Assuntos
Anemia Falciforme/patologia , Processamento de Imagem Assistida por Computador/métodos , Leucemia/patologia , Microscopia/métodos , Anemia Falciforme/complicações , Anemia Falciforme/diagnóstico , Eritrócitos/patologia , Humanos , Leucemia/complicações , Leucemia/diagnóstico , Leucócitos/patologia , SoftwareRESUMO
An evidence-based triage plan for cellular therapy distribution is critical in the face of emerging constraints on healthcare resources. We evaluated the impact of treatment delays related to COVID-19 on patients scheduled to undergo hematopoietic cell transplantation (HCT) or chimeric antigen receptor T-cell (CAR-T) therapy at our center. Data were collected in real time between March 19 and May 11, 2020, for patients who were delayed to cellular therapy. We evaluated the proportion of delayed patients who ultimately received cellular therapy, reasons for not proceeding to cellular therapy, and changes in disease and health status during delay. A total of 85 patients were delayed, including 42 patients planned for autologous HCT, 36 patients planned for allogeneic HCT, and 7 patients planned for CAR-T therapy. Fifty-six of these patients (66%) since received planned therapy. Five patients died during the delay. The most common reason for not proceeding to autologous HCT was good disease control in patients with plasma cell dyscrasias (75%). The most common reason for not proceeding to allogeneic HCT was progression of disease (42%). All patients with acute leukemia who progressed had measurable residual disease (MRD) at the time of delay, whereas no patient without MRD at the time of delay progressed. Six patients (86%) ultimately received CAR-T therapy, including 3 patients who progressed during the delay. For patients with high-risk disease such as acute leukemia, and particularly those with MRD at the time of planned HCT, treatment delay can result in devastating outcomes and should be avoided if at all possible.
Assuntos
COVID-19 , Transplante de Células-Tronco Hematopoéticas , Imunoterapia Adotiva , Pandemias , SARS-CoV-2 , Tempo para o Tratamento , Adulto , Idoso , Aloenxertos , Amiloidose/terapia , Anemia Aplástica/terapia , COVID-19/complicações , COVID-19/epidemiologia , COVID-19/transmissão , Defesa Civil , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/prevenção & controle , Progressão da Doença , Prática Clínica Baseada em Evidências/organização & administração , Feminino , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Humanos , Controle de Infecções/métodos , Transmissão de Doença Infecciosa do Profissional para o Paciente , Leucemia/mortalidade , Leucemia/patologia , Leucemia/terapia , Masculino , Pessoa de Meia-Idade , Doenças Mieloproliferativas-Mielodisplásicas/mortalidade , Doenças Mieloproliferativas-Mielodisplásicas/terapia , Neoplasia Residual , Neoplasias/mortalidade , Neoplasias/terapia , Cidade de Nova Iorque/epidemiologia , Alocação de Recursos , Tempo para o Tratamento/estatística & dados numéricos , Transplante Autólogo , Triagem/organização & administração , Adulto JovemRESUMO
The oxysterol-binding protein-related proteins (ORPs) have emerged as orchestrators of phosphatidylinositol-4,5-bisphosphate (PIP2) and cholesterol trafficking to the plasma membrane (PM). In this scenario, recent studies raised the prospect of ORPs cooperative behavior in sustaining leukemia stem cells (LSCs) survival by remotely enhancing ER-mitochondria Ca2+ communication. At the apex of the signaling cascade, the aberrantly upregulated LSC-ORP4L fosters PM-PIP2 extraction & cleavage, endoplasmic reticulum (ER)-Ca2+ release and mitochondrial energetics. The theoretical ember of draining fuel from the chemoresistant LSCs by restraining endoplasmic reticulum (ER)-mitochondria Ca2+ fluxes in a lipid-contingent fashion ensues. In light of relevant literature, this review briefly and critically discusses some key molecular ins & outs underlying such therapeutic opportunity in acute myeloid leukemia (AML).
Assuntos
Cálcio/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Colesterol/metabolismo , Humanos , Transdução de SinaisRESUMO
The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.
