Preparation of Bone sections

Two types of adult bones

-Compact bone known as the strong bone, supports the shaft of long bones like femur, tibia and external surface of the flat
bone and the skull.

- The trabecular or spongy bone is composed of a mesh of bone strands which forms the weight – bearing structures of the
diaphysis , epiphysis, or vertebrae.

-The three major components of the bone are the minerals cells, and organic extracellular matrix. ( 70% mineral and 30%
organic components)

1. Minerals – initial mineralization phase, the mineral in the bone is approximately 38% calcium deposited as amorphous
calcium phosphate.
-Later on, it is transformed into hydroxyapatite( addition of hydroxyl ion)
-Also forms crystal lattice of needle-like crystals (addition of calcium)
(carbonate, citrate, fluoride ions as well as magnesium, potassium, and strontium can be substituted or included)

2. Cells – Three types of bone cells are the osteoblast, osteocytes and osteoclast.
- Some osteoblasts are fully differentiated cells and form osteoids.
-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.
-some of the osteoblasts trapped in the osteoid matrix are immature cells that become mature osteocytes( these
osteocytes reside in the lacunae,of the bone which are connected to each other and to the vascular spaces by canaliculi.
- osteoclasts are responsible for bone resoprtion or erosion.
-they are multinucleated giant cells supported by a cytoplasm rich in mitochondria and alkaline phophatase .
-they appear singly or in small clusters on bone surfaces undergoing resorption.

3.organic extracellular matrix is composed of collagen fibers and ground substances. (bone collagens differs from other
collagen fibers and ground substances)
-it becomes mineralized and is laid down in bands or lamellae roughly parallel to one another.
-The organization of collagen lamellae is responsible for the distinctive micro- anatomical patterns of bone which are easily
seen with polarized light microscopy

Bone Decalcification
- 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 histological investigation by producing thin sections using a microtome.

- The first step in preparing the bone for decalcification is fixation using of 10% neutral buffer formalin (NBF).
- Failure to decalcify tissue results in torn and ragged sections and damages to the cutting edge of the microtome.
- A tissue must not exceed 4 or 5 mm in thickness for a good decalcification process.

Preparation of bone specimens for decalcification

- Bone biopsy is similar to soft tissue biopsy except that the former needs decalcification before processing.
- X-ray of the specimen may also help select samples for processing.
- Interpretation of biopsy requires assessment of the relationship between mineralized and umineralized bone or osteoid.
The methods used are:

1. Mineralized bone sections embedded in methyl methacrylate (MMA) plastics. (It is the preferred method for metabolic
bone disease work-up)

2. Frozen section from an undecalcified bone biopsy.

3. Silver impregnation method of Tripp and McKay demonstrating bone and osteiod in a decalcified, paraffin embedded
bone sections.

Types of Specimens
1. Amputated limbs – often sent to the laboratory immediately after surgery secondary to tumor, inflammation, and
gangrene.
-they are often delivered without fixative, must be taken care of as soon as possible.
-relevant portions should be immersed in a large volume of fixation 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°C -6°C.

2. Resected specimens
-Bones with benign or low- grade malignant tumors and arthritic femoral heads (less urgent)

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Selection of Specimens
- In urgent cases of suspected tumor or infection, a sample with the least mineralizartion should be selected.
-sample should be properly fixed
-rapidly decalcified
-processed for the quickest possible diagnosis.

For metabolic bone diseases,

- The iliac crest trephine biopsies are used; one-half of the tissue is submitted for decalcification and paraffin techniques
-the other half is for undecalcified bone MMA sections
-proper band saw should be used instead of scalpel blades,fretted wire or jewellers saws(theses can damage the bone by
crushing or causing fine trabeculae creating a “fracture artefact”

If an X-ray of the specimen is available, a representative sample can be selected through a “map” diagram lesion.
Main purpose:
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 of 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.

NOTE: both decalcification and processing depend on bone thickness. (Ideal thickness is 1 – 3mm)
1. Thicker – prolonged
2. Collagen – matrix prevents adequate paraffin wax penetration and is not held firmly in the block during microtomy
3. Bones pieces less than the ideal thickness are held loosely in the during cutting

Sawing of specimens
-A good band saw is essential equipment in a histopathology laboratory.
-the saws cuts through the cortical bone slowly with cuts not deeper than 7.5cm, the first cut is made through the
midplane, then approximately 3 -5 mm slabs are cut parallel to the first cut.
-if there are soft tissues and dense connective tissues they are removed before sawing.

