IMMUNOLOGY USERS HANDBOOK

Transcription

1 IMMUNOLOGY DEPARTMENT County Durham and Darlington NHS Trust University Hospital of North Durham IMMUNOLOGY USERS HANDBOOK July 2011 (reviewed annually or when major changes in repertoire) 1

2 CONTENTS: General Information, contact numbers, staff Autoantibody section Individual autoantibodies (alphabetical) Immunochemistry section Complement components Serum proteins Allergy testing Cellular section Lymphocyte phenotyping Lymphocyte function Neutrophil function Disease Index Alphabetical list of diseases with appropriate investigations 2

3 Immunology Department The Immunology Department was set up as a stand-alone department of the CDDAH NHS Trust (now CDDFT) in March It is based in Pathology, University Hospital of North Durham (UHND), Staircase C, Lower Ground Floor. The Immunology Department is a CPA accredited provider of Immunology Services. A clinical Allergy/ Immunology service has been set up within the General Medicine OPD, UHND. Weekly clinics are offered by Dr Desa Lilic, Consultant Clinical Immunologist Postal Address Immunology Department University Hospital of North Durham North Road Durham DH1 5TW Telephone: ext or (0191) Fax: ext or (0191) NB! All results are reported electronically and as printed reports. Please avoid telephoning for results wherever possible as this creates significant additional work, but do not hesitate to contact us for queries, consultations, interpretations & discussion. Medical staff Desa Lilic desa.lilic@cddft.nhs.uk Consultant Clinical Immunologist ext or (0191) Zoe Wilkinson zoe.wilkinson2@cddft.nhs.uk Secretary to Dr Lilic ext (0191) Scientific staff Patricia Smith patricia.smith@cddft.nhs.uk Lead Biomedical Scientist ext or (0191) Catherine Wedd Senior Biomedical Scientist ext or (0191) Debra Wilkinson, Rebecca Riant, Melanie Robson Biomedical Scientists BMS trainees Jonathan Bell Associate Practitioner 3

4 General Information Working hours The laboratory is open from 8.30am to 5.00pm Monday to Friday but samples can be sent in at any time. The Immunology Department does not provide an on-call service. For exceptional out-ofhours queries Dr D. Lilic, Consultant Immunologist or Mrs Patricia Smith, Lead Biomedical Scientist may be contacted at home via switchboard. Test repertoire We provide a comprehensive range of tests for the immunological investigation of patients. We aim to provide the highest quality of service with prompt delivery of accurate results, backed up by specialist medical and scientific expertise. Where specific tests are not available locally, we will refer samples on to colleagues in other centres. The quality of all our work is regularly and frequently monitored in National Quality Control Schemes (UKNEQAS). Requesting immunology tests Biochemistry/ haematology/ immunology request forms are in use. All in-house immunology tests are carried out at UHND; in BAGH and DMH, samples are registered by Pathology reception and sent to UHND. A list of immunology tests most frequently requested (both in-house and referred) is given in this handbook If you require tests that are not on the list, please contact the laboratory before sending sample to ensure that the test is available and the correct sample is taken. Referral Laboratory addresses: - Regional Immunology Department, RVI, Queen Victoria Road, Newcastle upon Tyne, NE1 4LP - Department of Immunology, PO BOX 894, Sheffield, S5 7YT - Regional Immunology Laboratory, Birmingham Heartlands Hospital, Bordesley Green East, Birmingham, B9 5SS - National Blood Service, Langley Lane, Sheffield, S5 7JN - Immunology Department, Churchill Hospital, Headington, Oxford, OX3 7LJ - Immunology Department, Camelia Botnar Laboratories, Great Ormond Street Hospital for Children, London, WC1N 3JH - Department of Neuro-Immunology, Room 917, Institute of Neurology, Queen s Square, London WC1N 3BG - Department of Clinical Biochemistry and Immunology, Level E4, Addenbrooks Hospital, Cambridge, CB2 2QQ - Neuroimmunology, Southern General Hospital, Glasgow, G51 4FT We check all reference laboratories annually to ensure they maintain standards required for Clinical Pathology Accreditation (CPA). 4

5 NB! NB! It is essential that full patient identification is provided. We are obliged to discard samples with inadequate identifying information. Clinical information is essential for interpretation of results thus ensuring highest quality service. If clinical details are lacking, interpretative comments cannot be given Interpretation of results The department is happy to assist in the interpretation of patient s test results. Interpretative comments are routinely added to test reports where appropriate. In the list of tests described in the following pages we have added a brief summary of the clinical application of each test that is intended to be helpful but is to assist in the selection of the most appropriate investigations. Comments / suggestions are appreciated & welcome! 5

6 Autoimmune Serology General information: Interpretation and comments are given subject to clinical details and reason for request being available. If further advice is required please contact the laboratory. Turn-around times (working days): o Routine Autoantibody Screen 3-5 days o ENAs 3-5 days o DNAs 3-5 days o RF 2-3 days o ANCAs 3-5 days o Send away tests days Includes: - (anti) nuclear factor (ANF) = antinuclear antibody (ANA) - mitochondrial antibody (AMA) - liver-kidney microsomal antibody (LKM) - gastric parietal cell antibody (GPC) - smooth muscle antibody (SMA) - rheumatoid factor (RF) - ribosomal antibody (reported if positive) - other antibodies (soluble liver antigens (SLA), liver-kidney microsomal (LKM) antibody, liver-pancreas antigen (LP) etc) if ANF is positive, sample will automatically be tested for extractable nuclear antigen antibodies (ENA) including Ro, La, Sm, RNP, Jo-1, ScL-70, Centromere antibody and double-stranded DNA antibodies (ds-dna); it is therefore sufficient to request autoantibodies only Samples positive for AMA and LKM antibodies will be automatically tested for M2 positivity if ANF is negative further testing will not be performed reticulin antibody is not reported but if positive, sample will automatically be tested for antiendomysial antibodies RF is done automatically as part of the autoantibody screen but can also be requested separately Repeat autoantibody screen should not be requested in less than 3-monthly intervals as existing antibody will not be degraded All the above tests are in-house tests. 6

