Slide Preparation Methods Research Articles

Pancreaticobiliary Cytology Practice in 2021: Results of a College of American Pathologists Survey.

The College of American Pathologists (CAP) surveys provide national benchmarks of pathology practice. To investigate pancreaticobiliary cytology practice in domestic and international laboratories in 2021. We analyzed data from the CAP Pancreaticobiliary Cytology Practice Supplemental Questionnaire that was distributed to laboratories participating in the 2021 CAP Nongynecologic Cytopathology Education Program. Ninety-three percent (567 of 612) of respondent laboratories routinely evaluated pancreaticobiliary cytology specimens. Biliary brushing (85%) was the most common pancreaticobiliary cytology specimen evaluated, followed by pancreatic fine-needle aspiration (79%). The most used sampling methods reported by 235 laboratories were 22-gauge needle for fine-needle aspiration (62%) and SharkCore needle for fine-needle biopsy (27%). Cell block was the most used slide preparation method (76%), followed by liquid-based cytology (59%) for pancreatic cystic lesions. Up to 95% (303 of 320) of laboratories performed rapid on-site evaluation (ROSE) on pancreatic solid lesions, while 56% (180 of 320) performed ROSE for cystic lesions. Thirty-six percent (193 of 530) of laboratories used the Papanicolaou Society of Cytopathology System for Reporting Pancreaticobiliary Cytology in 2021. Among all institution types, significant differences in specimen volume, specimen type, ROSE practice, and case sign-out were identified. Additionally, significant differences in specimen type, slide preparation, and ROSE practice were found. This is the first survey from the CAP to investigate pancreaticobiliary cytology practice. The findings reveal significant differences among institution types and between domestic and international laboratories. These data provide a baseline for future studies in a variety of practice settings.

Evaluation of automated sample preparation system for lymph node sampling.

Endobronchial ultrasound-guided transbronchial fine needle aspiration (EBUS-FNA) has revolutionized the diagnostic and staging approach to non-small cell carcinoma and thoracic lymphadenopathy. However, obstacles to efficacy of rapid on-site evaluation (ROSE) of the samples include variability in sample quality and slow and cumbersome process in the procedure room leading to extended procedure time. The purpose of this pilot study was to evaluate the feasibility and specimen quality of lymph node biopsies prepared through a novel automated system for automated fixation, drying and staining compared to standard slide preparation method. We performed a prospective, single-center pilot feasibility study of patients undergoing EBUS. Samples were split into conventional standard of care (SOC) slide preparation and preparation using the device ("instrument"). Pathologists compared the SOC slides to the slides prepared by the automated system and assessed the following metrics: nuclear and cytoplasmic quality, presence of debris/artifact, staining quality, creation of a monolayer, and ease of adequacy/diagnosis assessment. A score between 1 (lowest quality) and 3 (highest quality) was assigned to the above metrics. Sixty patients were recruited. One to three lymph nodes were sampled for each patient for a total of 72 samples collected. The mean scores of each assessment category showed no statistical difference between the two preparation techniques except for improved monolayer creation in the instrument samples. Thirty of thirty-one (96.8%) paired samples in the final analysis showed diagnostic equivalency between the automated slides and conventional slides; the discordant pairing was reported to be suspicious on the instrument sample and atypical on the SOC. Study results suggest that slides prepared by the automated system are of adequate quality for adequacy assessment with diagnostic concordance when compared to SOC slides.

Various technique of cell culture and slide preparation for human chromosome

The cell culture and slide preparation method of human chromosome is the mechanism that is a corresponding change in DNA content. In this research we used various techniques of cell culture and slide preparation for human chromosome and examined for 2 days, 3 days and 4 days cell culture because these are average days for approximately the good results and the treatment will still be good also the result has minimum bias. The research objectives were to determine the appropriate length of cell culture needed for human chromosome with different lengths and to determine the appropriate method for staining the slide for human chromosome with different lengths. Based on the data analysis, the result of this research shows that the appropriate length of cell culture needed for human chromosome with different lengths is 2 days of incubation. Although there were no significant differences between 2,3 and 4 days length based on statistical analysis, still there is little difference between them and the best is 2 days rather than 3 and 4 days based on the graph analysis. The appropriate method for staining the slide for human chromosome with different lengths is hot plate rather than another technique

