Oxford Handbook of Clinical Immunology and Allergy (3 edn)
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Introduction
There are many hundreds of reported autoantibodies, not all of clinical value. The repertoire of the typical regional immunology laboratory will cover most of those described below. Some will only be available through specialist referral laboratories or research laboratories.
When requesting tests, the following criteria should be used.
Decide in advance what clinically useful information will be obtained by carrying out the test.
Because a test is available does not mean that it is of value under a given circumstance.
If a test result does not affect clinical management in any way, testing is of no value.
Autoantibodies are divided broadly into two categories.
Organ-specific, where the target antigen has a restricted distribution, usually limited to one organ such as the thyroid gland.
Organ-specific, where the target antigen has a wide distribution.
Organ-non-specific antibodies may be associated with a disease of restricted organ involvement, e.g. primary biliary cirrhosis.
Target antigen is in the mitochondria, which are widely distributed, but disease is limited to the liver.
It is not clear why autoantibodies to a widely distributed antigen should be associated with an organ-specific disease.
It is now known that in some circumstances autoantibodies may cross intact cell membranes. Tissue-restricted passage may explain selective effects.
Autoantibodies may also be divided into two categories.
Primary pathogenic antibodies, where the antibody mediates a functional effect by:
interfering with a cellular or molecular function (e.g. blocking neuromuscular transmission by antibodies to the acetylcholine receptor on muscle endplates).
direct damage to tissues (e.g. anti-glomerular basement membrane antibodies).
Views on pathogenicity will be modified in the light of known penetration of whole antibodies into intact cells.
Secondary antibodies, which are not directly involved in the disease process, are markers for the existence of the process (e.g. anti-thyroglobulin antibodies).
These may still be useful diagnostic tools.
Not all autoantibodies are diagnostically useful, as they may have low sensitivity and specificity, e.g.
rheumatoid factor
gliadin antibodies.
Techniques: overview
Multiple techniques may be available to test for individual autoantibodies.
Comparison of different assays is often lacking.
Results may vary between different laboratories.
EQA may be widely discrepant.
No gold standard assay may be identified.
Establishment of a new test require the following.:
Review of existing methods (if any).
Evaluation by comparison with other assays:
tested in healthy controls
disease state
other confounding disease states.
Calculation of sensitivity and specificity.
Validation of clinical utility.
Establishment of internal QC material.
Validation against national/international standard reference material.
Participation in external QA (if available).
Evaluation of cost–benefit.
Diagnostic laboratories may use in-house or commercial assays.
Commercial assays must be CE (Conformité Européenne) marked (EU regulations).
Cost of CE marking has led manufacturers to withdraw low-volume commercial assays, restricting availability.
In-house assays may apply for CE marking.
This requires evidence of utility, a large application form, and money!
Non-CE-marked assays may not be sold for profit, but may be used within the NHS.
Type of antibodies
Autoantibodies can be of any class.
In most circumstances IgG antibodies are usually sought.
IgM autoantibodies are not normally significant unless persistent and of high titre.
IgM anti-cardiolipin antibodies are considered significant.
IgA autoantibodies are rare but may be diagnostically useful, e.g.
coeliac disease—IgA endomysial antibodies have the highest sensitivity and specificity.
Value of other IgA autoantibodies is unclear, e.g.
IgA rheumatoid factors
IgA ANCA (HSP?).
Autoantibodies commonly appear after infections, e.g.
EBV
adenovirus
HIV
acute and chronic bacterial infections
They will usually disappear after 6 months
They are not usually associated with clinical disease.
Most common associations:
rheumatoid factor—any infection
anti-nuclear antibodies—adenovirus (children), HIV, Gram-negative bacteria
smooth muscle antibodies—adenovirus
liver–kidney microsomal antibodies—HCV
cardiolipin antibodies—EBV
dsDNA antibodies—rare, HIV.
Drugs may also induce autoantibodies. These may cause disease and may persist after the drug is withdrawn.
Anti-nuclear antibodies (anti-histone): procainamide, hydralazine, ACE-inhibitor, chlorpromazine, minocycline.
Liver–kidney microsomal antibodies: tienilic acid.
Non-M2 mitochondrial antibodies: alcohol.
Particle agglutination assays
Technique is old and reliable.
It is cheap, but labour-intensive.
Use has almost disappeared, apart from low-level screening, e.g. rheumatoid factor, DCT.
Antigen (either pure or extract) is coated onto an inert carrier particle, usually gelatin or latex but originally tanned red cells.
When mixed with serum containing the appropriate antibody, the particles are agglutinated.
The principle is simple, but reading the endpoint of a particle agglutination titration requires skill.
IgM antibodies are picked up preferentially because of their pentameric shape, which allows better cross-linking.
Immunoprecipitation assays
These depend upon the formation of insoluble immune complexes where an antibody encounters the optimum concentration of antigen.
Prototype assay is the Ouchterlony double-diffusion assay.
Antigen and antibody are added to wells cut in agar gels and allowed to diffuse towards one another.
Line(s) of precipitation form at the point of equivalence, indicating the presence of an antibody against the antigen.
The process is slow and may take up to 72 hours to form lines.
The technique may be improved using electrolyte-containing agarose and applying a current across the gel, forcing the antibody and antigen together (countercurrent immunoelectrophoresis (CIE)).
CIE is used to detect of antibodies to extractable nuclear antigens.
Commercial assays are not available, and CIE tends to be used to complement commercial EIA tests for ENA in specialist centres.
It is labour-intensive and time-consuming.
If the immune complex formation takes place in the liquid phase, the light-absorbing/scattering properties of the solution will be altered and can be measured (nephelometry/turbidimetry).
Can be used for antibody detection on automated analysers.
Rheumatoid factor, thyroid antibodies.
Stability of the immune complex may be poor, and agents such as PEG may be added to ensure a stable reaction.
PEG can also be used in gels to enhance and stabilize the immune complex.
Indirect immunofluorescence
Tissue preparation
Standard technique for the detection of many serum autoantibodies.
Appropriate tissue block is snap-frozen and cut on a cryostat to provide sections (usually 4mm thick) that are mounted on a slide and air-dried.
Other fixation techniques may be used under special circumstances (e.g. acetone or ethanol).
Similar methods can be used on cell suspensions prepared on slides using a cytocentrifuge, e.g.
neutrophils for ANCA
HEp-2 cells for anti-nuclear antibodies.
Most laboratories use commercially produced slides (CE marked). The cost is outweighed by convenience:
no problems sourcing animal tissues
staff are not tied up on routine slide production
quality control of section cutting is better.
Technique
Slides are incubated with appropriate dilutions of test and control sera, washed, and then incubated with anti-human immunoglobulin (isotype-specific) antiserum which is conjugated with fluorescein isothiocyanate (FITC).
The technique allows the tissue and intracellular distribution of autoantibody binding to be visualized.
Alternatives may be used for FITC in the second stage, e.g. enzymes (immunoperoxidase) that will give a colour reaction when the slides are incubated with an appropriate substrate.
Slides can be fixed and counterstained to reveal the tissue structure.
An ordinary transmission light microscope is all that is required.
Processing has an extra incubation step.
Obtaining reliable results
The following are key features that are essential to obtaining reliable results.
Good tissue selection and processing.
Appropriate starting dilution (to avoid non-specific serum binding).
Use of serum not plasma, as fibrinogen causes non-specific fluorescence.
Appropriate FITC-conjugated antiserum selection.
Commercial antisera are usually used.
There may be considerable batch-to-batch variation.
Ratio of fluorescein molecule to protein needs to be between 1 and 4.5 to give reasonable results.
If it is too low, the intensity is inadequate.
If it is too high, the non-specific fluorescence swamps the specific staining.
Optimal dilution needs to be determined by a chequerboard titration.
New antisera are tested at serial dilutions on standard tissue sections incubated with serial dilutions of a standard control serum with previously identified titre.
Appropriate internal/external controls (quantitative as well as qualitative).
A good-quality fluorescence microscope, properly maintained, with a properly adjusted light source.
An experienced microscopist who is familiar with the relevant patterns.
Tissue multiblock
For laboratory convenience, it is standard practice to test for the basic autoantibodies using a tissue multiblock containing liver, stomach, and kidney (some laboratories also include thyroid), usually rat.
Commercial slides may have ‘chips’ containing the separate tissues and HEp-2 cells. This allows the detection of:
most anti-nuclear antibodies
smooth muscle antibodies
mitochondrial antibdies
reticulin antibodies—these are not usually reported but may indicate need to check endomysial or tissue transglutaminase antibodies
gastric parietal cell antibodies
ribosomal antibodies
liver–kidney microsomal (LKM) antibodies.
HEp-2 cells allow detection of cellular staining patterns.
HEp-2000 cells have been genetically engineered to express higher levels of Ro antigens: in practice there is little difference.
The disadvantage of the multiblock screen is that it encourages clinicians to request ‘autoantibody screens’ without thinking about what they are specifically looking for.
Always encourage clinicians to request the test they need for diagnosis.
Screening and titration
Screening is carried out at a single dilution with a conjugated antiserum that recognizes IgG, IgA, and IgM (polyvalent):
normal adult screening dilution is 1/20.
Positive samples are then titrated using a monospecific anti-IgG antiserum:
a limited number of steps only are required (twofold dilutions to 1/640; anything higher reported >1/640).
Screening dilutions need to be adjusted in children; 1/10 may be appropriate.
Not all antibodies need to be titrated (e.g. GPC, reticulin).
Titration is only semi-quantitative.
Other tissues
Same techniques as used for other tissues are used.
Monkey oesophagus (endomysial, epidermal antibodies).
Other tissues used are pancreas, adrenal, gonad, small intestine, pituitary, cerebellum, cerebrum, salivary gland.
Alternative techniques
Some laboratories are now using laser-based array systems or automated EIA screening tools for multiple antigens to weed out negative samples.
Array systems are also used to screen for extractable nuclear antigens.
These supplement but do not replace indirect immunofluorescence (IIF).
Performance characteristics are variable.
Systems are available for automation of the dilution and staining of slides.
Direct immunofluorescence
This technique is very similar to that used for indirect immunofluorescence, i.e. tissue is obtained directly from the patient, snap-frozen, and sectioned prior to incubation with the FITC-conjugated antiserum.
This allows the detection of tissue-bound antibody in the patient.
Tissue-bound antibody may be present even when there is insufficient antibody to be detected free in the serum.
Other tissue reactants such as complement and fibrinogen may be detected. Patterns of reaction may be absolutely diagnostic, e.g. in bullous skin diseases.
Direct immunofluorescence is used extensively in the diagnosis of skin diseases (because of the accessibility of the tissue for biopsy) and renal disease.
Radio-immunoassay (RIA)
These assays are highly sensitive.
They require pure antigen.
They require radio-isotopes.
Few assays are now done using these techniques as laboratories move away from isotopic tests to enzyme-linked immunoassays.
RIA is the gold standard for:
acetylcholine receptor antibodies
ds-DNA antibodies (Farr assay).
RIA is also used for intrinsic factor antibodies.
Enzyme-linked (EIA) and fluorescent immunoassays (FIA)
These have taken over from RIA, and to some extent from indirect immunofluorescence.
Antigen is bound on to a solid phase (bead or plate) which is reacted with serum, washed, and reacted with the antiserum against human immunoglobulin, which is coupled to either an enzyme (EIA) or a fluorescent dye (FIA).
The final stage in EIA is reaction with the substrate, either directly or via an amplification step, to give a colour that can be measured spectrophotometrically.
In FIA, the plate can be read directly using an appropriate exciting light source (which will be of a different wavelength to the emitted light).
Assays tend to be more sensitive than immunofluorescence, but may lose specificity.
Pure antigen is required; the source may be critical to value of tests.
Recombinant human tissue transglutaminase (tTG) gives better results than guinea pig tTG.
Commercial assays tend to be expensive.
Samples may need to be run in duplicate, increasing cost.
Assays are most cost-effective and accurate when performed on automated instruments.
Results may differ from those obtained by other methods:
dsDNA antibodies by Farr and EIA
ENA antibodies by CIE and EIA.
The clinical significance of the different results has not always been established.
Differences need to be considered when introducing new tests.
External quality assurance scheme data may help in comparing methods.
Immunoblotting
Antigens are either electrophoresed in a matrix or applied to the matrix at specific points, incubated with appropriate dilutions of sera, washed, then incubated with enzyme conjugated antisera, followed by substrate. This gives a coloured band.
Assays are often quick, and suitable for urgent screening.
Staining may be automated (slide-based kits).
Results are qualitative.
Commercial kits are available for qualitative detection of antibodies:
ENA
ANCA antigens (PR3, MPO)
liver antigens (M2, LKM, SLA, LC)
neuronal antigens
gangliosides.
Acetylcholine-receptor antibodies (AChRAb)
Units: mol/L.
Normal ranges: <2×10–10 mol/L.
Principles of test
Antibodies are detected by a quantitative competitive radio-immunoassay.
No EQA scheme exists.
Indications for testing
AChRAbs are the marker for myasthenia gravis.
Interpretation
Two types of antibodies have been described:
those binding to the receptor at sites distinct from the binding site for acetylcholine
those blocking the binding of the neurotransmitter or α-bungarotoxin.
Some antibodies are capable of modulating the removal of the receptors from the surface of the muscle through cross-linking followed by internalization.
Levels above 5×10–10mol/L are regarded as positive.
Levels of 2–5×10–10mol/L are regarded as equivocal, and may be seen in ocular myasthenia.
Some laboratories report the levels only semi-quantitatively (high, low, etc.).
Highest levels are seen in young patients (<40 years with generalized disease).
Lower levels are seen in:
older patients
thymoma
penicillamine-induced myasthenia.
Approximately 15% of typical myasthenic patients are negative for AChRAb.
Some may have IgM antibodies (not detected in routine assays).
In ocular myasthenia, about 20% of patients will be seronegative.
Antibodies persist in 60% of patients even if the disease is in remission.
AChRAbs have also been detected in the myasthenic syndrome associated with penicillamine usage (about 1% of treated patients). These antibodies disappear when the drug is stopped.
Very rarely, they may appear transiently during the immunological reconstitution phase of bone marrow transplantation.
Actin antibodies
See ‘Smooth muscle antibodies’, p.526.
Adrenal cortex autoantibodies
Units: qualitative.
Normal adult range: not detected.
Principles of test
IIF, using a multiblock of primate adrenal gland, ovary, testis, and pituitary.
EIA or RIA are used to look at individual antigensin the research setting.
No EQA scheme exists.
Commercial positive control sera are available.
Indications for testing
Suspected Addison’s disease.
Screening in polyglandular syndromes.
Interpretation
Autoantibodies to adrenal cortex (any or all of the three layers) are found in approximately 50% of patients with Addisonian adrenal insufficiency where there are other autoimmune diseases.
Prevalence drops when the autoimmune adrenalitis occurs alone.
They are virtually never found in patients with tuberculous adrenal destruction.
Target antigen is usually adrenal microsomes.
Antibodies to the ACTH receptor have also been described in a few patients with Cushing’s syndrome (paralleling thyroid-stimulating antibodies).
21-hydroxylase (P450c21) is the major target antigen in Addison’s disease and type I APGS.
Frequent cross-reactivity of the antibodies with the steroid-producing cells of the theca interna of the ovary (ovarian failure) and the Leydig cells of the testis.
Other antigenic enzymes in steroid-producing cells include the P450 side-chain cleavage enzyme (P450scc) and 17α-hydroxylase (P450c17).
P450c17 antibodies are associated with type I autoimmune polyendocrinopathy syndrome.
P450c21 antibodies are associated with type II autoimmune polyendocrinopathy syndrome.
P450scc antibodies are associated with premature ovarian failure.
Autoimmune adrenal disease is closely associated with other organ-specific autoimmune disease:
thyrogastric (Schmidt’s syndrome).
parathyroid autoimmune disease.
It is important to screen for thyroid antibodies and gastric parietal cell antibodies as well.
Multiple endocrine autoantibodies may be found in chronic mucocutaneous candidiasis with endocrinopathy.
Screening such patients is important, as the autoantibodies may appear before overt manifestations of endocrine insufficiency.
Amphiphysin antibodies
Anti-amphysin antibodies bind widely to presynaptic terminals in the brain, giving variable cytoplasmic staining.
Association is with a range of neurological disorders including:
subacute sensory neuropathy
sensorimotor peripheral neuropathy
paraneoplastic stiff person syndrome.
Commonly associated tumours are small cell lung cancer and breast cancer.
Anti-nuclear antibodies (ANA) and ANCA
Anti-nuclear antibodies (ANA)
See ‘Nuclear antibodies (ANA)’, p.514.
ANCA
See ‘Neutrophil cytoplasmic antibodies (ANCA)’, p.512.
Aquaporin antibodies
IgG antibodies to aquaporin 4 are found in neuromyelitis optica (Devic’s disease), a demyelinating disease with some similarities to multiple sclerosis.
Also known as NMO-IgG.
Found in 60–70% of patients and is a predictor of subsequent relapse.
Auerbach’s plexus antibodies
Antibodies against the myenteric plexus of the oesophagus have been reported to be detected by immunofluorescence in patients with achalasia of the cardia, a motility disorder of the oesophagus.
The diagnostic role of these antibodies remains to be confirmed.
β2-GPI antibodies
These antibodies have been reported as part of the anti-phospholipid antibody spectrum (see ‘Cardiolipin antibodies (ACA) and lupus anticoagulant’, p.485).
Provided that anti-cardiolipin antibody assays include β2-GPI as a cofactor, there is no clinical indication for separate measurement of anti-β2-GPI antibodies.
Some patients with the anti-phospholipid syndrome, negative for ACA and with a normal dRVVT, are positive for β2-GPI antibodies.
Also seen with M5 anti-mitochondrial antibodies.
C1q antibodies
Antibodies to C1q have been described in hypocomplementaemic urticarial vasculitis, rheumatoid vasculitis, and SLE.
70% of patients with Felty’s syndrome are positive for C1q antibodies.
IgA anti-C1q is found in rheumatoid vasculitis.
In SLE, quantitative measurement of C1q antibodies is now thought to be a measure of the activity of renal disease.
Antibodies to the neoantigen formed by activation of C1q have also been associated with types of glomerulonephritis.
EIA assays are available commercially.
There is no EQA scheme at present.
Cardiac antibodies
These antibodies are positive in:
a proportion of patients with Dressler’s syndrome after myocardial infarction
cardiac surgery
some cardiomyopathies
after acute rheumatic fever.
Multiple antigens have been identified.
Diagnostic value is low.
Detected by immunofluorescence.
Cardiolipin antibodies (ACA) and lupus anticoagulant
Units: GPLU/mL; MPLU.
Normal adult range:
IgG, <10 GPLU/mL (10–20 borderline);
IgM, <10 MPLU/mL (10–20 borderline).
Principles of testing
EIA.
International standards exist for IgG and IgM ACA.
A UK specific standard exists for IgG ACA.
An EQA scheme exists in the UK.
Indications for testing
Suspected anti-phospholipid syndrome:
recurrent DVT/PE (as part of thrombophilia screen), major arterial and venous thrombosis
recurrent miscarriages
premature stroke, multi-infarct dementia
severe and/or atypical migraine
vasculitis (Behçet’s syndrome)
connective tissue disease (SLE, Sjögren’s syndrome)
livedo reticularis
Sneddon’s syndrome (cerebral events and livedo)
Budd–Chiari syndrome
▶ Testing must include both ACA and a test for lupus anticoagulant:
either or both may be present
clinical significance is the same whichever is present.
Interpretation
Antibodies to cardiolipin form part of the spectrum of anti-phospholipid antibodies.
Other related antibodies include:
false-positive VDRL
lupus anticoagulants
antibodies to derived phospholipids.
Standardization and reproducibility of the assays continue to be a major problem.
This is related in part to the requirement for β2-GPI (apolipoprotein H) from serum as a cofactor for the binding of cardiolipin antibodies.
Autoantibodies have also been detected to the cofactor itself.
The cofactor binds anionic phospholipids in vivo and its normal function is to inhibit coagulation and platelet aggregation.
Presence of ACA may be found in the conditions listed in ‘Indications for testing’.
There is no strong correlation with premature myocardial infarction or with cerebral lupus (despite the fact that the brain is full of phospholipid!).
Symptoms are mainly associated with IgG-class antibodies.
Rare patients with typical symptoms will be encountered who have only IgM-class antibodies, and never make IgG antibodies.
Amount of the antibody in units does not seem to relate to the severity of the disease.
Immunosuppression does not have a significant effect on the level of ACA and does not affect thrombophilic tendency.
Transient positive antibodies may be found after viral infections (especially EBV).
Anti-phospholipid antibodies associated with syphilis and other infections do not usually react with β2-GPI and are rarely associated with a clotting disorder.
Lupus anticoagulants are antibodies that interfere with the clotting process in vitro and are usually detected by prolongation of the APTT:
test of choice is dilute Russell viper venom test (dRVVT).
Although it has been suggested that lupus anticoagulants are more specific for recurrent fetal loss than cardiolipin antibodies, both may be associated with the syndrome.
Women with lupus who are planning pregnancy should be screened for both anti-cardiolipin antibodies and lupus anticoagulants, in addition to testing for anti-Ro antibodies.
Cartilage antibodies
Antibodies to collagens types I, II, and III have been found in a range of inflammatory conditions where there is cartilage damage, including:
rheumatoid arthritis
relapsing polychondritis (collagen type II, 60% of patients positive)
a range of other connective tissue diseases, e.g. juvenile RhA (30–40%).
Specificity is low and they are of little diagnostic value.
Centriole antibodies
Centriole antibodies will only be detected if HEp-2 cells are used as the substrate for ANA detection.
Immunofluorescence will show two brightly staining polar dots.
Found very rarely in patients with scleroderma and related overlap syndromes.
May also occur commonly in mycoplasmal pneumonia.
Centromere (kinetochore) antibodies
Units: qualitative.
Normal adult range: not detected.
Principles of testing
Detected by immunofluorescence on HEp-2 cells.
It is essential that the HEp-2 cells contain adequate numbers of dividing cells.
EIA and immunoblot assays are available for three main antigens: CENP-A, CENP-B, CENP-C.
Indications for testing
Indicated in patients with suspected scleroderma, Raynaud’s phenomenon, cutaneous calcinosis.
Patients with severe Raynaud’s phenomenon and features of scleroderma should also be screened for the ENA Scl-70, associated with progressive systemic sclerosis (PSS).
Interpretation
Antibodies can only be detected on HEp-2 cells.
Also referred to as kinetochore antibodies as they react with antigens located at the inner and outer kinetochore plates.
Antigens are 17, 80, and 140kDa proteins involved in the attachment of the spindle fibres (CENP-A, CENP-B, CENP-C).
Minor centromere antigens may also be targets (CENP-D, CENP-E, CENP-F).
CENP-B appears to be the predominant antigen, with five epitopes, some of which are shared with CENP-A and CENP-C.
Antibodies show diagnostic condensation of fluorescence along the metaphase plate in dividing cells, which distinguishes the staining from other speckled-pattern ANA.
Found in the CREST syndrome (sometimes referred to as limited scleroderma):
about 70–80% of patients with features of CREST will have anti-centromere antibodies
1% of patients with PSS will be positive.
Detection of anti-centromere antibodies is of prognostic significance.
Titration of centromere antibodies is of no value.
Scl-70 and anti-centromere antibodies seem to be mutually exclusive.
Up to 12% of patients with primary biliary cirrhosis may be positive for anti-centromere antibodies, of whom about half will have clinical signs of scleroderma.
This may be a misinterpretation of the immunofluorescence pattern, as M2-antibody-negative PBC is often positive for a pattern of multiple nuclear dots (see ‘Multiple nuclear dot antibody’, p.509), which is sometimes referred to as pseudo-centromere because of its resemblance to centromere staining. However, the metaphase plate is not stained.
Cold agglutinins
Often confused with cryoglobulins.
They are autoantibodies that reversibly agglutinate erythrocytes in the cold.
Cause small vessel obstruction in the skin of the extremities, Raynaud’s phenomenon, and haemolytic anaemia.
Most common specificity is anti-i but other specificities such as anti-I or anti-Pr occur.
Often triggered by infections:
Mycoplasma pneumoniae
Rickettsia
Listeria monocytogenes
EBV.
Usually polyclonal IgMκ, although EBV may be associated with a polyclonal IgMλ anti-i response.
May also occur in association with lymphoproliferative diseases where the agglutinin is usually monoclonal (invariably IgMκ):
typically a disease of the elderly
cold agglutinins may precede the overt development of lymphoma by many years.
Paroxysmal cold haemoglobinuria is associated with anti-P antibody:
binds to the red cell and fixes complement in the cold
red cell lysis takes place when the cell is rewarmed
rare, and originally described in association with syphilis (Donath–Landsteiner antibody)
more commonly associated with viral infections such as mumps, measles, and chickenpox.
As for cryoglobulins, samples must be taken and transported to the laboratory at 37°C.
CV2/CRMP5 antibodies
Antibodies recognize the collapsin response-mediator brain protein (CRMP) family; CRMP5 is the dominant antigen.
Staining is seen in the cytoplasm of oligodendrocytes
Associated with small cell lung cancer (SCLC) in 77% and thymoma in 6%.
Syndrome can include:
subacaute sensory neuropathy
limbic encephalopathy
cereballar ataxia
extra-pyramidal syndromes
myopathy.
Presence of antibodies to CV2/CRMP5 in SCLC is associated with better prognosis than anti-Hu antibodies.
Cyclic citrullinated peptide (CCP), cytokeratin, and desmin antibodies
Cyclic citrullinated peptide (CCP) antibodies
Rheumatoid arthritis has been associated with antibodies to perinuclear factor, keratin, and filaggrin on buccal mucosa and rat oesophagus by immunofluorescence.
A synthetic peptic (CCP) can be used in EIA to detect these antibodies.
Specificity for RhA is said to be 96%.
It is being increasingly used for early diagnosis of RhA.
Cytokeratin antibodies
Antibodies to cytokeratin 18 are non-specific and associated with:
rheumatoid arthritis
psoriasis and psoriatic arthritis
Crohn’s disease
coronary artery disease.
