Complement proteins are mainly produced in the liver. Early complement components Cq, C1r, and C1s bind to immune complexes and start a cascade of reactions that result in the formation of the membrane attack complex. In clinical practice serum levels of C3 and C4, complement proteins are measured as a marker of complement activation. Due to consumption the levels are reduced in diseases like SLE, post-streptococcal glomerulonephritis, endocarditis-associated nephritis, etc. About two-third patients with SLE have low complement levels and the levels fluctuate with disease activity [5]. Some SLE patients have congenital deficiency of C4 and in them levels stay low despite control of disease activity.

In Henoch-Schonlein purpura (HSP), C4 is normal but C3 is low due to activation of alternative system of complement activation. CH50 is a functional assay to assess activity of complement proteins of the classical pathway. Its levels are very low in patients with congenital deficiencies of complement proteins like C1q, C2, or C4. AH50 is used to assess function of alternative complement pathway. These assays are rarely used in clinical practice.

The WHO international standard is most often used and the ASO values are reported as international units per milliliter (IU/mL). A rising titer 2–4 weeks apart is considered the best evidence of antecedent group A streptococcal infection. If this is not available, the upper limit of normal (ULN) of the ASO, if locally defined, may be used as the cutoff [39]. The ASO rises within a week of infection, maximum titer is seen in 6–8 weeks, and in the absence of reinfection, it is negative in 6–12 months [5].

The ASO titer is supportive criteria to diagnose acute rheumatic fever in the appropriate clinical setting, but of note is positive in children belonging to areas of high endemic zones for streptococcal infections without rheumatic fever, in children with Henoch-Schonlein purpura, IgA nephropathy, and systemic onset juvenile idiopathic arthritis [40–42].

Additionally, in the industrialized world, where the pretest probability of post-streptococcal syndromes is low, the ASO should be interpreted with care in the absence of a clear history of rheumatic fever or glomerulonephritis. In this context, it is likely to be a false positive, because of an unrelated polyclonal B cell activation [39].

Testing for tuberculosis (TB) is commonly done in pediatric rheumatology clinics, especially in countries such as India which have a high burden of TB.

It is done for several reasons:

The use of tuberculin strength of 1TU is recommended for standard TST in India, and most tuberculin surveys done in India have been carried out by using 1TU of PPD-RT 23 as per earlier recommendation of the World Health Organization (WHO) [43]. Mycobacterium tuberculosis-specific interferon-γ release assays (IGRAs) such as QuantiFERON Gold are designed to overcome problems with administration and reading of the tuberculin skin test (TST). A positive TST and QuantiFERON Gold have been found to be comparable in children with intrathoracic tuberculosis [44]. As the TST may sometimes be false negative, a combination of TST and QuantiFERON Gold is the preferred method to exclude tuberculosis [45]. A recent study has reported that the TST is useful in children who are in close contact with sputum-positive patients and those who are not BCG vaccinated. On the contrary, IGRAs are useful in children who are BCG vaccinated or in contact with lower-risk patients [46].

It is important to be vigilant for thyroid function abnormalities in children with juvenile idiopathic arthritis (JIA) as it has been reported to be abnormal in up to 12 % [47]. In addition, it should be checked for in children with lupus as well where up to 14 % have thyroid function abnormalities [48]. Any overt clinical features of thyroid function abnormalities, especially growth faltering, should also prompt the treating clinician to check the thyroid function.

The laboratory measures not 1–25 D3, but 25-hydroxy vitamin D 3 that most accurately reflects the synthesis and dietary intake of vitamin D. Though there are conflicting reports about the level that defines deficiency, levels below 25 nmol/l are the usual consensus [50].

It is a simple test and provides invaluable information in patients with monoarthritis. After collection of joint fluid, its color, consistency, and viscosity should be noted, and it should be sent to the laboratory for cell count, culture sensitivity, gram stain, and aerobic culture. In the adult patient, the synovial fluid is also sent for crystal analysis, but this is not needed for children as they rarely get crystal-induced arthritis except in a child with Down’s syndrome presenting with monoarthritis. If only a drop is obtained, after examining the wet preparation for type of cells, gram stain should be performed to exclude infection. These parameters can help in differentiating an inflammatory arthritis from noninflammatory arthritis [57] (Table 10.3). In a patient with chronic monoarthritis, culture and PCR for mycobacterial infection can help in excluding TB [58].

If the synovial fluid analysis is hemorrhagic, a possibility of coagulation defect, trauma, or pigmented villonodular synovitis should be considered.

HLA B27 is present in 65–80 % of patients with enthesitis-related arthritis subset of JIA and in about 60 % of children with reactive arthritis [59]. All boys above the age of 6 years with JIA should be tested for HLA B27. PCR has higher specificity as compared to flow cytometry and is the preferred method of detection. HLA B51 typing is used to support a diagnosis of Behcet’s disease though this association is best seen in European and Iranian population [60]. HLA-B51/B5 is present more often in males and is associated with genital ulcers, ocular, and skin disease [61].

Autoinflammatory diseases are monogenic diseases; thus, molecular diagnosis is most important but it is available only at a few centers across the world. However as all are accompanied by inflammation, the patients have elevated ESR and CRP. A normal CRP during an attack almost rules out autoinflammatory disease. Most important clue comes when common diseases are excluded and the patient has multiple self-limiting episodes of fever. Review of pattern of fever and associated symptoms helps to narrow down the diagnostic possibilities [62]. Patients with hyper-IgD syndrome have elevated IgD levels, and this may serve as an indicator to do genotyping for MVK gene; however, it can be rarely normal [63].

Biopsies of different tissues are done either for diagnosis or for assessing the severity of organ involvement. In rheumatology skin biopsy is used in diagnosis of vasculitis especially in an atypical case of HSP or cutaneous PAN. In cutaneous PAN deep dermal vessels show transmural inflammation and fibrinoid necrosis [64]. It is important to include subcutaneous fat if a diagnosis of cutaneous PAN or panniculitis is being considered.