Opinion on Current Use of Non-Blinded Versus Blinded Histopathologic Evaluation in Animal Toxicity Studies.

The toxicologic pathologist plays a vital role in the scientific community, using their unique blend of diagnostic and investigative skills to advance biomedical research, public health, drug discovery, or regulatory practices. But what exactly do toxicologic pathologists contribute? Where do these specialized professionals work? How can toxicologic pathologists maximize their efficiency and potential? To enlighten students and trainees, as well as early- or mid-career toxicologic pathologists, or even those approaching retirement, the Career Development and Outreach Committee of the Society of Toxicologic Pathology (STP) sponsored a career development workshop entitled "Practical Strategies for Navigating Toxicologic Pathology in One's Early Career…and Beyond!" in conjunction with the STP 37th annual symposium. The workshop featured toxicologic pathologists from contract research organizations and the pharmaceutical industry, who provided their perspectives on career preparation, evolving veterinary pathologist roles within various sectors of toxicologic pathology, the fundamentals of safety assessment, logistics of projects involving good laboratory practices, tools for effective interpretation and communication of anatomic and clinical pathology results, and a recap of scientific resources available to support the toxicologic pathologist in his or her journey. This article provides brief summaries of the talks presented during this career development workshop.

Identifying compounds that are neurotoxic either toward the central or the peripheral nervous systems (CNS or PNS) would greatly benefit early stages of drug development by derisking liabilities and selecting safe compounds. Unfortunately, so far assays mostly rely on histopathology findings often identified after repeated-dose toxicity studies in animals. The European NeuroDeRisk project aimed to provide comprehensive tools to identify compounds likely inducing neurotoxicity. As part of this project, the present work aimed to identify diagnostic non-invasive biomarkers of PNS toxicity in mice. We used two neurotoxic drugs in vivo to correlate functional, histopathological and biological findings. CD1 male mice received repeated injections of oxaliplatin or paclitaxel followed by an assessment of drug exposure in CNS/PNS tissues. Functional signs of PNS toxicity were assessed using electronic von Frey and cold paw immersion tests (oxaliplatin), and functional observational battery, rotarod and cold plate tests (paclitaxel). Plasma concentrations of neurofilament light chain (NF-L) and vascular endothelial growth factor A (VEGF-A) were measured, and histopathological evaluations were performed on a comprehensive list of CNS and PNS tissues. Functional PNS toxicity was observed only in oxaliplatin-treated mice. Histopathological findings were observed dose-dependently only in paclitaxel groups. While no changes of VEGF-A concentrations was recorded, NF-L concentrations were increased only in paclitaxel-treated animals as early as 7 days after the onset of drug administration. These results show that plasma NF-L changes correlated with microscopic changes in the PNS, thus strongly suggesting that NF-L could be a sensitive and specific biomarker of PNS toxicity in mice.

Pathology peer review verifies and improves the accuracy and quality of pathology diagnoses and interpretations. Pathology peer review is recommended when important risk assessment or business decisions are based on nonclinical studies. For pathology peer review conducted before study completion, the peer-review pathologist reviews sufficient slides and pathology data to assist the study pathologist in refining pathology diagnoses and interpretations. Materials to be reviewed are selected by the peer-review pathologist. Consultations with additional experts or a formal (documented) pathology working group may be used to resolve discrepancies. The study pathologist is solely responsible for the content of the final pathology data and report, makes changes resulting from peer-review discussions, initiates the audit trail for microscopic observations after all changes resulting from peer-review have been made, and signs the final pathologist's report. The peer-review pathologist creates a signed peer-review memo describing the peer-review process and confirming that the study pathologist's report accurately and appropriately reflects the pathology data. The study pathologist also may sign a statement of consensus. It is not necessary to archive working notes created during the peer-review process.

Use of whole slide images for primary pathology evaluation of non-clinical toxicology studies in a GLP-validated environment.