Assuntos
Medula Óssea/metabolismo , Medula Óssea/patologia , Leucemia/metabolismo , Leucemia/patologia , Metaboloma/fisiologia , Biópsia/métodos , Humanos , Leucemia/genéticaRESUMO
In a previous study, a formulation of methotrexate (MTX) incorporated in the lipid bilayer of 100-nm liposomes in the form of diglyceride ester (MTX-DG, lipophilic prodrug) was developed. In this study, first, the interactions of MTX-DG liposomes with various human and mouse tumor cell lines were studied using fluorescence techniques. The liposomes composed of egg phosphatidylcholine (PC)/yeast phosphatidylinositol/MTX-DG, 8:1:1 by mol, were labeled with fluorescent analogs of PC and MTX-DG. Carcinoma cells accumulated 5 times more MTX-DG liposomes than the empty liposomes. Studies on inhibitors of liposome uptake and processing by cells demonstrated that the formulation used multiple mechanisms to deliver the prodrug inside the cell. According to the data from the present study, undamaged liposomes fuse with the cell membrane only 1.5-2 hours after binding to the cell surface, and then, the components of liposomal bilayer enter the cell separately. The study on the time course of plasma concentration in mice showed that the area under the curve of MTX generated upon intravenous injection of MTX-DG liposomes exceeded that of intact MTX 2.5-fold. These data suggested the advantage of using liposomal formulation to treat systemic manifestation of hematological malignancies. Indeed, the administration of MTX-DG liposomes to recipient mice bearing T-cell leukemic lymphoma using a dose-sparing regimen resulted in lower toxicity and retarded lymphoma growth rate as compared with MTX.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Lipossomos/administração & dosagem , Linfoma de Células T/tratamento farmacológico , Metotrexato/administração & dosagem , Pró-Fármacos/administração & dosagem , Animais , Antimetabólitos Antineoplásicos/química , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Injeções Intravenosas , Leucemia/tratamento farmacológico , Leucemia/patologia , Bicamadas Lipídicas/química , Lipossomos/química , Lipossomos/metabolismo , Linfoma de Células T/patologia , Metotrexato/química , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Pró-Fármacos/químicaRESUMO
AIMS: Elimination of non-value added testing without compromising high-quality clinical care is an important mandate for laboratories in a value-based reimbursement system. The goal of this study was to determine the optimal combination of flow cytometric markers for a screening approach that balances efficiency and accuracy. METHODS: An audit over 9â months of flow cytometric testing was performed, including rereview of all dot plots from positive cases. RESULTS: Of the 807 cases in which leukaemia/lymphoma testing was performed, 23 were non-diagnostic and 189 represented bronchoalveolar lavage specimens. Of the remaining 595 cases, 137 (23%) were positive for an abnormal haematolymphoid population. Review of the positive cases identified minimum requirements for a screening tube as well as analysis strategies to overcome the diagnostic pitfalls noted. It is estimated that 38% fewer antibodies would be used in a screening approach, representing an opportunity for significant cost savings. CONCLUSIONS: We provide a framework for developing an evidence-based screening combination for cost-effective characterisation of haematolymphoid malignancies, promoting adoption of 'just-in-time' testing systems that tailor the evaluation to the diagnostic need.
Assuntos
Biomarcadores Tumorais/análise , Medicina Baseada em Evidências , Citometria de Fluxo , Imunofenotipagem/métodos , Leucemia/metabolismo , Linfoma/metabolismo , Redução de Custos , Análise Custo-Benefício , Diagnóstico Diferencial , Erros de Diagnóstico , Citometria de Fluxo/economia , Citometria de Fluxo/normas , Custos de Cuidados de Saúde , Humanos , Imunofenotipagem/economia , Imunofenotipagem/normas , Leucemia/patologia , Linfoma/patologia , Auditoria Médica , Valor Preditivo dos Testes , Indicadores de Qualidade em Assistência à Saúde , Reprodutibilidade dos Testes , Fluxo de TrabalhoRESUMO
Genotoxicity of Ceratonia siliqua extracts, was investigated by assessing their capacity to induce nucleus DNA degradation of murine leukaemia cells L1210, using the "Comet assay". The ability of total oligomer flavonoids (TOF) and aqueous extracts to protect cell DNA against oxidative stress induced by H2O2, was performed by pre- co or post-treatment of cells with the before mentioned extracts for different periods preceding exposure to H2O2 stress. No significant genotoxic effect was detected at different exposure times, except at the lowest concentration of TOF extract (16.25 µg/ml). It appears that extracts decreased DNA damage, induced by H2O2. Both of TOF and aqueous extracts exhibited cellular antioxidant capacity, with EC50 values of respectively <16.25 and < 35 µg/ml, as well as, a protective capacity against lipidperoxidation inducing using L1210 cells line as a cellular model. MDA inhibition percentages reached 88.43% and 90.52% with respectively 35.5 µg/ml of TOF extract and 70 µg/ml of aqueous extract. Antioxidant properties of carob leaf extracts revealed by our study make a good antioxidant protection and thus a good candidate as food addition component.
Assuntos
Antimutagênicos/farmacologia , Antioxidantes/farmacologia , Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Leucemia/genética , Mutagênicos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Antimutagênicos/isolamento & purificação , Antimutagênicos/toxicidade , Antioxidantes/isolamento & purificação , Antioxidantes/toxicidade , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fabaceae/química , Fabaceae/toxicidade , Peróxido de Hidrogênio/toxicidade , Leucemia/metabolismo , Leucemia/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Camundongos , Mutagênicos/isolamento & purificação , Mutagênicos/toxicidade , Fitoterapia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/toxicidade , Plantas Medicinais , Medição de RiscoRESUMO
In 1985, external quality assurance was initiated in the Netherlands to reduce the between-laboratory variability of leukemia/lymphoma immunophenotyping and to improve diagnostic conclusions. This program consisted of regular distributions of test samples followed by biannual plenary participant meetings in which results were presented and discussed. A scoring system was developed in which the quality of results was rated by systematically reviewing the pre-analytical, analytical, and post-analytical assay stages using three scores, i.e., correct (A), minor fault (B), and major fault (C). Here, we report on 90 consecutive samples distributed to 40-61 participating laboratories between 1998 and 2012. Most samples contained >20% aberrant cells, mainly selected from mature lymphoid malignancies (B or T cell) and acute leukemias (myeloid or lymphoblastic). In 2002, minimally required monoclonal antibody (mAb) panels were introduced, whilst methodological guidelines for all three assay stages were implemented. Retrospectively, we divided the study into subsequent periods of 4 ("initial"), 4 ("learning"), and 7 years ("consolidation") to detect "learning effects." Uni- and multivariate models showed that analytical performance declined since 2002, but that post-analytical performance improved during the entire period. These results emphasized the need to improve technical aspects of the assay, and reflected improved interpretational skills of the participants. A strong effect of participant affiliation in all three assay stages was observed: laboratories in academic and large peripheral hospitals performed significantly better than those in small hospitals. © 2015 International Clinical Cytometry Society.
Assuntos
Citometria de Fluxo , Imunofenotipagem , Leucemia/patologia , Linfoma/diagnóstico , Linfoma/patologia , Controle de Qualidade , Doença Aguda , Bélgica , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem/métodos , Países Baixos , Estudos RetrospectivosRESUMO
A new series of chalcones, flavanones and flavones with methylenedioxy group at the 3',4' position in chalcone, 7,8 position in flavanones and flavones with mono-, di- and trimethoxy groups in the benzaldehyde ring have been assessed for their effect on proliferation, cytotoxic potential and apoptosis in human leukemia cells. Among the tested compounds, the chalcone series showed the best activity and chalcone 3 (mono methoxy group at the ortho position in A-ring) showed a significant effect on down-regulation of cancer cell proliferation and viability in three different leukemia cell lines (K562, Jurkat, U937). The executioner caspase cleavage analyses indicated that the cytotoxic effect mediated by chalcone 3 is due to induction of apoptotic cell death. Interestingly, the cytotoxic effect was cell type-specific and targeted preferentially cancer cells as peripheral blood mononuclear cells (PBMCs) from healthy donors were less affected by the treatment compared to K562, Jurkat and U937 leukemia cells. Altogether our results indicate a potential drug candidate with interesting differential toxicity obeying Lipinski's rule of five.
Assuntos
Antineoplásicos/farmacologia , Citotoxinas/farmacologia , Flavonoides/química , Flavonoides/farmacologia , Leucemia/patologia , Antineoplásicos/síntese química , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/síntese química , Citotoxinas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Flavonoides/síntese química , Humanos , Células Jurkat , Células K562 , Leucemia/tratamento farmacológico , Estrutura Molecular , Relação Estrutura-Atividade , Células U937RESUMO
Discretization of a geographical region is quite common in spatial analysis. There have been few studies into the impact of different geographical scales on the outcome of spatial models for different spatial patterns. This study aims to investigate the impact of spatial scales and spatial smoothing on the outcomes of modelling spatial point-based data. Given a spatial point-based dataset (such as occurrence of a disease), we study the geographical variation of residual disease risk using regular grid cells. The individual disease risk is modelled using a logistic model with the inclusion of spatially unstructured and/or spatially structured random effects. Three spatial smoothness priors for the spatially structured component are employed in modelling, namely an intrinsic Gaussian Markov random field, a second-order random walk on a lattice, and a Gaussian field with Matérn correlation function. We investigate how changes in grid cell size affect model outcomes under different spatial structures and different smoothness priors for the spatial component. A realistic example (the Humberside data) is analyzed and a simulation study is described. Bayesian computation is carried out using an integrated nested Laplace approximation. The results suggest that the performance and predictive capacity of the spatial models improve as the grid cell size decreases for certain spatial structures. It also appears that different spatial smoothness priors should be applied for different patterns of point data.
Assuntos
Leucemia/epidemiologia , Linfoma/epidemiologia , Análise Espacial , Teorema de Bayes , Criança , Inglaterra/epidemiologia , Humanos , Leucemia/diagnóstico , Leucemia/patologia , Modelos Logísticos , Linfoma/diagnóstico , Linfoma/patologia , Cadeias de Markov , Neoplasia Residual , Distribuição Normal , Topografia MédicaRESUMO
The rapid technological development in diagnostic pathology, especially of immunohistochemical and molecular techniques, also has a significant impact on diagnostic procedures for the evaluation of bone marrow trephine biopsies. The necessity for optimal morphology, combined with preservation of tissue antigens and nucleic acids on one hand and the wish for short turnaround times on the other hand require careful planning of the workflow for fixation, decalcification and embedding of trephines. Although any kind of bone marrow processing has its advantages and disadvantages, formalin fixation followed by EDTA decalcification can be considered a good compromise, which does not restrict the use of molecular techniques. Although the majority of molecular studies in haematological neoplasms are routinely performed on bone marrow aspirates or peripheral blood cells, there are certain indications, in which molecular studies such as clonality determination or detection of specific mutations need to be performed on the trephine biopsy. Especially, the determination of B- or T-cell clonality for the diagnosis of lymphoid malignancies requires stringent quality controls and knowledge of technical pitfalls. In this review, we discuss technical aspects of bone marrow biopsy processing and the application of diagnostic molecular techniques.
Assuntos
Biópsia por Agulha , Exame de Medula Óssea/métodos , Medula Óssea/patologia , Hibridização in Situ Fluorescente , Leucemia/genética , Leucemia/patologia , Linfoma/genética , Linfoma/patologia , Técnicas de Diagnóstico Molecular , Doenças Mieloproliferativas-Mielodisplásicas/genética , Doenças Mieloproliferativas-Mielodisplásicas/patologia , Biomarcadores Tumorais/genética , Aberrações Cromossômicas , Análise Mutacional de DNA , Marcadores Genéticos/genética , Imuno-Histoquímica/métodos , PrognósticoRESUMO
Genome wide analysis of DNA methylation provides important information in a variety of diseases, including cancer. Here, we describe a simple method, Digital Restriction Enzyme Analysis of Methylation (DREAM), based on next generation sequencing analysis of methylation-specific signatures created by sequential digestion of genomic DNA with SmaI and XmaI enzymes. DREAM provides information on 150,000 unique CpG sites, of which 39,000 are in CpG islands and 30,000 are at transcription start sites of 13,000 RefSeq genes. We analyzed DNA methylation in healthy white blood cells and found methylation patterns to be remarkably uniform. Inter individual differences > 30% were observed only at 227 of 28,331 (0.8%) of autosomal CpG sites. Similarly, > 30% differences were observed at only 59 sites when we comparing the cord and adult blood. These conserved methylation patterns contrasted with extensive changes affecting 18-40% of CpG sites in a patient with acute myeloid leukemia and in two leukemia cell lines. The method is cost effective, quantitative (r ( 2) = 0.93 when compared with bisulfite pyrosequencing) and reproducible (r ( 2) = 0.997). Using 100-fold coverage, DREAM can detect differences in methylation greater than 10% or 30% with a false positive rate below 0.05 or 0.001, respectively. DREAM can be useful in quantifying epigenetic effects of environment and nutrition, correlating developmental epigenetic variation with phenotypes, understanding epigenetics of cancer and chronic diseases, measuring the effects of drugs on DNA methylation or deriving new biological insights into mammalian genomes.
Assuntos
Metilação de DNA , Leucemia/genética , Leucócitos/fisiologia , Análise de Sequência de DNA/métodos , Sequência de Bases , Linhagem Celular Tumoral , Cromossomos Humanos X , Sequência Conservada , Ilhas de CpG , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Feminino , Impressão Genômica , Humanos , Leucemia/patologia , Leucemia Mieloide Aguda/genética , Masculino , Valores de Referência , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA/economiaRESUMO
Despite having well-characterized disease-associated mutations, the mechanisms underlying the progressive bone marrow failure and cancer susceptibility of Fanconi anemia have been unclear. In this issue of Cell Stem Cell, Ceccaldi et al. identify an overactive p53/p21 stress response and cell cycle arrest as an underlying cause that starts during fetal development.
Assuntos
Medula Óssea/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Modelos Animais de Doenças , Anemia de Fanconi/terapia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia/patologia , CamundongosRESUMO
The purpose of the study was to characterize the involvement of reactive oxygen species (ROS) in mediating the cytotoxic effects of arsenic trioxide (ATO) in combination with sulindac or its metabolites: sulfide (SS) and sulfone (SF) on human leukemic cell lines. Jurkat, HL-60, K562, and HPB-ALL cells were exposed to the drugs alone or in combinations. Cell viability was measured using WST-1 or XTT reduction tests and ROS production by dichlorodihydrofluorescein diacetate staining (flow cytometry). Modulation of (a) intracellular glutathione (GSH) level was done by using L: -buthionine sulfoximine (BSO) or diethylmaleate (DEM), (b) NADPH oxidase by using diphenyleneiodonium (DPI), and (c) MAP kinases by using SB202190 (p38), SP600125 (JNK), and U0126 (ERK) inhibitors. ATO cytotoxicity (0.5 or 1 µM) was enhanced by sulindacs, with higher activity showed by the metabolites. Strong cytotoxic effects appeared at SS and SF concentrations starting from 50 µM. The induction of ROS production seemed not to be the major mechanism responsible for the cytotoxicity of the combinations. A strong potentiating effect of BSO on ATO cytotoxicity was demonstrated; DEM (10-300 µM) and DPI (0.0025-0.1 µM; 72 h) did not influence the effects of ATO. Some significant decreases in the viability of the cells exposed to ATO in the presence of MAPK inhibitors comparing with the cells exposed to ATO alone were observed; however, the effects likely resulted from a simple additive cytotoxicity of the drugs. The combinations of ATO with sulindacs offer potential therapeutic usefulness.
Assuntos
Proliferação de Células/efeitos dos fármacos , Leucemia/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Trióxido de Arsênio , Arsenicais/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Glutationa/metabolismo , Humanos , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Óxidos/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Sulindaco/administração & dosagem , Sulindaco/análogos & derivadosRESUMO
The segmentation of leukocytes and their components plays an important role in the extraction of geometric, texture, and morphological characteristics used to diagnose different diseases. This paper presents a novel method to segment leukocytes and their respective nucleus and cytoplasm from microscopic bone marrow leukemia cell images. Our method uses color and texture contextual information of image pixels to extract cellular elements from images, which show heterogeneous color and texture staining and high-cell population. The CIEL ( ∗ ) a ( ∗ ) b ( ∗ ) color space is used to extract color features, whereas a 2D Wold Decomposition model is applied to extract structural and stochastic texture features. The color and texture contextual information is incorporated into an unsupervised binary Markov Random Field segmentation model. Experimental results show the performance of the proposed method on both synthetic and real leukemia cell images. An average accuracy of 95% was achieved in the segmentation of real cell images by comparing those results with manually segmented cell images.
Assuntos
Leucócitos/ultraestrutura , Algoritmos , Células da Medula Óssea/patologia , Núcleo Celular/ultraestrutura , Separação Celular , Cor , Biologia Computacional , Citoplasma/ultraestrutura , Humanos , Interpretação de Imagem Assistida por Computador , Leucemia/patologia , Cadeias de Markov , Modelos Biológicos , Modelos Estatísticos , Reconhecimento Automatizado de Padrão/estatística & dados numéricos , Coloração e Rotulagem , Processos EstocásticosRESUMO
Participation in external quality assessment is an integral part of laboratory work and mandatory when the results have a clinical application, which is one of the requirements of standard 15189 for accreditation of medical laboratories. Institute of Clinical Chemistry, the first laboratory accredited for clinical cell analysis by flow cytometry in Croatia, participated in UKNEQAS for Leukocyte Immunophenotyping in 3 schemes: "Immune Monitoring", "CD34 Stem Cell Enumeration" and "Leukaemia Immunophenotyping". For sample processing on EPICS XL flow cytometer, lyse/no wash preparation technique with ammonium chloride (NH4Cl) or ImmunoPrep lysing reagent was employed. In "Immune monitoring" programme CD45/sideward light scatter (SSC) proposed gating strategy was adopted for lymphocyte subsets, while modified ISHAGE protocol was used for CD34+ cell enumeration. Absolute count determination was performed on flow cytometer using FlowCount beads solution. In the period from the beginning of 2006 until the middle of 2009 a total number of 100 stabilized whole blood samples were processed. The relative and absolute enumeration results for lymphocyte subsets were within tolerable limits, in 97.1 and 97.1% of cases, and 95 and 90% of CD34+ cell enumeration, respectively. In immune monitoring CD45/SSC proposed gating strategy is the most frequent analysis used (> 85% participants) and ISHAGE protocol for CD34+ cell determination with continuous rise from 76 to 83%. A number of participants who accept beads method for absolute count enumeration on flow cytometer get greater, 69 to 86%, while FlowCount was the second of bead-based techniques used (25 and 35%). Sample treatment in lyse/no wash technique using NH4Cl lysing solution was dominant procedure used by more than 1/3 participants, although its home made solution has replaced slowly by commercial reagents. The unacceptable results, 6 of 244, were obtained for 20 most frequently determined cell antigens in "Leukaemia Immunophenotyping" samples screened for leukaemia/lymphoma. Processing results of all participants showed that the deviation from laboratory guidelines and the use of older methods for cell identification, quantification of cell counting on haematology analyser, or usage an antibody conjugated with fluorochrome lesser fluorescence quantum often lead to an unacceptable result, although is noticeable trend to accept new referrals and protocols to reduce the inter-laboratory differences.
Assuntos
Citometria de Fluxo/normas , Imunofenotipagem/normas , Laboratórios Hospitalares/normas , Leucemia/patologia , Linfoma/patologia , Garantia da Qualidade dos Cuidados de Saúde , Antígenos CD34/metabolismo , Guias como Assunto , Antígenos HLA-DR/metabolismo , Humanos , Subpopulações de Linfócitos/metabolismo , Subpopulações de Linfócitos/patologia , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
Gene expression profiling studies have been employed to investigate prognostic subgroups in pediatric acute leukemia. Tissue microarrays (TMAs) are useful for high-throughput analysis of protein expression of target genes in acute leukemia samples and for validation of gene microarray analysis. Using cryopreserved samples of pediatric acute leukemia bone marrow aspirates, we constructed TMA from as few as 1 million cells. Bone marrow core biopsies from the same patients were included on the same TMA for comparison. A panel of 15 immunohistochemical markers typically used for diagnosis as well as those targeting recently characterized, prognostically relevant molecules of interest in pediatric acute leukemia was used to evaluate protein expression. Staining results confirm that suspension cells from bone marrow aspirates can be effectively used to derive protein expression data from multiple cases simultaneously with comparable efficacy to that of biopsy tissue. This method allows for new markers of diagnostic, prognostic, or therapeutic importance to be screened on large numbers of study patients. Furthermore, this technique may facilitate the inclusion of small samples, aspirates, and body fluids in large-scale studies of protein expression in clinical trials and protocols in which tissue biopsies are often unavailable.
Assuntos
Células da Medula Óssea/patologia , Perfilação da Expressão Gênica , Leucemia/metabolismo , Análise Serial de Tecidos/métodos , Doença Aguda , Adolescente , Biomarcadores Tumorais/metabolismo , Biópsia , Células da Medula Óssea/metabolismo , Criança , Pré-Escolar , Análise Citogenética , Feminino , Humanos , Imuno-Histoquímica , Lactente , Leucemia/genética , Leucemia/patologia , MasculinoRESUMO
There is a need to introduce cytometry into areas of the globe that have remained virtually untouched by modern laboratory medicine. With the demand to carry out tests on 100,000 s of individuals requiring antiretroviral therapy (ART), flow cytometry must remain simple and cost-effective - while being sustainable and industry supported as well as proven by quality assessment (QA). This outlook is referred to as "smart flow cytometry" (S-FC). There are five main areas where the power of S-FC is demonstrated. These are: (i) the use of CD45 to assist precise cell counting in blood and tissue samples; (ii) the primary CD4 gating to count CD4+ T cells in patients waiting for ART, including the combination (i) and (ii) in the panleucogating (PLG) protocol; (iii) monitoring of human immunodeficiency virus (HIV+) patients during ART by the decreasing levels of lymphocyte activation in a CD8/CD38 test - leading to economies of viral-load assays; (iv) in tuberculosis and HIV-TB coinfections the use of TB-antigen-stimulated cytokine-synthetic CD4+ T cells to identify active disease; and (v) the utilization of "minimal residual disease (MRD)-Lite" technology in patients 19 days after the start of antileukemic therapy to detect MRD. These methods of S-FC have been successfully introduced in "resource-restricted" countries with international and local QA.
Assuntos
Citodiagnóstico/métodos , Citometria de Fluxo/métodos , Infecções por HIV/patologia , Leucemia/patologia , Tuberculose/patologia , Citodiagnóstico/economia , Citometria de Fluxo/economia , Humanos , InternacionalidadeRESUMO
OBJECTIVE: Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution normal T-cell immunity. Measurement of T-cell receptor excision circles (TRECs) and T-cell receptor beta (TCRBV) CDR3 repertoire is a means of quantifying recent thymic T-cell production and reflecting antigen-specific T-cell clones proliferation. METHODS: We used real-time quantitative PCR to detect TRECs from 43 Chinese patients who underwent three kind of allo-HSCT without T-cell depletion. RT-PCR was performed to amplify 24 subfamily genes of TCRBV in 24 patients of them. RESULTS: For haploidentical-D group, the TRECs numbers were lower up to 24 months. For matched-sibling donor (MSD) group, the recovery of TRECs was faster than those of other two groups. TRECs values in matched-unrelated donor (MUD) were in the middle. During 2-19 months after transplantation, there were 6-16 BV subfamilies expressed and 33-48% of them were polyclones. The usage rate of TCRBV and percentage of polyclones in haploidentical-D were less than those of other two groups. Twenty-three CDR3 molecules were obtained from nine patients who were potentially associated with GVHD or CMV infection. CONCLUSIONS: Analyzing the changes of TCRBV repertoire and measuring TRECs during immune reconstitution would be useful to determine the host's current immune status and ability of T-cell immune reconstitution and also to find antigen-specific T-cell clones in the three kinds of HSCT.