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CP H58 Histology Competency Manual

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Histology competency manual

20th September 2011Histology competency manual R1

Histology competency manualJuly 18, 2011

Revision 1 | David Muskett

East Lancashire Hospitals NHS TrustCP/H58 - Histology competency manual

Histology Competence ManualWhat is cellular pathologyCellular Pathology is the study of DefinitionsSpecimenOften used interchangeably with sample, describes the material removed from the body and sent to the laboratory.

BiopsyOften used to describe a small piece of tissue removed, often for initial diagnosis

FixationThe process of tissue preservation with chemicals. Fixation acts to prevent the natural processes of cell death, autolysis and putrefaction.

AutolysisThe process of self digestion within a cell where the enzymes i.e. auto self, lysis splitting.

PutrefactionThe process of cellular degradation due to micro-organism

ProcessingThe removal of water from tissue and replacing it with another substance such as paraffin wax but sometimes tissues are also processed to other media (e.g. resin).

Request formThe document supplied with tissue samples giving details of the patient and of the specimen sent. The request form should also include clinical information.

ReagentsThe chemicals and solutions used in the histology laboratory

Haematoxylin & EosinThe standard staining method used in histology. It is able to show good nuclear and cytoplasm detail and gives general tissue architecture.

Special stainsA range of stains used to identify specific tissue elements. Usually a single element of tissue is investigated with special stains such as fibrin or glycogen, acid mucins or elastin.

HistochemistryThe process of staining cells using the enzymes within the cells to facilitate the investigation.

ImmunocytochemistryA range of antigen antibody related tests where antibodies are raised against specific tissue elements, which may be nuclear, cytoplasmic or membrane bound. The antibody reactions are identified by precipitating a coloured product via an enzyme reaction at the site of the antibody antigen reaction.

StainingHuman tissues do not look coloured under the microscope by themselves. Tissue sections require treating with chemicals which give colour. Some chemicals are coloured and others have colour when they react with the tissue.

SectionA thin piece of tissue. This is usually one or two cells thick, approximately 4m thick, so that the tissue is easy to analyse microscopically. Occasionally there is requirement to examine material in thinner sections e.g. renal biopsies and material for electron microscopy or as thicker sections e.g. neuropathology samples.

Frozen sectionA section cut from a piece of tissue which has not been processed and usually not fixed but has been snap frozen. The tissue freezes and goes firm. Once hard the tissue may be easily cut with a cryostat. Frozen sections are sometimes required for rapid real time diagnosis of patients undergoing surgery or for determining tumour margin clearance in cases of Mohs micrographic surgery.

Specimen potThe container in which a specimen is received. It should be labelled with the details of the patient demographics plus the name of the clinician performing the biopsy and the sender clinic details. In addition there should be information on the type of specimen taken.

ResectionA large tissue sample removed for analysis. It may be a kidney, a breast, and a length of bowel or a lobe of liver.

Incisional biopsyA skin biopsy showing some normal tissue and some of the lesion. It is not a complete removal due to the size of the lesion or its anatomical position.

Excisional biopsyA skin biopsy with the lesion completely removed There should be a margin of normal tissue removed as well.

MicrotomeA machine which cuts sections. The basic operation is a knife and a specimen holding mechanism. The specimen holding mechanism advances a set distance (usually 4-5 m) as the material moves up and down against the knife.

CryostatA microtome in a refrigerated unit. The cutting temperature is commonly -20oC.

BlocksThis word is used interchangeably with pieces of tissue used in histological analysis and cassette They are usually fixed and paraffin embedded. The blocks themselves usually include a plastic cassette containing the specimen number. The pieces may vary greatly in size from tiny diagnostic biopsies (a few millimetres across) to up to 2m in length. Larger pieces may be placed in mega cassettes or mega block.

Mega blocksLarger plastic cassettes containing tissue. They are big enough to contain a full cross section of bowel or a cross section of an enlarged prostate.

Tissue processorA machine which processes tissue. Tissues are moved through a variety of chemical reagents to dehydrate them and make them suitable for placing in the embedding media, which is usually wax.

Embedding mediaThe material which tissue is held within for cutting sections. Embedding media may be wax or may be resin. The selection of the embedding media is dependent on the hardness of the tissues, harder tissues need a harder embedding media , and the thinner the sections need to be cut then the harder the media needs to be.

LesionAn area of suspected pathology, that may be well or ill defined, pigmented or not, raised, smooth or textured.

Specimen receptionIt is essential that the material for investigation is received in the laboratory in a suitable condition for testing. For the laboratory to handle specimens correctly laboratories need quite a lot of information about what the sample is, which patient it is from, who is requesting the test and who wants the report.

The specimen request form should contain information about who m the results are to be sent to

The minimum specimen information should be Full surname Full forename One other identifier e.g. Date of birth, NHS number, Hospital number. What the sample is

The request form may also ask for all of these items (if appropriate) in addition to the minimum data. The laboratory will provide instructions to users on how the specimen should be taken and transferred to the laboratory. Figure 3.3 shows a typical specimen reception area in a busy histology laboratory.

When specimens are received into the laboratory it is important to check the details on the request form and the specimen pot. If there are any discrepancies for example in the name and date of birth of the patient on the pot and on the request form then the specimen should be returned to the person requesting the specimen for correction. A record of this error patterns/logs should be made so that any patterns of mistake can be followed up.

What patient information is required to accept a specimen in a histology laboratory? Clinical informationWhen samples arrive in the laboratory, the type of specimen and what the clinical history is dictates the way samples are handled. If some vital clinical information is not supplied then it is possible that diagnosis can be compromised, incomplete or incorrect.

The clinical history matters. Good clinical history helps get a good report

For example liver biopsies can be handled completely differently based on the clinical information provided. If patient A has had a previous colonic cancer and had lymph node involvement with tumour presents with decreased liver function tests. Immunocytochemistry will be required to investigate the presence of metastatic cancer as this is the most likely differential diagnosis.. Patient B who had a history of alcoholism and presents with decreasing liver function tests. Special stains will be required to investigate primary liver function. This is summarised below.

Figure 1 - Figure showing the most likely lab tests required based upon the clinical details provided for a liver biopsySubsequent tests within the histology laboratory follow depending on the information seen on the first slides viewed

Clinical correlationThe liver has many differed functions and a liver biopsy is a very valuable diagnostic tool for assessing a range of disease states. In the histology laboratory about 6 extra stains are performed. This gives lots of extra information

Full clinical information helps the laboratory to make a full diagnosis

A single patient may receive many biopsies. Therefore, it is important for the reporting pathologist to be aware of the previous clinical history. Often a small diagnostic biopsy - sometimes an endoscopic sample 2mm in diameter, or a needle core of tissue 2mm wide by 10mm long, will be taken to assess the nature of the patient cancer. This diagnostic sample will lead to a diagnosis and the planning of the patient treatment. The treatment plan itself may result in further surgical procedures, known as a therapeutic biopsy or resection.The storing of patient results enables any new pathology to be correlated with that of previous samples. This is of particular importance when monitoring patients with recurrent cancers.

Endoscopic biopsies are biopsies obtained by using a flexible tube (endoscope) with a grip function on the end. The endoscope is inserted into the mouth or anus and allows samples to be taken from the inside of the GI tract without the need for invasive surgeryTimelinessThe processing of samples, and return of results, can be affected by a number of time factors. For example, some laboratories can serve multiple hospitals, such that the sheer workload may place a limit on how quickly a sample can be processed. Also, while most laboratories provide a collection service for samples from GP surgeries within their service provider areas, the size of collection areas may mean that collections may only happen daily at best.Most histology samples will not be available for reporting until the next working day from receipt at the earliest. Any specimen requiring more urgent handling will need to be highlighted and discussed with the laboratory.

Clinical correlationPatients with suspected cancer in the UK can expect to receive their results back within 14 days of the first GP appointment. Included in this time is the hospital appointment and the laboratory diagnosis. Rapid results means the patients can be treated quickly which helps with their clinical management.

Histology takes timeResults are only available same day when specially requested

Urgent samplesUrgent samples are received where the patient needs urgent clinical intervention based upon the histology result. Samples may be urgent because a result is needed while the patient is on the operating table, as in the case of patients requiring rapid frozen sections, or they may have a rapidly declining medical condition which needs clinical intervention. Patients who present with suspected cancer require their results back within 2 weeks so treatment can be planned.

Urgent samples fall into two groups1 Frozen sections2 Urgent fixed specimensFrozen sectionsFrozen sections are a way of reporting histology specimens rapidly. The specimens are frozen to make them hard and then thin sections are cut, hence the name.Urgent fixed samplesHospitals deal with many patients. Some can be treated in a measured manner with planned surgery and treatment. Other patients present with symptoms only when they are very ill. Patients who present with late symptoms are often difficult to diagnose and therefore urgent histology samples are removed from the patient. They may be endoscopic biopsies or needle core specimens. Urgent fixed samples are lesions or nodules which can be excised as a discrete entity but clinicians are anxious to plan future surgery or treatment.

A 63 year old male patient presents at his GP with a slightly itchy darkly pigmented lesion with an irregular border. In recent months the lesion has grown significantly. See fig 3.8 The GP suspects a malignant melanoma. A rapid appointment is made at the local hospital dermatology department and the patient is biopsied 3 days later. The excised skin lesion is placed in formalin and sent to the lab for urgent reporting. After processing, 3 levels are cut on the 3 blocks and the reporting pathologist suspects malignant melanoma and immunocytochemistry is performed to confirm this. The case is reported and the results sent back to the consultant dermatologist and the GP. The who process from biopsy to report took 8 days, hence the process is urgent.Specimens need a request form with information about the patient and the tissue sample on it

What clinical circumstances might indicate an urgent specimen?Competency assessmentsSpecimen receptionYou will be able to: explain the purpose of the procedure accurately maintain records demonstrate completion of procedure

Mostly needed promptingSatisfactoryGood

1. State the purpose of the specimen reception procedure

2. State the health and safety risks of formalin

3. State the use of Michels transport media

4. Describe the specimen acceptance / rejection policy

5. Describe how specimens are allocated to consultants

6. Describe the purpose and limitations of frozen section diagnosis of histology specimens

7. Why are specimens sometimes sent to referral centres

8. What constitutes a high risk sample? How should they be handled?

9. Why are specimens from patients with a similar sounding name kept separate?

10. Why are specimens of the same type kept separate?

11. Why are gastro-intestinal specimens labelled in anatomical order?

12. Why are records of rejected specimens kept?

TaskDate AssessedAssessorAssessors signatureCandidatesSignature

Can clear up a small formalin spill

Can clear up a large formalin spills

Can use the respirator

Can explain / demonstrate how to date and time stamp specimens

Demonstrate the procedure for dealing with empty specimen pots

Demonstrate how specimens are labelled up

Demonstrate how private specimens are divided up amongst consultants

Demonstrate how specimens are labelled up

Can describe how red cross samples are handled

Demonstrate how specimens are prioritised for frozen sections

Demonstrate how specimens are sent to referral centres

TelepathTaskDate AssessedAssessorAssessors signatureCandidatesSignature

Can book a specimen in on telepath using RXR number match

Can HPROE a specimen to ensure block and slide details match

IBMS Portfolio cross-reference2a.1, 2a.2, 2a.3Specimen dissection roomFixationA majority of routine specimens received in the laboratory are suitable for formalin fixation, but not all of the subsequent tests required are compatible with fixation. Therefore before the sample is taken it is important to know what tests will be required and arrangements made to receive the samples either fresh or in Michels transport media in the case of skin samples for immunofluorescence. When cells are in the body they receive oxygen, food and have waste products removed. When they are removed from the body they decline and die; due to the absence of oxygen, absence of food and the build up of waste. Cells start to change, the DNA, proteins, sugars and fats all start to deteriorate initially through the build up of waste products and the uncontrolled release of proteases. This process is known as autolysis. Autolysed cells stained with haematoxylin and eosin show enhanced cytoplasmic staining or eosinophilia (i.e. more pink) and a reduced nuclei staining (less purple) this is due to the breakdown of RNA molecules in the cytoplasm and the breakdown of DNA molecules in the nuclei. Later bacterial or fungal contamination can cause further destruction of cells and tissues. This process is known as putrefaction. If the shape and structure of these cells are allowed to deteriorate then diagnostic analysis becomes impossible. The first part of the histology process is therefore making sure the cells do not deteriorate. Prevention of cellular deterioration is done by one of two processes - fixing or freezing. The nature of preservation in histology is to keep cells in as life-like state as possible. For example, onions are pickled in vinegar to preserve them and keep them edible. At a cellular level the process of autolysis is seen as karryorhexis and apoptosis. The freezing of histology samples occurs only in the minority of cases and will be due to the case being required for tests which are incompatible with the chemicals used in fixation. Examples of tissues which should be frozen on receipt are muscle biopsies, specimens for immunofluorescence and suspected rare tumours were molecular studies may be of interest.

Autolysis is the process of cellular degradation by the release of intracellular enzymes Eosinophilia is enhanced staining with eosin, sections will look much more pink than usual Michels transport media is a buffered aqueous salt solution used for transferring biopsies for immunofluorescence to the laboratory Putrefaction is decomposition of cells by bacterial or fungi. Karryorhexis - is the destructive fragmentation of thenucleusof a dyingcellwhereby its chromatin is distributed irregularly throughout the cytoplasm. Apoptosis is the process of programmed cell death. The nuclei is seen to reduce in size and stain much more intensely with attachment, cell shrinkage,nuclearfragmentation,chromatin condensation, and chromosomalDNAfragmentation. Pyknosis - Fixation - is the process of chemically preserving cells

Admiral Nelson was placed in a barrel of rum after being shot dead on HMS Victory. The artist Damian Hurst has placed sheep and sharks in formalin to preserve them. In histology formalin is also used as the main preservative of cells.

Fixation aims to preserve cells and tissue constituents in as close to a life-like state as possible. Essentially it is concerned with the stabilization of proteins within the tissue and arresting the effects of autolysis and bacterial decomposition. However, cell preservation is only part of the fixation effect. Fixatives impart additional benefits for the histologist as they allow the tissues to undergo further preparative procedures without change and make the cellular components more easily colourable by dyes. It is important to realise that the appearance of the tissue after fixation is artefactual. The stabilization of proteins is necessary to prevent their diffusion during subsequent processing but this also changes the structure and appearance of these proteins.

