Translational research has improved the diagnosis and follow-up of hematological diseases and malignancies. However, some classical diagnostics used for research and clinical practice that have remain practically unchanged for decades may be better addressed through advances in flow cytometry technology, whereby more precise measurements may be implemented in a straightforward manner. The current method for semiquantitative analysis of granulocytic alkaline phosphatase (GAP) activity is still based on observer-dependent color-intensity classification. Here, we describe a novel strategy for flow cytometric quantification of GAP activity in which staining and analytical flow cytometry facilitate the detection and quantification of subpopulations of leukocytes with different GAP activities. Our experiments demonstrate the potential of flow cytometry as a simple and highly sensitive approach for measuring GAP activity in unlysed whole blood. Liothyronine in vivo Notably, a comparison of flow cytometry and enzyme cytochemistry techniques showed that enzyme activity scores were not similar, indicating that results needs to be interpreted with caution, given that the enzyme-substrate binding affinities may differ, as well as the subjective evaluation of the intensity of the precipitated dye. © 2020 Wiley Periodicals LLC. Basic Protocol Protocol preparation, sample acquisition, and gating strategy for flow cytometric identification of alkaline phosphatase activity in granulocytes from whole blood samples Support Protocol 1 Sample preparation for granulocyte alkaline phosphatase determination by flow cytometry using no-lyse no-wash methods Support Protocol 2 Data analysis and formula to calculate the GAP score.The efficacy of tandem HDCT against high-risk neuroblastoma has been reported; however, an optimal regimen remains to be established. In this paper, we report our experience using tandem HDCT comprising the MEC and BuMel regimens in patients with high-risk neuroblastoma. We retrospectively analyzed four patients with stage M high-risk neuroblastoma who received HDCT with MEC followed by BuMel combined with autologous stem cell rescue. Although none of their metastatic lesions had disappeared after induction chemotherapy, three patients showed a CR after tandem HDCT. Gastrointestinal mucosal injuries and renal dysfunction were observed as non-hematologic adverse events of grade 3 or higher. Gastrointestinal mucosal injuries were observed in all four patients following the first HDCT and in one patient following the second HDCT and were treated with parenteral nutrition and analgesics. One patient experienced renal dysfunction during the first HDCT, which was alleviated by sufficient hydration and diuretics and resulted in the reduction of melphalan dosage for the second HDCT. SOS was not observed in any patient. The HDCT regimens examined in this study were observed to be feasible and did not result in any life-threatening adverse events. Our findings indicate that tandem HDCT comprising MEC and BuMel is a potentially effective regimen for patients with high-risk neuroblastoma, including for those who respond poorly to induction chemotherapy, although additional studies in a larger population should be conducted to verify any long-term outcomes and toxicity.Background Congenital sensorineural deafness (CSD) is the most common type of deafness in Dalmatian dogs. Objectives To use results of CSD screening in Dalmatian dogs in the United Kingdom in genetic analysis and to determine any changes in the prevalence of CSD in this breed over time. Animals A total of 8955 Dalmatian puppies undergoing hearing function screening using brainstem auditory evoked response (BAER) between July 1992 and February 2019. Methods Results of BAER testing and pigmentation phenotypic data were linked to the UK Kennel Club Dalmatian pedigree database. Mixed model analysis was used to estimate variance parameters. Results The overall prevalence of CSD was 17.8% (13.4%, unilateral; 4.4%, bilateral). Heritability of CSD was approximately 0.3 (across models) and significantly >0. Genetic correlations between CSD and blue irises (+0.6) and pigmented head patch (-0.86) were large in magnitude and significantly different form 0. Significant improving phenotypic and genetic trends were identified, likely as the result of selection against deafness, equivalent to avoiding breeding with the 4% to 5% of animals with the highest genetic risk of CSD. Conclusions and clinical importance A decrease in the prevalence and genetic risk of CSD implies breeders have been selecting for hearing dogs. Selective breeding based on estimated breeding values (EBVs) can help further decrease the prevalence of CSD in Dalmatians in the future.Despite the established benefits of long-term endocrine therapy, women with hormone receptor-positive breast cancer remain at risk for late relapse. The basis of this is multi-factorial including genetic, epigenetic, and host factors. In this study we have explored the epigenetic regulation of estrogen receptor (ER)-dependent molecular and cellular phenotype by hsa-miR-18a-5p using well-established human ER-positive (ER+) breast cancer cell lines. miR-18a was overexpressed in MCF7 and ZR-75-1 and this led to an increase in the proliferative ability of the cells and concurrently resulted in decreased expression of luminal markers and higher expression of the basal marker, cytokeratin 14. The cells became more migratory with a significant repression of E-cadherin and activation of the Wnt noncanonical pathway. We observed an activation of the planar cell polarity (PCP) pathway with increased activation of JNK pathway and eventually change in actin dynamics. There was increased F-actin polymerization in cells with higher expression of miR-18a. Examination of miR-18a expression in a set of human ER+ breast cancer specimens showed a negative correlation between miR-18a and ESR1 transcripts as well as ER protein. Kaplan-Meier survival analysis of the cohort stratified by tumor hsa-miR-18a-5p levels produced significant differences in disease-free survival (log rank P less then .05). This observation was independently validated in the METABRIC cohort. These data provide support for a role of hsa-miR-18a-5p in altering the proliferative and migratory behavior of ER+ cells and its potential utility as a prognostic marker in clinical ER+ breast cancers.Background Accumulating data support the role of flow cytometry (FCM) in diagnostic work-up of myelodysplastic syndromes (MDS). Changes in erythropoiesis are less documented than in granulopoiesis. However, most studies were performed on bone marrow samples (BMSs) after red blood cell lysis. We have established a FCM protocol for erythropoiesis, following a no-lysis approach and live gate acquisition of nucleated cells using DNA dye DRAQ5. link2 Methods The ERY tube consisted of CD36, CD71, CD105, CD117, CD13, and CD45. Comparison with cytomorphological differential counts was carried out in a learning cohort of 80 BMS. To detect aberrations, we analyzed 208 BMS from 135 patients and five normal donors, divided into three cohorts MDS (n = 68), nonclonal cytopenia (n = 43), and normal controls (n = 29). Radar plot (RP) was created for an overview of normal and aberrant patterns. Results The proportion of erythropoiesis in the ERY tube showed better agreement with the cytomorphology, compared to FCM panels on lysed BMS. We confirmed that aberrations in coefficient of variation (CV) of CD36 fluorescence intensity (p less then .001), mean fluorescence intensity of CD36 (p = .012), and CV of CD105 (p less then .001) can distinguish between MDS and nonclonal cytopenia. RP facilitated evaluation of erythropoietic maturation patterns and aberrant patterns were identified in 85% of MDS patients. Conclusion This study provides evidence that a no-lysis approach and RP analysis allow a more reliable evaluation of erythropoiesis and erythroid dysplasia, supporting the integration of FCM erythroid panels in the standard work-up of MDS.Background Intraoperative nerve monitoring (IONM) of the recurrent laryngeal nerve (RLN) predicts the risk of vocal cord palsy (VCP). IONM can be used to adapt the surgical strategy in order to prevent bilateral VCP and associated morbidity. link3 Controversial results have been reported in the literature for the effect of IONM on rates of VCP, and large multicentre studies are required for elucidation. Methods Patients undergoing first-time thyroidectomy for benign thyroid disease between May 2015 and January 2019, documented prospectively in the European registry EUROCRINE®, were included in a cohort study. The influence of IONM and other factors on the development of postoperative VCP was analysed using multivariable regression analysis. Results Of 4598 operations from 82 hospitals, 3542 (77·0 per cent) were performed in female patients. IONM was used in 4182 (91·0 per cent) of 4598 operations, independent of hospital volume. Postoperative VCP was diagnosed in 50 (1·1 per cent) of the 4598 patients. The use of IONM was associated with a lower risk of postoperative VCP in multivariable analysis (odds ratio (OR) 0·34, 95 per cent c.i. 0·16 to 0·73). Damage to the RLN noted during surgery (OR 24·77, 12·91 to 48·07) and thyroiditis (OR 2·03, 1·10 to 3·76) were associated with an increased risk of VCP. Higher hospital volume correlated with a lower rate of VCP (OR 0·05, 0·01 to 0·13). Conclusion Use of IONM was associated with a low rate of postoperative VCP.Object Revealed the spatial-temporal patterns of acquired immune deficiency syndrome (AIDS) incidences in Mainland China. Methods Empirical orthogonal function (EOF) technique was applied to analyze the major spatial distribution modes and the temporal changes of AIDS incidences in Mainland China during 2002-2017. Results The annual average AIDS incidences increased from 0.06 per 100 000 in 2002 to 4.15 per 100 000 in 2017, with an annual average increase of 0.31 per 100 000. The southwest regions were high-incidence areas, as well as Xinjiang province in the northwest. There were two typical spatial modes. EOF 1 represented an isodirectional spatial pattern that the incidences were relatively high in general, and the fluctuation ranges were relatively high in the southwest and northeast. EOF 2 represented a reverse spatial pattern that the incidences were relatively high (or low) in Guangxi, Yunnan, Xinjiang, Shanghai, and Henan, yet were relatively low (or high) in the remaining regions. Conclusion The AIDS incidences in Mainland China were relatively low during 2002-2010, yet were kept in a relatively high level since 2012. The prevention and control of AIDS need further development, especially in the southwest regions.Tirapazamine (TPZ) has been tested in clinical trials on radio-chemotherapy due to its potential highly selective toxicity towards hypoxic tumour cells. It was suggested that either the hydroxyl radical or benzotriazinyl radical may form as bioactive radical after the initial reduction of TPZ in solution. In the present work, we studied low-energy electron attachment to TPZ in the gas phase and investigated the decomposition of the formed TPZ – anion by mass spectrometry. We observed the formation of the (TPZ – OH) – anion accompanied by the dissociation of the hydroxyl radical as by far the most abundant reaction pathway upon attachment of a low-energy electron. Quantum chemical calculations suggest that NH 2 pyramidalization is the key reaction coordinate for the reaction dynamics upon electron attachment. We propose an OH roaming mechanism for other reaction channels observed, in competition with the OH dissociation.