Meeting Home > Abstracts

Conference Abstracts

Title: Gestational Bisphenol A Exposure May Shift B1a-B Cell Localization from the Peritoneal Cavity to the Bone Marrow Background: B1a-B cells are a distinct subset of innate-like B cells that predominantly originate during fetal development in the bone marrow. They migrate to the peritoneal and pleural cavities in adults, where they maintain relatively constant population size. B1a-B cell dysregulation and abnormal migrations have been linked to autoimmune diseases like Systemic Lupus Erythematosus and Rheumatoid Arthritis. Estrogen has been shown to directly alter the CXCR4/CXCL12 signaling axis, which plays a significant role in the migration and homing of hematopoietic stem cells, immune cells, and progenitor cells. Due to its structural similarity to 17ß-estradiol (E2), Bisphenol A (BPA) is classified as an endocrine disruptor capable of binding estrogen receptors and mimicking estrogen signaling. Objectives: To understand the effect gestational BPA and BPC exposure has on B1a population distribution.

Methods: As part of a broader investigation into the transcriptional and developmental effects of BPA and Bisphenol C (BPC) on B cells, pregnant BALB/c dams were exposed to BPA or BPC either orally or sub-q throughout their entire gestation period. Peritoneal lavage was performed on dams. Spleen, liver, bone marrow, and blood were removed from both dams and E18 pups.

Results: Preliminary flow cytometry data indicate B1a population in BPA dam bone marrow but not peritoneum.

Conclusions: BPA'sdisruption of B cell maturation checkpoints may contribute to a possible decrease in B1 peritoneal localization, causing retention in the adult bone marrow.

Background Flow cytometric immunophenotyping is routinely used for the evaluation of plasma cell dyscrasias. Conventional plasma cell identification relies on gating strategies incorporating CD38, CD138, and CD45. However, CD38 expression is variable in malignant plasma cells and is influenced by anti-CD38 targeted therapy. This study was to evaluate the feasibility of using cytoplasmic immunoglobulin light chain (cyILC)-based gating as an alternative to CD38-based gating for plasma cell identification. Methodology List-mode data from 52 bone marrow samples analysed using a 10-color flow cytometry panel were analysed. Plasma cells were identified using a conventional CD38/CD138/CD45-based approach and a modified strategy substituting CD38 with cytoplasmic kappa and lambda light chain expression. Total and aberrant plasma cell percentages were compared using the Wilcoxon signed rank test, Pearson correlation, Bland-Altman agreement analysis, and Lin's concordance correlation coefficient (CCC). Results Of the 52 samples analysed, 15 samples showed aberrant plasma cells by both methods. The median total plasma cell percentage was 0.52% by conventional gating and 0.49% by cyILC-based gating. Total plasma cell percentages identified by cyILC-based gating demonstrated excellent correlation with conventional CD38-based gating (r = 0.996, p<0.001) and near-perfect concordance (CCC = 0.979). cyILC-based gating yielded slightly lower total plasma cell percentages (Wilcoxon p= 0.0004). Bland-Altman analysis showed a mean bias of −0.25% in the cyILC method, with 95% limits of agreement from −2.24% to +1.75%. Aberrant plasma cell percentages showed near-perfect intermethod correlation (r = 0.999, p<0.001), no significant difference (p = 0.53), and excellent concordance (CCC = 0.984) between the methods. Bland-Altman analysis demonstrated a mean bias of −0.13% for the cyILC method, with 95% limits of agreement from −1.88% to +1.61%. Conclusion cyILC-based gating demonstrates excellent agreement with conventional CD38-based gating for both total and aberrant plasma cell enumeration, representing a practical, cost-neutral alternative for routine flow cytometric evaluation of plasma cells.

Background: Minimal residual disease (MRD) quantification in B-lineage acute lymphoblastic leukemia (B-ALL) is crucial for assessing treatment response. Frequently, forward-scatter (FSC) and side-scatter (SSC)-derived viable-singlet events are used as denominators in MRD calculations, but this approach is subjective. Syto-dyes, which label all nucleated cells, provide an objective alternative to enhance reproducibility.

Methods: We analysed 176 MRD-positive B-ALL samples to compare Syto13-based MRD% with those derived from FSC/SSC-defined viable singlets. Samples were categorized as <0.01%, 0.01%-<0.1%, 0.1%-<1%, and ≥1% MRD across both methods and compared. Pearson correlation, Bland-Altman analysis, and Lin's concordance correlation coefficient (CCC) were used to assess correlation, agreement, and interchangeability of MRD% between approaches.

Results: Overall, 13% (23 of 176) cases showed discordance between Syto13 and FSC/SSC-derived MRD categories. Notably, 15% (6 of 39) patients classified as <0.01% MRD by FSC/SSC-derived viable singlets were reclassified as >0.01% MRD by Syto-based assessment. Overall correlation between MRD% identified by Syto13 and FSC/SSC denominators was very strong (r=0.98, p<0.0001), but varied across categories (refer to Table-1 and Figure-1). At Syto13-based MRD categories of <0.01% and >1% (n=34&46), correlation was strong (r=0.892& 0.981, p<0.0001) along with substantial concordance (CCC=0.89&0.96), though these MRD-thresholds are of limited clinical relevance. In Syto13-based MRD of 0.01-0.1% category (n=51), correlation remained strong (r=0.674, p<0.0001) but limits of agreement (LoA) were wide (±0.03), poor concordance (CCC=0.66) and Syto13 consistently resulted in higher MRD% (+0.003 bias). Importantly, in the 0.01% (±0.005) MRD threshold (n=25), correlation was moderate (r=0.53, p<0.0001) with minimal bias (+0.00032), but with wider-LOA (+0.0076to-0.00070) and poor concordance (CCC=0.49), indicating marked disagreement between the methods in this crucial MRD threshold. Conclusion: Syto dye-based MRD yields higher values than FSC/SSC viable singlet-based MRD. Although correlations are strong, poor concordance indicates that the two methods cannot be used interchangeably for MRD% assessment.

Background: Staining preparation methods can influence an antibody's brightness (MFI) and staining index. The staining method previously used for the LGL tube for the BDFACS Canto II analyzers utilized a stain/lyse/wash/wash (SLW) method. Expression of the CD16 PerCP-Cy5-5 antibody on NK cells has historically appeared relatively dim compared to other antibodies. One sample showed dim to negative CD16 PerCP-Cy5-5 expression on the NK cells when a positive expression was expected. Using a different staining method of lyse/wash/stain/wash/wash (LSW), a strong positive CD16 PerCP-Cy5-5 expression was observed. Objectives: Determine if an alternative sample prep method will aid in detecting CD16 PerCPCy5-5 without negatively affecting other markers in the same assay.

Methods: A comparison experiment was done to determine if a change in staining methods was appropriate, which included running method comparisons on 5 patient samples. Acceptable changes in MFI for the other markers in the panel were ≤0.5log.

Results: The results showed a significant increase (an average of 0.7 log change) in the MFI value of CD16 PerCP-Cy5.5 when the sample was prepared using the lyse/wash/stain/wash/wash method. There was no significant change in MFI values of other antibodies in the panel. Conclusion: With the data from this study, we were able to change the SOP for this assay to implement the LSW staining method. There are other assays on the Canto II analyzer that still utilize the SLW staining method. Future projects include validating the LSW method on all pathology assays.

Background & Objectives The superRCA mutation assay is an ultra-sensitive molecular technology that utilizes flow cytometry for digital readout. Flow cytometry enables high-level multiplexing through detection of fluorescence emissions across multiple wavelengths. By leveraging fluorescence resonance energy transfer (FRET)-based tandem dye labeling, superRCA products can generate distinct fluorescence signatures, enabling simultaneous detection of multiple mutations in a single reaction while preserving assay sensitivity. Methods A multiplex panel targeting nine clinically relevant IDH variants was developed, including five IDH1 mutations (R132C, R132H, R132G, R132S, and R132L) and four IDH2 mutations (R140Q, R140L, R172K, and R172M). A corresponding multiplex genotyping probe set containing wild-type and mutation-specific probes was evaluated using single-positive reference samples. Fluorescent tandem dye labeling was applied to generate unique emission signatures for each target. Results Single-mutation probe sets generated 5×5 and 4×4 specificity matrices for IDH1 and IDH2, respectively, demonstrating highly specific detection with signals observed exclusively in mutation-positive samples. Multiplex analysis using the complete 9-plex probe set produced variant allele frequency (VAF) measurements consistent with singleplex assays. Tandem dye labeling enabled simultaneous detection of all nine IDH1/2 variants in a single reaction. Flow cytometry resolved 12 distinct populations, corresponding to three wild-type controls and nine mutant genotypes. Conclusions The tandem dye-labeled superRCA assay enables highly multiplexed detection of nine IDH1/2 variants from a single DNA sample without compromising sensitivity. This approach maximizes DNA utilization and provides a streamlined solution for mutation profiling and DNA-based MRD monitoring.

Background: Dried-down reagents are increasingly utilized in flow cytometry to reduce reagent handling variability, save time, and improve data reproducibility. The Cytek^® cFluor^® 14-Color Immunoprofiling Kit has been widely used to phenotype subsets of T, B, NK, and myeloid cells. Objectives:In this study, a dried-down 14-color cocktail was developed and evaluated for analytical performance, stability, and reproducibility on a spectral flow cytometry platform. Methods: 14 single-color control reagents and a 14-color cocktail were formulated and individually air-dried overnight. Accelerated stability of the dried single-color control reagents and cocktail was assessed at 37℃for 4 weeks. Peripheral blood samples from ten donors were stained in triplicate per sample with both liquid and dried-down reagents. The samples were acquired on a 5-laser Cytek Aurora™ system and analyzed with SpectroFlo^® software. The spectral signatures of each single-color reagent, cell population frequency, and median fluorescent intensity (MFI) for each marker were analyzed. Results: The spectral signatures of dried and liquid single-color reagents were identical. Comparable cell percentages, MFIs, and %CV from triplicates were observed across 10 donors using dried cocktails and liquid cocktails. Individual marker MFIs of the dried 14‑color cocktail remained stable after storage at 37 °C for four weeks. These results indicated that the dried-down cocktail showed equivalent performance to the liquid cocktail and high uniformity across replicate samples with excellent stability.

Conclusions: These results demonstrate that Cytek's dried-down 14-color cocktail is a robust and efficient tool that can simplify workflow and produce consistent results for high-dimensional immunophenotyping.

Introduction: CellMek SPS is an automated sample preparation system designed for IVD use in flow cytometry laboratories. Automation of routine and labor-intensive steps improves laboratory efficiency while allowing technologists to focus on analytical interpretation. The newly introduced Dry-Tube feature extends CellMek SPS functionality by enabling automated preparation using dry-down antibody, providing ready-to-use reagents with room-temperature stability and reduced hands-on time. This study evaluated the analytical performance of CellMek SPS Dry-Tube feature using a 10-color panel and compared results to manually prepared samples.

Methods:Peripheral whole blood from 40 donors was processed using a representative workflow consisting of washing, staining, lysing/fixing and post-lysis washing. Samples were prepared on CellMek SPS using a 10-color panel and were paired with matched-donor manually prepared using equivalent reagents. Flow cytometry acquisition was performed using Navios, and data were analyzed with Kaluza. Measurement procedure comparison and bias estimation analyses were performed on leukocyte subpopulations.

Results:For leukocyte-gated subpopulations exceeding 20%, total bias relative to manually prepared samples was within ±10%. For subpopulations at or below 20%, total bias was within ±5%. All evaluated subpopulations met these acceptance criteria, demonstrating strong agreement between CellMek SPS and manual methods across donors and instruments. Conclusion: CellMek SPS with Dry Tube functionality delivered sample preparation performance comparable to manual methods while reducing procedural variability associated with reagent handling and manual pipetting. The combination of automation, reagent stability, and consistent analytical results positions CellMek SPS as a reliable and efficient solution for standardized flow cytometry sample preparation in routine laboratory workflows.

Background Flow cytometry is routinely implemented in hematology laboratories for immunophenotypic MRD assessment. However, molecular MRD assays remain largely confined to specialized molecular laboratories due to their dependence on DNA purification and DNA quantification steps, which are typically not available in standard hematology lab workflows. This disconnect limits the adoption of highly sensitive mutation-based MRD assays in routine clinical practice. We present the superRCA assay, enabling direct use of blood samples and eliminating the need for DNA extraction and quantification, thereby bridging this operational gap. Methods Peripheral blood samples were used directly as assay input without DNA purification or quantification. Target sequences were amplified, circularized, and subjected to rolling circle amplification (RCA). Mutation-specific padlock probes enabled highly selective detection, followed by a second RCA step to generate superRCA products. Results The superRCA assay demonstrated ultra-high sensitivity, detecting mutations at frequencies as low as 1 in 10,000, even when starting directly from blood samples. In AML patient monitoring, mutation signals were reliably detected in peripheral blood, enabling identification of residual disease and early relapse. The removal of DNA purification and quantification steps significantly simplified the workflow, reduced turnaround time, and enabled seamless integration into existing hematology lab infrastructure. Conclusions superRCA uniquely enables molecular MRD testing within the hematology lab environment, where flow cytometry is already established but molecular preprocessing steps are absent. By eliminating DNA purification and quantification, the assay lowers operational barriers and supports broader clinical adoption of ultra-sensitive mutation-based MRD monitoring directly from blood samples.

Background Sensitive monitoring of CAR-T persistence is critical for next-generation allogeneic CAR-T therapies, where low-level engineered cells must be tracked to assess engraftment, expansion, persistence, and relapse risk. Current qPCR and ddPCR methods have analytical sensitivity limitations for rare-event detection. Conventional qPCR assays typically detect ~50 CAR copies from 400 ng DNA input, corresponding to ~0.04% (4 in 10,000 cells). ddPCR improves quantification precision but is limited to ~120 ng DNA input per reaction, restricting both sensitivity and throughput. Methods A superRCA assay targeting the FMC63 CAR construct was developed for genomic DNA analysis. Target-specific PCR products were amplified by rolling-circle amplification (RCA). FMC63-specific padlock probes interrogated repeated target sequences, followed by a second RCA step to generate superRCA particles for quantification by flow cytometry. Result superRCA assay detected the FMC63 CAR sequence down to 2 CAR copies in 200,000 GAPDH reference molecules (~0.001%). Assay background noise was approximately 1-2 false-positive events per million GAPDH reference molecules, indicating that assay chemistry is not the limiting factor for sensitivity. Increased DNA input is expected to improve sensitivity by at least 10-fold. Compared with qPCR and ddPCR, superRCA achieved substantially improved sensitivity through high DNA input tolerance and digital particle enumeration without droplet partitioning constraints. Conclusions The superRCA FMC63 CAR assay enables ultra-sensitive CAR-T monitoring with sensitivity substantially exceeding current qPCR and ddPCR approaches. Its ultra-low background and scalable DNA input make it particularly valuable for monitoring long-term persistence of allogeneic CAR-T therapies and other low-abundance engineered cell populations.

fdsa

Background: The primary aim of this work was to develop a classification model that integrates with FlowSOM clustering outputs for standardised B-cell and PC identification in research flow cytometry datasets.

Methods: Bone marrow aspirates were stained with standard BD OneFlow™ PC screening tube and acquired on a BD FACSLyric™ flow cytometer. FCS files from 40 samples (training dataset) were exported to CellEngine cytometry software and underwent FlowSOM clustering. Cluster-level MFIs were exported to an Excel "PC Trainer Classifier" that (i) normalises MFIs to an in-sample B-cell anchor, (ii) computes a PCscore (CD138+CD38+), plus secondary features (CD19↓, CD45↓, CD56↑), (iii) applies a forced core fallback (highest CD138/CD38 score) when strict criteria yield no PCs, and (iv) derives a NEOscore for neoplastic phenotype. The rule-based classifier was validated on a set of 52 samples, independent of the training dataset. Elements of the Excel formula design were co-developed with ChatGPT (OpenAI).

Results: Across validation cases, B-cell detection achieved: Sensitivity 0.902 (0.79 - 0.96), Specificity 0.984 (0.96 - 0.99), Precision 0.885 (0.77 - 0.94), Accuracy 0.973 (0.95 - 0.99) and F1 0.893. PC identification achieved: Sensitivity 0.651 (0.54 - 0.75), Specificity 0.828 (0.79 - 0.86), Precision 0.458 (0.37 - 0.55), Accuracy 0.796 (0.76 - 0.83) and F1 0.537.

Conclusions: A transparent Excel-based classifier integrated with FlowSOM enables reproducible B-cell identification, provides a structured approach to PC classification and will improve inter-case harmonisation for research flow cytometry datasets. The PC classification component is best interpreted as an augmented-intelligence tool intended to support expert assessment. Further optimisation may refine PC classification performance.

Presentation #1, Panel Design:

This presentation will discuss the Principles of Flow Cytometry Panel Design with the following objectives:

• Review the steps to introduce a new or reconfigured multi-color flow cytometry panel
• Review the Predevelopment Questions and Steps
• Review the components of panel construction
• List and review the steps to take prior to launching your new panel
• Put proof of the design into practice

Presentation #2 Fetal RBC Detection and Enumeration by Flow Cytometry:

This presentation will be an introduction to the application of Using Flow Cytometry for the Detection and Enumeration of Fetal Red Blood Cells in Maternal Circulation. It will include a description of the assay, the procedure for setting it up and analyzing the results including how to interprete the results for determination of anti-Rh Immunoglobulin dosage.

Plasmablastic lymphoma (PBL) is a rare, aggressive lymphoid neoplasm that is often immunodeficiency-associated. The diagnosis of PBL is challenging given its plasma cell immunophenotype, thus flow cytometry is critical. Here, we discuss the diagnosis of PBL in two immunocompetent males. First, a 64-year-old-male presented with a sinonasal mass which revealed large, round atypical cells with minimal cytoplasm and prominent nucleoli. Flow cytometry revealed larger cells expressing CD38 and CD56, without significant hematolymphoid marker expression. Subsequent flow cytometry revealed CD138 and cytoplasmic lambda expression, with dim CD45 and aberrant CD33. To corroborate, immunohistochemical (IHC) staining demonstrated MYC expression with IgM lambda restriction in tumor cells; EBV encoded RNA (EBER) in situ hybridization was positive. In this instance, classic morphology, flow immunophenotype, and IHC expression were diagnostic for PBL. Comparatively, an 80-year-old male presented similarly with a sinonasal mass, however, pathology reported a high-grade malignant neoplasm with primitive, small round blue cells; an atypical morphology for PBL. Flow cytometry detected an abnormal CD45-/CD20-/CD56+ population with increased size (54%) that expressed plasma cell markers (CD38 and CD138), without light chain expression. Upon staining, EBER in situ hybridization was negative and the tumor cells lacked kappa/lambda expression. Notably, the tumor cells showed diffuse MUM-1 and CD138 positivity, variable C-MYC and near-100% Ki67 proliferative index, supporting the diagnosis of PBL. As each case demonstrates, flow cytometry is instrumental in the diagnosis of PBL and it cannot be excluded in the immunocompetent population.

Background: Platelet P-selectin (CD62P) is the gold-standard flow cytometric marker for platelet activation, but rapid ex vivo activation post-phlebotomy restricts its clinical utility. Current guidelines require processing within 4 to 6 hours, hindering decentralized trials and centralized testing. Existing stabilizing reagents require manual pipetting, cold chains, or cause sample dilution. We evaluated a novel, vacuum-extractable Platelet Stabilization Tube designed to streamline pre-analytical workflows. Objectives: To evaluate a pre-filled vacuum stabilization tube for preserving baseline and agonist-induced CD62P expression in whole blood over 7 days under variable temperatures.

Methods: Whole blood from 10 healthy donors was collected into 3.2 percent sodium citrate and allocated into four groups: citrate control (resting), stabilization tube (resting), adenosine diphosphate (ADP)-activated control, and ADP-activated stabilization tube. Samples were stored at 25 or 4 degrees Celsius. Platelet CD62P expression was quantified via flow cytometry at baseline, 6 hours, and days 3, 5, and 7.

Results: In citrate controls, resting CD62P expression artifactually rose from 4.8 percent to 65.1 percent (4 degrees) and 41.4 percent (25 degrees) by day 7. Conversely, the stabilization tube maintained resting CD62P at 1.2 to 1.5 percent across all conditions (P > 0.05). For ADP-activated samples, the stabilization tube maintained CD62P expression at 70.9 to 71.9 percent for 7 days (P > 0.05).

Conclusions: The novel stabilization tube preserves resting and activated platelet phenotypes for up to 7 days independent of a cold chain, resolving critical pre-analytical barriers for multicenter clinical networks.

Background superRCA assay has demonstrated ultra-sensitive mutation detection from purified DNA using standard flow cytometers. However, conventional RNA analysis requires cell lysis, RNA purification, reverse transcription, and PCR amplification, resulting in complex workflows and loss of single-cell information. We developed a next-generation superRCA assay for direct detection of individual RNA molecules within intact cells, enabling molecular analysis by flow cytometry while preserving cellular context. Methods Intact cells were fixed and permeabilized before hybridization with RNA-specific probes. Target RNA molecules were converted into circular DNA templates and amplified by rolling circle amplification (RCA). A second RCA step generated fluorescent superRCA products that remained localized within individual cells. Fluorescent signals were quantified on a standard flow cytometer without RNA purification, reverse transcription, or PCR amplification. Results The assay was evaluated using the RUNX1::ETO fusion transcript in the RUNX1::ETO-positive Kasumi-1 cell line, with MOLM-14 cells serving as a negative control. Bright superRCA products enabled clear discrimination between positive and negative cells with minimal background. Individual RNA molecules were detected while preserving single-cell resolution. The assay was compatible with conventional flow cytometry workflows and can be combined with immunophenotyping for simultaneous RNA and protein analysis. Conclusions superRCA RNA assay extends the superRCA platform from purified DNA mutation analysis to single-RNA molecule detection in intact cells. Detection of the clinically relevant RUNX1::ETO fusion transcript demonstrates the potential for molecular diagnosis and MRD monitoring in Acute Myeloid Leukemia. This technology transforms standard flow cytometers into integrated platforms for simultaneous protein and RNA analysis at single-cell resolution.

Course welcome presented by Val and Mike

We will review inspection ready goals, discuss areas of focus for inspections and how to be inspection ready.

A collection of absolutely classic cases of mature B/T-LPDs, ALLs, and AMLs, and nomrals. Live demonstration at the front of the class.

Course wrap up presented by Val and Mike

Spectral flow cytometry is increasingly being adopted in clinical laboratories due to its expanded multiparametric capabilities. However, successful implementation requires careful consideration of instrument selection, validation strategies, panel design, quality management, and long-term standardization. Through case-based discussion and lessons learned from clinical implementation, attendees will gain practical tools to support the successful deployment and long-term operation of spectral flow cytometry in the clinical laboratory.

Introduction: The diagnosis of interstitial lung diseases (ILDs), including sarcoidosis and hypersensitivity pneumonitis (HP), remains challenging because of their overlapping clinical and radiological features. Both disorders are characterized by lymphocytic alveolitis and granulomatous inflammation, making their differential diagnosis difficult. This study evaluated the diagnostic value of bronchoalveolar lavage (BAL) immunophenotyping by flow cytometry in distinguishing these conditions. Materials and

Methods: We performed a retrospective descriptive study including 44 patients diagnosed with sarcoidosis (n=32) or HP (n=12). All patients underwent BAL as part of the diagnostic evaluation for suspected ILD. Paired BAL and peripheral blood samples were analyzed by flow cytometry (BD FACSCanto™ II). Cellular composition and lymphocyte subsets, including T cells, B cells, NK cells, and CD4⁺ and CD8⁺ T-cell populations, were assessed and correlated with the final diagnosis.

Results: BAL from patients with sarcoidosis showed 60.9% lymphocytes, 8.9% monocytes, and 30.1% polymorphonuclear cells. The mean CD4⁺/CD8⁺ ratio increased from 1.08 in peripheral blood to 5.34 in BAL, with 68.9% of patients presenting a BAL ratio >3.5. In HP, BAL contained 63.3% lymphocytes, 5.0% monocytes, and 31.7% polymorphonuclear cells. The mean CD4⁺/CD8⁺ ratio decreased from 2.08 in blood to 0.30 in BAL. Although both diseases exhibited lymphocytic alveolitis, they differed markedly in BAL T-cell subset distribution.

Conclusions: BAL immunophenotyping by flow cytometry provides valuable diagnostic information and may improve the differential diagnosis between sarcoidosis and HP, particularly through assessment of the BAL CD4⁺/CD8⁺ ratio.

CD123 (IL-3 receptor alpha) is highly expressed on leukemic blasts and leukemic stem cells in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and represents an important biomarker for disease characterization and therapeutic targeting. We validated a 12-color, 3-tube flow cytometry assay for quantitative assessment of CD123 expression in bone marrow aspirates. The assay incorporates a fluorescence-minus-one control and bead-based calibration to enable standardized quantification of CD123 using molecules of equivalent soluble fluorochrome (MESF). Analytical validation included assessment of precision, repeatability,reproducibility, and stability using AML cell lines and patient samples. The assay demonstrated robust identification of blast populations, with CD123 expression detected in the majority of blasts (>65-95% in high-burden samples) and minimal background signal (<2% in controls). Precision for primary populations met acceptance criteria, with most intra- and inter-assay coefficients of variation ≤25%, and inter-instrument/operator variability typically <10%. CD123 expression levels ranged approximately from 1,100 to 2,500 MESF across blast and stem cell populations. Increased variability was observed in rare populations (<5% of parent or <100 events), where %CV often exceeded 35%. Samples remained stable for key parameters up to 72-96 hours, with ≤20% deviation for major populations. Clinically, this assay enables standardized and reproducible quantification of CD123, supporting its use in patient stratification, pharmacodynamic monitoring, and evaluation of CD123-targeted therapies. Limitations in low-frequency populations should be considered when interpreting minimal residual disease or rare subset analyses.

Introduction: Acute leukemia (AL) is the most common pediatric malignancy worldwide and a leading cause of cancer-related mortality. In resource-limited settings, diagnosis often relies on morphology due to limited access to advanced diagnostic techniques. This study aimed to characterize acute leukemia among children below 15 years at Kilimanjaro Christian Medical Centre (KCMC), determine immunophenotypic subtypes, assess diagnostic concordance between morphology and flow cytometry, and evaluate two-year overall survival. Methods: A hospital-based analytical cross-sectional study was conducted among 118 children diagnosed with acute leukemia at KCMC between June 2025 and February 2026. Morphological assessment of peripheral blood and bone marrow smears was compared with flow cytometric immunophenotyping. Diagnostic concordance was assessed using Cohen's kappa statistic. Retrospective survival data were analyzed using Kaplan-Meier methods.

Results:Fever (99.2%), pallor (97.5%), and fatigue (97.5%) were the most common presenting features. Flow cytometry identified B-cell acute lymphoblastic leukemia (B-ALL) as the predominant subtype (52.5%), followed by acute myeloid leukemia (AML) (24.6%) and T-cell acute lymphoblastic leukemia (T-ALL) (20.3%). Morphology demonstrated high diagnostic accuracy (91.3%), with sensitivity of 94.2% and specificity of 82.8%, showing substantial agreement with flow cytometry (κ=0.769). The overall two-year survival rate was 66.9%. B-ALL had the most favorable survival outcome (77.8%), whereas AML demonstrated the poorest survival (36.2%). Conclusion:B-ALL was the predominant immunophenotypic subtype among children with acute leukemia at KCMC. Morphology showed substantial agreement with flow cytometry and remains a valuable frontline diagnostic tool in resource-limited settings. Expansion of advanced diagnostic services is essential to improve classification, risk stratification, and patient outcomes.

Background: Staining preparation methods can influence an antibody's brightness (MFI) and staining index¹. The staining method previously used for the LGL tube for the BDFACS Canto II analyzers utilized a stain/lyse/wash/wash (SLW) method. Expression of the CD16 PerCP-Cy5-5 antibody on NK cells has historically appeared relatively dim compared to other antibodies. One sample showed dim to negative CD16 PerCP-Cy5-5 expression on the NK cells when a positive expression was expected. Using a different staining method of lyse/wash/stain/wash/wash (LSW), a strong positive CD16 PerCP-Cy5-5 expression was observed. Objectives: Determine if an alternative sample prep method will aid in detecting CD16 PerCPCy5-5 without negatively affecting other markers in the same assay.

Methods: A comparison experiment was done to determine if a change in staining methods was appropriate, which included running method comparisons on 5 patient samples. Acceptable changes in MFI for the other markers in the panel were ≤0.5log.

Results: The results showed a significant increase (an average of 0.7 log change) in the MFI value of CD16 PerCP-Cy5.5 when the sample was prepared using the lyse/wash/stain/wash/wash method. There was no significant change in MFI values of other antibodies in the panel. Conclusion: With the data from this study, we were able to change the SOP for this assay to implement the LSW staining method. There are other assays on the Canto II analyzer that still utilize the SLW staining method. Future projects include validating the LSW method on all pathology assays.