Laboratory testing in autoimmune rheumatic diseases

1

Laboratory testing in autoimmune

rheumatic diseases

Joanna Sheldon* FIMLS, PhD, MRCPath

Director of the Protein reference unit, consultant clinical scientist and Honorary senior lectures

Protein Reference Unit, St George’s Hospital Medical School, Cranmer Terrace, London SW15 0RE, UK

There are a number of pathological conditions in which tissue damage occurs in association withimmune activation directed against components of normal tissue. The initial damaging eventsusually involve cells of the immune system, the T-cells, but the cell damage releases antigens thatbecome targets for an antibody response. The detection and quantification of autoantibodies hasbecome an important component in the diagnosis and management of autoimmune rheumaticdiseases such as rheumatoid arthritis, systemic lupus erythematosus, the systemic vasculitidesand systemic sclerosis.

Each of these diseases is associated with a particular autoantibody or group of autoantibodies.They are usually detected by their reaction against tissue components using subjective methodssuch as indirect immunofluorescence. Any positive samples are further analysed using morespecific and quantitative methods for the ‘quantification’ of the specific autoantibodyconcentration.

It is important that these autoantibodies are not considered to be ‘gold standard’ tests: they areno more than markers of the disease with significant limitations. They are best used as part of adiagnostic panel rather than as a marker indicating one particular disease. Techniques are graduallyimproving, giving numerical results rather than titres, but a lack of standardization makes theseresults extremely variable. Many of the markers show no correlation with disease activity. Their useshould be restricted to the initial investigation and not repeated every time the patient is followedup. Other markers do, however, correlate with disease activity and can be used to monitor disease.

When investigating patients who have symptoms associated with autoimmune rheumaticdiseases, analytes such as immunoglobulins, complement components and C-reactive protein mayall be measured.

Key words: autoantibodies; autoimmune diseases; detection; quantification.

The immune system has evolved to recognize and destroy invading pathogens withminimal damage to the host. We do, in fact, make immune responses to host antigensbut usually eliminate these self-reactive cells, giving us tolerance to the large variety ofhost antigens.

1521-6942/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.

Best Practice & Research Clinical RheumatologyVol. 18, No. 3, pp. 249–269, 2004

doi:10.1016/j.berh.2004.03.007available online at http://www.sciencedirect.com

* Tel.: þ44-208-725-5752; Fax: þ44-208-725-0025.E-mail address: [email protected] (J. Sheldon).

http://www.sciencedirect.com

The components of the immune system include cells (lympocytes, monocytes,macrophages, neutrophils, dendritic cells), soluble components (antibodies, comp-lement, cytokines, acute-phase proteins) and of course the lymphoid tissue, wheremany of these components are made and where the initial stages of an immuneresponse occur. Briefly, an antigen, usually encountered across mucosal surfaces, ispicked up by antigen-presenting cells (APCs). These APCs degrade the antigen intoshort sequences and insert them into the human lymphocyte antigens on their surface.The APCs travel around the body showing the antigens to the T- and B-lymphocytes inthe lymphoid tissue. Activation of the T- or B-cell occurs if their receptors can ligatewith the antigen. The production of cytokines is an integral part of immunologicalactivation and function. T-helper cells can be generated that will enhance B-cell functionand the production of specific antibodies. The binding of antibody to antigen willactivate the complement system, which will release inflammatory mediators to recruitneutrophils and cause an acute-phase response. Cytotoxic T-cells, generatedparticularly in response to viral antigens, can directly destroy targeted cells.

There are many suggestions for how this response, normally to a foreign antigen,results in a response to host tissue and possibly an autoimmune disease. Thisbreakdown of tolerance can be due to the following:

† Molecular mimicry: pathogen-derived antigens, when presented to the immunesystem, initiate an immune response that is cross-reactive with host tissue.

† Release of hidden antigens: tissue injury may release antigens that have previouslybeen ‘hidden’ from the immune system, and an autoimmune response occurs.

† T-cell bypass: autoreactive T-cells are usually deleted, but if an antigen becomesaltered, for example by infection, drugs or ultraviolet light, new autoreactive T-cellclones may be activated.

† Cytokine imbalances and defective immune regulation: cytokines are the mediators ofimmune responses. There is a complex interaction between various groups of cytokines thatgive an individual and immunological reactivity. Inappropriate cytokine production isassociated with a variety of immunological abnormalities.

Tissue damage may be caused by cellular responses, by antibody activity or by both.T-helper cells and cytotoxic T-cells are usually found in inflammatory tissue lesions andcontribute to the tissue destruction and perpetuation of the immunological andinflammatory reactions. Antibodies can react directly with tissue-specific cell surfaceantigens or with circulating antigens that deposit in tissue with the antibodies asimmune complexes. These immune complexes activate the complement cascade,causing tissue damage, the recruitment and activation of neutrophils and inflammation.

Self-reactive T-cells are the likely instigators of most autoimmune reactions, butdetecting these cells is not a practical possibility. Autoantibodies, however, are much morereadily detected in easily collected blood samples and are therefore likely to remain animportant part of the diagnosis and monitoring of autoimmune diseases for years to come.

LIMITATIONS TO THE USE OF AUTOANTIBODY TESTING

Autoantibodies should be considered to be no more than markers of disease. They arecommonly found in normal individuals in the absence of any definable disease and withincreasing prevalence in an ageing population. This lack of specificity makesautoantibody testing at best only a part of a diagnostic panel.

