Algorithm to identify circulating tumor cell clusters using in vivo flow cytometer

Recent studies in oncology have addressed the importance of detecting circulating tumor cell clusters because circulating tumor cell clusters might survive and metastasize more easily than single circulating tumor cells.Signals with larger peak widths detected by in vivo flow cytometer(IVFC)have been used to identify cell clusters in previous studies.However,the accuracy of this criterion might be greatly degraded by variance in blood°ow and the rolling behaviors of circulating tumor cells.Here,we propose a criterion and algorithm to distinguish cell clusters from single cells.In this work,we first used area-based and volume-based models for single°uorescent cells.Simulating each model,we analyzed the corresponding morphology of IVFC signals from cell clusters.According to the Rayleigh criterion,the valley between two adjacent peak signals from two distinguishable cells should be lower than 73.5%of the peak values.A novel signal processing algorithm for IVFC was developed based on this criterion.The results showed that cell clusters can be reliably identied using our proposed algorithm.Intravital imaging was also performed to further support our algorithm.With enhanced accuracy,IVFC is a powerful tool to study circulating cell clusters.

STUDYING THE ROLE OF MACROPHAGES IN CIRCULATING PROSTATE CANCER CELLS BY IN VIVO FLOW CYTOMETRY

Metastasis is a very complicated multi-step process and accounts for the low survival rate of the cancerous patients.To metastasize,t he malignant cells must detach from the primary tumor and migrate to secondary sites in the body through either blood or lymph circulation.Macrophages appear to be directly involved in tumor progression and metastasis.However,the role of macrophages in affecting cancer metast asis has not been fully elucidated.Here,we have utilized an emerging technique,namely in vivo flow cytometry(IVFC)to study the depletion kinetics of circulating prostate cancer cells in mice and determine how depletion of macrophages by the liposome encapsulated clodronate affects the depletion kinetics.Our results show diferent depletion kinetics of PC-3 cells between the macrophagedeficient group and the control group.The number of circulating tumor cells(CTCs)in the macrophage-deficient group decreases in a slower manner compared to the control mice group.The differences in depletion kinetics indicate that the absence of macrophages facilitates the stay of prostate cancer cells in circulation.In addition,our imaging data suggest that macrophages might be able to arrest,phagocytose and digest PC-3 cells.Therefore,phagocy tosis may mainly contribute to the de-pletion kinetic diferences.The developed methods elaborated here would be useful to study the relationship between macr ophages and tumor metastasis in small animal cancer models.

Short-term travel flow prediction method based on FCM-clustering and ELM

Short-term travel flow prediction has been the core of the intelligent transport systems(ITS). An advanced method based on fuzzy C-means(FCM) and extreme learning machine(ELM) has been discussed by analyzing prediction model. First, this model takes advantages of ability to adapt to nonlinear systems and the fast speed of ELM algorithm. Second, with FCM-clustering function, this novel model can get the clusters and the membership in the same cluster, which means that the associated observation points have been chosen. Therefore, the spatial relations can be used by giving the weight to every observation points when the model trains and tests the ELM. Third, by analyzing the actual data in Haining City in 2016, the feasibility and advantages of FCM-ELM prediction model have been shown when compared with other prediction algorithms.

Microchip imaging cytometer:making healthcare available,accessible,and affordable

The Microchip Imaging Cytometer(MIC)is a class of integrated point-of-care detection systems based on the combination of optical microscopy and flow cytometry.MIC devices have the attributes of portability,cost-effectiveness,and adaptability while providing quantitative measurements to meet the needs of laboratory testing in a variety of healthcare settings.Based on the use of microfluidic chips,MIC requires less sample and can complete sample preparation automatically.Therefore,they can provide quantitative testing results simply using a finger prick specimen.The decreased reagent consumption and reduced form factor also help improve the accessibility and affordability of healthcare services in remote and resource-limited settings.In this article,we review recent developments of the Microchip Imaging Cytometer from the following aspects:clinical applications,microfluidic chip integration,imaging optics,and image acquisition.Following,we provide an outlook of the field and remark on promising technologies that may enable significant progress in the near future.

