A semi-quantitative histochemical method for assessment of biochemical responses to osmotic stress in Coffea arabica leaf disks.

A simple method set for assessing biochemical changes associated with osmotic stress responses was developed using coffee (Coffea arabica L.) leaf disks. Stress was induced by polyethylene glycol (PEG) exposure. Quantitative evaluation of tissue physiological stress parameters was carried out using analytical methods to validate the conversion of classic qualitative histochemical tests for localizing lipid peroxidation, hydrogen peroxide, and total xanthine alkaloids into semi-quantitative assays. Relative electrolyte leakage (EL%) and chlorophyll content (SPAD index) were also recorded. EL% levels of treated disks were higher than those of control ones, whereas SPAD indexes were comparable. Histochemical localization indicated that levels of lipid peroxidation, H2O2, and total xanthines were also higher under osmotic stress than in control conditions. Semi-quantitative data obtained by image processing of histochemical staining consistently matched quantitative evaluations. Chromatographic analyses revealed that theophylline and caffeine concentrations increased in the presence of PEG, whereas theobromine remained constant in relation to the control. The methods herein described can be useful to rapidly acquire initial data regarding biochemical osmotic stress responses in coffee tissues based on simple staining and imaging steps. Moreover, it is likely that the same method may be applicable to other types of stresses and plant species upon minor adjustments.

Assessment of neurodegeneration and neuronal loss in aged 5XFAD mice.

Neuronal loss resulting from progressive neurodegeneration is a major pathological feature of Alzheimer's disease (AD). Here, we present a protocol to detect neurodegeneration, neuronal apoptosis, and neuronal loss in 5XFAD mouse strain, which is a well-established model for interrogating the molecular mechanism of neuronal death in AD. This protocol describes the use of the neurodegenerative marker Fluro-Jade C, cleaved caspase-3 immunofluorescent staining and Nissl staining for the analysis of neurodegeneration and neuronal loss in 5XFAD mice. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

Do pathologists agree with each other on the histological assessment of pT1b oesophageal adenocarcinoma?

Background: In early (T1) oesophageal adenocarcinoma (OAC), the histological profile of an endoscopic resection specimen plays a pivotal role in the prediction of lymph node metastasis and the potential need for oesophagectomy with lymphadenectomy. Objective: To evaluate the inter-observer agreement of the histological assessment of submucosal (pT1b) OAC. Methods: Surgical and endoscopic resection specimens with pT1b OAC were independently reviewed by three gastrointestinal pathologists. Agreement was determined by intraclass correlation coefficient for continuous variables, and Fleiss' kappa (κ) for categorical variables. Bland-Altman plots of the submucosal invasion depth were made. Results: Eighty-five resection specimens with pT1b OAC were evaluated. The agreement was good for differentiation grade (κ=0.77, 95% confidence interval (CI) 0.68-0.87), excellent for lymphovascular invasion (κ=0.88, 95% CI 0.76-1.00) and moderate for submucosal invasion depth using the Paris and Pragmatic classifications (κ=0.60, 95% CI 0.49-0.72 and κ=0.42, 95% CI 0.33-0.51, respectively). Systematic mean differences between pathologists were detected for the measurement of submucosal invasion depth, ranging from 297 µm to 602 µm. Conclusions: A substantial discordance was found between pathologists for the measurement of submucosal invasion depth in pT1b OAC. Differences may lead to an over- or underestimation of the lymph node metastasis risk, with grave implications for the treatment strategy. Review by a second gastrointestinal pathologist is recommended to improve differentiating between a favourable and an unfavourable histological profile.

Improved performance of quantitative collagen parameters versus standard histology in longitudinal assessment of nonadvanced liver fibrosis for chronic hepatitis B.

