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Effect of blood collection tube containing protease inhibitors on the pre-analytical stability of Alzheimer's disease plasma biomarkers.

Author(s): Chen Y, Zeng X, Diaz JL, Sehrawat A, Lafferty TK, Boslett JJ, Klunk WE, Pascoal TA, Villemagne VL, Cohen AD, Lopez OI, Yates NA, Karikari TK

Publication: J Neurochem, 2024, Vol. 168, Page 2736-2750

PubMed ID: 38814273 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the stability of Alzheimer’s disease markers (Neurofilament light, NfL; Glial fibrillary acidic protein, GFAP; amyloid beta -42 and -40, Aβ42 and Aβ40; and phosphorylated tau 181, ptau181) in p100 and EDTA plasma stored for up to 96 h at room temperature and in blood stored in P100 and EDTA tubes at room temperature for 24 h pre-centrifugation. Levels of Aβ42 and Aβ40 were measured using two different assays, immunoprecipitation-mass spectrometry (IP-MS) and a Simoa analyzer. Unless otherwise specified, blood was collected from patients at the Alzheimer's Disease Research Center into matched P100 and EDTA tubes and inverted 8-10 times. Plasma was obtained by immediate centrifugation at 2000 g for 15 min at 4°C and frozen in cryovials at -80°C until analysis.  Plasma levels of NfL, GFAP, Aβ42, and Aβ40 were quantified using a Neurology 4-Plex E assay, and p-tau181 was measured using the p-tau181 V2 Advantage kit on a Simoa instrument. Aβ42, and Aβ40 were also assayed using an IP-MS assay. To investigate the effects of storage duration, plasma specimens from 5 patients were pooled according to collection tube type and stored for 0, 24, 48 and 96 h before freezing. To investigate the effects of storing individual plasma specimens, plasma specimens from 26 patients were stored at room temperature for 0 and 24 h before freezing. To investigate the effects of storing blood before plasma separation, blood from 15 patients was stored for 24 h at room temperature before plasma separation.

   Summary of Findings:

   In pooled plasma specimens, levels of Aβ42, and Aβ40 declined with storage (post-separation) at room temperature, but the rate of decline was slower in plasma from P100 than EDTA tubes. After 96 h, Aβ42 in EDTA and P100 plasma declined by 39.2% and 28.6%, respectively, using the IP-MS method and by 72.9% and 57.5%, respectively, using the Simoa analyzer. After 96 h, Aβ40 in EDTA and P100 plasma declined by 78.1% and 60.1%, respectively, using the IP-MS method and by 74.2% and 45.3%, respectively, using the Simoa analyzer. Further, the increase in the ratio of Aβ42 to Aβ40 as observed by IP-MS and the decrease in the ratio of Aβ42 to Aβ40 by the Simoa analyzer was greater in EDTA than P100 plasma, but the differences depended on the analyzer used (larger by IP-MS). In the follow-up study that used individual plasma specimens, larger declines in Aβ40 and Aβ42 were observed after storing plasma for 24 h at room temperature in EDTA than P100 plasma by IP-MS (Aβ40 -16.4% versus -6.7%, P=0.008; Aβ42 -14.2% versus -2.7%, P=0.048) and by Simoa analyzer (Aβ40 -11.1% versus -1.2%, P<0.001; Aβ42 -21.1% versus -10.2%, P<0.001). Using individual plasma specimens, no significant difference in the change in the ratio of Aβ42 to Aβ40 after 24 h at room temperature was found between the tube types evaluated, regardless of analysis method. Levels of GFAP, NfL, and p-tau181 remained relatively stable (<25% changes) with room temperature, storage of pooled (96 h) and individual plasma (24 h), regardless of tube type.

   Aβ40 and Aβ42 declined with 24 h room temperature storage of whole blood (pre-centrifugation) in either EDTA (-34.8% and -23.0%, respectively, by IP-MS and -48.3% and -46.0%, respectively, by Simoa analyzer) or P100 tubes (-16.7% and -17.9%, respectively, by IP-MS and -25.6% and -31.3%, respectively, by Simoa analyzer). Generally, the declines in Aβ40 and Aβ42 after 24 h room temperature storage of whole blood were significantly larger when stored in EDTA than P100 tubes (P=0.008 and P=0.303, respectively by IP-MS and P<0.001, both by Simoa Analyzer). The increase in the ratio of Aβ42 to Aβ40 after 24 h at room temperature was significantly higher when blood was stored in EDTA than P100 tubes and analyzed by IP-MS (11.4% versus 6.0% P=0.018), but not when measured using by Simoa analyzer (-3.2% versus -8.1%, P=0.735). Levels of GFAP, NfL, and p-tau181 remained relatively stable (<6% changes) with 24 h room temperature storage of whole blood regardless of tube type.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Alzheimer's Disease

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	EDTA tube P100 tube
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Storage	Type of storage container	P100 tube EDTA tube
   Storage	Time at room temperature	0 h 24 h 48 h 96 h
   Storage	Storage conditions	EDTA plasma pool (all timepoints) P100 plasma pool (all timepoints) EDTA plasma (0 versus 24 h) P100 plasma (0 versus 24 h) Whole blood in EDTA tube (0 versus 24 h) Whole blood in P100 tube (0 versus 24 h)
   Clinical chemistry/auto analyzer Specific	Technology platform	IP-MS Simoa Autoanalyzer

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