Pre-analytical stability of serum biomarkers for neurological disease: neurofilament-light, glial fibrillary acidic protein and contactin-1.
Author(s): van Lierop ZYGJ, Verberk IMW, van Uffelen KWJ, Koel-Simmelink MJA, In 't Veld L, Killestein J, Teunissen CE
Publication: Clin Chem Lab Med, 2022, Vol. 60, Page 842-850
PubMed ID: 35333481 PubMed Review Paper? No
Purpose of Paper
This paper compared levels of Neurofilament-light (NfL), glial fibrillary acidic protein (GFAP) and contactin-1 (CNTN1) in serum isolated from blood collected in 6 different collection tube types, subjected to 30 min to 24 h of storage at room temperature or 2-8°C prior to serum separation, centrifugation at room temperature or 4°C , storage for 4-24 h post-separation at room temperature or 2-8°C, storage at -20°C or 2-8°C for 2 weeks before storage at -80°C, as well as among serum subjected to 1-5 freeze-thaw cycles.
Conclusion of Paper
Compared to serum from blood collected in the reference tube (serum clot activator tubes), plasma from blood collected in sodium citrate tubes had lower NfL, GFAP and CNTN1, and plasma from blood collected in EDTA and EDTA gel separator tubes had lower levels of GFAP. There was no effect of pre-centrifugation storage of blood for 30 min-24 h at room temperature or 2-8°C on GFAP or CNTN1 levels, but Nfl levels were 20% higher and more variable when blood was stored for 24 h at 2-8°C rather than for 30 min. Centrifugation temperature (4°C versus room temperature) and post-separation storage of serum (room temperature or 2-8°C for ≤24 h or 2 weeks at 2-8°C or -20°C) had no effect on levels of NfL, GFAP or CNTN1. While NfL and GFAP levels were unaffected by up to 4 freeze-thaw cycles, CNTN1 levels increased slightly when serum was subjected to 4 additional freeze-thaw cycles.
As expected, serum from multiple sclerosis (MS) patients had higher NfL and GFAP levels and lower CNTN1 levels than observed in healthy volunteers. Generally, preanalytical effects on all three markers were similar in serum from healthy volunteers and patients with MS, but serum from MS patients showed a larger increase in CNTN1 and more variability in response to freeze-thaw cycling than serum from healthy controls.
Studies
-
Study Purpose
This study compared levels of NfL, GFAP and CNTN1 in serum/plasma from blood collected in six different collection tube types, subjected to 30 min to 24 h of storage at room temperature or 2-8°C prior to serum separation , centrifugation at room temperature or 4°C, post-separation storage of serum for 4-24 h at room temperature or 2-8°C, storage at -20°C or 2-8°C for 2 weeks before storage at -80°C, as well as 1-5 freeze-thaw cycles. Blood was collected from thirty-five patients with multiple sclerosis (MS) and thirty-five volunteers (no specified diagnosis); 5 specimens from patients with MS and 5 specimens from healthy volunteers were used to test each preanalytical variable. Unless otherwise specified, blood was collected in BD Vacutainer clot activator tubes and allowed to clot for 30 min before serum separation by centrifugation at 1800 g for 10 min at room temperature. The resultant serum was then aliquoted and immediately frozen at -80°C until analysis. Before analysis, serum was thawed and centrifuged at 10,000 g for 10 min at room temperature. Levels of NfL and GFAP were quantified using an HDx analyzer and CNTN1 were quantified using the Human contactin-1 Magnetic Luminex Assay on a Bio-PlexTM 200 system. To test the effects of tube type, blood was collected into clot activator (reference), serum gel separator, EDTA, EDTA with gel separator, acid citrate and heparin vacutainer tubes. To investigate effects of prolonged clot time, blood from was allowed to clot in clot activator tubes for 30 min (reference), 3 h, 6 h, and 24 h at room temperature before serum separation. To test the effects of refrigerated clotting, blood s was allowed to clot in clot activator tubes for 30 min, 3 h, 6 h, and 24 h at 2-8°C before serum separation. To investigate potential effects of centrifugation, blood from was centrifuged at room temperature and 4°C. To test for effects of serum storage, matched serum specimens were directly frozen (reference), or stored for 4 h or 24 h at room temperature or at 2-8°C before freezing. To determine effects of short-term storage temperature, serum was stored directly at -80°C (reference), stored for 2 weeks at -20°C before transfer to -80°C, and stored for 2 weeks at 2-8°C before transfer to -80°C. To test the effects o freeze-thaw cycling of serum, serum aliquots from 5 patients with MS and 5 healthy volunteers were freeze-thaw cycled 1, 2, 3, and 5 times before analysis.
