Handling, storage and disposal of samples - Free Essay Example

Sample details Pages: 33 Words: 9774 Downloads: 2 Date added: 2017/06/26 Category Statistics Essay Did you like this example? Expectations of a Health Care Professional Handling, Storage and Disposal of Samples 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. Don’t waste time! Our writers will create an original "Handling, storage and disposal of samples" essay for you Create order 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 practitioners 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 ones 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 employers 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. Vertical Audit 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 Deis 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. Specimen reception/numbering 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). Specimen Registration 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 patients 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. Specimen Processing 1. Cut-Up 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. 3. 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. 4. Microtomy 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 18-22m. 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 3m. A series of sections forming a ribbon were cut and the first one was not used since it is usually thicker than 3m. 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. 5. Staining 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. Quality Control: 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. Results: Nucleus: Blue 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 Massons Trichome Stain Useful in liver fibrosis Iron Stain useful for haemosiderosis, haemochromatosis Title: Frozen Sections Aim 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 Introduction 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 (Karciolu, 2005, p.121). Principle 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, Procedure 1. Macro-examination 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. 3. Cryostat 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 5 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 30 (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 fixed in formalin for 10 seconds The slide is then rinsed in water (10 dips) It is then stained in haematoxylin (15 dips) The section stained with haematoxylin is rinsed in water Bluing is then performed by dipping the slide 15 times in Scots The slide is rinsed in 95% ethanol (10 dips) The section is counterstained in alcoholic eosin (10 dips) The stained section is dehydrated in absolute alcohol The slide is cleared in 3 baths of xylene The slide is immediately mounted with DPX 5. Microscopic examination: The stained section is immediately taken to the consultant who verifies if the section taken is appropriate or not. If the section is not appropriate a new section must be cut and stained (a second section is cut to play safe because time is precious for the patient and for the surgeon to make a decision). The consultant sees the stained tissue section under the microscope and sees the previously suspected tumour. If more than one slide are required due to a lymph node the consultant checks if there are tumour cells due to metastasis. The surgeon is immediately informed to see what to do next. Quality Control The slides are seen by the pathologist as quickly as possible. If the slides are not good then the pathologist orders other sections, example order thinner sections of sections with less stain intensity. More than one section is stained during processing of frozen sections so that if the pathologist orders a new slide, it is already available. Precautions Microtome should be working with a clean sharp blade knife Blade angle should be between 30 to 50 The anti-roll plate should be adjusted to prevent curling of the section. This means that blade edge must have correct height and angle, plate edge should not be damaged and the cabinet temperature must be correct sections. A sable hair brush manipulates section when anti-roll plate is not functioning properly (Bancroft Gamble, 2008, p.100) The blade should have sharp edges to obtain good quality sections (Bancroft et al., 2008, p.100) Optimum temperature according to the type of specimen being cut. Most tissues are cut a temperature maintained at -20oC 5C. Liver, brain, lymph node, spleen and uterine curettings which are sectioned at -10C. Fat and breast with fat are