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LABORATORY AND INTERVENTIONAL TESTS

A wide range of tests may be required in patients with cutaneous signs of systemic disease, and in monitoring therapy, but these will not be addressed here. This section discusses tests that are performed on the skin but that require some additional equipment or laboratory facilities.

Testing for contact hypersensitivity

Patch tests

Patch tests (Fig.2.24) are a frequently performed procedure in the investigation of type 4 hypersensitivity reactions (contact dermatitis), for which the tests are usually applied for 48h (Ch.6). The same basic technique, but with application times of a few hours, is used in investigation of contact urticaria .

Prick tests

These tests for type 1 hypersensitivity are less commonly performed in dermatology. The more common agents that give positive results are the aeroallergens involved in atopy, but these are generally more relevant to management of asthma than of eczema. Prick tests can be useful in some food reactions and in some immediate hypersensitivity reactions (e.g. to latex). They are of limited value in urticaria, as many cases are not allergic in mechanism, but may be useful to confirm a suspected allergen in some instances. Prick testing in urticaria is also limited by the fact that pricking the skin in such patients will often cause a weal regardless of the test agent.

Radio-allergo-sorbent test (RAST)

This test and similar immunologic tests are performed on blood samples to detect type 1 hypersensitivity. They are more expensive than prick tests, and the range of substances that can be tested is smaller, but they have the advantage of safety in patients with severe hypersensitivity reactions, such as that occurring with peanuts

Testing for fungal infection

Fungi of relevance to the skin include yeasts such as Candida species, dermatophytes (ringworm fungi), and less common organisms that cause deeper infections.

Skin swabs

These are performed primarily for identification of bacterial infection, but are also appropriate if candidal infection is present. In the case of deeper infections, culture of biopsy tissue is more helpful.

Skin scrapings

These are the pertinent investigation for cutaneous dermatophyte fungal infection (ringworm) and for pityriasis versicolor (a yeast infection). They are obtained by scraping the skin surface with a blade, ideally from the edge of a lesion where scaling is most apparent. Generally, in the case of dermatophytes, the evaluation of skin scrapings will consist of both initial direct microscopy (after softening the keratin with 20% potassium hydroxide) and culture to identify the precise type of dermatophyte present. Sometimes fungi are visualized using special stains as well. Culture is not routinely performed for suspected pityriasis versicolor.

Figure. 2.24 Patch testing. This patient has had test strips removed to reveal positive tests to nickel (in metal items) and phosphorus sequisulfide (in red match heads, a cause of facial eczema). The blue-black dots are ink marks adjacent to the site of test application, so that any positive reactions later can be localized and identified.

Hair and nails

Fungal infection of the scalp due to some dermatophytes, notably the infection acquired from cats and dogs, will fluoresce under a Wood's light. Hairs can also be plucked for microscopy and culture, as performed for skin scrapings. Nail clippings for identification of fungal infection are an important investigation, as treatment usually involves relatively long-term systemic therapy. In the most common types of infection, affecting the distal nail, detection rates are best if the nail is clipped back as far as possible to the proximal edge of the clinically abnormal zone.

Testing for viral infection

Direct identification of viral infection from skin samples is performed primarily for herpesvirus infection. Viral culture is infrequently used in routine practice now, due to improved immunologic techniques.

Tzank smear

This technique involves scraping the base of a viral vesicle, transferring the material on to a glass slide, staining as for a blood film, and observing cells with viral cytopathic changes by direct microscopy. It has the advantage of speed and simplicity but does not identify the virus if positive (e.g. it does not distinguish between herpes simplex and herpes zoster). It can also be used to confirm a diagnosis of molluscum contagiosum using material expressed from a lesion

Electron microscopy

This is also a rapid technique for identification of herpesvirus infection but is not widely available. It is also useful for other large viruses, for example to confirm a diagnosis of orf.

Immunological methods

In detection of herpesviruses, enzyme-linked immunosorbent assay (ELISA) is useful, as it does not require viable virus and can be performed on scrapings as described earlier, or even on crust from an older vesicle. More sophisticated immunologic methods, such as in situ hybridization and the polymerase chain reaction (PCR), are used to identify the presence and type of human papillomavirus in skin lesions.

Biopsy of lesions

This is not frequently required for diagnosis of viral diseases but may demonstrate characteristic histology in some instances, such as viral warts or molluscum contagiosum. Additionally, since the advent of PCR techniques, biopsy material may be used to detect infection with agents such as human papillomavirus or Epstein–Barr virus.

Obtaining samples for histology

Most histologic specimens submitted by dermatologists are those obtained during excision or other treatment of localized lesions such as skin cancers; surgical techniques are discussed in more detail in Chapter 5. However, there are also many situations where sampling part of a skin eruption is beneficial to guide or support a diagnosis. This is usually
to obtain a sample for histologic examination but may be to obtain specimens for microbial culture. For diagnostic purposes, there are various types of biopsy tissue-removal procedures (discussed in this section). Most are performed with injected local anesthesia, but some do not require this (e.g. fine-needle aspiration or small superficial shave biopsies to confirm a diagnosis of basal cell carcinoma), and some may be performed with topical anesthesia.

    When submitting specimens to a laboratory, it is helpful to indicate which technique has been used, and also whether the specimen is an excised lesion, a portion from within a lesion, or a biopsy into the edge of a lesion; all these may influence the orientation of the specimen during processing and the interpretation of the results. In some instances, fresh unfixed skin may be required; this may either be frozen for immunologic stains (e.g. in blistering eruptions, Fig.2.25, or for some lymphocyte markers), or be cultured (especially in mycobacterial or deep fungal infections). Immunofluorescence of blistering eruptions is discussed in more detail in Chapter 16.

    In the laboratory, the fixed tissue is processed as thin slices and stained, usually with hematoxylin (blue) and eosin (red). However, other special stains may also be used, depending on the likely pathologic process, to identify collagen, melanin, fungi, mucopolysaccharide, etc.

Figure. 2.25 Hemorrhagic bullae in a patient with pemphigoid. In this condition, direct immunofluorescence tests show deposits of IgG and complement C3 at the dermo–epidermal junction (see Ch.16).

    In the laboratory, the fixed tissue is processed as thin slices and stained, usually with hematoxylin (blue) and eosin (red). However, other special stains may also be used, depending on the likely pathological process, to identify collagen, melanin, fungi, mucopolysaccharide, etc.

    An important test in dermatology is the use of immunofluorescence, which is used primarily in diagnosis of blistering disorders and in some connective-tissue diseases and vasculitis. The most useful immunofluorescence test is known as direct immunofluorescence and uses frozen biopsy tissue sections. As an example, in pemphigoid (Fig. 2.19) deposits of IgG are expected to be seen at the dermo-epidermal junction, acting as an autoantibody against the bullous pemphigoid antigen, which is a normal constituent at this site. The section of biopsy tissue is therefore incubated in turn with an antibody (usually rabbit anti-human-IgG) raised against the protein under investigation (in this case human IgG), and then with a fluorescein-tagged antibody against the first antibody (such as fluorescein-labeled goat-anti-rabbit); examination under a fluorescence microscope then identifies the site of the protein deposits in the skin. This is discussed in more detail in Chapter 14.

    The main biopsy techniques are described here.

Excision biopsy

This is applied primarily to localized lesions, for which the aim is often both therapeutic and diagnostic. See Chapter 5.

Curettage

This involves scraping a lesion from the skin surface, usually with some form of hemostasis or cautery to the base. It is most commonly performed for treatment of localized lesions such as seborrheic keratosis or small basal cell carcinomas, and is discussed in Chapter 5. It is definitely not appropriate for diagnosis of rashes, and the pathologic specimen obtained is suboptimal generally if the issue is mainly diagnostic.

