment to the interstitium, reducing the plasma volume by 12% on average. This increases the apparent concentration of cells, macromolecules, and protein-bound small molecules, and consequently the platelet count and coagulation factor levels may be elevated and the PT and APTT shortened. Also, upon standing, vasoactive hormones are released which can result in platelet activation. Therefore, it is desirable to standardize patient position during phlebotomy. A broad category of variables related to phlebotomy technique and practices can introduce preanalytical variability. Placement of the tourniquet for longer than 1 minute causes migration of fluid and low molecular weight molecules from the intravascular to the interstitial space, resulting in elevation in cells and coagulation proteins. This can also lead to coagulation activation. Traumatic and/or prolonged phlebotomy accentuates the hemostatic activation that is inevitable in blood collection. This can produce an artificially short PT and/or APTT, if excessive vascular injury results in slight to moderate activation of coagulation factors and platelets. Alternatively, with greater difficulty in the phlebotomy, marked coagulation activation can lead to depletion of fibrinogen and other factors, resulting in prolonged clotting times. Underfilling or overfilling of the citrate tube, with resulting imbalances in the blood/additive © laboratorymedicine> january 2003> number 1> volume 34 ratio, can produce artificially prolonged or shortened clotting times, respectively.18,20 Finally, it is important to avoid obtaining blood samples from a site near infusion of intravenous fluids (particularly heparin). In the NCCLS guideline H21-A3, it is recommended to use evacuated blood collection systems into a tube containing a specified amount of sodium citrate.18 The use of a needle and syringe to draw blood samples for coagulation testing introduces a number of uncontrolled preanalytical variables. This is made worse if “homemade” citrate additives are used as anticoagulants to be added to glass or plastic tubes. This technique also increases risk of health care worker injury from bloodborne pathogen exposure. Particularly with larger syringes, there is a significant risk that clotting may occur. The volume of blood drawn is critical for accurate coagulation results. The blood:citrate ratio of 9:1 must be maintained. Use of a syringe makes it more difficult to consistently obtain the desired blood volume draw. Syringe draws also produce variability in the shear forces to which platelets are exposed, depending upon technique. This is particularly important for heparin monitoring because shear force activates platelets, and this can introduce clinically significant shortening of APTT results. Hemolysis is also significantly more common with use of needle and syringe compared with evacuated systems. The NCCLS recommended order of draw for obtaining multiple blood collection tubes is shown in T2.21 Citrate tubes 51 YLF1_Jan03.qxd 12/4/02 10:47 AM Page 52 NCCLS Recommended Order of Draw21 T2 Blood culture tubes Plain serum glass tubes Sodium citrate tubes Gel separator tubes/plain serum plastic tubes Heparin tubes/heparin gel separator tubes EDTA tubes Glucose preservative tubes 52 should be drawn after blood culture tubes and serum tubes lacking clot activator, but before other additive tubes. If only a coagulation sample is to be drawn, discard tubes are not necessary unless a winged blood collection set is to be used.18,22 In this case, a discard should be drawn before filling the citrate tube in order to compensate for the dead space in the tubing of the blood collection set. Prothrombin time, APTT, and other coagulation assays require a specified plasma concentration of sodium citrate, which in turn controls the amount of ionized calcium available for clotting. As described in the discussion of anemia and polycythemia, if the citrate tube is overfilled with blood, the blood:citrate ratio will be elevated, resulting in excessive ionized calcium availability and artificially short clotting times. Conversely, in an underfilled tube, the blood:citrate ratio is decreased, reducing the availability of ionized calcium and causing artifactually prolonged clotting times. The latter may be accentuated by dilution of the plasma by the liquid anticoagulant solution. Similarly, the citrate concentration in the tube itself can influence PT and APTT results because less ionized calcium is available with 3.8% sodium citrate (0.129M) than with 3.13% (0.105M) or 3.2% (0.109M). Adcock and colleagues examined the effect of underfilling tubes containing 0.129M citrate compared with underfilling 0.105M citrate tubes.23 They found that the prolongation of the APTT is more pronounced with underfilling in 0.129M citrate tubes than in 0.105M tubes; similar results were found with PT. In this sense, the lower citrate concentration is more “forgiving” with respect to underfilling (or overfilling) the tube; 0.105M citrate is