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一个非常感兴趣的领域。其中一种名为erlotinib的药物是表皮生长因子受体(EGFR)的抑制剂，目前正用于治疗非小细胞肺癌(NSCLC)，然而，即使在高度敏感的肿瘤中，这种反应也是不完全的，其基础尚不清楚。为了验证耐药细胞亚群可能导致不完全应答的假设，根据CD24和CD44表面标志的表达对H1650和H1975非小细胞肺癌细胞系进行了分类。在这两种情况下，在克隆形成试验中，CD24和CD44表达缺失(CD24-/CD44-)的细胞对厄洛替尼的耐药性最强。无论是CD24-/CD44-表型还是对厄洛替尼的相对耐药性都与Hoechst 33342染料外流增加有关，这表明这种对可能的干细胞的检测不能解释药物敏感性。然而，CD24-/CD44-细胞具有不同的EGFR信号通路，这支持了非小细胞肺癌可能由不同的细胞群体组成的假设，细胞信号活性不同，对厄洛替尼的反应也不同。 多色免疫表型分析具有潜在地解析新的临床输入淋巴细胞亚群的能力，同时最大限度地减少样品处理和在多个试管中使用多余抗体的相关成本。通常，选择合适的荧光色素组合的经验法则是将最亮的标记(PE、APC或PE-Cy5)分配给与具有最低表达的目的蛋白反应的抗体。多种因素可以影响对亮度的感知，包括荧光色素本身的生物物理性质和仪器相关变量。Percp是甲藻光合作用复合体的一个亚基，具有极高的量子效率和较大的斯托克斯漂移，但它很容易发生光漂白，从而限制了其在高功率气流细胞仪中的应用，并限制了其在低功率固定光束系统中的使用。PerCP用于多色分析，因为它与其他常见荧光染料的光谱重叠很小；然而，它通常被认为更适合于中密度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(CD4和CD8T细胞、B细胞和NK细胞)常规免疫表型分析的混合四色板的过程中，PerCP结合的抗体可以很容易地用于在相对低密度表达的共同抗原的准确性上，例如在B细胞中。然而，当PerCP结合的CD19抗体用于稳定的血液样本时，染色明显改变。此外，细胞的固定和通透性是测量细胞内抗原的必要条件，就像ZAP-70的表达一样，ZAP-70是慢性淋巴细胞性白血病疾病进展和生存的替代标记物。在这些条件下，无论是用PE、PerCP还是PEAF700标记的荧光染料测量，准确的B细胞计数都会优先和显著地改变。总体而言，这种敏感性的降低导致未标记(对照)细胞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记物的临床价值的有效性方面引入了错误。

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. 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.