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1 IMMUNOLOGY DEPARTMENT County Durham and Darlington NHS Trust University Hospital of North Durham IMMUNOLOGY USERS HANDBOOK January 2017 (reviewed annually or when major changes in repertoire)
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IMMUNOLOGY USERS HANDBOOK - cddft.nhs.uk handbook... · Immunology Department The Immunology Department was set up as a stand-alone department of the CDDAH NHS Trust (now CDDFT) in

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  • 1

    IMMUNOLOGY DEPARTMENT

    County Durham and Darlington NHS Trust University Hospital of North Durham

    IMMUNOLOGY USERS HANDBOOK

    January 2017

    (reviewed annually or when major changes in repertoire)

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    CONTENTS:

    General Information, contact numbers, staff

    Autoantibody section Autoantibody screen 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

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    Immunology Department

    The Immunology Department was set up as a stand-alone department of the CDDAH NHS Trust (now CDDFT) in March 2005. It is based in Pathology, University Hospital of North Durham (UHND), Staircase C, Lower Ground Floor. The Immunology Department is a CPA/UKAS ISO 15190:2012 accredited provider of Immunology Services.

    Postal Address Immunology Department

    University Hospital of North Durham North Road

    Durham DH1 5TW

    Telephone: ext 32635 or (0191) 333 2635

    Fax: ext 32115 or (0191) 333 2115

    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 Catherine Stroud c.stroud@nhs.net Consultant Clinical Immunologist ext 32781 or (0191) 333 2781 Anne Richardson anne.richardson7@nhs.net Immunology Secretary ext 32433 or (0191) 333 2433

    Scientific staff Nicola Sherriff nicola.sherriff@nhs.net Cellular Sciences service manager ext 32435 or (0191) 333 2435

    Catherine Wedd cwedd@nhs.net Senior Biomedical Scientist ext 32635 or (0191) 333 2635 Debra Swainston debra.swainston@nhs.net Senior Biomedical Scientist ext 32635 or (0191) 333 2635 Melanie Robson, Rebecca Haycox, Nicola McKeague, Specialist Biomedical Scientists ext 32635 or (0191) 333 2635 Roger Webster Biomedical Scientist ext 32635 or (0191) 333 2635

    mailto:c.stroud@nhs.net

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    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-of-hours queries Immunology Registrars at the RVI may be contacted on 0191 282 9572. For any urgent, out-of-hours requests senior BMS or medical staff can be contacted 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

    - Immunology Department, Churchill Hospital, Headington, Oxford, OX3 7LJ

    - Histocompatibility & Immunogenetics Diagnostic Laboratory at NHS Blood and Transplant, Holland Drive, Barrack Road, Newcastle upon Tyne, NE2 4NQ

    - Department of Neuro-Immunology, Room 917, Institute of Neurology, Queen’s Square, London WC1N 3BG

    Uncommon test requests may be referred to other specialist laboratories not listed here

    We check all reference laboratories annually to ensure they maintain standards required for Clinical Pathology Accreditation (CPA)/United Kingdom Accreditation Service (UKAS).

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    NB! It is essential that full patient identification is provided. We are obliged to discard samples with inadequate identifying information.

    NB! 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 not intended to be comprehensive or replace discussion of individual patients. Clinical advice is given on allergies, autoimmune disorders, suspected immune deficiency etc.

    Additional information:

    Complaints: We endeavour to do our best but if you are not satisfied with any aspects of our service please contact any of the Laboratory Staff or medical Consultant by phone or email given above.

    Disclosure of clinical information and patient family history to referral laboratory: When referring samples, it is a requirement to disclose clinical information and family history to referral laboratory where sample referral will be made to ensure appropriate interpretative comments can be given. If you have concerns about any aspects relating to privacy please visit our website http://intranet/Directorates/CorporateDirectorates/CS/HI/IG/Pages/default.aspx or contact Lisa Wilson, Head of Information Governance (lisa.wilson4@nhs.net; mobile 07980 726539)

    Comments / suggestions are appreciated & welcome!

    http://intranet/Directorates/CorporateDirectorates/CS/HI/IG/Pages/default.aspxmailto:lisa.wilson4@nhs.net

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    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.

    If you require additional tests to be done from samples already sent in, please contact Lab within 14 days of sending.

    Turn-around times (working days): o Routine Autoantibody Screen 3-5 days o ENAs 5-10 days o DNAs 5-10 days o RF 1-3 days o ANCAs 3-5 days o Send away tests approx. 20 days

    Autoantibody screen Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working 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

    Antinuclear antibodies can appear as homogenous (common in SLE), speckled (common in SLE, MCTD and other rheumatic diseases), nucleolar (common in scleroderma), multiple nuclear dots (seen in PBC/Sjogren’s and SLE) as well as other variants mostly seen in rheumatic diseases.

    Samples positive for AMA and LKM antibodies will be automatically tested for M2 positivity

    if ANF is negative further testing will not be performed

    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.

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    Individual Autoantibodies (alphabetical order)

    Acetylcholine receptor antibody (AChR) Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days 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 approx. 15 working days See anti-endocrine gland antibodies.

    Cardiac muscle antibody Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days 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 Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days 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: 0.4 – 340 U/mL; Reference range: Negative: < 7 U/mL Equivocal: 7 – 10 U/mL Positive: >10 U/mL

    Centromere antibody Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days Indicated in the investigation of unexplained Raynaud’s; positive in ~50% of patients with primary Raynauds, 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; NB! If patient is on gluten-free diet test may be negative in presence of Coeliac Disease; turn-around time 3-10 working days See Endomysial antibody (EMA); see also Gliadin, Tissue Transglutaminase (tTG test), Coeliac Disease (CD), Dermatitis Herpetiformis (DH).

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    Cytokine antibodies Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days 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; NB! If patient is on gluten-free diet test may be negative in presence of Coeliac Disease / Dermatitis Herpetiformis; turn-around time 3-10 working days See Endomysial antibody (EMA); see also Gliadin, Tissue Transglutaminas (tTG test ), Coeliac Disease (CD), Dermatitis Herpetiformis

    dsDNA antibody (dsDNA) Serum sample required; no special preparations needed prior to sampling; turn-around time 5-10 working days Assayed routinely on any serum with positive ANA i.e. this test is usually not performed as a stand-alone 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: 0.5 – 379 IU/mL; Reference range: Negative: < 10 IU/mL Equivocal: 10 – 15 IU/mL Positive: >15 IU/mL

    ENA antibodies Serum sample required; no special preparations needed prior to sampling; turn-around time 5-10 working days 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). Titers 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 10 0.3 – 240 U/mL

    La/SSB 10 0.3 – 320 U/mL

    SM 10 0.1 – 120 U/mL

    RNP 10 0.3 – 240 U/mL

    Scl-70 10 0.4 – 320 U/mL

    Jo-1 10 0.3 – 240 U/mL

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    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 approx. 15 working days

    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 Distrophy) 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

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    Endomysial antibody (EMA) Serum sample required; NB! If patient is on gluten-free diet test may be negative in presence of Coeliac Disease; turn-around time 5-10 working days (see also Gliadin, Tissue Transglutaminase (tTG test ), Coeliac Disease (CD), Dermatitis Herpetiformis (DH)) EMA is a diagnostic test for coeliac disease (CD) and dermatitis herpetiformis (DH). Current guidelines advise that samples are initially screened for tTG, followed by EMA confirmation of positive samples (>3). EMA-IgA antibodies are clinically relevant and are very sensitive and specific for CD (94-100%). EMA are important in investigation of malabsorption (especially paediatric), IgA deficiency, anaemia of unknown cause etc. Gliadin antibodies may be requested for young children (95% of cases. The majority of cases are associated with haplotype: DQA1*05:01 – DQB1*02:01 (DQ2 / DQ2.5), a minority of cases are associated with haplotype: DQA1*03:01 – DQB1*03:02 (DQ8) and a small percentage are associated with haplotype: DQA1*02:01 – DQB1*02:02 (DQ2.2). If a patient is positive for any of the above, CD cannot be excluded but if they are negative it is highly unlikely they have CD i.e. a negative finding has a high negative predictive value (excludes CD). However, the diagnostic value of HLA phenotyping in CD is limited by the fact that HLA-DQ2 and/or HLA-DQ8 are present in ~40% of healthy caucasians so that a positive finding does not confirm CD (referred test).

