Transfusion-associated graft-versus-host disease in a presumably immunocompetent patient after transfusion of stored packed red cells

A 54-year-old woman developed transfusion-associated graft-versus-host disease (TA-GVHD) after the transfusion of stored packed red cells obtained from unrelated donors. The patient was presumed to be immunocompetent. A diagnosis of TA-GVHD was made by clinical features and postmortem pathologic findings. Sex chromatin analysis of the patient's lymphocytes demonstrated chimerism. HLA typing of the blood donors revealed one to be HLA-homozygous for one of the patient's HLA haplotypes (A33-B44-Cblank). This case illustrates the risk in the general patient population of TA-GVHD after routine blood transfusion therapy. Workers should be aware of this possibility and should continue searching for an efficient way to prevent it.     

荧光标记复合扩增短串联重复序列基因分析骨髓移植物的植入

目的 为了准确地识别骨髓移植物的植入状态,探讨PCR扩增短串联重复序列（short tandem repeat,RCR－STR）在亲缘或无关供者骨髓移植的预后及白血病复发中的预警作用。方法 建立荧光标记PCR－STR等位基因分析技术,采用单克隆磁珠提取DNA,四色荧光标记,于移植前、移植后7天－6个月不等,采集供、受者血液（2例回顾性研究患者刮取口腔粘膜脱落细胞作为术前样本）DNA作PCR－STR分析。结果 ①12例患者骨髓移植后10例为完全供者型,其中同胞损髓8例,HLA全相合77例,半相合1例;无关供者损髓1例,HLA全相合,母亲供髓1例,HLA-Ⅰ全相合,HLA－Ⅱ半相合。10例受者术后出现Ⅰ度移植物抗宿主病（GVHD）,STR均完全和持续表达供者基因型,追求观察3－25个月,预后良好。②12例中1例由嵌合型转变为完全供者型,系同胞损髓,HLA－Ⅰ半相合,HLA－Ⅱ全相合;术后第30天PCR－STR表达供、受者双方基因型,第60天完全转变为供者基因型,而自身的基因在外周血中消失,预后良好。③12例中1例为持续未植入,HLA半相合同胞捐髓,术后虽然有血液学和临床情况的改善,但移植后7,14,21天STR分析始终仅显示受者基因型,移植后4个月死亡。结论 基于分子水平的移位点PCR－STR分析是骨髓移植后供者植入的精确标志。研究表明,PCR－STR植入分析的准确性优于任何传统技术,对移植效果有预警作用。     

A rapid molecular diagnosis of posttransfusion graft-versus-host disease by polymerase chain reaction

A woman with recurrent Paget's disease of the vulva developed acute graft-versus-host disease (GVHD) 12 days after radical surgery and massive blood transfusion. Molecular diagnosis of lymphocyte chimerism in the peripheral blood was made by polymerase chain reaction (PCR) directed against a Y chromosome-specific sex-determining region Y (SRY) gene. PCR with skin biopsy after onset of GVHD also revealed infiltration of SRY-positive donor lymphocytes. The diagnosis was confirmed by HLA-DNA typing with PCR-sequence-specific oligonucleotide that revealed the presence of complex HLA-DR chimerism in the peripheral lymphocytes collected after onset of GVHD. The use of SRY- directed PCR is a rapid technique for the early diagnosis of acute posttransfusion GVHD in female patients.     

Early diagnosis and successful treatment of a patient with transfusion-associated GVHD with autologous peripheral blood progenitor cell transplantation

BACKGROUND: Transfusion-associated GVHD (TA-GVHD) is an uncommon complication of blood transfusion. Diagnosis of TA-GVHD is difficult, and it is usually rapidly fatal. There are few documented sur- vivors of TA-GVHD. CASE REPORT: A 61-year-old woman with chronic lymphocytic leukemia (CLL) was treated with fludarabine followed by combination chemotherapy and high-dose radioimmunotherapy and peripheral blood progenitor cell (PBPC) rescue. She was transfused with nonirradiated blood components at an outside hospital and presented 10 days later with rash, elevated liver enzymes, and progressive pancytopenia. Skin biopsy was consistent with GVHD, and HLA typing of lymphocytes from the patient demonstrated mixed chimerism. The patient was treated with solumedrol and cyclosporin A, followed by high-dose cyclophosphamide and antithymocyte globulin and autologous PBPC infusion. She had rapid engraftment, resolution of skin rash, and normalization of liver function abnormalities. She is in good health with normal blood counts and no evidence of CLL 34 months after transplantation. CONCLUSION: TA-GVHD occurs in the setting of an immunocompromised recipient receiving nonirradiated blood components. A typical presentation includes skin rash, liver function abnormalities, and pancytopenia. Demonstration of mixed chimerism by HLA typing facilitated diagnosis in this patient. High-dose immunosuppression, facilitated by the availability of autologous PBPCs, resulted in a successful outcome.     

