METHODS AND SYSTEMS FOR DETECTION OF FIBRIN FORMATION OR REMOVAL AT THE NANO-SCALE

Abstract

Systems and methods for imaging and tracking fibrin formation via interaction of a test sample with a clotting agent or for imaging and tracking fibrin removal by an anti-clotting agent are described.In certain embodiments, the systems (200) comprise a planar reflective substrate (222, 224) comprising one or more capture agents and/or one or more fibrin reference regions; a mount for holding the substrate; an illumination light source (201) for directing illumination light toward a top surface of the substrate with fibrin (226)formed thereon; an image detector (232, 234) aligned with respect to the mount for detecting a portion of the illumination light that is scattered by the fibrin, and/or reflected by the reflective substrate, thereby obtaining a label-free image of fibrin formation or fibrin removal; a processor of a computing device (240); and a memory having instructions stored thereon, wherein the instructions, when executed by the processor, cause the processor to: receive and/or access data corresponding to the one or more label free images, and use the one or more label-free images to determine one or more measures of fibrin formation or fibrin removal.

Description

Claims

1. A method of imaging and tracking fibrin formation via interaction of a test sample with a clotting agent, the method comprising: (a) contacting the test sample with the clotting agent;(b) contacting a top surface of a substrate with the (i) the test sample and/or (ii) the clotting agent, thereby providing for formation of fibrin at the top surface of the substrate;(c) directing illumination light toward the top surface of the substrate, thereby illuminating the top surface of the substrate along with the fibrin formed thereon;(d) detecting, with one or more imaging detectors, a label-free scattering signal corresponding to a portion of the illumination light that is (A) scattered by the fibrin, and/or (B) reflected by the reflective substrate, thereby obtaining one or more label-free images of fibrin formation; and(e) using the one or more label-free images to determine one or more measures of fibrin formation.

2-3. (canceled)

4. The method of claim 1, wherein the top surface of the substrate comprises one or more primary capture agents, each specific to at least one of (i) the one or more components of the test sample, (ii) the clotting agent, and (iii) the one or more product components.

5. The method of claim 1, comprising drying the top surface of the substrate following step (b) and before performing step (c).

6. The method of claim 1, wherein step (b) comprises incubating the clotting agent with the test sample for a duration of about 5 minutes or less.

7. The method of claim 1, comprising performing steps (c) and (d) at one or more time points after contacting the clotting agent with the test sample, so as to obtain one or more label-free images of fibrin formation, each corresponding to a particular time point after the contacting the clotting agent with the test sample.

8. The method of claim 7, comprising performing steps (c) and (d) at a plurality of time points while incubating the clotting agent with the test sample, thereby obtaining a plurality of images tracking formation of fibrin.

9. The method of claim 7, comprising performing steps (c) and (d) at one or more times prior to and/or at the same time as step (b), thereby obtaining one or more reference images of the top surface of the substrate prior to formation of fibrin following the contacting the clotting agent with the test sample.

10. The method of claim 1, wherein step (d) comprises imaging the top surface of the substrate and/or any fibers formed thereon at a resolution better than 600 nm.

11. The method of claim 1, wherein the one or more measures of fibrin formation comprise one or more members selected from the group consisting of: a number of fibers;a density of fibers;a measure of fiber length;a measure of fiber thickness;a measure of branching capacity;a measure of fibrin cross-linking; anda measure of contrast.

12. The method of claim 1, wherein step (e) comprises: identifying, within at least a portion of the one or more label-free images, one or more point spread functions each corresponding to a piece of fibrin having a sub-diffraction limited length;determining, for each of the one or more point spread functions, a contrast value, thereby determining one or more contrast values; andusing at least a portion of the one or more determined contrast values to determine a length of the corresponding piece of fibrin.

13. The method of claim 1, comprising performing steps (c) and (d) at a plurality of time points while incubating the clotting agent with the test sample, thereby obtaining a plurality of images tracking formation of fibrin, and using the plurality of images to determine one or more time-dependent measures of fibrin formation.

14. The method of claim 1, comprising using the one or more measures of fibrin formation to determine one or more prognostic values for the test sample and/or a subject associated with the test sample.

15. The method of claim 14, wherein the one or more prognostic values comprise an activated partial thromboplastin time (APPT) and/or a prothrombin time (PT).

16. The method of claim 14, wherein the one or more prognostic values comprises a relative risk of one or more particular diseases and/or conditions.

17. (canceled)

18. The method of claim 1, wherein one or more fibrin forming components is/are fluorescently labeled.

19. (canceled)

20. The method of claim 1, wherein the top surface of the substrate comprises one or more secondary capture agents, each specific to one or more disease-associated biomolecules, each disease-associated biomolecule associated with a particular infectious disease, thereby providing for testing the test sample for the particular infectious disease.

21. The method of claim 1, wherein fibrin-capturing molecules are used to assess the presence of micro-clots within a plasma or blood sample.

22. (canceled)

23. The method of claim 1, wherein the top surface of the substrate comprises a material under test.

24. The method of claim 1, further comprising contacting the test sample with one or more secondary agents, thereby providing for assessing the influence of the one or more secondary agents on clotting and/or assessing removal of clots via the one or more secondary agents.

25. The method of claim 24, wherein the one or more secondary agents comprise anti-clotting agents.

26. The method of claim 24, wherein the one or more secondary agents comprise one or more clot promoting agents.

27-28. (canceled)

29. A method of imaging and tracking fibrin removal by an anti-clotting agent, the method comprising: (a) contacting a top surface of a substrate with an anti-clotting buffer comprising an anti-clotting agent, wherein the top surface of the substrate comprises one or more fibrin reference regions each comprising a known fibrin layer;(b) directing illumination light toward at least a portion of the one or more fibrin reference regions of the top surface of the substrate, thereby illuminating the top surface of the substrate along with fibrin formed thereon within the portion of the fibrin reference regions;(c) detecting, with one or more imaging detectors, a label-free scattering signal corresponding to a portion of the illumination light that is (A) scattered the fibrin, and/or (B) reflected by the reflective substrate, thereby obtaining one or more label-free images of fibrin formation; and(d) using the detected label-free scattering signal to determine one or more measures of fibrin formation for the test sample.

30-42. (canceled)

43. A system for imaging and tracking fibrin formation via interaction of a test sample with a clotting agent, the system comprising: (a) a planar reflective substrate comprising one or more capture agents and/or one or more fibrin reference regions;(b) a mount for holding a substrate;(c) one or more illumination light sources aligned with respect to the mount so as to and direct illumination light toward a top surface of the substrate, so as to provide for illumination of the top surface of the substrate along with fibrin formed thereon;(d) one or more detectors aligned with respect to the mount and operable to detect a portion of the illumination light that is (A) scattered by the fibrin, and/or (B) reflected by the reflective substrate, thereby providing for obtaining one or more label-free images of fibrin formation;(e) a processor of a computing device; and(f) a memory having instructions stored thereon, wherein the instructions, when executed by the processor, cause the processor to: receive and/or access data corresponding to the one or more label free images; anduse the one or more label-free images to determine one or more measures of fibrin formation.

44. The system of claim 43, wherein, the top surface of the planar reflective substrate comprises a plurality of capture agent spots, each capture agent spot comprising a particular capture agent specific to a particular clotting agent and/or component of fibrin.

45. The system of claim 43, wherein the top surface of the planar reflective substrate comprises a plurality of fibrin reference regions, each comprising a known fibrin layer.

46. The system of claim 43, comprising an objective lens aligned to (i) collect light (A) scattered by the fibrin and/or reflected by the reflective substrate and (ii) direct the collected light onto the one or more detectors.

47-51. (canceled)