Targeted transcriptomic analysis of pancreatic adenocarcinoma in EUS-FNA samples by NanoString technology.

Background: Integration of transcriptomic testing into EUS-FNA samples is a growing need for precision oncology in pancreatic ductal adenocarcinoma (PDAC). The NanoString platform is suitable for transcriptome profiling in low yield RNA samples. Methods: Inclusion of patients that underwent EUS-FNA cytological diagnosis of pancreatic ductal adenocarcinoma using 19G and/or 22G needles and subsequent surgical resection. Formalin-fixed, paraffin-embedded (FFPE) cytological and surgical samples underwent RNA extraction and transcriptomic analysis using a custom 52-gene NanoString panel of stromal PDAC features. Cell type abundance was quantified in FFPE specimens and correlated. Results: 18 PDAC patients were included. Mean EUS-FNA passes was 2 + 0.7. All FFPE passed the RNA quality control for genomic analysis. Hierarchical clustering on the global gene expression data showed that genes were differentially expressed between EUS and surgical samples. A more enriched cancer-associated fibroblasts and epithelial-mesenchymal transition transcriptomic profile was observed across surgical specimens whereas immunological biomarkers were more represented in EUS-FNA samples. Cytological examination confirmed a scanty representation of CAF and more immunological cell abundance in cytological samples in comparison to surgical specimens. Conclusion: Targeted transcriptomic NanoString profiling of PDAC samples obtained by EUS-FNA is a feasible approach for pre-surgical molecular analysis although stromal CAF/EMT mRNA biomarkers are underrepresented.

Comparative study of ethanol levels in blood versus bone marrow, vitreous humor, bile and urine.

Post-mortem ethanol levels in blood were compared to corresponding levels in rib bone marrow, vitreous humor, urine and bile. In forensic toxicology, a good correlation between blood and a tissue or body fluid is needed to estimate a blood alcohol concentration when blood is unavailable or contaminated. In this study, direct injection and headspace gas-chromatographic techniques were employed to quantitate the ethanol concentrations. Comparable findings by these two techniques showed a reproducibility of results. When the determined bone marrow ethanol levels were corrected for the lipid fraction, a consistent correlation could be established between ethanol levels in blood and bone marrow. The relationship (linearity and ratio range) between ethanol levels in blood and corrected levels in bone marrow was better than that between blood and vitreous humor, bile or urine. This study showed that blood ethanol levels can be predicted by extrapolating the corrected rib bone marrow ethanol level.

The stability of several compounds in formalin fixed tissues and formalin-blood solutions.

Buffered formalin solutions were added to spiked blood samples containing diazepam, phenytoin, carbon monoxide and cyanide to give formalin-whole blood solutions of 5 and 8%. Sections of liver positive for desipramine, phenobarbital and phenytoin were placed in separate 5 and 8% formalin-water solutions. The formalin-blood solutions were monitored daily for 30 days, while the fixed liver and formalin-water samples were analyzed once a week for 4 weeks. In the formalin-blood solutions losses were found for diazepam and phenytoin over the 30-day period of at least 41% and 33%, respectively. Cyanide detection was not possible immediately after the addition of formalin and the presence of carboxyhemoglobin was difficult to detect after 1 week. In the liver, losses of phenobarbital and desipramine were greater than 60% while phenytoin showed little change. This study has revealed that the drugs examined at toxic concentrations can be detected, with variable recoveries, for up to 30 days after fixation with formalin. However, quantitative analysis for cyanide and carboxyhemoglobin may be significantly impaired in the presence of formaldehyde.

An assay evaluation of the methylene blue method for the detection of anionic surfactants in urine.

Adding detergent to urine intended for drug testing is one of many ways to adulterate the specimen. This modified methylene blue procedure allows the detection and quantitation of anionic surfactants in urine. One-hundred urine specimens that exhibited normal foaming when shaken gave anionic surfactant values lower than 36 microg/mL with a mean of 8.73 microg/mL. Most of the suspected adulterated specimens and spiked samples with only 100 microL of detergent in 60 mL of urine had values greater than 750 microg/mL. Based on the analysis of negative samples, a urine specimen with an anionic surfactant level of 100 microg/mL or greater could be considered adulterated and most likely will have levels greater than 800 microg/mL.

Fatal strychnine ingestion.

A postmortem case involving ingestion of a strychnine-containing preparation is reported. Strychnine levels were determined in blood, urine, bile, liver, kidney, stomach contents, small and large intestines, and brain. The procedure, which is sensitive and specific, employs a gas-liquid chromatograph equipped with a flame ionization detector (FID). The extraction procedure using 5 mL of body fluids or tissue homogenate is described in detail. This is the first strychnine mortality in Allegheny County within a period of 20 years.

The validity of urine alcohol analysis in drunk drivers.

Blood and breath are the specimens of choice for determining alcohol levels. A random urine specimen may not reflect a blood level because of the influences due to the stage of absorption, the quantity of urine in the bladder, and the frequency of urination. A urine sample may accurately reflect a blood level only 30 minutes after the bladder is completely emptied. Individual states that permit urinalysis for alcohol must provide procedures for sample collection and statutory limit levels.

Blood alcohol concentrations: factors affecting predictions.

As a result of extensive alcohol research conducted on both humans and animals, it is possible to predict a BAC, given pertinent data. In addition, it is possible to estimate from a given BAC the quantity of alcohol consumed. Caution must be used in these predictions, for certain factors will affect the final estimation. Absorption of alcohol is influenced by gastrointestinal contents and motility, and also the composition and quantity of the alcoholic beverage. The vascularity of tissues influences the distribution of alcohol, and their water content will determine the amount of alcohol present after equilibrium. Elimination of alcohol begins immediately after absorption. The elimination rate varies for individuals but falls between .015 percent to .020 percent per hour, with an average of .018 percent per hour. In addition to these factors, a BAC will depend on the subject's weight, percentage of alcohol in the beverage, and the rate of drinking. The principal effect of alcohol in the body is on the central nervous system. Its depressant effect consists of impairment to sensory, motor and learned functions. When combined with some other drugs, a more intoxicated state occurs. Although tolerance to alcohol at low blood concentrations is possible, the tolerance most noted is a learned tolerance among chronic drinkers. contamination of antemortem blood samples collected for alcohol analysis is minimal when swabbing with an ethanolic antiseptic is performed with routine clinical technique; sloppy swabbing has been shown to increase the BAC determination significantly. The alcoholic content of blood used for transfusion does not contribute significantly to the BAC of the recipient, since extensive dilution occurs; nor does the alcohol present in injectable medication contribute significantly. Although many factors may alter the concentration of alcohol present in autopsy specimens, postmortem synthesis of alcohol receives the most attention. The microorganisms that cause postmortem ethanol production can be inhibited by adding a preservative to the samples and storing them under refrigeration. Should putrefaction be present, it is recommended that, in addition to blood, several different specimens be collected and analyzed for the presence of alcohol. Antemortem blood samples containing ethanol, collected using sterile tubes and techniques, may be analyzed up to 14 days later with reasonable certainty that the ethanol level reflects that which was present at the time of collection.