7 Individual Autoantibodies (alphabetical order) Acetylcholine receptor antibody (AChR) Serum sample required: no special preparations needed prior to sampling; turn-around time This antibody is positive in 80-90% of patients with myasthenia gravis (referred test); if AChR negative, check MuSK antibody if high clinical suspicion (referred test). Adrenal antibody Serum sample required: no special preparations needed prior to sampling; turn-around time See anti-endocrine gland antibodies. Cardiac muscle antibody Serum sample required: no special preparations needed prior to sampling; turn-around time Positive in some patients with Dressler s syndrome and post-cardiotomy syndrome (referred test). Cardiolipin antibody (ACL) Haematology test See Anti-phospholipid antibody; this test is always done together with Lupus Anticoagulant and clotting assessment (in-house test performed in the Haematology laboratory; send sample to Haematology). CCP antibodies Cyclic citrullinated peptide (CCP) antibodies are very specific (>95%) for RA i.e. will not be found in other patients but have a relatively low sensitivity i.e. will be found in only ~ 3-40% of patients with RA (in-house test) Analytical range: U/mL; Reference range: Negative: < 7 U/mL Equivocal: 7 10 U/mL Positive: >10 U/mL Centromere antibody Indicated in the investigation of unexplained Raynaud s; positive in ~50% of patients with primary Raynaud s, 60-70% of patients with the CREST variant of scleroderma and 20% of patients with generalised scleroderma. Centromere antibody is always tested as part of the autoantibody screen (in-house test). Coeliac Disease antibodies Serum sample required: N.B! If the patient is on gluten-free diet, test may be negative in presence of Coeliac Disease; turn-around time 3-5 See Endomysial antibody; see also Gliadin, Tissue Transglutaminase (ttg test ), Coeliac Disease (CD), Dermatitis herpetiformis Cytokine antibodies 7

8 Serum sample required: no special preparations needed prior to sampling; turn-around time Antibodies to GM-CSF and interleukin 17/ interferons type 1 (alpha and omega) may be found in immune deficiencies (pulmonary alveolar proteinosis and APS1 respectively) (referred tests). Dermatitis Herpetiformis antibodies Serum sample required: N.B! If the patient is on gluten-free diet, test may be negative in presence of Coeliac Disease; turn-around time 3-5 See Endomysial antibody; see also Gliadin, Tissue Transglutaminase (ttg test ), Coeliac Disease (CD), Dermatitis Herpetiformis dsdna antibody (dsdna) Assayed routinely on any serum with positive ANA i.e. this test is usually not performed as a standalone test. Strong positive test is strongly suggestive of systemic lupus erythematosus (SLE) particularly with kidney involvement or autoimmune chronic active hepatitis (AI-CAH) although it is present in only 40-60% of patients with SLE; weak positive tests may be found in other connective tissue diseases (scleroderma, MCTD, RA). Concentration of ds-dna correlates with disease activity and repeated assessments are indicated in disease monitoring (in-house test). Analytical range: IU/mL; Reference range Negative: < 10 IU/mL Equivocal: IU/mL Positive: >15 IU/mL ENA antibodies ENAs are of use in the diagnosis and prognosis of connective tissue diseases. Antibodies to extractable nuclear antigens (ENA) are assayed routinely on any serum with positive ANA i.e. this test is usually not performed as a stand-alone test. ENA antibody testing routinely includes: Ro/SSA, La/SSB, Sm, RNP, Scl-70 and Jo-1; rare ENA antibodies (PL-7, PL-12 etc) must be requested/discussed separately (referred tests). Titres of ENA antibodies are not tested as they do not correlate with disease activity. Repeat assessment of ENAs is not routinely necessary (in-house test). ENA antigen Negative Equivocal Positive Analytical range Ro/SSA < > U/mL La/SSB < > U/mL SM < > U/mL RNP < > U/mL Scl-70 < > U/mL Jo-1 < > U/mL 8

9 Major clinical associations of ENA antibodies are: Ro/SSA - SLE (particularly photosensitivity) - Cutaneous LE - Sjogren s syndrome - Recurrent miscarriage - Neonatal lupus and congenital heart block La/SSB - SLE - Sjogren s syndrome Sm - SLE RNP - SLE - Mixed connective tissue disease Scl-70 - PSS (generalised scleroderma) Jo-1 PL7 PL-12 - Polymyositis (anti-trna synthetase syndrome) Endocrine gland antibodies Serum sample required: no special preparations needed prior to sampling; turn-around time Includes adrenal, ovarian, testicular, thyroid, parathyroid, pancreatic, islet cell (ICA), insulin (IAA, IA2) and glutamic acid decarboxylase (GAD) antibodies Pituitary antibodies must be requested separately Each antibody must be requested separately. Thyroid antibodies are done in biochemistry as part of a thyroid screen. Clinical association: o These antibodies are found in Autoimmune polyendocrinopathy syndromes (APS) type I and II. Type I has recently been coined APECED syndrome (Autoimmune Polyendocrinopathy Candidiasis Ectodermal Dystrophy) and includes patients with chronic mucocutaneous candidiasis o Adrenal antibodies are positive in 60-70% of patients with idiopathic Addison s disease o Anti-steroid secreting cell antibodies are also detectable in the same assay and are found in cases of autoimmune premature ovarian failure o Pancreatic, ICA, IAA, IA2 and GAD antibodies are found in recent-onset IDDM-1 Apart from thyroid antibodies, all other endocrine antibodies are referred tests 9

10 Endomysial antibody Serum sample required: N.B! If the patient is on gluten-free diet, test may be negative in presence of Coeliac Disease; turn-around time 3-5 (see also Gliadin, Tissue Transglutaminase (ttg test ), Coeliac Disease (CD), Dermatitis Herpetiformis (DH)) Endomysial antibody is a diagnostic test for coeliac disease (CD) and dermatitis herpetiformis (DH). Current guidelines advise that samples are initially screened for ttg, followed by Endomysial antibody confirmation of positive samples (>3). Endomysial-IgA antibodies are clinically relevant and are very sensitive and specific for CD (94-100%). Endomysial antibodies are important in investigation of malabsorption (especially paediatric), IgA deficiency, anaemia of unknown cause etc. Gliadin antibodies may be requested for young children (<2yrs) as they are more sensitive than endomysial antibodies in this age group. Because only IgA-endomysial antibodies are relevant for CD, IgA-endomysial are routinely assessed and reported. However, in patients with total IgA deficiency (IgAD) who cannot produce IgA, IgG antibodies are relevant; all samples are therefore screened to exclude total IgAD; if IgAD is found, IgG-endomysial are assessed and reported. Please note that endomysial and gliadin antibodies will disappear if patient is on a gluten free diet. Tissue transglutaminase antibodies (ttg) are more sensitive but less specific and are used as a screening test which, if positive, is confirmed by endomysial antibodies. Reports of using ttg levels to monitor adherence to a gluten free diet have not been confirmed. See also gliadin antibodies (inhouse test). Gastric parietal cell antibody (GPC) GPC is done as part of the autoantibody screen. It is present in 95% of patients with pernicious anaemia but also occur in immune thyroid disease, up to 30% of patients with iron deficiency anaemia and 3% of the normal population (the incidence rising with increasing age). Positive sera should be tested for vitamin B12 levels (in-house test). Gliadin antibody Serum sample required: N.B! If the patient is on gluten-free diet, test may be negative in presence of Coeliac Disease; turn-around time Screening test for coeliac disease (CD) and dermatitis herpetiformis (DH) but is now largely replaced by anti-endomysial antibodies and anti tissue transglutaminase (ttg) antibodies as more specific (see above). However, in young children (<2yrs) gliadin antibodies may be more sensitive. IgA antibodies are clinically relevant although in patients with IgA deficiency, IgG antibodies are relevant. Gliadin antibodies are also associated with gluten induced cerebellar ataxia for which endomysial antibody and ttg antibodies are of no value; please note that in these circumstances, the request should clearly specify that gliadin antibodies are requested for assessment of a neurological condition (referred test). Glomerular basement membrane antibody (GBM) Serum sample required: no special preparations needed prior to sampling; turn-around time 3-10 Diagnostic test for Goodpasture s syndrome (>90% sensitivity). Please note that this is a referred test and there is no longer an urgent / out-of-hours service available (referred test). Histone antibodies 10

11 Serum sample required: no special preparations needed prior to sampling; turn-around time Present in SLE but if found without dsdna antibodies, characteristic of drug-induced lupus (referred test). Liver kidney microsomal antibody (LKM) Identifies a sub-group of patients with autoimmune chronic active hepatitis (CAH type 2). This is the most common form of CAH in childhood and has a poor prognosis. There is no known association with infection. LKM is done as part of the autoantibody screen (in-house test). Lupus anticoagulant (LA) Haematology test See Phospholipid antibodies. Send to Haematology M2 This is an automatic, follow-on confirmatory test for all samples found to be positive for antimitochondrial antibodies (AMA) (see below). AMA antibodies can be directed to a total of 9 different mitochondrial antigens (M1-M9) although in PBC AMA are directed toward mostly against the M2 antigen (rarely M9), now known to be the enzyme pyruvate dehydrogenase (in-house test). Mitochondrial antibody (AMA and M2) Serum sample required: no special preparations needed prior to sampling; turn-around time 3-10 AMA is done as part of the autoantibody screen. High titres of AMA are found in >95% of patients with Primary Biliary Cirrhosis (PBC). AMA antibodies can be directed to a total of 9 different mitochondrial antigens (M1-M9) although in PBC AMA are directed toward the M2 antigen, now know to be the enzyme pyruvate dehydrogenase. Low titres of AMA may also be found in chronic active hepatitis. There is an association of PBC with other autoimmune diseases particularly Sjogren s Syndrome, CREST and Systemic Sclerosis (in-house test).. All AMA positive samples will be automatically assessed for M2 confirmation (AMA and M2 are in-house tests). Myeloperoxidase antibody (MPO) Serum sample required: no special preparations needed prior to sampling; turn-around time 3-10 Confirmatory test used when ANCA are detected. MPO is the target antigen for the majority of panca associated with microscopic polyangitis. The detection of anti-mpo antibodies association with an ANCA increases the positive predictive value for primary vasculitic disorders to approximately 66% (in-house test). Analytical range: U/mL; Reference range: Negative: < 7 U/mL Equivocal: 7 10 U/mL Positive: >7 U/mL. 11

12 MuSK antibody Serum sample required: no special preparations needed prior to sampling; turn-around time May be positive in AChR seronegative patients with myasthenia gravis (referred test). (Anti) Nuclear antibody (ANA) or anti-nuclear factor (ANF) Serum sample required: no special preparations needed prior to sampling; turn-around time 3-10 ANA is done as part of the autoantibody screen. It is indicated in the investigation of suspected connective tissue disorders (see also under autoantibody screen). If positive, a sample will be automatically tested for ENA and dsdna. ANA are present in elevated titres, and usually of IgG class in >95% of untreated cases of SLE. The absence of an ANA virtually excludes this diagnosis. It is very sensitive but not very specific for SLE as ANA may also occur in a number of other conditions including all other connective tissue diseases, juvenile chronic arthritis, fibrosing alveolitis, chronic active hepatitis, viral infections particularly EBV and CMV and in drug reactions. The frequency of low titre ANA in normal individuals increases with increasing age. Titres of ANA do not correlate with disease activity; repeat assessment of ANA is not indicated in routine monitoring; if repeat assessment is required, it should not be requested in less than 3-monthly intervals as existing antibody will not be degraded. Neuronal antibodies Serum sample required: no special preparations needed prior to sampling; turn-around time Often also referred to as anti-ganglioside antibodies; not to be confused with paraneoplastic antibodies (see below). Reported in paraprotein-associated neuropathy, early Guillain-Barre syndrome, Miller-Fisher Syndrome (GM1, G1Qb, basal ganglia, NMDA, MAG, aquaporin 4 etc). Other neuronal antibodies such as Ribosomal P antibody are found in autoimmune diseases with CNS involvement (CNS Lupus) (referred test). (Anti) Neutrophil cytoplasmic antibody (ANCA) Indicated in the investigation of vasculitis. ANCA titres correlate with disease activity and are useful for disease monitoring. Two main patterns are recognised, cytoplasmic (canca) and perinuclear (panca). canca is associated with Wegener s granulomatosis, panca is associated with microscopic polyangitis, particularly the renal limited forms of the disease. Both types of antibodies have been reported in a wide range of other conditions which may / may not be associated with vasculitis, and close liaison with the laboratory is advised in their interpretation (inhouse test). ANCA tests positive by immunofluorescence (IF) are automatically assessed for PR3 and MPO specificity (see below/above). If other nuclear antibodies are present, IF test results may be reported as uninterpretable and the specimen automatically assessed for PR3 MPO specificity (in-house tests). An urgent out-of-hours service is no longer provided by the RVI, Newcastle. 12

13 Neutrophil autoantibodies Serum sample and 2 EDTA tubes required NB! This test requires live cells which must be assessed within <24h; please arrange previously with Laboratory Not to be confused with ANCA (see above). Anti-neutrophil antibodies are autoimmune antibodies seen in idiopathic autoimmune neutropenias or as part of other autoimmune diseases (e.g. SLE). They can be detected in serum as well as on neutrophil surfaces because of which they need to be assessed both on cells (EDTA tube) and serum (Serum sample required) (referred test). Ovarian antibodies See endocrine gland antibodies Pancreatic antibodies See endocrine gland antibodies Paraneoplastic antibodies Serum sample required: no special preparations needed prior to sampling; turn-around time Often - confusingly - also referred to as neuronal antibodies because they affect neurons. They are associated with neoplasms and cause associated paraneoplastic neurological syndromes (Lambert- Eaton, acquired neuromyotonia). Antibodies to nuclear antigens ANNA-1 (Hu) and ANNA-2 (Ri) are most frequently associated with small cell lung carcinoma (SCLC); antibodies to Purkinje cell cytoplasmic antigen (Yo/PCA-1) are associated with breast and gynaecological cancers; antibodies to plasma membrane proteins such as voltage gated Ca+ channel (VGCC) and K+ channel (VGKC) are associated with all of the above (referred test). Parathyroid antibodies See endocrine gland antibodies (Anti) Phospholipid antibody Haematology test Also known as anti-cardiolipin antibodies (see above). Found in the Anti-phospholipid or Hughes syndrome (APS), which may be primary or occur as a secondary complication of SLE or other connective tissue diseases. The major features of APS are recurrent spontaneous abortion, recurrent thromboses (arterial or venous) especially in young people ( young stroke ), typical skin rash (livedo reticularis) and thrombocytopenia. Patients with APS must always be checked for lupus anticoagulant (LA) (APS is an in-house test done in the Haematology Laboratory together with Lupus Anticoagulant and clotting). Proteinase 3 antibody (PR3) Confirmatory test used when ANCA are detected. PR3 is the major target antigen for canca. The detection of anti-pr3 in association with ANCA increases the positive predictive value of canca (in-house test). Analytical range: U/mL; Reference range: Negative: < 7 U/mL Equivocal: 7 10 U/mL Positive: >10 U/mL. 13

14 (Anti) Reticulin antibody (ARA) ARA is done as part of the autoantibody screen but is not reported. A positive finding may suggest coeliac disease because of which positive sera are routinely tested for endomysial and tissue transglutaminase (ttg) antibodies (in-house test). Rheumatoid Factor (RF) Serum sample required: no special preparations needed prior to sampling; turn-around time 2-3 RF is done as part of the autoantibody screen but can also be requested separately. It is indicated in the investigation of inflammatory arthropathies. Differentiates sero-negative arthritides from RA. High titres are associated with extra-articular manifestations in RA e.g. vasculitis and nodules. RF is not of value in laboratory monitoring of disease activity CRP should be used. RF is very nonspecific and low levels are often found in other connective tissue/autoimmune diseases, infections and malignancies. Semi-quantitative measurements of RF titres have been replaced by quantitative measurements expressed in international units (IU) / ml; although the negative cut-off level is 20 IU/ml, values of <100 IU/ml are considered to be low-moderate while much higher levels of >1000 IU/ml are often seen in rheumatoid arthritis (in-house test). Salivary gland antibody Serum sample required: no special preparations needed prior to sampling; turn-around time May be found in Sjogren s syndrome (see Ro/La antibodies) (referred test). Skeletal / striated muscle antibody Serum sample required: no special preparations needed prior to sampling; turn-around time Anti-skeletal muscle antibodies are characteristically found in patients with thymoma often associated with myasthenia gravis. They also occur in some patients with hepatitis, acute viral infections and polymyositis. Low titres may occur in viral infections, notably EBV and infectious hepatitis (referred test). Smooth muscle antibody (SMA) SMA is part of the autoantibody screen. Higher titres are found in patients with chronic active hepatitis both viral and autoimmune. Lower titres can also be seen in PBC or infections (in-house test). Autoimmune hepatitis (AIH) type 1 is characterised by antibodies ANA and SMA, AIH type 2 by liver-kidney microsomal (LKM) antibody and AIH type 3 by soluble liver antigen (SLA) and liverpancreas antibody (LP). Type 2 is usually seen in children and has a more severe course (In-house test). 14

15 Skin reactive antibodies Also known as anti-epidermal cell antibodies. - Anti-intercellular substance (anti-ics) antibodies are found in most patients with the blistering (bullous) skin disease pemphigus vulgaris. - Anti-basement membrane zone (anti-bmz) antibodies are found in the serum of most patients with bullous pemphigoid (in-house tests). Soluble Liver antigen (SLA) SLA is characteristic of autoimmune hepatitis (AIH) type 3. AIH type 1 is characterised by antibodies ANA and SMA, AIH type 2 by liver-kidney microsomal (LKM) antibody and AIH type 3 by soluble liver antigen (SLA) and liver-pancreas antibody (LP). Type 2 is usually seen in children and has a more severe course. The test is confirmed by immunoblotting (in-house test screen). Testicular antibodies See endocrine gland antibodies Thyroid antibody Biochemistry test Elevated titres of anti-thyroid microsomal (thyroperoxidase) antibodies are found in primary myxoedema, Hashimoto s thyroiditis and Graves s disease. Anti-thyroglobulin antibodies are no longer measured. In the absence of clinical and/or biochemical thyroid disease, elevated titres of these antibodies may be predictive of future thyroid disease and regular clinical and biochemical follow-up is advised; follow-up does not need to include repeat thyroid autoantibody testing (inhouse test which is done in the Biochemistry Laboratory as part of a thyroid screen ). Tissue Transglutaminase (ttg) Serum sample required: N.B! If the patient is on gluten-free diet, test may be negative in presence of Coeliac Disease; turn-around time 3-5 (see also Endomysial antibody, Coeliac Disease (CD), gliadin test and Dermatitis herpetiformis (DH) This test has the same indications as endomysial antibody testing but is less specific albeit more sensitive. It is therefore used as a screening test which is confirmed by endomysial antibody testing if positive. It is also done in patients with a clinical presentation very suggestive of celiac disease but negative endomysial antibody, which is very rare but does occur. Reports of using ttg levels to monitor adherence to a gluten free diet have not been confirmed (in-house test). 15

16 Immunochemistry section General information: Interpretation and comments are given subject to clinical details and reason for request being available. If further advice is required please contact the laboratory. Please note that specific sampling conditions are essential for some tests. Turn-around times (working days): o C3&C4 = 3-5 days o Serum electrophoresis (SEP) = 3-5 days o Immunoglobulins = 3-5 days o Immunofixation = 5-8 days o Beta-2-microglobulin (B2M) = 3-5 days o other complement tests = days o cryoglobulins = 5-10 days o Serum Free Light Chains (SFLC) = 3-5 days Complement C1q Serum sample required: no special preparations needed prior to sampling; turn-around time Anti-C1q in SLE are associated with renal involvement. Monitoring anti-c1q and their titers in SLE patients could be important for predicting renal flares (referred test). C3 and C4 Levels are useful in the investigation and monitoring of a wide range of inflammatory disorders caused by infection, autoimmunity or malignancy. Serial rather than one-point measurements are recommended. Low levels of C3 & C4 caused by increased consumption due to inflammation can be masked by increased production as part of the acute phase response. For monitoring complement consumption i.e. disease activity, C3d levels are more relevant (see below) (in-house test). C3d / C3dg EDTA tube required: no special preparations needed prior to sampling; turn-around time NB! EDTA sample (NOT serum!) must be assessed within 3hrs of being taken because of spontaneous breakdown. As this is an urgent, referred test to RVI, Newcastle please check transport arrangements with our Laboratory before sending to us or send directly to RVI. Please state on request form: For immediate Immunology attention This is a degradation product of C3 and is elevated in conditions in which an acute phase response masks complement consumption by increasing C3 levels. It is therefore of use in the interpretation of C3&C4 levels and in the long term monitoring of connective tissue disorders (e.g. disease activity in SLE, RA, Systemic Sclerosis). Elevated levels of C3d occur in those conditions associated with circulating immune complexes (e.g. vasculitis) and therefore this assay has superseded the measurement of immune complexes (referred test). 16

17 Classical haemolytic (CH100) & alternative haemolytic (APCH100) pathway activity Serum sample required: no special preparations needed prior to sampling; turn-around time NB! must be assessed within 3hrs of being taken because of spontaneous breakdown. As this is an urgent, referred test to RVI, Newcastle please check transport arrangements with our Laboratory before sending to us or send directly to RVI. Please state on request form: For immediate Immunology attention Measurement of complement haemolytic activity is a screening test for assessment of classical and alternative pathway function. This test is indicated in the investigation of suspected immunodeficiencies associated with recurrent pyogenic infections (e.g. C3 deficiency). Any suspected cases should be discussed with the Consultant Immunologist; assessment of other complement components is also available (referred test). C1 esterase inhibitor (C1-INH) level Serum sample required: no special preparations needed prior to sampling; turn-around time Low levels of C1-INH are found in 85% of patients with hereditary angioedema (HAE) but 15% can have normal levels of a non-functional protein see below). If HAE is suspected, C4 levels should always be checked as they will be low (always) or absent (during an attack) due to complement consumption; if C4 levels are normal there is no need to test C1-INH (referred test). C1 esterase inhibitor (C1-INH) function Serum sample required: no special preparations needed prior to sampling; turn-around time NB! must be assessed within 3hrs of being taken because of spontaneous breakdown. As this is an urgent, referred test to RVI, Newcastle please check transport arrangements with our Laboratory before sending to us or send directly to RVI. Please state on request form: For immediate Immunology attention Measures C1 esterase inhibitor activity. Approximately 15% of patients with hereditary angioedema have normal levels of a non-functional protein. Both types of hereditary angioedema are associated with low/absent serum C4 levels especially during an attack (see above). The rarer, acquired form of C1-INH deficiency can be associated with some lymphoproliferative disorders and SLE (referred test). C3 Nephritic Factor (C3nef) Serum sample required: no special preparations needed prior to sampling; turn-around time NB! must be assessed within 3hrs of being taken because of spontaneous breakdown. As this is an urgent, referred test to RVI, Newcastle please check transport arrangements with our Laboratory before sending to us or send directly to RVI. Please state on request form: For immediate Immunology attention C3nef is an autoantibody associated with membrano-proliferative glomerulonephritis (type II) and partial lipodystrophy (see complement section) (referred test). 17

18 Serum proteins / Immunochemistry Alpha-1-anti-trypsin (A1AT) This is an enzyme inhibitor whose levels are increased during inflammation as an acute phase reactant. Low levels are suggestive of A1AT deficiency but may be masked during inflammation (in-house test). Samples with low A1AT can be sent off for protein phenotyping if requested. Normal alleles are designated M; the two deficiency alleles are S and Z. Heterozygous individuals (MZ or MS) are usually not at clinical risk but SZ and ZZ individuals are prone to developing lung disease (and liver disease in children) (referred test). Bence-Jones proteins See urinary free light chains Beta 2 microglobulin (β 2 M) Indicated in the monitoring of patients with increased lymphocyte activity and turn-around such as multiple myeloma, Sjogren's Syndrome, HIV related disease, organ transplants (in-house test). Cryoglobulins Serum sample required: NB! Sample must be kept warm (at 37 o C); contact Laboratory to arrange collection (x32635) Cryoglobulins are proteins which precipitate and form complexes below body temperature, because of which blood samples must be kept warm to avoid precipitation and loss of cryoproteins. They are not to be confused with cold agglutinins (autoantibodies that lyse red blood cells at low temperatures). Cryoglobulins are not present in healthy individuals. Patients with cryoglobulinaemia may present with Raynaud s phenomenon, purpuric vasculitis, arthritis or nephritis. An unexpected, high-titre RF or low serum C4 may indicate the presence of a cryoglobulin. If detected, cryoglobulins will be routinely quantified and classified (in-house test). Cryofibrinogen EDTA tube: NB! EDTA sample (NOT serum) must be kept warm (at 37 o C); contact Laboratory to arrange collection (x32635). Cryoglobulins must always be assessed in parallel (see above). Indications are similar to cryoglobulins. Cryofibrinogen is of less clinical importance but may be mistaken for cryoglobulins (in-house test). Immunofixation Performed as a follow-on test to serum protein electrophoresis if monoclonal protein detected; no separate sample required: no special preparations needed prior to sampling; turn-around time 5-10 This technique is used to type paraprotein heavy and light chains detected by serum protein electrophoresis (SPE) (in-house test). 18

19 Immunoglobulins (IgG, IgA, IgM) For relevant interpretation of immunoglobulin results, serum protein electrophoresis (SPE) must be performed in parallel. Immunoglobulin quantitation is essential in the investigation of suspected immunodeficiency and lymphoproliferative diseases. Abnormally elevated levels in the absence of a paraprotein i.e. polyclonal elevations may occur in a number of disorders including chronic infectious/inflammatory conditions, liver disease and auto-immune diseases; isolated increases of IgA are associated with mucosal inflammation (guts, lungs), liver involvement (especially alcohol abuse) or rheumatic diseases (RA); isolated increases of IgM are associated with primary biliary cirrhosis (PBC) or lymphoproliferative diseases. Low levels of immunoglobulins may be secondary (protein loss, lymphoproliferative diseases) or primary immune deficiencies; total IgA deficiency (IgAD) is frequent in the normal population (1:800) but may predispose to allergy and autoimmune diseases; selective decrease in IgM may be associated with lymphoma/leukaemia. If immune deficiency is suspected, please contact Consultant Immunologist to discuss further investigations (in-house test). IgG subclasses Serum sample required: no special preparations needed prior to sampling; turn-around time The measurement of IgG subclasses is of limited value and should only be considered in the context of identifying primary immune deficiency or assessing hypergamaglobulinemia (but not normal IgG levels) in suspected Sjogren s Syndrome. Please discuss with Consultant Immunologist (referred test). Mannose binding lectin (MBL) Serum sample required: no special preparations needed prior to sampling; turn-around time This protein is involved in opsonisation and complement activation. Severe deficiency may contribute toward susceptibility to infections (referred test). Paraprotein quantitation (PPQ) Serum sample required: no special preparations needed prior to sampling; turn-around time 5-8 Paraproteins are monoclonal immunoglobulins most often seen in monoclonal gammopathies (myelomas, MGUSs, lymphoproliferative diseases) (see also SPE below). PPQ is useful in monitoring disease progression and response to therapy. Because paraproteins are abnormal immunoglobulins, PPQ is done by scanning densitometry and not by nephelometry, which is used for measuring normal immunoglobulins, so that results are not directly comparable. If a paraprotein is present, it is not possible to assess the proportion of normal to abnormal immunoglobulin (inhouse test). Serum protein electrophoresis (SPE) Serum sample required (+ EMU, no preservatives sample if paraproteinaemia is suspected for urine protein electrophoresis); : no special preparations needed prior to sampling; turn-around time SPE is performed on all specimens submitted for immunoglobulin quantitation to check whether the immunoglobulins are polyclonal or monoclonal proteins. Polyclonal increases are due to an 19

20 increased activity of numerous different lymphocytes usually triggered by an underlying inflammatory response (such as infection, malignancy, autoimmune diseases etc.). Monoclonal proteins (or paraproteins) are produced by only one clone of cells because of which they are identical and appear as a monoclonal band on serum electrophoresis. They may be benign (MGUS = monoclonal gammopathy of unknown significance) but are more often malignant with high concentrations of the monoclonal protein often accompanied by the presence of free monoclonal light chains in the serum and urine (Bence-Jones protein); levels of the normal immunoglobulins are often reduced. Malignant paraproteins occur in multiple myeloma and other lymphoproliferative diseases such as Waldenstrom s macroglobulinaemia, chronic lymphocytic leukaemia and non- Hodgkin s lymphoma (in-house test). Specific antibody responses (SPECs) Microbiology tests This test involves the quantitative measurement of antibody responses to specific antigens (SPECs) and is of major importance in investigating humoral immune deficiency. The test measures antibody responses to protein and polysaccharide antigens. The antigens to which responses are most frequently measured are Hemophilus influenzae B (HiB) and tetanus toxoid as protein antigens and Pneumovax (a mixture of 23 pneumococcal polysaccharide antigens) as a polysaccharide antigen. Before requesting SPECs for assessment of immune deficiencies please discuss with Consultant Immunologist; results require specialist interpretation (currently, send sample to Microbiology; referred test). Serum Free Light Chains (SFLC) This test is now available as an in-house test and has replaced urinary 24h free light chain (Bence- Jones) quantitation. SFLC are indicated as part of a new myeloma screen and on request for followup. New guidelines (2009) advise SFLC assessment for all new myeloma, follow-up of free-light chain myeloma, plasmocytoma, complete remission, amyloidosis and patients (in-house test; referred-in test) Urinary free light chains / urinary Bence-Jones proteins (UBJP) / myeloma screen (urine) UBJP detection: EMU, no added preservative: no special preparations needed prior to sampling; turn-around time 5-10 This test should always be done for ALL newly diagnosed paraproteins; it is essential for detecting the free light chain myeloma as well as for detecting free light chain production in cases of monoclonal protein presence. This test detects the presence of UBJP and is NOT quantitative (inhouse test). UBJP / free light chain quantitation and K/L ratio has been replaced by SFLC assay (see above) 24-hour urine, no added preservative This test has ceased to be provided routinely and will only be available following specific arrangements with the Laboratory (e.g. in amyloidosis). When requesting this test, in order to avoid confusion with UBJP detection, please clearly specify on request form that 24h QUANTITATION of UBJP/free light chains and KAPPA/LAMBDA RATIO is being requested (referred test). 20

21 Allergy testing Total serum IgE Measurement of total serum IgE is of little value in the assessment of patients with allergies as total IgE can be normal in patients with confirmed allergies and increased in patients without allergies. Total IgE is increased in atopic eczema, parasitic diseases, certain lymphomas (Hodgkin s), some vasculitidies (Churg-Strauss) and in some rare immunodeficiency disorders (in-house test). Allergen specific IgE (RAST) RAST tests measure IgE specific for a particular allergen because of which requests must specify which allergens should be tested based on clinical history and as much clinical information as possible. RAST to common inhaled allergens include house dust mite, cat/dog dander & moulds; common food allergens include cow s milk, eggs white, fish (cod), wheat, peanut and soya; nut allergens include peanut, hazelnut, brazil nut, almond, and coconut as an initial screen although many other nuts are available if specifically requested. ALWAYS specify allergen based on clinical finding. A positive RAST is not synonymous with clinical allergy, nor does failure to detect a positive RAST exclude the diagnosis. Acute urticaria is caused by allergy in only about a third of cases; chronic urticaria is almost never caused by allergy. RAST and total IgE testing is of no use following anaphylaxis. NB! A RAST for 1 allergen is 10 (in-house test). Tryptase EDTA tube: no special preparations needed prior to sampling; turn-around time 5-10 NB! Samples must be taken <3h following onset of reaction because of short tryptase half-life Mast cell tryptase levels are a specific measure of mast cell activation / degranulation, and thus indirectly of histamine release which is the main mediator of allergic/pseudoallergic reactions. Mast cell activation can be caused by allergic mechanisms (binding and crosslinking of specific IgE bound to mast cells) or pseudoallergic mechanisms (direct activation of mast cells without IgE). Tryptase has a short half-life (~1h) because of which there is a time limit for taking samples. If tryptase levels are found to be increased, we will require one more baseline sample to confirm that levels have gone back to normal after reaction. This sample can be taken any time in remission. Persistently high tryptase levels are suggestive of mastocytosis (referred test; currently send sample to biochemistry). Normal ranges: 2-14ug/L (mild increases e.g are often non-immune; type I anaphylactic reactions usually give levels of >50ug/l) (referred test). 21

