CHAPTER 9 – Preparation of Bone Sections

2 TYPES OF ADULT BONES

1. Compact bone – known as the strong bone, supports the shaft of long bones like femur, tibia,
and external surface of flat bones (e.g., the skull)
2. Trabecular or Spongy bones – is composed of a mesh of bone strands which forms the weight-
bearing structure of the diaphysis, epiphysis or vertebrae

3 MAJOR COMPONENTS OF THE BONE

o Mineral
o Cells
o Organic extracellular matrix

 Bone – undergoes remodeling or replacement through RESORPTION and DEPOSITION in

equilibrium during adulthood
 Remodeling – in this process, the volume and shape of the bone stay more or less constant
- Slows down and bones become brittle
 Cells – die later in life and are replaced, and the collagen and mineral erode and reform
continually
 Main bulk of the Bone – is approximately 70% mineral and 30% organic components by weight
with sparse bone cells
 Mineral – is approximately 38% calcium deposited as amorphous calcium phosphate during the
initial mineralization phase
 Hydroxyapatite – mineral is transformed into later on, with addition of HYDROXYL IONS
 Calcium – with addition of this, mineral forms a CRYSTAL LATTICE of needle-like crystals in which
carbonate, citrate, and fluoride ions as well as magnesium, potassium, and strontium can be
substituted or included
 Cells – composed of the 3 types of bone cells
3 TYPES OF BONE CELLS:
o Osteoblasts – some of these are fully differentiated cells and form OSTEOID
- On the surface of an actively forming bone, they appear as PLUMP CELLS with
eccentric nuclei distal to the bone surface and supported by a BASOPHILIC
CYTOPLASM
- Most of these revert to a QUIESCENT undifferentiated state and are found
among the heterogeneous cell population after a bone is formed
- Some of the osteoblasts trapped in the osteoid matrix are immature cells that
become mature osteocytes
 Lacunae – where the immature osteocytes that were trapped in the
osteoid matrix that became mature resides
– Are connected to each other and to the vascular spaces of
canaliculi
 o Osteocytes
o Osteoclasts – are responsible for bone resorption or erosion
- Are multinucleated giant cells supported by a cytoplasm rich in mitochondria
and alkaline phosphatase
- Appear singly or in small clusters on bone surfaces undergoing resorption
- Responsible for bone erosion
 Organic extracellular matrix – is composed of collagen fibers and ground substance
 Bone collagen – differs from other collagen in the body
- Becomes brittle and is laid down in bands or lamellae roughly parallel to one
another
 Collagen Fibers within each lamella – tend to lie next each other but an angle relative to the fibers
in adjacent lamellae
 Collagen lamellae – its organization is responsible for the distinctive microanatomical patterns of
bone which are easily seen with polarized light microscopy
 Bone decalcification – is the removal of calcium ions or lime salts from the organic extracellular
matrix, calcified collagen, and surrounding tissues of the bone
- This process makes the bone easy for histopathological investigation by
producing thin sections using a microtome
 Fixation using 10% neutral buffered formalin (NBF) – the first step in preparing the bone for
decalcification
- Failure to decalcify tissue results in torn and ragged sections and damages to
the cutting edge of the microtome
 4-5 mm – the tissue thickness for a good decalcification process
 Bone Biopsy – is similar to soft tissue biopsy except it needs decalcification before processing
 X-ray of specimen – may also help in selecting samples for processing

INTERPRETATION OF BIOPSY REQUIRES ASSESSMENT OF THE RELATIONSHIP BETWEEN MINERALIZED

AND UNMINERALIZED BONE OR OSTEIOD. THE METHODS ARE:

1. Mineralized bone sections embedded in METHYL METHACRYLATE (MMA) PLASTIC – is the

preferred method for metabolic bone-disease work-up
2. Frozen sections from undecalcified bone biopsy
3. Silver Impregnation method of TRIPP and MCKAY (1972) demonstrating bone and osteoid in a
decalcified, paraffin-embedded bone sections

