A new application to one-step nucleic acid amplification-discarded sample in breast cancer: Preliminary results.
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ABSTRACT:
SUBMITTER: Ribeiro C
PROVIDER: S-EPMC8408673 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The presence of peritoneal free cancer cells (FCC) in gastric cancer (GC) patients is a poor prognostic factor. D2 gastrectomy may induce exfoliated FCC spread from the primary tumour or involved lymph nodes (LN). Conventional cytology for FCC detection has several limitations, whereas prophylactic use of extensive intraoperative peritoneal lavage (IPL) does not improve survival. A prospective single-arm observational study was conducted to verify whether D2 gastrectomy causes an intraoperative increase of FCC in peritoneal fluid. Twenty-seven GC patients underwent D2 gastrectomy, followed by objective quantitative measurements of CK19 mRNA level reflecting FCC with One-Step Nucleic Acid Amplification (OSNA) assay. The IPL with 3000 mL of saline was performed twice: (1) after gastrectomy with D2 lymphadenectomy and (2) after alimentary tract reconstruction. The IPL samples were analysed by initial cytology and four (1-4) consecutive OSNA assays. Initial OSNA measurement (1) revealed positive results (≥24.6 cCP/μL) in 7 (29.6%) patients. Subsequent OSNA measurements showed a significant decrease in the FCC level after D2 gastrectomy (1 vs. 2; p = 0.0012). The first IPL induced a non-significant increase in the FCCs (2 vs. 3, p = 0.3300), but the second IPL reversed it to normal levels (3 vs. 4, p = 0.0.0574). The OSNA assay indicates a temporal intraoperative increase in the peritoneal FCC in advanced GC patients undergoing D2 gastrectomy. Two consecutive IPLs are necessary to reverse the increase of CK19 mRNA level in peritoneal washings.
| S-EPMC8621409 | biostudies-literature
Project description:ObjectiveTo evaluate the accuracy of the one-step nucleic acid amplification (OSNA) assay for the diagnosis of lymph node (LN) metastasis in uterine cancer.MethodsA total of 116 LNs from 30 patients with cervical and endometrial cancer, enrolled in this prospective study, were used. Excised LNs were cut into 4 to 6 blocks at 2 mm intervals, and nonadjacent blocks were alternately subjected to either histological examination or the OSNA assay.ResultsThe concordance rate between histological examination and the OSNA assay in cervical cancer and in endometrial cancer was 95.9% and 95.2%, respectively. The sensitivity, specificity, and negative predictive value of the OSNA assay were 80%, 97.7%, and 97.7% in cervical cancer, and 85.7%, 93.3%, and 98.2% in endometrial cancer, respectively. In cervical cancer, discordant results were observed in 2 out of 49 LNs (4.1%); 1 was OSNA assay-positive and histological examination-negative, and 1 was OSNA assay-negative and histological examination-positive. In endometrial cancer, discordant results were observed in 5 out of 67 LNs (7.5%); 4 were OSNA assay-positive and histological examination-negative, and 1 was OSNA assay-negative and histological examination-positive.ConclusionThe OSNA assay showed high concordance rate with histological examination, sensitivity, and specificity in uterine cancer, suggesting that it could enhance the accuracy of conventional pathological examination for the detection of LN metastasis by reducing false negative rate.
