Comparison of Thrombophilia Assay Results for the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee Plasma Standard from Different External Quality Assessment Providers-for the External Quality Assurance in Thrombosis and Haemostasis Group.

External quality assessment (EQA) is used to evaluate laboratory performance in tests of hemostasis; however, some esoteric tests are performed by too few centers in any one EQA program to allow valid statistical assessment. To explore the feasibility of pooling data from several EQA providers, an exercise was carried out by the External Quality Assurance in Thrombosis and Haemostasis group, using the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee (SSC) plasma standard for thrombophilia screening assays. Six EQA providers took part in this exercise, distributing the SSC plasma standard as a "blinded" sample to participants for thrombophilia tests between November 2020 and December 2021. Data were collected by each provider, anonymized, and pooled for analysis. Results were analyzed as overall results from each EQA provider, and by kit/method-specific comparisons of data from all providers pooled together. For each parameter, median results and range were determined. Over 1,250 sets of data were returned in the six EQA programs. The overall medians (all data pooled) were <4% of the assigned values for each parameter with the exception of protein C activity by clot-based assay. Method-related differences in median results were observed for free protein S antigen and protein S activity-a pattern seen across data from the different EQA providers. Antithrombin antigen results reported in mg/dL provided an example where small numbers of results for a single EQA provider may be supplemented by pooling data from multiple providers with good agreement seen among results reported by the different EQA providers. This study demonstrated that a multicenter EQA provider collaboration can be carried out and demonstrated benefit for assays with smaller number of participants. In addition, results showed good agreement with the assigned values of the SSC plasma standard. Further exercises for tests performed by only small numbers of laboratories can be planned.

Internal Quality Control in Hemostasis Assays.

Internal quality control (IQC) for routine and specialist hemostasis testing represents a mandatory requirement for assays offered by clinical laboratories under International Organization for Standardization, Code of Federal Regulations, and Clinical and Laboratory Standards Institute standards. The underlying principle is that regular IQC audits the analytical performance of automated, semiautomated, and manual methods. This review investigates IQC practices, including benefits, limitations, frequency per time period or batch, sources of material used, primary supplier, third party or in-house, plus troubleshooting when IQC falls outside acceptance criteria. To assess IQC practice, the UK National External Quality Assessment Scheme (NEQAS) Blood Coagulation distributed a questionnaire to 1,200 participants enrolled in our scheme that collected details of the local practices for IQC testing. We received returns from 127 centers that described their local practices for the frequency of IQC, the type of IQC material employed, acceptance criteria for IQC data, and troubleshooting protocols for IQC failures. The data collected as part of an NEQAS BC questionnaire confirmed that all the participants returning answers to the questionnaire meet the standards for regular IQC testing for the hemostasis assays they perform.

Anti-PF4 testing for vaccine-induced immune thrombocytopenia and thrombosis (VITT): Results from a NEQAS, ECAT and SSC collaborative exercise in 385 centers worldwide.

BACKGROUND: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following the administration of the AstraZeneca (AZ) ChAdOx1 nCOV-19 vaccine is a well recognized clinical phenomenon. The associated clinical and laboratory features have included thrombosis at unusual sites, thrombocytopenia, raised D-dimer levels and positivity for immunoglobulin G (IgG) anti-platelet factor 4 (PF4) antibodies. OBJECTIVES: A collaborative external quality assessment (EQA) exercise was carried out by distributing five lyophilized samples from subjects with VITT and one from a healthy subject to 500 centers performing heparin-induced thrombocytopenia (HIT) testing. METHODS: Participating centers employed their locally validated testing methods for HIT assays, with some participants additionally reporting results for VITT modified assays. RESULTS: A total of 385 centers returned results for anti-PF4 immunoassay and functional assays. The ELISA assays used in the detection of anti-PF4 antibodies for the samples distributed had superior sensitivities compared with both the functional assays and the non-ELISA methods. CONCLUSION: ELISA-based methods to detect anti PF4 antibodies have a greater sensitivity in confirmation of VITT compared with functional assays regardless of whether such functional assays were modified to be specific for VITT. Rapid immunoassays should not be employed to detect VITT antibodies.

External quality assessment for one-stage and chromogenic factor IX assays in samples containing Alprolix (rFIXFc) or Idelvion (rIX-FP) and evidence that UK National External Quality Assessment Scheme for blood coagulation samples are commutable with patient samples.

