Tissue Processing: Decalcification PDF

TISSUE PROCESSING: DECALCIFICATION 2021-2022 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES LEC 2nd Semester Instructor: Prof. Kimberly Pulga, RMT, MPH...

TISSUE PROCESSING: DECALCIFICATION 2021-2022 HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES LEC 2nd Semester Instructor: Prof. Kimberly Pulga, RMT, MPH WEEK 9 HPCT311 LEC Date: March 3, 2022 TOPIC OUTLINE Calcium may be removed by any of the following: Decalcification o Acids Acid Decalcifying Agents o Chelating Agents a. Nitric Acid o Ion exchange resins i. Aqueous nitric acid solution o Electrophoresis ii. Formol-nitric acid iii. Perenyi’s fluid Gregorios’ Histopathologic Technique pg. 150-153 iv. Phloroglucin-nitric acid ✓ Decalcification is the removal of calcium ions from a b. Hydrochloric acid bone or calcified tissue through a histological process i. Von Ebner’s fluid that makes them flexible and easier to cut. c. Formic acid ✓ Decalcification enables the histotechnologist to cut soft i. Formic Acid-Sodium Citrate solution sections of the bone using the microtome, so that they d. Trichloroacetic acid can be processed like any other soft tissue of the body. ✓ Decalcification is a lengthy procedure, as bone pieces e. Sulfurous acid have to be left in the decalcifying agent for several days f. Chromic acid or even weeks, depending on the size of the tissue. g. Chelating agents ✓ The principle of decalcification is fairly simple. Strong i. EDTA mineral acids, such as 10% hydrogen chloride (HCl), or ii. Neutral EDTA weak organic acids, such as 5-10% formic acid Ion exchange resin (HCOOH), form soluble calcium salts in an ion exchange Electrophoresis that moves calcium into the decalcifying solution. Factors affecting decalcification ✓ Decalcification should be done after fixation and before impregnation, to ensure and facilitate the normal cutting Measuring the extent of decalcification of sections and to prevent obscuring the microanatomic Post-decalcification detail of such sections by bone dust and other cellular debris. ***Please read the book (Gregorios’) for more information: ✓ Poorly-fixed specimens become macerated during pg. 150-167 decalcification and stain poorly afterwards. This is very * = side notes noticeable in areas containing bone marrow. ✓ = info from book ✓ High-quality fine tooth saws should be used to prepare bone slices. Learning Objectives: ✓ Cartilage does not require any softening, except if some At the end of the session, the students are expected to be calcified areas are present. It is a waste of time to put toenails in decalcification solution, because they are able to: composed of insoluble keratin filaments. Explain the basic concept of decalcification Identify the best decalcifying agent or method for Acid Decalcifying Agents decalcified tissue Most widely used for routine decalcification of large Analyze the factors that may affect the process of amounts of bony tissues decalcification Stable, readily available, inexpensive Demonstrate the process of decalcification * 3 Main Types of decalcifying agents * Based on Strong mineral acids Decalcification * Based on Weaker organic acids * Optional step * Composed of Chelating agents * Should be done after fixation and before impregnation for us to ensure and facilitate the normal cutting of tissue sections Strong Mineral Acid Weak Acid Removal of calcium ions from bone or calcified tissue Adjust the hard substance of bones to the softness of Nitric Acid Formic Acid paraffin embedding medium. Principle: strong mineral acids or weak organic acids, Hydrochloric Acid Trichloroacetic Acid they will form soluble calcium salts in an ion exchange that moves the calcium into decalcifying solution. Chromic Acid * Purpose: * To ensure and facilitate the normal cutting of Gregorios’ Histopathologic Technique pg.153 tissue sections. ✓ Acid decalcifying agents are the most widely used * To prevent the obscuring of the micro- agents for routine decalcification of large amounts of anatomic detail of the sections. bony tissues because they are stable, readily available, * In choosing a decalcifying agent, it should be capable and relatively inexpensive as compared to other of removing the calcium salts from the tissue decalcifying agents. completely and it should not cause any destruction to ✓ The acids make up a solution of calcium ions while the chelating agents take up the calcium ions. the cells and tissue components. It should also have a ✓ Nuclear and cytoplasmic detail are compromised if good staining capacity. specimens are exposed for too long to acidic decalcifying agents. M. LIONG & J. LABASTIDA– TRANSCRIBERS  A. Nitric Acid * Disadvantage: Most common & fastest * It is a slow decalcifying agent for dense bones; ✓ produce minimal distortion and is, therefore, hence, is not recommended for urgent diagnosis. recommended for routine purposes. Simple or compound/ combined d. Phloroglucin-Nitric Acid ✓ Utilized both as simple solution or combined Formula: Conc. nitric acid (10mL) + phloroglucin (1g) with other reagents. + 10% nitric acid (100 mL) * This may be used as simple aqueous solution with recommended concentration of 5% to Decalcification time: 12 to 24 hours 10%. Advantage: Most rapid for urgent cases ✓ It is the most rapid decalcifying agent so far, Disadvantage: Inhibiting nuclear stains & destroying recommended for urgent cases. tissues ✓ Prevent: combine it with Formaldehyde or Disadvantage: Alcohol o Poor nuclear staining o Prolonged decalcification produces extreme a. Aqueous Nitric Acid Solution (10%) tissue distortion. Formula: Concentrated nitric acid (10mL) + distilled B. Hydrochloric Acid water (100mL) Inferior to nitric acid: because it has slower action Decalcification time: 12 - 24 hours compared to nitric acid & has greater distortion of Advantages: tissue. o Rapid action * Remember: HCl produces good nuclear staining and o Minimum distortion if used in 1% solution with 70% alcohol, may be o Good nuclear staining (slower) recommended for surface decalcification of the tissue o Acid be easily removed by 70% Alcohol blocks. o Recommended for urgent biopsy, needle & small biopsy specimens to permit rapid a. Von Ebner’s Fluid diagnosis within 24 hours or less o For large or heavily mineralized cortical bone Formula: Salt aqueous solution of NaCl (50mL) + 36% specimen conc. HCL (8mL) + distilled water (50mL) Disadvantages: Advantages: o Imparts yellow color o Good for cytologic staining o Damage tissue enzymes * It is considered as a moderately rapid ✓ Prolonged decalcification may lead to tissue decalcifying agent. distortion. o Does not require washing before dehydration o Recommended for Teeth and small pieces b. Formol-Nitric Acid of bone ✓ Disadvantage: the extent of decalcification cannot be Formula: Conc. nitric acid (10mL) + 40% measured by a chemical test. formaldehyde (5mL) + distilled water (85mL) Decalcification time: 1 - 3 days C. ⭐️Formic Acid Advantages: * Considered as the best decalcifying agent, the best all- o Rapid; recommended for urgent biopsies around decalcifying agent. o Good nuclear staining * A moderate-acting decalcifying agent which produces o Less tissue destruction than 10% aq. nitric better nuclear staining with less tissue distortion. acid * Safer to handle than nitric acid or hydrochloric acid. Disadvantages: o Yellow discoloration 10% formic acid: Best all-around decalcifying agent * In which it can impair the staining reaction of the cell. Formula: Formic acid (10mL) + Normal saline (90mL) o Prevented by: 5% Sodium sulfate Decalcification time: 2 - 7 days neutralization or running water (for 12 hours) * It is recommended for routine decalcification or an addition of 0.1% Urea of postmortem research tissues, although not * To pure concentrated Nitric acid will suitable for urgent examinations. also make the discoloration Advantages: disappear. o It may be used both as a Fixative and decalcifying agent c. Perenyi’s Fluid o Excellent nuclear and cytoplasmic staining Formula: 10% nitric acid (40mL) + 0.5% chromic acid o Recommended for small pieces of bones & (30mL) + absolute ethanol (30 mL) teeth ✓ Suitable for most routine surgical specimens Decalcification time: 2 - 7 days (recommended for routine purposes) Disadvantage: * Advantages: o It is slow but may increase concentration to * It is recommended for routine purposes. 25 mL * It decalcifies and softens tissues at the same time. ✓ It is relatively slow; hence, is not * Nuclear and cytoplasmic staining is good. suitable for urgent specimens. * Maceration is avoided due to the presence of Decalcification may be hastened by chromic acid and alcohol. increasing the proportion of formic acid to 25 ml. However, such M. LIONG & J. LABASTIDA– TRANSCRIBERS  concentration may make the Chelating Agents solution opaque, thereby interfering with the staining results. a. EDTA (Ethylene diamine tetra acetic acid) o Requires neutralization with 5% Sodium * Are substances which combine with calcium ions and sulfate & washing out to remove acid from other salts (e.g. iron and magnesium deposits) to form tissue weakly dissociated complexes and facilitate removal of * But it makes the solution opaque calcium salt. and unsuitable for staining. ✓ Commercial name: Versene ✓ Recommended only for detailed microscopic studies a. Formic Acid-Sodium Citrate Solution Most commonly available chelating agent Formula: 20% aq. Na citrate (50 mL) + Formic acid * It combines with calcium ions and other salts (50mL) to form weakly dissociated complexes and to Decalcification time: 3 - 14 days facilitate the removal of calcium. Advantages: * Very slow decalcifying agent o Nuclear staining > nitric acid Binds metallic ions (calcium & magnesium) ✓ It permits better nuclear staining Captures calcium ions from the surface of apatite than nitric acid method crystals slowly reducing the size o Recommended for autopsy materials, BM, * Decalcification by EDTA takes much longer than cartilage, *and tissues studied for research decalcification by acids. It takes weeks rather than purposes. days. Disadvantages: * Compared to acids, EDTA takes more than or o Slow much longer than the acids. It takes days for o Not for routine purposes acid while EDTA is for weeks. ✓ not recommended for routine * If you process small specimens it takes 1 to purposes and for dense tissues. 3 weeks. o Neutralize with 5% Sodium sulfate * If you process dense cortical bones, it will ✓ It requires neutralization with 5% take 6-8 weeks to decalcify it. sodium sulfate pH= 6.5 - 7.4 Preferred for nucleic acids and enzymes D. Trichloroacetic Acid Formula: Trichloroacetic acid (5g) + 10% Formol b. Neutral EDTA saline (95mL) Advantage: Formula: EDTA disodium salt (250g) + distilled water (buffer: Sodium hydroxide; pH= 7.0) o Permits good nuclear staining o Does not require washing out Acts slow with little tissue damage * The excess acid may be removed by Conventional stains unaffected several changes of 90% alcohol, Disadvantage: Inactivates ALP (Alkaline thus improving tissue dehydration. Phosphatase Activity); resolved by adding Decalcification time: 4 - 8 days Magnesium Chloride. E. Sulfurous acid Ion Exchange Resin Very weak acid suitable only for minute pieces of bones Ammonium form of polystyrene resin that hastens decalcification by removing calcium ions Formic acid- F. Chromic Acid (Flemming’s Fluid) containing decalcifying solutions. Formula: Chromic acid (15mL) + osmium tetroxide Not recommended for fluids containing mineral (4mL) + 2% glacial acetic acid (1mL) acids such as nitric acid or hydrochloric acid. Advantage: A layer of ion exchange resin (1/2 thick) is spread over * May be used both as a Fixative & decalcifying the bottom of the container and the specimen is placed agent on top of it. Then the decalcifying agent is added (20- o Used for decalcifying minute bone spicules 30x the volume of the tissue). Disadvantage: The tissue may stay in solution for 1 - 14 days. The o Inhibition in nuclear staining with hematoxylin degree of decalcification may be measured by * It tends to undergo reduction and forms Physical or X-ray method. precipitates at the bottom of the container thus requiring frequent changes of solution. Electrophoresis * Degree of decalcification cannot be measured A process whereby positively charged calcium ions are by the routine chemical test. attracted to a negative electrode and subsequently o Environmental toxin removed from the decalcifying solution. * Chromic acid is highly corrosive to This method is satisfactory for small bone fragments, skin and mucous membranes. processing only a limited number of specimens at a * It is Carcinogenic. time * Suitable protective material is Good cytologic and histologic details are not always needed preserved. M. LIONG & J. LABASTIDA– TRANSCRIBERS  Factors affecting decalcification 3. Chemical Method (Calcium Oxalate Test) 1. Concentration Simple, reliable and convenient method for routine Concentrated = increase rate of purposes. decalcification = harmful to tissues Detect calcium in the decalcifying solution by ✓ In general, more concentrated acid precipitation of insoluble calcium hydroxide or calcium solutions decalcify bone more oxalates. rapidly, but are more harmful to the tissue. Post-decalcification Alcohols or buffers: decrease rate of The removal of acid from tissue or neutralized decalcification chemically by immersing the specimen either in ✓ This is especially true of aqueous saturated Lithium carbonate solution or 5-10% acid solutions, as various additives aqueous sodium bicarbonate solution for several such as alcohol or buffers that hours. protect the tissues may slow down the decalcification process. Simply rinse the decalcified specimens with running ✓ High concentrations and greater tap water. ✓ Generally, a short, effective wash in tap amount of fluid will increase the water should be sufficient as any remaining speed of the process. acid will be removed during processing. Fluid to tissue ratio: 20:1 (recommended) ✓ Adequate water rinsing can usually be accomplished in 30 minutes for small 2. Fluid access samples and 1-4 hours for larger specimens. ✓ Decalcification may be hastened by suspending the tissue in decalcifying solution for greater fluid References access. Prof. Kim Pulga’s ppt and notes Gregorios’ Histopathologic Techniques: 3. Size & consistency Chapter 8 pg. 150-167 Increase size= decrease rate of decalcification * Has inverse relationship ✓ Increase in size and consistency of tissues will require longer periods for complete decalcification. Dense bone= >14 days (decalcification time) 4. Agitation ✓ Gentle agitation may increase the rate of decalcification. ✓ Gentle fluid agitation is achieved by low-speed rotation, rocking, stirring or bubbling air into the solution. 5. Temperature Increase rate of decalcification= increase damaging effects of acid to tissues ✓ Increased temperature will hasten decalcification, but it will also increase the damaging effects of acids on tissue. ✓ Conversely, lower temperature decreases reaction rates. Recommended: Room temperature (18C - 30C) * Ideal time for decalcification: 24 - 48 hours Measuring the extent of decalcification 1. Physical or Mechanical Test Done by touching with the fingers to determine the consistency of tissue. Bending, needling or by use of scalpel if it bends easily that means decalcification is complete. Pricking causes damage and distortion of tissue. 2. X-Ray or Radiological Method Best method for determining complete decalcification * Not recommended for mercuric chloride-fixed tissues due to the latter's characteristic radio-opacity which will interfere with the correct interpretation of the plate. M. LIONG & J. LABASTIDA– TRANSCRIBERS