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Buchanan, Ruaridh, Stanley Fan i Caoimhe NicFhogartaigh. "Performance of Gram Stains and 3 Culture Methods in the Analysis of Peritoneal Dialysis Fluid". Peritoneal Dialysis International: Journal of the International Society for Peritoneal Dialysis 39, nr 2 (marzec 2019): 190–92. http://dx.doi.org/10.3747/pdi.2018.00087.
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Microbiological diagnosis of peritoneal dialysis (PD)-related peritonitis includes PD fluid cell count, Gram stain, and culture, as recommended by the International Society for Peritoneal Dialysis. In this retrospective study, we examined the utility of Gram stains and compared 3 culture methods. We examined a laboratory cohort (samples sent to the laboratory for any reason; n = 251) and a clinical cohort (samples sent from patients felt clinically to have peritonitis; n = 264). Culture positivity rates were higher in the clinical cohort (39.4%) than the laboratory cohort (21.5%), with no difference in the distribution of organisms between the cohorts; cell counts were significantly higher in culture-positive samples in both cohorts. Rates of positivity in the laboratory and clinical cohorts, respectively, were as follows: Gram stains 1.9% and 7.7%; direct plate culture 13% and 30.8% and “bedside” inoculated blood culture bottles 82.1% and 92.8%. Enrichment culture was never negative when another method was positive. Our data indicate that enrichment culture can be used as a single culture methodology for analyzing PD fluid without loss of sensitivity. They also suggest that Gram stains are of relatively low yield; consideration could be given to ceasing their routine performance provided that broad antimicrobial therapy is administered pending culture results.
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Yudianto, Ahmad, i Yeti Eka Sispitasari. "DNA ISOLATION FROM HUMAN URINE STAIN AS AN ALTERNATIVE MATERIAL FOR PERSONAL IDENTIFICATION EXAMINATION". Folia Medica Indonesiana 52, nr 4 (14.08.2017): 277. http://dx.doi.org/10.20473/fmi.v52i4.5476.
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Accurate determination of personal identity is crucial for an investigation since any inaccuracy may lead to fatal consequences in the judicial process. Identification through DNA analysis involves somatic chromosomes and mtDNA. Each part of the human body can be taken as a specimen since every nucleated cell in the body of an individual has identical DNA sequence. To date, samples for identification through DNA analysis are obtained from blood stains, semen stains, bones, vaginal swab, buccal swab etc. In certain cases, urine stains on the clothing have frequently been overlooked. So far, personal identification through DNA analysis by the use of urine stains has not been commonly carried out. The present study detected bands in the loci CSF1PO, THO1, TPOX and 106bp-112bp amelogenin in all samples visualized from the results of Polymerase Chain Reaction (PCR) with Polyacrylamid Agarose Gel Electrophoreses-silver staining for exposure durations of 1, 7 and 14 days. However, for exposure duration of 20 days (the maximum in the study), bands were only detected in the loci THO1 and TPOX in all samples (100%), whereas the loci CSF1PO and 50% amelogenin exhibited obvious bands. This indicated that DNA analysis of urine stains through detection of the locus STR CSF1PO, THO1, TPOX exhibited different detection responses for different exposure durations assigned to the samples of urine stain. Successful detection of these loci was supported by the differences in amplicon product and GC content at each locus. Of the loci studied, the ratio of GC content of the primers, sorted from the lowest, were as follows: locus CSF1PO of 42.6 1%, TPOX of 56.25%, and THO1 of 63.83%. In conclusion, the loci THO1 and TPOX had the same probability of success in the STR examination compared with the locus CSF1PO.
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Lacerenza, Daniela, Giorgio Caudullo, Elena Chierto, Serena Aneli, Giancarlo Di Vella, Marco Barberis, Samuele Voyron, Paola Berchialla i Carlo Robino. "Evaluation of the Effects of Different Sample Collection Strategies on DNA/RNA Co-Analysis of Forensic Stains". Genes 13, nr 6 (30.05.2022): 983. http://dx.doi.org/10.3390/genes13060983.
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The aim of this study was to evaluate the impact of different moistening agents (RNase-free water, absolute anhydrous ethanol, RNAlater®) applied to collection swabs on DNA/RNA retrieval and integrity for capillary electrophoresis applications (STR typing, cell type identification by mRNA profiling). Analyses were conducted on whole blood, luminol-treated diluted blood, saliva, semen, and mock skin stains. The effects of swab storage temperature and the time interval between sample collection and DNA/RNA extraction were also investigated. Water provided significantly higher DNA yields than ethanol in whole blood and semen samples, while ethanol and RNAlater® significantly outperformed water in skin samples, with full STR profiles obtained from over 98% of the skin samples collected with either ethanol or RNAlater®, compared to 71% of those collected with water. A significant difference in mRNA profiling success rates was observed in whole blood samples between swabs treated with either ethanol or RNAlater® (100%) and water (37.5%). Longer swab storage times before processing significantly affected mRNA profiling in saliva stains, with the success rate decreasing from 91.7% after 1 day of storage to 25% after 7 days. These results may contribute to the future development of optimal procedures for the collection of different types of biological traces.
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Sun, Mao-ling, Ji-long Zheng, Bao-jie Wang i Jun Yao. "Sperm Cell Capture Based on ABH Antigen Differences to Separate Two Men in Mixed Seminal Stains". BioMed Research International 2021 (27.11.2021): 1–5. http://dx.doi.org/10.1155/2021/7269237.
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Personal identification of two individuals in mixed semen samples in forensic DNA testing in general usually involves analysis using autosomal and Y chromosome short tandem repeats (STRs). Results may exclude unrelated donors but cannot identify individuals. In this study, sperm cell capture based on ABH antigen differences was used to obtain the cells with the single ABO blood type. Immunohistochemical staining using labeled anti-A, anti-B, and anti-H antibodies and the laser microdissection system can be used to enrich sperm with different ABO types in mixed seminal stains from two individuals. Then, PCR amplification and capillary electrophoresis were performed to genotype the STR loci. To some extent, after sperm cell capture based on ABH antigen differences, autosomal STR typing using enriched single blood group cells can be utilized to partially identify different individuals in a mixed seminal stain sample from two individuals.
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Alama, Khalid, i Prof/ Adam Dawoud Abakar. "THE ACCURACY OF LIQUID BASE PAP SMEAR VS CONVENTIONAL PAP SMEAR CYTOLOGY". European Journal of Health Sciences 4, nr 1 (7.10.2019): 32. http://dx.doi.org/10.47672/ejhs.414.
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The main aim of the present study was to assess the diagnostic accuracy of Liquid base versus Conventional smears (CS). The specific objective was to evaluate and compare efficacy liquid base cytology with conventional cytology (CS) as a screening tool and to assess the quality of immunohistochemical stain in conventional smears. A prospective study including 100 cervical samples over a period of six month. Split sample was obtained using cervex-brush. CS was prepared from the brush and the brush head was suspended in the LBC vial and processed by thin prep 5000 machine. The smears were stained with Pap stain and extra five conventional and thin prep slides prepared and stained with immunomarker. Results showed that there were 4.0% unsatisfactory (U/S) cases in CS and 1.0% in LBC; the main cause was ranging between obscuring blood and inflammation in CS and low squamous cellularity in LBC. About 5% split samples had epithelial abnormalities both in CS and LBC (3% atypical squamous cells of undetermined significance (ASCUS), devided between LBS 2% while CS1%.Low grade squamous intraepithelial lesion (LSIL) 2%, devided between LBC 1% and CS 1%. Infections as Trichomonas vaginalis (TV) and spores of candida species, 1% and 2% respectively detected only in LBC smear and missed in CS preparations of the same samples, considering 2-3 minutes for LBC screening and 5-6 minutes for CS screening following the international standards. Conventional smears did not appear to confer a cytomorphological advantage and has a lower diagnostic accuracy using IHC. The sensitivity of Thin Prep was significantly higher than that of CS due to cellular clumps and presence of marked inflammatory cells and blood which compete other epithelial cellular elements in staining affinity in addition to the length of the smear which need large volume of stains to cover the whole area. While the confined area of thin prep smear and homogenous cellular distribution support the advantages of thin prep over the conventional smear when using IHC stain. The study concluded that LBC technique leads to significant reduction of U/S rate. LBC samples offered better clarity, uniform spread of smears, less time for screening and better handling of hemorrhagic and inflammatory samples. In addition to feasibility to do further special stains and HPV tests. LBC had equivalent sensitivity and specificity to CS.
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Maryani, Maryani, Desvita Sari, Ridha Wahyutomo i Masfiyah Masfiyah. "Errors in Interpretation of Gram Stain in The First Notification from Positive BACTEC Blood Cultures in Clinical Microbiology Laboratory of Dr. Kariadi Hospital". Sains Medika : Jurnal Kedokteran dan Kesehatan 4, nr 1 (8.12.2012): 23. http://dx.doi.org/10.30659/sainsmed.v4i1.381.
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Background: Blood cultures in conjunction with the initial Gram stain of positive cultures have often been considered the “gold standard†for the diagnosis of bacteremia. When blood cultures turn positive, the attending physicians are usually notified immediately about Gram stain findings. However, information on the accuracy of Gram staining is very limited. We examined the error of preliminary blood culture reports provided by a local laboratory in an observational study.Design and Method: This was an observational study with a cross sectional approach. In this study, 369 blood cultures were examined. The positive blood cultures (135 samples) were then examined by Gram stain. Blood cultures handled on Bactec 9050, while the Gram stain was done in standard procedure Gram. Interpretation errors of Gram stain were confirmed by cultures result.Result: During one month (April 2011) we examined 369 blood cultures which 135 are positive (36.5%). Positive blood cultures were misread for 6 (4.4%) of 135 patients, they were two read as gram positive cocci had gram negative organisms by culture which were Acinetobacter baumannii, one read as gram positive bacilli had gram negative bacilli by culture which was Klebsiella pneumoniae. One isolate read as gram negative bacilli had gram positive bacilli which was Bacillus species, while two sample read as gram negative bacilli only had polymicrobial by culture, of these one isolate grew to be Enterobacter aerogenes and Staphylococcus aureus and the other were Escherichia coli and Acinetobacter spp.Conclusion: The overall 4.4% error rate of misinterpreted Gram stains from positive blood culture bottles is relatively high, so laboratory professionals and clinical microbiologist must be aware of the potential types of error that occur (Sains Medika, 4(1):23-29).
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Mosiiuk, E., i O. Karasova. "Features of establishing the presence of blood in old stains by the method of thin-layer vertical chromatography". Theory and Practice of Forensic Science and Criminalistics 23, nr 1 (27.07.2021): 400–409. http://dx.doi.org/10.32353/khrife.1.2021.31.