also less sensitive to variability in the hematocrit. For these reasons, the NCCLS recommends the use of 3.13% or 3.2% sodium citrate tubes,18 and in recent years there has been a significant shift away from 3.8% citrate to the lower concentrations. It is important to quickly invert the sodium citrate tube after it is filled in order to ensure adequate mixing and prevent clotting in the tube. However, an excessive number of inversions, or vigorous shaking of the tube, can lead to platelet activation and artificial shortening of clotting times, particularly in heparinized samples. Thus, it is optimal to invert the tube gently 3 to 4 times. Specimen Transportation and Handling Coagulation proteins, particularly factors V and VIII, undergo in vitro degradation that increases with time. This loss of cofactor activity is accelerated at increased temperatures, and it results in artifactual prolongation of PT and APTT in most patient populations when there are delays between sample acquisition and analysis. While elevated temperatures enhance factor V and VIII degradation, refrigeration can result in cold-induced activation of the intrinsic coagulation system, subsequent factor VII activation, and artifactual PT shortening if unsiliconized glass citrate tubes are used.24 The NCCLS recommends that specimens for PT assays uncentrifuged or centrifuged with plasma remaining on top of the cells in an unopened tube should be tested within 24 hours from the time of specimen collection.18 For APTT and other coagulation assays on nonheparinized patients, uncentrifuged samples or samples centrifuged with laboratorymedicine> january 2003> number 1> volume 34 © plasma remaining on top of the cells should be tested within 4 hours of specimen acquisition.18 However, in the latter case, if agitation of the specimens is likely after centrifugation (such as remote transportation of the sample), then the plasma should be removed within 1 hour of collection and tested within 4 hours.18 Adcock and colleagues reported that in patients undergoing unfractionated heparin therapy, citrate samples demonstrate a clinically significant shortening of the APTT when stored uncentrifuged at room temperature.25 This shortening was attributable to release of platelet factor 4. Therefore, for APTT specimens suspected to contain unfractionated heparin, centrifugation should be performed within 1 hour of collection, and testing should be performed within 4 hours from time of collection.18 If testing is not completed within these time periods, plasma should be removed from the cells and frozen at –20ºC for up to 2 weeks. If the samples are kept at –70ºC, they may be stored for up to 6 months. A frost-free freezer should not be used, and frozen plasma samples should be rapidly thawed at 37ºC while gently mixing, and tested immediately (the sample may be held for a maximum of 2 hours at 4ºC before testing).18 Platelets also rapidly lose their reactivity after storage, so platelet aggregation studies should typically be initiated within an hour after sample acquisition and completed within 2 hours. Traumatic specimen handling, such as in some older pneumatic tube systems, can lead to platelet activation and shortening of clotting times. Centrifugation conditions are critical for coagulation testing. The NCCLS H21-A3 guideline recommends that centrifugation be performed at room temperature at a speed and time that consistently produces platelet-poor plasma with a platelet count under 10,000/µl—eg, 1,500 g x 15 minutes or longer.18 Higher platelet counts can neutralize anti-phospholipid antibodies, resulting in false-negative lupus anticoagulant assays. Just as in the blood bank and other sections of the laboratory, unsuitable coagulation samples should be rejected for analysis; otherwise, erroneous results may be reported. When YLF1_Jan03.qxd 12/4/02 10:47 AM Page 53 testing is performed on aliquots derived from a primary citrate tube, errors in pipetting and sample misidentification can occur. Coagulation Tubes Citrate tubes need to be compatible with the holders, centrifuges, and coagulation analyzers present in the laboratory. It is also desirable that the tubes protect health care workers from injury from bloodborne pathogen exposure due to blood splatter and tube breakage. Safety closures and plastic tubes offer such protection; however, with the latter it is important to establish that the coagulation test results are not compromised. The tube’s stopper plays a key role in maintaining vacuum and liquid retention in the tube. It must be readily penetrated by the non-patient end of the needle within evacuated tube holders and remain seated in the tube, and it must not leach calcium, magnesium, and zinc into the citrate additive. This requires vigilant quality control since rubber preparations must be specially formulated to prevent divalent cation leaching.26 The draw volume of the tube must be adequate to support the plasma volume necessary for testing without leading to unnecessary