Summary of Findings:
Levels of NfL were comparable to reference (clot activator tubes) when collected in serum gel separator, plasma EDTA, plasma EDTA gel separator or lithium-heparin tubes, but NfL levels were lower when blood was collected in sodium citrate tubes (mean 63.3% of reference). Although, NfL levels in serum were unaffected by storage of blood in clot activator tubes for up to 24 h at room temperature or 6 h at 2-8°C, NfL levels were 20% higher and more variable when blood was stored for 24 h at 2-8°C rather than for 30 min. NfL levels in serum were unaffected by storage of serum at room temperature or 2-8°C for ≤24 h, storage of serum for 2 weeks at 2-8°C or -20°C, or up to 4 additional (5 total) freeze-thaw cycles.
Levels of GFAP were comparable to reference (clot activator tubes) when collection was in serum gel separator of lithium heparin tubes but were lower (66.4-98.1% of reference) when collected in EDTA plasma, EDTA gel separator or sodium citrate tubes. There was no effect of pre-centrifugation storage of blood for 30 min-24 h at room temperature or 2-8°C, centrifugation temperature, storage of serum at room temperature or 2-8°C for ≤24 h, storage of serum for 2 weeks at 2-8°C or -20°C, or up to 4 additional (5 total) freeze-thaw cycles on GFAP levels.
Levels of CNTN1 were comparable to reference (clot activator tubes) when collected in serum gel separator, plasma EDTA, plasma EDTA gel separator or lithium-heparin tubes, but CNTN1 levels were lower when blood was collected in sodium citrate tubes (mean 78.9% of reference). There was no effect of pre-centrifugation storage of blood for 30 min-24 h at room temperature or 2-8°C, centrifugation temperature, storage of serum at room temperature or 2-8°C for ≤24 h, or storage of serum for 2 weeks at 2-8°C or -20°C on CNTN1 levels. However, CNTN1 levels increased slightly when serum was subjected to 4 additional freeze-thaw cycles (a total of 5 freeze-thaw cycles).
As expected, serum from MS patients had higher NfL and GFAP levels and lower CNTN1 levels than that from healthy volunteers (P=0.02, P=0.03 and P=0.03, respectively). Generally, preanalytical effects on all three markers were similar in serum from healthy volunteers and patients with MS, but serum from MS patients showed a larger increase in CNTN1 and more variability in response to freeze-thaw cycling than serum from control patients.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Not specified
- Multiple Sclerosis
Platform:
Analyte Technology Platform Protein Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition MS
Volunteer (Not specified)
Biospecimen Acquisition Type of collection container/solution Serum clot activator tube
Serum gel separator tube
EDTA tube
EDTA tube with gel separator
Acid citrate tube
Heparin tube
Storage Storage conditions As serum
As blood
Storage Storage duration Clot 30 min
Clot 3 h
Clot 6 h
Clot 24 h
0 h
4 h
24 h
2 weeks
Storage Storage temperature Room temperature
2-8°C
-20°C
-80°C
Biospecimen Aliquots and Components Centrifugation Multiple temperatures compared
Centrifugation delays investigated