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ORIGINAL ARTICLE
Year : 2011  |  Volume : 29  |  Issue : 4  |  Page : 401-405
 

Evaluation of a dry format reagent for CD4+ and CD8+ T-cell enumeration with FACSCount and Guava polymerase chain reaction


Department of Clinical Virology, Christian Medical College, Vellore 632 004, India

Date of Submission23-Jan-2011
Date of Acceptance01-Jul-2011
Date of Web Publication24-Nov-2011

Correspondence Address:
R Kannangai
Department of Clinical Virology, Christian Medical College, Vellore 632 004
India
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Source of Support: None, Conflict of Interest: The ReaMetrix reagents were provided by the manufacturer.


DOI: 10.4103/0255-0857.90176

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 ~ Abstract 

Purpose: In all CD4+/CD8+ T-cell estimation systems, the reagents used are liquid in nature and have to be transported and stored at 2°-8°C. This causes problems in countries where the ambient temperature is high for most parts of the year or where the laboratories are at remote places. Materials and Methods: We evaluated a dry format of CD4/CD8 reagents from ReaMetrix (Bangalore, India) against the existing liquid reagents from Becton Dickinson (San Jose, CA, USA) and Guava PCA system (Guava Technologies, Hayward, CA, USA). Blood samples collected during March 2009 through May 2009 from 102 HIV-infected individuals and 31 normal healthy individuals in a tertiary care centre in India (south) were tested by Guava; EasyCD4™ System (PCA) and FACSCount using the respective reagents and the corresponding ReaMetrix reagents. Results: Overall, the correlation (r) of the new Rea T Count and FACSCount reagents for the CD4+ T-cell estimation was 0.98, while with ReaPan 3 4 G reagent in the Guava PCA system with the Guava reagent was 0.97. The mean bias for CD4+ T-cell measurements between Rea T count and BD reagent was -6 cells/ml, while the same with ReaPan 3 4 G reagent in the Guava PCA system was 78 cells/ml. The mean bias for the Rea T count and the ReaPan 3 4 G reagent tested in the FACSCount and Guava PCA system was 17 cells. Conclusions: The dry reagents were found to be reliable and cheaper compared to the existing liquid reagents. This allows the transportation of reagents in the absence of cold chain and will facilitate a more user-friendly CD4+ T-cell testing system.


Keywords: CD4, CD8, dry format, HIV-1, Reametrix


How to cite this article:
Sachithanandham J, Solomon K, Prasannakumar S, Nithyandham G, Kannangai R. Evaluation of a dry format reagent for CD4+ and CD8+ T-cell enumeration with FACSCount and Guava polymerase chain reaction. Indian J Med Microbiol 2011;29:401-5

How to cite this URL:
Sachithanandham J, Solomon K, Prasannakumar S, Nithyandham G, Kannangai R. Evaluation of a dry format reagent for CD4+ and CD8+ T-cell enumeration with FACSCount and Guava polymerase chain reaction. Indian J Med Microbiol [serial online] 2011 [cited 2019 Sep 15];29:401-5. Available from: http://www.ijmm.org/text.asp?2011/29/4/401/90176



 ~ Introduction Top


The management of human immunodeficiency virus (HIV)-infected individuals continue to be an uphill task in developing countries such as India. The majority of the infected individuals belong to the lower socioeconomic strata of society, and hence it is essential that the supporting health care systems operate in a cost-effective manner. The CD4+/CD8+ T-cell estimation continues to be the cost-effective indicator for monitoring disease progression and to decide on the initiation of treatment. [1] There are several systems available currently for the enumeration of CD4+/CD8+ T cells with flowcytometry being the gold standard. [2] In almost all these systems the reagents used are liquid in nature, and hence have to be transported and stored at 2°-8°C. This causes difficulty in geographic areas where the ambient temperature is high in most parts of the year and also in laboratories situated in remote areas. One of the solutions to this difficulty may be the usage of more stable dry format reagents in place of the liquid format reagents. Recently, dry format reagents have been developed by the ReaMetrix (Bangalore, India) which are compatible with both FACSCount (Becton Dickinson, San Jose, CA, USA) and Guava PCA system (Guava Technologies, Hayward, CA, USA). In this pilot study we evaluated the dry format reagents from ReaMetrix (Bangalore, India) with the original reagents from the manufacturer's Becton Dickinson (San Jose, CA, USA) and Guava Technologies (Hayward, CA, USA).


