This discussion will cover the principles and practical applications of the following topics:
The Embedding Station
The Faxitron
Introduction
The purpose of this essay is to explain the scientific and technological principles behind specific laboratory equipment. Additionally, we will examine how these instruments are used in practical applications and discuss any related health and safety considerations.
The embedding station is used to accurately position a histological specimen in a block of paraffin wax. This ensures that the tissue sample is securely held, allowing for precise cutting of thin sections for histological diagnosis. The necessary equipment includes the Tissue tek / R A LAMB embedding station, various sizes of tissue moulds, and heated speci-ceps forceps. The moulds are used to fully surround and support the tissue sample with enough embedding medium. Heated forceps are essential to keep any wax that may come into contact with them in a molten state, as cold wax would stick to the forceps and hinder the orientation of the tissue sample. Paraffin wax is used as the embedding medium in this laboratory, as it becomes liquid at temperatures above 60oC and solidifies at room temperature. Its hardness is ideal for providing support to cells and holding the tissue sample firmly during microtome sectioning.
The embedding station is made up of three components;
1) The Thermal Console 1
The rear chamber of this device is used for storing and heating the base moulds, while the front chamber is heated and is used for holding tissue specimens that have been processed and are awaiting paraffin embedment.
2) The Dispensing Console
The equipment consists of a heated 2 litre paraffin chamber, a heated orientation platform, a “cold spot” for cooling the specimen onto a position on the first paraffin pour, and a dispensing island that utilizes a finger operated control to dispense paraffin.
3) The Cryo Console
The specimens embedded in paraffin are cooled on a large refrigerated chiller plate maintained at approximately -6°C before they are sliced using a microtome.
All tissue cassettes are placed in the block reservoir on the embedding station’s right side, where molten wax is present. It is crucial to ensure that all cassettes are fully submerged in the wax to prevent the tissue from drying out and hardening. The temperature is regulated by an automatic time switch to prevent overheating, which can harm the tissue samples and pose health risks.
One cassette is taken out using heated forceps and moved to the “hot embedding stage”. The lid is carefully checked to ensure there is no tissue sticking to it. This is done to avoid losing tissue or transferring it to another patient’s cassette, which could result in an incorrect diagnosis. The lid of the cassette is then placed in a designated tray. Biopsy sponges also need to be examined. These sponges, which are small and oblong, are placed in the tissue cassettes during grossing. They are used to pack tiny pieces of tissue to prevent them from getting lost during processing as the cassette lids have holes that allow the processing reagents to go in and out.
Please check the specimen number on the cassette against the information on the biopsy card. This will provide details about the biopsy type and the expected number of tissue pieces. Choose an appropriate size mould for the tissue, and pour a small amount of melted wax into it. Transfer the tissue into the mould using heated forceps. The orientation of the tissue depends on its type. For example, skin should be arranged on its edge to ensure all layers (epidermis, dermis, and hypodermis) can be seen under the microscope. Tubes and arteries should be oriented to reveal their lumen. Ovarian tissue should be oriented flat in order to see the stratified squamous epithelium and endocervical cells under the microscope. Make sure that the base of the tissue is positioned at the bottom of the mould. Transfer the mould to a cold surface and apply slight pressure to fix the tissue in place as the wax sets. This is done to ensure that the tissue is as flat as possible, so that during microtome sectioning, a complete face of tissue is visible rather than an uneven or lopsided sample. By flattening out the surface of the tissue, it helps prevent important cells from being lost while removing excess wax during trimming. Finally, firmly place the lid on top of the cassette and fill it with melted wax.The cassette is transferred to the ice tray and left to solidify.
If there is a lack of tissue or less than indicated on the biopsy card, or if the tissue orientation is unclear, a senior staff member should be consulted. It is recommended to clean the forceps using a paper tissue after each blocking procedure to prevent any cross-contamination between patient blocks. The embedding process should prioritize small biopsies, followed by skin tissues, decalcified tissue, routine specimens, and post-mortem tissues.
After embedding all the specimens, the moulds are returned to the chamber. The cassette lids and mesh bases are cleaned, and more molten wax is added to the paraffin reservoir. The wax in the storage tray should be cleaned weekly, but it can be changed more frequently if it has become contaminated. Any excess wax should be removed from all surfaces, including the wax drip tray, which should be changed daily.
There are several potential sources of errors in the laboratory. One possibility is that dirty forceps or moulds may result in specimen carry over into other patients’ blocks, leading to a false diagnosis. Another issue is the careless disposal of lids and/or wrappings, which can cause specimens to become lost. Additionally, it is crucial to correctly orientate the tissue to prevent the diagnostic zone from being lost during microtome trimming.
When using the embedding console, there are health and safety concerns to take into account. One hazard is the heat associated with the process, as molten paraffin wax is used at temperatures of approximately 62oC, which can cause burning. It is also important to be cautious when handling the heated forceps, as they are very hot to touch. Failure to regularly empty the wax drip can lead to instrument failure or even a fire. To protect from hot wax spills, it is necessary to wear personal protective clothing during embedding. In this particular laboratory, a white plastic apron is worn. However, in other laboratories, a white Howie coat may be worn with or without gloves.
If the correct procedures and regulations for careful use, health and safety SOPs, and COSHH are followed, the likelihood of injury or harm to one’s health is minimal.
The Faxitron X-ray machine is used to detect micro calcification deposits in excised tissues. It is commonly used in breast specimen radiography to reveal micro calcifications in the range of 10 to 20 m, aiding in the detection of occult lesions (small carcinomas with no symptoms or metastases). This can lead to earlier and more curable diagnosis of breast cancer. The machine can also be used for high-resolution radiography of bone specimens, revealing fine trabecular detail. Additionally, it can assist in micro angiography of post mortem hearts to visualize coronary artery distribution and identify any alterations. However, this specific laboratory does not perform this application.
Fig.3 The Faxitron machine
The required specimens for testing include slices or pieces of tissue, as well as specimens preserved in wax blocks. The size of the tissue slices typically ranges from approximately 10mm to 30mm. To conduct the necessary procedures, specific equipment is needed, such as the Faxitron model no. 43855A. Additionally, Petri dishes (R&L Slaughter – 109), X-ray opaque letters and numbers, and Kodak X-ray minolar mammography cassettes and film from the X-ray department are also required.
Figs 4 & 5 depict both a Petri dish and film cassettes.
Before beginning operation of the machine, always make sure that the exposure chamber door is closed. Insert the key into the safety lock and turn it on in order to allow the machine to warm up before use. As a result, the digital tube voltage meter will illuminate.
To adjust the voltage on the tube, start by turning the control knob counterclockwise until the tube voltage meter displays a reading of 00 or 01. After that, you can set your desired voltage by turning the knob. Additionally, open the door and position the shelf at your desired level.
The tissue to be X-rayed can be placed in a Petri dish and then placed on the X-ray cassette with the tube facing up. To identify the specimen, the histology specimen accession number, kilovoltage, and exposure time (in seconds) should be placed using radio opaque numbers. For safety reasons, make sure the door is closed as the machine will not work otherwise. The exposure time can be set using the thumb wheels, usually around 6 seconds but may vary based on the thickness of the specimen.
To achieve the desired voltage, turn the control button of the kvp anti clockwise until the voltage meter shows the appropriate reading. The specific voltage needed depends on the thickness of the specimens being used. Typically, breast slices are cut to around 15mm thickness, although this can vary based on the pathologist’s technique. Once set, press the X-ray start switch. This will activate the X-ray stop switch and display tube current on the bar graph. At the same time, a digital time gauge will begin counting down from a specified number (in seconds) determined by adjusting the thumb wheel. Within this timeframe, x-ray and gamma rays are produced.
Once the time reading reaches zero, the display will switch off to indicate the cessation of X-ray generation.
The door can be opened and the cassette removed. Take the cassette to the X-ray department for processing on the Kodak dry developer. Upon arrival, check with a staff member to ensure availability of the processing machine. Set the size to 18″ x 24″ using the appropriate button. Insert the cassette with clip facing upwards and towards the front of the machine. Slowly push it in until grabbed by the machine, indicated by a beep. Around 30 seconds later, insert another cassette if safe to do so. If fault light appears or cassette is ejected, seek assistance. The machine will then place a fresh film back into same cassette before ejecting it for further use.
Once the film has been developed, it should be placed in the hopper below. It is important to consult with the pathologist to confirm if the film is acceptable or if it has been over or underexposed. If this is the case, the x-ray will need to be repeated.
If the film is approved, it can be handed over to the pathologist who requested it. The specimen should then be put back in the container and placed on the correct shelf inside the walk-in refrigerator for future use. The numbers and letters should also go back into their respective containers so that they can be easily identified by the next person using the machine. Lastly, ensure that any contamination on the specimen cassette, like formalin from the container, ink from the tissue, or even the tissue itself, is thoroughly cleaned.
If the X-ray is not acceptable, you should repeat the exposure using a higher or lower kvp setting depending on overexposure or underexposure. Afterward, reset all settings to zero and turn the safety lock to the off position while removing the key.
Health and Safety Issues – It is important to ensure that there are no X-rays being produced after an exposure has finished. The tube current bar graph should indicate zero. If it does not, the kvp control knob should be used to return it to zero. The power switch should then be turned off and the key removed. It is essential to inform a senior member of staff and a service engineer about this issue.
If the door remains open during the radiation exposure, the exposure will cease and be terminated. To continue the exposure, adjust the time set on the thumb wheels to match the digital time display. However, if the thumb wheels are not adjusted and do not match the digital time display, the timer circuit will reset itself to the number displayed on the thumb wheels. This can result in excessive exposure to the film.
When handling specimens that are fresh or fixed in formalin, it is always important to wear disposable gloves, a Howie coat, and a plastic apron. Before placing the specimen on the X-ray cassette, make sure to remove any excess formalin. Additionally, remember to reset all settings to zero and turn the safety lock key to the off position before removing the key.
If the control measures suggested in SOP’s (Standard Operating Procedures), COSHH (Control of Substances Hazardous to Health), and Risk Assessments are adhered to accurately, the likelihood of experiencing harm or detrimental health effects is minimal.
