Hemostasis Laboratory Research Articles

Assessing quality in the specialized hemostasis laboratory: review and critique of external quality assurance (EQA) programs in the US

In 1999 the National Committee for Clinical Laboratory Standards (NCCLS, now known as Clinical & Laboratory Standards Institute) introduced the concepts of quality practice for clinical laboratories, with the notion to improve the pre-analytical, analytical and post-analytical phases of laboratory testing. Hemostasis testing is particularly sensitive to pre-analytical issues, but most quality assurance measures are specific for the analytical phase of hemostasis testing. The external quality assessment (EQA) program is typically a bi-annual assessment using blinded samples that are intended to improve laboratory practice. We review the current EQA options in the US for specialized hemostasis laboratories to assess whether (I) they address all three phases of testing, and whether (II) the testing platforms are providing optimal assessment of hemostasis testing.

Standardization of Light Transmission Aggregometry for Diagnosis of Platelet Disorders: An Inter-Laboratory External Quality Assessment.

Several in vitro platelet function tests are available for the diagnosis of inherited platelet function disorders. Currently, the light transmission aggregometry (LTA) is recommended as one of the first-step tests. LTA is available in most specialized hemostasis laboratories. Although the LTA is accepted as a 'gold standard' assay for the evaluation of platelet function, its standardization in the clinical practice is still challenging. The GTH-based THROMKID-Plus Study Group has performed an inter-laboratory trial in Germany and Austria. Five different agonists were selected according to the Scientific and Standardization Committee/International Society on Thrombosis and Haemostasis recommendations and shipped in 3 different sets (one should represent a healthy control and two should simulate platelet function disorders) to 15 specialized laboratories in Germany and Austria. Agonists were analyzed by APACT or PAP4/8 aggregometer using platelet-rich plasma from healthy donors. In addition, laboratory-internal platelet agonists were tested in platelet-rich plasma from a healthy donor. All laboratories (9 used APACT, 6 used PAP4/PAP8) showed very consistent data regarding the maximum percentage of aggregation induced by the tested agonists and identified the differential diagnosis of the simulated platelet function disorders with one exception, which was due to technical problems. In contrast, there was a high variability of the laboratory-internal inductors regarding reagent type, concentrations and pathological cut-off values. Our study showed that the shipment of agonists is suitable for an inter-laboratory survey of LTA. However, there is still a remarkable need for standardization of agonist reagents and their concentration as well as for definition of reference ranges.

Pre-analytical quality control in hemostasis laboratories: visual evaluation of hemolysis index alone may cause unnecessary sample rejection

Abstract Background Visual inspection is the most widespread method for evaluating sample hemolysis in hemostasis laboratories. The hemolysis index (HI) was determined visually (visual index, VI) and measured on an ACL TOP 750 (IL Werfen) system with a hemolysis-icterus-lipemia index (HIL) module. These values were compared with those measured on clinical chemistry systems Unicel DXC600 and AU680 and with quantitation of free-hemoglobin (Hb) performed by a spectrophotometric measurement method (SMM). Methods The HI was measured in 356 sodium citrate plasma samples, 306 of which were visibly hemolyzed to varying degrees and 50 were not hemolyzed. The analytical performance of each method was evaluated. Results Linear regression analysis, calculated between SMM and the other systems in the study, returned coefficients of determination r2 = 0.853 (AU680), r2 = 0.893 (DXC600) and r2 = 0.917 (ACL TOP 750). An r2 = 0.648 was obtained for linear regression analysis between VI and ACL TOP 750. In addition, ACL TOP 750 showed an excellent correlation in multivariate analysis (r2 = 0.958), showing good sensitivity (0.939) and specificity (0.934) and a diagnostic accuracy of 94%. By comparison, DXC600 and AU680 showed coefficients of determination of 0.945 and 0.923, respectively. A cut-off was set at 0.15 g/L free-Hb, as determined by the automated method, such that any hemostasis samples measuring above this threshold should not be analyzed. Based on this criterion, samples were classified as accepted or rejected, and the number of samples discarded during VI or ACL TOP 750 measurements was compared. Conclusions This study confirmed that hemostasis laboratories should consider introducing an objective, automated and standardized method to check samples for hemolysis. By relying solely on visual inspection, up to 50% of samples could be unnecessarily rejected. The ACL TOP 750 system demonstrated a satisfactory analytical performance, giving results comparable to those of the reference method.

Reversal strategies in patients treated with direct oral anticoagulants.

