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The clinical importance of blood group (BG) antigens is related to their ability to induce immune antibodies that can cause hemolysis. Yet, ABO and D (Rh) are still considered to be the key antigens for healthy blood transfusion and secondary antigens are the next priority. Serological typing is the most widely used typing method. Rapid and accurate blood grouping plays an important role in some clinical conditions, rather than conventional techniques. Hence, developing a simple and economical model for rapid blood grouping would facilitate these tests. In recent decades, paper-based microfluidics such as μPADs has gained much interest in wide application areas such as point-of-care diagnostic. In this study, we evaluated μPADs that are performed for blood grouping and its recent progress. A comprehensive literature search was performed using databases including PUBMED, SCOPUS, Web of Science and Google Scholar. Keywords were blood grouping or typing, paper analytical device, rapid test, etc. After investigation of search results, 16 papers from 2010 to 2020 were included. Further information in detail was classified in Table 1. Generally, two principles for blood typing μPADs are introduced. The lateral chromatographic flow method and the vertical flow-through method that detects BG in a visual-based manner. To detect results with acceptable clarity many factors and challenges like paper, blood sample, buffer, Ab and RBC interaction and also μPADs stability need to be considered, which are discussed. In conclusion, the simplicity, stability, cheapness, portability and biocompatibility of μPADs for blood grouping confirming its utility and also they have the capability to robust, universal blood-grouping platform. Table 1 Summary of blood grouping tests using paper-based analytical devices Antigens Type of diagnosis Validation method Sample No Accuracy Action time Paper type Stability Sample dilution Buffer Ref A, B, Rh Forward volunteers records 5 - - Whatman No. 4 - 1/2 PBS* (Khan et al. 2010) A, B, Rh Forward gel assay test and conventional slide test 100 100% 1 min Whatman No. 4 and Kleeenex paper towel 7 Days in 4 °C 1/1 NSS (Al-Tamimi et al. 2012) A, B, Rh Forward gel card assay 99 100% 20 Sec + Washing Kleeenex paper towel - 1/1 NSS (Li et al. 2012) A, B, Rh Forward - - - - Kleeenex paper towel - 45/100 PSS (Li et al. 2013) A, B, Rh Forward gel card assay 98 100% 1.5 min Kleeenex paper towel - 85/100 PBS (Guan et al. 2014b) C, E, c, e, K, Jka, Jkb, M, N, S, P1, and Lea Forward gel card assay 266 100% - Kleeenex paper towel - 1/1 NSS (Li et al. 2014b) A, B, Rh Forward and Reverse conventional slide test 96 ≈ 91% 10 min Whatman No. 1 21 Days in 4 °C 1/2 NSS (Noiphung et al. 2015) C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, M, N, S and s, P1, Lea and Leb Forward - 478 - - Kleeenex paper towel - 1/1 NSS, PBS (Then et al. 2015) A, B Forward and Reverse conventional slide test 76 100% 5-8 min Whatman No. 4 38 Days in 4 °C 1/4, 1/1 NSS (Songjaroen and Laiwattanapaisal 2016) D, K Forward volunteers records 210 - 7.5 min Kleenex paper towel - 1/1 NSS (Yeow et al. 2016) A, B, c, e, D, C, E, M, N, S, s, P1, Jka, Jkb, Lea, Leb, Fya, and Fyb Forward and Reverse gel card assay 3550 ≈100% 30 s Fiber glass and cotton linter 180 Days in 25 °C 45/100, 1/1 PBS (Zhang et al. 2017) A, B Forward conventional slide test 598 100% 3 min Whatman No. 113 14 Day in 4 °C 1/1 NSS (Songjaroen et al. 2018) A, B, Rh Forward conventional slide test - - 30 Sec + Washing Unrefined sisal paper - 1/2 NSS (Casals-Terré et al. 2019) A, B, Rh Forward - - - - Whatman No.1 - 1/1 NSS (Ansari et al. 2020) ABORh Forward and Reverse conventional slide test - 100% Unrefined Eucalyptus papers - 1/2 NSS, PBS (Casals-Terré et al. 2020) A, B, Rh Forward - - - 30 Sec + Washing Whatman No. 4 modified with chitosan ≥ 100 days in 25 °C 1/1 NSS (Parween et al. 2020)

We report the use of bioactive paper for typing of secondary human blood groups. Our recent work on using bioactive paper for human blood typing has led to the discovery of a new method for identifying haemagglutination of red blood cells. The primary human blood groups, i.e., ABO and RhD groups, have been successfully typed with this method. Clinically, however, many secondary blood groups can also cause fatal blood transfusion accidents, despite the fact that the haemagglutination reactions of secondary blood groups are generally weaker than those of the primary blood groups. We describe the design of a user-friendly sensor for rapid typing of secondary blood groups using bioactive paper. We also present mechanistic insights into interactions between secondary blood group antibodies and red blood cells obtained using confocal microscopy. Haemagglutination patterns under different conditions are revealed for optimization of the assay conditions.