cut at lower temperatures (-25o to 30oC). Tissue must be fresh not dehydrated Cryospraying is not done directly on the section (especially if it is a thyroid section). This causes artifacts (round circles) When there are cutting problems the microtome is defrosted and maintenance is called (Bancroft Gamble, 2008, p.100) Results: Nucleus: Blue Cytoplasm and other eosinophilic structures: Pale pink Advantages of frozen sections Additional samples can be taken for additional samples. This avoids a second surgery (Brender et al., 2011) Quick diagnosis can be made (Brender et al., 2011) Cancerous tissue can be removed at the time of injury. Benign mass do not always need to be removed (Brender et al., 2011) Ensures that the removal of the tissue is that of the intended tissue (Brender et al., 2011) The whole mass and its boundaries are removed (Brender et al., 2011) Scientific research can be performed on the tissue samples (Brender et al., 2011) There is cooperation between patient and pathologist so that the patient can benefit as much as possible (Brender et al., 2011) Multiple regions from a single set of sections can be quantified (McKim et al., 2004) Costs are reduced (McKim et al., 2004) Time is reduced when few sections are required and when sectioning and staining go smooth (McKim et al., 2004) Limitations The time for the result can increase as the number of frozen sections required increase. The patient remains more under surgery. Complex and large specimens take more time. Therefore, sectioning and staining is rapid for intra-operative decision by the surgeon (Peters, 2009, p.14). When compared to permanent paraffin sections, frozen sections contain more artifacts. Sectioning and staining defects including wrinkles, non-smooth incomplete sections (lack of margin control), folds, staining condensations and cellular distortion (Karciolu, 2005, p.121). Poor sampling can cause inappropriate diagnosis (Karciolu, 2005, p.121). Very hard samples (calcified) cannot be cut and sectioned therefore they cannot be rapidly processed. A lower temperature in the cryostat produces a harder block and a higher temperature produces a softer tissue. Lack of consultation between biomedical scientists during processing Diagnostic Application Intraoperative diagnosis The sections are rapidly obtained so surgeon can decide what to do. Other applications include: Non-enzyme histochemistry demonstrates unsaturated lipids example in analysis of lipids of arterial walls, fetal colon, lung and muscle (Liadsky Woolf, 1967; Garbarsch, 1969). Protein-bound lipids removed from the tissue during routine processing of paraffin sections (Bancroft et al., 2008, p.191). Frozen sections can be stained for lipid by Oil Red O (Tracey, Kissling, Gandia Reynolds, 1989). Non-enzyme histochemistry: Frozen section can also be used to demonstrate mucopolysaccharidoses example in bone. These carbohydrate deposits are weak to formalin so there is little preservation of sections (Bancroft Cook, 1994, p.146). They are water soluble so special fixatives or frozen sections are performed (Stocker Dehner, 2001, p.186). Enzyme histochemistry: Muscle biopsies require enzyme methods to produce a definite diagnosis. A temperature of -70oC allows little enzyme loss so the muscle fibers are more preserved than they would be in fixation. Enzyme histochemistry allows a more and detailed localised reaction of the histochemical reaction product (Murray Ewen, 1989). Immunohistochemistry (IHC) and Immunofluorescence (IMF): Antigens are inactivated during fixation but preserved during preparation of frozen sections for IHC and IMF. Infective agents are also destroyed during preservation of the substrate antigens (Bancroft et al., 2008, p.151). Examples of tissue preparation for antigen demonstration are classification of lymphoproliferative disorders (IHC) and skin and renal biopsies (IMF) (Sheibani Winberg, 1987; Bancroft et al., 2008, p.518). Neuropathology: Can be used on central nervous system sections which are then stained with silver stain (Bancroft et al., 2008, p.98). The sections are sent wrapped inside a gauze in saline during surgery and cannot be fixed. Preservation of DNA and mRNA for molecular biology. Usually when fixed with formalin protein links between mRNA and proteins occur if left for more than one day. Alternative Methodologies Slow mohs: It is a method used for skin cancers such as lentigo maligna. After excision is performed the patient is sent home. A permanent paraffin section is made from full cuts of the specimen. In the paraffin block, the