Multi-site reproducibility of a human immunophenotyping assay in whole blood and peripheral blood mononuclear cells preparations using CyTOF technology coupled with Maxpar Pathsetter, an automated data analysis system.

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™.

Automated Data Cleanup for Mass Cytometry.

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.

Sometimes simpler is better: VLog, a general but easy-to-implement log-like transform for 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.

Probability state modeling theory.

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.

Automated analysis of flow cytometric data for measuring neutrophil CD64 expression using a multi-instrument compatible probability state model.

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%.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part III - analytical issues.

Clinical diagnostic assays, may be classified as quantitative, quasi-quantitative or qualitative. The assay's description should state what the assay needs to accomplish (intended use or purpose) and what it is not intended to achieve. The type(s) of samples (whole blood, peripheral blood mononuclear cells (PBMC), bone marrow, bone marrow mononuclear cells (BMMC), tissue, fine needle aspirate, fluid, etc.

Automated quantitation of fetomaternal hemorrhage by flow cytometry for HbF-containing fetal red blood cells using probability state modeling.

Background: Flow cytometric methods (FCMs) are the contemporary standard for fetal red blood cell (RBC) quantitation and fetomaternal hemorrhage (FMH) detection. FCM provides greater sensitivity and repeatability relative to manual microscopic Kleihauer-Betke methods. FCM assays are not totally objective, employing subjective manual gating of fetal RBCs with measureable interobserver imprecision.

Automated analysis of GPI-deficient leukocyte flow cytometric data using GemStone™.

Background: Flow Cytometry is the standard for the detection of glycosylphosphatidylinositol (GPI)-deficient clones in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders. Although the International Clinical Cytometry Society (ICCS) and the International PNH Interest Group (IPIG) have published guidelines for PNH assays, data analysis has not been standardized. Current analyses use manual gating to enumerate PNH cells.

Effects of resolution reduction on data analysis.

Background: There is often a need in flow cytometry to display and analyze histograms at resolutions lower than those native to the data. It is common, for example, to analyze DNA histograms at 256-channel resolution, even though the data were acquired at 1,024 channels or more. The most common method for reducing resolution, referred to as the consecutive summation (CS) method, can introduce distortions into the shape of histograms.

DNA and cell cycle analysis as prognostic indicators in breast tumors revisited.

Both DNA ploidy and S-phase ploidy are promising prognostic factors for node-negative breast cancer patients. Based largely on the analysis of one large study, much of the reported problems with these factors have been caused by some unappreciated complexities in categorizing DNA ploidy into low- and high-risk groups and the lack of some necessary adjustments to eliminate unwanted correlations between DNA S-phase and ploidy. When both DNA ploidy and S-phase are compensated properly, they become independent prognostic markers, forming a powerful prognostic model.

Optimizing flow cytometric DNA ploidy and S-phase fraction as independent prognostic markers for node-negative breast cancer specimens.

Developing a reliable and quantitative assessment of the potential virulence of a malignancy has been a long-standing goal in clinical cytometry. DNA histogram analysis provides valuable information on the cycling activity of a tumor population through S-phase estimates; it also identifies nondiploid populations, a possible indicator of genetic instability and subsequent predisposition to metastasis. Because of conflicting studies in the literature, the clinical relevance of both of these potential prognostic markers has been questioned for the management of breast cancer patients.

Becoming a parent: the relation between prenatal expectations and postnatal experience.

The relationship between individuals' prenatal expectations about parenthood and their postnatal experience of parenthood was examined. Seventy-three primiparous couples were interviewed during the 3rd trimester of pregnancy and asked open-ended questions regarding their expectations. A content analysis of these expectations identified several themes.

Thinking ahead: complexity of expectations and the transition to parenthood.

This study examined the integrative complexity of thinking in individuals making the transition to parenthood, and the relationship between complexity and adjustment during this period. Sixty-nine couples were interviewed 3 months before their babies were born, and 6 months after the birth. The prenatal interview focussed on individuals' expectations about what it would be like being a parent; the postnatal interview focussed on individuals' actual experiences as parents.

Moral and social reasoning and perspective taking in later life: a longitudinal study.

In this study 27 older adults (ages 64-80) and 23 middle-aged adults (ages 35-54) were tested for moral stage, integrative complexity of social reasoning, and perspective-taking levels twice over a 4-year period. Moral reasoning stage levels did not change over time for either age group. Older adults, but not the middle-aged, showed a significant decline over time in level of moral perspective taking.

Four pathways in the analysis of adult development and aging: comparing analyses of reasoning about personal-life dilemmas.

Four systems for analyzing thinking about 2 personal-life dilemmas, as discussed by 29 men and 35 women (ages 35-85), were compared. Kohlberg's (1976) moral judgment stages, Kegan's (1982) ego-development stages, Gilligan's (1982) moral orientation system, and Suedfeld and Tetlock's (1977) integrative complexity scoring were used. Subjects completed Kohlberg's (Colby & Kohlberg, 1987) standard moral judgment measure, a self-concept description, and several questionnaires.

Reasoning about the self and relationships in maturity: an integrative complexity analysis of individual differences.

This study examined age (35-55 versus 65-85), gender, and self-concept-orientation differences in reasoning about the self, relationships, and morality, on the basis of the theorizing of Gilligan (1982). Participants were interviewed about general relationship issues, a specific relationship, and about the self. Reasoning was scored for integrative complexity (Suedfeld & Tetlock, 1977).

Religion, age, life satisfaction, and perceived sources of religiousness: a study of older persons.

Eighty-five persons aged 65 to 88 years participated in this interview study of three issues. The present study supported previous findings of a tendency toward increased religiosity in older age. This was tempered, however, by the finding that, although highly religious older persons tended to report an increase in religiousness over the course of their lives, respondents who were low in religiosity tended to report a decrease.