Late-onset Alzheimer’s disease (AD), the leading cause of dementia worldwide, is projected to affect more than 150 million individuals globally by 2050, with the increase mainly driven by population growth and ageing [1]. AD diagnosis is made clinically based on a syndrome of progressive cognitive impairment, typically with a predominant amnestic profile, though the constellation of symptoms can vary considerably. Diagnosis can be supported with the use of biomarkers, such as those derived from biofluids and brain imaging. However, there is significant global variation in the use of these ancillary investigations partly due to the invasiveness of some tests, such as obtaining cerebral spinal fluid (CSF), and the accessibility of advanced imaging including magnetic resonance imaging (MRI) and positron emission tomography (PET) [2]. The clinical interpretation of these biomarkers within different disease stages and in the setting of comorbid cerebral disease also remains challenging. These barriers have thus far prevented the widespread implementation of biomarkers into a unified diagnostic pathway for AD, though there is a movement towards defining and diagnosing AD biologically through the presence of biomarkers [3].

2. Blood-Based Protein Biomarkers as a Measure of Dynamic Risk

While considerable progress has been made in uncovering the genetic architecture of AD through large GWASs, the resulting PRSs represent a static risk that is embedded within an individual’s genetics. The analysis of more dynamic biomarkers may provide further insights into the real-time risk of AD at a particular stage of life. Technological improvements in biomarker detection have led to improved utility for AD diagnosis. In particular, ultrasensitive detection is possible with the use of single molecule array (Simoa) and mass spectrometry, allowing for the quantification of ultralow concentrations (pico- and femtomolar ranges) ^[4][5][6][4,60,61]. Blood levels of AD-implicated proteins such as neurofilament light chain (NfL), Aβ and tau (including hyperphosphorylated species) demonstrate strong concordance with CSF levels ^[7][8][9][62,63,64], thereby increasing their utility in diagnosing AD. Potential applications of different protein blood biomarkers in assessing AD risk are discussed below.

2.1. Neurofilament Light Chain

NfL is a cytoskeletal protein found predominantly in neuronal axons with a role in axonal growth and stability, with CSF and blood levels increasing after axonal damage ^[10][65]. Elevated plasma NfL levels are detected in a wide range of neurodegenerative diseases ^[11][66] including AD ^[12][13][67,68], frontotemporal dementia (FTD) ^[14][69], amyotrophic lateral sclerosis ^[15][70] and HIV-associated dementia ^[16][71]. However, NfL can also be elevated in acute non-neurodegenerative causes of brain injury including stroke ^[17][72] and encephalitis ^[18][73], highlighting that clinical context is important for biomarker interpretation. The role of NfL may be in discriminating between neurological disorders and psychiatric disorders, both of which can present with cognitive and memory dysfunction, a common clinical dilemma ^[19][20][74,75]. The ‘real world’ utility of blood NfL levels for assessing AD risk has been studied in memory clinics. In one prospective study of over 100 patients assessed in a tertiary memory clinic, physicians found knowledge of serum NfL levels diagnostically useful in patients under 62 years of age (60% useful) and in male patients (62% useful), as well as in those with a diagnostic uncertainty (67% vs. 51% useful in those patients with no diagnostic uncertainty) ^[21][76]. These findings were independent of knowledge of CSF NfL levels, suggesting that there is growing confidence in the use of blood NfL levels amongst physicians. Analysis of plasma NfL levels in over 550 patients with established diagnoses in a retrospective memory clinic study also found higher values in those with neurodegenerative conditions compared to non-neurodegenerative conditions ^[22][77]. Moreover, the increase in NfL levels correlates with the degree of cognitive impairment (higher levels seen in dementia compared to those with MCI, which was in turn higher than those with subjective cognitive impairment) ^[22][23][24][77,78,79]. However, the addition of plasma NfL levels to a diagnostic model based on clinical factors (age, cognitive test scores and APOE status) does not significantly increase the area under the receiver operating characteristic curve (AUC, 0.83 [0.78–0.87] versus 0.81 [0.77–0.85] for a model without NfL levels) for discriminating neurodegenerative from non-neurodegenerative conditions in patients with established cognitive impairment, suggesting that knowledge of blood NfL level may not be as relevant once substantial symptoms have developed ^[22][77]. Further, longitudinal blood NfL levels are not predictive of conversion to AD ^[25][26][80,81]. While NfL is emerging as an attractive biomarker for neurodegenerative conditions, its low specificity reduces its ability to serve as an AD-specific biomarker ^[4][27][4,82]. Moreover, cut-off values have not been well established for clinical use, though NfL levels increase in an age-dependent manner ^[27][28][82,83] and age-adjusted models have been proposed ^[29][84]. Importantly, the effects of ancestry on normative values for NfL are unclear with conflicting results ^{[20][30][31][32]}[75,85,86,87]. These open questions need to be addressed before NfL can be used robustly in a clinical setting to identify neurodegeneration ^[33][88].

