Rapid Staining Technique Research Articles

Performance of Field's Stain Compared with Conventional Giemsa Stain for the Rapid Detection of Blood Microfilariae in Gabon.

ABSTRACT.Filarial infections caused by Loa loa and Mansonella perstans are a considerable public health burden in rural regions of Central Africa. Rapid diagnostic tools for the detection of microfilariae in the blood are needed. Field’s stain is a rapid staining technique for microscopic slides originally established for malaria diagnostics. It requires less than 1 minute of staining compared with conventional staining protocols requiring at least 15 to 20 minutes for staining and could thus significantly accelerate diagnostics for human filariasis. Here we evaluated Field’s stain as a rapid staining technique in comparison to Giemsa stain for the detection of microfilariae in peripheral blood. Blood smears were collected from 175 participants residing in the region of Lambaréné and Fougamou, Gabon. Each participant’s samples were stained in parallel with Field’s stain and conventional Giemsa stain. Slides were then microscopically assessed and compared for qualitative and quantitative results by a blinded assessor for the two endemic filarial blood pathogens M. perstans and L. loa. Field’s stain shows excellent diagnostic performance characteristics for L. loa microfilariae compared with Giemsa staining. Concordance was favorable for M. perstans although lower than for L. loa. Field’s stain offers a rapid alternative to Giemsa stain for detection of L. loa microfilariae in thick blood smears. This could help accelerate diagnostics of blood filarial pathogens in mass screening programs or resource constrained health care institutions with high patient load.

A study of cytopathological pattern of cervical papanicolaou smear examination in women between 20 - 75 years of age group

Background: Cancer of uterine cervix is a leading cause of mortality and morbidity among women worldwide. The primary cause of this cancer is infection with human papillomavirus. Pap smear is a simple, safe, non-invasive and cost effective method for the detection of pre-cancerous, cancerous and benign lesions of cervix.Aim: To study the cytological patterns of cervical Pap smears in 766 cases and to classify the cytological abnormalities based on the Bethesda system 2014.Materials and methods: A retrospective study of total 766 cases of cervical smears was done at Pathology department, GMERS Medical College and Civil Hospital, Sola, Ahmedabad over a period of 1.5 years starting from 1st January 2020 to 30th June 2021. Women between 20 -75 years of age group were included in the study. Cervical smears were taken by obstetrics and gynaecology department and were received at pathology department, stained by using rapid Papanicolaou staining technique and studied by using light microscopy.Results: we studied total 766 cases out of which 26(3.40%) smears were unsatisfactory for evaluation. Out of these 740 cases, most common were inflammatory lesions-382 cases (49.86%).Cervical epithelial cell abnormalities including ASCUS-5 cases (0.67%), ASC-H -2 cases (0.24%), LSIL- 6 cases (0.78%), HSIL-1 case (0.13%) and AGC-NOS – 1 case (0.13%) were detected in our study. Majority of the cases were belonging to 31-40 years of age group -364 cases(47.50%) followed by 41-50 years-260 cases (33.94%).Conclusion: Pap smear is an easy, simple, safe and noninvasive method of cervical examination. By using Pap smear as a screening test, it is possible to detect malignant lesions at an earlier stage.

A simple and rapid staining technique to confirm mating in Wistar rats

A simple and rapid staining technique was developed to confirm mating in Wistar rats. The study involves collection of vaginal smear from six adult female Wistar rats and the stages of the estrous cycle were determined by vaginal exfoliative cytology technique using Field stains. The Vaginal epithelial cells are classified as basal, intermediate, superficial intermediate, superficial and cornified by microscopical examination of stained vaginal smears. Proestrus females were co-habitat with adult Wistar male at the ratio of 2:1 and mating was confirmed by presence of sperms in the vaginal smear. The day on which sperm cells were observed in the vaginal smear was considered as day zero of pregnancy, after which the female rats was separated from the male rat. Out of the six female rats exposed to male, five female rats whose vaginal smears were positive for sperm cells were confirmed pregnant by ultrasonography. This study concludes that the Field staining can be used as a simple, rapid and less expensive technique to confirm mating by vaginal exfoliative cytology in rats.

A Simple and Rapid Staining Technique for Sex Determination of Trichinella Larvae Parasites by Confocal Laser Scanning Microscopy

The roundworms of Trichinella genus are worldwide distributed and their prevalence in nature is high. Trichinella genus parasites are the causative agents of foodborne zoonosis trichinellosis. The main prevention and control of the infection are meat inspection by the magnetic stirrer method for the detection of Trichinella larvae in muscle samples. The treatment can be effective if the parasite is discovered early in the intestinal phase. Once the Trichinella larva has reached the muscle tissue, the parasite remains therein and there is no treatment for this life cycle stage. The Trichinella species is dioecious with separate male and female individuals. The developed staining technique that uses confocal laser scanning microscopy (CLSM) displays sufficient results for Trichinella larvae examination and this protocol is applicable to study the internal and external structures and for the sex determination of T. britovi and T. spiralis larvae samples. In the present study, a luminescent derivative was synthesized and used for staining of T. spiralis and T. britovi larvae samples for the examination by CLSM. Various fixatives, such as AFA, 70% ethanol, and Bouin's and Carnoy's solutions were tested for sample preparation. The synthesized luminescent compound demonstrates best visualization results for samples fixed in Bouin's fixative.

A Rapid Special Staining Technique for Identification of Normal Ureter by Frozen Section in Children With Ureteropelvic Junction Obstruction.

The exact etiology of ureteropelvic junction obstruction (UPJO) is unknown, and inadequate excision of the narrow segment has been proposed as a cause of failure in 5% to 7% of cases of pyeloplasty. To study whether frozen section can be useful to detect normal ureter distal to UPJO during pyeloplasty. Histological sections from 31 patients with UPJO were analyzed for collagen to muscle ratio (CMR) on conventional (formalin) and rapid (frozen section) Masson's trichrome staining. Pathological findings were correlated with postoperative outcomes analyzed at 1-year follow-up and expressed as excellent, moderate, or mild improvement, static and deterioration based on ultrasound grade, differential renal function, and renogram drainage pattern. There was a very strong positive correlation (r = .94; P = .001) between CMR by conventional and rapid frozen Masson's trichrome staining. There was a very strong negative correlation between pyeloplasty outcomes and CMR on conventional staining (r = -.94; P = .001) or rapid frozen Masson's trichrome staining (r = -.91; P = .001). Regression analysis revealed that a CMR of 1.2 or less (95% confidence interval: 1.9-0.7) was associated with a successful outcome. It is feasible to intraoperatively identify normal ureter distal to UPJO using CMR analysis on the novel rapid frozen section technique reported.

Cardiotoxicity in African clawed frog (Xenopus laevis) sub-chronically exposed to environmentally relevant atrazine concentrations: Implications for species survival

The toxic effects of different atrazine concentrations on tadpoles and adult male African clawed frogs (Xenopus laevis) were assessed in a controlled laboratory environment following 90 days’ exposure. The aim was to elucidate the danger of atrazine exposure on the cardiac tissue relative to its critical function of rhythmic contractility, fundamental for optimal blood circulation and homeostasis.Tadpoles and adult frogs were exposed to 0 μg/L (control), 0.01 μg L−1, 200 μg L−1 and 500 μg L−1 concentrations of atrazine for 90 days. Mortality was concenration-dependent and significantly increased in juvenile group (77%, 43%, 23% and 0 respectively for 500 μg L−1, 200 μg L−1, 0.01 μg L−1, and control group). While the mean juvenile heart area decreased concentration-dependently, adult frog mean heart area significantly increased in the 200 μg L−1 group only and mean heart weight change was variable across all exposure levels.Light microscopy of hematoxylin and eosin (H&E) and Mallory-Heidenhain rapid one-step staining techniques on cardiac tissue sections of the juvenile and adult frogs revealed shrinkage of cardiac muscle cells into thin wavy myocytes. Additionally, disorganized branching of muscle fibres with reduced striations were observed in 0.01 μg L−1 and 200 μg L−1 but hypertrophied myocytes, thickened intensely staining myofibrils in the 500 μg L−1 group in juvenile and adult frogs. Significant increase in the mean percentage area of connective tissue in all the treated groups (p < 0.036) were also recorded. Immunohistochemistry analysis showed decreased eNOS localization in cardiac tissue in 200 μg L−1 and 500 μg L−1 of both juvenile and adult group, suggestive of decreased cardiac contractility due to atrazine exposure.The results indicate that environmentally relevant atrazine concentrations cause significant mortality in tadpoles while concentrations ≥200 μg L−1 adversely affect cardiac muscle morphology and may induce functional perturbations in cardiac tissue contractility and consequent dysfunction which generally may have an adverse impact on their survival and longevity.