INTRODUCTION: There may be clinically relevant differences between results of different FIX assays in samples containing extended half life FIX concentrates requiring regular surveillance of assay results through proficiency testing exercises. Control materials used in proficiency testing must be commutable, that is have the same inter-assay properties as those demonstrated by authentic clinical samples when measured by different analytical methods. METHODS: We assessed relationships between results with different FIX assays and commutability of UK National External Quality Assessment Scheme (NEQAS) materials containing rIX-FP (Idelvion) or rFIXFc (Alprolix) by comparing results obtained using widely used one-stage and chromogenic assays during a proficiency testing exercise with results obtained when analysing a series of individual patient samples using the same assay systems. NEQAS samples prepared by addition of either Idelvion or Alprolix to FIX deficient plasma were sent to 76 haemophilia centres in Europe. A total of 18 Idelvion and 22 Alprolix patient samples were assayed in a single centre. Two chromogenic and two one-stage assays were compared. RESULTS: The pattern of results obtained for NEQAS samples and patient samples was similar. In all cases, the NEQAS sample data point was within the scatter of patient sample data in plots of patient sample results showing one-stage assay results using Synthasil or Actin FS plotted against chromogenic assay results with Biophen or Rox chromogenic FIX kits. CONCLUSION: This indicates that the NEQAS samples containing Idelvion or Alprolix were commutable and therefore suitable for use in proficiency testing exercises.

Anti-PF4 testing for vaccine-induced immune thrombocytopenia and thrombosis and heparin induced thrombocytopenia: Results from a UK National External Quality Assessment Scheme exercise April 2021.

BACKGROUND: Vaccine-induced immune thrombocytopenia and thrombosis (VITT) following the administration of the AstraZeneca (AZ) ChAdOx1 nCOV-19 vaccine has recently been reported. The associated clinical and laboratory features have included thrombosis at unusual sites, thrombocytopenia, and raised D-dimers with positivity for IgG anti-platelet factor 4 (PF4) antibodies. OBJECTIVES: A UK National External Quality Control Assessment Scheme external quality control exercise was carried out by distributing liquid and lyophilized samples from a subject with VITT, a pool of samples from subjects with classical heparin-induced thrombocytopenia (HIT), and a non-VITT/non-HIT case to 85 centers performing HIT testing. METHODS: Participating centers employed their locally validated testing methods for HIT assays. RESULTS: The lyophilized and liquid samples were found to be commutable for the ELISA assays used in the detection of anti-PF4 antibodies. The Aeskulisa, Stago, Hyphen, and LIFECODES anti-PF4 ELISA assays successfully detected the VITT antibody, whereas the Acustar HIT, Werfen LIA, and the Stago STIC assays did not. CONCLUSION: It is important that clinical and laboratory teams are aware of the limitations of some anti-PF4 assays when using them to aid diagnosis of VITT syndrome.

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.

Clotting and chromogenic factor VIII assay variability in post-infusion and spiked samples containing full-length recombinant FVIII or recombinant factor VIII Fc fusion protein (rFVIIIFc).

INTRODUCTION: Variability in FVIII measurement is a recognized problem. There are limited data for samples containing recombinant Factor VIII Fc fusion protein (rFVIIIFc). Many studies use samples for which factor concentrate has been spiked into FVIII deficient plasma in vitro. This approach requires validation. AIM/METHODS: Four samples were distributed in a UK National External Quality Assessment Scheme for Blood Coagulation (NEQAS BC) survey. One contained Advate (full-length recombinant FVIII) (rFVIII) added to FVIII deficient plasma, one was from a severe haemophilia A patient after infusion of Advate, one was prepared by addition of rFVIIIFc (marketed as Elocta/Eloctate) to FVIII deficient plasma and the fourth was collected from a severe haemophilia A patient following rFVIIIFc (Eloctate) infusion. Fifty-three haemophilia centres (UK and Scandinavia) performed one-stage FVIII assays and 27 performed chromogenic FVIII assays. RESULTS/CONCLUSIONS: One-stage assays gave significantly lower results than chromogenic assays by 7% (P < 0.01) and 13%(P < 0.001) for post-Advate and Advate spiked samples, and by 22% (P < 0.001) and 23% (P < 0.001) for post-rFVIIIFc and rFVIIIFc spiked samples. The interlaboratory variation was similar for all samples, with CVs of 12%-16% (chromogenic) and 10%-13% (one stage). The data indicate that either product can be safely monitored by one-stage or chromogenic assay. Spiked samples behaved in a similar way to post-infusion samples for both products and could be substituted for post-infusion samples for use in proficiency testing exercises (ie, samples were commutable).

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.

Point of Care INR testing devices: performance of the Roche CoaguChek XS and XS Plus in the UK NEQAS BC external quality assessment programme for healthcare professionals: four years' experience.

BACKGROUND: Vitamin K antagonists have been used for many decades and have been traditionally monitored by the measurement of the International Normalised Ratio (INR) in the laboratory. Introduction of Point of Care (POC) testing devices to measure INR has resulted in many tests being undertaken in primary care. External Quality Assessment (EQA) of these POC devices is recommended to ensure accuracy and reliability of INR results outside a laboratory setting. AIM: To assess the quality of INR results for users of two POC devices (CoaguChek XS and CoaguChek XS Plus) over a four-year period. METHODS: Four surveys (two samples) were sent in each 12-month period. The median INR value of each sample was calculated and the percentage deviation from this median determined. Any results greater than 15% from the median were considered to be outside consensus which indicated a possible problem within the testing system. RESULTS: Variability of INR results in this UK National External Quality Assessment Scheme (NEQAS) programme was comparable to that in the UK NEQAS EQA programme for laboratory INR testing. Occurrence of persistent problems was lower in the POC programme than the laboratory programme. CONCLUSIONS: Utilisation of an EQA programme for POC devices in primary care is feasible and necessary. Our data suggest for those health professionals using EQA, the reliability and accuracy of INR testing matches the quality of laboratory testing.

SPIN (Scottish Pregnancy Intervention) study: a multicenter, randomized controlled trial of low-molecular-weight heparin and low-dose aspirin in women with recurrent miscarriage.

To assess whether treatment with enoxaparin and low-dose aspirin, along with intensive pregnancy surveillance, reduces rate of pregnancy loss compared with intensive pregnancy surveillance alone in women with history of 2 or more consecutive previous pregnancy losses, a parallel group, multicenter, randomized controlled trial was performed in the United Kingdom and New Zealand. Participants (n = 294) presenting for initial antenatal care at fewer than 7 weeks' gestation with history of 2 or more consecutive previous pregnancy losses at 24 or fewer weeks' gestation and no evidence of anatomic, endocrine, chromosomal, or immunologic abnormality were randomly assigned to receive either enoxaparin 40 mg subcutaneously and 75 mg of aspirin orally once daily along with intense pregnancy surveillance or intense pregnancy surveillance alone from random assignment until 36 weeks' gestation. The primary outcome measure was pregnancy loss rate. Of the 147 participants receiving pharmacologic intervention, 32 (22%) pregnancy losses occurred, compared with 29 losses (20%) in the 147 subjects receiving intensive surveillance alone, giving an odds ratio of 0.91 (95% confidence interval, 0.52-1.59) of having a successful pregnancy with pharmacologic intervention. Thus, we observed no reduction in pregnancy loss rate with antithrombotic intervention in pregnant women with 2 or more consecutive previous pregnancy losses. The trial was registered at http://www.controlled-trials.com as ISRCTN06774126.

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.

The GOAL study: a prospective examination of the impact of factor V Leiden and ABO(H) blood groups on haemorrhagic and thrombotic pregnancy outcomes.

Factor V Leiden (FVL) and ABO(H) blood groups are the common influences on haemostasis and retrospective studies have linked FVL with pregnancy complications. However, only one sizeable prospective examination has taken place. As a result, neither the impact of FVL in unselected subjects, any interaction with ABO(H) in pregnancy, nor the utility of screening for FVL is defined. A prospective study of 4250 unselected pregnancies was carried out. A venous thromboembolism (VTE) rate of 1.23/1000 was observed, but no significant association between FVL and pre-eclampsia, intra-uterine growth restriction or pregnancy loss was seen. No influence of FVL and/or ABO(H) on ante-natal bleeding or intra-partum or postpartum haemorrhage was observed. However, FVL was associated with birth-weights >90th centile [odds ratio (OR) 1.81; 95% confidence interval (CI(95)) 1.04-3.31] and neonatal death (OR 14.79; CI(95) 2.71-80.74). No association with ABO(H) alone, or any interaction between ABO(H) and FVL was observed. We neither confirmed the protective effect of FVL on pregnancy-related blood loss reported in previous smaller studies, nor did we find the increased risk of some vascular complications reported in retrospective studies.

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.

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.

Exogenous sex hormones and thrombophilia.

Worldwide, hundreds of millions of women use exogenous estrogens in contraceptives or for postmenopausal hormone replacement. Exogenous estrogens increase the risk for venous and arterial thrombosis. This article reviews the use of exogenous sex hormones in women with thrombophilia.

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.