Fixation of Specimen
-all bone specimens must be fixed before subjecting them to any decalcification and processing procedures.
-fixation is skipped when immediate diagnosis is needed and 10% NBF is suitable.
-fixation should be done immediately; the size should not be too small or too big for faster fixation.
-opening the bone and removing the excess skin and soft tissues surrounding the lesion are required.
-Large specimens can be bisected and cut into smaller pieces, and immersed into the fixative immediately
-they should not be longer than 48hrs after the initial fixation.

Methods of Decalcification
-Good decalcification means the agent is capable of completely removing the calcium without destroying the cells.
Acid method
-Most commonly used method in many laboratories, Acids are readily available in the market, relatively stable, and
inexpensive.
-The agents used are aqueous solutions of nitric acid, formic acid, and trichloroacetic acid. Alcoholic solutions
used when glycogen is to be preserved.
- In decalcification, the tissue is suspended in a piece of gauze in the upper part of a jar to allow the dissolve salts to sink to
the bottom.
-Acid solution should be change daily or even twice a day for better decalcification.
Strong Inorganic acids
- These acid are used as simple aqueous solutions usually with 5%-10% concentration.
-used for
1. Needle biopsy and small biopsy specimens.
2. Urgent diagnoses that requires diagnosis within 24hrs or less
3. Large or heavily large or heavily mineralized cortical bone specimens whose decalcification progress is
carefully monitored by a decalcification endpoint test.

1. Nitric acid – nitric acid is the most commonly used agent for decalcification. It is a very rapid decalcifying agent that also
produces minimal distortion.
- 5% aqueous solution is usually used; the contents are change once 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.
Ex. 5 - 10% aqueous Nitric acid solution (decalcification time is 12 – 24 hours)
Formalin- nitric acid (decalcification time is 1-3 days)

2. Hydrochloric acid - causes rapid decalcification even in dilute solutions

-used at 15% - 25%, H & E staining is satisfactory.

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 -Impaired if decalcification is prolonged for 24hrs in 8% solution, especially in high temperatures
-recommended for teeth and small pieces of bones.
Ex. 8% Aqueous Hydrochloric Acid(decalcification time is 1-3 days)

Weak Organic acids

1.Formic acid – for prolonged decalcification like large bones

-allows excellent nuclear and cytoplasmic staining
EX. 10% formalin- formic acid mixture is simultaneously used as a fixative and decalcifier.
-recommended for very small bones
-suitable for most routine surgical specimens, particularly for immunohistochemical staining

2. Tricholoracetic acid and picric acid

-Decalcification is carried out with large quantities of 10% aqueous solution. This permits good nuclear staining
And does not require washing.

Chelating methods
-Ethylenediamine tetraacetic acid(EDTA) is the most common chelating agent in the
market.
-It merges with calcium or magnesium ions to form a non-ionized soluble complex.
-A 10% solution of EDTA with a pH of 6.5 to 7.5 is recommended for best results.
-excellent bone decalcifier for immunohistochemical, enzyme staining, and electron
microscopy.
-slow decalcifier, not recommended for urgent and routine purposes.
Ex. Formalin –EDTA
EDTA aqueous, pH 7.0-7.4

Ion exchange Resin method

-most commonly used is ammonium form of polystyrene. ( Mixture of formic acid and
ion-exchange resin)
-calcium is rapidly removed by the decalcifying solution containing formic acid
-advantages are cellular details is well preserved, specimen can be kept in the
solution for 20 days without being damage.
-Ex. Ion –exhange resin solution ( Ion- exchange resin 100g, 10% formic acid,
aqueous 800ml)

Electrolyte method
-for rapid decalcification electrolysis is employed.
-Positively charge calcium ions are attracted to negative electrodes.
-very similar to chelating method. The only difference is that it applies heat and
electricity.
Ex. Electrolytic decalcifying solutions( 88% formic acid 100ml, Hydrochloric acid
80ml, Distilled water 820ml)

Factors affecting the rate of decalcification

1. Concentration and Volume of the Decalcifying Agent

- High concentration= rapid decalcification rate, but more harmful to the tissue
2. Temperature
- suggested temperature is 25°C (room temp)
-If EDTA is used, 60°C can the temperature employed if the bone is well fixed.
3. Agitation
- influences fluid exchanges within and around tissue with other reagents.
4. Suspension
- Bone samples should be suspended so that the decalcifying fluid makes complete contact with the surface of the
specimen

Determination of End point of decalcification

- The endpoint of decalcification can be checked by specimen X-ray examination, chemical testing, or physical testing.
-X-ray most accurate to determine if decalcification is complete.
1. Radiography
-Most sensitive and most reliable test for detecting calcium in bone and tissue calcification.
-it can spot the smallest focus of calcium seen as opaque material in the plate.
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 -expensive
2. Chemical Test
- When calcium is released from a bone in acid solution, insoluble calcium,
ammonium hydroxide, ammonium oxalates are precipitated by chemical
methods. (Occurs on the discarded fluid)
- When no calcium is found or the result is negative, decalcification is complete.
-One extra change of decalcifier after actual completion.
-Results: Decalcification is complete when no precipitate or turbidity is
observed.
3. Physical Test
- inaccurate and damages the tissue
Ex. Probing, bending the specimen, needling or inserting a pin, razor or scalpel
directly into the tissue, pricking
Or slicing the tissue, touching the specimen, squeezing the tissue.

Processing of Frozen section

- During surgical procedures, it is sometimes necessary for the surgeon to obtain a rapid diagnosis, more commonly for
the following reasons:
1.To guide the surgeon’s intraoperative and /or perioperative management of the patient’s tumor.
- malignant neoplasm need to be evaluated for clear margins.
- lymph node metastases
-unknown pathologic processes
-tissue identification
2. To process tissues for special studies in diagnosis, treatment, and research.
3. To confirm that the tissue taken intraoperatively is lesional.

Instructions for the surgeon

- Most regarded as the most definitive form of intraoperative consultation and should be done in collaboration
with the surgeon.
- A management tool rather than a faster way to obtain a diagnosis from the pathologist.

1. Lymph nodes
-primarily used for intraoperative consultation for purposes such as sentinel lymph node biopsy or if molecular studies
and cytogenetic analysis.
-lymph nodes are submitted fresh to the histopathology laboratory and must be placed in gauze damped with saline.
-Excess fat must be removed before freezing the tissue.
-The entire node should be thinly sectioned along its long axis, and all slices should be frozen to minimize false
Negativity of the specimen.

2. Fatty tissues
- Extremely difficult to section during frozen section processing, since it contains little water.
-tissues such as lymph nodes and breast specimens also contain some amount of fat. (recommended that the fat
Removed from the specimens prior to processing)

3. Colon
-for colon surgery (colon cancer and colonic polyps) , surgical margins are sampled to make sure that these are
Free from tumor.
- Segments of bowel should be opened entirely to determine if there is invasion at the serosal surface or muscularis layer.
- stapled ends are completely opened and cut as close as possible to the stapled area

Setting up the cryostat

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Specimen processing on the Cryostat
-refer to Basic Histopathologic Technique by Raymundo W. Lo page 116 – 118

Staining (H &E staining)

1. Rinse with tap water, until the freezing medium is barely visible (3 dips)
2. Stain the section in Harris hematoxylin for 90 seconds
3. Rinse with tap water (2 dips)
4. 0.25% ammonia water until it becomes blue
5. Rinse in tap water (2 dips)
6. Stain in eosin until the desired color intensity is achieved (15- 20 dips)
7. Dehydrate the specimen with 95% alcohol and absolute alcohol (10 dips)
8. Air- dry the specimen clean the slide
9. Clean the section with xylene in 10 dips of 3 changes
10. Mount the section.

Maintenance of the cryostat

-recommended that the cryostat be maintained at the temperature suggested by the manufacturer when not in use.
-Cleaning should be done daily by removing frozen section wastes with a cold brush
-storage shelves and waste trays should likewise be removed and cleaned.
-cleaning agents specified by the manufacturer should be used.
-Most cryostat machines use 70% alcohol as a disinfectant
- Cryostat is disinfected daily after each use.
-Before cleaning, the temperature value has to be reduced to -20°C
- The knife and the knife holder should be removed, as well as all samples, microscope slides and tools
-the cryostat chamber should reach the previously selected temperature.
-Once this temperature is reached, its contaminated surfaces should be cleaned either by evenly spraying them with a
disinfectant or by applying a disinfectant with a soaked cloth.

