Ophthalmic Pathology: An Illustrated Guide for Clinicians

Chapter 1

Basics

In order to achieve a better understanding of disease processes occurring in different regions of the eye, this section describes the technology currently employed by the histopathologist in the examination of tissue specimens referred by ophthalmologists. It is important to be aware of the range of laboratory services locally available. When there is a suspicion of infection, the relevant specialist (bacteriologist/mycologist/virologist) should be consulted for advice concerning appropriate transport media and therapy. The value of an accurate and concise history cannot be overestimated and good collaboration will be rewarding to both clinicians and laboratory specialists.

Examination of the enucleated eye

A formalin-fixed enucleated globe bears little resemblance to the in vivo appearance due to opacification of the cornea, lens, vitreous, and retina. Previous intervention, for example removal of keratoplasty tissue, can produce secondary damage to the anterior segment tissues (Figure 1.1). In routine practice, it is unwise to try to cut across the lens because this produces damage to the anterior segment but occasionally a suitable illustration can be provided (Figure 1.2). By dividing the globe in the coronal plane, the pathologist has the advantage of examination of the lens and ciliary body from the posterior aspect (Figure 1.3) and the retina from the anterior aspect (Figure 1.4). For demonstration purposes, it is possible to divide the optic nerve and the lens (Figure 1.5). In general, the globe is divided above the optic nerve and at the edge of the cornea to avoid traumatic artefact to the main axial structures. After paraffin processing, the microtomist cuts into the centre of the eye. The orientation of the extraocular muscles on the posterior aspect of the globe allows the pathologist to identify the side from which the globe was enucleated (Figure 1.6). Orientation of the specimen is vital if the correct plane of cut is to be made.

Microscopic features

These are described wherever relevant to pathology in the corresponding chapters and are therefore only illustrated briefly in this chapter. The histological features of each of the following tissues are annotated in detail:

cornea (Figure 1.7)

chamber angle (Figure 1.8)

iris (Figures 1.8, 1.9)

ciliary body (Figures 1.8, 1.10, 1.11)

lens (Figures 1.9, 1.11)

retina and choroid (Figure 1.12)

optic disc (Figure 1.13).

Features for identification of the age of a patient (in this case a child):

thin Descemet’s membrane

finger-like ciliary processes

intact, non-hyalinised ciliary muscle

absence of proliferations in the pars plana epithelium

absence of sub-RPE (retinal pigment epithelium) deposits (for example drusen).

Figure 1.1 In the current litigious climate, the only normal autopsy material available for study will be that used for donor keratoplasty. In this example, formalin fixation accounts for opacification in the cornea and lens. Damage to the iris is the result of the trephine.

Figure 1.2 The anatomical features of the anterior segment are easily recognised. Note that formalin fixation leads to opacification of those tissues (cornea, lens, zonules, and vitreous) which are normally transparent.

Figure 1.3 Dividing the eye in the coronal plane provides the opportunity to examine the ciliary body and lens in detail. In this case, there is a subcapsular cataract. The radial linear opacities in the lens substance are a common degenerative feature in the elderly globe. Note that in the pars plicata, there are ridges and troughs which explain the differing appearance of the ciliary processes in Figures 1.10 and 1.11.

Figure 1.4 In a globe removed at autopsy, there is often autolytic swelling of the macula due to delayed fixation. The opacification of the retina is the result of formalin fixation. After cessation of blood flow, the blood columns in the vessels tend to fragment (cattle-trucking).

Figure 1.5 This normal globe is part of an exenteration and is fixed in gluteraldehyde. For demonstration purposes, the section passes through the centre of the optic nerve, the lens, and the pupil (left). The macula is located on the temporal side of the optic nerve, which is confirmed by the adjacent scleral insertion of the inferior oblique muscle. The distance from the optic nerve to the ora is greater on the temporal side than on the nasal side. A higher magnification of the posterior pole of the globe is shown on the right. Myelination of the axons in the optic nerve ends at the lamina cribrosa.

Figure 1.6 The orientation of the extraocular muscles in relation to the optic nerve reveals that this specimen is a left globe.

Figure 1.7 A full thickness section of the cornea (left) demonstrates the relative thinness of the epithelium and endothelium in relation to the stroma. Both cell layers are shown in higher magnification (upper right and lower right). Note the artefactual separation of the corneal lamellae.

Figure 1.8 Hyalinisation and atrophy of the circular and oblique components of the ciliary muscle is a feature of ageing, but the longitudinal fibres inserting into the scleral spur persist. In an infant (inset), the components of the ciliary muscle are intact: note the thin ciliary processes.

Figure 1.9 In the pupillary portion of the iris, the sphincter pupillae is a prominent feature and the close relationship to the lens provides the opportunity to illustrate the anterior capsule and the epithelium of lens. The iris pigment epithelium terminates at the pupillary rim in the normal eye.

Figure 1.10 In this illustration of the normal ciliary body, the relative absence of hyalinisation in the ciliary muscle suggests the younger age of the patient. This section passes through one of the troughs in the pars plicata.

Figure 1.11 The ciliary processes are lined by a two-layered epithelium corresponding to the layers of the optic cup (see Chapter 6). The stroma of the ciliary processes contains blood vessels. The equator of the lens contains the nuclear bow. This section passes through a ridge in the pars plicata.

Figure 1.12 The normal histology of the retina, choroid, and sclera. ILM = inner limiting membrane, NFL = nerve fibre layer, GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer (bipolar cells), OPL = outer plexiform layer, ONL = outer nuclear layer (photoreceptor nuclei), PR = photoreceptors in inner segment (IS) and outer segment (OS) (cones have a distinctive pink inner segment), RPE = retinal pigment epithelium with underlying Bruch’s membrane and choriocapillaris. The choroid contains blood vessels, nerves, fibroblasts, and melanocytes with branching processes. The sclera is avascular and contains scattered scleral fibroblasts.

Basic pathology definitions

The following text describes the histological appearance in basic pathological processes and serves only as an introduction. Additional illustrations are provided throughout the text.

Inflammation

For a detailed description of the functions of inflammatory cells, the reader is advised to consult specialised immunology texts. Inflammatory disease entities relevant to ophthalmic pathology are described in Chapter 8.

Cellular constituents

Polymorphonuclear leucocytes

Neutrophilic polymorphonuclear leucocytes

Acute purulent inflammation occurs after pathogenic organisms, particularly bacteria or fungi, are introduced into the ocular or orbital tissues. The predominant cell is the neutrophilic polymorphonuclear leucocyte (PMNL) which contains intracytoplasmic granules encasing lysosomal enzymes capable of destroying pathogenic organisms (Figure 1.14). The proteolytic enzymes are also destructive of normal tissues adding to the lytic enzymes secreted by pathogenic organisms.

Eosinophilic polymorphonuclear leucocytes

These possess intracytoplasmic granules which, with an appropriate stimulus from interleukin 6, release major basic protein and ribonuclease (Figure 1.15). These enzymes are often associated with reactions against protozoal parasites, such as Toxocara canis, but this response is disadvantageous in allergic conditions (for example vernal conjunctivitis) because release of enzymes leads to vasodilatation and oedema.

Mast cells

Mast cells are large mononuclear cells with intracytoplasmic pink granules (in an H&E stain) containing heparin, histamine, and prostaglandin (Figure 1.16). These cells are an important component of allergic reactions (for example vernal conjunctivitis).

Lymphocytes and plasma cells

Lymphocytes and plasma cells predominate in chronic inflammatory processes, particularly in autoimmune disorders. Both types of cells are small (Figure 1.17). Lymphocytes have a homogeneous nucleus and very little cytoplasm. Plasma cells are oval with a cartwheel or clockface nucleus, and a paranuclear clear area in the pink cytoplasm.

Granulomatous inflammatory reaction

The classic reaction is characterised by an infiltration of macrophages accompanied by lymphocytes and plasma cells. Macrophages frequently fuse to form multinucleate giant cells. Granulomatous inflammatory reactions occur during chronic infections by bacteria (for example Mycobacteria sp.) and fungi (for example Aspergillus sp.). Foreign material also stimulates a granulomatous reaction (Figure 1.18).

A granulomatous inflammatory reaction must be distinguished from granulation tissue which is formed by fibrovascular proliferation occurring as part of a healing response.

Figure 1.13 A longitudinal section through the optic nerve demonstrates the different components. The nerve fibre layer (NFL) is thickened at the edge of the disc. The prelaminar part contains non-myelinated axons which pass through the lamina cribrosa. In the retrolaminar part of the optic nerve, the axons are myelinated.

Figure 1.14 This illustration was taken from a pooled collection of polymorphonuclear leucocytes in the anterior chamber (hypopyon). These cells are recognised by their distinctive multilobed nuclei. The pink-staining material around the cells is plasma.

Figure 1.15 Eosinophilic polymorphonuclear leucocytes (PMNLs) are often part of a reaction which contains macrophages. The eosinophils possess bilobed nuclei and bright red intracytoplasmic granules which are released into the tissue (degranulation). Macrophages have relatively larger oval nuclei and have a phagocytic function.

Figure 1.16 Mast cells are mononuclear cells and are larger than PMNLs. The red granules in the cytoplasm are characteristic. These cells are easily found in the normal iris stroma, as in this example.

Figure 1.17 This chronic inflammatory reaction in the choroid contains numerous lymphocytes and plasma cells. Note the clear area adjacent to the nucleus of the plasma cell.

Figure 1.18 Implantation of hair fragments into the conjunctiva induces a foreign body giant cell granulomatous reaction.

Figure 1.19 The term pyogenic granuloma is a misnomer as can be seen from this illustration: there is neither evidence of pus nor of a giant cell granulomatous reaction. The congested mass of tissue consists only of radiating blood vessels in loose fibrous connective tissue. A chalazion (see Chapter 2) is present beneath the pyogenic granuloma.

Pyogenic granuloma

Clinically this condition appears as a fleshy, granular, red mass over a pre-existing defect in the conjunctiva. Most commonly a pathologist will see a pyogenic granuloma over a chalazion (Figure 1.19) or over suture material in a muscle insertion after squint surgery.

Neoplasia

Knowledge of terminology used by pathologists is essential to a clinician in the interpretation of a pathological report.

Hypertrophy

An increase in the volume of tissue by enlargement of individual cells. While this is a common phenomenon in striated muscle following repetitive use (for example weight training), it is difficult to find a suitable example in ophthalmic pathology.

Hyperplasia

An increase in the volume of tissue due to the proliferation of cells (for example response of conjunctival epithelium or epidermis of the eyelid to an irritant – Figure 1.20).

Hyperkeratosis

Thickening of the keratin-containing layer in an epidermis (for example actinic keratosis – Figure 1.20).

Parakeratosis

Nuclear debris persists in a thickened keratin layer when the growth rate of the epithelium is accelerated (for example actinic keratosis – Figure 1.20).

Metaplasia

This is transformation of one cell type to another cell type (for example columnar epithelium of conjunctiva to stratified squamous in keratoconjunctivitis sicca). Two other forms of metaplasia of importance in ophthalmology are fibrous metaplasia of the retinal pigment epithelium (for example proliferative vitreoretinopathy following rhegmatogenous retinal detachment – see Chapter 10) and fibrous metaplasia of the lens epithelium (for example anterior subcapsular cataract – Figure 1.21).

Dysplasia

Irregular arrangement and loss of maturation of cells in an epithelium. The nuclei are irregular in size, shape, and chromatin distribution, and mitotic figures may be found in all levels. This can be regarded as a carcinoma-in-situ because the neoplastic cells have not penetrated the underlying basement membrane (Figure 1.22).

Mitotic figures

Mitotic division of cells involves the duplication of chromosomes and separation to form two daughter cells. A mitotic figure is most easily identified in metaphase when the nuclear material forms a broad line across the cell (Figure 1.22). Mitotic activity is a normal function of those cells which undergo wear-and-tear replacement (for example basal layer of epithelium). Pathologists attach great importance to the identification of mitotic figures, especially in neoplasias where an increase in mitotic activity reflects cell proliferation rates. Many examples of mitotic activity will be provided throughout this textbook to illustrate the varying appearances of the process.

