A systems serology approach to identifying key antibody correlates of protection from cerebral malaria in Malawian children.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins are expressed on the surface of infected erythrocytes, mediating parasite sequestration in the vasculature. PfEMP1 is a major target of protective antibodies, but the features of the antibody response are poorly defined. METHODS: In Malawian children with cerebral or uncomplicated malaria, we characterized the antibody response to 39 recombinant PfEMP1 Duffy binding like (DBL) domains or cysteine-rich interdomain regions (CIDRs) in detail, including measures of antibody classes, subclasses, and engagement with Fcγ receptors and complement. Using elastic net regularized logistic regression, we identified a combination of seven antibody targets and Fc features that best distinguished between children with cerebral and uncomplicated malaria. To confirm the role of the selected targets and Fc features, we measured antibody-dependent neutrophil and THP-1 cell phagocytosis of intercellular adhesion molecule-1 (ICAM-1) and endothelial protein C (EPCR) co-binding infected erythrocytes. RESULTS: The selected features distinguished between children with cerebral and uncomplicated malaria with 87% accuracy (median, 80-96% interquartile range) and included antibody to well-characterized DBLß3 domains and a less well-characterized CIDRγ12 domain. The abilities of antibodies to engage C1q and FcγRIIIb, rather than levels of IgG, correlated with protection. In line with a role of FcγRIIIb binding antibodies to DBLß3 domains, antibody-dependent neutrophil phagocytosis of ICAM-1 and EPCR co-binding IE was higher in uncomplicated malaria (15% median, 8-38% interquartile range) compared to cerebral malaria (7%, 30-15%, p < 0.001). CONCLUSIONS: Antibodies associated with protection from cerebral malaria target a subset of PfEMP1 domains. The Fc features of protective antibody response include engagement of FcγRIIIb and C1q, and ability to induce antibody-dependent neutrophil phagocytosis of infected erythrocytes. Identifying the targets and Fc features of protective immunity could facilitate the development of PfEMP1-based therapeutics for cerebral malaria.

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.

Pathogenicity and virulence of malaria: Sticky problems and tricky solutions.

Infections with Plasmodium falciparum and Plasmodium vivax cause over 600,000 deaths each year, concentrated in Africa and in young children, but much of the world's population remain at risk of infection. In this article, we review the latest developments in the immunogenicity and pathogenesis of malaria, with a particular focus on P. falciparum, the leading malaria killer. Pathogenic factors include parasite-derived toxins and variant surface antigens on infected erythrocytes that mediate sequestration in the deep vasculature. Host response to parasite toxins and to variant antigens is an important determinant of disease severity. Understanding how parasites sequester, and how antibody to variant antigens could prevent sequestration, may lead to new approaches to treat and prevent disease. Difficulties in malaria diagnosis, drug resistance, and specific challenges of treating P. vivax pose challenges to malaria elimination, but vaccines and other preventive strategies may offer improved disease control.

External Quality Assessment Data for Investigation of von Willebrand Disease: Focus on Relative Utility of Contemporary Functional von Willebrand Factor Assays. The United Kingdom National External Quality Assessment Scheme (UK NEQAS) Experience.

Von Willebrand disease (VWD) is one of the most common hereditary bleeding disorders. Effective management of patients and their families depends on accurate diagnosis and subtype classification, and quality assurance including participation in proficiency testing programs is essential to ensure the accuracy of the panel of assays required to achieve this diagnosis. We report here findings from recent external quality assessment (EQA) exercises, as well as from a questionnaire about diagnostic practices employed by centers in the United Kingdom National Quality Assessment Scheme (UK NEQAS) performing von Willebrand factor (VWF) assays. Plasma samples from donors with VWD, "normal" donors, the International Society for Thrombosis and Haemostasis Scientific Subcommittee (ISTH SSC) plasma standard, and whole blood samples were sent to participants in the UK NEQAS BC program for VWF investigation. Calibration of lot#5 of the ISTH SSC plasma standard was shown to give improved comparability between the recovered value from an EQA exercise and the assigned potency for VWF activity assays. Diagnostic accuracy and precision amongst UK NEQAS participants was good, with an average 99% of centers reporting the correct interpretation for normal, type 1 and type 2 VWD samples, 100% diagnostic accuracy for centers performing FVIII binding assays, and good agreement amongst centers performing multimeric analysis. Genetic analysis of the VWF gene by specialist centers demonstrated errors in the genotyping process in one center, but also demonstrated failings in the interpretation of results in other centers. Despite evidence of good laboratory accuracy and precision in their assays, a questionnaire identified marked variation in diagnostic criteria employed, underlining the importance of guidelines to support the diagnosis of VWD.

