Abstract

Blood biomarkers have emerged as accurate tools for detecting Alzheimer’s disease (AD) pathology, offering a minimally invasive alternative to traditional diagnostic methods such as imaging and cerebrospinal fluid (CSF) analysis. Yet, the logistics surrounding venipuncture for blood collection, although considerably simpler than the acquisition of imaging and CSF, require precise processing and storage specific to AD biomarkers that are still guided by medical personnel. Consequently, limitations in their widescale use in research and broader clinical implementation exist. The DROP-AD project investigates the potential of dried plasma spot (DPS) and dried blood spot (DBS) analysis, derived from capillary blood, for detecting AD biomarkers, including phosphorylated tau at amino acid 217 (p-tau217), glial fibrillary acidic protein and neurofilament light. Here, 337 participants from 7 centers were included, with 304 participants providing paired capillary DPS or DBS and venous plasma samples. We observed strong correlations between DPS p-tau217 and venous plasma p-tau217 (rS = 0.74, P < 0.001). DPS p-tau217 progressively increased with increasing disease severity, and showed good accuracy in predicting CSF biomarker positivity (area under the curve = 0.864). Similarly, we demonstrated the successful detection of glial fibrillary acidic protein and neurofilament light with strong correlations between DBS and DPS, respectively, using paired venous plasma samples. Notably, the method was also effective in individuals with Down syndrome, a population at high genetic risk for AD but in whom standard blood sampling by venipuncture may be more complicated, revealing elevated biomarkers in those with dementia compared with asymptomatic individuals. The study also explored unsupervised blood collection, finding high concordance between supervised and self-collected samples. These findings underscore the potential of dried blood collection and capillary blood as a minimally invasive, scalable approach for AD biomarker testing in research settings. Yet, further refinement of collection and analytical protocols is needed to fully translate this approach to be viable and useful as a clinical tool.

Main

In less than a decade, the development of blood biomarkers for the identification of AD pathology has transitioned from a promising research endeavor to a valued tool that is now included in research diagnostic criteria1 and is increasingly being adopted in clinical practice. Phosphorylated tau at amino acid 217 (p-tau217) has emerged as an early and accurate AD blood biomarker2,3, offering higher accuracy compared with other putative blood biomarkers for detecting cerebral amyloid-β (Aβ) pathology4, a required hallmark for an AD diagnosis5,6. Several blood p-tau217 assays, spanning different immunological detection methods and mass spectrometry techniques7 are now available with some—but not all—meeting the recommended criteria for clinical usefulness, approved for clinical use, or currently under regulatory evaluation8. Thus, a cost-effective and timely tool is now available to identify individuals who may benefit from approved and emerging treatments or to monitor disease progression9. Specifically, the most likely clinical applications of blood p-tau217 will be based on a two-cutoff approach10 aimed at identifying people at either very high or very low risk of brain amyloidosis and for whom additional biomarker investigations are unnecessary, thereby lowering the need for positron emission tomography (PET) or CSF testing11. Other supportive blood biomarkers also offer insights into disease pathophysiology. Specifically, glial fibrillary acidic protein (GFAP), a marker of astrogliosis, has been associated with the onset of Aβ deposition12,13; and neurofilament light (NfL), a marker for axonal degeneration across neurodegenerative diseases14, has also been developed and is widely deployed in research and some clinical and therapeutic settings.

Although current guidelines recommend AD blood biomarker testing for symptomatic individuals15, there is also the potential to screen cognitively unimpaired (CU) older adults using a simplified test in a research setting and prevention strategies. This is because of the expectation of improved effectiveness of disease-modifying therapies targeting amyloid pathology in earlier disease stages, with trials currently ongoing16. Moreover, broader implementation of blood biomarkers will likely substantially advance the treatment, management and biological understanding of AD and related disorders in populations and communities currently underrepresented in research; for example, in individuals with Down syndrome (DS).

