Histopath sem

Immunohistochemistry

- Identification of specific or selective tissue or cellular Ag found on tissues

- Used for detection of organisms in cytologic preparations (eg. Fluids, sputum samples, FNA)

- Makes use of antigen-antibody reactions

Antigen

found in cells

IgG

most commonly used antibody

Polyclonal antibodies

- produced by different cells; react with various epitopes

- Most common source: Rabbits

- Other animal sources: goat, pig, sheep, horse, guinea

Monoclonal antibodies

- most specific; single done of a plasma

- source: mice

Enzyme labels

- Use of Horse radish peroxidase with chromogen such as Diaminobenzidine (dark brown) and AEC (brick red)

- Counterstain: traditional nuclear stains such as Hematoxylin

- Optimum Incubation time for linking antibodies with peroxidase conjugates is 30-60 mins at room tempt

Fluorochrome labels

- use of fluorescein

- need of fluorescence microscope

Colloidal metals

such as gold (+) pink under light microscope;

Lectins

plant or animal proteins that can bind to tissue CHO. Can also be labelled like Ab.

Positive control

A section known and proven to have the antigen in question

Negative control

- Omit the primary antibody from the staining schedule

- Replace the specific primary antibody by an Ig that is directed against unrelated antigen

Internal tissue control

- "built-in" control

- Contains the target antigen but not in the tissue elements under investigation

Traditional Direct Technique

Primary Ab is labelled

EPOS

- direct labelling system; use of primary Ab conjugated with labeled long chain dextran polymer

Two step indirect technique

more sensitive than direct; increased signal amplification; Use of labeled secondary Ab

Polymer two step indirect technique

Secondary Ab conjugated to an enzyme labelled polymer chain

Keratin

- marker of Epithelial tissue

- Examples: CK7(+)(carcinomas of lung, breasts, uterus, ovarian cancers), CK20(+)(colon and stomach cancers); (CK7 and CK20 pos - transitional cell carcinomas of bladder and mucinous ovarian tumors); CK7 and CK20 neg - renal cell carcinomas, liver carcinomas, prostatic adenocarcinomas, thyroid and squamous cell carcinomas

EMA (Epithelial membrane antigen)

Helpful in determining the site of tumor; (+) for breast adenocarcinomas, lungs and kidneys

CEA (Carcinoembryonic Antigen)

- An oncofetal antigen present cárcinomas of GI tract, pancreas, lung, breast, ovary, uterus, cerix

- Differentiates adenocarcinoma (+) from mesothelioma (-)

TTF-1 (Thyroid transcription factor -1)

- Distinguishes lung adenoCA from mesothelioma

- Present in thyroid gland neoplasms

PSA (Prostate Specific Antigen)

(+) Prostate adenoCA, pancreatic and salivary tumors

ER/PR (Estrogen/Progesterone Receptor)

- Normally found in breast epithelial cells but not myoepithelial cells

- Prognosis and treatment of breast CA, identification of metastatic breast CA

Actin

- Sensitive Marker for muscle differentiation

- Identify tumors derived from smooth, skeletal and cardiac muscles

Vimentin

- Normally present in mesenchymal cells and neoplastic cells such as sarcomas, lymphomas, leukemias, seminomas, neural tumors

- Melanomas and schwannomas - always stain positive

Desmin

- Normally expressed by smooth, skeletal, cardiac muscle

- Highly Specific for myogenic tumors

GFAP (Glial Fibrillary Acidic Protein)

- Normally expressed by CNS glial cells especially astrocytes

- Diagnosis of astrocytoma

NF (Neurofilament)

Expressed by cells of neural origin: (+) inneuroblastomas, ganglioneuromonas, neuromas, chemodectomas

S100 protein

- calcium binding protein

- Expressed by CNS glial cells, Schwann cells, melanocytes, histiocytes, chrondrocytes, skeletal and cardiac muscles, myoepithelial cells, epithelial cells of the breast, salivary and sweat glands

NSE (Neuron-specific enolase)

(+) for tumors with neural or neuroendocrine differentiation

Chromogranin

- Found in neural secretory granules of endocrine tissues

- (+) neuroendoerine differentiation

- Keratin (+) and Chromogranin (+) = neuroendocrine carcinomas

Synaptophysin

Present in presynaptic vesicles of neurons, normal neurons and neuroendocrine cells

Human Chorionic Gonadotropin (HCG)

- Synthesized by placental syncytiotrophoblast

- (+) Germ cell tumors; choriocarcinoma

Alpha-Fetoprotein (AFP)

- Synthesized by normal liver

- (+) Hepatocellular CA

Placental-like Alkaline Antigen (PLAP)

- Produced by placental syncytiotrophoblasts in late pregnancy

- (+) Germ cell tumors, embryonal CA, chorioCA, endodermal sinus tumor, seminoma

Myogenic tumors

- Tumors of skeletal muscle origin

- (+) Actin, Desmin, Myo D-1, Myoglobin, Myogenin

Fibrohistiocytic tumors

- Malignant Fibrohistiosarcoma

- (+) CD68, FAM 56, Alpha-1 Antitrypsin, Alpha-1-antichymotrypsin

Vascular tumors

- Endothelial markers

- Angiosarcoma

- (+) Factor VII-related antigen, CD 31, Ulex europaeus (UEA)

Melanoma

- Melanocytes are derived from the neural crest

— (+) S100 protein

- Melanosome

— (+) HMB45

Lymphoma

- (+) LCA (Leukocyte Common Antigen) aka CD45

— Best Scrocning markerfor lymphoma

- T cells (+) CD3, CD4, CD8, B cells: (+) CD19, CD20, CD23

- Reed Sternberg: (+) CD15, CD30, Ig light and heavy chains

Ki-67 (MIB-1) and PCNA (Proliferating Cell Nuclear Antigen)

- Used to assess proliferation of tumors

- Greater aggressiveness and higher probability of metastasis.

p53

- Overexpression uncommon or absent in normal or benign cells

- (+) Malignant tumors but not specific for malignancy

- Other Cancer associated genes (esp. in breast cancer) |

— HER2/C-erbB-2, c-myc, ras,