Comparison of CD4 cell count by a simple enzyme-linked immunosorbent assay using the TRAx CD4 test kit and by flow cytometry and hematology

Abstract

Measurement of CD4 T-lymphocyte levels is clinically useful in monitoring immune status in a number of conditions, including human immunodeficiency virus (HIV) infection, in which the absolute CD4 count is used to guide therapy. The absolute CD4 count is obtained by multiplying the results of the leukocyte count and the differential with a hematology cell counter and the percentage of CD4 ؉ T lymphocytes determined by flow cytometry. These techniques require expensive, complex instrumentation, and interlaboratory results are difficult to standardize and reproduce. The rapid growth of HIV infection worldwide has increased the need for more-reproducible and cost-effective methods for CD4 T-cell monitoring. The TRAx CD4 test kit is based on a novel adaptation of conventional enzyme-linked immunosorbent assay (ELISA) and permits the simple quantitation of total CD4 protein from whole-blood lysates. In this study, the relationship between total CD4 protein measured in units per milliliter (TRAx) and in cells per microliter (flow cytometry and hematology) was defined in a multisite clinical study using linear regression analysis. Data from 230 HIV-seronegative and 321 HIV-seropositive specimens were used to calibrate the TRAx assay recombinant CD4 standards and controls in equivalent CD4 T lymphocytes per microliter (cells per microliter). The calibration of the TRAx CD4 assay in cells per microliter was validated with a second group of specimens from 17 healthy volunteers and 20 HIV-seropositive patients which were collected and tested under strictly controlled conditions intended to minimize the effects of specimen aging on the results of the reference method. These data were also used to estimate the variability of absolute CD4 count by cytometric methods as well as the precision of the TRAx CD4 result after it was calibrated in cells per microliter. Overall, correlations between the two methods ranged from 0.87 to 0.95. Additional studies demonstrated that the contribution of CD4 protein from monocytes and any soluble CD4 in sera are negligible in the TRAx assay and do not significantly affect results. This new method represents a promising alternative to absolute CD4 T-cell enumeration by flow cytometry and hematology.

Evaluation of a method for counting absolute numbers of cells with a flow cytometer

“…It is believed that the variability of this measurement (flow cytometry combined with WBC count and differential) could be lowered by using a direct measurement of the number of cells rather than a counting method with multiple steps. Other methods for performing CD4 cell counts require dedicated instruments (5,8) or kits that measure cell products or characteristics of groups of cells (3,9). Recently, methods for measuring cells directly from the flow cytometer have been developed (7) (Flow Count [Coulter Immunology], ImmunoCount [Ortho Diagnostics], and TruCount [Becton Dickinson Immunocytometry Systems]).…”

“…Single-platform tests for determining the number of CD4 ϩ T cells have included simple technologies dedicated to obtaining absolute numbers for CD4 and CD8 T cells (3,5,8,9). Recently, tests that permit these measurements to be made di-rectly on clinical flow cytometers have been developed.…”

AIDS in an HIV-seronegative Ghanaian woman with intersubtype A/G recombinant HIV-1 infection

“…After obtaining verbal informed consent, peripheral blood mononuclear cells (PBMCs) and plasma were isolated from venous blood collected in cell preparation CPT tubes (Becton Dickinson, Franklin Lakes, NJ) and stored at −70°C. CD4 + cell level was determined with the TRAx enzyme immunoassay as previously described [Paxton et al, 1995;Adu-Sarkodie et al, 1998].…”

Selection of lymphocyte gating protocol has an impact on the level of reliability of T‐cell subsets in aging specimens

“…The accurate enumeration of these cells both as absolute and percentage values remains crucial in providing care to HIV‐1–infected individuals and assessing new antiretroviral drugs and therapeutic vaccines. Previous studies on absolute counts have identified substantial variability in results between laboratories (4, 5). The introduction of multicolor immunophenotyping has made it possible to harness lineage‐specific markers (6, 7).…”

“…It has been known for over a decade that the age of the blood sample has a significant impact on the accuracy and reproducibility of the T‐cell subset measured with the traditional lymphocyte gating technology (5). In this study, we assessed the impact of specimen aging (up to 4 days) on the absolute number and percentage of T‐cell subsets with four gating protocols.…”