中国大陆地区发现一例罕见的血小板抗原HPA-10bw等位基因报告

采用序列特异性引物-聚合酶链式反应(PCR-SSP)为基础的人类血小板抗原(HPA)基因分型技术做群体调查, 在1,000例受检者中发现1例罕见的HPA-10w(a+b+)杂合子个体, 为了验证分型的可靠性, 使用PCR反应特异性扩增HPA-10基因片段, 然后测序分析。结果表明, nt263位G→A导致GPⅢa糖蛋白第62位精氨酸(CGA)→谷氨酰胺(CAA), 产生HPA-10bw抗原特异性。在中国人群中检测出HPA-10bw低频抗原, 提示在血小板同种免疫引起的新生儿同种免疫血小板减少症(NAIT)、输血后紫癜症(PTP)以及血小板输注无效症(PTR)的诊断中, 该抗原具有临床意义。     

Intrauterine transfusion with red cells and platelets.

Having direct access to the fetoplacental circulation by ultrasound-directed needle puncture has led to therapeutic interventions for fetal anemia and thrombocytopenia. Most cases of red cell alloimmunization associated with fetal anemia are caused by the antibody to the D red cell antigen. The intravascular transfusion of red cells to a hydropic fetus in such cases has notably improved survival. Nonimmune hydrops fetalis due to maternal parvovirus infection has also been treated successfully with the intravascular transfusion of red cells, whereas fetomaternal hemorrhage has not proved amenable to such therapy. Sensitization to the PLA-1 platelet antigen is the most common cause of fetal thrombocytopenia in maternal platelet alloimmunization. Fetal platelet transfusions have not proved to be a practical therapeutic modality for this disorder owing to the short half-life of the platelets. Platelets transfusions to the fetus just before delivery may avert the need for cesarean section in cases of severe thrombocytopenia.     

血小板输注无效的原因与对策的研究进展

血小板的主要生理作用是参与正常的止血,防止损伤后血液丢失。在临床治疗中,血小板的输注频率仅次于红细胞,是现代成分输血的一个重要组成部分,适用于预防和治疗血小板减少和/或血小板功能缺陷患者的出血,并且已成为各种血液病及肿瘤患者放、化疗的有效支持疗法[1],可以降低血液病患者及肿瘤患者放、化疗后因血小板减少而导致出血致死的死亡率。但是随着血小板的大量使用,血小板输血反应日渐增多,特别是血小板输注无效(refractoriness to platelet transfusion,RPT)是临床面临的一大难题。据文献报道,其发生率为30%-70%。产生原因包括免疫性因素和非免疫性因素。处理措施主要是分析血小板输注无效的主要原因,尽量减少输血次数,控制感染和发热,开展血小板组织配型为患者选择适用的血小板,提高输注疗效。     

Stimulation of lymphocytes by platelet-antibody-complexes and their role in the pathogenesis of idiopathic thrombocytopenia

The effect of washed human platelets, platelet lysates, and platelet antibody complexes on 14C-thymidine incorporation by human lymphocytes was studied. For sensitization of platelets, HLA-specific alloantibodies as well as platelet autoantibodies were used. Lymphocytes for in vitro cultures were collected from unsensitized individuals, healthy women with proven fetomaternal immunization against HLA antigens and patients with idiopathic thrombocytopenic purpura (ITP). Unsensitized platelets have a dose-dependent inhibitory effect on the in vitro proliferation of normal lymphocytes induced by mitogens (PHA, ConA, PWM). Platelet antibody complexes (allo- and autoantibodies; allogenic and autologous lymphocyte-platelet combinations) did not stimulate 14C-thymidine incorporation. Lymphocytes from ITP patients showed a significantly reduced stimulatory response toward PHA compared to normal persons. These findings are discussed in the light of our present knowledge regarding the role of cellular immune reactions in the pathogenesis of ITP.     

