USER GUIDE. Department of IMMUNOLOGY Directorate of Laboratory Medicine. 1 of 119

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1 USER GUIDE 1 of 119

2 Index About Us 3 Location and working hours 3 Quality Statement 3 Services Available 4 Accreditation 5 Quality Assurance 6 Scope of Service 7 How to Find Us 8 Staff Contact Details 9 Clinical conditions covered by the Immunology Department 11 Allergy / Hypersensitivity 13 Organ Specific Autoimmunity 17 Obstetrics and Gynaecology 20 Infection and Immunity 21 Malignancy 24 Neurology 26 Renal Disease 28 Rheumatology/Connective Tissue Disease Tests 31 Vasculitis 34 A-Z of clinical conditions and Immunological Tests 36 A-Z of Tests 39 Information for Patients of 119

3 About Us The laboratory offers a comprehensive test repertoire for the immunological investigation of patients. The four main areas covered include: Allergy, Autoimmunity, Cellular Immunology and Immunochemistry. We aim to provide a user-responsive service with rapid turn around of accurate results along with interpretive comments. Requests that are not covered by our service will be referred to other accredited specialist immunology laboratories. Location and working hours The laboratory is situated in the Manchester Royal Infirmary, within the Clinical Science Buildings, and is open from Monday Friday 08:30 to 17:00. It is closed on Bank Holidays. There is no out-of-hours service. Quality Statement The Immunological investigations performed within the CPA accredited laboratory are recognised standards of practice. Documentation relating to internal quality control and quality assurance are retained for scrutiny by users of the Immunology Service. 3 of 119

4 Services Available The Immunology Department aims to provide interpretative comments that are added to report forms. Referral for full clinical assessment is welcomed and is preferred for patients with suspected immune deficiency and those who are thought to have suffered an anaphylactic reaction. The laboratory is accredited with CPA and we participate in appropriate national quality assurance schemes. If you have any comments on the services that we provide please contact us by phone, letter or e- mail. Areas of clinical interest include: Allergy, Autoimmune Disorders and suspected Immune Deficiency. The Clinical Immunology staff provide a variety of specialist outpatient clinics as follows: Day/Frequency Speciality Clinical Support Provided Monday am Primary Immunodeficiency Dr Matthew Helbert Consultant Weekly Clinical Immunologist Monday am Weekly Tuesday am Fortnightly Tuesday am Weekly Wednesday pm Fortnightly Wednesday pm Weekly Thursday am Weekly Thursday pm Weekly Friday am - approx every 4 weeks Nurse Allergy Clinic Anaesthetic Clinic Paediatric Clinic Primary Immunodeficiency Follow-up Allergy Clinic Allergy Clinic, including Desensitisation Allergy Clinic including Latex Allergy Challenge day Day Case admissions Sister Janet Hanrahan, Immunology Specialist Nurse Dr Matthew Helbert, Consultant Clinical Immunologist Dr Nigel Harper, Consultant Anaesthetist Dr Peter Arkwright, Consultant Paediatric Immunologist Dr Steven Hughes, Consultant Paediatric Immunologist Dr Matthew Helbert Consultant Clinical Immunologist Sister Alex Farragher, Immunology Specialist Nurse Dr Matthew Helbert, Consultant Clinical Immunologist Sister Alex Farragher, Immunology Specialist Nurse Sister Alex Farragher, Immunology Specialist Nurse Dr Matthew Helbert Consultant Clinical Immunologist Sister Alex Farragher, Immunology Specialist Nurse Sister Janet Hanrahan, Immunology Specialist Nurse 4 of 119

5 Accreditation The Immunology Department is accredited by Clinical Pathology Accreditation (CPA), Health Professions Council (HPC) and the Conference of Post Graduate Medical Deaneries (COPMeD). CPA Accredited Medical Laboratory Reference No: of 119

6 Quality Assurance The laboratory participates in all relevant quality assurance schemes and aims to maintain standards in all aspects of its work; however, problems will occasionally occur. Any complaints should be directed to the Clinical or Laboratory Managers please make any reservations you may have about the quality of any aspect of the service known to us as soon as possible: we take your complaints very seriously. In particular, let us know of any untoward delay in receipt of reports, any discrepancies between results and clinical picture, and any errors in patient or clinician name on the report where samples have been referred to other laboratories for testing, this will be indicated on the report form. Please also let us know about new services you would wish to see developed. 6 of 119

7 Scope of Service The range of immunology tests offered, together with the specimens required, are described in the A-Z of tests. A brief summary of the clinical applications of each test is provided this is intended only as a guide. Additional background support is provided by links to clinical areas. Specimen Acceptance Policy The has a Specimen Acceptance Policy. A copy of the Directorate Specimen Acceptance policy is included in the DLM handbook obtained from the Client Services Office, CSB1 Request form The request form should contain a unique identifier, in addition to the minimum acceptance criteria listed below, so that the results of investigations can be accurately filed in the laboratory computer patient record. The request form data must match the three pieces of information that are on the samples surname, forename or initial, date of birth or record number. Where there are multiple samples, taken at different times, the request form should include when with the times recorded. The times should also match the times written on the tubes. Each request form should also contain: NHS or Hospital Number Address for the report Consultant or GP Name of requestor Test(s) required Date and time of sample when the time is important and when there are multiple samples Sex Contact number for requestor Relevant clinical information If the above items are omitted, then it may not be possible to issue a report or to interpret results. In addition, the laboratory may not conduct some analyses if this information is not given. Appropriate comments will be made on the report. Additional Examinations The time limit for requesting additional examinations is restricted to two weeks. This restriction excludes requests for the unmasking of ENA and DNA results following a 7 of 119

8 positive ANA result. The unmasking of ANA s is restricted to 28 days from the date of the original assay. How to Find Us The Immunology Department is located in the Manchester Royal Infirmary and is part of the Central Manchester University Hospitals NHS Foundation Trust (CMFT). The laboratory is situated within the Clinical Sciences Building III, and is open from Monday Friday 08:30 to 17:00. It is closed on Bank Holidays. For a full map of the hospital site please click on this link: or if you have a paper copy type the following into your browser. de%20aug.09.pdf The full postal address is: Department of Immunology Clinical Sciences Building Manchester Royal Infirmary Oxford Road Manchester M13 9WL 8 of 119

9 Consultant and Management Staff Contact Details Title: Name: Clinical Manager and Consultant Immunologist Dr Matthew Helbert Tel: CLINICAL INTERPRETATION Title: Laboratory Manager Name: Mr. Colin Dennett Tel: / MANAGERIAL INTERPRETATION Senior Staff Title: Chief Biomedical Scientist Name: Philippa Coles Tel: TECHNICAL Title: Chief Biomedical Scientist Name: John Hewitt Tel: TECHNICAL Title: Chief Biomedical Scientist Name: Andrew Moran Tel: TECHNICAL 9 of 119

10 Immunology Office Title: Name: Administration Manager Angela Bedford Tel: ADMINISTRATION Immunology Clinical Support Title: Name: Immunology Specialist Nurse Alex Farragher Tel: NURSING ADVICE Central Sample Reception Title: Name: Central Specimen Reception Manager Mary Hynes Tel: OPERATIONAL GUIDANCE Title: Name: Client Services Manager Colette McAlister Tel: OPERATIONAL GUIDANCE Results Hotline Tel: Other Contacts: Clinic Secretaries: /5653 Departmental Fax: of 119

11 Below is a list of clinical conditions covered by the Immunology department. In addition to this an A-Z list Immunological tests available is detailed below. Medical Category Clinical Conditions Covered Please click on a Medical Category for further information Allergy/Hypersensitivity Other Hypersensitivity Organ Specific Autoimmunity Obstetrics and Gynaecology Infection and Immunity Malignancy Neurology Anaphylaxis, Asthma, Atopic Eczema Bronchopulmonary eosinophilia Conjunctivitis Food Allergy and Intolerance Rhinitis Urticaria Bronchopulmonary aspergillosis, Aspergilloma Hypersensitivity pneumonitis Bird fancier s disease Farmer s lung Coeliac disease Adrenal failure Chronic active hepatitis Dermatitis herpetiformis, Dressler s Syndrome Gonadal failure Liver autoimmunity Pemphigus/Pemphigoid, Polyendocrine autoimmunity Primary biliary cirrhosis, Primary sclerosing cholangitis Thyroid disease Recurrent fetal loss Gonadal failure Screening for exposure to tuberculosis HIV Screening for primary immunodeficiency Secondary cases of immunodeficiency Acquired C1-inhibitor deficiency Hereditary angioedema Lymphoproliferative disorders: Acute and Chronic Leukaemias, Lymphoma Motor neuropathy, Myasthenia, Paraneoplastic syndromes Renal Disease Goodpasture s Syndrome IgA nephropathies 11 of 119

12 Membranoproliferative glomerulonephritis Necrotising crescentic glomerulonephritis, Nephrotic syndrome Rheumatology Vasculitis Polymyositis/Dermatomyositis Primary anti-phospholipid antibody syndrome Scleroderma/Systemic Sclerosis Screening Sjögren s syndrome, Systemic lupus Erythematosus Churg-Strauss Henoch-Schönlein Purpura Kawasaki s syndrome Microscopic polyarteritis Primary systemic vasculitides Small vessel (hypersensitivity) vasculitis Wegener s 12 of 119

13 Allergy / Hypersensitivity Brief Overview Requests for RAST are not acceptable. Requests should be for specific allergens as indicated by the history. Allergy tests may help identify which allergens suggested by the history could cause symptoms. However, the finding of allergy specific IgE in the serum does not mean that the allergen is responsible for the symptoms under investigation, nor does it necessarily indicate that avoidance measures will help the patient. Specific IgE provides similar, although not identical, information to skin testing but is particularly valuable in assessing some groups of patients (young children, eczema/dermographism, taking antihistamines, past history of anaphylaxis), is needed to interpret the significance of the specific IgE. For each clinical condition, the relevant immunological tests are listed in blue together with a short explanation of their use. More detail, including the required is available following the A-Z of tests. Patient information is available at: National Asthma Campaign: British Lung Foundation: National Eczema Society: Anaphylaxis Campaign: Conjunctivitis Allergen Specific IgE (limited association) When the allergic reaction is strictly limited to the conjunctiva allergy tests are frequently negative. When conjunctivitis is part of a more generalised allergy, specific IgE and skin prick tests are usually positive for the causative allergen. Rhinitis IgE and Allergen Specific IgE Allergy tests may help distinguish allergic from vasomotor or other causes of rhinitis. Total IgE is often in the normal range or slightly elevated. Specific IgE may be sought to inhalant allergens. While the range of allergens tested should be sensibly guided by a careful history this should generally include allergens to which most 13 of 119

14 people are exposed, such as cat and house dust mite (HDM). Investigation of seasonal rhinitis is only indicated if there is some doubt about the diagnosis or if desensitisation is being considered. The pollens involved in seasonal rhinitis or asthma are as follows: grasses (May-Sept), trees (March-May) and weeds (July- September). There is little point in finding the exact pollen allergen unless it is intended to give the patient immunotherapy when skin testing is mandatory. Asthma IgE and Allergen Specific IgE Total IgE is usually raised in extrinsic asthma; specific IgE to relevant allergens is also detectable. Specific IgE should invariably be sought against the house dust mite. Often this will suggest the appropriate animals (cats, dogs, horses etc). IgE to Aspergillus is associated with the need for closer monitoring and maybe more intensive steroid treatment. For seasonal asthma see seasonal rhinitis. Atopic Eczema IgE and Allergen Specific IgE Total IgE is often markedly elevated in widespread disease and Specific IgE may be present at high level to allergens that cause no overt symptoms. Any positive Specific IgE results therefore need careful interpretation. Specific IgE to house dust mite is often high and house dust mite allergy may exacerbate eczema in such patients. Anaphylaxis Mast cell tryptase and IgE and Allergen Specific IgE Please refer all patients experiencing anaphylaxis to the allergy clinic for assessment. Blood samples for mast cell tryptase taken within 1-2 hours of reaction can help indicate if the reaction was anaphylactic. Anaphylaxis and anaphylactoid reactions to anaesthetics etc. Patients will be invited to attend the special clinic run jointly by Immunology and the Department of Anaesthesia at which they will be assessed. Acute Urticaria IgE and Allergen Specific IgE 14 of 119

15 Total IgE and specific IgE may help identify the causal antigen involved in type I hypersensitivity. Bronchopulmonary eosinophilia Allergen Specific IgE (to Aspergillus) Aspergillus fumigatus precipitins IgE ANCA (See vasculitic section) Total IgE, specific IgE to Aspergillus, and Aspergillus precipitins (IgG) should identify cases due to hypersensitivity to the fungus. Total IgE may be raised in association with parasitic infestation. A positive ANCA may point to a vasculitic cause (Churg-Strauss). Food Intolerance IgE and Allergen Specific IgE Evidence of specific IgE antibodies may be consistent with a diagnosis of food allergy but, unfortunately, the presence of such antibodies does not prove clinical sensitivity. Elimination and food challenge testing may help confirm or rule out food intolerances. Laboratory immunology tests cannot help investigate non-allergic food intolerance. Measurement of total IgE will permit the ratio of specific to total IgE to be assessed in positive samples. Other Hypersensitivity Bronchopulmonary aspergillosis, Aspergilloma and Hypersensitivity pneumonitis Aspergillus precipitins In bronchopulmonary aspergillosis and aspergilloma a mixture of IgE and IgG antibodies are usually present. Bronchopulmonary aspergillosis can be caused by species other than A. fumigatus. In allergic rhinitis and asthma, where aspergillus is the relevant allergen, IgE antibodies dominate. In hypersensitivity pneumonitis, IgG antibodies are usually present alone. IgG antibodies, without IgE antibodies can also be seen in some cases of sub acute invasive aspergillosis. IgG antibodies are infrequent in healthy individuals. Aspergillus antibodies are not a recommended test for the investigation of the majority of cases of invasive aspergillosis in immunocompromised hosts. 15 of 119

16 Farmer s Lung Farmer s lung precipitins (thermophilic actinomyces) Hypersensitivity to Micropolysporium faeni is a common cause of farmer s lung hypersensitivity pneumonitis occurring 12 to 24 hours after exposure to mouldy hay. In some cases of farmer s lung, hypersensitivity to other fungal species implicated. The absence of precipitin (IgG) to Micropolysporium faeni does not rule the diagnosis out. These antibodies can be found in some healthy individuals: The presence of these antibodies supports, but does not confirm, a diagnosis of farmer s lung. There are numerous other causes of hypersensitivity pneumonitis including fungal spores and occupational antigens. Please discuss with the laboratory if these may be incriminated. The diagnosis is made by a combination of clinical features, X-ray and lung function tests. Bird fanciers Disease Avian Precipitins The symptoms are similar to farmer s lung but more commonly are of the chronic type. Precipitins (IgG) to avian proteins provide good evidence of the cause of the symptoms. Coeliac Disease (Gluten sensitive enteropathy GSE) Patient information is available at: Coeliac Society: Ab to tissue transglutaminase Immunoglobulin levels IgG, IgA, IgM (IgA may be required) IgA antibodies to tissue transglutaminase are found in active coeliac disease, and can be used to monitor compliance with treatment. Similar antibodies are seen in dermatitis herpetiformis. We may have to measure IgA to ensure that IgA deficiency (particularly common in these patients) is not causing a false negative result. IgG antibodies to tissue transglutaminase are utilized in IgA deficient patients. 16 of 119

17 Organ Specific Autoimmunity Brief Overview Requests for autoimmune screen are not acceptable. Requests should be for specific autoantibodies as indicated by the history. For each clinical condition, the relevant immunological tests are listed together with a short explanation of their use. More detail, including the required are available below and at the end of the guide. Tests indicated in black are not routinely available or are offered by other laboratories. Patient information is also available at the following web address: Thyroid goitre/nodule, hypo/hyperthyroidism Ab to thyroid peroxidase (TPO) The levels of antibodies to thyroid peroxidase are closely related to the degree of lymphocytic infiltration in the thyroid. They are raised in autoimmune thyroiditis (90% of hypo-, >60% of hyper-) and both in post-viral and post-partum thyroiditis. They are less frequently raised in thyroid neoplasia/nodules/cysts, but their presence does not exclude these conditions. Adrenal failure and Gonadal failure Ab to adrenal cortex Ab to ovary Ab to testis In this country, Addison s disease is most often due to autoimmunity; the presence of antibodies to adrenal cortex suggests an autoimmune cause. There may also be antibodies to steroid producing cells of ovary and testis. A small proportion of cases of premature menopause are due to autoimmune oophoritis. Some of these patients also have adrenal failure the same tests are done for both. Polyendocrine autoimmunity Ab to adrenal cortex Ab to Ovary Ab to Testis Ab to thyroid peroxidase (TPO) Ab to islet cells Ab to gastric parietal cells 17 of 119

