IMMUNOLOGY SERVICES SCOTTISH NATIONAL BLOOD TRANSFUSION SERVICES EDINBURGH

Transcription

1 IMMUNOLOGY SERVICES SCOTTISH NATIONAL BLOOD TRANSFUSION SERVICES EDINBURGH USER MANUAL; V3.3 Revised October,

2 CONTENTS GENERAL INFORMATION AUTOANTIBODIES IN ENDOCRINE DISORDERS AUTOANTIBODIES IN LIVER DISEASES AUTOANTIBODIES IN PRIMARY VASCULITIDES & GOODPASTURE S SYNDROME AUTOANTIBODIES IN CONNECTIVE TISSUE DISEASES MISCELLANEOUS AUTOANTIBODIES p3 p4 p7 p8 p10 p15 GUIDELINES FOR REPEAT TESTING OF AUTOANTIBODIES p19 AUTOANTIBODY CODES ON TRAK MEASUREMENT OF COMPLEMENT PROTEINS MEASUREMENT OF IgG SUBCLASSES GUIDELINES FOR ALLERGY TESTING SPECIFIC IgG ANTIBODY TEST AGAINST FUNGAL & AVIAN PRECIPITINS LABORATORY TURNAROUND TIME DETERMINATION OF UNCERTAINTY p20 p21 p25 p26 p29 p30 p30 2

3 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 Friday: 8.30 am to 5.00 pm There is no weekend working or out of hours service. Blood Samples 1 Specimens and request forms MUST be labelled with the patient's surname, forename, date of birth, and CHI number (or other previously agreed unique identifier) in accordance with SNBTS Zero Tolerance Policy. Location, clinical details and date of collection are desirable. Addressograph labels may be used on specimen tubes. Requests not complying with this policy will not be accepted. 2 Specimens should be accompanied by the appropriate Request Form completed, including all patient details as stated in para 1 above. It is essential to indicate where the Report of Test Results should be sent to. 3 Additional tests may be requested in retrospect on stored samples (where available). Samples are stored for 6-9 months dependent on availability of space. This laboratory has the responsibility for the investigation of patients suspected of suffering from disease consequent on abnormal functioning of the immune system, in particular, autoimmunity, immune deficiency and allergy. Tests for Autoantibodies, Complement Components, Total IgE, Specific IgE, IgG Subclasses, IgG Precipitins and Tryptase 5-10 ml of clotted blood should be sent in an anticoagulant free tube. For children, a 1-2ml clotted sample may be sent. Samples which are grossly haemolysed or excessively lipaemic or icteric may be rejected as these conditions can interfere with the immunoassay process and render the result useless. Transport All blood samples should be sent to the above address either by post, following current guidelines, or by using the LUHT delivery service. 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 Scientist is available by telephone for discussion of the clinical significance of test results. Patients requiring information on clinical management or therapeutic implications are requested to discuss such matters with their GP in the first instance. 3

4 Staff and Telephone Numbers Consultant Clinical Scientist...Dr M Kadlubowski BMS Mrs G Steven BMS2..Mr G McIntosh Immunology Laboratory, Reception & Results / (Internal 27525/27529) Dr Kadlubowski (Internal 27535) Secretary (Internal 27521) Abbreviations used: IIF Indirect Immunofluorescence ELISA Enzyme Linked Immunosorbent Assay AUTOANTIBODIES IN ENDOCRINE DISORDERS Anti-Thyroid Peroxidase Antibodies (anti-tpo) 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 diseases such as Graves disease, Hashimoto s Thyroiditis and Primary Atrophic Hypothyroidism. They are usually IgG and rarely respond to treatment. Anti-TPO in other autoimmune diseases (eg Pernicious Anaemia, Insulin-Dependent Diabetes Mellitus and Myasthenia Gravis) may indicate subclinical thyroid disease. s Diagnosis of Autoimmune Thyroid Disease. ELISA for IgG anti-tpo. Normal: 0-50 IU/ml Positive: >50 IU/ml Anti-TPO antibodies are detected in: % of patients with Hashimotos Thyroiditis 70-90% of patients with Graves Disease 60% of patients with Primary Hypothyroidism 60% of patents with Pernicious Anaemia % of patients with Insulin-Dependent Diabetes Mellitus 15-25% of patients with Myasthenia Gravis They are detected in low levels in 2-8% of normal individuals particularly the elderly and more often in women than men. 4

5 Anti-Gastric Parietal Cell Antibodies (anti-gpc or PCA)) 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 ATPdependent 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 (see below). 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 Mellitus or Addison s Disease. Diagnosis of pernicious anaemia. Indirect immunofluorescence (IIF) on rat gastric mucosa. This antibody is measured on a combined liver, kidney and stomach section and so the results for anti-mitochondrial antibodies and anti-smooth muscle antibodies will also be reported. Positive results are reported as +/- (borderline), + (positive) or ++ (strong positive) Anti-gastric parietal cell antibodies are detected in: >90% patients with Pernicious Anaemia. 30% patients with Addisons Disease 30% patients with Autoimmune Thyroiditis 30% patients with Insulin-Dependent Diabetes Mellitus 20% patients with Primary Ovarian Failure Approximately 5% of the normal population 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 asymptomatic patients with elevated levels should be followed up. PCA have also been reported in H.pylori-induced gastritis. The presence of these antibodies can be masked by anti-mitochondrial antibodies and so are not reported if the latter are present. 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 this disease. ELISA for IgG IFA. Normal: U/ml Positive >6.0 U/ml 5

6 Anti-GAD and anti-ia2 Islet cell antibodies (ICA) are helpful in the diagnosis and management of insulin dependent diabetes mellitus (IDDM) and have been measured by indirect immunofluorescence (IIF) on pancreas sections since Many antibody specificities have been detected but the most clinically relevant are autoantibodies to glutamate decarboxylase (GAD) and a tyrosine protein phosphatase known as IA-2. ICA measurements by immunofluorescence (known as pancreatic islet cell (PIC) testing) are less sensitive than combined anti-gad and anti-ia-2 measurements and are also more difficult to perform and standardise. For these reasons, the measurement of anti-gad and anti-ia-2 (together with anti-insulin antibodies in some labs.) is gradually replacing PIC testing. There are two isoforms of GAD known as GAD65 and GAD67. These are highly homologous but autoantibodies in IDDM are almost always restricted to GAD65. Several epitopes have been identified but IDDM binding is almost always restricted to conformation dependent epitopes. Patients with Stiff Person Syndrome (SPS) usually also have autoantibodies to GAD65 but these usually bind the linear epitopes the titres tend to be 100- to 500-fold higher than those found in IDDM. Diagnosis of Type I, Insulin-Dependent/Juvenile Diabetes Mellitus. ELISAs for IgG autoantibodies to GAD65 and IA2 Anti-GAD Normal: < 5 U/ml Positive > 5 U/ml Anti-IA2 Normal: < 7.5 U/ml Positive > 7.5 U/ml Autoantibodies to IA-2 are highly specific for IDDM especially in childhood. Anti-IA2 is detected in a high frequency at diagnosis in type 1 diabetic children whereas the frequency is lower in adult onset type 1 diabetic patients. When autoantibodies against GAD and IA2 are measured at diagnosis, antibody titres may be considered to identify slow (low titres) or rapid progression (high titres) to beta cell failure Anti-GAD autoantibodies are found in the majority of newly diagnosed IDDM but are also found in Type 2 diabetes mellitus and latent autoimmune diabetes in adults (LADA). In patients with type 2 diabetes or LADA the presence of anti-gad is strongly associated with future insulin dependency and a drift towards type 1 diabetes. Also anti-gad is high and persists for a longer time in the slowly progressive form of IDDM (SPIDDM) The need for insulin treatment during pregnancy and positivity for anti-gad confer a high risk of subsequent progression to type 1 diabetes in women affected by GDM. Anti-GAD are found in autoimmune polyendocrine syndrome Type 1 (APS-1), several rare neurological diseases (stiff person syndrome (SPS), cerebral ataxia, Batten disease, palatal myoclonus and drug-resistant epilepsy), autoimmune thyroid disease (AITD) and autoimmune polyendocrinopathy-candisiasis-ectodermal dystrophy (APACED). Because of the strong association of IDDM with autoimmune thyroid disease (AITD), testing for anti-gad and anti-ia2 in AITD is useful as a predictive marker for risk of progression to IDDM. In addition AITD patients positive for anti-gad are less likely to go into remission following treatment with anti-thyroid drugs. AUTOANTIBODIES IN LIVER DISEASES 6

