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PubMed1991

Elution of anti-Zwa (-PIA1) from autologous platelets after normalization of platelet count in post-transfusion purpura.

In two patients suffering from post-transfusion purpura, anti-Zwa was demonstrated in either eluates from autologous platelets harvested during the remission phase and after normalization of the platelet count. In both patients, platelet-associated immunoglobulin (PAIg) was demonstrated during the acute phase. PAIg disappeared concomitantly with recovery and, thus, seems to be associated with the thrombocytopenia. These data support the assumption that immunocomplexes are adsorbed nonspecifically to the platelets and cause destruction of autologous platelets lacking the corresponding antigen. An association between the IgG3 subclass of anti-Zwa antibodies and the destruction of autologous platelets was also seen.

Aged↗
PubMed1991

Brb, a platelet alloantigen involved in neonatal alloimmune thrombocytopenia.

Serum from a pregnant woman with the May-Hegglin anomaly contained a platelet-specific antibody. The serum reacted in the platelet indirect immunofluorescence test (PIIFT) with 97.6% of random donor platelets and those of the father but not with the mother's own platelets. This antibody induced a moderate thrombocytopenia in the infant that responded to infusion of intravenous immunoglobulin concentrates. The platelet phenotypes were PLA1+, Baka+, Bra+/Brb- for the mother, PLA1+, Baka+, Bra-/Brb+ for the father, and PLA1+, Bra+/Brb+ for the neonate. Analysis of the maternal serum with an immunoassay based on monoclonal antibody immobilization of platelet antigens (MAIPA) and immunoprecipitation techniques demonstrated the absence of antibodies directed against HLA class I antigens and that the antigen recognized was located on the platelet-GpIa/IIa complex. This antigen was present on 113/115 random donor platelets, in 7 of the 7 unrelated May-Hegglin platelets, and only absent in 3/24 Bra+ individuals, including the mother. No platelet-specific antibody was present in the serum of the 7 unrelated May-Hegglin subjects. The antigen recognized by this platelet-specific antibody thus meets the criteria defining the antithetic allele of Bra, i.e. the Brb alloantigen.

Adult↗
PubMed1992

A case of 'auto-immune thrombocytopenic purpura' serologically similar to post-transfusion purpura.

The report describes a HPA-1a (Zwa)-negative woman with thrombocytopenia and antibodies in serum and eluate from autologous platelets with an operational anti-HPA-1a specificity. The results of the serological investigations were similar to the findings in most patients suffering from post-transfusion purpura. However, the present patient had no history of blood transfusion prior to the unset of purpura and the thrombocytopenia had persisted 6 months before splenectomy.

Antigens, Human Platelet↗
PubMed1993

Evaluation of monoclonal antibodies with specificity for the human platelet HPA-1 allotypes.

Two mouse monoclonal antibodies have been characterized. Both antibodies specifically recognized the amino acid sequences which are responsible for the presence of the HPA-1a and HPA-1b alloantigens at the surface of human platelet. The monoclonality allied with an exquisite specificity of these antibodies allow a perfect typing of platelet HPA-1 status. A fast and reliable Elisa assay which fulfills all the needs and requirements of hematology laboratories has been set up and is described here. The Elisa assay has been tested on a population of 689 blood donors. The results agreed with the known phenotypic frequencies of the HPA-1 alloantigens.

Amino Acid Sequence↗
PubMed1995

The therapeutic value of HPA-1a-negative platelet transfusion in post-transfusion purpura complicated by life-threatening haemorrhage.

Post-transfusion purpura (PTP) was diagnosed in a female patient 1 week after transfusion for hysterectomy. Despite treatment with oral steroids and intravenous immunoglobulin, platelet counts remained low, and the patient developed profuse rectal bleeding. Random platelet transfusion is reported to be ineffective in PTP. We report a case in which the transfusion of HPA-la negative platelets provided good increment and clinical haemostasis before intravenous immunoglobulin could have had an effect.

Antigens, Human Platelet↗
PubMed1998

Novel diagnostic and therapeutic strategies with genetically engineered human antibodies.

Human antibodies can be generated by recombinant technology. The human immunoglobulin repertoire can be tapped in an effective manner by the so called V gene phage display technology. Using this technique the genes encoding the variable domains of an antibody of interest can be captured from the B cell repertoire. With these V genes tailor-made immune recognition molecules can be obtained by a 'design and build' strategy. These novel developments will lead to the introduction of a manifold of antibody based therapies into the clinic in coming decades. Recombinant antibody technology also provides unique opportunities to study the molecular structure of antibody variable domains against blood cell antigens. Such studies might possibly lead to the development of new therapies for antibody mediated blood cell destruction.

Antibodies↗
PubMed1998

Screening programmes for foetomaternal alloimmune thrombocytopenia.

Screening to detect pregnancies at risk of severe thrombocytopenia due to Foetomaternal alloimmunisation to Human Platelet Alloantigens (FAIT) is not yet a standard of care. Screening may be performed antenatally with the possibility of early intervention/therapy. Post natal measurement of cord platelet count may not always prevent fetal damage but may detect severe thrombocytopenia from other causes. Assessment of FAIT screening against World Health Organisation standards for screening programmes helps define areas where knowledge is still lacking and where research effort should be directed. Limitations to screening have been lack of reliable assays for typing and antibody detection, a limited understanding of the relationship between the presence of alloantibodies and clinical disease, inability to predict non-invasively which cases will have a severe outcome, and controversy as to which antenatal therapy is optimal for cases at high risk of a poor outcome.

Adult↗
PubMed2004

Platelet characteristic antigens of CD34+ cells in cryopreserved cord blood: a study of platelet-derived microparticles in transplant processing.

BACKGROUND AND OBJECTIVES: In previous studies, we found that platelet microparticles (PMPs) bind to cord blood (CB) CD34+ cells and transfer adhesion molecules to them, which enhances their engraftment. Before applying this phenomenon in actual transplants, we investigated the effect of PMPs on cryopreserved CD34+ cells in CB. MATERIALS AND METHODS: We cryopreserved 18 CB units, then evaluated the binding of PMPs to CD34+ cells after thawing, by varying the expression of platelet characteristic antigens (CD41a, CD61, CD62P and CXCR4) on these cells. Adherence of the CD34+ cells, coated with freeze/thaw-induced PMPs, to endothelium and fibronectin was also studied, as were the effects of thrombin-induced PMPs from both fresh and preserved CB platelets. RESULTS: PMPs induced by freezing and thawing adhered less well to CD34+ cells than did those from fresh CB, and cells coated with these PMPs had poor adherence. However, thrombin-induced PMPs from both fresh and preserved CB platelets bound equally well to cryopreserved CD34+ cells and improved their adhesion properties. CONCLUSIONS: PMPs could be a useful tool for enhancing engraftment after CB transplantation.

Annexin A5↗
PubMed2005

Collaborative study to establish the first international standard for quantitation of anti-HPA-1a.

BACKGROUND AND OBJECTIVES: This report describes the production of a freeze-dried preparation of pooled human plasma, containing immunoglobulin G (IgG) antibodies against the human platelet antigen 1a (HPA-1a). The material, coded 03/152, is proposed as an International Standard containing 100 arbitrary units of anti-HPA-1a for use in quantitative assays to determine the anti-HPA-1a activity in clinical samples. MATERIALS AND METHODS: Plasma samples containing potent anti-HPA-1a were pooled and freeze dried in 1-ml ampoules. In addition, three individual plasma samples were selected which had varying levels of anti-HPA-1a activity. The anti-HPA-1a activity of these three samples was determined by using a variety of quantitative assays with the proposed standard as a reference. RESULTS: An international collaborative study, which was part of the 2004 ISBT Platelet Immunology Workshop, involved 39 laboratories in 24 countries and showed that the anti-HPA-1a activity in three test samples could be reliably determined by using the proposed standard. CONCLUSIONS: Laboratories can use this standard to measure the anti-HPA-1a activity in patient's samples. Further studies are required to determine the relationship between anti-HPA-1a activity and clinical outcome in patients with neonatal alloimmune thrombocytopenia (NAIT).

Antibodies, Monoclonal↗
PubMed2005

Report on the 12th International Society of Blood Transfusion platelet immunology workshop.

BACKGROUND AND OBJECTIVES: The aims of the 12th International Society of Blood Transfusion (ISBT) Platelet Immunology Workshop were to evaluate the proficiency of molecular human platelet antigen (HPA) genotyping and detection of platelet antibodies of unusual specificity or reactivity, to assess whether quantification of anti-HPA-1a is practicable, and to determine the variability of reagents and components used in the monoclonal antibody immobilization of platelet antigens assay (MAIPA). MATERIALS AND METHODS: Forty participants from 23 countries were sent 10 samples for DNA typing, five samples for antibody detection, a freeze-dried anti-HPA-1a standard, three samples for anti-HPA-1a quantification and a MAIPA method questionnaire. RESULTS: The detection and identification of HPA antibodies varied from 2.7 to 95% of participants. The number of HPA genotyping errors per sample ranged from 0 to 3.96% per HPA loci. The majority of laboratories were able to assign an arbitrary number of units/ml of anti-HPA-1a activity to the unknown samples. The MAIPA questionnaire indicated a wide variation among participants, both in method and in reagents used. CONCLUSIONS: The results obtained from this workshop highlighted deficiencies in testing regimes and identified a need for internationally available reference materials.

Antibodies, Monoclonal↗
PubMed2005

Severe thrombocytopenia due to host-derived anti-HPA-1a after non-myeloablative allogeneic haematopoietic stem cell transplantation for multiple myeloma: a case report.

BACKGROUND AND OBJECTIVES: Host- or donor-derived alloimmune thrombocytopenia can develop after non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT). We report the first case of host-derived HPA-1a antibodies. CASE REPORT: A 52-year-old male patient received HSCT from his human leucocyte antigen (HLA)-A, -B, -C, -DR identical brother after reduced intensity conditioning. Bilinear engraftment around day 12 was accompanied by a continuous decrease of platelet counts. We investigated for platelet antibodies because of a progressive decline of platelet counts and refractoriness to platelet transfusions. METHODS: The patient's serum was tested by enzyme-linked immunosorbent assay (ELISA), a solid phase assay and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay. Recipient's DNA from the time before HSCT and donor's DNA were genotyped for human platelet antigens. RESULTS: Serum obtained on day 15 after HSCT reacted strongly with the donor's platelets due to host-derived anti-HPA-1a- and anti-HLA I antibodies. Serum samples from days 39, 45 and 65 after HSCT contained only anti-HLA I; no antibodies were detectable on day 149. Platelet counts increased on day 20 spontaneously. The decrease of the antibodies accompanied by the increase of the platelet counts suggests progressive elimination of residual host cells. CONCLUSIONS: The HPA-1a antibodies affected thrombopoietic engraftment and the success of platelet transfusions.

Antigens, Human Platelet↗
PubMed2006

A novel antigen-specific capture assay for the detection of platelet antibodies and HPA-1a phenotyping.

BACKGROUND AND OBJECTIVES: The antigen-specific assays currently used for the laboratory investigation of platelet antibodies and antigens are technically complex and cannot be used in most routine laboratories. Here, we describe a simple antigen-specific capture assay (ASCA) for the detection of serum platelet antibodies and for human platelet antigen-1a (HPA-1a) phenotyping. MATERIALS AND METHODS: For the detection of platelet antibodies, platelets from healthy blood donors were incubated with biotinylated monoclonal antibodies to platelet glycoprotein complexes (GP), then solubilized and mixed with superparamagnetic streptavidin particles. Serum samples from patients with autoimmune thrombocytopenia (n = 39), from patients with platelet alloantibodies (6 HPA-1a, 1 HPA-2b, 1 HPA-3a, 6 HPA-5b), and from healthy blood donors (n = 70), were tested. All serum samples from the patients were investigated in parallel by the indirect monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA). For HPA-1a phenotyping, superparamagnetic particles were coated with a monoclonal antibody to HPA-1a and mixed with diluted whole blood samples from healthy blood donors (n = 139), who had previously been genotyped for platelet alloantigens. Results The indirect MAIPA detected autoantibodies in 18%, and the direct MAIPA in 50% of patients tested. In contrast, the new ASCA demonstrated positive results in 77% of patients. All tested alloantibodies reacted positive by the ASCA, and all serum samples from healthy blood donors were negative. The results of HPA-1a phenotyping were in concordance with those of genotyping in all cases. CONCLUSION: In our opinion, the ASCA is easy to perform and much more sensitive than the currently available antigen-specific assays for the detection of platelet antibodies.

Antigens, Human Platelet↗
PubMed2005

6-Hydroxydopamine-induced alterations in blood-brain barrier permeability.

Vascular inflammation is well known for its ability to compromise the function of the blood--brain barrier (BBB). Whether inflammation on the parenchymal side of the barrier, such as that associated with Parkinson's-like dopamine (DA) neuron lesions, similarly disrupts BBB function, is unknown. We assessed BBB integrity by examining the leakage of FITC-labeled albumin or horseradish peroxidase from the vasculature into parenchyma in animals exposed to the DA neurotoxin 6-hydroxydopamine (6OHDA). Unilateral injections of 6OHDA into the striatum or the medial forebrain bundle produced increased leakage in the ipsilateral substantia nigra and striatum 10 and 34 days following 6OHDA. Microglia were markedly activated and DA neurons were reduced by the lesions. The areas of BBB leakage were associated with increased expression of P-glycoprotein and beta 3-integrin expression suggesting, respectively, a compensatory response to inflammation and possible angiogenesis. Behavioural studies revealed that domperidone, a DA antagonist that normally does not cross the BBB, attenuated apomorphine-induced stereotypic behaviour in animals with 6OHDA lesions. This suggests that drugs which normally have no effect in brain can enter following Parkinson-like lesions. These data suggest that the events associated with DA neuron loss compromise BBB function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Prenatal determination of human platelet antigen type using DNA amplification following amniocentesis.

OBJECTIVES: To demonstrate that fetal human platelet antigen (HPA1) type can be determined, without the need for fetal blood sampling, by amplification of fetal DNA from amniotic fluid cells using polymerase chain reaction and allele specific oligonucleotide hybridisation. DESIGN: Oligonucleotide DNA primers were designed to amplify a portion of the platelet glycoprotein GpIIIa gene which spans the site of the single base change which differentiates HPA1a from HPA1b. Specific oligonucleotides were designed to hybridise either to the amplified HPA1a allele or to the HPA1b allele. Amniotic cells were used as the DNA template both directly and following formal isolation of DNA. Fetal HPA1 type, determined by this method in fifteen pregnancies not at risk of perinatal alloimmune thrombocytopaenia, was compared to typing of fetal blood obtained following cordocentesis. The methodology was then used to HPA type the fetus in two pregnancies at risk of the disease. SETTING: Department of Molecular Biology and Centre for Fetal Care, Queen Charlotte's Hospital. SUBJECTS: Fifteen women undergoing amniocentesis and fetal blood sampling for other indications and two women at risk of perinatal allo-immune thrombocytopaenia whose partners were heterozygotes. RESULTS: In the 15 control cases and the two clinical cases, determination of fetal HPA1 type from amniotic fluid cells agreed with typing of fetal blood. There was no difference in the efficiency of amplification from amniotic fluid cells directly or from isolated DNA. CONCLUSIONS: Fetal HPA type may be reliably determined by amplification of DNA from amniotic fluid cells, eliminating the need for fetal blood sampling or immunoglobulin administration when the fetus is HPA1a negative.

Amniocentesis↗
PubMed1997

Antenatal screening for HPA-1a by flow cytometry.

Pregnant women who attended antenatal clinics at King George V Hospital, the Birth Centre or were referred by obstetricians from February 19 July, 1996 were screened for the platelet antigen HPA-1a by flow cytometry. Forty out of 2,300 (1.7%) were found to be negative for this antigen. Of the 28 women followed throughout their pregnancy, none developed antibody to HPA-1a. Platelet counts performed on samples from 17 babies born to 17 of these mothers were all normal. This study proves the simplicity and rapidity of flow cytometry for platelet antigen screening. The results were comparable with the Solid Phase Red Cell Adherence (SPRCA) method and with PCR. The lack of a plentiful supply of specific antibody and the rarity of fetomaternal alloimmune thrombocytopenia (FMAIT) argue against the introduction of routine screening for maternal HPA-1a status at the present time.

Adolescent↗
PubMed2001

Feto-maternal alloimmune thrombocytopenia: a literature review and statistical analysis.

Exploring prognostic factors that determine outcomes in fetomaternal alloimmune thrombocytopenia (FMAIT), a search of Medline was performed covering the years 1966 to April 1998. 376 articles were collected and reviewed; 140 articles contained the case histories of 297 mothers and 433 pregnancies that fulfilled entry criteria. More than 30 data variables were sought from these cases. The data were analysed using SPSS and Arcus Quickstat Biomedical. Nineteen different antigen incompatibilities were documented, the majority being human platelet antigen (HPA)-1a (77.3%), HPA-3a (3.5%) and HPA-5b (3.5%). The relative risk reduction (RRR) in mortality with any intervention was 57% (0.19-0.77) p = 0.009. Treatment of HPA-1a (PlA1) pregnancies with intravenous immunoglobulin (IVIG) increased the likelihood of a neurologically normal outcome, relative risk (RR) 1.68, confidence interval (1.3-2.2) p = 0.0003. Treatment of HPA-1a (PlA1) pregnancies with only antenatal complementary platelet transfusions increased the likelihood of a neurologically normal outcome, RR 1.63 (1.1-2.1) p = 0.01. Despite reviews of more than 400 cases of FMAIT, few prognostic variables are identifiable. Although IVIG appears to reduce the risk of intracranial haemorrhage (ICH), the dosage and timing of IVIG treatment was varied. This study highlights the need for standardised and directed research.

Antigens, Human Platelet↗