Diagnosis and management of heparin-induced thrombocytopenia: third edition

This guideline updates and widens the scope of the previ-ous British Society for Haematology (BSH) Clinical guide-lines for Diagnosis and Management of Heparin-Induced Thrombocytopenia: Second Edition1 to include functional assays in the diagnosis of heparin-induced thrombocytope-nia (HIT), when to use direct-acting oral anti-coagulants, and the role of intravenous (IV) immunoglobulins and plasma exchange in the management of HIT and spontane-ous HIT.HIT is an immune-mediated, highly pro-thrombotic dis-order of platelet activation caused by pathogenic antibodies against a platelet factor 4 (PF4)­heparin complex. It is the most frequent drug-induced immune thrombocytopenia and may lead to life-threatening thrombosis. There are two distinct forms of HIT: type I, also known as heparin-asso-ciated thrombocytopenia, which is a non-immunological response to heparin treatment, mediated by a direct interac-tion between heparin and circulating platelets causing plate-let clumping or sequestration, and type II, which is immune mediated.

Kidney transplant outcomes in patients with antibodies to human neutrophil antigen 3a.

BACKGROUND: Antibody mediated rejection (ABMR) of kidney transplants has been shown to occur in the absence of a known donor specific antibody to human leucocyte antigen (HLA). Antibodies to the human neutrophil antigen (HNA) system have been detected in kidney transplant recipients and linked to ABMR in the absence of an HLA donor specific antibody (DSA), but there remains limited literature regarding this. METHODS: Case series analysis was carried out examining three cases of HNA-3a antibody positive flow cytometry cross match (FC-XM) from two transplant centres in Scotland. RESULTS: All patients included were female and had been sensitised as a result of pregnancy. One live donor recipient with HNA-3a antibodies identified prior to transplant received ATG induction and has had a good outcome. The remaining two patients received deceased donor transplants. HNA-3a antibodies were indicated following a retrospective flow cytometry crossmatch. Both patients received Basiliximab induction and both have experienced ABMR requiring supplementary immunosuppression. CONCLUSIONS: The predicted rate of HNA-3a antibodies amongst patients awaiting kidney transplant in the UK is <1%. However, with increasing evidence to support a role for HNA-3a antibodies in the development of ABMR there may be value in screening at risk groups to allow for augmented immunosuppression to be considered at the time of kidney transplant.

World human neutrophil antigens investigation survey.

BACKGROUND AND OBJECTIVES: Human neutrophil antigens (HNAs) are categorized into five systems: HNA-1 to HNA-5. Given the importance of neutrophils in immunity, we sought to create awareness of the role of HNA diagnostic services in managing immune neutropenia and transfusion-related acute lung injury. To provide health communities all around the world with access to these services, we conducted a survey to create a directory of these HNA diagnostic services. MATERIALS AND METHODS: An Excel table-based survey was created to capture information on the laboratory's location and was emailed to 55 individuals with known or possible HNA investigation activity. The collected data were then summarized and analysed. RESULTS: Of contacted laboratories, the surveys were returned from 23 (38.2%) laboratories; 17 have already established HNA diagnostic (of them 12 were regular participants of the International Granulocyte Immunobiology Workshop [ISBT-IGIW]), 4 laboratories were in the process of establishing their HNA investigation and the remaining 2 responder laboratories, did not conduct HNA investigations. In established laboratories, investigation for autoimmune neutropenia (infancies and adults) was the most frequently requested, and antibodies against HNA-1a and HNA-1b were the most commonly detected. CONCLUSION: The directory of survey respondents provides a resource for health professionals wanting to access HNA diagnostic services. The present study offers a comprehensive picture of HNA diagnostics (typing and serology), identifying weak points and areas for improvement for the first time. Identifying more laboratories involved in HNA diagnostics with limited access to international societies in the field will globally improve HNA diagnostics.

Identification of the novel HLA-DRB1*15:184 allele by next-generation sequencing.

HLA-DRB1*15:184 differs from HLA-DRB1*15:01:01 by a single base substitution in exon 3 at codon 134.

