Neonatal alloimmune thrombocytopenia due to a new alloantigen Bl(a) defined by an Asp458Gly substitution in GPIIIa.

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) commonly arises due to antibodies against a small number of well-defined human platelet antigens (HPAs). A minority of NAIT cases occur due to maternal immunization against low-frequency polymorphisms in platelet glycoprotein that result in new immunogenic epitopes. Antibodies to these novel epitopes can be detected by the incubation of maternal serum with paternal platelets and is usually performed after initial investigation using HPA-typed panel platelets has failed to provide evidence of NAIT. STUDY DESIGN AND METHODS: The propositus and the parents from a case of suspected neonatal alloimmune thrombocytopenia (NAIT) were investigated using serologic and molecular techniques to detect and identify relevant platelet-specific antibodies and for HPA typing. Calculations of molecular dynamics were undertaken to explore potential variations in the molecular structure. RESULTS: Maternal antibodies were detected that were reactive only in crossmatch with paternal platelets using the platelet immunofluorescence test (PIFT) and a GPIIb/IIIa monoclonal antibody immobilization of platelet antigen (MAIPA) assay. In the propositus and father, a novel mutation c.1373 A > G was found in exon 10 of ITGB3 resulting in the substitution of an aspartic acid for a glycine (p.Asp458Gly). Recombinant GPIIIa glycoprotein mutated to contain the novel mutation and expressed in HEK293 cells with GPIIb was also specifically recognized by maternal antibodies. Calculations of molecular dynamics identified that the mutation was in a structurally constrained site. CONCLUSION: This case describes a low-frequency platelet antigen (Asp458Gly) that defines a further alloantigenic target in NAIT. The case emphasizes the role of the platelet crossmatch as the single most useful tool to establish evidence of immunization of low-frequency platelet glycoprotein polymorphisms. A crossmatch should always be performed where there is strong clinical evidence of NAIT but initial laboratory investigations are not confirmatory.

Human platelet antigen (HPA)-specific immunoglobulin M antibodies in neonatal alloimmune thrombocytopenia can inhibit the binding of HPA-specific immunoglobulin G antibodies.

BACKGROUND: A term baby with unexplained thrombocytopenia and a platelet (PLT) count of 14 × 109 /L (maternal PLT count was 200 × 109 /L) was investigated for neonatal alloimmune thrombocytopenia. STUDY DESIGN AND METHODS: Serologic investigations were performed using the PLT immunofluorescence test (PIFT), monoclonal antibody immobilization of PLT antigens (MAIPA), and a bead-based assay (BBA) with maternal sera taken up to 56 days postdelivery. One serum sample was also separated into "immunoglobulin (Ig)M-rich" and "IgM-depleted" fractions and tested for PLT-specific antibodies. The family was genotyped for HPA. RESULTS: HPA-3a-specific IgM antibodies were detected in the PIFT and confirmed in the BBA. PLT-specific IgG HPA-3a antibodies were not detected in the MAIPA assay and BBA in the initial sample but were detected in both techniques in subsequent serum samples. Testing of IgM-rich and IgM-depleted fractions in the MAIPA assay revealed that IgG antibody binding of the IgM-depleted fraction was inhibited by approximately 50% when it was reconstituted with the IgM-rich fraction suggesting that the IgM antibodies blocked the binding of the IgG antibodies. This effect was not observed when the IgM-depleted fraction or untreated serum was diluted with elution buffer. Incompatibility for HPA-3 was identified between the mother and the infant. The infant received one HPA-1a, -5b negative neonatal PLT transfusion, and one random PLT transfusion, with satisfactory outcomes. Both units were later found to be HPA-3b3b. CONCLUSION: HPA-3a IgM antibodies can inhibit PLT-specific HPA-3a IgG antibodies in the MAIPA assay.

Late onset cytopenias following haematopoietic stem cell transplant associated with viral infection and cell specific antibodies.

This report describes a patient who received an allogeneic haematopoietic stem cell transplant and who, following a viral infection, developed late onset cytopenias associated with antibodies against red cells, platelets and granulocytes. Investigation of these cytopenias revealed the presence of lineage specific auto- and allo-antibodies, which were not present in either the donor or in the recipient prior to the viral infection. This case provides further evidence for the concept that viral challenges following HSCT can result in the production of cell specific antibodies that can have significant implications for patient management.

A distinct plasmablast and naïve B-cell phenotype in primary immune thrombocytopenia.

Primary immune thrombocytopenia is an autoimmune disorder in which platelet destruction is a consequence of both B- and T-cell dysregulation. Flow cytometry was used to further characterize the B- and T-cell compartments in a cross-sectional cohort of 26 immune thrombocytopenia patients including antiplatelet antibody positive (n=14) and negative (n=12) patients exposed to a range of therapies, and a cohort of matched healthy volunteers. Markers for B-cell activating factor and its receptors, relevant B-cell activation markers (CD95 and CD21) and markers for CD4(+) T-cell subsets, including circulating T-follicular helper-like cells, were included. Our results indicate that an expanded population of CD95(+) naïve B cells correlated with disease activity in immune thrombocytopenia patients regardless of treatment status. A population of CD21-naïve B cells was specifically expanded in autoantibody-positive immune thrombocytopenia patients. Furthermore, the B-cell maturation antigen, a receptor for B-cell activating factor, was consistently and strongly up-regulated on plasmablasts from immune thrombocytopenia patients. These observations have parallels in other autoantibody-mediated diseases and suggest that loss of peripheral tolerance in naïve B cells may be an important component of immune thrombocytopenia pathogenesis. Moreover, the B-cell maturation antigen represents a potential target for plasma cell directed therapies in immune thrombocytopenia.

A multicenter validation of recombinant ß3 integrin-coupled beads to detect human platelet antigen-1 alloantibodies in 498 cases of fetomaternal alloimmune thrombocytopenia.

BACKGROUND: Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by human platelet (PLT) antigen (HPA) incompatibility. Beads coupled with recombinant ß3 integrins, displaying the biallelic HPA-1 epitopes (rHPA-1), have been shown to detect HPA-1a alloantibodies implicated in FMAIT. This report describes a multicenter validation of the beads using the results of well-characterized samples to define the optimum parameters for analysis of a large cohort of 498 clinical samples. STUDY DESIGN AND METHODS: Fifty-one blinded quality assurance (QA) samples were tested by six laboratories to standardize the rHPA-1 bead assay and to develop an algorithm for sample classification. Five laboratories retrieved samples from 498 independent FMAIT cases, previously tested by the monoclonal antibody-specific immobilization of PLT antigens (MAIPA) assay, from their local archives for testing with the rHPA-1 beads. The results were evaluated using a mathematical algorithm developed to classify the samples. RESULTS: The QA samples gave a mean concordance of 94% between the bead and MAIPA assays, while 97% concordance was observed with the FMAIT samples. Of the 15 discrepant samples, seven were positive by the beads but negative by MAIPA, while the contrary was observed for eight samples. Overall, the bead assay achieved 98% sensitivity for HPA-1a antibody detection in FMAIT and 98.7% specificity compared to the local MAIPA. CONCLUSION: The rHPA-1 bead assay is a rapid 3-hour assay for the sensitive detection of HPA-1 antibodies. Its ease of use would enable prompt detection of maternal HPA-1a antibodies in suspected FMAIT cases, which is important supportive evidence for treatment by transfusion with HPA-1b1b PLTs.

A Novel Homozygous c.800C>G Substitution in GP1BA Exon 2 in a Kuwaiti Family with Bernard-Soulier Syndrome.

BACKGROUND: Bernard-Soulier syndrome (BSS) is a congenital bleeding disorder characterised by thrombocytopenia, giant platelets and decreased platelet adhesion resulting from genetic alterations of the glycoprotein (GP) Ib/IX/V complex. OBJECTIVES: Three sisters with a lifelong bleeding history and a provisional diagnosis of BSS were referred for further characterisation of their bleeding diathesis. The siblings' symptoms varied in severity from skin and gum bleeding to menorrhagia associated with iron-deficiency anaemia requiring regular transfusion of red cells and platelets. The parents were consanguineous but did not demonstrate any bleeding disorder. METHODS: The family were investigated using standard haematological techniques, platelet aggregometry, platelet membrane GP analysis and DNA sequencing of the genes encoding the GPIb/IX complex. RESULTS: All 3 sisters had thrombocytopenia and giant platelets. Platelet aggregation and flow cytometry studies confirmed the lack of aggregation with ristocetin and a markedly reduced GPIb/IX surface expression. Molecular analysis demonstrated a novel homozygous c.800C>G substitution in GP1BA exon 2 leading to a serine 267 Ter stop codon in all 3 siblings. CONCLUSIONS: A novel, nonsense mutation was identified as the cause of the bleeding disorder in this family. This is the first reported BSS mutation identified in a family from Kuwait.

A V740L mutation in glycoprotein IIb defines a novel epitope (War) associated with fetomaternal alloimmune thrombocytopenia.

BACKGROUND: Most recently described human platelet antigens (HPAs) have been detected in cases of fetomaternal alloimmune thrombocytopenia (FMAIT) where the mother has been immunized against a low-frequency antigen that the fetus has inherited from the father. Low-frequency antigens are not represented in normal panel platelets (PLTs) and antibody detection and identification in such cases requires incubation of maternal serum with paternal PLTs and definition of the causative mutation. STUDY DESIGN AND METHODS: A suspected case of FMAIT was investigated for PLT-specific antibodies using a panel of both HPA-typed and paternal PLTs. HPA typing was performed by polymerase chain reaction with sequence-specific primers and further DNA analysis was performed using direct sequencing of the coding regions of the ITGA2B and ITGB3 genes. RESULTS: Maternal antibodies reactive only with paternal PLTs were localized to glycoprotein (GP)IIb/IIIa using the monoclonal antibody immobilization of PLT antibody assay. A single-nucleotide polymorphism was detected in Exon 23 of ITGA2B in the father and affected child, which predicted a V740L substitution in the mature protein. Recombinant V740L mutated GPIIb expressed in HEK293 cells was specifically recognized by maternal antibodies. The polymorphism was not detected either in the mother or in a cohort of 100 donors. CONCLUSION: The V740L polymorphism defines a new low-frequency antigen implicated in two cases of FMAIT in a single family. Low-frequency HPAs are clinically important and their elucidation requires both crossmatch studies and gene sequencing in cases where there is strong clinical evidence of FMAIT but initial laboratory investigations do not support the diagnosis.

The granulocytes in neutropenia 1 (GIN 1) study: a safety study of granulocytes collected from whole blood and stored in additive solution and plasma.

OBJECTIVE/AIM: To evaluate the safety of transfusing pooled, whole blood-derived granulocytes in additive solution and plasma (GASP) in 30 recipients. BACKGROUND: Demand for granulocytes in England has increased five-fold. With the advantages of reduced red cell, plasma and overall volume, GASP maintains function in vitro. METHODS AND MATERIALS: Observations were recorded prior to and post transfusion. Increments were recorded at 1 h and the following morning. Leucocyte antibody screening was undertaken prior to and at 1-6 months following transfusion. RESULTS: Thirty patients aged between 8 months and 68 years received 221 GASP in 148 transfusion episodes. GASP contained an average of 1.0 × 10(10) granulocytes in 207 mL. Adults usually received two packs and children 10-20 mL kg(-1). Children and adults received a median [interquartile range (IQR)] dose of 12.5 (9.1-25.3) and 19.7 (12.0-25.8) × 10(9) granulocytes per transfusion, respectively. There was one episode of transfusion-associated circulatory overload (TACO) in a patient with chronic cardiac failure following 600 mL of unpooled granulocytes, other fluids and one GASP. New leucocyte alloimmunisation occurred in 3/30 recipients 10%. No other significant reactions were reported. Median peripheral blood neutrophil increments at 1 h post transfusion were 0.06 (IQR, 0.01-0.17) in children and (0.03) (IQR, 0-0.16) in adults. CONCLUSION: GASP has a similar safety profile to other sources of granulocytes for patients with refractory infection or in need of secondary prophylactic transfusion. Further studies are required to clarify the role of GASP in the treatment of neutropenic patients.

Detection of HNA-3a and -3b antibodies using transfected cell lines and recombinant proteins.

BACKGROUND: HNA-3 is a diallellic system located on choline transporter-like protein 2 (CTL2), defined by a polymorphism at Amino Acid 154. HNA-3a antibodies are of clinical importance in transfusion-related acute lung injury but antibody detection requires labor-intensive granulocyte isolation from HNA-typed donors and the use of techniques such as the granulocyte agglutination test or granulocyte immunofluorescence test. Also, there is no commercial test for detection of HNA-3 antibodies. STUDY DESIGN AND METHODS: HEK293 cells were transfected to generate stable cell lines expressing CTL2 fragments (Amino Acids 55-230) and full-length membrane bound CTL2 with HNA-3a and -3b epitopes. Soluble fragments were used in enzyme-linked immunosorbent assays to detect HNA-3 antibodies. The cell lines expressing full-length proteins were trypsin treated to remove HLA antigens and frozen at -80°C. Thawed cells were then used to detect HNA-3 antibodies by flow cytometry. RESULTS: Glycosylated and soluble CTL2 fragments were correctly recognized by 15 of 31 anti-HNA-3a sera and by both available anti-HNA-3b sera. Twenty-one anti-HLA sera reacted variably with untreated cell lines expressing full-length CTL2. After trypsin treatment of the cell lines, reactivity with HLA antisera was abrogated and all 31 anti-HNA-3a and two anti-HNA-3b sera bound to the corresponding cell line. CONCLUSION: Whereas soluble, glycosylated CTL2 fragments cannot be used for the detection of HNA-3 antibodies, the HEK293 cells expressing full-length CTL2 proteins were useful in the detection of HNA-3 antibodies even in the presence of HLA antibodies. Moreover, the cell lines can be stored for at least 6 months before use.

Detection of human platelet antigen-1a alloantibodies in cases of fetomaternal alloimmune thrombocytopenia using recombinant ß3 integrin fragments coupled to fluorescently labeled beads.

BACKGROUND: Testing for alloantibodies against human platelet antigens (HPAs) is essential for the clinical diagnosis of fetomaternal alloimmune thrombocytopenia (FMAIT), posttransfusion purpura, and platelet (PLT) refractoriness. Most of the methods currently used for HPA alloantibody detection rely on the availability of panels of HPA-typed PLTs and some rely on validated monoclonal antibodies (MoAbs) against the PLT glycoproteins. Recombinant ß3 integrins displaying the HPA-1a (rHPA-1a) or HPA-1b (rHPA-1b) epitopes have been produced as an alternative source of antigen. The suitability of these integrin fragments was evaluated for the development of an HPA-1a alloantibody screening assay, using Luminex xMAP technology. STUDY DESIGN AND METHODS: A 3-plex bead assay was developed by coupling biotinylated rHPA-1a, rHPA-1b, and recombinant glycoprotein VI to LumAvidin microspheres. Forty patient samples referred for FMAIT diagnostic testing, which were previously screened by the MoAb-specific immobilization of PLT antigens (MAIPA) assay, were used to assess the assay. RESULTS: The rHPA-1a- and rHPA-1b-coupled beads were able to detect HPA-1a and HPA-1b alloantibodies in all patient samples tested that were previously confirmed to contain HPA-1-specific antibodies. Furthermore, HLA Class I antibodies did not cross-react with the coupled beads. CONCLUSION: The 3-plex bead assay can be used to detect HPA-1a antibodies with sufficient specificity and sensitivity for use in the clinical setting of FMAIT. The development of other recombinant integrin fragments with the use of Luminex xMAP technology may assist in providing more rapid HPA antibody detection, enabling prompt diagnosis of alloimmune PLT disorders.

Recipient-derived HPA-1a antibodies: a cause of prolonged thrombocytopenia after unrelated donor stem cell transplantation.

BACKGROUND: Patients with human platelet antigen (HPA) specific antibodies in cases of neonatal alloimmune thrombocytopenia and platelet (PLT) refractoriness derive clinical benefit from the use of HPA-selected PLTs. STUDY DESIGN AND METHODS: This study describes three patients with underlying diagnoses of acute myeloid leukemia, chronic lymphocytic leukemia, and myelodysplasia, respectively, who underwent allogeneic bone marrow transplantation (BMT) with unrelated donors matched at the HLA-A, B, C, Dr, and DQ loci but who failed to achieve an adequate PLT count. Investigation using PLT immunofluorescence test, monoclonal antibody immobilization of PLT antigens assay, and genotyping revealed the presence of recipient-derived HPA-1a antibodies. RESULTS: In two patients, anti-HPA-1a was detected post-BMT and in the third patient, anti-HPA-1a was detected during pre-BMT chemotherapy. Despite apparent 100% engraftment of donor cells, the patients' PLT counts failed to recover 9-10 months posttransplant. The patients remained PLT-transfusion dependent and failed to achieve satisfactory increments following random donor or HLA-matched PLT transfusions. After the identification of HPA-1a antibodies, the patients were supported by HPA-1a(-) PLTs and satisfactory posttransfusion PLT increments were obtained. These cases illustrate that HPA-1a antibodies may remain detectable for 10 months following apparently successful donor engraftment and the disappearance of recipient-derived HLA antibodies. The prolonged persistence of recipient-derived PLT-specific antibodies following BMT has to our knowledge not been described previously. CONCLUSION: HPA-1a antibodies were associated with protracted PLT-transfusion dependence and significant hemorrhagic complications. Appropriate and timely laboratory investigation for HPA-specific antibodies followed by transfusion support with HPA-selected PLTs provided the cornerstone of the hemostatic management in these cases.

A large kindred with X-linked neutropenia with an I294T mutation of the Wiskott-Aldrich syndrome gene.

X-linked neutropenia (XLN, OMIM #300299) is a rare form of severe congenital neutropenia. It was originally described in a three-generation family with five affected members that had an L270P mutation in the GTP-ase binding domain (GBD) of the Wiskott-Aldrich syndrome protein (WASP) [Devriendt et al (2001) Nature Genetics, Vol. 27, 313-317]. Here, we report and describe a large three-generation family with XLN, with 10 affected males and eight female carriers. A c.882T>C mutation was identified in the WAS gene, resulting in an I294T mutation. The infectious course is variable and mild in view of the profound neutropenia. In addition to the original description, low-normal IgA levels, low to low-normal platelet counts and reduced natural killer (NK)-cell counts also appear as consistent XLN features. However, inverted CD4/CD8 ratios were not found in this family, nor were cases identified with myelodysplastic syndrome or acute myeloid leukaemia. Female carriers exhibited a variable attenuated phenotype. Like L270P WASP, I294T WASP is constitutively active towards actin polymerization. In conclusion, this largest XLN kindred identified to date provides new independent genetic evidence that mutations disrupting the auto-inhibitory GBD of WASP are the cause of XLN. Reduced NK cells, low to low normal platelet counts and low to low-normal IgA levels are also features of XLN.

Ten years of hemovigilance reports of transfusion-related acute lung injury in the United Kingdom and the impact of preferential use of male donor plasma.

BACKGROUND AND METHODS: From 1996 through 2006, 195 cases were reported as transfusion-related acute lung injury (TRALI) to the Serious Hazards of Transfusion scheme and from 1999 onward classified by probability, using clinical features and HLA and/or HNA typing. From late 2003, the National Blood Service provided 80 to 90 percent of fresh-frozen plasma (FFP) and plasma for platelet (PLT) pools from male donors. RESULTS: Forty-nine percent of reports were highly likely/probable TRALI, and 51 percent possible/unlikely. Of 96 investigations, donor antibodies recognizing recipient antigens were found in 73 cases (65%), with HLA Class I in 25 of those (40%), HLA Class II antibodies in 38 (62%), and granulocyte antibodies in 12 (17%). A review in 2003 revealed that the TRALI risk/component was 6.9 times higher for FFP and 8.2 times higher for PLTs than for red blood cells, and that in donors of implicated FFP/PLTs, white blood cell antibodies were found 3.6 times more often than by chance (p

Ninety-six suspected transfusion related acute lung injury cases: investigation findings and clinical outcome.

Transfusion related acute lung injury (TRALI) is one of the complications of blood transfusion and can result in major morbidity or mortality. The diagnosis depends upon the application of strict clinical criteria defining acute lung injury (ALI) and a temporal relationship to blood transfusion. We present the clinical and immunogenetic findings of 96 suspected TRALI cases investigated between 1996 and 2004. During this time period the national haemovigilance scheme (UK) defined TRALI as a reaction occurring either during or within 24 h of blood transfusion. Using clinical, laboratory and post mortem evidence, 64/96 cases could be defined as TRALI in our series. Sensitive techniques were employed to screen for HLA class I, class II and granulocyte specific antibodies in donor serum. Donor derived antibodies were detected in 58/64 (90%) of cases. Recipient derived DNA or cells were not always available but incompatibility was confirmed by the presence of the cognate antigen on recipient leucocytes or by crossmatching in 47/64 (73%) of cases. Cases referred prior to 2001 were not tested for HLA class II antibodies. By applying strict clinical criteria and using sensitive techniques a white blood cell antibody mediated immunological pathophysiology can be implicated in the majority TRALI cases.

The impact of universal leukodepletion of the blood supply on hemovigilance reports of posttransfusion purpura and transfusion-associated graft-versus-host disease.

BACKGROUND: The pathogenesis of posttransfusion purpura (PTP) and transfusion-associated graft-versus-host disease (TA-GVHD) involves patient exposure to donor platelets (PLTs) and T lymphocytes, respectively, which are removed during blood component leukodepletion (LD). STUDY DESIGN AND METHODS: Reports of PTP and TA-GVHD to the UK hemovigilance scheme Serious Hazards of Transfusion (SHOT) from 1996 to 2005 were compared before and after implementation of universal LD during 1999. RESULTS: There were 45 reports of PTP, with a mean of 10.3 per year before universal LD and 2.3 per year afterward (p < 0.001). All patients had received red cells, but before universal LD, only 1 of 31 (3%) cases had also received PLTs, compared to 8 of 14 (57%) afterward (p < 0.001). Thirty-four cases (76%) had human platelet antigen (HPA)-1a antibodies, whereas 11 had antibodies to other HPA specificities, only 1 of which occurred after LD. Two cases reported before LD also had heparin-dependent PLT antibodies. There were 13 reports of TA-GVHD, all fatal, of which only 2 cases of undiagnosed immunodeficiency met current UK criteria for irradiated components. Eight others had one or more risk factors: B-cell malignancy (6), steroids (1), fresh blood (1), and donor-recipient HLA haplotype share (4). Eleven cases were due to non-LD and 2 to LD components (p < 0.001). No cases have been reported since 2001. In an additional 405 cases, nonirradiated components were transfused in error to high-risk recipients, mainly on fludarabine, but none developed TA-GVHD. CONCLUSIONS: These findings suggest that universal LD has further reduced the already low risk of TA-GVHD in immunocompetent recipients and has altered the profile of PTP cases.

Transfusion-related alloimmune neutropenia: an undescribed complication of blood transfusion.

Alloimmune neutropenia in neonates is rare. We describe severe and persistent neutropenia in a 4-week-old neonate, which arose within 2 h of a transfusion of blood that contained about 28 mL of plasma and in which strong antibodies against human neutrophil antigen 1b (HNA-1b) were subsequently identified. The infant was positive for HNA-1b. No other likely cause of neutropenia was discovered. We believe this complication of blood transfusion to be a previously unrecognised one, and have called the condition transfusion-related alloimmune neutropenia (TRAIN).