High‐dimensional mass cytometry data potentially enable a comprehensive characterization of immune cells. In order to positively affect clinical trials and translational clinical research, this advanced technology needs to demonstrate a high reproducibility of results across multiple sites for both peripheral blood mononuclear cells (PBMC) and whole blood preparations. A dry 30‐marker broad immunophenotyping panel and customized automated analysis software were recently engineered and are commercially available as the Fluidigm® Maxpar® Direct™ Immune Profiling Assay™. In this study, seven sites received whole blood and six sites received PBMC samples from single donors over a 2‐week interval. Each site labeled replicate samples and acquired data on Helios™ instruments using an assay‐specific acquisition template. All acquired sample files were then automatically analyzed by Maxpar Pathsetter™ software. A cleanup step eliminated debris, dead cells, aggregates, and normalization beads. The second step automatically enumerated 37 immune cell populations and performed label intensity assessments on all 30 markers. The inter‐site reproducibility of the 37 quantified cell populations had consistent population frequencies, with an average %CV of 14.4% for whole blood and 17.7% for PBMC. The dry reagent coupled with automated data analysis is not only convenient but also provides a high degree of reproducibility within and among multiple test sites resulting in a comprehensive yet practical solution for deep immune phenotyping.

SNE methods are a set of 9 to 10 interconnected algorithms that map high-dimensional data into low-dimensional space while minimizing loss of information. Each step in this process is important for producing high-quality maps. Cense′™ mapping not only enhances many of the steps in this process but also fundamentally changes the underlying mathematics to produce high-quality maps. The key mathematical enhancement is to leverage the Cauchy distribution for creating both high-dimensional and lowdimensional similarity matrices. This simple change eliminates the necessity of using perplexity and entropy and results in maps that optimally separate clusters defined in high-dimensional space. It also eliminates the loss of cluster resolution commonly seen with t-SNE with higher numbers of events. There is just one free parameter for Cen-se′ mapping, and that parameter rarely needs to change. Other enhancements include a relatively low memory footprint, highly threaded implementation, and a final classification step that can process millions of events in seconds. When the Cen-se′ mapping system is integrated with probability state modeling, the clusters of events are positioned in a reproducible manner and are colored, labeled, and enumerated automatically. We provide a step-by-step, simple example that describes how the Cen-se′ method works and differs from the t-SNE method. We present data from several experiments to compare the two mapping strategies on high-dimensional mass cytometry data. We provide a section on information theory to explain how the steepest gradient equations were formulated and how they control the movement of the low-dimensional points as the system renders the map Since existing implementations of the t-SNE algorithm can easily be modified with many of these enhancements, this work should result in more effective use of this very exciting and far-reaching new technology.

Mass cytometry is an emerging technology capable of 40 or more correlated measurements on a single cell. The complexity and volume of data generated by this platform have accelerated the creation of novel methods for high-dimensional data analysis and visualization. A key step in any high-level data analysis is the removal of unwanted events, a process often referred to as data cleanup. Data cleanup as applied to mass cytometry typically focuses on elimination of dead cells, debris, normalization beads, true aggregates, and coincident ion clouds from raw data. We describe a probability state modeling (PSM) method that automatically identifies and removes these elements, resulting in FCS files that contain mostly live and intact events. This approach not only leverages QC measurements such as DNA, live/dead, and event length but also four additional pulse-processing parameters that are available on Fluidigm Helios™ and CyTOF® (Fluidigm, Markham, Canada) 2 instruments with software versions of 6.3 or higher. These extra Gaussian-derived parameters are valuable for detecting well-formed pulses and eliminating coincident positive ion clouds. The automated nature of this new routine avoids the subjectivity of other gating methods and results in unbiased elimination of unwanted events. © 2019 International Society for Advancement of Cytometry.

The fundamental purpose of log and log-like transforms for cytometry is to make measured population variabilities as uniform as possible. The long-standing success of the log transform was its ability to stabilize linearly increasing gain-dependent uncertainties and the success of the log-like transforms is that they extend this notion to include zero and negative measurement values. This study derives and examines a transform called VLog that stabilizes the three general sources of variability: (1) gain-dependent variability, (2) photo-electron counting error, and (3) signal-independent sources of error. Somewhat surprisingly, this transform has a closed-form solution and therefore is relatively simple to implement. By including some quantitation elements in its formulation, the shape-dependent arguments, α and β, usually do not require optimization for different datasets. The simplicity and generality of the transform may make it a useful tool for cytometry and possibly other technologies. © 2016 International Society for Advancement of Cytometry.

