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Method Article
This protocol describes a standardized multicolor flow cytometry workflow for measurable residual disease assessment in acute leukemia, integrating controlled specimen processing, harmonized acquisition parameters, sequential gating strategies, and denominator-adjusted interpretation to improve analytical reproducibility in routine clinical laboratories.
Measurable residual disease (MRD) is an important prognostic indicator in the management of acute leukemia. However, routine multicolor flow cytometric (MFC) assessment remains vulnerable to pre-analytical and analytical variability, including hemodilution, inconsistent acquisition depth, and subjective interpretation. This article presents a standardized operational workflow for routine MRD evaluation in B-cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML). The protocol incorporates strict specimen acceptance criteria, with integrated hemodilution assessment, and uses a bulk red blood cell lysis method for bone marrow and peripheral blood specimens. Analytical standardization is achieved through disease-specific two-tube, eight-color antibody panels, harmonized acquisition targets requiring at least 500,000 CD45-positive events, and fixed sequential gating strategies. In addition, the workflow applies denominator-adjusted limits of detection (LOD) and quantification (LOQ) to support an objective three-tiered reporting system. Validation across 197 clinical specimens demonstrated strong repeatability, robust dilutional linearity, and high concordance with paired orthogonal molecular assays. Collectively, this protocol provides a practical framework for standardized MRD monitoring. for standardized MRD monitoring in routine clinical laboratories by integrating controlled pre-analytical handling and harmonized analytical parameters.
Even after achieving morphological remission, patients with acute leukemia require ongoing monitoring for measurable residual disease (MRD) to assess relapse risk accurately. Conventional morphologic and cytopathologic examinations lack the sensitivity required to detect low-level residual leukemic populations. Consequently, MRD assessment has become an essential component of disease monitoring in both acute myeloid leukemia (AML) and B-cell acute lymphoblastic leukemia (B-ALL). Recent recommendations from the European LeukemiaNet emphasize that reliable MRD assessment in AML depends on technical standardization and interobserver reproducibility rather than simple ass....
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All procedures involving human specimens were performed in accordance with institutional guidelines and approved by the Ethics Committee of The Second People’s Hospital of Neijiang (Approval No. PXTS887192). The study utilized de-identified residual clinical specimens obtained after routine diagnostic testing. Informed consent was waived because the study did not affect patient care, treatment allocation, or specimen collection procedures. The chemicals, reagents, and equipment used in the protocol are listed in the Table of Materials.
1. Specimen collection and pre-analytical assessment
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Characteristics of the routine implementation dataset
During the study period, 197 consecutive specimens passed eligibility screening and pre-analytical quality assessment and entered the reportable workflow. The study cohort included 98 patients, 62 (63.3%) with B-cell acute lymphoblastic leukemia (B-ALL) and 36 (36.7%) with acute myeloid leukemia (AML). The cohort consisted of 64 males (65.3%) and 34 females (34.7%), with a median age of 32 years (interquartile range [IQR], .......
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The primary objective of this study was to establish a standardized workflow for multicolor flow cytometric assessment of measurable residual disease (MRD) in acute leukemia. Reliable MRD evaluation depends not only on antibody panel selection, but also on consistent specimen handling, acquisition parameters, and data interpretation11. The results of this study demonstrate that harmonized specimen processing, controlled acquisition depth, and denominator-adjusted reporting criteria can improve ana.......
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The authors have nothing to disclose.
The author thanks the Department of Clinical Laboratory at The Second People’s Hospital of Neijiang for providing laboratory facilities and technical support for this study. The author also acknowledges the laboratory staff and clinical personnel for their assistance with specimen processing and data collection. Finally, the author thanks the patients whose residual clinical specimens contributed to this work. This research received no specific funding from public, commercial, or not-for-profit funding agencies.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Acquisition buffer | Laboratory-prepared | PBS containing 0.5% BSA and 2 mM EDTA; final resuspension volume 500 µL | Final resuspension before flow cytometric acquisition |
| AML Tube 1 anti-CD117-APC antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 104D2; fluorochrome: APC; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Early myeloid/blast identification |
| AML Tube 1 anti-CD13-FITC antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: L138; fluorochrome: FITC; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Myeloid lineage anchor |
| AML Tube 1 anti-CD33-PE antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: P67.6; fluorochrome: PE; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Myeloid lineage anchor |
| AML Tube 1 anti-CD34-PerCP-Cy5.5 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 8G12; fluorochrome: PerCP-Cy5.5; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Immaturity marker |
| AML Tube 1 anti-CD45-APC-H7 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 2D1; fluorochrome: APC-H7; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Leukocyte backbone marker and denominator definition |
| AML Tube 1 anti-CD56-V500 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: NCAM16.2; fluorochrome: V500; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Aberrant blast-associated phenotyping |
| AML Tube 1 anti-CD7-V450 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: M-T701; fluorochrome: V450; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Cross-lineage aberrancy detection |
| AML Tube 1 anti-HLA-DR-PE-Cy7 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: G46-6; fluorochrome: PE-Cy7; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Maturation and subtype discrimination |
