Ab Optimization & Validation

Antibody Optimizationand

Validation of FFPE Tissues

Jim Burchette, HT (ASCP)

Duke University Medical Center

Durham, NC

burch007@mc.duke.edu

Antibody Optimization

Chromogranin, Pheochromocytoma

It’s not all black and white

Getting Started

• Prepare a word document for lab notes

• Visit the manufacturers web site

• Read & print the product information

• Obtain information such as:– Ab. Species– Isotype– Clone– Ig concentration

Antibody Data Sheet

• Review the data sheet for:– Reaction pattern– Recommended dilution– Pretreatment recommendations– Reactivity in neoplastic tissues– Reactivity in normal tissues

References

• Accumulate references that use the same antibody clone

• Print copies for file

• Electronic links

• Include references in your lab notes

Tissues

• Pathologist involvement is needed

• Obtain needed tissues & cases– Cut fresh sections

• Use weak and strong expressing tissues

• Neoplastic and normal tissues that contain immunoreactive components

Antibody Dilutions

• Manufacturers recommendation– Bracket– Very dilute

HIER Pretreatment

• Manufacturers recommendation– Prove it!

• Try different HIER solutions– Citrate vs. high pH solutions

• Compare heat sources– High temp water bath– Pressure cooker– On line retrieval– Overnight retrieval

High Temp Water bath

Pressure Cooker

Pretreatment Module

PT Module Display

HIER Solutions

• 10 mM citrate, pH 6.0

• 10 mM EDTA, pH 8.0

• 10 mM Tris, pH 9.5

• 10 mM Tris & 1 mM EDTA, pH 9.0

E cadherin Tris / EDTA

Cyclin D-1 10 mM Tris PC

CD5 ER2 (20’) EDTA

ER1 (20’) Citrate

Parathyroid hormone

Citrate buffer DOG-1

Citrate bufferCD3 & CD20

Enzyme Pretreatment?

• Why enzyme? You never know

• 0.25% pepsin, pH 2.0

• 0.25% trypsin, pH 7.8

• Other proteolytic enzymes

Pepsin

• 0.25%, pH 2.0 prepared in TBS

• 15 minutes at 37-40° C

• Note: freeze aliquots

Respiratory Syncitial Virus Pepsin

PepsinCytomegalovirus

Varicella Zoster Virus Pepsin

Cytokeratin 20 Pepsin

Trypsin

Part A. 1% trypsin at pH 3.5

Part B. 0.1% CaCl2 TBST buffer, pH 7.8

Mix 1 part A and 3 parts B

Apply to sections for 15 minutes at

37-40° C

D2-40 Trypsin

D2-40 Trypsin

Adenovirus Trypsin

Pro-collagen type 1

Trypsin

Detection systems

• Research or clinical?

• Avidin / Biotin system

• Non-biotin polymer system

Verification

• Review the finished product for proper reactivity and pattern

• Always look for immunoreactive components

• Repeat testing for reproducibility

• Move test to the routine bench for consistency

Verification

• Prepare a bulk antibody supply and test for stability and performance

• Use the same control tissue of previous cut and fresh cut sections for weekly testing

Verification

• Continue adding and documenting positive cases

• Date your finished slides

• File validation slides under the run date

• Enter the run date & info in lab notes

The finished product

Renal cell carcinoma Citrate buffer

HBcAg & HBsAg No pretreatment

Cytomegalovirus Pepsin

SV40 Citrate buffer

Parvovirus B-19 Pepsin

Toxoplasma gondii Citrate retrieval

EGFr vlll ER2 (20’) EDTA

Derm CK cocktail Pepsin

Validation

3+ Her2Neu

CAP Guidelines

• ANP.22750 Has the laboratory documented evaluation of new antibody lots and new antibodies, prior to use in patient diagnosis?

“Based on FDA’s ruling on class reagents in IHC, the legal responsibility for validation and knowing the relevant parameters is put squarely on the shoulders of the lab director.”

Neal S. Goldstein, MD

Validation, Getting started

• Pathologist participation

• How many cases?

• Continue the documentation on your previously prepared word document

• Communication– Email– Sign off forms

Validation

• Automated IHC platforms

• Manual IHC methodology

• Both must be validated if test is performed with both technologies

Validation

• “Much of antibody test validation is dependant on the confidence of the pathologist and the quality of the IHC lab”

R Cartun, PhD

• Proper validation will give you confidence in the test and results

Validation

• Involvement by the pathologist and the technologist is vital to proper validation and subsequent trouble shooting

• CAP MK proficiency testing program

• Not all immunohistochemical tests are checked by the MK PT series, consider performing in house PT every 6 months

• How many cases?

Example: ER, PR & Her2 Neu

25 high expression

25 moderate expression

25 low expression

25 negative

Quantitative results may require more

The number of cases needed depends on the antibody being validated

Example:

CD246, ALK-1

Cyclin D-1

Antibody Comparison

• Compare different clones

• Same clone, different company

• IVD vs. ASR vs. RUO

New Antibody Lot QC

• Review spec sheet for Ig and protein concentration

• Run on a historically known positive control section

• Document your results

Control Block Validation

• Fixation of control blocks

• Not all tonsils are created equal

• Test for reactivity

• Test an early cut and a last cut

Attachements

• Trypsin SOP

• Pepsin SOP

• HIER formulas

• References

Thank you

Jim Burchette

919-681-3973

burch007@mc.duke.edu