blood loss for the patient. The tube’s sodium citrate concentration (eg, 3.2% versus 3.8%) affects coagulation results through its effect on ionized calcium availability, with relatively longer clotting times observed with 3.8% citrate.27 While early coagulation tubes were manufactured with untreated glass, current coagulation assays have been standardized using full-draw sodium citrate tubes made of siliconized glass. Siliconization was introduced because of the increased activation of the contact portion of the intrinsic coagulation cascade observed with unsiliconized glass, leading under certain conditions to shortened clotting times.24 Certain plastic formulations show similar properties. Clinical correlation studies in which outcomes such as hemorrhage and thrombosis are linked to PT and APTT values have used full draw siliconized glass tubes. This is critical because the bloodsurface interaction with tubes made of other materials, such as unsiliconized glass or various plastics (such as polyethylene terephthalate or polypropylene), may differ significantly from that observed with siliconized glass.28 Furthermore, the effect of the inner surface of coagulation tubes on test results is difficult to predict because the degree of hydrophobicity of surfaces has opposite effects on coagulation activation and platelet activation.29 Because of these complexities, the bias in coagulation test results exhibited by different plastic tubes, relative to siliconized glass tubes, is unpredictable and may differ for different assays.30-34 The magnitude, and even the direction, of the bias may differ according to the instrument and reagent used. Unlike with clinical chemistry and other laboratory disciplines, since clotbased coagulation tests are reported in seconds [or International Normalized Ratio (INR) units based upon seconds], there are no primary standards to use for calibration and validation of these assays. Instead, clinical validation of the PT and APTT is ultimately based upon the association of particular clotting time values with important clinical outcomes such as hemorrhage and thrombosis in randomized, controlled clinical trials. For example, Azar and colleagues studied 3,404 myocardial infarction patients receiving oral anticoagulant therapy.35 They found that as the PT increased, the incidence of thromboembolic events declined while the incidence of hemorrhage rose. The clinically optimized INR range is defined as that which minimizes the combined incidence of adverse events (ie, thromboembolic + hemorrhagic). Similar studies in venous thrombosis and pulmonary embolism patients established the range of 2.0 to 3.0 as the optimal INR range for anticoagulation of such patients, while for patients with mechanical heart valves and others at particularly high risk of thrombosis, the recommended INR therapeutic range is 2.5 to 3.5.36 These studies (as well as trials of heparin therapy monitored with the APTT) have all been conducted with full-draw sodium citrate tubes made of siliconized glass. If a laboratory uses any other type of coagulation © laboratorymedicine> january 2003> number 1> volume 34 tube (such as partial draw and/or plastic tubes), it is critical to demonstrate clinical equivalence of the PT or APTT results obtained with the new tube to those obtained with the clinically-defined “gold standard” full draw siliconized glass tubes. Otherwise the optimal range of INR or APTT results observed with the new tube may not be the same as found in standard recommendations in the literature derived from trials using the “gold standard” tubes, and patients could be at risk of over- or under-anticoagulation. The manufacturer is responsible for demonstration of equivalent clinical performance of newly introduced tubes. However, under CLIA ’88 and CAP requirements, the laboratory is responsible to obtain such documentation from the manufacturer. One should also be careful that the documentation provided by the vendor reflects patient populations actually at risk for hemorrhagic or thrombotic complications, particularly heparin patients. This is critical because the typical studies performed in normal subjects rarely detect analytical bias that occurs in anticoagulated patients. Recently, plastic coagulation tubes with a novel design permitting both a full draw configuration and lower blood draw volumes than previous full draw glass tubes (ie, 2.7 and 1.8 mL) have been introduced. In clinical trials with >1,000 patients, these tubes demonstrated equivalence to the siliconized glass “gold standard” tube in normal subjects, warfarin and heparin patients, and patients with a variety of coagulopathies, across the spectrum of instruments and reagents present in most clinical laboratories. Some of these comparative data are presented as an example of the kinds of clinical trials necessary to compare the clinical performance of new citrate tubes with that of full draw siliconized glass tubes [T3]. Since platelets are prone to activation during