Biopsy Fragments Research Articles

Phæohyphomycose à Roussoella percutanea

Phæohyphomycosis are opportunistic mycosis due to black (dematized) fungi. Although ubiquitous, these infections are more common in tropical countries. We report here the case of a 65-year-old man, of Congolese origin, in France for 40 years, who was consulting for a retro malleolar tumefaction evolving since 4 months. In his background, there was a kidney transplant for cancer a year ago. MRI revealed a mass of 3 cm in diameter without bone involvement. Histological examination revealed septate filaments. The absence of fistulas and grains ruled out the diagnosis of mycetoma. On a cutaneous biopsy fragment, pan-fungal PCR was performed. Sequencing analysis allowed identifying Roussoella percutanea.

CD8+ T cell infiltration in breast and colon cancer: A histologic and statistical analysis.

The prevalence of cytotoxic tumor infiltrating lymphocytes (TILs) has demonstrated prognostic value in multiple tumor types. In particular, CD8 counts (in combination with CD3 and CD45RO) have been shown to be superior to traditional UICC staging in colon cancer patients and higher total CD8 counts have been associated with better survival in breast cancer patients. However, immune infiltrate heterogeneity can lead to potentially significant misrepresentations of marker prevalence in routine histologic sections. We examined step sections of breast and colorectal cancer samples for CD8+ T cell prevalence by standard chromogenic immunohistochemistry to determine marker variability and inform practice of T cell biomarker assessment in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Stained sections were digitally imaged and CD8+ lymphocytes within defined regions of interest (ROI) including the tumor and surrounding stroma were enumerated. Statistical analyses of CD8+ cell count variability using a linear model/ANOVA framework between patients as well as between levels within a patient sample were performed. Our results show that CD8+ T-cell distribution is highly homogeneous within a standard tissue sample in both colorectal and breast carcinomas. As such, cytotoxic T cell prevalence by immunohistochemistry on a single level or even from a subsample of biopsy fragments taken from that level can be considered representative of cytotoxic T cell infiltration for the entire tumor section within the block. These findings support the technical validity of biomarker strategies relying on CD8 immunohistochemistry.

Is hematoxylin-eosin staining in rectal mucosal and submucosal biopsies still useful for the diagnosis of Hirschsprung disease?

BackgroundHematoxylin-eosin (HE) staining of a full-thickness rectal wall fragment is classically used for the diagnosis of Hirschsprung disease (HD). However, this technique requires large fragments for a better diagnosis. Additionally, the histochemical and immunohistochemical methods of staining small fragments of rectal mucosal and submucosal biopsies are not available in all centers. Therefore, the possibility of diagnosing HD through HE staining in these biopsies could be a valuable alternative for centers that do not have more specific techniques. The objectives of the current investigation were to evaluate the concordance of the results obtained by HE staining and the calretinin method with acetylcholinesterase (AChE) activity in fragments of mucosa and submucosa in the diagnosis of HD.MethodsFor this study, 50 cases from our laboratory were selected. The tissue material was embedded in paraffin. Sixty levels of each fragment were utilized for HE, and the other 3 levels were used for calretinin. These slides were analyzed under the microscope, photographed and classified as either positive for HD when no ganglion cells were found with nerve trunks present or as negative when ganglion cells were found. The results from reading the slides were compared with those of AChE.ResultsOf the 50 cases evaluated by the HE technique, only 5 contradicted the diagnosis based on AChE, with a Kappa value of 0.800 and an accuracy of 90%. In the comparison between calretinin and AChE, 8 cases were discordant, with a Kappa value of 0.676 and an accuracy of 84%.ConclusionsThe concordance of results from AChE and HE methods was satisfactory, allowing for the potential use of the HE method for fragments of mucosa and submucosa as a valid alternative in the diagnosis of HD. The immunohistochemical technique of calretinin did not show good agreement with the AChE activity in our study.

