Project description:INTRO: The ongoing challenge of accurately diagnosing infection in the ICU motivates a search for novel molecular diagnostics. In the current study, we tested the hypothesis that the informational content of circulating leukocytes differs, thereby allowing one to rank leukocyte populations on their potential to contribute to RNA diagnostics for pneumonia. METHODS: Sixteen patients (10 male, 6 female) at risk for VAP were entered into our IRB-approved study that collects blood and clinical data every 48 hours for up to 21 days. Four of the sixteen patients developed VAP as diagnosed and treated by the attending ICU physician. Previously reported blood protocols were used to isolate buffy coat, enriched neutrophil, and enriched monocyte populations by using negative selection. Cellular purity was assessed by FACS for one of the 4 VAP patients. Genome-wide expression analysis was performed on RMA-normalized signal from Affymetrix U133 2.0 Plus GeneChips. EDGE software (FDR=0.10) was used to determine changes in mRNA abundance over time for each cell population. RESULTS: During the 5-day window in which each of the four patients (all males) developed VAP, significant changes in gene expression were observed (Table 2), but the information content (number of genes altered) varied across leukocyte populations. These differences were not due to signal variance (coefficient of variation, CV) or differences in the number of samples available for analysis. Moreover, only 0.6% of the monocyte gene list overlaps with the neutrophil list, arguing that neutrophil contamination of monocyte populations is insufficient to explain the 40-fold difference in gene number. CONCLUSIONS: Enriched monocyte populations from circulating blood provide far more genes for study of the host response to VAP than the other 2 cell populations studied. This information should be considered when designing blood gene expression profiling experiments in patients at risk for sepsis. Keywords: time course Four patients over several timepoints measured three different cell types for VAP signal

Project description:INTRO: The ongoing challenge of accurately diagnosing infection in the ICU motivates a search for novel molecular diagnostics. In the current study, we tested the hypothesis that the informational content of circulating leukocytes differs, thereby allowing one to rank leukocyte populations on their potential to contribute to RNA diagnostics for pneumonia. METHODS: Sixteen patients (10 male, 6 female) at risk for VAP were entered into our IRB-approved study that collects blood and clinical data every 48 hours for up to 21 days. Four of the sixteen patients developed VAP as diagnosed and treated by the attending ICU physician. Previously reported blood protocols were used to isolate buffy coat, enriched neutrophil, and enriched monocyte populations by using negative selection. Cellular purity was assessed by FACS for one of the 4 VAP patients. Genome-wide expression analysis was performed on RMA-normalized signal from Affymetrix U133 2.0 Plus GeneChips. EDGE software (FDR=0.10) was used to determine changes in mRNA abundance over time for each cell population. RESULTS: During the 5-day window in which each of the four patients (all males) developed VAP, significant changes in gene expression were observed (Table 2), but the information content (number of genes altered) varied across leukocyte populations. These differences were not due to signal variance (coefficient of variation, CV) or differences in the number of samples available for analysis. Moreover, only 0.6% of the monocyte gene list overlaps with the neutrophil list, arguing that neutrophil contamination of monocyte populations is insufficient to explain the 40-fold difference in gene number. CONCLUSIONS: Enriched monocyte populations from circulating blood provide far more genes for study of the host response to VAP than the other 2 cell populations studied. This information should be considered when designing blood gene expression profiling experiments in patients at risk for sepsis. Keywords: time course

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National Cancer Institute "Cancer Moonshot Biobank"

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