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1.
Sci Rep ; 9(1): 4623, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874567

RESUMO

The knowledge of optical properties of biological cells is essential to interpret their interaction with light and to derive morphological information and parameters associated with cell function like the oxygen transport capacity of human red blood cells (RBCs). We present a method to determine the dependence between the refractive index (RI) of human RBCs and their intracellular hemoglobin (Hb) concentration from spectral extinction measurements of a cell suspension. The procedure is based on the analysis of the corresponding ensemble averaged extinction cross section [Formula: see text]. Thus far two complementary approaches have been taken to derive RIs of RBCs. The first one uses homogeneous macroscopic samples prepared by hemolysis for the destruction of the RBCs' membranes and subsequent centrifugation. A second approach is the determination of RIs of single intact cells by microscopic investigation. These techniques are limited to a few discrete wavelengths or a rather narrow wavelength range. In addition most of these techniques require additional information about the concentration dependence. In contrast, our approach yields the RI increment with Hb concentration of intact, reversibly isovolumetrically sphered, oxygenated RBCs over a wide wavelength range from 290 nm to 1100 nm from macroscopic measurements.

2.
Artigo em Inglês | MEDLINE | ID: mdl-30790450

RESUMO

BACKGROUND: Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of CD34 cells. However, volumetric flow cytometers have the ability to measure the acquisition volume, which removes the need for reference beads for calculation of cell concentrations. METHOD: In this study, we compared both methods for calculating CD34 cell concentrations in volumetric cytometers, using either the volume reading or the number of reference beads for calculation. In addition, the uncertainty of measurement for each method was estimated. RESULTS: The results show that both methods have similar uncertainties of measurement. Regression analysis showed low to no statistical difference in CD34 cell concentrations obtained with each method. CONCLUSIONS: Overall, this study suggests that the volumetric method is a valid approach but that the adoption of this technology may be hindered without some form of external calibration of volume readings to increase confidence in the measurement. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

3.
Appl Opt ; 57(2): 344-355, 2018 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-29328184

RESUMO

A method is presented to infer simultaneously the wavelength-dependent real refractive index (RI) of the material of microspheres and their size distribution from extinction measurements of particle suspensions. To derive the averaged spectral optical extinction cross section of the microspheres from such ensemble measurements, we determined the particle concentration by flow cytometry to an accuracy of typically 2% and adjusted the particle concentration to ensure that perturbations due to multiple scattering are negligible. For analysis of the extinction spectra, we employ Mie theory, a series-expansion representation of the refractive index and nonlinear numerical optimization. In contrast to other approaches, our method offers the advantage to simultaneously determine size, size distribution, and spectral refractive index of ensembles of microparticles including uncertainty estimation.

4.
Appl Opt ; 55(31): 8951-8961, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27828301

RESUMO

The real part of the refractive index of aqueous solutions of human hemoglobin is computed from their absorption spectra in the wavelength range 250-1100 nm using the Kramers-Kronig (KK) relations, and the corresponding uncertainty analysis is provided. The strong ultraviolet (UV) and infrared absorbance of the water outside this spectral range were taken into account in a previous study employing KK relations. We improve these results by including the concentration dependence of the water absorbance as well as by modeling the deep UV absorbance of hemoglobin's peptide backbone. The two free parameters of the model for the deep UV absorbance are fixed by a global fit.

5.
Lab Chip ; 16(12): 2326-38, 2016 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-27229300

RESUMO

We developed a microfluidic sensor for label-free flow cytometric cell differentiation by combined multiple AC electrical impedance and light scattering analysis. The measured signals are correlated to cell volume, membrane capacity and optical properties of single cells. For an improved signal to noise ratio, the microfluidic sensor incorporates two electrode pairs for differential impedance detection. One-dimensional sheath flow focusing was implemented, which allows single particle analysis at kHz count rates. Various monodisperse particles and differentiation of leukocytes in haemolysed samples served to benchmark the microdevice applying combined AC impedance and side scatter analyses. In what follows, we demonstrate that AC impedance measurements at selected frequencies allow label-free discrimination of platelets, erythrocytes, monocytes, granulocytes and lymphocytes in whole blood samples involving dilution only. Immunofluorescence staining was applied to validate the results of the label-free cell analysis. Reliable differentiation and enumeration of cells in whole blood by AC impedance detection have the potential to support medical diagnosis for patients with haemolysis resistant erythrocytes or abnormally sensitive leucocytes, i.e. for patients suffering from anaemia or leukaemia.


Assuntos
Difusão Dinâmica da Luz/métodos , Eritrócitos/citologia , Citometria de Fluxo/instrumentação , Dispositivos Lab-On-A-Chip , Leucócitos/citologia , Contagem de Células Sanguíneas/instrumentação , Contagem de Células Sanguíneas/métodos , Plaquetas/citologia , Diferenciação Celular , Difusão Dinâmica da Luz/instrumentação , Impedância Elétrica , Eletrodos , Hemólise , Humanos , Limite de Detecção , Técnicas Analíticas Microfluídicas/instrumentação , Razão Sinal-Ruído
6.
Cytometry A ; 87(3): 244-53, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25655255

RESUMO

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) µL(-1) CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment.


Assuntos
Linfócitos T CD4-Positivos , Fluoresceína-5-Isotiocianato , Leucócitos Mononucleares , Fenótipo , Anticorpos/análise , Contagem de Linfócito CD4/métodos , Contagem de Linfócito CD4/normas , Linfócitos T CD4-Positivos/química , Fluoresceína-5-Isotiocianato/análise , Liofilização/métodos , Humanos , Leucócitos Mononucleares/química , Projetos Piloto
7.
Sensors (Basel) ; 13(4): 4674-93, 2013 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-23571670

RESUMO

This study demonstrates the suitability of microfluidic structures for high throughput blood cell analysis. The microfluidic chips exploit fully integrated hydrodynamic focusing based on two different concepts: Two-stage cascade focusing and spin focusing (vortex) principle. The sample--A suspension of micro particles or blood cells--is injected into a sheath fluid streaming at a substantially higher flow rate, which assures positioning of the particles in the center of the flow channel. Particle velocities of a few m/s are achieved as required for high throughput blood cell analysis. The stability of hydrodynamic particle positioning was evaluated by measuring the pulse heights distributions of fluorescence signals from calibration beads. Quantitative assessment based on coefficient of variation for the fluorescence intensity distributions resulted in a value of about 3% determined for the micro-device exploiting cascade hydrodynamic focusing. For the spin focusing approach similar values were achieved for sample flow rates being 1.5 times lower. Our results indicate that the performances of both variants of hydrodynamic focusing suit for blood cell differentiation and counting. The potential of the micro flow cytometer is demonstrated by detecting immunologically labeled CD3 positive and CD4 positive T-lymphocytes in blood.


Assuntos
Citometria de Fluxo/instrumentação , Hidrodinâmica , Microfluídica/instrumentação , Células Sanguíneas/citologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Calibragem , Fluoresceína-5-Isotiocianato , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Fenômenos Ópticos , Ficoeritrina/metabolismo , Reologia
8.
Cytometry A ; 79(8): 613-24, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21618424

RESUMO

In this article, we demonstrate the potential of a microfluidic chip for the differentiation of immunologically stained blood cells. To this end, white blood cells stained with antibodies typically applied for the determination of the immune status were measured in the micro-device. Relative concentrations of lymphocytes and subpopulations of lymphocytes are compared to those obtained with a conventional flow cytometer. The stability of the hydrodynamic focusing and the optical setup was determined by measuring the variation of the signal pulse height of fluorescence calibration beads, being about 2% for the micro-device. This value and the overall performance of the micro-device are similar to conventional flow cytometers. It follows from our results that such microfluidic structures are well suited as modules in a compact, portable read-out instrument. The production process of the microflow cytometers, which we exploited for immunological cell differentiation, is compatible with mass production technology like injection molding and, hence, low cost disposable chips could be available in the future.


Assuntos
Diferenciação Celular , Citometria de Fluxo/instrumentação , Linfócitos/imunologia , Técnicas Analíticas Microfluídicas/instrumentação , Antígenos CD/análise , Humanos , Linfócitos/citologia
9.
J Fluoresc ; 15(3): 433-41, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15986162

RESUMO

We have determined the fluorescence yield of stained micro beads, used for calibration purposes in flow cytometry, as function of the irradiance of the exciting laser beam. A rate equation model has been applied to derive the number of fluorescence molecules carried by each micro bead. To derive in situ photo-physical properties of the specific dye, required for the rate equation model, we discuss an approach based on flow cytometric sorting of micro beads, which have passed two laser beams with properly chosen different irradiances, and subsequent observation of single molecule bleaching employing high sensitivity microscopy. The feasibility of our approach is demonstrated presenting first results concerning saturation of fluorescence of beads in flow and single molecule bleaching by high sensitivity microscopy.


Assuntos
Citometria de Fluxo/métodos , Fluorescência , Citometria de Fluxo/instrumentação , Citometria de Fluxo/estatística & dados numéricos , Corantes Fluorescentes , Humanos , Lasers , Microscopia de Fluorescência , Microesferas , Modelos Teóricos
10.
Appl Opt ; 42(31): 6388-97, 2003 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-14649283

RESUMO

We used a flow cytometer together with an intensified CCD camera to record spatially resolved light scattering from micrometer-sized single particles and single oriented particle agglomerates. Experimental differential cross sections of an oriented dumbbell made from two identical polystyrene spheres were compared with theoretical values calculated within the discrete dipole approximation, and good agreement was achieved. Furthermore, characteristic two-dimensional patterns of the scattered-light intensity were recorded for single blood cells, yielding information on the cells' shape and volume. Besides flow cytometry, we observed and analyzed differential light scatter of particle clusters of known size, shape, and orientation located within an optical trap.


Assuntos
Algoritmos , Eritrócitos/citologia , Citometria de Fluxo/instrumentação , Interpretação de Imagem Assistida por Computador/métodos , Leucócitos/citologia , Micromanipulação/instrumentação , Gravação em Vídeo/instrumentação , Tamanho Celular , Desenho de Equipamento , Análise de Falha de Equipamento , Citometria de Fluxo/métodos , Substâncias Macromoleculares , Micromanipulação/métodos , Microesferas , Conformação Molecular , Tamanho da Partícula , Poliestirenos/química , Rotação , Espalhamento de Radiação , Gravação em Vídeo/métodos
11.
Cytometry B Clin Cytom ; 55(1): 46-51, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12949959

RESUMO

BACKGROUND: Cell-Dyn automated blood cell analyzers use laser flow cytometry technology, allowing detection of malaria pigment (hemozoin) in monocytes. We evaluated the value of such an instrument to diagnose malaria in febrile travelers returning to Berlin, Germany, the relation between the instrument's performance and the patients' immune status, and the capacity to increase its sensitivity. METHODS: Malaria diagnosis was routinely established by thick-film microscopy. The patients' immune status was determined by an indirect fluorescent antibody test. Hemozoin detection was performed with a Cell-Dyn 3000. To assess the capacity for sensitivity increase, the relative frequencies of pigment-containing monocytes were determined for a subgroup of patients with the Cell-Dyn 3000 in comparison with a flow cytometer specifically adapted to rare-event analysis. RESULTS: Of 403 patients screened microscopically, 107 had malaria. Overall sensitivity with the Cell-Dyn 3000 reached 48.6% (73.7% in semi-immune and 28.6% in nonimmune individuals; P < 0.0001). Specificity was 96.2%. The detection limit was at a relative concentration of 2 x 10(-4) pigment-containing monocytes (PCMs). By employing rare-event flow cytometry, the detection limit decreased to 3.25 x 10(-5), thus yielding a considerably increased sensitivity for the subgroup studied. CONCLUSIONS: The correlation between immune status and relative concentration of PCMs explains the failure of the routine instrument for nonimmune patients and its significantly higher sensitivity for semi-immune individuals. The technique can be significantly improved by rare-event flow cytometry.


Assuntos
Citometria de Fluxo , Hemeproteínas/análise , Malária/diagnóstico , Malária/imunologia , Monócitos/química , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Separação Celular , Criança , Pré-Escolar , Feminino , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade
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