    Gastric parietal cell antibody (GPC) Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days GPC is done as part of the autoantibody screen. It is present in 95% of patients with pernicious anaemia but also occurs 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; NB! If patient is on gluten-free diet test may be negative in presence of Coeliac Disease; turn-around time approx. 20 working days Screening test for coeliac disease (CD) and dermatitis herpetiformis (DH) but is now largely replaced by anti-endomysial antibodies (EMA) and anti tissue transglutaminase (tTG) antibodies as they are more specific (see above). However, in young children (

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    associated with gluten induced cerebellar ataxia for which EMA 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 approx. 10 working days 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 Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days Present in SLE but if found without dsDNA antibodies, characteristic of drug-induced lupus (referred test).

    Liver kidney microsomal antibody (LKM) Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days 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 Serum sample required; no special preparations needed prior to sampling. Turn-around time 5-10 working days 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 working days 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).

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    All AMA positive samples will be automatically assessed for M2 confirmation (AMA and M2 are in-house tests).

    MuSK antibody Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days May be positive in AChR seronegative patients with myasthenia gravis (referred test).

    Myeloperoxidase antibody (MPO) Serum sample required; no special preparations needed prior to sampling; turn-around time 5-10 working days 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: 0.2 – 134 IU/mL; Reference range: Negative: < 3.5 IU/mL Equivocal: 3.5-5 IU/mL Positive: >5 IU/mL

    (Anti) Nuclear antibody (ANA) or anti-nuclear factor (ANF) Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days 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. Titers 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 and neuromyelitis optica antibodies Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days 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, MAG, aquaporin 4 etc). NMDAR antibodies are neuronal surface receptor antibodies which can cause severe but treatable immune-mediated encephalitis (viral prodrome to neuropsychiatric symptoms) (Titulaer et al, Lancet Neurol 2013). NMDAR antibody was first found in young female patients with ovarian tumours and prominent psychiatric symptoms, amnesia, seizures, dyskinesia’s, autonomic dysfunction and decreased level of consciousness (Delmau J et al. Lanet Neurol 2011). The antibodies are now known to be found in patients with behavioural and cognitive problems and seizures, in both males and females with no tumour and in children. Symptoms can progress over time to a movement

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    disorder, autonomic fluctuations and coma. Antibodies (in both serum and CSF) can be low or sub-threshold in first samples but rise subsequently. Sending further serum and CSF samples if clinical suspicion is high. The NMDAR assay will routinely be carried out on fixed cells (“fixed NMDAR ab”) and has lower sensitivity than the “live NMDARab” assay. Where clinical suspicion is high, ring Oxford University Hospitals, Pathology Laboratory on 01865 225995 to request the live cell assay. Other neuronal antibodies such as Ribosomal P antibody are found in autoimmune diseases with CNS involvement (CNS Lupus). Aquaporin 4 (AQP4) is a major autoantigen in neuromyelitis optica (NMO or Devic’s disease). Antibodies to AQP4 are found in >80% of NMO patients and ~ 50% of patients with longitudinal extensive transverse myelitis. They are infrequent in patients with optic neuritis without spinal cord involvement (reviewed in Jarius & Wildemann, Nat Rev Neurol 2010). Patients with AQP4 antibodies are at risk of relapsing and immunosuppressive treatment is recommended before waiting for further events. If further guidance is needed, contact National NMO Centres (Oxford: Jacqueline.palace@neuro.ox.ac.uk or maria.leite@neuro.ox.ac.uk; Liverpool: anu.jacob@thewaltoncentre.nhs.uk or www.nmouk.nhs.uk) (referred tests).

    (Anti) Neutrophil cytoplasmic antibody (ANCA) Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days Indicated in the investigation of vasculitis. ANCA titers 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 (in-house 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.

