Multiplex, megaplex, index, and complex: the present and future of laboratory diagnostics in rheumatology

In a recent issue of Arthritis Research & Therapy, Chandra and colleagues described the use of multiple multiplex immunoassays and complex computer algorithms to investigate the possibility of improved laboratory diagnosis and novel classification of rheumatoid arthritis on the basis of biomarkers. Such complex predictive tools in rheumatology can be guided by the experience of multiplex testing in oncology, which has demonstrated the importance of uniform specimen handling and prospectively collected specimen repositories. Although there are high expectations for these complex approaches, they require careful evaluation

B-lymphocyte stimulator/a proliferation-inducing ligand heterotrimers are elevated in the sera of patients with autoimmune disease and are neutralized by atacicept and B-cell maturation antigen-immunoglobulin

Abstract Introduction B-lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor (TNF) family that regulate B-cell maturation, survival, and function. They are overexpressed in a variety of autoimmune diseases and reportedly exist in vivo not only as homotrimers, but also as BLyS/APRIL heterotrimers. Methods A proprietary N-terminal trimerization domain was used to produce recombinant BLyS/APRIL heterotrimers. Heterotrimer biologic activity was compared with that of BLyS and APRIL in a 4-hour signaling assay by using transmembrane activator and CAML interactor (TACI)-transfected Jurkat cells and in a 4-day primary human B-cell proliferation assay. A bead-based immunoassay was developed to quantify native heterotrimers in human sera from healthy donors (n = 89) and patients with systemic lupus erythematosus (SLE; n = 89) or rheumatoid arthritis (RA; n = 30). Heterotrimer levels were compared with BLyS and APRIL homotrimer levels in a subset of these samples. Results The recombinant heterotrimers consisted mostly of one BLyS and two APRIL molecules. Heterotrimer signaling did not show any significant difference compared with APRIL in the TACI-Jurkat assay. Heterotrimers were less-potent inducers of B-cell proliferation than were homotrimeric BLyS or APRIL (EC50, nMol/L: BLyS, 0.02; APRIL, 0.17; heterotrimers, 4.06). The soluble receptor fusion proteins atacicept and B-cell maturation antigen (BCMA)-immunoglobulin (Ig) neutralized the activity of BLyS, APRIL, and heterotrimers in both cellular assays, whereas B-cell activating factor belonging to the TNF family receptor (BAFF-R)-Ig neutralized only the activity of BLyS. In human sera, significantly more patients with SLE had detectable BLyS (67% versus 18%; P < 0.0001), APRIL (38% versus 3%; P < 0.0002), and heterotrimer (27% versus 8%; P = 0.0013) levels compared with healthy donors. Significantly more patients with RA had detectable APRIL, but not BLyS or heterotrimer, levels compared with healthy donors (83% versus 3%; P < 0.0001). Heterotrimer levels weakly correlated with BLyS, but not APRIL, levels. Conclusions Recombinant BLyS/APRIL heterotrimers have biologic activity and are inhibited by atacicept and BCMA-Ig, but not by BAFF-R-Ig. A novel immunoassay demonstrated that native BLyS/APRIL heterotrimers, as well as BLyS and APRIL homotrimers, are elevated in patients with autoimmune diseases

Add Health Wave IV Documentation: Measures of Inflammation and Immune Function

During Wave IV, Add Health collected biological specimens from a large, nationally representative sample of young adults. Given the size of the Wave IV sample, its geographic distribution, and in-home setting of the respondent interviews, biological specimen collection involved practical, relatively non-invasive, cost-efficient and innovative methods. These methods included collection of capillary whole blood via finger prick by trained and certified field interviewers, its in situ desiccation, then shipment, assay and archival of dried blood spots. The collection of capillary whole blood followed the collection of cardiovascular and anthropometric measures (Entzel et al, 2009) and saliva (Smolen et al, 2012). It preceded the collection of data on respondent use of prescription and select over-the-counter medications (Tabor et al, 2010). Further details on the design of Add Health Waves I-IV, are available elsewhere (Harris, 2011). Included in the Add Health Wave IV data are two measures of inflammation and immune function based on assay of the dried blood spots: • High Sensitivity C-Reactive Protein (hsCRP, mg/L) and • Epstein Barr Viral Capsid Antigen IgG (EBV, AU/ml) To facilitate analysis and interpretation of hsCRP and EBV, the restricted-use Add Health Wave IV data also include two data quality flags and 11constructed measures: • CRP_FLAG • EBV_FLAG • Classification of hsCRP (Pearson et al, 2003) • Count of Common Subclinical Symptoms (Vaidya et al, 2006) • Count of Common Infectious or Inflammatory Diseases • NSAID/Salicylate Medication Use in the Past 24 Hours • NSAID/Salicylate Medication Use in the Past 4 Weeks • Cox-2 Inhibitor Medication Use in the Past 4 Weeks • Inhaled Corticosteroid Medication Use in the Past 4 Weeks • Corticotropin/Glucocorticoid Medication Use in the Past 4 Weeks • Anti-rheumatic/Anti-psoriatic Medication Use in the Past 4 Weeks • Immunosuppressive Medication Use in the Past 4 Weeks • Anti-inflammatory Medication Use. This document summarizes the rationale, equipment, protocol, assay, internal quality control, data cleaning, external quality control, and classification procedures for each measure listed above. Measures of glucose homeostasis and candidate genes are documented elsewhere 3 (Whitsel et al, 2012; Smolen et al, 2012). Documentation of lipids will be provided in a separate report

