DECALCIFICATION

Decalcification

Process of removing calcium or lime salts from tissues

Saw (fine-fret)

If tissue is very large what is used

Regularly

How often should decalcifying agent should be changed

20:1

Ratio of Decalcifying Agent

Grating

Sensation during cutting

Place block in 10% HCL for 1 hour

If “grating” is experienced, what should be done?

Rapid decalcification

produces effect on nuclear staining

Nitric Acid

● Most common

● Fastest

Formalin or Alcohol

Nitric Acid inhibits nuclear stain when combined with

Yellow

Nitric Acid imparts what color when combined with Nitrous Acid

Nitrous Acid

Nitric Acid imparts a yellow color when mixed with

10% Aqueous Nitric Acid

• Rapid, with minimal tissue distortion (if prolonged)

• Recommended for urgent biopsy, needle biopsy

• Yellow color imparted

10mL Conc. Nitric acid + 100 mL dH2O

Concentration of 10% Aqueous Nitric Acid

Formol-Nitric Acid

● Rapid acting

● Good nuclear staining

● Less tissue destruction than 10% Aqueous nitric acid

12-24 days

How many hours/days is 10% Aqueous Nitric Acid used?

Fume Hood

When using Formol-Nitric Acid, what should be used?

5% sodium sulfate or 0.1% Urea

To lessen yellow tissue discoloration from Formol-Nitric Acid, what should be added?

1-3 days

How many hours/days is Formol-Nitric Acid used

Conc. Nitric Acid + 40% Formalin + dH2O

Concentration of Formol-Nitric Acid

Perenyi’s Fluid

● Decalcifies and softens

● Good nuclear and cytoplasmic staining

● Disadvantages: slow, difficult to assess complete decalcification by chemical means

chromic/ethyl

Maceration from Perenyi’s Fluid is avoided by

2-7 days

How many hours/days is Perenyi’s Fluid used

10% Nitric acid + 0.5% Chromic acid + absolute ETOH

Concentration of Perenyi’s Fluid

Phloroglucin Nitric Acid

● Most rapid

● Poor nuclear staining

3 changes of 70 to 90% ethanol

When decalcification using Phloroglucin Nitric Acid is complete, acid must be removed by

12-24 hours

How many hours/days is Phloroglucin Nitric Acid used?

Conc. Nitric + Phloroglucin Æ dense white fumes are formed, add 10% Nitric acid after disappearance

Concentration of Phloroglucin Nitric Acid

Hydrochloric Acid

● Inferior compared to Nitric Acid as decalcifying agent

● Slower action, greater tissue distortion

● Good nuclear staining

● Recommended for surface decalcification

Von Ebner’s Fluid

● Good cytologic staining

● Recommended for teeth and small pieces of bones

NaCl, HCl, H20

Concentration of Von Ebner’s Fluid

Formic Acid

● Better nuclear staining with less tissue distortion

● Safer to handle than Nitric and HCl

● Recommended for postmortem research tissues upon the addition of sodium citrate

● Recommended for autopsy materials, BM, cartilage and tissue for research

10% Formic Acid

● Slow

● Fixative and decalcifying agent

● Permits excellent nuclear & cytoplasmic staining

2-7 days

How many days is 10% Formic Acid used

Formic Acid (SG 1.20) + 10% Formol saline

Concentration of 10% Formic Acid

Formic Acid Sodium Citrate Solution

● Slow, not recommended for routine purposes

● Permits better nuclear staining than Nitric Acid

3-14 days

How many days/hours is Formic Acid Sodium Citrate Solution

45% Formic Acid + 20% Na Citrate

Formic Acid Sodium Citrate Solution

Trichloroacetic Acid (TCA)

● Permits good nuclear staining

● Weak decalcifying agent

● Suitable only for small bone spicules

Small bone spicules

Trichloroacetic Acid (TCA) is suitable only for

Sulfurous Acid

● Very weak decalcifying agent

● Suitable only for minute pieces of bone

4-8 days

How many days is Trichloroacetic Acid (TCA) acting

Chromic Acid

● Fixative and decalcifying agent

● Nuclear staining with hematoxylin is inhibited

● Forms precipitate at the bottom

● Carcinogenic, corrosive to skin

CITRIC-ACID CITRATE BUFFER (pH 4.5)

● Permits good nuclear and cytoplasmic staining

Chromic acid + Osmium tetroxide + glacial HAc

Concentration of Chromic Acid (Flemming’s Fluid)

7% Citric Acid + 7.4% Ammonium Citrate + 1% Zinc sulfate + Chloroform (preservative)

Concentration of Citric-Acid Citrate Buffer

6 days

Days to use Citric-Acid Citrate Buffer

Chelating Agents

Substances which combine with Calcium ions and other salts

EDTA (Versene, Sequetrene)

most common chelating agent, will not bind Ca at pH below 3.0

1-3 weeks

How many days does Chelating Agents take to decalcify?

Add magnesium chloride

EDTA inactivates alkaline phosphatase

Ion Exchange Resins

● Ammonia form of Polystrene Resin

● Hastens decalcication by removing Ca ions from Formic acid-containing decalcifying solutions

● Artifacts produced, usually caused by CO2 bubbles

● Slow – 1-14 days

● Degree of calcication cannot be measured by chemical means

Electrophoresis

● process whereby positively charged Ca ions are attracted to a negative electrode and subsequently removed from the decalcifying agent, satisfactory for small bone fragments ● Uses electricity

● Solutions used for Electrolytic Decalcication: Formic Acid 88%, Concentration, Hydrochloric Acid, distilled water

● Shorter time of calcium removal

● Suited for small bone fragments

Physical/Mechanical

○ Bending or touching the tissue, using needle

○ Prone to produce artifacts and may destroy cellular details

X–ray/Radiological

Very expensive but not ideal, not applied w/ tissues w/ Mercuric Chloride

● Perenyi’s Fluid – 12-24 hours

● 4% Aqueous phenol – 1-3 days

● Molliex (swollen & soapy appearance)

● 2 % HCl

● 1 % HCl in 70 % alcohol

Tissue Softeners

saturated lithium carbonate solution or 5-10% aqueous NaHCO3

Remove acid by ___ for several hours