7 Anti-Smooth Muscle Antibodies (SMA) Sera from patients with Type 1 autoimmune 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 antigens recognised by anti-smooth muscle antibodies in sera of patients with Type 1 autoimmune hepatitis are usually actins and/or myosins. 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. Anti smooth muscle antibodies can be diagnostic of Type 1 autoimmune hepatitis, especially if ANA are also present, and are useful in differential diagnosis of liver diseases. IIF using rat liver, kidney and stomach tissues. This antibody is measured on a combined liver, kidney and stomach section and so the results for anti-mitochondrial antibodies and anti-parietal antibodies will also be reported. Positive results are reported as: +/- (borderline), + (positive) or ++ (strong positive) Smooth muscle antibodies are detected in: 40-70% of patients with Type 1 autoimmune hepatitis 50% of patients with primary biliary cirrhosis 28% of patients with cryptogenic cirrhosis Type 1 autoimmune 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) and they have been found in less than 2% of the normal population. In these cases SMA are usually present at low titre. Anti-Mitochondrial Antibodies (AMA) Nine different AMA patterns are recognised by IIF, termed M1 to M9. Of these, M2, M4, M8 and M9 are asscociated with primary biliary cirrhosis (PBC) but M2 are diagnostically the most useful. 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 enzymes and the related 45kDa protein. Differentiating these nine AMA patterns can be very difficult and therefore all AMA requests are initially screened by IIF. The finding of any AMA pattern leads to that serum being reflexed to an M2 specific ELISA Diagnosis of Primary Biliary Cirrhosis; differential diagnosis of liver disease. Sera are initially screened by IIF using sections of rat liver, kidney and stomach. This antibody is measured on a combined liver, kidney and stomach section and so the results for anti-parietal cell antibodies and anti-smooth muscle antibodies will also be reported. If AMA are observed the sera are further tested using an M2-specific ELISA. 7

8 Positive results are reported as: +/-(borderline), + (positive) or ++ (strong positive) ELISA results are reported as Non-M IU/ml M2 >10 IU/ml Anti-mitochondrial antibodies of the M2 type are present in at least 90% of patients with PBC where they are considered diagnostic but can also be present in other autoimmune disease such as 2 o Sjögrens syndrome, autoimmune thyroid disease, SLE or polymyositis. Can be associated with anti-smooth muscle antibodies in PBC/CAH overlap. Antibodies to nuclear dots or centromere proteins together with M2 in definite PBC are strongly indicative of poor prognostic outcome. Non-M2 AMA antibodies can also be associated with PBC (M4, M8 & M9) but may also be indicative of syphilis, connective tissue diseases, cardiomyopathy and drug-induced hepatitis. Please note that the laboratory does not subtype non-m2 anti-mitochondrial antibodies. Anti-Liver/Kidney Microsomal Antibody (anti-lkm1) Antibodies recognising this antigen are found in Type 2 autoimmune 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 autoimmune 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. These two subtypes are not reported. Diagnosis of Type 2 autoimmune hepatitis in the young. IIF using sections of rat kidney, liver and stomach sections Positive results are reported as: +/- (borderline), + (positive) or ++ (strong positive) LKM antibodies are strongly associated with Type 2 autoimmune hepatitis but are also found in a subset of patients with chronic hepatitis C infection. AUTOANTIBODIES IN PRIMARY VASCULITIDES & GOODPASTURE S SYNDROME Anti-Neutrophil Cytoplasmic Antibodies (ANCA) 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 8