 ~ Materials and Methods Top


Materials

Whole blood samples were collected from 102 HIV-infected individuals who had come to the Clinical Virology department of a tertiary care centre in India (south) during March 2009 through May 2009 for their CD4+/CD8+ T-cell estimation. In addition, blood samples were also collected from 31 normal healthy individuals, who had no major or minor illness for the past 6 months. All these healthy individuals were HIV negative by an HIV antibody-screening test. In order to avoid any diurnal variation in the T-cell subset counts, all the samples were collected between 8:00 AM and 10:00 AM in K 2 /K 3 EDTA vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, USA) after obtaining an informed consent.

Methods

The absolute CD4+ and CD8+ T-cell counts were estimated by the Guava® EasyCD4™ PCA System using the reagents from both Guava technology and Reamatirx (ReaMetrix, Bangalore, India). However, only absolute CD4+ T cell counts were enumerated by FACSCount using reagents from Becton Dickinson (San Jose, CA, USA) and ReaMetrix (ReaMetrix Bangalore, India). The enumeration of the T cell subsets by the FACSCount and Guava® EasyCD4™ PCA system using the respective reagents (liquid format) were performed as reported earlier. [3]

The Reametrix reagents were used as per the manufacturer's instruction. The reagents were formulated in buffered saline with sodium azide and stabilizers. For the Guava technology the ReaPan 3 4 G reagent is provided in dried-down format and was ready-to-use containing anti-CD4 monoclonal antibody and anti-CD3 monoclonal antibody. The CD8 reagent (ReaPan 3 8 G) consisted of anti-CD8 monoclonal antibody and anti-CD3 monoclonal antibody. Briefly 50 ml of the whole blood was added to the tubes, which containing the corresponding reagents, vortexed for 30 s and incubated at room temperature (RT) in the dark for 30 min. Subsequently, 450 ml of the lyzing solution was added, vortexed and incubated for 15 min. This was followed by the analysis of samples using EasyCD4™ System with CytoSoft™ software, version 2.2 in the Guava PCA system. The reagent used for the FACSCount was Rea T-Count CD3/CD4 Reagent. Briefly 50 ml of the whole blood was added to the tubes which contained the corresponding reagents, vortexed for 30 s and incubated at room temperature (RT) in the dark for 60 min. This was followed by the addition of 450 ml of the fixative solution, vortexed and incubated for 15 min. Subsequently, the sample was analyzed in FACSCount with CD4/CD3 soft ware version 1.

Based on the CD4+ T-cell counts obtained in FACSCount with the BD reagents, all the 133 individuals were classified into three groups, that is, group one with CD4 counts <200, group 2 with CD4 counts with 201-500 and group 3 with CD4 counts >500 cells/ml. One individual had the CD8+ T cell estimated using the two reagents showing a very high variation (outlier), this value was excluded from the analysis for CD8+ T-cell counts.

Statistical analysis

The correlation of cell counts estimated by the three different reagents on both Guava® EasyCD4™ System and FACSCount was assessed by the Pearson's correlation test using the Microsoft Excel software. The percentage coefficient of variation (%CV) for the mean cell counts estimated using the different reagents was calculated by the Microsoft Excel software. We also analyzed the data using Bland-Altman plots by displaying the average values of cell counts obtained by two methods using the above three reagents on the x-axis and the difference between any two sets of reagents shown on the y-axis. [4]


 ~ Results Top


In the HIV-infected group, there were 28 females and 74 males with an average age of 34.6 (SD=10.3) and 42.1 (SD=10.8), respectively. The number of males in the control group were 23 with an average age of 28 years (SD=5.9) and the average age of the 8 females was 28.6 (SD=7.2).

The means ± SD of the CD4+ T-cell count obtained by the BD reagents and T-Count CD3/CD4 reagent in the FACSCount were 436 ± 315 and 429 ± 306, respectively. The means ± SD of the CD4+ T-cell count obtained by the Guava® EasyCD4™ System using the Guava reagents and ReaPan 3 4 G reagent were 499 ± 355 and 412 ± 274, respectively. The mean, median, 10 th and the 90 th percentile differences for the absolute CD4+ T cells as determined by FACSCount and Guava® EasyCD4™ System with the 4 different reagents used are shown in [Table 1]. The means ± SD of the CD8+ T-cell count obtained by the Guava® EasyCD4™ System using the Guava reagents and ReaPan 3 8 G reagents were 890 ± 530 and 793 ± 505, respectively. The mean, median, 10 th and the 90 th percentile differences for the absolute CD8+ T cells estimated by the ReaPan 3 8 G reagent and Guava reagents are shown in [Table 2].
Table 1: The mean, median, 10th and 90th percentile differences for the absolute CD4+ T cells as determined by FACSCount and Guava Easy CD4 with their respective reagents and reagents from ReaMetrix among the study population


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Table 2: The mean, median, 10th and 90th percentile differences for the absolute CD8+ T cells as determined by Guava Easy CD4 with reagents from Guava and ReaMetrix among the study population


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The bias between the number of CD4 cells estimated by the FACSCount using the Rea T-Count CD3/CD4 Reagent and BD reagents was 6 cells. The bias between the Guava® EasyCD4™ System using ReaPan 3 4 G reagents and the Guava reagents in the Guava® EasyCD4™ System was 78 cells. The bias between the number of CD4+ T cells estimated by the FACSCount using the BD reagents and Guava reagents by Guava® EasyCD4™ System was 64 cells. The observed bias between the number of CD4 cells estimated using the ReaMetrix reagents with Guava® EasyCD4™ System (ReaPan 3 4 G) and FACSCount (Rea T-Count CD3/CD4) was 17 cells. Bland-Altman plots displaying the average values of cell counts obtained by two methods (FACSCount and Guava) using the three reagents (reagents from ReaMetrix, Guava technology and Becton Dickinson) are shown in [Figure 1].
Figure 1: Bland-Altman plots by displaying the average values of cells counts obtained by two methods using the three reagents on the x-axis and the difference between any two sets of reagents shown on the y-axis. (a) CD4+ T cell estimated by Guava PCA using reagents from Guava and Reametrix, (b) CD4+ T cell estimated by FACSCount using reagents from BD Biosciences and Reametrix, (c) CD4+ T cell estimated by Guava PCA and FACSCount by their respective reagents, (d) CD4+ T cell estimated by Guava PCA and FACSCount by Reametrix reagents, and (e) CD8+ T cell estimated by Guava PCA using reagents from Guava and Reametrix

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Overall, the correlation (r) of the Guava® EasyCD4™ System to the FACSCount for CD4+ T cells estimated by different reagents varied between 0.96 and 0.98. The same for the CD8+ T cells was 0.90. The scatter gram showing the correlation (r) for both CD4+ T cells and CD8+ T cells measured by both techniques with the three different reagents are shown in [Figure 2].
Figure 2: Correlation and 'r' values for CD4+ and CD8+ T-cell counts estimated by Guava PCA with FACSCount using different reagents: (a) CD4+ T cell estimated by Guava PCA using reagents from Guava and Reametrix, (b) CD4+ T cell estimated by FACSCount using reagents from BD Biosciences and Reametrix, (c) CD4+ T cell estimated by Guava PCA and FACSCount by their respective reagents, (d) CD4+ T cell estimated by Guava PCA and FACSCount by Reametrix reagents and (e) CD8+ T cell estimated by Guava PCA using reagents from Guava and Reametrix

Click here to view


The mean %CV calculated for the CD4+ T-cell counts estimated by Guava® EasyCD4™ System using the Guava reagent and ReaPan 3 4 G reagent was 13.6, while the mean %CV calculated for the CD4+ T-cell counts estimated by FACSCount using the BD reagent and Rea T-Count CD3/CD4 Reagent was 6.7. The mean %CV calculated for the CD4+ T-cell counts estimated by the FACSCount using the BD reagents and Guava® EasyCD4™ System by Guava reagents was 12.9. The mean %CV calculated for the CD4+ T-cell counts estimated by the FACSCount and Guava® EasyCD4™ System using the two ReaMetrix reagents was 9.6. The mean %CV calculated for the CD8+ T-cell counts estimated by Guava® EasyCD4™ System using the Guava reagent and ReaMetrix reagent was 13.7.


 ~ Discussion Top


Currently, more and more anti-retroviral treatment (ART) centres are being established in developing countries such as India and this has made the management of HIV-infected individuals more accessible. The success of this ART initiative also depends on the proper monitoring of patients. This requires minimum laboratory facilities to carry out CD4+ T-cell estimation. The quality of the CD4+ T-cell estimation depends on the proper transport and storage of the reagents. Our pilot study to evaluate the dry format reagents for CD4+ and CD8+ T-cell estimation using the Rea T Count, ReaPan 3 4 G and ReaPan 3 8 G reagents manufactured by Reametrix showed that it is a reliable alternative for CD4+/CD8+ T-cell estimation by the FACSCount and Guava® EasyCD4™ System. The correlation (r) of the ReaPan 3 4 G and Guava Easy 4 reagents was 0.97, while the same for the Rea T Count and BD reagents was 0.98.

Bergeron et al. (2009) have carried out an evaluation of the ReaMetrix CD4+ and CD8+ T-cell reagents using the FACSCount and the %CV was found to be 4.2% and 4.8%, respectively. [5] Our CD4+ T-cell results in the FACSCount also were comparable to their findings. In addition, our study also compared the performance of ReaMetrix CD4+ and CD8+ T-cell reagents with reagents from Guava technology. Furthermore, we also analyzed the correlation of cell counts estimated by the ReaMetrix reagents using FACSCount and Guava technology. The analysis and comparison of the CD4+ T-cell count obtained by FACSCount and Guava Technology by their respective reagents in this study also reiterated our earlier findings on the performance of Guava and FACSCount. [3]

The coefficient of variation (%CV) observed in our study with the ReaPan 3 4 G and the Guava reagents for CD4 estimation was 13.6%. Compared to this the %CV for the Rea T Count and BD reagents in the FACSCount was only 6.7%. The %CV observed for the CD8+ T-cell estimation was 13.7% in the Guava® EasyCD4™ System using Guava and ReaMetrix reagents. All %CVs observed for CD4+ and CD8+ T-cell counts were within the expected %CV for the "single platform" systems, that is, between varies between 10% and 18.3%. [6]

We have also analyzed the data using Bland-Altman plots to see the bias in testing as it indicates if the two methods/reagents used agree sufficiently so that they can be used interchangeably. The bias between the CD4+ T-cell count estimated by Guava® EasyCD4™ System using the two reagents was slightly higher (bias −78; 95% CI −272 to +116), than the one measured by the FACSCount using the BD and Rea T Count reagents (bias −6; 95% CI −146 to +134). The variations of the cell counts were found to be higher when the CD4+ T-cell values were high. Our earlier study on evaluation of the CD4+ T-cell counts estimated by Guava® EasyCD4™ System with the FACSCount showed a better bias than one observed now (bias was +25 cells; 95% CI −15 to −34.3). [3] This difference in the bias observed may be due to the larger number of samples with CD4+ T-cell count of >500 cells tested in this study. When we analyzed the bias in the group I of tested individuals, that is, individuals with <200 CD4+ T cells, the performance of the assays was found to be better. The bias between the CD4+ T-cell count estimated by Guava® EasyCD4™ System using the two reagents was only −4 cells (95% CI −40 to +32), while the one measured by the FACSCount using the respective two reagents was 7 cells (95% CI 29 to +15). The bias observed for the CD4+ T-cell counts estimated by Guava® EasyCD4™ System using the Guava reagent and the FACSCount using the BD reagent in this group of individuals was 8 cells (95% CI −21 to +6). The bias in cell number observed for the ReaPan 3 4 G and Rea T Count reagents by the two testing systems in individuals with <200 CD4+ T-cell count was 8 cells (95% CI, 21 to +6). As the cell counts increase the variation reported with CD4+ T-cell counts was also shown to be higher. [7] In this study reported here, the difference in the cell count for all the 133 samples tested by different methods or reagents varied between 6 and 78 cells. However, the difference in the cell counts for the 37 individuals with <200 CD4+ T cell was between 4 and 19 cells.

One of the important advantages of the ReaMetrix reagent is its stability as it is in a dry format. Compared to the reagents from Becton Dickinson and Guava technology, the ReaMetrix reagents are cheaper. The CD3/CD4 reagents from BD cost approximately 8 USD per test and that by Guava PCA is approximately 5 USD. However, the cost of the reagents from Reametrix is approximately 2.5 USD.

To conclude, the evaluation of the Rea T Count, ReaPan 3 4 G and ReaPan 3 8 G reagents manufactured by Reametrix (Bangalore, Karnataka, India) showed a reliable performance compared to the reagents from Becton Dickinson (San Jose, CA, USA) and Guava Technologies (Hayward, CA, USA). One of the important advantages of this new reagent is the dry format as it eliminates the cold-chain transportation and on-site refrigeration at storage without compromising the quality of the assay.

 
 ~ References Top

1.Kaplan JE, Masur H, Holmes KK. USPHS; Infectious Disease Society of America. Guidelines for preventing opportunistic infections among HIV-infected persons-2002. Recommendations of the U.S. Public Health Service and the Infectious Diseases Society of America. MMWR Recomm Rep 2002;51(RR-8):1-52.  Back to cited text no. 1
    
2.Didier JM, Kazatchkine MD, Demouchy C, Moat C, Diagbouga S, Sepulveda C, et al. Comparative assessment of five alternative methods for CD4+ T-lymphocyte enumeration for implementation in developing countries. J Acquir Immune Defic Syndr 2001;26:193-5.  Back to cited text no. 2
[PUBMED]  [FULLTEXT]  
3.Kandathil AJ, Kannangai R, David S, Nithyanandam G, Solomon S, Balakrishnan P, et al. Comparison of Microcapillary Cytometry Technology and Flow Cytometry for CD4+ and CD8+ T-cell Estimation. Clin Diagn Lab Immunol 2005;12:1006-9.  Back to cited text no. 3
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4.Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307-10.  Back to cited text no. 4
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5.Bergeron M, Ding T, Elharti E, Oumzil H, Soucy N, Harmouche H, et al. Evaluation of a dry format reagent alternative for CD4 T-cell enumeration for the FACSCount system: A report on a Moroccan-Canadian study. Cytometry B Clin Cytom 2010;78:188-93.  Back to cited text no. 5
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6.Barnett D, Granger V, Whitby L, Storie I, Reilly JT. Absolute CD4+ T-lymphocyte and CD34+ stem cell counts by single-platform flow cytometry: The way forward. Br J Haematol 1999;106:1059-62.  Back to cited text no. 6
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7.Kunkl A, Risso D, Terranova MP, Girotto M, Brando B, Mortara L, et al. Grading of laboratories on CD4+ T-lymphocyte evaluations based on acceptable data boundaries defined by the measurement error. Cytometry 2002;50:117-26.  Back to cited text no. 7
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