Figure 2.23 Sagittal T2 fat saturated MRI of the lumbar spine showing endplate destruction and a \202 uid collection in acute infective discitis. Appendicular skeleton In \202 ammatory disease of bone, joints and soft tissues in the per ipheral skeleton is well demonstrated on MRI. Examples include: \200 MRI in in \202 ammatory disease Spine MRI plays an important role in the investigation of in \202 am- matory spondyloarthropathy (Figure 2.22). Whilst plain \201 lms and CT can o nly detect established bony changes, the ability of MRI to detect marrow oedema makes it ve ry Hip pathologies such as avascular necrosis, synovitis and trochanteric bursitis (Figure 2.2 4); Tendinopathy and taenosynovitis, e.g. at the ankle; Subacromial bursitis and rotator cu ff pathology; In \202 ammatory arthropathy with synovitis (Figure 2.25); \200 \200 CHAPTER 2 Investigation of rheumatic disease 35 \200 Entheseal changes, e.g. tennis elbow Muscle abnormalities such as myositis or wasting with fatty in \201 ltration. Figure 2.26 Coronal and sagittal T2 weighted images of a left paracentral disc herniation a t L5/S1 compressing the traversing left S1 root in the lateral recess (arrowheads comparing both). Less severe degenerative changes affect the L4/5 disc. Appendicular skeleton MRI is also very sensitive in the early stages of the degen- erative process in the remainder of the skeleton. Areas commonly imaged include: \200 \200 \200 \200 \200 MR arthrograms are sometimes performed to assess cartilaginous structur es, such as the labrum in the shoulder and hip, and articular cartilage surfaces in other j oints, triangular \201 brocartilagenous complex in the wrist. Hips Knees Ankles Shoulders Wrist Figure 2.24 Coronal T2 fat saturated image of the left hip showing high signal adjacent to the greater trochanter indicating trochanteric bursitis and/or gluteal tendinopathy (arrow) . MRI is also commonly used in trauma, both acute and chronic. A good example is in the inves tigation of osteo- chondral lesions which are most commonly seen in the knee and ankle (Fig ure 2.27). Figure 2.25 Sagittal contrast-enhanced T1 fat saturated MRI of the elbow in rheumatoid arth ritis, showing prominent enhancing synovitis (high signal) with erosions of the capitellum (solid arrow) and cartilage thinning on the radius (broken arrow). Degenerative disease Spine MRI is a fundamental part of the assessment of back pain, the ma jority of which is due to degenerative change affecting the intervertebral disks and/or the facet joints, with or without subsequent neural compression. MRI \201 ndings include: \200 \200 Figure 2.27 An osteochondral lesion is shown in the medial aspect of the talar dome. There is chondromalacia of the overlying articular cartilage. Loss of signal in and height of the intervertebral disks. Herniation of disc material causi ng compression of nerve roots in the lateral recess or exiting nerves in the intervertebral foramen (Figure 2.26). Spinal stenosis, which may cause cord compression, cauda equina syn drome or myelopathy. Spondylolisthesis, which may be due to facet joint degen- eration or p ars interarticularis defects. Vertebral end plate reactive changes. RADIONUCLIDE IMAGING 36 Radionuclide imaging Gamma camera imaging using radionuclides in the investi- gation of musculoskeletal cases is long established. Positron emission tomography (PET) imaging is a recent development whose role is expanding rapidly. All methods involve ionizing radiation with a dose to each pati ent. For example the effective dose from a bone scan equates to 200 chest X-rays. The image s generated are physiological, rather than anatomical as provided by other modalities (i.e. a bone scan is an image of the distribution of osteo- blastic activity, rather than of the bones themselves). Improved specificity has been repor ted using 99mTc-labeled IgG. The radiation dose is high er than plain \201 lms. Limited anatomical detail. Lesions where there is not increased o steoblastic activity will not demonstrate increased activity may be missed, e.g. plasmacyto ma. \200\230Cold spots \200\231 are areas of photopenia and occur where there is reduced o steoblastic activity compared with the surrounding bone and may be difficult to detect. Exa mples include \201 brous dysplasia and prostheses. Interpretation in the immature skeleto n is hampered by normal increased activity at the growth plates. \200 \200 Nuclear medicine Bone scan (Figure 2.28) Technetium-99m-labeled methylene diphosphonate (99 mTc-MDP) is incorporated into bone at a rate depen- dant on blood \202 ow and osteoblasti c activity. Rheumatological indications Indications for bone scan in rheumatological practice include: \200 Advantages \200 \200 Elevated serum markers but no localizing information (e.g. in Paget \200\231s disease). Inv estigation of painful prostheses. In complex regional pain