重型地中海贫血造血干细胞移植后血细胞减少原因的临床研究

发布时间：2018-01-12 22:17

本文关键词：重型地中海贫血造血干细胞移植后血细胞减少原因的临床研究　出处：《南方医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：研究背景重型β地中海贫血(thalassemia major)是β-珠蛋白链合成减少或消失,导致a-珠蛋白链不平衡和无效红细胞被破坏产生溶血性贫血的一种遗传性疾病。虽然规律输血加去铁治疗已显著改善了患者预期寿命。然而与铁过载相关的并发症并不能通过去铁治疗治愈,同时一生输血很难遵守,唯一根治疗法依然是通过异基因造血干细胞移植替代缺陷红细胞生成。我中心使用独仓NF-08-TM新预处理方案包括加强预防急性移植物抗宿主病,通过克服异基因移植高排斥率,明显提高了无关供者外周血干细胞移植和HLA全相合同胞供者移植效果。然而移植后随诊过程中我们观察到少数病人出现血细胞减少,目前病因并不清楚。可能的病因包括预处理时使用骨髓毒性药物、病毒感染及与移植相关的一些因素,如供者年龄、HLA配型,供受者CMV状态,干细胞来源和慢性移植物抗宿主病。因此找出病因十分重要,有人认为可能与使用环胞素A导致自身免疫抑制有关,另有人认为可能与慢性移植物抗宿主病或其它病因有关。至目前,尚未有关于重型地中海贫血造血干细胞移植后血细胞减少的报道及其病因,因此,我们对我中心行造血干细胞移植重型地中海贫血187例患儿资料进行总结、分析。研究目的本课题旨在探讨重型地中海贫血患儿在异基因造血干细胞移植28天后发生血细胞少的原因,通过对与血细胞减少的相关危险因素进行分析,以便找出与血细胞减少相关的危险因素,以此为临床治疗提供指导。研究方法我们回顾性的分析2009年1月～2012年11月于我中心行造血干细胞移植重型β-地中海贫血患儿187例,其中男117例,女70例,年龄2.0～16岁,中位年龄6岁。中位随访时间19个月(范围1-44个月)。120例接受变更供者移植(alternative donor transplant ADT)(无关供者109例,父母供者11例),67例行同胞移植。其中发生血细胞减少的共39例,我们对39例发生血细胞减少病人与148例未发生血细胞减少病人进行比较、分析。根据地贫患儿年龄、肝脏大小和铁蛋白水平,将重型地中海贫血分为Ⅰ度、Ⅱ度和Ⅲ度。与血细胞减少的相关危险因素包括：供受者年龄、性别、血清中巨细胞病毒感染情况,HLA配型、ABO血型、干细胞来源、移植类型、急性移植物抗宿主病及慢性移植物抗宿主病。预处理方案、GVHD预防所有患儿均采用我中心NF-08-TM新预防处理方案,新方案主要由环磷酰胺CY(-10天～-9天),氟达拉滨Flu(-8天～-4天),静脉马利兰Bu(-8天～-6天),塞替哌TT(-5天)组成,其中马利兰的药物剂量根据患儿年龄制定。马利兰的稳态血液浓度维持在300-600ng/m1为宜,当小于300ng/ml或大于600ng/ml时亦进行药量调整,即总量再增加或减少1.2mg/kg,增加或减少的剂量在-6d分次给药。所有的病人在移植前-45天时开始服用硫唑嘌呤3mg/kg/d及羟基脲30mg/kg/d。GVHD的预防采用联合多种免疫抑制剂,包括环胞素A(Cs-A)、吗替麦考酚酯(MMF)及短疗程的甲氨喋呤,具体方法是静脉应用环胞素A(Cs-A)-10天～-2天、剂量为1.5mg/kg.d,在-1d天～+25天剂量为3mg/kg.d,之后口服给药,使血药浓度维持在200±50ng/ml。MMF在+1天到+30天序贯给药,剂量为15mg/kg.d、分两次口服,条件是移植后没有Ⅱ度以的GVHD(graft-versus-host disease), MTX是在移植后+1天、+3天、+6天静脉给药,剂量分别是10mg/m2、10mg/m2、10mg/m2。在变更供者移植组,所有病人均接受外周血干细胞移植,且大部分患儿接受的是无关供者。植入证据检测移植后常规于28天、第2月、第3月、第6月及移植后1年行植入证据检测,如果一个患儿植入不佳,可能随时进行植入证据检测。供受者间性别不同者以FISH检查染色体变化为依据,性别相同者进行HLA定量监测DNA短程串联重复序列多态性分析。植入成功的标准有间接指标和直接指标。(1)、间接指标：移植后临床存活时间超过21天；各造血系统恢复正常,包括红系、粒系、淋巴系、巨核系等；临床上发生GVHD。中大的肝脾回缩至正常大小；乳酸脱氢酶(LDH)降至正常水平。(2)、直接指标：红细胞抗原(ABO和Rh型转换成供者血型)、细胞遗传学标记(外周血或骨髓性染色体转变成供者型)、HLA抗原(在HLA配型不全相合的病例中,HLA抗原转换为供者型)、分子遗传学证据(应用PCR扩增短串联重复序歹PCR-STR分析移植后病例外周血中,供者的遗传学标记及所占的比例)。血细胞减少定义满足以下标准之一我们就定义为移植28天后血细胞减少；1)外周血白细胞计数小于3G/L,持续4周或4周以上；2)中性粒细胞计数小于0.5G/L,持续4周或4周以上；3)血红蛋白小于9.0g/dl,持续4周或4周以上；4)血小板小于20.0×107mm3,持续4周或4周以上；身免疫性溶血性贫血(AIHA)定义为临床上有明确的溶血,且实验室检查Coombs试验阳性。统计学分析单因素和多因素logistic回归模型评估了与上述定义的血细胞减少相关风险因素的优势比及其95%置信区间。协变量(与血细胞减少的相关危险因素)包括供者年龄、受者年龄、供者性别、受者性别、供受者ABO血型是否相合、供受者巨细胞病毒感染状态,HLA配型、干细胞来源、移植类型、急性移植物抗宿主病及慢性移植物抗宿主病。