Chloroquine-treated Platelets Research Articles

Slot blotting and flow cytometry: two efficient assays for platelet antibody screening among patients with platelet refractoriness.

Frequent platelet transfusion may lead to the formation of alloantibodies and immune-mediated platelet destruction. Currently, identifying economic and effective screening methods is necessary for the management of platelet transfusion while different tests were recommended. The present study aims to challenge the performance of slot blotting (SB) and flow cytometry (FC) assays in detecting immune platelet refractoriness. Sera from 118 patients who received blood components and were clinically suspected of platelet refractoriness were enrolled. Platelet-reactive antibodies were explored in parallel by SB, FC and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) techniques. In a further study, chloroquine-treated platelets were incubated with MAIPA-positive serum, and then, the results of the SB and FC techniques were compared. Using MAIPA as a reference, antibodies were detected in 51 sera, with specificity for human leucocyte antigens (HLA), human platelet antigens (HPA) or both HLA/HPA, in 27, 18 and 6 patients, respectively. The sensitivity and specificity of SB and FC were 86·3%, 88·1%, 82·4% and 95·5%, respectively. The Spearman correlation revealed significant (P<0·001) correlations between FC (r=0·763) and SB (r=0·738) with MAIPA. In respect to HPA antibody detection, SB had 83·3% sensitivity and 92·6% specificity compared to 91·7% and 96·3% for FC while both approaches are acceptable (P<0·001, r=0·69; P<0·001, r=0·773) and can be recommended. The present study acknowledges that among the used methods, the flow cytometry's performance is the most appropriate, but slot blotting, with acceptable sensitivity, can be used as an acceptable and convenient procedure for platelet antibody screening.

Fetal and neonatal alloimmune thrombocytopenia: a late or missed diagnosis disease in fetal and perinatal health-care settings

Introduction Even though Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT) has been recognized as the main cause of primary hemorrhagic morbidity and mortality in fetuses and newborns, screening programs to detect pregnancies at risk have not yet been implemented in any country. Moreover, in spite of increased concerns about maternal, fetal and neonatal health care in general, this potentially lethal disease is still underdiagnosed. The aim of this report is to highlight the importance of considering FNAIT in fetal and perinatal health-care settings and show the usefulness of molecular tools in early diagnosis of this clinical entity. Methods DNA was extracted from whole blood from parents and newborns; genotyping was performed by in house PCR using sequence-specific primers for typing Human Platelet Antigens (HPA)-1 to -6, -9, and -15, and with commercial HPA-TYPE (BAG HealthCare, Lich, Germany). Anti-HPA antibodies in the maternal serum were detected by the Monoclonal Antibody Solid Phase Platelet antibody Test (MASPAT). Chloroquine-treated platelets were used for the discrimination of platelet-specific antibodies from anti-HLA antibodies. Results Patients 1 and 2 had severe thrombocytopenia due to incompatibility in HPA-1 and HPA-15, respectively. The third case was a thrombocytopenic neonate with severe bleeding complications other than ICH and in whom differential diagnosis between FNAIT and Von Willebrand congenital disease was necessary; incompatibility in HPA-15 was also demonstrated. Case 4 represents a missed diagnostic opportunity. Conclusion This is the first report of FNAIT cases confirmed by molecular evidence and anti-HPA antibodies detection in Argentina. This report reinforces the relevance of early diagnosis of this clinical entity. Since the delay in FNAIT diagnosis could lead to severe consequences in the fetus and neonates, strategies to approach maternal, fetal, and perinatal health, as well as prevention policies aimed to reduce fetal and neonatal morbidity and mortality should focus on implementing programs to identify high-risk pregnancies and thus reduce thrombocytopenia-related complications in fetuses and newborns.

Modification of Solid Phase Red Cell Adherence Assay for the Detection of Platelet Antibodies in Patients With Thrombocytopenia

Platelet refractoriness is caused by HLA antibodies and platelet-specific antibodies. Current methods used to detect antiplatelet antibodies have limitations. Solid phase red cell adherence (SPRCA) lacks sensitivity and requires a second assay using chloroquine-treated intact platelets to specify the response due to anti-HLA. We modified SPRCA by using 2 types of antihuman platelet antibodies with different specificities toward platelet lysate and tested samples from 361 patients (69 with unexplained thrombocytopenia and 292 with poor response to platelet transfusions not explicable by alloimmunization or the clinical situation) and 50 from healthy volunteers. Our method compared favorably with platelet suspension direct immunofluorescence. All samples from healthy volunteers were negative; of the samples from the patient population, 240 were positive (147 samples had only antiplatelet and 3 samples had only anti-HLA antibodies). This modified technique had a sensitivity of 98% and a specificity of 91%.

