Department of Immunology - University of Birmingham of Immunology and Immunotherapy College of Medical & Dental Sciences Clinical Immunology Service Laboratory Handbook A brief guide

Institute of Immunology and Immunotherapy College of Medical & Dental Sciences

Clinical Immunology Service

Laboratory Handbook

A brief guide for clinical and laboratory staff

Updated: May 2017 Review date: May 2018

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CONTENTS 1. Introduction 3

2. Contact Details 4

3. Routine/Urgent assay processing 5

4. Specimen collection requirements: General 6

Surface markers 29

Cell function 33

5. A brief guide to common immunology tests 7

6. Myeloma screen guide 8

7. Completion of request forms 9

8. Specimen retention/additional tests 10

9.

10.

11.

Data protection

Complaints

Details of available assays/normal adult ranges

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11-29

12. Immunophenotyping 30-33

13. Cell function assays 34-35

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1. INTRODUCTION

The Clinical Immunology Service (CIS), in the Institute of Immunology and Immunotherapy,

provides a comprehensive range of laboratory services. In particular the CIS is a major

testing centre for multiple myeloma, leukaemia/lymphoma, immunodeficiency, autoimmunity,

renal and rheumatic diseases and allergy. The CIS laboratory liaises closely with other local

laboratories, particularly pathology, cytogenetics, haematology and chemistry.

This handbook provides contact details, information about turnaround times for assays, and

other information about the laboratory staff, working hours, results and their interpretation.

Normal working hours are 8:00am to 5:30pm from Monday to Friday. Clinical advice is

available from 8:00am to 8:00pm Monday to Friday via the mobile telephone numbers listed

on page 4. If you are unable to reach a clinician on their mobile, please leave a voicemail

and someone will contact you as soon as possible. There is no formal on-call service but

clinically urgent requests may be arranged through a clinician, clinical scientist or senior

biomedical scientist, by telephone.

Most analytes and autoantibodies are carried out on the same day, or the day following

receipt of the specimen. However, many tests are expensive when dealt with in small

numbers and in order to manage costs such assays are ‘batched’ on certain days of the

week (see page 4). If a faster turnaround time is required for clinical reasons, please phone

to discuss.

Postal delays can cause problems with turnaround times. Frequently we have despatched

results but the report has not reached its destination. We are able to offer an automated

email reporting system. Please contact the Laboratory Manager for information about

this service and to arrange its implementation for your reports. You will need to provide

an nhs.net email address to which reports can be sent.

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2. CONTACT DETAILS Postal Address:

Clinical Immunology Service

Institute of Immunology and Immunotherapy

The Medical School

University of Birmingham

Vincent Drive

Edgbaston

Birmingham

B15 2TT

Web address:

http://www.birmingham.ac.uk/facilities/clinical-immunology-services/index.aspx

Key contact telephone numbers:

Prefix last five digits with 7600 from UHB NHS Trust

General enquiries/results: (0121) 41 44069

Trials office: (0121) 41 44069

Laboratory Manager: (0121) 41 43092

Clinical enquiries:

General clinical enquiries:

Prof P.J.L. Lane: (mobile) 07831 681955, email: [email protected]

Dr A.G. Richter: (mobile) 07949 518773, email: [email protected]

Myeloma/Lymphoma/Leukaemia enquiries:

Prof M.T. Drayson (mobile) 07798 585319, email: [email protected]

Dr S.D. Freeman (mobile) 07884 310528, email: [email protected]

http://www.birmingham.ac.uk/facilities/clinical-immunology-services/index.aspx

mailto:[email protected]



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3. ROUTINE/URGENT ASSAY PROCESSING Daily assays:

As a general guide, most immunochemistry, indirect immunofluorescence, electrophoresis,

cell markers and some neuroimmunology assays are carried out on a daily basis.

Batched assays:

Non urgent, expensive or labour intensive assays which are batched on a less frequent basis

include:

Cardiolipin/B2GP1 antibodies – Wednesday

dsDNA antibody ELISA – 2 or 3 times a week

ENA antibodies – Thursday

Intrinsic factor antibodies – Wednesday

Functional C1Inh – Once a week

Organ specific antibodies – Friday

IgG subclasses – Twice a week

MPO/PR3 antibodies – 2 or 3 times a week

GAD antibodies – Once a week

Urgent assays:

Some assays are available with a reduced turnaround time on discussion with a member of

the senior laboratory staff. Urgent specimens must be discussed with the laboratory and

handed over to a member of staff in specimen reception within the CIS at the medical school.

The request form should be clearly marked “Urgent” as well as with which member of staff

the request was discussed. The sample should arrive before 2pm. Contact details (ideally a

mobile phone number) for the requesting clinician must also be supplied to enable results to

be communicated urgently.

Prior warning for urgent requests is essential. It should be noted that urgent requests

may incur an additional charge and where an initial qualitative result is provided these will be

followed up with quantitative assays when the next routine batch is processed.

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4. GENERAL SPECIMEN COLLECTION REQUIREMENTS When sending specimens to the laboratory the following should be noted:

Different samples require different blood tubes. For example; red topped tubes (in the

Vacutainer system used at UHB) allow blood to clot for tests that require serum.

Some complement components are unstable. For functional complement assays send 5ml

of blood in a red topped tube (for C3d use EDTA blood) immediately after venepuncture. For

distant clinics the serum or plasma should be separated within the hour, frozen and sent to

the laboratory to arrive frozen.

For cryoglobulins blood should be taken into a warm syringe, transferred to a warm tube

and brought to the laboratory whilst being maintained at not less than 37°C (and up to 40°C).

The laboratory will provide a pre-warmed container with electronic temperature tracking probe

and phlebotomy equipment and provide advice on sampling – contact lab prior to performing

this test to arrange.

Urinary free light chains (BJP). A 25ml aliquot of a random urine in a universal container

(without preservative) sent together with 10ml (clotted) blood in a red top tube.

T cell antigen receptor & Immunoglobulin gene rearrangement studies: Please supply

blood or bone marrow samples drawn into an EDTA bottle (Please note: heparinised material

may interfere with PCR and will NOT be processed).

Quantiferon (QFT): Two 6ml Lithium Heparin specimens are required for this assay. Use

Cell Markers request form. Samples must arrive in the laboratory before 2pm on the same

day as they are drawn. Testing is only carried out Monday to Thursday. (Please note:

samples sent on Fridays will NOT be processed).

T-SPOT TB: Two 6ml Lithium Heparin specimens are required for this assay. Use Cell

Markers request form. Samples must arrive in the laboratory before 2pm on the same day as

they are drawn. (Tuesdays only: On other days this assay may be available under special

circumstances and by prior arrangement only). This assay must be booked with laboratory

staff before sending.

For all cell markers and cell function assays please see the notes towards the end of this

handbook under ‘Immunophenotyping’ (p29) and ‘Cell function assays’ (p33).

All high-risk specimens and their accompanying forms must be clearly labelled. Samples are not

tested on site if they are from a patient with suspected CJD or vCJD. Where such samples are

forwarded to an alternative laboratory, turnaround times will be slightly longer.

Please also note special requirements for cell work and neuroimmunology requests both

of which have separate request forms.

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5. A BRIEF GUIDE TO COMMON IMMUNOLOGY TESTS Anti-nuclear antibodies (ANA):

This test is most useful in situations where SLE is suspected – as ANA negative SLE is rare.

ANA is also associated with a number of connective tissue diseases and therefore high titre

ANA results may be considered supportive evidence of this. However, positive ANA

(particularly at low titre) may also be seen after infection or even in asymptomatic individuals

(especially older people and females).

SLE in pregnancy or with planned pregnancy:

These patients should have anti-cardiolipin (ACL) antibodies tested and their Ro antibody

status determined. Ro antibodies are specific for a type of extractable nuclear antigen (ENA),

and are relevant in pregnancy because they are associated with congenital heart block.