TYPE OF SPECIMENS

1. Amputated limbs – are often sent to the laboratory immediately after surgery secondary to
tumor, inflammation and gangrene
- Often delivered without fixative, and must be taken care of as soon as possible
- The relevant portions should be immersed in a large volume of fixative to ensure
adequate fixation
- If it is not possible to treat the specimen for several hours after its arrival, it should
be refrigerated at 4° to 6° C
- After gross examination, the lesions are retained for final evaluation while the rest
of the limb is discarded
 2. Resected Specimens – these include bones with benign or low-grade malignant tumors and
arthritic femoral heads
- Are usually with established diagnosis and are considered less urgent
 Sample with least mineralization – should be selected in urgent cases of suspected tumor or
infection
- Samples should be properly fixed, rapidly decalcified, and are processed for the
quickest possible diagnosis
 Iliac Crest Trephine biopsies – are used for metabolic bone disease
- ½ of the tissue is submitted for decalcification and the other half is for
undecalcified bone MMA sections
 Proper band saw – should be used instead of scalpel blades, fretted wire, or jewelers’ saws
because these can damage the bone by crushing or causing fine trabeculae creating “FRACTURE
ARTIFACT”
 Map diagram of the lesion – where a representative sample can be selected if an X-ray of the
specimen is available

MAIN PURPOSE OF BONE RADIOGRAPHY:

1. To examine the nature of a lesion

2. To determine the extent of a lesion
3. To provide a map of a lesion prior to selection for processing
4. To check the progress of decalcification endpoint test
5. To confirm the presence of prosthetic devices, metal or glass fragments implanted by trauma

 Decalcification and Processing – depend on bone thickness

 1-3 mm – the ideal thickness
 If the bone is THICKER than the ideal thickness, decalcification and processing are prolonged.
This is because the COLLAGEN MATRIX prevents adequate paraffin wax penetration and is not
held firmly in the block during microtomy.
 Bone pieces LESS THAN the ideal thickness are held LOOSELY in the block during cutting
 Good band saw – is essential in a histopathology laboratory
 Not deeper than 7.5 – the saws cut through the cortical bone slowly
 Midplane – the first cut is made through
 3-5 mm slabs – are cut parallel to the first cut
 Soft tissues and dense connective tissues – should be removed before sawing or the sample will
drag through the blade
 Wood block held against the first cut edge – ensures an even slice
 Sawing – should be at a slow even rate to match the speed of the blade cutting into the bone
 Pushing the bones – produces uneven cuts and may jam or break a blade
 Fixation – is skipped when immediate diagnosis is needed and 10% buffered formalin is suitable
- The size should not be too small or too big for faster fixation

 Carnoy’s fixative – can cause extreme chemical toxicity because it contains chloroform
- Makes the bone radio-opaque and unsuitable for specimen radiography
  Large specimen – can be bisected and cut into smaller pieces and immersed into the fixative
immediately
- Should not be fixed longer than 48 hours after the initial fixation

METHODS OF DECALCIFICATION:

 Decalcification – is commonly carried out using chemical methods, either with acids to form
soluble calcium salts or with chelating agents that bind to calcium ions
- The tissue is suspended in a piece of gauze in the upper part of a jar to
allow the dissolved salts to sink to the bottom
- Is usually carried out in a 5% aqueous solution that contains of which are
changed everyday or twice a day for 1 to 4 days
- It can be combined with formaldehyde so long as its endpoint is
monitored to prevent tissue damage and impaired staining
1. Acid Method – is the most widely used decalcification method in many laboratories
o Acids – are readily available in the market, relatively stable, and inexpensive
- Is carried out at room temperature
- These solutions should be changed daily or even twice a day for better
decalcification

AGENTS USED ARE AQUEOUS SOLUTIONS OF:

 Nitric acid
 Formic acid
 Trichloroacetic acid
 Alcoholic solutions – are used when glycogen is to be preserved
 Excellent histologic details – cannot be expected if the tissue is decalcified for a long period of
time
 Rapid decalcifying acid agents – affect the quality of staining
- For instance, they can fail to stain the nucleus
 24-48 hours – the ideal length of time for decalcification
- Should not be prolonged beyond 4 days
 Very dense bone blocks – take 14 days or longer to be completely decalcified
 Material Safety Data Sheets (MSDS) – are provided by the manufacturers of decalcifying
solutions which include the type of chemicals included, concentration levels of acids,
instructions, and warnings against prolonged use
- These sheets are required in laboratory licensing, certification or
accreditation
 Trimming the paraffin-embedded block – a small foci of calcification occur, cause resistance,
and produce a “grating” sensation when sectioned
- In such situation, the block should be removed from the chuck and
exposed face down on a pad of cotton or gauze saturated with 10% HCl
for approximately 1 hour

DIFFERENT KINDS OF ACID DECALCIFYING SOLUTIONS:

i. Strong Inorganic Acids – are used as simple aqueous solutions usually with 5%-10%
concentrations
They are used for:
o Needle biopsy and small biopsy specimens
o Urgent diagnosis, that is, those which require diagnosis within 24 hours or less;
and
o Large or heavily mineralized cortical bone specimens whose decalcification
progress is carefully monitored by a decalcifying endpoint test

Disadvantages of using Strong Inorganic acids are as follows:

o They decalcify bones rapidly

o Concentrated agents destroy tissue by causing tissue swelling
o They can seriously affect tissue or nuclear tissue stainability if decalcified for
more than 24 hours
o Old stocks of reagents cause tissue distortion
o They damage tissue antigens for immunohistochemical staining
o Enzymes may be totally lost

Some of the acids that may be used for decalcification are:

a) Nitric acid – is the most commonly used agent for decalcification

- Is a very rapid decalcifying agent that also produces minimal distortion
- It develops good nuclear staining and is recommended for urgent biopsy
or needle biopsies
DISADVANTAGE: Tissue distortion after prolonged decalcification, impaired
staining reactions imparting a YELLOW color with nitrous acid, and destruction of
tissue antigens for immunohistochemical staining

AQUEOUS NITRIC ACID, 5%-10%

 Nitric acid, concentrated – 5 to 10 mL
 Distilled water up to – 100 mL
 Decalcification time – 12 to 24 hours

FORMALIN-NITRIC ACID
 Formaldehyde, 37%-40% - 10 mL
 Distilled water – 80 mL
 Nitric acid, concentrated – 10 mL
 Decalcification time – 1 to 3 days

b) Hydrochloric Acid – causes rapid decalcification even in dilute solutions

- It does not require washing out before dehydration and is
recommended for teeth and small pieces of bones
- Compared with nitric acid, it acts less quickly and causes greater
distortion to tissues
DISADVANTAGE: The measurement of the extent of decalcification
  H&E staining – is satisfactory when HCl is used at 15% to 25%
- Is markedly impaired if decalcification is prolonged fir 24 hours in 8%
solution, especially in high temperatures

AQUEOS HYDROCHLORIC ACID, 8%

 HCl, concentrated – 80 mL
 Distilled water – 920 mL
 Decalcification time – 1 to 3 days

ii. Weak Organic Acids

a) Formic Acids – its use allows excellent nuclear and cytoplasmic staining
- Can be used as 5%-10% aqueous or buffered or combined with formalin
- Are safer and easier to handle compared to nitric acid and HCl
- Suitable for routine most routine surgical specimens, particularly when
immunohistochemical staining
- However, it should still be end-point tested because it can damage
tissues, antigens, and enzyme histochemical staining
- Is relatively slow in decalcification, hence, it is not suitable for urgent
specimen
o Large bones – require prolonged decalcification making formic acid the most
suitable decalcifying agents
o 1 to 10 days – decalcification is usually completed, depending on the size and type
of bone and the concentration of the acid used
o 10% formalin-formic acid mixture – is simultaneously used as a fixative and a
decalcifier
- Recommended for very small bones
- Complete fixation is still advisable before use
 Sodium formate / Sodium citrate – buffer or salt to be used
- Forms “acidic” buffers to as low 4% to 5% concentration
- They are used to counteract the injurious effects of the acid or to
chelate the calcium as it is liberated from the bone
- Is used to counteract excessive swelling caused by the acid
 Nitric acid – will decalcify bone more rapidly than formic acid, but it produces a marked
decrease in the stainability of the tissue after 20 hours
AQUEOUS FORMIC ACID:

 90% stock formic acid – 5 to 10 mL

 Distilled water – 100 mL

Buffered formic acid with a pH of approximately 2.3

SOLUTION A 20% AQUEOUS SODIUM CITRATE

 Sodium Citrate – 200 mL

 Distilled water – 1000 mL
SOLUTION B 50% FORMIC ACID

 88% formic acid – 500 mL

 Distilled Water – 500 mL

 Stock solutions – equal parts of solutions A and B should be combined

FORMIC-ACID-FORMALIN

 90% stock formic acid – 5 to 10 mL

 Formaldehyde (37%-40%) – 5mL
 Distilled Water – 100 mL
 Dense Cortical or Large bones – have been effectively decalcified with 15% aqueous formic acid
and a 4% hydrochloric acid -4% formic acid mixture

b) Trichloroacetic acid and Picric acid – are weak decalcifying agents

- Stronger concentrations of them are needed for adequate
decalcification
 10% aqueous solution – quantity of which decalcification is carried out
- This permits good nuclear staining and does not require staining and
does not require washing because the excess washing may be removed
by several changes of 90% alcohol
 4 to 8 days – decalcification time
 1 hour or less – duration for needle biopsies
 48 hours – for thick sections

DISADVANTAGE: They only produce minimal decalcification

 Also causes tissue swelling
 They are used as components in Carnoy’s and Bouin’s fixatives
2. Chelating Method
 EDTA – is the most common chelating agent in the market
- Ironically, though it is an acid, but doe not act like one
- It merges with calcium or magnesium ions to form a non-ionized soluble
complex
- This solution can be adjusted to a specific pH for enzyme staining
- Does not blend with calcium below pH 3
- A higher pH may damage alkaline-sensitive protein linkages
- Washing is not necessary
- Is an excellent bone decalcifier for immunohistochemical or enzyme
staining and electron microscopy
 Saturated 10% solution of EDTA with a pH of 6.5 to 7.5 – is recommended for best
results
 Enzymes – require specific pH conditions in order to maintain activity
  1 to 3 weeks – small specimen tissues are placed in EDTA
DISADVANTAGE: It is a slow decalcifier
- Not recommended for urgent and routine purposes
- Also causes tissue hardening
 If the solution is CLOUDY, adjust to pH 7 with approximately 25 g Sodium Hydroxide. The
solution should become clearer
 6 to 8 weeks or longer – decalcification time may take

3. Ion-exchange Resin Method – calcium is rapidly removed by the decalcifying solution containing
formic acid, thereby increasing solubility from the tissue
- Cellular detail is well-preserved
- Produces minimal cell and tissue distortion
- Decalcification is faster and daily changing of solutions is not necessary
- This method is not recommended for urgent and routine purposes
because it is slow
- Also causes tissue hardening
- The degree of decalcification is only done through X-ray and cannot be
measured by the chemical method
 Ammonium form Polystyrene – the most commonly used ion-exchange resin
 20 days – the specimen can be kept in the solution without being damaged
4. Electrolytic Method – is a fast method that is also very similar to the chelating method, the only
difference is that it applies heat and electricity
- it is dependent on a supply or direct current to remove calcium deposits
- Good cytologic and histologic details are not always preserved in tissues
which have undergone this process
- Sections also do not stain well
- Only a limited number of specimens can be processed at a time
 Rapid decalcification – electrolysis is employed
 Prolonged decalcification of tissues – may produce maceration and destruction of
tissue components which are poorly stained
 Decalcification for a short period of time – may be incomplete, thereby interfering with
the normal cutting of sections and staining of specimens

FACTORS AFFECTING THE RATE OF DECALCIFICATION:

1. Concentration and Volume of the Decalcifying agent

 Generally, acid solutions of a higher concentration decalcify bone more rapidly, but they
are more harmful to the tissue
 Sometimes, a combination of fixative-acid decalcifying solutions is preferred
 Decalcification rate cannot exceed the Fixation rate or else the acid will damage or
macerate the tissue before fixation is complete
 Brain – points out that if a sufficiently large volume of fluid is used (100 mL/g of tissue),
it is not necessary to renew the decalcifying agent even though depletion is less
apparent in a larger volume
2. Temperature – the optimal temperature for acid decalcification has not been determined
 25° C – Smith suggested as the standard temperature
 RT 18° to 30° C – is acceptable
 Increased temperature – accelerates decalcification as well as tissue damage
 60° C – bones, soft tissues, and cell may become completely macerated almost as soon
as they are decalcified
- Also accelerates EDTA decalcification without the risk of maceration but
may not be acceptable for preservation of heat-sensitive antigens,
enzymes, or electron microscopy work
- If EDTA is used, it can be the temperature employed if the bone is well-
fixed
 Wallington – suggest that tissues not completely decalcified at the end of a working week
could be left in acid at 4 ° C over the weekend
 It is however recommended that decalcification be interrupted by briefly rinsing the acid
off the bone, immersing it in NBF, and resuming decalcification on the next working day
 If microwave, sonication and electrolytic methods are utilized in decalcification,
INCREASING the temperature must be monitored because it can damage the tissue
3. Agitation – there is an assumption that this speeds up decalcification but its effects remain
controversial
- Influences fluid exchange within and around tissues with other reagents
AGITATION METHOD INCLUDES:
 Low-speed rotation
 Rocking
 Stirring
 Bubbling air into the solution
4. Suspension
 Bone samples – should be suspended so that the decalcifying fluid makes complete
contact with the surface of the specimens
 Gauze or Cloth bag – where the specimen can be suspended in and tied with a thread
 They should not touch the bottom of the container to have good fluid access between
flat surfaces
 They must also be separated from one another
5. Other Factors
o Patient’s age
o Type of bone
o Size of the specimen
 Mature Cortical Bone – decalcifies more slowly than immature, developing cortical or trabecular
bone
 Dense bone tissues – require up to 14 days or longer in order to complete the process
Decalcification Endpoint
RECOMMENDATIONS FOR EACH OF THE DECALCIFYING AGENTS:

1. When using FORMIC, HCl, or NITRIC ACID, daily testing is recommended, except if the
procedure is near the endpoint, in which case one test should be done EVERY 5 HOURS when
possible
2. With EDTA, weekly tests are sufficient unless solution changes are more frequent
3. MINIMALLY-CALCIFIED TISSUES and NEEDLE BIOPSIES decalcified by a strong acid may be
tested only once.
 The endpoint of decalcified can be checked by: SPECIMEN X-RAY EXAMINATION, CHEMICAL
TESTING, or PHYSICAL TESTING
 Specimen X-ray exam and Chemical Testing – are the most reliable
 X-ray – is the most accurate way to determine if decalcification is complete
 Chemical Tests for the presence of residual calcium and EDTA – are recommended instead of
the less accurate and potentially damaging physical ones
i. Radiography – is the most sensitive and reliable test for detecting calcium in bone or tissue
calcification
- It can spot even the smallest focus of calcium seen as opaque material in
the plate
- It is expensive
ii. Chemical Test – precipitates insoluble calcium/ammonium hydroxide or calcium/ammonium
oxalate when calcium is released from a bone in acid solution
- When no calcium is found or the result is negative, decalcification is
complete and may entail using one extra change of decalcifier after
actual completion

Solutions that are needed to chemically test for residual calcium:

o 5% Ammonium Hydroxide Stock

o 5% Ammonium Oxalate stock

 Decalcification is COMPLETE when NO PRECIPITATE or TURBIDITY is observed

 White precipitate (calcium phosphate) – forms immediately after adding ammonium hydroxide,
a large quantity of calcium present
iii. Physical Test – are considered inaccurate and damaging to tissues.
- Can create artifacts (e.g., needle tracts), disrupt soft tumors from the
bone, or cause false-positive microfractures of fine trabeculae, a
potential misdiagnosis
They involve the following:
o Probing
o Bending the specimen
o Needling or inserting a pin, razor, or scalpel directly into the tissue
o Pricking or Slicing the tissue
o Touching the specimen
o Squeezing the tissue
 When decalcification is completed, acids should be removed from tissues or neutralized
chemically
 Chemical Neutralization – is accomplished by immersing the decalcified bone into
saturated lithium carbonate solution for several hours
 Culling – recommends washing in 2 changes of 70% alcohol for 12-18 hours before
continuing with dehydration in processing
 30 minutes – adequate water rinsing for small samples can be done
 1 to 4 hours – adequate water rinsing for large bones can be done
 Samples needing immediate attention (e.g., needle biopsies) can be blotted or quickly
rinsed to remove acid from their surfaces before proceeding to the first dehydrating fluid
 Tissues decalcified in EDTA solutions should NOT be placed directly in 70% alcohol as this
would cause residual EDTA to precipitate in the alcohol and within the tissue