Fixation aims to preserve cells and tissue constituents in as close to a life-like state as possible

Information is available when the nucleus is examined. The size shape, texture and staining properties of the nucleus alter in differing pathological conditions. .

Not all specimens received in the laboratory are suitable for fixation. This is because the subsequent tests required on some specimens are not compatible with fixation. Before the sample is taken it is important to know what tests will be required. Having said this, the majority of specimens received in the lab are received in fixative which is ideal for the vast majority of routine samples. The others are most probably received either fresh or in Michels transport media. Michels transport media is not a fixative but a solution of salts plus an antibacterial agent, which acts to sustain biopsies in an osmotically balanced solution until transported to the laboratory for treatment can be performed.

Clinical correlationMuch of the key clinical information is linked to the size and shape and staining properties of nuclei. Good fixation is critical for good preservation of nuclei.

Principles of fixationThere are a number of features that make a fixative ideal. They are:-Table 3.1 - Features of an ideal fixativeFactorReason

Kill cells quickly and evenlyThis stops the build up of harmful metabolites which disrupt the cellular appearance

Penetrates tissues quickly and evenlyThe solution diffuses through the tissue quickly

Prevents autolysis and putrefactionStops cellular functions immediately preventing self destruction and prevents microbial damage

Does not shrink or swell the cellMaintains the normal shape and size of the cell in relation to the size in situ. This allows for meaningful diagnostic and prognostic measurements.

Prepares the tissue for later treatmentsAllows the use of the tissue for a large number of further tests. This includes making the tissue harder

Prevent desiccation and drying of tissueKeeps the cells hydrated

Safe to useDoes not cause harm to the operator when using the fixative. Does not require expensive disposal regimes.

Reasonably pricedAs large quantities of fixative are used in the routine laboratory. The cost should not be prohibitive

Tolerant in useSpecimens are likely to be placed into fixative by non histology staff in many different ways. A tolerant fixative allows various permutations of usage not to prevent

No fixative is ideal for all circumstances

Often salt or buffer is included. The salts are added to adjust the osmolarity of the solution. Ideally the fixative solution should have the same salt levels as the cells being fixed. This ensures that cells neither shrink nor swell. Figure 3.6 shows how cells alter in solutions of differing tonicity.

Mechanisms of fixationFixation is a chemical process where the constituent molecules within the cells can be manipulated in several ways:-

Cross linking (or additive) fixatives act by binding amino acids within proteins to adjust their 3D structure, preventing autolysis and putrefaction. Formalin fixation is not suitable when handling specimens for enzyme histochemistry since the cross linking acts to denature many enzymes.

Precipitating or (non additive) fixatives act by removing water from the cellular matrix, this acts to disrupt the 3D (tertiary) protein structure and therefore, precipitating the protein..Table 3 Table showing the various types of fixative in common use and their mode of action.Cross linking PrecipitatingCompound

GlutaraldehydeFormaldehydeAlcoholAcetonePicric acid

The aim of fixation is to prevent the decomposition of cells by intracellular processes and external action of bacteria, preserving cells in a balanced isotonic solution.

The use of fixation reagents results in significant changes to the tissue structures and components. These changes are beneficial in terms of tissue preservation and arresting the decomposition effects described previously. However it is important to recognise that some tissue components are extremely labile and so would be readily inactivated by the use of these chemicals. As such it is important to know what investigations may be required for individual samples so that the appropriate processing regimes can be employed. Fixation is a reversible process and much effort is taken to undo the effects of fixation e.g. in immunocytochemistry, during antigen retrieval, you will read more about this in chapter 5.

Labile able to under go change easily i.e. unstable

Well fixed cells have good nuclear detail they are neither swollen nor shrunk, the integrity of the general architecture is not disturbed and cellular proteins are well preserved for future tests.Poorly fixed cells have poor nuclear detail; they may be swollen or shrunk. The general architecture of the piece of tissue may be distorted and cellular proteins may be lost. Tissue which is fatty is very vulnerable to poor fixation as the aqueous reagents do not penetrate the tissue well.Poor fixation can limit the possibility of a definitive diagnosis

Tissue fixation is usually achieved by means of chemicals but the same or similar effects can be achieved by the use of heat. Heat is used to precipitate proteins and to prevent degradation. When heating care must be taken not to overheat or damage the tissue. Microwave ovens can be controlled to heat at a steady temperature, neither too hot nor too cold. Heating also allows fixation to take place at a much faster rate, reducing the fixation time from hours to minutes. Heat can also be used in combination with chemical fixatives. More of this later in the chapter when we talk about automated processors.

Factors affecting fixationFixation is a chemical process and is affected by environmental conditions.

Table 3.4 Factors affecting the rate of fixationTemperatureThe hotter the fixative media the quicker the fixation. Microwave processors utilise the heating of fixatives to allow quick processing schedules.

Size of specimens and penetration of fixative Large specimens take longer to fix than small specimens. The penetration rate is the rate at which the chemicals permeate the tissue. This may be only a four or five millimetres a day, consequently large resections need slicing open before the processes of autolysis destroys the detail within the tissue.

Changes of volumeThe larger the volume to specimen the more rapidly fixation will take place. It is recommended that there is 20x as much fixative as specimen. Often this is not possible, due to the resection being large.

pH and buffersThe addition of buffers does slow the fixation process slightly, yet it does ensure that the chemicals are at the correct pH. Acidic formalin fixatives react with tissues and can create pigments.

OsmolarityFixatives should be the same osmolarity as the cells they are fixing. This will help to prevent shrinking or swelling.

Concentration of fixativesThe stronger the fixative the more rapidly fixation takes place i.e. the more active ingredients for the fixation reaction the more rapidly this can take place.

Duration of fixativesThe longer tissues are in fixative the more complete the fixation will be.

Table 3.4 near hereKey fixativesThere is not a single fixative that is suitable for all preparations. Many different fixatives exist although the number in routine use is now relatively small.The most commonly used fixative in the UK routine laboratory is formalin.

It is important that laboratories use similar fixation regimes as this allows laboratories to review each others cases more easily and to compare methodologies. Bouins solution is occasionally used for testicular biopsies due to enhanced nuclear detail.

FormalinFormalin is cheap, easy to make, keeps well on the shelf and is easily transported. Formalin is a solution of formaldehyde gas contained within water. Formalin is an aqueous acidic solution that contains a concentration of formaldehyde gas dissolved within it. Acid formalin causes formalin pigment, a brown residue seen in red blood cells.. When the pH is neutralised formalin pigment is not seen, hence in most circumstances buffering agents are added. When purchased commercially within the UK it is usually supplied at a concentration of 37-40% formaldehyde. Traditionally this solution has been considered to be 100% formalin. This solution is usually diluted 1/10 to give a formaldehyde concentration of 4%. This 10% formalin solution is the most commonly used fixative in routine laboratories.

Formalin has a number of health and safety considerations which impinge on the running of the laboratory.

