Panels and reagent kits for minimal residual disease measurement in pediatric b-cell precursor acute lymphoblastic leukemia via immunophenotyping

Abstract

The present invention relates to a reagent panels for the measurement of minimal residual disease (MRD) associated with pediatric B-cell precursor acute lymphoblastic leukemia (B- ALL) by multiparametric flow cytometry. The reagent panel of the invention comprises a combination of antibodies directed against markers, wherein the combination of antibodies comprises i) antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81, CD123, CD304, CD44, CD86, CD99 and CD371, and ii) antibodies targeting markers CD19 and / or CD22, wherein the antibodies are conjugated with fluorochromes. The invention further relates to the use of said panels for detecting MRD associated with B-ALL and / or for identifying a subject at risk of developing B-ALL relapse. The invention also relates to methods of detecting MRD associated with B-ALL. Key advantages of the invention include achieving high MRD detection sensitivity (e.g., 10⁻⁵ or 0.001%) and high specificity.

Description

[0001] Panels and Reagent Kits for Minimal Residual Disease Measurement in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia via Immunophenotyping

[0002] RELATED APPLICTAIONS

[0003] This application claims priority to BG Provisional Patent Application BG / P / 2024 / 113932, filed July 19, 2024, the entire disclosure of which is hereby incorporated herein by reference.

[0004] FIELD

[0005] The present invention refers generally to the field of minimal residual disease (MRD) measurement technology. Specifically, the invention pertains to the assessment of treatment effectiveness in patients afflicted with hematologic malignancies, particularly pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Provided herein are unique reagent compositions comprising specifically selected and rigorously validated antibody combinations suitable for, for example, 12-color, 14-color, and 16-color MRD measurements utilizing multiparametric flow cytometry. These compositions are capable of achieving a detection sensitivity of at least 10“4, and potentially extending to 10“5. The invention further provides diagnostic kits incorporating said compositions and methods for detecting MRD in BCP-ALL.

[0006] BACKGROUND

[0007] Acute lymphoblastic leukemia (ALL) represents the most prevalent malignancy diagnosed in the pediatric population, constituting approximately 25% of all such diagnoses. Approximately 60% of ALL cases manifest in children and adolescents below the age of 20, exhibiting an annual incidence exceeding 90 cases per one million individuals within this demographic. Among childhood ALL cases, B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the predominant subtype. Current cytostatic or cytotoxic treatment protocols, however, may not successfully eradicate all malignant cells in every patient. Consequently, a subpopulation of leukemic cells can persist within the bone marrow following therapy, often leading to subsequent disease relapse. These persisting cells constitute what is termed minimal residual disease (MRD). Presently, the quantification of MRD serves as the most potent prognostic indicator for predicting survival outcomes in pediatric ALL. Accordingly, MRD measurement has been integrated into contemporary treatment strategies as a fundamental criterion for patient risk stratification and the tailoring of therapeutic regimens, as evidenced in established protocols including, but not limited to, COG AALL1631 , BFM-ALL 2017, DFC! ALL, UKALL, FRALLE, and AIEOP [1 , 2],

[0008] Several methodologies for the measurement of residual disease are known in the art. Conventional microscopic examination of bone marrow aspirates typically possesses a detection limit ranging from 1% to 5% leukemic cells. MRD is defined as the presence of leukemic cells at levels below this detection threshold achievable by standard morphological analysis. Advanced genotypic and phenotypic methods, such as multiparametric flow cytometry and polymerase chain reaction (PCR)-based techniques, can attain significantly higher MRD detection sensitivities, often reaching 0.01% (equivalent to one leukemic cell per 10,000 nucleated cells). Certain genotypic approaches are capable of achieving even greater sensitivity, potentially detecting MRD at levels as low as 0.001% [3, 4], It is pertinent to note that these sensitivity percentages are expressed relative to the total count of nucleated cells within the analyzed clinical sample, necessitating accurate quantification thereof.

[0009] Genotypic methodologies are predicated upon the detection of specific genes or gene mutations characteristic of leukemic cells. Commonly employed genotypic techniques encompass realtime quantitative polymerase chain reaction (RQ-PCR), reverse transcription polymerase chain reaction (RT-PCR), droplet digital polymerase chain reaction (ddPCR), and next-generation sequencing (NGS) [5]. While genotypic methods offer the advantages of high specificity and sensitivity, they are subject to several limitations. These include the relatively low prevalence of requisite genetic markers (detectable in less than 50% of pediatric ALL patients), potential inaccuracies in quantification, the possibility of false-positive results (occurring in up to 20% of analyses), susceptibility to cross-contamination between samples, the prerequisite for identifying relevant chromosomal abnormalities at the time of initial diagnosis, considerable associated costs, and substantial processing time [4, 6, 7],

[0010] Phenotypic methodologies, conversely, are based upon the detection of specific macromolecules, termed leukocyte "antigens," which are characteristic features of leukemic cells. These antigens are systematically classified into clusters of differentiation and designated by the abbreviation "CD," followed by a unique numerical identifier. For instance, CD3 and CD4 are characteristically expressed by T lymphocytes, CD19 and CD20 by B lymphocytes, and CD13 and CD33 by myeloid lineage cells. The currently established CD nomenclature encompasses over 400 distinct leukocyte antigens. These entities are also commonly referred to as "markers." Within the context of the present invention, the terms "CD," "marker," and "antigen" are employed interchangeably.

[0011] Leukocytes typically co-express multiple distinct markers, the specific combination of which defines the cell's phenotype. This phenotype serves as a characteristic signature for different leukocyte subpopulations. By way of example, the phenotype of a memory T-helper lymphocyte is defined by the expression pattern including markers such as CD45, CD3, CD4, CD45RO, and CD27. Beyond the mere presence or absence of specific CD markers, phenotype characterization also incorporates the quantitative assessment of marker expression levels (e.g., low, medium, or high expression). The classification of distinct leukocyte subpopulations relies on their unique CD phenotypes, and the analytical process of determining these phenotypes is referred to as phenotyping.

[0012] Leukemic cells likewise exhibit characteristic phenotypes that enable their discrimination from their normal cellular counterparts. Certain markers function to identify the cell lineage (e.g., lymphoid, myeloid, erythroid). Other markers provide information regarding the cell's differentiation stage (e.g., CD10, CD38, CD20, CD34). A further category comprises "aberrant" markers, signifying expression patterns atypical for the determined lineage (e.g., the expression of myeloid markers CD13 or CD33, or markers such as CD56 or CD11 b, on B-lineage lymphoblasts). Furthermore, alterations in marker expression levels, known as antigen modulation, contribute to the distinct phenotypic profile of leukemic cells. The composite analysis of these features — lineage determination, differentiation stage assessment, identification of aberrant marker expression, and evaluation of antigen modulation — facilitates the differentiation of leukemic cells from normal hematopoietic cells, thereby establishing phenotyping as a pivotal methodology for MRD quantification.

[0013] The detection of phenotypic markers is typically accomplished utilizing immunological techniques employing antibodies engineered to specifically bind to target markers. This analytical approach is commonly referred to as “immunophenotyping”.

[0014] A principal method for visualizing the binding interaction between an antibody and its corresponding marker involves the conjugation of the antibody to a fluorochrome. Key characteristics defining a fluorochrome include its specific excitation wavelength and its corresponding emission wavelength, the latter manifesting as fluorescence. Presently, a wide array of fluorochromes exists, each possessing distinct excitation and emission spectral properties. Examples include, but are not limited to, BUV395, BUV496, BV421 , BV480, BV605, BV650, BV711 , BV786, BB515, FITC, PE, PE-CF594, PERCP-CY5.5, PE-CY7, APC, APC- R700, and APC-H7. This extensive palette of available fluorochromes facilitates the labelling of antibodies directed against various CD markers with distinguishable fluorescent reporters, thereby enabling the simultaneous detection and quantification of multiple phenotypic markers on individual cells.

[0015] The detection of fluorochrome-labeled antibodies bound to cells is most frequently performed using instrumentation such as fluorescence microscopes or, more commonly in this context, flow cytometers. A flow cytometer incorporates distinct detectors optimized for specific fluorescence emission spectra, often referred to as "channels," "parameters," or "colors." Flow cytometers equipped to measure eight or more parameters simultaneously are designated as "multiparametric" or "multicolor" instruments. Within the scope of the present invention, these terms, "multiparametric" and "multicolor," are used interchangeably.

[0016] Standard methodologies for MRD detection typically necessitate the capability to identify leukemic cells at frequencies below one cell per 10,000 normal bone marrow or peripheral blood cells (i.e., a sensitivity of 10“4). The reagent compositions and methods disclosed herein enable the detection of MRD cells at frequencies below one cell per 100,000 normal cells (i.e., achieving a sensitivity of 10“5).

[0017] Contemporary MRD measurement protocols mandate the simultaneous detection of numerous markers expressed by leukemic cells. This is accomplished through the use of precisely formulated combinations of antibodies, wherein each antibody targets a specific CD marker and is conjugated to a unique fluorochrome, allowing for spectral differentiation. Such a combination of fluorochrome-conjugated antibodies designed for a specific analytical purpose is conventionally termed an antibody "panel." An illustrative example of an established antibody panel, developed by the EuroFlow consortium, is presented in Table 1.

[0018] TABLE 1. EuroFlow Consortium One-Tube ALOT Panel (https: / / euroflow.org / )

[0019] Due to the limited number of parameters of the flow cytometer, antibodies in a single panel need to be distributed into several separate combinations. It is generally accepted that these combinations within the panel are referred to as "tubes." An example of such a four-tube panel from the EuroFlow consortium is presented in Table 2.

[0020] TABLE 2. A four-tube panel for detecting B-cell precursor ALL from the EuroFlow consortium (https: / / euroflow.org / )

[0021] A scientific publication by Muhsin, A.M. et al. (2024) [8] discloses a study conducted at the Jin Oncology Center from March 2019 to November 2023. This study evaluated the clinical significance of minimal residual disease (MRD) assessment in pediatric patients diagnosed with acute lymphoblastic leukemia (ALL), comparing morphological analysis with flow cytometric analysis at the conclusion of the induction therapy phase. The cohort comprised 58 patients under the age of 16, diagnosed via peripheral blood morphology, bone marrow examination, and / or flow cytometry. On day 29 of therapy, bone marrow aspirates were analyzed for MRD utilizing flow cytometry targeting the markers CD19, CD10, and TdT. Patients were stratified into three categories based on morphological assessment of blast percentage: C1 (<5% blasts), C2 (5-20% blasts), and C3 (>20% blasts). The findings indicated that while 46 patients achieved morphological remission, only 24 patients met the criteria for remission based on flow cytometric MRD assessment. Discrepancies between morphological and flow cytometric results were noted in 19 instances. Flow cytometry identified MRD positivity in 25 cases, representing 52.08% of the B-cell ALL patient subgroup. The publication concludes that incorporating MRD assessment alongside conventional morphological evaluation enhances the accuracy of remission status determination in pediatric ALL. U.S. Patent No. 5,047,321 , issued September 10, 1991 , describes a method for the flow cytometric analysis of cellular constituents within peripheral blood (PB) and bone marrow (BM) samples. This method employs a three-color fluorescence approach utilizing a combination comprising a fluorescently labeled anti-CD45 monoclonal antibody, a DNA-specific dye (LDS- 751 ), an RNA-specific dye (thiazole orange), in conjunction with forward light scatter (FSC) and side light scatter (SSC) measurements. The disclosed procedure enables the differentiation and quantification of various cellular components present in PB and BM, facilitating differential cell counting. Specifically, the methodology permits the identification of nucleated red blood cells, erythrocytes, reticulocytes, platelets, lymphocytes, monocytes, neutrophilic granulocytes, basophilic granulocytes, eosinophilic granulocytes, and progenitor cells of all nucleated hematopoietic lineages. However, the described multiparametric analysis is inherently limited in that it cannot discriminate between normal, reactive, regenerative, and neoplastic cell populations co-existing within a single sample, nor does it provide means for further characterization of these distinct cellular states. Consequently, this procedure is unsuitable for application in MRD measurement.

[0022] Chinese Patent No. CN116593699 (B), granted October 3, 2023, discloses an antibody composition formulated for flow cytometric applications, specifically targeting the detection of CD39 expression on T-lymphocyte surfaces. The disclosed composition comprises a mixture of monoclonal antibodies directed against CD45, CD3, CD4, CD8, and CD39, combined in a predefined 1 :1 :1 :1 :2 stoichiometric ratio. The stated purpose of this composition is to furnish comprehensive and precise cellular information pertinent to individual patients, thereby guiding subsequent clinical treatment strategies towards personalized and targeted therapeutic interventions. However, the design and intended application of this composition are focused on the identification and characterization of specific pathological T-cell populations. Therefore, it is not applicable to the measurement of MRD in B-cell acute lymphoblastic leukemia (B-ALL).

[0023] U.S. Patent No. 5,538,855, issued July 23, 1996, describes a procedure developed for achieving a more detailed analysis of lymphoid cell populations. This procedure enables the simultaneous identification of twelve distinct T-lymphocyte, B-lymphocyte, and Natural Killer (NK) cell subgroups within PB, BM, or lymph node specimens. The methodology utilizes a three-color antibody panel incorporating markers such as CD3, CD19, CD56 (and / or CD16), CD4, and CD8. These markers are distributed across six separate reaction tubes designed for two-color and three-color staining protocols. Notwithstanding the utilization of an increased number of markers compared to simpler panels, this approach is limited in its scope. It does not provide for further phenotypic characterization of the identified cell subgroups beyond basic identification, nor does it possess the capability to differentiate between normal and neoplastic cells within these populations. As a result, this procedure is inadequate for the purpose of MRD measurement.

[0024] The methodologies described in the aforementioned U.S. Patent Nos. 5,047,321 , CN116593699, and U.S. Patent No. 5,538,855 are limited in their capacity for detailed cellular characterization. Specifically, they lack the necessary resolution to effectively distinguish between normal, reactive, regenerative, and neoplastic or clonal cell populations within a given sample. Such differentiation necessitates the utilization of a substantially greater number of phenotypic markers analyzed concurrently via a flow cytometer capable of detecting and resolving multiple distinct fluorescent emissions simultaneously. Consequently, the procedures disclosed in these patents are unsuitable for the specific application of MRD measurement in BCP-ALL.

[0025] U.S. Patent No. 9,777,332 (B2), issued October 3, 2017, describes a seven-color panel designed for MRD measurement in B-cell ALL. This panel comprises a combination of antibodies directed against 22 distinct CD markers, distributed across seven separate reaction tubes. Notably, antibodies against CD10, CD19, CD34, and CD45 are replicated in all seven tubes, while an antibody against CD38 is replicated in three tubes. Implementation of this panel requires aliquoting the patient sample (bone marrow or peripheral blood) into each of the seven tubes. To achieve an MRD sensitivity level of 10s, the methodology mandates the acquisition of at least 4 x 10® cellular events per tube. This translates to a requirement of processing approximately 4.5 x 10® cells per tube, culminating in a total cellular input requirement of roughly 44 x 10® cells. Such a substantial sample volume requirement poses significant practical challenges, particularly in the context of pediatric MRD assessment where obtainable sample volumes are often limited. Furthermore, the repetitive inclusion of antibodies targeting CD10, CD19, CD34, CD45, and CD38 across multiple tubes inherently increases reagent consumption, thereby elevating the overall cost per test. Additionally, the described panel lacks specific reagents dedicated to the identification and enumeration of total nucleated cells within the bone marrow sample, a critical component for accurate MRD quantification. The present invention overcomes these limitations through the provision of at least 12-, at least 14-, and at least 16-color antibody panels wherein all necessary reagents are consolidated into a single reaction tube format, rendering the invention particularly advantageous for pediatric MRD applications.

[0026] U.S. Patent No. 10,656,144 (B2), issued May 19, 2020, discloses an eight-color panel developed for MRD measurement specifically in multiple myeloma. This panel incorporates 13 antibodies targeting markers including CD38, CD138, CD45, CD19, CD56, CD27, CD81 , CD117, Anti-kappa, and Anti-lambda, distributed across two reaction tubes. Four markers (CD38, CD138, CD45, CD19) are duplicated in both tubes. The specific marker combination employed is tailored for the detection of MRD in multiple myeloma and is, therefore, inappropriate for MRD assessment in B-cell precursor ALL, which necessitates a distinct set of markers relevant to B-lymphoblast immunophenotyping.

[0027] Australian Patent Application No. AU 2021203620 (A1), published May 19, 2020, describes an eight-color panel intended for the immunophenotyping of various hematologic neoplasms. The specific panel configuration proposed for B-ALL comprises antibodies against 19 CD markers (CD19, CD34, CD45, CD20, IgK, CD9, CD21 , CD58, CylgM, TdT, CD13, NG2, CD10, IgM, CD117, CD123, CD38, IgLambda, CD24, and CD81), allocated across four separate tubes. The inventors explicitly state that this panel is primarily designed for the classification of B-ALL at the time of diagnosis, rather than for MRD monitoring post-therapy. The requirement for four distinct tubes renders this approach less suitable for pediatric MRD measurement due to the associated demand for larger bone marrow sample volumes. Furthermore, this panel configuration lacks reagents for the crucial step of identifying total nucleated cells within the bone marrow matrix. It also omits several markers commonly recommended and utilized for sensitive MRD detection in BCP-ALL, such as CD73, CD304, CD44, CD86, CD99, CD371 , CD66c, and CD22. Consequently, this panel is considered suboptimal for robust BCP-ALL MRD measurement. In contrast, embodiments of the panels of the present invention incorporate additional informative markers and include a DNA dye in combination with an anti-CD36 antibody for accurate nucleated cell staining, thereby enhancing the precision of MRD quantification.

[0028] BG Utility Model Application No. 5932 (Registration No. 4690), dated 12 December 2023, which discloses an eight-color reagent panel for the quantification of Minimal Residual Disease (MRD) in B-cell Acute Lymphoblastic Leukemia (B-ALL). Said panel comprises seven antibodies directed against cell surface markers CD45, CD19, CD10, CD20, CD38, CD34, and CD58, intended for the identification of residual leukemic cells, referred to herein as MRD cells. Additionally, the panel incorporates a DNA-intercalating dye for staining cellular nuclei. The purpose of this DNA dye is to facilitate the discrimination of nucleated cells from anucleate cells and cellular debris, thereby enabling a more accurate calculation of the frequency of MRD cells, typically expressed as a percentage relative to total nucleated cells.

[0029] The aforementioned prior art panel exhibits certain limitations. Firstly, the number of markers employed is limited, primarily including core antigens for MRD assessment in pediatric B-ALL, while omitting other established ancillary markers known to enhance the sensitivity and specificity of MRD detection. Secondly, the included DNA dye, while enabling the identification of nucleated cells, does not facilitate the exclusion of contaminating platelet aggregates. The presence of such aggregates can interfere with the accurate enumeration of total nucleated cells, thereby potentially compromising the precision of the calculated MRD frequency.

[0030] The present disclosure provides compositions and methods designed to overcome the limitations of the prior art. Embodiments of the invention augment the foundational set of markers (CD45, CD19, CD10, CD20, CD38, CD34, CD58) with antibodies directed against a selection of additional informative markers, specifically including CD81 , CD73, CD304, CD44, CD86, CD99, CD371 , CD66c, and CD22. The incorporation of these additional markers serves to improve the sensitivity and specificity for identifying MRD populations.

[0031] Furthermore, embodiments of the invention incorporate an antibody directed against the CD36 marker. The inclusion of the anti-CD36 antibody specifically addresses the limitation related to platelet contamination by enabling the identification and subsequent analytical exclusion of platelets and platelet aggregates from the nucleated cell population gate. This ensures a more accurate determination of the total nucleated cell count, which serves as the denominator for calculating MRD frequency.

[0032] Consequently, owing to the increased total number of antibodies incorporated, the reagent compositions according to embodiments of the present invention are formulated for multiparameter flow cytometric analysis, for example as 12-color, 14-color, and 16-color panels, suitable for high-resolution MRD assessment in B-ALL.

[0033] Further relevant background is provided in the scientific publication by Manivannan, P. et al., dated 12 September 2022.

[0010] This publication discloses an evaluation of multicolor flow cytometry (MFC) for detecting Minimal Residual Disease (MRD) in B-cell Acute Lymphoblastic Leukemia (B-ALL). A primary objective reported therein was the development of a cost- effective, single ten-color antibody panel suitable for MRD detection, particularly in resource- constrained environments.

[0034] The study characterized the immunophenotypic profile of B-ALL at diagnosis in 82 newly diagnosed cases between October 2019 and April 2021 . This characterization utilized seven markers associated with leukemia-associated immunophenotypes (LAIPs), in conjunction with six established core markers. The results indicated that the markers CD73 and CD86 were the most frequently informative LAIPs identified at diagnosis, followed by the underexpression of CD81 . Based on these findings, Manivannan et al. proposed a ten-color panel comprising antibodies against CD45, CD19, CD34, CD10, CD20, CD38, CD73, CD86, CD81 , and CD44. This panel was suggested for utility in both initial diagnosis and subsequent MRD monitoring in post-therapy B-ALL samples, particularly within low- and middle-income countries.

[0035] In contrast to the ten-color panel disclosed by Manivannan et aL, embodiments of the reagent panels according to the present invention incorporate antibodies directed against seven additional markers, namely: CD58, CD304, CD99, CD371 , CD123, CD66c, and CD22. The inclusion of this expanded set of markers is intended to enhance the sensitivity and specificity of MRD quantification in B-ALL.

[0036] Furthermore, embodiments of the reagent panels according to the present invention incorporate both a DNA-intercalating dye and an antibody directed against the CD36 marker. The DNA dye facilitates the identification and enumeration of nucleated cells, while the anti-CD36 antibody enables the specific identification and subsequent analytical exclusion of platelet aggregates. This dual approach serves to increase the accuracy of the MRD measurement by refining the determination of the total nucleated cell population used as the denominator in MRD frequency calculations.

[0037] A scientific articie by Arunachalam, A.K., et al [9] published in October 2021 , details the development and validation of a 12-color single-tube panel for MRD assessment in B-ALL patients. The antibody selection for this panel was guided by prior institutional experience, existing published literature, and technical considerations including antigen expression intensity and management of fluorescence spillover between detection channels. Antibody concentrations were optimized through titration experiments. Panel validation involved analyzing samples from healthy controls alongside initial diagnostic and follow-up samples from B-ALL patients, with comparative analysis against an established 8-color MRD assay. Following successful validation, the 12-color panel was implemented for MRD assessment in 138 consecutive B-ALL patient samples. The study identified CD73, CD58, and CD38 as particularly useful markers due to frequent aberrant expression patterns, while CD304 was found to be the least informative marker within their specific panel configuration. While representing an advance, the present invention further enhances MRD detection capabilities by incorporating additional markers not included in the Arunachalam et al. panel, specifically CD99, CD371 , CD123, CD66c, and CD22. The inclusion of these markers increases the likelihood of identifying two or more leukemia-associated immunophenotypes (LAIPs), thereby augmenting the sensitivity and specificity of the MRD measurement methodology for pediatric BCP-ALL. Moreover, embodiments of the panels of the present invention integrate a DNA-binding dye and an anti-CD36 antibody specifically for accurate staining and gating of nucleated cells, contributing to improved overall measurement accuracy.

[0038] European Patent No. EP 3064942 A2 (published December 20, 2017) and U.S. Patent No. US 10,874,710 (B2) (issued December 29, 2020), which share common inventorship and title, disclose antibody panels configured for 8-color, 10-color, and 12-color flow cytometric analysis for Minimal Residual Disease (MRD) measurement in B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL). These panels comprise antibodies directed against markers including CD45, CD20, CD19, CD38, CD10, CD81 , CD66c, CD123, CD34, CD304, CD73, nuclear Terminal deoxynucleotidyl Transferase (NuTdT), surface membrane Immunoglobulin kappa (SmlgK), surface membrane Immunoglobulin lambda (SmlgA), and cytoplasmic Immunoglobulin mu heavy chain (CylgM). The described 8-color panel configuration necessitates the distribution of antibodies across three separate reaction tubes. Specifically, antibodies against CD45, CD20, and CD19 are included in all three tubes, whereas antibodies against CD38 and CD10 are included in two of the three tubes. To alleviate the requirement for large patient sample volumes and to reduce reagent consumption associated with the multi-tube 8-color format, the inventors further proposed 10-color and 12-color panel configurations. The 10-color panel configuration described omits antibodies targeting SmlgK, SmlgA, and CylgM. Within the 12- color panel configuration, specific antibody pairs (namely, anti-SmlgK combined with anti-SmlgA; anti-CD66c combined with anti-CD123; and anti-CD304 combined with anti-CD73) are conjugated to the identical fluorochrome, thereby requiring co-detection within a single fluorescence channel of the flow cytometer. However, these prior art disclosures exhibit certain limitations relevant to optimal MRD assessment:

[0039] 1 . They lack dedicated reagents for the precise identification and enumeration of total nucleated cells within the bone marrow or peripheral blood sample. Accurate quantification of the total nucleated cell population is essential for calculating the MRD cell frequency reliably, thus the absence of such reagents potentially reduces the accuracy of the final MRD result.

[0040] 2. The disclosed panels omit several antibodies directed against markers recognized as informative for MRD detection in pediatric BCP-ALL, including CD44, CD86, CD99, CD371 , and optionally CD22. The inclusion of an anti-CD22 antibody, in particular, is gaining significant importance for MRD monitoring due to the increasing clinical application of CD22-targeted therapies (e.g., anti-CD22 monoclonal antibodies or antibody-drug conjugates) in the treatment of B-ALL.

[0041] In contrast, the present invention addresses these deficiencies. The antibody panels disclosed herein incorporate antibodies directed against markers CD44, CD86, CD99, CD371 , and optionally CD22. Furthermore, the compositions explicitly include reagents facilitating the accurate measurement of total nucleated cells.

[0042] Analysis of the known solutions indicates a clear trend in the field towards increasing the number of simultaneously analyzed markers — consequently increasing the number of parameters or "colors" utilized in flow cytometric measurements — to enhance the sensitivity and specificity of MRD detection. Additionally, there is a recognized need to integrate reagents for the quantitative determination of total nucleated cells directly within the MRD panel or kit.

[0043] The present invention advances this field by providing improved antibody panels and diagnostic kits featuring optimized, well-defined combinations of antibodies as described herein. These combinations are specifically designed to enhance the sensitivity and specificity of MRD measurement, particularly in the context of pediatric BCP-ALL. Embodiments of the panels of the invention incorporate reagents, such as a DNA-binding dye and an anti-CD36 antibody, for the specific purpose of staining and accurately enumerating nucleated cells, thereby improving the overall precision of the MRD measurement. Description of Figures

[0044] Figure 1. Illustrate a template with bivariate plots for the analysis of flow cytometric data for MRD measurement in B-ALL.

[0045] A) Plot for assessing the homogeneity of the measurement.

[0046] B) Plot for eliminating granulocytes based on SSC and CD38.

[0047] C and D) Control plots for the presence of CD 19+ blasts and hematogones.

[0048] E) Plot for doublet removal. F) Plot for isolating CD19+ cells.

[0049] G) Plot for eliminating plasmacytes.

[0050] H) Plot for doublet removal and selection of single cells.

[0051] I and J) Plots for selecting common B-cells.

[0052] K) and subsequent plots Determination of marker co-expression.

[0053] Figure 2. Illustrates a template for determining the percentage of nucleated cells.

[0054] A) Plot for selecting SYT041 + cells.

[0055] B) Plot for selecting single SYT041 + cells.

[0056] C) Plot for selecting SYT041 + CD19+ cells.

[0057] Figure 3. Illustrates the specificity and sensitivity of an 8-color panel and a 14-color panel, obtained through ROC analysis.

[0058] SUMMARY OF THE INVENTION AND DETAILED DESCRIPTION

[0059] In one aspect, the invention relates to a reagent panel for the measurement of minimal residual disease (MRD) associated with pediatric B-cell precursor acute lymphoblastic leukemia (B-ALL) in a sample from a subject by multiparametric flow cytometry, said panel comprising a combination of antibodies directed against markers, wherein the combination of antibodies comprises i. antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99 and CD371 , and ii. antibodies targeting markers CD19 and / or CD22; wherein the antibodies are conjugated with fluorochromes. in some embodiments, CD19 may be replaced by CD22 when the subject has previously been treated with an anti-CD19 therapy, in some embodiments, both CD22 and CD19 are included in the reagent panel. in some embodiments, the combination of antibodies further comprises an antibody targeting CD36. in some embodiments, the reagent panel further comprises a DNA-binding dye. in some embodiments, the reagent panel comprises a combination of two tubes: a) a first tube (T ube 1 ) containing a first reagent composition comprising a combination of monoclonal antibodies directed against human leukocyte markers, wherein said first reagent composition comprises a combination of antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99, CD371 and CD19 and / or CD22 thereby facilitating enhanced identification of MRD leukemia cells, wherein the antibodies are conjugated with fluorochromes; and b) a second tube (Tube 2) containing a second reagent composition formulated for quantifying nucleated cells said second reagent composition comprising a DNA-binding dye and monoclonal antibodies directed against markers CD45, CD19, and CD36, wherein the antibodies are conjugated with fluorochromes.

[0060] In some embodiments, the panel is configured as at least a 12-colour panel, at least a 14-colour panel, at least a 16-colour panel, at least a 18-colour panel, or at least a 20-colour panel.

[0061] In some embodiments, the panel is configured as a 12-colour panel.

[0062] In some embodiments, the antibodies targeting markers comprise: a) anti-CD10, b) anti-CD45, c) anti-CD34, d) anti-CD20, e) anti-CD81 , f) anti-CD58 and anti-CD371 , g) anti-CD99 and anti-CD34, h) anti-CD44 and CD73, i) anti-CD38, j) anti-CD123 and CD66c, k) anti-CD19, and l) anti-CD86, wherein each of the antibodies of groups a)-l) are conjugated with spectrally distinct fluorochromes.

[0063] In some embodiments, the antibodies targeting markers comprise: a) anti-CD10, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD34, conjugated with fluorochrome excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD20, conjugated with one or more fluorochromes excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 710 / 50 nm, e) anti-CD81 , conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, f) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, g) anti-CD99 and anti-CD34, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm,

[0064] I) anti-CD86, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, h) anti-CD44 and CD73, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD38, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, j) anti-CD123 and CD66c, conjugated with one or more fluorochromes excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and k) anti-CD19, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0065] In some embodiments, the panel is configured as a 14-colour panel. in some embodiments, the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , b) anti-CD45, c) anti-CD304, d) anti-CD44, e) anti-CD86, f) anti-CD99, g) anti-CD58 and anti-CD371 , h) anti-CD66c and anti-CD123, i) anti-CD34, j) anti-CD10, k) anti-CD19, l) anti-CD22, m) anti-CD38, and n) anti-CD20, wherein each of the antibodies of groups a)-n) are conjugated with spectrally distinct fluorochromes. in some embodiments, the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , conjugated with one or more fluorochromes excitable by a violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, e) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, f) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, g) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by a blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, h) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, i) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, j) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, k) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, l) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, m) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and n) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0066] In some embodiments, the panel is configured as a 16-colour panel.