phlebotomy, tube inversion, and centrifugation, and with the attendant risk of interference with coagulation assays, coagulation tubes have been developed in which metabolic inhibitors have been added to sodium citrate for platelet stabilization. Mody and colleagues studied the 53 YLF1_Jan03.qxd 12/4/02 10:47 AM Page 54 Warfarin Donor Prothrombin Time Instrument: ELECTRA 1400C Coagulation Analyzer (Instrumentation Laboratory, Lexington, MA) Reagent Hemoliance Brain Thromboplastin (n=30) Draw volume Tube type Mean + SD 4.5 mL Siliconized glass 17.43+3.19 2.7 mL Plus Plastic 17.43+3.18 T3 Dade Innovin (n=30) 1.8 mL Plus Plastic 17.41+3.00 4.5 mL Siliconized glass 19.76+7.17 2.7 mL Plus Plastic 19.46+7.17 1.8 mL Plus Plastic 19.52+7.13 None of the differences was statistically significant. Unfractionated Heparin Donor Activated Partial Thromboplastin Time Instrument: ELECTRA 1400C Coagulation Analyzer (Instrumentation Laboratory, Lexington, MA) Reagent Hemoliance ThrombosilI (n=58) Draw volume Tube type Mean + SD 4.5 mL Siliconized glass 50.89+13.80 2.7 mL Plus Plastic 49.30+12.83 Dade ACTIN FSL (n=59) 1.8 mL Plus Plastic 48.86+12.85 4.5 mL Siliconized glass 46.19+7.17 1.8 mL Plus Plastic 45.67+13.83 None of the differences was statistically significant. Anticoagulated warfarin and unfractionated heparin donor samples were collected, transported, processed, and tested with the designated PT and APTT reagents and coagulation analyzer according to NCCLS Guideline H21-A3.18 Samples were collected in BD Vacutainer siliconized glass 4.5 ml 0.105 M sodium citrate tubes, and 2.7 and 1.8 mL Plus Plastic 0.109 M sodium citrate tubes, and test results with each of the Plus Plastic tubes were statistically compared for equivalence with the “gold standard” siliconized glass tubes.42 effect of one such additive, CTAD (containing sodium citrate, theophylline, adenosine, and dipyridamole) on platelet activation over time.37 They reported that for both uncentrifuged and centrifuged tubes, CTAD significantly inhibited platelet activation. This resulted in a significant reduction in preanalytical variability in APTT results in heparinized patients. 54 Greatest Susceptibility to Preanalytical Variability A number of coagulation assays that show high susceptibility to preanalytical variability are predominantly research tools. These include highly sensitive immunoassays for thrombin generation (the F1+2 prothrombin activation peptide and thrombin-antithrombin complex) and peptides indicative of thrombin activity (fibrinopeptide A), as well as markers of fibrinolysis (plasminogen activator inhibitor-1, tissue plasminogen activator, and plasmin-antiplasmin complex). The time-dependence of platelet aggregation studies was noted before, and flow cytometric analysis of platelets is also highly susceptible to preanalytical variability, as are assays for cytokines such as interleukin-1 and tumor necrosis factor. The coagulation assays in which the greatest clinical risk is associated with preanalytical variability are the following: PT monitoring of warfarin therapy, APTT monitoring of unfractionated heparin therapy, platelet count verification in pseudothrombocytopenia, and lupus anticoagulant testing. Warfarin Monitoring Introduction of the INR system significantly improved management of warfarin patient by removing a substantial proportion of the variability associated with thromboplastin sensitivity. However, the coagulation studies by which the manufacturers and contracted reference laboratories make International Sensitivity Index (ISI) assignments to a thromboplastin show the same sensitivity to preanalytical variables such as sodium citrate concentration, coagulation tubes, time between sample acquisition and analysis, temperature, etc.31-34,38 For example, it has been reported that ISI assignments are approximately 10% lower when determined with samples collected in 0.129M sodium citrate than with 0.109M citrate.38 Thus, if a laboratory is relying upon the manufacturer’s ISI assignment for INR determinations, it laboratorymedicine> january 2003> number 1> volume 34 should verify that its preanalytical practices are consistent with those used by the manufacturer. As noted, use of unsiliconized glass is potentially hazardous for warfarin monitoring, since cold-induced PT shortening can lead to inappropriate increases in patients’ warfarin dosages.24 Fortunately, warfarin monitoring using the PT is overall significantly less susceptible to preanalytical variability than heparin monitoring with the APTT, since the former is insensitive to platelet activation. Unfractionated Heparin Monitoring Intravenous heparin therapy has become standard practice for patients with acute coronary syndrome, in addition to its established role in the treatment of deep venous thrombosis and pulmonary embolism. However, this life-saving therapy is also associated with life-threatening risks of hemorrhage. Hirsh and Fuster reviewed a series of published studies of intravenous heparin