Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays
Abstract In a precarious world of rapidly growing pandemics, the field of vaccine production has witnessed considerable growth. Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine and a part of the immunization program in 157 countries. The quality control is based on a potency test through...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-023-38657-x |
| _version_ | 1797774272498761728 |
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| author | Hend M. Moghawry Mohamed E. Rashed Kareeman Gomaa Sameh AbdelGhani Tarek Dishisha |
| author_facet | Hend M. Moghawry Mohamed E. Rashed Kareeman Gomaa Sameh AbdelGhani Tarek Dishisha |
| author_sort | Hend M. Moghawry |
| collection | DOAJ |
| description | Abstract In a precarious world of rapidly growing pandemics, the field of vaccine production has witnessed considerable growth. Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine and a part of the immunization program in 157 countries. The quality control is based on a potency test through viable cell enumeration. The colony-forming unit (CFU) assay is the official method, however, it often yields fluctuating results, suffers from medium cracking, and requires lengthy analysis (~ 28 days). Flow cytometric analysis was proposed earlier, but it was coupled with a Coulter counter for measuring the entire bacterial population (live/dead). In the present study, thiazole orange/propidium iodide dyes supplemented with fluorogenic reference beads were employed for viable counting, eliminating the need for a Coulter counter. Both the flow cytometry and the colorimetric technique employing tetrazolium salt were validated and compared to the CFU assay. The colorimetric assay displayed high precision, accuracy, and a strong positive correlation with the CFU assay. The flow cytometry assay demonstrated high precision and a notable ability to distinguish different forms of BCG cells (live, injured, and dead). It also exhibited a perfect positive correlation with the CFU assay. Both methods reduced the analysis time by > 26 days and eliminated the need for human intervention by automating the test. |
| first_indexed | 2024-03-12T22:18:38Z |
| format | Article |
| id | doaj.art-3632641bc38042e88639fd150a5fa212 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-03-12T22:18:38Z |
| publishDate | 2023-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-3632641bc38042e88639fd150a5fa2122023-07-23T11:10:51ZengNature PortfolioScientific Reports2045-23222023-07-0113111110.1038/s41598-023-38657-xDevelopment of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assaysHend M. Moghawry0Mohamed E. Rashed1Kareeman Gomaa2Sameh AbdelGhani3Tarek Dishisha4Department of Pharmaceutical Microbiology and Immunology, Faculty of Pharmacy, Beni-Suef UniversityGeneral Administration of Biological Products, Central Administration of Biological and Innovative Products and Clinical Trials, Egyptian Drug Authority (EDA)Clinical and Chemical Pathology Department, Faculty of Medicine - Kasr Al-Ainy, Cairo UniversityDepartment of Pharmaceutical Microbiology and Immunology, Faculty of Pharmacy, Beni-Suef UniversityDepartment of Pharmaceutical Microbiology and Immunology, Faculty of Pharmacy, Beni-Suef UniversityAbstract In a precarious world of rapidly growing pandemics, the field of vaccine production has witnessed considerable growth. Bacillus Calmette-Guérin (BCG) is a live-attenuated vaccine and a part of the immunization program in 157 countries. The quality control is based on a potency test through viable cell enumeration. The colony-forming unit (CFU) assay is the official method, however, it often yields fluctuating results, suffers from medium cracking, and requires lengthy analysis (~ 28 days). Flow cytometric analysis was proposed earlier, but it was coupled with a Coulter counter for measuring the entire bacterial population (live/dead). In the present study, thiazole orange/propidium iodide dyes supplemented with fluorogenic reference beads were employed for viable counting, eliminating the need for a Coulter counter. Both the flow cytometry and the colorimetric technique employing tetrazolium salt were validated and compared to the CFU assay. The colorimetric assay displayed high precision, accuracy, and a strong positive correlation with the CFU assay. The flow cytometry assay demonstrated high precision and a notable ability to distinguish different forms of BCG cells (live, injured, and dead). It also exhibited a perfect positive correlation with the CFU assay. Both methods reduced the analysis time by > 26 days and eliminated the need for human intervention by automating the test.https://doi.org/10.1038/s41598-023-38657-x |
| spellingShingle | Hend M. Moghawry Mohamed E. Rashed Kareeman Gomaa Sameh AbdelGhani Tarek Dishisha Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays Scientific Reports |
| title | Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays |
| title_full | Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays |
| title_fullStr | Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays |
| title_full_unstemmed | Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays |
| title_short | Development of a fast and precise potency test for BCG vaccine viability using flow cytometry compared to MTT and colony-forming unit assays |
| title_sort | development of a fast and precise potency test for bcg vaccine viability using flow cytometry compared to mtt and colony forming unit assays |
| url | https://doi.org/10.1038/s41598-023-38657-x |
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