Exam #1: Medical Applications of Tissue Processing & Examination Flashcards
7 Steps of Tissue Preparation for Light Microscopy
1) Fixation
2) Dehydration
3) Clearing
4) Embedding
5) Sectioning
6) Mounting
7) Staining
Fixation (Purpose & Reagent)
- Preserve normal tissue after death
- Formalin &Glutaraldehyde
Dehydration (Purpose & Reagent)
- Remove water from tissue specimen
- Graded series of ethanol (50-100%)
Clearing (Purpose & Reagent)
- Prepare the tissue for embedding
- Xylene
Embedding (Purpose & Reagent)
- Infiltrate w/ Parafin so that it can be sectioned
Sectioning (Purpose & Reagent)
- Tissue is cut into sections w/ microtome
Mounting (Purpose & Reagent)
- Sections placed on glass slide
Staining (Purpose & Reagent)
- Provides contrast to tissue structures for visualization
Acid Dye
Net negative charge
Basic Dye
Net positive charge
Acidophilic
- Cellular structures w/ a net positive charge
- Have affinity for negatively charged dye
Basophilic
- Cellular structures w/ a net negative charge
- Have affinity for positively charged dye
Metachromasia
Phenomenon in which a stain imparts different colors to a tissue in a density dependent manner
Hematoxlyin (1. What does it stain? 2. What color? 3. Clinical Application?)
- RNA, DNA, ribosome, & ER affinity
- Stains Blue
- Basic
- Coagulative Necrosis
Eosin (1. What does it stain? 2. What color? 3. Clinical Application?)
- Secretory Vesicles, sER, Lysosomes, Mitochondria, Type I Collagen
- Stains Pink
- Acid
- Coagulative Necrosis
Feulgen Reaction (1. What does it stain? 2. What color? 3. Clinical Application?)
- DNA (Both Nuclear & Mitochondrial)
- Stains Magenta
-Nuclear changes in Cancer
Mallory Triple (1. What does it stain? 2. What color? 3. Clinical Application?)
Dye Cocktail that stains a spectrum of colors:
- Nuclei= Red
- Muscle= Red to Orange
- Collagen= Blue
- Hyaline Cartilage= Blue
-Fibrosis
Periodic Acid Schiff (PAS) Reaction (1. What does it stain? 2. What color? 3. Clinical Application?)
- Carbohydrates
- Magenta
- Thickened basement membranes in kidney disease
- Glycogen Storage Disease (Liver)
- Alpha 1-Antitrypsin Disease (Emphysema)
Osmic Acid (1. What does it stain? 2. What color? 3. Clinical Application?)
- Lipids
- Black
Verhoeff (1. What does it stain? 2. What color? 3. Clinical Application?)
- Elastic Fibers
- Black
- Elastic Fibers in Marfan Syndrome
Silver Methods (1. What does it stain? 2. What color? 3. Clinical Application?)
- Intermediate Filaments of Nerve Cells, Glial Cells, Reticular Fibers
- Black
Trypan Blue (1. What does it stain? 2. What color? 3. Clinical Application?)
- Stains Macrophages in “Vital Tissue”
- Blue
Prussian Blue (1. What does it stain? 2. What color? 3. Clinical Application?)
- Hemosiderin (Ferric Iron)
- Blue
-Excessive iron in hemochromatosis
Nissil (1. What does it stain? 2. What color? 3. Clinical Application?)
- Ribosomes
- Blue
Iron Hematoxylin (1. What does it stain? 2. What color? 3. Clinical Application?)
- Nuclear Elements, Chromosomes, Mitochondria, Centrioles & Muscle Striation
- Dark blue to black
Congo Red
Amyloid Deposits (e.g. Alzheimer’s Disease)
Frozen Tissue Section
- Preparation method when tissue examination is urgent
- Results in thicker slices with less detail
Immunocytochemistry
- Technique that uses tagged antibody as a marker directed to a specific antigen
- Direct & Indirect
Direct Immunocytochemistry
- Less sensitive w/ fewer antibodies
- Marker associated w/ antibody
Indirect Immunocytochemistry
- More sensitive w/ more antibodies that amplify the signal
- Marker associated with antibody to antigen/antibody complex
Clinical Utility of Immunocytochemistry
- Categorization of Tumor Origin e.g. Cytokeratins (Epithelial) & D2-40 (Lymph)
- Breast Cancer e.g. Estrogen Receptor, HER2/neu, & Carinoembryonic Antigen
In situ hybridization
- Uses complementary nucleic acid probes to specifically identify a nuclei acid sequence
- When probe has a fluorescent maker, called FISH
Clinical Utility of In situ hybridizatyion
- Id. cell infected w/ virus e.g. HPV
- Id. specific genes on a chromosome e.g. BRCA1 & 2
- Detection of gene amplification e.g. Myc & HER2/neu