Low‐cost enumeration of CD4<sup>+</sup> T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries

Bold, Angelika; Wurth, Rainer; Keller, Thomas; Trahorsch, Ulrike; Voigt, Peter; Schubert, Stefan; Sack, Ulrich

doi:10.1002/cyto.a.20494

Cited by 3 publications

(3 citation statements)

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“…Nowadays, negative selection methods are preferred for the isolation of rare cells of interest, e.g. erythrocyte rosetting methods by tetramer antibodies [5,6] or magnetic removal of undesired cells [15]. However, this isolation method requires a density gradient centrifugation step too.…”

Section: Discussionmentioning

confidence: 99%

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An Innovative Cascade System for Simultaneous Separation of Multiple Cell Types

et al. 2013

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Isolation of different cell types from one sample by fluorescence activated cell sorting is standard but expensive and time consuming. Magnetic separation is more cost effective and faster by but requires substantial effort. An innovative pluriBead-cascade cell isolation system (pluriSelect GmbH, Leipzig, Germany) simultaneously separates two or more different cell types. It is based on antibody-mediated binding of cells to beads of different size and their isolation with sieves of different mesh-size. For the first time, we validated the pluriSelect system for simultaneous separation of CD4+- and CD8+-cells from human EDTA-blood samples. Results were compared with those obtained by magnetic activated cell sorting (MACS; two steps -first isolation of CD4+, then restaining of the residual cell suspension with anti-human CD8+ MACS antibody followed by the second isolation). pluriSelect separation was done in whole blood, MACS separation on density gradient isolated mononuclear cells. Isolated and residual cells were immunophenotyped by 7-color 9-marker panel (CD3; CD16/56; CD4; CD8; CD14; CD19; CD45; HLA-DR) using flow cytometry. Cell count, purity, yield and viability (7-AAD exclusion) were determined. There were no significant differences between both systems regarding purity (MACS (median[range]: 92.4% [91.5-94.9] vs. pluriSelect 95% [94.9-96.8])) of CD4+ cells, however CD8+ isolation showed lower purity by MACS (74.8% [67.6-77.9], pluriSelect 89.9% [89.0-95.7]). Yield was not significantly different for CD4 (MACS 58.5% [54.1-67.5], pluriSelect 67.9% [56.8-69.8]) and for CD8 (MACS 57.2% [41.3-72.0], pluriSelect 67.2% [60.0-78.5]). Viability was slightly higher with MACS for CD4+ (98.4% [97.8-99.0], pluriSelect 94.1% [92.1-95.2]) and for CD8+-cells (98.8% [98.3-99.1], pluriSelect 86.7% [84.2-89.9]). pluriSelect separation was substantially faster than MACS (1h vs. 2.5h) and no pre-enrichment steps were necessary. In conclusion, pluriSelect is a fast, simple and gentle system for efficient simultaneous separation of two and more cell subpopulation directly from whole blood and provides a simple alternative to magnetic separation.

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Section: Discussionmentioning

confidence: 99%

“…However, they have limited specificity, thus specific cell types cannot be isolated. High cell specificity can be obtained by erythrocyte rosetting [5,6] in combination with density gradient centrifugation.…”

Section: Introductionmentioning

confidence: 99%

An Innovative Cascade System for Simultaneous Separation of Multiple Cell Types

et al. 2013

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“…The treatment of HIV‐infection by highly active antiretroviral therapy (HAART) requires regular monitoring of different blood parameters. Beside CD4 T‐cell number (1–3), which is described as the best surrogate marker (4) or for children, the more significant CD4% value (1, 5, 6) blood serum virus concentration (viral load) represents the most meaningful parameter to evaluate treatment success and to decide about the initiation of HAART (7). Virus detection is also important for evaluation of prevention of mother‐to‐child transmission, chemoprophylaxis, and for testing newborns and mother's milk (8) or in persons with atypical course of disease (9).…”

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confidence: 99%

A new affordable flow cytometry based method to measure HIV‐1 viral load

et al. 2008

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Most commercially available assays for diagnosis of HIV infection have shown shortcomings in the detection and quantification of rare genotypes of the virus. Most of the assays do not detect subtype O (outlier) and/or N (nonmajor, nonoutlier) or new circulating recombinant forms (CRFs), which are becoming more important in sub-Saharan Africa. Furthermore, the commonly available tests require costly measuring devices and expensive test kits, which are not easily affordable for developing countries. This study was designed to explore solutions to the problem of viral load assays in developing countries. Two forward primers, digoxygenin (DIG) and dinitrophenol (DNP) labeled, and one biotin (BIO) labeled reverse primer were used to amplify both, the HIV-1-5 0 LTR (long terminal repeat) region and an internal standard sequence. The two polymerase chain reaction (PCR)-products were captured by anti-DIG and anti-DNP antibody coated microparticles. Flow cytometric analyses were carried out after labeling with streptavidin-R-phycoerythrine. The primer system used recognized all HIV-1 subtypes.

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Microfluidic impedance‐based flow cytometry

Berardino²,

et al. 2010

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Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance-based characterization. Such systems are being developed to provide simple, low-cost, label-free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro-organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high-content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance-based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip-based cytometry can make a large impact. ' 2010International Society for Advancement of Cytometry Key terms microfluidics; impedance characterization; label free; single cell analysis; hydrodynamic focusing; sorting MICROFLUIDIC flow cytometers can bring many advantages to the field of flow cytometry (FCM). Compared to typical flow cytometry channel sizes, the miniaturized dimensions permit microfluidic systems to analyze single cells, to identify cellular variability in gene expression, or drug response within a cell population. Chipbased cytometers can have lower size and costs than conventional benchtop instruments, and may be portable. Today, the developmental aim for microfluidic systems is to reach the same sensitivity and capability for multiparametric analyses as delivered by conventional flow cytometers. Many efforts have been made to improve existing devices and to create new miniaturized high-end instruments. Microfluidic chips can incorporate on-chip cell preparation, cell culture, lysis, and modules for optical, electrophoretic, or genomic analysis (1). They are also suitable for analysis of cells in suspension as well as adherent cells.Miniaturized cytometry devices will have high impact in the development of point-of-care devices in developing countries. Accurate CD4 1 T-cell counts are used to monitor human immunodeficiency virus (HIV)-infected patients, and various thresholds of the number of CD4 1 T lymphocytes per ll of whole blood are used to start antiretroviral therapy (2-5). A simple, single-purpose CD4 cell counting device, which does not require standard laboratory equipment or trained laboratory personnel, could help some of the 33 million HIV-infected people worldwide monitor the stage of infection (6)(7)(8). In this application, increased analysis throughput is a secondary concern compared to increased sensitivity and specificity. Portable, miniatu...

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Low‐cost enumeration of CD4⁺ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries

Cited by 3 publications

References 25 publications

An Innovative Cascade System for Simultaneous Separation of Multiple Cell Types

An Innovative Cascade System for Simultaneous Separation of Multiple Cell Types

A new affordable flow cytometry based method to measure HIV‐1 viral load

Microfluidic impedance‐based flow cytometry

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Low‐cost enumeration of CD4+ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries

Cited by 3 publications

References 25 publications

An Innovative Cascade System for Simultaneous Separation of Multiple Cell Types

An Innovative Cascade System for Simultaneous Separation of Multiple Cell Types

A new affordable flow cytometry based method to measure HIV‐1 viral load

Microfluidic impedance‐based flow cytometry

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Product

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Low‐cost enumeration of CD4⁺ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries