Simultaneous Measurement of 25 Inflammatory Markers and Neurotrophins in Neonatal Dried Blood Spots by Immunoassay with xMAP Technology Kristin Skogstrand, 1,2 Poul Thorsen, 2 Bent Nørgaard-Pedersen, 1 Diana E. Schendel, 3 Line C. Sørensen, 4 and David M. Hougaard 1* 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 congen- ital 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 immu- noassays based on flowmetric Luminex ® xMAP technol- ogy to measure inflammatory markers and neutrophins in DBSS. Results: The high-capacity 25-plex multianalyte method measured 23 inflammatory and trophic cytokines, trig- gering 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 exog- enous analytes added at physiologic concentrations in DBSS models was 174%, mean intra- and interassay CVs were 6.2% and 16%, respectively, and the mean correla- tion between added and measured analytes was r 2  0.91. In DBSS routinely collected on days 5–7 from 8 newborns with documented inflammatory reactions at birth, the method detected significantly changed con- centrations 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 in- flammatory 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. © 2005 American Association for Clinical Chemistry Newborn screening programs are based on analysis of a single sample of capillary blood collected by heel-prick from infants 3–14 days of age. The samples are usually stored as dried blood spot samples (DBSS) 5 on special filter paper that has been demonstrated to be a robust, convenient medium for collection, transport, and storage (1). Most programs screen for phenylketonuria and con- 1 Department of Clinical Biochemistry, Statens Serum Institut, Copenha- gen, Denmark. 2 NANEA at Department of Epidemiology and Social Medicine, Aarhus University, Aarhus, Denmark. 3 National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention, Atlanta, GA. 4 Department of Pediatrics, Copenhagen University Hospital, Hvidovre, Denmark. *Address correspondence to this author at: Department of Clinical Bio- chemistry, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark. Fax 45-3268-3878; e-mail dh@ssi.dk. Received April 8, 2005; accepted July 12, 2005. Previously published online at DOI: 10.1373/clinchem.2005.052241 5 Nonstandard abbreviations: DBSS, dried blood spot sample(s); TREM-1, triggering receptor expressed on myeloid cells; CRP, C-reactive protein; IL, interleukin; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; BSA, bovine serum albumin; NT-3 and -4, neurotrophin-3 and -4, respectively; MMP, matrix metalloproteinase; sTNF RI, soluble tumor necrosis factor receptor I; BDNF, brain-derived neurotrophic factor; sIL-6r, soluble IL-6 receptor; IFN-, interferon-; MCP-1, monocyte chemoattractant protein-1; GM-CSF, granulocyte/macrophage colony-stimulating factor; MIF, macro- phage migration inhibitory factor; MIP, macrophage inflammatory protein; TGF, transforming growth factor; LOD, lower limit(s) of detection; and MFI, median fluorescence intensity. Clinical Chemistry 51:10 1854 –1866 (2005) Automation and Analytical Techniques 1854