| S-EPMC8899876 | biostudies-literature
Project description:One-step nucleic acid amplification (OSNA) is an intraoperative technique with a high sensitivity and specificity for sentinel node assessment. The aim of this study was to assess the impact of OSNA on micrometastases detection rates and use of adjuvant chemotherapy. A retrospective review of patients with sentinel node micrometastases over a five-year period was carried out and a comparison of micrometastases detection using OSNA and H&E techniques was made. Out of 1285 patients who underwent sentinel node (SLN) biopsy, 76 patients had micrometastases. Using H&E staining, 36 patients were detected with SLN micrometastases (9/year) in contrast to 40 patients in the OSNA year (40/year) (p < 0.0001), demonstrating a fourfold increase with the use of OSNA. In the OSNA group, there was also a proportional increase in Grade III, triple-negative, ER-negative, and HER-2-positive tumours being diagnosed with micrometastases. Also on interactive PREDICT tool, the number of patients with a predicted 10-year survival benefit of more than 3% with adjuvant chemotherapy increased from 52 to 70 percent. OSNA has resulted in an increased detection rate of micrometastases especially in patients with aggressive tumour biology. This increased the number of patients who had a predicted survival benefit from adjuvant chemotherapy.
| S-EPMC5485356 | biostudies-other
Project description:The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPR-mediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12b-mediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPR-top assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 °C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.
| S-EPMC8197615 | biostudies-literature
Project description:One-step nucleic acid amplification (OSNA) is an established method for intraoperative diagnosis of breast cancer metastasis in sentinel lymph nodes, based on quantification of CK19 mRNA, specific to breast epithelial cells. Inhibitors interfere with the PCR amplification process of PCR. Thus, OSNA, based on gene amplification without RNA purification, might be impacted by numerous factors persisting in a sample, and thereby potentially acting as PCR inhibitors. However, neither the characteristics of breast cancers showing inhibitory effects during OSNA, nor any of the possible inhibitors, have as yet been identified. Inhibitory effects detected during OSNA in 72 metastatic lymph nodes and the patients' clinicopathological features were examined. Left-over OSNA samples were analyzed with mass spectrometry to identify proteins possibly acting as inhibitors. Most tumors showed inhibitory effects, though to varying degrees. Large tumor, young age and high tumor-infiltrating lymphocyte counts were related to stronger inhibitory effects. Proteome analysis revealed elevations in RPB9 protein and EIF2 signaling upregulation in samples showing strong inhibitory effects. Tumors showing strong inhibitory effects had clinically relevant characteristics, including large size and extensive tumor-infiltrating lymphocyte involvement. Identifying inhibitors in OSNA might provide new insights into breast cancer biology as well as advancing the current technology.
| S-EPMC5954024 | biostudies-literature
Project description:Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.
| S-EPMC8588466 | biostudies-literature
Project description:Sentinel lymph node (SLN) examination is a standard in breast cancer patients, with several methods employed along its 20?years history, the last one represented by one-step nucleic acid amplification (OSNA). The latter is a intra-operative molecular assay searching for CK19 mRNA as a surrogate of metastatic cells. Our 3?years experience with OSNA (1122 patients) showed results overlapping those recorded in the same institution with a morphological evaluation (930 patients) of SLN. In detail, the data of OSNA were almost identical to those observed with standard post-operative procedure in terms of patients with positive SLN (30%) and micrometastatic/macrometastatic involvement of SLN (respectively, 38-45 and 62-55%). By contrast, when OSNA was compared to the standard intraoperatory procedure, it was superior in terms of accuracy, prompting the use of this molecular assay as a very valid, and reproducible for intra-operative evaluation of SLN. Further possibilities prompting the use of OSNA range from adhesion to quality control programs, saving of medical time, ability to predict, during surgery, additional nodal metastasis, and molecular bio-banking.
| S-EPMC4469115 | biostudies-literature
Project description:BackgroundThe World Health Organization (WHO) recommends parasite-based diagnosis of malaria. In recent years, there has been surge in the use of various kinds of nucleic-acid amplification based tests (NAATs) for detection and identification of Plasmodium spp. to support clinical care in high-resource settings and clinical and epidemiological research worldwide. However, these tests are not without challenges, including lack (or limited use) of standards and lack of reproducibility, due in part to variation in protocols amongst laboratories. Therefore, there is a need for rigorous quality control, including a robust external quality assessment (EQA) scheme targeted towards malaria NAATs. To this effect, the WHO Global Malaria Programme worked with the UK National External Quality Assessment Scheme (UK NEQAS) Parasitology and with technical experts to launch a global NAAT EQA scheme in January 2017.MethodsPanels of NAAT EQA specimens containing five major species of human-infecting Plasmodium at various parasite concentrations and negative samples were created in lyophilized blood (LB) and dried blood spot (DBS) formats. Two distributions per year were sent, containing five LB and five DBS specimens. Samples were tested and validated by six expert referee laboratories prior to distribution. Between 37 and 45 laboratories participated in each distribution and submitted results using the online submission portal of UK NEQAS. Participants were scored based on their laboratory’s stated capacity to identify Plasmodium species, and individual laboratory reports were sent which included performance comparison with anonymized peers.ResultsAnalysis of the first three distributions revealed that the factors that most significantly affected performance were sample format (DBS vs LB), species and parasite density, while laboratory location and the reported methodology used (type of nucleic acid extraction, amplification, or DNA vs RNA target) did not significantly affect performance. Referee laboratories performed better than non-referee laboratories.ConclusionsGlobally, malaria NAAT assays now inform a range of clinical, epidemiological and research investigations. EQA schemes offer a way for laboratories to assess and improve their performance, which is critical to safeguarding the reliability of data and diagnoses especially in situations where various NAAT methodologies and protocols are in use.
| S-EPMC7106789 | biostudies-literature
Project description:Peritoneal dissemination is a common form of gastric cancer (GC) recurrence, despite surgery with curative intent. This study aimed to evaluate the prognostic value of intraperitoneal lavage One-Step Nucleic Acid Amplification (OSNA) assay in advanced GC patients. OSNA assay targeting CK-19 mRNA was applied to detect free cancer cells (FCC) in intraperitoneal lavage samples obtained during gastrectomy. A total of 82 GC patients were enrolled to investigate the correlation between OSNA assay and patient's prognosis. Of the 82 patients, OSNA assay was positive in 25 (30.5%) patients. The median OS in OSNA positive patients was significantly lower than in OSNA negative patients (19 vs 45 months). Positive OSNA assay result was a significant unfavourable prognostic factor in both, univariable (HR 3.45, 95% CI 0.95-12.48; p = 0.0030) and multivariable analysis (HR 3.10, 95% CI 1.22-8.54; p = 0.0298). Positive OSNA assay in intraperitoneal lavage is a valuable indicator of poor survival in advanced GC patients after multimodal treatment. After further confirmation on larger sample size, OSNA assay of peritoneal washings could be considered an adjunct tool to conventional cytology, the current gold standard, to provide precise intraoperative staging and additional prognostic information.
| S-EPMC9304381 | biostudies-literature
Project description:As nucleic acid testing is playing a vital role in increasingly many research fields, the need for rapid on-site testing methods is also increasing. The test procedure often consists of three steps: Sample preparation, amplification, and detection. This review covers recent advances in on-chip methods for each of these three steps and explains the principles underlying related methods. The sample preparation process is further divided into cell lysis and nucleic acid purification, and methods for the integration of these two steps on a single chip are discussed. Under amplification, on-chip studies based on PCR and isothermal amplification are covered. Three isothermal amplification methods reported to have good resistance to PCR inhibitors are selected for discussion due to their potential for use in direct amplification. Chip designs and novel strategies employed to achieve rapid extraction/amplification with satisfactory efficiency are discussed. Four detection methods providing rapid responses (fluorescent, optical, and electrochemical detection methods, plus lateral flow assay) are evaluated for their potential in rapid on-site detection. In the final section, we discuss strategies to improve the speed of the entire procedure and to integrate all three steps onto a single chip; we also comment on recent advances, and on obstacles to reducing the cost of chip manufacture and achieving mass production. We conclude that future trends will focus on effective nucleic acid extraction via combined methods and direct amplification via isothermal methods.
| S-EPMC9930993 | biostudies-literature