(Centre for Adult Immunodeficiency)

Interpretation of Test Results

Allergy testing
Allergy tests may help identify which allergens, suggested by the patients clinical history, could cause symptoms. However, the finding of an antigen-specific IgE in the serum does not prove that the antigen is responsible for the symptoms under investigation, nor does it necessarily indicate that avoidance measures will help the patient. Specific IgE testing provides similar, although not identical, information to skin testing; but may be particularly valuable in assessing some groups of patients (young children, extensive eczema/dermographism, taking antihistamines, past history of anaphylaxis). Requests for specific IgE should be guided by the clinical history. Multiple requests are discouraged. Total IgE is needed to interpret the significance of the specific IgE. The severity of allergic reactions is not proportional to the grade of specific IgE.

Sample requirements:

5ml clotted blood (Red)

Normal ranges:

Total IgE 0-120 ku/L (adult) Paediatric ranges are age related

Specific IgE:
Grade Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 Grade 6 Interpretation Grade 0 negative Grade 1 borderline positive Grades 2-6 moderate to very strong positive KUA/L <0.35 0.35 - 0.70 0.70 - 3.50 3.50 - 17.5 17.5 - 50 50 - 100 >100

Asthma/rhinitis
Total IgE is often in the normal range or slightly elevated. Specific IgE may be sought to inhalant allergens: Skin prick testing is the method of choice. The range of allergens tested should be sensibly guided by a careful history but should generally include allergens to which most people are exposed such as house dust mite. In asthma, IgE to Aspergillus is associated with the need for closer monitoring and maybe more intensive steroid treatment.

Atopic eczema

Total IgE is often markedly elevated in widespread disease and Specific IgE may be present at high level to allergens that cause no overt symptoms. Any positive specific IgE results therefore need careful interpretation.

Anaphylaxis
Please refer all patients for full clinical assessment to an allergy clinic. Laboratory tests need careful interpretation. Blood samples for mast cell tryptase taken as soon as possible after the reaction and at 4 hours post-reaction will be helpful to confirm that the reaction was anaphylactic. A baseline sample should be taken 24 hours later.

Acute urticaria
Total IgE and Specific IgE may help identify the causal antigen involved in type I hypersensitivity reactions, if there is a suggestive clinical history. Skin prick testing with suspect antigens is usually the investigation of choice.

Allergic bronchopulmonary aspergillosis

Total IgE, specific IgE to Aspergillus, and Aspergillus precipitins should identify cases due to hypersensitivity to the fungus. Total IgE may be raised in association with parasitic infestation.

Food allergy and intolerance

Evidence of specific IgE antibodies may be consistent with a diagnosis of food allergy in the context of an appropriate clinical history, but the presence of such antibodies does not prove clinical sensitivity. Elimination and food challenge testing may be more directed assessments. Laboratory immunology tests cannot help investigate non-IgE mediated (type 1 hypersensitivity) food intolerances.

Other hypersensitivity diseases

Extrinsic allergic alveolitis
Precipitins to avian proteins (Bird Fancier's Disease) indicate exposure but are not invariably associated with disease.

Gastroenterology
Coeliac disease
IgA anti-endomysial antibodies/IgA anti-tissue transglutaminase (TTG) antibodies are found in active disease, and can be used to monitor compliance with treatment. Similar antibodies are seen in dermatitis herpetiformis. IgA levels (measured in Clinical Biochemistry) should be measured as part of a routine coeliac screen (IgA deficiency affects approximately 1 in 50 coeliac disease patients and could cause false negative results). IgA anti-gliadin and particularly IgG anti-gliadin are less sensitive and specific and are currently performed only on paediatric samples.

Normal range

IgA anti-TTG IgG anti-gliadin IgA anti-gliadin

0-3 U/ml 0-10 U/ml 0-10 U/ml

IgA Endomysial results: Negative, Weak positive, Positive.

Pernicious anaemia
Antibodies to gastric parietal cells are classically associated with type A atrophic gastritis and are found in up to 90% of patients with early stage pernicious anaemia. However, antibodies to GPC are non-specific and can be seen in a variety of autoimmune diseases. If positive suggest check B12 level.

Method

Indirect Immunofluorescence screened at 1/40

Results

Negative, Weak Positive, Positive (graded +/++/+++)

Antibodies to intrinsic factor are highly specific for pernicious anaemia and are found in 50-75% of patients. They are rarely seen in healthy individuals.

