Click on the following for more information: 


Skin swabs

  • For bacteriology, and virology if appropriate
  • In order to get a good sample it is important to dip the swab in the culture medium and then rub it against the skin 
  • Make sure that request forms are thorough and include a history of recent antibiotics and known allergies to antibiotics 

Mycology - skin scrapings and clippings

  • The sample depends on the site eg skin, nails or scalp
  • Refer to the investigation section in the chapter Tinea for more information

Blood tests

  • For individual conditions refer to the A-Z of clinical guidance section of this website 
  • For immunological tests please refer to the section on immunology on this page

Skin biopsies

Histology - light microscopy 

Light microscopy is used for pathological interpretation. Lesions sent for histology must be accompanied by a relevant history, description and a differential diagnosis to aid the histopathologist. It must be remembered that histology has its limitations:

  • It does not always help when diagnosing difficult rashes - such patients are often instead best managed by being referred for a specialist opinion
  • It may fail to detect some cases of early lentigo maligna and cutaneous t-cell lymphoma
  • It can be difficult to differentiate histologically between some cases of dysplastic naevi and melanoma, and to this end any incompletely excised ‘dysplastic naevus’ must be re-excised
Immunofluorescence and immunohistochemistry
  • Please refer to the section on immunology, which can be found lower down this page
    Tissue culture and PCR of skin biopsies
    • Tissue culture can be used when looking for a number of cutaneous infections such as mycobacteria, fungi and leishmaniasis, all of which are best cultured from biopsies as opposed to swabs
    • The sample needs to be sent in a plain white-capped universal bottle, with a small amount of normal saline or water
    • With specific regards to tuberculosis (either mycobacterium tuberculosis, or atypical mycobacteria), it is important to also request for PCR (polymerase chain reaction) testing, which aids in diagnosis and significantly reduces the number of false negative results

    The teaching of histology lies outside of the scope of this website. The best way to learn about histology is to attend local dermatopathology and other MDT meetings. Or if you do not understand a histological result pick up the phone and speak to the local histopathologist or dermatologist. Additional resources include Leeds Virtual Pathology.


    Allergy tests

    A thorough history and examination are needed to help identify allergy-related dermatological conditions, even then it may not always be possible to identify the precise allergen.

    The following provides an outline of the different clinical presentations that can result from allergic reactions - the links provide more detailed information including any recommended investigations:

    Acute (or intermittent) urticaria, angioedema or anaphylaxis

    • Have a multitude of causes of which food allergy is one
    • Chronic urticaria is very unlikely to be allergic in nature

    Latex allergy

    • Causes a contact urticaria, which causes acute swellings after contact with rubber gloves, balloons, condoms and certain medical / surgical equipment

    Swellings of the mouth / perioral skin

    • In infants and very young children, marked eczematous changes around the mouth after eating could suggest food allergy
    • Recurrent oral swellings (lips, mouth or throat) could suggest the Oral Allergy syndrome, caused by a food allergy to raw fruit, vegetable and nuts. This is mainly found in adults who may also have hay fever
    • Chronic eczematous changes to the lips could suggest a contact allergic dermatitis to a number of allergens including lipstick and toothpaste
    • Old amalgam fillings can occasionally cause a contact allergic dermatitis resulting in oral symptoms and occasionally swelling of the cheeks

    Childhood eczema

    • Food allergy has a role to play in approximately seven percent of patients with atopic eczema
    • Food allergy should be considered in infants and young children, normally under the age of three years, who have any of the following:
      • Moderate-severe eczema that responds poorly to appropriate topical treatments
      • Associated GI symptoms (reflux, vomiting, diarrhoea), failure to thrive
      • Wheeze

    Contact allergic dermatitis

    • Contact allergic dermatitis can result from allergic reactions to a wide range of household and workplace products, cosmetics and medicaments
    • The most common sites for reactions include the hands, face (including lips and eyelids), feet, peri-anal skin and around leg ulcers being treated with a variety of different topical medications and bandages
    • Certain pollens can cause a contact allergic dermatitis of the face in the spring and summer months

