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Jenivie Morillo Jose University of Santo Tomas National Childrens Hospital

is

the COMPLETE REMOVAL of CALCIUM SALTS from the tissue following fixation

I. ACID METHOD NITIRIC ACID (5-10%)


-DECALCIFICATION

TIME: 1-3 days - for URGENT BIOPSIES -yellow color due to FORMATION OF NITROUS ACOD -addition of 0.1% urea =temporarily arrests the DISCOLORATION -produces GOOD NUCLEAR STAINING -rapid in action

FORMIC ACID (5%)


-DECALCIFICATION

TIME:2-7 days -BEST for POST-MORTEM AND RESEARCH TISSUE -EXCELLENT STAINING RESULT -slow in action (unsuitable for urgent work)

FORMIC ACID-SODIUM CITRATE SOLUTION


-DECALCIFICATION

TIME: 2-7 days - for RESEARCH PURPOSE, BONE MARROW, AUTOPSY -BETTER nuclear staining -relatively slow in motion

HYDROCHLORIC ACID (1%)


-DECALCIFICATION

TIME: 1-2 days - for URGENT BIOPSIES -BETTER nuclear staining -DISTORTION OF THE CELLS -slow in action

TRICHLOROACETIC ACID (5%)


-DECALCIFICATION

TIME: 4-6 days - for SMALL SPICULE OF DELICATE TISSUES -GOOD nuclear staining -DOESNT REQUIRE washing out -WEAK DECALCIFYING FLUID -VERY SLOW in action

CHROMIC ACID(FLEMMINGS FLUID)


-DECALCIFICATION TIME: 2-6 days - for MINUTE BONES -used as both DECALCIFYING AND

FIXING

AGENT -inhibits NUCLEAR STAINING with HEMATOXYLIN -undergo reduction and forms PRECIPITATE AT THE BOTTOM OF THE CONTAINER -cant be measured by the ROUTINE CHEMICAL TESTS

PERENYIS FLUID
-DECALCIFICATION

TIME: 2-8 days - for ROUTINE WORK -DECALCIFIES and SOFTENS BONES at the same time -good result in staining of CYTOPLASM and NUCLEUS -cannot be determined by CHEMICAL TESTS -SLOW in action for DENSE TISSUES

II. CHELATING AGENTS -substances that combine with calcium ions, other salts (iron and magnesium deposits) to form weakly dissociated complexes and facilitate removal of calcium salts -RECOMMENDED FOR: DETAILED MICROSCOPIC STUDIES

CAL-EX
-RAPID

COMMERCIAL DECALCIFYING

AGENT -DECALCIFICATION TIME: 2-24 HOURS

VERSENE (EDTA)
-EDTA-ETHYLENE

DIAMINETETRA

ACETIC ACID -combines with calcium and forms soluble non-ionized complexes -DECALCIFICATION TIME: 2-4 days - EXCELLENT STAINING RESULTS

-MINIMAL

cell distortion, artifacts usually caused by the production of CO2 BUBBLES -VERY SLOW DECALCIFYING AGENT -REMOVES ALL OTHER METALS (lead, iron, magnesium) -expensive

III. ION EXCHANGE RESINS An AMMONIUM form of POLYSTYRENE RESIN to the decalcifying solution speeds up the process of decalcification. The calcium ions are removed over from the solution thus INCREASING the rate of solubility of the calcium from the tissue Is LIMITED to DECALCIFYING SOLUTION that have a NON-MINERAL ACID as their constituent, FORMIC ACID as the common choice.

IV. ELECTROLYTIC METHOD A PROCESS whereby insoluble calcium salts in the bone are changed to an IONIZABLE SALT and ACID by the ELECTRICAL FIELDS between the ELECTRODES causing calcium ion to migrate to the CATHODE -successful for SMALL FRAGMENTS, processing tissues on a LIMITED number at a time -DECALCIFICATION TIME: 1-4 hours

PHYSICAL OR MECHANICAL TEST By touch Pliability Resistance to fingernail Needling This is apt to produce NEEDLE TRACT ARTIFACT and DESTROYS CELLULAR details and small CALCIFIED FOCI may not be detected

X-RAY OR RADIOLOGICAL TEST -very EXPENSIVE -MOST IDEAL and MOST RELIABLE METHOD (can detect the SMALLEST FOCUS of calcium which remains opaque in any x-ray plate)

CHEMICAL TEST Procedure: 1. In a clean test tube, place 5 ml of decalcifying fluid that has been in contact with the bone for the last 3-12 hours 2. Alkalinize by carefully adding STRONG AMMONIA WATER, shaking after each addition

Add 0.5 ml saturated aqueous solution of AMMONIUM OXALATE. Mix and let stand for 15-30 minutes RESULTS: Fluid is clear-COMPLETE DECALCIFICATION Fluid is cloudy-INCOMPLETE DECALCIFICATION
3.

Thank

You

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