Hematoxylin Counterstain Research Articles

Detecting SARS-CoV-2 in bats of New Mexico using immunohistochemistry on formalin- fixed, paraffn-embedded tissues

Objectives: The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) and its zoonotic origin have raised critical questions regarding the potential role of local bat populations as reservoirs or intermediates for the transmission of this or related coronaviruses to humans. The goal of this project is to investigate the presence and diversity of coronaviruses in bat species native to the Southwest USA. Broadly, this study has two primary aims: (1) to detect antigens of coronaviruses in tissues of local bat populations via histological methods and (2) to perform a molecular screen of coronaviruses in these bats. Methods: Individuals from five bat species were evaluated: Artibeus jamaicensis, Myotis velifer, Antrozous pallidus, Tadarida brasiliensis, and Eptesicus fuscus. All species were local, wild-caught bats, with the exception of Artibeus jamaicensis, which is from a colony established at New Mexico State University (NMSU). Tissue was sourced from bats collected by TJO at NMSU. Bat tissue was fixed with formalin and samples subsequently taken of lung, kidney, nasal epithelium, and uterus. Tissue was further processed and embedded in paraffn wax. The embedded tissue was sectioned via a rotary microtome at 4 μm thickness and mounted onto slides. Slides were stained with general coronavirus anti-nucleocapsid primary antibody and a hematoxylin counterstain to visualize cellular structures. Slides were cover-slipped with DPX mounting medium and observed via light microscopy. Results: The immunohistochemistry protocol was performed on tissue from each of the five bat species investigated and on human placenta samples known to be positive for SARS-CoV-2. The latter was used as positive controls. Putative positive staining was seen in both human placenta and bat tissues. Interestingly, coronavirus was detected in Artibeus jamaicensis samples, an unexpected finding given its status as a laboratory colony. Verification of this putative finding is ongoing. Further work on our samples will determine the genetic identity of the coronaviruses detected and contribute to developing a molecular screen for more effcient detection of coronavirus antigens in local bat tissue. This work will serve as a springboard for further understanding coronavirus ecology and evolution in bats of the US southwest, and it can be used to further inform both local surveillance efforts and public policy surrounding the interaction between humans and animals in the natural environment and in controlled settings. Conclusions: Putative detection of coronavirus antigens was found in tissues from bat samples from the Southwestern USA. Implications for this work include broadening our understanding of the diversity and abundance of coronaviruses in the US. Burrell College; US National Science Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

51 A preliminary study of the effect of dietary cottonseed on the follicular reserve of does artificially infected with Haemonchus contortus.

Abstract Numerous studies have reported the negative effect of cottonseed gossypol on the ovarian reserve of the follicles, impairing female fertility. Injuries resulting in the depletion of primordial follicles can lead to permanent infertility. At birth, female mammals possess a fixed number of ovarian primordial follicles, which develop into primary, secondary, and antral follicles in a continuous and irreversible process. In this study, we evaluated the potential effects of dietary cottonseed gossypol in combination with parasitic infection on ovarian follicular reserve in meat goats. A total of 40 Boer x Spanish cross does were randomly subjected to cottonseed (CS) and parasite (P: Haemonchus contortus) in a 2x2 factorial arrangement of does (n = 8) per treatment groups (CS-/P-; CS+/P-; CS-/P+, and CS+/P+). Does were artificially infected with Haemonchus contortus for 2 d, and after 25 d, they began shedding parasite eggs. Thereafter, does were fed a hay diet supplemented with cottonseed (0.5% BW; CS+) or concentrate (CS-), to meet the nutritional requirements. Ten weeks later, animals were slaughtered, and ovaries of each treatment group (n = 4) were fixed with formalin and subjected to a standard procedure of histology staining (Periodic acid–Schiff and Hematoxylin counterstaining). Cuts of one left and one right ovary of two does were placed on microscope slides, and 48 slides were subjected to a microscope visualization (EVOS FL Auto; 20x magnification). Primordial follicles were classified as healthy (stained nucleus), unhealthy (pycnotic), and undetermined. Data were analyzed with ANOVA 1 and expressed as mean ± sem. P < 0.05 indicates a significant difference. A total of 20,562 primordial follicles corresponding to 10,410 (50.6%) healthy; 3,729 (18.1%) unhealthy; and 6,909 (33.6%) undetermined were examined; of which 26%, 20%, 31%, and 23% of total primordial follicles belonged to the CS-/P-, CS-/P+, CS+/P-, and CS+/P+ treatment groups, respectively. A significantly (P < 0.05) greater number of follicles were counted on left vs. right ovary sections (11,584 vs. 8,978) corresponding to 56.3% vs. 43.7% of total follicle counts. Averages of 121 ± 6 vs. 101±6 follicles were calculated per left and right ovary sections. Diets containing cottonseed (CS+/P-) increased the average number of follicles per ovary section, while parasite infections significantly (P < 0.05; CS-/P+ and CS+/P+) decreased these numbers. Regardless of the treatment group, the proportions of healthy follicles remained comparable between treatment groups. We concluded that dietary cottonseed and parasitic infections inversely affect ovarian follicle reserve, while the proportions of healthy follicles were unaffected across treatment groups. Nevertheless, higher microscopic magnification is needed to better characterize the large proportion of undetermined follicles.

Ki-67 Index as an Adjunct Histological Marker for Grading Cartilage Tumors: A Pilot Study

Objectives Distinguishing enchondromas (grade 0) from grade 1 chondrosarcomas, recently classified as atypical cartilaginous tumors (ACT), is challenging due to histologic and radiographic similarity. However, diagnostic ambiguity poses a management dilemma: enchondromas are observed, while ACTs are excised. Protein Ki-67 is a cellular proliferation nuclear marker used as a proxy for aggressiveness in other tumors. This retrospective pilot study aimed to characterize the association between grade and Ki-67 index and evaluate its ability to aid differentiation between critical cartilage tumor grades. Methods Tissue blocks from 26 patients treated for enchondromas or chondrosarcomas within the past ten years were exposed to anti-Ki-67 rabbit monoclonal antibody and hematoxylin counterstain. Counting two high-power fields per sample yielded a Ki-67 index. Results There was a strong positive correlation between tumor grade and the Ki-67 index (r = 0.704, P<0.001). One-way analysis of variance (ANOVA) showed significant differences in the Ki-67 index between grades (P<0.001). Tukey’s post-hoc test demonstrated that enchondroma Ki-67 indexes were not significantly different from ACT Ki-67 indexes (P=0.094). Receiver operator curve cutoff index value of 13.4% differentiated enchondroma vs. ACT with 67.9% sensitivity and 42.9% specificity. Conclusion Ki-67 index is correlated with cartilage tumor grade. Although our study did not show a significant difference in the Ki-67 index between enchondromas from ACTs, this study provided a protocol for future investigations. Study limitations include small sample size and reliance on historical tissue blocks of variable preservation quality.

Predicting IHC staining classes of NF1 using features in the hematoxylin channel

Immunohistochemistry (IHC) highlights specific cell types in tissues and traditionally involves antibody staining together with a hematoxylin counterstain. The intensity and pattern of hematoxylin staining differs between cell types and reveals morphological characteristics of cells. Here, we propose that features in the hematoxylin stain can be used to predict IHC labels, such as Neurofibromin (encoded by the gene NF1). The dataset consists of 7.2 million cells from benign and kidney cancer cores in a tissue microarray. Morphology and hematoxylin (H&M) features defined within QuPath are subjected to a clustering analysis in CytoMap. H&M features are also used to train 4 different XGBoost models to predict high, low, and negative NF1 stain classes in benign renal tubules, clear cell (ccRCC), papillary (PRCC), and chromophobe (ChRCC) renal carcinoma. The prediction accuracies of NF1 staining classes in benign, ccRCC, ChRCC, and PRCC range between 70% and 90% with areas under the precision recall curve PRAUCNF1-high = 0.82+0.12, PRAUCNF1-low = 0.62+0.25, and PRAUCNF1-negative = 0.83+0.16. The most important feature for predicting the NF1 class involves the minimum cellular hematoxylin staining intensity. Together, these results demonstrate the feasibility to predict NF1 expression solely from features in hematoxylin staining using open source software. Since the hematoxylin features can be obtained from regular H&E and IHC slides, the proposed workflow has broad applicability.

Initial Correlation Analysis of Diffusion Basis Spectrum Imaging of Alzheimer's Brain and Quantitative Histology

AbstractBackgroundDiffusion Basis Spectrum Imaging (DBSI), a novel MR imaging method, can be utilized as a biomarker in Alzheimer's disease (AD) staging and progression. By distinguishing diffusion effects from other concomitant disorders like axonal injury/loss and demyelination, it is capable of specifically evaluating cellularity and edema in AD brains. To test the hypothesis that DBSI cell fraction and mean cell apparent diffusion coefficient (ADC) correlate with measures of cell density and neuroinflammation, we employed high‐resolution ex vivo DBSI sequences and quantitative digital histology on post‐mortem human AD brain tissue.MethodA 15 × 20 × 6 mm3 sample of middle frontal gyrus (coronal plane) from a formalin‐fixed post‐mortem human AD brain (Knight ADRC) was incubated in phosphate‐buffered saline for 48 hours and then imaged with a Varian 11.7T MRI, using 2D spin‐echo sequence with 20‐direction diffusion encoding (max b = 3900 s/mm2, voxel = 0.25 × 0.25 × 1 mm). After MRI, it was precisely bisected along the plane of imaging, and processed into paraffin. Slide‐mounted 6 µm histologic sections were stained with H&E and DAB immunohistochemistry (IBA‐1 antibody, hematoxylin counterstain) and digitized (20× magnification, Hamamatsu NanoZoomer). Maps of nuclei and microglia density were computed by segmenting nuclei and microglia respectively using Orbit Image Analysis and in‐house MATLAB programs. These histology maps were registered and downsampled to the corresponding DBSI maps. We generated cell fraction maps and mean cell ADC maps from MR images using in‐house DBSI software by computing the fraction and mean ADC of restricted isotropic diffusion (0 – 0.4 ×10‐3 mm2/s in each image voxel, respectively.ResultAmong 71 evenly spaced regions of interest (ROIs, 2 × 2 mm2) drawn across the entire specimen, the mean DBSI cell fraction correlated with nuclei density (R = 0.74, P < 0.001). Additionally, among 42 ROIs drawn in the white matter region, the mean cell ADC in each ROI correlated with microglia density (R = 0.54, P < 0.001).ConclusionOur findings demonstrated the feasibility of DBSI technique in ex vivo AD brain, which could improve our understanding of neuroinflammation in AD pathogenesis, early diagnostics and new disease‐modifying therapies targeting the immune response.

