Interlaboratory comparison of high-throughput protein biomarker assay quantifications for radiation exposure classification.

In the event of a widespread radiological incident, thousands of individuals will require rapid assessment of exposure using validated biodosimetry assays to inform clinical triage. In this scenario, multiple biodosimetry laboratories may be necessary for large-volume sample processing. To meet this need, we have developed a high-throughput assay for the rapid measurement of intracellular protein biomarkers in human peripheral blood samples using an Imaging Flow Cytometry (IFC) platform. The objective of this work was to harmonize and validate the reproducibility of our blood biomarker assay for radiation exposure across three IFC instruments, two located at Columbia University (CU) and the third at Health Canada. The Center for Radiological Research (CRR) at CU served as the central laboratory and reference instrument, where samples were prepared in triplicate, labeled with two radiation responsive leukocyte biomarkers (BAX and phosphor-p53 (Ser37)), and distributed for simultaneous interrogation by each IFC. Initial tests showed that significantly different baseline biomarker measurements were generated on each instrument when using the same acquisition settings, suggesting that harmonization of signal intensities is necessary. Subsequent tests harmonized biomarker measurements after irradiation by modulating laser intensity using two reference materials: unstained samples and standardized rainbow beads. Both methods generated measurements on each instrument without significant differences between the new and references instruments, allowing for the use of one master template to quantify biomarker expression across multiple instruments. Deming regression analyses of 0-5 Gy dose-response curves showed overall good correlation of BAX and p53 values across new and reference instruments. While Bland-Altman analyses indicated low to moderate instrument biases, ROC Curve analyses ultimately show successful discrimination between exposed and unexposed samples on each instrument (AUC values > 0.85).

Development of high-throughput systems for biodosimetry.

Biomarkers for ionising radiation exposure have great utility in scenarios where there has been a potential exposure and physical dosimetry is missing or in dispute, such as for occupational and accidental exposures. Biomarkers that respond as a function of dose are particularly useful as biodosemeters to determine the dose of radiation to which an individual has been exposed. These dose measurements can also be used in medical scenarios to track doses from medical exposures and even have the potential to identify an individual's response to radiation exposure that could help tailor treatments. The measurement of biomarkers of exposure in medicine and for accidents, where a larger number of samples would be required, is limited by the throughput of analysis (i.e. the number of samples that could be processed and analysed), particularly for microscope-based methods, which tend to be labour-intensive. Rapid analysis in an emergency scenario, such as a large-scale accident, would provide dose estimates to medical practitioners, allowing timely administration of the appropriate medical countermeasures to help mitigate the effects of radiation exposure. In order to improve sample throughput for biomarker analysis, much effort has been devoted to automating the process from sample preparation through automated image analysis. This paper will focus mainly on biological endpoints traditionally analysed by microscopy, specifically dicentric chromosomes, micronuclei and gamma-H2AX. These endpoints provide examples where sample throughput has been improved through automated image acquisition, analysis of images acquired by microscopy, as well as methods that have been developed for analysis using imaging flow cytometry.

Monte Carlo modelling of experimental setup used for biodosimetry intercomparison.

When using biodosimetry techniques to assess absorbed dose from an ionising radiation exposure, a calibration curve is required. At Health Canada (HC), these curves are generated for a variety of radiation qualities and assays to translate biological damage into absorbed dose. They are produced by irradiating biological samples in custom-designed water-equivalent phantoms inside a cabinet X-ray machine. In the HC lab, two different phantoms can be used for irradiation that differs in material composition and internal geometry. To ensure consistency, the impact of using the phantoms interchangeably was investigated. This was done through lab measurements and the development of a Monte Carlo (MC) model. Differences up to 6.7% were found between the two experimental setups, indicating the need for careful consideration if using these setups interchangeably in the laboratory. Once validated, the MC model can be used to investigate different aspects of the experimental setup without the need for laboratory measurements.

Application of the Cytokinesis-Block Micronucleus Assay for High-Dose Exposures Using Imaging Flow Cytometry.

The cytokinesis-block micronucleus assay is a well-established method to assess radiation-induced genetic damage in human cells. This assay has been adapted to imaging flow cytometry (IFC), allowing automated analysis of many cells, and eliminating the need to create microscope slides. Furthermore, to improve the efficiency of assay performance, a small-volume method previously developed was employed. Irradiated human blood samples were cultured, stained, and analyzed by IFC to produce images of the cells. Samples were run using both manual and 96-well plate automated acquisition. Multiple parameter-based image features were collected for each sample, and the results were compared to confirm that these acquisition methods are functionally identical. This paper details the multi-parametric analysis developed and the resulting calibration curves up to 10 Gy. The calibration curves were created using a quadratic random coefficient model with Poisson errors, as well as a logistic discriminant function. The curves were then validated with blinded, irradiated samples, using relative bias and relative mean square error. Overall, the accuracy of the dose estimates was adequate for triage dosimetry (within 1 Gy of the true dose) over 90% of the time for lower doses and about half the time for higher doses, with the lowest success rate between 5 and 6 Gy where the calibration curve reached its peak and there was the smallest change in MN/BNC with dose. This work describes the application of a novel multi-parametric analysis that fits the calibration curves and allows dose estimates up to 10 Gy, which were previously limited to 4 Gy. Furthermore, it demonstrates that the results from samples acquired manually and with the autosampler are functionally similar.

The Imaging Flow Cytometry-Based Cytokinesis-Block MicroNucleus (CBMN) Assay.

The dose of ionizing radiation received by an individual can be determined using biodosimetry methods which measure biomarkers of exposure in tissue samples from that individual. These markers can be expressed in many ways, including DNA damage and repair processes. Following a mass casualty event involving radiological or nuclear material, it is important to rapidly provide this information to medical responders to assist in the medical management of potentially exposed casualties. Traditional methods of biodosimetry rely on microscope analysis, making them time-consuming and labor-intensive. To increase sample throughput following a large-scale radiological mass casualty event, several biodosimetry assays have been adapted for analysis by imaging flow cytometry. This chapter briefly reviews these methods with a focus on the most current methodology to identify and quantify micronuclei in binucleated cells within the cytokinesis-block micronucleus assay using an imaging flow cytometer.

Advanced airway management and drug-assisted intubation skills in an advanced critical care practitioner team.

INTRODUCTION: Airway management, including endotracheal intubation, is one of the cornerstones of care of critically ill patients. Internationally, health professionals from varying backgrounds deliver endotracheal intubation as part of their critical care role. This article considers the development of airway management skills within a single advanced critical care practitioner (ACCP) team and uses case series data to analyse the safety profile in performing this aspect of critical care. Skills were acquired during and after the ACCP training pathway. A combination of theoretical teaching, theatre experience, simulation and work-based practice was used. Case series data of all critical care intubations by ACCPs were collected. Audit results: Data collection identified 675 intubations carried out by ACCPs, 589 of those being supervised, non-cardiac arrest intubations requiring drugs. First pass success was achieved in 89.6% of cases. A second intubator was required in 4.3% of cases. Some form of complication was experienced by 42.3% of patients; however, the threshold for complications was set at a low level. CONCLUSIONS: This ACCP service developed a process to acquire advanced airway management skills including endotracheal intubation. Under medical supervision, ACCPs delivered advanced airway management achieving a first pass success rate of 89.6%, which compares favourably with both international and national success rates. Although complications were experienced in 48.3% of patients, when similar complication cut-offs are compared with published data, ACCPs also matched favourably.

Evaluation of the safety of inter-hospital transfers of critically ill patients led by advanced critical care practitioners.

INTRODUCTION: Ten thousand inter-hospital transfers of critically ill adults take place annually in the UK. Studies highlight deficiencies in experience and training of staff, equipment, stabilisation before departure, and logistical difficulties. This article is a quality improvement review of an advanced critical care practitioner (ACCP)-led inter-hospital transfer service. METHODS: The tool Standards for Quality Improvement Reporting Excellence was used as the format for the review, combined with clinical audit of advanced critical care practitioner-led transfers over a period of more than 3 years. RESULTS: The transfer service has operated for 8 years; ACCPs conducted 934 critical care transfers of mechanically ventilated patients, including 286 inter-hospital transfers, between January 2017 and September 2020. The acuity of transfer patients was high, 82.2% required support of more than one organ, 49% required more than 50% oxygen. Uneventful transfer occurred in 81.4% of cases; the most common patient-related complication being hypotension, logistical issues were responsible for half of the complications. CONCLUSION: This quality improvement project provides an example of safe and effective advanced practice in an area that is traditionally a medically led domain. ACCPs can provide an alternative process of care for critically ill adults who require external transfer, and a benchmark for audit and quality improvement.

