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Alloimmunization against human platelet antigen (HPA)-1a during pregnancy can cause foetal/neonatal alloimmune thrombocytopenia (FNAIT) and severe bleeding in the foetus or newborn and likely depends on several factors. HPA-1a alloimmunization is associated with DRB3*01:01, which is associated with several DR-DQ haplotypes. However, it is not known to what extent these haplotypes contribute to the prevalence of HPA-1a alloimmunization. HPA-1a-alloimmunized women, identified in a prospective study, and random donors were typed for selected DRB3, DRB4, DRB1, DQA1 and DQB1 alleles to determine allele and DR-DQ haplotype frequencies. DRB3*01:01 was carried by 94% HPA-1a-immunized women compared to 27% in the general population. In the first population, the DR3-DQ2 haplotype was overrepresented (P < .003). The prevalence of HPA-1a alloimmunization was estimated to be about twice as frequent with DR3-DQ2 compared to DR13-DQ6, together accounting for about 90% of DRB3*01:01-positive individuals. Further, we examined DQB1*02 and DRB4*01:01 alleles for their reported association with HPA-1a alloimmunization, in the context of DR-DQ haplotypes. Since ~ 80% of DQB1*02 alleles are linked to the DR3-DQ2 haplotype, the association might be coincidental. However, the DQB1*02:02-associated DR7-DQ2 haplotype was also overrepresented in alloimmunized women, suggesting a role for this allele or haplotype in HPA-1a alloimmunization. As DRB4*01:01 is predominantly associated with the DR7-DQ2 haplotype in HPA-1a-alloimmunized individuals, the reported association with FNAIT may be coincidental. Typing for DR-DQ haplotypes revealed important genetic associations with HPA-1a alloimmunization not evident from typing individual alleles, and the presence of different DRB3-associated DR-DQ haplotypes showed different prevalence of HPA-1a alloimmunization., (© 2020 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology.)

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., (© 2017 British Blood Transfusion Society.)

Emerging lipidomic technologies have enabled researchers to dissect the complex roles of phospholipases in lipid metabolism, cellular signaling and immune regulation. Host phospholipase products are involved in stimulating and resolving the inflammatory response to pathogens. While many pathogen-derived phospholipases also manipulate the immune response, they have recently been shown to be involved in lipid remodeling and scavenging during replication. Animal and plant hosts as well as many pathogens contain a family of patatin-like phospholipases, which have been shown to have phospholipase A 2 activity. Proteins containing patatin-like phospholipase domains have been identified in protozoan parasites within the Apicomplexa phylum. These parasites are the causative agents of some of the most widespread human diseases. Malaria, caused by Plasmodium spp., kills nearly half a million people worldwide each year. Toxoplasma and Cryptosporidium infect millions of people each year with lethal consequences in immunocompromised populations. Parasite-derived patatin-like phospholipases are likely effective drug targets and progress in the tools available to the Apicomplexan field will allow for a closer look at the interplay of lipid metabolism and immune regulation during host infection., (© 2017 John Wiley & Sons Ltd.)

Background: The development of specific antibodies against human leukocyte antigen (HLA) and/or human platelet antigen (HPA) could induce platelet transfusion refractoriness especially in patients receiving multiple platelet transfusions. A retrospective analysis was conducted to evaluate the prevalence of platelet-specific antibodies and the efficacy of crossmatch-compatible platelet transfusions in these recipients., Study Design and Methods: All enrolled patients were refractory to random single-donor apheresis Platelet (PLT) units. Enzyme-linked immunosorbent assay (ELISA) was used to detect anti-HLA and anti-HPA antibodies in serum. For those patients with antibodies, the PLT crossmatch assays were performed to select the compatible PLTs with a commercial solid-phase adherence kit., Results: A total of 193 patients were included and 29.02% of which was HLA and/or HPA antibody-positive. There were no significant differences in antibody-positive rates among AML/CML, ALL/CLL, MDS, SAA and ITP groups, but they are statistically significantly higher than other groups (P = 0.0035). Of those antibody-positive patients, there were 41 (73.21%) patients with only HLA antibodies, 11 (19.64%) patients with only HPA antibodies and 4 (7.14%) patients with both HLA and HPA antibodies. A total of 43 random PLT units and 88 crossmatch-compatible PLT units were administered. The mean (± SD) corrected count increment (CCI) was 8700 (± 4500) after crossmatch-compatible unit transfusion, significantly higher than 3600 (± 2400) for random PLT units (P < 0.001)., Conclusions: HLA and/or HPA alloimmunisation is an important factor to cause refractoriness to platelet transfusions. Crossmatch-compatible platelet transfusion is an effective method in those patients refractory to random platelet transfusions., (© 2014 British Blood Transfusion Society.)