[0067] In some embodiments, the antibodies targeting markers comprise: a) anti-CD58, b) anti-CD81 , c) anti-CD73, d) anti-CD45, e) anti-CD304,

[0068] Y1 f) anti-CD44, g) anti-CD86, h) anti-CD99, i) anti-CD371 , j) anti-CD66c and anti-CD123, k) anti-CD34, l) anti-CD10, m) anti-CD19, n) anti-CD22, o) anti-CD38, and p) anti-CD20, wherein each of the antibodies of groups a)-p) are conjugated with spectrally distinct fluorochromes.

[0069] In some embodiments, wherein the antibodies targeting markers comprise: a) anti-CD58, conjugated with a fluorochrome excitable by a UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 379 / 28 nm, b) anti-CD81 , conjugated with a fluorochrome excitable by the UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 515 / 30 nm, c) anti-CD73, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, d) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, e) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, f) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, g) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, h) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD371 , conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 530 / 31 nm, j) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, k) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, l) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, m) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, n) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, o) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and p) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0070] In some embodiments, the DNA-binding dye is selected from the group consisting of SYTO41 , DAPI, Hoechst or propidium iodide.

[0071] In some embodiments, the DNA-binding dye is SYTO41 .

[0072] In some embodiments, the antibodies are in liquid tubes.

[0073] In some embodiments, the antibodies are in dry tubes. in some embodiments, the sample comprises bone marrow cells. in some embodiments, the sample comprises peripheral blood cells. in some embodiments, the subject is a human. in some embodiments, the subject has previously been diagnosed with pediatric B-cell precursor acute lymphoblastic leukemia. in some embodiments, the subject has previously been treated for pediatric B-cell precursor acute lymphoblastic leukemia. in a further aspect, the invention relates to use of a reagent panel for detecting MRD associated with B-ALL in a sample from a subject, wherein the reagent panel comprises a combination of antibodies directed against markers, wherein the combination of antibodies comprises i. antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99 and CD371 , and ii. antibodies targeting markers CD19 and / or CD22; wherein the antibodies are conjugated with fluorochromes.

[0074] In some embodiments, CD19 may be replaced by CD22 when the subject has previously been treated with an anti-CD19 therapy. In some embodiments, both CD22 and CD19 are included in the reagent panel.

[0075] In some embodiments, the sample is MRD positive if at least two of CD58, CD81 , CD73, CD45, CD304, CD44, CD86, CD99, CD371 , CD123 or CD66c are present in the sample.

[0076] In some embodiments, the combination of antibodies further comprises an antibody targeting CD36.

[0077] In some embodiments, the reagent panel further comprises a DNA-binding dye.

[0078] In some embodiments, the reagent panel comprises a combination of two tubes: a) a first tube (T ube 1 ) containing a first reagent composition comprising a combination of monoclonal antibodies directed against human leukocyte markers, wherein said first reagent composition comprises a combination of antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99, CD371 and CD19 and / or CD22 thereby facilitating enhanced identification of MRD leukemia cells, wherein the antibodies are conjugated with fluorochromes; and b) a second tube (Tube 2) containing a second reagent composition formulated for quantifying nucleated cells said second reagent composition comprising a DNA-binding dye and monoclonal antibodies directed against markers CD45, CD19, and CD36, wherein the antibodies are conjugated with fluorochromes.

[0079] In some embodiments, the panel is configured as at least a 12-colour panel, at least a 14-colour panel, at least a 16-colour panel, at least a 18-colour panel, or at least a 20-colour panel.

[0080] In some embodiments, the panel is configured as a 12-colour panel.

[0081] In some embodiments, the antibodies targeting markers comprise: a) anti-CD10, b) anti-CD45, c) anti-CD34, d) anti-CD20, e) anti-CD81 , f) anti-CD58 and anti-CD371 , g) anti-CD99 and anti-CD34, h) anti-CD44 and CD73, i) anti-CD38, j) anti-CD123 and CD66c, k) anti-CD19, and l) anti-CD86, wherein each of the antibodies of groups a)-l) are conjugated with spectrally distinct fluorochromes.

[0082] In some embodiments, the antibodies targeting markers comprise: a) anti-CD10, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD34, conjugated with fluorochrome excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD20, conjugated with one or more fluorochromes excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 710 / 50 nm, e) anti-CD81 , conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, f) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, g) anti-CD99 and anti-CD34, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm,

[0083] I) anti-CD86, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, h) anti-CD44 and CD73, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD38, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, j) anti-CD123 and CD66c, conjugated with one or more fluorochromes excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and k) anti-CD19, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0084] In some embodiments, the panel is configured as a 14-colour panel.

[0085] In some embodiments, the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , b) anti-CD45, c) anti-CD304, d) anti-CD44, e) anti-CD86, f) anti-CD99, g) anti-CD58 and anti-CD371 , h) anti-CD66c and anti-CD123, i) anti-CD34, j) anti-CD10, k) anti-CD19, l) anti-CD22, m) anti-CD38, and n) anti-CD20, wherein each of the antibodies of groups a)-n) are conjugated with spectrally distinct fluorochromes.

[0086] In some embodiments, the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , conjugated with one or more fluorochromes excitable by a violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, e) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, f) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, g) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by a blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, h) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, i) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, j) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, k) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, l) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, m) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and n) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0087] In some embodiments, the panel is configured as a 16-colour panel.

[0088] In some embodiments, the antibodies targeting markers comprise: a) anti-CD58, b) anti-CD81 , c) anti-CD73, d) anti-CD45, e) anti-CD304, f) anti-CD44, g) anti-CD86, h) anti-CD99, i) anti-CD371 , j) anti-CD66c and anti-CD123, k) anti-CD34, l) anti-CD10, m) anti-CD19, n) anti-CD22, o) anti-CD38, and p) anti-CD20, wherein each of the antibodies of groups a)-p) are conjugated with spectrally distinct fluorochromes.

[0089] In some embodiments, wherein the antibodies targeting markers comprise: a) anti-CD58, conjugated with a fluorochrome excitable by a UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 379 / 28 nm, b) anti-CD81 , conjugated with a fluorochrome excitable by the UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 515 / 30 nm, c) anti-CD73, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, d) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, e) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, f) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, g) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, h) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD371 , conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 530 / 31 nm, j) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, k) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, l) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, m) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, n) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, o) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and p) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0090] In some embodiments, the DNA-binding dye is selected from the group consisting of SYTO41 , DAPI, Hoechst or propidium iodide.

[0091] In some embodiments, the DNA-binding dye is SYTO41 .

[0092] In some embodiments, the antibodies are in liquid tubes.

[0093] In some embodiments, the antibodies are in dry tubes.

[0094] In some embodiments, the sample comprises bone marrow cells.

[0095] In some embodiments, the sample comprises peripheral blood cells.

[0096] In some embodiments, the subject is a human.

[0097] In some embodiments, the subject has previously been diagnosed with pediatric B-cell precursor acute lymphoblastic leukemia.

[0098] In some embodiments, the subject has previously been treated for pediatric B-cell precursor acute lymphoblastic leukemia.

[0099] In a further aspect, the invention relates to use of a reagent panel for identifying a subject at risk of developing B-ALL relapse by detecting MRD in a sample from a subject, wherein the reagent panel comprises a combination of antibodies directed against markers, wherein the combination of antibodies comprises i. antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99 and CD371 , and ii. antibodies targeting markers CD19 and / or CD22; wherein the antibodies are conjugated with fluorochromes. in some embodiments, CD19 may be replaced by CD22 when the subject has previously been treated with an anti-CD19 therapy, in some embodiments, both CD22 and CD19 are included in the reagent panel. in some embodiments, the subject is considered to be at risk of developing B-ALL relapse if at least two of CD58, CD81 , CD73, CD45, CD304, CD44, CD86, CD99, CD371 , CD123 or CD66c are present in the sample. in some embodiments, the combination of antibodies further comprises an antibody targeting CD36. in some embodiments, the reagent panel further comprises a DNA-binding dye. in some embodiments, the reagent panel comprises a combination of two tubes: a) a first tube (T ube 1 ) containing a first reagent composition comprising a combination of monoclonal antibodies directed against human leukocyte markers, wherein said first reagent composition comprises a combination of antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99, CD371 and CD19 and / or CD22 thereby facilitating enhanced identification of MRD leukemia cells, wherein the antibodies are conjugated with fluorochromes; and b) a second tube (Tube 2) containing a second reagent composition formulated for quantifying nucleated cells said second reagent composition comprising a DNA-binding dye and monoclonal antibodies directed against markers CD45, CD19, and CD36, wherein the antibodies are conjugated with fluorochromes.

[0100] In some embodiments, the panel is configured as at least a 12-colour panel, at least a 14-colour panel, at least a 16-colour panel, at least a 18-colour panel, or at least a 20-colour panel.

[0101] In some embodiments, the panel is configured as a 12-colour panel.

[0102] In some embodiments, the antibodies targeting markers comprise: a) anti-CD10, b) anti-CD45, c) anti-CD34, d) anti-CD20, e) anti-CD81 , f) anti-CD58 and anti-CD371 , g) anti-CD99 and anti-CD34, h) anti-CD44 and CD73, i) anti-CD38, j) anti-CD123 and CD66c, k) anti-CD19, and l) anti-CD86, wherein each of the antibodies of groups a)-l) are conjugated with spectrally distinct fluorochromes.

[0103] In some embodiments, the antibodies targeting markers comprise: a) anti-CD10, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD34, conjugated with fluorochrome excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD20, conjugated with one or more fluorochromes excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 710 / 50 nm, e) anti-CD81 , conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, f) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, g) anti-CD99 and anti-CD34, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm,

[0104] I) anti-CD86, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, h) anti-CD44 and CD73, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD38, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, j) anti-CD123 and CD66c, conjugated with one or more fluorochromes excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and k) anti-CD19, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0105] In some embodiments, the panel is configured as a 14-colour panel.

[0106] In some embodiments, the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , b) anti-CD45, c) anti-CD304, d) anti-CD44, e) anti-CD86, f) anti-CD99, g) anti-CD58 and anti-CD371 , h) anti-CD66c and anti-CD123, i) anti-CD34, j) anti-CD10, k) anti-CD19, l) anti-CD22, m) anti-CD38, and n) anti-CD20, wherein each of the antibodies of groups a)-n) are conjugated with spectrally distinct fluorochromes.

[0107] In some embodiments, the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , conjugated with one or more fluorochromes excitable by a violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, e) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, f) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, g) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by a blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, h) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, i) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, j) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, k) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, l) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, m) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and n) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0108] In some embodiments, the panel is configured as a 16-colour panel.

[0109] In some embodiments, the antibodies targeting markers comprise: a) anti-CD58, b) anti-CD81 , c) anti-CD73, d) anti-CD45, e) anti-CD304, f) anti-CD44, g) anti-CD86, h) anti-CD99, i) anti-CD371 , j) anti-CD66c and anti-CD123, k) anti-CD34, l) anti-CD10, m) anti-CD19, n) anti-CD22, o) anti-CD38, and p) anti-CD20, wherein each of the antibodies of groups a)-p) are conjugated with spectrally distinct fluorochromes.

[0110] In some embodiments, wherein the antibodies targeting markers comprise: a) anti-CD58, conjugated with a fluorochrome excitable by a UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 379 / 28 nm, b) anti-CD81 , conjugated with a fluorochrome excitable by the UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 515 / 30 nm, c) anti-CD73, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, d) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, e) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, f) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, g) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, h) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD371 , conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 530 / 31 nm, j) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, k) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, l) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, m) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, n) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, o) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and p) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm. in some embodiments, the DNA-binding dye is selected from the group consisting of SYTO41 , DAPi, Hoechst or propidium iodide. in some embodiments, the DNA-binding dye is SYTO41 . in some embodiments, the antibodies are in liquid tubes. in some embodiments, the antibodies are in dry tubes. in some embodiments, the sample comprises bone marrow cells. in some embodiments, the sample comprises peripheral blood cells. in some embodiments, the subject is a human. in some embodiments, the subject has previously been diagnosed with pediatric B-cell precursor acute lymphoblastic leukemia. in some embodiments, the subject has previously been treated for pediatric B-cell precursor acute lymphoblastic leukemia.

[0111] In a further aspect, the invention relates to a method of detecting MRD associated with B-ALL in a sample from a subject, the method comprising detecting MRD in the sample from the subject by multiparametric flow cytometry using a reagent panel comprising a combination of antibodies directed against markers, wherein the combination of antibodies comprises i. antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99 and CD371 , and ii. antibodies targeting markers CD19 and / or CD22; wherein the antibodies are conjugated with fluorochromes. in some embodiments, CD19 may be replaced by CD22 when the subject has previously been treated with an anti-CD19 therapy, in some embodiments, both CD22 and CD19 are included in the reagent panel. in some embodiments, the sample is MRD positive if at least two of CD58, CD81 , CD73, CD45, CD304, CD44, CD86, CD99, CD371 , CD123 or CD66c are present in the sample. in some embodiments, the reagent panel further comprises a DNA-binding dye. in some embodiments, the combination of antibodies further comprises an antibody targeting CD36. in some embodiments, the reagent panel comprises a combination of two tubes: a) a first tube (T ube 1 ) containing a first reagent composition comprising a combination of monoclonal antibodies directed against human leukocyte markers, wherein said first reagent composition comprises a combination of antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99, CD371 and CD19 and / or CD22 thereby facilitating enhanced identification of MRD leukemia cells, wherein the antibodies are conjugated with fluorochromes; and b) a second tube (Tube 2) containing a second reagent composition formulated for quantifying nucleated cells said second reagent composition comprising a DNA-binding dye and monoclonal antibodies directed against markers CD45, CD19, and CD36, wherein the antibodies are conjugated with fluorochromes.

[0112] In some embodiments, the panel is configured as at least a 12-colour panel, at least a 14-colour panel, at least a 16-colour panel, at least a 18-colour panel, or at least a 20-colour panel.

[0113] In some embodiments, the panel is configured as a 12-colour panel.

[0114] In some embodiments, the antibodies targeting markers comprise: a) anti-CD10, b) anti-CD45, c) anti-CD34, d) anti-CD20, e) anti-CD81 , f) anti-CD58 and anti-CD371 , g) anti-CD99 and anti-CD34, h) anti-CD44 and CD73, i) anti-CD38, j) anti-CD123 and CD66c, k) anti-CD19, and l) anti-CD86, wherein each of the antibodies of groups a)-l) are conjugated with spectrally distinct fluorochromes.

[0115] In some embodiments, the antibodies targeting markers comprise: a) anti-CD10, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD34, conjugated with fluorochrome excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD20, conjugated with one or more fluorochromes excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 710 / 50 nm, e) anti-CD81 , conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, f) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, g) anti-CD99 and anti-CD34, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm,

[0116] I) anti-CD86, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, h) anti-CD44 and CD73, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD38, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, j) anti-CD123 and CD66c, conjugated with one or more fluorochromes excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and k) anti-CD19, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0117] In some embodiments, the panel is configured as a 14-colour panel.