They are identified by IIF on HEp-2 cells.
Desmin antibodies
Antibodies to desmin are non-specific and associated with:
autoimmune hepatitis
PBC
coronary artery disease
Crohn’s disease.
They are identified by IIF on HEp-2 cells.
dsDNA antibodies
Units: IU/mL.
Normal adult range:
negative, <30IU/mL
borderline, 30–50IU/mL
positive, 50–300IU/mL
strongly positive, >300IU/mL
may vary according to assay.
Principles of testing
Gold standard remains Farr assay (RIA): highly specific and sensitive.
dsDNA is precipitated using ammonium sulphate.
High-avidity antibodies are detected.
EIA assays are available: results may not be concordant with Farr assay because of presence of ssDNA and z-DNA.
EIA assays are very sensitive but not always specific.
Low-avidity antibodies of no clinical significance may be detected.
Staining of the kinetoplast of Crithidia lucilae is specific but not sensitive. It cannot be recommended as a screening test.
Indications for testing
Suspected connective tissue disease.
Suspected autoimmune hepatitis.
Follow-on test when homogeneous or peripheral fluorescent anti-nuclear antibodies detected.
Interpretation
Test is confirmatory for SLE.
Only antibodies to dsDNA are measured.
Elevated levels occur predominantly in SLE, but also in ‘lupoid’ chronic active hepatitis.
Antibodies are not found in other connective tissue diseases or in all patients with SLE.
Because the antibodies have a circulating half-life of 3 weeks, serial measurements are not useful for monitoring the activity of SLE.
A rising titre may predict clinical relapse, and treatment on a rising titre before symptoms reappear may reduce the total amount of immunosuppression required.
Titre of high-avidity antibody may be associated with progression of renal disease.
ssDNA antibodies
Antibodies to single-stranded DNA and other forms (z-DNA) occur in a wide range of connective tissue diseases.
They have a low sensitivity and specificity.
Antibodies to ssDNA may occur in drug-induced lupus as well as idiopathic lupus.
Other diseases in which there is a high prevalence of anti-ssDNA antibodies are:
rheumatoid arthritis
scleroderma
polymyositis.
The antibodies reduce the sensitivity and specificity of EIA assays for dsDNA because the substrate in assays for the latter may be contaminated with ssDNA produced during the purification process.
A number of commercial assays have been shown to be contaminated in this way, leading to erroneous diagnoses of lupus on the basis of false-positive reports of antibodies to dsDNA.
Assays for antibodies to ssDNA have no clinical role but are used for quality control of assays to dsDNA.
ENA antibodies
Units: qualitative.
Normal adult range: see individual antigens.
Principles of testing
No gold standard test available.
Originally detected by countercurrent immunoelectrophoresis of serum against saline extracts of cells (thymus, spleen).
Other techniques include:
Ouchterlony double diffusion
immunoblotting
EIA.
More than one technique may be required to identify relevant specificities.
EIA assays have increased sensitivity compared to CIE and double diffusion. The clinical significance of this is uncertain.
Testing should include a six-antigen screen (including Scl-70 and Jo-1). Four-antigen screening is not adequate.
EQA and international standards exist.
EQA performance can be quite diverse, depending on assays used.
Indications for testing
Suspected connective tissue disease.
Investigation of congenital complete heart block.
Follow-up testing when high-titre speckled ANA detected by IIF.
Interpretation
These antibodies recognize saline-extracted cellular antigens and cause speckled ANA staining.
Six major specificities are tested for routinely:
anti-Ro (associated with Sjögren’s, SLE, cutaneous lupus, neonatal lupus, and congenital complete heart block)
anti-La (associated with Sjögren’s, SLE, and neonatal lupus)
anti-Sm (specific for SLE, but common only in West Indians)
anti-RNP (associated with SLE and, when occurring alone, said to identify mixed connective tissue disease)
anti-Scl-70 (associated with progressive systemic sclerosis)
anti-Jo-1 (associated with polymyositis and dermatomyositis).
The individual antibodies are discussed separately.
Many other specificities have been identified. Clinical utility is variable.
Where a high-titre speckled ANA is seen but the 6-antigen screen is negative, further investigation by alternative methods may be appropriate if clinically indicated.
Endomysial antibodies (EMA)
Units: qualitative.
Normal adult range: negative.
Principles of testing
IIF for IgA EMA on primate oesophagus (preferred substrate) or human umbilical vein.
Gradually being replaced by EIA for antibodies to tTG (see ‘Tissue transglutaminase (tTG) antibodies’, p.530).
IgA EMA may be a better screening test that IgA tTG.
IgA tTG may be better for long-term follow-up.
Testing should include a screen for IgA deficiency (increased in coeliac disease).
Nephelometric assays do not confirm IgA deficiency at the lowest detection levels.
Ouchterlony double diffusion is more sensitive.
IgG EMA should be sought in IgA-deficient patients.
Indications for testing
Suspected coeliac disease.
Suspected dermatitis herpetiformis.
NICE also recommends regular screening of all children with type I IDDM on an annual basis.
Any patient with small bowel lymphoma must be screened.
Monitoring known coeliac patients for dietary compliance.
NICE have recommended the use of tTG assays for screening in primary care, but this test is too sensitive and generates a lot of false positives who then have to undergo a small intestinal biopsy.
Interpretation
IgA-EMA will be positive in 60–70% of patients with dermatitis herpetiformis and 100% of untreated coeliac patients.
Monitoring of IgA-EMA is valuable in confirming adherence to a gluten-free diet (GFD), as the antibody disappears, along with anti-gliadin antibodies, on a GFD and returns if there is a gluten challenge, even in the absence of overt symptoms.
IgA EMA and IgA tTG give entirely comparable results.
Sensitivity and specificity of IgA EMA and IgA tTG mean that jejunal biopsy is no longer compulsory.
Antibodies may be detected in patients without biopsy evidence of villous atrophy. These patients may be in the early stage of disease. Outcome of early intervention with gluten-free diet is unknown.
IgA EMA and IgA tTG may be negative in children <1 year old (incomplete development of IgA system). Testing for IgG antibodies may be helpful (unproven).
IgA deficiency increases risk of coeliac disease 15-fold.
Patients with coeliac disease may have autoantibodies to the crypt basement membrane of human fetal jejunum. These antibodies appear to be of identical specificity to those detected as endomysial antibodies.
Endothelial antibodies
Antibodies against a variety of endothelial antigens have been described in a variety of vasculitic syndromes:
SLE
rheumatoid vasculitis
systemic sclerosis
haemolytic uraemic syndrome
Kawasaki syndrome
Wegener’s granulomatosis
microscopic polyarteritis
during solid organ graft rejection.
Therefore diagnostic significance is low.
Endothelial cell antibodies have been detected by immunofluorescence on rodent kidney, cultured human endothelial cells, and human umbilical vein cell line (Eayh926).
EIA and immunoblotting and immunoprecipitation have also been used.
Techniques have been difficult to standardize.
Titres may correlate with disease activity in vasculitis.
Test is not routinely available and is of uncertain value clinically.
Epidermal antibodies (including direct immunofluorescence of skin)
These antibodies are useful in the diagnosis of blistering skin diseases.
Bullous pemphigoid
Autoantibodies are directed against the basement membrane.
Autoantibodies recognize two keratinocyte hemi-desmosomal proteins, BP230 and BP180.
On DIF up to 90% of cases have typical linear IgG deposition.
On IIF of serum only 70% will be positive (using monkey oesophagus as a substrate).
Herpes gestationis
Autoantibody is directed against basement membrane.
Antigen is the BP180 protein.
IgG deposition is seen on DIF in only 25% of cases.
100% will have C3 deposition.
Serum is rarely positive.
Epidermolysis bullosa acquisita
Autoantibody is directed against basement membrane.
Gives linear IgG and C3 on DIF that has no distinguishing features from other basement membrane staining.
Diagnostic test is splitting the biopsy between the dermis and epidermis by using high-salt incubation. Immunofluorescence appears on the dermal side.
Antigen is type VII procollagen.
Pemphigus vulgaris
Antibodies recognize the intercellular substance of the epidermis and give typical chickenwire staining by DIF and IIF.
Antigen is desmoglein-1, an intercellular adhesion molecule of the cadherin family.
Pemphigus foliaceus
Antibodies recognize the intercellular substance of the epidermis and give typical chickenwire staining by DIF and IIF.
The antigen in pemphigus foliaceus appears to be different from pemphigus vulgaris by immunoblotting.
The two conditions are not readily distinguishable by routine immunofluorescence.
Paraneoplastic pemphigus
A paraneoplastic form of pemphigus has been described with autoantibodies to desmoplakin I, a desmosomal protein.
Dermatitis herpetiformis (DH)
DH causes the deposition of granular IgA, and sometimes C3, along the dermal papillae on DIF.
Endomysial and gliadin antibodies may be present in serum.
DH must be distinguished from linear IgA disease (a bullous disease).
Linear IgA deposition, often with IgG and C3, at the dermo-epidermal junction.
SLE
DIF of the skin from patients with SLE usually shows coarse irregular granular deposition of IgG, IgM, C3, and C4 along the dermo-epidermal junction (lupus band test).
Similar features may be found in chronically sun-exposed skin from individuals without lupus.
Lichen planus
DIF of skin from patients with lichen planus shows characteristic flame-shaped deposits of fibrin and IgM in the epidermis.
Solid-phase assays are now available for some of the antigens. Salt-split skin direct immunofluoresence can be helpful in localizing the staining.
Erythrocyte antibodies
Anti-red cell antibodies are investigated to test for:
temperature of maximal activity
specificity for red cell antigens
complement binding
agglutination
haemolysis.
This involves looking at the patient’s red cells and serum, and the eluate of the cells.
Cells can also be treated with enzymes to enhance reactivity with certain antigenic systems (e.g. Ii or Pr).
In warm haemolytic anaemia the major target antigens are those of the rhesus system, although many other antigens have been reported as involved.
Warm haemolytic anaemia may be associated with idiopathic haemolysis or be secondary to:
SLE
CLL
lymphoma
viral infections.
In drug-induced haemolysis, there are often antibodies to drug–cell neoantigens (e.g. quinine, penicillins, and cephalosporins).
See also ‘Cold agglutinins’, p.488.
Ganglioside antibodies
Antibodies to gangliosides (sialylated glycolipids which form part of the myelin sheath) have been associated with a number of neurological diseases.
Diagnostic value is limited to supporting clinical diagnoses.
Antibodies to GM1 (and asialo-GM1) and other gangliosides have been associated with:
Guillain–Barré syndrome (GBS)
chronic demyelinating polyneuropathy
multifocal motor neuropathy
paraproteinaemic neuropathies (usually monoclonal IgM with anti-GM1 specificity)
(see Chapter 5).
It has been suggested that the presence of anti-GM1 antibodies may be a predictor of response to intravenous immunoglobulin.
Anti-GD1b is associated with:
GBS
sensory neuropathy.
Anti-GQ1b is associated with:
Miller Fisher variant of GBS (external ophthalmoplegia, ataxia, arreflexia)—most specifically with ophthalmoplegia
chronic ataxic neuropathy (IgM antibodies).
Anti-GT1a is associated with the Miller Fisher variant of GBS.
IgM antibodies to GD1b are highly specific for a rare chronic ataxic neuropathy termed CANOMAD.
Gastric parietal cell (GPC) antibodies
Units: semi-quantitative.
Normal adult range: negative.
Principles of testing
IIF using rodent stomach (tissue multiblock).
Heterophile antibodies (see ‘Heterophile antibodies’, p.500) may produce false positives on human and rat stomach, but do not react on mouse stomach.
EIA available.
There is no international standard.
In the UK, EQA is provided through the General Autoimmune Serology scheme.
Indications for testing
Suspected pernicious anaemia.
Most tests are done without requests as part of an ‘autoantibody screen’.
Interpretation
GPC antibodies are found in almost all patients with pernicious anaemia (PA) in the early stages.
Frequency diminishes with disease progression.
Target autoantigens are the α- and β-subunits of the H+, K+-ATPase (proton pump).
Antibodies are associated with atrophic gastritis (type A).
Antral gastritis (type B) is not associated with GPC antibodies, but may be associated with antibodies to the gastrin-producing cells.
GPC antibodies may be found in asymptomatic individuals. However, about 3% per annum will go on to develop PA.
There is a strong association of PA with thyroid disease:
50% of patients with PA will also have anti-thyroid antibodies
30% of patients with thyroiditis will have GPC antibodies.
There is no correlation of the titre of antibody with disease.
Antibodies to gastrin-producing cells and gastrin receptors (blocking gastrin binding) have also been described in patients with PA (8–30%), but these are of no routine clinical value at present.
Gliadin antibodies (AGA)
Units: qualitative.
Normal range: 15–20% of children have IgA antibodies to gliadin in the absence of coeliac disease.
Principles of testing
EIA for IgA and IgG antibodies against alcohol-soluble fraction of gluten.
Indications for testing
Higher sensitivity and specificity of IgA EMA and IgA tTG assays mean that these assays are the preferred tests for gluten-sensitive enteropathy.
Cerebellar ataxic syndromes have a possible weak association with gliadin antibodies. Routine testing is not recommended in view of the lack of specificity and sensitivity.
