Validation and Interpretation in Cellular Pathology

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Presentation transcript:

Validation and Interpretation in Cellular Pathology Sarah Dean

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

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

Type of Samples

Types of Stain H&E Special Stains IHC

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

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?

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

Scoring of IHC stained slides Prognostic vs Predictive

Examples for you to try

Cancer Grading E.g. Prostate cancer

Prostate cancer H&E

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

Computer Software interpretation? Your thoughts?

The evidence?

The evidence?

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

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?

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”

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

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

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

Validating Ab for IHC Antibody Validation Bordeaux J. et al. 2010 biotechniques.com

Blocking Peptide

Western Blotting

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.

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

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

UK NEQAS Scheme How do we ensure validity AND standardisation between clinical labs? http://www.ukneqas.org.uk/images/Flash/FlashPl ayer_High.asp http://www.ukneqasiccish.org/assessment- procedure/ an example.

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

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

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