DETECTION OF MINIMAL RESIDUAL DISEASE IN PATIENT WITH CHRONIC LYMPHOCYTIC LEUKEMIA USING BOTH FOUR-COLOR AND EIGHT-COLOR FLOW CYTOMETRY AND QUANTITATIVE REAL TIME PCR APPROACH

Sborník

Background. Minimal residual disease (MRD) is a routinely used term for small population of pathologic cells that remain during or after treatment when the patient is in remission. Rawstron et al. (2007) published four-color flow cytometric method, which made possible to standardize MRD assessment in patients with chronic lymphocytic leukemia (CLL). Application of this method enables to determine the MRD level in peripheral blood or bone marrow down to less then 0. 01% of leucocytes. The results can be compared to RQ-ASO IgH PCR (real-time quantitative allele-specific oligonucleotide Immunoglobulin Heavy chain gene PCR). In ASO-PCR individual patient-specific oligonucleotide primer are designed to detect MRD, its sensitivity reaches to 1 cell in 106. Aims. The aim of our study was to compare multiparametric flow cytometry to molecular biology approaches with detection of IgVH specific clones in patients with CLL diagnosis. Methods. Cohort of 22 CLL patients who underwent allogeneic stem-cell transplantation (SCT) was included in the study. MRD were analyzed employing four-color flow cytometry protocol (Rawstron, 2007) together with eight-color flow cytometry protocol. Cell surface were stained with fluorescence labeled monoclonal antibodies (anti-CD3, 5, 14, 19, 20, 22, 38, 43, 45, 79b and 81). Flow cytometric acquisition was performed on a FACSCantoII flow cytometer (Becton Dickinson,NJ,USA). Detection of clonal IgVH was performed by RQ-PCR with LNA probe (Locked Nucleic Acid, TaqMan technology). Results. Since RQ-ASO IgH PCR shows sensitivity attaining 1 cell in 106, 8. 6% of samples were found positive using RQ-PCR but negative using flow cytometry. However, 10 patients were simultaneously observed using flow cytometry and RQ-PCR method with strong correlation (r=0. 94). In 17 patients both flow cytometric protocols (four- and eight-color) were employed to observe the correlation of these two methods. It was found out that the correlation coefficient is getting closed to one (r=0. 96). Summary and Conclusions. We compared multiparametric flow cytometry to molecular biology approaches with detection of IgVH specific clones in patients with CLL diagnosis. Despite lower sensitiveness, flow cytometry is adequate method to MRD detection. Concurrently, we demonstrate potential application of eight-color flow cytometric protocol. The advantage of this protocol lies in simultaneous detection of more than two markers compared to four-color cytometry.

Acknowledgement.This work was supported by grant MUNI/A/0784/201.

Haematologica, 2012; 97(s1):  524