Flow rate calibration II: A clinical evaluation study using PanLeucoGating as a single‐platform protocol

Abstract

Background: CD4؉ T-lymphocyte enumeration is vital for monitoring disease progression in individuals positive for the human immunodeficiency virus (HIV), and as a result, there is a need to develop costeffective protocols that provide accuracy, precision, and affordability. Recently, PanLeucoGating has been shown to fulfill these requirements; however, although comparable to state-of-the-art single-platform protocols (SP), there is still a requirement for an accurate total white cell count. To overcome this limitation, we recently developed a flow-rate based calibration method that enables the PanLeucoGating protocol to be used as a SP approach, and in this study show that this approach can be used for CD4؉ T-lymphocyte enumeration.Methods: A total of 113 HIV samples were analyzed using three protocols: (a) state-of-the art SP bead-based method (MultiSet; predicate protocol), (b) PanLeucoGating protocol used as a dual-platform (DP) approach, and (c) the newly developed flow rate-based SP approach. We demonstrate that flow rate calibration can be achieved easily and that the method is highly comparable to the state-of-the-art SP method.Results

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

“…In addition, the bead-based technology is marginally less reproducible than volumetric SP technology (20), as errors may occur if the ratio of the number of beads added and the number of cells in the sample is altered with time, and/or if the beads are not equally dispersed through the samples (7,21). A recent breakthrough is the development of a flowrate based calibration method that obtains the absolute CD4 1 counts without the need for microsphere beads in the process (11)(12)(13). We have, therefore, adapted the use of the flow rate calibration method as an SP approach in our search for a cost-effective routine enumeration of CD4 1 T-lymphocyte counts using flow cytometers that have been in service for more than 10 years.…”

“…This can be assessed either by counting CD4 1 T-lymphocyte populations by using the known numbers of fluorescent microbeads admixed to a known volume of CD4 1 -stained blood, or by counting CD4 1 Tlymphocytes in a precisely determined blood volume. Unfortunately, the use of bead-based flow cytometry for the SP approach in Thailand is still expensive (US$ [15][16][17][18][19][20] when compared with the present DP approach using a hematological cell analyzer (US$ [10][11][12]. The use of volumetric methods, although simple and inexpensive, requires a new volumetric flow cytometer, thus making this approach impractical in Thailand where large numbers of nonvolumetric flow cytometers already exist.…”

“…The use of volumetric methods, although simple and inexpensive, requires a new volumetric flow cytometer, thus making this approach impractical in Thailand where large numbers of nonvolumetric flow cytometers already exist. Recently, a new flow-rate based flow cytometric method has been developed to use with the SP approach (11)(12)(13). This new method is designed to use the flow rate of the acquiring sample in the flow cytometer for enumeration of absolute CD4 1 T-lymphocytes.…”

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

“…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.…”