Investigations

Connective tissue disease

CTDs are a heterogeneous group of diseases characterized by abnormal structure or function of one or more of the elements of connective tissue. Differential diagnosis of CTDs is mainly based on clinical findings but is complicated by the similarity of their symptoms. Therefore, autoantibodies are useful markers to support the diagnosis or exclusion of CTDs. The most prominent CTDs are systemic lupus erythematosus (SLE; potentially affecting all organs), Sjögren’s syndrome (SS; characterized by diminished lacrimal and salivary gland secretions), scleroderma (systemic sclerosis; a chronic, progressive dermatosis), limited systemic sclerosis (CREST syndrome), with a more benign disease course), poly-/dermatomyositis (PM/DM; an acute or chronic inflammatory disease of muscle and skin), and mixed connective tissue disease (MCTD; a syndrome with features of scleroderma, rheumatoid arthritis, SLE and PM/DM). 

The CTD screen test contains the following tests: U1RNP, SS-A/Ro, SS-B/La, Centromere B, Scl-70, Jo-1, Fibrillarin, RNA Pol III, Rib-P, PM-Scl, PCNA, Mi-2, SmD and dsDNA 

A positive CTD screen is further investigated for ds-DNA and ENA antibodies. 

Coeliac Disease

Coeliac disease is an autoimmune condition in which the ingestion of gluten, the water soluble wheat-gliadin and the prolamins in rye and barley causes chronic inflammation and damage to the small intestinal mucosa. IgA TTG has been identified as the major auto-antigen in coeliac disease.  

IgA Tissue transglutaminase (TTG) is 90% specific for coeliac disease patients while endomysial antibodies (EMA) are 98% specific. Both have comparable disease sensitivity and both tests will detect >95% of coeliac patients 

TTG screening will detect those patients with coeliac disease and positive results are confirmed with IgA endomysial antibody testing. Known coeliacs can be monitored by detection of tissue transglutaminase antibodies for dietary compliance.  

It is important to ensure patients are on a gluten containing diet 6 weeks prior to testing to prevent false-negative results.

Patients with IgA deficiency can be tested for coeliac disease using the IgG tissue transglutaminase assay. Further IgG based coeliac disease testing can be performed determined on a case-by-case basis. 

Skin disease

Antibodies to antigens in the dermo-epidermal junction are found in pemphigoid disease 

These antibodies are also seen in Herpes Gestationis and in Dermatitis Herpetiformis 

High titre antibodies to intercellular antigens in the stratified epithelium are found in pemphigus disease 

These antibodies are also found in 5-10% of RA patients on penicillamine, the antibody disappears when the penicillamine is discontinued 

They are also seen in some patients with cutaneous fungal infection or thermal burns 

Though the diagnosis of DH is based on the appearance of the rash and IgA at the dermo-epidermal junction in the dermal papillac in biopsies, the presence of the antibodies associated with coeliac disease may point to the association of DH with gluten sensitivity (see Coeliac disease) 

Vasculitis Investigations

The term vasculitis refers to inflammation of blood vessels and represents a heterogeneous group of clinical disorders 

Immunopathological mechanisms may be involved in primary (e.g. Wegener’s) and secondary vasculitides (e.g. infection, neoplasia, connective tissue disease, cryoglobulinaemia) 

If there is evidence for more extensive visceral involvement, one of the primary systemic vasculitides may be involved 

Alternatively, the vasculitis may be secondary to autoimmune disease (e.g. SLE, chronic active hepatitis), neoplasia (e.g. lymphoma), cryoglobulinaemia (these are immunoglobulins that form precipitates in the cold) 

Some forms of systemic vasculitis are strongly associated with circulating antibodies to neutrophil cytoplasmic antigens (ANCA) 

The diffuse cytoplasmic pattern (C-ANCA) is seen most typically in Granulomatosis with Polyangiitis.  PR3 (proteinase 3) antibodies are thought to correspond to C-ANCA and act as a serological marker for disease activity with decline in the antibody level in remission 

MPO (myeloperoxidase) antibodies in a large proportion of cases correspond to the P-ANCA seen by immunofluorescence. 

