IMMUNOLOGY SERVICES

SCOTTISH NATIONAL BLOOD TRANSFUSION

SERVICES EDINBURGH

USER MANUAL; V2.2 Revised April, 2008

HTTP://WWW.SCOTBLOOD.CO.UK/DOCS/IMMUNOLOGY.DOC

CONTENTS

GENERAL INFORMATION p3

AUTOANTIBODIES IN ENDOCRINE DISORDERS p4 AUTOANTIBODIES IN LIVER DISEASES p7 AUTOANTIBODIES IN PRIMARY VASCULITIDES & p9 GOODPASTURE’S SYNDROME AUTOANTIBODIES IN CONNECTIVE TISSUE DISEASES p10 MISCELLANEOUS AUTOANTIBODIES p16 GUIDELINES FOR REPEAT TESTING OF AUTOANTIBODIES p19 ANA Q&A p20 MEASUREMENT OF COMPLEMENT PROTEINS p21 INVESTIGATIONS OF IMMUNODEFICIENCIES p25 GUIDELINES FOR ALLERGY TESTING p27 SPECIFIC IgG ANTIBODY TEST AGAINST FUNGAL & AVIAN p29 PRECIPITINS

GENERAL INFORMATION BTS Immunology Laboratory, Edinburgh & SE Scotland Blood Transfusion Service, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh, EH16 4SA. Opening Times Monday – Thursday: 8.30 am to 5.00 pm Friday: 8.30 am to 4.30 pm. There is no weekend working or out of hours service. Blood Samples 1 Specimens should be labelled with the patient's full name, date of birth, ward, outpatient department or general practice, and date of collection. The patient's CHI number must be given where available and Addressograph labels may be used. 2 Specimens should be accompanied by the appropriate Request Form completed, including all patient details as stated in para 1 above, relevant clinical information and previous report numbers (if available), signed by a member of the medical staff. It is essential to indicate where the Report of Test Results should be sent to. 3 Specimens that are incorrectly labelled or documented may not be accepted. This laboratory has the responsibility for the investigation of patients suspected of suffering from disease consequent on abnormal function of the immune system, in particular, autoimmunity, immune deficiency and allergy. Tests for Autoantibodies, Complement, total IgE & RAST (specific IgE tests)

5-10 ml of clotted blood should be sent in an anticoagulant free tube. For children, a 1-2ml clotted sample may be sent.

Transport All blood samples should be sent to the above address either by post, following current guidelines, or by using the LUHT delivery service. Reporting of Results All test requests are handled as expeditiously as possible but some specialised tests are redirected to other centres. Results can be obtained by telephoning the laboratory when needed urgently. The Consultant Clinical Immunologist is available by telephone for discussion of the clinical implications of test results

Staff and Telephone Numbers

Consultant Clinical Immunologist…………….……………………………………………. Dr PL Yap Head of Immunology Laboratory…..………………………………………………………. Mrs J Sawers Deputies …………………………………….……………………………………………… Mrs G Steven ……………………………………………………………………………………. Ms G McNab Prinicpal Clinical Scientist…………………………………………………………………. Dr M Kadlubowski Immunology Laboratory, Reception & Results…………………………………………….. 0131 242 7525/7529 …………………………………………………………………………………….. (Internal 27521/27529) Dr PL Yap………………………………………………………………………………….. 0131 242 7526 …………………………………………………………………………………….. (Internal 27526) Dr Yap’s Secretary…………………………………………………………………………..0131 242 7521 …………………………………………………………………………………….. (Internal 27521) Abbreviations used: IFF – Indirect Immunofluorescence ELISA – Enzyme Linked Immunosorbent Assay

AUTOANTIBODIES IN ENDOCRINE DISORDERS Anti-Thyroid Peroxidase Antibodies (anti-TPO) Introduction Thyroglobulin (MW 660 kD) is the major protein of the colloid of the thyroid follicular cells. Thyroid peroxidase (TPO), the thyroid microsomal antigen, is a 110kD peroxidase enzyme found on intracellular membranes of the epithelial cells of the follicles and its function is to incorporate iodine onto the benzene ring of thyroglobulin tyrosine residues. In response to thyroid stimulating hormone, iodinated tyrosines are cleaved by lysosomal proteases to eventually yield thyroxine (T4) and triiodothyronine (T3). Autoantibodies to TPO are predominantly directed against 2 immunodominant regions which reside in a region with structural homology to myeloperoxidase. They are frequently found in very high levels in sera of patients with autoimmune thyroid disease Graves’ disease, Ha\shimoto’s thyroiditis and primary atrophic hypothyroidism). They are usually IgG and rarely respond to treatment. Autoimmune thyroiditis may be associated with other autoimmune diseases especially pernicious anaemia or myasthenia gravis. Use of Tests Diagnosis of autoimmune thyroid disease. Method Used ELISA for IgG anti-TPO. Reporting of Results Results are expressed as IU/ml where >82 IU/ml is positive. A borderline result is in the range of 42-81.9 IU/ml. High levels (>5000 IU/ml) can be detected in patients with autoimmune thyroiditis. The turnaround time is 5 working days. Interpretation of Results Anti-TPO antibodies are detected in raised levels (>82u/ml) in:

85-100% of patients with overt autoimmune thyroiditis (Hashimotos thyroiditis) 70-90% of patients with Graves disease

60% of patients with primary hypothyroidism

30-60% of patents with pernicious anaemia but no overt thyroid disease

Anti-thyroid antibodies are also frequently found in patients with other autoimmune diseases eg

Pernicious anaemia 60% Myasthenia gravis 50% Lambert-Eaton syndrome 30%

They are detected in low levels in 2-8% of normal individuals particularly the elderly and more often in women than men. Anti-thyroglobulin autoantibodies (anti-Tg) are no longer assayed. However, this can be arranged by special request in patients with suspected thyroid cancer. It is very unusual to detect anti-Tg antibodies in the absence of anti-TPO antibodies but quite common to find anti-TPO antibodies in the absence of anti-Tg antibodies, especially in patients with small goitres. Anti-Tg requests are sent to an external laboratory for testing* (see p 18 ) Anti-Gastric Parietal Cell Antibodies (anti-GPC or PCA)) Introduction The parietal cells of the gastric mucosa secrete a protein, intrinsic factor, which is essential for the absorption of vitamin B12, a vitamin needed for normal erythropoiesis. The target antigen for GPC is the transmembrane ATP-dependent proton pump. The latter consists of an alpha catalytic subunit MW approx. 90 kDa) and a beta core subunit (MW approx. 35 kDa). The resulting destruction of the parietal cells leads to inability to absorb the vitamin B12 and hence to anaemia. Antibodies against gastric parietal cell antigens are diagnostic of chronic autoimmune gastritis whether or not the result is pernicious anaemia. Very severe gastric atrophy is necessary before anaemia is detected. . Antibodies to intrinsic factor can also be detected in the same diseases. Anti-gastric parietal cell antibodies (in the presence or absence of anaemia) are frequently associated with other autoimmune diseases such as autoimmune thyroiditis, insulin dependent diabetes or Addison’s disease. Use of Test Diagnosis of pernicious anaemia. Method Used Indirect immunofluorescence (IIF) on rat gastric mucosa. Reporting of Results Positive results are expressed from +/- (a borderline result) to +++ (strongly positive). The turnaround time is 7 working days. Interpretation of Results Anti-gastric parietal cell antibodies are found in >90% patients with pernicious anaemia. They are not associated with duodenal ulcer or gastritis which is not autoimmune. Anti-gastric antibodies are also found in: 30% patients with Addisons disease

30% patients with autoimmune thyroiditis 30% patients with insulin dependent diabetes 20% patients with primary ovarian failure

Around 5% of normals have low levels of these autoantibodies rising to 10% in older women. They are more common in relatives of patients with autoimmune thyroid disease or gastritis. The antibodies do appear many years before the onset of pernicious anaemia and therefore patients with high elevated levels should be followed up.

Anti-Intrinsic Factor Antibodies (IFA

)

Two types of IFA are recognised. Type I blocks the binding of vitamin B12 to intrinsic factor and Type II prevents attachment of intrinsic factor to the ileal mucosa. Both types are measured in the one method in the laboratory. These antibodies are specific for patients with pernicious anaemia but are only found in 50-60% of patients with pernicious anaemia. These tests are performed infrequently and only on patients with a low serum B12 level and high red cell mean corpuscular volume. Method Used ELISA for IgG IFA. Reporting of Results Results are reported as ‘Negative’ ' Borderline' or 'Positive'. The turnaround time is 10 days. Anti-Adrenal Cortex Antibodies/Anti-Steroid Cell Antibodies Introduction Antibodies to the adrenal cortex usually stain the glomerula, fasciculate and reticulosa cells and are directed against a number of enzymes involved in steroid metabolism (esp. Steroid 21-Hydroxylase, Steroid 17-alpha Hydroxylase and a number of Cytochrome P450 subtypes) . Such antibodies have been detected by a variety of procedures, but the method most commonly used is IIF. The antibodies belong predominantly to the IgG class. Use of Test Diagnosis of Addison’s disease. Method Used IIF using adrenal tissue sections, using a goat anti-human IgG. Reporting of Results Positive results are expressed from +/- (a borderline result) to +++ (strongly positive). The turnaround time is 10 working days. Interpretation of Results Anti-adrenal cortex antibodies are found in:

75% of patients with Addison’s disease at the time of diagnosis. Also found occasionally in IDDM and autoimmune

polyendocrinopathies... They are not found in tuberculosis associated adrenal failure or secondary carcinoma. They are very rare in normal individuals (0.6%), usually in older women. Anti-Pancreatic Islet Cell Antibodies (ICA) Introduction Islet Cell antibodies (ICA) occur in 65-85% of patients with Type I (insulin-dependent, juvenile) diabetes mellitus early in the course of the disease. The standard method for the detection of these antibodies is IIF. Islet cell antibodies, when detected by IIF, stain the cytoplasm of islet cells of the pancreas from humans, monkeys and rats. A large number of target antigens to ICA have been identified and these include glutamic acid decarboxylase (GAD), protein tyrosine phosphatase (ICA512), phogrin and glycolipids. 60 to 80% of individuals with new onset insulin dependent diabetes mellitus have anti-GAD antibodies.

Use of Test

Diagnosis of Type I, insulin dependent/juvenile, diabetes. Method Used IIF using monkey pancreas sections. Reporting of Results Positive results are expressed from +/- (a borderline result) to +++ (strongly positive). The turnaround time is 10 working days. Interpretation of Results Anti-islet cell antibodies are present in 65-85% of patients with Type I diabetes particularly around the time of diagnosis. They tend to disappear later unless there is multi-endocrine disorder. They are also found less commonly in non-diabetic relatives.

AUTOANTIBODIES IN LIVER DISEASES Anti-Smooth Muscle Antibodies (SMA) Introduction Sera from patients with Type 1 chronic active hepatitis contain antibodies to smooth muscle antigens that are detectable by IIF and stain the smooth muscle of several organs. The antibodies belong mainly to the IgG class, but they can also be found in the IgM class. The antigen recognised by anti-smooth muscle antibodies in sera of patients with Type 1 chronic active hepatitis is often reported to be actin. However, this is a very complex and incompletely characterised group of autoantibodies which in liver disease and in other diseases can recognise desmin, vimentin, tropomyosin, cytokeratins or other cytoskeletal proteins. Use of Test Anti smooth muscle antibodies can be diagnostic of Type 1 chronic active hepatitis and are useful in differential diagnosis of liver diseases. Method Used IIF using rat liver, kidney and stomach tissues. Reporting of Results Positive results are expressed from 1:10 (a borderline result) to >1:640 (strongly positive). The turnaround time is 7 working days. Smooth muscle antibodies are detected in:

40-70% of patients with Type 1 chronic active hepatitis 50% of patients with primary biliary cirrhosis 28% of patients with cryptogenic cirrhosis

Type 1 chronic active hepatitis is often associated with hypergammaglobulinaemia and with anti-nuclear antibodies. Anti-smooth muscle antibodies are also found in patients with acute viral hepatitis, infectious mononucleosis, asthma, yellow fever and malignant tumours (carcinomas of the ovary, malignant melanoma). They have been found in less than 2% of the normal population. Antibody titres are in the range of 80 to 320 in Type 1 chronic active hepatitis and are much lower, rarely over 80, in the other conditions listed above. They can be found in patients with rheumatoid arthritis, multiple sclerosis but probably not SLE.

Anti-Mitochondrial Antibodies (AMA)

Introduction Anti-mitochondrial antibodies are frequently associated with autoimmune liver disease. There are a number of antibody specificities which have been reported. These are termed M1-M9. M2 (the subtype reported here) are the most common ones observed and these are highly specific for Primary Biliary Cirrhosis. The M2 autoantibodies recognise mainly four closely related enzymes in the 2-oxoacid dehydrogenase complex; pyruvate dehydrogenase, branched chain 2-oxoacid dehydrogenase, 2-oxoglutarate dehydrogenase and an unknown protein. Most antibodies recognise the 74kDa acyltransferase E2 component of the enzymres and the related 45kDa protein. These autoantibodies are most frequently associated with primary biliay cirrhosis. Antibodies of other specificity are associated with other liver diseases. The other 8 specificities recognise a disparate number of antigens and are observed in a variety of disorders including syphilis (M1), coonective tissue diseases (M5) and cardiomyopathies (M7). Use of Test Diagnosis of primary biliary cirrhosis; differential diagnosis of liver disease. Method Used IIF using sections of rat liver, kidney and stomach. Reporting of Results Positive results are expressed from 1:10 (a borderline result) to >1:640 (strongly positive). The turnaround time is 7 working days. Interpretation of Results Anti-mitochondrial antibodies of the M2 type are present in at least 90% of patients with

PBC but can also be present in other autoimmune disease such as 2o Sjgrens syndrome,�

autoimmune thyroid disease, SLE or polymyositis. Can be associated with anti-smooth muscle antibodies in PBC/CAH overlap. Anti-Liver/Kidney Microsomal Antibody (anti-LKM1) Antibodies recognising this antigen are found in a rare subset of patients with Type 2 chronic active hepatitis occurring usually in young patients. The antibodies react with a 50-57kDa protein (Cytochrome P450 2D6) found on cells in the third portion of the proximal renal tubules and on hepatocytes. They may also react with cells in bronchial, oesophageal and duodenal epithelium. These autoantibodies are not associated with ANA or SMA which tend to be associated with the more common Type 1 chronic active hepatitis. There are also two much rarer LKM subtypes. LKM2 targets other cytochrome P450 subtypes and is associated with drug-induced hepatitis and LKM3 targets UDP-glucuronyltransferase and is associated with Hepatitis D. The much rarer LKM2 autoantibody is associated with drug-induced liver disease. Use of Test Diagnosis of Type 2 chronic active hepatitis in the young. Method Used IIF using sections of rat kidney, liver and stomach sections Reporting of Results Results are expressed as either ‘Positive' or ‘Negative' The turnaround time is 7 working days. Interpretation of Results

A positive result is diagnostic of this subset of patients with Type 2 chronic active hepatitis. L/K microsomal antibodies are also found in patients with chronic active hepatitis due to hepatitis C infection - usually in middle aged males of Mediterranean extraction.

AUTOANTIBODIES IN PRIMARY VASCULITIDES & GOODPASTURE’S SYNDROME

Anti-Neutrophil Cytoplasmic Antibodies (ANCA) Introduction Autoantibodies recognising antigens in the cytoplasmic granules of neutrophils (ANCA) were first detected in a number of patients with Wegener’s granulomatosis. There are two clinically relevant staining patterns found in ethanol-fixed granulocytes known as c-ANCA and p-ANCA. c-ANCA is a granular cytoplasmic staining pattern which in approx. 50% of cases is due to recognition of Proteinase 3 (PR3), a primary granule 29kDa protease. p-ANCA is a perinuclear staining pattern due to recognition of Myeloperoxidase (MPO), Cathepsin G, Elastase and other antigens which relocate to the perinuclear region during ethanol fixation of the cells. Other immunofluoresence staining patterns termed atypical-, x- and a-ANCA have been reported and are found in a wide range of diseases including vasculitides, connective tissue diseases, inflammatory bowel diseases and infections. They are the result of antibodies to at least 20 neutrophil proteins. Of these only autoantibodies to proteinase 3 (PR3-ANCA) and myeloperoxidase (MPO-ANCA) are considered to be of diagnostic and prognostic usefulness. The latter are measured by ELISA using purified human antigens.

