Handling, Storage and Disposal of Samples
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Published: Tue, 06 Feb 2018
Expectations of a Health Care Professional
In the histology laboratory all specimens arrive fixed in 10% buffered formalin. In the laboratory, the specimen and the request form are labeled with the same lab number. The specimens are left in the same order the lab number is given and processed. Safety gloves and an apron are worn when processing the specimen.
Unfixed specimens received in a plane container are fixed in 10% formalin which is commercially prepared and left for one day to process. This is done if the specimen requires fixation. Certain specimens are an exception to this rule. Lymph nodes are wrapped in gauze when lymphoma is suspected, skin sections for Immunofluorescence due to Pemphigus vulgaris are suspended in saline solution, and frozen sections are not fixed since fresh tissue is sectioned for microscopic examination.
Whether the result has been reported or not determines which samples are disposed and stored if the result has been reported or not:
After the samples are processed the pieces of the sample that were not inserted in the cassette are placed back inside the respective container. The fixed specimen in the container is refrigerated until the result is reported (figure1). 3 weeks after reporting the result a disposal list is created and the specimens to be disposed are packed inside boxes, labeled and then sent for incineration. Samples such as fetus are kept for burial.
Empty containers are left for a week and a half as a quality control and for human errors. In many cases the label on the container shows the type of specimen so the empty containers are left in case verification of type of specimen is required.
Blocks and slides are stored permanently inside a storage room. All blocks and slides are carefully and methodically filed so they are available for records or for future reference. As years pass blocks remain unchanged but stains on the slide tend to fade so there is sample deterioration. After cutting, the blocks are placed in numerical order according to the year and placed inside boxes. The first and the last number of the blocks in each box are written on the boxes. All sides and top box are labeled and sealed with tape. Slides are filed in numerical order after the report is issued. Slides are placed in a slide box and the lab number of the first and last slide are written on the box.
Effective self-management of time and workload
The opening hours for the histology are from 6.00 am till 5.00 pm. The lab is open from Monday till Sunday and time shifts are available so the laboratory remains more open and more service is given to the public. The laboratory does not open during night shift because results in the histology laboratory are not considered urgent. Results must first be seen by the pathologist so no immediate results are required so processing is done during the day.
Samples that are considered urgent
In histology, specimens are not considered urgent because they have to be viewed by the pathologist results are issued. Frozen sections are considered urgent since the sample must be quickly processed so an intra-operative decision can be taken by the surgeon. Samples can also be considered urgent when a pathologist needs the results in a quick time, due to surgery scheduled on that day or the following day.
Career-Long Self Directed Learning
What is CPD?
CPD stands for Continuing Professional Development, an ongoing free training programme in histopathology including histology and cytology (Institute of biomedical Science, 2011). It is defined as “The systematic maintenance, improvement and broadening of knowledge and skills, and the development of personal qualities, necessary for the execution of professional and technical duties throughout the practitioner’s working life” (The Chartered Institution of Highways & Transportation, 2011). This means that CPD allows the employer to improve and to widen knowledge, quality, competence and skills in his/her profession.
What constitutes CPD activity?
A CPD is constituted by meetings, short courses, conferences or workshops that are created to inform other members of stuff or even the public. Organization and participation are essential for a successful CPD. It must be transparent, accountable and visible (Fox & Fox, 2004, p.182). CPD can be done:
- To present one’s own research report
- With the aid of websites, journals, posters, books and other printed media
- To show something encountered during work, that can be of interest to rest of the workers
- To make and encourage new procedures and changes
- Introduce a new course that will be of interesting to the public or workers
How does the CPD scheme benefit Pathology employers?
A CPD scheme enables the biomedical scientist to develop the necessary knowledge, attitudes, personal effectiveness and skills for his/her professional practice. The employer must identify his/her and their employer’s learning needs. In order to improve patient care the employer must be up to date on facts, new concepts and most importantly on opinion and consensus (The Royal College of Pathologists, 2010). The employer can record activity and document all learning achieved (Academy of Medical Royal Colleges, 2010). All this is done not for only the present but also for future progression (Institute of biomedical Science, 2011).
What are the benefits to a biomedical scientist (the employee) participating in the CPD scheme?
- Keep up to date with current rapid and expanding knowledge (The Royal College of Pathologists, 2010).
- Increases job satisfaction, productivity and quality of working life (Chen, Chang & Yeh, 2006)
- Acquire new skills for safe and effective practice. This builds up confidence in the employee (Institute of biomedical Science, 2011).
- Promote professional ideas and new initiatives, increasing job satisfaction (Institute of biomedical Science, 2011).
- Documentation of all that is learned from the scheme is encouraged (The Royal College of Pathologists, 2010).
- Benefit from quality control measures (Academy of Medical Royal Colleges, 2010).
- Encourage reflective practice (Academy of Medical Royal Colleges, 2010).
- Reduce risk of clinical isolation (The Royal College of Pathologists, 2010).
- Prepare for new roles example managerial. Employers value employees that undergo continuous CPD since such employees show learning agility (Chen et al., 2006; Royal College of Pathologists, 2010).
- Maintain a reputation of the biomedical possession and public assurance (The Royal College of Pathologists, 2010).
Where is the information relating to CPD displayed in Pathology?
When a CPD meeting is going to be held all biomedical scientists are informed through an email. The email is sent to the principle to make sure that all the histology staff knows about the meeting.
Site of origin
The trucut samples were taken from the right breast upper outer quadrant (Figure 2)
Sample Taking and Description of sample
The trucut biopsy is taken after a mammography showed a suspicious result. To diagnose, a trucut biopsy was performed. A trucut (core) biopsy is mostly done to sample tissues from a solid mass or calcium deposits, increasing sensitivity (Youk, Kim, Kim, Lee & Oh, 2007). Very small masses or masses that are too deep are sampled using a guiding imaging technique. No scars are left after sampling. It has the advantage of being highly sensitive and specific (Sadler et al., 1994).
The biopsy was performed at Mater Dei’s surgical operating theater (SOP) (in the Breast Clinic). The patient was given local anesthetic and left for a few minutes. A 16mm gauge core needle (figure 3) was then used to obtain the tissue samples. The tissues sampled contain tissues from the mass and normal healthy tissues from the breast. The sections sampled contain also provide more diagnostic information than mammography and fine needle aspiration. The samples are larger than FNA therefore results are more accurate (Kasraeian, Allison, Ahlmann, Fedenko & Menendez, 2010).
The clinician or nurse localised the mass and its boundaries and the mass was then immobilised. The needle was inserted through the skin into the lump and the tissue section was taken. To increase the chances of diagnosis 6 trucut specimens were taken. Their length varied from 9mm to 14 mm. The needle was then detached. The trucut specimens were then introduced into a container contain 10% buffered formalin. The container and the request form where received in the histology laboratory the following day.
A courier brought the trucut specimen for histology processing into the histology laboratory. In the laboratory, the request form which comes together with the specimen was left for a day where registration and processing began. The following day, the receptionist used the HOE system to input data so they can be available only in the laboratory. The ID number of the patient was inputted followed by location the specimen was sampled example BOFFA, the name of the medical lab scientist, and the name of the pathologist/consultant. If available, the macro examination results were also included. A label containing the lab number, the letter on the cassette, the last two digits of the year, and the patient’ name and surname was prepared and printed. The label was prepared to label the slide after staining (in this case only one label was required).
The sample and its respective request form were both labeled with a barcode containing a specific laboratory number. The barcode was stuck on the top of the request form and at the back of the container (without covering any patient’s details). The laboratory number was also written with the aid of marker on top of the tap of the container. The request form was stamped at the top and at the bottom with the date in which it was received in the laboratory. The trucut specimen and the other histological specimens were left one after the other, according to the laboratory number. Specimen processing proceeded in this order.