therapy,39 and reported that while heparin is effective in preventing fatal pulmonary embolism and reducing recurrent thrombosis, major bleeding occurred in from 1.9% to 6.5% of patients (with a © YLF1_Jan03.qxd 12/4/02 10:47 AM Page 55 combined rate in the reviewed studies of 3.8%). A significant number of these life-threatening hemorrhagic events are likely attributable to problems in monitoring this therapy with the APTT, and of these errors, a substantial proportion may be preanalytical in origin. APTT reagent systems show significant variability in their sensitivity to heparin. However, despite multiple attempts, no standardization system comparable to the INR has been devised. While many laboratories continue to use the traditional therapeutic range for heparin of 1.5 to 2.5 times the midpoint of the population reference range, the College of American Pathologists Conference on Laboratory Monitoring of Anticoagulant Therapy made recommendations for defining a local therapeutic range that takes into consideration the varying sensitivity of APTT reagent systems.40 This involves studying a series of patients on a stable heparin dose (without accompanying oral anticoagulants) by measuring both the APTT using the local preanalytical and reagent/instrument systems and heparin activity using a chromogenic anti-factor Xa assay. Values for APTT associated with heparin levels between 0.3 and 0.7 U/mL define the therapeutic range. However, preanalytical variability complicates this system, as the APTT is much more sensitive to the sample quality than the PT. This difference in sensitivity is because the former assay is significantly affected by platelet activation and secretion, and it reflects many more biochemical reactions encompassing both the intrinsic and common coagulation pathways. This renders the APTT significantly more sensitive to sodium citrate concentrations as well as to sample handling (eg, tube inversion), specimen transport time and conditions, and centrifugation. In addition to these preanalytical factors, APTT results in the presence of heparin are highly sensitive to properties of coagulation tubes, such as their configuration (eg, full-draw versus partial-draw) and composition (ie, siliconized glass versus unsiliconized glass versus plastic). In response to increasing concerns about iatrogenic blood loss, sodium citrate tubes were designed to draw 2.7 mL or 1.8 mL blood into the same 13 x 75 mm configuration as traditional full-draw 4.5 mL tubes. This was achieved by creating a reduced vacuum in the tube. This design is referred to as a partial-draw tube. In the first several years after this product’s release, it performed equivalently to the standard full-draw “gold standard” sodium citrate tubes with the coagulation analyzers and reagents in clinical use. However, as APTT reagents were developed with greater sensitivity to heparin, lupus anticoagulants, and factor deficiencies, and as coagulation instrumentation became more robust, it became apparent that partial-draw tubes were associated with artifactual shortening of the APTT in heparinized patients with certain instrument/reagent combinations. Siegel and colleagues studied a series of heparinized patients by comparing APTT results on samples collected in full-draw and partial-draw sodium citrate tubes.41 They reported that there was a significant negative bias with the partialdraw tubes in heparinized patients that could result in excessive anticoagulation in these patients. No significant differences were observed with results within the reference range. Similar bias in heparin monitoring has been observed in plastic partial-draw citrate tubes. This negative bias in APTT results in partial-draw tubes has been shown to be due to platelet activation during the slower filling of these tubes. Shear stress is known to activate platelets. In partialdraw tubes, platelets are exposed for a longer period of time to altered shear forces related to the tubes’ increased headspace. This activates the platelets, leading to release of platelet factor 4 from the alpha granules, which in turn neutralizes the heparin in the sample and artificially shortens the APTT.25 Thus, if a laboratory is planning to use coagulation tubes other than the fulldraw siliconized glass tubes that were used in the clinical trials which formed the basis for the recommended heparin therapeutic range, it is critical for the manufacturer and/or laboratory to verify equivalent clinical performance of the new tubes to those of these “gold standard” tubes. © laboratorymedicine> january 2003> number 1> volume 34 Platelet Counts in Citrate Tubes A small proportion of inpatients (1% to 2%) and outpatients (0.1% to 0.2%) demonstrate platelet clumping in the presence of EDTA due to EDTA-dependent antibodies reactive with platelet antigens. Most cases of clumped platelets are due to phlebotomy technique or failure to adequately mix the specimen. Standard protocols designed to obtain reliable platelet counts in such