Histopathology

1. Introduction to Histopathology

An appreciation of systemic pathology is based on the knowledge of normal and general pathology; a sound understanding of the principles of pathology is essential for a sound physician, surgeon, and gynecologist. A firm foundation in general and systemic pathology is very essential for a teacher to impart teaching in pathology. The teaching and understanding of pathology is incomplete without understanding the morphological basis of the disease, and for this, the study heavily relies on histopathology. A clinical issue is well understood only if the underlying changes in cells and tissues are understood. A working knowledge, a critical and a logical approach to arrive at a diagnosis by a well-timed and a clinically oriented laboratory investigation form the basis of the practice of diagnostic pathology. This approach requires a thorough knowledge of the basic principles of various facets of diagnostic pathology, e.g. cytopathology, hematology, and immunopathology, but the most essential being understanding of histopathology.

The study of usual and unusual morphology of cells and tissues and the structural and functional disturbances which underlie the pathogenesis of disease come under the purview of Pathology. In recent years, the use of advanced technology has greatly enhanced the capacity of pathologists to accurately diagnose both the disease and the extent of the disease. Despite this, there has been no impact on the three basic sciences underlying diagnostic pathology, namely etiology, pathogenesis, and morphogenesis. The basic information required to make a diagnosis is contained largely in the morphologic and architectural disturbances of the cells and tissues that constitute the particular disease process, and this information is imparted by histopathology. Though there has been tremendous development in diagnostic radiology and imaging techniques, the final diagnosis requires tissue diagnosis in the vast majority of cases.

Histopathology is the gold standard for the diagnosis and prognosis of most diseases. It helps to analyze and understand the underlying causes and mechanisms of diseases, the extent and natural course of diseases, as well as the possible outcomes of diseases. Histopathology also helps to determine the efficacy of treatment or prevention measures and provides a scientific basis for the classification of diseases.

1.1. Importance of Histopathology in Pathology

Histopathology, a term derived from the Greek words historia "tissue" and "logos" knowledge, has been established as an important basic science which is essential in understanding the underlying causes of disease. In more recent years, it has become an integral part in the diagnosis of diseases and has become the basis of research into the causation of disease and the effects of specific disease processes. An understanding of pathology is critical for the physician setting up a diagnosis, for it is the knowledge of the changes in the normal state which help define the states of illness. An example to this is the physician diagnosing a patient with arthritis. With an understanding of pathology, the physician can deduce that the patient has inflammation of the joint, that this is the cause of the pain and knows to look at the joint under further examination. He can then go on to look at the precise causation of the inflammation and ways to prevent this, with the knowledge of the changes being the starting point for all this. In the modern day when an increasing number of diseases can be treated or prevented but there is a lack of understanding of the disease processes, there is an increasing requirement for a pathologist to define the changes in the diseased states and relay these to the clinician so that effective measures can be taken. 63% of the tests ordered by clinicians require some input from pathology and it has been estimated that 70% of medical decisions are based on pathology results. This shows the importance of pathology in disease diagnosis where histopathology is the cornerstone.

1.2. Role of Histopathology in Disease Diagnosis

The great increase in use of histopathology by practicing physicians. Much of the study of the pathology of disease is concerned with etiology and pathogenesis and with the mechanisms of injury or repair. Thus, the pathologist is as much concerned with the dynamic aspects of disease as is the biochemist or physiologist, and much information obtained from studies in these allied fields is of great significance in understanding the nature of morphologic changes. However, in providing a diagnosis from tissue examination, it is to be expected that most often the pathologist will be called upon to be the "finder of the fact", and it is this role which has led to the greatly increased demand for tissue diagnosis and to the realization by practicing physicians of the value of histopathology.

Histopathology deals with the examination of tissues for the ultimate purpose of arriving at a correct diagnosis of disease. For while it is true that the physician must at times make diagnoses and prescribe treatment solely on the basis of his immediate impressions and clinical findings (laboratory procedures being unavailable or too time consuming), the fact remains that in this scientific age, patient and physician alike demand an ever increasing degree of precision in the practice of medicine. Thus it is that the pathologist, who so often functions as the "physician's physician", frequently is asked to provide a tissue diagnosis in cases where clinical impressions are in conflict, where it is a question of therapy, or where the physician seeks to establish a prognosis. And the practice of obtaining tissue diagnoses - which once was confined largely to major hospital centers with a consultant service - now is quite common even among practitioners in the general field of medicine.

1.3. Basic Principles of Histopathology

Tissue is most commonly fixed using a 10% neutral buffered formalin solution. After fixation, the tissue is processed through a variety of dehydration stages using alcohols of increasing concentration to remove water from the tissue. It is then cleared using xylene before being infiltrated with wax. This enables the tissue to be embedded in wax blocks and then sliced into thin sections for mounting on slides. The wax is then removed from the tissue sections prior to staining.

Fixation is the term given to the preservation of tissue structure and is the first stage of tissue processing. It is essential to prevent autolysis of the tissue, which is the degradation of cellular material by enzymes released from cell organelles following cell death. Autolysis causes morphological changes in cell structure that can interfere with accurate interpretation. Fixation also needs to preserve the relationship between cells and prevent any changes in their appearance. The ideal fixative would be one that hardens the tissue, kills microscopic organisms, and preserves all cell components and their relationships without altering normal morphological appearances. This is rarely achieved, and different fixatives have specific actions on different types of tissues.

