1. Validation and Interpretation in Cellular Pathology
Sarah Dean

2. Lecture Overview Types of Samples Types of Stains
Interpretation of results
Clinical vs Research
How do we ensure those results are valid?
Use of controls
UK NEQAS scheme
Types of analyses/statistical tests

3. Lecture Overview Types of Samples Types of Stains
Interpretation of results
Clinical vs Research
How do we ensure those results are valid?
Use of controls
UK NEQAS scheme
Types of analyses/statistical tests

4. Type of Samples

5. Types of Stain
H&E
Special Stains
IHC

6. IHC ABC Method Biotin Biotin – Conjugated to Peroxidase Avidin
Avidin Biotin Complex (ABC)

7. Interpretation Can be considered subjective
Pathologist must be highly trained
Types of interpretation:
Scoring of IHC results
Grading of a cancer (H&E)
Presence of a marker (positive or negative?)
What about computer software interpretation?

8. Scoring of IHC stained slides
E.g. Oestrogen receptor status in breast cancer

9. Scoring of IHC stained slides
Prognostic vs Predictive

10. Examples for you to try

11. Cancer Grading
E.g. Prostate cancer

12. Prostate cancer H&E

13. Presence or not of a marker
E.g. Amyloid protein
Congo red stain
Apple birerefringence

14. Computer Software interpretation?
Your thoughts?

15. The evidence?

16. The evidence?

17. Lecture Overview Types of Samples Types of Stains
Interpretation of results
Clinical vs Research
How do we ensure those results are valid?
Use of controls
UK NEQAS scheme
Types of analyses/statistical tests

18. Clinical vs Research Interpretation
What do you think the differences are?
“In medicine and statistics, gold standard test refers to a diagnostic test or benchmark that is the best available under reasonable conditions. It does not have to be necessarily the best possible test for the condition in absolute terms.”
Clinical?
Research?

19. Standard Clinical Interpretation examples in cell path
Cancer Grading
Each cancer has it’s own grading system
Immunohistochemical Staining
Biomarkers used in clinical lab have defined scoring systems:
Allred
0-3+ for HER2 in breast cancer
H scoring
Biomarker dependent
Disease dependent
Are considered the “gold standard”

20. How do you decide in a research setting?
New IHC Biomarker
Has someone published with it?
YES NO
In the same disease?
Is their a suggested method from the manufacturer?
YES NO
Great- then use their method*
Is the expected staining pattern similar?
YES NO
Use a similar biomarker scoring system for your disease
Great- then use their method*
YES NO
Use a similar biomarker scoring system for your disease
Great- then use their method*
*Check their staining quality and use of controls

22. Lecture Overview Types of Samples Types of Stains
Interpretation of results
Clinical vs Research
How do we ensure those results are valid?
Use of controls
UK NEQAS scheme
Types of analyses/statistical tests

23. How do we ensure valid results?
How do we ensure scientific “vigour”?
Why Bother???

24. Validating Ab for IHC Antibody Validation
Bordeaux J. et al biotechniques.com

25. Blocking Peptide

26. Western Blotting

27. Use of Controls How do we prove a negative result?
Example: A breast cancer sample is stained for 3 different biomarkers using IHC. The sample is negative for all 3.

28. What can be used as a Positive control?
A piece of tissue known to contain that biomarker
E.g. Amyloid
Infectious agent
Cell line controls
With known expression
Over-expressing

29. We can “prove” a negative result beyond reasonable doubt.

30. UK NEQAS Scheme
How do we ensure validity AND standardisation between clinical labs?
ayer_High.asp
procedure/ an example.

31. Lecture Overview Types of Samples Types of Stains
Interpretation of results
Clinical vs Research
How do we ensure those results are valid?
Use of controls
UK NEQAS scheme
Types of analyses/statistical tests

32. Examples of tests
Does the new method give the same results as the current?
Cohen’s Kappa- level of agreement
Spearman’s or Pearson’s
Is this new method of prognostic value?
Retrospective study
Kaplan Meier survival plot
Correlation with tumour grade/stage
Does this new method predict response to therapy
Clinical Trials

33. Lecture Overview Types of Samples Types of Stains
Interpretation of results
Clinical vs Research
How do we ensure those results are valid?
Use of controls
UK NEQAS scheme
Types of analyses/statistical tests