Administration of direct oral anticoagulants (DOACs) for the treatment of venous thrombotic events (VTE) or non-valvular atrial fibrillation (AF) is now standard of care and has demonstrated clinical efficacy and safety in numerous clinical studies. Usually these substances have lower overall mortality and less risk of cerebral hemorrhage, but depending on the substance and study, they are more likely to cause gastrointestinal bleeding than vitamin K antagonists (VKA), the medication that used to be standard for VTE and AF. Since DOACs have very short plasma elimination half-lives compared to VKA, for most bleeding events, expert opinions suggest that withdrawal of DOACs and supportive care will likely suffice to stop a bleeding episode. Because there is a bleeding risk associated with DOACs, reversal strategies may be needed if a patient receiving DOAC therapy bleeds during surgery or an invasive procedure. So far, idarucizumab has been the only available antidote that binds specifically to dabigatran and safely and quickly reverses its anticoagulant effects. Idarucizumab has no effects on anti Xa inhibitors or other anticoagulants. To date, treatment of serious, life-threatening bleeds in patients with anti-Xa-inhibitor has involved 4 factor prothrombin complex concentrates (PCC). PCC restores normal hemostasis laboratory values in most patients with major bleeding events after anti Xa inhibitor intake. Recently, the US Food and Drug Administration (FDA) approved andexanet alfa as the first specific antidote for the anti-Xa inhibitors apixaban and rivaroxaban. So far clinical experience with this substance and data comparing it with PCC are lacking. Currently ciraparantag is under investigation as a universal reversal agent for all DOACs and low molecular weight heparin as well. Because it is so broadly applicable, ciraparantag might be a good future option for the management of most bleeding complications under anticoagulant treatment. The aim of this review is to summarize recent study data and recommendations on nonspecific and specific DOAC reversal strategies and to present the current evidence.

Optimization of von Willebrand factor multimer analysis in vertical mini-gel electrophoresis systems: A rapid procedure

Von Willebrand disease (VWD) is a common cause of bleeding worldwide. Analysis of von Willebrand factor (VWF) multimer distribution (VWF:MD) is essential to properly classify and treat different types of VWD, and it is performed using a SDS agarose gel electrophoresis followed by Western blotting, a handmade technique that demands days to be completed and requires skillful execution. Aiming both to facilitate gel production and to shorten the preparation time, we developed an uncomplicated technique to provide agility in the analysis of VWF:MD, so that it can be easily accomplished in the routine practice of hemostasis laboratories. On that account, we used a commercial vertical mini-gel electrophoresis system for SDS-PAGE and a semi-dry transfer system, which allowed us to analyze VWF:MD of various samples in a period shorter than 12 h. This technique differentiated VWF:MD in human and animal plasmas under normal, congenital and acquired (experimental envenomation by Bothrops jararaca snake) conditions. This optimized method is cheap, rapid, reproducible, easy to be performed, and uses electrophoresis and Western blotting systems available in most laboratories. All these advantages encourage hemostasis professionals to use it in their routine practices. In order to facilitate the setup and accomplishment of the whole procedure step by step, videos were appended to the article.

Modern aspects of the pathogenesis, diagnosis and therapy of hemostasis disorders in children with acute leukemias

Patients with oncohematological diseases, both children and adults, face high risks of thrombotic and hemorrhagic complications.About 40 % of pediatric patients with acute lymphoblastic leukemia develop bleedings, and the incidence of thrombosis in this disease ranges from 1 to 36 %. Most thromboses are associated with the use of central venous catheters and the use of L-asparaginase, which leads to a significant reduction in the synthesis of coagulation proteins.Massive hemorrhages account for two-thirds of all causes of early death in pediatric patients with acute myelogenous leukemia (AML). Absolute risks of death due to bleeding and leukostasis range from 1.8 % in the total population of children with AML to 14.3 % in a population with hyperleukocytosis more than 200 × 109 /l. The risk of thrombotic complications in children with AML varies between 3.4–11 %. In patients with AML, complex systemic coagulopathies may occur, such as disseminated intravascular coagulation (DIC), excessive fibrinolysis, or nonspecific proteolysis. This scale is not yet applicable due to the lack of research on its effectiveness in the pediatric population. The laboratory diagnostics of hemostasis is difficult due to the combined nature of thrombotic and hemorrhagic complications: bleeding, thrombosis and even DIC syndrome (combining both hyper- and hypocoagulation phases) can be expected in each specific patient with hemoblastosis. Because of the long-term nature of the treatment and the varying intensity of the various treatment units, the patient’s hemostasis during disease manifestation does not allow one to predict with any certainty the complications on induction or consolidation therapy. Involving all the components of the hemostasis system – vascular, platelet and plasma – into the pathological process makes prediction and diagnosis of thrombohemorrhagic complications impossible with the help of standard hemostatic tests and a general blood test, since these tests are designed to assess the concentrations of individual proteins and the functioning of individual components of the hemostatic system, and does not assess the balance between its procoagulant and anticoagulant components. Global hemostatic tests such as thromboelastography, thrombodynamics and thrombin generation test adequately reflect hypercoagulable conditions and can serve as a basis for the development of a new set of laboratory hemostasis tests.Conflict of interest. F.I. Ataullakhanov is co-founder of HemaCore LLC, which holds several patents and patent applications that are related to the diagnostic use of Thrombodynamics® (Ataullakhanov F.I., international patent applications: PCT/CH2007/000543 filing date 02.11.2007 and РСТ/RU2012/000570 filing date 16.07.2012). None of the other authors has any competing interests to declare.