Normal range
0-6 U/ml

Liver disease
Autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are associated with characteristic autoantibodies that are helpful in classifying the hepatitis and separating autoimmune hepatitis from other forms. Patterns may include antibodies to smooth muscle (actin) in Type I AIH, liver/kidney microsome (LKM) antibodies in Type II AIH and antibodies to mitochondria (M2) in PBC. ANA and anti-smooth muscle antibodies are also often moderately raised in viral hepatitis. Because of the overlap between the various different forms of hepatitis it is usually best to test for all the types of autoantibody - AMA, SMA, LKM and ANA. Serum protein

electrophoresis and quantification of the levels of IgG, IgA and IgM should be performed. AIH may be associated with polyclonal hypergammaglobulinemia. PBC may be associated with elevated IgM. Autoantibody testing currently has no place in the diagnosis of primary sclerosing cholangitis.

Method

Indirect Immunofluorescence screened at 1/40

Results
Negative, Weak positive, Positive (graded +/++/+++)

Endocrine disorders
Thyroid goitre / hypo / hyperthyroidism
The level of antibodies to thyroid peroxidase are raised in autoimmune thyroiditis (90% of hypo, >60% of hyper-) and also post-viral and post-partum thyroiditis. They are far less often raised in thyroid neoplasia/nodules/cysts, but their presence does not exclude these conditions. Testing of these antibodies in patients with normal thyroid function is not indicated.

Results

< 60 IU/ml 60-100 IU/ml > 100 IU/ml

negative borderline positive

Adrenal failure and gonadal failure

In this country Addison's disease is most often due to autoimmunity; the presence of antibodies to adrenal cortex strongly indicates an autoimmune cause. There may also be antibodies to steroid producing cells of ovary and testis. A small proportion of cases of premature menopause are due to autoimmune oophoritis. Some of these patients also have adrenal failure the same tests are done for both.

Results

Negative or Positive

Diabetes mellitus
Islet cell antibodies may be found early in the course of type I IDDM, but gradually disappear with time. They are not found in type II diabetes.

Results

Negative or Positive

Dermatology
Pemphigus / pemphigoid
Antibodies are found to the epidermal intercellular "cement" in pemphigus, and to the epidermal basement membrane in pemphigoid. The pemphigus-like pattern is also seen in some patients with leprosy, burns, penicillin rashes, SLE, MG with thymoma, dermatomycoses, erythema multiforme etc - that of pemphigoid in herpes gestationis and epidermolysis bullosa acquisita.

Results

Negative or Positive

Dermatitis herpetiformis
Though the diagnosis of DH is based on the appearance of the rash and IgA at the dermoepidermal junction in the dermal papillae in biopsies, the presence of IgA anti-endomysial, IgA anti-TTG, may point to the association of DH with gluten sensitivity - (see coeliac disease).

Rheumatoid factor and CCP

Rheumatoid factor
This is a non-specific test and is positive in a variety of conditions including viral infections, chronic bacterial infections, connective tissue diseases and lymphoid malignancy. The prevalence of rheumatoid factor increases with age. Although rheumatoid factor is often taken as an indicator of RA it is raised in 15% of the population without RA following chronic inflammation or infection and is not raised in 20-35% of adult RA and 95% of juvenile RA. Rheumatoid factor is a poor screening test and should not be ordered in patients with minimal or low symptoms. It is best ordered in patients with a high pretest probability of RA and in cryoglobulinaemia.

Normal Range

<20 IU/ml

CCP
Antibodies against citrullinated peptide (CCP) are found in approximately 70% of RA patients. This test is more specific for RA than rheumatoid factor. It is rarely positive in infectious diseases but is positive in a minority of patients with connective tissue diseases. Its main use is as a predictor of early RA.

Results

Negative Weak Positive Positive Strong Positive

Antinuclear antibody (ANA)

Antinuclear antibodies are found in connective tissue diseases, other autoimmune diseases, chronic infections, malignancy and in normal individuals. Approximately 5% of normal individuals have a positive ANA at a screening dilution of 1/100 with the prevalence of ANA increasing with age. If a positive ANA is found further characterisation is dependent on the clinical history, titre and immunofluorescent pattern. Low autoantibody titres are usually not significant. ANA is frequently positive in SLE, scleroderma, Sjogren's Syndrome, inflammatory myositis, discoid lupus, mixed connective tissue disease and autoimmune hepatitis.

Method

Indirect immunofluorescence using HEp-2 cell line screened at 1/100 and 1/1000

Result

Negative or Positive with grading (1/100+, 1/100++, 1/100+++, or >1/1000) and pattern (including anti-centromere).

