Impact of Paper Substrates and Storage Conditions on the Stability of Blood Grouping Assays

ABSTRACT Blood grouping assays are commonly performed using tube tests or column agglutination, which require meticulous procedures by trained personnel. Paper‐based assays offer simplicity and readability; however, they are typically made with cellulose‐based mediums and demonstrate poor long‐term stability and low background to signal contrast. Although nitrocellulose (NC) membranes improve stability through higher protein adsorption, very few are manufactured with sufficiently large pore sizes that prevent the non‐specific retention of blood cells. This study presents two new NC membrane‐based assays, including an NC vertical flow‐through membrane not previously applied for blood grouping, and compares their performance and stability against two cellulose‐based papers commonly studied. Stability was investigated at 4°C and room temperature (RT) over a convenient 9‐month period. Anti‐A antibodies were tested following 1/2 to 1/16 dilution with natural saline solution (NSS, 0.9% NaCl), 5% bovine serum albumin (BSA) in NSS, or 5% sucrose in NSS. Assays were characterized according to blood spot signal intensity in comparison to negative controls. NC‐based assays demonstrated superior antibody stability compared to cellulose, with the vertical flow NC membrane showing the highest performance. All papers were more stable following 4°C storage. Furthermore, sucrose significantly improved stability at RT compared to BSA and NSS alone.