LESSON 3: DECALCIFICATION Flashcards
✓ process that entails the removal of calcium or lime salts from tissue samples after fixation
✓ this process is also known as demineralization
DECALCIFICATION
✓ 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
✓ Distortion or damage to tissues
✓ Affects staining
✓ Sections Float-off During Staining
Consequences of Performing Decalcification
hematoxylin is inhibited
Basic dyes
eosin produces a deep brick red color without differential staining
Acid dyes
➢ 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
➢ Observed after immersion in acid alcohol
➢ Collodionize prior to staining
Consequences of Performing Decalcification
3. Sections Float-off During Staining
➢ 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)
Fixative of choice
10% neutral buffered formalin
partial or complete decalcification is required before cutting samples
Teeth
➢ 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
➢ Dense/Hard Bone: 2-5 mm thick
➢ Softer Tissue: 4-6 mm thick
Thickness of the Specimen
➢ Ideal: 24-48 hours
➢ Dense Cortical Bone: 14 days
Duration
✓ 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
Required temperature
18-30 degrees Celsius (ROOM TEMP)
Temp that impairs nuclear staining with Van Gieson’s
37 degrees Celsius
temp at w/c tissues will undergo complete digestion within 24-48 hrs
55 degrees Celsius
stains for muscles, for blood samples, and bone marrow samples
Van Gieson’s, Trichrome and PAS
❖ Concentration of Solutions
➢ Directly proportional to the rate of decalcification
❖ Strong Acids
➢ Affects the antigenicity of cells and tissue components
Solution Used
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
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
the more concentrated the reagent is, the faster the decalcification
Directly proportional to the rate of decalcification
❖ Protect tissues but slows down decalcification
Presence of Additives
❖ Tissues are to be suspended in the upper portion of the jar/container
Fluid Access
❖ 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
- Remove calcium salts completely.
- Does not produce considerable destruction of cells and tissue components.
- 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
pH
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