Use of Test The diagnosis of primary vasculitides (mainly small vessel) such as Wegener’s granulomatosis and microscopic polyangiitis and the monitoring of treatment. Method Used ANCAs are measured by IIF and/or an ELISA screen (which measures IgG antibodies to both PR3 and MPO). Both of these methods are used to screen patient samples. All positive samples are then tested for ANA by IIF (see below) and PR3-ANCA and MPO-ANCA by ELISA. Reporting of Results IIF Results are expressed as pANCA, cANCA, atypical pANCA, atypical cANCA, atypical ANCA or negative. ELISA screen results are expressed as negative, equivocal or positive. All equivocal and positive results are further assayed for IgG antibodies to PR3-ANCA and MPO-ANCA. The turnaoround time is 10 working days. Borderline 5.0 to 9.9 AU/ml Positive >10 AU/ml Interpretation of Results It is important to be aware that in an unselected population barely half of c-ANCAs are caused by autoantibodies to PR3 and that only ~25% of p-ANCAs are caused by autoantibodies to MPO. It should therefore never be assumed that c-ANCA and p-ANCA results are synonymous with PR3-ANCA and MPO-ANCA respectively. The majority of patients with Wegener’s granulomatosis (WG) and microscopic polyangiitis (MPA), however, will have antibodies to PR3 or MPO. PR3-ANCA are much more common in WG whereas MPO-ANCA are more common in MPA. ANCAs are also found in patients with segmental necrotising glomerulonephritis and in patients with crescentic glomerulonephritis. IgA anti-neutrophil antibodies have been detected in Henoch-Schnlein purpura associated with IgA rheumatoid factor. � Atypical ANCAs not due to PR3 or MPO antibodies are often found in patients with inflammatory bowel disease. The antibodies occur in some patients with ulcerative colitis and less commonly in patients with Crohn's disease

with colon involvement and also in patients with sclerosing cholangitis associated with Crohn's disease.

Granulocyte specific anti-nuclear antibodies are found in rheumatoid arthritis and must not be confused with ANCAs. Anti-Glomerular Basement Membrane Antibodies (anti-GBM) Introduction Antibodies recognising glomerular (and alveolar) basement membranes are characteristic of Goodpasture’s syndrome. The antigen is most frequently the non-collagenous domain (NC1) of the alpha 3 chain of collagen IV. These anti-glomerular basement (GBM) antibodies are pathogenic and are usually IgG. There appears to be some subclass restriction with IgG1 and IgG4 predominating. Use of Test Diagnosis of Goodpasture’s Syndrome. Method Used ELISA for IgG anti-GBM. Reporting of Results Results are expressed as AU/ml where > 20 is positive. The turnaround time is 10 working days. Interpretation of Results Anti-GBM antibodies are found in the sera of the majority (>90%) of patients with Goodpasture’s syndrome. The diagnosis must be confirmed by renal biopsy. They are also occasionally found in ANCA-associated disease (see above), usually with MPO-ANCA but very rarely with PR3-ANCA.. Less than 2% of patients with glomerulonephritis have these autoantibodies. ANCA/GBM Dot-Blot An emergency qualitative immunoblot assay for antibodies to PR3, MPO and GBM is available by request only with the approval of the consultant immunologist. The blood sample must reach the immunology laboratory by 1.00 pm at the latest . The result will be telephoned to the requesting physician between 4.00 and 4.30 pm on the same day and will be reported as positive or negative for each of the 3 specificities. Any positive result will be confirmed by the appropriate ELISA as above.

AUTOANTIBODIES IN CONNECTIVE TISSUE DISEASES Anti-Nuclear Antibodies (ANA) Introduction The detection of circulating antibodies to nuclear antigens is an important tool in the investigation of systemic connective tissue diseases. Many techniques have been developed to detect antinuclear antibodies (ANA), but the fluorescent-ANA (FANA) test continues to be the most widely used and accepted. We and many other laboratories use this test to screen sera before other techniques are used to define antibody specificity. Compared with these more elaborate techniques, the FANA test has the advantages of broad specificity, sensitivity, economy, reproducibility and relative ease of performance. A wide range of ANAs are detected by this technique because most nuclear antigens are represented in carefully prepared tissue substrates. The ANA of patients with systemic connective tissue diseases are not restricted by tissue specificity and will therefore bind to nuclear components from various species. Exceptions to this rule include sera that react specifically with human leukocyte nuclei and to Sjgren's syndrome antigen A (SS-A/Ro). � Use of Test

The major reason for ordering the FANA test is to confirm the clinical diagnosis of a systemic connective tissue disease such as systemic lupus erythematosus (SLE). A negative FANA test does not completely rule out the diagnosis of SLE, but alternative diagnoses should be considered. Anti-nuclear antibodies, particularly at low titres are associated with a number of autoimmune diseases. In addition, patients receiving a drug such as procainamide, phenytoin or hydralazine, should be tested if symptoms occur suggesting a diagnosis of drug-induced lupus erythematosus.

Method Used Indirect immunofluorescence (IIF) is performed on fixed Hep2000 cells. The titre of antibody and the pattern of staining is reported. This cell line has large nucleii which allows better definition of the staining pattern and preparations contain approx. 10% of cells engineered to contain elevated levels of Ro60. There are also significant numbers of dividing cells allowing detection of autoantibodies to cell cycle specific antigens such as centromere proteins. Reporting of Results Results are reported as 'Negative' or 'Positive' with the antibody titre on HEp2000 cells reported as 1:40 (borderline) to >1:640 (strongly positive). The turnaround time is 5 working days. The intensity of staining depends on the antigen which is being detected. This also has a marked effect on the staining pattern. Although a large number of staining patterns have been described, we classify the staining patterns into various groups. The major staining patterns are: Homogeneous Homogeneous staining of interphase nuclei plus positive staining in the chromosomal region of mitotic cells Speckled Large, coarse or fine speckled staining of the interphase nuclei with negative staining in the chromosomal region of mitotic cells Homogeneous/Speckled Very similar staining pattern to speckled but with positive staining of the chromosomal region of mitotic cells. Atypical Speckled This staining pattern is specific to HEp2000 and results from the fact that approx. 10% of the cells present are engineered to contain significant amounts of Ro. It is only these 10% of cells showing nucleolar and a very fine speckled pattern in the rest of the nucleus. Nucleolar Staining of nucleolus only. The appearance can vary depending on which of several nucleolar antigens are involved Homogeneous/Nucleolar Staining of the nucleoli only of interphase nuclei together with positive staining in the chromosomal region of mitotic cells. Centromere Approx 40 discrete dots caused by staining of the centromere regions of the chromosome which interact with the mitotic spindles. The dots are found in the condensed nuclear chromatin during mitosis. The centromere antigen is located at the point of attachment of the chromosome to the mitotic spindle. The major antigen recognised is an 80kDa DNA binding protein termed CENP-B.

Nuclear Membrane Similar to homogeneous but with more intense staining of the nuclear membrane (also called rim staining) Many minor staining patterns are observed and these include Mitotic Spindle, PCNA, Centriole, Nuclear Dots and a large number of mixed patterns (ie. 2 of the above in the same patient sample). Interpretation of Results All positive ANAs (>10) are titred out (40, 160, 640) A Positive ANA may trigger the measurement of anti-dsDNA if requested (dependent on pattern and titre).

A homogenous/nucleolar pattern triggers an ENA screen and a positive ATS pattern will trigger an ENA profile.

