Laboratory and the Pediatric Rheumatologist

Fig. 10.1
Indirect immunofluorescence assay: (a) homogeneous pattern of ANA, (b) cytoplasmic pattern of ANCA

Antinuclear antibodies target different antigens, and assessing the sub-specificities of ANA can give some clue to the diagnosis in association with clinical features. While anti-Sm is highly specific for SLE and anti-Scl70 for diffuse systemic sclerosis, others like anti-Ro and anti-RNP antibodies are seen in multiple diseases (Table 10.1).These antibodies have a pattern; anti-La usually occurs with anti-Ro, anti-Sm with anti-RNP, and anti-nucleosome with anti-dsDNA [34]. ELISA or immunoblotting (line assay) is used to identify antigenic specificities. With the availability of ELISA based on recombinant proteins, the sensitivity and specificity have improved significantly. Immunoblot or line assays are widely used in India and have good specificity. In practice, if the IIF test for ANA is negative, further testing for the other antibodies is of little value.

Table 10.1
Antinuclear antibody specificities in different diseases


ANA sub-specificity


dsDNA, nucleosomes, Sm, nRNP, Ro, La

Systemic sclerosis

Scl-70, RNA polymerase III, centromere

Mixed connective tissue disease


Sjogren syndrome

Ro, La


Jo-1 and other t-RNA synthetases, SRP, Mi2, TIF1γ, Ro

dsDNA double-stranded DNA, Sm Smith

Anti-neutrophil Cytoplasmic Antibody (ANCA)

As the name suggests, this antibody is directed against components of neutrophil cytoplasm. It is very useful in diagnosis of systemic vasculitis and glomerulonephritis. It is detected by indirect immunofluorescence assay using human neutrophils as the substrate. Two patterns, i.e., cytoplasmic-ANCA (c-ANCA, Fig. 10.1b) and perinuclear-ANCA (p-ANCA), can be identified. All samples positive by IIF should be tested for antibodies to proteinase 3 (PR3) and myeloperoxidase (MPO) [35] (Table 10.2).

Table 10.2
Antigenic specificities of ANCA and its clinical utility


Antigenic specificity

Clinical utility


Proteinase 3

Very specific for granulomatosis with polyangiitis

Active widespread disease (90 %)

Limited disease (60–80 %)

Inactive disease (40–60 %)



Seen in microscopic PAN, polyangiitis overlap syndrome, crescentic GN

In a large pediatric series of GPA, 67 % of 56 children had anti-PR3 ANCA [36]. The levels of antibodies have modest correlation with disease activity in GPA.

Antiphospholipid Antibodies

Antiphospholipid antibodies are directed against phospholipids present in the plasma, on platelets, and endothelial cells and are essential for diagnosis of antiphospholipid syndrome (APS). Among the various assays, anticardiolipin antibodies have high sensitivity, while antibodies to beta 2 glycoprotein, a cofactor for cardiolipin, have higher specificity. Due to variability in the different assay systems, it is suggested that instead of the exact value, it should be reported as normal, mild, moderate, or high positive [37].

For detecting lupus anticoagulant, kaolin clotting time, activated partial thromboplastin time, and Russell viper venom time are used. If the value is abnormal, mixing with normal plasma is done to see if the abnormality gets corrected or not. The absence of correction suggests the presence of lupus anticoagulant.

In the Ped-APS registry, anticardiolipin antibodies were detected in 81 %, anti-β2GPI antibodies in 67 %, and lupus anticoagulant in 72 % of children, respectively. One third patients had all three tests positive [38].

Complement Levels

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.

Miscellaneous Tests

Antistreptolysin O (ASO) Titer

This is a test that causes significant confusion with the interpretation, especially in countries with a high endemic burden of streptococcal disease. There are two methods to detect a streptococcal infection: a throat culture and tests that measure antibodies to the extracellular products of the bacteria. The antistreptolysin O (ASO) assay was the first such antibody test to be developed, and it measures the ability of human serum to neutralize the hemolytic activity of streptolysin O reagent.

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 [4042].

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].

Tuberculin Skin Test (TST) and QuantiFERON Gold

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:

  • To diagnose articular disease, such as tubercular arthritis or Poncet’s disease

  • To screen the child for latent tuberculosis, when he/she presents with a rheumatic disease and a positive family history of contact

  • To detect latent tuberculosis, as a routine screen, prior to using either high-dose steroids or biologic response modifiers (BRMs)

  • When tuberculosis is suspected, during the course of a treatment of a child with a known rheumatologic disease

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].

Thyroid Function Tests

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.

Vitamin D

The active form of vitamin D, 1–25-dihydroxycholecalciferol (calcitriol) (1,25 D3), is in fact a steroid hormone and has several effects other than calcium homeostasis. It is derived from 7-dehydrocholesterol after complex hydroxylation processes in the liver and kidney, and it is now referred to as “hormone D.” This hormone binds to vitamin D receptor (VDR) and has multiple immunomodulatory and epigenetic effects. Though vitamin D deficiency has been reported in several autoimmune conditions, the cause and effect relationship is not clear. All the same, in pediatric rheumatic diseases, the child should be supplemented with vitamin D, especially when on steroids [49].

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].

Von Willebrand Factor (vWF) Antigen

This is a plasma protein and is synthesized by megakaryocytes and endothelial cells. It is important for both platelet aggregation and adhesion and the plasma level increases with endothelial damage resulting from vasculitis. It has been studied therefore as a surrogate marker of vasculitis in children for several conditions such as Henoch-Schonlein purpura, Kawasaki disease, central nervous system angiitis, and granulomatosis with polyangiitis [51, 52]. The levels correlate with disease activity in children with Kawasaki disease [53].

Angiotensin-Converting Enzyme (ACE)

The ACE level is measured in suspected cases of sarcoidosis. The source of ACE is thought to be the epithelioid cells of the granulomas that characterize the disease. There are two main clinical scenarios where the ACE is ordered: in infants and children younger than 5 years who present with skin, joint, and eye involvement, without typical lung disease, and in older children who present with lungs and lymph nodes disease. It should be noted that normal children have higher ACE levels as compared to adults (118 (SD 30) vs. 100 (SD 35) U/IL). Measurement of ACE is important to diagnose the child with sarcoidosis and also to follow up during treatment [54]. It is elevated in 74 % of patients with childhood sarcoidosis [55]. As a substantial number of patients, especially the ones with early onset sarcoidosis may have a normal ACE, the gold standard remains tissue diagnosis [56].

Synovial Fluid Examination

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].

Table 10.3
Synovial fluid findings in different diseases







Straw to yellow













Mucin clot





Cell count/μL






<25 %

<25 %

>50 %

>75 %






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

HLA Typing

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].

Tests for Autoinflammatory Diseases

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].

Tissue Diagnosis

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.

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Oct 25, 2017 | Posted by in RHEUMATOLOGY | Comments Off on Laboratory and the Pediatric Rheumatologist
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