Concordance of CSF measures of Alzheimer's pathology with amyloid PET status in a preclinical cohort: A comparison of Lumipulse and established immunoassays

Abstract INTRODUCTION We assessed the concordance of cerebrospinal fluid (CSF) amyloid beta (Aβ) and tau measured on the fully automated Lumipulse platform with pre‐symptomatic Alzheimer's disease (AD) pathology on amyloid positron emission tomography (PET). METHODS In 72 individuals from the Insight 46 study, CSF Aβ40, Aβ42, total tau (t‐tau), and phosphorylated tau at site 181 (p‐tau181) were measured using Lumipulse, INNOTEST, and Meso Scale Discovery (MSD) assays and inter‐platform Pearson correlations derived. Lumipulse Aβ42 measures were adjusted to incorporate standardization to certified reference materials. Logistic regressions and receiver operating characteristics analysis generated CSF cut‐points optimizing concordance with 18F‐florbetapir amyloid PET status (n = 63). RESULTS Measurements of CSF Aβ, p‐tau181, and their ratios correlated well across platforms (r 0.84 to 0.94, P < .0001); those of t‐tau and t‐tau/Aβ42 correlated moderately (r 0.57 to 0.79, P < .0001). The best concordance with amyloid PET (100% sensitivity and 94% specificity) was afforded by cut‐points of 0.075 for Lumipulse Aβ42/Aβ40, 0.087 for MSD Aβ42/Aβ40 and 17.3 for Lumipulse Aβ42/p‐tau181. DISCUSSION The Lumipulse platform provides comparable sensitivity and specificity to established CSF immunoassays in identifying pre‐symptomatic AD pathology.

A decrease in CSF concentration of soluble Aβ1-42 (Aβ42) peptide is one of the earliest changes in preclinical AD, [5][6][7] likely reflecting the aggregation and deposition of Aβ into plaques in the brain. 8 CSF Aβ42/Aβ40 ratio has consistently shown better diagnostic value for AD than Aβ42 alone, 9 perhaps compensating for individual differences in the total production of Aβ and CSF turnover. 10 The Aβ42/Aβ40 ratio has also been found to mitigate the adsorption-related effects of low sample storage volume (< 1 mL) on measurements of Aβ concentrations by different platforms. 11,12 CSF Aβ42 has a high concordance of 89% to 92% with amyloid PET; 13,14 this is further improved when using CSF Aβ42/Aβ40 ratio, to 94% to 98%. 14 Both reduced CSF Aβ42/Aβ40 ratio 15 and increased uptake of amyloid PET tracers including 18F-florbetapir 16 have been shown to correlate with neuropathologically confirmed cerebral Aβ deposition.
Multiple analytical platforms are used for measuring core CSF AD biomarkers, for example, INNOTEST (Fujirebio) provides clinically validated enzyme-linked immunosorbent assays (ELISA) for Aβ42, total tau (t-tau), and phosphorylated tau at site 181 (p-tau181); the Meso Scale Discovery (MSD) Aβ triplex electrochemiluminescence assay simultaneously measures Aβ38, Aβ40, and Aβ42. However, despite efforts to standardize biomarker measurements among multiple platforms and laboratories, 17 differences in absolute values among platforms and coefficients of variation remain high, hampering the development of universal cut-points for use in clinical settings. Therefore, there is a drive toward validating fully automated platforms that reduce manual steps as a source for variation. One of these automated platforms is the Lumipulse G system (Fujirebio), on which chemiluminescent immunoassays for Aβ40, Aβ42, t-tau, and p-tau181 have been developed, using the same antibodies as the INNOTEST ELISAs.
Recent studies directly comparing measurements by Lumipulse with INNOTEST ELISAs have shown good concordance between the two platforms but reduced intra-and inter-assay variability on the Lumipulse. [18][19][20][21][22] However, systematic differences in absolute CSF Aβ42 concentrations between Lumipulse and INNOTEST platforms have been observed, 18,19 with one study reporting 27% lower concentrations measured by INNOTEST compared to Lumipulse. 18 When assessing the diagnostic accuracy of Lumipulse CSF Aβ and tau in classifying clinical AD from cognitively asymptomatic controls, Lumipulse ratios of Aβ42/Aβ40, Aβ42/t-tau, and Aβ42/p-tau181 were found to have a higher diagnostic accuracy than individual markers. 20,21 Other studies assessed the ability of Lumipulse assays to differentiate clinical AD from non-AD neurological conditions. No significant difference in diagnostic accuracy has been shown between Lumipulse and INNOTEST assays; 19 again, compared to using individual biomarkers, the Lumipulse Aβ42/t-tau ratio, 19 and the Aβ42/Aβ40 ratio 23 showed improved diagnostic performance.
A few studies have assessed the concordance of CSF Aβ and tau with amyloid PET. 13,24,25 Janelidze et al. 13 investigated individuals with mild cognitive complaints, comparing concordance of CSF Aβ42, Aβ40, and t-tau with visual amyloid PET status across five CSF assay platforms.
Newer immunoassays, including a modified INNOTEST and Lumipulse assays, showed improved agreement with visual amyloid PET when using Aβ42/Aβ40 or Aβ42/t-tau ratios (concordance 93% to 95%), compared to their respective Aβ42 assays (87%to 89%), but the classic INNOTEST Aβ42 assay gave a concordance of 92%. Spiked Aβ40 over a concentration range of 1 to 40 ng/mL led to progressive decrease in values of Aβ42 measured by the classic INNOTEST (with 60% reduction at the highest spiked concentration) and the MSD platform (with 20% at the highest spiked concentration), but not by the modified INNOTEST. 13 Taken together, these results suggest that the classic INNOTEST assay exhibits some non-specificity to Aβ42 measurement due to quenching of signal by Aβ40 levels.
When assessing CSF by Lumipulse in a mixed memory clinic cohort, Alcolea et al. also found a higher concordance with amyloid PET when using the CSF Aβ42/Aβ40 ratio (86%) than when using individual markers (76% to 84%). 24 Kaplow et al. used Lumipulse CSF Aβ42 and t-tau cut-points to predict amyloid PET status in multiple cohorts and reported the best performance in all cohorts when using the ttau/Aβ42 ratio (concordance 85% to 95%). 25 As yet, no single study has directly compared all four CSF Aβ and tau markers and ratios measured by the Lumipulse platform with established immunoassays and compared platforms according to concordance with amyloid PET in a preclinical setting. In the present study we extend the comparison of individual Lumipulse CSF Aβ40, Aβ42, t-tau, and p-tau181 markers to also include ratios, with direct comparison to the INNOTEST and MSD platforms. We supplement existing knowledge about the possible contribution of Aβ40 interference to differences in measurements of Aβ42 by evaluating all three platforms, and assess concordance of individual markers and ratios with amyloid PET imaging in a preclinical cohort.

