Development of a New Approach to Determine the Potency of Bacille Calmette–Guérin Vaccines Using Flow Cytometry

OBJECTIVES: To circumvent the limitations of the current golden standard method, colony-forming unit (CFU) assay, for viability of Bacille Calmette–Guérin (BCG) vaccines, we developed a new method to rapidly and accurately determine the potency of BCG vaccines. METHODS: Based on flow cytometry (FACS) and fluorescein diacetate (FDA) as the most appropriate fluorescent staining reagent, 17 lots of BCG vaccines for percutaneous administration and 5 lots of BCG vaccines for intradermal administration were analyzed in this study. The percentage of viable cells measured by flow cytometry along with the total number of organisms in BCG vaccines, as determined on a cell counter, was used to quantify the number of viable cells. RESULTS: Pearson correlation coefficients of FACS and CFU assays for percutaneous and intradermal BCG vaccines were 0.6962 and 0.7428, respectively, indicating a high correlation. The coefficient of variation value of the FACS assay was less than 7%, which was 11 times lower than that of the CFU assay. CONCLUSION: This study contributes to the evaluation of new potency test method for FACS-based determination of viable cells in BCG vaccines. Accordingly, quality control of BCG vaccines can be significantly improved.

Development of Cryopreserved Red Blood Cell Panels for Verifying ABO and D Blood Grouping Reagents / 대한수혈학회지

BACKGROUND: ABO blood grouping reagent verification is essential to ascertain safe blood transfusions. However, the research use of donated blood products has been hampered in Korea by the blood transfusion law and management policies. In this study, we developed cryopreserved red blood cell (RBC) panels utilizing the high glycerol method to verify the ABO and D blood grouping reagents. In addition, we evaluated the stability of ABO and D antigenicity. METHODS: Fresh blood was frozen by the high glycerol method, aliquoted and cryopreserved in 2 mL cryotubes. Twenty-four vials of bloods with types A (n=5), B (n=5), AB (n=4) and O (n=10) for ABO RBC panels, and eleven vials of blood types D positive (n=5), D negative (n=5) and D weak (n=1) for D RBC panels were established. Potency, avidity and specificity tests were carried out with four different commercial ABO and D blood grouping reagents. RESULTS: The potency of cryopreserved RBCs after thawing showed no statistical difference compared with pre-freezing RBCs. Avidity time measurements were 5 seconds in ABO blood and 20 seconds in D positive blood. Specificity test uniformly showed 100% specificity. When thawed RBCs were stored at 4degrees C for 7 days, the potency test measured at intervals of 2 days showed no variation. CONCLUSION: Cryopreserved RBC panels produced by the high glycerol method showed excellent results in stability test with reagents produced by manufacturers in Korea. Therefore, these panels can be utilized as a reliable method of verifying blood grouping reagents.