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Fecal immunochemical tests (FIT) are used to screen for colorectal cancer by detecting blood present in stool. Patients collect FIT specimens at home in a sampling kit and return them to the lab for testing. At our institution, patients are instructed to return their specimens to the laboratory within seven days from collection, which is shorter than the manufacturer stated room temperature (RT) stability of 15 days. The objective of this study was to assess and verify the stability of FIT specimens at RT and to determine if refrigerated storage improves stability. A series of experiments were performed with the OC-Sensor DIANA iFOB Test system between 2017 and 2019, using a positive clinical cut-off of 75 ng/mL (15 µg/g) hemoglobin (Hb). Specimens were collected and categorized based on their initially measured Hb concentration and had repeated measurements for up to 21 days following collection. FIT specimens were stored either at RT or refrigerated. Our results show that FIT specimens have reduced concentrations of Hb compared to baseline when stored at RT; refrigeration improved FIT specimen stability but did not completely prevent the reduction in Hb concentration. Additionally, specimens marginally above the cut-off (initial concentrations between 75-100 ng/mL (15-20 µg/g)) that were stored at RT showed 100% positivity on the day of collection (n=33), 63% on Day 3 (n=19), 46% on Days 4/5 (n=26), and 38% on Days 6/7 (n=26). Finally, specimens with Hb values near the clinical cut-off appear to be particularly susceptible to false negatives as a result of the reduction in Hb over time. Therefore, laboratories should verify the specifics of their FIT tests before offering it to patients to reduce false negatives.

Background Serial differences between intrapatient consecutive measurements can be transformed into Taylor series of variation vs time with the intersection at time = 0 (y0) equal to the total variation (analytical + biological + preanalytical). With small preanalytical variation, y0, expressed as a percentage of the mean, is equal to the variable component of the reference change value (RCV) calculation: (CVA2 + CVI2)1/2. Methods We determined the between-day RCV of patient data for 17 analytes and compared them to healthy participants’ RCVs. We analyzed 653 consecutive days of Dartmouth-Hitchcock Roche Modular general chemistry data (4.2 million results: 60% inpatient, 40% outpatient). The serial patient values of 17 analytes were transformed into 95% 2-sided RCV (RCVAlternate), and 3 sets of RCVhealthy were calculated from 3 Roche Modular analyzers’ quality control summaries and CVI derived from biological variation (BV) studies using healthy participants. Results The RCVAlternate values are similar to RCVhealthy derived from known components of variation. For sodium, chloride, bicarbonate calcium, magnesium, phosphate, alanine aminotransferase, albumin, and total protein, the RCVs are equivalent. As expected, increased variation was found for glucose, aspartate aminotransferase, creatinine, and potassium. Direct bilirubin and urea demonstrated lower variation. Conclusions Our RCVAlternate values integrate known and unknown components of analytic, biologic, and preanalytic variation, and depict the variations observed by clinical teams that make medical decisions based on the test values. The RCVAlternate values are similar to the RCVhealthy values derived from known components of variation and suggest further studies to better understand the results being generated on actual patients tested in typical laboratory environments.

Objectives The level of glycated hemoglobin A (HbA1C) in blood is the preferred marker for diabetes monitoring and treatment. Here, we evaluate the analytical performance of the Roche Diagnostics Cobas c 513, a stand-alone HbA1C immunoassay analyzer. Design and Methods Performance was assessed with regards to imprecision, accuracy, linearity, method comparison against the Roche Cobas Integra 800 CTS, specimen stability, interference from common hemoglobin variants and hemoglobin F, and throughput. Results Within-run and between-run precisions were 0.5–0.7 and 0.8–1.3%CV, respectively. An average bias of −1.6% to proficiency survey samples was observed. The c 513 correlated well with the Integra (slope = 0.94, y-intercept = 0.50, and correlation coefficient = 0.998). The effect of hemoglobin variants on this assay was negligible while specimens containing ≥10% HbF demonstrated a negative bias. The c 513 instrument can process up to 340 samples per hour. Conclusions The c 513 is a precise, accurate, automated high throughput analyzer for measuring HbA1C in large laboratories.

We used the intragastric feeding rat model for alcoholic liver disease to evaluate the relationship between morphologic and functional indicators of endothelial cell dysfunction.Twelve groups of rats (4-5 rats/group) were fed the following diets: saturated fat and dextrose (SD), saturated fat and ethanol (SE), corn oil and dextrose (CD), corn oil and ethanol (CE). Four of the 12 groups were sacrificed at 2 weeks, four groups at 4 weeks and remaining four groups at 8 weeks. The following were evaluated at sacrifice: pathologic changes in the liver, endothelial cell proliferation using a monoclonal antibody to proliferating cell nuclear antigen, factor VIII-related antigen staining of endothelial cells in liver, plasma endotoxin, hyaluronan and prostaglandin F2 alpha.Only CE rats at 4 and 8 weeks showed pathologic changes. The plasma levels of HA were significantly higher in the CE groups compared to the other groups at all time intervals studied. In the CE rats, a significant correlation was obtained between plasma endotoxin and hyaluronan (r = 0.84, p0.01). Endotoxin levels also correlated significantly with the number of G1/S arrested hepatic sinusoidal endothelial cell (r = 0.61, p0.05). A role for prostaglandin F2 alpha, in causing endothelial dysfunction, was suggested by a significant correlation between plasma hyaluronan and prostaglandin F2 alpha levels (r = 0.95, p0.01). Positive factor VIII related antigen staining of hepatic endothelial cells was seen in rats with high plasma hyaluronan levels.We propose that endotoxin, mediating part of its effect through prostaglandin F2 alpha, plays a role in hepatic sinusoidal endothelial cell G1/S arrest. This morphologic change, associated with increased plasma hyaluronan levels, precedes capillarization in this model of alcoholic liver injury.

Background & Aims: Regulation of blood flow and oxygen supply are important pathogenetic factors in alcoholic liver disease. Because nitric oxide may have an important role, its effects on alcoholic liver injury were investigated. Methods: Rats were fed ethanol intragastrically with either saturated fat or corn oil. Spontaneous production of NO by liver nonparenchymal cells was compared in the two dietary groups. Two additional groups of rats fed corn oil and ethanol were treated with either an NO inhibitor (l-NAME) or supplemented with l-arginine. Liver pathology and plasma NO production were evaluated. Results: In the corn oil and ethanol group, a progressive decrease in liver nonparenchymal cell NO production and increased plasma NO levels were associated with liver injury. Reduced nicotinamide adenine dinucleotide phosphate diaphorase staining showed increased centrilobular staining of hepatocytes in the corn oil and ethanol group and l-NAME-treated group. Moreover, l-NAME increased the severity, whereas l-arginine supplementation completely prevented liver injury. In the saturated fat and ethanol group, in which there was no liver injury, the levels of NO 2 − in nonparenchymal supernatant were 5–10-fold higher than in the corn oil and ethanol group. Conclusions: Decreased NO production by nonparenchymal cells may contribute to liver injury in ethanol-fed rats, and the compensatory increase in hepatocyte NO production may contribute to centrilobular liver injury.

Capillarization of the hepatic sinusoid occurs in alcoholic liver disease. Because endothelial cell proliferation is relevant to capillarization, we used the intragastric feeding rat model to evaluate the relationship between pathological liver injury and endothelial cell proliferation.Male Wistar rats (225-250 g) were fed liquid diets containing corn oil and ethanol for periods ranging between 1 week and 2 months. At the time the rats were killed, the severity of pathological injury and endothelial cell proliferation using anti-rat proliferating cell nuclear antigen/antibody was evaluated.Two distinctly different populations of proliferating sinusoidal lining cells were identified; one population showed relatively weak granular staining consistent with cells arrested at the G1/S boundary. The other population of cells showed strong granular staining of the nucleoplasm and nucleoli (cells in mid to late S phase). A strong correlation (r = 0.85; P0.01) was obtained between pathological severity and G1/S-arrested endothelial cells. There was no correlation between cells in S phase.The presence of an increased number of G1/S-arrested endothelial cells in animals with severe pathological change suggests that stimuli are present for both endothelial cell proliferation and G1/arrest. The identification of these stimuli could lead to a better understanding of the pathogenesis of alcoholic liver disease.

