DIRECT TECHNIQUE

MLS 013: HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES

○ The TRADITIONAL TECHNIQUE used that conjugates the primary
LESSON TITLE: IMMUNOHISTOCHEMISTRY antibody to the label such as fluorochrome or Horseradish peroxidase
enzyme

IMMUNOHISTOCHEMISTRY
INDIRECT TECHNIQUE

○ Use of antibodies as histological tools for identifying patterns of

○ MORE SENSITIVE than the traditional direct technique
antigen distribution within an organism and tissue
○ Immunohistochemical techniques are NOW ROUTINELY used for the
Horseradish peroxidase → is the most commonly used enzyme for direct
identification of specific or highly selective antigens/epitopes in
antibody enzyme-complex techniques
frozen or paraffin-embedded tissues
○ Two or three step procedure that involves the application of
IgG → is the most commonly used antibody for immunohistochemistry
unconjugated primary antibody, followed by a labeled antibody directed
against the first antibody

PEROXIDASE-ANTIPEROXIDASE TECHNIQUE

○ An INDIRECT ANTIBODY ENZYME-COMPLEX TECHNIQUE where

the soluble peroxidase-antiperoxidase complex is bound to
unconjugated primary antibody (anti-human IgG) by a second layer of
“bridging” antibody, usually a swine anti-rabbit antibody, that then binds
to both the primary antibody and the rabbit PAP complex

Horseradish peroxidase → is the most commonly used enzyme for labeling

○ Combining Horseradish peroxidase with the most common

chromogen, Diaminobenzidine (DAB), results in a stable, insoluble
dark brown reaction end product when antigen is present in the tissue

AVIDIN-BIOTIN COMPLEX (ABC) TECHNIQUE

○ Uses avidin (which is derived from egg white) because of its marked
affinity for biotin, a low molecular weight vitamin that can be easily
conjugated to antibodies and enzyme markers

○ Basic sequence of the staining procedure consists of primary

PREPARATION OF ANTIBODIES (REAGENT) antibody, biotinylated secondary antibody followed either by
performed Streptavidin-biotin-enzyme complex or by a labeled
streptavidin
1. Polyclonal antibodies
⇨ produced by immunizing an animal with an immunogen that
contains the antigen of interest LABELED STREPTAVIDIN AVIDIN BIOTIN (LSAB) TECHNIQUE
⇨ MOST FREQUENTLY USED ANIMAL:
★ Rabbit
○ A labeled avidin-biotin (LAB) method has been RECENTLY
★ Goat
INTRODUCED and is found to be 4 TO 8 TIMES MORE SENSITIVE
★ Sheep
than the old ABC method
★ Horse
★ Guinea pig
○ The staining sequence consists of primary rabbit or mouse antibody,
biotinylated anti-rabbit / anti-mouse immunoglobulin and
2. Monoclonal antibodies
streptavidin-enzyme conjugate. The color reaction is then developed
⇨ products of an individual clone of plasma cell
with appropriate substrate or chromogen, such as Horseradish
⇨ MOST FREQUENTLY USED ANIMAL:
peroxidase
★ Mice

IMMUNOFLUORESCENCE METHODS
IMMUNOHISTOCHEMISTRY TECHNIQUES

i. DIrect Fluorescence for solid tissue biopsies:

1. Direct technique
⇨ The target tissue is reacted with fluorescein-conjugated
2. Indirect technique
antibody specific for the material being sought within the tissue
3. peroxidase-Antiperoxidase technique
4. Avidin-Biotin Complex (ABC) technique
ii. IndIrect Fluorescence for solid tissue biopsies:
5. Labeled Streptavidin Avidin Biotin (LSAB) technique
⇨ Used for the detection of autoantibodies in the patient’s
serum including the anti-nuclear antibody (ANA),
anti-mitochondrial antibody and liver-kidney microsomal
antibody
 ANTIGEN RETRIEVAL INTERMEDIATE FILAMENT MARKERS

1. By proteolytic enzyme digestion 1. Actin

⇨ used commonly heavy chain immunoglobulins, complement, ⇨ muscle and some non-muscle tissues
and specific antigens
⇨ MOST COMMONLY USED ENZYMES: 2. Vimentin
➢ Trypsin: 0.1% Trypsin and 0.1% CaCl in distilled water ⇨ normal mesenchymal cells and their neoplastic counterparts
(pH is adjusted to 7.8 using Sodium hydroxide; preheated (sarcoma, melanoma, lymphoma, leukemia, seminoma and some
to 37⁰C) neural tumors)

