Lecture 4 - Decalcification

DECALCIFICATION

•PROCEDURE WHEREBY CALCIUM OR LIME SALTS ARE REMOVED FROM TISSUES (MOST ESPECIALLY BONES AND TEETH) FOLLOWING FIXATION.

DECALCIFICATION

THIS IS USUALLY CARRIED OUT BY THE USE OF CHEMICAL AGENTS, EITHER WITH ACIDS TO FORM SOLUBLE CALCIUM SALTS, OR WITH CHELATING AGENTS THAT BIND TO CALCIUM IONS.

INADEQUATE DECALCIFICATION

MAY RESULT IN POOR CUTTING OF HARD TISSUES
AND DAMAGE TO THE KNIFE EDGE DURING SECTIONING.

FINE FRET-SAW ; HAND RAZOR

BONES AND CALCIFIED TISSUES (SUCH AS TUBERCULOUS ORGANS AND ARTERIOSCLEROTIC VESSELS) ARE USUALLY CUT INTO SMALL PIECES WITH A _ AND TRIMMED WITH A _ TO PERMIT COMPLETE PENETRATION OF THE DECALCIFYING SOLUTION WITH MINIMAL SURFACE DAMAGE AND TISSUE DISTORTION.

DARK PURPLE GRANULAR MASSES WITH LIGHTER PURPLE HALOS

▪ OCCASIONALLY, SMALL FOCI OF MICROCALCIFICATION IN PARAFFIN-EMBEDDED OR FROZEN TISSUES CAN BE SECTIONED WITHOUT NOTICEABLE DAMAGE TO THE KNIFE OR
DISRUPTION OF SURROUNDING TISSUES. CAN BE SEEN AS _ AFTER HEMATOXYLIN STAINING.

ACIDS
CHELATING AGENTS
ION EXCHANGE RESINS
ELECTRICAL IONIZATION (ELECTROPHORESIS)

CALCIUM MAY BE REMOVED BY ANY OF THE FOLLOWING:

ACID DECALCIFYING AGENTS

MOST WIDELY USED AGENTS FOR ROUTINE DECALCIFICATION OF LARGE AMOUNTS OF BONY TISSUES FOR THEY ARE STABLE, EASILY AVAILABLE,

ACID DECALCIFYING AGENTS

NITRIC ACID
HYDROCHLORIC ACID
FORMIC ACID

ACID DECALCIFYING AGENTS

TRICHLOROACETIC ACID
SULFUROUS ACID
CHROMIC ACID
CITRIC ACID

NITRIC ACID

MOST COMMON AND THE FASTEST DECALCIFYING AGENT USED SO FAR, UTILIZED BOTH AS A SIMPLE SOLUTION OR COMBINED WITH OTHER REAGENTS.

NITRIC ACID

VERY RAPID DECALCIFYING AGENT, PRODUCING MINIMAL DISTORTION, THEREFORE RECOMMENDED FOR ROUTINE PURPOSES.

5-10%

NITRIC ACID: RECOMMENDED CONCENTRATION OF _ IF USED AS SIMPLE AQUEOUS SOLUTION.

A. AQUEOUS NITRIC ACID SOLUTION 10%
B. FORMOL-NITRIC ACID
C. PERENYI'S FLUID
D. PHLOROGLUCIN-NITRIC ACID

NITRIC ACIDS

CONCENTRATED NITRIC ACID 10 ML
DISTILLED WATER 100 ML

AQUEOUS NITRIC ACID SOL'N formula

12-24 HOURS

AQUEOUS NITRIC ACID SOL'N decalcification time

AQUEOUS NITRIC ACID SOLUTION 10%

■ RAPID IN ACTION
■ PRODUCES MINIMUM DISTORTION OF TISSUES

AQUEOUS NITRIC ACID SOLUTION 10%

■ PRODUCES GOOD NUCLEAR STAINING.
■ EASILY REMOVED BY 70% ALCOHOL

AQUEOUS NITRIC ACID SOLUTION 10%

RECOMMENDED BY URGENT BIOPSY, AND FOR
NEEDLE & SMALL BIOPSY SPECIMENS TO PERMIT
RAPID DIAGNOSIS WITHIN 24 HRS OR LESS.

AQUEOUS NITRIC ACID SOLUTION 10%

CAN BE USED FOR LARGE, HEAVILY MINERALIZED CORTICAL BONE SPECIMEN IF DECALCIFICATION PROGRESS IS CAREFULLY
MONITORED BY A DECALCIFICATION ENDPOINT TEST.

AQUEOUS NITRIC ACID SOLUTION 10%

IMPARTS YELLOW COLOR WITH NITROUS
ACID, THERE BY IMPAIRING THE STAINING
REACTION OF THE TISSUE.

STRONG ACIDS

_ TEND TO BE MORE DAMAGING TO TISSUE ANTIGENS FOR
IMMUNOHISTOCHEMICAL STAINING, AND
ENZYMES MAY BE TOTALLY LOST.

TISSUE DISTORTION.

PROLONGED DECALCIFICATION MAY LEAD TO

CONCENTRATED NITRIC ACID 10 ML
STRONG FORMALDEHYDE, 40% 5 ML
DISTILLED WATER 85 ML

FORMOL - NITRIC ACID
FORMULA:

1-3 days

FORMOL - NITRIC ACID decal time

FORMOL-NITRIC ACID

IT PRODUCES LESS TISSUE DESTRUCTION THAN
10% AQUEOUS NITRIC ACID

5% sodium sulfate and washing in running tap water for at least 12 hours.

THE YELLOW COLOR IMPARTED BY THE NITROUS ACID
FORMATION WILL IMPAIR STAINING REACTION OF THE
CELL. THIS MAYBE PREVENTED BY NEUTRALIZING THE
TISSUE WITH -

Fume hood

▪ THE formol-nitric acid SOLUTION SHOULD BE USED INSIDE A

NITRIC ACID 40 ML
CHROMIC ACID, 0.5% 30 ML
ABSOLUTE ETHYL ALCOHOL 30 ML

PERENYI'S FLUID
FORMULA:

PERENYI'S FLUID

(MIX SHORTLY BEFORE USE, CHROMIC ACID MUST BE COLLECTED FOR PROPER DISPOSAL):

2-7 days

DECALCIFICATION TIME of PERENYI'S FLUID

PERENYI'S FLUID

■ IT IS RECOMMENDED FOR ROUTINE PURPOSES
■ IT DECALSIFIES AND SOFTENS TISSUES AT THE SAME TIME

PERENYI'S FLUID

■ NUCLEAR AND CYTOPLASMIC STAINING IS GOOD
■ MACERATION IS AVOIDED DUE TO THE PRESENCE OF CHROMIC
ACID AND ALCOHOL

PERENYI'S FLUID

IT'S A RELATIVELY SLOW DECALSIFYING AGENT FOR DENSE
BONES; HENCE IS NOT RECOMMENDED FOR URGENT
WORKS

GLACIAL ACETIC ACID DROP BY DROP.

▪ COMPLETE DECALSIFATION CAN'T BE DETERMINED BY
CHEMICAL TESTS BECAUSE A PRECIPITATE IS FORMED UPON
THE ADDITION OF AMMONIA TO THE FLUID EVEN IN THE
ABSENCE OF CALCIUM ION, THIS MAY BE DISSOLVED BY
ADDING

0.5 ML OF SATURATED AQUEOUS AMMONIUM OXALATE

ABOUT - IS THEN ADDED TO THE SOLUTION. REAPPEARANCE OF WHITE PRECIPITATE WITHIN 30 MINUTES WILL REAFFRIM THE
PRESENCE OF CALCIUM IN THE AGENT, SIGNIFYING THAT
DECALCIFICATION IS INCOMPLETE

CONCENTRATED NITRIC ACID 10 ML
PHLOROGLUCIN 1 GM
NITRIC ACID, 10% 100 ML

D. PHLOROGLUCIN-NITRIC ACID
FORMULA:

D. PHLOROGLUCIN-NITRIC ACID

(TO BE ADDED AFTER DISAPPEARANCE OF DENSE WHITE FUMES FORMED BY COMBINING FIRST TWO INGREDIENTS

12-24 hours

D. PHLOROGLUCIN-NITRIC ACID decal time

PHLOROGLUCIN-NITRIC ACID

IT IS THE MOST RAPID DECALCIFYING AGENT SO FAR,
RECOMMENDED FOR URGENT WORKS.

PHLOROGLUCIN-NITRIC ACID

▪ NUCLEAR STAINING IS POOR
▪ PROLONGED DECALCIFICATION PRODUCES EXTREME TISSUE
DISTORTION
▪ COMPLETE DECALCIFICATION CANNOT BE DETERMINED BY
CHEMICAL MEANS

5% SODIUM SULFATE

YELLOW COLOR of PHLOROGLUCIN-NITRIC ACID MUST BE NEUTRALIZED WITH - AND THOROUGHLY WASHED WITH RUNNING TAP WATER FOR ATLEAST 24 HOURS

PHLOROGLUCIN-NITRIC ACID

• WHEN DECALCIFICATION IS COMPLETE, THE ACID MUST BE REMOVED BY 3 CHANGES OF 70% TO 90% ETHANOL, SINCE WASHING IN WATERY SOLUTIONS WILL LEAD TO EXCESSIVE SWELLING AND DETERIORATION OF TISSUE

PHLOROGLUCIN-NITRIC ACID

• WHEN THE SECTIONS ARE CUT, THE SLIDES ARE BROUGHT TO WATER AND PLACED IN 1% AQUEOUS LTHIUM CARBONATE FOR 1 HOUR, WASHED IN LATER FOR 15 MINUTES, AND THEN STAINED

HYDROCHLORIC ACID

INFERIOR COMPARED TO NITRIC ACID IN ITS ROLE AS A DECALCIFYING AGENT BECAUSE OF ITS SLOWER ACTION AND GREATER DISTORTION OF TISSUE PRODUCED ON THE SECTION DECALCIFIED.

HYDROCHLORIC ACID

HOWEVER, IT WILL PRODUCE GOOD NUCLEAR STAINING AND IF USED IN 1% SOLUTION WITH 70 % ALCOHOL , MAY BE
RECOMMENDED FOR SURFACE DECALCIFICATION OF THE
TISSUE BLOCKS

VON EBNER'S FLUID

HYDROCHLORIC ACID fluid

SATURATED AQUEOUS SOLUTION OF NaCl 36% 50 ML
CONCENTRATED HYDROCHLORIC ACID 8 ML
DISTILLED WATER 50 ML

VON EBNER'S FLUID FORMULA:

VON EBNER'S FLUID

■ IT PERMITS RELATIVELY GOOD CYTOLOGIC STAINING
■ IT IS A MODERATELY RAPID DECALCIFYING AGENT

VON EBNER'S FLUID

■ IT DOES NOT REQUIRE WASHING OUT BEFORE DEHYDRATION
■ IT IS RECOMMENDED FOR TEETH AND SMALL PIECES OF BONE

FORMIC ACID

MODERATE-ACTING DECALCIFYING AGENT WHICH
PRODUCES BETTER NUCLEAR STAINING WITH LESS TISSUE DISTORTION, AND IS SAFER TO HANDLE THAN NITRIC ACID OR HYDROCHLORIC ACID.

FORMIC ACID

RECOMMENDED FOR ROUTINE DECALCIFICATION OF POSTMORTERM RESEARCH TISSUES, ALTHOUGH NOT SUITABLE FOR URGENT EXAMINATIONS

FORMIC ACID

IT IS THE ONLY WEAK ACID USED EXTENSIVELY AS A
PRIMARY DECALCIFYING AGENT. ADDITION OF CITRATE PROBABLY ACCELERATE DECALCIFICATION BY CHELATING THE CALCIUM AS IT IS LIBERATED FROM THE BONE.

