From 169 patients, a total of 191 samples, for either one or two of four possible sample types (plasma, saliva, serum and urine) were purchased from Bioreclamation Inc (Hicksville, NY). All blood specimens were collected at clinical locations using standard vacutainer-type blood collection tubes and processed to plasma or serum by the vendor. The samples were aliquoted and stored frozen at −80 °C for single use. Bioreclamation Inc. collects every sample under IRB approved protocols, where ethical guidelines are followed to protect patient confidentiality and safety. Each sample has the patients consent for use in a wide range of research including the development of commercial products or services.
Patients were classified as either normal or as having one of seven possible disease states (COPD, Mononucleosis, Myeloma, Psoriasis, Rheumatoid Arthritis, and Type 2 Diabetes). Patient personal record and medical history were blinded and their sample identification was randomized prior to analysis. Samples were analysed across three 96-well plates, labelled here as Plate = (plate1, plate2, plate3), for the presences of 37 analytes (April, Baff, CD163, CD30, Chitinase, gp130, IFN-a2, IFN-b, IFN-g, IL-10, IL-11, IL-12p40, IL-12p70, IL-19, IL-2, IL-20, IL-22, IL-26, IL-27, IL-28, IL-29, IL-32, IL-34, IL-35, IL-6RA, IL-8, Light, MMP-1, MMP-2, MMP-3, OCN, OPN, Pentraxin, TNFR1, TNFR2, TSLP, and Tweak). The actual number of patients associations between sample types, conditions and plates are given inSupplementary Table S1 . Note, the 16 mononucleosis patients across plasma and serum are paired, as is the 6 myeloma patients across plasma and serum. All other patient groups represent different patients. All samples were diluted 4-fold with sample diluent prior to data acquisition.
The fluorescence responses and concentrations of analytes were obtained using a Bio-Plex Pro™ Human Inflammation Panel 37-Plex assay kit with magnetic beads (171AL1001M, Bio-Rad, Hercules, California, USA) and analysed with a Luminex100 system and the accompanying Bio-Plex ManagerTM Software 6.1(Bio-Rad, Hercules, California, USA).
The concentration values and detection limits were determined from standard curves generated from each kit’s standards using the Bio-Plex Software ManagerTM weighted 5PL curve fitting procedure. To maximize the number of concentrations values available for analysis we included the Bio-Plex extrapolated values. Therefore, the definition of out-of-range here, and unless otherwise stated, refers to concentration values that cannot be obtained from the 5PL logistic curve; that is beyond extrapolation.
All statistical analysis was performed using R version 3.1.0 (2014-04-10)45 via RStudio Version 0.98.50746 . The mixed-effects modelling were done using lmer47 (link). The visualization of regression results was done using visreg44 , and the significance of interactions terms and interaction means were determined using Phia package43 . Unless otherwise stated all p-values have been multiple test corrected according to Holm’s method48 . For simulation experiments normal distributions were obtained from rnorm and skewed distribution were obtained using skew normal distribution methods, rsn, from the R package sn49 .
Patients were classified as either normal or as having one of seven possible disease states (COPD, Mononucleosis, Myeloma, Psoriasis, Rheumatoid Arthritis, and Type 2 Diabetes). Patient personal record and medical history were blinded and their sample identification was randomized prior to analysis. Samples were analysed across three 96-well plates, labelled here as Plate = (plate1, plate2, plate3), for the presences of 37 analytes (April, Baff, CD163, CD30, Chitinase, gp130, IFN-a2, IFN-b, IFN-g, IL-10, IL-11, IL-12p40, IL-12p70, IL-19, IL-2, IL-20, IL-22, IL-26, IL-27, IL-28, IL-29, IL-32, IL-34, IL-35, IL-6RA, IL-8, Light, MMP-1, MMP-2, MMP-3, OCN, OPN, Pentraxin, TNFR1, TNFR2, TSLP, and Tweak). The actual number of patients associations between sample types, conditions and plates are given in
The fluorescence responses and concentrations of analytes were obtained using a Bio-Plex Pro™ Human Inflammation Panel 37-Plex assay kit with magnetic beads (171AL1001M, Bio-Rad, Hercules, California, USA) and analysed with a Luminex100 system and the accompanying Bio-Plex ManagerTM Software 6.1(Bio-Rad, Hercules, California, USA).
The concentration values and detection limits were determined from standard curves generated from each kit’s standards using the Bio-Plex Software ManagerTM weighted 5PL curve fitting procedure. To maximize the number of concentrations values available for analysis we included the Bio-Plex extrapolated values. Therefore, the definition of out-of-range here, and unless otherwise stated, refers to concentration values that cannot be obtained from the 5PL logistic curve; that is beyond extrapolation.
All statistical analysis was performed using R version 3.1.0 (2014-04-10)45 via RStudio Version 0.98.50746 . The mixed-effects modelling were done using lmer47 (link). The visualization of regression results was done using visreg44 , and the significance of interactions terms and interaction means were determined using Phia package43 . Unless otherwise stated all p-values have been multiple test corrected according to Holm’s method48 . For simulation experiments normal distributions were obtained from rnorm and skewed distribution were obtained using skew normal distribution methods, rsn, from the R package sn49 .