Citations

Abstract Background Platelet dense granule (DG) deficiencies, including Hermansky-Pudlak syndrome, are relatively common and frequently under-diagnosed hereditary platelet hypofunctional disorders. Among various laboratory tests, platelet whole mount (WM) transmission electron microscopy (TEM) is considered the gold standard method for diagnosing DG deficiencies. Nevertheless, platelet DG TEM testing is still largely a research tool and has not been thoroughly validated and standardized as a clinical test. Furthermore, reference ranges (RR) of DG count/platelet (DGC) have not been adequately established. Aim Our goals were to validate and standardize platelet WM TEM testing and establish reference ranges for platelet DGC. Methods Based on previously established methods (Blood, 33:598-606), we first optimized and standardized various pre-analytical, analytical and post-analytical procedures including sample matrix, sample stability, platelet-rich plasma (PRP) preparation, platelet mounting procedure and DG counting criteria. Then whole blood samples in ACD-B tubes were collected from a total of 128 healthy donors (64 males and 64 females). Platelet DGC, percentage of platelets without any DG (empty platelets), platelet count (PC), mean platelet volume (MPV) and immature platelet fraction (IPF) were measured. In addition, platelet aggregometry analyses were performed on citrated PRP samples from the same donors. Standard statistical analyses were employed in this study. Results DGC and platelet ultra-structure were stable when whole blood samples were stored at room temperature (RT) for up to 4 days. Reproducible DGC and adequate preservation of platelet ultrastructure depended on an optimized mounting method. The DG counting criteria were developed based on previous publications and guidance from Dr. James G. White. Using the same WM TEM images and DG counting criteria, agreements among different technologists (n=5) improved from 60% to 95%. Finally, DGC reference range study using 128 healthy donor samples (age range 18, 72 years) and scoring 100-200 platelets from each donor showed that the DGC distribution of each donor sample was left-skewed with a median of 15% of empty platelets (range: 3, 40). The median DGC was 2.6 with a range of 1.0—5.1. Reference ranges established at the lower 95th percentile for DGC empty platelets is ≤ 31% (95% CI 28, 34) and for the mid-95th percentiles of DGCs between 1.5 and 4.0. DGCs were weakly associated with MPV and IPF (ρ between 0.22 and 0.28); and inversely associated with the percentage of empty platelets (ρ between -0.26 and -0.25). DGC showed no significant association with PC, platelet maximum aggregation or primary slope of aggregation, nor age or gender. Conclusion We validated platelet WM TEM procedures and for the first time established an adult reference range for platelet DGC. The results of association analyses implied that immature platelets may have slightly more DGs. Disclosures: No relevant conflicts of interest to declare.

Objective Obesity is a risk factor for colorectal cancer (CRC), accounting for more than 14% of CRC incidence. Microbial dysbiosis and chronic inflammation are common characteristics in both obesity and CRC. Human and murine studies, together, demonstrate the significant impact of the microbiome in governing energy metabolism and CRC development; yet, little is understood about the contribution of the microbiome to development of obesity-associated CRC as compared to individuals who are not obese. Design In this study, we conducted a meta-analysis using five publicly available stool and tissue-based 16S rRNA and whole genome sequencing (WGS) data sets of CRC microbiome studies. High-resolution analysis was employed for 16S rRNA data, which allowed us to achieve species-level information to compare with WGS. Results Characterisation of the confounders between studies, 16S rRNA variable region and sequencing method did not reveal any significant effect on alpha diversity in CRC prediction. Both 16S rRNA and WGS were equally variable in their ability to predict CRC. Results from diversity analysis confirmed lower diversity in obese individuals without CRC; however, no universal differences were found in diversity between obese and non-obese individuals with CRC. When examining taxonomic differences, the probability of being classified as CRC did not change significantly in obese individuals for all taxa tested. However, random forest classification was able to distinguish CRC and non-CRC stool when body mass index was added to the model. Conclusion Overall, microbial dysbiosis was not a significant factor in explaining the higher risk of colon cancer among individuals with obesity.

