HPA-1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia

BACKGROUND: The antenatal management of fetomaternal alloimmune thrombocytopenia (FMAIT) due to HPA-1a antibodies remains controversial, and a test identifying pregnancies that do not require therapy would be of clinical value. STUDY DESIGN AND METHODS: The statistical correlation was analyzed between clinical outcome and 1) anti-HPA-1a potency in maternal serum samples determined by a monoclonal antibody immobilization of platelet (PLT) antigen assay with an international anti-HPA-1a potency standard and 2) anti-HPA-1a biological activity measured by a monocyte chemiluminescence (CL) assay. RESULTS: A total of 133 pregnancies with FMAIT due to anti-HPA-1a were analyzed. In 97 newly diagnosed cases, there was no difference in antibody potency or CL signal between cases with intracranial hemorrhage (ICH; n = 15), those with no ICH but a PLT count of less than 20 x 10(9) per L (n = 52), and those with a PLT count of at least 20 x 10(9) per L (n = 30). In 22 previously known pregnancies, the positive predictive value of maternal anti-HPA-1a of greater than 30 IU per mL for a PLT count of less than 20 x 10(9) per L was 90 percent, but the negative predictive value was only 66 percent. Antibody potency tended to stay stable throughout pregnancy (n = 16) and from one pregnancy to the next (n = 16). CONCLUSION: Neither severe thrombocytopenia nor ICH in HPA-1a-alloimmunized pregnancies can be predicted with sufficient sensitivity and specificity for clinical application from maternal anti-HPA-1a potency or bioactivity.     

HPA-5b (Bra) neonatal alloimmune thrombocytopenia in Quebec: incidence and clinical outcome in 31 cases

BACKGROUND: Clinical detection of neonatal allo-immune thrombocytopenia (NAITP) is often less than the incidence predicted in prospective studies. The aims of this study were to calculate the observed and expected incidence of NAITP in Quebec, Canada, and to evaluate the clinical outcome of infants with anti-HPA-5b NAITP. STUDY DESIGN AND METHODS: Records from 1998 to 2002 of the only PLT serology laboratory for the province of Quebec were reviewed, as were hospital charts of all HPA-5 cases. The number of expected NAITP cases was estimated with known alloantigen gene frequencies. RESULTS: Ninety cases of NAITP were identified. The clinical detection rate was 1 in 4100, with 9 to 30 percent of expected HPA-1a and 11 to 23 percent of expected HPA-5b NAITP cases detected. Seventy-eight percent of cases of HPA-5b NAITP were asymptomatic. Sixty-three percent of all deliveries but 94 percent of HPA-5b cases occurred in hospitals performing cord blood PLT counts. CONCLUSION: The NAITP detection rate was 25 to 50 percent of the rate reported in prospective trials. The less severe clinical presentation of NAITP owing to HPA-5b was confirmed. The frequent routine determination of cord blood PLT counts may explain increased detection of asymptomatic NAITP involving anti-HPA-5b.     

Maternal alloimmunization against the rare platelet-specific antigen HPA-9b (Max a) is an important cause of neonatal alloimmune thrombocytopenia

BACKGROUND: Neonatal alloimmune thrombocytopenia (NATP) is an important cause of morbidity and mortality in the newborn. Optimal management of subsequent pregnancies requires knowledge of the alloantigen that caused maternal immunization, but this is possible only in a minority of cases. This study investigated whether this can be explained in part by maternal immunization against the "rare" alloantigen HPA-9b (Max(a)), implicated previously only in a single NATP case. STUDY DESIGN AND METHODS: Archived paternal DNA from unresolved cases of NATP and normal individuals was typed for platelet (PLT)-specific antigens with real-time polymerase chain reaction and direct sequencing. PLT-specific alloantibodies were characterized by flow cytometry and solid-phase enzyme-linked immunosorbent assay. Recombinant GPIIb/IIIa was expressed in stably transfected Chinese hamster ovary cells. Clinical information was obtained directly from attending physicians. RESULTS: Six of 217 fathers were positive for the presence of HPA-9b (Max(a)), an incidence about seven times that in the general population. In each of five cases studied, maternal serum samples reacted with intact paternal PLTs and paternal GPIIb/IIIa. Only one of three serum samples tested recognized recombinant GPIIb/IIIa carrying the HPA-9b (Max(a)) mutation. These seemingly discrepant reactions may reflect different requirements for oligosaccharides linked to residues close to the mutation in GPIIb that determines HPA-9b (Max(a)). NATP in the affected children was severe and was associated with intracranial hemorrhage in three of six infants on whom information was obtained. CONCLUSIONS: Maternal immunization against HPA-9b (Max(a)) is an important cause of NATP and should be considered in cases of apparent NATP not resolved on the basis of maternal-fetal incompatibility for "common" PLT antigens.     

抗HPA-3a抗体所致新生儿同种免疫性血小板减少性紫癜一例并文献复习

目的 探讨抗血小板特异性抗原(HPA)-3a抗体所致新生儿同种免疫性血小板减少性紫癜(NAITP)的诊断和治疗.方法 采用多重PCR及基因测序技术检测1例出血伴血小板减少新生儿及其父母HPA-1~21bw系统基因型,采用流式细胞术(FCM)和血小板抗原单克隆抗体特异性免疫固定检测技术(MAIPA)检测患儿及其母亲血清血小板特异性抗体并进行特异性鉴定.结果 患儿出生后2 h出现全身多发皮下出血点、血尿及咖啡色呕吐物.基因分型显示患儿为HPA-3ab、母亲为HPA-3bb、父亲为HPA-3aa;患儿及母亲血清中均含与患儿父亲血小板反应的特异性抗体,经MAIPA技术鉴定为抗HPA-3a抗体.结论 发现1例抗HPA-3a抗体所致NAITP患者,通过临床特征分析为该病的诊断和治疗提供借鉴与参考.     

HPA-13bw neonatal alloimmune thrombocytopenia and low frequency alloantigens: case report and review of the literature

BACKGROUND: Fetal-neonatal alloimmune thrombocytopenia (FNAIT) linked to rare or private antigens is not a rare event. STUDY DESIGN AND METHODS: Such a case discovered during the follow-up of a second child with jaundice with mild thrombocytopenia is reported here. Platelet (PLT) genotyping was performed by polymerase chain reaction (PCR)-sequence-specific primers method and PCR-restriction fragment length polymorphism (RFLP) analysis. Serologic investigation was done with the monoclonal antibody-specific immobilization of PLT antigens technique. Glycoprotein Ia-specific amplification and sequencing were performed for the polymorphism 807 (exon 7). RESULTS: The mother was found to be HPA-13aaw, and the father HPA-13abw. A maternal alloantibody directed against HPA-13bw has been characterized, leading to the diagnosis of neonatal alloimmune thrombocytopenia. CONCLUSION: This report provides further evidence that NAIT associated with low-frequency antigens is not restricted to single families. Therefore, laboratory investigation of a suspected case should be carried out in a specialist laboratory well experienced in optimal testing to propose appropriate management for the index case and subsequent pregnancies.     

Strategies to develop a prophylaxis for the prevention of HPA-1a immunization and fetal and neonatal alloimmune thrombocytopenia

Anti-HPA-1a-antibodies are the main cause of fetal and neonatal alloimmune thrombocytopenia (FNAIT) which may result in intracranial hemorrhage (ICH) and death among fetuses and newborns. Advances in understanding the pathogenesis of FNAIT and proof of concept for prophylaxis to prevent immunization suggest that development of hyperimmune anti-HPA-1a IgG aimed at preventing immunization against HPA-1a and FNAIT is feasible. Anti-HPA-1a IgG can be obtained either by isolating immunoglobulin from already-immunized women or by development of monoclonal anti-HPA-1a antibodies.Here we discuss recent advances that may lead to the development of a prenatal and postnatal prophylactic treatment for the prevention of HPA-1a-associated FNAIT and life-threatening FNAIT-induced complications.     

Perinatal management of fetal hemolytic disease due to Rh incompatibility combined with fetal alloimmune thrombocytopenia due to HPA-5b incompatibility.

We report out experience in the perinatal management of a complex case of fetal hemolytic disease primarily due to Rhesus incompatibility combined with fetal alloimmune thrombocytopenia. The lowest fetal hemoglobin and platelet levels were 2.6 g/dl and 13,000/microliter, respectively. Intrauterine treatment consisted of six transfusions of packed red cells into the umbilical vein and one transfusion of platelets. The neonate required four transfusions of packed red cells to correct her hyporegenerative erythropoiesis. Postnatal management also included one platelet transfusion, intravenous immunoglobulins and erythropoietin. Although some degree of fetal thrombocytopenia may invariably be found in fetal red cell incompatibility, other rare causes need to be excluded.     

Implication of antibodies against human leukocyte antigen in simultaneous presentation of fetal and neonatal alloimmune thrombocytopenia and neutropenia

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) and neonatal alloimmune neutropenia (NAN) are two rare complications of newborns caused by antibodies against paternal inherited antigens. Human platelet (HPA) and neutrophil antigens (HNA) are the common targets. Human leukocyte antigen (HLA) class I proteins are also expressed on platelets and neutrophils and anti-HLA antibodies have occasionally been implicated in these complications. We report a premature twin infant who presented with severe thrombocytopenia and neutropenia clinically compatible with FNAIT and NAN, from a mother with no identifiable HPA or HNA antibodies, but with very high levels of complement-fixing antibodies against paternal inherited HLA. These antibodies were also detected in the infant. HLA antibodies are commonly present in multiparous women who deliver healthy infants. They can, however, be cytotoxic and cause clinical complications after blood products transfusion (TRALI and becoming refractory to platelets transfusion) and after organ transplantation (allogeneic organ rejection).     

Human platelet antigen typing of neonatal alloimmune thrombocytopenia patients using whole genome amplified DNA and a 5'-nuclease assay

BACKGROUND: A serious constraint in the investigation of the human platelet antigen (HPA) status of potential neonatal alloimmune thrombocytopenia (NAIT) cases is the limited amount of DNA available from the neonates. Whole genome amplification (WGA) of these DNA samples could overcome this problem, but requires validation to ensure that it is sufficiently sensitive and accurate before its application in a clinical diagnostic setting.
STUDY DESIGN AND METHODS: This study has validated the use of WGA DNA for HPA-1, -2, -3, -4, -5, and -15 genotyping with a panel of six controls and 13 previously HPA-typed samples from neonates together with parental DNA, using a 5'-nuclease (TaqMan) assay. WGA was performed using titrated amounts of genomic and WGA DNA template. HPA typing was performed on genomic and amplified DNA using a 5'-nuclease assay or polymerase chain reaction with sequence-specific primers (PCR-SSP).
RESULTS: WGA DNA yields were in the suggested range of 400× to 800×, as assessed by spectrophotometry and gel analysis, and did not require further purification. HPA genotyping showed 100 percent concordance when using down to 5 ng of genomic or WGA template.
CONCLUSION: This study demonstrates that WGA can be used for HPA typing using PCR-SSP or plate-based 5'-nuclease assays. The use of WGA for HPA typing in clinical samples from NAIT patients was validated with 100 percent concordance, and it is suggested that this technology can be used for other analyses where DNA amounts are limited.     

Metabolite-specific (IgG) and drug-specific antibodies (IgG, IgM) in two cases of trimethoprim-sulfamethoxazole-induced immune thrombocytopenia

Two cases of trimethoprim-sulfamethoxazole (TMP-SMX)-induced immune thrombocytopenia are reported in which unusual drug-dependent platelet antibodies were demonstrated by immunofluorescence and enzyme-linked immunosorbent assay. Whereas two distinct sulfamethoxazole-dependent antibodies of the IgG and IgM class were detectable in the serum of one patient, the serum of the other patient contained a platelet antibody exclusively reactive with N-4-acetyl-sulfamethoxazole, a metabolite of sulfamethoxazole. Urine from a healthy volunteer collected after administration of therapeutic doses of TMP-SMX proved to be an appropriate source of ex vivo metabolites for antibody testing. The results of this study stress the role of metabolite-specific antibodies in drug-dependent immune thrombocytopenia and underscore the necessity of including metabolite preparations of drugs in serologic analyses.     

Neutrophil antigen 5b is carried by a protein, migrating from 70 to 95 kDa, and may be involved in neonatal alloimmune neutropenia

BACKGROUND: Neutrophil antigen 5b has been described as involved in transfusion reactions and not in neonatal alloimmune neutropenia. CASE REPORT: Anti-5b was found in the serum of a mother of a persistently neutropenic newborn, who had several bacterial infections. The neutropenia responded to treatment with recombinant human granulocyte-colony-stimulating factor. Immunoprecipitation experiments performed with this and three other 5b antisera identified a protein, migrating from 70 to 95 kDa, as carrier of 5b. The observed pattern of migration may point to heavy glycosylation of this protein. RESULTS: Six 5b-negative donors were identified among 54 screened white donors, for a 5b gene frequency of 0.66. CONCLUSION: Alloimmunization to 5b in pregnancy is rare. In the patients with neonatal neutropenia analyzed in the last decade, this was the first case discovered.     

Collection tubes with or without gel separator did not interfere with detection of rubella virus antibodies IgM and IgG

Rubella infection is an exanthematic disease, with high prevalence in the adult population. The only modality of disease that causes serious consequences is congenital rubella syndrome (CRS), which happens when a pregnant woman seronegative to rubella virus acquires the infection during early pregnancy. Due to the lack of signals and characteristic symptoms of disease, diagnosis of rubella is based essentially on laboratory tests: antibodies detection and/or virus isolation. Results of serologic tests should always be interpreted with caution, because they can be affected by the quality of blood samples, processing and storage of sera, the equipment and reagents used to perform tests, and finally by the technical expertise and training of biologists. The collection tubes with gel seem to facilitate serum separation, but on the other hand gels can retain and consequently decrease antibody titers. Therefore, we decided to investigate whether the use of collection tubes containing gel separator might interfere with rubella virus antibody detection in blood samples from children. We did not observe statistically significant differences with respect to rubella virus antibody detection (immunoglobulin M [IgM] and immunoglobulin G [IgG]) for samples collected in tubes with or without gel separator, from the two evaluated manufacturers.     

国内首例抗HPA-5b引发的新生儿同种免疫性血小板减少性紫癜的检测、诊断及分析

本研究旨在探讨抗HPA-5b抗体引发的新生儿同种免疫性血小板减少性紫癜（NAITP）的实验检测及临床诊断。临床监测患儿血小板计数及其他临床表现;多重PCR及DNA测序检测患儿及其父母HPA-1—21bw系统基因型;流式细胞术（FCM）和血小板抗原单克隆抗体特异性免疫固定检测技术（MAIPA）检测及鉴定患儿及其母亲血清中的血小板特异性抗体。结果表明：患儿临床症状较轻,基因分析显示患儿为HPA-5ab,父亲为HPA-5ab,母亲为HPA-5aa;患儿母亲血清中含与父亲血小板反应的血小板特异性抗体为抗HPA-5b抗体。结论：该病例为抗HPA-5b抗体引起的新生儿同种免疫性血小板减少性紫癜。