Anti-neutrophil cytoplasmic antibodies (ANCA):

This test is most useful in situations where small vessel vasculitis is suspected. ANCA have

different specificities, of which the most clinically relevant are MPO and PR3 antibodies.

These are particularly associated with microscopic polyangiitis, eosinophilic granulomatosis

with polyangiitis (EGPA) and granulomatosis with polyangiitis (GPA). However, clinicians

should be mindful that vasculitis is not excluded by a negative ANCA result.

Allergy tests:

Specific IgE tests to individual allergens can support a diagnosis of allergy in a patient who

has compatible symptoms. However, specific IgE antibodies are not useful as a screening

test because they can be positive in asymptomatic patients. These patients may be

sensitized but not necessarily allergic. These tests only have a role in type I hypersensitivity

reactions, which are characterised by a rapid onset of symptoms following exposure.

Suspected Myeloma:

Immunoglobulins should be measured (this includes levels of IgG, IgA and IgM).

Electrophoresis is performed to identify whether there is a paraprotein. If present then the

type and size of the paraprotein is determined by immunofixation and densitometry

respectively. Not all myeloma patients will secrete an intact paraprotein and therefore kappa

and lambda serum free light chains are also typically requested.

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6. MYELOMA SCREEN GUIDE Myeloma (median age 68 yrs) should be suspected when there is a normochromic and

normocytic anaemia, unexplained renal impairment / osteoporosis / fractures, and severe /

recurrent bacterial respiratory tract infection.

The myeloma clone usually secretes a whole serum immunoglobulin paraprotein at high

levels (>30g/l in two thirds of patients): 60% of patients have an IgG paraprotein, 25% have

an IgA paraprotein. IgM, IgD & IgE paraproteins are very rare.

Most patients also secrete free immunoglobulin light chains (FLC) detectable as an abnormal

kappa : lambda ratio in serum of 85% of patients and / or as Bence Jones Protein in urine of

60% of patients. 15% of myeloma patients don’t secrete a whole paraprotein – they have light

chains only.

Polyclonal IgG, IgA and IgM levels are below the normal range in 80% of myeloma patients

Polyclonal IgG, IgA and IgM levels are above the normal range in <0.5% of myeloma patients

Monoclonal Gammopathy of Undetermined Significance (MGUS) is present in 3% of

healthy people aged over 50 years and 8% of people aged over 80 years. It is defined by the

presence of a paraprotein / abnormal FLC ratio and absence of the damage caused by

myeloma described above. MGUS is the most likely diagnosis if a paraprotein is <15g/l, FBC,

serum creatinine and calcium are normal. In this circumstances asymptomatic patients do not

usually need a bone marrow and skeletal survey.

70% of MGUS patients have an IgG paraprotein, 15% IgA and 15% IgM paraprotein.

In 90% of MGUS patients (only 10% myeloma patients) the paraprotein is <15g/l.

In only 25% of MGUS patients (80% myeloma patients) the polyclonal antibody levels are

below normal range.

In 50% of MGUS patients (85% myeloma patients) the FLC ratio is abnormal.

The clonal plasma cells in MGUS are benign but in a few patients the paraprotein secreted

by MGUS can cause damage (light chain amyloid, neuropathy).

There is a risk of progression from an IgM MGUS to lymphoma that needs treating and from

IgG, IgA and light chain only MGUS to myeloma that needs treating. That risk varies between

patients but averages 1% every year for the rest of the patient’s life.

If the paraprotein is IgG, <15g/l with a normal serum FLC ratio then the risk is only 2% over

25 years and the patient can be reassured.

If the paraprotein is IgM or IgA, and/or >15 g/l and/or FLC ratio is abnormal then consider

monitoring; if all three abnormal refer to haematology.

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7. REQUEST FORMS The clinical immunology service has three different request forms:

o General immunological investigations – REQ.G.1.2

o Cell markers/function studies – REQ.C.1.2

o Neuroimmunology requests – REQ.N.1.2

These forms (with integral specimen bags) can be obtained by contacting the laboratory or if

bags are not required can be printed from the departmental website.

Completion of the forms

Please ensure all information is completed on the front (and back, where appropriate) of the

forms. As a guide, the data required is:

Essential Desirable

Sample Patient’s Full Name

Date of birth and/or Registration Number or other unique identifier (e.g. referring lab number)

Date and time of collection

Patient location

Destination for Report

Form Patient’s Full Name.

Patient’s NHS/CHI number

Date of Birth and/or Reg. Number or other unique identifier (e.g. referring lab number)

Patient’s Sex

Destination for Report.

Requesting Consultant/GP

Signature of person taking the sample

Specimen type

Test(s) required

Relevant Clinical Information

Date and time of collection

Clinician’s Telephone/ Bleep Number

(essential for urgent requests)

Patient’s Address

Requesting Clinician’s Specialty

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8. SPECIMEN RETENTION / ADDITIONAL TESTS Most cell markers samples will be retained for one week, in case further tests are required.

Other samples are routinely retained for >2 years.

If you require additional tests please contact the department and we will endeavour to assist

wherever sufficient volume/correct sample type is available and storage requirements for the

test have been met.

9. DATA PROTECTION The department is compliant with the Data Protection Principles, which are set out in the

Data Protection Act 1998. Staff processing personal information do so in accordance with the

University’s Data Protection Policy, and training in data protection is mandatory for staff. The

University’s Data Protection officer is Carolyn Pike ([email protected]).

10. COMPLAINTS We welcome feedback on our service, and treat all complaints seriously. If you complaint

relates to something clinical then you may prefer to contact Dr Alex Richter, Consultant

Immunologist ([email protected]). If your complaint relates to a non-clinical matter then

you may prefer to contact Mr Tim Plant, Laboratory Manager ([email protected]).

Complaints may also be directed to the Head of Department, Professor Mark Drayson

([email protected]). If the person you want to contact is not available, please

telephone the department and you will be directed to the most appropriate person. Contact

numbers can be found on page 4.





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11. AVAILABLE ASSAYS/NORMAL RANGES

TEST: (Preferred sample: normal range) (Turnaround time – from receipt in department to result available)

Acetylcholine receptor antibodies (Serum: 0 – 5 x 10-10 moles/l)§ (Up to 28 days)

Test for Myasthenia Gravis. Also see MUSK antibodies. This assay is currently sent to Immunology, Churchill Hospital, Oxford.

Adrenal cortical antibodies (Serum: Negative) (Up to 14 days)

Test for autoimmune adrenal disease.

Anaphylaxis testing See mast cell tryptase

Anti-nuclear antibodies (Serum: Titre <1:40)

Low ANA titres of 1:40 (positive fluorescence at serum dilution of one in forty) are generally not significant in adults, but can be in children.

(Up to 4 days)

ANA’s are associated with a variety of conditions other than SLE including rheumatoid diseases, chronic active hepatitis, fibrosing alveolitis, viral infections and drug ingestion. Patterns of ANA are said to be significant: Nucleolar associated with scleroderma, centromere with CREST syndrome, and speckled pattern with MCTD, Sjögrens, SLE and Polymyositis. Rim or homogeneous has been associated with SLE but there is a considerable amount of pattern overlap. High titre ANA at 1:1600 are strongly suggestive of connective tissue disease.

Anti-C1q autoantibodies (Serum: 0 – 20 units/ml)* (Up to 28 days)

Autoantibodies against C1q are a major criterion in the diagnosis of hypocomplementaemic urticarial vasculitis. They are also found in up to 50% of SLE patients and 95% of patients with lupus nephritis. C1q antibodies may be useful for assessing the risk of renal flares, and also for monitoring the effectiveness of immunosuppressive treatment in active lupus nephritis.