Interpretation
Gliadin antibodies are found in coeliac disease and dermatitis herpetiformis, but are not specific.
AGA are also found in Crohn’s disease and ulcerative colitis.
In children, IgA-AGA may be seen in cow’s milk intolerance and post-infective malabsorption.
IgG anti-gliadin antibodies occurring alone are of no particular diagnostic significance, and may be present in a wide range of inflammatory and infective bowel conditions.
The sensitivity and specificity of IgA anti-gliadin antibodies are approximately 100% and 95%, respectively, while the figures for IgG antibodies are 50% and 60%, respectively.
IgA EMA and IgA tTG are the preferred tests for clinical use.
IgA anti-gliadin antibodies are also found in IgA mesangial glomerulonephritis, and may be useful as a diagnostic test.
Disease itself is not affected by adherence to a GFD.
Positive results are also seen in:
children and adults with diabetes mellitus (type I)
first-degree relatives of patients with coeliac disease
patients with Down’s syndrome (increased risk of developing coeliac disease).
Glomerular basement membrane antibodies
Units:
usually qualitative
quantitative assays available through PRUs.
Normal adult range: not detectable.
Principles of testing
Test of choice is EIA, using C-terminal peptide of type IV collagen.
IIF assays using rodent, primate of group O human kidney are not recommended because of high rate of false-positive and false-negative reactions:
only 75% of proven cases will show positive IIF.
Antibodies may be detected by DIF of biopsies of kidney and occasionally lung (linear staining on basement membrane):
IgG and C3 will be detected
occasionally IgA will be present.
Quantitation is useful when plasmapheresis is undertaken.
An EQA scheme exists in the UK.
Indications for testing
Investigation of rapid-onset glomerulonephritis.
Investigation of pulmonary haemorrhage and haemoptysis.
Interpretation
Anti-GBM antibodies are the marker for GBM disease or Goodpasture’s syndrome.
Antibodies are directed against the non-collagenous domains of type IV collagen.
There may be other target antigens (entactin/nidogen?).
Antibodies are examples of primary pathogenic antibodies directly involved in disease process.
Complement fixation takes place at sites of antibody localization.
The same antigen is present in both glomerular and alveolar basement membranes.
Alveolar haemorrhage is usually limited to patients who smoke or are exposed to other irritants (solvent fumes).
10–35% of patients with anti-GBM antibodies may also have ANCAs, with a P-ANCA pattern, usually due to myeloperoxidase antibodies. Significance is uncertain.
Wegener’s granulomatosis may present with pulmonary haemorrhage and glomerulonephritis.
The screen for all patients with glomerulonephritis and/or pulmonary haemorrhage must include both ANCA and GBM antibodies.
These patients require urgent immunological investigations—one of the few occasions on which this is necessary.
Glutamate receptor (mGluR1) antibodies
Identified in small numbers of patients with ataxia and cerebellar degeneration.
Associated with Hodgkin’s lymphoma.
Also identified in patients with stroke and head injury.
Glutamic acid decarboxylase (GAD) antibodies
Units: AU/mL.
Normal adult range: negative.
Principles of testing
EIA for GAD65 antibodies.
Epitopes for IDDM and stiff person syndrome are different and recognition in EIAs differs between assays.
Antibodies to GAD67 also occur but are cross-reactive with GAD65.
Separate assays not used clinically.
Immunoblotting may also be used.
No EQA or international standards.
Indications
Suspected type I IDDM.
Screening with islet cell antibodies may be quicker and cheaper.
Suspected stiff person syndrome.
Interpretation
GADI antibodies against conformational epitopes seen in IDDM.
Only react with native GAD65.
GADII antibodies against linear epitopes seen in stiff person syndrome.
Anti-GAD65 antibodies have been found in >60% of patients with stiff person syndrome.
It is known that the enzyme is concentrated in the GABAergic neurons involved in the control of muscle tone.
Antibodies to GAD67 are not found in IDDM.
Gut (enterocyte) antibodies
Antibodies to gut epithelium may occur:
in UC—antibodies to brush border have been described in UC and in some patients with Yersinia enterocolitis
in UC: antibodies to colonic goblet cells
in intractable diarrhoea of infancy and HIV-associated diarrhoea—antibodies to jejunal and ileal enterocytes
in IPEX syndrome—antibodies to jejunal and ileal enterocytes
in bile salt malabsorption—IgA ileal enterocytes
transiently post-BMT.
The significance of these antibodies is uncertain.
Detection is by IIF.
Heterophile antibodies
These are not true ‘autoantibodies’ but represent a source of confusion with real autoantibodies.
They are antibodies that may normally be present in serum and bind to tissue sections, particularly rodent tissues (see ‘Gastric parietal cell (GPC) antibodies’, p.496).
They are particularly common in patients who have been transfused or allo-immunized in other ways (multiple pregnancies, organ grafts).
Histone antibodies
Antibodies to histones are the marker for drug-induced lupus (95%).
Also seen in SLE (up to 50%).
Cause a homogeneous anti-nuclear staining pattern on IIF.
Target antigens are invariably the histones:
H2A–H2B in procainamide-induced lupus
Hs, H4 in hydralazine-induced lupus.
Virtually all procainamide-treated patients with lupus will have histone antibodies.
Most cases of drug-induced lupus are negative for antibodies to dsDNA, although antibodies to ssDNA may be present.
Commercial ELISA assays are available.
Histone antibodies can be detected by IIF after acid elution of the cellular substrate.
It is essential to ensure that the histone is entirely free from contaminating DNA. This is difficult to do, as the DNA has to be digested in the presence of protease inhibitors to prevent damage to the histones.
There is no EQA scheme.
Hsp-90 antibodies
Antibodies to the 90kDa mammalian heat shock protein have been described in up to 50% of lupus patients and in a few patients with polymyositis.
Antigen is located in the cytoplasm and on the surface membrane.
They are not sought routinely.
Hu antibodies
Antibodies specific for neuronal cell nuclei (anti-Hu, ANNA) have been described in some patients with SCLC accompanied by paraneoplastic syndromes of sensory neuropathies or encephalomyelitis.
Antibodies recognize a 36–42kDa protein of neuronal nuclei, especially of Purkinje cells, recognizing an RNA binding nuclear proteins (HuD, HuC, Hel-N1, and Hel-N2).
They must be distinguished from non-neuron-specific anti-nuclear antibodies. The immunofluorescence staining pattern is fine speckled on neuronal nuclei; glial cells are not stained.
Immunoblotting and EIA may also be used to detect anti-Hu.
Pathological significance is uncertain as removal by plasmapheresis does not improve disease and immunization of animals with the antigen does not elicit disease.
Inner ear antibodies
Rare cases of progressive deafness may be due to an autoimmune process directed against antigens of the inner ear.
These can be detected by immunofluorescence on sections of the inner ear (either bovine or guinea pig), although obtaining suitable material is exceptionally difficult.
Originally it was thought that antibodies to type II collagen formed the basis of this autoimmune process, but more recently it has been shown that the antigen is a heat shock protein (Hsp-2).
A commercial immunoblot system is now available for this antigen.
Insulin antibodies (IAA)
Insulin antibodies may be seen in the following.
Type I IDDM prior to treatment (40%):
they are seen in all young children with IDDM
they are seen in few adults with IDDM (4%)
they disappear with progressive islet cell destruction.
Autoimmune polyendocrinopathies.
Treatment with exogenous insulins.
Epitopes recognized differ from those found in IDDM pre-treatment.
Autoantibodies to insulin have been described as a cause of insulin resistance and are highly specific.
IgE anti-insulin antibodies may be associated with allergic reactions to administered insulin (RAST available).
Antibodies to the insulin receptor have also been described in insulin resistance, usually associated with acanthosis nigricans.
Insulinoma-associated autoantibodies (IA2)
IA2 antibodies are found in latent diabetes and type I diabetes in combination with other antibodies (islet cell antibodies, GAD-65 antibodies, and insulin antibodies) and may provide useful predictive information.
The presence of three or four antibodies gives a risk of progression of 60–100%, although this may be over a long time frame.
The optimum combination of autoantibodies is not defined.
Intrinsic factor antibodies
Units: qualitative.
Normal adult range: negative.
Principles of testing
RIA or EIA.
There is currently no reliable EQA scheme or international standards.
Indications for testing
Suspected pernicious anaemia.
Further investigation of positive gastric parietal cell antibodies.
Interpretation
Antibodies to intrinsic factor are highly specific for pernicious anaemia and are found in up to 75% of patients (see Chapter 4).
Two types of antibodies can be detected:
type I block the binding of B12 to intrinsic factor
type II block the uptake of the IF–B12 complex.
Exogenous vitamin B12 interferes with RIA for type I antibodies (as the assay is a competitive assay using radiolabelled B12).
EIA assays have not been entirely satisfactory.
Value of testing for IF antibodies is uncertain as the diagnosis of pernicious anaemia depends on demonstrating abnormal uptake of B12 in a Schilling test.
Owing to concerns about the use of human albumin and porcine intrinsic factor in the Schilling test, the test is less widely available.
Islet cell antibodies (ICA)
Units: JDF units.
Normal adult range: negative.
>40 JDF units gives 85% PPV for diabetes.
Principles of testing
Indirect immunofluorescence using serum on human group O pancreas.
There is an international standard with a system of units (JDF units).
No EQA scheme in the UK.
Indications for testing
Suspected type I diabetes mellitus.
Screening of first-degree relatives.
Interpretation
Antibodies react with both α- and β-cells:
staining for both is seen in IDDM
staining of β-cells alone is seen in autoimmune polyglandular syndrome
one of the antigens recognized is GAD65.
May be positive when GAD antibodies are negative.
May be found early in the course of type I diabetes mellitus.
Detectable within first year of diagnosis.
>85% newly diagnosed type I diabetics are positive.
Gradually disappear with time as islets are destroyed.
Found in type II diabetes (5–10%).
A small group of patients with multiple autoimmune endocrine disease maintain their antibody levels.
Useful for screening first-degree relatives for risk of developing diabetes. Their presence increases the relative risk of type I IDDM 75-fold.
Prevalence in first-degree relatives is 3–4%. If ICA are present and glucose tolerance impaired >50% risk of developing diabetes within 5 years.
There is no good evidence that quantitation offers any particularly useful clinical information.
The role of ICA vis-à-vis direct measurement of GAD-65, IA2, ZnT8, and IAA in the diagnosis of type I diabetes, latent diabetes, and screening of children with coeliac disease has not been defined.
Jo-1 and related anti-transferase antibodies
Antibodies are found in:
approximately 25% of adult patients with autoimmune myositis
68% in patients with myositis, Raynaud’s, arthritis, and interstitial lung disease (anti-synthetase syndrome).
In sera containing anti-Jo-1, the anti-nuclear antibody may be negative, without the speckled pattern seen with other antibodies to ENA.
Variable faint cytoplasmic staining may be seen on HEp-2 cells.
Therefore antibodies to Jo-1 should be included routinely in the 6-antigen ENA screen (see ‘ENA antibodies’, p.491).
Target antigens are aminoacyl-tRNA synthetases. Jo-1 is the major antigen but other specificities have been identified.
Jo-1: histidyl-tRNA synthetase.
PL-7: threonyl-tRNA synthetase (not reactive in immunoblotting, ?conformational epitope).
PL-12: alanyl-tRNA synthetase.
OJ: isoleucyl-tRNA synthetase.
EJ: glycyl-tRNA synthetase.
lysyl-tRNA synthetase.
All are associated with myositis. Only Jo-1 is common.
Antibodies to signal recognition particles (SRPs) may also be associated, especially in dermatomyositis and polymyositis without the additional features of the anti-synthetase syndrome.
Anti-SRP anti-bodies give a granular cytoplasmic staining on HEp-2 cells.
Jo-1 antibodies are detected by EIA or immunoblotting.
Other specificities are detected by immunoprecipitation from cell lysates followed by polyacrylamide gel electrophoresis.
EQA for ENA covers Jo-1 but not other antigens.
An international standard is available for Jo-1.
Ki and Ku antibodies
Ki antibodies
Ki antibodies recognize a 32kDa protein.
Ki used to be thought to be identical to Ku but is now known to be distinct.
Occur in 10% of SLE and are increased in CNS SLE.
May be detected by CIE or EIA.
Ku antibodies (also known as PL-2 and SL)
Ku antibodies recognize 66 and 86kDa DNA-binding proteins (DNA-dependent protein kinase involved in dsDNA repair).
Found in patients with SLE, MCTD, Sjögren’s syndrome, RhA, and scleroderma (often with myositis).
Patient likely to have Raynaud’s phenomenon.
Also found in primary pulmonary hypertension, polymyositis, Graves’ disease, ITP.
They are of little diagnostic value.
IIF shows fine speckled nuclear and nucleolar staining depending on the stage of the cell cycle.
La (SS-B) antibodies
Antibodies to the ENA La recognize a 48kDa protein complexed to small RNAs that is probably involved in processing of RNA Pol III transcripts.
Protein has sequence homology with a retroviral protein.
Antibody to La is found mainly in primary Sjögren’s syndrome.