Cribriform pattern in prostate tissues: Predictor for intraductal carcinoma of the prostate based on biopsy and radical prostatectomy pathology.

This study aims to investigate whether a cribriform pattern on prostate biopsy may be a factor in suspicion of intraductal carcinoma of the prostate after radical prostatectomy. This retrospective study assessed 100 men who underwent prostatectomy from 2015 to 2019. Participants were grouped as 76 patients with Gleason pattern 4 and 24 patients without this pattern. All 100 participants underwent retrograde radical prostatectomy and limited lymph node dissection. The same pathologist evaluated all specimens. The cribriform pattern was evaluated with haematoxylin and eosin counterstaining, and intraductal carcinoma of the prostate was evaluated with immunohistochemical analysis of cytokeratin 34βE12. Patients with intraductal carcinoma of the prostate on immunohistochemical analysis showed a significant tendency to relapse in the postoperative period, and those with the cribriform pattern on biopsy had a significant recurrence rate. In univariate and multivariate analyses, intraductal carcinoma of the prostate confirmed in biopsy tissue was an independent predictor of biochemical recurrence after prostatectomy. The rate of intraductal carcinoma of the prostate confirmation was 28% of cases with a cribriform pattern in biopsy tissue, which was increased to 62% in prostatectomy tissues. The cribriform pattern in the biopsy tissue may be a predictor for intraductal carcinoma of the prostate.

Abstract 2040: Invisible chromogens expand brightfield multiplexing and enable combined protein expression and morphological analysis

Abstract Anatomical pathology relies upon visual evaluation of clinical specimens using brightfield microscopy. This necessarily utilizes histological stains and immunohistochemical (IHC) chromogens that absorb visible light, between 430 and 690 nm. To complement the visible stains, we developed chromogens for use in IHC that absorb light outside the visible range, and imaging methods for detecting dyes absorbing from the ultraviolet (UV) through the near infrared (NIR). Invisible chromogens partnered with conventional visible stains on the same slide provides complementary information, enabling pathologists to gain deeper insights from a patient’s tissue. Methods: Invisible IHC was performed using conventional antibodies and antibody-enzyme conjugates utilizing covalently deposited chromogens absorbing in the UV and NIR. This was combined with IHC using visible chromogens or histological stains. Resulting stained specimens were evaluated interactively in real-time using a dual-camera microscope system comprising color and monochrome cameras, or by recording single microscope fields using multispectral imaging and the monochrome camera. Results: Multiplex IHC was performed with different combinations of visible and invisible chromogens, with the highest level of multiplexing - 7 biomarkers + hematoxylin counterstain (5 visible IHC chromogens plus a UV and an NIR chromogen) - demonstrated on a formalin-fixed paraffin-embedded (FFPE) prostate tumor. Images of each stain were recorded using multispectral imaging, and staining patterns were concordant with single stain DAB analysis on serial sections. In addition to increased IHC multiplexing capacity, multiplexing of IHC with histological stain was demonstrated by combining an NIR absorbing chromogen, identifying MART-1 expression, with H&E staining on FFPE melanoma specimens. The dual-camera microscope presented video of the visible H&E staining (color) next to the NIR IHC staining (monochrome) on the computer monitor as the specimen was manually scanned. Archiving was afforded by multispectral imaging which also enabled quantitative analysis. An additional advantage of the NIR chromogen was the suppression of melanin pigment, the absorbance of which was significantly reduced in the NIR. Other conventional stains multiplexed with invisible IHC on colon, lung, pancreas, and tonsil FFPE tissues also showed good separation of conventional and IHC staining. Conclusion: Taking advantage of invisible chromogens, IHC multiplexing capacity can be increased and conventional histological stains can be combined with IHC on a single slide. Clinical benefits include saving precious specimen, enabling differentiation of cell populations by simultaneous expression of multiple biomarkers, and providing biomarker expression within the cellular and morphological context of conventional stains. Citation Format: Larry E. Morrison, Mark R. Lefever, Lauren J. Behman, Monesh J. Kapadia, Daniel R. Bauer. Invisible chromogens expand brightfield multiplexing and enable combined protein expression and morphological analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2040.