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Due to the fact that the bloodstain pattern analysis takes the first place in the structure of the study of biological objects, the question of the study of blood patterns is relevant today. The main task (that will allow to solve the following, which puts the investigation before the experts), is to prove that these patterns, which are examined, contain blood, (regardless of their remoteness, attempts to destroy patterns of blood), find out its types or refute traces of blood. If the expert fails to prove the presence of blood, he must refuse to address the following issues. Otherwise, the expert may obtain results that will ultimately lead him to an erroneous conclusion.
The article considers options for extraction of old blood stains in order to establish the presence of blood by the method of thin-layer vertical chromatography. This question is still relevant, as biological material can change under the influence of environmental factors and time and this can be an obstacle to establishing the fact of its presence on physical evidence, and also it can do harm to the quality of the study and the correctness of the conclusions, made as a result of the study. In order to select substances, that could provide rapid extraction of old blood stains, a study was performed using weak solutions of alkalis and acids and subsequent determination of the presence of blood by thin layer vertical chromatography. In immunological studies, there have been cases where blood crusts on non-hygroscopic surfaces under the influence of natural factors (such as high temperatures and direct sun rays) have not been extracted in saline and distilled water for several days. In addition, when using known blood samples in the reactions, the dependence of the saturation of the extracts on the age of the samples and the time of their extraction. These observations prompted researches in the sector of biological research and accounting of the Cherkasy NDEKTs of the Ministry of Internal Affairs on methods for extracting old blood stains, which would allow to detect blood in the shortest possible time by the method of thin-layer vertical chromatography.
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Oliveira, Naila Francis Paulo de, Mary Anne Heidi Dolder i Selma Candelária Genari. "Light microscope observation of circulating human lymphocytes cultured in vitro". Brazilian Archives of Biology and Technology 53, nr 5 (październik 2010): 1097–100. http://dx.doi.org/10.1590/s1516-89132010000500013.
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The purpose of this work was to study the isolation and a light microscopy technique for cultured lymphocytes. Blood samples were obtained by venipuncture with an anticoagulant added and centrifuged in a Percoll density gradient to separate the leukocytes. Lymphocytes were placed in 25 cm ³ tissue culture flasks at 37ºC. After culturing, they were fixed and stained with the methods used for blood smears. Results showed that not all fixing solutions and stains were an equally good choice for cultured lymphocytes.
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Stazio, Alice, Juan G. Victores, David Estevez i Carlos Balaguer. "A Study on Machine Vision Techniques for the Inspection of Health Personnels’ Protective Suits for the Treatment of Patients in Extreme Isolation". Electronics 8, nr 7 (30.06.2019): 743. http://dx.doi.org/10.3390/electronics8070743.
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The examination of Personal Protective Equipment (PPE) to assure the complete integrity of health personnel in contact with infected patients is one of the most necessary tasks when treating patients affected by infectious diseases, such as Ebola. This work focuses on the study of machine vision techniques for the detection of possible defects on the PPE that could arise after contact with the aforementioned pathological patients. A preliminary study on the use of image classification algorithms to identify blood stains on PPE subsequent to the treatment of the infected patient is presented. To produce training data for these algorithms, a synthetic dataset was generated from a simulated model of a PPE suit with blood stains. Furthermore, the study proceeded with the utilization of images of the PPE with a physical emulation of blood stains, taken by a real prototype. The dataset reveals a great imbalance between positive and negative samples; therefore, all the selected classification algorithms are able to manage this kind of data. Classifiers range from Logistic Regression and Support Vector Machines, to bagging and boosting techniques such as Random Forest, Adaptive Boosting, Gradient Boosting and eXtreme Gradient Boosting. All these algorithms were evaluated on accuracy, precision, recall and F 1 score; and additionally, execution times were considered. The obtained results report promising outcomes of all the classifiers, and, in particular Logistic Regression resulted to be the most suitable classification algorithm in terms of F 1 score and execution time, considering both datasets.
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Shabbir, Sheeba, Nusrat Ali, Iftikhar ,. Ahmad, Aina Khurshid, Muhammad Farhan Siddiq Rao, Sabika Hussain i Muhammad Rizwan. "Enzyme Immunoassay for the Detection of Human Chorionic Gonadotropin (HCG) and Placental MRNA Marker a Practical Technique for Forensic Pregnancy Identification in Bloodstains". Pakistan Journal of Medical and Health Sciences 16, nr 10 (30.10.2022): 804–6. http://dx.doi.org/10.53350/pjmhs221610804.
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Introduction: The diagnosis of pregnancy from forensic bloodstains can be useful in cases of infanticide, criminal abortions and missing person identification. Objective: This research illustrated the use of a rapid, precise, and tremendously responsive enzyme immunoassay kit designed for medical usage, which is put to good use in our lab for qualitative HCG detection in blood stains and has proven to be a useful tool in forensic pregnancy identification. Methods: HCG concentrations had previously been generally known, and total eighty whole blood samples were taken: forty expectant females (every single one between months one to six of pregnancy), twenty healthy young males, and twenty postmenopausal females with good health ration has been cleared in all data. Results: Enzyme immunoassay is a useful forensic technique for detecting human chorionic gonadotropin hormone in pregnant women. In the forty sample batch that was dated for six months, 37 samples (92.5%) yielded positive results, 38 of which (95%) yielded good outcomes in the qualitative analysis, most of them within the sensitivities boundary. The samples that were diluted to 1/100 and 1/200 and kept at room temperature for one week and six months period, respectively, produced just two (5.0%) and five (12.5%) successful results and the samples showed negative results when diluted to 1/200 and stored for six months. Practical implication This paper reports on human chorionic gonadotropin (HCG) detection in bloodstains based on enzyme immunoassay. Conclusion: Enzyme immunoassay has proven to be a suitable method intended for detecting an hCG hormone in blood stains, allowing for the qualitative assessment of hCG, and making it particularly interesting for use in forensic science applications. Keywords: Enzyme immunoassay, human chorionic gonadotropin (hCG), pregnancy, bloodstain
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Patro, Jagganath, Swagatika Panda, Neeta Mohanty i Uma S. Mishra. "The Potential of Light Microscopic Features of the Oral Mucosa in Predicting Post-mortem Interval". Sultan Qaboos University Medical Journal [SQUMJ] 21, nr 1 (15.03.2021): e34-41. http://dx.doi.org/10.18295/squmj.2021.21.01.005.
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Objectives: The post-mortem interval (PMI) refers to the amount of time elapsed between death and discovery of the body. This study aimed to evaluate light microscopic cellular changes in the oral mucosa and identify the potential of this method for predicting PMI. Methods: This prospective study was conducted between July 2016 and January 2018 at the Institute of Dental Sciences, Siksha ‘O’ Anusandhan University, Bhubaneswar, India. A total of 150 post-mortem (including 75 gingival and 75 buccal mucosa samples) and 40 ante-mortem (including 20 gingival and 20 buccal mucosa samples) tissue samples were compared using haematoxylin and eosin, periodic acid-Schiff (PAS) and van Gieson stains. Microscopic changes in the epithelium and connective tissue were categorised according to PMI stage as early (<12.5 hours since death), intermediate (12.5–20.5 hours since death) or late (>20.5 hours since death). Results: Most epithelial cellular changes occurred early, except for arc-shaped nuclei and epithelial shredding which were intermediate and late changes, respectively. However, microscopic changes in the connective tissue were only observable at ≥12.5 hours. There was a progressive decrease in intensity in van Gieson stains and an increase in intensity in PAS stains as PMI increased. Several microscopic features were found to be significant predictors of PMI including epithelial homogenisation, cytoplasmic vacuolation, nuclear degeneration, arc-shaped nuclei, chromatin clumping, red blood cell clumping and lysis, melanin incontinency, myofibril degeneration, salivary gland acini degeneration and epithelial connective tissue separation (P <0.050 each). Conclusion: These findings indicate that microscopic evaluation of the oral mucosa may be helpful for PMI prediction. KEYWORDS Post-mortem Changes; Light Microscopy; Oral Mucosa; Epithelial Cells; Lamina Propria; Salivary Glands; Histocytochemistry; Periodic Acid-Schiff Reaction; India.
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Liu, Zhiyong, Qiangwei Wang, Nana Wang, Yu Zang, Riga Wu i Hongyu Sun. "A Comprehensive Characterization of Small RNA Profiles by Massively Parallel Sequencing in Six Forensic Body Fluids/Tissue". Genes 13, nr 9 (25.08.2022): 1530. http://dx.doi.org/10.3390/genes13091530.
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Body fluids/tissue identification (BFID) is an essential procedure in forensic practice, and RNA profiling has become one of the most important methods. Small non-coding RNAs, being expressed in high copy numbers and resistant to degradation, have great potential in BFID but have not been comprehensively characterized in common forensic stains. In this study, the miRNA, piRNA, snoRNA, and snRNA were sequenced in 30 forensic relevant samples (menstrual blood, saliva, semen, skin, venous blood, and vaginal secretion) using the BGI platform. Based on small RNA profiles, relative specific markers (RSM) and absolute specific markers (ASM) were defined, which can be used to identify a specific body fluid/tissue out of two or six, respectively. A total of 5204 small RNAs were discovered including 1394 miRNAs (including 236 novel miRNA), 3157 piRNAs, 636 snoRNAs, and 17 snRNAs. RSMs for 15 pairwise body fluid/tissue groups were discovered by differential RNA analysis. In addition, 90 ASMs that were specifically expressed in a certain type of body fluid/tissue were screened, among them, snoRNAs were reported first in forensic genetics. In brief, our study deepened the understanding of small RNA profiles in forensic stains and offered potential BFID markers that can be applied in different forensic scenarios.
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Hamada, Kisei, Ako Koreeda, Yuki Ohtsu, Kosei Yonemitsu i Shigeyuki Tsunenari. "A simple dot enzyme-linked immunosorbent assay for ABO blood typing of biological fluid and stains: effects of heating samples". Legal Medicine 4, nr 4 (grudzień 2002): 217–22. http://dx.doi.org/10.1016/s1344-6223(02)00048-2.
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Dülmer, Marlies, Gabriele Reker, T. Trang Nguyen, Lotte Henke i Jürgen Henke. "Human Orosomucoid (ORM1) Subtyping: Further Population Genetic Data and Reports on the Feasibility to Type Aged Blood Samples and Stains". Journal of Forensic Sciences 43, nr 2 (1.03.1998): 16159J. http://dx.doi.org/10.1520/jfs16159j.
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Sharma, Arun, V. K. Arora, M. P. Sachdeva i V. Bhalla. "Stability Studies on ACP1 Isozymes in Human Blood and Menstrual Blood Stains: Polymorphism in a Heterogeneous Indian Population Sample". Anthropologist 3, nr 1 (styczeń 2001): 69–71. http://dx.doi.org/10.1080/09720073.2001.11890690.
