A simple and rapid competitive enzyme-linked immunosorbent assay to identify HPA-1a (PlA1)-negative donor platelet units

BACKGROUND: Alloantibodies to HPA-1a (PlA1) are the major cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura and have been implicated in refractoriness to random-donor platelet transfusions. However, most assays used to phenotype platelets are cumbersome or time-consuming for large numbers of samples. STUDY DESIGN AND METHODS: A simple, competitive (inhibition) enzyme-linked immunosorbent assay for HPA-1a phenotyping of donor platelets was developed. A segment from the donor platelet unit transfer line was sealed to obtain a small aliquot of platelets. These platelets were washed once and added to a predetermined dilution of serum containing alloantibodies to HPA-1a. Residual anti-HPA-1a binding to the glycoprotein IIb/IIIa purified by lectin and high-performance liquid chromatography and coated on microtiter wells was detected with a conjugated antihuman IgG. A lack of inhibition equivalent to control (no platelets) was used to determine that the platelets were HPA-1b/b. RESULTS: Of the 557 platelet units tested, 14 (2.5%) were found to be HPA-1a negative, and they were confirmed to be HPA-1b/b by DNA genotyping. Two of the 14 HPA-1b/b units were also HPA-3b/b (approx. 0.35% of the random population). Use of the microtiter format allows 100 to 200 samples to be processed per day. CONCLUSION: This simple and inexpensive assay is useful for identifying HPA-1b/b units for platelet- compatible transfusions or for platelet antibody investigations.     

Enzyme-linked immunosorbent assay (ELISA) for direct quantification of surface-bound platelet immunoglobulins

Surface-bound platelet IgG and IgM were measured by an enzyme-linked immunosorbent assay (ELISA) using washed platelets and commercially available alkaline phosphatase anti-human immunoglobulins (Fc-specific). With this technique platelets from normal donors had small amounts of platelet-bound IgG ranging from 0.00 to 0.16 A405 (absorbance at 405 nm wavelength) (10(7) platelets)-1 (0 to 124 ng) and of platelet-bound IgM ranging from 0.00 to 0.05 A405 (10(7) platelets)-1. Eight out of 10 (80%) thrombocytopenic patients with idiopathic autoimmune thrombocytopenic purpura (IATP) had values of both IgG and IgM exceeding the normal range. In addition, one patient (8%) had platelet-bound IgM only. An inverse relationship was demonstrated in patients with IATP between the blood platelet count and the amount of both IgG and IgM. Increased values were also demonstrated in patients with SLE and patients with monoclonal hypergammaglobulinaemia. The direct ELISA is a useful and reproducible technique for platelet-bound IgG and IgM, which requires standard laboratory equipment only.     

Human platelet alloantigens.

Antibody formation against alloantigens of the human platelet membrane is responsible for clinical syndromes and transfusion related conditions as neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP), platelet transfusion refractoriness (PTR) and passive alloimmune thrombocytopenia. Moreover, rare cases of alloimmune reactions involving platelets have been observed after transplantation of hematopoietic stem cells. Among alloantigens of the platelet membrane shared with other cells (type I alloantigens) are the glycoconjugates of the ABO system and class I human leukocyte antigen (HLA) antigens. Antibodies against these structures are responsible for PTR and for febrile nonhemolytic transfusion reactions. Antibodies against type II antigens (formerly termed "platelet specific antigens") have been observed in NAIT, PTP and passive alloimmune thrombocytopenia. ABH antigens have been identified on intrinsic platelet membrane glycoproteins. Moreover, it is now clear that HLA class I antigens are an integral part of the platelet membrane. The quantity of both HLA and ABH-antigen expression on the platelet membrane varies considerably. Single point mutations account for almost all platelet specific alloantigens, but most antigenic determinants seem to depend upon glycoprotein conformation: generally, platelet specific alloantibodies fail to recognize synthetic peptides encompassing the polymorphic residues. Restriction fragment polymorphism analysis and allele-specific PCR have been implemented for genotyping of platelet alloantigens in many laboratories. Antigen specific assays using monoclonal antibodies (MAIPA, immunobead assay) became de facto standard for diagnosis of platelet antibodies in serum/plasma samples. It can be expected that innovative techniques as human alloantibody fragments produced by phage display technique and the production of recombinant antigens will allow rapid and reliable phenotyping and antibody detection in the future.     

Platelet antibodies of the IgM class in immune thrombocytopenic purpura

The clinical course and response to therapy of patients with immune thrombocytopenic purpura (ITP) are not completely determined by the level of IgG present on the platelet surface. It is possible that antibodies of other immunoglobulin classes also play a role in platelet destruction in some of these patients. Therefore, we studied 175 patients with ITP for the presence of IgM anti-platelet antibodies using radiolabeled polyclonal or monoclonal anti-IgM. We observed that 57% of patients with clinical ITP had increased levels of IgM on their platelets, compared with normal controls and patients with thrombocytopenia who did not have ITP (less than 10%), (P less than 0.01). We obtained similar results using either radiolabeled polyclonal or monoclonal anti-IgM, reagents whose integrity was first characterized using erythrocytes coated with defined amounts of IgM antibody. Among patients with increased platelet-IgM there was a significant correlation both with the presence of increased platelet-C3 as well as the amount of platelet-C3 (P less than 0.01, r = 0.53). We demonstrated the presence of warm-reacting IgM anti-platelet antibodies in the plasma of two of these patients who were further studied. The isolated IgM fraction from these two plasmas was able to activate complement and place 3H-C3 on normal platelets. These studies demonstrate the presence of warm-reacting IgM anti-platelet antibodies in some patients with ITP. They suggest that the binding of complement to platelets by IgM antibodies may initiate platelet clearance as well as enhance the effect of IgG antibodies in ITP.     

Rapid detection of HPA-1 alloantibodies by platelet antigens immobilized onto microbeads

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is one of the most common bleeding disorders in neonates. It occurs when alloantibodies from an immunized mother react with paternally inherited alloantigens, mostly human platelet antigen 1a (HPA-1a), on the fetal platelets (PLTs). Currently, monoclonal antibody-immobilized PLT antigen (MAIPA) assay represents the standard technique for the serologic diagnosis of NAIT. MAIPA is time-consuming, however, and limited by the availability of monoclonal antibodies (MoAbs). Here, a gel antigen-specific assay (GASA) was developed, which allows rapid detection of HPA-1 alloantibodies without the use of MoAbs. STUDY DESIGN AND METHODS: Glycoprotein (GP) IIb/IIIa was purified by affinity chromatography from outdated PLT concentrates derived from HPA-1aa or HPA-1bb donors. Purified GPs were biotinylated, immobilized onto streptavidin beads, and used for the analysis of HPA-1a alloantibodies by a microtyping system. HPA-1a serum samples derived from mothers with NAIT (n = 36) and from posttransfusion purpura patients (n = 2) as well as HPA-1b (n = 4), HPA-5b (n = 2), HPA-3a (n = 4), and HLA Class I (n = 2) alloantiserum samples from multitransfused patients were investigated in GASA and MAIPA assays. RESULTS: GASA was able to detect all HPA-1a and -1b alloantibodies recognized by MAIPA. Cross-reactivity with other PLT-reactive alloantibodies was not observed. Interestingly, 3 of 36 serum samples, which showed only moderate reactivity in MAIPA, reacted strongly in GASA. CONCLUSION: GASA has proved to be a rapid method for the detection of HPA-1a alloantibodies and maybe useful for PLT antibody screening, especially in initial assessment of suspected NAIT cases.     

Maternal immunization to Gov system alloantigens on human platelets

BACKGROUND: Immunization to platelet alloantigens can occur during pregnancy or after the transfusion of blood components. Platelet alloantibodies can cause neonatal alloimmune thrombocytopenia and posttransfusion purpura. Transfusion-induced alloimmunization to a novel platelet alloantigen system, Gov, expressed on the 175-kDa glycosyl phosphatidylinositol-anchored platelet glycoprotein, CD109, was previously described. This report describes three unrelated patients who were alloimmunized to Gov(a) or Gov(b) during pregnancy. STUDY DESIGN AND METHODS: Platelets were typed by using radioimmunoprecipitation for HPA-1a, -3a, -5a, -5b, Gov(a), and Gov(b) and by polymerase chain reaction-restriction fragment length polymorphism for HPA-1a, -1b, -3a, and -3b. Maternal sera were screened for platelet antibodies by using radioimmunoprecipitation and the antigen capture assay. RESULTS: Patients 1 and 2 were investigated after the diagnosis of neonatal alloimmune thrombocytopenia in their children, and alloantibodies specific for Gov(b) and Gov(a), respectively, were detected in maternal serum. Serum from patient 3, who had mild idiopathic thrombocytopenia purpura with no detectable autoantibody, was found to contain alloantibodies to Gov(b) and to HPA- 5b, presumably as a result of immunization during pregnancy. Platelet typings confirmed that the patients were at risk for alloimmunization to the respective antigen. CONCLUSION: This report of three cases of maternal alloimmunization to antigens in the Gov system indicates that immunization can occur via placental transfer of antigen and that Gov system alloantibodies may be associated with neonatal alloimmune thrombocytopenia.     

Heterogeneity of HPA-3 alloantibodies: consequences for the diagnosis of alloimmune thrombocytopenic syndromes

BACKGROUND: Immunization against the human platelet alloantigen (HPA)-3a residing on alphaIIbbeta3 integrin accounts for approximately 2 percent of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Anti-HPA-3a alloantibodies are sometimes difficult to detect and can be overlooked by standard antigen capture assays. STUDY DESIGN AND METHODS: The reactivity of 12 anti-HPA-3a and 2 anti-HPA-3b alloantibodies from patients with FNAIT and posttransfusion purpura was analyzed by serologic (monoclonal antibody-specific immobilization of platelet antigens [MAIPA] assay, flow cytometry) and immunochemical (immunoprecipitation, immunoblotting) techniques. The influence of platelet (PLT) age, storage conditions, recombinant antigens from Chinese hamster ovary (CHO) cells, and sialic acids (treatment with neuraminidase) were analyzed. RESULTS: The most sensitive anti-HPA-3 alloantibody detection in MAIPA assay could be achieved with fresh homozygous PLTs. During a PLT storage period of 14 days before use, three types of anti-HPA-3 alloantibodies were found: 1) complete loss of reactivity (n = 6), 2) considerably weakened reaction (> or =50% reduction; n = 3), and 3) minor reduction of reactivity (< or =40% decrease; n = 5). When cryopreserved PLTs were used, 10 of 12 anti-HPA-3a and all anti-HPA-3b alloantibodies reacted positive. Only 6 of 10 serum samples reacted with recombinant HPA-3a on CHO cells. Neuraminidase treatment of PLTs showed that some anti-HPA-3a alloantibodies require the presence of sialic acids. The storage lesion seems to be related to cleavage of sialic acids. Immunochemical analysis revealed evidence that most anti-HPA-3a alloantibodies require an intact three-dimensional alphaIIbbeta3 integrin structure. CONCLUSIONS: Anti-HPA-3 alloantibodies show considerable heterogeneity, which may hamper the serologic diagnosis of FNAIT. Preservation of the alphaIIbbeta3 integrin and protection from enzymatic degradation seem to be important during PLT storage.     

Platelet alloantibodies in transfused patients

BACKGROUND: Patients receiving cellular blood components may form HLA antibodies and platelet-specific alloantibodies. STUDY DESIGN AND METHODS: Serum samples from a cohort of 252 patients with hematologic or oncologic diseases who are receiving cellular blood components were studied for platelet-reactive antibodies. Specificity of platelet alloantibodies was determined with a panel of typed platelets RESULTS: Platelet-reactive antibodies were detected in the sera of 113 patients (44.8% of 252), HLA antibodies in the sera of 108 (42.9%), and platelet-specific antibodies in the sera of 20 (8%). The following platelet-specific antibodies were identified: anti-HPA-5b (n = 10), anti-HPA-1b (n = 4), anti-HPA-5a (n = 2), anti-HPA-1a (n = 1), anti-HPA-2b (n = 1), anti-HPA-1b+5b (n = 1), and anti-HPA-1b+2b (n = 1). Fifteen sera from the 108 patients with anti-HLA (13.9%) contained additional platelet-specific alloantibodies, while in 5 sera, platelet-specific alloantibodies only were detected: anti-HPA-5b (n = 4) and anti-HPA-1a (n = 1). Of the 108 sera with HLA antibodies, 29 (26.9%) showed discordant results when studied with the lymphocytotoxicity test and the glycoprotein-specific immunoassay. Ten sera contained panreactive antibodies against platelet glycoproteins (GP) IIb/IIIa, GPIa/IIa, and/or GPIb/IX. Alloimmunization occurred in 58.3 percent of female patients with previous pregnancies, but in only 23.3 percent of those without previous pregnancies (p = 0.0049). CONCLUSION: Platelet alloantibody specificities in transfused patients (predominantly anti-HPA-5b and -1b with antigen frequencies <30% among whites) differ significantly from those observed in patients with neonatal alloimmune thrombocytopenia or posttransfusion purpura, in whom anti-HPA-1a (antigen frequency >95%) is the most prevalent specificity. HLA antibody detection yields discordant results when the lymphocytotoxicity assay and a glycoprotein-specific immunoglobulin-binding assay are used.     

Low-avidity HPA-1a alloantibodies in severe neonatal alloimmune thrombocytopenia are detectable with surface plasmon resonance technology

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is mostly caused by maternal alloantibodies directed against the human platelet alloantigen (HPA)-1a. Currently, the serologic diagnosis of FNAIT is based on the characterization of the HPA alloantibodies in monoclonal antibody–based antigen-capture assays (e.g., MAIPA assay). Accumulated current evidence indicated that such assays may overlook some HPA-1a antibodies.
STUDY DESIGN AND METHODS: This study employed surface plasmon resonance (SPR) technology using immunoaffinity-purified glycoprotein IIb/IIIa isoforms immobilized on biosensor chips to study the binding kinetics of HPA-1a alloantibodies from different FNAIT cases in real time.
RESULTS: Analysis of HPA-1a alloantibodies from FNAIT cases (n = 9) in SPR showed a moderate relative response (22.2-69.7 resonance units [RU]) and slow antibody dissociation. After the dissociation phase, varying amounts of bound antibodies (41%-79%) remained on the chip. In contrast in HPA-1a alloantibodies from a patient suffering from posttransfusion purpura, a high relative response (∼490 RU) was observed at the end of the association phase and no dissociation of antibody binding was detectable. Of particular relevance, by the use of this SPR technique, HPA-1a alloantibodies were detected in two severe FNAIT cases that had determined as false negative by MAIPA assay. In SPR, these HPA-1a alloantibodies showed low-avidity nature characterized by gradual dissociation of antibody during the association phase and complete detachment of antibody binding after the dissociation phase. This high "off-rate" character of low-avidity HPA-1a alloantibodies indicates that such antibody binding is easily detachable by the extensive washing procedure of the MAIPA.
CONCLUSIONS: Our results demonstrated that the SPR method can facilitate the diagnosis of clinically relevant low-avidity HPA-1a antibodies.     

Assessment of platelet antibody by flow cytometric and ELISA techniques: a comparison study

Two different methods for evaluating platelet antibody were used to study 12 normal subjects and 24 patients consisting primarily of intravenous drug users (IVDUs) who were positive for human immunodeficiency virus (HIV). Total platelet-associated immunoglobulin G (IgG) and immunoglobulin M (IgM) were measured by enzyme-lined immunosorbent assay on platelet lysate, and platelet surface-associated IgG and IgM were measured by semiquantitative flow cytometry. IgG and IgM values showed significant correlations between the two measurement methods. Mean platelet surface IgG and total IgG were 3.6 and 4.3 times greater, respectively, in IVDUs than in controls, and platelet IgM was also significantly higher in IVDUs than in controls as measured by both techniques. Although mean platelet immunoglobulin levels were higher in the IVDUs with thrombocytopenia than in IVDUs with normal platelet counts, these differences did not achieve significance. These data show that platelet IgG and IgM levels are increased in IVDU-associated HIV infection and suggest that these increases are not confined to patients manifesting thrombocytopenia. The herein described platelet surface antibody and total platelet antibody measurements appear to be equally useful in studying this patient population. Specific details for generating platelet-associated immunofluorescence units are discussed.     

Relationship of platelet-associated immunoglobulin G and platelet protein to platelet size and density in normal individuals and patients with thrombocytopenia

Total platelet-associated immunoglobulin G (PA-IgG) and platelet volume and protein content in normal individuals and in patients with idiopathic thrombocytopenic purpura (ITP) and other thrombocytopenias of presumed nonimmune origin have been measured on platelets separated into subpopulations on the basis of density on continuous polyvinylpyrrolidone (Percoll) gradients. In all subjects PA-IgG per platelet was primarily found in the lightest platelets at levels up to sevenfold greater than in the heavier platelets. PA-IgG level per platelet was raised in light platelets in 29% of patients with thrombocytopenia and in heavy platelets in 60%. In almost half of these instances the PA-IgG level fell to within the normal range when considered in relation to either platelet volume or protein content. PA-IgG levels of patients with untreated ITP did not differ significantly from those with treated ITP or thrombocytopenia of other causes. Mean platelet volume and protein content of the total platelet population of all subjects showed significant linear correlation (P less than 0.01). Thus PA-IgG of both controls and patients with thrombocytopenia of all causes is preferentially located in the lightest platelets, but increases in PA-IgG in immune thrombocytopenias occur more frequently in the heavier platelets. These findings suggest that part of the process of IgG accumulation by platelets is the same in normal individuals as in patients with thrombocytopenia.     

Heparin-induced thrombocytopenia: a prospective study on the incidence, platelet-activating capacity and clinical significance of antiplatelet factor 4/heparin antibodies of the IgG, IgM, and IgA classes

INTRODUCTION: Platelet-activating antiplatelet factor 4/heparin (anti-PF4/heparin) antibodies are the major cause of heparin-induced thrombocytopenia (HIT). However, the relative utility of functional (platelet activation) vs. antigen [enzyme-immunoassay (EIA)] assays, and the significance of assay discrepancies remain unresolved. METHODS: Consecutive patient sera (n = 1650) referred for diagnostic HIT testing were screened prospectively by both the heparin-induced platelet activation (HIPA) test and anti-PF4/heparin EIA - including individual classes (IgG, IgA, IgM) - with clinical correlations studied. Platelet microparticle and annexin-V-binding properties of the sera were also investigated. RESULTS: Only 205 (12.4%) sera tested positive in either the HIPA and/or EIA: 95 (46.3%) were positive in both, 109 (53.1%) were only EIA-positive, and, notably, only one serum was HIPA-positive/EIA-negative. Of 185 EIA-positive sera, only 17.6% had detectable IgM and/or IgA without detectable IgG. Among sera positive for EIA IgG, optical density values were higher when the sera were HIPA-positive (1.117 vs. 0.768; P < 0.0001), with widely overlapping values. Two HIPA-positive but EIA-IgG-negative sera became HIPA-negative following IgG depletion, suggesting platelet-activating antibodies against non-PF4-dependent antigens. Clinical correlations showed that HIPA-negative/EIA-positive patients did not develop thrombosis and had reasons other than HIT to explain thrombocytopenia. IgM/A antibodies did not increase microparticle penetration, but increased annexin-V binding. CONCLUSIONS: The anti-PF4/heparin EIA has high ( approximately 99%) sensitivity for HIT. However, only about half of EIA-positive patients are likely to have HIT. Anti-PF4/heparin antibodies of IgM/A class and non-PF4-dependent antigens have only a minor role in HIT.     

Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells.

Heparin-induced thrombocytopenia/thrombosis (HITP) is thought to be mediated by immunoglobulins that activate platelets in the presence of pharmacologic concentrations of heparin, but the molecular basis for this relatively common and often serious complication of heparin therapy has not been established. We found that plasma from each of 12 patients with HITP contained high titer (> or = 1:200) antibodies that reacted with immobilized complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein contained in platelet alpha-granules. Recombinant human PF4 behaved similarly to PF4 isolated from platelets in this assay system. Complexes formed at an apparent heparin/PF4 molecular ratio of approximately 1:2 (fresh heparin) and approximately 1:12 (outdated heparin) were most effective in binding antibody. Immune complexes consisting of PF4, heparin, and antibody reacted with resting platelets; this interaction was inhibited by a monoclonal antibody specific for the Fc gamma RII receptor and by excess heparin. Human umbilical vein endothelial cells, known to express heparin-like glycosaminoglycan molecules on their surface, were recognized by antibody in the presence of PF4 alone; this reaction was inhibited by excess heparin, but not by anti-Fc gamma RII. Antibodies reactive with heparin/PF4 were not found in normal plasma, but IgG and IgM antibodies were detected at dilutions of 1:10 (IgG) and 1:50 (IgM) in 3 of 50 patients (6%) with other types of immune thrombocytopenia. These findings indicate that antibodies associated with HITP react with PF4 complexed with heparin in solution or with glycosaminoglycan molecules on the surface of endothelial cells and provide the basis for a new hypothesis to explain the development of thrombocytopenia with thrombosis or disseminated intravascular coagulation in patients sensitive to heparin.     

Platelet specific alloantigens.

The ability of platelets to aggregate and to form a platelet plug is central to the maintenance of normal hemostasis. When platelets have normal function, the severity of bleeding is related to the degree of thrombocytopenia. In patients with normal platelet production, the most common cause of thrombocytopenia is due to immune mechanisms that results in platelet injury and removal from the circulation. These mechanisms involve the binding of platelet-associated immunoglobulins and are classified as immune. Immune thrombocytopenias can be caused by autoantibodies (autoimmune thrombocytopenia), alloantibodies (isoimmune thrombocytopenia), or drug-induced immune complexes and conditions secondary to autoimmune disorders such as systemic lupus erythematosus. In this paper the focus is on alloimmune thrombocytopenias resulting from the formation of alloantibodies to platelet specific antigens. The clinical importance of the platelet alloantigens is due to their ability to elicit alloantibody production. Alloantigens, also referred to as isoantigens, are substances that induce the production of alloantibodies when they are infused into individuals of the same species who lack the specific alloantigen.     

Reticulated platelets predict platelet count recovery following chemotherapy

BACKGROUND: A laboratory measure that predicted the timing of platelet recovery after chemotherapy could guide prophylactic platelet transfusion. Reticulated platelets (RPs) are the youngest circulating platelets; an increased percentage of RPs is diagnostic of increased marrow platelet production, such as seen with idiopathic thrombocytopenic purpura, whereas a low percentage of RPs with thrombocytopenia indicates marrow suppression. This study examined whether the percentage of RPs, in combination with a newly devised measurement of "stress thrombopoiesis," the RP maturation index (RP-MI), could predict platelet count recovery following chemotherapy-induced thrombocytopenia. STUDY DESIGN AND METHODS: Platelet count nadirs were retrospectively determined in 35 chemotherapy-induced thrombocytopenia patients; percentage of RPs and RP-MI values were assayed at the early nadir (no imminent platelet recovery) and the late nadir (imminent platelet recovery). The latter was defined by a platelet count increase of 20 x 10(9) per L or more in the subsequent 48 hours without platelet transfusion. RESULTS: Early in the nadir (when platelet recovery did not occur in the subsequent 48 h after sampling), a low RP-MI and a low percentage of RPs were found in 29 of 35 patients. Late in the nadir, when recovery was imminent, 27 of 30 evaluable patients had elevated percentages of RPs or RP-MI values; the mean time from sampling to an increase of 20 x 10(9) per L or more was 42 hours. The positive and negative predictive values of this assay were 82 and 91 percent, respectively. Furthermore, when thrombocytopenia was severe (platelet count < or = 20 x 10(9)/L), an elevated RP-MI and/or percentage of RPs correctly predicted imminent platelet count recovery in five of five patients. CONCLUSION: This noninvasive, rapid, whole-blood assay of stress thrombopoiesis provides reproducible indices for timing platelet recovery following chemotherapy and the potential to optimize the use of prophylactic platelet transfusions in chemotherapy patients.     

人类血小板同种抗原研究进展

人类血小板同种抗原（human platelet alloantigens, HPA）是由血小板糖蛋白携带的一类特异性抗原，其基因具有单核苷酸多态性（SNP）。HPA可介导同种抗体的产生，引起同种免疫反应，与输血后血小板减少性紫癜（PTP）、血小板输注无效（PTR），新生儿同种免疫血小板减少性紫癜（NAITP）及移植排斥密切相关。因其在临床输血实践及相关疾病中的重要作用，而备受关注。本文就HPA抗原的命名、血小板糖蛋白多态性、HPA检测方法、血小板同种免疫反应机制以及相关疾病研究进展作一综述。     

Advances in the studies on human platelet alloantigen--review]

Human platelet alloantigens (HPA) are specific antigens carried by platelet glycoproteins, which genes showing single nucleotide polymorphism. HPA can induce alloantibodies bringing about alloimmune response. They play important roles in post-transfusion refractoriness to platelets, post-transfusion thrombocytopenic purpura, fetomaternal alloimmune thrombocytopenia, and graft-versus-host disease. Because of their side effects in clinical blood-transfusion, there were a great deal of studies on HPA during last few decades. This review focuses on the nomenclature of HPA, the polymorphisms of platelet glycoproteins, HPA typing of the serological and molecular technology, as well as the mechanism of alloimmunization to HPA and correlated diseases.     

Antiidiotype antibody against platelet anti-GPIIIa contributes to the regulation of thrombocytopenia in HIV-1-ITP patients

Patients with human immunodeficiency virus 1-associated immunological thrombocytopenia (HIV-1-ITP) have markedly elevated platelet-bound immunoglobulin (Ig)G, IgM, and C3C4, as well as serum circulating immune complexes (CICs) composed of the same. Affinity purification of IgGs from their CICs with fixed platelets reveals high-affinity antibody (Ab) against platelet glycoprotein (GP)IIIa 49-66, which correlates inversely with their platelet count. However, sera from these patients have little to no anti-GPIIIa activity. To investigate this, we assayed serum, purified serum IgG, and CIC-Ig from these patients. This revealed approximately 150-fold greater Ab activity in purified serum IgG, and approximately 4,000-fold greater reactivity in CIC-IgG. This was shown to be associated with the presence of antiidiotype Ab2 (both IgG and IgM) sequestered in the CIC-IgG. The IgM antiidiotype was predominantly blocking Ab, as demonstrated by specificity for F(ab')(2) fragments of anti-GPIIIa 49-66 of HIV-1-ITP patients and inhibition of reactivity with peptide GPIIIa 49-66, not with a control peptide. The IgM antiidiotype was not polyreactive. Similar measurements were made in nonthrombocytopenic HIV-1-infected patients. Their serum reactivity was not measurable, but serum Ig and CIC-IgG against platelet GPIIIa 49-66 was present, although considerably lower than that found in HIV-1-ITP patients (26- and 35-fold lower, respectively). In addition, their IgM antiidiotype reactivity was 12-fold greater than that found in HIV-1-ITP patients. The IgM antiidiotype Ab titer of both cohorts correlated with in vivo platelet count (r = 0.7, P = 0. 0001, n = 32). To test the in vivo effectiveness of the IgM antiidiotype, thrombocytopenia was induced in mice with 25 microgram of affinity-purified anti-GPIIIa 49-66 (mouse GPIIIa has 83% homology with human GPIIIa and Fc receptors for human IgG1). Maximum effect was obtained at 4-6 h after intraperitoneal injection into Balb/c mice with a platelet count of approximately 30% baseline value. Preincubation of the anti-GPIIIa Ab with control IgM at molar ratios of IgM/IgG of 1:7 before intraperitoneal injection had no effect on the in vivo platelet count, whereas preincubation with patient IgM antiidiotype improved the platelet count to 50-80% of normal. Thrombocytopenia could be reversed after addition of IgM antiidiotype 4 h after induction of thrombocytopenia. Thus, CICs of HIV-1-infected patients contain IgM antiidiotype Ab against anti-GPIIIa, which appears to regulate their serum reactivity in vitro and their level of thrombocytopenia in vivo.     

Detection and identification of platelet antibodies in clinical disorders.

Serologic assays to detect and identify platelet-reactive antibodies have progressed from less sensitive and specific Phase I tests based on platelet functional endpoints through more sensitive Phase II assays that detect platelet-associated immunoglobulins, to highly specific Phase III assays that detect antibodies bound to alloantigens located on isolated platelet surface glycoproteins. Phase II and III assays are useful in the evaluation of patients with suspected platelet alloimmune syndromes neonatal alloimmune thrombocytopenia (NATP) and post-transfusion purpura (PTP) as well as in platelet crossmatching. Flow cytometry, a Phase II assay, can be modified to detect drug-dependent platelet-reactive antibodies. 14C-serotonin release, a Phase I assay and the platelet factor 4 ELISA, a Phase III assay, are now used to diagnose patients with heparin-induced thrombocytopenia (HIT). A sufficiently sensitive and specific assay to diagnose idiopathic (autoimmune) thrombocytopenia (ITP) remains elusive.     

Heterogeneity of antibody response to human platelet transfusion.

To study the antibody response to human platelet transfusions, nine thrombocytopenia patients with bone marrow failure were given 6 U (3X10(11)) of random platelet concentrates twice a week. Before transfusion, none of the patients had preexisting antibodies detectable with lymphocytotoxicity, platelet aggregation, or capillary leukoagglutination techniques. After receiving 18-78 U of platelets, they became refractory to further transfusions of random platelets and alloantibodies were detectable. Two patterns of antibody response could be identified. In three patients, the sera were not lymphocytotoxic with a panel of standard cells in which all the known HLA antigens in the first and second series were represented at least once. Yet, they caused platelet aggregation with 30, 24, and 60%, respectively, of a donor population studied. The aggregating activities were inhibited by antihuman IgG but not by antihuman IgA or antihuman IgM antiserum. The aggregating antibodies could be absorbed out with donor platelets but not lymphocytes or granulocytes. Antibodies from two of these patients aggregated platelets of their respective siblings matched for both HLA haplotypes. Transfusion of platelets from these two siblings did not increase the platelet count while platelets obtained from aggregation-negative donors did. The sera from the remaining six patients were lymphocytotoxic with 15-100% of the panel of standard cells. They also had aggregating antibodies, which could be absorbed out by both platelets and lymphocytes, suggesting that they were HLA antibodies. These data suggest that the development of platelet-specific antibodies may play an important role in the immunological rejection of isologous platelets, and should be considered in the selection of donors for patients who are refractory to platelets from random donors.