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Article

Effects of Daratumumab (Anti-CD38) Monoclonal Antibody Therapy on Flow Cytometry Analysis in Multiple Myeloma

by
Sharon Koorse Germans
1,*,
Christine Wamsley Kahlow
2,
Weina Chen
1,3 and
Franklin Fuda
2,3
1
Department of Hematopathology, University of Texas Southwestern, 2330 Inwood Road, Dallas, TX 75390, USA
2
Department of Pathology, University of Texas Southwestern, 2330 Inwood Road, Dallas, TX 75390, USA
3
Department of Flow Cytometry, University of Texas Southwestern, 2330 Inwood Road, Dallas, TX 75390, USA
*
Author to whom correspondence should be addressed.
Therapeutics 2025, 2(1), 2; https://doi.org/10.3390/therapeutics2010002
Submission received: 12 December 2024 / Revised: 15 January 2025 / Accepted: 6 February 2025 / Published: 18 February 2025
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Abstract

:
Background: Plasma cell myeloma is an incurable malignancy of clonal plasma cells. Recent success in immunotherapeutic strategies has altered the landscape of myeloma treatment. Daratumumab is an anti-CD38 IgG kappa monoclonal antibody that has shown great efficacy in the treatment of myeloma. However, Daratumumab brought with it new challenges in post-therapeutic laboratory assessment, including therapeutic antibody interference with serum protein electrophoresis and serum immunofixation electrophoresis assays. In this study, we highlight the interference identified in post-therapeutic flow cytometry analysis related to bound Daratumumab on normal hematopoietic cells. We also highlight the methods of detection of residual plasma cell neoplasm, post-Daratumumab therapy.: A total of 28 patients with refractory plasma cell myeloma who received Daratumumab (2016–2018) were included in this study. Flow cytometry was performed using 4- or 10-color antibody panels (BD FASC Canto) and analyzed by cluster analysis (Cytopaint Classic software) using four tube panels including VS38c for measurable residual disease (MRD) testing. Pretreatment and post-Daratumumab follow-up bone marrow flow cytometry samples were analyzed. In addition, 10 multiple myeloma patient samples were reflexed to multi-epitope CD38 analysis by flow cytometric analysis of post-Daratumumab residual disease. When discussing CD38 expression, we will refer to CD38 as being detected by conventional reagents. Results: All post-Daratumumab-treated cases (100%) showed negative staining for CD38 using conventional reagents on all plasma cells in the specimens. MRD testing successfully identified small clonal plasma cell populations using VS38C and multi-epitope CD38 (meCD38) antibodies, despite the absence of demonstrable CD38 expression. Additionally, all cases exhibited weak kappa light chain staining on hematogones, attributed to the binding of Daratumumab kappa monoclonal antibody. This interaction can create the appearance of a CD10+ monotypic B-cell population. We also noted diminished CD38 staining on myeloblasts, resulting in an atypical CD34/CD38 staining pattern. This alteration could potentially be misinterpreted as indicative of a myelodysplastic neoplasm (MDS). Furthermore, decreased staining of CD38 was noted on T cells, natural killer (NK) cells, basophils, monocytes, and plasmacytoid dendritic cells. Conclusions: With the emergence of successfully targeted immunotherapies, such as anti-CD38 antibodies, it is important to understand and correctly interpret variations in flow cytometry that may arise from the therapy. Hematogones exhibit high-intensity levels of CD38 expression; thus, Daratumumab binds to them, creating the appearance of kappa expression on all hematogones. Stage I/early stage II hematogones normally lack surface immunoglobulin light chain expression, but in the presence of Daratumumab, they appear to be a CD10(+) monotypic population of B cells. The misinterpretation of these normal cells as a CD10(+) B-cell clone can lead to inaccurate assessment, unnecessary bone marrow immunohistochemical evaluation, and unwarranted anxiety. Additionally, artefacts on various other hematopoietic cells can result in inaccurate assessments of immunophenotypic aberrancy due to binding of the drug. This may lead to the false interpretation of a secondary/therapy-related myeloid neoplasm. This study highlights in detail the interferences that must be considered when assessing residual disease in the era of targeted drug therapies.

1. Introduction

Plasma cell myeloma accounts for 10% of all hematological malignancies, with a 5-year survival of 61.1% (2014–2022) [1]. It is a clonal plasma cell neoplasm with substantial morbidity and mortality, characterized by end organ damage—renal impairment, hypercalcemia, lytic bony lesions, and anemia. Great strides have been made in the treatment of this incurable disease, with the aim of reducing morbidity and increasing survival. The first major advancement was the development of autologous stem cell transplantation (ASCT) in the 1980s and 1990s [2], followed by the development of novel agents in the late 1990s and early 2000s, beginning with immunomodulatory drugs, “IMIDs” (thalidomide and lenalidomide), and then proteasome inhibitors [3]. Two novel therapeutic monoclonal antibodies were approved in 2015 by the US Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma: Elotuzumab, a humanized IgG1κ that targets signaling lymphocytic activation molecule F7, and Daratumumab [4,5].
Daratumumab is a human anti-CD38 IgG1-κ monoclonal antibody jointly developed by Genmab and Janssen Biotech (a Johnson & Johnson subsidiary) [6,7]. It has been used as monotherapy as well as combination therapy in treatment-naïve, transplant-eligible and -ineligible patients to improve overall survival.
The diagnosis of plasma cell myeloma includes the presence of blood/bone marrow monoclonal plasma cells, the elevation of serum and urine free light chains, biochemical evidence of end organ damage, and accompanying cytogenetic molecular analysis for prognostic markers [8]. The identification, characterization and measurement of serum monoclonal proteins are essential for diagnosing multiple myeloma and for monitoring patients after treatment. Flow cytometry also plays a crucial role in initially identifying plasma cell clones and in detecting the persistence of those clones during follow-up examinations in these patients.
Several studies and case reports have identified a major interference of Daratumumab in serum protein electrophoresis and immunofixation studies, which remain the first-line follow-up analyses for the identification of complete response by IMWG (International myeloma working group) criteria (CR) [9,10,11,12]. Daratumumab also interferes with antibody screening in the blood bank by causing pan-reactive antibody panels. Current studies highlight the role of techniques including dithiothreitol (DTT) to alleviate such interference [13].
This study highlights a significant interference caused by Daratumumab in the flow cytometry evaluation of samples for minimal residual disease (MRD) testing in plasma cell myeloma. Daratumumab attaches to the same epitope on CD38 as conventional flow cytometric anti-CD38 reagents, obstructing staining and creating the impression of decreased to negative expression levels. This interference poses major limitations in identifying residual plasma cell clones, potentially impacting remission status, ongoing treatment and prognosis. Additionally, we noted an artificial (artefactual) pseudo-light chain restriction of early-stage hematogones in all patients recently treated with Daratumumab. This could be mistakenly interpreted as a CD10+ B-cell clone. In addition, variations in normal immunophenotypic patterns on various hematopoietic cells were observed, which could be erroneously interpreted as aberrancy on those populations, indicative of a hematolymphoid malignancy such as a secondary B-lymphoblastic leukemia or a myeloid neoplasm. This critical study highlights various artefacts caused by Daratumumab in flow cytometry that pathologists must consider when analyzing samples post-therapy. Understanding these artefacts is essential for accurately classifying remission status and guiding treatment.