It is very rare in Sjögren’s syndrome secondary to rheumatoid arthritis, systemic sclerosis, or primary biliary cirrhosis.
About 15% of patients with SLE will have antibodies to La.
Antibodies to this specificity have been associated with neonatal congenital complete heart block. This is less common than with anti-Ro antibodies.
For methods of detection, see ‘ENA antibodies’, p.491.
Liver cytosol antibodies (LC1)
LC1 antibodies seen in:
autoimmune hepatitis type 2 associated with LKM antibodies
autoimmune cholangitis.
Antigen is forminimontransferase cyclodeaminase (FTCD).
Titre may correlate with disease activity.
Pattern on IIF similar to that of LKM antibodies.
Confirm with immunoblot against 58kDa protein.
Liver–kidney microsomal (LKM) and liver microsomal (LM) antibodies
Units: titre (semi-quantitative).
Normal adult range: negative.
Principles of testing
IIF on tissue multiblock.
Immunoblotting with purified antigen to confirm.
Indications
Suspected autoimmune hepatitis.
Interpretation
LKM antibodies bind to microsomes in the cytoplasm of hepatocytes and the cells of the proximal renal tubules, but do not stain distal renal tubules.
Often confused with mitochondrial antibodies, but the latter will also stain both the stomach and other tubules in the kidney.
Three types of LKM antibodies have been described: LKM-1, LKM-2, and LKM-3.
LKM-1 antibodies recognize the cytochrome P450IID6 and are associated with types 2a and 2b autoimmune chronic active hepatitis:
type 2a disease begins in childhood in 50% of cases and is associated with autoimmunity to thyroid and gastric parietal cells
type 2b is associated with antibodies to hepatitis C in addition to the LKM antibodies.
LKM-2 recognize the cytochrome P450IIC8, C9, C10 and are associated with hepatitis induced by the diuretic ticrynafen (tienilic acid).
LKM-3 recognize uridine diphosphate glucuronyl tranferase and have been reported in hepatitis.
Hepatitis-δ infection is also associated with antibodies to the lamins of the nuclear envelope.
Liver microsomes (LM antibodies) can be seen, where the target antigen is cytochrome P450IA2.
Fluorescence staining is most marked in perivenous hepatocytes.
Associated with hepatitis induced by the drug dihydralazine.
Loop of Henle antibodies and lupus anticoagulant
Loop of Henle antibodies
This pattern is extremely rare and is probably of low diagnostic value.
Reported cases have had renal tubular acidosis, pernicious anaemia, and primary biliary cirrhosis.
Lupus anticoagulant
See ‘Cardiolipin antibodies (ACA) and lupus anticoagulant’, p.485.
Lymphocytotoxic antibodies
Autoantibodies (as opposed to alloantibodies induced by pregnancy or transfusion) to lymphocytes have been detected in:
rheumatoid arthritis
systemic sclerosis
SLE.
Their role in the generation of lymphopenia is uncertain.
There is no evidence to support their role in the lymphopenia of common variable immunodeficiency.
Routine search for these antibodies is probably of little clinical value.
Ma antibodies
Antibodies to Ma2 are found in patients with limbic encephalitis in association with germ cell tumours of the testis (78%), and occur in young patients.
Ma1 and M3 antibodies are found in older patients with cerebellar syndromes in association with tumours (lung, parotid, breast, colon).
Antibodies react with neuronal nucleolar proteins.
Mi-2 antibodies
Antibodies to this ENA are typically found in patients with a steroid-responsive dermatomyositis.
Antibodies are rare in polymyositis (<1%).
Antibodies recognize a 240kDa nuclear antigen (helicase).
Homogeneous nuclear staining on HEp-2 cells is usually seen.
Mitochondrial antibodies (AMA)
Units: titre (semi-quantitative).
Normal adult range: titre <1/40.
Principles of testing
IIF using tissue multiblock.
Follow-up tests using EIA or immunoblots against purified antigens confirm specificity.
Indications for testing
Suspected autoimmune liver disease.
Interpretation (see Table 18.1)
AMA show typical granular staining on the cytoplasm of all cells in the tissue multiblock.
May be confused with LKM, ribosomal, and SRP antibodies.
Nine discrete reactivities have been described, although these are not all detectable on standard tissue sections.
For those patterns that are detectable, the distinction is dependent on recognizing quantitative differences in the level of staining of different cell populations.
Few laboratories report the different specificities by IIF.
M2 antibodies are strongly associated with primary biliary cirrhosis, and more than 95% of PBC patients will be positive.
New mitochondrial antibodies should be put up against the E2 antigen in an EIA or immunoblot (see mitochondrial M2 antibodies) to confirm the specificity.
M4 and M8 antibodies usually occur with M2 antibodies. This pattern is associated with severe disease.
M9 antibodies occurring alone are associated with mild disease.
Patients with type 3 autoimmune chronic active hepatitis (usually associated with antibodies to soluble liver antigens) will often have anti-mitochondrial antibodies and this entity may be an overlap syndrome of CAH and PBC.
Mitochondrial antibodies are often detected in patients with autoimmune thyroiditis, rheumatoid arthritis, scleroderma, and Sjögren’s syndrome, diseases that are associated clinically with PBC.
| Antibody . | Target antigen . | Staining characteristics . | Disease associations . |
|---|---|---|---|
M1 | Cardiolipin | Distal tubules ++ | Syphilis anti-phospholipid syndrome (1° and 2°) |
M2 | Pyruvate (2-oxo-acid) dehydrogenase complex | Distal tubules ++ Parietal cells ++ | Primary biliary cirrhosis (PBC) Also seen in RhA, SSc |
M3 | Not known | Proximal P1, P2, P3 ++ Distal tubules ++ Parietal cells ++ | Lupus-like disease |
M4 | Sulphite oxidase | Proximal tubules + | PBC (often with M2) |
Distal tubules ++ Parietal cells ++ | PBC–scleroderma overlap | ||
M5 | Not known | P1, P2 proximal tubules ++ P3 proximal tubules + Parietal cells + | Miscellaneous connective tissue diseases Occur with anti-β2-GPI antibodies in APS |
M6 | Monamine oxidase B | P1 proximal tubules++ Loop of Henle ++ APUD cells, stomach ++ | Iproniazid-induced hepatitis |
M7 | Sarcosine dehydrogenase | Distal tubules ++ P2, P3 proximal tubules + | Myocarditis cardiomyopathy |
M8 | Not known | Distal tubules ++ Collecting ducts ++ | PBC (often with M2) |
M9 | Glycogen phosphorylase | Distal tubules ++ Collecting ducts ++ | PBC (mild disease?) Autoimmune hepatitis |
| Antibody . | Target antigen . | Staining characteristics . | Disease associations . |
|---|---|---|---|
M1 | Cardiolipin | Distal tubules ++ | Syphilis anti-phospholipid syndrome (1° and 2°) |
M2 | Pyruvate (2-oxo-acid) dehydrogenase complex | Distal tubules ++ Parietal cells ++ | Primary biliary cirrhosis (PBC) Also seen in RhA, SSc |
M3 | Not known | Proximal P1, P2, P3 ++ Distal tubules ++ Parietal cells ++ | Lupus-like disease |
M4 | Sulphite oxidase | Proximal tubules + | PBC (often with M2) |
Distal tubules ++ Parietal cells ++ | PBC–scleroderma overlap | ||
M5 | Not known | P1, P2 proximal tubules ++ P3 proximal tubules + Parietal cells + | Miscellaneous connective tissue diseases Occur with anti-β2-GPI antibodies in APS |
M6 | Monamine oxidase B | P1 proximal tubules++ Loop of Henle ++ APUD cells, stomach ++ | Iproniazid-induced hepatitis |
M7 | Sarcosine dehydrogenase | Distal tubules ++ P2, P3 proximal tubules + | Myocarditis cardiomyopathy |
M8 | Not known | Distal tubules ++ Collecting ducts ++ | PBC (often with M2) |
M9 | Glycogen phosphorylase | Distal tubules ++ Collecting ducts ++ | PBC (mild disease?) Autoimmune hepatitis |
Mitochondrial M2 antibodies
Units: qualitative.
Normal adult range: negative.
Principles of testing
EIA using purified E2 antigen.
Immunoblot/line blot assay.
Indications
Confirmation of specificity of AMA detected by IIF.
Interpretation
Antigen of the M2 mitochondrial autoantibodies is now known to be the E2 component of the pyruvate dehydrogenase complex.
Specificity for PBC approaches 100%.
Other components, E1-α and E1-β, as well the E2 subunit of branched-chain dehydrogenase, are target antigens.
In view of the association of mitochondrial antibodies with diseases other than PBC, it is now advisable to screen AMA-positive sera for antibodies to the M2 antigen to confirm the specificity.
Screening for PBC in the first instance should be by IIF (non-M2 patterns may be identified).
MuSK antibodies
Antibodies to muscle-specific receptor tyrosine kinase may be found in acetylcholine receptor antibody negative myasthenia gravis.
Antibodies to MuSK interfere with signalling by the nerve released ligand, agrin. MuSK and agrin are required for the formation of the neuromuscular junction.
Patients are more likely to be female, to have less eye involvement, and likely to be African Caribbean.
Multiple nuclear dot antibody
Two patterns of multiple nuclear dots are recognized on HEp-2 cells.
Nsp-1: pattern of 2–6 dots located close to the nucleolus.
Antibodies to p80 coilin (anti-coiled body antibodies).
Seen in primary biliary cirrhosis.
Nsp-2: pattern of 5–10 dots (up to 30).
Antibodies to a soluble nuclear protein Sp100.
Often called pseudo-centromere, but there is no staining of the metaphase plate as in a true centromere antibody.
This pattern is associated with a subgroup of patients with primary biliary cirrhosis, especially those who are anti-M2 antibody negative.
Myelin-associated glycoprotein (MAG) antibodies
IgM anti-MAG antibodies are associated with paraproteinaemic polyneuropathies where the paraprotein is IgM (but not IgG or IgA).
Levels of antibody do not correlate with severity of the nerve disease, but removing the antibody by plasma exchange or immunosuppression can improve the neuropathy.
Rarely, anti-MAG antibodies are found in Guillain–Barré syndrome, MS, and myasthenia gravis.
Nephritic factors
Nephritic factors are autoantibodies of either IgG or IgM class that stabilize activated complement components and prevent their normal inactivation by the control proteins. Four types are recognized.
C3-nephritic factor
An autoantibody to the alternate pathway C3 convertase (C3bBb) that stabilizes the convertase and prevents its natural destruction by factors H and I.
Antibody recognizes the Bb component of the convertase, allowing continuous and unregulated activation of C3.
Detected by its effect on C3 mobility on electrophoresis, using immunofixation, and is reported as present or absent.
Patients with mesangiocapillary glomerulonephritis type II and/or partial lipodystrophy who have a markedly reduced C3 should be screened for the presence of a C3-nephritic factor.
Not all patients with partial lipodystrophy who have a C3-nephritic factor will have renal disease, although there is an increased risk of their developing it.
Not all patients with C3-nephritic factor have partial lipodystrophy.
C4-nephritic factor
A rare autoantibody that stabilizes the active form of C4 (C4bC2a) and leads to increased activation of the first part of the classical pathway.
Because the normal regulatory processes are intact at the level of C3, the reaction proceeds no further.
C4 and C3 are usually reduced.
Associated with SLE and other types of glomerulonephritis.
Detected by electrophoretic studies of activated serum using immunofixation.
Properdin-dependent nephritic factor
A nephritic factor of the alternate pathway that slowly cleaves C3, C5, and C9 and depends on the presence of properdin.
It is heat labile.
It occurs in other types of mesangiocapillary glomerulonephritis.
C1q antibodies
These antibodies occur in SLE and mesangiocapillary GN.
They recognize activated C1q (bound to antibody or solid phase).
Neuronal antibodies and neuronal nuclear antibodies (ANNA)
Neuronal antibodies
These antibodies react with a 96kDa surface antigen of neuronal cells and occur mainly in patients with neuropsychiatric lupus.
May be detected by immunofluorescence on cultured human neuroblastoma cell lines or by Western blotting.
May be found in serum of patients with SLE (11%) even in the absence of neuropsychiatric lupus.
Approximately 74% of patients with neuropsychiatric lupus will have these antibodies.
It has been suggested that the titre of antibodies correlates with degree of neuropsychiatric impairment.
More specific for neuropsychiatric lupus than ribosomal P antibodies.
Anti-Yo antibodies recognize the cytoplasm of Purkinje cells (see ‘Yo antibodies’, p.532).
Neuronal nuclear antibodies (ANNA)
ANNA-1 (anti-Hu) react with all neuronal cell nuclei (see ‘Hu antibodies’, p.501).
ANNA-2 (anti-Ri) react only with central not peripheral neuronal cell nuclei (see ‘Ri antibodies’, p.523).
ANNA-3 very rare recognizing 170kDa protein in terminally differentiated neurons and podocytes.
Anti-Ma1, Ma2 (Ta), Ma3 expressed on neuronal nucleoli (see ‘Ma antibodies’, p.507).
Anti-Zic4 (see ‘Zic4 antibodies’, p.532).
Neutrophil antibodies
Antibodies against neutrophil surface antigens may occur in autoimmune neutropenia.