epidermal and the deep margins are orientated in the same plane (Rutledge Chlipala, 2004). This helps to spare normal tissue. The patient comes back for a second excision only from those areas that are positive. This goes on until microscopic examination shows tumour clearance (Huang, 2004). Frozen sections are quicker and a decision can be done within minutes so, example, a tumour can be completely removed. Freeze Drying: Sections are quenched and dried. But these sections are allowed to reach temperature before fixation. Therefore, they take more time than frozen sections (Bancroft et al., 2008, p.102). Conclusion Frozen sections allow rapid processing and rapid microscopic examination of a biopsy/ies taken during surgery. Although it has a lot of limitations, it allows intra-operative decision making to the surgeon. Title: Immunohistochemistry Aim Cutting tonsil sections using a microtome for immunohistochemistry Performing antigen retrieval using sodium citrate buffer (pH 6.0) Performing immunohistochemistry on the sections, using the Avidin-Biotin Complex method Introduction Immunohistochemistry (IHC) is an advanced technique used to detect antigens on cellular or tissue constituents by means of antigen-antibody interactions. The site the antibody (marker) binds can be identified by directly labeling the antibody or by using a labeled secondary antibody (Bancroft Gamble, 2008, p. 435). Visualization can be made under light microscopy. IHC is important in diagnosis when a therapeutic decision for neoplasia leads to inconclusive results (Hayat, 2006, p.532). Principle After the sections are cut, using a microtome, the slides are collected on labeled APES coated glass slides. These glass slides enhance adhesion of the section on their surface and prevent the section from getting lost due to high temperatures. The slides must be dried before they are introduced in xylene (to prevent contamination). Antigen retrieval Fixation with neutral buffered formalin causes the formation of methylene bridges that cross-link proteins and cause masking of the antigenic sites. To retrieve antigens, a heat-induced epitope retrieval method with sodium citrate buffer (pH6.0) was performed. The buffer is placed inside a pressure cooker, followed by the slides (inside a metal rack) after the buffer boils. The methylene bridges break and the antigenic sites are exposed allowing binding of antibodies. The addition of diluted hydrogen peroxide to the slides will block enzyme activities of the tissue. Cells such as red blood cells contain endogenous peroxide that is able to react with the developing mixture. By pretreating tissues with hydrogen peroxide will prevent non-specific background staining when adding the enzyme (HRP) (Li, Ziesmer Lazcano-Villareal, 1987). Slides are adhered onto a cover slide and mounted inside a mounting chamber. Normal swine serum is added to block non-specific binding of antibody to the antigen on the tissue. The primary antibody (polyclonal anti-CD3) containing rabbit polyclonal then was left overnight, to bind with the CD3 antigen on the surface of the tissue section. Excess antibody is removed by washing with PBS without suppressing the antigen-antibody interaction. The solution also prevents non-specific binding and maintains a pH 7 environment. The addition of Biotinylated Goat Anti Rabbit (secondary antibody) is directed against the primary antibody. It is left to bind for 1 hour and excess unbound secondary antibody is washed, using PBS. ABC (Avidin-biotin peroxidase complex) is able to create a 3-D that contain a lot of biotinylated horseradish peroxidase molecules (HRP enzyme) that are cross-linked by avidin. Enzyme is located at the antigen site through these complexes, increasing sensitivity (Bratthauer, 1999). Glycoprotein Avidin has four binding sites per molecule (i.e. a high affinity for biotin) and biotin is able to bind with only one binding site with avidin. It is able to bind irreversibly to the secondary antibody. To the DAB solution, hydrogen peroxide is added before it is added on to the sections. DAB/H202 acts a colorimetric substrate solution. HRP catalyzes the hydrogen peroxide and electron transfer from DAB to HRP occurs to yield a brown insoluble product. The color intensity is seen under low power and stopped by phosphate buffered solution. When there is background the reaction is stopped immediately. Counterstaining is performed with haematoxylin since CD3 is a cytoplasmic and a membrane stain. This means that the counterstain will stain the nuclei blue. The sections