HISTOPATHOLOGY: DECALCIFICATION DEHYDRATION

20:1

Ratio of decalcifying agent to tissue

1-2 days

Duration of decalcification

Acid

Widely used for routine decalcification

Nitric acid

Most common acid decalcifying agent

5-10%

Most commonly used conc for nitric acid

Nitrous acid

Forms a yellow color (nitric acid)

Urea crystals, sodium thiosulfate

Remedy for nitrous acid

Formol nitric acid

Nitric acid + formalin

For rapid and urgent biopsies

Perenyis fluid

Decalcifies and softens tissue

Phloroglucin nitric acid

Most rapid decalcifying agent

HCl

Used for surface decalcification of tissue blocks

Formol nitric acid

Nitric acid + formalin

For rapid and urgent biopsies

Perenyis fluid

Decalcifies and softens tissue

Phloroglucin nitric acid

Most rapid decalcifying agent

HCl

Used for surface decalcification of tissue blocks

Von Ebner's

Nitric acid + 36% NaCl

Von Ebner's

Recommended for teeth and small pieces of bones

RT

Decalcification is performed at what temp?

Formic acid

Best decalcifying agent

Formic acid

Recommended for routine decalcification of postmortem research tissues

Citrate

Hastens decalcification if added to Formic acid

Formic acid

Fixative and a decalcifying agent

Formic acid sodium citrate

Recommended for autopsy mat, BM, cartilage, tissues for research

TCA

Weak decalcifying agent

Suitable for small bone spicules

Sulfurous acid

Weak decalcifying agent

For minute pieces of bone

Chromic acid

Flemming' fluid

Chromic acid

Fixative and decalcifying agent

For minute bone spicules

Chelating agents

Combine with calcium salts and other salts to form complexes and facilitate removal of calcium

EDTA (versene)

Most commonly used Chelating agents

1-3 weeks

Duration for small specimens (Chelating agents)

6-8 weeks

Duration for dense bones (Chelating agents)

Chelating agents

Excellent for immunohistochem or enzyme stain, and for EM

Ion exchange resin

Ammonium form of polystrene

Ion exchange resin

Hastens decalcification by using formic acid containing decalcifying sol.

(increases solubility of calcium)

20-30

Amount of Ion exchange resin added to tissue

Physical, xray

Used to measure degree of decalcification using Ion exchange resin

Electrophoresis

Electrical ionization

Electrophoresis

Positively charged calcium ions move towards the negative electrode using an electric charge

Electrophoresis

Suited for small bone fragments

Use 88% formic acid

Physical

Measure extent of decalcification:

-mechanical method

Physical

Measure extent of decalcification:

-bending or touching of the tissue using a needle

Physical

Measure extent of decalcification:

-prone to produce artifacts and may destroy cellular details

Rubbery

Consistency of tissue if its completely decalcified (using physical method)

X ray

Measure extent of decalcification:

-Radiologic method

X ray

Measure extent of decalcification:

-Very expensive

-Most ideal

X ray

Measure extent of decalcification:

-not for mercuric chloride fixed tissues

Radiopacity

Produced by mercuric chloride fixed tissues in x ray method

Chemical

Measure extent of decalcification:

-simple, reliable convenient

Chemical

Measure extent of decalcification:

-detection of calcium in acid sol by precipitation of CaOH or CaOx

Clear

In chemical method of detecting extent of decal.

-Indication of complete decalcification

Cloudy (precipitation)

In chemical method of detecting extent of decal.

-Indication of incomplete decalcification

Tissue softeners

Added to hard tissues to facilitate cutting and processing

Lendrum's

Tissue softeners: 4% phenol

Molliflex

Tissue softeners:

Tissues become swollen and soapy

decalcification

is the procedure whereby calcium or lime salts are removed from tissues following fixation

acids

chelating agents

usual chemical agents used for decalcification

acids

decalcifiers that form soluble calcium salts

chelating agents

decalcifiers that bind to calcium ions

after fixation and before impregnation

when should decalcification be done?

poor cutting of hard tissues

damage to the knife edge

effects of inadequate decalcification

tuberculous organs

arteriosclerotic vessels

other examples of calcified tissues

fine fret saw

used to to cut small pieces of calcified tissues

hand razor

used to trim small pieces of calcified tissues

to permit complete penetration of decalcifying solution without minimal surface damage and tissue distortion

purpose of trimming calcified tissues

sharp razor blade

used to trim teeth

dark purple granular masses with lighter purple halos

color of microcalcifications after hematoxylin staining

oligodendroglioma

name for brain tumor

cotton or gauze saturated with 10% HCl for 1 hr

remedy for resistantce or grating sensation of tissues when sectioned

staining properties

affected after decalcification is done

rapid decalcifying agents

agents that are more likely to adeversely affect any subsequent staining

cell nuclei

part of tissues where affected staining by the rapid decal agents are noticeable

acid dyes

dyes that will be less affected by decalcification

deep brick red

color produced by the eosin during decalcification

eosin

an acid dye that can produce a deep brick red stain without differential shading

post-decalcification

remedy for affected staining on H&E staining

must remove calcium salts from the tissues

must not destroy cell and tissue components

must not affect staining properties of the cell and tissues

characteristics of a good decalcifying agent

acids

chelating

ion exchange resins

electrical ionization (electrophoresis)

decalcifying agents

nitric acid

HCl

formic acid

TCA

H2SO4

chromic acid

citric acid

7 acid decalcifying agents

acids

most widely used agents for routine decalcification

acids

commonly used for large amounts of bony tissues

nitric acid

most common and the fastest decalcifying agent

5-10%

recommended concentration for nitric acid

nitric acid

it is a very rapid decalcifying agent

nitric acid

it inhibits nuclear stains and destroy tissues in concentrated solutions

addition of formaldehyde or alcohol

prevention for the inhibition of nuclear staining and destruction of tissues by nitric acid

10% aqueous nitric acid

formol nitric acid

perenyi's

phloroglucin-nitric acid

nitric acid decal agents

10% ANA

this acid may be easily removed by 70% alcohol

10% ANA

recommended for urgent biopsy and for needle and small biopsy

24 hours or less

decal time of nitric acid to needle and small biopsy

10% ANA

can be used for large or heavily mineralized cortical bone spx

tissue distortion

prolonged decal may lead to _______.

nitric acid

imparts a yellow color on tissues

old nictric acid solution

is particularly damaging and should be replaced with fresh stock solution

strong acids

tend to be more damaging to tissue antigens for immunohistochemical staining

formol nitric acid

nuclear staining is relatively good in this decal agent

formol nitric acid

produces less tissue destruction tham 10% aqueous nitric acid

5% sodium thiosulfate

running water

12 hrs

remedy for the yellow impart caused by formol nitric acid

0.1% urea

addition of this to pure conc. nitric acid will also make discoloration disappear

formol nitric acid

this solution should be used inside a fume hood

perenyi's

decalcifies and softens tissues at the same time