Pythium Insidiosum Keratitis: Histopathology and Rapid Novel Diagnostic Staining Technique.

To elucidate the histopathology of Pythium insidiosum keratitis and to describe a novel, simple, and rapid staining technique for identification of oomycete Pythium insidiosum and to differentiate it from fungi. This is a laboratory investigation study of 38 nonconsecutive cases (37 ocular samples and 1 colonic biopsy); 14 microbiologically diagnosed as Pythium insidiosum keratitis and 24 as fungal keratitis. Review of clinical, demographic details, microbiological results, and identification of cases that necessitated evisceration was performed. Reevaluation of histopathology slides was done using stains such as hematoxylin-eosin, Gomori methenamine silver (GMS), periodic acid-Schiff (PAS), potassium iodide-sulfuric acid (IKI-H2SO4). Morphology, degree, and nature of inflammation and load, distribution, and staining results of Pythium insidiosum and its comparison with fungi were studied. Delay in zoospore formation, failure of growth, and delay in identification of Pythium were the main cause of evisceration. Corneal pythiosis showed epithelial ulceration, stromal destruction, and varying inflammation; load and distribution of Pythium were inversely proportional to inflammation. The filaments were commonly wide, with admixed narrower structures and uncommonly involved Descemet membrane. The oomycete was not discretely discerned with PAS stain and stained distinctly with GMS stain and IKI-H2SO4 stain (100% sensitive). In comparison, fungal organisms stained well with PAS and GMS stain, but not with IKI-H2SO4 stain (100% specific). Pythium insidiosum keratitis is perhaps not more devastating than fungal keratitis but late diagnosis, misdiagnosis, and treatment as fungal infection are major heralds. Early diagnosis may markedly improve the patient outcome. IKI-H2SO4 is a cost-effective, simple, sensitive, and specific stain for the diagnosis of oomycete Pythium.

Comparison of Rapid Fungal Staining Technique 1% Chicago Blue 6B stain for the Diagnosis of Superficial Fungal Infections (Pityriasis Versicolor/ Tinea Versicolor) with 10% KOH Mount and Routine Fungal Culture on Sabouraud's Dextrose Agar with Olive Oil Overlay

1 Assistant Professor, Dept. of Microbiology, D Y Patil University School of Medicine, Nerul 2 Professor & H.O.D., Dept. of Microbiology, D Y Patil University School of Medicine, Nerul 3 Assistant Professor, Dept. of Microbiology, D Y Patil University School of Medicine, Nerul 4 Senior resident, Dept. of Microbiology, D Y Patil University School of Medicine, Nerul Corresponding Author Dr N. Kamath 601, Aston Tower, Plot No.9B, Sector 20.Kharghar, Navi Mumbai, 410210 Email: nktallur@gmail.com Abstract Pityriasis versicolor is a common skin infection all over the world caused by Malassezia furfur. 10% KOH mount is done for all clinically suspected Tinea versicolor by taking skin scrapings. 10% KOH has shown 66.66 % positives in this study. When we add 1% Chicago Blue 6B stain it enhances the results to 93.33 % which is as good as the Gold standard that is culture on SDA with a overlay of sterile Olive Oil. Culture has shown 96.66% positivity as Malassezia furfur grows well when there is oil overlay. So 1% Chicago Blue 6B stain can be routinely incorporated in Mycology laboratories and also in Skin clinics as the procedure is simple, rapid, reliable and can be identified with a good contrast and specificity/sesitivity is as good as culture. 1% Chicago Blue 6B is more efficacious than usual 10% KOH mount in diagnosing superficial fungal infections caused by Malassezia furfur.

