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Willem H Ouwehand - One of the best experts on this subject based on the ideXlab platform.
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an international reference reagent minimum sensitivity for the detection of anti Human Platelet Antigen 1a
Vox Sanguinis, 2009Co-Authors: Paul Metcalfe, M. De Haas, David Allen, Riitta Kekomaki, C Kaplan, Willem H OuwehandAbstract:Background and Objectives The Platelet-specific alloantibody anti-Human Platelet Antigen (HPA) 1a is involved in feto-maternal alloimmune thrombocytopenia, post-transfusion purpura and Platelet refractoriness. The existing minimum potency preparation for the detection of anti-HPA-1a (NIBSC code 93/710) was established by World Health Organization in 1997 and is used by laboratories to validate new assays or to calibrate ‘in-house’ controls. However, it has been well-used and a replacement is required. This report describes the production and comparative evaluation of a freeze-dried preparation of pooled Human plasma, coded 05/106, containing anti-HPA-1a. Materials and Methods Plasma containing anti-HPA-1a was obtained and 2974 1-ml aliquots were prepared and freeze-dried in glass ampoules. In order to characterize the material and compare it to the existing reference material, three collaborative studies were organized, involving a total of 50 different laboratories in 23 countries. Results As expected only anti-HPA-1a could be detected in the plasma and no additional HPA or Human leucocyte Antigen antibodies were detected. When tested in titration, there was a wide variation in the sensitivity of antibody detection by different laboratories, irrespective of the technique used. However, there was no significant difference between the two materials when compared using a t-test. Conclusions When diluted 1 in 2, most laboratories were able to detect the presence of anti-HPA-1a in both materials and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. In October 2007, the World Health Organization Expert Committee on Biological Standardization approved the material 05/106 as an International Reference Reagent.
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severe fetomaternal alloimmune thrombocytopenia due to anti Human Platelet Antigen hpa 1a in a mother with a rare and silenced itgb3 0101 gpiiia allele
Vox Sanguinis, 2007Co-Authors: Graham A Smith, Willem H Ouwehand, Angela Rankin, C Riddle, C Cheethamwilkinson, E Ranasinghe, Nicholas A WatkinsAbstract:Background and Objectives Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal antibodies against a Human Platelet Antigen (HPA) present on fetal, but absent from maternal Platelets. We identified and characterized a case of FMAIT due to anti-HPA-1a in a mother with an HPA-1a1b genotype. Materials and Methods The first child of a 29-year-old mother presented with a petechial rash and a Platelet count of 8 × 109 per l. Upon routine serological investigation, a discrepancy between the HPA-1a genotype and phenotype prompted the sequencing of the 15 exons of the ITGB3 (integrin β3, GPIIIa and CD61) gene in the mother. Results The mother was genotypically HPA-1a1b heterozygous but phenotyped as HPA-1a negative. Sequencing of the ITGB3 exons confirmed HPA-1a1b heterozygosity, but also identified a novel single nucleotide insertion in exon 10 leading to a frameshift and premature termination at amino acid 471 of ITGB3. Maternal anti-HPA-1a was detected but with a pattern typical for a low-affinity antibody. Three transfusions of HPA-1a and -5b negative neonatal Platelet concentrates were required to return to a safe Platelet count. Conclusion A rare ITGB3 allele was uncovered by the investigation of a severe case of alloimmune thrombocytopenia in a mother with HPA-1a antibodies who genotyped as HPA-1a1b.
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collaborative studies to establish the first world health organization international standard for detection of Human antibody against Human Platelet Antigen 3a
Vox Sanguinis, 2007Co-Authors: J Berry, M. De Haas, Willem H Ouwehand, David Allen, Riitta Kekomaki, C Kaplan, Leendert Porcelijn, Paul MetcalfeAbstract:Background and Objectives The Platelet-specific antibody anti-Human Platelet Antigen-3a (anti-HPA-3a) is involved in neonatal alloimmune thrombocytopenia, post-transfusion purpura, and Platelet refractoriness. However, HPA-3a antibodies are often difficult to detect, probably because the Antigen is labile. This report describes the production of a freeze-dried preparation of pooled Human plasma, coded 03/190, containing IgG antibodies against the HPA-3a. The material is intended for use as a minimum sensitivity reagent in glycoprotein-specific assays currently used for anti-HPA-3a detection. Laboratories can use it to assess the sensitivity of their ‘in-house’ assays for anti-HPA-3a and to calibrate local controls for routine use in each batch of tests. Materials and Methods Plasma containing anti-HPA-3a was obtained from a mother of two babies both born with severe thrombocytopenia, and following dilution it was freeze dried in glass ampoules. Results Two collaborative studies demonstrated that the candidate material contained anti-HPA-3a and Human leucocyte Antigen (HLA) class I antibodies, but no other HPA antibodies that might confuse the detection of the anti-HPA-3a. The minimum dilution that should give a positive result was determined to be 1 : 8 by two further international collaborative studies involving a total of 49 laboratories in 23 countries. Conclusion The material also contains HLA antibodies and is suitable for use only in techniques that are glycoprotein specific (i.e. monoclonal antibody immobilization of Platelet Antigens and enzyme-linked immunosorbent assay) where only HPA antibodies will be detected. This standard will allow laboratories to measure their sensitivity of detection of anti-HPA-3a and will also allow those laboratories with relatively insensitive techniques to monitor their performance as they improve their methodology.
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a modified rapid monoclonal antibody specific immobilization of Platelet Antigen assay for the detection of Human Platelet Antigen hpa antibodies a multicentre evaluation
Vox Sanguinis, 2007Co-Authors: K Campbell, Paul Metcalfe, Willem H Ouwehand, K Rishi, G J Howkins, D Gilby, R Mushens, Cedric Ghevaert, Geoff LucasAbstract:Background The monoclonal antibody-specific immobilization of Platelet Antigens (MAIPA) assay is the cornerstone technique for the detection and identification of Human Platelet Antigen (HPA) antibodies. However, the original technique described by Kiefel and colleagues requires approximately 8 h adding to diagnostic delay. Moreover, proficiency exercises indicate that there are substantial variations in the MAIPA protocol, and that these may account for interlaboratory differences in sensitivity and specificity. Study Design and Methods A review of current MAIPA assay protocols from six laboratories together with performance in quality-assessment schemes identified several key variables potentially affecting the assay results. An optimized protocol was derived and assay time reduced to 5 h. The modified rapid MAIPA (MR-MAIPA) assay was evaluated using 61 samples with a range of HPA antibodies typically encountered in cases of fetomaternal alloimmune thrombocytopenia (n = 22), post-transfusion purpura (n = 8), Platelet refractoriness (n = 7) and other Platelet immune conditions (n = 24). The sensitivity of the assay was assessed using three international standards and the recombinant HPA-1a antibody CamTran007. The results obtained were compared with the original findings obtained with the local MAIPA assays. In addition, four different glycoprotein IIb/IIIa capture monoclonal antibodies were evaluated for their effect on assay sensitivity. Results Complete concordance was found between the original MAIPA results and those obtained with the new assay when testing a selected panel of clinical samples. The modified assay had nanogram level sensitivity for the detection of HPA-1a antibodies and titration of HPA-1a and HPA-5b antibody sensitivity standards yielded end-points equal to or greater than the mean recorded in international workshops. Conclusion The MR-MAIPA assay offers improved turnaround for the detection of HPA antibodies without loss of sensitivity.
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collaborative studies to establish the first who reference reagent for detection of Human antibody against Human Platelet Antigen 5b
Vox Sanguinis, 2003Co-Authors: Paul Metcalfe, Willem H Ouwehand, D Sands, T W BarrowcliffeAbstract:Background and Objectives This report describes the production of a freeze-dried preparation of pooled Human plasma, coded 99/666, containing immunoglobulin G (IgG) antibodies against Human Platelet Antigen 5b (HPA-5b). Materials and Methods The material is intended for use as a minimum sensitivity reagent in the assays currently used for detection of antibodies to HPA-5b. Laboratories can use it to assess the sensitivity of their ‘in-house’ assays for antibodies to HPA-5b and to calibrate local controls for routine use in each batch of tests. Results Two collaborative studies demonstrated that the two candidate materials contained antibodies to HPA-5b and that there were no other HPA or Human leucocyte Antigen (HLA) antibodies which might confuse the detection of antibodies to HPA-5b. The two samples were pooled and freeze-dried in 1-ml ampoules. Conclusions The minimum dilution of the antibody against 5b required to yield a positive result was determined, by two international collaborative studies involving a total of 49 laboratories in 26 countries, to be 1 in 2.
Y. Shibata - One of the best experts on this subject based on the ideXlab platform.
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neonatal alloimmune thrombocytopenia due to an antibody against a labile component of Human Platelet Antigen 3b bakb
Transfusion Medicine, 2004Co-Authors: S Kataoka, Sachiyo Shinada, K. Chiba, M Nakamura, H. Kobayashi, S. Morita, Y. ShibataAbstract:Summary. We report the second case of neonatal alloimmune thrombocytopenia due to anti-Human Platelet Antigen (HPA)-3b (Bakb) antibody. The infant was severely affected with intracranial haemorrhage. Most importantly, we were able to detect this antibody only by using fresh, unfixed Platelets as Antigen. This antibody was detectable neither by conventional mixed passive haemagglutination, Platelet immunofluorescence test using fixed Platelets, nor by monoclonal Antigen immobilization of Platelet Antigen assay using solubilized Platelets. We assume that this antibody reacts only with fresh Platelets and that the Antigen is a labile component of HPA-3b (Bakb).
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Neonatal alloimmune thrombocytopenia associated with anti‐Human Platelet Antigen‐3a antibody
Pediatrics International, 1997Co-Authors: Hidetoshi Takada, S. Morita, Shigeyuki Nakamura, Toshihiro Nishiguchi, Kazuya Miyake, Keisuke Hamada, Hidetsugu Oshikawa, Toshimitsu Shingu, Y. ShibataAbstract:A sister and brother with neonatal alloimmune thrombocytopenic purpura (NAITP) caused by maternal anti-Human Platelet Antigen (HPA)-3a are reported. The children had transient severe thrombocytopenia in the newborn period, and were treated with intravenous gamma-globulin and Platelet concentrates from random donors. Although the sister had intracranial hemorrhage on day 2 postnatally, the development of the child has been normal and no neurological sequelae have been observed. The brother only had bloody stool when the Platelet count was low, and did not have severe hemorrhagic manifestations. The diagnosis of NAITP was made by the sera from the mother, which contained anti-HPA-3a antibody directed against Platelets of the children. The rate of recurrence might be high in this family, because the father of the patients was found to be homozygous for the HPA-3a gene.
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Genotype frequencies of the Human Platelet Antigen, Ca/Tu, in Japanese, determined by a PCR-RFLP method.
Vox Sanguinis, 1996Co-Authors: Shigenori Tanaka, Atsuko Taniue, Nobuo Nagao, Tadao Tomita, Shiro Ohnoki, Hirotoshi Shibata, Yasuto Okubo, Hideo Yamaguchi, Y. ShibataAbstract:Recently, the polymorphism of a new Human Platelet Antigen, Ca/Tu, was shown to be derived from a G-A nucleotide substitution at base 1564 of GPIIIa cDNA, which leads to a single amino acid difference, Arg/Gln at amino acid 489 of GPIIIa. We developed a PCR-RFLP method to determine the genotypes of Ca/Tu and their frequencies in a Japanese population. Fifteen Ca/Tua donors comprising 1 Ca/Tu(a/a) homozygous donor and 14 Ca/Tu(a/b) heterozygous donors were found among the 314 random donors analyzed. The frequencies of Ca/Tu genes were 0.025 (Ca/Tua) and 0.975 (Ca/Tu(b)). The present study showed that the frequency of Ca/Tua individuals in the Japanese (15/314) was approximately 7-fold higher than in the Finnish population (1/150) previously reported by Kekomaki et al. Therefore, attention must be given to the involvement of the Ca/Tu alloAntigen in neonatal alloimmune thrombocytopenia and the refractoriness of Platelet transfusion.
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DNA-Based Typing of Human Platelet Antigen Systems by Polymerase Chain Reaction- Single-Strand Conformation
1995Co-Authors: Koki Fujiwara, Y. Shibata, Katsushi Tokunagaa, Kazumi Isa, Masaki Mivamoto, Li Wanga, Tatsuya Akazaa, Kenji Tadokoro, Takeo JujiAbstract:Polymerase chain reaction-single-strand conformation polymorphism (PCR- SSCP) method has been established to discriminate genotypes for the Human Platelet Antigen (HPA) systems HPA-1, HPA-2, HPA-3, HPA-4, and HPA-5. Gene fragments which contain polymorphic sequences corresponding to the HPA-I, HPA-2, HPA-3, HPA-4, and HPA-5 systems were PCR-amplified with specific primers. The amplified DNA was denatured and subjected to non-denaturing polyacrylamide gel electrophoresis followed by silver staining. The results ob- tained by the PCR-SSCP method were in good agreement with those of the allo- types determined by serological typing. Furthermore, the results agreed with those obtained by other DNA-based typing methods such as PCR-allele-specific restriction enzyme analysis and PCR-sequence-specific primer. These results in- dicate that PCR-SSCP is a simple and sensitive method for determining HPA genotypes and identifying unknown polymorphisms.
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DNA-based typing of Human Platelet Antigen systems by polymerase chain reaction-single-strand conformation polymorphism method
Vox Sanguinis, 1995Co-Authors: Koki Fujiwara, Y. Shibata, Kazumi Isa, Kenji Tadokoro, Katsushi Tokunaga, M. Miyamoto, Li Wang, Tatsuya Akaza, Takeo JujiAbstract:Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method has been established to discriminate genotypes for the Human Platelet Antigen (HPA) systems HPA-1, HPA-2, HPA-3, HPA-4, and HPA-5. Gene fragments which contain polymorphic sequences corresponding to the HPA-1, HPA-2, HPA-3, HPA-4, and HPA-5 systems were PCR-amplified with specific primers. The amplified DNA was denatured and subjected to non-denaturing polyacrylamide gel electrophoresis followed by silver staining. The results obtained by the PCR-SSCP method were in good agreement with those of the allotypes determined by serological typing. Furthermore, the results agreed with those obtained by other DNA-based typing methods such as PCR-allele-specific restriction enzyme analysis and PCR-sequence-specific primer. These results indicate that PCR-SSCP is a simple and sensitive method for determining HPA genotypes and identifying unknown polymorphisms.
Dick Oepkes - One of the best experts on this subject based on the ideXlab platform.
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women s attitude towards routine Human Platelet Antigen screening in pregnancy
Acta Obstetricia et Gynecologica Scandinavica, 2017Co-Authors: Dian Winkelhorst, Rosanne M Loeff, Masja De Haas, Elske M Van Den Akkervan Marle, Dick OepkesAbstract:Introduction Fetal and neonatal alloimmune thrombocytopenia is a potentially life-threatening disease with excellent preventative treatment available for subsequent pregnancies. To prevent index cases, the effectiveness of a population-based screening program has been suggested repeatedly. Therefore, we aimed to evaluate women's attitude towards possible future Human Platelet Antigen-screening in pregnancy. Material and methods We performed a cross-sectional questionnaire study among healthy pregnant women receiving prenatal care in one of seven participating midwifery practices. Attitude was assessed using a questionnaire based on the validated Multidimensional Measurement of Informed Choice model, containing questions assessing knowledge, attitude and intention to participate. Results A total of 143 of the 220 women (65%) completed and returned the questionnaire. A positive attitude towards Human Platelet Antigen-screening was expressed by 91% of participants, of which 94% was based on sufficient knowledge. Attitude was more likely to be negatively influenced by the opinion that screening can be frightening. Informed choices were made in 87% and occurred significantly less in women from non-European origin, 89% in European women vs. 60% in non-European women (p = 0.03). Conclusions Pregnant women in the Netherlands expressed a positive attitude towards Human Platelet Antigen-screening in pregnancy. We therefore expect a high rate of informed uptake when Human Platelet Antigen-screening is implemented. In future counseling on Human Platelet Antigen-screening, ethnicity and possible anxiety associated with a screening test need to be specifically addressed.
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Women's attitude towards routine Human Platelet Antigen‐screening in pregnancy
Acta Obstetricia et Gynecologica Scandinavica, 2017Co-Authors: Dian Winkelhorst, Rosanne M Loeff, M. Elske Van Den Akker-van Marle, Masja De Haas, Dick OepkesAbstract:Introduction Fetal and neonatal alloimmune thrombocytopenia is a potentially life-threatening disease with excellent preventative treatment available for subsequent pregnancies. To prevent index cases, the effectiveness of a population-based screening program has been suggested repeatedly. Therefore, we aimed to evaluate women's attitude towards possible future Human Platelet Antigen-screening in pregnancy. Material and methods We performed a cross-sectional questionnaire study among healthy pregnant women receiving prenatal care in one of seven participating midwifery practices. Attitude was assessed using a questionnaire based on the validated Multidimensional Measurement of Informed Choice model, containing questions assessing knowledge, attitude and intention to participate. Results A total of 143 of the 220 women (65%) completed and returned the questionnaire. A positive attitude towards Human Platelet Antigen-screening was expressed by 91% of participants, of which 94% was based on sufficient knowledge. Attitude was more likely to be negatively influenced by the opinion that screening can be frightening. Informed choices were made in 87% and occurred significantly less in women from non-European origin, 89% in European women vs. 60% in non-European women (p = 0.03). Conclusions Pregnant women in the Netherlands expressed a positive attitude towards Human Platelet Antigen-screening in pregnancy. We therefore expect a high rate of informed uptake when Human Platelet Antigen-screening is implemented. In future counseling on Human Platelet Antigen-screening, ethnicity and possible anxiety associated with a screening test need to be specifically addressed.
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Noninvasive fetal genotyping of Human Platelet Antigen‐1a
BJOG: An International Journal of Obstetrics & Gynaecology, 2011Co-Authors: P. G. Scheffer, Dick Oepkes, A. Ait Soussan, O. J. H. M. Verhagen, Godelieve C. M. L. Page-christiaens, M. De Haas, C. E. Van Der SchootAbstract:Please cite this paper as: Scheffer P, Ait Soussan A, Verhagen O, Page-Christiaens G, Oepkes D, de Haas M, van der Schoot C. Noninvasive fetal genotyping of Human Platelet Antigen-1a. BJOG 2011;118:1392–1395. We describe a reliable noninvasive fetal Human Platelet Antigen (HPA)-1a genotyping assay on a real-time polymerase chain reaction (PCR) platform using cell-free fetal DNA isolated from maternal blood. Nonspecific amplification of maternal cell-free DNA is overcome by pre-PCR digestion of the cell-free DNA with the Msp1 restriction enzyme. Noninvasive fetal HPA-1a genotyping offers a safe method for alloimmunised pregnant women to determine whether their fetus is at risk of fetal or neonatal alloimmune thrombocytopenia (FNAIT) and whether interventions to prevent intracranial haemorrhage are required. The availability of this test is relevant to the ongoing debate on screening pregnancies for HPA-1a-mediated FNAIT.
Bjørn Skogen - One of the best experts on this subject based on the ideXlab platform.
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fetal exposure to maternal Human Platelet Antigen 1a does not induce tolerance an analytical observational study
PLOS ONE, 2017Co-Authors: Mette S Kjaer, Bjørn Skogen, Heidi Tiller, Goril Heide, Jens Kjeldsenkragh, Anne HusebekkAbstract:Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a disease that may cause severe bleeding complications with risk of perinatal death or lifelong disability. The main cause of FNAIT is maternal antibodies against Human Platelet Antigen (HPA)-1a. Both fetomaternal bleeding and transplacental trafficking of fetal cells during pregnancy could be the cause of alloimmunization. Persistence of fetal cells in the mother (fetal microchimerism) and maternal cells in the child (maternal microchimerism) are well-recognized phenomena. Thus, it could be envisaged that fetal exposure to the HPA-1a Antigen could tolerize an HPA-1a negative female fetus and prevent production of anti-HPA-1a antibodies later in life if she becomes pregnant with an HPA-1a positive fetus. The objective of the current study was to assess if the risk of producing anti-HPA-1a antibodies and the severity of neonatal thrombocytopenia in HPA-1a negative women with HPA-1a positive mothers (i.e. the mother is HPA-1a/b), was lower than in HPA-1a negative women with HPA-1a negative mothers. HPA-1a negative women with HPA-1a antibodies, identified from a Norwegian screening study (1996-2004), where HPA-1 genotype of their mothers was available, were included in the study. The frequency of HPA-1a positive mothers to HPA-1a immunized daughters were compared to the calculated frequency in the general population. We did not find any difference in the frequency of HPA-1ab among mothers to daughters with HPA-1a antibodies as compared with the general population. Furthermore, acknowledging sample-size limitations, we neither found an association between the mothers' HPA type and their daughters' anti-HPA-1a antibody levels or any difference between the two groups of mothers (HPA-1ab vs HPA-1bb), with respect to frequency of thrombocytopenia in the children of their daughters with HPA-1a antibodies. Hence, there was no indication of tolerance against fetal HPA-1a Antigen in HPA-1bb women who had been exposed to HPA-1a Antigen during fetal development.
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T cell responses to Human Platelet Antigen–1a involve a unique form of indirect allorecognition
JCI Insight, 2016Co-Authors: Maria Therese Ahlen, Anne Husebekk, Ida Løken Killie, Bjørn Skogen, Tor B. StugeAbstract:Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a pregnancy-related condition caused by maternal antibodies binding an alloAntigen on fetal Platelets. In most cases the alloAntigen is formed by a single amino acid, integrin β3 Leu33, referred to as Human Platelet Antigen-1a (HPA-1a). Production of anti-HPA-1a antibodies likely depends on CD4+ T cells that recognize the same alloAntigen in complex with the HLA-DRA/DRB3*01:01 molecule. While this complex is well characterized, T cell recognition of it is not. Here, to examine the nature of Antigen recognition by HPA-1a-specific T cells, we assayed native and synthetic variants of the integrin β3 peptide Antigen for binding to DRA/DRB3*01:01-positive Antigen-presenting cells and for T cell activation. We found that HPA-1a-specific T cells recognize non-allogeneic integrin β3 residues anchored to DRA/DRB3*01:01 by the allogeneic Leu33, which itself is not directly recognized by these T cells. Furthermore, these T cell responses are diverse, with different T cells depending on different residues for recognition. This represents a unique form of indirect allorecognition in which a non-allogeneic peptide sequence becomes immunogenic by stable anchoring to MHC by an allogeneic residue.
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t cell responses to Human Platelet Antigen 1a involve a unique form of indirect allorecognition
JCI insight, 2016Co-Authors: Maria Therese Ahlen, Anne Husebekk, Ida Løken Killie, Bjørn Skogen, Tor B. StugeAbstract:Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a pregnancy-related condition caused by maternal antibodies binding an alloAntigen on fetal Platelets. In most cases the alloAntigen is formed by a single amino acid, integrin β3 Leu33, referred to as Human Platelet Antigen-1a (HPA-1a). Production of anti-HPA-1a antibodies likely depends on CD4+ T cells that recognize the same alloAntigen in complex with the HLA-DRA/DRB3*01:01 molecule. While this complex is well characterized, T cell recognition of it is not. Here, to examine the nature of Antigen recognition by HPA-1a-specific T cells, we assayed native and synthetic variants of the integrin β3 peptide Antigen for binding to DRA/DRB3*01:01-positive Antigen-presenting cells and for T cell activation. We found that HPA-1a-specific T cells recognize non-allogeneic integrin β3 residues anchored to DRA/DRB3*01:01 by the allogeneic Leu33, which itself is not directly recognized by these T cells. Furthermore, these T cell responses are diverse, with different T cells depending on different residues for recognition. This represents a unique form of indirect allorecognition in which a non-allogeneic peptide sequence becomes immunogenic by stable anchoring to MHC by an allogeneic residue.
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foetal neonatal alloimmune thrombocytopenia in egypt Human Platelet Antigen genotype frequencies and antibody detection and follow up in pregnancies
Transfusion and Apheresis Science, 2012Co-Authors: Anne Husebekk, Bjørn Skogen, El M Ekiaby, G Gorgy, Mette Kjaer Killie, C Uhlinhansen, Wahida Salma, C Navarrete, El M Afandi, Maria Therese AhlenAbstract:Abstract Background and objectives Foetal and neonatal alloimmune thrombocytopenia (FNAIT) is studied mainly in Caucasian populations. Severe thrombocytopenia ( 9 /L) gives risk of haemorrhage and the most feared complication is intracranial haemorrhage (ICH). In Caucasian populations anti-Human Platelet Antigen (HPA)-1a antibodies are the cause of FNAIT in >80% of the cases. The aims of this project were to study the gene frequencies of HPA-1-5 and 15 alleles in an Egyptian population (Arabic), and to determine the frequency of HPA-1a and -5b immunisations in a cohort of Egyptian pregnant women. Materials and methods Altogether 6974 pregnant women were included in the study. Genotyping was performed by polymerase chain reaction and antibodies were detected by flow cytometry and enzyme-linked immunosorbent assay. HPA-1-5 and 15 alleles were studied in 367 individuals. Results The HPA genotypes differed from genotypes published from different Caucasian and Chinese (Han) populations in HPA-1, -2, -3, and -5 systems with significant higher frequency of HPA-1b, -2b and -5b. The rate of HPA-1a alloimmunisation was found comparable to Caucasian populations. Severe thrombocytopenia was found in two newborns. No bleeding complication was reported. Anti-HPA-5b antibodies were detected in 4.4% of the pregnant women. Clinical consequences of these antibodies were not studied. Conclusion The HPA-1bb and -5bb genotypes are more frequent in the Egyptian Arabic population studied compared to Caucasian populations. FNAIT due to anti-HPA-1a and -5b antibodies must be suspected in cases of neonatal thrombocytopenia. Further large prospective studies are needed to increase the knowledge of clinical complications related to HPA alloantibodies in populations with different genetic backgrounds.
