Impact of Preanalytical Procedures on Complement Biomarkers in Cerebrospinal Fluid and Plasma from Controls and Alzheimer's Disease Patients.
Author(s): Gutierrez J, Kurz C, Sandoval C, Edmonds R, Bittner T, Perneczky R, Biever A
Publication: J Alzheimers Dis, 2024, Vol. 101, Page 563-576
PubMed ID: 39213066 PubMed Review Paper? No
Purpose of Paper
This paper compared levels of complement proteins in cerebrospinal fluid (CSF) and plasma that remained unsupplemented or was supplemented with EDTA, EDTA and Futhan (FUT), or a protease inhibitor cocktail (PIC) prior to one to four freeze-thaw cycles. Plasma and CSF were acquired from 5 patients with Alzheimer’s disease and 4 healthy volunteers.
Conclusion of Paper
In plasma, levels of Complement 4a (C4a), Complement 4 (C4), Complement C3 (C3), Factor Bb (FBb), and Factor B (FB) were comparable in aliquots with supplements (EDTA, EDTA+ FUT, or PIC) and unsupplemented plasma after a single freeze-thaw cycle,, but C4a was highly variable, and C3a levels were slightly (<20%) lower in plasma with EDTA or EDTA+ FUT than in aliquots without. In CSF, levels of C4a, C4, and C3 were comparable in aliquots with supplements (EDTA, EDTA+ FUT, or PIC) and those without. In contrast, C3a levels in CSF were variable among the specimens that received a supplement: 40% lower with EDTA or EDTA+FUT and more than 2-fold higher with PIC compared to levels in the unsupplemented specimen. CSF levels of Bb and FB were 20% lower in CSF aliquots that received a EDTA+FUT supplement than unsupplemented CSF, but the authors state this is likely attributable to technical variability not a blockage of the in vitro complement activation. The authors conclude that supplementation of plasma or CSF with EDTA, EDTA+ FUT, or PIC has little effect on levels of most complement proteins, but that analysis of C3a in CSF benefits from supplementation with EDTA or EDTA+ FUT.
C3, C4 and FB in plasma were stable (<20% change) through four freeze-thaw cycles of plasma, regardless of supplementation, but C4a and C3a levels were higher in plasma aliquots that were freeze-thaw cycled two or more times than those that experienced one cycle, regardless of supplementation. Compared to unsuplemented plasma, plasma with EDTA, EDTA+ FUT or PIC had higher levels of C3a and C4a after two freeze thaw cycles, and plasma with EDTA+ FUT had lower levels of Bb after 4 freeze-thaw cycles. In CSF, levels of C4a, C4, C3, and FB were stable (<20% change) through four freeze-thaw cycles, regardless of supplementation, but C3a levels were higher after two freeze-thaw cycles than one, regardless of supplementation. The changes in C3a levels with freeze-thaw cycling were largest in unsupplemented plasma, but C3a levels were higher in plasma supplemented with PIC than the other plasma aliquots, with the greatest difference observed after two freeze-thaw cycles. While Bb was stable under most conditions, Bb levels were higher in CSF supplemented with PIC after two or more freeze-thaw cycles than the same sample type that experienced one freeze-thaw cycle. The correlation in rank ordering of complement proteins based on concentration was stronger when CSF that was freeze0thaw cycled once was compared to those cycled twice, and when plasma freeze-thaw cycled once was compared to those cycled twice. Importantly, none of the additives consistently improved the preservation of rank order in plasma or CSF freeze-thaw cycled twice versus once. The authors conclude that complement factor levels in plasma are affected by freeze-thaw cycling, regardless of supplementation; but, only C3a is affected by freeze-thaw cycling of CSF, and it is less affected in CSF supplemented with EDTA or EDTA+ FUT.
