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HORIZON DIAGNOSTICS
Research Use Only
Improving Immunohistochemistry Standardization in your Laboratory
1
Dr Farah Patell-Socha and Dr Vicky Spivey
Research Use Only2
What is the impact of assay failure in your laboratory and how do you monitor for it?
Research Use Only3
Variability in an IHC Workflow
IHC assay• Antigen retrieval methods • antibodies • automated platforms• Detection kits
Sample collection and fixation• Time of fixation• Sample heterogeneity
Accuracy of result interpretation• Quantification of ‘brownness’ • Quantitative assessment
Sectioning• Important to section at
consistent thickness
Antigen Retrieval
FFPE
Antibody Staining Microscopy
Tumor Sample Analysis
Quantitative Digital Pathology
Controls• Availability of material• Consistency• Validated reliable controls
Research Use Only4
Objective
To develop genetically defined IHC Reference Standards with consistent protein expression levels for analytic validation and quantitative assessment of immunohistochemistry assays.
Renewable Reproducible and Consistent Reference Standards
Research Use Only5
IHC HDx Reference Standards – Onslide Controls
Defined cores containing positive and negative protein expressing cell lines on the same slide Extensively characterized cell lines using molecular assays, IHC, Western Blot and FISH Quantitative Digital Pathology (QDP) assessed negative and positive cell line cores
(-)
Biomarker specific Multimarker
Research Use Only6
Production
Single Cell Dilution
Heterogeneous “Wildtype” cell line
Clonal “Wildtype” cell line
Generate a pair of isogenic cell lines that are characterized and validated for IHC and FISH
Clonal mutantcell line
Clonal Wildtypecell line
Evaluation
SNP 6.0
Sanger Sequencing
Droplet Digital PCR
RT-PCR
IHC and FISH
QDP
Research Use Only7
EML4-ALK IHC Staining Results
Core A: Negative by IHC
NSCLC ALK positive tissue control
Core B: Positive by IHC
Participant’s on slide tissue control
Ventana Benchmark XT – D5F3 Antibody (Optiview detection system)
Concordance between cell line and tissue staining results
Research Use Only8
The Importance of Understanding Expected Staining Results
Core A: Negative by IHC
Core B: Positive by IHC
DAKO Autostainer – D5F3 AntibodyVentana Benchmark XT – D5F3 Antibody (Optiview detection system)
99% of the cell were positive in the two laboratories
Highlights variation between methods and detection kits
Research Use Only9
Pre-pilot IHC study: ALK Expression in Reference Standards
Cell lines
Tissue
Concordance between cell line staining results and tissueHighlights need for standardisation across laboratories
Matched Tissue Samples
Research Use Only10
Quality Control
EML4-ALK IHC Reference Standard
H-score is out off a maximum of 300.The percentage of each group is added together to generate the scoreFor example 50% 3+ cells would score 150 (3 x 50 = 150)25% 1+ and 25% 2+ would score 75 (1 x 25 + 2 x 25 = 75)
Quantitative Digital Pathology (QDP) assessed negative and positive cell line cores
Research Use Only11
Key Points
Characterized and genetically defined cell line standards
Concordance between cell line and tissue staining results
Highlights variation between methods and detection kits and highlights the need for external controls
Quantitative Digital Pathology (QDP) assessed negative and positive cell line cores
Research Use Only12
Applications and Features
Confirm specificity and sensitivity of your antibody or probe staining everyday
Available as biomarker specific or multimarker cores mounted
on slides
Routinely monitor the performance of your workflows and assays with independent external controls
Allows you to test your multiplexing assay specificity
and sensitivity
Allows you to monitor the accuracy of your quantitative immunofluorescence assay
IHC HDx™ Reference Standards
Use for Routine Control of your IHC or FISH workflow alongside
your samples
Identify and Control Variability in your IHC
workflows
Provides a common reference point for routine use in ring
studies
Research Use Only13
Characterized consistent, reproducible and renewable Reference Standards are ideal as External Controls
Is your assay optimised?
What is the limit of detection of your
workflow?
Are you interested in achieving in
consistent results?
What is the impact of assay failure in your laboratory and how do you monitor for it?
Research Use Only14
Contact us!
Characterized consistent, reproducible and renewable Reference Standards are ideal as External Controls
Characterized consistent, reproducible and renewable Reference Standards are ideal as External Controls
Contact us:
[email protected]@horizondiscovery.com
