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Optimized multiparametric immunophenotyping of umbilical cord blood cells by flow cytometry

Nature Protocols volume 5, pages 1337–1346 (2010)Cite this article

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Abstract

Umbilical cord blood is a key source of stem cells for transplantation and regenerative medicine. To maximize each cord blood sample, it is important to analyze its cellular populations. Many cord blood banks focus on counts of total nucleated cells and/or cells that carry the CD34 antigen, but this limited focus does not give a true estimation of cord blood content, quality and appropriateness for use. This protocol is the first of its kind to enable a comprehensive investigation of cord blood cellular populations. Using multicolor flow cytometry it is possible to examine expression of 26 antigens—including hematopoietic markers CD45 and CD34, immune markers CD19 and CD3, and HLA—using a total of only 1 × 106 cells from each unit for both pre- and post-processing. The samples are stained, lysed, washed and analyzed flow cytometrically. This method also provides valuable information beyond cord blood composition and quality; for example, it can also be used to assess whether maternal factors affect CD34+ cell numbers. Collection, processing, cryopreservation and initial flow cytometry take ∼5 h.

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Figure 1: White blood cell populations within umbilical cord blood.
Figure 2: Analysis of CD34+ HSC cell numbers.
Figure 3: Expression of HLA in lineage-negative UCB-derived stem cells.
Figure 4: Average recovery of individual cell types within UCB (n = 80).

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Acknowledgements

We thank the staff at Newcastle's Royal Victoria Infirmary Woman's Directorate and Maternity Unit. We are also grateful to the One North East regional development agency of the British Government for their financial support to our laboratories. We thank I. Dimmick for his excellent advice during FACS analysis development and M. Lako for her supervision and laboratory role in the later stages of the project.

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Authors and Affiliations

  1. Stem Cell Group, Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, United Kingdom

    Christina Basford

  2. CTI-LYON, Cell Therapy Research Institute, Parc Technologique de Lyon–Saint Priest, Saint Priest, Lyon, France.,

    Nico Forraz & Colin McGuckin

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  1. Christina Basford
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Contributions

C.B., C.M. and N.F. conceived the protocol. C.M. and N.F. supervised the project. C.B. performed the experiments, analyzed the data and wrote the paper.

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Correspondence to Colin McGuckin.

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The authors declare no competing financial interests.

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Basford, C., Forraz, N. & McGuckin, C. Optimized multiparametric immunophenotyping of umbilical cord blood cells by flow cytometry. Nat Protoc 5, 1337–1346 (2010). https://doi.org/10.1038/nprot.2010.88

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