深圳地区无偿献血者群体CD36抗原缺失型的表型分析

目的分析深圳地区无偿献血者群体CD36抗原缺失型的筛查和表型情况。方法应用单克隆抗体和流式细胞技术在327名献血者中检测CD36抗原缺失型个体,应用血小板单克隆抗体捕获酶联免疫技术和流式细胞术筛查献血者血浆中的血小板抗体。结果此次筛查得到的深圳献血者的CD36抗原缺失型频率为3.06%(10/327),其中Ⅰ型占0.31%(1/327),Ⅱ型占2.75%(9/327);在3名女性CD36抗原缺失型献血者的血浆中,均未检出血小板反应性抗体,包括抗-CD36。结论深圳地区献血者群体中存在CD36抗原缺失型个体,其频率与亚洲其他地区人群的频率相当;对CD36的同簇免疫应该予以重视。     

苏州地区献血者血小板CD36抗原表型筛查及基因突变分析

目的 了解苏州地区血小板CD36抗原表达情况及其抗原缺失频率和基因多态性特征,为建立血小板CD36阴性表型资料库提供参考依据.方法 随机选取805例苏州市中心血站无偿献血者抗凝全血,流式细胞术筛查血小板和单核细胞表面CD36抗原的表达,分析抗原缺失类型,并结合基因组DNA测序,检测导致CD36抗原缺失的基因突变类型.结...     

南宁地区血小板输注无效患者血小板同种抗体检测及特异性分析

目的分析血小板输注无效(PTR)患者血小板同种抗体的特异性,以便更好的为临床服务。方法检测并鉴定72例PTR患者血小板同种抗体的特异性;采用流式细胞术(FCM)对其中4例患者进行血小板、单核细胞膜上的CD36抗原检测;基因测序法对上述4例患者进行CD36抗原缺失分型。结果 72例PTR患者中有22例产生血小板同种抗体,其中18例为抗HLA抗体,3例为抗CD36抗体,1例为抗CD36及抗HLA抗体并存;4例含抗CD36抗体的患者经证实均为Ⅰ型CD36缺失个体,基因检测也证实为CD36基因突变所致的CD36抗原缺失。结论 PTR患者血小板同种抗体以抗HLA多见,其次为抗CD36;应对CD36抗原给予足够重视。     

血小板CD36抗原Ⅱ型缺失与内含子序列多态性的相关性

目的 研究分析血小板CD36抗原Ⅱ型缺失与内含子序列多态性的相关性。方法 随机抽取辽宁省血液中心健康血小板捐献者516名,其中241例分别进行CD36抗原检测和CD36基因序列分析;剩余275例只进行CD36基因序列分析。CD36抗原检测采用流式细胞术法,基因序列分析采用PCR-SBT法。结果 在241例标本中检测到Ⅰ型缺失1例和Ⅱ型缺失4例,频率分别为0.41%和1.66%。3例Ⅱ型缺失者编码区无突变。所有Ⅱ型缺失个体在内含子3区域具有共同的多态性特征,即同时携带(TG)11和12个串联突变,且两者位于同一等位基因。(TG)11在516例随机人群中的基因频率仅为11.72%,远低于(TG)13的30.43%。12个串联突变在516例随机人群中的基因频率为8.81%。96.7%的12个串联突变都与(TG)11同时出现,但只有72.7%的(TG)11携带12个串联突变。流式细胞术检测发现,只携带(TG)11的标本,其血小板CD36抗原表达水平与正常标本相当,而93.1%的同时携带(TG)11和12个串联突变的标本血小板CD36抗原水平明显降低。结论 内含子3区域内的(TG)11不是血小板...     

