Multiparameter Flow Cytometry Research Articles

Clinical significance of flow cytometry in detection of minimal residual disease in cerebrospinal fluid.

Central nervous system leukemia (CNSL) is one of the main causes of recurrence and death in patients with acute leukemia. This study aims to dynamically monitor minimal residual disease (MRD) in cerebrospinal fluid and bone marrow of patients with different types of acute leukemia by flow cytometry (FCM), and to compare the timeliness and consistency of MRD detection between the 2 methods to further explore the application value of monitoring MRD in cerebrospinal fluid. A total of 199 patients with acute leukemia admitted to the Guangdong Provincial people's Hospital between October 2018 and January 2022 were retrospectively analyzed, and multiparametric FCM method was adopted to summarize and analyze MRD in cerebrospinal fluid of patients with different types of leukemia and MRD in cerebrospinal fluid and bone marrow specimens of the same patients, and its role in assessing the prognostic value of patients was discussed. Among the 199 acute leukemia cases, a total of 31 cases (15.58%) were positive MRD in the cerebrospinal fluid, of which 18 cases (58%) were detected earlier than the corresponding bone marrow specimens. Among the 19 patients with acute T lymphoblastic leukemia, 134 patients with acute B lymphoblastic leukemia, and 46 patients with acute myeloid leukemia counted, there were 4, 18, and 9 patients with positive MRD in the cerebrospinal fluid. The Kappa value of the concordance test between the results of cerebrospinal fluid MRD and bone marrow MRD in different types of acute leukemia was only 0.156, demonstrating a low concordance between them. Dynamic monitoring of cerebrospinal fluid MRD by FCM can be used as a monitoring index for central nervous system leukemia, and monitoring cerebrospinal fluid can detect MRD earlier compared with bone marrow, which complements each other as a sensitive index for evaluating prognosis with significant guidance in clinic.

Nickel Challenge In Vitro Affects CD38 and HLA-DR Expression in T Cell Subpopulations from the Blood of Patients with Nickel Allergy.

Nickel allergy is a major health problem and shows clinical manifestation of contact eczema. The response of specific lymphocyte subpopulations in sensitized patients after new challenge to nickel has until now not been studied in detail. To evaluate if nickel-based elicitation reaction could be objectively identified by multi-parametric flow cytometry, immunophenotyping of specific T cells was applied. White blood cells from 7 patients (4 positive in patch test, 3 negative) were challenged by nickel and in vitro short-term culture. Standardized antibody-dye combinations, specific for T helper(h)1, Th17 and cytotoxic T cell activation, were selected according to the recommendations of Stanford Human Immune Monitoring Center. In cytotoxic CD8+CCR7+CD45RA+ T cells from patients suffering from nickel allergy, CD38 and HLA-DR were elevated comparing to healthy donors. After challenge to nickel in vitro both markers decreased in CD8+CCR7+CD45RA+ T cells but found up-regulated in CD4+CCR7+CD45RA+CCR6-CXCR3+Th1 cells. Intracellular expression of T-bet and RORγt further indicated Th1 and Th17 cells. Finally, CD4+CD25+CCR4- T cells increased after challenge with nickel in PBMCs of patients with nickel allergy. Flow cytometry based quantification of T cell markers might be used as a specific and reliable method to detect chemical induced skin sensitization and confirm diagnostic patch testing in the clinics.

Combination of minimal residual disease on day 15 and copy number alterations results in BCR-ABL1-negative pediatric B-ALL: A powerful tool for prediction of induction failure

The current genomic abnormalities provide prognostic value in pediatric Acute Lymphoblastic Leukemia (ALL). Furthermore, Copy Number Alteration (CNA) has recently been used to improve the genetic risk stratification of patients. This study aimed to evaluate CNA profiles in BCR-ABL1-negative pediatric B-ALL patients and correlate the data with Minimal Residual Disease (MRD) results after induction therapy. We examined 82 bone marrow samples from pediatric BCR-ABL1-negative B-ALL using the MLPA method for the most common CNAs, including IKZF1, CDKN2A/B, PAX5, RB1, BTG1, ETV6, EBF1, JAK2, and PAR1 region. Subsequently, patients were followed-up by multiparameter Flow Cytometry for MRD (MFC-MRD) assessment on days 15 and 33 after induction. Data showed that 58.5 % of patients carried at least one gene deletion, whereas 41.7 % of them carried more than one gene deletion simultaneously. The most frequent gene deletions were CDKN2A/B, ETV6, and IKZF1 (30.5 %, 14.6 %, and 14.6 %, respectively), while the PAR1 region showed predominantly duplication (30.5 %). CDKN2A/B and IKZF1 were related to positive MRD results on day 15 (p = 0.003 and p = 0.007, respectively). The simultaneous presence of more than one deletion was significantly associated with high induction failure (p = 0.001). Also, according to the CNA profile criteria, the CNA with poor risk (CNA-PR) profile was statistically associated with older age and positive MRD results on day 15 (p = 0.014 and p = 0.013, respectively). According to our results, the combined use of CNAs with MRD results on day 15 can predict induction failure and be helpful in ameliorating B-ALL risk stratification and treatment approaches.

Impaired interferon response in plasmacytoid dendritic cells from children with persistent wheeze

Impaired interferon response and allergic sensitization may contribute to virus-induced wheeze and asthma development in young children. Plasmacytoid dendritic cells (pDCs) play a key role in antiviral immunity as critical producers of type I interferons. pDCs also express the high-affinity IgE receptor through which type I interferon production may be negatively regulated. Whether antiviral function of pDCs is associated with recurrent episodes of wheeze in young children is not well understood. We sought to evaluate the phenotype and function of circulating pDCs in children with a longitudinally defined wheezing phenotype. We performed multiparameter flow cytometry on PBMCs from 38 children presenting to the emergency department with an acute episode of respiratory wheeze and 19 controls. RNA sequencing on isolated pDCs from the same individuals was also performed. For each subject, their longitudinal exacerbation phenotype was determined using the Western Australia public hospital database. We observed a significant depletion of circulating pDCs inyoung children with a persistent phenotype of wheeze. The same individuals also displayed upregulation of the FcεRI on their pDCs. Based on transcriptomic analysis, pDCs from these individuals didnot mount a robust systemic antiviral response as observed inchildren who displayed a nonrecurrent wheezing phenotype. Our data suggest that circulating pDC phenotype and function are altered in young children with a persistent longitudinal exacerbation phenotype. Expression of high-affinity IgE receptor is increased and their function as major interferon producers is impaired during acute exacerbations of wheeze.

Inflammageing mediated by cytotoxic lymphocytes is associated with diabetes duration

AimsInflammageing, the age-related systemic increase of proinflammatory factors, has been linked to the development of cardiovascular disease, chronic kidney disease and cancer in the elderly. Chronic inflammation is believed to be a causative factor in the development of diabetic complications. However, exactly how type 2 diabetes impacts the inflammatory state of the immune system is incompletely characterised. MethodsBlood collection and anthropometric measurements were performed in patients with type 2 diabetes (n=49) and control subjects (n=30). The phenotype, proliferation capacity and cytokine production by cytotoxic lymphocytes were analysed using multiparametric flow cytometry. ResultsType 2 diabetes did not impact the phenotype or proliferation of the investigated cells. However, we observed a significantly increased production of tumour necrosis factor-α by CD8+ T cells and Granzyme B by natural killer cells and γδ T cells compared to controls. Hyperresponsiveness of cytotoxic blood lymphocytes did not correlate with glycaemia or body mass index, but instead was associated with older age and longer diabetes duration. ConclusionsType 2 diabetes is associated with an increased pro-inflammatory potential of cytotoxic blood lymphocytes correlating with age and diabetes duration. Further research is necessary to explore potential benefits of diabetes medications in reverting this effect.

FLAER as a standalone reagent for paroxysmal nocturnal hemoglobinuria: Do we need to reconsider the guidelines for testing?

Flow cytometry-based paroxysmal nocturnal hemoglobinuria (PNH) testing involves utilization of monoclonal antibodies against GPI-linked proteins and FLAER. The ability of FLAER to bind to a wide variety of GPI-linked structures and to be utilized across different leukocyte subsets is remarkable. We hypothesize that FLAER as a standalone reagent may be equally effective for detecting PNH clones. The present study intends to compare the results of a FLAER alone-based strategy to the recommended FLAER+GPI-linked protein-based approach for applicability in clinical settings. EDTA-anticoagulated blood samples from patients for PNH workup were tested for PNH by multiparametric flow cytometry. A conventional panel comprising gating markers (CD45 for WBC, CD15 for granulocytes, and CD64 for monocytes) and a combination of FLAER and GPI-linked markers, such as CD24 and CD14, henceforth referred to as the "routine panel," was employed. Second, a "FLAER-only panel" comprising the gating markers and FLAER alone (excluding the GPI-linked markers CD24 and CD14) was set up. The samples were processed using the lyse-wash-stain-wash technique, and events were acquired on BC Navios Ex flow cytometer (Beckman Coulter, Inc., USA) and analyzed on Kaluza Software 2.1. The presence of a PNH clone was reported at a value of ≥0.01%. A total of 209 patients were tested. Both panels found a PNH clone in 20.1% of patients (n = 42/209) with a 100% concordance rate. The PNH clone range for granulocytes was 0.01%-89.68%, and for monocyte was 0.04%-96.09% in the routine panel. The range in the FLAER-only panel for granulocytes was 0.01%-89.61%, and for monocytes, it was 0.01%-96.05%. Pearson correlation statistics revealed a significant correlation between the size of the PNH clone of granulocytes and monocytes among the two panels tested (granulocytes r = 0.9999, p < 0.0001, 95% CI = 0.9999 to 1.000; monocytes r = 0.9974, p < 0.0001, 95% CI = 0.9966-0.9980). Based on our results, FLAER as a standalone marker is specific and sensitive for identifying PNH clones in granulocytes and monocytes, even for high-sensitivity PNH assay. The proposed "FLAER-only panel" panel is efficient and cost-effective for highly sensitive PNH testing in two different cell lineages, especially in resource-limited clinical settings.

Prognostic significance of the frequencies of bone marrow lymphocyte subsets in adult acute myeloid leukemia at diagnosis.

Immune microenvironment plays an important role in the occurrence and development of acute myeloid leukemia (AML). Studies assessing the prognostic significance of bone marrow (BM) lymphocyte subsets' frequencies at diagnosis in patients with AML were limited. Fresh BM samples collected from 97 adult AML patients at diagnosis were tested for lymphocyte, T, CD4+ T, CD8+ T, γδT, NK, and B cell frequencies using multi-parameter flow cytometry. Low frequencies of lymphocytes, T, CD4+ T, and CD8+ T cells were associated with significantly lower rates of one-course complete remission (CR) (all p < 0.05). Moreover, the frequency of CD4+ T cells independently predicted one-course CR achievement (p = 0.021). Low frequencies of T and CD8+ T cells were significantly associated with lower relapse-free survival (RFS) rates (p = 0.032; 0.034), respectively, and a low frequency of CD8+ T cells was associated with a significantly lower overall survival (OS) rate (p = 0.028). Combination of frequency of CD8+ T cells and ELN risk stratification showed that patients with ELN-intermediate/adverse risk + high CD8+ T cell frequency had a similar RFS rate to those with ELN-favorable risk + high CD8+ T cell frequency and those with ELN-favorable risk + low CD8+ T cell frequency (p = 0.88; 0.76), respectively. The RFS rate of patients with ELN intermediate/adverse risk + low CD8+ T cell frequency was significantly lower than that of all aforementioned patients (p = 0.021; 0.0007; 0.028), respectively. The frequencies of BM lymphocyte subsets at diagnosis predicted clinical outcomes and could help improve risk stratification in AML.

Understanding differential technologies for detection of MRD and how to incorporate into clinical practice.

Patient- and leukemia-specific factors assessed at diagnosis classify patients with acute myeloid leukemia (AML) in risk categories that are prognostic for outcome. The induction phase with intensive chemotherapy in fit patients aims to reach a complete remission (CR) of less than 5% blasts in bone marrow by morphology. To deepen and sustain the response, induction is followed by consolidation treatment. This postremission treatment of patients with AML is graduated in intensity based on this favorable, intermediate, or adverse risk group classification as defined in the European Leukemia Network (ELN) 2022 recommendations. The increment of evidence that measurable residual disease (MRD) after induction can be superimposed on risk group at diagnosis is instrumental in tailoring further treatment accordingly. Several techniques are applied to detect MRD such as multiparameter flow cytometry (MFC), quantitative (digital) polymerase chain reaction (PCR), and next-generation sequencing. The clinical implementation of MRD and the technique used differ among institutes, leading to the accumulation of a wide range of data, and therefore harmonization is warranted. Currently, evidence for MRD guidance is limited to the time point after induction using MFC or quantitative PCR for NPM1 and core binding factor abnormalities in intermediate-risk patients. The role of MRD in targeted or nonintensive therapies needs to be clarified, although some data show improved survival in patients achieving CR-MRD negativity. Potential application of MRD for selection of conditioning before stem cell transplantation, monitoring after consolidation, and use as an intermediate end point in clinical trials need further evaluation.

A 19-color single-tube full spectrum flow cytometry assay for the detection of measurable residual disease in acute myeloid leukemia.

Multiparameter flow cytometry (MFC) has emerged as a standard method for quantifying measurable residual disease (MRD) in acute myeloid leukemia. However, the limited number of available channels on conventional flow cytometers requires the division of a diagnostic sample into several tubes, restricting the number of cells and the complexity of immunophenotypes that can be analyzed. Full spectrum flow cytometers overcome this limitation by enabling the simultaneous use of up to 40 fluorescent markers. Here, we used this approach to develop a good laboratory practice-conform single-tube 19-color MRD detection assay that complies with recommendations of the European LeukemiaNet Flow-MRD Working Party. We based our assay on clinically-validated antibody clones and evaluated its performance on an IVD-certified full spectrum flow cytometer. We measured MRD and normal bone marrow samples and compared the MRD data to a widely used reference MRD-MFC panel generating highly concordant results. Using our newly developed single-tube panel, we established reference values in healthy bone marrow for 28 consensus leukemia-associated immunophenotypes and introduced a semi-automated dimensionality-reduction, clustering and cell type identification approach that aids the unbiased detection of aberrant cells. In summary, we provide a comprehensive full spectrum MRD-MFC workflow with the potential for rapid implementation for routine diagnostics due to reduced cell requirements and ease of data analysis with increased reproducibility in comparison to conventional FlowMRD routines.

Biomarkers in Adult-Type Diffuse Gliomas: Elevated Levels of Circulating Vesicular Heat Shock Protein 70 Serve as a Biomarker in Grade 4 Glioblastoma and Increase NK Cell Frequencies in Grade 3 Glioma.

The presence of circulating Hsp70 levels and their influence on the immunophenotype of circulating lymphocyte subsets were examined as diagnostic/prognostic biomarkers for the overall survival (OS) in patients with IDH-mutant WHO grade 3 oligodendroglioma, astrocytoma, and IDH-wildtype grade 4 glioblastoma (GBM). Vesicular and free Hsp70 in the plasma/serum was measured using the Hsp70-exo and R&D Systems DuoSet® Hsp70 ELISAs. The immunophenotype and membrane Hsp70 status was determined by multiparameter flow cytometry on peripheral blood lymphocytes and single-cell suspensions of tumor specimens and cultured cells. Compared to healthy controls, circulating vesicular Hsp70 levels were significantly increased in patients with GBM, concomitant with a significant decrease in the proportion of CD3+/CD4+ helper T cells, whereas the frequency of NK cells was most prominently increased in patients with grade 3 gliomas. Elevated circulating Hsp70 levels and a higher prevalence of activated CD3-/CD56+/CD94+/CD69+ NK cells were associated with an improved OS in grade 3 gliomas, whereas high Hsp70 levels and low CD3+/CD4+ frequencies were associated with an adverse OS in GBM. It is assumed that a reduced membrane Hsp70 density on grade 4 versus grade 3 primary glioma cells and reduced CD3+/CD4+ T cell counts in GBM might drive an immunosuppressive tumor microenvironment.

Predictors of unsustained measurable residual disease negativity in patients with multiple myeloma

AbstractThe prognostic impact of achieving and in particular maintaining measurable residual disease (MRD) negativity in multiple myeloma is now established; therefore, identifying among MRD-negative patients the ones at higher risk of losing MRD negativity is of importance. We analyzed predictors of unsustained MRD negativity in patients enrolled in the FORTE trial (NCT02203643). MRD was performed by multiparameter flow cytometry (sensitivity of 10−5) at premaintenance and every 6 months thereafter. The cumulative incidence (CI) of MRD resurgence and/or progression was analyzed in MRD-negative patients. A total of 306 of 474 (65%) MRD-negative patients were analyzed. After a median follow-up of 50.4 months from MRD negativity, 185 of 306 (60%) patients were still MRD negative and progression free, 118 (39%) lost their MRD-negative status, and 3 patients (1%) died without progression. Amp1q vs normal (4-year CI, 63% vs 34), ≥2 concomitant high-risk cytogenetic abnormalities vs 0 (4-year CI, 59% vs 33%), circulating tumor cells at baseline (high vs low at 4-year CI, 62% vs 32%), and time-to-reach MRD negativity postconsolidation vs preconsolidation (4-year CI, 46% vs 35%) were associated with a higher risk of unsustained MRD negativity in a multivariate Fine-Gray model. During the first 2 years of maintenance, patients receiving carfilzomib-lenalidomide vs lenalidomide alone had a lower risk of unsustained MRD negativity (4-year CI, 20% vs 33%).

Immune checkpoints are predominantly co-expressed by clonally expanded CD4+FoxP3+ intratumoral T-cells in primary human cancers

BackgroundIn addition to anti-PD(L)1, anti-CTLA-4 and anti-LAG-3, novel immune checkpoint proteins (ICP)-targeted antibodies have recently failed to demonstrate significant efficacy in clinical trials. In these trials, patients were enrolled without screening for drug target expression. Although these novel ICP-targeted antibodies were expected to stimulate anti-tumor CD8 + T-cells, the rationale for their target expression in human tumors relied on pre-clinical IHC stainings and transcriptomic data, which are poorly sensitive and specific techniques for assessing membrane protein expression on immune cell subsets. Our aim was to describe ICP expression on intratumoral T-cells from primary solid tumors to better design upcoming neoadjuvant cancer immunotherapy trials.MethodsWe prospectively performed multiparameter flow cytometry and single-cell RNA sequencing (scRNA-Seq) paired with TCR sequencing on freshly resected human primary tumors of various histological types to precisely determine ICP expression levels within T-cell subsets.ResultsWithin a given tumor type, we found high inter-individual variability for tumor infiltrating CD45 + cells and for T-cells subsets. The proportions of CD8+ T-cells (~ 40%), CD4+ FoxP3- T-cells (~ 40%) and CD4+ FoxP3+ T-cells (~ 10%) were consistent across patients and indications. Intriguingly, both stimulatory (CD25, CD28, 4-1BB, ICOS, OX40) and inhibitory (PD-1, CTLA-4, PD-L1, CD39 and TIGIT) checkpoint proteins were predominantly co-expressed by intratumoral CD4+FoxP3+ T-cells. ScRNA-Seq paired with TCR sequencing revealed that T-cells with high clonality and high ICP expressions comprised over 80% of FoxP3+ cells among CD4+ T-cells. Unsupervised clustering of flow cytometry and scRNAseq data identified subsets of CD8+ T-cells and of CD4+ FoxP3- T-cells expressing certain checkpoints, though these expressions were generally lower than in CD4+ FoxP3+ T-cell subsets, both in terms of proportions among total T-cells and ICP expression levels.ConclusionsTumor histology alone does not reveal the complete picture of the tumor immune contexture. In clinical trials, assumptions regarding target expression should rely on more sensitive and specific techniques than conventional IHC or transcriptomics. Flow cytometry and scRNAseq accurately characterize ICP expression within immune cell subsets. Much like in hematology, flow cytometry can better describe the immune contexture of solid tumors, offering the opportunity to guide patient treatment according to drug target expression rather than tumor histological type.

Determining hemodilution in diagnostic bone marrow aspirated samples in plasma cell disorders by next-generation flow cytometry: Proposal for a bone marrow quality index

Hemodilution of bone marrow (BM) aspirates is a limitation of multiparameter flow cytometry (MFC) in plasma cell disorders. There is a need for a validated approach for assessing sample quality and the distribution of non-plasma cell BM populations by MFC could provide a solution. We evaluated BM-associated cell populations, assessed by next-generation flow cytometry (NGF) and white blood cell (WBC) count in 351 BM aspirated samples from 219 participants with plasma cell disorders in the Iceland Screens, Treats, or Prevents MM study (iStopMM), as markers of hemodilution by their discriminatory ability between first and (generally more hemodiluted) second pull BM aspirated samples. The most discriminating markers were used to derive a novel BM quality index (BMQI). Nucleated red blood cells and myeloid precursors provided the greatest discriminatory ability between first vs second pull samples (area under the curve (AUC): 0.87 and 0.85, respectively), significantly better than B cell precursors (AUC = 0.64; p < 0.001), mast cells (AUC = 0.65; p < 0.001), and the BM WBC count (AUC = 0.77; p < 0.05). We generated a novel BMQI that is intrinsic to current NGF protocols, for evaluating quality of diagnostic BM samples and suggest the use of a BMQI scoring system for interpreting results and guiding appropriate actions.

Abstract B054: Immunological control by PARP inhibitors for successful immunotherapy in metastatic ovarian carcinoma

Abstract Epithelial ovarian carcinoma (EOC) is among the top five causes of cancer-related death in women. Most women with EOC achieve indeed complete remission after primary or interval cytoreductive surgery combined with chemotherapy based on a platinum-taxane doublet. Homologous recombination (HR) defects imposed by germline or somatic BRCA1 DNA repair-associated (BRCA1) or BRCA2 mutations are not only key determinants of platinum sensitivity in EOC patients but also provide a strong rationale for maintenance therapy based on poly(ADP-ribose) polymerase (PARP) inhibitors, which is generally associated with improved progression-free survival (PFS). Besides direct cytotoxic and cytostatic properties, PARPi have been shown to mediate multipronged immunostimulatory effects, largely reflecting their ability to inhibit DNA repair in malignant cells and indicating the possibility to synergy with immune-check point inhibitors (ICIs). Here, using multiparametric flow cytometry, multiplex immunostaining, single cells transcriptomics and functional studies in experimental syngeneic BR5-Akt mouse model and EOC tumor samples, we discuss PARPi immunomodulatory activity, with a specific focus on molecular and cellular pathways that can be harnessed to develop superior combinatorial regimens for clinical management. Thus, PARPi might enhance the mutational load of EOCs as consequence of unrepaired DNA damage and DAMPs exposure, favoring T cell infiltration, but also appear to drive robust type I IFN secretion downstream of cyclic GMP-AMP synthase and stimulator of IFN response cGAMP interactor 1 activation. In addition, the combination of PARPi with ICIs positively regulate the balance between adaptive anti-tumor immunity and innate myeloid components and significantly improve the cytotoxic T cell profile as shown in both experimental mice model and HGSOC tumor samples. We surmise that rationally designed combinations of PARPi and immunotherapeutic agents might be critical to unlock immunosuppression in the EOC microenvironment in support of clinical efficacy. Citation Format: Sarka Vosahlikova, Peter Holicek, Irena Moserova, Sandra Orsulic, Romana Mikyskova, Michal Hensler, Lenka Kasikova, Tereza Lanickova, Ondrej Novotny, Milan Reinis, Jana Drozenova, Ales Ryska, Lukas Rob, Michael J Halaska, David Cibula, Radek Spisek, Jitka Fucikova. Immunological control by PARP inhibitors for successful immunotherapy in metastatic ovarian carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B054.

Increased SARS-CoV-2 reactive low avidity T cells producing inflammatory cytokines in pediatric post-acute COVID-19 sequelae (PASC).

A proportion of the convalescent SARS-CoV-2 pediatric population presents nonspecific symptoms, mental health problems, and a reduction in quality of life similar to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID-19 symptomatic. However, data regarding its clinical manifestation and immune mechanisms are currently scarce. In this study, we perform a comprehensive clinical and immunological profiling of 17 convalescent COVID-19 children with post-acute COVID-19 sequelae (PASC) manifestation and 13 convalescent children without PASC manifestation. A detailed medical history, blood and instrumental tests, and physical examination were obtained from all patients. SARS-CoV-2 reactive T-cell response was analyzed via multiparametric flow cytometry and the humoral immunity was addressed via pseudovirus neutralization and ELISA assay. The most common PASC symptoms were shortness of breath/exercise intolerance, paresthesia, smell/taste disturbance, chest pain, dyspnea, headache, and lack of concentration. Blood count and clinical chemistry showed no statistical differences among the study groups. We detected higher frequencies of spike (S) reactive CD4+ and CD8+ T cells among the PASC study group, characterized by TNFα and IFNγ production and low functional avidity. CRP levels are positively correlated with IFNγ producing reactive CD8+ T cells. Our data might indicate a possible involvement of a persistent cellular inflammatory response triggered by SARS-CoV-2 in the development of the observed sequelae in pediatric PASC. These results may have implications on future therapeutic and prevention strategies.

The effectiveness of blinatumomab in clearing measurable residual disease in pediatric B-cell acute lymphoblastic leukemia patients detected by next-generation sequencing.

Blinatumomab improved survival outcomes in B-cell acute lymphoblastic leukemia (B-ALL) patients with measurable residual disease (MRD) <10-4 . However, data on blinatumomab clearing MRD with high sensitivity of 10-6 remain scarce. This study evaluates the effectiveness of blinatumomab in eradicating extremely low level (up to <10-6 ) of MRD, as detected by next-generation sequencing (NGS), in children with B-ALL. Patients (n = 19) whose MRD was undetectable by multiparameter flow cytometry (MFC) (sensitivity of 10-4 ) but detectable by NGS after chemotherapy and followed by blinatumomab consolidation were included retrospectively. After one course of blinatumomab, 13/19 patients (68%) successfully achieved NGS-MRD clearance (undetectable). With a median follow-up of 13.3 months, three of patients who were NGS-MRD positive relapsed within 1.8 months, while another three remained complete remission. Our study was the first to demonstrate that blinatumomab could further eradicate MRD after patients achieve MFC-MRD undetectable in B-ALL patients.

Side-by-Side Comparison of Compensation Beads Used in Polychromatic Flow Cytometry.

Compensation or unmixing is essential in analyzing multiparameter flow cytometry data. Errors in data correction, either by compensation or unmixing, can completely change the outcome or mislead the researchers. Owing to limited cell numbers, researchers often use synthetic beads to generate the required single stains for the necessary calculation. In this study, the capacity of synthetic beads to influence data correction is evaluated. Corrected data for human peripheral blood cells were generated using cell-based compensation from the same cells or bead-based compensation to identify differences between the methods. These data suggest that correction with beads on full-spectrum and conventional cytometers does not always follow the basic flow compensation/unmixing expectations and alters the data. Overall, the best approach for bead-based correction for an experiment is to evaluate which beads and fluorochromes are most accurately compensated/unmixed.

Современные подходы к оценке минимальной остаточной болезни при множественной миеломе (плазмоклеточной миеломе)

The treatment of multiple myeloma is inextricably linked to the need for assessment and monitoring of the minimal residual disease (MRD). Assessment of the MRD allows evaluating the efficacy of therapy and obtaining significant prognostic information; it is an indicator of the degree of eradication of the tumor clone. The methods for detecting residual tumor cells evolve constantly, which translates into updates of the criteria reflecting the scale of response to therapy. There is no single MRD detection technique; common recommendations suggest seeking for pathological cells both intramedullary and extramedullary. This review describes current MDR determination methods, including imaging, next generation multiparametric flow cytometry, and methods based on DNA analysis — allele-specific oligonucleotide polymerase chain reaction and next generation sequencing. We compare their advantages, limitations, disadvantages, clinical significance, and show the necessary sensitivity thresholds of the described methods and the conditions that make this or that approach ideal in the context of detection of MRD.

Immunophenotypic portrait of leukemia-associated-phenotype markers in B acute lymphoblastic leukemia.

Multiparametric flow cytometry (MFC) is an essential diagnostic tool in B acute lymphoblastic leukemia (B ALL) to determine the B-lineage affiliation of the blast population and to define their complete immunophenotypic profile. Most MFC strategies used in routine laboratories include leukemia-associated phenotype (LAP) markers, whose expression profiles can be difficult to interpret. The aim of our study was to reach a better understanding of 7 LAP markers' landscape in B ALL: CD9, CD21, CD66c, CD58, CD81, CD123, and NG2. Using a 10-color MFC approach, we evaluated the level of expression of 7 LAP markers including CD9, CD21, CD66c, CD58, CD81, CD123, and NG2, at the surface of normal peripheral blood leukocytes (n = 10 healthy donors), of normal precursor B regenerative cells (n = 40 uninvolved bone marrow samples) and of lymphoblasts (n = 100 peripheral blood samples or bone marrow samples from B ALL patients at diagnosis). The expression profile of B lymphoblasts was analyzed according the presence or absence of recurrent cytogenetic aberrations. The prognostic value of the 7 LAP markers was examined using Maxstat R algorithm. In order to help the interpretation of the MFC data in routine laboratories, we first determined internal positive and negative populations among normal leukocytes for each of the seven evaluated LAP markers. Second, their profile of expression was evaluated in normal B cell differentiation in comparison with B lymphoblasts to establish a synopsis of their expression in normal hematogones. We then evaluated the frequency of expression of these LAP markers at the surface of B lymphoblasts at diagnosis of B ALL. CD9 was expressed in 60% of the cases, CD21 in only 3% of the cases, CD58 in 96% of the cases, CD66c in 45% of the cases, CD81 in 97% of the cases, CD123 in 72% of the cases, and NG2 in only 2% of the cases. We confirmed the interest of the CD81/CD58 MFI expression ratio as a way to discriminate hematogones from lymphoblasts. We observed a significant lower expression of CD9 and of CD81 at the surface of B lymphoblasts with a t(9;22)(BCR-ABL) in comparison with B lymphoblasts without any recurrent cytogenetic alteration (p = 0.0317 and p = 0.0011, respectively) and with B lymphoblasts harboring other cytogenetic recurrent abnormalities (p = 0.0032 and p < 0.0001, respectively). B lymphoblasts with t(1;19) at diagnosis significantly overexpressed CD81 when compared with B lymphoblasts with other recurrent cytogenetic abnormalities or without any recurrent alteration (p = 0.0001). An overexpression of CD58 was also observed in the cases harboring this abnormal cytogenetic event, when compared with B lymphoblasts with other recurrent cytogenetic abnormalities (p = 0.030), or without any recurrent alteration (p = 0.0002). In addition, a high expression of CD123, of CD58 and of CD81 was associated with a favorable prognosis in our cohort of pediatric and young adult B ALL patients. We finally built a risk score based on the expression of these 3 LAP markers, this scoring approach being able to split these patients into a high-risk group (17%) and a better outcome group (83%, p < 0.0001). The complexity of the phenotypic signature of lymphoblasts at diagnosis of B ALL is illustrated by the variability in the expression of LAP antigens. Knowledge of the expression levels of these markers in normal leukocytes and during normal B differentiation is crucial for an optimal interpretation of diagnostic cytometry results and serves as a basis for the biological follow-up of B ALL.

Monitoring Minimal Residual Disease in Patients with Multiple Myeloma By Tracking Serum M-Protein Using Mass Spectrometry

Objective： We performed this retrospective real-world study to evaluate the clinical utility of blood-based mass spectrometry for longitudinal M-protein monitoring during the disease course of patients of multiple myeloma（MM）. Methods：We initially analyzed serial serum samples of 56 patients with MM by clonal peptide MS Method named EasyM MS, then we compared the MS results with those determined using serum immunofixation electrophoresis (IFE),multiparameter flow cytometry (MFC), next-generation flow cytometry (NGF), to establish the concordance between different disease-monitoring protocols. A sensitivity for MFC MRD negativity of at least 1×10 -4 anda sensitivity for NGF MRD negativity of at least 1×10 -5.Additionally, we determined the optimal cutoff value of EasyM MS negativity to satisfy both the sensitivity and specificity between MS and NGF, and explored the prognostic impact of MS-MRD status. Results：The median diagnostic age of 56 NDMM patients was 56 years old, including 34 males (60.7%). There were 38 patients with IgG type MP and 18 patients with IgA type MP. 47 patients (83.9%) chose to receive proteasome inhibitor - based induction therapy, and a total of 44 patients (78.6%) received first-line ASCT treatment.Among 56 MM patients，38 patients with IgG type MP can further identify IgG subclasses through mass spectrometry. Additionally, 56 patients with MM have successfully used MS to identify their unique clonal peptides of MP. 56 patients provided a total of 447 samples of serum and all sequential serum samples were successfully measured by using the MS method. The initial comparison of the performance of blood-based MS-MRD with that of the conventional M-protein diagnostic method IFE used . Among the 397 comparable results collected at similar timepoints, 70.8% (281/397) of the samples were both M-MRD- and IFE-positive, 3.3% (13/397) were both MS-MRD- and IFE-negative, 25.7% (102/397) were MS-MRD-positive and IFE-negative.Flow cytometry MRD was performed on 218 bone marrow aspirates in this cohort, including 126 analyzed with MFC and 92 analyzed with NGF. MS-MRD detection was also performed on comparable serum acquired at the same time points. The sensitivity of MS-MRD was 100.0% in comparison with both MFC and NGF, with specificity of 1.9% and 22.2%, respectively. Considering the overly high sensitivity of EasyM at a cutoff of dotp &amp;gt;0.9, we used the sensitivity of NGF as the standard for detection (i.e., a detection sensitivity ≤10 -5) and set the EasyM MS negative cutoff value using ROC analysis, determined to be &amp;lt;1.86% of that at first diagnosis. Using these assumptions, we again performed the aforementioned analyses comparing the sensitivity and specificity between EasyM and IFE, MFC, and NGF. Under this scenario, the sensitivity of EasyM remained high in comparison with IFE (88.3%), MFC (85.1%), and NGF (93.2%), but the specificity was also greatly increased to 90.4%, 55.8%, and 93.2%, respectively. We then divided patients into MS-positive and MS-negative groups according to whether they achieved MS-MRD negativity, and median PFS and OS were calculated for each group. The median PFS of the MS-positive and MS-negative groups was 20.6 (95% CI: 16.6, 24.6) and 61.8 (39.7, 83.9) months, respectively . The median OS was 55.7 (95% CI:31.8, 79.5) months for the MS-positive group and not reached (NR) for the MS-negative group ,Both mPFS and mOS were significantly longer among the MS-negative group compared with the MS-positive group (both p &amp;lt; 0.001). Conclusion：EasyM exhibits high sensitivity and minimally invasive technological advantages, and also has important significance in efficacy evaluation and prognosis evaluation. In the future, EasyM MS detection can be used as a minimally invasive MRD monitoring method based on peripheral blood in clinical practice.