Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol.
BACKGROUND
Microparticles (MP) are small vesicles of 0.1-1 μm, released in response to activation or apoptosis. Over the past decade, they received an increasing interest both as biomarkers and biovectors in coagulation, inflammation and cancer. Clinical studies were conducted to assess their contribution to the identification of patients at cardiovascular risk. However, among the limitation of such studies, pre-analytical steps remains an important source of variability and artifacts in MP analysis.


OBJECTIVE
Because data from the literature are insufficient to establish recommendations, the objective of the present study was to assess the impact of various pre-analytical parameters on MP measurement. These parameters included the type of collection tube, phlebotomy conditions, transportation practices, centrifugation steps and freezing.


METHODS
MP were assessed by three methods: flow cytometry using a standardized approach, a thrombin generation test (Calibrated Automated Thrombogram(®)) and a procoagulant phospholipid-dependent clotting time assay (STA(®) -Procoag-PPL).


RESULTS
The main results show that the three major pre-analytical parameters which impact on MP-related data are the delay before the first centrifugation, agitation of the tubes during transportation and the centrifugation protocol.


CONCLUSIONS
Based on both this work and literature data, we propose a new protocol that needs to be validated on a larger scale before being applied for multicenter studies.
UMR1076 INSERM-Aix-Marseille Université, UFR de Pharmacie, Marseille, France.
R
R
Lacroix
C
C
Judicone
P
P
Poncelet
S
S
Robert
L
L
Arnaud
J
J
Sampol
F
F
Dignat-George
D000328:Adult; D000925:Anticoagulants / Q000494:pharmacology; D015415:Biomarkers / Q000097:blood; D001777:Blood Coagulation / Q000187:drug effects; D001780:Blood Coagulation Tests / Q000592:standards; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D002498:Centrifugation / Q000592:standards; D015925:Cryopreservation / Q000592:standards; D005260:Female; D005434:Flow Cytometry / Q000592:standards; D005615:Freezing; D006801:Humans; D008297:Male; D008875:Middle Aged; D018962:Phlebotomy / Q000592:standards; D017410:Practice Guidelines as Topic; D013048:Specimen Handling / Q000592:standards*; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D055815:Young Adult
D000925:Anticoagulants; D015415:Biomarkers; D013917:Thrombin