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Analysis of Whole Blood Samples: Optimization of Sample Preparation for Rapid Assays.
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Whole blood separation is a crucial step prior to the analysis of a whole blood sample in a lateral flow assay (LFA). While there are numerous diagnostic applications that utilize the technique for rapid and sensitive analysis, few are able to directly work with human whole blood samples and provide accurate and consistent results. The main objective of this project was to find and optimize a whole blood filter capable of effective whole blood filtration and plasma collection for the analysis of whole blood samples in the LFA format. The work focused on finding suitable filter materials that can be incorporated into any test strip in an LFA format, and explore the modifications required to enhance the filter membrane?s performance. Membranes examined for this purpose were polyvinylpyrrolidone (PVP)/ polyethersulfone (PES) Primecare membranes (Spectral Diagnostics), FUSION 5 (Whatman), and HanoRapid (Hanomy LLC.) Various factors evaluated included plasma yield, absorbance capacity, extent of hemolysis, leakage of red blood cells, unspecific binding, and wicking time. Based on the results, it was determined that the PVP/PES membrane X and NX, which separate and collect plasma from whole blood through a porosity gradient, were the most suitable membrane materials to be incorporated into the lateral flow strip test as whole blood filtering sample pads. By using the combination of X and NX as the sample pad for a lateral flow assay, diluted whole blood (95%) in PBS up to 30 ?L spiked with insulin was filtrated and analyzed correctly within 30 minutes. Other work done in this project included improving the sensitivity of a lateral flow assay on HanoRapid nitrocellulose membranes for the detection of serum insulin through optimization of blocking solution. By blocking the membrane with a solution containing 0.03% BSA, 0.02%PVP, 0.005%Casein, 1XTBS, 0.002% Tween20, the improved limit of detection for insulin on HanoRapid nitrocellulose membrane was achieved at 0.75 x 10E-6 g/mL, which was still above the serum insulin concentration. Based on these results, it was concluded that a traditional lateral flow assay with HanoRapid membranes is not sensitive enough for the detection of serum insulin.
whole blood separation; whole blood filtering device; lateral flow assay