250 J. Sheldon

A multitude of kits is now available for the detection and quantification ofautoantibodies. Unfortunately, there are few reliable national or internationalstandards, and there is a huge variation between reagent producers in the preparationand source of antigens and the methods. Results are often reported in arbitrary units,and every method will have different cut-off values, reference ranges and measuringrange. Overall, this makes comparing methods, interpretating published data andcarrying out multicentre studies difficult. Laboratories offering autoimmune serologytests should be accredited (in the UK, with clinical pathology accreditation (CPA) andparticipate, with satisfactory performance, in all relevant external quality assuranceschemes.

RHEUMATOID FACTOR

Rheumatoid factors (RFs) were initially described in 19401; they are antibodies withactivity directed against the Fc portion of immunoglobulin (Ig) G.2 The most commonlymeasured and clinically useful class of RF is IgM. It can be detected by sensitiveimmunoassay in approximately 90% of patients with rheumatoid arthritis (RA). IgG, IgAand IgE RFs are also reported and found by similar sensitive methods in approximately65% of patients with RA. IgA RF is reported to be associated with bone erosions andIgG RF with localized or monoarticular disease.3

Measurement of rheumatoid factor

RF can be measured by agglutination methods, but quantitative, analyser-basedimmuno-nephelometric or immuno-turbidimetric assays are increasingly being used.

Practice points

† none of the tests is perfect—most show a lack of clinical specificity andsensitivity

† get to know your laboratory† do not assume that results will be interchangeable between laboratories† if the results are completely inconsistent with your clinical findings, ask

(politely) for the test to be repeated, possibly using another method† if there is still doubt about a result, recheck it on a fresh sample† make sure that you are asking a specific question when you request laboratory

tests…and that the tests you request are capable of answering the question

Research agenda

† to establish and introduce international reference preparations that alldiagnostic companies will use

† to re-establish the clinical use of assays based on current clinical diseaseclassifications

† to evaluate current markers with respect to new therapies† to identify and validate new or novel markers to diagnose and monitor disease

Laboratory testing in autoimmune rheumatic diseases 251

Most methods available measure predominantly IgM RF, although many will also detectRFs of the other Ig classes. There is a reference preparation (WHO 1066), and theconcentration should be reported in IU/ml. Most methods are said to be calibratedagainst this standard, but external quality assurance schemes still show markedmethodological variations.

Clinical significance of rheumatoid factor

Positive or negative RF results neither confirm nor exclude RA. Table 1 shows thefrequency of IgM RF in autoimmune rheumatic diseases and in healthy individuals.4

Positive RF is also found in a number of other conditions, including infections such astuberculosis and osteomyelitis, associated with monoclonal proteins in B-cellmalignancy and with increasing frequency in an ageing population.

Rheumatoid arthritis

A raised concentration of IgM RF forms part of the classification of RA. There is acorrelation between higher RF concentrations and more severe disease and poorerlong-term prognosis. Concentrations of IgA and IgG RFs are reported to correlate withclinical parameters in RA patients.5 Reliable methods for detection and quantification ofclass-specific RFs are not routinely available.

There is a correlation between higher RF concentrations and systemic symptoms,vasculitis and more severe forms of the disease6, but the use of RF in monitoring diseaseactivity or response to treatment remains controversial. It is unlikely that RFconcentrations alone would be used for patient monitoring: C-reactive protein (CRP),erythrocyte sedimentation rate and clinical parameters are more useful.

Juvenile idiopathic arthritis

Pauci-articular-onset juvenile idiopathic arthritis is seen in approximately 50% ofpatients, who are generally RF negative but often anti-nuclear antibody (ANA)positive.7 Approximately 25% of children with polyarticular disease do showpositive RF.

Table 1. Table of the frequency of IgM rheumatoid factors (RF) in autoimmune rheumatic diseases and

healthy individuals.

Disease % RF

Rheumatoid arthritis 50–90

Systemic lupus erythematosus 15–35

Sjogren’s syndrome 20–30

Cryoglobulinaemia 40–100

Systemic sclerosis 20–30

Polymyositis/dermatomyositis 5–10

Healthy individuals 2–10

252 J. Sheldon

ANTI-CYCLIC CITRULLINATED PEPTIDE ANTIBODIES

The measurement of antibodies to cyclic citrullinated peptide (CCP) is gradually beingincorporated into the repertoire of many immunology laboratories. These antibodiesare directed against citrulline residues formed in post-translational modifications ofarginine8 and are reported to be highly specific (98%) and moderately sensitive (68%)for RA.9 The concentration of these antibodies may be of value, either alone or with RFmeasurements as markers of prognosis or of disease severity.9–11 These antibodieshave been incorporated into a newly proposed diagnostic criteria for RA.12

ANTI-NUCLEAR ANTIBODIES

The term ‘anti-nuclear antibodies’ refers to a diverse group of autoantibodies directedagainst antigens in the nucleus and cytoplasm. The antigens are common to all nucleatedcells and have functions in transcription or translation, in the cell cycle or as structuralproteins. A sketch of a cell showing the location of the important nuclear antigens isshown in Figure 1. The nomenclature of ANAs is complex. The antigens are named by:

† chemical structure (e.g. double-stranded [ds] DNA);† disease association (e.g. SS-A and SS-B in Sjogren’s syndrome);† the individual in whom they were first described (e.g. Ro, La, Sm);† their cytological location (e.g. nucleolar, centromere);† the particle where the antigen is found (e.g. U1 sn RNP).

This nomenclature is further complicated by some antigens having more than onename (e.g. Ro and La are also known as SS-A and SS-B, respectively), and many of theantibodies are known by their staining pattern in indirect immunofluoresence (IIF, e.g.homogeneous or speckled staining patterns).

Practice points

† if you are using anti-CCP antibodies, do not keep requesting RFmeasurements

† if you do not have access to anti-CCP antibodies, keep an eye on theliterature and see how their use develops

† there is likely to be a laboratory somewhere that can run an anti-CCPantibody so if you are desperate, ask your laboratory

† in a patient with back pain, do not forget to send serum and urine forparaprotein studies to exclude myeloma

Practice points

† CRP is more reliable than RF for monitoring RA† do not keep on requesting RF† encourage your laboratory to use quantitative RF methods


Detection of anti-nuclear antibodies

Screening tests for ANAs are most commonly carried out by IIF using either frozensections of animal tissue or, more appropriately, cultured cell lines (e.g. human epithelialHEp2 cells). Neither substrate is perfect for ANA screening, some antigens beingpoorly expressed in rodent tissue (e.g. Scl70 and centromeric proteins), and others(e.g. Ro/SS-A) not being conserved in their native form in cell lines or in rodent tissue.Laboratories should therefore be aware of the possibility of false-negative results in theANA screen and be prepared to run more specific follow-up tests if clinically indicated.

There are many methods available for the detection or quantification of specificantibodies to nuclear components; these include enzyme-linked immunosorbent assay(ELISA), Ouchterloney double immunodiffusion, Western blotting and countercurrentimmunoelectrophoresis. The different sensitivities and specificities of these methods islikely to influence the interpretation and clinical utility of many of the tests.13

Commercially produced ELISA-based ‘ANA screens’ are increasingly becomingavailable. The antigens used in these reagents range from crude cell preparations tomixtures of recombinant proteins, with a consequent high variability between differentmanufacturers. At present, there is considerable interest in these techniques but usuallyfor reasons of laboratory organization rather than scientifically driven improvement.

Clinical significance of anti-nuclear antibodies

The detection of a positive staining pattern should lead to a more specific assay for therelevant antigens. The standardization of these assays remains a problem for thefollowing reasons:

† variation in the antigen (used as a ‘substrate’ for the antibodies in the patients’serum) preparation;

† variations in antibody affinity between patients and between standards and patientsamples;

† difficulties and/or lack of standardization;† methodological differences, for example, the detection of IgG antibodies to an

antigen in some methods whereas others detect both IgG and IgM antibodies.

dsDNA (and ssDNA):homogeneously distributedacross the nucleus innon-dividing cells.Homogeneous staining seen with antibodiesto ssDNA and(with chromosomalstaining) withantibodies to dsDNA.

Extractable NuclearAntigens: clusters ofthe many ENAs arrangedin ranging sized 'speckles'across the nucleus

Chromosomes:organised from chromatin for celldivision. Stainingseen with antibodiesto dsDNA

Centromere:40_60 discrete finespeckles seen withcentromere antibodies

Nucleolar : 2-4 prominent nucleoli seen in the p2 cells

Figure 1. Sketch of a cell showing the location of the important nuclear antigens.

254 J. Sheldon

Because of these limitations, specific antibody results must always be interpreted withsome caution, particularly when laboratories have changed methods.

Anti-nuclear antibody patterns

The staining pattern seen on IIF gives some indication of the specificity of the antibodiesin the sample. Detecting subtle staining patterns has become rather an ‘art form’ insome laboratories; in reality, there are only a handful of well-defined staining patternsthat everyone testing for ANAs should be able to recognize. Typical ANA patterns areshown in Figure 2. Other specificities rarely have clinical significance: the antibodies arepresent in low concentrations and can be found non-specifically in many infections andinflammatory conditions. Table 2 shows the major staining patterns seen on IIF on HEp2cells, the related antigen and disease association.

Homogeneous staining patterns

Antibodies to single-stranded DNAAntibodies to DNA can be divided into three major groups:

† antibodies to the conformational epitopes inherent in the double helix structure ofthe native molecule;

† antibodies to the epitopes expressed on the deoxyribose phosphate backbone;† antibodies to epitopes expressed on the purine and pyrimidine bases.

Figure 2. Indirect immunofluorescence staining patterns of anti-nuclear antibodies seen on HEp2 cells. (a)Homogeneous staining pattern with chromosomal staining. (b) Speckled staining pattern. (c) Centromerestaining pattern. (d) Nucleolar staining pattern.


The antibodies to the purine and pyrimidine bases can only be made when theDNA is denatured to single-stranded DNA. These antibodies to single-strandedDNA are the most frequently encountered antibodies in systemic lupuserythematosus (SLE) but are also seen in most types of autoimmune rheumaticdisease, in patients receiving cytotoxic therapy and in viral infections. Their lack ofspecificity makes them of minimal value in the investigation of patients withautoimmune rheumatic diseases.

Antibodies to double-stranded DNAAntibodies to native or dsDNA are strongly associated with SLE, raisedconcentrations of anti-dsDNA being reported in approximately 60% of patientswith SLE.14 The Farr assay, based on radioimmunoassay, detects high-avidityantibodies, and has the highest specificity for SLE and the best correlation withdisease activity. Most laboratories have moved away from the Farr assay owing tothe safety concerns associated with the use of radiolabelled reagents. ELISA assaysare now the most commonly used method for quantifying antibodies to dsDNA,although these assays may detect low-affinity antibodies that are irrelevant todisease. IIF using Crithidia lucilae as the substrate shows high specificity for dsDNAantibodies but relatively low sensitivity; we use this method to confirm the presenceof anti-dsDNA antibodies when the IIF and ELISA results are discrepant. Theconcentration of anti-dsDNA antibodies is useful for both prognosis and monitoringpatients with SLE15 so serial measurements, for example, every 8–12 weeks, arejustified.

Table 2. Major staining patterns seen on indirect immunofluorescence on HEp2 cells, the related antigen

and disease association.

Pattern Antigen Disease association (%)

Homogeneous DNA SLE (60%)

Diffuse Histone Drug-induced lupus erythematosus (95%)

SLE (60%)

Topoisomerase-1 (Scl70)a Progressive systemic sclerosis (15–70%)

Peripheral (rim) dsDNA SLE

Speckled-coarse Sm SLE (20%)

RNP SLE-Sjogren’s overlap (100%)

SLE (25%)

Speckled-fine SS-A/Ro Sjogren’s syndrome (60%)

SLE (35%)

SS-B/La Sjogren’s syndrome (40%)

SLE (15%)

Centromere (46 dots) CENP Limited scleroderma (CREST) (7–21%)

Nucleolar-homogeneous PM-SC 1 Polymyositis (8%)

Myositis–scleroderma overlap (50%)

Nucleolar-speckled Nucleolar RNA Scleroderma (5–43%)

Cytoplasmic Histidyl-t-RNA synthetase (Jo-1) Polymyositis (2%)

SLE, systemic lupus erythematosus; see text for antigens.a May also appear as very finely speckled pattern.

256 J. Sheldon

Speckled staining patterns

Antibodies to extractable nuclear antigensThe extractable nuclear antigens (ENAs) are a group of antigens that leach from the cellwhen extracted with saline. There are many antigens within the nucleus, but only asmall number have any clinical utility. The original disease associations of the ENAswere defined with the relatively insensitive gel-phase assays, double immunodiffusionand countercurrent immunoelectrophoresis. Nowadays, more sensitive ELISA-basedmethods are more commonly used, with a consequent decrease in diseased specificity.Furthermore, these assays detect low-affinity antibodies in addition to the moreclinically significant high-affinity antibodies. In general, there is little association betweenthe concentration of antibody to the ENAs and severity of disease, and little fluctuationin antibody concentration with time or disease severity.

Anti SS-A (Ro) and Anti SS-B (La)The SS-A antigen consists of 52 and 60 kDa proteins (called Ro52 and Ro60,respectively) complexed with Y1–Y5 RNAs. Antibodies (mainly of the IgG isotype) tothe Ro52 antigen almost always appear in association with antibodies to the Ro60antigen, and few techniques can reliably distinguish between antibodies to the twodifferent antigens. Antibodies to Ro52 are more often seen in patients with primarySjogren’s syndrome, whereas antibodies to Ro60 are found more often in patientswith SLE.

Testing by countercurrent immunoelectrophoresis shows antibodies to SS-A(usually in association with antibodies to SS-B) in 60% of patients with primarySjogren’s syndrome and rarely in healthy individuals. There have been markedchanges in the methodologies used for detecting these antibodies, and the moresensitive ELISA methods may increase the ‘pick-up’ rate but with a consequentincrease in the number of false-positive results (reducing specificity). Anti-SS-Aantibodies are also found in a number of other autoimmune diseases, including RA,SLE and polymyositis.16

Anti-SS-A antibodies (Ro52 and Ro60) are seen in 35% of patients with SLE and canbe associated with neonatal lupus and with complete or partial fetal heart block. IgGcrosses the placenta in the last trimester of pregnancy; if anti-SS-A antibodies arepresent in the maternal serum, these antibodies will also cross the placenta and mayinteract with the conducting fibres of the heart and induce fetal heart block in utero.17

The development of heart block and neonatal lupus are, however, relatively uncommonevents. Of mothers who are known to be SS-A antibody positive, only 1 in 20 will givebirth to a baby with heart block, and 1 in 15 will have a baby who develops neonatallupus.

The SS-B protein is thought to participate in the termination of transcription ofRNA polymerase III.18 Antibodies to SS-B are seen in approximately 40% of patientssjogren’s syndrome with when tested by countercurrent immunoelectrophoresis. Aswith testing for anti-SS-A antibodies, new techniques have resulted in an increaseddetection rate but reduced disease specificity. Antibodies to SS-B are also seen inSLE, RA and polymyositis.19

The role of the anti-SS-A and anti-SS-B antibodies in disease pathogenesis remainscontroversial. Anti-SS-A antibodies are reported to show a close association withthe development of vasculitis, nephritis, lymphadenopathy and leucopenia.20 Anti-SS-B antibodies are reported to increase during disease flares in Sjogren’s syndrome.21

Anti-SS-A antibodies are often seen in patients with systemic sclerosis and occur


alongside the disease-specific antibodies. The presence of anti-SS-A antibodies(especially with anti-SS-B antibodies) may mark a subset of 5–10% of cases ofsystemic sclerosis with limited disease and a high prevalence of renal and lungdisease involvement.22

The detection of anti-SS-A and anti-SS-B antibodies is an important component ofthe investigation of patients with suspected Sjogren’s syndrome and SLE. Specific testsshould be used as a confirmatory investigation in samples showing a speckled ANApattern on HEp2 cells. There is, however, limited value in using these tests formonitoring disease progression.

Laboratories are occasionally criticized for not running the specific tests forantibodies to the ENAs; in my laboratory, we run anti-ENAs no more than once peryear on known patients unless there are good clinical indications. We will run thespecific tests, even on ANA-negative (on HEp2 immunoflourescence) samples, if thereis strong clinical suspicion.

Anti-SmThe Sm antigen is a complex group of four proteins (29 kDa protein B, 28 kDa proteinB, 16 kDa protein D, 13 kDa protein E) complexed with U1, U2, U4–U6 and U5snRNAs.23 Over 80% of serum samples showing antibodies to Sm will also be positivefor antibodies to ribonuclear protein (RNP), with which they share antigenicdeterminants. Antibodies to the U1–D1–3 snRNP complex, which forms part ofthe Sm complex, are highly specific for SLE although not very sensitive and areassociated with renal involvement and a poor prognosis.24

Anti-ribonuclear proteinThe RNP antigen is highly pleiomorphic, with three proteins (70, 33, and 22 kDa)complexed with U1 snRNA.23 The nomenclature of this complex is variable, U1-RNP,nRNP, snRNP, U-snRNP and U1-snRNP all being names given to the same antigen. Theantibodies show high specificity for mixed connective tissue disease, in which theyoccur as the sole antibody.25 They may also be seen without antibodies to the Smantigen in patients with SLE, a group of patients showing a lower frequency ofantibodies to dsDNA and clinically apparent renal disease.26

Antibodies associated with other staining patterns

Anti-Scl-70Antibodies to Scl-70 are directed againsts DNA topoisomerase 127, and are seen in 20–25% of patients with systemic sclerosis and rarely in other autoimmune diseases.Patients with anti-topoisomerase antibodies tend to be slightly younger than patientswith anti-centromere antibodies, and they are particularly affected by lung fibrosis.22

The anti-topoisomerase antibodies can give a variable staining pattern depending uponthe substrate; nucleolar staining with fine speckling is often seen. There are ELISAmethods available for quantifying anti-Scl-70 antibodies

Nucleolar antibodiesA nucleolar staining pattern (2–4 prominent nucleoli usually seen in HEp2 cells) is seenwith antibodies to a number of antigens. Fine speckled staining with nucleolar staining isseen with antibodies to RNA polymerase III and I. These are associated with diffusecutaneous systemic sclerosis and with a high incidence of lung fibrosis and renaldisease.28 Even, diffuse staining of the nucleoli is associated with antibodies to PM-Scl

258 J. Sheldon

and is seen with polymyositis–scleroderma overlap. This antibody is associated withmyositis and pulmonary fibrosis and a generally good prognosis.29 A clumpy nucleolarstaining pattern is seen with antibodies to the fibrillarin subunit of U3 snRNP.30 Theseare associated with a poor prognosis, an increased risk of heart and kidney involvementand life-threatening pulmonary hypertension.

Centromere antibodiesAntibodies to the centromeric proteins were described in 1980 and are directedagainst epitopes found in the kinetochore domain of the chromosome. There are threemain centromeric antigens—CENP-A, -B and -C—and the minor centromeric proteinsCENP-D, -E and -F. Antibodies to the three main centromeric proteins often occur inthe same sera, but antibodies to CENP-B are found in almost all patients with anti-centromere antibodies. Anti-centromere antibodies have a characteristic stainingpattern on IIF using HEp2 cells as a substate, and laboratories should report this patternif it is seen. A highly sensitive and specific ELISA assay for CENP-B is also available31,although the quantification of the antibody has little clinical application.

Anti-centromere antibodies are seen in systemic sclerosis in its limited cutaneousform and in the CREST variant. The antibody can also be found (at a low frequency) inother connective tissue diseases, for example RA, SLE and primary Sjogren’s syndrome.In patients with Raynaud’s phenomenon, the presence of anti-centromere antibodiessuggests an increased risk of developing rheumatic disease. Anti-centromere antibodiesare also found in a sub-group (approximately 20%) of patients with primary biliarycirrhosis, in whom the liver disease may predate the manifestations of systemicsclerosis.32

Anti-Jo-1 and antibodies to aminoacyl-tRNA synthetasesAnti-Jo-1 antibodies are part of a group of antibodies to a family of aminoacyl-tRNAsynthetases33, enzymes that complex amino acids with the cognate tRNA. The enzymeswithin this family that are associated with antibodies are shown in Table 3.34 Antibodiesto these enzymes, associated with polymyositis and dermatomyositis, are typicallydetected by IIF on HEp2 cells when a fine granular cytoplasmic staining pattern is seen.There are, however, a significant number of samples with antibodies to Jo-1 that do notshow any staining in HEp2 cells; therefore if there are clinical indications, the antibodyshould be detected and quantified by specific (ELISA-based) technology. These areclearly not ANAs, but the fact that they are seen in the screening test for ANAs oftenmeans that they are considered to be part of the ‘ANA group’.

Table 3. The family of aminoacyl-tRNA synthetase.

Name

Molecular

weight of

antigen (kDa)

Incidence

(%) Enzyme Disease association

Jo-1 50 18 Histidyl tRNA synthetase Interstitial lung disease, arthritis

PL-7 80 5 Threonyl tRNA synthetase Raynaud’s phenomenon

PL-12 110 3 Alanyl tRNA synthetase

EJ 75 1 Glycyl tRNA synthetase

OJ 145 1 Isoleucyl tRNA synthetase


Jo-1 antibodies are found in 20–40% of patients with aggressive polymyositis, usuallyin association with interstitial lung disease and arthralgia. The concentration of anti-Jo-1antibodies is reported to correlate with disease activity so sequential measurementsmay be of value in monitoring patients.35

A number of antibodies to other tRNA synthetases also show an association withmyositis. These antibodies are found with the highest frequency early in the disease butwill persist almost regardless of disease activity or treatment, so their measurementremains in the realms of specialized laboratories.

ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODIES

The function of neutrophils is to ingest and destroy antigens; the neutrophils aretherefore full of potent, damaging enzymes such as proteinase III (PR3), myeloperox-idase and elastase. Antibodies against these neutrophil enzymes have been described inassociation with primary systemic vasculitides. These heterogeneous disorders arecharacterized by widespread inflammation of the vessel walls, and their classification isbased on clinical and histological findings (Table 4).36 The small-vessel vasculitides ofWegener’s granulamotosis, Churg-Strauss syndrome and microscopic polyangiitis are

Practice points

† ask your laboratory whether you can have a look at how they carry out thesetests—you will be amazed at how much care they take and how hard some ofthe patterns can be to read

† a few laboratories have enormous experience of ANA testing and a variety oftechniques at their disposal for identifying odd patterns; good laboratoriesoften send samples on for confirmation or a second opinion

† laboratories should be able to run an ANA test within 1 working day if there isgood clinical justification; ask if necessary

† ask for an ANA rather than asking specifically for antibodies to dsDNA orENAs; this way, you will lessen the risk of missing something important

† use the concentration of anti-dsDNA antibodies to monitor but do not keeprequesting antibodies to ENAs

Table 4. The primary vasculitides.

Large-vessel vasculitis Giant cell (temporal) arteritis

Takayasu’s arteritis

Medium-sized vessel vasculitis Polyarteritis nodosa

Kawasaki disease

Small-vessel disease Wegener’s granulamotosisa

Churg-Strauss syndromea

Microscopic polyangiitisa

Henoch-Schonlein purpura

Essential cryoglobulinaemic vasculitis

Cutaneous leukocytoclastic angiitis

a Associated with anti-neutrophil cytoplasmic antibodies.

260 J. Sheldon

strongly associated with the presence of anti-neutrophil cytoplasmic antibodies(ANCA), in particular antibodies to PR3 and to myeloperoxidase.

Detection and quantification of anti-neutrophil cytoplasmic antibodies

In 1988, the First International Workshop on ANCA agreed to harmonize methods forthe detection of these antibodies. It was agreed that the basis of detection would be IIFusing washed human buffy coat leukocytes smeared or cytospun onto slides and thenfixed with ethanol or acetone and fluorescein isothiocyanate-conjugated anti-humanIgG as a detection antibody.37 This protocol was reiterated by the InternationalConsensus Statement38, which states that all new patients should be tested for IgGantibodies by IIF on ethanol-fixed human neutrophils and only positive results should beinvestigated with antigen-specific assays. Automated, ELISA-based screening assays forANCA are increasingly becoming available. Many laboratories are adopting thesetechniques in an attempt to ‘streamline’ their services. In the absence of internationalstandardization, however, results can be reported only in arbitrary units, the sensitivityof many of the techniques is questionable, and the diagnostic efficiency of these assays isunknown. Screening samples for ANCA using ELISA-based methods is notrecommended.

The IIF of ethanol-fixed neutrophils shows two major staining patterns: acytoplasmic granular pattern and a perinuclear pattern. These are now conventionallyknown as the c-ANCA pattern and the p-ANCA pattern, respectively. Typical c- and p-staining patterns are shown in Figure 3.

The c-ANCA pattern shows granular cytoplasmic fluorescence with centralinterlobular accentuation in the ethanol-fixed human neutrophils. This pattern isseen in association with Wegener’s granulomatosis.39,40 This pattern is usually causedby antibodies to the 29 kDa serine protease 3 (PR3) but can also be seen in associationwith other neutrophil cytoplasmic enzymes.

The p-ANCA pattern shows staining localized just around the nucleus. The ethanolfixation causes by redistribution of the positively charged neutrophil granule proteinstowards the negatively charged DNA in the nucleus. The p-ANCA staining pattern isassociated with antibodies to myeloperoxidase but is also seen with antibodies to otherneutrophil enzymes and with ANAs. Samples that show p-ANCA staining without

Figure 3. Indirect immunofluorescence staining patterns of anti-neutrophil cytoplasmic antibodies (ANCA)seen on ethanol-fixed neutrophils. (a) Cytoplasmic ANCA. (b) Perinuclear ANCA.


specificity for myeloperoxidase is associated with diseases such as ulcerative colitis,sclerosing cholangitis, autoimmune hepatitis and Felty’s syndrome.36

Other staining patterns can be seen and should be termed atypical ANCA. Thesepatterns are most likely caused by antibody binding to multiple antigenic targets in theneutrophils.38 In my laboratory, we always check samples showing atypical ANCAstaining with the specific assays for antibodies to PR3 and myeloperoxidase.

Clinical significance of anti-proteinase III antibodies

Antibodies to PR3 are predominantly seen in patients with Wegener’s granulomatosis(Table 5), although they are often seen in other vasculitic diseases.41 A c-ANCA stainingpattern combined with positive PR3 ANCA on enzyme immunoassay is 99% specific forsmall-vessel vasculitis.42 Patients with PR3 ANCA relapse more frequently thanpatients with myeloperoxidase ANCA-associated vasculitis43–45 and show morewidespread organ involvement, granuloma formation and active renal lesions. Studiessuggest that these antibodies are involved in the pathogenesis of the vasculitis46,47,although there are patients with active disease who are ANCA negative and somepatients in remission with high ANCA titres. Nevertheless, a relationship betweenANCA titre and disease activity has been reported48, and the regular measurement ofthe titre or concentration of ANCA antibodies is an important component ofmonitoring of the disease. Rising titres of ANCA or rising concentrations of PR3antibodies are frequently followed by relapses of Wegener’s granulomatosis49,50, andthe persistence of ANCA after induction of remission is reported to be a risk factor forrelapse.51

Clinical significance of anti-myeloperoxidase antibodies

Antibodies to myeloperoxidase are predominantly seen in patients with microscopicpolyangiitis, idiopathic crescentic glomerulonephritis and Churg-Strauss syndrome(Table 5). The clinical presentations of anti-myeloperoxidase-associated ANCAvasculitis are more diverse than those seen with anti-PR3-associated ANCA vasculitis.A p-ANCA staining pattern combined with positive myeloperoxidase ANCA onenzyme immunoassay is 99% specific for small-vessel vasculitis.52 There is evidence thatthe serial measurement of anti-myeloperoxidase antibodies has a role in monitoringdisease53, although this approach does not have such a well-established role as themeasurement of anti-PR3 antibodies.

Table 5. Disease associations of anti-proteinase III and myeloperoxidase antibodies.

Sensitivity of

Disease entity Anti-proteinase III (%) Anti-myeloperoxidase (%)

Wegener’s granulomatosis 85 10

Microscopic polyangiitis 45 45

Idiopathic cresentic glomerulonephritis 25 65

Churg-Strauss syndrome 10 60

Polyarteritis nodosa 5 15

262 J. Sheldon

ANTI-CARDIOLIPIN ANTIBODIES AND ANTIBODIES TO b2GLYCOPROTEIN-1

The term ‘anti-phospholipid antibodies’ is used to encompass a variety of differentphospholipid antigens, including the negatively charged cardiolipin and phosphatidyl-serine and the neutrally charged phosphatidylcholine and phosphatidylethanolamine.Beta-2 glycoprotein-1 (b2GP1) is the major phospholipid-binding protein, and ‘anti-cardiolipin antibodies’ may bind to epitopes on this protein rather than to cardiolipinepitopes.54

The detection and quantification of antibodies to cardiolipin and to b2GP1 are animportant component of the diagnosis of anti-phospholipid syndrome or Hughes’syndrome.55 The investigation of patients with recurrent venous or arterial thrombosis,recurrent fetal loss and thrombocytopenia should include a full blood count, clottingscreen and lupus anticoagulant, assays generally carried out in haematology departments.

Measurement of antibodies to cardiolipin and to b2 glycoprotein-1

Anti-cardiolipin antibodies are typically measured by ELISA-based techniques, but themethods are prone to significant problems and variability. This is because cardiolipinantibodies represent a small subset of low-affinity, highly heterogeneous antibodies whosein vitro binding may be influenced by many factors, for example, the nature of the antibody,antibody isotype, avidity and cross-reactivity. The nature of the reference preparation,antigen preparation and assay system further compound the variability.56 Methods for thequantification of IgG anticardiolipin antibodies are more robust than those for IgManticardiolipin antibodies, and some laboratories no longer offer IgM class antibodies.

Clinical significance of antibodies to cardiolipin and to b2 glycoprotein-1

Anti-phospholipid antibodies can be seen in infections and in chronic diseases; it is wellknown that these antibodies give false-positive results in the Wasserman reactionserological test for syphillis. False-positive results can also be seen with hepatitis C,leprosy, lyme disease, mycoplasma, tuberculosis, HIV, Legionnaire’s disease, Q fever andvaricella zoster infections. The anti-b2GP1 antibodies are usually negative in these non-specific reactions.

In 1999, preliminary classification criteria for the diagnosis of anti-phospholipidsyndrome were proposed.57 The laboratory criteria are of:

Practice points

† laboratories should be able to run an ANCA test within 1 working day if thereis good clinical justification; ask if necessary

† if you have a patient with deteriorating renal function, do not forget to ask forantibodies to glomerular basement membrane and ANAs, and send serum andurine samples for paraprotein studies

† specific anti-PR3I or anti-myeloperoxidase antibody concentrations may beuseful in monitoring

† check the CRP concentration


† anti-cardiolipin antibodies of the IgG and/or IgM isotype in the blood, present at amedium or high titre on two or more occasions at least 6 weeks apart, measured bya standardized ELISA assay for b2GP1-dependent cardiolipin antibodies

† lupus anticoagulant present in the plasma on two or more occasions at least 6 weeksapart, detected according to the guidelines of the International Society onThrombosis and Haemostasis.

These laboratory criteria emphasize the difference between lupus anti-coagulant andanti-cardiolipin antibodies, and the fact that they are neither synonomous nornecessarily co-existent. The diagnosis of anti-phospholipid syndrome can be madewhen at least one clinical and one laboratory criterion are met.

There is some correlation between anti-cardiolipin antibody concentration anddisease severity, so in addition to monitoring patients’ clotting profiles, a measurementof anti-cardiolipin antibodies may be useful.

IMMUNOGLOBULINS

The clinical indications for the measurement of serum IgG, IgA and IgM concentrationsare very limited. It is essential that they are measured if primary or secondary Igdeficiency or B-cell malignancy is being considered. Serum Ig quantifications shouldalways be interpreted and reported with reference to the serum proteinelectrophoresis. To exclude B-cell malignancy, the urine must always be checked forBence Jones protein. Ig concentrations may also be useful in the investigation of sometropical diseases and occasionally some liver diseases.

The concentrations of IgG, IgA and IgM show a markedly raised concentration inmany autoimmune diseases. The magnitude of the increase in concentration is notdirectly related to the severity of disease, and there is little relationship betweenconcentration and disease progression; repeated measurements are therefore a wasteof time and money. A sensible compromise is to exclude antibody deficiency at theinitial investigation and check the Ig concentrations no more than once per year andpreferably less frequently.

The antibodies will be directed against both organ- and non-organ-specific targets,and well as there being high amounts of circulating immune complexes andcryoglobulins.58

Practice points

† there is an enormous variability in the quantification of these antibodies† it is important to check the antibody concentrations on at least two occasions

to make a secure diagnosis

Practice points

† check the Ig concentrations in patients in whom immune deficiency or B-cellmalignancy needs to be considered

† do not keep repeating Ig measurement in patients with RA, SLE or systemicsclerosis; they may well be abnormal but add little to your diagnosis ormonitoring

264 J. Sheldon

CRYOGLOBULINS

Cryoglobulins are antibodies that precipitate in the cold and re-solubilize on warming;they are classified into three groups, shown in Table 6.

In general, for symptoms to be brought on by an exposure to cold, cryoproteins shouldexhibit their activity above 21 8C—the lowest physiological skin temperature. There aremany proteins that aggregate below 16 8C that never produce symptoms but do producelaboratory artefacts. The important exceptions are the mixed cryoglobulins, which aredetected after slow precipitation at 4 8C, precipitation sometimes taking up to 3 days tooccur. There is often little correlation between exposure to the cold and amount ofcryoprecipitate and patients’symptoms, although correlation can occurwithin an individual.

Type I cryoglobulins are monoclonal proteins that typically do not have RF activity andare not effective activators of the complement cascade. This group of proteins is morelikely to cause hyperviscosity and symptoms related to the precipitation of the protein,particularly in the extremities, at low temperatures. The mixed cryoglobulins (types IIand III) activate complement efficiently and are likely to cause vasculitis. The symptomsassociated with these types of protein are related to their immune complex behaviourrather than their precipitation in the cold. Their behaviour as cryoprecipitating proteinsis essentially a laboratory artefact.

Types I and II cryoglobulins are associated with B-cell malignancies such as myeloma,Waldenstrom’s macroglobulinaemia and chronic lymphocytic leukaemia. Type IIcryoglobulins also occurs with some chronic infections (especially hepatitis C) andrheumatic diseases (Sjogren’s syndrome, RA, SLE). Type III cryoglobulins occur withrheumatic diseases or chronic infections but not with B-cell malignancies.

Common symptoms associated with cryoglobulins include purpura, ulceration,Raynaud’s phenomenon, arthralgia, proteinuria, renal failure and vasculitis. It is vital thatsamples are collected, transported and separated at 37 8C to ensure that smallcryoproteins are not ‘lost’ into the cell pellet. Laboratories should have pre-warmedblood collection tubes and a warmed thermos flask; these should be brought to thepatient just before the blood is collected. The blood collected into the warmed tubes andimmediately put into the warm flask for immediate transport back to the laboratory.

Table 6. Classification of cryoglobulins.

Type I II III

Monoclonal Yes Yes No

Rheumatoid factor

activity present

No Yes (IgM) Yes

Clinical syndrome Hyperviscosity Vasculitis Vasculitis

Disease association Malignancy Hepatitis C, Rheumatic disease,

Malignancy, Idiopathic

Hepatitis C, Rheumatic disease,

Idiopathic

Practice points

† do not forget cryoglobulins—many of their symptoms overlap with those ofthe autoimmune rheumatic diseases

† if you are requesting cryoproteins, call the laboratory for proper instructions,tubes, etc.


C-REACTIVE PROTEIN

CRP is an acute-phase protein made in the liver under the control of cytokines such asinterleukins 6 and 1, and tumour necrosis factor-alpha. In acute inlammation, the plasmaCRP concentration increases within hours of the onset of the inflammatory responseand falls quickly on appropriate treatment. The magnitude of inflammation is related tothe magnitude of the CRP concentration.59 A raised CRP concentration is unequivocalevidence of an inflammatory process and may therefore be useful in distinguishingsimple mechanical damage from more serious organic disease.

CRP measurements are useful in the autoimmune rheumatic diseases. Patients withRA usually have a raised CRP concentration that falls with appropriate treatment andcorrelates better with radiological evidence of progressive joint inflammation thanother clinical of serological measurements.60

SLE, scleroderma, Sjogren’s syndrome and polymyositis can all be clinically activewithout a significant CRP response.61 Patients with these diseases occasionally haveraised CRP concentrations, and patients are capable of mounting a CRP response tointercurrent bacterial infections.62 CRP concentrations are raised in vasculitis and canprovide an objective marker of disease activity and response to treatment.63

COMPLEMENT

The complement proteins are a cascade of proteins that can be activated by a variety ofagents including immune or antigen-antibody complexes. Serum concentrations of C3and C4 are most commonly measured and low concentrations of C4 are associatedwith active immune complex disease and, in SLE, particularly with renal involvement.64

Patients with genetic deficiencies of complement (particularly C2 and C4) show anincreased risk of immune complex disease65, and if you are considering complementdeficiency, you should contact your laboratory for sample requirements for the totalhaemolytic complement (often called CH50) assay. Complement activity in vivo can bedetected by the marker C3d. This remains a useful test but lies very much in the realmsof the specialist laboratories.

Practice points

† CRP is a non-specific marker of inflammation† a raised CRP concentration is unequivocal evidence of an inflammatory

response† the measurement of CRP is useful in monitoring RA and in distinguishing

between infection and disease ‘flare’ in SLE, etc.

Practice points

† measure C3 and C4 concentrations in patients with active disease—a falling C4concentration is associated with an increased risk of renal complications

† if you are considering complement deficiency, check the total haemolyticcomplement level

266 J. Sheldon

SUMMARY

A huge range of laboratory tests is available to help in the diagnosis and management ofpatients with autoimmune rheumatic diseases. Not one of the tests is, however,perfect, and most tests have significant limitations. Nevertheless, when used properly,they provide a valuable minimally invasive way of diagnosing and monitoring disease.

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Laboratory testing in autoimmune rheumatic diseases