Validation of the LUMIPULSE automated immunoassay for the measurement of core AD biomarkers in cerebrospinal fluid

LUMIPULSE

For measurement of amyloid β 1-40, amyloid β 1-42, total tau and pTau 181, a LUMIPULSE^® G600II instrument was used (Fujirebio, Ghent, Belgium). It is a cartridge-based system that uses monoclonal antibody-coated beads for capture and monoclonal antibodies for detection. For amyloid β 1-42 and total tau, streptavidin-conjugated alkaline phosphatase (AP) is added after a washing step, which binds to the biotinylated monoclonal antibody. Amyloid β 1-40 and pTau 181, the antibodies are directly conjugated to AP instead, and 3-(2′-spiroadamantane)-4-methoxy-4-(3ʺ-phosphoryloxy) phenyl-1, 2-dioxetane disodium salt (AMPPD) is used as substrate and the resulting luminescence at 477 nm is measured.

Method comparisons

Method comparisons were performed at the neurochemistry laboratory, Sahlgrenska University Hospital/Mölndal, using de-identified CSF-samples from the hosptal routine analysis. Samples were selected based on initial INNOTEST results and re-analyzed on the same occasion by LUMIPULSE G and the respective assay. The use of surplus CSF from the routine analysis was approved by the Ethics Committee at the University of Gothenburg (EPN – Gothenburg, Aug 11, 2014).

INNOTEST β-amyloid (1-42), hTau Ag, and pTau 181 (Fujirebio) assays were performed according to the manufacturer’s kit insert instructions as described previously [22]. Samples with biomarker concentrations below the lower limit of quantification (LLoQ) as provided by the manufacturer were excluded (β-amyloid (1-42)<225 ng/L, hTau Ag<57 ng/L and pTau 181<20 ng/L). MSD electrochemiluminescence assay (MSD) was used according to the manufacturer’s instructions for measurement of Aβ 1-42/Aβ 1-40.

LC-MS analysis of Aβ 1-42 was performed using a certified reference method, as previously described [21, 23].

Reproducibility

CSF samples having low, medium, and high concentrations of the four analytes were analyzed by LUMIPULSE G in three laboratories. The experiments were performed in two separate rounds: in the first round, Total Tau was measured at the University of Gothenburg, Sweden (Lab “a” in Figure 2), at Radboud University, Netherlands (Lab “b”), and at FujiRebio, Belgium (Lab “c”). In the second round, pTau 181, β-amyloid 1-42, and β-amyloid 1-40 were measured at Radboud University, FujiRebio, and Sant Pau- Biomedical Research Institute, Spain (Lab “d”). The homogeneity of the sample aliquots was verified by repeated measurments by at the FujiRebio laboratory, Belgium. In each laboratory, the samples were analyzed in triplicate, twice per day over five days. For some measurements, there was insufficient sample volume avaliable; these measurements were then excluded from the comparison. The standard deviations and coefficients of variation (CV) for intra-laboratory repeatability and inter-laboratory reproducibility were calculated according to ISO 5725-2.

Inter- and intra-lab variation were also assessed by analyzing samples within the Alzheimer’s Association Quality Control (AA-QC) program, administered at the Clinical Neurochemistry Laboratory at the University of Gothenburg, Sweden [10]. In the AA-QC program, CSF samples with known concentrations of the AD biomarkers are periodically dispatched to laboratories around the world for method validation to monitor inter-lab variability of AD biomarkers. For LUMIPULSE G, only results of β-amyloid 1-42 and total tau were available. Individual CSF samples were analyzed on seven different occasion. For β-amyloid 1-42 a longitudinal sample consisting of pooled CSF was analysed at seven rounds with 4–19 participating laboratories. For tTau we used data from a pooled CSF sample analysed at four time points with 4–19 participating laboratories in each round.

Long-term consistency of measurments for all four assays, performed on a single LUMIPULSE G instrument was evaluated by analyzing aliquots of two CSF pools; one composed of patient samples with AD-like core biomarker profile, and one with normal biomarker levels. Samples were analyzed 71 times approximately once per week during 18 months in Gothenburg.