The fixation reaction is reversible and is exploited in immunocytochemistry in antigen retrieval.Methylene bridges are -CH2- groups which link proteins together

Health and safety - FormalinColourless liquidCauses burnsVery toxic by inhalation, ingestion and through skin absorptionReadily absorbed through the skinPossible human carcinogenMay cause allergic reactionsCauses watery eyes at over 20ppmNon formalin fixatives (Practical aspects)Despite its tolerance and general all round suitability, formalin is not the best fixative in all situations. Exceptions to the use of formalin as the initial choice of fixative are:Specimens requiring electron microscopy here the use of glutaraldehyde (despite its inherent health& safety issues) is still the agent of choice.GlutaraldehydeGlutaraldehyde provides excellent ultrastructural preservation and is the fixative of choice for electron microscopy. It fixes tissue rapidly but does not penetrate tissue deeply making it unsuitable for large samples. Specimens for electron microscopy are often cut up into 2mm cubes which allows for fixation throughout the whole tissue. Glutaraldehyde is a respiratory sensitiser and needs to be handled with care.

Ultrastructure refers to the preservation of the organelles within the cell i.e. mitochondria, golgi complex

Respiratory sensitiser is a chemical which irritates the lungs and may cause asthmaBouin'sBouin's solution is a mixture of picric acid, alcohol, formaldehyde. This yellow compound fixative is popular for the fixation of testicular biopsies, due to the improved nuclear chromatin appearance of the spermatogenic cells produced with this solution.

Others used even more rarely include Zenkers fixative, Industrial methylated spirits, methanol, acetone, formaldehyde vapour and commercial preparations.Microwave fixationThe stabilization of proteins can also be achieved by the use of microwave irradiation. This has the advantages of significantly improving the speed at which fixation can be completed and the absence of any noxious chemical fixing chemicals.An alternative approach is to supplement the usual chemical fixative with microwaves to speed up the fixation process. There is currently great interest in developing microwave based automated tissue fixation and processing machines. Specimens not for fixationNot all specimens to be investigated require fixation. Some specimens need to be handled more quickly than fixation can take place i.e. in urgent cases. Some tissue elements react with the fixatives in a way that is unsuitable for diagnosis, such as immunofluorescent samples and some cells are required to be kept alive i.e. cytogenetic samples.

CytogeneticsCytogenetics is the study of the structure of chromosomal abnormalities by analysing chromosome numbers. Tissue samples for cytogenetic analysis must be transferred in cytogenetic culture media directly to the cytogenetics laboratory.

Cytogenetics is a branch of laboratory science which looks at chromosome numbers. Cytogenetics is of particular interest in fetal and perinatal samples. A count of the chromosomes can aid the pathology diagnosis and explain the clinical presentation. In the cytogenetics laboratory tissue samples are separated into cells and cultured to propagate them - the resultant cell harvest has its DNA extracted. How do I know my tissue is well fixed?The quality of fixation within tissue is seen usually through two aspectsMorphology the shape of the cell, their constituents and the tissuesStaining quality the specificity of the staining seen

Red blood cells should look bi-concave. Chromatin within the nuclei is crisp and shows aggregation into clumps towards the cell membrane.DecalcificationAfter tissues have been fixed they are then ready for the next stage of laboratory investigation. Some tissues cut easily with a knife and require no further pre-treatment. Some tissue structures e.g. bone and teeth are extremely hard due to the presence of a calcium phosphate salt (hydroxyapatite). This mineral is essential in life for these structures to perform their role but for the histologist unless this mineral is removed then the production of thin paraffin sections for microscopy will be problematic. The method of achieving this is termed decalcification as it is primarily calcium salts which are removed. However, during the process materials other than calcium will also be removed so a more accurate term to apply would be demineralization.

Calcium crystals may also be present in other tissues as part of a pathological process. Tissues such as breast may be x-rayed prior to being processed) . The identification of this calcium has diagnostic significance and therefore it is important that the calcium remains in the tissue.

Decalcification is essential for the sectioning of paraffin processed tissue

There are certain bony samples which are not suitable for decalcification, they are samples where disturbances to calcium metabolism is suspected e.g.Osteomalacia (adults) or Rickets (children). In these conditions the amount of mineralized to non-mineralized bone is reduced. In order to be able to diagnose these conditions it is necessary to visualize the extent of both and if the specimen is decalcified then it will impossible to make this estimation. In these cases the sample must be treated by embedding the still mineralized bone into a hard embedding medium such as a plastic resin. This will permit suitable quality sections to be produced and stained.

Whichever decalcification method is employed there is a balance to be struck between speed of decalcification and preservation of cellular morphology. Generally the speedier the rate of decalcification the greater will be the rate of destruction (maceration) of cellular morphology. This effect is exaggerated if tissue fixation has not been completed satisfactorily prior to commencing decalcification. The choice of decalcifying method and chemical will be heavily influenced by whatever the sample is being decalcified for i.e. a patient's bone sample being decalcified for a possible clinical diagnosis of malignancy will be required much more rapidly than a similar sample being treated for training and education purposes only. The strength of the acid affects the speed of decalcification.

Factors affecting the rate of decalcification are:-Concentration of the acid within the decalcifying agentLevel of agitation of the tissueSaturation of calcium ions within the decalcifying solution

Maceration The loss of nuclear and cytoplasmic detail through autolysis.Excessive decalcification of tissues in acid can cause tissue damage or maceration. Decalcifying agentsDecalcifying agents fall in to two main categories: acid decalcifiers and chelating agents. Acid decalcifying agents act by reacting with the calcium in the tissue to form soluble salts.

Nitric acid very fast acting but produces the most tissue damage. It is suitable for the controlled decalcification of very hard bones such as skull.

Hydrochloric acid can be used alone or frequently as an ingredient of commercially available decalcifying solutions. Fast acting but produces less tissue damage than nitric acid.

Formic acid much slower than nitric or hydrochloric acids and so is gentler to the tissue. It is probably the most popular choice of decalcification fluid for most diagnostic histopathology applications.

Commercial solutions a number are available within the UK market. As mentioned above most use hydrochloric acid but some use formic acid. Recently recipes containing formic acid and formalin have been promoted which are claimed to permit 'concurrent fixation and decalcification' which contradicts the traditional edict mentioned previously.

Chelating agents offer alternatives to the use of acids. They act as 'calcium sponges' continuously mopping up the free ionic calcium that is always present around the mineralized bone. Chelating agents offer a more gentle approach and may be the decalcifying agent of choice in tissue which is likely to require immunocytochemistry.

Ethlenediaminetetraacetic acid (EDTA) this is the main chelating agent used. Calcium removal is very slow using this method and as a consequence there is minimal tissue damage. Probably the best option if there is no pressing demand for a speedy result. EDTA is the decalcifying agent of choice if immunocytochemistry is likely on the specimen.

Trisodium citrate has a similar rate of demineralization as with EDTA.

Specimen dissectionThe dissection of the specimen is carried out by trained biomedical scientists or pathologists depending on the specimen complexity. Each specimen is categorised A through to E for the complexity of the dissection (see table 1 for an explanation of the specimen categorisation groups). Sampling requires the tissue to be examined macroscopically (i.e. by eye), and only elements that appear to be of clinical significance are selected and submitted for histological examination. A robust knowledge of anatomy and pathology is required to select the correct tissue samples. The Royal College of Pathologists lays out the minimum assessment criteria to be reported on for most major cancer types e.g. bowel, breast, skin, liver, prostate and kidney. The assessment criteria are known as minimum data sets and each malignant sample should have each criteria of the minimum data set included in the final report. When specimens are dissected it is important that the selection of tissue leads to two points, firstly diagnosis of the disease and secondly the proximity of the disease to the margin, the depth of any invasion into the tissue and the involvement of any lymph nodes. Reporting on these criteria allows clinicians information on which to base patient management and decide whether they need further treatment or not.

