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JAIDS Journal of Acquired Immune Deficiency Syndromes:
Basic Science

An Inexpensive, Simple, and Manual Method of CD4 T-Cell Quantitation in HIV-Infected Individuals for Use in Developing Countries

Balakrishnan, Pachamuthu PhD*; Dunne, Mandy BS†; Kumarasamy, Nagalingeshwaran MBBS, PhD*; Crowe, Suzanne MD†; Subbulakshmi, Gangadharan BSc*; Ganesh, Aylur K. ACA*; Cecelia, Anitha J. MSc*; Roth, Patricia PhD‡; Mayer, Kenneth H. MD§; Thyagarajan, Sandras P. PhD, DSc||; Solomon, Suniti MD*

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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, Melbourne, Australia; ‡Beckman Coulter, Biomedical Research Division, Cell Analysis Development Center, Miami, FL; §Miriam Hospital/Brown University Medical School, Providence, RI; and ¶Dr. ALMPGIBMS, University of Madras, Taramani, Chennai, India.

Received for publication January 7, 2004; accepted April 28, 2004.

Supported by the Australia India Council.

Reprints: P. Balakrishnan, YRG CARE, Centre for AIDS Research and Education, Voluntary Health Services Campus, Taramani, Chennai 600 113, India (e-mail: bala@yrgcare.org).

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Abstract

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 investment for this technology and expensive reagents makes it unaffordable 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 individuals. 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 developing countries, for monitoring HIV progression and response to therapy.

Progressive 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 opportunistic 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 surveillance 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 infrastructure or technical expertise for performing flow cytometry 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 resource-poor world is of urgent need, because the rapid spread of HIV infection has already economically burdened the attempts to monitor infected populations in developing and underdeveloped 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 (Beckman 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 Administration. 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.

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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 individuals (randomly selected) attending an HIV clinic at the YRG CARE Medical Center, Voluntary Health Services campus, Taramani, Chennai, India. The study population included drug-naïve patients (n = 43) and those on treatment with anti-retroviral drugs (n = 80). The study subjects were randomly selected, and informed written consent was obtained. All the samples were processed within 24 hours by flow cytometry and within 6 hours by cytosphere assay in a blinded manner. The results were matched to samples after the completion of the cytosphere assay and analyzed statistically.

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Flow Cytometry

CD4+ T-lymphocyte enumeration was done for each specimen with a 2-color, single-platform, flow cytometer (FACSCount; Becton Dickinson Immunocytometry Systems) 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 (UKNEQAS) for leukocyte immunophenotyping and is certified for flow cytometry.

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Cytosphere Assay

The Coulter manual CD4+ count kit contains Coulter CD4+ cytosphere reagent, inert latex spheres coated with murine monoclonal antibody, which are used to identify and manually enumerate the absolute count of CD4+ T lymphocytes by visible light microscopy in fresh whole blood. Because monocytes and macrophages also express the CD4+ molecule, they must be identified and excluded from the absolute count. A blocking reagent with CD14 monoclonal antibody–labeled beads was added to the CD4 tube to achieve this objective. The assay was done per the manufacturer’s instructions. Briefly, 10 μL of Coulter MY4 cytosphere monocyte blocking reagent was added to 100 μL of whole blood in the test tube and mixed gently for 2 minutes. Then, 10 μL of Coulter CD4 cytosphere reagent was added to the same tube and again mixed gently for 2 minutes. Next, 10 μL of a blood–latex spheres mixture was pipetted and added to another tube containing Coulter lysing reagent and mixed gently for 10 to 15 seconds. Both chambers of the 0.1-mm deep hemacytometer were loaded with the processed sample. The hemacytometer was placed in a moisture chamber, and the cells were allowed to settle for 2 to 3 minutes. Using the light microscope, the cells having 3 or more large latex spheres attached to them were counted as CD4+ lymphocytes. Monocyte blocking reagent on the cytoplasmic membrane resulted in a golden-brown coloration to exclude the counting. The CD4+ T lymphocytes were counted in all 9 squares of both chambers of the hemacytometer. To get an absolute count, the count was multiplied by the number of CD4+ T lymphocytes counted by a factor 7.3.