2. Material and Methods

Plasma cell myeloma patients were identified from an Epic chart data search. The diagnosis and duration of Daratumumab therapy were noted for each patient. Morphologic and immunohistochemical analysis of peripheral blood and bone marrow samples were reviewed throughout diagnosis and follow-up for minimal residual disease. Concurrent flow cytometry analysis sets after the initiation of Daratumumab were included in this study to demonstrate this artifact. Pretreatment flow cytometry analysis for each patient was used to compare the absence of the artifact.
A total of 28 patients with resistant/refractory plasma cell myeloma (13 men, 8 women, age range: 30–77 years) received Daratumumab (2016–2018) with follow-up bone marrow hematogones > 0.10% (range 0.10–5.3%) were included in the study. In addition, 10 cases were reflexed to meCD38 (multi-epitope CD38, Cytognos, Salamanca, Spain) analysis by flow cytometry. Flow cytometry was performed using 4- or 10-color antibody panels (BD FASCCanto Clinical Flow Cytometry System, v 3.0, BD Biosciences, San Jose, CA, USA) and analyzed by cluster analysis (Leukobyte Cytopaint Classic software v1.1.x, Pleasanton, CA, USA). Panels included CD5, CD10, CD19, CD20, CD34, CD38, meCD38, CD45, CD56, CD138, Ig light chain kappa and lambda, and VS38C (Dako—Fluorescein isothiocyanate (FITC) conjugated monoclonal mouse antibody). Pretreatment and post-Daratumumab follow-up bone marrow flow cytometry samples were analyzed for persistent disease and Daratumumab interference on hematopoietic cells that express CD38.

Procedure for Flow Cytometry Analysis from Sample Collection in RPMI to Analysis

Bone marrow aspirate specimens were placed in RPMI (Roswell Park Memorial Institute) tissue culture medium for optimal cell viability and transported at room temperature within 12 h of collection. The specimens were processed using a whole blood lysing system with ammonium chloride and stained with fluorochrome-conjugated antibodies. Flow cytometric analysis in multiple myeloma patients was performed using a standard set of antibodies designed to detect plasma cells with a sensitivity of 0.01%. The permeabilization of plasma cells was performed to detect intracellular light chain expression. The list of antibodies used in our institute and the antibody combination in each tube are shown in Table 1. Plasma cells were gated based on their light scatter properties and expression of CD19, CD38, CD45, CD56, VS38C, meCD38, and cytoplasmic immunoglobulin light chains.

3. Results

All post-Daratumumab treated cases (100%) showed negative to diminished staining for CD38 on plasma cells, hematogones, progenitor cells/myeloblasts, natural killer (NK) cells, plasmacytoid dendritic cells, monocytes, granulocytes, and basophils in the specimens. In addition, all cases also showed a dim kappa light chain staining artefact on 100% of hematogones (Figure 1). In addition, diminished CD38 resulted in an atypical CD34/CD38 expression pattern on myeloblasts, which could be misinterpreted as a myelodysplastic neoplasm/syndrome (MDS). A decreased expression of CD38 was also observed on T cells, NK cells, basophils, monocytes, and plasmacytoid dendritic cells. The artefact was reproducible using different antibody tube designs with different anti-Kappa clones and fluorochromes. Fluorescence-minus-one (FMO) tubes showed positive kappa staining on 100% of hematogones (Figure 2). The interference was present right after the first dose of infusion and was detected up to 70 days (about 2 and a half months) after the drug was stopped. Daratumumab was used in multi-drug regimens in 97% of the patients and used as a single-drug salvage therapy in only 3%. We also found complete concordance between MRD detection by VS38C and meCD38 antibodies. However, VS38C expression patterns were brighter than those of meCD38 on the residual plasma cell clone.

4. Discussion

4.1. CD38

CD38 is a transmembrane glycoprotein of 300 amino acids, expressed in multiple tissues and highly expressed in hematopoietic cells, such as B-lineage cells (hematogones, germinal center cells, and plasma cells), macrophages, dendritic cells (DCs), innate lymphoid cells (ILC), NK cells, subsets of T cells, basophils, and monocytes. The role of CD38 in immune cells ranges from modulating cell differentiation to effector functions during inflammation, where CD38 may regulate cell recruitment, cytokine release, and Nicotinamide Adenine Dinucleotide (NAD) availability. CD38 is universally expressed by normal and neoplastic plasma cells [14,15]. The intensity on neoplastic plasma cells may be variable with a staining intensity like hematogones or activated T cells. Consensus guidelines on plasma cell myeloma—MRD analysis recommend the use of CD38, CD45, CD138, and light scatter to identify plasma cells by flow cytometry. Other markers such as CD19, CD27, CD56, CD81, CD117, and cytoplasmic kappa and lambda light chain immunoglobulins may also be included to further characterize the normal and neoplastic plasma cells [16,17].

4.2. Daratumumab in the Treatment of Plasma Cell Myeloma and MRD Testing

Plasma cell myeloma remains an incurable disease for the most part, and several targeted therapies have been tried for relapsed/resistant or refractory disease. Daratumumab is an FDA-approved humanized IgG Kappa anti-CD38 monoclonal antibody used for resistant/refractory plasma cell myeloma in patients who have failed three or more lines of therapy. Daratumumab acts by promoting apoptosis via antibody-mediated cellular cytotoxicity and complement-dependent cytotoxicity. It undergoes endocytosis and internalization [6,7]. CD38 antibodies have Fc-dependent immune effector mechanisms, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). The CD38 antibodies also improve host anti-tumor immunity by the elimination of regulatory T cells, regulatory B cells and myeloid-derived suppressor cells [18]. In the recent era, Daratumumab has been approved both as a monotherapy [19] and in combination with other therapies [20,21,22] for multiple myeloma, demonstrating significant improvements in progression-free (PFS) and overall survival (OS) [23]. There has been an increasing trend in the upfront use of Daratumumab-based quadruplets [24,25] to achieve minimal residual disease (MRD) negative status, which is considered a surrogate marker for improved OS and PFS [26]. Daratumumab is also used in other plasma cell disorders [27,28].
Plasma cells from patients treated with Daratumumab artificially appear to lack CD38 expression when analyzed by flow cytometric immunophenotyping. This phenomenon is due to masking of the antigen and may persist for 4–6 months after cessation of treatment based on data from Courville EL et al. [29]. This absence of detectable CD38 makes gating and detection of plasma cells difficult given the low number of cells seen in the MRD setting and the well-known instability of CD138 expression. This effect can cause a residual plasma cell clone to be missed, which can affect ongoing treatment and have major implications on the prognosis of these patients.
There are several strategies that can be utilized to overcome this challenge, including (1) utilizing CD38 antibodies that target several CD38 epitopes (meCD38) [30]; (2) utilizing other markers to replace CD38 in viewing plasma cells, such as CD54, CD150, CD200, CD229, and CD319; (3) utilizing intracytoplasmic VS38 (VS38 targets the rough endoplasmic reticulum cytoskeleton-linking membrane protein P63 and is strongly expressed by plasma cells) [31]; and (4) utilizing an alternative gating strategy utilizing aberrantly expressed marker(s) (such as CD56 or in comparison with previous testing) or light chain restricted expression (if present), among others. In our institute, successful MRD testing is based on alternate markers like VS38C/meCD38, along with side scatter properties, CD45, CD19, CD56, and immunoglobulin light chain expression (Figure 3).
Masking of CD38 by Daratumumab causes near absence of CD38 bright events on flow cytometry; however, there is positive immunohistochemical (IHC) staining for CD38 on the residual plasma cells in a bone marrow biopsy. The reason for the labeling discrepancy between flow cytometry and IHC is unknown. The different antibodies in the assays may target different epitopes; alternatively, tissue fixation/decalcification may dissociate the anti-CD38 therapeutic monoclonal antibody from its target.
VS38 recognizes cytoskeleton-linking membrane protein 63 (CLIMP-63) on the rough endoplasmic reticulum, and this protein may be expressed in secretory cells, with bright expression on normal and aberrant plasma cells. Mizuta and colleagues evaluated VS38c in 15 patients with plasma cell myeloma and showed that this antibody enabled the detection of plasma cells in all cases, including in 3 patients who had received Daratumumab [31]. Courville and colleagues evaluated VS38c in 38 patients with plasma cell myeloma; they showed that the percentage of multiple myeloma cells calculated using VS38c and standard panels (including CD38, clone HB7) did not differ, but that identification and quantitation using the VS38c antibody was easier [29].
The CD38 Fluorescein isothiocyanate (FITC) multi-epitope antibody is designed for flow cytometry use for the identification and enumeration of human CD38 antigen-expressing cells even when cells are treated with a monoclonal antibody against CD38. This multi-epitope antibody is manufactured by Cytognos (Salamanca, Spain) [32]. This allows testing for minimal residual disease in patients being treated with Daratumumab. In a study by Broijl et al. on 29 bone marrow samples, the authors concluded that both meCD38 and VS38c allow reliable MRD detection in plasma cell myeloma patients, but the high expression of VS38c allows easier identification of plasma cell myeloma cells, especially in Daratumumab-treated patients [33].
This finding is similar to ours, with concordant results between VS38C and meCD38; however, VS38C allowed easier detection due to brighter expression on the residual plasma cell clone. Both VS38C and meCD38 are cost-effective markers that can be validated for reflex testing in Daratumumab-treated samples. Our institute successfully uses both antibodies. However, due to its brighter expression and stability, we prefer VS38C over meCD38 for reflex MRD testing with successful identification of clones up to 0.01% sensitivity.