are dehydrated, cleared and mounted and viewed under the microscope. Materials Required Glassware rinsed in distilled water, single channel pipettes, cover slips, disposable pipettes, slide rack, Pressure Cooker, pH meter, Microtome, Oven set at 60C, Microscope. Reagents and Preparations Citrate buffer was prepared by adding 3.15g of citric acid and 1500mls distilled water Hydrogen peroxide 3mls 30% H2O2 and 300mls of distilled water Normal swine serum (NSS) A dilution of 1/20 was prepared. This required adding 250l of CD3 antibody and 4750 l of antibody diluent to make a total of 5000 l. Primary antibody (rabbit polyclonal to CD3) This required a dilution of 1/350. Since 5 slides were required, (one slide for this case and 3 routine slides and a control) 2 l of antibody and 698 l of antibody diluent were added. Biotinylated Goat Anti Rabbit (BGAR) A dilution of 1/300 was prepared. This required adding 16 l of concentrated BGAR and 4784 l of antibody diluent to make a total of 5000 l. Antibody diluent is made of made of albumin, sodium azide and phosphate buffered saline. Dilute Phosphate buffered Saline was prepared by adding 200mls of concentrated phosphate buffered saline and 1800mls of distilled water. Avidin-biotin peroxidase complex (ABC) solution: This was prepared by adding 50 l of avidin and 50 l of biotin to 2400 l of phosphate buffered saline. 3,3-Diaminobenzidine (DAB) A solution was prepared by adding 1 tablet of 3,3-Diaminobenzidine tetrahydrochloride stored in freezer 1 tablet of Tris-buffered saline 15mls of deionised water After left to dissolve, it was filtered and later 12l of hydrogen peroxide (H2O2) were pipetted (freshly prepared before use) Commercially prepared Haematoxylin, xylene, xylene alcohol, absolute alcohol, 75% alcohol, 50% alcohol, DPX mounting Medium Procedure (including precautions) Day 1 After the request form for immunohistochemistry was registered and the respective block was retrieved, the block was placed with the section touching the ice. One slide was marked with the patient number, date and test required, in this case CD3. Another slide was used as a control. (In total there were 5 slides for CD3 since one slide was used for this case, another was the control, and the other three were other slides from 3 different patients) The block was placed in the block holder of the microtome and the appropriate angle was found (so that there is one whole cut). The ribbon of wax containing the sections was then placed inside a water bath at 37oC and a section was collected on every slide. The slides were dried inside an oven at 60oC. Water would have contaminated the xylene during deparaffinisation. The measuring probe of the pH reader was inserted inside a liquid of pH 7 that read pH7.08. This served as a control, therefore it was working properly. The citrate solution was prepared and sodium hydroxide pellets were added. The measuring probe was immersed and the rise in pH was noted: pH6.10 The citrate buffer solution was then placed inside a pressure cooker. At the same time the dried slides were depraffinised in xylene for 2 minutes. The slides were then left for 2 minutes in 2 other xylene baths. The slides were then placed in xylene alcohol for 2 minutes, to start the hydration process. Hydration was then continued in a bath containing 75% alcohol, and a bath containing 30% alcohol, each for 2 minutes. The slides were then immersed in water. The pressure cooker, at the mean time, was powered on mark 4 until there was full pressure. It was left 3 minutes at full pressure and then adjusted left on mark 1. The slides were incubated with the buffer, inside the pressure cooker for 10 minutes for antigen retrieval. The pressure cooker was then placed inside a sink, the pressure release valve was activated and cold water was run outside the lid and then inside, for 10 minutes. The slides were treated with 12l hydrogen peroxide for 10 minutes, to block enzyme activity of the tissue, and washed. The slides were than attached to chamber slides under water (with the side of the section touching the chamber slide). They were tightly held and placed inside a mounting chamber. The slides together with chamber slides were placed in a straight position so that fluid reached the whole section and not part of it. This would have stained only one part of the section. The control was first placed and followed by the slides to be tested (CD3). The slides were washed with PBS. If it drained quickly, the slides and the cover slides were not attached appropriately. 