9 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. The diagnosis of primary vasculitides (mainly small vessel) such as Wegener s granulomatosis and microscopic polyangiitis and the monitoring of treatment. To avoid unnecessary testing, ANCA should be requested only if one or more of the following symptoms are present: Glomerulonephritis, especially RPGN Pulmonary haemorrhage, especially pulmonary renal syndrome Cutaneous vasculitis with systemic features Multiple lung nodules Chronic destructive disease of the upper airways Long-standing sinusitis or otitis Subglottic tracheal stenosis Mononeuritis multiplex or other peripheral neuropathy Retro-orbital mass ANCAs are measured by IIF and/or ELISAs which measure IgG antibodies to PR3 and MPO. Some ANCA IIF patterns can be confused with ANA and so the latter may be appended by the laboratory. Positive IIF results are reported as +/- (borderline), + (positive) and ++ (strongly positive). In addition the following ANCA patterns are reported: panca, canca, atypical panca, atypical canca or atypical ANCA. ANCA staining can be obscured by ANA staining, especially homogeneous ANA. In this case the pattern is reported as INA (not interpreted due to ANA staining). PR3-ANCA results are reported as Normal: 0-10 U/ml Positive: >10 U/ml. MPO-ANCA results are reported as Normal: 0-5 U/ml Positive: >5 U/ml It is important to be aware that in an unselected population only about 50% 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-Schönlein 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) 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. 9

10 Diagnosis of Goodpasture s Syndrome. ELISA for IgG anti-gbm. Normal: U/ml Positive: >20.0 U/ml 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. Urgent ANCA and/or GBM This is done using quantitative ELISAs. 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. This service is subject to staff availability. AUTOANTIBODIES IN CONNECTIVE TISSUE DISEASES Anti-Nuclear Antibodies (ANA) 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 Sjögren's syndrome antigen A (SS-A/Ro). The major reason for ordering the FANA test is as an aid to the diagnosis of a range of connective tissue diseases including systemic lupus erythematosus (SLE), Sjogren s syndrome and scleroderma. Anti-nuclear antibodies, particularly at low titres are associated with a number of other autoimmune diseases and are found frequently in the normal, especially elderly, population. 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. Indirect immunofluorescence (IIF) is performed on fixed HEp2 cells. The titre of antibody and the pattern of staining is reported. This cell line has large nuclei which allows better definition of the staining pattern and there are also significant numbers of dividing cells allowing detection of autoantibodies to cell cycle specific antigens such as centromere proteins. Results are reported as 'Negative' or 'Positive' with the antibody titre on HEp2 cells reported as 1:80 (borderline) to >1:640 (strongly positive). 10

11 A large number of staining patterns have been described but only the following most common patterns are reported here: Homogeneous Speckled Nucleolar Centromere Homogeneous staining of interphase nuclei plus positive staining in the chromosomal region of mitotic cells Large, coarse or fine speckled staining of the interphase nuclei with negative staining in the chromosomal region of mitotic cells Staining of nucleolus only. The appearance can vary depending on which of several nucleolar antigens are involved 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. Specific autoantibodies detected by the use of Hep2 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** Nucleolar** 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. Caused by many autoantibodies including PM-Scl, Th/To, RNA Polymerase III, RNA and RNA Helicase. 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 also in Sjogren s disease and Raynaud s (where it is likely to indicate future CREST). Its presence in Primary Biliary Cirrhosis is indicative of a more severe disease progression. Homogeneous/ Caused by antibodies to Scl-70, a specific marker for Scleroderma, PM-Scl which is associated Nucleolar with Polymyositis/Scleroderma overlap and U3RNP which is found in Scleroderma. Speckled/ Nucleolar PCNA Can be caused by antibodies to La which are found predominantly in SLE and SS and RNA Polymerase I which are associated with scleroderma. 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 connective tissue diseases Centriole Found rarely in a variety of connective tissue diseases Nuclear Dots*** Strongly associated with PBC but also found in CAH and sicca. Nucleolar/ Centromere May indicate connective tissue disease. Nuclear Membrane*** Midbody Found rarely in a variety of connective tissue diseases Found rarely in a variety of connective tissue diseases 11

12 * A positive homogeneous pattern of 1/80 or greater (in any combination) will trigger a dsdna ELISA ** A positive speckled and/or nucleolar pattern of 1/160 or greater (in any combination) will trigger an ENA Screen ELISA. *** A positive centromere, nuclear dot or nuclear membrane staining pattern will trigger an M2 mitochondrial ELISA Antibodies to Double-Stranded DNA (anti-dsdna) 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. 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, antidsdna antibodies are frequently found in patients with overlap symptoms between SLE and other autoimmune diseases. An ELISA for IgG anti-dsdna. Normal: IU/ml Positive: >20.0 IU/ml 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) 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, Sjögren's syndrome (SS), Rheumatoid Arthritis (RA) and other rarer CTDs.. In addition to determining ANA by IIF as described above the measurement of a subgroup of these antibodies known collectively as anti-enas is carried out. Most of these were initially identified by counterimmunoelectrophoresis and immunodiffusion as they were readily soluble in salt solution but they are now usually measured by ELISA. In this laboratory antibodies to Sm, RNP, Ro, La, Scl-70 & Jo-1 are measured. Assays to determine ENA specificities are indicated in the evaluation of any patient suspected of having a systemic connective tissue disease. 12

13 Samples for anti-ena are initially screened using an ELISA containing all six antigens. Positive samples are further investigated using a profile ELISA which can identify the individual specificities. Both the Screen and Profile ELISAs detect IgG autoantibodies.. ENA Profile: Normal: 0-25 U/ml Positive: >25 U/ml 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 for which it is a marker There is evidence to suggest that this antibody is associated with the more severe form of SLE, especially with renal involvement. 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 may not be 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 Sjögrens 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. Two forms of Ro are found, known as Ro60 and Ro52 and antibodies to both are clinically significant. ENA should always be requested if a Ro-associated disease is suspected as anti-ro52 will not be detected by IIF of Hep2 cells and anti-ro60 will frequently be missed. In addition it has recently been reported that anti-ro(52) in the absence of anti-ro(60) are frequently found in inflammatory myosists. Both specificities are detected in the ELISA used. 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 Sjögrens 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 is vital as it is associated with congenital heart block in the fetus from weeks gestation resulting in foetal death in approximately 2% of cases. 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. These antibodies target 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. 13

14 SUMMARY OF ANTI-NUCLEAR ANTIBODY PROFILE Percentage of Sera Positive for Autoantibodies Antibodies to SLE Drug induced LE MCTD Sjögrens Syndrome Progressive Systemic Sclerosis Dermato/ Polymyositis Rheumatoid Arthritis Nucleii dsdna ssdna Histones Ro La Sm RNP Scl Jo Centromere ~ CREST Nucleolar RA plus Sjögrens 14

15 Anti-Cardiolipin Antibodies (ACA) MISCELLANEOUS AUTOANTIBODIES 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 identical to the M1 anti-mitochondrial antibodies and are responsible for the false positive VDRL. 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. ELISA for IgG ACA Normal: 0-10 GPLU/ml Positive: >10 GPLU/ml 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. Anti-Citrullinated Cyclic Peptide (Anti-CCP) Citrullination is a post-translational process whereby arginine is deimidated by peptidyl arginine deiminase to form citrulline. This appears to happen to a large number of proteins in the synovium of patients with Rheumatoid Arthritis.. Many citrullinated proteins have been investigated as target antigens for the antibody but most laboratories now use a second generation cyclic peptide known as CCP2. This antibody appears to be more specific than RhF for Rheumatoid Arthritis and is gradually replacing RhF. Diagnosis of rheumatoid arthritis, differential diagnosis of rheumatic disease. Fluoroimmunoassay for IgG anti-ccp2 Normal: U/ml Positive: >4.8 U/ml 15

16 Anti-CCP antibodies are more specific than Rheumatoid Factor for diagnosing Rheumatoid Arthritis (RA) and are present in approximately 70% of early stage RA patients. They are good predictors of radiographic joint disease and may be useful in discriminating between erosive osteoarthritis and RA. These antibodies may be present up to 10 years prior to diagnosis and so are highly predictive of the future development of RA in both healthy subjects and patients with undifferentiated arthritis. Anti-CCP are only rarely found in patients with Juvenile Idiopathic Arthritis. and are not associated with Lupus Arthropathy but their presence in Sjogren s Syndrome is indicative of future RA. In patients with clinical symptoms of Polymyalgia Rheumatica their presence is strongly suggestive of elderly onset RA. Anti-CCP occur in a subgroup of patients with type 1 autoimmune hepatitis who have a greater occurrence of cirrhosis at presentation and higher incidence of death from hepatic failure. Rheumatoid Factors (RF) 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 Sjögrens syndrome or Bacterial endocarditis. This test is much less specific for Rheumatoid Arthritis than anti-ccp (see above) by which it has largely been replaced. Diagnosis of rheumatoid arthritis, differential diagnosis of rheumatic disease. ELISA for IgM Rheumatoid Factor Normal: 0-20 IU/ml Positive: >20.0 IU/ml High levels are most common in Rheumatoid Arthritis, Subacute Bacterial Endocarditis (SBE) and Sjögrens 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 C-Reactive Protein levels should be used. 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. Anti-Thyroglobulin Antibodies (anti-tg) 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. Consequently, this antibody is of no diagnostic usefulness in endocrine disorders and is no longer available. 16

17 Anti-Acetylcholine Receptor Antibodies (AChRAb)** Anti-acetylcholine receptor antibodies are found in 90% of patients with active adult onset generalised myasthenia gravis. They are IgG and are pathogenic. 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. 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-Hu, anti-yo & anti-ri Antibodies** Anti-Hu (ANNA-1), anti-yo (PCA-1) and anti-ri (ANNA-2) are found in a variety of paraneoplastic neurological syndromes. Anti-Steroid Cell Antibodies (Adrenal Cortex, Ovary & Testis)* These antibodies 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) and are usually detected by IIF. The antibodies belong predominantly to the IgG class. These antibodies are found in Addison s Disease, Premature Ovarian Failure, Polyglandular Autoimmune Syndromes and rarely in Insulin-Dependent Diabetes Mellitus. 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-Ganglioside Antibodies**** These are often found in a variety of autoimmune peripheral nervous system neuropathies such as multifocal motor neuropathy, Guillain-Barré and Miller-Fisher syndrome. IgG and IgM antibodies to GM1, GM2,GD1a, GD1b and GQ1b antibodies are measured. Anti-Parathyroid Antibodies* These are found in hypoparathyroidism and polyglandular endrocine syndrome, type 1 Anti-Histone Antibodies* These are helpful in the diagnosis of drug-induced lupus. Anti-Basal Ganglia Antibodies*** These are found in Paediatric Autoimmune Disorders Associated with Streptococcus (PANDAS) Anti-Aquaporin-4 Antibodies** These are directed at the water channels of astrocytes found in the glia limitans of patients with neuromyelitis optica. Anti-NMDA Receptor Antibody** 17

18 Found in limbic encephalitis, cognitive defects, epilepsy and the neuropsychiatric form of SLE.. Anti-Myelin-Associated Glycoprotein Antibody** These are found in a variety of neurological disorders but especially in sensory demyelinating neuropathies. Anti-Glycine Receptor Antibody** These are found in progressive encephalitis with rigidity and myoclonus (PERM) Anti-Insulin Antibody* These are found in insulin dependent diabetes mellitus and hyperinsulinaemic hypoglycaemia. They can also arise in response to insulin therapy. Anti-Skin Antibodies* Antibodies to desmosomes and to the basement membrane of skin are found in pemphigus (intraepidermal blistering) and pemphigoid (subepidermal blistering) respectively. External Referral Laboratories * 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 *** Department of Immunology, National Hospital for Neurology & Neurosurgery, University College London, Queen Square, London WC1M 3BG **** Department of Neurological Sciences, Southern General Hospital, 1345 Govan Road, Glasgow G51 4TF 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 OF AUTOANTIBODIES 18

19 Useful Limited Usefulness Not 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. C3/C4 & CH50. Useful for monitoring disease activity in RA and SLE and other immune complex diseases Specific IgE. Anaphylaxis can often result in the complete consumption of the pertinent specific IgE resulting in a false negative. Repeat testing after 2-3 weeks is therefore recommended where this is suspected. CCP: Levels of this antibody can fluctuate with disease activity. However, latter is probably better measured using CRP than CCP. RF: 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. 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 carry a higher risk of thrombosis. The following are considered not useful because: 1. levels remain persistently high/low regardless of clinical picture or treatment 2. disappear soon after diagnosis or 3. have not yet been shown to have any prognostic usefulness AMA, ANA, ENA, TPO, SMA, IFA, LKM, Adrenal/Ovary/Testis, GAD, IA2,PCA Given the above, the following autoantibodies will not be tested if a similar request has been received within the preceding 6 months: ANA, ENA, TPO, IFA, Ovary/Adrenal/Testis, GPC, AMA, LKM, GAD, IA2. The report will clearly indicate that there has been a test refusal. Should the requesting clinician, however, feel that a repeat test for a particular autoantibody is required for patient management then a phone call to the laboratory will ensure that it is carried out. 1. Profile Codes on TRAK AUTOANTIBODY CODES ON TRAK 19

20 Code IP2 IP4 IP6 IP7 IP11 IP16 IP17 IP18 IP19 Test Profile CH50, C3, C4 CH50, C3, C4, C1INH IgG Subclasses (Total IgG, IgG1, IgG2, IgG3, IgG4) Total and Specific IgE GPC, AMA, SMA Autoimmune Screen: GPC, AMA, SMA, ANA, TPO Vasculitis Screen: CH50, C3, C4, ANA, ANCA SLE Screen: CH50, C3, C4, ANA, dsdna, ENA CTD Screen: CH50, C3, C4, CCP, ANA, dsdna, ENA 2. Send Away Tests Test Code Destination Lab. (see above) AChReceptor Antibody IACH Oxford Anti-Gangliosides IGANG Glasgow Anti-Histones IHIST Sheffield Anti-Voltage-gated Calcium Channel IVGCC Oxford Anti-Voltage-gated Potassium Channel IVGKC Oxford Anti-Paraneoplastic Neuronal (Yo, Hu, Ri) INEUR Oxford Anti-Myelin-associated Glycoprotein IMAG Oxford Anti-Parathyroid IPARA Sheffield Anti-MUSK IMUSK Oxford Anti-Basal Ganglia IBAS London Anti-Aquaporin-4 IAQUA Oxford Anti-NMDA Receptor INMDA Oxford Anti-Steroid Cells (Adrenal,Ovary,Testis) IENDO Oxford Granulocyte-Specific Antibodies IGRAN Aberdeen Anti-Glycine Receptor IGLYC Oxford Anti-Insulin IINS Sheffield Anti-Pemphigoid & Anti-Pemphigus IPEMPH Sheffield Specific IgE Allergens Not Held in Stock ISALG Shefield MEASUREMENT OF COMPLEMENT PROTEINS 20

21 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 C1q C1r C1s C1INH C2 C4 C3 C5 C6 C7 C8 C9 Properdin of Factor I Disease Association 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 21

22 Component Disease Associations Increased levels C3, C4, Factor B Acute phase response (rises up to 100%) Decreased levels C1 Inhibitor C1q, C1r, C1s C2 C4 C3 Factor B 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 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. Diagnosis and monitoring of immune complex disease eg SLE, rheumatoid arthritis, immune mediated vasculitis and hereditary angioedema or other C1-inhibitor deficiencies. Turbidimetry The normal range is g/l. Low levels of C4 strongly suggest immune complex disease. They are found most commonly in cases of active 22

23 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. NOTE: C4 levels are very stable in healthy individuals and the test is very accurate. As a result serial determinations of C4 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 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 Diagnosis and monitoring of immune complex deposition, septicaemia or bacteraemia. They are useful in monitoring immune complex disease eg SLE or glomerulonephritis. Turbidimetry The normal range is g/l. 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. 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. 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 23

24 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. Diagnosis of hereditary angioedema, diagnosis of acquired C1-inhibitor deficiency, and monitoring of danazol therapy. Turbidimetry The normal range is g/l. 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. Classical Pathway Complement (CH50) Liposomes, encapsulating glucose-6-phosphate dehydrogenase (G6PDH) are used to mimic an invading organism. On addition of sample, antibodies in the reagent combine with dinitrophenyl groups on the surface of the liposomes. The resultant complex activates complement in the sample, which lyses the lipsomes releasing G6PDH which reacts with glucose-6-phosphate and NAD in the reagent to yield NADH. The change in absorbance is proportional to the complement activity in the sample. To detect deficiencies of any of the components in the classical complement pathway. Turbidimetry The normal range is U/ml In the event of a low CPC 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. 24

25 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: IgG %, IgG %, 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 in patients 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. Turbidimetry 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. g/l IgG1 IgG2 IgG3 IgG4 25

26 0-2 years years years years years years years years years 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 (<100KU/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). 26