Key pointCellular pathology reports on cancer cases are required to contain key information relating to the tumour type, involvement of lymph nodes with cancer and presence of metastases Minimum data set A set of information that each malignant case reported should contain. The information required includes size and grade of the tumour seen, distance from resection margins and presence of metastases in the lymph nodes or distant sites.

Table 3.5 - Table showing the various dissection categories of specimens with examplesSpecimen categoryProcessExamples

ASimple transfer of biopsies into a processing cassette. No dissection of the tissue is requiredGastric biopsies,

BSpecimens which require routine simple dissection. Usually no more than 2 3 blocksGall bladder, Appendix

CSpecimens which require a standard dissection i.e. set samples form set locationsUterus and cervix

DSpecimens which require interpretive sampling of the specimenColon

EComplex anatomical specimens which require extensive samplingNeck dissections, Whipples proceduresCystectomyVulvectomy

Whatever is being dissected it is important to realise that the selection of samples determines more than any other step the suitability and quality of the finished report.

Case studyA 51 year old lady presents at clinic with a firm breast lump, after an initial mammograph and core biopsy a ductal carcinoma is confirmed. A subsequent breast lump is excised. The specimen is inked intact so that the edge of the tissue or margin can be identified at a later date. Once the outer features have been described (i.e. what is it? How big is it? Is there skin attached? Is there a lesion easily identified?), then the specimen is sliced and any features are noted

Good selection of tissues for processing is also important, sometimes standard cassettes are not sufficient to allow for a whole cross section of tissue to be examined in one piece and therefore larger pieces of tissue are required to be sampled in a single piece i.e. colon specimens and specimens of larynx to be placed in their entirety in a single block. This can be achieved by loose processing of tissue or the use of Mega or Jumbo cassettes, which measure 6 x 5x 1.5cm. The advantage of this is it allows for much easier anatomical orientation of tissue.

Key pointCassettes that are overfull can present problems for processing embedding and sectioning

Additional cassettes may seem to be adding to the total workload but in all probability the subsequent embedding and microtomy will be much easier and so too will the quality of the end result. It is important to ensure that all stitches and staples are removed prior to processing as they can cause great difficulty when attempting to section blocks. The lids are added to the cassettes and they are ready for the next stage of the process. Each tissue block selected at specimen dissection should have an unique identifier on it stating the case number, the specimen pot identifier and the block number. This allows for unique identification of each block.

Health and safetyIt is essential that the specimen dissection area is cleaned of any bits of specimen after each case. Cross contamination of material can cause misdiagnosis!

The macroscopic specimen report should be sufficiently clear so that anyone reading the report can identify what features have been identified and where tissue blocks have been taken from

Specimen storage and disposalAfter dissection any remaining tissue is kept in an air extracted storage facility. The specimen is kept for a defined period of time, usually around 4 weeks after the sign out of the specimen report. The storing of specimens in this way allows subsequent investigations on the tissue for one of a number of reasonsInitial pathological lesion not identifiedUnexpected findings on the initial examination microscopicallyFurther clinical information becoming available

Competency assessmentAssisting with cut up

LevelCompetenceAssessed competentAssessorCountersigned by candidate

1Can perform patient demographic check

1Can describe and process an endoscopic biopsy

1Writes number of pieces on casette side

1Can set up for cut up

1Can demonstrate daily clean up

1Can demostrate end of week clean up

1Read SOP

2Can describe and process all other specimen types as per SOP CP/H2

2Completes competency assessment CP/H2C

2Can take a dictation of specimen at cut-up accurately using appropriate abbreviations

2Can mark cassettes for ribbons, levels, stains and orientation

2Can demonstrate what to do is specimens need further fixation

2Can accurately record details on request form

2Can trouble shoot discrepancies

2Can prepare racks for processing

BMS cut upLevelCompetenceAssessed competentAssesorCountersigned by candidate

1Read SOP

1Can perform patient demographic check

1Can describe and process an endoscopic biopsy

1Writes number of pieces on casette side

2Can describe and process all other specimen types as per SOP CP/H3

2Completes competency assessment CP/H3C


Macropath useLevelCompetenceAssessed competentAssesorCountersigned by candidate

1Has read SOP

1Can prepare 10% formic acid

1Can prepare block for decal

1Can maintain decal log book

1Can change decal reagent

2Completed competency questions

2Can demonstrate end point decalcification testing

X-ray useLevelCompetenceAssessed competentAssesorCountersigned by candidate

1Read SOP

1Can switch on and prepare x-ray machine

1Can prepare specimen for x-ray

1Can load specimen into x-ray machine

1Can select the appropriate time and KV

1Can perform X-ray

1Can safely remove speciem and x-ray casette

Can go to radiology to develop x-ray film (BGH only)

Can operate safely

Can switch on and prepare x-ray machine, PC and start faxitron software

Can enter patient details to start new case

Can perform x-ray of small specimen using faxitron software

Can perform x-ray of large specimen using large x-ray plates

Completed competency questions

Specimen storage and disposal

LevelCompetenceAssessed competentAssesorCountersigned by candidate

1Can select specimens from the shelf for discard

1Can discard of formalin and place specimens in clinical waste bag

1Can tag up specimens for anatomical waste

1Can remove waste from the lab

Can discard small specimen pots

Can clean buckets

Tissue Processing and embeddingTissue processingThe aim of tissue processing is to prepare tissue in a supporting media ready to be cut in to thin sections, so that very thin slices of the tissue can be cut in order to observe individual cells under the microscope. This means being able to produce slices (sections) at a thickness of between 2 and 6m, about the thickness of a single cell or 1/10th the thickness of a human hair. The most commonly used embedding media is paraffin wax. When solid, paraffin wax has a consistency very similar to that of the tissue itself and this assists with the subsequent sectioning process. Wax however is immiscible with water so the tissue can not simply be transferred from water to wax. Instead, the tissue must transfer through a number of intermediary steps until it is able to be impregnated with wax. This is termed paraffin processing.

The tissue blocks prepared during specimen dissection are immersed in a number of different chemical solutions. These solutions act to prepare the tissue for mixing with an embedding media, in most cases paraffin wax although resin is sometimes used for hard tissue and material which needs to be cut into sections thinner than about 2 m (about half the normal thickness). Paraffin wax is the embedding media of choice because it is easily handled, molten and solid, is non toxic, and is hard enough to support the cutting of thin sections.

Key pointParaffin wax is the embedding media of choice because it is easily handled, molten and solid, and is hard enough to support the cutting of thin sections.

Histology specimens are generally run in batches overnight although continuous throughput or Lean Technology is now being applied so that smaller batches can be processed, so that peaks and troughs of workload can be eliminated. This results in the service being more efficient and reduces result turnaround time for the results of some samples. Additional processing schedules exist for small urgent pieces of tissue or for larger pieces which require longer time in the reagents.

Processing is not always straight forward. Different types of tissue require slightly different processing regimes. In most laboratories this is not always possible as there will be a range of different tissue types appearing on any one day, a limited amount of tissue processors and limited time. Generally processing is a compromise of a number of different factors to give the best overall balance of quality of processing, timeliness and cost. Tissues are generally processed together in an overnight batch, but this may result in poor processing of fatty tissue and particularly hard tissue.

