IHC Troubleshooting Techniques

Immunohistochemical
Trouble Shooting &
Special Techniques
James Burchette, HT (ASCP)
Duke University Health System
PO Box 3712 Pathology
Durham, NC 27710
burch007@mc.duke.edu

Sponsored by the National Society for
Histotechnology
www.nsh.org

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 2

Common Pigments

Melanin

Anthracotic

Hemosiderin


Melanin Pigment

Hematoxylin
and Eosin


HMB-45 & DAB


HMB-45 with Giemsa


HMB-45 with Alk. Phos IHC


Lung H&E


Anthracotic and Hemosiderin
Pigment


Difficulty with CMV diagnosis

Pepsin
pretreatment


CMV with AEC

Pepsin
pretreatment


CMV with Alk. Phos.

Pepsin
pretreatment


HHV-8 and DAB

10 mM citrate
pH 6.1 @ 100° C


HHV-8 and Alk Phos

10 mM citrate
pH 6.1 @ 100° C


Enhancement of DAB

Why enhance?
Intensify a weak signal

Obtain better contrast


DAB Enhancing Reagents

Proprietary solutions
1% Copper sulfate
0.05% Nickel chloride
0.05% Cobalt chloride


Cyclin D-1
Rabbit Monoclonal Ab clone SP-4

Pressure cooker HIER w/ high pH Tris


Spirochetes with DAB

No enhancement


Spirochetes with enhanced DAB

DAB Sparkle


Spirochetes with Alk Phos

No pretreatment


EBV EBNA2

DAB Sparkle

Pressure cooker
HIER w/
citrate buffer


Double Stain
Applications

Primary antibody cocktails
and
Cocktails of detection reagents


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

CD3 and CD20


Mart-1 and Ki-67


‘Mart-67’


PIN4


Kappa / Lambda


Single Label
Antibody Cocktails

• AE1/AE3 and CAM 5.2 (CK 8/18)
• Dermpath Cytokeratin Cocktail
AE1/3, CK 8/18 & MNF116

• Mart-1 and HMB-45
• Commercial and ‘homebrew’ products

Cytokeratin cocktail
Breast Ca

AE1/AE3 & CK 8/18

Pepsin pretreatment

Cytokeratin Cocktail
Colon Ca

AE1/AE3 & CK 8/19


Dermatology Cytokeratin
Cocktail

Pepsin Pretreatment


CK 8/18 aka CAM5.2

Pepsin
pretreatment


Use of Proteolytic Enzymes
Why use them?

JB’s favorites

• 0.25% Pepsin

• 0.25% Trypsin


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


D2-40
 Lymphatic endothelium

Mesothelioma 

Trypsin pretreatment

Mart-1 and HMB-45

Broad spectrum of specificity
Easy to prepare

Retrieval is 10 mM citrate HIER
solution

Mart-1 & HMB-45


Antibody Improvements

• CD3 rabbit monoclonal, clone SP7
• Cyclin D-1 (Bcl-1) rabbit monoclonal,
clone SP4
• CD7-LP15 compared to CD7-BC272


CD3 Rabbit Monoclonal

Clone SP7


CD7

CD7 - 272

CD7 - 580


CD7 clone BC-272


CD7 clone LP15

NCL-CD7-580


Improved CD4 Detection
• Use less concentrated H202
• Endogenous peroxidase blocking can be
performed prior to detection or chromogen
• Use a chromogen enhancing solution


CD4

Skin

Tonsil


Antigen Retrieval
• Be consistent
◦ Start with room temperature solutions?
or
◦ Start with preheated solutions?

• Monitor your temperature
• Check solution pH


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


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


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


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


S100 Background

 Clean Buffer

Contaminated Buffer 


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


Positioning of Tissue
• Aesthetics

• Time saving for
Pathologist

• Shows
professionalism

• Centered sections
for automation


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


Metric Volume Review
1.0 liter = 1000 ml’s
1.0 liter = 1,000,000 ul’s
0.1 liter = 100 ml’s (one deciliter)
0.001 liter = 1.0 milliliter (ml)

1.0 ml = 1000 micro liters (µl)
0.1 ml = 100 µl
0.01 ml = 10 µl
0.001 ml = 1 ul

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


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

Preparation from 1:10 stock
Prepare a 1:10 antibody stock (10 µl ab +
90 µl diluent).
FVIII antibody working dilution is 1:36,000
Divide 36,000 ÷ 10 = 3600
Prepare a 1:3600 dilution from your 1:10
stock
Do the math: 10 x 3600 = 36,000


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 2007
http://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.


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.


Thank You
Support National Society for
Histotechnology
and
your state histology society
Questions?

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

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