Human neutrophil antigen 2 sequence-based typing: Joining the hunt for the CD177 answer.

BACKGROUND AND OBJECTIVES: Isoantibodies to human neutrophil antigen 2 (CD177) have been associated with several clinical conditions but to date the molecular basis for altered or non-expression has not been determined. Reliance on phenotyping and crossmatch to investigate these neutropenic clinical cases are inconvenient for the patients and demanding of resources within the laboratory. Therefore, a molecular approach has been introduced to address both issues. MATERIALS AND METHODS: A DNA panel of 100 randomly selected blood donors were collected and supplemented with 18 DNA samples from blood donors previously shown to be CD177 null. All DNA samples were sequence-based typed for all exons and observed polymorphisms recorded. The DNA from two families previously investigated for neonatal alloimmune neutropenia due to CD177 isoantibodies were also analysed. RESULTS: The incidence of CD177 null could be associated with a known exon 7 single-nucleotide polymorphism in 16/21 known CD177 null samples, which is consistent with previously published findings. Two additional mutations that may lead to null expression were also identified, of which one may be novel. In both family investigations, this same mutation could also be observed in the maternal DNA sample. CONCLUSION: Based on these observations, introduction of CD177 genotyping into routine use would identify null expression in over 75% (16/21) of associated cases. In turn, this could significantly reduce the need for supplementary testing and associated inconvenience to patients while permitting increased efficiency of laboratory testing. An added benefit would potentially elucidate other clinically relevant mutations and associated antigenic targets.

A first WHO reference reagent for the detection of anti-human platelet antigen-15b.

BACKGROUND AND OBJECTIVES: Alloantibodies to human platelet antigen-15b (anti-HPA-15b) have been detected in mothers with foetal-neonatal alloimmune thrombocytopenia and in multiply transfused patients. Assays used to detect this antibody, which aids in disease diagnosis, can be unreliable and vary in sensitivity. The objective was to generate a stable, lyophilized anti-HPA-15b preparation and evaluate its suitability as a World Health Organization (WHO) reference reagent for use in the quality control of platelet alloantibody detection assays. Results from an international collaborative study to evaluate the preparation were used to assign a minimum potency at which laboratories can be expected to detect the antibody. MATERIALS AND METHODS: Recalcified plasma containing anti-HPA-15b was aliquotted, lyophilized and coded 18/220. Twenty-five laboratories in 16 countries tested doubling dilutions of the reconstituted material in glycoprotein-specific assays such as the monoclonal antibody-specific immobilization of platelet antigen assay and reported the last positive (or endpoint) dilution. RESULTS: Twenty-four laboratories (96%) detected antibodies with HPA-15b specificity in preparation 18/220. Reported endpoint dilutions were normally distributed with a modal dilution of 1 in 16 and ranged from 1 in 2 to 1 in 128. Only two laboratories (8%) failed to detect anti-HPA-15b at 1 in 8 dilution. CONCLUSIONS: When diluted 1 in 8, most laboratories detected anti-HPA-15b in preparation 18/220 using HPA-15bb platelets but not with HPA-15aa platelets. The participants agreed this to be an appropriate dilution for assignment as the minimum potency. In October 2020, the WHO Expert Committee on Biological Standardization approved 18/220 as an International Reference Reagent.

Identification of the novel HLA-B*42:28 allele by next-generation sequencing.

HLA-B*42:28 differs from HLA-B*42:01:01 by a single base substitution in exon 4 at codon 267.2.

The first reported case of neonatal alloimmune thrombocytopenia due to low-frequency human platelet antigen-6b antibodies in the United Kingdom.

