Technical, gating and interpretation recommendations for the partitioning of circulating monocyte subsets assessed by flow cytometry.

The monocyte subset partitioning by flow cytometry, known as "monocyte assay," is now integrated into the new classifications as a supporting criterion for CMML diagnosis, if a relative accumulation of classical monocytes above 94% of total circulating monocytes is observed. Here we provide clinical flow cytometry laboratories with technical support adapted for the most commonly used cytometers. Step-by-step explanations of the gating strategy developed on whole peripheral blood are presented while underlining the most common difficulties. In a second part, interpretation recommendations of circulating monocyte partitioning from the dedicated French working group "CytHem-LMMC" are shared as well as the main pitfalls, including false positive and false negative cases. The particular flow-defined inflammatory profile is described and the usefulness of the nonclassical monocyte specific marker, namely slan, highlighted. Examples of reporting to the physician with frequent situations encountered when using the monocyte assay are also presented.

Metrological traceability in flow cytometry? Evaluation of a new volumetric method for lymphocyte subsets.

INTRODUCTION: Lymphocyte subset enumeration by flow cytometry is important for the therapeutic monitoring of a range of conditions. However, current bead-based methodologies do not produce metrologically traceable results. Here we compare an established bead-based methodology with a volumetric-based system traceable to an internationally recognised reference method. METHOD: A total of 118 samples received for lymphocyte subset analysis were tested using an established bead-based technique (BD Multitest™ 6-colour TBNK assay using Trucount™ tubes on a BD FACSLyric flow cytometer), followed by a volumetric method on the Sysmex XF-1600 flow cytometer using Exbio Kombitest 6-colour TBNK reagent. All samples were tested in accordance with the manufacturer's instructions. RESULTS: Absolute count values from both methodologies for CD3+, CD3 + CD4+, CD3 + CD8+, CD19+ and CD3-CD16+/CD56+ lymphocyte populations were compared using linear regression (R2 for all parameters >0.95) and Bland-Altman analysis. There was no significant bias (where p < 0.05) for absolute CD3 + CD4+ lymphocytes in the defined therapeutic range of 0-250 cells/µL (mean bias: 0.27 cells/µL). Although positive biases were seen for CD3 + CD4+ lymphocytes (over the entire range tested: 14-1798 cells/µL) and CD3-CD16+/CD56+ lymphocytes (mean bias: 10.83 cells/µL and 6.79 cells/µL, respectively). Negative biases were seen for CD3 + CD8+ and CD19+ lymphocytes (mean bias: -29.17 cells/µL and - 18.76 cells/µL, respectively). CONCLUSION: A high degree of correlation was found for results from both methodologies and observed bias was within the limits of clinical acceptability for all populations. This shows that the metrologically traceable lymphocyte subset absolute counts produced by the Sysmex XF-1600 are robust within clinically required limits.

Mimics and artefacts of measurable residual disease in a highly sensitive multicolour flow cytometry assay for B-lymphoblastic leukaemia/lymphoma: critical consideration for analysis of measurable residual disease.

High-sensitivity multicolour flow cytometry (MFC)-based B-lymphoblastic leukaemia (B-ALL) measurable residual disease (BMRD) assay is increasingly being used in clinical practice. Herein, we describe six consistently present low-level populations immunophenotypically mimicking abnormal B-ALL blasts in 441 BMRD samples from 301 children. These included CD19+ CD123+ plasmacytoid dendritic cells differentiating from lymphoid precursors, CD10+ transitional B cells with CD10+ /CD38dim-to-negative/CD20bright/CD45bright phenotype, CD19+ natural killer (NK) cells, CD73bright/CD10+ mesenchymal stromal/stem cells, CD73bright/CD34+ endothelial cells, and a CD34+ CD38dim-to-negative/CD10- /CD20bright/CD45bright subset of mature B cells. We provide the proportions, comprehensive immunophenotype, and practical clues for proper identification of these low-level populations. Knowledge regarding the presence and immunophenotype of these mimics is essential for accurate interpretation in high-sensitivity MFC-BMRD analysis.

Evaluation of measurable residual disease in multiple myeloma by multiparametric flow cytometry: Current paradigm, guidelines, and future applications.

Multiple myeloma (MM) is a heterogeneous group of mature B-cell diseases that are typically characterized by the presence and accumulation of abnormal plasma cells (PCs), which results in the excess production of monoclonal immunoglobulin and/or light chain found in the serum and/or urine. Multiparametric flow cytometry (MFC) is an indispensable tool to supplement the diagnosis, classification and monitoring of the disease due to its high patient applicability, excellent sensitivity and encouraging results from various clinical trials. In this regard, minimal or, more appropriately, measurable residual disease (MRD) negativity by MFC has been recognized as a powerful predictor of favourable long-term outcomes. Before flow cytometry can be effectively implemented in the clinical setting for MM MRD testing, sample preparation, panel configuration, analysis and gating strategies must be optimized to ensure accurate results. This manuscript will discuss the current consensus guidelines for flow cytometric processing of samples and reporting of results for MM MRD testing. We also discuss alternative approaches to detect plasma cells in the presence of daratumumab treatment. Finally, there is a lack of information describing the subclonal distribution of myeloma cells based on their protein expression. The advent of high-dimensional analysis may assist in following the evolution of antigen expression patterns on abnormal plasma cells in patients with relapsed/refractory disease. This in turn can help identify clonal subtypes that are more aggressive for potential informed decision. An analysis using t-SNE to identify the emergence of PCs subclones by MFC, along with the analysis of their immunophenotypic profiles are presented as a future perspective.

Utility of FLAER and CD157 in a five-color single-tube high sensitivity assay, for diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH)-A standalone flow cytometry laboratory experience.

BACKGROUND: FLAER-based flow cytometry assay is considered the gold standard for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). CD157 is a recently reported marker for GPI-anchored protein found both on neutrophils and monocytes. This study highlights the robustness of FLAER and CD157 combination to identify PNH clones in a high sensitivity assay. Though rare, the data shown highlight the presence of CD157 negativity in few cases re-emphasizing the importance of FLAER for PNH diagnosis. METHODS: A single 5-color tube-FLAER Alexa488/ CD157PE/ CD15PECy5/ CD64PE-Cy7 & CD45APCH7-was used for a high sensitivity PNH assay. RESULTS: Of 364 cases, 59(16.2%) cases had PNH clone in both granulocytes and monocytes. PNH clone sizes ranged from 0.02% to 96.6% in granulocytes and 0.07% to 96.3% in monocytes based on their FLAER-negative, CD157-negative phenotype. Twenty-two of the 59 PNH cases (37.3%) had WBC clone size of <1%. In addition, there were 10 cases which showed absence of CD 157 expression on both granulocytes and monocytes but on FLAER staining showed normal staining patterns. Three of these ten cases also showed a PNH clone based on absence of FLAER expression on both granulocytes and monocytes. CONCLUSION: FLAER and CD157 is a robust combination for diagnosis of clinical and subclinical PNH. Absence of CD157 expression in normal WBCs, though rare, should be kept in mind and re-emphasizes the importance of FLAER for the high sensitivity PNH assay.

Standardised immunophenotypic analysis of myeloperoxidase in acute leukaemia.

Given its myeloid-restricted expression, myeloperoxidase (MPO) is typically used for lineage assignment (myeloid vs. lymphoid) during acute leukaemia (AL) diagnostics. In the present study, a robust flow cytometric definition for MPO positivity was established based on the standardised EuroFlow protocols, the standardised Acute Leukaemia Orientation Tube and 1734 multicentre AL cases (with confirmed assay stability). The best diagnostic performance was achieved by defining MPO positivity as ≥20% of the AL cells exceeding a lymphocyte-based threshold. The methodology employed should be applicable to any form of standardised flow cytometry.

Reliable measurements of extracellular vesicles by clinical flow cytometry.

Extracellular vesicles (EVs) are cell-derived particles with a phospholipid membrane present in all body fluids. Because EV properties change in health and disease, EVs have excellent potential to become biomarkers for diagnosis, prognosis, or monitoring of disease. The only technique capable of detecting, sizing, and phenotyping a million of EVs within minutes is (clinical) flow cytometry. A flow cytometer measures light scattering and fluorescence signals of single EVs. Although these signals contain valuable information about the presence and composition of EVs, the signals are expressed in arbitrary units, which make the comparison of measurement results impossible between instruments and laboratories. Additionally, unintended and undocumented variations in the source, preparation, and analysis of the sample lead to orders of magnitude variations in the measured EV concentrations. Here, we will explain the basics, challenges, and common misconceptions of EV flow cytometry. In addition, we provide an overview of recent standardization initiatives, which are a prerequisite for comparison of clinical data and thus for clinical biomarker exploration of EVs.