We aimed to histopathologically evaluate the morphological spectrum, apoptotic index (AI), and mitotic index (MI) of endometrial lesions in patients presenting with abnormal uterine bleeding (AUB). A cross-sectional study was done over a period of 18 months where a total of 60 newly diagnosed cases of perimenopausal women presenting with AUB were included. All H and E stained pathology slides from the specimens were reviewed for initial histopathological evaluation and diagnosis. Immunohistochemistry for Bcl-2 and Bax was done. The study subjects were divided into two groups: Group 1 included 30 cases of histologically proven proliferative endometrium (PE) and endometrial polyps and Group 2 included 30 cases of hyperplasia, endometrial epithelial neoplasia (EIN)/or carcinoma. For all cases, AI and MI were calculated and compared among the two groups. Quantitative variables were compared using the Independent t-test/Mann-Whitney test between the two groups and Kruskal-Wallis test for comparison between more than two groups. Qualitative variables were correlated using the Chi-square test. P < 0.05 was considered statistically significant. In our study of 60 patients of AUB, the mean age was 45.87 years with a parity of 2 or more in most of the patients. Most of the patients in our study had heavy menstrual bleeding (66.67%) with associated complaints of weakness, pallor, and low backache. Out of 60 patients of AUB, Endometrial carcinoma was diagnosed in 13 (21.67%) patients, with the most common histopathological type being Endometrioid carcinoma. There was a significant difference in the AI, MI, and the ratio of AI/MI among various histopathological diagnoses. AI was highest for Endometrial carcinoma and lowest for hyperplasia and polyps. MI was also highest for Endometrial carcinoma and lowest for hyperplasia and polyps. However, the ratio was incongruent as it was highest for hyperplasia without atypia and lowest for PE with endometrial carcinoma being midway. The Bcl-2 expression of relatively benign conditions (Group 1) was significantly higher than Group 2 (Endometrial epithelial neoplasia/carcinoma). Bax intensity showed an almost inverse pattern, being highest in endometrial carcinoma and lower in hyperplasia and polyps with the lowest expression in PE. Even the Bcl-2:Bax ratio was also highest for PE and lowest for endometrial carcinoma with others falling in between them. It can be concluded that the combination of the proliferative and apoptotic markers and the ratio will help as a tool in aiding the diagnosis of endometrial lesions for patients presenting with AUB.

For registration of generic and similar drugs, it is necessary to carry out pharmaceutical equivalence (PE) tests and pharmaceutical bioequivalence (PB). To carry out these tests, duly qualified research centers are contracted, which need to be monitored by the sponsor who is legally responsible for the activities. To this end, it is the recommendation of the Document of the Americas, periodic monitoring to verify compliance with quality requirements, Standard Operating Procedures, Good Clinical Practices (GCP), Good Laboratory Practices (GLP), of the applicable regulatory framework, as well as of compliance with the study protocol. Thus, monitoring is a methodical and documented process to evaluate the degree of adhesion of the center to the planned design for the evaluation of the formulations. To this end, the implementation of a standardized and easily completed guideline is a very important tool to guarantee a consistent evaluation and maintain the organizational memory of the evaluated items by monitors designated by the sponsor, contributing to the constant improvement of the contracted centers and supporting traceability of the studies. This work provided a systemic view of the evidence process related mainly to pharmaceutical bioequivalence, with the monitoring guideline summarizing the items of greatest relevance to be verified.

Hepatoportal sclerosis (HPS) is a syndrome of obscure etiology, and is one of the causes of noncirrhotic portal hypertension (PH). We aimed to investigate this heterogeneous group of patients whose presentation showed cholestatic features, histopathologically. Between 1999 and 2009, 12 children diagnosed with HPS were retrospectively evaluated. HPS was diagnosed with evidence of PH, noncirrhotic liver biopsy with typical histopathologic findings, and exclusion of other possible causes of PH. The data was obtained from pathology reports and microscopic slides. In histopathological re-evaluation fibrosis state, aberrant portal vessels, portal tract dilation and inflammation, ductular reaction, regenerative nodular hyperplasia, acinar transformation, presence of bile pigment, and cholangitis were noted. Serum alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl, alkaline phosphatase, bilirubin, and albumin levels, presentation patterns, and radiologic findings were assessed. Familial relationship degrees were also investigated. Twelve patients (9 boys, 3 girls; 3-180 months) were re-evaluated. Two pairs of the patients were siblings. Parents of 7 patients were consanguine. The most common presenting symptom was abdominal distension. Histopathologically, all patients had hepatoportal sclerosis/intimal fibrous thickening of portal vein and periportal fibrosis, acinar transformation, and regenerative nodules not surrounded by fibrous septae. Eight patients had vascular aberrations, 7 had ductular reaction, 1 showed mild cholangitis, and 1 had canalicular bile pigment. We conclude that genetic predisposition might be a possible factor for HPS development in Turkish patients and it should be kept in mind that cholestatic features noticed in histopathological evaluation may represent a variant group in the spectrum of HPS.