This section is concerned with describing the basic processes of tissue preparation, including how the tissues are preserved to maintain their structure, then how they are prepared to be suitable for microscopic examination, and what methods are used to stain different tissues to specifically highlight the structures within them. An understanding of these processes is crucial for accurate interpretation of slide images and subsequent diagnosis of any pathological changes present.

2. Specimen Handling and Processing

Receiving and identification of specimens It is essential to have good communication with the surgical department in order to maintain an efficient and cost-effective service. With the introduction of pre-operative clinical diagnosis and special investigation, there has been a gradual increase in the amount of tissue received. The pathologist must decide whether a specimen should be dissected immediately or whether further communication with the clinician is required before processing the specimen. For example, a large hernia sac referred to as suspected carcinoma may warrant a discussion with the referring clinician before sectioning. A request form is usually completed by the clinician, which details the patient's name, hospital number, and the nature of the specimen. Unfortunately, these forms are often incomplete and it is not uncommon for a specimen to arrive without any clinical details. In an ideal situation, the pathologist and a member of the histopathology lab would meet with the surgical team in order to clarify any uncertainties about the specimen and to discuss special requirements for analysis.

2.1. Receiving and Identification of Specimens

The first step in proper tissue processing is accurate identification of the specimen in order to properly assess the clinical question being asked. Before accepting any specimen, it is important that the pathology department has a clear understanding with the surgical and clinical colleagues of what the specimen is and what tests are required on that specimen. It is not uncommon for a specimen to arrive in the laboratory with inadequate history or no clinical details at all. In these cases, it is perfectly acceptable to hold the specimen and contact the clinician for further information. However, it is important to ensure that doing this will not compromise patient welfare, and it might be wise to have a 'holding protocol' agreed with the clinical service so that the decision to hold a specimen can be quickly and easily reconciled. This is particularly important in cancer specimens where there may be a need to proceed quickly and 'holding' the specimen might compromise the patient's best interest. A decision to hold a specimen should always be documented, and this will include details of what was discussed and agreed with the clinician.

2.2. Specimen Fixation and Preservation Techniques

Formalin is an aqueous solution of formaldehyde gas, which is the simplest aldehyde. It polymerizes nucleic acids and proteins, and the onset of polymerization is rapid. It is a slow process which takes approximately 18 hours for a 5mm diameter specimen. Overfixation must be avoided, as this will cause tissue to become brittle and resistant to cutting, and will also lead to increased pigmentation of tissue in the H&E stain. The standard method of formalin fixation is generally regarded as the gold standard against which other fixation techniques are measured. Other fixation techniques are generally compared to formalin fixation to determine their effects on tissue structure, and ability to demonstrate different antigens in immunohistochemistry studies. This is because there is a wealth of data about the effects of formalin fixation on tissue and cellular morphology, and much of our understanding of normal and abnormal tissue structure is based on formalin-fixed, paraffin-embedded material.

Formalin fixation and paraffin embedding are universally established as the methods for anatomical pathology. A comprehensive overview of these techniques is critical to understanding the morphology of disease. While several alternatives exist, such as freezing tissue in liquid nitrogen and cryostat sectioning, these techniques are usually reserved for special circumstances or research. The most appropriate fixation and embedding methods for a particular project will depend on the goals of the study, and will be addressed in greater detail in later sections.

2.3. Tissue Processing: From Specimen to Slide

Once the tissue has been fixed, it must be processed in order to allow for efficient and thorough infiltration by liquid paraffin. This is the process by which tissue is taken from its original state (an in vivo or fixed whole tissue), dehydrated and then permeated with a medium that makes it possible to produce thin, interpretative slices. It is important that the initial specimen is undamaged and is representative of the lesion so that tissue taken for processing is orientationally correct and fit for interpretation. The most common method of tissue processing is automated and employs a machine that carries tissue through a series of time stages in which it is washed in alcohol to dehydrate it and then fixed in paraffin. This is a relatively hands-off approach with the exception of placing the tissue in cassettes prior to processing. Some laboratories may choose to manually process small biopsies and this can be achieved by using a tissue transfer processor in which tissue is carried in a cassette and connected to reservoirs that can be filled with liquid. This method is adequate for small laboratories but is not suitable for high volume laboratories due to the time constraints.

2.4. Embedding Techniques: Paraffin and Cryoembedding

Embedding is the process that involves orientating a specimen in a mould so as to allow easy cutting of tissue and support during the cutting. The choice of embedding material is important, as it determines the ease at which the block can be cut, the quality of the resulting ribbon, and the durability of the block for long-term storage. The two most common types of embedding used are paraffin and cryostat embedding. Paraffin embedding is the most common method used. The process involves dehydrating the tissue in alcohol followed by clearing in xylene. The tissue is then infiltrated with paraffin wax and embedded in a mould. The wax is allowed to solidify, and the block is trimmed, ready for cutting. This method allows storage of the block for long periods of time and is suitable for routine studies. However, during the process of infiltrating the tissue with wax, there is considerable shrinkage of the tissue. Cryostat embedding involves freezing the tissue in a suitable embedding medium and storing it at a temperature suitable for cutting. Optimal cutting is achieved when the tissue is very cold and hard, and this method is therefore useful when examining tissue with a view to frozen section diagnosis. Frozen section diagnosis allows rapid diagnosis during surgery and is often used to determine surgical margins. This can be important when deciding whether further excision of tissue is necessary. The frozen tissue is stored for varying lengths of time, and it is possible to freeze tissue for long-term storage. This makes cryostat embedding useful for both routine and research material. The method is particularly useful for avoiding tissue fixation, as this can cause masking of antigenic sites. At the ultrastructural level, frozen sections are useful for enzyme histochemistry and immunocytochemistry.