Laparoscopic ovarian biopsy pick-up method for goats

Biopsy pick-up (BPU) has been considered a safe method to harvest ovarian fragments from live animals. However, no studies have been reported on the use of BPU to collect in vivo ovarian tissue in goats. The goals of this study were: (i) to test different biopsy needle sizes to collect ovarian tissue in situ using the BPU method (Experiment 1), and (ii) to study ovarian tissue features such as preantral follicle density, morphology, class distribution, and stromal cell density in ovarian fragments obtained in vivo through a laparoscopic BPU method (Experiment 2). In Experiment 1, goat ovaries (n = 20) were collected in a slaughterhouse and subjected to in situ BPU. Three needles (16, 18, and 20G) were tested. In Experiment 2, the most efficient biopsy needle from Experiment 1 was used to perform laparoscopic BPU in goats (n = 8). In Experiment 1, the recovery rate was greater (P < 0.05; range 50–62%) with 16G and 18G needles than the 20G (17%) needle. The mean weight of ovarian fragments collected by the 16G needle was greater (P < 0.05) than the 18G and the 20G needle. In Experiment 2, 62 biopsy attempts were performed and 52 ovarian fragments were collected (90% success rate). Overall, 2054 preantral follicles were recorded in 5882 histological sections analyzed. Mean preantral follicular density was 28.4 ± 1.3 follicles per cm2. The follicular density differed (P < 0.05) among animals and ovarian fragments within the same animal. The mean stromal cell density in the ovarian fragments was 37.1 ± 0.5 cells per 2500 μm2, and differed (P < 0.05) among animals. Moreover, preantral follicle density and stromal cell density were associated (P < 0.001). The percentage of morphologically normal follicles was 70.1 ± 1.2, and differed (P < 0.05) among animals. The majority (79%) of the morphologically normal follicles was classified as primordial follicles, and differed (P < 0.05) among animals and between ovaries. In summary, a laparoscopic BPU method has been developed to harvest ovarian tissue in vivo with a satisfactory success rate in goats. Furthermore, this study described for the first time that goat ovarian biopsy fragments have a high heterogeneity in follicular density, morphology, class distribution, and stromal cell density.

Stool Ball Impaction and Obstipation: An Atypical Presentation of Clostridium difficile Infection

The hallmark manifestation of Clostridium difficile infections (CDI) has always been diarrhea and is a key point in identifying patients who may be infected. Throughout the years, a number of risk factors have been associated with CDI including antibiotic use and hospitalizations. Here we present an unusual case of CDI where a healthy patient without any identifiable risk factors presented with obstipation, and, whose diagnosis of CDI was critical to her improvement. A 35-year-old female with no significant past medical history of constipation, recent hospitalization, use of antibiotics or medications, or sick contacts, presented with left lower quadrant abdominal pain and constipation for four days. An abdominal X-ray was negative for obstruction and was initially prescribed magnesium citrate and enemas. Her symptoms worsened, as she developed a fever and leukocytosis of 19k/ul. A CT scan of the abdomen showed a 4cm circular mass in the sigmoid colon with proximal colon distended with stool, distal decompression, and fat stranding around the sigmoid colon (Img2). A flexible sigmoidoscopy showed sigmoid colitis with pseudomembranes, along with a dense, impacted stool ball that was not able to be moved or fragmented. A stool specimen taken was positive for clostridium difficile antigen and PCR toxin, confirming CDI. Biopsies also confirmed pseudomembranes colitis (Img1). Metronidazole was started with quick resolution of obstipation and leukocytosis. She was safely discharged home to complete the course of antibiotics. Our case seems to be the first of its kind in reporting this atypical presentation of community acquired CDI in a healthy young patient with obstipation related to a stool ball impaction without diarrhea or classical risk factors. While it is acknowledged that ileus is a complication of CDI and occasionally formed stool or a rectal swab may be needed to confirm the diagnosis, guidelines still focus on testing patients with diarrhea. Physicians should have an appreciation of the spectrum in which CDI may present and be prepared to initiate a work up even in the absence of diarrhea. Sigmoidoscopy was instrumental in this case and should also be considered as part of the diagnostic armamentarium. As our understanding of CDI is rapidly evolving, we hope to contribute to the current literature with this uncommon presentation of a common disease, thus increasing the role of physicians in making a timely diagnosis and delivering effective treatment.Figure: Mucosal biopsy fragment showing patchy mucosal injury with necrosis, and separate fragments of necroinflammatory debris suggesting pseudomembranous colitis.Figure: 4cm stool ball identified in the sigmoid colon with fat stranding along with proximal distension and distal compression.

HER2 testing in advanced gastric and gastro-oesophageal cancer: analysis of an Australia-wide testing program

This Australian human epidermal growth factor receptor 2 (HER2) testing program aimed to analyse >800 cases tested in a coordinated setting to further evaluate the criteria to establish HER2 status in advanced gastric and gastro-oesophageal junction (GOJ) cancer. Heterogeneity, and minimum number of biopsy fragments for reliable HER2 assessment were also examined in a subset of samples. Five laboratories tested 891 samples referred to determine HER2 status for potential anti-HER2 treatment. Cancer site, specimen type (endoscopic biopsy/resection/metastases), immunohistochemistry (IHC) score, HER2 gene and CEP17 copy number (CN) and HER2:CEP17 ratios were recorded. Samples were derived from stomach (53.1%), GOJ (28.2%) or metastases (18.5%). IHC for HER2 and dual probe HER2:CEP17 in situ hybridisation (ISH) were performed in parallel. A stringent definition (SD) of HER2 positivity was used (IHC2+/3+plus CN>6 and ratio>2) and compared with other published criteria. HER2 positive rate was 13.9% (114/820) by SD, and 12.9-16.0% using other definitions. There was higher concordance between IHC and HER2 CN by ISH than with ratio. The HER2 positive rate was significantly higher in GOJ samples than others (p=0.03) and in endoscopic biopsies than resections (p=0.047). In a subset of 98 positive cases, 39 (39.8%) showed heterogeneity, and in 282 endoscopic biopsies positivity rate plateaued at five tumour fragments, suggesting this is the minimum number of biopsies that should be examined.

Coeliac disease in infants: antibodies to deamidated gliadin peptide come first!

BackgroundThe onset of coeliac disease (CD) in the first year of life is uncommon and the diagnosis can be challenging due to the suboptimal sensitivity of tissue transglutaminase antibodies (tTG) at this age and the many other possible causes of malabsorption in infants. Antibodies to deamidated gliadin peptides (anti-DGPs), especially IgG, may appear earlier than IgA anti-tTG in very young children with CD.Case presentationWe report here on an 8-month-old child who was evaluated for failure to thrive, constipation and developmental delay. The symptoms started following gluten introduction in the diet. Laboratory tests showed high fecal elastase concentration, normal serum IgA levels with positive IgG and IgA anti-DGPs, whereas anti-tTG were not detected. The duodenal biopsy revealed a complete villous atrophy (Marsh-Oberhuber 3C). The culture of biopsy fragments in the presence of gliadin peptides did not stimulate the production of IgA anti-endomysial antibodies. Genetic testing proved the child was positive for HLA-DQ2 (DQA1*05; DQB1*02) and HLA-DQ8 (DQA1*03, DQB1*0302). Having initiated the gluten-free diet, the symptoms disappeared and the infant experienced rapid catch-up growth with normalization of psychomotor development.ConclusionsThis case report highlights the utility of anti-DGPs for screening infants with suspected CD. The pattern with positivity for IgG and IgA anti-DGPs only is rare in IgA-competent children with biopsy-proven CD. It could be explained in infancy as immaturity of the adaptive immune system.

Frequency of Celiac Disease in Children Presented with Liver Disease at a Tertiary Care Center

