Knowledge Of Treatment Group Research Articles

Pathology Working Groups

Pathology Working Groups (PWG) are a specialized form of review consisting of a panel of expert pathologists who provide an independent, unbiased assessment of specific questions concerning study results. PWGs may concentrate on pivotal studies with controversial end points, address questions that are of concern to a regulatory agency, or compare the results of multiple studies that may have been conducted and evaluated by different laboratories and/or pathologists. The PWG does not review the entire study but always includes a preliminary review by a peer review pathologist. The PWG chairperson needs to thoroughly understand the issue in question, reviews all relevant data and study results, and is responsible for the organization and conduct of the PWG. The members of the PWG examine coded slides without knowledge of treatment group or previous diagnosis and arrive at a consensus diagnosis by majority vote. Once the results are decoded, the PWG evaluates the results and provides discussion and conclusions that are reflected in the final PWG report. Specific examples of PWG issues are provided.

Industry–Contract Research Organization Pathology Interactions

This article provides observations on the features of sponsor–contract research organization communication that will achieve the best quality pathology report based on our collective experience. Information on the test article and any anticipated findings should be provided, and initial slide examination should be done with knowledge of treatment group (but may be followed by blinded review of target tissues to determine no-effect levels). Only a pathologist should write or revise the pathology report or the pathology section of the overall study report. To address concerns related to undue sponsor influence, comments by sponsors should be presented as suggestions rather than directives. Adversity should be defined for each finding by the study pathologist, but the no-observed adverse effect level should not be discussed in the pathology report. Board-certified pathologists are recommended, but are not essential. Sponsors that have a particular format or report preferences should make them known well in advance. Histologic processing “to glass” of protocol-specified tissues from all dosage groups is recommended for rapid evaluation of target tissues. Telepathology is beneficial in certain situations, but it is usually more efficient for the study pathologist and reviewing pathologist to be in the same physical location to review differences of opinion and reach a consensus.

Micro‐computed tomographic evaluation of fetal skeletal changes induced by all‐trans‐retinoic acid in rats and rabbits

Our laboratory has been conducting positive control studies to evaluate the utility of micro-computed tomography (micro-CT) for qualitative evaluation of fetal skeletal morphology. All-trans-retinoic acid (atRA) was used to produce a different spectrum of defects compared to our previous studies with boric acid and hydroxyurea. Groups of five mated Crl:CD(SD) female rats each were administered vehicle or atRA (2.5-50 mg/kg) on GD 10, and groups of four mated Dutch Belted rabbits each were dosed with vehicle or atRA (6.25-25 mg/kg) on GD 9. Cesarean sections were performed on GD 21 and 28, respectively. Following external examination the viscera were removed and fetuses scanned in a micro-CT imaging system. Fetuses were subsequently stained with alizarin red. Skeletal morphology was evaluated by each method without the knowledge of treatment group. Total bone mineral content (BMC) of each fetus was quantitated using the micro-CT images. In rats there were dose-related increases in the incidence of extra lumbar vertebra and non-dose-related increases in supernumerary ribs at all dose levels. There were decreases in mean number of ossified sacrocaudal vertebra at ≥ 5 mg/kg, and increases in skull bone malformations at ≥ 10 mg/kg. Rabbits were less sensitive on a mg/kg basis since skeletal malformations and a decrease in mean number of ossified sacrocaudal vertebra were observed only in the 25-mg/kg group. Micro-CT evaluation detected essentially the same incidence of skeletal abnormalities as seen in alizarin red-stained rat and rabbit fetuses. BMC analysis showed a trend toward slight decreases in atRA-treated rats, but no notable changes in rabbits. These results add support to our previous work that demonstrates that micro-CT imaging can effectively assess rat and rabbit fetal skeletal morphology.