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is a potentially serious clinical condition caused by maternal alloantibodies directed to human platelet antigens (HPA), inherited from the father and expressed on fetal/neonatal platelets. We report a case of an otherwise well, full term child, with a profound thrombocytopenia (33 x 109/L). There was no bleeding or obvious explanation for the low platelet count. Samples were sent for the investigation of NAIT. METHOD: Serological investigations were performed on maternal serum taken at day (D)+4 and D+78. The platelet immunofluorescence test (PIFT) and monoclonal antibody immobilization of platelet antigens (MAIPA) assays were performed with a panel of HPA typed donor platelets and against paternal platelets in a crossmatch. HPA 1-6, -9 and -15 and HLA genotyping was performed by in-house PCR-sequence based typing (SBT) and next generation sequencing (NGS). RESULTS: HPA antibody screening of D+4 maternal serum indicated that platelet-specific antibodies were absent. HPA genotyping of the father and child revealed the presence of the low frequency HPA antigen (LFHPA), HPA-6b, which was absent in the mother. Maternal samples were crossmatched against paternal platelets and were positive by PIFT and glycoprotein (GP) IIb/IIIa and HLA class I in the MAIPA assay. The infant required no platelet transfusion support as the thrombocytopenia resolved spontaneously. DISCUSSION: We conclude that the positive crossmatch reaction was due to anti-HPA-6b alloantibodies. This case further emphasizes the importance of platelet crossmatching and HPA genotyping of LFHPA in cases where there is a high clinical suspicion of NAIT but initial screening is negative.

Pathologic Antibodies to Platelet Factor 4 after ChAdOx1 nCoV-19 Vaccination.

BACKGROUND: The mainstay of control of the coronavirus disease 2019 (Covid-19) pandemic is vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Within a year, several vaccines have been developed and millions of doses delivered. Reporting of adverse events is a critical postmarketing activity. METHODS: We report findings in 23 patients who presented with thrombosis and thrombocytopenia 6 to 24 days after receiving the first dose of the ChAdOx1 nCoV-19 vaccine (AstraZeneca). On the basis of their clinical and laboratory features, we identify a novel underlying mechanism and address the therapeutic implications. RESULTS: In the absence of previous prothrombotic medical conditions, 22 patients presented with acute thrombocytopenia and thrombosis, primarily cerebral venous thrombosis, and 1 patient presented with isolated thrombocytopenia and a hemorrhagic phenotype. All the patients had low or normal fibrinogen levels and elevated d-dimer levels at presentation. No evidence of thrombophilia or causative precipitants was identified. Testing for antibodies to platelet factor 4 (PF4) was positive in 22 patients (with 1 equivocal result) and negative in 1 patient. On the basis of the pathophysiological features observed in these patients, we recommend that treatment with platelet transfusions be avoided because of the risk of progression in thrombotic symptoms and that the administration of a nonheparin anticoagulant agent and intravenous immune globulin be considered for the first occurrence of these symptoms. CONCLUSIONS: Vaccination against SARS-CoV-2 remains critical for control of the Covid-19 pandemic. A pathogenic PF4-dependent syndrome, unrelated to the use of heparin therapy, can occur after the administration of the ChAdOx1 nCoV-19 vaccine. Rapid identification of this rare syndrome is important because of the therapeutic implications.

Human neutrophil antigens: Nature, clinical significance and detection.

Granulocytes are an essential part of both the innate and adaptive immune systems. Human neutrophil antigens (HNAs) are a family of epitopes that are located on glycoproteins that are mostly expressed on human granulocytes. Antibodies that recognize these epitopes have been associated with neutropenia, transfusion complications, haematopoietic stem cell transplant nonengraftment and renal transplant rejection. Currently, there are fourteen recognized HNA alleles across five antigen systems (HNA-1 through HNA-5), the molecular basis of which are located on the genes FCGR3B, CD177, SLC44A2, ITGAM and ITGAL, respectively. Elucidation of the associated genes has permitted the development of testing strategies for HNA typing and aided understanding of the associated epitopes. This review will outline the associated clinical conditions that require HNA investigation and how these are performed in specialized laboratories. Investigations provided are both reactive for patients with a variety of existing or suspected neutropenias and proactive in the testing of blood component donors in order to reduce the potential risk to patients who require transfusion.

Passenger lymphocyte thrombocytopenia due to human platelet antigen 3a antibodies: Case report and review of literature.

