Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL

Abstract

Detection of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) is becoming increasingly important as treatments improve. An internationally harmonised four-colour (CLR) flow cytometry MRD assay is widely used but has limitations. The aim of this study was to improve MRD analysis by identifying situations where a less time-consuming CD19/CD5/κ/λ analysis would be sufficient for detecting residual CLL, and develop a six-CLR antibody panel that is more efficient for cases requiring full MRD analysis. In 784 samples from CLL patients after treatment, it was possible to determine CD19/CD5/κ/λ thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/κ/λ analysis was unsuitable for predicting iwCLL/NCI response status or identifying cases with no detectable MRD. For the latter cases requiring a full MRD assessment, a six-CLR assay was designed comprising CD19/CD5/CD20 with (1) CD3/CD38/CD79b and (2) CD81/CD22/CD43. There was good correlation between four-CLR and six-CLR panels in dilution studies and clinical samples, with 100% concordance for detection of residual disease at the 0.01% (10(-4)) level (n=59) and good linearity even at the 0.001-0.01% (10(-5)-10(-4)) level. A six-CLR panel therefore provides equivalent results to the four-CLR panel but it requires fewer reagents, fewer cells and a much simpler analysis approach.

The prognostic impact of minimal residual disease in patients with chronic lymphocytic leukemia requiring first-line therapy

“…48,49 Regarding the technique used to evaluate MRD in CLL, MRD-flow cytometry does not require pre-treatment samples and is becoming the technique of choice in view of its sensitivity and reproducibility, even in the context of multicenter trials. 19,50 Furthermore, several problems associated with four-color panels, such as the large amounts of time and sample required for an accurate analysis of patients with low level disease, have been partially circumvented with the advent of modern six-color combinations. With these new advances, MRD-flow could be implemented in most diagnostic laboratories with additional savings in terms of time, labor and reagents.…”

“…With these new advances, MRD-flow could be implemented in most diagnostic laboratories with additional savings in terms of time, labor and reagents. 50 There are, however, several considerations that should be highlighted. First, MRD-flow cytometry has changed several times over the years and has only been standardized in the last decade, even though its sensitivity has remained constant.…”

Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting

“…Therefore, the maximum number of events that could be considered to be below the limit of detection is 19 events, but due to the potential counting errors (95% confidence interval for a count of 19 events would be of 11 to 30 events) this implies that the LOD can be estimated as (30/total number of cells analyzed) 3 100%. Similarly, it is also widely accepted that more than 50 events is a standard threshold for reproducible enumeration of a cell population by experienced flow cytometer operators (28,29); consequently, the limit of quantification can be estimated as (50/total number of cells analyzed) 3 100%. Thus, the LOD and the LLOQ will be both typically dependent on the total number of cells analyzed; the expected limit of detection and lower limit of quantification for a range of cells analyzed are shown in Table 1.…”

The addition of ofatumumab to the conditioning regimen does not improve the outcome of patients with high-risk CLL undergoing reduced intensity allogeneic haematopoietic cell transplantation: a pilot trial from the GETH and GELLC (CLL4 trial)

“…We followed a standardised protocol of two tubes (six colours each) using CD5 PerCPCy5.5, CD19 PC7 and CD20 APCH7 as common markers. This protocol was defined by the European Research Initiative on CLL (ERIC) 9 and achieves a sensitivity of 10 − 4 . Whole blood and lineage-specific chimerism were locally assessed using conventional methods.…”