现今微小残留病检测方法的临床价值

现今微小残留病检测方法的临床价值同济医科大学附属协和医院季守新综述王辨明，李崇渔审校微小残留病（ＭｉｎｉｍａＲｅｓｉｄｕａｌＤｉｓｅａｓｅＭＲＤ）是指急性白血病经过有效地化疗或骨髓移植，达到临床和血液学的完全缓解，体内残存微量白血病细胞的状态。估计此...     

荧光原位杂交技术在白血病微小残留病灶检测中的应用

血液系统恶性疾病及实体肿瘤的现代化疗和造血干细胞移植治疗,使得患者获得较彻底的治疗。微小残留病（minimal residual disease,MRD）,是影响治疗效果的一个重要临床问题。在目前判断标准确定的MRD范围内,常规骨髓细胞形态学及细胞遗传学方法,不能敏感地检测MRD,难以客观地对病情转归进行科学预测,使治疗存在较大的盲目性。     

急性早幼粒细胞白血病患者微小残留病的检测与临床意义的研究

目的检测完全缓解后急性早幼粒细胞白血病患者微小残留病（MRD），同时分析其对预后判断的指导价值。方法建立逆转录PCR（RT／PCR）用以检测APL患者PML／RARα。结果RT-PCR方法敏感而可重复。治疗前APL患者的RT-PCR阳性率为92．8％（39／42），而维甲酸或化疗诱导完全缓解后MRD阳性率为56．7％（21／37）。另外，MRD阳性与缓解后APL患者的复发有关，并可以作为缓解后预测APL患者复发的指标。同时有助于指导临床上采取巩固治疗而防止复发。结论RT-PCR能够检测APL患者的MRD和作为预测复发的指标。     

微小残留病在急性淋巴细胞白血病中的意义

<正>急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)是一种累及骨髓早期淋巴前体细胞的克隆性疾病。该疾病从遗传背景、临床表现到预后都有很大的异质性。近年来,得益于儿童样化疗方案、异基因造血干细胞移植(allo-HSCT)和CAR-T技术的应用,ALL的生存率得到了较大的提高[1-3]。在ALL治疗中对微小残留病(minimal residual disease,MRD)的监测,有助于临床医师判断治疗反应,     

RT—PCR检测急性早幼粒细胞白血病微小残留病

用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术，检测了２２列早幼粒细胞白血病（ＡＰＬ）缓解患者ＰＭＬ－ＲＡＲα融合基因。结果显示性率在缓解时间１年内（１１／１４）较１￣２年（２／５）及３年以上（０／３）明显高，持续阳性多预示着复发。认为检测ＰＭＬ－ＲＡＲα融合基因，可作为监测ＡＰＬ病情转归、评价疗效的重要指标。     

急性B淋巴细胞白血病患者检测微小残留病的临床意义

采用多参数流式细胞仪对50例成人B淋巴细胞白血病(B-ALL)患者缓解后的微小残留病(M RD)进行检测,结果显示,B-ALL完全缓解组和正常对照组骨髓M RD分别为(5.9462±4.8964)%和(0.5845±0.2014)%,二者比较有显著性差异(P<0.01);B-ALL持续缓解与复发者的骨髓M RD分别为(4.1436±1.6125)%和(42.3514±20.6451)%,二者比较有显著性差异(P<0.01)。诱导缓解至治疗3个月时M RD阳性患者复发率高,预后不良。提示对B-ALL患者用流式细胞术进行缓解后M RD检测,有利于其复发预测及早期个体化治疗选择。     

白血病微小残留病的研究进展

本文对白血病微小残留病的定义、方法学及临床应用的最新研究进展进行综述,探讨其新的概念内涵,以维持其特有的独立术语。     

实时定量PCR检测血液系统恶性疾病微小残留病

临床治疗血液系统恶性疾病达到完全缓解(CR)后,体内仍残留少量恶性肿瘤细胞(106~108),即微小残留病(MRD)。微小残留病的存在是血液系统恶性肿瘤复发和移植失败的根本原因,因此MRD的动态检测,对血液系统恶性疾病的预后有极其重要的意义〔1〕。以往应用PCR技术体外扩增肿瘤细胞只能定性或半定量监测MRD,因此局限了其在临床判断愈后方面的应用。近年来出现的实时定量PCR(RQ-PCR)技术〔2〕实现了从定性到定量监测MRD的飞跃,能够准确、动态地反映患者体内的肿瘤负荷变化。本文对RQ-PCR在检测血液系统恶性疾病MRD中的应用原理、基因…     

CIK细胞对白血病微小残留病作用的研究

目的 观察CIK细胞对自体、异体原代白血病细胞的杀伤作用,对G_0期CD34~+白血病细胞的细胞毒作用,及对正常骨髓造血干细胞的影响.方法 取急性白血病(AL)化疗后、慢性粒细胞白血病(CML)慢性期及正常人外周血单个核细胞加入一系列细胞因子诱导培养CIK细胞,流式细胞仪检测CIK表型,G带法分析CIK细胞核型.冻存、复苏后作为靶细胞,MTT法测定CIK细胞对自体、异体原代白血病细胞杀伤作用.AO染色法测定复苏后G_0期急性粒细胞白血病(AML) CD34~+细胞比例及培养后CML CD34~+细胞中G_0期细胞比例.流式细胞仪检测CIK细胞杀伤前后G_0期CD34~+细胞比例变化.半固体培养法检测CIK细胞对正常骨髓CFU-GM、BFU-E集落形成的影响.结果 CIK细胞可从白血病患者外周血中获得,来源于正常淋巴细胞,对自体原代白血病细胞有明显杀伤作用,对G_0期白血病细胞有明显抑制作用,对正常造血干细胞无杀伤作用.结论 CIK细胞可作为清除残留白血病细胞的有效工具.     