2.2. Amyloid-Beta

Accumulation of Aβ plaques is a key component of AD pathology, with levels of Aβ1-42 changing decades before symptom onset. While a decrease in Aβ1-42 levels within the CSF has been robustly associated with AD ^[34][89], the association of blood levels was less clear due to concentrations being up to 100-fold lower in blood compared to CSF. Early meta-analyses of blood Aβ1-42 levels measured using plate-based immunoassays such as ELISA did not find significant differences between AD and healthy controls ^[35][90]. Measurements using mass spectrometry demonstrate a stronger correlation between plasma and CSF Aβ1-42 levels ^[36][37][91,92], suggesting that earlier equivocal results for Aβ likely reflect technological challenges rather than true pathobiology. Still, plasma Aβ levels and robustness differ significantly based on the assay used, limiting widespread uptake as a useful clinical blood biomarker ^[36][91]. To assess how plasma Aβ1-42 may serve as a biomarker for AD risk, a subgroup analysis of the Rotterdam study of over 450 older individuals (mean age of 68 years) found that lower plasma Aβ1-42 levels were associated with increased dementia incidence (HR = 1.27 [1.02–1.58]), especially among those individuals that were non-APOE ε4 carriers (HR = 1.47 [1.09–1.99]) ^[38][93]. In older individuals with subjective cognitive concerns but without a dementia diagnosis on the initial visit, a lower plasma Aβ42/40 ratio (value of Aβ1-42/Aβ1-40) demonstrated a steeper cognitive decline over a median follow-up of 3.9 years compared to those individuals with a higher Aβ42/40 ratio ^[39][40][94,95]. A lower plasma Aβ42/40 ratio is also seen in individuals with MCI who develop dementia compared to those who do not ^[41][96]. Moreover, individuals with the highest Aβ42/40 ratio have a significantly lower dementia risk (HR 0.52 [0.31–0.86]) over a 3-year period. Of note, plasma levels of Aβ (either as Aβ1-42, Aβ1-40 or Aβ42/40 ratio) do not significantly differ along the AD continuum (from cognitively unimpaired Aβ+ individuals to MCI to AD), indicating that it may not be useful in prognosticating disease progression ^[26][42][81,97]. As discussed above, Aβ deposition within the brain starts well before symptom onset. Detecting deposition non-invasively in vivo is possible with Aβ-PET emerging as a powerful imaging tool ^[43][98]. While diagnostically sensitive, Aβ-PET is resource intensive and not easily accessible in many countries. Plasma Aβ1-42 levels show excellent performance characteristics with high AUC values (above 0.9) in predicting Aβ-PET levels ^[44][45][99,100]. Consequently, new and efficient investigation pathways can be developed for individuals suspected for AD. For example, by using plasma Aβ to screen individuals with cognitive concerns and only proceeding to a Aβ-PET scan if plasma levels are abnormal, there would be over a 50% reduction in the number of PET scans needed to diagnose AD via PET imaging ^[46][47][101,102]. These findings highlight the potential role of blood Aβ measurements as a population-screening tool for AD, especially in those populations with a lower prevalence of AD. Aβ deposition is not restricted to AD and is detected in non-AD causes of dementia, including dementia with Lewy bodies (DLB), Parkinson’s disease dementia (PDD), FTD and vascular dementia (VaD). In these non-AD cases, Aβ levels, as measured by Aβ-PET, also increase with age and APOE ε4 carrier status but vary in cortical distribution relative to the underlying dementia diagnosis ^[48][49][103,104]. With respect to blood biomarkers, plasma Aβ levels also vary amongst non-AD dementia types and may be higher in VaD compared to AD ^{[50][51][52][53]}[105,106,107,108]. However, it is not well established how accurately blood Aβ levels, either as Aβ1-42 or as Aβ42/40, can discriminate between dementia subtypes early in the disease process and studies are needed to further investigate this.

