Automation histopathology

Automation in tissue processing in histopathology section

1. Automation in Histopathology

2. Automated tissue processing The basic principle for tissue processing requires the exchange of fluids using a series of solutions for a predetermined length of time in a controlled environment. To provide sufficient rigidity t o the tissue so that it can be cut into thin section f or microscopic examination. Recent advances now include: A. Tissue processors B. Microwave ovens C. Rapid Processors with multi-sectioned retorts

3. A. Tissue Processors

4. Carousal Type Processor ( Tissue Transfer ) Transports tissue blocks in baskets Through a series of reagents housed in stationary containers Submerged specimen’s length of time in each reagent container should be electronically controlled Vertical oscillation or the rotatory movement of the tissue basket provided the agitation

5. Automated tissue processor Basket (30-100 Cassettes) Rotating Head Reagent Containers (9-10) Controls & Indicators Wax containers (2-3) AGITATION by Vertical Oscillation or Rotatory Movement

6. Advantages : Allow maximum flexibility in the choice of reagents and schedules . Rapid turnaround time for day/night processing. In recent models, tissue basket is enclosed within an integrated fume hood – allow escape of fumes during agitation. Disadvantages: - Tissues dry , while being transferred.

7. Self-contained fluid exchange systems A microprocessor used in this instrument Tissue Cassettes are loaded into a retort chamber where they remain stationary throughout the process Reagents & melted paraffin wax moved sequentially into & out of the retort chamber by using vacuum & pressure Each step could be customized by controlling time , temperature , or pressure / vacuum Agitation is achieved by Tidal action

8. Touch Panel Retort for 100-300 Cassettes Reagents 10-12 reagent stations with adjustable temperature between 30 & 45⁰C Wax Station 3-4 Paraffin Wax stations with adjustable temperature between 48 & 60⁰C AGITATION by Tidal Action

9. Advantages : Vacuum & heat can be used at any stage – reduction in processing time & improved filtration of denser tissues. Retorts are sealed – prevent tissue drying . Divided retorts that allows for different programs to run simultaneously. Customized schedules for tissue processing are possible. There is a fluid spillage containment & elimination of fumes . Employ alarm systems & diagnostic programs for troubleshooting any instrumentation malfunction. Allows the user a safe, clean & easy reagent renewal . Newer instrumentation have : - Provides the opportunity to divide tissue by size. - Have solution management systems, allowing reagents to be monitored for purity & to be used for a greater period of time without adversely harming the tissue.

10. B. Microwave ovens or processors : Microwave ovens specially designed for tissue processing are now common in a recent tissue processing technique. This technique is 1 st used by Boon and Kok in 1985. It shortens the processing time from hours to minutes . Microwave ovens stimulates diffusion of the solutions into the tissue By increasing the internal heat of the specimen Thus, accelerating the reaction

11. That is in another words, the penetrative properties of microwave & conversion of this incident energy into heat, is made use of this processor so that it shortens the processing time . In processed tissue, The processing time depends on the thickness & density of the specimen.

12. AGITATION is provided by Air-Nitrogen system - Precise temperature controls, timers, and fume extraction systems. Built in regenerable Charcoal Filter Connection to fume extraction Software controlled stirring device 14 Cassettes per container Touchscreen Control Terminal

13. Reagents used for microwave processing include: - Ethanol, - isopropanol and - proprietary mixtures of alcohol & - paraffin.

14. Graded concentration of solutions is not required. Clearing agents are not necessary because the temperature of the final paraffin step facilitates evaporation of the alcohols from the tissue. Xylene & formalin are not used in this process, which eliminates toxic fumes & carcinogens. Properly controlled processing provides uncompromised morphology & antigenicity of the specimens.

15. Advantages : Increased efficiency through improved turnaround times, Shorter processing time Environmental friendly reagents Greater profitability due to reduction in number & volume of reagents. Lesser degree of denaturation of nucleic acids. Disadvantages : The process is labor intensive because the solutions are manually manipulated, Temperatures must be maintained between 70 and 85°C, The size of tissue sample is critical (2 mm). Costly

16. Manual TP Automated TP Microwave TP Reliability Less reliable as human error may occur More reliable More reliable Time consumption Time consuming Have customised schedules for TP Shortens the processing time Cost Inexpensive Costly More costly Chemicals like Xylene & formalin Employs noxious chemicals Vacuum & heat can be used Not used so eliminates toxic fumes & carcinogens Graded concentration of solution Monitored Monitored Not required Shrinkage of tissue Evaluated Less evaluated Lesser evaluated Comparison between tissue processers

17. C. Rapid Processors with multi-sectioned retorts : This processor uses : Microwave technology Vacuum infiltration ‘Molecular-friendly’ proprietary reagents A robotic arm moves the tissue cassettes through four stations which contain acetone , isopropanol , polyethylene glycol , mineral oil and paraffin .

18. Microwaves & agitation are used to accelerate the diffusion of solvents in tissue. The microwave technology utilized, operates at a continuous low power instead of pulsing high levels of microwave energy. The retort chamber is cylindrical . Microwaves circle around the cavity, taking advantage of the physical principle of the ‘ whispering chamber ’ effect that eliminates hot &cold spots.

19. Rapid Processors with multi-sectioned retorts

20. Advantages : Acceptance of tissues into the system in every 15 minutes , Improve turnaround time . The reagents used are environmentally safe . The morphology & quality of the specimens is consistent with that of traditional tissue processing. Disadvantages : The cost of the processor, The grossing of the tissue sample that requires standardization of specimen dissection.