Increasing Childhood Asthma Care Appointments on a Mobile Asthma Van.

Children in two communities of a large city in the Midwestern United States have higher rates of asthma than other areas of the city. The communities have barriers to accessing care, including high rates of unemployment and being uninsured and undocumented. A mobile van provides no-cost asthma care to children at schools in these communities, but use of these services has decreased more than 50% over the past 5 years. School nurses have the potential to improve asthma outcomes by collaborating with health-care providers. The purpose of the program was to increase the number of appointments scheduled and attended on the asthma van at both schools. For this program, we (a) implemented an unaccompanied minor consent, (b) enhanced care coordination, and (c) improved a respiratory health survey tool. Results showed an increased number of appointments scheduled and attended on the asthma van. The program was successful even though community-specific barriers existed.

The provision of central venous access, transfer of critically ill patients and advanced airway management.: Are advanced critical care practitioners safe and effective?

Advanced critical care practitioners are a new and growing component of the critical care multidisciplinary team in the United Kingdom. This audit considers the safety profile of advanced critical care practitioners in the provision of central venous catheterisation and transfer of ventilated critical care patients without direct supervision and supervised drug assisted intubation of critically ill patients. The audit showed that advanced critical care practitioners can perform central venous cannulation, transfer of critically ill ventilated patients and intubation with parity to published UK literature.

Automated Triage Radiation Biodosimetry: Integrating Imaging Flow Cytometry with High-Throughput Robotics to Perform the Cytokinesis-Block Micronucleus Assay.

The cytokinesis-block micronucleus (CBMN) assay has become a fully-validated and standardized method for radiation biodosimetry. The assay is typically performed using microscopy, which is labor intensive, time consuming and impractical after a large-scale radiological/nuclear event. Imaging flow cytometry (IFC), which combines the statistical power of traditional flow cytometry with the sensitivity and specificity of microscopy, has been recently used to perform the CBMN assay. Since this technology is capable of automated sample acquisition and multi-file analysis, we have integrated IFC into our Rapid Automated Biodosimetry Technology (RABiT-II). Assay development and optimization studies were designed to increase the yield of binucleated cells (BNCs), and improve data acquisition and analysis templates to increase the speed and accuracy of image analysis. Human peripheral blood samples were exposed ex vivo with up to 4 Gy of c rays at a dose rate of 0.73 Gy/min. After irradiation, samples were transferred to microtubes (total volume of 1 ml including blood and media) and organized into a standard 8 × 12 plate format. Sample processing methods were modified by increasing the blood-to-media ratio, adding hypotonic solution prior to cell fixation and optimizing nuclear DRAQ5 staining, leading to an increase of 81% in BNC yield. Modification of the imaging processing algorithms within IFC software also improved BNC and MN identification, and reduced the average time of image analysis by 78%. Finally, 50 ll of irradiated whole blood was cultured with 200 ll of media in 96-well plates. All sample processing steps were performed automatically using the RABiT-II cell: :explorer robotic system adopting the optimized IFC-CBMN assay protocol. The results presented here detail a novel, high-throughput RABiT-IFC CBMN assay that possesses the potential to increase capacity for triage biodosimetry during a large-scale radiological/nuclear event.

The potential for complete automated scoring of the cytokinesis block micronucleus cytome assay using imaging flow cytometry.

The lymphocyte Cytokinesis-Block Micronucleus (CBMN) assay was originally developed for the measurement of micronuclei (MN) exclusively in binucleated (BN) cells, which represent the population of cells that can express MN because they completed nuclear division. Recently the assay has evolved into a comprehensive cytome method to include biomarkers that measure chromosomal instability and cytotoxicity by quantification of nuclear buds (NBUDs), nucleoplasmic bridges (NPBs) and apoptotic/necrotic cells. Furthermore, enumeration of mono- and polynucleated cells allows for computation of the nuclear division index (NDI) to assess mitotic activity. Typically performed by manual microscopy, the CBMN cytome assay is laborious and subject to scorer bias and fatigue, leading to inter- and intra-scorer variability. Automated microscopy and conventional flow cytometry methods have been developed to automate scoring of the traditional and cytome versions of the assay. However, these methods have several limitations including the requirement to create high-quality microscope slides, lack of staining consistency and sub-optimal nuclear/cytoplasmic visualization. In the case of flow cytometry, stripping of the cytoplasmic membrane makes it impossible to measure MN in BN cells, calculate the NDI or to quantify apoptotic or necrotic cells. Moreover, the absence of cellular visualization using conventional flow cytometry, makes it impossible to quantify NBUDs and NPBs. In this review, we propose that imaging flow cytometry (IFC), which combines high resolution microscopy with flow cytometry, may overcome these limitations. We demonstrate that by using IFC, images from cells in suspension can be captured, removing the need for microscope slides and allowing visualization of intact cytoplasmic membranes and DNA content. Thus, mono-, bi- and polynucleated cells with and without MN can be rapidly and automatically identified and quantified. Finally, we present high-resolution cell images containing NBUDs and NPBs, illustrating that IFC possesses the potential for completely automated scoring of all components of the CBMN cytome assay.

Spatial and temporal variability in macroalgal blooms in a eutrophied coastal estuary.

All three macroalgal clades (Chlorophyta, Rhodophyta, and Phaeophyceae) contain bloom-forming species. Macroalgal blooms occur worldwide and have negative consequences for coastal habitats and economies. Narragansett Bay (NB), Rhode Island, USA, is a medium sized estuary that is heavily influenced by anthropogenic activities and has been plagued by macroalgal blooms for over a century. Over the past decade, significant investment has upgraded wastewater treatment from secondary treatment to water-quality based limits (i.e. tertiary treatment) in an effort to control coastal eutrophication in this system. The goal of this study was to improve the understanding of multi-year macroalgal bloom dynamics through intensive aerial and ground surveys conducted monthly to bi-monthly during low tides in May-October 2006-2013 in NB. Aerial surveys provided a rapid characterization of macroalgal densities across a large area, while ground surveys provided high resolution measurements of macroalgal identity, percent cover, and biomass. Macroalgal blooms in NB are dominated by Ulva and Gracilaria spp. regardless of year or month, although all three clades of macroalgae were documented. Chlorophyta cover and nutrient concentrations were highest in the middle and upper bay. Rhodophyta cover was highest in the middle and lower bay, while drifting Phaeophyceae cover was patchy. Macroalgal blooms of >1000g fresh mass (gfm)/m2 (max=3510gfm/m2) in the intertidal zone and >3000gfm/m3 (max=8555gfm/m3) in the subtidal zone were observed within a heavily impacted embayment (Greenwich Bay). Macroalgal percent cover (intertidal), biomass (subtidal), and diversity varied significantly between year, month-group, site, and even within sites, with the highest species diversity at sites outside of Greenwich Bay. Total intertidal macroalgal percent cover, as well as subtidal Ulva biomass, were positively correlated with temperature. Dissolved inorganic nitrogen concentrations were correlated with the total biomass of macroalgae and the subtidal biomass of Gracilaria spp. but not the biomass of Ulva spp. Despite seasonal reductions in the nutrient output of wastewater treatment facilities emptying into upper Narragansett Bay in recent years, macroalgal blooms still persist. Continued long-term monitoring of water quality, macroalgal blooms, and ecological indicators is essential to understand the changes in macroalgal bloom dynamics that occur after nutrient reductions from management efforts.

The Application of Imaging Flow Cytometry to High-Throughput Biodosimetry.

Biodosimetry methods, including the dicentric chromosome assay, the cytokinesis-block micronucleus assay and the γH2AX marker of DNA damage are used to determine the dose of ionizing radiation. These techniques are particularly useful when physical dosimetry is absent or questioned. While these assays can be very sensitive and specific, the standard methods need to be adapted to increase sample throughput in the case of a large-scale radiological/nuclear event. Recent modifications to the microscope-based assays have resulted in some increased throughput, and a number of biodosimetry networks have been, and continue to be, established and strengthened. As the imaging flow cytometer (IFC) is a technology that can automatically image and analyze processed blood samples for markers of radiation damage, the microscope-based biodosimetry techniques can be modified for the IFC for high-throughput biological dosimetry. Furthermore, the analysis templates can be easily shared between networked biodosimetry laboratories for increased capacity and improved standardization. This review describes recent advances in IFC methodology and their application to biodosimetry.

RENEB accident simulation exercise.

PURPOSE: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. MATERIALS AND METHODS: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. RESULTS: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). CONCLUSIONS: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested.

Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry.

Biodosimetry is an important tool for triage in the case of large-scale radiological or nuclear emergencies, but traditional microscope-based methods can be tedious and prone to scorer fatigue. While the dicentric chromosome assay (DCA) has been adapted for use in triage situations, it is still time-consuming to create and score slides. Recent adaptations of traditional biodosimetry assays to imaging flow cytometry (IFC) methods have dramatically increased throughput. Additionally, recent improvements in image analysis algorithms in the IFC software have resulted in improved specificity for spot counting of small events. In the IFC method for the dicentric chromosome analysis (FDCA), lymphocytes isolated from whole blood samples are cultured with PHA and Colcemid. After incubation, lymphocytes are treated with a hypotonic solution and chromosomes are isolated in suspension, labelled with a centromere marker and stained for DNA content with DRAQ5. Stained individual chromosomes are analyzed on the ImageStream®X (EMD-Millipore, Billerica, MA) and mono- and dicentric chromosome populations are identified and enumerated using advanced image processing techniques. Both the preparation of the isolated chromosome suspensions as well as the image analysis methods were fine-tuned in order to optimize the FDCA. In this paper we describe the method to identify and score centromeres in individual chromosomes by IFC and show that the FDCA method may further improve throughput for triage biodosimetry in the case of large-scale radiological or nuclear emergencies.

Quantitation of Chromosome Damage by Imaging Flow Cytometry.

Biodosimetry is a method for measuring the dose of radiation to individuals using biological markers such as chromosome damage. Following mass casualty events, it is important to provide this information rapidly in order to assist with the medical management of potentially exposed casualties. Currently, the gold standard for biodosimetry is the dicentric chromosome assay, which accurately estimates the dose from the number of dicentric chromosomes in lymphocytes. To increase throughput of analysis following a large-scale mass casualty event, this assay has been adapted for use on the imaging flow cytometer. This chapter describes the methods for the identification and quantification of mono- and multicentric chromosomes using the imaging flow cytometer.

Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry.

The cytokinesis-block micronucleus assay can be employed in triage radiation biodosimetry to determine the dose of radiation to an exposed individual by quantifying the frequency of micronuclei in binucleated lymphocyte cells. Partially automated analysis of the assay has been applied to traditional microscope-based methods, and most recently, the assay has been adapted to an automated imaging flow cytometry method. This method is able to automatically score a larger number of binucleated cells than are typically scored by microscopy. Whole blood samples were irradiated, divided into 2 mL and 200 µL aliquots, cultured for 48 h and 72 h, and processed to generate calibration curves from 0-4 Gy. To validate the method for use in radiation biodosimetry, nine separate whole blood samples were then irradiated to known doses, blinded, and processed. Results indicate that dose estimations can be determined to within ±0.5 Gy of the delivered dose after only 48 h of culture time with an initial blood volume of 200 µL. By performing the cytokinesis-block micronucleus assay using imaging flow cytometry, a significant reduction in the culture time and volume requirements is possible, which greatly increases the applicability of the assay in high throughput triage radiation biodosimetry.

Evolution of the Health Canada astronaut biodosimetry program with a view toward international harmonization.

Biodosimetry of astronaut lymphocyte samples, taken prior to- and post-flight, provides an important in vivo measurement of radiation-induced damage incurred during space flight which can be included in the medical records of the astronauts. Health Canada has been developing their astronaut biodosimetry program since 2007 and since then has analyzed data from 7 astronauts. While multiple cytogenetic endpoints may be analyzed for the astronauts, the Fluorescent in situ hybridization (FISH) assay is considered to be key for detecting long-lasting stable damage. It is believed that this long-lasting damage is most likely to lead to an increased risk to the health of the astronauts during long-term flights (lasting 6 months or more). The complexity of damage that results from protracted, non-homogeneous radiation exposure, like that found in the space environment, requires a detailed scoring schematic to capture as much information as possible. To that end, this paper outlines the efforts to harmonize the manner in which Health Canada's FISH data are recorded to better facilitate the comparison of results with other international biodosimetry programs.

Evaluation of the annual Canadian biodosimetry network intercomparisons.

PURPOSE: To evaluate the importance of annual intercomparisons for maintaining the capacity and capabilities of a well-established biodosimetry network in conjunction with assessing efficient and effective analysis methods for emergency response. MATERIALS AND METHODS: Annual intercomparisons were conducted between laboratories in the Canadian National Biological Dosimetry Response Plan. Intercomparisons were performed over a six-year period and comprised of the shipment of 10-12 irradiated, blinded blood samples for analysis by each of the participating laboratories. Dose estimates were determined by each laboratory using the dicentric chromosome assay (conventional and QuickScan scoring) and where possible the cytokinesis block micronucleus (CBMN) assay. Dose estimates were returned to the lead laboratory for evaluation and comparison. RESULTS: Individual laboratories performed comparably from year to year with only slight fluctuations in performance. Dose estimates using the dicentric chromosome assay were accurate about 80% of the time and the QuickScan method for scoring the dicentric chromosome assay was proven to reduce the time of analysis without having a significant effect on the dose estimates. Although analysis with the CBMN assay was comparable to QuickScan scoring with respect to speed, the accuracy of the dose estimates was greatly reduced. CONCLUSIONS: Annual intercomparisons are necessary to maintain a network of laboratories for emergency response biodosimetry as they evoke confidence in their capabilities.

Sources of variation in readmission rates, length of stay, and operative time associated with rotator cuff surgery.

BACKGROUND: Variation in readmission rates, length of stay, and operative time associated with rotator cuff surgery should be understood if cost-control strategies are to be considered. We hypothesized that there would be variation in resource utilization as measured in terms of these factors and that surgeon and hospital practice patterns, rather than patient characteristics, would explain this variation. METHODS: We conducted a retrospective analysis of the effects of surgeon, hospital, and patient-related factors on the readmission rates, length of stay, and operative time associated with 1077 rotator cuff repairs performed by thirty-two surgeons in eleven group-model health maintenance organization hospitals, two satellite centers, and one contract facility in southern California. RESULTS: The initial unadjusted analysis of covariance showed moderate-to-strong associations between surgeon and hospital variation and the rate of hospital readmission within thirty days (p = 0.0919 and p = 0.0209, respectively), extended length of stay (p = 0.0016 and p = 0.0016, respectively), and operative time (p < 0.0001 and p < 0.0001, respectively). The hospital effect was no longer significant when patient-related factors (i.e., sociodemographic characteristics and comorbidities) and the surgeon effect were taken into account. The surgeon effect was still significant (except with regard to the readmission rate) after adjustment for patient and hospital-related factors, explaining 23% of the variation in length of stay and 69% of the variation in operative time. There was a significantly increased risk of an extended stay (p = 0.0010) and readmission (p = 0.0260) following procedures performed at hospitals with an orthopaedic residency program. Increased operative time was significantly associated with decreased surgeon volume (p < 0.0001) and the absence of an orthopaedic residency program (p < 0.0001). CONCLUSIONS: Variation in length of stay and operative time associated with rotator cuff surgery is largely explained by surgeon practice patterns. Our results suggest that surgeons have the ability to affect these two factors, which are often identified as drivers of cost.