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Picking a path through the immunology testing maze

Consultant immunologist

Dr Gavin Spickett explains some of the basic tests available and how GPs can best make use of them

'm sure most GPs are mystified by immunology tests. What follows is for general guidance, but it is worth contacting your local immunology laboratory as certain details tend to vary.

Rheumatoid factor

This is the most abused test of all. It is not a test for rheumatoid arthritis and has no value as a screening test in patients with non-specific joint pain. It should only be used to differentiate sub-groups of patients with identified inflammatory arthritis ­ those who have a clinical diagnosis, based on history, physical examination and the presence of elevated acute phase markers such as CRP and ESR. In these patients, those who have high titres of rheumatoid factors are more likely to develop erosive disease.

Serial monitoring of rheumatoid factors has no prognostic value. Do not automatically refer patients with rheumatoid factors to the local rheumatologist, as most patients do not have rheumatoid arthritis.

The correct approach for a patient with joint pain is to check for evidence of acute inflammation and then to look for rheumatoid factors in those in whom there is a likelihood of an acute arthritis. Results may be reported in titres (semi-quantitative) or in international units (quantitative).

Autoantibody screen

Most laboratories will offer a fluorescent antibody screen on rodent tissue and a human cell line (Hep2 cells), which will detect a range of diverse antibodies. The detection of positive results may indicate underlying disease, and in some cases a need for further investigations, which may be initiated automatically by the laboratory. The indications for undertaking an autoantibody screen are:

lyoung patients with arthritis, photosensitive rashes, recurrent pleurisy, recurrent miscarriages, thrombocytopenia, impaired renal function (SLE and variants)

lpatients with Raynaud's (SLE, scleroderma, limited and diffuse)

lpatients with muscle pain and tenderness (myositis)

lpatients with dry eyes (Sjögren's syndrome and variants)

lpatients with pruritus, jaundice and/or abnormal liver function (primary biliary cirrhosis, autoimmune hepatitis, SLE)

lunexplained raised ESR (any of the above)

lmultisystem disease.

Just as with rheumatoid factor, false positive results can occur due to infection, particularly with chronic bacterial infections (SBE) and viral infections (adenovirus, herpes viruses, especially EBV). If an autoantibody is due to infection it will disappear gradually when the infection has resolved. If it is due to an underlying autoimmune disease it will persist. Antinuclear antibodies can also be induced by drugs, for example hydralazine, ACE inhibitors, minocycline and chlorpromazine. In this case a lupus syndrome may occur.

Antibodies are reported usually in titres: a significant titre for an adult is >1/80, but the significant level for a child is 1/20. Healthy elderly may have low titres (1/80 or less).

The tissue block also allows many other patterns to be identified, which give clues to other diseases. Liver-specific antibodies should prompt discussion with a gastroenterologist or hepatologist, and any such patient in whom LFTs are already abnormal should be referred at once. Low titre smooth muscle antibodies may be seen in many acute viral infections, not just with hepatitis viruses.

Identification of antibodies to gastric parietal cells is common, and is frequently associated with pernicious anaemia and thyroid autoimmunity. Most haematologists would not measure the B12, however, unless the FBC shows evidence of macrocytosis with or without anaemia. In true pernicious anaemia, the bilirubin and LDH are often raised due to low-grade haemolysis. Any patient with neuropathy who has gastric parietal cell antibodies should have their B12 checked.

IgA endomysial antibodies/IgA antibodies to tissue transglutaminase

These are equivalent antibodies and are specific and sensitive tests for coeliac disease. In treated coeliac disease, persistence of the antibody suggests there is gluten in the diet, and therefore non-compliance. However, it takes at least six months for the antibodies to disappear, so don't accuse your patient of cheating on their diet if antibodies are still present at three months. There is no place for antibodies to gliadin, as these are neither sensitive nor specific.

Particular symptom groups to target, other than those with steatorrhoea, are patients with persistent iron deficiency anaemia or osteoporosis. There is also an increased incidence of coeliac disease in patients presenting with chronic fatigue. IgA deficiency is common in coeliac disease and in these cases the laboratory will test for IgG endomysial antibodies, which have the same significance in IgA deficient patients as IgA endomysial antibodies in those with normal IgA levels.

Thyroid microsomal/peroxidase antibodies

Antibodies against thyroid microsomes have been recognised to be associated with thyroid disease, especially thyroiditis, for many years.

The target antigen has now been identified as thyroid peroxidase. Many laboratories still measure thyroid microsomal antibodies, and there is little clinical advantage from measuring thyroid peroxidase (although the assays can be automated).

Thyroid antibodies are present in all types of autoimmune thyroid disease, and do not reliably distinguish between the different types, although the highest levels are found in Hashimoto's thyroiditis. They will not be found in colloid or simple goitres. They are very strongly associated with antibodies to gastric parietal cell antibodies, as thyroid autoimmunity and pernicious anaemia often go together.

The serial monitoring of thyroid antibodies has no value except in acute severe thyroiditis, where the highest levels are predictive of subsequent hypothyroidism.

Detection of thyroid antibodies in pregnancy is important, as they increase the risk of post-partum hypothyroidism and may also be associated with neonatal hypothyroidism, although antibodies to the TSH receptor carry more risk in this regard.

Many patients in whom thyroid antibodies are detected by chance remain euthyroid for many years, but in the end are likely to develop overt thyroid disease. Presence of the antibodies therefore suggests that an annual check of thyroid function is desirable.

