3. DECALCIFICATION - SPC MLS 2B - HISTOPATH LEC.docx.pdf

HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE LESSON 3: DECALCIFICATION MIDTERMS | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD OUTLINE B. UNIQUE CHARACTERISTICS I. Decalci...

HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES - LECTURE LESSON 3: DECALCIFICATION MIDTERMS | A.Y. 2022-2023 | PROF. DOREN VENUS OTOD OUTLINE B. UNIQUE CHARACTERISTICS I. Decalcification A. Characteristics of a Good Decalcifying Stable Agent Easily available B. Unique Characteristics Inexpensive C. Factors Affecting Rate of Decalcification Easy to prepare II. Four Types of Decalcifying Agents A. Acid B. Chelating Agents C. FACTORS AFFECTING C. Ion Exchange Resin RATE OF DECALCIFICATION D. Electrophoresis Concentration I. DECALCIFICATION Less concentrated = slow acting Tissue-to-volume Ratio Purpose: 1:20 Removal of calcium and lime salts ○ 1 parts tissue and 20 parts decalcifying Done after fixation and before dehydration and agent impregnation Temperature Calcium might interfere with accurate evaluation Must be well regulated and examination Mechanical Agitation ○ Calcium should be removed in the tissue It is the key to a rapid acting or rapid decalcifying of samples because it can potentially the sample interfere with the accurate evaluation and Size and Consistency of Tissue Sample examination of the metabolic bone Large, thick, rigid = longer decalcification process disease, differentiate mineralized bone from osteoid, and morphometric III. FOUR TYPES OF DECALCIFYING AGENTS measurement of the tissue sample Significance: A. ACID Facilitate normal cutting of tissue in sectioning Prevent obscuring microanatomical detail of tissue Organs that require decalcification: Bones ○ Calcium is present in the bones Tuberculous Lungs ○ Calcified lungs due to TB bacteria Arteriosclerotic Vessels ○ Accumulated lipids, calcium, cellular debris in the lumen of the blood vessels Nitric Acid Teeth 5-10% Routine or most common decalcifying agent In order to facilitate good cutting and evaluation of your Fastest decalcifying agent in the market now tissue samples, they should undergo removal of calcium Imparts yellow coloration to the tissue sample and lime salts first. through nitrous acid formation ○ Remedy: A. CHARACTERISTICS OF A GOOD DECALCIFYING Add urea or Sodium AGENT Thiosulfate/sulfate Do not cause cell destruction 70% ROH (DHD) Rapid, cheap, inexpensive ROH is an alkyl alcohol ○ Should also render best and accurate Variation/s: result ○ 10% Aqueous Nitric Acid Solution Safe ○ Formol-Nitric Acid Readily available ○ Perenyi’s Fluid Acts as tissue softener Nitric acid + chromic acid + ROH FRONDA | SPC MLS 2B | 1 of 2  ○ Phloroglucinol-Nitric Acid Common brand: Fastest agent (simple or Cal-Ex and Versene compound) ○ Contains Na2EDTA Simple solution = 1 ingredient/component only C. ION EXCHANGE RESIN Compound solution = 2 or more ingredients/ components Hydrochloric Acid Provides good nuclear staining at 1% Slower and causes more distortion compared to nitric acid Variation/s: ○ Von Ebner’s Fluid Principle: For teeth and small bones Increase solubility HCL + 36% NaCl + distilled water ○ Uses formic acid with TSE : Formic acid of Formic Acid 1:20-30 Good for routine decalcification of post-mortem Remove calcium ions from formic acid containing research tissues, small pieces of bone and teeth, decalcifying solutions ISH staining Not recommended for hydrochloric acid and nitric If the formic acid contains a large amount of nitric acid fluid containing mineral acids acid, it produces better nuclear staining and less Decalcification Extent: tissue distortion Can be measured using physical method by simply Variation/s: bending or poking the tissue sample and/or X-ray ○ Formic Acid method ○ Formic Acid-Sodium Citrate Solution Trichloroacetic Acid D. ELECTROPHORESIS Best for small bone spicules Good nuclear staining Slow, weak Chromic Acid (Flemming’s Fluid) Best for minute bone spicules Sulfurous Acid Best for minute pieces of bone Weak Citric Acid Citrate Buffered Solution pH: 4.5 Principle: Attracting calcium ions going to the cathode part of B. CHELATING AGENTS the agarose gel Advantage: Time is shortened due to heat and electrolytic reaction produced in the process ○ Shorter time is needed to remove calcium ions or calcium salts ○ It utilizes 88% formic acid Principle: Procedure: Use of other salts to form complexes with Calcium Used for small bone fragments salts for ease of removal Decalcification Extent: Procedure: Best Measured Using: Utilized in Immunohistochemistry and enzyme ○ Physical or Mechanical Test staining with the help of electron microscope ○ X-ray or Radiologic Method Duration: ○ Chemical Method 1-3 weeks for small tissue samples 6-8 weeks for longer and dense bones pH: 7-7.4 ○ Should be maintained at this pH in order to work properly FRONDA | SPC MLS 2B | 2 of 2