Proposal for the standardization of flow cytometry protocols to detect minimal residual disease in acute lymphoblastic leukemia

Abstract

Minimal residual disease is the most powerful predictor of outcome in acute leukemia and is useful in therapeutic stratification for acute lymphoblastic leukemia protocols. Nowadays, the most reliable methods for studying minimal residual disease in acute lymphoblastic leukemia are multiparametric flow cytometry and polymerase chain reaction. Both provide similar results at a minimal residual disease level of 0.01% of normal cells, that is, detection of one leukemic cell in up to 10,000 normal nucleated cells. Currently, therapeutic protocols establish the minimal residual disease threshold value at the most informative time points according to the appropriate methodology employed. The expertise of the laboratory in a cancer center or a cooperative group could be the most important factor in determining which method should be used. In Brazil, multiparametric flow cytometry laboratories are available in most leukemia treatment centers, but multiparametric flow cytometry processes must be standardized for minimal residual disease investigations in order to offer reliable and reproducible results that ensure quality in the clinical application of the method. The Minimal Residual Disease Working Group of the Brazilian Society of Bone Marrow Transplantation (SBTMO) was created with that aim. This paper presents recommendations for the detection of minimal residual disease in acute lymphoblastic leukemia based on the literature and expertise of the laboratories who participated in this consensus, including pre-analytical and analytical methods. This paper also recommends that both multiparametric flow cytometry and polymerase chain reaction are complementary methods, and so more laboratories with expertise in immunoglobulin/T cell receptor (Ig/TCR) gene assays are necessary in Brazil.

Normal variation of bone marrow B‐cell precursors according to age – reference ranges for studies in myelodysplastic syndromes in Brazil

“…In practice, the criteria used comprised the increase in number of granulocytes with predominance of the more mature ones (CD10 + ). Furthermore, comparison of the smear collected for cytology and another made from the material sent for immunophenotyping was made, as recommended by our study Group .…”

Allogeneic Hematopoietic Stem Cell Transplantation for Children and Adolescents with Acute Myeloid Leukemia in Brazil: A Multicentric Retrospective Study

“…A lack of effective methods to measure residual disease after remission induction chemotherapy at many institutions also contributes to inadequate patient selection for HSCT and referral. Even though multiparametric flow cytometry laboratories are available at most Brazilian leukemia treatment centers, multiparametric flow cytometry processes must be standardized for MRD investigations in order to provide reliable and reproducible results that ensure quality in the clinical application of the method 38 .…”

How I investigate minimal residual disease in acute lymphoblastic leukemia

“…Table 1 summarizes the characteristics of all techniques described above regarding MRD detection in ALL. It is important to mention that discrepancy between the MFC and molecular assays may occur and is associated with some causes, such as: limited number of cells analyzed by MFC assays; presence of hematogones during therapy and related to age; drug‐induced immunophenotype modulation; quality of the PCR clonal markers; amplification of nonspecific DNA, oligoclonality and clonal evolution 61 . So, the laboratories need to seek continuous standardization for each technique, and apply all the parameters of the "EU in vitro diagnostic regulation" law and others national e international regulations, so that they work in a reproducible and harmonious way.…”