2.5. Microtomy: Sectioning of Tissue Blocks

Microtomy is the process by which a tissue specimen is cut into thin slices, usually between 5-10 microns thick, in order to be mounted on a slide and stained. The first step in microtomy is to select the block to be cut; ideally, this would be a paraffin block, as the material is easier to work with than frozen tissue and can be stored for years without damage. Before cutting, the block must be firmly attached to the microtome chuck on which it will be mounted. The microtome should then be set to the desired thickness (usually around 5 microns) and the blade positioned for cutting. If the block is particularly hard or soft, adjustment of the blade angle or temperature may be necessary. Step sections are thinner than routine sections and are taken in order to obtain the best section possible. Once cut, the ribbon of tissue produced by the microtome may be spread on a water bath at 45°C for a few seconds in order to flatten it and then picked up by means of a glass slide or warmed to the exact temperature at which the section will spread without creasing. Finally, a good section is picked up and floated onto the surface of a bath which has been previously warmed to the temperature at which the paraffin melts.

3. Staining Techniques

Hematoxylin and eosin staining is the most widely used staining technique in histopathology. It is the basic and primary method of staining done in the histopathology lab for the demonstration of general tissue structure. The hematoxylin is a basic dye which stains nuclei of the cell (basophilic staining) blue or purple in color. It imparts the morphological details of the nucleus and the intensity of the staining depends on the acidity of the nucleus. The hematoxylin which is used in the staining process exists in the form of alum hematoxylin which in turn exists as hematein. Hematein forms a complex with the acidic components of the cell and stains them blue in color. But pure hematoxylin is not useful as it gives a simple solution to staining the tissue. So mordants like aluminium potassium sulfate or ferric ammonium sulfate are often employed to help retain the dye on the tissue and intensify the color. Eosin on the other hand is an acidic dye and it stains the basic components (e.g. cytoplasm, RBC, collagen) of the cell in shades of pink and red (acidophilic staining). Eosin is less specific than hematoxylin. It provides only little information and stains the tissue in an ambiguous way. But it highlights the contrast provided by hematoxylin and gives a more 3-dimensional look to the tissue. Eosin also provides the color to the rest of the tissue so as to distinguish which has been stained by hematoxylin.

3.1. Hematoxylin and Eosin Staining

Despite the ubiquitous usage of H&E staining, what exactly constitutes a good H&E stained section is often difficult to define. The interpretability of stained sections is affected by many factors and both under and over staining can impede interpretation of tissue structure. The section may require some empirical tuning of staining conditions until optimal results are obtained. The skill of the histotechnologist has a marked effect upon the final product and histotechnique is often an under-appreciated art which directly affects the practice of surgical pathology. Within a pathology department, installation of an internal quality control program with regular review of H&E stains and feedback to the histotechnologist is a constructive method to ensure maintenance of standards in H&E histology. An example of a practical post analytic method to optimize interpretation of H&E sections is to photocopy the section on to acetate and use different coloured pens to mark different structural components. Difficult sections can then be traced back to the original block and an attempt can be made to restain it in a manner which enhances the interpretation.

Hematoxylin is a basic dye which stains basophilic components such as nuclei, endoplasmic reticulum and ribosomes. In the presence of acid, hematoxylin forms a complex with acidic tissue components and these complexes are then subsequently reduced to haematin within the tissue. There are many different formulations of hematoxylin and can be aluminium, ferric or mercury based. Different formulations and staining times can affect the colour of the final product. Eosin is an acidic dye which stains acidophilic components such as cytoplasm, extracellular matrix and intracellular proteins by forming ionic bonds.

A plethora of histochemical stains are available to demonstrate various cellular components presenting in tissues. Hematoxylin and eosin stains (H&E) are the most widely used stains in pathology and serve to demonstrate general tissue structure. Thus, understanding the principles of H&E staining and its limitations are crucial for the accurate interpretation of histologic sections.

3.2. Special Stains for Cellular Components

Another type of stain used for carbohydrates is the diastase periodic acid Schiff (dPAS). This is the same as the PAS stain, but the tissue is pretreated with diastase, which digests glycogen. This is useful to distinguish glycogen from other carbohydrate-like substances. In dPAS positive reactions, the substance is not glycogen.

PAS stain (Periodic Acid Schiff) is used mainly for the detection of glycogen in cells and tissues. Glycogen is a polysaccharide of glucose and is an important store of energy in the body. It is a normal finding in the liver and skeletal muscles, and its presence or absence in these tissues can be an indication of certain diseases. For example, glycogen is increased in liver damage, diabetes, and glycogen storage diseases. It is decreased in hypoglycemia. PAS reagent reacts with the aldehyde groups in glycogen to form Schiff reagent, which produces a pink to magenta color in the specimen.

Special stains are used to demonstrate specific components of cells, such as carbohydrates, lipids, and proteins. These are usually not visible with routine H&E staining. There are many different types of special stains, and each has its own principle and application. A few common ones will be discussed.

3.3. Immunohistochemistry: Detection of Antigens

Immunohistochemistry is commonly associated with the detection of cell type-specific markers which aid in determining the lineage of a malignant tumor. For example, cytokeratins are commonly used to aid in the diagnosis of carcinoma as well as being used to differentiate between the various subtypes. CD3, CD4, and CD8 are used to identify T-cells and determine their relationship to various skin disorders.