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Statistical Analysis

Using SPSS software (release 10.0.5; Chicago, IL), the intraclass correlation coefficient (ICC) 14 and analysis developed by Bland and Altman 15 were used to analyze the agreement between standard flow cytometry and the cytosphere assay.

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RESULTS

Overall, there was excellent correlation between standard flow cytometry and the cytosphere assay in estimating CD4+ T lymphocytes (Fig. 1), and outliers were removed for further analysis. The ICC between the cytosphere assay and flow cytometry of 122 samples was significant (r = 0.97, 95% confidence interval [CI]: 0.95–0.98; P < 0.0001) for CD4+ T lymphocytes. The mean absolute count ± standard deviation of CD4+ T lymphocytes by flow cytometry was 356.1 ± 265.3 cells/μL, and it was 359.9 ± 277.1 cells/μL by cytosphere assay.

Figure 1
Figure 1
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The ICC, CI, and mean absolute counts ± standard deviation by flow cytometry and cytosphere assay were determined for the study populations (Table 1).

Table 1
Table 1
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The accuracy of the cytosphere assay in identifying those individuals with less than 200 CD4+ T lymphocytes/μL was evaluated, because this count has been incorporated as part of the CDC case definition for AIDS to determine clinical levels of HIV disease. The cytosphere assay had a sensitivity of 94.9% and a specificity of 96.4%. The positive predictive value and negative predictive value were 92.5% and 97.6%, respectively, with flow cytometry as the gold standard method.

The determination of less than 200 CD4+ T lymphocytes/μL (r = 0.92, 95% CI: 0.85–0.95; P < 0.0001) by cytosphere assay was better correlated with the standard flow cytometry method than it was for counts from 201 to 499 cells/μL (r = 0.77, 95% CI: 0.64–0.86; P < 0.0001) and for those more than 500 cells/μL (r = 0.86, 95% CI: 0.72–0.93; P < 0.0001).

The 2 different sample processing periods were compared (0–3 hours and >3–6 hours), and there was no significant difference (Table 2), although raw data show drastic differences with some samples.

Table 2
Table 2
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The Bland-Altman analysis 15 showed that the difference between the 2 methods was greater for samples with CD4 counts >500 cells/μL (mean difference in CD4 count <500: 8 cells/μL; mean difference in CD4 count >500: 15 cells/μL). Overall, this manual assay yielded CD4+ T-lymphocyte counts greater than flow cytometry by a mean of 10 cells/μL (limit of agreement: –152 to 132 cells/μL;Fig. 2) as assessed by Bland-Altman analysis.

Figure 2
Figure 2
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DISCUSSION

The incidence of HIV infection and clinical disease continues to increase rapidly in the developing world. 8 As antiretroviral therapy is becoming more affordable and accessible, inexpensive laboratory tests are also needed to monitor the progression of disease in people with HIV infection living in resource-constrained environments most heavily affected by the epidemic. 16 Absolute CD4+ T-lymphocyte counts are used to monitor disease progression and institute prophylaxis against opportunistic infections. The gold standard for CD4+ T-lymphocyte enumeration remains flow cytometry. The easiest technique to use is single-platform volumetric flow cytometry (eg, FACSCount). This technology is quite expensive ($40,000), however, and the maintenance contracts ($1000 per year) and reagents ($15 per test) are not affordable for most countries; flow cytometry also requires highly trained personnel and a sophisticated laboratory. These factors necessitated alternative methods to enumerate CD4+ T-lymphocyte counts. Of the several new alternative methods, the Coulter cytosphere assay is of interest to developing countries because it is relatively inexpensive ($10 per test) and does not require any specialized equipment other than a simple light microscope and hemacytometer. The major expenditure involved for the cytosphere assay in CD4+ cell enumeration is the cost of the equipment, with only $500 estimated for a microscope; moreover, the test requires much less training of the operator than flow cytometry. The performance of the cytosphere assay in developing nations has been shown to be quite good, despite the lack of resources and highly trained personnel. 12