Analysis of Antibody Reactivity to Malaria Antigens by Microsphere-Based Multiplex Immunoassay.

Protein multiplex assays enable serological analysis of multiple target proteins simultaneously, using relatively small volumes of patient sample per assay. Here we present a detailed protocol to analyze antibody reactivity to malaria antigens by microsphere-based multiplex assay (xMAP technology). This method involves coupling of recombinant proteins to fluorescently labeled microspheres; simultaneous exposure of all microspheres to plasma or sera, and detection of antigen-specific antibodies with a fluorescent labeled anti-human Fc region antibody. In addition to total IgG, this assay can be adapted to measure multiple properties of the antibody Fc region, including subclass, isotype, and Fc receptor or complement C1q binding.

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.

Mental health among elite sportspeople: Lessons for medical education.

Leading sportspeople across 2021, such as Simone Biles (US gymnast), Naomi Osaka (Japanese tennis player) and Ben Stokes (English cricketer), have talked openly about the pressure of performing on the highest stage, including the challenge of managing mental health when engaged in elite competition. The withdrawal of Simone Biles midway through the women's team competition at the Tokyo 2020 Olympic Games propelled what was seemingly a debate within sport, into what is increasing becomingly wider societal conversation around mental health. The stories of sportspeople struggling to perform at the highest level with mental health contributing to their difficulties, has inevitably prompted much reflection within medical education among teachers and students alike, about parallels in our domain around assessment, feedback and support. The stories demonstrate that mental health problems affect everyone, including those who are at their peak physically, and those who are among the finest on the planet in terms of physical and sporting ability. The same is true within medical education of our students, who are also our future doctors. However, curriculum conversations about assessment, feedback and student support may not be as student-centred as they could be, or perhaps as they should be, with mental health possibly still being a taboo-subject or something associated with stigma within medical education. Here is another opportunity for medical education to learn from other disciplines, such as sports psychology, and now is the time for taking and applying those lessons: not just those around improving technical performance, but those around properly caring, being compassionate, and looking after our future Olympian equivalents.

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.

Factor VIII/IX inhibitor testing practices in the United Kingdom: Results of a UKHCDO and UKNEQAS national survey.

INTRODUCTION: Inhibitor formation is the greatest challenge facing persons with haemophilia treated with factor concentrates. The gold standard testing methodologies are the Nijmegen-Bethesda assay (NBA) for FVIII and Bethesda assay (BA) for FIX inhibitors, which are affected by pre-analytical and inter-laboratory variability. AIMS: To evaluate inhibitor testing methodology and assess correlation between self-reported and actual methodology. METHODS: Methodology was evaluated using a survey distributed alongside a UK National External Quality Assessment Service Blood Coagulation external quality assurance (EQA) exercise for FVIII and FIX inhibitor testing. RESULTS: Seventy four survey and EQA exercise responses were received (response rate 63.2%), with 50 paired survey/EQA results. 47.1% (33/70) reported using the NBA and 42.9% (30/70) the BA for FVIII inhibitor testing. Review of FVIII inhibitor assay methodology demonstrated discrepancy (self-reported to actual) in 64.3% (BA reporting) and 27.6% (NBA reporting). Pre-analytical heat treatment was used by 32.4%, most commonly 56°C for 30 minutes. Assay cut-offs of 0.1-1.0 BU/mL were reported. EQA samples (acquired FVIII and congenital FIX) demonstrated titres and coefficients of variation (CV) of 3.1 BU/mL (0.7-15.4 BU/mL; CV = 43%) and 18.0 BU/mL (0-117 BU/mL; CV = 33%), respectively. No significant assay or laboratory factors were found to explain this variance, which could have resulted in change in management for 6 patients (5 misclassified high-titre FVIII inhibitors and 1 false negative for a FIX inhibitor). CONCLUSIONS: Heterogeneity was seen at each stage of assay methodology. No assay-related factors were found to explain variation in inhibitor titres. Further standardization is required to improve inhibitor quantification to guide patient care.

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.

History of the West of Scotland Haemophilia Centre, Glasgow, 1950-2019.

Over 70 years, the West of Scotland Haemophilia Centre in the UK has played a leading role in research, education and training. Its staff studied the natural history of haemophilias, their complications, and their treatment complications, pioneered the use of fibrinolytic inhibitors to reduce the risk of receiving a blood transfusion and developed national audit. Collaborations across Scotland with other haemophilia centres and the Scottish National Blood Transfusion Service progressed self-suffi ciency in NHS-produced factor concentrates, heat treatments to prevent HIV and hepatitis transmission, and finally, replacement of human by recombinant factor concentrates.