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MIT HST 410J - A Microchip Counting Method for HIV Monitoring

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A Microchip CD4 Counting Method for HIVMonitoring in Resource-Poor Settings1,2,3* 4 4 3 4William R. Rodriguez , Nicolaos Christodoulides , Pierre N. Floriano , Susan Graham , Sanghamitra Mohanty ,1 1 5 5 5 4 4Meredith Dixon , Mina Hsiang , Trevor Peter , Shabnam Zavahir , Ibou Thior , Dwight Romanovicz , Bruce Bernard ,4 1,2 4*Adrian P. Goodey , Bruce D. Walker , John T. McDevitt1 Partners AIDS Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America, 2 Division of AIDS, Harvard Medical School, Boston,Massachusetts, United States of America, 3 Brigham and Women’s Hospital, Boston, Massachusetts, United States of America, 4 Department of Chemistry and Biochemistry,University of Texas, Austin, Texas, United States of America, 5 Botswana–Harvard AIDS Institute Partnership, Princess Marina Hospital, Gaborone, BotswanaCompeting Interests: WRR, NC,PNF, BDW, and JTM have applied fora patent for the application de-scribed here.Author Contributions: WRR, NC, PF,SG, BDW, and JTM designed thestudy. WRR, NC, PNF, SG, MD, SM, ST,IB, TP, MH, DR, BB, APG, BDW, andJTM collected and analyzed the data.WRR, BDW, NC, PNF, and JTMprepared the manuscript.Academic Editor: Zvi Bentwich,Rosetta Genomics, IsraelCitation: Rodriguez WR, Christo-doulides N, Floriano PN, Graham S,Mohanty S, et al. (2005) A microchipCD4 counting method for HIV mon-itoring in resource-poor settings.PLoS Med 2(7): e182.Received: January 31, 2005Accepted: April 26, 2005Published: July 19, 2005DOI:10.1371/journal.pmed.0020182Copyright: Ó 2005 Rodriguez et al.This is an ope n-access articledistributed under the terms of theCreative Commons AttributionLicense, which permits unrestricteduse, distribution, and reproductionin any medium, provided theoriginal work is properly cited.Abbreviations: CCD, charge-coupled device; ETC, electronic tastechip; PBS, phosphate buffered saline*To whom correspondence shouldbe addressed. E-mail: [email protected] (WRR), [email protected] (JTM)ABSTRACTBackgroundMore than 35 million people in developing countries are living with HIV infection. Anenormous global effort is now underway to bring antiretroviral treatment to at least 3 million ofthose infected. While drug prices have dropped considerably, the cost and technical complexityof laboratory tests essential for the management of HIV disease, such as CD4 cell counts,remain prohibitive. New, simple, and affordable methods for measuring CD4 cells that can beimplemented in resource-scarce settings are urgently needed.Methods and FindingsHere we describe the development of a prototype for a simple, rapid, and affordable methodfor counting CD4 lymphocytes. Microliter volumes of blood without further sample preparationare stained with fluorescent antibodies, captured on a membrane within a miniaturized flowcell and imaged through microscope optics with the type of charge-coupled device developedfor digital camera technology. An associated computer algorithm converts the raw digitalimage into absolute CD4 counts and CD4 percentages in real time. The accuracy of thisprototype system was validated through testing in the United States and Botswana, andshowed close agreement with standard flow cytometry (r ¼ 0.95) over a range of absolute CD4counts, and the ability to discriminate clinically relevant CD4 count thresholds with highsensitivity and specificity.ConclusionAdvances in the adaptation of new technologies to biomedical detection systems, such asthe one described here, promise to make complex diagnostics for HIV and other infectiousdiseases a practical global reality.PLoS Medicine | www.plosmedicine.org 0663 July 2005 | Volume 2 | Issue 7 | e182Open access, freely available onlinePLoSMEDICINEHarvard-MIT Division of Health Sciences and TechnologyHST.410J: Projects in Microscale Engineering for the Life Sciences, Spring 2007Course Directors: Prof. Dennis Freeman, Prof. Martha Gray, and Prof. Alexander Aranyosi13]. Low-cost microbead separation of CD4 cells from otherFluid Delivery Systemblood cells, followed by standard manual cell countingIn initial studies, we used a single peristaltic pump totechniques using a light microscope, offers significantly lowerdeliver sample and washes to the flow cell. Subsequently, areagent costs than flow cytometry. These methods, however,partially automated fluid delivery system was developed. Thisare low throughput and extremel y labor intensive, andfunctional adaptation uses two miniature OEM peristalticappear to be less accurate than traditional flow cytometry;pumps, each in conjunction with a pinch valve, and 0.031-in.thus, they have not been widely adopted [13–18].(0.79-mm) silicone tubing capable of delivering flow rates ofLess expensive CD4 counting methods that capitalize on46–920 ll/min to the flow cell. Integrated software (LabVIEW,low-cost microfabrication, efficient light sources, and afford-National Instruments, Austin, Texas, United States) directsable microelectronics and digital imaging hardware havedelivery of whole blood samples and washes to the flow cellbeen conceptualized, but never realized [19,20]. One of ususing the appropriate pumps and valves. Sample filtrate,(JTM) has previously reported the development of a novelincluding red blood cells, is captured in a waste reservoir.microchip-based detection system for measuring analytessuch as acids, bases, electrolytes, and proteins in solutionOptical Station and Image Capturephase [21–23]. This electronic taste chip (ETC) system carriesThe flow cell was positioned on the stage of a modified BX2out chemical and immunological reactions on microspheresOlympus (Tokyo, Japan) compound microscope equippedpositioned in the inverted pyramidal microchamber wells of awith a 103 objective lens and a high-pressure 100 W mercurysilicon or plastic microchip, which is housed in a miniature burner arc lamp as a light source. Focusing was maintainedPLoS Medicine | www.plosmedicine.org 0664 July 2005 | Volume 2 | Issue 7 | e182Microchip CD4 CountingIntroductionflow cell. Microfluidic channels deliver a series of small-volume reagents and washes to the flow cell, and hence to theMore than 35 million HIV-infected people live in develop-chip and to each one of the microspheres. Optical signalsing countries with significant resource limitations. Althoughgenerated by the reactions on the microspheres are visualized6 million people living in developing countries are in urgentand captured on


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