27 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 nonspecific 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 Chili Pepper Blue Mussel Lemon Paprika White Bean Green Pepper Omega-5 Gliadin Mixed Nuts 1 (peanut, hazelnut, brazil nut, almond, coconut) Mixed Nuts 2 (pecan, cashew, pistachio, walnut) Mixed Fish (cod, shrimp, blue mussel, tuna, salmon) Mixed Citrus (orange, lemon, grapefruit, mandarin, tangerine, clementine, satsuma) 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 <0.35 kua/l No specific IgE antibody detected kua/l Low Level kua/l Moderate Level 27

28 kua/l High Level kua/l Very High Level >100 0 kua/1 Extremely High Level Total IgE Reference Range <12 months 0-50 KU/l 1-2 years 0-80 KU/l 2-5 years KU/l 5-14 years KU/l years KU/l >18 years KU/l Determination of Tryptase Activity Activated human mast cells secrete preformed, granule derived, mediators including histamine, proteoglycans and the neutral proteases, tryptase and chymase, together with newly formed mediators. The latter 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 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. Normal range is ug/l 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. 28

29 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 semiquantitative 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 Gmx7 Ge91 Ge90 Ge92 Aspergillus fumigatus Combination of Micropolyspora faeni & Thermoactinomyces vulgaris Pigeon serum proteins, feathers & droppings Budgerigar serum proteins, feathers & droppings 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 0-58 mg/l Unlikely to indicate aspergillosis >58 mg/l Likely to indicate aspergillosis if patient has appropriate clinical features Note: In Cystic Fibrosis patients 90 mg/l is the upper limit of normal Farmers lung (Micropolyspora faeni & Thermactinomyces vulgaris precipitins) 0 13 mg/l Unlikely to indicate Farmers lung > 13 mg/l Likely to indicate Farmers lung if patient has appropriate clinical features Avian precipitins (Pigeon, Parrot and Budgerigar) Pigeon 0-21 mg/l Unlikely to indicate significant reaction to Pigeon Antigens >21 mg/l Likely to indicate significant reaction to Pigeon Antigens Parrot 0-26 mg/l Unlikely to indicate significant reaction to Parrot Antigens >26 mg/l Likely to indicate significant reaction to Parrot Antigens Budgerigar 0-16 mg/l Unlikely to indicate significant reaction to Bird Antigens >16 mg/l Likely to indicate significant reaction to Bird Antigens LABORATORY TURNAROUND TIME The target for turnaround time (TAT) is 80% within 10 working days. This is measured monthly. Each month, beginning on the first working day of that month, every 30 th sample received by the laboratory is chosen until 100 samples have been collected. Sample registration is Day 0 and completion is when all requested tests have been successfully carried out and authorised. The following should be noted when looking at the Table below: 29

30 1. No account is taken of how many tests/patient have been requested 2. Reflex testing is carried out and this will delay completion of the request. 3. The laboratory is closed at weekends and so only working days are counted. 4. Some test are carried out only once/week Date % Authorised in <10 Working Days November December January February March April May June July August DETERMINATION OF UNCERTAINTY Uncertainty can be divided into uncertainty of measurement and uncertainty of results. Uncertainty of Measurement (Type A) In any assay system there may be bias due to systemic effects leading to inaccuracy. These include errors induced by factors such as variations in incubation temperature, blockage of plate washer, change in the reagent batch or modifications in testing method etc. Measurement uncertainty therefore stems from imprecision as a result of random effects on the assay system. These can be identified by imprecision studies at assay verification resulting from the use of internal quality control (IQC) material. IQC materials are included in assays where possible and are treated as patient samples. In the case of qualitative assays, control charts (Shewart) are constructed and the results are interpreted using Westgard rules. Uncertainty of Results (Type B) This uncertainty includes errors arising from pre-examination (correct patient identification, sample preparation, sample condition, transport, sample storage), examination (calibrator errors, inaccurate equipment, laboratory environmental factors (eg. lab. too hot), differences between operators) and post-examination procedures (inappropriate reference ranges, incorrect report, incorrect interpretation, reporting of equivocal results). To determine the uncertainty of results within the Clinical Immunology Laboratory, all staff are made aware of factors which may cause variability in assay results. These factors are highlighted during training and at regular staff meetings. 30