Two primary methods of observation for immunohistochemistry are light microscopy and immunofluorescence. The former is widely used with diaminobenzidine. Compiled digital images of various stained slides can be subjected to computerized analysis. This is especially useful for quantitation of antigen expression levels.

6. Visualization. Dyes such as diaminobenzidine are then applied to the tissue. These dyes change color in the presence of the enzyme that is bound to the antibody complex. The reaction yields a colored product at the antigen site, allowing its identification under a microscope.

5. Incubation with secondary antibody. The secondary antibody is specific to the primary antibody's host. For example, if the primary antibody is a mouse antibody, a secondary antibody that is specific to mouse immunoglobulin is used. This step is followed by incubation with a third reagent in some cases. Avidin and biotin are commonly used for this purpose, as the avidin-biotin complex (ABC) has a very high binding affinity and is not easily disrupted.

4. Incubation with primary antibody. The primary antibody is the antibody that is specific to the antigen of interest. It is applied to the tissue and allowed to bind to the antigen.

3. Antigen retrieval. Because fixation of tissues with formalin and embedding in paraffin often masks antigens, this is a critical step for immunohistochemistry done on paraffin-embedded tissues. This can be done with enzymes or heat. Heat-induced epitope retrieval (HIER) with a microwave or conventional pressure cooker is widely used, as it is generally reproducible and can be optimized for different antibodies.

2. Sectioning. Tissues are embedded in paraffin and sectioned with a microtome. The sections are then placed on slides. Some studies have shown that freezing tissue rather than fixing with formalin yields better results. However, frozen tissue does not preserve well and the ability to do immunohistochemistry or any other type of staining is limited to a short period of time following resection.

1. Fixation. This step preserves the tissue of interest. Usually, formalin or paraformaldehyde is used as a fixative. These agents preserve tissue structure and antigenicity.

The basic steps in an IHC staining procedure are as follows:

Immunohistochemical (IHC) staining is used to determine the presence and distribution of antigens in tissues. IHC is based on the principle of antibodies binding specifically to antigens in biological tissues. This type of staining is widely used in the diagnosis of cancer.

3.4. In Situ Hybridization: Localization of Nucleic Acids

There are a number of possible ways in which the hybridization signal (or the position of the probe) can be detected and these include using either a radioactively, chemically or enzymatically labeled probe. The most sensitive method for probe detection is the use of a radioactive label and this can give excellent results, particularly when combined with autoradiography. However, increasingly stringent legislation concerning the use of radioactive materials in the laboratory and the disposal of radioactive waste is making non-radioactive detection methods more attractive. In situ hybridization will often be followed by the detection of antigens or cellular components in the same tissue and in these cases it is often useful to combine the results of the two procedures in a double label experiment. This can be achieved by using a radioactive probe together with an immunohistochemical detection method or by using two distinct non-radioactive labeling techniques.

In situ hybridization is a powerful technique used for the detection of specific nucleic acid sequences in morphologically preserved tissues or cells. The nucleic acids can be either DNA or RNA. In situ hybridization is achieved by allowing a labeled single-stranded RNA or DNA molecule (the probe) to hybridize to the specific RNA or DNA sequence of the cell or tissue under investigation. The basis of the methodology is similar to that of Southern or Northern blot analyses, where RNA or DNA is immobilized on a support (filter) and is then probed with a complementary sequence. In situ hybridization on tissue sections or whole mounts of tissue is more complex and the stringency of the reaction (or the condition necessary to give optimal hybridization specificity) is more difficult to control. Nevertheless, the ability to localize a specific nucleic acid sequence to a particular cell type or region within a tissue is what makes in situ hybridization so valuable.

3.5. Electron Microscopy: Ultrastructural Analysis

The greatest contribution of electron microscopy has been in the classification of acute myeloid leukaemia and the diagnosis of various renal diseases. Ultrastructural details help to classify myeloid leukemias and dysplastic, reactive and neoplastic changes of the myeloid or erythroid cell lines. The importance of the classification is that it provides some prognostic information and also gives an indication of the most appropriate therapy. For example, acute promyelocytic leukemia with the characteristic balanced translocation involving chromosomes 15 and 17 is very responsive to therapy with ATRA (all-trans retinoic acid). Electron microscopy is often useful in diagnosing renal disease to identify the precise histological nature of the condition and hence guide clinical management.

Tissue is fixed in glutaraldehyde or formalin, embedded in plastic and ultra-thin sections are cut and stained with heavy metals such as lead or uranium. The sections are examined in a transmission electron microscope that can resolve structures as small as 2 nanometres. Alternative methods include freeze-fracturing, freeze-etching and cytoskeleton extraction.

When the resolution provided by light microscopy is insufficient to make a diagnosis, the ultrastructural details of cells can be revealed by electron microscopy. However, the technique is expensive and time-consuming and its role has been considerably diminished by immunocytochemistry and other ancillary methods.

4. Microscopic Examination and Interpretation

After tissue sections have been prepared and observed grossly, they are processed and stained to be viewed under the light microscope. Light microscopy and the interpretation of findings is the cornerstone of anatomic pathology. Histopathologic diagnosis is based upon pattern recognition within the tissue seen at low magnification, followed by identification of cellular details on higher magnification. Color, pattern, and cellular abnormalities are well visualized with routine histologic staining, and excellent correlation between the clinical presentation and the radiologic findings and the histologic findings will usually be achieved. Hematoxylin and eosin (H&E) is the most commonly used stain in histopathology. It provides the basic information to diagnose most inflammatory, neoplastic and degenerative diseases and it serves as the starting point for further histochemical or immunocytochemical stains which may elucidate more specific information about a particular disease process. An example might include a suspected lymphoproliferative disorder such as Hodgkin disease. The abnormal cells are readily apparent with H&E staining, but immunostaining for CD markers is necessary to subtype the lymphoma and to decide on a prognosis and treatment. This is in contrast to other imaging modalities such as radiology, in which a specific diagnosis is often made based upon the morphologic appearance and it is then necessary to make an inference about prognosis and treatment. In summary, light microscopy is an inexpensive and relatively quick method to generate a wealth of information about the patient's disease state and it will remain the primary tool in diagnostic anatomic pathology.

