Application of analytical cytology to current issues in the biomedical sciences

多西紫杉醇是目前应用最广泛、最有效的抗肿瘤药物之一。虽然公认的紫杉烷药理活性机制包括稳定有丝分裂中的微管，但这种拟议的机制不能解释其许多临床和药理学特征。我们探索了乳腺癌细胞株中有丝分裂与多西紫杉醇活性的关系，这些细胞对药物表现出不同程度的敏感性。所有被检查的细胞株在用通常用于研究药物机制的多西紫杉醇水平(例如，10-100 NM)处理后，有丝分裂发生不可逆转的停滞。然而，剂量低得多的多西紫杉醇(例如，1 NM或更少)可以迅速引发高度敏感的乳腺癌细胞系，如HCC1806和MDA-MB-231的细胞死亡(具有凋亡特征)。早期细胞凋亡的典型变化(钝端DNA片段和膜联蛋白IV结合物的细胞表面暴露增加)在暴露后4小时内被检测到，尽管此时没有看到有丝分裂停止，出现在细胞周期的所有阶段。用延时照相法监测细胞证实，多西紫杉醇引发的细胞死亡不需要有丝分裂停止，甚至不需要通过有丝分裂过渡。这些数据表明，多西紫杉醇具有多种细胞杀伤机制，包括公认的涉及阻止细胞有丝分裂的机制。然而，重要的是，高敏感的乳腺癌细胞被相对较低水平的多西紫杉醇通过一种不涉及有丝分裂停止的机制杀死，并且是独立于细胞周期启动的。 随着能够常规检测8种或更多颜色的流动分析仪的不断开发和商业应用，适当的抗体-荧光色素组合的选择变得越来越复杂。除了仪器配置的不同之外，将最亮的荧光色素抗体结合物分配给表达最低的细胞抗原，选择减少光谱补偿需求的组合，以及避免明亮群体的信号扩散到用于解析产生假阳性事件的暗群体的检测器的荧光组合，是有效的一般性建议。在我们作为流式细胞仪核心的角色中，我们经常遇到用户他们的试剂板的设计和实施不是最优的。我们的经验似乎并不是独一无二的；例如，自2000年以来，在多色板中使用PerCP结合的CD19来识别和量化人类B细胞的引用已超过60次。这份清单还包括关于ZAP-70表达作为慢性淋巴细胞性白血病疾病进展和患者生存的替代标记物的价值的开创性论文(克雷斯波等人，2003年)。CD19-PerCP的使用似乎与直觉相反，似乎违反了最佳抗体-荧光色素选择的主要原则之一：CD19在细胞表面以低丰度表达，估计为27,000个分子/细胞，相比之下，CD45的表达是&gt；200,000个分子/细胞(Bikoue等人，1996)，PerCP通常被认为是一种暗淡的荧光色素，其相对强度通常为PE&gT；APC&gT；PE-Cy5&gT；PerCP&#8805；FITC。美国疾病控制和预防中心(CDC)和临床和实验室标准研究所(原NCCLS)的国际指南目前建议使用由CD3/CD4/CD8/CD45和CD3/CD19/CD56/CD45组成的四色板来计数正常人的CD4和CD8 T细胞、B和NK淋巴细胞亚群。在这种情况下，我们推测，在这样的四色试板中使用CD19-PerCP对提交给美国病理学家学会(CAP Flow Cytomology)和CDC(用于CD4+T细胞测定的模型性能评估程序)运行的性能评估计划的人类全血样本进行染色，将提供对使用不太理想的抗体-荧光色素组合构建的试剂板的现实限度的公正、准确的评估。我们将这些结果扩展到稳定的全血质量控制产品中的B细胞分析，这些产品模拟红细胞(RBC)裂解和光散射特性，最初是为临床新鲜全血的CD4+和CD8+T细胞亚群染色而开发的，以及在染色和分析之前用于稳定个别患者血液样本的专利试剂的效果。总体而言，这里的结果实质性地扩展了正常人类外周血B细胞计数所涉及的实际问题的有限知识库。

Docetaxel is one of the most widely used and most effective anti-neoplastic drugs currently available. While the generally accepted mechanism for pharmacological activity of taxanes involves stabilization of microtubules in mitosis, this proposed mechanism cannot explain many of its clinical and pharmacological characteristics. We explored the relationship between mitosis and activity of docetaxel in breast cancer cell lines that display variable levels of sensitivity to the drug. All cell lines examined show an irreversible arrest in mitosis after treatment with levels of docetaxel (e.g., 10-100 nM) that have been commonly used to study drug mechanisms. However, much lower doses of docetaxel (e.g., 1 nM or less) can rapidly trigger cell death (with features of apoptosis) in highly sensitive breast cancer cell lines, such as HCC1806 and MDA-MB-231. Changes typical of early apoptosis (blunt-end DNA fragments and increased cell-surface exposure of annexin IV conjugates) were detected within 4 hours of exposure, even though no mitotic arrest is seen at this time, appeared in all phases of cell cycle. Monitoring cells with time-lapse photography confirms that cell death initiated by docetaxel does not require mitotic arrest, or even the transition through mitosis. These data suggest that docetaxel has multiple mechanisms for cell killing, including the generally recognized mechanism involving arrest of cells in mitosis. Importantly however, highly sensitive breast cancer cells are killed by relatively low levels of docetaxel through a mechanism that does not involve mitotic arrest and is initiated independently of cell cycle. With the continued development and commercial availability of flow analyzers able to routinely detect 8 colors or more, the selection of the appropriate antibody-fluorochrome combinations has become increasingly complex. Aside from differences in instrument configurations, assigning the brightest fluorochrome-antibody conjugate to the cellular antigen with the lowest expression, choosing combinations that lessen the need for spectral compensation, and avoiding fluorochrome combinations where the signal of bright populations spreads into detectors used to resolve dim populations creating false positive events are effective general recommendations. In our role as a flow cytometry core, we frequently have encountered users where the design and implementation of their reagent panels is less than optimal. Our experience does not appear to be unique; for example, since 2000, there have been more than 60 citations using Peridinin chlorophyll protein (PerCP) conjugated CD19 in multicolor panels to identify and quantify human B-cells. This list also includes the seminal paper documenting the value of ZAP-70 expression as a surrogate marker in chronic lymphocytic leukemia disease progression and patient survival (Crespo et al., 2003). The use of CD19-PerCP appears counter-intuitive and would seem to violate one of the primary principles for optimal antibody-fluorochrome selection: CD19 is expressed at low abundance on the cell surface with estimates of 27,000 molecules/cell, by contrast CD45 expression is >200,000 molecules/cell (Bikoue et al, 1996) and PerCP is generally considered a dim fluorochrome, with the relative intensity generally PE > APC >PE-Cy5 >PerCP ≥FITC. International guidelines by the Centers for Disease Control and Prevention (CDC) and the Clinical and Laboratory Standards Institute (formerly NCCLS) currently recommend 4-color panels consisting of CD3/CD4/CD8/CD45 and CD3/CD19/CD56/CD45 for the enumeration of CD4 and CD8 T-cells, B, and NK lymphocyte subsets from normal individuals. In this context, we postulated that using CD19-PerCP in such a 4-color panel to stain human whole blood specimens submitted to performance evaluation programs run under the auspices of the College of American Pathologists (CAP Flow Cytometry Immunophentypic Analysis Program) and CDC (Model Performance Evaluation Program for CD4+ T-cell Determinations) would provide an unbiased, accurate assessment of the realistic limits of reagent panels constructed with presumably less than optimal antibody-fluorochrome combinations. We extend these results to B-cell analysis in stabilized whole blood quality control products that mimic red blood cell (RBC) lysis and light scatter properties, originally developed for clinical CD4+ and CD8+ T-cell subset staining of fresh whole blood and the effect of proprietary reagents used to stabilize individual patient blood specimens over prolonged periods of time prior to staining and analysis. Overall, the results here substantiality expand the limited knowledge base of practical issues involved in normal human peripheral blood B-cell enumeration.