Case ascertainment. We identified indi- viduals with major psychosis within the NE-NCPP cohorts through a two-stage diag- nostic assessment procedure (Figure 1). In the ﬁrst stage, we identiﬁed cohort members with possible psychotic illness through record link- ages with public hospitals, mental health clin- ics, and the Massachusetts and Rhode Island Departments of Mental Health, and from personal interviews. Using record linkage and personal inter- views, we identiﬁed 241 potential cases. In the second stage, we located these 241 subjects through a variety of methods, including searches of credit bureaus, address directories, death certiﬁcates, motor vehicle reports, and home visits. Those who consented to partici- pate in follow-up efforts were interviewed by a trained interviewer using the Structured Clinical Interview for the Diagnostic and Statistical Manual for Mental Disorders, 4th edition (DSM-IV) (American Psychiatric Association 2000), to determine lifetime prevalence of psychotic disorders as well as other mental disorders. Based on interview data and medical record review, trained PhD- and MD-level diagnosticians then completed best-estimate consensus diagnoses according to DSM-IV criteria. In the NE-NCPP, diagnos- tic interviews were completed for 167 subjects; medical charts alone were available for the remaining 74 subjects. We drew the samples used in this study from subjects with schizophrenic psychoses and matched controls. Of the 241 potential subjects with psychosis, 111 subjects were determined to have a major psychotic disorder. Of these subjects, 51 were diagnosed with schizophrenic psychoses [schizophrenia (n = 47) or schizoaffective disorder (n = 4)]. A total of 27 cases (25 diagnosed with schizophrenia and 2 diagnosed with schizoaffective disorder) had serum samples available for this analysis. For each case subject, we selected two healthy controls matched for sex, race/ethnicity, and date of birth. We selected controls from an unaffected subset of subjects with no Axis I psychiatric diagnoses. Overall, procedures for case ascertainment were similar to those used in the PDS study, including the use of consensus diagnostic pro- cedures based on DSM-IV criteria. In the PDS study, a broader case deﬁnition was used for schizophrenia spectrum disorder, whereas the NE-NCPP included only schizophrenia and schizoaffective disorder. However, matching criteria for selecting controls in the NE-NCPP differed from the PDS study. Specifically, although both the NE-NCPP and PDS study matched on sex and date of birth, only the NE-NCPP matched on maternal ethnicity, and only the PDS study included the length of time subjects were in the cohort and the avail- ability of maternal serum. Prenatal lead and schizophrenia: further evidence Serum samples in the NE-NCPP. Women were registered in their ﬁrst clinic or hospital visit, and in addition to detailed records on a variety of demographic characteristics of the parents and obstetric health, maternal blood was drawn every trimester and at birth (Buka et al. 2001). We obtained aliquots of 1 mL of maternal serum from both cases and controls, diluted 1:5 in phosphate-buffered saline and frozen at –20°C. We used third-trimester samples for 27 cases of schizophrenia spec- trum disorder and 54 matched controls. Although seasonal variation has been noted to cause interindividual variation in third- trimester BPb levels, published data indicate that variability between the second- and third-trimester BPb levels is generally limited. However, there is some indication that Pb levels may increase toward the end of the third trimester. These ﬂuctuations, when cor- rected using hematocrit levels to account for the increases in ﬂuid volume, range from 0 to 1 µg/dL (Gulson et al. 2004). Laboratory protocol for NE-NCPP. Procedures for analysis of δ-ALA were similar to those used previously in analysis of samples from the PDS study. In brief, we thawed frozen serum samples and centrifuged them for 30 min at 14,000 rpm at 4°C. We removed an aliquot of supernatant and added it to 3.0 mL of acetylacetone reagent and 0.45 mL of 37% formaldehyde. Aliquot volumes ranged from 50 to 500 µL, based on dilution and starting volume. We loosely capped tubes and heated them on an aluminum alloy block heater in a darkroom for 40 min and then removed them from the heat to a test tube rack to cool in the darkroom at room temper- ature for 4 hr. We removed supernatant from the test tubes to Eppendorf tubes and cen- trifuged the tubes for 30 min in a 4°C cool room. We then filtered the supernatant and analyzed 750 µL using a PerkinElmer model LC-250 equipped with an LC-600 autosam- pler and an LC-40 fluorescence detector (PerkinElmer, Norwalk, CT) with separation conditions as previously reported. 40 30 y c n ue20 q e r F 10 A Exposure definition and statistical methods. We classified subjects according to previously validated methods, that is, having a BPb of ≥ 15 µg/dL or < 15 µg/dL, based on a cutoff of 9.05 ng/mL δ-ALA (Opler et al. 2004). As discussed in our prior publication (Opler et al. 2004), we found this method to have a moderate correlation (0.64) when used as a continuous measure, but when used cate- gorically to predict BPb levels above or below 15 µg/dL, it was highly sensitive (90–91%) with positive predictive values of 89–91%. A calculated kappa statistic for duplicate samples was 0.89 with an SE of 0.23, indicating excellent agreement. Using this method and exposure defini- tion, we ﬁtted conditional logistic regression models and estimated odds ratios (ORs) relat- ing exposure status to a diagnosis of schizo- phrenia spectrum disorder. We combined results from the NE-NCPP with previously collected data from a nested sample of 119 subjects (44 cases and 75 controls) from the PDS study, described in detail elsewhere (Opler et al. 2004). The combined samples from the PDS study and NE-NCPP totaled 200 subjects (71 cases and 129 controls). To examine the relationship between δ-ALA and schizophrenia spectrum disorders in the com- bined samples, we used two approaches. First, we ﬁtted conditional logistic regression mod- els, including δ-ALA exposure as a predictor of schizophrenia spectrum disorder, while adjust- ing for covariates. Next, we ﬁtted a multilevel random intercept logistic regression model using generalized linear latent and mixed models (GLLAMM) software within the STATA statistical package (StataCorp, College Station, TX) to account for the multilevel structure of the data (Rabe-Hesketh et al. 2004). Thus, we took into account the poten- tial correlations within each “site” (i.e., PDS study vs. NE-NCPP). We assessed potential confounders based on their known association with Pb exposure and/or schizophrenia, including maternal age at delivery, paternal age at delivery, parental 40 30 y c n ue20 q e r F 10 B 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Prenatal sera δ-ALA (ng/mL) 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Prenatal sera δ-ALA (ng/mL) Figure 1. Serum δ-ALA distributions for NE-NCPP (mean ± SD = 11.6 ± 15.21; n = 81) (A) and PDS (mean ± SD = 9.0 ± 9.80; n = 119) (B) samples. Environmental Health Perspectives • VOLUME 116 | NUMBER 11 | November 2008 1587