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Yang, Xiaoxi, Julie Kanter, Nathaniel Z. Piety, Melody Benton, Seth M. Vignes i Sergey S. Shevkoplyas. "A Simple, Rapid, Low-Cost Test for the Diagnosis of Sickle Cell Disease Using a Paper-Based Hemoglobin Solubility Assay". Blood 120, nr 21 (16.11.2012): 245. http://dx.doi.org/10.1182/blood.v120.21.245.245.
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Abstract Abstract 245 Introduction: Sickle cell disease (SCD) is the most common inherited blood disorder caused by a mutation in the beta-globin chain of the hemoglobin (Hb) molecule. The result of this mutation, Hb S, polymerizes when deoxygenated, giving the red blood cells a characteristic, sickled shape. This polymerization causes in vivo vaso-occlusion and disrupts the vascular endothelium causing multi-organ complications in affected individuals. Nearly 70% of all affected births occur in Africa, where conservative estimates of SCD prevalence suggest a 10.68/1000 rate at birth (compared to 0.49/1000 in the U.S.). Although SCD causes significant morbidity and premature mortality, most affected persons born in the U.S. are able to survive into adulthood (primarily due to the implementation of universal newborn screening). In contrast, most affected individuals born in low-income countries die before the age of 5 years due to lack of early intervention. Current technology for diagnosing SCD relies on hemoglobin electrophoresis, isoelectric focusing or high performance liquid chromatography. This type of testing remains prohibitively expensive to low-income countries, where SCD is most prevalent and newborn screening is unavailable. Thus, many affected individuals are not diagnosed before severe complications or death can occur. The urgent need to develop a low-cost diagnostic test for SCD has been recently recognized as a priority by the World Health Organization. In this study, we describe the implementation of a paper-based hemoglobin solubility assay as a simple, low-cost and rapid test for definitive diagnosis of SCD. Materials and Methods: Normal human venous blood (Hb AA) was collected and compared to blood from patients with SCD (Hb SS) and SCT (Hb AS). To perform the test, a 20uL droplet of whole blood mixed with the components of the Hb solubility assay (SickleDex™, Streck) was deposited onto a paper-fluidic device fabricated using a previously published method. Polymerized deoxy-Hb S remained in the center of the blood stain entrapped by the paper substrate, while other forms of Hb remained soluble and were transported laterally to the periphery of the stain by capillary action. The resulting blood stain was digitized with a portable scanner and analyzed automatically. The red color intensity profiles were normalized by the total area under the curve to account for the differences in hematocrit among subjects. The SCD index was defined as the normalized color intensity 5 mm from the center of the blood stain. Data were presented as results of individual experiments and as mean ± SD. The values of the SCD index for samples from each Hb genotype were compared using a paired two-tailed t-test. Results: The entanglement of polymerized deoxy-Hb S by the fibers of the paper substrate resulted in the formation of a dark red spot in the center of the blood stain, while the wicking of the soluble forms of Hb from the center produced a pink ring on the periphery of the stain. The patterns of the blood stains produced on paper by normal (Hb AA), SCT (Hb AS) and SCD (Hb SS) samples were significantly different (Fig. A). The tight clustering of the normalized color intensity profiles for different subjects with the same Hb genotype enabled the use of the SCD index as a quantitative metric for distinguishing between the three types of samples (Fig. B). The difference between the SCD index measured for Hb AA, Hb AS and Hb SS samples was highly significant, with p<0.001 (Fig. C). Unlike the conventional, commercially available Hb solubility tests (e.g. SickleDex), the paper-based Hb solubility assay can conclusively differentiate between SCT and SCD samples using the characteristic blood stain patterns produced by each sample on the paper substrate. Conclusions: This proof-of-concept study demonstrated the feasibility of using the paper-based Hb solubility assay as a simple, low-cost, point-of-care diagnostic test for SCD. The ability to diagnose SCD quickly and inexpensively will be particularly useful for universal SCD screening in resource-limited settings, such as Africa. This test will also be very useful in the emergency room setting in high-income countries to enable healthcare professionals to objectively confirm suspected SCD at the bedside. Disclosures: No relevant conflicts of interest to declare.
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Strickland, Jaimie M., Doug Lyman, Lorraine M. Sordillo, Thomas H. Herdt i John P. Buchweitz. "Effects of Super Nutritional Hepatic Copper Accumulation on Hepatocyte Health and Oxidative Stress in Dairy Cows". Veterinary Medicine International 2019 (5.05.2019): 1–9. http://dx.doi.org/10.1155/2019/3642954.
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Concerns regarding excessive hepatic copper concentrations in dairy cows have increased. The objective of this study was to determine the association of hepatic copper concentrations with evidence of liver disease. Blood and liver samples were collected at the time of slaughter in cull dairy cows (n=100). Liver samples were analyzed for copper using inductively coupled plasma mass spectrometry and crude fat using liquid-liquid extraction and gravimetry. Serum samples were analyzed for glutamate dehydrogenase,γ-glutamyltransferase, sorbitol dehydrogenase, aspartate aminotransferase activities, and bile acid concentrations. Liver samples were examined histologically for inflammation, fibrosis, and rhodanine staining. Animals were stratified by hepatic copper concentration and samples in the highest and lowest quintiles (Q5 and Q1) were evaluated for oxidative stress. Systemic indices of oxidative stress included serum reactive oxygen and nitrogen species (RONS) and total antioxidant potential (AOP). Tissue-level oxidative stress was assessed by immunohistochemistry using 4-hydroxynonenal (4HNE) and 3-nitrotyrosine (3NIT) stains to score the relative abundance and distribution of oxidized lipid and protein products, respectively. Mean hepatic copper concentration was 496.83μg/g and median 469.72μg/g and ranged from 70.56 to 1264.27μg/g dry tissue. No association was found between hepatic copper concentrations and clinicopathological or histological evidence of hepatic damage or dysfunction. There was a significant increase in the amount of IHC staining of 4HNE and 3NIT in Q5 compared with Q1. Moreover, the IHC staining mirrored the distribution of the copper-specific stain rhodanine. These results demonstrate that cows with elevated hepatic copper concentrations had no evidence of active liver disease but had increased hepatic oxidative stress.
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Venkatesh, Veenaa, i Vinuta Malaichamy. "Role of special stains as a useful complementary tool in the diagnosis of renal diseases: a case series study". International Journal of Research in Medical Sciences 7, nr 5 (26.04.2019): 1539. http://dx.doi.org/10.18203/2320-6012.ijrms20191632.
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Background: Renal diseases constitute a major cause of morbidity in clinical practice and their incidence is on rise. Investigation usually requires division into even smaller samples to permit the application of specialized techniques.Methods: This is a prospective study conducted over a period of one year from January 2018 to December 2018 in the Department of Pathology, Coimbatore. A total of 58 renal biopsies were received from the Nephrology Department and the tissues were subjected to light microscopic and special stain studies.Results: Total 53 patients (91.38%) had high blood urea nitrogen value more than 20.0 mg/dl. 48 patients (82.76%) had high serum creatinine value more than 1.2 mg/dl. Out of 58 biopsy specimens, 46 (79.31%) showed primary glomerular lesions, 10 (17.24%) showed secondary glomerular lesion and 2 (3.45%) showed tubulointerstitial nephritis. Jones’s methanamine silver stain along with PAS stain helped in typing/staging of membranous glomerulopathy and membranoproliferative glomerulonephritis. Various changes in GBM like spike formation, thickening and moth-eaten appearance of GBM was noted which is seen in MGN stage II, IV and III respectively. Double contour and thickening of GBM was noted which is seen in type I and II MPGN respectively. In Myeloma cast nephropathy, tubular casts stained negative with Congo red which was used to differentiate it from amyloid deposition.Conclusions: The special stains used in this study helped in supplementing the light microscopic findings for diagnosis of kidney lesions. However, the use of other ancillary techniques like immunofluorescence and electron microscopy would help the pathologists in better and more accurate diagnosis.
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Kumsiri, Ratchanok, i Panan Kanchanaphum. "Comparison of Time Course Detection of Human Male DNA from Blood Stains on Various Objects on Surface in a Natural Environment and in a Laboratory Using Loop-Mediated Isothermal Amplification (LAMP)". Scientifica 2021 (25.11.2021): 1–7. http://dx.doi.org/10.1155/2021/4811608.
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In forensic study, the biological evidence can easily degrade, especially DNA. Degraded and environmentally challenged samples can produce numerous problems in forensic DNA analysis including loss of band product. Loop-mediated isothermal amplification or LAMP is one of the DNA analysis techniques used in forensic study. This study explores the limitations of the efficiency of the LAMP technique on abandoned DNA. For the DNA template, 8 male and 2 female blood-stained samples were taken from the surfaces, namely, brick, cloth, and tile from inside, and buried outside the laboratory. The LAMP reaction was used to amplify the SRY gene for detecting male DNA. All the blood-stained samples were stored for 1, 7, 15, 30, and 45 day (s). The LAMP product from the blood-stained samples on all the surfaces that were kept in a laboratory was detected using the gel electrophoresis technique from day 1 until day 45. However, the LAMP product on day 30 and 45 was smear and dim. The LAMP product from the blood-stained samples buried outside the laboratory was observed using the gel electrophoresis technique within day 30 (smear and dim). To increase the efficiency of detection, the qLAMP technique detected product on all the male samples from all the surfaces buried outside the laboratory for 45 days. The results indicate that this LAMP condition was possible detecting male DNA and the environmental factors are the main influence on the sensitivity of the LAMP technique. In addition, the qLAMP technique can increase the capacity and sensitivity of the detection.
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Zubakov, Dmitry, Eline Hanekamp, Mieke Kokshoorn, Wilfred van IJcken i Manfred Kayser. "Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples". International Journal of Legal Medicine 122, nr 2 (20.06.2007): 135–42. http://dx.doi.org/10.1007/s00414-007-0182-6.
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Anugrah, Puji, Citra Manela i Syamel Muhammad. "Pemeriksaan Bercak Darah pada Kain yang Direndam & Dikubur Menggunakan Tes Teichmann". Jurnal Ilmu Kesehatan Indonesia 2, nr 1 (30.07.2021): 29–32. http://dx.doi.org/10.25077/jikesi.v2i1.299.
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Background. Blood is the most important physical evidences and often found at crime scene. The Teichmann test is a confirmation test to check whether the spot really a blood.Objective. This study aims to identify the bloodstain on cloth soaked in water and buried in the ground using Teichmann test.Method. The research type is a descriptive study with a laboratory experimental study design. The sample of this study was a cloth dripped with blood, 27 samples are immersed in a bucket filled with water and 27 other samples are buried in the ground with a depth of 20 cm. The examination using the Teichmann test will be carried out on the 6th to the 14th day of exposure.Result. From the research that has been done, the results of the Teichmann test were positive on blood spots on cloth soaked in water and buried in the ground on the 6th to 9th day of exposure. Positive results indicate the formation of hemin hydrochloride crystals in the form of blackish-brown rods.Conclusion. The conclusion of this study is hemin hydrochloride crystals can still be found in blood stains on cloth soaked in water and buried in the soil using the Teichmann test but limited to the 9th day of exposure.
