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Immunohistochemical Trouble Shooting and Special Techniques
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ImmunohistochemicalTrouble Shooting & Special Techniques
James Burchette, HT (ASCP)Duke University Health System
PO Box 3712 PathologyDurham, NC 27710
Sponsored by the National Society for Histotechnology
www.nsh.org
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Objectives
Upon completion of this teleconference, participants will be responsible to:
♦ Implement IHC enhancement techniques
♦ Improve IHC trouble shooting
♦ Utilize this presentation for improving math and general lab science skills
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Common Pigments
Melanin
Anthracotic
Hemosiderin
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Melanin Pigment
Hematoxylin Hematoxylin and Eosinand Eosin
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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HMB-45 & DAB
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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HMB-45 with Giemsa
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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HMB-45 with Alk. Phos IHC
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Lung H&E
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Anthracotic and Hemosiderin Pigment
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Difficulty with CMV diagnosis
Pepsin Pepsin pretreatmentpretreatment
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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CMV with AEC
PepsinPepsinpretreatmentpretreatment
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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CMV with Alk. Phos.
Pepsin Pepsin pretreatmentpretreatment
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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CMV with Alk. Phos.
Pepsin Pepsin pretreatmentpretreatment
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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HHV-8 and DAB
10 mM citrate 10 mM citrate pH 6.1 @ 100° pH 6.1 @ 100° CC
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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HHV-8 and Alk Phos
10 mM citrate 10 mM citrate pH 6.1 @ 100° pH 6.1 @ 100° CC
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Enhancement of DAB
Why enhance?
Intensify a weak signal
Obtain better contrast
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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DAB Enhancing Reagents
Proprietary solutions
1% Copper sulfate
0.05% Nickel chloride
0.05% Cobalt chloride
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Cyclin D-1Rabbit Monoclonal Ab clone Rabbit Monoclonal Ab clone SP-4SP-4
Pressure cooker HIER w/ high pH Tris
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Spirochetes with DAB
No enhancementNo enhancement
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Spirochetes with enhanced DAB
DAB SparkleDAB Sparkle
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Spirochetes with Alk Phos
No pretreatmentNo pretreatment
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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EBV EBNA2
DAB SparkleDAB Sparkle
Pressure Pressure cooker cooker HIER w/ HIER w/ citrate buffercitrate buffer
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Double StainApplications
Primary antibody cocktails
and
Cocktails of detection reagents
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Antibody Cocktails for Double Staining
• Mouse and rabbit primary antibodies• Use the same control tissue for both ab’s• Work up each antibody separately• Prepare the cocktail solution of both ab’s• Trial IHC run• Adjust titer accordingly• Repeat on control and type of case
material used in double stain• Validate • Report disclaimer
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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CD3 and CD20
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Mart-1 and Ki-67
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‘Mart-67’
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PIN4
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Kappa / Lambda
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Single LabelAntibody Cocktails
• AE1/AE3 and CAM 5.2 (CK 8/18)
• Dermpath Cytokeratin CocktailAE1/3, CK 8/18 & MNF116
• Mart-1 and HMB-45
• Commercial and ‘homebrew’ products
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Cytokeratin cocktail
AE1/AE3 & CK 8/18AE1/AE3 & CK 8/18
Pepsin pretreatmentPepsin pretreatment
Breast CaBreast Ca
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Cytokeratin Cocktail
Colon CaColon Ca
AE1/AE3 & CK AE1/AE3 & CK 8/198/19
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Dermatology Cytokeratin Cocktail
Pepsin PretreatmentPepsin Pretreatment
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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CK 8/18 aka CAM5.2
Pepsin Pepsin pretreatmentpretreatment
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Use of Proteolytic Enzymes
Why use them?
JB’s favorites
• 0.25% Pepsin
• 0.25% Trypsin
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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0.25% Pepsin
• 398 ml Tris Buffered Saline w/ Tween-20
• pH to 2.0 with HCl
• Add 1 gram of porcine pepsin (Sig. P7012)
• Use at 37-40° C for 10-15 minutes
• For best results, apply at RT
• Store at 4° C.
• Stable at least 6 months
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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D2-40
Lymphatic endothelium
Mesothelioma
Trypsin pretreatment
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Mart-1 and HMB-45
Broad spectrum of specificity
Easy to prepare
Retrieval is 10 mM citrate HIER solution
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Mart-1 & HMB-45
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Mart-1 & HMB-45
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Antibody Improvements
• CD3 rabbit monoclonal, clone SP7
• Cyclin D-1 (Bcl-1) rabbit monoclonal,
clone SP4
• CD7-LP15 compared to CD7-BC272
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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CD3 Rabbit Monoclonal
Clone SP7
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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CD7
CD7 - 580CD7 - 580
CD7 - 272CD7 - 272
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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CD7 clone BC-272
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CD7 clone LP15
NCL-CD7-580NCL-CD7-580
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Improved CD4 Detection
• Use less concentrated H202
• Endogenous peroxidase blocking can be performed prior to detection or chromogen
• Use a chromogen enhancing solution
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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CD4
SkinSkin
TonsilTonsil
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Antigen Retrieval
• Be consistent◦ Start with room temperature solutions?
or
◦ Start with preheated solutions?
• Monitor your temperature
• Check solution pH
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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General Laboratory Trouble Shooting
• Be consistent with what you do
• Use a check list for dilutions and layout
• Run comparison techniques
• Trust your trained eye and follow up
• Listen to yourself and follow up
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Trouble Shooting
• Keep your stainers clean
• Wash buffer and water carboys regularly◦ Bad buffer causes background
• Use clean antibody vials
• Implement one change or variable
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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pH Meter
• Learn to properly use the pH meter
• Use proper electrode storage solution
• Learn to perform a slope adjustment
• Do not reuse calibrations more than 1 day
• Keep solution containers clean
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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pH Meter Use
• Use an electrode compatible with Tris based buffers
• Refillable pH electrodes can be rejuvenated
• Does not have to be a complex purchase
• Purchase a pH meter with a temperature compensation feature
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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S100 Background
Contaminated Buffer
Clean Buffer
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Presentation of Cut Sections
• Use your artistic talents
• Good microtomy makes good IHC slides
• Specimen orientation on the slide
• Properly dry your tissue sections!– Air drying prior to heating helps adhesion
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Positioning of Tissue
• Aesthetics
• Time saving for Pathologist
• Shows professionalism
• Centered sections
for automation
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Math Trouble Shooting
• Laboratory math is part of our job
• Develop good math skills
• Make a check list
• Learn basic formulas – it’s not hard
• Check your math for accuracy
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Metric Volume Review
1.0 liter = 1000 ml’s1.0 liter = 1,000,000 ul’s0.1 liter = 100 ml’s (one deciliter)0.001 liter = 1.0 milliliter (ml)
1.0 ml = 1000 micro liters (µl)0.1 ml = 100 µl0.01 ml = 10 µl0.001 ml = 1 ul
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Antibody Dilutions
Basic rule of thumb
Desired volume divided by the dilution factor gives you the amount of antibody needed. Subtract the ab amount from the volume for the amount of diluent needed.
Ab + diluent amounts = total volume
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Cytokeratin 1:50
1. Volume needed: 0.5 ml (500 µl)
2. Dilution factor divided into volume = 10
3. 10 µl subtracted from 500 µl = 490 µl
4. 490 µl (.490 ml) + 10 µl ab = 500 (0.5 ml)
5. Check your math, diluent volume + ab volume = total volume
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Preparation from 1:10 stock
Prepare a 1:10 antibody stock (10 µl ab + 90 µl diluent).
FVIII antibody working dilution is 1:36,000Divide 36,000 ÷ 10 = 3600
Prepare a 1:3600 dilution from your 1:10 stock
Do the math: 10 x 3600 = 36,000
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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REFERENCES• Antigen Retrieval Techniques: Immunohistochemistry and Molecular Pathology. S-
R Shi, J Gu and Clive R Taylor. Eaton Publishing, BioTechniques Books Division, 2000
• Immunohistopathology, A practical approach to Diagnostics. Jules Elias. Second edition 2003, ASCP Press, ISBN # 0-89189-300-8
• Handbook, Immunochemical Staining Methods, Fourth edition DAKO Corp, Carpentiera CA. Hard copy and available online http://pri.dako.com/08002_25may06_ihc_guide_book.pdf
• Antigen RetrievalTechniques. J Burchette. HistoLogic, November 2007http://www.sakura-americas.com/histologic/pdf/07_dec.pdf
• Diagnostic Atlas of Genitourinary Pathology, Chapter 6, Prostate, JF Madden, JL Burchette, M Tannenbaum., Churchill Livingston Elsevier, 2006
• Pathology of Parainfluenza Virus Infection in Patients with Immunodeficiency Syndromes. JF Madden, JL Burchette, LP Hale. Human Pathology. Vol. 35, No. 5, pages 594-603, May 2004.
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REFERENCES• The Spectrum of C-KIT (CD117) Immunoreactivity in Lung and Pleural Tumors: A
Study of 96 Cases Using a Single-Source Antibody With a Review of the Literature. KJ Butnor, JL Burchette, TA Sporn, SP Hammar, VL Roggli. Archives of Pathology and Laboratory Medicine, Vol. 128, No. 5, pages 538-543, May 2004.
• Fatal Disseminated Adenovirus Infections in Immunocompromised Patients. T Pham, JL Burchette, N Henshaw, LP Hale. American Journal of Clinical Pathology. Vol. 120, 2003. Pages 575-583.
• Immunohistochemical Detection of Helicobacter pylori. J Burchette. Histo-Logic, Vol. XXVII, No. 1, March 1997.
• Adherence of Helicobacter pylori to Areas of Incomplete Intestinal Metaplasia in the Gastric Mucosa. RM Genta, IE Gurer, DY Graham, B Krishnan, AM Segura, O Gutierrez, JG Kim, JL Burchette. Gastroenterology, Vol. 111, pages 1206-1211, 1996.
• Recent Advances in Automated Immunocytochemistry. JC Iezzoni, LJ Manahan, CS Park, DJ Brigati. Journal of Histotechnology, Vol. 16, No. 1, March 1993.
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Thank You
Support National Society for Histotechnology
andyour state histology society
Questions?
June 24, 2009 NSH Teleconference - Troubleshooting IHC
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Next 2009 NSH Teleconferences• July 22 – Staining and Identification of
Pigments and Minerals in Tissue – Debra Wood, MS, HT(ASCP)
• August 26 – Role of Immunohistochemistry in the Diagnosis of Lymphoma
• September – No teleconference due to NSH Symposium/Convention October 3-8 in Birmingham, Alabama