Prenatal exposure to di(n-butyl) phthalate delays the spermatogenic cycle in rats: Investigation using a BrdU-injection method

Di(n-butyl) phthalate (DBP) esters are plasticizers that are used to provide transparency and flexibility in household plastic products but can easily leach out to contaminate organisms and the environment. We investigated whether prenatal DBP exposure affects spermatogenesis in rats. Pregnant Sprague–Dawley rats were injected with DBP 10, 50, and 100 mg/kg, or vehicle, administered intragastrically, on gestation days 12–21. At 9 or 17 weeks, 5-bromodeoxyuridine (BrdU) 50 mg/kg was injected intraperitoneally, and one testis was removed 3 h later. The remaining testis was excised 12.95 days + 3 h after the BrdU injection. Immunohistochemical analysis of BrdU was performed with periodic acid-Schiff and hematoxylin counterstaining for a quantitative analysis of the delay in one cycle of spermatogenesis. The DBP 100 mg group showed that the ratio of the appearance of seminiferous tubules in stages VII and VIII were significantly decreased, but those of stages IX and X were significantly increased compared to the Vehicle group. The reference value for the duration of spermatogenesis per cycle was set at 310.8 h. The DBP 100 mg group showed a significant delay in the duration of one cycle of spermatogenesis (16.95 h at puberty and 19.01 h at adulthood) compared with the Vehicle group. This study determined that F1-generation rats with prenatal DBP 100 mg exposure revealed significant accumulation of spermatogenic cells at stages IX to X in the second and third cycles, and the significant delay in the duration of spermatogenesis was more prominent at adulthood than in puberty.

Identification of Vasculogenic Mimicry in Histological Samples.

Vasculogenic mimicry has been identified in several malignancies and is generally associated with aggressive tumor growth and increased risk of metastasis. Patterned matrix can be identified in light microscopy of tumor sections stained with periodic acid-Schiff (PAS) without hematoxylin counterstain. In this chapter, the process is comprehensively described including tissue sources, formalin fixation and paraffin embedding, staining protocols, and the method for pattern identification in the microscope. Specific pattern types are illustrated in figures, and a number of pitfalls are detailed. The text can be used as a guideline by any researcher or clinician that wishes to evaluate histological samples for the presence of vasculogenic mimicry.

Quality improvement Initiative to eliminate silver precipitate during Periodic Acid Silver Methenamine Stain (PAMS) automated protocol of renal biopsies at McGill University Health Centre

Abstract Introduction/Objective Annually, about 400 renal biopsies are processed at the McGill University Health Centre (MUHC) pathology laboratory located in Montreal, Canada. One of the stains used to visualize the glomerular basement membrane is Periodic Acid Silver Methenamine Stain (PAMS). In August 2020, a strong, granular precipitate of silver was noted during PAMS automated staining resulting in uninterpretable results and delay in the diagnosis. Based on a sample analysis, this problem affected 21 % of kidney biopsies. Methods/Case Report A cause-and-effect workflow was developed for systematic assessment of potential causes of the granular precipitate including pre-analytical and analytical factors. Some of the pre-analytical factors included length of time spent in transport before fixation and patient factors that predisposed precipitate formation. Analytical factors were categorized as fixation problems (temperature, pH, duration), embedding problems (parafilm temperature, cooling method, type of parafilm), slide preparation (temperature, water bath pH, dehydration and further processing steps), microtone parameters (microtone calibration, thickness, laboratory technologist expertise), automatic staining parameters (cartridge age, hematoxylin counterstain duration, wash-out period etc.) and coverslip placement (adhesive type, temperature, drying). Results (if a Case Study enter NA) Following our systematic approach, the cause of granular precipitate was identified as the timing of hematoxylin counterstain. A portion of renal biopsy tissue was taken from parafilm blocks of previouslly reported cases of patients with membranous glomerulonephritis to further test the hypothesis by introduction of various incubation times with the hematoxylin counterstain. Conclusion Best PAMS staining was attained when no hematoxylin counterstain was employed (instead, neutral red counterstain for 70 seconds was used). PAMS staining with hematoxylin counter stain for no more than 60 seconds was found to be acceptable for the interpretation of glomerular pathology.

Abstract 2813: A multiplexed, multispectral approach to analyzing the immune microenvironment of oral potentially malignant lesions