Background: Celiac disease is (CD) a genetically determined chronic inflammatory disease induced by an environmental precipitant. It is a multisystem disease and can develop at any point of time during life in genetically susceptible individuals upon ingestion of wheat gluten and related cereal proteins. The onset of symptoms in the atypical form generally occurs between 4-15 years of age. Diagnosis of CD with extraintestinal manifestations is frequently missed as it presents without diarrhoea.Objective: To observe the frequency of celiac disease in children with liver disease.Methods: This cross sectional study was conducted at BSMMU from January 2014 to June 2015. A total of 59 children (age 18 months to16 years) with clinical and biochemical features of liver disease were initially enrolled for the study. Their clinical history, examination findings and investigation reports were recorded in a data collection sheet and informed consent was obtained from parents. Routine investigations, liver function tests, tissue transglutaminase tTG (IgA), total IgA. were done. After exclusion of other causes of liver disease endoscopy of upper gastrointestinal tract (GIT) was done on patients who were tTG (titer of &gt;50 iu/mL) positive. Patients who were tTG negative but found IgA deficient (1 patient) was also selected for upper GI endoscopy and biopsy fragments were taken from second part of duodenum (D2) sent for histopathology.Results: Mean age of studied children was 8.33 ± 3.64 years. Out of 59 children with liver disease, 32.2% were tTG positive of whom female were 8 (13.6%) and male 11 (18.6%). Mean age at diagnosis of all patients with tTG positive group was 8.24 ± 2.78 (range 4-12) years. Among 19 sero-positive patients, short stature was found in 57.9% children. Mean Hb level in tTG positive group was 8.83 ± 2.64 gm/dl and in tTG negative group (10.27 ± 1.74 gm/dl). Sixteen (84.2%) out of 19 tTG positive patients had raised S. ALT. Out of 19 tTG positive children, endoscopy was done in 15 cases (endoscopy could not be done in 4 patients due to persistently raised PT) along with 1 patient who was IgA deficient. Endoscopic changes were mosaic and scalloping of D2 mucosa in 1 and 2 cases respectively. Histological changes compatible with CD were found in 5 (31.3%) patients. Marsh 3a category was found in 2 (12.5%) cases and 3b in 3 (18.8%) cases.Conclusion: In the present study, 32.2% liver disease cases were found tTG positive. Histological changes compatible with CD were found in about one-third cases. Screening for celiac disease may be included in the diagnostic tests for evaluation of liver disease in children.Bangladesh J Child Health 2016; VOL 40 (3) :144-148

Ki-67 labeling index of neuroendocrine tumors of the lung has a high level of correspondence between biopsy samples and surgical specimens when strict counting guidelines are applied

Optimal histopathological analysis of biopsies from metastases of neuroendocrine tumor (NET) of the lung requires more than morphology only. Additional parameters such as Ki-67 labeling index are required for adequate diagnosis, but few studies have compared reproducibility of different counting protocols and modalities of reporting on biopsies of lung NET. We compared the results of four different manual counting techniques to establish Ki-67 LI. On 47 paired biopsies and surgical specimens from 22 typical carcinoids (TCs), 14 atypical carcinoids (ACs), six large cell neuroendocrine carcinomas (LCNECs), and five small cell carcinomas (SCCs) immunohistochemical staining of Ki-67 antigen was performed. We counted, in regions of highest nuclear staining (HSR), a full ×40-high-power field (diameter=0.55mm), 500 or 2000 cells, or 2mm2 surface area, including the HSR or the entire biopsy fragment(s). Mitoses and necrosis were evaluated in an area of 2mm2 or the entire biopsy fragment(s). Between the four counting methods, no differences in Ki-67 LI were observed. However, a Ki-67 LI higher than 5% was found in only four cases when in an HSR, 500 cells were counted (18%), five (23%) when in an HSR 2000 cells were counted, four (18%) when 2mm2 were counted, and one (5%) TC case when the entire biopsy was counted. A 20% cutoff distinguished TC and AC from LCNEC and SCC with 100% specificity and sensitivity, while mitoses and necrosis failed to a large extent. Ki-67 LI in biopsy samples was concordant with that in resection specimens when 2000 cells, 2mm2, or the entire biopsy fragment(s) were counted. Our results are important for clinical management of patients with metastases of a lung NET.

