BACKGROUND: Rapid on-site evaluation (ROSE) is a technique used for simultaneous evaluation of biopsy specimens through rapid cytology staining. Diff-Quik (DQ) staining is the most commonly employed method for cytological rapid on-site evaluation (C-ROSE). However, the utilization of DQ staining for on-site cytological interpretation remains uncommon among pathologists in China, posing challenges to the implementation of C-ROSE. This study aims to assess the application of rapid hematoxylin-eosin (HE) staining and DQ staining for C-ROSE during percutaneous needle biopsy of peripheral lung cancer and evaluate the value of rapid HE staining in C-ROSE. METHODS: Computed tomography (CT)-guided lung biopsies were conducted on 300 patients diagnosed with peripheral lung cancer. The patients were randomly assigned to two groups for C-ROSE using either rapid HE staining or DQ staining, and subsequently the two methods were compared and evaluated. RESULTS: The concordance rate between C-ROSE and histopathological diagnosis was 96.7%. The median staining time for rapid HE staining was 160 s, while that for DQ staining was 120 s, representing a significant difference between the two groups (P<0.001). However, there were no significant differences observed in terms of total biopsy time, concordance rate with histopathology, cytology specimen peeling rate, and incidence of serious adverse reactions between the two groups (P>0.05). CONCLUSIONS Both staining methods comply with C-ROSE criteria in the biopsy setting of peripheral lung cancer. Rapid HE staining is more aligned with domestic clinical requirements and holds potential for further promotion and adoption in C-ROSE.
Humans ; Lung Neoplasms/pathology* ; Eosine Yellowish-(YS) ; Rapid On-site Evaluation ; Biopsy, Needle/methods* ; Staining and Labeling

OBJECTIVES: The advanced non-small cell lung cancer (NSCLC) patients with pleural effusion have no opportunity for surgery treatment. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the first-line drugs for these patients with EGFR-sensitive mutation. However, the disease progression and drug update during or after treatment of EGFR-TKIs bring more challenges and puzzles to clinical diagnosis and treatment, which inevitably requires archived pleural cell samples for EGFR re-examination or comparative study. Understanding the DNA quality of archived pleural fluid samples and effectively using archival data of pleural fluid cells are of great significance for tracing the origin of cases and basic medical research. This study aims to evaluate the consistency of EGFR mutant gene expression between the 2 methods, and to explore a reliable way for preserving cytological data and making full use of cytological archival data via cell HE staining smear and cell paraffin section. METHODS: A total of 57 pleural fluid cytology cases in the Department of Pathology of China Aerospace Center Hospital from October 2014 to April 2021 were selected. Tumor cells were detected by cell HE staining smears and immunohistochemical staining for TTF-1 and Napsin A in the paired cell paraffin sections. There were more than 200 tumor cells in cell HE staining smear and the proportion of tumor cells were ≥70% in matched cell paraffin sections. Patients with 2 cell smears (one for cell data retention and the other for DNA extraction) were selected as the research subjects, and 57 pleural fluid samples were enrolled. EGFR gene mutation was detected by amplification refractory mutation system-polymerase chain reaction in 57 paired cell HE staining smears and cell paraffin sections. DNA concentration was 2 ng/μL. Cell HE smear was amplified side-by-side with DNA samples from paired cell paraffin sections. Result determination was according to the requirements of the reagent instructions. The external control cycle threshold (Ct) value of the No. 8 well of the samples to be tested was between 13 and 21, which was considered as successful and reliable samples. When the Ct value of EGFR gene mutation was <26, it was considered as positive; when the Ct value was between 26 and 29, it was critical positive; when the Ct value was equal or more than 29, it was negative. ΔCt value was the difference between mutant Ct value and externally controlled Ct value. The smaller the ΔCt value was, the better the quality of DNA of the detected sample was. RESULTS: Among the 57 pleural effusion samples, 42 patients were hospitalized with pleural effusion as the first symptom, accounting for 73.7% (42/57). EGFR mutation was detected in 37 samples [64.9% (37/57)]. The mutation rate for 19del was 37.8% (14/37) while for L858R was 48.6% (18/37). Females were 56.7% (21/37) of mutation cases. The mutation consistency rate of cell HE staining smear and matched cell paraffin sections was 100%. The ΔCt values of cell HE staining smears were less than those of matched cell paraffin sections. The mutation Ct values of 37 cytological samples were statistically analyzed according to the preservation periods of the years of 2014-2015, 2016-2017, 2018-2019, and 2020-2021. There were significant differences in cell paraffin section in the years of 2014-2015 and 2016-2017 compared with the years of 2018-2019 and 2020-2021, while no significant differences were found in cell HE staining smear. Statistical analysis of externally controlled Ct values of 57 cytological samples showed that there were significant differences between cell HE staining smears and cell paraffin section in the years of 2014-2015 and 2016-2017, compared with the years of 2018-2019 and 2020-2021. The mutational Ct values of 37 paired cell blocks and smears were all <26, and the externally controlled Ct values of 57 paired cell paraffin sections and HE staining smears were all between 13 and 21. CONCLUSIONS The DNA quality of cell HE smears and matched cell paraffin section met the qualified requirements. Two methods possess show an excellent consistency in detecting EGFR mutation in NSCLC pleural fluid samples. The DNA quality of cell HE staining smear is better than that of cell paraffin sections, so cell HE staining smear can be used as important supplement of the gene test source. It should be noted that the limitation of cell HE staining smears is non-reproducibility, so multiple smears of pleural fluid are recommended to be prepared for multiple tests.
Carcinoma, Non-Small-Cell Lung/drug therapy* ; DNA Mutational Analysis/methods* ; ErbB Receptors/genetics* ; Female ; Humans ; Lung Neoplasms/drug therapy* ; Male ; Mutation ; Paraffin/therapeutic use* ; Pleural Effusion/genetics* ; Protein Kinase Inhibitors/therapeutic use* ; Staining and Labeling

In forensic traumatic pathology practice, immunohistochemistry and special staining technique play an important role in wound age estimation and complications of traumatic complication identification. They even play an important role in the identification of special cases, such as snakebites and insulin killings. This article reviews the application and value of immunohistochemistry and special staining techniques in forensic traumatic pathology based on the cases of forensic practice reported in literature.
Forensic Medicine ; Forensic Pathology/methods* ; Immunohistochemistry ; Staining and Labeling

BACKGROUND: Electromagnetic navigation bronchoscopy (ENB) has become the latest minimally invasive diagnostic and therapeutic technique due to its characteristics, e.g., non-invasion, accuracy, real-time positioning. In this study, we investigated the application of ENB biopsy combined with Massage staining in the diagnosis and treatment of peripheral pulmonary lesions (PPL). METHODS: The clinical data of 15 PPL patients undergoing ENB biopsy plus Massage staining between August 2017 and January 2018 were retrospectively reviewed. Among them, there were 12 male and 3 female, and the mean age was (51.3±2.1) years old. RESULTS: The diameter of PPLs ranged from 6 mm to 36 mm (mean: 14.0 mm). The successful biopsy rate was 66.7%. All patients successfully underwent Massage staining. The distance between the centers of staining and lesion was (1.0±0.4) cm, and the diameter of staining was (2.8±0.6) cm. The mean operation time was (26.7±5.3 ) min, and the mean blood loss during surgery was (3.3±1.5) mL. There was no pneumothorax, hemothorax and pulmonary vascular injury during the procedure. CONCLUSIONS The ENB biopsy plus Massage staining technique caused very few complications, and provided high precision, which warrants further application.
Bronchoscopy ; methods ; Electromagnetic Fields ; Female ; Humans ; Lung Diseases ; diagnosis ; surgery ; Lung Neoplasms ; diagnosis ; surgery ; Male ; Middle Aged ; Reproducibility of Results ; Retrospective Studies ; Sensitivity and Specificity ; Staining and Labeling ; methods

OBJECTIVE: To optimize the method for embedding multiple undecalcified mouse tibias in plastic blocks, improve the efficiency and stability of plastic embedding and reduce the detachment rate of plastic slides. METHODS: Thirty undecalcified tibias from 15 B6 mice were used for plastic embedding after calcein labeling, fixation, dehydration and infiltration. The tibias were embedded in cylindrical plastic blocks with a diameter of 4 mm. For each bone, the 1/4 proximal tibia was cut off, and the remaining 3/4 was used for re-embedding. Five bones were embedded in a single block with each bone standing closely on the surface of a flat plate. The samples were randomized into control and experimental groups in all the processes of embedding, sectioning and staining. In the 3 groups with modified embedment, flowing CO was added into the embedding solution, embedding solution was applied to the section surface, and the slides were heated at 95 ℃ for 15 min. The polymerization time, slide detachment rate, bone formation and osteoblast parameters were analyzed. RESULTS: We prepared 6 plastic blocks, each containing 5 tibias, whose cross sections were on the same plane. The blocks were completely polymerized and suitable for sectioning. Flowing CO into the embedding solution reduced the polymerization time and increased the rate of complete polymerization. Application of the embedding solution on the section surface significantly reduced the detachment rate of the sections ( < 0.05) without affecting bone formation analysis ( > 0.05). Heating the slides significantly lowered the detachment rate of the sections ( < 0.05) without affecting osteoblast analysis ( > 0.05). CONCLUSIONS The optimized method allows effective embedding of multiple undecalcified mice tibias in the same block and can be an ideal method for histological analysis of undecalcified bones.
Animals ; Mice ; Plastics ; Staining and Labeling ; Tibia ; Tissue Embedding ; methods

OBJECTIVES: We produced hematoxylin and eosin (H&E) staining-like color images by using confocal laser scanning microscopy (CLSM), which can obtain the same or more information in comparison to conventional tissue staining. METHODS: We improved images by using several image converting techniques, including morphological methods, color space conversion methods, and segmentation methods. RESULTS: An image obtained after image processing showed coloring very similar to that in images produced by H&E staining, and it is advantageous to conduct analysis through fluorescent dye imaging and microscopy rather than analysis based on single microscopic imaging. CONCLUSIONS: The colors used in CLSM are different from those seen in H&E staining, which is the method most widely used for pathologic diagnosis and is familiar to pathologists. Computer technology can facilitate the conversion of images by CLSM to be very similar to H&E staining images. We believe that the technique used in this study has great potential for application in clinical tissue analysis.
Diagnosis ; Eosine Yellowish-(YS) ; Fluorescence ; Hematoxylin* ; Image Processing, Computer-Assisted ; Methods* ; Microscopy ; Microscopy, Confocal* ; Staining and Labeling

OBJECTIVE: To assess magnetic resonance imaging (MRI) features of coronary microembolization in a swine model induced by small-sized microemboli, which may cause microinfarcts invisible to the naked eye. MATERIALS AND METHODS: Eleven pigs underwent intracoronary injection of small-sized microspheres (42 microm) and catheter coronary angiography was obtained before and after microembolization. Cardiac MRI and measurement of cardiac troponin T (cTnT) were performed at baseline, 6 hours, and 1 week after microembolization. Postmortem evaluation was performed after completion of the imaging studies. RESULTS: Coronary angiography pre- and post-microembolization revealed normal epicardial coronary arteries. Systolic wall thickening of the microembolized regions decreased significantly from 42.6 +/- 2.0% at baseline to 20.3 +/- 2.3% at 6 hours and 31.5 +/- 2.1% at 1 week after coronary microembolization (p < 0.001 for both). First-pass perfusion defect was visualized at 6 hours but the extent was largely decreased at 1 week. Delayed contrast enhancement MRI (DE-MRI) demonstrated hyperenhancement within the target area at 6 hours but not at 1 week. The microinfarcts on gross specimen stained with nitrobluetetrazolium chloride were invisible to the naked eye and only detectable microscopically. Increased cTnT was observed at 6 hours and 1 week after microembolization. CONCLUSION: Coronary microembolization induced by a certain load of small-sized microemboli may result in microinfarcts invisible to the naked eye with normal epicardial coronary arteries. MRI features of myocardial impairment secondary to such microembolization include the decline in left ventricular function and myocardial perfusion at cine and first-pass perfusion imaging, and transient hyperenhancement at DE-MRI.
Animals ; Coronary Angiography/*methods ; Coronary Vessels/*pathology ; Disease Models, Animal ; Embolism/*pathology ; Female ; Heart/radiography ; Image Processing, Computer-Assisted ; Magnetic Resonance Imaging/*methods ; Microspheres ; Myocardial Contraction/physiology ; Myocardial Infarction/*pathology ; Myocardium/pathology ; Nitroblue Tetrazolium ; Staining and Labeling ; Swine ; Troponin T/blood ; Ventricular Function, Left

OBJECTIVETo explore a method for combining Fluoro-Jade B (FJB) staining with immunofluorescent staining in rats with focal cortical infarction.

METHODPermanent distal middle cerebral artery occlusion (dMCAO) was induced in rats by electrocoagulation. The rat models were randomized into two groups, and frozen sections of the brain tissues from each group were stained with FJB followed by immunofluorescent staining or in the reverse order.

RESULTSFJB staining followed by immunofluorescence staining clearly visualized both FJB-positive and immunofluorescence-positive cells in the frozen sections, but the staining protocol in the reverse sequence failed to clearly show the immunofluorescence-positive cells.

CONCLUSIONFJB staining prior to immunofluorescence staining does not affect the staining effect of protein immunofluorescent staining and better visualizes the positive cells.

Animals ; Brain ; pathology ; Fluoresceins ; chemistry ; Fluorescent Antibody Technique ; methods ; Fluorescent Dyes ; chemistry ; Infarction, Middle Cerebral Artery ; Rats ; Staining and Labeling ; methods

Urokinase plasminogen activator receptor (uPAR) is a membrane protein which is attached to the cellular external membrane. The uPAR expression can be observed both in tumor cells and in tumor-associated stromal cells. Thus, in the present study, the human amino-terminal fragment (hATF), as a targeting element to uPAR, is used to conjugate to the surface of superparamagnetic iron nanoparticle (SPIO). Flowcytometry was used to examine the uPAR expression in different tumor cell lines. The specificity of hATF-SPIO was verified by Prussian blue stain and cell phantom test. The imaging properties of hATF-SPIO were confirmed in vivo magnetic resonance imaging (MRI) of uPAR-elevated colon tumor. Finally, the distribution of hATF-SPIO in tumor tissue was confirmed by pathological staining. Results showed that the three cells in which we screened, presented different expression characteristics, i. e., Hela cells strongly expressed uPAR, HT29 cells moderately expressed uPAR, but Lovo cells didn't express uPAR. In vitro, after incubating with Hela cells, hATF-SPIO could specifically combined to and be subsequently internalized by uPAR positive cells, which could be observed via Prussian blue staining. Meanwhile T2WI signal intensity of Hela cells, after incubation with targeted probe, significantly decreased, and otherwise no obvious changes in Lovo cells both by Prussian blue staining and MRI scans. In vivo, hATF-SPIO could be systematically delivered to HT29 xenograft and accumulated in the tumor tissue which was confirmed by Prussian Blue stain compared to Lovo xenografts. Twenty-four hours after injection of targeting probe, the signal intensity of HT29 xenografts was lower than Lovo ones which was statistically significant. This targeting nanoparticles enabled not only in vitro specifically combining to uPAR positive cells but also in vivo imaging of uPAR moderately elevated colon cancer lesions.
Cell Line, Tumor ; Colonic Neoplasms ; diagnosis ; Ferric Compounds ; Humans ; Magnetic Resonance Imaging ; Magnetite Nanoparticles ; chemistry ; Molecular Imaging ; methods ; Receptors, Urokinase Plasminogen Activator ; chemistry ; Staining and Labeling

Based on the principles of the in vitro staining technique, hypotonic swelling test, and water test, the Eosin Y-water test method was developed to simultaneously detect the integrity of the sperm head and tail and sperm membrane structure and function. As a widely used method in clinical laboratories in China, the Eosin Y-water test is methodologically characterized by three advantages. Firstly, both the sperm head and tail can be detected at the same time, which allows easy and comprehensive assessment of membrane damage in different parts of sperm. Secondly, distilled water is used instead of the usual formula solution to simplify and standardize the test by eliminating any potential effects on the water molecules through the sperm membrane due to different osmotic pressure or different sugar proportions and electrolyte solutions. Thirdly, the test takes less time and thus can be repeated before and after treatment. This article focuses on the fundamental principles and modification of the Eosin Y-water test and its application in sperm function examination and routine semen analysis for male infertility, assessment of the quality of sperm retrieved by testicular fine needle aspiration, semen cryopreservation program development, and evaluation of sperm membrane integrity after microwave radiation.
Cell Membrane ; China ; Cryopreservation ; Eosine Yellowish-(YS) ; Fluorescent Dyes ; Humans ; Infertility, Male ; diagnosis ; Male ; Osmotic Pressure ; Semen Analysis ; methods ; Sperm Head ; Sperm Motility ; Sperm Tail ; Spermatozoa ; Staining and Labeling ; Water