18 In addition: Antinuclear antibodies (includes anti- Centromere antibodies) Ab to smooth muscle/mitochondrial Ab to acetylcholine receptors (if indicated) Type 1: Usually presents under 10 yrs old, m=f. Hypoparathyroidism, adrenal failure and candidiasis also hepatitis, alopecia, delayed puberty, etc. Type 2: Occurs in adolescence/early adulthood, f>m, Addison s + thyroid failure + type 1 diabetes mellitus M maybe gonadal failure, vitiligo Type 3: Generally older, f>>m, autoimmune thyroiditis together with diabetes mellitus, gastric autoimmunity (GPC, anti-if) maybe other such as myasthenia. The spectrum of results may help confirm the diagnosis. Liver autoimmunity Antinuclear antibodies (includes anti- Centromere antibodies) Ab to smooth muscle/mitochondrial IgG IgA IgM electrophoresis Ab to LKM (Liver kidney Microsomal) Alpha-1-antitrypsin deficiency (Biochemistry) Primary biliary cirrhosis (PBC) and autoimmune chronic active hepatitis (CAH) are associated with characteristic autoantibodies that are helpful in classifying the hepatitis and separating autoimmune CAH from the other forms. Patterns may include antibodies to smooth muscle (actin), reported as SMA-T in CAH, liver/kidney microsomal antibodies in LKM-positive autoimmune hepatitis and antibodies to mitochondria PBC. Centromere antibodies are also found in some cases of primary biliary cirrhosis. Presence of autoantibodies does not exclude a viral cause for the hepatitis. Because of the overlap between various different forms of hepatitis, it is usually best to test for all the types of autoantibody AMA, SMA, LKM and ANA. There is a polyclonal increase in immunoglobulins in autoimmune and viral hepatitis, whilst a raised IgM is seen in primary biliary cirrhosis. Serum electrophoresis may reveal lack of alpha-1-antitrypsin if this is associated with the cirrhosis. 18 of 119

19 Primary sclerosing cholangitis has no definitive serological markers, but may be associated with ANCA (anti-neutrophil cytoplasmic antibodies) or ANA or SMA. Pemphigus/Pemphigoid Ab to skin Immunohistology Blistering skin conditions may involve autoimmunity antibodies are found to the epidermal intercellular cement in pemphigus, and to the epidermal basement membrane in pemphigoid. The pemphigus like pattern is also seen in some patients with leprosy, burns, penicillin rashes, SLE, MG with thymoma, dermatomycoses, erythema multiforme etc that of pemphigoid in herpes gestationis and epidermolysis bullosa acquisita. Dermatitis herpetiformis (DH) Ab to tissue transglutaminase (ttg) Immunohistology Though the diagnosis of DH is based on the appearance of the rash and IgA in the dermo-epidermal junction in the dermal papillae in biopsies, the presence of IgA antibodies to ttg may point to the association of DH with gluten sensitivity (see Coeliac disease). Dressler s syndrome, post cardiotomy syndrome Ab to cardiac muscle Post-myocardial injury syndromes, including Dressler s syndrome, are associated with the presence of anti-myocardial antibodies, which reflect the degree of cardiac damage. 19 of 119

20 Obstetrics and Gynaecology Brief Overview Recurrent foetal loss Anti-phospholipid antibodies Antibodies to cardiolipin (IgG and IgM) Mid-trimester foetal loss may be due to thrombosis of placental vessels in anti-phospholipid syndrome (primary or associated with SLE). For additional information refer to: Rheumatology and Connective Tissue Disease tests Gonadal Failure Ab to Ovary Ab to Testis Ab to adrenal cortex This may be due to autoimmune damage to the gonads in males or females associated with antibodies to ovary, testis and adrenal cortex. (This is found with adrenocortical failure in autoimmune polyendocrinopathy). 20 of 119

21 Infection and Immunity Please phone Immunology Clinical Staff ext to discuss the investigation of recurrent unusual infections Brief overview Primary immunodeficiency (PID) can cause preventable death or morbidity. Consider PID in patients with: - recurrent infection - severe infection - infection refractory to antibiotics - unusual infection Don t forget that PID can present in childhood and adulthood as well as in infancy. Guidelines for testing are available at: Patient information is available at: Primary Immunodeficiency Association: Relevant immunological tests are listed below. More detail, including the sample required, is available at the end of this guide. Primary Immunodeficiency The following panels of tests are available for investigating patients: Immunoglobulin levels: Antibody responses: Complement activity IgG, IgA, IgM electrophoresis, IgG subclasses Functional antibodies Hib/Pneumococcus CH50 activity, Classical Pathway C3, C4 Skin tests By arrangement T and B Cell subsets Tel: Neutrophil function Tel: of 119

22 Background Grouping is frequently based on the specific fault in the immune system. This may include: - B Cell antibody deficiency causes mainly recurrent bacterial infections - T cell deficiency causes viral, protozoal and fungal infections - Combined T and B cell deficiencies - Defective phagocytes - Complement deficiencies cause mainly bacterial infection. There are no screening tests for immune deficiency. In cases with recurrent bacterial infection it may be sensible to start by measuring immunoglobulins, but we advise discussing individual cases early on. Hereditary Angioedema C1 esterase inhibitor C1 esterase inhibitor (functional test) C4 Recurrent abdominal pain and/or deep subcutaneous swellings without urticaria (particular occurring after minor trauma), often with family history, may indicate HAE. C4 and C1 inhibitor will be low. Uncommonly there may be normal C1INH level with defective function. If C4 is very low without other explanation and C1INH normal, C1INH function will be measured. Secondary Immunodeficiency Immunoglobulins IgG, IgA, IgM, electrophoresis Monitoring absolute CD4 counts (for patients with known HIVserology) Secondary causes of immunodeficiency are more common than primary causes and levels of immunoglobulins might reflect decreased production (eg. lymphoproliferative disorders, drugs) or increased losses (nephritic syndrome). In HIV infection the CD4 count is used to monitor progress of disease and inform treatment decisions. Other acute and chronic diseases affect CD4 counts. In HIV infection they are best done at the same time of day each time and avoiding acute ill health. They should never be done as a substitute to HIV antibody testing to diagnose HIV infection. 22 of 119

23 Guidelines and information on CD4 counts are available at: Additional patient information is available at: National Aids Trust: Terrence Higgins Trusts: Acquired C1 esterase inhibitor deficiency C1 esterase inhibitor C1q C3 C4 Consumption/inactivation of C1INH may occur in SLE and lymphoproliferative disease. This may lead to episodes of angioedema as with the inherited form. C1q is low in acquired C1INH deficiency but usually normal in HAE. Patient information is available at: Primary Immunodeficiency Association: TB Exposure: Quantiferon This test measures gamma interferon produced by T cells in response to M. tuberculosis peptides. The test is used to help diagnose latent TB infection and is indicated in individuals with a positive delayed hypersensitivity skin test. The test may produce indeterminate results in patients with active TB and in immune deficiency states. Quantiferon 23 of 119

24 Malignancy Brief Overview Flow cytometry is a useful aid to the diagnosis of leukaemias and lymphomas. Cells from blood, marrow or fresh lymph node can be studied. Flow cytometry works when: - The sample is fresh (<12 hours old) - There are clear clinical details - Marrow samples should be placed in special medium, available from the laboratory - Lymph node biopsies should not be formalin fixed Flow cytometry may also be used to assess whether Leukaemia or lymphoma has gone into remission after chemotherapy. Finally, flow cytometry can be used to measure the number of circulating stem cells prior to stem cell transplantation. See: for more information and guidelines. The demonstration of paraproteins by electrophoresis is one of the criteria required for a diagnosis of multiple myeloma. Their quantification, the degree of associated immunosuppression of other immunoglobulins and the excessive proliferation of plasma cells in the bone marrow are all laboratory indicators of a malignant paraproteinaemia. Serum levels of β2-microglobulin are useful indicators of prognosis, partly reflecting the degree of renal damage (and are raised in other causes of renal failure, malignancies, and some autoimmune disorders). Serum free light chain estimation is a useful adjunct to the diagnosis and monitoring of myeloma. For each clinical condition, the relevant immunological tests are listed together with a short explanation of their use. More detail, including the required is available at the end of this guide. Tests indicated in black are not routinely available or are offered by other laboratories. Lymphoproliferative disorders Immunophenotyping for lymphoid and myeloid malignancies Chronic lymphocytic malignancies/lymphoma panel Acute Leukaemia panel 24 of 119

25 Stem cells Patient information is available at: Leukaemia Research Fund: Lymphoma association: Paraproteinaemia Serum protein electrophoresis/densitometry: Urinary Light Chains IgG, IgA, IgM electrophoresis, Serum Free Light Chains Bence-Jones protein Immunofixation Uses electrophoresis sample β2-microglobulin β2 microglobulin Patient information is available at: International Myeloma Federation (UK): 25 of 119

26 Neurology Brief Overview Myasthenia Ab to acetylcholine receptors Ab to thyroid peroxidase (TPO) Impaired neurotransmission in MG is caused by the presence of antibodies to acetylcholine receptor. Associated autoantibodies to skeletal muscle and thyroid microsomes are sometimes also detected. Although antibodies to acetylcholine receptor (AChR) are present in all patients with Myasthenia Gravis, they are detectable in only 90% of sera tested in the laboratory. They are particularly difficult to detect in patients with ocular myasthenia, where up to 40% of patients have undetected antibodies. Antibodies to striated muscle are present in 30% of patients with MG and 60% of these will also have a thymoma. This test is insufficiently reliable to help in management and is no longer performed. Motor Neuropathies - IgM antibodies to ganglioside GM1 Anti-ganglioside GM1 antibodies are present in 80% of patients with pure motor weakness with evidence of multifocal conduction block. Low titre anti-ganglioside antibodies are present in some sensori-motor neuropathies, SLE, other autoimmune disease and normal controls. Antibodies associated with other neurological conditions - Antibodies to Myelin associated glycoproteins (MAG) MAG antibodies are detected in 50-75% or patients with IgM paraproteinaemia and peripheral neuropathy. - Paraneoplastic antibodies (Yo, Hu and Ri) Anti-Yo antibodies have been found in paraneoplastic cerebellar degeneration Anti-Hu antibodies have been found in paraneoplastic encephalomyelitis and in sensory neuropathy. Anti-Ri antibodies can be observed in patients with rapidly progressive brainstem tumour. 26 of 119

27 A negative test result does not exclude the possibility of a paraneoplastic syndrome. 27 of 119

28 Renal Disease Brief Overview Acute renal presentations: Immunological processes are implicated in many renal diseases. In several situations, diagnosing and treating immunological processes can halt or reverse renal impairment. For this reason the following tests may be offered on an urgent basis, provided an urgent request is made by phone. Immunological test Clinical condition Turnaround time ANCA Vasculitis Same day Anti glomerular Goodpasture s Same day basement membrane ANA disease SLE and other connective tissue diseases Serum and urine Myeloma electrophoresis C3, C4 Immune complex nephritis Chronic renal presentations Same day Results next day Results next day The same tests listed above are also useful for the diagnosis of chronic renal disease. Cryoglobulinaemia may also be excluded in some patients with chronic renal failure. This test requires very careful specimen handling and it may be helpful to screen for cryoglobulinaemia by doing C4; if C4 is normal, cryoglobulinaemia is very unlikely. C3 nephritic factor can cause membranoproliferative disease. The laboratory can refer samples to a laboratory that measures C3 nef directly, but this is a complex test with delayed results. We recommend checking C3 first; if C3 is normal, C3 nef is very unlikely to be present. Some of these tests are also used to monitor renal disease (ANCA, C4). In SLE, monitoring anti-dna antibody levels and complement C4 is more useful than monitoring ANA. The half life of IgG antibodies is about three weeks, so most forms of immunosuppressive treatment will not produce rapid changes in 28 of 119

29 autoantibodies and repeat sampling more than every six weeks is probably unhelpful. The exceptions are plasmapheresis and administration of high dose IVIg, which cause rapid and clinically meaningful fluctuations in autoantibody level. In these situations, more frequent testing of ANCA or anti-gbm is appropriate. Relevant immunology tests for specific clinical conditions are listed below: Patient information is available at: National Kidney Federation: Screening for Glomerulonephritis First occasion: C3 C4 Antinuclear antibodies (includes Anti- Centromere antibodies ) Neutrophil cytoplasmic antibody (ANCA) Cryoglobulins Additional tests may help categorise the type of glomerulonephritis Membranoproliferative glomerulonephritis C3 C3 nephritic factor (only if C3 very low) Serum C3 levels are low in some forms of membranoproliferative glomerulonephritis reflecting the presence of the circulating autoantibody C3 nephritic factor which binds and activates C3 convertase. Glomerulonephritis Ab to glomerular basement membrane (GBM) Ab to proteinase3, PR3 (c-anca) Ab to myeloperoxidase, MPO (p-anca) p-anca associated glomerulonephritis is the common form of Necrotising crescentic glomerulonephritis, reflecting different vasculitic causes. 29 of 119

30 Goodpasture s syndrome Ab to glomerular basement membrane (GBM) A combination of renal and lung involvement may suggest Goodpasture s syndrome due to the presence of antibodies to the glomerular basement membrane (GBM). IgA Nephropathy Serum Immunoglobulins IgG, IgA, IgM, electrophoresis Serum IgA may be raised in IgA nephropathies including Henoch Schönlein purpura. Nephrotic syndrome Serum Immunoglobulins IgG, IgA, IgM, electrophoresis Serum immunoglobulins may be low in poorly selective proteinuric forms of nephrotic syndrome (focal glomerulonephritis). ATG monitoring: Patients receiving ATG therapy have T cell numbers (% and absolute counts) monitored daily. For the result to be available by noon, it is essential that the sample is received in the laboratory no later than 10:30am. 30 of 119

31 Rheumatology/Connective Tissue Disease Tests Background The tests described in this section are very non-specific. Some (ANA, rheumatoid factor) are found in completely healthy people, particularly older women. These can also be transiently positive in infection or as the result of drugs (particularly anti DNA). This means that these tests should not be used as a screen for investigating patients with raised ESR s or general malaise. Rheumatoid factor is especially prone to misinterpretation. It is resent in 15% of normal individuals and is not diagnostic of rheumatoid arthritis. Rheumatoid arthritis is diagnosed by finding symmetrical peripheral polyarthritis with erosions on X-ray. The half-life of IgG antibodies is about three weeks, so most forms of treatment will not produce rapid changes in autoantibody levels and repeat sampling more than every six weeks is probably unhelpful. We would advocate that it is nearly never useful to repeat positive ENA antibodies. Each specific test is highlighted in blue. More information including sample requirements and turnaround times can be found at the end of this guide Patient information is available at the following sites: British Society for Rheumatology: Lupus UK: Raynaud s and Scleroderma Association: Arthritis Research Campaign: Screening for Systemic Autoimmune Disease Antinuclear antibodies (includes anti Centromere antibodies) Rheumatoid factor Raised immunoglobulins, IgG, IgA, IgM, electrophoresis Systemic Lupus Erythematosus Antinuclear antibodies (includes anti Centromere antibodies) 31 of 119

32 Ab to double stranded DNA (IgG); Ab to Crithidia (if appropriate) Ab to ENA includes: Sm, Sm/RNP, RNP(RNP A, RNP68), Ro(SS-A52 SS-A60), La(SS-B), Ribo P, Scl-70, Jo-1 and Chromatin Ab to cardiolipin (IgG and IgM) IgG, IgA, IgM, electrophoresis Complement components C3, C4 Classical pathway activity CH50 B cell enumeration Criteria for the diagnosis of SLE include antinuclear antibodies (ANA), antibodies to ds DNA, (Crithidia assay if appropriate) antibodies to extractable nuclear antigens (ENA) and anti cardiolipin antibodies. The presence of particular groups of serum antibodies may be associated with different clinical patterns of disease activity. Other typical immunological findings include raised serum IgG, and low serum complement levels (C3, C4) as well as the presence of rheumatoid factor, and other autoantibodies. DNA antibody levels, C4 levels (and ESR) are of some help in monitoring disease activity. Sjögren s syndrome Antinuclear antibodies (includes anti Centromere antibodies) Ab to ENA includes: Sm, Sm/RNP, RNP(RNP A, RNP68), Ro(SS-A52 SS-A60), La(SS-B), Ribo P, Scl-70, Jo-1 and Chromatin Rheumatoid factor There may be considerable overlap with other autoimmune disorders, including SLE. Characteristically antinuclear antibodies and antibodies to extractable nuclear antigens (particularly antibodies to Ro or SS-A, and antibodies to La or SS-B) are found. Rheumatoid factor and raised immunoglobulins may be found. Scleroderma/Systemic sclerosis Antinuclear antibodies (includes anti Centromere antibodies) Ab to ENA includes: Sm, Sm/RNP, RNP(RNP A, RNP68), Ro(SS-A52 SS-A60), La(SS-B), Ribo P, Scl-70, Jo-1 and Chromatin 32 of 119

33 The Scl-70 antibody is associated with systemic sclerosis, whilst centromere antibodies are found in limited systemic sclerosis (CREST syndrome). Polymyositis/Dermatomyositis Antinuclear antibodies (includes anti Centromere antibodies) Ab to ENA includes: Sm, Sm/RNP, RNP(RNP A, RNP68), Ro(SS-A52 SS-A60), La(SS-B), Ribo P, Scl-70, Jo-1 and Chromatin Jo-1 antibodies are found in a subset of myositis patients, especially those with interstitial lung disease. Antiphospholipid antibody syndrome Antibodies to phospholipids Ab to cardiolipin (IgG and IgM) Lupus anticoagulant (Haematology) Recurrent arterial or venous thrombosis (or foetal loss) may be associated with antibodies to phospholipids including cardiolipin. Related antiphospholipid antibodies include the lupus anticoagulant. Cardiolipin antibodies may be found in other autoimmune disorders, particularly SLE and are also seen very frequently in acute and chronic infections. Guidelines suggest that 2 positive tests (anticardiolipin antibodies, lupus anticoagulant haematology) more than 6 weeks apart are required to diagnose antiphospholipid syndrome. More information is available at: 33 of 119

34 Vasculitis Brief Overview The term vasculitis refers to inflammation of blood vessels and represents a heterogeneous group of clinical disorders. Each form of vasculitis can produce a distinct clinical picture, but in many cases immunology testing can differentiate, confirm and monitor the presence of vasculitis. Other forms of vasculitis, for example, Behçet s disease and primary cerebral vasculitis are not associated with any specific blood tests. The main immunological tests are antinuclear antibody (ANA for SLE a connective tissue diseases) and anti-neutrophils cytoplasmic antibody (ANCA - Wegener s granulomatosis and other vasculitides). It is important to understand that neither of these tests are specific; they can both be positive in infection and in liver disease. This makes these tests very unsuitable as screens in patients with vague symptoms such as malaise, weight loss or fever. Other tests include complement C3 and C4, which are altered in immune complex disease and cryoglobulin. Small vessel hypersensitivity vasculitis Antinuclear antibodies Rheumatoid factor Complement C3, C4 Cryoglobulins Serum immunoglobulins Anti-neutrophil cytoplasmic antibodies (ANCA) CRP (Biochemistry) Infection, drugs, foreign proteins (as examples) may be causal factors in a vasculitis affecting predominantly the skin. Immunological findings may sometimes include raised ESR/CRP, depressed levels of complement factors suggesting consumption, and the presence of antinuclear antibodies and rheumatoid factor (low titre). General screening tests for vasculitis should also include serum immunoglobulins, and ANCA (see below). If there is evidence for more extensive visceral involvement, one of the primary systemic vasculitides may be involved (see below). Alternatively, the vasculitis may be secondary to autoimmune disease (eg. SLE, chronic active hepatitis see earlier section), 34 of 119

35 neoplasia (eg. lymphoma), cryoglobulinaemia (these are immunoglobulins that form precipitates in the cold). Primary systemic vasculitis Anti-Neutrophil cytoplasmic antibodies (ANCA) Anti-Myeloperoxidase (MPO) Anti-Proteinase 3 (PR3) Immunoglobulins (IgG, IgA, IgE) CRP (Biochemistry) Some forms of systemic vasculitis are strongly associated with circulating anti-neutrophil cytoplasmic antigens (ANCA). In Wegener s Granulomatosis, (WG) (lung, renal) there is a diffuse cytoplasmic pattern (c-anca), as well as polyclonal elevations of IgG, IgA, IgE and raised CRP. ANCAs with a perinuclear pattern (p-anca) are seen in some patients with polyarteritis nodosa, (PAN) (weight loss, musculoskeletal, renal) and Churg-Strauss ( asthma, eosinophilia, hypocomplemenaemia, raised IgE). Both types of ANCA may be seen in microscopic polyarteritis (MPA) (clinical overlap between classic PAN and WG). Atypical forms of ANCA reactivity may be seen in association with Henoch-Schönlein Purpura (sometimes IgA raised), Kawasaki s syndrome, and in other autoimmune disorders (eg. SLE, ulcerative colitis). 35 of 119

36 A-Z of clinical conditions and Immunological Tests. Clinical Condition A Acute leukaemia Adrenal failure Anaphylactoid reactions to drugs etc Anaphylaxis Angioedema (Hereditary) Asthma Atopic Eczema B-C B cell enumeration Bird Fancier s Disease Bronchopulmonary Aspergillosis C1 esterase inhibitor deficiency (acquired) Chronic lymphocytic malignancies/lymphoma Coeliac Disease Conjunctivitis Cryoglobulinaemia (mixed) D-G Dermatitis herpetiformis Dressler s syndrome Farmer s Lung Gonadal Failure Goodpasture s syndrome H-M IgA nephropathies Immunodeficiency (Primary) Immunodeficiency (Secondary), HIV Lymphoproliferative disorders Immunological Test Link(s) Cell marker panel CD2, CD10, CD13, CD14, CD19, CD33, CD34, CD79a, CD117, TdT and MPO Ab to adrenal cortex, Ab to Ovary, Ab to Testis Refer patients to the combined Immunology/Anaesthetic clinic IgE, Mast cell tryptase C1 esterase inhibitor. C1 esterase inhibitor functional IgE, Specific IgE IgE, Specific IgE Immune deficiency panel Avian precipitins Aspergillus precipitins (Aspergillus fumigatus) C1 esterase inhibitor, C1q, C3, C4 Cell marker panel: CD3, CD5, CD19, CD23, CD79b,Kappa chains, Lambda chains, FMC7, CD38 Ab to tissue transglutaminase Specific IgE Cryoglobulins Ab to tissue transglutaminase Ab to cardiac muscle Farmer s lung precipitins (Micropolysporium faeni) Ab to Ovary, Ab to Testis, Ab to adrenal cortex Ab to glomerular basement membrane Raised IgA, IgG, IgM electrophoresis Immunodeficiency panel Immunoglobulins IgG, IgA, IgM, electrophoresis; CD4 for patients with known HIV serology Immunophenotyping for lymphoid and myeloid malignancies (full clinical 36 of 119

37 Liver autoimmunity Lymphoid/myeloid malignancies Mixed connective tissue disease Motor neuropathy Myasthenia gravis N-P Necrotising crescentic glomerulonephritis Nephrotic syndrome Paraproteinaemia, Myeloma Pemphigus/Pemphigoid Polyendocrine Autoimmunity Polymyositis/Dermatomyositis Primary anti-phospholipid antibody syndrome R-S Recurrent foetal loss Rhinitis Scleroderma/systemic sclerosis information required) Ab to smooth muscle/mitochondrial; Liver kidney microsomal (LKM), IgG, IgA, IgM, electrophoresis Cell surface markers appropriate to diagnostic information provided Antibodies to ENA, particularly RNP Ab to ENA including Sm, Sm/RNP, RNP(RNP A, RNP68), Ro(SS-A52 SS- A60), La(SS-B), Ribo P, Scl-70, Jo-1 and Chromatin Anti-ganglioside antibodies (GM1) Antibodies to acetylcholine receptor (AchR) Ab to myeloperoxidase MPO (p- ANCA) IgG, IgA, IgM, electrophoresis IgG, IgA, IgM, electrophoresis (including immunofixation) Serum Free Light Chains Bence-Jones protein: β2 microglobulin Ab to skin, Immunohistology Ab to adrenal cortex, Ab to Ovary, Ab to Testis, Ab to islet cells, Ab to gastric parietal cells. Additional: Antinuclear antibody (includes anti-centromere Abs). Ab to smooth muscle/mitochondrial, Ab to acetylcholine receptors (if indicated) Antinuclear antibodies, antibodies to Jo-1 ENA includes: Sm, Sm/RNP, RNP(RNP A, RNP68), Ro(SS-A52 SS- A60), La(SS-B), Ribo P, Scl-70, Jo-1 and Chromatin Anti-phospholipid antibodies, Ab to cardiolipin (IgG, IgM) Ab to phospholipids, Ab to cardiolipin (IgG, IgM) IgE, specific IgE Antinuclear antibody includes anticentromere antibodies (CREST). Antibodies to Scl-70. ENA includes: Sm, Sm/RNP, RNP(RNP A, RNP68), 37 of 119

38 Sjögren s syndrome SLE Systemic Autoimmune Disease T-V TB (latent) Thyroid Disease Urticaria (Acute) Vasculitis (Primary systemic) Wegener s, Churg-Strauss, Microscopic polyarteritis, Henoch-Schönlein Purpura, Kawasaki s syndrome Vasculitis (small vessel hypersensitivity) W-Z Ro(SS-A52 SS-A60), La(SS-B), Ribo P, Scl-70, Jo-1 and Chromatin Antinuclear antibodies, antibodies to extractable nuclear antigens (ENA) includes Ro, La Antinuclear antibodies (includes anticentromere) Ab to double stranded DNA (IgG) Ab to ENA includes: Sm, Sm/RNP, RNP(RNP A, RNP68), Ro(SS-A52 SS- A60), La(SS-B), Ribo P, Scl-70, Jo-1 and Chromatin Other indicators include: Raised IgG IgG, IgA, IgM, electrophoresis, Low complement C3,C4 Antinuclear antibodies (includes anticentromere) Rheumatoid Factor, IgG, IgA, IgM, electrophoresis Gamma Interferon (Quantiferon) Ab to thyroid peroxidase (TPO) IgE, Specific IgE Antinuclear antibody (includes anticentromere) Ab to myeloperoxidase MPO (p- ANCA) Ab to proteinase 3 PR3 (c-anca) Serum immunoglobulins IgG, IgA, IgM electrophoresis IgE (Total), CRP (Biochemistry) Antinuclear antibodies (includes anticentromere) Rheumatoid factor, Complement C3, C4, Cryoglobulin Serum immunoglobulins IgG, IgA, IgM, electrophoresis Neutrophil cytoplasmic antibody (ANCA) 38 of 119

39 A-Z of Tests Acute Leukaemia panel CD2, CD10, CD13, CD14, CD19, CD33, CD34, CD79a, CD117, TdT, MPO Acetyl Choline Receptor Antibodies Myasthenia Gravis Adrenal antibody The results are positive in 60-7-% of patients with idiopathic Addison s disease. Antiadrenal antibodies are also present in 28% of patients with idiopathic hypothyroidism and 7% of patients with Hashimoto s disease. Allergy specific IgE (ImmunoCAP ) Measurement of allergens specific IgE is of value where skin testing is difficult for any reason. Over 100 specific allergens (request panel) are routinely available and can be viewed by selecting the link below. Any requests for allergens that are not on the list should be discussed with a clinician. Amphiphysin Anti-Basal Ganglia Antibodies Anti-C1q Anti-neutrophil cytoplasmic antibody (ANCA) Anti-neutrophil cytoplasmic antibodies (ANCA) are found in several types of vasculitis. Samples for ANCA are initially tested by immunofluorescence on neutrophils. Positive samples are then further tested by Multiplex assay for specific antibodies to either proteinase 3 (PR3) or myeloperoxidase (MPO). In general, a classical canca pattern corresponds to PR3 reactivity whilst a perinuclear panca pattern corresponds to MPO reactivity. Atypical ANCA patterns do not usually correlate with either antigen. These tests are used to diagnose vasculitis and to monitor disease activity. Infections and autoimmunity can produce positive tests reducing the specificity of ANCA testing. Antinuclear antibody (ANA), includes anti-centromere Indicated in the investigation of suspected connective tissue disorders. The absence of ANA almost excludes a diagnosis of SLE. Antinuclear antibodies are detected in other clinical conditions including: Sjögren s syndrome, systemic sclerosis (CREST), chronic active hepatitis, fibrosing alveolitis, juvenile chronic arthritis and infections. Aquaporin 4 39 of 119

40 Aspergillus fumigatus precipitins These tests detect presence of IgG antibodies to Aspergillus fumigatus. Aspergillus precipitins. See also: - Farmer s lung and Avian precipitins. Avian precipitins As for Aspergillus precipitins. B cell enumeration This can be used to monitor patients receiving Rituximab. We recommend a pretreatment baseline sample. See immunodeficiency panel. Bence Jones protein (Urinary light chains) Urine immunofixation Β2 Microglobulin High levels are seen in connective tissue disease e.g. Sjögren s, RA, Granulomatous disease like sarcoidosis, CVID. C1 esterase inhibitor Should always request C4 at the same time. C1 esterase inhibitor functional test C1q Reduced levels in acquired angiodema and urticarial vasculitis. C3 Nephritic Factor Mesangiocapilliary glomerulonephritis (only if C3 is low) Cardiac muscle antibodies Associated with Dressler s syndrome. Cardiolipin (IgG and IgM) antibodies Found in the anti-phospholipid syndrome, which may be primary or occur as a secondary complication of SLE. Raised levels are significantly associated with the presence of both venous and arterial thrombosis, thrombocytopaenia and recurrent foetal loss. CD34 (Stem cells) CD3 (T cells) CD4 count (T cell count) (for patients with known HIV serology) The CD4 count is used to monitor disease progression in HIV infection. For example, patients with counts below 200 cells/ul are at risk of pneumocystis pneumonia and should receive antibiotic prophylaxis. CD4 counts are also used to assist decision 40 of 119