Rapid,effective and user-friendly immunophenotyping of canine lymphoma using a personal flow cytometer

Background

Widespread use of flow cytometry for immunophenotyping in clinical veterinary medicine is limited by cost and requirement for considerable laboratory space, staff time, and expertise. The Guava EasyCyte Plus (Guava Technologies, Hayward, CA, US) is the first, personal, bench-top flow cytometer designed to address these limitations.

Objective

The aim of this study was to adapt the immunohistochemical protocol used for immunophenotyping of canine lymphoma to the personal flow cytometer for rapid, effective and user-friendly application to the diagnosis and prognosis of canine lymphoma and to demonstrate its practicality for widespread veterinary application. Performance of the personal flow cytometer for immunophenotyping T and B lymphocytes in blood and lymph nodes from normal dogs and dogs with lymphoproliferative disease, was assessed using only two monoclonal antibodies (against CD3 and CD21), and by comparison with analysis using two conventional flow cytometers.

Methods

26 dogs with lymphoproliferative disease (23 with lymphoma, 3 with lymphocytic leukaemia) were studied along with 15 controls (2 non-lymphoma lymph nodes and 13 non-leukemic bloods. Lymphocytes were immunostained with fluorescent-labeled, monoclonal antibodies against CD3 and CD21. To assess the effectiveness of the personal flow cytometer in discrimination between T and B cell immunophenotypes, T and B cell counts for half the samples (14 blood and 11 lymph node) were also determined using the same method and conventional flow cytometers (FACSCalibur, Cyan Dako). To assess the effectiveness of the personal flow cytometer in discriminating between leukocyte types, lymphocyte differential counts were determined for 21 blood samples and compared with those from automated hematology analyzers (CELL-DYN 3500, n=11 and ADVIA 2120, n=10). Quality and sub-cellular distribution of immunostaining was assessed using fluorescence microscopy.

Results

The protocol for immunophenotyping took 2 to 3 hours to complete from the point of receipt of sample to reporting of immunophenotype. The personal flow cytometer differential lymphocyte counts correlated highly (n=20; r=0.97, p<0.0001) with those of automated haematology analyzers. The personal flow cytometer counts consistently, but mildly, underestimated the percentages of lymphocytes in the samples (mean bias of -5.3%.). The personal flow cytometer immunophenotype counts were indistinguishable from those of conventional flow cytometers for both peripheral blood samples (n=13; r=0.95; p<0.0001; bias of -1.1%) and lymph node aspirates (n=11,r=0.98; p<0.001; bias of 1%). All but one leukemic and one lymphomatous lymph node sample, out of 26 samples of dogs with lymphoproliferative disease analyzed, could be immunophenotyped as either B or T cells.

Conclusions

We conclude that use of only 2 monoclonal antibodies is sufficient for immunophenotyping most cases of canine lymphoma by flow cytometry and enables rapid immunophenotyping. The personal flow cytometer may be as effectively used for immunophenotyping canine lymphoma as conventional flow cytometers. However, the personal flow cytometer is more accessible and user-friendly, and requires lower sample volumes.     

土壤细菌趋化性研究进展

土壤是地球生态系统中最具生命活性的组成部分,特有的孔隙结构承载着生物圈中最丰富多样的微生物生命形态,为动植物提供了大量的调控功能。土壤是一个不断演变和发展的生态系统,而微生物是土壤生态系统的核心,也是驱动碳、氮等元素以及能量循环的关键因子。趋化性是细菌在长期进化过程中形成的帮助其寻找食物或趋利避害的本能,结合其他的内在生理特征,细菌能够迅速适应动态变化的环境。营养物、异源污染物和水分条件等的不均匀分布致使土壤中细菌趋化现象普遍存在,并且时刻影响土壤微生物的群落组成及其时空分布。近年来,土壤细菌趋化性已成为国内外土壤微生物学研究的热点和重点。本文分析了土壤细菌趋化性研究的前沿问题和主要进展,阐述了细菌趋化行为模式、趋化信号传导通路和趋化性数学模型,探讨了土壤中普遍存在的细菌趋化现象及相关的主要研究技术手段(荧光原位杂交技术、微流控技术和光学显微技术),并对土壤细菌趋化性研究的发展趋势进行了展望,旨在为今后的相关研究和实际应用提供参考。     

基于培养池阵列微流控芯片的单线虫并行分离条件考察

秀丽隐杆线虫是一种重要的模式生物，已广泛应用于生物医药、农业和植物方面的研究，但线虫体态微小，常规方法难以实现对单条线虫的精准操控和长期追踪。本研究基于微流体控制技术，设计了培养池阵列微流控芯片，提出一种单线虫并行分离方法。通过考察液体培养时的线虫密度、线虫体宽及其分布等影响因素，分析在芯片上进行单线虫并行分离的条件。结果显示，采用无菌液体培养时，线虫密度对线虫体宽及其分布具有明显影响，高密度（约700条/mL）培养组和低密度（约300条/mL）培养组的线虫体宽分别为 （35.5±5.2） μm和（40.0±1.8） μm，且低密度下培养的线虫可获得较好的芯片分离效果，所得含线虫与含单线虫培养池的比例分别为83.3%和66.7%。该并行分离方法相对简便、可靠，结合其微流控芯片结构和流体控制方式，有望用于自动化单线虫长期培养和观测研究。