A highly-effective modified hard tick (Ixodes)-slide preparation method using lactophenol in comparison with a daily basis-used potassium hydroxide technique

A highly-effective modified hard tick (Ixodes)-slide preparation method using lactophenol in comparison with a daily basis-used potassium hydroxide technique

A Comparative Analysis of Deep Learning Models for Automated Cross-Preparation Diagnosis of Multi-Cell Liquid Pap Smear Images.

Routine Pap smears can facilitate early detection of cervical cancer and improve patient outcomes. The objective of this work is to develop an automated, clinically viable deep neural network for the multi-class Bethesda System diagnosis of multi-cell images in Liquid Pap smear samples. 8 deep learning models were trained on a publicly available multi-class SurePath preparation dataset. This included the 5 best-performing transfer learning models, an ensemble, a novel convolutional neural network (CNN), and a CNN + autoencoder (AE). Additionally, each model was tested on a novel ThinPrep Pap dataset to determine model generalizability across different liquid Pap preparation methods with and without Deep CORAL domain adaptation. All models achieved accuracies >90% when classifying SurePath images. The AE CNN model, 99.80% smaller than the average transfer model, maintained an accuracy of 96.54%. During consecutive training attempts, individual transfer models had high variability in performance, whereas the CNN, AE CNN, and ensemble did not. ThinPrep Pap classification accuracies were notably lower but increased with domain adaptation, with ResNet101 achieving the highest accuracy at 92.65%. This indicates a potential area for future improvement: development of a globally relevant model that can function across different slide preparation methods.

Effect of Romanowsky-Stained Concentrated Preparations versus Direct Smears on Veterinary Students' Ability to Identify Bacterial Sepsis in Fluid Cytology Samples from Dogs, Cats, and Horses.

Veterinary students' accuracy, confidence, and time required to diagnose bacterial sepsis in fluid cytology samples was evaluated using two different slide preparation methods: direct smears and cytocentrifuged concentrated preparations. We hypothesized veterinary students would diagnose fluids as septic on concentrated preparations more accurately and quickly than on direct smears. Thirty third- and fourth-year students who had previously participated in a clinical pathology course completed a survey regarding general cytology experience and reviewed 40 randomized Romanowsky-stained slides via microscopy. Slides consisted of 10 septic and 10 non-septic samples with matched direct and concentrated slides, prepared from fluids from dogs, cats, and a horse. Participants' slide evaluation time, diagnosis, confidence, and slide photographs of areas considered septic were recorded. No difference in diagnostic accuracy between direct and concentrated samples was identified (area under the curve: 57% for both preparations, p = 0.77), although students agreed with pathologist-determined diagnoses more often when viewing concentrated samples (M = 63%, SD = 11% for concentrated; M = 56%, SD = 21% for direct, p = .012). A positive relationship existed between accuracy of diagnosis (R2 = .59) and senior status (p = .002), comfort interpreting cytology slides (p < .03), and if the student had taken the senior pathology rotation (p = .02). Only 38% (121/319) of participant photographs correctly identified sepsis. Under experimental conditions, concentrated preparations did not increase the accuracy of veterinary students' bacterial sepsis diagnosis; however, since accuracy did increase with cytology experience and comfort level, additional pre-clinical and clinical cytology training may benefit students before entering practice.

The Co-Existence of Cryptosporidium parvum with Others Parasitic Protozoan Infection Among Children Under Five Years in Some Places of Mosul City

A hospital based, cross sectional study this was carried out at the period between the beginning of June 2020 to the end of September 2021. A total of 600 child Suffering from chronic diarrhea were examined. The co-existence of mixed infection of Cryptosporidium Parvum with others intestinal parasitic protozoan parasites as an etiology of chronic diarrhea was the aim of this study, Cryptosporidium Parvum, Entamoeba histolytica, Giardia lambilia was found to be the more common parasites as a cause of chronic diarrhea among children under five years, stool samples were collected form children who lived in some places of Mosul city which is divided into two sides as a result of the passage of the Tigris River from the center, a total of 400 (66.6%) positive cases were recorded, the highest infectious rate was found at the Right side of Mosul city 230 (38.3%) cases while the rest 170 (28.3%) cases was recorded in Left side. Direct slide preparation method with a Modified Zeihl Neelsen, cultivation of Protozoal parasites by using special media and ELISA method for stool samples were used to perform the diagnosis of infection of Cryptosporidium Parvum, Entamoeba histolytica and Giardia lambilia that was to be found in 130 (21.6%),150(25%),120(20%), respectively, The co-existence of mixed infection of Cryptosporidium Parvum with Entamoeba histolytica was recorded in 30 (5%)cases while with Giardia lambilia was found in 24(4%)cases these study also recorded the presence of mixed infection between Entamoeba histolytica and Giardia lambilia in 12(2%) cases.

Confocal microscopy 3D imaging of diesel particulate matter

To date, diesel particulate matter (DPM) has been described as aggregates of spherule particles with a smooth appearing surface. We have used a new colour confocal microscope imaging method to study the 3D shape of diesel particulate matter (DPM); we observed that the particles can have sharp jagged appearing edges and consistent with these findings, 2D light microscopy demonstrated that DPM adheres to human lung epithelial cells. Importantly, the slide preparation and confocal microscopy method applied avoids possible alteration to the particles’ surfaces and enables colour 3D visualisation of the particles. From twenty-one PM10 particles, the mean (standard deviation) major axis length was 5.6 (2.25) μm with corresponding values for the minor axis length of 3.8 (1.25) μm. These new findings may help explain why air pollution particulate matter (PM) has the ability to infiltrate human airway cells, potentially leading to respiratory tract, cardiovascular and neurological disease.

Comparative evaluation of centrifuged liquid-based cytology method over conventional brush cytology in normal oral mucosa

Introduction: Liquid-based cytology (LBC) is a new monolayer slide preparation method introduced to overcome the limitations of conventional Papanicolaou (PAP) smears which is used in cancer screening. The study was aimed to compare the efficacy of modified technique, centrifuged LBC (CLBC) with that of conventional brush cytology in normal oral mucosa. Materials and Methods: Two smears were obtained from 100 cases of normal mucosa using a cytobrush. One smear was spread on to the slide by conventional technique, and the other smear was prepared by CLBC. Regular PAP staining was done for both the smears. Both the smears were evaluated for cellularity, cell distribution, cellular overlapping, cell elongation, and cellular background and were graded using the grading criteria. Mann–Whitney U-test was used, and P ≤ 0.05 was considered statistically significant. Results: The mean score of the criterias was found to be more desirable in CLBC than conventional technique, but there was no statistical significance found. Conclusions: LBC offers a significant advantage over conventional smear preparation in most of the criteria to advocate its use for routine diagnostic and mass screening procedures.

Cytology reveals diverse cell morphotypes and cellin-cell interactions in normal collector sea urchins Tripneustes gratilla.

Echinoderms such as sea urchins are important in marine ecosystems, particularly as grazers, and unhealthy sea urchins can have important ecological implications. For instance, unexplained mortalities of Diadema antillarum in the Caribbean were followed by algal overgrowth and subsequent collapse of coral reef ecosystems. Unfortunately, few tools exist to evaluate echinoderm health, making management of mortalities or other health issues problematic. Hematology is often used to assess health in many animal groups, including invertebrates, but is seldom applied to echinoderms. We used a standard gravitometric technique to concentrate fixed coelomocytes from the collector sea urchin Tripneustes gratilla onto microscope slides, permitting staining and enumeration. Using Romanowsky stain and electron microscopy to visualize cell details, we found that urchin cells could be partitioned into different morphotypes. Specifically, we enumerated phagocytes, phagocytes with perinuclear cytoplasmic dots, vibratile cells, colorless spherule cells, red spherule cells, and red spherule cells with pink granules. We also saw cell-in-cell interactions characterized by phagocytes apparently phagocytizing mainly the motile cells including red spherule cells, colorless spherule cells, and vibratile cells disproportionate to underlying populations of circulating cells. Cell-in-cell interactions were seen in 71% of sea urchins, but comprised <1% of circulating cells. Finally, about 40% of sea urchins had circulating phagocytes that were apparently phagocytizing spicules. The coelomic fluid collection and slide preparation methods described here are simple, field portable, and might be a useful complementary tool for assessing health of other marine invertebrates, revealing heretofore unknown physiological phenomena in this animal group.