Retroperitoneal Fibrosis: Case Series of 20 Patients

Retroperitoneal fibrosis (RPF) is a rare disease. The objective of this paper is to study the prevalence, clinical, biological and radiological aspects of the RPFand to specify its therapeutic modalities. This study provides descriptive evidence for a series of 20 patients with RPF. We conducted a retrospective study of cases of RPF diagnosed in the Urology and Internal Medicine departments of the Ibn Sina Military Hospital in Marrakech over a five-year period from July 2010 to December 2015. The positive diagnosis of RPF was retained either on the basis of a histological analysis from a biopsy fragment or, in the absence of histological evidence, by the detection on a scan of an infiltrate or sleeve Regular and homogeneous tissue density, perivascular topography, surrounding the abdominal aorta, more or less extended to the iliac vessels and able to take the contrast after injection. They were 15 men and 5 women with an average age of 53 years with extremes of 41 to 74 years. Almost all patients had mainly lumbar and abdominal pain. An inflammatory syndrome existed in all cases and renal insufficiency in 15 cases. Radiological investigations showed unilateral or bilateral hydronephrosis in 16 cases. RPF was idiopathic in 14 patients. Secondary forms were present in 6 patients (three atheromatous diseases, one gastric tumor, one case associated with Riedel's thyroiditis and one case of systemic fibrosis associating RPF, Riedel's thyroiditis and liver fibrosis). The treatment consisted of corticosteroids in 19 cases, surgery in 17 cases associated with corticosteroid therapy or even immunosuppressant in the case of steroid-dependence. Two deaths were observed in this series; one of these two patients had developed cervix carcinoma with pleural and peritoneal metastases after 5 years of immunosuppressive therapy. The second died due to the complications of his gastric neoplasm. The general characteristics of patients included are similar to the other series of the literature. The frequency of secondary forms seems to be underestimated, hence the value of a rigorous etiological investigation. The therapeutic strategy and the use of the cortisone saving treatments should be specified by randomized therapeutic trials. The prognosis is generally good; however, the possibility of recurrences, the incidence of which is greater during the first five years, requires prolonged clinical, biological and radiological monitoring.

Intratumoral Genetic Heterogeneity in Rectal Cancer: Are Single Biopsies representative of the entirety of the tumor?

Demonstrate intratumoral genetic heterogeneity in rectal cancer. Several clinical management decisions in rectal cancer may be influenced by pretreatment biopsy information. However, in the setting of significant intratumoral heterogeneity, biopsies may not be representative of the entirety of the tumor and limit the reliability of the information provided from them for clinical decision management. Three fragments from a single rectal adenocarcinoma were chosen for whole-exome sequencing followed by mutation detection analysis. About 25 Gb of unambiguously mapped sequences were generated for each sample resulting in a median fold-coverage of 35x. Captured sequences mapped to the reference human genome were then used for the detection of somatic point mutations. Overall, 193 unique somatic point mutations were identified. Only 53 (27%) of these were shared by all three fragments, including known genes involved in early phases of the adenoma-carcinoma sequence (such as, APC). Approximately, 115 (59%) mutations were exclusively present in only one of the fragments, including mutations in "driver" genes (DNAH12). Jaccard distances showed a median distance of 0.603 for pair-wise comparison of fragments indicating significant heterogeneity between them. Considerable intratumoral heterogeneity is present among naive rectal cancers. The majority of point mutations detected in different fragments from rectal cancers are frequently unique to a single fragment. These findings support that gene mutations found on single pretreatment biopsies will not necessarily be representative of mutations present in the entirety of the tumor and therefore may limit the utility of the biological information provided by single biopsy fragments for clinical management decisions.

Cytocentrifuged biopsy fixative preparation: A simple cost-effective technique facilitating microscopic diagnosis of lumen-dwelling intestinal parasites.

Direct microscopic visualization is the most specific method for detecting intestinal parasites and is commonly achieved by stool examination or mucosal biopsy. However, postfixation, the intestinal biopsy fragment is often curled, and the entire surface of the biopsied mucosa is seldom viewed microscopically. Tissue processing further distorts morphology of the organisms and causes diagnostic difficulties. Examining multiple sections for parasite detection is time-consuming and often requires aid of special stains and/or immunohistochemistry. To overcome these disadvantages, we hypothesized that the fixative in which biopsies are transferred may provide a valid representation of the biopsied mucosal surface and therefore aid in the identification of mucosal surface parasites. Formalin in which biopsies were transferred was retained, stored at 4°C and processed with a cytocentrifuge. Totally, 120 consequent duodenal biopsy fixatives were processed in this way and the cytocentrifuged smears visualized after May-Grunwald-Giemsa staining. Findings of these smears were correlated with their corresponding formalin fixed paraffin embedded tissue sections. Cytocentrifuged formalin preparations were found to be representative of the mucosal surface contents. Giardia trophozoites were visualized in 10/120 preparations with distinct morphological characteristics which were seldom appreciable in tissue sections, eliminating the need for special stains. Furthermore, two of the corresponding histology sections did not demonstrate the parasites despite step sections, while in one case few parasites could be identified in the step sections. Cytocentrifuged fixative preparation is a simple and cost-effective technique which can be routinely employed for intestinal parasite characterization.

