Multiplexing immunoassays for cytokine detection in the serum of patients with rheumatoid arthritis: lack of sensitivity and interference by rheumatoid factor.

OBJECTIVES: Accurately measuring cytokines in clinical material remains an important challenge in the development of biomarkers. Enzyme-linked immunoabsorbent assays (ELISAs) are considered 'gold standard'; however, their use is limited by the relatively large sample volume required for multiple analyte testing. Several alternatives (including membrane or bead-ELISA) have been developed particularly to enable multiplexing. Concerns were raised regarding their use in rheumatology due to interference by heterophilic antibodies, notably rheumatoid factor (RF). In this report, we compared several multiplex assays using serum from rheumatoid arthritis (RA) patients with respect to the presence of residual RF following attempted removal employing commonly used procedures. METHODS: Healthy control and RF-positive/negative RA sera were used to compare 4 multiplex assays with ELISA: bead-based 'Luminex' immunoassay, cytometric bead assays (CBAs), membrane-based and Mosaic™ ELISAs. Sera were tested following Ig blockade (mixed species serum) or removal (using PEG6000 or sepharose-L). RESULTS: Ig removal was only partially efficient and residual RF was detected in most sera. RF had no impact on cytokine measurement by ELISA. In single and multiplex Luminex, cytokine levels associated with false positive results correlated directly with RF titres. Following Ig-blockade/removal, these relationship remained suggesting false positivity was still associated with the presence of residual RF. Conversely, detection of cytokines in multiplex membrane-based or Mosaic- ELISA were not affected by the presence of RF; however, levels of cytokines readily detected by ELISA were often below the detection threshold of these assays. CBA assays were also low on sensitivity but unaffected by RF. CONCLUSIONS: False positivity, due to the presence of heterophilic antibodies, mainly affected Luminex assays. Other assays however remained limited in their sensitivity. Multiplexing of cytokine measurement remains a challenge, particularly in rheumatological pathologies, until assays of adequate sensitivity are developed. ELISA remains the gold standard.

Bilingual language assessment: a meta-analysis of diagnostic accuracy.

PURPOSE: To describe quality indicators for appraising studies of diagnostic accuracy and to report a meta-analysis of measures for diagnosing language impairment (LI) in bilingual Spanish-English U.S. children. METHOD: The authors searched electronically and by hand to locate peer-reviewed English-language publications meeting inclusion criteria; the authors rated quality features, calculated accuracy metrics and confidence intervals, and generated forest plots. RESULTS: Of 771 citations (86 unique) located initially, accuracy metrics could be calculated for 17 index measures studied in a total of 100 children with LI and 109 with typical language. Most studies lacked clear descriptions of reference standards, procedures, and controls for subjective bias, making it difficult to rate specific quality features with confidence. Positive likelihood ratios (LR+) for most measures were at least diagnostically suggestive (pooled LR+ = 4.12; 95% CI [2.94, 5.78]). Negative likelihood ratios (LR-) were also generally suggestive, but heterogeneity precluded averaging. For every measure, confidence intervals for LR+ and LR- included diagnostically uninformative values. CONCLUSIONS: The available evidence does not support strong claims concerning the diagnostic accuracy of these measures, but a number appear promising. Several steps are suggested for strengthening future investigations of diagnostic accuracy.

Long bone defect models for tissue engineering applications: criteria for choice.

The replacement and repair of bone lost due to trauma, cancer, or congenital defects is a major clinical challenge. Skeletal tissue engineering is a potentially powerful strategy in modern regenerative medicine, and research in this field has increased greatly in recent years. Tissue engineering strategies seek to translate research findings in the fields of materials science, stem cell biology, and biomineralization into clinical applications, demanding the use of appropriate in vivo models to investigate bone regeneration of the long bone. However, identification of the optimal in vivo segmental bone defect model from the literature is difficult due to the use of different animal species (large and small mammals), different bones (weight-bearing and nonweight bearing), and multiple protocols, including the use of various scaffolds, cells, and bioactives. The aim of this review is to summarize the available animal models for evaluating long bone regeneration in vivo. We highlight the differences not only in species and sites but also in defect size, means of defect creation, duration of study, and fixation method. A critical evaluation of the most clinically relevant models is addressed to guide the researcher in his/her choice of the most appropriate model to use in future hypothesis-driven investigations.