Differences of soluble CD40L in sera and plasma: implications on CD40L assay as a marker of thrombotic risk.
BACKGROUND
Soluble CD40L (sCD40L) ELISA has emerged as a promising predictor of poor outcomes in acute coronary syndrome. Yet many blood processing techniques have been used with little consideration of their effect on the results.


METHODS
We measured sCD40L by ELISA in 10 patients with thrombocytopenia and 12 with normal or high platelet counts and 8 healthy controls using three sampling techniques: serum clotted on ice (serum-I) or at room temperature (serum-RT) and platelet poor plasma (PPP).


RESULTS
Serum-RT samples, compared to serum-I, gave significantly higher CD40L values (p=0.003), demonstrating that ex vivo sCD40L release by activated platelets is inhibited by cold temperature. Although serum-I and PPP were comparable in patients with normal platelet counts, serum-I gave significantly higher values than PPP in the thrombocytosis group (p=0.01), suggesting that cold inhibition is insufficient in the latter group. To estimate the fraction of sCD40L that was microparticle-bound CD40L (mp-CD40L), 16 samples underwent 0.1-microm filtration. 50.6% of sCD40L was mp-CD40L in serum-RT, whereas 21.3% and 29.9% were observed in serum-I and PPP, respectively. Lastly, plasma sCD40L was assayed in 46 patients with and 35 without thrombosis. Plasma sCD40L did not correlate with platelet count in non-thrombotic, non-inflammatory patients but did (p<0.01) in those with thrombosis.


CONCLUSIONS
Sample processing and temperature profoundly affect sCD40L assay. Serum-I and PPP minimize the release of sCD40L ex vivo and better represent sCD40L in vivo. However, PPP may be preferable particularly in patients with thrombocytosis. The existence of mp-CD40L highlights the importance of centrifuge conditions.
Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami School of Medicine, 1475 NW 12th Avenue, Miami, FL 33136, USA. eahn@med.miami.edu
Eugene R
ER
Ahn
Gabriella
G
Lander
Wenche
W
Jy
Carlos J
CJ
Bidot
Joaquin J
JJ
Jimenez
Lawrence L
LL
Horstman
Yeon S
YS
Ahn
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000097:blood / Q000737:chemistry; D023201:CD40 Ligand / Q000097:blood* / Q000737:chemistry; D004198:Disease Susceptibility / Q000097:blood / Q000175:diagnosis; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D018570:Risk Assessment / Q000379:methods*; D012307:Risk Factors; D012680:Sensitivity and Specificity; D044967:Serum / Q000737:chemistry*; D012995:Solubility; D013048:Specimen Handling / Q000379:methods*; D013223:Statistics as Topic; D013927:Thrombosis / Q000097:blood* / Q000175:diagnosis*
D015415:Biomarkers; D023201:CD40 Ligand