Predictive value of cross-matching for transfusion of platelet concentrates to alloimmunized recipients

Compatibility tests in which donor platelets were tested with recipient sera were performed retroactively after 64 transfusions of platelets from 59 unrelated donors to 10 alloimmunized patients. Techniques used were serotonin release, aggregometry, platelet factor 3 release, and lymphocytotoxicity, each of which has been advocated as a means of testing donor-recipient platelet compatibility. Although “false positive” reactions were few (positive crossmatch but satisfactory transfusion response), “false negative” reactions (negative crossmatch but poor transfusion response) were unacceptably high (43% by lymphocytotoxicity, 60% by serotonin release, 76% by platelet factor 3 release, and 83% by aggregometry). We conclude that current methods of detecting isosensitization to platelet alloantigens are less satisfactory than HLA phenotyping in selecting unrelated platelet donors for an alloimmunized patient population.     

A simplified [3H] serotonin release assay for the detection of platelet antibodies.

The [3H] platelet serotonin release assay is a sensitive means for detecting antiplatelet antibodies. We have found that by using prelabeled frozen platelets which have been treated with the cryoprotective agent dimethyl sulfoxide (DMSO) the performance of this assay can be facilitated without interfering with its sensitivity. There was 100% correlation between the standard [3H] platelet serotonin release assay using fresh platelets and the modified assay using prelabeled, frozen, cryopreserved platelets when sera from 12 patients with known antiplatelet antibodies were tested. When normal serum samples (n = 111) were tested against ten platelet donors, a false-positive rate of 3.9% was observed. This modification provides a simple means for quickly screening large numbers of potential platelet donors at one time.     

Cumulative experience with a simplified solid-phase radioimmunoassay for the detection of bound antiplatelet IgG, serum auto-, allo-, and drug-dependent antibodies

A simplified, sensitive, solid-phase radioimmunoassay employing 125I- staphylococcal protein A has been developed that is capable of detecting bound antiplatelet IgG as well as serum auto-, allo-, and drug-dependent antiplatelet antibodies. The simplified assay employs a ratio of test over control platelet counts per minute (cpm) for detection of positive results. All reagents are commercially available. The assay can be performed with as little as 10(6) washed platelets (10 microliters of whole blood) that have been stored for as long as 8 wk at 4 degrees C in microtiter plates. The assay time, employing stored platelets, is 4 hr. Bound platelet IgG is positive in 93% of 46 thrombocytopenic patients with autoimmune disease and correlates inversely with their platelet count, r = -0.65, p less than 0.001. The ability of this assay to detect serum antibody was studied with a rabbit anti-human platelet antibody capable of giving optimal immunoprecipitation with solubilized platelet membranes at a tier of 1:10. The present assay increases the sensitivity of antibody detection 256-fold to a titer of 1:2560. Human serum antiplatelet membrane antibody was positive in 2 of 2 patients with anti-PLA-1 antibody (titers of 1:256 and greater than 1:64); 7 of 12 multiply transfused patients who were refractory to platelet transfusion (2 had titers of greater than 1:256 and greater than 1:32); 5 of 19 patients with autoimmune thrombocytopenic purpura (2 had titers of 1:64 and 1:32); and 10 of 14 patients with clinical histories of drug-dependent antiplatelet antibody (2 had titers of 1:1280 for quinidine and 1:384 for phenazopyridine).     

Platelet antibodies in different forms of chronic thrombocytopenia.

Three techniques have been employed for the in vitro detection of circulating platelet antibodies in thrombocytopenic patients affected by 'idiopathic' form or by lupus erythematosus (SLE), the complement fixation test, the platelet factor 3 availability test and the serotonin release test. 29 of the 35 sera tested (82.8%) gave positive results for antiplatelet activity. In particular the serotonin release test allows to distinguish 4 groups of patients: a first group affected by idiopathic form; two groups with autoimmune thrombocytopenia and various degrees of serotonin release, and finally a fourth group which comprises subjects affected by SLE, with circulating immunocomplexes.     

Posttransfusion purpura: therapeutic failure of PlAl-negative platelet transfusion

The effect of platelet type-specific transfusion in posttranfusion purpura is reported. Seven days after receiving 4 units of whole blood during total hip replacement a 69-year-old woman developed fulminant thrombocytopenic purpura. Her undiluted serum inhibited the clot retraction of PlAl-positive but not PlAl-negative blood. Anti-PlAl titer of her serum, determined by 51Cr platelet lysis technique, was 1:64. The serum had no lytic activity against platelet-rich plasma from two PlAl-negative donors. No anti-HLA antibody was detectable in the serum by lymphocytotoxicity technique, and serum obtained prior to transfusions had no platelet lytic activity. Four units of PlAl-negative platelet concentrate were administered, the first instance in which this treatment has been used. No rise in platelet count ensued, and the patient succumbed to purpura. Exchange transfusion or plasmapheresis remain the treatments of choice.     

Platelet autoantibody in a case of infectious mononucleosis presenting as thrombocytopenic purpura

In this unusual case acute thrombocytopenic purpura was the sole clinical manifestation of infectious mononucleosis. An antiplatelet antibody was demonstrated in the serum by two newly described technics: release of radioactive serotonin from platelets and inhibition of platelet aggregation following exposure of platelets to serum containing antiplatelet activity. The antiplatelet activity was completely inactivated by adsorption to washed platelets and was located in the immunoglobulin G (IgG) gamma globulin fraction of serum. The patient's serum also reacted against her own platelets indicating that the antiplatelet factor behaved as a true autoantibody.     

Selection of donor platelets for alloimmunized patients using a platelet-associated IgG assay

A quantitative immunofluorescence platelet-associated immunoglobulins (PA-IgG) assay was used to detect alloimmunity to platelets in 8/12 multitransfused patients and to perform platelet crossmatching in the 8 alloimmunized patients. The correct separation of multitransfused patients into alloimmune and nonalloimmune groups was substantiated with chromium-51-labeled platelet survival studies. For 5 alloimmunized patients, compatible and incompatible donor platelets were demonstrated by PA-IgG crossmatching and were confirmed by platelet survival studies. With the other 3 alloimmunized patients, only studies with 5 of these incompatible donor platelets showed markedly reduced survival times on 4 occasions. PA-Igg compatible donor platelets survived 3.5- 8.7 days, while PA-IgG incompatible platelets showed survival times of 0.1-2.4 days. Overall, PA-IgG testing correctly indicated survival results on 15/17 occasions (88%), whereas platelet aggregation, serotonin release, and lymphocytotoxicity testing showed correct predictions for only 41%-59% of the survival studies. PA-IgG testing predicted which times, thus indication patients with platelet-specific alloantibodies. the PA-IgG assay provides a sensitive method to detect platelet alloantibodies and to perform platelet crossmatching, which can complement HLA typing in the selection of donor platelets for alloimmunized patients.     

Autoimmune thrombocytopenic purpura. Comparison of three different methods for the detection of platelet antibodies

An evaluation of three techniques for detection of antiplatelet antibodies in ATP is made. The platelet factor 3 availability after immunoinjury and the immunohystochemical technique using immunoperoxidase were more sensitive than ¹⁴C-5HT release from platelets for this purpose. The percentage of positive cases was similar in acute and chronic ATP patients while it significantly fell in cases in remission. A high proportion of patients in clinical remission had normal platelet aggregation, despite the persistence of antiplatelet antibodies.     

Platelets as target cells in rheumatoid arthritis and systemic lupus erythematosus: A platelet specific immunoglobulin inducing the release reaction

Summary Sera from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) were assessed for in vitro platelet activation as measured by serotonin release; 24% (30) of 124 tested RA sera and 51% (35) of 69 SLE sera induced a significant 3H serotonin release. Investigation of 17 synovial fluid samples from RA patients revealed significant release in 82%. Concomitant testing for lymphocytotoxic antibodies and immune complexes did not show any correlation to platelet activation. Upon gel filtration the release-inducing activity of positive sera was localized in the region of 160 000 Daltons. Further characterization by ion exchange chromatography, immune electrophoresis, chromatographic and SDS PAGE molecular weight determinations, as well as analytical ultracentrifugation all confirmed the IgG nature of the release-inducing protein. Negative blocking experiments performed by preincubation of platelets with Fc-IgG fragments prior to challenge with a release-inducing serum excluded the participation of Fc receptors in the reaction. It was concluded that the release was caused by a platelet reactive IgG antibody. This antibody may also cause release of platelet mediators in vivo and may thus contribute to the pathogenesis of the generalized vasculopathy in both diseases.     

In vitro Tests for Platelet Compatibility

Four methods were investigated to determine their suitability as platelet compatibility procedures: leukoagglutination, lymphocytotoxicity, platelet suspension immunofluorescence and platelet enzyme-linked immunosorbant assay. None of the tests were found to reliably predict the 24-hour-posttransfusion platelet increment in 8 refractory thrombocytopenic patients judged refractory to random donor platelet therapy.     

A diagnostic test for heparin-induced thrombocytopenia: detection of platelet microparticles using flow cytometry

Based on our previous observation that heparin-induced thrombocytopenia (HIT) sera can generate platelet microparticles from washed platelets in a heparin-dependent fashion, we developed a test for HIT using flow-cytometry to measure heparin-dependent platelet microparticle formation. During the developmental phase of the assay the optimal physical conditions for microparticle generation were defined. 133 sera were then evaluated using the microparticle assay and the serotonin release assay to determine the threshold for defining a positive result that gave optimal sensitivity and specificity. The microparticle assay was then prospectively evaluated against the serotonin release assay in 202 sera referred to our laboratory for HIT testing. Overall agreement between the two assays was 96% (Cohen's kappa = 0.91). When the clinical data were reviewed on patients whose sera gave discrepant results between the two assays, no case of HIT was detected by one assay and missed by the other. The platelet microparticle assay is as accurate as the serotonin release assay and may be a useful non-radioactive test for HIT.     

Specific antiplatelet glycoprotein autoantibodies are associated with the thrombocytopenia of primary antiphospholipid syndrome

Thrombocytopenia is a frequent complication of primary antiphospholipid syndrome (PAPL) and has been attributed to antibodies directed against platelet glycoproteins (Gp) and also to antiphospholipid antibodies. We tested patients with PAPL with and without thrombocytopenia for specific antiplatelet autoantibodies. Platelet autoantibodies were detected by means of platelet immunoassays which included MAIPA with a panel of monoclonal antibodies directed against all the platelet Gps known to be possible targets for platelet autoantibodies. A high prevalence of serum platelet antibodies was found in patients with thrombocytopenia (73%, 11/15 patients) whereas antiplatelet antibody was detected in only one of the 10 control patients ( P  < 0.01). The antibodies mainly recognized GpIIbIIIa ( n  = 7), but also CD9 ( n  = 5), GpIaIIa ( n  = 4), GpIbIX ( n  = 3) and GpIV ( n  = 3). Platelet-Gp antibodies eluted from the platelet surface had the same reactivity as those found in the original sera from three of the four patients tested, whereas no anticardiolipin activity was found in the platelet eluates, suggesting the absence of cross-reactivity between anticardiolipin and antiplatlet antibodies. The MAIPA assay was also performed with F(ab')2 fragments obtained by pepsin digestion of serum IgG from four patients. The same results were obtained with F(ab')2 fragments and the original serum, demonstrating that platelet antibodies specifically bind in vivo to platelet Gps via their F(ab')2 fragments. Our results suggest a link between specific platelet antibodies and the thrombocytopenia of PAPL.     

Flow cytometry detection of serotonin content and release in resting and activated platelets

Early detection of platelet activation is important for the diagnosis and follow-up of several pathological conditions that primarily or secondarily involve platelets in their pathogenesis. The golden standard assay to detect thrombocyte activation is represented by the release of serotonin, classically performed by demanding methodologies, such as high-performance liquid chromatography, 14C-labelling and enzyme-linked immunosorbent assay (ELISA). We developed a non-radioactive method, based on individual cells, for the detection of serotonin content in activated and resting platelets by flow cytometry. The assay was standardized on cells activated by Ca2+ ionophore or by sera from patients with heparin-induced thrombocytopenia (HIT). Cells were identified by CD41a surface staining and their serotonin content measured by specific antiserotonin intracytoplasmic staining, while their activation was independently shown by annexin V binding. Cellular degranulation was detected by flow cytometry in all the cases that were also positive by standard ELISA. Moreover, multiparametric flow cytometry analysis revealed that, although virtually all activated cells bind annexin V, serotonin was released only by the platelet subset that downmodulates surface CD41a.     

Leka, a New Platelet Antigen Absent in Glanzmann''s Thrombasthenia

The serum of a patient who developed a posttransfusion purpura contained antibodies directed against a previously undescribed platelet antigen Lek a. The antiplatelet activity was present in the IgG fraction and was detected by immunofluorescence, 51Cr lysis and 14C-serotonin release. The frequency of the Lek a phenotype in the French population is 98.18%. Lek a does not appear to be sex-linked and seems to be closely related to the Bak a antigen. The Lek a antigen is not expressed on thrombasthenic platelets but is found on platelets from patients with the Bernard-Soulier syndrome which suggests that this antigen is carried by platelet glycoproteins IIb and/or IIIa.     

Three Cases of Platelet Alloimmunisation Associated with the Presence of a Novel Platelet Specific Antibody

Background and Objectives : In three cases of platelet alloimmunisation, a platelet-specific alloantibody was detected which could not be classified within the known human platelet alloantigen or HLA systems. The first case was of a family in which two siblings suffered neonatal alloimmune thrombocytopenia at birth. In the second case, the newborn was suffering from phocomelia with hypoplastic thrombocytopenia. The third case was a male who became refractory to transfusions of HLA-matched platelets after a related bone marrow transplantation. Materials and Methods : The serum samples were investigated by: enzyme-linked immunosorbent assay, platelet suspension immunofluorescence test (PSIFT), monoclonal antibody immobilisation of platelet antigens assay (MAIPA), and by the lymphocytotoxicity test. Results : The antibody gave positive reactions with 26% of normal donor platelets. Surprisingly, no platelet-specific antibody was detected by PSIFT or by MAIPA and there was no evidence found to support classifying the antibody within the HLA system. Conclusion: The reactivity pattern of the antibody detected and the clinical presentation of the three cases described, strongly suggest the presence of an additional platelet specific alloantigen system.     

Interference of IgG, IgG aggregates and immune complexes in tests for platelet autoantibodies

Three techniques, based on the antiglobulin principle, used for the detection of autoantibodies against platelets, were compared; the antiglobulin consumption assay (QACA), the platelet radioactive antiglobulin test (PRAT) and the platelet suspension immunofluorescence test (PSIFT). Upon incubation of normal donor platelets with purified IgG, in concentrations higher than that in serum, an increased amount of platelet-associated IgG was demonstrated only in the QACA. Upon incubation with aggregated IgG, all three tests became positive, but the PSIFT only with high concentrations of aggregates. Binding of soluble C1q-binding immune complexes (IC), which consisted of tetanus toxoid and IgG antitetanus antibodies (TaT) to normal donor platelets, was only detectable in the QACA. However, a positive result was obtained in all three tests with platelets incubated with soluble DNA-IgG-antiDNA antibodies (DaD) IC. Fixation of the platelets with paraformaldehyde prevented the binding and the detection of the DaD-IC, but not of IgG, aggregated IgG or TaT-IC. Eluates from platelets incubated with aggregated IgG, TaT- or DaD-IC did not react with normal donor platelets in the three techniques, in contrast to eluates from platelets sensitized with platelet antibodies.     

The isolation and characterisation of antiplatelet antibodies

The isolation and characterisation of antiplatelet antibodies in autoimmune thrombocytopenia purpura patients (ITP) is described. Autoimmune thrombocytopenia purpura is an autoimmune disease, clinically defined by low platelet counts, normal or increased megakaryocytopoiesis and antiplatelet antibodies in serum. This study used phage display to isolate Fab antiplatelet antibodies to study the structure-function relationships of pathogenic antibodies in ITP. Out of six randomly selected colonies, four colonies reacted strongly with whole platelets in enzyme-linked immunosorbent assay (ELISA). Sequence analysis showed that all four colonies had the same DNA sequence and were the same antibody. Results of Western blotting against non-reduced human platelet lysate showed that the Fab reacted with platelet proteins with apparent molecular weights of 116, 92 and 39 kD. Furthermore, Western blotting assay against purified membrane glycoprotein IIIa demonstrated reactivity against a band with a molecular weight of 92 kD. Results from Western blotting against platelet lysate and pure platelet glycoprotein confirmed the Fab fragment recognised the platelet glycoprotein IIIa. Three out of the four phage colonies produced soluble Fab, which demonstrated reactivity against platelet autoantigens in ELISA. Further sequence analysis showed that the Fab was somatically mutated suggesting antigen drive and therefore T-cell assistance was important in the development of this antibody. One of the somatic mutations introduced an RSD amino acid sequence in the complementary determining region 1 (CDR1) of the light chain, which may mimic the RGD motif of fibrinogen which binds integrin GPIIb/IIIa. This raises the possibility that somatic mutation and antigen drive have produced a pathogenic autoantibody.     

Perinatal management of idiopathic thrombocytopenic purpura in pregnancy: risk factors for passive immune thrombocytopenia

Summary Thirty-nine pregnant women with idiopathic thrombocytopenic purpura (ITP) were studied in order to evaluate the influence of therapies for maternal ITP on fetal passive immune thrombocytopenia (PIT). Neonatal platelet counts were also compared with platelet counts, amount of PAIgG, and presence of circulating antiplatelet antibody in maternal blood. Eight of 41 neonates (19.5%) presented PIT without neonatal mortality. A higher incidence of PIT was observed in women with prior splenectomy than in women without splenectomy (66.7% vs 11.4%). Neither a therapeutic effect nor an increased risk of PIT was observed with steroids or gammaglobulin administration. No correlation was found between neonatal platelet counts and maternal platelet counts or maternal PAIgG, while positive cases for circulating antiplatelet antibody assay presented a higher incidence of PIT than negative cases. Additionally, a higher incidence of PIT was observed in women with a history of previous PIT than in women with a history of normal delivery. Prior splenectomy, presence of antiplatelet antibody in maternal blood, and a history of previous PIT seem to be risk factors for fetal PIT.     

The platelet serotonin‐release assay

Few laboratory tests are as clinically useful as The platelet serotonin‐release assay (SRA): a positive SRA in the appropriate clinical context is virtually diagnostic of heparin‐induced thrombocytopenia (HIT), a life‐ and limb‐threatening prothrombotic disorder caused by anti‐platelet factor 4 (PF4)/heparin antibodies that activate platelets, thereby triggering serotonin‐release. The SRA's performance characteristics include high sensitivity and specificity, although caveats include indeterminate reaction profiles (observed in ～4% of test sera) and potential for false‐positive reactions. As only a subset of anti‐PF4/heparin antibodies detectable by enzyme‐immunoassay (EIA) are additionally platelet‐activating, the SRA has far greater diagnostic specificity than the EIA. However, requiring a positive EIA, either as an initial screening test or as an SRA adjunct, will reduce risk of a false‐positive SRA (since a negative EIA in a patient with a “positive” SRA should prompt critical evaluation of the SRA reaction profile). The SRA also provides useful information on whether a HIT serum produces strong platelet activation even in the absence of heparin: such heparin‐“independent” platelet activation is a marker of unusually severe HIT, including delayed‐onset HIT and severe HIT complicated by consumptive coagulopathy with risk for microvascular thrombosis. Am. J. Hematol. 90:564–572, 2015. © 2015 Wiley Periodicals, Inc.     

A microplate ELISA for the detection of platelet alloantibodies: comparison with the platelet immunofluorescence test

An enzyme-linked immunoassay (ELISA) for the detection of platelet alloantibodies has been compared in detail with the platelet immunofluorescence test (PIFT). The ELISA appeared much simpler to perform than the PIFT. Both tests were comparable with regard to reproducibility and sensitivity. Alloantibodies were detected with ELISA and PIFT in 13 out of 14 patients who were refractory to random donor platelets. The value of these tests as a platelet crossmatch assay was determined in a retrospective comparison of the test results and the clinical transfusion responses expressed as the 1 h post-transfusion platelet recovery. 39/41 (95%) negative ELISA crossmatches and 30/33 (91%) negative PIFT crossmatches appeared to be associated with a successful platelet transfusion, whereas 7/10 positive ELISA crossmatches and 2/4 positive PIFT crossmatches appeared to be associated with transfusion failures. The high frequency of 'correct' negative tests indicates the importance of both assays in the prospective selection of compatible platelet donors for alloimmunized patients. However, because of its simplicity, the ELISA appears the method of choice for this purpose.