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Letters to the Editor |
1 Department of Neurology, and 2 Department of Clinical Chemistry, Alzheimer Centre, VU University Medical Centre, Amsterdam, The Netherlands
aAddress correspondence to this author at: Alzheimer Centre and Department of Neurology, VU University Medical Centre, Postbus 7057, 1081 HV Amsterdam, The Netherlands. Fax 31-20-4440715; e-mail femke.bouwman{at}vumc.nl.
To the Editor:
Measuring protein concentrations in cerebrospinal fluid (CSF) has gained wide acceptance for the differential and early diagnosis of dementia (1)(2)(3). Longitudinal changes in CSF biomarkers such as ß-amyloid1–42 (Aß1–42) are of potential use for studying the disease course and the effects of treatment, but they have rarely been studied. We evaluated changes in Aß1–42 concentrations with time and assessed the influence of assay variability and specimen storage on assay results.
At the Alzheimer Centre of the VU Medical Centre, 114 patients each underwent 2 lumbar punctures (LPs). Mean (SD) time between the first and second LP was 21 (9) months (i.e., follow-up time). CSF samples were collected in 12-mL polypropylene tubes, centrifuged within 2 h at 2100g for 10 min at 4 °C, aliquoted into 0.5- or 1-mL polypropylene tubes, and stored at –80 °C until further analysis. The study was approved by the ethics committee of the VU Medical Centre, and all participants gave informed consent.
Aß1–42 was measured with a sandwich ELISA (Innotest ß-amyloid1–42; Innogenetics) (4). Baseline samples were assayed twice: once shortly after the first LP (A1) and once, in a separately stored aliquot (A2), concomitant with the follow-up sample (B; note that storage time of the baseline sample equals follow-up time).
The intraassay CV [averaged (SD/mean) x 100%] was 2.8% for duplicate samples run in 4 different assays. The interassay CV was 6.9%–13% for 4 different quality-control samples run across 26 assays between January 2004 and December 2005. A paired-samples Student t-test was used to evaluate changes in Aß1–42 concentrations. The CVs for baseline and follow-up sample pairs were calculated; CVs were then graphed in Bland–Altman plots and compared by use of the Pitman test (5).
The demographic characteristics of the study population are summarized in Table 1 of the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol52/issue8/.
The mean (SD) Aß1–42 concentration in the baseline samples assayed shortly after collection (A1) was 485 (242) ng/L, and the mean concentration in the follow-up samples (B) was 520 (249) ng/L. The mean difference between these samples (A1 – B), measured in different analytical runs, was 35 (154) ng/L (P <0.01). In the stored baseline samples (A2), the mean Aß1–42 concentration was 477 (232) ng/L, and the difference between these stored baseline samples and the follow-up samples (A2 – B), measured in the same analytical run, was 43 (82) ng/L (P <0.01). The mean difference between the first and second values obtained for the baseline samples (A1 – A2) was 8 (123) ng/L (P = 0.50).
The CV for the baseline and follow-up CSF sample pairs measured in the same analytical run was 10% (Fig. 1A
). By contrast, the CV for the baseline/follow-up CSF sample pairs measured in different analytical runs was 18% (Fig. 1B
). The CV for the repeated Aß1–42 assessments in baseline CSF samples was 14% (Fig. 1C
). Analysis with the Pitman test (5) revealed a significant difference between these variances (P <0.001).
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Pearson correlation revealed that storage time was not associated with differences in Aß1–42 concentrations between baseline samples assayed shortly after collection and those assayed later (A1 – A2; r = 0.15; P = 0.12).
The main finding of this study is the higher variability of Aß1–42 concentrations in baseline and followup samples measured in different analytical runs compared with measurement in the same analytical run. This suggests that, even with acceptable within- and between-assay variation as judged from the results obtained for the quality-control pools, measurement error exceeds biological changes over time. Therefore, in case of repeated LPs, Aß1–42 concentrations should be measured in the same analytical run.
The variability of Aß1–42 concentrations may be caused by methodologic limitations of the Aß1–42 ELISA. Another possible cause is higher variability at higher Aß1–42 concentrations compared with lower concentrations, as suggested in Fig. 1
. There was no essential change of variability, however, after exclusion of the 12 highest Aß1–42 concentrations from analysis (results not shown). In addition, differences in follow-up time, and thus storage time, of CSF samples as a cause of variability was not likely because we found no association between follow-up time and differences in repeated Aß1–42 assessments measured in baseline samples. This confirms earlier data on the effects of processing and storage conditions on Aß1–42 concentrations (6)(7).
Only a few published studies have evaluated changes in Aß1–42 concentrations over time. One study showed that Aß1–42 concentrations decrease over time (8), whereas other studies showed no significant changes of Aß1–42 concentrations over time (9)(10)(11)(12)(13). Remarkable in all of these longitudinal studies is that only a few mention intra- and interassay variability and that no study explicitly reports that baseline and follow-up CSF samples were assayed in the same analytical run. All of the above-mentioned studies reported wide ranges and/or SD of Aß1–42 concentrations, which is in agreement with our finding of large variances.
The ultimate implication of our study may be that, with the methodologic limitations of the present ELISA, repeated Aß1–42 determinations are not useful in a clinical setting. The biological significance of repeated LPs in individual patients remains to be established.
References
The following articles in journals at HighWire Press have cited this article:
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  N A Verwey, W M van der Flier, K Blennow, C Clark, S Sokolow, P P De Deyn, D Galasko, H Hampel, T Hartmann, E Kapaki, et al. A worldwide multicentre comparison of assays for cerebrospinal fluid biomarkers in Alzheimer's disease Ann Clin Biochem, May 1, 2009; 46(3): 235 - 240. [Abstract] [Full Text] [PDF]
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 F. H. Bouwman, W. M. van der Flier, N.S.M. Schoonenboom, E. J. van Elk, A. Kok, F. Rijmen, M. A. Blankenstein, and P. Scheltens Longitudinal changes of CSF biomarkers in memory clinic patients Neurology, September 4, 2007; 69(10): 1006 - 1011. [Abstract] [Full Text] [PDF]
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 T. S.M. Reijn, M. O. Rikkert, W. J.A. van Geel, D. de Jong, and M. M. Verbeek Diagnostic Accuracy of ELISA and xMAP Technology for Analysis of Amyloid {beta}42 and Tau Proteins Clin. Chem., May 1, 2007; 53(5): 859 - 865. [Abstract] [Full Text] [PDF]
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