Transfusion-associated graft-versus-host disease in immunocompetent patients: report of a fatal case associated with transfusion of blood from a second-degree relative, and a survey of predisposing factors

A patient without evident immune deficiency who received a transfusion of blood from a second-degree family member developed fatal transfusion- associated graft-versus-host disease (TA-GVHD). The donor was homozygous for an HLA haplotype for which the recipient was heterozygous (one-way HLA match). All 39 reported cases of TA-GVHD in immunocompetent patients were reviewed to ascertain the predisposing factors and to define the indications for irradiating blood for this population. HLA typing was described in 15 cases; in 13, including seven related and six unrelated donors, a one-way HLA match was present. Thirty-one (79%) of the 39 cases were reported from Japan (and 196 other cases are cited in the Japanese literature), but a one-way HLA match among unrelated donors at HLA-A, -B, -DR loci is only approximately two to four times more likely in Japanese persons than in whites. Fresh blood (< 96 hours old) was used in 29 (94%) of the 31 cases reported from Japan and in 33 (87%) of 38 cases overall (in one case, the age of the blood used was not reported). Thus, factors that appear to predispose to TA-GVHD in immunocompetent patients are a one- way HLA match, fresh blood, and, possibly, Japanese ancestry. Irradiating cellular blood components from all blood relatives of transfusion recipients will not completely eliminate the risk of TA- GVHD.     

Potential for transfusion-associated graft-versus-host disease due to apheresis platelets matched for HLA class I antigens

Transfusion-associated graft-versus-host disease (TA-GVHD) has been reported in immunocompetent recipients of nonirradiated cellular blood components from donors who are homozygous for an HLA haplotype shared with the patient. In these cases, donor lymphocytes have no antigens foreign to the recipient, and this similarity in HLA antigens appears important for the development of TA-GVHD. Experience with 65 patients receiving apheresis platelets matched for class I HLA antigens was reviewed to determine the incidence of such a transfusion among HLA- matched, unrelated donor-recipient pairs. In 5 percent of transfusions (31/673), the patient received lymphocytes from a donor exhibiting no antigens foreign to the recipient, but the patient had additional HLA-A or -B antigens not present on donor lymphocytes. Twenty-three percent (n = 15) of patients received at least one such transfusion. In addition, most patients were immunosuppressed as a result of their underlying disease or therapy, which may decrease the degree of antigen matching required to initiate TA-GVHD. Until the pathogenesis of this disease is better understood, it is recommended that the transfusion of an HLA-matched cellular blood component be considered a risk factor for the development of TA-GVHD regardless of the patient's immune status, and that all such blood components be irradiated.     

The critical role of blood from HLA-homozygous donors in fatal transfusion-associated graft-versus-host disease in immunocompetent patients

Fatal transfusion-associated graft-versus-host disease developed in a 69-year-old woman with colon cancer who underwent elective hemicolectomy. During the perioperative period, she was transfused with 4 units of nonirradiated fresh whole blood less than 6 hours after the blood was donated by family members. She was immunocompetent and was not treated with any immunosuppressive agents such as corticosteroids, chemotherapy, or irradiation therapy. The implicated donor was thought to be her daughter, who was homozygous for an HLA haplotype that was shared with the recipient: A24, Bw52, CBL, DR2. This is the most common haplotype in the Japanese population. This case and others in the Japanese literature indicate that the transfusion of fresh, nonirradiated blood that contains immunocompetent lymphocytes and peripheral hematopoietic precursor cells from HLA-homozygous donors can be lethal to the recipient.     