Our study demonstrates that the cytosphere assay with flow cytometry had excellent correlation for CD4+ T-lymphocyte count (0.95 <r < 0.98). The correlation was more significant when the absolute count was less than 200 cells/μL (0.85 <r < 0.95) than when the absolute count was between 201 and 499 cells/μL (0.64 <r < 0.86) or more than 500 cells/μL (0.72 <r < 0.93). Hence, this assay is highly reliable in identifying those individuals with less than 200 CD4+ T lymphocytes/μL with a sensitivity and specificity of 94.9% and 96.4%, respectively. These results show that the cytosphere assay will enable developing countries to classify HIV patients accurately according to the CDC expanded surveillance case definition for AIDS among adolescents and adults. 3 The inclusion in the AIDS surveillance definition of persons with a CD4+ T-lymphocyte count of less than 200 cells/μL or a CD4+ percentage less than 14% will enable AIDS surveillance to reflect more accurately the number of persons with severe HIV-related immunosuppression and those at highest risk for severe HIV-related morbidity. More accurate reporting and analysis of CD4+ T-lymphocyte counts, together with HIV-related clinical conditions, should facilitate efforts to evaluate health care and referral needs of treatment of persons with HIV infection and to project future needs for these management services.

Although there are different methods available for the enumeration of CD4+ T lymphocytes, flow cytometry, the standard method for measuring the CD4+ T-lymphocyte count, is available only in a few centers in developing nations. 16 An earlier study in different centers using the cytosphere assay indicated that this assay can be used as an alternative to flow cytometry for the estimation of CD4+ T-lymphocyte subsets. The findings of the present study are in concordance with earlier findings of good correlation (Table 3). These findings emphasize the usefulness of the cytosphere assay as an alternative to flow cytometry for CD4+ cell count estimation to monitor the level of immune deterioration and as a prognostic test in individuals who are on treatment.

Table 3
Table 3
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In summary, the cytosphere assay proved to be easy to perform and highly reliable in identifying those individuals with CD4+ T-lymphocyte counts of 200 lymphocytes/μL and highly correlative to flow cytometry in cell counts of both antiretroviral-naïve and -treated HIV-infected patients. In developing countries like India, it is difficult to set up expensive technology such as flow cytometry for CD4+ T-lymphocyte counts, especially for care giving at the lower end of health care systems. The reagent cost, equipment required, and ease of use also make flow cytometry an impractical consideration for laboratories in resource-poor settings.

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REFERENCES

1. de Martini RM, Parker JW. Immunologic alterations in human immunodeficiency virus infection: a review. J Clin Lab Anal. 1989;3:56–70.

2. Fahey T, Taylor J, Detels R, et al. The prognostic value of cellular and serological markers in infection with human immunodeficiency virus type 1. N Engl J Med. 1990;322:166–172.

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4. Centers for Disease Control and Prevention. Revised guidelines for performing CD4+ T-cell determinations in persons infected with human immunodeficiency virus (HIV). MMWR Morb Mortal Wkly Rep. 1997; 46(RR-2):1–29.

5. 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. 1996;3:371–373.

6. Carella AV, Moss MW, Provost V, et al. A manual bead assay for the determination of absolute CD4+ and CD8+ lymphocyte counts in human immunodeficiency virus-infected individuals. Clin Diagn Lab Immunol. 1995;2:623–625.

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8. World Health Organization. Workshop on Flow Cytometry and Alternative Methodologies for CD4 Lymphocyte Determinations: Applications for Developing Countries. Washington, DC, April 16, 1992. Geneva: World Health Organization. 1992.

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11. Gernow A, Lisse IM, Bottiger B, et al. Determination of CD4+ and CD8+ lymphocytes with the cytosphere assay: a comparative study with flow cytometry and the immunoalkaline phosphatase method. Clin Immunol Immunopathol. 1995;76:135–141.

12. Landay A, Ho JL, Hom D, et al. A rapid manual method for CD4+ T-cell quantitation for use in developing countries. AIDS. 1993;7:1565–1568.

13. Zwerner RK, Hudson JC, Coulter WH, et al. A whole blood alternative to traditional methods for CD4+ T-lymphocyte determination. J Acquir Immune Defic Syndr Hum Retrovirol. 1997;14:31–34.

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