100 l of NSS were pipetted between the chamber slide and the slide with the section (tightly adhered together). It was left for 12 minutes so the next reagent does not dilute. Without rinsing, 100 l of the primary antibody were pipetted and left overnight refrigerated at 4oC. Day 2 The mounting chambers were removed from the refrigerator and all slides adhered to chamber slide were washed with PBS. This covered the whole section. The PBS was allowed to drain. 100 l of BGAR were then pipetted and left incubated for one hour on the working bench, closed with the mounting chamber lid Washing of sections was then performed using PBS. 100 l of ABC reagent were pipetted and incubated for 1 hour on the working bench, closed with the mounting chamber lid The sections were washed with PBS The slides were removed from the cover slides under water to prevent damage of the section. The slides were placed back to back on slide racks so that one section does not touch the other. They were placed in a bath containing PBS. 12 l of H202 were added to the DAB solution. The back and the sides of the slides were dried with a clean tissue and the section was covered with DAB/ H202 working solution. The slides were immediately observed under the microscope at lower power to assess the development of the colour. If the brown colour in the control is satisfactory it is stopped by dipping the slide in PBS. The rest of the slides for that same test are also stopped in PBS. If there is slight background in the section the slide is immediately stopped in PBS. The slides were rinsed in water. The slides were then counterstained in haematoxylin for 1 minute and placed in water bath. They were then placed in warm water. The slides were dipped 6-7 times in alcohol and rinsed with water. Bluing was performed in tap water for a few seconds The sections were viewed under the microscope to check that the nuclei were blue Dehydration was then started by placing the slides (2 minutes each) in 30% alcohol, 75% alcohol, and in 2 baths of absolute alcohol for 2 minutes each. The slides were cleared in xylene alcohol for 15 seconds followed in 2 baths of xylene (2 minutes each). This helps during mounting since DPX mountant is xylene based. The clearing agent is necessary because dehydrating agents are not miscible with impregnation medium. It acts an intermediate chemical. This removes alcohol. The high refractive index makes the tissue clear so that this is equal to that of the DPX mounting medium. The slides were mounted in DPX mounting medium and allowed to dry. Quality Control In immunohistochemistry a different control is used for every different stain test required. In this case, a tonsil section was used as a positive control for all CD3 tests required. Results: Cytoplasm and membrane Brown Nuclei Blue Interpretation of Results Control (Tonsil): The cytoplasm and the membrane stained brown, the nuclei stained blue. Therefore the control worked and showed diffuse positivity. Section (Patient tonsil tissue section): The cytoplasm and the membrane stained brown and the nuclei stained blue. But staining was not diffuse because there were areas in the cytoplasm and membrane that did not stain. The problem encountered could be that the DAB was not given a lot of chance to stain so the stain was very weak and areas did not stain. The stain was not very crispy, this is mostly possible because the section was big and not of very good quality. There was little staining in the mantle zone, and weak or very weak staining in the germinal center. The paracortical and interfollicular areas stained well. The capsule did not stain as expected. The CD3 antigen CD3 antigen is part of a polypeptide chain complex found on the surface membrane of T-lymphocytes, associated with the T-cell receptor (TCR). CD3 , CD3, CD3 and CD3 are all CD3 molecules forming part of the TCR and are stained with CD3. It is involved in signal transduction (Ioachim Medeiros, 2008, p.53). CD3 stains the cytoplasm and/or cell membrane (Law et al., 2002). Cytoplasmic positivity is in the early and late stage of development of thymocytes and membrane positivity is shown in T-cell lymphomas (Wang et al., 2009). CD3 stain can stain T-lymphocytes mostly in peri-follicular areas and to a lesser extent in germinal centers, mantle zones, stratified squamous epithelium and loose connective tissue (Harris, Meghji Speight, 1997; Ioachim et al., 2008). Diagnostic Application Besides normal tissue, CD3 stain is used to identify T cell neoplasms example: T-Cell Lymphomas and/or Natural Killer Cell lymphomas: T cells express CD3 so membranous staining is specific for T-cell lymphoma. Natural killer cells are able to show epsilon expression on their cytoplasm (Chu Weiss, 2009, p.486). Mycosis fungoides: A major subtype of cutaneous T-cell lymphoma (Pimpinelli et al., 2005). T cells are dominant in the dermis and epidermis. Infiltration of T-helper memory cells are a characteristic of mycosis fungoides (Cerroni, Gatter, Kerl Helmut, 2009, p.24). Alternative Methodologies Catalysed signal amplification Used to visualise rare or masked antigens that show weak Immunohistochemical signals. It is time consuming, and staining is complex and poorly reproducible (Hashizume, Hatanaka, Kamihara Tani, 2001). Peroxidase anti-peroxidase method (PAP) This is a 3 layer method considered sensitive (not as much as the ABC method) and eliminates non-specific binding. On the other hand, it has some disadvantages: primary antibody and PAP are raised from the same species, and it is expensive to obtain readymade complexes from different species (Bratthauer, 1995). ImmPress method: After addition of the primary antibody, there is addition of ImmPress reagent followed by peroxidase substrate. The result is fast due to the reduced incubation steps. It is very sensitive and produces discrete localisation of the antigen. Background staining is reduced because it pre-diluted (Vector Laboratories, 2010). Conclusion Immunohistochemistry is a very important technique in histology that demonstrates the localisation of the antigen on the surface of tissues. It has the advantages of efficiency (not time consuming), high sensitivity, stability and versatility and low background staining due to high pre-dilution. Although a positive result is obtained when an antibody is targeted towards the antigen, IHC is unable to define distinct cell populations. This can be achieved by flow cytometry. List of Hazardous Reagents Equipment Name: Year of Entry: Task: Date: Substance Associated Risk Actual risk to user Safety measures Action taken in case of incident Methylated Spirit Flammable Toxic Fire Eye Splash Ingestion Store in flammable cupboard Use PPE Eyes wash with water for 10min Skin wash with water Ingestion wash mouth with water, seek medical attention Chloroform Toxic Eye irritation Skin Irritation Ingestion Inhalation Store in safety storage cabinet away from heat and sources of ignition Eye wash station in the laboratory Use PPE Seek medical attention Eyes wash with water for 15 minutes Skin Wash with plenty of water and soap. Cover irritated skin with an emollient Ingestion Do not induce vomit, loosen tight clothing Inhalation rest in a well ventilated area 3,3-Diaminobenzidine tetrahydrochloride (DAB) Tablet (DAB) Tablet (continued) Flammable at high temperature Toxic Fire (at high temperature) Eye irritation Skin Irritation Ingestion Lung Irritant Store at -20oC in the dark Use PPE Seek medical attention Eyes wash with plenty of water for 15 minutes Skin Wash with plenty of water and cover irritated skin with an emollient Ingestion Wash out mouth with water Inhalation rest in a well ventilated area DPX Mountant Flammable Toxic Fire Eye Splash Skin Irritation Ingestion Inhalation Store in tightly closed labeled containers, store in a well ventilated area away from ignition Use PPE Seek medical attention Eyes wash with water for 15min Skin wash with water Ingestion wash mouth with water Inhalation move to fresh air away from exposure Ethanol Flammable Toxic Fire Eye Irritant Skin irritant Ingestion Inhalation Store in a segregated and approved area. Keep in a non-ventilated area tightly closed and not above 23oC Use PPE Seek Medical Attention Eyes flush with water for 15 min Skin Wash with plenty of water and cover irritated skin with an emollient Ingestion Do not induce vomit, loosen tight clothing Inhalation move to fresh air away from exposure Eosin Yellow Eosin Yellow (continued) Toxic Carcinogen Eye Irritant Mild skin irritant Digestive Tract irritation Inhalation (Poisonous) Store in a cool, dry, well-ventilated area away from incompatible substances. Use PPE Seek Medical Attention Eyes flush with water for 15 min Skin Wash with plenty of water and soap. Remove contaminated clothing. Ingestion Do not induce vomit, loosen tight clothing and give water Inhalation move to fresh air away from exposure Formalin buffered Solution Toxic Flammable when liquid vaporizes in air Corrosive Fire Eye Splash Skin Irritation Ingestion Inhalation Safety Storage cabinet room store in a well ventilated area away from ignition Use PPE Seek medical attention Eyes