MICROBIOLOGY SECTION - ORIGINAL ARTICLE
Year : 2008 | Volume
: 51 | Issue : 1 | Page : 127--129
Enumeration of CD 4 and CD 8 T lymphocytes in healthy HIV seronegative adults of northwest India: A preliminary study
Anita Rungta, Saroj Hooja, Nitya Vyas, Suman Rishi, Alaka Rao, Sonika Gupta
Department of Microbiology, SMS Medical College, Jaipur, India
A-29, Lal Bahadur Nagar, Opp. Hotel Clarks, Amer, Jaipur - 302 017
CD4 T lymphocyte count is used to measure the progression of HIV infection and to monitor the response to antiretroviral therapy. Information on reference CD4 and CD8 T cell counts in healthy individuals is lacking in northwest India. Samples from 65 HIV-seronegative healthy volunteers (males, 37; females, 28) aged 18 through 59 years were analyzed using FACS (Fluorescent Antibody Cell Sorter) Count TM System. The values of mean and standard deviation of each lymphocyte subpopulation were estimated. The mean ▒ SD of absolute numbers of CD4 and CD8 lymphocytes/Ál was 743.4 ▒ 307.8 and 541.7 ▒ 176.4 in males and 790.7 ▒ 280.4 and 497.03 ▒ 203.6 in females respectively. The range of CD4 counts was 379 to 1800 in males and 321 to 1265 in females. The mean CD4:CD8 ratio was 1.43 ▒ 0.56 in males and 1.78 ▒ 0.76 in females. The results of this study show a wide variability in CD4 counts in the Indian population. A large multicentric study would define normal ranges of CD4, CD8, and CD4:CD8 ratios among the Indian population.
|How to cite this article:|
Rungta A, Hooja S, Vyas N, Rishi S, Rao A, Gupta S. Enumeration of CD 4 and CD 8 T lymphocytes in healthy HIV seronegative adults of northwest India: A preliminary study.Indian J Pathol Microbiol 2008;51:127-129
|How to cite this URL:|
Rungta A, Hooja S, Vyas N, Rishi S, Rao A, Gupta S. Enumeration of CD 4 and CD 8 T lymphocytes in healthy HIV seronegative adults of northwest India: A preliminary study. Indian J Pathol Microbiol [serial online] 2008 [cited 2020 Jan 28 ];51:127-129
Available from: http://www.ijpmonline.org/text.asp?2008/51/1/127/40425
Reference ranges are essential for interpretation of hematological data and for deriving meaningful information in clinical laboratory.
The CD4 T cells are the main targets of HIV, and progressive destruction of these cells is characteristic of all stages of HIV disease. 
The enumeration of CD4 T lymphocytes in the peripheral blood is an essential tool for the laboratory monitoring of HIV-infected patients in order to predict the progression of disease and response to antiretroviral therapy. 
The mean values of CD4 cells need to be documented in order to help assess the degree of changes required for developing symptoms of immunodeficiency. Also, the lower normal limits of CD4 counts are important in the routine diagnosis for interpreting putative HIV-associated changes.  Reference ranges of different types of lymphocytes in peripheral blood of healthy individuals have been well laid out for western countries , and are being used in India.
Ranges of lymphocyte populations in Indians have been reported, but the information is based upon a relatively small number of blood samples generally confined to certain geographical areas of the country. 
Since the expected normal values may vary depending upon the age, sex, or race of the patient, it is necessary to determine the variations of T lymphocyte subsets encountered in normal healthy individuals of that geographical region.
The present study was undertaken to obtain baseline data of CD4 count, CD8 count, and CD4/CD8 ratio among the healthy population in this part of the country, for use in interpretation of these prognostic markers in HIV-infected persons and to assess the feasibility of extending it to a larger study group with the purpose of establishing a normal reference range in the Indian population.
Materials and Methods
This study was carried out in the Voluntary Counseling and Testing Centre (VCTC) in the Department of Microbiology, Sawai Man Singh Medical College (SMSMC), Jaipur, from January 2003 to December 2006. The VCTC also has a center for enumerating CD4 count, CD8 count, and CD4:CD8 ratio under National AIDS Control Programme.
