LESSON 3: DECALCIFICATION Flashcards by CANA GIFT CHAVEZ

✓ process that entails the removal of calcium or lime salts from tissue samples after fixation
✓ this process is also known as demineralization

DECALCIFICATION

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✓ Poor cutting of hard tissues
✓ Damage to the knife edge during sectioning
✓ Bone dust and other cellular debris obscures microanatomic details

Consequences of NOT Performing Decalcification

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✓ Distortion or damage to tissues
✓ Affects staining
✓ Sections Float-off During Staining

Consequences of Performing Decalcification

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hematoxylin is inhibited

Basic dyes

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eosin produces a deep brick red color without differential staining

Acid dyes

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➢ Failure of sections to stain properly is compounded by: (1) overtreatment in acid & (2) insufficient washing out of the acid
➢ Basic dyes: hematoxylin is inhibited
➢ Acid dyes: eosin produces a deep brick red color without differential staining

Consequences of Performing Decalcification 2. Affects staining

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➢ Observed after immersion in acid alcohol

➢ Collodionize prior to staining

Consequences of Performing Decalcification

3. Sections Float-off During Staining

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➢ cut into small pieces using fret-saw, trimmed with a hand razor and fixed with 10% neutral buffered formalin

Bones and other calcified samples (tuberculous organs, atherosclerotic vessels, teratomas)

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Fixative of choice

10% neutral buffered formalin

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partial or complete decalcification is required before cutting samples

Teeth

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➢ detected during sectioning or examination
➢ REMEDY IF DETECTED DURING SECTIONING: surface decalcification using a pad of cotton/gauze soaked with 10% HCl for 1 hour
➢ APPEARANCE UNDER THE MICROSCOPE: dark purple granular masses with lighter purple halos
➢ commonly found in malignancy

Microcalcified samples

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➢ Dense/Hard Bone: 2-5 mm thick

➢ Softer Tissue: 4-6 mm thick

Thickness of the Specimen

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➢ Ideal: 24-48 hours

➢ Dense Cortical Bone: 14 days

Duration

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✓ Required temperature: 18-30 degrees Celsius (ROOM TEMP)
✓ Heat enhances destructive action of acids on matrices
✓ 37 degrees Celsius: impairs nuclear staining with Van Gieson’s
→ reduced effectiveness of Trichrome and PAS
✓ 55 degrees Celsius: tissues will undergo complete digestion within 24-48 hrs

Temperature and Heat

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Required temperature

18-30 degrees Celsius (ROOM TEMP)

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Temp that impairs nuclear staining with Van Gieson’s

37 degrees Celsius

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temp at w/c tissues will undergo complete digestion within 24-48 hrs

55 degrees Celsius

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stains for muscles, for blood samples, and bone marrow samples

Van Gieson’s, Trichrome and PAS

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❖ Concentration of Solutions
➢ Directly proportional to the rate of decalcification
❖ Strong Acids
➢ Affects the antigenicity of cells and tissue components

Solution Used

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antigens which are tumor markers that can be used to detect malignancy or can detect cancer found in the surface of cells or tissue cells

antigenicity of cells

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bone sample can be destroyed is used acid is too strong; must be diluted so that reagent won’t be too concentrated in the sample

Strong Acids

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the more concentrated the reagent is, the faster the decalcification

Directly proportional to the rate of decalcification

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❖ Protect tissues but slows down decalcification

Presence of Additives

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❖ Tissues are to be suspended in the upper portion of the jar/container

Fluid Access

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❖ Once or twice a day – depends on the type of reagent (usually once or twice a day) because it causes turbidity

Changing of the Solution

Optimum: 20 times the volume of the tissue

Volume

❖ Mechanical agitation or moving of tissue in the solution which influences fluid exchange ❖ Gentle fluid agitation: low speed rotation, rocking, mechanical stirrer, bubbling air into the solution ❖ Vigorous agitation: sonication

Agitation

remove acid in the tissue so that it won’t affect staining soon

Removal of Decalcifying Solution

– use of heat and electricity for faster decalcification

Microwave and Electrolytic Methods

acids or chelating agents

DECALCIFYING AGENTS

1. Remove calcium salts completely. 2. Does not produce considerable destruction of cells and tissue components. 3. Does not adversely affect the staining capacity of the cell

Characteristics of a Good Decalcifying Agent:

use of acids, use of chelating agents, ion exchange resins and electrical ionization

DECALCIFYING METHODS

Injurious to the organic ground substance of tissues

USE OF ACID SOLUTIONS

✓ most common and fastest ✓ 5 to 10% is the recommended concentration when used as a simple solution ✓ rapid decalcifying agent: may inhibit nuclear stains and damage tissues ✓ formaldehyde or alcohol and chromic acid may be added as additives ✓ Washing of tissue: acid removed by 3 changes of 70-90% ethanol ✓ Washing slide: brought to water and placed in 1% aq. lithium carbonate for 1 hour→ wash for 15 min ✓ causes spontaneous yellow discoloration ➢ impairs staining reaction of the tissue ➢ IF PRESENT IN TISSUES: neutralize with 5% NaSO4 → wash in running tap water (at least 12 hours) ➢ IF PRESENT IN SOLUTION: add 0.1% urea to pure conc. nitric acid