2.3. Tau

Tau tangles, like Aβ, are a quintessential feature of AD, with tau-hyperphosphorylation leading to significant pathology ^[54][109]. Total-tau (t-tau) increases in the CSF and plasma following various causes of neuronal injury such as ischemic stroke and cardiac injury, as well as in neurodegenerative conditions including AD, DLB and FTD. The non-specific nature of elevated t-tau limits its ability to discriminate between AD and non-AD dementia. In contrast to CSF, blood t-tau levels reflect production from the central nervous system (CNS) as well as from peripheral tissues (such as liver, heart and kidney), explaining why blood t-tau levels are not considered diagnostic. Given that only one-fifth of plasma t-tau originates from the CNS ^[55][110], assays that specifically measure brain-derived tau levels are needed. By exploiting the fact that peripherally derived tau contains exon 4a, which is not found in CNS-derived tau109, a unique tau antibody has been developed to specifically measure plasma levels of brain-derived t-tau (BD-tau) ^[56][111]. Using this antibody, BD-tau levels correlate with CSF t-tau levels and are able to differentiate between autopsy-confirmed AD vs. non-AD cases (AUC = 0.86 [0.76–0.97]). When tested in memory clinics, BD-tau analysis is able to differentiate AD from non-AD neurodegenerative causes with AUC ranging from 0.78 (for progressive supranuclear palsy) to 0.99 (for the agrammatic variant of primary progressive aphasia due to a progranulin mutation). In addition to t-tau and BD-tau, there are nearly 100 known post-translational modifications of tau ^[54][109]. Some of the tau species are phosphorylated at unique threonine sites (p-tau) and have been found to be highly specific for AD ^[57][58][112,113]. The role of these p-tau species in AD prediction is highlighted below.

2.3.1. P-Tau181

Tau phosphorylated at threonine 181 (p-tau181) is one of the most studied tau species in AD. Using mass spectrometry and ultra-sensitive immunoassays, blood p-tau181 levels can differentiate between cognitively unimpaired individuals and those that have MCI or AD. In a prospective cohort study of 589 individuals from the Swedish BioFINDER cohort, plasma p-tau181 levels were strongly correlated with CSF p-tau181 levels, Aβ-PET and tau-PET ^[59][114]. Plasma p-tau181 was elevated in preclinical AD cases (individuals who were cognitively normal but with Aβ-PET positivity), and was able to discriminate between AD and non-AD dementia cases (AUC = 0.94 [0.90–0.99]). Similarly, in a retrospective North American cohort study of over 400 individuals, plasma p-tau181 levels were 3.5-fold higher in AD than cognitively unimpaired individuals and successfully discriminated between both clinically diagnosed and autopsy-confirmed AD and FTD cases (AUC = 0.87–0.89) ^[60][115]. The discriminatory power of blood p-tau181 was again demonstrated in a UK cohort of ^[60][115] individuals (AUC = 0.97 [0.94–1.00], autopsy-confirmed AD vs. non-AD dementia) ^[61][116] as well in separate North American and Swedish cohorts, with an AUC = 0.83–1.00 for AD vs. FTD and AUC = 0.92 for clinically diagnosed AD vs. VaD ^[62][117]. Importantly, p-tau181 tracks along the AD continuum (as measured by CSF Aβ levels and Aβ-PET load) and with cognitive decline, further supporting its role as a dynamic AD risk marker ^[26][42][81,97]. The robustness of plasma p-tau181 across ancestries was demonstrated by a prospective Spanish cohort study of 349 individuals (AUC = 0.96 for clinically diagnosed AD vs. cognitively unimpaired individuals) ^[63][118] and in a small Thai cohort study of 51 individuals (AUC = 0.84 [0.73–0.94]) ^[64][119]. However, p-tau181 performance was reduced among non-Hispanic White Americans (AUC = 0.69 [0.59–0.80]) and Black Americans (AUC = 0.63 [0.51–0.74]), and was considerably lower in Hispanic Americans (AUC = 0.51 [0.40–0.64]) ^[65][120]. More studies are needed to assess the generalizability of plasma p-tau181 amongst patients with different ancestral backgrounds.

