Limits...
Chasing the Effects of Pre-Analytical Confounders - A Multicenter Study on CSF-AD Biomarkers.

Leitão MJ, Baldeiras I, Herukka SK, Pikkarainen M, Leinonen V, Simonsen AH, Perret-Liaudet A, Fourier A, Quadrio I, Veiga PM, de Oliveira CR - Front Neurol (2015)

Bottom Line: Centrifugation conditions did not influence biomarkers levels, except for samples with a high CSF total protein content, where either non-centrifugation or centrifugation at RT, compared to 4°C, led to higher Aβ42 levels.Moreover, the concentration of Tau and pTau appears to be stable up to five freeze-thaw cycles, whereas Aβ42 levels decrease if CSF is freeze-thawed more than three times.This study contributes to the establishment of harmonized standard operating procedures that will help reducing inter-lab variability of CSF-AD biomarkers evaluation.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroscience and Cell Biology (CNC), University of Coimbra , Coimbra , Portugal.

ABSTRACT

Introduction: Core cerebrospinal fluid (CSF) biomarkers - Aβ42, Tau, and phosphorylated Tau (pTau) - have been recently incorporated in the revised criteria for Alzheimer's disease (AD). However, their widespread clinical application lacks standardization. Pre-analytical sample handling and storage play an important role in the reliable measurement of these biomarkers across laboratories.

Aim: In this study, we aim to surpass the efforts from previous studies, by employing a multicenter approach to assess the impact of less studied CSF pre-analytical confounders in AD-biomarkers quantification.

Methods: Four different centers participated in this study and followed the same established protocol. CSF samples were analyzed for three biomarkers (Aβ42, Tau, and pTau) and tested for different spinning conditions [temperature: room temperature (RT) vs. 4°C; speed: 500 vs. 2000 vs. 3000 g], storage volume variations (25, 50, and 75% of tube total volume), as well as freezing-thaw cycles (up to five cycles). The influence of sample routine parameters, inter-center variability, and relative value of each biomarker (reported as normal/abnormal) was analyzed.

Results: Centrifugation conditions did not influence biomarkers levels, except for samples with a high CSF total protein content, where either non-centrifugation or centrifugation at RT, compared to 4°C, led to higher Aβ42 levels. Reducing CSF storage volume from 75 to 50% of total tube capacity decreased Aβ42 concentration (within analytical CV of the assay), whereas no change in Tau or pTau was observed. Moreover, the concentration of Tau and pTau appears to be stable up to five freeze-thaw cycles, whereas Aβ42 levels decrease if CSF is freeze-thawed more than three times.

Conclusion: This systematic study reinforces the need for CSF centrifugation at 4°C prior to storage and highlights the influence of storage conditions in Aβ42 levels. This study contributes to the establishment of harmonized standard operating procedures that will help reducing inter-lab variability of CSF-AD biomarkers evaluation.

No MeSH data available.


Related in: MedlinePlus

Observed differences in Aβ42 levels between aliquots with 25 vs. 50 vs. 75% of total tube volume (75 vs. 50%, p = 0.03). Results expressed in relative percentage, facing the baseline condition (75% of tube volume representing 100%). Absolute concentration levels (mean ± SD): 25% – 651.9 ± 337.1; 50% – 636.7 ± 351.9; 75% – 657.6 ± 334.4.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4495343&req=5

Figure 2: Observed differences in Aβ42 levels between aliquots with 25 vs. 50 vs. 75% of total tube volume (75 vs. 50%, p = 0.03). Results expressed in relative percentage, facing the baseline condition (75% of tube volume representing 100%). Absolute concentration levels (mean ± SD): 25% – 651.9 ± 337.1; 50% – 636.7 ± 351.9; 75% – 657.6 ± 334.4.

Mentions: We hypothesized that the amount of CSF aliquoted in relation with total tube volume would have impact on protein concentration mainly because of the adhesive ability of Aβ42 and possibly Tau to tube walls, even though PP vials were always used throughout the study. Thus, different CSF volume percentages were tested in final aliquots and we found that decreasing the percentage of tube filling from 75% (baseline condition) to 50% resulted in a small but significant reduction of 3.7% in Aβ42 concentration (p = 0.03). This effect was indistinguishable from the analytical coefficient of variation of the assay. Moreover, when further decreasing the percentage of tube filling to 25%, Aβ42 levels increased to levels similar to the ones observed under baseline conditions (Figure 2). Neither Tau nor pTau proteins levels were influenced by the amount of CSF aliquoted in relation with total tube volume. Adding covariates to our tests showed influence of “CSF Total protein” (dichotomized as normal/abnormal) on pTau levels (p = 0.027), particularly in 25% filling volume aliquots presenting abnormal TP content (54.6 ± 30.5) vs. normal TP (47.1 ± 26.8) (Figure 3). Other covariates had no impact in any of the three biomarkers.


Chasing the Effects of Pre-Analytical Confounders - A Multicenter Study on CSF-AD Biomarkers.

Leitão MJ, Baldeiras I, Herukka SK, Pikkarainen M, Leinonen V, Simonsen AH, Perret-Liaudet A, Fourier A, Quadrio I, Veiga PM, de Oliveira CR - Front Neurol (2015)

Observed differences in Aβ42 levels between aliquots with 25 vs. 50 vs. 75% of total tube volume (75 vs. 50%, p = 0.03). Results expressed in relative percentage, facing the baseline condition (75% of tube volume representing 100%). Absolute concentration levels (mean ± SD): 25% – 651.9 ± 337.1; 50% – 636.7 ± 351.9; 75% – 657.6 ± 334.4.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495343&req=5

Figure 2: Observed differences in Aβ42 levels between aliquots with 25 vs. 50 vs. 75% of total tube volume (75 vs. 50%, p = 0.03). Results expressed in relative percentage, facing the baseline condition (75% of tube volume representing 100%). Absolute concentration levels (mean ± SD): 25% – 651.9 ± 337.1; 50% – 636.7 ± 351.9; 75% – 657.6 ± 334.4.
Mentions: We hypothesized that the amount of CSF aliquoted in relation with total tube volume would have impact on protein concentration mainly because of the adhesive ability of Aβ42 and possibly Tau to tube walls, even though PP vials were always used throughout the study. Thus, different CSF volume percentages were tested in final aliquots and we found that decreasing the percentage of tube filling from 75% (baseline condition) to 50% resulted in a small but significant reduction of 3.7% in Aβ42 concentration (p = 0.03). This effect was indistinguishable from the analytical coefficient of variation of the assay. Moreover, when further decreasing the percentage of tube filling to 25%, Aβ42 levels increased to levels similar to the ones observed under baseline conditions (Figure 2). Neither Tau nor pTau proteins levels were influenced by the amount of CSF aliquoted in relation with total tube volume. Adding covariates to our tests showed influence of “CSF Total protein” (dichotomized as normal/abnormal) on pTau levels (p = 0.027), particularly in 25% filling volume aliquots presenting abnormal TP content (54.6 ± 30.5) vs. normal TP (47.1 ± 26.8) (Figure 3). Other covariates had no impact in any of the three biomarkers.

Bottom Line: Centrifugation conditions did not influence biomarkers levels, except for samples with a high CSF total protein content, where either non-centrifugation or centrifugation at RT, compared to 4°C, led to higher Aβ42 levels.Moreover, the concentration of Tau and pTau appears to be stable up to five freeze-thaw cycles, whereas Aβ42 levels decrease if CSF is freeze-thawed more than three times.This study contributes to the establishment of harmonized standard operating procedures that will help reducing inter-lab variability of CSF-AD biomarkers evaluation.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroscience and Cell Biology (CNC), University of Coimbra , Coimbra , Portugal.

ABSTRACT

Introduction: Core cerebrospinal fluid (CSF) biomarkers - Aβ42, Tau, and phosphorylated Tau (pTau) - have been recently incorporated in the revised criteria for Alzheimer's disease (AD). However, their widespread clinical application lacks standardization. Pre-analytical sample handling and storage play an important role in the reliable measurement of these biomarkers across laboratories.

Aim: In this study, we aim to surpass the efforts from previous studies, by employing a multicenter approach to assess the impact of less studied CSF pre-analytical confounders in AD-biomarkers quantification.

Methods: Four different centers participated in this study and followed the same established protocol. CSF samples were analyzed for three biomarkers (Aβ42, Tau, and pTau) and tested for different spinning conditions [temperature: room temperature (RT) vs. 4°C; speed: 500 vs. 2000 vs. 3000 g], storage volume variations (25, 50, and 75% of tube total volume), as well as freezing-thaw cycles (up to five cycles). The influence of sample routine parameters, inter-center variability, and relative value of each biomarker (reported as normal/abnormal) was analyzed.

Results: Centrifugation conditions did not influence biomarkers levels, except for samples with a high CSF total protein content, where either non-centrifugation or centrifugation at RT, compared to 4°C, led to higher Aβ42 levels. Reducing CSF storage volume from 75 to 50% of total tube capacity decreased Aβ42 concentration (within analytical CV of the assay), whereas no change in Tau or pTau was observed. Moreover, the concentration of Tau and pTau appears to be stable up to five freeze-thaw cycles, whereas Aβ42 levels decrease if CSF is freeze-thawed more than three times.

Conclusion: This systematic study reinforces the need for CSF centrifugation at 4°C prior to storage and highlights the influence of storage conditions in Aβ42 levels. This study contributes to the establishment of harmonized standard operating procedures that will help reducing inter-lab variability of CSF-AD biomarkers evaluation.

No MeSH data available.


Related in: MedlinePlus