Platelet Antigens Research Articles

Blood group genotyping of blood donors: validation of a highly accurate routine method.

In the past century, blood group determination using serology has been the standard method. Now, molecular methods are gaining traction, which provide additional and easily accessible information. Here we designed and validated a high-throughput extended genotyping setup. We developed 35 competitive allele-specific polymerase chain reaction assays for genotyping of blood donors. Samples from 1034 Danish blood donors were genotyped, and 45,314 red blood cell antigens and 6148 platelet antigens were predicted. Predicted phenotypes were compared with 16,119 serologic phenotypes. We found 62 discrepancies of which 43 were due to serology. After exclusion of the discrepancies caused by serology, the accuracy of genotyping was 99.9%. Of 17 discrepancies caused by the genotype, three were incorrect antigen-negative predictions and could potentially, as the solitary analysis, have caused an adverse transfusion reaction. We have established a robust and highly accurate blood group genotyping system with a very high capacity for screening blood donors. The system represents a significant improvement over the former serotyping-only procedure. Almost all new technology in medicine incurs increased costs, but the presented efficient genotyping system is a rare example of a significant qualitative and quantitative technologic progress that is also more cost-efficient than previous technologies.

Novel method for simultaneously detecting HPA and HLA antibodies using Luminex microbeads

BackgroundAlloantibodies against human platelet antigens (HPAs) and human leukocyte antigen (HLA) are implicated in several immune-mediated platelet disorders. Detection of these antibodies is crucial in the diagnosis and management of these disorders. The aim of this study was to establish a novel method to simultaneously detect HPA-1, HPA-2, HPA-3, HPA-5 and HLA antibodies with Luminex microbeads technology.MethodsMonoclonal antibodies specific for platelet glycoproteins and HLA class I molecules were separately coupled to the Luminex microbeads. We validated specificity of the Luminex platform using the following antibodies: anti-HPA-1a, anti-HPA-2b, anti-HPA-3a, anti-HPA-5a, and anti-HLA positive samples. Sensitivity was evaluated by a serial dilution (from neat to 1/1024) using the following antibodies: anti-HPA-1a, anti-HPA-3a standard sera, and anti-HPA-5a positive serum. Serum samples were collected from 36 neonatal alloimmune thrombocytopenia (NAIT) patients suspected of having HPA or HLA antibodies and 8 samples from ISBT platelet workshop were tested using the Luminex assay.ResultsThe Luminex assay detected all antibodies tested from the known samples. The sensitivities of the Luminex assay detecting anti-HPA-1a, anti-HPA-3a, and anti-HPA-5a were 1:512, 1:64, and 1:128, respectively. The sensitivity of Luminex assay was higher than monoclonal antibody immobilization of platelet antigen method (MAIPA). No cross-reactivity was observed in the samples containing multi-platelet antibodies or mixture antibodies against HPA and HLA. The results of 44 samples with platelet disorders were consistent with those of the same samples processed with the MAIPA assay.ConclusionLuminex microbeads coupled with monoclonal antibodies could be successfully used to detect HPA and HLA antibodies simultaneously, especially with high sensitivity in detecting HPA antibodies.

Immune Thrombocytopenia-A Disease or a Group of Disorders? Where Do We Stand in 2019?

Immune thrombocytopenia (ITP) is an autoimmune disease that is characterized by a significant reduction in the number of circulating platelets and frequently associated with bleeding.[1] Although the pathogenesis of ITP is still not completely elucidated, it is largely recognized that the low platelet count observed in ITP patients is due to multiple alterations of the immune system leading to increased platelet destruction and impaired megakaryocytopoiesis and thrombocytopoiesis.[2] The clinical manifestations and the patients' response to different treatments are very heterogeneous suggesting that ITP is rather a group of disorders sharing common characteristics, namely, loss of immune tolerance toward platelet (and megakaryocyte) antigens[3] and increased bleeding tendency.[4]

Plasma levels and expression of interleukin-37 in patients with immune thrombocytopenia.

Interleukin (IL)-37 has an important role in autoimmune diseases by suppressing immunity and inflammation; however, the role of IL-37 in immune thrombocytopenia (ITP) has remained largely elusive. The present study aimed to investigate the expression of IL-37 and its potential role in the pathogenesis of ITP. The plasma levels and expression of IL-37 in the peripheral blood mononuclear cells of patients with active ITP, ITP patients in remission and healthy controls were measured by ELISA and reverse transcription-quantitative PCR, respectively. The levels of IL-37 in patients with ITP treated with and without glucocorticoids were also determined by ELISA. Specific anti-platelet glycoprotein (GP)IIb/IIIa and/or GPIb/IX autoantibodies were assayed by modified monoclonal antibody-specific immobilization of platelet antigens. The mean value of plasma IL-37 in ITP patients was slightly higher than that in healthy controls, but this was not statistically significant. There was no correlation between IL-37 and anti-platelet autoantibodies, and no significant difference in the IL-37 concentration was identified between patients treated with and without glucocorticoids. In addition, the correlation between IL-37 and the platelet count was analyzed, with no statistical significance observed. It was therefore concluded that IL-37 may not have a pivotal role in the development of ITP. However, the lack of significant differences may be due to the limited number of patients in different groups. A larger number of ITP patients should be enrolled in the future work and achieve more accurate results.

Fast and low-cost direct ELISA for high-throughput serological HPA-1a typing.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay. A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500. The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03. A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions.

Ocena dostępności dawców koncentratów krwinek płytkowych o oznaczonych antygenach leukocytarnych i płytkowych dla pacjentów z przeciwciałami anty-HLA i/lub anty-HPA

Clinical efficacy of PC transfusions in patients immunized with Human Leukocyte Antigens (HLA) and/or Human Platelet Antigens (HPA) can be improved by relying on platelets from donors typed for HPA and/or HLA. A specific group of PC recipients are newborns with alloimmune thrombocytopenia (FNAIT) resulting from maternal alloimmunization during pregnancy against specific fetal platelet antigens HPA (especially HPA-1a) produced by the mother as a result of fetomaternal HPA incompatibility. The study aim was to assess the demand and availability for HPA/HLA typed donors in Poland. Outcome of a survey distributed to 23 Polish blood transfusion centers (CKiK) supplemented by data acquired in direct contact (phone) revealed that: 1/ demand for PCs from donors with HLA and/or HPA typed antigens was reported in 7/23 (33.3%) CKiK; 2 / over a 1.5 year period, 1 788 PCs were prepared for patients with anti-HLA- antibodies, 63 PCs for patients with anti-HPA-antibodies and 58 PCs - for patients with anti-HLA and anti-HPA antibodies. FNAIT neonates were transfused with 35 PCs from register donors and 176 PCs from casual donors; 3 / in 13/23 CKiK (61.9%) PC selection for immunized patients was mainly based on crossmatching in LCT; 4 / registers report of 12,885 donors typed for HLA Class I antigens including 417 donors typed for HPA; 5 / nation-wide there are 133 HPA-1a negative donors available for immunized patients; they were selected after typing of 5 872 donors; The number of HPA/HLA typed donors for immunized patients is insufficient. as compared to other countries. Moreover, the demand for registered donors is steadily growing due to expension of FNAIT diagnostics currently performed at the Institute of Hematology and Transfusion Medicine (IHiT) but also in the laboratories of CKiK. One of the responsibilities of the blood transfusion service is to provide PCs for immunized patients.

Bidirectional graft-host hematological traffic in liver transplantation

The highly complex immuno-hematological system of the recipient has to rebalance itself when the liver is replaced with a graft that has its own system. This gives us an opportunity for observation. Here we consider the graft-to-recipient direction with passenger lymphocyte syndrome (PLS) as well as the recipient-to-graft direction with Factor VIII (FVIII) inhibitors, paroxysmal nocturnal hemoglobinuria (PNH) and graft endothelial replacement with liver transplantation. PLS extends beyond the ABO blood groups to any situation where the donor has been sensitized to a recipient antigen. PLS directed against ABO or minor blood group antigens is usually self limiting whereas Rhesus (Rh) PLS persists with life threatening immune hemolysis. Human platelet antigen (HPA) 1A PLS results in life threatening immune thrombocytopenia. Treatments of severe PLS may include reduction in immunosuppression, anti-B-cell therapy, plasmapheresis and splenectomy. Liver transplantation into recipients with FVIII inhibitors has been difficult. Donors with acquired hemophilia may transmit the capacity to make FVIII inhibitors by PLS and should be avoided. Patients with PNH have been transplanted successfully but a considerable cost in the continued use of high dose eculizumab. We speculate that combined bone marrow and liver transplantation would be a better option for recipients with FVIII inhibitors or PNH. Replacement of liver graft endothelium with recipient cells is common and may explain relative transplant tolerance that is believed to occur with liver transplantation.

PS1493 NEXT‐GENERATION SEQUENCING REVEALS DISTINCT EXPANDED T‐CELL RECEPTOR (TCR) B‐CHAIN CLONOTYPES IN PATIENTS WITH CHRONIC IMMUNE THROMBOCYTOPENIA (ITP)

Background:Immune thrombocytopenia (ITP) is an autoimmune disease characterised by isolated low platelet count. The pathophysiology of ITP is multifactorial and the role of CD8+ T‐cells remains largely unexplored. Our recent studies show that ITP patients have an increase in effector CD8+ T‐cell population. In order to further explore the nature of these expanded cells, we analysed the TCR β‐chain repertoires in PBMC and sorted CD8+ T‐cells of patients with ITP.Aims:To fully characterise the TCRβ repertoire of patients with chronic ITP.Methods:The study population consisted of 36 samples from 22 patients diagnosed with chronic ITP with a median age of 46yrs (IQR 33–58) and median platelet count of 36 × 109/L (IQR 21–142) at the time of sampling. Patients were either on no treatment (n = 10) or thrombopoietin‐receptor agonists (n = 12) and were compared to nine age and gender‐matched healthy controls (HC). DNA was extracted from 31 PBMC and five sorted CD8+ T‐cell samples. Next generation sequencing of TCRβ was carried out using the Illumina MiSeq platform. A multiplex of primers was used for PCR amplification of variable‐diversity‐joining (VDJ) gene segments of rearranged TCRβ gene. Analysis of the raw TCR sequences was performed using MiXCR, and data is presented as median values.Results:In total, 28,000,000 sequence reads were generated. When compared to HC, the number of unique TCRβ clonotypes in PBMC of ITP patients were significantly less (1556 vs 5757, p &lt; 0.0001; Fig1a). Principal component analysis of V and/or J gene usage demonstrated a difference in usage pattern between HC and patients as they clustered separately. When analysed by the differences in individual genes, Vβ 20.1 was significantly over‐represented in ITP patients (9.01% vs 5.37%; p &lt; 0.001) and Jβ 2.1 was significantly under‐represented (2.63% vs 12.63%; P &lt; 0.0001), compared to HC.In PBMC repertoires of ITP patients, there was a significantly higher prevalence of expanded clonotypes (defined as ≥1% frequency), compared to HC (81% vs 18%; p &lt; 0.0001; Fig 1b). Longitudinally, most expanded clones persisted over multiple years. The expanded clones in ITP patients were not present in HC. We also explored whether these expanded clonotypes were shared in ITP patients, i.e. present in more than one individual. We found two shared sequences present in two ITP patients in both sorted CD8+ T‐cell and PBMC repertoires at multiple timepoints (both absent in HC). For ITP patients, where repertoires of both PBMC and sorted CD8+ T‐cell samples were available, the top five expanded clones in PBMC were also the top five expanded clones in sorted CD8+ T‐cell population.Summary/Conclusion:Our findings reveal that the TCR repertoires of ITP patients display a significantly decreased diversity compared to HC and display a preferential use of unique V and J genes. The presence of the same expanded clonotypes in PBMC and CD8 T‐cells in ITP patients suggest that these expansions arise from CD8+ T‐cells rather than CD4+ T‐cells. The restricted TCR repertoire and stability of expanded clonotypes over time in ITP patients might be attributed to continuous CD8+ T‐cell responses to platelet antigens. Further exploration of these clonotypes, including isolation of shared clonotypes may allow us to determine disease causing cells in ITP, with potential prognostic and therapeutic value.image

PS1143 AUTOIMMUNIZATION TO PLATELET ANTIGENS IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background:The development of thrombocytopenia in chronic lymphocytic leukemia (CLL) can be enhanced by autoimmune cell destruction. The cause of anti‐platelet autoimmunization in CLL is associated with disturbance of the immune system tolerance to its own platelet antigens.Aims:To determine the importance of antiplatelet autoimmunization in thrombocytopenia at the stage of manifestation of CLL and to identify features of immunoreactivity in these patients.Methods:Under supervision there were 59 patients with CLL in the debut of the disease. All studies in patients performed before treatment. The content of anti‐platelet antibodies was determined using a FACS Canto II laser flow cytometer (Becton Dickinson). An excess of the permissible value of autoantibodies considered the binding of more than 3% of the patient's platelets with FITC‐conjugated monoclonal antibodies to human IgG during incubation with autologous serum. The content of T‐lymphocytes, their subpopulations, and natural killer cells was evaluated. The degree of expression of CD3, CD4, CD8 and CD45 molecules within lymphoid populations and subpopulations was taken into account in terms of the average intensity of immunofluorescence. The concentrations of IgG, IgA and IgM was determined in an enzyme immunoassay. The statistical significance of differences was assessed using the Mann‐Whitney test, differences were considered significant at p &lt; 0.05.Results:The group of patients with increased autoantibodies to platelets included 15 (25.4 %) people, the group without increase – 44 (74.6 %) people. The number of platelets in peripheral blood was significantly lower in patients with antibodies compared with patients without autoimmunization (121 [90; 167] x 109/l versus 187 [160; 235] x 109/l, p &lt; 0.01). A comparative analysis of cellular and humoral immunity parameters in groups of patients with CLL with different levels of autoantibodies was carried out. The values of the absolute content of T‐lymphocytes, T‐helper cells and cytotoxic T‐lymphocytes (phenotype: CD3+CD19‐, CD3+CD4+, CD3+CD8+), lymphocytes with the function of natural killer cells (phenotype: CD3‐CD8+, CD3‐CD(16+56)+ and CD3+CD(16+56)+), late activated T lymphocytes (CD3+HLA‐DR+) in groups of patients did not differ. Significantly lower levels of CD3 molecules on the membrane of T‐lymphocytes (4,9 [4,2; 5,5] conv. units against 5,7 [5,2; 6,7] conv. units p &lt; 0.01) and CD4 molecules on the t‐helper membrane (5.1 [4.2; 6.4] conv. units versus 7.8 [7.1; 8.4] conv. units, p &lt; 0.01) was registered in patients with CLL with increased antiplatelet antibodies. In the groups of patients there were no differences in the level of immunoglobulins of classes G and a in serum. Significantly higher content of immunoglobulin class M in patients with autoimmunization than in patients without antibodies (1,6 [1,3; 1,7] g/l versus 1,1 [1,0; 1,3] g/l, p &lt; 0,05) was detected.Summary/Conclusion:Antiplatelet autoimmunization is observed in a quarter of patients with CLL at the stage of diagnosis of the disease. If IgG is attached to the surface membrane of platelets, cells undergo accelerated damage in the reticular‐endothelial system, which, along with other causes, leads to a decrease in their number in the blood. It was found that in patients with CLL autoimmunization to platelet antigens is associated with increased anergy of T‐lymphoid elements, increased serum IgM levels.

Evaluation of immunohematology knowledge in hematology trainees.

Canadian hematology trainees are expected to attain clinical knowledge in the subject of red blood cell and platelet antigen systems and the principles of transfusion medicine. However, the relative degree of expertise required in blood bank serology is not well defined. A modified Delphi approach involving 10 Canadian hematology program directors was utilized to identify 12 relevant topics in immunohematology. A multiple-choice exam was developed and validated among hematology trainees from 13 hematology training programs across Canada. A Rasch analysis was used to determine fit of the examination before deploying the exam the following year to ascertain the level of knowledge in hematology trainees. The exam was piloted with 62 hematology trainees. The reliability of the exam was 0.93 with a mean item fit score of 1.01. The exam was able to discriminate between training years and self-rated expertise with better performance attained by more advanced trainees (p < 0.01). No differences were seen between geographic regions. A modified version of the exam was deployed the following year to 85 trainees, with a mean score of 58.9% ± 15.3%. Trainees scored poorest on topics concerning antibody investigations and D variants. A standardized exam for assessing hematology trainees on their expected expertise in transfusion immunohematology has been developed and can be used to assess the efficacy of educational resources provided in the subject. Trainees had a low overall mean score indicating additional educational initiatives are warranted.

Lower platelet count with increased density of platelet antigens in granulocyte colony-stimulating factor mobilized peripheral blood stem cell donors

Granulocyte colony-stimulating factor (G-CSF) is widely used for prophylaxis and treatment of neutropenia in cancer patients and also for peripheral blood stem cells (PBSC) mobilization. The aim of this study is to evaluate the possible changes of platelet surface antigens after G-CSF injection in PBSC donors compared with healthy control. Between January 1st and December 31st, 2014, 48 healthy voluntary PBSC donors were eligible for this study. Donors received G-CSF (Filgrastim) subcutaneously for five days, and then their whole blood was collected for complete blood count. Analysis of platelet antigens was performed by flow cytometry. Sixteen healthy controls were also included for comparison. Lower platelet counts were found in PBSC donors after G-CSF use and in comparison with health controls. The platelet size evaluated by forward scattering (FSC) showed smaller platelets in PBSC donors after G-CSF use compared with healthy controls (39.3 vs 46.7 mean fluorescence intensity, P=0.015). CD31 were higher in PBSC donor (203.2 vs. 120.7, P<0.001). Except CD31, other platelet surface antigens were not different between PBSC donors and healthy controls. After adjusting by FSC data, the mean antigen intensity/FSC of CD31, CD41a, CD42a, CD42b and CD61 showed 5.45 vs 2.78 (P<0.001), 4.35 vs 3.47 (P=0.007), 3.87 vs 3.17 (P=0.015), 20.45 vs 16.94 (P=0.045), and 5.98 vs 4.88 (P=0.018) respectively. We noted higher density of platelet surface antigens, lower platelet count and smaller platelet size after G-CSF injection.

Postnatal intervention for the treatment of FNAIT: a systematic review.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is associated with life-threatening bleeding. This systematic review of postnatal management of FNAIT examined transfusion of human platelet antigen (HPA) selected or unselected platelets, and/or IVIg on platelet increments, hemorrhage and mortality. MEDLINE, EMBASE and Cochrane searches were conducted until 11 May 2018. Of 754 neonates, 382 received platelet transfusions (51%). HPA-selected platelets resulted in higher platelet increments and longer response times than HPA-unselected platelets. However, unselected platelets generally led to sufficient platelet increments to 30 × 109/L, a level above which intracranial hemorrhage or other life-threatening bleeding rarely occurred. Platelet increments were not improved with the addition of IVIg to platelet transfusion. Overall, HPA-selected platelet transfusions were more effective than HPA-unselected platelets but unselected platelets were often effective enough to achieve clinical goals. Available studies do not clearly demonstrate a benefit for addition of IVIg to platelet transfusion.

HLA-DRB3*01:01 exhibits a dose-dependent impact on HPA-1a antibody levels in HPA-1a–immunized women

AbstractHLA-DRB3*01:01 is a predisposing factor for human platelet antigen 1a (HPA-1a) immunization, which is responsible for most cases of fetal and neonatal alloimmune thrombocytopenia. The aim of this study was to investigate if the HLA-DRB3*01:01 allele imposes a dose-dependent effect on anti-HPA-1a levels and neonatal platelet counts. One hundred and thirty HPA-1a–immunized women were divided into 3 groups: HLA-DRB3*01:01 negative, HLA-DRB3*01:01 hemizygous or heterozygous, and HLA-DRB3*01:01 homozygous. The dose of the HLA-DRB3*01:01 allele was determined by sequencing exon 2 of the HLA-DRB3 gene followed by HLA-DRB3 and HLA-DRB1 typing of selected samples. Anti-HPA-1a levels at time of delivery and neonatal platelet counts were compared among groups. There was a significant dose-dependent effect of the HLA-DRB3*01:01 allele on anti-HPA-1a levels (global P value [Pglobal] = .0032). Median (range) anti-HPA-1a levels were 1.5 IU/mL (0.0-19.0 IU/mL), 21.1 IU/mL (0.0-1967 IU/mL), and 43.7 IU/mL (1.0-980 IU/mL) in women with 0, 1, and 2 copies of the HLA-DRB3*01:01 allele, respectively. There was also a significant, but opposite, dose-dependent effect of the mother's HLA-DRB3*01:01 allele on the platelet count of the newborn (Pglobal = .0155). Median (range) neonatal platelet counts were 241 × 109/L (59 × 109/L to 393 × 109/L), 107 × 109/L (4 × 109/L to 387 × 109/L) and 32 × 109/L (4 × 109/L to 352 × 109/L) for newborns of mothers with 0, 1, and 2 copies of the HLA-DRB3*01:01 allele, respectively. Thus, the HLA-DRB3*01:01 allele exhibits a dose-dependent impact on maternal anti-HPA-1a levels in HPA-1a–immunized women.

Prenatal Management of Pregnancies at Risk of Fetal Neonatal Alloimmune Thrombocytopenia (FNAIT): Scientific Impact Paper No. 61.

WHAT IS IT?: Fetal neonatal alloimmune thrombocytopenia (FNAIT), also known as neonatal alloimmune thrombocytopenia (NAIT) or fetomaternal alloimmune thrombocytopenia (FMAIT), is a rare condition which affects a baby's platelets. This can put them at risk of problems with bleeding, particularly into the brain. One baby per week in the UK may be seriously affected and milder forms can affect one in every 1000 births. HOW IS IT CAUSED?: Platelets are blood cells that are very important in helping blood to clot. All platelets have natural proteins on their surface called human platelet antigens (HPAs). In babies, half of these antigens are inherited from the mother and half from the father. During pregnancy, some of the baby's platelets can cross into the mother's bloodstream. In most cases, this does not cause a problem. But in cases of FNAIT, the mother's immune system does not recognise the baby's HPAs that were inherited from the father and develops antibodies, which can cross the placenta and attack the baby's platelets. These antibodies are called anti-HPAs, and the commonest antibody implicated is anti-HPA-1a, but there are other rarer antibody types. If this happens, the baby's platelets may be destroyed causing their platelet count to fall dangerously low. If the platelet count is very low there is a risk to the baby of bleeding into their brain before they are born. This is very rare but if it happens it can have serious effects on the baby's health. HOW IS IT INHERITED?: A baby inherits half of their HPAs from its mother and half from its father. Consequently, a baby may have different HPAs from its mother. As the condition is very rare, and even if the baby is at risk of the condition we have no way of knowing how severely they will be affected, routine screening is not currently recommended. WHAT CAN BE DONE?: FNAIT is usually diagnosed if a previous baby has had a low platelet count. The parents are offered blood tests and the condition can be confirmed or ruled out. There are many other causes of low platelets in babies, which may also need to be tested for. As the condition is so rare, expertise is limited to specialist centres and normally a haematologist and fetal medicine doctor will perform and interpret the tests together. Fortunately, there is an effective treatment for the vast majority of cases called immunoglobulin, or IVIg. This 'blood product' is given intravenously through a drip every week to women at risk of the condition. It may be started from as early as 16weeks in the next pregnancy, until birth, which would be offered at around 36-37weeks. Less common treatments that may be considered depending on individual circumstances include steroid tablets or injections, or giving platelet transfusions to the baby. WHAT DOES THIS PAPER TELL YOU?: This paper considers the latest evidence in relation to treatment options in the management of pregnancies at risk of FNAIT. Specifically, we discuss the role of screening, when IVIg should be started, what dose should be used, and what evidence there is for maternal steroids. We also consider in very rare selected cases, the use of fetal blood sampling and giving platelet transfusions to the baby before birth. Finally, we consider the approaches to blood testing mothers to tell if babies are at risk, which is offered in some countries, and development of new treatments to reduce the risk of FNAIT.

Glycoprotein V is a relevant immune target in patients with immune thrombocytopenia.

Platelet autoantibody-induced platelet clearance represents a major pathomechanism in immune thrombocytopenia (ITP). There is growing evidence for clinical differences between anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib/IX mediated ITP. Glycoprotein V is a well characterized target antigen in Varicella-associated and drug-induced thrombocytopenia. We conducted a systematic study assessing the prevalence and functional capacity of autoantibodies against glycoprotein V. A total of 1140 patients were included. In one-third of patients, platelet-bound autoantibodies against glycoproteins Ib/IX, IIb/IIIa, or V were detected in a monoclonal antibody immobilization of platelet antigen assay; platelet-bound autoantiglycoprotein V was present in the majority of samples (222 out of 343, 64.7%). Investigation of patient sera revealed the presence of free autoantibodies against glycoprotein V in 13.5% of these patients by an indirect monoclonal antibody immobilization of platelet antigen assay, but in 39.6% by surface plasmon resonance technology. These antibodies showed significantly lower avidity (association/dissociation ratio 0.32±0.13 vs. 0.73±0.14; P<0.001). High- and low-avidity antibodies induced comparable amounts of platelet uptake in a phagocytosis assay using CD14+ positively-selected human macrophages [mean phagocytic index, 6.81 (range, 4.75-9.86) vs. 6.01 (range, 5.00-6.98); P=0.954]. In a NOD/SCID mouse model, IgG prepared from both types of anti-glycoprotein V autoantibodies eliminated human platelets with no detectable difference between the groups from the murine circulation [mean platelet survival at 300 minutes, 40% (range, 27-55) vs. 35% (16-46); P=0.025]. Our data establish glycoprotein V as a relevant immune target in immune thrombocytopenia. We would suggest that further studies including glycoprotein V will be required before ITP treatment can be tailored according to platelet autoantibody specificity.

Fetal and neonatal alloimmune thrombocytopenia: recommendations for evidence-based practice, an international approach.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) may result in severe bleeding, particularly fetal and neonatal intracranial haemorrhage (ICH). As a result, FNAIT requires prompt identification and treatment; subsequent pregnancies need close surveillance and management. An international panel convened to develop evidence-based recommendations for diagnosis and management of FNAIT. A rigorous approach was used to search, review and develop recommendations from published data for: antenatal management, postnatal management, diagnostic testing and universal screening. To confirm FNAIT, fetal human platelet antigen (HPA) typing, using non-invasive methods if quality-assured, should be performed during pregnancy when the father is unknown, unavailable for testing or heterozygous for the implicated antigen. Women with a previous child with an ICH related to FNAIT should be offered intravenous immunoglobulin (IVIG) infusions during subsequent affected pregnancies as early as 12weeks gestation. Ideally, HPA-selected platelets should be available at delivery for potentially affected infants and used to increase the neonatal platelet count as needed. If HPA-selected platelets are not immediately available, unselected platelets should be transfused. FNAIT studies that optimize antenatal and postnatal management, develop risk stratification algorithms to guide management and standardize laboratory testing to identify high risk pregnancies are needed.

Pathophysiology of Autoimmune Thrombocytopenia: Current Insight with a Focus on Thrombopoiesis.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by a significant reduction in the number of circulating platelets which is frequently associated with bleeding. The total count of platelets in the body is finely regulated by the balance between platelet production and destruction. Although the pathogenesis of ITP is still not completely elucidated, it is largely recognized that the low platelet count observed in ITP patients is due to alterations of both mechanisms. An abnormal proliferation of autoreactive T cells leading to the breakdown of immune tolerance to platelet antigens is suggested to be responsible for the up-regulated proliferation of autoantibody producing B cells. Consequently, the immune response induces enhanced T cell-mediated cytotoxicity and antibody-mediated platelet destruction through phagocytosis, complement activation and apoptosis. An additional contribution to the pathophysiology of ITP is given by alterations of thrombopoiesis caused by platelet-reactive autoantibodies or cytotoxic T cells leading to impaired megakaryocyte differentiation and platelet production. All these processes involved in ITP pathophysiology account for the complexity and heterogeneity in the clinical manifestation and therapy responsiveness of this disorder. For this reason, a better understanding of the different underlying mechanisms in ITP is necessary to develop more efficient therapeutic treatments in the future. In this review, we will provide an update on the pathophysiology of ITP with a particular focus on the impact of impaired thrombopoiesis.

Polymorphism of human platelet antigens in Tunisian population: Clinical and anthropological interests

Human Platelet Antigens (HPA) are of considerable interest in obstetric transfusion medicine and anthropological genetics. This study aims to provide clinicians with a detailed database of HPA antigenic variants, which allows them to estimate the probability of allo-immunisation of each antigen. In addition, it aims to make an interethnic comparison of the Tunisian population with other populations. The target population consists of 324healthy and unrelated Tunisian blood donors recruited from the National Blood Transfusion Center in Tunis. DNA extraction was performed by the Salting Out method and molecular genotyping was performed by the PCR-SSP technique. The statistical analysis was performed using two approaches: manual calculation and computerized calculation. Phylogenetic trees were constructed through the use of Standard Genetic Distances that were calculated from allelic frequencies. With the exception of the HPA-4 system, statistical analysis showed that all HPA systems are polymorphic especially the two systems HPA-3 and HPA-15. The inter-ethnic analysis showed that Tunisians are closer to North Africans and Caucasians than Sub-Saharan and Asian populations, which shows genetic mixing between Tunisians, Arabs, Europeans and Africans. The results of this study could be exploited to prepare a ready-to-use genotyping plate dedicated to HPA antigens, with the aim of ensuring better management, especially for polytransfused patients.

Neonatal Intracranial Hemorrhage with a Dramatic Outcome Due to Maternal Anti CD36 Antibodies

Fetal/neonatal allo-immune thrombocytopenia (FNAIT) results from maternal immunization against fetal platelet-specific antigens (HPA) inherited from the father. Most cases involve HPA located on glycoproteins (GP) IbIX, IaIIa and IIbIIIa. Iso-immunizations can also occur in the absence of expression of membrane proteins, such as GPIIb or GPIIIa in Glanzmann patients. CD36 (also called glycoprotein GPIV) deficiency is observed in 3 to 5% of Asian and African populations. We report here the case of a 41-year-old Canadian woman originated from Africa, who delivered a male dead new-born at 39 weeks of gestation. A massive intracranial haemorrhage was identified as being the obvious cause of death. No platelet antibody against GPIbIX, IaIIa, and IIbIIIa was identified by the gold-standard Monoclonal Antibody-specific Immobilization of Platelet Antigens (MAIPA) assay. Surprisingly, anti CD36 iso-antibodies were identified in the maternal serum with a new bead-based multiplex assay. The CD36 gene was sequenced for both parents, and a mutation was identified on Exon 10 of the mother’s CD36 gene, which was absent for the father: NM_000072.3:c.975T&gt;G inducing a STOP codon at position 325 of the mature protein. The absence of CD36 expression on the mother’s platelets was confirmed by flow cytometry.

Allele and haplotype frequencies of human platelet and leukocyte antigens in platelet donors.

ABSTRACTObjective To described the allele and haplotype frequencies of human leukocyte antigen genes at the -A, -B loci and human platelet antigen genes for human platelet antigen systems 1 to 9, 11 and 15 in blood.Methods We included 867 healthy unrelated volunteer donors who donated platelets between January 2011 and December 2014. Microarray genotyping was performed using a BeadChip microarray. Medium resolution typing of the human leukocyte antigen at loci A and B was carried out using sequence-specific oligonucleotide probe hybridization. We used multivariate analysis and our human leukocyte antigen population was compared to data from the United States national bone marrow donor program. Human platelet antigen results were compared to a literature review and data from around the world.Results Our human leukocyte antigen haplotype results were more similar to those of hispanics, followed by caucasians. Likewise, our human platelet antigen sample is more similar to those of Argentina, Rio Grande do Sul and Italy.Conclusion This was the first article that discusses human platelet antigen and human leukocyte antigen data together. Rare genotypes or antibody associations can make patient management difficult. A blood bank with genotyped donors allows for optimal transfusion and can contribute to better results. Our information can serve as basis for a database of platelet antigen polymorphisms.