Aquaporin 4 antibodies (Up to 28 days)

See NMO antibodies

Aspergillus - specific IgG antibodies (Serum: < 40 mgA/L)* Also see fungal antigens (Up to 5 days)

Specific IgG antibodies directed against aspergillus fumigatus demonstrate previous exposure.

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Autoimmune encephalitis screen. (Serum/Plasma/CSF: Negative) (For CSF samples a minimum volume of 250 microliters is required) (Up to 14 days) Amongst about 50% of cases of encephalitis (underlying cause unknown), some have been identified as being autoimmune, affecting children and young women with or without malignancy. The immune system produces antibodies against neuronal cell surface molecules, disrupting neuronal transmission, thus leading to abnormal CNS function. For completeness it is advisable to consider Thyroid, VGCC, VGKC, muscle AchR, ganglionic AchR (alpha-3), GAD and paraneoplastic encephalitic antibodies (Hu, CV2/CRMP5, amphiphysin and Ma2).

The neuronal cell surface molecules include:

Receptors Tumour

Glutamate receptor

NMDAR Anti-N-methyl-D-aspartate receptor Teratoma

AMPAR1 & 2 Alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic type 1 & 2

Lung, Breast, thymus

Voltage gated Potasssium Channel associated proteins

LGI1 Leucine-rich glioma inactivated protein 1 Lung, thymus

CASPR2 Contactin-associated protein 2 Thymus

Gamma-aminobutyric acid receptor

GABABR1 Gamma-aminobutyric acid receptor type B1 Lung (SCLC)

Clinical: severe neuropsychiatric symptoms.

With early diagnosis and treatment (immunotherapy and tumour removal) patients often improve.

Avian antigens - specific IgG antibodies (Serum: Negative) (Up to 5 days)

Specific IgG antibodies directed against budgerigar and pigeon antigens are currently available.

Basal ganglia antibodies (BGA) (Serum: Negative)§ (Up to 28 days)

Anti-BGA antibodies are reported in patients with post-streptococcal infection(s) and movement disorders associated with basal ganglia. This is a useful diagnostic marker of neurological disorders such as Sydenham’s chorea, tic and an encephalitis lethargica-like syndrome. This assay is currently sent to Immunology, Churchill Hospital, Oxford.

Bence Jones Protein Please see urinary free light chains

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B2 microglobulin (B2M) (Serum: 0 – 4.0 mg/l)‡ (Up to 4 days)

Useful for monitoring lymphocyte activation and turnover in myeloma and HIV related diseases. Because B2M is filtered by the glomeruli and metabolised in the renal tubules higher levels are seen in patients with renal dysfunction.

B2GP1 antibodies (Serum: 0 – 20 U/ml)* (Up to 7 days) [Also see cardiolipin antibodies]

B2GP1 is a 50kD plasma protein (apolipoprotein H) that inhibits the intrinsic coagulation pathway, ADP mediated platelet aggregation and the prothrombinase activity of activated platelets. “Anti cardiolipin antibodies” bind to an altered form of B2GP1 which may be reproduced by binding B2GP1 directly to an ‘ELISA’ plate. The detection of anti-B2GP1 antibodies is said to have enhanced specificity for Anti-phospholipid syndrome (APS) and related coagulation disorders over the traditional anti-cardiolipin assay, which may display some false positive results due to cross reactivity of these antibodies with some infectious disease related antigens. This is currently a quantitative IgG antibody assay.

Cardiac antibodies (Serum: Negative) (Up to 14 days)

Though the diagnostic value is low these antibodies are found in some patients with Dressler’s syndrome, following myocardial infarction, after cardiac surgery and in some cardiomyopathies.

Cardiolipin/Phospholipid antibodies (Serum: IgG: 0 – 15 GPLU/ml IgM: 0 – 12.5 MPLU/ml)* (Up to 7 days) [Also see B2GP1 antibodies]

Antibodies have been associated with SLE, recurrent miscarriages and arterial and venous thrombosis. Slightly elevated levels may be found in some infections and so only positive results at two time points at least 6 weeks apart are considered significant. IgG and IgM antibodies are assayed separately. Significant levels of antibodies do not necessarily correlate with the severity of the disease. Please note that lupus anticoagulant is performed in haematology.

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C-Reactive Protein (Serum: 0 – 10 mg/l)‡ (Up to 4 days)

Viral infection/AI disease: 11 – 49mg/l

Bacterial infection: 50 – 100mg /l

Major bacterial infection: >100mg/l

As CRP has a short serum half-life this acute phase protein is useful in distinguishing bacterial infections, inflammatory conditions, activity of rheumatoid arthritis and monitoring response to therapy. CRP may not be raised if a patient is on biological treatments such as anti-TNF.

CSF Tau protein: asialo-transferrin Present only in CSF (Up to 4 days) Please send whole blood/serum sample for control purposes. Ideal CSF volume = 250 microlitres, but not less than 50 microlitres.

Cerebrospinal rhinorrhoea is potentially serious due to risk from infection. In patients presenting with a nasal discharge of clear fluid it is important to identify the nature of the fluid. CSF is readily identified by the presence of asialo-transferrin (Tau protein). This laboratory offers a reliable, sensitive and simple electrophoretic method for the rapid identification of Tau protein.

Complement C3 and C4 (Serum: C3: 0.75 – 1.75 g/l)* C4: 0.14 – 0.54 g/l)† (Up to 4 days)

Measurement of C3 and C4 is of value in monitoring activity of SLE and in immune complex disease. C4 is of particular value in SLE and angioedema when levels are well below normal.

C1q complement component (Fresh serum: 0.08 – 0.15 g/l)§ (Up to 14 days)

Serum C1q is found to be lowered due to activation of immune complexes, which are deposited on capillary walls. This assay is currently sent to Immunology PRU, Northern General Hospital, Sheffield.

C1 (esterase) Inhibitor

Immunochemical levels: (Fresh serum: 0.18 – 0.30 g/l)‡ (Up to 10 days)

Functional activity (Fresh serum: 70 – 130%)* (Up to 28 days)

Please see page 5-6 for collection procedure

Hereditary Angioedema: Autosomal dominant. Most cases have reduced serum C1Inh levels. In 10% of cases there are normal or elevated levels of C1Inh but this is functionally inactive. In hereditary angioedema C4 levels are almost always reduced and C1q levels are normal.

Acquired angioedema: Have reduced C1Inh levels and usually reduced levels of both C4 and C1q. Associated with B cell neoplasia.

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C3d complement component (EDTA plasma : 0 – 3 mg/l)§ (Up to 28 days)

C3d is a 35kD product derived from protease activity on native C3b and may be detected in fresh plasma indicating in vivo activation of the complement cascade. It is essential that blood is collected in EDTA bottles and the sample transported to the laboratory without delay. This assay is currently sent to Immunology PRU, Northern General Hospital, Sheffield.

C3 Nephritic factor (Fresh Serum: Negative)§ (Up to 28 days)

C3 nephritic factor is an IgG antibody, which stabilises the alternative pathway C3 convertase leading to continuous C3 breakdown. It is associated with type II MPGN and also with partial lipodystrophy. Please note: C3 nephritic factor will not be carried out in the presence of normal levels of C3. This assay is currently sent to Immunology PRU, Northern General Hospital, Sheffield.

Complement function Classical pathway (CH50) (Fresh serum: Normal)§ (Up to 28 days)

Currently referred to Immunology at Birmingham Heartlands Hospital, this assay tests the integrity of the classical pathway of complement. Low levels are found when any one component is absent or non-functional. Assays for the individual complement components are available as follow up. Please see full list on page 37. Refer to important notes regarding collection of blood and its despatch to the laboratory (page 6).