Substantial efforts have been made to ensure that blood tests become widely accessible, rather than confined to specialized laboratories. A major advancement in this area is the development of high-performing commercial and fully automated immunoassays for AD blood biomarkers, in particular p-tau21717. These fully automated immunoassays demonstrate performance identical or almost identical to immunoprecipitation mass spectrometry, as shown in a series of papers17,18,19,20,21. Although immunoprecipitation mass spectrometry remains difficult for widespread research and clinical implementation because of its high costs and limited instrument availability, automated immunoassays offer reliable and scalable solutions that address these limitations. Yet, for their global adoption to be fully realized, logistical challenges surrounding blood collection—such as the need for timely and standardized sample handling and storage22, as well as limited access to phlebotomy services—must be overcome to avoid constraining the impact of blood-based biomarker testing.

The DROP-AD project aims to streamline blood sample collection for larger-scale research, therapeutic trial enrollment and, potentially, clinical care, by introducing an alternative method that addresses the logistical challenges of traditional venipuncture blood collection and processing. By using capillary blood collected on DBS or DPS cards, the latter needed for p-tau217, reliance on guided venipuncture, immediate centrifugation and temperature-controlled shipment is eliminated. This approach enables a simplified, and potentially self-administered, protocol using fingertip blood collection. We have previously demonstrated the feasibility of measuring AD biomarkers from dried blood spots23, with the blood source being venous, followed by manual transfer onto a card for shipping and storage advantages. Here we extend this previous work by evaluating the feasibility of remote biomarker assessment using capillary blood, obtained by fingerstick collection, thus potentially self-collected and remote. A total of 337 participants were recruited across 7 European centers to assess the quantification of key AD pathology and neurodegeneration biomarkers—plasma p-tau217, NfL and GFAP—from capillary-derived blood collected from the finger. These values were directly compared with those obtained by standard venous plasma sampling, as well as to CSF biomarker concentrations routinely used in clinical diagnostics.

Results

We recruited 337 participants (mean (s.d.) age, 70.8 (11.7) years; 167 women (53.4%) (Table 1) from 7 centers. Depending on the research site, and the evolution of the DROP-AD project, participants followed different capillary blood collection and testing procedures, which are summarized in Fig. 1. Cohort characteristics are shown in Supplementary Table 1.

Fig. 1: DROP-AD in-house collection and extraction protocol and testing procedures.

a, Collection and processing of venous plasma and capillary DPS and DBS samples. For DPS and DBS sample collection, a finger prick was carried out by trained study personnel and a few drops of capillary blood were spotted onto DPS and DBS collection devices. DPS and DBS were collected via semi-automated spot collectors and incubated with analyte-specific extraction buffer in a 96-well filter plate. After incubation and centrifugation, the eluate was immediately measured using ultrasensitive immunoassays on the single molecule array platform. b–f, Participant numbers and collection device numbers per cohort: capillary p-tau217 (b), capillary GFAP (c), capillary NfL (d), DS cohort (e) and self-sampling cohort (f). Panel a created using BioRender.com.

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Correlation of capillary DPS p-tau217 with venous plasma p-tau217

In total, 252 participants (mean (s.d.) age, 72.7 (9.0) years; 143 women (56.7%)) provided paired capillary DPS samples and venous plasma samples. We found a high correlation between DPS p-tau217 and venous plasma p-tau217 across all merged cohorts (Spearman’s rank correlation (rS) = 0.74, 95% confidence interval (CI) 0.678–0.791; P < 0.001) (Fig. 2a). The strength of this correlation varied among participating centers (Extended Data Fig. 1) and was highest in the Gothenburg cohort (rS = 0.904, 95% CI 0.731–0.968; P < 0.0001) and the Brescia cohort (rS = 0.838, 95% CI 0.694–0.917; P < 0.0001), followed by the Exeter (rS = 0.765, 95% CI 0.530–0.891; P < 0.0001) and Barcelona (rS = 0.735, 95% CI 0.646–0.805; P < 0.0001) cohorts, and the Malmö cohort (rS = 0.429, 95% CI 0.159–0.640; P < 0.001), the only cohort in which a different assay (Lilly2) for venous plasma was used. To further demonstrate the strength the relationship between capillary and venous blood, we stratified plasma p-tau217 concentrations into tertiles and computed Spearman correlations in each tertile, allowing us to assess agreement at low, medium and high concentrations (Extended Data Fig. 2). Significant correlations were observed for p-tau217 concentrations in tertile two (rS = 0.51; P < 0.0001) and tertile three (rS = 0.62; P < 0.0001), but no relationship in tertile one, where all participants were CSF Aβ42/p-tau181 negative. The strength of the relationship between capillary and venous blood was not confounded by age or sex (Supplementary Table 2).

Fig. 2: Correlations between capillary p-tau217 with venous plasma p-tau217, cognition and age.

a, Correlation between capillary p-tau217 and venous plasma p-tau217 (n = 252). Dots correspond to individual data points. b, Correlation between capillary p-tau217 and MMSE (n = 209). Left: capillary p-tau217. Right: venous plasma p-tau217. c, Correlation between capillary p-tau217 and age (n = 249). Left: capillary p-tau217. Right: venous plasma p-tau217. A mean regression line is presented in all panels, with ribbons representing 95% CI. For numerical representation of the correlation, we present Spearman coefficients alongside their P values. Statistical tests were two-sided.

Source data

Next, we investigated the association of capillary biomarkers with cognitive testing. DPS p-tau217 showed significant correlations with both Mini Mental State Evaluation (MMSE) (n = 209; rS = −0.374, 95% CI −0.485 to −0.251; P < 0.0001) (Fig. 2b) and age (n = 249; rS = 0.334, 95% CI 0.219–0.440; P < 0.0001) (Fig. 2c), which were similar to the correlations of venous plasma p-tau217 with MMSE (n = 209; rS = −0.410, 95% CI −0.517 to −0.290; P < 0.0001) (Fig. 2b) and age (n = 249; rS = −0.317, 95% CI 0.200 to 0.424; P < 0.0001) (Fig. 2c).

Diagnostic accuracy of capillary DPS p-tau217

DPS p-tau217 was significantly increased in clinically defined mild cognitive impairment (MCI) and AD (no biomarker classification) compared with CU participants and clinically defined non-AD dementias (Fig. 3a). Next, we investigated the discriminative accuracy of DPS p-tau217 to detect abnormal CSF biomarkers. In participants with DPS p-tau217, venous plasma p-tau217 and CSF Aβ42/p-tau181 (n = 176; mean (s.d.) age, 74.6 (7.9) years; 102 women (58.0%)), capillary DPS p-tau217 was significantly increased (+198%; P < 0.001) in the AD CSF biomarker-positive group (Fig. 3b). DPS p-tau217 had an area under the curve (AUC) of 0.863 (95% CI 0.809–0.917); however, this was significantly lower than venous plasma p-tau217 which had an AUC of 0.982 (95% CI 0.968–0.996; P < 0.0001) (Extended Data Fig. 3) in the same participant subsample. We also show the distribution of capillary p-tau217 across clinico-biological diagnostic groups (Fig. 3c), which shows a similar pattern to venous derived p-tau217, with similar statistical significance across groups (Extended Data Fig. 4). Results demonstrating DPS p-tau217 against CSF Aβ42/Aβ40 as the standard of truth are shown in Extended Data Fig. 5. Next, we tested the accuracy of capillary DPS p-tau217 to determine abnormal venous plasma p-tau217 (n = 252), which had predetermined cutoff validated against Aβ-PET (ALZpath single molecule array (Simoa) > 0.42 pg ml−1)3. DPS p-tau217 was more concordant with venous plasma p-tau217 and was increased by 217% in individuals with venous plasma p-tau217 > 0.42 pg ml−1, compared with individuals with venous plasma p-tau217 ≤ 0.42 pg ml−1, and had a discriminative accuracy to detect abnormal venous plasma p-tau217 of 0.868 (95% CI 0.825–0.911) (Extended Data Fig. 6).

