Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1-dependent apoptosis.

Flow cytometry was been widely used to measure apoptosis for many decades but the researcher has no definitive way of determining other forms of cell death using this technology. The use of Western Blot technology has numerous drawbacks in that all the cells in the sample whether live, dead or maybe undergoing multiple discrete forms of cell death are analysed as one population. Flow cytometry given that it can analyse different sub-populations of cells within a sample would reveal the expression of cell death markers within these sub-populations rather than just give a single result from the entire population. Here we describe a flow cytometric assay fully realising that potential by the use of anti-RIP-3 (Receptor-interacting serine/threonine-protein kinase 3) and anti-active caspase-3 fluorescently tagged antibodies and a fixable live dead fluorescent dye. This allows the determination of the degree of necroptosis, apoptosis and RIP1-dependent apoptosis within live and dead populations. Necroptosis was identified by the up-regulation of RIP3, while RIP1-dependent apoptosis was described by double positive for RIP3/active Caspase-3 events in live and dead populations. Apoptotic cells were defined by an active-Caspase-3+ve/RIP3-ve phenotype. Pan-caspase blocker zVAD and RIP1 inhibitors GSK'481 or necrostatin-1 revealed interesting modulations of such sub-populations of Jurkat cells. This novel flow cytometric assay employing two antibodies and a fixable viability probe provides the researcher with in-depth analysis of various forms of regulated forms of cell death beyond what is currently available and is a major methodological advancement in this field.

The dynamic change of circulating tumour cells in patients with operable breast cancer before and after chemotherapy based on a multimarker QPCR platform.

BACKGROUND: The possible presence of early tumour dissemination is the rationale behind the use of systemic adjuvant chemotherapy in patients with operable breast cancer. Circulating tumour cells (CTC) in peripheral blood may represent the possible presence of early tumour dissemination. However, relatively few studies were designed to investigate the relationship between the change of CTC status and the efficacy of adjuvant chemotherapy in operable breast cancer patients. METHODS: In a prospective study, we established a multimarker real-time quantitative PCR platform to detect CTC in peripheral blood of breast cancer patients. By using this platform, we detected CTC in peripheral blood of 94 operable breast cancer patients. Control group consisted of 20 patients with benign breast disease and 20 healthy volunteers. For 72 patients who underwent systemic adjuvant chemotherapy, the dynamic CTC status at three different time points (1 day before initiation of chemotherapy, 1 week after three cycles of chemotherapy and 1 week after all cycles of chemotherapy) was observed. RESULTS: Circulating tumour cells were detected in 56% (53 out of 94) of patients with operable breast cancer. The specificity was 95%. Seventy-two patients who received systemic adjuvant chemotherapy were followed up. After three cycles of chemotherapy, 47% (18 out of 38) of patients who were CTC-positive before chemotherapy changed into negative status. In addition, another 5% (2 out of 38) of patients had changed into negative status after all cycles of chemotherapy. CONCLUSION: Systemic adjuvant chemotherapy had a significant impact on CTC status, and this effect could be observed after three cycles of chemotherapy. Circulating tumour cells detection had the potential to be used to evaluate the efficacy of systemic adjuvant chemotherapy immediately after the chemotherapy was finished in operable breast cancer patients.

The effect of a pain management program on patients with cancer pain.

BACKGROUND: Pain is 1 of the most common symptoms that a cancer patient would experience. A significant barrier to positive pain management is patients' misconceptions regarding analgesics and inadequate use of nonpharmacological strategies as pain relief. OBJECTIVE: The purpose of this study was to investigate the effectiveness of a pain management program (PMP) on pain intensity, use of PRN drugs and nonpharmacological strategies as pain relief, and barriers to managing pain in cancer patients. METHODS: The study was conducted in the palliative care and hospice ward of a public hospital in Hong Kong. Patients were randomized to either an experimental group (receiving the PMP) or a control group (routine care). There were 38 hospitalized patients, with 20 (13 males and 7 females) in the experimental group and 18 (11 males and 7 females) in the control group; mean age was 61.95 years (experimental group) to 63.94 years (control group). RESULTS: Upon the completion of PMP, pain scores were significantly reduced in both groups, yet patients in the experimental group showed a significant increase in the use of PRN analgesics and nonpharmacological strategies to relieve pain (P < .05) and significantly reduce barriers to managing their cancer pain (P < .05) compared with the control group. CONCLUSION: Cancer patients should be empowered with pain management education to gain knowledge and correct misconceptions in managing their cancer pain. IMPLICATIONS FOR PRACTICE: Integration of the PMP into routine clinical work may help to improve the standard of care for cancer patients. It is recommended to provide pain management education to all cancer patients.