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Kanchanaphum, Panan. "Time Course of Detection of Human Male DNA from Stained Blood Sample on Various Surfaces by Loop Mediated Isothermal Amplification and Polymerase Chain Reaction". BioMed Research International 2018 (2018): 1–8. http://dx.doi.org/10.1155/2018/2981862.
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This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP), and LAMP-Lateral Flow Dipstick (LFD). For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, wood, clay, and tile. Then, the samples were stored at room temperature for 1, 7, 30, and 60 day(s). After the DNA amplification, the gel electrophoresis process was applied to detect LAMP product. The LFD was combined with the LAMP to detect LAMP product on the male cloth samples. For the male samples, the time course of detection on the first and seventh days indicated positive for both LAMP and PCR products on all the surfaces while no DNA amplification was found on any of the female samples. On day 30, positive LAMP product was still found on all the male samples. However, it had faded on the tiles. Moreover, all the male samples, which had tested positive for PCR product, were blurred and unclear. On day 60, LAMP product was still found on all the male samples. Conversely, the PCR method resulted in no bands showing for any of the male samples. However, the LAMP-LFD method detected product on all the male samples of cloth. The results show that the LAMP is an effective, practical, and reliable molecular-biological method. Moreover, the LFD can increase the efficiency and sensitivity of the LAMP, making it more suitable for field studies because gel electrophoresis apparatus is not required.
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Puri, Pooja, Naresh Kumar, Dhruv Sharma i SK Shukla. "Differential organic DNA extraction of semen sample contaminated with blood for the identification of a serial sexual offender: A case report". Medico-Legal Journal 87, nr 1 (2.08.2018): 32–35. http://dx.doi.org/10.1177/0025817218789569.
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In many cases of sexual assault, traces of semen are left behind on the victim’s body, clothes and the area in which the assault has taken place. The positive identification of semen is instrumental in supporting such cases. There are several methods of forensic examination of semen reported in literature, but the presence of blood complicates the identification of semen stains. This paper presents one such case study where the presence of blood makes DNA profiling more challenging as the PCR amplification becomes complicated, and the absolute differential isolation is the only way to get the clear profile using identifiler kits.
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Yudianto, Ahmad, i Nola Margaret. "Local Mapping Profile of Mitochondrial DNA (MtDNA)-Loop in Forensic Identification". Folia Medica Indonesiana 54, nr 3 (12.10.2018): 179. http://dx.doi.org/10.20473/fmi.v54i3.10008.
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To prove that mitochondrial DNA damage is not total or partial, as has been found in the preliminary study, studies need to be done to determine the opportunity of successful use of the mitochondrial DNA mini-primer set in an amplicon product below 250 bp. This is important because it can overcome quality problems in degraded DNA, which will complicate the process of DNA forensic identification. This was an observational analytic study with cross sectional design. The study material was DNA from blood and sweat stains taken from abandoned bodies. Samples consisted of 24 pieces of blood and sweat spots. The measurements of mean DNA levels and sample purity used UV-Visible Spectrophotometer, revealing mean DNA in blood samples of 152.89 ± 85.71 µg/ml and sweat samples of 89.19 ± 5.58 µg/ml, and sample purity of DNA and sweat were 1.89 ± 0.71 and 1.69 ± 0.76. Whereas, the result of D-Loop mtDNA: D-Loop I 143bp nt: 16268 -16410 and D-Loop HVS II 126bp nt: 34 -159, indicating blood spots were detected positively >95% and sweat was detected positively in 5%-20%. Results of DNA sequencing from mtDNA of blood spots and sweat spots in 126 bp and 143 bp amplicon revealed nucleotide damage marked with the letter 'N'. In conclusion, mini-primers of mitochondrial DNA in the amplification product mtDNA D-Loop HVS II 126 bp (nt 59-134) and D-Loop HVS I 143 bp (nt 16268-16410) were effectively used as support for DNA profiling in forensic medicine.
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Ancheyta-Palacios, Monserrat, Iris G. Velasco-Terán, Yojana J. P. Carreón i Jorge González-Gutiérrez. "Dried Droplets of Diluted Blood to Detect a High Concentration of Lipids". Processes 11, nr 7 (9.07.2023): 2047. http://dx.doi.org/10.3390/pr11072047.
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Hyperlipidemia is the elevated concentration of lipids in the blood, and it increases the probability of arterial obstruction, infarctions, and other complications of the circulatory system. While there are indications that qualitative analysis of blood stains could potentially identify patients with this pathology, the efficacy of this method remains uncertain. In this paper, we report an experimental study that investigates the formation of patterns in dried blood droplets with varying concentrations of ultrapure water. Two blood samples, one healthy and one with moderate hyperlipidemia, were examined to determine the ideal water and blood mixtures for detecting high lipid concentrations. Numerous intricate patterns were observed throughout the central region and periphery of the dried droplet. These patterns encompass various forms, such as plaques, bump-like patterns, and a range of cracks including random, radial, and ortho-radial configurations. By calculating the entropy of the Gray Level Co-occurrence Matrix (GLCM) and analyzing ROC curves, we determined that solutions with 4% and 12% hematocrit (indicating a high percentage of ultrapure water) exhibit over 95% accuracy in differentiating high lipid concentrations. These findings provide a promising outlook for the development of diagnostic methods based on the study of diluted blood coatings.
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Sciortino, C. V., Eric L. Xia i Alberta Mozee. "Assessment of a Novel Approach to Evaluate the Outcome of Endoscope Reprocessing". Infection Control & Hospital Epidemiology 25, nr 4 (kwiecień 2004): 284–90. http://dx.doi.org/10.1086/502393.
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AbstractObjective:To investigate and evaluate the use of a portable luminometer system for detecting contamination following the reprocessing and high-level disinfection of flexible endoscopes.Design:Random sampling of endoscopes spaced at 1- to 2-week intervals following normal use in patients.Methods:Portable luminometer system testing of 31 endoscopes undergoing reprocessing, 63 stored endoscopes, and 15 reprocessed endoscopes that underwent in-depth microbiological analysis. For testing with the portable luminometer system, samples were collected by swabbing a 100-cm2 shank surface area and the internal tip end orifice. Standardization of portable luminometer system results was performed in vitro by comparison of serial dilutions of known quantities of microorganisms and blood, tested before and after sterilization by autoclave. Microbiological analysis included Gram stain, culture for aerobic bacteria, and gene probes for Mycobacterium tuberculosis, herpes simplex viruses 1 and 2, and Cytomegalovirus. Trichrome and calcofluor white stains were used to detect parasites and fungi. Legionella was detected by stain with fluorescent-labeled monoclonal antibody.Setting:The gastroendoscopy unit of a Veterans Affairs hospital.Results:The portable luminometer system was capable of detecting microbial and cellular contamination of flexible endoscopes following high-level disinfection and reprocessing. The sensitivity of the assay was sufficient for detecting low-level contamination.Conclusions:The system provided a rapid microbiological outcome monitor for the cleaning and disinfection process. The system was easy to use and relatively accurate.
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Vele, Oana, Yale Nemerson i Viji Balasubramanian. "Local Shear Conditions and Platelet Aggregates Regulate the Incorporation and Activity of Circulating Tissue Factor in ex-vivo Thrombi". Thrombosis and Haemostasis 88, nr 11 (2002): 822–26. http://dx.doi.org/10.1055/s-0037-1613309.
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SummaryThe presence of thrombogenic blood-borne or circulating tissue factor (cTF) has recently been demonstrated. These observations have implicated cTF to be a key determinant of thrombus propagation by depositing on platelets in nascent thrombi. Previously, we detected cTF by detergent solubilization and addition of phospholipids. We now report the direct demonstration of TF activity in ex-vivo thrombi. Collagen-coated substrates were exposed to native blood at shear rates of 0, 650, and 2000 s-1 for 10 min in a modified rotating Teflon cone and plate viscometer. Substrates were then gently rinsed to remove ‘loose’ (unadherent) components of blood. cTF activity was measured by adding a solution containing 10 nM FVIIa, 100 nM FX, and 5 mM CaCl2 to the substrates exposed to blood. Samples of this mixture were obtained at intervals for 30 min and the amount of Xa generated was quantified by adding a chromogenic substrate, Spectrozyme Xa, and measuring the increase in OD at 405 nm. Our studies show that a minimal amount of generated Xa (∼ 1nM) can be measured from ex-vivo thrombi. Static and shear samples generated the same amount of Xa, with the exception of blood subjected to 650 s-1 shear. At 650 s-1 shear rate, the amount of Xa generated reached a maximum of 4 nM at 5 min and then decreased to ∼1 nM. Immunohistological stains and fluorescent images demonstrate the presence of cTF antigen at 650 s-1 wall shear rate.
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Źródłowski, Tomasz, Joanna Sobońska, Dominika Salamon, Isabel M. McFarlane, Mirosław Ziętkiewicz i Tomasz Gosiewski. "Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the “Gold Standard”?" Microorganisms 8, nr 3 (29.02.2020): 346. http://dx.doi.org/10.3390/microorganisms8030346.
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Standard blood cultures require at least 24–120 h to be reported as preliminary positive. The objective of this study was to compare the reliability of Gram staining and fluorescent in-situ hybridization (FISH) for detecting bacteria in otherwise negative blood culture bottles. Ninety-six sets were taken from patients with a diagnosis of sepsis. Six incomplete blood culture sets and eight blood cultures sets demonstrating positive growth were excluded. We performed Gram stain and FISH on 82 sets taken from post-operative septic patients: 82 negative aerobic blood cultures, 82 anaerobic blood cultures, and 82 blood samples, as well as 57 blood samples taken from healthy volunteers. From the eighty-two blood sets analyzed from the septic patients, Gram stain visualized bacteria in 62.2% of blood samples, 35.4% of the negative aerobic bottles, and in 31.7% of the negative anaerobic bottles. Utilizing FISH, we detected bacteria in 75.6%, 56.1%, and 64.6% respectively. Among the blood samples from healthy volunteers, FISH detected bacteria in 64.9%, while Gram stain detected bacteria in only 38.6%. The time needed to obtain the study results using Gram stain was 1 h, for FISH 4 h, and for the culture method, considering the duration of growth, 5 days. Gram stain and FISH allow quick detection of bacteria in the blood taken directly from a patient. Finding phagocytosed bacteria, which were also detected among healthy individuals, confirms the hypothesis that blood microbiome exists.
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Niimi, Yuta, Kyoko Baba, Masako Tsuchida i Akira Takeda. "A Histological Evaluation of Artificial Dermal Scaffold Used in Micrograft Treatment: A Case Study of Micrograft and NPWT Performed on a Postoperative Ulcer Formation after Tumor Resection". Medicina 58, nr 1 (4.01.2022): 73. http://dx.doi.org/10.3390/medicina58010073.