Skin thickness dimensions in histological section measurement during late‐fetal and neonatal developmental period: A systematic review

BackgroundThe development and maturation of the skin is a process that occurs during the gestation and neonatal period. Histological skin biopsy studies are relevant to improve knowledge on the skin protective barrier during the perinatal period. The thin skin of preterm newborns is unable to maintain homeostasis, thermal regulation through the skin, and is susceptible to infections. This study systematically reviewed the evidence regarding histological thickness dimensions of the skin and its layers during the late‐fetal and neonatal period.MethodsPubMed, Scopus, BVS, and e SciELO library databases, with no limits in the period of analysis or idiom. Eligibility criteria were as follows: studies describing the thickness of the entire skin or its layers during late‐fetal life or the neonatal period; human being; skin biopsy analysis; and any scientific report. Two independent reviewers screened the search and extracted the following standard data: fetal or neonatal age of assessment, biopsy site, technique used for preparation and staining of histological slides, measurement techniques, and values of skin thickness.ResultsFifty‐nine studies were screened, and eleven were identified from other sources. We recognized six studies that met the criteria for inclusion for proper extraction. Expressive differences between sites for sampling, methods of slide preparation, and number of layers measured made the thicknesses values summarization difficult. There were no reliable dimensions reported on this tissue.ConclusionDespite the importance of studying the human skin barrier, these findings confirmed limited evidence on skin thickness dimensions obtained by histology.

The use of two staining methods for identification of spermatozoon structure in roosters

Many of the methods used to stain semen result in very pronounced coloring of the sperm, but unfortunately they do not distinguish their individual structures, which play a key role in the fertilization process. Hence the aim of this study was to identify sperm structures using two staining techniques in the semen of roosters from breeding flocks. The subject of the study was the sperm of roosters from a Ross 308 breeding flock. To capture the differences in the dimensions of sperm subjected to the effect of different chemical substances in dyes, microscope slides were stained by two techniques: with an AgNO3 solution and by a differential method (eosin-nigrosin test). Assessment was made of the degree of coloration and the number of details that could be identified in the morphological structure of the sperm. The use of AgNO3 allowed accurate identification of the acrosome, nucleus, and midpiece, which were visible in the slides stained with eosin-nigrosin, but only in dead spermatozoa. The AGNO3 staining technique used in this study reveals the cell nucleus within the head and can be an alternative method to analysis with a scanning electron microscope. This staining technique can be used to stain sperm structures that cannot be seen in other methods of slide preparation, which means that it can be considered for routine use in assessing the fertility of roosters in breeder flocks.

Semi-automated scoring of triple-probe FISH in human sperm using confocal microscopy.

Structural and numerical sperm chromosomal aberrations result from abnormal meiosis and are directly linked to infertility. Any live births that arise from aneuploid conceptuses can result in syndromes such as Kleinfelter, Turners, XYY and Edwards. Multi-probe fluorescence in situ hybridization (FISH) is commonly used to study sperm aneuploidy, however manual FISH scoring in sperm samples is labor-intensive and introduces errors. Automated scoring methods are continuously evolving. One challenging aspect for optimizing automated sperm FISH scoring has been the overlap in excitation and emission of the fluorescent probes used to enumerate the chromosomes of interest. Our objective was to demonstrate the feasibility of combining confocal microscopy and spectral imaging with high-throughput methods for accurately measuring sperm aneuploidy. Our approach used confocal microscopy to analyze numerical chromosomal abnormalities in human sperm using enhanced slide preparation and rigorous semi-automated scoring methods. FISH for chromosomes X, Y, and 18 was conducted to determine sex chromosome disomy in sperm nuclei. Application of online spectral linear unmixing was used for effective separation of four fluorochromes while decreasing data acquisition time. Semi-automated image processing, segmentation, classification, and scoring were performed on 10 slides using custom image processing and analysis software and results were compared with manual methods. No significant differences in disomy frequencies were seen between the semi automated and manual methods. Samples treated with pepsin were observed to have reduced background autofluorescence and more uniform distribution of cells. These results demonstrate that semi-automated methods using spectral imaging on a confocal platform are a feasible approach for analyzing numerical chromosomal aberrations in sperm, and are comparable to manual methods. © 2017 International Society for Advancement of Cytometry.

A revised method for determining the absolute abundance of diatoms

Diatoms and other microfossils are used extensively in reconstructions of past climates and environments, in both terrestrial and marine settings. Both relative and absolute abundances of diatom taxa in sediment are important tools in these reconstructions. However, acquiring these data is a labor-intensive process. Settling-based diatom slide preparation techniques often bias samples through aliquot subsampling and sediment washing. Other techniques involve the use of added markers, which might obscure diatoms on the slide. This paper presents a revision to the widely adopted settling-based diatom slide preparation method presented by Scherer (J Paleolimnol 12:171–179, 1994) and provides a direct comparison to another widely used method. Evenly distributed diatom slides can be created by a settling process, which yields multiple statistically similar diatom slides without needing to clean sediment of salts or do aqueous subsampling, which may impart a bias in the sample when there is a wide range of particle shapes and sizes in the assemblage. Two samples originally utilized by Scherer (J Paleolimnol 12:171–179, 1994) were prepared via the updated method through a series of replicates. These results were compared to the same samples, processed with the method of Schrader and Gersonde (Utrecht Micropaleontol Bull 17:129–176, 1978), utilizing Petri dishes and the original results of Scherer (J Paleolimnol 12:171–179, 1994). The new modification presented here produces smaller standard deviations than the original Scherer method, and order of magnitude better statistics than the Schrader and Gersonde (Utrecht Micropaleontol Bull 17:129–176, 1978) method.

Web-based scoring of the dicentric assay, a collaborative biodosimetric scoring strategy for population triage in large scale radiation accidents

In the case of a large scale radiation accident high throughput methods of biological dosimetry for population triage are needed to identify individuals requiring clinical treatment. The dicentric assay performed in web-based scoring mode may be a very suitable technique. Within the MULTIBIODOSE EU FP7 project a network is being established of 8 laboratories with expertise in dose estimations based on the dicentric assay. Here, the manual dicentric assay was tested in a web-based scoring mode. More than 23,000 high resolution images of metaphase spreads (only first mitosis) were captured by four laboratories and established as image galleries on the internet (cloud). The galleries included images of a complete dose effect curve (0-5.0 Gy) and three types of irradiation scenarios simulating acute whole body, partial body and protracted exposure. The blood samples had been irradiated in vitro with gamma rays at the University of Ghent, Belgium. Two laboratories provided image galleries from Fluorescence plus Giemsa stained slides (3 h colcemid) and the image galleries from the other two laboratories contained images from Giemsa stained preparations (24 h colcemid). Each of the 8 participating laboratories analysed 3 dose points of the dose effect curve (scoring 100 cells for each point) and 3 unknown dose points (50 cells) for each of the 3 simulated irradiation scenarios. At first all analyses were performed in a QuickScan Mode without scoring individual chromosomes, followed by conventional scoring (only complete cells, 46 centromeres). The calibration curves obtained using these two scoring methods were very similar, with no significant difference in the linear-quadratic curve coefficients. Analysis of variance showed a significant effect of dose on the yield of dicentrics, but no significant effect of the laboratories, different methods of slide preparation or different incubation times used for colcemid. The results obtained to date within the MULTIBIODOSE project by a network of 8 collaborating laboratories throughout Europe are very promising. The dicentric assay in the web based scoring mode as a high throughput scoring strategy is a useful application for biodosimetry in the case of a large scale radiation accident.

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

A slide preparation technique for light microscopy analysis of ciliates preserved in acid Lugol's fixative

A slide preparation method for seawater samples preserved in acid Lugol's is presented here as an alternative to the traditional Utermöhl settling chamber method for microplankton analysis. This preparation maintains the integrity of fragile cells, such as ciliates, resolves issues associated with the transience of samples prepared in settling chambers, and allows the use of automated image acquisition methods. Samples are filtered onto polycarbonate membranes and analyzed with transmitted light microscopy. The visibility of pore outlines is minimized by using mounting oil (Cargille Series A immersion oil, Certified Refractive Index, nD25°C 1.5840 ± 0.0002) with a refractive index matching that of the membrane material. We assessed the efficacy of this new method by comparing abundance and biomass estimates for ciliates in settled and filtered samples. Acceptable results were found for the most delicate of samples stored long‐term in acid Lugol's. Some cell shrinkage occurred during the filtration and brief drying steps. Therefore, corrections for ciliate length and width measurements in filtered samples were determined to counteract this effect on total cell biovolume. Overall, the method provides a simple and stable alternative to settling chamber analysis for ciliates preserved in acid Lugol's.

P5.018 Improved Diagnosis of Non-Neoplastic Conditions of Cervix by Liquid Based Cytology, Cellbock in Comparison with Conventional Pap Smear Method

Background The Papanicolaou (Pap) smear is the most successful screening test for carcinoma of cervix, mainly as a result of its simplicity, low cost, low false-negative rate and its early detection of preneoplastic lesions. It is also used to diagnose several infectious agents that manifest with specific cytologic features. The diagnoses of candida, trichomonas, herpes simplex virus, HPV and bacterial vaginosis can be reliably rendered on Pap smears. Conventional pap smear (CPS) has its limitations. False negatives in CPS may be related to inadequate sampling, inadequate transfer of the sample onto the glass slide or deficiencies in the microscopic assessment of the slide. To overcome these problems, a new slide preparation method namely the Manual Liquid Based Cytology (MLBC) was introduced, where cells are uniformly dispersed by a membrane, from a suspension of cells in a polymer solution. MLBC helps in detecting number of infestations like Candida, Leptothrix, HPV with koilocytic atypia and bacterial vaginosis, as observed by many authors and us also. MLBC, which we are following is inexpensive, cost effective method which we have adapted and are comparing it with CPS for its adequacy and utility. The other advantages, is that the residual specimens can be used for ancillary testing like immunocytochemistry by cell block preparation. Results Number of cases of bacterial vaginosis diagnosed by CPS were 8, LBC 9 and cell block were zero. trichomonas were 4, by CPS and LBC,2by cell block., herpes was 1 by all three methods,inflammatory (NILM) cases were 22 by CPS,26 by LBC,12 by cell block. Inadequate smears were 5 by CPS, NIL by LBC,25 by cell block Conclusion CPS and LBC with cell block are useful methods in detection of non neoplastic conditions of cervix. LBC has increased the diagnostic accuracy by 13% in our study.

Bone marrow aspirate collection and preparation – A comparison of three methods

Preparing bone marrow smears using non-anticoagulated bone marrow aspirate is a traditional practice but many laboratories now use anticoagulated aspirate samples in K-EDTA. There are no published studies comparing the effectiveness of these two methods. This report compares the readability of slides, prepared using non-anticoagulated and anticoagulated methods, from three laboratories in Hamilton Ontario. A blinded set of 129 aspirate slides prepared using anticoagulated and non-anticoagulated methodologies (using K-EDTA) was reviewed by three reviewers. Slides were classified as unreadable if two of the three observers rejected them based on a standardized survey. The proportion of slides classed as unreadable varied widely (5.0% to 46.9%) depending on collection and slide preparation methods. Degree of coagulation did not affect readability. A measurable advantage to using non-anticoagulated bone marrow was not demonstrated. Immediate anticoagulation of bone marrow samples, with laboratory personnel at the bedside to assess sample quality, followed by slide preparation in the laboratory provided the best results.