Missing rate for gastric cancer during upper gastrointestinal endoscopy: a systematic review and meta-analysis.

Esophagogastroduodenoscopy (EGD) is considered a very effective method to identify gastric cancer (GC). However, the existence of missed lesions has been frequently discussed. This systematic review and meta-analysis aimed at assessing the magnitude of missing GC diagnosis with EGD and its predictive factors. MEDLINE was searched to identify all studies assessing and reporting the proportion of missed GC diagnosis with EGD. Pooled proportion and negative predictive values were computed using the random-effects model and heterogeneity was assessed using the Cochrane Q-test and I. The studies included (n=22) were grouped by study design. The pooled negative predictive value was 99.7% (95% confidence interval 99.6-99.9%). Missed GCs proportion was 9.4% (95% confidence interval 5.7-13.1%), being 10.0% in studies including patients with negative EGD followed over time, 8.3% in studies including patients with GC, and 23.3% in studies evaluating the proportion of missed synchronous lesions. Mainly, missed cancers were located in the gastric body both in Eastern and in Western studies (39 and 47%, respectively). The majority of missed GCs were adenocarcinomas. Younger age (<55 years), female sex, marked gastric atrophy, gastric adenoma or ulcer, and inadequate number of biopsy fragments were reported as predictive factors for diagnostic failure. EGD is a very effective method to rule out GC. However, missing GC with EGD is not uncommon, with one out of 10 cancers being potentially missed. Interestingly, lesions were more often missed in the body and therefore a more rigorous protocol for endoscopy and biopsy should be implemented worldwide.

Prevalence of Propionibacterium acnes in Intervertebral Discs of Patients Undergoing Lumbar Microdiscectomy: A Prospective Cross-Sectional Study.

BackgroundThe relationship between intervertebral disc degeneration and chronic infection by Propionibacterium acnes is controversial with contradictory evidence available in the literature. Previous studies investigating these relationships were under-powered and fraught with methodical differences; moreover, they have not taken into consideration P. acnes’ ability to form biofilms or attempted to quantitate the bioburden with regard to determining bacterial counts/genome equivalents as criteria to differentiate true infection from contamination. The aim of this prospective cross-sectional study was to determine the prevalence of P. acnes in patients undergoing lumbar disc microdiscectomy.Methods and FindingsThe sample consisted of 290 adult patients undergoing lumbar microdiscectomy for symptomatic lumbar disc herniation. An intraoperative biopsy and pre-operative clinical data were taken in all cases. One biopsy fragment was homogenized and used for quantitative anaerobic culture and a second was frozen and used for real-time PCR-based quantification of P. acnes genomes. P. acnes was identified in 115 cases (40%), coagulase-negative staphylococci in 31 cases (11%) and alpha-hemolytic streptococci in 8 cases (3%). P. acnes counts ranged from 100 to 9000 CFU/ml with a median of 400 CFU/ml. The prevalence of intervertebral discs with abundant P. acnes (≥ 1x103 CFU/ml) was 11% (39 cases). There was significant correlation between the bacterial counts obtained by culture and the number of P. acnes genomes detected by real-time PCR (r = 0.4363, p<0.0001).ConclusionsIn a large series of patients, the prevalence of discs with abundant P. acnes was 11%. We believe, disc tissue homogenization releases P. acnes from the biofilm so that they can then potentially be cultured, reducing the rate of false-negative cultures. Further, quantification study revealing significant bioburden based on both culture and real-time PCR minimize the likelihood that observed findings are due to contamination and supports the hypothesis P. acnes acts as a pathogen in these cases of degenerative disc disease.

