Label-free screening of brain tissue myelin content using phase imaging with computational specificity (PICS)

Inadequate myelination in the central nervous system is associated with neurodevelopmental complications. Thus, quantitative, high spatial resolution measurements of myelin levels are highly desirable. We used spatial light interference microcopy (SLIM), a highly sensitive quantitative phase imaging (QPI) technique, to correlate the dry mass content of myelin in piglet brain tissue with dietary changes and gestational size. We combined SLIM micrographs with an AI classifying model that allows us to discern subtle disparities in myelin distributions with high accuracy. This concept of combining QPI label-free data with AI for the purpose of extracting molecular specificity has recently been introduced by our laboratory as phase imaging with computational specificity (PICS). Training on nine thousand SLIM images of piglet brain tissue with the 71-layer transfer learning model Xception, we created a two-parameter classification to differentiate gestational size and diet type with an accuracy of 82% and 80%, respectively. To our knowledge, this type of evaluation is impossible to perform by an expert pathologist or other techniques.

Probe-based confocal laser endomicroscopy for in vivo assessment of histological healing in ulcerative colitis: development and validation of the ENHANCE index

Background and aims Histological healing may represent the ultimate therapeutic goal in ulcerative colitis (UC), but it requires biopsies. Our aim was to develop a non-invasive index able to assess histological disease activity in ulcerative colitis using probe based confocal laser endomicroscopy (pCLE). Methods One hundred patients with quiescent UC were prospectively included in 5 French centres. After fluorescein intravenous injection, during colonoscopy, the colorectal mucosa was analysed by white light imaging, pCLE and then biopsied in different locations. Five endoscopists performed central reading of pCLE images blindly to clinical, endoscopic and histological data. One expert pathologist performed a central histological reading (Nancy index: gold standard). An univariate and multivariate analysis were performed to identify the endomicroscopic items associated with the presence of histologically active disease. Results Over 1000 pCLE videos sequences performed in 100 UC patients in endoscopic remission (Mayo 0 and 1) were evaluated. We observed that vessel diameter > 20 µm, dilated crypt lumen, fluorescein leakage and irregular crypt architecture were statistically associated with histologically proven inflammation according to the Nancy index. Hence, we built a pCLE index of mucosal inflammation that overall accuracy was of 79.6% and overall sensitivity and specificity were respectively of 57.8% and 82.8%. Negative predictive value, especially when a pCLE index ≤ 1 is observed was high (93.1%). Conclusion Using a robust methodology, large vessel diameter, dilated crypt lumen, fluorescein leakage and irregular crypt architecture are reliable endomicroscopic items defining the ENHANCE index for real-time assessment of histological disease activity in UC.

Standardization of a Radiofrequency Ablation Tool in an Ex-Vivo Porcine Liver Model

(1) Background: Preclinical and clinical data about a novel radiofrequency ablation (RFA) system (STARmed Co, Ltd.; Koyang, Korea) designed to be used under endoscopic ultrasound (EUS) control for pancreatic lesion ablation, are limited, obtained with non-standardized procedures and heterogeneous results. The aim of this study is to standardize the RFA procedure of this system in order to define the optimal ablation power and time. (2) Methods: RFA was performed on an ex-vivo porcine liver at different powers (40, 30, 20, 10 Watts (W)) and times (1, 3, 5, 7, 15 min) with a 1-centimeter monopolar electrode (perfused by chilled solution) positioned on the distal tip of a 19-Gauge needle. A blinded expert pathologist histologically analyzed each ablation area. (3) Results: The size of the total macroscopic ablated area was negatively correlated with ablation power (R −0.74): the largest was obtained at 10 W (p = 4.7 × 10−4) for longer times (R 0.92; p = 8.9 × 10−8). Central histologic coagulative necrosis did not differ among ablation settings (mean size 3.25 mm). External “parenchymal hypochromia” or “diaphanization” resulted the widest at 10 W, for longer times (R 0.8, p = 3.6 × 10−4). (4) Conclusions: The RFA system can produce small sizes of coagulative necrosis, regardless of the setting. Larger areas of diaphanization surrounding the necrosis can be produced at lower powers for longer times.

