Balakrishnan, Pachamuthu PhD*; Solomon, Suniti MD*; Mohanakrishnan, Janardhanan MSc*; Cecelia, Anitha J. MSc*; Kumarasamy, Nagalingeswaran PhD*; Murugavel, Kailapuri G. PhD*; Venkatakrishnan, Bhavani BSc*; Solomon, Sunil S. MPH*; Crowe, Suzanne M. MD†; Ganesh, Aylur K. ACA*; Thyagarajan, Sadras P. DSc‡; Flanigan, Timothy MD§; Mayer, Kenneth H. MD§
From the *YRG Centre for AIDS Research and Education, Chennai, India; †Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia; ‡University of Madras, Chennai, India; and §Brown University, Providence, RI.
Received for publication August 4, 2005; accepted April 25, 2006.
Reprints: Pachamuthu Balakrishnan, PhD, YRG Centre for AIDS Research and Education (YRG CARE), Voluntary Health Services campus, Taramani, Chennai-600 113, India (e-mail: email@example.com).
Summary: Serial measurements of absolute CD4+ T-lymphocyte counts are required to initiate and gauge response to therapy and monitor disease progression. Hence, there is an urgent need to evaluate the accuracy and validity of low-cost CD4+ T-cell count assays. Tripotassium EDTA blood specimens from HIV-infected individuals were studied using a novel flow cytometric assay (EasyCD4 assay; Guava Technologies, Hayward, CA) in comparison with standard flow cytometry (FACSCount; Becton Dickinson Immunocytometry Systems, San Jose, CA). The sensitivity, specificity value by EasyCD4 assay in enumerating absolute CD4+ T-cell counts of less than 200 cells/μL were 95% and 100%, respectively. Bland-Altman analysis showed close agreement, with the EasyCD4 assay yielding CD4+ T-cell counts a mean difference of −26 cells/μL (95% confidence interval, −96 to 44 cells/μL) higher than by flow cytometry. Our data suggest that EasyCD4 assay could be a useful alternative assay to conventional flow cytometry, may be appropriate for use in resource-limited settings.
The CD4+ T-lymphocyte (CD4+ T-cell) count is an essential biological indicator used in the clinical follow-up of patients infected with HIV-1, commonly used to determine disease stage, and initiate and monitor highly active antiretroviral therapy. Accurate and reliable quantification of CD4+ T cells is essential for the assessment of the immune system in HIV-infected persons. The reagent costs for CD4+ T-cell counts using the currently available flow cytometric assays remain very high and make the assay too expensive for monitoring most of the persons with HIV infection in developing nations.
The rapid growth of HIV infection worldwide has increased the need for more reliable and cost-effective methods for CD4+ T-cell monitoring.1 Several investigators have evaluated different low-cost immunologic assays as an alternative to standard flow cytometric analysis.2-9 In the present study, we investigated the correlation of CD4+ T cells as estimated by the new alternative method, microcapillary flow cytometry (EasyCD4 assay, Guava Technologies, Hayward, CA) with a standard flow cytometry assay (FACSCount; Becton Dickinson, San Jose, CA) in patient populations in Chennai, India at different stages of HIV infection.
Whole blood specimens were collected using Vacutainer (Becton Dickinson) tubes containing tripotassium EDTA from 110 (78 men and 32 women) HIV-1-infected individuals with the median age of 35 years (range, 6-60 years) attending the HIV clinic at the YR Gaitonde Center for AIDS Research and Education in Chennai, India. Among HIV-infected cases, 62 patients (56%) were drug naive, and 48 (44%) were on highly active antiretroviral therapy. The patients who were attending the HIV clinic consecutively were included in the study, and informed written consent was obtained from all the participants. The study was approved by institutional review board of YR Gaitonde Center for AIDS Research and Education. All the samples were processed within 24 hours by standard flow cytometry and EasyCD4 assay in parallel.
Tripotassium EDTA whole blood was also collected from HIV-negative healthy volunteers (n = 118) and processed in an identical manner. The study population (72 men and 46 women) had a median age of 36 years (range, 21-52 years).
CD4+ T-cell enumeration was done for each specimen with 2-color single-platform flow cytometer FACSCount as per the manufacturer's instructions. Our laboratory has established ongoing participation in a quality control program with the United Kingdom National External Quality Assessment Schemes for Leucocyte Immunophenotyping since 2001 and is certified for the FACSCount.
The EasyCD4 reagent kit contains 2 direct immunofluorescence reagents (a murine monoclonal antihuman CD3 antibody [MAb] conjugated to phycoerythrin and Cy5) anti-CD4-phycoerythrin Mab. The assays in this study were performed according to manufacturer's instructions.10 Briefly, 10 μL of whole blood was added into 1.5-mL screw-capped centrifuge tubes containing 10 μL of EasyCD4 MAbs. The tubes were vortexed gently for 2 to 3 seconds and incubated for 15 minutes at room temperature (20-25 °C) in the dark. Lysing Reagent (180 μL) was pipetted into each tube to bring total sample volume to 200 μL. Sample tubes were vortexed for 2 to 3 seconds on medium speed and then incubated in the dark for 15 minutes at room temperature (20-25 °C). Samples were analyzed on the Guava Personal Cell Analysis instrument using the Guava EasyCD4 software and CytoSoft software version 2.2 (Hayward, CA).
Bland-Altman Analysis11,12 was used to analyze the agreement between the standard flow cytometry and EasyCD4 assay, using SPSS software (Release 10.0.5, SPSS Inc, Chicago, IL).
Agreement of the EasyCD4 with standard flow cytometry (FACSCount) was evaluated for 110 HIV-seropositive patient specimens. Demographic characteristics of the study subjects are described in Table 1. Agreement was evaluated at various clinically critical ranges of CD4+ T-cell count between the 2 methods (Table 2). Bland-Altman plot (Fig. 1) indicated a mean difference of −26 cells/μL (FACSCount-EasyCD4). This shows that EasyCD4 overestimates CD4+ T-cell count, and the limits of agreement were −96 to 44 cells/μL.
The mean difference was observed to be lesser for estimating CD4+ T-cell count less than 200 cells/μL and 350cells/μL, and −10 (−43 to 22) cells/μL and −13 (−53 to 27) cells/μL, respectively. The mean difference was −47 (−131 to −37) cells/μL for CD4+ T-cell count greater than 350cells/μL.
The EasyCD4 assay had a sensitivity of 95% (42/44) and a specificity of 100% (66/66) to correctly identify CD4+ T-cell count less than 200 cells/μL, when compared with FACSCount (the positive and negative predictive values were 100% and 97%, respectively). Sensitivity and specificity were 91% (21/23) and 98% (85/87), respectively, for correctly identifying CD4+ T cells between 200 to 350 cells/μL.
Also, EasyCD4 was compared with FACSCount in estimating CD4+ T cells with the 118 HIV-negative healthy volunteers. Bland-Altman plot (Fig. 2) showed a mean difference of −3 cells/μL, and the limit of agreement was −233 to 227 cells/μL. This range of difference is not clinically acceptable.
Several alternative methods to monitor CD4+ T-cell counts in HIV-infected individuals have been evaluated recently. Many of them show excellent correlation with the standard flow cytometric assays2-7 and have been successfully implemented in resource-poor settings such as west Africa.8 Although the manual immunobead-based methods such as Cytospheres (Fullerton, CA) and Dynabeads (Carlsbad, CA) have the advantage of only requiring routine light microscopy, any manual method may be associated with error and reproducibility remains questionable despite some supportive evaluations.9
Our independent study has shown that the EasyCD4 assay has very strong agreement for CD4+ T-cell count with the standard assay (FACSCount) with the HIV-infected individuals and also with HIV-negative healthy volunteers. Our results show similar accuracy and validity as some preliminary reports from South Africa and United States.13-15 The study from South Africa found less agreement with higher CD4+ T-cell counts (>500 cells/μL) which is to similar to the findings in our study where the mean difference was 60cells/μL.
In our study, the accuracy of the EasyCD4 assay in identifying correctly those individuals with the CD4+ T-cell count less than 200 cells/μL was excellent (95% sensitivity and 100% specificity). However, CD4+ T-cell count below 200 cells/μL is clinically more important to have a higher negative predictive value than a positive predictive value. Hence, EasyCD4 assay could be optimized to gain higher sensitivity. Because the present version of the EasyCD4 assay is overestimating even in the clinical decision-making count, the clinicians should be cautioned about overestimation while using this assay for the CD4+ T-cell monitoring.
Such as other new assays,2,8 EasyCD4 is less accurate at high absolute CD4+ T-cell counts, particularly when the count is more than 500 cells/μL. This may be due to the standardization and optimization of these alternative assays to provide better results at the lower critical CD4+ T-cell counts. However, the higher CD4+ T-cell counts are less relevant for clinical monitoring.
In the present study, the HIV-negative healthy population has been also included to evaluate the agreement between EasyCD4 assay and FACSCount for the higher CD4+ T-cell count. However, the Bland-Altman analysis (Fig. 2) has shown the higher variations (biases) in the higher CD4+ T-cell counts, making it as unsuitable for calculation (using absolute lymphocyte value from hematology analyzer) of CD4+% for pediatric monitoring.
EasyCD4 assay is a microcapillary cytometry technology requiring minimal volumes of blood (10 μL); therefore, it is of practical importance especially when drawing the blood from intravenous drug users. Another advantage of this method is that the procedures are technically simple with only 3 steps; a short incubation (15 minutes) period after the staining, an internal bead control and very little biowaste generated as sheath fluid are not required. Moreover, the assay costs are substantially less expensive (approximately US $5) than the standard assay cost (US $10-15). This assay requires much less training for the operator than flow cytometry, as the procedures are user-friendly; use of the software analysis requires only the positioning of a simple rectangular gate around the subsets (CD3+ and CD4+) population on the data acquisition screen. The shorter incubation period allows the operator to process more samples per day than standard assays.
Although EasyCD4 assay system has several advantages, it involves large initial capital investment for the instrument (US $25,000-30,000) and requires the same sophisticated infrastructure facilities and technical support as standard flow cytometry does. Our laboratory is currently further evaluating the instruments, including an assessment of interpersonal and intrapersonal reproducibility and comparing results with fresh versus aged samples before processing. Longitudinal samples analysis in clinical trials may further determine the suitability of this assay for the use in routine patient care.
The work is partially supported by NIH YRG CARE International CTU-ACTG site no. 11701, grant no. 5U01 AI 038858, and NIH grant nos. 5D43TW000237, P30-AI-42853-05, and 5UO1AI46381. All the clinical and laboratory staff, who assisted in this study are acknowledged. We are also thankful to Mr Edward Livant, Johns Hopkins University School of Medicine, Baltimore, Maryland for his assistance in analysis of the results.
2. Moss MW, Carella AV, Provost V, et al. Comparison of absolute CD4+
lymphocyte counts determined by enzyme immunoassay (TRAx CD4 test kit) and flow cytometry. Clin Diagn Lab Immunol
3. Didier JM, Kazatchkine MD, Demouchy C, et al. Comparative assessment of five alternative methods for CD4+
T-lymphocyte enumeration for implementation in developing countries. J Acquir Immune Defic Syndr
4. Nicholson JK, Velleca WM, Jubert S, et al. Evaluation of alternative CD4 technologies for the enumeration of CD4 lymphocytes. J Immunol Methods
5. Balakrishnan P, Dunne Mandy, Kumarasamy N, et al. An inexpensive, simple and manual method of CD4 T-cell quantitation in HIV-infected individuals for use in developing countries. J Acquir Immune Defic Syndr
6. Forum for Collaborative HIV Research. A meeting organized by the forum for collaborative HIV research: Transfer of HIV diagnostic and monitoring technologies into resource-poor settings. Washington, DC, April 22, 2002.
7. Kumarasamy N, Mahajan AP, Flanigan TP, et al. Total lymphocyte count (TLC) is a useful tool for the timing of opportunistic infection prophylaxis in India and other resource-constrained countries. J Acquir Immune Defic Syndr
8. Diagbouga S, Chazallon C, Kazatchkine MD, et al. Successful implementation of a low-cost method for enumerating CD4+
T lymphocytes in resource-limited settings: the ANRS 12-26 study. AIDS
9. Crowe S, Turnbull S, Oelrichs R, et al. Monitoring of human immunodeficiency virus infection in resource-constrained countries. Clin Infect Dis
. 2003;37(suppl 1):S25-S35.
10. Guava EasyCD4 Software, Version 2.2. Guava EasyCD4 System User's Guide. CA: Guava Technologies Inc; May 2004.
11. Jeyaseelan L, Rao PSS. Statistical measures of clinical agreement. Natl Med J India
12. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet
13. Scott L, Lawrie D, Harvey J, et al. A comparison of the guava personal cell analyser with PLG CD4 T-cell enumeration: a pilot evaluation. Paper presented at: 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, CA. Abstract 962.
14. Josefowicz S, Buchner L, Epling CL, et al. A simple, low-cost CD4+
T-cell assay: comparison of the Guava Easy CD4 and the BD Biosciences MultiTest Assays for the determination of CD4+
T-cell counts in HIV-1-seropositive and -seronegative volunteers. Paper presented at: 11th Conference on Retroviruses and Opportunistic Infections; February 8-11, 2004; San Francisco, CA. Abstract 961.
15. Josefowicz S, Louzao R, Lam L, et al. 5-Site evaluation of the Guava EasyCD4™ assay for the enumeration of human CD4+
T cells. Paper presented at: 12th Conference on Retroviruses and Opportunistic Infections; February 22-25, 2005; Boston, MA. Abstract 741.
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