Methods for detecting and measuring aggregation

• US 10,281,479 B2
• Filed: 01/16/2015
• Issued: 05/07/2019
• Est. Priority Date: 07/18/2012
• Status: Active Grant

• Current Assignee: Labrador Diagnostics LLC (SoftBank Group Corp.)
• Original Assignee: Theranos IP Company, LLC
• Inventors: Patel, Paul, Anekal, Samartha, Gibbons, Ian, Holmes, Elizabeth A., Joshi, Swapna
• Primary Examiner(s): Chen, Stacy B
• Application Number:
  US14/598,498
• Publication Number:
  US 20150198591A1
• Time in Patent Office: 1,572 Days
• Field of Search: None
• CPC Class Codes: B01L 3/5021 Test tubes specially adapted for centrifugation purposes centrifuges B04B5/04
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/556 Fixed or stabilised red blood cell
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors

Abstract

Methods, compositions, systems, and devices are provided for performing and analyzing agglutination assays. In one aspect, methods for image analysis of agglutination assays are provided. In another aspects, methods for performing agglutination assays are provided. In one aspect, the methods may be used for the detection of various molecules, including viruses or antibodies against a virus. In another aspect, the methods can be used to determine effective immunization of a subject.

Claims

1. A method of assaying for the agglutination of visualization particles in a mixture, comprising:

A) generating a mixture comprising visualization particles and agglutinating particles under conditions permitting agglutination of the visualization particles via interaction with the agglutinating particles, wherein the generating of the mixture uses only a single incubation step and without red cell reagent preparation;

B) obtaining at least one image of the mixture with an imaging device; and

C) analyzing said at least one image by use of a computer programmed to measure agglutination by analysis of the at least one image by identifying clusters in the image to obtain a measurement of the agglutination of the visualization particles in the mixture, wherein agglutination is evidenced by the formation of clusters comprising said visualization particles and said agglutinating particles, and wherein a cluster comprises a contiguous distribution of two or more visualization particles having center-to-center distances between said visualization particles of up to two times visualization particle diameter.

2. The method of claim 1, wherein the visualization particles are RBCs or microspheres.

3. The method of claim 1, wherein the agglutinating particles are viral particles or antibodies.

4. The method of claim 1, wherein the image is obtained with an imaging device comprising a CCD or CMOS image sensor.

5. The method of claim 1, wherein the agglutinating particles are provided in a biological sample from a subject.

6. The method of claim 5, wherein the biological sample comprises plasma or serum.

7. The method of claim 5, wherein the agglutinating particles are viral particles.

8. The method of claim 5, wherein the agglutinating particles are antibodies.

9. The method of claim 5, further comprising determining of the quantity of viral particles or antibodies in the sample.

14. The method of claim 1, wherein the image of the mixture is obtained within 1 hour of generating the mixture.

15. The method of claim 1, wherein the analyzing the image comprises identifying a region of interest (ROI) in the image.

16. The method of claim 15, wherein the ROI comprises imaged information of the mixture, said mixture comprising visualization particles and agglutinating particles.

17. The method of claim 1, wherein the measurement of the agglutination of the visualization particles in the mixture is a qualitative measurement of agglutination or non-agglutination of the visualization particles.

18. The method of claim 1, wherein the measurement of the agglutination of the visualization particles in the mixture is a quantitative measurement of degree of agglutination of the visualization particles.

19. The method of claim 1, wherein two or more images of the mixture are obtained.

20. The method of claim 1, further comprising determining the size or number of clusters in the image.

21. The method of claim 1, wherein the step of identifying clusters in the image comprises identifying a cluster of at least three visualization particles, wherein each of the three visualization particles is positioned such that the center of a first particle is located no further than 50 micrometers (μ

m) from the center of each of the other two visualization particles in the cluster.

22. The method of claim 21, wherein the center-to-center distance between said center of said first particle and the center of each of said other two visualization particles in the cluster is no greater than 40 micrometers (μ

m).

23. The method of claim 1, wherein the step of analyzing the image comprises determining the center-to-center distance between a first visualization particle and a second visualization particle in the image.