syndrome (CRPS). Investigation o f metastatic disease (Figure 2.29). Cheap compared to MRI. Readily available with most radiology departments having gamma camer as on site. Excellent sensitivity to bone turnover. Increased uptake often precedes plain \201 lm changes. Whole body imaging allows investigation of non-speci \201 c abnormalitie s such as raised serum alkaline phosphatase. Early synovitis is seen as increased activity in the affected joint(s) and can be useful as a \200\230one shot \200\231 view, although i t has largely been superseded by ultrasound or MRI. Early images visualize perfusion and bl ood pool phases, and so identify hyperaemia in infection/in \202 ammation. Serial imaging enables follow-up. Improved anatomical detail with single photon emission computed tomograp hy (SPECT) imaging, in which the gamma camera rotates 360* around the patient provides impr oved lesion conspicuity and spatial resolution at the expense of increased time for image a cquisition. \200 \200 \200 Figure 2.29 Bone scan showing multiple areas of increased uptake of 99mTc-MDP in a patient with extensive skeletal metastases. A urinary catheter is in place. Other nuclear medicine applications Other applications of nuclear medicine include: \200 Ventilation-perfusion (V/Q) scans using 99mTc-labeled MAA for perfusion and aerosolized 99m Tc-DTPA for ventilation in the investigation of pulmonary emboli. A normal chest X-ray is r equired to obtain adequate information, otherwise a CT pulmonary angiogram is performed. In dium-111 or 99mTc can be used to label white blood cells in imaging for infection or in \202 ammation, e.g. in pyrexia of unknown origin, in \202 ammatory bowel disease, infected grafts (Figure 2.30) or osteomyelitis. 99mTc-pertechnetate and occasionally Iodine-123 are used to image the thyroid in the investigation of a palpa- ble mass, hyperthyroidism or th yroid malignancies. Iodine-131 is much higher energy and is used to treat active nodules, d iffuse toxic goiter and thyroid cancer. Figure 2.28 Bone scan showing mildly increased uptake of tracer at the CMC joints of both t humbs in keeping with osteoarthritis, but otherwise normal distribution of tracer. Disadvantages \200 Although sensitive to osteoblastic activity, it is not spe- ci \201 c, with increased upta ke seen in any area of increased bone turnover, including healing fractures, OA and other a rthropathies, neoplastic lesions (both primary and sec- ondary), infection, and avascular n ecrosis. Correlation with plain \201 lms or other modalities is often required. CHAPTER 2 Investigation of rheumatic disease 37 Positron emission tomography (PET) PET scanning is a recent development and is a functional modality that most commonly uses 18F-FDG, a glucose analog tracer with a short half-life w hose distribution corresponds to glucose metabolism. The images obtained therefore identify areas of increased glycolysis, such as many malignancies. It is now usually combined with CT scanning (PET-CT) with co-registration of images to better localize the areas of increas ed activity improving accuracy of the technique. Advantages \200 The ability to assess function, rather than simple morphology as with other modalities. Thi s enables more accurate staging of tumors with uptake seen in the primary tumor, and involv ed lymph nodes or metastatic deposits. Whole body scans are currently most commonly used in non-small cell lung cancer, lymphoma, recurrent colorectal cancer, and melanoma. It is not effective in tumors with low meta- bolic activity, such as many bronchoalveolar cell and r enal cell carcinomas. Early serial imaging is increasingly used to assess response to treat ment and allow an early switch to second line chemotherapy in non-responders. Figure 2.30 Indium-111 labeled WBC scan showing increased uptake around an infected aortic graft. Normal hepatic and splenic reticuloendothelial uptake. \200 99mTc-sestamibi imaging is used in parathyroid imaging. 99mTc-sestamibi is used in cardiac imaging to assess myocardial perfusion. 99mTc-labeled red blood cells, 99mTc sulfur colloid or 99mTc pertechnetate are used in the investigation of gastrointestinal bleeding. 