Anticytoplasmic antibodies
Cytoplasmic staining is detected by the same immunofluorescence test as ANA. However, a positive cytoplasmic staining is NOT a positive ANA. Some antibodies to cytoplasmic components have clinical significance whereas the relevance of others is unknown. Antibodies to ribosomes may accompany ANA in SLE. Mitochondrial patterns are associated with Primary Biliary Cirrhosis. In polymyositis anti-Jo-1 antibodies have a discrete cytoplasmic speckled pattern. Cytoskeletal patterns can also be distinguished but are mainly non-specific. Anti-actin antibodies are associated with autoimmune hepatitis. Method and reporting as for ANA.

Method

Indirect immunofluorescence using HEp-2 cell line Screened at 1/100 and 1/1000

Result

Negative or Positive with grading (1/100+, 1/100++, 1/100+++, or >1/1000) and pattern (including anti-centromere).

Double stranded DNA (dsDNA)

Antibodies against dsDNA are present in 60% of SLE. In most instances it is pointless to request antibodies to dsDNA either without knowing the ANA result or if the ANA is negative. If the ANA is negative dsDNA antibodies are rarely indicated unless the clinical picture is exceptional. Please discuss with the clinical immunologist. Elevated levels of anti-dsDNA have also been reported at low frequency in other connective tissue diseases and in autoimmune hepatitis.

Normal Range

0-40 IU/ml

Extractable nuclear antigens (ENA)

These antibodies recognise soluble cellular (mainly nuclear) antigens and are useful in characterising the specificity of positive ANA in patients with features connective tissue diseases. If the ANA is negative ENAs are rarely indicated, unless the clinical picture is strongly suggestive of a connective tissue disease eg Sjogrens syndrome or discoid lupus. Certain ENA are predictive of the clinical course, particularly in Scleroderma. Further characterisation by radioimmunoprecipitation may be necessary in Scleroderma and Myositis, depending on the ANA pattern.

Results

Negative or Positive ENA Ro (SSA) ANA Fine speckled Disease Association [also seen in] Sjogrens (60-80%) SLE (35%) Subacute cutaneous lupus Scleroderma (1015%) Sjogrens (50%) SLE (15%) SLE (highly specific, 15-30%) MCTD (100%) SLE (40-60%) Scleroderma (10-15%) [viral hepatitis]

La (SSB) Sm (Smith) RNP

Fine Speckled Coarse speckled Coarse speckled

Scl-70 (antitopoisomerase-1)

Homogenous/intense Scleroderma (25%) speckling

Jo-1 PM-Scl Ku PCNA SL Mi-2

Cytoplasmic speckled Myositis (30%) Fine speckled + nucleolar Homogenous + nucleolar Cell cycle staining Homogeneous Polymyositis / scleroderma overlap (8-12%) Polymyositis/Scleroderma overlap SLE SLE [Sjogrens syndrome] Dermatomyositis

Other Scleroderma/Myositis specific antibodies

Antibodies to RNA polymerases and to aminoacyl-tRNA synthetases (Anti-PL7 &PL12) are available by prior consultation with the laboratory.

Antibodies in vasculitis
Immunopathological mechanisms may be involved in primary and secondary vasculitides (eg infection, neoplasia, connective tissue disease, cryoglobulinaemia). Pulmonary-renal syndromes are associated with Goodpasture's syndrome due to the presence of antibodies to the glomerular basement membrane (GBM) and vasculitis. Anti-neutrophil cytoplasmic antibodies ANCA - should be requested in all patients with vasculitis and rapidly progressing glomerulonephritis (RPGN). Positive cANCA (cytoplasmic staining) and pANCA (perinuclear staining) by immunofluorescence will be characterised by ELISA for the presence of antibodies to PR3 (proteinase-3) and MPO (myeloperoxidase) respectively. ANCA is positive in 70-80% of Wegeners Granulomatosis, Microscopic Polyangiitis and idiopathic RPGN, in 60% of Churg Strauss syndrome and less than 20% of polyarteritis nodosa (pauci-immune GN.