4.1. Light Microscopy: Basics and Techniques

The light microscope is a tool to enhance the visualization of objects that are otherwise difficult to see (i.e., the details of small structures and particles). This is achieved by magnification of the image (making the object appear larger) and resolution. Resolution is the ability to distinguish two very closely approximated objects as separate, or recognition of detail. As resolution increases, the observer is able to see smaller structures within cells and tiny aggregates of organisms, and to discriminate fine variations in cellular morphology that are signs of specific disease processes. In light microscopy, the upper limit of useful magnification and resolution is about 1250x and 0.2 μm respectively. These upper limits are rarely necessary for routine pathology work, but it is important to realize that not all compound light microscopes are capable of this level of performance.

While there are numerous special stains and techniques used on tissue sections, the basic tool of diagnostic surgical pathology is the light microscope. Yet light microscopy tends to be less standardized among pathologists than are many of the newer tests. Although one can look through the oculars of any microscope and see something, image clarity, quality, and especially the ability to make valid diagnostic interpretations are the results of understanding and skill in use of the instrument. This chapter provides a practical guide to choosing and using a microscope, techniques for creating good quality microscopic slides and for enhancing image clarity, and simple methods to measure the size and other features of microscopic objects. A proficiency in use of the light microscope and its accessory tools will contribute greatly to making consistent and valid interpretations.

4.2. Interpretation of Cellular Morphology

The microscopic appearance of cells offers much information to the diagnostic pathologist, since normal, preneoplastic, and neoplastic cells often can be distinguished on the basis of their morphology. At the light microscopic level, a pathologist observes the size and shape of cells, the size, shape and staining intensity of their nuclei, the character and color of the cytoplasm, and the architectural relationships among the different cells within a given tissue. Ultrastructural examination with the electron microscope provides additional information regarding the nature of intracytoplasmic or intranuclear inclusions, the fine structure of cellular organelles, etc. This information is essential, not only in identifying the type and grade of tumor, but also in determining its histogenesis and in predicting its biological behavior. In some instances, identification of neoplastic cells is based primarily on their exclusion of normal cells. For example, in diagnosing lymphoma, plasma cell myeloma, or metastatic carcinoma, the neoplastic population of cells is identified by their resemblance to one of these normal hematopoietic cells or by their site of involvement of tissue. In certain tumors, the identification of immature or undifferentiated cells is of diagnostic importance. An illustration of the impact of cell morphology on tumor diagnosis is provided by the myeloproliferative disorders. The original classification of these diseases was based on clinical, laboratory, and sometimes pathologic features, but without regard for morphologic characteristics of tumor cells. Subsequently, it was recognized that detailed morphologic examination of the peripheral blood and bone marrow could provide a reproducible means of diagnosing the different myeloproliferative diseases and assessing their prognosis. This subsequently led to a proposal to redefine or rename these diseases to reflect their morphologic features.

4.3. Grading and Staging of Tumors

Tumors are unusual masses of tissue created by an abnormal accumulation of cells. These masses can be created by a plethora of different mechanisms, and when it comes to identifying exactly what type of tumor a certain mass of cells represents, pathology is the definitive discipline. When diagnosing and classifying tumors, pathologists use two distinct processes to evaluate the tumors and reach a diagnosis - grading and staging. These processes are used for solid tumor classification - that is, neoplasms which have a mass form as opposed to liquid neoplasms which circulate in the blood, i.e., leukemias. The aim of grading and staging is to appraise the expected behavior of the tumor and to thus better plan the treatment and eventually predict the patient's prognosis.

Grading of tumors occupies the part of tumor classification concerning primarily the level of differentiation of a particular neoplasm. The essential objective of grading is to estimate the biological behavior of the tumor. Although there are a lot of different grading systems, criteria commonly feature: 1) the degree of structural disorder in neoplastic cells and their pleomorphism, 2) the mitotic activity of the neoplastic cell population, 3) the level of differentiation comparing neoplastic cells to their tissue of origin, and 4) the quantity of tumor cell necrosis. The last item is not always included, but necrosis suggests a less favorable outcome in the majority of neoplasms. Grading is not a simple task, and a certain degree of subjectivity often exists. It is said that grading may have up to 60% predictive accuracy in terms of neoplasm behavior, but this is, of course, variable depending on the specific neoplasm.

4.4. Identification of Infectious Agents

Identification of infectious agents, like the diagnosis of neoplasia, is something that should be attempted in all cases not just those where an infectious etiology is suspected. With an increasing number of patients with iatrogenic or disease-related immunocompromise and an increasing variety of emerging infections, identification of infectious disease in tissue sections will continue to be an important part of pathology practice.

4.4.4. Serology and molecular diagnostics These may be the only means of diagnosis with some infections because the organism is not locally present in the tissue being examined. An example is in the diagnosis of Lyme disease with detection of antibodies in the patient's serum.

