Jesper Lohse

Improved catalyzed reporter deposition, iCARD.

Novel reporters have been synthesized with extended hydrophilic linkers that in combination with polymerizing cross-linkers result in very efficient reporter deposition. By utilizing antibodies to stain HER2 proteins in a cell line model it is demonstrated that the method is highly specific and sensitive with virtually no background. The detection of HER2 proteins in tissue was used to visualize individual antigens as small dots visible in a microscope. Image analysis-assisted counting of fluorescent or colored dots allowed assessment of relative protein levels in tissue. Taken together, we have developed novel reporters that improve the CARD method allowing highly sensitive in situ detection of proteins in tissue. Our findings suggest that in situ protein quantification in biological samples can be performed by object recognition and enumeration of dots, rather than intensity-based fluorescent or colorimetric assays.

A novel quantitative immunohistochemistry method for precise protein measurements directly in formalin-fixed, paraffin-embedded specimens: analytical performance measuring HER2

In clinical routine pathology today, detection of protein in intact formalin-fixed, paraffin-embedded tissue is limited to immunohistochemistry, which is semi-quantitative. This study presents a new and reliable quantitative immunohistochemistry method, qIHC, based on a novel amplification system that enables quantification of protein directly in formalin-fixed, paraffin-embedded tissue by counting of dots. The qIHC technology can be combined with standard immunohistochemistry, and assessed using standard bright-field microscopy or image analysis. The objective was to study analytical performance of the qIHC method. qIHC was tested under requirements for an analytical quantitative test, and compared with ELISA and flow cytometry for quantitative protein measurements. Human epidermal growth factor receptor 2 (HER2) protein expression was measured in five different cell lines with HER2 expression from undetectable with immunohistochemistry to strong positive staining (IHC 3+). Repeatability, reproducibility, robustness, linearity, dynamic range, sensitivity, and quantification limits were evaluated. Reproducibility and robustness were assessed in a setup to resemble daily work in a laboratory using a commercial immunohistochemistry platform. In addition, qIHC was correlated to standard HER2 immunohistochemistry in 44 breast cancer specimens. For all evaluated parameters, qIHC performance was either comparable or better than the reference methods. Furthermore, qIHC has a lower limit of detection than both immunohistochemistry and the ELISA reference method, and demonstrated ability to measure HER2 accurately and precise within a large dynamic range. In conclusion, the results show that qIHC provides a sensitive, quantitative, accurate, and robust assay for measurement of protein expression in formalin-fixed, paraffin-embedded cell lines, and tissue.

Automated sequential chromogenic IHC double staining with two HRP substrates.

Automated IHC double staining using DAB and HRP Magenta is illustrated utilizing a new acid block with sulfuric acid to prevent cross-reactivity. Residual cross-reactivity in double staining is determined to arise from chromogenic-bound antibodies and amplification system during the first part of the double staining.

Abstract 4568: A new quantitative in-situ IHC method - validation of analytical performance

Background In routine pathology, in situ quantification of protein in formalin-fixed, paraffin-embedded (FFPE) tissue is limited to immunohistochemistry (IHC) which is semi-quantitative at best. Accurate and robust measurement of protein expression in FFPE samples, without loss of morphological information, is currently not achievable. Quantitative protein assessment of FFPE tissue can be destructive, and/or time consuming with risk of analyzing non-relevant tissue due to heterogeneity. We have developed a new reliable quantitative IHC (qIHC) method based on a novel amplification system that enables quantification of protein directly in FFPE tissue by counting dots using a standard bright field microscope. The qIHC method combines the ease, speed and morphological information gained from classical IHC staining together with quantitative assessment of protein expression in situ. The goal of this study was to assess the analytical performance of qIHC in a HER2 assay analyzing both FFPE cell pellets and breast cancer specimens. Methods Five different cell lines with HER2 expression ranging from undetectable with IHC to strongly positive (IHC 3+) were used as test samples. Flow cytometry, and ELISA were used as reference methods to confirm protein expression levels and stability of the cell lines. The qIHC assay was conducted using an automated Autostainer Link 48 stainer, and quantitative assessment was done by counting dots per cell using an image analysis algorithm. Repeatability, reproducibility, robustness, linearity, sensitivity, and quantification limits were evaluated. In addition, 44 breast cancer specimens were stained to compare standard HER2 IHC with qIHC. Results We validated the quantitative test through testing for accuracy, reproducibility, repeatability, robustness, and precision. Each of the different tests showed statistical significant results (p: The dynamic range and linearity of the assay were investigated by correlating the qIHC results to an in-house-developed flow cytometry assay as well as an FDA-approved ELISA kit for HER2 measurement. All tests demonstrated statistically significant correlation (p: 0.0002-0.0045). No overlap was seen between the different cell lines and a broad dynamic range could be demonstrated ranging from 1*103 receptors/cell up to 2.5*106 receptors/cell. Furthermore, HER2 was clearly detectable above background in the low expression cell line MDA-MB-468, thus showing the inherent high sensitivity of the qIHC assay. To demonstrate the capability of the qIHC assay in tumor tissue, we tested the qIHC concordance to HER2 IHC in 44 breast cancer specimens. We confirmed a strong correlation between IHC and qIHC (p: Conclusions The results show that qIHC provides a highly sensitive, accurate, quantitative, and reliable assay for protein expression in FFPE tissue, which could provide better and more precise diagnosis and thus improved personalized cancer treatment. Citation Format: Kristian Jensen, Rikke M. Jorgensen, Kenneth H. Petersen, Jesper Lohse, Helene Derand. A new quantitative in-situ IHC method - validation of analytical performance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4568. doi:10.1158/1538-7445.AM2014-4568