Lung intragraft donor-specific antibodies as a risk factor for graft loss

The effect of donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) on graft survival is recognized in lung transplantation, but not all serum DSAs appear to be harmful. We wondered whether in situ DSA detection from graft biopsy specimens could help in identifying lung transplant recipients (LTRs) at higher risk for graft loss. Class I and II HLA antibody single-antigen flow bead assays were performed in 53 LTRs to identify immunoglobulin G DSA in biopsy specimen eluates and in sera and to evaluate C1q binding ability of DSA in sera. Intragraft DSAs (gDSAs) were correlated with serum DSAs (sDSAs), clinical and histologic data, and graft survival. Twenty-eight (52.8%) LTRs had sDSAs, 12 (22.6%) had C1q-positive sDSAs, and 11 (20.8%) had gDSAs. Fifty sDSAs were found, among which 15 (30%) were C1q-positive and 14 (28%) were found in biopsy specimen eluates. One DSA was detected in the biopsy specimen only. Serum mean fluorescence intensity and biopsy fragment size were higher for sDSAs detected in biopsy specimens (p = 0.003 and p = 0.02, respectively). One-year post-biopsy graft survival was lower for LTRs with gDSAs (p = 0.008 by log-rank test). Presence of gDSA at the time of biopsy constituted a risk factor for graft loss in univariate (odds ratio, 6.67; 95% confidence interval [CI] 1.51-29.47; p = 0.008; hazard risk, 3.44; 95% CI, 1.47-8.01, p = 0.005) and multivariate (odds ratio, 5.85; 95% CI, 1.23-27.68; p = 0.03; hazard risk, 4.51; 95% CI, 1.83-11.13; p = 0001) analyses using logistic regression and a Cox proportional hazard model, respectively. In lung transplantation, gDSA appears to be a valuable biomarker to identify pathogenic DSA and LTRs with a higher risk for graft loss.

Effects of Cryoprotectant Agents on Equine Ovarian Biopsy Fragments in Preparation for Cryopreservation

The exposure effect of cryoprotectant agents (CPAs) on morphology of preantral follicles (PAFs), stromal cell and PAF densities, and area of equine ovarian fragments were evaluated. Three independent experiments with identical methodologies were performed. Each experiment was composed of one CPA (dimethyl sulfoxide, ethylene glycol, or propylene glycol) and was performed in three replicates. Ovarian biopsy fragments were harvested from six mares in each experiment and submitted to the cryoprotectants using four times of exposure (0, 10, 15, and 20 minutes). PAF and stromal cell densities, and area of the fragments were not affected (P > .05) by any of the CPAs throughout the time of exposure. However, the morphology of the PAFs was affected (P < .05) by the CPAs. In the propylene glycol and dimethyl sulfoxide, higher (P < .05) percentages of abnormal PAFs were observed at 10 and 20 minutes of exposure, respectively. The PAF morphology in the ethylene glycol treatments was not affected (P > .05) throughout the times of exposure. Positive correlations (r = 0.57–0.77; P < .001, power = 96%–99%) were identified between PAF density and stromal cell density in all experiments. In conclusion, (1) ethylene glycol seems to be a less harmful CPA to equine PAFs, (2) exposure to CPAs did not affect the cell density and area of ovarian fragments, (3) PAF density was positively correlated with stromal cell density, and (4) stromal cell density did not affect the morphology of PAFs.

Avaliações macroscópica e histológica do reparo da cartilagem articular equina tratada com microperfurações do osso subcondral associadas ou não à injeção intra-articular de cartogenina

Resumo O objetivo deste estudo foi avaliar o reparo da cartilagem hialina equina, por meio de análises macroscópica (através de videoartroscopia) e histológica (através de fragmentos de biopsia), em defeitos condrais induzidos na tróclea lateral do fêmur tratados pela técnica de microperfurações subcondral associada ou não com administração intra-articular de cartogenina. Foram utilizados seis equinos pesando em média (±DP) 342±1,58 kg, com a idade aproximada de 7,2±1,30 anos e escore corporal de 7,1±0,75, que foram submetidos a videoartroscopia para indução da lesão condral de 1 cm2 na tróclea lateral do fêmur e realização da técnica de microperfuração do osso subcondral de ambos os joelhos. Foram realizadas quatro aplicações semanais com 20 μM de cartogenina intra-articulares em um dos joelhos (grupo tratado) e solução de ringer com lactato na articulação contralateral (grupo controle). Após o período de 60 dias, foram feitas as avaliações macroscópicas, através de videoartroscopias, e histológicas, através de biopsia. Não foram observadas diferenças significativas nos escores macroscópicos e histológicos para reparação condral entre animais dos grupos tratados e não tratados (P&gt;0,05). De modo geral, a porcentagem média de cartilagem hialina no tecido de reparo (17,5%) foi condizente com a literatura internacional usando outros tipos de perfuração condral. Entretanto, não se observaram diferenças estatísticas entre grupos (P&gt;0,05). A terapia com cartogenina, segundo protocolo utilizado, não produziu melhora do processo cicatricial em lesões condrais induzidas e tratadas com microperfurações na tróclea lateral do fêmur em equinos.