4.4.3. Microbiologic culture Identification is the ultimate goal of culture and repeated attempts with different culture media may be needed. The use of molecular methods, especially gene sequencing is readily available in identification of microorganisms cultured in tissues.

4.4.2. Special stains for microorganisms Many different stains are available for the identification of microorganisms. Gram stain and acid-fast stains are still widely used and very useful. There are also many types of stains for identification of fungi. Immunoperoxidase stains and in-situ hybridisation are also available for identification of specific antigens or DNA sequences.

4.4.1. H&E stained sections Identification of inflammation, necrosis, viral inclusions, and other cytologic alterations indicative of possible infection. In some cases, especially with fungi, the only finding may be necrosis with little or no agent identified.

4.5. Ancillary Techniques for Diagnosis

Identification of specific types of infectious agents, being bacteria, fungi, parasites or viruses can often require specific tests which will not routinely be performed on histological sections. This may involve simple microbiological cultures from fresh tissue, or more complex techniques such as PCR, in-situ hybridization or serological tests. These tests may be performed by the pathologist but more often in conjunction with a microbiologist or virologist. They can often lead to a definitive diagnosis after the histology has been suggestive but not conclusive.

Immunohistochemistry: this technique has become the most widely used ancillary technique, often being referred to as a surrogate for doing an invasive or expensive test. It involves the use of antibodies which bind to antigens in tissue providing information on the distribution and localization of the antigens, and is essentially an extension of traditional enzyme and protein histochemistry but using specific antibodies. This can provide valuable diagnostic and prognostic information and has the greatest impact in tumor pathology.

Alternative techniques for a specific diagnosis can be necessary when routine morphology does not lead to a definitive answer. Newer, more sophisticated techniques are often highly targeted at a particular diagnosis, often described as "diagnostically targeted techniques". These can lead to a better and quicker diagnosis with less tissue from smaller biopsies and often overcome the limitations of light microscopic findings. For example, a common situation in diagnostic pathology is determining if a tumor is a primary or secondary from another site. Immunohistochemistry or molecular studies can be very helpful; another example is differentiating a non-Hodgkin lymphoma from a poorly differentiated carcinoma - here gene rearrangement studies can be the only way to reach a definitive answer. Ultrastructural studies have become somewhat less used as it requires fresh tissue and the cost effectiveness is often low, but can still be very valuable in certain situations.

5. Quality Assurance and Record Keeping

Standard operating procedures (SOPs) are defined as written instructions incorporating protocols, infrastructural and utilization decisions, and integrated in histopathology practice to standardize aspects of practice and improve consistency. The most significant area to standardize in histopathology practice is in the preparation of tissue for microscopic examination, its examination, and subsequent reporting of the results. Instituting and maintaining efficient SOPs will lead to improved turnaround time and higher quality reporting. For example, a complex surgical specimen (large bowel resection, cancer of the breast, lymph node biopsy) may be examined by several different laboratory staff, and clinicians may want to know when the tissue is expected to be reported on and any special instructions given. These preparation protocols may be used as a teaching tool for new laboratory or secretarial staff and are a source of information for laboratory staff. Routine procedures are often handed down from senior to junior staff members, and it is important to have a clear written record of what these are. Written instructions on the preparation of individual specimens may be stored with these specimens for retrieval and comparison with future specimens.

5.1. Standard Operating Procedures in Histopathology

Overall a well designed and appropriately followed SOP is an important tool to maintain and or improve the quality of the work.

- Discarding outdated and less effective work methods. - Adherence by all staff to the specific procedure will promote a team environment preventing isolation of work practices and it's also highly likely that there will be someone to take over a task or method that is thought to be effective. - Consistency of diagnostic tissue preparation between biomedical scientists and pathologists. - Reduction of error and the repetition of work. - Beneficial to the training of newcomers to the field.

Basically the creation of a set of standard practices will provide the facilitation and quality driven guidelines to produce quality pathology work. This is achieved in the following ways:

There are several published documents, which describe the use of SOPs in histopathology laboratories all with the same basic goal. That is, to lay down standard practices for the intended purpose of improving the quality of the work done. The NSW Health Department has produced a set of publications based on safe and quality work practices. This could be very beneficial to anatomic pathologists and laboratory staff as it provides guidelines for not only safe practices but also the hazardous and potentially harmful substances that they might use. The Royal College of Pathologists of Australasia has produced a document based around work practices and safety for pathology laboratories with similar goals.

Procedures are extensively used by the scientific community, and in the field of histopathology they are an adjunct to the clinical diagnosis. They are the means by which pathologists and biomedical scientists process tissue and prepare it for diagnosis. This can be via a variety of methods and ultimately procedures are aimed at the production of a diagnostic slide.

Standard Operating Procedure (SOP) is a written set of step by step instructions and techniques which provides consistency and facilitates the standardization of a specific function or operation. The aim is to achieve the operation of a specific process in a consistent and quality driven manner.

5.2. Quality Control and Assurance Measures

Control is the set of procedures and practical measures that ensure that the operations are achieved according to the plan. Step one, therefore, is the expression of the desired level of performance, usually documented by standard operating procedures (SOPs). In anatomic pathology, there is little agreement about what represents desirable performance for any particular technique or diagnosis, and some view it as an unattainable ideal. To avoid that, a plan should have enough flexibility to accommodate field conditions and what is feasible. The precise specification of error is useful in this step. Error can be defined as the difference in the performance of an operation from what was intended. The greater the disparity, the more successful the detection of the error. This concept is important because the first part of any QA system is error detection. The aim is to prevent errors that have a harmful effect and to correct or attenuate one's own errors in terms of the effects on the patient. Step two is to compare the actual performance of an operation with the desired one. This comparison is an ongoing process and has already been discussed in terms of monitoring. The third step is action to maintain the desired performance. Error occurs because of unwanted variation in the performance of an operation. Step three aims to reduce this variation and has a very close relationship to QA.

