A Microchip Approach for Practical Label-Free CD4+ T-Cell Counting of HIV-Infected Subjects in Resource-Poor Settings

Xuanhong Cheng, PhD; Daniel Irimia, MD, PhD; Meredith Dixon, BS; Joshua C Ziperstein, BS; Utkan Demirci, PhD; Lee Zamir, MS; Ronald G Tompkins, MD, DSc; Mehmet Toner, PhD; William R Rodriguez, MD

Disclosures

J Acquir Immune Defic Syndr. 2007;45(3):257-261. 

In This Article

Summary and Introduction

Summary

Simple affordable CD4 cell counting is urgently needed to stage and monitor HIV-infected patients in resource-limited settings. To address the limitations of current approaches, we designed a simple, label-free, and cost-effective CD4 cell counting device using microfluidic technology. We previously described the fabrication of a microfluidic system for high-efficiency isolation of pure populations of CD4+ T cells based on cell affinity chromatography operated under controlled flow. Here, we compare the performance of a microfluidic CD4 cell counting device against standard flow cytometry in 49 HIV-positive subjects over a wide range of absolute CD4 cell counts. We observed a close correlation between CD4 cell counts from the microchip device and measurements by flow cytometry, using unprocessed whole blood from HIV-positive adult subjects. Sensitivities for distinguishing clinically relevant thresholds of 200, 350, and 500 cells/µL are 0.86, 0.90, and 0.97, respectively. Specificity is 0.94 or higher at all thresholds. This device can serve as a functional cartridge for fast, accurate, affordable, and simple CD4 cell counting in resource-limited settings.

Introduction

As the cost of antiretroviral therapy (ART) continues to fall, HIV treatment has been made available to nearly 2 million people worldwide.[1] Nevertheless, delivering antiretroviral-based care on a global scale, and reaching an additional 4 million people with advanced HIV infection, presents critical challenges. Especially in sub-Saharan Africa, the delivery of HIV care is compromised by underresourced facilities and severe shortages of skilled health workers.[2] Deficiencies in laboratory infrastructure have become particularly critical bottlenecks to the scale-up of HIV care programs.[3] Accurate CD4 cell counts are needed to stage and monitor HIV-infected patients. As in the United States, according to World Health Organization (WHO) guidelines, a CD4 count less than 200 cells/µL establishes the diagnosis of AIDS and is used to initiate antiretroviral treatment. CD4 counts of 350 and 500 cells/µL are used to initiate cotrimoxazole prophylaxis and to increase patient monitoring intensity, respectively.[4] Given the widespread lack of viral load testing, the WHO also recommends that changes in CD4 counts be used as the primary means to monitor patient responses to ART.

The standard laboratory equipment and kits used for CD4 cell counting have limited penetration in resource-poor settings, however, despite ongoing international efforts to extend ART in these areas. A severe shortage of trained laboratory technicians may be the major barrier. Flow cytometers, including smaller single-purpose CD4 cell counting devices developed recently, require trained laboratory technicians to handle and process samples and to analyze and report data.[5,6,7,8] Because flow cytometry-based CD4 cell counting is beyond the reach of so many HIV-infected people, the WHO and others have urged the development of simple-to-use point-of-care CD4 cell count assays designed for resource-limited settings.[9,10,11]

To address the need for CD4 cell counting devices that are simple to use and inexpensive, we recently reported a new microfluidic system for isolating pure populations of CD4+ T cells that requires fingerprick samples of whole blood, with no sample processing or specific labeling.[12] This technology enables specific capture of CD4+ T lymphocytes with high efficiency; key elements of the approach include (1) a microfluidic channel functionalized with immobilized anti-CD4 antibodies for affinity isolation of CD4+ cells from unprocessed whole blood, (2) microliter volumes of sample and wash buffer, and (3) controlled sample and wash buffer delivery for efficient CD4+ T-cell capture with minimal monocyte contamination. CD4 cell counts are obtained by enumerating all cells isolated from a 10-µL volume of blood, using a standard light microscope. In this study, we evaluate the performance of our microchip CD4 cell counting system using whole-blood samples from 49 HIV-infected subjects at different stages of disease. We compare the accuracy and simplicity of CD4 cell counts obtained from the microchip system with standard flow cytometric measurements.

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