Because of the presence of Fc receptors for IgG on neutrophils, it is very difficult to prove unequivocally that anti-neutrophil antibodies are present, and none of the current techniques is entirely satisfactory.
Tests used include:
neutrophil agglutination
antibody-mediated phagocytosis of neutrophils by macrophages
antibody-dependent lysis
flow cytometry.
Antibodies specific for neutrophil nuclei, reacting with unknown antigens, are found in RhA with vasculitis, particularly in Felty’s syndrome (RhA, splenomegaly, vasculitis and leg ulceration, neutropenia).
Homogeneous nuclear staining is present only on neutrophils, and not on liver or HEp-2 cells.
Their importance is more in their potential to cause misinterpretation in tests for ANCA, where they may be confused with the perinuclear pattern by the inexperienced.
Neutrophil cytoplasmic antibodies (ANCA)
Units: titre.
Normal adult range: titre < 1/10.
Principles of testing
ANCA are detected on ethanol-fixed human granulocytes.
Other fixation methods are not recommended by the International Consensus Statement.
P-ANCA pattern results from a redistribution of certain cytoplasmic antigens when cold ethanol is used as a fixative for the human neutrophils.
Positive results should be followed up with antigen-specific EIA or immunoblots.
Serial monitoring of titre may be valuable.
Indications for testing
Rapidly progressive glomerulonephritis pulmonary haemorrhage.
New patients must always have both ANCA and GBM performed.
Interpretation (see Table 18.2)
90% of patients with Wegener’s granulomatosis will be C-ANCA+.
5–10% of WG patients will be ANCA–.
Antibodies are present in both the systemic and localized forms of WG.
As a test for WG, meta-analysis of all published data has shown a sensitivity of 66% and a specificity of 98%.
30% of patients with microscopic polyarteritis and Churg–Strauss syndrome will be C-ANCA+.
C-ANCA antigen is proteinase 3 (Pr3), a granule protein also expressed on the neutrophil surface.
Perinuclear (P-ANCA) pattern is found less commonly in WG and MPA, but mainly in other forms of severe vasculitis, including Churg–Strauss syndrome, SLE, and rheumatoid vasculitis.
Atypical ANCA (X-ANCA) may occur in inflammatory bowel disease, particularly where there is liver involvement (primary sclerosing cholangitis (PSC)).
Multiple antigens are involved in producing C-ANCA and P-ANCA.
Antibodies to nuclear antigens and GBM may coexist with ANCA, making diagnosis difficult.
Where there is doubt check fluorescent pattern on tissue multiblock to exclude ANA.
Serial monitoring of C-ANCA is useful as a rising titre may herald a relapse of WG.
Patients with treated vasculitis may remain weakly ANCA+ for years in clinical remission.
Whether there is any value in serial monitoring of P-ANCA is less certain.
IgA ANCA have been reported in Henoch–Schönlein purpura.
| Target antigen . | Staining pattern . | Clinical association . |
|---|---|---|
Proteinase 3 | C-ANCA (90%) P-ANCA (2%) | WG, microscopic polyarteritis, Churg–Strauss (30%) |
Myeloperoxidase | P-ANCA (70%) P-ANCA (5%) | WG, microscopic polyarteritis, Churg–Strauss (60%), GBM (30%) |
Azuocidin (CAP37) | P-ANCA | Uncertain |
β-glucuronidase | P-ANCA | Uncertain |
Bactericidal permeability-increasing protein (BPI) | C-ANCA (4%) atypical ‘flat’ ANCA | Cystic fibrosis (50%) |
Cathepsin G | P-ANCA (5%) X-ANCA | Sclerosing cholangitis, ulcerative colitis |
Elastase | P-ANCA (8%) X-ANCA | SLE (neuro-SLE), hydralazine-induced SLE, RhA, ulcerative colitis |
Lactoferrin | P-ANCA (10%) X-ANCA | Inflammatory bowel disease, PBC, AIH, SLE, RhA |
Lysozyme | C-ANCA P-ANCA | Uncertain |
| Target antigen . | Staining pattern . | Clinical association . |
|---|---|---|
Proteinase 3 | C-ANCA (90%) P-ANCA (2%) | WG, microscopic polyarteritis, Churg–Strauss (30%) |
Myeloperoxidase | P-ANCA (70%) P-ANCA (5%) | WG, microscopic polyarteritis, Churg–Strauss (60%), GBM (30%) |
Azuocidin (CAP37) | P-ANCA | Uncertain |
β-glucuronidase | P-ANCA | Uncertain |
Bactericidal permeability-increasing protein (BPI) | C-ANCA (4%) atypical ‘flat’ ANCA | Cystic fibrosis (50%) |
Cathepsin G | P-ANCA (5%) X-ANCA | Sclerosing cholangitis, ulcerative colitis |
Elastase | P-ANCA (8%) X-ANCA | SLE (neuro-SLE), hydralazine-induced SLE, RhA, ulcerative colitis |
Lactoferrin | P-ANCA (10%) X-ANCA | Inflammatory bowel disease, PBC, AIH, SLE, RhA |
Lysozyme | C-ANCA P-ANCA | Uncertain |
NMDA (N-methyl-d-aspartate) receptor antibodies
Antibodies to NR1 and NR2 have been associated with a limbic encephalitis, SLE, and variant epilepsies.
There is an association with ovarian or mediastinal teratomas expressing the NMDA receptor, although cases may occur without any evidence of malignancy.
Nuclear antibodies (ANA)
Units: semi-quantitative.
Normal ranges:
age <18, titre <1/20
age 18–65, titre <1/40
age >65, titre <1/8.
Principles of testing
Testing is undertaken by IIF using a tissue multiblock with HEp-2 cells.
Genetically modified HEp-2 cells (HEp-2000) are said to express higher levels of Ro antigen. In practice, there is little advantage when the microscopist is experienced.
Ro antigens may be eluted from rodent liver and give false-negative results (‘ANA-negative lupus’). These will be identified by parallel use of HEp-2 cells.
HEp-2 cells are more sensitive. Screening with HEp-2 cells will increase the false-positive rate compared with rodent liver.
HEp-2 cells are valuable for identification of staining pattern.
EIA screening assays are available to reduce the number of negative immunofluorescent screens performed.
Only IgG antibodies are of clinical significance. Screening should be carried out with anti-human IgG-FITC, not polyvalent antibodies.
Initial screening dilution for serum must be modified for children.
Appropriate titratable controls are required.
Reporting should include significant patterns.
EQA is available: performance is variable.
Modern fluorescent microscopes significantly affect titration results.
International standards are available.
Indications for testing
Suspected connective tissue disease.
Interpretation (see Table 18.3)
Nuclear antibodies are associated with the connective tissue diseases.
Only IgG antibodies are significant.
IgM ANA are non-specific and frequently occur after viral infections.
Occasional patients with connective tissue disease produce only IgM ANA.
IgM ANA occur in rheumatoid arthritis (cross-reactive IgM RhF).
Significance of the titre of IgG ANA is age-dependent.
Low-titre positive ANAs in the elderly must be carefully interpreted in the context of relevant clinical symptoms and signs.
Pattern of ANA identifies likely antigens and significance.
Follow-up testing with ENA and dsDNA assays is required to fully investigate antigenic specificities identified by IIF.
Because of the long circulating half-life of the autoantibodies, measurement does not need to be carried out more frequently than once every 3 weeks (unless the patient has been plasmapheresed).
Detection of a strong positive ANA is an indicator for confirmatory tests, including dsDNA, ENA, and, if clinically appropriate, histone antibodies.
| HEp-2 staining pattern . | Antigen(s) . | Disease associations . |
|---|---|---|
| Nuclear staining . | ||
Homogeneous nuclear/nucleolar | dsDNA, histones, topoisomerase 1, Ku | SLE |
Homogeneous nuclear (nucleoli negative) | Histone | SLE, drug-induced SLE |
Nuclear membrane | Lamins A, B, C | Autoimmune liver disease anti-phospholipid syndrome leucocystoclastic vasculitis |
Nuclear pore (punctate staining) | gp210 pore protein, lamin B receptor | Primary biliary cirrhosis |
Peripheral nuclear | dsDNA | SLE |
Nuclear matrix (coarse speckles) | hn-RNP | SLE |
Coarse speckled | U1-RNP, Sm, Ki | SLE, MCTD |
Fine speckled | Ro, La, Mi-2 | SLE, Sjögren’s, dermatomysositis |
Granular nuclear staining | Topoisomerase 1 (Scl-70) | Systemic sclerosis |
Coarse and fine speckles | PCNA | SLE |
Centromere (condensed in metaphase plates) | CENP-A, -B, -C | CREST, limited scleroderma |
Nuclear dots (2–6) Nsp-1 | p80 coilin | PBC |
Nuclear dots (6–10, up to 30): pseudo-centromere | Sp100 | PBC (AMA negative) |
Homogeneous nucleolar | Pm-Scl, Ku, nucleolin | Polymyositis–scleroderma overlap |
Clumpy nucleolar | Fibrillarin | Systemic sclerosis |
Speckled nucleolar | RNA Pol 1 | Systemic sclerosis |
Nucleolar dots | Nor-90 (nucleolar organizer region) | Scleroderma |
Centriole | Centriole | Non-specific (CTDs, post-infective) |
Nuclear mitotic spindle apparatus (NuMa, MSA-1) | NuMA protein | Non-specific CTD |
Mitotic spindle apparatus-2 (MSA-2) | Mid-body | Systemic sclerosis, Raynaud’s mid-body pattern |
Mitotic spindle apparatus -3 (MSA-3) | CENP-F | Respiratory tract tumours |
Cytoplasmic | ||
Mitochondria (large granule staining) | Multiple antigens (M1–M9) | PBC, autoimmune liver disease |
Ribosomes | Ribosomal P proteins | SLE |
Irregular granular staining | Lysosomes, peroxisomes | Non-specific |
Fine speckled | Jo-1, other tRNA synthetases | Polymyositis |
Golgi body | Giantin (50%) golgins 245, 160, 95, 97 | SLE, Sjögren’s, other CTDs, chronic CMV |
Actin filaments | Actin | Autoimmune hepatitis |
Vimentin | Vimentin | SLE, other CTDs |
Cytokeratin | Cytokeratin 18 | RhA |
| HEp-2 staining pattern . | Antigen(s) . | Disease associations . |
|---|---|---|
| Nuclear staining . | ||
Homogeneous nuclear/nucleolar | dsDNA, histones, topoisomerase 1, Ku | SLE |
Homogeneous nuclear (nucleoli negative) | Histone | SLE, drug-induced SLE |
Nuclear membrane | Lamins A, B, C | Autoimmune liver disease anti-phospholipid syndrome leucocystoclastic vasculitis |
Nuclear pore (punctate staining) | gp210 pore protein, lamin B receptor | Primary biliary cirrhosis |
Peripheral nuclear | dsDNA | SLE |
Nuclear matrix (coarse speckles) | hn-RNP | SLE |
Coarse speckled | U1-RNP, Sm, Ki | SLE, MCTD |
Fine speckled | Ro, La, Mi-2 | SLE, Sjögren’s, dermatomysositis |
Granular nuclear staining | Topoisomerase 1 (Scl-70) | Systemic sclerosis |
Coarse and fine speckles | PCNA | SLE |
Centromere (condensed in metaphase plates) | CENP-A, -B, -C | CREST, limited scleroderma |
Nuclear dots (2–6) Nsp-1 | p80 coilin | PBC |
Nuclear dots (6–10, up to 30): pseudo-centromere | Sp100 | PBC (AMA negative) |
Homogeneous nucleolar | Pm-Scl, Ku, nucleolin | Polymyositis–scleroderma overlap |
Clumpy nucleolar | Fibrillarin | Systemic sclerosis |
Speckled nucleolar | RNA Pol 1 | Systemic sclerosis |
Nucleolar dots | Nor-90 (nucleolar organizer region) | Scleroderma |
Centriole | Centriole | Non-specific (CTDs, post-infective) |
Nuclear mitotic spindle apparatus (NuMa, MSA-1) | NuMA protein | Non-specific CTD |
Mitotic spindle apparatus-2 (MSA-2) | Mid-body | Systemic sclerosis, Raynaud’s mid-body pattern |
Mitotic spindle apparatus -3 (MSA-3) | CENP-F | Respiratory tract tumours |
Cytoplasmic | ||
Mitochondria (large granule staining) | Multiple antigens (M1–M9) | PBC, autoimmune liver disease |
Ribosomes | Ribosomal P proteins | SLE |
Irregular granular staining | Lysosomes, peroxisomes | Non-specific |
Fine speckled | Jo-1, other tRNA synthetases | Polymyositis |
Golgi body | Giantin (50%) golgins 245, 160, 95, 97 | SLE, Sjögren’s, other CTDs, chronic CMV |
Actin filaments | Actin | Autoimmune hepatitis |
Vimentin | Vimentin | SLE, other CTDs |
Cytokeratin | Cytokeratin 18 | RhA |
Nuclear matrix antibodies
These antibodies give large coarse speckles on HEp-2 cells.
Specificity appears to be against heterogeneous nuclear RNA and matrix proteins. At least four proteins, of molecular weight 70, 31, 23, and 19kDa, are recognized by immunoblotting.
70kDa determinant is U1-RNP.
These antibodies are rarely seen but occur in lupus as well as in undifferentiated connective tissue diseases.
Nuclear mitotic spindle antibodies (MSA)
Antibodies will only be seen on HEp-2 cells. They are rare and not specific, being found in
SLE
rheumatoid arthritis
CREST
MCTD
Sjögren’s syndrome.
Described patterns
NuMA (nuclear mitotic apparatus protein, MSA-1) show fluorescence concentrated at the spindle poles:
non-specific (CTDs).
Anti-tubulin antibodies stain the spindle fibres:
post-infective and autoimmune disease
low titres in normal individuals.
Antibodies to MSA show two patterns of staining:
MSA-2 does not stain interphase cells and is sometimes referred to as the mid-body pattern:
associated with systemic sclerosis and Raynaud’s.
MSA-3 shows fine dense nuclear staining in some interphase cells and two sets of discrete granules on either side of the metaphase plate in dividing cells:
antigen is CENP-F (p330 protein)
seen rarely in respiratory tract tumours.
Nuclear pore antibodies
Antibodies to a 210kDa nuclear pore antigen have been described in up to 27% of cases of primary biliary cirrhosis (in addition to the multiple nuclear dot pattern).
IIF on HEp-2 cells gives a punctate perinuclear staining pattern.
Nucleolar antibodies
Nucleolar antibodies will be detected on routine screening on rodent liver. They are much easier to see on HEp-2 cells, where three discrete staining patterns can be identified.
Speckled nucleolar staining with fine speckled nuclear staining:
antibody recognizes RNA polymerase I
present in 4% of patients with systemic sclerosis (diffuse disease).
Clumpy nucleolar staining with nuclear dots:
antibody recognizes fibrillarin, component of U3-RNP
present in 8% of patients with systemic sclerosis, particularly with cardiopulmonary involvement.
Other nucleolar antigens to which autoantibodies have been detected include:
Nor-90, a 90kDa protein of the nucleolar organizer region (nucleolar dot staining on HEp-2 cells)
To, a 40kDa protein complexed to 7S or 8S RNA associated with pulmonary hypertension and bowel involvement in scleroderma
an RNP particle that includes RNA polymerase III.
Ovarian antibodies
Antibodies identified by IIF on multiblock (adrenal, ovary, testis, pituitary).
Useful for identifying primary autoimmune ovarian failure as a cause of infertility.
Found in 15–50% of patients with premature ovarian failure.
Patients may also be positive for adrenal antibodies (cross-reactive steroid cell antibodies) and thyroid antibodies.
Ovarian antibodies recognize P450scc enzyme.
Antibodies to 3β-hydroxysteroid dehydrogenase may be a more sensitive marker of premature (autoimmune) ovarian failure.
See also ‘Adrenal cortex autoantibodies’, p.483.
Parathyroid antibodies
Found in patients with:
idiopathic hypoparathyroidism
hypoparathyoidism associated with other endocrinopathies
chronic mucocutaneous candidiasis.
Detected on group O human parathyroid by immunofluorescence.
They are impossible to detect if ANA or mitochondrial antibodies are present.
Therefore testing should be carried out in parallel with testing on the standard tissue multiblock.
Control sera are extremely difficult to obtain.
PCNA (proliferating cell nuclear antigen) antibodies
Antigen recognized by the autoantibody is expressed only at certain times in the cell cycle.
It is readily detectable only on HEp-2 cells, where it gives variable-sized speckles in only some cells.
Antigen is a 33kDa auxiliary protein for DNA polymerase-δ.
PCNA has erroneously been referred to as cyclin.
It is seen in about 3% of patients with SLE, but does not appear to identify any particular clinical subgroup.
Pituitary gland antibodies
Antibodies to pituitary components have been described in:
a variety of centrally mediated endocrine disorders, including autoimmune lymphocytic hypophysitis (70% positive) and autoimmune hypopituitarism (30% positive)
empty sella syndrome
some pituitary tumours
some patients with insulin-dependent diabetes mellitus.
Antibodies are usually detected by immunofluorescence on pituitary sections or pituitary cell lines.
Antibodies recognize a cytoplasmic 49kDa antigen.
Diagnostic value is uncertain.
No commercial control sera are available.
Platelet antibodies
Platelet autoantibodies (as opposed to alloantibodies) are found in patients with ITP.
Also found in other conditions associated with thrombocytopenia, such as:
HIV
connective tissue diseases
thrombotic thrombocytopenic purpura (TTP)
heparin-induced thrombocytopenia.
Many techniques have been described to detect such antibodies including:
phagocytosis by neutrophils
lymphocyte activation
RIA
EIA
flow cytometry.
When sensitive techniques areed, >90% of patients with ITP will be shown to have platelet-associated IgG.
There is no consensus on the best technique or on the value of routine testing.
Target antigens are varied but include platelet GP Ib, IIb, and IIIa.
Alloantibodies to platelets react with PLA1 antigen.
PM-Scl antibodies (also known as PM-1)
PM-Scl antibodies are found in the polymyositis–scleroderma overlap syndrome.
May also be found in patients with dermatomyositis/polymyositis or systemic sclerosis occurring alone.
There may be an increased risk of renal disease in the overlap patients.
Target antigen appears to be a complex of 11 proteins occurring in the nucleolus and elsewhere:
two major antigens are PM/Scl-100 and PM/Scl-75.
The pattern of immunofluorescence on HEp-2 cells is variable, with homogeneous nucleolar staining with some atypical nuclear speckling and cytoplasmic staining.
Detection is usually by immunodiffusion or immunoblotting.
Poly-(ADP-ribose) polymerase antibodies
These antibodies to the 116kDa protein have been reported in Sjögren’s syndrome and in association with neuropathy.
They are not sought routinely.
Purkinje cell antibodies
See ‘Yo antibodies’, p.532.
RA-33 antibodies and rheumatoid-associated nuclear antibodies (RANA)
RA-33 antibodies
These antibodies have been described as specific for rheumatoid arthritis.
They react with a 33kDa non-histone nuclear antigen of HeLa cells.
There are insufficient data to evaluate their utility.
Rheumatoid-associated nuclear antibodies (RANA)
If EBV-transformed cell lines are used as a target, antibodies that react only with EBV-infected nuclei but not normal nuclei can be detected in serum of patients with RhA and other connective tissue diseases.
There is no routine value to this test.
Recoverin antibodies
See retinal S100 antibodies.
Renal biopsy (direct immunofluorescence)
Direct immunofluorescence of renal biopsies is an essential part of the evaluation of renal disease.
DIF should always take place in parallel with examination of biopsies with standard stains and by electron microscopy.
Staining should include the use of antisera to IgG, IgA, IgM, C3, C4, and fibrinogen.
Linear deposition of IgG in the glomeruli is a feature of anti-GBM disease.
IgA deposition is heavy in a segmental pattern in IgA nephropathy.
Henoch–Schönlein purpura is also associated with IgA deposition, often with fibrin deposition.
In type II MPGN associated with a nephritic factor, there is heavy C3 deposition in the GBM, without immunoglobulin.
Patchy IgG and C3, or C3 alone, is seen in post-streptococcal GN.
SLE may give any pattern of renal disease, and is usually accompanied by IgG, IgM, and complement deposition with a variable distribution.
In tubulo-interstitial nephritis, there may be antibodies to the tubular basement membrane.
Reticulin antibodies
These antibodies are non-specific.
They are found in:
coeliac disease
inflammatory bowel disease, particularly where there is concomitant liver disease.
They may occur in the absence of clinical disease in the elderly.
They will be detected as part of the ‘autoimmune screen’ on a tissue multiblock.
If coeliac disease is suspected, endomysial or tTG antibodies should be requested.
IgA reticulin antibodies are slightly more specific for coeliac disease than IgG antibodies, but sensitivity and specificity is far lower than for EMA and tTG antibodies.
Up to five different patterns of anti-reticulin antibodies have been described using immunofluorescence.
Only the R1 pattern is associated with coeliac disease.
IgA-R1-reticulin antibodies are reported to have a specificity of 98% but a sensitivity of only 25% for coeliac disease.
Laboratories no longer report reticulin antibodies routinely.
Detection of strong R1 ARA should trigger EMA or tTG testing automatically in the laboratory.
Retinal S100 and cancer-associated retinopathy (CAR) antibodies
Antibodies against retinal antigens (S100) have been identified in many types of chronic inflammatory eye disease affecting the uveal tract, including Vogt–Koyanagi–Harada syndrome.
Antibodies are not disease-specific.
Diagnostic value is uncertain at present.
The antibodies may induce demyelination of the optic nerve.
Other anti-retinal antibodies (CAR) have been described as a paraneoplastic phenomenon in patients with a cancer-associated retinopathy syndrome, seen rarely in association with SCLC.
Antibodies bind to small cell carcinoma cell nuclei.
Major antigen is recoverin, a calcium-binding protein.
a-enolase is a minor autoantigen.
CV2/CRMP5 antibodies may also be associated with CAR.
Lens-induced uveitis, a rare condition that may follow trauma or surgery, is associated with circulating antibodies to lens proteins.
Melanoma-associated retinopathy antigen is not well characterized. Patients present with dazzling photopsia. Autoantibody binds to bipolar cells of the retina.
Rheumatoid factor (RhF)
Units:
titre
IU/mL.
Normal adult range:
titre, <1/80
<30 IU/mL.
Principles of testing
Assays detect anti-immunoglobulin antibodies.
Originally identified by sheep-cell agglutination test (SCAT, Rose–Waaler test)—this test is no longer used.
Now detected by latex or particle agglutination assays, EIA, nephelometry, or turbidimetry.
Measurement in IU/mL is preferred, but latex agglutination assays, which are sensitive but not specific, provide a cheap screening tool.
Semi-quantitative particle agglutination assays are less desirable:
these assays may be susceptible to interference by serum fibronectin.
EQA and international standards exist.
Indications for testing
The only indication for testing is in patients with clinical RhA.
It is not a screening test for RhA.
There is no value in the elderly, as RhF may be found in healthy elderly.
Interpretation
RhF is a non-specific test as it detects immunoglobulins of any class reactive with the Fc region of other immunoglobulins.
RhFs occur in a wide variety of conditions, such as:
viral infections
chronic bacterial infections (SBE, TB)
myeloma
lymphomas
many connective tissue diseases
old age (not associated with disease).
In myeloma, paraproteins with RhF activity may cause a type II cryoglobulin.
In infections and connective tissue diseases, polyclonal RhFs may cause a type III cryoglobulin.
RhA patients may be positive or negative for RhFs.
Those with progressive disease, and with vasculitis, usually have high-titre RhFs.
There is little value in serial monitoring of RhF as the titre correlates poorly with disease activity. The CRP is more useful.
Requesting RhFs in the elderly is not helpful, as positive results do not necessarily indicate disease.
At present there is no conclusive evidence that detecting IgA RhFs is valuable.
Ri antibodies
Anti-Ri, a rare anti-neuronal nuclear antibody, has been documented in a few women with breast cancer or SCLC, associated with ataxia, myoclonus, and opsoclonus.
Antibodies recognize 55kDa and 80kDa proteins (Nova-1, Nova-2), found in all neuronal cell nuclei.
Antibodies can be detected by immunofluorescence and immunoblotting.
Ribosomal (ribosomal P) antibodies
Antibodies to ribosomes, particularly to ribosomal ribonucleoprotein (rRNP), are associated particularly with SLE (about 5–12% of patients).
Majority of patients with neuropsychiatric lupus will be positive for ribosomal P antibodies. Antibody level is reported to correlate with disease activity.
May also be found in RhA.
Antibodies recognize three phosphoproteins (P0, P1, P2):
antigens are distinct from antibodies to nuclear RNPs.
Ribosomal and ribosomal P antibodies are the same.
They frequently cause diagnostic confusion as they may be misinterpreted as mitochondrial antibodies by inexperienced microscopists.
They react particularly strongly with the chief cells of rodent stomach, and also with pancreatic tissue.
On HEp-2 cells there is fine speckled staining of the cytoplasm and some staining of nucleoli.
If seen on autoantibody screen, antibody should be titrated and reported.
RNP antibodies
These antibodies recognize nuclear, rather than ribosomal, RNPs.
There are a number of such complexes (U1–U6-RNPs), which comprise a number of proteins with small nuclear RNAs.
The same protein components may occur in more than one RNP complex.
The most important antibodies are those against the U1-RNP, which recognize the 70kDa A and C protein components.
They give rise to a coarse speckled pattern on IIF on HEp-2 cells.
Found in SLE and mixed connective tissue disease (MCTD).
When they occur in the absence of antibodies to dsDNA and Smith antibodies (Sm). This is supposed to indicate MCTD, although some authorities doubt that MCTD is truly a distinct entity and prefer to think of it as a subset of SLE.
There is no relation between anti-RNP antibody titres and disease activity.
Some sera from patients with SLE contain antibodies that react with both Sm and U1-RNP.
Antibodies that react with both U1- and U2-RNP, recognizing the U1-A and U2-B′ proteins, have been described.
These are seen in SLE and SLE overlap syndromes, particularly where myositis is a feature.
