RESUMEN

Commonly used methods for detection of melanoma cells in blood, including RT-PCR and immunocytochemistry, display only a limited sensitivity and specificity. Reliable detection of less than one melanoma cell per ml of blood is hardly possible using these methods. To obtain greater sensitivity so that a single melanoma cell in up to 25 ml of blood can be detected (5 x 10(7) peripheral blood mononuclear cells, or PBMC), we developed a new assay for combined enrichment and immunocytochemical detection of disseminated melanoma cells from PBMC of patients with malignant melanomas. Melanoma cells are directly magnetically labeled using colloidal superparamagnetic microparticles approximately 60 nm in diameter conjugated to the anti-melanoma monoclonal antibody 9.2.27, with no reactivity to normal cells in blood. Magnetically labeled melanoma cells are enriched from PBMC by magnetic cell separation and detected by a new approach for immunocytochemical staining with monoclonal mouse anti-melanoma antibodies (anti-MelanA and HMB-45). The efficiency of this assay was demonstrated in a model system in which 5-500 tumor cells from the melanoma cell line SK-MEL-28 were seeded into PBMC samples from healthy donors containing 5 x 10(7) leukocytes. Mean recovery of the seeded tumor cells was 47.4 +/- 13.99% (n = 15). Applying the assay to 20-50 ml blood samples of patients with stage III-IV malignant melanomas, we were able to detect melanoma cells in two of eight patients (25%).

Asunto(s)

Inmunohistoquímica/métodos , Separación Inmunomagnética/métodos , Leucocitos Mononucleares/patología , Melanoma/diagnóstico , Células Neoplásicas Circulantes/patología , Anticuerpos Monoclonales , Antígenos de Neoplasias , Humanos , Antígeno MART-1 , Metástasis de la Neoplasia/diagnóstico , Proteínas de Neoplasias/análisis , Estadificación de Neoplasias , Células Tumorales Cultivadas

RESUMEN

We have generated a panel of mAbs that identify three presumably novel human dendritic cell Ags: BDCA-2, BDCA-3, and BDCA-4. In blood, BDCA-2 and BDCA-4 are expressed on CD11c(-) CD123(bright) plasmacytoid dendritic cells, whereas BDCA-3 is expressed on small population of CD11c(+) CD123(-) dendritic cells. All three Ags are not detectable on a third blood dendritic cell population, which is CD1c(+) CD11c(bright) CD123(dim), or on any other cells in blood. BDCA-4 is also expressed on monocyte-derived and CD34(+) cell-derived dendritic cells. Expression of all three Ags dramatically changes once blood dendritic cells undergo in vitro maturation. BDCA-2 is completely down-regulated on plasmacytoid CD11c(-) CD123(bright) dendritic cells, expression of BDCA-3 is up-regulated on both plasmacytoid CD11c(-) CD123(bright) dendritic cells and CD1c(+) CD11c(bright) CD123(dim) dendritic cells, and expression of BDCA-4 is up-regulated on CD1c(+) CD11c(bright) CD123(dim) dendritic cells. BDCA-2 is rapidly internalized at 37 degrees C after mAb labeling. The three presumably novel Ags serve as specific markers for the respective subpopulations of blood dendritic cells in fresh blood and will be of great value for their further analysis and to evaluate their therapeutic potential.

Asunto(s)

Antígenos de Diferenciación/sangre , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunofenotipificación , Animales , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Antígenos CD , Antígenos CD1/biosíntesis , Antígenos CD34/biosíntesis , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/inmunología , Antígenos de Diferenciación/metabolismo , Antígenos de Superficie/biosíntesis , Biomarcadores/sangre , Separación Celular , Células Cultivadas , Células Dendríticas/citología , Endocitosis/inmunología , Femenino , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Inmunoglobulinas/biosíntesis , Linfocitos/citología , Linfocitos/inmunología , Linfocitos/metabolismo , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos BALB C , Monocitos/inmunología , Monocitos/metabolismo , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Coloración y Etiquetado , Antígeno CD83