patients include preparing blood smears from a fingerstick specimen and measuring the platelet count in a sodium citrate tube. However, preanalytical variability, such as excessive inversion or shaking of the tube, and/or use of tubes with a partial draw configuration, can result in erroneously low platelet counts in these patients. F1 shows the effect of platelet activation occurring in partial-draw citrate tubes on the platelet count in 30 healthy donors. The platelet counts with EDTA tubes and full-draw sodium citrate tubes were equivalent. However, platelet activation associated with 2.7 mL and 1.8 mL partial-draw tubes led to platelet aggregation and resulted in artificially reduced platelet counts. Consequently, use of partial draw citrate tubes to obtain accurate platelet counts in patients with EDTA-associated pseudothrombocytopenia would be associated with the risk of clinically significant preanalytical error. The hypothesis that platelet activation is responsible for this phenomenon is confirmed by use of the CTAD metabolic platelet inhibitor cocktail. Platelet counts were equivalent in full-draw 4.5 mL CTAD tubes and 2.7 mL and 1.8 mL partial draw CTAD tubes [F1]. Lupus Anticoagulant Testing Sensitive and accurate detection of the presence of the lupus anticoagulant is critical for the evaluation of patients with thrombotic disorders and recurrent spontaneous abortion. Assays to detect the lupus anticoagulant are very sensitive to the presence of platelets, since false negative results can occur if the antiphospholipid antibody activity present in the patient’s plasma is neutralized by platelet phosphoplipids. Thus, compliance with the NCCLS 55 YLF1_Jan03.qxd 12/4/02 10:47 AM Page 56 3. Emery JD, Leifer DW, Moura GL, et al. Whole blood platelet aggregation predicts in vitro and in vivo primary hemostatic function in the elderly. Arterioscl Thromb Vasc Biol. 1995;15:748-753. 4. Sagripanti A, Carpi A. Natural anticoagulants, aging, and thromboembolism. Exp Gerontol. 1998;33:891-896. 5. Lawrence JB, Leifer DW, Moura GL, et al. Sex differences in platelet adherence to subendothelium: Relationship to platelet function tests and hematologic variables. Am J Med Sci. 1995;309:201-207. 6. Campbell NR, Hull RD, Brant R, et al. Different effects of heparin in males and females. Clin Invest Med. 1998;21:71-78. [F1] Phlebotomy was performed on 30 healthy donors; samples were collected, transported, and processed according to NCCLS Guideline H21-A321; and platelet counts were measured on a Coulter STKS hematology analyzer. Samples were collected in the following BD Vacutainer tubes: • 4.5 mL glass K3EDTA tubes • Siliconized glass 0.105 M sodium citrate tubes: 4.5 mL full-draw, and 2.7 and 1.8 mL partialdraw tubes • Siliconized glass CTAD tubes (containing 0.109 M sodium citrate, 15 mM theophylline, 3.7 mM adenosine, and 0.198 mM dipyidamole): 4.5 mL full draw, and 2.7 and 1.8 mL partial draw tubes The platelet counts reported by the hematology analyzer with citrate and CTAD tubes were multiplied by 1.1 (to compensate for the dilution arising from the liquid citrate or CTAD additive) to give the values to be compared with the platelet counts obtained with K3EDTA tubes; the bars in the figure represent the mean of these platelet counts for the 30 healthy donor samples collected with each tubes type. The 2.7 and 1.8 mL partial draw citrate tubes gave statistically significantly lower mean platelet counts than the K3EDTA or 4.5 mL full draw citrate tubes. No significant differences were observed in the platelet counts obtained in full draw or partial draw CTAD tubes; these values were also statistically equivalent to the platelet counts obtained with the K3EDTA and 4.5 mL full draw citrate tubes. *:P<0.001. centrifugation guidelines18 is critical for lupus anticoagulant testing. 56 Implications for Clinical Laboratories The information described in this article suggests that of all possible approaches to improve the quality of coagulation test results standardizing preanalytical practices probably offers the greatest payoff because preanalytical variability is the greatest contributor to laboratory errors.1 Laboratories must ensure that all their preanalytical practices (including phlebotomy, specimen identification, transportation and handling, and citrate tubes) minimize bias. Because of the complexity of preanalytical operations within hospitals and other health care facilities, it is essential to maintain accountability for implementation of best practices at each step— from the patient to the coagulation analyzer—regardless of institutional reporting relationships. Furthermore, preanalytical educational efforts must be persistent and directed at all personnel involved, regardless of department. 7. Gill JC, Endres Brooks J, Bauer PJ, et al. The effect of ABO blood group on the diagnsosis of von Willebrand’s disease. Blood. 1987;69:16921695. 8. Tofler GH, Brezinski D, Schafer AI, et al. Concurrent morning increase in platelet aggregability and the risk of myocardial infarction and sudden cardiac death. N Engl J Med. 1987;316:1514-1518. 9. Mavri A, Guzic-Salobir B, Salobir-Pajnic B, et al. Seasonal variation of some metabolic and haemostatic risk factors in subjects with and without coronary artery disease. Blood Coagul Fibrinolysis. 2001;12:359-365. Finally, the requirements of good clinical and laboratory practice and our obligations under CLIA ’88 and CAP requirements teach us that laboratories have the right and duty to request documentation from manufacturers about citrate tubes in all relevant patient populations. Preanalytical variability can be critical for accurate coagulation results in patients receiving warfarin or heparin and those undergoing testing for lupus anticoagulants, while normal individuals and certain other patient populations are significantly less sensitive to preanalytical practices. Therefore, validation studies in healthy subjects are not necessarily predictive of results in patients with coagulopathies due to inherited or acquired conditions or anticoagulant therapy. Laboratories should require manufacturers to demonstrate clinical performance of their tubes in these key patient populations. 1. Plebani M, Carraro P. Mistakes in a stat laboratory: Types and frequency. Clin Chem. 1997;43:1348-1351. 2. Andrew M, Paes B, Johnston M. Development of the hemostatic system in the neonate and young infant. Am J Pediatr Hematol Oncol. 1990;12:95-104. laboratorymedicine> january 2003> number 1> volume 34 © 10. Ceriello A, Bortolotti N, Motz E, et al. Red wine protects diabetic patients from meal-induced oxidative stress and thrombosis activation: A pleasant approach to the prevention of cardiovascular disease in diabetes. Eur J Clin Invest. 2001;31:322-328. 11. Miller GJ, Bauer KA, Cooper JA, et al. Activation of the coagulation pathway in cigarette smokers. Thromb Haemost. 1998;79:549-553. 12. Zhang QH, Das K, Siddiqui S, et al. Effects of acute, moderate ethanol consumption on platelet aggregation in platelet-rich plasma and whole blood. Alcohol Clin Exp Res. 2000;24:528-534. 13. Walsh BW, Kuller LH, Wild RA, et al. The effects of raloxifene HCl on serum lipids and coagulation factors in healthy postmenopausal women. JAMA. 1998;279:1445-1551. 14. Kadir RA, Economides DL, Sabin CA, et al. Variations in coagulation factors in women: Effects of age, ethnicity, menstrual cycle and combined oral contraceptive. Thromb Haemost. 1999;82:1456-1461. 15. Weiss C, Bierhaus A, Kinscherf R, et al. Tissue factor-dependent pathway is not involved in exercise-induced formation of thrombin and fibrin. J Appl Physiol. 2002;92:211-218. 16. Von Kanel R, Mills PJ, Fainman C, et al. Effects of psychological stress and psychiatric disorders on blood coagulation and fibrinolysis: A biobehavioral pathway to coronary artery disease? Psychosom Med. 2001;63:531-544. 17. Koepke JA, Rodgers JL, Ollivier MJ. Preinstrumental variables in coagulation testing. Am J Clin Pathol. 1975;64:591-596. 18. NCCLS. Collection, Transport, and Processing of Blood Specimens for Coagulation Testing and General Performance of Coagulation Assays; Approved Guideline—Third Edition. NCCLS document H21-A3. 1998 NCCLS, Wayne, PA. YLF1_Jan03.qxd 12/4/02 10:47 AM Page 57 19. Valeri CR, Cassidy G, Pivacek LE, et al. Anemiainduced increase in the bleeding time: Implications for treatment of nonsurgical blood loss. Transfusion. 2001;41:977-983. 32. D’Angelo G, Villa C. Measurement of prothrombin time in patients on oral anticoagulant therapy: Effect of two different evacuated tubes. Haematologica 1999;84:656-672. 20. Reneke J, Etzell J, Leslie S, et al. Prolonged prothrombin time and activated partial thromboplastin time due to underfilled specimen tubes with 109 mmol/L (3.2%) citrate anticoagulant. Am J Clin Pathol. 1998;109:754-757. 33. van den Besselaar AMHP, Bertina RM, van der Meer, et al. Different sensitivities of various thromboplastins to two blood collection systems for monitoring oral anticoagulant therapy. Thromb Haemost. 1999;82:153-154. 21. NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard – Fourth Edition. NCCLS document H3-A4. 1998 NCCLS, Wayne, PA. 34. Biron-Adreani C, Mallol C, Seguret F, et al. Plastic versus siliconized glass tubes: Evaluation in current laboratory practice. Thromb Haemost. 2000;83:800-801. 22. Adcock DM, Kressin DC, Marlar RA. Are discard tubes necessary in coagulation studies? Lab Med. 1997;28:530-533. 35. Azar AJ, Cannegieter SC, Deckers JW, et al. Optimal intensity of oral anticoagulant therapy after myocardial infarction. J Am Coll Cardiol. 1996;27:1349-1355. 23. Adcock DM, Kressin DC, Marlar RA. Minimum specimen volume requirements for routine coagulation testing. Dependence on citrate concentration. Am J Clin Pathol. 1998;109:595-599. 24. Palmer RN, Gralnick HR. Inhibition of coldpromoted activation of the prothrombin time: Studies of new siliconized borosilicate collection tubes in normals and patients receiving warfarin. Am J Clin Pathol. 1985;83:492-494. 25. Adcock D, Kressin D, Marlar RA. The effect of time and temperature variables on routine coagulation tests. Blood Coag Fibrinolysis. 1998;9:463-470. 26. Cummings J. Evacuated tubes for monitoring heparin treatment. Thromb Haemost. 1981;34:939-940. 27. Adcock D, Kressin D, Marlar R. Effect of 3.2% vs. 3.8% sodium citrate concentration on routine coagulation testing. Am J Clin Pathol. 1997;107:105-110. 28. Vogler EA, Graper JC, Harper GR, et al. Contact activation of the plasma coagulation cascade. I. Procoagulant surface chemistry and energy. J Biomed Mater Res. 1995;29:1005-1016. 29. Hunt BJ, Parratt R, Cable M, et al. Activation of coagulation and platelets is affected by the hydrophobicity of artificial surfaces. Blood Coagul Fibrinolysis. 1997;8:223-231. 36. Hirsh J, Hoak J. Management of deep venous thrombosis and pulmonary embolism: A statement for healthcare professionals from the Council on Thrombosis (in consultation with the Council on Cardiovascular Radiology), American Heart Association. Circulation. 1996;93:2212-2245. 37. Mody M, Lazarus AH, Semple JW, et al. Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant. Transfus Med. 1999;9:147-154. 38. Duncan EM, Casey CR, Duncan BM, et al. Effect of concentration of trisodium citrate anticoagulant on calculation of the international normalized ratio and the international sensitivity index of thromboplastin. Thromb Haemost. 1994;72:84-88. 39. Hirsh J, Fuster V. Guide to anticoagulant therapy. Part 1: Heparin. Circulation. 1994;89:1449-1468. 40. Olson JD, Arkin CF, Brandt JT, et al. College of American Pathologists Conference XXXI on laboratory monitoring of anticoagulant therapy: Laboratory monitoring of unfractionated heparin therapy. Arch Pathol Lab Med. 1998;122:782-798. 30. Polack B, Barro C, Pernod G, et al. Impact of the blood collection tube on the activation of coagulation. Thromb Haemost. 1997;77:217-218. 41. Siegel JE, Bernard DW, Swami VK, et al. Monitoring heparin therapy: APTT results from partial- vs full-draw tubes. Am J Clin Pathol. 1998;110:184-187. 31. van den Besselaar AMHP, Meeuwisse J, Wineveen E, et al. Effect of blood collection tubes on thromboplastin calibration. Thromb Haemost. 1998;79:1062-1063. 42. Berger RL, Hsu JC. Bioequivalence trials, intersection-union tests, and equivalence confidence sets. Statistical Science. 1996;11:282302. 57 © laboratorymedicine> january 2003> number 1> volume 34 CE_ExamJan03.qxd 12/4/02 9:44 AM Page 69 Continuing Education Update Exams Earn 1 CMLE credit hour for each exam (maximum of 4 exams per issue). After processing your examination, ASCP will mail you a certificate of participation and the answer key. CE Update is approved to meet licensure requirements for California, Florida, and other states. To complete an exam, fill in the answer section found on the back of this page. Answers must be received by: March 30, 2003 Hematology 0301 Exam 1. Which of the following preanalytical factors would be expected to artifactually shorten the prothrombin time/INR in warfarin patients? a. Ethanol intake b. Delay between sample acquisition and testing c. Anemia d. Underfilling the coagulation tube e. Use of 3.8% citrate instead of 3.2% citrate 2. The following practice would be expected to increase preanalytical error in the coagulation laboratory: a. conversion to evacuated blood collection systems from a needle and syringe technique b. inversion of sodium citrate tubes vigorously 8 to 10 times for APTT monitoring of unfractionated heparin therapy c. centrifuging coagulation specimens to maintain a platelet count below 10,000/microliter d. requiring patients to rest comfortably for 30 minutes prior to phlebotomy e. use of CTAD tubes for unfractionated heparin monitoring 3. Partial-draw sodium citrate tubes can result in an increased hemorrhagic risk through which of the following mechanisms: a. artifactual elevation of the platelet count b. heparin neutralization by protein S activation c. inactivation of factor VII d. degradation of factors V and VIII e. platelet activation 4. Extended delays between phlebotomy and analysis could be expected to lead to which of the following: a. shortening of the prothrombin time/INR in warfarin patients b. enhanced activity of factor VIII c. APTT shortening in patients receiving unfractionated heparin therapy d. elevated platelet counts e. elevated blood-to-additive ratio 5. Cigarette smoking is associated with which of the following effects: a. thrombin generation b. depressed plasma fibrinogen levels c. platelet inhibition d. reduced hematocrit e. hemodilution 69 © laboratorymedicine> january 2003> number 1> volume 34 CE_ExamJan03.qxd 12/4/02 9:44 AM Page 70 Fax Mail Questions? 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The text was organized logically. 6. Illustrations, charts, and tables helped explain text and added to the article's value. 쏔쏔쏔쏔쏔 쏔쏔쏔쏔쏔 쏔쏔쏔쏔쏔 쏔쏔쏔쏔쏔 쏔쏔쏔쏔쏔 쏔쏔쏔쏔쏔 1 2 3 4 5 Comments: (Attach additional pages if necessary.) laboratorymedicine> january 2003> number 1> volume 34 ©