From Ink Pens to Computers: A Personal Look Back at Landmark Changes during 5 Decades as a Clinical Laboratory Scientist in U.S. Hemostasis Laboratories.

In 2023, Seminars in Thrombosis and Hemostasis will be celebrating its 50th anniversary, and similarly this will also mark my 5th decade of working in, or association with, laboratories that perform hemostasis testing. My career started at a large military medical center, but I also worked at several other facilities, including military dispensaries, community hospitals, and a large academic institution. The difference between each type of hemostasis laboratory was as expected, with larger facilities having better instrumentation and more prolific test menus. However, whether one worked in a large academic center, or a small rural hospital, regulatory changes affected every clinical laboratory to the same degree. Advances in technology also eventually affected every hemostasis laboratory, but these salient changes were more likely to occur earlier at the larger institutions. As Seminars in Thrombosis and Hemostasis celebrates its 50th anniversary, that milestone triggered recollection about those salient events that occurred during my own career in hemostasis testing. As such, I describe (my impression) the top ten landmark changes that altered laboratory practice at the facilities where I worked during the past 5 decades.

Factor VIII and Factor IX Activity Measurements for Hemophilia Diagnosis and Related Treatments.

Accurate measurement of clotting factors VIII (FVIII) or IX (FIX) is vital for comprehensive diagnosis and management of patients with hemophilia A or B. The one-stage activated partial thromboplastin time (aPTT)-based clotting assay is the most commonly used method worldwide for testing FVIII or FIX activities. Alternatively, FVIII and FIX chromogenic substrate assays, which assess the activation of factor X, are available in some specialized laboratories. The choice of reagent or methodology can strongly influence the resulting activity. Variation between one-stage FVIII or FIX activities has been reported in the measurement of some standard and extended half-life factor replacement therapies and gene therapy for hemophilia B using different aPTT reagents. Discrepancy between one-stage and chromogenic reagents has been demonstrated in some patients with mild hemophilia A or B, the measurement of some standard and extended half-life factor replacement therapies, and the transgene expression of hemophilia A and B patients who have received gene therapy. Finally, the measurement of bispecific antibody therapy in patients with hemophilia A has highlighted differences between chromogenic assays. It is imperative that hemostasis laboratories evaluate how suitable their routine assays are for the accurate measurement of the various hemophilia treatment therapies.

The Future of Laboratory-Developed Tests in Hemostasis Laboratories.

Laboratory-developed tests (LDTs) are widely used in clinical hemostasis laboratories. The extent to which LDTs are regulated varies greatly around the world, and proposed changes to regulations have raised concerns about the future of LDTs in clinical laboratories. It is increasingly difficult to justify the use of an LDT where a commercially available method with regulatory approval is available. Conversely, where there is no suitable test with regulatory approval and there is a compelling clinical need, using an LDT outweighs the risk associated with not performing the test. We argue that LDTs are still required in specialist clinical laboratories to fulfill unmet clinical needs, and in lower middle-income countries where they are a vital resource.

Activated Partial Thromboplastin Time and Prothrombin Time Mixing Studies: Current State of the Art.

Mixing studies have long been in the clinical laboratory armamentarium for investigating unexpected, prolonged activated partial thromboplastin time (aPTT) or prothrombin time (PT). The purpose of the mixing study is to identify whether the aPTT/PT prolongation is secondary to a factor deficiency versus an inhibitor, which would present as a "corrected" and "noncorrected" mixing study, respectively. The differentiation between a factor deficiency and inhibitor may likely further direct clinical decisions, including additional diagnostic testing or factor replacement therapy. While aPTT/PT mixing studies are simple tests to perform, there is a lack of standardization for both the testing protocol and the interpretation of what is considered to be a corrected or noncorrected mixing study result. This review will describe the common indications for the mixing test, preanalytic variables that may affect mixing study performance, and describe several methods for interpreting the results of aPTT and PT mixing tests.

International Council for Standardization in Haematology Guidance for New Lot Verification of Coagulation Reagents, Calibrators, and Controls.

The clinical laboratory uses commercial products with limited shelf life or certain expiry dates requiring frequent lot changes. Prior to implementation for clinical use, laboratories should determine the performance of the new reagent lot to ensure that there is no significant shift in reagent performance or reporting of patient data. This guideline has been written on behalf of the International Council for Standardization in Haematology (ICSH) to provide the framework and provisional guidance for clinical laboratories for evaluating and verifying the performance of new lot reagents used for coagulation testing. These ICSH Working Party consensus recommendations are based on good laboratory practice, regulatory recommendations, evidence emerged from scientific publications, and expert opinion and are meant to supplement regional standards, regulations, or requirements.

International Council for Standardization in Haematology Recommendations for Hemostasis Critical Values, Tests, and Reporting.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH), the aim of which is to provide hemostasis-related guidance documents for clinical laboratories. The current ICSH document was developed by an ad hoc committee, comprising an international collection of both clinical and laboratory experts. The purpose of this ICSH document is to provide laboratory guidance for (1) identifying hemostasis (coagulation) tests that have potential patient risk based on analysis, test result, and patient presentations, (2) critical result thresholds, (3) acceptable reporting and documenting mechanisms, and (4) developing laboratory policies. The basis for these recommendations was derived from published data, expert opinion, and good laboratory practice. The committee realizes that regional and local regulations, institutional stakeholders (e.g., physicians, laboratory personnel, hospital managers), and patient types (e.g., adults, pediatric, surgical) will be additional confounders for a given laboratory in generating a critical test list, critical value thresholds, and policy. Nevertheless, we expect this guidance document will be helpful as a framework for local practice.

Ecarin based coagulation testing.

Ecarin is derived from venom of Echis carinatus, and will activate prothrombin into meizothrombin which will then cleave fibrinogen to result in clot formation. Ecarin based testing has been described for decades, but these assays were typically restricted to reference or speciality coagulation laboratories. This test was initially described for the assessment of direct thrombin inhibitors (eg, bivalirudin lepirudin, or argatroban) and was not affected by heparins or heparinoids. Ecarin based assays were rarely used for anticoagulation monitoring until the emergence of the direct oral thrombin inhibitor dabigatran etexilate in 2010. As this test was mentioned in the prescribing information for dabigatran etexilate, there was increased interest for use by clinical laboratories as the preferred method for assessing the anticoagulant effect of this drug. The purpose of this document is to review the current status of ecarin based assays for assessing dabigatran. This is with the understanding that these methods can also be exploited for determining the anticoagulation effect of parenteral direct thrombin inhibitors, such as argatroban and bivalirudin.

Preanalytical Variables in Coagulation Testing: Setting the Stage for Accurate Results.

Many preanalytical variables may affect the results of routine coagulation assays. While advances in laboratory instrumentation have partially addressed the laboratory's ability to recognize some of these variables, there remains an increased reliance on laboratory personnel to recognize the three potential areas where coagulation testing preanalytical issues may arise: (1) specimen collection (including patient selection), (2) specimen transportation and stability, and (3) specimen processing and storage. The purpose of this article is to identify the preanalytical variables associated with coagulation-related testing and provide laboratory practice recommendations in an effort to improve the quality of coagulation testing and accuracy of result reporting.

Laboratory Assessment of Direct Oral Anticoagulants.

Oral vitamin K anticoagulation (warfarin, Coumadin, coumarin) has long been used for long-term treatment and prophylaxis in a variety of clinical settings. Given the unpredictable pharmacokinetic and food impact of warfarin, episodic monitoring is required. Since the early 2000s, direct oral anticoagulants (DOACs) entered into clinical trials as a potential alternative strategy to oral vitamin K antagonists for long-term anticoagulation. As these drugs have predictable pharmacokinetics and pharmacodynamics, there was no requirement for episodic or routine monitoring. However, shortly after their introduction into clinical use, it became apparent that certain emergent or acute situations may require some capacity to measure these drugs, especially in a bleeding patient with DOAC exposure. The scramble for literature and data to support or suggest laboratory methods which can rapidly and accurately quantify or estimate DOAC concentration soon began. This review describes the literature to date, and recommendations for laboratories to provide tests that will assure either the presence/absence of DOAC or the capacity to quantify DOAC using rapid methods that could be implemented on most clinical laboratory instruments.

Heparin-Calibrated Chromogenic Anti-Xa Activity Measurements in Patients Receiving Rivaroxaban: Can This Test Be Used to Quantify Drug Level?

BACKGROUND: Determination of plasma rivaroxaban concentration may be necessary in certain clinical situations. Rivaroxaban concentration can be accurately and rapidly determined using a chromogenic anti-activated factor X (factor Xa) assay with specific drug calibrator material. However, there are currently no Food and Drug Administration (FDA)-approved rivaroxaban calibrators available in the United States. OBJECTIVE: To determine whether FDA-approved commercial kits for measuring heparin anti-factor Xa activity can be used to assess rivaroxaban concentrations when calibrated for unfractionated heparin or low-molecular-weight heparins. METHODS: Trough and peak samples were taken from 30 patients taking rivaroxaban as part of their routine care for atrial fibrillation or venous thromboembolism. The samples were tested using 3 different FDA-approved commercial kits for measuring heparin anti-factor Xa activity. RESULTS: There was acceptable correlation between rivaroxaban levels and heparin anti-factor Xa activity using Berichrom and COAMATIC heparin kits. The STA liquid heparin method was the most sensitive to presence of rivaroxaban. CONCLUSION: This study demonstrates a strong correlation, but variability between kits, for assessing rivaroxaban concentrations using heparin anti-factor Xa assays. The extent of the heparin calibration curve significantly limits the measurable rivaroxaban range, and this application may be useful only for trough samples. The STA liquid heparin, being exquisitely sensitive to rivaroxaban, may be suitable for ruling out presence of the drug. The routine use of heparin-calibrated anti-factor Xa assays to quantify rivaroxaban is not advocated, and when applied, it must be used with caution and limitations clearly understood.

Algorithm for Rapid Exclusion of Clinically Relevant Plasma Levels of Direct Oral Anticoagulants in Patients Using the DOAC Dipstick: An Expert Consensus Paper.

BACKGROUND: With the widespread use of direct oral anticoagulants (DOACs), there is an urgent need for a rapid assay to exclude clinically relevant plasma levels. Accurate and rapid determination of DOAC levels would guide medical decision-making to (1) determine the potential contribution of the DOAC to spontaneous or trauma-induced hemorrhage; (2) identify appropriate candidates for reversal, or (3) optimize the timing of urgent surgery or intervention. METHODS AND RESULTS: The DOAC Dipstick test uses a disposable strip to identify factor Xa- or thrombin inhibitors in a urine sample. Based on the results of a systematic literature search followed by an analysis of a simple pooling of five retrieved clinical studies, the test strip has a high sensitivity and an acceptably high negative predictive value when compared with levels measured with liquid chromatography tandem mass spectrometry or calibrated chromogenic assays to reliably exclude plasma DOAC concentrations ≥30 ng/mL. CONCLUSION: Based on these data, a simple algorithm is proposed to enhance medical decision-making in acute care indications useful primarily in hospitals not having readily available quantitative tests and 24/7. This algorithm not only determines DOAC exposure but also differentiates between factor Xa and thrombin inhibitors to better guide clinical management.

Reversing dabigatran in life-threatening bleeding occurring during cardiac ablation with factor eight inhibitor bypassing activity.

OBJECTIVES: We report a case of a patient receiving dabigatran who developed a life-threatening bleeding complication during cardiac ablation that rapidly resolved after administration of Factor Eight Inhibitor Bypassing Activity (FEIBA). BACKGROUND: Therapeutic anticoagulation with warfarin during cardiac ablation has been shown to reduce the risk of stroke and systemic embolism. Cardiac tamponade is a potentially life-threatening procedural complication requiring emergent reversal of anticoagulation and pericardiocentesis. Dabigatran is superior to warfarin in preventing stroke and systemic embolism in nonvalvular atrial fibrillation, but has not been evaluated for use during cardiac ablation. Dabigatran is without a known reversal agent and, should tamponade occur during ablation, it is unclear what reversal strategy could be used to establish hemostasis. METHODS AND RESULTS: A 67-year-old man with history of atrial fibrillation with rapid ventricular rate, two previous atrial fibrillation ablations, and prescribed dabigatran 150 mg bid was admitted for an atrial fibrillation ablation procedure. The last dabigatran dose was 7 hours prior to procedure. During the procedure, a transseptal perforation occurred, requiring an emergent pericardiocentesis. Within 60 minutes, approximately 4.5 L of blood was removed via the pericardiocentesis. Low-dose FEIBA (3159 units, 26 U/kg actual body weight) over 15 minutes was administered. Hemostasis was noted within minutes of initiating the infusion with cessation of bleeding after administration was complete. CONCLUSION: This case report describes the potential ability of a low dose of the activated prothrombin complex concentrate, FEIBA, to reestablish hemostasis independent of the pharmacologic effects of dabigatran. Additional studies are warranted to confirm the findings of our observation.

Coagulation profile of liquid-state plasma.

BACKGROUND: Use of liquid plasma (LP) has been reported as early as the mid 1930s. Unlike fresh-frozen plasma (FFP), LP is maintained at 1 to 6°C for up to 40 days after collection and processing. Despite its approved use by the US Food and Drug Administration, the coagulation profile of LP is incompletely described. In this study we evaluate the coagulation profile of LP stored up to 30 days. STUDY DESIGN AND METHODS: LP was prepared by removing plasma from nonleukoreduced whole blood within 24 hours of collection. Three LP units from each ABO group were collected and stored at 1 to 6°C. Plasma aliquots were obtained at Postcollection Days 1 to 5, 10, 15, 20, 25, and 30 and then stored at -70°C. Each aliquot was tested for prothrombin time, activated partial thromboplastin time, and other coagulation and fibrinolytic factors. RESULTS: There was a significant decrease in Factor (F)V, FVII, FVIII, von Willebrand factor (VWF), protein S (PS) activity, and endogenous thrombin potential on Day 15 compared with Day 1. No significant difference was observed for PS antigen, D-dimer, or thrombin-antithrombin complex. At least 50% activity of all measured factors was noted on Day 15, compared to Day 1. Considerable heterogeneity was observed between the different blood groups for FVII, FVIII, and VWF. CONCLUSION: These data demonstrate that LP maintains at least 50% of factor activity and thrombin-generating capacity up to 15 days of refrigerated storage. It may be more appropriate to limit LP storage and supplement with FFP when used for management of massively bleeding patients.

Measuring dabigatran concentrations using a chromogenic ecarin clotting time assay.

BACKGROUND: Clinicians managing patients receiving the direct thrombin inhibitor dabigatran may benefit in being able to determine the amount of drug present in selected situations. This may include assessment of accumulation, concurrent drug interactions, or adequate removal from circulation. The ability to estimate the amount of dabigatran present using the chromogenic ecarin assay (ECA) requires further clarification. OBJECTIVE: To describe the reliability of dabigatran measurements using a chromogenic ECA. METHODS: This was an evaluation of the ECA method that incorporated assessment of imprecision, linearity, accuracy, carryover, and lower limits of detection or blank. Pooled normal plasma enriched with dabigatran at concentrations of 0, 25, 50, 75, 100, 125, 150, 200, 300, 400, and 500 ng/mL were sent blinded to 3 laboratories in the United States to compare our ECA results with those of laboratories reporting dilute thrombin time methods (HEMOCLOT thrombin inhibitor assay) for measuring dabigatran. Trough and peak levels from 35 patients were also compared with mass spectrophotometry for assessing ECA accuracy. RESULTS: The within-run or day-to-day imprecision was less than 10%, with high linearity (R (2) = 0.989) and high degree of accuracy (R (2) = 0.985; slope = 0.908) for levels ranging between 18 and 470 ng/mL and no carryover at 0 ng/mL noted. The ECA approach appeared to be more reliable at lower dabigatran concentrations. CONCLUSIONS: The chromogenic ECA appears to be an effective approach to determine the amount of dabigatran present. Further insights are necessary to determine how it can be used to reduce thromboembolic or bleeding complications in patients receiving dabigatran.

Case report: artificial elevation of prothrombin time by telavancin.

BACKGROUND: Methicillin-resistant Staphylococcus aureus infections are a well-documented risk of surgery and are becoming increasingly difficult to treat owing to continued acquired resistance. A new antibiotic for treatment of Staphylococcus aureus is telavancin. CASE DESCRIPTION: A patient at our institution was prescribed telavancin for multiple spinal abscesses before spinal surgery. Routine preoperative testing revealed an international normalized ratio (INR) of 2.05 with no clear cause. Careful review of the patient's medication history and prescriber information revealed that telavancin may interfere with prothrombin time (PT/INR) testing. In vitro testing by our laboratory confirmed an association between telavancin dose and an increase in PT/INR. An alternative reagent for PT/INR testing unaffected by telavancin dose revealed a PT/INR of 0.97. LITERATURE REVIEW: Telavancin interacts with artificial phospholipid surfaces used to monitor coagulation while having no actual effect on coagulation. PURPOSES AND CLINICAL RELEVANCE: All physicians, especially orthopaedic surgeons, should be aware of the effects of telavancin and ensure proper measures are taken to acquire the true INR by switching the reagent used to test PT/INR or ensuring the PT/INR is drawn before telavancin dosing.

Preanalytical Variables in Hemostasis Testing.

Hemostasis testing performed in clinical laboratories are critical for assessing hemorrhagic and thrombotic disorders. The assays performed can be used to provide the information required for diagnosis, risk assessment, efficacy of therapy, and therapeutic monitoring. As such, hemostasis tests should be performed to the highest level of quality, including the standardization, implementation, and monitoring of all phases of the testing, which include the preanalytical, analytical, and post-analytical phases. It is well established that the preanalytical phase is the most critical component of the testing process, being the hands-on activities, including patient preparation for blood collection, as well as the actual blood collection, including sample identification and the post-collection handling to include sample transportation, processing, and storage of samples when testing is not performed immediately. The purpose of this article is to provide an update to the previous edition of coagulation testing-related preanalytical variables (PAV) and, when properly addressed and performed, can reduce the most common causes of errors in the hemostasis laboratory.