FORMIC ACID (Sp. grav. 1.20) 10 ML
FORMAL SALINE 10% 90 ML

FORMIC ACID FORMULA:

2-7 days

Formic acid decal time

FORMIC ACID

■ IT MAY BE USED BOTH AS A FIXATIVE AND DECALCIFYING AGENT
■ IT PERMITS EXCELLENT NUCLEAR AND CYTOPLASMIC STAINING

FORMIC ACID

■ IT IS RECOMMENDED FOR SMALL BONES AND TEETH
■ IT IS SUITABLE FOR MOST ROUTINE SURGICAL SPECIMENS, PARTICULARLY WHEN IMMUNOHISTOCHEMICAL STAINING IS NEEDED

FORMIC ACID

IT IS RELATIVELY SLOW; HENCE, IS NOT SUITABLE FOR URGENT SPECIMENS. DECALCIFICATION MAY BE HASTENED BY INCREASING THE PROPORTION OF FORMIC ACID TO 25ML.
HOWEVER, SUCH CONCENTRATION MAY MAKE THE SOLUTION OPAQUE, THEREBY INTERFERING THE STAINING RESULTS.

FORMIC ACID

IT REQUIRES NEUTRALIZATION WITH 5%
SODIUM SULFATE, AND WASHING OUT TO
REMOVE THE ACID FROM THE TISSUE

AQUEOUS SODIUM CITRATE 20% 50 ML
FORMIC ACID 45% 50 ML

FORMIC ACID-SODIUM CITRATE SOLUTION formula

3-14 days

FORMIC ACID-SODIUM CITRATE decal time

FORMIC ACID-SODIUM CITRATE SOLUTION

■ IT PERMITS BETTER NUCLEAR STAINING THAN
NITRIC ACID METHOD
■ IT IS RECOMMENDED FOR AUTOPSY MATERIALS,
BONE MARROW, CARTILAGE AND TISSUES
STUDIED FOR RESEARCH PURPOSES

FORMIC ACID-SODIUM CITRATE SOLUTION

■ IT IS RELATIVELY SLOW; HENCE, IS NOT SUITABLE
FOR ROUTINE PURPOSES AND FOR DENSE
TISSUES
■ IT REQUIRES NEUTRALIZATION WITH 5%
SODIUM SULFATE

TRICHLOROACETIC ACID 5 GM
FORMOL SALINE 10% 95 ML

TRICHLOROACETIC ACID
FORMULA:

4-8 days

TRICHLOROACETIC ACID decal time

TRICHLOROACETIC ACID

■ IT PERMITS GOOD NUCLEAR STAINING
■ IT DOES NOT REQUIRE WASHING OUT; THE
EXCESS ACID MAY BE REMOVED BY SEVERAL
CHANGES OF 90% ALCOHOL, THUS IMPROVING
TISSUE DEHYDRATION

TRICHLOROACETIC ACID

■ IT IS A WEAK DECALCIFYING AGENT, NOT USED
FOR DENSE TISSUES, AND IS SUITABLE ONLY FOR
SMALL SPICULES OF BONE
■ IT IS VERY SLOW ACTING; HENCE, IS NOT
RECOMMENDED FOR URGENT EXAMINATIONS

SULFUROUS ACID

• IS A VERY WEAK DECALCIFYING SOLUTION SUITABLE
ONLY FOR MINUTE PIECES OF BONE

CHROMIC ACID % 15 ML
OSMIUM TETROXIDE 2% 4 ML
CLACIAL ACETIC ACID 1 ML

CHROMIC ACID (FLEMMING'S FLUID)
FORMULA:

CHROMIC ACID (FLEMMING'S FLUID)

■ IT MAYBE USED BOTH AS A FIXATIVE AND
DECALCIFYING AGENT
■ IT MAY BE USED FOR DECALCIFYING MINUTE
BONE SPICULES

CHROMIC ACID (FLEMMING'S FLUID)

■ NUCLEAR STAINING WITH HEMATOXYLIN IS
INHIBITED

CHROMIC ACID (FLEMMING'S FLUID)

IT TENDS TO UNDERGO REDUCTION AND FORMS
PRECIPITATES AT THE BOTTOM OF THE CONTAINER
THUS REQUIRING FREQUENT CHANGES OF SOLUTION

CHROMIC ACID (FLEMMING'S FLUID)

INSOLUBLE PIGMENTS ARE FORMED WHEN
DECALCIFIED TISSUE IS DEHYDRATED WITH
ALCOHOL; HENCE, TISSUES MUST BE WASHED PRIOR
TO DEHYDRATION

CHROMIC ACID

HIGHLY CORROSIVE TO SKIN AND MUCOUS MEMBRANES
SUITABLE PROTECTIVE MATERIAL IS NOT READILY AVAILABLE FOR PRACTICAL LABORATORY USE

DRAIN DISPOSAL

- IS NOT A LEGITIMATE OPTION FOR ANY SOLUTION CONTAINING CHROMIUM, INCLUDING SUBSEQUENT PROCESSING OF FLUIDS FOLLOWING FIXATION OR RINSES FOLLOWING STAINING PROCEDURES INVOLVING CHROMIUM

CITRIC ACID (MONOHYDRATE) AQUEOUS SOLUTION 5 ML
AMMONIUM CITRATE (ANHYDROUS) AQUEOUS SOLUTION 95 ML
ZINC SULFATE AQUEOUS SOLUTION 0.2 ML
CHLOROFORM (AS PRESERVATIVE) A FEW DROPS

CITRIC ACID-CITRATE BUFFER SOLUTION (ph 4.5)
FORMULA:

6 days

CITRIC ACID-CITRATE BUFFER SOLUTION (ph 4.5) decal time :

CITRIC ACID-CITRATE BUFFER SOLUTION (ph 4.5)

■ IT PERMITS EXCELLENT NUCLEAR AND
CYTOPLASMIC STAINING
■ IT DOES NOT PRODUCE CELL OR TISSUE
DISTORTION
■ ITS ACTION IS TOO SLOW FOR ROUTINE PURPOSES

CHELATING AGENTS

SUBSTANCES WHICH COMBINE WITH CALCIUM
IONS AND OTHER SALTS (e.g. IRON AND MANESIUM DEPOSITS) TO FORM WEAKLY DISSOCIATED COMPLEXES AND FACILITATE REMOVAL OF CALCIUM SALT

ETHYLENE DIAMINE TETRAACETIC ACID EDTA SALT,

THE MOST COMMON CHELATING AGENT IN THE MARKET
IS _ WITH THE COMMERCIAL NAME OF VERSENE, RECOMMENDED ONLY FOR DETAIL MICROSCOPIC STUDIES.

VERSENE

THE MOST COMMON CHELATING AGENT IN THE MARKET
IS ETHYLENE DIAMINE TETRAACETIC ACID EDTA SALT, WITH THE
COMMERCIAL NAME OF _, RECOMMENDED ONLY FOR DETAIL MICROSCOPIC STUDIES.

CALCIUM AND MAGNESIUM

ALTHOUGH EDTA IS TRADITIONALLY REFERRED
TO AS "ACID", IT DOES NOT ACT LIKE INORGANIC OR ORGANIC ACIDS BUT IT BINDS METALLIC IONS, NOTABLY

EDTA

COMBINES WITH CALCIUM FORMING AN INSOLUBLE NON-IONIZED COMPLEX (WHICH IS WHY IT IS ALSO USED AS AN ANTICOAGULANT AND WATER SOFTENER)

False

T/f EDTA is a very fast decal agent

1-3; 6-8

THE TISSUE IS PLACED IN EDTA FROM - WEEKS FOR SMALL SPECIMENS, BUT IT MAY TAKE - WEEKS OR LONGER TO TOTALLY DECALCIFY DENSE CORTICAL BONE

3

THE edta SOLUTION SHOULD BE CHANGED EVERY - DAYS,
AND IN THE FINAL STAGE, EVERYDAY, TO FACILITATE DECALCIFICATION.

3; 7-7.4; 8

EDTA WILL NOT BIND TO CALCIUM BELOW pH _ AND IS FASTER AT pH -. ALTHOUGH pH _ AND ABOVE WILL GIVE OPTIMAL BINDING, THE HIGHER pH MAY DAMAGE ALKALINE SENSITIVE PROTEIN LINKAGES

EDTA AND EDTA DISODIUM SALT (10%)

CAN BE SIMPLE AQUEOUS OR BUFFERED SOLUTIONS AT NEUTRAL pH of 7-7.4, OR ADDED TO FORMALIN.

EDTA TETRASODIUM SOLUTION

IS ALKALINE, AND pH SHOULD BE ADJUSTED TO
7.4 USING CONCENTRATED ACETIC ACID

EDTA SODIUM SALT 5.5 GM
DISTILLED WATER 90 ML
FORMALDEHYDE (37-40% STOCK) 10 ML

CHELATING AGENTS
FORMULA:

CHELATING AGENTS

■ IT PERMITS EXCELLENT STAINING RESULTS
■ IT PRODUCES MINIMAL CELL AND TISSUE
DISTORTION
■ IT FORMS MINIMAL HISTOLOGICAL ARTIFACTS,
USUALLY CAUSED BY PRODUCTION OF CO2 BUBBLES

CHELATING AGENTS

■ EXTENT OF DECALCIFICATION CAN BE MEASURED BY
ROUTINE CHEMICAL TEST
■ EDTA IS AN EXCELLENT BONE DECALCIFIER FOR
IMMUNOHISTOCHEMICAL OR ENZYME STAINING
AND FOR ELECTRON MICROSCOPY

CHELATING AGENTS

ENZYMES REQUIRE SPECIFIC pH CONDITIONS IN
ORDER TO MAINTAIN ACTIVITY, AND EDTA
SOLUTION CAN BE ADJUSTED TO A SPECIFIC pH FOR
ENZYME STAINING

CHELATING AGENTS

■ IT IS VERY SLOW, AND IS THEREFORE NOT
RECOMMENDED FOR URGENT AND ROUTINE
PURPOSES
■ IT CAUSES SLIGHT TISSUE HARDENING

EDTA

INACTIVATES ALKALINE PHOSPHATASE
ACTIVITY, WHICH CAN BE RESTORED BY
ADDITION OF MAGNESIUM CHLORIDE

ION EXCHANGE RESIN

(AMMONIUM FORM OF POLYSTERENE RESIN) HASTENS DECALCIFICATION BY REMOVING CALCIUM ION FOR FORMIC ACID CONTAINING DECALCIFYING SOLUTIONS, THEREBY INCREASING SOLUBILITY FROM THE TISSUE.

ION EXCHANGE RESIN

IT IS NOT RECOMMENDED FOR FLUIDS
CONTAINING MINERAL ACIDS SUCH AS NITRIC ACID OR HYDROCHLORIC ACID

ION EXCHANGE RESIN

ABOT ½ INCH THICK IS SPREAD OVER THE BOTTOM OF THE CONTAINER TO BE USED AND THE SPECIMEN IS PLACED ON TOP OF IT. THE DECALCIFYING AGENT IS THEN ADDED,
USUALLY 20-30 TIMES THE VOLUME OF THE TISSUE.

ION EXCHANGE RESIN

• THE TISSUE MAY BE ALLOWED TO STAY IN SOLUTION FOR
1-14 DAYS.

ION EXCHANGE RESIN

THE DEGREE OF DECALCIFICATION MAY THEN
BE MEASURED BY PHYSICAL OR X-RAY METHOD

N/10 HC1

• THE RESIN THAT HAS BEEN PREVIOUSLY USED MAY LATER
BE REACTIVATED BY IMMERSING IT IN _ TWICE
AND WASHING IT WITH DISTILLED WATER THRICE