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Background and Objectives: Wound healing (WH) is a complex natural process: the achieving of a proper WH with standard therapies sometimes is not fulfilled and it is often observed in aged and diabetic patients, leading to intractable ulcers. In recent years, autologous micrograft (AMG) therapies have become a new, effective, and affordable wound care strategy among both researchers and clinicians. In this study, a 72-year-old female patient underwent a combination of treatments using micrograft and negative pressure wound therapy (NPWT) on a postoperative skin ulcer after a benign tumor resection on the back with the aim to present an innovative method to treat skin ulceration using AMG combined with an artificial dermal scaffold and NPWT. Materials and Methods: A section of the artificial dermal scaffold, infused with micrografts, was sampled prior to transplant, and sections were collected postoperatively on days 3 and 7. Hematoxylin-eosin (HE) and immunohistochemical stains were employed for the evaluation of Cytokeratin AE1/AE3, desmin, and Factor VIII. Additionally, on postoperative day 3, NPWT dressing was evaluated using HE stains, as well. The resulting HE and immunostaining analysis revealed red blood cells and tissue fragments within the collagen layers of the artificial dermis prior to transplant. On postoperative day 3, collagen layers of the artificial dermis revealed red blood cells and neutrophils based on HE stains, and scattering of cytokeratin AE1/AE3-positive cells were detected by immunostaining. The HE stains on postoperative day 7 showed more red blood cells and neutrophils within the collagen layers of the artificial dermis than on day 3, an increase in cytokeratin AE1/AE3-positive cells, and tissue stained positively with desmin and Factor VIII. Results: Results suggest that the effects of both micrografts and migratory cells have likely accelerated the wound healing process. Furthermore, the NPWT dressing on day 3 showed almost no cells within the dressing. This indicated that restarting NPWT therapy immediately after micrograft transplant did not draw out cells within the scaffold. Conclusions: Micrograft treatment and NPWT may serve to be a useful combination therapy for complex processes of wound healing.
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Kuddus, Mohammed, i Pramod W. Ramteke. "Cold-active extracellular alkaline protease from an alkaliphilicStenotrophomonas maltophilia: production of enzyme and its industrial applications". Canadian Journal of Microbiology 55, nr 11 (listopad 2009): 1294–301. http://dx.doi.org/10.1139/w09-089.
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A novel psychro-tolerant bacterium Stenotrophomonas maltophilia (MTCC 7528) with an ability to produce extracellular, cold-active, alkaline, and detergent-stable protease was isolated from soil samples obtained from Gangotri glacier, Western Himalaya, India. The culture conditions for higher protease production were optimized with respect to incubation time, agitation, substrate, pH, and temperature. Maximum protease production of 56.2 U·mL–1was achieved in the medium at 20 °C and pH 9.0 after 120 h incubation. The protease was partially purified by ion-exchange chromatography and approximately 55-fold purification was achieved. The purified enzyme was a 75 kDa protease with maximum activity and stability at pH 10 and 20 °C. The activity of enzyme is stimulated by Mn2+and inhibited completely by metalloprotease inhibitors, indicating that it is a metalloprotease. The protease showed excellent stability and compatibility with commercial detergents and exhibited high efficiency for the removal of different types of protein-containing stains at low temperature. The wash performance analysis of blood and grass stains on cotton fabric showed an increase in reflectance by 26% and 23%, respectively, after treatment with enzyme in comparison to detergent only. These results indicate that it may be a potential component to use as a detergent additive for cold washing and in environmental bioremediation in cold regions.
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Hartina, Oktania, Ulil Amna i Rahmatul Fajri. "IDENTIFIKASI BAHAN PEWARNA NAPHTHOL YELLOW S (C10H6N2NaO8S+) DALAM SEDIAAN PERONA MATA SECARA KROMATOGRAFI LAPIS TIPIS (KLT)". QUIMICA: Jurnal Kimia Sains dan Terapan 2, nr 1 (5.10.2020): 5–8. http://dx.doi.org/10.33059/jq.v2i1.2612.
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Naphtol yellow S is a synthetic dye used for coloring batik. In cosmetics, naphtol yellow S is abused for eye shadow coloring to get more attractive colours, even though the use of naphtol yellow S can cause negative effects such as eye and respiratory tract irritation, dangerous if swallowed or inhaled will cause blood to become abnormal and followed liver and kidney damage. This study aims to identify naphtol yellow S in eye shadow. The coloring agent analysis was determined by Thin Layer Chromatography (TLC). Based on the result, there were no naphtol yellow S stains found in the sample. It can be concluded that the eye shadow samples used did not contain naphtol yellow S.
Keywords: Naphtol Yellow S, Cosmetics, Eye Shadow, TLC
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Acar, Kemalettin, Ayse Kurtulus Dereli, Esin Avci, Volkan Zeybek, Erdi Kutlu, Süleyman Demir i Hande Senol. "Determination of haemoglobin A1c levels using high-performance liquid chromatography of bloodstains". Medicine, Science and the Law 60, nr 1 (23.10.2019): 19–25. http://dx.doi.org/10.1177/0025802419879272.
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This study aimed to determine haemoglobin A1c (HbA1c) levels in bloodstains shed on glass and fabric surfaces on specified test dates. Blood samples were taken from 26 patients (13 diabetic and 13 non-diabetic). Initial HbA1c levels were detected by using high-performance liquid chromatography (HPLC), and bloodstains were created on both cotton fabric and glass surfaces. Samples were processed at different ages (0, 7, 14, 28 and 56 days) by diluting distilled water and then measuring HbA1c levels by HPLC again. In all stains, HbA1c levels could be determined by using HPLC, but there was a moderate rise in accordance with the age of the stains. A statistically significant difference was found for bloodstains on clothes compared to those on glass surfaces. Receiver operating curve analysis found a sensitivity of 1.0 and specificity of 0.923 (cut-off 6.55) for glass surfaces on the seventh day; a sensitivity of 1.0, a specificity of 0.846 (cut-off 6.45) for clothes on the seventh day; a sensitivity of 1.0 and a specificity of 0.923 (cut-off 6.85) for clothes on the 56th day; and a sensitivity of 1.0 and a specificity of 0.846 (cut-off 7.55) for glass surfaces on the 56th day. In conclusion, this study found that HbA1c levels could be measured with high reliability from forensic bloodstains by using HPLC. Thus, in cases where DNA data banks cannot identify individuals, it would make sense to turn to those who have a medical history of diabetes among the suspects with the results of high HbA1c levels.
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Ishwar Prasad Dubey, R. K. Kumawat, I. P. Tripathi i Pankaj Shrivastava. "A pilot study of DNA yield from bloodstains on various surfaces using Phenol chloroform isoamyl alcohol (PCIA) and Chelex DNA extraction methods". GSC Biological and Pharmaceutical Sciences 13, nr 2 (30.11.2020): 124–27. http://dx.doi.org/10.30574/gscbps.2020.13.2.0363.
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Presently, Short Tandem Repeats (STRs) based forensic DNA typing technology is being globally used in solving a diverse range of forensic cases such as paternity, identification of unknown dead bodies/skeletal remains, or suspect in a case of rape or mass rape. The technology has invaded its tentacles in almost all areas of criminal investigation in the last few decades. The present forensic DNA technology is based on capillary electrophoresis and utilizes short tandem repeats(STRs).On one hand, the technology is extensively used in the investigation of crime in highly sensitive cases, but on the another hand, obtaining DNA profile from forensic samples are highly challenging many times. Advent of PCR has been a boon for handling the challenging samples in forensic DNA analysis. The quality DNA profiles from challenging samples rely on the yield and quality of DNA, which is mainly dependent upon the method used for DNA extraction. Any specific method can never be thought of to be useful for all variety of samples. Still, Phenol Chloroform Isoamyl Alcohol (PCIA) organic extraction method has been proven to be useful for a wide variety of samples from the simplest saliva/blood to complex teeth and bone samples. In the present study, we compared the yield of DNA from blood stains recovered from various surfaces using the PCIA extraction method and Chelex DNA extraction methods and their compatibility with present-day STR based capillary electrophoresis typing. The mean value of DNA yield was found 50.5 ng/ µl and 32.25 ng/ µl by PCIA and Chelex DNA extraction methods, respectively. Overall, the highest yield was observed from all the tested samples from the PCIA method.
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Billett, Henny H., Kateryna Fedorov, Margarita Kushnir, David Yin i Morayma Reyes Gil. "Neutrophil Extracellular Traps (NETs) Are a Subset of Smudge Cells Identifiable By Peripheral Smear Autoanalyzers". Blood 134, Supplement_1 (13.11.2019): 2321. http://dx.doi.org/10.1182/blood-2019-127448.
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Background. Neutrophil Extracellular Traps (NETs) are composed of extracellular decondensed chromatin networks that play an important role in immune and inflammatory response regulation. Simple and reliable identification of NETs has been challenging. Automated analyzers such as the CellaVision® Hematology Autoanalyzer identify a subset of cell-derived entities as smudge cells. We hypothesize that, in addition to the typical degenerated lymphocytes (DLs) forming smudge cells, a proportion of smudge cells as identified by the Autoanalyzer are actually NETs. Since patients with high numbers of NETs have increased and specific morbidities, accurate and rapid identification of NETs may be clinically useful. Methods. To test our hypothesis, we analyzed peripheral blood smears from samples processed by the CellaVision® Hematology Autoanalyzer. To differentiate NETs from DLs we used morphologic characteristics, immunohistochemistry, and flow cytometry. After immunohistochemistry and flow cytometry informed our morphologic classification of NETs and DLs, we relied solely on morphology. Smears with >20% smudge cells were classified as the study group; the control group had < 5% smudge cells. Medical chart review was performed by investigators blinded to sample group designation. Data obtained from chart review included patient demographics; presence of bacterial and/or viral infection occurring <1 week of sample collection; myeloproliferative and lymphoproliferative disorders (LPD); solid organ malignancy and/or transplant; sickle cell disease; chronic kidney (CKD) and liver diseases; autoimmune disorders; HIV; thrombotic events. We reviewed the same sample laboratory data for hemoglobin, platelet counts, and white blood cell (WBC) counts. Statistical analyses were performed using two-sided t-test with α =0.05 for continuous variables, chi-square for categorical variables with samples size >10 and Fisher's exact tests for categorical variables with sample size <10. Results. 155 samples were used in the analysis: 96 in the study group, 59 in the control group. Cell-derived entities staining strongly with myeloperoxidase (MPO), neutrophil elastase (NE), and leukocyte alkaline phosphatase (LAP) were classified as NETs. On flow cytometry, these NETs are at least twice as large as WBCs, display extracellular DNA (as identified by Sytox dye) and stain positively for MPO [Figure 1]. On Wright-Giemsa stain, these appeared morphologically as cell remnants with decondensed nuclei, no intact cytoplasm, dispersed granules, and polarized chromatin projections that resemble spider webs [Figure 2]. Of 96 patient samples with >20% smudge cells, morphologic analyses designated 88 as NETs and only 8 as DLs. Comparing patients with >20% to <5% total smudge cells, the former had a higher incidence of bacterial infections (p=0.0091), as well as higher WBC, lymphocyte, monocyte counts and hemoglobin (p=0.014, 0.009, 0.004, 0.01 respectively) [Table 1]. High percentage of smudge cells correlated with a higher percentage of bacterial infection when compared to those with fewer smudge cells despite a non-significant difference in total neutrophil counts. Only 3 of the 36 patients with documented bacterial infection in the >20% smudge cell group had a neutrophils >8.5 x109/L. Of 13 infants <1yr of age with >20%smudge cells, 5 had a bacterial infection with normal neutrophil counts. When the smudge cells were separated according to their category, NETs vs DLs, the DL group was older, had more LPD, higher total WBC, lymphocyte and monocyte counts (p=0.000044, 0.00036, 0.018, 0.022, 0.033) [Table 2]. Slides with mostly NETs had higher LAP scores than those classified as mostly DLs (172 +30.5 vs 103.3 +30.8, p=0.0087). Conclusions: NETosis is a relatively newly discovered tool in the neutrophil's arsenal. This study is the first to identify NETs on peripheral smear evaluations as performed by a routine Hematology Autoanalyzer and to differentiate them from DLs using a reliable set of morphologic characteristics, immunohistochemical stains, and flow cytometry probes. This study also supports data that associate NETs with bacterial infections. This could be clinically useful in diagnosis of infection in the absence of leukocytosis. Disclosures Billett: Albert Einstein College of Medicine: Patents & Royalties: Patent application pending for NETs AI software. Kushnir:Janssen Pharmaceuticals: Research Funding. Reyes Gil:Albert Einstein College of Medicine: Patents & Royalties: Patent application pending for NETs AI software.
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Schade, Rogier P., Janke Schinkel, Freek W. C. Roelandse, Ronald B. Geskus, Leo G. Visser, Marc C. van Dijk, Joan H. C. Voormolen, Hans van Pelt i Ed J. Kuijper. "Lack of value of routine analysis of cerebrospinal fluid for prediction and diagnosis of external drainage–related bacterial meningitis". Journal of Neurosurgery 104, nr 1 (styczeń 2006): 101–8. http://dx.doi.org/10.3171/jns.2006.104.1.101.
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Object Routine microbiological and chemical analysis of cerebrospinal fluid (CSF) is often performed to diagnose external drainage–related bacterial meningitis (ED-BM) at an early stage. A cohort study was performed to investigate the value of several commonly used CSF parameters for the prediction and diagnosis of ED-BM. Methods In a cohort of 230 consecutive patients in whom external drains had been placed, CSF samples were collected daily, prospectively evaluated for the presence of bacteria using Gram stain and microbiological culture, and analyzed for leukocyte count, protein concentration, glucose concentration, and ratio of CSF glucose to blood glucose. In addition, the CSF concentration of interleukin-6 (IL-6) was determined. The definition of ED-BM was based on positive culture results in combination with clinical symptoms. A matched case–control study was performed to evaluate the cohort longitudinally and to control for biasing factors such as duration of external drainage. External drainage–related bacterial meningitis developed in 22 patients (9.6%). Results from analyses of 1516 CSF samples showed no significant differences between the patients in whom ED-BM developed and a control group without ED-BM during the first 3 days of infection or during the 3 days preceding the infection with regard to leukocyte count, protein concentration, glucose concentration, and CSF/blood glucose ratio. No significant difference between groups was found for the CSF IL-6 concentration during the 3 days preceding the infection. In the matched case–control study, none of the parameters had significant predictive or diagnostic value for ED-BM in analyses using absolute values, ratios, and differences between the current and previous day’s values. A comparison of the results from Gram stains and CSF cultures showed that the Gram staining had a very high specificity (99.9%) but a low sensitivity (18% [four of 22 patients] on the 1st day of infection and 60% [nine of 15 patients] on the 2nd day). Conclusions Severe disturbances in the CSF of patients with external drains limit the value of routine CSF analysis for prediction or diagnosis of ED-BM. Routine Gram stain of CSF has also limited predictive or diagnostic value due to its low sensitivity in screening for ED-BM.
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Taranushenko, T. Ye, A. K. Kostyuk, T. V. Leiman, S. A. Dogadin i I. I. Kalyuzhnaya. "Results of neonatal screening for congenital hypothyrosis in the Krasnoyarsk region". Problems of Endocrinology 43, nr 2 (15.04.1997): 19–21. http://dx.doi.org/10.14341/probl199743219-21.
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A total of 25130 newborns were examined, which was 91% of all babies born alive in the Krasnoyarsk region. For detecting hypothyrosis, TTH levels were measured in dry blood stains on special paper filters using Delfia diagnostic kit in accordance with the manufacturer’s instructions. Hyperthyrotropinemia was detected in 3.76% of all samples examined (primary positive results of screening), but subsequent examinations of newborns with suspected thyroid insufficiency confirmed congenital hypothyrosis in only 6 patients, or 0.99% of all infants examined repeatedly. In 99.01% of cases the detected hyperthyrotropinemia was transitory. According to the findings of neonatal screening, the incidence of congenital hypothyrosis was 1 per 4134 newborns. Congenital hypothyrosis is most often confirmed in cases with the primary positive value of TTH higher than 100 pU/ml. At lower TTH levels the probability of the diagnosis validation was lower, although not ruled out
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Juma, A., D. Oudit i M. Ellabban. "Patterns of Reinnervation and Blood Flow in Split-Skin Grafts". Canadian Journal of Plastic Surgery 13, nr 3 (sierpień 2005): 133–37. http://dx.doi.org/10.1177/229255030501300305.
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One of the most important functions of skin is thermoregulation. The alterations in the patterns of blood flow in skin is one of the main physiological processes responsible for thermoregulatory control. The mechanisms governing the thermoregulatory control of cutaneous blood flow are mainly neural and chemical in nature. At present, there is a lack of studies in the literature looking at the relationship between reinnervation and the blood flow pattern of skin grafts. The present study uses Laser Doppler flowmetry and the immunohistochemical stains protein gene product 9.5, calcitonin gene-related peptide and substance P to identify nerve fibres, and antibodies to CD31 and von Willebrand factor to identify endothelial tissues. The aim of the present study was to investigate the patterns of blood flow and nerve tissue regeneration in split-skin grafts up to 15 years following the original procedure. Thirty-two split-skin grafts were studied and these were placed into two groups based on the nature of the bed of excision: group I consisted of patients who underwent tangential excision and split-skin grafting (n=17), and group II consisted of patients with split-skin grafts placed onto fascial beds (n=15). Each subpopulation of patients was further divided into three groups based on the length of time following grafting: one to three years, four to six years and seven to 15 years. These divisions were arbitrarily chosen and called A1, A2 and A3, respectively. In the Laser Doppler flowmetry arm of the study, the grafts were assessed at various stages after heating, cooling and further reheating. The Laser Doppler flowmetry studies showed that, on subjecting the skin grafts in both groups I and II to heating and cooling followed by reheating, the overall response of the blood flow to changes in the temperature was slower. The immunohistochemical analysis showed that in all graft types and graft ages, protein gene product 9.5, calcitonin gene-related peptide and substance P stains demonstrated a relative lack of the presence of nerve fibres in the split-skin grafts compared with the control (‘normal’ skin). However, von Willebrand factor and CD31 immunological staining demonstrated that vessels were present in the split-skin grafts, with no significant difference in size or quantity from the control samples. It was found that the blood flow in the split-skin graft in response to thermal challenge, although present, was slower than that of normal skin, a finding which was independent of the age of the skin graft. It is thought that this was related to a lack of regeneration of nerve fibres and, hence, a deficiency in the neurally mediated reflexes of the blood vessels within the split-skin grafts.
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Roshdy Abouelkeir, Abrar, Abla Abdel Alrahman Ali, Mokhtar Fathi Abdelsatar, Laila Ahmed Rashed i Shimaa Ahmed Alsaeed. "Application of the Methylated Markers (Spectrin Beta and DEAD-Box Protein) for Definitive Differentiation Between Fresh and Aged Semen by evaluating Their Role in Identifying Semen From Mixed Body Fluids". International Journal of Medical Toxicology and Forensic Medicine 12, nr 4 (1.10.2022): 38615. http://dx.doi.org/10.32598/ijmtfm.v12i4.38615.
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Background: Semen identification is assumed a crucial proof of sexual assault. Moreover, body fluids at the crime scene of a human being, such as blood, semen, and saliva, are often mixed. Methods: Hence, in our study, we aimed to use methylation analysis targeting DNA epigenetic markers Spectrin beta chain (B_SPTB_03) and DEAD-box protein (DDX4) to differentiate between fresh semen (less than 4 hours) and aged semen (after 24 hours) as well as to differentiate between semen alone and semen mixed with other body fluids (blood and saliva) in the fresh and dried state. Results: Our findings showed statistically significant differences in the methylation patterns of the SPTB and DDX4 loci to distinguish semen from mixed body fluids in fresh and old samples. We were able to obtain two novel cutoff values to differentiate between fresh and aged semen, which are (52.25) with the SPTB marker and (70.75) with the DDX4 marker. Conclusion: It is concluded that the methylation approach based on the epigenetic markers of Spectrin beta chain and DEAD-box protein (B_SPTB_03 and DDX4) successfully identified fresh from aged semen and semen-derived alleles from mixed stains, hence it is recommended to be employed in forensic practice.
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Usman, Shireen J., Thomas Conley, Hannah E. Whitehead, Wojciech Wojciechowski, Kaye Thomas, Dan Bergstralh, Andrea M. Baran i in. "Characterizing Naked Nuclei Frequency and Movement in Primary AML Cell Culture Using an ECM-Based Model". Blood 138, Supplement 1 (5.11.2021): 2365. http://dx.doi.org/10.1182/blood-2021-149645.
