Universiity of Birmingham Clinical Immunology Service ...

CONTENTS Introduction 3 Contact address / telephone numbers 3-4 Routine/Urgent assay processing 4 Department of Immunology Division of Immunity & Infection Specimen collection requirements: General 4–5 Surface markers 38 – 39 Cell Function 43 Details of available assays/normal adult ranges 6 – 32 Clinical Immunology Service Guide to appropriate use of immunology assays 33 Laboratory Handbook and price list Assay turnaround times/costs 34 – 42 Cell function assays 43 A brief guide for clinical staff APRIL 2007 __________________________________________ 2

INTRODUCTION Departmental Website: The Clinical Immunology Service (CIS) of the Division of Immunity and Services: http://www.ii.bham.ac.uk/clinicalimmunology/ Infection provides a comprehensive range of clinical and laboratory services. In Image library: http://www.ii.bham.ac.uk/clinicalimmunology/CISimagelibrary/ particular the CIS is a major testing centre for multiple myeloma, leukaemia/lymphoma, immunodeficiency, autoimmunity and rheumatic Neuroimmunology: http://www.ii.bham.ac.uk/clinicalimmunology/Neuroimmunology/ diseases. The CIS laboratory liases closely with other on site laboratories, particularly pathology and cytogenetics. Daily assays: As a guide most immunochemistry, indirect immunofluorescence, electrophoresis, cell markers and some neuroimmunology This handbook provides information about turnaround times for assays, assays are carried out daily. Radial immunodiffusion (RIDs) though carried out together with test costs and other information about the laboratory staff, on a daily basis take three days to completion. working hours, results and their interpretation. Batched assays: Non urgent, expensive or labour intensive assays which are Normal working hours are 8:00am to 5:30pm from Monday to Friday. Clinical batched on a weekly basis are: Cardiolipin abs (Wednesday); dsDNA abs advice is available from 8:00am to 8:00pm Mon – Fri via mobile telephones: (Tuesday and Friday); ENA abs (Thursday); Intrinsic factor abs 07831 681955, 07798 585319, 07973 362423 or 07884 310528. If you are (Wednesday); GBM abs (Wednesday and Friday) Functional complement unable to reach a clinician on their mobile, please leave a voice mail and (Classical/Alternative pathways) (Thursday); Functional C1Inh (Friday); someone will contact you as soon as possible. There is no on call service but tTG/Gliadin abs (Monday); Organ specific abs (Friday); IgG subclasses clinically urgent requests may be arranged through a clinician/senior BMS by (Tuesdays); MPO/PR3 EIA (Tuesday & Thursday). telephone. Urgent assays: Certain assays are available with a rapid turnaround time. Most analytes and autoantibodies are carried out on the same day, or the day These include ANA, ANCA, dsDNA abs, glomerular basement membrane following receipt of the specimen. However many tests are expensive when (GBM) abs, myeloperoxidase and proteinase 3 abs (ANCA). Specimens dealt with in small numbers and in order to maintain an economic cost and should be sent, or preferably brought, to the laboratory with the request form acceptable turn around time such assays are ‘batched’ on certain days of the clearly marked “Urgent”. Prior warning for urgent requests is essential. It week. (See page 4). If in doubt, please phone for advice. should be noted that urgent requests will incur and additional charge and where a qualitative result is provided (GBM, MPO and PR3) these will be followed up Postal delays are notorious. Frequently we have despatched the results but the with quantitative assays when the next batch is processed. report has not reached its destination. Though telephone calls are disruptive to the workflow we would encourage you to enquire when delays seem unreasonable. Address: Clinical Immunology Service, Division of Immunity General specimen collection: and Infection, P.O. Box 1894, Medical School, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2SZ. When sending specimens to the laboratory the following should be noted: Telephone numbers: (University switchboard prefix 7600 from UHB Trust) • Some complement components are labile. For these assays send 10ml of Results: (0121) 414 3824/4069 whole blood (for C3d use EDTA blood) immediately after venepuncture. Clinical enquiries: For distant clinics the serum or plasma should be separated within the hour, (Mobile) 07831 681955 General clinical enquiries (Prof PJL Lane) frozen and sent to the laboratory to arrive frozen. Email: P.J.L.Lane@bham.ac.uk (Mobile) 07798 585319 Myeloma/Lymphoma/Leukaemia enq. (Dr MT Drayson) • For cryoglobulins whole blood to be taken into a warm syringe, transferred Email: M.T.Drayson@bham.ac.uk (Mobile) 07884 310528 Myeloma/Lymphoma/Leukaemia enq. (Dr SD Freeman) to a warm tube and brought to the laboratory whilst being maintained at Email: S.D.Freeman@bham.ac.uk body temperature. Prior warning that this test is required is appreciated. 3 4

List of assays available through the department: • For Von Willebrand factor (FVIII Rag) use citrated tubes (4ml). TEST: (Preferred sample: normal range) • Urinary free light chains (BJP). A 25ml aliquot of a random urine in a universal container (NO preservative) sent together with 10ml clotted Acetylcholine receptor abs Test for Myasthenia Gravis. This assay is blood. (Serum: 0 – 5 x 10-10 moles/l) currently sent to Immunology, Churchill Hospital, Oxford. • T cell antigen receptor & Immunoglobulin gene rearrangement studies: Please supply blood or bone marrow samples drawn into an EDTA Adrenal cortical abs Test for autoimmune adrenal disease. bottle (N.B. Heparinised material may interfere with PCR process). (Serum: Negative) Adverse Anaesthetic reactions In suspected cases where a patient has had For all cell markers and cell functional assays please see the notes at the back of symptoms of an adverse reaction we this book under ‘Cell Marker Tests’ (p38 – 39) and ‘Investigation of immune Mast cell tryptase recommend the following: A clotted blood deficiencies’ (p42 – 43). (Serum: 0 – 13.5 µg/l) sample should be taken immediately and at 24 hrs post reaction and sent to the clinical All high-risk specimens and accompanying form must be clearly labelled. immunology laboratory for measurement of serum tryptase. Please indicate time/date of adverse reaction and time/date of samples. Please also note special requirements for cell work and neuroimmunology Raised levels of this enzyme indicate requests both of which have separate request forms. systemic mast cell degranulation and substantiate clinical suspicions of anaphylactic reactions. To identify which drug precipitated the reaction and to find safe alternatives we recommend skin prick and intradermal testing. In conjunction with Heartlands Hospital, the University of Birmingham’s Clinical Immunology Service runs an outpatient clinic for patients with allergy at the Chest Clinic in Great Charles Street, Birmingham B3 3HX. All patients should be referred to Prof Peter Lane at the above address (phone 07831 681955) Please indicate all drugs administered to the patient prior to the allergic reaction. 5 6

Antinuclear abs ANA’s are associated with a variety of B2GP1 antibodies B2GP1 is a 50kD plasma protein (Serum: Titre <1:40) conditions other than SLE including (Serum: Negative) (apolipoprotein H) that inhibits the intrinsic rheumatoid diseases, chronic active hepatitis, coagulation pathway, ADP mediated platelet ANA titres of 1/40 are generally fibrosing alveolitis, viral infections and drug [Also see cardiolipin antibodies] aggregation and the prothrombinase activity not significant in adults, but can ingestion. Patterns of ANA are said to be of activated platelets. “Anti cardiolipin abs” be in children. significant: Nucleolar associated with bind to an altered form of B2GP1 which may Antibodies against ENA usually scleroderma, centromere with CREST be reproduced by binding B2GP1 directly to give a low titre ANA result but syndrome, and speckled pattern with MCTD, an ‘ELISA’ plate. The detection of anti- to exclude ENA abs an ENA Sjögrens, SLE and Polymyositis. Rim or B2GP1 abs is said to have enhanced screen should be performed homogeneous has been associated with SLE specificity for APS and related coagulation rather than relying upon a but there is a considerable amount of pattern disorders over the traditional anti-cardiolipin negative ANA result. overlap. assay, which may display some false positive results due to cross reactivity of these abs Aspergillus - specific IgG abs Specific IgG antibodies directed against with some infectious disease related (Serum: Negative) aspergillus fumigatus are currently available. antigens. This is currently a quantitative IgG antibody assay. Also see fungal antigens Cardiolipin/Phospholipid abs Antibodies have been associated with SLE, Avian antigens - specific IgG Specific IgG antibodies directed against (Serum: IgG: 0 – 12 GPLU/ml recurrent miscarriages and arterial and abs (Serum: Negative) budgerigar and pigeon antigens are currently IgM: 0 – 10 MPLU/ml) venous thrombosis. Slightly elevated levels available. may be found in some infections and so only [Also see B2GP1 abs] positive results at two time points at least 6 B2 microglobulin (B2M) Useful for monitoring lymphocyte activation weeks apart are considered significant. IgG (Serum: 0 – 4.0 mg/l) and turnover in myeloma and HIV related and IgM antibodies are assayed separately. diseases. Because B2M is metabolised in the Significant levels of abs do not necessarily renal tubules high levels are seen in patients correlate with the severity of the disease. with renal dysfunction. Please note that lupus anticoagulant is performed in haematology. Caeruloplasmin Copper binding serum protein, levels of (Serum: 0.15 – 0.60 g/l) which are reduced in Wilson’s disease, nephrotic syndrome and Menkes’ kinky hair syndrome. Raised levels are found in chronic active liver disease, pregnancy, leukaemia, post trauma, infectious diseases and after myocardial infarction. 7 8

Cardiac abs Though the diagnostic value is low these abs (Serum: Negative) are found in some patients with Dresslers C-Reactive Protein As CRP has a short serum half-life this acute syndrome, following myocardial infarction, (Serum: 0 – 10 mg/l) phase protein is useful in distinguishing after cardiac surgery and in some bacterial infections, inflammatory cardiomyopathies. Viral infection/AI disease: conditions, activity of rheumatoid arthritis 11 – 49mg/l and monitoring response to therapy. It is not affected directly by steroids or CSF pigments (scan) Important in the evaluation of the 14% of Bacterial infection: immunosuppressives. xanthochromia subarachnoid haemorrhage patients who are 50 – 100mg /l (Red cell free CSF: both orientated and CAT scan negative with Normal Spectroscopy no absence of xanthochromia by visual Major bacterial infection: pigments detected) inspection of CSF. Spectroscopic >100mg/l xanthochromia is defined by the presence of an oxyhaemoglobin peak at 413 nm in the Complement C3 and C4 Measurement of C3 and C4 is of value in supernatant after centrifugation of freshly (Serum: C3: 0.75 – 1.75 g/l) monitoring activity of SLE and in immune obtained CSF. NB: A traumatic tap leading C4: 0.14 – 0.54 g/l) complex disease. C4 is of particular value in to blood stained CSF will obfuscate SLE and angioedema when levels are well interpretation of xanthochromia. Pseudo- below normal. When taking blood care xanthochromia can result from in vitro should be taken to avoid unnecessary in vitro haemolysis of as little as 1,000x103 red cells activation of complement. per ml if CSF is left for 12 hrs or more at C3d complement component C3d is a 35kD product derived from room temperature. (EDTA plasma: 7 – 17 mg/l) protease activity on native C3b and may be detected in fresh plasma indicating in vivo CSF Tau protein: Cerebrospinal rhinorrhoea is potentially activation of the complement cascade. It is asialo-transferrin serious due to risk from infection. In patients essential that blood is collected in EDTA Present only in CSF presenting with a nasal discharge of clear bottles and the sample transported to the fluid it is important to identify the nature of laboratory without delay. This assay is Please send whole blood/serum the fluid. CSF is readily identified by the currently sent to Immunology PRU, sample for control purposes. presence of asialo-transferrin (Tau protein). Northern General Hospital, Sheffield This laboratory offers a reliable, sensitive and simple electrophoretic method for the C3 Nephritic factor C3 nephritic factor is an IgG antibody, rapid identification of Tau protein. (Fresh Serum: Negative) which stabilises the alternative pathway C3 convertase leading to continuous C3 breakdown. It is associated with type II MPGN and also with partial lipodystrophy. Please note: C3 nephritic factor will not be carried out in the presence of normal levels of C3. This assay is currently sent to Immunology PRU, Northern General Hospital, Sheffield 9 10

C1q complement component IgG antibodies to the collagen-like region of Complement function Carried out by RID in gels containing (Fresh serum: 0.08 – 0.15 g/l) C1q have been described in patients with Classical pathway (CH100) sensitised sheep red cells this assay tests the SLE and are said to be present in 30% of (Fresh serum: 24 – 40 IU/l) integrity of the classical pathway of patients with active disease. Serum C1q is complement. Low levels are found when any found to be lowered due to activation of one component is absent or non-functional. immune complexes, which are deposited on Assays for the individual complement capillary walls. components are available as follow up. Please see full list on page 37. Refer to Anti-C1q autoantibodies Autoantibodies against C1q are a major important notes regarding collection of (Serum: 0 – 15 units/ml) criterion in the diagnosis of blood and its despatch to the laboratory hypocomplementaemic urticarial vasculitis. (page 4). They are also found in up to 50% of SLE Complement function patients and 95% of patients with lupus Alternative pathway Carried out by RID in gels containing nephritis. C1q antibodies may be useful for (Fresh serum: 75 – 150%) chicken erythrocytes, this assay tests the assessing the risk of renal flares, and also for integrity of the alternative pathway of monitoring the effectiveness of complement and the terminal sequence (C3- immunosuppressive treatment in active lupus C9) components. Low levels are found nephritis. when any one component is absent or non- functional. Assays for the individual C1 (esterase) Inhibitor Hereditary Angioedema: Autosomal complement components are available as Immunochemical levels: dominant. Most cases have reduced serum follow up. Refer to important notes (Fresh serum: 0.18 – 0.30 g/l) C1Inh levels. In 10% of cases there are regarding collection of blood and its normal or elevated levels of C1Inh but this is despatch to the laboratory (page 4). Functional activity functionally inactive. In hereditary (Fresh serum: 70 – 130%) angioedema C4 levels are almost always Creatine kinase Quantitative measurement of serum Creatine reduced and C1q levels are normal. (phosphocreatine kinase): kinase (phosphocreatine kinase) in human (Serum) serum and plasma is useful for monitoring Please see page 4 for collection Acquired angioedema: Have reduced (Adult males: 0 - 190 IU/L) and evaluation of neuromuscular disease, procedure C1Inh levels and usually reduced levels of (Adult females: 0 - 165 IU/L) such as muscle weakness, muscle both C4 and C1q. Associated with B cell dystrophies, congenital myopathies and aids neoplasia. N.B. In children (up to 16 years the detection of Duchenne muscular of age) values may be up to 50% dystrophy carriers. higher than adults CK unreliable during pregnancy 11 12

Cryoglobulins When cryoglobulins are associated with ENA antibodies ENA antibodies recognise saline extracted (Serum: Negative) Waldenströms macroglobulinaemia, (Serum: 0 – 10 ELISA units) nuclear antigens. There are many myeloma or lymphoma they consist of one specificities recognised of which this Positives may be typed: immunoglobulin isotype but may be mixed laboratory currently offers six: Sm (a monoclonal, polyclonal or or polyclonal in other diseases such as marker for SLE); RNP (said to be present in mixed (IgG/A/M). connective tissue diseases. Patients with >95% MCTD); SSA [Ro] (associated with renal disease and a low C4 level or patients SLE, cutaneous lupus, neonatal lupus and with unexplained cutaneous vasculitis should congenital heart block); SSB [La] (SLE, be screened for presence of circulating Sjögrens syndrome); Jo1 (30% of cryoglobulin. Please refer to special polymyositis cases) and Scl70 (associated conditions of collection and despatch to with systemic sclerosis). Patients with SLE laboratory (page 4). or Sjögrens should be screened for ENA abs especially females considering pregnancy. DNA binding abs Assay of abs to native, double stranded DNA (Serum: (dsDNA abs), is carried out on all patients Functional antibodies Specific antibody production is Crithidia IIF: Negative with SLE, as a qualitative test by IIF on the recommended for first line investigation of EIA: 0 – 75 IU/ml) kinetoplast of crithidia lucillae which is then B-cell function. In patients with recurrent followed up with a quantitative assay by infections, functional antibody responses can EIA. dsDNA antibodies may be detected in be abnormal even if immunoglobulin and Ig the absence of ANA and are extremely subclass levels are normal. T-cell dependent useful in monitoring the activity of the protein antigens (e.g. Tetanus and disease. Diphtheria) produce an IgG1 response whilst polysaccharide antigens (e.g. Pneumovax Endomysial abs IgA abs directed against the endomysium are and unconjugated Haemophilus influenzae (Serum: Negative) detected in 70% of patients with dermatitis vaccine) elicit an IgG2 response. Normal herpetiformis and >90% of patients with antibody production would include a rise in [Also see Transglutaminase] untreated coeliac disease but are rarely specific antibody levels within two weeks for present in normal individuals or in patients protein antigens and within three weeks for with other enteropathies. Decreasing polysaccharide antigens. These assays are antibody titres correlate well with adherence currently sent to Immunology, City Hospital, to gluten free diet. See also gliadin abs. Birmingham. Fungal antigens – Specific IgG Specific IgG antibodies directed against (Serum: Negative) candida albicans, aspergillus fumigatus and micropolyspora faeni are currently available. Note: most adult women will have low levels of candida antibodies. 13 14

Ganglioside abs GD1b Antibody to the ganglioside GD1b has been Ganglioside abs sulphatide These abs are currently screened by this (Serum: IgG <1:500 associated with motor or sensorimotor (Sensory neuropathy) department and positives are sent to IgM <1:500) neuropathies. High titres of anti GM1 are (Serum: <1:10000 (IgG & IgM) specialist centres for confirmation. Please most typical of multifocal motor neuropathy see price list for full details of assays done. but abs to other gangliosides such as GD1b These assays are currently sent to and asialoGM1 may also be detected. Low Neurology, Southern General Hospital, titres of abs directed against GD1b, GM1 Glasgow. and asialoGM1 may also be detected in amyotrophic lateral sclerosis and Guillain - Barré syndrome. These assays are currently sent to Neurology, Southern General Hospital, Glasgow. Ganglioside abs GM1 The presence of abs directed against GM1 (Serum: IgG <1:500 (monosialoganglioside GM) has been IgM <1:500) associated with motor and sensorimotor neuropathies and in particular with multifocal motor neuropathies. Lower titre of GM1 abs may also be found in amyotrophic lateral sclerosis and Guillain - Barré syndrome. a’GM1 abs may occur as either polyclonal or IgM monoclonal abs. The carbohydrate moiety of GM1, in particular the galactose and sialic acid residues, is the site of antibody binding to gangliosides. Due to the presence of similar moieties on other gangliosides low levels of antibody cross-reaction may be experienced in tests for gangliosides other than GM1. These assays are currently sent to Neurology, Southern General Hospital, Glasgow. Ganglioside abs GQ1b These abs are currently screened by this (Miller Fisher syndrome) department and positives are sent to (Serum: <1:500 IgG & IgM) specialist centres for confirmation. Please see price list for full details of assays done. These assays are currently sent to Neurology, Southern General Hospital, Glasgow. 15 16

Gastric parietal cell abs These antibodies are present in up to 90% of Glutamic acid decarboxylase: Glutamic acid decarboxylase (GAD) is an (Serum: Negative) patients with atrophic gastritis and Stiff Man syndrome enzyme concentrated in neurons, which pernicious anaemia. Also present in gastritis (Serum: 0 - 10 IU/ml) control muscle tone and exteroreceptive Intrinsic factor Abs should be without anaemia (12%), autoimmune thyroid spinal reflexes. Abs to GAD are found in carried out in conjunction with disease (30%), Addison’s disease (25%) and ~60% of patients with Stiff man syndrome GPC Abs iron deficiency anaemia (20%). and also in IDDM where the titres are much lower. The contribution of GAD abs to Gliadin abs In susceptible individuals alpha gliadins are IDDM has not been proved. (Serum: IgA <10 mg/ml known to activate Coeliac Disease a gastro- IgG <70 mg/ml) intestinal disorder characterised by the IgA antibodies IgA abs occur in IgA deficient patients in flattening of the jejunal mucosa. The (Serum: zero titre (negative)) receipt of blood products containing IgA. Gliadin Abs are often seen in laboratory offers a quantitative enzyme Their presence is indicative of risk of patients with inflammatory immunoassay, which allows monitoring of adverse transfusion reactions. This assay is bowel and liver disease and they the patient for both IgG and IgA abs. The currently sent to NBS, Bristol. do not necessarily indicate titre of IgA abs decreases with gluten free underlying coeliac disease. diet, as does the level of endomysial abs and Immunoglobulins (IgG/A/M) Immunoglobulins are an essential request in Endomysial, tissue trans - TTG antibodies. (Serum IgG: 6.00 – 16.00 g/l recurrent infections, lymphoproliferative glutaminase (TTG), antibodies IgA Gliadin abs are more specific for (adult): IgA: 0.80 – 4.00 g/l diseases including myeloma and all cases of are much more specific tests for Coeliacs than IgG (~95% compared to IgM: 0.50 – 2.00 g/l) ‘failure to thrive’. IgA deficiency occurs in Coeliac Disease, but are ~60%) but IgG abs are more sensitive (~80- 1:500 individuals but, transfusion reactions negative in patients with IgA 90% c.f. ~50%). It has been reported that apart, may not be associated with disease. deficiency where gliadin abs are IgA deficient patients have a ten to fifteen Polyclonal increases of IgG occur in chronic most clinically useful. fold increased incidence of Coeliac Disease. infection and inflammation, chronic liver It is suggested that gliadin abs are carried out disease and connective tissue diseases. in all IgA deficient individuals. Raised levels of IgM are found in acute inflammation and in primary biliary Glomerular basement Test for Goodpastures syndrome. Abs to the cirrhosis. (Markedly elevated IgM in the membrane abs non collagenous portion of type IV collagen presence of mitochondrial abs is virtually (Serum: 0 – 3 EU/ml) are detected by EIA as indirect diagnostic of PBC) Low levels of IgG and immunofluorescence is both less sensitive IgA may be due to loss (protein losing A rapid qualitative test is now and less specific being positive in only 75%, enteropathy or nephrotic syndrome) reduced available or less, of proven cases. Urgent requests for synthesis (lympho - proliferative disorders GBM abs (as with ANA, ANCA and dsDNA or primary immunodeficiency) or excessive abs) must be arranged with the laboratory. catabolism. Low levels of immunoglobulins always indicate further investigation. Where appropriate details are supplied age and sex related normal levels are printed on the report. See also sub-classes (IgG and IgA) and functional abs. 17 18

IgG Subclasses IgG subclass deficiency is mainly related to IgA Subclasses IgA comprises two subclasses of which, in (Serum IgG1: 2.75 – 9.50 g/l IgG1 and IgG2 where individuals suffer (Serum: IgA1 0.64 – 3.40 g/l serum, IgA1 predominates (85%). adult: IgG2: 1.20 – 4.50 g/l recurrent infections because they are unable adult IgA2 0.11 – 0.60 g/l) Consequently deficiencies or even complete IgG3: 0.17 – 1.79 g/l to mount an antibody response against absence of IgA2 may go undetected with IgG4: 0.00 – 1.30 g/l) organisms. (This may be ascertained The clinical significance of IgA normal or near normal levels of total serum employing functional antibody assays using subclass deficiency is unknown. IgA. The functions of the two subclasses of Generally functional antibodies Tetanus toxoid requiring the presence of It may be a marker for IgA are comparable to IgG1 and IgG2 but to tetanus (a T-cell dependent IgG1 and pneumococcus requiring IgG2) underlying impaired antibody their activity is on mucosal surfaces whereas antigen), and Pneumovax (a T responses to bacterial IgG is intravascular. The immunology cell independent antigen) give a polysaccharide antigens. laboratory offers RID assays of IgA1 and much better picture of the IgA2 with a turnaround of up to seven days. capacity to mount protective antibody responses than total Intrinsic factor abs Detected in 70% of patients with pernicious subclass assays. (Serum: Negative) anaemia. Assayed by enzyme immunoassay this test should be carried out together with Immunoglobulin D (IgD) There is no known clinical significance for gastric parietal cell abs. (Serum: 2 – 100 mg/l) the measurement of serum IgD except in the case of IgD myeloma and periodic fever. Isoelectric focusing Oligobanding refers to discrete populations (Oligobanding) IgG of immunoglobulin detected by electro- Immunoglobulin E (IgE) Serum IgE may be helpful in diagnosing (Paired serum and CSF: phoresis in CSF, which are NOT paralleled (Serum: 0 – 90 IU/ml, adult) atopic diseases however the normal range is Clinical comment is supplied in serum from the same patient. Oligo very wide and levels do not always correlate with each report) banding is seen in ~85-95% of patients with N.B. Our normal range is given well with symptoms. A high level of specific clinically proven multiple sclerosis. The for Caucasians. Due to IgE to a single allergen may be seen with a Please enclose CSF protein assay is useful as a confirmatory test in environmental factors, and an normal level of IgE. Very high levels of IgE value with each request multiple sclerosis but bands are not specific inherited capacity to produce are seen both in atopic eczema and in for this disease as they also occur in higher quantities of Igs, other parasitic infestations (especially S Mansoni). cerebrovascular accidents, in infections of ethnic groups, notably Black Please note that paediatric values are the CNS and in pathological processes Africans, may have higher quoted on all reports provided that the age involving an immune response e.g.: normal levels compared to of the patient is given. encephalitis, neurosarcoid and SLE. Please Europeans. note that paired samples of CSF and serum are essential for this assay. Liver antigens (blot) Detection and confirmation of antigen specificity associated with primary biliary cirrhosis and autoimmune hepatitis. These include M2, LKM-1, LC-1, SLA/LP, cytokeratin 8/18 and glutathione-S- transferase. 19 20

LKM abs These abs, which stain the cytoplasm of Myeloperoxidase (MPO) abs Antibody to myeloperoxidase is associated (Serum: Negative) hepatocytes and proximal renal tubules, and (Serum: 0 – 8 EU/ml) with organ-limited vasculitis including hence, to the untrained eye are difficult to necrotising and crescentic glomerulo- distinguish from mitochondrial abs by IIF, A rapid qualitative test is now nephritis. The assay is useful in confirming are found in a subgroup of patients with available. MPO specific abs in sera which are positive ANA negative, autoimmune chronic active for anti neutrophil cytoplasmic abs of the hepatitis (CAH). LKM1 abs are positive in perinuclear type (pANCA). Typically the CAH type 2, which is the most common level of MPO abs parallel disease state with autoimmune liver disease of childhood and increasing levels when vasculitis is active. has a relatively unfavourable prognosis. Urgent requests must be arranged with Lymphocyte cell markers A wide range of lymphocyte markers for the laboratory. assessment of immunodeficiency and lymphoproliferative diseases is available to Neutrophil cytoplasmic abs c-ANCA is a test for Wegeners granulo- the West Midland Region. A separate cell (ANCA) matosis and for microscopic polyarteritis form is in use for cell marker requests but a (Serum: Negative) (see also test for proteinase 3). pANCA may clinician or a technical member of staff occur in other vasculitic disorders as well as within the laboratory MUST be contacted Pattern and titre reported on some forms of glomerulonephritis (see also prior to samples being sent in order that the positives test for myeloperoxidase). most appropriate studies are undertaken. [See MPO & PR3 antibodies] Lymphocyte function tests Special sample requirements. Please discuss with Consultant Immunologist. Neutrophil function test Special samples required. Please discuss with Consultant Immunologist. Mitochondrial abs Present in >95% of cases of primary biliary (Serum: Negative) cirrhosis usually in high titre (>1:200). Also Pancreatic islet cell abs At the time of diagnosis 75% of type I occasionally present in chronic active (Serum: Negative) diabetics have detectable levels of hepatitis and halothane induced hepatitis circulating islet cell abs. Such abs patients but with titres of <1:100. Serum decrease and eventually disappear with IgM levels are invariably increased. duration of disease. Some studies have indicated persistent levels of abs in Mitochondrial (M2) abs For those wishing to confirm the presence of association with polyendocrine disease (type (Serum: 0 – 10 EU/ml) mitochondrial abs or to monitor patients with Ib). There have been no reports of abs to a quantitative assay an EIA method is pancreatic islet cells in type II diabetics. available which distinguishes three separate mitochondrial abs (enzymes) and affords a quantitative assay in EU’s/ml. Currently abs to the major enzyme pyruvate dehydrogenase complex (M2) are assayed routinely. 21 22

Paraproteins Levels of monoclonal IgG, IgA, IgM, IgD Phytanic acid. Elevated levels of blood phytanic acid are Reported in g/l. and in some instances IgE are measured (Whole blood:0.0 – 1.0 mg/100ml) found in Refsums’ disease which is immunochemically and in all cases of follow characterised by any combination of retinitis up the previous specimen is run in parallel to pigmentosa, ataxia or polyneuropathy with confirm the change in paraprotein level. elevated phytanic acid levels. There may Immunofixation of presentation sample also be anosmia, sensorineural hearing loss, defines both the isotype and light chain type. icthyosis and cardiac abnormalities. Follow up specimens will be subjected only Elevated levels result from accumulation of to electrophoresis unless immunofixation is ingested phytanates in the absence of required to confirm complete response. (See phytanic acid hydroxylase. This assay is ‘guide to appropriate use of tests at the back currently performed in Biochemistry, of this booklet). The presence of a Birmingham Children’s Hospital. paraprotein should prompt investigation of B cell malignancy, particularly myeloma, (IgG, Proteinase 3 (PR3) abs PR3 antibody is a marker for Wegeners IgA) and lymphoplasmacytoid lymphoma (Serum: 0 – 10 EU/ml) granulomatosis and is occasionally detected (IgM). Monoclonal gammopathy of in microscopic polyarteritis. The quantity of uncertain significance (MGUS) is found in A rapid qualitative test is now PR3 antibody generally parallels disease one or more percent of the general available. activity with higher levels in the active state population over the age of 50 years. of the disease. EIA affords a quantitative assay which is useful when monitoring the disease. Abs to PR3, an elastinolytic neural Paraprotein neuropathies Myelin associated glycoprotein (MAG) is a serine protease, are responsible for the MAG; POEMS glycoprotein component of the myelin of characteristic granular cytoplasmic pattern of (Serum: Negative) central and peripheral nervous systems the neutrophils when stained by IIF. present in the periaxonal region, Schmidt- Lantermans incisures, lateral loops and outer Urgent requests must be arranged with mesaxon of the myelin sheath. A member of the laboratory. the immunoglobulin super- family MAG probably functions as an adhesion molecule and mediates cell-cell interactions. Monoclonal reactivities against MAG are detected in about 50-75% of patients with IgM paraproteinaemia and peripheral neuropathy. Sera from patients with neuropathy that are negative for MAG antibodies often exhibit reactivity against various gangliosides. 23 24

Paraneoplastic neurological antibodies: RAST (allergen specific IgE) Assays for the detection of circulating IgE (Serum: 0 – 0.35 kU/l) antibodies directed against specific antigens Paraneoplastic neurological antibodies are associated with paraneoplastic in sensitised patients are available to a wide neurological syndrome and systemic malignancies For clinical advice regarding range of allergens. Common substrates allergic diseases and reactions include animal fur or dander, house dust Most frequent phone 07831-681955. A mite, tree and grass pollens, moulds, feathers Antibody Neurological disorder(s) tumour(s) regional allergy clinic now and an extensive range of food substances paraneoplastic cerebellar operates from the Chest Clinic including a variety of nuts. Tests against Yo (PCA-1) Ovary, breast in the centre of Birmingham degeneration allergens in stock have a turnaround time of paraneoplastic neurological where skin prick and patch seven days. Ma (Ma1) disorder, brainstem Various, lung cancer testing is available. encephalomyelitis It is essential to supply as much information brainstem encephalomyelitis, N.B. Total IgE will be carried as possible with RAST tests, including Ta (Ma2) Testicular cancer out on all RAST requests clinical details and total IgE, if available. If limbic encephalomyelitis paraneoplastic cerebellar unless an IgE level is stated at in any doubt it is advisable to discuss the Hu degeneration, paraneoplastic small cell lung the time of request. patient with the Consultant Immunologist (ANNA1) encephalomyelitis, sensory carcinoma (07831 681955). neuropathy opsoclonus/myclonus, Breast, small cell lung Red cell transketolase [See transketolase] paraneoplastic cerebellar Ri (ANNA2) carcinoma, degeneration, brainstem gynaecological Rheumatoid factor Two tests are currently carried out to detect encephalomyelitis Breast, colon, small (Serum: Negative) these abs, which are directed against other GAD Stiff person syndrome immunoglobulins. A latex test which is cell lung carcinoma paraneoplastic encephalomyelitis / small cell lung more sensitive but less specific for RF and CV2/CRMP5 an haemagglutination test which is less sensory neuropathy carcinoma, thymoma Stiff person syndrome, sensitive but highly specific. Breast cancer, small Amphiphysin paraneoplastic cell lung carcinoma Approximately 70% of patients with encephalomyelitis rheumatoid arthritis are sero positive and paraneoplastic cerebellar antibodies may occur in other conditions Tr Hodgkin’s lymphoma degeneration including many infections, myeloma, lymphomas, cryoglobulinaemia and The presence of Yo, Hu (ANNA1) and Ri (ANNA2), Ma/Ta, CV2/CRMP5 and connective tissue diseases. Antibodies (RF) amphiphysin antibodies are confirmed by Western blot. may also be found in allegedly normal individuals aged over 75. Titre of rheumatoid factor is less sensitive than sequential assay of CRP when monitoring activity of rheumatoids. 25 26

Salivary Duct abs Salivary duct abs are present in Sjögrens Serum immunoglobulin free Normal plasma cells make more (Serum: Negative) syndrome with arthritis (65%) and in SICCA light chains immunoglobulin light than heavy chains and syndrome (10%) but SSA (Ro) and SSB (La) Kappa 7.0 – 22.6 mg/l secrete free light chains in amounts are more sensitive and more specific for Lambda 9.9 – 34.4 mg/l detectable in serum (estimated to be Sjögrens syndrome. Kappa / Lambda ratio 0.30 – 0.5g/day). Serum free light chains are 1.57) removed by glomerular filtration with a half- Salivary IgA and secretory This is a quantitative test for the presence of life of a few hours. They are not easily piece. mucosal, saliva antibody (secretory IgA, This assay may be inaccurate at levels detectable in urine until the threshold for <0.9mg/l. In <1% of patients with high (Saliva: Clinical comment will sIgA) used in the work up of suspected cases tubular reabsorption is exceeded (10 – 20g / levels of serum FLC these are missed by accompany each report) of immunodeficiency. Serum levels of this assay because of antigen excess. day). Serum flc measurements are immunoglobulins should be carried out in Any anomaly between the serum FLC recommended in diagnosis and management parallel. results and other laboratory tests and/or of light chain amyloid, plasmacytoma, non- clinical evidence should be reported to secretory myeloma and light chain only the laboratory for re-testing the serum Normal age related levels will be shown on FLC. myeloma. the form and will be accompanied by a clinical comment. Skin abs. Abs are found in (i) intercellular substance Serum electrophoresis. Sera are screened for qualitative (Serum: Negative) of the epidermis (desmosome), which (Serum: Clinical comment will abnormalities in proteins especially of the strongly suggest a diagnosis of pemphigus accompany each report) immunoglobulins. Scans demonstrating a though these abs may also be found in monoclonal band are automatically followed patients with severe burns or a trichophyton up using immunofixation to determine both infection. (ii) dermal-epidermal basement the isotype and the light chain of the membrane which is highly specific for monoclonal protein. Other typical patterns bullous pemphigoid and is present in 80% of seen on electrophoresis may indicate these patients. A titre is useful in monitoring evidence of acute phase response, the disease. immunodeficiency and absence of alpha-1- antitrypsin. Smooth muscle abs. Present in high titre in up to 70% of patients (Serum: Negative) with autoimmune hepatitis who may also be Where myeloma is suspected urine and positive for mitochondrial, nuclear and serum should be sent together. dsDNA abs (25%) Striated muscle abs In patients with Myasthenia Gravis with (Serum: Negative) thymoma these abs are invariably positive but in such patients without thymoma the abs occur in only 60% of cases. This assay is usually carried out with a test for acetyl choline receptor abs but as this latter test is sent away to Oxford for quantitative assay it will not be carried out as a routine unless specifically requested. 27 28

Thyroid peroxidise abs. Present at high levels in 95% of patients with Tryptase [See Adverse Anaesthetic reactions] (microsomal [TPO]) Hashimotos thyroiditis, 20% of patients with (Serum: <35 IU/ml) Graves disease and 90% of patients with Urinary free light chains. As a National centre for myeloma trials this primary myxoedema. Abs may also be (Urine: Kappa or Lambda laboratory quantitates the level of free light present at low levels in colloidal goitre, 0 – 0.04 g/g of creatinine) chains in urine. Expressed in terms of g/g of thyroid carcinoma, De Quervains thyroiditis, creatinine it is an accurate method for the other organ specific auto-immunities and in assessment of light chain output in the allegedly normal people. If persistent in presence of circulating paraprotein; 24hr euthyroid individuals it may indicate See serum immunoglobulin urine collections are not required. autoimmune thyroiditis and predisposition to free light chains Polyclonal free light chains may occur in future thyroid failure. healthy individuals particularly following strenuous exercise and also in patients with Thyroglobulin abs Thyroglobulin antibodies will be assayed for chronic infections or inflammatory diseases (Serum) specific patients if requested. For example, such as rheumatoid arthritis. Unlike where thyroglobulin levels are used as a laboratories where concentration of urine is tumour marker. This assay is currently sent carried out before assay, thereby introducing to Immunology PRU, Northern General a source of error, this laboratory measures Hospital, Sheffield. free light chain directly on urine by RID on commercial diffusion plates. TSH receptor abs Hyperthyroidism in Grave’s disease is due to (Serum) autoantibodies to the TSH receptor and measurement of these autoantibodies can be Vasculitis screen Several laboratory tests may be useful in the useful in disease diagnosis and management. (ANA/ANCA and CRP) diagnosis of clinical disorders involving This assay is currently sent to Immunology inflammation of vessel walls. These include PRU, Northern General Hospital, Sheffield. ANA, anti-neutrophil cytoplasmic antibody and measurement of immune complexes, Tissue Transglutaminase abs The endomysial autoantigen has been Von Willebrand factor (FVIII Rag) and (Serum: 0 – 20 ELISA Units) identified as the protein cross-linking CRP. The laboratory offers a vasculitis enzyme tissue transglutaminase (tTG). screen of the three most commonly requested Positive tTG abs will be tested Antigen specific EIA assays provide an tests as first line investigation of this for endomysial abs by IIF alternative to the conventional indirect condition. immunofluorescence assay based on primate [Also see anti-Gliadin abs] oesophagus. The EIA assay provides a Vasculitis screen (Urgent) A vasculitis screen is available as an urgent quantitative measurement of IgG and IgA request with a turnaround time of just two anti-tTG antibodies and is not adversely hours. However as this disrupts the normal influenced by the presence of other planned laboratory work there will be an autoantibodies such as ANA or ASMA. extra, but moderate, charge for this service. 29 30

Viscosity Serum or plasma viscosity is an essential test GUIDE TO THE APPROPRIATE USE OF IMMUNOLOGICAL (Serum: 1.25 – 1.70 (c.f. to when monitoring Waldenstroms’ macro- ASSAYS. water) globulinaemia and also when investigating Plasma: 1.40 – 1.90 (c.f. to an unexplained retinal or cerebrovascular Diagnosis of SLE: water)) occlusion. In such patients a cryoglobulin may also be present. It is therefore essential Request ANA, dsDNA, ENA, C3/C4, GAM and CRP. that the precautions outlined for cryoglobulins are observed and blood Follow up of SLE: collected and transported to the laboratory at body temperature. This assay is currently Request dsDNA, C3/C4, and CRP. (GAM and ENA at 12 monthly intervals.) sent to Immunology, George Eliot Hospital, Nuneaton. SLE in pregnancy or with planned pregnancy. These patients should also have their cardiolipin antibodies assayed. Volt gated Ca++ channel abs The Lambert Eaton myasthenic syndrome (LEMS) is a form of myasthenia often Monitoring Infections: associated with small cell lung carcinoma. In ~50% of cases there is IgG mediated Alternate day samples for CRP will give adequate information regarding reduction in presynaptic voltage gated response to antibacterial therapy. CRP, an acute phase protein, has a half-life calcium channels. This assay is currently of 4-6 hrs. sent to Immunology, Churchill Hospital, Oxford. Diagnosis of Myeloma: Full characterisation (Blood and Urine) Volt gated K+ channel abs Antibodies to voltage gated potassium IgG, IgA and IgM; Immunofix; B2M; Cryoglobulin; Viscosity; Kappa and channel are found in ~40% of patients with Lambda light chains and creatinine. acquired neuromyotonia. This assay is currently sent to Immunology, Churchill Monitoring Myeloma: Follow up (Blood and Urine). Hospital, Oxford. IgG, IgA or IgM; electrophoresis; B2M; Kappa or Lambda light chains; creatinine. Autoantibody screen: ANA on Hep2 cells; GPC; LKM; AMA and SMA. Arthritis screen: ANA on Hep2 cells; CRP and RF. Vasculitis screen: ANA on Hep2 cells; ANCA and CRP. 31 32

