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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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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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ys04 recombinant mini β3 integrin fragments for the detection of hpa antibodies
Transfusion Medicine, 2006Co-Authors: Prachi Stafford, K Campbell, Cedric Ghevaert, G Smith, Lorna M Williamson, James A Huntington, Nicholas A Watkins, Willem H OuwehandAbstract:Background The β3 integrin [Glycoprotein (GP) IIIa] gene is highly polymorphic and encodes eight of the Human Platelet Antigen (HPA) systems. HPA immunisation is of clinical relevance in neonatal alloimmune thrombocytopenia (NAIT), Post-Transfusion Purpura (PTP) and platelet refractoriness. However, the current methods of antibody detection are cumbersome and rely upon the use of platelets from genotyped donors. In the case of the rare HPA antigens, obtaining donor platelets is particularly problematic. We therefore set out to develop an antibody screening assay that uses recombinant, soluble β3-integrin fragments expressing the HPA-1, –4, –6, –7, –10, –11 and –16 epitopes. Methods A total of four integrin fragments of different lengths (ΔSDL, ΔβA, PSI-Hybrid and PSI) were designed using structural data and expressed in Drosophila S2 cells. HPA-1a (33) and HPA-1b (P33) forms of all four proteins were expressed as were the fragments ΔSDL-4b and ΔSDL-SuperRare, the latter two for the detection of HPA-4b and the rare HPA antigens (HPA-6, 7, 10, 11 and 16) respectively. The recombinant proteins were then screened for reactivity with monoclonal antibodies (mAb) and polyclonal antisera containing anti-HPA in an ELISA based assay. Results The reactivity pattern seen with murine mAbs Y2/51, SZ21 and CRC54 was as expected. Two of three human monoclonal HPA-1a antibodies (19–7, 23–15, both from a PTP patient) reacted with the four L33 fragments but the third (CamTran007, derived from a NAIT patient) only reacted with the ΔSDL-L33 and ΔβA-L33 fragments, defining an epitope split. The reactivity of both potency (NIBSC-03/152) and sensitivity (NIBSC-93/710) standards for anti-HPA-1a detection were comparable or better than those seen by MAIPA. In addition, samples from 132 NAIT patients with anti-HPA-1a [15 with cerebral haemorrhage (CH), 49 with platelet counts <20 × 109 L−1 but no CH, 35 with counts ≥ 20 × 109 L−1 and 33 with antenatal treatment] were tested. Anti-HPA-1a was detected in 126 samples including all CH cases. The reactivity patterns against the four different β3-integrin fragments did not correlate with clinical outcome. Finally, mini-β3 integrins Δ SDL-4b and ΔSDL-SuperRare allowed the detection of 14 of the 15 anti-HPA samples tested (5 HPA-4a, 5 HPA-4b, 1 HPA-6b, 1 HPA-7b and 2 of 3 HPA-11b). Conclusions Recombinant fragments of the β3-integrin expressing HPA antigens can be used to successfully detect platelet alloantibodies. These fragments with be of use in the screening and diagnosis of suspected cases of NAIT.
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the immunogenicity of gov alloantigens is comparable to that of the hpa 5 system
Transfusion Medicine, 2000Co-Authors: G Smith, Paul Metcalfe, Willem H Ouwehand, J Berry, E Ranasinghe, C MurphyAbstract:The 175 kDa glycosylphosphatidylinositol (GPI)-linked glycoprotein CD109 carries the bi-allelic platelet system, Gov. To date, detection of Gov antibodies has relied on the immunoprecipitation of radio-labelled CD109. We have modified the monoclonal antibody-specific immobilization of platelet antigen assay for the detection of Gov antibodies. Screening of 605 archived patient samples referred for neonatal alloimmune thrombocytopenia (NAITP, n = 305); Post-Transfusion Purpura (PTP, n = 60); and platelet refractoriness (PR, n = 240) revealed 14 Gov antibodies (10 anti-Gova, 4 anti-Govb) which equals the number of HPA-5 antibodies detected in the 605 samples. There is no obvious linkage between the HLA class II type and the ability to mount an immune response. Phenotyping of 113 donors by MAIPA showed frequencies of 19·5%, 40·7% and 39·8% for ‘aa, ab and bb’, respectively, with ‘a’ and ‘b’ allele frequencies of 0·40 and 0·60. Cloning of the CD109 gene and determination of the molecular basis of the Gov antigens has become relevant now that it has been established that Gov alloantibodies are more frequent than previously thought.
Paul Metcalfe - 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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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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the immunogenicity of gov alloantigens is comparable to that of the hpa 5 system
Transfusion Medicine, 2000Co-Authors: G Smith, Paul Metcalfe, Willem H Ouwehand, J Berry, E Ranasinghe, C MurphyAbstract:The 175 kDa glycosylphosphatidylinositol (GPI)-linked glycoprotein CD109 carries the bi-allelic platelet system, Gov. To date, detection of Gov antibodies has relied on the immunoprecipitation of radio-labelled CD109. We have modified the monoclonal antibody-specific immobilization of platelet antigen assay for the detection of Gov antibodies. Screening of 605 archived patient samples referred for neonatal alloimmune thrombocytopenia (NAITP, n = 305); Post-Transfusion Purpura (PTP, n = 60); and platelet refractoriness (PR, n = 240) revealed 14 Gov antibodies (10 anti-Gova, 4 anti-Govb) which equals the number of HPA-5 antibodies detected in the 605 samples. There is no obvious linkage between the HLA class II type and the ability to mount an immune response. Phenotyping of 113 donors by MAIPA showed frequencies of 19·5%, 40·7% and 39·8% for ‘aa, ab and bb’, respectively, with ‘a’ and ‘b’ allele frequencies of 0·40 and 0·60. Cloning of the CD109 gene and determination of the molecular basis of the Gov antigens has become relevant now that it has been established that Gov alloantibodies are more frequent than previously thought.
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detection of gov system antibodies by maipa reveals an immunogenicity similar to the hpa 5 alloantigens
British Journal of Haematology, 2000Co-Authors: J Berry, Paul Metcalfe, C M Murphy, G Smith, E Ranasinghe, Robert W Finberg, J Walton, Juliette Brown, Cristina Navarrete, Willem H OuwehandAbstract:The glycosylphosphatidylinositol-linked platelet protein CD109 carries the biallelic alloantigen system Gov. There is limited information on the incidence of Gov alloantibodies in neonatal alloimmune thrombocytopenia (NAITP), Post-Transfusion Purpura (PTP) and platelet refractoriness. We adapted the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay to the detection of Gov antibodies and determined their incidence in 605 archived samples (112 with HPA antibodies) referred for the aforementioned conditions. Here, we show that CD109 expression was reduced upon platelet storage in saline or by cryopreservation, but was stable when stored as whole blood or therapeutic platelet concentrate. Fourteen of the 605 samples contained Gov alloantibodies (anti-Gova, n = 10; anti-Govb, n = 4), with the majority in platelet refractoriness (n = 9) and, of the remaining five, four in NAITP and one in PTP. In seven cases, no other HPA antibodies were detected, three being NAITP cases. The incidence of Gov antibodies was significantly lower than HPA-1 system antibodies (n = 87), but equalled the number of HPA-5 system antibodies (n = 14) and outnumbered HPA-2 and -3 system antibodies (10 altogether).
C Kaplan - 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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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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genetic susceptibility and anti human platelet antigen 5b alloimmunization role of hla class ii and tap genes
Human Immunology, 1996Co-Authors: Gilbert Semana, Thierry Zazoun, Mehdi Alizadeh, Mariechristine Morelkopp, Bernard Genetet, C KaplanAbstract:Abstract Platelet alloimmunization may result in Post-Transfusion Purpura, and during pregnancy may cause neonatal alloimmune thrombocytopenia (NAIT), with a frequency estimated at 1.3 per 1000 live births. The risk of morbidity is significant: 20% of affected infants have neurologic sequelae and the death rate is about 10%. A better understanding of the immune response to platelet alloantigens would allow for a better definition, and thus better management of pregnant women at high risk. Limited data are available on the immune response against HPA-5b, the second most frequent antigen, after HPA-la, implicated in NAIT. We studied HLA class II and TAP gene polymorphism in 50 women immunized against HPA-5 system antigens. Our results suggest a strong association of alloimmunization with a cluster of HLA DR molecules sharing a particular polymorphic amino acid sequence at position 69–70 (Glu-Asp encoded by GAA-GAC nucleotide sequence) of the DRβl chain (RR = 2.95, RR = 5.70 when patients were homozygous for this sequence), and a negative association with the DRB1 ∗ 0301 allele (2.1% vs. 28%; RR = 0.08). Furthermore, increased frequency of a TAP2 dimorphism at position 379 was observed in immunized women against the HPA-5 antigens (RR = 4.7).
