Introduction To Decalcification & Frozen Section Techniques PDF

Introduction to Decalcification & Frozen Section Techniques HLAB 1260  What is Decalcification? Any Tissue containing “CALCIUM” must undergo calcium removal prior to processing, but only after fixation. Tissues containing calcium cannot be sectioned after paraffin embedding...

Introduction to Decalcification & Frozen Section Techniques HLAB 1260  What is Decalcification? Any Tissue containing “CALCIUM” must undergo calcium removal prior to processing, but only after fixation. Tissues containing calcium cannot be sectioned after paraffin embedding In pathology most evaluations are made on delcalcifed sections. “undecalcified sections are examined primarily for the diagnosis of “Metabolic Bone Disease”  Best Practice “Grossing & Fixation” To ensure proper processing of bone, specimens are cut into thin “4mm” slices for adequate fixation. Fixation must occur before decalcification! Decalcification of specimens that are not adequately fixed may adversely effect tissue morphology!!! There are a few different ways to accomplish decalcification.  Acid Method One of the most popular methods. Several advantages & disadvantages Despite buffered contents, The Acid method will have some effect on staining. The stronger the Acidity of the acid or the longer time in an acid solution, the more detrimental the effect on staining will become. Overdecalcified tissue can lead to lack of nuclear staining, destruction of IHC/IF/Molecular Epitopes. Proper endpoints times are a must!  Acid Method Works by releasing bone calcium from phosphate & carbonate anions. The released calcium ionizes with the acid producing a soluble calcium salt. Calcium Salts are soluble at a pH of 4.5 Most Acid Decalcifiers have a pH of 0.5-3.0, therefore must be buffered to 4.5 prior to use.  Simple Acids Used as 5%-10% Solution Hydrochloric Acid Nitric Acid Formic Acid  Simple Acids Continued Formalin must be washed out before placing tissue in Hydrochloric acid solution. Bis-chloromethyl ether “A known carcinogen” can be formed is Hydrochloric acid & formaldehyde cross-react. Nitric Acid: Most deterior effect on enzymes & other morphology if tissue remains longer than 48 hours! Formic Acid : Works by “Ion exchange Resins” involes ammonium ion exchange for calcium ions. Electric Method Can be used with formic acid & hydrochloric acid. Bone is attached to a positive pole “Anode”. Calcium ions are transferred to negative pole “Cathode” Requires 2-6 hours Not really used due to destruction & loss of cellular detail  Chelating Agents EDTA is the primary regaent Work by binding to calcium ions Work by a pH of 5.0-7.4, Slightly alkaline Gentle, only eats away at calcium, leaves other morphology alone Great choice for enzyme studies  3 ways to determine endpoint Mechanical: Simple test that checks the flexibility of the specimen with a needle or pin. Least desirable because inaccuracy. Chemical: Dependent on the precipitation of calcium oxalate fluid. The fluid is placed on litmus paper treated with ammonium oxalate. ( Turbidity or cloudy appearance” indicates presence of calcium. Radiography: Uses X-rays to determine calcium removal. Most accurate & easy to use.????  Washing in Water All decalcified specimens should go through a rise of deionized water after decalcification to remove any excess acid. An Alkaline solution “lithium carbonate” may be used to neutralized any remaining acid.  Surface Decal Should you come across a specimen that is under decalcified, A surface decal may be used 1% Hydrochloric Acid Can be identified by sectioning bone that is hard, damages blade, and creates deep artifacts in the tissue sections. Bone & Cartilage Von kossa on undecalcified bone Under decalcified Specimen  Introduction to Frozen Sectioning Used for the pathological diagnosis of a specimen processed rapidly for microscopic examination Done on patients having surgery, on the examination table. High stress Time sensitive environment CAP standard of 15 minutes for rapid diagnosis  Frozen Section Fixatives Sectioning frozen tissue requires fresh unfixed tissue The required fixative is 95% Alcohol Acetone for Immunofluorescence For Immunohistochemistry studies, 10% NBF may be used. Antigen retrieval is required after formalin fixation to unmask “cross-links” that mask “epitope markers”  Sectioning The medium used to hold tissue in place is known as “OCT” The Tissue grossed, inked, mapped, and orientated on a metal chuck The Tissue is covered with a layer of OCT, then frozen Liquid Nitrogen spray or bath is used to rapidly freeze the specimen. The tissue is rough cut & faced until a full face of the surface is visible Routine Specimen mounted in OCT Frozen Specimen, section being rolled with brush onto faceplate, to be picked up on clean glass slide.  Rapid Stains 1. Rapid HE Stain 2. Toluidine Blue 3. Methyl Blue 4. Mehtyl Violet “Amyloid” 5. Oil Red O “Fat” 6. Diff Quick “Touch Prep”