Comparison of minimal residual disease detection in multiple myeloma between the DuraClone and EuroFlow methods

In this study, the minimal residual disease (MRD) levels in patients with multiple myeloma (MM) were assessed by comparing the new 8-color single-tube multiparameter flow cytometry method (DuraClone), which reduces the cost of antibodies and labor burden of laboratories, with the EuroFlow next-generation flow (NGF) method. A total of 96 samples derived from 69 patients with MM were assessed to determine the total cell acquisition number (tCAN), percentages of total and normal plasma cells (PCs), and MRD levels using two methods. We found that the tCAN was significantly higher with EuroFlow-NGF than with DuraClone (median 8.6 × 106 vs. 5.7 × 106; p < 0.0001). In addition, a significant correlation in the MRD levels between the two methods was noted (r = 0.92, p < 0.0001). However, in the qualitative analysis, 5.2% (5/96) of the samples showed discrepancies in the MRD levels. In conclusion, the DuraClone is a good option to evaluate MRD in multiple myeloma but it should be used with caution.

www.nature.com/scientificreports/ mixed, measured, and split evenly (2 mL each). One sample was analyzed with the DuraClone 8-color singletube panel at Kameda Medical Center, and the other was analyzed with the EuroFlow-NGF 8-color 2-tube panel at Kanazawa University Hospital. Within 48 h after collection, up to 20 million cells or the entire volume of the sample was used. All study methods were carried out in accordance with the Declaration of Helsinki.
On the other hand, the DuraClone method 15 was performed with fixative-free erythrocyte lysis (VersaLyse Lysing Solution, Beckman Coulter), followed by surface staining with the prefixed dry reagent of antibodies against CD138-APC, CD38-PB (non-ME antibody), CD56-APC-A750, CD19-PC5.5, CD45-KrO, CD200-PC7, CD81-FITC, and CD27-PE in a single tube to identify aPCs. The Navios flow cytometer (Beckman Coulter, USA) was used to acquire the samples. Cell doublets were excluded by omitting the events with high time of flight (TOF) 15 (Fig. 1A) and debris were excluded by omitting the events with lower forward scatter peak signals than lymphocytes (Fig. 1B). Furthermore, dye aggregates were eliminated with excluding the high fluorescent events with CD81-FITC and CD56-APC-A750 (Fig. 1C). Following these procedures, primary identification of plasma cells was performed by gating the events with CD38-PB high and CD138-APC high (Fig. 1D). Additionally, plasma cells were selected with the gating of CD38 and CD45 (Fig. 1E). The samples following anti-CD38 monoclonal antibody treatment, removal of doublets ( Fig. 2A), debris (Fig. 2B), and aggregates ( Fig. 2C) was www.nature.com/scientificreports/ performed as well as the samples without using anti-CD38 monoclonal antibody, but primary identification of plasma cells was performed CD38-PB negative and CD138-APC high (Fig. 2D). Additionally, plasma cells are selected with the gating of CD138 and CD45 (Fig. 2E), and identification of aPCs was manually performed using a radar plot, which is a projection of the intensity of the released light energy from each eight fluorochrome on a 2-dimensional series of spokes projecting from a central point analyzed by the Kaluza v1.5a software (Beckman Coulter) as previously described in both samples with or without using anti-CD38 monoclonal antibody 15 (Figs. 1F, 2F). Using DuraClone, the analysis strategy bases on the different-from-normal approach as there is no specific marker of abnormality known yet but only deviations from the normal expression pattern which are CD45med, CD38high, CD138med/high, CD19positive, CD56dim/neg, CD27high, CD81high, CD200dim/neg. Abnormal plasma cells can deviate by a diversity of abnormal patterns, e.g. CD45low/neg, CD38med, CD138 all high, CD19neg, CD56dim/neg, CD27dim, CD81high, CD200high, or as another example, CD45med, CD38high, CD138med/high, CD19neg, CD56high, CD27med, CD81dim/neg, CD200dim/neg 16   www.nature.com/scientificreports/ analysis, total cell acquisition number (Tube 1 + Tube 2, 2 mL-BM), percentages of the total and normal PCs, and MRD levels of Tube 1 (1 mL-BM) was adopted for comparison with EuroFlow-NGF. In the MRD level analysis, detection of at least 20 aPCs was required for their precise identification in both methods. When 1 × 10 7 cells were analyzed using EuroFlow-NGF, the lower limit of detection (LOD) was set at 2.0 × 10 −6 and LOD of Dura-Clone was set at 4.0 × 10 −6 . Matching of target channels was verified daily with a new calibration run to prevent target mismatch and all instruments underwent daily verification of optical alignment and fluidics using other calibration bead particles (Flow Check beads, Beckman Coulter

Consent for publication. All patients provided written consent in accordance with the Declaration of
Helsinki.