Quality assurance (QA) is a term that has been used in a variety of fields and applications. The aims of QA are to prevent errors, avoid mistakes, and improve the performance of the particular system or service. In a clinical laboratory, QA is vital. An accurate diagnosis is based on reliable and relevant data provided by the laboratory. If such evidence is poor, unreliable, or compromised by error in any way, the consequences can be catastrophic. The requirement for increased regulation and accreditation, the trend towards subspecialization, and the increasing complexity of modern medicine have all highlighted the importance of implementing effective QA within a laboratory. QA is best viewed as a cycle with a series of interrelated steps: monitoring performance by the use of internal audit and the development of performance indicators, addressing any identified problems, and improving performance.

5.3. Maintenance of Laboratory Records

There are various types of documents generated in a histopathology laboratory, ranging from patient request forms, tissue cassettes, blocks, slides, stained sections, reports, and quality control records. It is essential for the pathology service that all these records are retained for an appropriate length of time. Patient request forms and report records should be retained for the patient's lifetime. Other records should be retained for a minimum of 10 years, or in the case of a child, until the age of 25. These retention times are in line with The Royal College of Pathologists' guidelines. Specific diseases or types of analysis may necessitate longer retention times for some records. All documents and other records need to be stored in a manner that allows easy retrieval. This applies to both active and archived records, which need to be kept in conditions that prevent deterioration. In the case of paper records, this means storage in a clean, dry environment with protection from vermin and destruction by fire or water. Archived records should be boxed, and the box clearly labeled with the contents and the date range for the records. In some cases, it may be appropriate to scan documents and store them in a digital format. This has benefits in terms of ease of retrieval and protection of the original documents, but there are legal issues, and these documents would still need their minimum retention periods. It is important to continuously monitor all documents and records to ensure their quality and to prevent loss or damage. This should be built into the laboratory's quality management system with regular internal audits of records management.

5.4. Digital Imaging and Archiving of Slides

A major step was the development of affordable and versatile digital cameras for photomicrography, although these have limited application for routine diagnosis and are not ideal for capture of an entire slide. More recently, scanning devices specifically for the purpose of creating a digital version of a whole slide have become available. The concept is analogous to the digitization of music into a file which can be played back with no loss of quality, even though the file is much smaller than the original source. Digital slides have the advantage that they can be viewed on a computer monitor and easily shared via the internet.

In modern day histopathology, archiving and management of slides is an important issue. Many pathologists and laboratories maintain thousands, even millions of glass slides and reports. These require storage space and there are issues relating to retrieval and movement of slides. Maintenance of slide quality over decades is critical for the diagnosis and understanding of disease processes. Furthermore, in today's multidisciplinary practice, cases are reviewed and reported many years after the original diagnosis. The use of digital technology provides a potential solution to many of these problems without compromising the quality of the material.

5.5. Ethical Considerations in Histopathology

Ethical considerations in histopathology are especially important as pathologists are dealing with patient material and information which is often irreplaceable. For example, a surgical resection which results in the histological diagnosis of benign tissue will mean that no further treatment is required. However, if a different pathologist re-examining the same specimen interprets the findings as malignant, the consequences are considerable (Okonkwo et al., 2002). It is important in this situation that the patient's best interests are maintained. This can occur through documentation of the macroscopic and histological features of the specimen which ideally should be in the form of photographs and drawings, as descriptions alone can be vague and open to misinterpretation. These images can then be stored within a clinico-pathological database for future reference. An internal second opinion can also be sought as to whether the material should be sent to an external source for consultation.

6. Future Directions in Histopathology

Advancements in technology have significantly influenced the practice of histopathology and are likely to continue to do so. One of the most notable changes in pathology practice has been the development of digital pathology. This involves the capture of a glass slide (whole slide image; WSI) into a digital format, allowing the image to be viewed on a computer screen. Its potential applications are wide ranging and it is envisaged that digital pathology will eventually replace the microscope in surgical pathology. Initial developments focused on utilizing digital images for teaching and it is now possible to build image banks to aid undergraduate and postgraduate learning. Diagnostic work has been revolutionized with the possibility of utilizing telepathology, enabling pathologists to view and report on cases from any location with an internet connection. This has particular benefits for pathologists working in different hospitals or laboratories, and for those seeking expert opinion on difficult cases. The main drive behind the development of digital pathology is the potential improvement in working practices and patient care that can be achieved by the increased efficiency in handling and accessing digital images when compared to conventional glass slides. This includes the automation of image analysis and the development of computer aided diagnosis (see Section 6.2). Although the technology has been available for over a decade, issues regarding validation, standardization, and cost have meant that its uptake has been slow. However, recent years have seen greater awareness of the technology and a realization of its potential benefits, and it seems likely that digital pathology will eventually become an integral part of pathology practice.

6.1. Advancements in Digital Pathology

A National Health Service (NHS) health technology assessment reported that there were compatibility issues with existing systems and it was difficult to calculate the costs and benefits of implementing digital pathology. It was concluded, however, that the capabilities and predicted advancements in the near future made information technology in pathology inevitable.

Most recently, digital pathology has been the forerunner in the transition from an analogue practice to a digital one. More recently, cell analysis has been done by acquiring digital images (virtual slides) of the histopathological glass slides. This is usually done with an automated device that is able to scan the slides. Virtual slides can be viewed on a computer using software that simulates a microscope. This technology is already widely used for educational purposes and is set to replace the optical microscope as the primary tool for diagnosis. The ability to share digital images between professionals has opened up interesting possibilities for telepathology. This is a practice where a pathologist can interpret the images at a remote location. This could be a very beneficial service for overseas labs as well as to obtain an expert opinion.

Pathology and its sub-specialty, histopathology, are the study and diagnosis of disease. This involves describing and interpreting the cause and effect of disease. The best results are often obtained by meticulous observation of cells and tissues, often with the use of a microscope. Recent advances in computer technology have opened an array of possibilities to further improve results as well as streamline the workload of pathologists.

6.2. Automation and Artificial Intelligence in Histopathology

Automation in histopathology involves the use of either robotic technology or computer-controlled devices to perform tasks normally done by pathologists. Robotics research in histopathology is still in its infancy; however, automated vision technologies are already allowing cell-by-cell analysis to be carried out in a manner that frees the human raters from this tedious task. Artificial intelligence is the capability of a device to imitate intelligent human behavior. It is a diverse subject covering a wide range of machine capabilities and applications. Expert systems are already being developed to provide diagnostic assistance; some are no more than algorithmic pathways with no true AI, while others are based on machine learning and complex data analysis. The potential exists for AI to automate diagnostic processes in histopathology, and although it still remains an emerging concept, its implications are already creating unease among pathologists who fear for the security of their jobs.

6.3. Integration of Molecular Pathology

Histopathology is a complex discipline that involves the use of many different techniques to make a diagnosis. It seeks to observe the manifestations of the disease process in tissues in order to understand the pathobiology and to arrive at a diagnosis. The integration of molecular pathology into the framework of the standard practices of surgical pathology is now an essential feature of a histopathologist's work. It is rare now to see a research paper published that does not have some form of molecular study and these results are always interpreted in the context of the disease process. In the clinical setting, the findings often have therapeutic and predictive implications and in many cases can be used to tailor the treatment of an individual patient. It is not our role to describe the techniques of molecular pathology, but rather to consider how the molecular information relates to the tissue findings and how it can be integrated into diagnostic practice. Many disease processes occur as a result of an abnormality in gene transcription and protein synthesis within cells and molecular studies are often aimed at understanding these processes. The traditional approach of the histopathologist has been to study the abnormal cells in situ and to interpret the changes in the context of the morphology and the clinical findings. The identification of specific morphological changes often leads to further hypothesis-driven studies aimed at understanding the underlying pathobiology and it is at this point that the information from molecular studies becomes highly relevant. In some cases, the molecular information will directly elucidate the morphological changes observed. An understanding of the link between human papilloma virus infection and overexpression of p16 protein leading to cervical intraepithelial neoplasia is one such example and it is now common for histopathologists to order immunohistochemistry for specific molecular markers in order to aid diagnosis.

6.4. Emerging Techniques for Tissue Analysis

6.4.3 Microscale technologies New microscale technologies such as microfluidics and microarrays offer the ability to break down complex biological systems into more simple and analyzable components. This could potentially bring analysis close to the in-vivo state of the tissue and reduces the amount of tissue required for analysis. Although these techniques are not specific to tissue analysis, their impact on the study of disease and disease models will inevitably change the way we look at and understand disease processes in tissues.

6.4.2 In-vivo molecular imaging Imaging techniques are a key diagnostic tool in modern medicine and in-vivo molecular imaging is a natural progression in medical imaging. It is a rapidly evolving field that can be broadly defined as visualization of endogenous molecular processes in living organisms. Though its primary use is in preclinical research, the prospect of correlating imaging findings with histological and molecular information is an attractive one. This should eventually lead to tissue specific diagnoses and the ability to monitor specific disease processes in an individual patient.

6.4.1 MALDI mass-spectrometry imaging Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry (MS) is a powerful proteomic technique for the discovery of protein and peptide biomarkers between normal and diseased tissues. MALDI MSI extends the capability of MALDI MS analysis to provide the preservation and analysis of the spatial distribution of biomolecules directly in tissue, providing a direct correlation of locational pathology with molecular pathology.

The potential impact of a better understanding of disease and drug mechanism through the discovery of new biomarkers, drug targets, and efficacy/toxicity assessment is enormous. New technologies are driving a major change in the way tissue analysis will be performed in the not too distant future. As the traditional model of histopathologist as sole analyser of tissue on read only glass slides gives way to pathologists as integrators of information from multiple sources, there will be a greater need for interdisciplinary interactions. Molecular biologists, chemists, information technologists and engineers will all be part of the new multidisciplinary team involved in tissue analysis.

6.5. Challenges and Opportunities in Histopathology

There are many challenges and opportunities in histopathology, some of which have been discussed in previous sections. The challenge to the discipline is to maintain a focus on the tissue and morphological diagnosis in the face of increasing complexities ranging from molecular biology to big data derived from clinical trials. An important opportunity lies in the potential to integrate findings from other methods of tissue analysis with the light microscope-based diagnosis. This should enable tissue diagnosis to remain the key reference point in anatomic pathology offering a durable linkage between clinical and basic science. The implications of this linkage will be the subject of ongoing study and debate. The rapid pace of technological change will continue, and histopathology is likely to evolve in a range of different subspecialised areas. Some new techniques may not be disseminated widely, and it will remain important to critically appraise their utility while ensuring that the core disciplines of anatomic pathology are maintained.