Comparison of Microcapillary Cytometry Technology and Flow Cytometry for CD4 ⁺ and CD8 ⁺ T-Cell Estimation

Abstract

An alternative technology for the estimation of T cells based on a microcapillary technique (Guava Technologies, Hayward, CA) was compared to FACSCount (Becton Dickinson, San Jose, CA). Samples from 51 human immunodeficiency virus-infected and 21 healthy individuals were tested. The correlation (r) of the two systems for CD4؉ T cells was 0.994, and the coefficient of variation was 6.5%, establishing equable performance between the two technologies.The number of human immunodeficiency virus (HIV)-infected individuals continues to rise in India ever since it was first detected in Vellore, India (6). A high proportion of these infected individuals belong to the economically lower strata of society, and hence it is essential that the supporting health care systems operate in a cost-effective manner. CD4 ϩ T-cell counts are essential for monitoring disease progression and as indicators of when treatment should be started (4). Flow cytometry is the widely used method for analyzing CD4 counts, including in India. More recently a system based on a new technology developed by Guava Technologies (Hayward, CA) has been introduced. This technology has been used only in a small number of centers and requires more widespread testing for user acceptability. In this study, we investigated the performance of the Guava EasyCD4 System in relation to FACSCount.Blood samples were collected between 8:00 a.m. and 10:00 a.m. from 51 HIV-infected individuals who had come to the Clinical Virology department of a tertiary care center in India (south) for their CD4 ϩ /CD8 ϩ T-cell estimation and also from 21 normal healthy individuals from October 2004 through December 2004 after informed consent was obtained in K 2 EDTA vacutainer tubes. Of the 51 samples from HIV-infected individuals, 8 were done in duplicates to investigate the reproducibility of results. In addition to these eight samples, samples from four HIV-infected individuals were tested in quadriplicates. Samples from another seven HIV-infected individuals were stored at room temperature (20 to 25°C) and were retested after 24 h to look for any difference in the count. In order to examine the interpersonnel variation in testing, samples from two healthy individuals were processed and tested by five different laboratory personnel only for CD4 counts.Absolute CD4 ϩ /CD8 ϩ T-cell counts were estimated by the Guava EasyCD4 System, which allows enumeration of CD4 ϩ and/or CD8 ϩ lymphocytes in human peripheral blood. The instrument used two fluorescence parameters in combination with forward scatter (FSC) to identify cells. The reagents consisted of a monoclonal anti-human CD3 antibody conjugated to the tandem dye phycoerythrin (PE)-Cy5 and monoclonal anti-human CD4 and CD8 antibodies conjugated to PE. The antibody staining solutions were used to stain 10-l portions of the blood samples. For each sample, two separate tubes were used: one for determining the absolute CD4 ϩ T-cell count (monoclonal antibody CD3PE-Cy5 and CD4PE were added) and the other for determining the CD8 ϩ T-cell c...

Evidence for lower CD4^<b>+</b> T cell and higher viral load in asymptomatic HIV-1 infected individuals of India: Implications for therapy initiation

“…We had earlier analysed the correlation of the CD4 + T cells estimated by the GuavaEasy CD4 system and the ß ow cytometry and had shown that both systems are usable interchangeably as there was a very signiÞ cant correlation. [5] The CD4 + T cells and CD3 + cell values of the all the quality control samples were within the expected acceptable range. In the present study, we have also analysed the association of CD4 + T cell estimated by GuavaEasyCD4 system and the viral load level estimated by artus HIV-1 RG RT-PCR assay and found to be signiÞ cant (r = −0.55).…”

“…[5] The CD4 evaluation in our laboratory by this instrument is a part of a regular external quality assurance program carried out by WHO and a laboratory in Thailand under the auspices of NACO, India. The HIV-1 viral load was estimated using real-time PCR, RotorGene 3000 (Corbet Research ScientiÞ c, Australia) with artus HIV-1 RG RT-PCR assay (Qiagen GmbH, Germany).…”

“…[2][3][4] Recently we have evaluated the Guava EasyCD4 TM system (Guava Technologies, Hayward, CA, USA) for the T cell estimation with ß ow cytometry. [5] The aim of this study was to investigate further the previous suggestion on new CD4…”

Usefulness of Alternate Prognostic Serum and Plasma Markers for Antiretroviral Therapy for Human Immunodeficiency Virus Type 1 Infection

“…The CD4 ϩ T-cell counts were estimated with the Guava EasyCD4 System (Guava Technologies, Hayward, CA) as reported earlier (12). The HIV-1 load was estimated using real-time PCR equipment (Rotor Gene 3000; Corbett Research Scientific, Australia) with reagents form Artus (Qiagen, GMBH, Germany).…”

Drug Resistant Mutations Detected by Genotypic Drug Resistance Testing in Patients Failing Therapy in Clade C Hiv-1 Infected Individuals From India

“…[3] Previous reports from India have reported M184V Technologies, Hayward, CA, USA), as described earlier, was used for CD4 estimation. [6] Viral load estimation was done using Artus RealArt™ HIV-1 kits (Qiagen Hamburg Gmbh, Germany) using Rotor-Gene™ 3000. [7] The genotypic resistance assay was carried out by sequencing the Pr and RT genes.…”