基因扩增技术检测急慢性B—淋巴细胞白血病免疫球蛋白基因重排

应用聚合酶链反应(PCR)扩增IgH重排产生的CDR-Ⅱ序列,检测23例急慢性B-淋巴细胞白血病患者。20例发生IgH重排,其中4例出现两种基因扩增产物。6例完全缓解的患者,3例检出异常重排的IgH基因,并先后复发。本文应用Southern blot法和PCR进行了对照性研究。     

Quantitative Assessment of Minimal Residual Disease in Childhood Lymphoid Malignancies Using an Allele-Specific Oligonucleotide Real-Time Quantitative Polymerase Chain Reaction

We developed an assay using a real-time quantitative polymerase chain reaction (RQ-PCR) for the quantitative assessment of minimal residual disease (MRD) in childhood lymphoid malignancies by using a consensus V-region probe combining a allele-specific oligonucleotide (ASO) reverse primer. Our strategy employs a set consisting of a consensus V-region probe, an ASO reverse primer, and a patient-specific forward primer for clonal antigen-receptor (IgH, immunoglobulin heavy chain; TCR, T-cell receptor) gene rearrangements (IgH-ASO and TCR-ASO RQ-PCR assays). The limit of detection in both assays was 5 copies of the target/10(5) cell equivalents. We tested the assays in 17 childhood malignancies (14 cases of acute lymphoblastic leukemia and 3 of non-Hodgkin's lymphoma). High correlation coefficients of the standard curves (>0.980) and PCR efficiency (>0.95) were achieved with all primer/probe sets. In 2 (12%) of the 17 patients, ASO primers could not be designed because there was no junctional N-sequence. The quantitative data suggest that the copy number of clonal antigen receptors markedly decreased after induction therapy in 15 of 17 patients and that 1 patient relapsed and died of the disease. Consensus probes make it possible to examine a large number of patients with only a limited number of probes. The strategy used for IgH-ASO and TCR-ASO RQ-PCR assays is accurate and reliable in the clinical prospective study of MRD in childhood lymphoid malignancies.     

Principles of the Polymerase Chain Reaction in Haematology

The polymerase chain reaction (PCR) is an automated process that specifically amplifies selected DNA sequences that are usually chosen to reflect the presence of genes, or parts of genes, in the sample material. Since many genetic alterations resulting in the onset of disease are now detectable using PCR, it can be used as both a diagnostic and prognostic tool. Genetic changes can occur in DNA due to mutation, deletion, inversion or chromosomal translocation. As a consequence, genes become either non functional or are aberrantly expressed. Research in recent years has now associated many defined genetic abnormalities with specific diseases. Therefore detection and surveillance of such lesions has led to disease diagnosis and subsequent monitoring of disease progression, for example, during and after administration of therapeutic regimens. The major effect of PCR in this area is that genetically abnormal cells can be detected within a normal cell population at a far lower incidence level than any other existing technology. Moreover, PCR can be used to identify naturally existing genetic polymorphisms that can be used as personal identifiers, or tags, for a given individual. Where these polymorphisms occur within a genetically modified region resulting in disease, identification of the polymorphism within families can be used as a predictor of disease carrier status or likelihood of inheritance of the disease.     

定量监测慢性粒细胞白血病患者治疗过程中BCR-ABL融合基因转录本水平变化的临床意义

目的：探讨实时定量逆转录聚合酶链反应（RQ-PCR）在监测慢性粒细胞白血病（CML）患者微小残留病、考核疗效以及预测疾病预后方面的作用。方法：应用RQPCR技术对25例CML患者伊马替尼治疗或异基因造血干细胞移植前后BCR-ABL融合基因转录本水平的变化进行监测。结果：CMI。急变期患者BCR-ABL转录本水平明显高于慢性期和加速期患者。7例异基因造血干细胞移植后6个月BCR-ABL转录本水平较6个月前明显降低,4例检测不到BCR-ABL转录本水平,3例极低。伊马替尼治疗的前6个月,BCR-ABL转录本水平下降最明显,治疗12个月后,疗效与造血干细胞移植者类似。结论：RQPCR方法在监测CML患者骨髓微小残留病及考核疗效方面,具有重要的临床应用价值。

Abstract：

Objective： To explore the application of real-time quantitative polymerase chain reaction （RQ- PCR） in detecting minimal residual disease （MRD）, assessing curative effects and predicting the prognosis of chronic myeloid leukemia （CML）. Methods： Changes of levels of BCR ABL were detected by the method of RQPCR in samples of bone marrow （BM） obtained from 25 patients with CML receiving imatinib or allogenie hematopoietic stem cell transplantation （allo-HSCT）. Results： The levels of BCR-ABL mRNA were obviously higher in patients with CML- blastie phase than those in patients with CML-chronic phase or CML-aecelerated phase. The BCR-ABL mRNA levels in the later 6 months were obviously lower than those in the former 6 months after HSCT in 7 cases. The BCR- ABL mRNA levels declined clearly in 3 cases, and even to zero in 6 months in 4 eases. Patients treated with imatinib had a significant decrease in the expression level of BCR ABL during the previous 6 months. It showed similarity in curative effects between patients receiving imatinib and HSCT in 12 months after treatment. Conclusions： RQ-PCR targeted at BCR ABL can be used in the quantitative detection of MRD in CML, and provide gist for instructing clinical therapy.     

PCR检测血清中HBV—DNA及其临床意义

应用聚合酶链反应(PCR)结合Southern blot核酸杂交和DNA直接杂交技术,检测50例乙型肝炎病毒(HBV)感染后不同血清学标志的血清标本及5例无任何HBV血清学标志的血清标本中的HBV DNA,并与血清学检查结果比较。结果发现PCR结合Southern blot核酸杂交的灵敏度明显高于DNA直接杂交技术,而且还能直接反映患者血液是否有感染性。此外,前者还能直接反映病毒在体内的复制情况,澄清模棱两可的血清学结果。     

急性髓系白血病H-ras表达和点突变─流式细胞术和聚合酶链反应比较研究

目的：了解急性髓系白血病细胞H－rasP21表达与点突变的相关性，以及免疫组化法的临床实用价值。方法：用流式细胞术（FCM）分析67例初治急性髓系白血病（AML）H－rasp21表达；对所有P21阳性和部分P21阴性者作聚合酶链反应（PCR），观察H－ras15位密码子点突变。结果：67例AMLFCMP21阳性者45例（67．2％），P21阴性者22例（32．8％）。P21阳性45例中高表达者22例（48．9％），16例（72．7％）检出H－ras15位密码子点突变，11例（50％）完全缓解（CR）。10例P21阴性和23例P21＋低表达均无H－ras15位密码子点突变，CR率分别为77．2％和87．2％。结论：FCMP21高表达者常有H－ras15位密码子点突变，CR低，H－ras点突变与免疫组化P21＋高表达相关良好，提示AML转归不良。     

DNA聚合酶链反应检测石蜡包埋人脑组织中弓形虫的研究

孕妇感染弓形虫是胎儿先天性畸形的危险因素。本文运用DNA聚合酶链反应检测30例先天性畸形儿尸检石蜡包埋脑组织中弓形虫。结果表明,30例先天性畸形儿石蜡脑组织切片经体外扩增后出现阳性条带者13例,而对照组无1例阳性(P<0.05)。研究结果进一步支持弓形虫感染与胎儿先天性畸形有密切关系。     

Clinical Significance of Fecal Lactoferrin and Multiplex Polymerase Chain Reaction in Patients with Acute Diarrhea

Background/AimsThe diagnostic yield of fecal leukocyte and stool cultures is unsatisfactory in patients with acute diarrhea. This study was performed to evaluate the clinical significance of the fecal lactoferrin test and fecal multiplex polymerase chain reaction (PCR) in patients with acute diarrhea.MethodsClinical parameters and laboratory findings, including fecal leukocytes, fecal lactoferrin, stool cultures and stool multiplex PCR for bacteria and viruses, were evaluated prospectively for patients who were hospitalized due to acute diarrhea.ResultsA total of 54 patients were included (male, 23; median age, 42.5 years). Fecal leukocytes and fecal lactoferrin were positive in 33 (61.1%) and 14 (25.4%) patients, respectively. Among the 31 patients who were available for fecal pathogen evaluation, fecal multiplex PCR detected bacterial pathogens in 21 patients, whereas conventional stool cultures were positive in only one patient (67.7% vs 3.2%, p=0.000). Positive fecal lactoferrin was associated with presence of moderate to severe dehydration and detection of bacterial pathogens by multiplex PCR (21.4% vs 2.5%, p=0.049; 100% vs 56.5%, p=0.032, respectively).ConclusionsFecal lactoferrin is a useful marker for more severe dehydration and bacterial etiology in patients with acute diarrhea. Fecal multiplex PCR can detect more causative organisms than conventional stool cultures in patients with acute diarrhea.     

生殖器疱疹患者的单纯疱疹病毒PCR实验诊断研究

目的探讨对生殖器疱疹患者进行单纯疱疹病毒PCR实验诊断的必要性和实用性.方法采用聚合酶链反应(PCR)对生殖器疱疹患者组82例及对照组30例进行HSV检测.结果 GH患者组检出阳性75例,占91.46%,检出率明显高于对照组(P＜0.01).结论 PCR作为HSV感染的实验诊断技术具有很好的应用价值.