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. 2008 Dec 31;339(2):185-94.
doi: 10.1016/j.jim.2008.09.013. Epub 2008 Oct 12.

Determination of thymic function directly from peripheral blood: a validated modification to an established method

A R Lorenzi  1 , A M Patterson, A Pratt, M Jefferson, C E Chapman, F Ponchel, J D Isaacs
Affiliations

Determination of thymic function directly from peripheral blood: a validated modification to an established method

A R Lorenzi et al. J Immunol Methods. .

Erratum in

  • J Immunol Methods. 2009 May 15;344(1):84

Abstract

The thymus contributes naïve, self MHC reactive, self tolerant T cells to the peripheral immune system throughout life, albeit with a log-linear decline with age. Quantification of thymic function is clinically relevant in the setting of lymphoablation, but a phenotypic marker distinguishing recent thymic emigrants from long lived naïve T cells remains elusive. T cell receptor excision circles (TREC) are present in thymocytes exiting the thymus and quantification of the most frequent of these, the deltarec-psiJalpha rearrangement has been widely used as a measure of recent thymic function. However, interpretation of results presented as TREC per cell has been criticised on the basis that extra-thymic cellular proliferation impacts on peripherally determined TREC numbers. TREC/ml is now considered to be more representative of thymic function than TREC/cell, especially where significant cellular proliferation occurs (e.g. during reconstitution following stem cell transplantation). Here we describe the validation of a novel variation to the established assay, directly quantifying TREC/ml from 300 microl whole blood. We show the assay to be reproducible, robust and stable longitudinally and we show equivalence of performance when compared with more standard assays. This assay particularly lends itself to the measurement of thymic function in children and where monitoring clinical variables is limited by tissue availability.

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Figures

Fig. 1
Fig. 1
Determination of assay reproducibility. DNA was extracted from ten 300 µl replicate aliquots of whole blood from six healthy donors (A). WBTREC/ml was then calculated for five of these replicates in three donors (B). To examine the potential effect of a delay in sample processing, DNA was extracted from five replicates of a single sample at 1 h, 6 h and 24 h (C). WBLogTREC/ml was then quantified in all five aliquots for each time point (D). All plotted values are mean ± SD.
Fig. 2
Fig. 2
WBLogTREC/ml is positively correlated with lymphocyte count (A) (Pearson's correlation coefficient r = 0.67; p < 0.01) and weakly with white cell count (r = 0.38; p < 0.05) (B) but not neutrophil count (p = n.s.) (C) when directly quantified from whole blood.
Fig. 3
Fig. 3
Duplicate estimation of the number of TREC molecules per sample was performed for 175 individuals. Each data point represents the average value obtained from two simultaneous PCRs performed on two (i and ii) occasions for a single donor. Overall, the assay was shown to be highly reproducible as evidenced by the regression line's closeness to the diagonal and Pearson's correlation coefficient r = 0.96 (p < 0.01). However, as the number of TREC determined per reaction approached 10, the reproducibility of the assay was less. The vertical dashed line represents the average final value of WBLogTREC/ml, calculated according to Eq. (1), for samples in which 10 TREC were measured. Values to the left of the line thus represent samples with less than 10 TREC and those to the right those with more than 10 TREC.
Fig. 4
Fig. 4
(A) Comparison of TREC values derived from WB DNA (filled bars), PBMC DNA (striped bars) and CD4+ T cell DNA (white bars) in blood drawn from ten patients with rheumatoid arthritis. The age (yrs) of the patients is given below each. TREC values are those derived from the assay standard curve to give raw data values for comparison. Where there are no bars for individual subsets TREC were not detected in the PCR reaction (or insufficient DNA extracted in the case of the two samples where CD4+ samples were tested). Each reaction was performed using 200 ng of DNA. (B) TREC values derived from the PCR reactions were compared in these ten patients plus a further six patients from DNA extracted from PBMC and WB DNA. The values derived are strongly correlated (r = 0.92; p < 0.01).
Fig. 5
Fig. 5
Biological variation in WBLogTREC/ml in HC over 3 months. Nine individuals had blood drawn on more than two occasions over a three month period. On each occasion DNA was extracted and stored. TREC values were determined for each individual in a single real time experiment. Age and gender for each individual is given. The average value for each individual was taken and plotted against age to check that the group was a representative one (Pearson's r for the correlation with age = − 0.904; p < 0.01). The standard deviation about the mean for each individual was independent of age (data not shown).
Fig. 6
Fig. 6
Healthy control samples were divided into 5 year groupings and WBLogTREC/ml determined. The mean (thick bar), inter-quartile range (boxes) and 95% confidence intervals (whiskers) are plotted. Outliers are plotted as stars (⁎). Age was negatively correlated with WBLogTREC/ml (Pearson's r = − 0.86; p < 0.01).
Fig. 7
Fig. 7
Gender differences are detectable in WBLogTREC/ml when females (open circles/ dashed regression line) are compared with males (closed circles—solid regression line) at any age. There is a statistically significant difference between the two groups after correcting for age (p = 0.043). The negative correlation with age is maintained in each population. (A single sample (male; TREC/ml—2.58) was removed from this analysis because it was an outlier exerting significant influence on the result when included).
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References

    1. Arstila T.P., Casrouge A., Baron V., Even J., Kanellopoulos J., Kourilsky P. A direct estimate of the human alphabeta T cell receptor diversity. Science. 1999;286(5441):958. - PubMed
    1. Bland M. 3 ed. Oxford University Press; 2000. An Introduction to Medical Statistics.
    1. de Villartay J.P., Hockett R.D., Coran D., Korsmeyer S.J., Cohen D.I. Deletion of the human T-cell receptor delta-gene by a site-specific recombination. Nature. 1988;335(6186):170. - PubMed
    1. Douek D.C., McFarland R.D., Keiser P.H., Gage E.A., Massey J.M., Haynes B.F. Changes in thymic function with age and during the treatment of HIV infection. Nature. 1998;396(6712):690. - PubMed
    1. Douek D.C., Vescio R.A., Betts M.R., Brenchley J.M., Hill B.J., Zhang L. Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of T-cell reconstitution. Lancet. 2000;355(9218):1875. - PubMed

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