The trucut specimen was first processed in the laboratory at the cut-up laboratory. The name and surname of the patient and the lab number on the request form and on the specimen container were checked. The trucut biopsies in 10% buffered formalin were taken out from the container, using forceps, on the working bench. A macroscopic examination was performed on the 6 trucut biopsies obtained. Their length ranged in length from 9mm to 14mm. They were all embedded in one printed cassette labeled A1. Blue foam was also placed and the cassette was covered with a medal lid. It was then was placed in eosin with the other specimens. The trucut biopsies were then ready for further laboratory processing. After all specimens were cut, a histopathology worksheet was filled in. This included the case number of the patient, the number of tissues taken (6) , the tissue type (breast trucut), the number of blocks (A1), any comments such as left to fix (not applicable), the name of the pathologist who will examine the slides, and the name of the medical laboratory scientist (in this case who performed the cut up).
2. Tissue Processing (Impregnation)
The biopsies were processed in an automated processing machine. This was performed in a closed system for trucut specimens using program A. It is important that the specimen is not larger than 3mm since it will not fit and cannot be cut afterwards. The closed system has 14 baths and it provides pressure, waving, bubbling and rotation to the tissues so the reagent can penetrate better. This is performed overnight, therefore the processor is programmed. When time for embedding is prolonged, the fixation time is prolonged to compensate.
- The tissues were first fixed in 10% buffered formalin so that the fixation was continued
- They were then dehydrated in 2 baths of 70% alcohol, in 1 bath 95% alcohol and then in 2 baths of absolute alcohol.
- The dehydrated sections were then moved into the chloroform and xylene. This step was done for clearing. Chloroform is a carcinogen and it affects the nervous system.
- The tissues were automatically moved in wax for tissue impregnation. This caused the tissues to harden. A temperature of less than 60oC was necessary because a higher temperature would have affected elasticity of the wax.
Fumes go in a waste bottle and charcoal filter is present to filter leak. The tissues were now ready for embedding.
During embedding, the formalin fixed processed tissues are surrounded by wax so a solid paraffin block is obtained. This will enable the medical lab scientist to obtain thin sections from the block so that they can be stained and later viewed by the pathologist. The procedure involved was as follows:
- The cassette was taken from the processor to the warm compartment of the histocentre. The histocentre is an embedding center that facilitates paraffin embedding. It is equipped with a dispenser, specimen handling tank, warmed embedding moulds, warmed forceps wells and warm plate for orientation of the specimen in the melted paraffin.
- After checking the wax tank was properly filled, the cold plate and light were switched on. The cold plate helps in transferring of the melted paraffin.
- The tissue cassette was opened and the number on the labeled cassettes was checked with that on the worksheet entry.
- A suitable mould compartment corresponding to the size of the tissues in the cassette was chosen.
- The mould was filled with paraffin wax
- The tissue was placed at the bottom of the mould, correctly orientated. Incorrect orientation ruins the first section taken
- The trucuts were placed centrally aligned across long axis of the mould, and not placed at random. Adequate border of embedding medium must surround all sides of the tissue to give maximum cutting support.
- The mould placed in its cassette was placed on a cold plate and allowed to solidify.
- The block was scraped along a para trimmer to trim excess wax on surface. Tissues embedded must be perfectly flat to ensure that a complete section will be obtained.
After the block was trimmed, thin sections were now cut using a microtome. The block was first trimmed to expose the area to be sectioned. A sharp non-rusted blade was used not to cause damage to the tissue by scoring. The microtome was cleaned from staples and sutures that remained to avoid damage of the blade. Microtomy was then started.
- The tightly screwed blade was checked and adjusted in the correct position. The micrometer gauge was set at a thickness of 18-22µm.
- The block was placed inside the block holder of the microtome and secured. The block holder was in parallel to the edge of the blade so a straight ribbon of sections was obtained.
- The block holder was moved using the couae trimming device until the wax block was almost touching the edge of the blade. The fine trimming rotator device was when the block touched the edge of the blade and trimming of the block was started. Excess wax from the surface of the block was removed until the surface of the tissue was exposed. Debris due to coarse cutting was removed using a Camel hairbrush.
- The block was then placed on ice to cool giving the tissue and the wax similar consistency. Water absorbed by the tissue, slightly swelling it, so cutting is easier later on. If this does not occur sections tend to crease.
- The block was reattached to the microtome, leaving the left-hand rotator device. The micrometer gauge was then set at 3µm. A series of sections forming a ribbon were cut and the first one was not used since it is usually thicker than 3µm.
- 4 layers were taken. This means that the after the first section was achieved, the next few layers were ignored and then a second section was taken. The same was done for the third and fourth. This is done so that the pathologist can study many layers from the site taken so that diagnosis is more accurate.
- The appropriate ribbon section (for all the four sections obtained) was gently transferred into a water bath using forceps. The water bath is set a few degrees below the melting point of the wax.
- The sections were floated onto a glass slide containing 20% alcohol. The ribbon section was then released on the surface of a water bath (at a temperature less than that of melting point of wax i.e. 60oC.
- The sections were collected on an APES-coated glass slide. They were placed on near the other. Coated APES facilitates adhesion of the sections onto the glass slide. 4 slides were obtained (a slide for each layer taken).
- The number of the block was written on the glass slide using a diamond pen and placed on a slide rack. It was dried in an oven at about 60oC for 10 minute.
Together with the other blocks from the other specimens the slides were dried and now ready for staining. The routine gold standard stain in histology is Haematoxylin and Eosin stain. This was done in an automated staining machine which followed the regressive method. This allowed overstaining of the tissues and removal excess dye by differentiation. The staining procedure was programmed as followed:
- The slides were left in the heating station so that all water is removed.
- The slides were dewaxed in a xylene for 4 minutes. This removed the surrounding wax from the tissues.
- They were then placed in xylene alcohol for 15 seconds. This started the gradual hydration process and prepared the tissues to be stained by haematoxylin dissolved in an aqueous solvent.
- The hydration process is followed by 2 baths of absolute alcohol (15 seconds each). The slides were then passed into four baths: 95% alcohol, 70% alcohol, 50% alcohol, and 30% alcohol (15 seconds each). They were then passed for 15 seconds in distilled water since haematoxylin stain is water based.
- The slides were then passed for 10 minutes in haematoxylin stain. The time inside the haematoxylin bath varies according to the properties of the stain. The prolonged use of the stain increases the time the slides pass in the bath.
- The slides were rinsed in two baths of distilled water, the first bath for 30 seconds and the second bath for 10 seconds.
- Differentiation then occurred in acid alcohol for 1 second. This allowed the nucleus to retain the stain and to decrease the pH (acidic) so colour changes to light purple.
- The slides were rinsed in distilled water for 15 seconds.
- Bluing occurred in tap water for 5 minutes. This raised the pH so sections became light blue.
- The slides were then passed into a bath containing distilled water for 15 seconds. The slides were passed in absolute alcohol for 15 minutes for dehydration and because this favours alcoholic eosin staining since it is alcohol based.
- Counterstaining was performed in a bath containing alcoholic eosin for 3.15 minutes. The time in alcoholic eosin varies according to the properties of the stain. The prolonged use of the stain increases the time the slides pass in the bath. Prolonging time allows the cytoplasm to take up the pink eosin stain.
- The slides were then dehydrated in four baths of acid alcohol, 15 seconds each.
- The slides were cleared in xylene alcohol for 15 seconds followed in 2 baths of xylene for 5 minutes each. This helps during mounting since DPX mountant is xylene based.
- The slides were left in the heating station so that all water is removed.
- The slides were taken out from the rack and mounted with DPX mountant.
Two slides are stained with H & E stain using the automated machine in the morning before starting routine staining. Errors in staining such as weak stains and contamination (example of eosin) can be detected so they can be solved. The 4 slides of the patient were therefore well stained since the machine passed QC on that day.
Cytoplasm and other eosinophilic structures: Pale pink
After processing, the number on the slides was checked with that of the cassette and the block. The slides were then labelled with their respective label. The cassette was placed on top of the slide to see if all the stained sections present on the block were sectioned. All the stained sections agreed with those on the block.
