Monoclonal Antibody Immobilization Of Platelet Antigens Research Articles

Platelets retain function and can be stored following disruption of human leucocyte antigens.

Antibodies to human leucocyte antigen (HLA) Class-I antigens can lead to refractoriness to platelet transfusion. Although this can be overcome by transfusion of HLA-compatible platelets, they are not always available. Disruption of HLA antigens on platelets by acid treatment may be a suitable alternative when no other components are available. The aim of this study was to assess the effect of HLA disruption and subsequent storage of platelet components. Platelet components were treated with 0.9% saline or citric acid solution (pH 3.0), and then stored until expiry (Day 7). HLA and platelet glycoprotein expression, platelet viability, activation and sialylation were measured by flow cytometry. Release of soluble factors was measured by ELISA and metabolism by biochemistry analyser. Reactivity to patient anti-sera containing anti-HLA antibodies was measured using platelet immunofluorescence tests (PIFTs) and monoclonal antibody immobilization of platelet antigen (MAIPA) assays. Platelet function was measured using aggregometry and thromboelastography (TEG). Acid treatment reduced detection of HLA Class-I on platelets by 75%, with significant reductions in reactivity to patient anti-sera. Acid treatment reduced platelet content and viability, increased platelet activation and accelerated metabolism. Glycan cleavage was increased by acid treatment. Treatment reduced platelet activation following agonist stimulation by ADP and TRAP-6, but platelets remained functional, displaying increased aggregation response and reduced time to clot formation by TEG. Although HLA disruption had some detrimental effects, acid-treated platelets remained functional, retaining their capacity to respond to agonists and form clots, and with further development could be used to support refractory patients.

Detection of alloimmunization in Glanzmann Thrombasthenia and Bernard-Soulier Syndrome: Data from a Brazilian Center

IntroductionThe Glanzmann Thrombasthenia (GT) and Bernard-Soulier Syndrome (BSS) are rare hereditary disorders of platelet function. Their treatment often requires platelet transfusion, which can lead to the development of alloantibodies. ObjectiveIn this study, we aim to develop a strategy for alloantibody detection and to describe the frequency of alloimmunization in a patient population from a single center in southeastern Brazil. MethodsSamples from patients with GT or BSS were tested using the Platelet Immunofluorescence Test (PIFT). If a positive result was obtained, a confirmatory step using the Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) and Luminex bead-based platelet assay (PAKLx) was executed. Main results: Among 11 patients with GT, we detected the presence of alloantibodies in 5 using PIFT, with confirmation through MAIPA and PAKLx in 2 (1 anti-HLA and 1 anti-HPA), resulting in a frequency of 18.1%. Among 4 patients with BSS, PIFT was positive in 3, with confirmation by MAIPA and PAKLx in 1 (anti-HLA), showing a frequency of 25%. The two patients with anti-HLA antibodies exhibited a panel reactive antibody (PRA-HLA) testing greater than 97%. ConclusionOur study highlights the importance of identifying platelet alloimmunization in this patient population. The proposed algorithm for platelet alloantibodies detection allows resource optimization.

Sequestration Pattern in ITP Liver/Spleen Scan Correlates with the Presence of Anti-Glycoprotein V Antibodies in ITP Patients

