clinical immunology service - labroratory handbook · the clinical immunology service (cis), in the...

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Updated: February 2020 Review planned: February 2022

Institute of Immunology and Immunotherapy College of Medical & Dental Sciences

Clinical Immunology Service

Laboratory Handbook

A brief guide for clinical and laboratory staff

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CONTENTS

Section Page 1 Introduction 3 2. Contact Details 4 3. Routine/Urgent assay processing 5 4. General Specimen collection requirements: 6 5 Request forms 6 6 Specimen acceptance/rejection 7 7 A brief guide to common immunology tests 9

8 Myeloma screen guide 10 9 Specimen retention/additional tests 11 10 Data protection 11

11 Complaints 11 12 General assays/adult reference ranges 12 13 Immunophenotyping assays including specimen collection

requirements Haematological malignancies Investigation of immunodeficiency

30 31 33

14 Cell function assays 34

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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 histopathology, genetics and haematology.

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

during working hours Monday to Friday via the telephone numbers listed on page 4. There is

no formal on-call service but clinically urgent requests may be arranged through a clinician,

clinical scientist or senior biomedical scientist, by telephoning the laboratory. On university

closed days (see website for details www.birmingham.ac.uk/staff/employeebenefits/closed-

days.aspx ), only urgent assays will be performed by the laboratory.

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 5). 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.

http://www.birmingham.ac.uk/staff/employeebenefits/closed-days.aspx

http://www.birmingham.ac.uk/staff/employeebenefits/closed-days.aspx

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1. CONTACT DETAILS

Postal Address: Clinical Immunology Service Institute of Immunology and Immunotherapy Medical School University of Birmingham Vincent Drive Edgbaston Birmingham B15 2TT

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

or search the internet for: “Clinical Immunology Birmingham”

Key contact numbers: General telephone enquiries/results: (0121) 414 4069 Fax: (0121) 414 3069 Laboratory Manager: (0121) 414 3092

Clinical enquiries: General immunology enquiries: Dr A.G. Richter: email: [email protected] Dr A.M. Shields: email: [email protected]

Myeloma/Lymphoma/Leukaemia enquiries: Prof M.T. Drayson: email: [email protected] Prof S.D. Freeman: email: [email protected]

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3. ROUTINE/URGENT ASSAY PROCESSING

Daily assays: As a general guide, most immunochemistry, electrophoresis and immunophenotyping 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 – Monday Functional C1Inh – Once a week (see page 6 for collection requirements) Organ specific antibodies – Friday IgG subclasses – Twice a week MPO/PR3 antibodies – 2 or 3 times a week GAD antibodies – Every 2 weeks M2 mitochondrial ELISA – Once a week

Urgent assays: (e.g. Tau protein, MPO/PR3/GBM abs, acute leukaemia

immunophenotyping). 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 must be clearly marked “Urgent” and with which

member of staff the request was discussed. The sample must 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) allow blood to clot for tests that require serum. Some complement components are unstable. For Functional C1Inh please send 5ml blood

in a sodium citrate (blue top) tube. For distant clinics the plasma should be separated within

the hour, frozen and sent to the laboratory to arrive frozen. 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).

All samples MUST be shipped to the department in appropriate packaging. For transport via

road, rail and/or air regulations must be followed and UN3373 compliant packaging used. For all immunophenotyping and cell function assays please see the notes towards the end of

this handbook under ‘Immunophenotyping’ (page 30) and ‘Cell function assays’ (page 34).

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 longer.

Please also note special requirements for cell work and neuroimmunology requests

both of which have separate request forms (see section 5)

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

General immunological investigations – REQ.G.1.3

Haemato-oncology requests – RF001 MIRHO Request form v3.0

Neuroimmunology requests – REQ.N.1.3

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.

www.birmingham.ac.uk/facilities/clinical-immunology-services/handbook.aspx

Where minimum volumes are stated, this is the absolute minimum volume required

for that assay. Preferred volumes will be greater.

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

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6. SPECIMEN ACCEPTANCE/ REJECTION 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 hospital

number or other unique identifier

(e.g. referring lab number)

Date and time of collection

Form Patient’s full name.

Patient’s NHS/CHI number

number or other unique identifier

(e.g. referring lab number)

Date and time of collection

Patient’s sex

Destination for report

Requesting consultant/GP

Signature of person taking the

sample

Specimen type

Test(s) required

Relevant clinical information

Clinician’s telephone/bleep

number (essential for urgent

requests)

Patient’s address

Requesting clinician’s specialty

Clinical information MUST be provided for all requests received by the laboratory.

Only correctly and clearly labelled samples with matching request forms will be accepted.

Sample Rejection Criteria.

Rejection of requests will be made in circumstances where there is a failure to provide essential

or mandatory details. This may represent a risk to the patient and may compromise their safety.

Samples may be rejected in the following circumstances:

The minimum essential information is missing from the sample or request.

The sample and request form information do not match.

The sample is unlabelled or otherwise unsuitable (e.g. wrong tube type).

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Where essential information is missing from a sample or request form, the laboratory will

attempt to contact the requesting medical officer/practitioner identified on the request using the

contact number, where this is given.

The laboratory may require the requesting medical officer/practitioner to attend the laboratory

to complete or amend details before the request is accepted.

If the laboratory is unable to contact the requesting medical officer/practitioner or colleague the

sample will be rejected or analysis deferred until contact is made.

When samples are rejected due to insufficient information, a report will be issued through the

laboratory information system as soon as practicable, stating that the sample has not been

processed and giving details.

Some assays are sensitive to interferences from icterus, haemolysis or lipaemia. If this is the

case, the assay may not be possible and the sample will be rejected. This will be indicated on

the report issued through the laboratory information system, stating that the sample has not

been processed and giving details.

Lipaemic or haemolysed samples are known to affect electrophoresis resulting in falsely

positive results so any affected samples will be rejected prior to analysis.

Samples that have been rejected and not processed may be stored in the laboratory for up to

one week to allow the requesting practitioner time to get in touch. This storage will be at the

discretion of individual departments.

Failure to provide clinical information with the request may result in reporting delays or

in some cases, assays will not be carried out without clinical justification of the work.

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7. 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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8. MYELOMA SCREEN GUIDE

Myeloma (median age 68 years) 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 reference range in 80% of myeloma patients

Polyclonal IgG, IgA and IgM levels are above the reference 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 reference 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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9. SPECIMEN RETENTION / ADDITIONAL TESTS

Most immunophenotyping 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.

Where repeat tests are requested within an inappropriate timescale the department will issue

a report detailing the previous result and will store the sample in case other investigations are

required. This includes:

Test

Timescale for intervention

(days)

SFLC 2

ANCA, dsDNA 7

Complement C3/C4 30

MUSK, NMO, VGC, VGK 90

ANA 180

CCP, ENA, M2, mitochondrial, rheumatoid factor, TPO 330

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

Protection Act 1998 and General Data Protection Regulation (EU) 2016/679 (GDPR). Staff

processing personal information do so in accordance with the University’s Data Protection

Policy (https://www.birmingham.ac.uk/privacy/index.aspx), and training in data protection is

mandatory for staff. To contact the University’s Data Protection team or to make a complaint

about how your data is or has been processed, email: [email protected]

or telephone +44 (0) 121 414 3916

11. COMPLAINTS

We welcome feedback on our service, and treat all complaints seriously. If your 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 departmental quality manager by phoning (0121) 414

4069. If the person you want to contact is not available, please telephone the department and

you will be directed to the most appropriate person.

https://www.birmingham.ac.uk/privacy/index.aspx

mailto:[email protected]



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12. AVAILABLE ASSAYS/REFERENCE RANGES TEST: (Preferred sample: reference range) (Turnaround time – from receipt in department to result available)

Adrenal cortical antibodies (Serum : Negative) Minimum sample volume 500µl Preferred sample volume 2ml (Up to 14 days)

Test for autoimmune adrenal disease. Also see endocrine antibodies

Anaphylaxis testing See mast cell tryptase

Anti-nuclear antibodies (Serum: Titre <1:100) Low ANA titres of 1:100 (positive Fluorescence at serum dilution of one in one hundred) are generally not significant in adults but can be in children. Minimum sample volume 500µl Preferred sample volume 2ml (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)* Minimum sample volume 500µl Preferred sample volume 2ml (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 14 days)

See NMO antibodies

Aspergillus - specific IgG antibodies (Serum: < 40 mgA/L)* Minimum sample volume 500µl Preferred sample volume 2ml Also see fungal antigens (Up to 5 days)

Specific IgG antibodies directed against aspergillus fumigatus demonstrate previous exposure.

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

Autoimmune encephalitis screen. (Serum/Plasma: Negative) Minimum sample volume 500µl Preferred sample volume 2ml Minimum volume for CSF is 250µl: (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**

NMDAR Teratoma

AMPAR1 & 2 Lung, Breast, thymus

Voltage gated Potassium Channel associated proteins

LGI1 Leucine-rich glioma inactivated protein 1

Lung, thymus

CASPR2 Contactin-associated protein 2 Thymus

Gamma-aminobutyric acid receptor

GABABR1/2 Gamma-aminobutyric acid receptor type B1/2

Lung (SCLC)

** The cancer association is variable

** Clinical: severe neuropsychiatric symptoms.

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

Avian antigens - specific IgG antibodies Serum: Negative) Minimum sample volume 500µl Preferred sample volume 2ml (Up to 5 days)

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

B2 microglobulin (B2M) (Serum: 0 – 4.0 mg/l)‡

Minimum sample volume 500µl Preferred sample volume 2ml (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.

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B2GP1 antibodies (Serum: 0 – 20 U/ml)* Minimum sample volume 500µl Preferred sample volume 2ml (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) Minimum sample volume 500µl Preferred sample volume 2ml (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)* Minimum sample volume 500µl Preferred sample volume 2ml (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.

C-Reactive Protein (Serum: 0 – 10 mg/l)‡

Minimum sample volume 500µl Preferred sample volume 2ml (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.

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CSF Tau protein: asialo-transferrin Present only in CSF (Up to 4 days) Ideal suspected CSF volume 250µl but not less than 50µl.

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)†


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.

C1 (esterase) Inhibitor Immunochemical levels: (Fresh serum: 0.18 – 0.30 g/l)‡

Minimum sample volume 500µl Preferred sample volume 2ml (Up to 10 days) Functional activity (Fresh plasma (citrate-blue topped tube): 70 – 130%)* Minimum sample volume 500µl Preferred sample volume 2ml (Up to 28 days) Please see page 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.

Cyclic citrullinated peptide antibodies (CCP) (Serum: < 7 U/ml)* Minimum sample volume 500µl Preferred sample volume 2ml (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

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dsDNA antibodies (Serum: EIA: < 30 IU/ml * Crithidia IIF: Negative) Minimum sample volume 500µl Preferred sample volume 2ml (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.

Endocrine abs (Adrenal, Ovary, Testis) (Serum: Negative) Minimum sample volume 500µl Preferred sample volume 2ml (Up to 14 days)

Adrenal antibodies can be associated with autoimmune Addison’s disease where a gradual destruction of the adrenal gland leads to adrenocortical insufficiency. Steroid cell antibodies can also be associated with premature ovarian failure and premature testicular failure. They are also associated with the autoimmune polyglandular syndrome types 1, 2 and 3. Autoimmune endocrinopathy may be seronegative in a minority of cases.

Endomysial abs (Serum: Negative) Minimum sample volume 500µl Preferred sample volume 2ml (Up to 14 days)

IgA abs directed against the endomysium are detected in 70% of patients with dermatitis herpetiformis and >90% of patients with untreated coeliac disease but are rarely present in normal individuals or in patients with other enteropathies. Decreasing antibody titres correlate well with adherence to gluten free diet.

Extractable Nuclear Antigen (ENA) antibodies. (Serum: 0 – 20 EU/ml)* Minimum sample volume 500µl Preferred sample volume 2ml (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.

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Specific microbial antibodies (Functional antibodies) (Serum required) Minimum sample volume 2ml (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)* Minimum sample volume 500µl Preferred sample volume 2ml (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 (Serum) At least 2ml sample required.

Ganglioside antibodies GD1b (Serum: IgG <1:500, IgM <1:500)§

(Up to 14 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-Barre 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 14 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 14 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 14 days)

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

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Ganglioside antibodies – further information:

Clinical presentation Associated Ganglioside antibodies Isotype

Guillain-Barré syndrome GM1, GD1a, GD1b, GT1a, GT1b, GQ1b IgG (IgM)

Miller-Fisher syndrome GQ1b, GT1a IgG

Multifocal muscular neuropathy

GM1, GM2, GM3, GD1a, GD1b IgM

Chronic inflammatory demyelinated polyneuropathy (CIDP)

GM2, GM3, GD1a, GD1b IgM

Chronic-ataxic neuropathy (CANOMAD)

GM3, GD1b, GD2, GD3, GT1b, GQ1b IgM

Acute sensory ataxic neuropathy GD1b, GD3

IgG

Acute muscular axonal neuropathy

GM1, GD1a IgG

IgM paraproteinemia, demyelinating neuropathy

Sulphatides IgM (IgG)

GM1, GM2, GD1a, GD1b and GQ1b are tested as these will capture majority of the neuropathy related positivity. The additional antigens are also reported as these share the same epitopes. GM4 can be associated with some neuromuscular disease & inflammatory neuropathies.

Gastric parietal cell antibodies (Serum: Negative) Minimum sample volume 500µl Preferred sample volume 2ml (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)* Minimum sample volume 500µl Preferred sample volume 2ml (Up to 7 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 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.

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Glomerular basement membrane (GBM) antibodies (Serum: < 7 U/ml)* Minimum sample volume 500µl Preferred sample volume 2ml (Up to 4 days)

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)* Minimum sample volume 500µl Preferred sample volume 2ml (Up to 14 days) GAD index GAD Index is available to determine CSF specific GAD synthesis (requires CSF and serum).

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 implicated in autoimmune encephalitis.

High sensitivity (ultrasensitive) C- Reactive Protein (Serum :) Normal range: 0-5 mg/L* Minimum sample volume 500µl Preferred sample volume 2ml (Up to 4 days) Risk assessment guidelines: hsCRP <1.0 mg/L = Low risk. hsCRP 1.0-3.0 mg/L = Average risk. hsCRP >3.0 mg/L = High risk.

CRP measurement by high sensitivity methods can indicate the risk for future cardiovascular and peripheral vascular disease. Elevated values may be indicative of the prognosis of individuals with acute coronary syndromes or stable coronary disease. High sensitivity CRP (hsCRP) measurement should not be used as a substitute for assessment of traditional cardiovascular risk factors. Individuals with evidence of active infection, inflammation or trauma should not be tested for cardiovascular disease risk assessment by hsCRP measurement until these conditions have abated.

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Immunoglobulins (IgG/A/M)

(Serum (adult)

IgG: 6.00 – 16.00 g/l †

IgA: 0.80 – 4.00 g/l †

IgM: 0.50 – 2.00 g/l) †

Minimum sample volume 500µl Preferred sample volume 2ml (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. lymphoproliferative 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: adult:

IgG1: 3.2 – 10.2 g/l †

IgG2: 1.2 – 6.6 g/l †

IgG3: 0.2 – 1.9 g/l †

IgG4: 0.0 – 1.3 g/l)†

Minimum sample volume 1ml (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)‡


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) Minimum sample volume 500µl Preferred sample volume 2ml N.B. Reference ranges are age specific and may differ between ethnic groups

Serum IgE may be helpful in diagnosing atopic diseases however the reference 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)* Minimum sample volume 500µl Preferred sample volume 2ml (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 250µl. 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) (Serum) Minimum sample volume 500µl Preferred sample volume 2ml (Up to 14 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, SP100, GP210 and f-Actin. M2 is also measured quantitatively by ELISA

LKM antibodies (Serum: Negative) Minimum sample volume 500µl Preferred sample volume 2ml (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 (EDTA) Minimum sample volume4ml (Up to 4 days)

A wide range of lymphocyte markers for assessment of immunodeficiency and lymphoproliferative diseases are available. Use MIRHO request form (RF001 MIRHO Request form v3) see section 5 of this handbook.

Lymphocyte function tests (Lithium Heparin sample) Minimum sample volume 6ml (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)‡

Minimum sample volume 500µl Preferred sample volume 2ml 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) Minimum sample volume 2ml Preferred volume 4ml (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)‡


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.

MOG antibodies (Serum: Negative) Preferred sample volume 2ml (Up to 14 days)

Another autoimmune cause for neuromyelitis optica is an oligodendropathy attributed to MOG antibody. MOG antibodies target myelin sheath thus causing demyelination. MOG and NMO antibodies are run as a combine test.

Myeloperoxidase (MPO) antibodies (Serum) Negative: <3.5 IU/ml* Equivocal: 3.5 – 5.0 IU/ml* Positive: >5.0 IU/ml* Minimum sample volume 500µl Preferred sample volume 2ml (Up to 7 days)

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.

http://www.nice.org.uk/guidance/cg134

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Anti-Neutrophil cytoplasmic antibodies (ANCA) (Serum: Negative) Minimum sample volume 500µl Preferred sample volume 2ml (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 proteinase3). p-ANCA may occur in other vasculitic disorders as well as some forms of Glomerulonephritis (see also test for myeloperoxidase).

Neutrophil respiratory burst test (Lithium Heparin sample) Minimum sample volume 6 ml. Discuss with Laboratory. (4 days)

This is a diagnostic test for chronic granulomatous disease. Please arrange with laboratory. Please send a time matched healthy control (1 x 6ml Lithium heparin) with patient bloods.

NMO antibodies (Serum: Negative)§


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. NMO and MOG antibodies are run as a combined test.

Pancreatic islet cell antibodies (Serum: Negative) Minimum sample volume 500µl Preferred sample volume 2ml (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 (Serum) Minimum sample volume 1ml Preferred sample volume 2ml 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 samples 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 handbook). 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)* Minimum sample volume 500µl Preferred sample volume 2ml (Up to 14 days) Positive samples are sent away for quantitation by ELISA

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 Indirect immunofluorescence (Serum : Negative)* Minimum sample volume 500µl Preferred sample volume 2ml (Up to 14 days) ELISA (Serum) Negative: <14 RU/ml* Borderline: ≥14 – <20 RU/ml* Positive: ≥20 RU/ml* (Up to 28 days)

Autoantibodies to the M-type phospholipase A2 receptor (PLA2R) are sensitive and specific for 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. The ELISA assay will be run on specific request or when the serum sample is “sticky” and prevents processing by IIF

Proteinase 3 (PR3) antibodies (Serum) Negative: <2 IU/ml* Equivocal : 2.0 – 3.0 IU/ml* Positive : >3.0 IU/ml* Minimum sample volume 500µl Preferred sample volume 2ml (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.

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Paraneoplastic neurological antibodies: (serum)

Minimum sample volume 500µl Preferred sample volume 2ml 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

SOX1 Lambert-Eaton myasthenic syndrome small cell lung carcinoma

Tr paraneoplastic cerebellar degeneration

Hodgkin’s lymphoma

Zic4 paraneoplastic cerebellar degeneration

small cell lung carcinoma

The presence of paraneoplastic antibodies are confirmed by Western blot.

Rheumatoid factor (Serum: 0-14 IU/ml)* Minimum sample volume 500µl Preferred sample volume 2ml (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: 200µl per allergen: 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) Minimum sample volume 500µl Preferred sample volume 2ml (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 seem 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 (Serum) Kappa 3.30 – 19.40 mg/l * Lambda 5.71 – 26.30 mg/l * Kappa / Lambda ratio 0.26 – 1.65 * Minimum sample volume 500µl Preferred sample volume 2ml (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.

Skin antibodies (Serum: Negative) Minimum sample volume 2ml (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.

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Smooth muscle antibodies (Serum: Negative) Minimum sample volume 500µl Preferred sample volume 2ml (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) Minimum sample volume 500µl Preferred sample volume 2ml (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)* Minimum sample volume 500µl Preferred sample volume 2ml (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.

TSH receptor antibodies (Serum: Negative)§


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) Negative: < 7 U/ml* Equivocal : 7 – 10 U/ml* Positive : >10 U/ml* Minimum sample volume 500µl Preferred sample volume 2ml (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. We also add immunoglobulins to check for IgA deficiency. IgA deficiency (partial or complete) is about 1:400 blood donors and 1:40 patients with coeliac disease.

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Viscosity (EDTA Plasma: 1.50 – 1.72 (c.f. to water))* Minimum sample volume 2ml whole blood (or 1ml plasma) (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.

Key for reference ranges Where a quantitative reference 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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13. IMMUNOPHENOTYPING

Immunophenotyping tests performed by the Clinical Immunology Service

All queries and requests for urgent investigations to be addressed to Miss Sarah Terjesen

or Mr Steven Dix in the Clinical Immunology laboratory (0121 414 4069)

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 (0121 414 4069)

Bone marrow * 4ml bone marrow in EDTA and 2 unfixed marrow smear slides

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.

T cell antigen receptor & immunoglobulin gene rearrangement studies

Blood or bone marrow drawn into an EDTA bottle (Heparinised material is unsuitable for the PCR process)

Immunodeficiency studies *** (inc. T cell subsets)

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 leukaemia including lineage determination. It can be processed to provide an urgent, telephoned report to the requesting clinician. The information derived will be used to select a more appropriate secondary panel / additional markers if appropriate. Kappa, Lambda, CD3, CD4, CD7, CD13, CD19, CD33, CD34, CD45, CD117,

HLA-DR Plus cytoplasmic panel (see below for markers).

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.

WBC tube: CD15, CD24, FLAER on

neutrophils CD64, CD14, FLAER on

monocytes RBC tube: CD59, CD235a on red blood cells (only added on if a known patient or a positive WBC tube result).

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Investigation of Immunodeficiency

T cell subset markers CD3, CD4, CD8 - expressed as percentile and absolute values

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: CD19, CD20, CD21, CD27, CD38, CD45, IgD, IgM. Autoimmune lymphoproliferative disease (ALPS) screening: CD3, CD4, CD8, CD45RA, TCR alpha/beta. Leukocyte adhesion deficiency surface markers (research test): CD18, CD11b, CD45 (screening test undertaken in CIS and second confirmatory sample sent to Great Ormond Street Hospital).

Immunoglobulin/T cell receptor gene studies This assay is carried out by the West Midlands Genetic Service and reported by the Clinical Immunology Service in conjunction with immunophenotyping results.

See Cell Function section below for functional immunodeficiency investigations.

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14. CELL FUNCTION ASSAYS Functional studies and cytokine assays

We perform a number of basic investigations to test defects in cell function including neutrophil oxidative burst and cytokine measurement. These investigations are labour intensive and technically demanding. To identify which tests are most appropriate please discuss with one of the clinicians on the telephone numbers listed on page 4. 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. For request form, use General immunological investigations (REQ.G.1.3)

Functional Assays: Functional studies MUST be booked in advance with the laboratory. Please call 0121 414 4069. Samples should be labelled ‘URGENT !! and addressed to: Clinical Immunology Service Medical School, Vincent Drive Edgbaston, Birmingham, B15 2TT Contact 0121 414 4069 (external) or 44069 (internal) on arrival at the Medical School foyer. Include the telephone number, or bleep, of the doctor to be contacted in case of query. Neutrophil function studies (respiratory burst) (1 x 6ml Lithium heparin) Performed by flow cytometric dihydrorhodamine (DHR) assay. Results within 24 hours if urgent. Please send a time matched healthy control (1 x 6ml Lithium heparin) with patient bloods. Samples must arrive in the laboratory on the same day as venepuncture.

Cytokine measurement Cytokine measurement in serum or plasma: Collect blood into a serum separation, plain, or EDTA tube. If sending sample by courier: 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, IFN-g, VEGF. Other cytokine measurements may be available, on a research use only basis, on request.

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