非清髓异基因外周造血干细胞移植治疗高龄血液病临床研究

目的 探讨非清髓异基因外周造血干细胞移植(NAST)在治疗高龄血液病中的作用。方法 采用NAST、治疗高龄血液病患者5例。5例患者平均年龄55岁(53～59岁)。预处理方案主要由环孢素、抗淋巴细胞球蛋白和环磷酰胺等组成。结果 5例患者均顺利度过移植后造血抑制期，3例供者细胞完全植入，2例为供受者混合嵌合体。2例出现移植物抗宿主病(GVHD)，治疗后好转。结论 NAST简便安全，并发症少，疗效好，为高龄血液病的治疗开辟了新途径。     

单倍相合异基因外周血联合骨髓造血干细胞移植的干细胞采集及移植效果评价

目的采集单倍相合异基因供者外周血和骨髓中的造血干细胞,分析不同采集方式的差异,并进行移植后的效果评价。方法单倍相合异基因造血干细胞移植的受者为2006年6月-2008年12月在第三军医大学新桥医院血液科就诊的56例白血病患者,健康供者为患者的父母或兄弟姐妹。在供者皮下注射重组人粒细胞集落刺激因子进行造血干细胞动员,当外周血单个核细胞(MNC)>4.0×109/L时用血细胞分离机采集外周血中的造血干细胞,采集后1h内输给受者。56例供者根据骨髓造血干细胞的采集方法不同分为3组:第1组(n=5)采用注射器针头半密闭式过滤法,第2组(n=6)采用Thomas不锈钢开放式过滤法,第3组(n=45)采用本课题组的改进方法(用过滤输血器替换注射器针头及不锈钢网)。如若供、受者ABO血型不合,则对采集后的骨髓液进行处理:主侧不合用羟乙基淀粉沉降法去除红细胞,次侧不合采用低温低速离心去除血浆。骨髓液干细胞采集后4h内输给受者。结果56例供者外周血采集量的中位值为56ml,MNC中位值5.15×108/kg,CD34+细胞中位值5.80×106/kg;骨髓液采集量中位值950ml,有核细胞(NC)中位值10.31×108/kg...     

异基因外周血造血干细胞移植后造血细胞嵌合状态的分析

为评估异基因外周血造血干细胞移植（ALLO－PBSCT）后的植入状况，采用细胞遗传学分析结合短串联重复序列－聚合酶链反应（STR－PCR），对30例Allo-PBSCT病人进行了1－25个月的随访观察。结果表明，不同性别移植的23例，21例完全嵌合（CC），1例混合嵌合（MC），1例为完全受者核型；相同性别移植的慢性髓性白血病（CML）7例，除2例外，其余均未检测到Ph染色体；STR－PCR检测结果与染色体分析结果完全一致。说明两种方面具有互补性，可结合使用。     

造血干细胞移植患者人巨细胞病毒糖蛋白gB的基因多态性分析

目的阐明造血干细胞移植患者人巨细胞病毒（human cytomegalovirus,HCMV）糖蛋白gB的基因多态性。方法提取造血干细胞移植患者外周血标本DNA,采用巢式PCR对DNA进行扩增,利用RasⅠ与HinfⅠ对扩增片段进行分型,并对不同型别的扩增片段进行序列测定和序列同源性分析。结果 60.3%（38/63）的患者为HCMV核酸检测阳性,在28例PCR检测强阳性的病例标本中,gB-1型占60.7%（17/28）,gB-2型占17.9%（5/28）,gB-3型占21.4%（6/28）,未见gB-4型。gB-1和gB-2型之间的核苷酸同源性较高,在98%以上,而gB-3型与gB-1型（或gB-2型）相比,核苷酸同源性仅为81.2%~84.3%,并且缺失6个核苷酸,即在蛋白质水平上缺失2个氨基酸。结论我中心造血干细胞移植患者HCMV病毒感染较为复杂,存在变异较高的gB-3型,但以gB-1型为主。     

自体混合HLA半相合异基因骨髓移植后的移植物抗宿主病

目的:为了观察自体骨髓混合HLA半相合异基因骨髓移植治疗恶性血液病后的GVHD发生情况.方法:观察16例混合移植GVHD的发生情况及其对疗效的影响.结果:16例中无1例发生急性GVHD(aGVHD),但在ABMT后1h输入异基因骨髓的8例皆有程度不同的慢性GVHD(cGVHD),主要表现在皮肤,粘膜炎、全血细胞减少、肝功异常、发热、体重减轻及易感冒等.中位随访8月无1例复发,除2例在移植后3和8月分别因爆发性肝炎和急性阑尾炎穿孔死亡外.余6例皆存活.最长1例已无病存活12月余.其中供受者性别不同的5例移植后性染色体观察4例形成嵌合体,3例已超过半年,最长者已1年余.而ABMT移植后6h输异基因骨髓的8例,则未观察到急慢性GVHD.结论:移植过程是安全的,部分可诱发cGVHD,形成嵌合体,白血病的复发较少.     

