Graham A. Smith

Management and outcome of 200 cases of fetomaternal alloimmune thrombocytopenia

BACKGROUND: Fetomaternal alloimmune thrombocytopenia (FMAIT) is the commonest cause of severe thrombocytopenia in term neonates but its management remains controversial.

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.

Platelet αIIbβ3 recombinant autoantibodies from the B‐cell repertoire of a post‐transfusion purpura patient

Summary. The biallelic human platelet alloantigen (HPA) 1 system encodes a leucine to proline substitution at position 33 in the β3 integrin. Homozygous individuals can be immunized by the non‐self allele‐encoded protein following transfusion or during pregnancy. In post‐transfusion purpura (PTP), a subsequent recall alloantibody response against the non‐self form of β3 is paralleled by the destruction of autologous platelets, leading to profound thrombocytopenia. Although serological evidence suggests platelet autoantibodies are responsible, such autoantibodies are poorly defined. We used variable gene phage display to isolate αIIbβ3 autoantibodies formed in the acute phase of PTP and determined the epitopes recognized. An immunoglobulin G (IgG)‐encoded variable heavy‐chain domain (VH) gene repertoire containing 4·7 × 107 single‐chain Fv fragments was cloned and three αIIbβ3 antibodies were isolated (clones 2F2, E3 and B12). All three used different VH genes with a low level of somatic mutation for genes derived from γ‐encoding mRNA. Two (2F2 and E3) recognized an overlapping epitope and their binding was inhibited by sera from patients with PTP; all three recognized Ca2+‐dependent compound epitopes on αIIbβ3. Our results support the theory that a transient loss of tolerance for αIIbβ3 with autoantibody formation occurs in PTP.

Immunologic and structural analysis of eight novel domain-deletion beta3 integrin peptides designed for detection of HPA-1 antibodies.

Summary.  Background: The single‐nucleotide polymorphism (SNP) rs5918 in the ITGB3 gene defines the human platelet antigen‐1 (HPA‐1) system encoding a Leu (HPA‐1a) or Pro (HPA‐1b) at position 33. HPA‐1 antibodies are clinically the most relevant in the Caucasoid population, but detection currently requires αIIbβ3 integrin from the platelets of HPA‐genotyped donors.Objectives: We set out to define the β3 integrin domains required for HPA‐1a antibody binding and produce recombinant soluble β3 peptides for HPA‐1 antibody detection.Methods: We designed two sets (1a and 1b) of four soluble β3 domain‐deletion peptides (ΔSDL, ΔβA, PSIHybrid, PSI), informed by crystallography studies and computer modeling. The footprints of three human HPA‐1a‐specific phage antibodies were defined by analyzing binding patterns to the β3 peptides and canine platelets, and models of antibody–antigen interfaces were derived. Specificity and sensitivity for HPA‐1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAIT). Results: Fusion of recombinant proteins to calmodulin resulted in high‐level expression in Drosophila S2 cells of all eight β3 peptides. Testing of FMAIT samples indicated that ΔβA‐Leu33 is the superior peptide for HPA‐1a antibody detection, with 96% sensitivity and 95% specificity. The existence of type I and II categories of HPA‐1a antibodies was confirmed by the study of HPA‐1a phage antibody footprints and the reactivity pattern of clinical samples with the four β3‐Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. Conclusions: Soluble recombinant β3 peptides can be used for detection of clinical HPA‐1a antibodies.

Rapid phenotyping of HPA-1a using either diabody-based hemagglutination or recombinant IgG1-based assays

BACKGROUND: The HPA‐1 system is carried on the β3 integrin. HPA‐1a (Zwa, PlA1) is immunogenic in an HPA‐1b homozygote (HPA‐1b1b). In pregnancy, 1 of 365 women forms anti‐HPA‐1a, which causes severe thrombocytopenia in 1 in 1100 neonates. Identification of women at risk of forming anti‐HPA‐1a and the screening of donors to obtain HPA‐1a‐negative platelets for therapy need reliable, low‐cost, automated assays.

Alloantibodies against low-frequency human platelet antigens do not account for a significant proportion of cases of fetomaternal alloimmune thrombocytopenia: evidence from 1054 cases

BACKGROUND: Maternal alloantibodies against the five common human platelet antigen (HPA) systems (HPA‐1 to ‐3, ‐5, and ‐15) are found in only 20% of cases referred for fetal and neonatal thrombocytopenia (FMAIT) investigations. The question asked was whether mismatches for the remaining 11 low‐frequency HPAs (HPA‐4 and ‐6bw to ‐17bw) might in part explain the remaining 80% of cases.

The importance of using multiple techniques for detection of platelet antibodies.

In red cell immunology, it has long been known that no one technique will detect all clinically significant antibodies. The same appears to be true for platelet immunology, and we highlight this fact by showing four examples of anti‐human platelet antigen‐1a that were not detected by the monoclonal antibody‐specific immobilization of platelet antigen test, the most commonly used technique. Each antibody was found in a case of fetomaternal alloimmune thrombocytopenia in which the fetus or neonate was severely affected.

Severe fetomaternal alloimmune thrombocytopenia due to anti-human platelet antigen (HPA)-1a in a mother with a rare and silenced ITGB3*0101 (GPIIIa) allele

Background and Objectives  Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal antibodies against a human platelet antigen (HPA) present on fetal, but absent from maternal platelets. We identified and characterized a case of FMAIT due to anti‐HPA‐1a in a mother with an HPA‐1a1b genotype.

Molecular characterization of the variable domains of an αIIbβ3‐specific immunoglobulin M κ platelet cold agglutinin in a follicular lymphoma patient with treatment refractory autoimmune thrombocytopenia: idiotypic overlap between αIIbβ3 integrin antibodies

BACKGROUND: Cold hemagglutinins are generally immunoglobulin M (IgM) κ antibodies reactive at temperatures below 37°C and if of high titer may cause hemolysis. Platelet (PLT) cold agglutinins (CAs) are rare and poorly characterized. A detailed molecular characterization of the variable domains of a pathologic, PLT‐reactive, CA is presented.