Immunoglobulins and


The immunoglobulins that are measured routinely are IgG, IgA and IgM. There are no indications for measuring IgD in general practice. IgE is covered below. The indications for requesting serum immunoglobulins are:

lsuspected immunodeficiency in children or adults

lknown or suspected liver disease

lknown or suspected Sjögren's syndrome

lknown or suspected myeloma, lymphoma or other paraprotein associated condition.

You should be aware that the normal serum immunoglobulins do not exclude immunodeficiency, and if the history is a strong one (recurrent infections) you should discuss the case with an immunologist.

Low immunoglobulins are, however, extremely suspicious and such cases should be referred immediately to an immunologist. The laboratory should always quote age-related ranges for serum immunoglobulins as there is considerable variation, especially in children.

When investigating suspected myeloma, send both serum and urine to the laboratory, as free light chain myelomas (Bence Jones myeloma) will only have abnormal proteins in the urine in the early stages.

Serum electrophoresis should always be requested with serum immunoglobulins, as the electrophoresis is the only way the presence of abnormal immunoglobulins can be detected.

Allergy tests

There is little value in measuring IgE. The normal range is complex and a normal result does not exclude the possibility of severe allergy. Conversely, a level above normal does not imply that symptomatic allergic disease will occur. Specific IgE ('RAST') testing is expensive.

The test of choice for identifying allergic disease is skin-prick testing, which needs to be carried out by an experienced operator to ensure consistent and reproducible results. RAST tests are helpful where skin-prick testing is not available, or inappropriate (small children, those on antihistamines, those with severe eczema or skin disease, dermographic patients, and those with severe reactions).

Requests to the laboratory must specify the allergens to be tested (blanket requests for 'RAST testing' will be returned. RAST testing is not suitable for many allergens, particularly foods, drugs and latex. It is satisfactory for most inhalant allergens (dust mites, pets, pollens, moulds), some foods (fish, nuts, peanuts) and some occupational allergens.

Causes of a positive rheumatoid factor

Bacterial and viral infection: TB, SBE, EBV, adenovirus, Common

other bacterial and viral infections

Healthy elderly Common

Connective tissue disease (RA, SLE, Sjögren's syndrome) Common

Haematological malignancy (myeloma, lymphoma), Less common

other malignancy

effective investigation

Interpretation of anti-nuclear and related antibodies

Pattern Disease association Further tests to confirm diagnosis (often done by lab)

Homogenous antinuclear SLE, drug-induced lupus Double-stranded DNA antibodies (dsDNA)

antibody; peripheral Antibodies to extractable nuclear antigens (ENA)

nuclear antibody Antihistone antibodies (drug-induced lupus)

Nucleolar antibody Scleroderma, Double-stranded DNA antibodies (dsDNA)

polymyositis, SLE Antibodies to extractable nuclear antigens

(ENA), including Scl-70, Jo-1

Speckled antinuclear SLE, Sjögren's syndrome, Double-stranded DNA antibodies (dsDNA)

antibody undifferentiated connective Antibodies to extractable nuclear antigens (ENA)

tissue disease

Centromere antibody Limited scleroderma (CREST) Diagnostic in its own right

Ribosomal antibody SLE Diagnostic in its own right

Interpretation of other antibodies identified in screen

Antibody Disease association Further tests

Mitochondrial antibody Primary biliary cirrhosis LFTs, M2 antibody (more specific marker;

(95 per cent of cases) S100 and gp210 antibodies) ­ none of these

Autoimmune hepatitis (rare) need serial monitoring

Smooth muscle antibodies Autoimmune hepatitis LFTs, antinuclear antibody and dsDNA

antibody; LC-1, SLA antibodies

Liver-kidney microsomal Autoimmune and LFTs (in children associated with a poorer prognosis)

antibody drug-induced hepatitis

Gastric parietal Pernicious anaemia, FBC, TFTs, thyroid microsomal/peroxidase antibody

cell antibody thyroid disease

Interpretation of immunoglobulins (common patterns)

Results Interpretation Further action

Low/absent IgG, IgA, IgM Immunodeficiency; Check urine electrophoresis to ensure that there

Bence Jones myeloma is not a Bence Jones protein; discuss with


Low IgG, IgA, normal IgM Immunodeficiency; loss Check urine for protein, check for diarrhoea, check

of immunoglobulins through serum albumin; discuss with immunologist

kidney, bowel

Low/absent IgA, Selective IgA deficiency ­ Check endomysial antibody; if symptomatic,

normal IgG, IgM common (may be associated discuss with immunologist

with coeliac disease, autoimmune

disease and immunodeficiency)

Raised single immunoglobulin Paraprotein present, consider Ensure urine sent for electrophoresis; discuss with

with monoclonal band myeloma, lymphoma and haematologist regarding criteria for referral

on electrophoresis; other monoclonal gammopathy of (usually all patients <60 years="" and="">

classes normal/low uncertain significance (MGUS) >60 years with paraproteins levels >10 g/l or symptoms)

IgG, IgA, IgM raised, Infection, inflammation pattern; Recheck in three months

electrophoresis shows myeloma unlikely

multiple small monoclonal

bands on a polyclonal


Raised IgM, electrophoresis Consider primary biliary cirrhosis Check LFTs (raised alkaline phosphatase)

shows no monoclonal band

Raised IgG, IgA, normal IgM; Consider liver disease Check LFTs

electrophoresis shows (autoimmune hepatitis),

no monoclonal band sarcoidosis

Raised IgG, normal IgA and Sjögren's syndrome Check ESR, autoantibodies

IgM; electrophoresis shows

no band or small band on

a polyclonal background

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