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.

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.

Induced pluripotent stem cell-derived neuronal cells from a sporadic Alzheimer's disease donor as a model for investigating AD-associated gene regulatory networks.

BACKGROUND: Alzheimer's disease (AD) is a complex, irreversible neurodegenerative disorder. At present there are neither reliable markers to diagnose AD at an early stage nor therapy. To investigate underlying disease mechanisms, induced pluripotent stem cells (iPSCs) allow the generation of patient-derived neuronal cells in a dish. RESULTS: In this study, employing iPS technology, we derived and characterized iPSCs from dermal fibroblasts of an 82-year-old female patient affected by sporadic AD. The AD-iPSCs were differentiated into neuronal cells, in order to generate disease-specific protein association networks modeling the molecular pathology on the transcriptome level of AD, to analyse the reflection of the disease phenotype in gene expression in AD-iPS neuronal cells, in particular in the ubiquitin-proteasome system (UPS), and to address expression of typical AD proteins. We detected the expression of p-tau and GSK3B, a physiological kinase of tau, in neuronal cells derived from AD-iPSCs. Treatment of neuronal cells differentiated from AD-iPSCs with an inhibitor of γ-secretase resulted in the down-regulation of p-tau. Transcriptome analysis of AD-iPS derived neuronal cells revealed significant changes in the expression of genes associated with AD and with the constitutive as well as the inducible subunits of the proteasome complex. The neuronal cells expressed numerous genes associated with sub-regions within the brain thus suggesting the usefulness of our in-vitro model. Moreover, an AD-related protein interaction network composed of APP and GSK3B among others could be generated using neuronal cells differentiated from two AD-iPS cell lines. CONCLUSIONS: Our study demonstrates how an iPSC-based model system could represent (i) a tool to study the underlying molecular basis of sporadic AD, (ii) a platform for drug screening and toxicology studies which might unveil novel therapeutic avenues for this debilitating neuronal disorder.

High incidence of heparin-induced allergic delayed-type hypersensitivity reactions in pregnancy.

BACKGROUND: Among the most frequent adverse effects of subcutaneous heparin treatment, heparin-induced skin lesions occur with an incidence of 10.3% in nonpregnant female patients. Clinical observations suggest an even higher risk during pregnancy. OBJECTIVES: We sought to determine the incidence and causes of heparin-induced skin reactions during pregnancy in a prospective cohort study. METHODS: Pregnant women with subcutaneous heparin treatment were prospectively examined for skin reactions. If a skin lesion was observed, further diagnostics were performed (skin biopsy, subcutaneous provocation, clinical/laboratory assessment for thrombosis, bleeding, and heparin-induced thrombocytopenia [HIT]). Safety parameters were also analyzed (cross-allergies, frequency of thromboembolic and bleeding complications, HIT, and pregnancy outcome). RESULTS: Among 111 pregnant patients, 22 (19.8%) had heparin-induced skin reactions (95% CI, 13% to 29%). All lesions were caused by allergic delayed-type hypersensitivity (DTH) reactions and not by HIT or other rare conditions. The median time of onset was 50.5 days (range, 5-184 days). The cross-reactivity rate was 33.3%. While nadroparin treatment exhibited a higher DTH risk than dalteparin (hazard ratio [HR], 26.7; 95% CI, 3.4-211.0; P = .00187), enoxaparin treatment was not significantly different from dalteparin treatment (HR, 5.6; 95% CI, 0.3-96.1; P = .238). Three thromboembolic events and 1 major bleeding event occurred. CONCLUSIONS: Among patients receiving long-term heparin anticoagulation during pregnancy, heparin-induced skin lesions are frequent (incidence, 19.8%) and are all caused by allergic DTH reactions. Nadroparin has the highest frequency of skin lesions (approximately 65% at 100 days), which is significantly higher than that of dalteparin (HR, 26.7). Therefore nadroparin use should be avoided in pregnancy when possible.