Abstract Objectives: Tissue sections of oral potentially malignant lesions (OPML) can be used not only to provide pathological assessment (diagnosis) but could also be used to analyze the interactions between cellular populations, signaling molecules, and structural proteins that impact the clinical course of disease. However, much of this information remains unpacked due to methodological limitations. Traditional immunohistochemistry (IHC) limits the number of proteins able to be simultaneously analyzed within a tissue section and is also prone to human error in its analysis. Newer multiplexed IHC (mIHC) methods involving repeated cycles of staining allow for quantification of a greater number of markers; however, tissue integrity may be compromised, and complexity is added to the interpretation of the results. There is a need to develop an immunostaining method that overcomes these barriers. Hypothesis: mIHC and an in-house Hyperspectral Cell Sociology (HCS) platform will allow for robust and detailed investigation of the immune microenvironment of OPML compared to traditional IHC staining and scoring techniques. Methods: Automated mIHC staining with a seven immune marker panel was completed on annotated formalin-fixed paraffin-embedded OPML tissue. A cocktail of three primary antibodies was applied, then antigens and chromogens stripped using SDS-glycine before second round staining with four antibodies and a hematoxylin counterstain was completed. Slides were then digitally imaged, regions of interest selected in conjunction with an oral pathologist and staining quantified computationally using the HCS platform. Traditional IHC was completed on a randomized subset of cases using sequentially cut tissue sections and a double-staining technique. Scoring was completed by two blinded clinicians. Results: One cycle of multiplexed staining and de-staining allowed for the detection of seven markers on one section while minimizing loss of tissue integrity. The HCS platform captures a single tissue section at multiple wavelengths, enabling the unmixing of multiple overlapping, colocalized chromogens. The resulting set of images displayed each stain separately, allowing for nuclei segmentation and the generation of a map of the epithelium and underlying connective tissue. True positive cells for each stain were demarcated on this map, allowing for investigation of marker positivity, co-positivity, cell to cell spatial relationships, and layer-based analysis, compared to cell count and density analyses obtained with traditional IHC. Conclusion: The immune microenvironment contains a wealth of information pertaining to the biology, pathogenesis, and outcome of disease. Multiplexed staining and imaging methods to analyze and unpack this information is of great clinical utility. Citation Format: Iris Lin, Kouther Noureddine, Paul Gallagher, Martial Guillaud, Lewei Zhang, Leigha Rock, Miriam Rosin, Denise Laronde. A multiplexed, multispectral approach to analyzing the immune microenvironment of oral potentially malignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2813.

Overexpression of D-amino acid oxidase prevents retinal neurovascular pathologies in diabetic rats.

Diabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy. Rats were used as a model of diabetes by i.p. injection of streptozotocin at the age of 2months and blood glucose was monitored with a glucometer. Quantitative real-time PCR was used to examine Dao mRNA and western blotting to examine targeted proteins in the retinas. Bisulphite sequencing was used to examine the methylation of Dao mRNA promoter in the retinas. Intravitreal injection of DAAO-expressing adenovirus (AAV8-DAAO) was conducted one week before streptozotocin administration. Brain specific homeobox/POU domain protein 3a (Brn3a) immunofluorescence was conducted to indicate retinal ganglion cells at 3months after virus injection. The permeability of the blood-retinal barrier was examined by Evans blue leakage from retinal capillaries. Periodic acid-Schiff staining and haematoxylin counterstaining were used to indicate retinal vasculature, which was further examined with double immunostaining at 7months after virus injection. At the age of 12months, DAAO mRNA and protein levels in retinas from diabetic animals were reduced to 66.2% and 70.4% of those from normal (control) animals, respectively. The Dao proximal promoter contained higher levels of methylation in diabetic than in normal retinas. Consistent with the observation, DNA methyltransferase 1 was increased in diabetic retinas. Injection of DAAO-expressing virus completely prevented the loss of retinal ganglion cells and the disruption of blood-retinal barrier in diabetic rats. Diabetic retinas contained retinal ganglion cells at a density of 54 ± 4/mm2, which was restored to 68 ± 9/mm2 by DAAO overexpression, similar to the levels in normal retinas. The ratio between the number of endothelial cells and pericytes in diabetic retinas was 6.06 ± 1.93/mm2, which was reduced to 3.42 ± 0.55/mm2 by DAAO overexpression; the number of acellular capillaries in diabetic retinas was 10 ± 5/mm2, which was restored to 6 ± 2/mm2 by DAAO overexpression, similar to the levels in normal retinas. Injection of the DAAO-expressing virus increased the expression of occludin and reduced gliosis, which were examined to probe the mechanism by which the disrupted blood-retinal barrier in diabetic rats was rescued and retinal neurodegeneration was prevented. Altogether, overexpression of DAAO before the onset of diabetes protects against neurovascular abnormalities in retinas from diabetic rats, which suggests a novel strategy for preventing diabetic retinopathy. Graphical abstract.

S2852 Tissue Invasive Cytomegalovirus Enteritis With Gastric Outlet Obstruction in an Immunocompetent Patient