4.3. Daratumumab and the Dilemma of Interference in Flow Cytometry in the Era of MRD Testing

4.3.1. Artefactual Kappa Restriction of Hematogones

We observed a uniform interference on flow cytometry in all patients treated with Daratumumab. A dim kappa restriction of early-stage (I/II) CD38 (strong +) hematogones was noticed in these patients. This IgG kappa monoclonal antibody binds and coats hematogones which have high CD38 surface expression. This causes the false appearance of a kappa monotypic CD10+ B-cell population (Figure 1) [34].
Hematogones were first described in 1937 by Peter Vogel. Hematogones are bone marrow precursors which can be seen in the peripheral blood of neonates and in adult bone marrow. They can also be observed in patients recovering from bone marrow transplantation or chemotherapy. An increased number of hematogones can be seen in a variety of benign and malignant conditions, including autoimmune conditions, idiopathic thrombocytopenic purpura (ITP), and certain viral infections. Hematogones are markedly decreased in patients with myelodysplastic neoplasm/syndrome and severe aplastic anemia [35,36].
The positive prognostic significance of increased hematogones has been studied in several disorders including leukemia, autoimmune disorders, and regenerating marrow [37]. There are few studies on hematogones in plasma cell myeloma. However, plasma cell myeloma patients with increased hematogones post-transplant were shown to have a worse overall survival and progression-free survival in a study by Santigo V. et al. [38].
Three distinct stages of hematogones have been described with characteristic immunophenotypic expression patterns (Figure 4). Stage I hematogones are the most immature and express the following markers: terminal deoxynucleotidyl transferase (TdT), CD34, CD10, CD38, CD45 (dim), and CD19 (slightly dim). In contrast, Stage II hematogones exhibit a loss of TdT and CD34, but retain CD38. They also demonstrate slightly brighter CD45 and CD19, along with dimmer CD10 compared to Stage I hematogones. Additionally, stage II hematogones gradually acquire CD20, showing increased intensity as they mature. The most mature hematogones, namely, those in stage III, have the same intensity of CD20 expression as the mature B cells and progressively decreasing dim CD10 expression. Hematogones share immunophenotypic and morphological features with B-lymphoblasts, requiring multiparametric flow cytometry to distinguish minimal residual disease from response [39]. The masking of CD38 antigen or the destruction/loss of antigen makes identifying specific immunophenotypic aberrancies cumbersome and makes the distinction of hematogones from B-lymphoblasts difficult. It is important to identify Daratumumab-related artefacts in hematogones to prevent misinterpreting them as neoplastic B-lymphoblasts. This is especially crucial for patients who suffer from both plasma cell neoplasm and B-lymphoblastic leukemia/lymphoma (B-ALL), as well as for myeloma patients treated with lenalidomide, who are at risk of developing secondary B-ALL [40].

