Antibody Optimization and Validation of FFPE Tissues Jim Burchette, HT (ASCP) Duke University Medical Center Durham, NC [email protected]
Antibody Optimizationand
Validation of FFPE Tissues
Jim Burchette, HT (ASCP)
Duke University Medical Center
Durham, NC
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