广州地区伊朗献血人群CD36表型筛查及基因型鉴定

目的了解广州地区伊朗籍献血者CD36表型及基因型。方法对2016年在广州血液中心捐献机采血小板的53例伊朗人献血者标本,应用流式细胞仪分别检测血小板和单核细胞上CD36抗原的表达。对CD36抗原表达异常者提取DNA,采用PCR-SBT测序做CD36的基因型鉴定并做基因突变分析。结果本组伊朗人群的CD36抗原表达异常率7.55%(4/53),其中2例的CD36抗原在血小板上表达含量低,1例为血小板和单核细胞上均无表达(即CD36Ⅰ型缺失),1例为单核细胞上CD36表达量低,由此得出CD36缺失表达的频率为1.89%(1/53)。PCRSBT法分析:CD36抗原表达异常4例的CD36基因型中,血小板上低表达CD36抗原的2例发生基因突变,分别是exon 10-975T/G和exon11 del CTA,且均为新的基因突变;2例CD36基因型正常。结论本组伊朗献血人群里CD36抗原表达异常者中存在新的基因突变,其对CD36抗原表达的影响尚需进一步认证。     

合肥地区无偿献血人群CD36抗原缺失的调查

目的 研究合肥地区无偿献血人群CD36抗原的缺失频率.方法 随机留取合肥市中心血站2020年3月～2020年7月血小板无偿捐献者血样973例,流式细胞术检测CD36抗原.EDTA抗凝全血离心制备富血小板血浆,进行血小板CD36抗原检测,下层细胞经红细胞裂解液处理后,进行单核细胞CD36抗原检测.结果 973例血小板无偿...     

孕妇CD36抗原表达筛查及基因测序分析

目的了解孕妇CD36抗原的表达及其基因型。方法对在本中心做抗体筛查及血小板抗体检测的200名孕妇,采用流式细胞术检测其血小板及单核细胞上的CD36表达,并对其中血小板或单核细胞上CD36抗原缺失者做基因测序分析。结果本组孕妇CD36缺失表达率为1.5%(3/200),3例CD36缺失均为Ⅱ型缺失(只有血小板上CD36缺失),未见CD36Ⅰ型缺失(血小板和单核细胞上CD36都缺失)。3例CD36缺失表达者基因测序分析:2例发生CD36基因突变,其突变位点分别是Exon12 A1163T和Exon5 329-330del AC,为常见的基因突变类型;1例为正常CD36基因型。结论孕妇CD36表达缺失率与中国普通人群相似,缺失者CD36基因发生了基因突变。     

血小板配合性输注的分析和展望

血小板配合性输注可有效解决免疫性血小板输注无效(PTR)并节约血小板资源、提升血液安全性。本文对血小板配合性输注中的HLA、HPA和CD36配合性方式,HLA抗体效价和抗原免疫原性以及血小板配合性输注发展作了述评和展望。HLA配合性方式涉及等位基因、抗原、表位水平的配合以及规避供者特异性抗体(DSA)对应抗原的策略,需要探索的是设定规避DSA强度(MFI)的阈值。PTR患者可检出HLA等位基因特异性抗体,因此患者和献血者HLA-A、-B位点宜做高分辨水平的基因分型,以规避相应特异性的抗-HLA。HLA-A、-B位点不同抗原或表位的免疫原性存在差异,选择抗原低表达或免疫原性低的献血者血小板或成为1种相容性配合方式。不同人群抗-HPA产生的概率和种类不同,HPA配合性方式有等位基因的配合和规避抗体对应抗原的配合。CD36配合性方式为规避抗体对应抗原的配合,原则上优先选择CD36抗原Ⅰ型缺失的献血者,次选CD36抗原Ⅱ型缺失的献血者。今后在血小板配合性输注中,应关注供者血小板基因库的库容量提升和信息化建设;在库存和待检血小板检索和配合,将会明显缩短血小板配合性输注所需时间。     

抗-CD36患者造血干细胞移植前后血小板输注1例

目的对1例疑为CD36抗体引起的血小板输注无效患者进行CD36抗体鉴定、CD36缺失表型及CD36基因突变类型分析,借助血小板稀有血型库为该患者寻找相匹配的血小板输注,确保移植成功。方法采用商品化的ELISA试剂盒进行血小板抗体检测;对患者血小板和单核细胞上CD36的表达进行流式分析;运用PCR-SBT方法分析患者CD36基因突变类型;利用查询功能在血小板稀有血型库中查询与患者ABO血型相符且CD36缺失表达的供者血小板给予配型输注。结果血小板抗体检测显示该患者呈CD36抗体阳性(移植前CD36+4.2%,移植后CD36+22.1%),且血小板和单核细胞均缺乏表达CD36抗原(Ⅰ型CD36缺失);基因测序分析发现该患者存在纯合A1163T基因突变。在造血干细胞移植前后,给予CD36-/-血小板配型输注可明显提高患者的血小板数量,从而确保移植成功。结论由CD36抗体引起的血小板输注无效患者(尤其是需要进行造血干细胞移植的患者)应给予CD36-/-血小板输注以确保血小板输注疗效。     

深圳地区血小板捐献者CD36抗原缺失型的基因多态性研究

目的:分析58名CD36抗原缺失型血小板捐献者的CD36基因分子变异情况。方法:选取2019年9月至2020年12月本实验室筛查到的58名CD36抗原缺失型献血者,并将其作为研究组,其中男性39名,女性19名,均为汉族。另随机挑选2020年12月深圳市血液中心血小板无偿献血者120名,其中男性76名,女性44名,均为汉族,经流式细胞术检测CD36抗原阳性者作为对照组。采用PCR-SBT检测所有样本CD36基因的编码区,分析献血者CD36抗原缺失型的基因突变情况,并与抗原阳性对照者的基因突变类型进行比较。结果:58名CD36抗原缺失型献血者中,有32名检测出CD36基因突变,Ⅰ型71.43%(5/7),Ⅱ型52.94%(27/51)。120名CD36抗原阳性对照者中有12名检测出CD36基因突变,突变个体比例为10%。在CD36抗原缺失型献血者中发现16种基因突变,发生突变频率最高的为329-330 del AC(20.69%),其次为1228-1239 del ATTGTGCCTATT(15.52%)和1156 C>T(10.34%)。198-205 del GATCTTTG和2...     

Posttransfusion purpura-like syndrome associated with CD36 (Naka) isoimmunization

BACKGROUND: CD36 deficiency, which could lead to CD36 isoimmunization, has been reported in the Japanese population. CD36 isoantibody has been involved in platelet transfusion refractoriness. CASE REPORT: A 50-year- old woman originally from Corsica developed severe acute thrombocytopenia after massive transfusion. She was found to be CD36 deficient, and platelet immunoassays revealed a CD36 (Naka) platelet isoantibody. Although the involvement of another mechanism could not be entirely ruled out, the thrombocytopenia was attributed to posttransfusion purpura-like syndrome. The antibody was also involved in platelet transfusion refractoriness. CD36 deficiency was present in two members of the patient's family as well. Flow cytometry studies demonstrated the absence of CD36 expression on the surface of blood monocytes and cultured erythroblasts and megakaryocytes from one of the two CD36-deficient family members studied, but, in the absence of previous immunization, these CD36-deficient patients were not isoimmunized. In contrast, CD36 deficiency was not found in a population of 808 healthy blood donors in the Paris, France, area. CONCLUSION: CD36 isoantibody might be involved in some cases of posttransfusion purpura and platelet transfusion refractoriness. These findings also confirm the extremely low frequency of CD36 deficiency among whites.     

鼠抗人血小板CD36分子单克隆抗体的制备及活性分析

本研究旨在制备具有功能活性的重组人血小板表面CD36抗原的鼠抗人单克隆抗体。提取人肝细胞总RNA,经RT-PCR扩增编码人血小板CD36抗原胞外区（Gly30-Asn439）氨基酸残基cDNA,构建于原核表达载体pMDl8并转化大肠杆菌DH5α,筛选获得阳性重组子pMD18-CD36,提取质粒。经序列测定后,将该基因插入到真核细胞瞬时表达载体pTE2上,构建成为pTE2-s-CD36-10His真核瞬时表达载体。采用lipofectamine2000转染法,将重组质粒转染至HEK293细胞,表达产物经Ni^2＋2NTA柱层析纯化。以制备的重组CD36蛋白免疫BALB／c小鼠后,取脾与小鼠骨髓瘤细胞融合,筛选出阳性克隆,行Western blot检测抗体结合活性。结果显示：RT-PCR扩增获得了1．4kb的片段。经测序,该序列分析结果与GenBank中的NM_001001547．2完全一致。SDS-PAGE证实转染的HEK293细胞表达了人CD36抗原胞外区蛋白片段。鼠单克隆抗体在Westernblot中可以识别重组CD36蛋白,灵敏度达到8ng。结论：成功制备了抗人血小板CD36单克隆抗体,为临床筛选CD36阴性患者及献血员、深入研究人血小板CD36表面抗原对血小板输注无效的影响提供了实验基础。     

血小板抗体检测及配型对血小板输注无效的临床意义

目的 通过对比血小板配型前后血小板的输注效果,评估血小板抗体检测及配型对血小板输注无效的临床意义.方法 以出血症状改善情况、血小板计数增高指数(CCI)、血小板恢复百分率(PPR)为标准,对比配型前后血小板的输注效果.结果 25例血小板输注无效患者的血小板抗体筛查阳性9例; 9例血小板抗体阳性患者血小板交叉配型前后血小板输注有效率差异有统计学意义(P<0.01),配型后输注的 1 h和24 h CCI、PPR数值明显高于配型前输注的.结论 血小板抗体检测及血小板配型输注可以为患者选择适用的血小板,提高单采血小板的输注有效率,避免滥用血小板.     

CD36 deficiency is frequent and can cause platelet immunization in Africans

BACKGROUND: CD36 is expressed on several cell lineages. About 5 to 10 percent of Asians lack platelet membrane CD36 (pCD36), but the frequency of pCD36 deficiency in other ethnic groups is not known. Persons who are pCD36-negative are apparently healthy but can develop CD36 isoimmunization. STUDY DESIGN AND METHODS: The pCD36 phenotype was studied in 1885 subjects belonging either to a group of 1127 healthy French blood donors (almost all of whom were white Europeans) or to a group of 758 patients of known ethnic origin. RESULTS: No pCD36-negative persons were found among the blood donors. Only 1 of the 301 white European patients was pCD36-negative. In contrast, 16 of the 206 sub-Saharan Africans was pCD36-negative, a proportion higher than that among that black Caribbeans (1/148, p<0.01). The frequency of pCD36-negative patients was similar in blacks with and without sickle cell disease. Monocyte CD36 (mCD36) expression was studied in 15 of 22 pCD36-negative individuals: it was <10 percent in 7 subjects (type I deficiency) and between 12 and 100 percent in 8 others (type II deficiency). Thirteen pCD36-negative individuals had risk factors for immunization, and 4 had anti-CD36. Some had a history resembling posttransfusion purpura (n = 2), platelet transfusion refractoriness (n = 1), and recurrent miscarriage (n = 1). No correlation was found between immunization and the amount of mCD36. Anti-CD36 from an immunized type II-deficient woman reacted with monocytes from normal controls but not with monocytes from type I- or type II-deficient individuals, and thus it is postulated that mCD36 could be structurally different in normal and type II CD36-deficient individuals. CONCLUSION: CD36 deficiency is frequent in sub-Saharan Africans; development of anti-CD36 can lead to serious complications in multiply transfused patients, such as those with sicke cell disease.     

中山地区血小板供者HLA基因分型资料库的建立

目的 建立血小板供者HLA基因分型资料库,探讨HLA-A、B基因频率及单体型频率,为临床患者提供HLA匹配的血小板,解决血小板输注无效(PTR)患者的血小板输注.方法 采用聚合酶链反应-序列特异寡核苷酸探针杂交技术 (PCR-SSOP)对568名中山地区单采血小板捐献者进行HLA-I类(A、B位点)基因分型并形成数据库,根据HLA表型群体资料,使用最大数学预期值算法(EM)计算HLA-A、B单体型频率.结果 HLA-A位点共检出17种等位基因,HLA-B位点共检出29种等位基因,单体型数153种.结论 血小板供者HLA分型资料库的建立可以为PTR患者迅速寻找到HLA相合血小板,提高血小板输注效果.     