INTRODUCTION: Cytomegalovirus (CMV) usually causes asymptomatic or mononucleosis-like syndrome in immunocompetent individuals. We present a case of severe tissue invasive gastrointestinal CMV in an immunocompetent individual complicated by gastric outlet obstruction. CASE DESCRIPTION/METHODS: A 72-year-old man with a history of hypertension presented with vomiting, postprandial abdominal pain, and hematochezia for one week. He had also lost 40lbs in three months. Physical examination was normal except for epigastric tenderness. The previous colonoscopy done 5 years ago was normal. No personal or family history of inflammatory bowel disease, gastrointestinal malignancy, or lymphoma. He had never had immunosuppressive therapy, organ transplantation, or any other biological agent. Initial blood work revealed Hemoglobin 10.9 g/dl, AST 64 IU/l, ALT 83 IU/l, and ALP 350 IU/l. His HIV and Tuberculosis screening were negative. Ultrasound and CT scan of the abdomen and pelvis were normal except for increased gallbladder wall thickness (3mm). An Esophagogastroduodenoscopy (EGD) revealed esophagitis, pyloric channel ulcer, and duodenal ulcer. Colonoscopy revealed an ulcer at the ileocecal valve. The biopsy of all the ulcers revealed ulcerated mucosa, architectural distortion, and reactive epithelial atypia suspicious of CMV infection which was later confirmed by immunohistochemical staining (IHC) [Figure 1]. Quantitative CMV DNA PCR analysis showed levels of 1739 IU/ml. He was started on intravenous ganciclovir while inpatient and sent home on oral valganciclovir for 21 days. His symptoms improved significantly after treatment. CMV DNA levels decreased to 1230 IU/ml post-treatment. The patient returned with symptoms of gastric outlet obstruction a few months later which were getting worse. His barium X-ray revealed a delayed gastric emptying time with pyloric stenosis and partial malrotation of the small bowel [Figure 2]. A repeat EGD revealed pyloric stenosis with an inner diameter of 11 mm, which was dilated endoscopically. The biopsy taken from an antral ulcer was negative for CMV on IHC staining. There were no other signs of prior CMV infection on the EGD. DISCUSSION: The gastric outlet obstruction in the patient is likely a long term sequela of CMV. Due to the undertreatment and delay in diagnosis, CMV infections can lead to long term and potentially life-threatening complications. Therefore, gastrointestinal CMV should be considered in immunocompetent patients and aggressively treated.Figure 1.: Medium power (100×) Immunohistochemical staining for CMV with hematoxylin counter-stain. A representative high power view demonstrating strong focal CMV immunoreactivity.Figure 2.: Barium Xray reveals pyloric stenosis (black arrow) and malrotation with the first loop of jejunum to the right.

Neuronal Membrane Disruption Occurs Late Following Diffuse Brain Trauma in Rats and Involves a Subpopulation of NeuN Negative Cortical Neurons.

The repercussions of traumatic brain injury (TBI) endure years following the initial insult and involve chronic impairments/disabilities. Studies indicate that these morbidities stem from diffuse pathologies, however, knowledge regarding TBI-mediated diffuse pathologies, and in particular, diffuse neuronal membrane disruption, is limited. Membrane disruption has been shown to occur acutely following injury, primarily within neurons, however, the progression of TBI-induced membrane disruption remains undefined. Therefore, the current study investigated this pathology over a longer temporal profile from 6 h to 4 w following diffuse TBI induced using the central fluid percussion injury (CFPI) model in rats. To visualize membrane disruption, animals received an intracerebroventricular infusion of tagged cell-impermeable dextran 2 h prior to experimental endpoints at 6 h, 1 d, 3 d, 1 w, 2 w, or 4 w post-CFPI. The percentage of total neurons demonstrating dextran uptake, indicative of membrane disruption, was quantified within the lateral neocortex layers V and VI from 6 h to 4 w post-injury. We found that membrane disruption displayed a biphasic pattern, where nearly half of the neurons were membrane disrupted sub-acutely, from 6 h to 3 d post-TBI. At 1 w the membrane disrupted population was dramatically reduced to levels indistinguishable from sham controls. However, by 2 and 4 w following CFPI, approximately half of the neurons analyzed displayed membrane disruption. Moreover, our data revealed that a subset of these late membrane disrupted neurons were NeuN negative (NeuN-). Correlative western blot analyses, however, revealed no difference in NeuN protein expression in the lateral neocortex at any time following injury. Furthermore, the NeuN- membrane disrupted neurons did not co-label with traditional markers of astrocytes, microglia, oligodendrocytes, or NG2 cells. Immunohistochemistry against NeuN, paired with a hematoxylin and eosin counter-stain, was performed to quantify the possibility of overall NeuN+ neuronal loss following CFPI. A NeuN- population was observed consistently in both sham and injured animals regardless of time post-injury. These data suggest that there is a consistent subpopulation of NeuN- neurons within the lateral neocortex regardless of injury and that these NeuN- neurons are potentially more vulnerable to late membrane disruption. Better understanding of membrane disruption could provide insight into the mechanisms of diffuse pathology and lead to the discovery of novel treatments for TBI.

Applicability of eucalyptus wood waste extracts for the histological staining of connective tissue

ABSTRACT Biological staining of tissue is a crucial procedure in histotechnology. Rudimentary methods for section preparation have often used stains from natural products, although use of synthetic dyes has become the contemporary standard. Artificial dyes increase the operating costs of a laboratory as well as increase the environmental and personnel risks during manufacturing and usage. These considerations have stimulated research to find alternative natural stains from the wide diversity of plant species. The present study investigated the effect of Eucalyptus sp. (Myrtaceae) wood waste extract on histological staining of animal tissues, using a series of pigment concentrations, pH conditions, and temperatures. Eucalyptus wooden slivers were dried, milled, and 1 g, 2 g, and 4 g of the fine powder was subjected to 50% ethanol extraction for 2 days. Staining tests were then performed on formalin fixed paraffin embedded (FFPE) sections. Increasing acetic acid concentrations (1%, 2% and 4%) were added to the extracts and compared to an acid-free extract. Staining was performed at both ambient room temperature (RT) and 60°C. Connective tissue acidophilic components were well-contrasted and a hematoxylin counterstain demonstrated distinct structural differences between matrix and cell nuclei. Therefore, the present findings demonstrate the potential utility of the eucalyptus wood extracts application as a natural stain alternative for routine histology.

