Quantifiable Internal Reference Standards for Immunohistochemistry and Uses Thereof

• US 20080038771A1
• Filed: 06/29/2007
• Published: 02/14/2008
• Est. Priority Date: 06/30/2006
• Status: Active Grant

• Current Assignee: University of Southern California
• Original Assignee: University of Southern California
• Inventors: Taylor, Clive, Shi, Shan
• Granted Patent: US 8,785,150 B2
• Time in Patent Office: Days
• US Class Current: 435/40.5
• 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 2001/2893 Preparing calibration standards
• CPC Class Codes: G01N 33/96 involving blood or serum control standard
• CPC Class Codes: G01N 35/00693 Calibration

Abstract

Methods for identifying Quantifiable Internal Reference Standards (QIRS) for immunohistochemistry (IHC). Also disclosed are methods for using QIRS to quantify test antigens in IHC.

Claims

1. A method of identifying a Quantifiable Internal Reference Standard (QIRS) for immunohistochemistry (IHC), comprising:

providing multiple samples of cells or tissues;

determining the amount of a first antigen and the amount of a second antigen in each of the cell or tissue samples;

preparing a formalin-fixed, paraffin-embedded (FFPE) sample from each of the cell or tissue samples; and

determining the amount of the first antigen and the amount of the second antigen in each of the FFPE samples by IHC,

wherein the first antigen is identified as a QIRS for the second antigen in IHC if the ratio of the amount of the first antigen to the amount of the second antigen in the cell or tissue samples is at least 95% identical among the cell or tissue samples and the ratio of the amount of the first antigen to the amount of the second antigen in the FFPE samples is at least 95% identical among the FFPE samples.

2. The method of claim 1, wherein the cells or tissues are of the same type or different types.

3. The method of claim 1, wherein the cells or tissues are normal or pathologic cells or tissues.

4. The method of claim 1, wherein the cells are lymphocytes or the tissues contain lymphocytes.

5. The method of claim 4, wherein the cells are Raji or HL60 cells.

6. The method of claim 1, wherein the cells are endothelial cells or the tissues contain endothelial cells.

7. The method of claim 6, wherein the cells are HuVEC cells.

8. The method of claim 1, wherein the cells are fibroblasts or the tissues contain fibroblasts.

9. The method of claim 8, wherein the cells are LD419 cells.

10. The method of claim 1, wherein the cells are epithelial cells or the tissues contain epithelial cells.

11. The method of claim 10, wherein the cells are breast cells.

12. The method of claim 11, wherein the cells are MCF7, MDA, or MB468 cells.

13. The method of claim 1, wherein the cells or tissues are from prostate or spleen.

14. The method of claim 1, wherein the amount of the first antigen in the FFPE samples is at least 50% of the amount of the first antigen in the cell or tissue samples.

15. The method of claim 1, wherein the amount of the first antigen in the FFPE sample is determined using a first quantifiable label and the amount of the second antigen in the FFPE sample is determined using a second quantifiable label.

16. The method of claim 1, wherein the first antigen is detectable by a first antibody to the first antigen or the second antigen is detectable by a second antibody to the second antigen.

19. The method of claim 17, wherein the QIRS is identified according to the method of claim 1.

32. The method of claim 16, wherein the QIRS is PSA, p53, Rb, or ER.

17. A method of quantifying a test antigen by IHC, comprising:

providing an FFPE cell or tissue sample prepared from an original cell or tissue sample;

determining the amount of a QIRS for a test antigen in the FFPE sample by IHC; and

calculating the amount of the test antigen in the FFPE sample from the amount of the QIRS in the FFPE sample.

18. The method of claim 17, further comprising calculating the amount of the test antigen in the original cell or tissue sample from the amount of the QIRS in the FFPE sample.

20. The method of claim 17, wherein the original cell or tissue is a normal or pathologic cell or tissue.

21. The method of claim 17, wherein the original cell is a lymphocyte or the original tissue contains lymphocytes.

22. The method of claim 21, wherein the QIRS is selected from the group consisting of CD45, CD20, actin, B2 microglobulin, vimentin, histone H1, and MIB1.

23. The method of claim 17, wherein the original cell is an endothelial cell or the original tissue contains endothelial cells.

24. The method of claim 23, wherein the QIRS is selected from the group consisting of CD31, actin, B2 microglobulin, vimentin, factor VIII, histone H1, MIB1, Fli 1, CD34, and VWF.

25. The method of claim 17, wherein the original cell is a fibroblast or the original tissue contains fibroblasts.

26. The method of claim 25, wherein the QIRS is selected from the group consisting of fibroblast surface protein, actin, B2 microglobulin, vimentin, desmin, histone H1, and MIB1.

27. The method of claim 17, wherein the original cell is an epithelial cell or the original tissue contains epithelial cells.

28. The method of claim 27, wherein the original cell is a breast cell or the original tissue contains breast cells.

29. The method of claim 28, wherein the QIRS is selected from the group consisting of Her2, EGFR, actin, B2 microglobulin, vimentin, histone H1, and MIB1.

30. The method of claim 17, wherein the original cell or tissue is from prostate or spleen.

31. The method of claim 17, wherein the QIRS is a cell surface protein, a cytoplasmic protein, or a nuclear protein.