[0118] In some embodiments, the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , b) anti-CD45, c) anti-CD304, d) anti-CD44, e) anti-CD86, f) anti-CD99, g) anti-CD58 and anti-CD371 , h) anti-CD66c and anti-CD123, i) anti-CD34, j) anti-CD10, k) anti-CD19, l) anti-CD22, m) anti-CD38, and n) anti-CD20, wherein each of the antibodies of groups a)-n) are conjugated with spectrally distinct fluorochromes.

[0119] In some embodiments, the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , conjugated with one or more fluorochromes excitable by a violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, e) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, f) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, g) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by a blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, h) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, i) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, j) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, k) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, l) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, m) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and n) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0120] In some embodiments, the panel is configured as a 16-colour panel.

[0121] In some embodiments, the antibodies targeting markers comprise: a) anti-CD58, b) anti-CD81 , c) anti-CD73, d) anti-CD45, e) anti-CD304, f) anti-CD44, g) anti-CD86, h) anti-CD99, i) anti-CD371 , j) anti-CD66c and anti-CD123, k) anti-CD34, l) anti-CD10, m) anti-CD19, n) anti-CD22, o) anti-CD38, and p) anti-CD20, wherein each of the antibodies of groups a)-p) are conjugated with spectrally distinct fluorochromes.

[0122] In some embodiments, wherein the antibodies targeting markers comprise: a) anti-CD58, conjugated with a fluorochrome excitable by a UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 379 / 28 nm, b) anti-CD81 , conjugated with a fluorochrome excitable by the UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 515 / 30 nm, c) anti-CD73, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, d) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, e) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, f) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, g) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, h) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD371 , conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 530 / 31 nm, j) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, k) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, l) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, m) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, n) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, o) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and p) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0123] In some embodiments, the DNA-binding dye is selected from the group consisting of SYTO41 , DAPI, Hoechst or propidium iodide.

[0124] In some embodiments, the DNA-binding dye is SYTO41 .

[0125] In some embodiments, the antibodies are in liquid tubes.

[0126] In some embodiments, the antibodies are in dry tubes.

[0127] In some embodiments, the sample comprises bone marrow cells.

[0128] In some embodiments, the sample comprises peripheral blood cells.

[0129] In some embodiments, the subject is a human.

[0130] In some embodiments, the subject has previously been diagnosed with pediatric B-cell precursor acute lymphoblastic leukemia.

[0131] In some embodiments, the subject has previously been treated for pediatric B-cell precursor acute lymphoblastic leukemia.

[0132] In a further aspect, the invention relates to a method for detecting MRD associated with B-ALL in a sample from a subject, the method comprising: a. contacting the sample with a reagent panel, wherein the reagent panel comprises a combination of antibodies directed against markers, wherein the combination of antibodies comprises i. antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123 CD304, CD44, CD86, CD99 and CD371 , and ii. antibodies targeting markers CD19 and / or CD22; wherein the antibodies are conjugated with fluorochromes, b. contacting the sample with a DNA-binding dye, c. detecting the presence of the antibody binding targets by multiparametric flow cytometry, wherein detection of at least two of CD58, CD81 , CD73, CD45, CD304, CD44, CD86, CD99, CD371 , CD123 or CD66c determines the presence of MRD.

[0133] In some embodiments, CD19 may be replaced by CD22 when the subject has previously been treated with an anti-CD19 therapy. In some embodiments, both CD22 and CD19 are included in the reagent panel.

[0134] In some embodiments, the combination of antibodies further comprises an antibody targeting CD36.

[0135] In some embodiments, the reagent panel comprises a combination of two tubes: a) a first tube (T ube 1 ) containing a first reagent composition comprising a combination of monoclonal antibodies directed against human leukocyte markers, wherein said first reagent composition comprises a combination of antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99, CD371 and CD19 and / or CD22 thereby facilitating enhanced identification of MRD leukemia cells, wherein the antibodies are conjugated with fluorochromes; and b) a second tube (Tube 2) containing a second reagent composition formulated for quantifying nucleated cells said second reagent composition comprising a DNA-binding dye and monoclonal antibodies directed against markers CD45, CD19, and CD36, wherein the antibodies are conjugated with fluorochromes.

[0136] In some embodiments, the panel is configured as at least a 12-colour panel, at least a 14-colour panel, at least a 16-colour panel, at least a 18-colour panel, or at least a 20-colour panel.

[0137] In some embodiments, the panel is configured as a 12-colour panel.

[0138] In some embodiments, the antibodies targeting markers comprise: a) anti-CD10, b) anti-CD45, c) anti-CD34, d) anti-CD20, e) anti-CD81 , f) anti-CD58 and anti-CD371 , g) anti-CD99 and anti-CD34, h) anti-CD44 and CD73, i) anti-CD38, j) anti-CD123 and CD66c, k) anti-CD19, and l) anti-CD86, wherein each of the antibodies of groups a)-l) are conjugated with spectrally distinct fluorochromes.

[0139] In some embodiments, the antibodies targeting markers comprise: a) anti-CD10, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD34, conjugated with fluorochrome excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD20, conjugated with one or more fluorochromes excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 710 / 50 nm, e) anti-CD81 , conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, f) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, g) anti-CD99 and anti-CD34, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm,

[0140] I) anti-CD86, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, h) anti-CD44 and CD73, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD38, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, j) anti-CD123 and CD66c, conjugated with one or more fluorochromes excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and k) anti-CD19, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0141] In some embodiments, the panel is configured as a 14-colour panel.

[0142] In some embodiments, the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , b) anti-CD45, c) anti-CD304, d) anti-CD44, e) anti-CD86, f) anti-CD99, g) anti-CD58 and anti-CD371 , h) anti-CD66c and anti-CD123, i) anti-CD34, j) anti-CD10, k) anti-CD19, l) anti-CD22, m) anti-CD38, and n) anti-CD20, wherein each of the antibodies of groups a)-n) are conjugated with spectrally distinct fluorochromes.

[0143] In some embodiments, the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , conjugated with one or more fluorochromes excitable by a violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, e) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, f) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, g) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by a blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, h) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, i) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, j) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, k) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, l) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, m) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and n) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0144] In some embodiments, the panel is configured as a 16-colour panel.

[0145] In some embodiments, the antibodies targeting markers comprise: a) anti-CD58, b) anti-CD81 , c) anti-CD73, d) anti-CD45, e) anti-CD304, f) anti-CD44, g) anti-CD86, h) anti-CD99, i) anti-CD371 , j) anti-CD66c and anti-CD123, k) anti-CD34, l) anti-CD10, m) anti-CD19, n) anti-CD22, o) anti-CD38, and p) anti-CD20, wherein each of the antibodies of groups a)-p) are conjugated with spectrally distinct fluorochromes.

[0146] In some embodiments, wherein the antibodies targeting markers comprise: a) anti-CD58, conjugated with a fluorochrome excitable by a UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 379 / 28 nm, b) anti-CD81 , conjugated with a fluorochrome excitable by the UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 515 / 30 nm, c) anti-CD73, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, d) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, e) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, f) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, g) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, h) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD371 , conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 530 / 31 nm, j) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, k) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, l) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, m) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, n) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, o) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and p) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

[0147] In some embodiments, the DNA-binding dye is selected from the group consisting of SYTO41 , DAPI, Hoechst or propidium iodide.

[0148] In some embodiments, the DNA-binding dye is SYTO41 .

[0149] In some embodiments, the antibodies are in liquid tubes.

[0150] In some embodiments, the antibodies are in dry tubes.

[0151] In some embodiments, the sample comprises bone marrow cells.

[0152] In some embodiments, the sample comprises peripheral blood cells.

[0153] In some embodiments, the subject is a human.

[0154] In some embodiments, the subject has previously been diagnosed with pediatric B-cell precursor acute lymphoblastic leukemia.

[0155] In some embodiments, the subject has previously been treated for pediatric B-cell precursor acute lymphoblastic leukemia.

[0156] In a further aspect, the invention relates to a method for identifying a subject suitable for treatment with an anti-cancer therapy, wherein the method comprises a method as described herein.

[0157] Definitions

[0158] The term “treating” or “treatment”, as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or slowing or delaying the progression or reoccurrence of, the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term “treating” also refers to prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. The term “minimal residual disease”, as used herein, unless otherwise indicated, refers to the presence of a subpopulation of leukemic cells persisting within the bone marrow following therapy or treatment. Said cells can lead to subsequent disease relapse.

[0159] The term “monoclonal antibody”, as used herein, unless otherwise indicated, refer to immunoglobulins derived form a monoclonal cell line and which have a defined specificity to a target of interest.

[0160] The term “sensitivity”, as used herein, unless otherwise indicated, refers to the ability of the invention as descried herein to correctly identify true positives e.g. to correctly identify MRD in cell populations containing a population of MRD cells. As described herein, sensitivity may be represented as a percentage. In instances wherein sensitivity is represented as a percentage value, the percentage value indicates the percentage of instances in which the invention is capable of detecting true positive cell populations e.g. the percentage of patients in which the invention is capable of correctly identifying MRD in a patient containing a population of MRD cells. As used herein, sensitivity may also be referred to by the limit of detection. The “limit of detection”, as described herein, unless otherwise stated, refers to the lowest concentration of a substance that can be reliably detected e.g. the lowest concentration of MRD cells that can be reliably detected in a cell population.

[0161] In some embodiments, the reagent panels and methods of the invention are capable of achieving a limit of detection of at least 10“4. In some embodiments, the reagent panels and methods of the invention are capable of achieving a limit of detection of 10“5.

[0162] In some embodiments, the reagent panels and methods of the invention are capable of achieving a sensitivity of at least 60%, at least 70%, at least 80%, a least 90% or at least 100%. In some embodiments, the reagent panels and methods of the invention are capable of achieving a sensitivity of at least 82%. In some embodiments, the reagent panels and methods of the invention are capable of achieving a sensitivity of at least 82.1% or 82.2% or 82.3% or 82.4%, or 82.5%, or 82.6%, or 82.7% or 82.8%.

[0163] In some embodiments the reagent panels and methods of the invention are capable of achieving improved sensitivity as compared to reagent panels and / or methods comprising an 8- colour panel. In some embodiments, the reagent panels and methods of the invention are capable of achieving a sensitivity at least 5%, 10%, 15% or 20% higher than the specificity of a reagent panel or method comprising an 8-colour panel.

[0164] The term “specificity”, as used herein, unless otherwise indicated, refers to the ability of the invention as described herein to correctly identify true negatives e.g. to correctly not identify MRD cell populations in cell populations which do not contain a population of MRD cells. As described herein, specificity may be represented as a percentage. In instances wherein specificity is represented as a percentage value, the percentage value indicates the percentage of instances in which the invention is capable of detecting true negative cell populations e.g. the percentage of patients thar the invention is capable of correctly identifying as MR D-negative (i.e. containing no MRD cells).

[0165] In some embodiments the reagent panels and methods of the invention are capable of achieving an improved specificity as compared to reagent panels and / or methods comprising an 8-colour panel. In some embodiments, the reagent panels and methods of the invention are capable of achieving a specificity at least 5% or 10% higher than the specificity of a reagent panel and / or method comprising an 8-colour panel. In some embodiments, the reagent panels and methods of the invention are capable of achieving a specificity at least 5%, 6%, 7%, 8%, 9% or 10% higher than the specificity of a reagent panel and / or method comprising an 8-colour panel. In some embodiments, the reagent panels and methods of the invention are capable of achieving a specificity at least 8% higher than the specificity of a reagent panel and / or method comprising an 8-colour panel.

[0166] The term “spectrally distinct fluorochromes”, as used herein, unless otherwise indicated, refers to fluorochromes which are excitable at different wavelengths and detectable at different wavelength. The term “spectrally equivalent fluorochromes”, as used herein, unless otherwise indicated, refers to fluorochromes which are excitable at the same wavelength and detectable at the same wavelength. As will be understood by the skilled person, a degree of variation in the excitation and detection wavelengths of spectrally equivalent fluorochromes as described herein should be allowed for. Accordingly, the term “spectrally equivalent fluorochromes”, as used herein, unless otherwise indicated also refers to fluorochromes which are excitable at about the same wavelength and detectable at about the same wavelength.

[0167] The objective of the present invention is achieved through the development and formulation of optimized antibody panels comprising carefully selected CD markers pertinent to MRD assessment in pediatric BCP-ALL. This involves the selection of appropriate fluorochromes based on parameters such as fluorescence intensity (brightness), spectral characteristics (to minimize compensation requirements), and stability, followed by the design of effective combinations of antibodies conjugated to these fluorochromes. Additionally, the invention provides means for accurately quantifying the total nucleated cell count in the analyzed sample, incorporating, for example, a nuclear staining reagent (e.g., a DNA dye) and optionally a reagent facilitating the exclusion of platelets or other interfering particles (e.g., anti-CD36).

[0168] Specifically, three distinct panel configurations are provided:

[0169] 1 . Panel-1 : Configured for 12-color immunophenotyping via multiparametric flow cytometry.

[0170] 2. Panel-2: Configured for 14-color immunophenotyping via multiparametric flow cytometry.

[0171] 3. Panel-3: Configured for 16-color immunophenotyping via multiparametric flow cytometry.

[0172] These configurations enable laboratories to implement the invention based on the specific multicolor capabilities of their available flow cytometry instrumentation.

[0173] Panel-1 Configuration (Referenced in Table 3)

[0174] Panel-1 is designed for MRD measurement utilizing 12-color flow cytometry instrumentation and comprises two distinct reagent tubes:

[0175] 1 . Tube 1 (MRD Detection Tube): Formulated for the identification and quantification of MRD cells within bone marrow or peripheral blood samples. This tube contains a premixed composition comprising the following monoclonal antibodies:

[0176] 1 .1 . An anti-CD10 antibody conjugated to the fluorochrome BV421 , or a spectrally equivalent fluorochrome. This conjugate is excitable by a violet laser (nominally 405 nm) and its fluorescence emission is detectable within a corresponding violet fluorescence channel (e.g., utilizing a 450 / 40 nm bandpass filter, or an equivalent detection range).

[0177] 1 .3. An anti-CD45 antibody conjugated to the fluorochrome BV480, or a spectrally equivalent fluorochrome, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 525 / 50 nm bandpass filter, or equivalent detection optics).

[0178] 1 .4. An anti-CD34 antibody conjugated to the fluorochrome BV605, or a spectrally equivalent fluorochrome, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 610 / 20 nm bandpass filter, or equivalent detection optics). 1 .5. An anti-CD20 antibody conjugated to the fluorochrome BV711 , or a spectrally equivalent fluorochrome, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 710 / 50 nm bandpass filter, or equivalent detection optics).

[0179] 1 .6. An anti-CD81 antibody conjugated to the fluorochrome BV786, or a spectrally equivalent fluorochrome, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 780 / 60 nm bandpass filter, or equivalent detection optics).

[0180] 1 .7. An anti-CD58 antibody conjugated to the fluorochrome BB515, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 530 / 31 nm bandpass filter, or equivalent detection optics).

[0181] 1.8. An anti-CD371 antibody conjugated to the fluorochrome BB515, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within the same blue fluorescence channel as item 1.8 (e.g., using a 530 / 31 nm bandpass filter, or equivalent detection optics).

[0182] 1 .9. An anti-CD99 antibody conjugated to the fluorochrome PE (Phycoerythrin), or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 585 / 42 nm bandpass filter, or equivalent detection optics).

[0183] 1 .10. An anti-CD304 antibody conjugated to the fluorochrome PE, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within the same blue fluorescence channel as item 1.9 (e.g., using a 585 / 42 nm bandpass filter, or equivalent detection optics).

[0184] 1 .11 . An anti-CD10 antibody conjugated to the fluorochrome PERCP-CY5.5 (Peridinin- Chlorophyll-Protein complex conjugated to Cyanine 5.5), or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 695 / 40 nm bandpass filter, or equivalent detection optics).

[0185] 1 .12. An anti-CD86 antibody conjugated to the fluorochrome PE-CY7 (Phycoerythrin conjugated to Cyanine 7), or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 780 / 60 nm bandpass filter, or equivalent detection optics).

[0186] 1.13. An anti-CD38 antibody conjugated to the fluorochrome APC (Allophycocyanin), or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 670 / 30 nm bandpass filter, or equivalent detection optics).

[0187] 1.14. An anti-CD123 antibody conjugated to the fluorochrome Red718, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 730 / 45 nm bandpass filter, or equivalent detection optics).

[0188] 1.15. An anti-CD66c antibody conjugated to the fluorochrome Red718, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within the same violet fluorescence channel as item 1.14. (e.g., using a 730 / 45 nm bandpass filter, or equivalent detection optics).

[0189] 1.16. An anti-CD19 antibody conjugated to the fluorochrome APC-H7, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 780 / 60 nm bandpass filter, or equivalent detection optics).

[0190] 2. Tube 2 (Nucleated Cell Quantification Tube): Formulated for the quantitative determination of total nucleated cells within the patient's bone marrow or peripheral blood sample. This tube contains a mixture comprising the following reagents:

[0191] 2.1. A monoclonal antibody directed against CD19, conjugated to the fluorochrome BB515, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 530 / 31 nm bandpass filter, or equivalent detection optics).

[0192] 2.2. A monoclonal antibody directed against CD45, conjugated to the fluorochrome PE, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 585 / 42 nm bandpass filter, or equivalent detection optics).

[0193] 2.3. A monoclonal antibody directed against CD36, conjugated to the fluorochrome APC, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 670 / 30 nm bandpass filter, or equivalent detection optics).

[0194] 2.4. A DNA-binding dye, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 525 / 50 nm bandpass filter, or equivalent detection optics), suitable for staining the nuclei of all nucleated cells.

[0195] TABLE 3: Composition of the 12-color panel for measuring minimal residual disease in B- ALL.

[0196] Panel-2 Configuration (Referenced in Table 4)

[0197] Panel-2 is formulated for Minimal Residual Disease (MRD) measurement utilizing 14-color flow cytometry instrumentation, it comprises two distinct reagent tubes:

[0198] 1 . Tube 1 (MRD Detection Tube): Formulated for the identification and quantification of MRD cells within bone marrow or peripheral blood samples. This tube contains a premixed composition comprising the following monoclonal antibodies:

[0199] 1 .1 . An anti-CD73 antibody conjugated to the fluorochrome BV421 , or a spectrally equivalent fluorochrome. This conjugate is excitable by a violet laser (nominally 405 nm) and its fluorescence emission is detectable within a corresponding violet fluorescence channel (e.g., utilizing a 450 / 40 nm bandpass filter, or equivalent detection optics).

[0200] 1 .2. An anti-CD81 antibody conjugated to the fluorochrome BV421 , or a spectrally equivalent fluorochrome, excitable by the violet laser (nominally 405 nm) and detectable within the same violet fluorescence channel as item 1.1 (e.g., using a 450 / 40 nm bandpass filter, or equivalent detection optics).

[0201] 1 .3. An anti-CD45 antibody conjugated to the fluorochrome BV480, or a spectrally equivalent fluorochrome, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 525 / 50 nm bandpass filter, or equivalent detection optics).

[0202] 1 .4. An anti-CD304 antibody conjugated to the fluorochrome BV605, or a spectrally equivalent fluorochrome, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 610 / 20 nm bandpass filter, or equivalent detection optics).

[0203] 1 .5. An anti-CD44 antibody conjugated to the fluorochrome BV650, or a spectrally equivalent fluorochrome, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 660 / 20 nm bandpass filter, or equivalent detection optics).

[0204] 1 .6. An anti-CD86 antibody conjugated to the fluorochrome BV711 , or a spectrally equivalent fluorochrome, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 710 / 50 nm bandpass filter, or equivalent detection optics).

[0205] 1 .7. An anti-CD99 antibody conjugated to the fluorochrome BV786, or a spectrally equivalent fluorochrome, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 780 / 60 nm bandpass filter, or equivalent detection optics).

[0206] 1 .8. An anti-CD58 antibody conjugated to the fluorochrome BB515, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 530 / 31 nm bandpass filter, or equivalent detection optics).

[0207] 1.9. An anti-CD371 antibody conjugated to the fluorochrome BB515, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within the same blue fluorescence channel as item 1.8 (e.g., using a 530 / 31 nm bandpass filter, or equivalent detection optics). 1.10. An anti-CD66c antibody conjugated to the fluorochrome PE (Phycoerythrin), or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 585 / 42 nm bandpass filter, or equivalent detection optics).

[0208] 1 .11 . An anti-CD123 antibody conjugated to the fluorochrome PE, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within the same blue fluorescence channel as item 1.10 (e.g., using a 585 / 42 nm bandpass filter, or equivalent detection optics).

[0209] 1 .12. An anti-CD34 antibody conjugated to the fluorochrome PE-CF594, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 616 / 23 nm bandpass filter, or equivalent detection optics).

[0210] 1.13. An anti-CD10 antibody conjugated to the fluorochrome PERCP-CY5.5, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 695 / 40 nm bandpass filter, or equivalent detection optics).

[0211] 1.14. An anti-CD19 antibody conjugated to the fluorochrome PE-CY7, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 780 / 60 nm bandpass filter, or equivalent detection optics).

[0212] 1 .15. An anti-CD22 antibody conjugated to the fluorochrome APC, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 670 / 30 nm bandpass filter, or equivalent detection optics).

[0213] 1 .16. An anti-CD38 antibody conjugated to the fluorochrome APC-R700, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 730 / 45 nm bandpass filter, or equivalent detection optics).

[0214] 1 .17. An anti-CD20 antibody conjugated to the fluorochrome APC-H7, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 780 / 60 nm bandpass filter, or equivalent detection optics). Tube 2 (Nucleated Cell Quantification Tube): Formulated for the quantitative determination of total nucleated cells within the patient's bone marrow or peripheral blood sample. This tube contains a mixture comprising the following reagents:

[0215] 2.1. A monoclonal antibody directed against CD19, conjugated to the fluorochrome BB515, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 530 / 31 nm bandpass filter, or equivalent detection optics).

[0216] 2.2. A monoclonal antibody directed against CD45, conjugated to the fluorochrome PE, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 585 / 42 nm bandpass filter, or equivalent detection optics).

[0217] 2.3. A monoclonal antibody directed against CD36, conjugated to the fluorochrome APC, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 670 / 30 nm bandpass filter, or equivalent detection optics).

[0218] 2.4. A DNA-binding dye, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 525 / 50 nm bandpass filter, or equivalent detection optics), suitable for staining the nuclei of all nucleated cells.

[0219] TABLE 4. Composition of the 14-color panel for Minimal Residual Disease measurement in B-ALL.

[0220] Panel-3 Configuration (Referenced in Table 5): a 16-Color Flow Cytometry Assay for MRD Measurement

[0221] This panel is specifically designed for Minimal Residual Disease (MRD) measurement employing 16-color flow cytometry instrumentation. The configuration comprises two distinct reagent tubes:

[0222] 1 . Tube 1 (MRD Detection Tube): Formulated for the identification and quantification of MRD cells within bone marrow or peripheral blood specimens. This tube contains a premixed composition comprising the following monoclonal antibodies:

[0223] 1 .1 . An anti-CD58 antibody conjugated to the fluorochrome BUV395, or a spectrally equivalent fluorochrome. This conjugate is excitable by a UV or near-UV / violet laser (e.g., nominally 355 nm or 405 nm) and its fluorescence emission is detectable within a corresponding UV / violet fluorescence channel (e.g., utilizing a 379 / 28 nm bandpass filter, or equivalent detection optics).

[0224] 1 .2. An anti-CD81 antibody conjugated to the fluorochrome BUV496, or a spectrally equivalent fluorochrome, excitable by a UV or near-UV / violet laser (e.g., nominally 355 nm or 405 nm) and detectable within a violet / blue fluorescence channel (e.g., using a 515 / 30 nm bandpass filter, or equivalent detection optics).

[0225] 1 .3. An anti-CD73 antibody conjugated to the fluorochrome BV421 , or a spectrally equivalent fluorochrome, excitable by a violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 450 / 40 nm bandpass filter, or equivalent detection optics).

[0226] 1 .4. An anti-CD45 antibody conjugated to the fluorochrome BV480, or a spectrally equivalent fluorochrome, excitable by a violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 525 / 50 nm bandpass filter, or equivalent detection optics).

[0227] 1 .5. An anti-CD304 antibody conjugated to the fluorochrome BV605, or a spectrally equivalent fluorochrome, excitable by a violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 610 / 20 nm bandpass filter, or equivalent detection optics).

[0228] 1 .6. An anti-CD44 antibody conjugated to the fluorochrome BV650, or a spectrally equivalent fluorochrome, excitable by a violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 660 / 20 nm bandpass filter, or equivalent detection optics).

[0229] 1 .7. An anti-CD86 antibody conjugated to the fluorochrome BV711 , or a spectrally equivalent fluorochrome, excitable by a violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 710 / 50 nm bandpass filter, or equivalent detection optics).

[0230] 1 .8. An anti-CD99 antibody conjugated to the fluorochrome BV786, or a spectrally equivalent fluorochrome, excitable by a violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 780 / 60 nm bandpass filter, or equivalent detection optics).

[0231] 1.9. An anti-CD371 antibody conjugated to the fluorochrome BB515, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 530 / 31 nm bandpass filter, or equivalent detection optics).

[0232] 1 .10. An anti-CD66c antibody conjugated to the fluorochrome PE, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 585 / 42 nm bandpass filter, or equivalent detection optics).

[0233] 1 .11 . An anti-CD123 antibody conjugated to the fluorochrome PE, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within the same blue fluorescence channel as item 1.10 (e.g., using a 585 / 42 nm bandpass filter, or equivalent detection optics).

[0234] 1 .12. An anti-CD34 antibody conjugated to the fluorochrome PE-CF594, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 616 / 23 nm bandpass filter, or equivalent detection optics). 1.13. An anti-CD10 antibody conjugated to the fluorochrome PERCP-CY5.5, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 695 / 40 nm bandpass filter, or equivalent detection optics).

[0235] 1.14. An anti-CD19 antibody conjugated to the fluorochrome PE-CY7, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 780 / 60 nm bandpass filter, or equivalent detection optics).

[0236] 1 .15. An anti-CD22 antibody conjugated to the fluorochrome APC, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 670 / 30 nm bandpass filter, or equivalent detection optics).

[0237] 1 .16. An anti-CD38 antibody conjugated to the fluorochrome APC-R700, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 730 / 45 nm bandpass filter, or equivalent detection optics).

[0238] 1 .17. An anti-CD20 antibody conjugated to the fluorochrome APC-H7, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 780 / 60 nm bandpass filter, or equivalent detection optics). Tube 2 (Nucleated Cell Quantification Tube): Formulated for the quantitative determination of total nucleated cells within the patient's bone marrow or peripheral blood sample. This tube contains a mixture comprising the following reagents:

[0239] 2.1. A monoclonal antibody directed against CD19, conjugated to the fluorochrome BB515, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 530 / 31 nm bandpass filter, or equivalent detection optics).

[0240] 2.2. A monoclonal antibody directed against CD45, conjugated to the fluorochrome PE, or a spectrally equivalent fluorochrome, excitable by the blue laser (nominally 488 nm) and detectable within a blue fluorescence channel (e.g., using a 585 / 42 nm bandpass filter, or equivalent detection optics).

[0241] 2.3. A monoclonal antibody directed against CD36, conjugated to the fluorochrome APC, or a spectrally equivalent fluorochrome, excitable by the red laser (nominally 640 nm) and detectable within a red fluorescence channel (e.g., using a 670 / 30 nm bandpass filter, or equivalent detection optics).

[0242] 2.4. A DNA-binding dye, excitable by the violet laser (nominally 405 nm) and detectable within a violet fluorescence channel (e.g., using a 525 / 50 nm bandpass filter, or equivalent detection optics), suitable for staining the nuclei of all nucleated cells.

[0243] TABLE 5. Composition of the 16-color panel for Minimal Residual Disease measurement in B-ALL. Development of 12-, 14-, and 16-Color Flow Cytometry Kits for MRD Assessment

[0244] Based on the aforementioned 12-, 14-, and 16-color panels, three kits have been developed for laboratory application:

[0245] 1. Kit for Minimal Residual Disease (MRD) Measurement in B-cell Acute Lymphoblastic Leukemia (B-ALL) using a 12-Color Immunophenotyping Panel:

[0246] This kit includes all reagents necessary for conducting the assay. It comprises the following components:

[0247] 1.1. Monoclonal antibodies against selected human CD markers, provided in opaque dark vials as follows.

[0248] 1.1.1. Vial 1 : Anti-CD73, conjugated with BV421 or an equivalent fluorochrome.

[0249] 1 .1 .2. Vial 2: Anti-CD81 , conjugated with BV421 or an equivalent fluorochrome.

[0250] 1 .1 .3. Vial 3: Anti-CD45, conjugated with BV480 or an equivalent fluorochrome.

[0251] 1 .1 .4. Vial 4: Anti-CD99, conjugated with BV605 or an equivalent fluorochrome.

[0252] 1 .1 .5. Vial 5: Anti-CD304, conjugated with BV605 or an equivalent fluorochrome.

[0253] 1 .1 .6. Vial 6: Anti-CD44, conjugated with BV650 or an equivalent fluorochrome.

[0254] 1 .1 .7. Vial 7: Anti-CD86, conjugated with BV711 or an equivalent fluorochrome.

[0255] 1 .1 .8. Vial 8: Anti-CD34, conjugated with BV786 or an equivalent fluorochrome.

[0256] 1.1.9. Vial 9: Anti-CD58, conjugated with BB515 or an equivalent fluorochrome.

[0257] 1 .1.10. Vial 10: Anti-CD371 , conjugated with BB515 or an equivalent fluorochrome.

[0258] 1.1.11. Vial 11 : Anti-CD66c, conjugated with PE or an equivalent fluorochrome.

[0259] 1.1.12. Vial 12: Anti-CD123, conjugated with PE or an equivalent fluorochrome.

[0260] 1 .1.13. Vial 13: Anti-CD10, conjugated with PERCP-CY5.5 or an equivalent fluorochrome.

[0261] 1.1.14. Vial 14: Anti-CD19, conjugated with PE-CY7 or an equivalent fluorochrome.

[0262] 1.1.15. Vial 15: Anti-CD22, conjugated with APC or an equivalent fluorochrome.

[0263] 1.1.16. Vial 16: Anti-CD38, conjugated with APC-R700 or an equivalent fluorochrome.

[0264] 1.1.17. Vial 17: Anti-CD20, conjugated with APC-H7 or an equivalent fluorochrome.

[0265] 1 .1.18. Vial 18: Anti-CD45, conjugated with BV510 or an equivalent fluorochrome.

[0266] 1 .1.19. Vial 19: Anti-CD19, conjugated with FITC or an equivalent fluorochrome.

[0267] 1 .1 .20. Vial 20: Anti-CD36, conjugated with APC or an equivalent fluorochrome. 1 .1 .21 . Vial 21 : DNA dye, excited by the 405nm violet laser and detected in the violet channel of the flow cytometer (525 / 50nm or equivalent). This vial is supplied separately in a frozen state.

[0268] 1.2. Auxiliary buffers and solutions.

[0269] 1.2.1. Vial 22: Buffer for fluorochrome-labeled antibodies.

[0270] 1 .2.2. Vial 23: Cell lysis and fixation solution.

[0271] 1 .2.3. Vial 24: Wash buffer.

[0272] 2. Kit for Minimal Residual Disease (MRD) Measurement in B-cell Acute Lymphoblastic Leukemia (B-ALL) using a 14-Color Immunophenotyping Panel:

[0273] This kit includes all reagents necessary for conducting the assay. It comprises the following components:

[0274] 2.1. Monoclonal antibodies against selected human CD markers, provided in opaque dark vials as follows:

[0275] 2.1 .1 . Vial 1 : Anti-CD73, conjugated with BV421 or an equivalent fluorochrome.

[0276] 2.1 .2. Vial 2: Anti-CD81 , conjugated with BV421 or an equivalent fluorochrome.

[0277] 2.1 .3. Vial 3: Anti-CD45, conjugated with BV480 or an equivalent fluorochrome.

[0278] 2.1 .4. Vial 4: Anti-CD304, conjugated with BV605 or an equivalent fluorochrome.

[0279] 2.1 .5. Vial 5: Anti-CD44, conjugated with BV650 or an equivalent fluorochrome.

[0280] 2.1 .6. Vial 6: Anti-CD86, conjugated with BV711 or an equivalent fluorochrome.

[0281] 2.1 .7. Vial 7: Anti-CD99, conjugated with BV786 or an equivalent fluorochrome.

[0282] 2.1.8. Vial 8: Anti-CD58, conjugated with BB515 or an equivalent fluorochrome.

[0283] 2.1.9. Vial 9: Anti-CD371 , conjugated with BB515 or an equivalent fluorochrome.

[0284] 2.1.10. Vial 10: Anti-CD66c, conjugated with PE or an equivalent fluorochrome.

[0285] 2.1.11. Vial 11 : Anti-CD123, conjugated with PE or an equivalent fluorochrome.

[0286] 2.1.12. Vial 12: Anti-CD34, conjugated with PE-CF594 or an equivalent fluorochrome.

[0287] 2.1.13. Vial 13: Anti-CD10, clone HI10a, conjugated with PERCP-CY5.5 or an equivalent fluorochrome.

[0288] 2.1.14. Vial 14: Anti-CD19, conjugated with PE-CY7 or an equivalent fluorochrome.

[0289] 2.1.15. Vial 15: Anti-CD22, conjugated with APC or an equivalent fluorochrome.

[0290] 2.1.16. Vial 16: Anti-CD38, conjugated with APC-R700 or an equivalent fluorochrome.

[0291] 2.1.17. Vial 17: Anti-CD20, conjugated with APC-H7 or an equivalent fluorochrome. 2.1.18. Vial 18: Anti-CD45, conjugated with BV510 or an equivalent fluorochrome.

[0292] 2.1 .19. Vial 19: Anti-CD19, conjugated with FITC or an equivalent fluorochrome.

[0293] 2.1 .20. Vial 20: Anti-CD36, conjugated with APC or an equivalent fluorochrome. 2.1 .21 . Vial 21 : DNA dye, excited by the 405nm violet laser and detected in the violet channel of the flow cytometer (525 / 50nm or equivalent). This vial is supplied separately in a frozen state.

[0294] 2.2. Auxiliary buffers and solutions:

[0295] 2.2.1. Vial 22: Buffer for fluorochrome-labeled antibodies.

[0296] 2.2.2. Vial 23: Cell lysis and fixation solution.

[0297] 2.2.3. Vial 24: Wash buffer.

[0298] 3. Kit for Minimal Residual Disease (MRD) Measurement in B-cell Acute Lymphoblastic Leukemia (B-ALL) using a 14-Color Immunophenotyping Panel:

[0299] This kit is designed for the measurement of Minimal Residual Disease (MRD) in B-cell Acute Lymphoblastic Leukemia (B-ALL) using a 16-color immunophenotyping panel. The kit includes all necessary reagents for conducting the assay and comprises the following components:

[0300] 3.1. Monoclonal Antibodies: A panel of monoclonal antibodies targeting selected human CD markers, provided in opaque dark vials as follows:

[0301] 3.1 .1 . Vial 1 : Anti-CD58, conjugated with BUV395 or an equivalent fluorochrome.

[0302] 3.1 .2. Vial 2: Anti-CD81 , conjugated with BV421 or an equivalent fluorochrome.

[0303] 3.1 .3. Vial 3: Anti-CD73, conjugated with BUV395 or an equivalent fluorochrome.

[0304] 3.1 .4. Vial 4: Anti-CD45, conjugated with BV480 or an equivalent fluorochrome.

[0305] 3.1.5. Vial 5: Anti-CD304, clone U21 -1283, conjugated with BV605 or an equivalent fluorochrome.

[0306] 3.1 .6. Vial 6: Anti-CD44, conjugated with BV650 or an equivalent fluorochrome.

[0307] 1 .1 .7. Vial 7: Anti-CD86, conjugated with BV711 or an equivalent fluorochrome.

[0308] 3.1 .8. Vial 8: Anti-CD99, conjugated with BV786 or an equivalent fluorochrome.

[0309] 3.1.9. Vial 9: Anti-CD371 , conjugated with BB515 or an equivalent fluorochrome.

[0310] 3.1.10. Vial 10: Anti-CD66c, conjugated with PE or an equivalent fluorochrome.

[0311] 3.1.11. Vial 11 : Anti-CD123, conjugated with PE or an equivalent fluorochrome.

[0312] 3.1.12. Vial 12: Anti-CD34, conjugated with PE-CF594 or an equivalent fluorochrome.

[0313] 3.1 .13. Vial 13: Anti-CD10, conjugated with PERCP-CY5.5 or an equivalent fluorochrome. 3.1.14. Vial 14: Anti-CD19, conjugated with PE-CY7 or an equivalent fluorochrome.

[0314] 3.1.15. Vial 15: Anti-CD22, conjugated with APC or an equivalent fluorochrome.

[0315] 3.1.16. Vial 16: Anti-CD38, conjugated with APC-R700 or an equivalent fluorochrome.

[0316] 3.1.17. Vial 17: Anti-CD20, conjugated with APC-H7 or an equivalent fluorochrome.

[0317] 3.1.18. Vial 18: Anti-CD45, conjugated with BV510 or an equivalent fluorochrome.

[0318] 3.1 .19. Vial 19: Anti-CD19, conjugated with FITC or an equivalent fluorochrome.

[0319] 3.1 .20. Vial 20: Anti-CD36, conjugated with APC or an equivalent fluorochrome.

[0320] 3.1 .21 . Vial 21 : DNA dye, excited by the 405nm violet laser and detected in the violet channel of the flow cytometer (525 / 50nm or equivalent). This vial is supplied separately in a frozen state.

[0321] 1.2. Auxiliary Buffers and Solutions:

[0322] 3.2.1. Vial 22: Buffer for fluorochrome-labeled antibodies.

[0323] 3.2.2. Vial 23: Cell lysis and fixation solution.

[0324] 3.2.3. Vial 24: Wash buffer.

[0325] Additional Equipment (Not Included): The following equipment is required but not included in this kit: micropipettes, vortex mixer, and refrigerated centrifuge with swinging bucket rotor.

[0326] EXAMPLES

[0327] The present invention is further illustrated by the following non-limiting examples. The following examples are offered by way of illustration, and not by way of limitation.

[0328] Example 1 : Implementation of the Kit for Measuring Minimal Residual Disease in B-ALL Utilizing the 14-Color Immunophenotyping Panel (Panel-2)

[0329] The composition of the kit designed for measuring Minimal Residual Disease (MRD) in B-cell Acute Lymphoblastic Leukemia (B-ALL) employing the 14-color immunophenotyping panel (designated as Panel-2) is detailed in the "SUMMARY OF THE INVENTION" section and corresponds to the configuration presented in Table 4.

[0330] Intended Use:

[0331] The ready-to-use kit is designed for utilization in specialized clinical settings, including hospitals, clinics, or diagnostic centers focused on hematological diseases such as acute lymphoblastic leukemia, for diagnostic purposes and monitoring of treatment response. The procedure necessitates obtaining a biological specimen, typically bone marrow aspirate or peripheral blood, from the patient.

[0332] Procedure:

[0333] 1 . Preparation: A qualified operator (e.g., physician, biologist, medical laboratory technician) prepares two flow cytometry tubes suitable for the available instrument. Prior to staining, the total leukocyte count within the patient sample is determined using standard laboratory methods (e.g., manual counting via hemocytometer or automated analysis).

[0334] 2. Tube Labeling: Label the first tube as "Tube 1" and the second tube as "Tube 2," including appropriate patient identifiers on each.

[0335] 3. Reagent Addition (Tube 1 - MRD Detection):

[0336] 3.1. Into Tube 1 , dispense 10 pL of 'Buffer for antibodies labeled with fluorochromes' (e.g., corresponding to Vial 22 of the kit).

[0337] 3.2. Sequentially add the individual monoclonal antibody reagents specified for Panel-2, Tube 1 (corresponding to Vials 1 through 17, as detailed in Table 4). The volume of each antibody added is typically between 5 pL and 20 pL, determined according to optimized protocols and the quantity of cells being stained. (Note: Alternatively, if provided as a pre-mixed cocktail, add the specified volume of the cocktail according to manufacturer instructions).

[0338] 4. Reagent Addition (Tube 2 - Nucleated Ceil Quantification):

[0339] Into Tube 2, sequentially add the monoclonal antibody reagents specified for Panel-2, Tube 2 (corresponding to Vials 18, 19, and 20 - anti-CD19, anti-CD45, anti-CD36 respectively), typically at a volume of approximately 1 pL per reagent, following optimized protocols. (Note: DNA dye, e.g., Vial 21, is added later).

[0340] 5. Ceil Addition:

[0341] 5.1 . Add a minimum of 5 x 10® total cells from the patient sample to Tube 1 . Gently mix (e.g., by vortexing).

[0342] 5.2. Add approximately 0.5 x 10® total cells from the patient sample to Tube 2. Gently mix (e.g., by vortexing).

[0343] 6. incubation: Incubate both Tube 1 and Tube 2 for 20 minutes at ambient room temperature (e.g., 18-25°C), protected from light. Lysis and Fixation: Add 1 mL of 'Cell Lysis and Fixation Solution' (e.g., Vial 23) to each tube. Mix well (e.g., vortex) and incubate for 10 minutes at ambient room temperature, protected from light. Centrifugation : Centrifuge both tubes in a refrigerated centrifuge at 4°C for 10 minutes at approximately 1400 rpm (or equivalent RCF). Carefully remove the supernatant by aspiration or decantation, ensuring the cell pellet remains intact. Washing: Add 4 mL of 'Wash Buffer' (e.g., Vial 24) to each tube. Resuspend the cell pellets by gentle mixing. Centrifuge the tubes again under the same conditions (4°C, 10 minutes, -1400 rpm). Carefully remove the supernatant. Resuspension: Resuspend the final cell pellet in Tube 1 and Tube 2 in 250 pL of 'Wash Buffer' (Vial 24) by gentle mixing. DNA Staining: immediately prior to flow cytometric analysis, add 1 -2 pL of a working solution of SYTO41 DNA dye (prepared by diluting the stock solution, e.g., Vial 21 , at a 1 :250 ratio in Tris buffer or another suitable buffer) to both Tube 1 and Tube 2 and mix gently. Data Acquisition: Acquire data using a flow cytometer configured for 14-color analysis. Acquire a minimum target of 4 x 10® events from Tube 1 and a minimum target of 0.4 x 10® events from Tube 2. Data Anaiysis (Tube 1): Analyze data from Tube 1 using appropriate flow cytometry software and a validated gating strategy (template), potentially similar to that illustrated in Figure 1 . identify and enumerate the MRD cell population and the total relevant nucleated single cell population (denominator). Calculate the percentage of MRD cells using Formula 1 :

[0344] (Formula 1) % MRD cells = (Number of MRD cells I Total Number of Analyzed Single Nucleated Cells) x 100 Data Analysis (Tube 2) & Correction Factor: Analyze data from Tube 2 using a relevant gating template (e.g., Figure 2). Calculate the B-cell Recovery Correction Factor using Formula 2:

[0345] (Formula 2) B-cell Recovery Correction Factor = (% CD19+cells in Tube 21 % CD19+cells in Tube 1 ) 15. Final MRD Calculation: Calculate the percentage of corrected MRD using Formula 3: (Formula 3) % Corrected MRD = (% MRD cells from Formula 1 ) x (B-cell Recovery Correction Factor from Formula 2)

[0346] Calculation Example:

[0347] Initial Data:

[0348] 1 . Number of MRD cells identified in Tube 1 = 75

[0349] 2. Total Number of Analyzed Single Nucleated Cells in Tube 1 = 535,938

[0350] 3. Percentage of CD19+cells identified in Tube 1 = 9.35%

[0351] 4. Percentage of CD19+cells identified in Tube 2 = 16.7%

[0352] Calculations:

[0353] 1. % MRD cells = (75 / 535,938) x 100 = 0.014%

[0354] 2. B-cell Recovery Correction Factor = 16.7% 19.35% = 1 .78

[0355] 3. % Corrected MRD = 0.014% x 1 .78 = 0.025%

[0356] Example 2: Determination of Analytical Sensitivity (Limit of Detection)

[0357] To determine the analytical sensitivity, often referred to as the Limit of Detection (LoD), of the described panel for detecting Minimal Residual Disease (MRD) cells, a series of spiking experiments were conducted. Samples were meticulously prepared by introducing known, varying quantities of leukemic cells into a background matrix of bone marrow presumed negative for leukemic infiltration, thereby creating samples with target leukemic cell frequencies of 1% (10’2), 0.1% (10“3), 0.01% (10’4), 0.001% (10’5), and 0.0001% (10“6).

[0358] Materials and Methods:

[0359] • Leukemic Cell Source: Bone marrow aspirate obtained at the time of initial diagnosis from a patient with B-cell Acute Lymphoblastic Leukemia (B-ALL), characterized by a blast count of 80%.

[0360] • Background Cell Matrix: Pooled bone marrow aspirate samples obtained from pediatric patients diagnosed with Ewing's sarcoma, confirmed negative for B-ALL involvement.

[0361] • Procedure: 1 . into each of five separate flow cytometry tubes, an aliquot containing 4.5 x 106cells from the background bone marrow matrix was dispensed.

[0362] 2. Leukemic cells from the B-ALL source sample were then added (spiked) into these tubes in calculated amounts to achieve the following final target concentrations within the total cell population:

[0363] ■ Tube 1 : Approximately 45,000 leukemic cells added, targeting a final concentration of 1% (10“2).

[0364] ■ Tube 2: Approximately 4,500 leukemic cells added, targeting a final concentration of 0.1% (10“3).

[0365] ■ Tube 3: Approximately 450 leukemic cells added, targeting a final concentration of 0.01% (10“4).

[0366] ■ Tube 4: Approximately 45 leukemic cells added, targeting a final concentration of 0.001% (10“5).

[0367] ■ Tube 5: Approximately 5 leukemic cells added, targeting a final concentration of 0.0001% (10“6). (Note: Actual cell numbers adjusted based on the 80% purity of the source sample).

[0368] 3. These prepared spiked samples were subsequently processed and analyzed utilizing the "Kit for measuring minimal residual disease in B-ALL by 14-color immunophenotyping panel" (Panel-2), adhering strictly to the methodology detailed in Example 1 .

[0369] Results:

[0370] The results of the sensitivity analysis are summarized in Table 6. Analysis revealed that the measured percentages of detected leukemic cells in Tubes 1 through 4 closely approximated the target spiked concentrations. However, no distinct leukemic cell population could be reliably identified and enumerated above background in Tube 5 (target 0.0001%).

[0371] Conclusion:

[0372] Based on these experimental findings, the analytical sensitivity (LoD) of the 14-color immunophenotyping panel assay described herein is established at 0.001% (equivalent to 1 leukemic cell per 100,000 total nucleated cells, or 10“5). This represents an improvement in sensitivity, potentially by one order of magnitude, compared to the commonly cited sensitivity of 0.01% (10“4) often associated with conventional or lower-plex MRD panels. TABLE 6. Percentage of Detected Leukemic Cells in the Sensitivity Experiment (Table content assumed to be provided elsewhere)

[0373] Example 3: Comparative Assessment of Specificity and Sensitivity of the 14-Color Panel

[0374] Objective:

[0375] The objective of this example was to conduct a comparative evaluation of the diagnostic specificity and sensitivity of the novel 14-color panel (Panel-2) for MRD measurement, as described in the present invention, against an established 8-color panel, previously described in BG Utility Model Application No. 5932 (Protection Number 4690).

[0376] Methodology:

[0377] • Samples: Bone marrow aspirate samples were collected from a cohort of 77 pediatric patients diagnosed with B-ALL and confirmed to be in clinical remission following therapy.

[0378] • Parallel Processing: Each patient sample was divided and processed concurrently using two distinct panel configurations: o (A) Comparator Panel: An 8-color panel formulated according to the kit described in BG Utility Model Application No. 5932 (Protection Number 4690). This panel incorporated antibodies against the following core CD markers commonly used for B-ALL MRD assessment: CD45, CD58, CD10, CD34, CD38, CD19, CD20, along with the DNA dye SYTO41 . o (B) Inventive Panel: The "Kit for measuring minimal residual disease in B-ALL using a 14-color immunophenotyping panel" (Panel-2, Table 4), as described herein, which includes the aforementioned core markers plus additional markers selected to enhance detection.

[0379] • Procedure: Sample preparation and staining for both panels followed the general procedures outlined in Example 1.

[0380] • Instrumentation and Analysis: Sample acquisition was performed using a BD FACS Aria™ III flow cytometer. Subsequent data analysis was conducted utilizing BD FACSDiva™ software, employing standardized gating templates analogous to those depicted in Figure 1 and Figure 2 for MRD population identification and quantification.

[0381] • Statistical Analysis: The performance characteristics (specificity and sensitivity) of each panel were evaluated using Receiver Operating Characteristic (ROC) curve analysis, performed with the MedCalc® statistical software package (referencing MedCalc Software Ltd, Ostend, Belgium; medcalc.org).

[0382] Results:

[0383] The results of the comparative ROC curve analysis are visually represented in Figure 3.

[0384] • For the comparator 8-color panel (A), the analysis yielded a calculated sensitivity of

[0385] 61 .7% and a specificity of 91 .7% (p < 0.001 ) for distinguishing MR D-positive from MRD- negative remission samples within this cohort.

[0386] • For the inventive 14-color panel (B), the analysis demonstrated significantly improved performance, achieving a sensitivity of 82.8% and a specificity of 100% (p < 0.001).

[0387] Conclusion:

[0388] The results derived from this comparative study clearly demonstrate the superior diagnostic performance of the 14-color immunophenotyping panel described in the present invention compared to the conventional 8-color panel. The significantly enhanced sensitivity (82.8% vs. 61.7%) and specificity (100% vs. 91.7%) underscore the technical advantages of the inventive panel configuration for the accurate and reliable measurement of minimal residual disease in pediatric patients with B-cell precursor acute lymphoblastic leukemia.

[0389] The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures.

[0390] Such modifications are intended to fall within the scope of the appended claims.

[0391] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

[0392] References

[0393] Non patent references:

[0394] 1 . Brown, P., et al., Pediatric Acute Lymphoblastic Leukemia, Version 2.2020, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Cane Netw, 2020. 18(1 ): p. 81-112.

[0395] 2. Brown, P., et aL, Pediatric Acute Lymphoblastic Leukemia, Version 2.2020, NCCN Clinical Practice Guidelines in Oncology. Journal of the National Comprehensive Cancer Network, 2020. 18: p. 81 -112.

[0396] 3. Della Starza, I., et aL, Minimal Residual Disease in Acute Lymphoblastic Leukemia: Technical and Clinical Advances. Frontiers in oncology, 2019. 9: p. 726-726.

[0397] 4. Abou Dalle, I., E. Jabbour, and N.J. Short, Evaluation and management of measurable residual disease in acute lymphoblastic leukemia. Ther Adv Hematol, 2020. 11 : p. 2040620720910023.

[0398] 5. van Dongen, J.J.M., et aL, Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies. Blood, 2015. 125(26): p. 3996- 4009.

[0399] 6. Szczepahski, T., Why and how to quantify minimal residual disease in acute lymphoblastic leukemia? Leukemia, 2007. 21 (4): p. 622-626.

[0400] 7. GleiBner, B., et aL, Prospective BCR-ABL analysis by polymerase chain reaction (RT- PCR) in adult acute B-lineage lymphoblastic leukemia: reliability of RT-nested-PCR and comparison to cytogenetic data. Leukemia, 2001. 15(12): p. 1834-1840. 8. Abdulrahman Muhsin, Akrem M. Atrushi, and Adnan A. Al-Doski. "The significance of minimal residual disease in acute lymphoblastic leukaemia: A single centre study." Science Journal of University of Zakho 12, no. 2 (April 2024): 144-148. https: / / doi.Org / 10.25271 / sjuoz.2024.12.2.1240..

[0401] 9. Arunachalam, A.K., at al. A single tube, 12 color panel for detection of measurable residual disease with a sensitivity of 3 in 10A6 in B cell acute lymphoblastic leukemia. B: Proceedings of the 2021 International Clinical Cytometry Meeting, October 2021.

[0402] 10. Manivannan, P., Mazumder, S., Bhandary, C., Kar, R., Kayal, S., & Basu, D. (2022). Proposed Single-Tube Ten-Color Antibody Panel to Optimize Resources for Measurable Residual Disease Detection in B-Acute Lymphoblastic Leukemia Based on Leukemia Associated Immunophenotype Evaluation at Diagnosis: A Single Center Experience from Southern India. Journal of Leukemia, August 2022. https: / / doi.org / 10.35248 / 2329-6917.22.10.308.

[0403] Patent references:

[0404] US 5047321 A, Method for analysis of cellular components of a fluid.

[0405] US 5538855 A, Procedure for the simultaneous quantification, in a single measurement, of the major types of human lymphocytes and their subsets.

[0406] US 9777332 B2, Methods and compositions for identifying minimal residual disease in acute lymphoblastic leukemia.

[0407] US 10656144 B2, Immune profiling and minimal residue disease following stem cell transplantation in multiple myeloma

[0408] AU 2021203620, Al Methods, reagents and kits for flow cytometric immunophenotyping.

[0409] BG Utility Model Application No. 5932 (Registration No. 4690), A kit for sampling biological material and its pre-analytical processing.

[0410] EP 3064942 A2, Methods, reagents and kits for detecting minimal residual disease.

[0411] US10874710 (B2) , Methods, reagents and kits for detecting minimal residual disease.

[0412] CN1 16593699 (B), Antibody composition capable of being applied to flow cytometry for detecting CD39 molecules on surfaces of T cells. Exemplary clauses

[0413] Clause 1 : The invention relates to reagent panels for the measurement of minimal residual disease (MRD) in pediatric B-cell precursor acute lymphoblastic leukemia by multiparametric flow cytometry, said panels comprising a combination of two tubes: a) a first tube (T ube 1 ) containing a first reagent composition comprising a combination of approximately 19 monoclonal antibodies directed against human leukocyte markers conjugated with spectrally distinct fluorochromes, wherein said combination includes antibodies targeting markers CD45, CD19, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, and CD304, supplemented by antibodies targeting additional markers CD44, CD86, CD99, CD371 , and CD22, thereby facilitating enhanced identification of MRD leukemia cells; and b) a second tube (Tube 2) containing a second reagent composition formulated for quantifying nucleated cells within a sample, said second composition comprising a DNA-binding dye and monoclonal antibodies directed against markers CD45, CD19, and CD36 conjugated with spectrally distinct fluorochromes.

[0414] Clause 2: The reagent panel according to clause 1 , wherein the first tube (Tube 1 ) is configured as a twelve-color panel formulation for MRD measurement and contains the following antibodies: a) anti-CD10, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, or a spectral equivalent thereof; b) anti-CD45, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, or a spectral equivalent thereof; c) anti-CD34, conjugated with fluorochrome excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 610 / 20 nm, or a spectral equivalent thereof; d) anti-CD20, conjugated with one or more fluorochromes excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 710 / 50 nm, or a spectral equivalent thereof; e) anti-CD81 , conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, or a spectral equivalent thereof; f) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, or a spectral equivalent thereof; g) anti-CD99 and anti-CD34, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, or a spectral equivalent thereof. c) anti-CD86, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, or a spectral equivalent thereof; h) anti-CD44 and CD73, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, or a spectral equivalent thereof; i) anti-CD38, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, or a spectral equivalent thereof; j) anti-CD123 and CD66c, conjugated with one or more fluorochromes excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, or a spectral equivalent thereof; and k) anti-CD19, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, or a spectral equivalent thereof;

[0415] Clause 3: The reagent panel according to clause 1 , wherein the first tube (Tube 1 ) is configured as a fourteen-color panel formulation for MRD measurement and contains the following antibodies: a) anti-CD73 and anti-CD81 , conjugated with one or more fluorochromes excitable by a violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 450 / 40 nm, or a spectral equivalent thereof; b) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, or a spectral equivalent thereof; c) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, or a spectral equivalent thereof; d) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, or a spectral equivalent thereof; e) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, or a spectral equivalent thereof; f) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, or a spectral equivalent thereof; g) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by a blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, or a spectral equivalent thereof; h) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, or a spectral equivalent thereof; i) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, or a spectral equivalent thereof; j) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, or a spectral equivalent thereof; k) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, or a spectral equivalent thereof; l) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, or a spectral equivalent thereof; m) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, or a spectral equivalent thereof; and n) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, or a spectral equivalent thereof. Clause 4: The reagent panel according to clause 1 , wherein the first tube (Tube 1 ) is configured as a sixteen-color panel formulation for MRD measurement and contains the following antibodies: a) anti-CD58, conjugated with a fluorochrome excitable by a UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 379 / 28 nm, or a spectral equivalent thereof; b) anti-CD81 , conjugated with a fluorochrome excitable by the UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 515 / 30 nm, or a spectral equivalent thereof; c) anti-CD73, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, or a spectral equivalent thereof; d) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, or a spectral equivalent thereof; e) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, or a spectral equivalent thereof; f) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, or a spectral equivalent thereof; g) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, or a spectral equivalent thereof; h) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, or a spectral equivalent thereof; i) anti-CD371 , conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 530 / 31 nm, or a spectral equivalent thereof; j) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, or a spectral equivalent thereof; k) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, or a spectral equivalent thereof; l) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, or a spectral equivalent thereof; m) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, or a spectral equivalent thereof; n) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, or a spectral equivalent thereof; o) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, or a spectral equivalent thereof; and p) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, or a spectral equivalent thereof.

Claims

Claims1. A reagent panel for the measurement of minimal residual disease (MRD) associated with pediatric B-cell precursor acute lymphoblastic leukemia (B-ALL) in a sample from a subject by multiparametric flow cytometry, said panel comprising a combination of antibodies directed against markers, wherein the combination of antibodies comprises:

1. antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99 and CD371 , and ii. antibodies targeting markers CD19 and / or CD22; wherein the antibodies are conjugated with fluorochromes.

2. The reagent panel according to claim 1, wherein the combination of antibodies further comprises an antibody targeting CD36.

3. The reagent panel according to claims 1 or 2, wherein the reagent panel further comprises a DNA-binding dye.

4. The reagent panel according to claims 1-3, wherein the reagent panel comprises a combination of two tubes: a) a first tube (Tube 1) containing a first reagent composition comprising a combination of monoclonal antibodies directed against human leukocyte markers, wherein said first reagent composition comprises a combination of antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81, CD123, CD304, CD44, CD86, CD99, CD371 and CD19 and / or CD22 thereby facilitating enhanced identification of MRD leukemia cells, wherein the antibodies are conjugated with fluorochromes; and b) a second tube (Tube 2) containing a second reagent composition formulated for quantifying nucleated cells said second reagent composition comprising a DNA-binding dye and monoclonal antibodies directed against markers CD45, CD19, and CD36, wherein the antibodies are conjugated with fluorochromes.

5. The reagent panel according to any of the preceding claims, wherein the panel is configured as at least a 12-colour panel, at least a 14-colour panel, at least a 16-colour panel, at least a 18-colour panel, or at least a 20-colour panel.

6. The reagent panel according to any of the preceding claims, wherein the panel is configured as a 12-colour panel.

7. The reagent panel according to any of the preceding claims, wherein the panel is configured as a 14-colour panel.

8. The reagent panel according to any of the preceding claims, wherein the panel is configured as a 16-colour panel.

9. The reagent panel according to any of claims 1-6, wherein the antibodies targeting markers comprise: a) anti-CD10, b) anti-CD45, c) anti-CD34, d) anti-CD20,e) anti-CD81 , f) anti-CD58 and anti-CD371 , g) anti-CD99 and anti-CD34, h) anti-CD44 and CD73, i) anti-CD38, j) anti-CD123 and CD66c, k) anti-CD19, and l) anti-CD86, wherein each of the antibodies of groups a)-l) are conjugated with spectrally distinct fluorochromes.

10. The reagent panel according to claim 9, wherein the antibodies targeting markers comprise: a) anti-CD10, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD34, conjugated with fluorochrome excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD20, conjugated with one or more fluorochromes excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 710 / 50 nm, e) anti-CD81 , conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, f) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, g) anti-CD99 and anti-CD34, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm,I) anti-CD86, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, h) anti-CD44 and CD73, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD38, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, j) anti-CD123 and CD66c, conjugated with one or more fluorochromes excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, andk) anti-CD19, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

11. The reagent panel according to any of claims 1-5 or 7, wherein the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , b) anti-CD45, c) anti-CD304, d) anti-CD44, e) anti-CD86, f) anti-CD99, g) anti-CD58 and anti-CD371, h) anti-CD66c and anti-CD123, i) anti-CD34, j) anti-CD10, k) anti-CD19, l) anti-CD22, m) anti-CD38, and n) anti-CD20, wherein each of the antibodies of groups a)-n) are conjugated with spectrally distinct fluorochromes.

12. The reagent panel according to claim 11, wherein the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , conjugated with one or more fluorochromes excitable by a violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, e) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm,f) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, g) anti-CD58 and anti-CD371, conjugated with one or more fluorochromes excitable by a blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, h) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, i) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, j) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, k) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, l) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, m) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and n) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

13. The reagent panel according to claims 1-5 or 8, wherein the antibodies targeting markers comprise: a) anti-CD58, b) anti-CD81 , c) anti-CD73, d) anti-CD45, e) anti-CD304, f) anti-CD44, g) anti-CD86, h) anti-CD99, i) anti-CD371, j) anti-CD66c and anti-CD123, k) anti-CD34, l) anti-CD10, m) anti-CD19, n) anti-CD22,o) anti-CD38, and p) anti-CD20, wherein each of the antibodies of groups a)-p) are conjugated with spectrally distinct fluorochromes.

14. The reagent panel according to claim 13, wherein the antibodies targeting markers comprise: a) anti-CD58, conjugated with a fluorochrome excitable by a UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 379 / 28 nm, b) anti-CD81 , conjugated with a fluorochrome excitable by the UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 515 / 30 nm, c) anti-CD73, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, d) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, e) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, f) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, g) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, h) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD371, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 530 / 31 nm, j) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, k) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, l) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, m) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, n) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, o) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, andp) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

15. The reagent panel according to any preceding claim, wherein the DNA-binding dye is SYTO41.

16. The reagent panel according to any of claims 1-15, wherein the sample comprises bone marrow cells.

17. The reagent panel according to any of claims 1-15, wherein the sample comprises peripheral blood cells.

18. The reagent panel according to any of the preceding claims, wherein the subject is a human.

19. The reagent panel according to claim 18, wherein the subject has previously been diagnosed with pediatric B-cell precursor acute lymphoblastic leukemia.

20. The reagent panel according to claim 18 or 19, wherein the subject has previously been treated for pediatric B-cell precursor acute lymphoblastic leukemia.

21. Use of a reagent panel for detecting MRD associated with B-ALL in a sample from a subject, wherein the reagent panel comprises a combination of antibodies directed against markers, wherein the combination of antibodies comprises: i. antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99 and CD371 , and ii. antibodies targeting markers CD19 and / or CD22; wherein the antibodies are conjugated with fluorochromes.

22. Use of a reagent panel according to claim 21, wherein the sample is MRD positive if at least two of CD58, CD81 , CD73, CD45, CD304, CD44, CD86, CD99, CD371 , CD123 or CD66c are present in the sample.

23. Use of a reagent panel for identifying a subject at risk of developing B-ALL relapse by detecting MRD associated with B-ALL in a sample from a subject, wherein the reagent panel comprises a combination of antibodies directed against markers, wherein the combination of antibodies comprises: i. antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99 and CD371 , and ii. antibodies targeting markers CD19 and / or CD22; wherein the antibodies are conjugated with fluorochromes.

24. The use of a reagent panel according to claim 23, wherein the subject is considered to be at risk of developing B-ALL relapse if at least two of CD58, CD81 , CD73, CD45, CD304, CD44, CD86, CD99, CD371 , CD123 or CD66c are present in the sample.

25. The use of a reagent panel according to any of claims 21-24, wherein the combination of antibodies further comprises an antibody targeting CD36.

26. The use of a reagent panel according to any of claims 21-25, wherein the reagent panel further comprises a DNA-binding dye.

27. The use of a reagent panel according to any of claims 21-26, wherein the reagent panel comprises a combination of two tubes: a) a first tube (Tube 1) containing a first reagent composition comprising a combination of monoclonal antibodies directed against human leukocyte markers, wherein said first reagent composition comprises a combination of antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81, CD123, CD304, CD44, CD86, CD99, CD371 and CD19 and / or CD22 thereby facilitating enhanced identification of MRD leukemia cells, wherein the antibodies are conjugated with fluorochromes; and b) a second tube (Tube 2) containing a second reagent composition formulated for quantifying nucleated cells said second reagent composition comprising a DNA-binding dye and monoclonal antibodies directed against markers CD45, CD19, and CD36, wherein the antibodies are conjugated with fluorochromes.

28. The use of a reagent panel according to any of claims 21-27, wherein the panel is configured as at least a 12-colour panel, at least a 14-colour panel, at least a 16-colour panel, at least a 18-colour panel, or at least a 20-colour panel.

29. The use of a reagent panel according to any of claims 21-28, wherein the panel is configured as a 12-colour panel.

30. The use of a reagent panel according to any of claims 21-28, wherein the panel is configured as a 14-colour panel.

31. The use of a reagent panel according to any of claims 21-28, wherein the panel is configured as a 16-colour panel.

32. The use of a reagent panel according to any of claims 21-29, wherein the antibodies targeting markers comprise: a) anti-CD10, b) anti-CD45, c) anti-CD34, d) anti-CD20, e) anti-CD81 , f) anti-CD58 and anti-CD371 , g) anti-CD99 and anti-CD34,I) anti-CD86, h) anti-CD44 and CD73, i) anti-CD38, j) anti-CD123 and CD66c, and k) anti-CD19, wherein each of the antibodies of groups a)-l) are conjugated with spectrally distinct fluorochromes.

33. The reagent panel according to claim 32, wherein the antibodies targeting markers comprise: a) anti-CD10, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD34, conjugated with fluorochrome excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD20, conjugated with one or more fluorochromes excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 710 / 50 nm, e) anti-CD81 , conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, f) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, g) anti-CD99 and anti-CD34, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm,I) anti-CD86, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, h) anti-CD44 and CD73, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD38, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, j) anti-CD123 and CD66c, conjugated with one or more fluorochromes excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and k) anti-CD19, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

34. The use of a reagent panel according to any of claims 21-28 or 30, wherein the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , b) anti-CD45, c) anti-CD304, d) anti-CD44, e) anti-CD86,f) anti-CD99, g) anti-CD58 and anti-CD371, h) anti-CD66c and anti-CD123, i) anti-CD34, j) anti-CD10, k) anti-CD19, l) anti-CD22, m) anti-CD38, and n) anti-CD20, wherein each of the antibodies of groups a)-n) are conjugated with spectrally distinct fluorochromes.

35. The reagent panel according to claim 34, wherein the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , conjugated with one or more fluorochromes excitable by a violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, e) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, f) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, g) anti-CD58 and anti-CD371, conjugated with one or more fluorochromes excitable by a blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, h) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, i) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, j) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm,k) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, l) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, m) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and n) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

36. The use of a reagent panel according to any of claims 21-28 or 31, wherein the antibodies targeting markers comprise: a) anti-CD58, b) anti-CD81 , c) anti-CD73, d) anti-CD45, e) anti-CD304, f) anti-CD44, g) anti-CD86, h) anti-CD99, i) anti-CD371, j) anti-CD66c and anti-CD123, k) anti-CD34, l) anti-CD10, m) anti-CD19, n) anti-CD22, o) anti-CD38, and p) anti-CD20, wherein each of the antibodies of groups a)-p) are conjugated with spectrally distinct fluorochromes.

37. The reagent panel according to claim 36, wherein the antibodies targeting markers comprise: a) anti-CD58, conjugated with a fluorochrome excitable by a UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 379 / 28 nm,b) anti-CD81 , conjugated with a fluorochrome excitable by the UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 515 / 30 nm, c) anti-CD73, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, d) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, e) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, f) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, g) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, h) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD371, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 530 / 31 nm, j) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, k) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, l) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, m) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, n) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, o) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and p) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

38. The use of a reagent panel according to any of claims 21-37, wherein the DNA- binding dye is SYTO41.

39. The use of a reagent panel according to any of claims 21-38, wherein the sample comprises bone marrow cells.

40. The use of a reagent panel according to any of claims 21-38, wherein the sample comprises peripheral blood cells.

41. The use of a reagent panel according to any of claims 21-40, wherein the subject is a human.

42. The use of a reagent panel according to claim 41, wherein the subject has previously been diagnosed with pediatric B-cell precursor acute lymphoblastic leukemia.

43. The use of a reagent panel according to claim 41 or 42, wherein the subject has previously been treated for pediatric B-cell precursor acute lymphoblastic leukemia.

44. A method of detecting MRD associated with B-ALL in a sample from a subject, the method comprising detecting MRD in the sample from the subject by multiparametric flow cytometry using a reagent panel comprising a combination of antibodies directed against markers, wherein the combination of antibodies comprises: i. antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123, CD304, CD44, CD86, CD99 and CD371 , and ii. antibodies targeting markers CD19 and / or CD22; wherein the antibodies are conjugated with fluorochromes.

45. The method according to claim 44, wherein the sample is MRD positive if at least two of CD58, CD81 , CD73, CD45, CD304, CD44, CD86, CD99, CD371, CD123 or CD66c are present in the sample.

46. The method of claim 44 or 45, wherein the reagent panel further comprises a DNA- binding dye.

47. A method for detecting MRD associated with B-ALL in a sample from a subject, the method comprising: a. contacting the sample with a reagent panel, wherein the reagent panel comprises a combination of antibodies directed against markers, wherein the combination of antibodies comprises: i. antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81 , CD123 CD304, CD44, CD86, CD99 and CD371, and ii. antibodies targeting markers CD19 and / or CD22; wherein the antibodies are conjugated with fluorochromes, b. contacting the sample with a DNA-binding dye, c. detecting the presence of the antibody binding targets by multiparametric flow cytometry, wherein detection of at least two of CD58, CD81, CD73, CD45, CD304, CD44, CD86, CD99, CD371, CD123 or CD66c determines the presence of MRD.

48. The method according to any one of claims 44-47, wherein the combination of antibodies further comprises an antibody targeting CD36.

49. The method according to any of claims 44-48, wherein the reagent panel comprises a combination of two tubes: a) a first tube (Tube 1) containing a first reagent composition comprising a combination of monoclonal antibodies directed against human leukocyte markers, wherein said first reagent composition comprises a combination of antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81, CD123, CD304, CD44, CD86, CD99, CD371 and CD19 and / or CD22 thereby facilitating enhanced identification of MRD leukemia cells, wherein the antibodies are conjugated with fluorochromes; andb) a second tube (Tube 2) containing a second reagent composition formulated for quantifying nucleated cells said second reagent composition comprising a DNA-binding dye and monoclonal antibodies directed against markers CD45, CD19, and CD36, wherein the antibodies are conjugated with fluorochromes.

50. The method according to any of claims 44-49, wherein the panel is configured as at least a 12-colour panel, at least a 14-colour panel, at least a 16-colour panel, at least a 18- colour panel, or at least a 20-colour panel.

51. The method according to any of claims 44-50, wherein the panel is configured as a 12-colour panel.

52. The method according to any of claims 44-50, wherein the panel is configured as a 14-colour panel.

53. The method according to any of claims 44-50, wherein the panel is configured as a 16-colour panel.

54. The method according to any of claims 44-51 , wherein the antibodies targeting markers comprise: a) anti-CD10, b) anti-CD45, c) anti-CD34, d) anti-CD20, e) anti-CD81 , f) anti-CD58 and anti-CD371 , g) anti-CD99 and anti-CD34,I) anti-CD86, h) anti-CD44 and CD73, i) anti-CD38, j) anti-CD123 and CD66c, and k) anti-CD19, wherein each of the antibodies of groups a)-l) are conjugated with spectrally distinct fluorochromes.

55. The method according to claim 54, wherein the antibodies targeting markers comprise: a) anti-CD10, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm,c) anti-CD34, conjugated with fluorochrome excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD20, conjugated with one or more fluorochromes excitable by the violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 710 / 50 nm, e) anti-CD81 , conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, f) anti-CD58 and anti-CD371 , conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, g) anti-CD99 and anti-CD34, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm,I) anti-CD86, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, h) anti-CD44 and CD73, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD38, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, j) anti-CD123 and CD66c, conjugated with one or more fluorochromes excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and k) anti-CD19, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

56. The method according to any of claims 44-50 or 52, wherein the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , b) anti-CD45, c) anti-CD304, d) anti-CD44, e) anti-CD86, f) anti-CD99, g) anti-CD58 and anti-CD371, h) anti-CD66c and anti-CD123, i) anti-CD34, j) anti-CD10,k) anti-CD19, l) anti-CD22, m) anti-CD38, and n) anti-CD20, wherein each of the antibodies of groups a)-n) are conjugated with spectrally distinct fluorochromes.

57. The method according to claim 56, wherein the antibodies targeting markers comprise: a) anti-CD73 and anti-CD81 , conjugated with one or more fluorochromes excitable by a violet laser (nominally 405 nm) and co-detectable within a fluorescence channel corresponding to approximately 450 / 40 nm, b) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm, c) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, d) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, e) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, f) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, g) anti-CD58 and anti-CD371, conjugated with one or more fluorochromes excitable by a blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 530 / 31 nm, h) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, i) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, j) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, k) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, l) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, m) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, andn) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

58. The method according to any of claims 44-50 or 53, wherein the antibodies targeting markers comprise: a) anti-CD58, b) anti-CD81 , c) anti-CD73, d) anti-CD45, e) anti-CD304, f) anti-CD44, g) anti-CD86, h) anti-CD99, i) anti-CD371, j) anti-CD66c and anti-CD123, k) anti-CD34, l) anti-CD10, m) anti-CD19, n) anti-CD22, o) anti-CD38, and p) anti-CD20, wherein each of the antibodies of groups a)-p) are conjugated with spectrally distinct fluorochromes.

59. The method according to claim 58, wherein the antibodies targeting markers comprise: a) anti-CD58, conjugated with a fluorochrome excitable by a UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 379 / 28 nm, b) anti-CD81 , conjugated with a fluorochrome excitable by the UV or violet laser (e.g., 355 nm or 405 nm) and detectable in a fluorescence channel corresponding to approximately 515 / 30 nm, c) anti-CD73, conjugated with a fluorochrome excitable by a violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 450 / 40 nm, d) anti-CD45, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 525 / 50 nm,e) anti-CD304, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 610 / 20 nm, f) anti-CD44, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 660 / 20 nm, g) anti-CD86, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 710 / 50 nm, h) anti-CD99, conjugated with a fluorochrome excitable by the violet laser (nominally 405 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, i) anti-CD371, conjugated with a fluorochrome excitable by a blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 530 / 31 nm, j) anti-CD66c and anti-CD123, conjugated with one or more fluorochromes excitable by the blue laser (nominally 488 nm) and co-detectable within a fluorescence channel corresponding to approximately 585 / 42 nm, k) anti-CD34, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 616 / 23 nm, l) anti-CD10, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 695 / 40 nm, m) anti-CD19, conjugated with a fluorochrome excitable by the blue laser (nominally 488 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm, n) anti-CD22, conjugated with a fluorochrome excitable by a red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 670 / 30 nm, o) anti-CD38, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 730 / 45 nm, and p) anti-CD20, conjugated with a fluorochrome excitable by the red laser (nominally 640 nm) and detectable in a fluorescence channel corresponding to approximately 780 / 60 nm.

60. The method according to any of claims 44-59, wherein the DNA-binding dye is SYTO41.

61. The method according to any of claims 44-60, wherein the sample comprises bone marrow cells.

62. The method according to any of claims 44-60, wherein the sample comprises peripheral blood cells.

63. The method according to any one of claims 44-62, wherein the subject is a human.

64. The method according to claim 63, wherein the subject has previously been diagnosed with pediatric B-cell precursor acute lymphoblastic leukemia.

65. The method according to claim 63 or 64, wherein the subject has previously been treated for pediatric B-cell precursor acute lymphoblastic leukemia.

66. A method for identifying a subject suitable for treatment with an anti-cancer therapy, wherein the method comprises a method according to any one of claims 44-65.

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