Monitoring longitudinal nonadvanced fibrosis is a more common scenario in management of chronic hepatitis B (CHB), for which, however, current evaluation methods generally lack sufficient performance. We conducted a proof-of-concept study to evaluate the performance of quantitative fibrous collagen parameters (q-FP) in the assessment. Data sets from a prior CHB trial (NCT00962533) with mostly mild-to-moderate fibrosis participants were used for this study. 301 subjects with paired liver biopsies were consecutively included. Of these, 139 subjects were used to establish the test and the rest for internal validation. Fibrosis change between baseline and week 104 of treatment was blindly assessed with q-FP and was compared with Ishak fibrosis staging. There were 70% and 93% subjects with Ishak F0-2 at baseline and week 104, respectively. For the test of the subjects, q-FP and Ishak staging showed no difference in determining the incidence of fibrosis regression (68% vs 67%; difference = 0.7%, P = 1.00). Q-FP demonstrated that the regression was independently associated with the antiviral efficacy endpoint (OR 3.0, 95% CI 1.4-6.5, P = .005), but Ishak failed the detection (OR 0.6, 95% CI 0.3-1.3, P = .24). Moreover, q-FP directly revealed a higher fibrosis-resistance to antiviral treatment in virus genotypes C vs B and in males vs females. These results were confirmed in the validation subjects. Additionally, a functional model built on the test subjects showed an accuracy of 82% in stratifying fibrosis reversibility of the validation subjects. In conclusion, q-FP could have improved efficiency and accuracy in the longitudinal assessment of mild-to-moderate CHB fibrosis, indicating a potential alternative to current evaluation methodologies.

Elastogram quality assessment score in vibration-controlled transient elastography: Diagnostic performance compared to digital morphometric analysis of liver biopsy in chronic hepatitis C.

Vibration-controlled transient elastography (VCTE) is widely used for noninvasive fibrosis staging in chronic hepatitis C. However, internal validation is based solely on variability and success rate and lacks reproducible quality indicators. We analysed the graphic representation of shear wave propagation in comparison with morphometric results of liver biopsy, eliminating observer variability bias. Individual elastograms were classified according to two morphologic criteria: extension of wave propagation (length of the graphic representation) and shear wave dispersal (level of parallelism displayed in the elastogram). Then, a score based on these criteria stratified the elastogram in classes I through III (highest to lowest technical quality). Liver stiffness results of each measurement were compared with collagen contents in liver biopsy by morphometric analysis. A total of 3243 elastograms were studied (316 patients). Digital morphometry in liver biopsy showed significant fibrosis in 66% of samples and advanced fibrosis in 31%. Elastogram quality analysis resulted in 1438 class I measurements (44%), 1070 class II (34%) and 735 class III. Area under the receiver operating curve (AUROC) for severe fibrosis according to class (I, II and III) was 0.941, 0.887 and 0.766, respectively. For advanced fibrosis, AUROCs were 0.977, 0.883 and 0.781, respectively. Spearman's correlation testing for all classes and levels of fibrosis demonstrated significant independent association (r2  = -.95, P < .01). Our study is the first to propose measurable quality criteria for VTCE and to validate them against objective assessment of liver biopsy through digital morphometric imaging analysis. We concluded that VCTE performance is significantly influenced by quality assessment of individual measurements. Considering these criteria in clinical practice may improve accuracy.

Immunohisto- and Cytochemistry Analysis of Connexins.

Immunohistochemistry (IHC) is a ubiquitous used technique to identify and analyze protein expression in the context of tissue and cell morphology. In the connexin research field, IHC is applied to identify the subcellular location of connexin proteins, as this can be directly linked to their functionality. The present chapter describes a protocol for fluorescent IHC to detect connexin proteins in tissues slices and cells, with slight modifications depending on the nature of biological sample, histological processing, and/or protein expression level. Basically, fluorescent IHC is a short, simple, and cost-effective technique, which allows the visualization of proteins based on fluorescent-labeled antibody-antigen recognition.

Overview and assessment of the histochemical methods and reagents for the detection of ß-galactosidase activity in transgenic animals.

Bacterial ß-galactosidase is one of the most widely used reporter genes in experiments involving transgenic and knockout animals. In this review we discuss the current histochemical methods and available reagents to detect ß-galactosidase activity. Different substrates are available, but the most commonly used is X-gal in combination with potassium ferri- and ferro-cyanide. The reaction produces a characteristic blue precipitate in the cells expressing ß-galactosidase, and despite its efficiency in staining whole embryos, its detection on thin tissue sections is difficult. Salmon-gal is another substrate, which in combination with ferric and ferrous ions gives a reddish-pink precipitate. Its sensitivity for staining tissue sections is similar to that of X-gal. Combining X-gal or Salmon-gal with tetrazolium salts provides a faster and more sensitive reaction than traditional ß-galactosidase histochemistry. Here, we compare the traditional ß-galactosidase assay and the combination of X-gal or Salmon-gal with three tetrazolium salts: nitroblue tetrazolium, tetranitroblue tetrazolium and iodonitrotetrazolium. Based on an assessment of the sensitivity and specificity of the different combinations of substrates, we are proposing an optimized and enhanced method for ß-galactosidase detection in histological sections of the transgenic mouse brain. Optimal staining was obtained with X-gal in combination with nitroblue tetrazolium, which provides a faster and more specific staining than the traditional X-gal combination with potassium ferri- and ferro-cyanide. We recommend the X-gal/nitroblue tetrazolium staining mixture as the first choice for the detection of ß-galactosidase activity on histological sections. When faster results are needed, Salmon-gal/nitroblue tetrazolium should be considered as an alternative, while maintaining acceptable levels of noise.

In vivo imaging techniques: a new era for histochemical analysis.

In vivo imaging techniques can be integrated with classical histochemistry to create an actual histochemistry of water. In particular, Magnetic Resonance Imaging (MRI), an imaging technique primarily used as diagnostic tool in clinical/preclinical research, has excellent anatomical resolution, unlimited penetration depth and intrinsic soft tissue contrast. Thanks to the technological development, MRI is not only capable to provide morphological information but also and more interestingly functional, biophysical and molecular. In this paper we describe the main features of several advanced imaging techniques, such as MRI microscopy, Magnetic Resonance Spectroscopy, functional MRI, Diffusion Tensor Imaging and MRI with contrast agent as a useful support to classical histochemistry.

External quality assessment for acid fast bacilli smear microscopy in eastern part of Ethiopia.

BACKGROUND: External quality assessment (EQA) of sputum smear microscopy is essential and indispensable component of any tuberculosis program. This study assessed the EQA of acid fast bacilli (AFB) smear microscopy through onsite evaluation, blinded rechecking and panel test. A one year study was conducted on eight health institution laboratories from December 2011 to December 2012. Onsite evaluation, blinded rechecking and panel tests were used to collect data. Data were analyzed using SPSS version 16. Sensitivity, specificity, predictive values, and proportions of false readings were calculated. The level of agreement was measured using Kappa (κ) value. RESULTS: Problems observed during onsite evaluation include shortages of materials, disinfectant, and poor storage and working condition. A total of 578 slides were collected for blinded rechecking, of which 102 (17.6%) were reported as positive by peripheral laboratories. The panel test revealed an overall error of 17 (25.25%) of which 14 (17.5%) were minor errors [low false negative 6 (7.5%) and low false positive 8 (10%)], and 3 (3.75%) were major errors (high false positive). The sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) of the peripheral laboratories were 83.5, 97.8, 91.7, and 95.7, respectively. The false readings at the peripheral laboratories were 32 (5.5%). Agreement on reading the slides was observed on 546 (94.5%) slides (K = 0.84, SE = 0.054). CONCLUSIONS: Lack of reagents, supplies, favorable working environment and AFB related technical problems were identified in the peripheral laboratories. High false negative error was found to be the predominant major error. A continuous and strong EQA scheme should be implemented to avoid reporting errors and produce quality sputum results.

Use of eriochrome cyanine R in routine histology and histopathology: is it time to say goodbye to hematoxylin?

Eriochrome cyanine R (ECR) is a synthetic anionic dye that forms complexes with cations such as iron. We found that an iron-ECR (Fe-ECR) mixture provided either nuclear or myelin staining depending on the differentiator used. Selective nuclear staining was obtained by differentiation in an aqueous HCl solution, pH 0.95, followed by a wash in slightly alkaline tap water; the pH difference facilitated control of differentiation. When used with an eosin B counterstain, results were nearly indistinguishable from standard hematoxylin and eosin (H & E) staining. Nuclear staining with Fe-ECR provides tinctorial features similar to regressive aluminum-hemateins as well as resistance to acidic solutions such as those of iron hemateins. Fe-ECR also stained selectively intestinal cells of the diffuse neuroendocrine system (DNES). In addition to its use as an H & E substitute, acid differentiated Fe-ECR produced acid-resistant and selective nuclear counterstaining in combination with Alcian blue, and in the Papanicolaou and van Gieson techniques. With alkali differentiation, Fe-ECR produced selective myelin staining, which was compatible with neutral red counterstaining. Myelin sheaths were stained aqua blue. Fe-ECR could be used for both cytological and histological samples, and was suitable for use in automated tissue stainers. ECR also is less expensive than hematoxylin. Hematoxylin still may be preferred as a nuclear counterstain for some immunostaining methods for which Fe-ECR mixtures probably are too acidic.

Segmentation of histopathology HER2/neu images with fuzzy decision tree and Takagi-Sugeno reasoning.

The Human Epidermal Growth Factor Receptor 2 (HER2/neu) is a biomarker, recognized as a valuable prognostic and predictive factor for breast cancer. In approximately 20% of primary breast cancers, the HER2/neu protein is over-expressed. By recent clinical research, a treatment procedure, with corresponding monoclonal antibodies specifically designed to target the HER2/neu receptor, was confirmed. Therefore, in modern breast cancer diagnostics, it is critical to provide accurate recognition of the HER2/neu positive breast cancer. This can be done by segmentation of the membranes of cancer cells that are visualized as HER2/neu over-expressed on images acquired from corresponding histopathology preparations. In our research, we propose an accurate segmentation process of these structures using an appropriately defined fuzzy decision tree. Moreover, we introduce a new reasoning concept based on the Takagi-Sugeno inference model.

Crystal violet stain as a selective stain for the assessment of mitotic figures in oral epithelial dysplasia and oral squamous cell carcinoma.

BACKGROUND: Assessment of mitotic figures (MFs) is routinely practiced as prognostic indicator in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), but identification of MFs poses a problem in terms of staining characteristics. AIM: To evaluate effectiveness of crystal violet stain for staining of MFs and its comparison with hematoxylin and eosin (H and E) stain. MATERIALS AND METHODS: Study sample includes archival tissues embedded in paraffin blocks diagnosed as OED (n = 30) and OSCC (n = 30). The control group comprised of tissue specimen from oral mucosa of healthy volunteers (n = 30). Two serial sections of each tissue specimen were stained separately with H and E stain and 1% crystal violet stain. The stained sections were observed under microscope for identification and counting of MFs. Data obtained was statistically analyzed by using the Man-Whitney U test. RESULTS: A significant increase in number of MFs was observed in OED and OSCC in comparison with normal oral mucosa. There was a highly significant increase in number of MFs in crystal violet stained tissue sections when compared with H and E stain. Metaphase is the most commonly observed phase of mitosis in crystal violet stain when compared with H and E stain for all three groups. CONCLUSION: Crystal violet stain can be considered as selective stain for mitotic figures.

A rapid lactate dehydrogenase histochemical method for the intraoperative assessment of Hirschsprung's disease.

PURPOSE: The aim was to assess the ability of a fast lactate dehydrogenase (LDH) staining technique to evaluate the boundary of the abnormal bowel segment in Hirschsprung's disease (HD) as a guide for surgical resection. METHODS: Seventy children diagnosed with HD were equally divided into two groups. For the study group, fast LDH staining was used to confirm the diagnosis and determine the boundary of abnormal bowel. Frozen H&E staining was applied to the control group. Postoperatively, bowel samples were examined by paraffin H&E staining to confirm the intraoperative diagnosis. Patients received a follow-up analysis, and bowel function was scored and compared between the two groups. RESULTS: In the study group, 19 children were diagnosed with isolated HD, and the remaining had HD in combination with HD-allied disorders (HAD). The diagnosis was identical to the post-operative H&E staining, and the ganglia cells at the proximal end of the resected bowel were normal. In the control group, 30 children were diagnosed with isolated HD. However, the paraffin H&E staining showed that only 16 cases had isolated HD, and the remaining had a combined diagnosis of HAD. Moreover, 12 of these allied disorders were found at the proximal end of the resected bowel. Patients received follow-up for 6-15 months. The bowel function score of the study group was significantly higher than the control group. CONCLUSIONS: Fast LDH staining can clearly identify ganglion cells and rapidly diagnose HD and HAD intraoperatively. In addition, this method is helpful for improving patient prognosis.

[The application of a "pixel method" for the quantitative assessment of the results of the histochemical studies].

The results of the application of a "pixel method" for the automated measurement of the intensity of histochemical reaction in the neurons are presented. The method is based on measuring principle, which includes the automatic counting the sum of brightnesses of all the pixels forming the image with the help of standard computer programs. The potential of this method is demonstrated on the example of NADPH-diaphorase activity study in the nitroxidergic neurons of sensory and motor nuclei of the medulla of healthy rats.

Weighted maximum posterior marginals for random fields using an ensemble of conditional densities from multiple Markov chain Monte Carlo simulations.

The ability of classification systems to adjust their performance (sensitivity/specificity) is essential for tasks in which certain errors are more significant than others. For example, mislabeling cancerous lesions as benign is typically more detrimental than mislabeling benign lesions as cancerous. Unfortunately, methods for modifying the performance of Markov random field (MRF) based classifiers are noticeably absent from the literature, and thus most such systems restrict their performance to a single, static operating point (a paired sensitivity/specificity). To address this deficiency we present weighted maximum posterior marginals (WMPM) estimation, an extension of maximum posterior marginals (MPM) estimation. Whereas the MPM cost function penalizes each error equally, the WMPM cost function allows misclassifications associated with certain classes to be weighted more heavily than others. This creates a preference for specific classes, and consequently a means for adjusting classifier performance. Realizing WMPM estimation (like MPM estimation) requires estimates of the posterior marginal distributions. The most prevalent means for estimating these--proposed by Marroquin--utilizes a Markov chain Monte Carlo (MCMC) method. Though Marroquin's method (M-MCMC) yields estimates that are sufficiently accurate for MPM estimation, they are inadequate for WMPM. To more accurately estimate the posterior marginals we present an equally simple, but more effective extension of the MCMC method (E-MCMC). Assuming an identical number of iterations, E-MCMC as compared to M-MCMC yields estimates with higher fidelity, thereby 1) allowing a far greater number and diversity of operating points and 2) improving overall classifier performance. To illustrate the utility of WMPM and compare the efficacies of M-MCMC and E-MCMC, we integrate them into our MRF-based classification system for detecting cancerous glands in (whole-mount or quarter) histological sections of the prostate.

Design and validation of the biliary atresia research consortium histologic assessment system for cholestasis in infancy.

BACKGROUND & AIMS: Pathologists participating in the National Institutes of Health-sponsored Biliary Atresia Research Consortium (BARC) developed and then evaluated a standardized system for histologic reporting of liver biopsies from infants with cholestasis. METHODS: A set of 97 anonymous liver biopsy samples was sent to 10 pathologists at BARC centers. A semiquantitative scoring system that had 16 histologic features was developed and then used by the pathologists, who had no knowledge of clinical history, imaging results, or laboratory data. Interobserver agreement was evaluated statistically. Agreement on scoring of each feature and on the pathologists' diagnosis, compared with the final clinical diagnosis, was evaluated by using weighted kappa statistics. RESULTS: There was moderate to substantial interobserver agreement in identification of bile plugs in ducts, giant-cell transformation, extramedullary hematopoiesis, and bile duct proliferation. The pathologists' diagnosis of obstruction in clinically proven cases of biliary atresia (BA) ranged from 79%-98%, with a positive predictive value of 90.7%. Histologic features that best predicted BA, on the basis of logistic regression, included bile duct proliferation, portal fibrosis, and absence of sinusoidal fibrosis (each P<.0001). CONCLUSIONS: The BARC histologic assessment system identified features of liver biopsies from cholestatic infants, with good interobserver agreement, that might be used in diagnosis and determination of prognosis. The system diagnosed BA with a high level of sensitivity and identified infants with biliary obstruction with reasonable interobserver agreement. However, distinguishing between BA and disorders such as total parenteral nutrition-associated liver disease and alpha(1)-antitrypsin deficiency is not possible without adequate clinical information.

