SLIDE PRESENTATION ON AUTOMATION IN HISTOPATHOLOGY

Automation in histopathology

1. BY: Dr Bhavya MODERATORS: Dr Kishan Dr Hemalatha AUTOMATION IN HISTOPATHOLOGY

2. TABLE OF CONTENTS INTRODUCTION ADVANTAGES OF AUTOMATION WHAT IS TISSUE PROCESSING? AUTOMATION – GROSSING TISSUE PROCESSING EMBEDDING MICROTOMY H & E STAINING AND COVERSLIP POST H&E TECHNIQUES SLIDE DIGITIZATION

3. INTRODUCTION Providing high quality and efficiency is needed for the laboratory including histopathology section. One of the ways to achieve this is by automation. AUTOMATION is the technique of operating or controlling a process by highly automated means. The word automation is used to describe the maximum use of instruments to perform laboratory work with minimum involvement of laboratory personale .

4. ADVANTAGES OF AUTOMATION Rapid than manual methods and turn around time is minimum by automation. Less man power required than manual processing. Clean , healthy and safer laboratory environment. High quality results. Reduces exposure to hazardous chemicals.

5. WHAT IS TISSUE PROCESSING ? ( i )   FIXATION  – This is the process of preserving or fixing tissues by passing them through chemicals called  fixatives . The fixatives will help protect the tissue from decay and autolysis. Routine fixative of use is  10% formalin. (ii)  DEHYDRATION  – This is the process of removing water molecules from the tissue by passing the tissue through ascending grades of alcohol. E.g methanol, acetone, 70-100% alcohol. (iii)  CLEARING  – This is the process of removing alcohol from the tissue by passing it through chemicals that will remove the alcohol molecules. These agents are called  clearing agents . Xylene is mostly used for clearing.

6. (iv)  INFILTRATION  – This is the process of filling intracellular spaces left in the tissue by paraffin wax. This will help confer a bit of rigidity to the processed tissue. (v)  EMBEDDING - This last step is  manually done . This has to do with immersing the processed tissue into a mould containing liquid paraffin wax. This is for external support so that the tissue won’t crumble during  microtomy

7. BARCODING Automation in reception of tissue sample. Tissue arrives at the pathology laboratory. Barcode scanner accesses the specimen and barcode cassette generated. A barcode scanner can be utilized to read the code and transfer the information between source and the laboratory information system.

8. GROSSING Laser based devices are used to measure thickness of specimens. It raises processing quality by making sure that the thickness of specimens meets chosen protocols (3-4mm). Special grossing tools are available making it easy to produce sections of uniform thickness on a consistent basis. The tools set includes a board with an adjustable depth tissue well and a guide for the knife or surgical blade.

9. The tissue wells provide an area for the placement of the tissue and knife guide keeps the cutting blade level.

10. AUTOMATED TISSUE PROCESSING A tissue processor is a device that prepares tissue samples for sectioning and microscopic examination in the diagnostic laboratory. The ATP machine plays a big role in the preparation of the tissue by passing them through various chemicals; a major process called  TISSUE PROCESSING.

11. TYPES OF TISSUE PROCESSORS CAROUSEL TYPE STATIONARY CHAMBER PROCESSOR VACCUM TISSUE PROCESSOR MICROWAVE PROCESSORS

12. Automated carousel type In the instrument, tissue samples were placed in a basket which revolved from one reagent-containing dish to the next and remained in each for an established set period of time. Current automated carousel-type tissue processors, such as those manufactured by Leica and Thermo Shandon.

13. Stationary-Chamber Processor . In this type of instrument, the tissue samples are placed in a retort into which the reagents are pumped in and drained out following a time schedule. Stationary-retort processors are made by a number of companies, including Leica , Sakura Finetek , Thermo Shandon, and Vision BioSystems .

14. The elements used for processing in carousel or stationary-chamber processors are Solvents: Formalin Alcohol Xylene and Paraffin Physical agents : Vacuum , Convective heat Agitation

15. OPEN TYPE LINEAR TYPE Electromechanical mechanism for station to station transfer with transfer arm Microcomputer control Programmable 24 hrs clock Fume hood facility Variable delay time Capacity: 110 casettes 2L Capacity glass beakers 10 reagent container areas 2 wax baths

16. AUTOMATED TISSUE PROCESSOR LEICA TP 1020 AUTOMATED VACCUM TISSUE PROCESSOR

17. PARTS OF THE ATPM – using TP 1050 Leica model

18. ATPM – processing time schedule Processing schedule varies and it depends oh the following: ( i ) Nature and size of tissue (ii) Urgency Beaker I – fixative (formalin) 1-2 hours Beaker II – fixative 1 hour Beaker III – fixative. 30- 45 minutes Beaker IV – 70% alcohol. 30 minutes Beaker V – 90% alcohol. 30 minutes Beaker VI – Absolute alcohol. 1 hour Beaker VII – Absolute alcohol. 1 hour Beaker VIII – Methanol 30 minutes Beaker IX – Xylene . 1-2 hours Beaker X – Xylene 45 minutes – 1 hour Wax bath I (done at 45°c) 2 hours Wax bath II. 2 hours

19. Most machines consists of ten beakers and two wax baths thermostatically controlled to 56⁰C±3⁰C. Beakers are filled with appropriate fluids and the wax baths are filled with wax .the main switch is switched on in order to keep the wax in a molten state. The tissue present perforated tissue basket is moved through the different processing fluids according to the time adjustment made on the machine.

20. Advantages of vaccum tissue processor Tissue cassettes are loaded into static chamber and tissue remain in retort chamber through out the process. Static single reaction chamber helps in keeping the potentially hazardous solvents in special cabinet separate from reaction chamber . Facilitates to process tissues with application of heat vaccum at all stages of processing .

21. Using vaccum and pressure, reagents and melted paraffin wax moves into and out of the retort chamber. Each step is customized by controlling time, temperature or pressure/ vaccum . Turnaround time is short . No evaporation of reagents.

22. DISADVANTAGES Because of the time required by these automated instruments (8 hrs or longer), most processing is carried out overnight, small biopsies can be done in approximately 2.5 hrs on the regular processor. Laboratory problems: The need to monitor the instruments The safety concerns for instruments operating unattended overnight. The requirement for batch processing, which interrupts the work f low .

23. MICROWAVE PROCESSORS Shortening the procedure is greatly facilitated by replacing convective heat with microwave heating. Remove the need for hazardous chemicals such as xylene . Currently available microwave based tissue processors: Energy beam science Milestone Sakura fineteck Tissue tek express120.

24. Tissue- Tek ® Xpress® x120 Efficiency •Continuous efficient workflow/ Loading every 20 minutes. •Fewer human errors and rework , Reduced cost •Reagent volume reduction of more than 80% Speed •Standardized 1-hour processing/ Loading every 20 mins •Throughput of 120 cassettes/ hour leading to 960 cassettes in an 8-hour shift •Reduced patient waiting time Quality •Excellent morphology and sharp nuclear details due to gentle microwave technology •Standardized reproducible results: QC ready to use reagent kits •Formalin- and xylene free processing

25. Leica PELORIS The Leica PELORIS is a dual retort processor capable of traditional or xylene -free processing. Xylene -free protocols use two sets of dehydrants : industrial denatured alcohol (IDA) and isopropyl alcohol (IPA) rather than separate dehydration and clearing steps. This allows the use of higher temperature paraffin wax impregnation steps leading to faster processing.

26. MICROWAVE PROCESSORS ADVANTAGES DISADVANTAGES Environment friendly reagents: ethanol, isopropanol , mixtures of alcohol and paraffin. Reduce the number and volume of reagents: No need of concentration of solutions and no need of clearing agent. Morphology antigenicity of specimen is preserved. Improved turn around time. Size of tissue sample is critical.(2mm) Expensive Required careful calibration and monitoring. Solutions are manually manipulated and temperatures must be maintained(70-85c)

27. AUTOMATION IN EMBEDDING The paraffin-impregnated samples taken from the tissue processor must be suspended in molten paraffin and then cooled to form a hard paraffin block for microtomy . This step, called embedding, is tedious and cumbersome because the tissue must be removed from the cassettes, visually oriented and placed in a mold , bathed with melted paraffin, and transferred to a cold plate. Special care is necessary to ensure that the tissue is appropriately oriented for sectioning, particularly for small biopsies, and that pressure is applied on the sample to flatten the surface and prevent air bubbles from collecting in the block.

28. Two examples of automated embedding equipment are the Sakura Finetek Tissue- Tek ® AutoTEC ® and Milestone’s SYNERGY system Central to this unique instrument is the development of a sectionable paraform cassette, which is used instead of the regular plastic cassettes. The Paraform cassettes’ plastic mesh is the same density as the solid paraffin so the resulting block can be sectioned using routine microtomy techniques. The system is capable of embedding up to 120 cassettes per hour with continuous loading of 4 magazines

29. Tissue- Tek ®   Paraform ®  Cassettes, are capable of locking in and preserving the orientation of very tiny or difficult to orient tissues from grossing through to imaging, helping to further extend the range of tissues suitable for automated embedding .   Tissue- Tek ®   Paraform ®  biopsy gels.

30. Tissue- Tek ® AutoTEC ®

31. MICROTOMY In this step, very thin sections of tissue are cut with long, flat blades from a paraffin block, floated on a water bath, and then transferred to a glass slide. there is not a single functional, fully-automated microtome available to assist in standardizing this highly-technical and variation-rich task of producing thin tissue sections.

32. TYPES OF MICROTOMES ROTARY MICROTOME Most commonly used Rotation of hand wheel by 360degree cause the movement of the specimen vertically past the cutting surface Ability to cut thin 2-3 micrometer sections. Can easily cut all types of hard, fragile, fatty tissues. ideal instrument for cutting serial sections Large block of tissues may be cut. Cutting angle and knife angle can be adjusted.

33. They align and trim the tissue blocks with optimal precision. They reduce the risk of losing tissue. Blocks are ready for sectioning in only 10 seconds. This enables laboratories to work faster, better and more efficiently. Leica RM2255 Fully Automated Rotary Microtome

34. SLEDGE MICROTOME The specimen is held stationary and knife slides or moves across the top of the specimen during sectioning. Cutting sections of very large blocks of tissue and hard tissues. Knife holding clamps are adjustable.

35. SLIDING MICROTOME FREEZING MICROTOME

36. AUTOMATED MICROTOME KNIFE SHARPNERS They perform the actual honing process automatically. They consists of a plate on which knife held by arm rests at an angle so that the cutting facet is in contact with the plate. The plate moves in an oscillatory and rotatory fashion which produces sharpening of knife edge.

37. STAINING AND COVERSLIP Alongside tissue processing, H & E stain is another major component of histology which is automated in a majority of labs. Linear style stainer . X-Y stainer design.

38. Linear style stainer The slide progress along a conveyer individually or with in a rack being submerged in various solvents and dye pots. Time in each container is constant. Disadvantages: Constant use of the same reagent pots can lead to variation in staining as the day progresses and frequent reagent changes or monitoring may be required. These systems are not fully enclosed. Issue arising from evaporation , humidity and increase user contact with the chemicals

39. X-Y STAINER DESIGN This is usually enclosed and can attach to ovens and cover slipper . These machines feature a robotic arm which moves the racks along, following a predesigned protocol . Protocols can be designed by dictating the sequence of reagents, and the time in each can be modified independently, allowing multiple stains to be performed in single unit. . User contact can be minimized as the rack can be loaded into an empty input area . Disadvantage : they can cause batching of work and delay.

