Date Published: October 3, 2011
Publisher: BioMed Central
Author(s): Madhuri R Thakar, Philip R Abraham, Sunil Arora, Pachamuthu Balakrishnan, Bhaswati Bandyopadhyay, Ameeta A Joshi, K Rekha Devi, Ravi Vasanthapuram, Madhu Vajpayee, Anita Desai, Janardhanan Mohanakrishnan, Kanwar Narain, Krishnangshu Ray, Shilpa S Patil, Ravinder Singh, Anuj Singla, Ramesh S Paranjape.
CD4+ T lymphocyte counts are the most important indicator of disease progression and success of antiretroviral treatment in HIV infection in resource limited settings. The nationwide reference range of CD4+ T lymphocytes was not available in India. This study was conducted to determine reference values of absolute CD4+ T cell counts and percentages for adult Indian population.
A multicentric study was conducted involving eight sites across the country. A total of 1206 (approximately 150 per/centre) healthy participants were enrolled in the study. The ratio of male (N = 645) to female (N = 561) of 1.14:1. The healthy status of the participants was assessed by a pre-decided questionnaire. At all centers the CD4+ T cell count, percentages and absolute CD3+ T cell count and percentages were estimated using a single platform strategy and lyse no wash technique. The data was analyzed using the Statistical Package for the Social Scientist (SPSS), version 15) and Prism software version 5.
The absolute CD4+ T cell counts and percentages in female participants were significantly higher than the values obtained in male participants indicating the true difference in the CD4+ T cell subsets. The reference range for absolute CD4 count for Indian male population was 381-1565 cells/μL and for female population was 447-1846 cells/μL. The reference range for CD4% was 25-49% for male and 27-54% for female population. The reference values for CD3 counts were 776-2785 cells/μL for Indian male population and 826-2997 cells/μL for female population.
The study used stringent procedures for controlling the technical variation in the CD4 counts across the sites and thus could establish the robust national reference ranges for CD4 counts and percentages. These ranges will be helpful in staging the disease progression and monitoring antiretroviral therapy in HIV infection in India.
CD4 + T lymphocytes play a central regulatory role in the immune response. The decrease in CD4+ T cell numbers can compromise the normal immune functions of the body. The number of CD4+ T cells in circulation provides important information about the immune competence of an individual. Hence estimation of CD4+ T cells is an important parameter in immune deficiency disorders. The clinical applications of immunophenotyping of CD4+ T cells include the diagnosis of immunodeficiency disorders, the evaluation of immune-mediated diseases, the assessment of immune reconstitution following stem cell transplantation and the monitoring of disease progression in Human Immunodeficiency Virus (HIV) infection.
We have established the national reference range for CD4+ T cell subset, both for CD4 percentages and absolute CD4+ T cell counts, using the uniform protocol at eight centres across the country and enforcing strict quality control for a single-platform method. We have used the lyse-no wash procedure for CD4+ T cell count estimation which has been reported to control the inter laboratory variation . This has reduced the chances of the variability. All the seven sites used FACSCalibur for absolute CD4 count enumeration while the EPIC XL flow cytometer from Coulter was used at one centre from South India. Before initiation, the inter equipment variability between these two equipment was determined using the stabilized blood samples. The percent CV was found to be very good, below 10%. The variability was also determined during the study using the samples received from UKNEQAS for proficiency and the %CV was found to be less than 10% making the results from both the equipments comparable. The blood sample was uniformly collected in the morning to reduce the diurnal variation as the diurnal variation is reported to add as high as 20% variation in the CD4+ T cell counts .
The authors declare that they have no competing interests.
MT designed the study, implemented at the respective site, performed the data analysis and drafted the manuscript. PA designed the questionnaire, developed SOP’s for all sites, implemented study and observed quality control at NARI. SA, PB, BB, AJ, KR, RV, MV, KR implemented the study at the respective sites. AD, JM, KN, SP, and RS & AJ carried out the flow cytometry and observed the quality control at the respective sites. RP participated in designing the study and writing the manuscript.