    Photo-allergic reactions

    • Some photo-dermatoses are allergic in nature
    • Certain plants cause a phytophotodermatitis resulting in a streaky and sometimes bullous eruption
    • Sunscreens and topical NSAIDs are occasionally associated with a photo-contact allergic dermatitis
    • Systemic drugs can be associated with photo-allergic reactions

    Adverse cutaneous drug reactions (ACDR)

    • ACDR can present in a variety of ways

    Screening tests for connective tissue disorders, vasculitis, and pathological livedo reticularis

    Given that there can be significant overlap between these conditions, most patients require the investigations listed below (exceptions are several of the disorders that only have cutaneous pathology eg morphoea, discoid lupus erythematosus, and exercise-induced vasculitis):

    General screen 
    • full history and physical examination, including checking for lymphadenopathy and hepatosplenomegaly (lymphoproliferative disorders and other malignancies can sometimes be associated with a number of connective tissue disorders and vasculitis)
    • Renal involvement
      • In SLE, nephritis is common. Renal damage can also occur in other connective tissue disorders and/or vasculitis
      • Relevant findings can include:
        • New or deteriorating hypertension
        • Proteinuria and haematuria
        • Dysmorphic / fragmented red cells, casts
        • An abnormal urine protein:creatinine ratio (a morning random sample)
        • Abnormal U&Es / creatinine 
      • If findings suggest renal involvement patients should be referred urgently to a renal department   
      • Persistent microscopic haematuria can also point to a urological malignancy 
    • Blood tests
      • FBC - to exclude lymphoproliferative disorders, which can present with numerous cutaneous features. A raised eosinophil count can be seen in drug-induced reactions and the Churg-Strauss syndrome
      • Inflammatory markers such as CRP and ESR - in SLE the ESR level is generally raised, whereas the CRP is normal
      • U&Es / creatinine 
      • LFT - raised LFT could suggest systemic involvement, a drug reaction, or hepatitis
      • Other standard biochemistry 
      • Autoantibodies - ANA as a screening tool. See notes below for details on when other autoantibodies are required  
      • Immunoglobulins and plasma electrophoresis - myeloma and other causes of paraproteinaemia can be associated with cryoglobulinaemia and some other types of vasculitis
      • Complement levels (C3 and C4) - low complement levels can be found in SLE, urticarial vasculitis, and cryoglobulinaemia
    • CXR 
      • To exclude paraneoplastic causes of connective tissue disorders and vasculitis 
    Additional investigations - required in some cases
    • Connective tissue disorders
      • Refer to the sections below on autoantibodies 
    • Vasculitis
      • Bloods - all patients require Hepatitis B & C screen, HIV screen, and an ANCA antibody screen. Check an ASO titre in cases of suspected Henoch–Schönlein purpura
      • Skin biopsies of an active lesion for both for histology (light microscopy) and direct immunofluorescence (a positive IgA suggests additional renal involvement), and occasionally tissue culture:
        • Punch biopsies for some cases of small vessel vasculitis 
        • Deep-incisional biopsies are needed for medium-large vessel vasculitis
        • In cases of nodular vasculitis samples are also required for tissue culture and PCR (polymerase chain reaction) testing to look for mycobacteria 
    • Consider cryoglobulinaemia in patients with cold-induced urticaria and severe Raynaud's phenomenon associated with tissue necrosis (refer below for more information) 
    • A thrombophilia screen should be checked in patients with SLE and/or pathological livedo reticularis (which is persistent, as opposed to physiological livedo in which the signs improve when the skin warms), as well as cases of livedoid vasculopathy to look for coagulation disorders such as the antiphospholipid syndrome, and fibrinolytic disorders such as protein C deficiency and factor V mutation (Leiden)