24. The method of claim 23, further comprising determining the location of the first visualization particle and the second visualization particle in the image.

25. The method of claim 1, wherein the step of identifying the clusters comprises identifying a first visualization particle and a second visualization particle, wherein the center-to-center distance between the first visualization particle and the second visualization particle is no greater than 1.5 times the diameter of either the first or second visualization particle.

26. The method of claim 1, wherein the step of identifying the clusters comprises identifying a first visualization particle and a second visualization particle, wherein the center-to-center distance between the first visualization particle and the second visualization particle is no greater than 12 micrometers (μ

m).

27. The method of claim 1, wherein the step of identifying the clusters comprises identifying at least three visualization particles, wherein each of the three visualization particles is positioned such that the center of the visualization particle is located no further than 15 micrometers (μ

m) from the center of each of the other two visualization particles.

28. The method of claim 1, wherein the step of identifying the clusters comprises selecting a cut-off value distance, wherein visualization particles situated at a distance from each other which is less than the cut-off value distance are considered to be associated with each other.

29. The method of claim 28, wherein the step of selecting a cut-off value distance comprises calculating a radial distribution function for two or more visualization particles in the image.

30. The method of claim 1, wherein the step of analyzing the image comprises extracting a feature from a region of the image of the mixture.

31. The method of claim 30, wherein the step of extracting the feature comprises analyzing the texture of the mixture.

32. The method of claim 31, wherein the step of analyzing the texture of the mixture comprises analyzing the image with a local binary patterns operator.

33. The method of claim 30, wherein the step of extracting the feature comprises analyzing the intensity of a signal in one or more color channels in a region of the image.

34. The method of claim 32, wherein the step of analyzing the texture of the mixture comprises inputting information obtained from the local binary patterns operator into an algorithm which has been previously trained with local binary patterns information from one or more images of a mixture of known agglutination status.

36. The method of claim 1, wherein steps A)-C) are performed in one hour or less.

10. A method of assaying for the agglutination of visualization particles in a mixture, comprising:

A) generating a mixture comprising visualization particles and agglutinating particles under conditions permitting agglutination of the visualization particles via interaction with the agglutinating particles, wherein the generating of the mixture uses only a single incubation step and without red cell reagent preparation;

B) obtaining at least one image of the mixture with an imaging device; and

C) analyzing said at least one image by use of a computer programmed to measure agglutination by analysis of the at least one image by identifying clusters in the image to obtain a measurement of the agglutination of the visualization particles in the mixture, wherein agglutination is evidenced by the formation of clusters comprising said visualization particles and said agglutinating particles, and wherein a cluster comprises a contiguous distribution of two or more visualization particles having center-to-center distances between said visualization particles of up to two times visualization particle diameter;

wherein the mixture further comprises an antibody which specifically binds to at least one agglutinating particle of said agglutinating particles and inhibits the interaction of the agglutinating particle with a visualization particle of said visualization particles.

11. The method of claim 10, wherein the antibody is provided to the mixture in a biological sample from a subject.

12. The method of claim 11, wherein the biological sample comprises plasma or serum.

13. The method of claim 11, further comprising determining the quantity of the antibody in the sample.

35. A method of assaying for the agglutination of visualization particles in a mixture, comprising:

A) generating a mixture comprising visualization particles and agglutinating particles under conditions permitting agglutination of the visualization particles via interaction with the agglutinating particles, wherein the generating of the mixture uses only a single incubation step and without red cell reagent preparation;

B) obtaining at least one image of the mixture with an imaging device; and

C) analyzing said at least one image by use of a computer programmed to measure agglutination by analysis of the at least one image by identifying clusters in the image to obtain a measurement of the agglutination of the visualization particles in the mixture, wherein agglutination is evidenced by the formation of clusters comprising said visualization particles and said agglutinating particles, and wherein a cluster comprises a contiguous distribution of two or more visualization particles having center-to-center distances between said visualization particles of up to two times visualization particle diameter;

wherein the step of analyzing the image comprises comparing feature extraction information from the image of the mixture with feature extraction information from an image of a control mixture of known agglutination status, said control mixture comprising visualization particles and agglutinating particles.