ObjectiveTo determine whether using image guidance technology with 3‐dimensional image segmentation increases the endoscopic surgeon's accuracy, efficiency, and confidence in identifying the anterior ethmoidal artery.MethodsThis is a cross‐sectional study of attending physicians and residents at an academic medical center. Because identification of the anterior ethmoidal artery during image‐guided surgery can be challenging, we studied the effect of anterior ethmoidal artery image segmentation (ie, partitioning and coloring) on surgeon test performance. A computerized test was administered to 16 surgeons who were asked to identify the anterior ethmoidal artery on multiplanar computed tomographic images and to answer multiple‐choice questions. Half the questions showed segmented images of the anterior ethmoidal artery, and half showed images without segmentation. Efficiency and accuracy of identification and subjective surgeon confidence were determined for each question. Descriptive statistics were used to compare test performance for identification of the anterior ethmoidal artery on images with or without segmentation.ResultsPercentage of correct answers (P <. 001), efficiency (P <. 001), and confidence (P <. 001) in identification of the anterior ethmoidal artery were significantly better with segmented computed tomographic images.DiscussionWe demonstrated that use of segmented images improves surgeons’ accuracy, confidence, and efficiency for identification of the anterior ethmoidal artery.Implications for PracticeWe describe how segmentation can allow surgeons to improve the surgical course by increasing their accuracy, confidence, and efficiency in identifying the anterior ethmoidal artery.

Platelet-arterial wall interaction appears to be important in the genesis of atherosclerosis. Pigs with homozygous von Willebrand’s disease (vWd) appear resistant to atherosclerosis. We investigated whether there is impairment in platelet-arterial wall interaction in porcine vWd. Superficial endothelial injury was produced in a 4 cm segment of carotid artery by drying for 3 minutes in a stream of air which entered one end (100 ml/min) and exited through a small hole in the other end. This procedure was performed in 9 normal and 6 vWd pigs 24 hours after the I.V. infusion of III-Indium-labeled platelets. Carotid blood flow was re-established and the pigs were sacrificed 24 hours later and the carotids fixed in glutaraldehyde.The ratio of radioactivity (cpm/g) of injured/noninjured carotid arteries was markedly decreased in the vWd pigs. In addition, transmission and scanning electron microscopy confirmed superficial endothelial damage in all injured segments, but only significant platelet deposition in the normal pig injured segments.After a mild endothelial injury, this “in vivo” evidence of impaired platelet-arterial wall interaction in vWd may be related to the resistance to atherosclerosis in these animals.

Abstract Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier function and increases susceptibility to infections. Herein, we hypothesized that somatic mutations together with cigarette smoke create a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from controls (n=33) or cancer cases (n=143), we conducted 16S rRNA gene sequencing (MiSeq), with RNA-seq data from lung cancer cases in The Cancer Genome Atlas (n=1112) serving as the validation cohort. We demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue, indicating a shift in the overall microbial community in lung cancer patients as compared to those without cancer. Lung cancer cases were classified by the relative abundance of two taxa, Variovorax and Streptococcus, with an increase in Variovorax abundance in tumors as compared to non-tumor adjacent paired lung tissue (FDR corrected P=0.013). The species of Variovorax was identified histologically, and also by two additional 16S rRNA strategies (Resphera Insight analysis and PacBio sequencing). A group of taxa were associated with squamous cell carcinoma (SCC), of which Acidovorax were enriched in smokers (P =0.0013). Further, these taxa, including Acidovorax, exhibited higher abundance among the subset of SCC cases with TP53 mutations, an association not seen in adenocarcinomas (AD). Therefore, we observed a microbiome-gene and a microbiome-exposure interaction in SCC lung cancer tissue. Together, these results open the door for future biomarker research that could be used to improve screening and direct mechanistic studies of lung cancer therapy. Citation Format: Leigh Greathouse, James White, Valery Bliskovsky, Ashley Vargas, Eric Polley, Elise Bowman, Mohammed Khan, Ana Robles, Brid Ryan, Amiran Dzutsev, Giorgio Trinchieri, Marbin Pineda, Sven Bilke, Paul Meltzer, Marina Walther-Antonio, Garth Ehrlich, Joshua Mell, Joshua Earl, Sergey Balashov, Archana Bhat, Alex Valm, Clayton Deming, Sean Conlan, Julia Oh, Julie Segre, Curtis Harris. Microbiome-TP53 gene interaction in human lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4925. doi:10.1158/1538-7445.AM2017-4925