Understanding the "philosophy" of laboratory hemostasis.

Unlike many other areas of laboratory medicine, laboratory hemostasis has some peculiarities, which makes it one of the most complex diagnostic domains in clinical medicine. The inherent complexity of the hemostasis process, the components of which have not yet been thoroughly unravelled, is mirrored by a large number of hemostasis disturbances, which can involve single or multiple components. Although laboratory diagnostics represents an unavoidable part of the diagnostic reasoning in patients with bleeding or thrombotic disorders, the basic concept beneath the assumption that in many human pathologies, one single test may be sufficient for the diagnosis, does not hold true in hemostasis. There are in fact many aspects that would lead us to conclude that laboratory hemostasis can be considered a very challenging arena for many clinicians and perhaps also for some laboratory professionals. The most challenging aspects typically include the following concepts; that hemostasis is an intricate and multifaceted process, that more than one test is typically needed to achieve a final diagnosis, that results of screening tests depend on many biological factors and do not allow making a final diagnosis, that harmonization of techniques is still an unmet target, and that the calculations used vary widely among laboratories. This article is hence aimed at discussing many of these aspects, with the hope of presenting a useful contribution to better understand the "philosophy" of laboratory hemostasis.

Возможность получения реагента для определения ристоцетин-кофакторной активности фактора Виллебранда (ФВ:РКФА) из тромбоцитарных концентратов длительного срока хранения

Введение. Фактор Виллебранда (ФВ) является важным компонентом системы гемостаза, и отклонение от нормы его содержания или состава вызывает развитие различных типов болезни Виллебранда. Определение ристоцетин-кофакторной активности ФВ (ФВ:РКФА) является особенно значимым при выявлении таких типов болезни Виллебранда как 2А, 2В и 2М, при которых содержание антигена ФВ находится в нормальных пределах, а ФВ:РКФА значительно снижена. Цель исследования: получение реагента для измерения ФВ:РКФА из концентратов тромбоцитов с длительным сроком хранения, обеспечивающего правильность диагностики. Материалы и методы. Функционально полноценные тромбоциты получали из донорских тромбоцитарных концентратов со сроком хранения 7–14 суток. Результаты. При создании реагента для измерений ФВ:РФКА нами был подобран метод отбора функционально полноценных донорских тромбоцитов для получения фиксированных формальдегидом клеток. На основе таких тромбоцитов был разработан реагент, позволяющий измерять ФВ:РКФА с использованием как агрегационного, так и агглютинационного метода. Выявлено соответствие результатов определения ФВ обоими методами с коэффициентом корреляции 0,96. Проведена оценка агрегационного метода измерения ФВ:РКФА по результатам участия в международной программе внешнего контроля качества UK NEQAS for Blood Coagulation (Великобритания). Заключение. Показано, что использование полученного из фиксированных тромбоцитов реагента позволяет правильно измерять ФВ:РКФА. Introduction. Von Willebrand factor (vWF) is an important component of hemostatic system and deviations from the norm of its content or composition are the cause of various types of von Willebrand disease development. Determination of ristocetin-cofactor activity of vWF (vWF:RCo) is particularly important for identifying 2A, 2B and 2M types of von Willebrand disease, in which the content of vWF antigen is within the normal range, and vWF:RCo signifi cantly reduced. Aim: to obtain a reagent for the measurement of vWF:RCo from platelet concentrates with a long shelf life, providing correct diagnostics. Materials and methods. Fully functional platelets were obtained from donor platelet concentrates with a shelf life of 7–14 days. Results. We discovered the method for selection of fully functional donor platelets to produce formaldehyde-fi xed cells. On the basis of these platelets we developed a reagent that allows to measure vWF:RCo using both aggregation and agglutination methods. The appropriateness was found between the results of vWF determination by both methods with a correlation coeffi cient 0.96. The aggregation method of measuring vWF:RCo was evaluated by a result of participation in the international program of external quality control UK NEQAS for Blood Coagulation (United Kingdom). Conclusion. It is shown that the use of a reagent obtained from fi xed platelets allows for the correct measurement of vWF:RCo.

Differences in Protein S, Protein C, and Antithrombin Levels and Risk of Venous Thromboembolism Among American Black and White Enrollees in the Gate Study

Introduction: Black Americans have been reported to have a higher incidence of venous thromboembolism (VTE) compared to white Americans. The underlying cause of this disparity is not well characterized. Protein S, protein C, and antithrombin deficiency are known risk factors for VTE. There is inconsistent data regarding racial variation on the effect of protein S, protein C, and antithrombin levels and/or deficiency and risk of VTE. Most prior studies have not included a large black study population. The aim of this investigation is to compare levels of protein C, protein S, and antithrombin between VTE cases and controls by race among enrollees in the Genetic Attributes and Thrombosis Epidemiology (GATE) study, accounting for genetic differences such as hemoglobin genotype that may contribute to differences in protein levels by race.Methods: Testing for protein S, protein C, and antithrombin deficiency was performed on a subset of participants enrolled in the GATE study, a case-control study comprised of VTE cases and controls frequency-matched on age, sex, and race (self-identified). Controls had no history of VTE and did not receive anticoagulant therapy. Antigen and activity levels in cases were measured at least one month after completion of anticoagulation therapy. A priori cutoff levels for determining deficiency status were standardized per Centers for Disease Control Hemostasis Laboratory reference ranges. Beta hemoglobin genotype was determined by either a multiplex genotyping assay or sequencing of the beta hemoglobin gene. Differences in protein levels between cases and controls were compared using the Wilcoxon rank sum test. Conditional logistic regression analysis was performed to evaluate the association between protein S, protein C, or antithrombin deficiency and VTE. Crude models were conditioned on the matching factors, and adjusted models were conditioned on the matching factors and controlled for patient characteristics found to be associated with VTE and protein S, protein C, or antithrombin deficiency. Potential effect modification was assessed using likelihood ratio tests.Results: Among 2,454 subjects enrolled in the GATE study, 1,436 subjects (63.6%) had samples available for analysis. Protein S activity and total protein S antigen levels were significantly lower among VTE cases compared to controls in both white and black participants; however, antithrombin levels were higher among VTE cases compared to controls in white participants only. (Table 1) The prevalence of protein S and protein C deficiency was higher among VTE cases compared to controls in both white and black participants in the GATE study (Table 2). The prevalence of antithrombin deficiency was similar between VTE cases compared to controls. Adjusting for the effect of family history of VTE, history of cancer, and hemoglobin genotype, the effect of protein S and protein C deficiency on odds of VTE was stronger among black participants compared to white participants (Table 2).Conclusions: The effects of protein S and protein C deficiency on the odds of being a VTE case were higher among black participants in the GATE study, controlling for confounding variables including hemoglobin genotype. While these results indicate protein S and protein C deficiency may be important contributors to disparities in VTE occurrence, additional investigations using a larger study populations and different study designs are warranted. [Display omitted] DisclosuresPatel:Daiichi Sankyo: Other: DSMB member. Payne:Bioverativ: Other: treatment product donation; Novo Nordisk: Other: treatment product donation; Shire: Other: treatment product donation; Genentech: Membership on an entity's Board of Directors or advisory committees; Bayer: Other: treatment product donation.

Hemostasis testing and therapeutic plasma exchange: Results of a practice survey.

Performing therapeutic plasma exchange (TPE) with albumin replacement decreases coagulation factor and platelet levels. No defined guidelines exist regarding laboratory testing to assess hemostasis in patients undergoing TPE. A survey to evaluate hemostasis testing with TPE was distributed using online survey software. One response per institution was analyzed based on a hierarchical algorithm, excluding membrane filtration users, resulting in a maximum of 120 respondents per question. Descriptive analysis was performed with results reported as the number and/or frequency (%) of respondents to each question. The practices represented vary by institution type, number of apheresis procedures per year, and performance of TPE on children. Prior to TPE planned with albumin replacement, many respondents obtain laboratory studies for almost all patients (54.9% outpatients and 68.7% inpatients); however, some do not routinely obtain laboratory studies (9.7% outpatients and 4.4% inpatients). Hemoglobin/hematocrit, platelet count, fibrinogen, partial thromboplastin time (aPTT), and international normalized ratio (INR) are obtained prior to all TPE by 62.5%, 53.4%, 31.0%, 18.1%, and 17.7% of respondents, respectively; however, 1.0%, 8.7%, 29.0%, 38.3%, and 35.4%, respectively, do not routinely obtain these studies. Variation was observed in laboratory threshold values for action; the most common reported were hemoglobin/hematocrit <7 g/dL or 21% (31.0%), platelet count <50 × 109 /L (24.1%), fibrinogen <100 mg/dL (65.3%), aPTT >reference range and >1.5 times reference range (tied, 28.1%), and INR >1.5 (20.7%). Practice variation exists in hemostasis laboratory testing and threshold values for action with TPE. Further studies are needed to determine optimal hemostasis testing strategies with TPE.

A collaborative study by the Working Group on Hemostasis and Thrombosis of the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) on the interference of haemolysis on five routine blood coagulation tests by evaluation of 269 paired haemolysed/non-haemolysed samples

IntroductionHaemolysis is the leading cause of sample rejection in laboratory haemostasis. Most studies focused on artificially haemolysed samples. The aim of this study was a prospective assessment of spontaneous haemolysis on haemostasis tests, by comparing results of haemolysed (H) versus new, non-haemolysed (NH) specimens, collected within 4hrs. As new coagulometers can identify interfering substances, visual assessment of haemolysis was also compared with instrumental haemolysis index and stratified in subclasses.Materials and methodsTwo hundred and sixty nine paired samples were collected and analysed using ACL TOP750-CTS (Instrumentation Laboratory, Bedford, USA), for prothrombin time (PT), activated partial thromboplastin time (aPTT), D-Dimer (DD), fibrinogen (Fib) and antithrombin (AT). Bias between H and NH was calculated and compared with the respective critical difference (CD).ResultsMean bias was - 0.1 s for PT (P = 0.057), - 1.1 s for aPTT (P < 0.001), 1025 ng/mL for DD (P < 0.001), - 0.04 g/L for Fib (P = 0.258) and 1.4% for AT (P = 0.013). Bias exceeding the CD varied according to the method, with larger differences for aPTT (36.1%) and DD (17.1%) and < 8% for PT, Fib and AT. No correlation emerged between free haemoglobin values and difference in haemostasis tests in H and NH samples for any tests. Moderate/severe haemolysis involved > 95% of samples. The agreement between visual assessment and instrumental evaluation of haemolysis was 0.62.ConclusionSpurious haemolysis deeply influences aPTT and DD, and to a lesser extent AT and Fib. Prothrombin time seems only slightly influenced, suggesting that PT can be accepted also in haemolysed samples. Although a good inter-observer correlation of haemolysis evaluation was found, the instrumental assessment of haemolysis seems recommendable.

Аspects of the methodology of laboratory studies of hemostasis in pediatric hematology-oncology and general approaches in the pathology of hemostasis in leukemia

Children with acute leukemia are faced with high risks of thrombotic and hemorrhagic complications. The pathogenesis of haemostasis disorders in hemoblastoses is complex because, in addition to the disease itself, the aggressiveness of the therapy and the need for numerous invasive manipulations also make a significant contribution. Patients with hemoblastoses are equally susceptible to thrombosis and hemorrhage, which makes it possible to speak of multidirectional shifts in the balance of the hemostatic system in each individual patient. Standard laboratory hemostasis tests (clotting times, marker tests) are designed to assess the concentrations of individual proteins and the functioning of individual components of the hemostasis, and in do not assess the balance between its procoagulant and anticoagulant components. Global hemostatic tests designed to assess the coagulation balance, such as thromboelastography, thrombin generation test, and thrombodynamics, can be the alternative for the standard coagulation assays. The review focuses on the mechanisms of various laboratory hemostasis tests, as well as an assessment of their informative value in frequent complications of the underlying disease (sepsis leading to the development of disseminated intravascular coagulation (DIC) syndrome, thrombocytopenia) and catheterization, which is present in the majority of patients with hemoblastosis. General screening tests of the blood coagulation system have little diagnostic value in the DIC syndrome in patients with acute leukemia, mainly due to their insensitivity to hypercoagulability. Standard markers (for example, D-dimers) are non-specific and only confirm the clinical manifestations of clotting disorder in sepsis and septic shock, but are unable to predict the dynamics of this process at earlier stages of the inflammatory response. In this case, the thrombin generation test and thrombodynamics make it possible to reveal the hypercoagulable phase of the DIC syndrome. Thrombocytopenia accompanies almost all protocols of chemotherapy. In this case, the degree of bleeding does not always depend only on the concentration of platelets, since chemotherapeutic drugs can affect not only the quantity, but also the functional characteristics of platelets, which are not determined by standard examination of patients. The catheterization that accompanies the treatment of hemoblastoses is the leading cause of thrombosis in children with acute leukemia. Thromboembolism of the pulmonary artery due to thrombosis in the central vein system occurs in 8–15 % of patients. The prediction of catheter-associated thromboses using standard laboratory methods for assessing the state of the hemostasis is not possible. Absence of sensitive tests in modern diagnostic schemes leads to the fact that the attending physician is forced to focus exclusively on the clinical picture of thrombosis or bleeding. The development of new functional methods of hemostasis allows one to think that today the existing standard panel of coagulation tests can be expanded and made much more informative in terms of the prediction of thrombohemorrhagic complications in pediatric hematology-oncology.

Benefits and Pitfalls of Point-of-Care Coagulation Testing for Anticoagulation Management

Point-of-care (POC) testing is generally less precise and has higher reagent costs per test than laboratory-based assays. However, POC hemostasis testing can offer significant advantages in particular situations: patient-managed warfarin therapy as well as rapid turnaround time heparin management for intraoperative patients. Of note, POC hemostasis testing is generally approved for the purposes of anticoagulation monitoring and is inferior to laboratory coagulation testing for the diagnosis of congenital or acquired coagulopathy. The frequently used POC coagulation instruments for POC international normalized ratio and activated clotting time are reviewed, as well as their typical performance relative to central laboratory testing (where available). Several cases are discussed that highlight the benefits, as well as pitfalls, of POC coagulation testing. POC coagulation testing for anticoagulation monitoring offers advantages in particular situations. Clear policies and protocols must be developed to guide proper use of POC versus central laboratory hemostasis testing.

Laboratory hemostasis: from biology to the bench.

Physiological hemostasis is an intricate biological system, where procoagulant and anticoagulant forces interplay and preserves blood fluidity when blood vessels are intact, or trigger clot formation to prevent excessive bleeding when blood vessels are injured. The modern model of hemostasis is divided into two principal phases. The first, defined as primary hemostasis, involves the platelet-vessel interplay, whilst the second, defined as secondary hemostasis, mainly involves coagulation factors, damaged cells and platelet surfaces, where the so-called coagulation cascade rapidly develops. The activation and amplification of the coagulation cascade is finely modulated by the activity of several physiological inhibitors. Once bleeding has been efficiently stopped by blood clot formation, dissolution of the thrombus is essential to restore vessel permeability. This process, known as fibrinolysis, also develops through coordinate action of a vast array of proteins and enzymes. An accurate diagnosis of hemostasis disturbance entails a multifaceted approach, encompassing family and personal history of hemostatic disorders, accurate collection of clinical signs and symptoms, integrated with laboratory hemostasis testing. Regarding laboratory testing, a reasonable approach entails classifying hemostasis testing according to cost, complexity and available clinical information. Laboratory workout may hence initiate with some rapid and inexpensive "screening" tests, characterized by high negative predictive value, then followed by second- or third-line analyses, specifically aimed to clarify the nature and severity of bleeding or thrombotic phenotype. This article aims to provide a general overview of the hemostatic process, and to provide some general suggestions to optimally facilitate laboratory hemostasis testing.