The implementation of surface plasmon resonance technique in monitoring pregnancies with expected fetal and neonatal alloimmune thrombocytopenia.

BACKGROUND: Maternal anti-HPA-1a alloantibodies are responsible for most cases of severe fetal and neonatal alloimmune thrombocytopenia (FNAIT). The presence of HPA-1a alloantibodies in maternal blood alone does not predict the fetal platelet (PLT) count, and the predictivity of antibody titers determined by enzyme immunoassays (EIAs) is debated. In contrast to EIA, surface plasmon resonance (SPR) provides information on antibody-binding properties. STUDY DESIGN AND METHODS: Sequential sera from pregnant women with expected FNAIT were assessed for HPA-1a alloantibodies using SPR. Group I (n = 6) was treated with intravenous immunoglobulin (IVIG) and steroids beginning at 19 weeks of gestation (w.g.), and Group II (n = 4) received intrauterine PLT transfusions (IUT) beginning at 22 w.g. Maternal alloantibodies were quantified using an HPA-1a monoclonal antibody (MoAb) as a standard. Antibody avidity was determined as the ratio of B700 (end of the dissociation phase) to B350 (end of the association phase); the area under the curve (AUC) was calculated to determine overall antibody binding. RESULTS: After 22 w.g., alloantibody characteristics remained stable in both groups, while there was a steep decrease in B700 and B350 values between 16 and 22 w.g. (assessed only in Group I), indicating a decrease in anti-HPA-1a alloantibody concentrations. Interestingly, the AUCs of the last maternal sample before elective delivery appeared to be correlated with fetal and neonatal PLT counts (p = 0.014 and 0.017, respectively). CONCLUSION: SPR provides quantitative information on HPA-1a alloantibody characteristics in addition to monoclonal antibody-specific immobilization of platelet antigens. SPR results can be calibrated using a MoAb standard and should be further assessed for a potential correlation with fetal PLT count.

Application, tolerance and safety of fondaparinux therapy in a German hospital: a prospective single-centre experience.

INTRODUCTION: The pentasaccharide fondaparinux is widely approved for prophylaxis and treatment of thromboembolic diseases and therapy of acute coronary syndrome. It is also used off-label in patients with acute, suspected or antecedent heparin-induced thrombocytopenia (HIT). The aim of this prospective observational cohort study was to document fondaparinux' prescription practice, tolerance and therapy safety in a representative mixed German single-centre patient cohort. PATIENTS AND METHODS: Between 09/2008 - 04/2009, 231 consecutive patients treated with fondaparinux were enrolled. Medical data were obtained from patient's records. The patients were clinically screened for thrombosis (Wells score), sequelae of HIT (4T's score), and bleeding complications (ISTH-criteria) and subjected to further assessment (i.e. sonography, HIT-diagnostics), if necessary. The mortality rate was assessed 30 days after therapy start. RESULTS: Overall, 153/231 patients had a prophylactic, 74/231 patients a therapeutic, and 4/231 patients a successive prophylactic/therapeutic indication. In 11/231 patients fondaparinux was used due to suspected/antecedent HIT, in 5/231 patients due to a previous cutaneous delayed-type hypersensitivity to heparins. Other indications were rare. Three new/progressive thromboses were detected. No cases of HIT, major bleedings, or fatalities occurred. CONCLUSIONS: Fondaparinux was well tolerated and was safe in prophylaxis and therapy; prescriptions mostly followed the current approval guidelines and were rarely related to HIT-associated indications (<5% of prescriptions), which is in contrast to previous study results in the U.S. (>94% of prescriptions were HIT-associated). A trend towards an individualised fondaparinux use based on the compound's inherent properties and the patients' risk profiles, i.e., antecedent HIT, bone fractures, heparin allergy, was observed.

Implication of transfected cell lines for the detection of alloantibodies against human neutrophil antigen-3.