We report a case of severe acute thrombocytopenia occurring within days after a cadaveric liver transplant, received from a female patient with aplastic anemia who died of intracranial bleeding. The donor, who was homozygous for the ITGA2B*002 (HPA-3b) gene, had developed human platelet antigen (HPA)-3a antibodies, whereas the recipient was homozygous for the ITGA2B*001 (HPA-3a) gene. Thrombocytopenia responded to an infusion of immunoglobulin G. This is the first report of a passenger lymphocyte syndrome manifesting with thrombocytopenia due to anti-HPA-3a. We review the literature on thrombocytopenia in the setting of PLS and discuss the differential diagnosis.

Rapid, highly accurate and cost-effective open-source simultaneous complete HLA typing and phasing of class I and II alleles using nanopore sequencing.

Accurate rapid genotyping of the genes within the HLA region presents many difficulties because of the complexity of this region. Here we present the results of our proof of concept nanopore-based long read polymerase chain reaction (PCR) solution for HLA genotyping. For 15 HLA anthropology-based samples and 13 NHS Blood and Transplant derived samples 40 ng of genomic DNA underwent long-range PCR for class I and II HLA alleles. Pooled PCR products were sequenced on the Oxford Nanopore MinIoON R9.4.1 flow cell. Sequenced reads had HLA genotype assigned with HLA-LA. Called genotypes were compared with reference derived from a combination of short-read next-generation sequencing, Sanger sequence and/or single-site polymorphism (SSP) typing. For concordance, accuracy was 100%, 98.4%, 97.5% and 95.1% for the first, second, third and fourth fields, respectively, to four field accuracy where it was available, otherwise three field in 28 samples for class I calls and 17 samples for class II calls. Phasing of maternal and paternal alleles, as well as phasing based identification of runs of homozygosity, was shown successfully. Time for assay run was 8 hours and the reconstruction of HLA typing data was 15 minutes. Assay cost was £55 ($80USD)/sample. We have developed a rapid and cost-effective long-range PCR and nanopore sequencing-based assay that can genotype the genes within HLA region to up to four field accuracy, identify runs of homozygosity in HLA, reconstruct maternal and paternal haplotypes and can be scaled from multi-sample runs to a single sample.

HLA-C expression level in both unstimulated and stimulated human umbilical vein endothelial cells is defined by allotype.

Human leukocyte antigens (HLA) are present on the surface of all nucleated cells, with the level of expression dependent on the particular HLA locus, the cell type and cellular activation state. Human umbilical vein endothelial cells (HUVECs) are easily isolated from umbilical cords and may aid our understanding of HLA expression on the vascular endothelium in the setting of transplantation. Endothelial cells on the donor-recipient interface form the barrier between transplanted organs and the host immune system. Increased knowledge of the variation in levels of individual HLA specificities may inform the assessment of transplant risk. HUVECs from 48 full term babies born consecutively following planned caesarean section were isolated, HLA typed and grown on gelatin coated culture wells. Once confluent, cells were stimulated with optimal concentrations of the cytokines TNF-α and IFN-γ for 24 hours and HLA-C expression on both unstimulated and stimulated cells was quantified by flow cytometry using the fluorescent labelled monoclonal antibody DT-9 PE. Unstimulated HLA-C expression varied by over 60% between allotypes (ANOVA, P = .004). Following stimulation, HLA-C levels increased over 15-fold and showed the same variation of expression between allotypes (P < .001). Cell surface HLA-C expression increases between 500% and 3125%, after stimulation for 24 hours. HLA-C level varies between allotypes and cells expressing more HLA-C at baseline tended to have corresponding higher levels of HLA-C following cytokine stimulation (Pearson's correlation coefficient between unstimulated and stimulated expression, P = .002).

HLA expression levels of unstimulated and cytokine stimulated human umbilical vein endothelial cells.

