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Coagulation and Transfusion Medicine / aPTT Reagent Selection and Utilization

Recommendations for Appropriate Activated Partial Thromboplastin Time Reagent Selection and Utilization George A. Fritsma, MS, MLS,1,2 Francine R. Dembitzer, MD,3 Ankush Randhawa, PhD,4 Marisa B. Marques, MD,1 Elizabeth M. Van Cott, MD,5 Dorothy Adcock-Funk, MD,6 and Ellinor I. Peerschke, PhD3 Key Words: aPTT; Lupus anticoagulant; Activated partial thromboplastin time; Reagent; Routine coagulation DOI: 10.1309/AJCP3J1ZKYBFQXJM

Abstract The activated partial thromboplastin time (aPTT) is widely used as a screening coagulation test and for monitoring unfractionated heparin therapy. Various commercial reagents are available, with different performance characteristics, particularly responsiveness to the lupus anticoagulant (LA). Because aPTT reagent selection significantly affects the interpretation of results, we reviewed College of American Pathologists proficiency testing data involving approximately 4,000 coagulation laboratories, and conducted a survey of coagulation laboratories (n = 93) using The Fritsma Factor hemostasis Web site to determine the basis for aPTT reagent selection. The data demonstrate that for routine aPTT testing, most laboratories use reagents with high/moderate responsiveness to LA. Significant misunderstanding was apparent regarding the use of appropriate aPTT reagent for routine testing and LA identification. We recommend aPTT reagents with low LA responsiveness to screen for coagulation factor deficiencies and heparin monitoring, and suggest continued education of laboratory professionals and reagent manufacturers about appropriate aPTT reagent use.

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Am J Clin Pathol 2012;137:904-908 DOI: 10.1309/AJCP3J1ZKYBFQXJM

The clot-based activated partial thromboplastin time (aPTT) was first developed in 1954 as the recalcification time assay1 and evolved to become the partial thromboplastin time (PTT) with the addition of a “platelet substitute,” an extract of rabbit brain tissue composed of phospholipids called “cephalin.” This was considered a “partial thromboplastin,” because there was no tissue factor included. The assay became designated as “activated,” when negatively charged particulate contact activators such as kaolin, silica, ellagic acid, or celite were added to improve responsiveness and reproducibility.2 Many current aPTT reagents use soy-based synthetic phospholipids and nonsettling activators such as colloidal silica.3 When an aPTT reagent is added to citrated platelet-poor plasma, it triggers in vitro clot formation by activating factor XII.4 The aPTT is responsive to deficiencies in the intrinsic and/or common pathways, and will prolong with a significant single factor deficiency or multiple deficiencies of the common pathway. Manufacturers generally formulate reagents so that the aPTT will prolong when the activity of factors VIII, IX, or XI is reduced to approximately 30% (0.3 IU) or below. Fibrinogen levels usually have to fall below 100 mg/dL (2.94 μmol/L) before the aPTT will prolong. Laboratory operators may confirm aPTT responsiveness to factor deficiencies as a quality assurance practice. From 1970 to the present, the aPTT has also been used to monitor the anticoagulant effect of unfractionated heparin, which decelerates coagulation by binding to antithrombin and inhibits most serine proteases, but most importantly, activated factor X (Xa) and thrombin (IIa).5 To guide heparin therapy, laboratory operators establish a reagent-specific aPTT therapeutic range equivalent to 0.3 to 0.7 anti-Xa heparin units/mL. The target range must be verified or updated with each new © American Society for Clinical Pathology

Coagulation and Transfusion Medicine / Original Article

lot of aPTT reagent, because the responsiveness to inhibition of factors Xa and IIa varies among reagents and reagent lots.6 Lupus anticoagulant (LA) belongs to a family of “antiphospholipid antibodies,” immunoglobulins that recognize phospholipid-bound proteins. LA has a prevalence of 1% to 2% in the unselected patient population. Although most LAs are transient and disappear within 12 weeks, chronic LA is associated with the potential for venous and arterial thrombosis.7 Because of the heterogeneity of LA reactivity, complex laboratory testing strategies involving a panel of screening and confirmatory tests are required.8-10 Screening for LA with an isolated aPTT is not recommended. LA may prolong the aPTT because it partially neutralizes reagent phospholipids required for optimal clot formation. Thus, some manufacturers formulate low-phospholipid aPTT reagents to enhance detection of LA.9 For instance, Siemens Actin FSL (Siemens Healthcare Diagnostics, Tarrytown, NY), Beckman-Coulter HemosIL aPTT-SP (Beckman Coulter, Brea, CA), and Stago PTT-LA (Diagnostica Stago, Parsippany, NJ) are low-phospholipid reagents that are especially responsive to LA, whereas Siemens Actin FS and Stago C.K. Prest are considered to exhibit relatively low responsiveness to LA; Stago STAPTT-LA and Beckman-Coulter HemosIlSynthASil, both formulated for automation, possess intermediate responsiveness to LA. When LA-responsive reagents are used to screen for intrinsic factor deficiency or to monitor heparin, prolongation secondary to LA will interfere with result interpretation. Importantly, prolongation in response to LA may be misinterpreted as a possible coagulation factor deficiency or, for patients receiving heparin or anticoagulation therapy with direct thrombin inhibitors (eg, lepirudin, dabigatran, and argatroban), can mislead clinicians to overestimate the level of anticoagulation, and erroneously provide insufficient anticoagulation therapy for the patient. Because a single aPTT reagent most often does not meet the clinical requirements of heparin/anticoagulation monitoring, coagulopathy screening, and LA screening, the present study was developed to learn

whether laboratories select aPTT reagents based on their most common clinical needs.

Materials and Methods Proficiency testing data from the College of American Pathologists (CAP) from 2008 to 2011 for a coagulationlimited survey were analyzed for aPTT reagent use. Approximately 3,800 to 4,000 laboratories participated in each testing event. In addition, a survey of coagulation laboratories was conducted in 2010 through a hemostasis Web site, The Fritsma Factor (www.fritsmafactor.com) to determine whether low, intermediate, and high LA-responsive aPTT reagents are being used appropriately. The Fritsma Factor is an interactive Web site that provides an open forum to access and share knowledge, information, and insight in the area of hemostasis. Content is managed independently by one of us (G.F.). Site design and infrastructure are supported by Precision BioLogic, Dartmouth, Canada. A total of 93 laboratories responded to the survey. Finally, a regional discussion was conducted among coagulation laboratory directors (n = 5) from New York and New Jersey in September 2010 to solicit opinions regarding the most appropriate and cost-effective use of aPTT reagents that have varying responsiveness to LA.

Results Results analyzed from CAP proficiency tests conducted from 2008 to 201111 demonstrate that most laboratories performing automated aPTT tests use reagents with either high or intermediate responsiveness to LA ❚Table 1❚. Selection of high LA-responsive aPTT reagents predominated in 2008. A slight shift to aPTT reagents with intermediate LA responsiveness was noted in subsequent years, such that by 2011, approximately equal numbers of laboratories reported

❚Table 1❚ aPTT Reagent Selection in North America LA Responsiveness

Example Reagents

CAP 2008*

CAP 2009*

CAP 2010*

CAP 2011*

Fritsma Factor Survey†

High

Siemens Actin FSL Beckman-Coulter HemosIL aPTT-SP Stago STA-PTT-LA Beckman-Coulter HemosILSynthASil Siemens Actin FS Stago C.K. Prest

2,019 (53)

2,015 (51)

2,023 (48)

1,902 (46)

9 (10)

1,491 (39)

1,647 (41)

1,854 (44)

1,954 (47)

39 (43)

150 (4)

127 (3)

132 (3)

125 (3)

17 (18)

—

—

—

—

26 (29)

Intermediate Low Don’t know

aPTT, activated partial thromboplastin time; CAP, College of American Pathologists; LA, lupus anticoagulant. * Number (%) of laboratories reporting results for CAP proficiency testing exercises CGL-A (coagulation limited survey). The reagents listed were used by more than 95% of proficiency testing participants. † Number (%) of Fritsma Factor survey respondents.

© American Society for Clinical Pathology

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using high and intermediate LA-responsive reagents. In comparison, data obtained from the Fritsma Factor survey indicate that only 10% of respondents use reagents with high LA responsiveness, while 43% use reagents with intermediate LA responsiveness and 18% use reagents with low LA responsiveness. The remaining respondents (29%) were unaware of the LA responsiveness of their laboratory’s reagent. The low utilization rate of aPTT reagents with high LA responsiveness among Fritsma Factor survey respondents may reflect a higher level of understanding about the effect of LA interference on interpretation of screening aPTT test results. These respondents included a self-selected group of laboratory professionals who are familiar with the Fritsma Factor coagulation Web site and chose to respond to the online survey, compared with the broader group of laboratories subscribing to CAP limited coagulation proficiency testing modules. The Fritsma Factor survey attracted responses from a cross-section of laboratory supervisors, managers, directors, and technologists, of whom 67% indicated specialization in hematology/coagulation ❚Table 2❚. Respondents were affiliated with laboratories performing a range of aPTT test volumes (Table 2). More than one third of laboratories reported an aPTT test volume exceeding 150 tests per day. When asked during the Web-based survey, “Does your laboratory use aPTT reagents with different responsiveness to lupus anticoagulant?” 66% of participants indicated a negative response, 32% positive, and 2% did not know. The subgroup of laboratories (32%) that used both LA-responsive reagents and aPTT reagents with low LA responsiveness further answered the following questions: “How do you use the aPTT reagent with low responsiveness to LA?” and “How do you use the aPTT reagent with high responsiveness to LA?” As summarized in ❚Table 3❚, low responsiveness reagents were appropriately used by most participants to screen for intrinsic coagulation factor deficiency and heparin monitoring. Reagents with high LA responsiveness were used appropriately, predominantly to screen for LA. However, even among the subgroup of laboratories that apparently recognized the advantage of using separate aPTT reagents with different LA responsiveness, a small number inappropriately

❚Table 2❚ Demographics of Fritsma Factor Survey Respondents (n = 93) % of Respondents

Job Classification Supervisor, manager, pathologist, laboratory director Technologist, lead technologist, technical specialist Laboratory specialty Hematology/coagulation Administration, medical director, pathologist Daily test volume 150 tests

56 44 67 33 33.3 33.3 33.3

used aPTT reagents with low LA responsiveness to screen for LA and reagents with high or moderate LA responsiveness to monitor for heparin and/or screen for factor deficiencies. Given the revised International Society on Thrombosis and Haemostasis (ISTH) guidelines for the detection of LA by clinical laboratories,10 the survey further asked the opinion of all participants regarding the statement “A normal aPTT rules out the presence of a LA.” As shown in ❚Table 4❚, most respondents (69%) appropriately disagreed with this statement. However, 31% of laboratories appear not to understand the specific requirements for LA testing, and thus are not able to provide proper consultation to clinicians whom they serve. Many laboratories across North America (based on CAP proficiency testing exercises and study survey) use only a single reagent with moderate or high responsiveness to LA

❚Table 4❚ Survey Responses to “A Normal aPTT Rules Out LA” Response

% of Respondents

Strongly agree Agree Strongly disagree* Disagree* Neutral No opinion

3 12 25 44 8 8

aPTT, Activated partial thromboplastin time; LA, lupus anticoagulant. *Correct responses.

❚Table 3❚ aPTT Reagent Usage Among Laboratories That Use Both Low and High LA Responsive Reagents: Fritsma Factor Survey Respondents No. of Laboratories* Low LA Responsiveness Screen for intrinsic coagulation factor deficiency to predict bleeding risk Monitor standard unfractionated heparin therapy Screen for LA to predict thrombosis risk All 3

20 17 2 7

High LA Responsiveness 1 2 18

aPTT, Activated partial thromboplastin time; LA, lupus anticoagulant. *Laboratories represent a subgroup (32%) of all survey respondents.

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for automated aPTT testing. Therefore a regional discussion among laboratory directors from New York and New Jersey was convened to understand the reasons for this practice. Conference participants cited reagent availability and difficulty troubleshooting mismatched instrument/reagent combinations because of a lack of adequate peer group for comparison as well as reagent/analyzer compatibility as predominant concerns. A prolonged aPTT often leads to the performance of additional tests, including an aPTT mixing study with pooled normal plasma to discriminate between the presence of a circulating anticoagulant and factor deficiency. Therefore the Fritsma Factor survey asked participants whether mixing studies were performed by their laboratories and what the turnaround time was for such testing. Of the respondents, 19% performed mixing studies reflexively when the aPTT was prolonged, whereas 54% performed them only when ordered by a physician, and 26% did not perform mixing studies (1% did not know). Among laboratories performing mixing studies, 60% provided results within 8 hours, 7% within 8 to 12 hours, and 17% within 12 to 24 hours. The remaining 16% of laboratories tested batches of samples and reported results after 24 hours or longer.

Discussion LA testing challenges laboratory scientists with a unique problem. Transient LA is an innocent finding, but one that forces laboratory operators to follow up with expensive, complex, and time-consuming assays. Positive screening results also raise patient anxiety. Conversely, chronic LA is an established thrombosis risk factor associated with antiphospholipid syndrome, a cause of venous and arterial thrombosis, and obstetric complications.7 A 2008 survey conducted by the North American Specialized Coagulation Laboratory Association demonstrated variable responsiveness of screening tests to LA,12 underscoring the recommendation by the ISTH8,10 that at least 2 different screening tests be performed to rule out the presence of an LA. In the current study, nearly 70% of study survey respondents agreed that an isolated normal aPTT result does not rule out the presence of an LA, and nearly all were aware that LA-responsive aPTT reagents should be used for LA testing. However, despite the coagulation laboratory community’s general appreciation of LA interference with aPTT screening and heparin monitoring, results of our analysis show that most laboratories select moderate or high LA-responsive reagents on the basis of availability and adaptability to instrumentation without reference to reagent clinical use and efficacy. Based on the findings of the present study, the following consensus recommendations are made:

1. The routine laboratory aPTT is useful for evaluating the intrinsic pathway of coagulation and some anticoagulants, and should be performed with a reagent that exhibits low responsiveness to LA. 2. A greater selection of commercial aPTT reagents with low LA responsiveness is needed to accommodate a greater variety of instrument/reagent combinations. 3. Special testing panels, including multiple phospholipidbased clotting assays, some of which may be aPTTbased, are required when LA screening is undertaken to assess thrombosis risk. Screening using a single aPTT assay is inadequate to rule out the presence of LA. 4. Further education of laboratory professionals and coagulation reagent manufacturers is needed to address clinical need and to increase awareness regarding the appropriate use and selection of aPTT reagents to improve patient care and laboratory efficiency. From the 1Department of Pathology and Division of Laboratory Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL; 2The Fritsma Factor, Your Interactive Hemostasis Resource, Trussville, AL; 3Department of Pathology, The Mount Sinai School of Medicine, New York, NY; 4Precision BioLogic, Dartmouth, Canada; 5Department of Pathology, Harvard Medical School and Massachusetts General Hospital, Boston, MA; and 6Esoterix, Englewood, CO. Address reprint requests to Dr Peerschke: Department of Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065; [email protected]. Acknowledgment: The authors thank Diagnostica Stago, Parsippany, NJ, for financial support of the regional coagulation roundtable focused on aPTT reagent selection and utilization.

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