A new statistical method for quantitative analyses: application to the precise quantification of T cell receptor repertoires.

In experimental immunology, a situation quite commonly arises in which there are a large number of potential events but the probability of any individual event is small and one wishes to measure the number of events which actually occur. We present a new general statistical method, denoted Continuous Poisson Method (COPOM), for estimating the number of events underlying a quantitative measurement. This situation is well illustrated in the case of quantitative analyses of the immune receptor repertoire in a diverse population of cells. We show that repetition of T cell receptors (TCRs) complementarity determining region 3 (CDR3) length measurements by Immunoscope, on independent samples containing the similar numbers of cells prepared from splenocytes, results in variable profiles. When analyzed by COPOM, this variability provides direct quantification of the lymphocytes expressing any antigen receptor with a given V, J and CDR3 length inside the cell population. Using COPOM, a single dilution was sufficient to cover events over a 100-fold variation in frequency and the sensitivity of the assay was such that a single cell inside a pool of 5 x 10(4) lymphocytes could be quantified. A comparison of the frequency of splenocytes using either Vbeta14-Jbeta or the specific Vbeta8.3-Jbeta1.1 rearrangement, determined either by our or other approaches, revealed the accuracy and convenience of our method. This approach provides the first precise method able to measure the diversity of the antigen receptor repertoire inside a complex cell population by the use of a single straightforward technique.