Maintenance of the cryostat microtome

- Annual inspection by a maintenance engineer is recommended for proper maintenance of the cryostat microtome
-In the event of long periods of power failure, the microtome can be removed from the cryostat for extensive drying
- The microtome must be thoroughly dry before applying a lubricating solution and re-cooling.
-If the microtome becomes stiff and difficult to operate, it should be treated with absolute alcohol and allowed to
evaporate.
-All routine cleaning and maintenance procedures should be regularly scheduled and documented when performed.

Safety in the Histopathology Laboratory

-hazards includes electrical equipment, combustible materials, sharp objects, chemical reagents, human tissue may harbour
infection, repetitive motions and with the advent of sentinel lymph nodes biopsy and radiation exposure.
-safety begins with the design and planning of the histopathology section.
-there should be more than enough space for the staff and equipment, including future additions.
-proper ventilation, lighting, exhaust system, and fume hoods.
-electrical installation should be checked.
-sinks and faucets should have ample of running water
-waste disposal contingencies must be prepared

Universal precautions
- All blood and body fluids are considered potentially infectious.

Work practice Controls

- Good housekeeping should include regular are cleaning and daily trash emptying.
- Ergonomically – designed and computer sections and setting the workflow minimize interpersonnel impedance and
facilities safe but effective human- machine interaction.
- Mouth – pipetting is prohibited.
- Centrifuges must be kept closed and well- balanced when used.
-Laboratory work surfaces should be decontaminated with sodium hypochlorite (bleach).

Personal hygiene
- Proper hand washing (before and after work)
-Personal hygienic habits
-smoking, eating, and drinking are prohibited in technical work areas.
-Laboratory coats must be worn while working in the laboratory
-shoes should cover the toes
-hair must be secured

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 Labelling and Warning signs
-All primary and secondary containers of hazardous materials must have appropriate warning labels with the substance
Name, its manufacturer’s name and address, physical and health hazards.
-Signs should be posted on entrances of laboratories to indicate the hazardous biological, radioactive, or carcinogenic
materials used inside these areas.

Exits and aisles

-must be conspicuous and not be blocked, bolted or obstructed in any way to prevent egress .

Personal protective equipment, Emergency fixtures, Barriers, spill kits, and first aid kits.
-Glove must be worn when handling liquids that can cause harm or be absorbed through the skin.
-Face shields or goggles and mask must be worn when handling materials which may spill or splatter
such as acids and formalin.
-Eye washing station, safety shower showers, fire extinguisher, smoke detectors and alarms
levers must be installed in strategic places and checked regularly.
-Physical barriers, such as doors cabinets, and locks, must be present.
-Spill kits for biological or chemical hazards must be stored in designated areas.
-First aids should be available and fully stocked.
-vaccination against hepatitis B.
-Staff training on Awareness.

Documentation and record keeping

-All maintenance and troubleshooting activated should be recorded in a dedicated logbook.
-Meeting, incident report injuries, and sentinel events should be filed.
-Temperature of refrigerators and other machines should be monitored and recorded.
-Material Safety Data sheets (MSDS) of all reagents should be compiled and made accessible.

Machines
-automation contributes to efficient service, but equipment should be properly maintenance.
-Document periodic maintenance and calibration extend the life of the equipment and prevent mechanical malfunctions
which can cause problem.

Infection control
-The reported agents that easily infect laboratory staff include Mycobaterium tuberculosis, via aerosols, hepatitis B and C
and HIV via needle sticks and cuts and Creutzfeldt-Jakob (CJD) or Prion disease.
-A fresh tissue received in the histopathology laboratory can be a source of infectious agents such as bacteria and viruses.
- Universal precaution is the most effective way of preventing infection.
-An N95 mask, face shield, apron and glove should be worn as needed.
-for those preparing cytology of body fluids, centrifugation must be under a fume hood or a biologic safety cabinet.

Chemical safety
Various reagents used in histopathology may be grouped as follows:
1. Caustic or corrosive: Acid and alkalis that may cause burns in the skin, mouth or eyes and damage to equipment and
storage areas.
2. Irritant or allergen
3. Poisons substances
4. Carcinogenic
5. Flammable
6. Explosive
-it is good practice to avoid unnecessary exposure to these chemicals.

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 -Appropriate PPEs must be worn by all persons, including visitors, whenever chemicals are used or stored.
- The available ventilation system must be able to handle the chemical used.
-Toxic chemical must be vented into the local exhaust devices.
-work area must be clean and uncluttered, with chemical and equipment properly labelled and stored.

Disposal
Handling Hazardous Waste
-waste is any solid, liquid, or contained gas that is no longer used and is designated for disposal or recycling .
- If the waste can cause injury or death, or pollute land, water or air, it is deemed to be hazardous.

