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

Lorenzi AR, Patterson AM, Pratt A, Jefferson M, Chapman CE, Ponchel F, Isaacs JD - J. Immunol. Methods (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle University, Newcastle-upon-Tyne, NE2 4HH, UK.

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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Related in: MedlinePlus

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.
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fig1: 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.

Mentions: From Eq. (1), four sources of experimental variability were identified that could contribute to measurement error in the assay. Such methodological considerations are true for most systems of this nature but infrequently described in the literature. We therefore sought to confirm that we could a) reproducibly extract the same quantity of DNA from a single donor sample on multiple occasions; b) that we could reproducibly quantify TREC molecules from single donor samples on multiple occasions; c) that there was no effect of time from sample donation to sample preparation on values obtained and d) that TREC values were independent of total white cell count—since granulocytes, B cells and NK cells are all nucleated cells present in whole blood. Fig. 1(A) shows that DNA was reproducibly extracted from whole blood samples and Fig. 1(B) that TREC/ml was reproducibly calculated in three of six of these samples. Over a 24 h period there was no effect of time from the sample being drawn to being processed on the quantity of DNA extracted—Fig. 1(C) or the determined TREC value for that sample—Fig. 1(D). The coefficient of variation for the DNA extraction was calculated to be 5.07% from six samples extracted 10 times each.


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

Lorenzi AR, Patterson AM, Pratt A, Jefferson M, Chapman CE, Ponchel F, Isaacs JD - J. Immunol. Methods (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2593795&req=5

fig1: 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.
Mentions: From Eq. (1), four sources of experimental variability were identified that could contribute to measurement error in the assay. Such methodological considerations are true for most systems of this nature but infrequently described in the literature. We therefore sought to confirm that we could a) reproducibly extract the same quantity of DNA from a single donor sample on multiple occasions; b) that we could reproducibly quantify TREC molecules from single donor samples on multiple occasions; c) that there was no effect of time from sample donation to sample preparation on values obtained and d) that TREC values were independent of total white cell count—since granulocytes, B cells and NK cells are all nucleated cells present in whole blood. Fig. 1(A) shows that DNA was reproducibly extracted from whole blood samples and Fig. 1(B) that TREC/ml was reproducibly calculated in three of six of these samples. Over a 24 h period there was no effect of time from the sample being drawn to being processed on the quantity of DNA extracted—Fig. 1(C) or the determined TREC value for that sample—Fig. 1(D). The coefficient of variation for the DNA extraction was calculated to be 5.07% from six samples extracted 10 times each.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Musculoskeletal Research Group, Institute of Cellular Medicine, Newcastle University, Newcastle-upon-Tyne, NE2 4HH, UK.

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.

Show MeSH
Related in: MedlinePlus