Shave biopsy

This technique uses a scalpel or razor blade to obtain a superficial sample of a localized lesion. Some small lesions can be removed completely by a saucer-shaped shave biopsy. The base is treated as for a curettage. It is mainly used for treatment rather than for diagnosis but can be of diagnostic value; it is particularly useful in confirming a diagnosis of basal cell carcinoma prior to radiotherapy, as many basal cell carcinomas can be painlessly and quickly biopsied in this way without need for local anesthesia.

Incisional biopsy

Incisional biopsies may be of various types. The most frequent and easiest to interpret histologically is an ellipse-shaped biopsy through the edge of a rash or lesion, consisting of two-thirds lesion and one-third adjacent normal skin. This has the advantage of providing the pathologist with the edge of the lesion, which is often informative, and also with adjacent normal skin as a control (particularly useful if immunohistochemistry is required). Note that the laboratory workers need to be told what has been done, as they will process a diagnostic ellipse of this type by cutting along the length of the specimen (Fig. 2.26).

    A more rapid technique, but one which provides smaller specimens and generally is limited to lesional skin, is use of punch biopsy.

Figure. 2.26 Diagram demonstrating how the laboratory will process a diagnostic ellipse into the edge of lesional skin. The aim is to have about one-third normal and two-thirds lesional skin, cut so that the histopathologist can see the interface between the two. By contrast, excision of a localized lesion will be processed perpendicular to the axis shown here (compare with Fig. 5.9). As specimens will shrink, and are generally pale after formalin fixation compared with the in vivo appearance, it is essential to tell the laboratory what technique has been used. A simple diagram is often best. (After an original drawing by Dr. N. H. Cox.)

PRACTICE POINTS

Hair disorders

Culture for fungal infection has already been discussed. Other investigations are discussed in this section.

Hair microscopy

This may involve light and electron microscopy, and is performed for identification of hair shaft defects, especially congenital hair shaft disorders with easily broken hair.

Hair pluck analysis

The ratio between growing (anagen) and involuting (telogen) hair roots can be determined using plucked samples of 20–50 hairs, and can be diagnostically useful in some situations (Fig. 2.27).

Scalp biopsy

This can give useful information about scalp disorders with effects on hair growth. The processing of scalp biopsies for histologic examination may be either along the line of the hair root or, in some instances, transversely.

Testing for disorders of sweating

Special techniques, such as pilocarpine stimulation, can be used to quantify sweat production but are primarily research tools. Identifying discrete areas of increased or decreased sweat production can be performed in a semiquantitative manner and is important in some genodermatoses with abnormal sweating, or in neurologic disorders with damage to sympathetic nerve fibers to the skin (such as reflex sympathetic dystrophy, Frey syndrome, or some cases of carpal tunnel syndrome). The starch–iodine test is the most frequently used in these situations (Fig. 2.28).

Phototesting for photosensitivity disorders

This specialized area is discussed in Chapter 28.

Figure. 2.27 Hair microscopy.

Figure. 2.28 Starch–iodine test. Starch in anhydrous oil is applied to the skin and then painted with iodine in anhydrous alcohol. In the presence of water, starch and iodine form a blue-black reaction product, so active sweating can be identified as small black dots over the sweat duct orifices. In this case, the patient complained of asymmetric color, sweating, and grip; this was due to sympathetic nerve damage from previous thoracic surgery, most easily demonstrated by the asymmetry of sweating illustrated. The sweating of his ‘good’ hand is so marked that the black reaction product of the test has pooled.

Figure. 2.29 ( a ) Radiology is not used extensively in dermatology but can be helpful, as in this patient with tophi due to gout. ( b ) The punched-out erosions occurring in the phalanges of the case shown in (a).

Radiology and ultrasound

These are rarely used other than for investigation of systemic features of disease. However, radiology may be useful in detection of cutaneous calcinosis (e.g. childhood dermatomyositis) and calcification of cartilage (e.g. of the ear in recurrent cold injury), or for illustrating bony abnormalities (Fig.2.29). Ultrasound of skin is used primarily as a research tool but may be useful in monitoring sclerodermatous processes or in the assessment of angiomas.

White/Cox: Diseases of the Skin, 2ed.(c) 2006, Elsevier Inc. All rights reserved.