Comparison of malaria &lt;i&gt;p.f/Pan&lt;/i&gt; rapid diagnostic test device with field microscopy for the detection of &lt;i&gt;Plasmodium&lt;/i&gt; species in Nigerian children

Malaria remains a serious public health and life threatening problem to children under five years in Nigeria despite the tremendous efforts made to control the disease in recent years. This study was conducted todetermine the prevalence of malarial infection, identify the species of Plasmodium and determine the performance of the Malaria P.f/Pan Rapid Diagnostic Test Device as compared to microscopy in screening clinical cases of malaria among children under five years in Gboko, Benue State, Nigeria. Malaria P.f/Pan Rapid Diagnostic Test Devices and staining technique were used to determine Plasmodia antigens and malaria parasites in blood respectively. The occurrence of malaria irrespective of the Plasmodia species was 20.4% among the children examined. There was a predominance of Plasmodium falciparum malaria 14.5% among the malaria cases diagnosed (x2 =8.93, p=0.002). No significant difference was observed between Malaria P.f / Pan RDTs (17.7%; 39/220) and microscopy (20.4%; 45/220) in screening malaria (x2 with Yates’ correction =178.5, p=0.57). The Malaria P.f/Pan RDT device recorded a sensitivity and specificity of 86.7%, CI (73.2%-94.9%) and 100%, CI (90.9%-100.0%) respectively when compared to microscopy the Gold Standard Technique. The study revealed that malaria is endemic with a predominance of P. falciparum among children under five years in Gboko, Nigeria. Both malaria P.f/Pan RDT devices and microscopy were comparable in screening malaria among the children. It is recommended that the use of Malaria P.f/Pan RDT devices should be encouraged in hospitals, though the combination of both methods will provide more accurate diagnosis.Keywords: malaria, RDT, children, Gboko, Nigeria

Exploratory development of rapid immunohistochemical staining technique in prostate biopsy tissue frozen section diagnosis

Objective To investigate the application value in the diagnosis of prostate biopsy tissue frozen section with rapid immunohistochemical detection of p40(ΔNp63),34βE12,p504s. Methods The expression of p40, 34βE12,p504s in 118 cases of prostate biopsy tissue frozen section detected by Max Vision rapid immunohistochemical staining technique. Conventional paraffin section immunohistochemistry of frozen remaining tissue served as a control. Results 118 cases of prostate biopsy histopathological diagnosis was divided into 79 cases of benign prostatic hyperplasia and 39 cases of prostate cancer. Max Vision rapid immunohistochemical staining technique was completed in 30minutes.Antibody expression located accurately. Background stained clearly and no interfering signal.The background was better than conventional immunohistochemistry of frozen remaining tissue. The positive expression rate of p40,34βE12,p504s with rapid immunohistochemistry in prostatic hyperplasia was 97.4%(77/79),93.7%(74/79), 0%(0/79),and in prostatic carcinoma was 0%(0/39),0%(0/39),97.4%(38/39).The positive expression rate of p40,34βE12,p504s with conventional immunohistochemistry in prostatic hyperplasia was 96.2%(76/79),93.7%(74/ 79),2.5%(2/79),and in prostatic carcinoma was 0%(0/39),0%(0/39),92.3%(36/39).The difference of expression between prostatic hyperplasia and prostatic carcinoma with rapid immunohistochemical detection p40 group:χ2= 109.402,P=0.000,34βE12 group:χ2=97.971,P=0.000,p504s group:χ2=113.537,P=0.000;The difference of expression between prostatic hyperplasia and prostatic carcinoma with conventional immunohistochemical detection p40 group:χ2=105.410,P=0.000,34βE12 group:χ2=97.971,P=0.000,p504s group:χ2=96.388,P=0.000;The expression of prostatic hyperplasia with between rapid immunohistochemical detection and conventional immunohistochemical detection 34βE12 group was identical,p40 group:χ2=0.207,P=0.649,p504s group:χ2=2.026,P=0.155; The expression of conventional immunohistochemical detection with between rapid immunohistochemical detection and conventional immunohisto-chemical detection p40 group and 34βE12 group were identical,p504s group:χ2=1.054, P=0.305.The expression of three markers between prostatic hyperplasia and prostatic carcinoma had statistical significance(P 0.05). Conclusion Max Vision rapid immunohisto-chemical staining technique has the advantages of rapid, accurate, timely. It could be used to rapid diagnosis of prostate biopsy tissue.The combined detection of p40,34βE12,p504s has very high practical value in the differential diagnosis of benign and malignant lesions of the prostate. Key words: Rapid immunohistochemistry; Prostate; p40; 34βE12; p504s; Diagnosis

Postmortem cytology: Alive and well in the practice of autopsy and forensic pathology.

