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).


CONCLUSIONS
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.
Nantakomol
Duangdao
D
Department of Medical Technology, Faculty of Allied Health Science, Naresuan University, Phitsanulok, Thailand. nantadao@gmail.com
Imwong
Malika
M
Soontarawirat
Ingfar
I
Kotjanya
Duangporn
D
Khakhai
Chulalak
C
Ohashi
Jun
J
Nuchnoi
Pornlada
P
D000293:Adolescent; D002138:Calibration; D002452:Cell Count; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D004912:Erythrocytes / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D011786:Quality Control; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D055815:Young Adult