Introduction Immune thrombocytopenia (ITP) is characterized by low platelet counts due to high clearance and low production. Better insight in the pathophysiology of ITP is needed to personalize management regimes, especially in patients with persisting and chronic ITP. In the Haga Teaching Hospital, we reproduced and validated the ITP liver/spleen scan from St Bartholomew's Hospital in London. With this scan the in vivo sequestration patterns of platelets in either spleen, liver or a combination of both (mixed sequestration) can be assessed, by using Indium-111 labelled autologous platelets . The ITP liver/spleen scan may provide better insights in autoantibody-mediated platelet destruction in ITP patients. Autoantibodies directed against glycoprotein (GP) IIb/IIIa, Ib/IX and V namely are found in a majority of ITP patients and can be detected using specific assays, like platelet immunofluorescence test (PIFT) and Monoclonal Antibody Immobilization of Platelet Antigen (MAIPA). To the best of our knowledge no studies have investigated the possible association between sequestration patterns and glycoproteins. In this study we investigated associations between the sequestration patterns from the ITP liver/spleen scan and the presence of different anti-GP platelet antibodies.Methods All ITP patients receiving an ITP liver/spleen scan between 2014 and 2016 in the Haga Teaching Hospital, a large tertiary referral centre in the Netherlands, are collected from our electronic patient files. We retrospectively collected demographic and clinical characteristics, including duration of ITP, previous management of ITP (corticosteroids, rituximab, thrombopoietin receptor agonists (TPO-RA)), antibody assays (PIFT, direct and indirect MAIPA) and the results from the ITP liver/spleen scan. MAIPA is used as a continuous variable. To investigate the association between anti-GP antibodies and the sequestration pattern from the ITP liver/spleen scan we used linear regression models for the continuous variables and logistic regression models for the binary data. Observational data and scatter plots aid in the interpretation of the possible associations. Both univariate and multivariate regression models including age and sex will be performed.Results 55 ITP patients with a median age of 41 years (IQR 29-57) and 67% females underwent an liver/spleen scan in the study period. The median age of the cohort at diagnosis of ITP was 32 (IQR 22-54) years. Treatment history consisted of corticosteroid therapy in 96% of cases, 44% IVIG, 48% TPO-RA and 21% rituximab. 10% of patients received only 1 treatment line, 23% received 2 lines, 33% received 3 lines, 21% 4 lines, 10% 5 lines and 1 patient received 7 lines (2%). Clinical significant bleedings (WHO bleeding grade ≥2) were reported by 53%, and 8% of the patients received a transfusion in the past. Sequestration pattern of the ITP liver/spleen was in 51% of cases a splenic pattern vs. 49% a mixed/hepatic pattern. A significantly higher prevalence of a splenic pattern was present in patients under 30 years at diagnosis while patients older than 30 years showed a more mixed/hepatic pattern. Anti-glycoprotein antibodies were found in 54% of the patients. Interestingly, linear regression models showed no association between either anti-GPIIb/IIIa or anti-GPIb/IX antibody detection and sequestration pattern. However a significant association was observed between the presence of anti-GPV antibodies and sequestration pattern. A higher anti-GPV antibody level and/or affinity was associated with a higher level of splenic sequestration.Conclusions This study has shown that the presence of anti-GPV antibodies is associated with a splenic sequestration pattern in the ITP liver/spleen scan in patients suffering from persistent and chronic ITP. GPV is usually found as a complex with GPIb/IX in several other bleeding disorders. Surprisingly no association was found in our data between a splenic sequestration pattern and the presence of anti-GPIIb/IIIa and especially anti-GPIb/IX. Further studies are needed to clarify the possible role of anti-GPV antibodies in thrombocyte clearance by the spleen in ITP patients. DisclosuresAmini:AMGEN: Research Funding. Schutgens:Baxalta/Shire: Research Funding; Novo Nordisk: Research Funding; Bayer: Research Funding; CSL Behring: Research Funding; Pfizer: Research Funding; Uniqure BV: Research Funding. Schipperus:AMGEN: Research Funding; Novartis: Research Funding.

Platelet antibodies identification: comparison between two laboratory tests

IntroductionPlatelet antibody identification is indispensable for diagnosing the human platelet antigen (HPA) or human leukocyte antigen (HLA) immunization, mostly because it can restrict the compatibility and results of transfusions. Correct detection of these antibodies is of utmost importance for the diagnosis and treatment. MethodWe present 16 platelet alloimmunization results, comparing two tests with different technologies: the MAIPA (monoclonal antibody immobilization of platelet antigens), as a reference technique, and a bead-based assay, the Pak-Lx. ResultsEleven samples (68.75%) showed agreement in both techniques. Two tests were false negatives in the Pak-Lx: a pan-reactivity in GPIIbIIIa and an anti-HPA-9b. On the other hand, the Pak-Lx was more sensitive to detect a decreasing anti-HPA-5b. The Pak-Lx found an anti-HPA-2b positive, but with a low median fluorescent intensity (MFI), suggesting a false-positive result. Moreover, in one case, the MAIPA was negative for a positive Pak-Lx HLA. ConclusionAntibody platelet diagnosis can sometimes be challenging. The methods seemed similar, the Pak-Lx being faster and simpler than the MAIPA, and they can be complementary to solve clinical issues.

Circulating antiplatelet antibodies in pregnant women with immune thrombocytopenic purpura as predictors of thrombocytopenia in the newborns

Newborns from mothers with immune thrombocytopenic purpura (ITP) have a risk of thrombocytopenia due to passage of maternal antiplatelet antibodies into fetal/neonatal circulation. We looked for predictors of neonatal thrombocytopenia (nTP) in pregnant women with ITP. One hundred pregnant women with platelet count <100 × 109/l, no non-immune causes of thrombocytopenia and increased platelet associated IgG (PA-IgG) were included in the study. Thirty seven and 63 of them gave birth to babies with and without nTP, respectively (nTP+ and nTP− groups). Platelet count, mean platelet volume, PA-IgG, antiplatelet circulating antibodies (cAB), time of ITP onset (before or during pregnancy), and frequency of corticosteroid treatment were compared in these groups. There were no differences in all test parameters between nTP+ and nTP− groups except cAB. These antibodies were detected in 33 out of 37 in nTP+ group and in 2 out of 63 mothers in nTP− group (p < 0.001). The sensitivity of this test was 89% and its specificity was 97%. A strong reverse correlation (r = −0.749, p < 0.001) was established between maternal cAB titer and neonatal platelet count. Antibodies against glycoproteins IIb–IIIa and/or Ib were identified in antigen specific MAIPA (Monoclonal Antibody Immobilization of Platelet Antigen) assay only in 10 out of 19 (53%) test sera with cAB. Antiplatelet cAB in pregnant women with ITP could serve as reliable predictors of nTP in their babies.