Sixty-five apparently healthy HIV-seronegative volunteers aged 18 through 59 years were included. Subjects willing to participate in the study were enrolled after obtaining the basic personal information. Informed verbal consent was obtained from each subject. The exclusion criteria included :
i. History of infections/communicable diseases including viral infections, gastrointestinal infections, accident, trauma, etc., within the past six months
ii. Any major surgery within the past six months
iii. Pregnancy/Lactation within the past six months
iv. Allergy to drugs within the past six months
v. Active drug abuse
vi. Vaccination within the past six months
vii. Any chronic infection (autoimmune diseases)
Sample collection and processing
Four milliliters of blood was collected at a fixed time between 10 AM and noon. Two milliliters of blood was transferred to a sterile vial for HIV serology, and 2 mL to a K2/K3 EDTA vacutainer tube for T lymphocyte subset counts.
Screening test for HIV: After separating the serum, all samples were subjected to a screening ELISA test (Microlisa HIV J. Mitra and Co. Pvt. Ltd.) for HIV antibodies. All 65 volunteers were HIV negative.
Enumeration of CD4 and CD8 lymphocytes and CD4:CD8 ratio
Immunophenotyping of lymphocytes was carried out by FACS (Fluorescent Antibody Cell Sorter, Becton Dickinson, Singapore, BD) Count system by using 100 Ál of whole blood. It is a microbead-based single-platform instrument designed specifically for enumerating the absolute CD4, CD8, and CD3 T cell counts in no-lyse, no-wash whole blood. All samples were stained on the same day and tested within two hours.
Quality Control: All tests were performed according to the manufacturer's recommendations. Manufacturer's guidelines were strictly adhered to with regard to biosafety practices, trouble-shooting, and maintenance of equipment. The laboratory also participates in the external quality assurance scheme (EQAS) conducted by National AIDS Research Institute (NARI), Pune.
The values of mean and standard deviation of each lymphocyte subpopulation were generated using a Monte Carlo procedure for non-Gaussian distribution. Test of significance was done by calculating the standard error of difference between two means.
Of the 65 subjects, 37 (56.92%) were males and 28 (43.07%) were females. The maximum number of subjects were males aged >30 years. The mean age was 33.56 ▒ 7.15 years (range, 18-59 years). The mean age of men was 33.83 ▒ 6.35 years (range, 22-45 years). The mean age of women was 33.21 ▒ 8.21 years (range, 18-59 years).
The mean CD4 count in the study population was 763.81 ▒ 297.51cells/Ál. The mean CD4 count in men and women was 743.4 ▒ 3078 cells/Ál and 790.78 ▒ 280.4 cells/Ál respectively. The CD4 counts in 13 (20%) of the 65 controls were et al.  reported a mean of 1036 cells/Ál ( n = 26) in healthy individuals, while Bofill M et al.  reported a mean of 830 cells/Ál ( n = 676). In a study in Africa, Serge Diagbough et al.  reported a mean of 943 cells/Ál ( n = 39).
Wide variations in mean CD4 count have also been reported from studies conducted in different parts of India. Kannangai et al.  reported a mean of 1048 cells/Ál ( n = 46), while Singh et al.  reported a mean of 848 cells/Ál ( n = 14) in normal healthy individuals. Our study reported a mean of 763.81 cells/Ál, which compares well to a study reported in South India, who have reported a mean of 799cells/Ál ( n = 99).  The mean CD4 counts among normal controls in our study were significantly lower than those documented in the Western population and parallelled those of the Chinese. 
The CD4 counts in 13 (20%) of the controls in the present study were 
Thus in populations with inherently low CD4 counts, in healthy controls, a lower cut off value would have important clinical implications.
The absolute CD8 counts in our study were comparable with those of the West ,, but were significantly lower than a study in south India that reported a CD8 cell count of 919.6 cells/Ál. 
Categorization of data based upon sex of the volunteers indicated a significantly higher CD4 count in females than in males. Our findings are consistent with a multicentric study done by the Indian Council of Medical Research.  It is possible that sex hormones influence lymphocyte subpopulations and may be responsible for these sex-related differences.
On stratification of volunteers into two age groups, 30 years, CD4 counts were higher in both males and females in the younger age group. Age had no influence on the distribution of CD8 cells in men; but in women, the CD8 cells decreased with age, leading to a higher CD4:CD8 ratio.
The mean CD4:CD8 ratio in the present study was 1.43 ▒ 0.56 in men and 1.78 ▒ 0.76 in women. In a multicentric study done in India, the CD4:CD8 ratio was significantly lower in the southern parts than in the northern and western parts of the country. This lower ratio was attributed to lower values of CD4 count and higher values of CD8 cells among healthy individuals seen in these states. Healthy controls from northern and western parts of the country had CD4:CD8 ratios above 1.3, whereas the ratio was 0.91 and 0.99 for Tamil Nadu and Kerala respectively. These results suggest that significant geographical differences may occur in lymphocyte subpopulations, and a relatively lower value or CD4:CD8 ratio may be normal for subjects from some of the southern states. The reasons for these geographical differences are not clear but may be related to environmental factors. 
The mean CD4:CD8 ratio in our study was 1.58 ▒ 0.68, which is higher than the national average of 1.16 ▒ 0.41. 
The results of this study emphasize that there is heterogeneity in the data even in the Indian population.
Thus, it is important to have baseline data on normal healthy individuals in a particular region, facilitating the interpretation of results in HIV-infected and other immunodeficient patients. These reference ranges can be used to guide clinical decisions. A larger group should be studied to thoroughly assess the variations in lymphocyte subpopulations in people residing in different states of India.
|1||Singh YGK, Dar L, Singh, Brajachand NG. Levels of CD4 and CD8 among the inhabitants of Manipur, India. J Commun Dis 2000;32:201-6.|
|2||Pattanapanyasat K, Tharar RM. CD4 T cell count as a tool to monitor HIV progression and antiretroviral therapy. Indian J Med Res 2005;121:539-49.|
|3||Bofill M, Janossy G, Lee CA, MacDonald-Burns D, Phillips AN, Sabin C, et al . Laboratory control values for CD4 and CD8 lymphocytes: Implications for HIV diagnosis. J Clin Exp Immunol 1992;88:243-52.|
|4||Reichert T, Debruyere M, Deneys V, T φtterman T, Lydyard P, Yuksel F, et al . Lymphocyte subset ranges in adult Caucasians. Clin Immunol Immunopathol 1991;60:190-208.|
|5||Saxena RK, Choudhry V, Nath I. Normal ranges of some select lymphocyte sub- populations in peripheral blood of normal healthy Indians. Curr Sci 2004;86:969-75.|
|6||Tollerud DJ, Clark JW, Brown ML, Neuland CY, Pankiw-Trost LK, Blattner WA, et al . The Influence of age, race and gender on peripheral blood mononuclear cell subsets in healthy non-smokers. J Clin Immunol 1989;9:214-22.|
|7||Diagbouga S, Durand G, Sanou TP, Dahourou H, Ledru E. Evaluation of a quantitative determination of CD4 and CD8 molecules as an alternative to CD4 and CD8 lymphocyte counts in Africans. Trop Med Int Health 1999;4:79-84.|
|8||Kannangai R, Prakash KJ, Ramalingam S, Abraham OC, Mathews KP, Jesudason MV, et al . Peripheral CD4/CD8 T- lymphocyte counts estimated by an immunocapture method in the normal healthy South Indian adults and HIV seropositive individuals. J Clin Virol 2000;17:101-8.|
|9||Ramalingam S, Kannangai R, Zachariah A, Mathai D, Abraham C. CD4 counts of normal and HIV infected South Indian adults: Do we need a new staging system. Nat Med J India 2001;14:335-9.|
|10||Kam KM, Leung WL, Kwok MY, Hung MY, Lee SS, Mak WP. Lymphocyte sub-population reference ranges for monitoring human immunodeficiency virus-infected Chinese adults. Clin Diagn Lab Immunol 1996;3:326-30.|