MPO antibodies are seen in patients with a variety of vasculitic disorders, most notably patients with small vessel arteritis with renal impairment 

In these patients MPO antibodies appear to be a good serological marker for disease activity as the antibody levels decline in remission 

Sera of patients with acute pulmonary-renal syndrome or GPS like presentation will also be subjected to measurement of anti-glomerular basement membrane (GBM) antibodies 

Urgent requests include: 

• Pulmonary renal syndrome, i.e. Good Pasteur’s syndrome (GPS) like presentation 

• Suspected Granulomatosis with Polyangiitis in kidney 

• Cerebrovascular events in young patients with clinical suspicion of vasculitic involvement 

• ANCA and GB tests can be performed urgently. Please contact the department if an urgent sample is being sent.

Allergy Investigations

Allergy is a type I hypersensitivity reaction mediated by IgE together with inflammatory cells

Such a reaction could occur systemically (as in anaphylaxis) or could be localised to target organs (eye, airway mucosa etc)

Genetic predisposition to produce excessive amounts of IgE leading to allergy is termed atopy

Measurement of serum total IgE is a simple way to determine an individual’s atopic status

Diagnosis of allergy must be made in conjunction with clinical history and where possible supplemented with skin prick testing (SPT)

Specific IgE

The Immunology department uses an automated system for allergy testing

It is an extremely specific and sensitive method and is used by the majority of laboratories in the UK

Low levels of IgE are normally accompanied by lower levels of specific IgE levels

Comparatively high levels of IgE often present higher levels of specific antigen reaction

This Department reports the total protein measured in kU/L.

The class is measured on a scale of 0 to 6. Class 0 and class 1 reactions represent an insignificant reaction whereas 2-6 have increasing significance, however there is no correlation between the grade and severity of the reaction since the presence of allergen specific IgE is a marker of exposure only, therefore a positive result can be observed in the absence of a clinical reaction

In addition levels of IgE fall when exposure is reduced, therefore low or negative results are shown in sensitised patients or in patients that have not had recent exposure

It should be noted also that if a reaction is highly localised there may be insufficient circulating IgE for detection

It is therefore crucial that allergy results are regarded with the clinical history of the patient

Some drug Specific IgE tests are available but if the determinants are limited (such as with penicillin) a negative result should not exclude allergy

Specific IgE investigations are conducted through the use of panel testing such as the common food panel or common airborne allergen panel

Where panels are positive individual allergens will be analysed

Adverse Drug Reaction Investigations

Mast cell tryptase indicates the level of mast cell degranulation that has occurred in serum

It is therefore a useful marker to establish whether an anaphylactic or adverse drug reaction has occurred

Three clotted specimens are required to assess the reaction (yellow top)

The first should be taken at the event, the second must be taken three hours post event and the final specimen should be taken 24 hours post event to measure baseline levels

Please ensure that all three specimens are clearly labelled with the respective time of phlebotomy

Immunodeficiency Investigations

It is advised that all patients with suspected primary immunodeficiency (PID) be referred to the Immunology Clinic

Please telephone the Department to discuss appropriate investigations and patient referral if required

Anti-phospholipid syndrome

Antiphospholipid antibodies, including anticardiolipin antibodies, are frequently detected in sera from patients with SLE.  Numerous reports have associated these antibodies with various venous and arterial thrombosis disorders, including cerebral infarction, deep venous thrombosis, thrombocytopaenia, pulmonary embolism and recurrent foetal loss with placental infarction.

Cardiolipin antibody positive assay results require confirmation by repeat testing at least 12 weeks, to account for false-positive results (due to infection).

Autoimmune screen

Primary Biliary Cholangitis (PBC) is a liver disease that damages bile ducts. Early treatment can help prevent liver failure. 90% of PBC patients are female middle aged and older. Smoking has also been found to be a risk factor.

Chronic hepatitis is an inflammation of the liver that lasts more than 6 months. Common causes of chronic hepatitis are Hep-B and C viruses, fatty liver disease, alcohol-related liver disease and certain drugs.

Positive liver autoantibody results will be called through for children (>18 years old) with raised LFTs.