Dabigatran etexilate in the treatment of localized intravascular coagulopathy associated with venous malformations

IntroductionVenous malformations (VM) are congenital slow-flow vascular malformations. A common complication is localized intravascular coagulopathy (LIC), which results from chronic, localized, intraluminal activation and consumption of clotting factors and is characterized by elevated D-dimer and, in severe cases, decreased fibrinogen levels. Therapeutic options include anti-coagulation by Low Molecular Weight Heparin (LMWH) which has been reported to improve pain, decrease thrombosis, improve laboratory parameters and reduce hemorrhagic complications. However, the use of subcutaneous injections of LMWH has several limitations and the indications for therapy remain controversial. Materials and methodsWe present the first small case series of 3 patients with VM-LIC in whom a long-term treatment by dabigatran etexilate, a direct thrombin inhibitor, was initiated in the setting of ambulatory hemostasis consultations. ResultsClinically, drug tolerance was excellent in all 3 patients. Moderate hemorrhagic complaints occurred in 2 out of 3 patients but there was no thromboembolic event under dabigatran therapy. Dabigatran provided symptomatic relief on pain, swelling and heaviness feeling. Biologically, dabigatran treatment showed various modifications in D-dimers level but the fibrinogen level and platelet count remained unchanged. ConclusionIn conclusion, dabigatran etexilate may provide a viable and more convenient alternative to LMWH in the long-term treatment of VM-LIC. The dose has to be tailored in each patient in function of the bleeding tendency and laboratory hemostasis parameters.

Usefulness of multiple electrode aggregometry as a screening tool for bleeding disorders in a pediatric hospital

Platelet function testing is a cornerstone in the diagnostic investigation of patients with a bleeding history. Multiple electrode aggregometry (MEA) has been shown to detect von Willebrand disease (VWD), platelet function disorders, and drug-induced bleeding disorders. However, there are few studies supporting its successful use in children. We have implemented and used MEA over 3 years in our hemostasis laboratory in order to study its usefulness to supplement and expedite diagnosis. This is a retrospective, single-center, cohort study of 109 hospitalized children who underwent a laboratory investigation of hemostasis and either had a reported bleeding history or an abnormal bleeding episode. Plasmatic coagulation testing, blood counts, plasmatic von Willebrand testing, platelet function analyzer (PFA-100), and impedance aggregometry (MEA) were performed in all children. Light transmission aggregometry testing was performed as needed. In 41 cases (37.6%), a working diagnosis was made; a primary hemostatic disorder was detected in 35 children (VWD (n = 16), platelet disorder (n = 15), and valproic acid therapy-induced bleeding disorder (n = 3), acetylsalicylic acid-related bleeding (n = 1). In patients diagnosed with VWD, MEA ristocetin-induced platelet aggregation test (RISTO) high test revealed abnormally low aggregation in six patients (43.8%); whereas in patients diagnosed with a platelet function disorder, abnormally low values were found by MEA in only three children (20%). Three of the four children with laboratory evidence of drug-induced platelet dysfunction had abnormalities on MEA. There were no cases in which an abnormal MEA result was used to make a previously undetermined diagnosis. Retrospectively, MEA has demonstrated limited additional diagnostic value beyond standard laboratory testing for detecting defects of primary hemostasis in children.

Guidelines for certification of International Normalized Ratio (INR) for vitamin K antagonists monitoring according to the EN ISO 22870 standards

Point of care testing (POCT) must comply with regulatory requirements according to standard EN ISO 22870, which identify biologists as responsible for POCT. INR for vitamin K antagonists (VKAs) monitoring is a test frequently performed in haemostasis laboratories. Bedside INR is useful in emergency room, in particular in case of VKAs overdosage but also for specific populations of patients like paediatrics or geriatrics. INR POCT devices are widely used at home by the patients for self-testing, but their use in the hospital by the clinical staff for bedside measurement is growing, with devices which now comply with standard for POCT accreditation for hospital use. The majority of point of care devices for INR monitoring has shown a good precision and accuracy with results similar to those obtained in laboratory. With the aim to help the multidisciplinary groups for POCT supervision, the medical departments and the biologists to be in accordance with the standard, we present the guidelines of the GFHT (Groupe français d'étude sur l'hémostase et la thrombose, subcommittee "CEC et biologie délocalisée") for the certification of POCT INR. These guidelines are based on the SFBC guidelines for the certification of POCT and on the analysis of the literature to ascertain the justification of clinical need and assess the analytical performance of main analysers used in France, as well as on a survey conducted with biologists.

Team-Based Learning Is Effective in Teaching Appropriate Utilization of Hemostasis Laboratory Tests Using Storyline Case-Authoring Software.