➢ 0.005-0.1% protease in distilled water 3. Desmin

(pH is adjusted to 7.8 using Sodium hydroxide; faster rate ⇨ smooth and striated muscle; highly specific for myogenic tumors,
of digestion) including leiomyoma (smooth muscle tumor) and
rhabdomyosarcoma (skeletal muscle tumor)
2. Microwave antigen retrieval
⇨ boiling of formalin fixed deparaffinized section in certain 4. Glial Fibrillary acidic protein
solutions such as: ⇨ central nervous system glial cells, particularly astrocytes
➢ 0.01 M Citrate buffer (pH 6.0)
➢ EDTA (pH 8.0) 5. Neurofilament
➢ Tris-EDTA (pH 9.9 or 10) ⇨ expressed in cells of neural origin

3. Pressure cooker antigen retrieval 6. S100

⇨ Alternative method that is less time consuming and allows for
more consistent recovery of many antigens
⇨ Heat induced epitope retrieval method
⇨ Some labs use a water bath set to 60⁰C and incubate the slides
in retrieval solution overnight
⇨ CNS glial cells, Schwann cells, melanocytes, histiocytes,
chondrocytes, skeletal and cardiac muscle, myoepithelial cells
and some epithelial cells of the breast, salivary and sweat gland
epithelium

GERM CELL TUMOR MARKERS

EPITHELIAL TUMOR MARKERS
1. HCG (Human Chorionic gonadotropin)
1. Keratin ⇨ synthesized by placental syncytiotrophoblasts; marker for
⇨ HIGHLY SENSITIVE MARKER FOR EPITHELIAL CELLS and is choriocarcinoma
present in epithelial tumors (carcinoma)
⇨ are intermediate filaments forming proteins that provide 2. AFP (Alpha-fetoprotein)
mechanical support and fulfill a variety of additional functions in ⇨ embryonal carcinomas and teratomas containing these elements
epithelial cells as well as hepatocellular carcinomas will stain positive
⇨ They are PART OF THE CYTOSKELETON and the LARGEST
FAMILY OF INTERMEDIATE FILAMENT PROTEINS 3. PLAP (Placenta-like ALP)
⇨ is used as a marker for germ cell tumors particularly
germinomas; also positive in majority of seminomas

MESENCHYMAL TUMOR MARKERS

1. Myogenic tumors
⇨ Positive for muscle-specific actin and desmin and / or other
muscle markers such as Myo-D1, myoglobin and myogenin

2. Fibrohistiocytic tumors
⇨ CD68 or FAM56, combined with nonspecific proteolytic enzymes
such as alpha-1-antitrypsin and alpha-1-antichymotrypsin

3. Vascular tumors
⇨ Factor VII related antigen, CD31 and Ulex Europaeus I (UEA)

2. EMA (Epithelial Membrane antigen) 4. Melanomas

⇨ Positive in adenocarcinomas of the breast, lung and kidneys ⇨ S100, HMB-45, Melan-A (MART-1); HMB-45 (Melanosome)
widely used, highly sensitive marker for the diagnosis of
3. CEA (Carcinoembryonic antigen) melanoma
⇨ Positive in carcinomas of the gastrointestinal tract, lungs, breast,
ovary, uterus and cervix 5. Lymphomas
⇨ LCA (Leukocyte Common Antigen / CD45)
4. TTF-1 (Thyroid Transcription Factor 1)) ⇨ Markers used include those for T-cells (CD3, CD4, CD8), B-cells
⇨ Positive in thyroid, lung and neuroendocrine tumors (CD19, CD20, CD23), Reed-Sternberg cells (CD15, CD30), and
immunoglobulin light and heavy chains
5. PSA (Prostate Specific antigen)
⇨ Useful in the diagnosis of prostatic adenocarcinoma
 NEUROENDOCRINE MARKERS

1. NSE (Neuron-specific enolase)

⇨ Isoenzyme marker whose presence in tissue provides strong
evidence of neural or neuroendocrine differentiation

2. Chromogranin
⇨ found in neural secretory granules of endocrine tissues, and is
recognized as a marker of neuroendocrine differentiation

3. Synaptophysin
⇨ Transmembrane protein associated with presynaptic vesicles of
neurons; identified in normal neurons and neuroendocrine cells

4. Cell proliferation markers-Ki67

⇨ MIB-1 is the reference monoclonal antibody for the demo of Ki67

5. PCNA (Proliferating Cell Nuclear antigen)

INFECTIOUS AGENT MARKERS

■ Hepa A virus
■ Hepa B virus
■ Hepa C virus
■ Human papillomavirus
■ Cytomegalovirus
■ Epstein-barr virus
■ Toxoplasma
■ Pneumocystis carinii
■ H. pylori
■ Cryptosporidium
■ C. neoformans
■ Histoplasma
■ E. histolytica
■ Mycobacteria