    Neutrophil autoantibodies NB! This test requires live cells which must be assessed within

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    reported in 70% to 80% of MS patients. A finding of 4 or more cerebrospinal fluid (CSF)-specific bands (ie, bands that are present in CSF but are absent in serum) is consistent with multiple sclerosis (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 approx. 20 working days 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. NB! VGKC antibodies have been renamed VGKC-complex antibodies as it is now recognised that the VGKCs are tightly complexed with other proteins and that the radioimmunoprecipitation assay recognises these complex proteins as well as the VGKCs themselves. The most common complex proteins discovered so far are LGI1 and CASPR2. CASPR2 (Contactin-2) antibodies are found in Morvan’s Sy (Irani et al Ann Neurol 2012) and cerebellar ataxia. Glycine receptor antibodies are found in siff persons sy (usually GAD negative) and progressive encephalomyelitis. LGI1 (leucin-rich glioma inactivated 1) is one of the VGKC complex antibodies, found in limbic encephalitis with low plasma Na+, often associated with faciobrachia dystonia type seizures. However, as not all the complex proteins have been identified, we recommend testing for VGKC-complex antibodies as a first step (advised by Oxford Radcliffe Hospitals Pathology Laboratory). See also website: http://www.ouh.nhs.uk/immunology/documents/EquivocalVGKC.pdf. (referred test).

    Parathyroid antibodies See endocrine gland antibodies

    (Anti) Phospholipid antibody Haematology test – send to Haematology 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 thrombocytopoenia. Patients with APS must always be checked for lupus anticoagulant (LA) (APS is an in-house test done in the Heamatology Laboratory together with Lupus Anticoagulant and clotting).

    http://www.ouh.nhs.uk/immunology/documents/EquivocalVGKC.pdf

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    Proteinase 3 antibody (PR3) Serum sample required; no special preparations needed prior to sampling; turn-around time 5-10 working days 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: 0.2 – 177 IU/mL; Reference range: Negative: < 2 IU/mL Equivocal: 2 – 3 IU/mL Positive: >3 IU/mL.

    Rheumatoid Factor (RF) Serum sample required; no special preparations needed prior to sampling; turn-around time 1-3 working days 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 non-specific and low levels are often found in other connective tissue/autoimmune diseases, infections and malignancies. Semi-quantitative measurements of RF titers have been replaced by quantitative measurements expressed in international units (IU/mL); although the negative cut-off level is 20 IU/mL, values 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 approx. 20 working days May be found in Sjogrens syndrome (see Ro/La antibodies) (referred test).

    Skeletal / striated muscle antibody Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days 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).

    Skin reactive antibodies Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days 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).

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    Smooth muscle antibody (SMA) Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days SMA is part of the autoantibody screen. Higher tit are found in patients with chronic active hepatitis both viral and autoimmune. Lower titers can also be seen in PBC or infections. 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 liver-pancreas antibody (LP). Type 2 is usually seen in children and has a more severe course (In-house test).

    Soluble Liver antigen (SLA) Serum sample required; no special preparations needed prior to sampling; turn-around time 5-10 working days 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).

    Testicular antibodies See endocrine gland antibodies

    Thyroid antibody Biochemistry test – send to Biochemistry 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 (in-house test which is done in the Biochemistry Laboratory as part of a “thyroid screen”).

    Tissue Transglutaminase (tTG) (see also Endomysial antibody (EMA), Coeliac Disease (CD), gliadin test and Dermatitis Herpetiformis (DH)) Serum sample required; NB! If patient is on gluten-free diet test may be negative in presence of Coeliac Disease; turn-around time 3-5 working days This test has the same indications as endomysial antibody (EMA) testing but is less specific albeit more sensitive. It is therefore used as a screening test which is confirmed by EMA testing if positive. It is also done in patients with a clinical presentation very suggestive of coeliac disease but negative EMA, 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).