99mTc-l abeled DMSA or MAG-3 are used to investigate the renal tract in obstructive uropathy, renal failure, renal masses, or vesico-ureteric re \202 ux. 99mTc-labeled albumin is injected i ntradermally and the activity traced proximally in the investigation of lymphoedema (Figure 2.31). Disadvantages \200 Expensive modality with cyclotrons needed to produce the radionuclide. Short half-life of m ost of the radionuclides means these need to be sited close to the hospital. This has meant that scanners have tended to be located at specialist centers. FDG has a relatively long h alf-life allowing remote imaging, e.g. with the increasing utilization of mobile PET/CT ser vices in many areas. High radiation dose to patients, with a PET scan of the head equivalen t to 250 chest X-rays. Major radiation protection issues in relation to staff manning the f acility. Applications in rheumatology The role of PET is expanding and includes both oncological and other patient groups in whom the accurate detection of in \202 ammatory processes is impo rtant. Examples that may be encountered in rheumatological practice include: \200 Investigation of unexplained lymphadenopathy, e.g. in sarcoidosis, TB or lymphoma. In large vessel vasculitis, where the in \202 ammatory process is detected before the structural c hanges that are required for detection using conventional modalities. Assessment of bone an d soft tissue tumors. Acknowledgements My thanks to Dr Duncan MacIver for his help in preparing this chapter. Figure 2.31 Lymphangoscintigram with tracer injected intradermally in both feet, showing hy poplasia of the lymphatics on the left. AUTOIMMUNE SEROLOGY 38 Autoimmune serology Antinuclear antibodies The identi \201 cation of antinuclear antibodies (ANA) is an import ant part of clinical rheumatology. ANA testing is one of the most frequently performed anti body tests. Table 2.1 Principal associations of HEp-2 positive sera Disease % Incidence Chronic discoid lupus 5 \200\22350 The traditional substrate was rodent tissue, but over the past 20 years their use has been superseded by HEp-2 cells. These are immortalized cells, which originate from a human laryn geal carcinoma (human epithelioma type 2 cells) and are grown as monolayers. Chronic infections 10 \200\22350 Dermatomyositis/polymyositis 30 \200\22340 Drug induced SLE <50 Felty \200\231s syndrome 95 \200\223100 HEp-2 cells advantages \200 \200 Healthy population <5 More sensitive enabling identi \201 cation of more patterns. Human origin ensures better s peci \201 city than animal tissues. Nuclei are larger. Cell monolayer ensures all nuclei a re visible. Cell division rates are higher so that antigens produced only in cell division are easily located. No obscuring intracellular matrix. Antigen distribution is uniform. Healthy relatives SLE patients 25 Juvenile idiopathic arthritis 15 \200\22330 \200 \200 Lupoid hepatitis 95 \200\223100 Neonatal lupus syndrome <90 Neoplastic diseases 10 \200\22330 Normal elderly <30 \200 Polyarteritis nodosa 15 \200\22325 Pregnancy 5 \200\22310 Methods of detection ANA in serum are detected using an indirect immuno \202 uo- rescence (IIF) assay. The intensity and patterns of \202 uores- cence are reported. Many centers o nly report the presence or absence of ANA with a 3- point scale \200\223 negative, weak po sitive, strong positive. Semi-quantitative evaluation can be obtained using serial dilution s to endpoint \202 uorescence (the titre = reciprocal of dilution. A titre >1:80 is sugge stive of an autoimmune connective tissue disease. Primary biliary cirrhosis 95 \200\223100 Rheumatoid arthritis >95 Rheumatic fever <5 Sj gren \200\231s syndrome >95 SLE 95 \200\223100 Scleroderma (SSc) >90 From Bradwell et al. (1995). Standard reference sera are available for laboratory standardization. Disease and autoantibodies are associated with HEp-2 Clinical association Common in SLE and SLE overlap syndromes. patterns. Speci \201 c ANA patterns Homogeneous nuclear Uniform diffuse \202 uorescence of the ent ire nucleus of interphase cells. Surrounding cytoplasm is negative. Antigen Particularly U1-snRNP or Sm proteins, but also produced by other antinuclear antibo dies. Antigens are located in the small ribonuclear proteins (snRNPs). Fine speckled Fine to discrete speckled staining of interphase cells in a uniform distribut ion. Clinical association Common in systemic lupus erythematosus (SLE), drug induced rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), and systemic sclerosis. Clinical association Common pattern found in SLE, Sj gren \200\231s syndrome, subacute LE, and scleroderma. Antigen Antibodies to dsDNA (con \201 rm by ELISA or Crithidia luciliae IIF), histones mai nly [H2A-H2B]-DNA complex, Ku. Antigen Common to many nuclear proteins including SS-A (Ro) and SS-B (La) and RNA polymeras es II and III. Homogeneous nuclear with rim Similar to homogeneous with uniform diffuse \202 uorescence of the entire nucleus of interphase cells, but with greater intensity at the nuclear rim. S urrounding cytoplasm is negative. Centromere 40 \200\22360 discrete speckles distributed throughout the inter- phase nuclei a nd characteristically found in the condensed nuclear chromatin during mitosis. Clinical association Common in SLE especially if active. Clinical association Found almost exclusively in limited cutaneous systemic sclerosis. Antigen Antibodies to dsDNA and other antigens producing nuclear staining. Antigen Centromere proteins are located at the inner and outer kinetochore plates, which in teract with mitotic spindle ap- paratus during cell division. Antibodies recognize three pr o- teins CENT-B, CENT-A, and CENT-C. Kinetochore antigens Coarse speckled Dense, intermediate-sized particles, together with large speckles. Metaphas e cells show no staining of the condensed chromatin region. CHAPTER 2 Investigation of rheumatic disease 39 of the kintoplast. The assay is less sensitive than ELISA but is more speci \201 c. duplicate during G2 of interphase leading to the typical pattern seen in mitosis. ELISA Solid phase ELISA is the most widely used test to detect dsDNA antibodies. It is chea p, easily automated and is quanti \201 able. Nucleolar homogeneous IIF staining of the entire nucleolus. Clinical association Found in 50% of patients with polymyositis-scleroderma overlap syndrom e. Clinical association Antibodies against dsDNA are the characteristic autoanti- bodies in SL E, with a speci \201 city of 95%, occurring in 70% of patients at some stage of their dise ase. They are associated with renal involvement. dsDNA antibody levels measured by ELISA \202 uctuate with disease activity and can be used to guide treatment decisions, but change s in treatment should not be made solely on the basis of changes in dsDNA levels. It is unc lear whether a rise in anti-dsDNA antibody levels predicts a \202 are. Immunosuppressant treatment aimed solely at normalizing anti-dsDNA antibody levels is associ- ated with high drug toxicity. Antigen The target antigen is PM-SCl. Fine speckled Fine granules condensed around the nucleus which reduces towards the peripher y is typical of the tRNA synthetases. Clinical association Seen in around 20 \200\22340% of patients with polymyositis, espe- cia lly associated with polyarthralgia and interstitial lung disease (tRNA synthetase syndrome) . Extractable nuclear antigens Detection of antibodies against the extractable nuclear antige ns (ENA) is useful in the diagnosis of several autoim- mune rheumatic diseases. These antib odies were tradition- ally detected using immunodiffusion techniques, although ELISAs are n ow very widely used. Enzyme digestion studies showed that these antigens were sensitive to RNase and trypsin and are composed of ribonucleoprotein (RNP). The Sm (or Smith antigen; Sm , Ro, and La antigens were each named after the \201 rst two letters of the last name of the patients in whom antibodies to them were \201 rst found) is resistant to RNase and tr ypsin. The RNP antigenic deter- minants are found in association with U1 RNA, whereas Sm de terminants are present on U1, U2, and U4 \200\2236 RNA. The other two important nuclear ant igens are Ro and La, these are identical to the independently identi \201 ed antigens Sj gren \200\231s syndrome A (SS-A) and Sj gren \200\231s syndrome B (SS-B). Antigen Jo-1 antibodies are targeted against the reactive site of histydyl tRNA synthetase, the cytoplasmic enzyme respon- sible for complexing histidine to its cognate transfer RNA. Other tRNA synthetase antibodies are PL7 (threonyl- tRNA), PL12 (alanyl-tRNA synthetase), EJ (glycyl-tRNA synthetase, OJ (isoeucyl-tRNA synthetase). Signal recogni- tion particle an tibodies give a similar pattern. Mitochodrial Numerous course granular \201 lamentous cytoplasmic speckles extending aroun d the nucleus and throughout the cytoplasm. Rodent kidney sections are more sensitive and s hould be used to con \201 rm the pattern. Clinical association Commonly found in primary biliary cirrhosis, scleroderma (40%) and rar ely in overlap syndromes. Antigen M2 is the commonest target antigen. M2 comprises a cluster of mitochondrial inner m embrane proteins of which pyruvate dehydrogenase is the antigen in primary biliary cirrhosi s. Clinical association The clinical associations of the ENA are given in Table 2.2. Antiphospholipid (anticardiolipin) antibodies Antibodies to phospholipids particularly card iolipin (ACA) are responsible for the false positive Wasserman reaction and lupus anticoagu lant seen in patients with the primary antiphospholipid antibody syndrome (APS) and in some patients with SLE. The interaction of phospholipid and a co-factor beta 2-glycoprotein is necessary for the antienic site to be available for binding of antibody. DNA antibodies The detection of antibodies to double stranded (ds) DNA is central to the di agnosis of SLE. Their presence in the serum of patients with SLE was \201 rst described 5 0 years ago. Very early it was recognized that they might be patho- genic as they could be eluted from the kidneys of patients with lupus nephritis. Antibodies to single stranded DNA are found in wide variety of autoimmune and infectious conditions. Methods of detection ACA are typically detected by an ELISA method. A positive IgG anticard iolipin test present on more than one occasion at least 6 weeks apart is most speci \201 c for APS, but some patients are only IgM antibody positive. Methods of detection A number of different assays have been described for detection of DNA antibodies (reviewed in Isenberg & Smeenk, 2002). Described assays include IIF using HEp2 c ells, Crithidia luciliae IIF, radioimmunoassay and ELISA. The technique used in most routin e laboratories is ELISA, but the crithidia assay has a role in con \201 rming the presence of dsDNA antibodies. Clinical association Antiphospholipid antibodies are associated with an increased risk of v ascular thrombosis, recurrent fetal loss, livedo reticularis, and thrombocytopaenia. Anti-C1q antibodies C1q is the \201 rst component of the classical pathway of complement activation, hereditary de \201 ciency is a risk factor for SLE. Crithidia luciliae assay Crithidia luciliae is a haemo \202 agellate protozoan with a kine - toplast, which contains pure circular dsDNA, and no histone or other common autoantigens. This is considered the \200\230gold standard \200\231 immuno \202 uorescence shows brigh t staining AUTOIMMUNE SEROLOGY 40 Table 2.2 Antibody Disease Prevalence Speci \201 city Clinical associations dsDNA SLE 70% High Lupus nephritis Sm SLE 5% (Caucasian) High Lupus vasculitis 30 \200\22350% Afro-Caribbean CNS lupus Ro (SS-A) SLE 40% Low Photosensitivity, subacute cutaneous. LE, congenital heart block, neonatal LE. Sj gren \200\231s 80% High Extraglandular disease La (SS-B) SLE 15% Low as for Ro Sj gren \200\231s 50% High as for Ro U1RNP SLE 30% Low Raynaud \200\231s phenomenom, swollen \201 ngers rRNP SLE 15% High CNS lupus (psychosis, depression) PCNA (cyclin) SLE 5% High Proliferative glomerulonephritis Phospholipid SLE High Thrombosis, fetal loss, thrombocytopaenia C1q SLE 40% Moderate Lupus nephritis Centromere SSc 30% Moderate Limited cutaneous variant Topoisomerase 1 SSc 30% Moderate Limited cutaneous variant. Absence of lung disease RNA-polymerases SSc 20% High Diffuse variant, visceral involvement PM-SCL SSc 5% High Scleroderma/polymyositis overlap Jo-1 Polymyositis 30% High Polymyositis with interstitial lung disease SRP Polymyositis 4% High Severe myositis Mi-2 Polymyositis 10% High Dermatomyositis CCP RA 80% High Erosive RA PR3 Vasculitis 90% High Wegener \200\231s granulomatosis MPO Vasculitis 50% Moderate Microscopic polyangiitis M2 Liver 80% High Primary biliary cirrhosis SS = systemic sclerosis; SLE = systemic lupus erythematosus; SRP = signal recognition parti cle; PR3 = proteinase 3; MPO = myeloperoxidase; ccp = cyclic citrullinated peptide. (HCV) associated cryoglobulinaemia. Cryoglobulins are formed of HCV core protein plus IgG a ntibody, which are then precipitated by IgM RF. Clinical association Antibodies against C1q are present in the serum of patients with SLE ( up to 47%) and are associated with renal involvement. Monitoring anti-C1q may be useful to detect renal \202 ares. Anti-cyclic citrullinated peptide (CCP) antibodies The relatively poor speci \201 city of traditional RF assays for RA has led to the development of new serological markers. The mos t promising of these are antibodies directed against citrullinated antigens. The citrulline moiety the key component of the antigenic determinant is post- translationally generated b y peptidylarginine deaminases from arginine. Proteins that normally include citrulline resi dues include myelin basic protein, filaggrin and some histones. Proteins such as fibrin and vimentin may citrullinated following in \202 ammation, cell death or injury. Rheumatoid factor The detection of rheumatoid factor (RF) has been a key diagnostic tool in the diagnosis of RA. It pre-eminence has recently been challenged by the development of an ti-CCP antibodies (vide infra). RFs are directed against the Fc part of IgG. Conventional a ssays detect IgM RF but IgG and IgA RF can be detected using specialized assays (see Plate 25). Methods of detection The presence of RF can be detected by agglutination of IgG-sensitized sheep red cells or latex coated with human IgG. Other assays are radioimmunoassays, ELISA o r nephelometry. Methods of detection Several ELISA assays have been developed and the second generation ass ays are being increasingly used. Increasingly ELISAs are being used because they are cheap, reliable and easily automated. Clinical association Anti-CCP antibodies are present in 80% of patients with established RA . The speci \201 city is around 95 \200\22398% with a sensitivity of 70 \200\22380% (Avoua c et al, 2006). The prevalence in other diseases, which may be confused with RA is low and they are present in 0.4% of the general population. Anti-CCP antibody positivity is strongl y associated with the shared epitiope. Clinical association RF has relatively low speci \201 city for RA, however it is a good pr edictor of persistence of disease. RF is often detected in other chronic in \202 ammatory conditions such as SLE, Sj gren \200\231s syndrome (60%), chronic osteomyelitis, and up to 10% of the general population especially the elderly. RF activity is often detected in pat ients with hepatitis C virus CHAPTER 2 Investigation of rheumatic disease 41 recommended that a positive result for ANCA by IIF is con \201 rmed by antigen speci \201 c ELISA. This is especially important in pANCA positive sera where there may be confusion with a rim pattern ANA (Savige et al., 1999). A positive anti-CCP test appears to predict the develop- ment of erosive RA and this predic tive value is independent of RF. Anti-neutrophil cytoplasmic antibodies Antineutrophil cytoplasmic antibodies (ANCA) were \201 rst detected in the sera of patients with systemic vasculitis in the early 1980s and h ave become an important tool in the diagnosis of patients with systemic vasculitis. Clinical associations The combination of cANCA and PR3 is highly speci \201 c (>90%) for W egener \200\231s granulomatosis, whilst pANCA- MPO occurs in microscopic polyangiitis and C hurg \200\223Strauss syndrome, but is less speci \201 c (Hagan et al., 1998). Methods of detection ANCA in serum are detected using an IIF assay on ethanol \201 xed hu man neutrophils. The intensity and patterns of \202 uorescence are reported. Many centers only report the presence or absence of ANCA with a three point scale \200\223 negative, w eak positive, strong positive. Semi-quantitative evaluation can be obtained using serial di lutions to end- point \202 uorescence (the titre = reciprocal of dilution. A titre >1:80 is suggestive of a systemic vasculitis. Levels of PR3-ANCA and MPO-ANCA \202 uctuate with disease activity, but an increase in le vel should not alone be used as indication to change treatment. Further reading Avouac J, Gossec L, Dougados M. Diagnostic and predicitive value of anti-cy clic citrullinated protein antibodies in rheumatoid arthritis: a systematic review of the l iterature. Ann Rheum Dis 2006; 65: 845 \200\22351. Bradwell AR, Stokes RP, Johnson Gd. Atlas of HEp-2 cell patterns. Standard reference sera are available for laboratory Binding Site, Birmingham, 1995. standardization. Hagen EC, Daha MR, Hermans J, Andrassy K, Csernok E, Gaskin G, et al. Diagnostic value of s tandardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vascul itis. EC/BCR Project for ANCA Assay Standardization. Kidney Int 1998; 53: 743 \200\22353. I senberg DA, Manson JJ, Ehrenstein MR, Rahman A. Fifty years of anti-ds DNA antibodies: are we approaching journey \200\231s end? Rheumatology 2007; 46: 1052 \200\2236. Speci \201 c ANCA patterns Cytoplasmic There is uniform staining of the cellular cytoplasm . Antigen The cytoplasmic pattern of staining on IIF (cANCA) is associated with the antigen p roteinase 3 (PR3). Isenberg DA, Smeenk R. Clinical laboratory assays for measuring anti-ds DNA antibodies. Whe re are we now? Lupus 2002; 11: 797 \200\223800. Perinuclear The staining is located around the rim of the nucleus- perinuclear pattern (pAN CA). Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ, et al. International consensus statement on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA). Am J Cl in Pathol 1999; 111: 507 \200\22313. Antigen The major target of pANCA is myeloperoxidase (MPO). There are a number of other ant igens associated with pANCA (e.g. lactoferrin), but with less clinical signi \201 cance. A ntigen speci \201 c ELISAs have been developed for the detection of the two main antigenic targets of ANCA. It is Symmons DPM. Classi \201 cation criteria for rheumatoid arthritis \200\223 time to abando n rheumatoid factor? Rheumatology 2007; 46: 755 \200\2236. Westwood OMR, Nelson PN, Hay FC. Rheumatoid factors: what \200\231s new? Rheumatology 2006; 45: 379 \200\22385. SYNOVIAL FLUID ANALYSIS 42 Synovial \202 uid analysis De \201 nition Normal synovial fluid is a transudate of plasma that additionally contains high molecular weight sugar-rich molecules mainly hyaluronans. Highly viscous \202 uid can be obtained from ganglia and mucous cysts. Nucleated cell count A nucleated cell count can be performed simply using a haemocytometer chamber. The number of cells can be used to classify an effusion. \200 Normal synovial \202 uid In the normal joint synovial \202 uid production is balanced b y drainage via synovial lymphatics. Macrophage derived type A synoviocytes keep the \202 uid clear of debris. Fibroblast derived type B synoviocytes produce hyaluronans. Normal synovial \202 uid contains <200 cell/mm3 (<0.2 \227 109/l). Non-in \202 ammatory effusions contain 200 \200\2231000 cells/ mm3 (0.2 \200\2231.0 \227 109/l). In \202 amma tory effusions contain >1000 cells/mm3 (>1.0 \227 109/l). Cell counts > 50 000 cells/mm3 ( 50 \227 109/l) raise the suspicion of infection but can occur in RA, crystal arthritis and reactive arthritis. Synovial \202 uid in the diseased joint In the diseased joint there is a change in the vo lume and composition of the synovial \202 uid. Synovial \202 uid examination Simple exa mination of the \202 uid may be performed in the consulting room or at the bedside; more detailed analysis requires access to the laboratory. Examination of synovial \202 uid com prises four parts. visual analysis, nucleated cell count, \200\230wet \200\231 preparation , cytocentrifuge preparation, Wet preparation Direct microscopy of an unstained fresh specimen enables cells and crystals to be detected. Routine light microscopy Erythrocytes and white cells may be observed and counted. Ragocyte s containing intracytoplasmic granules are seen in RA and other in \202 ammatory arthritid es. Crystals of mono- sodium urate, and oxalate can also be seen. Visual analysis Volume Volume is usually increased in active disease. A low volume does not exclude signi \201 cant disease. Effusions may be dif- \201 cult to aspirate because \201 brin clots rice bodies or other debris are present and clog the needle. Long-standing effusions may be become loculated and inaccessible through the chosen route of access. Polarizng light microscopy Polarizing light microscopy is required to distinguish various t ypes of crystal. Monosodium urate crystals are needle shaped and highly birefringent. The c rystals when viewed through crossed polarizers with an interposed fourth wave plate appear yellow or blue against a red background. Urate crystals when intracytoplasmic are diagnosti c of gout. Calcium pyrophosphate dihydrate crystals are also bire- Color Normal synovial \202 uid is a pale yellow color. A diffusely red or orange effusion suggests a haemarthrosis. Damage to cartilage or a vessel during aspiration will result in an initially clear effusion, which becomes blood stained. A in- \202 ammatory effusion w ill appear white or creamy in color. A septic effusion may be pigmented by bacterial produc ts. fringent, but are rhomboid in shape Hydroxyapatite crystals are too small to be with plain light microscopy, but if stained wit h alizarin red can be seen as red birefringent clumps. Clarity Normal synovial \202 uid is clear and print can be read through it. As the cell c ount increases the \202 uid becomes progres- sively more opaque. Lipid crystals may be seen as Maltese cross-shaped deposits. Cytocentrifuge preparation Detailed cell analysis must be performed on a cytocentri- fuge p reparation. Neutrophils account for 60 \200\22380% of cells in in \202 ammatory arthritide s. In septic arthritis neutrophil counts may be 95% of nucleated cells present. Macro- phag es are predominant in viral arthritis. Viscosity Normal synovial \202 uid is viscous because it contains com- plex sugars. In in \202 ammatory disease there is enzymatic breakdown of these sugars and consequent fall in viscosity. Table 2.3 Feature of synovial \202 uid crystals Size (mm) Shape Crystals Birefringence Disease associations MSU 2 \200\22310 Needle, rod Strongly negative Acute and chronic gout CPPDD 2 \200\22310 Rhomboids, rods Weakly positive CPPDD crystal deposition disease, OA Apatite-like clumps 5 \200\22320 Round, irregular None Periarthritis, OA, clumps Calcium oxalate 2 \200\22310 Polymorphic, Positive Renal failure dipyramidal Cholesterol 10 \200\22380 Rectangles, needles Negative or positive Chronic RA or OA Depot glucocorticoids 4 \200\22315 Irregular rods, rhomboids Strongly positive or negative Iatrogenic post-injection \202 are Lipid liquid crystals 2 \200\2238 Maltese crosses Strongly positive Acute in \202 ammation, bursitis MSU = monosodium urate; CPPDD = calcium pyrophosphate dehydrate. CHAPTER 2 Investigation of rheumatic disease 43 \200 Identi \201 cation of crystal arthropathy. Identi \201 cation of septic arthritis. Micro-organisms can be identi \201 ed in 85% of clinically infected aspirates. Gram staini ng should be performed on aspirates to exclude infection by Gram-positive organisms, which account the majority of joint infections. Gram nega- tive organisms and organisms in previo usly or partially treated effusion may be dif \201 cult to detect. Further reading Dieppe P, Swan A. the identi \201 cation of synovial crystals in synovial \202 uid. Ann Rheum Dis 1999; 58: 261 \200\2233. Use of synovial \202 uid examination Examination of synovial \202 uid is most useful. \200 To distinguish non-inflammatory joint disease from in \202 ammatory causes. CLINICAL NEUROPHYSIOLOGY 44 Clinical neurophysiology De \201 nition Clinical neurophysiology is the diagnostic study of evoked and spontaneous bioelectrical signals from the central and peripheral nervous systems and from skeletal mus cle. It encompasses nerve conduction studies (NCS), needle electromyography (EMG), electroe ncephalography (EEG) and evoked central, and cortical responses (SSEPs, VEPs and BAEP). In the context of rheumatology only those aspects related to the study of the peripheral nervo us sys- tem and neuromuscular function are most often required. motor response distally. Any delay in the time taken for this re \202 ex (F latency) with normal distal conduction implies proximal slowing and demyelination. H re \202 ex The Hoffman re \202 ex (H re \202 ex) is used in a similar way, but is a ph ysiological response dependent on the stretch re \202 ex arc. It is the neurophysiological counterpart of the tendon re \202 ex. Other techniques Repetitive motor nerve stimulation is used to study the function of the ne uromuscular junction. Stimulation at a rate of 3 Hz normally elicits a compound muscle acti on potential of near identical amplitude with each successive stimulus. If there is failure of transmission at the neuromus- cular junction, as occurs in myasthenia gravis, the ampli tude of the compound action potential progressively falls with each stimulus, producing an amplitude \200\230decrement \200\231. Nerve conduction study Action potentials and conduction velocity Action potentials are evok ed by stimulating a peripheral nerve with a supra-maximal electrical square wave current of 0 \200\223100 mA usually of 0.1 ms duration (in some circum- stances the duration may be i ncreased up to 1 ms). The resultant evoked action potential is recorded with surface electr odes at a measured distance from the stimulating surface electrode. The amplitude of the ev oked response is recorded and the conduction velocity calculated. The action potential reco rded is a compound action potential generated by the summation of all the individual axon a ction potentials in a particular nerve or muscle. Needle electromyography With a needle electrode placed in a muscle the individual muscle \201 bre or motor unit action potentials can be recorded in response to a voluntary contrac tion (a motor unit is the group of muscle \201 bres supplied by one axon). The shape, dur ation, \201 ring rate, and amplitude of the individual action potentials are assessed. At maximal voluntary contraction the number of motor units is assessed by measuring the ampli tude of the overall envelope of electrical activity referred to as the interference pattern . Although there are objective quantitative techniques for this, in routine clinical practi ce this assessment is most often performed subjec- tively by observation of the action pote ntial representation on a display and through a loudspeaker. EMG is used to determine if a de \201 cit of motor function is secondary to nerve dysfunction (neurogenic) or due to pri mary muscle disease (myopathic). It also allows one to work out the anatomy of a nerve lesi on. Both sensory nerves and motor nerves can be studied. The amplitude of the compound action p otential gives an estimate of the number of axons stimulated. Each nerve and muscle has its own normal reference range for ampli- tude. Sensory amplitudes are measured in microvolts ( V) and motor amplitudes in millivolts (mV). The sensory ref- erence ranges are very wide and dependent on body weight, occupation, age and race. Conduction velocities are measured to the onset of the action potential and are those of the fastest axons stimulated, giving information about the integrity of the myelin. Conduction velocities are much less variabl e between patients and nerves. In the upper limb velocities are in the range of 50 \200\223 60 m/s, whereas in the lower limb the velocities are generally slower, around 40 \200\22350 m/s. Only the largest myelinated \201 bres can be studied by routine nerve conduction. Spontaneous activity is initially recorded. The muscle being studied must be completely rel axed. Normally, a muscle should be near silent on insertion of the needle. If one is near t he neuromuscular junction, a characteristic \200\230end plate noise \200\231 is heard. In acutely denervated muscle, myositis and many myopathies, muscle \201 bres generate sponta neous low amplitude very brief potentials called \201 brillations, which are a relatively non-speci \201 c feature of disturbed muscle physiology. Fibrillations may occur from mon ths to years if a denervated muscle does not reinnervate. Sensory action potentials (SAP) are measured with surface electrodes over the sensory nerve at a measured distance from the stimulating electrode allowing calcula- tion of the conduc tion velocity (CV). Motor function is studied by stimulating a nerve and measuring the elic ited compound muscle action potential (CMAP) amplitude recorded with surface electrodes ove r a muscle. The time taken between stimulus and response is the distal motor latency (DML). Motor conduction velocity (MCV) is deter- mined between any two accessible points along a nerve, which can be divided into multiple segments along the limb from the most distal stim ulus point up to the nerve root. This allows the demonstration of any focal slowing, motor conduction block or patchiness of abnormality.