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Escuela Tecnología Medica

Análisis Tisular III
Alumna Nicole Cotrina Vera

THE IMPORTANCE OF EPITOPE
DENSITY IN SELECTING A
SENSITIVE POSITIVE IHC CONTROL
Kodela Vani, Seshi R. Sompuram, Anika K. Schaedle, Anuradha Balasubramanian, Monika Pilichowska, Stephen Naber, Jeffrey D. Goldsmith,
Kueikwun G. Chang,* Farzad Noubary, and Steven A. Bogen*
Medical Discovery Partners LLC, Boston, Massachusetts (KV, SRS, AKS, AB, SAB); The Department of Pathology & Laboratory Medicine (MP, SN, SAB)
and The Institute for Clinical Research and Health Policy Studies (FN), Tufts Medical Center, Boston, Massachusetts; Tufts Clinical and Translational
Science Institute, Tufts University, Boston, Massachusetts (FN); The Department of Pathology, Boston Children’s Hospital, Boston, Massachusetts (JDG);
and The Department of Pathology, Lahey Hospital and Medical Center, Burlington, Massachusetts (KGC)

INTRODUCCIÓN Control Analito Epitopo Positivo .

. INTRODUCCIÓN • In this study. we address the question of how these uncontrolled analyte concentrations affect the sensitivity of IHC controls in detecting primary antibody degradation.

The analyte coated microbeads bear covalently linked peptid epitopes for HER-2. and PR tests are represented HER2 PR ER • Photomicroscopy . peptide analytes for all of the major clinical HER-2. Among all of the various IHControls products used in this study. MATERIALES Y MÉTODOS • IHControls IHControls are comprised of two different microbeads: analyte-coatedglass test microbeads (7–8 micron diameter) and colorstandard microbeads (4. ER. ER.5 micron diameter). and/or PR.

• Tissue Controls For ER and PR.0 means that the test microbeads. A score ≥1 represents a strong stain intensity. stained for HER- 2. HER-2 controls were comprised of two different breast carcinomas. are equally intense in color (expressed in mean pixel intensity) as the color standard microbeads. paraffin . MATERIALES Y MÉTODOS • IHControls Stain Intensity Image Quantification IHControls stain intensity is expressed as a ratio. A score of 1. or PR.embedded. • ER/PR Image Quantification of Tissue Sections • HER2 Image Quantification of Tissue Sections . normal uterine endometrium was used. archival formalin-fixed. one expressing HER-2 at a 3+ level and another expressing HER-2 at a 1+ level. ER. For ER and PR tissue controls. as they express lower levels of ER and PR than the glandular epithelium. We measured only the stromal cells.

For HER-2. • Pathologist’s Assesment of Controls • Statistical Analysis . the manufacturer’s reagents. MATERIALES Y MÉTODOS • Inmunohistochemistry Staining Antigen retrieval was performed using Dako’s antigen retrieval solutions provided with the HER-2 or ER/PR kits. buffers. Pacheco. as per the manufacturer’s instructions. For all subsequent steps. the slide were processed for 25 min in a Biocare Medical Decloaking Chamber (Biocare Medical. and instructions were followed. For ER/PR antigen retrieval. antigen retrieval was performed in a 97C to 98C water bath for 40 min. CA) pressure cooker.

correlating immunohistochemical stain intensity (y-axis) as a function of HER-2 concentration (x-axis). Both tissue controls and IHControls are mounted on the same slide. that is. (B) Graphical representation of IHControls stain intensity after diluting the HercepTest primary antibody by the factor indicated on the x-axis. . (C) Graphical representation of tissue section control stain intensity after the same primary antibody dilutions. The rate of decline in stain intensity is apparent as the slope of the regression lines. molecules of equivalent fluorochrome. (A) HercepTest response curve with IHControls. Abbreviations: HER-2. three independent staining replicates. Each data point is the mean ± SD of triplicate slides. traceable to units of MEF. RESULTADOS Figure 1. HER-2 concentration (x-axis) is expressed in units of molecules per microbead. MEF. human epidermal growth factor receptor type II.

