scholarly journals Precise RNA Quantification by Counting Individual RNA Molecules Using High-Sensitivity Capillary Flow Cytometry

2022 ◽  
Author(s):  
Hee-Bong Yoo ◽  
Sang-Ryoul Park ◽  
Kee-Suk Hong ◽  
Inchul Yang
Keyword(s):  
Flow Cytometry ◽  
Capillary Flow ◽  
High Sensitivity ◽  
Rna Molecules ◽  
Rna Quantification

scholarly journals Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 964
Author(s):  
Sarka Benesova ◽  
Mikael Kubista ◽  
Lukas Valihrach
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
High Sensitivity ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Liquid Biopsies ◽  
Comprehensive Overview ◽  
Rna Molecules ◽  
Novel Mirna ◽  
The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

scholarly journals Evaluation of an Alternative Staining Method Using SYTO 13 to Determine Reticulated Platelets

2019 ◽  
Vol 119 (05) ◽  
pp. 779-785 ◽  
Author(s):  
Laura Hille ◽  
Marco Cederqvist ◽  
Julia Hromek ◽  
Christian Stratz ◽  
Dietmar Trenk ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Reactivity ◽  
Dependent Manner ◽  
Reticulated Platelets ◽  
Automated Assay ◽  
Rna Quantification ◽  
Time Period ◽  
Immature Platelet Fraction ◽  
Syto 13

AbstractReticulated platelets reflect the rate of platelet turnover and represent the youngest circulating platelets in peripheral blood. Reticulated platelets contain residual ribonucleic acid (RNA) from megakaryocytes which is lost in a time-dependent manner and can be transcribed into proteins even in the absence of a nucleus. An increased proportion of reticulated platelets is associated with higher platelet reactivity, cardiovascular events and mortality. At present, a fully automated assay system (SYSMEX haematology analyser) is available for analysis. This method, however, is not suitable for extended laboratory investigations like subsequent cell sorting. Flow cytometry analysis after staining with thiazole orange (TO) is frequently used in such settings despite several limitations. Here, we describe a new assay for determination of reticulated platelets by flow cytometry using the nucleic acid staining dye SYTO 13 and compare it with SYSMEX and TO staining as current standards. A significant correlation between immature platelet fraction (IPF) determined by SYSMEX XE-2100 analyser and results obtained with the SYTO 13-based assay was observed (r = 0.668, p < 0.001) which was stable during a reasonable time period. In contrast, the correlation between TO staining and IPF was weaker (r = 0.478, p = 0.029) and lost after 90 minutes of staining. SYTO 13 staining of platelets enabled sorting of RNAlow and RNArich platelets which was confirmed by RNA quantification of sorted platelets. Except for fixation of platelets, sorting of these platelet sub-populations was stable under various experimental settings. In summary, determination of reticulated platelets with the new SYTO 13 assay offers distinct technical advantages enabling further laboratory processing.

scholarly journals Flow Cytometry Can Diagnose Classical Hodgkin Lymphoma in Lymph Nodes With High Sensitivity and Specificity

2009 ◽  
Vol 131 (3) ◽  
pp. 322-332 ◽  
Author(s):  
Jonathan R. Fromm ◽  
Anju Thomas ◽  
Brent L. Wood
Keyword(s):  
Flow Cytometry ◽  
Lymph Nodes ◽  
Hodgkin Lymphoma ◽  
Sensitivity And Specificity ◽  
High Sensitivity ◽  
Classical Hodgkin Lymphoma

Evaluation of multiple myeloma measurable residual disease by high sensitivity flow cytometry: An international harmonized approach for data analysis

2022 ◽  
Author(s):  
Kah Teong Soh ◽  
Neil Came ◽  
Gregory E. Otteson ◽  
Dragan Jevermovic ◽  
Min Shi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Data Analysis ◽  
Residual Disease ◽  
High Sensitivity ◽  
Measurable Residual Disease

scholarly journals Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

2016 ◽  
Vol 14 (3) ◽  
pp. 366-373 ◽  
Author(s):  
Rodolfo Patussi Correia ◽  
Laiz Cameirão Bento ◽  
Ana Carolina Apelle Bortolucci ◽  
Anderson Marega Alexandre ◽  
Andressa da Costa Vaz ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Retrospective Study ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Laboratory Data ◽  
High Sensitivity ◽  
Laboratory Diagnosis ◽  
Clone Detection ◽  
Technical Advances ◽  
Sensitive Determination

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones.

