Grossing involves macroscopic inspection, measurement, and dissection.
Adhesive glass slides enable more efficient adhesion of tissue sections.
Tissue grossing
Non-adhesive glass slides require additional heating to ensure sufficient tissue adhesion.
Common patient body samples include biopsies, surgical specimens, and tissue samples from various organs.
Extra care should be taken when handling sharp equipment, and personal protective equipment (PPE) should be worn.
Molten paraffin, hot forceps, and histo-embedder are examples of hot equipment.
Microtomy is used to section tissue blocks for examination under a microscope.
Tissue floaters refer to foreign tissues that come from the floatation water bath.
You will need 4 tissue sections on 4 adhesive glass slides.
A good tissue block must be clearly labelled with patient identifiers, embedded firmly within the paraffin, have smooth and uniform paraffin (no splitting or holes), fully represent the tissue surface and be correctly orientated, and have the tissue located in the center surrounded completely by paraffin.
Xylene is the most preferred as it clears alcohol rapidly.
Fixative volume should be 15 to 20 times more than the tissue volume.
Possible causes include cutting board metastasis during tissue grossing, tissues floating out of the tissue cassette holes during ATP, dirty forceps and mould during embedding, and tissue sections left floating on the water bath.
A cryostat microtome is used to obtain frozen tissue sections for immediate or urgent diagnosis.
Criteria include time, cost, viscosity, disposal, storage, rapid removal of dehydrating agent, flammability, toxicity, minimal tissue damage, and ease of removal by molten paraffin.
A good tissue section should have even thickness (4-8 microns), no tears, no splits or chatters, minimum wrinkles, contain embedded tissue (full face), and be consistent and continuous.
The temperature inside the cryostat is about -20 to -30 degrees Celsius.
Fatty tissues require longer processing times to ensure sufficient removal of water and clearing of alcohol.
Embedding is a histotechnique in which processed tissue specimens are enclosed in a solid embedding medium.
Factors affecting ATP outcomes include lipid content, density, and tissue type, as well as technical factors like thickness or size, reagent freshness and quantity, cassette load, reagent volume, and the correct ATP program and duration.
Over-processed bloody tissues can become dry and powdery.
Embedding is performed to support the tissue and enable thin sectioning for microscopic examination using a microtome.
Patient identification
Knife angles play important roles in obtaining the perfect tissue sections.
Regular-sized metal moulds come in various sizes to accommodate different sized tissues.
Medical technologists.
Incorrect water bath temperature, poor floatation technique, section too thin.
Tissue grossing
The doctor orders an ultrasound to confirm the presence of the lump, which is then removed for histological examination.
Laboratory accession numbers.
A tissue microarray allows many different tissues to be represented in a single tissue block.
Microtome, microtome blades or knives, forceps, and a brush for cleaning paraffin waste.
Microtomy is the process of slicing thin tissue sections using a microtome.
Crisp, crunchy, or brittle tissues; cooked tissues from extreme heat; dry and powdery tissues.
Grossing is performed to gather diagnostically critical macroscopic information about the disease and to dissect and obtain crucial parts from the specimen for further processing.
10% neutral buffered formalin (NBF).
Some labs may prefer to use disposable plastic moulds instead of metal ones.
Large cassette, large metal mould, and large tissue block are used for embedding large tissues.
Dense tissues such as those from the uterus and prostate need time to be sufficiently dehydrated.
The clearance angle can be adjusted by the microtome user.
Re-adjust water bath temperature, re-adjust micrometer section thickness.
Patient identification
The rat organs are being fixed in formalin.
To prepare tissue samples for microtomy
Tissue fixation is important to prevent autolysis or putrefaction, or cell destruction, as tissue specimens can be irreversibly damaged if preservation is not done effectively.
10 microns for the initial section and 4 microns for subsequent sections.
Formalin has a strong odour, is carcinogenic, and flammable.
You will observe a tissue processor.
All anatomical structures present in the specimen.
Tissue fixation preserves the tissue structure for further processing.
Tighten or change blade, adjust cutting speed, re-adjust micrometer section thickness, re-calibrate microtome.
Modern enclosed tissue processors are found in hospital labs where many tissue blocks need to be processed in one run.
Used for measuring specimens.
Formalin-fixed specimen.
The Leica TP1020 is used for the 'dip and dunk' processing of tissue samples, facilitating dehydration, clearing, and infiltration.
A frozen section is used for rapid diagnosis for intraoperative management to know the extent of the lesion.
The key steps are dehydration, clearing, and infiltration.
Clean the microtome regularly after use.
The sections are placed onto a glass slide and stained for viewing by the pathologist.
Commercial household cleaners, soap, water, alcohol or xylene should not be used.