Complement function Alternative pathway (Fresh serum: Normal)§ (Up to 28 days)

Currently referred to Immunology at Birmingham Heartlands Hospital, this assay tests the integrity of the alternative pathway of complement and the terminal sequence (C3-C9) components. Low levels are found when any one component is absent or non-functional. Assays for the individual complement components are available as follow up. Refer to important notes regarding collection of blood and its despatch to the laboratory (page 6).

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Cryoglobulins (Serum: Negative) (Up to 7 days)

Positives may be typed: monoclonal, polyclonal or mixed (IgG/A/M).

When cryoglobulins are associated with myeloma, Waldenströms macroglobulinaemia, or lymphoma they consist of one immunoglobulin isotype but may be mixed or polyclonal in other diseases such as connective tissue diseases. Patients with renal disease and a low C4 level or patients with unexplained cutaneous vasculitis should be screened for presence of circulating cryoglobulin. Please refer to special conditions of collection and despatch to laboratory (page 6).

Cyclic citrullinated peptide (CCP) antibodies (Serum: < 7 U/ml)* (Up to 4 days)

Anti-CCP antibodies are potentially important surrogate markers for diagnosis and prognosis in rheumatoid arthritis (RA), because they:

are as sensitive as, and more specific than, IgM rheumatoid factors (RF) in early and fully established disease

may predict the eventual development into RA when found in undifferentiated arthritis

are a marker of erosive disease in RA

may be detected in healthy individuals years before onset of clinical RA

dsDNA antibodies (Serum: EIA: < 30 IU/ml * Crithidia IIF: Negative) (Up to 7 days)

Assay of antibodies to native, double stranded DNA (dsDNA antibodies), is carried out on all patients with SLE, as a qualitative test by IIF on the kinetoplast of crithidia lucillae which is then followed up with a quantitative assay by EIA. dsDNA antibodies may be detected in the absence of ANA and are extremely useful in monitoring the activity of the disease.

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Extractable Nuclear Antigen (ENA) antibodies (Serum: 0 – 20 EU/ml)* (Up to 7 days)

ENA antibodies recognise saline extracted nuclear antigens. There are many specificities recognised of which this laboratory currently offers six:

Sm (a marker for SLE);

RNP (said to be present in >95% MCTD);

SSA [Ro] (associated with cutaneous lupus,

SLE, neonatal lupus & congenital heart block);

SSB [La] (SLE, Sjögrens syndrome);

Jo1 (30% of polymyositis cases) and

Scl70 (associated with systemic sclerosis).

Patients with SLE or Sjögrens should be screened for ENA antibodies especially females considering pregnancy.

Specific microbial antibodies (Functional antibodies) (Serum) (Up to 28 days) Pneumococcal ab protective level is 0.35 ug/ml for each serotype. 7/12 serotypes tested (4, 6B, 9V, 14, 18C, 19F, 23F) are present in both pneumovax II and Prevnar whilst a further 5 are present only in pneumovax II (1, 3, 5, 7f, 19a). A normal adult response to Pneumovax II is >0.35 ug/ml in 8/12 serotypes (6/12 in children aged 2 to 5 years). Meningococcal C antibody protective level is 2.0 ug/ml. Hib ab protective levels - 1.0 ug/ml (long-term) and 0.15ug/ml (short-term). Tetanus antibody protective levels are 0.1 IU/ml (long-term) and 0.01 IU/ml (short-term).†

Specific antibody testing can be helpful in patients with recurrent infections. Specific antibody responses can be abnormal even if immunoglobulins are normal. Antibody responses are normally assessed 4 – 6 weeks after vaccination.

Fungal antigens – Specific IgG antibodies (Serum: species specific ranges)* (Up to 5 days)

Specific IgG antibodies directed against candida albicans, aspergillus fumigatus and micropolyspora faeni are available. Note: most adult women will have low levels of candida antibodies. IgG antibodies indicate previous exposure.

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Ganglioside antibodies GD1b (Serum: IgG <1:500 IgM <1:500)§ (Up to 28 days)

Antibody to the ganglioside GD1b has been associated with motor or sensorimotor neuropathies. High titres of anti GM1 are most typical of multifocal motor neuropathy but antibodies to other gangliosides such as GD1b and asialoGM1 may also be detected. Low titres of antibodies directed against GD1b, GM1 and asialoGM1 may also be detected in amyotrophic lateral sclerosis and Guillain -Barré syndrome. These antibodies are currently screened in house and positives are sent to Neurology, Southern General Hospital, Glasgow, for quantitation.

Ganglioside antibodies GM1 (Serum: IgG <1:500 IgM <1:500)§ (Up to 28 days)

The presence of antibodies directed against GM1 (monosialoganglioside GM) has been associated with motor and sensorimotor neuropathies and in particular with multifocal motor neuropathies. Lower titre of GM1 antibodies may also be found in amyotrophic lateral sclerosis and Guillain - Barré syndrome. a’GM1 antibodies may occur as either polyclonal or IgM monoclonal antibodies. The carbohydrate moiety of GM1, in particular the galactose and sialic acid residues, is the site of antibody binding to gangliosides. Due to the presence of similar moieties on other gangliosides low levels of antibody cross-reaction may be experienced in tests for gangliosides other than GM1. These antibodies are currently screened in house and positives are sent to Neurology, Southern General Hospital, Glasgow, for quantitation.

Ganglioside antibodies GQ1b (Miller Fisher syndrome) (Serum: <1:500 (IgG & IgM))§ (Up to 28 days)

These antibodies are currently screened in house and positives are sent to Neurology, Southern General Hospital, Glasgow, for quantitation.

Ganglioside antibodies sulphatide (Sensory neuropathy) (Serum: <1:10000 (IgG & IgM))§ (Up to 28 days)

These antibodies are currently screened in house and positives are sent to Neurology, Southern General Hospital, Glasgow, for quantitation.

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Gastric parietal cell antibodies (Serum: Negative) (Up to 4 days) Intrinsic factor antibodies should be carried out in conjunction with GPC antibodies

These antibodies are present in up to 90% of patients with atrophic gastritis and pernicious anaemia. Also present in gastritis without anaemia (12%), autoimmune thyroid disease (30%), Addison’s disease (25%) and iron deficiency anaemia (20%).

Gliadin deamidated peptide (DP) antibodies (Serum: IgG < 7 U/ml)* (Up to 4 days)

IgA anti-tissue transglutaminase (tTG), antibodies are specific for coeliac disease, but can be negative in patients with IgA deficiency. In this situation IgG anti-gliadin DP antibodies can be clinically useful. The titre of these antibodies decreases with gluten free diet, as does the level of endomysial antibodies and tTG antibodies.

It has been reported that IgA deficient patients have a ten to fifteen fold increased incidence of coeliac disease. It is therefore suggested that IgG anti-gliadin DP antibodies are carried out in all IgA deficient individuals.

Glomerular basement membrane (GBM) antibodies (Serum: < 7 U/ml)* (Up to 4 days)

A rapid qualitative test is now available

Test for Goodpastures syndrome. Antibodies to the non-collagenous portion of type IV collagen are detected by ELISA method as indirect immunofluorescence is both less sensitive and less specific being positive in only 75%, or less, of proven cases. Urgent requests for GBM antibodies (as with ANA, ANCA and dsDNA antibodies) must be arranged with the laboratory.

Glutamic acid decarboxylase antibodies: Stiff Man syndrome (Serum: 0 - 10 IU/ml)* (Up to 14 days)

Glutamic acid decarboxylase (GAD) is an enzyme concentrated in neurons, which control muscle tone and exteroreceptive spinal reflexes. High levels of antibodies to GAD are found in ~60% of patients with Stiff man syndrome; in IDDM the titres are much lower. The contribution of GAD antibodies to IDDM has not been proved. GAD has also been implicated in autoimmune encephalitis.