Paraconsistent analysis network applied in the treatment of Raman spectroscopy data to support medical diagnosis of skin cancer.

Paraconsistent logic (PL) is a type of non-classical logic that accepts contradiction as a fundamental concept and has produced valuable results in the analysis of uncertainties. In this work, algorithms based on a type of PL-paraconsistent annotated logic of two values (PAL2v)-are interconnected into a network of paraconsistent analysis (PANnet). PANnet was applied to a dataset comprising 146 Raman spectra of skin tissue biopsy fragments of which 30 spectra were determined to represent normal skin tissue (N), 96 were determined to represent tissue with basal cell carcinoma, and 19 were determined to be tissue with melanoma (MEL). In this database, paraconsistent analysis was able to correctly discriminate 136 out of a total of 145 fragments, obtaining a 93.793% correct diagnostic accuracy. The application of PAL2v in the analysis of Raman spectroscopy signals produces better discrimination of cells than conventional statistical processes and presents a good graphical overview through its associated lattice structure. The technique of PAL2v-based data processing can be fundamental in the development of a computational tool dedicated to support the diagnosis of skin cancer using Raman spectroscopy.

The Mare Model to Study the Effects of Ovarian Dynamics on Preantral Follicle Features.

Ovarian tissue collected by biopsy procedures allows the performance of many studies with clinical applications in the field of female fertility preservation. The aim of the present study was to investigate the influence of reproductive phase (anestrous vs. diestrous) and ovarian structures (antral follicles and corpus luteum) on the quality, class distribution, number, and density of preantral follicles, and stromal cell density. Ovarian fragments were harvested by biopsy pick-up procedures from mares and submitted to histological analysis. The mean preantral follicle and ovarian stromal cell densities were greater in the diestrous phase and a positive correlation of stromal cell density with the number and density of preantral follicles was observed. The mean area (mm2) of ovarian structures increased in the diestrous phase and had positive correlations with number of preantral follicles, follicle density, and stromal cell density. Biopsy fragments collected from ovaries containing an active corpus luteum had a higher follicle density, stromal cell density, and proportion of normal preantral follicles. In conclusion, our results showed: (1) the diestrous phase influenced positively the preantral follicle quality, class distribution, and follicle and stromal cell densities; (2) the area of ovarian structures was positively correlated with the follicle and stromal cell densities; and (3) the presence of an active corpus luteum had a positive effect on the quality of preantral follicles, and follicle and stromal densities. Therefore, herein we demonstrate that the presence of key ovarian structures favors the harvest of ovarian fragments containing an appropriate number of healthy preantral follicles.

Preantral follicle density in ovarian biopsy fragments and effects of mare age

The aims of the present study were to: (1) evaluate preantral follicle density in ovarian biopsy fragments within and among mares; (2) assess the effects of mare age on the density and quality of preantral follicles; and (3) determine the minimum number of ovarian fragments and histological sections needed to estimate equine follicle density using a mathematical model. The ovarian biopsy pick-up method was used in three groups of mares separated according to age (5-6, 7-10 and 11-16 years). Overall, 336 preantral follicles were recorded with a mean follicle density of 3.7 follicles per cm2. Follicle density differed (P<0.05) among animals, ovarian fragments from the same animal, histological sections and age groups. More (P<0.05) normal follicles were observed in the 5-6 years (97%) than the 11-16 years (84%) age group. Monte Carlo simulations showed a higher probability (90%; P<0.05) of detecting follicle density using two experimental designs with 65 histological sections and three to four ovarian fragments. In summary, equine follicle density differed among animals and within ovarian fragments from the same animal, and follicle density and morphology were negatively affected by aging. Moreover, three to four ovarian fragments with 65 histological sections were required to accurately estimate follicle density in equine ovarian biopsy fragments.