BACKGROUND: Alloantibodies against human neutrophil antigen-3 (HNA-3) are responsible for the fatalities reported in transfusion-related acute lung injury. Consequently, reliable detection of these alloantibodies is mandatory to improve blood transfusion safety. In this study, we developed stable cell lines for the detection of HNA-3 antibodies. STUDY DESIGN AND METHODS: HEK293T were transfected with HNA-3a or HNA-3b constructs and sorted by flow cytometry according to high surface expression. Transfected cells were tested with sera containing HNA-3 antibodies in flow cytometry and antibody capture assay (ACA). The results were compared with granulocyte agglutination test and granulocyte immunofluorescence test. RESULTS: In flow cytometry, 12 of 14 HNA-3a sera reacted specifically with HNA-3aa cells. One serum sample showed positive reaction with HNA-3bb cells. All HNA-3b sera recognized HNA-3bb cells. No reaction was observed with broad reactive antibodies against HLA Class I. In ACA, all HNA-3a sera (12/12) showed positive reactivity with HNA-3aa cells with no cross-reactivity with HNA-3bb cells. Again, all HNA-3b sera reacted with HNA-3bb cells only. Furthermore, genotyping of 249 individuals detected a new HNA-3 allele caused by a nucleotide substitution C>T at Position 457 leading to L(153)F mutation in choline transporter-like protein-2. This mutation impairs polymerase chain reaction with sequence-specific primers based HNA-3a typing. However, analysis with cells expressing F(153) isoform showed that this mutation did not alter the binding of HNA-3 antibodies. CONCLUSIONS: This study demonstrated that HEK293T cells expressing stable recombinant HNA-3 are suitable for the detection of HNA-3 alloantibodies allowing reliable screening of blood products.

A new platelet alloantigen, Swi(a) , located on glycoprotein Ia identified in a family with fetal and neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disorder caused by transplacental passage of maternal antibodies to fetuses whose platelets (PLTs) express the corresponding human PLT antigen (HPA). STUDY DESIGNS AND METHODS: We observed a fetus with FNAIT who died from a severe intracranial hemorrhage. Analysis of maternal serum in antigen capture assay with paternal PLTs showed reactivity with PLT glycoprotein (GP)IIb/IIIa (α(IIb) ß(3) ) and GPIa/IIa (α(2) ß(1) integrin), indicating the presence of anti-HPA-1a and an additional alloantibody against GPIa (termed anti-Swi(a) ). RESULTS: By immunochemical studies, the localization of the Swi(a) antigen on GPIa/IIa could be confirmed. Analysis of paternal GPIa full-length cDNA showed a single-nucleotide substitution C(3347) T in Exon 28 resulting in a Thr(1087) Met amino acid substitution. Testing of family members by polymerase chain reaction-restriction fragment length polymorphism using MslI endonuclease showed perfect correlation with phenotyping. Extended family and population studies showed that 4 of 10 members of the paternal family but none of 500 unrelated blood donors were Swi(a) carriers. Expression studies on allele-specific transfected Chinese hamster ovary (CHO) cells confirmed that the single-amino-acid substitution Thr(1087) Met was responsible for the formation of the Swi(a) epitope. Adhesion of CHO cells expressing the Swi(a) alloantigen to immobilized collagens was not impaired compared to the wild-type control and was not inhibited by anti-Swi(a) alloantibodies. CONCLUSION: In this study we defined a new PLT alloantigen Swi(a) that was involved in a case of additional immunization against HPA-1a. Our observations demonstrate that combinations of PLT-specific alloantibodies may comprise low-frequency alloantigens.

Low allergenic potential with fondaparinux: results of a prospective investigation.

