# US 8,785,150 B2 · Quantifiable internal reference standards for immunohistochemistry and uses thereof

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- Number: US 8,785,150 B2
- Document ID: US8785150B2
- Status: Granted
- Kind: B2
- Application number: 11/772,042
- Filed: 2007-06-29
- Published: 2008-02-14
- Granted: 2014-07-22
- Earliest claimed: 2006-06-30
- Assignees: University of Southern California
- Applicants: Clive R. Taylor; Shan Rong Shi
- Inventors: Clive R. Taylor; Shan Rong Shi
- Web page: https://patentgrep.com/patent/US8785150B2

## CPC

- G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis}
- G01N 33/96: involving blood or serum control standard
- G01N 35/00693: {Calibration}
- G01N 2001/2893: {Preparing calibration standards}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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

### Claim 1

A method of determining the amount of a test antigen by immunohistochemistry (IHC), comprising:
providing a formalin-fixed, paraffin-embedded (FFPE) cell or tissue sample comprising the test antigen and a quantifiable internal reference standard (QIRS) for the test antigen, the QIRS being a second antigen different from the test antigen, the FFPE sample having been prepared at a time T1 from an original cell or tissue sample having an original amount of the test antigen and an original amount of the QIRS;
providing a reference calibration curve comprising at least a ratio that relates the amount of the test antigen to the amount of the QIRS in a reference FFPE sample at a time T2 after T1;
measuring a first IHC signal corresponding to the QIRS in the FFPE sample at time T2, wherein the first IHC signal having a magnitude that varies in proportion to at least the concentration of the QIRS;
measuring a second IHC signal corresponding to the test analyte in the FFPE sample at time T2, wherein the second IHC signal having a magnitude that varies in proportion to at least the concentration of the test analyte; and
calculating the amount of the test antigen in the FFPE sample by applying the ratio(s) of the calibration curve as a conversion factor.

### Claim 2 (depends on 1)

The method of claim 1, wherein the calibration curve provides a ratio, A, of the original amount of the test antigen to the original amount of the QIRS and a ratio, C, of the original amount of the QIRS to the amount of the QIRS in the FFPE sample at time T2 is known, and the amount the test antigen in the original sample is calculated by multiplying the amount QIRS in the FFPE sample by the ratio A and by the Ratio C.

### Claim 3 (depends on 1)

The method of claim 1, wherein the original cell is an endothelial cell or the original tissue contains endothelial cells.

### Claim 4 (depends on 3)

The method of claim 3, 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.

### Claim 5 (depends on 1)

The method of claim 1, wherein the QIRS is a cell surface protein, a cytoplasmic protein, or a nuclear protein.