Results
A total of 96 samples derived from 69 patients were analyzed, and 21 patients were assessed repeatedly in different states of disease. The patient characteristics (diagnosis and clinical status by the IMWG response criteria for MRD evaluation) are summarized in Table 1. The number of cells obtained using EuroFlow-NGF (median 8.6 × 10 6 ; range 1.1 × 10 6 -11.7 × 10 6 ) was significantly higher than that obtained by the DuraClone panel (median 5.7 × 10 6 ; range 0.5 × 10 6 -18.3 × 10 6 ; p < 0.0001), and the correlation was not so high (r = 0.40; p < 0.0001) (Fig. 4A). In the   18 . In addition, 81 (84.3%) and 56 (58.3%) of the 96 samples assessed by the EuroFlow-NGF and DuraClone methods, respectively, achieved an acquisition of 5 million cells as recommended by the IMWG 10 . Next, we compared the percentages of the total and normal PCs between the EuroFlow-NGF and DuraClone methods. Relatively a high-correlation was noted in the percentages of the total (r = 0.79; p < 0.0001) and normal (r = 0.75; p < 0.0001) PCs (Fig. 4B-C).
Finally, we compared the efficiency of MRD detection between the two methods. In the quantification of the MRD levels, a high-correlation in MRD levels was observed between the two methods (r = 0.92; p < 0.0001)  www.nature.com/scientificreports/ (Fig. 4D). Conversely, in the qualitative study of MRD detection, 5 samples (5.2%) showed a disagreement of MRD negativity when a cutoff value of 10 −5 was applied (Fig. 4E, Table 2). Among these samples, three were found to be MRD positive by EuroFlow-NGF and MRD negative by DuraClone, and the total number of cells acquired by EuroFlow-NGF (median 8.4 × 10 6 , range 7.3 × 10 6 -8.7 × 10 6 ) was almost the same with that acquired by the DuraClone panel except for sample No. 5 (median 8.7 × 10 6 , range 2.2 × 10 6 -10.0 × 10 6 ; p = 0.7). In contrast, two samples were found to be MRD negative by EuroFlow-NGF and MRD positive by DuraClone, judged as such using the following antigens: 1 sample by CD56 only and 1 sample by CD56 and CD200 ( Fig. 4E and Table 2).

