1 Introduction

Generally the diagnosis of rheumatic diseases is based on a set of clinical, serological, and radiological measures. The discovery of a novel test that appears to be considerably more disease-specific and preferably sensitive would be of value for the early diagnosis and immediate, effective therapy to prevent joint deterioration, functional disability, and unfavorable disease outcome [1].

However, components of acute phase reaction proteins such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) or rheumatoid factor (RF) lack specificity and sensitivity and could not reach the expectation of earlier diagnosis of specific rheumatic diseases. Therefore, the discovery of immunologic laboratory tests has occupied a valued position in the practice of rheumatology and has helped define the pathophysiology of various rheumatic conditions such as the immunologic basis of rheumatoid arthritis (RA) [2, 3] and explain the contribution of genetic basis to autoimmune disease via the association of ankylosing spondylitis (AS) with HLA-B27 and RA with certain HLA-DR alleles [4, 5].

Hence the salient existence of such immunologic laboratory tests has assisted the more precise diagnosis of diverse rheumatologic conditions that may share some clinical characteristics. In addition, these tests can provide valuable evidence concerning disease manifestation, activity and prognosis, and therapeutic monitoring.

Essential terms concerning the laboratory tests are needed to be defined such as sensitivity, specificity, and positive and negative predictive values. Sensitivity refers to the ability of the test to detect the proportion of patients with a disease which usually have a positive test result. However, specificity refers to the ability of the test to detect the proportion of patients without the disease which usually have a negative test result. Predictive value refers to the likelihood of disease or non-disease based on a positive or negative test result. A high positive predictive value test indicates that the patient with a positive test result most probably has the disease in question. Similarly, a high negative predictive value test indicates that the patient with a negative test result most likely does not have the disease in question.

Unlike with sensitivity and specificity of the test, the predictive value is markedly affected by disease prevalence. For instance, the predictive value of a positive rheumatologic test in patients with polyarthralgia is likely to be higher in a rheumatology clinic than in a family physician’s clinic [6].

The subsequent sections will discuss the stepwise approach to the diagnostic workup of rheumatic diseases and are presented as follows:

  • Inflammatory markers (ESR and CRP)

  • Rheumatoid factor (RF)

  • Antinuclear antibody (ANA) profile, for instance, anti-double-stranded DNA antibodies (anti-dsDNA) and anti-ribonucleic protein (RNP) antibodies

  • Other disease-specific antinuclear antibodies and cytoplasmic antibodies

  • Complement deficiencies and decreased complement activity in certain medical conditions

  • Components and classification of synovial fluid analysis

  • Other biochemical tests: renal function tests and urine analysis (this section is not in the scope of the current chapter but it will be discussed in details in the chapter of “Renal System and Rheumatology”)

1.1 Objectives

By the end of the current chapter, the candidates should be able to:

  • Identify the rule of acute phase reaction proteins in rheumatic diseases.

  • Interpret the auto-antibodies’ results based on clinical findings.

  • Classify various types of joint effusions based on clinical and laboratory analysis of synovial fluid.

2 Acute Phase Reactants

Acute phase reactants (APRs) or proteins are defined as those proteins whose serum concentrations increase or decrease by at least 25% during inflammatory states. Changes in levels of APR largely result from the effects of cytokines, including interleukin (IL)-6, IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), and interferon gamma.

Serum APR level measurements are useful because they frequently reflect the presence and intensity of an inflammatory process. The assessment of APR may be most helpful in patients with RA, polymyalgia rheumatica, and giant cell arteritis.

However, APR measurements in clinical use are not specific to any particular disease.

The most widely used indicators of the acute phase response are the ESR and CRP [7].

ESR and CRP definitions, measurements, uses, and other important aspects are addressed in Table 3.1.

Table 3.1 ESR versus CRP
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3 Rheumatoid Factor (RF) and Anti-citrullinated Protein Antibody (ACPA)

3.1 Definition

RF is an antibody directed against the Fc fragment of immunoglobulin G (IgG). It may be of any isotype: IgG, IgA, IgE, and IgM. RF-IgM is the only one measured in clinical practice. The origin of RF is incompletely understood [7]. ACPAs are antibodies that are targeted against citrulline which is situated on proteins. Important clinical features of RF including measurement and common issues while dealing with it in clinical practice are all addressed in Table 3.2.

Table 3.2 Characteristics of RF
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4 Antinuclear Antibodies (ANAs)

4.1 Definition

ANAs are serologic hallmarks of patients with systemic autoimmune disease. These antibodies should be ordered when the clinical assessment of the patient suggests the presence of an autoimmune or connective tissue diseases [7]. Clinical aspects of ANAs are discussed in Table 3.3.

Table 3.3 ANA characteristics
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4.2 Methods of Measurement

  • Indirect immunofluorescence method using “fluorescence microscope” is the gold standard method to detect ANAs. Currently most laboratories use human epithelial cell tumor line (HEp2 cells) as a substrate to detect antibodies that bind to various nuclear antigens (ANAs) instead of frozen section of rodent organ cells.

  • Other methods that can be used for detection of specific ANA include ELISA, immuno-blotting, and Western-blotting methods.

5 ANA Profile

5.1 Definition

An ANA profile consists of many antibodies to measure specific ANAs for certain nuclear antigens. It should be performed when the screening test for ANA is positive and when further information is needed regarding the type of autoimmune disorder [7].

ANA profile antibodies and their specific uses are elaborated on Table 3.4.

Table 3.4 The standard ANA profile
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6 Other Disease-Specific Antinuclear Antibodies and Cytoplasmic Antibodies

These antibodies have to be ordered individually according to the set-up diagnosis based on patient’s symptoms and clinical presentations, and they include:

  1. 1.

    Anti-histone antibodies: sensitive (70%) for drug-induced lupus but nonspecific and have limited diagnostic utility because they may also be present in patients with SLE. The best test to conduct in patient with suspected drug-induced lupus is antichromatin antibody test, not anti-histone antibody test [7]. However, anti-histone antibody test might be of value in patients having a positive ANA test with history of exposure to medications-induced lupus, such as procainamide (Pronestyl) and isoniazid (INH) [27].

  2. 2.

    Anti-Th/To antibodies: crest syndrome 20% [7].

  3. 3.

    Anti-SCL-70 antibodies (topoisomerase1): diffuse systemic sclerosis (scleroderma) 22–40% [7]. They are highly specific but not sensitive for scleroderma [29].

  4. 4.

    Anti-tRNA synthetase antibodies (anti-Jo-1, other): polymyositis 20–30% [7].

  5. 5.

    Anti-neutrophil cytoplasmic antibodies (ANCAs):

    • Cytoplasmic anti-neutrophil cytoplasmic antibodies (C-ANCA), the most common c-ANCA target is serine proteinase-3: granulomatous polyangiitis (GPA) (Wegener granulomatosis) 90%, microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA) (rare). Its titer can correlate with GPA disease activity [30].

    • Perinuclear anti-neutrophil cytoplasmic antibodies (P-ANCA), the most common p-ANCA target is myeloperoxidase: MPA 70%, pauci-immune glomerulonephritis, and EGPA, or myeloperoxidase (−)—ulcerative colitis, chronic infection, and neoplasm (rare) [30].

  6. 6.

    Anti-mitochondrial antibodies (AMAs): primary biliary cirrhosis 80% [7].

  7. 7.

    Antibodies to the gp210 and p62 proteins of the nuclear pore complex: primary biliary cirrhosis 10–40% [7].

7 Circulatory Complement Components

Complement is an important effector pathway of innate immunity and has a role in the pathogenesis of some of rheumatic conditions, namely, SLE.

Causes of Decreased Circulatory Complement Components

  • Hereditary complement deficiencies (decreased production)

  • Secondary complement deficiencies (acquired) [31]

7.1 Mechanism of Acquired Complement Deficiencies

  1. 1.

    Increased level of circulatory immune complexes (increased consumption of complements) due to:

    • Infectious causes

    • Glomerulonephritis

    • Rheumatic diseases:

      1. (a)

        SLE: Low C4 and C3 levels occur in about 50% of patients with SLE. Levels of C3 and C4 are decreased with increased severity of SLE, especially renal disease. A return to normal levels with treatment is a good prognostic sign. Serial observations reveal decreased levels preceding clinical exacerbation.