Neural cell adhesion molecule (NCAM) and polysialic acid–NCAM expression in developing ICR mice

Abstract Background Coexpression of polysialic acid (PSA)–neuronal cell adhesion molecule (NCAM) with immature neuronal markers is used to indicate the developmental state of neurons generated in the subgranular zone (SGZ) of adult hippocampus. PSA–NCAM is highly expressed throughout the embryonic and juvenile mammalian brain, but heavily downregulated in adult brain. Objective To visualize the expression profiles of NCAM/PSA–NCAM in the dentate SGZ of the hippocampus in developing ICR mice. Methods Cellular distribution, expression, and developmental changes of NCAM/PSA–NCAM were studied in ICR mice at embryonic age 17 days (E17); and similarly at postnatal ages P3, P5, and P7. The SGZ was studied using NCAM and PSA–NCAM immunoreactive staining with or without hematoxylin counterstaining. Western blotting was used to confirm protein expression levels. Results NCAM expression was localized to the surface of neurons and glia and was higher in postnatal mice than it was in embryonic mice. PSA–NCAM was found in cytoplasm and membrane of neural cells, more densely staining in the dentate SGZ at P7, but no staining found at E17. Western blotting of brain tissues also showed expression of both PSA–NCAM and NCAM increased significantly at P5 and P7 compared with expression at P3. Conclusions Progressive increase in NCAM expression occurs in the SGZ during embryogenic and postnatal development. PSA–NCAM was not expressed in embryonic ICR mice, but was increased after birth and highly localized in the SGZ at P7. This NCAM expression pattern in the developing brain indicating structural plasticity and neurogenesis may be useful for study of brain repair.

Hematoxylin and Eosin Counterstaining Protocol for Immunohistochemistry Interpretation and Diagnosis.

Hematoxylin and eosin (H&E) staining is a well-established technique in histopathology. However, immunohistochemistry (IHC) interpretation is done exclusively with hematoxylin counterstaining. Our goal was to investigate the potential of H&E as counterstaining (H&E-IHC) to allow for visualization of a marker while confirming the diagnosis on the same slide. The quality of immunostaining and the fast-technical performance were the main criteria to select the final protocol. We stained multiple diagnostic tissues with class I IHC tests with different subcellular localization markers (anti-CK7, CK20, synaptophysin, CD20, HMB45, and Ki-67) and with double-staining on prostate tissues with anti-high molecular weight keratins/p63 (DAB detection) and p504s (alkaline phosphatase detection). To validate the efficacy of the counterstaining, we stained tissue microarrays from the Canadian Immunohistochemistry Quality Control (cIQc) with class II IHC tests (ER, PR, HER2, and p53 markers). Interobserver and intraobserver concordance was assessed by κ statistics. Excellent agreement of H&E-IHC interpretation was observed in comparison with standard IHC from our laboratory (κ, 0.87 to 1.00), and with the cIQc reference values (κ, 0.81 to 1.00). Interobserver and intraobserver agreement was excellent (κ, 0.89 to 1.00 and 0.87 to 1.00, respectively). We therefore show for the first time the potential of using H&E counterstaining for IHC interpretation. We recommend the H&E-IHC protocol to enhance diagnostic precision for the clinical workflow and research studies.

Tumor-collagen digital proximity signature to indicate prognosis in diffuse large B-cell lymphoma.

e19073 Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous tumor that originates from normal B-cells. Despite the use of combination chemotherapy, around 40% of DLBCL patients die (de Jonge, et al. European Journal of Cancer, 2016). Limited studies have investigated the role of collagen in the acellular tumor microenvironment. In this study, we present a novel digital signature of the proximity of tumor cells and collagen-VI (COL6) that can predict overall survival (OS) in DLBCL patients. To the best of our knowledge, this is the first study of its kind to employ automated image analysis. Methods: The proposed digital proximity signature (DPS) aggregates summary-level statistics from the entire whole slide image (WSI) and serves as a marker of regions, categorizing weak, moderate, significant, and strong tumor-collagen proximity and can be described as a surrogate for signaling. To accomplish this, we developed a novel artificial intelligence (AI) based multi-task model for simultaneous detection and classification of tumor cells and another bespoke method for automatically identifying COL6 fiber. The tumor-collagen proximity analysis was then performed by aggregating tumor cell statistics within the vicinity of COL6 fibres. Finally, the prognostic significance of DPS for OS in DLBCL was investigated with Kaplan-Meier analysis, stratifying patients into two groups based on the median of the DPS values. Results: We took WSIs of DLBCL tissue slides for 32 cases immunohistochemically stained with COL6 and Hematoxylin counterstain. The AI model for tumor cell identification achieved a high F1-score of 0.84, outperforming recent single-task learning models. Our results show that strong proximity of COL6 and tumor cells is linked to better OS in DLBCL patients ( p = 0.03). Conclusions: Our novel digitally computed COL6-tumor proximity signature shows prognostic significance for overall survival on a pilot dataset of 32 DLBCL patients. We are further validating the utility of this novel signature as a prognostic biomarker in larger cohorts of DLBCL patients.

