Handheld point-of-care devices for snakebite coagulopathy: a scoping review.

Venom induced consumption coagulopathy (VICC) is a common complication of snakebite that is associated with hypofibrinogenaemia, bleeding, disability, and death. In remote tropical settings, where most snakebites occur, the 20-minute whole blood clotting test is used to diagnose VICC. Point-of-care (POC) coagulation devices could provide an accessible means of detecting VICC that is better standardised, quantifiable, and more accurate. In this scoping review, the mechanistic reasons that previously studied POC devices have failed in VICC are considered, and evidence-based recommendations are made to prioritise certain devices for clinical validation studies. Four small studies have evaluated a POC international normalised ratio (INR) device in patients with Australian Elapid, Daboia russelii and Echis carinatus envenoming. All of these studies used POC INR devices that rely on a thrombin substrate endpoint, which, unlike laboratory-based INR measurement, is known to underestimate INR in patients with hypofibrinogenaemia. Seventeen commercially available POC devices for measuring INR, activated clotting time (ACT), activated partial thromboplastin time (aPTT), fibrinogen, D-dimer, and fibrin(ogen) degradation products (FDP) have been reviewed. POC INR devices that detect fibrin clot formation, as well as a novel POC device that quantifies fibrinogen were identified, that show promise for use in patients with VICC. These devices could support more accurate allocation of antivenom, reduce the time to antivenom administration, and provide improved clinical trial outcome measurement instruments. There is an urgent need for these promising POC coagulation devices to be validated in prospective clinical snakebite studies.

Pre-analytical variables in haemostasis: Findings from the United Kingdom National External Quality Assessment scheme for Blood Coagulation (UK NEQAS BC) haemolysis exercise.

INTRODUCTION: Haemolysis is considered one of the major contributors of nonconformities and sample rejection in coagulation testing. MATERIALS AND METHODS: Two lyophilized plasmas were distributed to 800 centres registered for prothrombin time (PT), activated partial thromboplastin time (APTT) and either Clauss fibrinogen or thrombin time (TT) in the UK NEQAS BC programme. The same pool of normal plasma was used to prepare both samples, to one of which red blood cell haemolysate was added to mimic haemolysis at 3 g/L haemoglobin concentration. Participants were asked to complete a questionnaire about their laboratory approach to dealing with haemolysed samples, including strategies used to deal with different levels of haemolysis. RESULTS: Results for tests performed did not show great differences between the two samples. It should be noted that artificially constructed haemolysed samples may not behave in the same way as patient samples (ie, may not be commutable). However, the possibility of carrying out a large multicentre study for detection of haemolysis was demonstrated. Inconsistency in practice was observed with 226/551 (41%) of centres indicated they reject haemolysed samples solely on visual checks, and 163 (30%) using initial visual checks with further sample rejection evaluation by analyser flags. Furthermore, 333 (72%) of centres indicated that the level of haemolysis affects sample rejection decisions, while 132 (28%) stated it did not. CONCLUSION: Variability of responses for dealing with haemolysed samples reflects a lack of clear consistency in the pre-analytical area of sample processing.

Effects of Emicizumab on APTT, FVIII assays and FVIII Inhibitor assays using different reagents: Results of a UK NEQAS proficiency testing exercise.

INTRODUCTION: Emicizumab (Hemlibra: Roche Switzerland) is a, humanized, bi-specific monoclonal modified immunoglobulin G4 (IgG4) which binds human FX, FIX and activated FIX (FIXa) to mimic activated FVIII activity. AIM: Evaluate the effects of emicizumab on the APTT, surrogate FVIII activity and FVIII inhibitor results. METHODS: Two samples were provided, one obtained from an emicizumab treated severe haemophilia A patient with FVIII inhibitors and one constructed by in vitro addition of emicizumab using plasma from a severe haemophilia A patient without FVIII inhibitors. An APTT screen, surrogate FVIII and FVIII inhibitor tests were performed on both samples by participating centres. RESULTS: APTT results were below the lower limit of normal range. Chromogenic FVIII assay results with the Hyphen/Biophen human component-based assay gave higher than expected coefficient of variation (CV) results, 38%-40%. The modified one-stage FVIII assay with emicizumab calibrators showed similar results regardless of the APTT reagent. Inhibitor assay median results for sample S18:23 = 1.43 BU (range 0.9-3.0 BU/ml, CV 38%). S18:24 was classified as below the lower limit of detection. CONCLUSION: APTT screens showed a consistent shortening. Unmodified one-stage Factor VIII assay results were remarkably high. APTT-based assays are unsuitable for measurement of coagulation factors or inhibitors in patients treated with emicizumab. Bovine origin chromogenic assays are insensitive to emicizumab and should be used to monitor FVIII levels/FVIII inhibitors in emicizumab treated patients. Product-specific calibrators should be implemented to reduce result variability.