      2. (b)

        Cryoglobulinemia: The complement profile shows decreased levels of C4 and C2 with normal or slightly lowered C3.

      3. (c)

        Systemic vasculitis especially polyarteritis nodosa, urticarial vasculitis: 50% of patients with polyarteritis may have decreased serum complement levels. Its values can be helpful in assessing the clinical course, especially the response to therapy.

      4. (d)

        RA with extra-articular manifestation (rare) [7, 32].

  2. 2.

    Reduced hepatic synthesis (uncommon)

  3. 3.

    Loss of complement components in the urine (rare) [30]

8 Synovial Fluid Analysis

The presentation of one or more hot, swollen joints is a common medical emergency, and synovial fluid aspiration, the so-called arthrocentesis, is the single most important test helping in the diagnosis of different types of arthropathies [33].

Therefore, specialized laboratories analyze synovial fluid to either confirm the diagnosis of crystal-associated arthropathies, support the diagnosis of septic arthritis, or establish other rheumatologic diagnoses such as mono-arthritis or joint effusion [34].

The complete analysis of synovial fluid includes macroscopic (gross appearance), microscopic, and specific stain tests to provide detailed information about the joint’s condition and helps in establishing the diagnosis and treatment [35]. Description of macroscopic analysis of synovial fluid includes color, clearance, volume, and viscosity. However, the microscopic analysis can differentiate between inflammatory and infectious processes by measuring a complete leukocyte count. In addition, a differential of the synovial WBC count should be ordered, so that if the results came positive for infectious process, the performance of Gram-stain and culture tests will provide guidance to diagnosis and/or antibiotic therapy [36].

Microscopic examination specifically can also allow the detection and identification of various types of crystal by using polarized light microscope. Refer to Table 3.5 for an overview on important issues as regards arthrocentesis and synovial fluid analysis. However, Table 3.6 shows the classification of joint effusions into normal, inflammatory, non-inflammatory, and septic effusion based on clinical and laboratory analysis of synovial fluid with the causes of each type [37, 38]. Indications, contraindications, complications, and specimen analysis of synovial fluid are presented in Table 3.5. Classification and causes of joint effusions based on laboratory analysis of synovial fluid are presented in Table 3.6. Fig. 3.1 is the clinical diagnostic approach for painful peripheral joint.

Table 3.5 Overview on arthrocentesis and synovial fluid analysis
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Table 3.6 Classification and causes of joint effusions based on laboratory analysis of synovial fluid
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Fig. 3.1
figure 1

Clinical approach for painful peripheral joint

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9 Key Notes

  • The likelihood diagnosis of septic arthritis is markedly increased with higher synovial WBC counts. It has been illustrated that for synovial WBC count the likelihood ratio (LR) of having septic arthritis is as follows [34]:

    • WBC count <25,000/μL, the LR is 0.32 at 95% CI.

    • WBC count ≥25,000/μL the LR is 2.9 at 95% CI.

    • WBC count >50,000/μL, the LR is 7.7 at 95% CI.

    • WBC count >100,000/μL, the LR is 28.0 at 95% CI.

  • Polymorphonuclear (PMN) cells of 90% are associated with increasing likelihood of septic arthritis of 3.4, while if the percentage of PMN cells is less than 90%, the likelihood decreases down to 0.34 (95% CI) that supports the clinician’s suspicion of bacterial arthritis [38, 39].

  • Eosinophilic cells in the synovial fluid suggest parasitic infection, allergy, Lyme disease, or neoplasm [40].

  • If there is a suspicion of joint involvement by a neoplasm or hematologic malignancy, formal cytological examination should be ordered [38].

  • Hemorrhagic effusions may be caused by hemophilia, anticoagulation or other bleeding diathesis, scurvy, trauma, neuropathic arthropathy, and tumors [38].

9.1 Gram Stain

  • It is used to identify common bacterial organisms (Gram-positive versus Gram-negative coverage) for the diagnosis and treatment of septic arthritis.

  • It may be the only evidence of infection with fastidious organisms that are not able to grow in culture [41].

  • The sensitivity is not known precisely.