| AML Tube 2 anti-CD117-APC antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 104D2; fluorochrome: APC; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Early myeloid/blast identification |
| AML Tube 2 anti-CD11b-V450 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: ICRF44; fluorochrome: V450; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Maturation-discordance assessment |
| AML Tube 2 anti-CD13-FITC antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: L138; fluorochrome: FITC; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Myeloid lineage anchor |
| AML Tube 2 anti-CD15-V500 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: HI98; fluorochrome: V500; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Different-from-normal myeloid pattern assessment |
| AML Tube 2 anti-CD33-PE antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: P67.6; fluorochrome: PE; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Myeloid lineage anchor |
| AML Tube 2 anti-CD34-PerCP-Cy5.5 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 8G12; fluorochrome: PerCP-Cy5.5; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Immaturity marker |
| AML Tube 2 anti-CD45-APC-H7 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 2D1; fluorochrome: APC-H7; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Leukocyte backbone marker and denominator definition |
| AML Tube 2 anti-HLA-DR-PE-Cy7 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: G46-6; fluorochrome: PE-Cy7; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Maturation and subtype discrimination |
| Ammonium chloride-based red blood cell lysis buffer | Laboratory-prepared / commercial supplier | Ammonium chloride-based lysis buffer; 10 mL/test; 10 min at room temperature | Bulk red blood cell lysis |
| Analysis software | Beckman Coulter (Brea, CA, USA) | Kaluza 2.1 | Data analysis and sequential gating |
| Automated hematology analyzer | Commercial supplier | Automated hematology analyzer used for nucleated cell concentration and viability assessment | Nucleated cell concentration and viability assessment |
| B-ALL Tube 1 anti-CD10-PE antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: HI10a; fluorochrome: PE; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Precursor B-cell definition |
| B-ALL Tube 1 anti-CD19-FITC antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: SJ25C1; fluorochrome: FITC; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | B-lineage identification and entry into precursor compartment |
| B-ALL Tube 1 anti-CD20-APC antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: L27; fluorochrome: APC; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Maturation pattern assessment |
| B-ALL Tube 1 anti-CD34-PerCP-Cy5.5 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 8G12; fluorochrome: PerCP-Cy5.5; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Immaturity marker |
| B-ALL Tube 1 anti-CD38-PE-Cy7 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: HIT2; fluorochrome: PE-Cy7; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Separation from regenerating normal precursors |
| B-ALL Tube 1 anti-CD45-APC-H7 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 2D1; fluorochrome: APC-H7; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Leukocyte backbone marker and denominator definition |
| B-ALL Tube 1 anti-CD58-V450 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: TS2/9; fluorochrome: V450; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Aberrancy detection in residual leukemic precursors |
| B-ALL Tube 1 anti-CD81-V500 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: JS-81; fluorochrome: V500; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Support for abnormal precursor recognition |
| B-ALL Tube 2 anti-CD10-PE antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: HI10a; fluorochrome: PE; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Precursor B-cell definition |
| B-ALL Tube 2 anti-CD123-V450 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 9F5; fluorochrome: V450; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Aberrant precursor phenotyping |
| B-ALL Tube 2 anti-CD19-FITC antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: SJ25C1; fluorochrome: FITC; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | B-lineage identification and entry into precursor compartment |
| B-ALL Tube 2 anti-CD20-APC antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: L27; fluorochrome: APC; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Maturation pattern assessment |
| B-ALL Tube 2 anti-CD34-PerCP-Cy5.5 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 8G12; fluorochrome: PerCP-Cy5.5; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Immaturity marker |
| B-ALL Tube 2 anti-CD45-APC-H7 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: 2D1; fluorochrome: APC-H7; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Leukocyte backbone marker and denominator definition |
| B-ALL Tube 2 anti-CD66c-PE-Cy7 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: KOR-SA3544; fluorochrome: PE-Cy7; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Leukemia-associated aberrancy support |
| B-ALL Tube 2 anti-CD73/CD304*-V500 antibody | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | Clone: AD2/AZ1; fluorochrome: V500; staining volume: 5 µL/test in the routine validated panel unless antibody-specific titration differs | Additional aberrancy confirmation according to diagnostic phenotype |
| Bone marrow / peripheral blood specimens | The Second People’s Hospital of Neijiang | Residual clinical specimens | MRD assessment |
| Compensation beads | Commercial supplier | Single-stained compensation beads used on the same day as specimen acquisition | Same-day compensation control preparation |
| Fixable viability dye | Commercial supplier | Fixable viability dye included in every staining tube | Exclusion of non-viable cells; viability gate referenced in Protocols 3.7 and 5.4 |
| Flow cytometer | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | FACSCanto II / Navios EX | Multicolor flow cytometric acquisition |
| Manual hemocytometer | Commercial supplier | Manual hemocytometer count | Manual nucleated cell counting |
| PBS/BSA/EDTA wash buffer | Laboratory-prepared | Phosphate-buffered saline containing 0.5% bovine serum albumin and 2 mM EDTA | Cell washing and resuspension |
| Trypan blue solution | Commercial supplier | Trypan blue exclusion | Manual viability assessment by trypan blue exclusion |
| Validated antibody cocktails | BD Biosciences (Franklin Lakes, NJ, USA) / Beckman Coulter (Brea, CA, USA) | See antibody-specific rows below and Supplementary Table S1 | MRD immunophenotypic detection |
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