Calculating potential doubling time using monoclonal antibodies specific for two halogenated thymidine analogues

A new flow cytometric technique that allows for two-incorporated thymidine analogues to be measured simultaneously and independently has been used to improve the accuracy of in vivo cell kinetic estimates, i.e., the length of S-phase (TS) and potential doubling time (Tpot).

The analogues chlorodeoxyuridine and iododeoxyuridine were injected at different times into mice bearing the mouse mammary tumor MCaK. At different times after labeling, the tumors were harvested and prepared for three color flow cytometric analysis of DNA, chlorodeoxyuridine, and iododeoxyuridine. Control experiments showed that similar estimates of Tpot were obtained from each label when administered singly, or as staggered pulses. Comparisons were made between TS and Tpot calculated from a single label (single point), from the averaged result of the two labels from the same tumor (two point-ave), and from the simultaneous nonlinear fitting of the measured parameters from the two labels, from the same tumor (two point-fit). These estimates of TS and Tpot were then compared to reference values obtained by fitting the pooled measured parameters from all the tumors, that were labeled for different periods of time.

While all of the methods resulted in similar mean estimates of TS and Tpot that were close to the reference values, the fewest assumptions, and the least variability in the results, were obtained using the two point-fit data.

The estimation of Tpot using two thymidine analogues is more accurate than that obtained from a single label.