Histopathology and Cytologic Techniques: Automated Tissue Processing

Study Notes

Automated tissue processing machines ("tissue processors") are used for large volumes of tissues to be processed, increasing efficiency and convenience.
The machine allows specimens to be infiltrated with a sequence of different solvents, finishing in molten paraffin wax.
Principle: Specimens start in an aqueous environment, then pass through multiple changes of dehydrating and clearing solvents (typically ethanol and xylene) before being placed in molten wax.
Molten wax is hydrophobic and immiscible with water, and the processing schedule depends on the nature and size of the specimens.

Tissue density and thickness affect infiltration and subsequent sectioning of tissues.
Agitation (manual or automated) increases the flow of fresh fluids in and around the tissues.
Temperature (37°C to 45°C) affects fluid penetration and tissue processing, but high temperatures can cause tissue shrinkage and hardness.
Vacuum and pressure can increase infiltration rate and decrease processing time, especially for dense and fatty tissues.

Modern processors have a chamber where the specimens are held and the different solutions are pumped in and out.
The whole process typically takes around 6 hours and is usually set up to run overnight.
Tissues must be manually aligned and oriented in the block of paraffin wax after processing.

RTP is a dramatic change in modern histotechnology, allowing for faster processing times (60% to 80% faster than conventional processing).
RTP uses a dedicated microwave oven with precise temperature control and a timer, allowing for rapid dehydration and infiltration.
Advantages of RTP include:
Faster processing time
Excellent morphology
Sharp nuclear detail and crisp cellular images
Ability to control temperatures
Ability to accelerate fixation, decalcification, tissue processing, and staining

Microwave technology is used to accelerate fixation, decalcification, tissue processing, and staining.
Microwave excitation of molecules increases movement in both solutions and tissues, improving tissue penetration and fixation.
Laboratory microwave ovens are designed for precise temperature control, unlike domestic microwave ovens.
Microwave processing is used to unmask or retrieve antigens, and is commonly employed in modern histotechnology.

Rapid tissue processors run in 'batch' mode, with the ability to add new specimens to the processor every 15 minutes.
Size constraints apply, with large tissue sections requiring special handling and small blocks (e.g., endoscopic biopsies) being processed in a shorter period.
Current RTPs use innovative, low-wattage microwave technology, molecular-friendly reagents, and traditional vacuum infiltration techniques to provide consistent, high-quality results in record time.

Staining methods take much shorter time in a microwave oven compared to conventional tissue processing.
Microwaved slides have better contrast, more intense staining, and less non-specific staining.
Calibrated laboratory microwaves equipped with temperature probes and air-bubble agitation devices have addressed the earlier problems of inconsistent or uneven heating.

Several studies have shown that the quality of tissue sections after rapid tissue processing is comparable, and in some cases, superior to conventional tissue processing methods.
Rapid tissue processing has been used to achieve same-day turnaround time from receiving the biopsy until microscopic evaluation and completion of surgical pathology reports.### Alternative Fixatives
An alcohol-based fixative has been introduced, which can be used with a formalin-free microwave-based rapid tissue processor (RTP) to recover DNA, RNA, and proteins for molecular analyses.
UMFIX is a molecular fixative made of a mixture of methanol and polyethylene glycol, which is an excellent and cost-effective alternative to formalin.
UMFIX is non-volatile and active at room temperature.

Mechanical or electrical faults can occur in tissue processors, resulting in processing mishaps and compromised tissues.
Human error, such as mistakes in replenishing or sequencing of processing reagents, can also lead to compromised tissues.
Thin sections are necessary to allow fixative and dehydrating solutions to penetrate completely (about 1.5 mm).
Additional steps are required for certain tissues, such as brain and large tissue blocks, before placing them into the rapid tissue processor.
Continuous flow processing requires ongoing attention to the instrument and removal of samples as they complete the processing cycle.

Paraffin must be added to the microwave in liquid form, as microwave energy will not melt paraffin pellets.
Tissue must be brought to the boiling point of the intermedium (82°C for ethyl alcohol and 78°C for isopropanol) in the last step to "flash evaporate" the remaining ethyl alcohol or propanol.