Studies
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Study Purpose
This study compared levels of complement proteins in cerebrospinal fluid (CSF) and plasma that remained unsupplemented with those that were supplemented with EDTA, EDTA and Futhan (FUT), or a protease inhibitor cocktail (PIC) prior to one to four freeze-thaw cycles. Blood-free CSF was collected via lumbar puncture at L4/L5 and case-matched K2EDTA blood was collected by standard venipuncture from 5 patients with Alzheimer’s disease and 4 healthy volunteers. Tubes were mixed by inversion (2-3 times for CSF and 8-10 times for blood) and placed on wet ice until centrifugation (<10 min). CSF and blood were centrifuged at 2000 g and 15000 g, respectively, for 10 minutes at 4°C and the supernatant was aliquoted into polypropylene screw top microtubes containing water or; EDTA, EDTA+FUT, or PIC to achieve a concentration of 20 mM EDTA. CSF and plasma aliquots were mixed and stored frozen at -80°C until analysis. CSF and plasma aliquots were subjected to 1-4 cycles of thawing on wet ice, before refreezing at -80°C for >16 h. Aβ1-40 and Aβ1–42 were quantified by immunoassay; a ratio of Aβ1–42/1-40 <5.5% was considered amyloid positive. Levels of C3, C3a, C4, and Factor B were quantified by ELISA. The authors established the sensitivity, precision and specificity of each assay.
Summary of Findings:
The CSF from each Alzheimer’s disease patient was confirmed to have amyloid positivity (ratio of Aβ1–42/1-40 <5.5%). After a single freeze-thaw cycle, levels of C4a, C4, C3, Bb, and FB in plasma were comparable in aliquots with supplements (EDTA, EDTA+ FUT, or PIC) and unsupplemented plasma; notably, C4a was highly variable, and C3a levels were slightly (<20%) lower in plasma with EDTA or EDTA+ FUT than in aliquots without a supplement. In CSF, levels of C4a, C4, and C3 were comparable among aliquots with supplements (EDTA, EDTA+ FUT, or PIC) and those without. In contrast, C3a levels in CSF were variable among aliquots with supplements, 40% lower with EDTA or EDTA+FUT and more than 2-fold higher with PIC than in unsupplemented CSF. CSF levels of Bb and FB were 20% lower in CSF aliquots with EDTA+FUT than in unsupplemented CSF, but the authors state this is likely attributable to technical variability not a blockage of the in vitro complement activation. The authors conclude that supplementation of plasma or CSF with EDTA, EDTA+ FUT, or PIC has little effect on levels of most complement proteins, but that analysis of C3a in CSF benefits from supplementation with EDTA or EDTA+ FUT.
C3, C4 and FB in plasma were stable (<20% change) through four freeze-thaw cycles of plasma, regardless of supplementation, but C4a and C3a levels were higher in plasma that was freeze-thaw cycled two or more times than that aliquots that were freeze-thaw cycled once, regardless of supplementation. Compared to unsupplemented plasma freeze-thaw cycled once, plasma with EDTA, EDTA+ FUT or PIC had higher levels of C3a and C4a after two freeze-thaw cycles, and plasma with EDTA+ FUT had lower levels of Bb after 4 freeze-thaw cycles. In CSF, levels of C4a, C4, C3, and FB were stable (<20% change) through four freeze-thaw cycles, regardless of supplementation; but C3a levels were higher after 2 freeze thaw cycles than one, regardless of supplementation. The changes in C3a levels that were observed with freeze-thaw cycling were largest in unsupplemented plasma, but C3a levels were higher in plasma supplemented with PIC than the other supplements examined, with the greatest difference observed following two freeze-thaw cycles. While Bb was stable under most conditions, Bb levels were higher in CSF supplemented with PIC after two or more freeze-thaw cycles than CSF that was freeze-thaw cycled once. The correlation in rank ordering of complement proteins based on concentration was stronger when CSF freeze-thaw cycled once was compared to those cycled twice than when plasma that was freeze-thaw cycled once was compared to those cycled twice. Importantly, none of the additives/supplements consistently improved the preservation of rank order in plasma or CSF aliquots that were freeze-thaw cycled twice versus once. The authors conclude that complement factor levels in plasma are affected by freeze-thaw cycling, regardless of supplementation; however, in CSF, only C3a is affected by freeze-thaw cycling and it is less affected when CSF is supplemented with EDTA or EDTA+ FUT.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Alzheimer's Disease
Platform:
Analyte Technology Platform Protein ELISA Protein Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
4 cycles
Storage Short-term storage solution None
EDTA
EDTA+FUT
Protease inhibitor cocktail