Fig. 3: Capillary p-tau217 levels and their relationship to clinical diagnosis and CSF AD pathology status.

a, Relationship between capillary p-tau217 level and diagnostic groups (CU, n = 37; MCI, n = 92; AD, n = 45; non-AD, n = 30). Horizontal solid-line bars represent group-wise comparisons alongside P values, obtained from post-hoc contrasting of a linear model adjusted for age and sex. b, Relationship between capillary p-tau217 level and CSF p-tau181/Aβ42 status (CSF-negative, n = 71; CSF-positive, n = 93). In addition to the P value, the mean fold-change between groups is presented. c, Relationship between capillary p-tau217 level and clinico-biological groups (CU Aβ−, n = 13; CU Aβ+, n = 1; MCI Aβ+, n = 49; AD Aβ+, n = 37; non-AD Aβ+, n = 6; MCI Aβ−, n = 39; AD Aβ−, n = 2; non-AD, n = 17), defined by clinical syndrome in conjunction with the CSF p-tau181/Aβ42 status, which is a validated metric for Aβ-positivity. In all panels, individual data points for each participant are shown and an overlaid boxplot represents group-wise distributions. Boxplots show the median (center line), interquartile range (IQR; box limits, 25th–75th percentiles), whiskers extending to the most extreme values within 1.5× IQR from the quartiles. A mean regression line is presented with ribbons representing 95% CI. Statistical tests were two-sided, and for group comparisons Tukey’s adjustment was used.

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Capillary DPS cutoffs based on CSF biomarker positivity

Exploratory diagnostic accuracy metrics were derived in a subset of individuals (n = 176) with paired capillary and CSF Aβ42/p-tau181 metrics, at a prevalence of 56.3% of CSF biomarker positivity. A capillary p-tau217 cutoff of 0.01 pg ml−1, with 90% sensitivity for abnormal CSF Aβ42/p-tau181, led to a positive predictive value (PPV) of 0.738 (95% CI 0.653–0.808) and a negative predictive value (NPV) of 0.833 (95% CI 0.713–0.910), at a specificity of 58.4%. A capillary cutoff of 0.02 pg ml−1, with 90% specificity for abnormal CSF Aβ42/p-tau181, led to a PPV of 0.884 (95% CI 0.789–0.940) and an NPV of 0.645 (95% CI 0.551–0.729), at a sensitivity of 66.7%. A Youden’s J statistic capillary cutoff (0.016 pg ml−1) led to a PPV of 0.849 (95% CI 0.758–0.909) and an NPV of 0.711 (95% CI 0.610–0.795), at a sensitivity of 73.7% and specificity of 83.2%. When combining the 90% sensitivity cutoff (0.01 pg ml−1) with the 90% specificity cutoff (0.02 pg ml−1) in a two-cutoff approach, the NPV of the lower cutoff was, as above, 0.833 (95% CI 0.713–0.910) and the PPV of the upper cutoff was 0.884 (95% CI 0.789–0.940), reaching an overall accuracy for those below the lower cutoff and above the upper cutoff of 86.2% (95% CI 78.8–91.7%), with 53 of the 176 individuals falling in the intermediate zone, which corresponded to 30.1% of the evaluated participants.

GFAP and NfL in capillary DBS

Based on 240 individuals (mean (s.d.) age, 69.9 (10.8) years; 99 women (48.8%)) measured using at least one of the three candidate DBS methods, we found that B50 and Telimmune collection cards were most compatible for GFAP and were combined for this analysis (Extended Data Fig. 7B). When comparing GFAP levels from capillary samples with venous plasma, a strong correlation was found (r = 0.773, 95% CI 0.710–0.823; P < 0.0001) (Fig. 4a). We also observed a similar correlation of capillary GFAP and venous plasma GFAP with age (capillary GFAP, r = 0.392, 95% CI 0.261–0.509; P < 0.0001; venous plasma GFAP, r = 0.276, 95% CI 0.135–0.405; P < 0.0001) (Fig. 4b) and MMSE (capillary GFAP, r = −0.448, 95% CI −0.558 to −0.324; P < 0.0001; venous plasma, r = −0.436, 95% CI −0.547 to −0.310; P < 0.0001) (Fig. 4c).