2.3.2. P-Tau217

Other phosphorylated tau species are being investigated for their ability to discriminate between AD and non-AD dementia along the disease continuum. One such species is p-tau217 (phosphorylated at threonine 217), which has demonstrated improved performance in CSF, compared to CSF p-tau181, in distinguishing clinically diagnosed AD from non-AD dementia ^[66][67][121,122]. Similarly, plasma p-tau217 has also been found to better discriminate between autopsy-confirmed AD and non-AD cases (AUC = 0.89 [0.81–0.97]) compared to plasma p-tau181 (AUC = 0.72 [0.60–0.84]) ^[68][123]. Similarly, p-tau217 was shown to be superior to p-tau181 in discriminating between clinically diagnosed AD and non-AD cases (p-tau217 AUC = 0.96 [0.93–0.98], p-tau181 AUC = 0.81 [0.74–0.87], p < 0.001) as well as being more specific for Aβ-PET positivity than p-tau181 ^[55][110]. The specificity of p-tau217 for AD compared to other neurodegenerative tauopathies is further supported by a North American multicohort study where p-tau217 differentiated clinically diagnosed AD from FTD, with an AUC = 0.93 (0.91–0.96), compared to an AUC = 0.91 (0.88–0.94) for p-tau181 (p = 0.01) ^[69][124]. With respect to p-tau performances amongst individuals from different ancestral backgrounds, similar to p-tau181, plasma p-tau217 performed better in non-Hispanic White Americans (AUC = 0.71 [0.61–0.82]) and Black Americans (AUC = 0.68 [0.57–0.78]), but had a poor performance in Hispanic Americans (AUC = 0.52 [0.40–0.64]), considerably lower accuracy than those seen in European studies ^[65][120]. With respect to PET imaging, plasma p-tau217 levels can distinguish cognitively unimpaired individuals who are Aβ-PET positive from Aβ-PET negative cognitively unimpaired individuals even when the former individuals’ tau-PET scans are negative in the entorhinal cortex, a region involved early in AD-related tau pathology ^[70][125]. Furthermore, individuals with negative tau-PET imaging within the entorhinal cortex demonstrate a 2.2% increase per year of tracer uptake if they have higher baseline levels of plasma p-tau217. Interestingly, plasma p-tau217 levels in PSEN1 E280A mutation carriers (a form of autosomal dominant Alzheimer’s disease) are significantly altered at 24.9 years of age compared to non-carriers, which is approximately 20 years earlier than the expected age for symptom onset within the PSEN1 mutation carrier population ^[68][123]. This finding has been replicated in another cohort of cognitively unimpaired PSEN1 E280A mutation carriers, where plasma p-tau217 levels are higher compared to non-carriers and predict a higher burden of Aβ and tau as measured using PET imaging ^[71][126]. P-tau217 is also associated with AD progression risk. In cognitively unimpaired individuals who are Aβ-PET positive, a higher baseline plasma p-tau217 level confers an increased risk of conversion to clinically defined AD over a median follow-up time of 6 years (HR = 2.03 [1.57–2.63], p < 0.001]) ^[72][127]. Longitudinal plasma p-tau217 measurements demonstrate a greater increase in individuals with MCI who convert to AD compared to those who did not ^[73][128]. These studies demonstrate the potential utility of p-tau217 in identifying individuals at risk for AD well before symptoms and PET imaging changes emerge.

2.3.3. P-Tau231

P-tau231 (phosphorylated at threonine 231) is emerging as another potential blood biomarker which is highly sensitive for AD. Using a newly developed ultra-sensitive Simoa assay, p-tau231 was detected in all clinical stages of AD, including in individuals with MCI and sub-threshold signals in Aβ-PET, with levels increasing alongside disease progression ^[74][129]. Plasma p-tau231 is also seen in the early stages of AD and able to differentiate between Braak 0 (no deposition of Aβ plaque) and Braak I-II (Aβ plaque confined to the transentorhinal region) stages, which has not been observed with p-tau181 ^[74][75][129,130]. However, plasma p-tau231 is similar to p-tau181 in differentiating between AD and non-AD neurodegenerative cases, including limbic age-related TDP-43 encephalopathy and hippocampal sclerosis, both of which can clinically present similarly to AD ^[75][76][130,131]. Further studies are needed to elucidate the role of p-tau231 in dynamic AD risk profiling and how it differs from the other p-tau species. These studies highlight that novel blood tau antibody development, including different assay systems that target known p-tau species ^[77][132], can further improve the utility of p-tau and brain-specific tau as sensitive blood biomarkers for AD prediction.

