In studies utilizing IMR published so far [7,8,9,10,11,12,13,14], the concentration of plasma samples of Aβ1–40, Aβ1–42 or T-Tau represented the averaged value of duplicate measurements. It has also been observed that plasma T-Tau is negatively correlated with hippocampal volume [12, 13]. Aβ1–42 × T-Tau or Aβ1–42/Aβ1–40 in plasma has been shown to discriminate NCs from subjects with aMCI and AD [8,9,10]. These results reveal the impacts of plasma biomarkers on clinical applications, such as in assisting in the diagnosis of AD, the prediction of cognitive decline in aMCI [23, 24], assessment of results from neurological imaging studies [11, 25] and the monitoring of interventions, among other impacts. The authors of an earlier study reported that there was a negative correlation between plasma and cerebral spinal fluid Aβ1–42 levels in patients with AD (r = − 0.352) , suggesting that IMR is no less useful than single molecule array (SIMOA; r = 0.288) testing in clinical applications . According to the results shown in Figs. 1 and 2, reliable measurements of plasma Aβ1–40, Aβ1–42 or T-Tau concentrations were achieved in a single test by using IMR. This result was the underlying motivation to investigate the associations of plasma biomarkers that were obtained in a single test with the use of MRI and clinical diagnoses.
Two subjects withdrew their consent after blood sampling and diagnosis based on the MRI scan. A total of 240 enrolled subjects underwent MRI scans to analyze the right hippocampal volume and left hippocampal volume (Table 2). Two rounds of single-test T-Tau plasma concentrations with the use of IMR (referred to as the first and second measurement , respectively) were performed. The relationships between the left hippocampal volume and the first measurement of T-Tau concentration are plotted in Fig. 3a. The measured plasma T-Tau concentration was found to increase with decreasing hippocampal volume. Additionally, we observed a negative correlation between hippocampal volume and plasma T-Tau levels. This result is consistent with that reported in a previous study  and indicates that atrophy of the hippocampus results in an elevation of plasma T-Tau. The Spearman correlation coefficient in Fig. 3a was found to be − 0.4723 (p < 0.0001). Figure 3b shows the relationship between the left hippocampal volume and the second measurement of T-Tau concentration. The Spearman correlation coefficient was found to be − 0.4446 (p < 0.0001). The measured T-Tau concentrations of the first and second measurements for a subject were averaged and referred to as duplicate measurements. The averaged T-Tau concentrations versus the left hippocampal volume are plotted in Fig. 3c. The Spearman correlation coefficient was found to be − 0.4767 (p < 0.0001). The values of the Spearman correlation coefficient of the first measurement, second measurement and duplicate measurements were similar to each other (approximate range − 0.4446, − 0.4767), which indicates that the plasma T-Tau concentration obtained in a single test has the same clinical significance for left hippocampal atrophy as that obtained in duplicate tests.
Figure 3d–f plot the relationships between the right hippocampal volume and plasma T-Tau in the first measurement, second measurement and duplicate measurements. Negative correlations were found in Fig. 3d–f, with Spearman correlation coefficients of − 0.4267, − 0.4081 and − 0.4306 (p < 0.0001), respectively. Thus, the measured plasma T-Tau concentrations obtained in a single test showed equivalent significance in monitoring hippocampal atrophy to that obtained in duplicate tests.
Among the enrolled subjects, 132 were NCs (mean age 67.6 ± 7.7 years), 34 were patients with MCI due to AD (aMCI; mean age 71.5 ± 8.9 years) and 74 were patients with AD dementia (mean age: 77.2 ± 8.9 years). For the purposes of this study, we combined the aMCI and AD patients to represent the patient group (Table 3). According to a previously published study , the cutoff value of plasma Aβ1–42 × T-Tau using IMR that discriminates patients from NCs is 455.49 pg/ml2. Using the single-test concentrations of Aβ1–42 and T-Tau in the plasma, the subjects showing Aβ1–42 × T-Tau values that are not lower than 455.49 pg/ml2 were regarded as being positive, whereas subjects showing Aβ1–42 × T-Tau values that are lower than 455.49 pg/ml2 were regarded as being negative. The agreement between the clinical diagnosis and the plasma biomarker diagnosis using single-test Aβ1–42 × T-Tau was analyzed, as shown in Table 3. Two rounds of single tests were performed (1st and 2nd measurements, respectively). Both the agreements of these two rounds of a single test and the duplicate tests are listed in Table 3. The Aβ1–42 and T-Tau concentrations in the duplicate measurements are presented as the averaged values of those concentrations in the 1st measurement and 2nd measurement. Although the duplicate tests showed slightly higher agreements than those of the first measurement and second measurement, the agreements in all of the cases were > 0.85 (or 85%). Thus, the single-test plasma Aβ1–42 × T-Tau assay using IMR is a promising parameter to discriminate NCs from patients with MCI due to AD and AD.
In addition to discriminating between patients and NCs, plasma Aβ1–42 × T-Tau showed promise to differentiate AD from aMCI, with a cutoff value of 642.58 pg/ml2, as has been previously reported . The data used in Table 3 were further analyzed to investigate the agreement between the clinical diagnosis and the plasma biomarker diagnosis using Aβ1–42 × T-Tau for discriminating AD from aMCI. In Table 4, the overall percentage agreement in the duplicate measurements was 82.4%, which was higher than those in the first measurement and second measurement by 5.5%. In fact, the overall percentage agreement in a single test was 76.9%, which is sufficient for the clinical purpose of discriminating AD from aMCI.
Another plasma biomarker index to differentiate NCs from patients with AD and aMCI is Aβ1–42/Aβ1–40. According to a previously published study, the cutoff value to discriminate NCs from patients was 0.325 . Hence, subjects showing Aβ1–42/Aβ1–40 values that were not < 0.325 were regarded as being positive, and subjects with Aβ1–42/Aβ1–40 values that were < 0.325 were regarded as being negative. The agreements between the clinical diagnosis and the plasma biomarker diagnosis using single-test Aβ1–42/Aβ1–40 were also analyzed, as shown in Table 5. Notably, two rounds of single tests were performed in this study ( 1st measurement and 2nd measurement). The averaged values of those tests in the first measurement and second measurement were reported as Aβ1–42 and T-Tau concentrations in the duplicate tests. In Table 5, the agreement between the two rounds of single tests (1st measurement and 2nd measurement), duplicate tests and clinical diagnosis are shown. Regardless of whether a single test or duplicate tests were performed, the agreements were > 0.85 (or 85%). Similar to the case of plasma Aβ1–42 × T-Tau, single-test plasma Aβ1–42/Aβ1–40 using IMR represents a promising parameter for differentiating NCs from patients with MCI due to AD and AD.
There are several limitations in this study. First, the clinical samples were re-analyzed using the original duplicate data. More subjects with single measurements should be enrolled to further validate the discrimination in patients with AD and NCs. Second, this is a cross-sectional study. Therefore, a longitudinal study is needed to confirm whether the feasibility is as good as suggested by the results of this cross-sectional study.