Histology and Cytology Techniques: Automated Tissue Processing PDF

HISTOPATHOLOGY AND CYTOLOGIC TECHNIQUES | LECTURE L22: AUTOMATED TISSUE PROCESSING AND RAPID PROCESSING...

HISTOPATHOLOGY AND CYTOLOGIC TECHNIQUES | LECTURE L22: AUTOMATED TISSUE PROCESSING AND RAPID PROCESSING FINALS | 2ND SEMESTER BSMLS 2N Guidelines: The entirety of this topic can be read on: Pages 70-80 of ○ If the processor is to be run overnight, it Bruce-Gregorios should be programmed to hold on the first ethanol bath and not finish until the next AUTOMATIC TISSUE PROCESSING morning When there is a large volume of tissues that need to so the specimens do not sit in hot be processed, an Automated tissue processing paraffin longer than the time machine (“tissue processor”) is more convenient indicated and more efficient to use. ○ If spx are fresh, they may incubate in ○ This machine allows the specimens to be formalin in the first stage on the machine infiltrated with a sequence of different ○ IMPORTANT: Do not keep the tissues in hot solvents finishing in molten paraffin wax. paraffin for too long or else they may Principle: become hard and brittle ○ Specimens are in a aqueous environment to ○ Processed tissues can be stored in start with (water-based) cassettes at room temp indefinitely. ○ Passed through multiple changes of Vacuum infiltration is the impregnation of tissues by dehydrating and clearing solvents (typically a molten medium under reduced pressure. ethanol and xylene) prior to placing it in ○ The procedure assists the complete and molten wax rapid impregnation of tissues with wax and Molten wax is hydrophobic and reduces the time tissues are subjected to immiscible with water high temperatures ○ NOTE: The duration and step details of the Thereby, minimizing heat-induced “processing schedule” chosen for a particular tissue hardening, batch of specimens will depend on the facilitating complete removal of nature and size of the specimens. transition solvents, and prolonging the life of wax by reducing solvent contamination Older The older design of an automatic tissue processors processor is a carousel FACTORS THAT IMPACT THE DURATION OF TISSUE Carousel - contains a cage in which PROCESSING AND EXTENT OF INFILTRATION the tissue cassettes are placed. Tissue density and thickness ○ Has a number of glass Agitation beakers w/ solvents and Temperature solutions Vacuum and pressure ○ Vertically agitates the cage in each solution before moving TISSUE DENSITY AND THICKNESS on to the next solution in the Variable tissue density affects infiltration and dehy/clearing method. subsequent sectioning of tissues. Spongy tissues (like lungs) are usually more rapidly Modern The modern processors have a chamber infiltrated than hard and dense tissues. processors Chamber - in which the specimens ○ Thickness of the tissue also influences the are held and the different solutions rate of reagent diffusion and hence are pumped in and out of the processing time. chamber Tissue thickness should be optimized for particular In general, the whole process takes around six (6) processing schedules, or alternatively, processing hours and is usually set up to run overnight times should be adjusted to accommodate thick, thin Tissues that come off the tissue processor are still in or large tissue blocks. the cassettes and must be manually put into the blocks by a technician who must pick the tissues out AGITATION of the cassette and pour molten paraffin over them Agitation using manual or automated processors (embedding). increases the flow of fresh fluids in and around the ○ This "embedding" process is very important, tissues. because the tissues must be aligned, or ○ Most tissue processing protocols utilize oriented, properly in the block of paraffin. automated processors - w/ vertical or rotary oscillation mechanisms to speed fluid exchange Without agitation: ○ Tissues tend to settle to the bottom of the processing device ○ Tissues become too tightly packed (reduced surface area for fluid exchange) Tissue should be loosely packed in baskets to facilitate exchange of reagents and increase diffusion. BSMLS 2N | Page 1  TEMPERATURE RAPID TISSUE PROCESSING Ranges 37° to 45°C Rapid Tissue Processing (RTP) has introduced ○ Speeds up fluid penetration and tissue dramatic changes into the practice of modern processing protocols histotechnology However, heat must be carefully monitored. In comparison to conventional tissue processors, ○ High temp can cause tissue shrinkage and microwave assisted processing allows the makes it hard and brittle following: ○ Low temp increases viscosity of reagents ○ Dehydration to be done in only one step and used in tissue processing = reducing the rate employs a four step process involving a of diffusion and increasing processing time single change each in ethyl alcohol and Maintain embedding waxes 2-3°C above their melting isopropanol points ○ Followed by two changes in paraffin When exposed to high microwave temperatures: VACUUM AND PRESSURE ○ Alcohols quickly heat and dissipate Reduced pressure = increased infiltration rate and ○ Paraffin remains inert, allowing the paraffin decreases the time needed to complete steps in tse to fully infiltrate the spx (eliminates the use of processing xylene from the process) High pressure = facilitates infiltration of dense spx w/ RTP is undertaken in a dedicated microwave oven more viscous embedding media ○ Fitted w/ precise temperature control and Vacuum application during tse infiltration has the ff. timer advantages: ○ Interlocked fume extraction system (to ○ Improves processing quality prelude ignition of solvent vapor) ○ Can aid in removal of trapped air from ○ A turntable or in-built radiation dispenser porous tse (facilitates even reagent heating) ○ Reduce infiltration time when dealing w/ dense and fatty tissue MICROWAVE PROCESSING However, duration of wax infiltration is dependent 60% to 80% faster than conventional processing time upon viscosity and is not generally reduced by ○ Hourly throughput of up to 120 specimens applying a vacuum. Implementation of RTP allows for rapid TAT (turnaround) of tissue biopsies TECHNICAL CONSIDERATIONS ○ As effective as slower conventional Baskets and metal cassettes should be clean and processing wax-free. ○ Provides excellent morphology Tissues should not be packed too tightly in baskets so ○ Sharp nuclear detail and crisp cellular as to impede fluid exchange. images Processors must be free of spilled fluids and wax Microwave technique is now commonly employed to accumulations. unmask or retrieve antigens, using 10mm. citrate Fluid levels must be higher than the specimen buffer (pH 6.0) containers. Timing and delay mechanism must be correctly set MICROWAVE IRRADIATION and checked against the appropriate processing Used successfully to expedite tissue fixation and schedule. processing A processor log should be kept in which the number Microwave, a form of electromagnetic of specimens processed, processing reagent wave-induced heat yields histologic material or changes, temperature checks on the wax baths and superior quality to that provided by conventional the completion of the routine maintenance schedule processing methods. are recorded as part of the quality assurance ○ Saves time and better results program. ○ Ability to control temperatures makes it Gentle washing and minimal thickness of cell layers possible to achieve results that were not will prevent the cells from detaching during staining. formerly available in conventional systems. Make sure that there are enough sections to make a This technology makes it possible to employ ANY diagnosis, and routinely used reagents for paraffin processing and Ensure that the reagents have been applied evenly to can be applied to accelerate the following stains: the slides. ○ Routine stains Quality of staining can be compromised by ○ Special stains inadequate fixation and similarly by poor tissue ○ Metallic stains processing. ○ Immunofluorescent stains A good technician must evaluate and determine the Microwave works as a physical agent that accelerates processing of choice for each purpose, i.e., special fixation, decalcification, tissue processing, and stains on paraffin, frozen or cell smear preparations. staining for IHC and electron microscopy. Make sure glass slides are clean and free from debris. EQUIPMENT In general, needle biopsies and bloody specimens Vary from kitchen type microwave ovens to should be incubated conservatively, whereas fatty sophisticated semi-automated or automated specimens can be processed for longer than average. instruments. ○ Microwave excitation of molecules = increases movement in both solutions and BSMLS 2N | Page 2  tissues = improved tissue penetration and RAPID TISSUE PROCESSORS fixation Rapid tissue processors run in 'batch' mode; once the cycle has begun no additional tissues can be added for processing until the cycle is completed. Size constraints: ○ Large tissue section (greater than 1.5 mm. thick) = may show uneven penetration of reagents = incomplete clearing and paraffin impregnation. Large tissue blocks will require special handling ○ Small blocks (i.e., endoscopic biopsies) can be processed in a shorter period using tailor-specific protocol Domestic Precise maintenance of temp is microwave ovens not possible (output power not (kitchen-type) precise) = overheating Produces hotspots (uneven heating) Toxic and flammable vapors cannot be vented from domestic ovens (ignition hazard) Laboratory Requires very precise microwave ovens maintenance of temperature Specifically designed microwave units has This system of continuous throughput rapid tissue removable reaction vessel (to processing allows for the addition of new specimens hold cassettes), with manual to the processor every 15 minutes, as a reaction reagent exchanges according to chamber becomes available a predetermined schedule Other characteristics: Does not produce hotspots or ○ Utilizes computer controlled microwave uneven heating in tissues due delivery to the presence of magnetic ○ Temperature controls in a formalin free stirrer kept beneath (provides environment an even field of irradiation) ○ Reagent volumes are small Adequate ventilation is created ○ All reagents are relatively inexpensive and for the escape of fumes non-toxic Equipped with thermocouple ○ Xylene has also been eliminated temperature probe and ○ Vacuum assisted paraffin impregnation controller to maintain allows for a complete processing cycle of temperature within a very 60-70 minutes. narrow range of +/- 1°C Current RTPs use an innovative, low-wattage microwave technology, molecular-friendly reagents and traditional vacuum infiltration techniques to STAINING METHODS provide consistent, high quality results in record time. Staining methods take much shorter time in a ○ Has a continuous efficient workflow microwave oven compared to conventional tissue ○ Consistent high quality 1-hour processing processing. up to 120 specimens per hour. In general, microwaved slides have better contrast, ○ Formalin- and xylene-free processing more intense staining, and less non-specific staining ○ 960 cassettes in an 8-hour shift Calibrated laboratory microwaves equipped with Loads up to 40 cassettes every 20 temperature probes and air-bubble agitation device mins. have largely addressed the earlier problems of Immerses specimens for 15 mins in inconsistent or uneven heating observed when each of the 4 enclosed processing kitchen-type microwaves were originally used. retorts w/ the standard program Optimum temperature/s: ○ Metallic stains: 75° to 95°C SPECIAL STUDIES ○ Non-metallic stains: 55° to 60°C Several studies have shown that the quality of tissue Researchers have found microwave technology to be sections after rapid tissue processing is comparable, beneficial in achieving epitope recovery in and in some cases, superior to conventional tissue formalin-fixed tissues for many markers within 10 min processing methods, with no detrimental effects on to 15 min. the commonly used special staining procedures IHC stains can be performed on sections without antigen retrieval in some cases IMPACT: Rapid tissue processing has been in the same day turnaround time from receiving the biopsy BSMLS 2N | Page 3  until microscopic evaluation and completion of surgical pathology reports ○ A significant departure from conventional surgical pathology practice 60% faster than conventional processing time so that urgent cases can be signed out on the same day. Other studies: ○ An alcohol-based fixative has recently been introduced which, when combined with a formalin-free microwave based RTP, has been demonstrated to permit the recovery of DNA, RNA, and proteins for molecular analyses. ○ UMFIX - a molecular fixative is a mixture of methanol + polyethylene glycol, is an excellent and cost-effective alternative to formalin Non-volatile and active at room temp. Mechanical or electrical faults occasionally occur in tissue processors, processing mishaps where tissues are actually compromised, mainly occurring because of human error (mistake in replenishing or sequencing of processing reagents) = will not provide any useful microscopic information to the pathologist. PRECAUTIONARY MEASURES Sections must be thin enough so fixative and dehydrating solutions can penetrate completely (about 1.5 mm.). This may increase the time required in grossing of specimens. Certain tissues (i.e., brain and large tissue blocks) require additional steps before placing into the rapid tissue processor. Continuous flow processing eliminates batching of specimens and necessitates ongoing attention to the instrument. As samples complete the processing cycle, they must be removed from the instrument to accommodate the next basket of cassettes. 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. Reference: Bruce-Gregorios, J.H. (2016). Histopathologic Techniques. (2nd Revised Edition). Makati, PH: Katha Publishing (611.0182/B83) BSMLS 2N | Page 4