Decalcification can be achieved either by acids or chelating agents.
Well-processed tissues should be firm and adequately infiltrated with paraffin wax.
Tissue contaminants refer to foreign tissue particles that end up on a particular section.
Tissues sampled from the dead body or living patient.
To determine the cause of death via histological examination.
The patient visits a doctor with signs or symptoms that may indicate a condition requiring diagnosis.
The water bath is used for floating tissue ribbons.
TEM allows observation of cellular organelles that cannot be viewed using optical microscopes.
Trim or shave excess paraffin to expose the tissue.
Used for larger specimens and comes in various sizes.
You will prepare ONE tissue cassette.
Change blade, cool the block.
ATP provides firmness to tissues, making it easy to slice them for microscopic examination.
Sections cut by an ultramicrotome for TEM are typically 60 to 100 nanometers thick.
Poorly processed tissue, poorly embedded tissue block, microtome not accurately calibrated, blade is blunt, inconsistent cutting speed, inappropriate section thickness setting, blade not clamped securely.
Re-embed with filtered wax or clean/change the blade.
Harder knives such as diamond and glass knives are needed to cut harder embedding media.
Tissue is embedded into molten paraffin and oriented while molten/semi-molten.
Poorly processed tissue, tissues are hard or calcified (e.g. bone, cartilage, nails), dirt particles or paraffin on knife cutting edge or between the blade and clamping plates, vigorous handling of sections in the water bath, bubbles under the section, section floated for too long in the hot water bath.
They lead to difficulty during microtomy, resulting in poor quality sections.
To hold and protect tissue samples during processing.
1800 ml of 70% alcohol is used.
Xylene dissolves alcohol and is miscible with both alcohol and paraffin wax.
Always remove the microtome blade before cleaning.
You should be alert for harmful reagents, sharp equipment, and hot equipment.
Sludgy, mushy, or greasy tissues; tissues that smell of clearing agent; soft and compressible tissue.
Tissue sections are cut and picked up on a glass slide, and then they are ready for staining.
Histological grade paraffin is the most preferred and widely used embedding medium around the world.
Automated Tissue Processing
Sections for TEM are much thinner, measured in nanometers, compared to paraffin tissue sections which are measured in micrometers (microns).
In microtomy, the blade remains stationary while the clamped tissue block moves vertically towards the blade.
Wear PPE and work in a biosafety cabinet. Properly dispose of blades and handle with extra care.
Embedding media such as resins and plastics (epon, araldite, etc.) are used for TEM.
Processed tissue and heated metal mould is ready for embedding.
The most common fixative used in histology is 10% neutral buffered formalin, as it penetrates tissues well and creates cross-links to preserve tissue structures effectively.
The smooth-turning handwheel is rotated 360 degrees clockwise.
Filter paraffin or replace paraffin after clearing out the paraffin reservoir and clean the moulds.
Correct paraffin temperature is crucial for ensuring proper embedding of the tissue samples.
Microtomy
Colour, bleeding, oozing, and odour.
Missing tissue components and inaccurate diagnosis.
4 sections
3D measurements and weight.
Handle sections gently when removing folds, adjust water bath temperature.
It is important for paraffin to be of histological grade because it should not alter the specimen integrity or the microscopic features.
Alcohol replaces water in all cells.
3600 ml of 95% alcohol is used (1800 ml twice).
Immerse cassettes into 10% NBF sufficiently prior to ATP.
Remove paraffin waste using a dry brush.
Follow manufacturer’s recommendations with regard to inspection and lubrication of microtome parts.
Adhesive glass slides should be used to prepare your tissue sections.
In the storage room of any hospital-based histology lab.
The size of the storage container is dependent on the size of the tissue.
Specimen container.
It is crucial because even one wrong reagent can cause severe tissue damage.
No, the recommended clearance angle is not the same for all microtomes available in the market.
To diagnose conditions based on histological examination of the samples.
They can be used to archive patient samples, preserve test reagents, or for drug discovery and cancer research.
Blade is blunt, uneven surface of block, block is warm.
Areas of interest from each tissue block are punched into holes in a paraffin block to create a microarray tissue block.
The block clamp distances forward as per the set micrometer thickness.
Unstable clamping during microtomy and interference with block alignment.
Brain biopsy.
Molten paraffin is dispensed into heated metal mould.
Well-preserved small intestinal tissue shows viable cells and a clearly seen villi structure.
Refer to the Safety Data Sheet (SDS) provided.
Tissue grossing.
Be alert and stay safe while handling them with care.
The preferred melting point range for paraffin used in histopathology laboratories is 52°C - 58°C.
