Biomedicine today is experiencing a shift towards decentralized data collection, which promises enhanced reproducibility and collaboration across diverse laboratory environments. This inter-laboratory study evaluates the performance of biocytometry, a method utilizing engineered bioparticles for enumerating cells based on their surface antigen patterns. In centralized and aggregated inter-lab studies, biocytometry demonstrated significant statistical power in discriminating numbers of target cells at varying concentrations as low as 1 cell per 100,000 background cells. User skill levels varied from expert to beginner capturing a range of proficiencies. Measurement was performed in a decentralized environment without any instrument cross-calibration or advanced user training outside of a basic instruction manual. The results affirm biocytometry to be a viable solution for immunophenotyping applications demanding sensitivity as well as scalability and reproducibility and paves the way for decentralized analysis of rare cells in heterogeneous samples.

• MeSH
• Single-Cell Analysis * methods   MeSH
• Immunophenotyping methods   MeSH
• Laboratories standards   MeSH
• Humans MeSH
• Flow Cytometry methods   MeSH
• Reproducibility of Results MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. OBJECTIVE: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. METHODS: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. RESULTS: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. CONCLUSION: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.

• Keywords
• CD marker, cluster of differentiation (CD), expression profiling, flow cytometry, surfaceome,
• MeSH
• Antigens, Surface * metabolism   MeSH
• Killer Cells, Natural metabolism   MeSH
• Antigens, CD metabolism   MeSH
• Leukocytes MeSH
• Humans MeSH
• Antibodies, Monoclonal MeSH
• Immunity, Innate * MeSH
• Workflow MeSH
• Flow Cytometry methods   MeSH
• Reference Standards MeSH
• Reproducibility of Results MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Surface * MeSH
• Antigens, CD MeSH
• Antibodies, Monoclonal MeSH

Reproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these lymphomas by flow cytometry and compared it to the WHO gold standard diagnosis. Overall, 662 samples from 662 patients representing 9 disease categories were analyzed at 9 laboratories using the previously published EuroFlow 5-tube-8-color B-cell chronic lymphoproliferative disease antibody panel. Expression levels of all 26 markers from the panel were plotted by B-cell entity to construct a univariate, fully standardized diagnostic reference library. For multivariate data analysis, we subsequently used canonical correlation analysis of 176 training cases to project the multidimensional space of all 26 immunophenotypic parameters into 36 2-dimensional plots for each possible pairwise differential diagnosis. Diagnostic boundaries were fitted according to the distribution of the immunophenotypes of a given differential diagnosis. A diagnostic algorithm based on these projections was developed and subsequently validated using 486 independent cases. Negative predictive values exceeding 92.1% were observed for all disease categories except for follicular lymphoma. Particularly high positive predictive values were returned in chronic lymphocytic leukemia (99.1%), hairy cell leukemia (97.2%), follicular lymphoma (97.2%), and mantle cell lymphoma (95.4%). Burkitt and CD10+ diffuse large B-cell lymphomas were difficult to distinguish by the algorithm. A similar ambiguity was observed between marginal zone, lymphoplasmacytic, and CD10- diffuse large B-cell lymphomas. The specificity of the approach exceeded 98% for all entities. The univariate immunophenotypic library and the multivariate expert-independent diagnostic algorithm might contribute to increased reproducibility of future diagnostics in mature B-cell neoplasms.

• MeSH
• Lymphoma, Large B-Cell, Diffuse * MeSH
• Adult MeSH
• Lymphoma, Follicular * diagnosis   MeSH
• Immunophenotyping MeSH
• Humans MeSH
• Flow Cytometry methods   MeSH
• Reproducibility of Results MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH

• Keywords
• diagnosis, flow cytometry, functional studies, immunophenotyping, primary immunodeficency,
• MeSH
• Immunophenotyping methods   MeSH
• Humans MeSH
• Primary Immunodeficiency Diseases diagnosis   MeSH
• Flow Cytometry methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Introductory Journal Article MeSH
• Editorial MeSH