Methods of analyzing an H and E stained biological sample

• US 9,176,032 B2
• Filed: 03/06/2013
• Issued: 11/03/2015
• Est. Priority Date: 12/23/2011
• Status: Active Grant

• Current Assignee: Leica Microsystems CMS GmbH (Danaher Corporation)
• Original Assignee: General Electric Company
• Inventors: Sood, Anup, Kenny, Kevin Bernard, Natarajan, Arunkumar, Kaanumalle, Lakshmi Sireesha
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US13/786,747
• Publication Number:
  US 20130178392A1
• Time in Patent Office: 972 Days
• US Class Current: 1/1
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Methods comprising the use probing multiple targets in a H&E stained biological sample are provided. The methods include the steps of providing a hematoxylin and eosin stained biological sample containing multiple targets, observing the sample, removing the hematoxylin and partially removing the eosin by washing the sample, contacting the sample with a borate salt, and irradiating the sample to remove the residual eosin fluorescence. The method further includes the optionally performing the additional steps of binding at least one probe to one or more targets to the sample, observing a signal from the probe and contacting the sample with a bleaching agent. The process of binding, observing and bleaching may be iteratively repeated.

Claims

1. A sequential method of preparing a hematoxylin and eosin stained biological sample for analysis comprising:

(a) providing a hematoxylin and eosin stained biological sample containing multiple targets;

(b) observing the hematoxylin and eosin si al from the stained biological sample;

(c) removing the hematoxylin and partially removing the eosin by washing the sample;

(d) contacting the sample with an electron transfer reagent where the electron transfer reagent is a borate salt represented by the following structural formula;

2. The method of claim 1, wherein irradiating the sample in step (e) is accomplished by exposing the sample to light of 350 nm-1.3 μ

M in wavelength.

3. The method of claim 2, wherein irradiating the sample in step (e) is accomplished by exposing the sample to light of 400-700 nm in wavelength.

4. The method of claim 1, wherein irradiating the sample in (e) is carried out in the presence of a buffer at pH of 5-9.

5. The method of claim 1 wherein step (e) is carried out at the temperature of 4-50°

C.

6. The method of claim 1, wherein step (e) is performed for about 20 seconds to about 15 minutes.

7. The method of claim 1 wherein each R₁, R₂, and R₃ is each independently an optionally substituted aryl and R₄ is an optionally substituted alkyl.

8. The method of claim 7 wherein each R₁, R₂, and R₃ is phenyl and R₄ is butyl, or benzyl.

9. The method of claim 1 wherein M⁺ is an inorganic cation selected from the group consisting of Li⁺, Na⁺ or K⁺.

10. The method of claim 1 further comprising the steps of:

(f) binding at least one probe capable of signal generation to one or more targets to the sample of step (e);

(g) observing a signal from the probe bound in step (f); and

(h) optionally contacting the sample with a bleaching agent and repeating steps (f) through (g).

11. The method of claim 10 wherein the bleaching agent is a photoactivated chemical bleaching agent and the step (h) further comprises irradiating the sample by exposing the sample to light of 350 nm-1.3 μ

M in wavelength.

12. The method of claim 11, wherein irradiating the sample is accomplished by exposing the sample to light of 400-700 nm in wavelength.

13. The method of claim 10, wherein the probe in step (f) comprises a fluorescent signal generator, and the signal observed in step (g) is a fluorescent signal.

14. The method of claim 10, wherein steps (f)-(h) are repeated two or more times.

15. The method of claim 10, further comprising measuring one or more intensity values of the signal observed in observing step (g).

16. The method of claim 15 further comprising correlating the intensity value with an amount of target present in the sample.

17. The method of claim 10, wherein the electron transfer agent in step (d) is the same as the bleaching agent in step (h) and step (h) further comprises irradiating the sample by exposing the sample to light of 350 nm-1.3 μ

M in wavelength.

18. A sequential method of probing multiple targets in a hematoxylin and eosin stained biological sample comprising:

(a) providing a hematoxylin and eosin stained biological sample containing multiple targets;

(b) observing the hematoxylin and eosin signal from the stained biological sample;

(c) optionally removing the hematoxylin and partially removing the eosin by washing the sample;

(d) subjecting the sample to an antigen retrieval process to expose one or more antigens in the region of interest;

(e) optionally applying a blocking reagent to block against nonspecific binding of one or more antigens;

(f) contacting the sample with an electron transfer agent reagent where the electron transfer reagent is a borate salt represented by the following structural formula;

19. The method of claim 18 wherein step (h) wherein the probe is a protein stain.

20. The method of claim 18 wherein the bleaching agent is a photoactivated chemical bleaching agent and the step (j) further comprises irradiating the sample by exposing the sample to light of 350 nm-1.3 μ

M in wavelength.

21. The method of claim 18, wherein each R₁, R₂, and R₃ is an independently optionally substituted aryl and R₄ is an optionally substituted alkyl.

22. The method of claim 21, wherein each R₁, R₂, and R₃ is phenyl and R₄ is butyl or benzyl.

23. The method of claim 18 wherein M⁺ is an inorganic cation selected from the group consisting of Li⁺, Na⁺ or K⁺.

24. The method of claim 18 wherein the electron transfer agent in step (f) is the same as the bleaching agent in step (j) and step (j) further comprises irradiating the sample by exposing the sample to light of 350 nm-1.3 μ

M in wavelength.

25. A sequential method of probing multiple targets in a hematoxylin and eosin stained biological sample comprising:

(a) providing a hematoxylin and eosin stained biological sample containing multiple targets;

(b) observing the hematoxylin and eosin signal from the stained biological sample;

(c) removing the hematoxylin and partially removing the eosin by washing the sample;

(d) contacting the sample with an electron transfer agent reagent where the electron transfer reagent is a borate salt represented by the following structural formula;

26. The method of claim 25, wherein each R₁, R₂, and R₃ is each independently an optionally substituted aryl and R₄ is an optionally substituted alkyl.

27. The method of claim 26, wherein each R₁, R₂, and R₃ is phenyl and R₄ is butyl or benzyl.

28. The method of claim 25 wherein M⁺ is an inorganic cation selected from the group consisting of Li⁺, Na⁺ or K⁺.

29. The method of claim 25 wherein the electron transfer agent in step (d) is the same as the bleaching agent in step (h) and step (h) further comprises irradiating the sample by exposing the sample to light of 350 nm-1.3 μ

M in wavelength.

30. A method according to claim 27 wherein the at least one probe is a FISH probe.