Assessment of platelet activation in several different anticoagulants by the Advia 120 Hematology System, fluorescence flow cytometry, and electron microscopy

Abstract

In vivo platelet activation results are often confounded by activation induced in vitro during the preparative procedures. We measured ex vivo (basal) and in vitro (thrombin-induced) platelet activation in sodium citrate, ethylenediaminetetraacetic acid (EDTA), and Citrate Theophylline Dipyridamole Adenosine (CTAD) whole blood specimens. Determinations were made by measurements of platelet density (mean platelet component: MPC concentration) on the Advia 120 Hematology System. The MPC has been previously shown to correlate with a fluorescence flow cytometric method, also determined in this study, using the surface expression of CD62P. Moreover, platelet shape and structure changes in EDTA and CTAD anticoagulated whole blood specimens were characterized by transmission electron microscopy (TEM). Observations made using the Advia 120 Hematology System platelet density parameter, MPC, in the absence of thrombin were 25.7 +/- 0.9 g/dl, 27.9 +/- 0.9 g/dl and 24.8 +/- 1.2 g/dl in sodium citrate, EDTA and CTAD whole blood specimens, respectively. Addition of thrombin induced a significant change in platelet MPC for sodium citrate (21.9 +/- 1.9 g/dl; p<0.0001) and EDTA (23.2 +/- 0.9 g/dl; p<0.0001) whole blood specimens. In contrast, thrombin had no effect on MPC measured in whole blood taken into CTAD tubes. In vitro fluorescence flow cytometric platelet activation experiments measuring the percentage of platelets expressing anti-CD62P showed increase in sodium citrate specimens from 9.2 +/- 7.0 to 55.5 +/- 23.1 % (p<0.0001) and in EDTA specimens from 1.9 +/- 1.7 to 64.6 +/- 12.4 % (p<0.0001) after addition of thrombin. However, in blood taken into CTAD tubes, there was no significant change. Studies on platelets isolated from whole blood in CTAD showed activation by thrombin indicating that platelets in CTAD, while protected in its presence remained functional upon its removal. When observed by TEM over time, platelets in EDTA appear more activated and contain fewer granules than platelets in CTAD. We conclude that CTAD demonstrates in vitro platelet activation inhibition and may be useful in stabilizing ex vivo platelet activation. The novel platelet activation parameter, MPC, measured by an automated routine hematology system, using customized proprietary software, may be used in conjunction with CTAD, a stabilizing anticoagulant, to measure the ex vivo platelet activation state in whole blood specimens. TEM studies verify shape modifications and simultaneous retention of intracellular granules at early post-venipuncture time periods in CTAD specimens.

Comparison of two methods to assess variability of platelet response to anti-platelet therapies in patients with acute coronary syndrome undergoing angioplasty

“…In this study and others, MPC has been shown to be increased in inactivated platelets and decreased in activated platelets (20,22). The measurement of MPC does not require specimen preparation, platelet activation specific receptors, or activation-specific receptor labels.…”

“…The patients were characterized based on the combination of the baseline platelet activation parameters, MPC and CD62 expression, and the response to the anti-platelet therapy. For our population, we have already reported a normal MPC value, 27.9 ± 0.9 (22). In a larger set of data (n = 51), the MPC in normal healthy donors was 28.1 ± 1.0 g/dL (data not published).…”

“…The measurement of MPC does not require specimen preparation, platelet activation specific receptors, or activation-specific receptor labels. The MPC values showed very good inverse correlation with the percentage of platelets expressing CD62P antigen as detected by fluorescent flow cytometry after activation by thrombin (22). The present study was designed to establish the correlation and to evaluate the clinical usefulness of these parameters to measure the magnitude of baseline platelet activation, as well as to follow anti-platelet therapy in patients with acute coronary syndrome undergoing coronary angioplasty.…”

“…In a larger set of data (n = 51), the MPC in normal healthy donors was 28.1 ± 1.0 g/dL (data not published). The CD62P-PE % positive events was < 2% for normal healthy donors (22).…”

“…A decrease in platelet density, measured by a reduction in MPC is indicative of platelet activation. MPC also shows, in vitro, excellent correlation with the well-defined fluorescence marker, CD62P-Phycoerythrin (CD62P-PE) (20)(21)(22).…”