Anti-human platelet antigen-5b antibodies and fetal and neonatal alloimmune thrombocytopenia; incidental association or cause and effect?

Most cases of fetal and neonatal thrombocytopenia (FNAIT) are caused by maternal anti-human platelet antigen-1a antibodies (anti-HPA-1a). Anti-HPA-5b antibodies are the second most common antibodies in suspected FNAIT cases. Given the high prevalence of anti-HPA-5b antibodies in pregnant women delivering healthy newborns, the association with FNAIT may be coincidental. This review of the literature related to FNAIT using the MEDLINE database was conducted according to PRISMA guidelines. A retrospective analysis of a single-centre cohort of 817 suspected FNAIT cases was conducted. The pooled prevalence of anti-HPA-5b antibodies in unselected pregnant women of European descent was 1.96% (n = 3113), compared with 3.4% (n = 5003) in women with suspected FNAIT. We found weak evidence that a small proportion of pregnant women presenting with anti-HPA-5b antibodies will give birth to a newborn with mild thrombocytopenia. The neonatal platelet counts were not different between suspected FNAIT cases (n = 817) with and without maternal anti-HPA-5b antibodies. The prevalence of maternal anti-HPA-5b antibodies was not different between neonates with intracranial haemorrhage and healthy controls. The current experimental and epidemiological evidence does not support the hypothesis that anti-HPA-5b antibodies cause severe thrombocytopenia or bleeding complications in the fetus or newborn.

Evaluation of a new microbeads assay for granulocyte antibody detection.

BACKGROUND: To reduce the risk of transfusion-associated acute lung injury (TRALI), a high number of plasma donors were tested for human leukocyte antigen (HLA) and human neutrophil antigen (HNA) antibodies. For HNA antibody detection, the gold standard is a combination of the granulocyte immunofluorescence test (GIFT) and the granulocyte agglutination test (GAT). However, these tests are not suitable for a high-throughput of samples. STUDY DESIGN AND METHODS: To evaluate the new generation of the LABScreen MULTI assay (One Lambda, Inc.), which has special new beads for all the known HNA specificities, including HNA-3a, 97 sera samples containing well-defined HNA antibodies were used. For background testing, we used 91 samples from plasma donors previously identified by GAT, GIFT, and the monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) assay. RESULTS: Compared with previous tests, the new LABScreen MULTI assay was highly specific for the HNA-1a, HNA-1b, HNA-2, and HNA-3a antibody specificities required to prevent TRALI. Ninety-eight percent of the HNA-1a, HNA-1b, and HNA-2 antibodies could be detected as true positive; and 90% of the HNA-3a antibodies were recognized correctly as positive. False-positive reactions were identified in 5.5% of samples that previously tested negative. CONCLUSION: The detection of HNA-3a antibody specificities could be integrated into the new LABScreen MULTI assay; however, we detected only 90%. In addition, we detected further HNA antibodies, such as HNA-1c, HNA-1d, and some HNA-3b and HNA-4a antibodies. The new generation of LABScreen MULTI is a great step toward feasible high-throughput testing for HNA antibodies. Nevertheless, GIFT and GAT remain the gold-standard methods for the differentiation of rare and currently unknown HNA specificities.

Antiendothelial αvß3 Antibodies Are a Major Cause of Intracranial Bleeding in Fetal/Neonatal Alloimmune Thrombocytopenia.

