Elsevier

Biosensors and Bioelectronics

Volume 67, 15 May 2015, Pages 350-355
Biosensors and Bioelectronics

Simple and rapid CD4 testing based on large-field imaging system composed of microcavity array and two-dimensional photosensor

https://doi.org/10.1016/j.bios.2014.08.051Get rights and content

Highlights

  • Simplified CD4 testing method based on large-field fluorescent imaging was developed.

  • Desk-top-sized device consists of light source, CMOS sensor and single-cell array.

  • Leukocyte recovery, staining, imaging and cell count were performed within a device.

  • Image of leukocytes was acquired in dual color by one-shot imaging (64 mm2) in 2 s.

  • CD4/CD8 ratio was correctly measured from a few microliters of whole blood.

Abstract

This study presents a novel method for CD4 testing based on one-shot large-field imaging. The large-field imaging system was fabricated by a microcavity array and a two-dimensional (2D) photosensor within the desk-top-sized instrument. The microcavity array was employed to separate leukocytes from whole blood based on differences in the size of leukocytes and other blood cells. The large-field imaging system with lower side irradiation enabled acquisition of cell signatures with high signal-to-noise ratio, because the metallic substrate of the microcavity array obstructed excessive excitation light. In this setting, dual-color imaging of CD4+ and CD8+ T cells was achieved within the entire image area (64 mm2) in 2 s. The practical performance of the large-field imaging system was demonstrated by determining the CD4/CD8 ratio in a few microliter of control whole blood as small as those obtained by a finger prick. The CD4/CD8 ratios measured using the large-field imaging system correlated well with those measured by microscopic analysis. These results indicate that our proposed system provides a simple and rapid CD4 testing for the application of HIV/AIDS treatment.

Introduction

Approximately 35 million people worldwide are living with human immunodeficiency virus (HIV), which causes acquired immune deficiency syndrome (AIDS) (McCune, 2001). Through the analysis of lymphocyte subsets based on cluster of differentiation (CD) antigen, CD4+ T cell count is the indicator for the initiation of antiretroviral therapy (ART) in patients diagnosed with HIV and for monitoring the therapeutic effect of the treatment (Boyle et al., 2012). There are several useful measurements to determine the levels of CD4+ T cells in blood (CD4 testing), including the number of CD4+ T cells per microliter of blood (absolute CD4 counts), the percentage of CD4+ T cells among all lymphocytes (CD4/lymphocyte), and the ratio of CD4+ T cells to CD8+ T cells (CD4/CD8 ratio) (O’Gorman and Zijenah, 2008).

Flow cytometry is commonly used in developed countries as a standard method for CD4 testing. However, it is not suitable for widespread use in resource-limited settings owing to its technical requirements, difficulty of operation, and lack of portability. Therefore, simplified CD4 counting technologies are needed to provide widespread treatment (Willyard, 2007, Yager et al., 2006, Zachariah et al., 2011). Recently, the Pima Analyzer (Alere Inc.), which is a microscopic image-based analyzer, has come onto the market. The performance of the Pima system has been evaluated in resource limited regions (Jani et al., 2011, Mtapuri-Zinyowera et al., 2010, Wade et al., 2013). The CD4 counts obtained by this method were found to be comparable to those obtained using flow cytometry. However, negatively-biased CD4 counts at high cell concentrations may limit the use of this system for long-term immunological monitoring of ART progression in patients (Manabe et al., 2012). At high concentrations, blood components may interfere with measurements. Therefore, leukocyte counting generally requires prior separation of leukocytes from whole blood and their enrichment in order to attain acceptable sensitivity.

Our research group has demonstrated a novel microfluidic device equipped with a size-controlled microcavity array for CD4 testing (Hosokawa et al., 2012, Hosokawa et al., 2013). The microcavity array can separate leukocytes from a few microliters of whole blood as small as those obtained by a finger prick based on differences in the size of leukocytes and other blood cells. Leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. This device enabled us to recover leukocytes efficiently from whole blood without pretreatment. However, microscopic scanning was required to observe the entire cell population. Alternatively, we have developed a large-field imaging system for high-throughput cell profiling using a two-dimensional (2D) photosensor (Saeki et al., 2014, Tanaka et al., 2010a, Tanaka et al., 2010b). The 2D photosensor allows large-field imaging of 30 mm2 in a single capture. These systems have the potential to provide simple and rapid cell counting platforms through simultaneous imaging of thousands of individual cells.

In this study, we developed simple and rapid imaging system integrated with a microcavity array and a 2D photosensor for CD4 testing. Dual color imaging of a 64-mm2 array area was achieved using a simple optical design with a color-complementary metal oxide semiconductor (CMOS) sensor equipped with a relay lens. The acquired image was used to count fluorescence-labeled CD4+ T cells and CD8+ T cells to determine the CD4/CD8 ratio. The CD4/CD8 ratio is a critical parameter in pediatric HIV-infected patients, because absolute count of lymphocyte subsets is age-dependent and more variable than percentages of lymphocyte subsets in children less than 5 years of age (O’Gorman and Zijenah, 2008). In this platform, sample introduction, cell entrapment, immunostaining and rapid detection were accomplished within an integrated device. Furthermore, the measurement accuracy was evaluated using control blood. Our proposed system will provide a simple and rapid CD4 testing method for the application of HIV/AIDS treatment.

Section snippets

Materials

A CMOS sensor (DFK72BUC02; Imaging Source Europe GmbH; Bremen, Germany) composed of 2592×1944 pixels (pixel size: 2.2 μm) with an area of 5.70×4.28 mm2 was used as a 2D photosensor. A prepolymer of polydimethylsiloxane (PDMS), Silpot 184, was purchased from Dow Corning Toray Co., Ltd. (Tokyo, Japan). IMMUNO-TROL cells (high sample) or IMMUNO-TROL low cells (low sample) were obtained from Beckman Coulter, Inc. (CA, USA) as control blood samples. Immunostaining of leukocytes was performed using rat

Single-cell imaging by the large-field imaging system

Fig. 2 shows the fluorescence images of JM cells stained with ethidium homodimer-1 acquired using the large-field imaging system with an exposure time of 0.25 s. Light with a wavelength of 530–550 nm was used for cell imaging. The stained cells were visualized as red-colored dots. The fluorescent dots were more clearly recognized with increasing light intensities. When the light was irradiated from the upper side of the microcavity array (Fig. 1A and B), the fluorescent dots were recognized as

Discussion

This study represents a design on a new method based on the one-shot image capture to achieve a simple and rapid cell population analysis. Our proposed system was designed to accomplish total process for CD4 test consisting of leukocyte recovery from whole blood, immunostaining, fluorescent imaging and cell counting within an integrated device. The device was fabricated using a microcavity array and a 2D photosensor. The microcavity array enabled leukocyte recovery from a few microliters of

Conclusions

This study focused on the development of a large-field imaging system for simple and rapid CD4 testing. A 2D photosensor integrated with a microcavity array allowed one-shot imaging of single-cells on an entire 64-mm2 array for 2 s. Furthermore, the CD4/CD8 ratio in whole blood was successfully determined using dual-color imaging. Therefore, our proposed system enables high-throughput cell profiling based on fluorescence detection and cell counting within the desk-top-sized instrument. This

Acknowledgments

This work was funded in part by a support program for technology development based on academic findings from the New Energy and Industrial Technology Development Organization (P03040). Tatsuya Saeki thanks the Japan Society for the Promotion of Science for the financial support (12J03043).

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