22 Cellular section Investigation of the cellular immune system should only be undertaken after discussion with Consultant Immunologist who will advise on type of tests indicated and specimen requirements All cellular investigations of the immune system require live cells which must be assessed within <24h of sampling All cellular tests are referred tests and must be arranged in advance Send sample to Immunology; Please state on request form: For immediate Immunology attention Lymphocyte subsets for suspected primary immune deficiency (not to be confused with HIV lymphocyte subsets!) EDTA tube: no special preparations needed prior to sampling; turn-around time working days. NB! Please arrange previously with Laboratory (x2635); Please state on request form: For immediate Immunology attention Lymphocyte subsets phenotyping for assessing immune deficiency includes CD3 (pan T cell), CD4 (T helper subset), CD8 (T cytotoxic/suppressor subset), CD19 (pan B cell), CD 16/56 (pan NK cell) and other cell surface markers as required based on clinical data. This analysis is indicated in diagnosis and monitoring of immunodeficiencies and immunotherapy in children and adults (referred test; send sample to Immunology). Lymphocyte function EDTA tube: no special preparations needed prior to sampling; turn-around time working days. NB! Must be previously arranged with Laboratory (x2635) Please state on request form: For immediate Immunology attention Indicated in further definition of humoral and/or cellular immunodeficiency. Live cells are required and proliferative response to mitogens and/or specific antigens are performed as a measure of lymphocyte function. This test is only available upon consultation with Consultant Immunologist and must be previously arranged (referred test; send sample to Immunology). Neutrophil function tests EDTA tube: no special preparations needed prior to sampling; turn-around time working days. NB! Must be previously arranged with Laboratory (x2635) Please state on request form: For immediate Immunology attention The nitroblue-tetrazolium test (NBT) is a measure of the respiratory oxidative burst capacity which is compromised in patients with childhood or adult onset chronic granulomatous disease. A newer method with a similar principle but using a different dye is called the dihydro-rodhamine assay (DHR) and is performed on the flow-cytometer. It is indicated in investigation of recurrent skin infections, chronic gingivitis, recurrent deep seated bacterial and fungal infections. Live cells are required so that this test must be previously arranged following consultation with Consultant Immunologist (referred test; send sample to Immunology). 22

23 CD40-ligand expression EDTA tube: no special preparations needed prior to sampling; turn-around time working days. NB! Must be previously arranged with Laboratory (x2635) Please state on request form: For immediate Immunology attention This test is used for the diagnosis of Hyper IgM syndrome due to CD40 ligand deficiency. This test is only available upon consultation with Consultant Immunologist and must be previously arranged (referred test). 23

24 Disease Index: A to C Disease Investigations Addison s Disease Allergy Anaphylaxis Angioedema / urticaria (acute) Anti-Phospholipid Syndrome (APS) Bullous skin disorders C1 Esterase Inhibitor Deficiency Cerebellar Ataxia Chronic Active Hepatitis Chronic Granulomatous Disease Coeliac Disease Congenital Heart Block Connective Tissue Disease Screen CREST Cryoglobulinaemia Anti-endocrine antibodies (adrenal) IgE RAST (suspected allergens) Tryptase IgE RAST (suspected allergens) C3/C4 Thyroid screen (Biochemistry) FBC/ESR(Haematology) Anti-phospholipid Ab (Haematology) Anti-cardiolipin Ab (Haematology) Lupus Anticoagulant (Haematology) Anti-endomysial Ab Anti-gliadin Ab Skin reactive Ab C1 esterase inhibitor C3/C4 Anti-gliadin Ab Anti-smooth muscle Ab Anti-mitochondrial Ab Anti-nuclear Ab Neutrophil function test Anti-tissue transglutaminase Ab Anti-endomysial Ab Anti-gliadin Ab for <2 yr ENA (Anti-Ro) Anti-nuclear Ab ENA DNA C3/C4 CRP (Biochemistry) Anti-centromere Ab Cryoglobulins C3/C4 Immunoglobulins Serum protein electrophoresis RF 24

25 Disease Index: D to H Disease Investigations Deep Venous Thrombosis Dermatitis Herpetiformis Dermatomyositis Diabetes (recent onset) DLE Dressler s Syndrome Endocrinopathies - autoimmune Fibrosing Alveolitis Glomerulonephritis Goodpasture s Syndrome Graves Disease Guillain-Barre Syndrome Hashimoto's Thyroiditis Hereditary angioedema (HAE) Anti-cardiolipin / phospholipid Ab Lupus Anticoagulant Anti-tissue transglutaminase Ab Anti-endomysial Ab Ant-Jo-1 Anti-PL7 Anti-PL12 Islet cell Ab (ICA) Insulin antibodies (IAA) Glutamic acid decarboxylase Ab (GAD) Anti-nuclear Ab Anti-cardiac muscle Ab Adrenal, parathyroid, gonadal, insulin, pancreatic etc Anti-nuclear Ab ANCA (MPO / PR3) Anti-glomerular basement membrane Ab Anti-glomerular basement membrane Ab Anti-thyroid peroxidase (TPO) Ab (Biochemistry) Anti-neuronal Ab Anti-thyroid peroxidase (TPO) Ab (Biochemistry C3/C4 C1 esterase inhibitor C3/C4 C1 esterase inhibitor 25

26 Disease Index: I to N Disease Investigations Infection Immune Deficiency Screen Juvenile Chronic Arthritis Lymphoproliferative disorders Membrano-Proliferative Glomerulonephritis (MPGN) Microscopic Polyangitis Mixed Connective Tissue Disease (MCTD) Monoclonal Gammopathy Uncertain Significance (MGUS) Myasthenia Gravis Myeloma / Paraprotein screen Nephritic Syndrome (acute) Non-Hodgkin's Lymphoma Immunoglobulins Serum protein electrophoresis Immunoglobulins Serum protein electrophoresis Specific antibody responses IgG subclasses C3/C4 Anti-nuclear Ab Immunoglobulins Serum protein electrophoresis C3 nephritic factor C3/C4 ANCA (MPO / PR3) Anti-ENA Ab Anti-nuclear Ab Serum protein electrophoresis Immunofixation Urinary Bence-Jones protein β2-microglobulin Anti-acetylcholine receptor Ab Serum protein electrophoresis Immunoglobulins Immunofixation Paraprotein quantitation (PPQ) Urinary Bence-Jones proteins (UFLC) Serum free light chains (SFLC) β2-microglobulin Serum protein electrophoresis Immunoglobulins ANCA (MPO / PR3) Anti-glomerular basement membrane Ab C3/ C4 Serum protein electrophoresis Urinary Bence-Jones protein 26