如果单因素分析单个协变量时,其P值小于0.05,此协变量即纳入多因素分析,所有P值均为双侧检验。此外,我们采用混合效应模型进一步探讨了外周血白细胞计数与环胞素A血药浓度、甲强剂量之间的关系,同时x2比较了变更供者移植组与同胞供者移植组两组血细胞减少发病率。我们采用统计学软件SPSS13.0和SAS9.2进行的分析。结果 187例重型地中海贫血造血干细胞移植的患儿,共39例在移植28天后发生血细胞减少,累积一年发病率20.9%(95%置信区间11.5%-26.7%)。血细胞减少发生的中位时间为移植后102天(范围48天～189天),血细胞减少持续的中位时间70天(范围28天～308天)。39例发生血细胞减少的患儿,白细胞计数小于3G/L、2G/L、1G/L,分别有24例、9例、3例,其中有34例仅白细胞减少、2例AIHA、1例血小板减少、1例白细胞减少合并AIHA,还有1例白细胞+血小板减少。我们比较了ADT组与MST组两组的发病率,结果显示ADT组发病率明显高于MST组(25.8%vs.11.9%,P=0.025),具有统计学意义,这可能与ADT组中HLA不相合的例数多有关。我们进一步分析了ADT组中HLA配型相合与否的发病率,结果显示HLA不相合的发病率明显高于相合发病率(31/120,36.4%vs8/67,19.7%,P=0.045)。我们进一步比较了C1C1纯合子受者与C1C2杂合子受者发病率,结果显示前者发病率明显高于后者(P=0.018)。单因素分析结果显示,与血细胞减少相关的危险因素有HLA配型不相合(尤其A、DR位点不相合)和干细胞来源(同胞移植与变更供者移植相比,同胞移植倾向于不易发生血细胞减少,OR=0.389,P=0.028),因HLA配型B、C位点不相合例数太少,并没有纳入分析。多因素分析结果显示仅HLA不相合具有统计学意义(OR=2.561,P=0.023),同时混合效应模型结果显示白细胞计数与环胞素A(β=0.002037P0.001)、甲强剂量(β=0.6053P0.001)具有显著统计学差异,与患儿年龄亦有统计学差异(β=0.1357P=0.0364),但甲强剂量与患儿年龄之间存在交互效应(β=-0.04345P0.001),与患儿性别没有统计学差异(P=0.7241)。 34例仅有白细胞减少的病人,我们通过调整环胞素A和(或)甲强剂量,或者改环胞素A为他克莫司胶囊,有些病人需要联合甲氨喋呤治疗。经过治疗后,其中有30例病人经过中位时间70天(范围28天-308天)白细胞均恢复正常。39例中其中有2例白细胞减少病人予兔抗人胸腺细胞免疫球蛋白(Thymoglobuline)(总量5mg/kg),1例病人白细胞已恢复正常,另1例血象仍在2G/L左右,此外还有3例病人移植后已3月余,至此次研究结束时白细胞减少未恢复正常。39例血细胞减少患儿仅1例于移植后5月余死于继续性糖尿病和肺部感染。结论 ADT供者、HLA不相合、对免疫抑制剂治疗反应良好、减Cs-A和MP剂量过程中出现皮疹、白细胞计数与Cs-A浓度相关、C1C1受者血细胞减少的高发病率及血细胞减少发生的时间)这些通常与恶性疾病造血干细胞移植后cGVHD有关。基于这些原因,我们认为移植28天后血细胞减少这一并发症,可能是年龄较小患儿在接受异基因造血干细胞移植后一种cGVHD的表现,尽管按现在美国西雅图或美国国立卫生研究院的标准并不能诊断为cGVHD。
[Abstract]:Research background
Beta thalassemia major (thalassemia major) is the beta globin chain synthesis to reduce or disappear, resulting in a- globin chain imbalance and ineffective erythropoiesis destroyed a hereditary disease of hemolytic anemia. Although regular blood transfusion and iron therapy has significantly improved the life expectancy. However not associated with complications and iron overload by iron treatment. At the same time, it is difficult to comply with the life of blood transfusion, the only method is still the root treatment by allogeneic hematopoietic stem cell transplantation is defective erythropoiesis. Use only the new warehouse NF-08-TM pretreatment program I center including the strengthening of the prevention of acute graft-versus-host disease, by overcoming the high rejection rate of allogeneic transplantation and obviously improves the unrelated HLA transplantation and peripheral blood stem cells matched sibling