Discuss the principles behind, and the use of:
a) The Embedding Station
b) The Faxitron
This essay will attempt to explain the scientific, technological principles and the practical uses of the above mentioned pieces of laboratory equipment highlighting health and safety issues where appropriate.
The principle of the embedding station is to accurately and precisely orientate a histological specimen into a block of paraffin wax; this will allow the support and firm hold of the tissue sample so that fine cutting of thin sections for histological diagnostic purposes can ensue. The equipment required is the Tissue tek / R A LAMB embedding station, tissue moulds (assorted sizes) and speci-ceps heated forceps. The tissue moulds are of various sizes to allow enough embedding medium to fully surround and support the tissue sample. Heated forceps are used so that any wax coming into contact with them is kept at a molten state which can be easily removed using a paper tissue, cold wax will adhere to the forceps making it very difficult to manoeuvre tissue into the correct orientation as there would be a build up of solid wax. The wax used as the embedding medium in this particular laboratory is paraffin wax. This type of wax is molten above 60oC and is in the solid state at room temperature. Its hardness is ideal to support the cells and to hold the tissue sample firmly for microtome sectioning.
Fig.1 Embedding station
The embedding station consists of 3 parts;
1) The Thermal Console 1
This stores and warms the base moulds in its rear chamber, and holds processed tissue specimens in cassettes awaiting paraffin embedment in the heated front chamber.
2) The Dispensing Console
This includes a heated 2 litre paraffin chamber, a heated orientation platform, a “cold spot” for chilling the specimen onto a position on the first paraffin pour, and a dispensing island which dispenses paraffin using a finger operated control.
3) The Cryo Console
This is composed of a large, refrigerated chiller plate which is kept around -6oC, on which paraffin embedded specimens are kept cool prior to microtome sectioning.
The tissue cassettes are placed into the block reservoir (in molten wax) on the right on the embedding station. It is important to ensure that all the cassettes are well submerged in molten wax to prevent the tissue from drying and becoming hard. The temperature is controlled by an automatic time switch this prevents the temperature from becoming too hot which would have a detrimental effect on the tissue samples and may also be hazardous to health.
One cassette is removed at a time using the heated forceps and transferred to the “hot embedding stage. The lid is carefully removed which is then checked to make sure no tissue is adhering to it. This is to prevent any tissue either being lost or transferred to another patients’ cassette which could lead to an incorrect diagnosis. The lid of the cassette is then placed in an appropriately located tray. Also to be checked are biopsy sponges. Biopsy sponges are small oblong sponges which are placed in the tissue cassettes at the time of grossing, they are used to pack very small pieces of tissues to prevent any of these tiny fragments from being lost during processing as the cassette lids have holes in them which are needed to allow the processing reagents in and out.
Fig. 2 Tissue cassettes
Check the patient specimen number on the cassette against the details on the biopsy card. This will give details about the type of biopsy and the number of tissue pieces that should be present. A suitably sized mould should be selected for the tissue size this needs to have a small amount of molten wax poured into it. The tissue should be transferred into a mould using the heated forceps. The orientation of the tissue depends on the tissues type i.e. skin should be arranged on edge to ensure all layers (epidermis, dermis and hypodermis) are revealed under the microscope. Tubes and arteries need to be orientated so that lumen is revealed. Ovarian tissue is orientated flat so that under the microscope this should enable the stratified squamous epithelium and endocervical cells to be seen. The tissue should be arranged so that the base of the tissue is held at the bottom of the mould. The mould then needs to be transferred to the cold surface and slight pressure should be applied to the tissue so that it is fixed in position this will occur as the wax is setting. This is done so that tissue is a flat as possible which during microtome sectioning a full face of tissue is seen rather than the sample being uneven or possibly lopsided which would mean during the trimming away of excess wax important cells could be lost by trying to flatten out the surface of the tissue. The lid is then firmly placed on top of the cassette and filled with molten wax. The cassette is then transferred to the ice tray where it is left to solidify.
If there is an absence of tissue or less than recorded on the biopsy card or the orientation of the tissue is confusing, a senior member of staff must be consulted. It is advisable to clean the forceps with a paper tissue between each blocking to prevent any tissue being transferred into another patients block. Small biopsies are embedded first, followed by skin tissues, decalcified tissue, routine specimens and then post-mortem tissues.
After all the specimens have been embedded, the moulds are replaced into the chamber, cassette lids and mesh bases are cleaned and the paraffin reservoir is topped up with molten wax. The wax in the storage tray should be cleaned at least once a week but can be changed more often if it has become contaminated. Excess wax should be removed from all surfaces along with the wax drip tray which needs to be changed daily.
There are possible sources of errors which include specimen carry over into other patients’ blocks via dirty forceps or moulds. This could lead onto a false diagnosis. If lids and /or wrappings are carelessly discarded specimens may become lost. It is also very important to orientate the tissue in the correct fashion to prevent the diagnostic zone from being lost possibly via microtome trimming.
There are health and safety issues to consider when using the embedding console; heat is a hazard, molten paraffin wax is used at temperatures of approximately 62oC and may cause burning. Also the heated forceps are very hot to the touch and need to be handled carefully. If the wax drip isn’t emptied regularly instrument failure may occur or lead to a fire. Personal protective clothing must be worn during embedding, in this particular lab this is a white plastic apron to protect from hot wax spilling onto the person or clothing. In other laboratories a white Howie coat maybe worn with or without gloves.
If careful use and health and safety SOPs and COSHH regulations are followed correctly then the risk of injury or damage to health is low.
The principle of the Faxitron X-ray machine is to determine the presence or absence of micro calcification deposits in excised tissues. Typical clinical applications include; breast specimen radiography, specimen X-rays can reveal micro calcifications in the range of 10 to 20 m. This can assist in the detection of occult lesions. An occult lesion is a small carcinoma, either asymptomatic or giving rise to metastases without symptoms due to the primary carcinoma. Therefore breast cancer can be diagnosed at an earlier and more curable stage. High resolution radiography can be applied to bone specimens which can reveal fine trabecular detail. Another example of clinical use is micro angiography of post mortem hearts which helps to visualise the distribution of coronary arteries and to locate any alternations in them also this is not an application carried out in this particular laboratory.
Fig.3 Faxitron machine
The specimen requirements are slices or pieces of tissue or specimens in wax blocks. The sizes of the slices tissue can range from 10mm to 30mm approximately. The equipment required is the Faxitron model no. 43855A, 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 dept.)
Figs 4 & 5 Petri dish and film cassettes.
Always ensure the exposure chamber door is closed before starting to operate the machine. Insert the key into the safety lock and turn on this allows the machine to warm up before use. At this point the digital tube voltage meter will light.
Turn the kvp control knob fully anti clockwise and ensure that the tube voltage meter indicates 00 or 01 then it is possible to turn the knob to the require voltage. Open the door and position shelf on the required level.
The tissue to be X-rayed may be placed in a Petri dish which is then placed on the X-ray cassette, tube facing upwards. The specimen must be identified by placing the histology specimen accession number, kilovoltage and exposure time (secs) using the radio opaque numbers. Ensure door is closed otherwise the machine will not work, this is a safety measure. Set the exposure time using the thumb wheels, typically 6 seconds although a different time may be used depending on the thickness of the specimen.
The kvp control button is turned anti clockwise until the voltage meter indicates the required voltage this again depends on the thickness of the specimens. Typically breast slices will be sliced at around 15mm but it will depend on which pathologist has cut the specimens. Press X-ray start switch. The X-ray stop switch will light up and the tube current bar graph will show tube current. The digital time gauge will count down from the number (time on secs) set of the thumb wheel during this time x-ray and gamma rays are being produced.
When the time reading indicates zero, the display will blank out indicating that the X-rays are no longer being produced.
The door may be then opened and the cassette removed. The cassette is taken to the X-ray department to be processed on the Kodak dry developer. On arrival to the X-ray department, check with a member of staff that the processing machine is available for use. Using the appropriate button set the size to 18″ x 24″. The cassette is inserted with the clip facing uppermost and facing the front of the machine. Push slowly the cassette until the machine “grabs” it. When the machine beeps it is safe to enter a further cassette this is in the region of 30 seconds after insertion of the first one. If the fault light appears or the cassette is ejected ask for assistance. The machine will replace a fresh film back into the same cassette; it will then be ejected for further use.
When the film has been developed it will be fed into the hopper below. Check with pathologist that the film is acceptable it may have been over or underexposed and if this is the case the x-ray will need to be re done.
If the film is acceptable, the film may then being given to the requesting pathologist, the specimen needs to be returned to the pot and placed back on the appropriate shelf in the walk-in fridge so that it can be easily found should it be needed for future use. The numbers and letters should be returned to their respective containers so that they are easy to find by whomever uses the machine next, and then clean the cassette of any contamination from the specimen, contamination may be in the form of formalin from the specimen pot, ink from the tissue or tissue itself.
If the X-ray is not acceptable, repeat the exposure with a higher or lower kvp setting depending on over or under exposure. Return all setting to zero and turn safety lock to off position and remove key.
Health and Safety Issues – The tube current bar graph is evidence that X-rays are being produced, when exposure has finished, the tube current needs to show zero. If this does not happen, when exposure is complete, use kvp control knob to return to zero. Turn the power switch to off and remove the key. Notify senior member or staff and service engineer regarding this issue.
If the door is open during the exposure, radiation will stop and exposure will be terminated. It is possible to continue exposure by altering the time set on the thumb wheels to agree with the digital time display. However, if the thumb wheels are not changed, and disagree with the digital time display, the timer circuit will automatically reset to the number shown on the thumb wheels. This may cause over exposure to the film.
Always wear disposable gloves, Howie coat, and plastic apron when handling specimens that are fresh or fixed in formalin. Remove excess formalin from the specimen before placing on the X-ray cassette, return all settings to zero and turn safety lock key to off position and remove key.
If control measures recommended in SOP’s, COSHH and if Risk Assessments are followed correctly, the risk of injury or damage to health is low.