Value Of Flow Cytometric Analysis Research Articles

Abstract 13285: Short-term Low-saturated Fat Diet Compared to High-saturated Fat Diet Improves Monocyte Phenotypes in Subjects With Hypertriglyceridemia

Introduction: Clinical trials suggest that low-saturated fat diet (LSFD) may reduce the risk of atherosclerotic cardiovascular disease (ASCVD) in subjects with hypertriglyceridemia (HTG). Monocytes play crucial roles in atherogenesis. Hypothesis: LSFD vs high-saturated fat diet (HSFD) improves monocyte phenotypes, thereby reducing ASCVD risk, in subjects with HTG. Methods: Subjects with HTG and metabolic syndrome (MetS, n=19) received isocaloric LSFD (~25% of calories from fat, 5% from saturated fat) and HSFD (~52% of calories from fat, 25% from saturated fat) in randomized order for 4 days (days 1-4) plus a breakfast on day 5, separated by a 4- to 6-week washout period. Blood was drawn on day 1 fasting before the diets and 3 times on day 5 (fasting before the breakfast and 4 and 6 hours postprandial) for measurement of lipid profile and analyses of monocyte phenotypes by flow cytometry and monocyte adhesion by a lab-on-a-chip microfluidic assay. Results: On day 5, LSFD, compared to HSFD, induced lower plasma levels of postprandial total triglyceride and LDL-triglyceride and fasting and postprandial total cholesterol, LDL-cholesterol, and small dense LDL-cholesterol. Compared to HSFD, LSFD reduced fasting and postprandial intracellular lipid accumulation in classical and intermediate monocytes examined by nile red staining and indicated by side scatter value of flow cytometric analysis. LSFD versus HSFD also reduced ex vivo uptake of oxidized LDL by classical monocytes at 4 hours postprandially and by intermediate monocytes in fasting state. Surface levels of molecules involved in monocyte adhesion/migration, including CD11c, CD81, and CCR2, were lower on monocytes with LSFD than with HSFD. Consistently, LSFD compared to HSFD reduced monocyte adhesion to VCAM-1. Intracellular levels of cytokines such as IL-1β, TNFα, and IL-6 in monocytes showed no difference between the two diets. Conclusions: In subjects with HTG and MetS, short-term LSFD compared to HSFD reduces monocyte intracellular lipid accumulation and improves monocyte phenotypes with reductions in monocyte adhesion and oxidized LDL uptake. These findings highlight the importance of diet composition in monocyte phenotypes and possibly atherosclerosis risks in patients with HTG and MetS.

Can flow cytometrically determined DNA ploidy and S-phase fraction predict regional metastasis in squamous cell carcinoma of the oral cavity?

The value of flow cytometric analysis of DNA ploidy and S-phase fraction (SPF) as an indicator of regional metastasis in oral cancer is currently being debated. Intratumoral heterogeneity makes this problem complex. Intratumoral DNA ploidy heterogeneity and intratumoral SPF variation were examined using multiple specimens from 31 surgically resected specimens taken from patients with oral cancer without preoperative therapy. Flow cytometric analysis of single biopsy specimens from 79 patients with oral cancer was also undertaken to ascertain their value as indicators of regional metastasis. Forty-five percent (14 of 31) of tumors showed intratumoral ploidy heterogeneity. Intratumoral SPF variation in the 31 tumors ranged from 0.2% to 6.9% (mean, 3.3%). Multivariate analysis showed that a SPF greater than 27% was the most important parameter for predicting regional metastasis. DNA ploidy is heterogeneous within a tumor, whereas SPF is relatively stable and can be correlated with regional metastasis in oral cancer.

Predictive value of flow cytometric analysis in DNA contents in patients with locally advanced head and neck carcinoma.

A retrospective study was performed on 61 eligible patients with stage III and IV (AJC/UICC Staging System) squamous carcinomas of the head and neck region who were treated with definitive radiotherapy with, or without, surgery. DNA contents were measured by flow cytometric analysis of archival paraffin blocks and were correlated with clinicopathological findings, tumour response and patient survival. Comparison of variables including treatment modality was performed for identification of significant prognostic factors. There were 28 diploid, 27 aneuploid tumours and the remaining six were questionable. All patients were followed-up for at least two years or until death. Aneuploid tumours had a significantly higher S-phase fraction (percentage S-phase) (p < 0.001). Neither ploidy nor percentage S-phase were found to have predictive value in tumour response or patient survival within the power of a sample size of 61. Twenty of the 27 (74 per cent) aneuploid tumours had a complete response (CR) whereas 19 out of 28 (68 per cent) diploid tumours achieved CR. Five-year survival by the Kaplan-Meier method was 33 per cent for both aneuploid and diploid tumours. However, nodal stage (N stage) was found to have significant predictive value in both tumour response and patient survival. The complete response for stage N0 patients was 96 per cent, N1 patients 61 per cent, N2 patients 60 per cent and 43 per cent for N3 patients (p < 0.002). Similarly, the five year survival for the N0 and N3 groups of patients was 53 per cent and 29 per cent respectively (p < 0.05).

