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In large-scale radiation exposure events, the ability to triage potential victims by the received radiation dosage is crucial. This can be evaluated by radiation-induced biological changes. Radiation-responsive mRNA is a class of biomarkers that has been explored for dose-dependency with methods such as RT-qPCR. However, these methods are challenging to implement for point-of-care devices. We have designed and used molecular beacons as probes for the measurement of radiation-induced changes of intracellular mRNA in a microfluidic device towards determining radiation dosage. Our experiments, in which fixed TK6 cells labeled with a molecular beacon specific to BAX mRNA exhibited dose-dependent fluorescence in a manner consistent with RT-qPCR analysis, demonstrate that such intracellular molecular probes can potentially be used in point-of-care radiation biodosimetry. This proof of concept could readily be extended to any RNA-based test to provide direct measurements at the bedside.
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After a large-scale radiological or nuclear event, hundreds of thousands of people may be exposed to ionizing radiation and require subsequent medical management. Acute exposure to moderate doses (2-6 Gy) of radiation can lead to the hematopoietic acute radiation syndrome, in which the bone marrow (BM) is severely compromised, and severe hemorrhage and infection are common. Previously, we have developed a panel of intracellular protein markers (FDXR, ACTN1, DDB2, BAX, p53 and TSPYL2), designed to reconstruct absorbed radiation dose from human peripheral blood (PB) leukocyte samples in humanized mice up to 3 days after exposure. The objective of this work was to continue to use the humanized mouse model to evaluate biomarker dose-/time- kinetics in human PB leukocytes in vivo, at an earlier (day 2) and later (day 7) time point, after exposure to total-body irradiation (TBI) doses of 0 to 2 Gy of X rays. In addition, to assess hematological sensitivity and radiation-induced injury, PB leukocyte cell counts, human BM hematopoietic stem cell (HSC) and progenitor cell [multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte myeloid progenitor (GMP), megakaryocyte/erythrocyte progenitor (MEP) and multi-lymphoid progenitor (MLP)] levels were measured, and their correlation was also examined as the BM damages are difficult to assess by routine tests. Peripheral blood B-cells were significantly lower after TBI doses of 0.5 Gy on day 2 and 2 Gy on days 2 and 7; T-cells were significantly reduced only on day 2 after 2 Gy TBI. Bone marrow HSCs and MPP cells showed a dose-dependent depletion after irradiation with 0.5 Gy and 2 Gy on day 2, and after 1 Gy and 2 Gy on day 7. Circulating B cells correlated with HSCs, MPP and MLP cells on day 2, whereas T cells correlated with MPP, and myeloid cells correlated with MLP cells. On day 7, B cells correlated with MPP, CMP, GMP and MEP, while myeloid cells correlated with CMP, GMP and MEP. The intracellular leukocyte biomarkers were able to discriminate unirradiated and irradiated samples at different time points calculated by receiver operating characteristic (ROC) curve. Using machine learning algorithm methods, combining ACTN1, p53, TSPYL2 and PB-T cell and PB-B cell counts served as a strong predictor (area under the ROC >0.8) to distinguish unirradiated and irradiated samples independent of the days after TBI. The results further validated our biomarker-based triage assay and additionally evaluated the radiation sensitivity of the hematopoietic system after TBI exposures.
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Female hypogonadism (FH) is a relatively common endocrine disorder in women of premenopausal age, but there are significant uncertainties and wide variation in its management. Most current guidelines are monospecialty and only address premature ovarian insufficiency (POI); some allude to management in very brief and general terms, and most rely upon the extrapolation of evidence from the studies relating to physiological estrogen deficiency in postmenopausal women. The Society for Endocrinology commissioned new guidance to provide all care providers with a multidisciplinary perspective on managing patients with all forms of FH. It has been compiled using expertise from Endocrinology, Primary Care, Gynaecology and Reproductive Health practices, with contributions from expert patients and a patient support group, to help clinicians best manage FH resulting from both POI and hypothalamo-pituitary disorders, whether organic or functional.
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In cytogenetic biodosimetry, assessing radiation exposure typically requires over 48 hours for cells to reach mitosis, significantly delaying the administration of crucial radiation countermeasures needed within the first 24 hours post-exposure. To improve medical response times, we incorporated the G0-Premature Chromosome Condensation (G0-PCC) technique with the Rapid Automated Biodosimetry Tool-II (RABiT-II), creating a faster alternative for large-scale radiation emergencies. Our findings revealed that using a lower concentration of Calyculin A (Cal A) than recommended effectively increased the yield of highly-condensed G0-PCC cells (hPCC). However, integrating recombinant CDK1/Cyclin B kinase, vital for chromosome condensation, proved challenging due to the properties of these proteins affecting interactions with cellular membranes. Interestingly, Cal A alone was capable of inducing chromosome compaction in some G0 cells even in the absence of mitotic kinases, although these chromosomes displayed atypical morphologies. This suggests that Cal A mechanism for compacting G0 chromatin may differ from condensation driven by mitotic kinases. Additionally, we observed a correlation between radiation dose and extent of hPCC chromosome fragmentation, which allowed us to automate radiation damage quantification using a Convolutional Neural Network (CNN). Our method can address the need for a same-day cytogenetic biodosimetry test in radiation emergency situations.
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BACKGROUND: Bone metastasis is a common consequence of advanced prostate cancer. Bisphosphonates can be used to manage symptoms, but there are currently no curative treatments available. Altered tumour cell glycosylation is a hallmark of cancer and is an important driver of a malignant phenotype. In prostate cancer, the sialyltransferase ST6GAL1 is upregulated, and studies show ST6GAL1-mediated aberrant sialylation of N-glycans promotes prostate tumour growth and disease progression. METHODS: Here, we monitor ST6GAL1 in tumour and serum samples from men with aggressive prostate cancer and using in vitro and in vivo models we investigate the role of ST6GAL1 in prostate cancer bone metastasis. FINDINGS: ST6GAL1 is upregulated in patients with prostate cancer with tumours that have spread to the bone and can promote prostate cancer bone metastasis in vivo. The mechanisms involved are multi-faceted and involve modification of the pre-metastatic niche towards bone resorption to promote the vicious cycle, promoting the development of M2 like macrophages, and the regulation of immunosuppressive sialoglycans. Furthermore, using syngeneic mouse models, we show that inhibiting sialylation can block the spread of prostate tumours to bone. INTERPRETATION: Our study identifies an important role for ST6GAL1 and α2-6 sialylated N-glycans in prostate cancer bone metastasis, provides proof-of-concept data to show that inhibiting sialylation can suppress the spread of prostate tumours to bone, and highlights sialic acid blockade as an exciting new strategy to develop new therapies for patients with advanced prostate cancer. FUNDING: Prostate Cancer Research and the Mark Foundation For Cancer Research, the Medical Research Council and Prostate Cancer UK.
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Neoplasias Óseas , Ácido N-Acetilneuramínico , Neoplasias de la Próstata , Sialiltransferasas , Masculino , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Humanos , Sialiltransferasas/metabolismo , Sialiltransferasas/genética , Animales , Neoplasias Óseas/secundario , Neoplasias Óseas/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Ratones , Ácido N-Acetilneuramínico/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Antígenos CD/metabolismo , Polisacáridos/farmacología , Glicosilación , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
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).
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Biomarcadores , Exposición a la Radiación , Humanos , Exposición a la Radiación/análisis , Citometría de Flujo/métodos , Reproducibilidad de los Resultados , Ensayos Analíticos de Alto Rendimiento/métodos , Proteína p53 Supresora de TumorRESUMEN
OBJECTIVE: The risk of aortic dissection (AoD) is increased in Turner syndrome (TS) but predicting those at risk is difficult. Based on scarce evidence, preventive aortic surgery is recommended when aortic diameter increases >5 mm/year. To investigate the aortic growth rate in TS and TS-related conditions associated with aortic growth. We also reported our experience of women who suffered aortic dissection (AoD), and who had preventive aortic replacement. METHODS: 151 adult TS were retrospectively identified. Women who had more than one transthoracic echocardiogram (TTE) after age 16 years were included in the aortic growth study. Aortic diameters at sinuses of Valsalva (SoV) and ascending aorta (AA) were analysed by two experts. RESULTS: 70/151 women had more than one TTE (interscan interval 4.7 years). Mean aortic growth was 0.13 ± 0.59 mm/year at SoV and 0.23 ± 0.82 mm/year at AA. Known risk factors for aortic dilatation and TS-related conditions were not associated with aortic growth. 4/151 women experienced AoD (age 25±8 years): two had paired scans for aortic growth, which was 0.67 mm/year at both SoV and AA in the first woman, and 11 mm/year (SoV) and 4 mm/year (AA) in the second. Only 1/4 of women with AoD survived; she used a TS cardiac-alert card to inform emergency personnel about her risk of AoD. 5/151 had a preventive aortic replacement, but one died post-operatively. CONCLUSIONS: Mean aortic growth in our TS population was increased compared to non-TS women and was not associated with currently known risk factors for AoD, suggesting that aortic growth rate itself could be a useful variable to stratify who is at risk for AoD.
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Enfermedades de la Aorta , Disección Aórtica , Síndrome de Turner , Adulto , Femenino , Humanos , Adolescente , Adulto Joven , Síndrome de Turner/complicaciones , Síndrome de Turner/epidemiología , Estudios Retrospectivos , Enfermedades de la Aorta/complicaciones , Enfermedades de la Aorta/epidemiología , Medición de RiesgoRESUMEN
The cytokinesis-block micronucleus (CBMN) assay is an established method for assessing chromosome damage in human peripheral blood lymphocytes resulting from exposure to genotoxic agents such as ionizing radiation. The objective of this study was to measure cytogenetic DNA damage and hematology parameters in vivo based on MN frequency in peripheral blood lymphocytes (PBLs) from adult and pediatric leukemia patients undergoing hematopoietic stem cell transplantation preceded by total body irradiation (TBI) as part of the conditioning regimen. CBMN assay cultures were prepared from fresh blood samples collected before and at 4 and 24 h after the start of TBI, corresponding to doses of 1.25 Gy and 3.75 Gy, respectively. For both age groups, there was a significant increase in MN yields with increasing dose (p < 0.05) and dose-dependent decrease in the nuclear division index (NDI; p < 0.0001). In the pre-radiotherapy samples, there was a significantly higher NDI measured in the pediatric cohort compared to the adult due to an increase in the percentage of tri- and quadri-nucleated cells scored. Complete blood counts with differential recorded before and after TBI at the 24-h time point showed a rapid increase in neutrophil (p = 0.0001) and decrease in lymphocyte (p = 0.0006) counts, resulting in a highly elevated neutrophil-to-lymphocyte ratio (NLR) of 14.45 ± 1.85 after 3.75 Gy TBI (pre-exposure = 4.62 ± 0.49), indicating a strong systemic inflammatory response. Correlation of the hematological cell subset counts with cytogenetic damage, indicated that only the lymphocyte subset survival fraction (after TBI compared with before TBI) showed a negative correlation with increasing MN frequency from 0 to 1.25 Gy (r = -0.931; p = 0.007). Further, the data presented here indicate that the combination of CBMN assay endpoints (MN frequency and NDI values) and hematology parameters could be used to assess cytogenetic damage and early hematopoietic injury in the peripheral blood of leukemia patients, 24 h after TBI exposure.
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Leucemia , Irradiación Corporal Total , Adulto , Humanos , Niño , Irradiación Corporal Total/efectos adversos , Pruebas de Micronúcleos/métodos , Citocinesis/genética , Citocinesis/efectos de la radiación , LinfocitosRESUMEN
Testing and validation of biodosimetry assays is routinely performed using conventional dose rate irradiation platforms, at a dose rate of approximately 1 Gy/min. In contrast, the exposures from an improvised nuclear device will be delivered over a large range of dose rates with a prompt irradiation component, delivered in less than 1 µs, and a protracted component delivered over hours and days. We present preliminary data from a large demographic study we have undertaken for investigation of age, sex and dose rate effects on dicentric and micronucleus yields. Our data demonstrate reduced dicentric and micronucleus yields at very high dose rates. Additionally, we have seen small differences between males and females, with males having slightly fewer micronuclei and slightly more dicentrics than females, at high doses.
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Bioensayo , Núcleo Celular , Femenino , Masculino , Humanos , Citogenética , Análisis CitogenéticoRESUMEN
BACKGROUND AND OBJECTIVE: The risk of aortic dissection (AoD) is increased in women with Turner syndrome (TS) but predicting those with this heightened risk is difficult. In response to this, we sought to create a pathway to monitor TS patients to improve efficiency and resource utilisation in our dedicated TS clinic, and to monitor more closely those women thought to be at increased risk of AoD. DESIGN AND PATIENTS: Our pathway was designed based on evidence derived from International Guidelines for the management of aortic disease in women with TS. Women were divided according to those with known risk factors for AoD, and those with no known risk factors. These groups were further subdivided into 4 pathways depending on ascending aortic size which in-turn determined the frequency of outpatient appointments and imaging. RESULTS: Out of the 168 patients included in the analysis, 7 have had ascending aorta replacements, all in the highest risk group. Of the remaining 4 patients in the highest risk groups: 1 dissected whilst awaiting planned aortic surgery, 1 is currently awaiting surgery, 1 has low body mass index, therefore, making her aorta proportionally larger but not necessitating surgery and one has declined surgery. No women changed pathways. CONCLUSION: The risk-stratified pathway safely allowed consolidation of resources to women perceived to be at highest risk of AoD (excluding pregnancy), supporting the efficacy of the pathway and allowing the diversion of resources to those most at risk of AoD.
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Enfermedades de la Aorta , Disección Aórtica , Síndrome de Turner , Embarazo , Humanos , Femenino , Síndrome de Turner/complicaciones , Aorta Torácica , Aorta , Enfermedades de la Aorta/etiologíaRESUMEN
In the event of a widespread radiological incident, thousands of people may be exposed to a wide range of ionizing radiation. In this unfortunate scenario, there will be a need to quickly screen a large number of people to assess the amount of radiation exposure and triage for medical treatment. In our earlier work, we previously identified and validated a panel of radiosensitive protein biomarkers in blood leukocytes, using the humanized-mouse and non-human primate (NHP) models. The objective of this work was to develop a high-throughput imaging flow-cytometry (IFC) based assay for the rapid measurement of protein biomarker expression in human peripheral blood samples irradiated ex vivo. In this assay design, peripheral human blood samples from healthy adult donors were exposed to 0-5 Gy X-irradiation ex vivo and cultured for up to 2 days. Samples were stained with a cocktail of surface antigens (CD66b, CD20, and CD3), fixed and permeabilized, and intracellularly stained for BAX (Bcl-2-associated X) protein, used here as a representative biomarker. Samples were interrogated by IFC, and a uniform analysis template was created to measure biomarker expression in heterogeneous and specific leukocyte subtype populations at each time point. In this human blood ex vivo model, we show that within gated populations of leukocyte subtypes, B-cells are highly radiosensitive with the smallest surviving fraction, followed by T-cells and granulocytes. Dose-dependent biomarker responses were measured in the lymphocytes, B-, and T-cell populations, but not in the granulocytes, with dose-response curves showing increasing fold changes in BAX protein expression up to Day 2 in lymphocyte populations. We present here the successful use of this ex vivo model for the development of radiation dose-response curves of a candidate protein biomarker towards future applications of dose reconstruction and biodosimetry.
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Linfocitos , Primates , Adulto , Humanos , Animales , Ratones , Relación Dosis-Respuesta en la Radiación , Dosis de Radiación , Linfocitos/metabolismo , Biomarcadores/metabolismo , Radiometría/métodosRESUMEN
Following a mass-casualty nuclear/radiological event, there will be an important need for rapid and accurate estimation of absorbed dose for biological triage. The cytokinesis-block micronucleus (CBMN) assay is an established and validated cytogenetic biomarker used to assess DNA damage in irradiated peripheral blood lymphocytes. Here, we describe an intercomparison experiment between two biodosimetry laboratories, located at Columbia University (CU) and Health Canada (HC) that performed different variants of the human blood CBMN assay to reconstruct dose in human blood, with CU performing the assay on isolated lymphocytes and using semi-automated scoring whereas HC used the more conventional whole blood assay. Although the micronucleus yields varied significantly between the two assays, the predicted doses closely matched up to 4 Gy - the range from which the HC calibration curve was previously established. These results highlight the importance of a robust calibration curve(s) across a wide age range of donors that match the exposure scenario as closely as possible and that will account for differences in methodology between laboratories. We have seen that at low doses, variability in the results may be attributed to variation in the processing while at higher doses the variation is dominated by inter-individual variation in cell proliferation. This interlaboratory collaboration further highlights the usefulness of the CBMN endpoint to accurately reconstruct absorbed dose in human blood after ionizing radiation exposure.
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Citocinesis , Radiometría , Humanos , Radiometría/métodos , Triaje/métodos , Linfocitos , Pruebas de Micronúcleos/métodosRESUMEN
Aberrant glycosylation is a universal feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer type. A common change in tumour cell glycosylation is an increase in α2,6 sialylation of N-glycans, a modification driven by the sialyltransferase ST6GAL1. ST6GAL1 is overexpressed in numerous cancer types, and sialylated glycans are fundamental for tumour growth, metastasis, immune evasion, and drug resistance, but the role of ST6GAL1 in prostate cancer is poorly understood. Here, we analyse matched cancer and normal tissue samples from 200 patients and verify that ST6GAL1 is upregulated in prostate cancer tissue. Using MALDI imaging mass spectrometry (MALDI-IMS), we identify larger branched α2,6 sialylated N-glycans that show specificity to prostate tumour tissue. We also monitored ST6GAL1 in plasma samples from >400 patients and reveal ST6GAL1 levels are significantly increased in the blood of men with prostate cancer. Using both in vitro and in vivo studies, we demonstrate that ST6GAL1 promotes prostate tumour growth and invasion. Our findings show ST6GAL1 introduces α2,6 sialylated N-glycans on prostate cancer cells and raise the possibility that prostate cancer cells can secrete active ST6GAL1 enzyme capable of remodelling glycans on the surface of other cells. Furthermore, we find α2,6 sialylated N-glycans expressed by prostate cancer cells can be targeted using the sialyltransferase inhibitor P-3FAX -Neu5Ac. Our study identifies an important role for ST6GAL1 and α2,6 sialylated N-glycans in prostate cancer progression and highlights the opportunity to inhibit abnormal sialylation for the development of new prostate cancer therapeutics. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Neoplasias de la Próstata , Sialiltransferasas , Masculino , Humanos , Glicosilación , Polisacáridos/química , Polisacáridos/metabolismo , Reino Unido , beta-D-Galactósido alfa 2-6-Sialiltransferasa , Antígenos CD/metabolismoRESUMEN
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.
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Citocinesis , Radiometría , Humanos , Citocinesis/genética , Pruebas de Micronúcleos/métodos , Citometría de Flujo/métodos , Radiometría/métodosRESUMEN
Background: Standard Breast Cancer (BC) risk prediction models based only on epidemiologic factors generally have quite poor performance, and there have been a number of risk scores proposed to improve them, such as AI-based mammographic information, polygenic risk scores and pathogenic variants. Even with these additions BC risk prediction performance is still at best moderate. In that decreased DNA repair capacity (DRC) is a major risk factor for development of cancer, we investigated the potential to improve BC risk prediction models by including a measured phenotypic DRC assay. Methods: Using blood samples from the Breast Cancer Family Registry we assessed the performance of phenotypic markers of DRC in 46 matched pairs of individuals, one from each pair with BC (with blood drawn before BC diagnosis) and the other from controls matched by age and time since blood draw. We assessed DRC in thawed cryopreserved peripheral blood mononuclear cells (PBMCs) by measuring γ-H2AX yields (a marker for DNA double-strand breaks) at multiple times from 1 to 20 hrs after a radiation challenge. The studies were performed using surface markers to discriminate between different PBMC subtypes. Results: The parameter Fres, the residual damage signal in PBMC B cells at 20 hrs post challenge, was the strongest predictor of breast cancer with an AUC (Area Under receiver-operator Curve) of 0.89 [95% Confidence Interval: 0.84-0.93] and a BC status prediction accuracy of 0.80. To illustrate the combined use of a phenotypic predictor with standard BC predictors, we combined Fres in B cells with age at blood draw, and found that the combination resulted in significantly greater BC predictive power (AUC of 0.97 [95% CI: 0.94-0.99]), an increase of 13 percentage points over age alone. Conclusions: If replicated in larger studies, these results suggest that inclusion of a fingerstick-based phenotypic DRC blood test has the potential to markedly improve BC risk prediction.
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Oregon has a lack of primary care providers in rural areas. To address this issue, employers have indicated they plan to hire greater numbers of advanced practice registered nurses (APRNs). Oregon Health & Science University (OHSU) School of Nursing (SoN) responded to this need by developing a statewide delivery model to educate APRN students in their communities. A performance improvement work group including practice faculty, statewide academic leaders, and staff created a project charter with scope of work, timelines, and outcomes with the goal of improving the systems supporting APRN education. An initial distance APRN education delivery model emerged from this effort and was refined over the following year. Strategies were implemented to address identified challenges using small cycles of change. The final model has three main principles: being learner-centered, equitable, and sustainable. The central outcome is graduating students committed to practicing in rural and urban underserved communities to meet workforce needs in Oregon.
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Bachillerato en Enfermería , Educación de Postgrado en Enfermería , Estudiantes de Enfermería , Humanos , Población Rural , Estudiantes , EscolaridadRESUMEN
The γ-H2AX assay is a sensitive and reliable method to evaluate radiation-induced DNA double-strand breaks. The conventional γ-H2AX assay detects individual nuclear foci manually, but is labor-intensive and time-consuming, and hence unsuitable for high-throughput screening in cases of large-scale radiation accidents. We have developed a high-throughput γ-H2AX assay using imaging flow cytometry. This method comprises (1) sample preparation from small volumes of blood in the Matrix™ 96-tube format, (2) automated image acquisition of cells stained with immunofluorescence-labeled γ-H2AX using ImageStream®X, and (3) quantification of γ-H2AX levels and batch processing using the Image Data Exploration and Analysis Software (IDEAS®). This enables the rapid analysis of γ-H2AX levels in several thousand of cells from a small volume of blood with accurate and reliable quantitative measurements for γ-H2AX foci and mean fluorescence levels. This high-throughput γ-H2AX assay could be a useful tool not only for radiation biodosimetry in mass casualty events, but also for large-scale molecular epidemiological studies and individualized radiotherapy.
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Ensayos Analíticos de Alto Rendimiento , Histonas , Histonas/genética , Citometría de Flujo/métodos , Núcleo Celular , Roturas del ADN de Doble CadenaRESUMEN
BACKGROUND: Population studies suggest cancer morbidity may be different in Turner syndrome (TS) compared to the background female population. However, significant variability is observed in cancer associations likely due to heterogeneity in patient cohorts. We explored the prevalence and patterns of cancer amongst a cohort of women with TS attending a dedicated TS clinic. METHODS: Retrospective analysis of the patient database was performed to identify TS women who developed cancer. Population data (available before 2015) from the National Cancer Registration and Analysis Service database were used for comparison. RESULTS: Out of 156 TS women, median age of 32 (range 18-73) years, 9 (5.8%) had a recorded cancer diagnosis. Types of cancers were, bilateral gonadoblastoma, type 1 gastric neuroendocrine tumour (NET), appendiceal-NET, gastrointestinal stromal tumour, plasma cell dyscrasia, synovial sarcoma, cervical cancer, medulloblastoma and aplastic anaemia. Median age at cancer diagnosis was 35 (range 7-58) years and two were detected incidentally. Five women had 45,X karyotype, three received growth hormone treatment and all except one received oestrogen replacement therapy. The cancer prevalence of the background age-matched female population was 4.4%. CONCLUSIONS: We confirm the previous observations that women with TS do not appear to be at overall increased risk of common malignancies. Our small cohort showed a spectrum of rare malignancies that are not typically associated with TS, except for a single patient with a gonadoblastoma. The slightly higher prevalence of cancer in our cohort might simply represent increased cancer prevalence in the background population, or might be related to small sample size and regular monitoring of these women due to TS per se.