Measurable Residual Diseases in Multiple Myeloma

Measurable Residual Diseases in Multiple Myeloma

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Validation of a High-Sensitivity 10C Flow Cytometric Measurable Residual Disease (MRD) Assay for Multiple Myeloma using Sysmex XF-1600 Flow Cytometer and VenturiOne (Applied Cytometry) Software

Background

Flow cytometry (FC) is pivotal in detecting measurable residual disease (MRD) in multiple myeloma (MM), serving as a predictor of therapeutic response. CD38 and CD138 are commonly used markers for effective discrimination of plasma cells (PCs) from other cells. However, the monoclonal antibody drug for MM daratumumab, binds to cell-surface CD38, which may hinder PC detection. One of the strategies to overcome this interference is to stain PCs with cVS38, an antibody that recognizes cytoskeleton-linking membrane protein 63 (CLIMP-63), which is found on the rough endonlasmic reticulum in secretory cells, including PCs.

Methods

We develoned and validated a high-sensitivity, single-tube 10-color myeloma MRD flow cytometry assay using cVS38c and CD138 as backbone markers for plasma cell detection, combined with surface and cytoplasmic markers in a single tube after bulk lyse method (lyse, wash, stain, wash).

Panel: cVS38c FITC. cLambda PE. CD117 ECD, CD138 PerCP-Cv5.5, CD56 PC7, cappa APC, CD19 A700, CD27 APC-A750, CD81 PB, CD45 PO.
Instrument: Sysmex XF-1600 flow cytometer.
Analysis Software: VenturiOne and Kaluza 2.1.

We performed instrument PMT optimization compensation, antibody titrations, cocktail stability studies, as well as evaluated the performance characteristics of the assay, including carryover studies, sensitivity (Limit of Blank [LOB], Limit of Detection [LOD], and Lower Limit of Quantification ILLOQI, selectivity, accuracy, precision, and reference range studies.

Results

  1. Sensitivity: LOB: 10 events, LOD: 20 events (0.5 x10-5) LLOQ: 50 events (1.2 x10-5) out of 4 x10-6 acquired nucleated cells.
  2. Correlation: The cell recovery percentages were found to be acceptable, with a 98% concoreance with comparative methods
  3. Selectivity: Compared VS38, CD38ME, and JK36 for PC identification in post-therapy patients. Effective identification of targeted populations was achieved using VS38.
  4. Precision: Demonstrated an acceptable coefficient of variation (CV) of <30% across runs, days, and instruments.
  5. Reference Range: Verified by 25 normal bone marrow aspirate (BMA) samples.
  6. Carryover: Calculated as <0.0001%


Analysis and Gating Strategy

Evaluated Kaluza and VenturiOne software regarding FCS data upload and analysis time. Adopted a gating strategy in which the percentages of abnormal PCs were determined using VS38, CD138, and side scatter (SSC), followed by immunoglobulin light chain restriction. Evaluated mast cells for specimen adequacy.

Step 1: gated total number of analyzed nucleated cells after excluding debris and doublets.
Step 2: identified plasma cells.
Step 3: identified abnormal plasma cells.
Step 4: Assessed specimen adequacy by evaluating mast cells and either hematogones, or myeloid or erythroid precursors.

Conclusion

The total acquisition time for 5 million events averaged approximately ~10 minutes on the XF-1600 flow cytometer. FCS data upload and analysis were observed to be faster with VenturiOne compared to Kaluza. Moreover. cVS38c was confirmed as a reliable marker for plasma cell detection in MRD testing. Overall, the MM MRD panel, with a functional sensitivity of 105 was comprehensively evaluated for cocktail stability, accuracy, precision, sensitivity, selectivity, and reference range studies, and the assay's performance characteristics met acceptable standards for MRD detection in MM.

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