    Immunology: antinuclear antibodies 

    • The ANA test identifies autoantibodies that target substances contained inside cells. Although the name implies that the test detects only autoantibodies directed against components of the nucleus, the test can also be used to detect antibodies directed against cellular components that are contained within the cell cytoplasm, outside of the nucleus
    • There are many antinuclear antibodies including dsDNA, histones, centromere (as well as antibodies against the antigens of the extractable nuclear antigens ie ENA - see below
    • Patient samples are often screened for ANA after being diluted 1:40 and 1:160 in a buffered solution. If staining is observed at both the 1:40 and 1:160 dilutions, then the laboratory continues to dilute the sample until staining can no longer be seen under the microscope. The level to which a patient's sample can be diluted and still produce recognisable staining is known as the ANA "titre." The ANA titre is a measure of the amount of ANA in the blood. A higher titre, resulting from more autoantibodies, means that the result is more likely to be relevant 
    • ANA titres: 
      • 1:40 may be found in 32% of the normal population - age, smoking and previous chronic illness can contribute to ANA positivity
      • Only 5% of a healthy population have an ANA titre of 1:160
      • Titres above 1:160 are considered positive (eg 1:320)
    • ANA pattern:
      • There are several patterns of anti-nuclear antibodies. The ANA staining patterns are loosely associated with underlying autoimmune diseases. The patterns seen are as follows:
        • Homogenous - the entire nucleus is stained with ANA. This is the most common pattern and can be seen with any autoimmune disease. Homogenous staining can result from antibodies to DNA and histones. Homogenous is the most common and the least concerning pattern of ANA
        • Speckled - fine and coarse speckles of ANA staining are seen throughout the nucleus. This pattern is more commonly associated with antibodies to extractable nuclear antigens (ENA). This pattern can be associated with systemic lupus erythematosus, Sjögren’s syndrome, systemic sclerosis, dermatomyositis, rheumatoid arthritis, and mixed connective tissue disease
        • Nucleolar - the ANA staining is seen around the nucleoles inside the nucleus. This can be seen in systemic sclerosis
        • Centromere - the ANA staining is seen along the chromosomes. This pattern can be associated with limited systemic sclerosis, primary biliary cirrhosis, and other autoimmune diseases like Raynaud’s Phenomenon
    • Patients with the following autoimmune diseases may have a positive ANA test:
      • Lupus erythematosus (LE) 
      • Morphoea
      • Systemic sclerosis 
      • Dermatomyositis
      • Sjögren's syndrome 
      • Mixed connective tissue disease
      • Rheumatoid arthritis 
      • Oligoarticular juvenile chronic arthritis
      • Polyarteritis nodosa
    • Patients with organ-specific autoimmune diseases can have a positive ANA. These diseases include:
      • Thyroid diseases (Hashimoto thyroiditis, Grave disease)
      • Gastrointestinal diseases (autoimmune hepatitis, primary biliary cholangitis [also known as primary biliary cirrhosis], inflammatory bowel disease)
      • Pulmonary diseases (idiopathic pulmonary fibrosis)
    • Patients with infectious diseases may also test positive for ANA. These diseases include:
      • Viral infections (hepatitis C, parvovirus)
      • Bacterial infections (tuberculosis) 
      • Parasitic infections (schistosomiasis)
    • Other associations with positive ANA tests have been noted, including:
      • Various forms of cancer (rarely)
      • As a harbinger of the future development of autoimmune disease
      • Various medications, without causing an autoimmune disease
      • Having one or more relatives with an autoimmune disease

    If the ANA is positive at a titre suggestive of underlying pathology, labs will normally proceed with other tests to identify which protein(s) the antibodies are attacking such as the anti-dsDNA test and the ENA panel.


    Immunology: other autoantibodies relevant to dermatology including ENA (extractable nuclear antigens) and ANCA

    ENA - extractable nuclear antigens
    • ENA are specific antigens within the nucleus
    • The main antigens are:
      • RNP (U1-snRNP) - found in both SLE and mixed connective tissue disease (MCTD). In MCTD the presence of these antibodies is required for diagnosis, whereas they occur in only 30-40% of SLE patients
      • Scl-70 (topoisomerase-1) - characteristic and specific for systemic sclerosis. High titres may be more specific for MCTD, and are reported to occur less frequently in SLE
      • Sm - these are a highly specific but comparatively insensitive clinical marker for SLE
      • Ro60 (SS-A) - occur in SLE (prevalence 40-50%) and Sjogren's syndrome (prevalence 60-75%). They have been reported to occur in association with certain disease subsets such as subacute cutaneous LE and neonatal lupus
      • La (SS-B) - the serological hallmark of Sjogren's syndrome, however a small proportion of patients do remain negative. Also present in mothers of newborns with neonatal lupus
      • Jo-1 - can be found as markers of dermatomyositis
    • Patients with cutaneous lupus or systemic sclerosis occasionally have negative ANA levels but positive ENA levels. When testing for suspected cases of cutaneous lupus and systemic sclerosis it is important to request both ANA and ENA, even if the ANA test is negative 
      ANCA (anti-neutrophil cytoplasmic antibodies)
      • These tests should be requested in patients presenting with vasculitis, although they can be found connective tissue disease without vasculitis
      • PR3-ANCA (c-ANCA pattern) is positive in 90% of patients with Wegener's granulomatosis (syn. granulomatosis with polyangiitis), and much less commonly in microscopic polyangiitis
      • MPO-ANCA (p-ANCA) is positive in most patients with microscopic polyangiitis, and in 50-80% of patients with the Churg-Strauss syndrome (syn. allergic granulomatous angiitis)
      Other autoantibodies
      • The number of autoantibody tests continues to increase
      • For more detail refer below to the section on specific findings in connective tissue disorders 

