Ocean microbes are critical to both ocean ecosystems and the global climate. Flow cytometry, which measures cell optical properties in fluid samples, is routinely used in oceanographic research. Despite decades of accumulated data, identifying key microbial populations (a process known as ``gating'') remains a significant analytical challenge. To address this, we focus on gating multidimensional, high-frequency flow cytometry data collected {\it continuously} on board oceanographic research vessels, capturing time- and space-wise variations in the dynamic ocean. Our paper proposes a novel mixture-of-experts model in which both the gating function and the experts are given by trend filtering. The model leverages two key assumptions: (1) Each snapshot of flow cytometry data is a mixture of multivariate Gaussians and (2) the parameters of these Gaussians vary smoothly over time. Our method uses regularization and a constraint to ensure smoothness and that cluster means match biologically distinct microbe types. We demonstrate, using flow cytometry data from the North Pacific Ocean, that our proposed model accurately matches human-annotated gating and corrects significant errors.

This paper presents FlowCyt, the first comprehensive benchmark for multi-class single-cell classification in flow cytometry data. The dataset comprises bone marrow samples from 30 patients, with each cell characterized by twelve markers. Ground truth labels identify five hematological cell types: T lymphocytes, B lymphocytes, Monocytes, Mast cells, and Hematopoietic Stem/Progenitor Cells (HSPCs). Experiments utilize supervised inductive learning and semi-supervised transductive learning on up to 1 million cells per patient. Baseline methods include Gaussian Mixture Models, XGBoost, Random Forests, Deep Neural Networks, and Graph Neural Networks (GNNs). GNNs demonstrate superior performance by exploiting spatial relationships in graph-encoded data. The benchmark allows standardized evaluation of clinically relevant classification tasks, along with exploratory analyses to gain insights into hematological cell phenotypes. This represents the first public flow cytometry benchmark with a richly annotated, heterogeneous dataset. It will empower the development and rigorous assessment of novel methodologies for single-cell analysis.

We consider the problem of assigning class labels to an unlabeled test data set, given several labeled training data sets drawn from similar distributions. This problem arises in several applications where data distributions fluctuate because of biological, technical, or other sources of variation. We develop a distributionfree, kernel-based approach to the problem. This approach involves identifying an appropriate reproducing kernel Hilbert space and optimizing a regularized empirical risk over the space. We present generalization error analysis, describe universal kernels, and establish universal consistency of the proposed methodology. Experimental results on flow cytometry data are presented.

In the complex landscape of hematologic samples such as peripheral blood or bone marrow, cell classification, delineating diverse populations into a hierarchical structure, presents profound challenges. This study presents LeukoGraph, a recently developed framework designed explicitly for this purpose employing graph attention networks (GATs) to navigate hierarchical classification (HC) complexities. Notably, LeukoGraph stands as a pioneering effort, marking the application of graph neural networks (GNNs) for hierarchical inference on graphs, accommodating up to one million nodes and millions of edges, all derived from flow cytometry data. LeukoGraph intricately addresses a classification paradigm where for example four different cell populations undergo flat categorization, while a fifth diverges into two distinct child branches, exemplifying the nuanced hierarchical structure inherent in complex datasets. The technique is more general than this example. A hallmark achievement of LeukoGraph is its F-score of 98%, significantly outclassing prevailing state-of-the-art methodologies. Crucially, LeukoGraph's prowess extends beyond theoretical innovation, showcasing remarkable precision in predicting both flat and hierarchical cell types across flow cytometry datasets from 30 distinct patients. This precision is further underscored by LeukoGraph's ability to maintain a correct label ratio, despite the inherent challenges posed by hierarchical classifications.

Flow cytometry mainly used for detecting the characteristics of a number of biochemical substances based on the expression of specific markers in cells. It is particularly useful for detecting membrane surface receptors, antigens, ions, or during DNA/RNA expression. Not only can it be employed as a biomedical research tool for recognising distinctive types of cells in mixed populations, but it can also be used as a diagnostic tool for classifying abnormal cell populations connected with disease. Modern flow cytometers can rapidly analyse tens of thousands of cells at the same time while also measuring multiple parameters from a single cell. However, the rapid development of flow cytometers makes it challenging for conventional analysis methods to interpret flow cytometry data. Researchers need to be able to distinguish interesting-looking cell populations manually in multi-dimensional data collected from millions of cells. Thus, it is essential to find a robust approach for analysing flow cytometry data automatically, specifically in identifying cell populations automatically. This thesis mainly concerns discover the potential shortcoming of current automated-gating algorithms in both real datasets and synthetic datasets. Three representative automated clustering algorithms are selected to be applied, compared and evaluated by completely and partially automated gating. A subspace clustering ProClus also implemented in this thesis. The performance of ProClus in flow cytometry is not well, but it is still a useful algorithm to detect noise.

ABSTRACT Typical state of the art flow cytometry data samples consists of measures of more than 100.000 cells in 10 or more features. AI systems are able to diagnose such data with almost the same accuracy as human experts. However, there is one central challenge in such systems: their decisions have far-reaching consequences for the health and life of people, and therefore, the decisions of AI systems need to be understandable and justifiable by humans. In this work, we present a novel explainable AI method, called ALPODS, which is able to classify (diagnose) cases based on clusters, i.e., subpopulations, in the high-dimensional data. ALPODS is able to explain its decisions in a form that is understandable for human experts. For the identified subpopulations, fuzzy reasoning rules expressed in the typical language of domain experts are generated. A visualization method based on these rules allows human experts to understand the reasoning used by the AI system. A comparison to a selection of state of the art explainable AI systems shows that ALPODS operates efficiently on known benchmark data and also on everyday routine case data. KEYWORDS: Explainable AI, Expert System, Symbolic System, Biomedical Data 1. INTRODUCTION State of the art machine learning (ML) artificial intelligence (AI) algorithms are effectively and efficiently able to diagnose (classify) high-dimensional data sets in modern medicine, e.g., for multiparameter flow cytometry data [Hu et al., 2019; Zhao et al., 2020]. These are systems that, after a training (learning) phase using learning data, perform well on data that are not part of the training data, i.e., the test data. This is called supervised learning [Murphy, 2012].

Flow cytometry is a high-throughput technology used to quantify multiple surface and intracellular markers at the level of a single cell. This enables to identify cell sub-types, and to determine their relative proportions. Improvements of this technology allow to describe millions of individual cells from a blood sample using multiple markers. This results in high-dimensional datasets, whose manual analysis is highly time-consuming and poorly reproducible. While several methods have been developed to perform automatic recognition of cell populations, most of them treat and analyze each sample independently. However, in practice, individual samples are rarely independent (e.g. longitudinal studies). Here, we propose to use a Bayesian nonparametric approach with Dirichlet process mixture (DPM) of multivariate skew $t$-distributions to perform model based clustering of flow-cytometry data. DPM models directly estimate the number of cell populations from the data, avoiding model selection issues, and skew $t$-distributions provides robustness to outliers and non-elliptical shape of cell populations. To accommodate repeated measurements, we propose a sequential strategy relying on a parametric approximation of the posterior. We illustrate the good performance of our method on simulated data, on an experimental benchmark dataset, and on new longitudinal data from the DALIA-1 trial which evaluates a therapeutic vaccine against HIV. On the benchmark dataset, the sequential strategy outperforms all other methods evaluated, and similarly, leads to improved performance on the DALIA-1 data. We have made the method available for the community in the R package NPflow.

We consider the problem of assigning class labels to an unlabeled test data set, given several labeled training data sets drawn from similar distributions. This problem arises in several applications where data distributions fluctuate because of biological, technical, or other sources of variation. We develop a distribution-free, kernel-based approach to the problem. This approach involves identifying an appropriate reproducing kernel Hilbert space and optimizing a regularized empirical risk over the space. We present generalization error analysis, describe universal kernels, and establish universal consistency of the proposed methodology. Experimental results on flow cytometry data are presented.