IgA antibodies (Serum: zero titre (negative))§ (Up to 28 days)

Anti-IgA antibodies occur in IgA deficient patients in receipt of blood products containing IgA. Their presence may increase risk of adverse transfusion reactions. This assay is currently sent to the National Blood Service, Sheffield, S5 7JN.

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Immunoglobulins (IgG/A/M) (Serum: IgG: 6.00 – 16.00 g/l † (adult): IgA: 0.80 – 4.00 g/l † IgM: 0.50 – 2.00 g/l) † (Up to 4 days)

N.B. Reference ranges are age specific and may differ between ethnic groups

Immunoglobulins are an essential request in recurrent infections, lymphoproliferative diseases including myeloma and all cases of ‘failure to thrive’. IgA deficiency occurs in 1:500 individuals but, transfusion reactions apart, may not be associated with disease. Polyclonal increases of IgG occur in chronic infection and inflammation, chronic liver disease and connective tissue diseases. Raised levels of IgM are found in acute inflammation and in primary biliary cirrhosis. (Markedly elevated IgM in the presence of mitochondrial antibodies is virtually diagnostic of PBC) Low levels of IgG and IgA may be due to loss (protein losing enteropathy or nephrotic syndrome), reduced synthesis (e.g. lymphopro-liferative disorders or primary immunodeficiency) or excessive catabolism. Low immunoglobulins always require further investigation. Where appropriate details are supplied age and sex related normal levels are printed on the report. See also IgG sub-classes and functional antibodies.

IgG Subclasses (Serum: IgG1: 3.2 – 10.2 g/l † adult: IgG2: 1.2 – 6.6 g/l † IgG3: 0.2 – 1.9 g/l † IgG4: 0.0 – 1.3 g/l)† (Up to 7days)

IgG subclass deficiency is mainly related to IgG1 and IgG2 where individuals may suffer recurrent infections.

Immunoglobulin D (IgD) (Serum: 0.05-0.20 g/L)‡ (Up to 14 days)

Serum IgD is measured in the case of IgD myeloma and some forms of periodic fever syndrome.

Immunoglobulin E (IgE) (Serum: 0 – 90 IU/ml, adult)‡ (Up to 7 days) N.B. Reference ranges may differ between ethnic groups

Serum IgE may be helpful in diagnosing atopic diseases however the normal range is very wide and levels do not correlate well with symptoms. Very high levels of IgE are seen both in atopic eczema and in parasitic infestations (especially S Mansoni) and may result in false positive specific IgE to a single allergen.

Intrinsic factor antibodies (Serum: < 6 U/ml)* (Up to 7 days)

Detected in 70% of patients with pernicious anaemia. This test should be carried out together with gastric parietal cell antibodies.

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Isoelectric focusing (Oligobanding) IgG (Paired serum and CSF: Clinical comment is supplied with each report) (Up to 14 days)

Please enclose CSF protein value with each request

Volume of CSF required: Ideally 1-2ml but minimum of 250 microlitres. Contact the lab if insufficient volume.

Oligobanding refers to discrete populations of immunoglobulin detected by electrophoresis in CSF, which are NOT matched in serum from the same patient. Oligobanding is seen in ~85-95% of patients with clinically proven multiple sclerosis. The assay is useful as a confirmatory test in multiple sclerosis but bands are not specific for this disease as they also occur in cerebrovascular accidents, in infections of the CNS and in pathological processes involving an immune response e.g. encephalitis, neuro-sarcoid and SLE. Please note that paired samples of CSF and serum are essential for this assay.

Liver antigen antibodies (blot) (Up to 28 days)

Detection and confirmation of antigen specific antibodies associated with primary biliary cirrhosis and autoimmune hepatitis. These include M2, LKM-1, LC-1, SLA/LP and f-Actin. M2 is also measure quantitatively by ELISA

LKM antibodies (Serum: Negative) (Up to 4 days)

These antibodies, which stain the cytoplasm of hepatocytes and proximal renal tubules are found in a subgroup of patients with ANA negative, autoimmune chronic active hepatitis (CAH). LKM1 antibodies are positive in CAH type 2, which is the most common autoimmune liver disease of childhood.

Lymphocyte cell markers (Up to 4 days)

A wide range of lymphocyte markers for assessment of immunodeficiency and lymphoproliferative diseases are available. A separate request form is in use for cell markers.

Lymphocyte function tests (Up to 28 days)

Special sample requirements. Please discuss with Consultant Immunologist.

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Mast cell tryptase

(Serum: 0 – 13.5 g/l)‡ Samples may be kept at room temperature for shipping purposes for 2 days.

(Up to 5 days – unless agreed as urgent by telephone)

For suspected cases of anaphylaxis. A clotted blood sample should be taken immediately, four hours and at 24 hrs post reaction. It is often difficult to give an immediate sample if patient is not in hospital. Tryptase release in IgE mediated allergy peaks at 4 hours and then decline. Please indicate time/date of adverse reaction and time/date of samples.

All patients who have suffered anaphylaxis should be referred to a specialist allergy clinic (NICE guideline http://www.nice.org.uk/guidance/cg134).

A persistently raised tryptase may indicate mastocytosis or a mast cell activation disorder.

Mitochondrial antibodies (Serum: Negative) (Up to 4 days)

Present in >90% of cases of primary biliary cirrhosis, often at high titre (>1:200). Also occasionally present in chronic active hepatitis and halothane induced hepatitis patients but with titres of <1:100. Serum IgM levels are invariably increased.

Mitochondrial (M2) antibodies (Serum: 0 – 10 EU/ml)‡ (Up to 7 days)

For those wishing to confirm the presence of mitochondrial antibodies or to monitor patients with a quantitative assay an EIA method is available which distinguishes antibodies to the major enzyme pyruvate dehydrogenase complex (M2) and affords a quantitative assay in EU/ml.

MuSK antibodies (Serum: Negative)§ (Up to 28 days)

Muscle specific tyrosine kinase (MuSK) is a surface membrane enzyme that is thought to be essential in aggregating AChR during the development of the neuromuscular junction. Anti-MuSK antibodies assist in confirming the diagnosis in myasthenia gravis when anti-AChR antibodies are absent. This assay is currently sent to Immunology, Churchill Hospital, Oxford.

Myeloperoxidase (MPO) antibodies (Serum: 0 – 9 EU/ml)* (Up to 7 days)

A rapid qualitative test is now available.

Antibody to myeloperoxidase is associated with organ-limited vasculitis including necrotising and crescentic glomerulonephritis. The assay is useful in confirming MPO specific antibodies in sera which are ANCA-positive. Typically the level of MPO antibodies parallel disease state with increasing levels when vasculitis is active.

Urgent requests must be arranged with the laboratory.

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Anti-Neutrophil cytoplasmic antibodies (ANCA) (Serum: Negative) (Up to 4 days)

Pattern and titre reported on positives [See MPO & PR3 antibodies]

c-ANCA is a test for granulomatosis with polyangiitis and microscopic polyarteritis (see also test for proteinase 3). p-ANCA may occur in other vasculitic disorders as well as some forms of glomerulonephritis (see also test for myeloperoxidase).

Neutrophil respiratory burst test (4 days)

This is a diagnostic test for chronic granulomatous disease. Please arrange with laboratory.

NMO antibodies (Serum: Negative)§ (Up to 28 days)

Anti-NMO antibodies are associated with neuromyelitis optica (NMO) also known as Devic’s disease and optic-spinal multiple sclerosis. It is a severe inflammatory demyelinating disease that affects optic nerves and spinal cord without affecting the brain. Aquaporin 4 has been identified a major NMO antigen and the test offer is against this antigen. This test is used to distinguish NMO from multiple sclerosis. This assay is currently sent to Immunology, Churchill Hospital, Oxford.

