BASIC SCIENCE An Inexpensive, Simple, and Manual Method of CD4 T-Cell Quantitation in HIV-Infected Individuals for Use in Developing Countries Pachamuthu Balakrishnan, PhD,* Mandy Dunne, BS,† Nagalingeshwaran Kumarasamy, MBBS, PhD,* Suzanne Crowe, MD,† Gangadharan Subbulakshmi, BSc,* Aylur K. Ganesh, ACA,* Anitha J. Cecelia, MSc,* Patricia Roth, PhD,‡ Kenneth H. Mayer, MD,§ Sandras P. Thyagarajan, PhD, DSc,  and Suniti Solomon, MD* Summary: CD4 + T lymphocytes are currently the most common surrogate marker indicating immune status and disease progression with HIV infection. The cost of monitoring disease progression and response to therapy is still prohibitively expensive. Flow cytometry is the gold standard for the estimation of CD4 + , but the high initial in- vestment for this technology and expensive reagents makes it unaf- fordable for developing countries like India. We evaluated the Coulter cytosphere assay for quantifying CD4 + T lymphocytes in comparison with the standard method, flow cytometry, in 122 HIV-infected indi- viduals. The correlation coefficient of the cytosphere assay compared with that of flow cytometry for CD4 + T lymphocytes was 0.97 (P< 0.0001), with a confidence interval of 0.95 to 0.98. The sensitivity, specificity, positive predictive value, and negative predictive value of the cytosphere assay in enumerating absolute CD4 + T-lymphocyte counts of less than 200/μL were 94.9%, 96.4%, 92.5%, and 97.6%, respectively. This is a simple inexpensive method and has a strong correlation with flow cytometry. Hence, the cytosphere assay can be an alternate to flow cytometry for the estimation of CD4 + T- lymphocyte counts, especially in resource-poor settings of develop- ing countries, for monitoring HIV progression and response to therapy. (J Acquir Immune Defic Syndr 2004;36:1006–1010) P rogressive clinical and immunologic decline in HIV- infected patients is usually correlated with a falling CD4 + T-lymphocyte count, and the absolute CD4 + count has been used to decide when to initiate antiretroviral therapy and op- portunistic infection prophylaxis. 1 Changes in CD4 + T- lymphocyte count have also been used as part of therapeutic monitoring of patients with HIV. 2 The expanded AIDS sur- veillance case definition of the Centers for Disease Control and Prevention (CDC) includes all HIV-infected individuals with CD4 + T-lymphocyte absolute counts of less than 200 cells/μL. 3 Many of the developing nations do not have the in- frastructure or technical expertise for performing flow cytom- etry to determine CD4 + T-lymphocyte counts as a means of evaluating HIV disease progression. 4 A simple, reliable, and cost-effective immunologic marker within the reach of the re- source-poor world is of urgent need, because the rapid spread of HIV infection has already economically burdened the at- tempts to monitor infected populations in developing and un- derdeveloped countries. 5 Several investigators have evaluated different low-cost immunologic assays as alternatives to flow cytometric analysis of CD4 + cell counts. 5–10 A manual cytosphere method (Beck- man Coulter Corporation, Miami, FL) using a hemocytometer with simple light microscopy has been evaluated in different countries with encouraging results, 6,11–13 and this method has already been approved by the US Food and Drug Administra- tion. In the present study, we investigated the correlation of CD4 + T-lymphocytes as estimated by the cytosphere method with flow cytometry in patient populations at different stages of HIV infection. METHODS Patients Whole blood samples were obtained using Vacutainer (Becton Dickinson Immunocytometry Systems, San Jose, CA) tubes containing tripotassium ethyl diamine tetraacetate (K3 EDTA) from 123 (103 male and 20 female) HIV-infected in- dividuals (randomly selected) attending an HIV clinic at the YRG CARE Medical Center, Voluntary Health Services cam- pus, Taramani, Chennai, India. The study population included Received for publication January 7, 2004; accepted April 28, 2004. From the *YRG Centre for AIDS Research and Education, Voluntary Health Services, Taramani, Chennai, India; †Clinical Research Laboratory, Mac- farlane Burnet Institute for Medical Research and Public Health, Mel- bourne, Australia; ‡Beckman Coulter, Biomedical Research Division, Cell Analysis Development Center, Miami, FL; §Miriam Hospital/Brown University Medical School, Providence, RI; and ¶Dr. ALMPGIBMS, Uni- versity of Madras, Taramani, Chennai, India. Supported by the Australia India Council. Reprints: P. Balakrishnan, YRG CARE, Centre for AIDS Research and Edu- cation, Voluntary Health Services Campus, Taramani, Chennai 600 113, India (e-mail: bala@yrgcare.org). Copyright © 2004 by Lippincott Williams & Wilkins 1006 J Acquir Immune Defic Syndr • Volume 36, Number 5, August 15 2004