Ann Reed

Statistical analysis of real-time PCR data

BackgroundEven though real-time PCR has been broadly applied in biomedical sciences, data processing procedures for the analysis of quantitative real-time PCR are still lacking; specifically in the realm of appropriate statistical treatment. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. Based on the standard curve method and other useful data analysis methods, we present and compare four statistical approaches and models for the analysis of real-time PCR data.ResultsIn the first approach, a multiple regression analysis model was developed to derive ΔΔCt from estimation of interaction of gene and treatment effects. In the second approach, an ANCOVA (analysis of covariance) model was proposed, and the ΔΔCt can be derived from analysis of effects of variables. The other two models involve calculation ΔCt followed by a two group t- test and non-parametric analogous Wilcoxon test. SAS programs were developed for all four models and data output for analysis of a sample set are presented. In addition, a data quality control model was developed and implemented using SAS.ConclusionPractical statistical solutions with SAS programs were developed for real-time PCR data and a sample dataset was analyzed with the SAS programs. The analysis using the various models and programs yielded similar results. Data quality control and analysis procedures presented here provide statistical elements for the estimation of the relative expression of genes using real-time PCR.

Toddlers’ Food Preferences: Concordance with Family Members’ Preferences

Abstract To determine the food preferences of toddlers and the concordance with preferences of their family members, 118 children, ages 28 to 36 months, were assessed with a written questionnaire completed by their mothers. The questionnaire included 196 foods commonly eaten across the U.S. Response categories were [food] never offered, never tasted, [child] likes and eats, dislikes but eats, likes but does not eat, and dislikes and does not eat. Similar questionnaires were completed by mothers (n = 117), fathers (n = 96), and an older sibling (n = 47) of the child. Results indicated strong concordance (82.0–83.3%) of food preferences between the child and other family members. Similarity between foods never offered to the child and the mothers dislikes was significant at p = .005. On the average, children had been offered 77.8% of the 196 foods and liked 81.1% of the foods offered to them. Foods liked and eaten by over 95% of the children included French fries, pizza, potato chips, apple juice, bananas, saltine crackers, spaghetti, biscuits, rolls (white), and popcorn. No single food was disliked and not eaten by a majority of the children. Results suggest that the most limiting category related to food preferences were those foods never offered to the child.

Mealtime Communication Patterns of Infants from 2 to 24 Months of Age

Abstract The purposes of this study were to document mealtime communication behaviors used by 98 Caucasian infants who were studied longitudinally from 2 to 24 months of age and to describe how these behaviors changed in the group over time. Using both closed- and open-ended questions in personal interviews, these middle and upper socioeconomic status mothers reported how their infants communicated hunger, satiety, and food likes and dislikes. Mothers also reported their own response behaviors when they believed that their infants had not eaten enough. Only a few mealtime communication behaviors were common to all infants, suggesting that infants use various behaviors to communicate similar messages. Food likes were most often communicated by mouth/eating behaviors, such as opening the mouth as food approached, eating readily, or eating a large amount of food. Food dislikes were communicated by mouth/eating behaviors, by facial expressions, and by body movements, such as turning the head or body away from food or throwing disliked food. When the mother perceived that the child had not eaten enough, most mothers offered alternative choices, either at mealtime or shortly thereafter.The results indicate that most infants are communicating via behaviors in feeding situations throughout the 2- to 24-month period.

Elevation of alanine transaminase and gallbladder wall abnormalities as biomarkers of anaphylaxis in canine hypersensitivity patients.