OBJECTIVE: Fetal/neonatal alloimmune thrombocytopenia is a severe bleeding disorder, which can result in intracranial hemorrhage (ICH), leading to death or neurological sequelae. In whites, maternal anti-human platelet antigen-1a (HPA-1a) antibodies are responsible for the majority of cases. No predictive factors for ICH are available to guide prophylactic treatment during pregnancy. In this study, we investigated antibodies from mothers with ICH-positive fetal/neonatal alloimmune thrombocytopenia and with ICH-negative fetal/neonatal alloimmune thrombocytopenia to identify serological and functional differences between the groups. APPROACH AND RESULTS: In an antigen capture assay, we observed a stronger binding of +ICH antibodies to endothelial cell (EC)-derived αvß3. By absorption experiments, we subsequently identified anti-HPA-1a antibodies of anti-αvß3 specificity in the +ICH but not in the -ICH cohort. Only the anti-αvß3 subtype, but not the anti-ß3 subtype, induced EC apoptosis of HPA-1a-positive ECs by caspase-3/7 activation, and mediated by reactive oxygen species. In addition, only the anti-αvß3 subtype, but not the anti-ß3 subtype, interfered with EC adhesion to vitronectin and with EC tube formation. CONCLUSIONS: We conclude that the composition of the anti-HPA-1a antibody subtype(s) of the mother may determine whether ICH occurs. Analysis of anti-HPA-1a antibodies of the anti-αvß3 subtype in maternal serum has potential in the diagnostic prediction of ICH development and may allow for modification of prophylactic treatment in fetal/neonatal alloimmune thrombocytopenia.

Longitudinal Changes in the Blood Supply and Demand in North-East-Germany 2005-2015.

BACKGROUND: Securing future blood supply is a major issue of transfusion safety. In this prospective 10-year longitudinal study we enrolled all blood donation services and hospitals of the federal state Mecklenburg-Western Pomerania. METHODS AND RESULTS: From 2005 to 2015 (time period with major demographic effects), whole blood donation numbers declined by 18%. In male donors this paralleled the demographic change, while donation rates of females declined 12.4% more than expected from demography. In parallel, red cell transfusion rates/1,000 population decreased from 2005 to 2015 from 56 to 51 (-8.4%), primarily due to less transfusions in patients >60 years. However, the transfusion demand declined much less than blood donation numbers: -13.5% versus -18%, and the population >65 years (highest transfusion demand) will further increase. The key question is whether the decline in transfusion demand observed over the previous years will further continue, hereby compensating for reduced blood donation numbers due to the demographic change. The population structure of Mecklenburg-Western Pomerania reflects all Eastern German federal states, while the Western German federal states will reach similar ratios of age groups 18-64 years / ≥65 years about 10 years later. CONCLUSIONS: Regular monitoring of age- and sex-specific donation and transfusion data is urgently required to allow transfusion services strategic planning for securing future blood supply.

A population-based longitudinal study on the implications of demographics on future blood supply.

BACKGROUND: Changes in demographics with increases in older age groups and decreases in younger age groups imply an increased demand for blood transfusions paralleled by a decrease in the population eligible for blood donation. However, more restrictive transfusion triggers and the patient blood management initiative also reduce the demand for red blood cells (RBCs). Eastern Germany is a model region for the impact of demographic changes, which manifest in this region approximately 10 years earlier than in other regions due to the 50% birth rate decline after 1989. STUDY DESIGN AND METHODS: We report the 2010 longitudinal 5-year follow-up of the study assessing all whole blood donations and RBC transfusions in Mecklenburg-West Pomerania. We compared the projections that were made 5 years ago with: 1) the current age structure of the blood donor and transfusion recipient populations and 2) its impact on blood demand and blood donation numbers in specific age groups. RESULTS: Transfusion rates were lower and blood donation rates were higher than predicted in 2005. Although transfusion rates/1000 decreased in nearly all age groups, the overall annual transfusion rate increased to 66.4 RBC units/1000 (in 2005, 62.2/1000) due to the absolute increase in the elderly population. Despite a 7.4% decline in the population 18 to 65 years of age, whole blood donations increased by 11.7% between 2005 and 2010, but thereafter decreased by 21% (first-time donors by 39.4%), reflecting the effect of the post-1990 birth rate decline on the donor population. CONCLUSION: Changes in demography and medical practice impact the delicate balance between available blood supply and potential future transfusion needs. In times of pronounced demographic changes, regular monitoring of the blood demand and age structure of blood recipients and donors is required to allow strategic planning to prevent blood shortages or overproduction.

Flucloxacillin-induced immune thrombocytopenia.

BACKGROUND: Drug-induced immune thrombocytopenia (DITP) is an adverse drug reaction associated with platelet (PLT) destruction by drug-dependent antibodies. Demonstration of drug-dependent PLT antibodies is often difficult and can only be rendered by extensive laboratory testing. In this report, we present the first serologically confirmed case of DITP caused by the antibiotic flucloxacillin. CASE REPORT: A 68-year-old man developed severe thrombocytopenia that was first suspected to be related to heparin-induced thrombocytopenia. The absence of PLT-activating heparin-dependent antibodies and the abrupt decrease in PLT count to fewer than 20 × 10(9) /L raised the suspicion of DITP. DISCUSSION: Flucloxacillin-dependent antibodies were detected in patient serum using whole PLT enzyme immunoassay and flow cytometry. The glycoprotein (GP) specificity was identified to be against GP IIb/IIIa complexes using the monoclonal antibody immobilization of PLT antigens assay. Interestingly, antibody binding was abolished when EDTA plasma was used and restored after adding calcium. CONCLUSION: In summary, DITP should be considered in cases of acute thrombocytopenia during treatment with flucloxacillin.

'Chameleonic' Serological Findings Leading to Life-Threatening Hemolytic Transfusion Reactions.

BACKGROUND: The phenomena of co-incidence of transfusion-induced allo- and autoantibodies, blockage and/or loss of red blood cell (RBC) antigens are conspicuous and may result in confusion and misdiagnosis. CASE REPORT: A 67-year-old female was transferred to the intensive care unit due to hemolysis which developed 2 days following transfusion of three Rh(D)-negative RBC units in the presence of strongly reactive autoantibodies. Standard serological testing and genotyping were performed. Upon arrival, the patient was typed as Ccddee. Her hemolysis was decompensated, and an immediate blood transfusion was required. In addition, direct and indirect antiglobulin tests (DAT and IAT) as well as the eluate were strongly positive. Emergency transfusion of Rh(D)-negative RBCs resulted in increased hemolysis and renal failure. An exhaustive testing revealed anti-D, anti-c, CCddee phenotype and CCD.ee genotype. Three units of cryopreserved CCddee RBCs were transfused, and the patient's condition immediately improved. The discrepancy between Rh-D phenotyping and genotyping was likely caused by masking of the D-epitopes by the autoantibodies. In fact, further enquiry revealed that the patient had been phenotyped as Rh(D)-positive 6 months ago and had been transfused at that time following hip surgery. CONCLUSION: The phenomena of transfusion-induced autoantibodies, masked alloantibodies, antigen blockage and/or loss are rare but important features which should be considered in patients presenting with autoimmune hemolytic anemia and/or hemolytic transfusion reactions.

Management of infants born with severe neonatal alloimmune thrombocytopenia: the role of platelet transfusions and intravenous immunoglobulin.

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is a fetomaternal incompatibility most commonly induced by maternal anti-HPA-1a alloantibodies. Transfusion of immunologically compatible platelets (PLTs) to prevent cerebral hemorrhage, the most severe complication in affected newborns, is usually recommended. Such PLT concentrates, however, are often not readily available. STUDY DESIGN AND METHODS: The efficacy of random-donor PLT transfusions and intravenous immunoglobulin (IVIG) for the management of 17 neonates across four centers with unexpected, severe NAIT was evaluated. Neonates were treated with random-donor PLTs alone (n=7), random-donor PLTs with IVIG (n=8), or matched HPA-1bb PLTs (n=2). RESULTS: All but one patient (treated with random PLTs and IVIG) achieved a posttransfusion PLT count of higher than 30 × 10(9) /L after the first PLT transfusion. The PLT count remained higher than 30 × 10(9) /L for longer than 24 hours in five of seven, seven of eight, and two of four newborns who received random-donor PLTs alone, random-donor PLTs with IVIG, or matched HPA-1bb PLTs, respectively. None of the newborns developed major bleeding or intracranial hemorrhage. IVIG did not appear to improve either posttransfusion PLT counts or total PLT transfusion requirements. CONCLUSION: Transfusion of random-donor PLTs alone was effective at correcting critically low PLT counts and should be considered as first-line treatment of newborns with unexpected severe NAIT.

