Precision of DNA flow cytometry in inter‐institutional analyses

A Bladder Cancer Flow Cytometry Network study has been carried out to further identify and quantify sources of inter‐ and intra‐laboratory variability. Replicate samples containing four mixtures of peripheral blood lymphocytes and aneuploid cell lines were distributed together with reference standards to six laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Two of each of the four sample types and a reference standard were analyzed by each laboratory on 3 separate days to obtain cellular DNA distributions. DNA index (DI) and hyperdiploid fraction (HDF) were calculated for each histogram using an automated technique. The results showed significant inter‐ and intra‐laboratory differences. Results were evaluated by a two‐way analysis of variance to estimate components of the overall variation attributable to individual sources. Error variation was found to be the major component of random variation. Specimen means were also compared for each laboratory. No significant differences were noted in mean DI for similar specimens; however, agreement in HDF between similar specimens was lacking in most laboratories. Prediction intervals were computed to estimate the range of values expected for a single specimen based on the analysis of the previous six. Prediction intervals for DI were quite good while those for HDF were troublesome due to wide variation. The results of these studies indicate that intra‐ and inter‐laboratory variability are high enough that results for a single sample may not be sufficiently precise to allow comparison to results obtained in other laboratories. This study demonstrates the need for measurement and control of laboratory variability and for the routine use of laboratory standards and check samples if the results from one laboratory are to be compared with the results of another or if classification criteria are to be transferred between laboratories.