Reduction of autofluorescence in whole adult worms of Schistosoma japonicum for immunofluorescence assay.

Immunofluorescence assay is one of methods to understand the spatial biology by visualizing localization of biomolecules in cells and tissues. Autofluorescence, as a common phenomenon in organisms, is a background signal interfering the immunolocalization assay of schistosome biomolecules, and may lead to misinterpretation of the biomolecular function. However, applicable method for reducing the autofluorescence in Schistosoma remains unclear. In order to find a suitable method for reducing autofluorescence of schistosomes, different chemical reagents, such as Sudan black B (SBB), trypan blue (TB), copper sulfate (CuSO4), Tris-glycine (Gly), and ammonia/ethanol (AE), at different concentrations and treatment time were tested, and SBB and CuSO4 were verified for the effect of blocking autofluorescence in immunofluorescence to localize the target with anti-SjCRT antibody. By comparing the autofluorescence characteristics of different conditions, it was found that SBB, TB and CuSO4 had a certain degree of reducing autofluorescence effect, and the best effect in females was using 50 mM CuSO4 for 6 h and in males was 0.5% SBB for 6 h. Furthermore, we have applied the optimized conditions to the immunofluorescence of SjCRT protein, and the results revealed that the immunofluorescence signal of SjCRT was clearly visible without autofluorescence interference. We present an effective method to reduce autofluorescence in male and female worm of Schistosoma japonicum for immunofluorescence assay, which could be helpful to better understand biomolecular functions. Our method provides an idea for immunofluorescence assay in other flukes with autofluoresence.

Digital analysis of hormonal immunostaining in pituitary adenomas classified according to WHO 2017 criteria and correlation with preoperative laboratory findings.

OBJECTIVE: The authors sought to evaluate clinical and laboratory data from pituitary adenoma (PA) patients with functioning PA (associated with acromegaly [n = 10] or Cushing disease [n = 10]) or nonfunctioning PA (NFPA; n = 10) that were classified according to 2017 WHO criteria (based on the expression of the transcription factors pituitary-specific positive transcription factor 1 [Pit-1], a transcription factor member of the T-box family [Tpit], and steroidogenic factor 1 [SF-1]) and to assess the immunostaining results for growth hormone (GH) and adrenocorticotropic hormone (ACTH) in the corresponding tumors. METHODS: Clinical and laboratory data were collected retrospectively. The percentage of tumoral cells positive for Pit-1, Tpit, or SF-1 was assessed and ImageJ software was used to evaluate immunopositivity in PAs with 2 different antibodies against GH (primary antibody 1 [AbGH-1] and primary antibody 2 [AbGH-2]) and 2 different antibodies against ACTH (primary antibody 1 [AbACTH-1] and primary antibody 2 [AbACTH-2]). RESULTS: Cells with positive Pit-1 staining were more frequently observed in lesions from patients with acromegaly (acromegaly group) than in lesions from patients with Cushing disease (Cushing group; p < 0.001) and those from patients with NFPA (NFPA group; p < 0.001). The percentage of Tpit-positive cells was higher in the Cushing group than in the acromegaly (p < 0.001) and NFPA (p < 0.001) groups. No difference was detected regarding SF-1 frequency among all groups (p = 0.855). In acromegalic individuals, GH immunostaining levels varied depending on the antibody employed, and only one of the antibodies (AbGH-2) yielded higher values in comparison with the values for NFPA patients (p < 0.001). For all of the antibodies employed, no significant correlations were detected between GH tissue expression and the laboratory data (serum GH vs AbGH-1, p = 0.933; serum GH vs AbGH-2, p = 0.853; serum insulin-like growth factor-1 [IGF-1] vs AbGH-1, p = 0.407; serum IGF-1 vs AbGH-2, p = 0.881). In the Cushing group data, both antibodies showed similar ACTH tissue expression, which was higher than that obtained in the NFPA group (p < 0.001). There were no significant associations between ACTH immunohistochemical findings and ACTH serum levels (serum ACTH vs AbACTH-1, p = 0.651; serum ACTH vs AbACTH-2, p = 0.987). However, ACTH immunostaining evaluated with AbACTH-1 showed a significant correlation with 24-hour urinary cortisol (24-hour cortisol vs AbACTH-1, p = 0.047; 24-hour cortisol vs AbACTH-2, p = 0.071). CONCLUSIONS: Immunostaining for Pit-1 and Tpit accurately identified lesions associated with acromegaly and Cushing disease, respectively. Conversely, SF-1 did not differentiate NFPA from lesions of the other two groups. Regarding hormonal tissue detection, results of the current investigation indicate that different antibodies may lead not only to divergent immunohistochemical results but also to lack of correlation with laboratory findings. Finally, PA classification based on transcription factor expression (Pit-1, Tpit, and SF-1), as proposed by the 2017 WHO classification of pituitary tumors, may avoid the limitations of PA classification based solely on digital immunohistochemical detection of hormones.

Comparison of verdeluz orange G and modified Gallego stains.

Tumors of the oral cavity include combinations of hard and soft tissues that may be difficult to identify using routine hematoxylin and eosin (H & E) staining. Although combination stains can demonstrate hard and soft tissues, trichrome stains, such as VanGieson and Masson, cannot differentiate dental hard tissues, such as dentin, cementum and osteoid. Modified Gallegos (MGS) and verdeluz orange G-acid fuchsin (VOF) stains can differentiate components of teeth. We used 10 tissue sections of decalcified bone and 10 pathologic tissue sections that contained different calcified tissues including peripheral ossifying fibroma, odontoma, central ossifying fibroma and cemento-ossifying fibroma. Sections were stained with H & E, VOF or MGS. H and E stained both hard tissues pink. VOF stained bone purple-red, cementum red and collagen blue. MGS stained bone green-blue, cementum red and collagen blue. VOF staining intensity and differentiation was better than MGS staining. VOF staining demonstrated hard tissue components distinctly and exhibited good contrast with the surrounding connective tissue. VOF also is a simple, single step, rapid staining procedure.

Comparison between live and photographed slit lamp grading of corneal staining.

PURPOSE: To compare live and photographic (still) grades of corneal staining of the same eyes and the repeatability of grading between two investigators. METHODS: Thirty patients were recruited to participate in a contact lens study, and their level of corneal staining was graded by two investigators in situ (live images), using slit lamp biomicroscopy. Digital still images of the corneal staining were also captured during the study visits. An independent observer selected 105 of the still images graded by investigator 1 and another 105 images graded by investigator 2 and presented them to the original investigator in a random order, on three separate occasions. Grading was performed at the time of the live grading and the three still image sessions, using the Centre for Contact Lens Research corneal staining scale that combines grades of both extent and type to provide an overall "global staining score" from 0 to 10,000 for corneal staining. A comparison was made between live and still grades as well as the intrainvestigator repeatability for the multiple grading of the still images. RESULTS: The mean (±SD) of corneal staining grades recorded for the same eyes examined live and then later on three occasions was 1795 (±1083) and 714 (±974), respectively, for participants examined by investigator 1 (p < 0.001) and 1854 (±1075) and 461 (±411) for those examined by investigator 2 (p < 0.001). There was a significant difference over the three repeated still grading sessions for each investigator (p < 0.001), although there was a high degree of consistency among the three still grading sessions for each of the investigators: the intraclass correlation for investigator 1 was 0.91 (confidence interval, 0.87 to 0.93) and that for investigator 2 was 0.82 (confidence interval, 0.77 to 0.87). DISCUSSION: Digital still image grading of corneal staining significantly underrepresented the amount of corneal staining observed through a slit lamp. Clinical investigators graded corneal staining with a high degree of consistency.

External validation of a classification for methylene blue magnification chromoendoscopy in premalignant gastric lesions.

BACKGROUND: Conventional endoscopy has low sensitivity, specificity, and interobserver agreement for the diagnosis of gastric atrophy, intestinal metaplasia, and dysplasia. Magnification chromoendoscopy (ME) may optimize the evaluation of premalignant gastric lesions. OBJECTIVE AND DESIGN: As part of a multicenter trial, we aimed at validating a previously proposed classification for gastric methylene blue ME at a different center. SETTING, PATIENTS, AND INTERVENTIONS: A sample of patients (n = 42) with previously diagnosed chronic atrophic gastritis with or without intestinal metaplasia underwent ME (Pentax EG-3430Z) with 1% methylene blue by 2 endoscopists. MAIN OUTCOME MEASUREMENTS: A simplified version of a previously published ME classification (group I, group II [further divided into subgroups IIE and IIF], and group III) was used for macroscopic lesions (n = 203) with Sydney-Houston and Vienna classifications being used for histologic analysis (n = 479 biopsy specimens). RESULTS AND LIMITATIONS: Excellent reproducibility (wK = 0.92 [95% CI, 0.88-0.96]) was observed for classification in groups and substantial reproducibility (wK = 0.78 [95% CI, 0.72-0.84]) was found for classification in subgroups. Global validity was 82% (range 78%-86%), showing no false negatives (sensitivity of 100% [1/1 biopsy]) and a very low rate of false positives (specificity 99% [297/299 biopsies]) for dysplasia detection. CONCLUSIONS: This classification for methylene blue ME was highly reproducible and valid for the diagnosis of premalignant gastric lesions when used in a center different from that involved in its conception. Despite requiring an unconventional endoscope and a longer procedure, these results could reinforce ME as a valuable technique in the surveillance of patients at risk for gastric cancer.

Reliability of grading lissamine green conjunctival staining.

PURPOSE: To assess the reliability of a lissamine green grading scale for conjunctival images. METHODS: A 20-second video clip of the right eye of 288 contact lens-wearing individuals was recorded using a digital slip-lamp camera after instilling liquid lissamine green. A single nasal and temporal still image were selected. A masked reader used the Oxford grading scale to grade the images on two occasions whereas a second masked reader graded each image on 1 occasion. kappa statistics and 95% confidence intervals (CIs) were used to determine the within- and between-grader reliability overall and when the sample was stratified by age, sex, contact lens type, and disease severity. RESULTS: There was substantial within-grader reliability for both the nasal (kappasimple = 0.69, 95% CI, 0.63-0.75) and temporal (kappasimple = 0.73, 95% CI, 0.67-0.79) images. There was moderate between-grader reliability for both the nasal (kappasimple = 0.51, 95% CI, 0.44-0.58) and temporal (kappasimple = 0.51, 95% CI, 0.44-0.58) images. Age, sex, and contact lens type did not affect within- or between-examiner reliability. There may have been an influence of disease severity on within-examiner reliability, because grading of the temporal images was significantly less reliable in the images with more significant staining. CONCLUSION: Within- and between-grader reliability of lissamine green staining seems to be at least substantial to moderate. Because the extent of conjunctival staining may influence reliability, this should be considered when studies may include patients with significant staining.