Test Turn around time Cost Autoantibodies Test Turn around time Cost Allergy Assays Autoantibody Screen 24h (ANA [Hep 2]; GPC; AMA; SMA; LKM) Adrenal antibodies up to 7d 9.75 Total IgE up to 7d 12.25 Anti-C1q antibodies up to 28d 16.10 Mast cell tryptase up to 28d 14.30 B2GP1 antibodies (IgG) up to 7d 9.30 Specific IgE antibodies (RAST) per allergen up to 7d 14.30 Cardiolipin antibodies (IgG and IgM) up to 7d 14.20 Common allergies: Centromere antibodies 24h 7.65 Cat/House dust mite/grass up to 7d 35.45 CCP antibodies up to 28d 8.25 Egg/Milk/Wheat up to 7d 35.45 Endomysial antibodies up to 7d 7.65 Aspergillus/Cladosporium up to 7d 23.65 Epidermal antibodies up to 7d 7.65 Mixed allergens (eg trees, nuts) up to 7d 14.85 Gastric parietal cell antibodies 24h 7.65 Other specific allergens (Many available) up to 28d 14.30 Gliadin antibodies (IgG and IgA) up to 7d 14.20 Neuroimmunology Assays Glomerular basement membrane abs (quantitative) 3-4d 18.50 GBM - rapid test for urgent samples 2 hours, from receipt 17.25 CSF Samples Intrinsic factor antibodies up to 7d 12.25 Oligoclonal IgG bands (paired blood and CSF) 7-14d 31.90 Islet cell antibodies up to 7d 9.75 Spectrophotometric scan/xanthochromia same day* 13.60 LKM/Mitochondrial/Smooth muscle antibodies 24h 7.65 Tau protein (blood must be provided with CSF) 1-2d 29.50 Liver antibody blot (M2, LC1, LKM, CYT, GST, SLA) up to 28d 36.05 Mitochondrial M2 antibodies (quantitative) up to 7d 12.25 Blood Samples Myocardial antibodies up to 7d 9.75 Caeruloplasmin 3-7d 8.25 Salivary duct antibodies up to 7d 9.75 Skeletal muscle antibodies up to 7d 9.75 Autoimmune diseases (Serum Samples) Thyroid microsomal antibodies (TPO) 3-4d 8.00 Acquired neuromyotonia Volt. gated K+ channel 30d 41.35 Tissue transglutaminase antibodies (tTG) up to 7d 12.25 Lambert-Eaton syndrome Volt. gated Ca+ channel 30d 41.35 Myasthenia gravis. Acetylcholine receptor 30d 23.95 Arthritis screen (RF/ANA/CRP) 24h* Paraneoplastic syndromes: PCA1 (Yo), ANNA1 (Hu), 24h 18.55 ANNA2 (Ri), CV2/CRMP5, Ma/Ta, & amphiphysin ANA (Hep2 cells; includes titre/pattern on positives) 24h 7.65 Brain antibody western blot (Yo, Hu, Ri, CV2/CRMP5, up to 28d 36.05 Centromere antibodies 24h 7.65 Ma/Ta, and amphiphysin) ds(native)DNA antibodies (Crithidia + EIA up to 7d 14.10 Paraprotein neuropathies: MAG, POEMS 7-10d 12.25 quantitation) Miller-Fisher syndrome GQ1b, GT1a Done as a panel of )4 ENA antibodies Screen up to 7d 9.30 Sensory motor neuropathies (e.g. Guillan-Barré syn) assays taking up to ) 3. ENA " Type: (SSA,SSB,Sm,RNP,Jo-1,Scl-70) up to 7d 29.45 GM1, Asialo-GM1, GDa+b 28 days and charged ) 0 Sensory neuropathies. Sulphatide at £43.00 ) 0 Rheumatoid factor by turbidimetry (RALX) 24h 6.80 Stiff-man syndrome. Glut. decarboxylase 23.95 Further testing on positive RALX samples up to 28d 10.30 * Same day if received before 3.15p.m. (otherwise next day) 33 34

Test Turn around time Cost Test Turn around time Cost Precipitin Assays Antibody Deficiency Aspergillus fumigatus 7 - 10d 10.70 Immunoglobulins (IgG, A and M) 24h 13.25 Avian (Pigeon & budgerigar) 7 - 10d 18.00 IgG subclasses (IgG1,2,3 and 4) up to 7d 18.10 Candida albicans 7 - 10d 10.70 IgA " (IgA1 and 2) up to 7d 17.70 Mycopolyspora faeni 7 - 10d 10.70 Functional antibodies: (Tetanus toxoid, Pneumococcus and H.Influenza b) up to 14d 30.00 Immunochemistry Free Secretory component up to 7d 14.90 Secretory IgA up to 7d 14.90 Paraprotein/Myeloma Screen up to 7d 47.00 (IgG,A,M; Elec; Urine immunofixation) Complement Assays Paraprotein/Myeloma (full characterisation, blood up to 7d 75.75 and urine) Complement C1q up to 7d 9.45 (IgG,A and M; Imfix; B2M; Cryo; Visc; K/L light C1r up to 7d 9.45 chains) C1s up to 7d 9.45 Paraprotein/Myeloma (follow up, blood and urine) up to 7d 35.65 C2 up to 7d 9.45 (Paraprotein; Elec; B2M; K or L light chain, creatinine) C3/C4 24h 9.25 B2Microglobulin 24h 5.35 C3d up to 28d 9.45 CRP (C reactive protein) 24h 4.60 C5 up to 7d 9.45 Cryoglobulin (qualitative screen) up to 7d 3.70 C6 up to 7d 9.45 (type and quantitation) up to 7d 22.50 C7 up to 7d 9.45 Cryofibrinogen up to 7d 3.70 C8 up to 7d 9.45 Serum protein electrophoresis 24h 4.70 C9 up to 7d 9.45 Immunofixation (serum) 24h 18.80 Factor-B up to 7d 9.45 Immunofixation (urine – kappa/lambda) 24h 7.10 Factor-D up to 7d 9.45 Immunoglobulins (IgG,A and M) 24h 13.25 Factor H(ß1H) up to 7d 9.45 IgD up to 7d 9.45 Factor I (KAF) up to 28d 9.45 Plasma/serum viscosity up to 14d 5.60 Paraprotein (G or A or M with previous specimen in 24h 8.90 C1 esterase inhibitor parallel) (Immunochemical and functional assays) up to 14d 18.50 Urinary free light chain: Kappa or Lambda quantitation: C3 Nephritic factor (C3 Nef) up to 28d 21.40 as part of the myeloma workup* up to 7d 12.35 Functional complement: Classical pathway up to 7d 9.00 as an individual quantitative assay* up to 7d 25.20 Alternate pathway up to 7d 9.00 Serum free kappa/lambda & ratio 24h 25.00 35 36

Test Turn around time Cost Tissue biopsy Freshly excised and put in Vasculitis cold tissue culture medium or saline. Vasculitis screen (ANA/ANCA/CRP) 24h Neutrophil cytoplasmic antibodies (ANCA) 24h 7.65 T cell antigen receptor & Blood or bone marrow Von Willebrand factor (FVIII Rag) up to 7d 7.65 Immunoglobulin gene drawn into EDTA bottle CRP 24h 4.60 Rearrangement studies (Heparinised material may ANCA (EIA) Myeloperoxidase (Quantitative) up to 7d 12.25 interfere with the PCR Proteinase III (Quantitative) up to 7d 12.25 process) Myeloperoxidase (urgent screen) 2 hours (from receipt 17.25 Proteinase III (urgent screen) 2 hours of specimen) 17.25 * When taking blood for marker studies a sample must also be sent to the local haematology department for a white cell count and differential. A clotted blood sample and urine may be needed for assessment of immunoglobulin concentrations Cell marker tests performed by the and paraprotein analysis. Clinical Immunology Service Neoplastic Diseases All queries and requests for urgent investigations to be addressed to Mr Peter Panels available (all 3- or 4-colour analysis) Richardson in the Clinical Immunology laboratory. Myeloid markers – (MDS/MPD/some AML) Specimen collection for Cell Marker Work CD33/CD117/CD45, CD15/CD117/CD34, CD13/CD11b/HLADR, £87.00 CD64/CD14/CD45, GlyA/CD41/CD45, surface kappa/lambda/CD19, All samples must arrive in the laboratory by 2.00 p.m. on the day of sampling CD4/CD8/CD3 accompanied by clinical details Acute leukaemia rapid screen. £47.30 Immunodeficiency studies: please telephone for clinical discussion and advice CD13/CD33/CD34/CD117. CD79a/CD10/CD34/CD19, CD1a/CD7/CD34/CD3 regarding appropriate tests and samples required Followed by appropriate additional panel/tests for AML, B-ALL, T-ALL Bone marrow 1ml bone marrow in EDTA (including 4 colour analysis if appropriate to identify aberrant phenotype and an unfixed for subsequent detection of Minimal Residual Disease) marrow smear AML – 4 colour – (some AMLs/appropriate tubes selected for MRD ) £87.00 Blood * CD45/CD13/CD34/CD33, CD38/CD56/CD34/CD33, Immunodeficiency marker studies 5mls blood in EDTA CD15/CD117/CD34/CD33, DR/CD117/CD34/CD33, CD13/CD11b/DR/CD14, CD64/CD117/CD45/CD14, CD45/CD4/CD3/CD19, CD33/CD13/CD2/CD8/CD117, CD33/CD13/CD7/CD3/CD34, CD33/CD13/CD22/CD34/CD19, CD71/GlyA/CD45/CD34, Effusions At least 20mls sample in CD36/CD41a/CD61/CD34 EDTA or lithium heparin Additional Tests (where appropriate) Myeloperoxidase /Tdt/cytCD79a/cytCD3, PML antigen. C.S.F. As much as possible in a universal bottle containing AML 4 colour follow up (MRD detection) £77.10 tissue culture medium (Appropriate tubes selected from AML 4 colour panel for FU ) 37 38

AML (CSF) 4 colour panel Hodgkins (CSF) 4 colour panel CD13/CD33/CD34/CD117, CD4/CD8/CD45/CD3 CD15/CD30/CD45/CD19, CD4/CD8/CD45/CD3 B-ALL-4 colour - (diagnosis/follow-up) £87.00 CD20/CD10/CD34/CD19, CD22/CD10/CD34/CD19, CD34/CD10/CD45/CD19, Myeloma (myeloma/MGUS/paraprotein) £77.10 CD38/CD10/CD34/CD19 skappa/slambda/CD19, CD20/CD5/CD19, CD22/CD23/CD19, CD13/CD33/CD10/CD34/CD19, skappa/slambda/CD45/CD19 CD10/CDCD19/CD34, CD138/CD45/CD38, CD56/CD19/CD38 cytoplasmic kappa/lambda/CD38 B-ALL (CSF) 4 colour panel £25.60 ZAP-70 – CLL only CD79b/CD10/CD34/CD19, CD4/CD8/CD45/CD3 ZAP70/CD19/CD3/CD5 T-ALL-4 colour - (diagnosis/follow-up) £87.00 ........................................................................................................... CD4/CD8/CD45/CD3, CD7/CD2/CD3/CD5, CD7/CD38/CD34/CD3, CD7/CD117/CD3,CD5 If appropriate - Tdt/CD34/CD7/CD3 T-ALL (CSF) 4 colour panel CD7/CD2/CD34/CD5, CD4/CD8/CD45/CD3 PNH screen CD16/CD15/CD45, CD59/CD55/CD45, CD14/CD64/CD45, £77.10 CD66b/CD15/CD45, CD24/CD15/CD45 Lymphoproliferative diseases- General panel (LPD) skappa/slambda/CD19, CD20/CD5/CD19, CD79b/CD23/CD19, £77.10 CD103/CD22/CD19, CD10/CD19/CD34, CD19,CD38,CD45, CD7/CD56/CD3, CD4/CD8/CD3, TCRgammadelta/CD2/CD3 LPD (CSF) panel kappa/lambda/CD19/CD5, CD4/CD8/CD45/CD3 T-NHL LPD + CD25/CD4/CD3, CD16/CD56/CD3, CD7/CD57/CD3, DR/CD69/CD3, CD45RA/CD45RO/CD4, CD45RA/CDRO/CD8 £47.30 Hairy cell – (hairy cell/SLVL diagnosis/FU) CD103/CD22/CD19, CD25/CD11c/CD19, skappa/slambda/CD19, CD10/CD5/CD19 £47.30 Hodgkins/Anaplastic lymphoma (diagnosis/FU) CD4/CD8/CD3, skappa/slambda/CD19, CD23/CD5/CD19, CD15/CD30/CD45, EMA/CD30/CD45 £47.30 39 40

Investigation of Immunodeficiency Neutrophil/lymphocyte functional studies B/NK cell markers £25.60 We perform a number of basic investigations to test defects in neutrophil function including oxidative burst and phagocytosis, and lymphocyte proliferation and function including cytokine production. These investigations are labour intensive Full Immunodeficiency surface markers panel £109.35 and technically demanding. To identify which tests are most appropriate please Similar panel as for T/NK-Lymphorproliferative Disease discuss with one of the clinicians on the mobile telephone numbers listed below. Specialist staff and facilities are booked to carry out detailed functional assessments Short Immunodeficiency surface markers panel £47.30 of neutrophils and lymphocytes. Only a limited number of appointments can be CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD45RA, CD45RO, CD56, made. It is essential that the blood arrives in the laboratory in good condition and HLA-DR with sufficient time to carry out the assays. We cannot guarantee to carry out investigations if samples are received after 10am. Leukocyte adhesion deficiency surface markers £60.00 CD11a, CD11b, CD11c, CD15, CD18. Functional studies must be booked in advance. To obtain advice please contact: HIV monitoring £25.60 Prof P.J.L.Lane (0121) 414 4078 (mobile 07831 681955) CD3, CD4, CD8 Dr M.T.Drayson (0121) 414 4069/4074 (mobile 07798 585319) Immunoglobulin/T cell receptor gene studies £224.95 Please carefully follow the guidelines below: Lymphocyte proliferation studies £59.00 - 1. The following samples should be taken and sent by taxi without delay to £148.15 arrive by 10am Neutrophil function studies (respiratory burst) £47.30 10ml blood in Lithium heparin (50ml for type I cytokine studies) 10ml clotted blood 5ml blood in EDTA. Full type 1 cytokine studies £375.00 In the case of children the minimum blood volume required should be confirmed with the laboratory. Cytokine measurement (per cytokine) £10.70 Available cytokines include: - 2. A full blood count and white cell differential should be done on the same IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, TNF-a, IL-12p70, IL- day at your hospital. (We will telephone your laboratory for the results). 13, IL-17, IFN-g, G-CSF, GM-CSF, MCP-1, MIP-1b, Eotaxin, FGF basic, IL-1ra, IL-9, IL-15, IP-10, MIP-1a, PDGFbb, Rantes, VEGF 3. A concise clinical summary including results of preliminary tests should be sent with the specimen. Interferon gamma receptor antibodies £24.35 4. Samples should be addressed to: Anti-cytokine antibodies and antibodies to biological therpeutics POA Clinical Immunology Service, Medical School, Birmingham. By arrangement with the laboratory and labelled “URGENT!!” contact 44069 on arrival. 5. Include the telephone number, or bleep, of the doctor to be contacted in case of any query. 41 42

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