2.4. YKL-40

The role of neuroinflammation in AD is becoming increasingly recognized ^[78][133]. YKL-40, also known as chitinase 3-like protein 1 (CHI3L1), is a highly conserved acute-phase glycoprotein involved in inflammation-activated remodeling and may be an indicator of neuroinflammation, but its exact function within the brain is not completely understood ^[79][134]. Interest in YKL-40 as a biomarker for AD was generated by early studies that found higher CSF YKL-40 levels in those with MCI and mild AD compared to cognitively unimpaired individuals; these levels also correlated well with CSF Aβ and p-tau181 ^[80][135]. Similarly, blood YKL-40 levels are higher in individuals with AD compared to healthy control, with levels increasing with disease severity ^{[81][82][83][84]}[136,137,138,139]. Interestingly, CHI3L1-associated SNPs correlated with blood protein levels and AD risk in a Han Chinese population ^[81][136]. Blood YKL-40 levels are also negatively correlated with structural (regional volume and cortical thickness) MRI changes in individuals with AD, but not with cognitive decline, suggesting it may serve as a generic marker of neurodegeneration ^[83][85][86][138,140,141]. The exact association of blood YKL-40 and AD needs further study as prospective analyses in cognitively unimpaired individuals suggest higher levels may be potentially protective (with reduced Aβ accumulation and improved cognitive testing) ^[87][142], while other studies suggest YKL-40 as a detrimental marker ^[88][89][143,144] and possibly specific to certain ethnic groups ^[90][145].

2.5. Soluble Triggering Receptor Expressed on Myeloid Cells 2

Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane receptor expressed in many immune-related cells, including CNS microglia ^[91][146]. Rare SNPs in this gene are second only to APOE in terms of magnitude of associated genetic risk of AD, with the R47H variant of TREM2 increasing AD risk two- to three-fold ^[92][93][147,148]. While there has been immense interest in TREM2 with respect to the pathogenesis of AD, its use as a peripheral biomarker has been recently proposed given the finding of an increase in blood TREM2 expression in AD patients compared to those cognitively unimpaired ^[94][95][149,150]. However, the association of AD status with levels of the soluble form of TREM2 (sTREM2) within the CSF and blood is inconsistent, possibly reflecting technological challenges in detecting this protein ^{[96][97][98][99][100]}[151,152,153,154,155]. Considerably more work is needed to determine if TREM2 and/or sTREM2 are robust biomarkers for AD risk.

2.6. Glial Fibrillary Acidic Protein

Glial fibrillary acidic protein (GFAP) is an abundant intermediate filament cytoskeletal protein highly expressed in astrocytes, with a role in neuro-inflammation and astrocyte reactivity. As such, GFAP’s use as an AD blood biomarker is promising as higher levels are found in individuals with AD compared to those cognitively unimpaired ^[101][102][156,157] and MCI ^[103][158]. Moreover, plasma GFAP levels predict conversion to AD in individuals with MCI over a 5 year period, independent of APOE ε4 status and age (AUC = 0.84 [0.77–0.91]) ^[104][159]. Plasma GFAP levels also correlate with Aβ-PET positivity ^[105][106][160,161], but not tau-PET ^[107][108][162,163], and can more accurately reflect CNS Aβ levels than other markers of inflammation such as YKL-40 or sTREM2 ^[107][162]. Interestingly, plasma GFAP may discriminate Aβ-PET positivity better than CSF GFAP (plasma AUC = 0.69–0.86 vs. CSF AUC = 0.59–0.76), as well as demonstrating a higher magnitude of change along the AD continuum ^[109][164]. Of note, a rise in plasma GFAP levels is also seen in Lewy body dementia ^[102][157] and FTD ^[110][165], suggesting that plasma GFAP levels are reflective of the reactive astrogliosis occurring in neurodegeneration more broadly. Nonetheless, the growing importance of GFAP in AD risk is evidenced by its potential inclusion in revised diagnostic criteria for AD [3].

2.7. Comparing Blood-Based Protein Biomarkers as Risk Predictors

As blood biomarkers demonstrate improved diagnostic performance, a natural question arises as to which of these protein biomarker(s) is/are most powerful in identifying AD risk. Recent studies have compared the various biomarkers at different stages along the AD continuum (Table 1). With the increasing rate of publications in this field, publicly available databases such as AlzBiomarker ^[111][166] provide updated biomarker meta-analyses, allowing for comparisons of multiple proteins of interest.