Cerebrospinal Fluid Aβ 42 /Aβ 40 as a Means to Limiting Tube- and Storage-Dependent Pre-Analytical Variability in Clinical Setting

Abstract

Background: Alzheimer’s disease (AD) cerebrospinal fluid (CSF) biomarkers have recently been included in the criteria for AD diagnosis. Unfortunately, their wider use in routine and interpretation require more standardization, particularly for the pre-analytical steps. In particular, amyloid-β (Aβ)₄₂ peptide measurement is strongly influenced by the type of collection tube and by repeated freeze/thaw cycles.

Objective: The objectives of this study were to compare, in clinical setting, the impact of collection tubes and the repetition of freeze/thaw cycles on Aβ₄₂ and Aβ₄₀ concentrations and consequently determine if the Aβ₄₂/Aβ₄₀ ratio could resolve the diagnosis difficulties related to these pre-analytical parameters.

Methods: CSF from 35 patients was collected in different polypropylene (PP) and stored at – 80°C after sampling. For CSF collected in the reference tube, three successive freeze-thaw cycles were done. Aβ₄₂ and Aβ₄₀ concentrations were determined in each condition in order to calculate the Aβ₄₂/Aβ₄₀ ratio.

Results: Our results showed that CSF Aβ₄₂ and Aβ₄₀ values were significantly different according to the type of collection tube and the number of freeze/thaw cycles. Although the calculation of the Aβ₄₂/Aβ₄₀ ratio eliminated the effect of PP tube-dependent variation, this was not the case for freeze-thaw cycle-associated variation.

Conclusion: The use of Aβ₄₂/Aβ₄₀ ratio rather than Aβ₄₂ alone could contribute toward pre-analytical standardization, thus allowing the general use of CSF AD biomarkers in routine practice.

Keywords

Alzheimer’s disease cerebrospinal fluid pre-analytical standardization tube freeze-thaw cycles

INTRODUCTION

Alzheimer’s disease (AD) diagnosis criteria were revised in 2011 to assimilate the advances in the understanding of AD [1]. Although clinical symptoms still constitute the core of the diagnosis, new criteria now include biomarkers related to the AD pathophysiological processes such as cerebrospinal fluid (CSF) and imaging biomarkers. In CSF, AD is biologically characterized by a reduction in CSF Aβ₄₂ peptide levels, which is correlated with the density of neuritic plaques [2], and by an increase in CSF tau and phosphorylated tau (P-tau) levels associated with neurodegeneration and neocortical neurofibrillary pathology [3]. The combination of these three parameters have shown high levels of sensitivity and specificity as well as diagnostic accuracy in discriminating AD versus controls [4].

In 2004, Lewczuk et al. [5] was the first to propose using the Aβ₄₂/Aβ₄₀ ratio rather than the absolute value of Aβ₄₂ peptide to improve the percentage of appropriately diagnosed patients. Since this publication, several studies have largely demonstrated the interest of using Aβ₄₂/Aβ₄₀ ratio for better interpretative CSF analysis and increase the level of evidence of AD process [6 –13]. Aβ₄₀ peptide is the most abundant Aβ peptide in the brain and its CSF level might therefore be considered to most closely reflect the total Aβ load in the brain. CSF Aβ₄₀ was characterized by a great inter-individual variability [14] and was long considered as unchanged in AD. Today, findings are less clear-cut. Indeed, Bousiges et al. [15] confirmed that CSF Aβ₄₀ level was not significantly different in AD than in the control group, whereas it was decreased in patients with Lewy body dementia. Thus, Aβ₄₂/Aβ₄₀ allowed better discrimination of AD from Lewy body dementia than Aβ₄₂ alone [15]. On the contrary, others studies showed lower CSF Aβ₄₀ levels in AD patients than in controls [13, 16]. More recently, Dorey et al. demonstrated that absolute value of CSF Aβ₄₀ levels allowed the correct classification of AD patients with non-pathological Aβ₄₂ levels and control patients with pathological Aβ₄₂ levels [11].

Various papers have pointed out pre-analytical and analytical variability between laboratories for Aβ₄₂ peptide [17 –21]. Tubes for collection, sample handling, and sample storage conditions were especially noted as critical factors [22, 23]. In the clinical setting, despite numerous recommendations, the use of a non-recommended tube to collect CSF and the thawing of samples are two abnormalities frequently encountered. This is particularly the case when CSF AD biomarkers are analyzed for a large geographical area and samples are aliquoted and frozen by a local laboratory close to the applicant hospital before being transferred to a central laboratory. Transport conditions are unfortunately not always respected, leading to premature thawing of the samples. These pre-analytical confounding factors strongly influence Aβ₄₂ peptide analysis, potentially leading to misinterpretation of biological profiles. A clear understanding of how these factors influence Aβ levels would therefore represent a major step for a general widespread clinical application.

