Abstract
Detection of platelet activation through conventional blood analyzers appears as a promising approach for a wide population of patients subjected to elevated cardiovascular risk. The mean platelet component (MPC) parameter calculated by the new generation of blood cell analyzers (ADVIA 120 and ADVIA 2120) provides direct information on density and granularity of platelets. Recent studies suggest that this parameter could correlate with the platelet activation state and become a potential predictive parameter for acute ischemic complications or thrombotic risk. Currently, the MPC parameter is largely affected by time and storage conditions. Standard anticoagulants based on K3EDTA are known to posses a deleterious effects on platelet morphology and granule content. It would be desirable that the stability of the MPC in blood specimens should be improved, to facilitate a more standardized use in different laboratories.
Blood from healthy controls was collected into K3EDTA plus fixatives, additives, and stored at different conditions that could prolong the stability of the MPC. MPC and the mean platelet volume (MPV) were assessed 30 min and at 1, 3, 6 and 24 hours after blood drawing on the ADVIA 2120 system. Flow cytometry techniques were used to identify platelet-activation proteins. Impact of these strategies on ultrastructural morphology of platelets was morphometrically evaluated using electron microscopy (EM).
Strategies based on addition of low concentrations of fixatives (paraformaldehyde or glutaraldehyde) that have proved useful for flow cytometry were unsuccessful to guarantee the stability of MPC. A solution based on K3EDTA containing wortmanin and tyrphostin (ED-WORTY) provided good stability for most of the parameters tested up to 6 hours at room temperature. Ultrastructural morphometry revealed a progressive statistically significant reduction in the number of alpha-granules per platelet section in samples stored in EDTA (from 12.3±6.07 to 6.4±2.3 and to 3.5±1.4; baseline, 6 and 24 h respectively, p<0.001). ED-WORTY prevented the degranulation observed in samples stored in EDTA with numbers of alpha-granules per platelet varying from 12.5±4.3 to 10.7±3.5 and to 10.9±4.7 at baseline, 6 and 24 h respectively. Storage at lower temperatures seemed to produce more favorable results, for all parameters measured, though they resulted in an enhancement of platelets positive for P-selectin. ED-WORTY solutions preserved adequate morphology and had minimal influence on other current parameters provided by the ADVIA 120/2120 Systems.
Storage in current anticoagulants based on EDTA result in a progressive increase in MPV, a decrease in MPC, with a significant reduction in the number of platelet alpha-granules as revealed by EM. Addition of inhibitors of protein phosphorylation provide stability for the MPV and for the MPC parameters, and significantly prevented the degranulation caused by EDTA. These additives could be useful to guarantee the stability of MPC for prolonged periods and to facilitate the transport and exchange of samples among institutions and laboratories.
Disclosures: Partially supported by a grant from Bayer Diagnostics.
