Autoantibodies



Autoantibodies







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. antithyroglobulin 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.





    • adenovirus




    • 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 (isotypespecific) 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 FITCconjugated 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:



  • 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:





    • 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-bungaroto×in.


  • 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.



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 17A-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)




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





  • 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 (Behcet’s syndrome)


    • connective tissue disease (SLE, Sjogren’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-GP (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




  • 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




    • 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




  • 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 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:





    • 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 dermoepidermal 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 flameshaped 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:







    • lymphoma


    • viral infections.


  • In drug-induced haemolysis, there are often antibodies to drug-cell neoantigens (e.g. quinine, penicillins, and cephalosporins).




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:





    • 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 GD1 b 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

Jul 22, 2016 | Posted by in GENERAL SURGERY | Comments Off on Autoantibodies

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