Polymorphisms in Canine Platelet Glycoproteins Identify Potential Platelet Antigens

Human alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness. More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system; however, there is no previous information on canine platelet-specific alloantigens. Using the HPA system as a model, we evaluated the canine ITGB3, ITGA2B, and GP1BB genes encoding GPIIIa (β₃), GPIIb (α_IIb), and GPIbβ, respectively, which account for 21 of 27 HPA, to determine whether amino acid polymorphisms are present in the orthologous canine genes. A secondary objective was to perform a pilot study to assess possible association between specific alleles of these proteins and a diagnosis of idiopathic thrombocytopenic purpura (ITP) in dogs. By using genomic DNA from dogs of various breeds with and without ITP, sequencing of PCR products encompassing all coding regions and exon–intron boundaries for these 3 genes revealed 4 single-nucleotide polymorphisms in ITGA2B resulting in amino acid polymorphisms in the canine genome, 3 previously reported and 1 newly identified (Gly[GGG]/Arg[AGG] at amino acid position 576 of ITGA2B. Of 16 possible ITGA2B protein alleles resulting from unique combinations of the 4 polymorphic amino acids, 5 different protein isoforms were present in homozygous dogs and explain all of the genotype combinations in heterozygous dogs. There was no amino acid polymorphism or protein isoform that was specific for a particular breed or for the diagnosis of ITP.Abbreviations: HPA, human platelet antigen; ITP, idiopathic thrombocytopenic purpura; PTR, platelet transfusion refractoriness; SNP, single-nucleotide polymorphismHuman alloimmune thrombocytopenic conditions caused by exposure to a platelet-specific alloantigen, through pregnancy or transfusion, include neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet transfusion refractoriness (PTR). More than 30 platelet-specific alloantigens have been defined in the human platelet antigen (HPA) system, with 12 alloantigens grouped into 6 biallelic systems (HPA1 through HPA5 and HPA15) in which alloantibodies against both the common (designated ‘a’) and rare (designated ‘b’) alleles have been identified.^5,12 For the remaining 21 platelet alloantigens (HPA6bw through HPA14bw and HPA16bw through HPA27bw), only antibodies against the rare allele (designated ‘bw’) have been detected. Although HPA traditionally were defined by using immune sera, the molecular basis of these antigens has now been characterized.^5,12 The HPA reside in platelet membrane glycoproteins, the most common being GPIIIa, which accounts for 14 HPA. In all but one (HPA14bw), the platelet alloantigens are defined by a single amino-acid substitution caused by a single-nucleotide polymorphism (SNP) in the gene encoding the relevant membrane glycoprotein.^5,12 The Platelet Nomenclature Committee, which includes members of both the International Society of Blood Transfusion and the International Society of Thrombosis and Haemostasis, has set guidelines for defining new platelet antigens, which include: 1) determining the genetic basis of the alloantigen by genomic DNA sequence analysis, and 2) demonstrating an association between the genetic mutation and the reactivity of alloantibodies with the allelic forms of the protein.¹² For patients suspected of having alloimmune thrombocytopenia, well-defined platelet genotyping methods and serologic assays for the detection of platelet antibodies are available to guide case management.In contrast to the well-characterized HPA system and wealth of information on alloimmune thrombocytopenic conditions in humans, there is no information on canine platelet-specific alloantigens in the literature. However, early serologic studies using antiHPA1a alloantiserum resulted in direct immunoprecipitation of a 90-kDa protein from canine platelets, suggesting that the antigenic determinant for HPA1a has been conserved between humans and dogs.¹⁰ Neonatal alloimmune thrombocytopenia has not yet been documented in dogs, and there is a single case report of suspected posttransfusion purpura in a dog with hemophilia A, in which severe thrombocytopenia was noted 1 to 2 wk after transfusion (cryoprecipitate, fresh whole blood), with an increased amount of platelet surface-associated IgG and rapid resolution (less than 1 wk) of the thrombocytopenia.³⁰ PTR, however, has been well documented in dogs for more than 25 y, because this species has been used as an experimental model to evaluate a variety of methods of preventing platelet alloimmunization.^21-23 The occurrence of platelet alloimmunization in dogs receiving platelets from dog leukocyte antigen-identical littermates indicates that nondog leukocyte antigen immunizing platelet antigens, perhaps platelet-specific antigens, are responsible for the development of PTR.²¹Using the HPA system as a model, our main objective in this study was to evaluate the canine ITGB3, ITGA2B, and GP1BB genes encoding the GPIIIa (β₃), GPIIb (α_IIb), and GPIbβ, respectively, proteins that account for 21 of 27 HPA, to determine whether there are amino acid polymorphisms that could define canine platelet-specific alloantigens. A secondary objective was to perform a pilot study to assess the possible association between canine platelet antigen protein alleles or single amino acid substitutions and primary immune-mediated thrombocytopenia, also referred to as idiopathic thrombocytopenic purpura (ITP), in dogs. The identification of a canine platelet antigen system would improve our understanding of the molecular basis of alloimmune thrombocytopenic conditions in dogs and help guide effective platelet transfusion support of these critical patients.     

基于免疫性PTR的血小板膜糖蛋白及T/B淋巴细胞表达水平研究

摘要 目的：探究血小板膜糖蛋白及T/B淋巴细胞免疫在免疫性血小板输注无效发生机制中的重要作用。方法：采用血小板特异性抗体检测试剂盒及流式细胞技术检测免疫性PTR患者血小板特异性抗体（抗GPIIb/IIIa、抗GPIa/IIa、抗GPIb/IX、抗GPIV）、血小板膜糖蛋白（CD36、CD61、CD41a）表达情况及外周血T/B淋巴细胞数量,运用SPSS19.0软件及R软件分析免疫性PTR与血小板膜糖蛋白、外周血T/B淋巴细胞的相关性。结果：免疫性PTR与血小板输注有效者比较,血小板特异性抗体的产生无显著差异,血小板膜糖蛋白CD36和CD61的表达具有显著差异,CD36对免疫性PTR发生风险具有极大的预测价值,外周血CD8⁺T细胞比例增高,而CD4⁺T细胞比例减低。结论：免疫性PTR患者产生血小板抗体具体机制不明确,了解患者细胞免疫状态有助于明确免疫性PTR的发生机制,为患者提供更优质的诊疗策略。     

HLA compatibility and survival of 51 chromium-labeled allogeneic thrombocytes

In 37 patients with thrombocytopenia (mostly with ITP) the survival time of 51Cr-labeled allogeneic platelets was investigated. The HLA antigens were typed in donors and recipients and the presence of HLA cytotoxins and specific thrombocyte antibodies in sera of patients were examined. In 7 cases the identity of 2 HLA antigens, in 15 cases that of 1 HLA antigen and in 15 cases the HLA incompatibility between donor and patient were found. The survival of platelets did not depend on the degree of HLA compatibility, unless the HLA cytotoxins in sera of patients appeared. The HLA, as well the specific platelet antibodies brought about the shortened platelet survival to 1 day and less. The importance of these observations for platelet kinetics is discussed.     

血小板输注无效与血小板抗原(HPA)多态性的相关性

目的:探讨血小板输注无效(PTR)与血小板抗原(HPA)多态性的相关性。方法:2017年5月到2018年9月选择在首都医科大学附属北京同仁医院输血科进行血小板输注的患者187例,检测所有患者血液的HPA多态性,判断PTR发生情况并进行相关性分析。结果:在187例患者中,发生PTR 32例,发生率为17.1%。PTR患者的HPA1、HPA2、HPA3、HPA4、HPA5的b基因频率都显著高于非PTR患者(P<0.05),也都呈基因多态性分布;非PTR患者都呈aa纯合子单性态分布。在187例患者中,直线相关分析显示HPA1、HPA5基因多态性与PTR有显著相关性(P<0.05),与HPA2、HPA3、HPA4基因多态性无相关性(P>0.05)。Logistic回归分析显示HPA1基因多态性、HPA5基因多态性、再生障碍性贫血、骨髓增生异常综合症为导致PTR的影响因素(P<0.05)。结论:血小板输注中PTR比较常见,多伴随有HPA多态性,HPA1和HPA5基因多态性、再生障碍性贫血、骨髓增生异常综合症为导致PTR的影响因素。     

Prevention of HLA alloimmunization: role of leukocyte depletion and UV-B irradiation

HLA alloimmunization is a major cause of the platelet refractory state. The stimulus for HLA alloimmunization is believed to derive from incompatibility between the recipient's lymphocytes and the passenger donor lymphocytes contained in transfused red cells or platelet concentrates. Two techniques to prevent post-transfusion HLA alloimmunization include filtration, which physically removes the donor lymphocytes, and UV-B irradiation, which renders the donor leukocytes biologically inactive. The role of these two techniques in the prevention of HLA alloimmunization is the focus of this review.     

Platelet transfusion refractoriness after T-cell-replete haploidentical transplantation is associated with inferior clinical outcomes

Haploidentical stem cell transplantation (haplo-SCT) has been an alternative source of bone marrow for patients without human leukocyte antigen (HLA)-matched donors. The aim of this study was to investigate the relationships between platelet transfusion refractoriness (PTR) and clinical outcomes in the setting of haplo-SCT. Between May 2012 and March 2014, 345 patients who underwent unmanipulated haplo-SCT were retrospectively enrolled. PTR occurred in 20.6% of all patients. Patients in the PTR group experienced higher transplant-related mortality (TRM, 43.7% vs. 13.5%, P<0.001), lower overall survival (OS, 47.9% vs. 76.3%, P<0.001) and lower leukemia-free survival (LFS, 47.9% vs. 72.3%, P<0.001) compared to patients in the non-PTR group. The multivariate analysis showed that PTR was associated with TRM (P=0.002), LFS (P<0.001), and OS (P<0.001). The cumulative incidences of PTR in patients receiving >12 platelet (PLT) transfusions (third quartile of PLT transfusions) were higher than in patients receiving either >6 (second quartile) or >3 (first quartile) PLT transfusions (56.1% vs. 41.6% vs. 28.2%, respectively; P<0.001). The multivariate analysis also showed that PTR was associated with the number of PLT transfusions (P<0.001). PTR could predict poor transplant outcomes in patients who underwent haploidentical SCT.     

The clearance mechanism of chilled blood platelets

Platelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior to transfusion. This peculiarity requires blood services to store platelets at room temperature, limiting platelet supplies for clinical needs. Here, we describe the mechanism of this clearance system, a longstanding mystery. Chilling platelets clusters their von Willebrand (vWf) receptors, eliciting recognition of mouse and human platelets by hepatic macrophage complement type 3 (CR3) receptors. CR3-expressing but not CR3-deficient mice exposed to cold rapidly decrease platelet counts. Cooling primes platelets for activation. We propose that platelets are thermosensors, primed at peripheral sites where most injuries occurred throughout evolution. Clearance prevents pathologic thrombosis by primed platelets. Chilled platelets bind vWf and function normally in vitro and ex vivo after transfusion into CR3-deficient mice. Therefore, GPIb modification might permit cold platelet storage.     

Relevance of blood groups in transfusion of sickle cell disease patients

Blood groups are clinically significant in sickle cell disease (SCD) as transfusion remains a key treatment in this pathology. The occurrence of a delayed haemolytic transfusion reaction (DHTR) is not rare and is a life-threatening event. The main cause of DHTR is the production of alloantibodies against red blood cell antigens. The high rate of alloimmunization in SCD patients is mainly due to the differences of red blood groups between patients of African descent, and the frequently Caucasian donors. From an immuno-haematological point of view, DHTR in SCD patients has specific features: classical antibodies known to be haemolytic can be encountered, but otherwise non significant antibodies, autoantibodies and antibodies related to partial and rare blood groups are also frequently found in individuals of African descent. In some cases, there are no detectable antibodies. As alloimmunization remains the main cause of DHTR, it is extremely important to promote blood donation by individuals of African ancestry to make appropriate blood available.     

Induction of differentiation of human stem cells ex vivo: Toward large-scale platelet production

Platelet transfusion is one of the most reliable strategies to cure patients suffering from thrombocytopenia or platelet dysfunction. With the increasing demand for transfusion, however, there is an undersupply of donors to provide the platelet source. Thus, scientists have sought to design methods for deriving clinical-scale platelets ex vivo. Although there has been considerable success ex vivo in the generation of transformative platelets produced by human stem cells (SCs), the platelet yields achieved using these strategies have not been adequate for clinical application. In this review, we provide an overview of the developmental process of megakaryocytes and the production of platelets in vivo and ex vivo, recapitulate the key advances in the production of SC-derived platelets using several SC sources, and discuss some strategies that apply three-dimensional bioreactor devices and biochemical factors synergistically to improve the generation of large-scale platelets for use in future biomedical and clinical settings.     

Computerization of a bank of platelets with HLA phenotype and preserved at -196 degrees C

In order to complete the logistics of the unit cytapheresis, we have developed a decision helping system which rests upon the computer management of -196 degrees C cryopreserved HLA type platelets. The hardware used is a micro-computer equipped with two floppy disks and a printer. Two files have been created: namely "Product" and "Patient". Data relating to 800 platelet concentrates may be recorded on a floppy disk. The software which has been developed has several functions: 1 - The input of the parameters of a HLA type concentrate with the possibility of reservation for the given recipient; 2 - The print out of the bank's stock; 3 - The selection and the reservation of HLA matched platelets according to the recipient; 4 - The print out of concentrates allocated to each patient, it is possible to thaw out chosen platelet concentrate and a thawing report is printed out, thus enabling one to locate the concentrate in the bank. Thanks to this "aid in decision making", the management of - 196 degrees C cryopreserved HLA type platelets may be carried out. The proposed software allows an one line answer for any emergency transfusion case. Considering the recipient HLA typing the suitable best match platelet concentrates are instantly transfused or save.     

In Vitro and In Vivo Characterization of Ultraviolet Light C-Irradiated Human Platelets in a 2 Event Mouse Model of Transfusion

UV-based pathogen reduction technologies have been developed in recent years to inactivate pathogens and contaminating leukocytes in platelet transfusion products in order to prevent transfusion-transmitted infections and alloimmunization. UVC-based technology differs from UVA or UVB-based technologies in that it uses a specific wavelength at 254 nm without the addition of any photosensitizers. Previously, it was reported that UVC irradiation induces platelet aggregation and activation. To understand if UVC-induced changes of platelet quality correlate with potential adverse events when these platelets are transfused into animals, we used a 2-event SCID mouse model in which the predisposing event was LPS treatment and the second event was infusion of UVC-irradiated platelets. We analyzed lung platelet accumulation, protein content in bronchoalveolar lavage fluid as an indication of lung injury, and macrophage inflammatory protein-2 (MIP-2) release in mice received UVC-irradiated or untreated control platelets. Our results showed UVC-irradiated platelets accumulated in lungs of the mice in a dose-dependent manner. High-doses of UVC-irradiated platelets were sequestered in the lungs to a similar level as we previously reported for UVB-irradiated platelets. Unlike UVB-platelets, UVC-platelets did not lead to lung injury or induce MIP-2 release. This could potentially be explained by our observation that although UVC treatment activated platelet surface αIIbβ3, it failed to activate platelet cells. It also suggests lung platelet accumulation and subsequent lung damage are due to different and separate mechanisms which require further investigation.     

Comparison of posttransfusion recoveries achieved with either fresh or stored platelet concentrates

The effectiveness of platelet concentrate transfusion depends on such variables as blood bag material, donor--recipient compatibility, and time elapsed between donation and transfusion. To study the latter a corrected thrombocyte increment for recovery in the recipients was evaluated with 108 platelet transfusions in 31 patients. In 83 treatment programs, the mean recovery at the one-hour post-transfusion time point was 8.6 X 10(9) platelets/l with fresh platelets and 5.9 X 10(9) platelets/l with stored platelets. Significantly better recovery was achieved with freshly prepared platelet over the total of platelet concentrates stored for up to 96 hours; however, if the recoveries in different patient groups given stored platelets were considered separately in terms of storage times of up to 48 h or 48-96 h, the good recovery with fresh platelets was significantly better only when compared to the older (p = 0.034) but not to the younger group of stored platelets. In patients with signs indicating enhanced platelet destruction (fever, splenomegaly, disseminated intravascular coagulation) the transfusion with fresh platelet concentrates gave a significantly better recovery compared to stored platelet concentrates (p = 0.028), whereas in the absence of such signs the recovery produced by fresh concentrates was not significantly higher than with stored concentrates. These findings may be relevant for the logistics in blood banking.     

Crossmatch difficulties following the prophylactic use of Rh immune globulin.

With increasing demand for platelet transfusion the need to use platelets from Rh-positive persons for Rh-negative individuals often arises. The administration of Rho(D) immune globulin (human) in this situation has been recommended, but may cause serologic difficulties owing to the recipient''s passive acquisition of antibodies other than anti-D.     

Evidence that platelet-derived microvesicles may transfer platelet-specific immunoreactive antigens to the surface of endothelial cells and CD34+ hematopoietic stem/ progenitor cells--implication for the pathogenesis of immune thrombocytopenias

The pathogenesis and tissue damage that accompanies destruction of platelets in immune thrombocytopenias (IT) is still not understood very well and in addition to platelets, other cells (e.g. endothelial cells, CD34+ hematopoietic stem/progenitors) may also become affected. Based on our previous work that platelet antigens (e.g., CD41) may be transferred by platelet-derived microvesicles (PMV) to the surface of other cells, we asked if platelet derived-antigens, especially those that are involved in the formation of anti-platelet antibodies in IT (e.g., against antigen HPA 1 a) could be also transferred by similar mechanism. To address this issue normal human CD34+ cells, human umbilical vein-endothelial cells (HUVEC) and monocytic cell line THP-1 were incubated with PMV derived from HPA1a+ donors. We noticed that the HPA1a antigen is highly expressed on PMV-derived from the HPAla positive platelets and is transferred in PMV-dependent manner to the surface of CD34+ cells, HUVEC and monocytic THP-1 cells. These cells covered with HPA1a positive PMV but not by PMV derived from HPAla negative platelets reacted with anti-HPA1a antibodies derived from the alloimmunized pregnant women. More importantly, human hematopoietic cells that were preincubated with HPA1a+ PMV and subsequently exposed to anti-HPA 1 a serum and human NK cells, become subject to elimination by antibody dependent cell cytotoxicity ADCC. Thus, we postulate that PMV-dependent transfer of antigens may playing an important role in "expanding" the population of target cells that may be affected by anti-platelet antibodies and explain several pathologies that accompany IT (e.g. damage of endothelium, cytopenias).     

Sensitive detection of idiotypic platelet-reactive alloantibodies by an electrical protein chip

To prevent and treat immune-mediated platelet disorders (e.g. neonatal allo-immune thrombocytopenia and platelet transfusion refractoriness) the causative idiotypic platelet-reactive antibodies have to be detected with high sensitivity and specificity. The "Monoclonal Antibody Immobilization Platelet Assay" (MAIPA) is the diagnostic gold standard for immunotyping sera with respect to alloantibodies against human platelet antigens (HPA). However, it is labor-intensive and time-consuming. In this work, an automated protein chip assay (enzyme-linked sandwich immunoassay) based on interdigitated gold microelectrodes in combination with an electrical read-out system was developed and optimized. For this purpose, specific capture antibodies were immobilized on the gold electrodes. The binding of the target is detected via an enzyme-labeled detection antibody by a redox-recycling process that corresponds to the amount of bound target molecule. With this electrical chip assay it is possible to detect antibodies against HPA-1a, HPA-5b and HLA with high sensitivity and specificity in less than half the duration of the MAIPA protocol with similar intra- and interassay variance.     

Platelet autoantigens: identification and characterization using immunoblotting

Antiplatelet autoantibodies are important in the etiology of idiopathic (or immune) thrombocytopenic purpura (ITP). Studies using immunoblotting techniques have been helpful in identifying the antigenic target proteins for the antibodies. Antibodies against the glycoprotein (GP) IIIa portion of the GPIIb/IIIa complex were the first to be demonstrated by this approach. Similar GPIIIa autoantigens have also been found to be the most frequent targets of ITP antibodies. Not all anti-GPIIIa antibodies are directed against the same epitope on GPIIIa. A subset of anti-GPIIIa antibodies found in patients with an acquired qualitative platelet dysfunction actually interfere with fibrinogen binding to normal platelets. Antibodies directed against targets on GPV have been found in patients with acute ITP of childhood. In patients with ITP associated with lupus erythematosus, antibodies which bind to intracellular proteins of apparent molecular weights of 66 and 108 kDa have been detected. Thus, ITP antibodies can have a variety of target antigens. Study of larger series of patients will determine whether identification of platelet autoantigens correlates with clinical course of ITP.