Validation of a hybrid approach to standardize immunophenotyping analysis in large population studies: The Health and Retirement Study.

Traditional manual gating strategies are often time-intensive, place a high burden on the analyzer, and are susceptible to bias between analyzers. Several automated gating methods have shown to exceed performance of manual gating for a limited number of cell subsets. However, many of the automated algorithms still require significant manual interventions or have yet to demonstrate their utility in large datasets. Therefore, we developed an approach that utilizes a previously published automated algorithm (OpenCyto framework) with a manually created hierarchically cell gating template implemented, along with a custom developed visualization software (FlowAnnotator) to rapidly and efficiently analyze immunophenotyping data in large population studies. This approach allows pre-defining populations that can be analyzed solely by automated analysis and incorporating manual refinement for smaller downstream populations. We validated this method with traditional manual gating strategies for 24 subsets of T cells, B cells, NK cells, monocytes and dendritic cells in 931 participants from the Health and Retirement Study (HRS). Our results show a high degree of correlation (r ≥ 0.80) for 18 (78%) of the 24 cell subsets. For the remaining subsets, the correlation was low (<0.80) primarily because of the low numbers of events recorded in these subsets. The mean difference in the absolute counts between the hybrid method and manual gating strategy of these cell subsets showed results that were very similar to the traditional manual gating method. We describe a practical method for standardization of immunophenotyping methods in large scale population studies that provides a rapid, accurate and reproducible alternative to labor intensive manual gating strategies.

FACSCanto II and LSRFortessa flow cytometer instruments can be synchronized utilizing single-fluorochrome-conjugated surface-dyed beads for standardized immunophenotyping.

BACKGROUND: Multiparameter flow cytometry is the preferred method to determine immunophenotypic features of cells present in a wide variety of sample types. Standardization is key to avoid inconsistencies and subjectivity of interpretations between clinical diagnostic laboratories. Among these standardization requirements, synchronization between different flow cytometer instruments is indispensable to obtain comparable results. This study aimed to investigate whether two widely used flow cytometers, the FACSCanto II and LSRFortessa, can be effectively synchronized utilizing calibration bead-based synchronization. METHOD: Two FACSCanto II and two LSRFortessa flow cytometers were synchronized with both multicolor hard-dyed and single-fluorochrome-conjugated surface-dyed beads according to the manufacturer's instructions. Cell staining was performed on five whole-blood samples obtained from healthy controls and were analyzed upon synchronization with the respective synchronization protocols. RESULTS: Comparability criteria (defined as <15% deviation from the reference instrument) were met with both bead sets when synchronizing different FACSCanto II or LSRFortessa instruments. However, we observed that the criteria could not be met when synchronizing FACSCanto II with LSRFortessa instruments with multicolor hard-dyed beads. By utilizing single-fluorochrome-conjugated surface-dyed beads to determine and adjust PMT voltages, the accepted comparability criteria were successfully met. The protocol has been validated using five different eight-parameter stained samples. CONCLUSION: We show that FACSCanto II and LSRFortessa instruments can effectively be synchronized using single-fluorochrome-conjugated surface-dyed beads in case deviation criteria cannot be met using multicolor hard-dyed beads. Synchronization with single-fluorochrome-conjugated surface-dyed beads results in decreased deviations between instruments, allowing comparability criteria to become stricter.

Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.

BACKGROUND: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. METHODS: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. RESULTS: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. CONCLUSION: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.

Refractory celiac disease type II: An atypical case highlighting limitations of the current classification system.