Methamphetamine (METH) is a widely used psychostimulant that severely impacts the host's innate and adaptive immune systems and has profound immunological implications. T cells play a critical role in orchestrating immune responses. We have shown recently how chronic exposure to METH affects T cell activation using a murine model of lymphocytic choriomeningitis virus (LCMV) infection. Using the TriCOM (trinary state combinations) feature of GemStone™ to study the polyfunctionality of T cells, we have analyzed how METH affected the cytokine production pattern over the course of chronic LCMV infection. Furthermore, we have studied in detail the effects of METH on splenic T cell functions, such as cytokine production and degranulation, and how they regulate each other. We used the Probability State Modeling (PSM) program to visualize the differentiation of effector/memory T cell subsets during LCMV infection and analyze the effects of METH on T cell subset progression. We recently demonstrated that METH increased PD-1 expression on T cells during viral infection. In this study, we further analyzed the impact of PD-1 expression on T cell functional markers as well as its expression in the effector/memory subsets. Overall, our study indicates that analyzing polyfunctionality of T cells can provide additional insight into T cell effector functions. Analysis of T cell heterogeneity is important to highlight changes in the evolution of memory/effector functions during chronic viral infections. Our study also highlights the impact of METH on PD-1 expression and its consequences on T cell responses.

As the technology of cytometry matures, there is mounting pressure to address two major issues with data analyses. The first issue is to develop new analysis methods for high-dimensional data that can directly reveal and quantify important characteristics associated with complex cellular biology. The other issue is to replace subjective and inaccurate gating with automated methods that objectively define subpopulations and account for population overlap due to measurement uncertainty. Probability state modeling (PSM) is a technique that addresses both of these issues. The theory and important algorithms associated with PSM are presented along with simple examples and general strategies for autonomous analyses. PSM is leveraged to better understand B-cell ontogeny in bone marrow in a companion Cytometry Part B manuscript. Three short relevant videos are available in the online supporting information for both of these papers. PSM avoids the dimensionality barrier normally associated with high-dimensionality modeling by using broadened quantile functions instead of frequency functions to represent the modulation of cellular epitopes as cells differentiate. Since modeling programs ultimately minimize or maximize one or more objective functions, they are particularly amenable to automation and, therefore, represent a viable alternative to subjective and inaccurate gating approaches.

BackgroundHuman progenitor and B-cell development is a highly regulated process characterized by the ordered differential expression of numerous cell-surface and intracytoplasmic antigens. This study investigates the underlying coordination of these modulations by examining a series of normal bone marrow samples with the method of probability state modeling or PSM.ResultsThe study is divided into two sections. The first section examines B-cell stages subsequent to CD19 up-regulation. The second section assesses an earlier differentiation stage before and including CD19 up-regulation.Post-CD19 Antigenic Up-RegulationStatistical analyses of cytometry data derived from sixteen normal bone marrow specimens revealed that B cells have at least three distinct coordinated changes, forming four stages labeled as B1, B2, B3, and B4. At the end of B1; CD34 antigen expression down-regulates with TdT while CD45, CD81, and CD20 slightly up-regulate. At the end of B2, CD45 and CD20 up-regulate. At the end of B3 and beginning of B4; CD10, CD38, and CD81 down-regulate while CD22 and CD44 up-regulate.Pre-CD19 Antigenic Up-RegulationStatistical analysis of ten normal bone marrows revealed that there are at least two measurable coordinated changes with progenitors, forming three stages labeled as P1, P2, and P3. At the end of P1, CD38 up-regulates. At the end of P2; CD19, CD10, CD81, CD22, and CD9 up-regulate while CD44 down-regulates slightly.ConclusionsThese objective results yield a clearer immunophenotypic picture of the underlying cellular mechanisms that are operating in these important developmental processes. Also, unambiguously determined stages define what is meant by normal B-cell development and may serve as a preliminary step for the development of highly sensitive minimum residual disease detection systems. A companion article is simultaneously being published in Cytometry Part A that will explain in further detail the theory behind PSM. Three short relevant videos are available in the online supporting information for both of these papers. (c) 2015 International Clinical Cytometry Society

