The kappa/lambda ratio of surface immunoglobulin light chain as a valuable parameter for MRD assessment in CLL with atypical immunophenotype.

In recent years, the significance of detecting minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) has increased due to the availability of highly effective therapeutic agents. Flow cytometry provides notable cost-effectiveness and immediacy, with an expected sensitivity level of approximately 10-4. The critical aspect of MRD detection via flow cytometry lies in accurately defining the region containing tumor cells. However, a subset of CLL, known as CLL with atypical immunophenotype, exhibits a distinct cell surface marker expression pattern that can make MRD detection challenging, because these markers often resemble those of normal B cells. To enhance the sensitivity of MRD detection in such atypical cases of CLL, we have capitalized on the observation that cell surface immunoglobulin (sIg) light chains tend to be expressed at a higher level in this subtype. For every four two-dimensional plots of cell surface markers, we used a plot to evaluate the expression of sIg kappa/lambda light chains and identified regions where the kappa/lambda ratio of sIg light chains deviated from a designated threshold within the putative CLL cell region. Using this method, we could detect atypical CLL cells at a level of 10-4. We propose this method as an effective MRD assay.

OMIP-102: 50-color phenotyping of the human immune system with in-depth assessment of T cells and dendritic cells.

We report the development of an optimized 50-color spectral flow cytometry panel designed for the in-depth analysis of the immune system in human blood and tissues, with the goal of maximizing the amount of information that can be collected using currently available flow cytometry platforms. We established and tested this panel using peripheral blood mononuclear cells (PBMCs), but included CD45 to enable its future use for the analysis of human tissue samples. The panel contains lineage markers for all major immune cell subsets, and an extensive set of phenotyping markers focused on the activation and differentiation status of the T cell and dendritic cell (DC) compartment. We outline the biological insight that can be gained from the simultaneous measurement of such a large number of proteins and propose that this approach provides a unique opportunity for the comprehensive exploration of the immune status in human samples with a limited number of cells. Of note, we tested the panel to be compatible with cell sorting for further downstream applications. Furthermore, to facilitate the wide-spread implementation of such a panel across different cohorts and samples, we established a trimmed-down 45-color version which can be used with different spectral cytometry platforms. Finally, to generate this panel, we utilized not only existing panel design guidelines, but also developed new metrics to systematically identify the optimal combination of 50 fluorochromes and evaluate fluorochrome-specific resolution in the context of a 50-color unmixing matrix.

Performance of a novel eight-color flow cytometry panel for measurable residual disease assessment of chronic lymphocytic leukemia.

BACKGROUND: Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored. METHODS: Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard." RESULTS: The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples. CONCLUSIONS: Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability.

Flow Cytometric Assessment of Myelodysplastic Syndromes/Neoplasms.

Myelodysplastic syndromes/neoplasms (MDS) are a heterogeneous class of hematopoietic stem cell neoplasms characterized by ineffective hematopoiesis leading to peripheral cytopenias. This group of diseases is typically diagnosed using a combination of clinical, morphologic, and genetic criteria. Many studies have described the value of multiparametric flow cytometry (MFC) in the diagnosis, classification, and prognostication of MDS. This review summarizes the approach to MDS diagnosis and immunophenotypic characterization using MFC and describes the current state while highlighting future opportunities and potential pitfalls.

Analytical assay validation for acute myeloid leukemia measurable residual disease assessment by multiparametric flow cytometry.

BACKGROUND: Measurable residual disease (MRD) assessed by multiparametric flow cytometry (MFC) has gained importance in clinical decision-making for acute myeloid leukemia (AML) patients. However, complying with the recent In Vitro Diagnostic Regulations (IVDR) in Europe and Food and Drug Administration (FDA) guidance in the United States requires rigorous validation prior to their use in investigational clinical trials and diagnostics. Validating AML MRD-MFC assays poses challenges due to the unique underlying disease biology and paucity of patient specimens. In this study, we describe an experimental framework for validation that meets regulatory expectations. METHODS: Our validation efforts focused on evaluating assay accuracy, analytical specificity, analytical and functional sensitivity (limit of blank (LoB), detection (LLoD) and quantitation (LLoQ)), precision, linearity, sample/reagent stability and establishing the assay background frequencies. RESULTS: Correlation between different MFC methods was highly significant (r = 0.99 for %blasts and r = 0.93 for %LAIPs). The analysis of LAIP specificity accurately discriminated from negative control cells. The assay demonstrated a LoB of 0.03, LLoD of 0.04, and LLoQ of 0.1%. Precision experiments yielded highly reproducible results (Coefficient of Variation <20%). Stability experiments demonstrated reliable measurement of samples up to 96 h from collection. Furthermore, the reference range of LAIP frequencies in non-AML patients was below 0.1%, ranging from 0.0% to 0.04%. CONCLUSION: In this manuscript, we present the validation of an AML MFC-MRD assay using BM/PB patient specimens, adhering to best practices. Our approach is expected to assist other laboratories in expediting their validation activities to fulfill recent health authority guidelines.

Computational flow cytometry provides accurate assessment of measurable residual disease in chronic lymphocytic leukaemia.

Undetectable measurable residual disease (MRD) is associated with favourable clinical outcomes in chronic lymphocytic leukaemia (CLL). While assessment is commonly performed using multiparameter flow cytometry (MFC), this approach is associated with limitations including user bias and expertise that may not be widely available. Implementation of unsupervised clustering algorithms in the laboratory can address these limitations and have not been previously reported in a systematic quantitative manner. We developed a computational pipeline to assess CLL MRD using FlowSOM. In the training step, a self-organising map was generated with nodes representing the full breadth of normal immature and mature B cells along with disease immunophenotypes. This map was used to detect MRD in multiple validation cohorts containing a total of 456 samples. This included an evaluation of atypical CLL cases and samples collected from two different laboratories. Computational MRD showed high correlation with expert analysis (Pearson's r > 0.99 for typical CLL). Binary classification of typical CLL samples as either MRD positive or negative demonstrated high concordance (>98%). Interestingly, computational MRD detected disease in a small number of atypical CLL cases in which MRD was not detected by expert analysis. These results demonstrate the feasibility and value of automated MFC analysis in a diagnostic laboratory.

Utility of CD229 as novel marker in measurable residual disease assessment in multiple myeloma-An evidence-based approach.

INTRODUCTION: CD229 has been found to be a useful plasma cell (PC) gating marker in multiple myeloma (MM). This study analyses the expression profile of CD229 on various bone marrow compartments namely, PC, non-PC and hematogones (HGs) using Multiparameter flow cytometry (MFC). Furthermore, it evaluates the ability of CD229 to delineate normal PC (NPC) from aberrant PC (APC) in measurable residual disease assessment (MRD) in MM. METHODS: Bone marrow aspirates from patients diagnosed with MM (per standard IMWG criteria) were collected in EDTA and processed for MFC using a single tube 14-color antibody panel as per standard operating procedure. RESULTS: A total of 74 patients with a diagnosis of MM (26 treatment naïve and 48 on therapy) were evaluated. The expression of CD229 was homogenous on both the PC and HG compartments as compared to CD138 and CD38. On comparing the expression of individual markers, it was found to be statistically significant between PC, HGs and non-PC for all three markers (p < 0.001). APC showed lower median expression of CD38 and higher median expression of CD138 and CD229 as compared to NPC and was found to be statistically significant for all markers (p < 0.001). In terms of differential expression on NPC and APC; CD38 was found to be the most aberrantly expressed (70%; 52/74) followed by CD229 (7%; 5/74) and CD138 (5%; 4/74). CONCLUSIONS: CD229 can be used for the identification of PC and due to relatively homogenous expression; it can be used as a suitable marker for targeted therapies. However, precise discrimination of NPC from APC cannot be reliably achieved with CD229, limiting its utility as a useful marker of diagnostic relevance and MRD assessment in MM.

A concise review of flow cytometric methods for minimal residual disease assessment in childhood B-cell precursor acute lymphoblastic leukemia.

Minimal residual disease refers to a leukemia cell population that is resistant to chemotherapy or radiotherapy and leads to disease relapse. The assessment of MRD is crucial for making an accurate prognosis of the disease and for the choice of optimal treatment strategy. Here, we review the advantages and disadvantages of the available genetic and phenotypic methods and focus on the multiparametric flow cytometry as a promising method with greater sensitivity, speed, and standardization options. In addition, we discuss how the application of automated data analysis outweighs the use of complex combinations of windows and gates in classical analysis, thus eliminating subjective evaluation.

Immunophenotypic assessment of PNH clones in major and minor cell lineages in the peripheral blood of patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Flow cytometric immunophenotyping is essential for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Most cases have easy to interpret flow cytometry profiles with red cells, neutrophils and monocytes showing complete deficiency of glycophosphatidylinositol (GPI) linked antigen expression. Some cases are more challenging to interpret due to the presence of multiple populations of PNH cells and variable levels of GPI antigen expression. METHODS: We studied 46 known PNH patients, many with complex immunophenotypic profiles using a novel, single tube, multi-parameter 7-color immunophenotyping assay that allowed simultaneous detection and assessment of PNH clones within multiple lineages of peripheral blood leucocytes. Red cell PNH clones were also assessed in total and immature (CD71+) components by CD59 expression. RESULTS: For individual patients, total PNH clones in each cell lineage were highly correlated. Monocytes, eosinophils and basophils showed the highest proportions of PNH cells. Red cell PNH clones were typically smaller than monocyte and neutrophil PNH clones. In most cases, PNH clones were detectable in minor leucocyte populations where multiple populations of PNH cells were present, variability in the proportions of type II and type III cells was seen across different cell lineages, even though total PNH clones remained similar. CONCLUSIONS: This study shows that PNH patients with multiple PNH clones do not always display the same abnormality across all cell lineages routinely tested. There is no simple explanation for this but is likely due to a combination of complex molecular, genetic and biochemical dysfunction in different blood cell types.

Flow cytometry assessment of reactive T-cells distinguishes classic Hodgkin lymphoma from benign lymphadenopathy in children.

BACKGROUND: Detection of classic Hodgkin lymphoma (cHL) neoplastic cells using flow cytometric immunophenotyping (FCI) remains limited. We hypothesized that characterization of the reactive infiltrates could assist in diagnosing cHL in children. METHODS: FCI using four-color staining approaches was performed on 156 lymph node specimens with the following histopathologic diagnoses: cHL (25 cases), reactive lymphoid hyperplasia (RLH, 44 cases), and non-Hodgkin lymphoma (87 cases). RESULTS: The overall concordance of FCI data with the histopathologic results of these cases was 81.4%. A reactive expansion of T-cells with increased expression of CD45RO was present in the reactive infiltrate of cHL (CD45RO/CD3, 67.5%) and Epstein-Barr virus (EBV) infected RLH (62.7%) but not in EBV-negative RLH (28.0%). The mean fluorescence intensity (MFI) of CD7 was higher for cHL and differed significantly from EBV-positive RLH (138.5 vs. 63.8). A proposed diagnostic algorithm markedly elevated the overall concordance rate from 81.4% to 97.4%. CONCLUSIONS: Immunophenotyping the reactive infiltrate of lymphoid tissue using flow cytometry is a reliable supplement to histopathology for the rapid diagnosis of pediatric cHL.

CD200 expression on Sezary cells: A valuable tool for flow cytometric assessment of peripheral blood T-cell neoplasms.

BACKGROUND: CD200 (OX-2) is a valuable marker in the diagnosis of B-cell neoplasms and is commonly used in the screening panels for assessment of peripheral blood B-cell lymphoproliferative disorders. However, there is limited understanding about CD200 expression in T-cell neoplasms. A previous study has shown that CD200 is expressed on the neoplastic cells of angioimmunoblastic T-cell lymphoma (AITL) by immunohistochemistry, but no study has evaluated CD200 expression in T-cell neoplasms by flow cytometry. METHODS: We assessed CD200 expression in peripheral blood T-cell lymphoproliferative disorders by retrospective analysis of our institutional flow cytometry screening database over a 6-year period. RESULTS: In addition to AITL, we identified CD200 expression in a significant number of mycosis fungoides/Sezary syndrome cases (58%, 19 of 33 samples), while most other T-cell neoplasms were negative for CD200. These findings were confirmed by CD200 immunohistochemical staining of tissue specimens from our patient cohort. CONCLUSIONS: CD200 is commonly expressed on circulating Sezary cells, a feature that can potentially improve the diagnostic value of flow cytometry for assessment of T-cell neoplasms.

Measurable Residual Disease Assessment as a Surrogate Marker in New Drug Development in Acute Myeloid Leukemia.

ABSTRACT: Response criteria for patients treated for acute myeloid leukemia (AML) based on cytomorphology are inadequate. Many patients achieving a complete remission by such criteria will later relapse. Patients with AML in such remissions who test negative using higher sensitivity measures of residual disease burden (measurable residual disease [MRD]) have on average lower relapse rates and better survival than those testing positive. This association has raised the possibility that these technological advances in measurement of tumor burden could be used to optimize the drug development and regulatory approval processes in AML. The heterogeneous genetic etiology, diverse immunophenotypic profiles, related precursor states and polyclonal architecture however combine to make the development of standardized and validated MRD assessments for AML challenging. Current and future methods to measure residual disease in AML, performance characteristics of testing currently in use, and potential uses for optimized AML MRD tests including as a surrogate endpoint are discussed.

Determining the immune environment of cutaneous T-cell lymphoma lesions through the assessment of lesional blood drops.

Detailed analysis of the cells that infiltrate lesional skin cannot be performed in skin biopsy specimens using immunohistochemistry or cell separation techniques because enzyme treatments applied during the isolation step can destroy small amounts of protein and minor cell populations in the biopsy specimen. Here, we describe a method for isolating T cells from drops of whole blood obtained from lesions during skin biopsy in patients with cutaneous T-cell lymphoma. Lesional blood is assumed to contain lesional resident cells, cells from capillary vessels, and blood overflowing from capillary vessels into the lesion area. The lesional blood showed substantial increases in distinct cell populations, chemokines, and the expression of various genes. The proportion of CD8+CD45RO+ T cells in the lesional blood negatively correlated with the modified severity-weighted assessment tool scores. CD4+CD45RO+ T cells in the lesional blood expressed genes associated with the development of cancer and progression of cutaneous T-cell lymphoma. In addition, CD8+CD45RO+ T cells in lesional blood had unique T-cell receptor repertoires in lesions of each stage. Assessment of lesional blood drops might provide new insight into the pathogenesis of mycosis fungoides and facilitate evaluation of the treatment efficacy for mycosis fungoides as well as other skin inflammatory diseases.

Necessity of flow cytometry assessment of circulating plasma cells and its connection with clinical characteristics of primary and secondary plasma cell leukaemia.

Plasma cell leukaemia (PCL) is a rare and very aggressive plasma cell disorder. Preventing a dismal outcome of PCL requires early diagnosis with appropriate analytical tools. Therefore, the investigation of 33 patients with primary and secondary PCL was done when the quantity of circulating plasma cells (PCs) using flow cytometry (FC) and morphology assessment was evaluated. The phenotypic profile of the PCs was also analysed to determine if there is an association with clinical outcomes and to evaluate the prognostic value of analysed markers. Our results revealed that FC is an excellent method for identifying circulating PCs as a significantly higher number was identified by FC than by morphology (26·7% vs. 13·5%, P = 0·02). None of secondary PCL cases expressed CD19 or CD20. A low level of expression with similar positivity of CD27, CD28, CD81 and CD117 was found in both PCL groups. A decrease of CD44 expression was detected only in secondary PCL. Expression of CD56 was present in more than half of PCL cases as well as cytoplasmic nestin. A decreased level of platelets, Eastern Cooperative Oncology Group score of 2-3 and lack of CD20+ PC were associated with a higher risk of death. FC could be incorporated in PCL diagnostics not only to determine the number of circulating PCs, but also to assess their phenotype profile and this information should be useful in patients' diagnosis and possible prognosis.

Immunophenotyping assessment in a COVID-19 cohort (IMPACC): A prospective longitudinal study.

The IMmunoPhenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective longitudinal study designed to enroll 1000 hospitalized patients with COVID-19 (NCT04378777). IMPACC collects detailed clinical, laboratory and radiographic data along with longitudinal biologic sampling of blood and respiratory secretions for in depth testing. Clinical and lab data are integrated to identify immunologic, virologic, proteomic, metabolomic and genomic features of COVID-19-related susceptibility, severity and disease progression. The goals of IMPACC are to better understand the contributions of pathogen dynamics and host immune responses to the severity and course of COVID-19 and to generate hypotheses for identification of biomarkers and effective therapeutics, including optimal timing of such interventions. In this report we summarize the IMPACC study design and protocols including clinical criteria and recruitment, multi-site standardized sample collection and processing, virologic and immunologic assays, harmonization of assay protocols, high-level analyses and the data sharing plans.

Critical evaluation of the utility of pre- and post-therapy immunophenotypes in assessment of measurable residual disease in B-ALL.

Measurable residual disease (MRD) is an important parameter to predict outcome in B-cell acute lymphoblastic leukemia (B-ALL). Two different approaches have been used for the assessment of MRD by multiparametric flow cytometry that include the "Leukemia Associated Aberrant Immunophenotype (LAIP)" and "Difference from Normal (DFN)" approach. In this retrospective study, we analyzed 539 samples obtained from 281 patients of which 258 were paired samples and the remaining 23 samples were from post-induction time point only, to explore the utility of baseline immunophenotype (IPT) for MRD assessment. Single-tube 10-color panel was used both at diagnosis and MRD time points. Out of 281 patients, 31.67% (n = 89) were positive and 68.32% (n = 192) were negative for MRD. Among 258 paired diagnostic and follow-up samples, baseline IPT was required in only 9.31% (24/258) cases which included cases with hematogone pattern and isolated dim to negative CD10 expression patterns. Comparison of baseline IPT with post-induction MRD positive samples showed a change in expression of at least one antigen in 94.04% cases. Although the immunophenotypic change in expression of various antigens is frequent in post-induction samples of B-ALL, it does not adversely impact the MRD assessment. In conclusion, the baseline IPT is required in less than 10% of B-ALL, specifically those with hematogone pattern and/or dim to negative expression of CD10. Hence, a combination of DFN and LAIP approach is recommended for reliable MRD assessment.

Potency Assessment of Dendritic Cell Anticancer Vaccine: Validation of the Co-Flow DC Assay.

For many years, oncological clinical trials have taken advantage of dendritic cells (DC) for the design of DC-based cellular therapies. This has required the design of suitable quality control assays to evaluate the potency of these products. The purpose of our work was to develop and validate a novel bioassay that uses flow cytometry as a read-out measurement. In this method, CD3+ cells are labeled with a fluorescent dye and the DC costimulatory activity is measured by the degree of T cell proliferation caused by the DC-T cell interaction. The validation of the method was achieved by the evaluation of essential analytical parameters defined by international guidelines. Our results demonstrated that the method could be considered specific, selective, and robust. The comparison between measured values and estimated true values confirmed a high level of accuracy and a lack of systematic error. Repeated experiments have shown the reproducibility of the assay and the proportionality between the potency and the DC amount has proven its linearity. Our results suggest that the method is compliant with the guidelines and could be adopted as a quality control assay or batch-release testing within GMP facilities.

Non-transplantable cord blood units as a source for adoptive immunotherapy of leukaemia and a paradigm of circular economy in medicine.

Advances in immunotherapy with T cells armed with chimeric antigen receptors (CAR-Ts), opened up new horizons for the treatment of B-cell lymphoid malignancies. However, the lack of appropriate targetable antigens on the malignant myeloid cell deprives patients with refractory acute myeloid leukaemia of effective CAR-T therapies. Although non-engineered T cells targeting multiple leukaemia-associated antigens [i.e. leukaemia-specific T cells (Leuk-STs)] represent an alternative approach, the prerequisite challenge to obtain high numbers of dendritic cells (DCs) for large-scale Leuk-ST generation, limits their clinical implementation. We explored the feasibility of generating bivalent-Leuk-STs directed against Wilms tumour 1 (WT1) and preferentially expressed antigen in melanoma (PRAME) from umbilical cord blood units (UCBUs) disqualified for allogeneic haematopoietic stem cell transplantation. By repurposing non-transplantable UCBUs and optimising culture conditions, we consistently produced at clinical scale, both cluster of differentiation (CD)34+ cell-derived myeloid DCs and subsequently polyclonal bivalent-Leuk-STs. Those bivalent-Leuk-STs contained CD8+ and CD4+ T cell subsets predominantly of effector memory phenotype and presented high specificity and cytotoxicity against both WT1 and PRAME. In the present study, we provide a paradigm of circular economy by repurposing unusable UCBUs and a platform for future banking of Leuk-STs, as a 'third-party', 'off-the-shelf' T-cell product for the treatment of acute leukaemias.

Assessment of the Neuroprotective and Stemness Properties of Human Wharton's Jelly-Derived Mesenchymal Stem Cells under Variable (5% vs. 21%) Aerobic Conditions.

To optimise the culture conditions for human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) intended for clinical use, we investigated ten different properties of these cells cultured under 21% (atmospheric) and 5% (physiological normoxia) oxygen concentrations. The obtained results indicate that 5% O2 has beneficial effects on the proliferation rate, clonogenicity, and slowdown of senescence of hWJ-MSCs; however, the oxygen level did not have an influence on the cell morphology, immunophenotype, or neuroprotective effect of the hWJ-MSCs. Nonetheless, the potential to differentiate into adipocytes, osteocytes, and chondrocytes was comparable under both oxygen conditions. However, spontaneous differentiation of hWJ-MSCs into neuronal lineages was observed and enhanced under atmospheric oxygen conditions. The cells relied more on mitochondrial respiration than glycolysis, regardless of the oxygen conditions. Based on these results, we can conclude that hWJ-MSCs could be effectively cultured and prepared under both oxygen conditions for cell-based therapy. However, the 5% oxygen level seemed to create a more balanced and appropriate environment for hWJ-MSCs.

The diagnostic and prognostic significance of flow cytometric bone marrow assessment in myelodysplastic syndromes according to the European LeukemiaNet recommendations in single-centre real-life experience.

INTRODUCTION: This analysis attempts to determine the diagnostic and prognostic value of bone marrow (BM) evaluation by multiparameter flow cytometry in patients with myelodysplastic syndrome (MDS). MATERIALS AND METHODS: The study group consisted of patients who underwent diagnostic process in the years 2008-2017 due to cytopenia and finally were diagnosed with MDS (n = 71). The comparative group included patients with cytopenia diagnosed in the same period, whose definitive diagnosis was other than MDS (n = 39). Flow cytometric evaluation of BM was performed following the recommendations of the European LeukemiaNet (ELN) in all patients. RESULTS: The median number of immunophenotypic abnormalities found on granulocytes in the MDS group was significantly higher compared to the comparative group [2 (range 0-5) vs 0 (range 0-2); P < .0001]. Similarly, the median Ogata score was significantly higher in the MDS group [2 (range 0-4) vs 1 (range 0-3); P < .0001]. Since the disturbances of the CD11b/HLA-DR and CD11b/CD13 on granulocytes were significantly more common in MDS patients, the Ogata score was extended by these abnormalities, what resulted in its higher diagnostic sensitivity (82%) while preserving high specificity (87%). The positive correlation was found between risk score determined by the Revised International Prognostic Scoring System and the number of the BM immunophenotypic abnormalities (P = .017). CONCLUSIONS: Our results indicate that the diagnostic usefulness of the Ogata score may be increased by including the abnormal expression of CD11b/HLA-DR and CD11b/CD13 on granulocytes. Moreover, our findings suggest the prognostic significance of the number of BM cytometric abnormalities in MDS.