Knowledge of Treatment Group Does Not Bias Assessment of Time to Seizure in an Animal Model of Cocaine Poisoning

Blinded outcome assessment decreases bias in human clinical trials. The necessity of blinded outcome assessment on animal studies is unknown. The authors determined the effect of knowledge of treatment group on assessment of time to seizure in an animal model of cocaine poisoning. Four subjects observed 20 animal experiments where all animals were administered a high dose of cocaine and placebo. For each experiment, two of the observers were told the animal had been treated with placebo and two were told the animal had been treated with a medication expected to delay the onset of seizures. Each observer recorded the time from cocaine administration to onset of seizure. The median time to seizure was compared between observers told the animal received placebo and those told the animal received active treatment. Seizures were reported by all subjects in 12 animals and by no subjects in five animals, and there was disagreement in three animals. The reported median time to seizure was similar for observers told that the animals were treated with placebo and those told they were treated with study medication. It is feasible to determine whether unblinded assessments are biased in an animal study. Knowledge of treatment group did not bias the assessment of time to seizure in this animal model.

Evaluation of hydroxyurea‐induced fetal skeletal changes in Dutch belted rabbits by micro‐computed tomography and alizarin red staining

This laboratory has been investigating the utility of X-ray micro-computed tomography (micro-CT) to produce high-resolution, 3D images of skeletal structures in common laboratory species. The present investigation uses micro-CT evaluation of skeletons from rabbit fetuses exposed to the known teratogen, hydroxyurea. Groups of 4-6 mated Dutch Belted female rabbits each were administered vehicle or hydroxyurea (62.5 to 500 mg/kg) once on GD 12. On GD 28, all live fetuses were weighed, euthanized, and viscera removed. Up to 7 fetuses per litter were placed into a custom-made polystyrene holder and scanned in the micro-CT imaging system. Raw projection data were acquired in approximately 15 seconds, and reconstructed images at 100-micron cubic voxel dimension could be viewed as early as 20 minutes later. Fetuses were subsequently stained with alizarin red, and findings recorded separately for each method without knowledge of treatment group. Except for a few isolated cases, micro-CT evaluation detected the same skeletal malformations, variations, and incomplete ossifications as seen by the staining method. Skeletal elements that are very small (e.g., caudal-most vertebrae, metacarpal no. 1) or those with a minimal degree of ossification were occasionally not observed with micro-CT. However, this difference did not impact the overall study conclusions. Femur length was easily measured by micro-CT. These results indicate that micro-CT imaging can effectively assess rabbit fetal skeletal structures, and for those laboratories with this resource, may be used to significantly reduce time prior to skeletal evaluation and hazardous wastes associated with staining.

Micro‐computed tomography and alizarin red evaluations of boric acid–induced fetal skeletal changes in Sprague‐Dawley rats

Assessment of developmental toxicity has historically included assessment of fetal skeletal morphology after alizarin red staining. X-ray micro-computed tomography (micro-CT) produces high-resolution images of skeletal structures and was investigated as an alternative method. Groups of 5 mated Crl:CD (SD) female rats each were administered vehicle or boric acid (40 to 500 mg/kg/day) from GD 6 through 11. On GD 21, all live fetuses were weighed, euthanized, and viscera removed. Each litter was placed into a custom-made polystyrene holder and scanned in the micro-CT imaging system. Raw projection data were acquired in approximately 15 sec ( approximately 20 litters per hour) and reconstructed images at 100-micron cubic voxel dimension could be viewed as early as 20 min later. Fetuses were subsequently stained with alizarin red, and findings recorded separately for each method without knowledge of treatment group. Micro-CT evaluation of fetal rat skeletons detected essentially the same skeletal malformations, variations, and incomplete ossifications as seen by the staining method. The specific skeletal abnormalities that did not match exactly involved the smallest skeletal elements with minimal degrees of ossification (i.e., cervical ribs, hypoplastic 13(th) ribs, supernumerary ribs, the 5(th) sternebra, and numbers of caudal vertebrae), but the differences did not impact the overall conclusions. Additional measures such as femur length were easily measured by micro-CT. These results indicate that micro-CT imaging can effectively assess rat fetal skeletal structures, and for those laboratories with this resource, it may be used to significantly reduce time prior to skeletal evaluation and hazardous wastes associated with staining.

