Evaluation of the anticoagulants EDTA and citrate, theophylline, adenosine, and dipyridamole (CTAD) for assessing platelet activation on the ADVIA 120 hematology system.
BACKGROUND
Monitoring of platelet activation by the ADVIA 120 Hematology System requires an anticoagulant and protocol that ensures that platelets are sphered and their activation status is not altered artifactually in vitro.


METHODS
Blood from healthy controls was collected into tripotassium EDTA; citrate, theophylline, adenosine, and dipyridamole (CTAD); or a combination of both (E/C) and stored at ambient temperature or at 4 degrees C (E/C only) and then analyzed between 0 and 180 min later on the ADVIA 120. In addition, immunofluorescent flow cytometry was used to identify activated platelets and platelet-leukocyte aggregates.


RESULTS
In blood stored with all three anticoagulants, the platelet count changed little, but the mean platelet volume (MPV) at first decreased and then increased, whereas the mean platelet component (MPC; an indicator of activation) changed in a reciprocal manner. The changes in MPV and MPC, which reflect platelet sphering and swelling, were greatest between 30 and 60 min in blood stored at ambient temperature, irrespective of which anticoagulant was used, and between 60 and 180 min when blood anticoagulated with E/C was stored at 4 degrees C. In all anticoagulants, the percentages of platelets expressing CD62P and of leukocytes in platelet-leukocyte aggregates increased significantly (P <0.01) over 180 min at ambient temperature. Only minimal (<2%) increases occurred when blood with E/C was stored at 4 degrees C.


CONCLUSIONS
When determining platelet activation ex vivo on the ADVIA 120, blood should be collected into E/C, stored at 4 degrees C, and analyzed between 60 and 180 min later; these conditions ensure maximum platelet sphering without concurrent artifactual platelet activation.
Macey
Marion
M
Department of Haematology, The Royal London Hospital, Whitechapel, London E1 1BB, United Kingdom. M.G.Macey@mds.qmw.ac.uk
Azam
Urooj
U
McCarthy
Desmond
D
Webb
Lee
L
Chapman
E Sabrinah
ES
Okrongly
David
D
Zelmanovic
David
D
Newland
Adrian
A
D000241:Adenosine / Q000494:pharmacology; D000328:Adult; D000925:Anticoagulants / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D001800:Blood Specimen Collection / Q000379:methods*; D002449:Cell Aggregation; D048429:Cell Size; D002951:Citrates / Q000494:pharmacology; D004176:Dipyridamole / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005434:Flow Cytometry; D006403:Hematologic Tests / Q000379:methods; D006801:Humans; D066298:In Vitro Techniques; D007962:Leukocytes / Q000502:physiology; D019007:P-Selectin / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects*; D010976:Platelet Count; D013696:Temperature; D013806:Theophylline / Q000494:pharmacology
D000925:Anticoagulants; D002951:Citrates; D019007:P-Selectin; D004176:Dipyridamole; D004492:Edetic Acid; D013806:Theophylline; D000241:Adenosine