Preanalytical evaluation of serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 measurements using LC-MS/MS.

Author(s): Mochizuki A, Kodera Y, Saito T, Satoh M, Sogawa K, Nishimura M, Seimiya M, Kubota M, Nomura F

Publication: Clin Chim Acta, 2013, Vol. 420, Page 114-20

PubMed ID: 23123828 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of collection tube type, anticoagulant type, delayed blood separation, storage, freeze-thaw cycling, patient food consumption, and analysis method on the measurement of 25OHD3 in plasma and serum from healthy patients. Plasma and serum were stored at -80 degrees C until analysis.

   Summary of Findings:

   Neither storage of blood for up to 6 h at room temperature prior to centrifugation nor storage of plasma or serum impacted the measurement of 25OHD3 in serum or plasma, regardless of light exposure during storage. Further, 25OHD3 levels in serum and plasma were not affected by up to 5 freeze-thaw cycles. While no significant effects were found based on the time of specimen collection, a trend toward lower 25OHD3 levels was noted in specimens collected 1 h after patients ate a standardized meal compared to levels in specimens collected before eating or 2 h after eating. Collection tube and anticoagulant type had no effects on 25OHD3 measurements. 25OHD3 levels measured by LC-MS/MS and RIA were very strongly correlated (r=0.947) with RIA giving a positive bias of 4 ng/mL. Average 25OHD3 levels were higher in Japanese males than in females (25.5 ng/mL and 20.9 ng/mL, respectively). Interference of the LC-MS/MS assay by bilirubin C, bilirubin F, hemolytic hemoglobin, chyle, and IgM-RF was less than 10%.

   Biospecimens

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

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Steroid	LC-MS or LC-MS/MS
   Steroid	Radioimmunoassay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	15 min 30 min 1 h 2 h 4 h 6 h
   Storage	Freeze/thaw cycling	1 cycle 2 cycles 3 cycles 4 cycles 5 cycles
   Storage	Storage conditions	Covered tube Uncovered tube
   Biospecimen Aliquots and Components	Biospecimen components	Spiked with bilirubin C Spiked with bilirubin F Spiked with hemolytic hemoglobin Spiked with chyle Spiked with IgM-RF
   LC-MS or LC-MS/MS Specific	Technology platform	RIA
   Preaquisition	Patient gender	Female Male
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum Whole blood
   Biospecimen Acquisition	Time of biospecimen collection	Before eating 1 h after eating 2 h after eating 3 h after eating
   Biospecimen Acquisition	Type of collection container/solution	VENOjectII plain serum tube Insepack serum tubes with separating gel and silica Insepack serum tubes for rapid coagulation Insepack plasma tubes with dipotassium EDTA and sodium fluoride Insepack plasma tubes with disodium EDTA Insepack plasma tubes with dipotassium EDTA Insepack plasma tubes with sodium heparin Microtainer serum tubes for neonates (gel and thrombin, accelerating clotting agent as the additive) Microtainer plasma tubes for neonates with dipotassium EDTA Serum clot activator Serum separator (SST)
   Biospecimen Acquisition	Anticoagulant	Potassium EDTA None Sodium EDTA Sodium fluoride Sodium heparin
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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