The pattern of IIF will often provide a clue to the category of nuclear antigens involved. For example, antibodies binding nonhistone proteins (Smith [Sm], nuclear ribonuclear protein [nuclear RNP], La antigen [SS-B]) give a speckled pattern of immune fluorescence, antibodies to DNA and histone give a homogenous or rim pattern, and antibodies to 4 to 6S RNA and nucleolar proteins give a nucleolar pattern. In the last few decades, many nuclear antigens that react with connective tissue disease sera have been described and characterized. Along with the description of new ANA has come differing and overlapping nomenclature for the respective nuclear antigens. Generally, the nuclear antigens may be divided into three categories: the nucleic acids (DNA and RNA), the histone (basic) proteins, and the nonhistone (acidic) proteins. Detailed characterisation of the autoantibodies is important since different autoantibodies can be associated with specific diseases. Specific autoantibodies detected by the use of Hep2000 cells include: Homogeneous Indicates presence of antibodies to dsDNA, ssDNA or histones. Suggestive of SLE but also found in other CTDs, drug-induced lupus and rarely CAH. Speckled Caused mainly by antibodies to Sm, RNP, Ro and La (see anti-ENA below). Pattern found in SLE, MCTD and SS but also other CTDs. Nucleolar High titre associated with scleroderma, low titre found in other CTDs. Centromere Most frequently found in CREST (Calcinosis, Raynaud's phenomenon, Oesophageal dysmotility, Sclerodactyly and Telangiectasia) variant of Progressive Systemic Sclerosis but alos occasionally in other CTDs. Homogeneous/ Caused by antibodies to Scl-70. These antibodies are very specific to scleroderma and are Nucleolar frequently associated with the more severe diffuse form of the disease. Speckled/ Caused by antibodies to La which are found predominantly in SLE and SS Nucleolar PCNA These are found in 4% of patients with SLE, but they are not found in patients with rheumatoid arthritis, scleroderma or polymyositis. Mitotic Spindle Found rarely in a variety of CTDs Centriole Found rarely in a variety of CTDs Nuclear Dots Strongly associated with PBC but also found in CAH and sicca. Antibodies to Double-Stranded DNA (anti-dsDNA) Introduction Antibodies to DNA were initially detected in sera of patients with systemic lupus erythematosus (SLE), and their unique relationship to this disease was immediately apparent. However, it has also become evident that DNA is a molecule with multiple epitopes and that antibodies to DNA may include a heterogeneous group of immunoglobulins with a variety of specificities. Probably most common among the various antibodies reactive with DNA are those directed against antigenic determinants found on single-stranded DNA (purine and pyrimidine nucleotide determinants). Detection of such antibodies has little diagnostic specificity; they are found in a wide variety of autoimmune and connective tissue diseases. In contrast, antibodies reactive primarily or exclusively with native, double-stranded DNA (herein referred to as anti-dsDNA) show strong association with SLE. These antibodies are probably directed against deoxyribose-phosphate determinants. Numerous clinical investigations have confirmed both the relative diagnostic specificity of these antibodies for SLE and the consistent correlation of their levels with disease activity in many patients.

The practical utility of measuring anti-DNA has led to the development of a number of techniques for their quantitation. We use an anti-dsDNA ELISA which is more sensitive but the increased sensitivity does appear to mean that the antibodies are detected in a wider range of diseases.

Use of Test An anti-dsDNA test is often useful diagnostically for patients who have antinuclear antibodies or clinical findings suggestive of SLE. The presence of anti-dsDNA is one of the American Rheumatism Association criteria for SLE and the antibody is rarely found in high levels in patients with other connective tissue diseases. However, anti-dsDNA antibodies are frequently found in patients with overlap symptoms between SLE and other autoimmune diseases. Method Used Two methods are used: 1. An ELISA for IgG anti-dsDNA. 2. IIF using fixed Crithidia luciliae as

substrate. Reporting of Results ELISA: The results are reported as IU/ml (using the W.H.O. standard WO80). The turnaround time is 7 working days. IFF: The results are reported as ‘positive’, ‘borderline’ or ‘negative’. Interpretation of Results The absence of anti-dsDNA antibodies by ELISA is strongly indicative of a disease other than SLE. Antibodies to dsDNA are not usually found in cases of drug induced lupus. In SLE actual levels do not correlate well with disease activity though rapid rises in antibody levels do suggest increases in disease activity. Antibodies to Extractable Nuclear Antigens (anti-ENA) Introduction Different systemic connective tissue diseases have distinct ANA profiles. Thus, the ANA profile can be helpful in the differential diagnosis of patients with systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), scleroderma, Sjgren's� syndrome (SS), rheumatoid arthritis (RA) and other rarer CTDs.. In addition to determining ANA by IIF as described above the measurement of a group of antibodies known collected as anti-ENAs has recently become routine. Most of these were initially identified by counterimmunoelectrophoresis and immunodiffusion but they are now usually measured by ELISA. In this laboratory antibodies to Sm, RNP, Ro, La, Scl-70 & Jo-1 are measured. Use of Test Assays to determine ENA specificities are indicated in the evaluation of any patient suspected of having a systemic connective tissue disease. In most cases ENA determination is indicated only when the ANA is positive. However, there is a group of patients who have mainly skin involvement SLE, but who have a negative ANA. They have been found to be positive for antibody to SS-A/Ro antigen. Method Used Both the Screen and Profile are performed using ELISA for IgG autoantibodies.. Reporting of Results ENA Screens are reported as ‘Negative’, ‘Borderline’ or ‘Positive’. ENA profile results are reported in U/ml with >25 is positive.

Interpretation of Results The clinical interpretation of the ELISA assays depends on the ENA specificities that are detected. Antibodies to Sm and RNP Antigens Antibody to Sm antigen is found in approximately 25% of patients with SLE. There is considerable disagreement about incidence of this antibody (there appear to be significant racial differences and reports differ on whether or not it varies with the progress of the disease) but all are agreed that it is strongly associated with SLE. The presence of antibody to RNP is found in patients with a variety of systemic connective tissue diseases, including SLE, discoid LE, RA, and SS. It is frequently associated with antibodies to Sm. However, if a high titre of RNP is found and other ANA specificities are absent, this ANA profile strongly suggests MCTD even though the full range of clinical involvement is not apparent at that time. Antibodies to Ro (SS-A) and La (SS-B) Antigens There are several clinical associations between anti-Ro antibody and certain systemic connective tissue diseases. This antibody is found in 60 to 70% of patients with Primary Sjgrens syndrome and 30 to 40% of patients with SLE. There seems to be a close� association between the presence of this antibody system and photosensitivity. Antibody to La antigen is detected in approximately 15% of SLE (70-85% SLE sicca) and 5% RA sicca sera. It is detected in a higher percentage (60-70%) of sera of patients with Primary Sjgrens syndrome often in the absence of Ro. Except for the overlapping presence� of SS in patients with SLE, there do not appear to be any distinguishing clinical features which are associated with the presence of anti-La. Ro and La are frequently detected together. The screening of pregnant lupus patients for anti-Ro can be important as it is associated with congenital heart block in the fetus from 18-20 weeks gestation. Antibodies to Scl-70 Anti-Scl-70 identifies DNA Topoisomerase-1 and is one of several antibodies which give rise to nucleolar staining. This antibody is associated with the more diffuse form of scleroderma and with impaired pulmonary diffusion. It also identifies a subgroup of SLE with pulmonary hypertension and nephritis. Antibodies to Jo-1 Anti-Jo-1 is the most common of a group of myositis-specific autoantibodies (MSAs) which includes anti-PL-12, anti-OJ, anti-Mi-2 and anti-SRP. The antibody targets are all aminoacyl-tRNA synthetases (histidyl-in the case of Jo-1). Jo-1 occurs in polymysositis and dermatomyosistis and is usually associated with interstitial lung disease.