异基因脐血干细胞移植后STR基因座位的表达分析

本研究分析杜氏型肌营养不良（DMD）病人异基因脐带血干细胞移植后STR基因座位的表达。利用PCR—SSO方法检测患者、供者HLA-A、B、DR位点，并利用STR—PCR方法检测术后各个器官的STR-基因位点的表达情况。结果表明：患者与供者在HLA中、低分辨情况下A、B、DR位点全相合，患者的胸骨骨髓显示为供者独立植入，脾、左上肺、前臂、肌舌、左肝、胃、右颞叶、膈肌、右支气管、左心室、右肾均为嵌合状态。结论：造血干细胞移植术后，供者的基因可在实体器官表达，形成嵌合状态.     

Dispermic chimerism identified during HLA typing for stem cell transplantation

BACKGROUND: Chimerism is defined as the presence of two genetically distinct cell populations in an organism. Few cases of phenotypically normal dispermic chimeras have been reported and most showed abnormalities on blood typing. CASE REPORT: A 32-year-old man was diagnosed with acute myelomonocytic leukemia. He clearly typed as group A, D-. No abnormalities of sexual development were identified on multiple physical exams, previous exploratory surgery, or CT scans. Molecular HLA typing (sequence-specific primers) in preparation for stem cell transplant showed the patient to have three HLA-B* and three HLA-Cw* alleles. Initial serologic HLA typing reported two haplotypes, but on subsequent review reactions for a third HLA-B antigen that were initially deemed to be false-positive reactions were identified. Two of 10 microsatellite short tandem repeat (STR) loci also showed three distinct alleles in blood and buccal samples. In all studies the third allele was attributable to a dual paternal contribution. CONCLUSION: This case represents dispermic chimerism, with one maternal and two paternal haplotypes variably distributed throughout body tissues in a phenotypically normal man without abnormalities in blood typing. The presence of additional alleles that may have been undetected or dismissed by serologic typing should be carefully investigated and verified by molecular techniques. Molecular HLA typing may increase the accurate identification of phenotypically normal chimeras and aid in selecting proper donors for transplantation to reduce graft-versus-host disease and transplant rejection in these patients.     

Major histocompatibility restriction of antigen recognition by T cells in a recipient of haplotype mismatched human bone marrow transplantation.

Immune T cells proliferate in response to antigen that is recognized in association with self-Ia determinants. T cells from a patient with severe combined immunodeficiency that has been successfully reconstituted with haplotype-mismatched, maternal bone marrow were studied in an attempt to understand the development of Ia restriction of antigen recognition in man. All the patient's T cells were of maternal origin as determined by HLA typing. The patient received a series of three immunizations with tetanus toxoid (TT) antigen between the 6th and 14th week posttransplant. TT-specific T cell lines were established from the patient's peripheral blood at 6 and 8 mo posttransplantation and were maintained in culture in the presence of irradiated monocytes from the patient, TT antigen, and interleukin-2. HLA typing of the two T cell lines revealed them to be exclusively of donor origin. Both T cell lines could proliferate to TT in the presence of monocytes derived from either the patient's mother or father. In contrast, a TT-specific T cell line obtained from the patient's mother proliferated to TT in the presence of autologous monocytes, but not in the presence of monocytes derived from the patient's father. Studies using monocytes from a panel of HLA-typed donors indicated that the patient's T cell lines proliferated to TT in the presence of monocytes that expressed the paternal DR antigen (HLA-DR4) inherited by the patient but not in the presence of monocytes that expressed the paternal DR antigen (HLA-DR1) not inherited by the patient or in the presence of monocytes bearing irrelevant DR antigens. Monocytes that expressed either one of the two maternal DR antigens (HLA-DR3 and DR5) could support the proliferation of the patient's T cell lines in response to TT antigen. HLA typing of the patient's monocytes at 6 mo post-transplant revealed only recipient HLA-DR antigens (HLA-DR3 and DR4). At 12 mo posttransplant, the patient's monocytes expressed recipient HLA-DR antigens as well as the non-shared HLA-DR5 antigen of donor origin. The results of the present study indicate that T cells of human bone marrow chimera recognized antigen in the context of Ia determinants of recipient origin. The apparent recognition of antigen by the chimera's T cells in the context of donor Ia determinants that were not shared with the recipient is discussed.     

