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HIV-infected patient blood samples were tested at the point-of-care using a portable and label-free microchip CD4 count platform that we have developed. A total of 130 HIV-infected patient samples were collected that included 16 de-identified left over blood samples from Brigham and Women's Hospital (BWH), and 114 left over samples from Muhimbili University of Health and Allied Sciences (MUHAS) enrolled in the HIV and AIDS care and treatment centers in the City of Dar es Salaam, Tanzania. The two data groups from BWH and MUHAS were analyzed and compared to the commonly accepted CD4 count reference method (FACSCalibur system).
More than 30 million human immunodeficiency virus (HIV)-infected people live in the sub Saharan Africa, yet it is estimated that only one in ten persons infected with HIV has been tested and knows their HIV status , , , , , , . Effective antiretroviral therapy (ART) for HIV has been available in developed countries for more than a decade and free of charge through philanthropic resources such as Bill and Melinda Gates Foundation, Clinton and Davis Duke, and governmental resources such as President's Emergency Fund for AIDS Relief (PEPFAR) . However, in Africa, less than 4 out of 10 people who need a treatment are actually receiving ART . Part of the problem associated with existing ART delivery services are the limitations of conventional methods to diagnose and monitor HIV-infected individuals living in rural communities.
HIV infection has reached epidemic proportions in Tanzania with an estimated 1.3 million patients living with HIV/AIDS. Effective antiretroviral therapy (ART) for HIV has been available in Tanzania for more than a decade. However, it is estimated that less than 20% of all the infected individuals in Tanzania are currently receiving treatment, the most affected persons are living in rural and hard to reach communities. A microchip test that is portable and affordable has potential to impact HIV monitoring at all levels possibly with a higher impact at the dispensary level.
The challenges with delivering healthcare at the POC in developed world settings include ease-to-use, sample processing, need for skilled health care workers, portability, and test turnaround time (Table 1) . These factors have long constituted a bottleneck for recent microfluidic and lab-microchip type platforms to be translated to the bed-side as applicable diagnostic methodologies. These POC challenges adopt an insurmountable silhouette at the resource-constrained settings, where additional challenges are encountered. The difficulties associated with resource-limited POC healthcare delivery involve undependable electricity, demanding portability requirements on devices and readers, and limitations on the use of peripheral devices , , . These interdependent challenges alter the parameters for POC device design. Therefore, we need to design new methodologies with practical thinking strategies to address problems that go beyond the developed world geared engineering and healthcare system . These factors all broadly define the poor performance encountered by well-operating, lab-designed machinery in such resource-limited settings. Furthermore, fluorescent labeling of cells for detection and enumeration present significant challenges in these settings , , , . Therefore, label-free approaches with simple imaging methods are needed at the resource limited POC settings. Taking on these intriguing biomedical, engineering and design challenges , , we here report clinical results from Tanzania using a POC CD4 count microchip, which is an inexpensive (
(a) Shadow images of captured cells were obtained using a large area CCD image sensor (2434 mm, 10 mega pixel). A pinhole LED (Light Emitting Diode) was used as a light source. The POC platform has following two main processes. First, a microfluidic chip captured target CD4+ cells from unprocessed fingerprick volume of HIV-infected whole blood by anti-CD4 antibody which was immobilized on the microchip surface. Second, the captured cells were imaged using the wide field of view (FOV) lensless CCD platform within a second. White light generated from LED light source went through a 100 µm pinhole and illuminated captured cells. Cell shadows were automatically detected and rapidly counted by automated image recognition software on a portable laptop computer. (b) Schematic representation and drawing of the lensless imaging system for microfluidic CD4 count, (c) Photograph of entire POCT CD4 imaging system, (d) microfluidic CD4 chip on top of CCD sensor inside a black box, (e) lensless imaging and magnified image represents the diffracted shadow signal of the cell shape. Scale bar is 200 µm. Entire platform setup and operational details were shown in Text S1.
Detailed description and illustration of device fabrication (Table S1 and Fig. S1), preparation (Fig. S3 and Table S2), and the standard operating procedures (Table S3) can be found in Text S1, which were based on our previous studies . The microfluidic whole blood CD4 counting operation is outlined in Figure 2, which was performed by minimally trained personnel at MUHAS (Table S3). Briefly, the microfluidic chips were removed from their vacuum sealed packages (Fig. S2 and Fig. 2a), the capture antibody was injected into the chips followed by a brief washing step (Table S2 and Fig. 2b), a one step whole blood injection was performed (Fig. 2c), and the microfluidic chip was imaged using a portable CCD based imaging platform (Fig. 2d). The gravitational flow required filling 50 µL of whole blood into the pipette directly on top of the inlet of the device without additional sample handling or processing (Fig. 2b&c). The pipette was removed from the inlet as soon as the channel got fully filled with the whole blood indicated by the red color. This process led to an estimated 8 µL of whole blood to pass through the device in MUHAS tests. In BWH tests, the volume of blood introduced into the channels was controlled with automated pipettes to be 6 µL per channel. The cell counts obtained from the microchips were normalized with the total blood volume to convert the results to number of cells per microliter of whole blood.
(a) Unpacking of the microfluidic chips, (b) antibody injection, (c) one step blood injection, (d) microfluidic CD4 count chip imaging using a point-of-care portable, battery operated lensless CCD based imaging platform.
The microchip platform presented here weighed 1 kg, and it was attached to a laptop system to acquire the shadow images of captured cells and count the CD4 T cells using automated software without the need for cell labelling. This whole system was battery operable and portable. The CD4 count system has the potential to be further miniaturized by replacing the laptop and the imaging setup with smaller handheld units such as cell phones with cameras indicating the future potential of such a system. Further, the microchip can be integrated with a simple aspiration system, where a single drop of blood can be introduced into the microchip. These developmental stages can bring this portable system closer to a commercially available product that can be broadly used in resource-limited settings. Further, we anticipate that these cost reductions in medical testing and enhancements in POC diagnostics and monitoring technologies will also impact the developed world healthcare delivery posing a broad interest for the general public.
In conclusion, we have performed for the first time label-free (fluorescent-free) CD4 T-lymphocyte counts from HIV-infected patient blood with a disposable microchip system in the USA and in Tanzania. Our results showed that portable CD4 capture and counting devices are feasible and applicable at the POC settings. The capture efficiency of the developed microchips was observed to be dependent on cell concentration, environmental factors and operational variations. Therefore, detection systems specifically designed for POC must be tested under the full conditions of resource limited settings for reliable evaluation and assessment.
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