Strategies of waste handling

1. Recycling: alcohol, xylenes, xylene substitutes, and formalin are example of chemicals in the histopathology laboratory
that can be recycled.
2. Scaling down: attempts should be made to “scale down” the quantities of chemicals used.
3. Re-use or treat- and release: Non-mercury hematoxylin is an excellent candidate to be re-constituted and re-used after
the first use.
4. Storage for transportation and disposal by third party waste management companies.

Substituting for hazardous chemicals

1. Fixation, spent formalin is considered to be a hazardous waste. It can be recycled. Many formalin substitutes are
available for fixation of tissue.
2. Dehydration of tissue during processing is still carried out by a graded alcohol series; laboratories usually employ ethanol
for this procedure. Iso-propanol is also used for “non-xylene” processing.
3. Clearing is done using xylene, proper ventilation must be used when handling, and there are many xylene subtitues
available for use.

Classification of Proper waste disposal (Colour Coding)

1. Black: general waster, non – infectious and dry.
2. Green: General waste, non – infectious and wet.
3. Yellow; infectious pathological, pharmaceutical waste.
4. Orange: radioactive waste in leaded containers.

Quality Management System of the histopathology laboratory

-In the histopathology laboratory, the customer requires courteous and skilled staffs who release results on time as per the
requirements of the surgeon and patient with a short turnaround time, and with the correct diagnosis and no specimen mix up.

-Quality assurance (QA) aims to generate the confidence of the patient in the final report, it covers all the aspects of the
histopathology laboratory from:
1. The receipt of the request and specimen to the release of the report
2. Availability of reagents and supplies
3. Preventive maintenance and monitoring performance of equipment
4. Evaluation of the quality of services
NOTE: In short, QA is a means of getting the right test at the right time on the right specimen from the right patient with the right
diagnosis and at the right price.

-Quality management system (QMS) is a set of coordinated activities to regulate a laboratory in order to continually improve the
efficiency of its performance.
QMS concerns with
1. Good sampling
2. Tissue processing
3. Quality reagents
4. Providing supplies and equipment
5. Receiving, documenting, and validating results.
6. Releasing of reports to make certain that the necessary and relevant steps have been taken to ensure excellent quality

To ensure an effective QMS, the histopathology laboratory should have the following
1. Skilled histotechnologist/ histotechnicians
2. Proper specimen collection
3. Proper processing of specimen
4. Efficient processing of results
5. High quality of reagents and equipment
6. Preventive maintenance of equipment
7. Continuous professional education of staff
8. Documentation and control
9. Proper coordination
10. Timely customer’s feedback

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Factors affecting the three phases of examination
-The reliability of histopathology results depends on many pre-examination procedures, and post – examination procedures. Pre-
and post – examination factors may be sources of error that are beyond the control of the laboratory staff.

Pre –examination (Pre- analytical Factors)

-The collection of specimen starts in the operating room
- Re-examination factors includes
1. Collection of the right specimen
2. The proper fixation of the specimen
3. The correct identification of the specimen
4. The timely transportation of the specimen

Examination (analytical factors)

-Examination factors are as important as pre-examination factors in the laboratory
1. Grossing of tissues
2. Processing
3. Procedure reliability using technical manuals
4. Reagent integrity and efficiency
5. Cutting of paraffin sections
6. Staining
7. Slide labelling
8. Equipment reliability
9. Adequate calibration
10. Proficiency of personnel and continuous updating of their knowledge
11. Good internal quality control

Post- examination (post- analytical factors)

-After the proper examination, the histotechnologist/ histotechnician prepare the slides for evaluation by the pathologist.
- The pathologist records the findings and diagnosis on the correct requisition slip clearly. Again these guidelines should be kept in
mind:
1. Render histopathologic diagnosis (hard copy or electronic) free from clerical errors by checking for literality
2. Ensure that the report reaches the appropriate clinicians/ surgeons.
3. Filling of paraffin blocks must be done in a cool area to prevent them from melting together and being consumed by vermins.
4. Slides are stored for 10 years, while reports can be stored for even longer
5. Any possible remarks on the diagnosis obtained should be also be indicated
6. Frequent dialogues between the pathologist and the surgeons for the right diagnosis of results obtained in the laboratory should
also be done.

Reference:
Basic Histopathologic Techniques by: Raymundo W. Lo, FCAP,FASCP,FPSP

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