Characterization of Microvesicles in Septic Shock Using High-Sensitivity Flow Cytometry

Shock ◽  
2016 ◽  
Vol 46 (4) ◽  
pp. 373-381 ◽  
Author(s):  
Georg Franz Lehner ◽  
Ulrich Harler ◽  
Viktoria Maria Haller ◽  
Clemens Feistritzer ◽  
Julia Hasslacher ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Septic Shock ◽  
High Sensitivity

Screening for Leptomeningeal Disease by High-Sensitivity Flow Cytometry in High Risk Patients with Aggressive Non-Hodgkin’s Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4397-4397
Author(s):  
Joy Mangel ◽  
Jazmin Marlinga ◽  
Mike Keeney ◽  
Jan Popma ◽  
Anargyros Xenocostas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Risk ◽  
High Sensitivity ◽  
B Cell Lymphoma ◽  
Leptomeningeal Disease ◽  
Cns Involvement ◽  
Lymphoma Cells ◽  
High Risk Patients ◽  
Csf Analysis ◽  
Risk Patients

Abstract Background: Central nervous system (CNS) involvement by non-Hodgkin’s lymphoma (NHL) portends a very poor prognosis. There is no consensus in the literature on the “high- risk” features that predict for leptomeningeal disease, and no standardized clinical guidelines exist regarding CNS surveillance, prophylaxis or treatment for patients at increased risk. 2–4 colour flow cytometry (FCM) has been reported to be more sensitive than standard cytology in detecting occult leptomeningeal disease (Blood 2005,105:496). The current study evaluates the utility of a high-sensitivity (5-colour) flow cytometry technique for detecting occult lymphoma cells in the cerebrospinal fluid (CSF) of high-risk patients with NHL. Method: Patients with a new diagnosis of histologically aggressive B or T cell NHL were included in this study if they displayed one or more “high-risk” features for CNS involvement. Patients suspected of CNS relapse of NHL were also eligible for participation. Patients underwent routine staging investigations, with the addition of a diagnostic lumbar puncture (LP) during initial assessment. CSF was tested by standard cytology, cell count and biochemistry, and an additional 5 ml was obtained for analysis by high-sensitivity FCM on a Beckman Coulter FC500. The antibody panel (5 antibodies per tube) was customized according to the phenotype of the lymphoma. The key markers for B cell lymphoma were CD19/kappa/lambda with CD5 or CD10. CD45 was used to identify all white blood cells in the sample. Results: Seventeen patients (8M/9F) with a median age of 59 (range 36–85) have been tested. Patients displayed anywhere from 2–6 “high-risk” features for CNS involvement. These included: HIV positivity (2), primary mediastinal B-cell lymphoma (4), bone marrow (5), multifocal bone (2), paraspinal (1), nasopharyngeal (2) or orbital (1) involvement, elevated serum LDH (12), multiple extranodal sites of disease (5), poor performance status (2), high IPI (3), B-symptoms (9), stage IV disease (11), and otherwise unexplained neurological symptoms (3). 14 patients underwent CSF analysis at time of initial diagnosis, one of whom had cranial nerve palsies secondary to a nasopharyngeal mass extending to the skull base. The other 3 were tested at relapse, transformation, and suspected CNS relapse ultimately diagnosed as a stroke. Despite the presence of these features, CSF analysis was negative for lymphoma cells by both cytology and FCM in all but one of the patients tested. However this patient had very high numbers of circulating lymphoma cells in the peripheral blood (PB), and the positive result was felt to be due to PB contamination of the CSF during a “bloody tap.” One patient with vague neurological symptoms had a negative LP at diagnosis, and later developed frank CNS involvement by lymphoma, but was too unwell to undergo a repeat LP. Conclusions: Given the limited number of patients enrolled thus far and the low prevalence of patients with NHL and CNS involvement (2/17), it is difficult to fully assess the utility of high-sensitivity FCM in the diagnosis of occult leptomeningeal disease. It is of interest that CSF analysis was negative even in the patient with cranial nerve palsies and in the patient who later developed multiple CNS lesions secondary to lymphoma, suggesting that this technique may have limited sensitivity in diagnosing leptomeningeal disease. The systematic screening of high-risk patients cannot yet be recommended as standard clinical practice.

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