Photomicrographs of the IHControls bearing 8187 molecules (MEF) HER-2. Scale bar 10 µm. after immunostaining with HercepTest at various primary antibody dilutions. Examples of a stained microbead bearing HER-2 and a color standard microbead are highlighted. The photomicrographs also illustrate the smaller color standard microbeads to which the stained test microbeads are compared. Figure 2A illustrates the appearance under normal conditions. The faint microbead outlines of unstained microbeads are due to light refraction.Figure 2. An example of an unstained microbead that bears an antigenically irrelevant analyte is highlighted. molecules of equivalent fluorochrome. Figure 2B and C illustrate representative immunostaining with the indicated primary antibody dilution. . not staining. S1. HER-2. A similar set of images using a slightly shorter photomicrographic exposure is shown in Fig. human epidermal growth factor receptor type II. Abbreviations: MEF. without primary antibody reagent dilution.

Photomicrographs of tissue control immunostaining with HercepTest after the indicated primary antibody dilutions. The images chosen for illustration were selected as representing the dilutions showing both extremes of staining and an intermediate dilution. . 1C.Figure 3. Scale bar 10 µm. These images are representative examples from the data set associated with Fig. Panels D to F are of the HER-2 low (1+) tissue control. These dilutions differ for the 3+ and 1+ tissue controls. Panels A to C are of the human epidermal growth factor receptor type II (HER-2) high (3+) tissue control.

Panel A: Pass rate of the IHControls (y-axis) after increasing primary antibody dilutions (x-axis). without any visually appreciable decrement. Pathologists’ interpretation of the HER-2 IHControls and tissue controls for detecting immunostaining problems. The low (1+) control fails at a lower primary antibody dilution. Each data point in both panels is the mean ± SD of triplicate slides. Panel B: Pass rate of the high (3+) and low (1+) tissue controls after increasing primary antibody dilutions. “Pass” means that the stain intensity appears normal.Figure 4.913 molecules IHControl group (green line). The pathologists fail the 8187 molecules IHControl group (purple line) at lower dilutions than the 77. human epidermal growth factor receptor type II. Abbreviation: HER-2. .

Figure 5. The dilution is indicated along the x-axis. correlating immunohistochemical stain intensity (y-axis) as a function of ER (Panel A) or PR (Panel D) concentration (x-axis). Panels C and F: Graphical representation of a tissue controls’ stain intensity after the same primary antibody dilutions. Panels D to F represent the PR immunostain using mAb 1294. each data point is the mean ± SD of triplicate slides. For all panels. Sensitivity analysis of ER and PR controls. . Panels A to C represent the ER immunostain using mAbs 1D5 and 2-123. Panels B and E: Graphical representation of stain intensity after diluting the ER (Panel B) or PR 1294 (Panel E) primary antibodies. Panels A and D: Analytic response curves. Abbreviation: MEF. molecules of equivalent fluorochrome.

Each data point is the mean ± SD of triplicate slides. . Panel A: Pass rate of the 77. Pathologists’ interpretation of the PR IHControls and tissue controls for detecting immunostaining problems.Figure 6. Panel B: Pass rate of the tissue control after increasing primary antibody dilutions.913 molecules IHControl group (y-axis) after increasing primary antibody dilutions (x-axis).

This is the region where the stain intensity exhibits the greatest change in response to increases or decreases in analyte concentration. . DISCUSIÓN • This article highlights the importance of selecting positiveIHC controls having an optimal concentration of analyte. • Our data indicatethat the most sensitive positive IHC controls have analyteconcentrations close to the concentration-dependent region of the analytic response curve.

DISCUSION • The important parameter affecting the sensitivity of a control appears to be the amount of immunoreactive analyte. We were then able to compare the changes in stain intensity for the IHControls and tissue controls. . regardless of the solid phase support to which it is attached. • IHControls and tissue controls were mounted on the same slides during the experiments. They were exposed to the same immunostaining conditions. • We were able to compare the sensitivities of IHControls and tissue controls by comparing them side by side.

. and prepare. validate. -Are labor-intensive to procure. Advantages Drawbacks -Tissue controls suffer from unpredictable and unknown analyte concentrations. -Tissue controls most closely resemble the matrix of patient samples.