Lumbar punctures and pre-analytical CSF processing
Exclusion criteria for lumbar puncture (LP) were clinical/neuroimaging safety concerns for raised intracranial pressure, known/suspected thrombocytopenia or coagulopathy, use of antiplatelet or anticoagulant medications (apart from aspirin 75 mg daily), congenital spinal malformation, lumbar fixation surgery, active skin inflammation overlying the proposed LP site, or lignocaine allergy. Participants were not instructed to fast, and LP was timed between 08:30 and 10:30 hrs. After local anesthesia with lignocaine, a 22G atraumatic spinal needle was used to collect up to 20 mL of CSF, without active withdrawal, into 2 × 10 mL polypropylene screw top containers (Sarstedt 62.610.018), which were transported on ice within 30 minutes to the laboratory. CSF was centrifuged at 1750 g for 10 minutes at 4 • C and the supernatant placed in 0.5 mL aliquots into polypropylene screw top cryovials, to be stored at -80 • C within 60 minutes of LP.

2.3
Imaging procedures between a pre-defined composite neocortical region of interest and an eroded white matter reference region was calculated, and a SUVR cutpoint of 0.61 was used to define amyloid PET status, as derived by mixture modeling generated at the first study time point. 28 Eighty-five percent of participants had their amyloid PET scan on the day before LP, but in the remaining 15%, either due to lack of tracer availability or participant choice, LP-scan delay ranged between -13 and +110 days.

CSF assays
For each of the four analytes of interest, CSF measurements were undertaken using the Lumipulse platform and at least one other established immunoassay platform that uses manual steps in the measurement protocol (Table 1).
For measuring Aβ peptides, three assay platforms were used: INNOTEST Samples were assayed after a single thaw to room temperature.
On each platform, a single batch of reagents was used for all samples. Measurements by INNOTEST and MSD assays were performed in duplicate, and sample measurements accepted if coefficients of variation across duplicates were < 30%. Given that the Lumipulse platform required a larger total volume of CSF due to dead volume, measurements by Lumipulse were made once per sample.
Two run validation controls (provided with each assay kit) and two control CSF samples (provided by the Neuroimmunology and CSF Laboratory at the National Hospital for Neurology and Neurosurgery) with low and high values of the analyte(s) of interest were used. Intra-run variation for the run validation controls and inter-run variation using the control CSF samples are shown in Table S1 in supporting information. Measurements were performed according to the manufacturers' instructions.