Postmortem cytology: Alive and well in the practice of autopsy and forensic pathology.

Immunofluorescence laser micro-dissection of specific nephron segments in the mouse kidney allows targeted downstream proteomic analysis.

Laser micro-dissection (LMD) is a very useful tool that allows the isolation of finite areas from tissue specimens for downstream analysis of RNA and protein. Although LMD has been adapted for use in kidney tissue, the use of this powerful tool has been limited by the diminished ability to identify specific tubular segments in the kidney. In this study, we describe a major improvement in the methodology to isolate specific cells in the mouse kidney using immunofluorescence LMD (IF-LMD). Using IF-LMD, we can reproducibly isolate not only glomeruli, but also S1–S2 proximal segments, S3 tubules, and thick ascending limbs. We also demonstrate the utility of a novel rapid immunofluorescence staining technique, and provide downstream applications for IF-LMD such as real-time PCR and cutting-edge proteomic studies. This technical breakthrough may become an invaluable tool for understanding cellular and molecular events in the heterogeneous kidney milieu.

Advantages of the rapid double-staining method for intraoperative detection of micrometastasis in sentinel lymph nodes

For rapid intraoperative diagnosis of lymph node micrometastasis, we refined the rapid immunohistochemistry method by combining anti-cytokeratin antibody-labeled nanocrystal beads with rapid hematoxylin and eosin (H&E) staining on the same section, referred to as the rapid double staining (RDS) technique. Two frozen-section slices each were obtained from 372 lymph nodes of 100 breast cancer patients. We performed RDS for 1 slide and rapid H&E staining for the other. The results were compared with the corresponding final pathological data obtained from the permanent specimens. For specimens from patients with pN1(mi) as determined by final pathological examination, the false‑negative rate was 33.3% for rapid H&E staining and 16.7% for RDS. For specimens from patients with pN0(i+) as determined by final pathological examination, the false‑negative rate was 80% for rapid H&E staining and 0% for RDS. These results indicate that RDS is superior to conventional rapid H&E staining for intraoperative diagnosis of nodal micrometastasis and isolated tumor cells.

Four weeks lithium treatment alters neuronal dendrites in the rat hippocampus

A large body of evidence from molecular, cellular and human studies suggests that lithium may enhance synaptic plasticity, which may be associated with its therapeutic efficacy. However, only a small number of studies have directly assessed this. To determine whether lithium treatment alters structural synaptic plasticity, this study examined the effect of 4wk lithium treatment on the amount and distribution of dendrites in the dentate gyrus (DG) and hippocampal area CA1 of young adult rats. Following 4wk lithium or control chow feeding, animals were decapitated, the hippocampi were prepared and stained using a rapid Golgi staining technique and the amount and distribution of the dendritic branching was evaluated using Sholl analyses (method of concentric circles). In the DG, lithium treatment increased the amount and distribution of dendritic branches in the proximal half of dendritic trees of the granule cells and reduced branching in the distal half. In area CA1, the same treatment also increased the number of dendritic branches in the proximal half of apical dendritic trees of CA1 pyramidal cells and reduced branching in the distal half of apical dendritic trees but had no effect on basilar dendritic trees. The lithium treatment altered the total density of dendritic trees in neither the DG nor area CA1. These findings suggest that, in the DG and apical CA1, chronic lithium treatment rearranges neuronal morphology to increase dendritic branching and distribution to where major afferent input is received.

A rapid differential staining technique for Fusarium pseudograminearum in cereal tissues during crown rot infections

A novel fluorescence‐based differential staining procedure has been developed for the rapid visualization of Fusarium pseudograminearum hyphae in cereal tissues. Infected tissues are stained with safranin, which binds to host cell walls, while hyphal tissues are stained with the fluorescent dye solophenyl flavine 7GFE. The method is rapid, does not require clearing of culm cross‐sections, and provides high contrast informative images of fungal and plant structures during pathogenesis.