BACKGROUND:Leuko64™ (Trillium Diagnostics) is a flow cytometric assay that measures neutrophil CD64 expression and serves as an in vitro indicator of infection/sepsis or the presence of a systemic acute inflammatory response. Leuko64 assay currently utilizes QuantiCALC, a semiautomated software that employs cluster algorithms to define cell populations. The software reduces subjective gating decisions, resulting in interanalyst variability of <5%. We evaluated a completely automated approach to measuring neutrophil CD64 expression using GemStone™ (Verity Software House) and probability state modeling (PSM). METHODS:Four hundred and fifty-seven human blood samples were processed using the Leuko64 assay. Samples were analyzed on four different flow cytometer models: BD FACSCanto II, BD FACScan, BC Gallios/Navios, and BC FC500. A probability state model was designed to identify calibration beads and three leukocyte subpopulations based on differences in intensity levels of several parameters. PSM automatically calculates CD64 index values for each cell population using equations programmed into the model. GemStone software uses PSM that requires no operator intervention, thus totally automating data analysis and internal quality control flagging. Expert analysis with the predicate method (QuantiCALC) was performed. Interanalyst precision was evaluated for both methods of data analysis. RESULTS:PSM with GemStone correlates well with the expert manual analysis, r(2) = 0.99675 for the neutrophil CD64 index values with no intermethod bias detected. The average interanalyst imprecision for the QuantiCALC method was 1.06% (range 0.00-7.94%), which was reduced to 0.00% with the GemStone PSM. The operator-to-operator agreement in GemStone was a perfect correlation, r(2) = 1.000. CONCLUSION:Automated quantification of CD64 index values produced results that strongly correlate with expert analysis using a standard gate-based data analysis method. PSM successfully evaluated flow cytometric data generated by multiple instruments across multiple lots of the Leuko64 kit in all 457 cases. The probability-based method provides greater objectivity, higher data analysis speed, and allows for greater precision for in vitro diagnostic flow cytometric assays.

The clinical management of patients with acute lymphoblastic leukemia (ALL) relies on accurate prediction of relapse hazard to determine the intensity of therapy and to avoid over- or under- treatment. Minimal residual disease (MRD) is a very effective method for predicting response to treatment and potential relapse in patients with ALL. The most validated method used to assess MRD in ALL is flow cytometric (FCM) analysis of leukemia-associated immunophenotypes which can detect up to 1 leukemic cell per 10,000 normal cells (0.01%). There is no standard method for detecting MRD by FCM in ALL patients. Our institutional approach has been to evaluate 20,000- 50,000 events using a specific immunophenotypic panel and to analyze the data using WinList software with a concurrent comparison with the diagnostic immunophenotype.

We recently reported on the use of cDNA subtraction combined with microarray based expression analysis for identifying genes that are differentially over-expressed in small cell lung carcinoma. One of the several hundred genes identified using this approach was termed L985P and its molecular characterization is described in this report. The differential over-expression of L985P mRNA in SCLC, as determined by microarray analysis, was confirmed by real-time RT-PCR and Northern blot analysis. Immunohistochemical analyses show that L985P protein is highly expressed in SCLC with very restricted expression observed in normal lung, which was confined to the apical region of the ciliated bronchiolar epithelium. Flow cytometric and immunohistochemical analysis showed that L985P was localized to the cell surface. Sequence homology comparison indicated that L985P is identical to MS4A8B, a member of the recently described membrane-spanning 4-domain family, subfamily A (MS4A) gene family. The MS4A gene family currently consists of greater than 20 distinct human and mouse proteins that include CD20 and FcepsilonRIbeta. Both CD20 and FcepsilonRIbeta are involved in signaling events that regulate diverse cellular functions including cell growth regulation and differentiation. Collectively, the results presented herein demonstrate that L985P is differentially over-expressed in SCLC and may have potential clinical utility as an immunotherapeutic target for the treatment of SCLC.

BackgroundHistone deacetylases (HDAC) are associated with a variety of transcriptional repressors that control cellular differentiation and proliferation. HDAC inhibitors such as trichostatin A, trapoxin and chlamydocin could be useful tools to modulate these cellular processes. We investigated their effect on the self-renewal of hematopoietic stem cells (HSC) during ex vivo culture.MethodsPurified murine HSC with the phenotype c-Kit(+), Thy-1.1(lo), Lin(-/lo), Sca-1(+) were cultured for 4 days with IL-3, IL-6 and c-Kit ligand without or with HDAC inhibitors, after which their degree of phenotypic differentiation in culture was assessed by flow cytometric analysis. To explore whether HDAC inhibitors could have a beneficial role in human HSC transplantation, mobilized peripheral blood CD34(+) cells were cultured with thrombopoietin mimetic peptide, flt3 ligand, and c-Kit ligand, without or with various HDAC inhibitors. The fluorescent dye, carboxyfluorescein-diacetate succinimidylester ( CFSE), was used to track division of cell subsets, and engrafting ability was evaluated in a non-obese diabetic (NOD) - SCID xenotransplantation model.ResultsMurine HSC cultured with HDAC inhibitors maintained a more primitive phenotype than control cultures. The number of human HSC expressing Thy-1 increased up to seven-fold during a 5-day culture with HDAC inhibitors compared with control cultures. Chlamydocin was the most effective of the HDAC inhibitors tested at promoting Thy-1 expression on human cells. CFSE tracking showed that the increase in Thy-1(+) cells resulted from cell division. In a NOD-SCID repopulation assay, cells exposed to chlamydocin for 24 h displayed an average four-fold higher engrafting ability over control cells.DiscussionOur studies suggest that HDAC inhibitors can induce ex vivo expansion of human HSC, and may improve engraftment in hematopoietic transplant patients when cell dose is limiting.

OBJECTIVE:The aim of this study was to determine the identity of the cell surface molecule on primitive hematopoietic cells recognized by monoclonal antibody HCC-1. MATERIALS AND METHODS:Screening of a cDNA expression library prepared from human bone marrow stromal cells with HCC-1 yielded a single cDNA, which when expressed in FDCP-1 cells, resulted in the specific acquisition of HCC-1 binding. The cDNA demonstrated complete identity with CD59, a phosphoinositol glycan-linked membrane protein that protects cells against autologous complement attack. The ubiquitous expression of CD59 is in marked contrast to the restricted reactivity of HCC-1. Studies were performed to examine the basis for the novel specificity of HCC-1 for CD59. The epitope on CD59 identified by HCC-1 was mapped using a series of rat/human CD59 chimeric proteins. Immunoprecipitation analyses were performed to determine whether CD59 associates with other membrane proteins. RESULTS:Mutagenesis of Asn18 did not alter the binding of HCC-1 to CD59, suggesting that N-linked carbohydrates are not responsible for the binding specificity of HCC-1. The epitope for HCC-1 was shown to differ from that identified by previously described CD59 antibodies, encompassing residues A31, L33, R55, and L59. An 80 kDa protein co-immunoprecipitated with CD59 in the HCC-1(-) cell line HL-60 but not in HCC-1(+) K562 cells. CONCLUSION:Collectively, these data support the hypothesis that the unique specificity of HCC-1 for CD59 is due in part to recognition of a novel epitope, which is masked as a result of association with an as yet unidentified 80 kDa protein.

BACKGROUND:Strategies for transplanting primitive hematopoietic progenitor (PHP) cells are under development that require in vitro manipulation of cells for several hours to several days prior to transplantation. This applies to gene-therapy protocols involving transduction with adenoviral or lentiviral vectors (typically 1 day of ex vivo culture) or retroviral vectors (up to 3 days of culture).METHODS:Human mobilized peripheral blood (MPB) CD34(+) cells were cultured with the cytokines thrombopoietin mimetic peptide (mTPO), flt3 ligand (FL), and c-kit ligand (KL). Equal numbers of CD34(+) cells, either uncultured or cultured for various time periods up to 5 days, were tested for engraftment in sublethally irradiated 8-10 week-old NOD/SCID mice. Cells were also compared for expression and function of several key surface molecules.RESULTS:At a limiting dose of 1 million cells, mice receiving uncultured cells had a mean of 20% CD45(+) (human) cells in their BM 6 weeks post-transplantation, versus 3% for mice receiving 3-5 day cultured cells. Analysis of 10 surface molecules, CD11a, CD18, CD29, CD49d, CD49e, CXCR-4, CD62L, CD31, CD43, and CD44 over a 5-day culture period showed that their expression levels were either maintained or up-regulated on CD34(+) cells and the primitive Thy-1(+) subset. Similar percentages of uncultured and 3-day cultured MPB CD34(+) cells bound to plates coated with vascular cell adhesion molecule-1 (VCAM-1) under both static and physiological flow conditions, and chemotaxis of cultured cells towards stromal-derived factor-1 (SDF-1) was not impaired, suggesting that VLA-4 and CXCR-4 were functional on cultured cells. CD34(+) Thy-1(+) MPB cells cultured with cytokines expressed increasing levels of Fas receptor beginning at 20 h in culture, with peak expression levels after 3 days (mean Day 0 expression, 39%; mean Day 3 expression, 86%), without increased apoptosis. Including inhibitors of caspases in the media of cells cultured for 24-48 h significantly improved their engraftment in a SCID-hu bone-engraftment model.DISCUSSION:Increased susceptibility to apoptosis upon in vivo injection may contribute to impaired engraftment of in vitro manipulated cells. Inhibitors of apoptosis may increase their engrafting capacity in clinical settings.

BACKGROUNDIncreasing use of phenotypically-enriched stem cell populations for clinical hematopoietic transplants has led to an urgent demand for a reliable, rapid and simple functional assay which would provide an estimation of the reconstituting potential of cells prior to transplantation.METHODSWe have developed a 2-week quantitative, stroma-free assay to measure the frequency of primitive progenitors within hematopoietic cell samples. This relatively short-term assay provides frequency information which correlates with that measured by a 5-week stroma-dependent CAFC assay. Cells with the phenotype CD34+Thy-1+ were purified by fluorescence-activated cell sorting from peripheral blood apheresis products of multiple myeloma patients mobilized with cytoxan and GM-CSF. CD34+Thy-1+ cells were plated at limiting dilution into microtiter wells and cultured in an Iscove's based serum-deprived culture medium, supplemented with the cytokines, interleukin (IL)-3, IL-6, G-CSF, Flk2/Flt3 ligand (FL) and Kit ligand (KL). After 2 weeks, cell proliferation in individual wells was quantified by microscopy and bright-field imaging, or by using a fluorescent nucleic acid-binding dye and fluorimetry. Poisson statistics were used to calculate the frequency of wells containing cells with high proliferative potential (wells containing > or = 500 cells).RESULTSProgenitor cell frequencies generated using this assay were compared by linear regression analysis to those generated from 32 parallel CAFC and CFU-C assays performed on the same patient samples. Correlations were r = 0.80, r2 = 0.65, and r = 0.76, r2 = 0.58, respectively; these correlations were highly significant (p < 10(-7)).DISCUSSIONThis limiting dilution assay should more directly quantitate the potential of primitive hematopoietic cells than a CFU-C assay. It also has advantages over both the CAFC and the CFU-C assay, in that scoring has been automated, making it simple, rapid, and objective compared with manual cobblestone area or colony counting. The described limiting dilution assay may provide a useful alternative to assays currently used to evaluate the viability and proliferative potential of purified hematopoietic cells intended for transplant.

Various combinations of cytokines have profoundly different effects on inhibition of apoptosis and stimulation of self-renewal division of hematopoietic stem cells (HSC) in short-term, ex vivo culture. Our goal was to quantitate expansion of cells with a primitive CD34+ Thy-1+ phenotype, as well as cell cycling, division history, differentiation, and apoptosis of CD34+ cells enriched from normal donor mobilized peripheral blood (MPB) cells. The balance of these parameters determines the net number of transplantable HSC produced in ex vivo cultures. Comparing several different combinations of cytokines added to 90-hour cultures of MPB CD34 cells, thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL) gave the best result, with the lowest percentage of apoptotic cells and a mean 1.2-fold increase in the number of CD34+ Thy-1+ cells. A combination of interleukin 3 (IL-3), interleukin 6 (IL-6), and leukemia inhibitory factor (LIF) gave the worst outcome, including a decrease of CD34+ Thy-1+ cell number to a mean of 30% of the starting cell number. Cell division history was tracked using the dye 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE). Division of CD34+ Thy-1+ cells was faster and more synchronous in TPO, FL, and KL than in IL-3, IL-6, and LIF, which left a significant proportion of CD34+ cells undivided. Such detailed analyses of short-term, ex vivo cultures generated "replication scores," which allowed prediction of a sixfold improvement of the efficiency of gene transduction of primitive hematopoietic progenitors from MPB, using TPO, FL, and KL to replace IL-3, IL-6, and LIF. Analysis of retroviral transduction efficiency confirmed the increase of transgene expression from MPB primitive hematopoietic progenitors assayed after stromal culture was fivefold, validating the usefulness of multiparameter analysis of short-term cultures for survival and replication of CD34+ Thy-1+ cells.

Inefficient retroviral-mediated gene transfer to human hematopoietic stem cells (HSC) and insufficient gene expression in progeny cells derived from transduced HSC are two major problems associated with HSC-based gene therapy. In this study we evaluated the ability of a murine stem cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintain gene expression in transduced human hematopoietic cells. CD34(+) cells lacking lineage differentiation markers (CD34(+)Lin-) isolated from human bone marrow and mobilized peripheral blood were transduced using an optimized clinically applicable protocol. Under the conditions used, greater than 75% of the CD34(+) cell population retained the Lin- phenotype after 4 days in culture and at least 30% of these expressed a high level of NGFR (NGFR+) as assessed by fluorescence-activated cell sorter analysis. When these CD34(+)Lin-NGFR+ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term stromal cultures, sustained reporter expression was observed in differentiated erythroid and myeloid cells derived from transduced progenitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34(+)Lin-NGFR+ cells were used to repopulate human bone grafts implanted in severe combined immunodeficient mice, MSCV-directed NGFR expression could be detected on 37% +/- 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC.