Evaluation of T Cell Subsets by an Immunocytochemical Method Compared to Flow Cytometry in Four Countries

Abstract

The authors tested an alternative method for CD4 and CD8 T lymphocytes enumeration, the immunoalkaline phosphatase method (IA), in three African countries and in Denmark. The IA determinations from 136 HIV antibody positive and 105 HIV antibody negative individuals were compared to the corresponding results obtained by flow cytometry (FC) performed in the respective countries. The authors found good correspondence between the two methods for measurements of CD4 and CD8 T lymphocytes independent of serological status and geographical site. However, the CD4 and CD8 T lymphocyte values obtained by the two methods are not interchangeable as IA compared to FC consistently gives higher percentage of CD4 T lymphocytes, and lower percentages of CD8 T lymphocytes. Mean differences between the two methods did not differ between the three African countries indicating that the IA method provides systematic results. Replicate measurements suggested good correspondence between results obtained by IA. By using an IA level of < 300 CD4 T lymphocytes/ml, the sensitivity was 81% and specificity 96% for detecting an FC level of < 200 CD4 T lymphocytes/ml. Using an IA level of < 20% CD4 T lymphocytes, the sensitivity was 89% and specificity 95% for detecting an FC level of <14% CD4 T lymphocytes, The FC and IA methods had the same internal correspondence between low absolute CD4 T cell counts and low CD4 percentages; the sensitivity and specificity for detecting a low absolute CD4 T cell count with a low CD4 percentage was 92% and 68% for FC and 91% and 73% for IA, respectively. The IA method is 10-fold cheaper than FC, is independent of advanced laboratory facilities, and does not need immediate processing of samples as blood smears can be stored for long periods. The IA method is therefore suitable for use in areas with limited resources and laboratory facilities where there is a need for immunological surveillance in hospital or community studies.

Life history theory and the immune system: Steps toward a human ecological immunology

“…Immune factors in blood provide more information, and a number of methods have been developed for small quantities of whole blood collected from a simple finger stick. Blood collected on filter paper can be used to analyze C-reactive protein (acute-phase protein) and antibodies against Epstein-Barr virus (indirect measure of cellmediated immunity) (McDade et al, 2000a,b), and whole blood smears on slides can be used to quantify white blood cell fractions and lymphocyte subsets (Lisse et al, 1990(Lisse et al, , 1997b. In addition, lymphocyte proliferation protocols have been developed that require less than a drop of whole blood, and recent advances in immunoassay technology support the simultaneous quantification of multiple cytokines (Bloemena et al, 1989;Carson and Vignali, 1999;Elsasser-Beile et al, 1991).…”

Enumeration of CD4 and CD8 T-cells in HIV infection in Zimbabwe using a manual immunocytochemical method

“…4 Nonetheless, because of the higher CD4 and lower CD8 Tcell percentage, we found that the IA method consistently gave higher CD4/CD8 ratios than those derived from FC. The wide limits of agreement suggest wide variation in individual measurements by the IA method and would likely be much lower with larger sample sizes.…”

“…However, the two methods are not interchangeable because the IA method gives consistently higher CD4 counts and percentages as has been observed by others. 3,4 There is no obvious explanation for the higher counts but this has been attributed to a qualified identification of lymphocytes by the manual method, whereas FC could miss CD4 T-cells weakly stained by the fluorochrome.3,15 Alternatively, in the IA method, CD4 positive monocytes could be mistaken for CD4 T-cells.…”

Thymic function and T cell parameters in a natural human experimental model of seasonal infectious diseases and nutritional burden

“…Persistent infectious burden rather than low thymic output may also be responsible for the lower TRECs; since elevated cell proliferation from antigenic exposure is known to dilute TREC concentrations[49]. Our findings, at the population level, that the T cell subsets are comparable to those of healthy individuals from the sub region, [50-53] imply that poor T cell immunity may be common here. The lack of observable differences arising from the further analyses by birth weight category (higher/lower than the population median) is consistent with the overall findings but may have been confounded by the resultant reductions in numbers.…”