Update on Immunohistochemical Methods Relevant to Dermatopathology

Dermatopathology covers a large variety of entities, some having very similar histologic appearances. Immunohistochemistry is an incredibly helpful tool that is useful in diagnosis as well as prognosis of selected skin tumors. To provide a comprehensive review of recent trends and immunohistochemical stains used by dermatopathologists. Emphasis is placed on new stains as well as novel uses of existing stains. All data were gathered from published journal articles available through the National Center for Biotechnology Information PubMed database. New immunohistochemical targets are continually being found, contributing to more accurate diagnosis and classification of skin tumors. The presence of specific markers can be used to determine the aggressiveness of malignancies and design treatment strategies. In addition, application of existing stains can help determine intravascular spread of malignancy in primary cutaneous lesions. And use of rapid immunohistochemical staining techniques on frozen sections can assist in more complete excision of tumor margins. Immunohistochemistry is a highly versatile and growing tool of dermatopathologists.

Automated Microscopy to Evaluate Surgical Specimens Via Touch Prep in Breast Cancer

Breast conservation therapy is the standard treatment for breast cancer; however, 20-50% of operations have a positive margin leading to secondary procedures. The standard of care to evaluate surgical margins is based on permanent section. Imprint cytology (touch prep) has been used to evaluate surgical samples, but conventional techniques require an experienced cytopathologist for correct interpretation. An automated image screening process has been developed to discern cancer cells from normal epithelial cells. This technique is based on cellularity of the imprint specimen and does not require expertise in cytopathology. A rapid immunofluorescent staining technique coupled with automated microscopy was used to classify specimens as cancer vs. noncancer based on the density of epithelial cells captured on touch prep of tumor cross-sections. The results of the automated analysis vs. a manual screen of ten 20x fields were compared to the pathology interpretation on permanent section. A total of 34 consecutive cases were analyzed: 10 normal cases, and 24 cancer cases. The cross-section specimens for invasive cancer were correctly classified in at least 65% of the cases by using manual microscopy and at least 83% by using automated microscopy. The manual and automated microscopy correlated well for measurements of epithelial cell density (R(2)=0.64); however, the automated microscopy was more accurate. This preliminary study using an automated system for intraoperative interpretation does not require a cytopathologist and shows that rapid, low-resolution imaging can correctly identify cancer cells for invasive carcinoma in surgical specimens. Therefore, automated determination of cellularity in touch prep is a promising technique for future margin interpretation of breast conservation therapy.

Electron tomography of negatively stained complex viruses: application in their diagnosis

BackgroundElectron tomographic analysis can be combined with the simple and rapid negative staining technique used in electron microscopy based virus diagnosis.MethodsStandard negative staining of representative examples of parapoxviruses and paramyxoviruses was combined with electron tomographic analysis.ResultsDigital sectioning of reconstructions of these viruses at a selected height demonstrated the viral ultrastructure in detail, including the characteristic diagnostic features like the surface threads on C-particles of a parapoxvirus and individual glycoproteins and the internal nucleoprotein strand of Newcastle disease virus. For both viruses, deformation and flattening were observed.ConclusionThe combination of negative staining of complex viruses with electron tomographic analysis, allows visualizing and measuring artifacts typical for negative staining. This approach allows sharp visualisation of structures in a subnanometer-thick plane, avoiding blurring due to superposition which is inherent to TEM. In selected examples, such analyses can improve diagnosis of viral agents.

Electrophoretic separation of serum esterases in Willow Ptarmigan (Lagopus lagopus lagopus).

A simple method for colleetion and storage of blood samples from Willow Ptarmigan under field conditions with a minimum of haemolysis and no harmful effect on the chicks, is presented. Samples are stored in batches of 50pl or 100 pi. Partial desalting of the plasma is carried out on ultrafilter floating on glycerol for 30 min. Casting of 0.12-0.48 mm polyacrylamide gels with linear pH gradients containing ampholytes and immobilines is described. Isoelectric focusing with carrier ampholytes is shown to depend upon available ampholytes, concentration of ampholytes and thickness of the gel. With a low cost, rapid staining technique using 25 ml staining solution at 42T, up to 35 bands were made visible on the gel. In vitro hybridization, showing, both qualitatively and quantitatively, expected band patterns, verified true original phenotypes.

E12-03: Intra-operative pathology consultation - novel approaches to frozen section

E12-03: Intra-operative pathology consultation - novel approaches to frozen section