NITRIC ACID

not too concentrated because too concentrated samples destroy the antigenic properties of tissue

5 to 10%

acid removed by 3 changes of 70-90% ethanol

Washing of tissue

used in dehydration which is the next step after decalcification

Ethanol

alkaline solution for neutralization; not too acidic slide

1% aq. lithium carbonate

silver impregnation of nerve fibers

De Castro’s Fluid

nitric acid, 40% formaldehyde

Formol-Nitric Acid:

nitric acid, 0.5% chromic acid, absolute alcohol

Perenyi’s Fluid

- slow-acting | - complete decalcification cannot be determined by chemical testing

DISADVANTAGES of Perenyi’s Fluid

conc. nitric acid, phloroglucin, 10% nitric acid ✓ most rapid decalcifying agent ✓ recommended for urgent work

Phloroglucin-Nitric Acid

changes of 70 to 90% ethanol

WASHING OF TISSUES using Phloroglucin-Nitric Acid

o bring slides to water o place in 1% aqueous lithium carbonate for 1 hour o wash for 15 minutes

WASHING OF SECTIONS using Phloroglucin-Nitric Acid

✓ fixative and decalcifying agent ✓ for small and large pieces of bones, and teeth ✓ gentler on tissues How will you know if your tissue is completely decalcified? 1&2. PHYSICAL AND CHEMICAL TESTING – bend or prick and touch the bone 3. RADIOLOGICAL: x-ray bone sample then the radtech will interpret if there is a calcified portions or completely decalcified sample or will be interpreted by the use of chemicals ✓ post-mortem research tissues ✓ concentrated reagent: 90% ➢ Aqueous Formic Acid: formic acid, formalin ➢ Formic Acid-Sodium Citrate ❖ WASHING: neutralize with 5% sodium sulfate

FORMIC ACID

concentrated reagent of FORMIC ACID

90%

formic acid, formalin

Aqueous Formic Acid

✓ slower action, with greater distortion ✓ provides good nuclear staining ✓ surface decalcification: 1% HCl with 70% alcohol ✓ cannot be measured by chemical testing

HYDROCHLORIC ACID

36% saturated aqueous NaCl, concentrated HCl | ✓ for teeth and small pieces of bones

Von Ebner’s Fluid

✓ for minute samples ✓ provides good nuclear staining ✓ does not require washing-out ✓ very slow and weak decalcifying acid

Trichloroacetic Acid (TCA)

✓ also for minute samples | ✓ fixative and decalcifying agent

Flemming’s Fluid

✓ provides excellent nuclear and cytoplasmic staining | ✓ does not produce distortion

Citric Acid-Citrate Buffer

✓ very weak decalcifying agent, thus it is recommended for very minute samples only

Sulfurous Acid

- H and E - Masson’s Hematoxylin-Phloxine-Safran - Giemsa Stain

STAINS USED AFTER ACID DECALCIFICATION

NITRIC ACID DECALCIFYING AGENTS - | Most commonly used decalcifying agent

5 to 10% Nitric acid

NITRIC ACID DECALCIFYING AGENTS | -For silver impregnation of nerve fibers

De Castro’s fluid

NITRIC ACID DECALCIFYING AGENTS | Nitric acid + 40% formaldehyde

Formol-Nitric acid

NITRIC ACID DECALCIFYING AGENTS | Slow-acting decalcifying agent

Perenyi’s fluid

NITRIC ACID DECALCIFYING AGENTS | Most rapid decalcifying agent

Phloroglucin-Nitric acid

NITRIC ACID DECALCIFYING AGENTS | For small and large pieces of bones and teeth

FORMIC ACID

HYDROCHLORIC ACID DECALCIFYING AGENTS | For surface decalcification

- 1% HCl with 70% alcohol | - 10% HCl

HYDROCHLORIC ACID DECALCIFYING AGENTS For teeth and small pieces of bones

Von Ebner’s fluid

OTHER ACIDS | For minute samples

- Trichloroacetic acid | - Flemming’s fluid

OTHER ACIDS | Excellent nuclear and cytoplasmic staining

Citric acid-Citrate buffer

OTHER ACIDS | For very minute samples

Sulfurous acid

✓ excellent bone decalcifying agent for immunohistochemistry and enzyme studies ✓ acts as both decalcifying agent and water softener ✓ does not interfere with staining ✓ does not distort tissues and enzymes ✓ available in 2 formulations

EDTA: ethylenediaminetetraacetic acid

- Binds to calcium and removes it in bones | - Used for non-urgent processing and for research purposes only

EDTA: ethylenediaminetetraacetic acid

❖ 5-10% EDTA Disodium | ❖ EDTA Tetrasodium

2 formulations of EDTA

pH adjusted to 7.4 using concentrated Hac

EDTA Tetrasodium

➢ 1 to 3 weeks: small specimens | ➢ 6 to 8 weeks: dense cortical bone

DURATION

➢ pH 3: inhibits calcium binding – cannot remove calcium ➢ pH 8: optimum binding ➢ pH 7.0 to 7.4: allows binding and does not destroy tissue components

DURATION of chelating small specimens

1 to 3 weeks

DURATION of chelating dense cortical bone

6 to 8 weeks

pH w/c inhibits calcium binding – cannot remove calcium

pH 3

pH for optimum binding

pH 8

pH w/c allows binding and does not destroy tissue components

pH 7.0 to 7.4

✓ uses ammonium-sulfonated polysterene + formic acid ✓ volume of the acid solution: 20-30 times the volume of the sample ✓ Resin and Formic Acid (RAF) ➢ Cellular detail is well preserved ➢ Decalcification is faster ➢ Daily changing of solution is eliminated

ION EXCHANGE RESIN

➢ cancellous bone 2-3 mm thick (2-3 hours) | ➢ 5-6 mm thick (4-8 hours)

10 % and 20 % RAF (Resin and Formic Acid)

pieces of dense bone (24 hrs)→trimmed to 3 mm thick→40 % RAF | 24 hrs

40 % RAF

→ 2 washings with N/10 HCl | → final washing with distilled water

REACTIVATION OF USED RESIN:

fastest way to decalcify hard tissue and bone samples bec it adds electricity and heat ✓ positively charged calcium ions are attracted to a negative electrode ✓ uses heat and electrolytic reaction ✓ dependent on electricity for the removal of calcium ✓ temperature: 30 to 45 degrees Celsius ✓ uses 90%/88% formic acid and concentrated HCl as the acid solution

ELECTROPHORESIS

temperature for ELECTROPHORESIS

30 to 45 degrees Celsius

not recommended bec it can damage bone ✓ done by touching or bending to determine consistency or pricking with fine needle or probe - can destroy sample and may have a needle crash artifacts ✓ vague and not a reliable method – only surfaces only you will feel decalcified and not the inner portions of the bone

PHYSICAL TEST / MECHANICAL WAY

✓ calcium and mineral salts produce opaque areas (interpreted by trained radtechs) ✓ very expensive, most ideal method, most sensitive and most reliable method ✓ DO NOT use for mercuric chloride fixed tissues mercuric chloride – produce opaque appearance in

RADIOLOGIC TEST / X-RAY METHOD

✓ Simple, reliable and convenient method for routine purposes Why is it called calcium oxalate test? Bec the one causing turbidity and precipitation is the formation of calcium and oxalate → formation of calcium oxalate REAGENTS USED: ammonia water or ammonium hydroxide, and ammonium oxalate – calcium oxalate is the one formed when calcium is present upon addition of ammonium or sodium oxalate ❖ PROCEDURE: ➢ Aliquot 5 ml of used reagent. ➢ Alkalinize with ammonia water → (+) precipitate = (+) for calcium = INCOMPLETE DECALCIFICATION → if clear, proceed to the next step ➢ Add 0.5 ml ammonium oxalate or 1% sodium oxalate ➢ Stand for 15 to 30 minutes → (+) cloudiness or precipitate = (+) for calcium = INCOMPLETE DECALCIFICATION ❖ COMPLETE DECALCIFICATION – clear solution after 15-30 mins

CHEMICAL TEST or CALCIUM OXALATE TEST

done to remove acid for succeeding procedure which is staining

POST-DECALCIFICATION TREATMENT

``` POST-DECALCIFICATION TREATMENT Water Rinsing ➢ 30 minutes for small samples ➢ 1 to 4 hours for larger samples ➢ quick rinsing and blotting for small needle biopsies ```

WASHING-OUT

POST-DECALCIFICATION TREATMENT ❖ 2% Lithium Carbonate ❖ 5 to 10% Aqueous Sodium Bicarbonate

NEUTRALIZATION

``` POST-DECALCIFICATION TREATMENT ❖ thorough washing in water ❖ storage in any of the following: ➢ formol saline with 15% sucrose ➢ phosphate-buffered saline (PBS) with 15 to 20% sucrose at 4 degrees Celsius ```

FROZEN SECTIONING

❖ wash in water NEVER IN ALCOHOL ❖ storage in formol-saline or PBS overnight PBS (Phosphate Buffered Saline)

EDTA

✓ surface blocks submerged for 1 to 2 hours | ✓ tissues immersed for 12 to 24 hours

Perenyi’s Fluid

✓ may cause swelling or make tissues soapy

Molliflex

``` Perenyi’s Fluid 4% Aqueous Phenol Molliflex 2% HCl 1% HCl in 70% alcohol ```

TISSUE SOFTENERS