Continuous control of endotracheal cuff pressure and tracheal wall damage: a randomized controlled animal study

BackgroundIntubation is frequently performed in intensive care unit patients. Overinflation of the endotracheal tube cuff is a risk factor for tracheal ischemia and subsequent complications. Despite manual control of the cuff pressure, overinflation of the endotracheal cuff is common in intensive care unit patients. We hypothesized that efficient continuous control of the endotracheal cuff pressure using a pneumatic device would reduce tracheal ischemic lesions in piglets ventilated for 48 hours through a high-volume, low-pressure endotracheal tube.Materials and methodsTwelve piglets were intubated and mechanically ventilated for 48 hours. Animals were randomized to manual control of the endotracheal cuff pressure (n = 6) or to continuous control of the endotracheal cuff pressure using a pneumatic device (n = 6). In the two groups, we inflated the endotracheal cuff with 50 ml air for 30 minutes, eight times daily. This hyperinflation of the endotracheal cuff aimed at mimicking high-pressure periods observed in intubated critically ill patients. In all animals, the cuff pressure and the airway pressure were continuously recorded for 48 hours. After sacrifice of the study animals, the trachea was removed and opened longitudinally for gross and histological examination. A pathologist evaluated the slides without knowledge of treatment group assignment.ResultsThe cuff pressure was significantly lower in piglets with the pneumatic device than in piglets without the pneumatic device (median (interquartile range), 18.6 (11–19.4) cmH2O versus 26 (20–56) cmH2O, P = 0.009). No significant difference was found in the percentage of time spent with a cuff pressure <15 cmH2O and that with a cuff pressure between 30 and 50 cmH2O. The percentage of time between 15 and 30 cmH2O of cuff pressure, however, was significantly higher in piglets with the pneumatic device than in piglets without the pneumatic device (98% (95–99%) versus 65% (44–80%), P = 0.002). In addition, the percentage of time with cuff pressure >50 cmH2O was significantly lower in piglets with the pneumatic device than in piglets without the pneumatic device (0% versus 19% (12–41%), P = 0.002).In all animals, hyperemia and hemorrhages were observed at the cuff contact area. Histological examination showed no difference in tracheal lesions between animals with and without the pneumatic device. These lesions included deep mucous ulceration, squamous metaplasia and intense mucosal inflammation. No cartilage lesions were observed.ConclusionThe pneumatic device provided effective continuous control of high-volume, low-pressure endotracheal cuff pressure in piglets mechanically ventilated for 48 hours. In the present model, however, no significant difference was found in tracheal mucosal lesions of animals with or without a pneumatic device. Further studies are needed to determine the impact of continuous control of cuff pressure over a longer duration of mechanical ventilation.

DIHYDROTESTOSTERONE (DHT) STIMULATES BRANCHING MORPHOGENESIS IN EMBRYONIC LUNG EXPLANTS. ▴ 361

DHT affects late gestation lung development by increasing the proliferation of fibroblasts and Type II cells and delaying surfactant production. Based on these findings, it has been suggested that DHT prolongs late gestation (fetal) lung growth resulting in males having larger lungs than females. We postulated that DHT also affects the early gestation (embryonic) lung growth and morphogenesis. We tested this hypothesis by examining the effect of DHT on branching morphogenesis and thymidine incorporation into DNA in embryonic day 11 1/2 mouse lung explants. Lungs were cultured for 72 hours in serum free BGJb medium with either 10-8M DHT or an equal volume (10 μl/ml) of diluent as control. Branching morphogenesis was evaluated at 0, 24, 48, and 72 hours by counting the total number of terminal buds of the left lung under light microscopy. These evaluations were done without knowledge of treatment group. At 48 hours 3H-thymidine (1μCi/ml) was added to all cultures. At 72 hours lung explants were harvested and thymidine incorporation into DNA was determined. The number of terminal buds was similar in the two groups at time zero. Thereafter, the number of terminal buds in the DHT treated group was significantly increased compared to control at 24 hours (DHT: 6.3±0.7 buds; Control: 4.0±0.4 buds; mean±SEM, P=0.006), at 48 hours (DHT: 10.8±0.7; Control 6.4±0.6, P=0.0001), and at 72 hours (DHT: 16.5±1.7; Control 11.1±1.1, P=0.01). The velocity of terminal bud formation, defined as the increase in buds within a given lung over time, was significantly greater in the DHT treated group for the time intervals of zero to 24 hours (DHT: 2.6±0.5; Control: 1.1±0.3; mean±SEM, P=0.02), zero to 48 hours (DHT: 7.1±0.6; Control: 3.5±0.7, P=0.0008), and zero to 72 hours (DHT: 12.8±1.3; Control: 8.2±1.2, P=0.01). Thymidine incorporation into DNA between 48 and 72 hours was decreased in DHT treated lungs (DHT 76%±15% of control; mean±SEM). These data suggest that DHT increases the early velocity of terminal bud formation in the embryonic lung. We speculate that DHT has divergent effects on the growth of airway epithelium versus mesenchymal tissue in embryonic lung, resulting in increased terminal bud formation but decreased overall thymidine incorporation. Supported by HL37930

