流式细胞术的临床应用

流式细胞术(flowcytometry,FCM)是20世纪70年代发展起来的对单细胞定量分析的一种新技术。它借鉴了荧光标记技术、激光技术、单抗技术和计算机技术,具有极高的检测速度与统计精确性,而且从单一细胞可以测得多个参数,为生物医学与临床检验提供了全新视角和强有力手段。目前,随着单克隆抗体技术的发展,流式细胞仪检测技术已经广泛使用在基础研究和临床实践的各个方面,在肿瘤学、血液学,免疫学、微生物学、细胞生物学-遗传学及临床检验学等诸多领域内发挥着重要作用[1]。本文就其在临床上的应用综述如下。1流式细胞仪的工作原理流式细胞仪主要由细胞流动室、激光聚焦区、检测系统、数据处理系统等4部分组成,其工作原理是将待测标本制成单细胞悬液,经染色后进入流动室,流动室充满流动的鞘液,鞘液压力与样品压力是不同的,当两者的压力差异达到一定程度时,鞘液裹挟着的样品流中细胞排成单列逐个经过激光聚焦区。若将细胞中感兴趣的部分特异性地标上荧光染料,那么这些染料将在细胞通过激光检测区时受激光产生特定波长的荧光,通过一系列信号转换、放大、数字化处理,就可以在计算机上直观地统计染上各种荧光染料的细胞各自的百分率。选择不同的单克隆抗体及荧光染料...     

The role of flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria in the clinical laboratory

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder closely related to aplastic anemia. Hemolytic anemia and life-threatening thromboses are common features in many patients. Rapid diagnosis is highly desirable and flow cytometry plays a key role in the laboratory investigation of PNH. By demonstrating absence of cell membrane glycosylphosphatidylinositol-anchored proteins from granulocytes or red cells, a definitive diagnosis of PNH can be established. This can have a considerable impact on patient management and outcome. As with all rare diseases, internal and external quality assurance is essential for good laboratory practice and to fulfill the requirements of national laboratory accreditation schemes.     

Quality control in clinical flow cytometry

Immunophenotyping by flow cytometry is a robust and highly complex technology used in the enumeration and characterization of leukocytes, including normal lymphocyte subsets and hematologic neoplasms. Samples consist of peripheral blood and many times irreplaceable samples, such as bone marrow and fresh tissue. These samples are collected, transported, prepared, analyzed, and interpreted, resulting in diagnostic and prognostic information. Such information is critical to treatment decisions for patients. In order to obtain accurate and reproducible results, it is essential to have optimized and standardized procedures, rigorous quality control, and assurance programs encompassing preanalytic, analytic, and postanalytic processes.     

流式细胞术的研究进展及临床应用前景

自从商品化的流式细胞仪在20世纪70年代进入市场以来,流式细胞术(flow cytometry,FCM)检测的项目不断地由科研转向临床应用领域,并在临床免疫学、血液肿瘤学及微生物学等疾病诊断和治疗等方面起到不可或缺的作用.现对该技术的进展概述如下.     

The role of flow cytometry in improving biocompatibility in transfusion medicine

In transfusion medicine, blood and blood components, donors and patients are increasingly confronted with biomaterials. The need to understand the response of human blood to contact with these artificial surfaces has led to multiple studies on the biocompatibility of biomaterials. Up to this time, these investigations have predominantly been performed using physical, immunological and biochemical methods. Many of these approaches are useful in investigating the multiple factors involved in blood-biomaterial interactions. However, they always reflect the overall behaviour of whole cellular populations in local or systemic reactions. The application of multiparameter flow cytometry, on the other hand, provides insight into antigenic expression and changes at the single-cell level. Therefore, the technique of flow cytometry represents a new and powerful way of analysing and improving the biocompatibility of these materials in blood-contacting applications in this field.     