Aβ42 measurement standardization to certified reference material
After the measurements of Aβ peptides were performed in this study, certified reference materials (CRM) were produced for Aβ1-42 and data have been published on the commutability of different assay platforms in measuring against these CRM. 29 The values obtained by measuring Aβ42 on the Lumipulse platform were retrospectively adjusted to incorporate standardization against the CRM, using information kindly provided by Dr Nathalie Le Bastard (Fujirebio) for the Lumipulse assay. 30 The INNOTEST and MSD calibrators have not been re-standardized to the CRM, but the CRM has been shown to be commutable for assays on both platforms. 29

Aβ40 interference
Investigation of Aβ40 interference with Aβ42 measurements is detailed in the supporting information.

Statistical analysis
All analyses used Stata v14.2 (Stata Corporation, College Station, Texas, USA). As individual biomarkers have a positively skewed distribution, log-transformation was undertaken before assessing Pearson correlations between individual biomarker values across platforms.
Such transformation was not required before assessing correlations between ratios. All individuals with available CSF data were included in correlation analyses.
Spearman correlation was used to assess the impact of spiking    Figure 1A-C). CSF Aβ40 was also highly correlated across the Lumipulse and MSD platforms (r = 0.936, P < .0001; Figure 1D). Measurements on the Lumipulse and INNOTEST platforms of p-tau181 were better correlated than those of t-tau (p-tau181 r = 0.935, t-tau 0.786, both P < .0001; Figure 1E

Spiked Aβ40 interference
Spiked Aβ40 did not significantly interfere with Aβ42 measurements by the Lumipulse platform ( Figure S1A and S1D in supporting information). However, significant negative correlations between mea-

Concordance of CSF biomarkers with amyloid PET
The performance of the three platforms in classifying amyloid PETnegative/positive status is shown in Table 3 Notes: The area under the receiver operating characteristics curve (AUC), its 95% confidence interval, the Youden index (at which the combination of sensitivity and specificity is maximized), and the corresponding optimal cut-point are shown for each of CSF Aβ42, t-tau, p-tau181, and their ratios in predicting amyloid PET status (n = 63). a Higher than AUC for Lumipulse t-tau, De Long test P = .005. b Lower than AUC for Lumipulse p-tau181, De Long test P = .024.
Abbreviations: Aβ, amyloid beta; AUC, area under the curve; CSF, cerebrospinal fluid; IQR, interquartile ratio; PET, positron emission tomography; p-tau181, phosphorylated tau at site 181; t-tau, total tau. Concordance of CSF biomarker ratios with amyloid PET SUVR as a continuous variable is shown in Figure 2A-F. The percentage of discordant individuals was low (4% to 11%) and all discordantly classified individuals were CSF-positive and PET-negative, except when the Lumipulse Aβ42/t-tau ratio was used (one individual was classified as CSF-negative but PET-positive). All discordantly-classified individuals were male, and 57% to 67% were APOE ε4 carriers. Despite this, incorporating age, sex, and APOE ε4 carrier status as covariates into predictive models did not significantly change the percentage of discordantly classified individuals or type of discordance (Table S2 and Figure S3 in supporting information).

DISCUSSION
In this study we build on previous validations of Lumipulse measure- It is unclear whether this reflects altered performance of one or both of the t-tau assays in this part of the measurement range, but it is important to note that the values were well above the published lower limits of quantification for both assays.
We found differences in absolute biomarker values between platforms, as reported by others. 18 Lumipulse is less susceptible to matrix effects, based on the optimal minimal required dilution of sample in the conjugate solution. We did find significant Aβ40 interference with MSD measurements; these could be due to similar matrix effects as found on the INNOTEST, or due to differences in antibody specificity between the two assays.
We report 100% sensitivity across all three platforms for CSF Aβ42 in predicting cortical amyloid load, but the specificity of the Lumipulse measurements (74%) was lower than that of the INNOTEST (86%) or MSD (80%). In contrast to other studies, which show CSF t-tau and p-tau181 to be good individual predictors of amyloid PET, 24 diction of amyloid PET status were higher for CSF Aβ42 and its ratios than that obtained by the model using age, sex, and APOE ε4 carrier status, the differences did not reach statistical significance, likely due to this being an interim dataset of samples collected by this point of the ongoing study. This cohort consists mostly of cognitively healthy individuals of the same age. It is possible that some individuals classified as "CSF-positive" (through the use of the ratio cut-points) but "PETnegative" do actually have subthreshold cerebral amyloid deposition, as CSF changes may precede PET changes. 34 However, in the absence of neuropathological data to date in this cohort, the use of amyloid PET as an in vivo "gold standard" is a necessary limitation.
In summary, this study supports the use of the fully automated Lumipulse platform, particularly for measuring CSF Aβ42/Aβ40 and Aβ42/p-tau181, to identify cerebral amyloid deposition with excellent sensitivity and high specificity, without Aβ40 interference, even in cognitively normal individuals.

ACKNOWLEDGMENTS
The authors are very grateful for the ongoing participation of the mem-