Methods for analyzing rare circulating cells

• US 10,545,151 B2
• Filed: 02/19/2015
• Issued: 01/28/2020
• Est. Priority Date: 02/21/2014
• Status: Active Grant

• Current Assignee: Epic Sciences, Inc.
• Original Assignee: Epic Sciences, Inc.
• Inventors: Marrinucci, Dena
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/120,502
• Publication Number:
  US 20170010268A1
• Time in Patent Office: 1,804 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: G01N 2333/70589 CD45
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/57423 of lung
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells

Abstract

The disclosure provides methods for analyzing rare circulating cells (RCCs) at cellular and molecular level following their detection in non-enriched blood samples, methods of this disclosure serve as diagnostic methods for several disease conditions, including cardiovascular diseases and cancer.

Claims

1. A method for analyzing rare circulating cells (RCCs) in a non-enriched blood sample, comprising:

(a) detecting RCCs in the non-enriched blood sample, comprising i) determining presence or absence of one or more immunofluorescent RCC detection markers in nucleated cells in the non-enriched blood sample, and ii) assessing the morphology of the nucleated cells, wherein RCCs are detected among the nucleated cells based on a combination of distinct immunofluorescent staining and morphological characteristics;

(b) quenching the immunofluorescence of the one or more immunofluorescent RCC detection markers comprising contacting the RCCs with a quenching buffer, wherein the quenching buffer comprises a chaotropic salt and wherein the immunofluorescence is quenched by more than 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 99.99%; and

(c) analyzing the detected RCCs, comprising determining presence or absence of one or more fluorescent RCC analysis markers, wherein analyzing the detected RCCs comprises assessing the morphology of the detected RCCs.

2. The method of claim 1, wherein the fluorescent RCC analysis markers are fluorescence in situ hybridization (FISH) markers.

3. The method of claim 1, wherein more than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of RCCs detected in (a) are retained in (c).

4. The method of claim 1, wherein the fluorescent RCC analysis markers are positive control markers.

5. The method of claim 4, wherein the positive control markers are present in more than 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of RCCs analyzed in (c).

6. The method of claim 4, wherein the positive control markers are chromosomal markers.

7. The method of claim 4, wherein the positive control markers are centromere markers or telomere markers.

8. The method of claim 1, wherein the RCC analysis markers are genetic mutations selected from the group consisting of gene translocation, gene amplification, gene deletion, gene aneuploidy and chromosomal aneuploidy.

9. The method of claim 1, wherein the RCCs are circulating tumor cells (CTCs).

10. The method of claim 1, wherein the RCCs are circulating epithelial cells (CECs).

11. The method of claim 1, wherein the RCCs are CTC mimics.

12. The method of claim 1, wherein the RCCs are CTC candidates.

13. The method of claim 1, wherein the quenching buffer comprises a chaotropic agent.

14. The method of claim 13, wherein the concentration of the chaotropic agent is at least 2M, 3M or 4M.

15. The method of claim 1, wherein the quenching buffer comprises guanidine or a guanidinium salt.

16. The method of claim 1, wherein the quenching buffer comprises guanidinium thiocyanate (guanidine thiocyanate) or guanidinium chloride (guanidine hydrochloride).

17. The method of claim 1, wherein the RCCs were contacted with the quenching buffer for a period of time of more than 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes or more than 20 minutes.

18. The method of claim 1, wherein the analyzed RCCs comprise RCC aggregates.