Acetylcholine receptor ab’s Serum: <2x10-10 moles/l

Adrenal cortical ab’s

Serum: Positive/Negative

Antinuclear ab’s

Aspergillus precipitins

B2 microglobulin (B2M)

Serum: <3.0mg/l.

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

CSF Tau protein:

asialo-transferrin.

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.

Cardiac ab’s

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

C-Reactive Protein

Serum: <10mg/l

Viral infection/AI disease:

11-49mg/l

Bacterial infection:

50-100mg /l

Major bacterial infection:

>100mg/l

C3d Complement component EDTA plasma: 7-17mg/l

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.

C3 Nephritic factor

Fresh Serum: Positive/Negative

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.

Cardiolipin/Phospholipid ab’s

Serum: IgG <12GPLU/ml IgM <6 MPLU/ml

Antibodies have been associated with SLE, recurrent miscarriages and arterial and venous thrombosis. Slightly elevated levels may be found in some infections and other vasculitides but these are not considered clinically relevant. Both IgG and IgM antibodies are assayed. Significant levels of ab’s do not necessarily correlate with the severity of the disease. Please note that lupus anticoagulant is performed in haematology.

Complement (Functional)

(CH100)

Carried out by RID in gels containing sensitised red cells this assay tests the integrity of the classical pathway of complement. Low levels are found when any one component is absent. Assays for the individual complement components are available as follow up. Please see full list on page 27. Refer to important notes regarding collection of blood and its despatch to the laboratory.

Complement C3 and C4

Serum: C3: 75-175mg/dl C4: 12-48 mg/dl

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. When taking blood care should be taken to avoid unnecessary in vitro activation of complement.

Cryoglobulins

Serum: Positive*/Negative

*These may be typed:

When cryoglobulins are associated with Waldenströms macro globulinaemia, myeloma 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.

DNA binding ab’s

Serum:

Crithidia Positive/Negative

Assay of native, double stranded DNA (dsDNA), 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 is extremely useful in monitoring the activity of the disease.

Endomysial ab’s

Serum: Positive/Negative

IgA ab’s 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. See also gliadin ab’s.

ENA:

(Extractable nuclear binding ab’s)

ENA recognises 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 SLE, cutaneous lupus, neonatal lupus and 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 ab’s especially females considering pregnancy.

Fungal precipitins

Serum: Positive/Negative

Precipitins directed against candida albicans, aspergillus fumigatus and micropolyspora faenii are currently available. Note: most adult women will have low levels of candida precipitins.

Gastric parietal cell ab’s

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%), Addisons disease (25%) and iron deficiency anaemia (20%).

Gliadin ab’s

Serum: IgA <10 mg/ml

IgG <50 mg/ml

In susceptible individuals alpha gliadins are known to activate coeliac disease a gastro- intestinal disorder characterised by the flattening of the jejunal mucosa. The laboratory offers a quantitative enzyme immunoassay which allows monitoring of the patient for both IgG and IgA ab’s. The titre of IgA ab’s decreases with gluten free diet as does the level of endomysial ab’s and reticulin type I ab’s.

IgA Gliadin ab’s are more specific for Coeliacs than IgG (~95% compared to ~60%) but IgG ab’s are more sensitive (~100% cf ~50%). It has been reported that IgA deficient patients have a ten to fifteen fold increased incidence of coeliac disease. It is suggested that gliadin ab’s are carried out in all IgA deficient individuals.

Glomerular basement membrane ab’s

Test for Goodpastures syndrome. Ab’s to the non collagenous portion of type IV collagen are detected by EIA as indirect immunofluorescence is both less sensitive and less specific being positive in only 75%, or less, of proven cases. Urgent requests should be discussed with our clinical staff.

Glutamic acid decarboxylase

(Stiff Man syndrome)

Immune Complexes (CIC)

Serum: <1:20

It has been suggested that circulating immune complexes, which may be present in sera of patients with any of a variety of autoimmune diseases, should be assayed by a combination of techniques such as C1q binding, conglutinin solid phase assay, poly ethylene (PEG) precipitation and Raji cell assay. Detection and characterisation of CIC, which are often composed of polyreactive human natural ab’s, will continue to be largely of research interest until studies of more robust design yield definitive data on clinical significance. This laboratory, like other regional laboratories offers a platelet agglutination technique which, though it may give false positives in the presence of platelet ab’s, has proved of value over many years of use.

