Affordable Technology for Enumeration of the Absolute CD4 T-Lymphocyte Count by Cell Bead Assay

The quantitative BD Trucount (San Jose, CA) tube method is the conventional but expensive method to quantitate CD4+T-lymphocyte (CD4) counts, and this may be beyond the means of countries with limited resources. In this study, we validated a quantitative method known as a cell-bead (CB) assay to quantitate CD4 counts in the peripheral blood of healthy subjects. The absolute CD4 count obtained from the CB method was highly correlated with those obtained from the Trucount tube (r2=0.98, y=26.73+1.01x, P<0.0001 and a mean bias of 34.8 cell/μL, limit of agreement [LOA] -34.8-104.4 cell/μL) and flow rate-based assay method (r2=0.97; y=69.51 + 0.88x, P<0.0001 and a mean bias -53.5 cell/μL, LOA -149.4-42.3 cell/μL). This study demonstrates that the CB method is suitable and more affordable for CD4 quantitation. This method is inexpensive and interchangeable with the latex bead-based methods for generating absolute counts in resource-limited areas

The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay.

BACKGROUND: Activation of red blood cell is associated with the formation of red cell-derived microparticles (RMPs). Analysis of circulating RMPs is becoming more refined and clinically useful. A quantitative Trucount tube method is the conventional method uses for quantitating RMPs. In this study, we validated a quantitative method called "flow rate based assay using red cell bead (FCB)" to measure circulating RMPs in the peripheral blood of healthy subjects. METHODS: Citrated blood samples collected from 30 cases of healthy subjects were determined the RMPs count by using double labeling of annexin V-FITC and anti-glycophorin A-PE. The absolute RMPs numbers were measured by FCB, and the results were compared with the Trucount or with flow rate based calibration (FR). Statistical correlation and agreement were analyzed using linear regression and Bland-Altman analysis. RESULTS: There was no significant difference in the absolute number of RMPs quantitated by FCB when compared with those two reference methods including the Trucount tube and FR method. The absolute RMPs count obtained from FCB method was highly correlated with those obtained from Trucount tube (r(2) = 0.98, mean bias 4 cell/microl, limit of agreement [LOA] -20.3 to 28.3 cell/microl), and FR method (r(2) = 1, mean bias 10.3 cell/microl, and LOA -5.5 to 26.2 cell/microl). CONCLUSION: This study demonstrates that FCB is suitable and more affordable for RMPs quantitation in the clinical samples. This method is a low cost and interchangeable to latex bead-based method for generating the absolute counts in the resource-limited areas

Increase membrane vesiculation in essential hypertension

Background: A hypertensive condition is known to be an important risk factor for arterial disease and thrombotic events. The detailed understanding of the pathophysiology in thrombotic events is crucial for the development of both preventive measures and the treatment during the early stage. Microparticles are submicron cell membrane vesicle shed from the cell surface in response to cell injury or apoptosis, and they are an essential element in the process leading to the pathogenesis of thrombosis and hemostasis dysfunction in patients. Activation of blood cells can result in the formation of microparticles, which carry a negatively charged, phosphatidylserine (PS), with a diameter of <1.0 micron. The biological and clinical functions of microparticles have been highlighted in coronary artery disease and heart failure. Objective: To elucidate the functional role of microparticles in essential hypertension. Methods: We quantitated the total number of circulating PS+ microparticles and studied the role of PS+ microparticles to see whether they can shorten the plasma recalcification time in patients with essential hypertension. Results: The PS+ microparticles were detectable at a low level in healthy blood and significantly increased in the patients with essential hypertension. With regard to PS+ microparticles affecting prothrombotic state in hypertension, we enriched microparticles and determined their procoagulant activity with a plasma recalcification time. The clotting time was significantly reduced after the addition of enriched microparticles to plasma poor microparticles (PPMP). A significant negative correlation was detected between numbers of enriched-PS+ microparticles and plasma-clotting time (r=-0.43, P=0.01). Conclusion: Taken together, high levels of PS+ microparticles are present in the circulating blood of essential hypertension and may contribute to the generation and perpetuation of a thrombotic state