Discussion
This study demonstrated that the DuraClone method was not comparable to EuroFlow-NGF in total cell acquisition number, but comparable in percentage of total/normal PCs, and MRD levels. Although at least 3 million cells and ideally 5 million cells should be analyzed to achieve the lower limit of detection, i.e., 1.0 × 10 −5 according to the recent guidelines 19 , only 58.3% (56/96) of the samples achieved over 5 million total nucleated cells using DuraClone. On the contrary, 84.3% (81/96) achieved this number using EuroFlow-NGF. This difference likely resulted because the DuraClone method utilized the fixative-free erythrocyte lysis process, which tends to lose the nucleated cells, contrary to the bulk lysis in the EuroFlow-NGF method, as previously reported 11 . In addition to that reason, in our modified DuraClone analysis, we used intracellular staining including cyIgκ and cyIgλ (Tube 2) for the confirmation of clonality, which cannot merge with surface staining (Tube 1) as EuroFlow-NGF can. We compared 1 mL sample of Tube 1 of DuraClone with 2 mL sample of EuroFlow-NGF for percentages of total and normal PCs and MRD, because Tube 1 of DuraClone was designed for detection of MRD, but Tube 2 was our institutional original method for just confirmation of clonality, not for detection of MRD. Confirmation of clonality by using cyIgκ and cyIgλ is helpful for the detection of MRD.
An inconsistent MRD negativity between the EuroFlow-NGF and DuraClone methods was noted in 5 samples (5.2%). We consider that one of reasons of inconsistent MRD negativities of these samples is that these inconsistencies are recognized in the samples with low MRD level and 3/5 of MRD positive samples (all three samples are MRD positive in EuroFlow-NGF) are MRD level of 1-2 × 10 −5 as shown in Table 2. Regarding the difference of tCAN, because tCAN of sample No.5 by DuraClone was much less than that by EuroFlow-NGF, the inconsistency of MRD could be due to the little tCAN by DuraClone. The difference between the antibody panels, in addition to tCAN, may also have resulted in the inconsistent cases between the EuroFlow-NGF and DuraClone methods. The DuraClone method adopted antibodies against CD200 instead of CD117, cyIgκ, and cyIgλ, which were used in the EuroFlow-NGF panel 11 . CyIgκ and cyIgλ are extremely important to confirm the clonality of myeloma cells, and thus, ruling out the polyclonal cells, which express aberrant antigens such as CD56 and CD200, is impossible without anti-cyIgκ and anti-cyIgλ antibodies. For example, Flores-Montero et al. reported that anti-CD200 antibody demonstrated low contribution as a potential MRD marker because it was heterogeneously expressed in the normal bone marrow PCs from 28/28 samples (median percentage of CD200 + normal PCs, 49%; range 40-68%) as well as in 8/20 (40%) cases with abnormal PC populations (median percentage of CD200 + aPCs, 100%; range 58-100%) 11 , thus using CD200 for detecting aPCs has the potential to overestimate MRD. Recently, Roshal et al. reported that the MSKCC 10-color single-tube and EuroFlow-NGF 8-color 2-tube methods exhibited similar efficiency in assessing MRD 20 ; in both methods, anti-cyIgκ and anti-cyIgλ antibodies were adopted to confirm the clonality of aPCs. In our analysis, 2 of 5 samples were MRD positive by DuraClone method and negative by EuroFlow-NGF (samples No. 1 and 2 in Table 2). CD19 negative/CD56 positive cells in sample No. 1 and CD200 positive/CD19 negative/CD56 positive cells in sample No. 2 were detected as aPCs by DuraClone analysis. In both samples, we performed cyIgκ and cyIgλ analysis for confirmation of clonality in Tube 2 of DuraClone, but no clonality was observed. In addition to the reason that CD200 would overestimate aPCs in Tube 1, we hypothesized that the reason of difference of MRD might be owing to the difference of approach for detecting aPCs between both methods. EuroFlow-NGF used cyIgκ and cyIgλ for counting aPCs in Table 2. Overview of the samples showing discrepancy of MRD negativity between DuraClone and EuroFlow-NGF methods. tCAN total cell acquisition number, PC plasma cells, MRD minimal residual disease, NGF next-generation flow, CR complete response, sCR stringent CR, VGPR very good partial response, PFS progressionfree survival, NA not assessed due to be transferred to another hospital. a The thresholds of MRD negativity was 1 × 10 −5 in both DuraClone and EuroFlow-NGF. www.nature.com/scientificreports/ addition to surface staining by the fusion of both surface and cytoplasmic staining tubes, however, DuraClone could not fuse surface and cytoplasmic staining cells together, thus counting of aPCs was performed only by surface staining tube using the radar plot. For these reasons, DuraClone has the potential to overestimate aPCs. To date, three methods for monitoring MRD have been developed, namely, MFC, allele specific quantitative PCR, and next-generation sequencing, and emerged as attractive and sensitive approaches 6,12,13 . Among these methods, EuroFlow-NGF is currently the most cost-effective and widely available method. Compared with the EuroFlow 2-tube method (< 350 USD/sample), the DuraClone single-tube method (< 100 USD/sample) is expected to be more cost-effective and can be used under public medical insurance in countries such as Japan.
In summary, we found Duraclone is a good option to evaluate MRD as the EuroFlow 8-color 2-tube method in MM but it should be used with caution and some discrepancies between them may have resulted because of the difference in the number of cells analyzed and antibody panels. Therefore, a sufficient cell acquisition number (> 5 × 10 6 ) is essential to achieve high sensitivity in MRD detection in MM.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.