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Influence of pre-analytical and analytical factors on soluble CD40L measurements.

Author(s): Varo N, Nuzzo R, Natal C, Libby P, Schönbeck U

Publication: Clin Sci (Lond), 2006, Vol. 111, Page 341-7

PubMed ID: 16856875 PubMed Review Paper? No

Suggested by: ISBER

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of anticoagulant choice, delay to centrifugation, centrifugation speed (200 x g, 400 x g, 1000 x g, 2000 x g, 13000 x g), storage, freeze-thaw cycling, and blood components on the measurement of sCD40L.

   Summary of Findings:

   sCD40L levels were higher in serum than in plasma (p=0.001) and were not correlated with one another. There were no significant differences in sCD40L levels when heparin, EDTA, or citrate was used for anticoagulation. When blood was stored at room temperature for 2 h or 6 h prior to centrifugation, levels of sCD40L in serum were 15 and 22% higher, respectively than when blood was stored for 30 min. Increasing the centrifugation speed led to a gradual decrease in sCD40L levels measured in plasma as the platelet and leukocyte levels decreased, but no additional effect was noted at centrifugation speeds greater than 1000 g. Up to 3 freeze-thaw cycles did not affect sCD40L levels in plasma with a low platelet count but increased sCD40L in platelet rich plasma. After centrifugation, storage of serum at 4 degrees C for 48 h did not affect sCD40L levels, but when serum was stored at room temperature, sCD40L levels declined with storage. Bilirubin and hemoglobin interfered with the measurement of sCD40L at all levels. While normal physiological concentrations of cholesterol and triglycerides (<250 mg/dL and <200 mg/dL respectively) did not affect measurement of sCD40L, pathological levels of triglycerides and cholesterol led to decreases in measured sCD40L levels. sCD40L levels were higher in plasma from diabetic patients than healthy controls.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Diabetes Type 2
   • Diabetes Type 1
   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	ELISA

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles
   Storage	Time at room temperature	30 min 2 h 6 h
   Storage	Storage temperature	4 degrees C Room temperature
   Storage	Storage duration	0 h 6 h 24 h 48 h
   Preaquisition	Diagnosis/ patient condition	Normal Diabetes
   Preaquisition	Biomarker level	Low sCD40L High sCD40L Triglycerides (50-400 mg/dL) Cholesterol (150-300 mg/dL) Hemoglobin (12-36 mg/mL) Bilirubin (50-200 mg/mL)
   Biospecimen Aliquots and Components	Blood and blood products	Platelet-poor plasma Platelet-rich plasma Serum
   Biospecimen Aliquots and Components	Centrifugation	Multiple speeds compared Centrifugation delays investigated
   Biospecimen Acquisition	Anticoagulant	EDTA Heparin None Citrate

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2. Study Purpose

   The purpose of this study was to determine the effects of pH on the recovery of sCD40L from spiked urine of normal and diabetic patients.

   Summary of Findings:

   Endogenous sCD40L was not detected in urine specimens, but recovery of spiked sCD40L was highest in urine specimens with a pH of 5-8.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Diabetes Type 1
   • Diabetes Type 2
   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	ELISA

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	pH	2 3 4 5 6 7 8 9

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