Labelling indices in human tumours: to apply corrections or not--that is the question

The advent of halogenated pyrimidines (bromodeoxyuridine, BrdU; idoxuridine, IdU) and antibodies to recognize them has opened new horizons for the measurement of proliferation in human tumours. These precursors of DNA can be given to patients and a single biopsy can be taken to measure in a flow cytometer both the fraction of labelled cells and their rate of movement through the S phase. From these two parameters the potential doubling time, T(POT), can be calculated. To measure both parameters simultaneously a compromise is made in the time of assessing the labelling index (LI). LI should ideally be assessed after a very short interval, e.g. 0.5-1 h, to avoid the contaminating influence of any cells dividing between injection and biopsy. However, an interval of 4-8 h is considered necessary to assess T(S) from the relative movement of cells through the S phase. Several techniques exist to correct for cell division if the interval is long. The simplest correction, which only corrects for the division of labelled cells, is most widely used. Downward correction factors of at least 10% are commonly applied, reducing the observed LI values. In this paper we illustrate graphically the dependence of the appropriate correction factor on various cell kinetic parameters. The duration of G2 is the most critical parameter for both the size and direction of any correction factor. The G2 phase has previously been shown to be about three times longer in human tumours than in rodents. If G2+M is as long as 6 h, the main artefact of the intervals between injection and biopsy up to 7 h is that the observed LI is too low because of division of unlabelled G2 cells. A correction of up to 10% is needed but in an upward direction. A nomogram of probable correction factors as a function of sampling interval is provided. We show from flow cytometric data that G2+M may be shorter than 4 h for head and neck tumours. It is recommended that the correction factor established by gating the flow histogram should always be checked against this nomogram, or that no correction factor should be applied. We have used this mathematical approach to re-evaluate two sets of published LI data for rectal and colorectal tumours. We show that the mathematical correction of each data point leads to a 30% increase in the median value, compared to the simple gating procedure. We question whether other of the published series of LI values gained with BrdU or IdU may also substantially underestimate the true LI values, if a simple gating procedure has been used in an attempt to reduce the impact of divided S phase cells.