Tiny tissues or cassettes with multiple bits pose challenges in embedding due to their size and complexity.
Size, colour, consistency, and odour.
The first step is receiving an unfixed fresh specimen for accessioning.
Histopathology laboratories purchase paraffin with a preferred melting point to suit the regional weather conditions.
5400 ml of xylene is used (1800 ml three times).
A paraffin repellant can help remove remnants of paraffin waste.
Yes, non-bony tissues can become calcified as a result of disease.
Dirty slides, lack of adhesives, water bath water too cold, contaminated water in water bath, poor quality sections, slides not dried properly, low drying temperature.
An ultramicrotome is used for sectioning tissue specimens for TEM.
ATP is a procedure to remove water within the tissue specimens and replace it with solid embedding or infiltration media such as paraffin wax.
Biopsies, Skin Biopsy, Breast Biopsy, Large surgical specimens, Mastectomy, Colectomy, Fetal miscarriage specimens, Post-mortem specimens, Brain samples.
Grossing knife.
Sections from two different tissue blocks must NOT be floated out simultaneously.
Place it into a water bath.
Sharp equipment such as scalpel blades and forceps.
To ensure a clean and precise sectioning of the tissue.
Slow orientation of tissues when paraffin has solidified or non-continuous flow of paraffin due to blockage or low volume.
Choosing the right mould size ensures accurate representation of tissue bits during the embedding process.
Re-embed the tissue and check the paraffin temperature control panel, refilling paraffin if necessary.
Molten paraffin is added up to the brim of the cassette.
Causes include a blunt blade, improper clamping of the blade, or the temperature of the tissue block.
Re-adjust the angle, change the blade, or cool the tissue block surface.
The hardness of paraffin will vary depending on its melting point.
Absolute alcohol is used three times, each time with 1800 ml.
Gradual dehydration is important to prevent osmotic shock, which can cause cell shrinkage.
Fluid should not enter the microtome during cleaning.
The manufacturer indicates the recommended clearance angle.
Read through the lab protocol thoroughly and watch the video on tissue sectioning if needed.
Too much or too little paraffin, or the tissue cassette being improperly placed on top of the mould or placed when paraffin has solidified.
Tissues that require TEM examination are commonly embedded in resin, such as Epon.
Automated Tissue Processing
Possible causes include dirt particles in the tissue block, a blunt blade in certain parts, or dirt on the knife edge.
TEM blocks need to be harder in consistency to support the tissue during sectioning.
Forceps.
Incorrect orientation or the orientation of tissue bits not being quick enough.
Colectomy.
Autolyzed small intestinal tissue shows non-viable cells and an absence of villi structure.
Remove them from the water bath.
Cassette is placed on top of the metal mould.
The experience and skill of the medical technologist affect the ease and speed of placing and orientating tissue during embedding.
Firm, solid, soft, or friable (comes apart easily).
2000 ml of 10% formalin is used.
Biopsy specimens are usually small and thus entirely sampled.
The specimen is grossed by the pathologist and then placed on a chuck and rapidly cooled within the cryostat.
The paraffin wax infiltration step takes 2 hours.
The diagnosis is given directly to the requesting surgeon.
Bony or calcified tissues need to be decalcified, and small biopsies also need to be decalcified.
Clean slides, drain water from slides completely, heat slides to ensure they are completely dry, replace contaminated water in water bath.
Staining
Incorrect temperature setting or a blocked paraffin dispenser with un-melted paraffin.
Factors include correct equipment settings (paraffin temperature), paraffin purity and quantity, uniformity of paraffin flow, keeping the mould in a hot embedding tray, choosing the right mould size, specimen size and number of bits per mould, and the ease and speed of the technique.
To prepare tissues for microscopic evaluation
ATP helps to firm up tissues internally and supports them externally through tissue embedding.
Glutaraldehyde is commonly used as a fixative for tissues that need TEM examination.
Used for cutting tissues.
Causes include tissue being too hard or calcified, debris in paraffin wax, poorly processed tissue, or a damaged blade.
Use filter wax or fresh wax for processing/embedding, treat tissue with a softening agent, or change the damaged blade.
Xylene is preferred as it clears alcohol rapidly.
Tissue blocks are placed on a cold plate to solidify.
Plasticizers can be added to paraffin to make subsequent cutting procedures easier.
To mark margins or areas of interest and to help visualize tiny tissues more easily.
OCT is an embedding medium meant for frozen tissues that helps in the rapid cooling of the specimen.
3600 ml of paraffin wax is used (1800 ml twice).
Microtome parts should be handled carefully to prevent damage.
Specimens should ideally be well-fixed before decalcification to protect surrounding soft tissue from the damaging effects of the acid or chelating agent.