Pancreatic islet cell antibodies (Serum: Negative) (Up to 14 days)

At the time of diagnosis 75% of type I diabetics have detectable levels of circulating islet cell antibodies. Such antibodies decrease and eventually disappear with duration of disease. Some studies have indicated persistent levels of antibodies in association with polyendocrine disease (type Ib). There have been no reports of antibodies to pancreatic islet cells in type II diabetics.

Paraprotein (Monoclonal protein, M-protein) quantitation Reported in g/l. (Up to 4 days)

Levels of monoclonal IgG, IgA, IgM, IgD and in some instances IgE are measured immunochemically. Immunofixation of presentation sample defines both the isotype and light chain type. Follow up specimens will be subjected only to electrophoresis unless immunofixation is required to confirm complete response. (See ‘guide to appropriate use of tests at the front of this booklet). The presence of an M-protein (paraprotein) should prompt investigation of B cell malignancy, particularly myeloma, (IgG, IgA) and lymphoplasmacytoid lymphoma (IgM). Monoclonal gammopathy of uncertain significance (MGUS) is found in one or more percent of the general population over the age of 50 years.

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MAG antibodies - paraprotein neuropathies (Serum: Negative)* (Up to 14 days)

Myelin associated glycoprotein (MAG) is a glycoprotein component of the myelin of central and peripheral nervous systems. Monoclonal reactivities against MAG are detected in about 50-75% of patients with IgM paraproteinaemia and peripheral neuropathy. Sera from patients with neuropathy that are negative for MAG antibodies often exhibit reactivity against various gangliosides.

Phospholipase A2 (PLA 2) receptor antibodies (Serum : Negative)* (Up to 14 days)

Autoantibodies to the M-type phospholipase A2 receptor (PLA2R) are sensitive and specific for idiopathic membranous nephropathy (IMN), an organ specific autoimmune disease of the glomeruli. The test is helpful both in the diagnosis of IMN and monitoring response to treatment. These antibodies are specific and are found in up to 70% of the patients with IMN.

Proteinase 3 (PR3) antibodies (Serum: 0 – 3.5 EU/ml) (Up to 7 days)

PR3 antibody is a marker for granulomatosis with polyangiitis and is occasionally detected in microscopic polyarteritis. The quantity of PR3 antibody generally parallels disease activity with higher levels in the active state of the disease. EIA affords a quantitative assay which is useful when monitoring the disease. Antibodies to PR3, are responsible for the characteristic granular cytoplasmic pattern of the neutrophils when stained by IIF. Urgent requests must be arranged with the laboratory.

Paraneoplastic pemphigus antibodies (Serum: Negative) (Up to 28 days)

Paraneoplastic pemphigus is a severely debilitating blistering disease affecting skin and mucous membranes in patients with malignancy, such as haematologic (lymphoma and leukaemia), sarcomas, thymomas and Castleman syndrome. As in other types of pemphigus, IgG is deposited on the cell surfaces of epidermal and epithelial cells in and around affected areas. IgG antibodies to basement membrane zone may also be present.

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Paraneoplastic neurological antibodies:

Paraneoplastic neurological antibodies are associated with paraneoplastic neurological syndrome and systemic malignancies (Up to 7 days)

Antibody Neurological disorder(s) Most frequent tumour(s)

Yo (PCA-1) paraneoplastic cerebellar degeneration Ovary, breast

Ma (Ma1) paraneoplastic neurological disorder, brainstem encephalomyelitis

Various, lung cancer

Ta (Ma2) brainstem encephalomyelitis, limbic encephalomyelitis

Testicular cancer

Hu (ANNA1) paraneoplastic cerebellar degeneration, paraneoplastic encephalomyelitis, sensory neuropathy

small cell lung carcinoma

Ri (ANNA2) opsoclonus/myclonus, paraneoplastic cerebellar degeneration, brainstem encephalomyelitis

Breast, small cell lung carcinoma, gynaecological

GAD Stiff person syndrome Breast, colon, small cell lung carcinoma

CV2/CRMP5 paraneoplastic encephalomyelitis / sensory neuropathy

small cell lung carcinoma, thymoma

Amphiphysin Stiff person syndrome, paraneoplastic encephalomyelitis

Breast cancer, small cell lung carcinoma

Tr paraneoplastic cerebellar degeneration Hodgkin’s lymphoma

The presence of Yo, Hu (ANNA1) and Ri (ANNA2), Ma/Ta, CV2/CRMP5 and amphiphysin antibodies are confirmed by Western blot.

Rheumatoid factor (Serum: 0-14 IU/ml)* (Up to 4 days)

Also see Cyclic citrullinated peptide (CCP) antibodies

Rheumatoid factors are antibodies which are directed against other immunoglobulins. A latex enhanced turbidimetric assay is used to detect these. Approximately 70% of patients with rheumatoid arthritis are sero positive and antibodies may occur in other conditions including many infections, myeloma, lymphomas, cryoglobulinaemia and connective tissue diseases. Antibodies (RF) may also be found in allegedly normal individuals aged over 75. Titre of rheumatoid factor is less sensitive than sequential assay of CRP when monitoring activity of rheumatoids.

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Serum specific IgE (allergen specific IgE) (Serum: 0 – 0.35 kU/l)* (Up to 7 days, if in stock)

N.B. Total IgE will be carried out on all Serum specific IgE requests unless an IgE level is stated at the time of request.

Assays for the detection of circulating IgE antibodies directed against specific antigens are available to a wide range of allergens. Tests for common substrates include animal fur or dander, house dust mite, tree and grass pollens, moulds, feathers and an extensive range of food substances including a variety of nuts and are performed in house. Requests for more unusual allergens are sent to Sheffield

Serum electrophoresis (Serum: Clinical comment will accompany each report) (Up to 4 days)

Sera are screened for qualitative abnormalities in proteins especially of the immunoglobulins. Scans demonstrating a monoclonal band are automatically followed up using immunofixation to determine both the isotype and the light chain of the monoclonal protein. Other typical patterns seen on electrophoresis may indicate evidence of acute phase responses, immunodeficiency, etc.

Where myeloma is suspected urine and serum should be sent together.

Serum immunoglobulin free light chains (FLC) Kappa 3.30 – 19.40 mg/l * Lambda 5.71 – 26.30 mg/l * Kappa / Lambda ratio 0.26 – 1.65 * (Up to 4 days) This assay may be inaccurate at levels <0.9mg/l. In a small proportion of patients with high serum FLC levels, false negative results may occur as a result of “antigen excess”. Any anomaly between the serum FLC results and other laboratory tests and/or clinical evidence should be reported to the laboratory for re-testing the serum FLC.

Normal plasma cells make more immunoglobulin light than heavy chains and secrete free light chains in amounts detectable in serum (estimated to be 0.5g/day). Serum free light chains are removed by glomerular filtration with a half-life of a few hours. They are not easily detectable in urine until the threshold for tubular reabsorption is exceeded (10 – 20g / day).

Serum FLC measurements are recommended in assessment of all plasma cell dyscrasias and in B cell lymphoproliferative diseases. They are particularly important in diagnosis and management of light chain only myeloma.

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Skin antibodies (Serum: Negative) (Up to 14 days)

Antibodies are found in (i) intercellular substance of the epidermis (desmosome), which strongly suggest a diagnosis of pemphigus though these antibodies may also be found in patients with severe burns or a trichophyton infection. (ii) dermal-epidermal basement membrane which is highly specific for bullous pemphigoid and is present in 80% of these patients. A titre is useful in monitoring the disease.

Smooth muscle antibodies (Serum: Negative) (Up to 4 days)

Present in high titre in up to 70% of patients with autoimmune hepatitis who may also be positive for mitochondrial, nuclear and dsDNA antibodies (25%)

Striated muscle antibodies (Serum: Negative) (Up to 14 days)

In patients with Myasthenia Gravis with thymoma these antibodies are typically positive but in such patients without thymoma the antibodies occur in only 60% of cases. This assay is usually carried out with a test for acetyl choline receptor antibodies but as this latter test is sent away to Oxford for quantitative assay it will not be carried out as a routine unless specifically requested.

Thyroid peroxidise antibodies (microsomal [TPO]) (Serum: < 60 IU/ml)* (Up to 4 days)

Present at high levels in 95% of patients with Hashimotos thyroiditis, 20% of patients with Graves disease and 90% of patients with primary myxoedema. Antibodies may also be present at low levels in colloidal goitre, thyroid carcinoma, De Quervains thyroiditis, other organ specific auto-immunities and in healthy individuals. If persistent in euthyroid individuals it may indicate autoimmune thyroiditis and predisposition to future thyroid failure.

Thyroglobulin antibodies (Serum: Negative)§ (Up to 28 days)

Thyroglobulin antibodies will be assayed for specific patients if requested. For example, where thyroglobulin levels are used as a tumour marker. This assay is currently sent to Immunology PRU, Northern General Hospital, Sheffield.

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TSH receptor antibodies (Serum: Negative)§ (Up to 28 days)

Hyperthyroidism in Grave’s disease is due to autoantibodies to the TSH receptor and measurement of these autoantibodies can be useful in disease diagnosis and management. This assay is currently sent to Immunology PRU, Northern General Hospital, Sheffield.

Tissue Transglutaminase antibodies (Serum: < 20 U/ml)* (Up to 4 days)

[Also see gliadin deamidated peptide antibodies]

The endomysial autoantigen has been identified as the protein cross-linking enzyme tissue transglutaminase (tTG). Antigen specific assays provide an alternative to the conventional indirect immunofluorescence assay using primate oesophagus.

We screen for coeliac disease with an IgA anti-TTG assay. A very low result suggests that the patient may be IgA deficient and therefore we proceed to an IgG anti-gliadin deamidated peptide assay which is more sensitive than IgG anti-TTG .

IgA deficiency (partial or complete) is about 1:400 blood donors and 1:40 patients with coeliac disease.

Quantiferon (QFT) (Non-reactive) (7 days)

Or

T-SPOT TB

Also called Elispot (Non-reactive) (2 days if sent on a Tuesday)

Interferon gamma release assay (IGRA) that measures T cells specific to Mycobacterium tuberculosis antigens. The test can be performed on all individuals including those who have had BCG vaccination and the immunocompromised. A positive test indicates previous exposure to TB and cannot distinguish between latent and active infection.

IGRAs are routinely performed using the Quantiferon assay method. In certain circumstances (such as lymphopeania) a TB Elispot may be performed by prior arrangement.

Refer to important notes regarding collection of blood and its despatch to the laboratory (page 4-5).

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Urinary free light chains and Bence Jones Protein (BJP) (Urine: undetectable)‡ (Up to 4 days)

Monoclonal free light chains in the urine (Bence Jones protein) are currently detected by immuno-fixation. This is not a quantitative method, and 24 hour urine collections are not required.

A quantitative method for detecting free light chains in the urine is being developed for use in clinical trials.

Viscosity (Plasma: 1.50 – 1.72 (c.f. to water))* (Up to 10 days)

Plasma viscosity is an essential test when monitoring Waldenstroms’ macro-globulinaemia and also when investigating an unexplained retinal or cerebrovascular occlusion. In such patients a cryoglobulin may also be present.

Blood samples can be transported at room temperature but separated plasma should not be refrigerated. Please send EDTA blood.

Voltage gated calcium channel antibodies (Serum: 0 – 45 pM)§ (Up to 28 days)

The Lambert Eaton myasthenic syndrome (LEMS) is a form of myasthenia often associated with small cell lung carcinoma.

In ~50% of cases there is IgG mediated reduction in presynaptic voltage gated calcium channels. This assay is currently sent to Immunology, Churchill Hospital, Oxford.

Voltage gated potassium channel antibodies (Serum: 0 – 100 pM)§ (Up to 28 days)

Antibodies to voltage gated potassium channels are found in ~40% of patients with acquired neuromyotonia. This assay is currently sent to Immunology, Churchill Hospital, Oxford.

Key for normal ranges Where a quantitative normal range is provided the origin of that range is as follows: * Manufacturers reference range (verified in-house) † National (or international) reference laboratory range (verified in-house) ‡ Internally generated reference range § Send away test with accompanying (referral laboratory derived) range

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12. IMMUNOPHENOTYPING

Cell marker tests performed by the Clinical Immunology Service

All queries and requests for urgent investigations to be addressed to Mr Peter Richardson or Mr Steven Dix in the Clinical Immunology laboratory (0121 414 3824)

Specimen collection for Cell Marker Work

All samples must arrive in the laboratory by 5.00 p.m. on the day of sampling accompanied by clinical details. Samples received on working day 1 will normally be processed working day 2 and reported working day 3 (except samples received on a Friday). At present there is no weekend or Bank Holiday service.

Urgent samples (which have been arranged and agreed with the lab in advance via telephone) will be processed and reported on the day of receipt (Monday to Friday) provided they reach the lab before 2.00pm. Results will be telephoned to the requesting clinician if a mobile, or direct landline, telephone number is provided at the time of requesting. Turnaround time data for urgent requests will be available on request.

Immunodeficiency studies: please telephone for clinical discussion and advice regarding appropriate tests and samples required (07831 681 955 or 0121 414 3824) Bone marrow * 4ml bone marrow in EDTA and an unfixed marrow smear Blood ** 5mls blood in EDTA Effusions At least 20mls in EDTA C.S.F. As much as possible in a universal bottle ideally containing tissue culture medium. Please discuss with the laboratory if only small volume as quality of testing is reduced. Tissue biopsy Freshly excised and put in cold tissue culture medium or saline. NB no preservative T cell antigen receptor & Blood or bone marrow drawn into an EDTA bottle immunoglobulin gene (Heparinised material is unsuitable for the PCR process) rearrangement studies Immunodeficiency studies (inc HIV+) *** 5ml EDTA blood * Results from haemodilute bone marrow samples will be unreliable as not representative ** Send additional clotted blood and urine samples if assessment of immunoglobulin concentrations and M-protein (paraprotein) analysis is also requested. *** For paediatric and adult cases of congenital immunodeficiency please discuss with a clinician in order to ensure the most appropriate assays are booked in and performed.

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Haematological Malignancies (Turnaround time 3 days for most (75%) of samples)

Panels currently available

All include morphological appraisal and a written report. Lymphoproliferative disease /LPD Screen

Appropriate for the investigation of unexplained lymphocytosis, mature B cell neoplasms and mature T cell neoplasms.

Kappa, Lambda, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD16, CD19, CD20, CD23, CD25, CD27, CD30, CD34, CD38, CD45, CD49d, CD56, CD79b, CD103, CD200, TCR-gamma/delta.

CSF -LPD

Kappa, Lambda, CD2, CD3, CD4, CD5, CD8, CD7, CD19, CD20, CD23, CD45, CD200, TCR-gamma/delta

Myeloma panel

Appropriate for the investigation of known or suspected cases of myeloma, MGUS, lymphoplasmacytoid lymphoma and amyloid.

Cytoplasmic and surface Kappa and Lambda, CD3, CD5, CD10, CD11c, CD19, CD20, CD23, CD25, CD27, CD34, CD38, CD45, CD49d, CD56, CD79b, CD103, CD117, CD138, CD200,

Myeloid screen

Appropriate for ?MDS, ?MPD, and as part of acute leukaemia screen for ?AML

Kappa, Lambda, CD3, CD4, CD7, CD11b, CD13, CD14, CD16, CD19, CD33, CD34, CD45, CD56, CD71, CD117, CD123, CD235a, HLA-DR.