Incidence of the Nak(a)-negative platelet phenotype in African Americans is similar to that of Asians

BACKGROUND: About 5 to 10 percent of Asians have platelets that lack the major membrane glycoprotein (GP) IV (CD36, GPIIIb) that carries the isoantigen Naka. The GPIV-negative platelet phenotype is extremely rare among whites, but its frequency in persons of African ancestry has not yet been determined. Isoimmunization against GPIV can occur in GPIV- negative persons and can lead to platelet transfusion refractoriness. Therefore, the expression of GPIV on platelets from unrelated African Americans was studied. STUDY DESIGN AND METHODS: Platelets were obtained from 250 African American and 280 white blood donors. Flow cytometry was used to determine the ability of these platelets to bind a monoclonal antibody that reacted with GPIV. Platelets that failed to react with this probe were tested with other GPIV-specific monoclonal antibodies and with anti-Naka, an isoantibody that recognizes an epitope on GPIV. RESULTS: Platelets from 6 of the 250 African American donors (2.4%) lacked GPIV and failed to bind anti-Naka, whereas platelets from all of the white donors were GPIV positive (p>0.05). No platelet-reactive antibodies were identified in the serum of the GPIV- negative donors. CONCLUSION: The frequency of the GPIV-negative platelet phenotype in African Americans is comparable to that in Asians and much greater than that in whites. Studies are needed to determine the frequency with which African Americans become isoimmunized to GPIV by transfusions and the possible contribution of this isoimmunization to platelet transfusion refractoriness in this population.     

Establishment of platelet donor registry improves the treatment of platelet transfusion refractoriness in Guangzhou region of China

Platelet transfusion refractoriness (PTR) is the major complication of long‐term platelet supportive care. To improve the effectiveness of platelet transfusion therapy in PTR patients, we aimed to establish a platelet donor registry in our region (Guangzhou, China) by typing the human leukocyte antigen (HLA) and human platelet antigen (HPA). Blood donors (n = 864) from our population were genotyped for HLA‐A, HLA‐B and HPA systems by polymerase chain reaction amplification with sequence‐specific primer(PCR‐SSP) techniques. Using this cohort, we compared the results of platelet transfusions (matched vs. random) in 23 patients with PTR. Matched platelets were selected either by HLA antigen matching or by HLA antibody matching, as predicted by antibody specificity prediction (ASP) analysis. Significantly higher platelet recovery (PPR) values were obtained with HLA‐matched platelets in comparison with random platelets. No significant difference in PPR was observed between HLA matching and ASP methods. In two patients, platelet‐specific alloantibodies (alloabs) (anti‐HPA‐3b and anti‐HPA‐5b) were detected besides HLA class I alloabs. Transfusion with HLA‐ and HPA‐compatible platelets in both the patients resulted in significantly higher PPR when compared with HLA‐compatible platelet transfusion alone. In this study, we demonstrated that the establishment of an HLA‐ and HPA‐typed platelet aphaeresis donor registry is useful to improve the treatment outcome of PTR patients and to maintain a long‐term platelet transfusion strategy.     

改良血小板添加液低温液态保存血小板的形态及功能研究

本研究探讨使用改良的血小板添加液替代70％自体血浆在低温（10℃）液态条件下对血小板的保存效果。采集6名供者单采血小板,每一名供者单采血小板平均分为3组,A组（常规保存组）加入100％血浆、22℃保存：B组（添加液10℃保存组）加入70％PAS—ⅢM／30％血浆、10℃保存;C组（冰冻保存组）加入100％血浆和海藻糖、-85℃保存;另设D组（血浆4℃保存组）加入100％血浆、4℃保存,作为扫描电子显微镜对照组。A、B组在第1、5、7、9天取样,C组在20天后复苏取样,分别检测CD62p、HSR、PAgT、LDH的变化。血浆常温组、添加液低温组于保存后第3、9天取样扫描电镜观察,血浆低温组于保存后第3天取样观察．冰冻组在20天后复苏取样观察,同时使用新鲜血小板作为对照。结果表明,保存期内随保存时间延长,A组和B组CD62p表达增加,HSR和最大聚集率降低;A组的LDH释放、CD62p表达、HSR、血小板最大聚集率与B组比较有显著统计学差异（P〈0．05）。C组LDH释放明显较其他两组增多,但CD62p表达较少（P〈0．05）。A、B组血小板形态保持较好,C组血小板形态保持差。结论：改良的PAS—ⅢM保存液能够替代血浆用于血小板的保存,在低温条件下能够很好地保护血小板的功能,保存效果优于血浆常温保存。