Measurement batch differences and between-batch conversion of Alzheimer's disease cerebrospinal fluid biomarker values

Abstract

Introduction: Batch differences in cerebrospinal fluid (CSF) biomarker measurement can introduce bias into analyses for Alzheimer's disease studies. We evaluated and adjusted for batch differences using statistical methods.

Methods: A total of 792 CSF samples from 528 participants were assayed in three batches for 12 biomarkers and 3 biomarker ratios. Batch differences were assessed using Bland-Altman plot, paired t test, Pitman-Morgan test, and linear regression. Generalized linear models were applied to convert CSF values between batches.

Results: We found statistically significant batch differences for all biomarkers and ratios, except that neurofilament light was comparable between batches 1 and 2. The conversion models generally had high R ² except for converting P-tau between batches 1 and 3.

Discussion: Between-batch conversion allows harmonized CSF values to be used in the same analysis. Such method may be applied to adjust for other sources of variability in measuring CSF or other types of biomarkers.

Keywords: Alzheimer's disease; batch difference; biomarker; cerebrospinal fluid; conversion; generalized linear model.

FIGURE 1

Distribution of cerebrospinal fluid (CSF) samples between measurement batches. CSF samples included in this analysis were from participants of six National Institutes of Health (NIH)–funded Alzheimer's disease (AD) studies: the Wisconsin Alzheimer's Disease Research Center (ADRC) Clinical Core, ⁴⁵ the Wisconsin Registry for Alzheimer's Prevention (WRAP), ⁴⁶ the Longitudinal Early Alzheimer's Detection (LEAD) study, The Longitudinal Course of Neural Function and Amyloid in People At Risk for Alzheimer's Disease (PREDICT) study, the PIB Imaging in People at Risk for Alzheimer's Disease (PIPR) study, and the Statins in Healthy, At Risk Adults: Impact on Amyloid and Regional Perfusion (SHARP) study. ⁴⁷ A total of 792 CSF samples collected from 528 participants were assayed in three batches, with a subset of samples re‐assayed, which resulted in 977 batch measures. Groups {1}, {2}, {3} represent the CSF samples assayed in a single batch 1, 2, and 3, respectively. Group {1‐2} represents the CSF samples assayed in both batches 1 and 2, and group {1‐3} represents the CSF samples assayed in both batches 1 and 3. The groups with CSF samples assayed in two batches had smaller sample sizes than the groups with CSF samples assayed in a single batch. The mean age for each group ranged from 55.5 to 65.2 years. The clinical diagnoses associated with the CSF samples were all cognitively unimpaired in groups {1‐2}, {1‐3}, {3}, and were mostly cognitively unimpaired in groups {1}, {2}. Types and counts (percentages) of impaired diagnoses for these two groups are summarized in Table S1

FIGURE 2

Bland‐Altman plot for evaluating between‐batch differences. We present this example to illustrate how to evaluate and interpret between‐batch differences using the Bland‐Altman plot. The differences between the two batch values of the biomarker sAPP‐α for the same cerebrospinal fluid (CSF) sample (Batch 2–Batch 1) are plotted against the average of the two batch values. The dashed line represents the linear regression fit of the difference on the average. Mean ± 2 SD are limits of agreement (LOA).The mean of the differences is above zero, which indicates that batch 2 on average yielded higher values than batch 1. The negative slope of the linear fit indicates less variability in batch 2, and its crossing over the zero line indicates that batch 2 measurements exceeded batch 1 at the lower end of the range, but at higher levels, batch 1 exceeded batch 2. The variance of these batch differences was non‐constant at different biomarker levels, with the largest variance observed in the middle of the biomarker level range. Under ideal circumstances, one can evaluate the significance of batch differences by comparing the LOA against an a priori threshold for a clinically significant difference. However, in this case we had no such a priori thresholds. Furthermore, the observed non‐constant variance of the differences and its marked trend complicate such an interpretation. Consequently we focus on these descriptive aspects in our report

FIGURE 3

AβX‐42 converting from batch 2 to batch 1 and checking out of range values. Only samples assayed in batch 2 but not batch 1 (group {2}) were converted. Conversion was done by applying the mean structure from the final generalized linear model (GLM): [converted AβX‐42 batch 1 value, represented with red dot] = 171 + 1.82 × [raw AβX‐42 batch 2 value, represented with blue dot]. The final GLM model was developed using the raw batch 1 and raw batch 2 values of the group {1‐2} (represented with black dot), with 5% outliers excluded (represented with triangle). The final GLM line deviated from the identity line Y = X, which indicated that raw batch 1 and batch 2 values were not comparable and conversion was important. We use x to represent the cerebrospinal fluid (CSF) value being checked, and use min and max to represent the minimum and maximum values of the range being compared to, that is, the range of the raw batch values used to fit the final GLM. POR indicates the extent a value is out of the range, calculated as a proportion of that range's width. A negative value represents below the range, a positive value represents above the range, and zero represents in the range. A larger absolute value indicates being further beyond the range. Thus, if $\min \le x\le max,\mathbf{then}POR=0$ $\mathbf{If}x<min,\mathbf{then}POR=\frac{(x-min)}{(max-min)}\times 100\%,POR<0$ $\mathbf{If}x>max,\mathbf{then}POR=\frac{(x-max)}{(max-min)}\times 100\%,POR>0$ For each CSF value being converted, there are two sets of POR values, one for the raw value being converted and the other for the converted value. The two horizontal dotted lines parallel to the x‐axis represent the minimum (Min) and maximum (Max) of the raw batch 1 values of the group {1‐2}. If a converted value of the group {2} (represented with red dot) is between these two lines, it is in the range of the raw batch 1 values of the group {1‐2}, Converted POR value = 0. If a converted value is below the Min line, it is below the range, Converted POR value < 0. If a converted value is above the Max line, it is above the range, Converted POR value > 0. The two vertical dotted lines parallel to the y‐axis represent the Min and Max of the raw batch 2 values of the group {1‐2}. If a raw value of the group {2} (represented with blue dot) is between these two lines, it is in the range of the raw batch 2 values of the group {1‐2}, Raw POR = 0. If a raw value is below the Min line, it is below the range, Raw POR < 0. If a raw value is above the Max line, it is above the range, Raw POR > 0