Abstract

1. BACKGROUND

Cerebrospinal fluid (CSF) amyloid beta (Aβ) and tau, and quantification of cortical amyloid burden by positron emission tomography (PET) remain among the best‐established biomarkers of Alzheimer's disease (AD). Guidelines for their use in the clinical setting include the UK National Institute of Clinical Assessment Guideline 2018, 1 and the Alzheimer's Association's appropriate use criteria for CSF testing 2 and for amyloid PET. 3 Biomarkers are key components of research criteria for AD, which are defined by the presence of AD pathology even in asymptomatic individuals, 4 and are widely used as inclusion criteria and outcome measures for clinical trials.

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 (less than 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 (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 (ELISAs) 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 between multiple platforms and laboratories, 17 differences in absolute values between 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 individuals with clinical AD from cognitively normal 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 the 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 with their respective Aβ42 assays (97% 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 t‐tau/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.

2. METHODS

2.4. 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).

TABLE 1

Immunoassay platforms used and respective measured biomarkers

	Biomarker measured
Platform	Aβ42	Aβ40	t‐tau	p‐tau181
Lumipulse
MSD
INNOTEST

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The ticks show the biomarkers measured by each platform.

For measuring Aβ peptides, three assay platforms were used: INNOTEST β‐amyloid 1‐42 (Fujirebio) for Aβ42, Lumipulse G600II automated assay (Fujirebio) for Aβ42 and Aβ440, and MSD Multi‐spot Aβ 6E10 Triplex assay (Meso Scale Diagnostics) for Aβ42 and Aβ40. A single 500 µL aliquot of neat CSF was used to perform the INNOTEST and Lumipulse assays in parallel. INNOTEST required 25 µL per replicate for measuring Aβ42 alone. Lumipulse required 100 µL of dead volume, 50 µL per replicate for measuring Aβ42, and 40 µL per replicate for measuring Aβ40. A different aliquot of CSF from the same individuals was used to perform the MSD assay, with 15 µL per replicate of neat CSF (diluted 1:2 in assay diluent) used to measure all three peptides Aβ38, Aβ40, and Aβ42 together.

T‐tau and p‐tau181 were measured in parallel on the INNOTEST and Lumipulse platforms, using the same aliquot of CSF. The INNOTEST hTau Ag assay required 25$\mu$L per replicate and the INNOTEST Phospho‐tau (181P) assay 75 µL per replicate. The Lumipulse assays required 100 µL of dead volume, 75 µL per replicate for measuring t‐tau, and 40 µL per replicate for measuring p‐tau181.

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 less than 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 Supplementary Table S1. Measurements were performed according to the manufacturers’ instructions.

3. RESULTS

3.2. Correlations between CSF biomarker measurements across platforms

CSF A$\beta$42 measurements were highly correlated across the three platforms (Lumipulse vs INNOTEST r = 0.891; Lumipulse vs MSD r = 0.905; MSD vs INNOTEST r = 0.887; all P < .0001; Figure 1A‐D). Measurements on the Lumipulse and INNOTEST platforms of p‐tau181 were better correlated than those of t‐tau (p‐tau181 r = 0.935, t‐tau 0.786, both P < .0001; Figure 1E,F). This was also reflected in correlations of ratios between biomarkers (Figure 1G‐I); only a modest correlation was observed for Lumipulse versus INNOTEST A$\beta$42/t‐tau (r = 0.569, P < .0001) but correlations were stronger for Lumipulse versus INNOTEST A$\beta$42/p‐tau181 (r = 0.840, P < .0001) and Lumipulse versus MSD A$\beta$42/A$\beta$40 (r = 0.952, P < .0001).