Figure 1.20 Sun damage to skin results in hyperplasia of the epidermis with excessive keratin formation (hyperkeratosis) and migration of epithelial cell nuclei into the surface keratin (parakeratosis). There is still maturation of the epidermal layers from basal to surface, which makes the distinction between hyperplasia and dysplasia (see Figure 1.22).

Figure 1.21 The formation of an anterior subcapsular fibrous plaque is one complication of anterior uveitis. The fibrous tissue staining green with the Masson stain (lower) is formed by metaplasia of lens epithelial cells to fibroblasts

Atrophy

A decrease in size or number of cells. This process may be physiological or pathological. The lacrimal gland (see Chapter 5) becomes atrophic in later life (physiological) and the nerve fibre layer becomes atrophic in glaucoma (pathological).

Apoptosis

Apoptosis describes the programmed cell death which occurs in normal tissue (for example embryogenesis of the retina) and in disease. In the absence of inflammatory cell infiltration, individual cells undergoing apoptosis appear shrunken and the nuclei are fragmented (Figure 1.23). Programmed cell death is common in malignant tumours and explains the slow growth of basal cell carcinomas and uveal melanomas.

Necrosis

By comparison, in necrosis there is widespread cell death as observed in rapidly proliferating tumours such as retinoblastoma in which tumour growth exceeds the blood supply (see Chapter 11). When a population of cells dies simultaneously, the nuclei and cytoplasmic membranes disappear leaving a pale pink staining area of tissue in an H&E section.

Type of specimens

Pathological services are now used more frequently due to the importance of accountability, audit, and research.

The following types of specimen may be presented for pathological examination:

eyelid (including lacrimai sac) – biopsy*

conjunctiva – biopsy, impression cytology, and scrape

cornea – lamellar or full thickness (penetrating keratoplasty), impression cytology, and scrape

orbit (including lacrimal gland) – biopsy + exenteration

optic nerve – biopsy or enucleation

temporal artery biopsy

globe – biopsy, evisceration, enucleation, exenteration

aqueous and vitreous tap

vitrectomy including membrane peeling

subretinal membrane – excision.

Tissue preparation

Fixatives

Fixation is essential to good histopathology because excised tissue undergoes rapid autolysis and desiccation.

Formalin

This is the traditional universal fixative solution in ophthalmic pathology because it has prolonged chemical stability and is most appropriate for immunohistochemistry.

Gluteraldehyde

If scanning or transmission electron microscopy is required for diagnosis, gluteraldehyde fixation is essential. One advantage of this fixative is that the macroscopic appearances are closer to those observed in vivo, although it is less suitable for immunohistochemistry.

Figure 1.22 This is an example of metaplasia and dysplasia in the conjunctival epithelium (upper). The normal appearance is shown below for comparison. In the upper figure, the conjunctival epithelial cells have undergone metaplasia to squamous cells. Dysplasia is characterised by a failure of maturation in the cell population which exhibits the cytological characteristics of malignancy (e.g. marked variation in nuclear size and shape and mitotic figures located outside the basal layer). Although this is a premalignant change, there is no evidence of invasion into the underlying stroma and this change is classified as carcinoma-in-situ.

Figure 1.23 Programmed cell death in individual cells occurs in both physiological and pathological processes. In this orbital rhabdomyosarcoma, the apoptotic cells can be recognised by the small fragmented nuclei in comparison with large irregular nuclei of the viable cells.

Alcohol

Alcohol (for example gin) can be used for specimen fixation in countries where formalin is not available.

Preparation

Paraffin embedding

Currently, tissue is cut to provide histological preparations after it is embedded in paraffin wax. To achieve impregnation of the tissue by wax, it is necessary to remove water (ascending concentrations of alcohol) and lipid (xylene). The wax supports the tissue during sectioning (5–10 μm in thickness) and acts as an adhesive when the section is mounted on a glass slide. A reverse process takes place to remove the wax (xylene) and rehydrate the tissue (descending concentrations of alcohol) prior to tissue staining with water-soluble conventional stains or the application of immunohistochemistry.

Paraffin blocks and sections can be stored indefinitely.

Fresh/frozen sections

If an urgent diagnosis is required during a surgical procedure, the tissue can be rapidly frozen and sections cut on a freezing microtome. Section preparation is easier when the tissue is not fixed so the specimen should be transferred to the laboratory in saline or transport media.

Fresh tissue is also more suitable for the study of fat within cellular components (for example lipid keratopathy or sebaceous carcinoma) and for immunohistochemical studies in which a full exposure of antigenic epitopes is required.

For the best preservation of tissue, specially designed transport media (for example Michel’s transport medium) should be used. Consultation with the pathologist is essential.

Plastic embedding

For transmission electron microscopy, it is necessary to embed tissue in hard plastic material (Araldite). Plastic material can be cut on a microtome to achieve the thin sections (0.05–0.06 μm) necessary for high-resolution imaging and are able to resist electron beam bombardment. Thin plastic sections (1 μm) are cut for light microscopy and stained with toluidine blue.

Macroscopic examination or specimen grossing

In ophthalmic pathology, specimens are often small and the initial examination requires magnification with a dissecting microscope. The specimen is measured and a description recorded before division into blocks for paraffin embedding. The methods for each tissue sample are described at the beginning of the relevant chapter. It is however appropriate to describe the methodology for the examination of a globe at this point.

Measurements

The maximum dimensions in the globe in the following sequence are measured using a calliper:

1 Antero-posterior (normal 24 mm).

2 Horizontal (normal 23.5 mm).

3 Vertical (normal 23 mm).

The following are examples of conditions that may alter the dimensions:

Increase (25–30 mm): axial myopia, adult glaucomatous enlargement due to uveoscleral bulging (staphyloma formation) or buphthalmos resulting from infantile glaucoma.

Decrease (15–18 mm): axial hypermetropia when the globe is shortened. Shrinkage (for example atrophia bulbi or phthisis bulbi) occurs after prolonged loss of pressure in the eye. Ocular hypotonia may be the consequence of inflammatory damage to the ciliary processes or to leakage of intraocular fluids through a defect in the corneoscleral envelope.

Transillumination

The shadow from a powerful light source behind the globe is used to locate intraocular masses.

Different cuts and terminology

The plane of section through an eye is extremely important in revealing the pathological features in a paraffin section. Cuts are carefully chosen to include all the pathological features and relationships in one plane of section.

In the horizontal plane, the paraffin sections should include the centre of the pupil, the lens, the macula, and the optic nerve (Figure 1.24). The temporal side of the eye is longer than the nasal side, so that a horizontal cut can be recognised in a section even when the retina and macula are atrophic. The inferior oblique muscle when present is useful to identify the posterior temporal sclera and overlies the macula.

The vertical plane is favoured for displaying surgery for glaucoma and cataract. However, in the case of a tumour or a foreign body, an oblique section may be required to cut across the feature of interest (Figure 1.25).

Anatomical location

For accurate clinical correlation, the ocular abnormalities should be described in their correct quadrants–referred to as superior, inferior, temporal, and nasal.

Calotte

The term calotte (French: cap) is used for the two hemispheres which are cut from the globe before the central pupil-optic nerve block (PO block) is processed through paraffin. NB: This should not be confused with culotte (French: knickers/panties)!

Stains for microscopy

Conventional histopathology

All of the conventional stains and the more sophisticated diagnostic techniques summarised in Tables 1.1 and 1.2 will be referred to in detail in subsequent chapters.

Figure 1.24 The primary cuts in a globe are important if all the features are to be displayed in a paraffin section. Upper left: a second cut in a globe is made with a large dermatome blade. The first cut reveals a small tumour adjacent to the optic nerve (right). The paraffin section passes through the centre of the nerve, the centre of the tumour, and the centre of the anterior segment. The clinical diagnosis was a retinoblastoma but pathologically the tumour was a small benign glial tumour (astrocytic hamartoma, see Chapter 11).