J. V. Villalba - One of the best experts on this subject based on the ideXlab platform.
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Post-Transfusion Purpura as the main manifestation of a trilineal transfusion reaction, responsive to steroids: flow-cytometric investigation of granulocyte and platelet antibodies
Annals of Hematology, 1991Co-Authors: N. Puig, M. J. Sayas, J. A. Montoro, J. V. VillalbaAbstract:We report a typical case of Post-Transfusion Purpura (PTP) due to anti-Pl^Al in a 65-year-old woman. Serological studies were carried out using flow cytometry (FCM). The patient also developed red cell alloantibodies that produced a delayed hemolytic transfusion reaction (DHTR) and broad HLA antibodies. Treatment with high-dose intravenous IgG (HDIgG; a first-generation preparation) was ineffective, but a course of steroids resulted in a rapid increase in the the platelet count.
J Berry - One of the best experts on this subject based on the ideXlab platform.
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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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the immunogenicity of gov alloantigens is comparable to that of the hpa 5 system
Transfusion Medicine, 2000Co-Authors: G Smith, Paul Metcalfe, Willem H Ouwehand, J Berry, E Ranasinghe, C MurphyAbstract:The 175 kDa glycosylphosphatidylinositol (GPI)-linked glycoprotein CD109 carries the bi-allelic platelet system, Gov. To date, detection of Gov antibodies has relied on the immunoprecipitation of radio-labelled CD109. We have modified the monoclonal antibody-specific immobilization of platelet antigen assay for the detection of Gov antibodies. Screening of 605 archived patient samples referred for neonatal alloimmune thrombocytopenia (NAITP, n = 305); Post-Transfusion Purpura (PTP, n = 60); and platelet refractoriness (PR, n = 240) revealed 14 Gov antibodies (10 anti-Gova, 4 anti-Govb) which equals the number of HPA-5 antibodies detected in the 605 samples. There is no obvious linkage between the HLA class II type and the ability to mount an immune response. Phenotyping of 113 donors by MAIPA showed frequencies of 19·5%, 40·7% and 39·8% for ‘aa, ab and bb’, respectively, with ‘a’ and ‘b’ allele frequencies of 0·40 and 0·60. Cloning of the CD109 gene and determination of the molecular basis of the Gov antigens has become relevant now that it has been established that Gov alloantibodies are more frequent than previously thought.
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detection of gov system antibodies by maipa reveals an immunogenicity similar to the hpa 5 alloantigens
British Journal of Haematology, 2000Co-Authors: J Berry, Paul Metcalfe, C M Murphy, G Smith, E Ranasinghe, Robert W Finberg, J Walton, Juliette Brown, Cristina Navarrete, Willem H OuwehandAbstract:The glycosylphosphatidylinositol-linked platelet protein CD109 carries the biallelic alloantigen system Gov. There is limited information on the incidence of Gov alloantibodies in neonatal alloimmune thrombocytopenia (NAITP), Post-Transfusion Purpura (PTP) and platelet refractoriness. We adapted the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay to the detection of Gov antibodies and determined their incidence in 605 archived samples (112 with HPA antibodies) referred for the aforementioned conditions. Here, we show that CD109 expression was reduced upon platelet storage in saline or by cryopreservation, but was stable when stored as whole blood or therapeutic platelet concentrate. Fourteen of the 605 samples contained Gov alloantibodies (anti-Gova, n = 10; anti-Govb, n = 4), with the majority in platelet refractoriness (n = 9) and, of the remaining five, four in NAITP and one in PTP. In seven cases, no other HPA antibodies were detected, three being NAITP cases. The incidence of Gov antibodies was significantly lower than HPA-1 system antibodies (n = 87), but equalled the number of HPA-5 system antibodies (n = 14) and outnumbered HPA-2 and -3 system antibodies (10 altogether).