OBJECTIVE: To determine the incidence and causes of skin reactions to the synthetic pentasaccharide fondaparinux. PATIENTS AND METHODS: Patients who received prophylactic/therapeutic subcutaneous fondaparinux treatment for more than 7 days were prospectively examined for cutaneous adverse effects between September 1, 2008, and April 30, 2009. When indicated, other procedures, such as skin biopsy, allergy testing, and clinical/laboratory assessment for thrombosis and heparin-induced thrombocytopenia, were performed. RESULTS: Overall, 231 patients were enrolled. No patient developed typical delayed type IV hypersensitivity (DTH) erythematous skin lesions. However, one female patient experienced abdominal pruritus at sites of injection. Histology revealed a mild lymphohistiocytic infiltrate, confirming a DTH reaction. Heparin-induced thrombocytopenia, as another possible underlying pathomechanism for cutaneous lesions, was ruled out clinically and serologically. Hence, the overall incidence of fondaparinux-induced allergic skin lesions was 0.4% (95% confidence interval, 0.01%-2.4%). No cross-allergies were observed in patients with DTH reaction to heparins. CONCLUSION: Fondaparinux has a low allergenic potential. The incidence of allergic cutaneous DTH reactions is almost 20 times lower compared to that with commonly used heparins. These results, together with the known low prevalence of secondary thrombotic events or heparin-induced thrombocytopenia during fondaparinux therapy, suggest that in selected patients fondaparinux might substantially improve patient care, therapeutic safety, and cost-effectiveness of anticoagulant therapy. TRIAL REGISTRATION: clinicaltrials.gov identifier: NCT00510432.

Safety and tolerability of inhaled heparin in idiopathic pulmonary fibrosis.

BACKGROUND: Abnormalities in alveolar coagulation occur in idiopathic pulmonary fibrosis (IPF). Anticoagulants attenuate bleomycin-induced lung fibrosis in animals. In this study, we first examined the pharmacokinetics of inhaled heparin in healthy subjects. Second, we investigated the safety and tolerability of heparin inhalation in IPF patients. METHODS: Coagulation assays were performed in blood and bronchoalveolar lavage fluid samples from 19 healthy volunteers after inhalation of increasing amounts of unfractionated heparin. The acute effects of heparin inhalation on lung function and exercise capacity and the safety and tolerability of chronic heparin inhalation for 28 days were assessed in 20 IPF patients in an open-label exploratory pilot study. RESULTS: In healthy subjects, inhalation of 150,000 IU heparin ("filled dose") significantly increased the partial thromboplastin time and anti-factor Xa activity in blood samples indicating the threshold dose. The local alveolar anticoagulant effect was detectable up to 72 h, and the alveolar half-life was estimated at 28 h. In IPF-patients, no acute deleterious effects on pulmonary function, gas exchange, or exercise capacity were noted after inhalation of the threshold dose. During chronic treatment, where one-fourth of the threshold dose was inhaled every 12 h for 28 days to obtain a steady-state anticoagulant activity in the alveolar space approximating the anticoagulant activity observed after threshold dose inhalation, no heparin-related side effects, such as hemoptysis or heparin-induced antibodies and thrombocytopenia, were detected in any patient, and median lung function values, exercise capacity, and quality of life scores appeared largely unaltered. Three adverse and one serious adverse events were noted; however, the relation of these events to the heparin inhalation was assessed as "unlikely" or "no relation" in each case. CONCLUSIONS: Inhaled heparin appears to be safe and well tolerated in IPF patients. Future clinical trials are required to demonstrate the long-term safety and efficacy of inhaled heparin in IPF.

Incidence and causes of heparin-induced skin lesions.

BACKGROUND: Little is known about the incidence and causes of heparin-induced skin lesions. The 2 most commonly reported causes of heparin-induced skin lesions are immune-mediated heparin-induced thrombocytopenia and delayed-type hypersensitivity reactions. METHODS: We prospectively examined consecutive patients who received subcutaneous heparin (most often enoxaparin or nadroparin) for the presence of heparin-induced skin lesions. If such lesions were identified, we performed a skin biopsy, platelet count measurements, and antiplatelet-factor 4 antibody and allergy testing. RESULTS: We enrolled 320 patients. In total, 24 patients (7.5%, 95% confidence interval [CI] 4.7%-10.6%) had heparin-induced skin lesions. Delayed-type hypersensitivity reactions were identified as the cause in all 24 patients. One patient with histopathologic evidence of delayed-type hypersensitivity tested positive for antiplatelet-factor 4 antibodies. We identified the following risk factors for heparin-induced skin lesions: a body mass index greater than 25 (odds ratio [OR] 4.6, 95% CI 1.7-15.3), duration of heparin therapy longer than 9 days (OR 5.9, 95% CI 1.9-26.3) and female sex (OR 3.0, 95% CI 1.1-8.8). INTERPRETATION: Heparin-induced skin lesions are relatively common, have identifiable risk factors and are commonly caused by a delayed-type hypersensitivity reaction (type IV allergic response).

Rapid screening of granulocyte antibodies with a novel assay: flow cytometric granulocyte immunofluorescence test.

BACKGROUND: White blood cell (WBC)-associated antibodies can lead to severe pulmonary transfusion reactions (transfusion-related acute lung injury [TRALI]). Investigation of a large number of blood donor samples using the standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) proved to be difficult to perform due to the time-consuming process and the large quantity of test cells required. This study describes the novel flow cytometric GIFT (Flow-GIFT) method for a rapid detection of granulocyte antibodies by flow cytometric analysis. STUDY DESIGN AND METHODS: A total of 141 sera were analyzed for the presence of granulocyte antibodies that were previously associated with suspected TRALI. As test cells whole blood samples from human neutrophil antigen (HNA)-typed donors were isolated using cell sedimentation in a ficoll density gradient. WBCs were incubated with the respective serum and binding of antibodies to the test cells was detected using fluorescein isothiocyanate-conjugated anti-human antibody. Standard GIFT and GAT were performed as reference methods. RESULTS: Seven sera containing anti-HNA-3a, CD16, and HLA Class I were negative in the standard GIFT and eight sera containing anti-HNA-2a, anti-CD16, and anti-HLA Class I were not detected in the GAT. The novel Flow-GIFT was able to detect all granulocyte antibodies, which were only detectable in a combination of standard GIFT and GAT. In serial dilution tests, the Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. CONCLUSION: The Flow-GIFT method permits rapid detection of granulocyte antibodies requiring fewer donor test cells. This method is ideal for automation and will potentially open the way for screening of granulocyte antibodies in a large donor population.

Low-avidity HPA-1a alloantibodies in severe neonatal alloimmune thrombocytopenia are detectable with surface plasmon resonance technology.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is mostly caused by maternal alloantibodies directed against the human platelet alloantigen (HPA)-1a. Currently, the serologic diagnosis of FNAIT is based on the characterization of the HPA alloantibodies in monoclonal antibody-based antigen-capture assays (e.g., MAIPA assay). Accumulated current evidence indicated that such assays may overlook some HPA-1a antibodies. STUDY DESIGN AND METHODS: This study employed surface plasmon resonance (SPR) technology using immunoaffinity-purified glycoprotein IIb/IIIa isoforms immobilized on biosensor chips to study the binding kinetics of HPA-1a alloantibodies from different FNAIT cases in real time. RESULTS: Analysis of HPA-1a alloantibodies from FNAIT cases (n = 9) in SPR showed a moderate relative response (22.2-69.7 resonance units [RU]) and slow antibody dissociation. After the dissociation phase, varying amounts of bound antibodies (41%-79%) remained on the chip. In contrast in HPA-1a alloantibodies from a patient suffering from posttransfusion purpura, a high relative response (approximately 490 RU) was observed at the end of the association phase and no dissociation of antibody binding was detectable. Of particular relevance, by the use of this SPR technique, HPA-1a alloantibodies were detected in two severe FNAIT cases that had determined as false negative by MAIPA assay. In SPR, these HPA-1a alloantibodies showed low-avidity nature characterized by gradual dissociation of antibody during the association phase and complete detachment of antibody binding after the dissociation phase. This high "off-rate" character of low-avidity HPA-1a alloantibodies indicates that such antibody binding is easily detachable by the extensive washing procedure of the MAIPA. CONCLUSIONS: Our results demonstrated that the SPR method can facilitate the diagnosis of clinically relevant low-avidity HPA-1a antibodies.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for genotyping of human platelet-specific antigens.

BACKGROUND: Genotyping of single-nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technique, where finally tools for end users have become available to design primers and analyze SNPs of their own interest. This study investigated the potential of this technique in platelet (PLT) genotyping and developed a validated method for genotyping of clinical relevant human PLT antigens (HPAs). STUDY DESIGN AND METHODS: A multiplex assay using MALDI-TOF MS to analyze six HPA loci (HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, and HPA-15) simultaneously in a single reaction was applied for the genotyping of 100 DNA samples from a cohort of plateletpheresis donors and a patient population (n = 20) enriched for rare alleles. The genotyping results using MALDI-TOF MS were validated by the comparison with the results from typing by polymerase chain reaction with sequence-specific primers and conventional DNA sequencing. RESULTS: Both homozygous and heterozygous genotypes of HPA-1 to -5 and -15 of the 120 individuals were easily identified by a six-plexed assay on MALDI-TOF MS. The three approaches achieved a 100 percent concordance for the genotyping results of the six HPA loci. CONCLUSION: Compared to conventional methods, the MALDI-TOF MS showed several advantages, such as a high velocity, the ability to perform multiplexed assays in a single reaction, and automated high-throughput analysis of samples. This enables cost-efficient large-scale PLT genotyping for clinical applications.

Heterogeneity of HPA-3 alloantibodies: consequences for the diagnosis of alloimmune thrombocytopenic syndromes.

BACKGROUND: Immunization against the human platelet alloantigen (HPA)-3a residing on alphaIIbbeta3 integrin accounts for approximately 2 percent of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Anti-HPA-3a alloantibodies are sometimes difficult to detect and can be overlooked by standard antigen capture assays. STUDY DESIGN AND METHODS: The reactivity of 12 anti-HPA-3a and 2 anti-HPA-3b alloantibodies from patients with FNAIT and posttransfusion purpura was analyzed by serologic (monoclonal antibody-specific immobilization of platelet antigens [MAIPA] assay, flow cytometry) and immunochemical (immunoprecipitation, immunoblotting) techniques. The influence of platelet (PLT) age, storage conditions, recombinant antigens from Chinese hamster ovary (CHO) cells, and sialic acids (treatment with neuraminidase) were analyzed. RESULTS: The most sensitive anti-HPA-3 alloantibody detection in MAIPA assay could be achieved with fresh homozygous PLTs. During a PLT storage period of 14 days before use, three types of anti-HPA-3 alloantibodies were found: 1) complete loss of reactivity (n = 6), 2) considerably weakened reaction (> or =50% reduction; n = 3), and 3) minor reduction of reactivity (< or =40% decrease; n = 5). When cryopreserved PLTs were used, 10 of 12 anti-HPA-3a and all anti-HPA-3b alloantibodies reacted positive. Only 6 of 10 serum samples reacted with recombinant HPA-3a on CHO cells. Neuraminidase treatment of PLTs showed that some anti-HPA-3a alloantibodies require the presence of sialic acids. The storage lesion seems to be related to cleavage of sialic acids. Immunochemical analysis revealed evidence that most anti-HPA-3a alloantibodies require an intact three-dimensional alphaIIbbeta3 integrin structure. CONCLUSIONS: Anti-HPA-3 alloantibodies show considerable heterogeneity, which may hamper the serologic diagnosis of FNAIT. Preservation of the alphaIIbbeta3 integrin and protection from enzymatic degradation seem to be important during PLT storage.

Marked hyperbilirubinemia associated with the heme oxygenase-1 gene promoter microsatellite polymorphism in a boy with autoimmune hemolytic anemia.

Mild hyperbilirubinemia is a clinical feature of hemolysis. Here we describe a boy with marked elevation of serum bilirubin values (maximum: 70 mg/dL) during an acute episode of autoimmune hemolytic anemia, which returned to within the reference range after clinical improvement. The boy was a homozygous carrier of short alleles of the heme oxygenase-1 (HO-1) gene GT dinucleotide-repeat promoter polymorphism, which is associated with increased activity and inducibility of the heme-degrading enzyme HO-1, which catalyzes the production of bilirubin. In addition, heterozygosity of the uridine 5'-diphosphate-glucuronosyl-transferase 1A1 promoter polymorphism that is linked with Gilbert syndrome was found in this patient. Because bilirubin production plays a critical role during the neonatal period, the HO-1 promoter polymorphism may be an important genetic factor for the clinical outcome of neonatal hyperbilirubinemia.

A prospective study on the prevalence and risk factors for neonatal thrombocytopenia and platelet alloimmunization among 9332 unselected Brazilian newborns.

BACKGROUND: Neonatal thrombocytopenia (NT) occurs in 0.5 to 0.9% of unselected Caucasian newborns. However, the prevalence of this complication in other populations is unknown. In this study the prevalence/causes of NT was determined in Brazilian newborns, a population characterized by admixture among Indigenous, Africans, and Caucasians. STUDY DESIGN: A prospective study was carried out in a 3-year period, to determine the prevalence and causes of thrombocytopenia in cord blood samples. Genotyping for HPA 1-5 systems was performed in pairs of mother/neonates with and without thrombocytopenia. All mothers with genotypic mismatches from each group were tested for HPA-specific antibody using the MAIPA technique to identify alloimmunization. RESULTS: Platelet counts <100 x 10(9)/L were detected in 1.5% of 9,332 unselected newborns. In 0.15%, platelet count was <50 x 10(9)/L. Clinically significant bleeding was rare. Underlying diseases were present in 48% of the thrombocytopenic cases. HPA 1-5 system genotype mismatches occurred in 50% of gestations, but did not predict the risk for thrombocytopenia. Notably, mismatched genotypes for HPA-5 were slightly increased in the thrombocytopenic group. The presence of anti-HPA-5b antibodies was observed in 0.05% of unselected pregnancies, but increased to 12% among mothers of neonates with thrombocytopenia and mismatched genotype (N = 51). CONCLUSIONS: Neonatal thrombocytopenia is common among Brazilian newborns at rates comparable with those described among Caucasians. These data suggest that screening for genotypic HPA mismatch, followed by an HPA-specific immunoassay system, particularly for the HPA-5 system, among mothers of newborns with thrombocytopenia in our population would allow the identification of pregnancies at risk of alloimmune thrombocytopenia.

A variable number of tandem repeats polymorphism influences the transcriptional activity of the neonatal Fc receptor alpha-chain promoter.

The neonatal Fc receptor, FcRn, plays a central role in immunoglobulin G (IgG) transport across placental barriers. Genetic variations of FcRn-dependent transport across the placenta may influence antibody-mediated pathologies of the fetus and the newborn. Sequencing analysis of 20 unrelated individuals demonstrated no missense mutation within the five exons of the FcRn gene. However, a variable number of tandem repeats (VNTR) region within the FcRn promoter was observed, consisting of five different alleles (VNTR1-VNTR5). Alleles with two (VNTR2) and three (VNTR3) repeats were found to be most common in Caucasians (7.5 and 92.0%, respectively). Real-time polymerase chain reaction revealed that monocytes from VNTR3 homozygous individuals express 1.66-fold more FcRn transcript than do monocytes from VNTR2/VNTR3 heterozygous individuals (P = 0.002). In reporter plasmid assays, the VNTR3 allele supported the transcription of a reporter gene twice as effectively as did the VNTR2 allele (P = 0.003). Finally, under acidic conditions, monocytes from VNTR3 homozygous individuals showed an increased binding to polyvalent human IgG when compared with monocytes from VNTR2/VNTR3 heterozygous individuals (P = 0.021). These data indicate that a VNTR promoter polymorphism influences the expression of the FcRn receptor, leading to different IgG-binding capacities.