Clinical cutpoint determination

For determination of clinical cutpoints for tTau, pTau, and Aβ42, CSF samples from three cohorts were used; from McGill University (Translational Biomarkers for Aging and dementia (TRIAD), Canada), University of Perugia (Italy), and Brno/Praha (Czech Republic). Demographics and biomarker data of the samples are listed in Table 5.

For the TRIAD cohort (n=101), lumbar puncture (LP) was performed under local anesthesia, using an 18 ga. “introducer” to penetrate the interspinous ligaments, followed by dural puncture using the 24 ga. Sprotte atraumatic needle. Twenty nine milli-liter of fluid was collected with polypropylene syringes into 10 mL polypropylene tubes (Sarstedt, part no. 62.9924.294). The first four ml was sent to the clinical laboratory for determination of albumin, total protein, glucose and cells. The remaining 25 mL was transferred to polypropylene tubes and centrifuged at 20 °C for 10 min at 2,200×g. The CSF was then rapidly frozen for permanent storage at −80 °C until analysed on the LUMIPULSE instrument.

The Perugia cohort was sampled at the Center of Memory Disturbances of the University of Perugia. CSF samples were obtained from subjects that were consecutively recruited between January 2012 and June 2016 and followed up for at least two years. The cohort consisted of 58 patients with probable AD diagnosed according to the NIA-AA criteria [24], regardless of CSF biomarker profile, and 37 non-demented controls. All patients underwent a baseline clinical examination by experienced neurologists, detailed neuropsychological assessment including Mini-Mental State Examination (MMSE), blood chemistry, MRI and lumbar puncture (LP). Neurological controls included cognitively normal subjects, with other neurological diseases such as headache, epilepsy and polyneuropathies, who showed no evidence of progression to dementia after at least two years of follow-up. Patients with subjective memory complaints were not included in the control group. CSF samples were collected via LP from 8:00 to 10:00 a.m. after overnight fasting, following a standardized procedure [12] and according to international guidelines [25, 26]. CSF (10–12 mL) was taken from the L3–L4 or L4–L5 interspace, immediately collected in sterile polypropylene tubes (Sarstedt cat. nr. 62.610.201), and gently mixed to avoid possible gradient effects. The samples were centrifuged at 2000×g for 10 min at room temperature, aliquoted (0.5 mL) in polypropylene tubes (Sarstedt cat. nr. 72.730.007) and stored at −80 °C pending analysis.

The Czech cohort was collected within the Czech Brain Aging Study (CBAS) [27]. CBAS is a prospective longitudinal memory clinic-based multicentre study recruiting non-demented adults 55+ years of age. Both CBAS centres in Prague and Brno work as a low-threshold facility; hence, the participants are mostly volunteers who enter by self-referral, with memory complaints expressed by themselves or the family or who were referred by general practitioners, local specialists or the Czech Alzheimer Society to one of the memory clinics. All study participants underwent a standard set of procedures, including neurological and comprehensive neuropsychology examinations, as well as laboratory and vital function assessments. Sociodemographic, personal, pharmacological and family history data were collected. Participants and their informants completed multiple questionnaires about cognitive complaints and lifestyle factors. MRI scans of 1.5 or 3 T were performed every 24 months or earlier when a participant converted to dementia or progressed towards cognitive impairment at an unusual rate. Genotyping was carried out at baseline. In a subset, CSF and/or amyloid PET was performed. The CBAS is complemented by a biological sample bank linked to data from the CBAS and CBAS Plus cohorts. The cerebrospinal fluid (CSF) collection and storage were carried out according to the widely recognised consensus protocol for the standardisation of CSF collection and biobanking. CSF was collected in polypropylene 10 mL tubes (Gama, part no. 400 942). Eighteen aliquots of 0.2 mL CSF were stored in polypropylene tubes for each participant. All samples were stored at −80 °C.

The Czech and Italian cohorts were analyzed by LUMIPULSE G at the respective sites where the samples were collected, while the Canadian cohort and the clinical routine samples used to establish clinical cutpoints for Aβ 1-42/Aβ 1-40 were analyzed at the University of Gothenburg in Sweden. Use of the samples for these studies was granted by the respective local ethics boards: the Czech cohort: No. EK-701/16, Date of EC Session: 25.5.2016; the Italian cohort: Prot. N. 1936908/AV del 09/10l2O08; the Canadian cohort: REB from Douglas Hospital Research Centre – Montreal – Canada, no. IUSMD-16-60.

Determination of clinical cutpoint for Aβ 1-42/Aβ 1-40 was based on mixture model analysis of data from 2,782 de-identified patient samples analyzed at the Neurochemistry Laboratory at Sahlgrenska University Hospital between December 2019 – October 2020.

Statistical analysis

Statistical data analysis was performed in R (version 3.6.1; http://www.R-project.org/). Passing-Bablok regression analysis was performed using the R package mcr (https://CRAN.R-project.org/package=mcr). Clinical cutpoints for tTau, pTau, and Aβ42 were determined by ROC curve analysis, using the ROC01 algorithm [28, 29], implemented in the R package “pROC” [30]. For Aβ42/Aβ40, the cutpoint was determined by mixture model analysis, using the expectation-maximization (EM) algorithm for mixtures of normal distributions (normalmixEM), implemented in the R package “mixtools” [31].

Method comparisons

The LUMIPULSE G assays β-amyloid 1-42, β-amyloid 1-40, total tau, and pTau 181 were compared to the corresponding INNOTEST assays, β-AMYLOID (1-42), β-AMYLOID (1-40), hTAU Ag, and PHOSPHO TAU (181P) (Figure 1A–C); to MSD (V-PLEX Plus Aβ Peptide Panel 1 (6E10)) for Aβ 1-42, Aβ40, and Aβ 1-42/Aβ40 (Figure 1D–F); an LC-MS reference method for Aβ 1-42 (Figure 1G), using regression analysis. The results are summarized in Table 1. The LUMIPULSE G assays showed strong correlation to all other immunoassays (r>0.93 for all assays). The results for LUMIPULSE G amyloid 1-42 and total tau were similar to the corresponding INNOTEST ELISA assays, with small intercept and slope close to one in the correlation plots. LUMIPULSE pTau 181 also had a small intercept but a positive bias (slope=1.6) compared to INNOTEST PHOSPHO TAU(181P). Compared to the MSD V-PLEX assays for Aβ 1-42 and Aβ 1-40, LUMIPULSE G also gave significantly higher values for both β-amyloid 1-42 (slope=1.38, intercept=16.8) and β-amyloid 1-40 (slope=2.07, intercept=−1,724.6), and approximatley 20% lower Aβ 1-42/Aβ 1-40 ratio. LUMIPULSE G β-amyloid 1-42, which has been adjusted according to an LC-MS based reference method [21, 23], gave similar values to LC-MS in the range of 400–800 ng/L but showed a slight negative bias at lower concentrations, and a positive bias at higher concentrations.

Figure 1:

Regression analysis (Passing-Bablok) comparing LUMIPULSE G and: INNOTEST (A) β-AMYLOID (1-42), (B) hTAU Ag and (C) PHOSPHO TAU(181P); MSD (D) β-amyloid 1-42, (E) β-amyloid 1-40, and (F) 10 × β-amyloid 1-42/1-40; LC-MS (G) β-amyloid 1-42.

The regression line is indicated in solid blue, and the identity line in dashed orange. The shaded blue area indicates a 95% confidence interval.

Reproducibility

The intra-laboratory repeatability CVs (Figure 2, Table 2). were below 6% for all four analytes, with the largest variation for a single analyte was observed for β-amyloid 1-40 (6.2%), while the β-amyloid 1-42/1-40 ratio had a CV of 6.2%. Inter-laboratory reproducibility CVs were similar in magnitude, with the largest CV observed for β-amyloid 1-42 (6.5%).

Figure 2:

Inter- and intra-lab variation of LUMIPULSE G (A) total tau, (B) pTau 181, (C) β-amyloid 1-42, (D) β-amyloid 1-40, and (E) β-amyloid 1-42/1-40, measured at three (out of four) different laboratories designated a–d.

The black circle indicates value of the mean and the bars represent ±1 standard deviation.

Reproducibility was also studied within the AA-QC program. β-amyloid 1-42 and total tau were measured in (different) CSF samples, analysed on seven occasions in different laboratories (Figure 3A, B, Table 3). The inter-laboratory variation varied significantly between measurement rounds, from 7.6–21% for β-amyloid 1-42, and from 3.5–10.6% for Total Tau. Longitudinal measurement of a single sample indicated no significant drift over time for β-amyloid 1-42 (Figure 3C, Table 3), but again high CV variation between rounds (7.1–18.9%). For total tau (Figure 3D, Table 3), there was a slight downward trend over time, with the last measurement 20% lower than the first, while the CVs were more uniform between the measurement rounds (4.3–8.9%) compared to β-amyloid 1-42.

Figure 3:

Inter-laboratory variation in the AA-QC program.

(A and B) Variation of (A) β-amyloid 1-42 and (B) total tau for different samples measured in different rounds. The mean for each round is indicated by a circle and the whiskers indicate ±1 standard deviation. (C and D) Variation for (C) β-amyloid 1-42 and (D) total tau for a longitudinal sample analysed in seven and four rounds, respectively. The shaded gray area indicates a 95% confidence interval.

Long-term assay stability was also assessed by measuring aliquots of two CSF sample pools, analyzed on a single LUMIPULSE G instrument in Gothenburg twice per week over an 18-month period (Table 4). For the AD pool (CSF Pool 2), the CV:s for all analytes were below 4%, while for the normal pool (CSF Pool 1) they were slightly higher, but for all analytes within 8%.

Diagnostic performance and cut-off values

To determine the diagnostic performance of the LUMIPULSE G β-amyloid 1-42, β-amyloid 1-42/1-40, total tau, and pTau 181 assays and establish cut-off values for use in a clinical setting, we analyzed CSF from three cohorts of AD patients and non-demented controls (Table 5). Cut-off values were determined by ROC curve analysis of data from all cohorts combined. Histograms depicting the distributions of the biomarker concentrations in the groups and ROC curves are shown in Figure 4, and the established cut-off values, sensitivities and specificities are listed in Table 6, including also values for each cohort separately. The optimal cut-off values were <409 ng/L för total tau, <50.2 ng/L for pTau 181, and >526 ng/L for β-amyloid 1-42. Because of the high sensitivity and specificity of the Aβ42/Aβ40 ratio, this biomarker displays a clear bimodal distribution in patient populations. Therefore, it was possible to use an alternative approach to determining the cut-off value, by using mixture model analysis of data from a large number of patient CSF samples (n=2,782) analyzed in a clinical routine laboratory setting (Figure 5, Table 6), resulting in a cut-off of >0.72.

Figure 4:

Diagnostic performance of LUMIPULSE and cutpoints.

Histograms and ROC curves for (A) tTau, (B) pTau 181, (C) β-amyloid 1-42, (D) 10 × β-Amyloid 1-42/1-40 for separating Alzheimer patients and controls, in three combined cohorts.

Figure 5:

Mixture model analysis of β-amyloid 1-42/1-40 of data from patient CSF samples (n=2,782) analyzed in a routine laboratory setting.

Total tau and pTau 181 had AUC:s of 80 and 86%, respectively, with sensitivity and specificity of 72%/80% for total tau of, and 82%/81% for pTau 181. β-amyloid 1-42 had AUC of 79% with sensitivity and specificity figures of 85%/84%. For amyloid 1-42/1-40, the sensitivity and specificity, as determined by mixture modeling [31], was 92%/71%.