Immunoglobulins (IgG/A/M) Serum: {IgG: 6.00-16.00 g/l

(adult) {IgA: 0.75-4.00 g/l

Immunoglobulins is 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 ab’s is virtually diagnostic of PBC) Low levels of IgG and IgA may be due to loss (protein losing enteropathy or nephrotic syndrome) reduced synthesis (lympho proliferative disorders or to primary immunodeficiency) or excessive catabolism. Low levels of immunoglobulins always indicate further investigation. Where appropriate details are supplied age and sex related normal levels are printed on the report. See also sub-classes (IgG and IgA).

IgA ab’s

Serum: zero titre (negative)

IgA ab’s occur in IgA deficient patients in receipt of blood products containing IgA. Their presence is indicative of risk of adverse transfusion reactions.

IgA Subclasses

IgA comprises two subclasses of which, in serum, IgA1 predominates (85%). Consequently deficiencies or even complete absence of IgA2 may go undetected with normal or near normal levels of total serum IgA. The functions of the two subclasses of IgA are comparable to IgG1 and IgG2 but their activity is on mucosal surfaces where IgG is intravascular. The immunology laboratory offers RID assays of IgA1 and IgA2 with a turnaround of up to seven days. (Subclasses of IgG are measured nephelometrically on a daily basis).

IgG Subclasses

Serum: IgG1: 3.20 - 10.20 g/l

IgG2: 1.20 - 6.60 g/l

IgG subclass deficiency is mainly related to IgG1 and IgG2 where individuals suffer recurrent infections because they are unable to mount an immune response against organisms. (This may be ascertained employing functional antibody assays using Tetanus toxoid requiring the presence of IgG1 and pneumococcus requiring IgG2)

Intrinsic factor ab’s

Detected in 70% of patients with pernicious anaemia. Assayed by enzyme immunoassay this test should be carried out together with gastric parietal cell ab’s.

Isoelectric focusing (Oligobanding)

Oligobanding refers to discrete populations of immunoglobulin detected by electrophoresis in CSF which are NOT paralleled in serum from the same patient. Oligo banding 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, neurosarcoid and SLE. Please note that paired samples of CSF and serum are essential for this assay.

LKM ab’s

Serum: Positive/Negative

These ab’s, which stain the cytoplasm of hepatocytes and proximal renal tubules, and hence, to the untrained eye are difficult to distinguish from mitochondrial ab by IIF, are found in a subgroup of patients with ANA negative, autoimmune chronic active hepatitis (CAH). LKM1 ab’s are positive in CAH type 2 which is the most common autoimmune liver disease of childhood and has a relatively unfavourable prognosis. LKM2 ab’s were found in drug induced hepatitis but tienilic acid, ticrynafen, has now been withdrawn. LKM3 ab’s are found in 10% of chronic delta virus hepatitis and, in contrast to LKM1 and 2, are both organ and species specific.

A wide range of lymphocyte markers including HIV status screening is available to the West Midland Region. A separate cell form is in use for cell marker requests but a clinician or a technical member of staff within the laboratory MUST be contacted prior to samples being sent in order that the most appropriate studies are undertaken.

Mitochondrial ab’s

Serum: Positive*/Negative

Present in >95% of cases of primary biliary cirrhosis usually in 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 ab’s EIA:

(MT2/MT4 and MT9)

Serum: Positive*/Negative

*EU/ml

For those wishing to confirm the presence of mitochondrial ab’s or to monitor patients with a quantitative assay there is available an EIA method which distinguishes three separate mitochondrial ab’s (enzymes) and affords a quantitative assay in EU’s/ml.

Currently ab’s to three enzymes are assayed: pyruvate dehydrogenase complex (M2), sulphite oxidase (M4) and glycogen phosphorylase (M9).

Myeloperoxidase (MPO)

Serum: <10 Eu/ml

Neutrophil cytoplasmic ab’s (ANCA)

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

Pancreatic islet cell ab’s

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

Paraproteins

Levels of monoclonal IgG, IgA, IgM, IgD and in some instances IgE are measured immunochemically and in all cases of follow up the previous specimen is run in parallel to confirm the change in paraprotein level. Immunofixation of presentation sample defines both the isotype and light chain type. Follow up specimens will be subjected only to electrophoresis. (See ‘guide to appropriate use of tests’ at the back of this booklet). Healthy adults do not have detectable levels of paraproteins though benign increases do occur in the elderly and in some cases of chronic infections in immunosuppressed patients.

Paraprotein neuropathies

MAG; POEMS

Myelin associated glycoprotein (MAG) is a glycoprotein component of the myelin of central and peripheral nervous systems present in the priaxonal region, Schmidt-Lantermans incisures, lateral loops and outer mesaxon of the myelin sheath. A member of the immunoglobulin super- family MAG probably functions as an adhesion molecule and mediates cell-cell interactions. Monoclonal reactivities against MAG are detected in about 50-75% of patients with IgM paraproteinaemia and peripheral neuropathy. Sera from patients with neuropathy that are negative for MAG antibodies often exhibit reactivity against various gangliosides.