Method

Indirect Immunofluorescence on ethanol fixed neutrophils screened at 1/10

Results

Negative or positive (graded +/++/+++ with pattern cANCA or pANCA)

Characterisation of positives
Normal Range Anti-PR3 ELISA Anti-MPO ELISA Anti-GBM ELISA 0-10 EU 0-10 EU 0-10 EU Positives 10-600 EU 10-100 EU 10-600 EU

Antiphospholipid syndrome
Definite antiphospholipid syndrome is diagnosed when at least one clinical and one laboratory criteria are met: Clinical criteria: Recurrent venous or arterial thrombosis or foetal loss. Laboratory criteria: IgG and/or IgM anti-cardiolipin in medium/high titre on two separate

occasions at least three months apart. Lupus anticoagulant positive on two occasions at least three months apart (Lupus anticoagulant investigation is performed on citrated plasma in the Haemophilia department). Antibodies against 2-glycoprotein 1 (2GP1) are also associated with this syndrome. Cardiolipin antibodies may be found in other autoimmune disorders, particularly SLE. Transient positive results may be found after infections.

Normal range

IgG cardiolipin 0-10 GPLU/ml IgM cardiolipin 0-15 MPLU/ml IgG 2GP1 Negative, Weak positive, Positive, Strong positive

Neurology
Sample requirement

5ml clotted blood (Red)

Neuronal (paraneoplastic) antibodies (Hu, Ri, Yo) are screened for on monkey cerebellum by indirect immunofluorescence. All positives are sent to a referral centre for characterisation. Anti-ganglioside (GM-1, GQ1b), basal ganglia, GAD and voltage gated channel antibodies are also sent to a referral laboratory (see section on referred tests). Clinical biochemistry offers a forwarding service for anti-acetylcholine receptor antibodies.

Complement
Sample requirements
C3/C4, C1 inhibitor, CH100, AP50, Functional C1 inhibitor: 5ml clotted blood (Red)

Method (C3/C4)

Rate nephelometry

Normal adult range

C3 70 165 mg/dl C4 16 45 mg/dl Low C3, Low C4 Low C3, Normal C4 Normal C3, Low C4

Severe sepsis SLE (active) Malnutrition

Post streptococcal GN Genetic deficiency C3 nephritic factor Sepsis SLE Hereditary angioedema Hypocomplementemic urticarial vasculitis Type II cryoglobulinaemia Eclampsia

Liver cirrhosis / failure SBE

Increased complement levels are associated with acute phase responses. Normal levels may reflect increased production as well as consumption. Serum C3 levels may remain low in some forms of membranoproliferative glomerulonephritis, due to the circulating autoantibody C3 nephritic factor which binds and activates C3 convertase. Hereditary angioedema (HAE) (C1 esterase inhibitor (C1INH) deficiency): Recurrent abdominal pain and/or deep subcutaneous swellings without urticaria (particularly occurring after minor trauma), often with family history, may indicate HAE. Patients can present with respiratory compromise (if laryngeal oedema occurs) and can mimic the presentation of anaphylaxis. It is important to consider the possibility of C1 INH deficiency in this group as standard therapy for anaphylaxis will not work. C4 and C1 esterase inhibitor will be low. Uncommonly there may be normal C1INH level with defective function. If C4 is very low without other explanation and C1INH normal, C1INH function should be measured. Acquired C1INH deficiency: Consumption / Inactivation of C1INH may occur in SLE and lymphoproliferative disease. This may lead to episodes of angioedema as with the inherited form. C1q is low in acquired C1INH deficiency but usually normal in HAE.

Method

Single radial immunodiffusion

Normal range

C1 inhibitor (antigenic) C1 inhibitor (functional)

15-35 mg/ml sent to reference laboratory

Complement deficiencies
CH100 and AP50 test the integrity of the classical and alternate pathways of complement activation. Their use is limited to the investigation of suspected complement deficiencies. Early classical pathway complement component deficiencies are associated with SLE and recurring bacterial infections. Deficiencies in the alternative and terminal pathways are associated with recurrent neisserial (meningococcal) infection. To avoid misinterpretation due to the effects of complement consumption by immune complex formation or infection, the test should be requested when the patient has recovered. It follows that if the antigenic level of C3 or C4 is low the CH100/AP50 is also likely to be low

Method

Complement mediated red cell lysis

Normal range

CH100 AP50

392-1019 CH100 Units/ml 50-125% of Normal Human Pooled Serum

Investigation of immunodeficiency
Please phone the clinical immunologist to discuss the investigation of recurrent unusual infection. The nature of the organism, the site, severity and frequency of infection may give clues into the nature of the immune defect. Investigation is required in the following circumstances: 1. 2. family history of immunodeficiency infant or young child (an important warning sign in an infant is a low total lymphocyte count on the FBC) with failure to thrive, opportunistic infections, persistent infections with low virulence organisms, severe diarrhoea, unusual extensive skin rashes, hepatosplenomegaly recurring/persisting sinopulmonary infections recurring skin infections, abscesses or periodontitis recurring meningitis

3. 4. 5.

Screening tests for primary immunodeficiency should include FBC, including differential WBC, serum immunoglobulins, occasionally specific antibody response to vaccinations and lymphocyte subsets. Further tests should be directed towards the suspected arm of defence considered deficient, and include tests of neutrophil function and the measurement of total haemolytic complement CH100, and the alternative complement pathway AP50. Lymphocyte proliferation assays require prior discussion with the clinical immunologist. CD4 levels are monitored in patients with HIV as a marker of disease progression and response to therapy. CD38 is the activation marker of most prognostic relevance. Increased percentages of CD8+CD38+ T cells can predict a faster rate of decline of CD4 T cells and progression to AIDS.

Investigations
Age related normal ranges are reported where appropriate.

Lymphocyte disorders
Lymphocyte subset analysis T cell activation markers in HIV

Sample requirement

4ml EDTA blood (Lavender)

Lymphocyte proliferation assay MHC Class I and II expression

Sample requirement

10ml Lithium Heparin blood (Green)

Lymphocyte activation markers Serial monitoring of lymphocyte activation markers is useful in titrating immunosuppression dose reduction (ISDR) in patients with post-transplant lymphoproliferative disease (PTLD). The aim is to see a three-fold rise in the immunosuppression. The rise in activation markers correlates with the generation of an immune response to the PTLD.

Neutrophil disorders
NBT (nitroblue tetrazolium) test of respiratory burst pathway Adhesion marker expression: CD11, CD18.

Sample requirement

4ml EDTA blood (Lavender) The NBT test is indicated in any patient in whom chronic granulomatous disease is suspected often a child with deep-seated abscesses or fungal infections. This test needs to be discussed in advance with the laboratory (ext 33473) Adhesion molecules CD11 and CD18 are indicated in children in whom a leukocyte adhesion deficiency is suspected. Please phone the clinical immunologist to discuss such cases.

Reference ranges
Lymphocyte subsets
Adult reference ranges Lymphocyte count Absolute CD3 count Absolute CD4 count Absolute CD8 count Absolute CD19 count 1.0-3.2 x 109/L 0.8-2.5 x 109/L 0.4-1.5 x 109/L 0.2-1.1 x 109/L 0.05-0.50 x 109/L

Absolute CD16 count CD3+ T cell CD4+ T cell CD8+ T cell CD19+ B cell CD16+ NK cell CD4/CD8 ratio % of lymphocytes % of lymphocytes % of lymphocytes % of lymphocytes % of lymphocytes

0.08-0.65 x 109/L 58-91 27-61 14-46 12-22 9-16 0.7-3.1

Reference ranges are age-related. Paediatric reference ranges are available on request.

Lymphocyte proliferation assay

Spontaneous proliferation PHA proliferation Anti-CD3 proliferation Anti-CD3/CD28 proliferation

% of control % of control % of control % of control

50-175 50-175 50-175 50-175

MHC class I and II expression

Normal or abnormal expression

T cell activation markers in HIV

% of CD38+ within CD8+ T cells % of CD38+ within CD8+ T cells

3-22

0.2-0.8 x 109/L

NBT

% of unstimulated granulocytes % of stimulated granulocytes

0-20

95-100

MHC class I and II expression

Normal or abnormal expression

Functional (antigen-specific) antibodies / IgG subclasses

Sample requirement

5ml clotted blood (Red)

Functional IgG antibodies (anti-tetanus toxoid, anti-pneumococcal capsular polysaccharide and anti-haemophilus influenzae B antibodies).

Minimal protective level for anti-tetanus toxoid antibodies: 0.1 U/ml Interpretation of anti-PCP and anti-HIB antibody results is response-dependent. IgG subclasses please measure total IgG before requesting IgG subclasses. Total immunolglobulins (IgG, IgA and IgM) are performed in clinical biochemistry. In a patient with normal IgA levels, IgG subclasses will not be measured if total IgG >11g/l as audit has shown that subclass deficiency cannot be detected in these cases. Furthermore, due to difficulties in establishing paediatric reference ranges, IgG subclasses will not be measured in patients less than 18 months of age.

Adult normal ranges (paediatric ranges are age-related)

IgG1 IgG2 IgG3

3.2-10.2 g/l 1.2-6.6 g/l 0.2-1.9 g/l

Complement deficiency
CH100, AP50

See section on complement.