      Immunology: complement and cryoproteins 

      Complement - C3 and C4
      • Hypocomplementaemia (low complement levels) can be found in a number of conditions such as urticarial vasculitis, SLE and cryoglobulinaemia
      • Complement levels should also be tested in patients with angioedema without urticaria -  reduced levels would prompt further investigations looking for C1 esterase inhibitor deficiency
      Cryoproteins
      • There are several cryoproteins including cryoglobulins, cryoagglutinins and cryofibrinogens 
      • Cryoglobulinaemia is associated with a number of conditions such as haematological malignancies, connective tissue disorders and hepatitis C. Cryoglobulinaemia can present in the skin as severe Raynaud's phenomenon associated with tissue necrosis, cold-induced urticaria and some cases of cutaneous vasculitis
      • Cryoglobulins are very unlikely to be present if complement levels and rheumatoid factor are normal. If levels need to be checked thy can only be tested in Secondary Care as stringent temperature control, maintaining the sample at 37 degrees Celsius, is needed from the time the sample is collected until separation of the serum

      Immunology: specific findings in connective tissue disorders

      Lupus erythematosus
      • If lupus is suspected ask for both ANA and ENA as some cases of cutaneous lupus can be ANA negative and ENA positive
      • ​SLE - more than 98% of patients will be positive for ANA, which must be a titre of 1:80 or greater. 50-70% have raised levels of double-stranded DNA (dsDNA), which are quite specific to SLE, as is the Sm antibody. Other positive results can include Ro (very photosensitive),  La (neonatal), and RNP (Raynaud’s overlap) 
      • Subacute cutaneous LE - 80% positive for ANA, 60% positive for Ro60/SSA
      • Discoid LE - 35% positive for ANA
      • ANA can be positive for years before lupus manifests
      Morphoea
      • Up to 50% of patients have elevated levels of ANA, anti-histone antibodies, or anti-ssDNA antibodies (single-stranded DNA). The presence of two or more of these autoantibodies appears to have a cumulative effect on correlation to disease affecting the deep muscle, fascia and tendons, resulting in joint contracture and limited mobility. RF also appears to be a strong indicator of deeper tissue disease when arthritis is associated 
      • Other autoantibodies are observed at frequencies below 10%
      Systemic sclerosis 
      • Centromere, Scl-70, RNA polymerase III, Fibrillarin (U3RNP), and Pm-Scl are the antibodies most commonly associated with systemic sclerosis 
      • Individual antibodies can help predict organ-specific complications:
        • SCL-70 - a very high risk of early lung fibrosis
        • RNA polymerase III - a 10 times increased risk of renal crisis
        • Fibrillarin (U3RNP)- a much increased risk of pulmonary hypertension
      Dermatomyositis (DM) and related conditions  

      Additional testing for myositis specific antibodies is helpful as they can help predict different clinical patterns. The following list continues to evolve:

      • Mi-2 - florid cutaneous changes, mild myositis
      • SAE1 - cutaneous changes
      • J0-1 - an increased frequency of interstitial lung disease, joint involvement, and “mechanic’s hands”
      • MDA-5 - untreated, this has a very poor prognosis with rapidly progressing lung disease and ulcerating skin lesions 
      • TIF1 and NXP-2 - a much higher association with malignancy
      • 5NT1A - associated with inclusion body myositis 
      • HMGCR and SRP - associated with necrotising myopathy (the former is related to statins)
      Sjögren's syndrome 
      • ANA - positive in most cases
      • Rheumatoid factor (RF) - often positive
      • Ro and La are often positive. As well as Ro60, which has a much stronger association with primary Sjogren’s, Ro52 is being increasingly recognised in secondary Sjogren’s and may also be a marker for a subset of patients with systemic sclerosis
      Mixed connective tissue disorders 
      • Almost 100% positive for ANA, many positive for RNP

      It is important to remember that although the antibody patterns referred to above are characteristic, the individual antibodies are not always found in the conditions mentioned and their absence does not rule necessarily exclude the condition.


      Immunology: the Antiphospholipid (APL) syndrome

      • The antiphospholipid (APL) antibodies are the anticardiolipin (ACL) antibody, the lupus anticoagulant (LAC), and the anti-beta2 glycoprotein-1 antibody. The LAC correlates most strongly with clinical manifestations
      • APL antibodies may be found periodically in 1-5% of apparently healthy individuals. Their prevalence increases with age and may be influenced by chronic disease, infections, malignancies, and certain drugs. Positivity in these patients usually occurs with low titres and is seldom persistent   
      • The antiphospholipid syndrome consists of persistently positive APL antibodies (two tests conducted 12 weeks apart) associated with arterial / venous thrombosis and / or adverse outcomes in pregnancy (mother or foetus). 
      • Cutaneous features may include thrombophlebitis, purpura, non-palpable livedo reticularis, leg ulcers and necrosis
      • The antiphospholipid syndrome occurs in isolation (primary APL syndrome) in more than 50% of patients, but it can be associated with other autoimmune disease - 20 to 35% of patients who have SLE will develop the secondary APL syndrome
      • Management
        • Patients with suspected APL syndrome need to be referred to an appropriate specialist
        • Those with a history of thrombosis, and those at a higher risk of thrombosis (eg all 3 antibodies positive) are considered for warfarin (as opposed to other anticoagulants)
        • Other patients need to modify risk factors for thrombosis
        • Women planning pregnancy need particular counselling from an obstetrician and are considered for prophylactic treatment with heparin

      Immunology: histology for light microscopy, direct immunofluorescence (immunohistology), and immunohistochemistry

      Light microscopy
      • The standard histological examination for most biopsy specimens
      Direct immunofluorescence
      • Some skin conditions benefit from an additional biopsy for direct immunofluorescence
      • Relevant conditions include suspected cases of pemphigus, pemphigoid, epidermolysis bullosa acquisita, linear IgA bullous dermatosis, dermatitis herpetiformis, discoid lupus erythematosus, systemic lupus erythematosus, porphyria, amyloid, and is some circumstances lichen planus
      • Patients suspected of having the conditions listed above are best referred to a specialist for further investigation. The way in which a biopsy is taken for direct immunofluorescence varies between conditions. Samples needs to be placed in Michel's medium before being sent for analysis (alternatively put the sample on a small piece of gauze soaked in normal saline and place in a dry specimen pot - if collected this way, the sample needs to be examined by the pathologist the same day). For more information refer to the St John's Institute of Dermatology
      Immunohistochemistry (IHC)
      • On occasions a pathologist will go on to perform additional tests on biopsy specimens. The use of immunohistochemistry is increasing in the diagnosis of various conditions, and is used especially in cases where it may not be possible to differentiate entities with overlapping clinical and histopathological features
      • IHC is the method of localisation of antigens in tissue sections using labelled antibodies, visualized by markers (chromogen). Theoretically, any antigenic cellular component, which can be retained (even if partially) in tissue section, should be demonstrable by IHC. The antibody is aimed to locate a specific antigen expression site (epitope) which remains masked normally. The marker used may be a fluorescent dye, enzyme-system, radioactive element or colloidal gold
      • The principles of immunohistochemical analysis were first conceptualized by Coons et al. Since then it has been in use to detect abnormal antigens in various malignant tumours, especially lymphoma and melanoma. The technique is also of value in immunobullous and hereditary or acquired mechanobullous disorders, and a range of genetic conditions