Immunology

This section will discuss the following:

• Autoantibodies – ANA, ENA and ANCA
• The antiphospholipid (APL) syndrome
• Hypocomplementaemia
• Cryoproteins
• Histology for light microscopy, electron microscopy and direct immunofluoresence

Autoantibodies

ANA - antinuclear antibodies

• There are many ANAs including dsDNA, histones, centromere and Mi-2 as well as antibodies against the six antigens of the ENA screen (see below)
• ANA at a titre of 1:40 may be found in 32% of the normal population - age, smoking and previous chronic illness can contribute to ANA positivity
• As the ANA figure increases so does the relevance such that only 5% of a normal population have an ANA titre of 1:160. Titres above 1:160 are considered positive
• If ANA is positive the labs normally go on to test ENA

ENA - extractable nuclear antigens

• These are specific antigens within the nucleus, which may be responsible for elevated ANA titres
• The six main antigens are Ro, La, RNP, Sm, Jo and DNA topoisomerase-1 (also known as Scl-70)
• Positive Ro antibodies can be seen in lupus (normally Ro 60, also Ro 52), Sjogrens, rheumatoid arthritis and primary bilary cirrhosis. They are occasionally found in scleroderma and dermatmyositis
• Jo-1 antibodies can be positive in dermatomyositis
• Scl-70 antibodies can be positive in scleroderma
• Patients with lupus or scleroderma can sometimes have negative ANA levels but positive ENA levels. When testing for suspected cases of lupus it is important to request ANA and Ro/La, when testing for suspected cases of scleroderma request ANA and Scl-70

Relevant ANA / ENA autoantibody profiles

• Lupus erythematosus 
  • SLE - up to 100% of patients will be positive for ANA and moderate to high levels of double-stranded DNA (dsDNA), which are quite specific to SLE. Other positive results can include Sm, Jo-1, RNP, Ro and La
  • Subacute cutaneous LE - 80% positive for ANA, 60% positive for Ro
  • Discoid LE -  35% of cases positive for ANA
  • Drug induced lupus - anti-histone antibodies can be found in 95% of cases of drug-induced lupus compared with 50% of cases of idiopathic lupus
• Mixed connective tissue disorders – almost 100% positive for ANA, many positive for RNP
• Sjogrens syndrome – Ro and La together (Ro without La can be found in lupus)
• Scleroderma – not all will be positive for ANA. Anti-centromere antibody (ACA) is the most common finding in localised morphoea, it is also found in 82-96% of patients with limited systemic sclerosis (previously known as CREST), and less frequently in progressive systemic sclerosis and other connective tissue disease. Scl-70 is the most frequent finding in diffuse morphoea, early internal organ involvement and a worse prognosis. RNP may also be positive
• Polymyositis / dermatomyositis – characterised by Jo-1 and, in dermatomyositis particularly MI-2. ANA often negative

ANCA (anti-neutrophil cytoplasmic antibodies)

• These are of particular importance when vasculitis is suspected but they can also be found in connective tissue disease without vasculitis
• ANCA positivity is found in around 90% of cases of Wegener’s granulomatosis, 70% of microscopic polyangiitis, 50% of Churg –Strauss syndrome and less than 20% of cases of polyarteritis nodosa. ANCA positivity can occur in inflammatory bowel disease

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

The antiphospholipid (APL) syndrome

• The most notable antiphospholipid (APL) antibodies are the anticardiolipin (aCL) antibody and lupus anticoagulant (LAC). The other is the anti-beta2 glycoprotein 1 antibody. The LAC correlates most strongly with clinical manifestations 
• APL antibodies may be found periodically in I-5 % of apparently healthy individuals. The 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 (2 tests carried out 12 weeks apart) associated with arterial/venous thrombosis and/or adverse outcomes in pregnancy (mother or foetus)
• The antiphospholipid syndrome occurs in isolation (primary APL syndrome) in more than 50% of patients, but it can be associated with other autoimmune disease - 20-35% of patients who have SLE will develop a secondary APL syndrome
• Patients with suspected APL syndrome need to be referred to an appropriate specialist. Those with a history of thrombosis require warfarin. Those who have not had a thrombotic events but who have SLE should be considered for aspirin, although the benefits vs. the risks are not clearly defined. Patients with the APL syndrome planning a pregnancy need particular councelling from an obstetrician and are considered for prophylactic treatment with heparin

Hypocomplementaemia

Complement levels should be checked in a number of dermatological presentations:

• Hypocomplementaemia (reduced complement levels) can be found in a number of conditions such as urticarial vasculitis, cutaneous vasculitis and SLE

• C4 (complement 4) levels are often used as a screening tool to help detect other conditions:
  • A low C4 level in a patient with angioedema without urticaria needs further testing to rule out C1 esterase inhibitor deficiency (see chapter on urticaria and angioedema)
  • Reduced C4 levels can also be found in some cold-induced disorders (see below)

Cold-induced disorders and cryoproteins

• There are several cryoproteins including cryoglobulins, cryoagglutinins and cryofibrinogins 
• Patients with significant cold-induced disorders such as cold urticaria or Raynauds associated with tissue damage should be screened as follows:
  • FBC, PV, routine biochemistry, ANA, C4, Rheumatoid factor, immunoglobulins and plasma electrophoresis
  • Depending on the results of the above it may then be necessary to check for cryoproteins – this is best performed in the hands of a dermatologist
  • In order to test for cryoproteins contact must be made with the local immunology department as the sample needs to be kept at 37 degrees C, which can be achieved by placing the sample inside sand in a vacuum flask
    

Histology for light microscopy, electron microscopy and direct immunofluoresence (immunohistology)

Certain conditions such as bullous disorders and lupus lend themselves to immunological investigations of skin biopsies. Patients suspected of having these disorders are best managed by a dermatologist

• Most bullous disorders (except dermatitis herptiformis) and lupus:
  • Excise an ellipse of affected skin; if bullous an intact lesion is preferable. The specimen should be sent in a standard histology bottle for electron microscopy if bullous, otherwise request routine light microscopy
  • A punch biopsy of normal peri-lesional skin also needs to taken, preferably from a non-sun exposed site if the rash allows. The specimen should be placed on a small piece of gauze dampened by sodium chloride and put in a dry pot. Request direct immunofluoresence on the histology form – the lab must be informed of the request immediately and the specimen tested the same day to prevent it from drying out
    
• Dermatitis herpetiformis:
  • Excise a lesion as above and send for electron microscopy
  • A punch biopsy also needs to be taken from unaffected buttock skin and sent as above for direct immunofluoresence