Figure 1.25 Oblique cuts are made in this globe to pass through a superonasal tumour in the ciliary body. The central pupil-optic nerve block is subsequently processed for paraffin histology and the calottes are retained for specialised investigation.

Table 1.1 A summary of the special stains used in routine diagnostic histopathology.

Figure 1.26 In macular dystrophy of the cornea, mucopolysaccharides accumulate in the endothelium, in the keratocytes, and in clumps beneath the epithelium. The best way to demonstrate the presence of mucopolysaccharides is to use the colloidal iron (left) or the Alcian blue (right) stains. NB: Mucopolysaccharides do not stain with H&E.

Figure 1.27 Alizarin red stain is used to identify calcium salts. This example shows calcified bone spicules within an osteosarcoma which arose in the orbit of a child who was previously treated by irradiation for a retinoblastoma. The inset shows the appearance of the tumour in an H&E section.

Figure 1.28 In an H&E section (see Figure 1.13) it is not possible to identify individual axons. The Bodian stain is one of the stains used for this purpose. Normal axons are so fine that they are not easily identified at low magnification (left). At high magnification, only segments of the sinuous axons are seen (right).

Figure 1.29 Examples of common pathogenic bacteria as seen in a Gram stain. (A) Gram positive staphylococci are dark blue in colour and occur in clumps. (B) Gram positive diplococci (Streptococcus pneumoniae) are smaller than staphylocci and possess a capsule. Both A and B can be the cause of postoperative endophthalmitis. (C) Gram negative diplocci (Neisseria gonorrhoeae) can be found in conjunctival swabs in adults with unresolving conjunctivitis and in neonates infected during birth (ophthalmia neonatorum). (D) Gram negative bacilli (Moraxella sp.) may be identified in a corneal scrape from a chronic ulcer.

Figure 1.30 Myelin sheaths are demonstrated by the Loyez stain. The inset is a transverse section through the optic nerve of a patient suffering from tobacco-alcohol amblyopia: the centre of the nerve is pale (axial demyelination). The high power view shows the transition from bundles containing sparse myelin sheaths on the left to more densely packed myelin sheaths on the right.

Figure 1.31 Masson trichrome stain is frequently used in corneal pathology. In this example, previous trauma has disrupted the Bowman’s layer and the adjacent stroma is replaced by an irregular fibrous scar tissue. The epithelium stains pink (and red cells are red!).

Figure 1.32 By comparison with an H&E stain (left), a PAS stain (right) is used to demonstrate basement membranes. In this example, there is a post-traumatic detachment of Descemet’s membrane which is incarcerated in the posterior corneal stroma. The stain also demonstrates a thickened epithelial basement membrane secondary to corneal oedema. Clefts in the stroma are artefactual and do not represent corneal oedema.

Figure 1.33 In a standard H&E section, the presence of iron in metallic foreign material in fibrous tissue cannot be determined. The Prussian blue stain demonstrates iron salts within the metallic particles and the widespread diffusion into the surrounding tissue.

Figure 1.34 The van Gieson stain is also a trichrome stain and is used to differentiate between muscle (yellow) and connective tissue (red). When combined with a stain for elastic tissue (black), fragmentation of the internal elastic lamina can be demonstrated in degenerative disease of the temporal artery.

Figure 1.35 The von Kossa stain reacts with phosphates to form a black precipitate. It is used to identify calcium phosphate complexes in tissues. In this example, the patient suffered from alkali burns which were intensively treated with phosphate buffered solutions. The extent of calcium phosphate deposition is not apparent in the H&E stained section (inset).

