Kinetic Approach Extends the Analytical Measurement Range and Corrects Antigen Excess in Homogeneous Turbidimetric Immunoassays.

BACKGROUND: Homogeneous turbidimetric immunoassays are widely used in the clinical laboratory and offer short assay times, reduced reagent costs, and the potential for full automation. However, these assays have a limited analytical measurement range (AMR) above which antigen excess leads to falsely low estimates of the analyte concentration (i.e., the hook effect). Traditional methods for correction of antigen excess require sample dilution, compromising time and cost-efficiency. Therefore, novel methods that extend the AMR are needed. METHODS: A kinetic model of a generic homogeneous turbidimetric immunoassay was built and then parameterized using a genetic algorithm. Kinetic features that could be used to extend the AMR were identified and subsequently validated with clinical data from consecutive measurements of 2 homogeneous turbidimetric immunoassays: κ serum free light chain and rheumatoid factor. RESULTS: A novel kinetic parameter, the area under the curvature (AUCU), was derived that increases in proportion to the analyte concentration in a range beyond the AMR of conventional end point methods. When applied to clinical data, the AUCU method provided a log-linear calibration curve in the zone of antigen excess extending the AMR by >10-fold for 2 different immunoassays. CONCLUSIONS: The AUCU method detects and corrects antigen excess, extending the AMR in homogeneous turbidimetric immunoassays. The advantage of this method over conventional methods would be a reduction in the number of repeated samples, resulting in significant time and cost savings.

Increased rheumatoid factor interference observed during immunogenicity assessment of an Fc-engineered therapeutic antibody.

Protein therapeutics may elicit an anti-therapeutic antibody (ATA) response in patients. This response depends on a number of factors including patient population, disease state, route of delivery or characteristics specific to the product. Therapeutics for immunological indications often target relatively young and healthy patients with hyperactive immune systems who have periodic flares and remissions. The hyperactive immune system of these patients can add several levels of bioanalytical complexity due to the presence of cross reactive molecules such as autoantibodies. In addition, the long-term chronic dosing regimen often necessary in this patient population can increase their risks of immunogenicity against the therapeutic and lead to safety concerns. Therefore, development of a sensitive and drug-tolerant ATA method is important. Bridging ATA assays are usually very sensitive and drug-tolerant methods for immunogenicity assessment; however these methods are particularly vulnerable to any factor that is able to bridge the conjugated therapeutics used as reagents and can generate false positive signal. Although there are many potential interfering factors in serum, rheumatoid factors (RFs), autoantibodies associated with rheumatoid arthritis (RA), are of particular concern in this type of assay. MTRX1011A is a non-depleting anti-CD4 monoclonal antibody therapeutic that was clinically tested in RA patients. This paper will discuss the bioanalytical challenges encountered during development of a clinical ATA assay for MTRX1011A. These challenges highlight interference due to patient disease state, in this case presence of RF in RA patients, as well as specific molecule-related interference caused by an engineered mutation in the Fc region of MTRX1011A designed to enhance its binding to the neonatal Fc receptor (FcRn). We will discuss the characterization work used to identify the cross-reactive epitope and our strategy to overcome this interference during development of an effective ATA assay to support clinical evaluation of MTRX1011A.

Are anti-citrulline autoantibodies better serum markers for rheumatoid arthritis than rheumatoid factor in Thai population?

The aim of the study is to evaluate the prevalence of anti-citrulline antibodies (anti-CCP) versus rheumatoid factor (RF) in a cohort of Thai patients with rheumatoid arthritis (RA), a variety of rheumatic diseases other than RA and healthy controls. The association between anti-CCP and RA disease activity was also examined. Serum from 125 RA patients, 60 from other rheumatic diseases (non-RA) and 60 from healthy controls were tested for IgM RF and second generation anti-CCP. The association between anti-CCP, RF, the Disease Activity Score (DAS 28) and other relevant laboratory tests (CBC, ESR and CRP) were assessed. The sensitivity and specificity of anti-CCP antibody were 58.7 and 100% when compared with 63.5 and 98.3% for RF. These differences were not statistically significant. The anti-CCP outperformed RF in terms of the positive-predictive values (100 vs. 97.6%); however, the negative-predictive values were 72.4% for RF and 69.6% for anti-CCP. The sensitivity when either anti-CCP or RF was positive increased to 71.2%. Nine out of 45 RF-negative patients had a positive anti-CCP test. Anti-CCP was significantly correlated with parameters of inflammation, but not with DAS 28. In conclusion, although anti-CCP is better than RF in distinguishing RA from other rheumatic diseases, its cost, which is 3.3 times higher than the RF test precludes it from replacing RF as a serum marker for Thai patients with RA. The treatment decisions cannot be based on the test alone, as it has no correlation with DAS 28. Its usefulness is in patients with suspected RA who have had a negative RF test.

High disease activity disability burden and smoking predict severe extra-articular manifestations in early rheumatoid arthritis.

OBJECTIVES: To identify patients with severe extra-articular RA (ExRA) in an early RA cohort and to investigate potential risk factors. METHODS: From a cohort (n = 2900) in a structured programme for newly diagnosed RA, 40 patients with severe ExRA after RA diagnosis were identified. Disease activity score (DAS28), functional disability (HAQ) and treatment with glucocorticosteroids (GCs) and DMARDs were assessed regularly. Cases with ExRA were compared with RA controls from the same cohort matched for age, sex and duration of symptoms at inclusion. RESULTS: Patients who developed severe ExRA were more often current smokers and had higher mean DAS28, HAQ and CRP at baseline. Among the ExRA cases, 93% had a positive RF vs 59% of the controls. The area under the curve (AUC) of DAS28 odds ratio (OR) 7.79/S.D.; 95% CI 3.04, 19.95, HAQ (OR 2.30/S.D.; 95% CI 1.37, 3.88) and CRP (OR 3.05/S.D.; 95% CI 1.77, 5.26) during the first 2 years of follow-up were strong predictors of subsequent development of ExRA. The most frequently used DMARDs were MTX and SSZ, with similar frequency and duration of treatment among cases and controls. The cases were treated with GC before onset of ExRA more frequently (73 vs 47%; P = 0.005) and with higher mean cumulative dose (3667 vs 2037 mg, P = 0.015). CONCLUSIONS: High levels of disease activity and disability during the first 2 years after RA diagnosis, smoking and RF predict the development of severe extra-articular RA.

Laboratory assessment in musculoskeletal disorders.

Autoimmune-mediated musculoskeletal disorders feature the presence and pathogenic role of circulating autoantibodies and autoreactive T cells. Determination of these autoantibodies provides crucial information to establish the diagnosis of these diseases. In addition, the determination of these antibodies may have prognostic value or may be used to monitor response to treatment or to predict relapse of disease. We first address the main characteristics of several autoantibody assays that are considered to be clinically most relevant. These include rheumatoid factor (RF), anti-cyclic citrullinated antibody (anti-CCP), antinuclear autoantibodies (ANA), anti-double-stranded DNA antibodies, antibodies to extractable nuclear antigens (ENA), and antineutrophil cytoplasmic autoantibodies (ANCA). Subsequently we provide a brief overview of the most important musculoskeletal disorders, such as rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis/CREST, polymyositis/dermatomyositis and vasculitis. Our main goal was to address the role of the determination of autoantibodies in the diagnosis and follow-up of musculoskeletal disorders.

Soluble interleukin-2 receptors and autoantibodies in the serum of healthy elderly individuals.

Recently, we reported an increased incidence of various autoantibodies in a healthy elderly population (Group A, 64 subjects). Presently we examined whether there is variability in the expression of the age-associated immunological aberrations between different geriatric populations by extending our observations in another healthy elderly population (Group B, 119 subjects). We also determined the serum levels of soluble IL-2 receptors (sIL-2R) attempting to define the activation status of the immune system during senescence. Compared to non-elderly controls, healthy elderly individuals exhibited a significantly higher incidence of autoantibodies as well as significantly higher levels of sIL-2R in serum (p less than 0.001), the latter possibly suggesting the occurrence of lymphocytic activation during the ageing process. The overall prevalence of autoantibodies was statistically associated with the presence of raised sIL-2R levels in serum (p less than 0.005). These aberrant immunological phenomena were more frequent among the elderly of group A, compared to group B (p less than 0.005). In contrast to the uniform expression of various autoantibodies previously observed in group A, the autoantibody profile of group B consisted mainly of rheumatoid factor and antibodies to single-stranded DNA. Finally, no association could be demonstrated between the presence of autoantibodies and HLA antigens in 42 elderly studied.

Clinical and laboratory assessment of outcome in rheumatoid arthritis.

The management of rheumatoid arthritis would be assisted by an ability to assess prognosis in the individual patient. A number of studies have investigated the role of various clinical and laboratory measurements as predictors of prognosis, but all are hindered by the lack of adequate measures of outcome. Age, sex, initial radiograph and functional grades, the presence of rheumatoid factor or nodules may all be useful indicators. Mode of onset may have a bearing on outcome. The relationship between these variables and the clinical and biochemical assessment of disease activity is discussed in relation to prognosis.

Environmental and social factors in rheumatoid factor epidemiology.

The effect of social and environmental conditions on the epidemiology of Rheumatoid Factor (RF) in healthy population was studied. For this purpose 2 sample of 828 subject was studied using 5 tests for RF. Our sample included: 419 randomized subjects from a quarter at the outskirts of Bologna (high rate immigration, non homogeneous habits and health background; 409 randomized subjects from one rural small town on the Romagna's hills (no immigration, no change in residence or profession for generations, uniform, habits and health background). These tests included: three on slide with binded human (2) and rabbit (1) IgGs and sheep-cells tests-one on slide (Scat) and the other in tube (W.-Rose by Mizuoka). The most frequent positive test was Scat test, in urban population and one human IgG latex-test in the rural population. No urban subject reacted at the same time to 4 or 5 tests. Associated positive results for 2, 3 or 4 tests are usually observed in rural subjects. These results are the expression of the different and more uniform social and biological background acquired by rural people.

Cricoarytenoiditis: CT assessment in rheumatoid arthritis.

The cricoarytenoid (CA) joint is a true diarthrodial joint that can be affected by rheumatoid disease. Its strategic location in the airway anatomy makes its evaluation of clinical importance. Direct fiberoptic laryngoscopy (DFL) and high-resolution computerized tomography (HRCT) were used to assess the larynx in 32 rheumatoid patients. Abnormalities were seen in 75% of patients at endoscopic examination. HRCT studies showed abnormalities in 72%. Erosion-luxation of the CA joint and surrounding soft-tissue swelling can be demonstrated on HRCT scans. A radiologic grading of the rheumatoid larynx is proposed, stressing that accurate evaluation of the larynx should be part of the diagnostic evaluation of every rheumatoid arthritic patient, given the high frequency of occurrence of rheumatoid laryngitis.

An assessment of the diagnostic value of quantitative measurements of IgA rheumatoid factor.

IgA rheumatoid factor (RF) was found in a significant number of the normal population. As a consequence, the presence of IgA RF was not more specific for a diagnosis of rheumatoid arthritis than IgM RF. However, further investigation of IgA RF as a prognostic indicator is important. Our results, which show an association between the presence of IgA RF and higher levels of IgM RF, suggest that careful differentiation between the predictive values of the presence of IgA RF and a high level of IgM RF will be necessary.

Problems in the assessment of disease activity in ankylosing spondylitis.

Serial assessments of disease activity using clinical, laboratory and thermographic indices were made on 20 patients with ankylosing spondylitis (AS) before and after active in-patient exercise classes and two months after discharge. Clinical measurements and the erythrocyte sedimentation rate suggested decreased activity by the time of the final assessment but plasma viscosity and thermography suggested increased activity and levels of C-reactive protein were unchanged. Functional improvements occurred mostly during the in-patient period. A wide range of complement levels was found but did not change, and IgG rheumatoid factor levels were negative throughout. The problems of laboratory assessment in AS are stressed.

Assessment of immune response during chrysotherapy. Comparison of gold sodium thiomalate vs. auranofin.

Auranofin (AF) differs significantly from gold sodium thiomalate (GST) in formulation, i.e., aurous gold is stabilized by dual sulfur and phosphorus ligands, has hydrophobic rather than hydrophilic characteristics, and lacks ionic charge. These attributes facilitate: oral absorption of AF, plasma membrane penetration, increase in intracellular lymphocyte gold concentration and perhaps thereby influence lymphocyte function. AF therapy was observed to affect primarily T rather than B lymphocyte function in 16 RA subjects receiving 6 mg of AF per day for an average of 45 weeks (range 20-74 weeks) compared with GST-treated RA subjects. Lymphocytes from AF-treated subjects manifested prompt and sharp declines in mitogen-induced lymphoproliferative response (LPR); suppressed response to skin testing with dinitrochlorobenzene (DNCB); and blebbing of lymphocyte membranes as shown by scanning electron microscopy. Suppression of LPR with AF was approximately 60% after the first week and 80% after 20 weeks of therapy, contrasting with 0% and 30% for the respective intervals in GST-treated subjects. DNCB skin testing of AF patients, indicated 11 of 14, failed to respond, whereas all GST patients responded. Local or systemic fungal, bacterial and/or opportunistic infections were not encountered. The effect of AF on B cell effector function, e.g., suppression of immunoglobulins and rheumatoid factor titer, was less marked when contrasted with GST therapy in RA subjects, as previously reported.

Rheumatoid arthritis in the Pima Indians of Arizona: an assessment of the clinical components of the New York criteria.

When a prevalence study of Rheumatoid Arthritis (RA) was made in the adult Pima Indian population living on the Gila River Reservation, a high prevalence was found using the New York criteria (5-9 per cent). This was mainly due to the high frequency of limitation of motion which brought in many undesirable subjects. After excluding it as a component of New York criteria we found a prevalence for RA of 3 per cent with a predominance among the females (3-8 as against 2-0 per cent in males). The group so defined fulfilled the requirements of the Rome criteria, showed a higher concordance with serological or radiological evidence of RA, and appeared to identify subjects in whom the experienced clinical rheumatologist would more often agree with the diagnosis.