Hemogram: Anemia is seen in most rheumatological disorders and is usually indicative of chronicity or disease activity. Anemia is generally normocytic and normochromic but can be microcytic. A normal hemoglobin level on follow-up in JIA is reassuring as it indicates reasonable disease control. Autoimmune disorders like SLE results in associated autoimmune hemolytic anemia and a positive direct Coombs test [2]. Total leucocyte count (TLC) is usually increased in inflammatory disorders like JIA. SLE causes leucopenia specifically, lymphopenia. A high platelet count is usually indicative of ongoing inflammation seen in KD, JIA and TA. Fresh onset thrombocytopenia may indicate macrophage activation syndrome (MAS). Thrombocytopenia in active lupus disease is commonly associated with antiphospholipid syndrome (APS) [3].

C-reactive protein (CRP) and Erythrocyte sedimentation rate (ESR): The CRP and ESR are acute phase reactants and provide useful information regarding disease activity and prognostication. Increased ESR may be seen in severe anemia. In inflammatory disorders it is a reflection of elevated fibrinogen which is an acute phase reactant. An ESR of more than 100 mm/hour is usually indicative of a serious underlying disorder like rheumatologic disease, malignancy or infection. Children with JDM may show a discordance between disease activity and ESR levels [4]. A rapid fall in ESR with underlying rheumatological disorder may have sinister connotation as it may herald the onset of MAS [3]. CRP is a sensitive marker for inflammation which is synthesized in the liver. It has low specificity and may be elevated in infections. It reflects changes in disease activity earlier than ESR. CRP is elevated in active lupus when associated with ongoing infection, serositis or arthritis [5]. CRP is elevated in MAS where ESR is usually normal or decreased (Fig. 1) [5,6].

Fig. 1 Role of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in rheumatological disorders.

Urinalysis: Urinalysis is important for assessment of patients with several rheumatological disorders. Renal involvement can be a primary manifestation of HSP, SLE, antineutrophilic cytoplasmic antibodies (ANCA) associated vasculitis and secondary amyloidosis due to sJIA. KD is the commonest cause of sterile pyuria in children which can lead to an erroneous diagnosis of a urinary tract infection [7]. Microscopic hematuria, proteinuria and casts are suggestive of active disease in SLE nephritis. A 24-hour urinary protein quantification indicates severity of lupus nephritis. Frequent urine examination is recommended for early identification of amyloidosis in patients with sJIA and polyarticular JIA.In HSP, significant renal involvement is usually seen only in children above the age of 7 years. In some children with HSP, the renal involvement may not manifest at first presentation, and may become apparent only on follow-up [8]. Urine examination in HSP is recommended weekly for first 1 month and every 2-4 weeks thereafter till 6 months [9].

Serum ferritin: Ferritin is an acute phase reactant and correlates with disease activity. It can differentiate sJIA and other inflammatory disorders like KD where serum ferritin levels are significantly elevated in sJIA with normal or slightly raised levels in KD [10]. Serum ferritin levels are helpful in early identification of MAS [11].

Rheumatoid factor (RF): RF is an IgM antibody (but may be IgG, IgA or IgE as well) directed against the Fc portion of IgG. It is a nonspecific inflammatory marker that can be elevated in infections like tuberculosis, infective endocarditis, hepatitis C infection, osteomyelitis and in connective tissue disorders. The usual methods for estimation of RF are nephelometry and enzyme-linked immunosorbent assays (ELISA). It is useful in classification of JIA and prognostication of polyarticular JIA. A positive RF may suggest onset of erosive disease. According to International League of Associations for Rheumatology (ILAR), RF should be positive on two separate occasions for the diagnosis of RF positive JIA (polyarthritis) as transient elevation of RF is seen in infectious illnesses. A positive RF does not indicate rheumatoid arthritis .

Anti-Citrullinated peptide antibodies (ACPA): These antibodies were originally identified in rheumatoid arthritis and are a marker of severe disease course . While the role of these antibodies in diagnosis and prognostication of adult rheumatoid arthritis is well established their role in JIA is still not clear. Approximately 20% children with JIA can have raised ACPA titres. ACPA titres are high in RF positive polyarticular JIA. ACPA positivity indicates more aggressive form of JIA and earlier onset of RF negative JIA [12,13].

Muscle enzymes: Muscle enzymes can be elevated in inflammatory myopathies like JDM and include creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and aldolase. All muscle enzymes may not be elevated in a patient at the same time. Serum levels of these enzymes usually decrease 3-4 weeks prior to clinical improvement in muscle strength. Similarly, an increase in muscle enzymes can predict disease relapse 5-6 weeks prior to clinical manifestations. CK is the first to rise and first to fall. A combination of LDH and AST is the best predictor for disease activity in children with JDM [17].

The presence of autoantibodies provide important clues to diagnosis of rheumatological disorders. Tests that are commonly used in clinical practice include ANA, anti-dsDNA, Immunoblot assay, Anti-neutrophil cytoplasmic antibody (ANCA) and anti-phospholipid antibodies (APLs).

Anti-nuclear antibody (ANA): Most commonly used methods for detection of ANA are indirect immunofluorescence (IIF) and ELISA based assays. Gold standard for ANA detection is IIF based assay which is performed by incubation of the test serum with a substrate that allows antibody binding. Anti-human IgG antisera labelled with a flurochrome is added (Fig. 2). The preferred substrate is human epithelial (HEp-2) cells, although in the past this test was carried out on rat liver cells. The IIF test also shows the pattern and staining intensity of ANA positivity. The ANA pattern is dependent on the specific nuclear antigens that provide the substrate (Fig. 3) [18]. ELISA is not the preferred method for detection of ANA and can be negative in at least a third of patients who test positive by IIF [18,19]. It allows high throughput of samples. The results are dependent upon specific nuclear antigens in the test system and can be false negative when a patient has an ANA that is not present in the commercial kit being used. A positive ANA is not synonymous with a diagnosis of lupus. Studies have shown that 9-15% healthy children can have positive ANA [20]. Results of ANA, therefore, must be interpreted in context of clinical findings [18]. ANA has very good negative predictive value but carries low specificity. Patients with other rheumatological disorders (e.g. scleroderma, mixed connective tissue disorders and overlap syndrome) can be ANA positive. In patients with oligoarticular JIA, positive ANA predicts risk of uveitis [21]. ANA positivity can also predict the risk of development of underlying connective tissue diseases in children presenting with Raynaud phenomenon [22].

Fig. 2 Indirect immunofluorescence method for measuring Anti-nuclear antibody (ANA).

Fig. 3 Different ANA patterns and association with disorders.

Anti-double-stranded DNA (Anti dsDNA) antibodies: These antibodies are highly specific and confirmatory for SLE. Measurement of these antibodies can be performed by various methods which include IIF and ELISA. Most specific method for estimation of anti-dsDNA is Crithidia lucilae based IF. It is highly specific for SLE but has poor negative predictive value. Elevated anti-dsDNA titers correlate well with active lupus nephritis and can be used to monitor disease activity [18,23,24].

Immunoblot assay: Immunoblotting is based on the principle of Western blotting where protein antigens (including nuclear and cytoplasmic antigens) are separated by using polyacrylamide gel electrophoresis. These antigens are then transferred to a nitrocellulose strip that is incubated with test serum. If antibody is present in the serum, it binds to a specific antigen over the membrane strip and that can be recognized by comparison with control results. The test will miss an antigen if not present in the commercial kit unlike IIF [18].

Anti-Neutrophil cytoplasmic antibodies (ANCA): ANCA are auto-antibodies against different antigenic components of azurophilic granules in neutrophils. ANCA is performed by IIF and ELISA based assays [25]. IIF is a more sensitive while ELISA is more specific. There are different IIF patterns of ANCA- cytoplasmic ANCA (c-ANCA); perinuclear ANCA (p-ANCA); and atypical ANCA. c-ANCA positivity results for binding to proteinase 3 (PR3) while p-ANCA positivity results for binding to Myeloperoxidase (MPO) enzyme. ELISA assay is used to confirm presence of specific antibody to PR-3 and MPO [18,26]. c-ANCA positivity is associated with granulomatosis with polyangiitis (GPA) while p-ANCA is positive in microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA).

HSP: Renal involvement in HSP is vital for long term outcome. A rapid decline of factor XIII in HSP with severe gastrointestinal involvement is associated with greater risk of renal involvement [34]. Elevated D-dimers, elevated urinary monocyte chemotactic protein-1/creatinine ratio, and HLA-B35 has been found to associated with higher chances of development of HSP nephritis [35-37].

SLE: Several blood (e.g. C4d, C5a) and urinary (e.g. monocyte chemoattractant proteins, NGAL, TWEAK, FOXP3) markers predict lupus nephritis in children with SLE. As most of these biomarkers are not specific for lupus nephritis, clinical extrapolation of these findings remains conjectural.

JDM: Patients with JDM can be classified on the basis of myositis-specific autoantibodies (MSA) and myositis associated autoantibodies (MAA) which predict treatment response, clinical course and long term outcome in these patients (Table II) [38,40].

TABLE II Important Myositis Specific Autoantibodies in Association with Juvenile Dermatomyositis

TA: Assessment of disease activity in patients with TA is fraught with problems because disease activity does not correlate with inflammatory markers like ESR and CRP. Levels of IL-6, MMP-2, -3 and -9, CCL5 chemokine, vascular cell adhesion molecules (VCAM) and Pentra-xin-3 correlate well with disease activity [41].

JIA: Several HLA association have been identified for subtypes of JIA. HLA-B27 has a high degree of genetic polymorphism and encompasses more than 100 known subtypes. Most common subtype include HLA-B*27:05 followed by HLA-B*27:04 [43]. HLAB27 is assayed in the laboratory by ELSIA, flow cytometry, microcytotoxity or by polymerase chain reaction. Flow cytometry is the most widely used method though less specific than PCR which is the gold standard [43]. There is well established relationship between HLAB27 and juvenile spondyloarthropathies and ankylosing spondylitis. HLAB27 is also present in 5-10% of normal individuals. HLAB27 can also be seen in reactive arthritis, psoriatic arthritis and inflammatory bowel disease associated arthritis in variable frequency [44]. Testing for HLAB27 is usually considered in older children who present with asymmetrical large joint arthritis, enthesitis, sacroiliitis, acute symptomatic uveitis, arthritis with family history of ankylosing spondylitis, psoriatic arthritis and inflammatory bowel disease. RF negative polyarthritis shows genetic similarity to oligoarticular JIA, while RF positive polyarthritis is similar to adult onset rheumatoid arthritis (Table III). Other important gene associations with oligoarticular JIA and RF negative polyarticular JIA include protein tyrosine phosphatase nonreceptor 22 (PTPN22), and signal transducer and activator of transcription factor 4 (STAT4) loci. Single nucleotide polymorphism in the IL-1 ligand gene, and TNFRSF1A, mevalonate kinase (MVK), and MEFV genes have been identified in patients with sJIA [45].

TABLE III	Important HLA Associations in Pediatric Rheumatology 

SLE: Monogenic forms of lupus are seen in approximately 1% of patients with SLE [16]. Strongest association has been seen with C1q deficiency, where the risk of developing SLE is 90%. Other complement deficiencies that have variable association with SLE include C4 deficiency (75%), C1s or C1r deficiency (50%), and homozygous C2 deficiency (10-30%). Lupus has been linked to several HLA haplotypes as well (Table III) [46].

Behcet disease: HLAB*5101 has the strongest genetic association with BD. However, the frequency of HLA-B51 varies from population to population. In patients where typical features of Behcet disease are not present, the presence of HLA-B51 helps in diagnosis [47].

Treatment goals in rheumatological disorders include control of inflammation and maintenance of functional integrity. Monitoring is largely clinical and is supported by laboratory investigations. Several scoring systems have been developed for monitoring of disease activity and damage in these disorders [48,49] (Web Table I).

The diagnosis of rheumatological disorders in pediatric practice is based on a detailed clinical assessment and a judicious use of relevant laboratory investigations. It is imprudent to order ‘panel’ of rheumatological investigations without keeping the clinical context in mind.

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