21. TISSUE MICROARRAY Tissue microarray (TMA) was developed as a method to evaluate numerous samples of tissue in a short period. The automated arrayer is easy to use and includes a specimen tracking software system. The instrument marks, edits, and saves punch coordinates using an on-screen display and software tools. Ideal for a laboratory with a high volume of TMAs, as 120–180 cores can be ‘punched’ per hour

22. Automated tissue arrayer ATA27 .

23. AUTOMATED EMBEDDING STATION EMBEDDING - the tissue is surrounded in a molten medium by using a mould . Subsequently this medium is solidified to make a block for cutting thin section of tissue. Aims of embedding 1. To give support of the tissue 2. To prevent distortion of the tissue during cutting 3. To preserve the tissue for archival use

24. Tissue- tek system is the combination of 1. Dispenser of liquid paraffin in a constant temperature 2. A metal plate to make the tissue block 3. Cold plate

25. Microtome Microtome is a mechanical instrument used to cut biological specimens into very thin segments for microscopic examination.

26. Parts Microtomes consist of three main parts: Base (microtome body) Knife attachment and knife Material or tissue holder Automated Microtome Setting Block Holder Blade Stage

27. Microtomes can be classified as: – Manual microtomes – Semi- automatic microtomes – Automatic microtomes

28. Laser Microtome Laser microtome is used for precise, non-contact sectioning and was designed to slice samples with high precision . It’s equipped with state-of-the-art femtosecond laser technology. It enables non-contact cutting inside biological tissues and various materials without causing thermal damage .

29. ADVANTAGES : Non- contact processing Sub micrometer precision Cutting of the tissue in its native state No thermal damage Fewer artifacts Less time consumption in tissue preparation

30. Computerized Microtome Computerized microtome is equipped with the advanced rapid thermostatic switch, semiconductor freezing, cryo-scalpel and cryoplate . Can carry out the rapid freezing section or routine paraffin section ( dual-purpose ). Advanced retraction function makes better sectioning. The retraction value is also adjustable. Automatic memorized coarse feed and reload function can significantly improve cutting efficiency.

31. Cryostat The cryostat is the instrument that has the arrangement to freeze the tissue and also to cut the frozen tissue for microscopic section. First cryostat was introduced in 1954. The cryostat is a machine in which a slicer called a microtome is placed in a freezer. The temperature may be regulated between-10ºc to -40ºc.

32. Control Panel Glass door Lock Hand wheel Freezing Chamber Waste Collection Bottle

33. Parts Cryocabinet ( -5 to -30 ⁰C ) – Most tissues section properly between −15 °C and −25 °C . Microtome ( Rotary / Sliding/Rocking) of any type but preferably rustproof, which is enclosed and operated within a deep freeze cabinet. Knife or blade : low- or high-profile disposable blades /profile C steel blade. The angle of the knife is kept in between 5° and 7° . Antiroll plate : just in front of the knife - prevents the rolling of the cut tissue. Specimen Holder : Small, round, metal CHUCKS Fluorescent light lamp Electronic contro l panel

34. The specimens are mounted on a cutting medium on a metal chuck and frozen to a cutting temperature Once frozen(-24 ⁰ C) the specimen is mounted on the microtome . Once the specimen is cut to a satisfactory quality, it is mounted on a warm dry glass slide , where it melts and dries. Principle When the tissue is frozen, the interstitial water in the tissue turns to ice, and in this state the tissue is firm with the ice acting as the embedding medium .

35. INDICATIONS • Rapid production of sections for intra-operative diagnosis. Immunofluorescence study. • Diagnostic and research enzyme histochemistry for labile enzymes. Diagnostic and research non-enzyme histochemistry, e.g. Lipids and some carbohydrates Silver demonstration methods, particularly in neuropathology

36. Advantages : Both tissue and knife are maintained at same low temperature. Capable of slicing sections as thin as 1μm . Serial sectioning is possible. Automatic defrosting and sterilisation . Antifogging air circulatory system. Disadvantages : Constant supervision and maintenance of temperature . Special lubricants with a low congealing point have to be used. Freeze artefacts . If temperature is too low – tissue becomes hard & crumbles – difficult to cut . Costly

37. WATER BATH Water bath is used to float the tissue after cutting – to to remove folds and wrinkles of ribbons . The temperature of the water bath is usually controlled automatically by a thermostat . The temperature of water in the water bath should be 10 °c below the melting point of the embedded paraffin wax and is usually kept in 40–50 °C. It is necessary to prevent formation of any air bubbles within the water bath.

38. DRYING The small amount of water held under the section will allow further flattening to occur when heat is applied to dry the section. The temperature should be at the melting point of the paraffin . Automated stainers have drying ovens as part of the instrumentation. Electrothermal Slide Drying Bench

39. AUTOSTAINER High Throughput stainer Compact stainer

40. PAP, AFB, Haematology IHC

41. Cover slipping Suctioning mechanism for picking up a coverglass from a stack of coverglasses . Exert a force onto the coverglass to insure that it is released from the selecting device and placed onto the slide. After placement of the coverglass onto the slide, capillary action pushes air bubbles out from underneath the coverglass .

42. LABELING Imprints - resistant to chemical exposure and physical wear. Easier to locate. Alphanumeric characters, barcodes or logos.

43. Automation in histopathology Automated Tissue processing Tissue processing Microwave Tissue processing Multi Sectioned Retorts Tissue processing Embedding Cryostat Labeling Water Bath Microtome Drying Autostainer Tissue Microarray Tissue Transfer Computerised Fluid exhange Laser Cover slipping

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