donor transplantation. However in the process of follow-up after transplantation, we observed a few patients less blood cells, At present, the etiology is not clear. The possible causes include the use of bone marrow toxicity drug pretreatment, virus infection and some factors associated with transplantation, such as donor age, donor recipient HLA matching, CMV, source of stem cells and chronic graft-versus-host disease. Therefore, to find out the cause is very important, some people think that may be related to the use of cyclosporin A leads to autoimmune inhibition, others believe that may be associated with chronic graft-versus-host disease or other causes. At present, there has not been a severe Mediterranean anemia after hematopoietic stem cell transplantation hematocytopenia reports and etiology, therefore, our analysis of our center for hematopoietic stem cell transplantation for thalassemia 187 cases of data summary.
research objective
The purpose of this study is to investigate thalassemia major in allogeneic hematopoietic stem cell transplantation after 28 days causes less blood cells, the relevant risk reduction and blood cell factor analysis, in order to find out the risk factors associated with blood cells decreased, so as to provide guidance for clinical treatment.
research method
We conducted a retrospective analysis of January 2009 ~ November 2012 in our center for hematopoietic stem cell transplantation in children with beta thalassemia major in 187 cases, male 117 cases, female 70 cases, aged 2 to 16 years old, the median age was 6 years. The median follow-up time was 19 months (range 1-44 months).120 patients undergoing change donor transplantation (alternative donor transplant ADT (109 cases), unrelated donor parents for 11 cases), 67 cases which occurred compatriots transplantation. Blood cells to reduce the total of 39 cases, we in 39 cases of pancytopenia patients and 148 patients without hematocytopenia were compared, according to the analysis of children with thalassemia. Age, liver size and ferritin levels, the thalassemia major is divided into first degree, second degree and third degree. Including the related risk factors and reduce blood cells: donor age, sex, human cytomegalovirus infection in serum, HLA type, ABO type, source of stem cells, migration Graft type, acute graft versus host disease and chronic graft versus host disease.
Preprocessing scheme, GVHD prevention
All patients were treated with the new scheme to prevent the center of NF-08-TM, the new scheme is mainly composed of CY (cyclophosphamide -10 days to -9 days), Flu (-8 ~ -4 days of fludarabine, intravenous busulfan (Bu days) -8 days to -6 days), tespamin TT (-5 days), the dosage of busulfan based on children age set. Steady blood concentration of busulfan maintenance in 300-600ng/m1 is appropriate, when less than 300ng/ml or greater than 600ng/ml for dose adjustment, the total increase or decrease 1.2mg/kg, increase or decrease the dose of -6d in divided doses. All patients started taking azathioprine 3mg/kg/d and hydroxyurea prevention by 30mg/kg/d.GVHD combined with a variety of immunosuppressive agents in the -45 days before transplantation, including cyclosporine A (Cs-A), mycophenolate mofetil (MMF) and short course methotrexate, the specific method of intravenous cyclosporin A (Cs-A) -10 days to -2 days, the dose of 1.5mg/kg.d, in -1d days ~ +25 day dose of 3mg/kg.d after oral administration, the blood concentration was maintained at 200 + 50ng/ml.MMF in +1 days to +30 days sequential therapy, the dose of 15mg/kg.d, two times a day, there is no second degree in the condition of GVHD after transplantation (graft-versus-host disease), MTX + 1 days after transplantation +3 days, +6 days intravenous dose were 10mg/m2,10mg/m2,10mg/m2. changes in the donor transplantation group, all patients received peripheral blood stem cell transplantation, and most of the children accept is unrelated donors.
Implantable evidence detection
On the 28 day after transplantation routine, second months, third months, sixth months and 1 years after transplantation for implantation of evidence detection, if a patient implanted poor may be detected at any time. Evidence of implantation between donor and recipient gender difference in FISH examination of chromosomal changes on the basis of the same sex were HLA DNA short tandem quantitative monitoring repeat polymorphism analysis. Implantation of the standard of success has indirect index and direct index. (1), indirect index: transplantation survived more than 21 days; the normal hematopoietic system, including erythroid, myeloid, lymphoid, megakaryocyte; clinical occurrence of GVHD. retraction to normal size; lactate dehydrogenase (LDH) decreased to normal level. (2), a direct indicator: red cell antigens (ABO and Rh into the donor's blood type), cytogenetic markers (change of peripheral blood or myeloid chromosomes of donor HLA antigen (HLA), in the match is not full Matched cases, HLA antigen into donor type), molecular genetic evidence (PCR amplification of short tandem repeat sequence or PCR-STR analysis after transplantation of peripheral blood disease, genetic markers of the donor and the proportion).
Definition of hemocyte reduction
Meet the following criteria is defined as one of our 28 days after transplantation of blood cells decreased; 1) peripheral white blood cell count is less than 3G/L, for 4 weeks or 4 weeks; 2) neutrophil count is less than 0.5G/L, for 4 weeks or 4 weeks; 3) hemoglobin less than 9.0g/dl, for 4 weeks or 4 weeks 4); platelet is less than 20 * 107mm3, 4 weeks or 4 weeks; autoimmune hemolytic anemia (AIHA) is defined as a clear clinical and laboratory examination of hemolytic, positive Coombs test.