Implications of demographics on future blood supply: a population-based cross-sectional study.

BACKGROUND: Data on blood recipients are sparse and unconnected to data on blood donors. The objective was to analyze the impact of the demographic change on future blood demand and supply in a German federal state. STUDY DESIGN AND METHODS: A population-based cross-sectional study was conducted. For all in-hospital transfused red blood cells (RBCs; n = 95,477), in the German federal state Mecklenburg-Pomerania in 2005, characteristics of the patient and the blood donor (118,406 blood donations) were obtained. Population data were used to predict blood demand and supply until 2020. RESULTS: By 2020 the population increase of those aged 65 years or more (+26.4%) in Mecklenburg-Pomerania will be paralleled by a decrease of the potential donor population (18-68 years; -16.1%). Assuming stable rates per age group until 2020, the demand for in-hospital blood transfusions will increase by approximately 25% (24,000 RBC units) while blood donations will decrease by approximately 27% (32,000 RBC units). The resulting, predicted shortfall is 47% of demand for in-hospital patients (56,000 RBC units). Validation using historical data (1997-2007) showed that the model predicted the RBC demand with a deviation of only 1.2%. Demographic changes are particularly pronounced in former East Germany, but by 2030 most European countries will face a similar situation. The decrease of younger age groups requires an increase of blood donation rates and interdisciplinary approaches to reduce the need for transfusion to maintain sufficient blood supply. CONCLUSIONS: Demography is a major determinant of future transfusion demand. All efforts should be made by Western societies to systematically obtain data on blood donors and recipients to develop strategies to meet future blood demand.

An international external quality assessment for laboratory diagnosis of heparin-induced thrombocytopenia.

Essentials A pilot study for External Quality Assessment for testing of HIT is described. The qualitative accordance for the PF4/heparin IgG test was 97.6%. The qualitative accordance for functional HIT tests was considerably lower. External Quality Assessment for functional HIT tests is required. SUMMARY: Objective Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening complication of heparin exposure. Diagnosis is most reliable using a combination of an enzyme immunoassay (EIA) that detects antibodies against platelet factor 4 (PF4)/heparin complexes ("antigen" assay) and a "functional" assay that detects platelet-activating properties of the pathogenic HIT antibodies. No External Quality Assessment (EQA) is available for a combination of the tests. Here we report on the results of the first international EQA. Methods The pilot EQA was organized by the Department of Transfusion Medicine, Universitätsmedizin Greifswald, Germany. Six serum samples of patients, which were referred to Greifswald for HIT diagnosis, and one negative control sample were distributed to seven participants in Germany, Canada, and Singapore. Participants were asked to report the optical density (OD) values of their local EIA test for IgG-specific antibodies against the PF4/heparin complexes and the results for a functional assay (HIPA or SRA). Consensus was defined as a minimum 70% agreement, i.e., agreement among at least five of the seven participating laboratories. Results and conclusion Six out of seven participants reported results for EIA, with a high quantitative accordance (97.6%). For the functional assay, consensus was reached for all samples except the negative control, for which some participants reported nonspecific reactivity. All HIT-negative samples were correctly diagnosed by all participants; for HIT-positive samples, consensus of 70% was reached. Although the limited availability of sample material is an obstacle to overcome, an EQA combining both EIA and functional testing is feasible.

Reactions Induced by Platelet Transfusions.

SUMMARY: Platelet transfusions play a central role in therapeutic regimens for patients with hematologic/oncologic diseases who develop severe thrombocytopenia either in the course of their disease or following cytostatic therapy. Like other blood components, platelet transfusions have achieved a high degree of safety as far as transmission of viral diseases is concerned. However, transfusion of platelet concentrates is accompanied by a high frequency of febrile and anaphylactoid reactions. In rare cases, recipients of platelet concentrates are threatened by severe reactions as septic complications due to bacterial contamination of platelet concentrates, transfusion-related acute lung injury and severe anaphylactic episodes.