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Abstract INTRODUCTION Naked nuclei (NN) are observed upon examining bone marrow aspirate slides from healthy individuals and from those with hematologic malignancies, but are not well understood. Without characteristic findings like cytoplasm or plasma membrane, NN are considered remnants of slide preparation or are discounted as cells of undetermined significance. NN have been associated with poor prognosis in several solid cancers. Understanding the significance of NN in hematologic malignancies such as acute myelogenous leukemia (AML) may elucidate valuable diagnostic and prognostic knowledge. Decellularized Wharton's jelly matrix (DWJM) is an extra cellular matrix (ECM)-based in vitro model that shares similar elements with the bone marrow ECM and can be used as scaffolding to culture leukemia cells. We hypothesize that NN exist in AML and interact with other cells, suggesting potential biological relevance within the bone marrow microenvironment. METHODS Primary AML samples obtained by leukapheresis were cultured in suspension with growth media or in the presence of DWJM submerged in growth media. In samples grown with DWJM, cells that were non-adherent to the matrix were collected first and then adherent cells were isolated by treating DWJM with collagenase. Live cells were stained with CellVue Maroon (CVM) for membrane, CellTracker Green (CTG) for cytoplasm, and Hoechst 33342 for nucleus, followed by analysis with Amnis/Luminex ImageStream-X imaging flow cytometer (Figure 1A). NN were defined as events positive for nuclear stain and negative for cytoplasmic and membrane stains (Figure 1B). 3-D movement of adherent AML cells in DWJM was captured in real time using confocal microscopy. Fixed cells from leukemia cell line K562 served as a control for movement. NN (Hoechst positive only), non-nucleated (Hoechst negative/CTG positive), and nucleated cells (Hoechst and CTG positive) were identified by fluorescent labeling. NN were also observed after isolation by cell sorting. Cell speed, cell displacement from origin, and change in distance to closest neighboring cell over time were measured. Additionally, flow sorted NN were examined by immunohistochemistry (IHC). RESULTS Adherent populations contained significantly more NN than non-adherent and suspension populations. The frequency of NN in matrix adherent cells ranged from 0.4-2.4% at day 3 and 0.5-5.4% at day 7 of culture (Figure 1C). Through confocal microscopic analysis, we observed NN, nucleated, and non-nucleated cells moving at speeds ranging from 0.002-0.08 µm/sec. Fixed cells showed no discernible movement in DWJM. The average speed of NN [0.019 mm/s, SD 0.011] significantly differed from the average speed of nucleated and non-nucleated cells [0.027 mm/s, SD 0.016] (p=0.004) (Figure 1E). To demonstrate directional movement, we measured change in distance between NN and closest neighboring nucleated or non-nucleated cells over time. Cells (nucleated and non-nucleated) and NN moved closer to each other over time suggesting directional movement (p=0.001) (Figure 1D). NN also showed movement in DWJM after isolation by cell sorting. IHC analysis showed sorted NN stained positive for nuclear lamin A/C, which are considered markers of nuclear membrane (Figure 1F). CONCLUSIONS Our findings confirm that NN are present in primary AML cells cultured in vitro using ourECM-based model and that they can be isolated using flow cytometry. Additionally, NN display directional movement in DWJM suggesting that they interact with other cells and may be biologically relevant structures in the bone marrow microenvironment. Figure 1 Figure 1. Disclosures Baran: AstraZeneca/Acerta: Research Funding. Chu: Pfizer: Current equity holder in publicly-traded company; Acerta/AstraZeneca: Research Funding; TG Therapeutics: Research Funding.
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El-Khouly, Fatma E., Rianne Haumann, Marjolein Breur, Sophie E. M. Veldhuijzen van Zanten, Gertjan J. L. Kaspers, N. Harry Hendrikse, Esther Hulleman, Dannis G. van Vuurden i Marianna Bugiani. "DIPG-33. CHARACTERIZING THE NEURO-VASCULAR UNIT IN DIFFUSE INTRINSIC PONTINE GLIOMA". Neuro-Oncology 22, Supplement_3 (1.12.2020): iii293. http://dx.doi.org/10.1093/neuonc/noaa222.081.
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Abstract Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a median overall survival of eleven months. Lack of chemotherapy efficacy may be related to an intact blood-brain-barrier (BBB). In this study we aim to compare the neuro-vascular unit (NVU) of DIPG to healthy pons tissue. End-stage DIPG autopsy samples (n=5) and age-matched healthy pons samples (n=22), obtained from the NIH NeuroBioBank, were immunohistochemically stained for tight-junction proteins claudin-5 and zonula occludens-1 (ZO-1), basement membrane component laminin, and pericyte marker PDGFRβ. Claudin-5 stains were also used to determine vascular density and diameters. In DIPG, expression of claudin-5 and ZO-1 was reduced, and claudin-5 was dislocated to the abluminal side of endothelial cells. Laminin expression at the glia limitans was reduced in both pre-existent vessels and neovascular proliferation. In contrast to healthy pons, no PDGFRβ expression was detected. The number of blood vessels in DIPG was significantly reduced compared to healthy pons, 13.9±11.8/mm2 versus 26.3±14.2/mm2, respectively (P<0.01). Especially the number of smaller blood vessels (<10µm) was significantly lower (P<0.01). Distribution of larger blood vessels (≥10µm) did not differ between groups (P=0.223). Mean vascular diameter was 9.3±9.9µm for DIPG versus 7.7±9.0µm in healthy pons (P=0.016). Our study demonstrates evidence of structural changes in the NVU in end-stage DIPG. Chemotherapeutic inefficacy could be the result of reduced vascular density. However, further research is needed to determine meaning and extent of these changes and to determine whether these observations are caused by the tumor or the result of treatment.
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Cooley-Andrade, O., DE Connor, DDF Ma, JW Weisel i K. Parsi. "Morphological changes in vascular and circulating blood cells following exposure to detergent sclerosants". Phlebology: The Journal of Venous Disease 31, nr 3 (17.02.2015): 177–91. http://dx.doi.org/10.1177/0268355515573686.
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Objectives To investigate morphological changes in vascular and circulating blood cells following exposure to detergent sclerosants sodium tetradecyl sulfate and polidocanol. Methods Samples of whole blood, isolated leukocytes, platelets, endothelial cells, and fibroblasts were incubated with varying concentrations of sclerosants. Whole blood smears were stained with Giemsa and examined by light and bright field microscopy. Phalloidin and Hoechst stains were used to analyze cytoplasmic and nuclear morphology by fluorescence microscopy. Endothelial cell and fibroblasts were analyzed by live cell imaging. Results Higher concentrations of sclerosants induced cell lysis. Morphological changes in intact cells were observed at sublytic concentrations of detergents. Low concentration sodium tetradecyl sulfate induced erythrocyte acanthocytosis and macrocytosis, while polidocanol induced Rouleaux formation and increased the population of target cells and stomatocytes. Leukocytes showed swelling, blebbing, vacuolation, and nuclear degradation following exposure to sodium tetradecyl sulfate, while polidocanol induced pseudopodia formation, chromatin condensation, and fragmentation. Platelets exhibited pseudopodia with sodium tetradecyl sulfate and a “fried egg” appearance with polidocanol. Exposure to sodium tetradecyl sulfate resulted in size shrinkage in both endothelial cell and fibroblasts, while endothelial cell developed distinct spindle morphology. Polidocanol induced cytoplasmic microfilament bundles in both endothelial cell and fibroblasts. Patchy chromatin condensation was observed following exposure of fibroblasts to either agent. Conclusion Detergent sclerosants are biologically active at sublytic concentrations. The observed morphological changes are consistent with cell activation, apoptosis, and oncosis. The cellular response is concentration dependent, cell-specific, and sclerosant specific.
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Ridhawati, Ridhawati, Riva Ambardina Pradita, Mulyati Mulyati i Robiatul Adawiyah. "Diagnosis Cepat Kandidemia Neonatus dengan Pemeriksaan Spesimen Darah Pulasan Giemsa". Jurnal Ilmu Kedokteran 13, nr 2 (1.09.2019): 16. http://dx.doi.org/10.26891/jik.v13i2.2019.16-20.
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Candidemia is one of the major problems in neonates with low birth weight (LBW). Neonatal candidemia has a high rateof morbidity and mortality. Early initiation of antifungal therapy could be decreasing the mortality, but this managementoften hampered due to late diagnosis. The gold standard for diagnosing candidemia is blood culture, but it takes a longtime about 5 days. A rapid alternative method is needed to decrease candidemia morbidity and mortality. The methodchosen in this study is Giemsa stain of blood smear which result could be read within one hour. The study wasconducted in the Department of Parasitology, Faculty of Medicine Universitas Indonesia with a total blood samples of170 from 2009-2012. Blood samples are devided in two, first is made thick blood smear than stain with giemsa, than readby microscope by 400×and 1000× magnification. The second is cultured on Sabauraud agar media and incubated inroom temperature for ten days. Thirty four patients (20%) were positive for Candida, 28 (82.4%) positive samples withboth giemsa staining and culture, while 6 (17,6%) positive culture samples but negative giemsa staining. The values ofthe sensitivity and specificity of the Giemsa stained blood smear examination were 82%, and 100%. This result showsthat Giemsa staining has a good diagnostic value for detecting neonatal candidemia.
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Hussin, Amer M. "Histological study of the constituents that related to the immune defensive mechanism in the vagina of ewes". Iraqi Journal of Veterinary Medicine 35, nr 1 (29.06.2011): 88–99. http://dx.doi.org/10.30539/iraqijvm.v35i1.608.
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In order to study the vaginal defensive mechanisms in ewes, vaginal smears and biopsies were collected from eight adult Awasi ewes. The biopsies samples were processed by the routine histological methods and stained by H & E and PAS stains, while the smears samples were stained with MB. Samples were examined under light microscope. The resent study revealed that the vaginal wall lacks many important constituents, among these were the vaginal glands, goblet cells, muscularis mucosa and lymphatic nodules. And as the vagina was the nearest organ to the external environment and as it receives the male copulatory organ, so it is more liable organ to be infected by external pathogens. On the contrary, the vagina has special compensatory histological mechanisms i: e, its epithelium was thrown into deep folds which serve to increase the surface area and in turn raise the epithelial efficiency. The study suggested that these folds were a remnants formed as a result of a failure during embryonic development of the glands as the gland formed by invagination of the epithelium. Moreover, the vaginal wall had a thick basement membrane. It appeared segmented due to accumulation of the defensive cells on some parts of it during their migration from the blood vessels to the epithelium. Besides, the vagina contained a great numbers of defensive cells, such as neutrophils, macrophages, lymphocytes, plasma cells and mast cells. In spite of the relation of dendritic cells with immune defense, this study couldn't recognize them by using only the general stains. On the other hand, the vaginal smears demonstrated that the vagina had many defensive cellular mechanisms among these, the process of keratinization of the vaginal epithelium; the process of sheet formation which lining the vaginal lumen; the presence of apparent junctional complexes which coalesce the cells of the sheet formation. These junctional complexes close completely the intercellular spaces leading to prevent any entrance of any foreign materials and pathogens to the underlying tissue. Some vaginal cells showed a pale foamy (vacuolated) appearance. Vacuolation was another defensive phenomena, it was indicative of increase cellular activity.
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Klezl, Petr, Eliska Pospisilova, Katarina Kolostova, Jindrich Sonsky, Ondrej Maly, Robert Grill, Ireneusz Pawlak i Vladimir Bobek. "Detection of Circulating Tumor Cells in Renal Cell Carcinoma: Disease Stage Correlation and Molecular Characterization". Journal of Clinical Medicine 9, nr 5 (7.05.2020): 1372. http://dx.doi.org/10.3390/jcm9051372.
