Primary objectives of laboratory investigations

Laboratory investigations are requisitioned for the following reasons:

• a)
  

  assisting the diagnostic process

  
  
• b)
  

  monitoring the activity of disease

  
  
• c)
  

  detecting adverse effects of treatment

Role of laboratory tests in the diagnosis of new-onset polyarthritis

It must be acknowledged that laboratory evidence cannot substitute for sound clinical assessment. A test carried out without clinical justification is likely to produce false-positive results. The performance of a test depends heavily on disease prevalence, that is, pretest probability . A test that is reasonably sensitive and specific can fail if the disease prevalence is low. For example, hyperuricaemia has a sensitivity of 80% and a specificity of 95% for gout (i.e., false-positive rate of 5%). The prevalence of gout is approximately 0.5%, that is, one in 200. Thus, if the test is ordered indiscriminately on 200 subjects, 10 will test positive and only one of them will be a true case of gout. Thus, 90% of positive results will occur in people without gout. On the other hand, if we perform this test on a middle-aged male with a history of a second episode of podagra, a positive result will confirm the diagnosis of gout. Synovial fluid examination for the presence of urate crystals will validate the result. Here, the pretest probability of gout is already raised and hyperuricaemia will raise it further. Normal uric acid level does not rule out gout, but it considerably reduces the likelihood to 20%.

Table 2 presents the list of laboratory tests commonly used in the investigation of polyarthritis.

Differentiating inflammatory from non-inflammatory disorders using routine tests

Laboratory plays a very important role in delineating inflammatory from non-inflammatory disorders. There is a whole list of parameters, which become abnormal in inflammatory disorders. These inflammatory markers include raised erythrocyte sedimentation rate (ESR), plasma viscosity, C-reactive protein (CRP), platelet count, total leucocyte count (TLC), serum globulins, alkaline phosphatase and gamma glutamyl transferase (GGT). The extended list includes elevated serum haptoglobin, fibrinogen, ceruloplasmin and complement components C3 and C4, although these are not recommended for this purpose in day-to-day practice. Anaemia of chronic inflammation and reversal of albumin globulin ratio are also considered markers of inflammation. Plasma viscosity is used in some parts of Europe, but ESR and CRP are widely used as inflammatory markers in rheumatology clinics. However, neither of them is completely reliable. Up to 40% of patients with RA may have normal ESR and/or CRP levels in the presence of clinical evidence of inflammation . It is helpful to obtain complete blood count (CBC), ESR, CRP, liver function test (LFT), creatinine, thyroid-stimulating hormone (TSH) and routine urine examination at presentation in all patients.

Differential diagnosis of inflammatory polyarthritis

Differential diagnosis of inflammatory polyarthritis requires careful choice of tests depending on the clinical picture. Two broad subgroups include one without any extra-articular symptoms and the other with prominent extra-articular symptoms.

• A.
  

  Inflammatory polyarthritis without any extra-articular symptoms: Some of the typical case scenarios are described as follows:

  
  
  
  
  • I.
    

    Polyarthritis in a young adult involving at least one small joint (proximal interphalangeal, PIP; metacarpophalangeal, MCP; metatarsophalangeal, MTP; and wrist), symmetrical/asymmetrical with no history of extra-articular symptoms: RA is likely to be the primary diagnosis. The clinician should request rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP). If anti-CCP is positive with or without RF at a titre of three times the upper limit of normal or more, the diagnosis is RA. .

    
    
  • II.
    

    In the previous example, if both RF and anti-CCP are negative, four important possibilities include psoriatic arthritis, peripheral spondyloarthritis (SpA), reactive arthritis and seronegative RA. The presence of psoriasis in the form of plaques, numerous ‘pinholes’ on nail plates, onycholysis and dactylitis as well as a family history of psoriasis are important clues to the diagnosis of psoriatic arthritis. Peripheral SpA will require documentation of human leucocyte antigen (HLA) B27 by the polymerase chain reaction (PCR) technique along with a typical clinical picture comprising lower extremity, asymmetrical large joint arthritis . This clinical picture is also consistent with reactive arthritis if there is a preceding history of diarrhoea or genitourinary infection in the past 4 weeks . It may be noted that reactive arthritis may or may not be accompanied by extra-articular features. ‘ Seronegative RA ’ is a diagnosis made by a process of exclusion. A close follow-up should be maintained and diagnosis revised as and when a new clue appears. At onset of RA, about 60% are seronegative but after 2 years this proportion is reduced to 20–30% . Therefore, the diagnosis of ‘seronegative RA’ in disease of <2 years’ duration is only tentative.

    
    
  • III.
    

    If there is a history of inflammatory back pain and unilateral or bilateral pain in the gluteal region along with peripheral arthritis, axial SpA is the likely diagnosis and HLA B27 (by PCR) should be requested. A positive result supports the diagnosis. However, if B27 is negative, it can still be axial SpA provided imaging favours the diagnosis .

    
    
  • IV.
    

    If the patient belongs to the elderly age group, the possibility of underlying malignancy must be ruled out by appropriate investigations. Paraneoplastic polyarthritis has no characteristic picture to provide a clue . A high index of suspicion is required.

  
  
  
  
• B.
  

  Inflammatory polyarthritis with extra-articular features:

This is a large group comprising connective tissue disorders, vasculitides, infections and others. The choice of laboratory tests should be in accordance with the working diagnosis.

• I.
  

  Connective tissue disorders

  
  
  
  
  • a.
    

    Systemic lupus erythematosus: This is a multisystem autoimmune disorder with protean clinical manifestations. Polyarthritis of the non-erosive type can occur, but often there is only polyarthralgia. For the purpose of diagnosis of systemic lupus erythematosus (SLE), antinuclear antibody (ANA) by indirect immunofluorescence using HEp-2 cell monolayers as a substrate is recommended. Four classical staining patterns are described: homogeneous, peripheral, nucleolar and speckled. The first two are commonly seen in SLE, the nucleolar pattern is typical of scleroderma and the speckled pattern is seen more often in overlap syndromes . A positive result supports the diagnosis while a negative result almost rules out the diagnosis of SLE. It must be remembered that ANA is a highly sensitive test for SLE but it is not very specific for this disease. Thus, it is also found to be positive in scleroderma, idiopathic inflammatory myopathies (IIMs), Sjögren’s syndrome, mixed connective tissue disease (MCTD), RA, autoimmune hepatitis, Hashimoto’s thyroiditis, Grave’s disease and primary pulmonary hypertension .

  
  

Anti-dsDNA (double-stranded DNA) test is important if the diagnosis of SLE is being considered. This autoantibody is positive in 60–80% of patients with SLE. It is has high specificity for SLE . False-positive results are possible if the enzyme-linked immunosorbent assay (ELISA) technique is used. Crithidia luciliae and radioimmunoassay (RIA) are superior techniques (more specific but less sensitive than ELISA), hence not as popular as ELISA . The latter has advantages of automation and lower cost. RIA is avoided by many commercial laboratories because it involves handling of radioactive material.

Another test with high specificity for SLE is anti-Sm . This autoantibody is part of the anti-ENA (extractable nuclear antigen) family. ENAs include >100 different soluble cytoplasmic and nuclear antigens. Commercially available tests utilise the double immunodiffusion technique and comprise antibodies to the following six antigens: Sm, U1RNP (ribonucleoprotein), SS-A, SS-B, Scl-70 and Jo-1. The utility of anti-Sm in clinical practice is limited because of its low sensitivity for SLE (∼30%) .

Antibodies to SS-A (Ro) and SS-B (La) are associated with subacute cutaneous lupus and photosensitivity. They are also associated with neonatal lupus and complete heart block in the foetus. Anti-histone antibodies are associated with drug-induced SLE. In cases where antiphospholipid syndrome (APS) is suspected because of venous or arterial thrombosis, bad obstetric history, thrombocytopenia or certain central nervous system (CNS) symptoms (chorea and memory deficits), anticardiolipin (aCL) antibodies (both immunoglobulin G (IgG) and IgM type), anti-beta2glycoprotein 1 and lupus anticoagulant should be obtained. The diagnosis of APS requires at least one of these tests to be positive on two occasions, 3 months apart .

• b.
  

  Scleroderma: This disease is easily diagnosed clinically by its characteristic extra-articular features. Polyarthritis of peripheral, symmetrical distribution occurs in 25% of cases. ANA is present in 95% of patients. The nucleolar pattern of staining is characteristic. Anticentromere antibody can be recognised by indirect immunofluorescence method on the Hep-2 cell line. This antibody correlates with the limited cutaneous variant of scleroderma . The diffuse cutaneous variant of scleroderma is associated with anti-Scl-70 antibody .

  
  
• c.
  

  Idiopathic inflammatory myopathies: These disorders are less likely to appear in the differential diagnosis of polyarthritis because muscle weakness rather than joint pain is the dominant feature. Numerous myositis-specific autoantibodies have been described, and they help in delineating various subsets . ANA is found in 60% of patients with IIM .

  
  
• d.
  

  Mixed connective tissue disease: When clinical features such as polyarthralgia, puffy hands, Raynaud’s phenomenon, proximal muscle weakness and interstitial lung disease point to this possibility, the test that clinches the diagnosis is anti-U1RNP in high titres (typically, 1: 1000) . The ANA positivity in MCTD is 100% .

  
  
• e.
  

  Primary Sjögren’s syndrome: Patients with primary Sjögren’s syndrome may present with rheumatoid-like polyarthritis at onset or this may occur during the course of disease . On the other hand, well-established RA can develop secondary Sjögren’s syndrome. Anti-Ro, anti-La, ANA, RF and anti-CCP are recommended. The latter is absent in primary Sjögren’s syndrome . The case definition of primary Sjögren’s syndrome according to ACR criteria requires at least two of the following three: (a) positive serum anti-SSA and/or anti-SSB or (positive RF and ANA titre >1:320), (b) ocular staining score >3 or (c) the presence of focal lymphocytic sialadenitis with a focus score >1 focus/4 mm ² in labial salivary gland biopsy samples .

  
  
• II.
  

  Systemic vasculiti s :

IgA vasculitis (Henoch–Schönlein purpura (IgAV)) may present with polyarthritis as one of its clinical features. Arthritis may precede the onset of the purpura by up to a week in 15–25% of patients . It is the most common vasculitis of childhood. The condition is recognised clinically and supported by histological evidence of leucocytoclastic vasculitis in the skin biopsy. There is no specific autoantibody test for HSP. However, serum IgA levels are elevated in 50% of cases. IgA (more specifically, IgA1)-containing immune complex deposits are found on kidney biopsy on immunofluorescence . The condition needs to be differentiated from microscopic polyangiitis (MPA), which is a more serious disease, belonging to the ANCA-associated vasculitis (AAV) group. Other members of the AAV group include granulomatosis with polyangiitis (GPA, Wegener’s granulomatosis) and eosinophilic granulomatosis with polyangiitis (EGPA, Churg–Strauss syndrome). ANCA (antineutrophil cytoplasmic antibody) is assayed by two methods, indirect immunofluorescence (IIF) and ELISA. Both should be performed . There are three patterns on IIF: cytoplasmic or C-ANCA, perinuclear or P-ANCA and atypical or X-ANCA . C-ANCA typically occurs in GPA while P-ANCA is seen in MPA. The ELISA method identifies anti-proteinase-3 (which correlates with C-ANCA) and anti-myeloperoxidase (which correlates best with P-ANCA). The diagnosis of GPA, MPA and EGPA relies mainly on the clinical picture supported by ANCA and biopsy of the involved tissue.

Behçet’s disease and Takayasu’s disease may sometimes present with polyarthritis. Diagnosis of Behcet’s disease is made clinically. There are no diagnostic laboratory tests. Takayasu’s disease is suspected clinically on the basis of arterial bruits and absent peripheral arterial pulses in association with an elevated acute phase response. Diagnosis depends entirely on imaging.

• III.
  

  Infective disorders:

Infections can present with polyarthritis, either by direct invasion of joint tissue or by causing an immunological reaction. The latter mechanism produces sterile arthritis in multiple joints. The aetiology of infective polyarthritis is usually viral or bacterial. Invasive bacterial infections generally cause a monoarthritis; polyarthritis and oligoarthritis are uncommon.

• a.
  

  Invasive infections of joints

  
  
  
  
  • i.
    

    Viral polyarthritis: A number of viruses can cause polyarthritis: parvovirus B19; rubella; hepatitis A, B and C viruses; arboviruses; retroviruses (including HIV); and others (Epstein–Barr (EB) virus, Echo, coxsackie, mumps, adenovirus, varicella zoster and herpes simplex virus and cytomegalovirus (CMV)) . Viral arthritis is generally a self-limited brief illness frequently associated with fever at onset. In many instances, there is no specific treatment and hence detailed workup is not worthwhile. Table 3 gives the clinical setting in which to suspect different types of viral arthritis and the laboratory tests to confirm the diagnosis.

    
    
    
    

    Table 3

    
    

    New-onset polyarthritis caused by viruses.

    
    

    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    

    Name	When to suspect?	Laboratory diagnosis
    Parvovirus B19	History of frequent contact with children, for example, school teachers, nurses in paediatric wards/clinics; also spreads vertically (mother to foetus); respiratory secretions and blood products (clotting factors)	Positive qPCR + presence of IgM antibody
    Rubella	More common in women; spreads through nasopharyngeal secretions	Positive IgM antibody with or without IgG antibody
    Hepatitis A	Spreads via faecal–oral route; skin rash accompanies arthritis	Presence of IgM antibody + elevated ALT and AST
    Hepatitis B	Parenteral and sexual transmission	Positive HBsAg, IgM antibody, elevated ALT and AST
    Hepatitis C	Parenteral transmission, uncommonly sexual	Positive anti-HCV antibody + HCV RNA in blood
    HIV	Arthritis can occur at any stage; not different from other types of viral arthritis; psoriatic and reactive arthritis can also occur	Anti-HIV ELISA + confirmatory Western blot
    HTLV-1	Transmitted through breast milk, sexual intercourse and blood products; causes oligo/polyarthritis with nodules and tenosynovitis, Sjögren’s and transverse myelitis	Anti-HIV ELISA + confirmatory Western blot
    Chikungunya	Fever, severe polyarthralgia/arthritis, rash and history of travel to endemic area; dengue co-infection can occur	IgM antibody to chikungunya in serum is diagnostic
    Adenovirus, Coxsackie A9, B2, B3, B4 and B6	Recurrent episodes of polyarthritis, pleuritis, myalgia, rash, pharyngitis, myocarditis and leucocytosis	Antigen detection or PCR or IgM antibody
    Echo	Polyarthritis, fever and myalgia	RT-PCR
    EB virus	Polyarthralgia, usually part of infectious mononucleosis syndrome	IgM antibody to VCA, IgG antibody to EA
    Mumps	Polyarthritis may precede or follow parotitis by up to a month	If needed, IgM antibody in blood or saliva
    Varicella zoster	Rarely, oligoarthritis develops in children with this disease	Tzanck smears from skin lesions, IgM antibody in serum

     
    
    
    
    
  • ii.
    

    Bacterial polyarthritis: These are classified under the term ‘septic arthritis’. Septic arthritis is further divided into gonococcal and non-gonococcal. The latter group comprises numerous bacteria. Table 4 presents details of the various causes of septic arthritis and their laboratory diagnosis .

    
    
    
    

    Table 4

    
    

    New-onset polyarthritis caused by bacterial infection.

    
    

    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    
    

    Name	When to suspect?	Laboratory diagnosis
    Gonococcus	History of sexual exposure followed by fever with chills, migratory/additive polyarthritis, tenosynovitis and tiny pustules/vesicles with a red base	Gram stain and culture of synovial fluid/pus; blood culture; organism is difficult to culture; needs special medium; PCR can be used
    Meningococcus	Polyarthritis/tenosynovitis occurs as part of meningococcaemia or may occur as primary suppurative arthritis	Synovial fluid and blood cultures
    Staphylococcus , Streptococcus (Group A, B and D)	Immunosuppressed patients likely to develop oligo-/polyarthritis; old age, diabetes, underlying RA and cancer	Synovial fluid – Gram stain and culture
    Brucella	Febrile illness with back pain following consumption of unpasteurised milk or contact with infected cattle; peripheral joint pain is more common than arthritis	Blood/synovial fluid culture on BACTEC system, PCR for Brucella , IgM and IgG antibodies to Brucella in serum
    Lyme disease	If erythema migrans is present, polyarthralgia and myalgia could occur in this early disseminated phase	IgM antibody to Borrelia and then confirmatory Western blot
    M. leprae	Polyarthritis in the setting of neurocutaneous features of leprosy; very often part of lepra reaction or swollen hands and feet syndrome	Demonstration M. leprae in synovial fluid or synovial biopsy; Antibodies to M. leprae -specific phosphoglycolipid-1

     
    
    

  
  
  
  
• b.
  

  Infections without joint invasion or reactive arthritis

Here, the primary site of infection is distant from the joints, which are inflamed as a result of the immune response mounted by the host. Typically, the patient presents with a triad of arthritis, conjunctivitis and urethritis. There are two main types: sexually acquired and enterocolitic. The former is caused by Chlamydia trachomatis and Ureaplasma urealyticum, while the latter is caused by Campylobacter , Shigella , Salmonella and Yersinia . The infection in the genitourinary tract or bowel precedes the arthritis by 1–4 weeks. Chlamydia can be cultured from urethral or vaginal swabs. Alternatively, a PCR can be used to establish the specific diagnosis. Stool cultures are recommended for the gut organisms. The utility of serology for the diagnosis of Campylobacter , Shigella , Salmonella and Yersinia depends on the prevalence of these infections in the community.

A few other causes of reactive arthritis need to be mentioned here. Poncet’s arthritis often presents with constitutional features such as fever, malaise, anorexia and night sweats along with a polyarthritis, usually involving large joints. Underlying tuberculosis is demonstrable usually in the lungs or lymph nodes. Joints do not show any evidence of tuberculosis . Similarly, infective endocarditis is known to get complicated by an oligo-/polyarthritis . No infection is demonstrable in the synovial fluid, which is sterile. The polyarthritis results from the immune response of the host to the infection. Rheumatic fever is a well-known cause of reactive arthritis. Streptococcal aetiology can be established by throat-swab culture or streptococcal serology (ASLO titre). Post-streptococcal reactive arthritis is another condition belonging to this group. Although the entity is well described in children as being distinct from rheumatic fever, in adults doubts have been raised about its existence .

• IV.
  

  Miscellaneous: This is a heterogeneous group that includes sarcoidosis-associated polyarthritis, drug-induced arthritis/arthralgia, serum sickness syndrome and crystal-induced arthritis. Sarcoidosis may be accompanied by polyarthritis, oligoarthritis or monoarthritis . Clinical clues often come from bilateral ankle swelling of recent onset with or without erythema nodosum lesions. Bilateral hilar lymphadenopathy on X-ray or contrast computed tomographic (CT) scan further corroborates the diagnosis. Non-caseating granuloma on histopathology is further supportive of diagnosis. Tuberculosis must always be considered as a close differential diagnosis. Effective options for obtaining tissue from hilar/mediastinal lymph nodes include endobronchial ultrasound (EBUS)-guided aspiration biopsy, endoscopic ultrasound (EUS)-guided needle aspiration biopsy, CT-guided needle biopsy and video-assisted thoracoscopic surgery . Serum levels of angiotensin-converting enzyme (ACE) are elevated in 60% of patients with sarcoidosis. As a diagnostic tool for sarcoidosis, it has poor discriminatory value. However, if the baseline level is high, it can be useful in monitoring response to treatment .

Drug-induced polyarthritis occurs as part of drug-induced SLE. Criteria for the diagnosis of drug-induced rheumatic syndrome mandate that the patient have exposure to an offending drug, clinical and laboratory evidence for a rheumatic syndrome without prior evidence of such a disorder and rapid improvement of symptoms and serologic findings with the discontinuation of the drug. About 80 drugs have been incriminated . Common drugs include isoniazid, hydralazine, phenytoin, anti-tumour necrosis factor (TNF) agents, sulfasalazine, methyldopa and minocycline. ANA is found positive in all such cases, but anti-dsDNA is usually negative with the exception of lupus induced by anti-TNF drugs and minocycline . Usually, these are mild syndromes and they subside on withdrawal of the offending drug. Serious organ involvement is rare. Anti-histone antibody is often positive.

Serum sickness syndrome is a well-recognised self-limited syndrome comprising fever, joint pains, urticaria and lymphadenopathy, originally described as a type 3 hypersensitivity reaction to a foreign protein such as horse serum . A similar illness can follow the intake of certain antibiotics and other drugs. Usually, there is a delay of 1–2 weeks after exposure to the drug. However, on subsequent exposures, the reaction is much quicker.

Crystal arthritis such as gout classically presents with an acute monoarthritis. However, as many as 12% of gout patients may present with acute polyarthritis, and this may masquerade as septic arthritis or some other rheumatic disease . Fever and leucocytosis occur frequently. Furthermore, the serum uric acid level may be normal, adding to the confusion. Diagnosis requires demonstration of negatively birefringent, needle-shaped, monosodium urate crystals in the synovial fluid using polarising light microscopy. Other common crystal-induced arthropathies include calcium pyrophosphate dihydrate and calcium apatite deposition disease.

Differential diagnosis of non-inflammatory causes of polyarticular joint pain

This group includes conditions such as osteoarthritis of hands, BJHS, fibromyalgia, vitamin D deficiency, hyperparathyroidism, hyperthyroidism, hypothyroidism, ochronosis, fluorosis, amyloidosis and haemochromatosis. Inflammatory markers by definition are not raised in these disorders.

OA of hands can be recognised by its typical joint distribution. Distal interphalangeal and PIP joints are involved along with first carpometacarpal joint. Heberden’s and Bouchard’s nodes are often present. The bony hypertrophy is obvious as opposed to soft-tissue swelling in inflammatory joint disease. Plain X-rays show typical degenerative changes of OA in the involved joints mentioned previously.

BJHS is recognised clinically using Brighton criteria . It may present with localised soft-tissue pain or a polyarthralgia. One must remember that the mere presence of hypermobility does not imply that symptoms can be ascribed to it. Other possibilities must be considered and excluded. BJHS has a strong association with fibromyalgia . The latter is recognised clinically and, it requires careful exclusion of any other rheumatic disease. Here, the laboratory is helpful in excluding other possibilities. Patients with fibromyalgia often complain of joint swellings, which are not present on examination. Vitamin D deficiency is an important cause of chronic widespread pain. Polyarthralgia is a common presentation. Serum 25-hydroxyvitamin D level defines the vitamin D status of an individual (and not 1,25-hydroxyvitamin D). The normal level ranges from 30 to 75 ng/ml.

Endocrine and metabolic causes include hypothyroidism, hyperthyroidism, hyperparathyroidism, amyloidosis, ochronosis, haemochromatosis and skeletal fluorosis. Table 5 describes the clinical clues to the diagnosis and laboratory investigations needed .

Table 5

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