    • In non-gonococcal bacterial arthritis, it is in range from 50% to 70%.

    • In gonococcal arthritis, it is <10% [41].

  • The specificity is high when performed and interpreted by an experienced clinician or technician [41].

9.2 Synovial Fluid Culture

  • The synovial fluid samples should be routinely sent for culture for staphylococci followed by streptococci and Gram-negative bacteria (non-gonococcal causes).

  • Antibiotics should generally not be given prior to joint aspiration [42, 43].

  • The specificity: Positive synovial culture should be indicative of septic arthritis in 100% of cases with exclusive of contamination and laboratory error [42, 43].

  • The sensitivity: It is not known precisely because of the lack of an alternative gold standard. The joint aspirate should be cultured for N. gonorrhoeae or unusual organisms (TB, Lyme disease, or fungal infections) when the history is suggestive [42, 43].

9.3 Diagnostic Approach

  • It should be noted that the absence of organisms on Gram stain or a negative subsequent synovial fluid culture does not rule out the diagnosis of septic arthritis especially if clinical suspicion is high. In such condition, an empirical treatment of the case as septic arthritis should be implemented [44,45,46].

  • Moreover, it has been suggested that the “gold standard” for the diagnosis of septic arthritis is the level of clinical suspicion by an expert physician in the management of patients with musculoskeletal disease [35, 45].

  • Similarly, another study had concluded that combining Gram stain and culture of synovial fluid with clinical follow-up is the best approach used to detect patients missed by Gram stain and culture alone [36].

9.4 Crystal Search Using Polarized Light Microscopy

Polarized light microscope (PLM) is a fundamental tool for detection and identification of various types of crystals present in synovial fluid depending on their shape (needle, rhomboid, cigar-shaped, etc.) birefringence, location (intracellular or extracellular), and quantity (scarce or plentiful). The obtained results of PLM help the clinicians in diagnosing and managing a case of monoarthritis. However, the presence of artifacts in microscopic analysis can confuse the inexperienced observer; therefore, a suitable interpretation of the synovial fluid analysis using PLM requires at least two experienced observers [47]. The microscopic features of common types of crystals that can differentiate between clinical cases of gout and pseudogout are illustrated in Table 3.7.

Table 3.7 Gout versus pseudo-gout
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10 Summary

Due to the fact that musculoskeletal symptoms are exceedingly common compared with the prevalence of systemic rheumatic disease, the pretest probability of systemic rheumatic disease in the population is rather low compared with musculoskeletal symptoms that are nearly ubiquitous. Therefore, establishing the diagnosis of a rheumatic disease may require exclusion of other differential diagnoses that present in a similar fashion. Even the disease established-guidelines, which are often used by clinicians, perform poorly during the assessment of a patient presenting with new polyarthritis [49]. As a consequence, widely used laboratory tests can be very specific and permit rapid diagnosis and appropriate management. However, clinicians should be aware of the false-positive tests that may result in inappropriate management and unnecessary concern.

Generally, serum rheumatologic tests are most helpful for confirming a clinically suspected diagnosis. For instance, testing for RF is appropriate when suspecting RA, Sjögren’s syndrome, or cryoglobulinemia, whereas ANA testing is highly sensitive for SLE and drug-induced lupus. Although an elevated ESR is a sensitive test for polymyalgia rheumatica and temporal arteritis, its specificity is quite low. In addition, ESR levels are frequently linked to the disease activity in rheumatoid arthritis and may found to be of value for monitoring therapeutic response. However, anti-double-stranded DNA antibodies are usually associated with lupus nephritis, and their titer often correlates with disease activity in SLE. On the other hand, cytoplasmic anti-neutrophil cytoplasmic antibody test is highly sensitive and specific for GPA.

In order to increase the utility and decrease the cost-effectiveness of the laboratory testing of rheumatic disease, these tests should be used more selectively and avoid absolute overreliance on lab results. However, a logic combination of the clinical background and the testing results would provide the appropriate diagnosis of the rheumatic conditions. Finally, as Shmerling RH has stated, “the passage of time is one of most useful diagnostic tests as many patients with musculoskeletal symptoms improve over time without a clear diagnosis” [50].