RESUMEN

The primary objective of this study was to evaluate the safety of infusion of CD34+ cells, selected using a clinical scale magnetically activated cell sorting device, assessed by time to hematological engraftment and incidence of adverse events. Secondary objectives included evaluation of device performance in terms of purity and recovery of the CD34+ cell product. Breast cancer patients suitable for transplantation received cyclophosphamide and filgrastim for mobilisation, followed by three leukaphereses. The products of the first two leukaphereses underwent CD34+ cell selection. The product of the third leukapheresis was cryopreserved unmanipulated. Following high-dose cyclophosphamide, thiotepa and carboplatin, selected CD34+ cells were infused. In 54 patients who received selected cells only, the median time to platelet recovery and neutrophil recovery was 11 days (range 5-51) and 9 days (range 5-51), respectively. There were no adverse events associated with infusion of selected cells. A total of 126 leukapheresis samples was available before and after selection for central CD34+ analysis. The median purity was 96.1% (27.4-99.4) and the median recovery was 52. 3% (15.2-146.3). These data show that cells selected using magnetically activated cell selection provide safe and rapid engraftment after high-dose therapy. Bone Marrow Transplantation (2000) 25, 243-249.

Asunto(s)

Antígenos CD34/sangre , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/terapia , Separación Inmunomagnética , Trasplante Autólogo/normas , Adolescente , Adulto , Anciano , Animales , Anticuerpos/sangre , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Neoplasias de la Mama/sangre , Relación CD4-CD8 , Supervivencia Celular , Falla de Equipo , Femenino , Supervivencia de Injerto , Humanos , Separación Inmunomagnética/instrumentación , Separación Inmunomagnética/normas , Leucaféresis/normas , Recuento de Linfocitos , Ratones/inmunología , Persona de Mediana Edad , Tasa de Supervivencia , Factores de Tiempo , Trasplante Autólogo/efectos adversos

RESUMEN

Following appropriate antigen-specific stimulation, CD4(+) and CD8(+) T lymphocytes rapidly express cytokines. Based on this stimulation-induced cytokine secretion and using cell surface affinity matrix technology we have developed a new method that permits specific, rapid and efficient detection, isolation and characterization of live antigen-specific CD4(+) and CD8(+) T lymphocytes. The power of this technique is demonstrated here for HLA-A0201-restricted influenza matrix protein peptide 58-66-specific CD8(+) cytotoxic T lymphocytes, influenza A virus- and recombinant tetanus toxin C fragment-specific Th1 cells and tetanus toxoid-specific Th2 cells.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Presentación de Antígeno , Antígenos/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Humanos , Separación Inmunomagnética/métodos

RESUMEN

Recent studies in mice have indicated that the long-lasting specific antibody responses seen after vaccination are probably due to the existence of long-lived plasma cells. Therefore, because the maintenance of humoral immunity does not necessarily reflect continuous restimulation of long-lived memory B cells, the question arises as to what degree antibody immunity, as determined by measuring serum immunoglobulin titers against a particular antigen, and memory B cell immunity, as determined by counting circulating memory B cells with specificity for that same antigen, correlate. Here, using a new assay combining two-step immunomagnetic enrichment with multiparameter flow cytometry to detect, enumerate and characterize antigen-specific memory B cells, we show for tetanus toxin C-fragment in blood of normal tetanus toxoid vaccinized donors, and for wasp venom phospholipase A1B in blood of wasp venom-allergic donors undergoing an immune therapy with wasp venom, that there is no statistically significant linear correlation between the frequencies of circulating antigen-specific IgG-bearing memory B cells and the serum titers of antigen-specific IgG. This lack of a statistically significant linear correlation is in accordance with the idea that B memory cells and plasma cells represent independently controlled forms of immunological memory.

Asunto(s)

Linfocitos B/inmunología , Inmunoglobulina G/sangre , Memoria Inmunológica , Humanos , Separación Inmunomagnética , Inmunofenotipificación , Lisofosfolipasa/inmunología , Fosfolipasas A/inmunología , Toxina Tetánica/inmunología

RESUMEN

Commitment of Th lymphocytes to the Th1 phenotype, as characterized by the expression of the major proinflammatory cytokine IFN-gamma, may be critically involved in the establishment of chronic inflammation and inflammatory autoimmune disease. To date, it has been shown that in IL-12-stimulated murine Th cell lines containing a major fraction of Th1 cells, Th2 cells can be induced by IL-4 until about 2 wk after initial activation, but not later. Here we analyze, based on the magnetic isolation of viable Th1 cells according to their specific expression of IFN-gamma, the cytokine commitment of individual Th1 cells. After activation of naive Th cells with Ag and IL-12 for up to 5 wk, isolated IFN-gamma-producing cells were restimulated with Ag and IL-4. Within the first 3 to 4 wk of IL-12 stimulation, some IFN-gamma+ cells stopped expression of IFN-gamma when restimulated with IL-4. However, within only 1 to 2 wk of IL-12 stimulation, few IFN-gamma+ cells could be converted to produce IL-4. Others continued to express IFN-gamma and thus were already committed to a proinflammatory, Th1-like phenotype. Surprisingly, within 3 wk of IL-12 stimulation, many of the IFN-gamma-producing cells responded to IL-4 restimulation by expression of IL-10, but neither IFN-gamma nor IL-4, i.e., by conversion to a suppressive, anti-inflammatory phenotype.

Asunto(s)

Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-12/farmacología , Interleucina-4/biosíntesis , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Células TH1/metabolismo , Animales , Antígenos CD4/análisis , Diferenciación Celular/inmunología , Polaridad Celular/inmunología , Separación Celular , Citometría de Flujo , Selectina L/análisis , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células TH1/inmunología

RESUMEN

Disseminated epithelial tumor cells have been detected in the bone marrow and blood of cancer patients by means of immunocytochemical or immunofluorescent staining of cytocentrifuge slides, multiparameter flow cytometry, and reverse transcriptase-polymerase chain reaction. However, it is hardly possible using such methods to detect tumor cells at a frequency below 10(-6). To increase the sensitivity of these detection techniques we have developed a new technology for the enrichment of disseminated epithelial tumor cells from hematopoietic cell samples by high-gradient magnetic cell sorting (MACS). Cells are permeabilized and fixed and carcinoma cells are magnetically labeled specifically with an anti-cytokeratin 8 monoclonal antibody (mAb) directly conjugated to superparamagnetic microbeads. Magnetically labeled cells are enriched on high-gradient magnetic columns. Tumor cells are detected in the enriched cell fraction by flow cytometry, fluorescence microscopy, or immunocytochemisty. In this study we demonstrated the method using a model system in which five to 5,000 cells from a breast cancer cell line were seeded into blood cell samples from a healthy donor containing 1.2 x 10(8) leukocytes. Tumor cells were 10,477+/-4242 (n=25)-fold magnetically enriched, and 57.7%+/-16.9% (n=33) of the initially seeded tumor cells were recovered. Applying the method to 20-40 mL blood samples from patients with advanced carcinomas of the breast, prostate, colon, rectum, or lung, we were able to detect between one and 6.8 x 10(4) cytokeratin-expressing tumor cells in 21 of 34 patients. This corresponds to frequencies of tumor cells between 6.8 x 10(-9) and 1.1 x 10(-3) among nucleated cells in the original sample. Enriched tumor cells were further analyzed for expression of tissue-specific and prognostic markers such as breast mucin glycoproteins, erbB2, and CD44v6 for additional characterization and to confirm their tumor origin. The technique described could become a valuable tool for the quantification and molecular characterization of metastatic carcinoma cells in hematopoietic tissue, and may ultimately prove useful in the diagnosis, prognosis, and monitoring of patients with carcinoma.

Asunto(s)

Separación Celular/métodos , Técnicas Inmunológicas , Adulto , Anciano , Neoplasias de la Mama/patología , Carcinoma/patología , Neoplasias Colorrectales/patología , Células Epiteliales/citología , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Inmunohistoquímica , Queratinas/inmunología , Neoplasias Pulmonares/patología , Magnetismo , Masculino , Persona de Mediana Edad , Mucinas/análisis , Metástasis de la Neoplasia , Neoplasias de la Próstata/patología , Receptor ErbB-2/metabolismo , Células Tumorales Cultivadas

RESUMEN

It has been considered before that human naive and memory/effector CD4+ T-cells cannot be subdivided solely according to the differential expression of CD45 isoforms. By the lack of expression of CD31 we have identified a subset of CD4+ CD45RA+ CD31- cells which show distinct features of antigen-experienced Th1 cells. Short term stimulation of highly purified human peripheral blood CD4+ T-cells with PMA/ionomycin, followed by the cytometric analysis of intracellular cytokines, showed that a minor subpopulation of CD4+ CD45RA+ CD45RO- cells is able to produce interferon-gamma (IFN-gamma) rapidly, a characteristic of antigen-experienced Th1 cells. Whereas among CD45RA+ CD4+ T-cells both CD31+ and CD31- subsets produce interleukin-2 (IL-2) upon PMA/ionomycin stimulation, only the CD31- subpopulation is able to produce IFN-gamma. Thus, our phenotypic and functional characterization of CD45RA+ CD45RO- Th cells shows that CD45RA+ CD45RO- cells do not represent a homogeneous population of antigen-unexperienced, naive T-cells. We speculate that a certain subset of human CD4+, CD45RO+ memory T-cells reverts to expression of the CD45RA isoform, and that this subset can be identified by the lack of CD31 expression.