Distinction of the fine specificities of antibodies to nuclear RNPs is not available routinely, but can be achieved with Western blotting.
Normally, antibodies to RNP will be detected by immunodiffusion, countercurrent immunoelectrophoresis, ELISA, or immunoblotting.
Ro (SS-A) antibodies
Antibodies to the ENA antigen Ro recognize two proteins of 60 and 52kDa complexed with Y1–Y5 RNAs.
Antibodies have been described that recognize either protein.
Antibodies are associated with:
Sjögren’s syndrome, with the highest levels in primary disease
SLE
cutaneous LE
C2-deficient lupus.
Antibodies are of particular significance in pregnancy, as they are usually of IgG class and cross the placenta.
Ro antigen is expressed transiently in the fetal cardiac conducting system.
Maternal anti-Ro binds to the antigen and causes fibrosis, leading to congenital complete heartblock.
Antibodies are also responsible for neonatal lupus.
Antigen is also inducible in keratinocytes by UV light, explaining strong association of anti-Ro with photosensitivity and with cutaneous disease.
Anti-Ro is the specificity detected in so-called ‘ANA-negative lupus’.
Ro antigens are easily eluted from rodent tissue, causing false-negative results.
HEp-2 (and HEp-2000) cells have higher levels of Ro antigen and are not susceptible to this problem.
Antibodies show a fine nuclear speckled staining on HEp-2 cells.
Commercial assays for Ro52 and Ro65 are available.
Salivary gland antibodies
It is not possible to test for these in the presence of anti-nuclear or anti-mitochondrial antibodies.
They are associated with Sjögren’s syndrome and are more likely to occur in secondary than primary disease.
Detected by IIF on rodent salivary gland.
Scl-70 antibodies
Found in 20–40% of patients with progressive systemic sclerosis (PSS).
Found in 20% of patients with limited scleroderma.
Associated particularly with severe skin disease, musculoskeletal disease, and cardiopulmonary disease.
Antibody may be a marker for the development of carcinoma of the lung in PSS.
Do not predict the development of renal disease, although their presence in patients with isolated Raynaud’s predicts the subsequent development of PSS and hence is a poor prognostic marker.
Antigen recognized is topoisomerase I, an enzyme involved in supercoiling DNA:
autoantibody interferes with the function of the target antigen.
Scl-70 and anti-centromere antibodies appear to be mutually exclusive, as only two cases have ever been reported of coexistence of both specificities.
Scl-70 antibodies give an atypical granular nuclear speckled pattern on HEp-2 cells.
Definitive proof of their presence is obtained by mmunodiffusion, ELISA, or immunoblotting.
Signal recognition particle (SRP) antibodies
These antibodies produce a cytoplasmic staining pattern on HEp-2 cells that may be mistaken for that of mitochondrial antibodies.
The antigen is a 54kDa protein complexed with RNA.
It is associated with polymyositis and dermatomyositis.
Skin and mucosal biopsies (direct immunofluorescence)
See ‘Epidermal antibodies (including direct immunofluorescence of skin)’, p.494.
Sm (Smith) antibodies
These antibodies, named after the patient in whom they were first described, are specific for SLE.
They are seen most frequently in West Indians with SLE.
Rare in Caucasians.
Antibodies react with the B′/B and D proteins shared by U1-, U2-, and U4–U6-RNPs.
These specificities are often seen with antibodies to U1-RNP.
Whether occurring alone or with anti-RNP, these antibodies are accepted as a diagnostic criterion for lupus.
They are usually detected by the same techniques as used for other ENAs.
See ‘ENA antibodies’, p.491.
Smooth muscle antibodies (SMA)
Units: titre (semi-quantitative).
Normal adult range: titre, <1/40.
Principles of testing
IIF on tissue multiblock.
Specific subpatterns may be identified on HEp-2 cells.
Indications for testing
Suspected autoimmune hepatitis.
Interpretation
SMA typically stain the muscular coats of arteries and the muscular layer of the stomach section, where there is also staining of the intergastric gland fibres. On HEp-2 cells, a meshwork of fine cytoplasmic fibres may be seen.
SMA are present in high titre in 50–70% of patients with autoimmune ‘lupoid’ hepatitis (type 1):
25% of patients may also be positive for nuclear and dsDNA antibodies.
Also seen in association with hepatitis B surface antigenaemia and adenovirus infection.
High-titre SMA antibodies against F-actin are seen in type 4 hepatitis, which affects predominantly young children.
Antibodies to F-actin also seen in a subset of PBC patients.
Other SMA directed against tropomyosin may be found.
Soluble liver antigen (SLA) antibodies and Sp100 antibodies
Soluble liver antigen (SLA) antibodies
Predominant marker for type 3 hepatitis.
May be the only autoantibody in up to 25% of cases.
Target antigen for SLA antibodies is thought to be a unique enzyme that is a member of the pyridoxal phosphate-dependent transferases.
SLA is the same as liver/pancreas antibodies
Testing is by immunoblot.
Sp100 antibodies
Antibodies to this antigen of nucleus give rise to the multiple nuclear dot (6–10 dots) pattern of staining on HEp-2 cells (Nsp-2, pseudo-centromere).
It is found in AMA-negative primary biliary cirrhosis.
A commercial ELISA assay for antibodies to this antigen is available.
See ‘Nuclear antibodies (ANA)’, p.514.
Sperm antibodies
Sperm antibodies
Both agglutinating and immobilizing antibodies have been described.
Direct agglutination, indirect agglutination, and EIA are used to detect antibodies.
An international standard exists and there is an EQA scheme in the UK.
Multiple antigens seem to be involved and only some seem to be important in interfering with fertility.
They are common after vasectomy (50% of men).
May occur after trauma to the testes.
Detected in:
1–12% of women
10–20% of women with infertility.
Antibodies may be of IgG or IgA class and may be found in serum or seminal/cervical secretions.
Steroid cell antibodies
Striated muscle antibodies
Striated muscle antibodies
Present in 25–40% of patients with myasthenia gravis.
Almost all (80–100%) patients with myasthenia with thymoma are positive.
A subgroup of myasthenic patients <40 years of age may be positive in the absence of thymoma.
May be seen in patients on penicillamine.
May be seen in graft-versus-host disease after BMT.
Constitute an important simple screening test in myasthenia for the presence of thymoma and should form part of the diagnostic work-up.
Antigen is thought to be a protein of the I-band of the myocyte (titin).
Detected by IIF on primate striated muscle.
Rodent striated muscle may give false-positive results.
Sympathetic nervous system antibodies
Antibodies reactive with sympathetic ganglia and adrenal medulla may be seen in type I IDDM.
Not routinely available.
Thyroid microsomal (peroxidase) antibodies
Units:
semiquantitative (particle agglutination)
KU/L (EIA).
Normal adult range:
titre, <1/800 (particle agglutination)
0–60 kU/L (EIA may vary between assays).
Principles of testing
Particle agglutination assays (thyroid microsomal antibodies) give semi-quantitative results:
particle agglutination assays use either tanned red cells or latex particles.
EIA for thyroid peroxidase antibodies is preferred assay and gives quantitative results.
Immunofluorescence using thyroid tissue in a multiblock can no longer be recommended.
Ideally, TPO antibodies should be integrated into thyroid testing strategy, linked to TSH results, on main biochemistry analysers.
An international standard exists and there is an EQA scheme in the UK.
Indications for testing
Suspected thyroid disease.
Autoimmune polyglandular syndromes.
Interpretation
Present in high titre in:
95% of patients with Hashimoto’s thyroiditis
18% of patients with Graves’ disease
90% of patients with primary myxoedema.
Present in low titres in patients with:
colloidal goitre
thyroid carcinoma
transiently in de Quervain’s thyroiditis
occasionally in normal people.
Positive TPO antibodies have also been associated with increased risks of pre-eclampsia, post-natal depression, and peri-menopausal depression.
They may also be found in patients with other organ-specific autoimmune diseases such as pernicious anaemia, Addison’s disease, etc.
Antigen is now known to be thyroid peroxidase.
There is no diagnostic value in simultaneous testing of thyroglobulin and thyroid microsomal/TPO antibodies.
Thyroglobulin antibodies
Normal adult range: titre <1/400 by particle agglutination.
Occurrence is similar to that of thyroid microsomal (peroxidase) antibodies but thyroglobulin antibodies are less sensitive and specific.
Little additional information over and above testing for thyroid microsomal/TPO antibodies.
Main use is to detect interfering assays in thyroglobulin assays (used to monitor thyroid cancer).
EQA scheme in UK has been wound up, but an international standard exists.
Thyroid-binding (stimulating/blocking) antibodies
Three classes of functional antibodies have also been described:
thyroid-stimulating antibodies that increase cAMP levels in thyrocytes
thyroid growth-promoting antibodies that increase tritiated thymidine uptake into DNA by isolated thyrocytes
thyroid-blocking antibodies that block the binding of TSH to its receptor on thyrocytes.
Several sites of antibody binding to the TSH-R have been demonstrated.
All three types of antibody have been strongly associated with Graves’ disease and rarely occur in other types of thyroid disease.
They may be of clinical significance through correlation with response to therapy and outcome.
Their detection involves bioassay with in vitro culture of thyrocytes, or competitive RIA.
Commercial RIA for thyrotropin receptor antibody (TRAB) does not distinguish the different types of antibodies.
An international standard exists.
Thyroid orbital antibodies
These are found in Graves’ disease.
They bind to the retro-orbital fat or fibroblasts, causing hypertrophy and resulting in exophthalmos.
The antigen(s) is unknown.
Antibodies may persist even when the thyroid disease is treated (exophthalmos may progress).
There is no routine screen for these antibodies.
Tissue transglutaminase (tTG) antibodies
tTG is the major autoantigen responsible for anti-endomysial antibody staining pattern by IIF on monkey oesophagus.
EIA assays for tTG have high sensitivity and specificity for coeliac disease.
Human recombinant antigen-based assays have superior performance to guinea-pig-derived tTG assays.
EIA assays have the advantage of automation and rapid throughput.
The test is too sensitive for routine screening in primary care, but is excellent for monitoring compliance with a gluten-free diet.
EIA assays will give quantitative results in arbitrary values.
See ‘Endomysial antibodies (EMA)’, p.492.
Tr antibodies
Anti-Tr antibodies are found in paraneoplastic cerebellar ataxia associated with Hodgkin’s lymphoma.
Antibodies can be found in both serum and CSF.
Staining pattern is similar to anti-Yo antibodies on Purkinje cell cytoplasm.
Tubulin and ubiquitin antibodies
Tubulin antibodies
IgM antibodies to tubulin are associated with:
EBV infection
visceral leishmaniasis
liver disease (PBC, AIH, HBV infection).
Identified on HEp-2 cells.
Ubiquitin antibodies
These antibodies occur in up to 80% of cases of SLE and are said to be specific.
At present, they are not measured routinely.
Vimentin antibodies
Non-specific pattern, associated with:
PBC, AIH
infection
SLE
Behçet’s syndrome
angioimmunoblastic lymphadenopathy
Crohn’s disease.
Detected by screening on HEp-2 cells.
Voltage-gated calcium-channel (VGCC) antibodies
IgG antibodies against the presynaptic calcium channel (P/Q-type) on the nerve terminal cause the Lambert–Eaton myasthenic syndrome (LEMS).
The number of channels is reduced because of cross-linking and internalization, and this impairs release of acetylcholine. Complement is also involved.
LEMS is usually associated with SCLC.
90% of SCLC-associated LEMS will be positive for antibodies to VGCC.
40% of patients with LEMS have no detectable tumour at presentation, but it may become obvious subsequently.
LEMS may also occur in patients who do not have SCLC.
Several types of calcium channel exist, and the antibody is specific for the neuronal (N) type.
Similar antibodies have also been documented in amyotrophic lateral sclerosis, although the significance is uncertain (anti-ganglioside antibodies may also occur in these patients).
Antibodies are detected by RIA using precipitation of VGCC with labelled ω-conotoxin.
Voltage-gated potassium-channel (VGKC) antibodies
Antibody has been associated with acquired neuromyotonia.
Detected by RIA using precipitation of I125-α-dendrotoxin labelled VGKCs.
Reported in pM-bound toxin:
>200, pM positive
<100, pM negative.
Potassium channels are located on the nerve terminal and control nerve excitability.
Yo antibodies
Yo antibodies have been associated with paraneoplastic cerebellar degeneration, seen typically with ovarian cancer and, less commonly, with breast cancer or Hodgkin’s lymphoma.
Development of cerebellar syndrome may precede the diagnosis of ovarian tumour.
Do not occur in the cerebellar syndrome seen in SCLC.
Antigen is found in the cytoplasm of Purkinje cells, and gives a coarse granular staining by immunofluorescence.
The putative target antigen (CDR34) is expressed on epithelial tumours as well as neuronal tissue (cross-reactive anti-tumour antibody).
Antibodies are detectable in the CSF as well as serum and disappear with successful treatment of the primary tumour.
Zic4 antibodies
IgG antibodies to Zic4 are found in association with SCLC in both cerebrospilnal fluid and serum.
Binds to neuronal nuclei on immunofluorescence.
May occur with anti-Hu, which makes detection difficult.
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