成人移植双份脐血植入状态的定量监测

背景:脐血因所含的有核细胞数量有限,主要应用于儿童患者,近年来,人们尝试将两份脐血混合输入,用于成人血液系统疾病的治疗。目的:定量监测双份异基因脐血用于成人白血病患者移植后两份脐血的植入状态、嵌合体类型、相对数量的动态变化及演变规律。设计:以脐血干细胞移植的供受者为观察对象,供受者移植前及受者移植后不同时间段的系列血样提取DNA作为检测标本,短串联重复序列遗传位点为观察指标。单位:深圳市血液中心输血医学研究所免疫遗传重点实验室。对象:纳入2005-06在北京大学深圳医院住院治疗的急性髓细胞白血病患者,男性,43岁,体质量75kg。首次化疗完全缓解后6个月移植两份人类白细胞抗原(HLA)各一个位点不合的非血缘脐血,脐血1有核细胞数为2.5×107kg-1;脐血2有核细胞数为1.53×107kg-1。脐血来源于广州脐血库。患者对治疗方案知情同意。方法:采用荧光标记复合扩增短串联重复位点嵌合体定量检测技术,对急性髓细胞白血病成人患者移植两份(脐血1有核细胞数为2.5×107kg-1,脐血2有核细胞数为1.53×107kg-1)HLA各一个位点不相合的异基因脐血前后的序列血样进行了9个短串联重复序列位点的检测,利用供、受者之间的差异位点定性判断脐血是否植入以及嵌合体类型;而后根据377XLDNA测序仪上荧光扫描后两供者差异基因检出峰的峰面积计算脐血植入后患者体内两份脐血的相对数量,定量分析供体细胞植入程度及演变规律。并与采用HLA差异基因对植入状态的分析结果进行对比。主要观察指标:在成人移植双份脐血后,观察患者及两供者9个位点的短串联重复序列基因在患者体内的转变过程,对植入状态进行定量及定性的描述。结果:移植后15d两份脐血植入状态为完全双份供者嵌合体,患者体内脐血1的相对细胞数量占51.3%,脐血2占48.7%;30d时脐血1上升为70.0%,脐血2下降为30.0%。52d时只检测到脐血1的基因,植入状态转为完全单份供者嵌合体,有核细胞数少的一份脐血被排斥,有核细胞数多的一份长期植入。结论:荧光标记复合扩增短串联重复序列嵌合体定量检测可精确地描述两份脐血的植入程度及变化过程,为临床脐血的应用及供者的选择提供了一个准确、可靠的实验依据,证明双份HLA各一个位点不合的脐血同时用于成人的移植是可行的。     

FISH Diagnosis of Acute Graft-Versus-Host Disease Following Living-Related Liver Transplant

Acute graft-versus-host disease (GVHD) is an uncommon but often fatal complication following liver transplant. We describe a GVHD case in which a female patient with primary biliary cirrhosis underwent a living-related liver transplant from her son. The human leukocyte antigen typing of the donor was homozygous at all loci. The recipient''s human leukocyte antigen type was haplo-identical to that of the donor. A bone marrow aspirate performed for pancytopenia revealed a severely hypoplastic marrow. Fluorescent in situ hybridization (FISH) using X- and Y-chromosome probes demonstrated that 80% of marrow cells were of donor origin. Comparison of Giemsa-stained cell morphology and FISH showed that the erythroid precursor cells were predominantly of male pattern (XY). This report is one of only a few studies that prove the migration of a donor''s hematopoietic stem cells to a recipient''s bone marrow. We demonstrated that FISH analysis using sex chromosome probes is useful to confirm a diagnosis of GVHD following organ transplantation from a donor of the opposite sex. We also showed that donor hematopoietic stem cells in a liver graft can migrate to the recipient''s bone marrow. We suggest that FISH is a rapid and reliable test for confirming the diagnosis of GVHD in a peripheral blood or skin biopsy sample.Acute graft-versus-host disease (GVHD) following liver transplantation is a rare complication with a high mortality rate.^1,2 GVHD occurs when immunocompetent donor lymphocytes originating from the transplanted organ undergo activation and clonal expansion, reacting against the recipient antigens. The clinical course begins with fever or skin rash as an early sign, followed by pancytopenia, overwhelming sepsis, and death.³The diagnosis of GVHD is a major challenge and thus the condition often goes unrecognized. Detection of donor lymphocytes in peripheral blood or bone marrow in high-level (>1%) is a key to early detection of GVHD.^1,3     

HLA gene amplification and hybridization analysis of polymorphism. HLA matching for bone marrow transplantation of a patient with HLA-deficient severe combined immunodeficiency syndrome

The treatment of choice for certain immunodeficiency syndromes and hematological disorders is bone marrow transplantation (BMT). The success of BMT is influenced by the degree of HLA compatibility between recipient and donor. However, aberrant expression of HLA sometimes makes it difficult, if not impossible, to determine the patient's HLA type by standard serological and cellular techniques. We describe here the application of new molecular biological techniques to perform high resolution HLA typing independent of HLA expression. A patient with HLA-deficient severe combined deficiency was HLA typed using in vitro amplification of the HLA genes and sequence-specific oligonucleotide probe hybridization (SSOPH). Two major advances provided by this technology are:detection of HLA polymorphism at the level of single amino acid differences; and elimination of a requirement for HLA expression. Although the patient's lymphocytes lacked class II HLA proteins, polymorphism associated with DR7,w53;DQw2;DRw11a (a split of DR5), w52b (a split of DRw52);DQw7 were identified. The patient's class I expression was partially defective, and typing was accomplished by a combination of serological (HLA-A and -C) and SSOPH analysis (HLA-B). Complete patient haplotypes were predicted after typing of family members [A2;B35(w6); Cw4; DRw11a(w52b);DQw7 and A2;B13(w4); Cw6;DR7(w53); DQw2]. Potential unrelated donors were typed and a donor was selected for BMT.     

混合脐血成人移植定量监测植入状态的实验研究

为了定量监测和研究双份异基因脐血用于成人白血病患者移植后两份脐血的植入状态、嵌合体类型、供者细胞相对数量的动态变化及演变规律 ,采用荧光标记复合扩增短串联重复 (STR)位点嵌合体定量检测技术 ,对 1例急性髓细胞白血病成人患者移植两份 (脐血 1有核细胞数为 2 .5× 10 7/kg ,脐血 2有核细胞数为 1.5 3× 10 7/kg)HLA各 1个位点不相合的异基因脐血前后的序列血样进行 9个STR位点的检测 ;利用供、受者之间的差异位点定性判断脐血是否植入以及嵌合体类型 ;而后根据 377XLDNA测序仪上荧光扫描后两供者差异基因检出峰的峰面积计算脐血植入后患者体内两份脐血的细胞相对数量 ,定量分析供体细胞植入程度及演变规律 ;并与采用HLA差异基因对植入状态的分析结果进行对比。结果表明 :移植后 15天两份脐血同时植入 ,植入状态为完全双份供者嵌合体 ,患者体内脐血 1的相对细胞数量占 5 1.3% ,脐血 2占 4 8.7% ;30天时脐血 1嵌合体细胞上升为 70 .0 % ,脐血2嵌合体细胞下降为 30 .0 %。 5 2天时只检测到脐血 1的基因 ,植入状态转为完全单份供者嵌合体 ,有核细胞数少的一份脐血被排斥 ,有核细胞数多的一份长期植入。结论 :荧光标记复合扩增STR嵌合体定量检测可精确地描述两份脐血的植入程度及变化过程 ,为临床脐     

深圳市健康无偿献血者外周血淋巴细胞表面人类白细胞抗原-E的基因型特异性表达研究

目的 探讨深圳市无偿献血个体的外周血淋巴细胞表面人类白细胞抗原(HLA)-E表达水平,并分析HLA-E基因型与HLA表达水平的相关性.方法 采用简单随机抽样法选择2015年8月至10月于深圳市血液中心无偿献血的82例献血者作为研究对象.采用Sepmate淋巴细胞分离管分离82例献血者的外周血淋巴细胞,然后采用流式细胞技术检测淋巴细胞表面HLA-E的表达水平;同时提取研究对象的外周血的DNA,采用PCR-直接测序法(SBT)进行序列测定,判定其HLA-E基因型;并且采用方差分析和t检验的统计学方法,分析比较不同HLA-E基因型献血者外周血淋巴细胞表面HLA-E表达水平的差异.结果 本研究82例献血者中,检出的HLA-E*01∶01/*01∶01基因型频率为20.7％(17/82),HLA-E* 01∶01/* 01∶03基因型频率为41.5％(34/82),HLA-E*01∶03/*01;03基因型频率为37.8％(31/82),3种基因型献血者的外周血淋巴细胞表面HLA-E相对表达水平分别为2.57±0.66、3.32±1.33和3.77±1.26,三者比较,差异有统计学意义(F=6.062,P＜0.05).其中,HLA-E* 01∶03/* 01;03基因型献血者外周血淋巴细胞表面HLA-E相对表达量最高,其次为HLA-E*01∶01/*01∶03基因型献血者,HLA-E*01∶01/* 01∶01基因型献血者外周血淋巴细胞表面HLA-E相对表达量最低,并且两两比较,差异均有统计学意义(HLA-E*01∶03/*01∶03基因型与HLA-E*01∶01/* 01∶03基因型比较,t=2.402,P＜0.05;HLA-E*01∶03/*01∶03基因型与HLA-E* 01;01/*01∶01基因型比较,t=4.212,P＜0.001;HLA-E*01∶01/* 01∶03基因型与HLA-E*01∶01/*01∶01基因型比较,t=3.054,P＜0.01).结论 深圳市健康献血人群外周血淋巴细胞表面HLA-E表达水平与其基因型密切相关.     

