Assessment of HNA alloimmunisation risk in Northeastern Thais, Burmese and Karen.

OBJECTIVES: This study aimed to determine human neutrophil antigen (HNA) frequency, estimate possible HNA incompatibilities and predict the risk of HNA alloimmunisation in the Northeastern Thai, Burmese and Karen populations. BACKGROUND: Alloantibodies against HNA are implicated in a number of clinical conditions, including immune-mediated neutropenia and transfusion reactions. METHODS: A total of 400 unrelated healthy Thais, 261 Burmese and 249 Karen was included in this study. DNA samples were typed for HNA-1, -3, -4 and -5 systems using polymerase chain reactions with sequence-specific primers (PCR-SSP). RESULTS: In this cohort, HNA-1a was more prevalent than HNA-1b. Accordingly, the possible risk of HNA-1a alloimmunisation against HNA-1a is lower than HNA-1b (0·0802-0·1351 vs 0·2293-0·2497). This is in contrast to the situation reported in Caucasian and African populations. The predicted risk of HNA-3 incompatibility in Thais, Burmese and Karen were 28·09%, 30·66% and 22·77%, respectively. The possible risks of HNA-3a alloimmunisation were 0·0493 in Thais, 0·0608 in Burmese and 0·0196 in Karen, respectively. No individuals were found to be homozygous for HNA-4bb. The probability of developing alloantibodies against HNA-4a was low in these populations and every population in Asia. In contrast, the overall frequency of HNA-5bb homozygous individuals was high in this study, peaking at 0·192. CONCLUSIONS: This is the first study that reported the allele frequencies of HNA-1, -3, -4, and -5 in a large sample of healthy unrelated individuals from ethnic Thais, Burmese and Karen. Our results indicated the high possible risk of HNA-1, -3 and -5 alloimmunisation in these populations.

Analysis of platelet-reactive alloantibodies and evaluation of cross-match-compatible platelets for the management of patients with transfusion refractoriness.

BACKGROUND: Cross-match-compatible platelets can improve corrected count increments (CCIs) in alloimmunised patients with transfusion refractoriness. However, only a few studies mentioned that the specificities of platelet-reactive alloantibodies can predict high reactivity in cross-match assays among these patients. METHODS: A total of 204 medical records of patients who were refractory to random single-donor apheresis platelets between January 2014 and December 2014 were enrolled. Platelet-reactive antibodies in patients' serum were screened by an enzyme-linked immunosorbent assay (ELISA).The platelet cross-match assays were performed by a solid-phase adherence assay. The specificities of human leukocyte antigen (HLA) class I and human platelet antigens (HPAs) alloantibodies were determined by Luminex Single Antigen and Monoclonal Antibody-specific Immobilization of Platelet Antigens (MAIPA) assays, respectively. RESULTS: Anti-HLA and anti-HPA alloantibodies were found in 114 of 204 (55.88%) patients, including 110 (96.49%) with anti-HLA alloantibodies only, 2 (1.75%) with anti-HPA alloantibodies (anti-GPIIb/IIIa) only and 2 (1.75%) with both anti-HLA and anti-HPA alloantibodies (anti-HPA-3a and anti-HPA-5b). The most common HLA class I alloantibody phenotypes in cross-match-incompatible patients were HLA-A23 (59.38%), -A24 (50.00%), -A02 (43.75%), -B27 (65.63%), -B40 (50.00%), -B18 (46.88%) and -B07 (43.75%). A total of 480 cross-matched platelet units were administered in 82 of 114 alloimmunised patients with a mean CCI of 7800 ± 5200, a significant improvement over random platelet units (P < 0.001). CONCLUSIONS: No development of additional platelet alloantibodies was observed during this platelet transfusion regiment. This study showed that transfusion of cross-match-compatible platelet units offers effective and safe management of platelet transfusion refractoriness (PTR). The finding of alloantibodies among cross-match-incompatible cases can be used as predictors for platelet donor selection.

The prevalence of leucocyte alloantibodies in blood donors from South China.

BACKGROUND: Several studies had demonstrated that leucocyte antibodies including anti-human leucocyte antigen (anti-HLA) antibodies (class I and class II) and anti-human neutrophil antigen (anti-HNA) antibodies (HNA-1, -2 and -3) present in the blood products are responsible for transfusion-related acute lung injury (TRALI). Therefore, selection of blood products exclusive of anti-HLA and anti-HNA antibodies may lower the risk of TRALI reaction. However, the prevalence of leucocyte antibodies among blood donors in China is currently not known. STUDY DESIGN AND METHODS: Blood samples were collected from 454 male and 560 female donors (143 nulliparous and 417 multiparous female). HLA class I and II antibodies were analyzed by bead assays. Anti-HNA-1 and -2 antibodies were screened by the LABScreen assay (One Lambda Inc.), and HNA-3 were detected by antigen capture assay, and confirmed by the granulocyte agglutination test (GAT). RESULTS: Screening of the total cohort showed higher prevalence of HLA antibodies in female compared with male donors (19.64 vs. 4.63%). We found antibodies against HLA class I (13.21%) and HLA class II (11.43%) in 560 female donors. The most frequent antibodies against HLA class I and II in parous females (n = 69) reacted with were A*11 (28.81%), B*07 (42.37%), Cw*07 (20.34%) and DRB1*04 (40.43%) molecules. Among 778 donors (randomly selected from 1014 donors), we found three donors with neutrophil reactive antibodies, two against HNA-2 and one without known specificity. Anti-HNA-3 antibodies were not found so far. CONCLUSION: In this study, we found alloimmunization against HLA class I, II and HNA in 4.63, 24.70 and 0.39%, respectively, in our female blood donors, indicating that the use of plasma containing blood products from parous female blood donors without HLA antibodies pre-testing may increase the risk of TRALI reaction. Although immunization against HNA seems to be a rare event in China, further observation is necessary to decide the necessity of HNA antibodies screening in our blood donors.

[Diagnosis of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)]. / Diagnose angeborener Störungen der Thrombozytenfunktion. Interdisziplinäre S2K-Leitlinie der Ständigen Kommission Pädiatrie der Gesellschaft für Thrombose- und Hämostaseforschung e. V.

Congenital disorders of platelet function are a heterogeneous group of disorders that are often not detected until bleeding occurs. In clinical settings only a few methods have proven to be useful for identification and classification of inherited platelet disorders. For a rational diagnostic approach, a stepwise algorithm is recommended. Patient history and clinical investigation are mandatory. Von Willebrand disease and other coagulation disorders should always be ruled out prior to specific platelet testing. Platelet count, size, volume (MPV) and morphology may guide further investigations. The PFA-100® CT is suited for screening for severe platelet defects. Platelet aggregometry allows assessment of multiple aspects of platelet function. Flow cytometry enables diagnosis of thrombasthenia Glanzmann, Bernard-Soulier syndrome and storage pool defects. Molecular genetics may confirm a putative diagnosis or pave the way for identifying new defects. We present an unabridged version of the interdisciplinary guideline.

Human platelet-specific antigen frequencies in Indonesian population.

BACKGROUND: Alloantibodies against human platelet antigens (HPAs) are responsible for the development of alloimmune thrombocytopenia including platelet transfusion refractoriness (PTR) and neonatal alloimmune thrombocytopenia (NAIT). Therefore, transfusion of HPA-compatible platelets is of importance for the management of these diseases. AIM: Determination of the allele frequency of the major HPA systems for Indonesian blood donors and the development of the first HPA-typed donor registry in Indonesia. METHODS: DNA derived from 500 Indonesian healthy blood donors was genotyped for HPA-1 to HPA-6 and HPA-15 alleles by the use of polymerase chain reaction sequence-specific primer method. RESULTS: The gene frequencies of the rare allelic variants HPA-1b, -2b, -3b, -4b, -5b, -6b and -15b were 0·023, 0·060, 0·493, 0·052, 0·032, 0·044 and 0·049, respectively. However, donors homozygous for the HPA-1b, -2b and -6b were not found in this cohort, indicating that the risks of alloimmunisation caused by incompatibility of these three HPA systems are extremely low. In contrast, alloimmunisation against HPA-3, -4, -5 and -15 systems is anticipated. CONCLUSION: The development of an HPA-genotyped registry for donors homozygous for HPA-1b, -2b and -6b is desired for the optimum management of PTR patients and children with NAIT.

The frequencies of human neutrophil alloantigens among the Japanese population.

