Impact of Different Cell Counting Methods in Molecular Monitoring of Chronic Myeloid Leukemia Patients

Abstract

Background: Detection of BCR-ABL1 transcript level via real-time quantitative-polymerase-chain reaction (Q-PCR) is a clinical routine for disease monitoring, assessing Tyrosine Kinase Inhibitor therapy efficacy and predicting long-term response in chronic myeloid leukemia (CML) patients. For valid Q-PCR results, each stage of the laboratory procedures need be optimized, including the cell-counting method that represents a critical step in obtaining g an appropriate amount of RNA and reliable Q-PCR results. Traditionally, manual or automated methods are used for the detection and enumeration of white blood cells (WBCs). Here, we compared the performance of the manual counting measurement to the flow cytometry (FC)-based automatic counting assay employing CytoFLEX platform. Methods: We tested five different types of measurements: one manual hemocytometer-based count and four FC-based automatic cell-counting methods, including absolute, based on beads, based on 7-amino actinomycin D, combining and associating beads and 7AAD. The recovery efficiency for each counting method was established considering the quality and quantity of total RNA isolated and the Q-PCR results in matched samples from 90 adults with CML. Results: Our analyses showed no consistent bias between the different types of measurements, with comparable number of WBCs counted for each type of measurement. Similarly, we observed a 100% concordance in the amount of RNA extracted and in the Q-PCR cycle threshold values for both BCR-ABL1 and ABL1 gene transcripts in matched counted specimens from all the investigated groups. Overall, we show that FC-based automatic absolute cell counting has comparable performance to manual measurements and allows accurate cell counts without the use of expensive beads or the addition of the time-consuming intercalator 7AAD. Conclusions: This automatic method can replace the more laborious manual workflow, especially when high-throughput isolations from blood of CML patients are needed.