Delaby et al. Alzheimer’s Research & Therapy (2022) 14:20 https://doi.org/10.1186/s13195-022-00967-z RESEARCH Open Access The Aβ1–42/Aβ1–40 ratio in CSF is more strongly associated to tau markers and clinical progression than Aβ1–42 alone Constance Delaby1,2, Teresa Estellés1,3, Nuole Zhu1,3, Javier Arranz1,3, Isabel Barroeta1,3, María Carmona‑Iragui1,3, Ignacio Illán‑Gala1,3, Miguel Ángel Santos‑Santos1,3, Miren Altuna1,3, Isabel Sala1,3, M. Belén Sánchez‑Saudinós1,3, Laura Videla1,3,4, Sílvia Valldeneu1,3, Andrea Subirana1,3, Mireia Tondo5,6,7, Francisco Blanco‑Vaca5,6,7, Sylvain Lehmann2, Olivia Belbin1,3, Rafael Blesa1,3, Juan Fortea1,3, Alberto Lleó1,3 and Daniel Alcolea1,3* Abstract Background: Cerebrospinal fluid (CSF) Aβ1–42 levels and the Aβ1–42/Aβ1–40 ratio are markers of amyloid pathol‑ ogy, but previous studies suggest that their levels might be influenced by additional pathophysiological processes. Aims: To compare Aβ1–42 and the Aβ1–42/Aβ1–40 ratio in CSF in different neurodegenerative disorders and study their association with other biomarkers (tTau, pTau181, and NfL) and with cognitive and functional progression. Methods: We included all participants from the Sant Pau Initiative on Neurodegeneration (SPIN) with CSF Aβ1–42 and Aβ1–42/Aβ1–40. Participants had diagnoses of Alzheimer’s disease (AD), dementia with Lewy bodies, fronto‑ temporal lobar degeneration-related syndromes, non-neurodegenerative conditions, or were cognitively normal. We classified participants as “positive” or “negative” according to each marker. We compared CSF levels of tTau, pTau181, and NfL between concordant and discordant groups through ANCOVA and assessed differences in cognitive (MMSE, FCSRT) and functional (GDS, CDR-SOB) progression using Cox regression and linear-mixed models. Results: In the 1791 participants, the agreement between Aβ1–42 and Aβ1–42/Aβ1–40 was 78.3%. The Aβ1–42/ Aβ1–40 ratio showed a stronger correlation with tTau and pTau181 than Aβ1–42 and an agreement with tTau and pTau181 of 73.1% and 77.1%, respectively. Participants with a low Aβ1–42/Aβ1–40 ratio showed higher tTau and pTau181 and worse cognitive and functional prognosis, regardless of whether they were positive or negative for Aβ1–42. The results were consistent across stages, diagnostic categories, and use of different cutoffs. Conclusion: Although Aβ1–42 and Aβ1–42/Aβ1–40 are considered markers of the same pathophysiological path‑ way, our findings provide evidence favoring the use of the Aβ1–42/Aβ1–40 ratio in clinical laboratories in the context of AD. Keywords: Amyloid, Aβ1–40, Aβ1–42, Cerebrospinal fluid, Tau, Biomarkers Introduction Cerebrospinal fluid (CSF) biomarkers of Alzheimer’s disease (AD) have changed the management of patients *Correspondence: dalcolea@santpau.cat 3 Centro de Investigación Biomédica en Red en Enfermedades with cognitive impairment [1]. In particular, CSF levels of Neurodegenerativas, CIBERNED, Madrid, Spain β-amyloid 1–42 (Aβ1–42), total tau (tTau), and its phos- Full list of author information is available at the end of the article phorylated form on threonine 181 (pTau181) have shown © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Delaby et al. Alzheimer’s Research & Therapy (2022) 14:20 Page 2 of 11 very high accuracy for the diagnosis of AD [2–4]. They non-neurodegenerative conditions such as psychiatric are consequently being implemented in clinical labora- etiology, vascular cognitive impairment, inflammatory, tories [5–9] both for diagnosis and research settings, as and those with uncertain etiology. Details about the SPIN well as in clinical trials. cohort have been reported previously [23]. The role of Aβ1–42 in CSF as a marker of amyloid pathology is widely accepted [6, 9, 10]. However, a series CSF collection and analysis of studies have shown that reduced levels of CSF Aβ1- CSF was obtained by lumbar puncture, collected, and 42 can also be found in a variety of conditions different processed in polypropylene tubes following international from AD, such as inflammatory diseases, prionopathies, recommendations [32, 33]. The same pre-analytical han- amyloid angiopathy, or frontotemporal dementia [10– dling was followed in all samples [23]. Concentrations of 15]. The Aβ1–42/Aβ1–40 ratio has proven to be of great Aβ1–42, Aβ1–40, total tau (tTau), and 181-phosphoryl- value in detecting amyloid pathology both in CSF [16, 17] ated tau (pTau181) in CSF were measured using com- and plasma [18, 19] and has shown a better correlation mercially available kits in the Lumipulse fully automated with amyloid burden in PET than Aβ1–42 alone [20–22]. platform (Fujirebio-Europe), as previously described [22, However, Aβ1–40 levels are not systematically assessed 34], and following provider’s instructions in line with in many clinical laboratories alleging that this marker Global Biomarker Standardization [35, 36]. For each alone has no diagnostic value. A large-scale head-to-head sample, Aβ1–42, Aβ1–40, tTau, and pTau181 were quan- comparison between Aβ1–42 and the Aβ1–42/Aβ1–40 tified simultaneously in the same run immediately after ratio would address the question of whether these two the first freeze-thaw cycle of each sample using pristine markers are equally tracking the same pathophysiologi- aliquots containing 500 μL of CSF [22, 34]. The results cal process. It would also inform laboratories on whether of the Lumipulse G β-amyloid 1–42 have been standard- to implement the Aβ1–42/Aβ1–40 ratio into the clinical ized according to certified reference material developed routine. by the International Federation of Clinical Chemistry and In the present work, we studied the CSF markers Aβ1– Laboratory Medicine as recommended by their work- 42 and Aβ1–42/Aβ1–40 ratio in a large cohort of partici- ing group for CSF proteins. Our laboratory participates pants with a variety of neurodegenerative disorders. We in the Alzheimer’s Association Quality Control Program compared the agreement between both measures in dif- led by the University of Gothenburg [37]. Three levels of ferent contexts and studied their association with other internal quality controls provided by the manufacturer CSF biomarkers (tTau, pTau181, and NfL) and with cog- were assessed for each analyte to assess the reproducibil- nitive and functional progression. This information is ity of the assays. We included at least one level of quality highly relevant in the implementation and interpretation control per analyte in each run. Inter-assay coefficients of of these markers in clinical routine. variation (CV%) were between 1.7% and 6.8% for all lev- els and analytes. Material and methods Neurofilament light (NfL) levels in CSF were meas- Study participants and clinical classification ured using a commercially available ELISA kit (NF-light, We included all participants in the Sant Pau Initiative UMAN DIAGNOSTICS, Umea, Sweden) as previously on Neurodegeneration (SPIN) cohort [23] that under- described [24, 25]. The mean inter- and intra-assay coef- went lumbar puncture for CSF biomarkers between ficients of variation were 3.4% and 11.4%, respectively. November 2013 and August 2021. The diagnostic groups included patients with mild cognitive impairment (MCI) Definition of amyloid profile or dementia and with either pathophysiological evidence To ensure that the cutoffs for Aβ1–42 and Aβ1–42/Aβ1– of Alzheimer’s disease (AD), frontotemporal lobar degen- 40 had comparable levels of sensitivity and specificity, we eration (FTLD)-related syndromes [24, 25], or probable applied cutoffs corresponding to one-sided 95% quan- dementia with Lewy bodies (DLB). Diagnoses were estab- tile (Q95%) values in a middle-aged cognitively healthy lished following internationally accepted diagnostic crite- population (age range 23–60 years, 67% female). This age ria [6, 7, 9, 26–28]. Clinical symptoms, neuroimaging and range was selected to minimize the presence of preclini- CSF biomarkers were considered for the diagnostic clas- cal AD in the reference population. We used these Q95% sification of patients. We also included participants with cutoffs for Aβ1–42 (637 pg/mL) and Aβ1–42/Aβ1–40 Down syndrome (DS) [29, 30] and cognitively normal (0.070) to classify all participants in four different pro- controls (CN). All CN participants had normal cognitive files: two concordant profiles in which both Aβ1–42 and scores in a formal neuropsychological evaluation [23, 31]. Aβ1–42/Aβ1–40 ratio were above (Aβ1–42[−]Ratio[−]) Patients with other diagnoses were grouped as “others” or below (Aβ1–42[+]Ratio[+]) their respective cutoffs, and included participants with prionopathy and other and two discordant profiles, in which only one of the two Delaby et al. Alzheimer’s Research & Therapy (2022) 14:20 Page 3 of 11 amyloid parameters, Aβ1–42 or the Aβ1–42/Aβ1–40 (v.3.1-12), “survminer” (v.0.4.6), “nlme” (v.3.1-147), ratio, was abnormal (Aβ1–42[+]Ratio[−] and Aβ1– “multcomp” (v.1.4-13), “ggplot2” (v.3.3.0), and “ggpubr” 42[−]Ratio[+], respectively). The objective of classifying (v.0.3.0), as implemented in the R statistical software (v participants in four amyloid profiles is to assess the par- 3.6.2). The alpha threshold was set at 0.05 for all analyses. ticularities of those groups where Aβ1–42 and the Aβ1– 42/Aβ1–40 ratio are discordant (Aβ1–42[−]Ratio[+] and Ethical approval and consent to participate Aβ1–42[+]Ratio[−]) and compare them to those that are All procedures in the study were approved by the Sant clearly amyloid negative (Aβ1–42[–]Ratio[−]) or clearly Pau Ethics Committee following the standards for medi- amyloid positive (Aβ1–42[+]Ratio[+]) according to both cal research in humans recommended by the Declaration markers. More details about the cognitively healthy refer- of Helsinki. All participants or their legally authorized ence population and results after applying other cutoffs representatives gave written informed consent. can be found in Additional file 1. Results Measures of cognitive and functional impairment Demographics and core CSF biomarkers Cognition was assessed by the Mini-Mental State Exami- We included a total of 1791 participants from the SPIN nation (MMSE) and the free and cued selective remind- cohort. The demographic characteristics and biomarker ing test (FCSRT). Global functional impairment was results are summarized in Table 1. There were differences assessed by the Clinical Dementia Rating Scale Sum of in age and male/female proportion between the groups. Boxes (CDR-SOB) and the Global Deterioration Scale As expected, the APOEε4 allele was more frequent in (GDS). Outcomes for cognitive and functional impair- AD patients (50%; p < 0.001), and no differences were ment were defined as MMSE < 24 and GDS ≥ 4, respec- observed among the other groups. MMSE scores were tively [31]. lower in all symptomatic groups compared to CN (p < 0.001). APOE genotyping DNA was extracted from whole blood using standard Aβ1–42 and the Aβ1–42/Aβ1–40 ratio in CSF show high procedures and APOE was genotyped according to previ- but not perfect agreement ously described methods [38]. Figure 1 shows the distribution of participants based on their CSF Aβ1–42 and Aβ1–42/Aβ1–40 ratio measures. Statistical analysis The correlation between these two parameters was rho = Non-normally distributed variables were log-trans- 0.71, p < 0.001. Using Q95% cutoffs as described in the formed. Differences in the frequency of categorical vari- “Material and methods” section, Aβ1–42 and Aβ1–42/ ables were assessed by the χ2 test, and we used age- and Aβ1–40 ratio had an overall agreement of 78.3% in the sex-adjusted analysis of covariance (ANCOVA) to com- whole sample, as both measures were normal (Aβ1– pare CSF levels of tTau, pTau181, and NfL between 42[−]Ratio[−]) in 41.6% and both were abnormal (Aβ1– concordant and discordant groups. We determined 42[+]Ratio[+]) in 36.7% of participants. Within each Spearman’s correlation coefficients between biomark- diagnostic category, the agreement ranged from 69.9% ers in the whole sample and after stratification by diag- (AD group) to 87.9% (CN group). More details about the nostic category, clinical stage, and amyloid profile. We agreement after applying other cutoffs values and with assessed the association with cognitive and functional other biomarkers can be found in Additional file 1. progression in patients with mild cognitive impairment through Kaplan-Meier survival curves and age- and Aβ1–42/Aβ1–40 ratio is more strongly associated with tau sex-adjusted Cox regression analysis. We studied the markers than Aβ1–42 association of amyloid profiles with a cognitive decline We next studied the association of Aβ1–42 and the through linear-mixed models. The initial model included Aβ1–42/Aβ1–40 ratio with the other CSF biomarkers by baseline MMSE score, baseline age, sex, years of educa- assessing Spearman correlations in the whole sample and tion, pTau181 levels, diagnosis, time, APOE4 status, and within diagnostic groups. In the whole sample, Aβ1–42 amyloid profile together with its interaction with time showed a significant correlation with tTau (rho = − 0.25, and with APOE4 status as fixed factors. We defined ran- p < 0.001) and pTau181 (rho = − 0.32, p < 0.001). These dom intercepts for diagnosis and at the participant level correlations were stronger for the Aβ1–42/Aβ1–40 to account for repeated measures and modeled residual ratio (rho = − 0.69 and rho = − 0.75, respectively, both errors per diagnostic group. The alpha threshold was set p < 0.001). Both Aβ1–42 and the Aβ1–42/Aβ1–40 ratio at 0.05, and all analysis were performed using MEDCALC showed a similar correlation with NfL (rho = − 0.26 (MEDCALC software ver 15.2.2) and packages “survival” and rho = − 0.32, respectively, both p < 0.001). These Delaby et al. Alzheimer’s Research & Therapy (2022) 14:20 Page 4 of 11 Table 1 Demographics, clinical information, and biomarkers across diagnostic categories CN AD DLB FTLD Down Others N 197 518 128 186 225 536 AGE, years Mean (SD) 53.5 (12.5) 73.1 (6.88) 75.7 (5.49) 70.8 (8.58) 45.1 (10) 70 (9.18) Median [IQR] 55 [46–62] 74 [69–78] 76 [71–80] 72 [66–77] 48 [40–52] 71 [65–77] SEX, females/males (% 132/65 (67%) 311/207 (60%) 64/64 (50%) 77/109 (41.4%) 103/122 (45.8%) 310/226 (57.8%) females) MMSE score Mean (SD) 29.2 (0.889) 23.6 (4.52) 24 (4.09) 24 (5.08) NAa 25.4 (4.12) Median [IQR] 29 [29–30] 25 [22–27] 24.5 [22–27] 25 [21–28] NAa 27 [24–28] Education, years Mean (SD) 15.6 (3.99) 10.7 (4.72) 9.18 (5.05) 12 (5.13) 15.3 (3.07) 10.7 (4.93) Median [IQR] 16 [12–20] 10 [8–13] 8 [7–12] 12 [8–16] NAa 9 [8–13] APOEε4, APOEε4−/APOEε4+ 46/151 (23.4%) 254/251 (50.3%) 33/93 (26.2%) 37/139 (21%) 44/177 (19.9%) 115/413 (21.8%) (%APOEε4+) Follow-up, years Mean (SD) 2.02 (1.99) 1.09 (1.49) 3.33 (1.93) 1.71 (1.47) 1.76 (2.09) 0.59 (1.23) Median [IQR] 1.71 [0–2.82] 0 [0–2.06] 3.46 [2.07–4.52] 1.52 [0.242–2.66] NAa 0 [0–0.383] Aβ1–42, pg/ml Mean (SD) 1148 (397) 562 (165) 817 (399) 938 (446) 715 (417) 1000 (500) Median [IQR] 1118 [849–1371] 556 [432–673] 703 [542–1009] 850 [569–1229] 583 [430–892] 896 [609–1323] Aβ1–40, pg/ml Mean (SD) 11,694 (3595) 12,790 (3781) 11,506 (4189) 10,806 (4357) 11,673 (4678) 11,399 (4390) Median [IQR] 11,329 [9238– 12,541 [10,125– 10,885 [8882– 10,140 [7710– 11,035 [8346– 10,638 [8112– 13,777] 15,122] 14,234] 13,334] 14,594] 13,986] Aβ1–42/Aβ1–40 Mean (SD) 0.0991 (0.0181) 0.0453 (0.0108) 0.0727 (0.0263) 0.0877 (0.0221) 0.0615 (0.0226) 0.0862 (0.0225) Median [IQR] 0.104 [0.0986– 0.0445 [0.0376– 0.0662 [0.0503– 0.0964 [0.0731– 0.0562 [0.0422– 0.0943 [0.0665– 0.109] 0.0514] 0.0996] 0.103] 0.078] 0.103] tTau, pg/ml Mean (SD) 255 (152) 748 (358) 456 (334) 387 (260) 644 (520) 334 (231) Median [IQR] 230 [174–291] 656 [488–915] 361 [253–525] 322 [222–456] 489 [262–870] 292 [213–378] pTau181, pg/ml Mean (SD) 37.3 (27.3) 122 (60.3) 70.5 (55.5) 49.9 (39.2) 100 (96.8) 45 (27.4) Median [IQR] 31.6 [24.9–42] 105 [78.7–145] 51 [35.7–83] 39.7 [29.2–54.1] 63.6 [29.8–151] 41 [29.3–52.7] NfL, pg/ml Mean (SD) 475 (256) 1330 (1824) 1108 (570) 2079 (1836) 815 (773) 1488 (1340) Median [IQR] 458 [320–533] 981 [791–1254] 918 [719–1297] 1412 [884–2767] 614 [357–1014] 1089 [595–1878] Clinical stage, CN/MCI/demen- 177/0/0 0/296/208 2/60/64 5/90/80 NAa 27/348/125 tia (% MCI) Amyloid profile (Aβ1–42[−] 165/13/11/8 4/154/2/358 50/29/7/42 120/8/23/35 61/37/13/114 345/45/47/100 Ratio[−]/Aβ1–42[−]Ratio[+]/ Aβ1–42[+]Ratio[−]/Aβ1–42[+] Ratio[+]) MMSE Mini-Mental State Examination, CSF cerebrospinal fluid, CN cognitively normal, AD Alzheimer’s disease, DLB dementia with Lewy bodies; FTLD frontotemporal lobar degeneration-related syndrome, MCI mild cognitive impairment a Due to specific particularities of the clinical and cognitive assessment in the context of intellectual disability, participants with Down syndrome were excluded from the prognostic analysis stronger associations of Aβ1–42/Aβ1–40 ratio with tTau NfL (Tukey post hoc p = 0.001). But we also found and pTau181 were observed within all symptomatic diag- differences in tTau and pTau181 levels between the nostic categories (Additional file 1). two discordant profiles. As seen in Fig. 2, Aβ1–42[−] Next, we compared CSF levels of tTau, pTau181 Ratio[+] participants showed higher levels of tTau and NfL between all four amyloid profiles. The objec- (Tukey post hoc p < 0.001) and pTau181 (Tukey post tive of this analysis was to assess the particularities hoc p < 0.001) compared to those with Aβ1–42[+] of those groups where the Aβ1–42 and the Aβ1–42/ Ratio[−]. These differences were also observed within Aβ1–40 ratio are discordant. As expected, compared all diagnostic categories and in all clinical stages to the Aβ1–42[−]Ratio[−] group and after adjusting (Additional file 1). These results indicate that reduced by age and sex, the Aβ1–42[+]Ratio[+] profile was levels of the Aβ1–42/Aβ1–40 ratio are associated with associated with higher levels of tTau (Tukey post hoc high levels of CSF tau markers, regardless of the status p < 0.001), pTau181 (Tukey post hoc p < 0.001), and of Aβ1–42 alone. Delaby et al. Alzheimer’s Research & Therapy (2022) 14:20 Page 5 of 11 Fig. 1 Distribution of participants according to CSF levels of Aβ1–42 and amyloid ratio within diagnostic categories. CN, cognitively normal; AD, Alzheimer’s disease; DLB, dementia with Lewy bodies; FTLD, frontotemporal lobar degeneration-related syndrome. Dashed lines indicate 95% quantile values (Q95%) for Aβ1–42 and Aβ1–42/Aβ1–40 in a middle-aged cognitively normal population as described in the “Material and methods” section Delaby et al. Alzheimer’s Research & Therapy (2022) 14:20 Page 6 of 11 2000 400 10000 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p=0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 p<0.001 1500 300 7500 pTau181, pg/ml tTau, pg/ml NfL, pg/ml 1000 200 5000 500 100 2500 0 0 0 A 1-42(-) A 1-42(-)A 1-42(+)A 1-42(+) A 1-42(-) A 1-42(-)A 1-42(+)A 1-42(+) A 1-42(-) A 1-42(-)A 1-42(+)A 1-42(+) Ratio(-) Ratio(+) Ratio(-) Ratio(+) Ratio(-) Ratio(+) Ratio(-) Ratio(+) Ratio(-) Ratio(+) Ratio(-) Ratio(+) Fig. 2 Levels of tTau (A), pTau181 (B), and NfL (C) in CSF according to their amyloid profile Aβ1–42/Aβ1–40 ratio is more strongly associated measures by amyloid profiles. As shown in Fig. 4, after with cognitive and functional progression than Aβ1–42 adjusting by baseline MMSE score, baseline age, sex, We studied the association of amyloid profiles with cog- years of education, pTau181 levels, APOEε4 status and nitive and functional progression in patients with MCI diagnosis, participants with low Aβ1–42/Aβ1–40 ratio (n = 794) through Kaplan-Meier survival curves and presented larger decreases in MMSE scores. The model age- and sex-adjusted Cox regression analysis. Sup- estimated an annual decrease of − 1.32 (− 1.55 to − 1.09) plementary Table 3 describes the characteristics of this points when Aβ1–42 was low and of − 0.86 (− 1.18 to subgroup. Due to specific particularities of the clinical − 0.55) points when Aβ1–42 was in the normal range. and cognitive assessment in the context of intellectual However, the annual change in MMSE scores in the two disability, participants with Down syndrome were not groups with normal Aβ1–42/Aβ1–40 ratio was not sig- included in this analysis. As displayed in Fig. 3, we found nificantly different from zero. As displayed in Fig. 4, we that patients with MCI with a low Aβ1–42/Aβ1–40 ratio also found that both groups with low Aβ1–42/Aβ1–40 had worse cognitive outcomes reflected by an earlier had larger annual decreases in the FCSRT total score, decline in MMSE scores. Compared to the Aβ1–42[−] estimated in − 1.21 in Aβ1–42[−]Ratio[+] and − 1.5 in Ratio[−] group, the adjusted risk of presenting a MMSE Aβ1–42[+]Ratio[+], compared to 0.05 in Aβ1–42[−] score lower than 24 during follow-up was 1.77 (1.25– Ratio[−] and 0.1 in Aβ1–42[+]Ratio[−]. Positivity in 2.49) times higher in the Aβ1–42[−]Ratio[+] group, 1.78 the amyloid ratio was also associated to larger annual (1.33–2.39) times higher in the Aβ1–42[+]Ratio[+], but increases in the cognitive-functional scale CDR-SOB, of not different in the Aβ1–42[+]Ratio[−] group (p = 0.28). 0.46 (0.22–0.71) in Aβ1–42[−]Ratio[+] and 0.65 (0.46- Similarly, the adjusted risk of progression to demen- 0.85) in Aβ1–42[+]Ratio[+], compared to 0.27 (0.16– tia was 1.55 (0.96–2.50) times higher in the Aβ1–42[−] 0.39) in Aβ1–42[−]Ratio[−] and no significant changes Ratio[+] group and 2.07 (1.43–2.99) times higher in in Aβ1–42[+]Ratio[−]. the Aβ1–42[+]Ratio[+] group compared to that of the Aβ1–42[−]Ratio[−]. The adjusted risk of progression to Discussion dementia in the Aβ1–42[+]Ratio[−] group was not sig- The results of our study indicate that, compared to nificantly different from the Aβ1–42[−]Ratio[−] group Aβ1–42 alone, the Aβ1–42/Aβ1–40 CSF ratio is more (p = 0.26). strongly associated with tau markers and with cognitive We also fitted linear-mixed models to assess the and functional progression. Regardless of the Aβ1–42 changes in longitudinal cognitive and functional status, participants with low CSF Aβ1–42/Aβ1–40 ratio Delaby et al. Alzheimer’s Research & Therapy (2022) 14:20 Page 7 of 11 A Kaplan-Meier curves (outcome: MMSE<24) B Adjusted Cox curves (outcome: MMSE<24) 100% 1.00 Participants that remain stable (%) 75% 0.75 Probability of remaining stable 50% 25% 0.50 0% 0 1 2 3 4 5 Follow-up time (years) 0.25 Number at risk (censored) 332 (161) 105 (186) 61 (225) 37 (248) 18 (262) 9 (271) 141 (67) 36 (71) 24 (76) 9 (85) 2 (91) 1 (92) 28 (19) 6 (20) 2 (24) 0 (26) 0 (26) 0 (26) 0.00 285 (122) 87 (138) 50 (157) 18 (175) 11 (181) 4 (185) 0 1 2 3 4 5 0 1 2 3 4 5 Follow-up time (years) Follow-up time (years) A 1-42(-) A 1-42(-) A 1-42(+) A 1-42(+) Profile Ratio(-) Ratio(+) Ratio(-) Ratio(+) Fig. 3 Cognitive progression in patients with mild cognitive impairment according to their amyloid profile. A Kaplan-Meier curve and B age- and sex-adjusted Cox regression display the risk of cognitive progression of all four amyloid profiles (outcome defined as MMSE < 24) Fig. 4 Estimation of the annual change in cognitive and functional scores across amyloid profiles. Estimations of the annual change in MMSE (A), CDR-SOB (B), and FCSRT total score (C) were calculated through linear-mixed models adjusted by baseline MMSE score, baseline age, sex, years of education, pTau181 levels, APOEε4 status, and diagnosis. MMSE, Mini-Mental State Examination; CDR-SOB, Clinical Dementia Rating Sum of Boxes; FCSRT, free and cued selective reminding test Delaby et al. Alzheimer’s Research & Therapy (2022) 14:20 Page 8 of 11 showed a biochemical and clinical profile character- pathology, low Aβ1–42/Aβ1–40 ratio values, but not ized by increased levels of CSF tTau and pTau181, worse low Aβ1–42 levels alone, were associated with high con- functional prognosis and larger cognitive decline. Inter- centrations of markers of neurofibrillary pathology and estingly, when the Aβ1–42/Aβ1–40 ratio was normal, neurodegeneration (pTau181, tTau, and NfL). In other participants with low Aβ1–42 levels had a biochemical contexts (DLB, FTLD, and other diagnoses), low levels and clinical profile similar to that of participants with of Aβ1–42 were only associated with markers of neu- normal Aβ1–42. Our findings provide evidence suggest- rofibrillary pathology in the presence of reduced Aβ1– ing that the use of the Aβ1–42/Aβ1–40 ratio is less con- 42/Aβ1–40 ratio. Our findings indicate that the ratio is founded by other comorbidities or processes, and thus, more strongly associated to the AD pathophysiological this work favors the use of the Aβ1–42/Aβ1–40 ratio as process (both as main and comorbid pathology) and also a marker of AD in clinical laboratories and in clinical support the idea that the isolated reduction of Aβ1–42 trials. levels in CSF might reflect additional processes (such as The use of CSF Aβ1–42 alone as a marker of amyloid neuronal or synaptic loss) or that the chrono pathology plaques entails some limitations. Numerous studies of their changes along the disease is not identical. These have reported that low concentrations of Aβ1–42 can be results are in line with our previous study showing that found in some non-AD conditions such as prionopathies, the Aβ1–42/Aβ1–40 ratio presents a stronger correla- bacterial meningitis, inflammatory diseases, amyloid tion with cerebral amyloid burden than Aβ1–42 alone angiopathy, or frontotemporal dementia [10–14, 39], thus [22]. limiting its diagnostic accuracy. Different hypotheses Baseline levels of the Aβ1–42/Aβ1–40 ratio were also have been suggested to explain these findings. Among influential in the cognitive and functional outcomes of other possibilities, low levels of Aβ1–42 in these contexts participants in our study. We found that participants with could be in relation with a decrease in Aβ generation due low Aβ1–42/Aβ1–40 ratio had faster cognitive and func- to neuronal or synaptic loss [10, 15] or the consequence tional worsening, especially in the group with low Aβ1– of abnormal clearance through the blood-brain barrier 42 but also when Aβ1–42 was in the normal range. The [10]. As Aβ1–40 would be similarly affected by these pro- group with a low Aβ1–42/Aβ1–40 ratio also presented a cesses, the use of the Aβ1-42/Aβ1-40 ratio could com- more rapid decline in episodic memory measured by the pensate the reduction in these situations to some extent, free and cued selective reminding test. These findings thus being less influenced by these processes and reflect- support the use of the Aβ1–42/Aβ1–40 ratio over Aβ1– ing more accurately the presence of amyloid plaques. 42 alone in the prognostic assessment of patients with The stronger association of the Aβ1–42/Aβ1–40 ratio cognitive decline. to other AD markers (tau markers) is in line with these The major strengths of our study are the large sample hypotheses. Another limitation for the use of Aβ1–42 size and the inclusion of a variety of diagnoses. Another alone is that it is particularly sensitive to preanalytical relevant strength is the fact that the same standard oper- and analytical variations, such as changes in the mate- ating procedures were used in the processing and analy- rial of collection or storage tubes, number of freeze-thaw sis of all samples. Aβ1–42 and Aβ1–40 were measured cycles, and volume of aliquoted CSF for storage [33, 40, simultaneously, and we followed the same preanalyti- 41]. In our study, we took advantage of a large cohort of cal and analytical protocol in all samples, thus minimiz- subjects with various neurodegenerative and non-neuro- ing the impact of confounders, which are critical in the degenerative conditions where CSF was collected using case of amyloid-β peptides. Lastly, we replicated our the same preanalytical protocol and analyzed under the results by using different levels of cutoffs, defined as same standard operating procedures. We found that the percentiles from a cognitively normal population. This overall agreement between Aβ1–42 and the Aβ1–42/ approach allowed us to match pairs of cutoffs for Aβ1–42 Aβ1–40 ratio did not exceed 85% in the whole sample, and Aβ1–42/Aβ1–40 ratio that had comparable levels of regardless of the cutoffs definition, suggesting that both sensitivity and specificity. But we also acknowledge some markers might be tracking similar but not identical pro- limitations. cesses or that they are influenced differently by other factors. Limitations of the study We found that CSF concentrations of tTau and Despite the large sample size, extensive cognitive pTau181 were higher in the presence of low Aβ1–42/ repeated measures were not available for all participants, Aβ1–40 ratio, regardless of the Aβ1–42 status. This thus limiting the statistical power in the longitudinal association was present in the whole sample but also analysis. On the other hand, amyloid PET imaging was within each diagnostic group. Thus, in the groups of CN, only available in a reduced group of participants previ- AD, and Down syndrome, less likely affected by non-AD ously reported [22]. Delaby et al. Alzheimer’s Research & Therapy (2022) 14:20 Page 9 of 11 Conclusions of the study Availability of data and materials Raw anonymized data and code for the statistical analysis are available upon The present work highlights the importance of routinely reasonable request. All requests should be sent to the corresponding author measuring Aβ1–40 in the CSF in combination with Aβ1– detailing the study hypothesis and statistical analysis plan. The steering 42 to assess the Aβ1–42/Aβ1–40 ratio, as this measure committee of this study will decide whether data/code sharing is appropriate based on the novelty and scientific rigor of the proposal. All applicants will be reflects more accurately the presence of amyloid plaques asked to sign a data access agreement. and is a useful and robust tool for the diagnostic and prognostic evaluation of patients with cognitive decline. Declarations The fact that the Aβ1–42/Aβ1–40 ratio shows a stronger association than Aβ1–42 with markers of neurofibril- Ethics approval and consent to participate The ethics committee of Hospital Sant Pau approved all procedures included lary pathology and with cognitive and functional decline in this study following the standards for medical research in humans recom‑ strengths the utility of this ratio in the clinical context of mended by the Declaration of Helsinki. All participants or their legally author‑ symptomatic and preclinical AD but also to detect con- ized representatives gave written informed consent before enrolment in the study. comitant pathology in other neurodegenerative diseases. Consent for publication Supplementary Information All authors revised the manuscript for content and provided critical feedback. The online version contains supplementary material available at https://​doi.​ Competing interests org/​10.​1186/​s13195-​022-​00967-z. Daniel Alcolea is employed by Hospital de la Santa Creu i Sant Pau and received research grants from Pla Estratègic de Recerca i Innovació en Salut Additional file 1. Supplementary tables and figures. (PERIS SLT006/17/125) and from Instituto de Salud Carlos III (PI18/00435 and INT19/00016). He participated in the advisory boards from Fujirebio-Europe and Roche Diagnostics and received speaker honoraria from Fujirebio-Europe, Acknowledgements Roche Diagnostics, Nutricia, Zambon S.A.U., Esteve, and from Krka Farmacéu‑ We are grateful to all participants in the study and their families. We also tica S.L. thank all the clinical team members that were involved in the selection and Constance Delaby is employed by Université de Montpellier and CHU de assessment of participants in the SPIN cohort and the laboratory teams for Montpellier. Declarations of interest: none sample handling, biomarker analyses, and structural support. We also thank Teresa Estellés is employed by Biomedical Research Institute Sant Pau. Declara‑ Fujirebio for providing technical support and part of the reagents necessary to tions of interest: none complete the study. Nuole Zhu is employed by Hospital de la Santa Creu i Sant Pau. Declarations of interest: none Authors’ contributions Javier Arranz is employed by Biomedical Research Institute Sant Pau. Declara‑ DA and AL designed the study. TE, NZ, JA, IB, MC-I, II-G, MAS-S, MA, IS, MBS- tions of interest: none S, LV, SV, AS, MT, FB-V, RB, JF, AL, and DA acquired the data relevant for the Isabel Barroeta is employed by Hospital de la Santa Creu i Sant Pau. Declara‑ study. MC-I, II-G, MAS-S, OB, RB, JF, AL, and DA contributed vital reagents/ tions of interest: none tools/patents. MC-I, II-G, MAS-S, OB, RB, JF, AL, and DA obtained funding María Carmona-Iragui is employed by Hospital de la Santa Creu i Sant Pau. for the study. DA and CD performed the statistical analysis. DA, CD, and Declarations of interest: none AL contributed to the analysis and interpretation of the data. DA and AL Ignacio Illán-Gala is supported by the Global Brain Health Institute (Atlantic participated in the study supervision or coordination. CD and DA drafted Fellow for Equity in Brain Health and pilot award for global brain health lead‑ the first version of the manuscript. The authors read and approved the final ers GBHI ALZ UK-21-720973) and the “Juan Rodés” grant from the Institute of manuscript. Health Carlos III (JR20/00018). Miguel Santos-Santos is employed by Hospital de la Santa Creu i Sant Pau. He Funding is funded by a “Juan Rodés” research grant from the Institute of Health Carlos This study was supported by the Fondo de Investigaciones Sanitario (FIS), III. Instituto de Salud Carlos III (PI14/01126, PI17/01019, and PI20/01473 to Miren Altuna is employed by Biomedical Research Institute Sant Pau. Dr. JF; PI13/01532 and PI16/01825 to RB; PI18/00335 to MCI; PI18/00435 and Altuna is funded by a “Río Hortega” research grant from the Institute of Health INT19/00016 to DA; PI17/01896 and AC19/00103 to AL) and the CIBERNED Carlos III. program (Program 1, Alzheimer Disease to AL), jointly funded by Fondo Isabel Sala is employed by Hospital de la Santa Creu i Sant Pau. Declarations of Europeo de Desarrollo Regional, Unión Europea, “Una manera de hacer interest: none Europa.” M. Belén Sánchez-Saudinós is employed by Biomedical Research Institute Sant This work was also supported by the National Institutes of Health (NIA grants Pau. Declarations of interest: none 1R01AG056850-01A1, R21AG056974, and R01AG061566 to JF), by Generalitat Laura Videla is employed by Fundació Catalana Síndrome de Down. Declara‑ de Catalunya (2017-SGR-547 and SLT006/17/125 to DA, SLT006/17/119 to JF, tions of interest: none SLT002/16/408 to AL) and “Marató TV3” foundation grants 20141210 to JF, Sílvia Valldeneu is employed by Biomedical Research Institute Sant Pau. Decla‑ 044412 to RB, and 20142610 to AL. This work was also supported by a grant rations of interest: none from the Fundació Bancaria La Caixa to RB (DABNI project). Fundació Catalana Andrea Subirana is employed by Biomedical Research Institute Sant Pau. Síndrome de Down and Fundació Víctor Grífols i Lucas partly supported this Declarations of interest: none work. The Horizon 21 Consortium is partly funded by Jérôme Lejeune Founda‑ Mireia Tondo is employed by Hospital de la Santa Creu i Sant Pau. Declarations tion (Clinical and trial outcome measures for dementia in individuals with of interest: none Down syndrome). Francisco Blanco-Vaca is employed by Hospital de la Santa Creu i Sant Pau. Part of the reagents necessary to complete the study was funded by Declarations of interest: none. Fujirebio-Europe. Sylvain Lehmann is employed by the University and the Hospital of Montpel‑ The sponsors of the study did not take part in the design and conduct of lier. He participated in advisory boards from Fujirebio-Europe and Roche. the study; collection, management, analysis, and interpretation of the data; Olivia Belbin is employed by Biomedical Research Institute Sant Pau. Dr. Belbin writing and review of the report; or the decision to submit the article for is funded by a “Miguel Servet” research grant from the Institute of Health publication. Carlos III. Delaby et al. Alzheimer’s Research & Therapy (2022) 14:20 Page 10 of 11 Rafael Blesa is employed by Hospital de la Santa Creu i Sant Pau and received in Psychiatry of the World Federation of Societies of Biological Psychiatry. research grants from Institute of Health Carlos III, Fundació Bancària Obra World J Biol Psychiatry. 2018;19:244–328. Social La Caixa, and Fundació La Marató de TV3. He participated in the 11. Iadanza MG, Jackson MP, Hewitt EW, Ranson NA, Radford SE. A new era advisory boards from Lilly and Nutricia, and he received speaker honoraria and for understanding amyloid structures and disease. Nat Rev Mol Cell Biol. travel funding from Novartis and Nutricia. 2018;19(12):755–73. Juan Fortea is employed by Hospital de la Santa Creu i Sant Pau and received 12. Frost B, Diamond MI. Prion-like mechanisms in neurodegenerative research grants from Institute of Health Carlos III, National Institutes of Health, diseases. Nat Rev Neurosci. 2010;11:155–9. Fundació La Marató de TV3, and Pla Estratègic de Recerca i Innovació en Salut 13. Spies PE, Slats D, Sjögren JMC, Kremer BPH, Verhey FRJ, Rikkert MGMO, (PERIS). Dr. Fortea has served as a consultant for Novartis and Lundbeck; has et al. The cerebrospinal fluid amyloid beta42/40 ratio in the differentia‑ received honoraria for lectures from Roche, NovoNordisk, Esteve, and Biogen; tion of Alzheimer’s disease from non-Alzheimer’s dementia. Curr Alzhei‑ and served at advisory boards for AC Immune, Zambon, and Lundbeck. mer Res. 2010;7:470–6. Alberto Lleó is employed by Hospital de la Santa Creu i Sant Pau and received 14. Llorens F, Schmitz M, Karch A, Cramm M, Lange P, Gherib K, et al. research grants from CIBERNED, Institute of Health Carlos III, Generalitat de Comparative analysis of cerebrospinal fluid biomarkers in the differ‑ Catalunya (PERIS and AGAUR), and Fundación BBVA. He participated in the ential diagnosis of neurodegenerative dementia. Alzheimers Dement. advisory boards from Fujirebio-Europe, Novartis, Roche Diagnostics, Otsuka 2016;12:577–89. Pharmaceutical, Nutricia, Zambón S.A.U., and Biogen and received speaker 15. Spies PE, Verbeek MM, van Groen T, Claassen JAHR. Reviewing reasons honoraria from Lilly, Biogen, KRKA, and Zambon. for the decreased CSF Abeta42 concentration in Alzheimer disease. Front Biosci (Landmark Ed). 2012;17:2024–34. Author details 16. Lehmann S, Delaby C, Boursier G, Catteau C, Ginestet N, Tiers L, et al. 1 Sant Pau Memory Unit, Department of Neurology, Institut d’Investigacions Relevance of Aβ42/40 ratio for detection of Alzheimer disease pathology Biomèdiques Sant Pau - Hospital de Sant Pau, Universitat Autònoma de in clinical routine: the PLMR Scale. Front Aging Neurosci. 2018;10:138. Barcelona, Sant Antoni Maria Claret 167, 08025 Barcelona, Spain. 2 IRMB, 17. Hansson O, Lehmann S, Otto M, Zetterberg H, Lewczuk P. 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