Clinical relevance and effect of surgical wound classification in appendicitis: Retrospective evaluation of wound classification discrepancies between surgeons, Swissnoso-trained infection control nurse, and histology as well as surgical site infection rates by wound class

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

To The Editor The existence of ovarian dysplasia is controversial, in part because of the rarity with which preinvasive ovarian lesions are identified. Some of the earliest reports of ovarian dysplasia were by Graham and colleagues (1–3) from our institution in the early 1960s, and the findings of their studies are occasionally cited in support of the existence of ovarian epithelial dysplasia or carcinoma in situ(4,5). For this reason we decided to review the pathology reports and histopathology slides of six cases reported as exhibiting abnormal ovarian epithelium (1) in oophorectomy specimens removed for abnormal cul-de-sac aspiration cytology. The cul-de-sac aspiration cytologies themselves could not be found. In the orginal study, slides of the ovaries were separately evaluated by a pathologist employed by the Department of Gynecologic Oncology, in addition to the evaluation of record by the surgical pathologist in the Department of Pathology. Both pathologists were aware of the reason for oophorectomy, but the histopathologic evaluation described in the paper by Graham and colleagues was that of the former pathologist. The clinical characteristics of the patients described in this study are shown in Table 1. Two patients (patients 7 and 8) were said to have had their ovaries incompletely examined, but with no evidence of pathology in the material that was examined. A cytologic specimen obtained by scraping the ovarian surface before processing in patient 8 reportedly showed malignant cells similar to those seen on cul-de-sac aspiration. Of the remaining four patients, three exhibited papillary surface growths and the fourth exhibited an “abnormal proliferation of the germinal epithelium” showing “all the characteristics of malignancy.”TABLE 1: Ovarian pathology of patients with positive cul-de-sac aspirationOn review, we interpret one of the three papillary lesions (patient 4) as being fallopian tube mucosa adherent to the ovarian surface, and the other two (patients 5 and 6) as papillary growths similar to those that we have found on the surface of normal ovaries (6). In the case with an abnormal proliferation exhibiting characteristics of malignancy (patient 3), it is our opinion that the appearance of the surface epithelial cells is within the range of normal variation. The original illustration and our photomicrograph of this case are shown in Figure 1. The official surgical pathology reports noted normal ovarian epithelium in all cases.FIG. 1.: Patient 3, originally reported as “malignant-appearing cells without invasion.”A: Reproduction from original publication (reproduced with permission, John Wiley and Sons, Inc., New York, NY). B: Photomicrograph by current authors (original magnification, 1000×).Figure 1: ContinuedThis report is not intended as a criticism of the original authors of these papers, who were highly regarded experts in the field. Many earlier pathologic studies failed to include control groups or to mask (blind) histopathologic interpretations, which we believe may have led to misinterpretation of normal ovarian pathology as pathologic in this case. Because this study is occasionally cited in support of the existence of ovarian dysplasia, we believed that it was worth reporting our reexamination of these cases. In our opinion, the cases classified as dysplastic or borderline lesions were variations of normal ovarian histology. Although ovarian dysplasia may exist, these studies do not provide evidence regarding its existence or morphology. Bruce A. Werness M.D.* Gamal H. Eltabbakh M.D.†

BackgroundCitric acid (CA) and sodium hypochlorite (NaOCl) have been used to disinfect animals to protect them against avian influenza and foot-and-mouth disease.ObjectivesWe performed a good laboratory practice (GLP)-compliant animal toxicity study to assess the acute toxic effects of CA and NaOCl aerosol exposure in Sprague-Dawley rats.MethodsGroups of five rats per sex were exposed for 4 h to four concentrations of the two chemicals, i.e., 0.00, 0.22, 0.67, and 2.00 mg/L, using a nose-only exposure. After a single exposure to the chemicals, clinical signs, body weight, and mortality was observed during the observation period. On day 15, an autopsy, and then gross findings, and histopathological analysis were performed.ResultsAfter exposure to CA and NaOCl, body weight loss was observed but recovered. Two males died in the CA 2.00 mg/L group and, two males and one female died in the 2.00 mg/L NaOCl group. In the gross findings and histopathological analysis, discoloration of the lungs was observed in the CA exposed group and inflammatory lesions with discoloration of the lungs were observed in the NaOCl exposed group. These results suggest that the lethal concentration 50 (LC50) of CA is 1.73390 mg/L for males and > 1.70 mg/L for females. For NaOCl, the LC50 was 2.22222 mg/L for males and 2.39456 mg/L for females.ConclusionsThe Globally Harmonized System is category 4 for both CA and NaOCl. In this study, the LC50 results were obtained through a GLP-based acute inhalation toxicity assessment. These results provide useful data to reset safety standards for CA and NaOCl use.

