Methodology Exploration Of Microparticles Detection In Peripheral Blood And Clinical Researches With The Application

Objective:(1) To establish the proper protocol for microparticles(MPs) detection in peripheral blood through flow cytometry.(2) To investigate the expression and clinical significance of four kinds MPs in peripheral blood in the healthy and gastric adenocarcinoma patients.(3) To determine the effect of D2 radical gastrectomy on MPs level through researches on peroperative MPs expression. EMPs expression in patients with acute pancreatitis(AP) was also detected during treatment.Methods:(1) Establishment of MPs detection protocols: Peripheral blood was drawn on an empty stomach in the morning, anticoagulated with sodium citrate(with prostaglandin E1 final concentration 300 n M). The platelet-poor plasma(PPP) was obtained through centrifugation at 3000 rpm for 15 min twice(4℃) and then stored at-80℃. Four kinds of phycoerythrin labeled antibodies with CD specificity were used to mark the homologous MPs. During FCM detection, the forward scatter was set under matched control with fluorescent 1.0um microspheres. PE labeled-vesicles with a diameter ranging from 0 to 1.0um were recognized as the aiming MPs.(2) MPs detection: Thirty healthy volunteers and 85 patients with histological diagnosed gastric adenocarcinoma were recruited. Expression of PMPs, TF-MPs, EMPs, and RMPs were detected.(3) Evolution of MPs expression during treatment: PPP specimens were collected prior to operation and on the 1st, 7th and 30 th postoperative day. Sequential detections were carried out. EMPs expression in patients with acute pancreatitis was detected during the treatment course.Results:(1) Expression levels of PMPs, TF-MPs, EMPs, and RMPs in healthy people are(92.1 ± 65.5)/ul,(37.6 ± 18.6) per ul,(97.2 ± 80.3)/ul, and(27.6 ± 12.0)/ul respectively. PMPs and EMPs constitute almost 80% of all MPs.(2) Levels of the four MPs in gastric adenocarcinoma patients are(169.9 ± 81.4)/ul,(65.4 ± 36.7)/ul,(178.06±119.42)/ul, and(46.2 ± 32.8)/ul. There are statistical differences between PMPs, TF-MPs, and RMPs comparisons(P<0.05). However, no statistical difference is seen between EMPs levels. Higher levels of PMPs and TF-MPs are correlated with higher TNM stages in gastric adenocarcinoma patients(P<0.05). Higher TF-MPs level is also correlated with vessel invasion.(3) Levels of four kinds of MPs on the 1st postoperative day are(608.9 ± 407.1)/ul,(135.2 ± 151.8)/ul,(733.5 ± 744.4)/ul, and(78.0 ± 73.6)/ul. Significant statistical differences are seen compared with preoperative levels(P<0.01). The augmented levels are seen to gradually decrease, with decreased levels of(110.7 ± 90.7)/ul,(42.2 ± 29.5)/ul,(186.9 ± 104.2)/ul, and(25.8 ± 13.4)/ul on 30 th postoperative day, which contrast statistically different with preoperative levels(P<0.05). Yet no differences are seen through comparison between postoperative levels and healthy people(P>0.05). EMPs level increases significantly under AP pathology(584.0 ± 317.5/ul) and it decreases to a normal level of(227.4 ± 105.0/ul) after recovery.Conclusion:(1) FCM is a feasible approach for MPs detection. The standard approach may be valuable for scientific research and clinical application.(2) Elevated levels of PMPs and TF-MPs are correlated with higher TNM stages in gastric adenocarcinoma patients(P<0.05). Higher TF-MPs level is also correlated with vessel invasion.(3) Surgical trauma is an identified stimulus that could increase MPs expression. D2 radical gastrectomy is effective to reduce the high preoperative levels of MPs.(4) Elevated level of EMPs is seen in the pathological process of AP and decreases to a normal level after recovery.