This document discusses the development of immunohistochemistry (IHC) reference standards using genetically defined cell lines to improve standardization and quality control in IHC laboratories. The reference standards consist of cell line cores containing positively and negatively expressing proteins mounted on the same slide. The cell lines are extensively characterized and shown to produce consistent staining results across laboratories and detection methods. Using quantitative digital pathology, the reference standards allow laboratories to routinely monitor assay performance and identify variability in their IHC workflows.
Analytical Profile of Coleus Forskohlii | Forskolin .pptx
HORIZON IHC STANDARDIZATION
1. HORIZON DIAGNOSTICS
Research Use Only
Improving Immunohistochemistry
Standardization in your Laboratory
1
Dr Farah Patell-Socha and Dr Vicky Spivey
2. Research Use Only2
What is the impact of assay failure in
your laboratory and how do you
monitor for it?
3. 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
4. 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
5. 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
6. 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 mutant
cell line
Clonal
Wildtype
cell line
Evaluation
SNP 6.0
Sanger Sequencing
Droplet Digital PCR
RT-PCR
IHC and FISH
QDP
7. 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
8. 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
9. Research Use Only9
Pre-pilot IHC study: ALK Expression in Reference Standards
Cell lines
Tissue
Concordance between cell line staining results and tissue
Highlights need for standardisation across laboratories
Matched Tissue Samples
10. 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 score
For 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
11. 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
12. 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
13. 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?
14. 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:
f.patell-socha@horizondiscovery.com
v.spivey@horizondiscovery.com