Best practices for user consultation in flow cytometry shared resource laboratories.

This "Best Practices in User Consultation" article is the result of a 2022 International Society for the Advancement of Cytometry (ISAC) membership survey that collected valuable insights from the shared research laboratory (SRL) community and of a group discussion at the CYTO 2022 workshop of the same name. One key takeaway is the importance of initiating a consultation at the outset of a flow cytometry project, particularly for trainees. This approach enables the improvement and standardization of every step, from planning experiments to interpreting data. This proactive approach effectively mitigates experimental bias and avoids superfluous trial and error, thereby conserving valuable time and resources. In addition to guidelines, the optimal approaches for user consultation specify communication channels, methods, and critical information, thereby establishing a structure for productive correspondence between SRL and users. This framework functions as an exemplar for establishing robust and autonomous collaborative relationships. User consultation adds value by providing researchers with the necessary information to conduct reproducible flow cytometry experiments that adhere to scientific rigor. By following the steps, instructions, and strategies outlined in these best practices, an SRL can readily tailor them to its own setting, establishing a personalized workflow and formalizing user consultation services. This article provides a pragmatic guide for improving the caliber and efficacy of flow cytometry research and aggregates the flow cytometry SRL community's collective knowledge regarding user consultation.

Multicompartmental analysis of the murine pulmonary immune response by spectral flow cytometry.

Studies of pulmonary inflammation require unique considerations due to the complex structure and composition of the lungs. The lungs have multiple compartments and diverse immune cell populations, with inherently high autofluorescence, and are involved in the host response to pulmonary pathogens. We describe a protocol that accounts for these factors through a novel combination of methodologies-in vivo compartmental analysis and spectral flow cytometry with a broad panel of antibodies. In vivo compartmental analysis enables the precise localization of immune cells within the marginated vasculature, lung interstitium, nonlavageable airways, and lavageable airways of the lungs, as well as the pulmonary lymph nodes. Spectral flow cytometry with a broad panel of antibodies supports an unbiased exploratory approach to investigating diverse immune cell populations during pulmonary inflammation. Most importantly, spectral flow uses cellular autofluorescence to aid in the resolution and identification of immune cell populations. This methodology enables the acquisition of high-quality data compatible with informed gating and dimensionality reduction algorithms. In addition, our protocol emphasizes considerations for compartmentalization of the inflammatory response, spectral flow panel design, and autofluorescence spectra analysis. These methodologies are critical for increasing the rigor of pulmonary research. We apply this protocol for the precise characterization and localization of leukocytes in the pulmonary host response to influenza A virus in C57BL/6J mice. In particular, we demonstrate that this protocol improves the quantification and localization of alveolar macrophages within the airways. The methodology is modifiable and expandable to allow for further characterization of leukocyte populations of special interest.NEW & NOTEWORTHY We describe a novel combination of methodologies that incorporates dual in vivo compartmental analysis using intravascular and intratracheal CD45 labeling, a broad panel of antibodies for identifying lymphoid and nonlymphoid cells, and spectral flow cytometry that uses cellular autofluorescence to aid in resolving and identifying immune cell populations. This methodology allows precise localization of immune cells in the lavageable airways, nonlavageable airways, interstitial lung tissue, and marginated in the lung vasculature.

Critical Steps in Data Management During a Crisis.

Data management is essential in a flow cytometry (FCM) shared resource laboratory (SRL) for the integrity of collected data and its long-term preservation, as described in the Cytometry publication from 2016, ISAC Flow Cytometry Shared Resource Laboratory (SRL) Best Practices (Barsky et al.: Cytometry Part A 89A(2016): 1017-1030). The SARS-CoV-2 pandemic introduced an array of challenges in the operation of SRLs. The subsequent laboratory shutdowns and access restrictions brought to the forefront well-established practices that withstood the impact of a sudden change in operations and illuminated areas that need improvement. The most significant challenges from a data management perspective were data access for remote analysis and workstation management. Notably, lessons learned from this challenge emphasize the importance of safeguarding collected data from loss in various emergencies such as fire or natural disasters where the physical hardware storing data could be directly affected. Here, we describe two data management systems that have been successful during the current emergency created by the pandemic, specifically remote access and automated data transfer. We will discuss other situations that could arise and lead to data loss or challenges in interpreting data. © 2020 International Society for Advancement of Cytometry.

Nrf2/antioxidant pathway mediates ß cell self-repair after damage by high-fat diet-induced oxidative stress.

Many theories have been advanced to better understand why ß cell function and structure relentlessly deteriorate during the course of type 2 diabetes (T2D). These theories include inflammation, apoptosis, replication, neogenesis, autophagy, differentiation, dedifferentiation, and decreased levels of insulin gene regulatory proteins. However, none of these have considered the possibility that endogenous self-repair of existing ß cells may be an important factor. To examine this hypothesis, we conducted studies with female Zucker diabetic fatty rats fed a high-fat diet (HFD) for 1, 2, 4, 7, 9, 18, or 28 days, followed by a return to regular chow for 2-3 weeks. Repair was defined as reversal of elevated blood glucose and of inappropriately low blood insulin levels caused by a HFD, as well as reversal of structural damage visualized by imaging studies. We observed evidence of functional ß cell damage after a 9-day exposure to a HFD and then repair after 2-3 weeks of being returned to normal chow (blood glucose [BG] = 348 ± 30 vs. 126 ± 3; mg/dl; days 9 vs. 23 day, P < 0.01). After 18- and 28-day exposure to a HFD, damage was more severe and repair was less evident. Insulin levels progressively diminished with 9-day exposure to a HFD; after returning to a regular diet, insulin levels rebounded toward, but did not reach, normal values. Increase in ß cell mass was 4-fold after 9 days and 3-fold after 18 days, and there was no increase after 28 days of a HFD. Increases in ß cell mass during a HFD were not different when comparing values before and after a return to regular diet within the 9-, 18-, or 28-day studies. No changes were observed in apoptosis or ß cell replication. Formation of intracellular markers of oxidative stress, intranuclear translocation of Nrf2, and formation of intracellular antioxidant proteins indicated the participation of HFD/oxidative stress induction of the Nrf2/antioxidant pathway. Flow cytometry-based assessment of ß cell volume, morphology, and insulin-specific immunoreactivity, as well as ultrastructural analysis by transmission electron microscopy, revealed that short-term exposure to a HFD produced significant changes in ß cell morphology and function that are reversible after returning to regular chow. These results suggest that a possible mechanism mediating the ability of ß cells to self-repair after a short-term exposure to a HFD is the activation of the Nrf2/antioxidant pathway.

International Society for Advancement of Cytometry (ISAC) flow cytometry shared resource laboratory (SRL) best practices.

The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. © 2016 International Society for Advancement of Cytometry.

Prevalence of Sexual Violence Against Women in 23 States and Two U.S. Territories, BRFSS 2005.

Sexual violence (SV) is a significant public health problem. Using data from the 2005 Behavioral Risk Factor Surveillance System (BRFSS), this article provides state-specific 12-month SV prevalence data for women residing in 23 states and two territories. Overall, more than 500,000 women in the participating states experienced completed or attempted nonconsensual sex in the 12-month period prior to the survey. The collection of state-level data using consistent, uniform, and behaviorally specific SV definitions enables states to evaluate the magnitude of the problem within their state and informs the development and evaluation of state-level SV programs, policies, and prevention efforts.

Prevalence of rural intimate partner violence in 16 US states, 2005.

