Christian Breukers

A single platform image cytometer for resource-poor settings to monitor disease progression in HIV infection

For resource‐poor countries, affordable methods are required for enumeration of CD4+ T lymphocytes of HIV‐positive patients. For infants, additional determination of CD4/CD8 ratio is needed.

CD4+ T lymphocytes enumeration by an easy-to-use single platform image cytometer for HIV monitoring in resource-constrained settings

HIV monitoring in resource‐constrained settings demands affordable and reliable CD4+ T lymphocytes enumeration methods. We developed a simple single platform image cytometer (SP ICM), which is a dedicated volumetric CD4+ T lymphocytes enumeration system that uses immunomagnetic and immunofluorescent technologies. The instrument was designed to be a low‐cost, yet reliable and robust one. In this article we test the instrument and the immunochemical procedures used on blood from HIV negative and HIV positive patients.

CD4 and CD8 Enumeration for HIV Monitoring in Resource-Constrained Settings

We developed a volumetric single platform image cytometer (SP ICM) that is dedicated to count CD4+ and CD8+ T lymphocytes for HIV monitoring in resource‐constrained settings. The instrument was designed to be low‐cost, yet reliable, easy‐to‐use, and robust.

Clinical evaluation of a simple image cytometer for CD4 enumeration on HIV‐infected patients

Affordable, easy‐to‐use, and reliable CD4+ T lymphocyte enumeration systems are needed in resource‐constrained settings to monitor HIV.

DNA Detection by Flow Cytometry using PNA-Modified Metal-Organic Framework Particles

Abstract A DNA‐sensing platform is developed by exploiting the easy surface functionalization of metal–organic framework (MOF) particles and their highly parallelized fluorescence detection by flow cytometry. Two strategies were employed to functionalize the surface of MIL‐88A, using either covalent or non‐covalent interactions, resulting in alkyne‐modified and biotin‐modified MIL‐88A, respectively. Covalent surface coupling of an azide‐dye and the alkyne–MIL‐88A was achieved by means of a click reaction. Non‐covalent streptavidin–biotin interactions were employed to link biotin–PNA to biotin–MIL‐88A particles mediated by streptavidin. Characterization by confocal imaging and flow cytometry demonstrated that DNA can be bound selectively to the MOF surface. Flow cytometry provided quantitative data of the interaction with DNA. Making use of the large numbers of particles that can be simultaneously processed by flow cytometry, this MOF platform was able to discriminate between fully complementary, single‐base mismatched, and randomized DNA targets.

Temperature-Switch Cytometry—Releasing Antibody on Demand from Inkjet-Printed Gelatin for On-Chip Immunostaining

Complete integration of all sample preparation steps in a microfluidic device greatly benefits point-of-care diagnostics. In the most simplistic approach, reagents are integrated in a microfluidic chip and dissolved upon filling with a sample fluid by capillary force. This will generally result in at least partial reagent wash-off during sample inflow. However, many applications, such as immunostaining-based cytometry, strongly rely on a homogeneous reagent distribution across the chip. The concept of initially preventing release (during inflow), followed by a triggered instantaneous and complete release on demand (after filling is completed) represents an elegant and simple solution to this problem. Here, we realize this controlled release by embedding antibodies in a gelatin layer integrated in a microfluidic chamber. The gelatin/antibody layer is deposited by inkjet printing. Maturation of this layer during the course of several weeks, due to the ongoing physical cross-linking of gelatin, slows down the antibody release, thereby reducing antibody wash-off during inflow, and consequently helping to meet the requirement for a homogeneous antibody distribution in the filled chamber. After inflow, complete antibody release is obtained by heating the gelatin layer above its sol-gel transition temperature, which causes the rapid dissolution of the entire gelatin/antibody layer at moderate temperatures. We demonstrate uniform and complete on-chip immunostaining of CD4 positive (CD4+) T-lymphocytes in whole blood samples, which is critical for accurate cell counts. The sample preparation is realized entirely on-chip, by applying temperature-switched antibody release from matured gelatin/antibody layers.

All-printed cell counting chambers with on-chip sample preparation for point-of-care CD4 counting

We demonstrate the fabrication of fully printed microfluidic CD4 counting chips with complete on-chip sample preparation and their applicability as a CD4 counting assay using samples from healthy donors and HIV-infected patients. CD4 counting in low-income and resource-limited point-of-care settings is only practical and affordable, if disposable tests can be fabricated at very low cost and all manual sample preparation is avoided, while operation as well as quantification is fully automated and independent of the skills of the operator. Here, we show the successful use of (inkjet) printing methods both to fabricate microfluidic cell counting chambers with controlled heights, and to deposit hydrogel layers with embedded fluorophore-labeled antibodies for on-chip sample preparation and reagent storage. The maturation process of gelatin after deposition prevents antibody wash-off during blood inflow very well, while temperature-controlled dissolution of the matrix ensures complete antibody release for immunostaining after the inflow has stopped. The prevention of antibody wash-off together with the subsequent complete antibody release guarantees a homogeneous fluorescence background, making rapid and accurate CD4 counting possible. We show the successful application of our fully printed CD4 counting chips on samples from healthy donors as well as from HIV-infected patients and find an excellent agreement between results from our method and from the gold standard, flow cytometry, in both cases.