    HLA-DQ2 & HLA-DQ8 typing for CD: EDTA sample required; not affected by gluten in diet; turn-around-time 2-3 week. This test should only be requested in special circumstances. CD patients will have one of these haplotypes in >95% of cases. The majority of cases are associated with haplotype: DQA1*05:01 – DQB1*02:01 (DQ2 / DQ2.5), a minority of cases are associated with haplotype: DQA1*03:01 – DQB1*03:02

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    (DQ8) and a small percentage are associated with haplotype: DQA1*02:01 – DQB1*02:02 (DQ2.2). If a patient is positive for any of the above, CD cannot be excluded but if they are negative it is highly unlikely they have CD i.e. a negative finding has a high negative predictive value (excludes CD). However, the diagnostic value of HLA phenotyping in CD is limited by the fact that HLA-DQ2 and/or HLA-DQ8 are present in ~40% of healthy caucasians so that a positive finding does not confirm CD (referred test).

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    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 and C4 = 1-3 days o Immunoglobulins = 1-3 days o Serum electrophoresis (SEP) = 3-5 days o Immunofixation = 5-10 days o Beta-2-microglobulin (B2M) = 1-3 days o other complement tests = 10-15 days o cryoglobulins = 8-10 days o Serum Free Light Chains (SFLC) = 5-10 days

    Complement

    C1q Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days Anti-C1q in SLE are associated with renal involvement. Monitoring anti-C1q and their titres in SLE patients could be important for predicting renal flares (referred test).

    C3 and C4 Serum sample required; no special preparations needed prior to sampling; turn-around time 1-3 working days 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 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 EDTA tube; no special preparations needed prior to sampling; turn-around time approx. 20 working days 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).

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    Classical haemolytic (CH100) & alternative haemolytic (APCH100) pathway activity 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 Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days 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 approx. 20 working days 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 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 Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days Measures C1 esterase inhibitor activity. Approximately 15% of patients with hereditary angiodema have normal levels of a non-functional protein. Both types of hereditary angiodema 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) 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 Serum sample required; no special preparations needed prior to sampling; turn-around time approx. 20 working days NB! This test is rarely required and must be previously discussed and arranged with the Laboratory; C3nef is an autoantibody associated with membrano-proliferative glomerulonephritis (type II) and partial lipodystrophy (see complement section) (referred test).

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    Serum proteins / Immunochemistry (alphabetical order)

    Alpha-1-anti-trypsin (A1AT) Serum sample required; no special preparations needed prior to sampling; turn-around time 1-3 working days 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) (Biochemistry referred test).

    Bence-Jones proteins See urinary free light chains

    Beta 2 microglobulin (2M) Serum sample required; no special preparations needed prior to sampling; turn-around time 1-3 working days 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

    oC); contact Laboratory to

    arrange collection (x32635); turn-around time 8-10 working days 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-titer 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

    oC); contact

    Laboratory to arrange collection (x32635). Cryoglobulins must always be assessed in parallel (see above); turn-around time 8-10 working days Indications are similar to cryoglobulins. Cryofibrinogen is of less clinical importance but may be mistaken for cryoglobulins (in-house test).

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    Immunofixation Serum sample required; no special preparations needed prior to sampling; turnaround time 5-10 working days Performed as a follow-on (reflex) test to serum protein electrophoresis if monoclonal protein is detected. This technique is used to type paraprotein heavy and light chains detected by serum protein electrophoresis (SPE) (in-house test).

    Immunoglobulins (IgG, IgA, IgM) Serum sample required; no special preparations needed prior to sampling; turn-around time 1-3 working days 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 approx. 15 working days 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 approx. 20 working days This protein is involved in opsonisation and complement activation. Severe deficiency may contribute toward susceptibility to infections (referred test).

    Myeloma screen For new myeloma, this request includes serum protein electrophoresis (SPE), immunoglobulins, immunofixation and urinary BJP. SFLC will be done only if specified (e.g. for light chain myeloma, amyloidosis, complete remission etc). See also “Paraproteins” below.

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    Paraproteins / paraprotein quantitation (PPQ) Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days 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 (in-house 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 3-5 working days 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 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 test – send to microbiology 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 requestings 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) Serum sample required; no special preparations needed prior to sampling; turn-around time 5-10 working days This test is now available as an in-house test and has replaced urinary 24h free light chain (Bence-Jones) quantitation. SFLC kappa (κ) and lambda (λ) will be done only if requested. New guidelines (2009) advise SFLC assessment for all new myeloma, follow-up of free-

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    light chain myeloma, plasmocytoma, complete remission and amyloidosis patients. Please note that the normal ranges for κ/λ ratio are affected by kidney failure: Normal kidney function Kidney failure κ/λ Ratio 0.26-1.65 0.37-3.10 (in-house 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 3-10 working days 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 (in-house 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).

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    Allergy testing

    Total serum IgE Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days 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) Serum sample required; no special preparations needed prior to sampling; turn-around time 3-5 working days unless referred approx. 15 working days (unusual allergens) 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). Repeat testing is rarely required in less than 6 months. For best results, clinical details must accompany RAST requests. Results interpretation: 0.70-49 kU(A)/L: confirms presence of specific IgE

    50-100 kU(A)/L: strongly suggestive of allergy.

    Allergen components testing will be carried out where available and indicated. Plant protein families are shared between species; the closer the species are related the more similar the proteins can be. This increases the potential for IgE antibodies directed against pollen allergen epitopes to bind to similar allergen epitopes in food. There are five main types of allergen groups indicated. These are storage proteins, LTP, PR-10, profilin proteins and CCDs (cross-reactive carbohydrate determinants). Storage & lipid transfer (LTP) proteins are heat stable (are not destroyed by cooking) and often cause systemic reactions. PR-10 proteins & prolifins are heat unstable (are destroyed by cooking) and mainly cause local reactions. Currently available components are: Peanut Ara h 1 – risk of severe reactions (storage protein) Ara h 2 - risk of severe reactions (storage protein) Ara h 3 - risk of severe reactions (storage protein) Ara h 9 – risk of both severe and local reactions (Oral Allergy Syndrome – OAS) (Lipid Transfer Protein - LTP); risk of cross reactivity to pollen and foods

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    Ara h 8 - risk of reactions to peanut due to cross reactivity to pollen and foods (birch pollen Bet v 1 , Bet v 2 and soy protein Glym 4)(PR-10 protein) Hazelnut Cor a 1 – risk of local reactions (OAS) (PR-10 protein) Cor a 8 - risk of both severe and local reactions (OAS) (LTP) Cor a 9 - risk of severe reactions (storage protein) Cor a 14 - risk of severe reactions (storage protein) Egg white Gal d 1 (ovomucoid) - risk of severe reaction to both cooked and raw egg Wheat Tri a 19 (omega-5-gliadin) – risk of WDEIA (wheat dependent exercise induced anaphylaxis) Bee rApi m 1+10 Common wasp rVes v1 rVes v5 rPol v5 For further information see website: http://allergyeducation-ma.com/

    Tryptase Serum sample required (EDTA can also be used if necessary); no special preparations needed prior to sampling; turn-around time approx. 15 working days NB! Samples must be taken 50ug/l) (referred test).

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    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

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    previously arranged following consultation with Consultant Immunologist (referred test; send sample to Immunology).

    CD40-ligand expression NB! This test requires live cells which must be assessed within

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    Disease Index: A to C

    Disease Investigations

    Addison’s Disease Anti-endocrine antibodies (adrenal)

    Allergy IgE RAST (suspected allergens & components)

    Anaphylaxis Tryptase

    Angioedema / urticaria (acute)

    IgE RAST (suspected allergens) Autoantibody screen C3/C4 Thyroid screen (Biochemistry) FBC/ESR(Haematology)

    Anti-Phospholipid Syndrome (APS)

    Autoantibody screen Anti-phospholipid Ab (Haematology) Anti-cardiolipin Ab (Haematology) Lupus Anticoagulant (Haematology)

    Bullous skin disorders / Dermatitis Herpetiformis (DH) Anti-endomysial Ab (if ?DH) Anti-gliadin Ab (if ?DH) Skin reactive Ab

    Bone density screen

    FBC + ESR (Haematology) U&E, LFT, TFT (Biochemistry) Bone screen (including Ca+) (Biochemistry) Testosterone (in men) (Biochemistry) IGs + paraproteins Urine Bence Jones proteins

    C1 Esterase Inhibitor Deficiency C1 esterase inhibitor C3/C4

    Cerebellar Ataxia Anti-gliadin Ab

    Chronic Active Hepatitis

    Autoantibody screen Anti-smooth muscle Ab Anti-mitochondrial Ab Anti-nuclear Ab

    Chronic Granulomatous Disease Neutrophil function test

    Coeliac Disease

    Anti-tissue trangslutaminase Ab Anti-endomysial Ab Anti-gliadin Ab for

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    Disease Index: D to H

    Disease Investigations

    Deep Venous Thrombosis Autoantibody screen Anti-cardiolipin / phospholipid Ab Lupus Anticoagulant

    Dermatitis Herpetiformis Anti-tissue transglutamuinase Ab Anti-endomysial Ab

    Dermatomyositis

    Autoantibody screen Ant-Jo-1 Anti-PL7 Anti-PL12

    Diabetes (recent onset) Islet cell Ab (ICA) Insulin antibodies (IAA) Glutamic acid decarboxylase Ab (GAD)

    DLE Autoantibody screen Anti-nuclear Ab

    Dressler’s Syndrome Anti-cardiac muscle Ab

    Endocrinopathies - autoimmune Anti-adrenal, parathyroid, gonadal, insulin, pancreatic etc autoantibodies

    Fibrosing Alveolitis Autoantibody screen Anti-nuclear Ab

    Glomerulonephritis ANCA (MPO / PR3) Anti-glomerular basement membrane Ab

    Goodpasture’s Syndrome Anti-glomerular basement membrane Ab

    Graves Disease Anti-thyroid peroxidase (TPO) Ab (Biochemistry)

    Guillain-Barre Syndrome Anti-neuronal Ab

    Hashimotos Thyroiditis Anti-thyroid peroxidase (TPO) Ab (Biochemistry C3/C4 C1 esterase inhibitor

    Hereditary angioedema (HAE) C3/C4 C1 esterase inhibitor

    Disease Index: I to N

    Disease Investigations

    Infection Immunoglobulins Serum protein electrophoresis

    Immune Deficiency Screen

    Immunoglobulins Serum protein electrophoresis Specific antibody responses IgG subclasses C3/C4

    Juvenile Chronic Arthritis Autoantibody screen Anti-nuclear Ab

    Lymphoproliferative disorders Immunoglobulins Serum protein electrophoresis

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    Membrano-Proliferative Glomerulonephritis (MPGN) C3 nephritic factor C3/C4

    Microscopic Polyangitis ANCA (MPO / PR3)

    Mixed Connective Tissue Disease (MCTD) Autoantibody screen Anti-ENA Ab Anti-nuclear Ab

    Monoclonal Gammopathy Uncertain Significance (MGUS)

    Serum protein electrophoresis Immunofixation (reflex tested) Urinary Bence-Jones protein

    -Microglobulin

    Myasthenia Gravis Anti-acetylcholine receptor Ab

    Myeloma / Paraprotein screen

    Serum protein electrophoresis Immunoglobulins Immunofixation (reflex tested) Paraprotein quantitation (PPQ) UrinaryBence-Jones proteins (UFLC) Serum free light chains (SFLC)

    -Microglobulin

    Nephritic Syndrome (acute)

    Serum protein electrophoresis Immunoglobulins Autoantibody screen ANCA (MPO / PR3) Anti-glomerular basement membrane Ab C3/ C4

    Non-Hodgkin's Lymphoma Serum protein electrophoresis Urinary Bence-Jones protein

    Disease Index: N to R

    Disease Investigations

    Nephritic Syndrome (acute)

    Serum protein electrophoresis Immunoglobulins Autoantibody screen ANCA (MPO / PR3) Anti-glomerular basement membrane Ab C3/ C4

    Neurological – autoimmune diseases GM1, GQ1, MAG, NMDA, aquaporin4 etc

    Non-Hodgkin's Lymphoma Serum protein electrophoresis Urinary Bence-Jones protein

    Paraprotein / myeloma screen

    Serum protein electrophoresis Immunoglobulins Immunofixation (reflex tested) Paraprotein quantitation (PPQ) UrinaryBence-Jones proteins (UFLC) Serum free light chains (SFLC)

    Partial Lipodystrophy C3 nephritic factor

    Pernicious Anaemia Anti-gastric parietal cell Ab

    Pemphigus Skin-reactive Ab

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    Pemphigoid Skin-reactive Ab

    Polymyositis Anti-Jo-1 Anti-PL12

    Premature Ovarian Failure Anti-endocrine Ab

    Primary Biliary Cirrhosis Anti-mitochondrial Ab

    Primary Immune deficiencies Lymphocyte subsets and functions, Immunoglobulins, complement, MBL, anti-GM-CSF etc

    Progressive System Sclerosis Autoantibody screen ENA (Anti-Scl 70)

    Raynaud’s Autoantibody screen

    Rheumatoid Arthritis Autoantibody screen C3d C3/C4

    Disease Index: S to Z

    Disease Investigations

    Sjogren’s Syndrome Autoantibody screen ENA (Anti-Ro / La) Anti-nuclear Ab

    Systemic Lupus Erythematosus

    Autoantibody screen Anti-dsDNA Ab Anti-ENA Ab C3d C3/C4 Anti-phospholipid Ab (Haematology) Anti-cardiolipin Ab (Haematology) Lupus Anticoagulant (Haematology)

    Vasculitis screen

    Autoantibody screen ANCA (MPO / PR3) C3/C4 Serum protein electrophoresis Immunoglobulins CRP (Biochemistry)

    Waldenstrom’s Macroglobulinaemia

    Serum protein electrophoresis Immunoglobulins Immunofixation (reflex tested) Paraprotein quantitation (PPQ) Bence-Jones proteins (UFLC) Serum free light chains (SFLC)

    microglobulin Cryoglobulins

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    INFORMATION SHEET ON PARAPROTEIN BANDS

    GUIDELINES FOR MONITORING

    Paraprotein bands are a relatively common incidental finding on routine biochemical tests and represent a clonal expansion of plasma cells. Although this is the pathological process in the development of multiple myeloma, a large number of patients with a paraprotein band do not have multiple myeloma but instead have a monoclonal gammopathy of undetermined significance (MGUS) for which they do not require any treatment.

    Features that suggest a paraprotein band may be associated with multiple myeloma include:

    IgG paraprotein of >30g/L; IgA paraprotein of >20g/L; IgM paraprotein of >10g/L

    The presence of immunoparesis (reduced levels of normal immunoglobulin).

    Presence of Bence Jones protein in early morning urine specimen.

    Anaemia.

    Renal impairment.

    Hypercalcaemia.

    Progressive increase in the size of the paraprotein band.

    Bone pain or pathological fracture.

    If none of the above features are present the paraprotein band is likely to be an MGUS. A small percentage of patients with MGUS on long-term follow-up will go on to develop multiple myeloma or other plasma cell malignancy (10% on eight years follow-up and 25% on 20-25 year follow-up) and it is advisable for such patients to have a full blood count and serum protein electrophoresis (SPE) checked every six months.

    A rising paraprotein level or the development of any of the above features would raise the possibility of MGUS progressing to multiple myeloma and such cases should be referred to the Haematology Department for further investigation. Patients with MGUS do not necessarily need to be seen in the Haematology Department provided they have a full blood count and SPE checked every six months.

    A polyclonal increase in globulins (raised total globulins with no paraprotein band) is not indicative of multiple myeloma and is of similar significance as a raised ESR. Such patients should be investigated for the usual infective, inflammatory and malignant conditions which may be associated with a raised ESR.

    Other conditions can occasionally be associated with paraprotein bands such as lymphoma, primary AL amyloidosis or neuropathy and the possibility of such an association should be considered in these patients.

    - Guidelines were composed by Dr Iqbal and Dr Keenan, Consultant Haematologists, UHND - For any queries regarding assays contactlilinsultant Immunologist, UHND

    Reviewed January 2017 (DL)