CD4⁺ T‐lymphocyte enumeration with a flow‐rate based method in three flow cytometers with different years in service

Abstract

Background: CD4 1 T-lymphocyte count remains the most important surrogate marker for management of HIV patients in resource-poor countries. The standard single-platform (SP) bead-based flow cytometric method for CD4 1 testing is expensive; more affordable methods are needed. We evaluated the SP flowrate-based calibration method for determining CD4 1 counts, using three flow cytometers of varying ages.Methods: CD4 1 counts from 103 HIV-1 infected Thai patients were determined using a SP flow-rate method in flow cytometers with 2, 12, and 16 years of service. Results were compared to the bead-based method. Correlation and agreement were analyzed using linear regression and Bland-Altman analysis.Results

Enumeration of the absolute CD4 T‐lymphocyte count by cell‐bead assay

“…Although this method is accurate, it is labor-intensive and expensive (US$ 19-20, a range of cost per one sample), because the samples have to be added to every CLB tube. FR was introduced by Storie and colleagues for determination of absolute CD4þ T-lymphocyte counts and was found to be userfriendly, reliable and reproducible with low-interlaboratory variation (7)(8)(9). However, a limitation with this method is that the calibration factors needs to be applied to adjust for differences in viscosity between the latex-bead suspension and clinical samples, a fact that increases the cost of analysis (US$ 10-11, a range of cost per one sample).…”

“…This CB was used as a counting bead for generating the reference of a precise sample acquiring volume on the flow rate-based assay as described earlier (9). RBCs were obtained aseptically from a healthy volunteer by venipuncture into a sterile EDTA tube.…”

“…Recently, a volumetric-based method called ''flow ratebased calibration (FR)'' for measuring the absolute number of CD4þ T-lymphocyte has been proposed (7)(8)(9). This approach removes the need for the use of latex bead to each sample, which significantly reduces the costs of analysis.…”

“…This approach removes the need for the use of latex bead to each sample, which significantly reduces the costs of analysis. Although FR method is accurate, it is still labor intensive and expensive, because the CLB need to be used for assessment of the calibration factor to adjust for differences in viscosity between latex-bead suspensions and clinical samples (7)(8)(9). With the burden of high numbers of HIV-infected patients, especially in the countries that has the limitation of the resources, the availability of a better method should allow better and more cost-effective monitoring of HIV infection in such countries.…”

A routinely applicable way for using FCM in cell enumeration with CFSE‐labeled CellBeads as internal standard

“…The third and most recently developed method uses the flow-rate of samples in FCM to enumerate the absolute number of relevant cells (7). Flow-rate based SP can be accomplished using conventional FCM; however, it is still unconfirmed whether or not this protocol is appropriate for wide and routine use (8).…”