In this study, we sought to determine if the Aβ₄₂/Aβ₄₀ ratio, which might be less affected by the CSF collection tube and the repetition of freeze-thaw cycles, could be more largely used to limit variability due to these two major pre-analytical confounding parameters.

MATERIAL AND METHODS

Patients

Thirty-five subjects needing CSF AD biomarkers (Tau, P-Tau, Aβ₄₂, and Aβ₄₀ peptides) testing were recruited in the Clinical and Research Memory Center of Nancy (France), i.e., patients with atypical clinical presentation or early-onset dementia [24].

Testing for CSF biomarkers was carried out according to the European Federation of Neurological Societies guidelines (diagnosis doubt, atypical clinical presentations, or early-onset dementia) or for amnesic mild cognitive impairment according to research criteria for the diagnosis of AD published in 2007 [25].

Each subject signed a written informed consent for CSF assessment, analysis, and storage, and protocol was approved by a local ethical committee.

Samples

CSF was collected by lumbar puncture of the L3-L4 or L4-L5 intervertebral space after local anesthesia in non-fasting patients. Two mL CSF were collected in three different polypropylene (PP) tubes, always in the same order (1, R, 2). Ten mL Sarstedt PP tube (ref 62.610.201, PP + polyethylene copolymer) was chosen as the reference tube (tube R), most commonly used as the standard tube in French laboratories. Five mL Gosselin PP tube (ref TP 10–03, PP), and 15 mL Biosciences Discovery Labware Falcon tube (ref 352096, PP + polyethylene copolymer) were respectively named tube 1 and tube 2.

All CSF samples were transported to the Biochemistry Laboratory of the University Hospital of Nancy, at 4°C in less than 4 h after the lumbar puncture. There they were centrifuged at 4,000×g for 10 min at 4°C (FROILABO^® centrifuge). Samples with blood contamination were excluded. CSF was sampled in aliquots of 250μL each in 500μL PP storage tubes (ref 045513, Dutscher^®) and stored at – 80°C until assay.

Freeze-thaw cycles procedure

The freeze-thaw cycles study was carried out with CSF collected in tube R but only 24 samples contained enough CSF to prepare at least 2 samples (250μL each). Cycles 1 and 2 were done with two different 250μL samples. Cycle 3 was done with the residual 120μL CSF after a second thawing step. Time at room temperature during a freeze-thaw cycle was 45 min. Aβ₄₂ and Aβ₄₀ concentrations were determined at each step in order to calculate Aβ₄₂/Aβ₄₀ ratio. For the freezing step, samples were stored at – 80°C for at least 24 h.

Biochemical analyses

Aβ₄₂ and Aβ₄₀ concentrations were determined using commercially available sandwich ELISA procedures (INNOTEST^®, Fujirebio, Ghent, Belgium) according to the manufacturer’s instructions. For each sample, Aβ₄₂/Aβ₄₀ ratio was calculated. A CSF pool stored at – 80°C in PP tubes was used as internal quality control for each assay. Standards, controls, and samples were run in duplicate. Measures were done at 450 nm with a BIO-TEK^® spectrophotometer (MWGt Lambda Scan 200, BIO-TEK^®, USA). Data were analyzed with KC4 software v.3.3 (BIO-TEK^®, USA). In cases of variation coefficients exceeding 10%, the sample was re-assayed.

Statistical analysis

Variables were expressed, according to their distributions, as number, percent, mean±standard deviation or median with Inter Quartile Range (IQR) and minimum maximum (range). Comparisons of CSF biological parameters for the three tubes and for the three repeated freeze-thaw cycles were performed by non-parametric test (Friedman test) with 5% level of significance. When the Friedman test concluded to a significant difference, a Dunn test was performed in order to identify the differences among the tubes or among the freeze-thaw cycles. To study the stability of the ratio between different tubes, we used the Spearman’s rank correlation coefficient (Spearman’s rho; r_s) and the Bland-Altman plot to analyze correlation and agreement.

RESULTS

Demographical characteristics

Thirty-five subjects were included in the study. Mean age was 74±10 years (range: 53–89 years) and gender ratio (men/women) was 0.7.