We evaluated the expression of interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha and transforming growth factor-beta mRNAs in the intragastric-feeding rat model of alcoholic liver disease. Rats were fed different diets for periods of 2 or 4 wk. Animals fed saturated fat and ethanol and the corn oil-dextrose control group had no liver injury, whereas animals fed corn oil and ethanol showed pathologic changes. RNA was extracted from the livers at the time of killing, reverse-transcribed and amplified; polymerase chain reaction products were subjected to electrophoresis on agarose gel. Interleukin-1 alpha mRNA was present in all groups at 2 and 4 wk; interleukin-1 beta and transforming growth factor-beta mRNAs were present in all groups at 4 wk. Tumor necrosis factor-alpha mRNA was absent in all groups at 2 wk but was present in the corn oil-ethanol group only at 4 wk. Because pathological liver injury was evident in the corn oil-ethanol group by 4 wk, the presence of tumor necrosis factor-alpha mRNA at this time suggests a pathogenetic role for tumor necrosis factor-alpha in alcohol-induced liver injury.

We investigated the association between vitamin E, lipid peroxidation and eicosanoid production in experimental alcoholic liver injury. We used the intragastric feeding rat model in which animals were fed corn oil and ethanol (CO+E) and corn oil and dextrose (CO+D) for 2 and 4 week periods. At sacrifice, we measured plasma levels of alpha-tocopherol, 8-isoprostane, thromboxane B2 (TXB2) and 6-ketoprostaglandin F1 alpha (6-KetoPGF1 alpha). Animals fed CO+E had significantly lower concentrations of alpha-tocopherol and higher concentrations of 8 isoprostane at both 2 and 4 weeks. a significant inverse correlation was seen between alpha-tocopherol concentrations and the TXB2: PGF1 alpha ratio (r = 0.72, p0.01). A positive correlation was seen between the TXB2: PGF1 alpha ratio and 8 isoprostane levels (r = 0.84, p0.001). These results suggest that vitamin E depletion and enhanced lipid peroxidation may affect eicosanoid metabolism in experimental alcoholic liver disease in such a way so as to increase the thromboxane to prostacyclin ration.

We evaluated the role of changes in microsomal phospholipases (A and C) and arachidonic acid in the intragastric rat feeding model. The experimental animals (male Wistar rats), divided into 4-5 rats/group, were fed the following diets: corn oil and ethanol and corn oil plus dextrose. One set of groups was killed after 2 weeks of feeding, and the second set was killed after 1 month. For each animal, microsomal analysis of cytochrome P-450 2E1 (CYP 2E1) and fatty acids was done. Fourteen animals had analyses of phospholipase C (PLC) and phospholipase A (PLA), and 10 animals had measurements of conjugated dienes. A significant correlation was obtained between the level of CYP 2E1 and the decrease in arachidonic acid (AA) from baseline levels (r = 0.69, p < 0.01). The decrease in AA also correlated with the increase in conjugated dienes (r = 0.70, p < 0.05). PLA and PLC activities were both significantly increased in the corn oil and ethanol groups. The activity of PLC correlated with the decline in AA (r = 0.69, p < 0.01). The correlations noted between the decrease in microsomal AA and CYP 2E1 induction and conjugated diene formation suggest that these processes may be interlinked especially in regard to generation of lipid peroxides that may play a role in alcoholic liver injury.

