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Open Access Highly Accessed Methodology article

Quantification of total T-cell receptor diversity by flow cytometry and spectratyping

Stanca M Ciupe1*, Blythe H Devlin2, Mary Louise Markert23 and Thomas B Kepler4

Author Affiliations

1 Department of Mathematics, Virginia Tech, 460 McBryde Hall, Blacksburg, VA 24060, USA

2 Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA

3 Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA

4 Department of Microbiology, Boston University School of Medicine, Boston MA 02118, USA

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BMC Immunology 2013, 14:35  doi:10.1186/1471-2172-14-35

Published: 6 August 2013



T-cell receptor diversity correlates with immune competency and is of particular interest in patients undergoing immune reconstitution. Spectratyping generates data about T-cell receptor CDR3 length distribution for each BV gene but is technically complex. Flow cytometry can also be used to generate data about T-cell receptor BV gene usage, but its utility has not been compared to or tested in combination with spectratyping.


Using flow cytometry and spectratype data, we have defined a divergence metric that quantifies the deviation from normal of T-cell receptor repertoire. We have shown that the sample size is a sensitive parameter in the predicted flow divergence values, but not in the spectratype divergence values. We have derived two ways to correct for the measurement bias using mathematical and statistical approaches and have predicted a lower bound in the number of lymphocytes needed when using the divergence as a substitute for diversity.


Using both flow cytometry and spectratyping of T-cells, we have defined the divergence measure as an indirect measure of T-cell receptor diversity. We have shown the dependence of the divergence measure on the sample size before it can be used to make predictions regarding the diversity of the T-cell receptor repertoire.