Application of analytical cytology to current issues in the biomedical sciences

癌症干细胞的概念及其对化疗反应的潜在影响是癌症研究中的一个非常感兴趣的领域。一种这样的药物，厄洛替尼，一种表皮生长因子受体（EGFR）的抑制剂，目前用于治疗非小细胞肺癌（NSCLC），然而，即使在高度敏感的肿瘤中，反应也是不完全的，其基础尚不清楚。 为了检验细胞的耐药亚群可能导致不完全应答的假设，基于CD 24和CD 44表面标志物表达对H1650和H1975 NSCLC细胞系进行分选。 在这两种情况下，缺乏CD 24和CD 44表达的细胞（CD 24-/CD 44-）在克隆形成试验中对厄洛替尼治疗最具抗性。 无论是CD 24-/CD 44-表型还是对厄洛替尼的相对耐药性都与Hoechst 33342染料的外排增加无关，这表明该推定干细胞的测定无法解释药物敏感性。然而，CD 24-/CD 44-细胞的特征在于EGFR信号通路的差异，支持非小细胞肺癌可能由异质性细胞群组成的假设，具有可变的细胞信号活性和对厄洛替尼的可变反应。 多色免疫表型分析具有潜在地分辨新的临床输入淋巴细胞亚群的能力，同时最小化样品处理和与在多个管中使用冗余抗体相关的成本。通常，用于选择适当荧光染料组合的经验法则是将最亮的标记（PE、APC或PE-Cy 5）分配给与具有最低表达的目标蛋白反应的抗体。多种因素可以影响亮度的感知，包括荧光染料本身的生物物理特性和仪器相关变量。多甲藻素叶绿素蛋白（PerCP）是甲藻Glenodinium sp.光合复合物的一个亚基，具有极高的量子效率和大的斯托克斯位移，但它容易光漂白，从而限制了它在高功率空气中喷射流式细胞仪中的有用性，并限制了它在低功率固定光束系统中的使用。PerCP用于荧光分析，因为它与其他常见荧光染料的光谱重叠最小;然而，它通常被认为更适合于以中等或高密度表达的抗原。在设计和测试用于主要淋巴细胞亚群（CD 4和CD 8 T细胞、B和NK细胞）的常规免疫表型分析的混合4色面板的过程中，可以容易地使用PerCP缀合的抗体，而不会牺牲以相对低密度表达的常见抗原（如B细胞）的准确度。然而，当在稳定的血液样品中使用PerCP结合的CD 19抗体时，染色显著改变。此外，细胞固定和透化是测量细胞内抗原的必要条件，如在ZAP-70表达的情况下，ZAP-70表达是慢性淋巴细胞白血病疾病进展和存活的替代标志物。在这些条件下，准确计数的B细胞优先和显着改变，无论是测量与PE，PerCP，或PEAF 700共轭荧光染料。总体而言，这种灵敏度降低导致未标记细胞（对照）和标记细胞的背景荧光之间的分离较差，从而混淆了B细胞数量的准确测量，并在这些条件下染色的这些标志物的临床价值的有效性中引入了误差。除本研究外，我们还使用市售的细胞稳定化试剂检查了B细胞标志物的稳定性，以稳定细胞形态并保持细胞抗原表达。

The concept of cancer stem cells and their potential impact on chemotherapeutic responses is an area of intense interest in cancer research. One such agent, erlotinib, an inhibitor of the epidermal growth factor receptor (EGFR), is currently used for treatment of non-small cell lung cancer (NSCLC), however, the response is incomplete in even highly sensitive tumors and the basis for this is not known. To test the hypothesis that resistant sub-populations of cells of may be responsible for the incomplete response, the H1650 and H1975 NSCLC cell lines were sorted on the basis of CD24 and CD44 surface marker expression. In both cases, cell lacking both CD24 and CD44 expression (CD24-/CD44-), were the most resistant to erlotinib treatment in clonogenic assays. Neither the CD24-/CD44- phenotype nor relative resistance to erlotinib was associated with increased efflux of Hoechst 33342 dye, suggesting that this assay for putative stem cells could not explain drug sensitivity. However, CD24-/CD44- cells are characterized by differences in EGFR signaling pathways, supporting the hypothesis that non-small cell lung cancers may consist of heterogeneous populations of cells, with variable cell signaling activity and variable responses to erlotinib.We are currently awaiting the results from xenograft transplantation into NOD/SCID mice. Polychromatic immunophenotypic analysis has the capability to potentially resolve new clinically import lymphocyte subsets, while at the same time minimizing sample processing and the costs associated with the use of redundant antibodies in multiple tubes. Generally, the rule of thumb for selecting the appropriate fluorochrome combination is to assign the brightest label (PE, APC, or PE-Cy5) to the antibody that reacts with the protein of interest having the lowest expression. Multiple factors can affect the perception of brightness including the biophysical properties of the fluorochrome itself and instrument-dependent variables. Peridinin chlorophyll protein (PerCP), a subunit of the photosynthetic complex of the dinoflagellate Glenodinium sp., has an extremely high quantum efficiency and large Stokes shift, yet it photobleaches easily, thus limiting it usefulness in high power jet-in-air flow cytometers and restricting its use to low-power, fixed beam systems. PerCP is used for multicolor analysis since its spectral overlap is minimal with other common fluorochromes; however it commonly thought to be more suited for antigens expressed at medium or high densities. In the course of designing and testing a hybrid 4-color panel for routine immunophenotypic analysis of the major lymphocyte subsets (CD4 and CD8 T-cells, B, and NK cells), PerCP-conjugated antibodies could be readily utilized without any sacrifice in accuracy for common antigens expressed at relatively low densities, as in B-cells. However, staining was significantly altered when PerCP-conjugated antibodies to CD19 were used in stabilized blood samples. Additionally, cell fixation and permeabilization is a requirement for the measurement of intracellular antigens, as in the case of ZAP-70 expression, a surrogate marker in chronic lymphocytic leukemia disease progression and survival. Under these conditions, the accurate enumeration B-cells is preferentially and significantly altered, whether measured with PE-, PerCP, or PEAF700-conjugated fluorochromes. Overall, this diminished sensitivity leads to a poorer separation between the background fluorescence of unlabeled- (control) and labeled-cells, thereby confounding the accurate measurement of B-cell numbers and introducing error in the validity of the clinical value of these markers stained under these conditions. In addition to this study, we have examined the stability of B-cell markers using commercially available cell stabilization reagents to stabilize cell morphology and preserve cellular antigen expression.