Effect of long-term frozen storage on antibodies in blood samples: immunoglobulin, tetanus toxin antibody, and ABO antibody.

The Japanese Red Cross Blood Services archives donor blood samples from all donations for look-back surveys. These samples, approximately 5 million per year, are stored frozen at -30°C for 11 years and are subsequently discarded. Because such samples may also be valuable for antibody-related research and other purposes after the mandated storage period, we evaluated their potential applicability, with a particular focus on the preservation of anti- body levels. Fresh samples were compared with frozen-stored samples (-30°C for 14 years: 11 years of mandatory good manufacturing practice storage period + 3 years until measurement) for immunoglobulin levels (IgM, IgG, IgA, and IgE) as a general indicator of humoral immunity, tetanus toxin antibody titers as a long-lived vaccine-induced antibody, and ABO antibody titers (saline-direct agglutination test and saline-indirect antiglobulin test [saline-IAT]), which are factors in blood group typing and hemolytic transfusion reactions. For immunoglobulin levels, comparisons of non-paired fresh and frozen-stored samples were made from a random donor population (IgM, IgG, IgA: 105 cases each, IgE: 32 cases). For tetanus toxin antibody and ABO antibody, comparisons were conducted from the same donors with fresh and frozen-stored samples (tetanus toxin antibody: 47 cases, ABO antibody: 12 cases). No significant differences were observed in immunoglobulin levels, tetanus toxin antibody titers, or ABO antibody titers measured by the saline-direct agglutination method. Saline-IAT-measured ABO antibody titers were significantly lower in frozen-stored samples. Detailed results were as follows: (1) Median immunoglobulin levels (fresh vs. frozen-stored): IgM, 78.5 mg/dL versus 86.7 mg/dL; IgG, 1240.2 mg/dL versus 1280.2 mg/dL; IgA, 241.0 mg/dL versus 245.3 mg/dL; and IgE, 0.24 μg/mL versus 0.12 μg/mL. (2) Tetanus toxin antibody titers-geometric mean titer (GMT) (95% confidence interval [CI]): fresh 0.23 (0.12-0.43) IU/mL versus frozen-stored 0.28 (0.17-0.46) IU/mL. (3) ABO antibody titers-GMT (95% CI): saline-direct agglutination, fresh 65.4 (33.3-128.5) versus frozen-stored 51.7 (24.9-107.4); saline-IAT, fresh 391.0 (160.3-953.6) versus frozen-stored 248.7 (107.8-573.6). The evaluated immunoglobulin levels were largely preserved, suggesting that archived blood samples retain sufficient stability for antibody research. However, careful study design is necessary to minimize potential sample deterioration.