Effect of tube type on Aβ peptide concentrations and Aβ₄₂/Aβ₄₀ ratio

Each comparison was done between median values because Aβ peptide concentrations and Aβ₄₂/Aβ₄₀ ratio were not normally distributed. Aβ₄₂ and Aβ₄₀ concentrations ranged from 252.5 to 1506 pg/mL and from 2379 to 18052 pg/mL, respectively. Median and extreme Aβ peptide concentrations and Aβ₄₂/Aβ₄₀ ratio are summarized in Table 1 for each tube. Box-plots of Aβ₄₂ and Aβ₄₀ concentrations and Aβ₄₂/Aβ₄₀ ratios are presented in Fig. 1. Concentrations were quite variable and extended all over the range of measure. Detectable Aβ₄₂ and Aβ₄₀ concentrations of samples in different PP tubes were significantly different (p < 0.001 for both peptides). Results from these data showed lower median values of Aβ₄₂ and Aβ₄₀ peptides in tubes 1 [–6.4% (–40.2 to 10.9), p < 0.01 and – 12.9% (–59.5 to – 6.5), p < 0.01, respectively] and 2 [–26.7 (–47 to 21.7) p < 0.001 and – 29.9% (–62.6 to 52.2), p < 0.001, respectively] compared to tube R. However, no significant difference in the median of Aβ₄₂/Aβ₄₀ ratio was globally observed between the three tubes in the whole population (Friedman test, p = 0.549) (Table 1, Fig. 1).

Furthermore, individually, all the correlations between the ratios were significant for T1 versus TR, T1 versus T2, and T2 versus TR (r_s = 0.957 p = 0.0001; r_s = 0.913 p = 0.0001; r_s = 0.899 p = 0.0001, respectively). Bland-Altman plots showed that the number of observations located outside the corresponding interval (differences within mean±1.96 SD) is 2 (5.7%) for T1 versus TR; 3 (8.6%) for T1 versus T2; and 2 (5.7%) for T2 versus TR. Overall, it should be noted that two observations located outside the intervals were the same for T1 versus T2 and T2 versus TR. Thus 30 observations among the 35 (85.7%) are located within the 95% limits of agreement (Fig. 2).

Finally, median CSF protein and glucose concentrations were 0.47 g/L (range from 0.19 to 0.81 g/L) and 3.55 mmol/L (range from 1.27 to 6.2 mmol/L), respectively. In this range of CSF protein concentrations and after one freeze/thaw cycle, variations in CSF Aβ₄₂ or Aβ₄₀ according to the tube were not influenced by CSF protein level (Table 2). On the contrary, Aβ₄₂ or Aβ₄₀ mean values were weakly correlated with variations in peptide levels according the type of tube used (Table 3).

Effect of free-thaw cycles on Aβ peptide concentrations and Aβ₄₂/Aβ₄₀ ratios

Twenty-four CSF were used for the freeze-thaw cycles study. Aβ peptide concentrations and Aβ₄₂/Aβ₄₀ ratio were not normally distributed. Median and extreme Aβ peptide concentration and Aβ₄₂/Aβ₄₀ ratios are summarized in Table 4. Box-plots of Aβ₄₂ and Aβ₄₀ concentrations and Aβ₄₂/Aβ₄₀ ratios are presented in Fig. 2. Median values of Aβ₄₂ and Aβ₄₀ concentrations were observed to be significantly different with the number of freeze/thaw cycles. Results from these data showed lower Aβ₄₂ level after two freeze-thaw cycles compared to one [–17.6% (–27.9 to – 14), p < 0.001] whereas Aβ₄₀ level was not significantly changed [–3.1% (–23.9 to 17.4), NS]. Consequently, a significant difference for Aβ₄₂/Aβ₄₀ ratio [–15.8% (–18.2 to 1.4), p < 0.001] was observed.

On the contrary, after the third freeze-thaw cycle, Aβ₄₂ levels were similar to those measured after one cycle and were significantly increased comparatively to the levels obtained after two cycles [+21.6% (11.1 to 40.7), p < 0.001]. Similarly, Aβ₄₀ levels were statistically higher after the third cycle than after both first and second cycles [+17.8% (6.9 to 35.5), p < 0.001 and + 21.6% (10.8 to 52.4), p < 0.001]. Consequently, Aβ₄₂/Aβ₄₀ ratio was similar after two and three free-thaw cycles [–6.6% (–9.1 to 11.5), NS]. Freeze-thaw cycles-induced variations in Aβ peptide concentrations and Aβ₄₂/Aβ₄₀ ratios led to biological misinterpretations in 4 patients among 24 (15%) since A₄₂/Aβ₄₀ ratio was lower or higher than reference threshold (0.07) according the freeze-thaw cycle.

DISCUSSION

In this study, we showed that the use of the CSF Aβ₄₂/Aβ₄₀ ratio rather than Aβ₄₂ alone could solve some pre-analytical difficulties which currently limit the standardization process and thus wider routine use of CSF biomarkers. We demonstrated that while Aβ₄₂/Aβ₄₀ ratio eliminated PP tube-dependent analytical variability, it did not completely resolve freeze-thaw cycle-associated variations.

Currently, PP is clearly recommended as the best material for CSF collection tubes, due to limited variability in Aβ₄₂ values [26]. However, heterogeneous results were also obtained with PP collection tubes from different suppliers [18]. Indeed, by investigating the composition of 11 different PP tubes, Perret-Liaudet et al. revealed the presence of co-polymers such as polyethylene which are reported to largely influence Aβ₄₂ adsorption [27] and led to more than 50% disparity in Aβ₄₂ values depending on the tube used [18]. This study led to the conclusion that Aβ₄₂ cut-off needed to be established by the laboratory using a single type of sampling tube [28]. Recommendations were made for the least adsorbent tube (Tube R in our study) to be used routinely in French labs [29]. This tube is composed of PP and polyethylene. Here we chose to use a commonly used tube (tube 1, PP only) and another tube containing PP and polyethylene similar to tube R. As in Perret-Liaudet study [18], we observed that tube 2 was the most adsorbent for Aβ₄₂ but also for Aβ₄₀ suggesting that information about tube composition provided by the supplier was insufficient to anticipate the adsorption potential of the tube.

Aβ₄₀ is known to be less hydrophobic than Aβ₄₂ peptide [30] suggesting a lesser tendency to interact with sampling tubes, but no study is available in the literature. We demonstrated here that variations in Aβ₄₀ concentrations according to the tube type were equivalent to those observed with Aβ₄₂ concentrations leading to Aβ₄₂/Aβ₄₀ ratios that were not globally significantly different, and independent of the tube type. Individual analysis showed that variations of the ratio according to the tube type were close to the mean except for 5 patients, mostly with ratio values very high relative to the threshold of 0.07 and without influence on the biological interpretation. These results support those of Lewczuk et al. [26] and Vanderstichele et al. [31], which concluded that Aβ₄₂/Aβ₄₀ ratio was comparable between PP and polystyrene, polycarbonate tubes [26] or PP and low protein binding tubes [31]. Thus, the use of Aβ₄₂/Aβ₄₀ ratio rather than Aβ₄₂ alone could also represent a solution toward minimizing pre-analytical variability related to the various collection PP tube. In daily practice, CSF collection in a non-recommended tube is a frequent mistake that leads to rejection of the sample and exposing the patient a second time to lumbar puncture-associated adverse effects and discomfort. The absence of variation in the Aβ₄₂/Aβ₄₀ ratio could be used as an alternative solution, rather than carrying out another CSF sampling.

Freeze/thaw cycles before Aβ₄₂ peptide analysis represent another critical pre-analytical step, especially for CSF transferred from others hospitals since CSF AD biomarkers are analyzed in one laboratory for a given geographic area. Unfortunately, data from published studies are extremely discordant. Several studies shown that samples could be frozen and thawed up to two times without any effect on the AD biomarkers in CSF [20, 32], whereas other demonstrated a decrease in Aβ₄₂ values after one [33] or two freeze/thaw cycles [34]. Taken together, these data led to imprecise recommendations suggesting the avoidance of repeated freeze/thawing of samples [28]. Finally, the use of Aβ₄₂/Aβ₄₀ ratios rather than Aβ₄₂ alone could also have been an interesting solution. Only one publication showed that Aβ₄₀ level was stable for two freeze/thaw cycles [32]. Otherwise Vanderstichele et al. recently reported that an additional freeze-thaw cycle in a PP tube reduced both CSF Aβ₄₂ and Aβ₄₀ levels whereas the Aβ₄₂/Aβ₄₀ ratio was not significantly affected [31]. Here, we observed a significant decrease in Aβ₄₂ levels between the first and the second cycle while Aβ₄₀ levels remained stable. Interestingly, both Aβ₄₂ and Aβ₄₀ were significantly increased between the second and the third freeze/thaw cycles. These results led to two major conclusions. Firstly, Aβ₄₂/Aβ₄₀ ratio did not prevent freeze-thaw cycle-associated variability in Aβ peptide concentrations, which could lead to misdiagnosis. Indeed, 4 samples out of 24 included (15%) had ratio values above or below the pathological cut-off, depending on the considered freeze/thaw cycle. This result demonstrates the need to work with a defined number of freeze/thaw cycles and to establish a ratio cut-off according selected conditions. Secondly, the Aβ₄₂ and Aβ₄₀ level re-increase in CSF after three freeze/thaw cycles suggested that amyloid degradation was not the unique cause of variability. Aliquot volume was identified as a potential confounding factor in CSF Aβ₄₂ peptide measurement [35, 36]. In our study, an extra variation factor occurred, since the total CSF volume was lower in cycle 3 than cycles 1 and 2. This could contribute to the variability between freeze/thaw cycles. However, it is known that tube Aβ₄₂ adsorption is rapid, and is a saturable phenomenon [18]. Here, the volume was effectively lower in cycle 3 than cycle 1, but CSF was left in the initial storage tube during the entire experimental period. In studies on the influence of aliquot volume such as that of Toombs et al. [35], different storage volumes (50–1500μL) were tested in independent storage tubes. No data about the impact of the variation of volume of CSF in the initial storage tube are available in literature. However, the decrease in CSF volume in storage tube could modify the equilibrium state between CSF and tube walls, and eventually lead to the release of Aβ peptide from tube to CSF. Further investigation would be necessary to clarify this issue. Other processes could be taken into account such as conformational changes of Aβ peptides, oligomerization, protein binding, or modification in adsorption of amyloid on the tube wall which could depend on delay to freezing and changes in freezing or thawing temperatures [37]. Moreover, a protein desorption processing could occur and it could be dependent on several parameters such as temperature, pressure [38], or eventually CSF volume variations. Finally, the impact of repeated freeze/thaw cycles on amyloid level variability seems to be a very complex process that requires further investigation to determine the respective involvement of amyloid species, storage conditions and nature of the storage tube.

The systematic use Aβ₄₂/Aβ₄₀ ratio could limit variability associated with some pre-analytical aspects, but results should be confirmed by further investigations with a larger cohort and by testing PP tubes from other suppliers. The effects of freezing seem to be dependent on the storage tube type and a cross-study exploring the link between PP tubes and repeated freeze/thaw cycle on amyloid pre-analytical variability could be relevant. Moreover, few data are available about amyloid level in fresh CSF. Blennow et al. [39] showed that Aβ₄₂ concentrations were highly consistent between fresh and frozen CSF samples. It would be interesting to know if similar results would be obtained with Aβ₄₀ and Aβ₄₂/Aβ₄₀ ratio.

There are several limitations to note in this study. Firstly, patients were not selected according to their diagnosis, whereas Aβ₄₂ and Aβ₄₀ levels seem to influence tube-associated variations. However, this choice was deliberate because this study was voluntarily conducted in a clinical setting before knowing final diagnosis and Aβ values. Secondly, time delay between lumbar puncture and centrifugation was very broad (<4 h) and could affect the amyloid peptide adsorption process. Again, the aim of this study was to evaluate the CSF Aβ₄₂/Aβ₄₀ as a means to limiting variability in clinical settings. Consequently, reducing this delay to less than 4 h would be very difficult. Today, AD biomarkers are not only research biomarkers, but also are largely integrated in diagnostic tools. It is thus very important to have information about pre-analytical confounding factors in daily practice. Moreover, Aβ adsorption occurred quickly, measurable within 15 min of storage, and did not increase with longer incubation times, suggesting that this delay should have little influence [18].

In conclusion, our study demonstrates that the use of Aβ₄₂/Aβ₄₀ rather than Aβ₄₂ could contribute to pre-analytical standardization which will allow generalizing the use of CSF AD biomarkers. If this ratio seems to be interesting to solve PP tube-dependent variability, it is not relevant or even inappropriate to limit freeze/thaw cycle associated variability. Further investigation must be carried out to better understand complex interactions between these two critical pre-analytical factors.

Footnotes

ACKNOWLEDGMENTS

The authors gratefully acknowledge all geriatricians and neurologists from CMRR. They also thank Dr. Frances T. Yen for her useful suggestions after reading the manuscript.

Authors’ disclosures available online ().

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Cerebrospinal Fluid Aβ 42 /Aβ 40 as a Means to Limiting Tube- and Storage-Dependent Pre-Analytical Variability in Clinical Setting

Abstract

Keywords

INTRODUCTION

MATERIAL AND METHODS

Patients

Samples

Freeze-thaw cycles procedure

Biochemical analyses

Statistical analysis

RESULTS

Demographical characteristics

Effect of tube type on Aβ peptide concentrations and Aβ42/Aβ40 ratio

Effect of free-thaw cycles on Aβ peptide concentrations and Aβ42/Aβ40 ratios

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Effect of tube type on Aβ peptide concentrations and Aβ₄₂/Aβ₄₀ ratio

Effect of free-thaw cycles on Aβ peptide concentrations and Aβ₄₂/Aβ₄₀ ratios