Design of limiting dilution analysis experiments for helper T lymphocyte precursor frequency determination in the context of allogeneic bone marrow transplantation

Helper, interleukin 2 (IL-2) producing, T lymphocyte precursor (HTLp) frequ ency determination by limiting dilution analysis (LDA) is of value for quan tifying alloreactivity in allogeneic bone marrow transplantation (BMT). LDA assays are labour-intensive and time-consuming to perform and the numbers of donor and recipient cells available are limited. It is therefore importa nt that the design of the experiment yields reliable frequencies with a min imum of effort and a realistic cell requirement. We have critically evaluat ed the methods proposed for LDA design by Strijbosch et al. [Strijbosch, L. W., Buurman, W.A., Does, R.J., Zinken, P.H., Groenewegen, G., 1987. Limitin g dilution assays. Experimental design and statistical analysis. J. Immunol . Methods 97, 133] and by Blackett and Gordon [Blackett, N.M., Gordon, M.Y. , 1996. Optimizing limiting dilution assays: frequency and 'ability' measur ements of haemopoietic progenitor cells. Br. J. Haematol. 92, 507 (see comm ents)] and found them inadequate for this application. The estimation of th e HTLp frequency is traditionally based on the single-hit Poisson model and the adequacy of this model was compared with that of a double-hit model. T he results were in favour of the single-hit model. Ten different LDA experi mental designs were explored by Monte Carlo simulations. The optimal design exploits the maximal numbers of cells that can be obtained for analysis to estimate HTLp frequencies in the range 1:1,000,000-1:20,000 with a coeffic ient of variation of 10-20% and with a minimum of manual labour. (C) 1999 E lsevier Science B.V. All rights reserved.