Transplant rejection occurs following recipient recognition of mismatched HLA on donor tissue, but active rejection is dependent not only upon the severity of the T cell or alloantibody response, but also upon the cell surface expression of target HLA molecules. To investigate the variation in HLA expression using a model of endothelium, human umbilical vein endothelial cell (HUVEC) cultures were generated from 48 umbilical cords donated consecutively following planned caesarean section. HUVECs were stimulated using the cytokines tumour necrosis factor alpha and interferon gamma and HLA expression of unstimulated and stimulated cells determined using flow cytometry. HLA-A2, HLA-A3 and HLA-C antigens all showed a modest increase in expression for 12 hours post cell activation, followed by a more pronounced response over the next 24 to 36 hours. Each of these antigens increased by up to 40 times over unstimulated levels and in addition cells homozygous for specific HLA antigens on average had twice the amount of antigen expressed compared with cells heterozygous for that antigen, both when unstimulated and following cytokine stimulation. Cell activation is an important consideration in the assessment of transplant risk and may help progress towards understanding why rejection does not always occur in the presence of significant donor specific antibody. This data also confirms guidelines for transplantation, which recommend doubling the specific antibody level when considering immunological risk for homozygous donors.

Factors affecting HLA expression: A review.

The detection and semiquantitative measurement of circulating human leucocyte antigen (HLA)-specific antibodies is essential for the management of patients before and after transplantation. In addition, the pretransplant cross-match to assess the reactivity of recipient HLA antibody against donor lymphocytes has long been the gold standard to prevent hyperacute rejection. Whilst both of these tests assume that recipient HLA-specific antibody is the only variable in the assessment of transplant risk, this is not the case. Transplant immunologists recognize that some HLA antigens are expressed at levels a magnitude lower than others (e.g., HLA-C, HLA-DQ), but within loci, and between different cell types there are many factors that influence HLA expression in both resting and activated cells. HLA is not usually expressed without the specific promoter proteins NLRC5, for HLA class I, and CIITA, for class II. The quantity of HLA protein production is then affected by factors including promoter region polymorphisms, alternative exon splice sites, methylation and microRNA-directed degradation. Different loci are influenced by multiple combinations of these control mechanisms making prediction of HLA regulation difficult, but an ability to measure the cellular expression of each HLA antigen, in conjunction with knowledge of circulating HLA-specific antibody, would lead to a more informed algorithm to assess transplant risk.

Neonatal alloimmune thrombocytopenia due to a new alloantigen Bl(a) defined by an Asp458Gly substitution in GPIIIa.

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) commonly arises due to antibodies against a small number of well-defined human platelet antigens (HPAs). A minority of NAIT cases occur due to maternal immunization against low-frequency polymorphisms in platelet glycoprotein that result in new immunogenic epitopes. Antibodies to these novel epitopes can be detected by the incubation of maternal serum with paternal platelets and is usually performed after initial investigation using HPA-typed panel platelets has failed to provide evidence of NAIT. STUDY DESIGN AND METHODS: The propositus and the parents from a case of suspected neonatal alloimmune thrombocytopenia (NAIT) were investigated using serologic and molecular techniques to detect and identify relevant platelet-specific antibodies and for HPA typing. Calculations of molecular dynamics were undertaken to explore potential variations in the molecular structure. RESULTS: Maternal antibodies were detected that were reactive only in crossmatch with paternal platelets using the platelet immunofluorescence test (PIFT) and a GPIIb/IIIa monoclonal antibody immobilization of platelet antigen (MAIPA) assay. In the propositus and father, a novel mutation c.1373 A > G was found in exon 10 of ITGB3 resulting in the substitution of an aspartic acid for a glycine (p.Asp458Gly). Recombinant GPIIIa glycoprotein mutated to contain the novel mutation and expressed in HEK293 cells with GPIIb was also specifically recognized by maternal antibodies. Calculations of molecular dynamics identified that the mutation was in a structurally constrained site. CONCLUSION: This case describes a low-frequency platelet antigen (Asp458Gly) that defines a further alloantigenic target in NAIT. The case emphasizes the role of the platelet crossmatch as the single most useful tool to establish evidence of immunization of low-frequency platelet glycoprotein polymorphisms. A crossmatch should always be performed where there is strong clinical evidence of NAIT but initial laboratory investigations are not confirmatory.

Human platelet antigen (HPA)-specific immunoglobulin M antibodies in neonatal alloimmune thrombocytopenia can inhibit the binding of HPA-specific immunoglobulin G antibodies.

BACKGROUND: A term baby with unexplained thrombocytopenia and a platelet (PLT) count of 14 × 109 /L (maternal PLT count was 200 × 109 /L) was investigated for neonatal alloimmune thrombocytopenia. STUDY DESIGN AND METHODS: Serologic investigations were performed using the PLT immunofluorescence test (PIFT), monoclonal antibody immobilization of PLT antigens (MAIPA), and a bead-based assay (BBA) with maternal sera taken up to 56 days postdelivery. One serum sample was also separated into "immunoglobulin (Ig)M-rich" and "IgM-depleted" fractions and tested for PLT-specific antibodies. The family was genotyped for HPA. RESULTS: HPA-3a-specific IgM antibodies were detected in the PIFT and confirmed in the BBA. PLT-specific IgG HPA-3a antibodies were not detected in the MAIPA assay and BBA in the initial sample but were detected in both techniques in subsequent serum samples. Testing of IgM-rich and IgM-depleted fractions in the MAIPA assay revealed that IgG antibody binding of the IgM-depleted fraction was inhibited by approximately 50% when it was reconstituted with the IgM-rich fraction suggesting that the IgM antibodies blocked the binding of the IgG antibodies. This effect was not observed when the IgM-depleted fraction or untreated serum was diluted with elution buffer. Incompatibility for HPA-3 was identified between the mother and the infant. The infant received one HPA-1a, -5b negative neonatal PLT transfusion, and one random PLT transfusion, with satisfactory outcomes. Both units were later found to be HPA-3b3b. CONCLUSION: HPA-3a IgM antibodies can inhibit PLT-specific HPA-3a IgG antibodies in the MAIPA assay.

A reliable method for avoiding false negative results with Luminex single antigen beads; evidence of the prozone effect.

Luminex single antigen bead (SAB) assays have become an essential tool in monitoring the status of antibody to the Human Leucocyte Antigen (HLA) of patients both before and after transplantation. In addition SAB data is used to aid risk stratification to assess immunological risk of humoral rejection in solid organ transplantation (CTAG/BTAG guidelines) [1]. Increasingly laboratories are reporting false negative results at high antibody titre due to a prozone effect. Here we report a case study where the prozone effect led to a false negative antibody result that could have resulted in adverse outcome. We describe a method to reliably remove the prozone effect through heat inactivation and the addition of Ethylenediaminetetraacetic acid (EDTA) to the Luminex wash buffer.

Further observations on the clinical significance and inheritance of the low-frequency platelet antigen HPA-28bw.

BACKGROUND: Most recently described human platelet antigens (HPAs) have been low-frequency polymorphisms identified in cases of fetomaternal alloimmune thrombocytopenia (FMAIT). There is limited opportunity to study the clinical significance or different antenatal management strategies in cases involving low-frequency HPA antibodies because many are single pregnancies. We have previously described a low-frequency platelet (PLT) antigen, HPA-28bw, implicated in FMAIT in two of the three infants in the same family. This report describes the outcome of an additional two pregnancies in this family. STUDY DESIGN AND METHODS: The fourth and fifth pregnancies in a HPA-28bw-alloimmunized mother with a heterozygous partner were investigated to determine the risk of FMAIT. The presence of anti-HPA-28bw was assessed by paternal crossmatch studies. Prenatal HPA genotyping of amniocytes was performed to inform antenatal management. RESULTS: GPIIb/IIIa antibodies reactive only with paternal PLTs were detected. These antibodies had been previously identified as HPA-28bw specific using recombinant GPIIb glycoprotein mutated to contain the HPA-28bw (V740L) mutation. The fetus in the fourth pregnancy did not inherit the HPA-28bw mutation, no antenatal management was required, and the baby had a normal PLT count. The fetus in the fifth pregnancy did inherit the HPA-28bw mutation. The mother received IVIG (2 g/kg/week) and prednisolone during pregnancy, and the baby was born with a normal PLT count. CONCLUSION: Study of this family has provided a unique opportunity to assess the clinical significance of antibodies against the low-frequency PLT antigen (HPA-28bw) during five pregnancies and to compare the outcomes of different antenatal treatments.