Bridging the gap between point-of-care testing and laboratory testing in hemostasis.

Point-of-care (POC) testing within hemostasis is an expanding field, with the most widely used test being POC international normalized ratio (INR). Many of these devices are being used in a nonlaboratory setting by staff with no laboratory training. In the United Kingdom, external quality assessment (EQA) is provided by the organization UK National External Quality Assessment Scheme for Blood Coagulation (UK NEQAS BC). Participants within the UK NEQAS BC POC INR program are largely based in primary care (77%), with the majority of EQA samples and patients tests being performed by nurses (70%). Many of these centers do not have support from the laboratory staff and may, therefore, not understand the requirement for a robust quality control (QC) system comprising both internal quality control (IQC) and EQA. From data acquired through a questionnaire of these UK NEQAS BC users, we observed that 2% of the centers never perform IQC tests, only 29% perform IQC tests when starting a new batch of test strips, and just 15% carry out IQC with each clinic as recommended by the UK guidelines. The imprecision of EQA tests was greater for POC users than in the UK NEQAS BC hospital laboratory program, with average coefficients of variation for a 2-year period of 11.0 and 7.3%, respectively. This may reflect the handling of EQA samples rather than the imprecision of the method, due to the lack of laboratory training amongst POC staff. POC INR in the UK could greatly benefit from more interaction and support from laboratories to these POC testers.

Problems and solutions in laboratory testing for hemophilia.

A diagnosis of hemophilia A or hemophilia B begins with clinical assessment of the patient and is facilitated by laboratory testing. The influence of the latter on a diagnosis of hemophilia A or hemophilia B is clear-a diagnosis cannot be made without laboratory confirmation of a deficiency of factor FVIII (FVIII) or factor IX (FIX), respectively. Moreover, the degree of hemophilia severity is specifically characterized by laboratory test results. In turn, patient management, including choice and application of therapies, is influenced by the diagnosis, as well as by identification of respective disease severity. An incorrect diagnosis may lead to inappropriate management and unnecessary therapy, and thus to adverse outcomes. Moreover, identification of factor inhibitors in hemophilia will lead to additional and differential treatments, and incorrect identification of inhibitors or inhibitor levels may also lead to inappropriate management. Problems in hemophilia diagnosis or inhibitor detection can occur at any stage in the clinical diagnosis/laboratory interface, from the "pre-preanalytical" to "preanalytical" to "analytical" to "postanalytical" to "post-postanalytical." This report outlines the various problems in laboratory testing for hemophilia and provides various strategies or solutions to overcome these challenges. Although some outlined solutions are specific to the potential errors related to hemophilia, others are general in nature and can be applied to other areas of laboratory hemostasis. Key to improvement in this area is adoption of best practice by all involved, including clinicians, phlebotomists, and laboratories. Also key is the recognition that such errors may occur, and thus that clinicians should assess laboratory test results in the context of their patient's clinical history and follow-up any potential errors, thus avoid misdiagnoses, by requesting repeat testing on a fresh sample.

Quality assurance and quality control of thrombelastography and rotational Thromboelastometry: the UK NEQAS for blood coagulation experience.

Global hemostasis devices are currently being employed in operating rooms to assess the bleeding risk and outcomes for patients undergoing surgery. Two devices currently available are the TEG (Thromboelastograph; Haemoscope Corp., Niles, IL) and the ROTEM (Rotation Thromboelastometer; Pentapharm GmbH, Munich, Germany). Both measure the speed of clot formation, the strength of the clot when formed, and clot fibrinolysis kinetics. The two devices use different parameters so no cross comparisons of results can be made. The devices are usually operated by a member of the operating team and not a laboratory scientist; thus their testing and performance is generally not laboratory controlled, despite quality control being required to ensure reliable results. The UK National External Quality Assessment Scheme (NEQAS) for Blood Coagulation has undertaken a series of exercises evaluating the provision of External Quality Assessment (EQA) material for these devices. A series of four studies have taken place using lyophilized plasmas as the test material. Up to 18 TEG users and 10 ROTEM users have been involved in testing two samples per study, for a total of eight samples tested. The samples were normal plasmas, factor VIII or XI deficient samples, or normal plasmas spiked with heparin. The precision of the tests varied greatly for both devices, with coefficients of variances ranging from 7.1 to 39.9% for TEG and 7.0 to 83.6% for ROTEM. Some centers returned results that were sufficiently different from those obtained by other participants to predict alterations in patient management decisions. Our data indicate that regular EQA/proficiency testing is needed for these devices.

Interlaboratory variation in factor VIII:C inhibitor assay results is sufficient to influence patient management: data from the UK national quality external assessment scheme for blood coagulation.

We report the results of external quality assessment exercises in which 60 to 120 centers performed factor VIII (FVIII) inhibitor testing on a series of samples over a 13-year period. Samples from seven different subjects were distributed for analysis comprising the following: four different subjects with severe hemophilia A with antibodies following replacement therapy, one subject with acquired hemophilia A and antibodies to FVIII, one subject with normal FVIII and an easily detected lupus anticoagulant, and one subject with mild hemophilia A and a difficult-to-detect lupus anticoagulant but without antibodies to FVIII. In all of the surveys the results obtained in different centers analyzing the same sample varied to an extent that would influence patient management decisions. In the UK National External Quality Assessment Scheme surveys reported here, there was considerable interlaboratory variation in the results of FVIII inhibitor testing that did not improve over the survey period. The coefficient of variation of results in different centers was between 33% and 106% in samples from patients with severe congenital hemophilia A. In some cases, results were affected by assay components. For one plasma, the mean FVIII inhibitor results in centers using one source of normal plasma was 3.9 Bethesda unit (BU)/mL compared with a mean of 5.7 BU/mL in centers using a different normal plasma source ( P = 0.04). Our data indicate that the detection of FVIII inhibitors is not the same in different centers, and the degree of variability noted makes it likely that assay variability has contributed to the lack of international consensus in relation to the real incidence of FVIII inhibitors in different clinical settings. Improvements in assay standardization are urgently needed.

Quality assessment in point-of-care coagulation testing.

Point-of-care (POC) testing in the field of hemostasis is rapidly expanding in many countries. This includes use of global tests of hemostasis in operating theaters and especially use of POC monitors for determination of the international normalized ratio (INR) for monitoring oral anticoagulant therapy. Issues related to internal quality control and external quality assessment for these devices are reviewed. Data from external quality assessment exercises involving users of several different POC-INR devices is described, and use of split samples where a patient sample is analyzed by both a POC device and by a conventional laboratory method is described.

Laboratory D-dimer measurement: improved agreement between methods through calibration.

Accurate and precise measurement of plasma D-dimer levels is important in the diagnosis and management of venous thromboembolism. Considerable variability in D-dimer results obtained using different methods has, however, been reported in multicentre studies. This study explored in two separate multicentre exercises the degree of precision amongst laboratory D-dimer measurements, and the degree by which inter-method agreement could be improved using a calibration curve model. The first exercise demonstrated generally good within-centre precision, with 82% of the centres reporting results for two identical but differently coded samples within 10% of each other. However, six centres reported results which would have excluded deep vein thrombosis (DVT) for one sample but failed to exclude DVT for the other, identical sample. In the second exercise, overall between-method precision of D-dimer results for two samples was shown to improve markedly when a calibration model was applied, using the consensus median values obtained by all participants for three "calibration plasmas" to recalculate D-dimer values. For centres reporting results in fibrinogen equivalent units (FEUs), between-centre coefficients of variation (CVs) fell from 25.9% to 11.6% and 22.4% to 7.7%, respectively, for the two samples. For centres reporting in ng/ml, CVs fell from 45.3-21.6% and 40.8-11.6%, respectively. In conclusion, improved harmonisation of D-dimer results by different methods may be achieved by a calibration model and common calibrant plasmas.

Multicentre ISI assignment and calibration of the INR measuring range of a new point-of-care system designed for home monitoring of oral anticoagulation therapy.

The new CoaguChek XS system is designed for use in patient selftesting. It is the successor of the current CoaguChek S system. The detection principle is based on the amperometric measurement of the thrombin activity initiated by starting the coagulation cascade using a human recombinant thromboplastin. This study was performed to assign the International SEnsitivity Index (ISI) to the new test according to the WHO guidelines for thromboplastins and plasmas used to control anticoagulant therapy, and to establish the measuring range of the new system. At four study sites a total of 90 samples of normal donors and 291 samples of warfarin-, phenprocoumon- or acenocoumarol-treated patients were included in the study. The ISI value of the new test was assigned against the human recombinant reference thromboplastin rTF/95 at each site using the samples from stabilized patients in the International Normalized Ratio (INR) range between 1.5 and 4.5 only. The new point-of-care system's measuring range between 0.8 and 8 INR was calibrated against the mean INR of rTF/95 and AD149 using polynomial regression. ISIs were (CV of the slope): Site 1: ISI 0.99 (1.1%); Site 2: ISI 1.02 (2.0%); Site 3: ISI 1.03 (1.1%); Site 4: ISI 1.00 (1.4%). All regression lines calculated from patient-only data pass through the normal donor data points. All CVs of the slopes of the orthogonal regression lines are well below 3%, thus fulfilling the requirements of the WHO guidelines. The mean ISI for the new CoaguChek XS PT Test is 1.01.

Emerging technologies and quality assurance: the United Kingdom National External Quality Assessment Scheme perspective.

The pattern of tests employed and technologies developed within hemostasis laboratories has changed considerably within the last 10 years. These changes have presented challenges to external quality assessment (EQA) providers, including the United Kingdom National External Quality Assessment Scheme (NEQAS). EQA for point-of-care devices used for monitoring oral anticoagulant therapy has focused on provision of suitable material to assess performance of devices designed for capillary blood testing, and on education of a user group not usually trained in laboratory quality control procedures. Development of novel therapeutic agents for hemophilia has presented challenges regarding standardization of assays for monitoring treatment, whereas advances related to laboratory testing and automation have not always been accompanied by improved accuracy and precision. EQA provision has also been shown to be of value in molecular genetic screening tests for thrombophilia, and in highlighting standardization issues related to D-dimer measurement in the exclusion of deep vein thrombosis. The increasing prevalence of screening tests of global hemostasis, such as thrombin generation tests and thromboelastography, presents additional challenges to EQA providers in the attempt to standardize these new and potentially beneficial technologies.

Point-of-care International Normalised Ratios: UK NEQAS experience demonstrates necessity for proficiency testing of three different monitors.

External quality assessment (EQA) or proficiency testing is widely considered to be necessary for International Normalised Ratio (INR) determinations performed in conventional laboratory settings. There is increasing use of near-patient-test (NPT) or point-of-care (POC) INR devices and it is not known whether EQA is also necessary for these monitors. We report here on six years experience of proficiency testing for POC monitors used by health care professionals. Three devices were used by >10 centres who participated in the programme, the CoaguChek (CUC), the CUC-S and the TAS or Rapidpoint Coag. Not all users of the same type of monitor obtained the same INR result when analysing the same plasma sample. For the three monitors the CV of results in different centres was 11-14%. The variation between results in different centres could relate to inappropriately handled proficiency testing material, inaccuracies in the calibration of the system by the manufacturer or deterioration during transport/storage of the test strips. In each survey 10-11% of centres using POC monitors obtained INR results which were >15% different from those in other centres using the same monitors. For hospital laboratories using conventional INR techniques this figure was 12%. The relationship between INR results obtained by users of the Rapidpoint Coag or TAS monitor and results obtained by conventional techniques was not constant over the period of study. During one period INRs with TAS were 13.7% greater than with conventional methods. For the remaining three time periods results were similar. Our data suggest that the variation between INR results determined with three POC monitors show similar variation to that observed in hospital laboratories using conventional methods. Based on our data we recommend that users of these POC monitors participate regularly in an independent external proficiency testing programme.

Laboratory tests for measurement of von Willebrand factor show poor agreement among different centers: results from the United Kingdom National External Quality Assessment Scheme for Blood Coagulation.

In recognition of the importance of von Willebrand factor (vWF) testing in the diagnosis of von Willebrand disease (vWD), the United Kingdom National External Quality Assessment Scheme for Blood Coagulation regularly distributes samples for determination of vWF:antigen (vWF:Ag). Data from 10 separate surveys performed between 2001 and 2005 are reviewed. These include results from ~200 different centers, of which 55% are within the United Kingdom and the remainder are from other countries. During the period of the surveys, the use of immunoelectrophoresis for determination of vWF:Ag practically disappeared and was largely replaced by latex agglutination assays. The coefficient of variation (CV) of results in different centers was approximately 15 to 20% for most vWF:Ag techniques, with CVs of approximately 7% for a fluorescence-based assay. Several different techniques were used for determination of vWF ristocetin cofactor activity (vWF:RCo), all of which were associated with poor agreement among centers as indicated by CVs of 40 to 50%. Several centers calculated the ratio of vWF:Ag/vWF:RCo but with variable success. Ratios compatible with either type 1 or type 2 vWD were obtained on samples from subjects with type 1 vWD, as well as on samples from subjects with genetically confirmed type 2 vWD. Overall, our data show that laboratory testing for vWD remains problematic. It remains to be seen whether newer techniques will offer consistently improved precision.

Variability in factor V:C assays in UK National External Quality Assessment Scheme surveys: there is a need for an international standard.

Severe familial factor V:C deficiency is a rare, recessively inherited coagulation disorder but there is little information in respect of the accuracy and reliability of factor V:C assays that are required for diagnosis and treatment monitoring. We present here the results of three External Quality Assessment exercises in respect of factor V:C assays undertaken by 192--225 participating laboratories performed over a 2-year period. Consistent significant differences were observed between results obtained using different reference plasmas and different thromboplastins. The relationship between results obtained with different reference plasmas was not constant and varied between the surveys. In-house studies confirmed the observation derived from the External Quality Assessment surveys that the choice of commercial reference plasma significantly affects the results of factor V:C assays. These results clearly indicate the necessity for an international standard for factor V:C.