Nsh Teleconference 6.24.09

ImmunohistochemicalTrouble Shooting & Special Techniques

James Burchette, HT (ASCP)Duke University Health System

PO Box 3712 PathologyDurham, NC 27710

burch007@mc.duke.edu

Sponsored by the National Society for Histotechnology

www.nsh.org

June 24, 2009 NSH Teleconference - Troubleshooting IHC

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

Common Pigments

Melanin

Anthracotic

Hemosiderin

Melanin Pigment

Hematoxylin Hematoxylin and Eosinand 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 Pepsin pretreatmentpretreatment

CMV with AEC

PepsinPepsinpretreatmentpretreatment

CMV with Alk. Phos.

HHV-8 and DAB

10 mM citrate 10 mM citrate pH 6.1 @ 100° pH 6.1 @ 100° CC

HHV-8 and Alk Phos

10 mM citrate 10 mM citrate pH 6.1 @ 100° pH 6.1 @ 100° CC

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-1Rabbit Monoclonal Ab clone Rabbit Monoclonal Ab clone SP-4SP-4

Pressure cooker HIER w/ high pH Tris

Spirochetes with DAB

No enhancementNo enhancement

Spirochetes with enhanced DAB

DAB SparkleDAB Sparkle

Spirochetes with Alk Phos

No pretreatmentNo pretreatment

EBV EBNA2

DAB SparkleDAB Sparkle

Pressure Pressure cooker cooker HIER w/ HIER w/ citrate buffercitrate buffer

Double StainApplications

Primary antibody cocktails

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’

Kappa / Lambda

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

Cytokeratin cocktail

AE1/AE3 & CK 8/18AE1/AE3 & CK 8/18

Pepsin pretreatmentPepsin pretreatment

Breast CaBreast Ca

Cytokeratin Cocktail

Colon CaColon Ca

AE1/AE3 & CK AE1/AE3 & CK 8/198/19

Dermatology Cytokeratin Cocktail

Pepsin PretreatmentPepsin Pretreatment

CK 8/18 aka CAM5.2

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

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 - 580CD7 - 580

CD7 - 272CD7 - 272

CD7 clone BC-272

CD7 clone LP15

NCL-CD7-580NCL-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

SkinSkin

TonsilTonsil

Antigen Retrieval

• Be consistent◦ Start with room temperature solutions?

◦ 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

Contaminated Buffer

Clean 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’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

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 1:36,000Divide 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 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.

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

andyour 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

Nsh Teleconference 6.24.09

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