Concentration of white blood cells from whole blood by dual centrifugo-pneumatic siphoning with density gradient medium

Due to the pervasiveness of HIV infections in developing countries there exists a need for a low-cost, user-friendly point-of-care device which can be used to monitor the concentration of T-lymphocytes in the patient’s blood expressing the CD4+ epitope. As a first step towards developing a microfluidic “lab-on-a-disc” platform with this aim we present the concentration of white blood cells from whole blood using a density medium in conjunction with centrifugo-pneumatic siphon valves [1]. Two such valves are actuated simultaneously, removing the bulk of plasma through the upper valve and the bulk of WBCs through the lower valve while leaving the vast majority of red blood cells in the centrifugal chamber

A 3d-printed optical reader for cost-efficient enumeration of cd4 cells for point-of-care diagnostics of hiv in resource-poor settings

Here we present a low cost, 3D printed optical reader which is compatible with a disposable, finger actuated chip which can be used for rapid CD4+ enumeration. This portable and widely autonomous device provides the next step in a workflow which can meet the WHO ASSURED requirements for assessment of HIV status in resource-poor settings. The reader is composed of 3D printed parts where the microfluidic chip can be moved along the optical axis of the objective lens for focusing using a screw adapted from a masonry bolt. Performance of the reader shows good agreement with measurements made using a conventional inverted microscope

CD4 cell isolation from blood using finger-actuated on-chip magnetophoresis for rapid HIV/AIDS diagnostics

With timely diagnosis and correct treatment, people living with HIV/AIDS can consider the disease as a chronic rather than a terminal illness. Still, in regions were HIV is endemic, rapid diagnosis is a challenge due to the complexity of the instrumentation required, the poor infrastructure in these countries, as well as the technical expertise required to carry out the diagnosis. This paper presents a microfluidic chip based approach allowing semi-quantitative CD4+ cell coun¬ting on a cheap, rapid, highly portable and instrumentation-free Point-of-Care HIV diagnostic device. Flow is driven by finger-pressing a flexible reservoir, and the target cells are immobilized through magnetophoresis. The fluidic test completes within ca. 30 seconds of sample application to the chip

Isolation of white blood cells using paper-triggered dissolvable-film valves on a centrifugal platform

The inherent centrifugation capability of the so-called ‘Lab-on-a-Disc’ (LoaD) platforms is widely used for blood processing during sample preparation. Here we introduce a valving technique which ena-bles rotational control of paper wetting to actuate dissolvable film (DF) valves. This mechanism is applied to the separation of whole blood into its chief constituents; plasma, leukocytes and erythrocytes

A hybrid microfluidic platform for cell-based assays via diffusive and convective trans-membrane perfusion

We present a novel 3D hybrid assembly of a polymer microfluidic chip with polycarbonate track-etched membrane (PCTEM) enabling membrane-supported cell culture. Two chip designs have been developed to establish either diffusive or convective reagent delivery using the integrated PCTEM. While it is well suited to a range of cell-based assays, we specifically employ this platform for the screening of a common antitumor chemotoxic agent (mitomycin C – MMC) on the HL60 myeloid leukemia cell line. The toxic activity of MMC is based on the generation of severe DNA damage in the cells. Using either mode of operation, the HL60 cells were cultured on-chip before, during, and after exposure to MMC at concentrations ranging from 0 to 50 lM. Cell viability was analysed off-chip by the trypan blue dye exclusion assay. The results of the on-chip viability assay were found to be consistent with those obtained off-chip and indicated ca. 40% cell survival at MMC concentration of 50 lM. The catalogue of capabilities of the here described cell assay platform comprises of (i) the culturing of cells either under shear-free conditions or under induced through-membrane flows, (ii) the tight time control of the reagent exposure, (iii) the straightforward assembly of devices, (iv) the flexibility on the choice of the membrane, and, prospectively, (v) the amenability for large-scale parallelization

Cluster size distribution of cancer cells in blood using stopped-flow centrifugation along scale-matched gaps of a radially inclined rail

There is increasing evidence that, in addition to their presence, the propensity of circulating tumour cells to form multi-cellular clusters bears significant information about both cellular resistance to chemotherapy and overall prognosis. We present a novel two-stage, stopped-flow, continuous centrifugal sedimentation strategy to measure the size distributions of events (defined here as cells or clusters thereof) in a blood sample. After off-chip removal of red blood cells, healthy white blood cells are sequestered by negative-immunocapture. The purified events are then resolved along a radially inclined rail featuring a series of gaps with increasing width, each connected to a designated outer collection bin. The isolation of candidate events independent of target-specific epitopes is successfully demonstrated for HL60 (EpCAM positive) and sk-mel28 (EpCAM negative) cells using identical protocols and reagents. The propensity to form clusters was quantified for a number of cell lines, showing a negligible, moderate or elevated tendency towards cluster formation. We show that the occupancy distribution of the collection bins closely correlates with the range of cluster sizes intrinsic to the specific cell line

Laboratory unit operations on centrifugal lab-on-a-disc cartridges using dissolvable-film enabled flow control

The suitability of the centrifugal “lab-on-a-disc” (LoaD) platform for point-of-use / point-of-care deployment, where ruggedness, portability, rapid turn-around times and ease-of-use are key, has resulted in increased interest from the academic community over the last decade. Recently, a new, so-called event-triggered valving paradigm was introduced which circumvented a number of the limitations of commonly used, rotationally actuated valves and complex instrument-controlled valving. In the dissolvable-film based event-triggered approach, it is the liquid movement about a disc which actuates a valve; thus enabling the concatenating of a number of liquid handling operations into an automated cascade. Functioning broadly independent of spin rate, the number of discrete valving operations is only limited by the available disc real-estate. In this work we present this valving paradigm to control a network of discrete Laboratory Unit Operations (LUOs) which, with experienced design, can be integrated together to implement complex fluidic assays. We describe how these valves can be configured, using Boolean-like network relationships, to implement LUOs such as sequential washing steps. We also describe how these valves can be configured to enable metering, mixing and selective routing of liquid flows. Finally, we describe how these valves can be configured to provide accurate temporal control of LUOs; thus providing an entire suite of process control technology which can be used to enable many bio-assays

Electromagnetic Trapping of Cancer Cells on an Array of Thin-Film Permalloy Microfeatures for Single Cell Analysis

Isolation of individual cells from a bulk sample is vital in cell based diagnostics. Previous work has established the ability to arrange target cells into ordered arrays, using physical barriers such as micropillars and cups [1]. We here present a magnetic separation device that allows the tagged cells of interest to align into a highly ordered array where they are selected based on both immuno-markers and size. But, unlike the physical barrier methods, our device is able to trap the cells using an only 160-nm thin film of permalloy (80% Nickel, 20% Iron) micropatterned on the base of the chamber (Fig. 1). Maxima of the magnetic field arise in the vicinity of the sharp edges of these localized micro-spots (Fig. 2), thereby creating potential wells to localize paramagnetic bioparticles such as tagged cells or beads during analysis or successive washing steps (Fig. 3). Compared to a complex network of physical barriers, the trapped cells can simply be released by turning off the external magnet, allowing for further downstream analysis or removal to waste

Baking-powder driven centripetal pumping controlled by event-triggering of functional liquids

This paper reports radially inbound pumping by the event-triggered addition of water to on-board stored baking powder in combination with valving by an immiscible, high-specific weight liquid on a centrifugal microfluidic platform. This technology allows making efficient use of precious real estate near the center of rotation by enabling the placement of early sample preparation steps as well as reagent reservoirs at the spacious, high-field region on the perimeter of the disc-shaped rotor. This way the number of process steps and assays that can be integrated on these of this “Lab-on-a-Disc” (LoaD) cartridge can be significantly enhanced while maintaining minimum requirements on the intrinsically simple, spindle-motor based instrumentation

Staging the clinical status from blood of cancer patients by chip-based cell enumeration following targeted removal of normal cells

Even though an agreed phenotypic definition of circulating tumor cells (CTCs) remains elusive in the literature, many current detection technologies isolate candidate cells based on molecular recognition of cellular epitopes that may not accurately predict CTC load. Rather than using such an epitope specific “positive-capture” strategy, we present a chip-based, centrifugal microfluidic platform integrating “negative-capture” magnetophoretic removal of normal white blood cells (WBCs) from a sample and subsequent, array-based enumeration of individualized, (untagged) abnormal cells. We compared the numerical recovery of cells on the array with the status of the donor patient, showing that the chip can has the potential to indicate the oncogenic severity of the blood donor