Preanalytical sample handling recommendations for Alzheimer's disease plasma biomarkers.
Author(s): Rózga M, Bittner T, Batrla R, Karl J
Publication: Alzheimers Dement (Amst), 2019, Vol. 11, Page 291-300
PubMed ID: 30984815 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of circadian rhythms, anticoagulant type, tube type and volume, partial tube filling, delayed centrifugation, plasma tube transfers, plasma storage (fresh, thawed of frozen), and freeze-thaw cycling on levels of the amyloid β peptides(1-40) [Aβ(1-40)] and Aβ(1-42) and total Tau (tTau) in plasma.
Conclusion of Paper
Levels of Aβ(1-40), Aβ(1-42), and tTau in plasma were affected by the time of day blood was collected, anticoagulant type, and delayed centrifugation. Levels of Aβ(1-40) and Aβ(1-42) were affected by the storage of fresh or thawed plasma but tTau levels were unaffected. tTau was affected by partial filling of the tubes and freeze-thaw cycling of plasma at -80 ˚C, but there were only minimal effects on levels of Aβ(1-40) and Aβ(1-42). There was no effect of the tube material [polypropylene (S-Monovette) versus polyethylene terephthalate (Vacutainer)], tube volume (2.7, 4.9, 8.5, 9, or 10 mL), EDTA cation (K2 or K3), presence of gel separator, frozen storage temperature, or transfer of plasma between tubes.
Studies
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Study Purpose
The purpose of this study was to determine if collection day or time of day affect levels of Aβ(1-40), Aβ(1-42), and tTau in plasma. Blood was collected from eight healthy volunteers into 9 mL K3EDTA tubes at 8 AM, noon, and 3 PM on Day 1 and at 8 AM only on Days 2 and 7. Within 10 min of collection, blood was centrifuged for 10 min at 2000 x g. Plasma was transferred to a polypropylene tube, inverted several times, and aliquoted. Levels of Aβ(1-40), Aβ(1-42), and tTau were quantified using a Roche Cobas e 601 analyzer and were normalized to levels at 8 AM on Day 1.
Summary of Findings:
Levels of Aβ(1-40), and Aβ(1-42) were 5-9% higher and tTau approximately 8% lower in specimens collected at noon and 3 PM on Day 1 than in specimens collected at 8 AM, but levels of all three were comparable at 8 AM on the three days.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Clinical chemistry/auto analyzer Peptide Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection 8 AM on day 1
12 PM on day 1
3 PM on day 1
8 AM on day 2
8 AM on day 3
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Study Purpose
The purpose of this study was to investigate the effects of anticoagulant type, tube volume, tube type (before and after freezing), EDTA cation, and incomplete filling on Aβ(1-40), Aβ(1-42), and tTau levels in plasma. To test the effect of anticoagulant type, blood was collected from 10 healthy patients into tubes containing K3EDTA, lithium heparin, or sodium citrate. To test the effect of tube material and volume, blood was collected from 8-11 individuals into polypropylene K3EDTA S-Monovette tubes (9 mL, 4.9 mL, and 2.7 mL), 4.9 mL polypropylene K2EDTA S-Monovette tubes, 2.7 mL K2EDTA polypropylene S-Monovette tubes with a gel separator, 10 mL K2EDTA polyethylene terephthalate Vacutainer tubes, and 8.5 mL K2EDTA polyethylene terephthalate Vacutainer tubes with a gel separator. To test the effects of partial filling, 4.9 mL (1 specimen) and 9 mL K3EDTA (5 specimens) polypropylene K3EDTA S-Monovette tubes and both types of Vacutainer tubes (9 specimens) were filled only half-way. Tubes were inverted five times after filling. Blood was centrifuged for 10 min at 2000 x g within 10 min of collection. Plasma was transferred to a polypropylene tube, inverted several times, and then aliquoted. Levels of Aβ(1-40), Aβ(1-42), and tTau were quantified using a Roche Cobas e 601 analyzer. The effect of a single freeze-thaw cycle was investigated using aliquots of plasma from the collection tube study.
Summary of Findings:
Levels of Aβ(1-40) and Aβ(1-42) were 10-20% higher in lithium heparin plasma than in K3EDTA plasma, but slightly lower in Na Citrate plasma than K3EDTA plasma. The ratio of Aβ(1-40) to Aβ(1-42) was comparable in lithium heparin plasma and K3EDTA plasma but higher in Na Citrate plasma than K3EDTA plasma. tTau levels were much lower in lithium heparin and sodium citrate plasma than K3EDTA plasma (approximately 30% and 50% K3EDTA levels, respectively). While Aβ(1-40), Aβ(1-42), and the ratio of Aβ(1-40) to Aβ(1-42) were comparable in plasma from full and half-full tubes, levels of tTau were 12-14% lower in plasma obtained from half-full tubes than full tubes. There was no effect of tube material [polypropylene (S-Monovette) versus polyethylene terephthalate (Vacutainer)], tube volume (2.7, 4.9, 8.5, 9, or 10 mL), EDTA cation (K2 or K3), or presence of gel separator on the levels of Aβ(1-40), Aβ(1-42), or tTau levels in EDTA plasma. Further, tube type had no effect on the recovery of Aβ(1-40), Aβ(1-42), or tTau after freezing.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Clinical chemistry/auto analyzer Peptide Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Lithium heparin
Sodium citrate
Potassium EDTA
Multiple forms evaluated
Gel-barrier
Biospecimen Aliquots and Components Aliquot size/volume 2.7 mL
4.9 mL
8.5 mL
9 mL
10 mL
Tube filled
Tube half-filled
Biospecimen Acquisition Type of collection container/solution S-Monovette (polypropylene)
Vacutainer (polyethylene terephthalate)
With gel separator
Without gel separator
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Study Purpose
The purpose of this study was to investigate the effects of delayed centrifugation, storage of fresh or thawed plasma, and repeated transfer of plasma to new tubes on Aβ(1-40), Aβ(1-42), and tTau levels in plasma. Blood was collected from healthy patients into 9 mL K3 EDTA S-Monovette tubes. To investigate the effects of processing delay, specimens from 6-8 individuals were centrifuged after 30 min, 1 h, 2 h, 6 h, or 24 h. The effects of storage of fresh plasma were investigated by storing plasma from six healthy patients for 0, 1, 3, 6, or 24 h at 4˚C after centrifugation. The effects of storage of thawed plasma was investigated by storing plasma from eight healthy individuals for 0, 1, 6, or 24 h after thawing at 4˚C or room temperature. The effects of transferring plasma to new polypropylene tubes was investigated by transferring plasma from four individuals 1-5 times. Plasma was obtained by centrifugation of blood for 10 min at 2000 x g. Plasma was transferred to a polypropylene tube, inverted several times, and then aliquoted. Levels of Aβ(1-40), Aβ(1-42), and tTau were quantified using a Roche Cobas e 601 analyzer and normalized to specimens that were not stored or transferred after the initial transfer.
Summary of Findings:
Levels of Aβ(1-40) and Aβ(1-42) in plasma declined progressively with pre-centrifugation storage of blood (4-5% after 2 h, 8-10% after 6 h, and 50% after 24 h), but tTau levels remained unchanged when blood was stored for 6 h and declined by less than 20% when stored for 24 h. Levels of Aβ(1-40) and Aβ(1-42) were stable in thawed plasma for 1 h at 4˚C and room temperature and in fresh plasma for 3 h at 4˚C. With further storage, levels of Aβ(1-40) and Aβ(1-42) declined in thawed plasma stored at 4˚C (5-7% after 6 h and 8-10% after 24h) and room temperature (15-18% after 6 h and 40% after 24 h), but Aβ(1-40) declined 3-5% after 6h and 10% after 24 h at 4˚C and Aβ(1-42) declined by 3-5% after 6 and 24 h at 4˚C in fresh plasma. In contrast, tTau was unaffected by storage of thawed plasma. There was no effect of transferring plasma between tubes 1-5 times on levels of Aβ(1-40), Aβ(1-42), or tTau.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Clinical chemistry/auto analyzer Peptide Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Freeze/thaw cycling 0 cycles
1 cycle
Storage Storage temperature Room temperature
4˚C
Storage Storage duration 0 h
30 min
1 h
2 h
3 h
6 h
24 h
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Study Purpose
This study investigated the effects of plasma storage temperature and freeze-thaw cycling on Aβ(1-40), Aβ(1-42), and tTau. Blood was obtained from 10-12 (freeze-thaw cycling experiments or 6 (storage temperature experiments) healthy individuals in 9 mL K3 EDTA S-Monovette tubes. Plasma was obtained by centrifugation of blood for 10 min at 2000 x g. Plasma was analyzed immediately and again after one, two, and three freeze-thaw cycles consisting of freezing for ≥8 h at -80˚C, -20˚C, and -25˚C followed by thawing at room temperature. Plasma aliquots were stored frozen at -80˚C, -20˚C, and -25˚C for 14 days. Levels of Aβ(1-40), Aβ(1-42), and tTau were quantified using a Roche Cobas e 601 analyzer and normalized to levels in the respective fresh specimen.
Summary of Findings:
Storage of plasma at -80˚C, -20˚C, and -25˚C for 14 days had no effect on the levels of Aβ(1-40), Aβ(1-42), or tTau. While levels of Aβ(1-40) and Aβ(1-42) remained within 4% of that in fresh plasma when subjected to up to 3 freeze-thaw cycles at -80˚C, levels of tTau were 7% lower after 2 freeze-thaw cycles at -80˚C than in fresh plasma. The authors report that 3 freeze-thaw cycles at -20˚C or -25˚C resulted in 2-3% decrease in tTau, no change in Aβ(1-40), and a 4-5% increase in Aβ(1-42).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Clinical chemistry/auto analyzer Peptide Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
0 cycles
Storage Storage temperature -20˚C
-25˚C
-80˚C