Impact of Prolonged Storage on the Blood Group Antigenicity of Reagent Red Blood Cells

Background:
Preserving reagent red blood cells (RBC) is critical for ensuring reliable results in Immunohematology testing prior to blood transfusion. Prolonged RBC storage can lead to hemolysis and loss of blood group antigens, compromising results in diagnostic testing. To mitigate this, RBCs are suspended in a solution that minimizes membrane lesions and maintain antigen integrity during long-term storage. Loss of blood group antigens can affect antigenicity, which may lead to false-negative results and potentially result in incorrect transfusion therapy. Some clinical departments produce reagent RBCs in-house, while others rely on commercially available reagent RBCs. These products have varying shelf lives depending on the storage solution used. Our in-house reagent RBCs are preserved in ID-Cellstab (BioRad Laboratories) and has a durability of 33 days. Given the challenges of obtaining RBCs with rare antigen profiles and the time-consuming preparation process, extending the shelf life would improve their availability and reliability for immunohematology testing.

Aims:
To evaluate the effect of prolonged storage on the antigenicity of Rh, Duffy, Kell and MNS blood group antigens, as well as the level of hemolysis in in-house reagent RBCs.
To determine whether the shelf life of reagent RBCs preserved in ID-Cellstab could be extended to 75 days.

Methods:
The stability of reagent RBCs was assessed on days 0, 32, 39, 45, 60, and 75. The degree of hemolysis was measured using the Vitros 3600 (Ortho Clinical Diagnostics) and HemoCue Plasma/Low Hb Photometer, while antigenicity loss was evaluated through antibody titration. Two reagent RBCs (double dose) were selected for each of the blood group antigens: Rh (D, c, E, e), Duffy (Fya, Fyb), MNS (M, N, S, s) and Kell (K). These RBCs were suspended in ID-CellStab (Bio-Rad Laboratories) at final concentrations of 0.8%, 5 % and papain treated 0.8%. They were stored at 4°C during the study period and tested towards their corresponding antibodies. RBC concentrations were adjusted during storage using the Sysmex XP-300 analyzer to maintain target concentrations of 0.8% and 5%. Manual titration of 11 patient plasma samples with single alloantibodies was performed using tube LISS-IAT, BioRad Column Coombs anti-IgG ID, and Neutral Cards (Bio-Rad Laboratories). End point titers and titer scores (Marsh) were compared across all storage days.

Results:
No decline in antibody titers was observed for antibodies targeting the Duffy, Kell, and MNSs and most Rh antigens throughout the 75-day storage period. However, the titer of anti-c declined by two titer steps on day 75 using 5% RBCs. The titer scores of anti-M, -N, -S, -s, -e and anti-Fy remained unaffected. However, the titer scores of anti-Fy, -K and anti-c decreased by more than 10 points. Hemolysis assessment revealed no increase in either 0.8% RBCs or
papain-treated 0.8% RBCs, while a slight gradual increase in 5% RBCs was observed throughout the 75-day storage period.

Summary / Conclusions:
Our findings suggest that extending the shelf life of reagent RBCs suspended in ID-CellStab to 75 days does not compromise the antigenicity of Rh, Duffy, Kell and MNs blood group antigens. Furthermore, the level of hemolysis remained minimal, indicating that the quality is maintained over the extended storage period.