- MeSH
- Biopsy methods MeSH
- DNA, Neoplasm analysis MeSH
- Carcinoma drug therapy genetics radiotherapy MeSH
- Humans MeSH
- Breast Neoplasms drug therapy genetics radiotherapy MeSH
- Flow Cytometry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Comparative Study MeSH
Genome analysis in many plant species is hampered by large genome size and by sequence redundancy due to the presence of repetitive DNA and polyploidy. One solution is to reduce the sample complexity by dissecting the genomes to single chromosomes. This can be realized by flow cytometric sorting, which enables purification of chromosomes in large numbers. Coupling the chromosome sorting technology with next generation sequencing provides a targeted and cost effective way to tackle complex genomes. The methods outlined in this article describe a procedure for preparation of chromosomal DNA suitable for next-generation sequencing.
- MeSH
- Chromosomes, Plant ultrastructure MeSH
- Genome Size MeSH
- Microscopy, Fluorescence MeSH
- Genome, Plant * MeSH
- In Situ Hybridization, Fluorescence MeSH
- Hordeum cytology genetics MeSH
- Germination genetics MeSH
- Metaphase genetics MeSH
- Polyploidy MeSH
- Flow Cytometry methods MeSH
- Triticum cytology genetics MeSH
- Sequence Analysis, DNA MeSH
- Seeds genetics MeSH
- Chromosomes, Artificial, Bacterial MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Secale cytology genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish "simple" from bilineal/"complex" mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter-laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society.
- MeSH
- Precursor Cell Lymphoblastic Leukemia-Lymphoma diagnosis MeSH
- Acute Disease MeSH
- Child MeSH
- Phenotype MeSH
- Immunophenotyping standards MeSH
- Consensus MeSH
- Humans MeSH
- Flow Cytometry standards MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Publication type
- Journal Article MeSH
Smíšená lymfocytární reakce (MLC) T-lymfocytů s dendritickými buňkami je jedním ze základních nástrojů studia imunologických mechanismů. Tato práce přináší popis nové metodiky hodnocení alogenní a autologní MLC pomocí průtokové cytometrie a diferenciálního gatingu. Metoda je založena na časově omezené akvizici událostí a na jejich rozdělení podle hodnot forward a side scatteru. Gatována je jednak populace všech živých buněk (R1), dále populace živých neproliferujících buněk (R2) a populace mrtvých/apoptotických buněk (R4). Pomocí fluorescenčních mikrosfér (R3) je navíc možno vypočítat absolutní množství buněk ve stanoveném gatu. Pomocí 7-AAD exkluze bylo ověřeno, že zhruba 90 % 7-AAD pozitivních buněk se promítá do gatu R4, navíc poměr R1: R2 výtečně koreluje s procentem buněk pozitivních na aktivační/proliferační marker CD71. Příklady dalších výsledků dosažených touto metodikou jsou uvedeny v textu. Vzhledem k tomu, že metoda diferenciálního gatingu ve své základní podobě nevyžaduje užití žádné fluorescenční protilátky, umožňuje rychlý a levný skríning velkého množství vzorků bez použití radioaktivně značených nukleotidů.
Mixed lymphocyte reaction (MLC) of T-lymphocytes with dendritic cells is one of the basic tools for studying of immune reaction mechanisms. This work describes a new method of evaluation of allogeneic and autologous MLC by flow cytometry and differential gating. This method is based on fixed time acquisition of events and their sorting according to their forward and side scatter properties. Differential gating distinguishes populations of all living cells (R1), living nonproliferating cells (R2) and dead/apoptotic cells (R4). Addition of fluorescence microspheres (R3) enables the calculation of absolute number of cells in a given sample volume. Using the method of 7-AAD exclusion, we have shown that approximately 90% of 7-AAD positive cells project into the R4 gate. Moreover, there was an excellent correlation between the R1: R2 ratio and the percentage of cells positive for activation/proliferation marker CD71. The method of differential gating in its basic version does not require use of any fluorescent antibody and therefore is suitable for rapid screening of large number of samples at reasonable cost and without the use of radiolabeled nucleotides.
A new computational framework for FLow cytometric Analysis of Rare Events (FLARE) has been developed specifically for fast and automatic identification of rare cell populations in very large samples generated by platforms like multi-parametric flow cytometry. Using a hierarchical Bayesian model and information-sharing via parallel computation, FLARE rapidly explores the high-dimensional marker-space to detect highly rare populations that are consistent across multiple samples. Further it can focus within specified regions of interest in marker-space to detect subpopulations with desired precision.
- MeSH
- Automation, Laboratory methods MeSH
- Probability MeSH
- Flow Cytometry methods MeSH
- Models, Theoretical * MeSH
- Publication type
- Journal Article MeSH
Prognóza pacientů s chronickou lymfocytární leukemií (CLL) může být upřesněna některými biologickými rizikovými faktory, jejichž stanovení ale musí být před zavedením do klinické praxe dostatečně standardizováno. V této studii se u 73 pacientů stanovoval mutační stav variabilních segmentů genů pro těžký řetězec imunoglobulinu (IgVH), mutace genu p53 a pomocí flow-cytometrie (FCM) exprese CD38 a tyrozinkinázy ZAP-70. Nemutovaný stav IgVH mělo 58 % nemocných, mutaci p53 19 %, pozitivní expresi CD38 26 % a pozitivní expresi ZAP-70 dle FCM 62 %. Pacienti s nemutovaným IgVH, pozitivní expresí ZAP-70 dle FCM a mutací p53, měli statisticky významně kratší čas od diagnózy do zahájení terapie. Celková konkordance mezi expresí ZAP-70 a mutačním stavem IgVH byla 85 %. Validace FCM analýzy exprese ZAP-70 byla provedena srovnáním s imunohistochemickou detekcí ZAP-70 na histologických preparátech a kvantitativní Real-Time PCR na úrovni ZAP-70 mRNA, kdy celková shoda byla 86 %. Exprese ZAP-70 se ukázala být stabilní v průběhu onemocnění; při mediánu 12 měsíců mezi odběrem prvního a posledního vzorku byla zaznamenána kvalitativní změna exprese ZAP-70 pouze u jednoho pacienta, kdy se stala pozitivní při progresi onemocnění.
Individual prognosis in patients with chronic lymphocytic leukemia (CLL) may be specified by biological risk factors, detection of which must however be sufficiently standardized prior to their introduction into clinical practice. In the presented study of 73 patients we have assessed immunoglobulin variable heavy-chain gene (IgVH) status, presence of mutation in p53 gene and with the use of flow cytometry (FCM) expression of CD38 and ZAP-70 tyrosine kinase. Unmutated IgVH status was present in 58 % patients, p53 mutation in 19 %, positive expression of CD38 in 26 % and positive expression of ZAP-70 using FCM in 62 %. Patients with unmutated IgVH, positive expression of ZAP-70 assessed by FCM and p53 mutation had significantly shorter time from diagnosis to initiation of therapy. Overall concordance between ZAP-70 expression and IgVH mutational status was 85 %. Validation of the FCM ZAP-70 detection was performed by comparison to the immunohistochemical analysis on histological sections and quantitative real-time PCR on the mRNA level; overall concordance was 86 %. Expression of ZAP-70 showed to be stable over time; with median time of 12 months from the acquisition of the first sample to the last, qualitative change of ZAP-70 expression was noted in only one patient, who became positive upon disease progression.
- MeSH
- ADP-ribosyl Cyclase 1 genetics MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell genetics MeSH
- Financing, Organized MeSH
- Genes, p53 genetics MeSH
- Genes, Immunoglobulin Heavy Chain MeSH
- Immunohistochemistry utilization MeSH
- Humans MeSH
- Mutation MeSH
- Prognosis MeSH
- Flow Cytometry MeSH
- Risk Factors MeSH
- Protein-Tyrosine Kinases genetics MeSH
- Check Tag
- Humans MeSH
Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.
Programmed cell death (PCD) is a crucial process required for the normal development and physiology of metazoans. The three major mechanisms that induce PCD are called type I (apoptosis), type II (autophagic cell death), and type III (necrotic cell death). Dysfunctional PCD leads to diseases such as cancer and neurodegeneration. Although apoptosis is the most common form of PCD, recent studies have provided evidence that there are other forms of cell death. The high majority of classical apoptotic hallmarks can be rapidly examined by flow cytometry. Cytometry thus became a technology of choice in diverse studies of cellular demise. A large variety of cytometric methods designed to identify apoptotic cells and probe mechanisms associated with this mode of cell demise have been developed during the past two decades.