供体外周血干细胞在活体肾脏移植中的应用研究

目的研究活体肾移植受体接受全身淋巴结照射(TLI)预处理基础上输注供体外周血干细胞(PBSCs)后嵌合体形成情况及其与肾移植的相关性。方法5对供受体进入本研究。供体平均年龄40岁,受体平均年龄27岁。HLA-A、B、DR6个抗原中4个抗原错配1例,3个抗原错配2例,1个抗原错配2例。TLI治疗从移植手术前5天开始,90cGy/d,共5天。术中和术后的免疫抑制药物方案与同时期的活体或尸体肾移植相同,但剂量略减少。供体的PBSCs动员使用瑞雪新,150μg/d,共5天,PBSCs采集和输注共2次,分别在移植手术后的第4、7天。术后应用PCR-SSP检测外周血嵌合体阳性率和持续时间。观察免疫抑制药物的使用情况、移植肾状态和急性排斥反应频率及GVHD等。结果供体经5天的G-CSF动员后,采集的PBSCs平均数为34×106。移植术后1个月5例受体外周血中均可检测到供体的HLD-DR抗原成分。2个月后4个HLA抗原错配,其中HLA-DR均错配的受体外周血中嵌合体PCR检测阴性,3个月后HLA-DR均错配和1个HLA-B错配的受体外周血嵌合体检测阴性,受体3、4和5的外周血中供体DNA仍为阳性。PBSCs输注后没有发生GVHD,没有任何感染发生。观察期内没有发生移植肾排斥反应,移植肾功能正常。免疫抑制剂使用量低于同时期的其他活体肾脏移植和尸体肾脏移植受体。结论非清髓性的TLI治疗可以促进供体PBSCs植入受体造血系统,诱导产生造血嵌合体,而造血嵌合体的形成可以减少肾脏移植的排斥反应,减少免疫抑制剂的应用,提高移植物的长期存活率。PBSCs的采集和输注简便、有效,痛苦小,是一种理想的造血干细胞输注源。     

自体骨髓混合HLA半相合异基因骨髓移植治疗恶性血液病的临床研究

目的:观察混合BMT治疗恶性血液病的临床疗效,方法:用自体骨髓混合HLA半相合异基因骨髓移植,同时体外高热及液体培养净化联合IL-2激活骨髓细胞,移植后又注射IL-2,以提高体内对残留肿瘤细胞的净化效应,减弱GVHD反应和增强GVL效应,结果:①MBMT是安全的,16例中无1例发生急性GVHD,②近期疗效较好,移植后16例中5例复发,11例未复发,其中7例中位随访12月仍存活且CCR,余4例死于并发症和其它非血液病;③16例除1例因早期死于VOD外,余皆成功造血重建;④对6例供受者性别不同患者性染色体分析发现3例移植后形成嵌合体,最长者已15月余.结论:本研究为临床恶性血液病治疗可能提供了一个新的途径.     

非清髓造血干细胞移植后移植排斥原因初探

为研究探讨非清髓异基因造血干细胞移植（NAST）后发生移植排斥的原因及促进供体细胞植入的措施，对NAST后发生移植排斥的8例患者进行了初步分析。结果表明，早期减停环孢霉素A（CSA）等免疫抑制剂、移植早期供体细胞植入率低及慢性粒细胞白血病（CML）等较易发生移植排斥。二次NAST是解决移植排斥的有效方法。     

非清髓异基因造血干细胞移植后急性白血病复发5例报告

目的 研究分析非清髓异基因外周造血干细胞移植(NAST)后急性白血病复发的相关因素。方法 29例急性白血病患者NAST后5例复发，男2例，女3例，中位数年龄36岁(18～59岁)。非清髓预处理方案：环磷酰胺、阿糖胞苷及CD3单克隆抗体，3例患者在此基础上加用氟达拉滨。结果 5例均顺利渡过造血抑制期。2例早期形成供者造血细胞完全嵌合体(FDC)并在FDC状态下复发。3例移植早期形成供受者混合造血细胞嵌合体(MC)，2例转为FDC后复发，1例在稳定MC状态下复发。5例中4例复发前无急性或慢性移植物抗宿主疾病(GVHD)，另1例NAST后发生Ⅱ度aGVHD并在皮肤慢性GVHD未治愈状态下复发。5例中1例行2次NAST治疗，在第2次完全缓解，但于2个月后再次复发，放弃治疗。另4例未再继续治疗，2例分别于复发后1和2个月死亡，2例仍带病存活。结论 NAST简便安全，并发症少，白血病复发率无明显升高，为治愈白血病提供了新手段。     

异基因造血干细胞移植后慢性期肺部病变的CT分析

目的 分析异基因造血干细胞移植后慢性期肺部病变的CT表现及治疗后变化的影像特点.资料与方法 对近4年行异基因造血干细胞移植后生存期超过3个月的受者中发现胸部异常的35例患者的CT影像资料进行回顾性分析.结果 移植后慢性期胸部CT表现:9例旱局限叶或多叶的磨玻璃样改变,同时伴网状或网织结节状影;16例为散在胸膜下肺内团片状或斑点状影.伴支气管气像5例;5例表现为胸膜下肺内多发球形不均匀密度影与厚壁空洞并存;4例表现为下肺间质条索状密度影;1例为肺气囊.治疗过程中累计出现19个空洞,2例纵隔气肿.2例气胸,治疗时间约2～6个月.结论 异基因造血干细胞移植后慢性期肺部病变以肺间质病变为主,实变病灶好发于胸膜下且易合并真菌感染出现空洞,上肺较下肺好发.     

Application of droplet digital PCR method for DNA methylation-based age prediction from saliva

The recent studies reported that DNA methylation markers show changes with age, and expected that the DNA methylation markers can be effectively used for estimation of age in forensic genetics. In this study, we applied droplet digital PCR (ddPCR) method to investigate the DNA methylation pattern in the CpG sites, and we constructed an age prediction model based on the ddPCR method. The ddPCR is capable of highly sensitive quantitation of nucleic acid and detection of sequence variations in gene by separating the sample into large number of partitions and clonally amplifying nucleic acids in each partition. We extracted DNA from saliva samples collected from several age groups. The DNA was bisulfite converted and subjected to ddPCR using specifically designed primers and probes. The methylation ratio of each sample was calculated and correlation between the methylation ratio and the chronological age was analyzed. In the results, methylated DNA ratio at the 4 CpG sites (cg14361627, cg14361627, cg08928145 and cg07547549) showed strong correlation with chronological age. Percent-methylation values at 4 CpG markers and chronological ages of the 76 individuals were analyzed by multiple regression analysis, and we constructed an age prediction model. We observed a strong correlation (Spearman’s rho = 0.922) between predicted and chronological ages of 76 individuals with a MAD from chronological age of 3.3 years. Collectively, the result in this study showed the potential applicability of ddPCR to predict age from saliva.     

非清髓异基因造血干细胞移植——造血干细胞移植的新方向

简述了国内外刚刚开展的非清髓异基因造血干细胞移植（NAST）的主要研究进展和研究方向。对NAST的概念、预处理方案、移植物抗宿主病预防、移植物抗白血病效应及临床应用等问题作了讨论和介绍。并提出NAST是对传统异基因造血干细胞移植理论和观念的发展及更新,为血液病、恶性肿瘤及免疫、遗传性疾病的治疗开辟了新路径。并建议加强对NAST有关问题的协作研究。     

Molecular characterisation of the nucleic acids recovered from aged forensic samples

The molecular composition of the genetic substrate recovered from seven aged forensic samples has been extensively investigated. A simple enzymatic test based on DNAseI incubation of the extracts showed that the UV-fluorescent material from the forensic specimens is composed of nucleic acids, with the DNA fraction representing at least 90% of the total amount. Since spectrophotometric determinations of the extracts showed unreliable results due to anomalous OD(260)/OD(280) ratios, quantification of the nuclease-sensitive genetic material was performed by a slightly modified agarose plate method. The first quantitative data on exogenous contamination in aged forensic samples are provided by slot-blot hybridisation of the extracts to human, bacterial and fungal probes. Only limited amounts of human and contaminant DNA were detected in the samples. The molecular integrity of the primary structure of these aged DNA samples was analysed by reversed-phase HPLC/MS. The data show a good correlation between the degree of chemical damage and the ability to hybridise to molecular probes. The ability to achieve specific genetic profiles was assessed by multiplex PCR amplification of STR loci. Our data show that accurate determination of the molecular composition of the DNA recovered from forensic samples can be extremely useful for a reliable evaluation of the PCR typing results.     

Quantitative evaluation of acute mucosal reaction to interstitial brachytherapy using color histograms

PURPOSE: Color histograms of tongue photographs were used to make a quantitative evaluation of acute mucosal reaction to high-dose-rate fractionated interstitial brachytherapy. METHODS AND MATERIALS: Six patients with oral cavity cancer were analyzed with two methods (color histogram and vinyl plate). Using a photograph of the tongue, we calculated the histogram of each pixel of 1mm(2) of the tongue surface. The results were compared with the physician's inspection and the radiation dose. RESULTS: The histogram showed a difference between the reactive area (erythema and pseudomembranous mucositis [PM]) and the nonreactive area. The relationship between inspection and the histogram showed a good correlation, although the histogram tended to change earlier than the subjective judgment. The peak time was 12-13 days after delivery of the final fraction of brachytherapy. The minimum dose required to cause PM was 3.3-3.6Gy per fraction (total: 29.7-32.4Gy). CONCLUSIONS: Our new method of using color histograms for quantitative evaluation is helpful in the analysis of the relationship between mucosal reaction and radiation dose. The minimum dose causing PM was considered to be about 30Gy per nine fractions.     

Feasibility of PET Template-Based Analysis on F-18 FP-CIT PET in Patients with De Novo Parkinson’s Disease

Purpose

The aim of this study was to evaluate the feasibility of FP-CIT PET template-based quantitative analysis on F-18 FP-CIT PET in patients with de novo Parkinson’s disease (PD), compared with MR-based and manual methods. We also assessed the correlation of quantitative parameters of those methods with clinical severity of the disease.

Methods

Forty patients with de novo PD underwent both MRI and F-18 FP-CIT PET. Images were spatially normalized to a standardized PET template. Mean counts of 4 ROIs: putamen, caudate, occipital cortex and cerebellum, were obtained using the quantification program, Korean Statistical Probabilistic Anatomical Map (KSPAM). Putamen-to-caudate ratio (PCR), asymmetry index (ASI), specific-to-nonspecific ratios with two different references: to occipital cortex (SOR) and cerebellum (SCR) were compared. Parameters were also calculated from manually drawn ROI method and MR-coregistrated method.

Results

All quantitative parameters showed significant correlations across the three different methods, especially between the PET-based and manual methods. Among them, PET-based SOR and SCR values showed an excellent correlation and concordance with those of manual method. In relationship with clinical severity, only ASI achieved significantly inverse correlations with H&Y stage and UPDRS motor score. There was no significant difference between the quantitative parameters of both occipital cortex and cerebellum in all three methods, which implied that quantitation using PET-based method could be reproducible regardless of the reference region.

Conclusions

Quantitative parameters using FP-CIT PET template-based method correlated well with those using laborious manual method with excellent concordance. Moreover, PET-based quantitation was less influenced by the reference region than MR-based method. It suggests that PET-based method can provide objective and quantitative parameters quickly and easily as a feasible analysis in place of conventional method.     

Fractal analysis of bone architecture at distal radius

Bone strength depends on bone quality (architecture, turnover, damage accumulation, and mineralization) as well as bone mass. In this study, human bone architecture was analyzed using fractal image analysis, and the clinical relevance of this method was evaluated. The subjects were 12 healthy female controls and 16 female patients suspected of having osteoporosis (age range, 22-70 years; mean age, 49.1 years). High-resolution CT images of the distal radius were acquired and analyzed using a peripheral quantitative computed tomography (pQCT) system. On the same day, bone mineral densities of the lumbar spine (L-BMD), proximal femur (F-BMD), and distal radius (R-BMD) were measured by dual-energy X-ray absorptiometry (DXA). We examined the correlation between the fractal dimension and six bone mass indices. Subjects diagnosed with osteopenia or osteoporosis were divided into two groups (with and without vertebral fracture), and we compared measured values between these two groups. The fractal dimension correlated most closely with L-BMD (r=0.744). The coefficient of correlation between the fractal dimension and L-BMD was very similar to the coefficient of correlation between L-BMD and F-BMD (r=0.783) and the coefficient of correlation between L-BMD and R-BMD (r=0.742). The fractal dimension was the only measured value that differed significantly between both the osteopenic and the osteoporotic subjects with and without vertebral fracture. The present results suggest that the fractal dimension of the distal radius can be reliably used as a bone strength index that reflects bone architecture as well as bone mass.     

Semi-quantitative evaluation of gallium-67 scintigraphy in lupus nephritis

Within nuclear medicine there is a trend towards quantitative analysis. Gallium renal scan has been reported to be useful in monitoring the disease activity of lupus nephritis. However, only visual interpretation using a four-grade scale has been performed in previous studies, and this method is not sensitive enough for follow-up. In this study, we developed a semi-quantitative method for gallium renal scintigraphy to find a potential parameter for the evaluation of lupus nephritis. Forty-eight patients with lupus nephritis underwent renal biopsy to determine World Health Organization classification, activity index (AI) and chronicity index (CI). A delayed 48-h gallium scan was also performed and interpreted by visual and semi-quantitative methods. For semi-quantitative analysis of the gallium uptake in both kidneys, regions of interest (ROIs) were drawn over both kidneys, the right forearm and the adjacent spine. The uptake ratios between these ROIs were calculated and expressed as the "kidney/spine ratio (K/S ratio)" or the "kidney/arm ratio (K/A ratio)". Spearman's rank correlation test and Mann-Whitney U test were used for statistical analysis. Our data showed a good correlation between the semi-quantitative gallium scan and the results of visual interpretation. K/S ratios showed a better correlation with AI than did K/A ratios. Furthermore, the left K/S ratio displayed a better correlation with AI than did the right K/S ratio. In contrast, CI did not correlate well with the results of semi-quantitative gallium scan. In conclusion, semi-quantitative gallium renal scan is easy to perform and shows a good correlation with the results of visual interpretation and renal biopsy. The left K/S ratio from semi-quantitative renal gallium scintigraphy displays the best correlation with AI and is a useful parameter in evaluating the disease activity in lupus nephritis.     

Semi-quantitative evaluation of gallium-67 scintigraphy in lupus nephritis

Within nuclear medicine there is a trend towards quantitative analysis. Gallium renal scan has been reported to be useful in monitoring the disease activity of lupus nephritis. However, only visual interpretation using a four-grade scale has been performed in previous studies, and this method is not sensitive enough for follow-up. In this study, we developed a semi-quantitative method for gallium renal scintigraphy to find a potential parameter for the evaluation of lupus nephritis. Forty-eight patients with lupus nephritis underwent renal biopsy to determine World Health Organization classification, activity index (AI) and chronicity index (CI). A delayed 48-h gallium scan was also performed and interpreted by visual and semi-quantitative methods. For semi-quantitative analysis of the gallium uptake in both kidneys, regions of interest (ROIs) were drawn over both kidneys, the right forearm and the adjacent spine. The uptake ratios between these ROIs were calculated and expressed as the "kidney/spine ratio (K/S ratio)" or the "kidney/arm ratio (K/A ratio)". Spearman's rank correlation test and Mann-Whitney U test were used for statistical analysis. Our data showed a good correlation between the semi-quantitative gallium scan and the results of visual interpretation. K/S ratios showed a better correlation with AI than did K/A ratios. Furthermore, the left K/S ratio displayed a better correlation with AI than did the right K/S ratio. In contrast, CI did not correlate well with the results of semi-quantitative gallium scan. In conclusion, semi-quantitative gallium renal scan is easy to perform and shows a good correlation with the results of visual interpretation and renal biopsy. The left K/S ratio from semi-quantitative renal gallium scintigraphy displays the best correlation with AI and is a useful parameter in evaluating the disease activity in lupus nephritis.     

Does percutaneous liver biopsy of hepatocellular carcinoma cause hematogenous dissemination? An in vivo study with quantitative assay of circulating tumor DNA using methylation-specific real-time polymerase chain reaction

OBJECTIVE: Our purpose was to find out whether percutaneous biopsy of hepatocellular carcinoma will cause significant dissemination of tumor into the circulation by quantitative analysis of circulating tumor DNA. SUBJECTS AND METHODS: In this prospective study of 32 patients with suspected hepatocellular carcinoma who underwent sonographically guided liver biopsy, a peripheral venous blood sample was obtained before and 5 min after the procedure. Biopsy was performed using an 18-gauge biopsy gun. DNA was extracted from the plasma of the blood samples for methylation-specific polymerase chain reaction. Quantitative measures of the plasma tumor DNA were determined with real-time quantitative polymerase chain reaction, and the amount was expressed as a methylation index (%) in plasma. RESULTS: Nineteen (59.4%) of 32 patients did not have detectable p16 tumor suppressor gene marker (p16M) in plasma before biopsy, and they showed no detectable plasma p16M after biopsy. Thirteen (65%) of 20 patients had p16M identified in the plasma before liver biopsy. Quantitative analysis of the plasma tumor DNA in these 13 patients showed no statistically significant difference in the methylation index before and after biopsy (p = 0.345, Wilcoxon's signed rank test). CONCLUSION: No evidence exists that percutaneous liver biopsy results in hematogenous dissemination of hepatocellular carcinoma as shown by quantitative analysis of circulating tumor DNA (p16M) using methylation-specific real-time polymerase chain reaction.