Refractory celiac disease (RCD) is a rare condition associated with high morbidity that develops in individuals with celiac disease. It is known to be biologically heterogeneous, and currently two types are recognized based on immunophenotypic and molecular features, type I (RCD I) and type II (RCD II). Differentiating between RCD I and RCD II is critical, as patients with RCD II have substantially worse outcomes and a high risk of developing enteropathy-associated T-cell lymphoma. However, the current RCD classification is limited in scope, and atypical presentations and immunophenotypes are not recognized at present. Herein, we describe a unique case of RCD II with atypical clinical (primarily neurologic manifestations and lack of significant gastrointestinal symptoms), histopathologic (no villous atrophy), immunophenotypic (virtual absence of cytoplasmic CD3 expression), and molecular features (absence of clonal TR rearrangement and identification of pathogenic STAT3 and KMT2D mutations). This case highlights limitations of the current RCD classification system and the utility of next generation sequencing (NGS) studies in the diagnostic workup of RCD. Future algorithms need to recognize extraintestinal manifestations and incorporate atypical histopathologic and immunophenotypic features, as well as results of NGS analysis for RCD II classification.

Multi-site reproducibility of a human immunophenotyping assay in whole blood and peripheral blood mononuclear cells preparations using CyTOF technology coupled with Maxpar Pathsetter, an automated data analysis system.

High-dimensional mass cytometry data potentially enable a comprehensive characterization of immune cells. In order to positively affect clinical trials and translational clinical research, this advanced technology needs to demonstrate a high reproducibility of results across multiple sites for both peripheral blood mononuclear cells (PBMC) and whole blood preparations. A dry 30-marker broad immunophenotyping panel and customized automated analysis software were recently engineered and are commercially available as the Fluidigm® Maxpar® Direct™ Immune Profiling Assay™. In this study, seven sites received whole blood and six sites received PBMC samples from single donors over a 2-week interval. Each site labeled replicate samples and acquired data on Helios™ instruments using an assay-specific acquisition template. All acquired sample files were then automatically analyzed by Maxpar Pathsetter™ software. A cleanup step eliminated debris, dead cells, aggregates, and normalization beads. The second step automatically enumerated 37 immune cell populations and performed label intensity assessments on all 30 markers. The inter-site reproducibility of the 37 quantified cell populations had consistent population frequencies, with an average %CV of 14.4% for whole blood and 17.7% for PBMC. The dry reagent coupled with automated data analysis is not only convenient but also provides a high degree of reproducibility within and among multiple test sites resulting in a comprehensive yet practical solution for deep immune phenotyping.

Methodological considerations for the high sensitivity detection of multiple myeloma measurable residual disease.

BACKGROUND: Recent advances in therapeutic interventions have dramatically improved complete response rates in patients with multiple myeloma (MM). The ability to identify residual myeloma cells (e.g., measurable residual disease [MRD]) can provide valuable information pertaining to patient's depth of response to therapy and risk of relapse. Multiparametric flow cytometry is an excellent technique to monitor MRD and has been demonstrated to correlate with patient outcome post-treatment. To achieve the high sensitivity (one abnormal cell in 105 -106 cells) required for MRD evaluation, millions of cells have to be acquired and conventional immunophenotyping protocols are unable to attain these numbers, indicating the needs for alternative flow cytometric staining procedures. A bulk, "Pre-lysis" method is the consensus approach for staining large number of cells, requires two red blood cell lysis steps, and can adversely affect epitope density. In this study, we tested the "Pooled-tube" and "Dextran Sedimentation" staining procedures and correlated them with the "Pre-lysis" method as potential alternative approaches. METHODS: A total of 22 bone marrow aspirates from patients with plasma cell (PC) dyscrasia were processed in parallel using the "Pre-lysis," "Pooled-tube," and "Dextran Sedimentation" techniques. Stain indices were calculated and compared to assess their impacts on staining performance for each antibody used in the consensus panel. The recovery of normal and abnormal PCs, mast cells, and B cell precursors was enumerated and compared after their counts were normalized using fluorescent beads. The limit of blank, limit of detection, and lower limit of quantification were established using serial dilution experiments. RESULTS: The staining performances of CD19 PECy7, CD27 BV510, CD81 APCH7, and CD138 BV421 were improved using the "Pooled-tube" method when compared to "Pre-lysis." "Pre-lysis" was better at resolving CD56 using clone C5.9 but our results demonstrated similar improvement can also be achieved by "Pooled-tube" when alternative CD56 PE clones were used. "Dextran sedimentation" yielded similar staining results when compared to "Pre-lysis" for all the markers analyzed. The "Pooled-tube" method, when normalized to "Pre-lysis," recovered higher numbers of total PCs (1.2 ± 0.2 times higher; p = .049), normal PCs (1.4 ± 0.26; p = .007), mast cells (1.46 ± 0.27; p = .003), and B cell precursors (1.42 ± 0.3; p = .011), but not abnormal PCs (1.09 ± 0.2; p = .352). There was no evidence that the recovery of cells was different between "Pre-lysis" versus "Dextran Sedimentation." All three flow cytometric assays achieved a minimum sensitivity of 10-5 and approached that of 10-6 for detecting rare events. CONCLUSION: Both "Pooled-tube" and "Dextran Sedimentation" staining procedures were comparable to the "Pre-lysis" method and are suitable high sensitivity flow cytometric approaches that can be used to process bone marrow samples for MM MRD testing.