Human neutrophil antigens (HNAs) play an important role in a variety of clinical conditions including immune-mediated neutropenia, non-hemolytic transfusion reactions, and transfusion-related acute lung injury. The aim of this study was to investigate the frequency distribution of HNAs-1 to -5 among the Japanese population. We analyzed samples from 570 healthy Japanese by molecular and serologic techniques to estimate the gene frequencies of HNAs-1 to -5. DNA samples were obtained and typed for the HNA-1 (n = 523), -3 (n = 570), -4 (n = 570), and -5 (n = 508), by molecular techniques. The HNA-1 genotype was determined by using a commercial polymerase chain reaction-reverse sequence-specific oligonucleotide probes (PCR-rSSOP) kit. The HNA-3 to -5 genotypes were determined by the PCR-sequence specific primer (PCR-SSP), previously described, with a small modification. The HNA-2a phenotype was determined in 301 donors by granulocyte immunofluorescence test. In Japanese, the gene frequencies of HNA-1a, -1b, and -1c were 0.623, 0.377, and 0.000, respectively. The frequency of HNA-2a phenotype was 0.987, and the gene frequencies of HNA-3a and -3b were 0.654 and 0.346, respectively. HNA-4a and -4b were found at 1.000 and 0.000, respectively, and HNA-5a and -5b at 0.840 and 0.160, respectively. We describe, for the first time, the frequencies of all HNAs (HNA-1 to -5) among the Japanese population. This study will be helpful for the prediction of the risk of alloimmunization to HNA, especially to determine the risk of HNA alloantibody production by transfusion of HNA incompatible blood and feto-maternal incompatibility.

Report on the 15th International Society of Blood Transfusion platelet immunology workshop.

BACKGROUND AND OBJECTIVES: The aim of the 15th ISBT Platelet Immunology Workshop was to evaluate the detection of free platelet-reactive autoantibodies from ITP patients by the use of a standardized MAIPA protocol, to compare sensitivity and specificity of antibody detection for anti-HPA-1a and serologically difficult-to-assess antibodies against HPA-3, to identify whether anti-HPA-1a titration results can be compared between laboratories, and to evaluate HPA genotyping methods. MATERIALS AND METHODS: Workshop materials were shipped from the organizing laboratory in Giessen, Germany. Thirty laboratories from 19 countries participated. RESULTS: Results for the detection of autoantibodies differed greatly between the laboratories and no consensus was reached for one of the two sera. Detection and titration of antibodies against HPA-1a, in contrast, gave largely congruent results. Serologically difficult-to-assess antibodies recognizing HPA-3a and HPA-3b were not detected by many laboratories. For genotyping, good agreement was achieved. CONCLUSIONS: Detection of HPA-1a antibodies, titration of anti-HPA-1a, and HPA genotyping are well performed in most participating laboratories. The workshop has identified two specific areas with room and need for improvement: the detection of autoantibodies and the detection of HPA-3 alloantibodies. Recommendations of the Working Party on techniques that can help to overcome these problems are desirable.

Novel homozygous mutation (c.175delG) in platelet glycoprotein ITGA2B gene as cause of Glanzmann's thrombasthenia type I.

BACKGROUND: Glanzmann's thrombasthenia (GT), is a rare autosomal recessive bleeding disorder. Platelets from patients with GT show quantitative or qualitative defects of the platelet membrane glycoprotein (GP) IIb/IIIa complex. A variety of genetic defects in ITGA2B and ITGB3 (genes for GPIIb and GPIIIa) has been described causing the clinical entity of GT. PATIENTS: A newborn with bleeding symptoms (petechiae) platelet analyses revealed an inherited primary hemostasis disorder. METHODS/RESULTS: Analyses of patient's platelets using flow cytometry and immunoblotting showed absence of GPIIb protein and reduced amount of GPIIIa. Using restriction fragment length polymorphism heterozygosity for the deletion could be identified in the parents and in two siblings. Expression studies in mammalian cells revealed that the mutant GPIIb is missing and additionally affects the expression of wildtype GPIIIa. This deletion leads to a truncation at the very N-terminal region of the GPIIb protein. CONCLUSION: The present study describes a patient with GT associated with a novel homozygous deletion (c.175delG) in exon 1 of ITGA2B. This deletion led to a reading frameshift and caused a severely truncated form of GPIIb.

Establishment of platelet donor registry improves the treatment of platelet transfusion refractoriness in Guangzhou region of China.

