A reproducible strategy for analysis of minimal residual disease measured by Standardized multiparametric flow cytometry in b acute lymphoblastic leukemia

We have read with great interest the article published by Theunissen and co-workers (1) concerning the design of a fully standardized EuroFlow 8-color antibody panel to assess minimal residual disease (FC-MRD) in Bcell precursor acute lymphoblastic leukemia (BCP-ALL) (2). The role of multiparametric flow cytometry as an alternative approach to polymerase chain reaction (PCR) is a subject of growing importance in scientific literature. The authors have revised recent studies based in eight-color flow cytometry by Karawejew (3), Shaver (4), and others, and propose a new fully standardized EuroFlow combination of two tubes with five backbone markers as well as CD81, CD38, CD66c/CD123, and CD73/CD304. This strategy is applicable to >98% of patients reaching a sensitivity at least similar to PCR when enough cells (>4 × 10 (5)) are analyzed. We have routinely been using EuroFlow standardization and immunophenotyping protocols for BCP-ALL (2,6) since March 2013. Euroflow diagnostic panels include markers for identification of maturation stage as well as markers for identification of aberrant phenotypes, for example, CD123 or CD66c (2). Given the lack of fully standardized protocols for FC-MRD, we decided to reproduce the same EuroFlow diagnostic combinations, standardization, and sample preparation procedures during the successive FCMRD determinations. First, we proceeded to create a database that represented the normal B counterpart using regenerating bone marrow samples obtained from 10 nonleukemic patients. Tube 1, crucial for lymphoid maturation, was used for the elaboration of six databases that included total B population (CD19+) and five maturation stages according to CD34, CD10, CD20, and CD45 expression (5) (stage I: CD19+/CD34+/CD10-/ CD20-, stage II: CD19+/CD34-/CD10+ +/CD20-; stage III CD19+/CD34-/CD10 +/CD20-, stage IV CD19+/CD34-/CD10 +/CD20+,++, and stage V mature B cells CD19+/CD34-/CD10-/CD20++/CD45++. Similarly, databases for three different maturation stages were created using a combination of backbone markers for the remaining tubes (CD19+/CD34+, CD19+/CD34-/CD45weak, and CD19 +/CD34-CD45+). At diagnosis, the leukemic population was compared with the normal counterpart using automatic population separator (APS, Infinicyt software 1.8) in order to achieve the best possible separation, revealing the most discriminative markers. Then we selected the best combination for follow-up between the standardized tubes used for diagnosis. When assessing FC-MRD we analyzed at least 5 million cells per sample after excluding debris and doublets (1 million in day +15). At this point, it was necessary to merge two to six tubes to obtain the required cellularity. Stability of all fluorochromes was checked throughout the acquisition process following the fluorescence for each marker. The minimum cluster size was 50 events over a total of 5 million analyzed reaching an expected 10 sensitivity with a coefficient of variation according to Poisson distribution (100/√n) of 15% (7). Between March 2013 and November 2016, 41 patients were diagnosed of different BCP-ALL subtypes, 27 common B ALL, 9 pro-B and 5 pre-B ALL, 23 were males, and 18 females between ages of 1 and 71. We used BCP-ALL EuroFlow panels (four proposed combinations and ALOT combination for leukemic line orientation). The identification of FC-MRD was carried out in two steps. First, the B progenitors were compared with the reference image of the normal maturation stage (Fig. 1A,B) and then with the original leukemic population (Fig. 1C). FC-MRD was considered positive when the suspect cluster was clearly differentiable from the maturation patterns or the normal B counterpart and its immunophenotypic profile was consistent with, but not necessarily identical to the original leukemic population. During the follow-up process, the most frequently used combinations for FC-MRD were tubes 1 and 4 (24 of 41 patients), only tube 1 (8 of 41 patients), both tubes 1 and 3 (3 of 41 patients), tubes 1, 2, and 4 (2 of 41 patients), and all four tubes (1 patient). The more discriminative markers were CD10 (overexpression or underexpression in 38/41 patients), CD38 (underexpression in 34/41), CD123 (overexpression in 34/41), CD66c (overexpression in 26/41), CD45 (underexpression in 25/41), CD58 (overexpression in 24/41), CD34 (underexpression in 17/41), and CD21 (overexpression in 11/41). FC-MRD was determined at days +15, +33, +78, and +180 and at months +12 and +18 in two different centers (Hospital General de Alicante and Hospital La Fe de Valencia) with a 98% concordance in the results analyzed by independent researchers. Three and four marker templates were frequently used for follow up (Fig. 1). However, we could observe a significant variation of intensity in marker expression when compared to the original leukemic population, with particular mention, the decrease of intensity of CD10, CD66c, or CD34 and increase of intensity of CD20, CD10, and CD21. Similar findings have been reported by others researchers (8). This can be identified early, even al day +15 (35 of 41 cases) and +33 (Fig. 2). In these cases, new images were configured and added to Cytometry Part B (Clinical Cytometry) 96:12–15 (2019)