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The presence of circulating tumor cells (CTCs) in patients with solid tumors is associated with poor prognosis. However, there are limited data concerning the detection of CTCs in renal cell cancer (RCC). The aim of this study is to evaluate the presence of CTCs in peripheral blood of patients with RCC undergoing surgery (n = 186). CTCs were tested before and after surgery as well as during the follow-up period afterwards. In total 495 CTC testing in duplicates were provided. To enrich CTCs, a size-based separation protocol and tube MetaCell® was used. CTCs presence was evaluated by single cell cytomorphology based on vital fluorescence microscopy. Additionally, to standardly applied fluorescence stains, CTCs viability was controlled by mitochondrial activity. CTCs were detected independently on the sampling order in up to 86.7% of the tested blood samples in patients undergoing RCC surgery. There is higher probability of CTC detection with growing tumor size, especially in clear cell renal cell cancer (ccRCC) cases. Similarly, the tumor size corresponds with metastasis presence and lymph node positivity and CTC detection. This paper describes for the first-time successful analysis of viable CTCs and their mitochondria as a part of the functional characterization of CTCs in RCC.
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Castelló, Ana, Francesc Francés i Fernando Verdú. "DNA Evidence Uncompromised by Active Oxygen". Scientific World JOURNAL 10 (2010): 387–92. http://dx.doi.org/10.1100/tsw.2010.47.
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Currently, forensic sciences can make use of the potential of instrumental analysis techniques to obtain information from the smallest, even invisible, samples. However, as laboratory techniques improve, so too should the procedures applied in the search for and initial testing of clues in order to be equally effective. This requires continuous revision so that those procedures may resolve the problems that samples present. As far as bloodstains are concerned, there are methods available that are recognized as being both highly sensitive and effective. Nevertheless, the marketing of new cleaning products, those that contain active oxygen, has raised doubts about the ability of those procedures to detect blood. It has been shown that stains washed with these detergents (and still visible) invalidated both the presumptive test (reduced phenolphthalein, luminol, and Bluestar®) and that applied for determining human hemoglobin. These findings have caused considerable concern both within the forensic and scientific community, and among the general public, so obliging us to seek solutions. In this work, the effect of these new cleaning products on DNA analyses is studied. The results, encouraging ones, show that these detergents, despite invalidating all other tests, do not hinder the extraction, or the subsequent analysis, of DNA.
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Haumann, Rianne, Fatma El-Khouly, Marjolein Breur, Sophie Veldhuijzen van Zanten, Gertjan Kaspers, Harry Hendrikse, Esther Hulleman, Dannis van Vuurden i Marianna Bugiani. "PATH-04. THE BLOOD-BRAIN BARRIER IN DIFFUSE MIDLINE GLIOMA AND ITS IMPLICATIONS FOR DRUG DELIVERY". Neuro-Oncology 22, Supplement_2 (listopad 2020): ii164. http://dx.doi.org/10.1093/neuonc/noaa215.686.
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Abstract INTRODUCTION Chemotherapy has been unsuccessful for pediatric diffuse midline glioma (DMG) most likely due to an intact blood-brain barrier (BBB). However, the BBB has not been characterized in DMG and therefore its implications for drug delivery are unknown. In this study we characterized the BBB in DMG patients and compared this to healthy controls. METHODS End-stage DMG pontine samples (n=5) were obtained from the VUmc diffuse intrinsic pontine glioma (DIPG) autopsy study and age-matched healthy pontine samples (n=22) were obtained from the NIH NeuroBioBank. Tissues were stained for BBB markers claudin-5, zonula occludens-1, laminin, and PDGFRβ. Claudin-5 stains were used to determine vascular density and diameter. RESULTS In DMG, expression of claudin-5 was reduced and dislocated to the abluminal side of endothelial cells. In addition, the expression of zonula occludens-1 was reduced. The basement membrane protein laminin expression was reduced at the glia limitans in both pre-existent vessels and neovascular proliferation. PDGFRβ expression was not observed in DMG but was present in healthy pons. Furthermore, the number of blood vessels in DMG was significantly (P< 0.01) reduced (13.9 ± 11.8/mm2) compared to healthy pons (26.3 ± 14.2/mm2). Markedly, the number of small blood vessels (< 10µm) was significantly lower (P< 0.01) while larger blood vessels (> 10µm) were not significantly different (P= 0.223). The mean vascular diameter was larger for DMG 9.3 ± 9.9µm compared to 7.7 ± 9.0µm for healthy pons (P= 0.016). CONCLUSION Both the BBB and the vasculature are altered at end-stage DMG. The reduced vascular density might have implications for several drug delivery methods such as focused ultrasound and convection enhanced delivery that are being explored for the treatment of DMG. The functional effects of the structurally altered BBB remain unknown and further research is needed to evaluate the BBB integrity at end-stage DMG
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Fedorov, Kateryna, Mohammad Barouqa, David Yin, Margarita Kushnir, Henny H. Billett i Morayma Reyes Gil. "Identifying Neutrophil Extracellular Traps (NETs) in Blood Samples Using Peripheral Smear Autoanalyzers". Life 13, nr 3 (23.02.2023): 623. http://dx.doi.org/10.3390/life13030623.
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Neutrophil Extracellular Traps (NETs) are large neutrophil-derived structures composed of decondensed chromatin, cytosolic, and granule proteins. NETs play an important role in fighting infection, inflammation, thrombosis, and tumor progression processes, yet their fast and reliable identification has been challenging. Smudge cells (SCs) are a subcategory of white cells identified by CellaVision®, a hematology autoanalyzer routinely used in clinical practice that uses digital imaging to generate “manual” differentials of peripheral blood smears. We hypothesize that a proportion of cells identified in the SC category by CellaVision® Hematology Autoanalyzers are actually NETs. We demonstrate that NET-like SCs are not present in normal blood samples, nor are they an artifact of smear preparation. NET-like SCs stain positive for neutrophil markers such as myeloperoxidase, leukocyte alkaline phosphatase, and neutrophil elastase. On flow cytometry, cells from samples with high percent NET-like SCs that are positive for surface DNA are also positive for CD45, myeloperoxidase and markers of neutrophil activation and CD66b. Samples with NET-like SCs have a strong side fluorescent (SFL) signal on the white count and nucleated red cells (WNR) scattergram, representing cells with high nucleic acid content. When compared to patients with low percent SCs, those with a high percentage of SCs have a significantly higher incidence of documented bacterial and viral infections. The current methodology of NET identification is time-consuming, complicated, and cumbersome. In this study, we present data supporting identification of NETs by CellaVision®, allowing for easy, fast, cost-effective, and high throughput identification of NETs that is available in real time and may serve as a positive marker for a bacterial or viral infections.
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Hassan, Siti Nazihahasma, Suharni Mohamad, Rosline Hassan, Selamah Ghazali i Wan Suriana Wan Ab Rahman. "A Comprehensive Comparison of DNA Extraction Using Fresh and Stored Bloods in Molecular Hematology Diagnostic Study". Bangladesh Journal of Medical Science 17, nr 3 (29.06.2018): 424–32. http://dx.doi.org/10.3329/bjms.v17i3.36998.
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Objective: Blood is the main source of DNA in molecular biology. It provides a high DNA quality and quantity. In this study, we compare the quality and quantity of DNA isolated from stored blood that has been kept at -40°C for one-year to that of fresh blood.Materials and Methods: Twelve fresh and stored blood samples were randomly selected for this study. Nucleo Spin® Blood L kit was used to isolate the DNA from the samples. The integrity and intensity of DNA were examined through 1.6% agarose gel precast with SYBR® safe DNA stain. The DNA samples were further examined through PCR amplification and Sanger sequencing.Results: There was no significant difference in quality and quantity of isolated DNA from fresh blood and stored blood samples. The high intensity of an intact DNA band as well as the success in PCR amplification and sequencing are indicators of high DNA quality.Conclusion: Proper storage of patients’left-over whole blood sample at -40°C offers an acceptable alternative for DNA resources in molecular study.Bangladesh Journal of Medical Science Vol.17(3) 2018 p.424-432
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Götz, Philipp, Sharon O. Azubuike-Osu, Anna Braumandl, Christoph Arnholdt, Matthias Kübler, Lisa Richter, Manuel Lasch, Lisa Bobrowski, Klaus T. Preissner i Elisabeth Deindl. "Cobra Venom Factor Boosts Arteriogenesis in Mice". International Journal of Molecular Sciences 23, nr 15 (30.07.2022): 8454. http://dx.doi.org/10.3390/ijms23158454.
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Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU+) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68+/MRC-1+ M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth.
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Ledesma, Carlo, i Ma Gina Sadang. "Asymptomatic Malaria Infection and Their Antibodies Against Malaria: A Study in Abra de Ilog, Occidental Mindoro, Philippines". American Journal of Clinical Pathology 152, Supplement_1 (11.09.2019): S116. http://dx.doi.org/10.1093/ajcp/aqz121.028.
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Abstract Human malaria, caused by four species of Plasmodium, namely P falciparum, P vivax, P malariae, and P ovale, remains a health problem of global concern, with one to two million deaths annually and risking about two billion people worldwide. Alternative ways of controlling the incidence of malaria through understanding the host’s immune response to monoinfection and the detection of the presence of asymptomatic malaria infection are the factors being addressed in this study. The determination of the possible existence of cross-antigenic stimulation is a matter of great significance for future research and development. The isolation of these antigenic structures may give the first step to the development of better vaccines that may protect the general population who are at risk of developing malaria. Prior to blood collection, a memorandum of agreement was signed between the researcher and the Iraya-Mangyan leaders of Abra de Ilog, Occidental Mindoro. A Certificate Precondition was issued by the National Commission of Indigenous Peoples, which was required by the Graduate School Ethics Review Committee. Determination of the presence of malaria parasite on blood samples of residents of two barangays in Abra de Ilog, Occidental Mindoro, was performed using two methods: microscopic examination of stained blood smears for the presence of malaria parasite and polymerase chain reaction. Blood smears were prepared and eventually stained using Giemsa and Dip Quick stains. The detection of 5 positive cases of malaria infection with ring/schizont stage among the 53 cases was a clear indication of positive asymptomatic cases. Nested PCR using Plasmodium spp.–specific primer as well as P falciparum–specific and P vivax–specific primers showed the absence of bands so that one of the recommendations in this study is the performance of real-time PRC using more sensitive primers. Levels of P falciparum and P vivax–specific immunoglobulin were measured using an enzyme-linked immunosorbent assay revealing a higher level of PF-specific IgG than PV-specific IgG. Whole blood samples were saved for future determinations such as real-time PCR, immunophenotypic analysis, and possible parasitic culture. Further similar studies may also be done by increasing the number of respondents as well as the areas of concern for a more extensive scope.