Platelet transfusion refractoriness (PTR) is the major complication of long-term platelet supportive care. To improve the effectiveness of platelet transfusion therapy in PTR patients, we aimed to establish a platelet donor registry in our region (Guangzhou, China) by typing the human leukocyte antigen (HLA) and human platelet antigen (HPA). Blood donors (n = 864) from our population were genotyped for HLA-A, HLA-B and HPA systems by polymerase chain reaction amplification with sequence-specific primer(PCR-SSP) techniques. Using this cohort, we compared the results of platelet transfusions (matched vs. random) in 23 patients with PTR. Matched platelets were selected either by HLA antigen matching or by HLA antibody matching, as predicted by antibody specificity prediction (ASP) analysis. Significantly higher platelet recovery (PPR) values were obtained with HLA-matched platelets in comparison with random platelets. No significant difference in PPR was observed between HLA matching and ASP methods. In two patients, platelet-specific alloantibodies (alloabs) (anti-HPA-3b and anti-HPA-5b) were detected besides HLA class I alloabs. Transfusion with HLA- and HPA-compatible platelets in both the patients resulted in significantly higher PPR when compared with HLA-compatible platelet transfusion alone. In this study, we demonstrated that the establishment of an HLA- and HPA-typed platelet aphaeresis donor registry is useful to improve the treatment outcome of PTR patients and to maintain a long-term platelet transfusion strategy.

Prospective evaluation of PF4/heparin immunoassays for the diagnosis of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies that recognize platelet factor 4-heparin (PF4/hep) complexes leading to platelet activation. Several methods are available for the identification of HIT antibodies. OBJECTIVES: To evaluate the clinical usefulness of different antigen-binding assays for detection of antibodies against PF4/hep complexes in a prospective study. PATIENTS/METHODS: A prospective cohort of 500 surgical and medical patients suspected of having HIT was evaluated. The laboratory assessment included particle gel immunoassay (PaGIA), polyspecific ELISA recognizing IgG, IgM and IgA antibodies (Poly-ELISA), IgG-specific ELISA (IgG-ELISA) and the HIPA test. The pretest probability of HIT was determined using the 4T's model. Positive and negative predictive values (PPV, NPV) of each immunoassay were determined depending upon the heparin-induced platelet activation (HIPA) results and the clinical scoring. The operating characteristics of each immunoassay were determined using the receiver-operation characteristic (ROC) curve. RESULTS: Platelet-activating antibodies were identified in 35/500 patients, all of whom had intermediate to high clinical probability. PF4/hep antibodies were detected in 124, 86 and 90 sera using Poly-ELISA (PPV = 28), IgG-ELISA (PPV = 40.6) and PaGIA (PPV = 36.6). NPV was 100% for Poly- and IgG-ELISA, but only 99.5% for PaGIA. ROC analysis revealed that PaGIA is less informative than ELISA. The IgG-ELISA provides better diagnostic information than the other assays. In addition, there is a clear correlation between optical density (OD) value and the probability of having HIT. CONCLUSIONS: Our observation indicates that an IgG-ELISA provides the best diagnostic information of all antigen-binding assays.

What's happening--probing of HPA-1a antigen-antibody interaction by surface plasmon resonance technology.

This brief report summarizes the use of surface plasmon resonance technology (SPRT) in probing HPA-1a antigen-antibody interactions, based on a poster presented at the 60th meeting of the American Association of Blood Banks. It was concluded that the GP purification method could affect the performance of antigen in SPRT. It also highlighted that chips immobilised with Monoclonal antibody (Mab)-purified GP-IIb/IIIa work satisfactorily with both monoclonal and recombinant Abs with the appropriate concentration and binding affinity, while determination of the avidity and concentration of maternal polyclonal antibodies in respect to clinical severity on NAIT warrants further development.

Three novel beta3 domain-deletion peptides for the sensitive and specific detection of HPA-4 and six low frequency beta3-HPA antibodies.

BACKGROUND: Antibodies against human platelet antigens (HPA) are clinically important in fetal-maternal alloimmune thrombocytopenia, refractoriness to platelet transfusions and post-transfusion purpura. Of the 16 HPAs, nine are located on the beta3 subunit of the alphaIIb beta3 integrin. Antibody detection is generally based on platelet-derived alphaIIb beta3 from HPA-genotyped donors. Recombinant allelic beta3 peptides, expressed at high levels would improve consistency in antibody detection, but the expression of soluble and monomeric integrins expressing complex dependent epitopes has previously proved challenging. OBJECTIVES: We aimed to generate three recombinant beta3 peptides for the detection of antibodies against HPA-4, HPA-8bw and five of the six remaining low frequency beta3 alloantigens. METHODS: The removal of the specificity-determining loop from the betaA domain and fusion of truncated beta3 to calmodulin was exploited to obtain expression of monomeric protein. Using site-directed mutagenesis, the mutations for HPA-4b and HPA-8bw were introduced in the ITGB3*001 haplotype. A third peptide for the detection of antibodies against HPA coded by non-synonymous single nucleotide polymorphisms of low frequency was generated by the introduction of five mutations forming the basis of HPA-6bw, -7bw, -10bw, -11bw, and -16bw antigens. RESULTS: Reactivity of the three peptides with beta3-specific murine monoclonal antibodies and human HPA-1a phage antibodies confirmed the structural integrity of the recombinant fragments, and reactivity with a unique panel of polyclonal anti-HPA sera confirmed expression of the relevant HPA epitopes. CONCLUSIONS: These data demonstrate that beta3 integrin domain-deletion fragments are suitable molecular targets for HPA antibody detection.

Genotyping of human neutrophil antigen-5a (Ond).

Antibodies to human neutrophil alloantigens (HNA) can cause immune-mediated neutropenias and transfusion complications. The diagnosis of antibodies to the HNA-5a (Ond) isoform of the alphaLbeta2 integrin is hampered by the lack of reliable methods for HNA-5a antigen typing. We have devised a polymerase chain reaction sequence-specific primer method (PCR-SSP) and used it to determine the HNA-5a gene frequencies in 320 individuals from different ethnicities. 15.3% were found to be HNA-5a negative, with no significant deviation between the populations. Results of HNA-5a genotyping were in accordance with phenotyping. Availability of HNA-5a PCR-SSP will facilitate the diagnosis of Ond antibody-mediated clinical conditions.

The role of junctional adhesion molecules in cell-cell interactions.

Cell-cell-interactions are important for the regulation of tissue integrity, the generation of barriers between different tissues and body compartments thereby providing an effective defence against toxic or pathogenic agents, as well as for the regulation of inflammatory cell recruitment. Intercellular interactions are regulated by adhesion receptors on adjacent cells which upon extracellular ligand binding mediate intracellular signals. In the vasculature, neighbouring endothelial cells interact with each other through various adhesion molecules leading to the generation of junctional complexes like tight junctions (TJs) and adherens junctions (AJs) which regulate both leukocyte endothelial interactions and paracellular permeability. In this context, emerging evidence points to the importance of the family of junctional adhesion molecules (JAMs), which are localized in tight junctions of endothelial and epithelial cells and are implicated in the regulation of both leukocyte extravasation as well as junction formation and permeability.

Human platelet alloantigens.

Antibody formation against alloantigens of the human platelet membrane is responsible for clinical syndromes and transfusion related conditions as neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP), platelet transfusion refractoriness (PTR) and passive alloimmune thrombocytopenia. Moreover, rare cases of alloimmune reactions involving platelets have been observed after transplantation of hematopoietic stem cells. Among alloantigens of the platelet membrane shared with other cells (type I alloantigens) are the glycoconjugates of the ABO system and class I human leukocyte antigen (HLA) antigens. Antibodies against these structures are responsible for PTR and for febrile nonhemolytic transfusion reactions. Antibodies against type II antigens (formerly termed "platelet specific antigens") have been observed in NAIT, PTP and passive alloimmune thrombocytopenia. ABH antigens have been identified on intrinsic platelet membrane glycoproteins. Moreover, it is now clear that HLA class I antigens are an integral part of the platelet membrane. The quantity of both HLA and ABH-antigen expression on the platelet membrane varies considerably. Single point mutations account for almost all platelet specific alloantigens, but most antigenic determinants seem to depend upon glycoprotein conformation: generally, platelet specific alloantibodies fail to recognize synthetic peptides encompassing the polymorphic residues. Restriction fragment polymorphism analysis and allele-specific PCR have been implemented for genotyping of platelet alloantigens in many laboratories. Antigen specific assays using monoclonal antibodies (MAIPA, immunobead assay) became de facto standard for diagnosis of platelet antibodies in serum/plasma samples. It can be expected that innovative techniques as human alloantibody fragments produced by phage display technique and the production of recombinant antigens will allow rapid and reliable phenotyping and antibody detection in the future.