Almost forty years have passed since flow cytometry was developed in the late 1960s. It has been used in cell biology, particularly the two major fields of cell surface antigens and cellular DNA content. Its rapid analysis of the latter led to the detection of quantitative choromosomal abnormality, which would otherwise require much time, experience, and especially mitotic cells. It was adopted initially for haematological disorders, but its use expanded to solid tumours, and even fixed tissues for retrospective analysis. Although DNA aneuploidy was proven to be a prognostic factor for limited tumours, its clinical significance has not been established yet for most tumours. Inappropriate sampling and flow cytometry determining procedures might have generated confusing evaluations. The advantages of flow cytometric analysis of DNA aneuploidy include: 1) removal of the requirement for mitotic cells, 2) rapid determination (a rate of thousands per second), 3) qualitative determination (more than five thousand cells per sample), 4) labour-saving determination, and 5) objective determination by an instrument. Conversely, disadvantages might include: 1) impossibility of detecting qualitative chromosomal abnormality, 2) difficulty with detecting subtle quantitative abnormalities, and 3) impossibility of identifying relevant specific chromosomes. However, the advantages clearly outweigh the disadvantages. The Standardization Committee of the Japan Cytometry Society has recently finished creating the guidelines for flow cytometric analysis of DNA aneuploidy. These include sampling, providing free cell suspension, staining with fluorochromes, determining by flow cytometry, evaluating, and outlining the terminology of DNA aneuploidy. It is hoped that all researchers will adopt and comply with these guidelines whenever they analyse DNA aneuploidy with flow cytometry, and that they will find the appropriate clinical significance of DNA aneuploidy with its strict determination and definition.
Many previous studies attempted to associate DNA ploidy with prognosis and malignant behavior for lung carcinomas as well as other carcinomas, but recently, the reports which are related to cellular DNA quantification in lung carcinomas decreased much more than before. Why did researchers stop having interest in the study of DNA ploidy? First of all, authors mentioned an outline of the history of DNA ploidy in lung carcinomas. We were anguished about many problems of DNA ploidy, and a difficult question was solved with patience. When it looks back on the history, authors found out that the analysis of DNA index is interesting in the malignant behavior of lung carcinomas which a distant metastasis (lung, brain, bone, et al) is high frequency. Furthermore, an questionable fact of DNA ploidy may be solved when DNA ploidy is combined with FISH and CGH methods. The authors emphasizes that interesting research is still left in the study of cellular DN Acontent.
The DNA ploidy and DNA indices (DI) of 412 colorectal cancers underwent curative operation were analyzed by flow cytometry. The aneuploid tumors were detected more frequently according to the tumor progression. The incidence of hematogenic recurrence (liver metastasis and lung metastasis) was found to be significantly higher in those with aneuploid tumors (DI > 1.5) than in those with diploid tumors or aneuploid tumors (DI < 1.5). These findings indicate that the flow cytometric analysis of DNA indices of colorectal tumors may be a good indicator of hematogenic recurrence. However, it is still difficult to prevent liver metastasis after curative operation by the adjuvant chemotherapy through peripheral venous, portal vein, or hepatic artery. Recent advancement of chemotherapy made it possible to prolong the survival of patients who had liver metastatic recurrence. But, these new chemotherapeutic drugs including monoclonal antibodies do not work sufficiently in some patients. The recurrent tumors of these patients may be chemo-resistance. In the future, the characteristics of recurrent tumors should be analyzed by flow cytometry.
In 1976, one of authors (Kawamoto) first introduced the flow cytometry in to Japanese for DNA analysis in brain tumors and studied the cell kinetics of brain tumor for 30 years. Different patterns of DNA histogram were classified according to their DNA ploidy patterns. Diploid patterns was classified as Type I, monomodal aneuploidy as, Type II, and multimodal aneuploidy as Type III with lym phocyte internal standard. Most benign tumors belonged to Type I, while most malignant ones belonged to Type II and Type III, and clinical prognosis of Type III was poor. LSC was applied to decide the surgical area in malignant glioma and analysis of DNA-ploidy could be useful for the diagnosis of malignancy and decision of removal area. Cytometric analysis of DNA ploidy could be useful for information of malignancy in brain tumor and will be applied to basic and clinical field. Cytometry will be widely advance and contribute to the cell analysis by simultenously multistaining of nucleus, cytoplasm and cell surface in future.
Flow cytometric determination of DNA ploidy in gynecologic cancer has made progress due to the development of sample treatment procedures and special initiatives by the Japan Cytometry Society in standardizing the determination of DNA ploidy in solid cancers. Although a strategy for the clinical application of DNA ploidy in gynecologic cancer has not yet been formulated, it is expected that DNA ploidy studies would serve to elucidate cell-biological features of gynecologic cancer and provide an entrance to the study of analytical cytology.
CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily of molecules and is known to play an important role in normal dendritic cell and B-cell maturation. However, the function of CD40 in malignant neoplasms, especially solid tumors, remains largely unknown. We previously reported CD40-CD40L stimulation had the anti-tumor effect in non-small cell lung cancer cell lines which expressed CD40 on their surface in vitro study. Detection of CD40 expression of the tumor cell is usually examined by FACS, not by immunohistochemical staining (IHC). As far as we know, IHC of CD40 has not been reported yet. Therefore, if IHC of CD40 could be examined from paraffin embedded specimens, the role of CD40 in malignant tumors might become clear from the clinicopathological findings. CD40 expression could be detected by IHC from the paraffin embedded specimens of lung adenocarcinomas. We observed enhanced IHC of CD40 in all specimens of N2 cases, in contrast none of N0 cases was expressed. CD40 expression rate was much higher in N2 cases than N0 cases from our experiment. Thus, CD40 may play an important role in lymph node metastasis in lung cancer. Further investigations are required to clarify the mechanism of CD40 on lung cancer.
Some studies suggest that the immunoreceptor NKG2D expression on CD8+ T cells is downregulated and this reduction may be involved in immune evasion in cancer patients. The present study was designed to investigate NKG2D expression on CD8+ T lymphocytes and it's relationship to immune evasion in esophageal and gastric cancer patients. NKG2D expression on circulating CD8+ T cells was evaluated by multicolor flow cytometry. Transwell experiments were carried out to determine the effect of cancer cells on NKG2D expression. NKG2D expression on circulating CD8+ T cells was downregulated and significantly correlated with IFN-gamma production in both esophageal and gastric cancer patients (r=0.68, p=0.007). Downregulated NKG2D expression was closely related to disease progression. Complete removal of tumor by surgery restored NKG2D expression on CD8+ T cells. Transwell experiments showed that this downregulation was induced by direct contact between cancer cells and CD8+ T cells, and that soluble factors did not affect the NKG2D expression. This phenomenon was blocked by the addition of anti-MICA antibodies. Decreased NKG2D expression may be one of the key mechanisms responsible for immune evasion by tumors in both esophageal and gastric cancer.
Hypercytokinemia and systemic inflammatory response syndrome (SIRS) is found in patients with shock due to sepsis. However complete set of cytokine profile as the hidden circumstances that led up to the case was not inquired. We measured 23-kind circulating cytokines simultaneously in virulent strain, Plasmodium bergheiinfected mice sera by suspension-array analysis. We had previously reported the induction of IL-18 production in malaria patients, as well as malaria-model mice. This report also discusses possible physiological roles of IL 18 induced by Plasmodium infection.
After organ transplantation, anti-donor alloreactivity, defined as the number and phenotype of alloreactive precursors in the recipient, could be used to monitor rejection or tolerance or for withdrawal of immunosuppression. For this purpose, we used intracellular fluorescent dye, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in mixed lymphocyte reaction (CFSE-MLR). The CFSE-MLR enables determination of the number of proliferating cells in response to allogeneic stimulation and simultaneous determination of the phenotype of proliferating cells by using of multiparameter FCM analysis. In our previous study, remarkable proliferation of CD8+ T cells in association with CD25 expression on anti-donor CFSE-MLR reflected the greater susceptibility to acute cellular rejection after liver transplantation. In addition to the determination of the phenotype of the proliferating T cells in response to allogeneic stimulation, cytokine-secreting activity in those T cells could be simultaneously determined by combining intracellular cytokine immunofluorescence staining (ICIS). Quantification of cell populations expressing specific cytokine proteins and identification of the cytokinesecreting cell phenotype in MLR would be much more informative than just the detection of cytokines in culture supernatants. We have demonstrated the utility of a method combining CFSE-MLR, ICIS, and multiparameter FCM for the simultaneous determination of proliferation and cytokine-secreting activity in T cells responding to allogeneic stimulation in mice. In allogeneic MLR utilizing a mouse model, CFSE-labeled responder splenocytes were cultured with irradiated stimulator splenocytes, followed by ICIS. In fully allogeneic combinations, interleukin (IL)-2 secreting cells and interferon (IFN)-γsecreting cells were identified predominantly in proliferating CD4+ and CD8+ T cell fractions, respectively. Thus, for monitoring the immune response after organ transplantation, the CFSE-MLR analysis combined with the ICIS assay would be an excellent tool to monitor the host T cell reactivity in response to allogeneic stimulation.