The role of denial in clinical practice

Denial is typically viewed as maladaptive in clinical practice, despite ample evidence of its adaptive role in personal and health cases The failure of health professionals to embrace the positive aspects of denial lies deeply rooted in cultural beliefs, which have been reinforced in more recent times by misapplication of clinical theory Denial is a form of self-deception that protects the individual from threats to the self and involves exaggerated perceptions of control and self-efficacy Social-psychological research suggests that such biases in cognitive appraisal are the norm and not exclusive reactions to cases It is proposed that denial has the following functions during cases denial protects the integrity of the self-concept by distorting reality in a self-enhancing way, promoting a sense of mastery and control, this in turn leads to lower levels of anxiety, which may enhance decision-making under conditions of stress Psychological health appears to be contingent, at least in part, on an ability to see the world through 'rose-tinted glasses' Health professionals should be aware, therefore, that whilst trying to promote psychological well-being we do not ask patients to be unrealistically realistic     

流式细胞分析在阿霉素耐药性研究中的重要作用

目的探讨流式细胞仪（ＦＣＭ）在肿瘤耐药细胞系（Ｓ－１８０Ｒ与ＢＧＣ－８２３／ＤＯＸ）阿霉素耐药性检测中的应用价值。方法应用细胞药物毒性分析（ＭＴＴ）、ＤＮＡ分子杂交（ｓｏｕｔｈｅｒｎｂｌｏｔ）、逆转录聚合酶链反应（ＲＴ－ＰＣＲ）、免疫组化、ＦＣＭ的检测方法，在ＤＮＡ、ＲＮＡ、蛋白质水平以及耐药倍数、细胞内药物含量等方面对肿瘤耐药细胞系进行分析。结果Ｓ－１８０Ｒ耐药倍数６６；耐药细胞多药耐药基因出现ＤＮＡ扩增、ＲＮＡ转录、蛋白水平的过度表达，亲本细胞为阴性。亲本细胞与耐药细胞药物荧光含量为２５９２．９，耐药培养２年后，耐药细胞峰半峰宽／峰高比值由０．５６变为０．２３。ＢＧＣ－８２３／ＤＯＸ耐药倍数为６．４倍；耐药和亲本细胞均有多药耐药基因ＤＮＡ扩增、ＲＮＡ转录、蛋白过度表达；亲本与耐药细胞药物荧光含量为６３８．５３２６．１。结论ＦＣＭ可以直观地检测阿霉素耐药细胞，具有灵敏、准确、定量的特点，是肿瘤耐药性灵敏的、功能性检测方法     

全自动图象细胞仪在膀胱移行细胞癌尿细胞学诊断中的应用及临床意义

目的探讨全自动图像细胞仪对尿脱落细胞DNA异倍体的检测在膀胱肿瘤诊断中的价值及临床意义。方法采用全自动图像细胞仪对40例膀胱移行细胞癌(BTCC)患者和20例良性血尿患者尿样中脱落细胞进行DNA定量分析,同时行常规尿细胞学检查。结果BTCC患者尿液脱落细胞经常规尿细胞学和全自动图像细胞仪检查,阳性率分别为27.5%(11/40)和67.5%(27/40),20例良性血尿患者尿液标本经2种方法检查均为阴性。DNA倍体在膀胱移行细胞癌患者的尿液中为(7.08±2.34)c,在非膀胱移行细胞癌患者的尿液中为(2.95±3.06)c,P<0.01;临床分期Tis~T1为(6.29±3.61)c,T2~T3为(8.55±4.16)c,病理分级Ⅰ级为(5.85±1.72)c,Ⅱ级为(7.24±2.01)c,Ⅲ级为(9.25±1.81)c,DNA含量随着分期分级的增加而增加。结论应用全自动图像细胞仪对BTCC患者尿脱落细胞DNA的定量分析能够反映患者尿细胞中的恶性变化,可以作为BTCC诊断的辅助技术。     

Clinical laboratory automated urinalysis: comparison among automated microscopy, flow cytometry, two test strips analyzers, and manual microscopic examination of the urine sediments

