Simultaneous Measurement of 25 Inflammatory Markers and Neurotrophins in Neonatal Dried Blood Spots by Immunoassay with xMAP Technology

Abstract

Background: Inflammatory reactions and other events in early life may be part of the etiology of late-onset diseases, including cerebral palsy, autism, and type 1 diabetes. Most neonatal screening programs for congenital disorders are based on analysis of dried blood spot samples (DBSS), and stored residual DBSS constitute a valuable resource for research into the etiology of these diseases. The small amount of blood available, however, limits the number of analytes that can be determined by traditional immunoassay methodologies. Methods: We used new multiplexed sandwich immunoassays based on flowmetric Luminex® xMAP technology to measure inflammatory markers and neutrophins in DBSS. Results: The high-capacity 25-plex multianalyte method measured 23 inflammatory and trophic cytokines, triggering receptor expressed on myeloid cells-1 (TREM-1), and C-reactive protein in two 3.2-mm punches from DBSS. It also measured 26 cytokines and TREM-1 in serum. Standards Recovery in the 25-plex method were 90%–161% (mean, 105%). The low end of the working range for all 25 analytes covered concentrations found in DBSS from healthy newborns. Mean recovery of exogenous analytes added at physiologic concentrations in DBSS models was 174%, mean intra- and interassay CVs were 6.2% and 16%, respectively, and the mean correlation between added and measured analytes was r2 = 0.91. In DBSS routinely collected on days 5–7 from 8 newborns with documented inflammatory reactions at birth, the method detected significantly changed concentrations of inflammatory cytokines. Measurements on DBSS stored at −24 °C for >20 years showed that most cytokines are detectable in equal concentrations over time. Conclusions: The method can reliably measure 25 inflammatory markers and growth factors in DBSS. It has a large potential for high-capacity analysis of DBSS in epidemiologic case–control studies and, with further refinements, in neonatal screening.