THE CLINICAL SIGNIFICANCE OF FLOW CYTOMETRIC DEOXYRIBONUCLEIC ACID MEASUREMENT OF DEPARA-FFINIZED SPECIMEN IN BLADDER TUMOR

A retrospective study of flow cytometric measurements on paraffin-embedded tumor specimens from 188 patients with bladder tumor was conducted. The results were analyzed in combination with the morphological variation of bladder tumors. It was found that the DNA ploid pottern, degree of infiltration and the multiplicity of bladder tumor were closely related with tumor recurrence, among which the DNA ploid pattern was most significant. In aneuploid bladder tumors the recurrent rate and mean annual recurrence frequency were 76.7% and 1.46, and those in the diploid bladder tumors were 18.7% and 0.33 respectively. Aneuploid was the most indicative parameter of the recurrence in bladder tumors. In addition, according to the DNA ploid pattern and DNA index (DI), the aneuploid tumors in our group were divided into 4 types, namely, tetraploid tumors, npn-euploid with DI(?)1.5, non-euploid tumors with DI>1.5 and two-aneuploid tumors. The results showed that the recurrent rate of tetraploid tumors was relatively lower and it became higher and higher in the following order: non-euploid tumors with DI(?)1.5, non-euploid tumors with DI>1.5, and two-aneuploid tumors. This indicates that there are different biological behaviors in tumors with different ploid pattern. Finally, the correlation between DNA ploid pattern and tumor metastasis was also discussed.

The Role of Kappa and Lambda in Subclassification of B Cell Lymphoblastic Leukemia in Sudanese Patients Using Flow Cytometry

Background: B-cell Acute lymphoblastic leukemia (B-ALL) is a neoplasm of lymphoblasts which are of B-cell lineage typically composed of small to medium sized blast cells, moderately condensed to dispersed chromatin with scanty cytoplasm and inconspicuous nucleoli, involving the bone marrow and/or blood. Methods and materials: This is a prospective cross-sectional study in which 50 blood and/or bone marrow samples of newly diagnosed patients (B-ALL) were tested for immunophenotyping. All samples were prepared for surface and cytoplasmic markers including kappa and lambda human antibody for 10 minutes in dark place and then run by the Flow Cytometer. Results: 64% of the study populations were males and 36% were females. Cases were classified according to immunophenotype and the age into different subtypes and showed the following frequencies: Pro B-ALL (8%), early pre B-ALL (56%), common B-ALL (16%), Pre-B-ALL (14%) and Mature B-ALL (only 6%). Surface immunoglobulin was positive in 10% and negative in 90% of all patients, showing 100% positivity in mature B-ALL and totally negative in other subtypes. While cytoplasmic immunoglobulin was positive in 16% and negative in 84% of all patients and was positive in 100% of Pre-B-ALL and in 50% of mature B-ALL. Surface kappa was more expressed in mature B-ALL than lambda giving a ratio of 2:1, while cytoplasmic kappa:lambda was 6:1 in Pre-B-ALL. Conclusion: Kappa and lambda have important role in B-ALL classification which necessitates their presence in immunophenotyping of B-ALL.

Temporal change of plankton size structure preserved by Lugol's solution:a FlowCAM study

Plankton size structure is crucial for understanding marine ecosystem dynamics and the associated biogeochemical processes.A fixation step by acid Lugol’s solution has been commonly employed to preserve plankton samples in the field.However,the acid Lugol’s solution can bias the estimation of size structure and the preserved plankton size structure can vary with time.Here,we explore the impact of sample storage time on the size-structure of the plankton community preserved by Lugol’s solution.Two short-term experiments and one long-term experiment were conducted to explore the change of plankton community size structure with the storage time:covering from a week to a month,and to nearly seven months based on particle-size data obtained by continuous Flow Cytometer and Microscope(FlowCAM)measurements.We found a linear change of plankton size with the storage time in short-term periods(less than 3 months)with a decrease of the slope but an increase of the intercept for the normalized biomass size spectrum(NBS S).However,there were opposite trends for NBSS with increasing slope but decreasing intercept after3 months.The potential causes of the distinct patterns of the NBSS parameters are addressed in terms of the interplay between particle aggregation and fragmentation.We found large changes in plankton biovolume and abundance among different size classes,which may indicate a distinct effect of acid Lugol’s solution on various plankton size classes.The mechanism driving temporal change in the size-structure of the Lugolfixed plankton community was further discussed in terms of particle aggregation and fragmentation.Finally,we emphasize that the effect of storage time should be taken into account when interpreting or comparing data of plankton community acquired from samples with various storage durations.

流式细胞术检测细胞周期影响因素及不同免疫细胞亚群周期分析

细胞周期检测对于了解细胞增殖状态、细胞功能研究、药物筛选与评估等领域具有重要意义。流式细胞仪分析细胞周期是目前较为经典且广泛应用的检测手段之一。碘化丙啶(propidium iodide,PI)单染是最常用的基于流式的检测细胞周期方法,然而,该方法在使用过程有诸多处理因素会对检测结果造成影响。此外,不同免疫细胞亚群在不同细胞周期阶段的分布差异也有助于研究免疫反应,了解疾病状态。本文以B16-F10细胞系为例,采用PI单染法,从固定条件、上机条件和软件分析3个角度,评价多种处理因素对细胞周期检测结果的影响。根据结果分析,在进行细胞周期检测时,建议取3×10^(6)个细胞,用300μL预冷PBS重悬细胞后逐滴滴入700μL预冷的无水乙醇,放置于4℃或-20℃过夜固定,低速收样,收样速率为每秒400~600颗粒数,去黏连后需收集至少3000个细胞。另外,通过EdU、PI双指标染色可以精确划分细胞周期,而细胞表面标志物染色联合Ki-67和PI染色法,可在不进行细胞分选的情况下进行不同免疫细胞亚群周期分析。本文较为系统的探讨了PI单染法检测细胞周期的影响因素,提供了标准的实验操作流程。建立了EdU联合PI检测细胞周期和针对不同免疫细胞亚群周期分析方案,拓宽了细胞周期检测方式。

流式细胞仪平台支撑本科细胞生物学实验课教学改革的实践

在流式细胞术检测细胞凋亡实验中,借助流式细胞仪平台,从教学模式、实验内容、仪器准备、教学素材等方面对细胞生物学实验课进行改革,取得了较好的教学效果。这种仪器平台与教学团队共同探索教学改革的模式,充分发挥大型仪器设备在人才培养方面的潜能,可为其他高校仪器平台建设提供参考。

流式细胞仪样品预处理系统的设计与实现

根据流式细胞仪对样品“均一化”的需求,研制了一套基于微流控细胞分选技术的流式细胞仪样品预处理系统,装置可以实现对粒径小于100µm细胞或微粒的驱动和分选.系统主要包含微流控分选芯片和样品驱动模块两部分,通过聚焦不同位置,实现了对不同粒径细胞/颗粒的有效分离.系统无需对细胞进行标记处理,经分选的细胞便于后续流式细胞仪检测.经验证,系统能够有效去除牡蛎血淋巴细胞样品中的大粒子杂质,提高细胞样品的稳定性和均一性,增加流式细胞仪检测结果的准确性.

利用流式细胞术鉴定3种李属植物基因组大小及染色体倍性检测

【目的】研究适合不同李属资源的FCM检测体系,估测其基因组大小及倍性,为李属资源的鉴定、分类及基因组和转录组等深入研究提供依据。【方法】利用FCM对3种常用的细胞核裂解液进行筛选与优化,选取最适宜的裂解液鉴定3种李属植物进行基因组大小和检测染色体倍性。【结果】LB01、Marie’s和WPB 3种裂解液对樱桃李检测效果均较好,而WPB裂解液检测效果最佳;Marie’s和LB01裂解液与欧洲李不匹配,改良的WPB裂解液最适合欧洲李;杏李与LB01裂解液不匹配,WPB裂解液对杏李的裂解效果好于Marie’s裂解液,而改良的WPB裂解液对杏李的裂解效果均最好。樱桃李和杏李基因组大小范围分别为491.22~566.40 Mb和514.46~573.83 Mb,欧洲李资源平均基因组大小为1678.37 Mb。【结论】樱桃李和杏李资源均为二倍体,欧洲李资源全部为六倍体,李属植物每个种内不同品种之间的基因组大小存在一定差异。

基于Sub_FCM聚类算法的交通流量段自动划分方法

基于FCM算法提出一种结合减法聚类与聚类有效性评判的模糊c均值聚类算法Sub_FCM,该算法能自动确定合理划分类数并初始化聚类原型,建立一种基于交替优化策略的无监督机器学习自动分类模型,并详细阐述了该模型在怀柔交叉路口流量段自动划分中的具体应用.实例分析表明,该算法能很好地反映路口交通流数据的内在结构,自动划分出合理流量段,为进一步实施合理的控制算法奠定基础.

改进FCM的交通流缺失数据修复方法

针对传统模糊C均值(FCM)算法中聚类数目和模糊度指数由先验知识人为确定而影响聚类结果精度的问题,提出一种改进FCM的交通流缺失数据修复方法。首先根据模糊决策理论,使用FCM目标函数和划分熵共同确定最优模糊度指数;其次利用模拟退火算法优化FCM的聚类数目;最后结合交通流数据的时空相关性提升修复效果。以美国加州运输局运行监测系统提供的高速公路交通数据为研究对象,实验结果表明:该方法克服了传统FCM算法全局搜索能力不足的问题,与传统FCM算法相比,其均方根误差降低了约30%。

多参数流式细胞技术分析髓系白血病细胞相关免疫表型

目的 探讨常见的急性髓系白血病细胞相关免疫表型,并阐述其在临床中的应用价值。方法 应用多参数流式细胞技术分析47例急性髓系白血病(AML)患者骨髓标本中白血病细胞相关免疫表型,并对其免疫分型结果与治疗、预后的相关性进行统计分析,进而探讨AML免疫分型在临床中的应用。结果 47例AML患者阳性高表达的髓系抗原有CD33(93.62%)、CD13(89.36%)、MPO(89.36%)、CD117(82.98%)、CD64(38.30%);造血干/祖细胞CD分子阳性表达以CD38(93.62%)、CD34(65.96%)、HLA-DR(51.06%)为主;其他抗原以CD56(48.94%)、CD7(23.40%)、CD19(19.15%)为主。经相同时间治疗后,23例(48.94%)AML患者病情得到完全缓解,患者阳性表达T细胞系抗原CD7的完全缓解率显著低于阴性患者(P<0.05)。6例(26.09%)患者在1 a内复发,没有任何一种抗原表达情况与之有关(P>0.05)。随访结束后,有26例(55.32%)患者死亡,阳性表达NK细胞系抗原CD56和T细胞系抗原CD7患者的死亡率显著性高于阴性表达的患者,阳性表达B细胞系抗原CD19的患者死亡率显著性低于阴性表达的患者(P<0.05),幼稚标志与成熟标志不同步的患者死亡率高于幼稚标志与成熟标志同步的患者(P<0.05)。结论 髓系抗原CD33、CD13、MPO、CD117、CD64是AML诊断中可靠的抗原标志物,AML患者的免疫分型结果可为临床医生确定治疗方案和判断预后提供参考。

FITC-AnnexinV/PI双染色FCM检测非小细胞肺癌药物敏感性

目的 检测非小细胞肺癌 ( NSCLC)体外化疗药物敏感性。方法 采用 FITC-Annexin V/PI双染色双变量流式细胞分析法。结果 49例非小细胞肺癌对化疗药物的敏感度由高至低依次为泰素、顺铂、丝裂霉素、诺维本、长春地辛和依托泊苷。结论 本试验是一种新的化疗药物敏感性检测方法。 6种化疗药物对 NSCLC标本的敏感度与临床单剂化疗疗效基本一致 ,提示该检测对提供临床个体化化疗方案的选择具有重要的参考价值。

依托大型科研仪器开展创新型本科实验教学的设计与研究

大型科研仪器是先进科学技术的体现,是培养创新型人才的重要基础条件和硬件保障,是开展高水平实验教学的宝贵资源。为促进创新型研究人才的培养,拓展生物医学本科实验教学内容,该研究设计了一套依托流式细胞仪开展的本科实验教学的具体方案,针对经典的“细胞增殖/细胞周期”问题,分别设计两个不同原理的流式细胞术进行对比实验。实践表明,依托流式细胞仪开展对比实验教学,能加深本科生对大型科研仪器的了解,培养学生扎实的动手能力,激发学生的创新性思维和科研热情。

应用流式细胞技术(FCM)检测儿童APL微小残留病

目的:研究应用流式细胞技术(FCM)检测APL病人的白血病微小残留病(MRD),评价其临床意义.方法:应用FCM检测和分析36例APL病儿在初发期和完全缓解期的CD13、CD33、CD34和TdT表达,全部APL病人每日输入AS2O3治疗,直到完全缓解(CR).结果:36例APL病人在初发期CD13、CD33表达均明显高于对照组(P<0.05),其阳性表达率分别为63.88%(23/36)和80.55%(29/36),CD34和TdT均为低表达,经AS2O3治疗的CR期有34例APL病人CD13和CD34阳性细胞为10‰左右,其余2例仍为高表达(CD13和CD33抗原阳性细胞>10%～20%),并于3个月内复发.结论:研究结果推断,应用FCM检测APL病人CD13和CD33表达可预测复发,FCM分析结果可提高检测MRD的敏感性并可估计APL病人的预后,CD13和CD33双表达可能预后不良.

基于MALDI-TOF MS与FCM联合应用对临床血流感染病原体鉴定和药敏试验新方法的建立和应用

目的探讨基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF MS)与流式细胞学(FCM)联合应用在临床血流感染(BSI)病原体快速鉴定和药敏试验中的意义及应用价值。方法采用分离胶-吸附法菌体分离系统直接将菌体从血培养阳性瓶中分离,通过流式细胞学(FCM)与基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF MS)联合应用,快速对病原体进行鉴定和药敏试验。结果 MALDI-TOF MS法和VITEK2系统对常见菌都有超过90%的鉴定到种的能力(t=12.70,P=0.213)。MALDI-TOF MS较VITEK2有更高的鉴定到群和属的能力(t=6.31,P=0.027;t=8.64,P=0.031)。FCM法的药敏结果与VITEK2系统结果几乎完全一致(t=4.30,P=0.127),且FDA的加入可以很好地发现感染菌群中的耐药异质性。结论联合应用MALDI-TOF MS与FCM对临床BSI病原体进行鉴定和药敏试验的结果与传统方法间有很好的一致性,且报告时间可由传统的36,72h缩短至3h以内。该方法对BSI的快速诊断有较大意义和较好的临床应用价值。