A resect and discard strategy would improve cost-effectiveness of colorectal cancer screening.

BACKGROUND & AIMS: A "resect and discard" policy has been proposed for diminutive polyps detected by screening colonoscopy, because hyperplastic and adenomatous polyps can be distinguished, in vivo, by using narrow-band imaging (NBI). We modeled the cost-effectiveness of this policy. METHODS: Markov modeling was used to compare the cost-effectiveness of universal pathology evaluations with a resect and discard policy for colonoscopy screening. In a resect and discard approach, diminutive lesions (≤5 mm), classified by endoscopy with high confidence, were not analyzed by a pathologist. Base case assumptions of an 84% rate of high-confidence classification, with a sensitivity and specificity for adenomas of 94% and 89%, respectively, were used. Census data were used to project outputs of the model onto the US population, assuming 23% as the current rate of adherence to a colonoscopy screening. RESULTS: With universal referral of resected polyps to pathology, colonoscopy screening costs an estimated $3222/person, with a gain of 51 days/person. Endoscopic polypectomy accounted for $179/person, of which $46/person was related to pathology examination. Adoption of a resect and discard policy for eligible diminutive polyps resulted in a savings of $25/person, without any meaningful effect on screening efficacy. Projected onto the US population, this approach would result in an undiscounted annual savings of $33 million. In the sensitivity analysis, the rate of high-confidence diagnosis and the accuracy for endoscopic polyp determination were the most meaningful variables. CONCLUSIONS: In a simulation model, a resect and discard strategy for diminutive polyps detected by screening colonoscopy resulted in a substantial economic benefit without an impact on efficacy.

Microwave histoprocessing versus conventional histoprocessing.

The aim of the study is to compare the histologic quality of the microwave histoprocessing with that of conventional method and to determine its positive impact on turnaround times and reduction of costs of tissue processing. One hundred and eighty-five paired tissue sections from different organs were taken. Each tissue sections were of size of 15 mm x 10 mm x 3 mm and divided into two; one set as experimental group and the other as control group. The tissues in the experimental group were further divided into six groups and processed by vacuum-microwave method according to six protocols from I to VI. Other tissues in the control group were processed by the conventional method and compared. Overall, the quality of microscopic tissue from both the methods was identical. Microwave processing shortened the time of processing without compromising the overall quality of the histologic section and was cost-effective.

Quantitative assessment of succinate dehydrogenase activity of rat lateral pterygoid muscle in undecalcified fresh-frozen section.

The quantitative assessment of enzyme activities in situ in single muscle fibres is essential for understanding the functions of skeketal muscles. The function of the lateral pterygoid muscle (LPM) is not fully understood because it is a deeply located masticatory muscle and cannot be dissected in an intact configuration. Here we report how to measure the activities of mitochondrial succinate dehydrogenase (SDH) in single muscle fibres in situ in the LPM in sections of rat heads. Unfixed head sections were incubated on gel films containing SDH substrate and nitroblue tetrazolium. During incubation, images of the section due to deposition of the final reaction products, formazans, were captured at intervals of 10 s using a real-time image analysis system for absorbance measurements. We found that the belly of the LPM consisted of four areas with different mean activities of SDH. The lateral and upper areas of the muscle showed similarly high SDH activities. Mean activity in the lower area was the lowest, about half of those of the lateral and upper areas. These results agree with the hypothesis that the superior head of the LPM participates in more continuous contraction and is more resistant to fatigue than the inferior head.