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

Ciupe SM, Devlin BH, Markert ML, Kepler TB - BMC Immunol. (2013)

Bottom Line: Spectratyping generates data about T-cell receptor CDR3 length distribution for each BV gene but is technically complex.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Mathematics, Virginia Tech, 460 McBryde Hall, Blacksburg, VA 24060, USA. stanca@vt.edu

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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Related in: MedlinePlus

CD4 T-cell spectratype data. Spectratype histograms show the number of CD4 T-cells bearing receptors versus CDR3 length for each TCR BV families tested.
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Figure 5: CD4 T-cell spectratype data. Spectratype histograms show the number of CD4 T-cells bearing receptors versus CDR3 length for each TCR BV families tested.

Mentions: CD3 T-cells from the peripheral blood of patients were isolated. RNA was prepared and used for cDNA synthesis. The cDNA was used as a template for 23 TCR BV specific primer pairs to amplify the complete CDR3 region by PCR [10]. Each PCR product, representing a different TCR BV family, was size separated by electrophoresis and the product lengths were identified using the GeneScan software (Applied Biosciences). An example of spectratype data in a healthy adult is presented in FigureĀ 5, which shows the histograms of the number of CD4 T-cells versus CDR3 length for each TCR BV family.


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

Ciupe SM, Devlin BH, Markert ML, Kepler TB - BMC Immunol. (2013)

CD4 T-cell spectratype data. Spectratype histograms show the number of CD4 T-cells bearing receptors versus CDR3 length for each TCR BV families tested.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750526&req=5

Figure 5: CD4 T-cell spectratype data. Spectratype histograms show the number of CD4 T-cells bearing receptors versus CDR3 length for each TCR BV families tested.
Mentions: CD3 T-cells from the peripheral blood of patients were isolated. RNA was prepared and used for cDNA synthesis. The cDNA was used as a template for 23 TCR BV specific primer pairs to amplify the complete CDR3 region by PCR [10]. Each PCR product, representing a different TCR BV family, was size separated by electrophoresis and the product lengths were identified using the GeneScan software (Applied Biosciences). An example of spectratype data in a healthy adult is presented in FigureĀ 5, which shows the histograms of the number of CD4 T-cells versus CDR3 length for each TCR BV family.

Bottom Line: Spectratyping generates data about T-cell receptor CDR3 length distribution for each BV gene but is technically complex.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Mathematics, Virginia Tech, 460 McBryde Hall, Blacksburg, VA 24060, USA. stanca@vt.edu

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus