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.

PFA-100 System: A New Method for Assessment of Platelet Dysfunction.

This is a celebratory reprint of a historical paper published in STH in 1998. The original Abstract follows.The PFA-100 system is a platelet function analyzer designed to measure platelet-related primary hemostasis. The instrument uses two disposable cartridges: a collagen/epinephrine (CEPI) and a collagen/ADP (CADP) cartridge. Previous experience has shown that CEPI cartridges detect qualitative platelet defects, including acetylsalicylic acid (ASA)-induced abnormalities, while CADP cartridges detect only thrombocytopathies and not ASA use. In this seven-center trial, 206 healthy subjects and 176 persons with various platelet-related defects, including 127 ASA users, were studied. The platelet function status was determined by a platelet function test panel. Comparisons were made as to how well the defects were identified by the PFA-100 system and by platelet aggregometry. The reference intervals for both cartridges, testing the 206 healthy subjects, were similar to values described in smaller studies in the literature (mean closure time [CT] of 132 seconds for CEPI and 93 seconds for CADP). The use of different lot numbers of cartridges or duplicate versus singleton testing revealed no differences. Compared with the platelet function status, the PFA-100 system had a clinical sensitivity of 94.9% and a specificity of 88.8%. For aggregometry, a sensitivity of 94.3% and a specificity of 88.3% were obtained. These values are based on all 382 specimens. A separate analysis of sensitivity by type of platelet defect, ASA use versus congenital thrombocytopathies, revealed for the PFA-100 system a 94.5% sensitivity in identifying ASA users and a 95.9% sensitivity in identifying the other defects. For aggregometry, the values were 100% for ASA users and 79.6% for congenital defects. Analysis of concordance between the PFA-100 system and aggregometry revealed no difference in clinical sensitivity and specificity between the systems (p > 0.9999). The overall agreement was 87.5%, with a Kappa index of 0.751. The two tests are thus equivalent in their ability to identify normal and abnormal platelet defects. Testing 126 subjects who took 325 mg ASA revealed that the PFA-100 system (CEPI) was able to detect 71.7% of ASA-induced defects with a positive predictive value of 97.8%. The overall clinical accuracy of the system, calculated from the area under the receiver operating characteristic curve, was 0.977. The data suggest that the PFA-100 system is highly accurate in discriminating normal from abnormal platelet function. The ease of operation of the instrument makes it a useful tool to use in screening patients for platelet-related hemostasis defects.

MRI-based synthetic CT of the hip: can it be an alternative to conventional CT in the evaluation of osseous morphology?

OBJECTIVES: MRI is the gold standard for soft tissue evaluation in the hip joint. However, CT is superior to MRI in providing clear visualization of bony morphology. The aim of this study is to test the equivalency of MRI-based synthetic CT to conventional CT in quantitatively assessing bony morphology of the hip. MATERIALS AND METHODS: A prospective study was performed. Adult patients who underwent MRI and CT of the hips were included. Synthetic CT images were generated from MRI using a deep learning-based image synthesis method. Two readers independently performed clinically relevant measurements for hip morphology, including anterior and posterior acetabular sector angle, acetabular version angle, joint space width, lateral center-edge angle, sharp angle, alpha angle, and femoral head-neck offset on synthetic CT and CT. Inter-method, inter-reader, and intra-reader reliability and agreement were assessed using calculations of intraclass correlation coefficient, standard error of measurement, and smallest detectable change. The equivalency among CT and synthetic CT was evaluated using equivalency statistical testing. RESULTS: Fifty-four hips from twenty-seven participants were included. There was no reported hip pathology in the subjects. The observed agreement based on reliability and agreement parameters indicated a strong degree of concordance between CT and synthetic CT. Equivalence statistical testing showed that all synthetic CT measurements are equivalent to the CT measurements at the considered margins. CONCLUSION: In healthy individuals, we demonstrated equivalency of MRI-based synthetic CT to conventional CT for the quantitative evaluation of osseous hip morphology, thus obviating the radiation exposure of a pelvic CT examination. KEY POINTS: •MRI-based synthetic CT images can be generated from MRI using a deep learning-based image synthesis method. •MRI-based synthetic CT is equivalent to CT in the quantitative assessment of bony hip morphology in healthy individuals. •MRI-based synthetic CT is promising for use in preoperative diagnosis and surgery planning.

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.

Current and Emerging Direct Oral Anticoagulants: State-of-the-Art.

Anticoagulant drugs comprise a specific subcategory of antithrombotic agents that act to inhibit blood coagulation at various stages, reducing clot development and ultimately lowering the risk of developing new-onset or recurrent thrombosis. Although the long history of anticoagulant drugs has been characteristically shaped by coumarin and heparin derivatives, a new generation of direct oral anticoagulants (DOACs), which specifically inhibit thrombin or activated factor X, combine many advantages of their progenitor drugs, and hence are prepotently revolutionizing the landscape of antithrombotic therapy. Several drugs (apixaban [BMS-562247], dabigatran [BIBR953], edoxaban [DU-1766], rivaroxaban [BAY 59-7939]) have already received widespread approval by national or supranational medicinal agencies. This narrative article provides a state-of-the-art for these and for several other DOACs at different stages of clinical evaluation (betrixaban, darexaban, eribaxaban, letaxaban, nokxaban), and certain others whose development has been discontinued (AZD-0837, fidexaban, LY517717, odiparcil, otamixaban, TTP889, and ximelagatran). What clearly emerges from our analysis is that DOACs sharing very similar mechanisms of action are still characterized by different efficacy and safety profiles. This not only depends on biochemical, biological, and pharmacokinetic characteristics, but also on lack of standardization between different clinical trials in terms of targeted disease, patient recruitment, sample size, duration and endpoints, as well as lack of harmonization around procedures used for drug licensing. These factors contribute to challenging the minds of physicians, who may find difficulty navigating their way through multiple indications, different pharmacological profiles, various side effects, and specific drug-to-drug interactions. Such considerations also burden laboratory professionals, who may face organizational and economic challenges in developing and/or implementing multiple assays to assess the pharmacodynamics (effect on coagulation) or pharmacokinetics (drug levels) of DOACs.

Towards harmonization of external quality assessment/proficiency testing in hemostasis.

Quality in diagnostic testing represents a key target of laboratory medicine, for which an assurance around the quality of testing is expected from all involved in the process. Laboratories attempt to assure the quality of their testing by various processes, but especially by performance of internal quality control and external quality assessment (EQA). This is especially true for tests of hemostasis and coagulation. EQA in general provides information on test accuracy and on evaluation of long-term laboratory performance. EQA providers support laboratory performance by various means, including distribution of material for testing of analytes ("proficiency testing"), educational support through expert advice, distribution of publications or case series. Participation in EQA is often a laboratory accreditation requirement. This review aims to identify some of the strengths and weaknesses of EQA, and targets attempts towards harmonization of EQA practice, in order to achieve the best outcome for participant laboratories and, thus, for patients and their clinical care providers.

Factor Activity Assays for Monitoring Extended Half-Life FVIII and Factor IX Replacement Therapies.

The advent of modified factor VIII (FVIII) and factor IX (FIX) molecules with extended half-lives (EHLs) compared with native FVIII and FIX represents a major advance in the field of hemophilia care, with the potential to reduce the frequency of prophylactic injections and/or to increase the trough level prior to subsequent injections. Monitoring treatment through laboratory assays will be an important part of ensuring patient safety, including any tailoring of prophylaxis. Several approaches have been used to extend half-lives, including PEGylation, and fusion to albumin or immunoglobulin. Some of these modifications affect factor assays as routinely performed in hemophilia centers; so, laboratories will need to use FVIII and FIX assays which have been shown to be suitable on a product-by-product basis. For some products, there are marked differences between results obtained using one-stage or chromogenic assays and results obtained using different reagents in the one-stage assay. The laboratory should use an assay in which the recovery of the product closely aligns with the assay used by the pharmaceutical company to assign potency to the product, so that the units reported by the laboratory agree with those used to demonstrate efficacy of the product during clinical trials. Reported assay differences in relation to several of the EHL FVIII and FIX molecules will be reviewed in this article.

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.