Government Officials Clarify the Situation

Tanzania Peace Corps director Paul Sack and Peace Corps doctor Tom McHugh fly to Mwanza, meet with attorneys, then fly to Maswa. In Maswa, they meet Bill, who appears to be suffering from a severe stress reaction; McHugh performs a postmortem on Peppy; and they fly back to Mwanza, where McHugh assists an eminent pathologist from Nairobi in a second and more complete autopsy. In Dar es Salaam, Peace Corps officials meet with Tanzania police officers to find out how the police plan to proceed, while Peace Corps medical staff deal with the complex issues of obtaining a coffin suitable for withstanding the heat and humidity, the proper medical exit authorization, and where to find an expert pathologist.

Preoperative prediction of non-invasive follicular thyroid neoplasm with papillary-like nuclear features: a Canadian single-Centre experience

Background An international group of experts recommended reclassifying non-invasive follicular variant of papillary thyroid cancers (FVPTC) as ‘non-invasive follicular thyroid neoplasm with papillary-like nuclear features’ (NIFTP) in April 2016. The purpose of this study was to establish preoperative clinical, laboratory, ultrasonographic, and cytological variables, which can differentiate NIFTP from FVPTC. Methods We conducted a retrospective chart review of consecutive patients from a single institution evaluated between January 2012 and December 2017. 203 adult patients underwent lobectomy or total thyroidectomy for a FVPTC during that period. Each patient’s medical chart was reviewed and information on pre-operative variables was recorded. An expert pathologist reviewed all surgical specimens and reclassified a subset of FVPTC as NIFTP according to the specific criteria. Results Overall, 44 patients were included in the NIFTP group and 159 in the non-NIFTP group. Mean age was 50.1 years in the NIFTP group and 50.7 in the non-NIFTP group. Most patients were female (86.4% (38/44) in the NIFTP group vs 79.8% (127/159) in the non-NIFTP group). More patients underwent lobectomy in the NIFTP group (50% (22/44) vs 16.4% (26/159) in the non-NIFTP group, p = < 0.0001). Less patients received radioactive iodine in the NIFTP group (31.8% (14/44) vs 52.2% (83/159) in the non-NIFTP group, p = 0.0177). Preoperative thyroglobulin levels were lower in NIFTP patients (Median 25.55 mcg/L +/− 67.8 vs 76.06 mcg/L +/− 119.8 in Non-NIFTP, p = 0.0104). NIFTP nodules were smaller (Mean size 22.97 mm +/− 12.3 vs 25.88 mm +/− 11.2 for non-NIFTP, p = 0.0448) and more often solid than non-NIFTP (93.2% (41/44) vs 74.8% (119/159) for non-NIFTP, p = 0.0067). 2017 ACR TIRADS nodule category of 1–4 on ultrasound had a negative predictive value and a sensitivity of 100% for NIFTP. ROC Curve Analysis demonstrated that a preoperative thyroglobulin level of 31.3 mcg/L had a sensitivity of 75% and a specificity of 62.5% to differentiate NIFTP from non-NIFTP cancers. Conclusion Lower preoperative thyroglobulin levels, smaller nodule size, solid texture and 2017 ACR TIRADS Category of 1–4 are more strongly associated with NIFTP than FVPTC and can favour less invasive surgical options such as lobectomy.

P067 Proteins citrullination and Crohn’s disease: PAD4 but not PAD2 is a strong marker of ileal inflammation

Background Citrullination is a post-translational modification of proteins, mediated by enzymes called PAD (peptidylarginine deiminases). The immune system can attack citrullinated proteins, leading to autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and ulcerative colitis, and the activity of PAD2 and PAD4 in innate immune cells has been demonstrated for these disorders. Recently, high levels of PAD2 have been described in activated fibroblasts in the context of liver fibrosis. We therefore investigated the role of PAD2 and PAD4, both in inflammatory and fibrotic contexts of ileal Crohn’s disease (CD). Methods We obtained ileal transmural samples from patients operated for stricturing ileal CD. Three different macroscopic areas within each resection specimen (i.e. proximal normal ileum, inflamed ileum and fibrotic ileum) were selected and histologically confirmed by an expert pathologist. Patients undergoing ileocolic resection for other conditions (e.g. right colon cancer) and with healthy terminal ileum were used as controls. For each region (normal CD, inflamed CD, fibrotic CD and control), immunohistochemistry (IHC), RNA and protein evaluations for PAD2 and PAD4 were performed. Multiplex immunofluorescence (IF) for PAD2, PAD4, myeloperoxidase, neutrophil elastase, CD68, vimentin and α-smooth muscle actin were carried out to investigate the enzymes-expressing cells. Additional IF was performed to study citrullinated histone 3 (H3cit) expression, the product of PAD4 activity in neutrophils and component of neutrophil extracellular traps (NETs). Statistical analysis was carried out with Kruskal–Wallis test and post hoc Mann–Whitney test. Results Resection specimens from 13 CD and 11 controls were included. IHC and IF showed an increased expression of both PAD2 and PAD4 in the neutrophils of inflamed areas, in cytoplasm and nucleus, respectively (Figure 1). Activated fibroblasts (vimentin+ and α-smooth muscle actin+) were negative for both enzymes. PAD4 mRNA expression was increased in inflamed tissue (p = 0.001, p = 0.008 and p = 0.028 vs. normal CD, fibrotic CD and controls, respectively), and confirmed using Western Blot (Figure 2). H3cit was increased in the ileal inflammatory infiltrates too (Figure 3), confirming high PAD4 expression. For PAD2, no significant changes were observed at RNA and protein level, mainly due to its reduced expression in epithelial cells from normal to diseased tissue (Figure 4). Conclusion Both PAD2 and PAD4 are strongly expressed in neutrophils of CD ileal resection specimens, but only PAD4 shows a significantly higher expression in the inflammatory context which translates in the formation of NETs. No direct correlation was observed between PAD enzymes and intestinal fibroblasts.

Consultative Interpretation for Lupus Anticoagulant by Expert Pathologist Reduces False-Positive Rates in the Era of Direct Oral Anticoagulants

Background The diagnosis of antiphospholipid syndrome requires detection of antiphospholipid antibodies (aPL). A retrospective review of our testing practices revealed that societal recommendations for lupus anticoagulant (LA) testing as part of aPL testing are largely not followed by clinicians, and there was a high proportion of positive LA results. Increasing direct oral anticoagulant (DOAC) usage creates additional challenges in identifying LA. This prompted us to establish an order set with pathologist consultation (“LA panel”) and testing algorithm to reduce false-positive LA and to ensure optimal LA identification and best practices for interpretation and follow-up. Methods The laboratory database was reviewed to determine the number of LA tests ordered and rate of LA positivity before and after the LA panel was instituted. We assessed the impact of pathologist consultation to minimize false-positive findings and on following diagnostic guidelines. Results LA panels were ordered for 1146 patients. LA was detected in 10% (111 of 1146) by dilute Russel viper venom time (dRVVT) normalized ratio [includes dRVVT screen (dRVVTs) positive/lupus-sensitive partial thromboplastin time (PTT-LA) positive and dRVVTs positive/PTT-LA negative] and 20% (228 of 1146) by Staclot-LA (includes dRVVTs negative/PTT-LA positive and dRVVTs positive/confirm negative). There was a reduction of false-positive LA by Staclot-LA; previously, 48% positive. We saw increased cancellation of LA testing for interfering anticoagulants [6.8% (16 of 236) vs 14.4% (55 of 383); P = 0.0061]. There was also increased adherence to follow-up LA testing [3% (8 of 236) vs 13.8% (53 of 383); P ≤ 0.001]. Conclusions Creating a predetermined order set and testing algorithm with pathologist consultation improved LA testing interpretation and diagnostic follow-up testing.

Automatic Detection of Granuloma Necrosis in Pulmonary Tuberculosis Using a Two-Phase Algorithm: 2D-TB

Granuloma necrosis occurs in hosts susceptible to pathogenic mycobacteria and is a diagnostic visual feature of pulmonary tuberculosis (TB) in humans and in super-susceptible Diversity Outbred (DO) mice infected with Mycobacterium tuberculosis. Currently, no published automated algorithms can detect granuloma necrosis in pulmonary TB. However, such a method could reduce variability, and transform visual patterns into quantitative data for statistical and machine learning analyses. Here, we used histopathological images from super-susceptible DO mice to train, validate, and performance test an algorithm to detect regions of cell-poor necrosis. The algorithm, named 2D-TB, works on 2-dimensional histopathological images in 2 phases. In phase 1, granulomas are detected following background elimination. In phase 2, 2D-TB searches within granulomas for regions of cell-poor necrosis. We used 8 lung sections from 8 different super-susceptible DO mice for training and 10-fold cross validation. We used 13 new lung sections from 10 different super-susceptible DO mice for performance testing. 2D-TB reached 100.0% sensitivity and 91.8% positive prediction value. Compared to an expert pathologist, agreement was 95.5% and there was a statistically significant positive correlation for area detected by 2D-TB and the pathologist. These results show the development, validation, and accurate performance of 2D-TB to detect granuloma necrosis.