CONTEXT: Intimate partner violence (IPV) is a public health problem that affects people across the entire social spectrum. However, no previous population-based public health studies have examined the prevalence of IPV in rural areas of the United States. Research on IPV in rural areas is especially important given that there are relatively fewer resources available in rural areas for the prevention of IPV. METHODS: In 2005, over 25,000 rural residents in 16 states completed the first-ever IPV module within the Behavioral Risk Factor Surveillance System (BRFSS). The BRFSS is a Centers for Disease Control and Prevention-sponsored annual random-digit-dialed telephone survey. The BRFSS provides surveillance of health behaviors and health risks among the non-institutionalized adult population of the United States and several US territories. FINDINGS: Overall, 26.7% of rural women and 15.5% of rural men reported some form of lifetime IPV victimization, similar to the prevalence found among men and women in non-rural areas. Within several states, those living in rural areas evidenced significantly higher lifetime IPV prevalence than those in non-rural areas. CONCLUSION: IPV is a significant public health problem in rural areas, affecting a similar portion of the population as in non-rural areas. More research is needed to examine how the experience of IPV is different for rural and non-rural residents.

Physical and sexual intimate partner violence and reported serious psychological distress in the 2007 BRFSS.

OBJECTIVES: We sought to determine the relationship between intimate partner violence (IPV) and serious psychological distress (SPD) as measured by the Kessler-6 (K6) among U.S. adults. We used data from the 2007 Behavioral Risk Factor Surveillance System (BRFSS) to determine whether individuals who reported multiple forms of IPV also reported higher prevalences of SPD compared with those who reported: 1) no physical or sexual IPV; 2) physical or sexual IPV only; and 3) threatened or attempted physical IPV. We also obtained adjusted prevalences for lifetime physical or sexual IPV. METHODS: We analyzed responses from three states that administered both the IPV and the K6 optional modules of the BRFSS in 2007. Respondents (5,985 men; 9,335 women) were categorized as experiencing threatened or attempted physical violence, physical violence, sexual violence, or both physical and sexual violence. We calculated lifetime IPV prevalence by demographic characteristics and performed adjusted and unadjusted logistic regressions of the relationship between level of IPV and SPD. RESULTS: 15.5 % of the sample reported some form of IPV. The prevalence of any IPV was almost twice as high in women (19.9 %) as in men (10.9 %). IPV was also associated with age, marital status, employment status, and income. Overall, the estimated prevalence of SPD was 2.9 % (95 % CI: 2.5-3.5). Among women, it was 2.1 % (95 % CI: 1.16-2.8) among those with no lifetime IPV and 15.4 % (95 % CI: 10.9-21.3) among those who reported both physical and sexual IPV. CONCLUSIONS: IPV is a serious public health problem associated with multiple adverse health outcomes, including SPD. In our study, the odds of SPD increased when respondents experience multiple forms of IPV. Medical and mental health practitioners should consider assessing exposure to IPV when patients have signs or symptoms of SPD or other conditions that might be consistent with IPV. Similarly, practitioners should consider assessing for IPV among patients with SPD. States should consider obtaining population-based IPV prevalence via the BRFSS to better plan for the health needs of their residents.

Characteristics of perpetrators in homicide-followed-by-suicide incidents: National Violent Death Reporting System--17 US States, 2003-2005.

Homicide-followed-by-suicide (referred to as "homicide-suicide") incidents are rare events but can have a profound impact on families and communities. A better understanding of perpetrator characteristics and how they compare with those of other homicide suspects and suicide decedents might provide insight into the nature of these violent acts. This report is based on 2003-2005 data from 17 US states participating in the National Violent Death Reporting System, a unique, incident-based, active surveillance system that integrates data on violent deaths from multiple sources. Of the 408 homicide-suicide incidents identified, most incidents were committed with a firearm (88.2%) and perpetrated by males (91.4%), those over 19 years of age (97.6%), and those of white race (77.0%); however, just over half of filicide (killing of children)-suicides (51.5%) were perpetrated by females. Over 55% of male homicide-suicide perpetrators versus 26.4% of other male suicide decedents had prior intimate partner conflicts (P < 0.001). In fact, having a history of intimate partner conflicts was even common among homicide-suicide perpetrators who did not victimize their intimate partners. Recognition of the link between intimate partner conflicts and homicide-suicide incidents and strategies involving collaboration among the court/legal and mental health systems might prevent these incidents.