4.3.2. Immunophenotypic Variation Progenitor Cells/Blasts and Myelomonocytic Cells

All cells that express CD38 may exhibit artefactually altered expression patterns when bound by Daratumumab. We observed a decrease in CD38 staining on progenitor cells/myeloblasts in cases after Daratumumab treatment, resulting in an atypical CD34/CD38 expression pattern (Figure 5). This unusual staining pattern could be misinterpreted as indicative of myelodysplastic neoplasms or secondary myeloid neoplasms in these patients. These cells exhibited positive expression of meCD38, confirming that the loss of demonstrable CD38 was an artefact rather than a true aberration. While most studies have documented lower levels of circulating CD34+ progenitor cells in patients treated with anti-CD38 therapy, collection targets for stem cell transplants were eventually achieved in similar proportions regardless of anti-CD38 treatment. This suggests that while Daratumumab may interfere with the mobilization of progenitor cells for autologous stem cell transplantation, it does not appear to affect overall progenitor cell counts or functionality of these progenitor cells [41]. Our study also observed a decreased staining of CD38 on granulocytes and monocytes (Figure 6). This immunophenotypic variation could similarly be misinterpreted as supportive evidence for the misdiagnosis of myelodysplastic syndrome or a secondary myeloid neoplasm.

4.3.3. Immunophenotypic Variation in Immune Cells and Effects on Immunity

The ability of Daratumumab to target CD38-expressing cells has prompted an evaluation of Daratumumab’s effects on other CD38+ cells. Krejcik et al. reported a marked reduction in CD38 expression on normal CD38+ white blood cell subsets, including NK cells, B cells, T cells, and monocytes, in multiple myeloma patients receiving Daratumumab therapy, as similarly seen in our study. Importantly, the CD38 reduction on tumor cells and immune cells occurs within hours after the initiation of the Daratumumab infusion and is present during the whole period of treatment as well as several months after Daratumumab has been stopped. CD38 levels were restored to baseline levels within six months of the last Daratumumab infusion [14]. These findings are corroborated by our study with reflex testing to VS38C and/or meCD38 applied to samples with a history of Daratumumab up to 6 months prior.
In addition to attenuating CD38 expression, Daratumumab has also been shown to affect total immune cell counts and function. Several studies have reported decreased NK cell counts following Daratumumab therapy [42,43,44]. Adams et al. reported that the remaining NK cells showed increased CD69 and CD127 levels; decreased CD45RA levels and trends for increased CD25, CD27 and CD137 levels, and decreased granzyme B levels. Additionally, they noted that after 2 months of Daratumumab, the T-cell population shifted towards a CD8+ prevalence, with an increase in granzyme B positivity. Finally, a reduction in CD38+ basophils in patients receiving Daratumumab monotherapy was observed [42]. Stocker et al. also reported that Daratumumab induced a strong depletion of both plasmacytoid dendritic cells and NK cells, with the absolute monocyte count remaining unchanged [44]. In our study, we observed a diminished CD38 expression on basophils and plasmacytoid dendritic cells (Figure 7). In another study by Krejcik et al., the authors observed that CD19+ B-cell counts did not change significantly with Daratumumab treatment [43]. Using in vitro B-cell differentiation assays, Verhoeven et al. determined that while Daratumumab did not change the overall percentage of B cells, it induced a dose-dependent decrease in B-cell proliferation and in vitro B-cell differentiation [45]. A recent study by Chen et al. demonstrated the kappa light chain restriction of CD19+ B cells and CD3+ T cells in patients treated with Alemtuzumab [46]. We observed a similar effect on activated T and B cells, which showed a decreased expression of CD38 post-Daratumumab. (Figure 8).
This study highlights our current reflex flow cytometry panels using VS38C and multi-epitope CD38 with great success in post-Daratumumab samples. In the modern era of targeted medicine, it is important to understand the effects of monoclonal antibodies in laboratory testing to arrive at an accurate diagnosis.