Objective: To study the clinical usefulness of maternal anti-HPA-1a antibody levels in predicting severe foetomaternal alloimmune thrombocytopenia (FMAIT)., Background: Recent studies using an international anti-HPA-1a standard have shown a correlation between maternal antibody levels and neonatal thrombocytopenia. Cut-off values for identifying high-risk pregnancies have also been suggested., Materials: In 1986-2010, HPA-1a alloimmunisation was confirmed in 84 women with 129 pregnancies. Maternal samples were obtained at delivery and during subsequent pregnancies. Anti-HPA-1a was quantified using a MAIPA assay with a detection limit of 0·8 IU mL(-1) (WHO reference serum 03/152). Antibody levels were compared with the severity of neonatal disease in the index and in the subsequent pregnancies., Results: In the index cases, the correlation between an anti-HPA-1a level and neonatal platelet count did not reach statistical significance (n = 77, P = 0·074). However, the platelet counts and antibody levels in cases with cutaneous (n = 45) or intracranial haemorrhage (n = 7) were significantly different from cases with no evidence of bleeding (n = 20). In the subsequent pregnancies, there was a stronger association between the second trimester anti-HPA-1a level and the foetal platelet count (n = 16, P = 0·046). The positive predictive value of the maternal antibody level for a foetal platelet count <20 × 10(9) L(-1) was 90%, but the negative predictive value only 31%., Conclusion: Although a higher anti-HPA-1a level correlated with a more severe neonatal disease, barely detectable antibody levels were also observed in severely affected pregnancies. Cut-off values with sufficient sensitivity and specificity to identify these foetuses could not be found. A previous obstetric history still remains the most useful predictive parameter for severe FMAIT in clinical practice., (© 2013 The Authors. Transfusion Medicine © 2013 British Blood Transfusion Society.)

Introduction: The HPA-15 antigen system is characterized by a low antigen expression on platelets. The antibodies against this antigen are implied in fetal/neonatal alloimmune thrombocytopenia (F/NAIT), post-transfusion purpura, and refractoriness to platelet transfusions. Detection of these antibodies appears to be related to the level of HPA-15 expression on the platelets used in the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay., Methods: We performed genotyping of 300 healthy blood donors for HPA-15 by TaqMan real-time PCR technology, and the HPA-15 antigen expression was investigated in 13 HPA-15aa and 19 HPA-15bb individuals. We also investigated the relevance of HPA-15 antigen expression on donor platelets used in MAIPA for antibody detection in 223 multitransfused hematological patients and 271 women with suspected F/NAIT., Results: In Polish donors, the HPA-15a allele frequencies were lower than the HPA-15b (0.480 vs. 0.515). We identified three HPA-15 expression groups: high (36.7 ± 8.36 MFI - eight cases), medium (19.5 ± 6.2 MFI - 21 cases), and low (6.5 ± 5.9 MFI - three cases). The HPA-15 expression was stable over time. The HPA-15aa and HPA-15bb platelets with high antigen expression were used for anti-HPA-15 antibody detection; anti-HPA-15 antibodies were detected in 4/223 (1.8%) patients receiving multiple transfusions but in none of the 271 women with suspected F/NAIT. Further examination of the four sera by MAIPA with various platelets revealed the optical density in the assay to be closely related to the level of HPA-15 antigen expression., Conclusion: Anti-HPA-15 antibody detection should be based on carefully selected platelets with high HPA-15 expression level., (© 2011 Blackwell Publishing Ltd.)

Pregnancy may allow alloimmunization against human platelet antigens (HPA), which can lead to neonatal alloimmune thrombocytopenia (NAIT). The specificities of alloantibodies are closely related to the distribution of the HPA systems. A total of 281 Tunisian multiparous women (mean number of pregnancies: 4.5) were phenotyped for the HPA-1, -3 and -5 systems, by monoclonal antibody immobilization of platelet antigens (MAIPA). We searched for antibodies against HPA-1a, HPA-3a, HPA-5b and HPA-5a in HPA-1b1b, HPA-3b3b, HPA-5a5a and HPA-5b5b individuals, respectively. The gene frequencies were: 0.83 for HPA-1a, 0.17 for HPA-1b, 0.78 for HPA-3a, 0.22 for HPA-3b, 0.82 for HPA-5a and 0.18 for HPA-5b. Anti-HPA-5b antibodies were present in eight sera and anti-HPA-3a antibodies were present in one serum. The anti-HPA-5b system is the most frequently involved in platelet alloimmunization in Tunisian multiparous women. However, prospective trials are required to confirm this result and to determine the exact frequencies and clinical relevance of platelet alloantibodies in pregnant Tunisian women.

In this study, we evaluated the response to treatment of 409 idiopathic thrombocytopenic purpura (ITP) patients who were tested for the presence of platelet-associated autoantibodies by direct-platelet immunofluorescence test (PIFT) and for the presence of plasma antibodies directed against the GPIIb/IIIa, GPIb and GPIa/IIa by monoclonal antibody immobilization of platelet antigens (MAIPA). In patients with platelet autoantibodies in comparison with patients without antibodies more frequently were observed the chronic form of disease (83.5%vs. 68.5%) and severe symptoms of haemorrhage diathesis (17.3%vs. 6.9%). Evaluation of the treatment response (to corticosteroids, immunosuppressive drugs and splenectomy) referred to patients with complete response, e.g. complete remission defined as platelet count of >100 x 10(9)/l for at least 2 years. The percentage of complete response in the whole population of ITP patients, both with and without autoantibodies regardless of the method of treatment, was similar (about 54%). However, the presence of platelet autoantibodies had effect on patients treated with corticosteroids: complete response approximately 71% (36/51) of patients with autoantibodies and in 60% (72/120) of patients without antibodies, as well as in patients treated with immunosuppressive drugs (cyclophosphamide, azathioprine, vincristin and vinblastin); complete response approximately 51% (11/21) of patients with autoantibodies and in 34.8% (6/17) of patients without autoantibodies. The presence of autoantibodies had no effect on the response of splenectomy patients.

To study the relationship between antibodies detected in patients' and/or donors' sera and the clinical features of acute non-haemolytic transfusion reactions (ANHTRs), and to determine any gender-related difference. ANHTRs range from urticaria to transfusion-related acute lung injury (TRALI). Antibodies to human leukocyte antigen (HLA), granulocytes, platelets, and/or plasma proteins are implicated in some of the ANHTRs. A higher antibody positivity is expected for females than for males. A comparative study of ANHTRs for antibody positivity and their clinical features between females and males for both patients and donors is helpful for characterizing ANHTRs including TRALI more clearly, but such studies are few and outdated. Two hundred and twenty-three ANHTR cases reported by 45 hospitals between October 2000 and July 2005 were analysed. The patients and 196 donors of suspect blood products were screened for antibodies to HLA Class I, HLA Class II, granulocytes, and platelets. The patients were also screened for anti-plasma protein antibodies. The types and severity of ANHTR did not differ significantly between female and male patients. The frequency of the anti-HLA antibodies, but not that of the non-HLA antibodies, was significantly higher in females. Non-HLA antibodies were significantly associated with severe reactions in females. All the TRALI cases had predisposing risk factors for acute lung injury, and 60% of the cases showed anti-leucocyte antibodies. Although the anti-HLA antibodies were detected more frequently in females than males, no significant association of ANHTRs including TRALI with gender, not only for patients, but also for donors, could be shown in this study.

Post-transfusion purpura is a rare bleeding disorder characterized by severe and sudden thrombocytopenia within 3-12 days after blood transfusion. Typically, preformed antibodies directed against human platelet antigens, especially HPA-1a, are associated with the clinical symptoms. A 46-year-old female presenting to the hospital with acute progressive kidney insufficiency and anaemia received two units of packed red blood cells (RBC) within 2 days. On day 7, platelet count felt from 414 to 189 x 10(9) L(-1) and 1 day later dropped to 4 x 10(9) L(-1). Four platelet concentrates were applied without success. After serological confirmation of an HPA-1a antibody, the patient was treated with intravenous gamma immunoglobulin (ivIgG), and the platelet count increased to normal values on day 17. In addition to the persisting HPA-1a alloantibody, an antibody reactive with GPIa/IIa of HPA-5a- and HPA-5b-positive platelets was detected during the acute phase of thrombocytopenia. After complete remission, the patient was transfused with four units of packed RBC from HPA-1a-negative donors, and platelet counts remained normal.

Anti-human platelet antigens (HPA) alloantibodies are seldom involved in febrile nonhaemolytic reactions (FNHTRs). We describe a case in which anti-HPA-5a alloantibodies are related to an FNHTR. We studied the specificity of the alloantibodies by flow cytometry, ELISA and MACE. Typing of donors and the patient was performed by sequence-specific polymerase chain reaction. The alloantibodies were found reactive with HPA-5a antigens. The patient was HPA-5b/b, whereas the donor of the platelet apheresis involved in the FNHTR was HPA-5a/a. Despite the low frequency of anti-HPA-5a antibodies, they might be responsible for FNHTR.

Post-transfusion purpura (PTP) is a rare transfusion reaction almost exclusively observed in female patients. Affected patients develop severe immune-mediated thrombocytopenia in the course of a strong anamnestic alloimmune reaction against a platelet-specific antigen. The pathophysiology of thrombocytopenia has remained elusive. Immunological analysis in the HPA-1a-alloimmunized patient described in this report revealed an antibody with features considered typical of PTP: not only was anti-HPA-1a detectable in plasma, but it could also be eluted from the patients' (alloantigen negative) platelets, and anti-HPA-1a could be detected in eluates from both antigen positive and negative test platelets, which had been incubated in the patient's serum. This is in contrast to two sera with HPA-1a alloantibodies obtained from mothers of children with neonatal alloimmmune thrombocytopenia which were strictly HPA-1a specific. It is proposed that alloantibodies with HPA-1a-like specificity explain the patient's immune thrombocytopenia. The technique described in this report is proposed for further investigation, as it might be useful for discrimination of alloantibodes in PTP and alloantibodies of transfused thrombocytopenic patients.

The optimal treatment of neonatal alloimmune thrombocytopenia (NAIT) is the transfusion of compatible donor platelets. The National Blood Service in England has established panels of "accredited" donors negative for human platelet antigens HPA-1a and HPA-5b, the most commonly implicated alloantigens. We have retrospectively surveyed the frequency of use and clinical effectiveness of donations collected over a 13-month period from the Oxford accredited panel. Ninety-five per cent of hyperconcentrated platelets (HPCs) collected were issued, all for intrauterine transfusion to fetuses at risk of NAIT due to the presence of maternal platelet alloantibodies and previously affected siblings. Thirty-one per cent of paediatric platelet concentrates (PPCs) collected were issued, of which 57% were used for cases of suspected NAIT. Fifty-four per cent of adult therapeutic doses collected were issued; 5% of these were used in cases of suspected NAIT or proven post-transfusion purpura (PTP). Good increments were seen in most NAIT cases transfused with HPCs or PPCs, and a moderate increment in the one PTP case. We conclude that the establishment of accredited panels is justified and enables delivery of a clinically effective treatment for NAIT. Increased use and cost-effectiveness could be achieved by the delivery of an educational programme to neonatal unit clinical staff to increase the awareness and appropriate treatment of NAIT.

We report the second case of neonatal alloimmune thrombocytopenia due to anti-human platelet antigen (HPA)-3b (Bak(b)) 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 (Bak(b)).

Foetomaternal alloimmune thrombocytopenia (FMAIT) occurs when maternal antibodies of an antigen-negative mother cause destruction of sensitized foetal platelets. In Caucasian populations, 6-12% of human platelet antigen (HPA)-1a-negative women develop anti-HPA-1a, and the incidence of clinically affected cases is estimated to be 10-20% of immunized women. This study was performed in order to elucidate the rate of maternal immunization, incidence of FMAIT and the likely outcome of the condition in Asians. Excluding two or more pregnancies during the period, serum samples from 24 630 pregnant women, mainly Japanese, were screened for antibodies against platelet alloantigens by means of mixed passive haemagglutination (MPHA) (Anti-HPA-MPHA, Olympus, Tokyo). Antibodies were detected in 0.91% (223/24 630) of the women's samples and the immunization rate was correlated with the number of pregnancies. Antibody specificity included anti-HPA-4b (49), anti-HPA-5a (three), anti-HPA-5b (168), anti-HPA-4b + 5b (one) and anti-Nak(a) (CD36) (two). No alloimmunization was observed within the HPA-1, HPA-2, HPA-3 or HPA-6 systems. Among HPA-4b- or HPA-5b-negative women, 24% or 14% estimated, respectively, had antibodies and 26% (10/38) or 10% (12/125) of neonates, respectively, born to these mothers developed thrombocytopenia. Two neonates born to mothers having anti-HPA-4b developed generalized purpura. No cases of intracranial bleeding or death due to FMAIT were recorded. Generalized purpura due to FMAIT occurs in one in 9359 (95% CI: 1 in 77 519-1 in 2591) pregnancies solely because of HPA-4b incompatibility.

The performance of an anti-IgG/A/M and two anti-IgG alkaline phosphatase (AP) conjugates in a commercial antigen capture ELISA (ACE) were compared for their ability to detect antibodies to human platelet antigens (HPAs) contained in 11 sera. Three anti-HPA-1a and one anti-HPA-3a were not detected by the anti-IgG/A/M conjugate, but both anti-IgG conjugates detected all HPA antibodies as a consequence of increased sensitivity in detecting specific antibody binding. This increase varied from 10% to 500%, depending on the serum being tested. The increased sensitivity in some instances was also accompanied by an apparent increase in nonspecific binding, as measured by activity against irrelevant glycoproteins that should not have been recognized by the relevant HPA antibodies in the sera in question. Hence, anti-IgG conjugates would appear to be preferable for detecting platelet-reactive antibodies in many clinical situations; however, the choice of anti-IgG conjugate should be made judiciously (and appropriately validated), in order to avoid false positive results arising from increased nonspecific binding, which may otherwise be erroneously attributed to the presence of auto-antibodies in the serum under investigation.

Sera of 500 female blood donors after pregnancy were tested for platelet-specific antibodies (HPA-1, 3, 5) using the MAIPA assay. Twenty-one sera (4.2%) were found to be positive: four anti-HPA-1a, one anti-HPA-5a and 16 anti-HPA-5b. Sera were positive up to 30 years after the last pregnancy. All women with antibodies were HLA-DR52 positive and 79% of the women with anti-HPA-5b were HLA-DR6 positive.

We describe a human platelet alloantigen (HPA) 5a-alloimmunized HPA-5b5b mother. The children were obligatory heterozygotes for HPA-5a but despite IgG class maternal anti-HPA-5a antibodies only two (second and fifth) of the six children developed neonatal thrombocytopenia. Throughout the 4-year follow-up the mother had anti-HPA-5a antibodies (confirmed in the 8th Platelet serology workshop of International Society of Blood Transfusion in 1996). Antibodies against glycoproteins (GP) IIbIIIa or IbIX were not detected. Differences in the children's HPA type (HPA-1, -2, -3, -5) did not correlate with thrombocytopenia. We hypothesized that different expression of GPIaIIa recently associated with two silent polymorphisms (C807T and G873A) of GPIa could explain the unpredictable recurrence pattern of neonatal alloimmune thrombocytopenia (NAIT). Both parents were homozygous for the silent polymorphisms (C807 and G873) associated with the low expression of GP Ia. Thus, the inheritance pattern of the silent polymorphisms (C807T and G873A) did not help in predicting the recurrence risk of thrombocytopenia in the offspring. More detailed comprehension of the natural history of NAIT would be necessary to enable directing fetal blood sampling to the cases at the highest risk of thrombocytopenia.

Twenty-six serum samples from 24 patients were investigated for the presence of platelet-specific antibodies in a partly retrospective (n = 15) and partly prospective (n = 9) study. The sera contained either alloantibodies to human platelet antigens (HPA) (n = 23) or were from clinically suspected cases of fetomaternal alloimmune thrombocytopenia (FMAITP) in which platelet-specific antibodies had not been detected (n = 3). Three techniques were used to detect platelet antibodies: the platelet immunofluorescence test, the monoclonal antibody immobilization of platelet antigens (MAIPA) assay and a commercially available enzyme-linked immunosorbent assay--GTI PakPlus (GTI kit). Two alkaline phosphatase-conjugated antiglobulin reagents provided by the manufacturer were used in the GTI kit: an antihuman IgG/IgA/IgM (IgGAM) conjugate and an antihuman IgG conjugate. The GTI kit with the anti-IgGAM conjugate failed to detect eight antibody specificities in seven sera (anti-HPA-1a [n = 3], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1] and anti-HPA-5b [n = 3]). Greater signal-to-background ratios were achieved in the GTI kit with the anti-IgG conjugate but five antibody specificities (anti-HPA-1a [n = 1], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1], anti-HPA-5b [n = 2]) remained undetectable. All the sera were detected by MAIPA assay and, furthermore, the MAIPA assay achieved the greatest signal-to-background ratio in the majority of sera tested. These findings re-emphasize the value of the MAIPA assay in reference laboratories and illustrate that the GTI kit may either fail to detect or incorrectly identify clinically significant HPA antibodies.

We describe an alloimmunized female patient who developed serious adverse reactions when receiving HPA-incompatible platelet transfusions. She had received 13 transfusions with random platelets before the first allergic reactions. Antibodies against both the human leucocyte antigens (HLA) and several human platelet antigens (HPA) were detected at the time of transfusions. When the patient received HLA- and HPA-compatible platelets, no reactions followed the transfusions and platelet count increments were good. When she was transfused with platelets from donors with one foreign HLA antigen, her reactions were fever, chills and headache and the response to platelet transfusions was poor. When the platelets were HLA compatible but HPA incompatible, the reactions were repeatedly rapid pulse, shortness of breath, tightness of chest and wheezing interpretable as anaphylactoid reactions. Platelet count increments were satisfactory. When rare side-effects occur after transfusion, detailed immunohaematological studies are indicated.

The serum from an 85-year-old man with a clinical diagnosis of post-transfusion purpura (PTP) was investigated for the presence of platelet-specific antibodies. Clinically, the case was typical of PTP but, unusually, the serum was found to contain multiple platelet-specific alloantibodies. Anti-HPA-1a, anti-HPA-2b and anti-HPA-3a antibodies were detected together with multispecific anti-HLA (class I) antibodies. Additional (but weaker) antibody reactivity was also observed with platelet glycoproteins (Gp) IIb/IIIa, GpIb and GpIa/IIa which lacked the antigens recognized by the alloantibodies, suggesting the presence of auto- or cross-reacting antibodies. The patient's genotype was HPA-1b/1b, HPA-2a/2a, HPA-3b/3b, HPA-5a/5b and was consistent with the platelet alloantibodies detected. The patient made a complete recovery following treatment with intravenous gamma-globulin.

An enzyme immunoassay (EIA) method based on solubilized human leucocyte antigens (HLA) derived from single donor platelets is described. The EIA results on these solubilized single donor HLA antigens (SDszHLA) correlated well with the complement dependent cytotoxicity (CDC) results on the lymphocytes of the same donors and also with the panel reactivity (PRA) in CDC. A concordancy rate of 78% was found for individual HLA specificities. The EIA+/CDC- ('false positive') discrepancies were more pronounced than EIA-/CDC+ ('false negative') discrepancies and varied for the different donors. To confirm discrepancies, our method was compared with a commercial PRA-STAT EIA method (based on secreted soluble HLA antigens). The same discrepancies between CDC and PRA-STAT EIA were found and are probably due to the higher and different sensitivity (e.g. non complement fixing antibodies) of EIA methods. A SDszHLA EIA method allows the identification of HLA specificities of HLA-antisera. The possibility of using individual and selected donors for the production of SDszHLA allows the directed search for well defined HLA specificities in order to confirm anti-HLA specificities found in other anti-HLA screening methods. An individualized HLA panel can be established with the support of blood banks that have HLA typed blood and platelet donors.

Blood group ABO antigens are known to be carried by several platelet glycoproteins (GP), e.g. GPIb, GPIIa, GPIIb, GPIIa and PECAM. Beside these proteins, we recently observed that blood group A antigen was also expressed on some other uncharacterized platelet proteins (70-90 kDa) having electrophoretic mobilities closely resembling those of GPIV and GPV. These findings prompted us further to characterize these latter ABO-expressing platelet proteins. By antigen capture ELISA, wherein the monoclonal antibodies (mAbs) CLB-IVC7 and CLB-SWI6 were used to hold the corresponding antigens GPIV and GPV, human anti-A specifically bound to these proteins derived from A1-platelets; neither GPIV nor GPV derived from A2-, B- or O-platelets bound anti-A. In a Western blot assay using immunoprecipitated GPIV and GPV as antigens, mAb anti-A immunostained GPIV and GPV precipitated from A1, but not from A2 and O platelets. These results conclusively demonstrate that blood group A antigen is expressed on platelet GPIV and GPV.

The frequency and specificity of platelet-alloantibodies to human platelet antigens (HPA) -1, -3 and -5 was investigated in 59 multitransfused, HLA-immunized patients. Using the MAIPA test (monoclonal antibody specific immobilization of platelet antigens) platelet alloantibodies could be demonstrated in 10 (17%) patients. In one patient the antibody was present prior to any transfusions and probably induced by multiple previous pregnancies. This antibody was directed to HPA-5b. The remaining nine antibodies were found in patients (n = 36) with HLA-antibodies reacting with over 95% of unselected lymphocytes. In these patients the target antigens were HPA-1b in six, HPA-3a in one and both antigens in two patients. Our findings demonstrate platelet alloimmunization induced by transfusions to be restricted to patients with high HLA-immunization. 25% of these patients (9/36) show platelet-specific antibodies, primarily HPA-1b.

Cryptic antigens are detected by antibodies present in a wide spectrum of patients with or without thrombocytopenia, and even in healthy individuals. They are produced for unknown reasons and do not react with antigens of native platelets, but only with altered platelets. Cryptantigen antibodies may not only result in spuriously low platelet counts, but also in 'falsely' positive tests for platelet antibodies. We report our experience in the characterization of the different types of antibodies directed against cryptantigens of platelets: EDTA-dependent antibodies, PFA-dependent antibodies, EDTA-PFA-dependent antibodies and cold agglutinins. These antibodies were detected in the course of the serological study of 37 patients from a group of 356 (10%) whose blood was sent to our laboratory for platelet antibody testing. Pseudothrombocytopenia was diagnosed in 24 cases. Twenty-one of these showed EDTA-dependent or EDTA-PFA-dependent platelet agglutination and three were due to the presence of cold agglutinins. In 13 patients the thrombocytopenia was genuine. Eleven of these presented EDTA-dependent or EDTA-PFA-dependent antibodies in their serum and in the two remaining cases PFA-dependent antibodies were found. Cryptantigen antibodies were also detected in 9 out of 228 (4%) blood donors who were used as healthy controls in the platelet immunofluorescence test. In the light of the results obtained we put forward some guidelines to detect the presence of these antibodies and establish an accurate serological and clinical diagnosis of the autoimmune thrombocytopenias.

We report a male patient who developed post-transfusion purpura. Thrombocytopenia did not recur when intravenous IgG was given before the transfusion of platelet-depleted washed red-cell concentrates.

Glanzmann's thrombasthenia (GT) is a recessive autosomal bleeding disorder characterized by the abnormality of aggregation due to a platelet glycoprotein (GP) IIb-IIIa deficiency or a dysfunctional complex. Molecular abnormalities have been localized on the gene coding for GP IIb or IIIa. The aim of our work was an attempt to obtain indirectly information on the putative localization of the molecular defect in patients with GT type I or II by the determination of the HPA-1 (GP IIIa) and HPA-3 (GP IIb) alloantigenic systems' expression in GT carriers. If GT results from a defective GP IIb gene, a GT carrier would appear homozygous for HPA-3 by serology, because the normal gene product will be expressed while the abnormal GP IIb gene product will not be present. Conversely, if the abnormality is in the GP IIIa gene, such an individual would appear homozygous for HPA-1. Therefore, the heterozygous status for HPA would result from the normal expression of the two genes for the considered alloantigenic system. Among the four families studied with informative members, our presumptions were strengthened by the preliminary genetic results in one family showing a mutation in the GP IIb gene. Thus, serology could be a simple screening test for the possible defective gene responsible for GT allowing molecular investigation focusing only on GP IIb or IIIa gene.

In fetal alloimmune thrombocytopenia (FAIT) the fetus is threatened by intracranial haemorrhage (ICH); therefore early diagnostic and therapeutic intervention is required. We followed the clinical course of a 30-year-old woman during her fifth pregnancy after she had given birth to a child with alloimmune thrombocytopenia due to anti-Zwa. The fetus was monitored by 13 fetal blood samplings (FBS) always followed by transfusion of either maternal or compatible donor platelets. Intravenous immunoglobulin (ivIg) treatment of the mother was begun at 20 weeks of gestation when the fetal platelet count was 36 x 10(9)/l. The fetal platelets were typed Zwa positive by DNA analysis. Despite 11 weeks of maternal ivIg treatment fetal platelet counts progressively declined to 6 x 10(9)/l and ICH occurred. Subsequently, the fetus was successfully managed by intrauterine platelet transfusions at shorter intervals (3-5 days) and elective Cesarean section was carried out at 35 weeks of gestation. We conclude that maternal ivIg treatment does not prevent ICH in FAIT. The treatment of choice for severely affected cases is serial FBS combined with transfusion of compatible platelets.

The recognition that spontaneous intracranial haemorrhage (ICH) may occur in utero in fetomaternal alloimmune thrombocytopenia (FMAIT) led us to attempt to prevent this in 15 pregnancies of 11 women who had previously affected infants with FMAIT due to anti-HPA-1a. The antenatal management included fetal platelet transfusions and maternal steroids and/or high-dose intravenous immunoglobulin (IVIgG). In the first pregnancy, ICH occurred between 32 and 35 weeks' gestation before any treatment had been given, emphasizing the need for earlier intervention. Five of the 14 subsequent pregnancies in this study were considered to be severely affected (severe haemorrhagic complications in a previous infant and initial fetal platelet count < 20 x 10(9)/L in this study); four were managed successfully with weekly fetal platelet transfusions started between 18 and 29 weeks and continued until delivery at 33-35 weeks, and one severely affected case who was referred at 36 weeks was managed successfully with a single platelet transfusion prior to delivery. Five pregnancies were considered to be mildly affected (previous infants were unaffected by severe bleeding and initial fetal platelet count > 50 x 10(9)/L in this study). The platelet counts were maintained in one case with steroids and in three with IVIgG without the need for repeated platelet transfusions, but in the fifth the fetal platelet count fell despite steroids and IVIgG and serial platelet transfusions were required. Four pregnancies were unsuccessful; two pregnancies were terminated after severe ICH occurred at an early stage before fetal blood sampling had been carried out, one fetus died after the mother had a severe fall despite the successful initiation of fetal platelet transfusions and one died due to a cord haematoma which occurred at the time of the initial fetal blood sampling. The optimal management of FMAIT to reduce the risk of antenatal ICH remains uncertain. Steroids and IVIgG may be effective in some mildly affected cases but serial fetal platelet transfusions are the preferred therapy for those who are severely affected.

Infants with neonatal alloimmune thrombocytopenia are at risk of severe intracranial haemorrhage. Placental transfer of maternal immunoglobulin G (IgG) directed against fetal platelet antigens is known to be the underlying mechanism. Since breast milk contains IgG it is theoretically possible that breast feeding of these infants could cause thrombocytopenia. The following case report shows that an infant with neonatal alloimmune thrombocytopenia may be safely breast fed, even when the breast milk contains the platelet specific antibody (HPA-1a).

The clinical and immunological data of 12 Danish cases of post-transfusion purpura (PTP) are summarized. All patients but one were women. All except one had thrombocytopenic purpura which occurred 4-11 days after transfusion, usually with a nadir of the platelet count below 10 x 10(9) 1(-1). The typical haemorrhagic symptoms were cutaneous bleedings, melena and haematuria lasting from 3 to 12 days. The time until normalization of platelet count varied from 3 to 50 days after onset. One patient had recurrence of PTP and one patient died due to intracranial haemorrhage. Ten of the HPA-1a negative patients (83%) had platelet-specific HPA-1a antibodies and two HPA-1a positive individuals had anti-HPA-1b antibodies. In 10 patients, HLA antibodies were also detectable and in one patient a delayed haemolytic transfusion reaction due to anti-E was seen. All of seven patients investigated had anti-HPA antibodies of both IgG1 and IgG3 subclasses during the thrombocytopenic period while all of 10 patients had anti-HPA of only the IgG1 subclass after recovery from PTP. Thus, the destruction of autologous platelets in PTP seems to be associated with the presence of anti-HPA of the IgG3 subclass which may be of importance in the pathogenesis of PTP.

Prenatal typing for the human platelet antigens-1 (HPA) permits identification of a fetus at risk for neonatal alloimmune thrombocytopenia (NAITP) in cases of HPA-1 incompatibility in which the father is heterozygous for the HPA-1a antigen. Diagnostic cordocentesis and phenotyping of the fetal platelets are used for this purpose. We applied allele-specific restriction enzyme analysis on polymerase chain reaction (PCR)-amplified DNA purified from amniocytes. This assays allows early second trimester typing for HPA-1 alleles. We were able to determine the genotype of three fetuses at risk. Iatrogenic fetal loss is lower with amniocentesis than with cordocentesis. Therefore, this technique is a welcome addition to the antenatal management of NAITP.

Neonatal alloimmune thrombocytopenia (NAIT) is caused by platelet antigen incompatibility between the mother and fetus. NAIT is mainly due to alloimmunization; the frequency varying among ethnic groups. In Caucasians HPA-1a is the antigen most frequently implicated. In Japan, NAIT due to anti-HPA-4b antibody has already been described, but this is the first case to be reported in Caucasians.

Neonatal thrombocytopenia affects 20-40% of the infants in intensive care units. The frequency of neonatal alloimmune thrombocytopenia (NAIT) is estimated at 1/1500 to 1/5000 live births. The risk of morbidity is significant with 20% neurological sequelae and the death rate is estimated at 10% of affected infants. During recent years considerable efforts have been made to prevent fetal bleeding and to avoid birth trauma, which have significantly changed the natural history of NAIT.

Investigation of the maternal serum in a case of suspected alloimmune neonatal thrombocytopenia by conventional, second-generation platelet serological assays (platelet radioactive antiglobulin test [PRAT], platelet suspension immunofluorescence test [PSIFT] and solid-phase adherence assay (SPAA, 'Capture-P') demonstrated only the presence of HLA class-I antibodies of limited specificities: no platelet-specific antibodies were detectable. The use of a third generation, glycoprotein capture assay (monoclonal antibody-specific immobilization of platelet antigens, MAIPA) revealed the additional presence of anti-HPA-5b with a titre of 1 in 32. Despite this relatively high titre, and the fact that it was able to induce a prolonged thrombocytopenia, this antibody was not detectable by conventional assays. In view of these findings we conclude that the use of MAIPA is essential when investigating cases of suspected alloimmune neonatal thrombocytopenia.

We describe platelet alloimmunization which caused severe thrombocytopenia in a neonate and could only be detected by testing the father's platelets. The platelet-specific antibodies were identified by a monoclonal antibody-immobilized platelet protein assay (MAIPA) using monoclonal antibodies against glycoprotein (GP) IIbIIIa complex (AP2 and 2G12). The previously described alloantigen systems on the GPIIbIIIa complex (HPA 1, HPA 3 or HPA 4) were not responsible for the reaction. In addition the newly described private platelet antigen Sra was not identical to the antigen. The antigen is therefore different from all known platelet alloantigens and was designated Vaa. The antigen was present on the platelets of the affected child. Family studies showed that the platelet antigen was transmitted as an autosomal dominant trait in three generations. No Va (a+) individuals were found in a population study of 250 blood donors, which indicates that the antigen is of low frequency in the Finnish population. The Va antigen was not detectable by immunoblot analysis, which suggests that the epitope may not be a linear peptide structure. The antigen was also destroyed by solubilization of platelets. Thrombin activation of platelets, known to increase the expression of GPIIbIIIa on platelets, did not increase the number of binding sites for anti-Vaa antibodies to the extent observed with anti-HPA 1a binding.

The isolated perfused lobule of human placenta was used as an in-vitro model to study the effect of intravenous immunoglobulin (IVGG) on the placental transfer of a human platelet-specific antibody (anti-P1A1). Normal human IgG was shown to transfer from the maternal to the fetal circulation of the placental model after a lag period of 2-3 h. IVGG also transferred across the placenta but only after a longer lag period (3-4 h) than normal human IgG at the same concentration, which suggests that IVGG may contain a factor that inhibits the transfer of its own component IgG. The sensitive Western immunoblotting technique was used to demonstrate progressive transfer of anti-P1A1 antibody to the fetal circulation after a 2-3 h lag period. When IVGG and anti-P1A1 antibody were added simultaneously to the maternal circulation, the transfer of platelet-specific antibody was strongly inhibited by IVGG. The inhibitory effect of IVGG on anti-P1A1 antibody transfer was consistent for three different batches of the same IVGG product (Sandoglobulin). These studies provide the first scientific data to support the use of IVGG to inhibit antiplatelet antibody transfer as part of the antenatal management of neonatal alloimmune thrombocytopenia.

Solid-phase red-cell adherence (SPRCA) techniques in platelet serology are used mainly for crossmatching. A SPRCA method for general diagnostic application was evaluated in parallel with the platelet suspension immunofluorescence test (PIFT). Of 149 patient sera sent for investigation of thrombocytopaenia, 76 were negative and 59 positive when studied by both methods, eight positive by PIFT only and six positive by SPRCA only. The reactivity observed for 24 sera containing HLA antibodies tested with chloroquine-treated and untreated platelets was similar for both methods. All of 14 sera containing quinine-associated antibodies reacted strongly to both techniques in the presence of added quinine. In comparison, however, whereas all sera were nonreactive to SPRCA in the absence of added quinine, and with PIFT, seven of the sera reacted weakly. Titration studies with three examples of anti-PlA1 and five sera containing HLA antibodies generally showed a one doubling dilution lower titre with the SPRCA procedure. End-point interpretation, however, was more readily achieved with the SPRCA method. The SPRCA technique displays similar sensitivity and specificity to the PIFT and is recommended for use by routine hospital laboratories to screen platelet antibodies.

Antibodies to the HPA-1a antigen can elicit a condition in the new-born known as neonatal alloimmune thrombocytopenia (NAITP). Retrospective and prospective studies have shown that there is a strong correlation between the presence of HLA-DR3, HLA-DRw52 in the mother and the antibody response to HPA-1a. HLA Class II molecules play an important role in the initiation of the immune response and it has been postulated that HPA-1a antibody production could be determined by the presence of a specific HLA Class II molecule at the surface of the antigen-presenting cell. Thirty-one HPA-1a negative women with HPA-1a antibodies (responders) and nine HPA-1a negative women without HPA-1a antibodies (non-responders) were recruited. They were studied using serological HLA Class I and Class II typing and RFLP analysis with a DR beta probe. We found that all responders had the HLA-DRw52a sub-specificity confirming recently published data. Moreover, two of the nine non-responders were also found to be HLA-DRw52a. These results suggest that the HLA-DRw52a molecule is necessary for HPA-1a antibody responsiveness but not sufficient. The results also indicate that in HPA-1a negative women the absence of HLA-DRw52a is associated with a very low risk of being antibody producers and hence, is associated with a very low risk for NAITP in their new-borns.