SUMMARY OF ANTI-NUCLEAR ANTIBODY PROFILE

Percentage of Sera Positive for Autoantibodies

Antibodies to

SLE

Druginduced LE

MCTD

PrimarySjg�rens Syndrome

Progressive Systemic Sclerosis

Dermato/ Polymyositis

Rheumatoid Arthritis

RA plus Sjg�rens

Nucleii

90-95

95 100

70 55-75

30-40

25-30

30

dsDNA

60-80

- - - - - 10

ssDNA

60-70

15-30

10-20

10-30

10-20

10-20

35-40

Histones

40-70

95-100

- - - - 15-20

Ro 30-50

- - 60-701¼ 2¼

- - - 9

La 15 - - 45-601¼ 2¼

- - - 3-5

Sm 25-30

- - - - - -

RNP

35-45

20 95-100

30 20 - -

Others

- - - Scl-70 20-30

Jo-1 25

-

Centromere

- - - 25-30CREST 90

- -

Nucleolar

- - - 5-10

50-60

5-10

-

Rheumatoid Factor

>50

>70

>70

MISCELLANEOUS AUTOANTIBODIES Anti-Cardiolipin Antibodies (ACA) Introduction Anti-cardiolipin antibodies are one of a group of anti-phospholipid antibodies. There is considerable overlap between these autoantibodies and lupus anticoagulant. They are found in approx 50% of patients with SLE in which they are associated with venous and arterial thrombosis. They are also associated with anti-phospholipid antibody sysndrome in which there is arterial or venous thrombosis, recurrent foetal loss, thrombocytopoenia and neurological disorders. Anti-cardiolipin antibodies are the anti-M1 anti-mitochondrial antibodies and are responsible for the false positive VDRL. Use of Test Determination of possible cause for myocardial infarct, stroke, peripheral arterial and venous thrombosis especially in patients under 50 years of age. May contribute to spontaneous abortions especially around 10 weeks duration and is a cause of neurological events especially in cerebral lupus. Method Used ELISA for IgG ACA Reporting of Results Results are reported as U/ml where > 10 IU/ml is positive. Interpretation of Results Positive results correlate with a predisposition for thrombosis, foetal loss, and thrombocytopoenia in patients with SLE but levels do not correlate well with disease activity. Anti-cardiolipin antibodies, the lupus anticoagulant test and the VDRL detect overlapping but not identical populations of antibodies. Therefore both anti-cardiolipin antibodies and lupus anticoagulant activity should be measured. Low levels are also found in infections and some vasculitides. Rheumatoid Factors (RF) Introduction Rheumatoid factors are autoantibodies of IgM, IgG, IgA or even IgE class which recognise an antigenic determinant on the Fc region of IgG. The exact nature of the determinant is a subject of some debate. Since the rheumatoid factor is detected in the presence of a vast excess of IgG in the serum, the antigen which is detected is often referred to as altered IgG. An alternative explanation is that the anti-IgG antibodies are of low affinity and are only detected when the IgG is aggregated upon coating plates or latex particles. Although high levels of rheumatoid factors are frequently found in rheumatoid arthritis especially in cases with extra- articular diseases, they are present, sometimes in even higher levels in a number of other diseases such as Sjgrens syndrome or Bacterial� endocarditis. Use of Test Diagnosis of rheumatoid arthritis, differential diagnosis of rheumatic disease. Method Used ELISA for IgM Rheumatoid Factor

Reporting of Results Positive results are those greater than the Reference Range quoted in the Test Report IgM RF are frequently found in the absence of other classes. It is very rare to detect IgA or IgG RF in the absence of IgM RF ie in the absence of a positive RA latex test. However, very high levels of IgA or IgG RF can be detected in the presence of moderate levels of IgM RF. Interpretation of Results Raised RF values are very occasionally found in healthy individuals but low levels are common in a wide range of autoimmune or infectious diseases especially those associated with hypergammaglobulinaemia including viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy, pulmonary fibrosis. Healthy people over 75 frequently have elevated RF. High levels are most common in Rheumatoid Arthritis, Subacute Bacterial Endocarditis (SBE) and Sjgrens syndrome. A very high level of all classes of RF in RA is often associated� with extra articular activity although the titres do not correlate well with disease activity. RF are of little use in monitoring disease activity in RA where CRP levels should be used. Anti-Citrullinated Cyclic Peptide (Anti-CCP) Anti-CCP kits are currently being evaluated with the intention of replacing RF testing by Summer/2008 or as soon as possible thereafter.

Anti-Acetylcholine Receptor Antibodies (AChRAb)** Introduction Antibodies to the acetylcholine receptor are almost invariably associated with myasthenia gravis. The receptor is a complex of proteins found in skeletal muscle motor endplates. There are several kinds of antibodies to AchR, ie, some that react to determinants other than the neurotoxin-binding site, others that react with the neurotoxin-binding site or the extrajunctional receptors, and others that are species specific. The antibodies are IgG and are pathogenic. Use of Test Diagnosis of myasthaenia gravis. Interpretation of Results Anti-acetylcholine receptor antibodies are found in 90% of patients with active adult onset generalised myasthenia gravis. The correlation between the concentration of receptor antibody and the severity of clinical symptoms appears to be rather low. However, the correlation is much higher in the individual patient, where changes in symptoms and signs very often coincide with changes in the concentration of antibodies to AChRAb These autoantibodies are only present in 50% of patients with ocular myasthesia alone. The test should be used for diagnosis only. Follow up is according to severity of symptoms. Myasthenic syndromes of childhood seldom show anti-acetylcholine receptor antibodies. The antibodies are invariably absent in polymyositis. Anti-Hu , anti-Yo & anti-Ri Antibodies** These are found in paraneoplastic neurological syndromes and are detected using sections of monkey cerebellum. Anti-Hu antibodies are associated with encephalomyelitis, sensory neuropathy and rarely autonomic neuropathies with gastrointestinal dysmotility.

Small cell lung carcinomas are found in 80% of cases. Anti-Yo antibodies are associated with subacute cerebellar ataxia. 90% of cases have carcinoma of ovary or breast. Anti-Voltage Gated Calcium & Potassium Channel Antibodies** Anti-calcium channel antibodies are found in the paraneoplastic Lambert-Eaton syndrome anti-anti-potassium channel antibodies are found in Isaac’s neuromyotonia syndrome.. Anti-Glutamic Acid Decarboxylase Antibodies (Anti-GAD)** These can be found in patients with “Stiff-Man Syndrome” in approximately 40 % of cases as well as a majority of patients with newly diagnosed IDDM. Anti-Ganglioside Antibodies** These are often found in a variety of autoimmune peripheralnervous system neuropathies such as multifocal motor neuropathy, Guillain-Barr and Miller-Fisher syndrome. � Anti-MuSK Antibodies** Antibodies to muscle-specific kinase are often found together with AChRAb in myasthenia gravis (MG) but also in a majority of patients with AChRAb-negative MG. They are strongly associated with the ocular form of MG. Anti-Pituitary Antibodies** There are a variety of anti-pituitary antibodies associated with such disorders as idiopathic panhypopituitarism, GH deficiency, ACTH deficiency and acromegaly. Anti-Parathyroid Antibodies* These are found found in hypoparathyroidism and polyglandular endrocine syndrome, type 1 Anti-Histone Antibodies* External Referral Laboratories * Mr Kevin Green, Laboratory Manager, Supraregional Protein reference Unit, Department of Immunology, PO Box 894, Sheffield, S5 7YT ** Neurosciences Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU The results of all these tests that are sent to specialised laboratories are reported directly to the requesting physicians.

It should be noted that this whole process is very expensive and time consuming .

GUIDELINES FOR REPEAT TESTING

Useful ANCA- Very useful for longitudinal monitoring of individual vasculitis patients. Successful treatment usually results in disappearance of antibody. Relapse is frequently preceded by re-appearance of antibody. GBM- Similar to ANCA but in Goodpasture’s dsDNA- Evidence is more controversial but there is frequently a good correlation between SLE flares and elevation of antibody.

Limited Usefulness RF: A very popular antibody but of disputed importance. There is some evidence that persistently positive patients have a more severe form of the disease whereas those that become RF-negative have a more benign outcome. This autoantibody is likely to be replaced in the near future by anti-CCP which appears to be of greater prognostic significance. Cardiolipin: This is used for the monitoring of pregnant women with SLE as there is a risk of miscarriage associated with this antibody. In addition, persistent elevation in SLE patients has been reported to crry a higher risk of thrombosis. PCA: Post-partum thyroiditis leads to a much greater risk of autoimmune gastritis and this is associated with PCA. PCA also appears to be involved in

ANA Q&A

Q.

What does the test detect?

A. Serum antibodies (usually IgG) to antigens on or inside the nucleus Q. How is the test performed? A. Usually by immunofluorescence on fixed cells. These days the cell of choice is almost always HEp2 (a human epithelioma cell line) which has very large nuclei enabling many ANA subtypes to be identified. Q. How should the blood sample be collected? A. The sample should be clotted so that serum can be used for the test. Fibrinogen may interfere with the test. Q. When should an ANA be requested? A. Any patient suspected of having a connective tissue disease or any autoimmune disease. Q. What is the significance of a positive result? A. This will depend very much on the subtype. The most commonly seen patterns are: 1. Homogeneous. Strongly

associated with SLE 2. Speckled. Found most frequently in SLE, Sjogren’s and MCTD 3. Centromere. Strongly associated with CREST syndrome 4. Nucleolar. Frequently founded in scleroderma 5. Nuclear Dots. This is a rare pattern but if found in PBC suggests a poor prognosis

Q. When should a patient be referred to a specialist? A. All positive samples are titred and a titre of 160 or greater is considered positive. It should be remembered, however, that the incidence of ANAs increases markedly with age and that the test is only an aid to diagnosis. Q. What are the pitfalls of ANA testing? A. Methodological: There are many antibodies that can give rise to a similar ANA pattern. eg. antibodies to Sm, RNP and La will all give a speckled pattern but are associated (but not diagnostic) for different connective tissue diseases (SLE, MCTD and Sjogren’s in this case). A speckled ANA should always be further subtyped.

Diagnostic: These antibodies are associated with a wide variety of connective tissue diseases but are also found in other diseases and in apparently normal people. There presence must therefore always be interpreted in the light of a full diagnostic investigation.

MEASUREMENT OF COMPLEMENT PROTEINS Introduction

Serum complement components are measured for the detection of:

1 Inherited deficiencies of complement components (which are very rare) 2 Complement activation during infectious diseases or immune complex diseases (which is much more common) Complement deposition in tissues is also detected in biopsy material (skin, kidney) to diagnose immune complex disease. Inherited Deficiencies Genetic deficiencies of almost all of the complement components have been detected very rarely in individuals. Deficiency of C3 and control proteins of the alternative pathway which lead to acquired deficiency of C3 are associated with recurrent bacterial infection with a variety of organisms. Deficiencies of the late components are also associated with recurrent infections, usually Neisserial. Deficiencies of the classical pathway are more commonly associated with immune complex disease rather than infection, demonstrating the key role of complement in the handling of immune complexes. Deficiency of the control protein C1-inhibitor is the cause of a unique disease, hereditary angioedema, which is an autosomal dominant trait characterised by intermittent swelling of limbs and internal viscera. Disease Associations of Complement Deficiencies

Component Deficiency Disease Association

C1q C1r C1s C1INH C2 C4 C3 C5 C6 C7 C8 C9 Properdin of Factor I

Recurrent infection, immune complex disease SLE like disease SLE like disease Hereditary angioedema (autosomal dominant) One of the commonest deficiencies. Frequently asymptomatic occasional SLE like disease SLE like disease* Recurrent bacterial infection Recurrent bacterial infection Recurrent gonococcal or meningococcal infection Variable, immune complex disease or no symptoms Recurrent gonococcal or meningococcal infection Recurrent gonococcal or meningococcal infection Recurrent pyogenic infection as C3

*C4 levels are the result of two genes C4A and C4B, both show codominant expression of alleles. 15% of the population have lack of one of the four alleles. Some are associated with SLE.

Disease Associations of Acquired Changes in Levels of Complement Components

Component Disease Associations

Increased levels C3, C4, Factor B Decreased levels C1 Inhibitor C1q, C1r, C1s C2 C4 C3 Factor B

Acute phase response (rises up to 100%) Hereditary angioedema, Acquired C1 inhibitor deficiency Immune complex disease especially SLE Acquired C1 Inhibitor deficiency Immune complex disease especially SLE Acquired and hereditary C1-Inhibitor deficiency Immune complex disease especially SLE Rheumatoid vasculitis, cryoglobulinaemia Acquired and hereditary C1 Inhibitor deficiency Acute glomerulonephritis Immune complex disease especially SLE with nephritis Acute poststreptococcal glomerulonephritis Liver disease Mesangiocapillary glomerulonephritis or partial lipodistrophy associated with nephritic factor (Sub) Acute infective endocarditis, bacteraemia, septicaemia Infection with gram negative organisms, bacteraemia, septicaemia

C3 and C4 levels are routinely measured and C1-Inhibitor levels in suspected cases of deficiency. Other components are not routinely measured. Determination of C4 Introduction C4 is a protein of the classical pathway of complement. Activation of this pathway occurs predominantly because of the presence of deposited IgG or IgM containing immune complexes. The products of C4 activation are rapidly removed from the circulation which results in lowered C4 levels. Depletion of C4 is thus frequently an indication of immune complex disease. One complication is that C4 is an acute phase protein whose concentration will rise during the acute phase of an infective or autoimmune disease. Use of Test Diagnosis and monitoring of immune complex disease eg SLE, rheumatoid arthritis or immune mediated vasculitis. Diagnosis of hereditary angioedema or other C1-inhibitor deficiencies. C1 inhibitor deficiency does not occur in the absence of a low C4 level. Method Used Nephelometry Reporting of Results

Clinically significant results are those < 0.12

–

0.30 g/l. The turnaround time is 5 working days.

Interpretation of Results Low levels of C4 strongly suggest immune complex disease. They are found most commonly in cases of active SLE, rheumatoid vasculitis (but not uncomplicated rheumatoid arthritis where C4 levels are usually normal). Very low levels are found in association with normal C3 levels in acquired or hereditary C1-inhibitor deficiency (vide infra). NOTE: C4 levels are very stable in healthy individuals and the test is very accurate. As a result serial determinations of C3 are sensitive monitors of disease activity. Since C4 is an acute phase protein, its synthesis will be stimulated in those diseases where it is consumed. It is thus possible for levels to remain in the "normal range" whilst fluctuating dramatically. In some long standing SLE patients C4 levels remain low. This does not necessarily denote active disease, however a sudden fall in levels does usually indicate exacerbation of disease activity. Serial determinations are always a better guide to disease activity. Determination of C3 Introduction C3 is the central protein of the complement system. It is involved in both the classical and alternative pathways. Activation of either pathway by immune complex or infectious disease results in activation of C3. The products of C3 activation are rapidly removed from the circulation which results in lowered C3 levels. Depletion of C3 is thus a useful indicator of immune function. One complication is that C3 is an acute phase protein whose concentration will rise during the acute phase of an infective or autoimmune disease. Uses of Test Serial C3 determinations are a powerful indicator of immune complex deposition, septicaemia or bacteraemia. They are useful in monitoring immune complex disease eg SLE or glomerulonephritis. C3 is dramatically depleted in acute bacterial infection, levels normalise on appropriate antibiotic treatment. C3 is also depleted in glomerulonephritis or partial lipodystrophy associated with nephritic factor (vide infra). Method Used Nephelometry. Reporting of Results Clinically significant results are those <0.73 – 1.4 g/l. The turnaround time is 5 working days. Interpretation of Results Low levels of C3 associated with low levels of C4 demonstrate classical pathway activation and strongly suggest immune complex disease. They are found most commonly in cases of active SLE. Low levels of C3 associated with normal levels of C4 demonstrate alternative pathway activation suggestive of infectious disease or nephritic factor activity. NOTE: C3 levels are very stable in healthy individuals and the test is very accurate. As a result serial determinations of C3 are sensitive monitors of disease activity.

Since C3 is an acute phase protein, its synthesis will be stimulated in those diseases where it is consumed. It is thus possible for levels to remain in the "normal range" whilst fluctuating dramatically. Serial determinations are always a better guide to disease activity. In some long standing SLE patients C3 levels remain low. This does not necessarily denote active disease, however a sudden fall in levels does usually indicate exacerbation of disease activity and a risk of renal damage.

Determination of C1-Inhibitor C1-inhibitor is a protein of the complement system which controls the activation of C1 and thus activation of the classical pathway. In C1-inhibitor deficiency there is uncontrolled activation of the classical pathway and depletion of C4. Since the activation occurs in the fluid phase C3 consumption does not occur. C1-inhibitor deficiency is rare. It can be hereditary or acquired. Hereditary C1-inhibitor deficiency is associated with hereditary angioedema a disease characterised by sporadic swelling of any part of the body but also with gastrointestinal problems associated with internal swelling. Acquired C1-inhibitor deficiency is associated with lymphoproliferative disease where an autoantibody to C1-inhibitor is produced. Symptoms can be identical to the inherited form of the disease. A low or absent C4 level is always found in C1 inhibitor deficiency. Use of Test Diagnosis of hereditary angioedema, diagnosis of acquired C1-inhibitor deficiency, and monitoring of danazol therapy. Method Used Nephelometry. Reporting of Results Clinically significant results are those <0.22 – 0.36 g/l. The turnaround time is 10 working days. Interpretation of Results Low levels of C1-inhibitor are only associated with acquired or genetic deficiency. C1-inhibitor is not consumed as a result of normal complement activation eg in immune complex disease. Low C1-inhibitor levels are invariably associated with very low C4 levels. Marginally reduced C1-inhibitor levels are very unlikely to be significant if the C4 level is normal. A second type of C1-inhibitor deficiency is associated with normal levels of an inactive form of C1-inhibitor. Here, again C4 levels are low. Functional tests can be carried out to identify inactive C1-inhibitor. NOTE: Hereditary angiodema is a rare disease which seldom presents as urticaria. It does present as unexplained abdominal pain and a family history may be present. Though hereditary, it is frequently diagnosed only later in life, in the late teen years to the early years of the third decade of life. Determination of Total Haemolytic Complement Activity The haemolytic activity of complement depends on the presence of all components in sufficient concentration. The test for the total complement classical pathway is the most useful and is routinely available. Although they can be used to measure complement depletion, these are better monitored by immunochemical determination of C3 and C4. Use of Test The main use of these tests are in the preliminary identification of the very rare genetic deficiencies of complement components. Method Used Radial Immunodiffusion. Reporting of Results The normal range is between 392 – 1019 U/ml Results are reported as normal or low. The turnaround time is 10 working days.

Interpretation of Results

In the event of very low haemolytic complement being detected in the absence of any evidence of complement activation determined by C3 and C4 levels, the possibility of primary or secondary complement deficiency should be considered.

INVESTIGATIONS OF IMMUNODEFICIENCIES Inherited (Primary) Immunodeficiancy Diagnosis of Immunodeficiency The investigaton of primary immunodeficiency can be complicated and expensive. It should only be undertaken where there is clear evidence of recurrent infection or relevant family history. Primary immunodeficiencies are very rare and are usually detected in childhood. However, they do not all result in repeated life threatening infection and can remain undetected until adult life. The pattern of infections provides key information for diagnosing the type of immunodeficiency: Repeated bacterial infections, particularly with gram-positive encapsulated bacteria, suggest antibody or complement deficiency. Useful initial investigations are IgG, IgA and IgM levels, C3 and C4 and functional antibodies. Repeated viral and/or fungal infections suggest a T cell abnormality. A useful initial investigation in addition to those above is a lymphocyte count. Lymphocyte phenotyping and function tests are required to fully assess T cell immunity. These tests are now not routinely available in the Immunology laboratory but lymphocyte phenotyping may be carried out by the Haematology laboratory by prior arrangement. Staphylococcal skin sepsis, deep-seated fungal infections, poor wound healing and severe periodontal problems suggest a neutrophil abnormality. Neutrophil function tests and surface marker analysis are required to fully assess neutrophil activity but these tests are now not routinely available in the Immunology laboratory. Prior to any detailed analysis of leucocyte function and phenotype it is obviously important that a full blood count is obtained. Specific protocols are used for the monitoring of acquired immunodeficiencies resulting from viral infection. These should only be applied following confirmation of HIV status. Lymphocyte phenotyping cannot be used as a surrogate marker for HIV infection. Amongst the tests which can be carried out are: (a) Lymphocyte Subsets and Function Tests

Unfortunately lymphocyte function tests are not routinely available from the Immunology laboratory.

(b) Granulocyte function

Primary deficiencies are extremely rare. Always associated with recurrent deep-seated bacterial or fungal infections. Deficiencies in opsonic activity, ie failure to trigger granulocyte function due to low complement or IgG levels are more common. Primary deficiencies can be studied by phagocytosis, phenotypic or respiratory burst measurements. Unfortunately, these tests are not routinely available in the Immunology Laboratory although in children it may be possible to test for some of the primary deficiencies of granulocyte function in another laboratory.

(c) Complement measurement see above

It is suggested that the Consultant Immunologist be contacted before initiating any specialised investigations.

Immunoglobulin G (IgG) Subclass Measurement

There are four subclasses of IgG (IgG1, IgG2, IgG3 & IgG4) defined on the basic of molecular characteristics. Their relative contributions to the total IgG in adults are: IgG1 60-70%, IgG2 14-20%, IgG3 4-8% and IgG4 2-6%. IgG1 is the predominant subclass so its presence may mask deficiencies of one or more of the other three subclass if only the total IgG is measured. The IgG subclasses demonstrate age, sex and genetic variation, IgG1 and IgG3 values attain 'adult' concentrations in early childhood whilst IgG2 and IgG4 do not attain 'adult' concentrations until the end of the first decade. Antibody responses show subclass restriction and it is important to note that polysaccharide antibody responses are mainly in the IgG1 subclass in children maturing to the IgG2 subclass in adults. Antibody responses are mainly of IgG1 and IgG3 subclass whilst antibody to parasitic antigen is usually IgG4. Clinical Use Isolated or compound deficiencies of all four subclasses have been described but, whilst some patients with deficiency exhibit clinical symptoms, others demonstrate no evidence of disease. With the exception of IgG1 deficiency, the total serum IgG is usually within the age-related reference range, or even increased, in both primary and secondary forms of IgG subclass deficiency. This suggests that the other subclasses may attempt to compensate for an isolated defect. IgG subclass assay may be of value inpatients with multiple recurrent infections in whom there is no overt immunoglobulin deficiency. The total lack of an IgG subclass may be see in healthy individuals. IgG1 deficiency occurs most commonly in combination with defects in synthesis of other immunoglobulin isotypes and probably represents a form of common variable immunodeficiency. IgG2 deficiency is the most common of the subclass deficiencies with an incidence approaching 1:1000 and can be associated with recurrent lower respiratory tract infections due to gram positive encapsulated bacteria. It may be associated with IgA deficiency. It is this group of IgA deficient subjects who may benefit from replacement immunoglobulin therapy. IgG2 deficiency has also been reported in children with multiple recurrent otitis media. A combination IgG2 and IgG3 deficiency may be associated with ataxia telangiectasia. IgG1 concentrations are usually raised, thus masking the deficiency and giving normal total IgG concentrations. In the absence of compensatory IgG1 increases, the combined deficiency is associated with severe pyogenic infections. There is some suggestion the IgG2-IgG4 deficiency may be a contributing factor in 10% of cases of 'idiopathic' bronchiectasis. IgG2-IgG4 deficiency may also be associated with IgA and/or IgE deficiency. IgG3 deficiency is not usually associated with severe disease but a minority of patients show progressive and recurrent respiratory infections with obstruction lung disease. IgG3 deficiency may be seen in association with IgG1 deficiency. Levels are reduced in some patients with juvenile diabetes mellitus and in the Wiskott Aldrich syndrome. IgG4 deficiency as an isolated event is exceedingly rare. Method Used Nephelometry Reporting of Results The reference range for each individual subclass depends on the age of the patient (see Table below) and will be shown on the Report Form for the relevant age group. The turnaround time is 5 working days.

g/l IgG1 IgG2 IgG3 IgG4 0-2 years 1.94-8.42 0.22-0.30 0.18-0.85 0.005-0.78 2-4 years 3.15-9.45 0.36-2.25 0.17-0.67 0.010-0.53 4-6 years 3.06-9.45 0.60-3.45 0.099-1.22 0.018-1.12 6-8 years 2.88-9.18 0.44-3.75 0.15-0.85 0.019-0.93 8-10 years 4.32-10.6 0.76-3.55 0.17-1.73 0.016-1.15 12-14 years 3.42-11.5 1.00-4.55 0.28-1.25 0.037-1.36 14-18 years 3.15-8.55 0.64-4.95 1.30-1.96 0.11-1.57 18-120 years 3.82-9.28 2.41-7.00 0.218-1.76 0.039-0.86

Subclasses ( all in g/l )

