Haploidentical stem cell transplantation: T cell depleted and repleted

Allogeneic hematopoietic stem cell transplantation (AHSCT), is a curative treatment option for many hematological diseases. Donor availability is the major limiting factor in the transplantation setting. More than half of the patients do not have a HLA fully matched related/unrelated donor but almost all patients have an haploidentical donor. Haploidentical transplantion can be performed either as T cell depleted or repleted. Despite the promising results in T cell depletion methods, expensive T cell selection and the necessity for experienced staff seem to be the limiting factors. However, a T cell repleted haploidentical stem cell transplantation strategy is simple, cheap and has been preferred by different transplant centers worldwide.     

Blood bank issues associated with red cell exchanges in sickle cell disease

Sickle cell disease (SCD) patients are prone to develop complications that include stroke, acute chest syndrome, and other crises. Some of these complications require chronic transfusion therapy or red cell exchange (RCE), either for therapeutic or prophylactic reasons. Due to a discrepancy of red cell antigens between African Americans and Caucasians (majority blood donors), the incidence of alloantibody formation is very high, which makes it difficult to find compatible red cell units, especially for urgent RCE. Some of the above conditions require immediate oxygen delivery to the tissues. Thus, SCD patients undergoing RCE should receive red blood cells with special attributes that include matching for Rh and Kell blood group antigens; RBCs should be fresh in order to provide (1) immediate oxygen delivery and (2) longer surviving cells to reduce the interval between RCE. Also, these units should be pre-storage leukoreduced to prevent febrile non-hemolytic reactions and screened for sickle cell traits to avoid transfusing red cells containing HbS. This requires a concerted effort between the apheresis unit, the local blood bank, and the central blood supplier.     

XT-4000i血细胞分析仪在体液细胞计数的应用分析

目的：探讨 XT-4000i 血细胞分析仪（简称 XT-4000i）在体液细胞计数中的应用价值。方法收集2013年9月份至2014年2月份住院患者体液标本113份,将收集的标本1 h 内分别在 XT-4000i 血细胞分析仪上和手工检测红细胞（RBC）和白细胞（WBC）,并将 RBC 值分为3个浓度水平 L1：100～1000×106／L;L2：1001×106～100000×106／L;L3：＞100000×106／L, WBC 分为2个浓度水平 L1：1～50×106／L;L2：＞50×106／L 并对2种检验方法进行相关性分析。结果 XT-4000i 血细胞分析仪与手工法检测 RBC、WBC 的结果具有较高的相关性,相关系数分别为0.931、0.996、0.865、0.942和0.988。结论 XT-4000i血细胞分析仪可以在临床体液细胞计数中应用。     

血细胞分析仪自动计数外周血有核红细胞的应用研究

目的评价血细胞分析仪自动计数外周血有核红细胞(NRBC)的方法学特点,并探讨其临床应用价值。方法选择115例血液病及非血液病患者,用SysmexXE2100全自动血细胞分析仪及显微镜计数法计数其外周血中NRBC数量。结果SysmexXE2100与显微镜计数法计数外周血中NRBC数量有极好的相关性(r>0.97),并且2种方法的计数结果差异无显著意义(P=0.2018)。高值、中值和低值NRBC标本绝对计数的批内CV平均<6.5%。在(0～15)×109/L范围内计数NRBC有极好的线性(r=0.9998)。SysmexXE2100自动计数外周血中NRBC的灵敏度和阴性预测值均为100%。外周血中NRBC增高时,WBC显著高于去除NRBC的计数结果(P=0.0168)。结论SysmexXE2100血细胞分析仪可灵敏、准确、精密地自动计数外周血中NRBC,并且可在NRBC增高的标本中准确计数白细胞数量。除白血病、贫血等血液病患者外,非血液病(如肿瘤化疗)等患者外周血中也可出现NRBC。血细胞分析仪自动计数外周血中NRBC,有助于一些血液病及非血液病患者的筛查与诊断。     

N-乙酰基转移酶10在透明细胞性肾细胞癌中的表达及意义

目的检测N-乙酰基转移酶10（NAT10）在透明细胞性肾细胞癌（CCRCC）中的表达情况,并探讨其意义。 方法对100例CCRCC临床病理标本进行NAT10抗体的免疫组织化学染色,并对染色结果进行观察。 结果100例CCRCC患者中,NAT10的表达率为100%,染色强度高于肿瘤旁正常组织,其着色部位为细胞核及胞浆,并在核仁区有特异着色。 结论NAT10在CCRCC中高表达,并在核仁区有特异着色,NAT10有望成为临床辅助CCRCC诊断及核仁的新标记物。     

姜黄素诱导Hep-2细胞凋亡及其对细胞周期各时相的影响

目的探讨姜黄素对Hep-2（人喉癌细胞株）细胞诱导分化机制，为喉癌治疗提供理论依据。方法应用MTT（塞唑兰）比色法和流式细胞仪检测Hep-2细胞增殖抑制率及细胞周期各时相的变化和凋亡率，并通过电子显微镜观察经姜黄素诱导分化后的亚细胞结构改变。结果姜黄素对Hep-2细胞增殖有明显的抑制作用，且呈剂量依赖性，流式细胞仪分析，G1期细胞增多，S期细胞减少，C2／M期细胞相对增多，亚细胞结构、细胞空化，染色质趋边凝集。结论姜黄素能够抑制Hep-2细胞增殖，阻止G1期细胞向S期的进程，促进Hep-2细胞凋亡。     

简易细胞块和细胞芯片的制作方法及应用价值

目的介绍一种石蜡包埋细胞块及细胞芯片的简易制作方法及验证其应用价值。方法收集临床送检细胞学标本,采用简易方法制作成石蜡包埋的细胞块及细胞芯片,HE染色、免疫细胞化学染色及TUNEL检测法验证其应用价值。结果此方法能简易制作出细胞块及60个点阵左右的细胞芯片,经HE染色、免疫组化染色及TUNEL凋亡染色证实,染色效果良好,结果可靠。结论本方法制作的石蜡包埋细胞块及细胞芯片的操作容易,设备简单,细胞形态和抗原保持良好,值得推广应用。     

体外诱导骨髓CD34+细胞生成血管内皮细胞的方法

目的：体外分离狗骨髓CD34^ 细胞，建立诱导分化血管内皮细胞的方法，并进行生物学鉴定。方法：利用免疫磁珠法分离狗骨髓CD34^ 细胞，在体外经血管内皮细胞生长因子（VEGF）、内皮细胞生长因子（EGF）、碱性成纤维细胞生长因子（hFGF)诱导后，观察细胞生长状况，以光镜、电镜进行形态学鉴定，应用免疫组化方法检测冯维靳布兰德因子（Von Willebrand factor,vWF)表达。结果：光镜下细胞单层融合生长，呈铺路石样形态，单个核位子中央，细胞群体倍增时间为35h。电镜下可见到Weibel-Palade小体，在细胞胞浆vWF染色阳性。结论：采用免疫磁珠法可从骨髓获得高纯度的CD34^ 细胞，在体外诱导分化成血管内皮细胞。     

主要ABO血型不合异基因造血干细胞移植后纯红细胞再生障碍

目的 研究主要ABO血型不合异基因造血干细胞移植（allo-HSCT）后患者纯红细胞再生障碍（PRCA）的发病情况及危险因素。方法 分析移植后患者PRCA的发病危险因素，比较抗A凝集素与抗B凝集素对红系造血恢复的影响。结果 100例ABO血型主要及主次要均不合allo-HSCT患者中，12例发生PRCA。A供O者9例，A供B者1例，B供O者2例。有抗A凝集素的患者（10例）较有抗B凝集素的患者（2例）易发生PRCA（P〈0．05）。PRCA的发生不影响急性移植物抗宿主病（GVHD）或巨细胞病毒（CMV）感染的发生。发生PRCA时血型转换的中位时问为150．5d，显著长于无PRCA发生患者（60．0d）（P〈0．05）；红系恢复的中位时间为203．5d，显著长于无PRCA发生患者（76．0d）（P〈0．05）。有抗A凝集素的患者血型转换中位时间为90．0d，显著长于有抗B凝集素的患者（55．0d）（P〈0．05）；红系恢复中位时间为98．0d，长于有抗B凝集素者（80．0d）（P〉0．05），但差异无统计学意义。结论 PRCA是ABO血型不合移植的合并症之一。A供O是主要ABO血型小合allo-HSCT后PRCA发病的危险因素。     

PEG诱导间充质干细胞与胃癌细胞融合的研究

目的探讨聚乙二醇(polyethylene glycol,PEG)1500诱导人脐带间充质干细胞(human umbilical cord mesechymal stem cell,huc MSC)与胃癌细胞系(HGC-27)融合的可能性及融合细胞(hybrids)的活力。方法荧光染料DIO、DID分别标记HGC-27与huc MSC;细胞计数法观察标记前后细胞增殖情况;PEG1500诱导细胞融合后,用荧光显微镜观察双色的融合细胞;CCK-8法检测融合细胞的活性;流式细胞术分析融合细胞的细胞周期。结果 DIO与DID荧光染料可分别标记HGC-27(表达绿色荧光)与huc MSC(表达红色荧光),且标记对细胞增殖无明显影响;荧光显微镜可观察到双色双核的融合细胞;流式细胞术结果表明,与HGC-27细胞相比,融合细胞活性增强,细胞周期中G1期比例降低,S期比例升高(P0.01)。结论 PEG可诱导huc MSC与HGC-27细胞融合,且融合后的细胞表现很强的体外增殖活性。     

XE-2100全自动血细胞分析仪对有核红细胞分析的应用评价

目的评价Sysmex XE-2100全自动血细胞分析仪对有核红细胞（NRBC）分析结果的准确性。方法采用仪器法和显微镜计数法分别对2006年1月至2011年2月间139例外周血NRBC阳性的临床血液样本进行测定。结果仪器法对NRBC的检测结果与显微镜计数法比较,中、高值差异无统计学意义（P〉0.05）,低值差异有统计学意义（P〈0.05）。仪器的重复性良好。结论 Sysmex XE-2100全自动血细胞分析仪分析结果准确、快速,可为临床医生提供准确血细胞监测数据。     

Improved method for automated red cell exchange in sickle cell disease

An improved method for intermittent-flow erythrocytapheresis in patients with sickle cell disease is reported. The method, a modification of the standard red cell exchange procedure for the Haemonetics 30S unit, dilutes with physiologic saline the patient's blood as it flows from the draw line and before it reaches the centrifugation bowl. The blood dilution (approximately 1.6 parts saline to 1 part blood) is used only during the first two passes, when the proportion of sickle erythrocytes in the patient's blood is still high. Only that amount of bowl supernatant (saline-diluted plasma) necessary to maintain extracorporeal volume below 500 ml is returned to the patient. The method described largely prevents the clumping of sickle erythrocytes in the centrifugation bowl, a complication frequently encountered with the Haemonetics 30S unit. Thus, changing the bowl between passes is avoided. Furthermore, the sickle red cells can be collected with the first pass and cryopreserved for possible future uses including the option of autotransfusion.     

A novel closed cell culture device for fabrication of corneal epithelial cell sheets

Automation technology for cell sheet‐based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell‐culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm‐thick gas‐permeable membrane, which was similar to the results with the cell‐culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100–300 µm‐thick gas‐permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial‐specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin‐1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets. Copyright © 2012 John Wiley & Sons, Ltd.     

Peripheral blood stem cell collection for allogeneic hematopoietic stem cell transplantation: Practical implications after 200 consequent transplants

Background

Proper stem cell mobilization is one of the most important steps in hematopoietic stem cell transplantation (HSCT). The aim of this paper is to share our 6 years’ experience and provide practical clinical approaches particularly for stem cell mobilization and collection within the series of more than 200 successive allogeneic HSCT at our transplant center.

成熟T细胞肿瘤与T细胞反应性增生抗原表达对比研究

目的 比较成熟T细胞肿瘤与T细胞反应性增生白细胞共同抗原CD45、全T细胞抗原CD2、CD3、CD5、CD7及CD4、CD8等的表达差异,探讨上述抗原表达变化在成熟T细胞肿瘤诊断中的价值.方法 对36例成熟T细胞肿瘤及36例T细胞反应性增生流式细胞免疫分型资料进行回顾性分析.根据抗原表达及其与正常T细胞的比较,将T细胞抗原表达模式分为抗原丢失及抗原强度变化.强度变化包括表达减弱、表达增强、双峰表达、表达不均匀等.结果 63.9%的成熟T细胞肿瘤有1项或多项全T细胞抗原丢失(23/36),明显高于对照组的8.3%(3/36),两者差异具有显著性(χ2=21.772,P<0.0001).36.1%的成熟T细胞肿瘤病例有3项或4项全T抗原强度变化(13/36),明显高于对照组的8.3%(3/36).两者差异具有显著性(χ2=6.525,P=0.0106).97.2%的成熟T细胞肿瘤CD4/CD8比例异常(35/36),明显高于对照组的22.2%(8/36),两者差异具有显著性(χ2=39.024,P<0.0001).27.8%的成熟T细胞肿瘤CD45表达异常(10/36),明显高于对照组的2.8%(1/36),两者差异具有显著性(χ2=6.859,P<0.0088).结论 成熟T细胞肿瘤与T细胞反应性增生在全T细胞抗原、CD4、CD8及CD45表达方面具有明显不同的特点,上述抗体的检测有助于成熟T细胞肿瘤的诊断及鉴别诊断.     

杀伤细胞免疫球蛋白受体配型在造血干细胞移植中的应用

自然杀伤(NK)细胞在造血干细胞移植(HSCT)对于移植物植入、抵抗感染,以及抗肿瘤效应方面均起着重要作用.杀伤细胞免疫球蛋白受体(KIR)作为NK细胞上的重要受体家族,具有基因组成多样性、等位基因多态性的特点.KIR与人类白细胞抗原(HLA)-Ⅰ类分子结合后,对移植后NK细胞增殖起重要调控作用.笔者从KIR家族基因系统,相应配体及对各类型HSCT的影响方面进行综述,旨在探讨KIR配型对HSCT的临床意义.     

TYMP基因在肾透明细胞癌中的表达及临床意义

目的:阐述胸苷磷酸化酶(thymidine phosphorylase,TYMP)基因在肾透明细胞癌(clear cell renal cellcarcinoma,CCRCC)中的表达及临床意义。方法:利用Oncomine及GEPIA数据库分析T YMP基因在正常肾组织及CCRCC组织中的表达差异;通过GEPIA数据库进行TYMP基因表达程度与病理分期的相关性分析;利用OncoLnc数据库对TYMP基因的表达水平与CCRCC患者生存率作Kaplan-Meier生存分析和log-rank检验;经MethHC数据库分析CCRCC组织和正常肾组织中T YMP启动子区DNA甲基化水平的差异;利用String-DB数据库分析与TYMP蛋白相互作用的蛋白;最后通过The Human Protein Atlas数据库分析TYMP蛋白在正常肾组织及CCRCC组织中的表达差异。结果:在mRNA及蛋白水平上,TYMP在CCRCC组织较正常肾组织中显著高表达,且TYMP mRNA表达水平越高,CCRCC病理分期越高;与TYMP基因低表达组相比,TYMP基因高表达的CCRCC患者总体生存率明显降低;CCRCC组织中TYMP启动子区DNA甲基化水平较正常组织甲基化水平显著降低;同时,与TYMP蛋白相关的蛋白有TK2,NT5M,CDA,UPRT,NT5C,UPP2,UMPS,TK1,UPP1和DPYD等,主要参与嘧啶核苷的合成、分解、代谢,转移戊糖基、蛋白质同源聚合等生物过程。结论:大样本数据挖掘能迅速获取CCRCC组织中TYMP基因表达的相关信息,为深入研究TYMP基因在CCRCC发生发展中的作用机制及预后奠定基础。     

朗格汉斯细胞源性肿瘤研究进展

朗格汉斯细胞肿瘤是一类起源于朗格汉斯细胞(LC)并保持其特定表型谱及超微结构特征的肿瘤,根据细胞形态学、免疫组化和超微结构特征,主要分为2个亚类:朗格汉斯组织细胞增生症(LCH)和朗格汉斯细胞肉瘤(LCS)。LCH是一种LC的良性克隆增殖性疾病,而LCS则是一种极其罕见的以LC恶性增殖及播散为主要特征的恶性肿瘤,被认为是LCH的高级别恶性型。两者均可累及全身各组织器官,临床表现复杂多样,程度不同。诊断主要依靠病理组织细胞形态学及免疫组织化学,必要时电子显微镜辅助诊断。治疗方面包括手术切除、放化疗、免疫治疗及造血干细胞移植等,目前尚缺乏公认最优治疗方案,治疗需个体化。LCH预后主要与受损器官数目有关,而LCS侵袭性强,进展迅速,总体预后差。本文分别就LCH和LCS的发病机制、临床表现、诊断、治疗及预后等方面进行综述。     

Haploidentical stem cell transplantation for patients with sickle cell disease: current status

For patients with high-risk sickle cell disease (SCD) without any available matched sibling or unrelated donor, haploidentical stem cell transplantation (haploHCT) expands the availability of this life-saving intervention to nearly all patients who may benefit from HCT. The greatest challenge in haploHCT has been the significant risk of graft failure. Developing a treatment modality which sustains engraftment without increasing the incidence of debilitating graft-versus-host disease (GvHD) remains the ultimate goal. A number of modifications have been explored to overcome the high incidence of graft rejection and severe GvHD including: (1) ex-vivo T-cell depletion (via CD34⁺ selection, CD3⁺/CD19⁺, or TCRαβ⁺/CD19⁺ depletion), and (2) in vivo T-cell depletion using unmanipulated grafts followed by post-transplant cyclophosphamide (PTCy) for GvHD prophylaxis. Furthermore, the presence of donor-specific anti-HLA antibodies (DSA) has been associated with an increased risk of both graft failure and poor graft function. Several approaches for desensitization ameliorate this risk when a suitable donor without DSA is not available. In addition to advances in supportive care, the recent demonstration that stable mixed chimerism post-HCT sufficiently sustains symptom-free status has opened the door for less toxic treatment approaches yielding excellent survival outcomes. Though late effects remain uncertain, the goal of finding the least toxic conditioning regimen while providing the highest rate of donor engraftment draws closer within reach. In this review, the authors aim to present the latest findings, challenges, and treatment modalities of this life-saving modality.