Statistical analysis
Univariate and multivariate logistic regression models were evaluated and defined above hematocytopenia related risk factors of odds ratio and 95% confidence intervals (covariate related risk reduction and blood cell factors) including donor age and recipient age, donor recipient gender, gender, donor recipient ABO blood is consistent and the state of cytomegalovirus infection in donor, HLA typing, source of stem cells, transplantation, acute graft-versus-host disease and chronic graft-versus-host disease. If the single factor analysis of individual covariates, the P value is less than 0.05, the covariates that included in the multivariate analysis, all P values are bilateral test. In addition, we use the mixed effect model to further explore the peripheral white blood cell count and cyclosporine A blood concentration, the relationship between a strong dose of X2, and compared the change of donor transplantation group and sibling donor transplantation blood cells of two groups decreased The incidence of the disease. We used statistical software SPSS13.0 and SAS9.2 analysis.
187 cases of children with thalassemia major hematopoietic stem cell transplantation, 39 cases occurred after 28 days of blood cells decreased, a year cumulative incidence rate of 20.9% (95% confidence interval 11.5%-26.7%). Blood cells decreased as the median time of occurrence in the 102 day after transplantation (range 48 ~ 189 days), median hematocytopenia the duration of 70 days (range 28 ~ 308 days).39 cases of pancytopenia in children, white blood cell count is less than 3G/L, 2G/L, 1G/L, there were 24 cases, 9 cases, 3 cases, of which 34 cases only leukopenia, 2 cases AIHA, 1 cases of thrombocytopenia, 1 cases of white the cell associated with a reduction in AIHA, there are 1 cases of white blood cells + thrombocytopenia. We compared ADT group and MST group the incidence of patients in the two groups, results showed that the incidence of ADT group was significantly higher than that of MST group (25.8%vs.11.9%, P=0.025), with statistical significance, which may be related to ADT and HLA in the group of mismatched cases we more. Further analysis of the ADT group Consistency in HLA typing and the incidence, the results showed that the onset of mismatched HLA was significantly higher than that of the matched incidence (31/120,36.4%vs8/67,19.7%, P=0.045). We compared C1C1 homozygous recipients with C1C2 heterozygous recipients of incidence, the results suggest that the incidence rate is significantly higher than the latter (P=0.018).
Univariate analysis showed that blood cells decreased and related risk factors of HLA mismatch (especially A, unrelated DR) and the source of stem cells (donor transplantation and transplantation compatriots change compared to sibling transplants tend not to occur hematocytopenia, OR=0.389, P=0.028, HLA) for type B unrelated C cases, too few, and not included in the analysis. The results of multivariate analysis showed that only HLA was not consistent with statistical significance (OR=2.561, P=0.023), and mixed effects model showed that white blood cell count and cyclosporin A (beta =0.002037P0.001), a high dose (beta =0.6053P0.001) with significant statistical differences. The age of children, and there were significant differences (P =0.1357P=0.0364), but there is a strong interaction between dose and age (P =-0.04345P0.001), no statistically significant differences between gender and children (P=0.7241).
Only 34 patients with leukopenia, we adjust the cyclosporin A and (or) a strong dose, or cyclosporin A for Tacrolimus Capsules, some patients need combined with methotrexate treatment. After treatment, the 30 patients after a median of 70 days (range, 28 days -308 days) of white blood cells were normal in.39 cases, including 2 cases of leukopenia treated with rabbit antithymocyte globulin (Thymoglobuline) (total 5mg/kg), 1 cases of white blood cells the patient has returned to normal, the other 1 cases of blood is about 2G/L, in addition to 3 cases of patients after transplantation was 3 times more than a month, at the end of the study when leukopenia did not restore normal.39 patients hypocytosis were only 1 cases in 5 months after transplantation to diabetic and died of pulmonary infection.
conclusion
ADT donor, HLA mismatched, good response to immunosuppressive therapy, rash Cs-A and MP dose reduction process, white blood cell count and Cs-A concentration, C1C1 recipients of blood cells to reduce the high incidence and blood cells to reduce the occurrence of time) these are usually malignant diseases and hematopoietic stem cell transplantation on cGVHD. Based on these reasons, we believe that 28 days after transplantation of blood cells to reduce the complications, may be smaller children in an expression of a cGVHD after allogeneic hematopoietic stem cell transplantation, although by now the United States of Seattle or the National Institutes of health standards and can not be diagnosed as cGVHD.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R725.5

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