Other techniques

Immunohistochemistry

This technology has brought about a revolution in diagnostic and research pathology and has superseded electron microscopy. Precise identification of cells by type is achieved by applying a specific antibody to an antigenic epitope within the cell. The antibody is subsequently labelled with a chromogen which can be visualised by light or fluorescence microscopy. The number of specific antibodies which are commercially available is ever increasing (Table 1.2). For example, there are at least 25 antibodies to specifically identify T- and B-cell subsets and macrophages in benign and malignant states (Figure 1.37). Similarly, a battery of immunohistochemical reagents is applied to poorly differentiated tumours when the H&E appearance is inconclusive (for example in metastatic disease). In ophthalmic pathology, the standard brown chromogen (peroxidase-antiperoxidase: PAP) is of limited value in the study of pigmented tissues; as an alternative, red chromogens (alkaline phosphatase) are more helpful (Figure 1.37).

Electron microscopy/immunoelectron microscopy

The transmission electron microscope (TEM) focuses electrons to resolve cell structures at a high magnification (for example up to ×100 000). This was a valuable tool prior to immunohistochemistry and was mainly used to identify cell organelles (for example melanosomes) and viral particles. The principles applied in immunohistochemistry can also be applied at the ultrastructural level. Antibodies are labelled with very small gold particles that appear as black dots in micrographs. The technique can localise epitopes within cellular organelles and membranes – this is essentially a research tool.

The scanning electron microscope (SEM) focuses a raster of electrons on tissue surfaces. It is particularly useful for the study of the corneal endothelium.

Polymerase chain reaction (PCR)

Specific DNA or RNA sequences can characterise pathogenic organisms or cellular constituents. Only very small samples of tissue are required (for example aqueous or vitreous tap). This technique breaks down nuclear chromatin into sequences and a particular sequence (for example unique constituents of viruses or bacteria) under investigation is amplified to a level that allows rapid detection using gel electrophoresis.

In situ hybridization

This technique also relies on the ability to cut segments of nuclear proteins with specific enzymes. The fragments are identified by immunohistochemical techniques in routine light microscopy. The advantage is that the precise location of the protein fragments can be visualised within the tissue.

Flow cytometry

This research tool is used to identify cells types within a population (for example lymphoid proliferations). A suspension of cells is labelled with fluorescent antibodies specific for antigen determinants on the surfaces of the different cell types. The flow cytometer differentiates and quantifies the different cell types (for example B and T cells).

Figure 1.36 An immunosuppressed patient who succumbed to tuberculosis. Acid-fast organisms stained with Ziehl–Neelsen were plentiful in a choroidal microabscess.

Figure 1.37 Immunohistochemistry is helpful in the diagnosis of benign and malignant conditions. In heavily pigmented tissues, it is necessary to bleach the melanin prior to application of a specific antibody labelled with a red chromogen. In this case, the angle in the trabecular meshwork is blocked by macrophages (CD68 positive) laden with melanin pigment derived from a necrotic melanoma of the ciliary body (melanomalytic glaucoma – left upper and lower). In the case of a malignant T-cell lymphoma, a brown chromogen (peroxidase-antiperoxidase: PAP) is used to label the anti-T-cell antibody (CD5, right). Note that not all the malignant cells express the epitope.

Table 1.2 A summary of the antibodies used in immunohistochemistry.

Figure 1.38 In poorly differentiated spindle cell tumours, an H&E section will not provide a specific diagnosis. In this example, an antibody against myoglobin demonstrates the protein within the cytoplasm of some of the tumour cells to confirm a diagnosis of embryonal rhabdomyosarcoma. Note that not all the tumour cells express the epitope.

Figure 1.39 In metastatic tumours, the primary site may not be evident on first presentation so that immunohistochemistry can be helpful in suggesting the origin. In this example of an orbital biopsy, CAM 5.2 labelled with peroxidase-antiperoxidase (PAP) demonstrates the characteristics of squamous epithelium. A primary bronchial carcinoma was the source. This specimen was negative for carcinoembryonic antigen which excluded a primary gastrointestinal carcinoma.

Table 1.3 A summary of routine media used in bacteriology.

Microbiology

The microbiology is here presented in context with the pathology commonly encountered in ophthalmic practice. It is important for ophthalmologists to be aware of basic microbiological techniques and the selection of media for an accurate diagnosis of infective conditions (Table 1.3). This text is not intended to be comprehensive, and for more