A population-based longitudinal study on the implication of demographic changes on blood donation and transfusion demand.

Transfusion safety includes the risk of transmission of pathogens, appropriate transfusion thresholds, and sufficient blood supply. All industrialized countries experience major ongoing demographic changes resulting from low birth rates and aging of the baby boom generation. Little evidence exists about whether future blood supply and demand correlate with these demographic changes. The ≥50% decline in birth rate in the eastern part of Germany after 1990 facilitates systematic study of the effects of pronounced demographic changes on blood donation and demand. In this prospective, 10-year longitudinal study, we enrolled all whole blood donors and all patients receiving red blood cell transfusions in the state of Mecklenburg-West Pomerania. We compared projections made in 2005 based on the projected demographic changes with: (1) number and age distribution of blood donors and transfusion recipients in 2015 and (2) blood demand within specific age and patient groups. Blood donation rates closely followed the demographic changes, showing a decrease of -18% (vs projected -23%). In contrast, 2015 transfusion rates were -21.3% lower than projected. We conclude that although changes in demography are highly predictive for the blood supply, transfusion demand is strongly influenced by changes in medical practice. Given ongoing pronounced demographic change, regular monitoring of the donor/recipient age distributions and associated impact on blood demand/supply relationships is required to allow strategic planning to prevent blood shortages or overproduction.

Surface Modification of Biodegradable Polymers towards Better Biocompatibility and Lower Thrombogenicity.

PURPOSE: Drug-eluting stents (DES) based on permanent polymeric coating matrices have been introduced to overcome the in stent restenosis associated with bare metal stents (BMS). A further step was the development of DES with biodegradable polymeric coatings to address the risk of thrombosis associated with first-generation DES. In this study we evaluate the biocompatibility of biodegradable polymer materials for their potential use as coating matrices for DES or as materials for fully bioabsorbable vascular stents. MATERIALS AND METHODS: Five different polymers, poly(L-lactide) PLLA, poly(D,L-lactide) PDLLA, poly(L-lactide-co-glycolide) P(LLA-co-GA), poly(D,L-lactide-co-glycolide) P(DLLA-co-GA) and poly(L-lactide-co-ε-caprolactone), P(LLA-co-CL) were examined in vitro without and with surface modification. The surface modification of polymers was performed by means of wet-chemical (NaOH and ethylenediamine (EDA)) and plasma-chemical (O2 and NH3) processes. The biocompatibility studies were performed on three different cell types: immortalized mouse fibroblasts (cell line L929), human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC). The biocompatibility was examined quantitatively using in vitro cytotoxicity assay. Cells were investigated immunocytochemically for expression of specific markers, and morphology was visualized using confocal laser scanning (CLSM) and scanning electron (SEM) microscopy. Additionally, polymer surfaces were examined for their thrombogenicity using an established hemocompatibility test. RESULTS: Both endothelial cell types exhibited poor viability and adhesion on all five unmodified polymer surfaces. The biocompatibility of the polymers could be influenced positively by surface modifications. In particular, a reproducible effect was observed for NH3-plasma treatment, which enhanced the cell viability, adhesion and morphology on all five polymeric surfaces. CONCLUSION: Surface modification of polymers can provide a useful approach to enhance their biocompatibility. For clinical application, attempts should be made to stabilize the plasma modification and use it for coupling of biomolecules to accelerate the re-endothelialization of stent surfaces in vivo.

A systematic review and survey of the management of unexpected neonatal alloimmune thrombocytopenia.

BACKGROUND: Unexpected neonatal alloimmune thrombocytopenia (NAIT) may have devastating consequences and its management is challenging. To design future trials, evidence from the literature and existing best practice need review. STUDY DESIGN AND METHODS: This study was a cross-sectional survey of neonatal units in Germany and Canada to determine management strategies of NAIT and a systematic search for randomized controlled trials (RCTs). RESULTS: Management of NAIT differs substantially between countries with regard to platelet (PLT) thresholds for screening, initiation of therapy, and treatment. Seventy-seven percent of Canadian physicians versus 68 percent of German physicians screen preterm and term infants, at a PLT threshold of 30 x 10(9) to 100 x 10(9) per L. In preterm infants, 60 percent of Canadian neonatologists commence treatment at a PLT count of between 30 x 10(9) and 50 x 10(9) per L. In Germany 32 percent of the physicians start treatment at this level and 25 percent use a threshold of between 10 x 10(9) and 20 x 10(9) per L. In term infants, 6 percent of the Canadian physicians and 16 percent of the German physicians use even lower treatment triggers of between 5 x 10(9) and 10 x 10(9) per L. In the presence of bleeding, 61 percent of German physicians await the arrival of antigen-negative PLTs, while 64 percent of Canadian neonatologists prefer intravenous immunoglobulin or random-donor PLTs (81%). Maternal PLTs are utilized by 31 percent of physicians in both countries. No RCTs were identified. CONCLUSION: In the absence of RCTs, management of unexpected NAIT differs between countries. Clinicians and transfusion services may use the results of our study to develop collaborative protocols, redefine preferred hospitalwide strategies, and design future controlled trials.

Antigen-positive platelet transfusion in neonatal alloimmune thrombocytopenia (NAIT).

Neonatal alloimmune thrombocytopenia (NAIT) is a fetomaternal incompatibility most commonly induced by maternal anti-HPA-1a, IgG alloantibodies against a polymorphic epitope of the glycoprotein IIb/IIIa complex in approximately 97.5% of white patients. Current guidelines recommend transfusion of immunologically compatible platelets to prevent cerebral hemorrhage, the most severe complication in affected newborns. Such platelet concentrates, however, are often not readily available. In a retrospective analysis in German and Canadian centers, 27 newborns with NAIT were identified who received platelets from random donors. Unexpectedly, 24 of 27 newborns showed an increase above a threshold of 40 x 10(9) platelets per liter, with moderate (n = 8) or significant (n = 16) platelet count increments (more than 80 x 10(9)/L). We conclude that transfusion of platelet concentrates from random donors is an appropriate strategy in the management of unexpected, severe NAIT predominantly in first pregnancies, pending the availability of compatible platelets.

A functional platelet fibrinogen receptor with a deletion in the cysteine-rich repeat region of the beta(3) integrin: the Oe(a) alloantigen in neonatal alloimmune thrombocytopenia.

This report describes a new low-frequency alloantigen, Oe(a), responsible for a case of neonatal alloimmune thrombocytopenia (NAIT). In a population study none of 600 unrelated blood donors was an Oe(a) carrier. By immunochemical studies the Oe(a) antigen could be assigned to platelet glycoprotein (GP) IIIa. Sequencing of GPIIIa complementary DNA from an Oe(a) (+) individual showed deletion of a lysine residue at position 611 (DeltaLys(611)). Analysis of 20 Oe(a) (-) and 3 Oe(a) (+) individuals showed that the DeltaLys(611) form of GPIIIa was related to the phenotype. Anti-Oe(a) reacted with the DeltaLys(611), but not with the wild-type isoforms on stable transfectants expressing GPIIIa, indicating that DeltaLys(611) directly induces the expression of Oe(a) epitopes. Under nonreducing conditions the Pro(33)DeltaLys(611) variant migrated with a slightly decreased molecular weight compared to the Pro(33)Lys(611) isoform suggesting that DeltaLys(611) has an influence on the disulfide bonds of GPIIIa. The Pro(33)DeltaLys(611) GPIIIa could undergo conformational changes and bind to fibrinogen in a similar manner as the Pro(33)Lys(611) isoform. No difference was found in the tyrosine phosphorylation of pp125(FAK), suggesting that DeltaLys(611) has no effect on integrin function. In contrast to all other low-frequency antigens, the DeltaLys(611) isoform was associated with the HPA-1b, but not with the high frequency HPA-1a allele. Comparison with GPIIIa DNA from nonhuman primates indicated that the HPA-1a allele represents the ancestral form of GPIIIa. It can be assumed that the Oe(a) form did arise as a result of a mutational event from an already mutated GPIIIa allele.