Add Health Wave IV Documentation: Measures of Glucose Homeostasis

During Wave IV, Add Health collected biological specimens from a large, nationally representative sample of young adults. Given the size of the Wave IV sample, its geographic distribution, and in-home setting of the respondent interviews, biological specimen collection involved practical, relatively non-invasive, cost-efficient and innovative methods. These methods included collection of capillary whole blood via finger prick by trained and certified field interviewers, its in situ desiccation, then shipment, assay and archival of dried blood spots. The collection of capillary whole blood followed the collection of cardiovascular and anthropometric measures (Entzel et al., 2009) and saliva (in preparation). It preceded the collection of data on respondent use of prescription and select over-the-counter medications (Tabor et al., 2010). Further details on the design of Add Health Waves I-IV, are available elsewhere (Harris, 2011). Included in the Add Health Wave IV data are two measures of glucose homeostasis based on assay of the dried blood spots: • Glucose (mg/dl) and • Hemoglobin A1c (HbA1c, %). To facilitate analysis and interpretation of HbA1c, the restricted-use Add Health Wave IV data also include a trichotomous flag distinguishing the original (0) from two types (1,2) of inter-converted assay results (see Section 4.2.3.3): • Convert (0,1,2) Moreover, the restricted-use Add Health Wave IV data include six constructed measures: • Fasting duration (h) • Classification of fasting glucose (ADA, 2011) • Classification of non-fasting glucose (ADA, 2011) • Classification of HbA1c (ADA, 2011) • Anti-diabetic medication use • Joint classification of glucose, HbA1c, self-reported history of diabetes, and anti-diabetic medication use. This document summarizes the rationale, equipment, protocol, assay, internal quality control, data cleaning, external quality control, and classification procedures for each measure listed above. Documentation of other (metabolic; inflammatory; immune; genetic) measures based on assay of the dried blood spots and genotyping of DNA extracted from salivary buccal cells will be provided in separate reports

Toxicology evaluation of radiotracer doses of 3'-deoxy-3'-[18F]fluorothymidine (18F-FLT) for human PET imaging: Laboratory analysis of serial blood samples and comparison to previously investigated therapeutic FLT doses

Background: 18F-FLT is a novel PET radiotracer which has demonstrated a strong potential utility for imaging cellular proliferation in human tumors in vivo. To facilitate future regulatory approval of 18F-FLT for clinical use, we wished to demonstrate the safety of radiotracer doses of 18F-FLT administered to human subjects, by: 1) performing an evaluation of the toxicity of 18F-FLT administered in radiotracer amounts for PET imaging, 2) comparing a radiotracer dose of FLT to clinical trial doses of FLT. Methods: Twenty patients gave consent to a 18F-FLT injection, subsequent PET imaging, and blood draws. For each patient, blood samples were collected at multiple times before and after 18F-FLT PET. These samples were assayed for a comprehensive metabolic panel, total bilirubin, complete blood and platelet counts. 18F-FLT doses of 2.59 MBq/Kg with a maximal dose of 185 MBq (5 mCi) were used. Blood time-activity curves were generated for each patient from dynamic PET data, providing a measure of the area under the FLT concentration curve for 12 hours (AUC12). Results: No side effects were reported. Only albumin, red blood cell count, hematocrit and hemoglobin showed a statistically significant decrease over time. These changes are attributed to IV hydration during PET imaging and to subsequent blood loss at surgery. The AUC12 values estimated from imaging data are not significantly different from those found from serial measures of FLT blood concentrations (p = 0.66). The blood samples-derived AUC12 values range from 0.232 ng*h/mL to 1.339 ng*h/mL with a mean of 0.802 � 0.303 ng*h/mL. This corresponds to 0.46% to 2.68% of the lowest and least toxic clinical trial AUC12 of 50 ng*h/mL reported by Flexner et al (1994). This single injection also corresponds to a nearly 3,000-fold lower cumulative dose than in Flexner's twice daily trial. Conclusion: This study shows no evidence of toxicity or complications attributable to 18F-FLT injected intravenously.This study was supported by NIH grant R01 CA115559, 1R01 CA107264, and 1R01 CA80907

Add Health Wave IV Documentation: Lipids

During Wave IV, Add Health collected biological specimens from a large, nationally representative sample of young adults. Given the size of the Wave IV sample, its geographic distribution, and in-home setting of the respondent interviews, biological specimen collection involved practical, relatively non-invasive, cost-efficient and innovative methods. These methods included collection of capillary whole blood via finger prick by trained and certified field interviewers, its in situ desiccation, then shipment, assay and archival of dried blood spots. The collection of capillary whole blood followed the collection of cardiovascular and anthropometric measures (Entzel et al. 2009) and saliva (Smolen et al. 2013). It preceded the collection of data on respondent use of prescription and select over-the-counter medications (Tabor et al. 2010). Further details on the design of Add Health Waves I-IV, are available elsewhere (Harris 2012; Harris et al. in press). Included in the Add Health Wave IV restricted use and public use data are thirteen constructed measures designed to facilitate analysis and interpretation of lipids results: • Total cholesterol decile • High-density lipoprotein cholesterol decile • Triglycerides decile • Total cholesterol measurement method • High-density lipoprotein cholesterol measurement method • Triglycerides measurement method • Low-density lipoprotein cholesterol decile • Non-high-density lipoprotein cholesterol decile • Total to high-density lipoprotein cholesterol ratio decile • Fasting duration • Fasted for nine hours or more • Antihyperlipidemic medication use • Hyperlipidemia. This document summarizes the rationale, equipment, protocol, assay, internal quality control, data cleaning, external quality control, and classification procedures for each measure listed above. Measures of glucose homeostasis, inflammation, immune function, and candidate genes are documented elsewhere (Whitsel et al. 2012a, 2012b; Smolen et al. 2013)

Blood spot–based measures of glucose homeostasis and diabetes prevalence in a nationally representative population of young US adults

We investigated under-studied, biomarker-based diabetes among young U.S. adults, traditionally characterized by low cardiovascular disease risk

Dominant-negative variant in SLC1A4 causes an autosomal dominant epilepsy syndrome.

SLC1A4 is a trimeric neutral amino acid transporter essential for shuttling L-serine from astrocytes into neurons. Individuals with biallelic variants in SLC1A4 are known to have spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM) syndrome, but individuals with heterozygous variants are not thought to have disease. We identify an 8-year-old patient with global developmental delay, spasticity, epilepsy, and microcephaly who has a de novo heterozygous three amino acid duplication in SLC1A4 (L86_M88dup). We demonstrate that L86_M88dup causes a dominant-negative N-glycosylation defect of SLC1A4, which in turn reduces the plasma membrane localization of SLC1A4 and the transport rate of SLC1A4 for L-serine

Low serum albumin and the acute phase response predict low serum selenium in HIV-1 infected women

BACKGROUND: Low serum selenium has been associated with lower CD4 counts and greater mortality among HIV-1-seropositive individuals, but most studies have not controlled for serum albumin and the presence of an acute phase response. METHODS: A cross-sectional study was conducted to evaluate relationships between serum selenium concentrations and CD4 count, plasma viral load, serum albumin, and acute phase response markers among 400 HIV-1-seropositive women. RESULTS: In univariate analyses, lower CD4 count, higher plasma viral load, lower albumin, and the presence of an acute phase response were each significantly associated with lower serum selenium concentrations. In multivariate analyses including all four of these covariates, only albumin remained significantly associated with serum selenium. For each 0.1 g/dl increase in serum albumin, serum selenium increased by 0.8 μg/l (p < 0.001). Women with an acute phase response also had lower serum selenium (by 5.6 μg/l, p = 0.06). CONCLUSION: Serum selenium was independently associated with serum albumin, but not with CD4 count or plasma viral load, in HIV-1-seropositive women. Our findings suggest that associations between lower serum selenium, lower CD4 count, and higher plasma viral load may be related to the frequent occurrence of low serum albumin and the acute phase response among individuals with more advanced HIV-1 infection