Chronic disease and health risk behaviors associated with intimate partner violence-18 U.S. states/territories, 2005.

PURPOSE: Few studies have examined the association between intimate partner violence (IPV) and health outcomes for both women and men. The current study examined this relationship for women and men as part of a large cross-sectional public-health survey that collected information on a range of health behaviors and health risks. METHODS: In 2005, over 70,000 respondents in 16 states and 2 territories were administered the first-ever IPV module within the Behavioral Risk Factor Surveillance System (BRFSS). The BRFSS, sponsored by the Centers for Disease Control and Prevention, is an annual random-digit-dialed telephone survey. Lifetime IPV was assessed by four questions that asked about threatened, attempted, or completed physical violence, as well as unwanted sex. RESULTS: Women and men who reported IPV victimization during their lifetime were more likely to report joint disease, current asthma, activity limitations, HIV risk factors, current smoking, heavy/binge drinking, and not having had a checkup with a doctor in the past year. CONCLUSIONS: Experiencing IPV is associated with a number of adverse health outcomes and behaviors. There remains a need for the development of assessment opportunities and secondary intervention strategies to reduce the risk of negative health behaviors and long-term health problems associated with IPV victimization.

Prevalence and risk factors of intimate partner violence in eighteen U.S. states/territories, 2005.

BACKGROUND: Intimate partner violence (IPV) has been shown to have serious health consequences for both women and men, including poor general health, depressive symptoms, substance use, and elevated rates of chronic disease. Aside from crime surveys, there have been no large-scale IPV prevalence studies since the 1996 National Violence Against Women Survey. The lack of regular, ongoing surveillance, using uniform definitions and survey methods across states has hindered efforts to track IPV. In addition, the lack of state-specific data has hampered efforts at designing and evaluating localized IPV prevention programs. METHODS: In 2005, over 70,000 respondents were administered the first-ever IPV module within the Behavioral Risk Factor Surveillance System (BRFSS). The BRFSS is a Centers for Disease Control and Prevention-sponsored annual random-digit-dialed telephone survey, providing surveillance of health behaviors and health risks among the non-institutionalized adult population of the United States and several U.S. territories. RESULTS: Approximately 1 in 4 women and 1 in 7 men reported some form of lifetime IPV victimization. Women evidenced significantly higher lifetime and 12-month IPV prevalence, and were more likely to report IPV-related injury than men. IPV prevalence also varied by state of residence, race/ethnicity, age, income, and education. CONCLUSIONS: State-level data can assist state health officials and policy planners to better understand how many people have experienced IPV in their state. Such information provides a foundation on which to build prevention efforts directed toward this pervasive public health problem.

Are nitrate levels in groundwater stable over time?

Epidemiologists often use a retrospective study design to examine for associations between an exposure and the occurrence of adverse health effects. Several of these studies used this approach to examine for an association between elevated levels of nitrate in drinking water and related health effects such as methemoglobinemia, cancer, neural tube effects, or spontaneous abortions. Often, data on exposures that occurred before these health outcomes were not available. Consequently, researchers use measurements of exposures at the time of the study to represent exposures that occurred before people developed these conditions. An opportunity to examine the stability of nitrate in water occurred during a survey of private water wells in nine Midwestern states. In this survey, water samples from 853 homes with drilled wells were collected in May 1994 and in September 1995 and nitrate-nitrogen (nitrate-N) was measured by the colorimetric cadmium reduction method. Nitrate-N levels from the same well over time were assessed by a mixed-effects analysis of variance. Analysis showed no significant difference in between the initial level and those measured 16 months later. Furthermore, analysis showed that most of the variance in the nitrate concentrations in well water was due to well-to-well variation (89%) rather than to residual error (12%). This observation showed that a single measurement of nitrate in water from drilled wells could represent longer periods of exposure.

Prevalence and characteristics of sexual violence victimization among U.S. adults, 2001-2003.

This article provides the most recent U.S. prevalence estimates of forced sex and unwanted sexual activity. Results of a national telephone survey conducted in 2001-2003 indicate that 1 in 59 U.S. adults (2.7 million women and 978,000 men) experienced unwanted sexual activity in the 12 months preceding the survey and that 1 in 15 U.S. adults (11.7 million women and 2.1 million men) have been forced to have sex during their lifetime. There were 60.4% of females and 69.2% of males who were 17 years old or younger at the time the first forced sex occurred. This study provides an update to the National Violence Against Women Survey with more recent national data. Findings suggest that victimization rates have remained consistent since the 1990s. These findings suggest that a continued effort toward primary prevention of sexual violence, particularly rape of children and adolescents, is needed.

Sensitive topics, survey nonresponse, and considerations for interviewer training.

This paper discusses current challenges in achieving higher survey participation rates in random-digit-dial telephone surveys and proposes steps to address them through interviewer training to avoid refusals. It describes features of surveys that contribute to respondent reluctance to participate and offers a brief overview of current refusal aversion training methods to reduce nonresponse. It then identifies what challenges that unique features of random-digit-dial telephone surveys on sensitive topics might contribute to nonresponse. Recommendations are then proposed for changes in refusal aversion training, standard survey introductions, and informed consent procedures. Finally, further research is called for to identify which methods best balance the need to improve response rates with respondent safety and privacy in surveys with sensitive questions.

The association between self-reported lifetime history of forced sexual intercourse and recent health-risk behaviors: findings from the 2003 National Youth Risk Behavior Survey.

PURPOSE: To expand the understanding of the association between recent health-risk behaviors and a history of forced sexual intercourse, using a nationally representative sample of female and male high school students. METHODS: Data were from the 2003 National Youth Risk Behavior Survey, a nationally representative biennial survey of U.S. high school students. Lifetime history of forced sex, recent physical dating violence, and health-risk behaviors (substance use, diet-related behaviors, violence-related behaviors, and health promoting behaviors) were assessed. Analyses were stratified by gender and controlled for grade and race/ethnicity. RESULTS: Of students surveyed, 8.9% reported ever being forced to have sex. One in eight females and one in 16 males experienced forced sex in their lifetime. For females and males, a history of forced sex was associated with experiencing physical dating violence and suicidal ideation in the 12 months preceding the survey and with substance use in the previous 30 days. Female victims were not as likely as female nonvictims to have participated in team sports during the previous 12 months. Male victims were more likely than male nonvictims to have fasted for more than 24 hours to lose weight during the previous 30 days. CONCLUSIONS: A lifetime history of forced sex is associated with recent dating violence and participation in unhealthy behaviors. Services and intervention programs for victimized youth should address health concerns that have been linked to sexual assault. Such programs would provide opportunities for early intervention with lasting implications for improved health.

Telephone survey respondents' reactions to questions regarding interpersonal violence.

Concerns have been raised regarding the appropriateness of asking about violence victimization in telephone interviews and whether asking such questions increases respondents' distress or risk for harm. However, no large-scale studies have evaluated the impact of asking such questions during a telephone interview. This study explored respondents' reactions to questions regarding violence in two large recently completed telephone surveys. After respondents were asked about violence, they were asked if they thought surveys should ask such questions and whether they felt upset or afraid because of the questions. In both surveys, the majority of respondents (regardless of their victimization history) were willing to answer questions about violence and were not upset or afraid because of the questions. More than 92% of respondents thought such questions should be asked. These results challenge commonly held beliefs and assumptions and provide some assurance to those concerned with the ethical collection of data on violent victimization.