The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays
The assessment of nonviable haematopoietic cells by Annexin V staining method in flow cytometry has recently been published by Duggleby et al. Resulting in a better correlation with the observed colony formation in methylcellulose assays than the standard ISHAGE protocol, it presents a promising met...
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| Format: | Article |
| Language: | English |
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Wiley
2013-01-01
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| Series: | Stem Cells International |
| Online Access: | http://dx.doi.org/10.1155/2013/823912 |
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| _version_ | 1850179174979862528 |
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| author | Teja Falk Radke David Barbosa Richard Charles Duggleby Riccardo Saccardi Sergio Querol Gesine Kögler |
| author_facet | Teja Falk Radke David Barbosa Richard Charles Duggleby Riccardo Saccardi Sergio Querol Gesine Kögler |
| author_sort | Teja Falk Radke |
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| description | The assessment of nonviable haematopoietic cells by Annexin V staining method in flow cytometry has recently been published by Duggleby et al. Resulting in a better correlation with the observed colony formation in methylcellulose assays than the standard ISHAGE protocol, it presents a promising method to predict cord blood potency. Herein, we applied this method for examining the parameters during processing which potentially could affect cord blood viability. We could verify that the current standards regarding time and temperature are sufficient, since no significant difference was observed within 48 hours or in storage at 4°C up to 26°C. However, the addition of DMSO for cryopreservation alone leads to an inevitable increase in nonviable haematopoietic stem cells from initially 14.8% ± 4.3% to at least 30.6% ± 5.5%. Furthermore, CFU-assays with varied seeding density were performed in order to evaluate the applicability as a quantitative method. The results revealed that only in a narrow range reproducible clonogenic efficiency (ClonE) could be assessed, giving at least a semiquantitative estimation. We conclude that both Annexin V staining method and CFU-assays with defined seeding density are reliable means leading to a better prediction of the final potency. Especially Annexin V, due to its fast readout, is a practical tool for examining and optimising specific steps in processing, while CFU-assays add a functional confirmation. |
| format | Article |
| id | doaj-art-bc6cdb9ebef140298c47523ddd0da9e0 |
| institution | OA Journals |
| issn | 1687-966X 1687-9678 |
| language | English |
| publishDate | 2013-01-01 |
| publisher | Wiley |
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| series | Stem Cells International |
| spelling | doaj-art-bc6cdb9ebef140298c47523ddd0da9e02025-08-20T02:18:33ZengWileyStem Cells International1687-966X1687-96782013-01-01201310.1155/2013/823912823912The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU AssaysTeja Falk Radke0David Barbosa1Richard Charles Duggleby2Riccardo Saccardi3Sergio Querol4Gesine Kögler5Institute of Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, Moorenstraße 5, 40225 Düsseldorf, GermanyInstitute of Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, Moorenstraße 5, 40225 Düsseldorf, GermanyAnthony Nolan Research Institute, Royal Free Hospital, Fleet Road, London NW3 2QG, UKCord Blood Bank, Haematology Department, Careggi University Hospital, Via delle Oblate 1, 50141 Firenze, ItalyAnthony Nolan Research Institute, Royal Free Hospital, Fleet Road, London NW3 2QG, UKInstitute of Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, Moorenstraße 5, 40225 Düsseldorf, GermanyThe assessment of nonviable haematopoietic cells by Annexin V staining method in flow cytometry has recently been published by Duggleby et al. Resulting in a better correlation with the observed colony formation in methylcellulose assays than the standard ISHAGE protocol, it presents a promising method to predict cord blood potency. Herein, we applied this method for examining the parameters during processing which potentially could affect cord blood viability. We could verify that the current standards regarding time and temperature are sufficient, since no significant difference was observed within 48 hours or in storage at 4°C up to 26°C. However, the addition of DMSO for cryopreservation alone leads to an inevitable increase in nonviable haematopoietic stem cells from initially 14.8% ± 4.3% to at least 30.6% ± 5.5%. Furthermore, CFU-assays with varied seeding density were performed in order to evaluate the applicability as a quantitative method. The results revealed that only in a narrow range reproducible clonogenic efficiency (ClonE) could be assessed, giving at least a semiquantitative estimation. We conclude that both Annexin V staining method and CFU-assays with defined seeding density are reliable means leading to a better prediction of the final potency. Especially Annexin V, due to its fast readout, is a practical tool for examining and optimising specific steps in processing, while CFU-assays add a functional confirmation.http://dx.doi.org/10.1155/2013/823912 |
| spellingShingle | Teja Falk Radke David Barbosa Richard Charles Duggleby Riccardo Saccardi Sergio Querol Gesine Kögler The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays Stem Cells International |
| title | The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays |
| title_full | The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays |
| title_fullStr | The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays |
| title_full_unstemmed | The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays |
| title_short | The Assessment of Parameters Affecting the Quality of Cord Blood by the Appliance of the Annexin V Staining Method and Correlation with CFU Assays |
| title_sort | assessment of parameters affecting the quality of cord blood by the appliance of the annexin v staining method and correlation with cfu assays |
| url | http://dx.doi.org/10.1155/2013/823912 |
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