Anti-Glycoprotein V Autoantibodies in Patients with Immune Thrombocytopenia: Regularly Detectable and Functionally Relevant

Immune thrombocytopenia (ITP) results from autoimmunization against platelet antigens. Autoantibodies (aabs) are considered to represent a major mechanism of thrombocytopenia in ITP by inducing platelet clearance from the circulation. Several lines of evidence demonstrate that aabs against glycoproteins (GP) IIb/IIIa and Ib/IX are predominant in ITP patients. Both disease severity and treatment response rates to specific therapeutics have been associated with aabs patterns. GP V is a well characterized immune target in Varicella-associated and drug-induced thrombocytopenia, but has never been studied systematically in ITP.In this study, patients with a suspected diagnosis of primary ITP were included once they met pre-defined clinical inclusion criteria. The presence of GP IIb/IIIa-, GP Ib/IX, and GP V-specific aabs was investigated by monoclonal antibody immobilization of platelet antigens (MAIPA) assay, both on patients' autologous platelets (direct MAIPA) and in serum (indirect MAIPA). In addition, serum IgG fractions were prepared from all patients with a positive direct MAIPA. IgG fractions were tested by surface plasmon resonance (SPR) technology for the presence of anti-GP V aabs. Complete data sets were obtained from 1,140 qualified patients. Platelet-bound aabs were detected in 343/1,140 patients (30.1%). Of these, 222 (64.7%) had platelet-bound anti-GP V aabs, either alone (10/222), or together with other specificities (211/222). Free anti-GP V aabs were detected in 30/222 patients by indirect MAIPA, but in 88/222 by SPR. The avidity of aabs detected by both methods (n=29; R700/R350=0.73±0.14) was significantly higher than the avidity for aabs detected by SPR only (n=59; R700/R350=0.32±0.13, p < 0.001).In order to study the potential biological relevance of anti-GP V, a phagocytosis assay using CD14+ positively-selected macrophages from spleen specimens from splenectomized ITP patients was performed. Anti-GPV aabs induced a modest amount of platelet uptake above the normal human serum-incubated control. No difference was observed between high avidity and low avidity anti-GP V. The effect of anti-GPV on platelet clearance was further studied in a NOD/SCID mouse model. Freshly isolated human platelets were injected into the lateral mouse tail vein; after 30 min, IgG fractions isolated from human sera containing anti-GPV antibodies or control sera from healthy donors were injected into the other lateral tail vein, and the survival of human platelets in the mouse circulation was analyzed by taking murine blood 60, 120, 300 min and 24h after baseline. High avidity and low avidity anti-GP V aabs (n=3 per group) eliminated human platelets with no detectable difference between the groups (mean platelet survival at t=300 min: 40% [range 27-55] versus 35% [range, 16-46]). A comparable, dose-dependent platelet clearance was also obtained with monoclonal antibody SW16 against GP V.In summary, we have demonstrated that anti-GP V autoantibodies are regularly detectable in ITP patients; and that they are able to induce phagocytosis and platelet clearance. Our findings have implications for both, further development of laboratory testing, and guidance for clinical decision making. First, comparison between MAIPA and SPR reveals that free aabs may be more frequent than reported, since aabs appear to escape detection by standard laboratory methods because of low avidity. Better test methods are required. Second, predicting disease severity and/or tailoring ITP therapy can possibly not be restricted to the postulated difference between anti-GP IIb/IIIa and anti-GP Ib/IX. Prospective studies are required to understand the impact of different GP-specific platelet aabs on the clinical course of ITP including, anti-GP V. DisclosuresRummel:Celgene: Honoraria; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Mundipharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Symbio: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Honoraria. Bakchoul:Aspen Germany gGmbH, CLS Behring, Stago gGmbH: Honoraria; German Research Society (DFG): Research Funding; Robert Bosch gGmbH: Research Funding.

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

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. 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. 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. 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.

Antiplatelet Antibody Testing in Immune Thrombocytopenia and Evans Syndrome: Longitudinal Serologic Evolution and Relation to Clinical Features

Introduction: Antiplatelet antibody (APA) testing is considered an adjunct laboratory test in the diagnosis of immune thrombocytopenia (ITP). While it is not routinely obtained owing to inconsistent sensitivity and specificity in prior studies (Neunert et al, 2011), the definition of ITP used in these studies was not standardized. Additionally, potential clinical utility of this testing beyond diagnosis has been suggested, such as correlations between certain serologic patterns and response to IVIG (Peng et al, 2014). In consideration of these prior findings, we undertook a retrospective analysis of APA testing [glycoprotein-specific testing done by the commercial PakAuto assay (Immucor, Brookfield, WI), a monoclonal antibody immobilization of platelet antigens (MAIPA) assay, with all testing performed by the same laboratory] utilizing standardized ITP and Evans syndrome (ES) diagnostic criteria for patient inclusion. We examined serologic evolution over time and relation of antibody positivity to disease severity and response to therapies.Methods: Data collected for analysis included dates and results of APA testing (including disease status and platelet count at time of testing), patient demographics, and disease characteristics. Satisfaction of the 2011 American Society of Hematology (ASH) ITP diagnostic criteria were required for ITP patient inclusion and standard definitions of disease severity and response to treatment from the ASH guidelines were used in analysis. Logistic regression was used to model the probability of disease severity (non-severe, severe, or refractory) and treatment response (to corticosteroids or IVIG) based on serologic findings. Longitudinal serologic evolution in patients with multiple APA assays were analyzed.Results: A total of 214 APA assays from 115 ITP patients and 12 ES patients were collected; results from eluate testing (direct assay) only were used in analysis. Baseline patient characteristics are listed in Table 1. Of 7 possible positive test serologic patterns, only 4 were seen (Figure 1); antibodies against both GPIIb/IIIa and GPIb/IX were required for the presence of antibodies against GPIa/IIa. A multinomial logistic regression model including disease severity, age, sex, duration of disease, and platelet count at time of APA assay found a statistically significant predictive relationship between an increasing number of positive antibodies and disease severity [relative to non-severe ITP, relative risk ratio for severe ITP and refractory ITP was 1.89 (P=0.001) and 2.38 (P=0.004), respectively, per one additional positive antibody, Figure 2]. Multiple logistic regression models including antibody positivity to each platelet glycoprotein (GPIIb/IIIa, GPIb/IX, or GPIa/IIa), age, sex, disease duration and splenectomy status found a significant predictive relation between presence of anti-GPIa/IIa and non-response to corticosteroid treatment (odds ratio, 0.082, P=0.008). No significant relation was found between an antibody and non-response to IVIG. Fifty patients had multiple (2 to 5) APA assays performed over months to years. In evaluation of serologic evolution over time, all 7 patients who entered clinical remission also converted from a positive to a negative serology; 22 patients had stable serologic findings over time; 5 patients had a reduction in number of positive antibodies; and 18 patients demonstrated evidence for epitope spreading, with an increase in the number of positive antibodies (Figure 3). The sensitivity of a positive APA assay for the presence of active ITP was 91%. The sensitivity and specificity of a negative APA assay for remission in a patient with previously confirmed ITP (N=40 assays in patients with clinical remission in the study) were 88% and 91%, respectively.Conclusions: The MAIPA-based direct APA assay is sensitive for the presence of active ITP in patients satisfying 2011 ASH diagnostic criteria. To our knowledge, this is the first study demonstrating that a higher number of positive glycoprotein-specific antibodies may predict for more severe disease as defined by 2011 ASH disease severity criteria. Anti-GPIa/IIa antibodies only occur in the setting of pre-existing positivity for both anti-GPIIb/IIIa and anti-GPIb/IX antibodies, possibly due to a distinctive sequence of epitope spreading. Serologic testing typically turns negative when a patient enters clinical remission. [Display omitted] DisclosuresAl-Samkari:Agios: Consultancy. Kuter:Novartis: Consultancy; Pfizer: Consultancy; Syntimmune: Consultancy; Argenx: Consultancy; Dova Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; ONO: Consultancy; Amgen Inc.: Consultancy; Bioverativ: Consultancy, Research Funding; Principia: Research Funding; Rigel: Consultancy, Research Funding; BMS: Research Funding; Protalex: Research Funding.

Fcγ Receptors I and III on Splenic Macrophages Mediate GPIIb/IIIa Autoantibody-Dependent Phagocytosis of Platelets in Human Immune Thrombocytopenia

Autoantibody-opsonized platelets in immune thrombocytopenia (ITP) are thought to be destroyed primarily by macrophage Fc gamma receptor (FcγR)-mediated phagocytosis in the spleen. Blockade of splenic macrophage FcγRs has been proposed as a therapeutic mechanism for ITP intervention. Unfortunately, the contribution of specific FcγRs to disease in ITP remains unknown. Our objective was to determine which FcγRs are responsible for the phagocytosis of ITP autoantibody-opsonized platelets by splenic macrophages. Splenic macrophages were purified by CD14 positive selection from spleens of splenectomized ITP patients, and were treated with blocking antibodies to FcγRI, FcγRIIa, FcγRIIa/b/c, and FcγRIII. Blocking antibodies were deglycosylated to prevent non-specific blocking effects by their Fc region. Two separate ITP sera confirmed positive for anti-GPIIb/IIIa autoantibodies by the monoclonal antibody immobilization of platelet antigens (MAIPA) assay were used to opsonize healthy donor human platelets. Phagocytosis was determined by confocal microscopy and non-phagocytosed (external) platelets were differentiated by an anti-platelet antibody stain following macrophage fixation. Human ITP splenic macrophages were found to express FcγRI, FcγRIIa, FcγRIIa/b/c, and FcγRIII, and expression was not significantly different compared to healthy (trauma) controls (n=5). The two anti-GPIIb/IIIa-positive ITP sera induced a mean 3.7- and 4.2-fold increase of platelet uptake by ITP splenic macrophages relative to normal human serum controls (n=3 each, p<0.001). Blockade of all FcγRs significantly reduced phagocytosis of serum-opsonized platelets for both ITP sera as compared to an IgG control (p<0.001) down to background (non-opsonized) levels. Using single blocking antibodies, inhibition of FcγRI reduced splenic macrophage phagocytosis of platelets by 45% and 37% for the two respective ITP sera as compared to IgG controls (n=3 for each patient sera, p<0.01), while inhibition of FcγRIII reduced phagocytosis of platelets by 43% and 44% (n=3 each, p<0.05). Blockade of FcγRIIa or FcγRIIa/b/c only marginally inhibited splenic macrophage phagocytosis and was not significant. An Fab-like FcγRIII-blocking antibody with a mutant Fc region completely deficient for FcγR binding demonstrated equal FcγRIII blockade compared to the deglycosylated anti-FcγRIII antibody. In comparison to ITP sera-opsonized platelets, FcγRI had a greater involvement in ITP splenic macrophage phagocytosis of anti-D-opsonized human erythrocytes as FcγRI blockade inhibited phagocytosis by 70% (n=5, p<0.001), while FcγRIII blockade inhibited phagocytosis by only 30% (p<0.001) and FcγRIIa or FcγRIIa/b/c blockade had no significant effect. This work demonstrates that FcγRI and FcγRIII are the primary phagocytic receptors on splenic macrophages for anti-GPIIb/IIIa autoantibody-opsonized platelets in human ITP and suggests that FcγRI and FcγRIII are the best targets for FcγR blockade as a potential therapeutic maneuver in the treatment of ITP. DisclosuresNo relevant conflicts of interest to declare.

Maternal HPA-1a antibody level and its role in predicting the severity of Fetal/Neonatal Alloimmune Thrombocytopenia: a systematic review.

In Caucasians, fetal/neonatal alloimmune thrombocytopenia (FNAIT) is most commonly due to maternal HPA-1a antibodies. HPA-1a typing followed by screening for anti-HPA-1a antibodies in HPA-1bb women may identify first pregnancies at risk. Our goal was to review results from previous published studies to examine whether the maternal antibody level to HPA-1a could be used to identify high-risk pregnancies. The studies included were categorized by recruitment strategies: screening of unselected pregnancies or samples analyzed from known or suspected FNAIT patients. Three prospective studies reported results from screening programmes, and 10 retrospective studies focused on suspected cases of FNAIT. In 8 studies samples for antibody measurement, performed by the monoclonal antibody immobilization of platelet antigen (MAIPA) assay, and samples for determining fetal/neonatal platelet count were collected simultaneously. In these 8 studies, the maternal antibody level correlated with the risk of severe thrombocytopenia. The prospective studies reported high negative predictive values (88-95%), which would allow for the use of maternal anti-HPA-1a antibody level as a predictive tool in a screening setting, in order to identify cases at low risk for FNAIT. However, due to low positive predictive values reported in prospective as well as retrospective studies (54-97%), the maternal antibody level is less suited for the final diagnosis and for guiding antenatal treatment. HPA-1a antibody level has the potential to predict the severity of FNAIT.

Establishment of a Multivariate Analysis Based on Photonic Crystal Hydrogel Bead Arrays and Its Application for Detecting Platelet-specific Antibodies

ABSTRACTPhotonic crystals are nanomaterials with unique structural coloration, and they have been used in multiple analyses, gene function analyses, and nanoarray biosensors. Here, we used agarose hydrogel microcarriers with characteristic reflection peak codes to improve the biocompatibility of the material and the efficiency of detection, which may be helpful for further clinical applications. The paper has two parts: condition optimization and product application. Goat antihuman immune globulin G, human immune globulin G, and fluorescein isothiocyanate-labeled goat antihuman immune globulin G are commonly used materials in double-antibody sandwich techniques, which are used for optimization in the application of advanced composite materials. Goat antihuman immune globulin G was immobilized on the surfaces of photonic crystal hydrogel beads through chemical bonding. When the target immune globulin G was captured, the fluorescence-labeled antibody could combine with photonic crystal hydrogel beads. The reaction conditions were optimized using the gray-scale value of fluorescent intensities. Photonic crystal hydrogel beads with different reflection spectra were coated with purified platelet glycoprotein IIb/IIIa and glycoprotein Ib/IX. A total of 64 clinical samples including sera from patients with immune thrombocytopenia (n = 32) and healthy human sera (n = 32) were tested using photonic crystal hydrogel beads and monoclonal antibody immobilization of platelet antigen (MAIPA). Photonic crystal hydrogel bead array reaction conditions were established for multiple analysis, showing similar sensitivity and specificity with MAIPA in testing different platelet antibodies and shortening the analysis time. The features of these composite materials make them suitable for potential applications in clinical screening assays.

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients

BackgroundPlatelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation.MethodsCapture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs.ResultsThe coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%.ConclusionsA quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

Anti-Human Platelet Antigen-1a Immunoglobulin G Preparation Intended to Prevent Fetal and Neonatal Alloimmune Thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe disease that is caused by maternal alloantibodies generated during pregnancy or at delivery as a result of incompatibility between maternal and fetal human platelet antigens (HPAs) inherited from the father. Antibody-mediated immune suppression using anti-HPA-1a immunoglobulins is thought to be able to prevent FNAIT caused by HPA-1a. A fractionation process to prepare anti-HPA-1a immunoglobulin (Ig) G (IgG) from human plasma was therefore developed. Anti-HPA-1a plasma was obtained from volunteer mothers who underwent alloimmunization against HPA-1a during a previous pregnancy. Plasma was cryoprecipitated and the supernatant treated with caprylic acid and solvent/detergent (S/D), purified by chromatography, nanofiltered, concentrated, and sterile-filtered. The anti-HPA-1a immunoglobulin fraction was characterized for purity and safety. PAK12 and quantitative monoclonal antibody immobilization of platelet antigen (MAIPA) assays were used to detect anti-HPA-1a IgG. Hepatitis C virus (HCV) removal during nanofiltration was assessed by spiking experiments, using cell culture-derived reporter HCV and luciferase analysis. The caprylic acid treatment precipitated non-Ig proteins yielding a 90% pure Ig supernatant. S-HyperCel chromatography of the S/D-treated supernatant followed by HyperCel STAR AX provided high IgG recovery (>80%) and purity (>99.5%), and efficient IgA and IgM removal. Concentrations of complement factors C3 and C4 were < 0.5 and < 0.4 mg/dL, respectively. The final IgG could be nanofiltered on Planova 20N under conditions removing more than 3 log HCV infectivity to baseline mock infection level, and concentrated to ca. 30 g/L. Proteolytic activity and thrombin generation were low in the final fraction. The Pak12 and MAIPA assays showed good recovery of anti-HPA-1a throughout the process. Clinical-grade HPA-1a IgG can be prepared using a process compliant with current quality requirements opening perspectives for the prevention of FNAIT.

Detection of Platelet Autoantibodies Revisited to Identify Immune Thrombocytopenia

Introduction: Immune Thrombocytopenia (ITP) is still diagnosed by exclusion of many other causes for thrombocytopenia. In order to prevent misdiagnosis, an ITP-specific diagnostic test would be very helpful. In addition, characterization of glycoprotein specificity of platelet autoantibodies may explain (the severity of) bleeding symptoms and response to therapy. In this regard, we optimized the cut-off value of the direct monoclonal antibody immobilization of platelet antigens (MAIPA) assay for detection of platelet glycoprotein directed autoantibodies and re-evaluated its sensitivity and specificity for the diagnosis of ITP.Materials and Methods: The MAIPA was performed as part of our routine protocol, described by Kiefel et al. (1985). For the determination of a new cut-off value, and to calculate the sensitivity and specificity, blood samples were tested from 462 healthy blood donors and 43 non-immune-mediated thrombocytopenic patients, suffering from either hematological malignancies or aplastic anemia (n=20), hepato-splenomegalic pooling (n=3), drug-induced thrombocytopenias (n=4), viral infections (n=6), pregnancy related thrombocytopenia (n=7), pseudothrombocytopenias (n=2) and microangiopathy (n=1) and from 60 known ITP patients. We then have tested 120 prospectively collected samples from thrombocytopenic patients, sent for diagnostic tests to our laboratory, and categorized these samples based on subsequently obtained clinical evaluation into 'most likely ITP' (n=64) or 'most likely non-ITP' (n=56).Results: The calculated direct MAIPA sensitivity and specificity, using a cut-off value of E=0.130, in the ITP and non-ITP control groups (n=103) were 85% (95% CI, 73-93%) and 100% (95% CI, 92-100%), respectively (see Figure). The platelet auto-antibodies in the ITP control group (n=60) were directed against glycoprotein (GP)IIb/IIIa (66.7%), GPIb/IX (60%), GPV (51.7%), GPIa/IIa (40.6%) and/or GPIV (26.9%). The calculated sensitivity and specificity for detection of platelet auto-antibodies in the prospective diagnosed ITP and non-ITP patient control groups (n=120) were 75% (95% CI, 63-85%) and 96% (95% CI, 88-100%), respectively (see Figure). For this group of patients, the direct MAIPA showed, for diagnosis of ITP, a negative predictive value (NPV) of 77% (95% CI, 66-86%) and a positive predictive value (PPV) of 96% (95% CI, 86-100%).Furthermore, in 23 ITP patients the sequential sampling in a rituximab-treatment protocol showed platelet counts that were significantly and inversely correlated with the direct-MAIPA extinctions (p=0.006). In this respect, we excluded that higher platelet counts impaired the detection of platelet autoantibodies - e.g. by diluting them over an higher platelet mass-since autoantibodies were successfully detected in samples from ITP patients (n=4) with, as a result of splenectomy, platelet counts above 100 x 109/L and in untreated ITP patients with platelet counts between 75 and 100 x 109/L. These findings may implicate that response to rituximab as reflected by a rise in platelet counts is dependent on antibody presence, but the mechanism of effective lowering of platelet autoantibody levels by rituximab is still unclear.In conclusion, the revisited direct MAIPA showed to be a valuable technique for the detection of platelet autoantibodies both at diagnosis and during treatment and can possibly become a guide for optimizing therapy towards a more personalized treatment of ITP.Direct MAIPA O.D. above 0.13 is considered positive. Control samples: historically well characterized ITP patients. Prospective study: requests for serological ITP diagnostics, after final clinical evaluation classified as ITP or non-ITP. [Display omitted] DisclosuresSchipperus:Novartis: Consultancy.

Report of the First Workshop of the ISBT International Platelet Immunobiology Working Party, Asia Regional

Background and ObjectivesThe aims of the first workshop of the International Platelet Immunology Working Party, Asia Regional (IPIWP‐AR), were to identify antibodies against human platelet antigen (HPA), to compare laboratory performance in the detection of the antibodies and to help laboratories to audit and standardize the own in‐house platelet DNA genotyping methods.Materials and MethodsFourteen laboratories from eight countries participated, which were provided with six DNA samples for genotyping, six serum samples, monoclonal antibodies for monoclonal antibody immobilization of platelet antigens (MAIPA) and the mixed passive hemagglutination (MPHA) kits for antibody detection.ResultsMost of the laboratories could detect the anti‐HPA‐2b, anti‐HPA‐4b, anti‐HPA‐5b and Naka antibodies, by the combination of MAIPA and MPHA. However, some had difficulties with the HPA‐1a antibody, possibly dependent on the rarity of HPA‐1b alloantigen among Asian. Although a good concordance was obtained in genotyping, a significant inter‐laboratory variation was observed, possibly dependent on the methods used.ConclusionsThe IPIWP‐AR and the workshop were successfully established, and most laboratories were able to identify majority of HPA antibodies and genotype HPA correctly.

Detection of clinically relevant platelet antibodies in the Asian population

BackgroundThe detection and identification of the clinically relevant platelet antibodies are important for the diagnosis and management of the affected patients with immune thrombocytopenia.AimsThe aim of this study was to investigate the characteristic of clinically relevant platelet antibodies in the Asian population, and to evaluate the ability of platelet immunology laboratories in Asia to detect and identify these platelet antibodies.MethodsTotal of 635 cases that were diagnosed and studied as NAITP, PTR and PTP by Asian platelet laboratories were reviewed for the clinical and experimental data from the publications.ResultsExcept the anti‐HLA and unknown‐specificity antibody, the most frequently identified platelet antibody was anti‐CD36 (18/635, 18·35%), and then anti‐HPA‐3 and anti‐HPA‐4 (each 5/635, 0·78%), and anti‐HPA‐5 (4/635, 0·62%). Only two cases of anti‐HPA‐1 antibodies (2/635, 0·31%) were reported. In 274 cases, including four cases contained anti‐HLA antibody, the laboratories could not identify the specificity of platelet antibodies. The methods of detection and identification of antiplatelet antibody, such as monoclonal antibody immobilization of platelet antigens (MAIPA), mixed passive hemagglutination assay (MPHA), platelet immunofluorescence test (PIFT), modified antigen‐capture ELISA (MACE) and solid‐phase red‐cell adherence (SPAA), have been used in Asian laboratories.ConclusionWith an increasingly heterogeneous patient population in Asia, diagnostic laboratories should be aware of the ethnic origin of a particular patient to consider the possibility of immunization to antigens. The most important clinical relevant platelet antibodies implicated in immune‐mediated thrombocytopenia among the Asian population are anti‐CD36, then anti‐HPA‐3, anti‐HPA‐4 and others.

A new bead‐based human platelet antigen antibodies detection assay versus the monoclonal antibody immobilization of platelet antigens assay

The performance of a newly developed Luminex bead-based platelet (PLT) antibody detection method (PAKLx) was compared with the monoclonal antibody immobilization of PLT antigens (MAIPA) assay and the LifeScreen Deluxe Luminex bead-based HLA Class I antibody detection method (LMX). Six sera containing anti-human PLT antigen (HPA)-1a (n=2), HPA-1b, HPA-2b, HPA-3a, or HPA-5b were tested in titration. A total of 194 sera, including HPA-1a, -1b, -2a, -2b, -3a, -5a, and -5b antibodies with or without HLA antibodies (n=63); glycoprotein (GP) IV antibodies (n=1); PLT autoantibodies (n=3); HLA antibodies (n=45); and samples with no PLT-reactive antibodies (n=82), were tested in both assays. Comparable levels of sensitivity were obtained for the MAIPA and PAKLx. The PAKLx showed four (6%) false-negative results in 67 sera with HPA or GP-reactive antibodies: anti-HPA-3a (n=1) or anti-HPA-5b (n=3). The PAKLx showed in 10 of the total 194 samples (5%) the presence of antibodies not detected by the MAIPA. This concerned broadly GP-reactive antibodies (n=7), anti-GPIIb/IIIa combined with anti-HPA-3a (n=1), anti-HPA-1a (borderline, n=1), and anti-GPIV (n=1). Testing 175 sera for anti-HLA Class I antibodies in the PAKLx and LMX showed four discrepant results: PAKLx negative and LMX positive, n=3 and n=1, respectively. For the vast majority of the specimens tested (93%) the results of the PAKLx were in concordance with the MAIPA. The PAKLx is a fast, easy to perform, and sensitive PLT antibody screening method.

Detection of autoantibodies against platelet glycoproteins in patients with immune thrombocytopenic purpura by flow cytometric immunobead array

BackgroundThe goal of this study is to develop a flow cytometric immunobead array (FCIA) assay to detect platelet autoantibodies commonly present in bleeding patients with immune thrombocytopenic purpura (ITP). MethodsPolystyrene microbeads coated with antibodies against human platelet glycoproteins (GPs) IX (SZ1), Ib (SZ2), IIIa (SZ21), IIb (SZ22), and P-selectin (SZ51) were incubated with platelet lysate from 50 ITP patients and 86 controls. The platelet antigen–autoantibody complexes were detected by flow cytometry using an FITC-labeled antibody. The results were compared with that of a monoclonal antibody immobilization of platelet antigen (MAIPA) assay. ResultsBy FCIA, platelet autoantibodies against GPIb, GPIIb, GPIIIa, GPIX and P-selectin were detected in ITP patients. Mean fluorescent intensity values with antibodies SZ1, SZ2, SZ21, SZ22 and SZ51 were all higher in ITP patients than controls (p values<0.01). In ROC analysis, values of the area under the curve were 0.89, 0.82, 0.93, 0.94 and 0.95, respectively. In ITP diagnosis, the FCIA assay with these five antibodies had better sensitivity and accuracy than the MAIPA assay (96% vs. 44% in sensitivity; 80.9% vs. 64.7% in accuracy, p<0.01). ConclusionFCIA assays with multiple antibodies against platelet GPs may be used to improve the diagnosis of ITP in hospitals.

The first case of alloantibody against human platelet antigen‐15b in Japan: possible alloimmunization by a hydatidiform mole

The involvement of the human platelet antigen (HPA)-15 system in neonatal alloimmune thrombocytopenia (NAIT) has been reported in various populations, but not in the Japanese population. In Japan, the mixed passive hemagglutination assay (MPHA) is used for detection of HPA alloantibodies. However, most of the reported cases of HPA-15 incompatibility are based on the monoclonal antibody immobilization of platelet antigen (MAIPA) assay or immunoprecipitation; thus there is a possibility that HPA-15 alloantibodies are not efficiently detected by the MPHA, and currently, the causative antibody is not detectable in approximately half of the suspected NAIT cases in Japan. We examined the sera of mothers from NAIT cases, previously with undetected HPA antibodies by MPHA, using the MAIPA technique. Sera from 90 mothers of suspected NAIT were tested by MAIPA for the presence of anti-HPA-15 alloantibodies. Anti-HPA-15b was detected in one case. This case was a mother in the first pregnancy diagnosed as hydatid mole-coexisting fetus, and the baby was born with suspected NAIT. The familial analysis revealed compatibility of HPA-15 genotype between the mother and the baby (both HPA-15a/a), but incompatibility with the paternal one (HPA-15a/b). The hydatid mole's tissue was genotyped as HPA-15b positive. Besides anti-HPA-15b, maternal sera contain strong HLA Class I antibody Here we reported the first case of anti-HPA-15 in Japan. Alloimmunization against the hydatid mole seems to be responsible for the production of HPA-15b alloantibody. This antibody, however, did not apparently involve in the development of NAIT of the newborn, the coexisting anti-HLA Class I being the possible cause.

PAKTM-Lx: A New Luminex*®-Based Assay to Detect and Identify Antibodies to Human Platelet Antigens (HPA) in Patient Serum.

Abstract 4187Platelets express a variety of glycoproteins (GPIIb-IIIa, GPIa-IIa, GPIb/IX, GPIV, CD109 and Class I HLA). Human Platelet Antigens (HPA) represent polymorphisms of these platelet surface glycoproteins. While these polymorphisms do not alter the function of the proteins, they can become target of the immune system in mismatched individuals. Alloantibodies against HPA are involved in the pathogenesis of Neonatal Allo-Immune Thrombocytopenia (NAIT), Post-Transfusion Purpura (PTP) and refractoriness to random donor platelets. In addition to generating anti-HPA antibodies, these patients also develop antibodies to Class I Human Leukocyte Antigen (HLA). Most reference laboratories now use a combination of a sensitive screening assay such as the Platelet Immunofluorescence Test (PIFT) along with a solid phase assay for antibody identication, usually either the ELISA and Monoclonal Antibody Immobilization of Platelet Antigens (MAIPA) as well as genotyping techniques for at least HPAs 1, 2, 3, and 5. Currently, MAIPA is considered the gold standard in detection of antibodies to HPA. However, all existing methods have significant limitations in terms of sample requirements, time and labor involved. These factors are a major impediment as to how many specificities can be resolved in a rapid, efficient and reproducible manner. Development of microbead-based assays like the xMAP (Luminex®) has provided a technology that can resolve these questions. GTI Diagnostics® and Gen-Probe have developed PAKTM -Lx, a bead-based assay for the Luminex® platform to detect and determine the specificity of anti-HPA antibodies. PAKTM -Lx is capable of resolving the specificities of antibodies to HPA 1, 2, 3, 4 and 5 in addition to detecting antibodies against GPIV and HLA Class-I antigens. Furthermore, it is capable of detecting antibodies to multiple HPA and HLA with only 10 μl of sample. With a total time of less than two hours from addition of samples to end results, PAKTM -Lx is sensitive, rapid and simple to perform. Focused, flexible multiplexing in the range of 1 to 100 analytes provided by Luminex® technology meets the needs to expand the panel to include more HPA combinations, thereby reducing sample requirements, labor and costs. Disclosures:No relevant conflicts of interest to declare.