IMMUNOHISTOCHEMISTRY

Ansarie P. Salpin, MD, RMT

Outline
Definition

Applications
Procedure Technical Problems and Troubleshooting Factors Affecting Immunogenicity Markers of Differentiation Actual Case

Immunohistochemistry
HISTOCHEMISTRY + ANTIBODY

A method of localizing specific

antigens in tissues or cells based on antigen-antibody recognition

Applications of IHC
1. Diagnosis of Tumors Classification of poorly differentiated neoplasm Diagnosis of Carcinoma of Unknown Primary Diagnosis of Invasion 2. Assessment of Marker Reflecting Prognosis (prognostic markers) 3. Assessment of Markers Reflecting Therapeutic Response (predictive or theranostic markers) 4. Detection of Micrometastasis 5. Identification of Infectious Organism

Steps in IHC
1.Deparaffinization with xylene 2. Rehydration 3. Antigen Retrieval 4. Washing with PBS 5. Blocking 6. Primary Antibody 7. Washing 8. Secondary Antibody 9. Chromogen + Substrate 10. Washing 11. Counterstain with Hematoxylin

FIXATION
The tissue must be fixed within 30

minutes after removal. 6-48 hours Type of fixatives 1. Coagulant 2. Crosslinking- 10% neutral buffered formalin

Advantages of 10% NBF

Good preservation of morphology even after

prolonged fixation Economical Sterilizes tissue specimens in a more reliable way than precipitating fixatives, particularly for viruses Carbohydrate antigens are well preserved Crosslinking of protein in situ avoids leaching out of proteins that may diffuse in water or alcohol. - LMW antigen are extracted in alcohol, but preserved in formalin.

ANTIGEN RETRIEVAL
Unmasking the antigenic site or epitope Three factors- temperature, length of time and pH Heating method- microwave, microwave and

pressure cooker, steam or autoclave heating method -100 degress Celsius for 10 minutes Antigen Retrieval Solution - 0.05% citraconic anhydradide solution at pH 7.4, heating at 98 degrees Celsius for 45 minutesuniversal AR - Nuclears antigens- use low pH - HMB45 and others- high pH

Estrogen Receptor Assay

BLOCKING NONSPECIFIC BINDING

Polyclonal Antibody

Endogenous Enzyme Activity (Label)

Blocking Agents

Methanol-H2O2 H2O2 in sodium azide

DETECTION SYSTEM
Direct Conjugate-Labeled Antibody Method

Indirect or Sandwich Method

Unlabeled Antibody Methods/ Enzyme Bridge

Method

Direct Conjugate-Labeled Ab Method

Advantage- rapidity and ease of performance Disadvantage- needs to conjugate each primary Ab separately

Sandwich Method

Enzyme Bridge Technique

-Widely used -Avoids inherent problems with chemical conjugation -100-1000x more sensitive

Technical Problems and Troubleshooting

ABSENCE OF STAINING OF

BOTH SPECIMEN AND CONTROL 1. Check if the procedure is followed correctly- omitted rgt 2. Check the expiration dates and storage of reagents

Absence or Weak Staining of Specimen with Appropriate Staining of Positive Control

PROBLEM
Inadequate fixation Incomplete dehydration Paraffin too hot

SOLUTION
Avoid delay of fixation or over-fixation Perform regular rgt change (alcohol) Monitor paraffin temperature (<60 degrees Celsius) Optimize Antigen Retrieval Time

Prolonged or Excessive Heating

Chromogen Deposits

Melanophage

Factors Affecting Immunogenicty

Type of Fixative

Length of Time of Fixation

Prior Decalcification with HCl- affects nuclear

antigen Temperature Length of Time Since the Block was Cut Antigen Retrieval Procedure Type of Antibody

Markers of Differentiation

CASE HISTORY
41 y.o./female G4P4 10 month Hx of vulvar mass,

associated with Dyspareunia Vaginal Bleeding Weight loss (35%)

Physical examination

gross appearance

Microscopic appearance

Microscopic appearance

Microscopic appearance

Microscopic appearance

Microscopic appearance

LYMPH NODE METS

LYMPH NODE METS

 HIGH GRADE NEOPLASM,

CONSIDERATIONS ARE: 1. AMELANOTIC MELANOMA 2. SARCOMATOID CARCINOMA 3. HIGH GRADE SARCOMA

cytokeratin

vimentin

S-100

Hmb-45