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Impact of pre-analytical sample handling factors on plasma biomarkers of Alzheimer's disease.

Author(s): Kurz C, Stöckl L, Schrurs I, Suridjan I, Gürsel SÜ, Bittner T, Jethwa A, Perneczky R

Publication: J Neurochem, 2023, Vol. 165, Page 95-105

PubMed ID: 36625424 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared levels of Aβ40, Aβ42, apolipoprotein E4, GFAP, NFL,  phospho-tau-181, and phospho-tau-217 among EDTA plasma obtained after pre-centrifugation storage of blood at 4°C and room temperature for up to 24 h, between fresh and frozen EDTA plasma, among EDTA plasma aliquots stored at 4°C and room temperature for up to 24 h, among  EDTA plasma aliquots subjected to up to five cycles of freezing at -20°C or -80°C and thawing at room temperature, among plasma aliquots transferred between tubes one to five times, and between plasma transferred to low-density polyethylene tubes rather than polypropylene tubes. Analyte levels in EDTA plasma were also compared to those in sodium citrate and lithium heparin plasma. Blood was collected from sixteen patients with cognitive impairment due to possible or probable Alzheimer’s disease in to K₃ EDTA S-Monovette Tubes and inverted five times. Unless otherwise specified, blood was stored at room temperature for <1 h before plasma separation by centrifugation at 2,000 g for 10 min. Unless otherwise specified, plasma was aliquoted in polypropylene tubes before analysis. To test the effects of freezing, plasma stored for <6 h at 4°C was compared to plasma stored for 30 min at room temperature before frozen storage at -20°C. To test the effects storage, blood and plasma from 6 patients were stored for <1, 2, 6, and 24 h at room temperature and 4°C; and plasma was analyzed immediately or after storage at -20°C. To test the effects of freeze thaw cycling, plasma from six patients was aliquoted and subjected to 1, 2, 3 and 4 cycles of freezing at -20°C or -80°C for >8 h and thawing at room temperature and were compared to matched aliquots that were never frozen. To investigate the effect of anticoagulant, blood from four patients was collected into S-Monovette lithium heparin (LiHep) and S-Monovette sodium citrate tubes; plasma was separated by centrifugation and frozen at -20°C before analysis.  To investigate the effect of tube transfers, frozen plasma from five patients were transferred from one tube to the next 1, 3, or 5 times before analysis and compared to plasma in the original tube. To investigate the effects of transfer to low-density polyethylene tubes prior to analysis, plasma from an unspecified number of patients was transferred to a new polypropylene tube and low-density polyethylene tubes immediately before analysis.

   Summary of Findings:

   Levels of all evaluated analytes were comparable in plasma stored for <6 h at 4°C and plasma stored for 30 min at room temperature before frozen storage at -20°C. Consequently, fresh and frozen plasma were used interchangeably in all further experiments. Plasma Aβ42 and Aβ40 levels were lower when whole blood (pre-centrifugation) or plasma was stored for more than 2 h at room temperature or when plasma was freeze-thaw cycled three times at either -20 or -80°C, but Aβ42 and Aβ40 levels were unaffected by storage of whole blood or plasma for 24 h at 4°C. The Aβ42/Aβ40 ratio in plasma was lower when whole blood (pre-centrifugation) or plasma were stored for more than 2 h at room temperature but was stable through four freeze-thaw cycles (either temperature) and storage of whole blood or plasma for 24 h at 4°C. Plasma phospho-tau-217 increased when plasma was stored for 24 h at room temperature but were stable during storage of whole blood or plasma for 24 h at 4°C, storage of blood at room temperature for 24 h, and through four freeze-thaw cycles (-20 or -80°C) of plasma. Levels of phospho-tau-181, GFAP, and NFL were stable when blood or plasma were stored for ≤24 h at room temperature or 4°C and through four freeze-thaw cycles (-20 or -80°C).

   Compared to EDTA plasma, lithium heparin plasma had higher levels of Aβ42 and Aβ40 (10% and 9%, respectively), sodium citrate plasma had lower levels of Aβ42 and Aβ40 (6% and 4%, respectively), and a nonsignificant trend toward lower GFAP and NFL was observed in both lithium heparin and sodium citrate plasma. Although a slight decrease in recovery of Aβ42 and Aβ40 was noted when plasma was transferred between tubes up to five times, no significant effects were noted. Levels of all analytes evaluated were comparable in EDTA plasma that were transferred to low-density polyethylene tubes or polypropylene tubes immediately before analysis.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Alzheimer's Disease

   Platform:

   Analyte	Technology Platform
   Peptide	Clinical chemistry/auto analyzer
   Lipoprotein	Clinical chemistry/auto analyzer
   Protein	Clinical chemistry/auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)
   Biospecimen Acquisition	Anticoagulant	Potassium EDTA Lithium heparin Sodium citrate
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Whole blood
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Type of storage container	Transferred between tubes 1-5 times Transferred to low-density polyethylene tubes rather than polypropylene tubes
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles 4 cycles
   Storage	Storage temperature	4°C Room temperature
   Storage	Storage duration	0 h 2 h 6 h 24 h
   Storage	Storage conditions	As blood As plasma
   Biospecimen Preservation	Temperature of fixation/preservation	At -20 degrees C At -80 degrees C

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