Proteinase 3 (PR-3) ab’s.

Serum: <10 Eu/ml

Purkinje cell ab’s (Yo)

Serum: Positive*/Negative

Ab’s characteristically stain Purkinje cell cytoplasm in a coarse granular pattern in patients with subacute or paraneoplastic cerebellar degeneration which is a rare complication of some gynaecological cancers (ovarian and breast) and Hodgkin's disease. Other ab’s which react with neuronal cell nuclei (ANNA1 and, more rarely ANNA2) are found in some patients with small cell lung cancer with subacute sensory neuropathy or paraneoplastic encephalomyelitis.

RAST tests (allergen specific IgE)

Assays for the detection of circulating IgE ab’s directed against specific antigens in sensitised patients are available to a wide range of allergens. 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. Tests against allergens in stock have a turnaround time of seven days. It is essential to supply as much information as possible with RAST tests. If in any doubt it is advisable to discuss the patient with the consultant immunologist.

Rheumatoid factor

Two tests are carried out to detect these ab’s which are directed against other immunoglobulins. A latex test which is more sensitive but less specific for RF and an haemagglutination test which is less sensitive but highly specific.

Approximately 70% of patients with rheumatoid arthritis are sero positive and antibodies may occur in other conditions including many infections, myeloma, lymphomas 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.

Salivary Duct ab’s

Salivary duct ab’s are present in Sjögrens syndrome with arthritis (65%) and in SICCA syndrome (10%) but SSA (Ro) and SSB (La) are more sensitive and more specific for Sjögrens syndrome.

Salivary IgA and secretory piece.

This is a qualitative test for the presence of mucosal, saliva antibody (secretory IgA, sIgA) used in the work up of suspected cases of immunodeficiency. Serum levels of immunoglobulins should be carried out in parallel.

Normal age related levels will be shown on the form and will be accompanied by a clinical comment.

Serum electrophoresis.

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 response, immunodeficiency and absence of alpha-1-antitrypsin.

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

Skin ab’s.

Ab’s are found in (i) intercellular substance of the epidermis (desmosome) which strongly suggests a diagnosis of pemphigus though these ab’s 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 ab’s.

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

Striated muscle ab’s

In patients with Myasthenia Gravis with thymoma these ab’s are invariably positive but in such patients without thymoma the ab’s occur in only 60% of cases. This assay is usually carried out with a test for acetyl choline receptor ab’s 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 ab’s.

(microsomal [TPO])

Serum: Positive*/ "Negative" Males: <1:100

Present in high titres in 95% of patients with Hashimotos thyroiditis, 20% of patients with Graves disease and 90% of patients with primary myxoedema. Ab’s may also be present in low titre in colloidal goitre, thyroid carcinoma, De Quervains thyroiditis, other organ specific auto-immunities and in allegedly normal people. If persistent in euthyroid individuals it may indicate autoimmune thyroiditis and predisposition to future thyroid failure.

Thyroid ab’s.

(thyroglobulin)

Total IgE

Serum IgE may be helpful in diagnosing atopic diseases however the normal range is very wide and levels do not always correlate well with symptoms. A high level of specific IgE to a single allergen may be seen with a normal level of IgE. Very high levels of IgE are seen both in atopic eczema and in parasitic infestations. Please note that paediatric values are quoted on all reports provided that the age of the patient is given.

Urinary free light chains.

As a National centre for myeloma trials this laboratory is one of the very few which quantitates the level of free light chains in urine. Expressed in terms of g/g of creatinine it is a very accurate method for the assessment of light chain output in the presence of circulating paraprotein.

Polyclonal free light chains may occur in healthy individuals particularly following strenuous exercise and also in patients with chronic infections or inflammatory diseases such as rheumatoid arthritis. Unlike laboratories where concentration of urine is carried out before assay, thereby introducing a source of error, this laboratory measures free light chain directly on urine by RID on commercial diffusion plates.

Viscosity

Serum: 1.40-1.90 (cf to water)

Serum viscosity is an essential test when monitoring Waldenstroms macro- globulinaemia and when investigating an unexplained retinal or cerebrovascular occlusion. In such patients a cryoglobulin may also be present it is therefore essential that the precautions outlined for cryoglobulins is observed and blood collected and transported to the laboratory at body temperature.

Volt gated Ca++

(Lambert Eaton syndrome)

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