The Value of Flow Cytometric Analysis of Platelet Glycoprotein Expression on CD34+ Cells Measured Under Conditions That Prevent P-Selectin–Mediated Binding of Platelets

In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these cells was found to be reversible, dependent on divalent cations, and mediated by P-selectin. Enzymatic characterization showed the involvement of sialic acid residues, protein(s). The demonstration of mRNA for the P-selectin glycoprotein ligand 1 (PSGL-1) in the CD34+ cells by polymerase chain reaction (PCR) analysis suggests that this molecule is present in these cells. Under conditions that prevent platelet adhesion, a small but distinct subpopulation of CD34+ cells diffusely expressed the platelet GPIIb/IIIa complex. These cells were visualized by immunochemical studies. Furthermore, synthesis of mRNA for GPIIb and GPIIIa by CD34+ cells was shown using PCR analysis. The semiquantitative PCR results show relatively higher amounts of GPIIb mRNA than of PF4 mRNA in CD34+CD41+ cells in comparison with this ratio in platelets. This finding is a strong indication that the PCR results are not caused by contaminating adhering platelets. MoAbs against GPIa GPIb alpha, GPV, P-selectin, and the alpha-chain of the vitronectin receptor did not react with CD34+ cells. The number of CD34+ cells expressing GPIIb/IIIa present in peripheral blood stem cell (PBSC) transplants was determined and was correlated with platelet recovery after intensive chemotherapy in 27 patients. The number of CD34+CD41+ cells correlated significantly better with the time of platelet recovery after PBSC transplantation (r = .83, P = .04) than did the total number of CD34+ cells (r = .55). Statistical analysis produced a threshold value for rapid platelet recovery of 0.34 x 10(6) CD34+CD41+ cells/kg. This study suggests that if performed in the presence of EDTA the flow cytometric measurement of GPIIb/IIIa on CD34+ cells provides the most accurate indication of the platelet reconstitutive capacity of the PBSC transplant.

Flow cytometric analysis of nuclear DNA content of renal cell carcinoma correlated with histologic and clinical features

The prognostic value of flow cytometric analysis of DNA content is contradictory in renal cell carcinoma (RCC). Flow cytometric analysis was performed on 72 patients with RCC, and the relationship between the DNA content and pathologic or clinical features was investigated. Aneuploidy was observed in 36 (50%) of these patients. Two tissue samples from primary tumors of 12 patients were analyzed, and heterogeneity was found in 7 (58%) of these patients. The incidence of aneuploidy was significantly higher in high-grade than in low-grade tumors (P = 0.0328). All five tumors with a maximum diameter of 2.5 cm or smaller (T1N0M0) were diploid, and a trend for more aneuploid tumors among high-stage diseases was observed. The survival rate of patients with diploid tumors was not significantly different from that of those with aneuploid tumors in the low- or high-stage group. DNA ploidy assessed with one sample is not a prognostic factor, and heterogeneity between different samples of a given tumor indicates that one sample is not enough for DNA flow cytometry in RCC.

The value of flow cytometric analysis in patients with gastric cancer.

Tumors in the gastric cardia and the body/antrum were studied to determine the effect of ploidy and S-phase fractions on patient survival. Forty-two percent of tumors were located in the cardia and 58% in the body/antrum. Nodal metastases occurred more often with cardia tumors than with body/antrum tumors (86% vs 65%). Aneuploidy occurred more frequently in patients with cardia tumors than in patients with body/antrum tumors (39% vs 20%). Metastasis to lymph nodes was more common in patients with aneuploidy than in patients with diploidy (31% vs 7%). S-phase fractions were not different between aneuploid and diploid tumors. More patients with diploid cancer were alive at 5 years than were patients with aneuploid tumors (90% vs 10%). We conclude that primary cellular differences in gastric tumors of the cardia or body/antrum determine patient survival.