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FIGURE 1

Correlations between measurements of the same biomarkers on different platforms. Individual biomarkers were natural log‐transformed before assessing Pearson correlations and performing linear regression. Ratios of biomarkers were not transformed. The Pearson correlation coefficient r and its P value are shown. The linear regression coefficient $\beta$ and the intercept of the regression line c are shown with their 95% confidence intervals in brackets

3.4. Concordance of CSF biomarkers with amyloid PET

The performance of the three platforms in classifying amyloid PET‐negative/positive status is shown in Table 3, for those individual biomarkers and ratios that performed better than chance. For CSF A$\beta$42 and all ratios incorporating it, AUCs were 0.89 or above, with no statistically significant differences between methods. However, specificity at the Youden index was improved by the use of ratios (86% to 94%) compared to using A$\beta$42 alone (74% to 86%) measured on any platform. P‐tau181 performed better when measured by Lumipulse (AUC Lumipulse 0.879 vs INNOTEST 0.791, De Long test P = .024) but t‐tau performed better when measured by Innotest (AUC Lumipulse 0.665 vs INNOTEST 0.825, P = .005). Supplementary Figure 2 shows scatter plots of the raw data, demonstrating the superiority of the ratios compared with individual biomarkers.

TABLE 3

Comparison of CSF biomarkers for prediction of amyloid PET status

Biomarker	Platform	AUC	95% CI for AUC	Youden index	Cut‐point (pg/mL)	Specificity (%)	Sensitivity (%)
Aβ42	Lumipulse	0.891	0.811–0.970	0.740	1423	74	100
	MSD	0.897	0.821–0.973	0.800	586	80	100
	INNOTEST	0.948	0.895–1.000	0.860	936	86	100
t‐tau	Lumipulse	0.665	0.479–0.851	0.358	443	82	54
	INNOTEST	0.825a	0.708–0.941	0.572	442	88	69
p‐tau181	Lumipulse	0.879	0.787–0.970	0.660	49	66	100
	INNOTEST	0.791b	0.654–0.927	0.458	77	92	54

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Biomarker	Platform	AUC	CI for AUC	Youden index	Cut‐point	Specificity (%)	Sensitivity (%)
Aβ42/Aβ40	Lumipulse	0.966	0.922–1.000	0.940	0.110	94	100
	MSD	0.966	0.921–1.000	0.940	0.087	94	100
Aβ42/t‐tau	Lumipulse	0.955	0.906–1.000	0.823	3.167	90	92
	INNOTEST	0.960	0.912–1.000	0.900	2.611	90	100
Aβ42/p‐tau181	Lumipulse	0.966	0.920–1.000	0.940	25.25	94	100
	INNOTEST	0.934	0.873–0.995	0.860	17.71	86	100

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The area under the receiver‐operating characteristic 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).

^aHigher than AUC for Lumipulse t‐tau, De Long test P = .005.

^bLower than AUC for Lumipulse p‐tau181, De Long test P = .024.

Concordance of CSF biomarker ratios with amyloid PET SUVR as a continuous variable is shown in Figure 2. 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 A$\beta$42/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 $\epsilon$4 carriers. Despite this, incorporating age, sex, and APOE $\epsilon$4 carrier status as covariates into predictive models did not significantly change the percentage of discordantly classified individuals or type of discordance (Supplementary Table 2 and Supplementary Figure 3).

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FIGURE 2

Scatter plots of CSF biomarker ratios (y axis) against SUVR (x axis) (n = 63). Dashed horizontal lines show the Youden index cut points for the CSF ratios, below which an individual was classified as CSF–positive; dashed vertical lines show the ¹⁸F‐florbetapir amyloid PET SUVR cut point, to the right of which an individual was classified as amyloid PET–positive

4. DISCUSSION

In this study we build on previous validations of Lumipulse measurements of CSF Aβ and tau biomarkers against two other established CSF assay platforms. We report good correlations of measurements of individual biomarkers of CSF Aβ40, Aβ42, t‐tau, and p‐tau181 between platforms, in agreement with other studies. 18 , 19 , 20 We found a stronger correlation between Lumipulse and MSD measurements of Aβ42/Aβ40 ratio compared to Aβ42 alone. The INNOTEST and MSD platforms showed interference by spiked Aβ40 in measurements of Aβ42, but the Lumipulse platform did not. All ratios incorporating Aβ42 were more concordant with amyloid PET than individual biomarkers; the Lumipulse and MSD Aβ42/Aβ40 and Lumipulse Aβ42/p‐tau181 ratios produced the highest accuracy. The Lumipulse Aβ42/Aβ40 cut point of 0.11 pg/mL and Aβ42/p‐tau181 cut point of 25.25 pg/mL demonstrated 100% sensitivity and 94% specificity for distinguishing PET‐positive from PET‐negative individuals.

Measurements of p‐tau181 correlated better than t‐tau between Lumipulse and INNOTEST, and this was also reflected in the Aβ42/p‐tau181 and Aβ42/t‐tau ratios. Our findings for t‐tau contrast with high correlations (r >0.9) reported in other studies between Lumipulse and INNOTEST measurements 19 , 29 but cannot be explained in our study by any differences in pre‐analytical handling, as both t‐tau and p‐tau181 were measured on both platforms from the same aliquot of CSF from each individual. Values of t‐tau at the lower end of the range of samples measured were less well correlated, whereas values of p‐tau181 were very well correlated throughout the range measured (Figure 1E,F). It is unclear as to 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 , 19 For MSD values this could in part be due to different antibodies used for Aβ measurements; other reasons could include differences in the technology and calibrators used. None of the three Aβ42 assays had been calibrated against the CSF Aβ42 certified reference material (CRM), 30 which was developed to provide an international standard for this analyte, and CRMs are not yet available for the other analytes. Furthermore, it is possible that native Aβ40 leads to inaccurate Aβ42 estimates in the INNOTEST assay as reported previously. 13 , 31 Lumipulse was the only platform of the three that did not show significant interference of spiked A$\beta$40 with A$\beta$42 measurements. We found that both the INNOTEST and MSD platforms showed about 25% reduction of measurements of A$\beta$42 when spiking up to 40 ng/mL of A$\beta$40, in contrast to Janelidze et al, 13 who showed 60% reduction for INNOTEST and 20% reduction for MSD with similar A$\beta$40 spiking concentrations. Although the Lumipulse A$\beta$42 assay uses the same monoclonal antibodies as INNOTEST, it is likely that Lumipulse is less susceptible to matrix effects, based on the optimal minimal required dilution of sample in the conjugate solution. We did find significant A$\beta$40 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 A$\beta$42 in predicting cortical amyloid load, but the specificity of the Lumipulse measurements (74%) was lower than that of the INNOTEST (86%) or MSD (80%). In contrast to other studies, which show CSF t‐tau and p‐tau181 to be good individual predictors of amyloid PET, 24 , 25 we found that performance varied by platform; p‐tau181 performed better when measured by Lumipulse compared to INNOTEST, and the converse was found for t‐tau. Furthermore, in line with previous studies 13 , 14 , 24 , 25 and extended to incorporate all platforms assessed, we show that all ratios incorporating A$\beta$42 improved concordance with amyloid PET. Lumipulse cut points of 0.11 for Aβ42/Aβ40 and 25.25 for A$\beta$42/p‐tau181 both produced a specificity of 94% and sensitivity of 100% for detecting amyloid PET status. Our optimal cut points are higher than those derived by Alcolea et al, who examined Lumipulse CSF biomarker concordance with ¹⁸F‐florbetapir PET (0.062 for Aβ42/Aβ40 and 0.068 for p‐tau181/A$\beta$42, which is equivalent to 14.7 for A$\beta$42/p‐tau181). 24 Our cut point of 3.167 for Lumipulse A$\beta$42/t‐tau is also higher than the cut point of 1.852 (equivalent to 0.54 for Lumipulse t‐tau/A$\beta$42) derived by Kaplow et al. 25 Possible explanations for these differences include differing definitions of amyloid PET positivity (Alcolea et al used the cerebellum as the SUVR reference region and Kaplow et al used a variety of PET tracers), a number of individuals in our cohort being close to the SUVR cut point, lack of calibration in our study to CRMs, and participants in the other studies having a wider range of cognitive performance and overall higher prevalence of APOE $\epsilon$4 carriage than the participants of our cohort.

Advantages of the Lumipulse platform over conventional assays include reduction in labor‐intensive steps and manual error, reduced total analysis time due to testing all four biomarkers on the same CSF sample, and improved accuracy for analyte detection, due to the measurement by photon‐counting of direct light emitted rather than wavelength‐based colorimetric absorbance. Furthermore, the Lumipulse platform can process small numbers of CSF samples, without needing to collect enough samples to use in batched assays (as is required for the INNOTEST or MSD). However, a disadvantage of the Lumipulse is its requirement for a large dead volume of 100 μL, relative to sample volumes per replicate for the four biomarkers of 40 to 75 μL, whereas the INNOTEST and MSD assays use similar volumes per replicate (25 to 75 μL) but have no dead volume requirement.

This study has some limitations that might be assessed in future research. MSD analysis took place on a separate day using a separate sample aliquot to that used for INNOTEST and Lumipulse analysis. Lumipulse measurements were performed in singleton due to CSF volume requirements, so precision of the Lumipulse assays was not assessed. We did not compare measurements of all four biomarkers on all platforms, and we focused on comparing Lumipulse measurement with other immunoassays but not with other methods of measurement like mass spectrometry. Although the AUC obtained for prediction of amyloid PET status were higher for CSF Aβ42 and its ratios than that obtained by the model using age, sex, and APOE $\epsilon$4 carrier status, the differences did not reach statistical significance, likely due to this being an interim data set 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 “PET–negative” do actually have sub‐threshold cerebral amyloid deposition, as CSF changes may precede PET changes. 32 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.

CONFLICTS OF INTEREST

Avid Radiopharmaceuticals, a wholly owned subsidiary of Eli Lilly, kindly provided the ¹⁸F‐florbetapir tracer (Amyvid) free of cost but had no role in the design, conduct, analysis, or reporting of Insight 46 study findings. We are particularly indebted to the support of the late Chris Clark of Avid Radiopharmaceuticals who championed this study from its outset.

Fujirebio provided and set up the Lumipulse platform free of cost but had no role in the design, conduct, analysis, or reporting of this study.

The National Survey of Health and Development is funded by the Medical Research Council (MC_UU_00019/1, MC_UU_00019/3). TDP was supported by a Wellcome Trust Clinical Research Fellowship (200109/Z/15/Z). NCF is supported by UK Dementia Research Institute at University College London, Medical Research Council, National Institute for Health Research (Senior Investigator award), and Engineering and Physical Sciences Research Council. HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (#2018‐02532), the European Research Council (#681712), Swedish State Support for Clinical Research (#ALFGBG‐720931), the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809‐2016862), and the UK Dementia Research Institute at UCL. HZ has served at scientific advisory boards for Denali, Roche Diagnostics, Wave, Samumed, and CogRx; has given lectures in symposia sponsored by Fujirebio, Alzecure, and Biogen; and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program (all outside the submitted work). JMS is supported by Engineering and Physical Sciences Research Council (EP/J020990/1), British Heart Foundation (PG/17/90/33415), and EU's Horizon 2020 research and innovation programme (666992). MH and JMS are supported by the University College London Hospitals Biomedical Research Centre. NCF and JMS are supported by the National Institute for Health Research Queen Square Dementia Biomedical Research Unit and the Leonard Wolfson Experimental Neurology Centre.

Supporting information

ACKNOWLEDGMENTS

Notes

Keshavan A, Wellington H, Chen Z, et al. Concordance of CSF measures of Alzheimer's pathology with amyloid PET status in a preclinical cohort: A comparison of Lumipulse and established immunoassays. Alzheimer's Dement. 2020;12:e12097 10.1002/dad2.12097 [Europe PMC free article] [Abstract] [CrossRef] [Google Scholar]

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