Urinalysis is one of the habitual clinical laboratory procedures, which implies that one of the largest sample volumes currently requires significant labor to examine microscopic sediments. Different analyzers currently used to perform this task have been compared with the manual microscopic sediment examination. The Atlas Clinitek 10 (Bayer Corporation, Diagnostics Division, Tarrytown, NY) and Urisys 2400 (Hitachi Science Systems Ltd., Ibaraki, Japan) test strips analyzers and two automated urinalysis systems, Sysmex UF-100 (Sysmex Corporation Kobe, Japan) and IRIS iQ200 (International Imaging Remote Systems, Chatsworth, CA), have been considered. We assessed the concordance between the results obtained from 652 freshly collected urine samples for erythrocytes (RBC), leukocytes (WBC), squamous epithelial cells (EC), nitrites/bacteria, and crystals using the methodologies mentioned. A principal components analysis was performed in order to examine the correlation between these parameters. Instrument accuracy was also assessed. The Spearman's statistic (p) showed an adequate agreement between methods for RBC (iQ200=0.473; UF-100=0.439; Atlas=0.525; Urisys=0.539), WBC (iQ200=0.695; UF-100=0.761; Atlas=0.684: Urisys=0.620), and bacteria/nitrites (iQ200=0.538; UF-100=0.647; Atlas=0.532; Urisys=0.561) counts. By applying the Wilcoxon and McNemar tests, a concordance degree was found between 82-99 and 52-95% for the values obtained from the two test strips analyzers considered and from the iQ200 and UF-100 systems, respectively. From these results, we can conclude that both test strips analyzers are similar and, on the other hand, that automated urinalysis is needed to improve precision and the response time; but sometimes manual microscopic revisions are required, mainly when flags, because of crystals, are detected.     

再谈尿流式有形成分分析在尿路感染诊断中的应用

编辑同志:拜读贵刊2009年第6期刊载的<尿流式有形成分及干化学分析在尿路感染诊断中的应用评价>[1]、[2]及2010年第4期顾兵等[3]的读者来信<对尿流式有形成分用于尿路感染诊断的思考>,收获很大.笔者就尿流式有形成分在尿路感染(UTT)诊断中的应用及顾兵等来信中提出的诸多问题,浅谈一下自己的看法和观点,仅供广大读者参考.     

再谈尿流式有形成分分析在尿路感染诊断中的应用

编辑同志:拜读贵刊2009年第6期刊载的<尿流式有形成分及干化学分析在尿路感染诊断中的应用评价>[1]、[2]及2010年第4期顾兵等[3]的读者来信<对尿流式有形成分用于尿路感染诊断的思考>,收获很大.笔者就尿流式有形成分在尿路感染(UTT)诊断中的应用及顾兵等来信中提出的诸多问题,浅谈一下自己的看法和观点,仅供广大读者参考.     

再谈尿流式有形成分分析在尿路感染诊断中的应用

编辑同志: 拜读贵刊2009年第6期刊载的<尿流式有形成分及干化学分析在尿路感染诊断中的应用评价>[1]、[2]及2010年第4期顾兵等[3]的读者来信<对尿流式有形成分用于尿路感染诊断的思考>,收获很大.笔者就尿流式有形成分在尿路感染(UTT)诊断中的应用及顾兵等来信中提出的诸多问题,浅谈一下自己的看法和观点,仅供广大读者参考.     

The role of blood microfilters in clinical practice

Blood filters have been available since the 1930s. In this review we evaluate the role of microaggregate filters (MF) in certain transfusion complications, namely non-haemolytic febrile transfusion reactions (NHFTR), pulmonary injury, thrombocytopenia, fibronectin depletion and histamine release. We review the latest generation of leucocyte depleting filters and discuss their role in preventing alloimmunisation, immunosuppression and CMV transmission. Finally, we provide a rationale for the role of blood microfiltration in the present day practice of intensive care medicine.     

Use of quality control standards in clinical flow cytometry

Flow cytometry is important in the clinical laboratory, especially in hematology and cancer diagnosis. With newer applications being continuously developed clinicians who will use the results and laboratory staff who produce them should be aware of the instrumental quality control procedures required. This will allow evaluation of the reliability as well as the comparison of results between different laboratories. While information about sample preparation as well as staining procedures is easily available, information about instrumental quality control procedures is less so. Various standards and controls are available commercially and their correct choice and usage requires an understanding of the methodology of instrumental quality control. Methods to evaluate the precision, sensitivity and accuracy of measurements are discussed. Most laboratories report immunophenotyping data as the percentage of antibody positive cells and make no attempt to quantitate the amount of antibody binding per cell. Methods for quantitating the number of bound antibody molecules per cell along with a simple technique to compare results between laboratories are discussed. These techniques should encourage laboratory staff and clinicians to standardise methodology and to exchange patient data between laboratories.     

Flow cytometry of urine

Flow cytometry is becoming an increasingly important tool for the evaluation of urine cytology. Although it has established a strong foothold in oncologic DNA analysis, studies of whole cells and cell markers are only in their infancy. In addition, potential diagnostic applications such as those involving binding of E. coli strains to uroepeithelial cells will move flow cytometry from a research tool to a real diagnostic tool.