Aims We sought to investigate the prevalence of Transthyretin amyloid (ATTR) deposits in the carpal ligament of patients undergoing surgery for carpal tunnel syndrome (CTS) and the frequency of concomitant ATTR-cardiomyopathy (CM) in a prospective study with long-term follow-up. Methods and results Our prospective exploratory study enrolled 551 patients treated for CTS. All resection specimens of carpal ligament underwent histopathological evaluation. Patients with confirmed amyloid deposits were referred to cardiac evaluation, including cardiac MRI (CMR) scans, as well as follow-up examinations. Amyloid deposits were found in 52 (9%) specimens (1 light chain amyloid, 51 ATTR). Among patients with positive biopsy ATTR-CM was diagnosed in 4 (8%) patients, 1 patient was diagnosed at initial evaluation, and 3 patients were diagnosed during the follow-up. All patients had ATTRwt at an early stage of disease. Extracellular volume (ECV) measured by CMR was significantly higher in amyloid-positive CTS patients compared to healthy controls (30 ± 2 vs. 24 ± 1%, p < .01). Conclusion Histopathological evaluation of resected carpal ligament should be considered in patients at least older than 70 years. Even though absolute numbers were low, cardiac screening of patients with evidence of amyloid deposits in in carpal tunnel tissues might facilitate early diagnosis of ATTR-CM in some patients.

To compare re-epithelialization rates of superficial partial-thickness burns treated with octylcyanoacrylate (OCA), silver sulfadiazine (SSD), and dry gauze (controls) in swine. This was a prospective, longitudinal, blinded, controlled, experimental trial using anesthetized swine. Sixty-three standardized burns were created by applying an aluminum bar preheated to 80 degrees C for 20 seconds to the flanks of four young pigs. Three equal sets of 21 burns were randomly treated with OCA spray, SSD, or dry gauze (controls). Full-thickness biopsies were taken after 30 minutes and at seven and 14 days for blinded histopathologic evaluation by two dermatopathologists using hematoxylin and eosin staining. The percent of wound re-epithelialization was measured at days 7 and 14, calculated by dividing the length of the regenerated epidermis by the measured width of the biopsy. Analysis of variance (ANOVA) and repeated-measures ANOVA controlling for the individual pig were used for comparisons among groups. This study had 80% power to demonstrate a large between-group difference in percent re-epithelization (alpha = 0.05). There were 63 burns and 126 biopsies. Ten biopsies were excluded for technical reasons. At seven days, there was a significant between-group difference in percent re-epithelialization. Percent re-epithelialization was greatest in the OCA group (65.0%), followed by the SSD group (37.6%), and lowest in the control group (8.8%). At 14 days, all wounds demonstrated near complete re-epithelialization and there was no significant difference in the percent of re-epithelialization among the groups. There was only one wound infection in the OCA group. Under these study conditions, treatment of partial-thickness burns with OCA spray resulted in a higher percent of re-epithelialization at seven but not 14 days when compared with both SSD and control, with no significant increase in infection rates. Future studies should evaluate the use of OCA for the treatment of burns in humans.

Previously, in a murine model of blunt thoracic trauma, we provided evidence of primary pulmonary thrombosis associated with increased expression of the cell adhesion molecule, P-selectin. In this study, mice are treated with P-selectin blocking antibody after injury to investigate the clinical viability of this antibody for the prevention of pulmonary thrombosis. In addition, viscoelastic testing is performed to investigate if P-selectin inhibition has a detrimental impact on normal hemostasis. A murine model of thoracic trauma was used. Mice were divided into sham control and experimental injury groups. Thirty minutes after trauma, mice were treated with the following: P-selectin blocking antibody, isotype control antibody, low-dose heparin, high-dose heparin, or normal saline. At 90 minutes, whole blood was collected for characterization of coagulation by viscoelastic coagulation monitor (VCM Vet; Entegrion, Durham, NC). Mean clotting time, clot formation time, clot kinetics (α angle), and maximum clot firmness were compared between each treatment group. Mice that received P-selectin antibody 30 minutes after blunt thoracic trauma had four- to fivefold less (p < 0.001) arterial fibrin accumulation than those that received the isotype control. In both sham and trauma groups, compared with vehicle (normal saline) alone, no statistical difference was noted in any coagulation parameters after injection with P-selectin antibody, isotype control, or low-dose heparin. In addition, blinded histopathological evaluation yielded no difference in hemorrhage scores between injured mice treated with P-selectin blocking antibody and those treated with isotype antibody control. This study supports the clinical use of P-selectin blocking antibody for the prevention of pulmonary thrombosis by confirming its efficacy when given after a blunt thoracic trauma. In addition, we demonstrated that the administration of P-selectin antibody does not adversely affect systemic coagulation as measured by viscoelastic testing, suggesting that P-selectin antibody can be safely given during the acute posttraumatic period.

Primary cicatricial alopecia: Histopathologic findings do not distinguish clinical variants