A complementary method to CD4 counting: measurement of CD4+/CD8+ T lymphocyte ratio in a tandem affinity microfluidic system

We describe a tandem affinity microfluidic separation that measures the ratio of CD4+/CD8+ T lymphocytes from blood samples. It is performed by injecting 2 μL of lysed blood samples at 1800–2700 cells μL^-1 into a microfluidic device containing two serially linked affinity regions, followed with a stop flow incubation that captures CD4+/CD8+ T lymphocytes on the corresponding affinity regions. Fluorophore conjugated antibodies are then injected at a controlled shear stress of 1.7 dyn cm^-2 to label target cells while eluting non-specific cells; and at last the CD4/CD8 ratio is calculated after the cell enumeration. The ratio of CD4+/CD8+ T lymphocytes achieved by our tandem affinity microfluidic system was in close agreement with that performed using conventional flow cytometry (R² = 0.97) over a wide range (0.4–2.5) that covered the reference values from immune deficient patients to healthy people. This approach may represent an inexpensive and powerful tool in diagnosis of immunodeficiency disorders including HIV or mycobacterium tuberculosis.