Daily Quality Control in CD3⁺and CD4⁺T Cell Estimation by the FACSCount System at a Tertiary Care Center in South India

Abstract

CD4؉ T cell count estimations are subject to high variations; hence, in this study, the previous day's tested samples were included routinely as the internal quality controls. The percentages of variation of the 2-day values were analyzed for 280 observations and the mean variation for CD4؉ and CD3 ؉ T cell counts ranged from 5.21% to 9.66%. This method is a good internal quality control (IQC) procedure for the estimation of CD3 ؉ and CD4 ؉ T cell counts in resource-poor settings.T he absolute CD4 ϩ T-cell count is an important laboratory tool for monitoring HIV-infected individuals (5). Frequent monitoring of CD4 ϩ T lymphocytes is essential to assess the immune suppression and the disease progression of HIV-infected individuals (10, 3). This also helps physicians to decide when to initiate antiretroviral therapy (ART) (4, 2) and when to change therapy due to immunologic failure in resource-poor settings (7, 11). There are several methods used to estimate the absolute CD4 ϩ T-cell count, with the standard technique used being flow cytometry (1). There are numerous factors that are associated with variation in the absolute lymphocyte subset counts estimated (6). The reported mean variation of CD4 ϩ T cell counts in a "singleplatform" methodology is about 13.7% (range, 10% to 18.3%). The variation reported for "double-platform" systems ranges from 14.5% to 43.4% (mean, 23.4%) (1).Since the CD4 ϩ T cell estimation is subject to a large amount of variation, it is very important that daily quality control measures are carried out in the laboratory. In its guidelines, the National AIDS Control Organization (NACO), India, has suggested for all the laboratories to do daily quality control testing that incorporates two samples (the samples with the lowest and highest CD4 ϩ cell counts) that had been tested the previous day.In this study, we investigated the performance of CD4 ϩ T cell count estimation using the FACSCount system (Becton, Dickinson) by retrospectively analyzing the results of the two daily quality control samples.The CD3 ϩ and CD4 ϩ T cell estimations were carried out using the FACSCount system (Becton, Dickinson) as reported earlier (10). In our laboratory, the testing was carried out from Monday through Friday and two internal quality control (IQC) samples were run every day except Mondays. The previous day's samples with the lowest and the highest CD4 ϩ T cell counts were included as the controls. The samples were stored at room temperature (between 20 and 28°C). The IQC values of a given day were compared with the previous day's values, and percent variation values were calculated. Percent variation was calculated as follows: (count on the first day/count on the second day) Ϫ 1 ϫ 100. The data from July 2010 through January 2012 were analyzed. In the last month of the study, the laboratory also started using the Multi-Check control (Becton, Dickinson, San Jose, CA) for routine quality control testing. The data were also analyzed. A sample showing more than 20% variation from the previous day's value w...

Duplicate analysis method: a cheaper alternative to commercial IQC materials in limited resource settings for monitoring CD4 testing

“…The CD4 values were compared with the previous day values and percent variation between the values was calculated by using following formula: observed (day 2 value)/expected (day 1 value) × 100 − 100. Percent variation more than ±20 was considered as non acceptable [ 7 ].…”

“…Commercially available stabilized blood samples are recommended for daily QC for CD4 testing as they give information regarding precision, biases and accuracy of the results. Alternative method for QC is duplicate analysis method in which patient samples tested on previous day are used as QC material [ 7 ]. This method has two distinct advantages as the QC materials match with the exact nature of the sample (fresh whole blood), and the materials are readily available at any laboratory at no extra cost [ 6 ].…”

Implementation of Highly Sophisticated Flow Cytometry Assays in Multicenter Clinical Studies: Considerations and Guidance

“…Although traditional quantitative QCs are difficult to implement, there are several uses of qualitative or relative QCs, such as those used by Ramalingam et al in which each days' test for CD4+ T cell counts included the low and high samples from the previous day as an internal control and the percent variation over the 2-day period was analyzed [25]. While providing some proof of consistency throughout the assay, this method of internal QCs would not detect overarching issues from the method itself or potential mistakes in the initial settings of the flow cytometer provided these settings were used throughout the study.…”