Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Circulating tumor DNA dynamics and recurrence risk in patients undergoing curative intent resection of colorectal cancer liver metastases: A prospective cohort study.

BackgroundIn patients with resectable colorectal liver metastases (CRLM), the role of pre- and postoperative systemic therapy continues to be debated. Previous studies have shown that circulating tumor DNA (ctDNA) analysis, as a marker of minimal residual disease, is a powerful prognostic factor in patients with nonmetastatic colorectal cancer (CRC). Serial analysis of ctDNA in patients with resectable CRLM could inform the optimal use of perioperative chemotherapy. Here, we performed a validation study to confirm the prognostic impact of postoperative ctDNA in resectable CRLM observed in a previous discovery study.Methods and findingsWe prospectively collected plasma samples from patients with resectable CRLM, including presurgical and postsurgical samples, serial samples during any pre- or postoperative chemotherapy, and serial samples in follow-up. Via targeted sequencing of 15 genes commonly mutated in CRC, we identified at least 1 somatic mutation in each patient's tumor. We then designed a personalized assay to assess 1 mutation in plasma samples using the Safe-SeqS assay. A total of 380 plasma samples from 54 patients recruited from July 2011 to Dec 2014 were included in our analysis. Twenty-three (43%) patients received neoadjuvant chemotherapy, and 42 patients (78%) received adjuvant chemotherapy after surgery. Median follow-up was 51 months (interquartile range, 31 to 60 months). At least 1 somatic mutation was identified in all patients' tumor tissue. ctDNA was detectable in 46/54 (85%) patients prior to any treatment and 12/49 (24%) patients after surgery. There was a median 40.93-fold (19.10 to 87.73, P ConclusionsWe confirmed the prognostic impact of postsurgery and posttreatment ctDNA in patients with resected CRLM. The potential utility of serial ctDNA analysis during adjuvant chemotherapy as an early marker of treatment efficacy was also demonstrated. Further studies are required to define how to optimally integrate ctDNA analyses into decision-making regarding the use and timing of adjuvant therapy for resectable CRLM.Trial registrationACTRN12612000345886

Figure S5 from The Origin of Highly Elevated Cell-Free DNA in Healthy Individuals and Patients with Pancreatic, Colorectal, Lung, or Ovarian Cancer

Supplemental Figure 5. Methylation profiles using quadratic programming vs. non-negative least- squares regression using the reference matrix described in Moss et al. (43). Pearson’s correlation coefficient and p values are presented at the bottom of this figure, showing the derived contributions from each of the 25 tissue types that could be assessed.</p

Figure S1 from The Origin of Highly Elevated Cell-Free DNA in Healthy Individuals and Patients with Pancreatic, Colorectal, Lung, or Ovarian Cancer

Supplemental Figure 1. Overview of the patient samples included in the present study.</p

Figure S8 from The Origin of Highly Elevated Cell-Free DNA in Healthy Individuals and Patients with Pancreatic, Colorectal, Lung, or Ovarian Cancer

Supplemental Figure 8. The relative contribution of cfDNA from various tissues before and ~24 hours after surgery for pancreatic cancer. * p < 0.05, ** p < 0.01, *** p < 0.001</p

Figure S7 from The Origin of Highly Elevated Cell-Free DNA in Healthy Individuals and Patients with Pancreatic, Colorectal, Lung, or Ovarian Cancer

Supplemental Figure 7. The amount of total cfDNA before and after surgery in patients with pancreatic cancer.</p