Cooperation between major histocompatibility complex mismatched mononuclear cells from a human chimera in the production of antigen-specific antibody.

Fetal liver and thymus transplantation can be successfully employed for the treatment of severe combined immunodeficiency disease. In virtually all cases, donor and recipient cells are HLA mismatched. In a patient suffering from a severe combined immunodeficiency disease, full immunological reconstitution was obtained after fetal liver and thymus transplantation. HLA typing revealed that the patient's T cells were of donor origin, while the B cells and monocytes were of host origin. Despite this complete HLA mismatch, the patient was found to mount a subnormal to normal antibody response in vivo. This finding is in contrast with the concept that antigen recognition by T cells is major histocompatibility complex (MHC) restricted. To define the mechanism responsible for this in vivo antibody response, antibody production by peripheral blood mononuclear cells from the patient was tested in vitro after in vivo booster. The in vitro anti-tetanus toxoid antibody production was similar to that of the control group. In addition, specific proliferative responses to tetanus toxoid were obtained. Immunoglobulin allotype determination showed that antibodies were synthetized by host B cells. The results of the present study indicate that transplanted T lymphocytes and recipient cells cooperate despite complete HLA mismatch.     

Transfusion‐associated graft‐versus‐host disease in a liver transplant recipient: an unusual presentation and review of the literature

BACKGROUND: Transfusion‐associated graft‐versus‐host disease (TA‐GVHD) is a rare, nearly universally fatal complication from transfusion of nonirradiated cellular blood components, occurring when a recipient's immune system is unable to recognize and destroy transfused T lymphocytes. Irradiation of cellular components eliminates this risk. We present an unusual case of a liver transplant recipient developing TA‐GVHD 13 weeks after transfusion of a random unit of nonirradiated red blood cells (RBCs) that happened to be from a donor homozygous for an HLA haplotype shared by the patient. STUDY DESIGN AND METHODS: This study was a single case review of a liver transplant recipient who developed skin GVHD and marrow aplasia. Clinical course and the chimerism studies involving the patient, the liver donor, and the blood donor are detailed. RESULTS: The patient presented 3 months posttransplant with GVHD of his skin and marrow aplasia. In addition to standard antigraft immunosuppression, this patient had started the interleukin‐1 receptor antagonist anakinra on Posttransplant Day 13 for an acute gout flare. Chimerism studies on the patient's peripheral blood identified a population of CD3 cells that did not originate with either the patient or his liver donor. HLA studies and microsatellite profiling of the unknown CD3 population identified the source of the patient's TA‐GVHD, a unit of nonirradiated, nonleukoreduced apheresis RBCs. CONCLUSION: Use of an immunomodulating agent may have contributed to the development of TA‐GVHD in a liver transplant patient who received a random unit of nonirradiated RBCs by chance from an unrelated haploidentical donor.     

Resolution of a serum sample mix-up through the use of short tandem repeat DNA typing

BACKGROUND: A sample mix-up occurred in a tissue procurement laboratory in which aliquots of serum from two tissue donors were accidentally mislabeled. The clues to the apparent mixup involved discrepant Hepatitis C test results. In an attempt to resolve the apparent mix up, DNA typing was performed using serum samples as a possible source of genomic DNA. STUDY DESIGN AND METHODS: Two hundred microliter aliquots of two reference sera and aliquots prepared from them were subjected to DNA extraction. PCR amplification of 9 STR loci was performed on the extracts and amplicons were analyzed by capillary electrophoresis. RESULTS: About 1 microg/ml of DNA was recovered from all serum samples and was of sufficient quality to direct the amplification of most, if not all STR loci allowing the mislabeled specimens to be traced to the proper tissue donor. CONCLUSIONS: Serum is a useful source of genomic DNA for STR analysis in situations in which such samples are the only source of DNA for testing. Interestingly, one of the tissue donors on life support and repeatedly receiving blood products, exhibited a mixed DNA profile indicative of the presence of DNA from multiple individuals in the bloodstream.     

FBC预处理方案在HLA半相合异基因外周血干细胞移植中的应用

目的　观察降低预处理强度对HLA半相合异基因干细胞植入的影响。方法　用氟达拉宾 (30mg m2 × 6d)、白消安 (4mg kg× 2d)、环磷酰胺 [(30～ 6 0 )mg kg× 2d]组成FBC方案。将 12例白血病患者分成两组 ,HLA完全相合移植组 4例 ,移植前患者处完全缓解状态 ;HLA半相合移植组 8例 ,皆为难治性白血病 ,其中 1例HLA 3个位点不合 ,6例二个位点不合 ,1例一个位点不合 ,接受本处理方案后 ,进行G CSF动员的异基因外周血干细胞移植 ,并于移植后第 30天 (+30天 )、+6 0天、+90天输注供者淋巴细胞 ,观察异基因干细胞植入和长期植入情况。结果　HLA半相合组患者平均接受 4.87× 10 8 kg供者外周血单个核细胞 ,其中CD3 4+细胞平均数为 4.5 8× 10 6 kg,HLA全相合组平均接受 4.85× 10 8 kg供者外周血单个核细胞 ,其中CD3 4+细胞 4.47× 10 6 kg。HLA半相合组 7例白细胞升至 1.0×10 9 L的平均时间为 2 9d(11～ 90d) ,血小板升至 2 0× 10 9 L的时间为 36d(14～ 96d) ,1例HLA 3个位点不合的患者移植失败 ,但该患者于 +5 0天恢复自体造血。而HLA全相合的 4例患者白细胞升至 1.0×10 9 L平均需 14d(11～ 18d) ,血小板升至 2 0× 10 9 L平均需 15d(11～ 18d)。经STR PCR检测 ,两组患者除 1例表现为混合嵌合体 ,其余均呈完     

异基因造血干细胞移植植活状态的变性高效液相色谱检测研究的首次报告

异基因造血干细胞移植后动态检测供者嵌合状态对判断移植效果和实施临床早期干预治疗具有重要意义。在临床上已有多种方法用于植活检测，本研究用变性高效液相色谱(DHPLC)技术结合聚合酶链反应扩增短串联重复序列(STR—PCR)检测造血干细胞移植植活状态的特点及可靠性。用非亲缘个体的系列DNA混合体在体外检测此测定方法的可行性和半定量结果的准确性。结果表明，体外模拟混合嵌合体检测实验，显示扩增前供者DNA嵌合百分率与扩增后供受者DHPLC色谱峰峰面积比值呈直线相关，最小检出DNA百分比为5％。用该方法分析51例移植对的结果显示：45例均至少有2个可以区分彼此的有信息住点(另外5例无移植前受者标本，1例为同卵双生双胞胎)；39例与荧光标记多重PCR扩增STR结合毛细管电泳方法进行对照，准确性为100％；29例与性染色体FISH分析，27例与特殊基因标记的分析结果一致；所有病例均检测到完全供者细胞嵌合状态(FDC)；对3例患者观察到临床复发的同时，检测到供者嵌合体的比例明显下降，其中2例为混合嵌合体，1例转变为受者自身型。在此基础上，我们将该方法首次用于对8例人类白细胞抗原(HLA)配型不同一单倍型亲缘供者干细胞及无血缘关系脐带血混合移植患者植活情况的动态检测。结果显示，8例混合移植患者STR—PCR检测结果均为FDC，来源均为亲缘供者。结论：用DHPLC技术结合短串联重复序列具有快速、经济、无污染和自动化高等特点，用于检测异基因造血干细胞移植物植活状态是可靠的。