Abstract 5652: Validation of novel InSituPlex™ technology utilizing standard chromogenic IHC and multiplexed tyramide-based immunofluorescence

Abstract Introduction: The demands of today's immuno-oncology research has driven the development of more robust tools to interrogate the tumor microenvironment, specifically using multiplex immunofluorescence (mIF). Multiplex immunofluorescence allows investigators to visualize multiple biomarkers in tissue sections while preserving spatial context. The current gold standard of immunohistochemistry is “brown” staining using DAB with a hematoxylin counterstain. In recent years, multiplex immunofluorescence has been accepted in the field using tyramide molecules (TSA) that permanently fix fluorescent dyes to the tissue. Consequently, tissue integrity is compromised and cannot be utilized in additional forms of analysis. In addition, high-level plexing using TSA requires the use of highly specialized and costly imaging equipment. Here we validate a novel technology, InSituPlex™, utilizing DNA barcoding of antibodies and the detection of markers using simple DNA hybridization techniques. InSituPlex allows for high-leveling of multiplexing, preservation of tissue integrity for additional analyses, and implementation on a variety of commonly used imaging platforms. Methods: Performance of the InSituPlex technology was assessed using 15 immuno-oncology relevant markers (CD3, CD4, CD8, CD20, CD25, CD68, CD163, FoxP3, Granzyme B, Ki67, PD1, PD-L1, pan-cytokeratin, Sox10, and TTF1). Each marker was benchmarked by performing a single-plex DAB chromogenic IHC and an immunofluorescence assay. Antibodies to be used for InSituPlex testing were first conjugated to unique DNA barcodes and the resulting conjugates were assessed utilizing the previously described assays to ensure equivalent performance of the InSituPlex conjugate to unconjugated antibodies. The performance of the complete InSituPlex assay was evaluated using InSituPlex HRP labelled imaging strands and the newly barcoded antibodies. InSituPlex vs. DAB/TSA performance was compared across platforms. All staining was performed manually and on the Leica BOND RX autostainer. Images were acquired across multiple imaging platforms, including the Leica VERSA whole slide scanner, and image analysis was performed using HALO from Indica Labs. Results: All antibodies performed equally well across all the staining methodologies as assessed qualitatively. Quantitatively, cell counts were performed on serial sections. Statistical analysis comparing InSituPlex to standard chromogenic and immunofluorescent assays across all 15 antibodies did not yield any significance (p-value > 0.1). Conclusions: Detection of relevant immune and cancer related markers utilizing InSituPlex technology is equivalent to standard chromogenic IHC and immunofluorescent assays. Citation Format: Katir Patel, Courtney Hebert, Jamie Buell, Sean Downing. Validation of novel InSituPlex™ technology utilizing standard chromogenic IHC and multiplexed tyramide-based immunofluorescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5652.

MEDI0382, a GLP-1/Glucagon Receptor Dual Agonist, Shows Differential Brain Activation vs. Liraglutide in Mice

MEDI0382 is under development for the treatment of type 2 diabetes. MEDI0382 reduces food intake and body weight in obese mice and primates. In rodents, the weight-reducing effects of MEDI0382 are superior to those of the GLP-1 analogue, liraglutide. The current study characterised the activation of distinct neuroanatomical areas following peripheral administration of MEDI0382, relative to liraglutide and the glucagon receptor agonist, G1432. C57Blk6 mice were fasted for 4 h and then injected with a single dose (3 nmol/kg; s.c) of test agent or vehicle (n=4 each). After 2 h, mice were sacrificed and brains were dissected out and immersion-fixed in formalin (24-48 h). The brains were then cut into 3 mm serial slices and processed to paraffin blocks. Each block was sectioned entirely, sampling a 4µm step section every 100µm. Each section was immunohistochemically stained for c-fos, using a rabbit anti-fos polyclonal, with haematoxylin counterstain. Stained sections were digitally scanned at 20x magnification (Aperio AT2). The appropriate brain sections containing the area postrema (AP), the nucleus of the solitary tract (NTS), and the hypothalamic parabrachial (PBN) and arcuate nuclei were selected. These areas were manually delimited using the online Allen mouse brain atlas. The number of positive and negative nuclei were automatically counted (Halo), and reported as % of total positive cells and number of cells per tissue area. MEDI0382 significantly increased c-fos activation in the AP and NTS vs. vehicle, whereas liraglutide and G1432 showed no difference from vehicle. There was no significant increase in c-fos activation in the arcuate nucleus or PBN in any treatment group when compared to vehicle. In summary, the neuronal activation by MEDI0382 shows that distinct differences were observed compared to GLP-1 and glucagon agonists alone, suggesting that a different pattern of activation exists for the dual agonist, MEDI0382 vs. the monotherapies. Disclosure L.K. Burke: None. L. Brown: Stock/Shareholder; Self; AstraZeneca. J. Lapointe: Employee; Self; MedImmune. M. Sulikowski: None. A.R. Lewis: Employee; Self; MedImmune. Stock/Shareholder; Self; MedImmune. J. Trevaskis: Employee; Self; MedImmune. Stock/Shareholder; Self; AstraZeneca. D. Baker: Stock/Shareholder; Self; AstraZeneca.