Differences of soluble CD40L in sera and plasma: Implications on CD40L assay as a marker of thrombotic risk

Eugene R. Ahn, Gabriella Lander, Wenche Jy, Carlos J. Bidot, Joaquin J Jimenez, Lawrence L. Horstman, Yeon Ahn

Research output: Contribution to journalArticle

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Abstract

Introduction: 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-μm 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.

Original languageEnglish
Pages (from-to)143-148
Number of pages6
JournalThrombosis Research
Volume114
Issue number2
DOIs
StatePublished - Aug 27 2004

Fingerprint

CD40 Ligand
Serum
Blood Platelets
Platelet Count
Thrombocytosis
Temperature
Thrombosis
Enzyme-Linked Immunosorbent Assay
Ice
Acute Coronary Syndrome
Thrombocytopenia

Keywords

  • CD40L
  • ITP
  • microparticle-associated CD40L
  • mp-CD40L
  • Plasma
  • platelet-poor plasma
  • Platelets
  • PPP
  • sCD40L
  • Serum
  • serum from blood clotted at room temperature
  • serum from blood clotted on ice
  • serum-I
  • serum-RT
  • soluble CD40L
  • Thrombosis

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Hematology

Cite this

Differences of soluble CD40L in sera and plasma : Implications on CD40L assay as a marker of thrombotic risk. / Ahn, Eugene R.; Lander, Gabriella; Jy, Wenche; Bidot, Carlos J.; Jimenez, Joaquin J; Horstman, Lawrence L.; Ahn, Yeon.

In: Thrombosis Research, Vol. 114, No. 2, 27.08.2004, p. 143-148.

Research output: Contribution to journalArticle

Ahn, Eugene R. ; Lander, Gabriella ; Jy, Wenche ; Bidot, Carlos J. ; Jimenez, Joaquin J ; Horstman, Lawrence L. ; Ahn, Yeon. / Differences of soluble CD40L in sera and plasma : Implications on CD40L assay as a marker of thrombotic risk. In: Thrombosis Research. 2004 ; Vol. 114, No. 2. pp. 143-148.
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abstract = "Introduction: 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-μm 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.",
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AU - Lander, Gabriella

AU - Jy, Wenche

AU - Bidot, Carlos J.

AU - Jimenez, Joaquin J

AU - Horstman, Lawrence L.

AU - Ahn, Yeon

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N2 - Introduction: 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-μm 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.

AB - Introduction: 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-μm 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.

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KW - Serum

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KW - serum from blood clotted on ice

KW - serum-I

KW - serum-RT

KW - soluble CD40L

KW - Thrombosis

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