Background & Aims: Lipid peroxidation may be important in the pathogenesis of alcoholic liver injury. The purpose of this study was to determine whether a saturated fatty acid-based therapy (palm oil) could decrease lipid peroxidation and alcoholic liver injury during ethanol withdrawal. Methods: Three groups of male Wistar rats (5 rats/group) were studied. Rats in group 1 were fed a fish oil-ethanol diet for 6 weeks; rats in groups 2 and 3 were fed a fish oil-ethanol diet for 6 weeks before treatment with fish oil-dextrose (group 2) or palm oil-dextrose (group 3) for 2 weeks. Liver samples were analyzed for histopathology, lipid peroxidation, fatty acid composition, cytochrome P450 2E1 activity, and tocopherol levels. Results: By 6 weeks, all rats had developed fatty liver, inflammation, and necrosis. Group 2 showed minimal histological improvement, whereas group 3 showed near normalization of the histology. The improvement in group 3 was associated with decreased lipid peroxidation and P450 2E1 activity. Higher levels of ω-3 fatty acids were detected in group 2 than group 3. Tocopherol levels were similar among the groups. Conclusions: A diet enriched in saturated but not unsaturated fatty acids reversed alcoholic liver injury. This effect may be explained by down-regulation of lipid peroxidation.

We evaluated the role of changes in cytochrome P-450 2E1 (CYP 2E1) and lipid peroxidation in relation to development of severe liver injury in fish oil-ethanol-fed rats. The experimental animals (male Wistar rats) were divided into 5 rats/group and were fed the following diets for 1 month: corn oil and ethanol (CO+E) or corn oil and dextrose (CO+D), and fish oil and ethanol (FO+E) or fish oil and dextrose (FO+D). For each animal, microsomal analysis of CYP 2E1 protein, aniline hydroxylase activity, fatty acid composition, and conjugated dienes was conducted. Also, evaluation of severity of pathology was done for each rat. The mean +/- SD of the pathology score was significantly higher (p < 0.01) in the FO+E (6.0 +/- 1.3) group than in the CO+E group (3.0 +/- 0.5). No pathological changes were evident in the dextrose-fed controls. The CYP 2E1 protein levels (mean +/- SD) were significantly higher (p < 0.01) in the FO+E group (13.1 +/- 2.0) compared with the CO+E (4.7 +/- 1.2) and FO+D (1.8 +/- 0.5) groups. Higher levels of eicosapentaenoic and docosahexaenoic acids and lower levels of arachidonic acid were detected in liver microsomes from rats fed fish oil compared with corn oil. A significant correlation was obtained between CYP 2E1 protein and conjugated diene levels (r = 0.78, p < 0.01). Our results showing markedly increased CYP 2E1 induction and lipid peroxidation in the FO+E group provides one possible explanation for the greater severity of liver injury in this group.

The influence of dietary fat and alcohol on hepatic microsomal levels of cytochromes P-450 2E1, 2B, and 4A; phospholipases A and C; and UDP-glucuronosyltransferase was studied in the intragastric feeding rat model for alcoholic liver injury. Eight groups of animals were evaluated. Control and ethanol fed rats received either saturated fat or corn oil and were killed after 2 weeks and 1 month of feeding. All animals were pair-fed by continuous infusion of liquid diet through permanently implanted gastric cannulas. Alcoholic liver injury developed only in the corn oil-ethanol-fed groups and was manifest by 1 month. Livers were subjected to the following analyses: pathologic evaluation of liver injury; levels of cytochromes P-450 2E1, 2B, and 4A protein and mRNA; aniline hydroxylase activity; and phospholipase A and C and UDP-glucuronosyltransferase activities. Ethanol-induced increases in cytochromes P-450 2E1 and 2B protein determined by Western blotting were greatest in the corn oil-ethanol-fed group, which developed pathologic changes in the liver. Cytochromes P-450 2E1 and 2B1 mRNA levels were unaffected, suggesting that posttranscriptional mechanisms are responsible for the increase in the corresponding P-450 proteins. In contrast, cytochrome P-450 4A levels were higher in the saturated fat-ethanol groups compared with the corn oil-ethanol groups. Phospholipase A and phospholipase C levels were higher in the corn oil-ethanol groups compared with pair-fed dextrose controls and the saturated fat-ethanol groups. UDP-glucuronosyltransferase levels declined with time in the ethanol-fed groups. These observations are discussed in the context of a model whereby the induction of phospholipases A and C and cytochromes P-450 2E1 and 2B1 in corn oil-ethanol-fed rats provide arachidonic acid substrate and induce lipid peroxidation, respectively. These changes may account for the more severe pathologic changes that develop in corn oil-ethanol-fed animals compared with animals fed saturated fat and ethanol.

We evaluated whether fish oil or vitamin E administration affected ethanol-induced changes in membrane ATPases. Male Wistar rats (225–250 g) were fed, through a gastric tube a liquid diet containing fish oil (25% of calories) and ethanol for one month. Another group of animals was given supplemental vitamin E (300 u/kg). In the pair-fed control animals, ethanol-derived calories were replaced with dextrose. The blood ethanol levels were maintained between 150 and 350 mg/dL. At sacrifice, the red cells were immediately washed with ice-cold saline, membranes were prepared and ATPases measured. These was no difference in the Na + K + ATPase, Ca 2+ ATPase and Mg 2+ ATPase activities between the fish oil-dextrose and corn oil-dextrose groups. A decrease in Ca 2+ ATPase and an increase in Na + K + ATPase was seen with ethanol feeding; these change are similar to those seen in corn oil-ethanol fed rats. In contrast, Vitamin E administration prevented the ethanol-induced changes in ATPases. This observation provides support for the role of lipid peroxidation in alcohol-induced changes in cell membrane ATPase activities.

We used the intragastric feeding rat model for alcoholic liver disease to investigate the relationship between prostacyclin and liver injury. Rats were fed the following diets for periods ranging from 1 to 8 weeks: corn oil plus ethanol (CO+E), corn oil plus dextrose (CO+D), saturated fat plus ethanol (SF+E) and saturated fat plus dextrose (SF+D). Prostacyclin production (assessed by 6-ketoprostaglandin F1 alpha) by liver non-parenchymal cells decreased steadily over the 8 week period in animals fed CO+E (liver injury present) whereas in animals fed SF+E (no liver injury) there was no change in prostacyclin production. Plasma levels of 6-ketoprostaglandin F1 alpha were also significantly lower in the CO+E group compared to the other groups studied. We propose that decreased prostacyclin production by liver non-parenchymal cells may contribute to the hepatotoxic effect of ethanol.

The present study evaluated the role of endotoxin, prostaglandin E 2 (PGE 2 ), leukotriene B 4 (LTB 4 ) and thromboxane B 2 in alcoholic liver injury. Animals (4–5 per group per time period) were fed ethanol and either saturated fat (SE group) or corn oil (CE group) and sacrificed at various time intervals: 1, 2, and 4 weeks. Non parenchymal cells (NPC) were isolated and the spontaneous and LPS-stimulated production of eicosanoids was evaluated. In addition, severity of pathology, plasma levels of endotoxin, TXB 2 , LTB 4 and PGE 2 were compared in the two groups. The severity of fatty, liver, necrosis and inflammation was significantly greater in the CE group compared to the SE group after 1 month. Plasma endotoxin levels were significantly higher in the CE group vs. SE group at all time periods studied. PGE 2 levels in NPCS and plasma were in general higher in the SE group. LTB 4 levels in NPCS declined with time in the SE group; in contrast the levels rose in the CE group. There was no difference in LTB 4 levels in plasma between the two groups. TXB 2 levels in plasma and NPCS were significantly higher in the CE group at all times. The pattern of LPS-stimulated eicosanoid production by NPC in the two groups (SE and CE) suggested the development of endotoxin tolerance in the CE group. However, an additional modulatory effect of ethanol was also suggested by our findings.