Lean technology is the concept of derived from manufacturing industry. It is the process of analysing the steps of the process to ensure the most efficient work patterns. Within tissue processing this is the concept of reducing batch size to the smallest number possible with the aim of ensuring work moves progressively through the laboratory without any waiting steps.

Key pointsThe size of the tissue dictates how quickly a block may be processed, small tissue pieces can be processed quicker than larger pieces

The embedding media should be the same hardness as the tissues being cut. If the tissue is particularly soft then a softer wax will be used. Brain is a particularly soft tissue, with a consistency of solid custard and therefore, neuropathology laboratories commonly use a mixture of paraffin wax and dental wax (a much softer wax dentists use to get patients to bite in to when taking a dental impressions) in their infiltrating stage, this will give the wax a pink colour but also makes it more appropriate for sectioning brain tissue.

For tissue processing it is convenient to consider tissue processing in 3 distinct stages:

Table 3.6 - Table showing the three stages of tissue processingDehydrationRemoves water from the section,

Clearingto act as a link between the dehydrant and wax. The solution must be miscible with both the dehydrating agent and the wax. Clearing is the term that was applied due to the observed effect that some of these chemicals had upon the tissues. Many have a similar refractive index to the tissues and this caused a resultant transparency or 'clearing' of the tissues after immersion for a suitable time. Not all of the reagents used for this purpose actually clear the tissue so other terms have been suggested e.g. ante-media but the use of clearing agents has remained steadfastly popular. Most of these chemicals are organic solvents and all are toxic to a greater or lesser degree. The purpose of clearing agents is to act as a reagent which is miscible with both alcohol and wax. Xylene is probably the most widely used agent but it suffers from rendering the tissue more brittle and harder than many of its alternatives, particularly if incubation is protracted.

Impregnation to infuse all parts of the tissue structure with molten wax in preparation for subsequent embedding into a solid block.

ReagentsSeveral containers of each reagent are used to reduce the effect of contamination from previous reagents. A number of dehydrants are available but the most popular are industrial methylated spirit (IMS) which is primarily ethanol and iso-propyl alcohol (synonyms iso-propanol, propan-2-ol and 2-propanol).

Some authors have described the use of iso-propyl alcohol as both dehydrant and clearing agent see xylene free processing.Whichever option is chosen it is preferable to start with a more dilute alcohol and gradually increase the concentration rather than to immediately introduce a higher alcohol concentration as the tissue is more adversely affected by the latter option by displaying increased shrinkage artefacts.

Health and safety Industrial methylated spiritHighly flammableHarmful by inhalation and if swallowedRequires specialist disposal

Tissue processing is a reversible process and by placing wax blocks in the processing reagents in reverse order allows tissue to be taken back to an aqueous state.

Health and safety xyleneFlammable. Harmful by inhalation and in contact with skin. Requires specialist disposalIrritating to eyes and skin.

Xylene-free processingInterest in the use of processing regimes which do not require the use of xylene or similar clearing agents is largely due to the health and safety concerns associated with the use of these chemicals and a desire to eliminate their use in laboratory practice. Advances in processor technology have reduced the effect of problems that were encountered with this concept in previous years. Iso-propyl alcohol (IPA) can be used either in combination with ethanol, where the IPA is used in place of xylene, or as both dehydrant and clearing agent. In either situation the ability to use high temperatures within the modern tissue processor retort improves the rate at which IPA is driven from the tissue as molten wax is introduced.

Processing fatty tissueBecause of their high lipid content these types of specimens may need extended times in processing reagents, particularly dehydrant and clearing agents in order to achieve the required effect. This may mean that these types of specimen are segregated from others and processed by a separate regime created specifically for this purpose. In addition, if xylene free processing is being considered then additional time may be required to ensure complete removal of iso-propanol prior to wax impregnation.

Hard tissuesHard tissues such as tendon, nail, decalcified bony structures etc, and may benefit from pre-treatment with tissue softening agents, either phenolic based solutions or surfactants before final processing. This helps to soften the tissues for easier sectioning.

Resin processingResin processing is an alternative to paraffin wax processing. Tissue is fixed and dehydrated in the same way, instead of xylene a dilute resin mix is used and progressively an increased amount of the resin is increased until the tissue is immersed in pure resin. At this point the resin is cured and a hard supportive media surrounds the tissue.

Key pointThe harder the supporting embedding media, the thinner the tissue can be cut.

Types of processing equipmentActivated charcoal filters are a constituent part of all enclosed tissue processors. The purpose of the filters is to remove solvent and formalin vapours that are a by-product of the pumping of reagents in and out of the reaction chamber. The activated charcoal filters act to react with the solvent vapours released by the chemicals. The vapours are locked into the filter and are rendered safe.

The quality of tissue processing is directly related to the reagents used.

Types of tissue processorHow does tissue processing take place? Tissue processing may be performed manually, with the histologist moving the material by hand or may be automated. There are a number of different types of processor, carousel, enclosed process flow and microwave, all of which work on the same principle i.e. bringing tissues into contact with processing solutions for a fixed period of time then transfer to the next solutions. We will look at each processing type in turn.

Hand processing requires the tissues to be moved by hand between the reagents. This is a very time consuming process and in practice, this does not happen often. Generally it is only used for small urgent diagnostic biopsies. The reagents can be warmed to enhance processing speed.

Factors affecting processingHeat MixingVacuumWax Processing

Automated processing schedules are either carousel, enclosed, process-flow or microwave. Most laboratories operate routine processing with enclosed processors but process flow machines are becoming of increasing interest in large laboratories. The major drawback with any automated processor is that the flexibility to adapt regimes for different tissue types is lost as all specimens undergoing a particular protocol will all be treated identically.

Carousel tissue processors originated in the 1950s and represented the first attempts to automate tissue processing. In essence they copied the manual processing regime and automated the transit from one processing reagent to the next using timer clocks. Their use meant that many more samples could be processed at any one time and in particular this automated transfer facility meant that processing schedules could be run overnight and timer delays incorporated into processing cycles so that schedules could be operated over weekends and public holidays. A major drawback with carousel processors is that the tissue samples are extremely vulnerable whilst being transferred from one processing reagent to the next. If an untoward event occurs during this process e.g. power failure then the sample may be left out of fluid for some considerable time and the processing solvents will rapidly evaporate from the tissue causing it to dry out and be unsuitable for diagnosis. In addition when transferring from one reagent to another, there is considerable release of flammable vapours with a carousel system, posing a considerable fire risk. Many diagnostic samples are irreplaceable and so it is probably because of this limitation more than any other that during the 1980s manufacturers began to develop enclosed tissue processors.

Enclosed processors contain all the reagents within the machine. The tissue blocks are contained within a chamber and the reagents are pumped in. Figure 3.17 shows a diagram of an enclosed batch processor. The length of time each reagent is in contact with the specimens is computer controlled. Enclosed processors have developed increasingly complex safety systems to ensure the integrity of samples and to monitor reagents. If a blockage prevents the entry of a solution the processor may skip to the next reagent or incubate further with the previous reagent. Figure 3.18 shows a photograph of an enclosed tissue processor

The process flow tissue processor allows material to be added to the processor at varying points during the day. This way of handling tissues offers a Lean way of processing material when it is needed, usually in more frequent, smaller batches. The principle of the machine is very similar to the carousel processor where the specimen moves through a series of reagents.

Microwave processing is a way of increasing the speed of tissue processing by using the microwave process. Standard processing reagents of industrial methylated spirit and xylene can not be used. Microwave processing technology is still in its infancy and some processing machines require the manual changing of reagents.

Hand processingTissues can be processed manually. In practice this does not happen often and generally it is only used for small urgent diagnostic biopsies. The reagents can be warmed to enhance the processing speed.

Method hand processing

All reagents are warmed to 45oC before use

Formalin (15minutes)70% alcohol (15 minutes)90% alcohol (15 minutes)100% alcohol (15 minutes)100% alcohol (15 minutes)Xylene (15 minutes)Xylene (15 minutes)Wax (15 minutes)Wax (15 minutes)

Embedding Once tissue has been processed it needs embedding in a manner to allow sectioning. The aim of embedding is to orientate tissue to allow for the maximum amount of diagnostic information to be retrieved. This involves ensuring the macroscopic cut surfaces are flat on the base of the embedding mould and the tissue is correctly orientated. Most material is embedded into a mould and has the plastic cassette which the tissue is placed in attached to the back. This allows the material to be held in a manner which allows the tissue to be gripped firmly in a clamp and thin sections to be made. Figure 3.20 shows a photograph of embedding. The way in which tissue is placed for embedding is very important. Correctly orientated tissue allows viewing and interpretation to happen easily. Incorrectly orientated tissue prevents this. Large square samples from resections must be placed flat within the tissue mould to allow for an even cross section of tissue to be prepared

Method embeddingVessels always need to be orientated in cross section e.g. Vasa deferentia, Fallopian tubes, Temporal arteriesSkin sections need to be orientated so that the skin surface and dermis can be seen in cross section.Endoscopic biopsies should be embedded orientated.Some colorectal biopsy samples. The orientation of specimens at embedding can have a direct consequence on the ability to diagnose the pathology of the specimen Embedding equipmentFor most laboratories, the volume of work requires the use of a designated embedding centre (see figure 3.22 for a photo of the embedding centre). Most histological equipment suppliers produce their own equipment centres but all have the same basic requirements:

An electrically heated storage well to store the tissue cassettes waiting embedding and a separate electrically heated storage well to store the base moulds.An electrically heated and thermostatically controlled storage tank for paraffin wax. There is also an outlet nozzle from this tank which allows the operator to dispense the required amount of molten wax as required. An electrically heated 'warm' area which allows the operator to move the blocks around the machine without everything sticking together.An electrically controlled chilled area which allows the operator to orientate tissue samples in the appropriate way prior to completing embedding.An electrically cooled area to speed up setting of the tissue blocks. This may be a fixed part of the embedding centre or purchased as a separate unit.A timer control to switch the machine on and off at appropriate times in the day to suit laboratory workflows.

The operator should be able to vary the temperatures of these heated and chilled parts of the equipment to suit local needs. Frequently the equipment is supplemented by the purchase of additional electrically heated forceps to permit the easy manipulation of specimens and to reduce the carry over of specimen fragments from one specimen to the next. The embedding area is the place in which the tissue is placed together for the creation of tissue blocks. It is essential that the work area is kept spotless to ensure that only the material from which the case is being worked upon is placed in the tissue block.

Embedding is the process of orientating and fixing tissue in a hard media

Competency assessmentsTissue processing LevelCompetenceAssessed competentAssesorCountersigned by candidate

1Can load and unload tissue processors with tissue blocks

1Can flush machines to prepare for a new run

1Can change reagents on processor

1Can update audit log documentation for reagents

EmbeddingLevelCompetenceAssessed competentAssesorCountersigned by candidate

1Embed large 'slabs'

1Read SOP

1Select the correct mould

1Top up wax

1Put cassette on mold flat

1Clean wax drains

1Wipe work area clean

1Insert bead

2Orientate biopsies

2Orientate skins

2Orientate tubes

2Can change fuses on embedder

2Complete CP/H9C

Routine sectioningSectioning is the part of the histology process in which microscope slide preparations are made. The produced slide preparations are translucent and can vary in thickness from 0.1m to 50m. The thinnest sections are required for electron microscopy and the thickest sections are required for neuropathology techniques.

Microtome Tissue sections are prepared on a machine called a microtome. This word comes from the Greek Micro small, tome cut.

Good section cutting is facilitated by tissue blocks being cut from a media which is about the same hardness as the tissue. Wax embedding is not really suitable for tissue which is very hard. Tissues which are very hard either need to be softened or embedded in a harder media such as resin. The microtome is an essential piece of equipment in the histology laboratory. Microtomes are available in a number of different forms. The rotary microtome is by far the most popular, and can be semi automated. The sledge and sliding microtome are popular in some laboratories for the simplicity of use and quality of sections. Whatever the type of microtome, the basic function is the same, to prepare tissue sections of a known thickness in a consistent manner.

For routine diagnostic specimens embedded in paraffin wax the objective is to cut a slice from the tissue that is approximately one cell in thickness. In practice this means that samples are routinely sliced (sectioned) at approximately 4m. Multiple sections are often required from the same block and the microtome must be able to provide ribbons of sections.

Hard material such as bone may need a different approach to softer tissues. Brittle material such as heavily keratinized skin or thyroid colloid may need softening prior to sectioning; this can be done with water phenolic based reagents or surfactant containing softening agents. Recent comparative studies suggest that softening agents containing surfactants perform better on a cross section of hardened tissue types.

Each block contains many sections worth of material and it is important to go sufficiently deeply into the block to obtain an appropriate cross section of material also it is equally important not to waste material. It is quite possible that numerous additional tests will be required. The way each block is sectioned depends on a number of factors; the type of tissue, the size of the tissue or the clinical history. Some blocks will require a single section for H&E, some require a number of sequential sections, to follow a lesion or tissue element through the tissue, and this is known as serials. Some require a number of sections distant to each other through the block to look at random parts sequentially, this is known as levels. Looking at various sections through the material allows you to see the clinical pictures in three dimensions. Often an H&E slide alone is required but there are specimens which need multiple different types of stain to obtain a diagnosis e.g. liver biopsy, renal biopsy.

Why are most routine sections cut at 4m?

Tissue blocks need to be cut with care so that sections are deep enough to reveal any pathology but shallow enough to allow further sectioning.

Health and safetyMicrotome blades are extremely sharp and it is easy to injure yourself, particularly if concentration wanes. If you feel your concentration flagging, take a short break and return later. Factors affecting section cuttingThere are a number of factors which affect the quality of cut sections. These factors includeSharpness of the cutting bladeRigidity of the knife and specimen holderHardness of the tissueBlood within the tissueThe coldness of the blocksSharpness of the cutting blade Rigidity of the knife in the specimen holder

The cutting blade needs to be sharp and free from defects. Most laboratories use disposable blades which are clamped within a knife holder. As more tissue is progressively cut the knife becomes blunt. Any pieces of calcium can nick the knife causing sections to score.Hardness of the tissueIf the tissue is very hard it can cause the tissue to cut thick and thin. This is because the difference between the hardness of the embedding media and the tissue causes a speeding up and slowing down of block through the blade.Blood within the tissueBlood does not process well and dries out. When dry the blood cracks on cutting. Blocks containing blood cut much better when they are soaked or cut from wet ice.Coldness of blocksBlocks need to be cool in most circumstances. The colder the block the harder the wax will be, the harder the supporting matrix, the easier it is to cut thin sections. There is a point that if blocks are too cold then the tissue itself becomes too brittle to section and ribbon easily. This means that the integrity of the overall section is spoiled, the sections may become chattered. Special considerations when cutting blocks Lymph nodesThese are very cellular specimens and so tend to look very crowded under the microscope when sectioned at 4m. Far better cellular resolution is achieved if these are sectioned routinely at 2m.Renal biopsiesAs with lymph nodes, the resolution of the glomerular basement membrane is much improved if sectioned routinely at 2m.AmyloidThe demonstration of amyloid variants with Congo red solutions is improved if thicker sections than usual are obtained. Ideally these should be sectioned at around a thickness of 8mNeuropathology sectionsAgain thicker sections are most suited for many of the tinctorial and metallic impregnation methods used. Thicknesses of between 15m and 50m are frequently encountered to be able to view and follow nerves through the section.MicrotomyMaterial is processed to paraffin embedded tissue blocks, at this point key parts of tissue have been sampled, fixed and processed so that all the water has been removed and replaced with an embedding media. The embedding media is the same hardness as the tissue. All these steps are to produce tissue blocks which are capable of having thin sections cut, this is the process of microtomy. Microtomy is the process of producing thin tissue sections from tissue blocks, using a specialist piece of equipment called a microtome. Microtomes exist in a number of forms -rotary, sledge and sliding.

All equipment handled during the microtomy process should be kept scrupulously clean. It is important that cells from one block are not transferred to the slides belonging to another block. This form of cross contamination is known as carry over.

Microtomy equipmentThe knifeFor routine paraffin sectioning traditional steel knives have almost completely been replaced by disposable carbon steel blades. For all but the toughest of tissues they provide a superior edge to steel knives and have also permitted the production of consistent sections of high quality at thicknesses less than 4m which has improved the morphological assessment of very cellular specimens. However the blades are not inexpensive and so represent a significant drain upon the budgetary resources of a department. In addition disposable blades do not require re-sharpening so the acquisition of sharpening instruments is no longer necessary and the health and safety considerations associated with the sharpening of steel knives has been avoided.

Knives lose their sharpness with use due to general wear and tear issue. Knives that are blunt will score sections and eventually fail to ribbon blocks. If this happens the knife should be sharpened or the blade disposed of and replaced which us expensive.

Rotary MicrotomesIn this design the tissue block and knife are held in the vertical plane. Turning the handle (flywheel) one complete revolution advances the tissue towards the blade at whatever thickness has been set. As the two make contact a sliver of wax, containing the tissue, is shaved from the surface of the block. See figure 3.25Sledge MicrotomesThis version works similarly to the rotary microtome with the exception that both block and knife are positioned horizontally and the block is slid backwards and forwards in this plane making contact with the knife. As the block is slid back towards the operator on runners (or a sledge) there an advance mechanism is operated which raises the block towards the knife by the required section thickness. Sliding microtomesUnlike the previous two with this instrument it is the knife that moves towards the stationary block in a reverse of the situation encountered with the sledge microtome. Like the sledge both block and specimen are orientated in the horizontal plane. The blade cannot be clamped at both ends as is possible with the rotary and sledge microtomes so this instrument is not particularly suited for cutting harder material. Also the moving blade makes it less satisfactory from a user's health and safety perspective! Whereas the previous two would commonly be seen in routine histopathology departments the sliding microtome is more suited to specialist applications, in particular for the sectioning of nitrocellulose blocks in neuropathology facilities. Freezing microtomesThe sample is frozen onto the cassette holder. The blade is then drawn over the tissue sample to produce the section.Cambridge rockerAlthough not used in many establishments, the Cambridge rocker has had an important role in the development on microtomy. The tissue block rocks on a stand against a blade. The resultant sections are cut in an arc from the block. The simple design of the Cambridge rocker has made them long lasting and easy to repair.

Health and safetyMuch microtomy work involves the use of knives. Care must be taken to ensure that they are appropriately guarded. When knives are not in use they should be covered or if finished with disposed of.

The waterbathSections are floated out onto waterbaths at a temperature just below the melting point of the wax, often about 50oC. This allows folds in the sections to be relaxed out of the tissue before being picked up onto microscope slides. Some tissues require adhesive slides to maintain the tissue sections on the slides. The adhesives may be electrostatically charged or be natural sticky products such as albumen. Figure 3.46 show tissue sections being floated out on a waterbath. The knife on a microtome needs to be well clamped to obtain evenly thick sections. The floating out is an important part of the process, sections need to be floated out for the appropriate length of time, if they are floated out for too short a length of time then sections may be creased if they are floated out for too long then sections may disaggregate or disintegrate.

Health and safetyThe waterbath is a potential source of cross contamination between cases. To ensure cleanliness the waterbath should be regularly skimmed of any debris using a tissueSlidesIt is important when you prepare good quality sections that you dont spoil all your effort by the use of inappropriate microscope slides. It is important slides should be clean and free from specs of dust so that the tissue section is not disrupted or torn. Commonly slides are either uncoated or coated to aid adhesion of sections on to the slide. The coating of slides can be from a number of sources, such as, albumin (egg white), 3-aminopropyltriethoxysilane (APES) and positive charged coating.

Competency assessmentsMicrotomyLevelCompetenceAssessed competentAssesorCountersigned by candidate

1`Has read SOP

1Can trim large blocks

1Can cut large block section with no artefact

1Can use microtome safely

1Can float sections on waterbath

1Can write slides with correct patient details and own symbol

1Can trim, cut and float 20 large blocks in 1 hour

2Can cut and orientate ribbons

2Can cut mega blocks

2Can cut and orientate levels

2Has completed CP/H10c

2Can trouble shoot microtomy problems

2Can dismantle and set up microtome

CryotomyCryotechniques are laboratory methods using tissues that have been frozen solid. Freezing techniques may be used for a variety of reasons, speed, and preservation of cell enzymes or avoidance of chemical fixatives due to the interference with the method under investigation. Tissues may be frozen by liquid nitrogen, card-ice ( solid CO2), electrical plates or ozone depleting fluorocarbon spray. It is important to freeze tissue quickly and evenly to prevent ice crystal artefact, see box for more details.

Ice crystal artefactIce crystal artefact is when tissues are damaged as they are frozen by ice crystals in their tissue. The water within the tissue freezes as large crystals which tear at the tissue when they freeze. Figure 3.x shows an H&E frozen section with ice crystal artefact. Note how the tissue has large ragged holes in it.

Frozen sections are cut on a cryostat (see figure 3.25). Cryostats are microtomes which are housed in freezer units. They are chilled to about -20oC. The front housing has a glass panel which closes off the unit from the warm air of the room. Tissue handled in cryostats may contain high risk micro-organisms or viruses such as TB, HIV or Hepatitis B. It is therefore essential that a cryostat can be decontaminated and fumigated to disinfect the microtome and cooling chamber. Frozen sections are also more difficult to cut thinly i.e.