CD71 improves delineation of PNH type III, PNH type II, and normal immature RBCS in patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. METHODS: We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). RESULTS: We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. CONCLUSIONS: Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

Applications and efficiency of flow cytometry for leukemia diagnostics.

Introduction: Multiparametric flow cytometry immunophenotype (MFCI) plays a crucial role in the diagnosis of acute leukemia (AL). Through the comprehensive assessment of surface and intracellular antigens expressed by blasts, MFCI permits to distinguish myeloid or B/T lymphoid AL, or AL of ambiguous lineages. By means of MFCI, the blasts can be characterized in bone marrow, peripheral blood, and body fluids, such as cerebrospinal fluid.Area covered: This review discusses how MFCI is currently applied in the diagnostic evaluation of AL; it also focuses on 'peculiar' issues such as the role of MFCI for the diagnosis of central nervous system leukemic involvement.Expert commentary: Despite the improved knowledge about the biology of AL, MFCI remains a fundamental tool to make a prompt and accurate diagnosis. MFCI also provides prognostic information for some antigens are associated with specific cytogenetic/genetic abnormalities and, recently, it became a powerful tool to evaluate the quality and depth of response (the so called 'measurable residual disease'). Its role as an efficient detector of residual disease paved the way to the investigation of tissues other than bone marrow and peripheral blood, demonstrating that even small amounts of AL appear to have a prognostic impact and may require personalized intervention.

Comments on EuroFlow standard operating procedures for instrument setup and compensation for BD FACS Canto II, Navios and BD FACS Lyric instruments.

This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.

Quality Control of Immunophenotyping.

Applications of immunophenotyping using flow cytometry offer precise and accurate means for providing information used to both diagnose and monitor disease; they serve as a standard platform for many research endeavors that study discrete populations of biological entities. The proper use of this highly sophisticated technology requires daily and ongoing monitoring of both the instrument and the methodology. Best practices for this begin with quality control (QC) procedures designed to set up and monitor the instrument performance, the reagents, and the results to ensure that they are working properly both on the day of use and over time. If the results of those QC procedures are outside of acceptable then recording the corrective action taken must also be included in the quality control records. Quality assurance (QA) is a way to know that the three phases of testing, namely, preanalytic, analytic, and postanalytic procedures, are being followed. This chapter describes the procedures used to assess quality control as it pertains to flow cytometry and immunophenotyping in all three phases of testing.

A Modified Injector and Sample Acquisition Protocol Can Improve Data Quality and Reduce Inter-Instrument Variability of the Helios Mass Cytometer.

Mass cytometry is a powerful tool for high-dimensional single cell characterization. Since the introduction of the first commercial CyTOF mass cytometer by DVS Sciences in 2009, mass cytometry technology has matured and become more widely utilized, with sequential platform upgrades designed to address specific limitations and to expand the capabilities of the platform. Fluidigm's third-generation Helios mass cytometer introduced a number of upgrades over the previous CyTOF2. One of these new features is a modified narrow bore sample injector that generates smaller ion clouds, which is expected to improve sensitivity and throughput. However, following rigorous testing, we find that the narrow-bore sample injector may have unintended negative consequences on data quality and result in lower median and higher coefficients of variation in many antibody-associated signal intensities. We describe an alternative Helios acquisition protocol using a wider bore injector, which largely mitigates these data quality issues. We directly compare these two protocols in a multisite study of 10 Helios instruments across 7 institutions and show that the modified protocol improves data quality and reduces interinstrument variability. These findings highlight and address an important source of technical variability in mass cytometry experiments that is of particular relevance in the setting of multicenter studies. © 2019 International Society for Advancement of Cytometry.