OBJECTIVE The purpose of the study was to evaluate alanine transaminase (ALT) and gallbladder wall abnormalities as possible biomarkers for anaphylaxis in dogs presented for acute hypersensitivity reactions. DESIGN Pilot study. SETTING A private practice, small animal, 24-hour emergency and specialty hospital. ANIMALS Ninety-six dogs presenting 101 times on an emergency basis for hypersensitivity reactions from March 2007 through March 2009. INTERVENTIONS Veterinarians acquired a history, physical exam, serum chemistry panel, blood pressure, and ultrasound image of the gallbladder. MEASUREMENTS AND MAIN RESULTS Dogs were then divided into 2 groups: dogs fulfilling the definition for anaphylaxis (moderate and severe systemic hypersensitivity) and dogs that did not fulfill the definition and were classified as allergic reactions (local hypersensitivity and mild systemic hypersensitivity). Elevated ALT was significantly associated with anaphylaxis (P<0.001). Increased gallbladder wall thickness and a striated wall pattern were significantly associated with anaphylaxis (P<0.001) and these changes were readily apparent to first-responder veterinarians. Decreased body temperature (P<0.001) and hypothermia (P=0.006) were significantly associated with anaphylaxis. There was no significant difference between groups regarding age, heart rate, or presence of respiratory signs. Lower blood pressure was significantly associated with anaphylaxis (P<0.001) but hypotension was not significantly different (P=0.09) between groups. Cutaneous signs were significantly associated with the allergic reactions group (P<0.001) and, when seen with anaphylaxis, were subtle. CONCLUSIONS This study showed an elevated ALT and an abnormal gallbladder wall to be biomarkers significantly associated with anaphylaxis in dogs with acute hypersensitivity reactions.

Association of magnetic resonance imaging findings and histologic diagnosis in dogs with nasal disease : 78 cases (2001-2004)

OBJECTIVE-To determine whether magnetic resonance imaging (MRI) features correlated with histologic diagnosis in dogs with nasal disease. DESIGN-Retrospective case series. ANIMALS-78 Dogs undergoing MRI for evaluation of nasal disease. PROCEDURES-Medical records and MRI reports of dogs were reviewed to identify MRI features associated with histologic diagnosis. Features evaluated were presence of a mass effect, frontal sinus involvement, sphenoid sinus involvement, maxillary recess involvement, nasopharyngeal infiltration by soft tissue, nasal turbinate destruction, vomer bone lysis, paranasal bone destruction, cribriform plate erosion, and lesion extent (ie, unilateral vs bilateral). RESULTS-33 Dogs had neoplastic disease, 38 had inflammatory rhinitis, and 7 had fungal rhinitis. Lesion extent was not significantly associated with histologic diagnosis. Absence of a mass effect was significantly associated with inflammatory disease. However, presence of a mass was not specific for neoplasia. In dogs with evidence of a mass on magnetic resonance (MR) images, nasal turbinate destruction, frontal sinus invasion, and maxillary recess invasion were not useful in distinguishing neoplastic from nonneoplastic disease, but cribriform plate erosion, vomer bone lysis, paranasal bone destruction, sphenoid sinus invasion, and nasopharyngeal invasion were. CONCLUSIONS AND CLINICAL RELEVANCE-Results suggested that in dogs with nasal disease, the lack of a mass effect on MR images was significantly associated with inflammatory disease. In dogs with a mass effect on MR images, vomer bone lysis, cribriform plate erosion, paranasal bone destruction, sphenoid sinus invasion by a mass, and nasopharyngeal invasion by a mass were significantly associated with a diagnosis of neoplasia.

Evaluation of the Effect of Orally Administered Acid Suppressants On Intragastric pH in Cats

Background Acid suppressant drugs are a mainstay of treatment for cats with gastrointestinal erosion and ulceration. However, clinical studies have not been performed to compare the efficacy of commonly PO administered acid suppressants in cats. Hypothesis/Objectives To compare the effect of PO administered famotidine, fractionated omeprazole tablet (fOT), and omeprazole reformulated paste (ORP) on intragastric pH in cats. We hypothesized that both omeprazole formulations would be superior to famotidine and placebo. Animals Six healthy adult DSH colony cats. Methods Utilizing a randomized, 4‐way crossover design, cats received 0.88–1.26 mg/kg PO q12h fOT, ORP, famotidine, and placebo (lactose capsules). Intragastric pH monitoring was used to continuously record intragastric pH for 96 hours beginning on day 4 of treatment. Plasma omeprazole concentrations at steady state (day 7) were determined by high performance liquid chromatography (HPLC) with ultraviolet detection. Mean percentage time that intragastric pH was ≥3 and ≥4 were compared among groups using ANOVA with a posthoc Tukey‐Kramer test (α = 0.05). Results The mean percentage time ± SD that intragastric pH was ≥3 was 68.4 ± 35.0% for fOT, 73.9 ± 23.2% for ORP, 42.8 ± 18.6% for famotidine, and 16.0 ± 14.2% for placebo. Mean ± SD plasma omeprazole concentrations were similar in cats receiving fOT compared to those receiving ORP and in a range associated with acid suppression reported in other studies. Conclusions and Clinical Importance These results suggest that both omeprazole formulations provide superior acid suppression in cats compared to famotidine or placebo. Fractionated enteric‐coated OT is an effective acid suppressant despite disruption of the enteric coating.

Agreement between values for arterial and end-tidal partial pressures of carbon dioxide in spontaneously breathing, critically ill dogs

OBJECTIVE To determine agreement between arterial partial pressures of carbon dioxide (PaCO(2)) and end-tidal partial pressures of carbon dioxide (PETCO(2)) measured with a nasal catheter in spontaneously breathing, critically ill dogs. DESIGN Validation study. ANIMALS 26 client-owned dogs admitted to an intensive care unit for various conditions. PROCEDURES PaCO(2) was measured with a commercial blood gas analyzer, and PETCO(2) was measured with a sidestream capnograph attached to a nasal catheter. Measurements were obtained twice (ie, with and without supplemental oxygen). Paired values were compared by means of the Pearson correlation method. Level of agreement was assessed by means of the Bland-Altman method. RESULTS Mean difference between PaCO(2) and PETCO(2) when dogs did not receive supplemental oxygen (mean +/- SD, 3.95 +/- 4.92 mm Hg) was significantly lower than mean difference when dogs did receive supplemental oxygen (6.87 +/- 6.42 mm Hg). Mean difference in dogs with a condition affecting the respiratory system (8.55 +/- 5.43 mm Hg) was significantly higher than mean difference in dogs without respiratory tract disease (3.28 +/- 3.23 mm Hg). There was a significant linear correlation and good agreement between measured values of PaCO(2) and PETCO(2). Catheter size, ventilatory status, and outcome were not significantly associated with mean difference between PaCO(2) and PETCO(2). CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that nasal capnography is a clinically relevant method of estimating PaCO(2) in spontaneously breathing, critically ill dogs, but that values should be interpreted with caution in dogs receiving supplemental oxygen and in dogs with conditions affecting the respiratory system.

Host utilization of field-caged native and introduced thistle species by Rhinocyllus conicus.

ABSTRACT Rhinocyllus conicus Fröelich was introduced from Europe into North America as a biological control agent of the exotic weed Carduus nutans L. Concern exists over the feeding of this weevil on at least 25 species of native Cirsium thistles. Beginning in 2008, cage studies isolating adults of R. conicus on buds and flower heads of all eight thistle species (native and introduced) recorded from Tennessee were conducted to test if R. conicus could use these species for reproduction and what impacts larval feeding of R. conicus may have on seed production. Larvae of R. conicus completed development in heads of the native species C. carolinianum (Walter) Fernald and Schubert, and C. horridulum Michaux, and significant reductions in seed numbers of both species occurred during 2008. Rhinocyllus conicus oviposited on both C. carolinianum and C. horridulum at significantly greater levels than the introduced species C. arvense (L.) Scopoli and C. vulgare (Savi) Tenore. Infested heads of C. carolinianum contained numbers of R. conicus per centimeter of plant head width similar to Ca. nutans in 2008, and both native species contained numbers of R. conicus per centimeter of plant head width similar to C. arvense and C. vulgare in 2009. Body length was similar between R. conicus reared on native thistles and its target host Ca. nutans. This report is the first documentation of R. conicus feeding and reproducing on C. carolinianum and C. horridulum. Although R. conicus has been observed only on introduced thistles in naturally occurring populations in this region, the utilization of C. carolinianum and C. horridulum as host species in controlled conditions warrants continued monitoring of field populations and further investigation into factors that may influence nontarget feeding in the future.

Diagnostic Accuracy of Tissue Impedance Measurement Interpretation for Correct Veress Needle Placement in Canine Cadavers

OBJECTIVE To establish the diagnostic accuracy of tissue impedance measurement interpretation (TIMI) for determining correct versus incorrect Veress needle placement. STUDY DESIGN Prospective, randomized, blinded trial. STUDY POPULATION Canine cadavers (n = 20). METHODS Two laparoscopists (1 experienced, 1 novice), blinded to tissue impedance measurement results, placed reusable Veress needles in canine cadavers in a randomized order. A 3rd individual interpreted impedance measurements as consistent with correct or incorrect Veress needle placement. Veress needle tip locations were marked by injecting contrasting colors of India ink. Tissue dissection was performed to localize ink. Sensitivity, specificity, accuracy, precision, and kappa statistics for TIMI for placements by the experienced and novice laparoscopist were determined. P < .05 was considered significant. RESULTS TIMI identified 29/33 correct and 7/7 incorrect placements, respectively. Impedance measurement interpretation had 87.9% sensitivity, 100% specificity, 90% accuracy, and 100% precision overall. Agreement between TIMI and Veress needle location was moderate (kappa 0.50, P = .01) for placements by the experienced laparoscopist and very high (kappa 0.88, P < .01) for the novice laparoscopist. CONCLUSIONS Diagnostic accuracy of TIMI for Veress needle placement was higher than has been reported for other tests, and TIMI had a shallow learning curve. TIMI successfully detected all incorrect Veress needle placements. Further prospective evaluation of Veress needle placement with and without TIMI is warranted to determine if its use increases operator detection of inappropriate Veress needle placements or decreases installment phase complication rates.

A Prospective, Placebo‐Controlled Pilot Evaluation of the Effect of Omeprazole on Serum Calcium, Magnesium, Cobalamin, Gastrin Concentrations, and Bone in Cats

Background Chronic proton pump inhibitor administration has been associated with electrolyte and cobalamin deficiency, disrupted bone homeostasis, hypergastrinemia, and rebound acid hypersecretion in humans. It is unknown if this occurs in cats. Objectives Prolonged oral omeprazole results in altered bone mineral density or content, serum calcium, magnesium, cobalamin, and gastrin concentrations in healthy cats. Animals Six healthy adult DSH cats. Methods In a within subjects, before and after design, cats received placebo followed by omeprazole (0.83–1.6 mg/kg PO q12h) for 60 days each. Analysis of serum calcium, magnesium, cobalamin, and gastrin concentrations was performed on days 0, 30, and 60. Bone density and content were evaluated on days 0 and 60 of each intervention. Continuous data were analyzed using a two‐way ANOVA (α = 0.006). On day 60 of omeprazole administration, continuous intragastric pH monitoring was performed in 2 cats to evaluate the effects of abrupt withdrawal of omeprazole. Results No significant changes were detected between treatments for any variables, except serum gastrin, which was significantly higher during omeprazole treatment in comparison to placebo (P = 0.002). Evidence of gastric hyperacidity was seen in both cats in which intragastric pH monitoring was performed following cessation of omeprazole. Conclusions and Clinical Importance Although further studies with larger populations of cats will be needed to draw any definitive conclusions, these preliminary results suggest that prolonged PPI treatment results in hypergastrinemia and abrupt PPI withdrawal might result in RAH in cats.