Relationships of p16 Immunohistochemistry and Other Biomarkers With Diagnoses of Cervical Abnormalities: Implications for LAST Terminology

Context.— Lower Anogenital Squamous Terminology (LAST) standardization recommended p16INK4a immunohistochemistry (p16 IHC) for biopsies diagnosed morphologically as cervical intraepithelial neoplasia (CIN) grade 2 (CIN2) to classify them as low-grade or high-grade squamous intraepithelial lesions (HSILs). Objective.— To describe the relationships of p16 IHC and other biomarkers associated with cervical cancer risk with biopsy diagnoses. Design.— A statewide, stratified sample of cervical biopsies diagnosed by community pathologists (CPs), including 1512 CIN2, underwent a consensus, expert pathologist panel (EP) review (without p16 IHC results), p16 IHC interpretation by a third pathology group, and human papillomavirus (HPV) genotyping, results of which were grouped hierarchically according to cancer risk. Antecedent cytologic interpretations were also available. Results.— Biopsies were more likely to test p16 IHC positive with increasing severity of CP diagnoses, overall (Ptrend ≤ .001) and within each HPV risk group (Ptrend ≤ .001 except for low-risk HPV [Ptrend &lt; .010]). All abnormal grades of CP-diagnosed biopsies were more likely to test p16 IHC positive with a higher HPV risk group (Ptrend &lt; .001), and testing p16 IHC positive was associated with higher HPV risk group than testing p16 IHC negative for each grade of CP-diagnosed biopsies (P &lt; .001). p16 IHC–positive, CP-diagnosed CIN2 biopsies were less likely than CP-diagnosed CIN3 biopsies to test HPV16 positive, have an antecedent HSIL+ cytology, or to be diagnosed as CIN3+ by the EP (P &lt; .001 for all). p16 IHC–positive, CP-diagnosed CIN1 biopsies had lower HPV risk groups than p16 IHC–negative, CP-diagnosed CIN2 biopsies (P &lt; .001). Conclusions.— p16 IHC–positive, CP-diagnosed CIN2 appears to be lower cancer risk than CP-diagnosed CIN3. LAST classification of “HSIL” diagnosis, which includes p16 IHC–positive CIN2, should annotate the morphologic diagnosis (CIN2 or CIN3) to inform all management decisions, which is especially important for young (&lt;30 years) women diagnosed with CIN2 for whom surveillance rather than treatment is recommended.

Prognostic Factors in a Large Nationwide Cohort of Histologically Confirmed Primary and Secondary Angiosarcomas

Angiosarcoma (AS) is a rare sarcoma of endothelial origin, arising spontaneously (primary AS) or after external damage such as radiation therapy or UV exposure (secondary AS). To date, reliable assessment of prognostic factors has proven difficult, due to disease rarity and heterogeneity of study cohorts. Although large registries provide relatively large AS patient series, these cases often lack histological confirmation. This study aimed to analyze AS prognostic factors in a large nationwide cohort of histologically confirmed cases, established through linkage of clinical data from the Netherlands Cancer Registry and pathology data from the Dutch pathology registry (PALGA). All cases were reviewed by an expert pathologist, showing a 16% discordance rate. Multivariable Cox regression survival analysis among 479 confirmed AS patients revealed remarkably poorer overall survival (OS) for primary AS compared to secondary AS (7 vs 21 months, Hazard ratio (HR) = 1.5; 95% confidence interval (CI) = 1.2–1.9). Age above 65 years, male gender, and no surgical treatment also significantly correlated to worse OS. Overall, OS was relatively poor, with a median of 13 months (95% CI = 10–16 months) and 22% five-year survival rate. With this study, we illustrate AS heterogeneity in clinical behavior and show for the first time better survival for secondary AS compared to primary AS.