Optimized automated data analysis for the cytokinesis-block micronucleus assay using imaging flow cytometry for high throughput radiation biodosimetry.

The cytokinesis-block micronucleus (CBMN) assay is a well-established technique that can be employed in triage radiation biodosimetry to estimate whole body doses of radiation to potentially exposed individuals through quantitation of the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using traditional microscope-based methods and most recently has been modified for application on the ImageStream(X) (IS(X) ) imaging flow cytometer. This modification has allowed for a similar number of BNCs to be automatically scored as compared to traditional microscopy in a much shorter time period. However, the MN frequency measured was much lower than both manual and automated slide-based methods of performing the assay. This work describes the optimized analysis template which implements newly developed functions in the IDEAS(®) data analysis software for the IS(X) that enhances specificity for BNCs and increases the frequency of scored MN. A new dose response calibration curve is presented in which the average rate of MN per BNC is of similar magnitude to those presented in the literature using automated CBMN slide scoring methods. In addition, dose estimates were generated for nine irradiated, blinded samples and were found to be within ±0.5 Gy of the delivered dose. Results demonstrate that the improved identification accuracy for MN and BNCs in the IS(X) -based version of the CBMN assay will translate to increased accuracy when estimating unknown radiation doses received by exposed individuals following large-scale radiological or nuclear emergencies. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

Multi-parameter dose estimations in radiation biodosimetry using the automated cytokinesis-block micronucleus assay with imaging flow cytometry.

The cytokinesis-block micronucleus (CBMN) assay is an established technique in radiation biological dosimetry for estimating the dose to an individual by measuring the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using slide-scoring algorithms, but an automated multiparameter method without the need of the slide-making procedure would be advantageous to further increase throughput for application in mass casualty events. The development of the ImageStreamX (ISX) imaging flow cytometer has made it possible to adapt the CBMN assay to an automated imaging flow cytometry (FCM) method. The protocol and analysis presented in this work tailor and expand the assay to a multiparameter biodosimetry tool. Ex vivo irradiated whole blood samples were cultured, processed, and analyzed on the ISX and BNCs, MN, and mononuclear cells were imaged, identified, and enumerated automatically and simultaneously. Details on development of the method, gating strategy, and dose response curves generated for the rate of MN per BNC, percentage of mononuclear cells as well as the replication index are presented. Results indicate that adapting the CBMN assay for use in imaging FCM has produced a rapid, robust, multiparameter analysis method with higher throughput than is currently available with standard microscopy. We conclude that the ISX-CBMN method may be an advantageous tool following a radiological event where triage biodosimetry must be performed on a large number of casualties.

HIV Tat protein affects circadian rhythmicity by interfering with the circadian system.

OBJECTIVES: Sleep disorders are common in patients with HIV/AIDS, and can lead to poor quality of life. Although many studies have investigated the aetiology of these disorders, it is still unclear whether impaired sleep quality is associated with HIV itself, social problems, or side effects of antiretroviral therapy (ART). Moreover, despite its known neurological associations, little is known about the role of the trans-activator of transcription (Tat) protein in sleep disorders in patients with HIV/AIDS. The purpose of this study was to test the hypothesis that the sleep quality of patients with HIV/AIDS affected by an altered circadian rhythm correlates with cerebrospinal HIV Tat protein concentration. METHODS: Ninety-six patients with HIV/AIDS between 20 and 69 years old completed the Pittsburgh Sleep Quality Index. Their circadian rhythm parameters of blood pressure, Tat concentration in cerebrospinal fluid, melatonin concentration, CD4 cell count and HIV RNA viral load in serum were measured. RESULTS: The circadian amplitude of systolic blood pressure and the score for sleep quality (Pittsburgh Sleep Quality Index) were negatively correlated with HIV Tat protein concentration, while the melatonin value was positively correlated with Tat protein concentration. CONCLUSIONS: The HIV Tat protein affects circadian rhythmicity by interfering with the circadian system in patients with HIV/AIDS and further increases the melatonin excretion value. A Tat protein-related high melatonin value may counteract HIV-related poor sleep quality during the progression of HIV infection. This study provides the first clinical evidence offering an explanation for why sleep quality did not show an association with progression of HIV infection in previous studies.

Automated analysis of the cytokinesis-block micronucleus assay for radiation biodosimetry using imaging flow cytometry.

The cytokinesis-block micronucleus (CBMN) assay is employed in biological dosimetry to determine the dose of radiation to an exposed individual from the frequency of micronuclei (MN) in binucleated lymphocyte cells. The method has been partially automated for the use in mass casualty events, but it would be advantageous to further automate the method for increased throughput. Recently, automated image analysis has been successfully applied to the traditional, slide-scoring-based method of the CBMN assay. However, with the development of new technologies such as the imaging flow cytometer, it is now possible to adapt this microscope-based assay to an automated imaging flow cytometry method. The ImageStream(X) is an imaging flow cytometer that has adequate sensitivity to quantify radiation doses larger than 1 Gy while adding the increased throughput of traditional flow cytometry. The protocol and analysis presented in this work adapts the CBMN assay for the use on the ImageStream(X). Ex vivo-irradiated whole blood samples cultured for CBMN were analyzed on the ImageStream(X), and preliminary results indicate that binucleated cells and MN can be identified, imaged and enumerated automatically by imaging flow cytometry. Details of the method development, gating strategy and the dose response curve generated are presented and indicate that adaptation of the CBMN assay for the use with imaging flow cytometry has potential for high-throughput analysis following a mass casualty radiological event.

RENEB Inter-Laboratory Comparison 2021: Inter-Assay Comparison of Eight Dosimetry Assays.

Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses <2.5 Gy or >2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.

RENEB Inter-Laboratory Comparison 2021: The Dicentric Chromosome Assay.

After large-scale radiation accidents where many individuals are suspected to be exposed to ionizing radiation, biological and physical retrospective dosimetry assays are important tools to aid clinical decision making by categorizing individuals into unexposed/minimally, moderately or highly exposed groups. Quality-controlled inter-laboratory comparisons of simulated accident scenarios are regularly performed in the frame of the European legal association RENEB (Running the European Network of Biological and Physical retrospective Dosimetry) to optimize international networking and emergency readiness in case of large-scale radiation events. In total 33 laboratories from 22 countries around the world participated in the current RENEB inter-laboratory comparison 2021 for the dicentric chromosome assay. Blood was irradiated in vitro with X rays (240 kVp, 13 mA, ∼75 keV, 1 Gy/min) to simulate an acute, homogeneous whole-body exposure. Three blood samples (no. 1: 0 Gy, no. 2: 1.2 Gy, no. 3: 3.5 Gy) were sent to each participant and the task was to culture samples, to prepare slides and to assess radiation doses based on the observed dicentric yields from 50 manually or 150 semi-automatically scored metaphases (triage mode scoring). Approximately two-thirds of the participants applied calibration curves from irradiations with γ rays and about 1/3 from irradiations with X rays with varying energies. The categorization of the samples in clinically relevant groups corresponding to individuals that were unexposed/minimally (0-1 Gy), moderately (1-2 Gy) or highly exposed (>2 Gy) was successfully performed by all participants for sample no. 1 and no. 3 and by ≥74% for sample no. 2. However, while most participants estimated a dose of exactly 0 Gy for the sham-irradiated sample, the precise dose estimates of the samples irradiated with doses >0 Gy were systematically higher than the corresponding reference doses and showed a median deviation of 0.5 Gy (sample no. 2) and 0.95 Gy (sample no. 3) for manual scoring. By converting doses estimated based on γ-ray calibration curves to X-ray doses of a comparable mean photon energy as used in this exercise, the median deviation decreased to 0.27 Gy (sample no. 2) and 0.6 Gy (sample no. 3). The main aim of biological dosimetry in the case of a large-scale event is the categorization of individuals into clinically relevant groups, to aid clinical decision making. This task was successfully performed by all participants for the 0 Gy and 3.5 Gy samples and by 74% (manual scoring) and 80% (semiautomatic scoring) for the 1.2 Gy sample. Due to the accuracy of the dicentric chromosome assay and the high number of participating laboratories, a systematic shift of the dose estimates could be revealed. Differences in radiation quality (X ray vs. γ ray) between the test samples and the applied dose effect curves can partly explain the systematic shift. There might be several additional reasons for the observed bias (e.g., donor effects, transport, experimental conditions or the irradiation setup) and the analysis of these reasons provides great opportunities for future research. The participation of laboratories from countries around the world gave the opportunity to compare the results on an international level.

The myelin-associated glycoproteins: membrane disposition, evidence of a novel disulfide linkage between immunoglobulin-like domains, and posttranslational palmitylation.

The myelin-associated glycoproteins (MAG) are members of the immunoglobulin gene superfamily that function in the cell interactions of myelinating glial cells with axons. In this paper, we have characterized the structural features of these proteins. The disposition of MAG in the bilayer as a type 1 integral membrane protein (with an extracellularly disposed amino terminus, single transmembrane segment, and cytoplasmic carboxy terminus) was demonstrated in protease protection studies of MAG cotranslationally inserted into microsomes in vitro and in immunofluorescent studies with site specific antibodies. A genetically engineered MAG cDNA, which lacks the putative membrane spanning segment, was constructed and shown to encode a secreted protein. These results confirm the identify of this hydrophobic sequence as the transmembrane segment. Sequencing of the secreted protein demonstrated the presence of a cleaved signal sequence and the site of signal peptidase cleavage. To characterize the disulfide linkage pattern of the ectodomain, we cleaved MAG with cyanogen bromide and used a panel of antibodies to coprecipitate specific fragments under nonreducing conditions. These studies provide support for a novel disulfide linkage between two of the immunoglobulin domains of the extracellular segment. Finally, we report that MAG is posttranslationally palmitylated via an intramembranous thioester linkage. Based on these studies, we propose a model for the conformation of MAG, including its RGD sequence, which is considered with regard to its function as a cell adhesion molecule.

The large-scale capture of eastern grey kangaroos (Macropus giganteus) and red kangaroos (Osphranter rufus) and its application to a population management project.

OBJECTIVE: A large-scale capture method was developed to enable sterilisation of a macropod population in western Sydney from 2005 to 2018. METHODS: Until March 2007, free ranging eastern grey kangaroos and red kangaroos were herded into purpose-built 15 m diameter capture yards (CYs) for darting with a projectile syringe. From March 2007 onwards, animals were free-range darted in large areas without herding. Kangaroos were darted with 1.33-5.10 mg/kg tiletamine/zolazepam and 0.01-0.02 mg/kg medetomidine, ± 0.03 mg/kg acepromazine. Deaths were monitored. Population counts were performed annually. RESULTS: There were 5825 capture events involving 3963 kangaroos. Over 85% of all captures occurred from 2005 to 2008. Of all reported deaths (n = 523), 135 were attributed to ill health. Musculoskeletal injuries incurred during capture were the main project-related cause of death (n = 116). Post capture myopathy was uncommonly diagnosed following capture (n = 19). CONCLUSION: The herding and capture method enabled a large number of kangaroos to be mobilised and captured with low mortality rates, and the use of CYs resulted in fewer capture-related injuries and deaths than free-range capture. The drug doses and combinations used for darting were safe and effective, and the capture technique was successfully applied to a population management project.

Laparoscopic ovariectomy in eastern grey kangaroos (Macropus giganteus) and red kangaroos (Macropus rufus).

OBJECTIVE: To develop a technique for permanent sterilisation of female eastern grey kangaroos (Macropus giganteus) and red kangaroos (M. rufus) as part of a large-scale macropod management program on an enclosed 1545-ha site in western Sydney. METHODS: Free-ranging female kangaroos (n = 1409: 1285 eastern grey kangaroos, 124 red kangaroos) were anaesthetised via remote anaesthetic drug delivery of tiletamine/zolazepam, medetomidine and acepromazine prior to inhalational anaesthesia using isoflurane-oxygen. A laparoscopic ovariectomy technique was developed using standard laparoscopic equipment to effect permanent sterilisation of the kangaroos. The technique described was also adapted for use on immature animals weighing as little as 1 kg. No direct post-surgical care was provided once the animals had recovered from the anaesthetic. RESULTS: The procedure was simple to perform and had a very high success rate, with an overall project mortality rate of 2.13% (n = 30). Seven kangaroos (0.05% of all operated kangaroos) were euthanased as a direct result of the surgical procedure. Surgical complications were rare but included inadvertent gastrointestinal tract puncture with the trocar, intraoperative haemorrhage and subcutaneous emphysema leading to pouch eversion following surgery. CONCLUSION: The procedure described is a rapid and effective method of permanent fertility control in macropods and carries a low mortality rate.

Studies of the pharmacokinetic profile, in vivo efficacy and safety of injectable altrenogest for the suppression of oestrus in mares.

OBJECTIVE: To investigate the efficacy and safety of the long-acting altrenogest injection (NV Readyserve® injection) for horses. DESIGN: A single-dose pharmacokinetic (PK) study was conducted. The in vivo efficacy study was a blinded, repeated measures design evaluating behaviour scores. The safety study was a non-blinded, controlled, parallel-group, randomised-block design as per the VICH protocol. METHODS: In the PK study, serial blood samples were obtained for analysis of plasma altrenogest for 150 h following the injection and a non-compartmental PK analysis was performed. For the efficacy study, 12 mares in oestrus were treated; they were monitored daily for 10 days for signs of oestrus during teasing and given a behaviour score that was compared with pretreatment scores. A standard safety study was conducted at 1-, 3- and 5-fold the recommended dosage for 84 days. Physical, haematological and biochemical examinations were performed. RESULTS: Mean plasma altrenogest concentrations were greater than ≈0.5 ng/mL for 148 h following administration. Oestrous behaviour was suppressed in all mares within 24 h of administration. Two mares returned to oestrus by day 6 and the rest on days 7-10. In the safety study there were no significant differences in the physical and haematological examinations, but minor biochemical changes in muscle enzymes. There was a low incidence of injection site reactions following the 3- and 5-fold dose, predominantly for pectoral injections. CONCLUSION: These studies support the efficacy and safety of a single dose of Readyserve® injection for the suppression of the signs of oestrus in mares for 5-7 days.

Retrospective Biodosimetry of an Occupational Overexposure-Case Study.

In 2014, Health Canada was approached by the Canadian Nuclear Safety Commission to conduct biodosimetry for a possible overexposure 4 y prior to assessment. Dose estimates were determined by means of two cytogenetic assays, the dicentric chromosome assay (DCA) and translocations as measured by the fluorescent in situ hybridization (FISH). As dicentrics are considered to be unstable over time, the results of the DCA were adjusted to account for the time elapsed between the suspected exposure and sampling. The frequency of damage was then compared to Health Canada's calibration curves, respectively, to calculate dose. In addition, the translocation data were corrected for age-related increases in background. With a half-life of 36 months for dicentric chromosomes taken into consideration, the dose estimates from both assays were in agreement. Due to the uncertainty in the half-life of dicentrics, the FISH assay is considered to be more reliable as a technique for retrospective biodosimetry.

THE EFFECT OF AN OPTIMIZED IMAGING FLOW CYTOMETRY ANALYSIS TEMPLATE ON SAMPLE THROUGHPUT IN THE REDUCED CULTURE CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY.

In cases of overexposure to ionizing radiation, the cytokinesis-block micronucleus (CBMN) assay can be performed in order to estimate the dose of radiation to an exposed individual. However, in the event of a large-scale radiation accident with many potentially exposed casualties, the assay must be able to generate accurate dose estimates to within ±0.5 Gy as quickly as possible. The assay has been adapted to, validated and optimized on the ImageStreamX imaging flow cytometer. The ease of running this automated version of the CBMN assay allowed investigation into the accuracy of dose estimates after reducing the volume of whole blood cultured to 200 µl and reducing the culture time to 48 h. The data analysis template used to identify binucleated lymphocyte cells (BNCs) and micronuclei (MN) has since been optimized to improve the sensitivity and specificity of BNC and MN detection. This paper presents a re-analysis of existing data using this optimized analysis template to demonstrate that dose estimations from blinded samples can be obtained to the same level of accuracy in a shorter data collection time. Here, we show that dose estimates from blinded samples were obtained to within ±0.5 Gy of the delivered dose when data collection time was reduced by 30 min at standard culture conditions and by 15 min at reduced culture conditions. Reducing data collection time while retaining the same level of accuracy in our imaging flow cytometry-based version of the CBMN assay results in higher throughput and further increases the relevancy of the CBMN assay as a radiation biodosimeter.

Mechanism of cytoskeletal regulation (I): functional differences correlate with antigenic dissimilarity in human brain and erythrocyte spectrin.

Human erythrocyte and brain spectrin (fodrin, calspectin) have been compared quantitatively with respect to the extent and sites of antigenic and functional similarity. Brain spectrin cross-reacts strongly with approx. 1% of the epitopes in erythrocyte spectrin, but weakly with at least 50%. The distribution of shared determinants is not uniform. Brain spectrin is most deficient in epitopes characteristic of the 80 kDa and 52 kDa domains of the alpha-subunit (alpha-I and alpha-III) and of terminal portions of the 28 kDa and 74 kDa domains of the beta-subunit (beta-I and beta-IV). The functions associated with these domains also differ between the two proteins. Brain spectrin does not undergo extensive polymerization and binds calmodulin at a different site. The unique ability of erythrocyte spectrin to oligomerize beyond the tetramer reflects its role in the membrane skeleton. Non-erythroid spectrins probably function as specific linkers between membrane receptors and the filamentous cytoskeleton. In this sense, they may act as regulated transducers of information flow between the membrane and the cytoplasmic matrix.

The phospholipid requirement for Rho(D) antigen activity: mode of inactivation by phospholipases and of protection by anti-Rh0(D) antibody.

Previous studies have suggested a membrane phospholipid requirement for Rho(D) antigen activity. Isolated erythrocyte membranes incubated with phospholipase A2 from both bee venom and porcine pancreas undergo loss of Rh antigen activity. The mode of attenuation of this antigen activity as indicated by double-reciprocal binding plots suggests substantial loss of sites accompanied by an apparently increased association constant. In the presence of anti-Rho(D), but not anti-A, bound to group A Rho(D)-positive membranes prior to hydrolysis, there is marked protection: almost complete preservation of sites at the expense of a decreased association constant. This pattern of protection is not seen with phospholipase C, which cleaves the polar headgroup in contrast to the A2-enzymes, which hydrolyze the fatty acid in the 2-position. Analysis of the products of digestion shows a trend to protection of bulk phospholipids of all major classes in the presence of bound specific antibody. The hydrophobic fatty acid chain may be the site with which the bound anti-Rho(D) antibody is in closest proximity.

Experience with a cross-study endpoint review committee for AIDS clinical trials. Terry Beirn Community Programs for Clinical Research on AIDS.

OBJECTIVES: To describe the methods and results of a standardized system for clinical endpoint determination for defining and reviewing endpoints in clinical trials for HIV-infected individuals. DESIGN: A system was developed utilizing standard definitions for the 24 diagnoses or clinical events that serve as trial endpoints and together define the combined endpoint 'progression of HIV disease. A common set of case report forms were used for all trials. Thus, an event of Pneumocystis carinii pneumonia (PCP), for example, for a subject co-enrolled in an antiretroviral trial and a PCP prophylaxis trial was only reported once. METHODS: A central committee was established to define clinical events and review endpoints across all studies. Events were classified according to established criteria for confirmed, probable and possible levels of certainty. RESULTS: This report describes the methods used to ascertain and review endpoints, and summarized 2299 clinical events for 8097 subjects enrolled in one or more of nine clinical trials. Data on the diagnostic certainty of events and agreement between site clinicians and the endpoint committee are presented. CONCLUSIONS: Uniform classification of endpoints across AIDS clinical trials can be accomplished by multicenter, multitrial organizations with standardized definitions and review of endpoint documentation. Our experience suggests that nurse coordinators reviewing all submitted endpoints for every trial are warranted and the need for external review by a clinical events committee may depend on the type of trial conducted.

Enhanced unscheduled DNA synthesis in UV-irradiated human skin explants treated with T4N5 liposomes.

Epidermal keratinocytes cultured from explants of skin cancer patients, including biopsies from xeroderma pigmentosum patients, were ultraviolet light-irradiated and DNA repair synthesis was measured. Repair capacity was much lower in xeroderma pigmentosum patients than in normal patients. The extent of DNA repair replication did not decline with the age of the normal patient. Treatment with T4N5 liposomes containing a DNA repair enzyme enhanced repair synthesis in both normal and xeroderma pigmentosum keratinocytes in an irradiation- and liposome-dose dependent manner. These results provide no evidence that aging people or skin cancer patients are predisposed to cutaneous malignancy by a DNA repair deficiency, but do demonstrate that T4N5 liposomes enhance DNA repair in the keratinocytes of the susceptible xeroderma pigmentosum and skin cancer population.

Noninvasive methods for quantitating blood time-activity curves from mouse PET images obtained with fluorine-18-fluorodeoxyglucose.

UNLABELLED: The mouse model is currently being explored for various applications with PET imaging. Low resolution of current animal scanners relative to mouse size leads to difficulty in quantitating data from mouse PET images. We have, therefore, investigated methods for determining blood time-activity curves (TACs) from mouse PET studies done with fluorine-18-fluorodeoxyglucose (FDG). METHODS: Eight mice were fasted, the tail vein was injected with 150-300 microCi of FDG and dynamic images were acquired with a CTI/Siemens (Knoxville, TN) animal tomograph for 64.5 min. Concurrently, 11-14 left ventricle (LV) blood samples were drawn directly from the LV chamber. Organ TACs were obtained by drawing circular regions of interest (ROIs) of various sizes on images of the heart, liver and brain. For each mouse, the FDG model parameter K = (K1 x k3)/(k2 + k3) was estimated by a Patlak algorithm with various estimates of the blood TAC and, as a reference tissue TAC, the brain TAC. RESULTS: Most partial-volume-corrected heart ROI TACs overestimated the LV samples. Blood TACs from heart images produced statistically different estimates of K than did the LV samples. The liver image-derived blood TACs yielded estimates of K that were comparable to those yielded by the LV samples. Estimates of K determined with two directly sampled LV points in conjunction with the liver image-derived TAC were not statistically different from the estimates obtained with the LV samples. The size and location of ROIs on images of the liver minimally affected the TACs. CONCLUSION: We have shown that it is experimentally possible to obtain a blood TAC from mouse studies by repeatedly sampling from the LV. We have also shown that images of the liver can be used to reliably estimate the blood TAC. Future FDG PET studies with the mouse model will benefit from this demonstrated ability to noninvasively quantitate blood TACs directly from FDG PET images.

Imaging of adenoviral-directed herpes simplex virus type 1 thymidine kinase reporter gene expression in mice with radiolabeled ganciclovir.

UNLABELLED: We are developing procedures to repeatedly and noninvasively image the expression of transplanted reporter genes in living animals and in patients, using PET. We have investigated the use of the Herpes Simplex Virus type 1 thymidine kinase gene (HSV1-tk) as a reporter gene and [8-14C]-ganciclovir as a reporter probe. HSV1-tk, when expressed, leads to phosphorylation of [8-14C]-ganciclovir. As a result, specific accumulation of phosphorylated [8-14C]-ganciclovir should occur almost exclusively in tissues expressing the HSV1-tk gene. METHODS: An adenoviral vector was constructed carrying the HSV1-tk gene along with a control vector. C6 rat glioma cells were infected with either viral vector and uptake of [8-3H]-ganciclovir was determined. In addition, 12 mice were injected with varying levels of either viral vector. Adenovirus administration in mice leads primarily to liver infection. Forty-eight hours later the mice were injected with [8-14C]-ganciclovir, and 1 hr later the mice were sacrificed and biodistribution studies performed. Digital whole-body autoradiography also was performed on separate animals. HSV1-tk expression was assayed, using both normalized HSV1-tk mRNA levels and relative HSV1-TK enzyme levels, in both the cell culture and murine studies. RESULTS: Cell culture, murine tissue biodistribution and murine in vivo digital whole-body autoradiography all demonstrate the feasibility of HSV1-tk as a reporter gene and [8-14C]-ganciclovir as an imaging reporter probe. A good correlation (r2 = 0.86) between the [8-14C]-ganciclovir percent injected dose per gram tissue from HSV1-tk positive tissues and HSV1-TK enzyme levels in vivo was found. An initial study in mice with [8-18F]-fluoroganciclovir and microPET imaging supports further investigation of [8-18F]-fluoroganciclovir as a PET reporter probe for imaging HSV1-tk gene expression. CONCLUSION: These results demonstrate the feasibility of using [8-14C]-ganciclovir as a reporter probe for the HSV1-tk reporter gene, using an in vivo adenoviral mediated gene delivery system in a murine model. The results form the foundation for further investigation of [8-18F]-fluoroganciclovir for noninvasive and repeated imaging of gene expression with PET.

Seismocardiographic changes associated with obstruction of coronary blood flow during balloon angioplasty.

Seismocardiography, a new noninvasive technique, detects low-frequency cardiac vibrations on the chest wall during ventricular contraction and during both early and late ventricular filling. To evaluate the ability of seismocardiography to detect ischemia caused by decreased coronary blood flow, 35 patients were studied during coronary angioplasty. Seismocardiograms and electrocardiograms were recorded twice at baseline, with the catheter across the lesion before first inflation (n = 15), every 30 seconds during the first inflation, 1 and 2 minutes after the first inflation and greater than or equal to 5 minutes after the final inflation. For comparison, sequential seismocardiograms were also obtained from 15 healthy volunteers. Electrocardiograms were blindly scored for ST change from baseline (0 = none, 1 = 0.5 mm ST depression, 2 = greater than or equal to 1.0 mm ST depression, 3 = ST elevation). Seismocardiograms were blindly scored for change from baseline (0 = none, 1 = mild, 2 = moderate, 3 = marked) for both the systolic and diastolic waves. The average maximal systolic seismocardiographic score was 2.5 +/- 0.8 for patients who had undergone angioplasty and 1.0 +/- 0.9 for volunteers (p less than 0.001). The average maximal diastolic seismocardiographic score was 2.3 +/- 0.8 for angioplasty patients and 0.7 +/- 0.9 for volunteers (p less than 0.001). The percentage of angioplasty patients with electrocardiographic, systolic and diastolic seismocardiographic scores greater than or equal to 2 was, respectively: 0, 11 and 14% at second baseline; 23, 67 and 53% with catheter across the lesion; 44, 75 and 59% after 30 seconds of inflation; 42, 71 and 61% after 60 seconds of inflation; 23, 74 and 61% after 1 minute of deflation; and 0, 71 and 47% 5 minutes after final inflation.(ABSTRACT TRUNCATED AT 250 WORDS)

A preliminary taxonomy of medical errors in family practice.

OBJECTIVE: To develop a preliminary taxonomy of primary care medical errors. DESIGN: Qualitative analysis to identify categories of error reported during a randomized controlled trial of computer and paper reporting methods. SETTING: The National Network for Family Practice and Primary Care Research. PARTICIPANTS: Family physicians. MAIN OUTCOME MEASURES: Medical error category, context, and consequence. RESULTS: Forty two physicians made 344 reports: 284 (82.6%) arose from healthcare systems dysfunction; 46 (13.4%) were errors due to gaps in knowledge or skills; and 14 (4.1%) were reports of adverse events, not errors. The main subcategories were: administrative failure (102; 30.9% of errors), investigation failures (82; 24.8%), treatment delivery lapses (76; 23.0%), miscommunication (19; 5.8%), payment systems problems (4; 1.2%), error in the execution of a clinical task (19; 5.8%), wrong treatment decision (14; 4.2%), and wrong diagnosis (13; 3.9%). Most reports were of errors that were recognized and occurred in reporters' practices. Affected patients ranged in age from 8 months to 100 years, were of both sexes, and represented all major US ethnic groups. Almost half the reports were of events which had adverse consequences. Ten errors resulted in patients being admitted to hospital and one patient died. CONCLUSIONS: This medical error taxonomy, developed from self-reports of errors observed by family physicians during their routine clinical practice, emphasizes problems in healthcare processes and acknowledges medical errors arising from shortfalls in clinical knowledge and skills. Patient safety strategies with most effect in primary care settings need to be broader than the current focus on medication errors.