Asunto(s)

Interferón gamma/biosíntesis , Antígenos Comunes de Leucocito/biosíntesis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Células TH1/inmunología , Animales , Humanos , Memoria Inmunológica/inmunología , Ionomicina/farmacología , Ratones , Ratones Endogámicos BALB C , Mitógenos/farmacología , Fitohemaglutininas/farmacología , Células TH1/efectos de los fármacos

RESUMEN

The Amgen Cell Selection Device (ACSD) is a fully automated system based on the research scale magnetic-activated cell separation (MACS) system (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) for the selection of CD34+ cells. Leukapheresis products (LP) (n = 30) from normal donors mobilized with recombinant human granulocyte colony-stimulating factor (rhG-CSF) were selected with the ACSD to evaluate the performance of this system. The starting LP contained a median of 0.51% CD34+ cells (range 0.21%-1.54%) and a median WBC count of 3.0 x 10(10) (range 1-4.7 x 10(10) cells). After selection on the ACSD a mean purity of 91.5% +/- 0.6% CD34+ cells was obtained, with a median purity of 95.5% CD34+ cells. A median of 98 x 10(6) total CD34+ cells were recovered postselection, with a range of 31-323 x 10(6) cells collected from the LP. This represented a mean recovery of 81.7% +/- 6% of CD34+ cells and a median of 78% compared with starting CD34+ cell numbers in the LP. FACS analysis of the selected products demonstrated a 4-5 log depletion of T cell subsets, including CD3, CD4, CD8, and CD56 subsets. These data demonstrate the high performance obtained with the ACSD resulting in a final product of greater than 90% purity of CD34+ cells. CD34+ cells selected with the ACSD represent an ideal product for clinical applications, such as tumor cell purging, T cell depletion for allogeneic transplant, ex vivo expansion, and gene therapy.

Asunto(s)

Antígenos CD34 , Antígenos CD , Separación Celular/instrumentación , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/citología , Biomarcadores , Separación Celular/métodos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Diseño de Equipo , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Hematopoyesis/efectos de los fármacos , Humanos , Leucaféresis/instrumentación , Leucaféresis/métodos , Depleción Linfocítica , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Linfocitos T

RESUMEN

The hematopoietic system consists of a network of stem and progenitor cells of varying degrees of maturity interacting with other cells that act in supportive and regulatory capacities. Many cell types have been identified by their in vivo or ex vivo growth potential, i.e., colony assays, or by physical characteristics like cell-surface markers which can be identified by monoclonal antibodies. One of the most studied antibody markers is the CD34 antigen which is present on the most primitive hematopoietic progenitor cells.

RESUMEN

Interferon (IFN)-gamma is a potent immunoregulatory protein secreted by CD4+ and CD8+ T cells and by natural killer cells. Here, we show that IFN-gamma is specifically displayed at a low concentration on the cell surface of those activated T cells from mouse and man which express IFN-gamma. It is transiently expressed on the cell surface with kinetics similar to those of intracellular IFN-gamma expression. Detectable surface IFN-gamma is not expressed by activated T helper (Th) cells producing other cytokines but which do not express IFN-gamma. Thus, surface IFN-gamma is the first available marker for live T lymphocytes expressing IFN-gamma, e.g. Th1 cells.

Asunto(s)

Interferón gamma/análisis , Interferón gamma/biosíntesis , Proteínas de la Membrana/biosíntesis , Linfocitos T/metabolismo , Animales , Humanos , Interleucina-4/biosíntesis , Interleucina-5/biosíntesis , Líquido Intracelular/química , Activación de Linfocitos , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Linfocitos T/clasificación

RESUMEN

BACKGROUND: Immunofluorescence and immunomagnetism are important technologies for analysis and sorting of cells according to specific marker molecules. Due to the limited sensitivity at least several thousands of antigens per cell are required for optical detection. Molecules expressed at low copy numbers cannot be analysed, although they may be of considerable functional importance. OBJECTIVES: Development of a magnetic and fluorescent staining reagent for analysis and sorting of cells according to antigens expressed at low number. To this end, uniformly sized, antibody-conjugated liposomes loaded with large amounts of dye molecules and small magnetic particles were generated. STUDY DESIGN: A method for the preparation of homogeneously sized large unilamellar liposomes which contain carboxyfluorescein, magnetic particles and surface-bound antibodies had to be developed. These liposomes were then tested for their ability to enhance immunofluorescence compared to conventional staining in a model system and by staining of CD25 on resting B and T cells. RESULTS AND CONCLUSION: Large unilamellar liposomes, homogeneous in size and loaded with fluorescein and magnetic beads can be prepared by combining membrane extrusion and magnetic filtration. Hapten-specific antibodies conjugated to their surface make them a universal tool for immunofluorescence. With such liposomes, intensity of fluorescent staining can be increased 100-1000-fold without increased background fluorescence, compared to conventional fluorochrome-conjugated antibodies. Due to the simultaneous magnetic labelling, stained cells can easily be isolated by MACS. The magnetofluorescent liposomes proved to be useful for improvement of sensitivity of detection and physical separation in general and to visualize and sort cells according to antigens expressed at low levels. The high affinity IL2 receptor CD25 is expressed in low copy number on a significant fraction of resting B and T lymphocytes in human peripheral blood, as can be shown exclusively by the magnetofluorescent liposomes.

Asunto(s)

Técnica del Anticuerpo Fluorescente , Liposomas/inmunología , Magnetismo , Anticuerpos/sangre , Antígenos CD19/inmunología , Haptenos/inmunología , Humanos , Receptores de Interleucina-2/sangre , Sensibilidad y Especificidad

RESUMEN

We have developed a technology for analysis and sorting of live cells according to secreted molecules. An artificial affinity matrix, specific for the secreted product of interest, is created on the cell surface, and the cells are allowed to secrete for a defined time period. The secreted molecules bind to the affinity matrix on the secreting cell and are subsequently labeled with specific fluorescent or magnetic staining reagents for cytometric analysis and cell sorting. Crossfeeding of the secreted products to other cells is prevented by decreasing the permeability of the incubation medium. This approach will have a wide range of applications in biotechnology and biomedical research. Here, we describe analysis and sorting of hybridoma cells, according to secreted antibodies, and of activated T lymphocytes, according to secreted cytokines.

Asunto(s)

Separación Celular/métodos , Hibridomas/citología , Inmunoglobulina M/biosíntesis , Linfocitos T/citología , Animales , Anticuerpos Monoclonales , Agregación Celular , Cromatografía de Afinidad/métodos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo/métodos , Hibridomas/inmunología , Inmunoglobulina M/análisis , Cadenas kappa de Inmunoglobulina/análisis , Interferón gamma/análisis , Interferón gamma/biosíntesis , Interleucina-2/análisis , Interleucina-2/biosíntesis , Cinética , Activación de Linfocitos , Ratones , Sensibilidad y Especificidad , Linfocitos T/inmunología

Asunto(s)

Citometría de Flujo/métodos , Separación Celular , Técnica del Anticuerpo Fluorescente , Humanos

RESUMEN

For simple and effective isolation of fetal cells from peripheral maternal blood, we combined depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell separation (MACS). First CD45+ and CD14+ cells were depleted from maternal peripheral blood mononuclear cells by MACS. From the depleted fraction, CD71+ erythroid cells were enriched up to 80 per cent by MACS. This double-MACS' procedure yielded an average depletion rate of 780-fold and an average enrichment rate of 500-fold, with approximate recovery rates of 40-55 per cent. For paternity testing, cells from unseparated blood and the various fractions were analysed for polymorphism of the HLA-DQ-A1 locus and D1S80 locus by the polymerase chain reaction (PCR). In CD45-/CD71+ sorted cells from maternal blood, but not in unfractionated cells from maternal blood or CD45-/CD14- cells, paternal alleles could be detected. In the CD45-/CD71+ fraction, the relative frequency of paternal alleles compared with maternal alleles ranged from 1 in 20 to 1 in 200 (determined by titration and depending on the quality of separation and biological variation). In 7 out of 11 cases, between weeks 12 and 25 of gestation, we could identify paternal alleles by PCR, either HLA-DQ-A1 or D1S80. This double-MACS procedure is simple, fast, efficient, and reliable for non-invasive prenatal diagnosis.

Asunto(s)

Separación Celular/métodos , Sangre Fetal/citología , Magnetismo , Reacción en Cadena de la Polimerasa , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos B/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Secuencia de Bases , ADN/química , ADN/aislamiento & purificación , Femenino , Sangre Fetal/inmunología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Genotipo , Antígenos HLA-DQ/genética , Cadenas alfa de HLA-DQ , Humanos , Inmunofenotipificación , Antígenos Comunes de Leucocito/análisis , Receptores de Lipopolisacáridos , Masculino , Datos de Secuencia Molecular , Polimorfismo Genético , Embarazo , Receptores de Transferrina

Asunto(s)

Antígenos CD , Separación Celular/métodos , Citometría de Flujo/métodos , Magnetismo , Animales , Anticuerpos , Separación Celular/instrumentación , Citometría de Flujo/instrumentación , Sistema Hematopoyético/citología , Sistema Hematopoyético/inmunología , Humanos , Leucosialina , Monocitos/citología , Sialoglicoproteínas/metabolismo , Coloración y Etiquetado/métodos , Esterilización , Linfocitos T/citología

RESUMEN

Human lymphocytes were labeled with biotinylated anti-lymphocyte-directed monoclonal antibodies, to which streptavidin and subsequently biotinylated dextran-magnetite particles were coupled. This labeling resulted in a strong and selective negative contrast enhancement of lymphocyte suspensions at 2.0 T, caused predominantly by the specific increase of R2 with a small but significant specific increase of R1. The R1 was found to decrease with increasing field strength. The immunolabeling procedure described here may be used for the selective signal depletion of target cells in MR imaging.

Asunto(s)

Anticuerpos Monoclonales , Medios de Contraste , Dextranos , Hierro , Linfocitos/patología , Imagen por Resonancia Magnética/métodos , Óxidos , Proteínas Bacterianas , Óxido Ferrosoférrico , Humanos , Técnicas In Vitro , Estreptavidina

RESUMEN

A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was developed and tested. Cells stained sequentially with biotinylated antibodies, fluorochrome-conjugated avidin, and superparamagnetic biotinylated-microparticles (about 100 nm diameter) are separated on high gradient magnetic (HGM) columns. Unlabelled cells pass through the column, while labelled cells are retained. The retained cells can be easily eluted. More than 10(9) cells can be processed in about 15 min. Enrichment rates of more than 100-fold and depletion rates of several 1,000-fold can be achieved. The simultaneous tagging of cells with fluorochromes and very small, invisible magnetic beads makes this system an ideal complement to flow cytometry. Light scatter and fluorescent parameters of the cells are not changed by the bound particles. Magnetically separated cells can be analysed by fluorescence microscopy or flow cytometry or sorted by fluorescence-activated cell sorting without further treatment. Magnetic tagging and separation does not affect cell viability and proliferation.

Asunto(s)

Separación Celular/métodos , Magnetismo , Animales , Biotina , Separación Celular/instrumentación , Citometría de Flujo , Ratones , Microesferas , Bazo/citología , Coloración y Etiquetado

RESUMEN

A method is described for the efficient purification of human B lymphocytes from peripheral blood by magnetic separation. Biotinylated, superparamagnetic particles were coupled to target cells by fluorescein isothiocyanate conjugated avidin and biotinylated monoclonal antibodies directed against cell surface antigens. This combination permitted flow cytometric control of the magnetic separation. Ficoll-Paque-separated peripheral blood mononuclear cells were first eliminated from monocytes by leucine-methyl ester treatment. B cells were enriched to 97% after magnetic depletion of CD3-positive T cells and magnetic enrichment of CD20-positive B cells. The separated B cells could be induced to proliferation and antibody production by various in vitro stimuli.

Asunto(s)

Células Productoras de Anticuerpos/citología , Antígenos de Diferenciación de Linfocitos B/análisis , Linfocitos B/citología , Separación Celular/métodos , Células Productoras de Anticuerpos/inmunología , Antígenos CD20 , Linfocitos B/inmunología , Diferenciación Celular , División Celular , Humanos , Técnicas In Vitro , Magnetismo , Monocitos/citología , Linfocitos T/citología