in india , the national aids control organization ( naco ) has initiated the introduction of inexpensive and generic highly active anti - retroviral therapy ( haart ) . current recommendations in western countries for initiation and monitoring of haart are based on cd4 t - cell counts and plasma human immunodeficiency virus ( hiv ) rna levels . however , these methods are expensive . due to this , the world health organization ( who ) stipulates that cd4 count testing is  desirable  but not essential for haart use in resource - limited settings . several studies have demonstrated the usefulness of absolute lymphocyte count ( alc ) or total lymphocyte count ( tlc ) , ( i.e. alc plus all large lymphocytes such as lymphoblasts or reactive lymphocytes ) in identifying patients who would benefit from initiating prophylaxis for acquired immunodeficiency syndrome ( aids)-related opportunistic infections.[47 ] we conducted this study to evaluate the correlation of tlc and alc to cd4 count and to determine a range of tlc and alc cut - offs for initiating haart in hiv - infected patients , as there are fewer published studies on this subject , from resource - limited settings . a prospective observational cohort study involving 108 hiv - positive patients , attending our art centre , from august 20 2006 onwards were selected . the duration of study period was 1 year from august 20 , 2006 to august 19 , 2007 . after taking an informed consent ( for hiv testing ) , these individuals , voluntarily attending our ictc at the department of microbiology ( or any of the government designated ictcs ) , underwent pre - test counseling by male or female ictc counselors , followed by hiv testing as per the strategy iii of the naco guidelines ( for hiv testing ) . after post - test counseling , those found hiv positive were referred to the art centre , k.r . hospital , mysore medical college and research institute , mysore , where they underwent pre - art counseling . after clinical evaluation , informed consent was taken from these patients and they were enrolled into the study if they satisfied the inclusion criteria . those found eligible for art as per the who guidelines were started on anti - retroviral therapy . the individuals should be above 18 years of age , they should be proven to be hiv - positive and they should not be on prior anti - retroviral therapy ( art ) . using standard precautions , 4 ml of venous blood was collected between 9 am to 12 noon using two 2 ml k3-edta vacutainers ( bd ) , one for cd4 testing and the other for complete blood counts ( cbcs ) . the cd4/cd3 enumeration was done using the single platform bd facs calibur machine ( becton , dickinson and company , san jose , united states of america ) , by strictly following the manufacturer 's instructions . internal quality control was performed with process controls using the manufacturer 's recommendations . external quality control was performed through an external quality assurance program with nari ( national aids research institute ) , pune , india . cbcs with differential were performed by using a sysmex k21 hematology analyzer ( sysmex corporation , kobe , japan ) . tlc was derived from the cbc by multiplying lymphocyte percentage by the white blood cell count . spearman correlations between alc and cd4 cell count and tlc and cd4 cell count were assessed . sensitivity , specificity , positive predictive value , and negative predictive values of various alc and tlc cut - offs were computed for cd4 count < 200 cells / cu.mm . all statistical analyses were performed using spss software ( version 16.0 , spss , chicago , usa ) . a prospective observational cohort study involving 108 hiv - positive patients , attending our art centre , from august 20 2006 onwards were selected . the duration of study period was 1 year from august 20 , 2006 to august 19 , 2007 . after taking an informed consent ( for hiv testing ) , these individuals , voluntarily attending our ictc at the department of microbiology ( or any of the government designated ictcs ) , underwent pre - test counseling by male or female ictc counselors , followed by hiv testing as per the strategy iii of the naco guidelines ( for hiv testing ) . after post - test counseling , those found hiv positive were referred to the art centre , k.r . hospital , mysore medical college and research institute , mysore , where they underwent pre - art counseling . after clinical evaluation , informed consent was taken from these patients and they were enrolled into the study if they satisfied the inclusion criteria . those found eligible for art as per the who guidelines were started on anti - retroviral therapy . the individuals should be above 18 years of age , they should be proven to be hiv - positive and they should not be on prior anti - retroviral therapy ( art ) . the individuals should be above 18 years of age , they should be proven to be hiv - positive and they should not be on prior anti - retroviral therapy ( art ) . using standard precautions , 4 ml of venous blood was collected between 9 am to 12 noon using two 2 ml k3-edta vacutainers ( bd ) , one for cd4 testing and the other for complete blood counts ( cbcs ) . the cd4/cd3 enumeration was done using the single platform bd facs calibur machine ( becton , dickinson and company , san jose , united states of america ) , by strictly following the manufacturer 's instructions . external quality control was performed through an external quality assurance program with nari ( national aids research institute ) , pune , india . cbcs with differential were performed by using a sysmex k21 hematology analyzer ( sysmex corporation , kobe , japan ) . tlc was derived from the cbc by multiplying lymphocyte percentage by the white blood cell count . spearman correlations between alc and cd4 cell count and tlc and cd4 cell count were assessed . sensitivity , specificity , positive predictive value , and negative predictive values of various alc and tlc cut - offs were computed for cd4 count < 200 cells / cu.mm . all statistical analyses were performed using spss software ( version 16.0 , spss , chicago , usa ) . the mean age of this cohort was 34.69 years . of the 108 patients , 71 ( 65.74% ) were males and 37 ( 34.26% ) were females . 90 ( 83.4% ) of the patients stated that they acquired hiv through heterosexual intercourse . during the study period , the mean cd4 count and mean tlc at baseline were 129.6576.84 cells / cu.mm and 1262.96738.98 cells / cu.mm , respectively . the correlations between alc and a cd4 cell count , and between tlc and a cd4 cell count were highly significant ( spearman correlation coefficients of r=0.5604 , p<0.0001 and 0.3497 , p<0.001 respectively ) . the positive predictive value ( ppv ) , negative predictive value ( npv ) , sensitivity , specificity , and likelihood ratio of alc and tlc for cd4 count < 200 cells / mm in all paired counts are given in tables 1a and 1b , respectively . positive predictive value , negative predictive value , sensitivity and specificity of absolute lymphocyte count for cd4 count < 200 cells / cu.mm in all paired counts ( n=108 ) positive predictive value , negative predictive value , sensitivity , and specificity of total lymphocyte count for cd4 count < 200 cells/ cu.mm in all paired counts ( n=108 ) an alc of 1400 cells / cu.mm or less had maximal combined sensitivity , 71.08% ( 95% ci : 60.11 - 80.51% ) , and specificity , 78.26% ( 95% ci : 56.26 - 92.53% ) , for a cd4 cell count of less than 200 cells / cu.mm . however , a tlc cut - off value of 1200 cells / cu.mm l had a sensitivity of 63.41% [ ( 95% ci : 52.09 to 73.74% ) and a specificity of 69.57% ( 95% ci : 47.10 to 86.80% ) , with p=0.0081 , considered very significant ] . notably , alc and tlc of less than 800 cells / cu.mm had a positive predictive value of 100.00% and 96.15% , respectively , for a cd4 cell count < 200 cells / cu.mm . similarly , the ( ppv , npv , sensitivity , specificity , and likelihood ratio of alc and tlc for cd4 count 200 - 350 cells / cu.mm in all paired counts are given in tables 2a and 2b , respectively . positive predictive value , negative predictive value , sensitivity and specificity of absolute lymphocyte count for cd4 count 200 to 350 cells / cu.mm in all paired counts positive predictive value , negative predictive value , sensitivity and specificity of total lymphocyte count for cd4 count 200 to 350 cells / cu.mm in all paired counts in this study , we have demonstrated that both tlc and alc can be used in the place of cd4 count as a routine marker of immune status . in this cohort of indian patients , there was a good correlation between tlc and cd4 count by spearman rank order correlation ( r=0.3497 ) , indicating a moderately positive association between tlc and cd4 counts . however , spearman correlations between tlc and cd4 count reported in north america ( r=0.77 ) , england ( r=0.76 ) , and india ( r=0.744 ) were higher , when compared with this study . this difference could be due to the small size of the study sample . however , the correlation between alc and cd4 counts in this study was higher ( r=0.5604 , p<0.0001 ) , indicating that alc was a better marker when compared with tlc as a surrogate for cd4 counts . we found that an alc of 1400 cells / cu.mm was 71.08% sensitive and 78.26% specific for a cd4 cell count < 200 cells / cu.mm . however , jacobson et al . reported similar findings at a slightly higher alc cut - off of 1500 cells / cu.mm . but , we found that all the four statistical indices ( ppv , npv , sensitivity , and specificity ) maximally aggregated at tlc < 1200 cells / cu.mm for cd4 < 200 cells / cu.mm , thus proving that the who recommended cut - off ( of 1200 cells / cu.mm ) was adequate though the sensitivity was less . a critical issue in taking an alc cut - off of 1400 cells / cu.mm is that a proportion of patients with a cd4 cell count < 200 cells / cu.mm who would be misclassified by alc as having a cd4 cell count greater than 350 cells / cu.mm , and who would thus mistakenly have antiretroviral treatment deferred . using an alc of 1400 cells / cu.mm or less as the threshold to initiate haart , 24 of the 108 patients ( 25.92% ) with a cd4 cell count < 200 cells / cu.mm would have been misclassified as not being in need of antiretroviral treatment , by having a simultaneous alc greater than 1400 cells / cu.mm . of note is the fact that 7 of these 108 ( 7.56% ) patients had a cd4 cell count < 100 cells / cu.mm and although at very high risk of opportunistic infection , would have been misclassified as not needing antiretroviral treatment , by an alc greater than 1400 cells / cu.mm . alc and tlc cut - off values could be defined in such a way that ppv is very high , so that cd4 cell enumeration might not be needed to initiate haart . however , lower alc and tlc values of < 800 cells / cu.mm can not be taken as cut - offs [ in spite of high ppv ( 100% for alc and 96.15% for tlc respectively ) ] because of the low sensitivity . the latest who guidelines on anti - retroviral therapy recommend that all adolescents and adults including pregnant women with hiv infection and cd4 counts of 350 cells / cu.mm should start art , regardless of the presence or absence of clinical symptoms . we therefore analysed the data from those patients with cd4 counts of 200 - 350 cells / cu.mm to evaluate the performance of alc and tlc . the positive predictive values ( ppv ) for alc and tlc at this higher cd4 count of 200 - 350 cells / cu.mm were poor [ tables 2a and 2b ] . we were also not able to draw any further conclusions as the results obtained were not statistically significant though the sensitivity and npv maximally aggregated at alc of < 1600 cells / cu.mm and tlc of < 1400 cells / cu.mm , respectively . we believe that this discrepancy was mainly due to the small size of the study sample . hence , more studies with larger sample sizes need to be done to analyse this aspect . the main limitations of this study were the small size of the study sample , which has an implication on determining the cut - offs for both alc and tlc . the second limitation was that we could not rule out the latent cases of intercurrent infections like tuberculosis ( tb ) and malaria , which might affect the interpretation of the alc / tlc . tb - related immune activation and anti - tb therapy may lead to fluctuations in cd4 cell count . malaria is another prevalent endemic disease known to have specific interactions with hiv infection . though its effect on cd4 cell count is less well documented , patients with hiv infection are known to have an increased likelihood of developing malaria , as well as a decreased response to prophylaxis . thus , further studies are needed to examine this aspect of impact of tb and malaria based on the correlation between tlc / alc and cd4 cell counts . our findings suggest that both alc and tlc could have clinical utility in determining when hiv - infected patients in resource - poor settings should initiate haart although alc is a better marker than tlc . however , more studies are required in resource - limited settings with larger study groups to ascertain the usefulness of alc / tlc as a surrogate for cd4 counts both before and after haart initiation .