免疫学的血小板輸血不応症例の解析―血小板輸血効果の向上のために

Refractoriness to platelet transfusions is considered one of the major obstacles in treating patients with hematological disorders. Although various transfusion procedures have been tried to prevent HLA alloimmunization, some patients still develop HLA and/or HPA antibodies and become refractory to platelet transfusions. We investigated the AHG-LCT, MPHA, LIFT and FlowPRA methods for detecting HLA antibodies and MPHA using chloroquine-treated platelets for HPA antibodies to analyze immunological factors in platelet transfusion-refractory (PTR) patients. Results showed that HLA antibodies were detected in 55 patients, HPA antibodies in 2 and both HLA and HPA antibodies in 2 patients. HLA-A and -B-compatible platelet transfusions were successful in boosting platelet levels in 27 of the patients. Nine patients developed not only HLA-A and/or -B antibodies but also HLA-C antibodies and became refractory to HLA-A and -B-compatible platelet transfusions. Matching of the HLA-C antigens was required in these patients to ensure the effectiveness of platelet transfusions. In some patients, ABH incompatibility reduced the effectiveness of transfusions. In these patients, platelet crossmatching was useful for predicting platelet survival. Of the 68 ABH-incompatible transfusions evaluated at 24 hours post-transfusion, 20 with positive crossmatches had corrected platelet count increments of 3.1±4.1 (×109)/L. In contrast, 48 transfusions with negative crossmatches had corrected platelet count increments of 18.1±7.6 (×109)/L. Other weak HLA antibodies in ten patients were not detected by the conventional methods but were detected by the FlowPRA method. Owing to screening and crossmatch tests, platelet transfusion efficacy was improved in 59 immunological PTR patients.

Platelet-specific antibodies in HLA-immunized patients receiving chronic platelet support.

In HLA-alloimmunized patients, the unexpected failure of HLA-matched platelet transfusions usually raises the suspicion about concomitant platelet-specific antibodies. As the reported frequency of platelet-specific antibodies in multitransfused patients varies widely, the aim of this study was to determine the prevalence of such antibodies in a population of chronic thrombocytopenic patients with HLA antibodies. From 1985 to 1997, 11,777 determinations of HLA antibodies were performed in 1330 hematologic patients receiving chronic platelet support. Fifty-two patients with HLA alloimmunization that lasted more than 1 month were selected. The search for platelet-specific antibodies was performed by using a monoclonal antibody immobilization of platelet antigens assay, thus allowing the identification of platelet-specific antibodies directed against the platelet glycoproteins (GP) Ib/IX, GPIIb/IIIa, and GPIa/IIa. Specificity of the platelet-specific antibodies was further investigated by using a solid-phase assay with chloroquine-treated platelets. Only 2 (3.8%) of the 52 patients had platelet-specific antibodies. One antibody reacted with an epitope of the GPIIb/IIIa that was present in all the panel platelets, and that probably was an autoantibody. The other was an anti-HPA-5b. The prevalence of platelet-specific antibodies in patients with HLA alloimmunization is very small. The search for concomitant platelet-specific antibodies would be indicated only when other causes of refractoriness to HLA-matched platelets are ruled out.

A survey of platelet serology in UK laboratories (1987): An assessment of the efficacy of using chloroquine-treated platelets to distinguish between platelet-specific and anti-HLA antibodies

A survey of platelet serology in UK laboratories (1987): An assessment of the efficacy of using chloroquine-treated platelets to distinguish between platelet-specific and anti-HLA antibodies

Acidified chloroquine treatment for the removal of class I HLA antigens.