wash with water for 15min, Skin wash with water Ingestion wash mouth with water, seek medical attention Inhalation move to fresh air away from exposure Freezer Spray Flammable (only when heated) Toxic Explosive (only when heated) Eye Splash Skin irritation Ingestion Inhalation Store in a well cool ventilated area away from light Use PPE Seek medical attention Eye wash with water for 15min Skin Wash with cold water and soap, remove contaminated clothing Ingestion Do not induce vomit, loosen tight clothing Inhalation: move to fresh air away from exposure Harris Haematoxylin Toxic Eye Irritation Skin irritant when in long contact GIT irritant Inhalation Store in a cool place and out of direct sunlight and heat. Use PPE Seek medical attention Eye wash with water for 15min Skin Wash with cold water and soap, remove contaminated clothing Ingestion Do not induce vomit, loosen tight clothing Inhalation: move to fresh air away from exposure Hydrochloric acid (HCl) Flammable (negligible) Toxic Corrosive Fire (negligible) Eye splash Skin irritation Inhalation Store in a dry cool area Use PPE Seek medical attention Eye wash with water for 15min Skin Wash contact areas with soap and water, wash contaminated clothing Inhalation: move to fresh air away from exposure Paraffin wax/Paraffin wax fumes Combustible at high temperatures Toxic (Acute) Fire at high Temperatures Eye irritant Skin irritant Inhalation Ingestion Store away from heat and ignition. Store in a dry cool place. Seek medical attention Eyes flush with plenty of water for 15 minutes Skin Wash with plenty of water and soap. Cover irritated skin with an emollient Ingestion Do not induce vomit, loosen tight clothing Inhalation rest in a well ventilated area Xylene Mod. Flammable Toxic Fire Eye irritation Skin Irritation Ingestion Inhalation Safety Storage cabinet room, store in a well ventilated area away from ignition Use PPE Seek medical attention Eyes flush with water for 15 minutes Skin Wash contact areas with soap and water, wash contaminated clothing Ingestion Do not induce vomit, loosen tight clothing Inhalation -move to fresh air away from exposure Other Hazards Blood borne pathogens present in fresh human tissue Pathogenic in frozen sections since other specimens are fixed ex: HIV, HBV or TB cannot be excluded Eye contact, Mouth contact or any other mucous membrane. Cut by glass slide Use PPE Prevent spraying with cryospray preventing splashing of organisms Use Class III Safety Cabinet example for lymph nodes In case of known exposure take vaccines Sharp Objects Skin cuts Can contain infectious agents such as HIV, TB, HPV Sharps such as glass slides, scalpel, knives (for cut up), blades can penetrate the skin. Use PPE Go to infectious control unit for immunization for tetanus titer Burns caused by improper handling of hot items Chemical and Heat burns Body contact Acidic or alkaline chemicals and heat can burn the skin increasing risk for infection Use PPE Handle with care and away from the body Use appropriate cream In case of fire use fire extinguishers and fire blankets as discussed in the following fire section. Seek medical. Electrical injury (burns, shock, or death) Electrocution Flammable Death Electric shock user, equipment and user can catch fire. In both cases the user can die Inspect wires and replaced hazardous cords Use fused adaptors, prevent long extension wires Remove water from near wire Use fire equipment (as discussed in the following section), Do not touch electrocuted users or machinery Call medical help Fire Flammable Body burns Handle with care and away from the body For small burns use appropriate cream, Remove burning clothing if possible, Use fire extinguishers In histology CO2 type B extinguisher is good for flammable liquids and safe for electrical equipment. A Foam spray type A extinguisher is good for textiles, paper and wood. A fire blanket can be used or even a fire hose. A map showing the fire exits is available in the lab in case of fire alarms and uncontrollable fire in the laboratory Chemical Spillages Toxic or non-toxic Flammable Possibly corrosive Fire Eye contact Skin contact Inhalation Ingestion Use PPE Place in a safe place whilst handling chemicals Store flammable chemicals away from source of ignition Seek Medical Attention Eyes flush with water for 15 minutes Skin Use cellulose pads and alcohol on the area affected. Inhalation move to fresh air away from exposure Ingestion Do not induce vomit, loosen tight clothing and seek medical attention Decontaminate work surfaces FOR OFFICIAL USE ONLY Authorised: Date: