Twenty-one healthy NCs and 21 patients diagnosed with BPD according to the criteria of DSM-IV15 (Table 1) provided informed consent as approved by the institutional review board at McLean Hospital, Belmont, Mass. Samples from patients with BPD and NCs were collected over the course of 6 months, and batches of BPD samples were matched with NC samples. Lymphocytes from 10 to 30 mL of freshly drawn blood were separated by centrifugation using Histopaque columns (Sigma-Aldrich, St Louis, Mo), washed 3 times, and split into 3 batches. One batch, containing two thirds of all cells, was frozen at −80°C and later subjected to gene expression microarray analysis, whereas 1 smaller batch each was cultured in either regular RPMI-1640 medium or low-glucose RPMI-1640 medium (25% normal glucose content; 0.5 g/L) for a period of 5 days, after which cells were frozen at −80°C. After sample collection was concluded, RNA was extracted from each batch (RNagents kit; Promega, Madison, Wis), complementary DNA (cDNA) was synthesized from 4 μg of RNA from fresh lymphocytes (SuperScript double-stranded cDNA synthesis kit; Invitrogen Corp, Carlsbad, Calif) or 1 μg of RNA from cultured lymphocytes (MessageAmp II-96 kit; Ambion, Austin, Tex), and biotinylated RNA was synthesized from cDNA (for fresh lymphocytes, Enzo IVT kit; Enzo Biochem, Farmingdale, NY; for cultured lymphocytes, MessageAmp II-96 kit). Biotinylated RNA was fragmented and hybridized to the HG-U133A 2.0 array (Affymetrix, Santa Clara, Calif) overnight at 45°C and stained on a washing station with 2 rounds of streptavidin-phycoerythrin (Molecular Probes, Eugene, Ore) separated by a round of biotinylated antistreptavidin antibody (Vector Laboratories, Burlingame, Calif) as described previously.9,16 All of the fresh-frozen lymphocytes were worked up in 1 batch for gene array experiments. All of the cultured lymphocytes were worked up together in a separate batch with an improved protocol developed during the course of this project, for which the amount of input RNA could be lowered from 4 μg to 1 μg. Because of the small sample sizes and the variable amount of lymphocytes yielded from individual probands, a number of samples did not yield enough mRNA for gene array analysis (Table 1). The number of samples per group ranged from 10 to 17.