40. LINEAR STYLE STAINER X-Y STAINER

41. AUTOMATED COVERSLIPPERS Leica’s ST5020/CV5030 Sakura’s Prisma and Ventana’s Symphony accept glass slides mounted with blank sections and return H&E slides ready for microscopic evaluation. The two widely used histochemical stainers are The dako artisan link system Ventana benchmarkSS

42. Leica ST5020-CV5030 The Leica ST5020 reagent management system (RMS) allows your lab to minimize reagent changes and maintenance time by maximizing reagent life. . 

43. Dako’s Artisan Link First instrument to provide true walk-away automation for complex special stains. A slide barcode reader standardizes and increases the speed of slide processing, reduces errors and improves your laboratory productivity. The software allows Artisan Link to be operated either as a stand-alone or in a networked configuration system using a LAN System ( DakoLink ) or a Laboratory Information System (LIS) providing 24/7 access to stainer processing status. 

44. A COMPARISON OF TWO COMMERCIALLY AVAILABLE AUTOMATED TINCTORIAL STAINING PLATFORMS TO MANUAL STAINING Manual Bench Mark SS Artisan Slide capacity Limited by space, time and reproducibility 1-20 1-48 Reagent capacity Limited by space and safety 25 50 Unmanned runs No Yes Requires advance deparaffinization of section Overnight runs No Yes Yes, but reduces life span of reagents recommended to be Refrigerated Reduces contact with harmful components No Yes Yes

45. Post-H&E Techniques Post-H&E techniques performed on histologic material fall into the 2 general categories of “tissue-homogenate-based” and “slide-based” procedures. The post-H&E slide-based methods include histochemistry , immunohistochemistry and in situ hybridization. Automated histochemical stainers improve workflow in the histology laboratory by reducing the turnaround time for special stains. Some of the current systems available include Artisan ( Dako ), Autostainer RSt (Vision BioSystems ), and NexES SS ( Ventana ).

46. The following is a list of automated immunohistochemical systems marketed in the United States: Nemesis 7200 ( Biocare Medical), i6000 ( BioGenex ), Autostainer Plus and Eridan ( Dako ), Autostainer 720 ( LabVision ), Bench-Mark XT and BenchMark LT ( Ventana ), and BondMax ( VisionBioSystems ).

47. Semi automatic autostain ers Fully automated Autostainers They have facility to stain the slides after antigen retreival . Deparaffinization and antigen retrieval steps are done outside the equipment. They execute complete processing of slides ,drying to counter staining and continous batch processing . Automation is also provide with centralized slide programming by connecting equipment to computer.

48. MOLECULAR TECHNIQUES Insitu hybridization and other molecular tests are increasingly used techniques, where specific nucleic acids are targeted with formalin fixed tissue sections . Individual gain or loss of gene expression can be veiwed with both temporal and spatial information,and the targets can either be RNA or DNA . THE BIOGenex Xmatrix Infinity is a staining system which can can provide either IHC,ISH,IF . This system fully automates the run from dewax to final coverslip , producing upto 100 slides per day.

49. Slide digitization Once a slide has been produced and stained, automated slide scanners can scan it and produce high resolution images. most software and hardware can be used to scan at multiple levels, allowing ‘z-stacking’ of images to duplicate the effect of focusing up and down through the tissue. Omnyx flatbed scanner with a ‘load-and-go’ operation which enables continuous scanning (overnight if required) to produce images in a timely manner

50. REFERENCES Automation of the Histology Laboratory Azorides R. Morales, MD, Mehdi Nassiri , MD Bancroft’s THEORY and PRACTICE of HISTOLOGICAL TECHNIQUES 8 th edition. Histopathology Techniques and its Management, Ramdas Naik MD. Internet

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