Fig. 4: Associations between capillary GFAP and NfL with venous plasma assay, cognition and age.

a, Association between venous plasma GFAP and capillary GFAP (n = 203). b,c, Association between MMSE score and blood GFAP (n = 181) (b), and between age and blood GFAP (n = 181) (c). Left: capillary GFAP levels. Right: venous plasma GFAP levels. d, Association between venous plasma NfL and capillary NfL for the Barcelona cohort (n = 71). e,f, Association between MMSE score and blood NfL (n = 71) (e), and between age and blood NfL (n = 71) (f). Left: capillary NfL levels. Right: venous plasma NfL levels. A mean regression line is presented in all panels, with ribbons representing 95% CI. For numerical representation of the correlation, we present Spearman coefficients alongside their P values. Statistical tests were two-sided, and for group comparisons Tukey’s adjustment was used.

Source data

Based on a set with 237 individuals measured with at least one of the three DBS method candidates, only Telimmune DPS cards were useful in examining capillary NfL using our protocol (Extended Data Fig. 7C). Therefore, we examined 72 participants for NfL using Telimmune DPS cards (mean (s.d.) age, 76.4 (7.0) years; 45 women (62.5%)). When comparing NfL levels from capillary DPS to venous plasma, a strong correlation was observed (r = 0.83, 95% CI 0.743–0.892; P < 0.0001) (Fig. 4d). Similarly to GFAP, we observed similar correlations between DPS and venous plasma NfL in relation to age (capillary DPS, r = 0.429, 95% CI 0.219–0.601; P < 0.001; venous plasma, r = 0.524, 95% CI 0.333–0.674; P < 0.0001) (Fig. 4e) and MMSE (capillary DPS, r = −0.269, 95% CI −0.471 to −0.039; P = 0.02; venous plasma, r = −0.367, 95% CI −0.552 to −0.148; P < 0.001) (Fig. 4f).

DPS p-tau217 and DBS GFAP in individuals with Down syndrome

We examined 31 participants with DS and DBS biomarker data. As with the euploid participants, we found a significant relationship between biomarkers measured in capillary blood and venous blood (p-tau217, r = 0.875, 95% CI 0.503–0.973 (Fig. 5a) GFAP, r = 0.629, 95% CI 0.347–0.806 (Fig. 5c)). Capillary biomarker levels were, as also shown in venous plasma (Fig. 5e,f), increased in DS with dementia (dDS), compared with DS without AD-related cognitive impairment (aDS), for both p-tau217 (Fig. 5b) and GFAP (Fig. 5d). Participants positive for CSF p-tau181/Aβ42 more often had higher levels of capillary GFAP (Fig. 5f), although there were not sufficient participants with DS and DBS p-tau217 and CSF biomarker data (n = 5, CSF-negative only). Telimmune cards were not collected in this study, so no NfL results were obtained.

Fig. 5: Associations between capillary p-tau217 and GFAP in participants with DS.

a, Scatterplot representing the association between capillary and venous plasma p-tau217 in the DS Barcelona cohort, alongside their Spearman correlation coefficient and associated P value (n = 9). b, Boxplots of capillary p-tau217 based on cognitive diagnosis (aDS, n = 4; pDS, n = 1; dDS, n = 4). c, Scatterplot representing the association between capillary and venous plasma GFAP, with a Spearman correlation coefficient and its associated P value presented (n = 30). d, Boxplots of capillary GFAP based on cognitive diagnosis (aDS, n = 18; pDS, n = 2; dDS, n = 9). e, Boxplots of venous plasma GFAP based on cognitive diagnosis (aDS, n = 21; pDS, n = 3; dDS, n = 9). f,g, Boxplots of capillary GFAP (f) and venous plasma GFAP (g) based on CSF Aβ42/p-tau181 status (CSF-negative, n = 6; CSF-positive, n = 7). For scatterplots, a mean regression line is presented with 95% CI. All boxplots show the median (center line), IQR (box limits, 25th–75th percentiles), whiskers extending to the most extreme values within 1.5× IQR from the quartiles. When group comparisons are presented with boxplots, horizontal solid-line bars represent group-wise comparisons alongside P values, obtained from post-hoc contrasting of a linear model adjusted for age and sex. Statistical tests were two-sided, and for group comparisons Tukey adjustment was used.

Source data

Supervised and unsupervised capillary DPS or DBS collection

In the previous result sections, all capillary DPS or DBS collection was supervised and guided by trained personnel. Here we evaluated the within-person difference if collection was supervised compared with unsupervised. In 30 participants, capillary blood guided by study personnel and self-collected unsupervised samples showed a very high concordance with little difference between timepoints (DPS p-tau217, 0.014 pg ml−1 versus 0.013 pg ml−1, P = 0.57 (Extended Data Fig. 8A); DBS GFAP, 10.1 pg ml−1 versus 11.0 pg ml−1, P = 0.26 (Extended Data Fig. 8B)). Because only one Telimmune card was sampled per participant dedicated to p-tau217 quantification, no NfL data were obtained.

Discussion

The DROP-AD project, constituting an effort to assess biomarkers for AD-type pathology and neurodegeneration from capillary blood, showcases the capability of quantifying p-tau217, GFAP and NfL protein levels. The study evaluated straightforward capillary blood collection methods, a new extraction protocol and ultrasensitive immunoassay biomarker determination. Biomarker levels from capillary blood correlated well with conventional venipuncture-collected plasma measures, and in the case of p-tau217, predicted with good accuracy, abnormal AD CSF biomarkers, as demonstrated in individuals classified as asymptomatic, MCI, dementia, as well as in individuals with DS, who are at high-risk for AD.

In blood, p-tau217 is the principal blood biomarker for determining AD pathology8 and is increasingly adopted as a reliable metric in research, clinical trials and clinical practice. It has the capabilities of high diagnostic accuracy to detect AD pathology, primarily amyloid3,24, but is also tightly associated with severity of tau pathology assessed by tangle counts at post-mortem examination2 and by tau PET during life25 not only in the symptomatic phase of the disease4, but also in the asymptomatic phase26. Therefore, p-tau217 holds promise not only for clinical use, but also population-level screening, identifying at-risk individuals in preclinical phases and enabling early intervention strategies27. Plasma p-tau217 has already been used to assess outcomes in secondary preventive trials16,28. A drawback in expanding blood biomarker testing outside specialized centers, is the strict protocol and guided venipuncture collection, sample handling and shipment. Dried blood sampling23 overcomes this limitation by enabling simplified, minimally invasive and potentially, remote self-collection, reducing the need for specialized personnel and facilitating broader population access to biomarker testing.

The DROP-AD project, conducted across multiple centers, highlights the strong potential of using dried capillary blood samples to accurately quantify plasma p-tau217. We observed robust correlations between p-tau217 concentrations measured from DPS and matched venous plasma samples, although the strength of these correlations varied by site. Importantly, p-tau217 levels showed a stepwise increase across clinical stages—CU, MCI and AD—and demonstrated good accuracy in predicting CSF biomarker-confirmed AD pathology. In addition to p-tau217, we successfully quantified GFAP and NfL using DBS and DPS matrices, respectively. Both GFAP and NfL showed high concordance between capillary and venous samples, and were similarly associated with cognitive performance and age, reinforcing the validity of these remote sampling methods. Although our primary focus was on biomarkers of AD neuropathology, the reliable detection of NfL from DPS samples has broader implications. Given its established role as a diagnostic, prognostic and monitoring biomarker, capillary-based NfL measurement could be transformative for other neurodegenerative and neurological conditions—including frontotemporal dementia, atypical parkinsonian syndromes, multiple sclerosis, amyotrophic lateral sclerosis and acute neurological injuries. Biomarker levels extracted from DPS or DBS cards, for all analytes of interest, were substantially lower than those quantified from venous plasma, which we believe is attributable to the elution of dried blood or plasma with buffer, resulting in dilution. Protein concentrations were not adjusted using a uniform dilution factor, because we cannot currently estimate the volume of plasma that is dried onto a card. Attempts to measure Aβ42 and Aβ40 using this technique yielded mixed results. Although Aβ40 was readily quantifiable, Aβ42 levels were mainly below the limit of detection and could not be included in the analysis, limiting the utility of this approach for this biomarker.

Imaging, CSF and blood-based biomarkers for AD pathology have shown strong translational applicability in individuals with DS, which represents the most common genetically determined form of AD29. Given the near-universal risk of AD in this population, there is a critical need for scalable and accessible methods to enable longitudinal biomarker monitoring, particularly in the context of preventive and disease-modifying clinical trials. Collection of blood samples by standard venipuncture may be complicated in individuals with DS—for example, due to relatively high rates of institutionalization and a lack of professionals—and remote blood collection thus offers a promising solution by reducing reliance on in-clinic visits and facilitating broader participation across diverse spectrum of intellectual disability. To evaluate the feasibility of this approach, we conducted a pilot study in which capillary blood samples were successfully collected from individuals with DS across a spectrum of cognitive stages. Our results revealed significantly elevated levels of capillary-derived GFAP and p-tau217 in participants with symptomatic AD compared with those who were cognitively asymptomatic. Importantly, biomarker concentrations derived from capillary samples showed strong concordance with those obtained from matched venous plasma, supporting the reliability and translational potential of remote sampling for biomarker quantification and ultimately, AD diagnosis, in this high-risk population.

This study has limitations. First, we have indicated that capillary blood collection may be useful in an unsupervised fashion, remotely. This has not been fully examined in this proof-of-principle study, where all capillary sampling was performed in research centers guided by trained staff. To gain some initial insights, we conducted a pilot in 30 participants who provided two capillary samples: one sampled by research staff and one unsupervised—1 h later. These initial findings demonstrate the reproducibility of both the collection method and the laboratory extraction procedure. Further, our venous plasma analyses were performed in single-batch analysis for all study sites, and this is particularly important to consider when comparing results directly to capillary testing, which was analyzed prospectively in multiple batches (less than 4 weeks from collection) throughout the 24-month study period. The observed lower accuracies to determine AD pathology by capillary p-tau217 could be partially attributed to this key difference in analytical design. This 24-month period also reflects a time of protocol optimization, in sample collection at multiple study sites and biomarker determination in the laboratory. Despite this optimization, we do experience a proportion of unsuccessful collections of capillary samples because of insufficient capillary blood flow, coagulation or technical issues during plasma separation in 15–25% of cases. This may, in part, reflect the inherent challenges of fingertip capillary blood sampling in clinical practice, where achieving consistent blood flow from a fingerstick collection is difficult and often complicated by hemolysis or admixture of interstitial fluid due to external compression of the fingertip30. We believe that diligent training of the study personnel and patients and/or caregivers and the provision of informational material is essential for successful collection of dried blood; however, alternative capillary blood collection methods—other than fingerstick—should be considered and examined given the encouraging finding from this study. Moreover, studies with larger cohorts are needed to investigate the impact of confounders on DPS or DBS biomarker levels.

In conclusion, our findings demonstrate that dried blood analysis offers a feasible and scalable approach for detecting AD pathology, particularly in research, population-based and epidemiological contexts. This minimally invasive method has the potential to substantially broaden our understanding of the prevalence and distribution of AD pathology across the general population, while also facilitating the inclusion of historically underrepresented populations and geographically diverse regions in AD research. However, despite the promise shown, we do not currently recommend the use of dried blood analysis for clinical use, decision-making or patient management, because of observed differences in analytical performance and diagnostic accuracy between capillary-derived and venous blood samples. Further methodological refinement and validation will be essential before clinical translation can be considered.

Methods

Study design

To evaluate the feasibility of capillary-derived blood as a simplified collection method compatible with AD biomarker analysis, paired venous plasma and capillary blood samples obtained by fingerstick were collected from CU and cognitively impaired individuals across seven European study centers. Capillary blood collection was conducted by trained study personnel at each site. Dried blood cards were shipped without temperature control to the Neurochemistry Laboratory at the University of Gothenburg, Sweden, within 1–40 days of collection. In parallel, venous plasma samples were stored at −80 °C at the respective study sites and shipped on dry ice to the same laboratory at the end of the study. Complementary CSF data (total n = 227; Barcelona, n = 131; Barcelona (DS), n = 13; Brescia, n = 7; Gothenburg, n = 13; Malmö, n = 40; Copenhagen, n = 23) and cognitive assessments (total n = 244; Barcelona, n = 130; Brescia, n = 32; Copenhagen, n = 24; Gothenburg, n = 14; Malmö, n = 44) were obtained from each site as part of routine clinical evaluations or existing research protocols.

Cohort characteristics

At each study site, all participants provided written informed consent before enrollment, and the studies were approved by local ethical review authorities. The inclusion criteria for each cohort are depicted below and summarized in Supplementary Table 1. Participants were not compensated for participation in this study. Biological sex was determined based on self-identification.

The Ace Alzheimer Center Barcelona, Spain (the ‘Barcelona’ cohort) included participants under investigation for cognitive complaints recruited between September 2022 and April 2024. At Fundació ACE, clinical diagnosis was carried out through a comprehensive neuropsychological evaluation using the NBACE battery31, assessment of functional status with the Clinical Dementia Rating (CDR) scale, and supported by biological diagnosis through CSF biomarkers following the AT(N) classification framework32. Individuals with MCI and dementia were offered a voluntary (and informed consented) lumbar puncture in accordance with established consensus recommendations. Venous plasma, CSF and capillary DPS or DBS samples were collected on the same day under fasting conditions. All biospecimens obtained were part of the ACE collection, which was registered in Instituto de Salud Carlos III (ISCIII, Ministry of Health of Spain) under the code C.0000299. Capillary DPS and DBS samples were stored and shipped at room temperature between 1 and 3 days after the collection. The study was approved by the Ethics Committees of the Hospital Universitari de Bellvitge, Barcelona (Ref. PR148/22). The H70 Clinical Studies (the ‘Gothenburg’ cohort) consecutively recruited participants under investigation for cognitive symptoms from the memory clinic at the Sahlgrenska University Hospital in Gothenburg, Sweden between June 2023 and April 2024. There were no exclusion criteria. Capillary DPS and DBS samples, venous plasma and CSF samples were collected at the same study visit. Cognitive testing (MMSE and CDR) was performed in each participant. Capillary DPS and DBS samples were stored at room temperature and delivered to the Neurochemistry Laboratory between 1 and 7 days after the collection. Ethical approval for H70 Clinical Studies was provided by The Swedish Ethical Review Authority (Etikprövningsmyndigheten; EPM: 2023-06137-02). In the BioFINDER Primary Care (NCT06120361) and BioFINDER Preclinical AD (NCT06121544) studies (the ‘Malmö’ cohort), cognitively asymptomatic volunteers (asymptomatic AD or healthy controls) and individuals with cognitive symptoms undergoing cognitive diagnostic evaluation in primary care were included between December 2023 and November 2024. The exclusion criteria were (1) not undergoing CSF or blood sampling as part of clinical practice and (2) not undergoing cognitive testing as part of clinical practice. Cognitive testing (MMSE) and CSF samples were available for each participant. Capillary DPS and DBS were collected at the same day as venous plasma samples, stored at room temperature and shipped to the Neurochemistry Laboratory between 1 and 7 days after the collection. The studies were approved by Swedish Ethical Review Authority (Dnr. 2021-05724-01 and 2019-04320). Participants enrolled at the Center for Neurodegenerative Disorders at the University of Brescia, Italy (the ‘Brescia’ cohort) met current clinical criteria for the diagnosis of fontotemperal dementia33,34 or AD35, or were healthy individuals recruited among spouses or family members. Consecutive recruitment took place between October 2023 and June 2024. Each participant underwent an extensive clinical and neuropsychological evaluation and simultaneous venous EDTA plasma and dried blood spot collection. Cognitive testing (MMSE and CDR) was available for each participant and CSF samples were collected in a subgroup. Capillary DPS and DBS samples were stored at room temperature and shipped to the Neurochemistry Laboratory between 1 and 30 days after the collection. The study was approved by the local ethics committee (NP2189 and NP1965). Participants enrolled at the University of Exeter Medical School (the ‘Exeter’ cohort) were adults aged 50 years or above with a body mass index >25 kg m−2 and within 2 h tra