GUIDELINES FOR ALLERGY TESTING

Allergic diseases probably affect around 25% of the population. They are an increasing cause of illness. Laboratory tests can play an important part in diagnosing allergy and identifying specific allergens. However, the tests are expensive and have to be interpreted with caution. This laboratory can measure the total level of IgE in a serum sample and can measure IgE against many different specific allergens (often collectively called RAST tests). The tests are done on serum obtained from a clotted sample of blood. The blood must be put in a tube with no anti-coagulant. The doctor requesting the tests should fill in relevant clinical details on the request form and should indicate the specific tests that are required, e.g. total IgE or antibodies to cat, grass, or milk, eggs etc. Specific IgE tests are most useful to confirm a clinical history of a suspected “trigger allergen” by demonstrating the presence of IgE antibodies to that allergen. They are not useful as blind screening tests. When the total IgE level is raised, this will confirm that the patient is allergic (atopic) provided they have no other cause for a raised IgE eg parasitic infection. A very low level (<20Ku/L) of IgE suggests that the patient is not atopic. However, occasionally an atopic patient may have a normal total IgE level (<100k/L) but have a high level of IgE to one or more specific allergens. Determination of total IgE levels is probably of most use in the wheezy child to differentiate an infectious (viral) cause from an allergic one. A high level of IgE to a specific allergen may confirm clinical reactions to the allergen, but the presence of IgE antibodies does not necessarily mean that the patient will have symptoms related to that allergen. Conversely a negative specific allergen IgE antibody test does not always exclude allergy and a patient may have symptoms when no specific IgE can be detected and it does not exclude the potential for developing allergy or resolution of a previous allergic condition. There are specific tests which can be used to detect IgE antibodies to many hundreds of different allergens but our laboratory only has the most commonly requested allergens. If a patient has had an allergic reaction to a specific allergen several years previously and has not been exposed to that allergen since, a negative specific IgE antibody level does not exclude allergy. If a patient has an anaphylactic reaction to a specific allergen IgE antibodies to the allergen may be “used up” in the reaction. Blood samples to test for specific IgE in the patients serum should therefore be taken 2-3 weeks after the anaphylactic reaction so the patient has time to build up the level of antibody. An atopic subject may have a high level of total IgE (up to 5000Ku/L) with very high IgE antibodies to many different specific allergens. Therefore, there is no point in these cases in doing multiple tests. The specific allergens causing symptoms need to be determined clinically from the history and if appropriate may be confirmed by testing for IgE specific to that allergen. The aim of laboratory allergy testing is to identify potentially harmful substances which can be avoided. For instance,.if a patient has symptoms suggestive of allergy to nuts but uncertain which nut is responsible, then their blood can be tested against mixed nuts (peanut, hazelnut, brazil nut, almond, coconut). If the text for ‘mixed nuts’ is positive, then the individual components should be tested for, depending on the patient symptoms. Similarly there are mixtures of animal fur (cat, dog, horse and cow dander) feathers from different birds (goose, chicken, duck and turkey feathers) and from different rodents (guinea pig, rabbit, hamster epithelium, rat and mouse).

In all case testing for specific IgE antibodies against the specific allergen is preferred as these tests are relatively costly and

‘

screening

’

by testing against a large number of allergens should be avoided in patients with non-specific symptoms.

It should be noted that is general the level of specific IgE antibodies does not indicate the severity of the allergic reaction following exposure and a negative result does not necessarily exclude an adverse reaction following exposure to be allergen. Similarly, a ‘false positive’ results do occur and the Consultant Clinical Immunologist should be contacted for discussion of the clinical significance of the results if necessary. Available Allergens Foods Egg White Barley Pea Almond Beef Salmon Apple Melon Peach Lentil Cow’s Milk Oat Peanut Crab Orange Strawberry Gluten Mutton Pecan Walnut Cod Maize Soya Shrimp Potato Yeast Hard Cheese Banana Cashew Turkey Wheat Rice Hazelnut Tomato Coconut Garlic Chicken Cocoa Pistachio Rye Sesame Brazil Nut Pork Tuna Onion Kiwi Pear Pineapple Mixed Nuts (peanut, hazelnut, brazil nut, almond, coconut) Mixed Fish (cod, shrimp, blue mussel, tuna, salmon) Animals & Insects House Dust Mite Honey Bee Venom Common Wasp Venom Horse Dander Dog Dander Cat Dander Mixed Animal (cat, dog, horse & cow dander) Mixed Feathers (goose, chicken, duck, turkey) Mixed Rodent (guinea-pig, rabbit, hamster epithelium, rat, mouse) Mixed Cagebird (budgerigar, parakeet, parrot, finch, canary) Pollens Mixed Grass Pollen (sweet vernal, rye, timothy, cultivated rye, velvet grass) Mixed Tree Pollen (box-elder, silver birch, oak, elm, walnut) Drugs Penicilloyl G Penicilloyl V Ampicilloyl Amoxycilloyl Suxamethonium Occupational Latex Mouse Epithelium Mouse Urine Rat Epithelium Rat Urine Moulds Aspergillus Mixed Moulds (penicillium notatum, cladosporium herbarum, aspergillus fumigatus, alternaria alterata Other allergens may be available on request Reporting of Results <0.35 kUA/l No specific IgE antibody detected 0.35-0.70 kUA/l Low Level 0.70-3.50 kUA/l Moderate Level 3.50-17.5 kUA/l High Level 17.5-100 kUA/l Very High Level >100 0 kUA/1 Extremely High Level

The turnaround time for these tests is 10 working days.

Total IgE Reference Range <12 months 0-50 KU/l 1-2 years 0-80 KU/l 2-5 years 0-140 KU/l 5-14 years 0-550 KU/l 14-18 years 0-380 KU/l >18 years 0-250 KU/l

Determination of Tryptase Activity Activated human mast cells secrete preformed, granule derived, mediators including histamine, proteoglycans and the neutral preteases, tryptase and chymase, together with newly formed mediators. The later include prostglandins, leucotrienes and platelet activating factor. Since tryptase is specific marker of the secretory granule, its presence in various body fluids is a reflection of mast cell degranulation. Tryptase is a serine esterase and basal concentrations are in the order of 0.2 - 8 ug/L with concentrations of more than 13.5 ug/L following allergic stimulation or mast cell degranulation. Levels as high as 100 ug/L may be seen in the extreme clinical situations, such as a severe reaction to anaesthetic agents. Tryptase is catabolised by the liver with an in vivo half-life of 3 hours compared to the 2-3 minutes of histamine. They decay curve after allergic stimulation and mast cell degranulation is observed over 24 hours compared with the 20 minutes for histamine. Tryptase is stable in isolated plasma or serum. Isolated tryptase concentrations may be of value in the assessment of allergic disorders and mast cell syndromes e.g. mastocytosis. Investigation of anaphylactic reactions would be better conducted with serial samples collected within one hour of reaction and subsequently at 3, 8 and 24 hours, or at similar intervals, if it is not possible to collect the samples at these times. Note that it is essential that all samples and accompanying Request Forms state the time of the adverse reaction and also have the precise time and date of the sample so that the results can be interpreted in relation to the event thought to trigger off mast cell degranulation. The turnaround time is 5 working days.

SPECIFIC IgG ANTIBODY TEST AGAINST FUNGAL & AVIAN PRECIPITINS IgG antibodies are part of the natural defence system in the body and are produced in response to contact with foreign substances. If intense exposure to these antigens continues over a long period of time, clinical disease may develop in some people. Clinical symptoms of Extrinsic Allergic Alveolitis (EAA), Farmer's Lung or Bird Fancier's Lung may include shortness of breath, aching joints and muscles and a general feeling of being unwell. EAA can occur alone or with Allergic Broncho- Pulmonary Alveolitis (ABPA). If left untreated ABPA can lead to permanent lung damage (fibrosis). Aspergilloma, a fungus ball, may develop with chronic lung disease. Although the incidence of ABPA is low, patients with asthma or cystic fibrosis are more susceptible to this disease and early diagnosis is essential to limit lung damage. Traditionally, the Ouchterlony immunodiffusion method has been used to demonstrate qualitatively, the presence of precipitin antibody to a specific antigen. These tests are technically demanding to perform and only semi-quantitative The automated test that we have adopted (solid phase immunoassay) provides a quantitative result (in mg/l) which can be used to monitor patients and the clinical effectiveness of treatment over a period of time Measuring Range

The measuring range for specific IgG antibodies is 2-200mg/l

Quantitative tests for IgG antibodies against the following antigens are available Gm3 Aspergillus fumigatus Gmx7 Combination of Micropolyspora faeni & Thermoactinomyces vulgaris Ge91 Pigeon serum proteins, feathers & droppings Ge90 Budgerigar serum proteins, feathers & droppings Ge92 Parrot serum proteins, feathers & droppings Note – this is not an exhaustive list, please contact the Immunology Laboratory for additional tests. Interpretation of Specific IgG Antibody levels Aspergillus precipitins 0 - 39 mg/l Unlikely to indicate aspergillosis 40 – 89 mg/l Likely to indicate aspergillosis if patient has appropriate clinical features 90 mg/l or greater Highly likely to indicate aspergillosis Note: In non-CF patients 40 is the cutoff whilst in CF patients 90 is the cut off Farmers lung (Micropolyspora faeni & Thermactinomyces vulgaris precipitins) 0 – 39 mg/l Unlikely to indicate Farmers lung 40 - 89 mg/l Likely to indicate Farmers lung if patient has appropriate clinical features 90 mg/l or greater Highly likely to indicate Farmers lung Avian precipitins (Pigeon, Parrot and Budgerigar) 0 - 9.9 mg/l Unlikely to indicate significant reaction to Bird Antigens 10 mg/l or greater Likely to indicate significant reaction to Bird Antigens