5. Conclusions

Daratumumab has become an important drug that plays a pivotal role in the management of not only resistant/refractory plasma cell myeloma, but also as a first-line therapeutic agent for improving progression-free survival. Non-invasive monoclonal antibody treatments and their lower cost compared to Chimeric Antigen Receptor (CAR) T-cell therapy make them a first-line option for many cases of plasma cell neoplasms. With its widespread use, Daratumumab treatment has brought with it several interferences in various branches of laboratory testing. It is important to be aware of these interferences and accurately follow up with patients, as the effect of the drug in circulation can last for a long period of time, even after its discontinuation. The interference of Daratumumab in serum protein electrophoresis and blood bank testing has become well-established. This study highlights Daratumumab’s interference in flow cytometry, which causes the artefactual light chain restriction of B-cell precursors and significant immunophenotypic variation in different hematolymphoid cells, including progenitor cells/blasts and various immune cells. Recent studies have shown that other monoclonal antibody drugs can also cause the artefactual light chain restriction of T- and B-cell populations. The light chain pseudo-restriction of hematogones and other cell populations is also possible following lymphoproliferative disorders.
Thus, monoclonal antibody drugs that are IgG kappa or IgG lambda may coat cells and cause this interference. A pseudo-kappa light chain restricted B-precursor cell population may signal workup for a B-cell monoclonal disease/lymphoma. A history of monoclonal antibody therapy and the awareness of this interference will avoid misinterpreting results and unnecessary testing. The advent of meCD38 and VS38c markers further improves MRD testing in post-Daratumumab samples and confirms the interference on several hematopoietic cells.
Although our study highlights important gating strategies and artefacts in samples with monoclonal antibody drug therapy, the limitations of this study include its single-center analysis, small representative sample size with reproducible findings, antibody panels designed and validated based on institutional needs, and the lack of plasma cell-enriched flow cytometry for MRD testing.
This important study highlights the efficacy of flow cytometric MRD testing in plasma cell myeloma patients who have been treated with Daratumumab, utilizing alternative markers and gating strategies. Additionally, we provided a thorough discussion on the various interferences that daratumumab can have on different hematopoietic cells, some of which have not been previously documented. Recognizing these artefacts is crucial for preventing misdiagnosis of secondary malignancies. Future studies highlighting gating strategies with other emerging monoclonal antibodies will help guide adequate reflex tubes to allow for precise MRD testing.

Author Contributions

Design, writing and editing: S.K.G.; Writing and editing: C.W.K.; Design, writing, review and editing: F.F.; Review and editing: W.C. All authors have read and agreed to the published version of the manuscript.

Funding

This study received no external funding.

Institutional Review Board Statement

This study was conducted in accordance with the declaration of Helsinki and approved by the Institutional Research Ethics Board (IRB no-122013-023, approved 20 July 2018). De-identified samples and data were used for this study.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The datasets analyzed during the current study are not publicly available but are available from the corresponding author upon reasonable request.

Acknowledgments

We acknowledge the department of Hematology & Oncology and the Multiple Myeloma group at the University of Texas Southwestern.

Conflicts of Interest

S.K.G., W.C., F.F. and C.K. declare no conflicts of interest.

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Figure 1. Polyclonal lg light chain preparation.
Figure 1. Polyclonal lg light chain preparation.
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Figure 2. Fluorescence-Minus-One using Polyclonal lg light chain preparation. Hematogones are labelled blue and B-lymphocytes are labelled black.
Figure 2. Fluorescence-Minus-One using Polyclonal lg light chain preparation. Hematogones are labelled blue and B-lymphocytes are labelled black.
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Figure 3. MRD testing in post-Daratumumab multiple myeloma. Malignant Plasma cells are colored red. Normal, polytypic plasma cells are colored black. Mature B-lymphocytes are colored blue. The positive staining for light chains on the B-lymphocytes on the cytoplasmic plot is likely due to surface membrane staining.
Figure 3. MRD testing in post-Daratumumab multiple myeloma. Malignant Plasma cells are colored red. Normal, polytypic plasma cells are colored black. Mature B-lymphocytes are colored blue. The positive staining for light chains on the B-lymphocytes on the cytoplasmic plot is likely due to surface membrane staining.
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Figure 4. This figure illustrates the stages of maturation for B-lineage cells in the bone marrow. Hematogones are depicted in blue, with the corresponding numbers indicating the various stages of maturation (1,2,3). Mature B-lymphocytes are represented in red.
Figure 4. This figure illustrates the stages of maturation for B-lineage cells in the bone marrow. Hematogones are depicted in blue, with the corresponding numbers indicating the various stages of maturation (1,2,3). Mature B-lymphocytes are represented in red.
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Figure 5. Daratumumab effect on progenitor cells/blasts (colored black) with an atypical CD34/CD38 expression pattern and dim kappa light chain restriction.
Figure 5. Daratumumab effect on progenitor cells/blasts (colored black) with an atypical CD34/CD38 expression pattern and dim kappa light chain restriction.
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Figure 6. Daratumumab binds to monocytes (purple) and granulocytes (green), causing decreased expression of CD38 and variation in immunophenotypic patterns.
Figure 6. Daratumumab binds to monocytes (purple) and granulocytes (green), causing decreased expression of CD38 and variation in immunophenotypic patterns.
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Figure 7. Dara effect on Basophils and PDCS. Plasmacytoid dendritic cells are colored red, and basophils are colored blue.
Figure 7. Dara effect on Basophils and PDCS. Plasmacytoid dendritic cells are colored red, and basophils are colored blue.
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Figure 8. Dara effect on B cells, T and NK cells: B-lymphocytes are colored blue, T-lymphocytes green, and NK-lymphocytes black.
Figure 8. Dara effect on B cells, T and NK cells: B-lymphocytes are colored blue, T-lymphocytes green, and NK-lymphocytes black.
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Table 1. Antibody and fluorochrome panels used to test for multiple myeloma.
Table 1. Antibody and fluorochrome panels used to test for multiple myeloma.
TubeFITCPEPerCp5.5PE-Cy7APCAPC-R700APC-H7BV421V500cBV605
#1CD2CD3CD5CD56CD4CD64CD8CD14CD45CD7Buffer
#2mLCD22CD20CD19CD34CD45 AF700CD38CD10CD5 BV480mKBuffer
#3ICKICL CD56CD38 CD45CD19
#4ICVS38ICLCD38CD56ICK CD19CD45
#5meCD38ICLCD38CD56ICK CD138CD45CD19
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MDPI and ACS Style

Germans, S.K.; Kahlow, C.W.; Chen, W.; Fuda, F. Effects of Daratumumab (Anti-CD38) Monoclonal Antibody Therapy on Flow Cytometry Analysis in Multiple Myeloma. Therapeutics 2025, 2, 2. https://doi.org/10.3390/therapeutics2010002

AMA Style

Germans SK, Kahlow CW, Chen W, Fuda F. Effects of Daratumumab (Anti-CD38) Monoclonal Antibody Therapy on Flow Cytometry Analysis in Multiple Myeloma. Therapeutics. 2025; 2(1):2. https://doi.org/10.3390/therapeutics2010002

Chicago/Turabian Style

Germans, Sharon Koorse, Christine Wamsley Kahlow, Weina Chen, and Franklin Fuda. 2025. "Effects of Daratumumab (Anti-CD38) Monoclonal Antibody Therapy on Flow Cytometry Analysis in Multiple Myeloma" Therapeutics 2, no. 1: 2. https://doi.org/10.3390/therapeutics2010002

APA Style

Germans, S. K., Kahlow, C. W., Chen, W., & Fuda, F. (2025). Effects of Daratumumab (Anti-CD38) Monoclonal Antibody Therapy on Flow Cytometry Analysis in Multiple Myeloma. Therapeutics, 2(1), 2. https://doi.org/10.3390/therapeutics2010002

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