(Additional markers CD38, CD123, CD45RA, CLL-1 may be included)

CSF-Myeloid

CD3, CD11b, CD13, CD33, CD34, CD45, CD117

Acute leukaemia screen

This panel may be used for the diagnosis of a possible acute leukemia including lineage determination. It can be processed to provide an urgent, telephoned report to the requesting clinician. The information thus derived will be used to select a more appropriate secondary panel / additional markers (e.g. CD123 for blastic plasmacytoid dendritic cell neoplasm) if appropriate.

Kappa, Lambda, CD2, CD3, CD4, CD7, CD8, CD10, CD11b CD11c, CD13, CD14, CD16, CD19, CD20, CD23, CD25, CD27, CD33, CD34, CD45, CD49d, CD56, CD71, CD103, CD117, CD200, CD235a, TCR-gamma/delta, HLA-DR.

Cytoplasmic / Intracellular panel

Kappa, Lambda, MPO, TdT, CD3, CD22, CD79a, CD38

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AML

For diagnosis and follow up of non-trial AML patients.

CD7, CD11b, CD13, CD14, CD19, CD33, CD34, CD38, CD45, CD56, CD117, HLA-DR.

The cytoplasmic/intracellular panel, and/or additional markers (CD38, CD123 and CD45RA) may also be included.

If AML M3 (APML) is suspected, a fixed cytospin may be stained for PML protein

B-ALL

Appropriate for the diagnosis and follow-up of precursor B lineage neoplasms.

CD10, CD13, CD19, CD20, CD22, CD27, CD33, CD34, CD38, CD45, surface Kappa, surface Lambda.

CSF- B-ALL

CD10, CD19, CD27, CD34, CD38, CD45, CD79b

T-ALL

Appropriate for the diagnosis and follow-up of precursor T lineage neoplasms

CD1a, CD2, CD3, CD4, CD8, CD5, CD7, CD34, CD38, CD45, CD117

The cytoplasmic/ intracellular panel (see above) may be added to confirm lineage.

CSF- T-ALL

CD2, CD3, CD4, CD7, CD34, CD45, CD117

PNH screen

Appropriate for the investigation of suspected or known PNH cases. N.B. This assay requires freshly drawn EDTA blood and should be received within 48 hours of collection. Bone marrow samples are not suitable.

CD15, CD24, FLAER on neutrophils

CD64, CD14, FLAER on monocytes

CD59, CD235a on red blood cells.

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Investigation of Immunodeficiency

T, B and NK lymphocyte markers

CD3, CD4, CD8, CD19, CD16/56 expressed as percentile and absolute values Immunophenotyping for immunodeficiency

B cell immunophenotyping, based on the Euroclass panel: CD5, CD19, CD20, CD21, CD27, CD38, CD45, IgD, IgM.

Research Tubes:

1. T cell immunophenotyping and autoimmune lymphoproliferative disease (ALPS) screening: CD3, CD4, CD8, CD25, CD27, CD28, CD45RA, CD69 CD127, TCR alpha/beta, TCR gamma/delta

2. MHC class I and II

Leukocyte adhesion deficiency surface markers: CD18, CD15 (screening test undertaken in CIS and second confirmatory sample sent to Great Ormond Street Hospital)

HIV monitoring

Expressed as percentile and absolute values: CD3, CD4, CD8

Immunoglobulin/T cell receptor gene studies

This assay is carried out by the West Midlands Genetic Service.

The following functional investigations for immunodeficiency are also performed, please see Cell function section below for more information;

Lymphocyte proliferation studies

Neutrophil function studies (respiratory burst)

Anti-cytokine antibodies and antibodies to biological therapeutics

Cytokine measurement

Full type 1 cytokine studies

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11. CELL FUNCTION ASSAYS

Neutrophil/lymphocyte functional studies

We perform a number of basic investigations to test defects in cell function including neutrophil oxidative burst lymphocyte proliferation and function including cytokine production. These investigations are labour intensive and technically demanding. To identify which tests are most appropriate please discuss with one of the clinicians on the mobile telephone numbers listed on page 4. Specialist staff and facilities are booked to carry out detailed functional assessments of neutrophils and lymphocytes. Only a limited number of appointments can be made. It is essential that the blood arrives in the laboratory in good condition and with sufficient time to carry out the assays. We cannot guarantee to carry out investigations if samples are received after 10am. Functional studies must be booked in advance with the laboratory. Please call 0121 414 4069. Please carefully follow the guidelines below:

1. The following samples should be taken and sent by taxi without delay to arrive by 10am:

o 10ml blood in Lithium heparin o 10ml clotted blood o 5ml blood in EDTA.

In the case of children the minimum blood volume required should be confirmed with the laboratory.

2. A full blood count and white cell differential should be done on the same day at your hospital if enumeration of cell populations required.

3. A concise clinical summary including results of preliminary tests should be sent with the

specimen.

4. Samples should be addressed to: Clinical Immunology Service, Medical School, Birmingham and labelled “URGENT!!” contact 0121 414 4069 (external) or 44069 (internal) on arrival at the medical school foyer or CIS reception. Include the telephone number, or bleep, of the doctor to be contacted in case of any query. Available Assays:

Lymphocyte proliferation studies (1 x 6ml Lithium heparin) This assay can only be run on Tuesdays or Fridays due to incubations required. Proliferation stimulants include PHA, PWM, candida. Other stimulants may be available on request. Please send a time matched healthy control with patient bloods.

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Neutrophil function studies (respiratory burst) 5ml blood in EDTA. Performed by flow cytometric dihydrorhodamine assay. Results within 24 hours if urgent. Please send a time matched healthy control with patient bloods. Anti-cytokine antibodies (research test) : 6ml clotted blood Antibodies to biological therapeutics (Sent to St Thomas ViaPath, London) Collect blood into a serum separation or plain tube, preferably shortly before drug administration (trough levels). Centrifuge sample at 3000 rpm for 10 minutes, aliquot serum or plasma and keep in fridge until transport. If transport is going to be delayed by > 5 days, freeze at -20°C and send by first class post at a later date. By arrangement with the laboratory. Cytokine measurement: Collect blood into a serum separation, plain, or EDTA tube, Allow serum 45 minutes to clot or separate plasma from EDTA straight away. Centrifuge sample at 3000 rpm for 10 minutes, aliquot serum or plasma and freeze at -80°C until ready to send on dry ice via courier. Available cytokines include: - IL-6, TNF-a, IL-17, IFN-g, VEGF. Other cytokine measurements may be available on request. Full type 1 cytokine studies Contact Addenbrooke’s Hospital (Cambridge) for sample requirements. Please send blood directly to Addenbrooke’s Hospital, Cambridge. T-SPOT TB (ELISPOT): Two 6ml Lithium Heparin specimens are required for this assay. Use Cell Markers request form. Samples must arrive in the laboratory before 2pm on the day that they are drawn (Tuesdays only: On others days this assay may be available under special circumstances and by prior arrangement only). This assay must be booked with laboratory staff before sending. (Please note: samples sent on Fridays will NOT be processed).

Quantiferon (QFT): Two 6ml Lithium Heparin specimens are required for this assay. Use Cell Markers request form. Samples must arrive in the laboratory before 2pm on the same day as they are drawn. Testing is only carried out Monday to Thursday. (Please note: samples sent on Fridays will NOT be processed).

Documents

Department of Immunology - University of Birmingham of Immunology and Immunotherapy College of Medical & Dental Sciences Clinical Immunology Service Laboratory Handbook A brief guide