Team-based learning (TBL) is an effective way to teach medical students a challenging topic: coagulation. This TBL requires students to discuss a differential diagnosis, order and analyze laboratory tests, and decide upon appropriate treatment. The coagulation TBL was utilized in a hematology/oncology system-based medical course. The TBL began with Individual and Group Readiness Assurance Tests (IRAT and GRAT, respectively) consisting of the same 10 multiple-choice questions. Next came a team application activity with the goal of evaluating a bleeding patient. Each team was given the clinical case and a whiteboard. Each team recorded a differential diagnosis and chose relevant laboratory tests from 20 hematology/coagulation assays placed in case-authoring software. Next, teams recorded the laboratory results, final diagnosis, and treatment on the whiteboards. Teams then voted for the best whiteboard. After discussing the highly voted whiteboard, instructors provided case discussion and elaboration about all 20 laboratory tests and their interpretation. The IRAT average score was 77.0% compared to the GRAT group average of 99.5% for the year 2016-2017. Instructors noted great enthusiasm and teamwork. Institutional module evaluation feedback results showed that students were pleased and felt competent analyzing laboratory tests in a bleeding patient. TBL provides a powerful way of teaching students the clinical reasoning approach to a bleeding patient and the appropriate use of laboratory test ordering and analysis. It is enjoyable and interactive and teaches students how to narrow their differential diagnosis by effective laboratory utilization.

International comparative field study evaluating the assay performance of AFSTYLA in plasma samples at clinical hemostasis laboratories

Introduction AFSTYLA (antihemophilic factor [recombinant] single chain) is a novel B-domain truncated recombinant factor VIII (rFVIII). For AFSTYLA, an approximate 50% discrepancy was observed between results of the one-stage (OS) and chromogenic substrate (ChS) FVIII activity assays. An investigation was undertaken to test whether there is a linear relationship between ChS and OS assay results that would allow reliable clinical interpretation of results independent of the assay method used. Aims To provide confidence in future clinical monitoring, this field study investigated the performance of AFSTYLA and a full-length rFVIII (Advate® ) in FVIII activity assays routinely performed in clinical laboratories. Methods The comparison of AFSTYLA and Advate was performed in an international, multicenter and blinded field study of simulated post-infusion samples. The study documented the extent of variability between methods and laboratories and characterized the relationship between the ChS and OS assays. Results Results from 23 laboratories demonstrate that intra and interlaboratory variability in OS assays were similar for both products. When comparing within the OS assay format, there was a similar and reagent-correlated variability in response to different activators for both AFSTYLA and Advate. The OS underestimation was highly predictable and consistent across the complete range of FVIII plasma concentrations. Conclusion Post-infusion plasma AFSTYLA levels can be monitored in patients by the OS and ChS assays. The consistent and predictable difference between the two assay formats provides clinicians with adequate guidance on how to interpret the results of the OS assay using a single conversion factor.

Evaluation of chromogenic factor IX assays by automated protocols

Chromogenic substrate assays (CSA) to measure Factor IX (FIX) have recently become commercially available. However, information on their performance characteristics and use in diagnostic haemostasis laboratories remains limited. To evaluate the Hyphen Biomed (Hyphen) and Rossix FIX CSAs on fully automated coagulation analysers and compare them to the FIX one-stage assay (OSA). This study was conducted in a tertiary referral haemostasis laboratory associated with a haemophilia treatment centre. Automated CSA protocols were adapted to the Sysmex CS2500 (CS2500) and Diagnostica Stago STA-R (STA-R) analysers. Samples assayed were from healthy volunteers, haemophilia B patients and FIX deficient plasma spiked with either plasma derived, recombinant or extended half-life FIX products. Reference intervals for Hyphen and Rossix assays were 73 IU/dL to 164 IU/dL and 73 IU/dL to 168 IU/dL, respectively, on the CS2500 analyser; and 84 IU/dL to 165 IU/dL for the Rossix assay on the STA-R. Repeatability across all method/analyser combinations resulted in CVs ranging from 0.8% to 5.4%. Between run reproducibility gave CVs <6.7% for all method/analyser combinations. In spiked samples, FIX recoveries were mostly within an acceptable limit of 100±25% for BeneFIX® , Rixubis® and Alprolix® with some differences between CSAs. Both commercial factor FIX CSA kits can be adapted for Stago and Sysmex automated coagulation analysers. Reagent cost and workflow practices will need to be considered. These assays are potentially more consistent than OSA in measurement of replacement FIX products in haemophilia B patients.