CONVERGENCE OF AGGREGATION RATE WITH VALIDATED PERIPHERAL DIAGNOSTIC FOR ALZHEIMER'S DISEASE
The present disclosure describes diagnostics for Alzheimer's disease based on the discovery of an increased aggregation rate with increasing cell density in cells from Alzheimer-disease patients versus controls. The aggregation rate was cross-validated with two more mature assays: AD-Index and Morphology diagnostic assays. Also disclosed is a simple-majority rule for diagnosing AD using at least three diagnostic assays, such as the Aggregation Rate, AD-Index, and Morphology assays.
1 . A method of diagnosing Alzheimer's Disease in a human subject comprising the steps of
(a) obtaining one or more cells from a human subject;
(b) culturing the one or more cells for a time period;
(c) determining an average area of cell aggregates and dividing the average area by the number of aggregates to obtain an average area per number of aggregates;
(d) determining an aggregation rate by evaluating the rate of change of the average area per number of aggregates determined in step (c) as a function of cell density;
(e) comparing the determination of step (d) with an aggregation rate determined using non-Alzheimer's disease cells; and
(f) diagnosing the presence or absence of Alzheimer's Disease based on the comparison in step (e), wherein the diagnosis is positive for Alzheimer's disease if the aggregation rate determined in step (d) is increased compared to the aggregation rate determined using the non-Alzheimer's disease cells.
2 . The method of claim 1 , wherein the diagnosis is confirmed using at least one additional diagnostic assay.
3 . The method of claim 2 , wherein the at least one additional diagnostic assay is chosen from AD-Index and Morphology diagnostic assays.
4 . The method of claim 1 , wherein the non-Alzheimer's disease cells are chosen from an aged-matched control.
5 . The method of claim 1 , wherein the time period is chosen from about 12 hours to about 72 hours.
6 . The method of claim 5 , wherein the time period is chosen from about 24 hours to about 48 hours.
7 . The method of claim 6 , wherein the time period is about 48 hours.
8 . The method of claim 1 , further comprising imaging the cultured cells at the end of the time period.
9 . The method of claim 8 , wherein the cell density is measured by measuring the number of cells per 10× image.
10 . The method of claim 8 , wherein the rate of change of the average area per number of aggregates as a function of cell density is evaluated within the boundaries of 320 to 550 cells/10× image.
11 . The method of claim 1 , wherein the rate of change of the average area per number of aggregates as a function of cell density is evaluated by determining the slope of a linear fit between the average area per number of aggregates and cell density.
12 . The method of claim 1 , wherein the one or more cells are fibroblast cells.
13 . The method of claim 12 , wherein the fibroblast cells are skin fibroblast cells.
14 . The method of claim 1 , wherein the one or more cells are cultured in a preparation comprising extracellular matrix proteins.
15 . The method of claim 14 , wherein the preparation comprises laminin, collagen, heparin sulfate proteoglycans, entactin/nidogen, and/or combinations thereof.
16 . The method of claim 14 , wherein the preparation further comprises a growth factor.
17 . The method of claim 14 , wherein the preparation is extracted from a tumor.
18 . The method of claim 17 , wherein the tumor is the EHS mouse sarcoma.
19 . A method of diagnosing Alzheimer's disease in a human subject comprising
i) performing at least three diagnostic assays;
ii) evaluating whether each assay indicates the presence or absence of Alzheimer's disease; and
iii) diagnosing the presence or absence of Alzheimer's disease based on whether a simple majority of the diagnostic assays indicate the presence or absence of Alzheimer's disease,
wherein one of the diagnostic assays comprises:
(a) obtaining one or more cells from a human subject;
(b) culturing the one or more cells for a time period;
(c) determining an average area of cell aggregates and dividing the average area by the number of aggregates to obtain an average area per number of aggregates;
(d) determining an aggregation rate by evaluating the rate of change of the area per number of aggregates determined in step (c) as a function of cell density; and
(e) comparing the determination of step (d) with an aggregation rate determined using non-Alzheimer's Disease cells, wherein the presence of Alzheimer's disease is indicated if the aggregation rate determined in step (d) is increased compared to the aggregation rate determined using the non-Alzheimer's Disease cells.
20 . The method of claim 19 , wherein two of the at least three diagnostic assays are AD-Index and Morphology assays.
21 . The method of claim 19 , wherein the one or more cells are fibroblast cells.
22 . The method of claim 21 , wherein the fibroblast cells are skin fibroblast cells.