Role of the Biomedical Scientist
The role of the biomedical scientist is to perform all the above procedures. The medical lab scientists are divided into different sections throughout the histology laboratory: in the cut-up room and in the embedding and staining section of the laboratory (excluding immunohistochemistry laboratory). In addition, the biomedical scientist must also fill several worksheets. The initials of the biomedical lab scientists performing the cutup, macro-examination, LID and embedding are written in the histopathology worksheet. The MLS must monitor any changes example in reagents. Any injuries or misshapen occurring in the laboratory must be recorded.
Pathologist Role/Result Reporting
After staining, the pathologist viewed the slides under the microscope and performed a microscopic examination. The observed results were noted. The microscopic examination results were sent to the secretary who typed the result in the results form. The pathologist then read the results form for any errors and once the result was verified the pathologist authorised the result.
Result Entering and Authorisation
After the pathologist viewed the slides under the microscope he took the fully written request form to the secretary. The secretary separated the forms into different piles, according to the pathologist. The form was typed in a result form and printed as a result sheet. The written and the print result form were separated into 2 different racks. The report sheet was taken to the respective consultant/pathologist who reviewed the printed result sheet for any mistakes. This includes patient details, clinical details, and examination results. Once the pathologist verified the data written, he used the software to authorise the result. Once the pathologist authorised the result, this was available in the LIS of the cytology and histology laboratories. The CMI system allowed the results to be available to the wards. The result sheet was taken to the secretary where the result form was piled with other results forms according to the pathologist/consultant. Copies were made and sent to ward and patient.
Result Issuing (Describe the results form)
The results form contains the details of the patient, including the name and surname, address, date of birth, sex and the hospital number. The name of the clinician and the site from where trucut biopsy was taken (SOP) are included. The date the specimen was taken and the date and time it was received are also included. The lab number associated to the specimen is important to be included because besides identifying the patient it can be used for future reference. If the slides or block containing the sections are required they are labelled (including lab number) and stored and easily retrievable.
The specimen type and site from where the biopsy was taken, the macroscopic examination and the microscopic examination are all included. The included, in this case “Benign breast parenchyma of the right breast”. The pathologist and the date and time the result was reported and authorised (by pathologist) and the date and time the result form was printed are also included.
Benign Breast Parenchyma: The breast parenchyma forms part of the normal breast tissue. It was reported as benign during microscopy because of few scattered (not clustered) lobules seen in breast sections. Since no atypical features were observed, no special stains or immunohistochemistry staining (example ER or Her-2 stains) were required. It is ideal the patient undergoes regular breast screening.
Sample Collection and Specialist Preparation
The containers to process routine surgical specimens vary from small to large received in 10% buffered formalin. Very large containers are rare. The container used depends on the size of the specimens. Small specimens such as polyps, prostate scrapings, appendix, trucuts, and trephines are received in small containers containing 10% buffered formalin. Some specimens such as fetus vary in size such as fetus and colon so they received in larger specimens (medium when compared to small containers). Large specimens such as lung, breast, and colon are received in large containers containing 10% buffered formalin. Large specimens require more than one day to be cut. First the specimen is opened and left for an additional day or more for further fixation.
The following are types of specimen the laboratory receives that require specialist preparation techniques and the actions taken:
Trephine and Bone specimens: – Decalcification with EDTA or formic acid. EDTA is used example for bone marrow trephine and formic acid is used example on bone sternum for one day
Figure 4 showing a femur bone undergoing decalcification in EDTA.
- Infective specimen example with HIV – Over fixation in formalin to kill infective cells*
- Lymph node –The time of fixation depends on the thickness of the specimen. More time the more the fixative is allowed to penetrate the lymph node.* It is left for two or three days depending on the thickness of the specimen.
- Over fixation will destroy the surface antigens causing artifacts and a false negative result during immunohistochemistry.
- Sural nerve: Sent from operation inside a gauze soaked with saline. The request from and case summary are required. The cut up laboratory gives the lab number and send the specimen to the immunohistochemistry laboratory. The tubular sural nerve is wet, and the two ends of the nerve are cut. One end is sent to a pathologist to get an idea of diagnosis and the centre part of the nerve and the other cut end are sent abroad.
- Muscle: This is received in saline and a lab number is given in the cut up laboratory and then sent to immunohistochemistry laboratory. It is frozen at -70oC and cut by a cryostat at -20oC. The thin sections are then stained with a series of special stains example Oil Red O and with immunohistochemistry stains example myosin. APES coated glass slides are used to prevent the tissue section from sliding off.
- Imprints: Example lymph node: A slide is pressed on the lymph node and the imprint is sent abroad. The lymph node is then worked normally in formalin. Imprints are used for genetic studies.
Liver with no tumour: A series of special stains are performed:
- PAS – useful if there is a high glycogen content upon staining
- Reticulin Stain – useful in liver cirrhosis and liver fibrosis
- Masson’s Trichome Stain – Useful in liver fibrosis
- Iron Stain – useful for haemosiderosis, haemochromatosis
Title: Frozen Sections
- Performing a macroscopic examination by the pathologist
- Cut up of the specimen
- Obtaining sections at -17oC using a micrometer, inside a cryostat
- Staining the section/s by haematoxylin and eosin stain
- Performing microscopic examination of the stained section/s by the pathologist
A frozen section is a specific type of biopsy performed during surgery so that a rapid diagnosis of the tissue extracted is made (Brender, Burke & Glass, 2011). The tissue can be sectioned and stained in the laboratory for microscopic examination by the pathologist. The surgeon is given flexible intra-operative decision making according to the result given by the pathologist after the rapid processing (KarcioÄŸlu, 2005, p.121).
A surgery is booked and a biopsy is taken and sent to the laboratory. As soon as the fresh specimen arrives in the histology laboratory the pathologist and the selected biomedical scientists start processing the specimen. The pathologist performs a macroscopic examination on the specimen and the observed features are written down by the pathologist. The MLS then start cutting thin sections according to the specimen, using a microtome inside a cryostat at -17oC. The sections are then quickly stained with haematoxylin and eosin stain. In contrary to routine H & E, the sections are not passed through xylene and dehydrated down to water. This is because the frozen sections are not embedded in paraffin wax prior staining. Since the stain is very fast there differentiation with acid alcohol is also not performed. After mounting the pathologist checks if the stained slide is satisfactory and after performs a microscopic examination. This lets the surgeon decide what to do next.
Materials and Equipment required
Cryostat, OCT medium, cryospray, Glass slides, cover slips, disposable pipettes,
The pathologist opens the container/s containing the specimen/s. A macro examination is performed on the specimen/s and the pathologist starts a description so that the medical lab scientist writes on the request form. The description includes the size dimension (length x width x height) in centimeters, the shape of the specimen and if it is soft or hard. The consultant suspects carcinoma and sampling is them performed.
2. Cutting the specimen
The consultant cuts piece of the specimen that covers the whole area of the specimen. It is important the most suspicious is included in the segmented section so that the consultant can find and detect the tumour during microscopy. If required, multiple sections can be taken to make a diagnosis. The size cut depends on the size of the sample and tumour. More than one pieces of the specimen can be cut example: two sections from a liver (due to liver transplantation), and from a lymph node attached to the liver.
- The cut specimen/s is/are placed, with the aid of tweezers, in the center of a cryostat object disk containing OCT medium.
- The cryostat object disk with the tissue is placed on the cryobar (holder) inside the -17oC set cryostat.
- The tissue is left to settle so it gets cold and this is enhanced by using a cryospray.
- When the tissue solidifies it is placed onto an object disk holder. The machine is set at 5µ on the control panel and the block is moved towards the edge of the blade. After making sure it is properly clamped trimming is started.
- The rotator on the right of the cryostat is turned. The section begins to curl as the block comes in contact with the blade. The section is held down slowly and gently with tweezers and cut until the surface of the tissue is visible. The cryostat is now quickly set at 30µ (this is the thickness used for most of the specimens in histology).
- A good section is detached and taken onto a glass slide placed opposite of the block. As the tissue comes in contact with the glass slide it sticks onto it since it melts and adheres to it.
- The glass slide is immediately in the staining station found adjacent to the cryostat. Haematoxylin and eosin staining is performed.
4. Haematoxylin and Eosin Staining
- The glass slide with tissue section is f
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