RESUMEN
The challenges associated with analyzing rare cells are dependent on a series of factors, which usually require large numbers of cells per sample for successful resolution. Among these is determining the minimum number of total events needed to be acquired as defined by the expected frequency of the target cell population. The choice of markers that identify the target population, as well as the event rate and the number of aborted events/second, will also determine the statistically significant detection of rare cell events. Sample preparation is another important but often overlooked factor in rare cell analysis, and in this study we examine Poisson theory and methods to determine the effect of sample manipulation on rare cell detection. After verifying the applicability of this theory, we have evaluated the potential impact of red cell lysis on rare cell analysis, and how cell rarity can be underestimated or overestimated based on erythrolytic sensitivity or resistance of healthy leukocytes and pathological rare cells.
Asunto(s)
Eritrocitos , Leucocitos , Muerte Celular , Manejo de Especímenes , Citometría de FlujoRESUMEN
Doublet discrimination is usually based on pulse analysis of light scatter parameters. A combination of two pulse parameters (Area, A; Height, H; or Width, W) can be used to discriminate a pulse originated in a single cell from a pulse originated from cells stuck together. Fluorescence signals can be also used to discriminate aggregates, being essential to identify cells in the G2/M phase from doublets in the G0/G1 phase in cell cycle/DNA applications. The most used method combines FSC-A versus FSC-H, whereas other strategies combine FSC-H versus FSC-W, SSC-H versus SSC-A and SSC-H versus SSC-W. However, when studying activated or proliferating cells, scatter discrimination can be difficult. In this study, we have compared the use of light scattering with fluorescence measurement techniques for successful doublet discrimination for single cells. Effective use of FSC and SSC height, area and width are commonly used to eliminate aggregates. However, fluorescence-based methods using viable DNA stains provide a good compromise between performance and accurate manual gating methods, especially for highly concentrated cell products and pathological specimens. Viable DNA dyes, such as Vybrant™ DyeCycle™ Violet stain or Hoechst 33342, can be used to detect nucleated cells in blood and in bone marrow, or to discriminate cell aggregates and debris based on no-lyse no-wash assays, where scatter degradation is a dominant component of the measured data, which increases with event rate.
Asunto(s)
ADN , Citometría de Flujo/métodos , Ciclo Celular , ADN/análisisRESUMEN
Flow cytometry (FCM) enumeration of peripheral blood dendritic cells (PBDCs) is a minimally invasive procedure extremely useful for immunological studies. Numbers of PBDCs vary depending on age, lifestyle, or in pathologies like cancer, leukemia or immunodeficiencies. Conventional methods for PBDC identification by FCM involve red blood cell lysis using either formaldehyde or ammonium chloride-based solutions. This specific procedure has been widely reported to cause a detrimental effect as well as an artifactual detection of target populations. Alternatively, minimal sample perturbation assays that avoid the use of erythrolytic solutions with centrifugation steps and preserve the native cellular state are simpler and more robust than conventional methods. In this study, we aimed to evaluate how conventional FCM assays can alter dendritic cell (DC) counting when compared with minimal sample perturbation protocols, in terms of absolute cell counting, percentage and stain index (SI) of PBDC subsets. We evaluated the use of three different erythrolytic solutions (CyLyse, OptiLyse C, and Pharm Lyse) on a series of n = 20 peripheral blood specimens for conventional and plasmacytoid DCs detection as well as for leukocyte and basophil detection. Our results showed a significant reduction of leukocytes and specifically, of DCs and basophils in terms of absolute number when using erythrolytic solutions. In conclusion, our study shows that PBDC counting is heavily affected when lysing solutions are used, indicating that these stellate-shaped populations appear to be more labile.
Asunto(s)
Células Sanguíneas , Eritrocitos , Citometría de Flujo/métodos , Recuento de Células , Células DendríticasRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.12497.].
RESUMEN
Distortions of the normal bi-concave disc shape for red blood cells (RBCs) appear in a number of pathologies resulting from defects in cell membrane skeletal architecture, erythrocyte ageing, and mechanical damage. We present here the potential of acoustic cytometry for developing new approaches to light-scattering based evaluation of red blood cell disorders and of the effects of storage and ageing on changes or damage to RBCs membranes. These approaches could be used to immediately evaluate the quality of erythrocytes prior to blood donation and following transfusion. They could also be applied to studying RBC health in diseases and other pathologies, such as artificial heart valve hemolysis, thermal damage or osmotic fragility. Abnormal distributions of erythrocytes can typically be detected after just 30 to 45 seconds of acquisition time using 1-2 µL starting blood volumes.
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Eritrocitos , Citometría de Flujo/métodos , Sonido , Anexina A5 , Conservación de la Sangre/métodos , Envejecimiento Eritrocítico , Deformación Eritrocítica , Índices de Eritrocitos , Membrana Eritrocítica/ultraestructura , Transfusión de Eritrocitos , Eritrocitos/ultraestructura , Citometría de Flujo/instrumentación , Colorantes Fluorescentes , Hemólisis , Humanos , Hidrodinámica , Luz , Lípidos de la Membrana/sangre , Fosfatidilserinas/sangre , Dispersión de Radiación , Esferocitos/ultraestructura , Esferocitosis Hereditaria/sangreRESUMEN
With the aim to detect candidate malignant primitive progenitor populations, we modified an original alkaline phosphatase (ALP) stem cell detection method based on the identification of alkaline phosphatase fluorescent cells in combination with flow cytometry immunophenotyping. Over a period of one year, we have been using this technique to study its activity in patients with leukemia and lymphoma, showing that changes in the alkaline phosphatase levels can be used to detect rare populations of highly refractory malignant cells. By screening different blood cancers, we have observed that this activity is not always restricted to CD34+ leukemic cells, and can be overexpressed in CD34 negative leukemia. We have verified that this method gives accurate and reproducible measurements and our preliminary results suggest that CD34+/ALPhigh cells appear to sustain leukemogenesis over time.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Pruebas de Enzimas/métodos , Células Madre Neoplásicas/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Adulto , Anciano , Antígenos CD34/metabolismo , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/metabolismo , Médula Ósea/enzimología , Médula Ósea/patología , Transformación Celular Neoplásica/metabolismo , Niño , Resistencia a Antineoplásicos , Detección Precoz del Cáncer/métodos , Pruebas de Enzimas/instrumentación , Femenino , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Inmunofenotipificación/instrumentación , Inmunofenotipificación/métodos , Masculino , Proteína de la Leucemia Mieloide-Linfoide/genética , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas c-myc/genética , Reproducibilidad de los ResultadosRESUMEN
INTRODUCTION: Studies are needed to understand the role of CD34 expressing cells with regard to efficient engraftment, especially in the adjuvant treatment of cancer. MATERIALS AND METHODS: In this study we have used a modified method in our laboratory for routinely counting CD34+ cells. Unlysed whole blood samples were stained with the DNA-selective and cell membrane-permeant Vibrant DyeCycle Violet stain. RESULTS: CD34+ cells exhibit a consistent and differential Vybrant Dye Cycle Violet staining pattern. Based on their different DCV intensity, we classified these subpopulations as CD34+/DCV(high) and CD34+/DCV(low) cells. In general, DCV(high) cells are about 12-times brighter than DCV(low) cells. CONCLUSION: DCV staining may be used to discriminate subsets of CD34+ cells similarly to other methods which have previously defined different functional properties that can be related to the characterization, resolution, and purification of primitive hematopoietic stem cells in combination with specific useful markers for multicolor flow cytometric measurements.