Avian model for 13-cis-retinoic acid embryopathy: morphological characterization of ventricular septal defects.

In developing an avian model for 13-cis-retinoic acid (13cisRA) embryopathy, we found 13cisRA induced cardiovascular defects, especially Type I ventricular septal defects (VSDs) (Hart et al.: Teratology 41:463-472, '90). As the first step of investigating possible mechanisms, we have examined the light microscopic morphology of RA-induced cardiovascular defects in chick embryos. Fertilized eggs were injected via yolk sac with 150 micrograms 13cisRA in dimethylsulfoxide (DMSO), DMSO or mock injection on embryonic day 5 (E5). On E6, E7, or E8, surviving embryos were sacrificed and fixed in 10% formalin. Thoracic blocks were exised, embedded in paraffin and serially sectioned through the heart, base to apex. Slides were stained, screened for tissue orientation, then coded and evaluated without knowledge of treatment group. Examination of serial sections permitted qualitative evaluation of conotruncal ridge volume, mesenchymal organization, necrosis and extent of fusion. Extent of fusion was the only parameter influenced by 13cisRA treatment. On E6, ridge fusion was incomplete in all groups at comparable levels. On E7, ridge fusion in 13cisRA-treated embryos had not progressed as far proximally as in controls. By E8, there was a significant difference in the extent of fusion between 13cisRA-treated and non-RA-treated groups. We conclude 13cisRA-induced VSDs resulted from defective conotruncal ridge fusion and that the fusion defects did not result from decreased tissue volume, altered mesenchymal organization or increased necrosis.

Radiographic Findings Associated with Surfactant Treatment

Radiographs of the chest (CXR) were evaluated in 35 of 41 infants enrolled in a randomized controlled trial of modified bovine surfactant extract (Surfactant-TA Tokyo-Tanabe) treatment. Infants between birthweight 1000 and 1500 gm with respiratory distress syndrome requiring mechanical ventilation and an inspired oxygen concentration 0.4 or greater were randomly assigned to either a single intratracheal dose of saline or surfactant-TA prior to 8 hours of age. Radiographs obtained prior to treatment and 24 hours after treatment were reviewed by a radiologist (N.T.G.) without knowledge of treatment group. Evaluation consisted of a score including criteria for inflation of the lungs, density of the lungs, and extent of air bronchograms. Pneumothorax, pulmonary interstitial emphysema, and asymmetric parenchymal involvement were noted as well. No significant difference in CXR scores were noted in the two groups, before or after treatment. There was a greater incidence of pneumothorax and pulmonary interstitial emphysema in the control infants, which supports the role of surfactant in preventing barotrauma. Increased incidence of asymmetric parenchymal involvement was noted in the surfactant-treated infants. Further study of the possibility of drug maldistribution is warranted.

Differentiation of lesions caused by mycotoxin T-2 from autolytic morphologic change in CD-1 mice

Experiments with topically applied T-2 trichothecene mycotoxin were undertaken to determine whether lesions caused by this toxin could be differentiated from autolysis. Two pathologists, who had previously seen lesions caused by T-2 toxin, graded lesions without knowledge of treatment group and stated whether the animal had received the toxin or not. Both pathologists differentiated T-2 toxin-treated mice up to 6 h post-mortem. Failure to distinguish between treated and control mice resulted in false-negative diagnoses only. It was concluded that the diagnosis of trichothecene mycotoxicosis would probably be missed more than 6 h post-mortem.