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Human Platelet Antigen 1 hpa 1 genotyping with 5 nuclease assay and sequence specific primers reveals a single nucleotide deletion in intron 2 of the hpa 1a allele of Platelet glycoprotein iiia
British Journal of Haematology, 2002Co-Authors: Killie Mette Kjaer, Anne Husebekk, Sissel Jaegtvik, Bjørn SkogenAbstract:Summary. We have established a 5′ nuclease assay (5′ NA) for Human Platelet Antigen (HPA) 1a/b allelic discrimination. The assay is based on the simultaneous amplification and detection of the two targets in a one-tube system. The results are read optically, immediately after termination of the polymerase chain reaction (PCR), and no post-PCR processing is necessary. This genotyping procedure is less time-consuming and cheaper than our conventional sequence-specific primer PCR (SSP-PCR), which is run as a two-tube test, with verification of the results after electrophoresis in agarose gel. The reduction of analytical steps, simplification of the procedure and potential for automation were important advantages for our choice of system. This test system is more suitable for large-scale testing and fits better for our screening programme for HPA 1bb determination. DNA from 1093 individuals were tested in parallel with the SSP-PCR and the 5′ NA. One thousand and ninety-one samples gave identical results in SSP-PCR and 5′ NA. Upon repeated testing, two samples consistently came out as HPA 1bb in SSP-PCR and HPA 1ab in 5′ NA. DNA sequencing revealed a defect located in an intronic area that corresponds to the consensus primer used for the SSP-PCR HPA 1a typing.
Nicholas A Watkins - One of the best experts on this subject based on the ideXlab platform.
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severe fetomaternal alloimmune thrombocytopenia due to anti Human Platelet Antigen hpa 1a in a mother with a rare and silenced itgb3 0101 gpiiia allele
Vox Sanguinis, 2007Co-Authors: Graham A Smith, Willem H Ouwehand, Angela Rankin, C Riddle, C Cheethamwilkinson, E Ranasinghe, Nicholas A WatkinsAbstract:Background and Objectives Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal antibodies against a Human Platelet Antigen (HPA) present on fetal, but absent from maternal Platelets. We identified and characterized a case of FMAIT due to anti-HPA-1a in a mother with an HPA-1a1b genotype. Materials and Methods The first child of a 29-year-old mother presented with a petechial rash and a Platelet count of 8 × 109 per l. Upon routine serological investigation, a discrepancy between the HPA-1a genotype and phenotype prompted the sequencing of the 15 exons of the ITGB3 (integrin β3, GPIIIa and CD61) gene in the mother. Results The mother was genotypically HPA-1a1b heterozygous but phenotyped as HPA-1a negative. Sequencing of the ITGB3 exons confirmed HPA-1a1b heterozygosity, but also identified a novel single nucleotide insertion in exon 10 leading to a frameshift and premature termination at amino acid 471 of ITGB3. Maternal anti-HPA-1a was detected but with a pattern typical for a low-affinity antibody. Three transfusions of HPA-1a and -5b negative neonatal Platelet concentrates were required to return to a safe Platelet count. Conclusion A rare ITGB3 allele was uncovered by the investigation of a severe case of alloimmune thrombocytopenia in a mother with HPA-1a antibodies who genotyped as HPA-1a1b.