Algorithm-assisted diagnosis of Hirschsprung's disease - evaluation of robustness and comparative image analysis on data from various labs and slide scanners.

BACKGROUND: Differences in the preparation, staining and scanning of digital pathology slides create significant pre-analytic variability. Algorithm-assisted tools must be able to contend with this variability in order to be applicable in clinical practice. In a previous study, a decision support algorithm was developed to assist in the diagnosis of Hirschsprung's disease. In the current study, we tested the robustness of this algorithm while assessing for pre-analytic factors which may affect its performance. METHODS: The decision support algorithm was used on digital pathology slides obtained from four different medical centers (A-D) and scanned by three different scanner models (by Philips, Hamamatsu and 3DHISTECH). A total of 192 cases and 1782 slides were used in this study. RGB histograms were constructed to compare images from the various medical centers and scanner models and highlight the differences in color and contrast. RESULTS: The algorithm was able to correctly identify ganglion cells in 99.2% of cases, from all medical centers (All scanned by the Philips slide scanner) as well as 95.5% and 100% of the slides scanned by the 3DHISTECH and Hamamatsu brand slide scanners, respectively. The total error rate for center D was lower than the other medical centers (3.9% vs 7.1%, 10.8% and 6% for centers A-C, respectively), the vast majority of errors being false positives (3.45% vs 0.45% false negatives). The other medical centers showed a higher rate of false negatives in relation to false positives (6.81% vs 0.29%, 9.8% vs 1.2% and 5.37% vs 0.63% for centers A-C, respectively). The total error rates for the Philips, Hamamatsu and 3DHISTECH brand scanners were 3.9%, 3.2% and 9.8%, respectively. RGB histograms demonstrated significant differences in pixel value distribution between the four medical centers, as well as between the 3DHISTECH brand scanner when compared to the Philips and Hamamatsu brand scanners. CONCLUSIONS: The results reported in this paper suggest that the algorithm-based decision support system has sufficient robustness to be applicable for clinical practice. In addition, the novel method used in its development - Hierarchial-Contexual Analysis (HCA) may be applicable to the development of algorithm-assisted tools in other diseases, for which available datasets are limited. Validation of any given algorithm-assisted support system should nonetheless include data from as many medical centers and scanner models as possible.

Short Training Significantly Improves Ganglion Cell Detection Using an Algorithm-Assisted Approach.

CONTEXT.­: Medical education in pathology relies on the accumulation of experience gained through inspection of numerous samples from each entity. Acquiring sufficient teaching material for rare diseases, such as Hirschsprung disease (HSCR), may be difficult, especially in smaller institutes. The current study makes use of a previously developed decision support system using a decision support algorithm meant to aid pathologists in the diagnosis of HSCR. OBJECTIVE.­: To assess the effect of a short training session on algorithm-assisted HSCR diagnosis. DESIGN.­: Five pathologists reviewed a data set of 568 image sets (1704 images in total) selected from 50 cases by the decision support algorithm and were tasked with scoring the images for the presence or absence of ganglion cells. The task was repeated a total of 3 times. Each pathologist had to complete a short educational presentation between the second and third iterations. RESULTS.­: The training resulted in a significantly increased rate of correct diagnoses (true positive/negative) and a decreased need for referrals for expert consultation. No statistically significant changes in the rate of false positives/negatives were detected. CONCLUSIONS.­: A very short (<10 minutes) training session can greatly improve the pathologist's performance in the algorithm-assisted diagnosis of HSCR. The same approach may be feasible in training for the diagnosis of other rare diseases.

Tissue microarrey: a potential cost-effective approach for mismatch repair testing in colorectal cancer.

BACKGROUND: Deficiencies in Mismatch Repair (MMR) proteins are one of the major pathways in the development of colorectal cancer (CRC). MMR status evaluation is recommended in every new CRC patient. However, this is not fully implemented due to high costs. Tissue microarray (TMA) enables allocating tissue cores from few specimens to a single paraffin block. The primary objective of this study was to evaluate the accuracy of TMA MMR immunohistochemistry (IHC) compared to whole slide. The secondary objective was to evaluate and validate automatic digital image analysis software in differentiating pathological and normal TMA cores. METHODS: Pathological cores were defined if at least one MMR protein was unstained. Tumoral and normal tissue of 11 CRC patients with known MMR status was used to obtain 623 TMA cores. The MMR staining of each core was evaluated by a pathologist and compared to the whole slide result. Digital analysis software by 3DHistech Ltd. was used to identify cell nucleus and quantify nuclear staining in 323 tissue cores. To identifying pathological tissue, cores the cohort was divided into a test (N = 146 cores) and validation sets (N = 177 cores). A staining intensity score (SIS) was developed, and its performance compared to the pathologist review of each core and to the whole slide result. RESULTS: Compared to the whole slide, the pathologist's assessment had 100% sensitivity (n/N = 112/112) and 100% specificity (n/N = 278/278) with 95% lower limit of 97 and 99% respectively. The area under the receiver operating characteristic (ROC) curve of SIS was 77%. A cutoff of 55 was obtained from the ROC curve. By implementing the cutoff in the validation dataset, the SIS had sensitivity and specificity of 98.2% [90.1-100%] and 58.5% [49.3-67.4%] respectively. CONCLUSIONS: The MMR status of CRC can be evaluated in TMA tissue cores thus potentially reducing MMR testing costs. The SIS can be used as triage indicator during pathologic review. TRIAL REGISTRATION: Institutional ethical approval was granted for the performance of this study (Emek Medical Center Ethics ID: EMC-19-0179).

Lung targeted liposomes for treating ARDS.

Acute Respiratory Distress Syndrome (ARDS), associated with Covid-19 infections, is characterized by diffuse lung damage, inflammation and alveolar collapse that impairs gas exchange, leading to hypoxemia and patient' mortality rates above 40%. Here, we describe the development and assessment of 100-nm liposomes that are tailored for pulmonary delivery for treating ARDS, as a model for lung diseases. The liposomal lipid composition (primarily DPPC) was optimized to mimic the lung surfactant composition, and the drug loading process of both methylprednisolone (MPS), a steroid, and N-acetyl cysteine (NAC), a mucolytic agent, reached an encapsulation efficiency of 98% and 92%, respectively. In vitro, treating lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages with the liposomes decreased TNFα and nitric oxide (NO) secretion, while NAC increased the penetration of nanoparticles through the mucus. In vivo, we used LPS-induced lung inflammation model to assess the accumulation and therapeutic efficacy of the liposomes in C57BL/6 mice, either by intravenous (IV), endotracheal (ET) or IV plus ET nanoparticles administrations. Using both administration methods, liposomes exhibited an increased accumulation profile in the inflamed lungs over 48 h. Interestingly, while IV-administrated liposomes distributed widely throughout the lung, ET liposomes were present in lungs parenchyma but were not detected at some distal regions of the lungs, possibly due to imperfect airflow regimes. Twenty hours after the different treatments, lungs were assessed for markers of inflammation. We found that the nanoparticle treatment had a superior therapeutic effect compared to free drugs in treating ARDS, reducing inflammation and TNFα, IL-6 and IL-1ß cytokine secretion in bronchoalveolar lavage (BAL), and that the combined treatment, delivering nanoparticles IV and ET simultaneously, had the best outcome of all treatments. Interestingly, also the DPPC lipid component alone played a therapeutic role in reducing inflammatory markers in the lungs. Collectively, we show that therapeutic nanoparticles accumulate in inflamed lungs holding potential for treating lung disorders. SIGNIFICANCE: In this study we compare intravenous versus intratracheal delivery of nanoparticles for treating lung disorders, specifically, acute respiratory distress syndrome (ARDS). By co-loading two medications into lipid nanoparticles, we were able to reduce both inflammation and mucus secretion in the inflamed lungs. Both modes of delivery resulted in high nanoparticle accumulation in the lungs, intravenously administered nanoparticles reached lung endothelial while endotracheal delivery reached lung epithelial. Combining both delivery approaches simultaneously provided the best ARDS treatment outcome.

A novel purgative protocol for capsule endoscopy of the small bowel produces better quality of visibility than 2 l of PEG: Timing is of the essence.

INTRODUCTION: The ideal way of preparing patients for small-bowel capsule endoscopy has been controversial. Previous studies have shown that ingestion of 2 l of polyethylenglycol (PEG) 12 hours prior to capsule ingestion leads to improved visibility in comparison to no preparation at all. We speculated that using a post-ingestion (PI), booster-based cleansing protocol might provide an alternative to the PEG cleansing protocol. METHODS: This randomized, blinded, prospective study enrolled 45 individuals. Patients were allocated to either of two groups. The PEG group ingested 2 l PEG 12 hours prior to the exam (n = 22) and the PI group ingested one sachet of Picolax® dissolved in 250 ml of water one hour after swallowing the capsule with 500 ml of water (n = 18). Primary endpoints were overall small bowel and distal third of small bowel cleansing levels. Secondary endpoints were average gastric and small-bowel transit time. RESULTS: Forty-five patients participated in this study. Five individuals were excluded because of incomplete study. Percentage of patients with adequate visibility in the distal third of the small bowel in the PEG group was 9% vs 72% in the PI group (p < 0.0001). Average gastric time and total transit time were shorter in the PI group vs the PEG group (p = 0.0065). CONCLUSION: Timing of ingestion of the Picolax® purgative 60 minutes after swallowing the capsule endoscopy delivers better visibility in the distal third of the small bowel than the accepted cleansing protocol of ingesting 2 l PEG 12 hours prior to the capsule endoscopy procedure.

The inhibitory effect of Mycoplasma fermentans on tumour necrosis factor (TNF)-alpha-induced apoptosis resides in the membrane lipoproteins.

Mycoplasma have been shown to be involved in the alteration of several eukaryotic cell functions, such as cytokine production, gene expression and more. We have previously reported that infection of human myelomonocytic U937 cell line with live Mycoplasma fermentans (M. fermentans) inhibited tumour necrosis factor (TNF-alpha)-induced apoptosis. Mycoplasmal membrane lipoproteins are considered to be the most potent initiators of inflammatory reactions in mycoplasmal infections. The aim of this study was to clarify whether the inhibitory effect on TNFalpha-induced apoptosis is exerted by M. fermentans lipoproteins (LPMf). A significant reduction in TNFalpha-induced apoptosis was demonstrated by stimulation of U937 cells with M. fermentans total proteins, LPMf or MALP-2 (M. fermentans synthetic lipopeptide), but not with M. fermentans hydrophilic protein preparation (AqMf). To investigate the mechanism of M. fermentans antiapoptotic effect, the reduction of mitochondrial transmembrane potential (delta psi m) was measured. M. fermentans total proteins LPMf and MALP-2, but not AqMf, inhibited the reduction of delta psi m. In addition, M. fermentans total proteins LPMf and MALP-2, but not AqMf, downregulated the formation of active caspase-8. NF-kappaB was transactivated in cells treated with M. fermentans lipoproteins, and was essential for host cell survival, but not for the inhibition of TNFalpha-induced apoptosis by LPMf. Our results suggest that the inhibitory effect exerted by M. fermentans on TNFalpha-induced apoptosis in U937 cells is due to the membrane lipoproteins of these bacteria.

Rapid and reactive nitric oxide production by astrocytes in mouse neocortical slices.

Nitric oxide (NO), a cellular signaling molecule, is produced in the brain by both neurons and astrocytes. While neurons are capable of rapid release of small amounts of NO serving as neurotransmitter, astrocytic NO production has been demonstrated mainly as a slow reaction to various stress stimuli. Little is known about the role of astrocyte-produced NO. Using the NO indicator 4,5-diaminofluorescein-2 diacetate (DAF-2DA) and acute slices from mouse brain, we distinguished neurons from astrocytes based on their different fluorescence kinetics and pattern, cellular morphology, electrophysiology, and responses to selective nitric oxide synthase (NOS) inhibitors. Typically, astrocytic fluorescence followed neuronal fluorescence with a delay of 1-2 min and was dependent on the inducible NOS isoform (iNOS) activity. Western blot analysis established the presence of functional iNOS in the neocortex. An assay for cell death revealed that most DAF-2DA-positive neurons, but not astrocytes, were damaged. Whole cell recordings from astrocytes confirmed that these cells maintained their membrane potential and passive properties during illumination and afterward. Induction of excitotoxicity by brief application of glutamate triggered an immediate and intense astrocytic response, while high-frequency electrical stimulation failed to do so. The present study demonstrates, for the first time, rapid and massive iNOS-dependent NO production by astrocytes in situ, which appears to be triggered by acute neuronal death. These data may bear important implications for our theoretical understanding and practical management of acute brain insults.