With the aim of reducing the damage to platelets while effectively removing class I HLA antigens from their surfaces, we developed a new method using acidified chloroquine diphosphate. Platelets were treated with a 0.2 M solution of chloroquine diphosphate (pH 4.0). More than 90% of the platelets remained viable after treatment. While a marked reduction in reactions of acidified chloroquine-treated platelets with multispecific HLA antisera was noted in comparison with phosphate-buffered-saline-(PBS)-treated platelets, reactions with platelet-specific antibodies were preserved. This was demonstrated by immunofluorescence tests and solid-phase and monoclonal antibody immobilization of platelet antigen assays. Aggregation responses, though reduced in comparison with PBS-treated platelets, were still preserved after acidified chloroquine treatment. Ultrastructural analysis did not show any significant difference from PBS-treated platelets. We conclude that treatment of platelets with acidified chloroquine diphosphate is a simple and effective method for removing class I HLA antigens from their surfaces with minimal damage to their structure and function.

Antibodies to platelet glycoprotein V in polytransfused patients with haematological disease.

Polytransfused patients often develop platelet-reactive antibodies (PRAb). These give positive reactions in the platelet immunofluorescence test (PIFT) and may be either lymphocytotoxic (LCTAb) or platelet-specific antibodies (PSAb). The latter may be detected in the PIFT using chloroquine-treated platelets (Chl-PIFT) or by immunoblotting. Serial samples from 106 multiply transfused patients with bone marrow failure were screened by PIFT using a microplate method and flow-cytometric analysis. PSAb activity was confirmed by Chl-PIFT. In 45 (42%) of the patients studied PSAb were detected; 37 (35%) formed LCTAb and 19 (51%) had co-existent PSAb. Sera from 25 of 27 patients with a positive Chl-PIFT, retested by immunoblotting, recognised determinants of Mr 82-160 kD on whole platelets. A large group became sensitised to a component of Mr 105-115 kD reduced (99 kD non-reduced) with similar electrophoretic mobility to GPIIIa using a monoclonal anti-GPIIIa and two human polyclonal anti-HPA-1a sera; some also produced anti-GPIIb. The largest group recognised a determinant of Mr 80-83 kD, probably glycoprotein V (GPV). Three sera were immunoblotted against thrombin-treated platelets and the results confirmed GPV specificity.

Antibodies to Platelet Glycoprotein V in Polytransfused Patients with Haematological Disease

Polytransfused patients often develop platelet-reactive antibodies (PRAb). These give positive reactions in the platelet immunofluorescence test (PIFT) and may be either lymphocytotoxic (LCTAb) or platelet-specific antibodies (PSAb). The latter may be detected in the PIFT using chloroquine-treated platelets (Chl-PIFT) or by immunoblotting. Serial samples from 106 multiply transfused patients with bone marrow failure were screened by PIFT using a microplate method and flow-cytometric analysis. PSAb activity was confirmed by Chl-PIFT. In 45 (42%) of the patients studied PSAb were detected; 37 (35%) formed LCTAb and 19 (51%) had co-existent PSAb. Sera from 25 of 27 patients with a positive Chl-PIFT, retested by immunoblotting, recognised determinants of Mr 82-160 kD on whole platelets. A large group became sensitised to a component of Mr 105-115 kD reduced (99 kD non-reduced) with similar electrophoretic mobility to GPIIIa using a monoclonal anti-GPIIIa and two human polyclonal anti-HPA-la sera; some also produced anti-GPIIb. The largest group recognised a determinant of Mr 80-83 kD, probably glycoprotein V (GPV). Three sera were immunoblotted against thrombin-treated platelets and the results confirmed GPV specificity.

Localization of the Platelet-Specific HPA-2 (Ko) Alloantigens on the N-Terminal Globular Fragment of Platelet Glycoprotein Ibα

The human platelet-specific alloantigens HPA-2a and HPA-2b (= Kob and Koa) together constitute a biallelic antigen system. The HPA-2 antigens have not, to date, been located on a particular platelet membrane molecule. Here, we describe the localization of these antigens on platelet glycoprotein (GP) Ib alpha. Platelets from two patients with the Bernard-Soulier syndrome (BSS) were HPA-2(a-,b-) in the immunofluorescence test with HPA-2 alloantibodies on chloroquine-treated platelets. With monoclonal antibody (MoAb) immobilization of platelet antigen assay (MAIPA), positive reactions were obtained only when MoAbs against the platelet GPIb/IX complex were used in combination with anti-HPA-2a or -2b alloantibodies and normal donor platelets. By immunoprecipitation under nonreducing and reducing conditions a protein of 160 Kd and 145 Kd, respectively, was precipitated by the anti-HPA-2a serum. A protein migrating identically to this was precipitated by anti-GPIb MoAb. Normal donor platelets became HPA-2(a-,b-) after elastase treatment, suggesting that anti-HPA-2 antibodies bind to the N-terminal elastase-sensitive part of GPIb alpha. Anti-HPA-2a antibodies inhibited the ristocetin-induced agglutination of HPA-2a-positive platelets but not of HPA-2a-negative platelets, indicating that the epitopes recognized by these alloantibodies are localized in the proximity of the von Willebrand-factor-binding domain. Together, these data provide evidence that the HPA-2 alloantigens are located on the N-terminal globular elastase-sensitive part of GPIb alpha. Furthermore, we show that the recently described Siba antigen is probably identical to HPA-2a.

Acid Treatment of Platelets as a Simple Procedure for Distinguishing Platelet‐Specific Antibodies from Anti‐HLA Antibodies: Comparison with Chloroquine Treatment

The identification of antibodies to platelet-specific antigens is important for correctly diagnosing neonatal alloimmune thrombocytopenia, posttransfusion purpura and refractoriness due to platelet-specific antibodies. However, the serologic identification of these platelet-specific antibodies is complicated by the presence of anti-HLA antibodies. We examined and compared the diagnostic usefulness of acid-treated and chloroquine-treated platelets for the discrimination of platelet-specific antibodies from anti-HLA antibodies. The viability of acid-treated platelets is 83.4%, which is better than that of chloroquine-treated platelets (52.6%). The antigenicity of HLA class I antigens of acid-treated platelets was significantly reduced compared with that of PBS- or chloroquine-treated platelets. On the other hand, platelet surface glycoprotein Ib and glycoprotein IIb/IIIa, and platelet-specific antigens were stable following acid or chloroquine treatment. Chloroquine-treated platelets were not suitable targets for analysis by immunofluorescence flow cytometry because of nonspecific fluorescence derived from platelet damage. We conclude that acid-treated platelets are more suitable targets than chloroquine-treated platelets for screening for platelet-specific antibodies and also for analyses of the specificity of platelet-specific antibodies.

A survey of platelet serology in UK laboratories (1987): An assessment of the efficacy of using chloroquine-treated platelets to distinguish between platelet-specific and anti-HLA antibodies

This survey was designed to determine the ability of participants to distinguish between platelet specific and anti-HLA antibodies. Four well characterized reference reagents and 10 unknown samples were sent to 29 laboratories affiliated to the UK Platelet and Granulocyte Serology Working Group and to three overseas reference laboratories. Results were obtained from 27 Working Group laboratories and from 2 reference laboratories. Overall, the results indicate that the use of chloroquine-treated platelets alone, without an independent test for anti-HLA antibodies, was insufficiently reliable to elucidate mixtures of platelet specific and anti-HLA antibodies. Laboratories using chloroquine-treated platelets were no more successful in elucidating the antibody composition of sera containing platelet-specific and anti-HLA antibodies than those laboratories which did not use this technique. The results may indicate a lack of experience with the chloroquine technique and suggest that further studies into the performance of the technique are required. The survey also provided insights into other areas of platelet serology: (1) There was a decrease in the incidence of false-positive results compared to earlier UK surveys. [2) All laboratories detected potent platelet-specific antibodies but weakly reactive antibodies were less readily detected. (3) Laboratories using fluorescent antiglobulin techniques recorded fewer errors than laboratories using ELISA techniques. (4) Laboratories using paraformaldehyde (PFA)-treated platelets in fluorescent antiglobulin techniques did not perform better than laboratories using untreated platelets.

New approach to eliminate HLA class I antigens from platelet surface without cell damage: acid treatment at pH 3.0.

A new method was studied for eliminating HLA class I antigens from the surface of platelets without damaging the cells. Platelets were exposed to an acid solution (pH 3.0) to eliminate the antigenicity of HLA class I antigens. The reduction in antigenicities of HLA class I common antigen and individual HLA class I antigens by acid treatment was marked. Patients' sera which contained multispecific HLA antibodies reacted with PBS-treated platelets, but not with acid-treated platelets. No changes were observed in the antigenicities of glycoprotein Ib or glycoprotein IIb/IIIa. The viability of acid-treated platelets was 83%. Ultrastructural investigations revealed no significant difference between the PBS-treated platelets and acid-treated platelets. The platelet function studies showed that the aggregation of acid-treated platelets induced by various agonists was only slightly reduced compared with PBS-treated platelets. We propose that acid-treated platelets are promising for clinical use in patients refractory to platelet transfusions and may be superior to chloroquine-treated platelets for analysis of the specificity of antiplatelet antibodies.

Quantitation of platelet antigens after chloroquine treatment

The effect of chloroquine treatment on the expression of various platelet antigens was studied. For this purpose, the binding of several human platelet-reactive antibodies (polyspecific HLA antibodies; platelet-specific PlA1, PlA2, Baka antibodies; platelet autoantibodies) and of murine monoclonal antibodies specific for epitopes on the glycoprotein (Gp) complexes IIb/IIIa or Ib/IX were quantitated by means of a competitive enzyme-linked immunoassay (CELIA) using fresh and stored chloroquine-treated platelets. We found that HLA antigens were substantially removed from platelets following chloroquine treatment (confirming earlier results) while platelet-specific antigens on the Gp complex IIb/IIIa were but little affected. Epitopes on the Gp complex Ib/IX did not show any alteration. Storage of chloroquine-treated panel platelets known to interfere with immunofluorescence had no effect on quantitative IgG determinations by CELIA. We conclude that chloroquine treatment of platelets is practicable for platelet antibody analysis, but exerts its effect unspecifically and requires cautious interpretation.

Quantitative analysis of platelet surface HLA by W6/32 anti-HLA monoclonal antibody

Class I molecules of human major histocompatibility complex (HLA) are the most important antigenic system in determining the survival of transfused platelets in alloimmunized patients. Platelets with reduced expression of a specific type of HLA antigen may escape specific anti- HLA antibody-mediated destruction. By using 125I-labeled Fab fragments of W6/32 anti-HLA monoclonal antibody and competitive protein binding assays, we measured the range of total HLA concentrations on platelets. In 12 individuals examined, the mean number of HLA-A, B, and C molecules per platelet was 81,587 +/- 20,016 (mean +/- SD); its range was between 54,782 to 116,185 molecules per platelet. After treatment with chloroquine, 79.9 +/- 7.0% (mean +/- SD, n = 6) of HLA antigens were removed from platelets as determined by binding of 125I-W6/32 Fab. A similar result was obtained when HLA antigens on chloroquine-treated platelets were evaluated with immunofluorescence flow cytometry. In contrast, chloroquine treatment did not remove integral membrane protein such as P1A1 antigens on platelets. The presence of HLA antigens in the chloroquine eluate of platelets could be demonstrated to contain HLA antigens similar in mol wts to intact class I molecules by an immunoblotting technique. These data suggest that 70% to 80% of platelet HLA antigens are adsorbed and that such HLA antigens are not proteolytic products of integral membrane class I molecules. The origin of the adsorbed platelet HLA-antigens remains to be determined.

Quantitative Analysis of Platelet Surface HLA by W6/32 Anti-HLA Monoclonal Antibody

Class I molecules of human major histocompatibility complex (HLA) are the most important antigenic system in determining the survival of transfused platelets in alloimmunized patients. Platelets with reduced expression of a specific type of HLA antigen may escape specific anti-HLA antibody-mediated destruction. By using 125I-labeled Fab fragments of W6/32 anti-HLA monoclonal antibody and competitive protein binding assays, we measured the range of total HLA concentrations on platelets. In 12 individuals examined, the mean number of HLA-A, B, and C molecules per platelet was 81,587 +/- 20,016 (mean +/- SD); its range was between 54,782 to 116,185 molecules per platelet. After treatment with chloroquine, 79.9 +/- 7.0% (mean +/- SD, n = 6) of HLA antigens were removed from platelets as determined by binding of 125I-W6/32 Fab. A similar result was obtained when HLA antigens on chloroquine-treated platelets were evaluated with immunofluorescence flow cytometry. In contrast, chloroquine treatment did not remove integral membrane protein such as P1A1 antigens on platelets. The presence of HLA antigens in the chloroquine eluate of platelets could be demonstrated to contain HLA antigens similar in mol wts to intact class I molecules by an immunoblotting technique. These data suggest that 70% to 80% of platelet HLA antigens are adsorbed and that such HLA antigens are not proteolytic products of integral membrane class I molecules. The origin of the adsorbed platelet HLA-antigens remains to be determined.