Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds.

Flow cytometric immunophenotyping has become essential for accurate diagnosis, classification, and disease monitoring in hemato-oncology. The EuroFlow Consortium has established a fully standardized "all-in-one" pipeline consisting of standardized instrument settings, reagent panels, and sample preparation protocols and software for data analysis and disease classification. For its reproducible implementation, parallel development of a quality assurance (QA) program was required. Here, we report on the results of four consecutive annual rounds of the novel external QA EuroFlow program. The novel QA scheme aimed at monitoring the whole flow cytometric analysis process (cytometer setting, sample preparation, acquisition and analysis) by reading the median fluorescence intensities (MedFI) of defined lymphocytes' subsets. Each QA participant applied the predefined reagents' panel on blood cells of local healthy donors. A uniform gating strategy was applied to define lymphocyte subsets and to read MedFI values per marker. The MedFI values were compared with reference data and deviations from reference values were quantified using performance score metrics. In four annual QA rounds, we analyzed 123 blood samples from local healthy donors on 14 different instruments in 11 laboratories from nine European countries. The immunophenotype of defined cellular subsets appeared sufficiently standardized to permit unified (software) data analysis. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%, average MedFI in each QA round ranged from 86 to 125% from overall median. Calculation of performance scores was instrumental to pinpoint standardization failures and their causes. Overall, the new EuroFlow QA system for the first time allowed to quantify the technical variation that is introduced in the measurement of fluorescence intensities in a multicentric setting over an extended period of time. EuroFlow QA is a proficiency test specific for laboratories that use standardized EuroFlow protocols. It may be used to complement, but not replace, established proficiency tests. © 2014 International Society for Advancement of Cytometry.

Decreased entropy modulation of EEG response to novelty and relevance in schizophrenia during a P300 task.

The analysis of the interaction between novelty and relevance may be of interest to test the aberrant salience hypothesis of schizophrenia (SCH). In comparison with other neuroimaging techniques, such as functional magnetic resonance imaging, electroencephalography (EEG) provides high temporal resolution. Therefore, EEG is useful to analyze transient dynamics in neural activity, even in the range of milliseconds. In this study, EEG activity from 31 patients with SCH and 38 controls was analyzed using Shannon spectral entropy (SE) and median frequency (MF). The aim of the study was to quantify differences between distractor (i.e., novelty) and target (i.e., novelty and relevance) tones in an auditory oddball paradigm. Healthy controls displayed a larger SE decrease in response to target stimulus than in response to distractor tones. SE decrease was accompanied by a significant and widespread reduction of MF (i.e., a significant slowing of EEG activity). In comparison with controls, patients showed a significant reduction of changes in SE in response to both target and distractor tones. These differences were also observed in patients that only received a minimal treatment prior to EEG recording. Furthermore, significant changes in SE were inversely correlated to positive and total symptoms severity for SCH patients. Our findings support the notion that SCH is associated with a reduced response to both novelty and relevance during an auditory P300 task.

Predictive factors for mortality in Fournier' gangrene: a series of 59 cases.

AIMS: Fournier's gangrene (FG) is the necrotizing fasciitis of the perineum and genital area and presents a high mortality rate. The aim was to assess prognostic factors for mortality, create a new mortality predictive scale and compare it with previously published scales in patients diagnosed with FG in our Emergency Department. METHODS: Retrospective analysis study between 1998 and 2012. RESULTS: Of the 59 patients, 44 survived (74%) (S) and 15 died (26%) (D). Significant differences were found in peripheral vasculopathy (S 5 [11%]; D 6 [40%]; P=.023), hemoglobin (S 13; D 11; P=.014), hematocrit (S 37; D 31.4; P=.009), white blood cells (S 17,400; D 23,800; P=.023), serum urea (S 58; D 102; P<.001), creatinine (S 1.1; D 1.9; P=.032), potassium (S 3.7; D 4.4; P=.012) and alkaline phosphatase (S 92; D 133; P=.014). Predictive scores: Charlson index (S 1; D 4; P=.013), severe sepsis criteria (S 16 [36%]; D 13 [86%]; P=.001), Fournier's gangrene severity index score (FGSIS) (S 4; D 7; P=.002) and Uludag Fournier's Gangrene Severity Index (UFGSI) (S 9; D 13; P=.004). Independent predictive factors were peripheral vasculopathy, serum potassium and severe sepsis criteria, and a model was created with an area under the ROC curve of 0.850 (0.760-0.973), higher than FGSIS (0.746 [0.601-0.981]) and UFGSI (0.760 [0.617-0.904]). CONCLUSIONS: FG showed a high mortality rate. Independent predictive factors were peripheral vasculopathy, potassium and severe sepsis criteria creating a predictive model that performed better than those previously described.

Decreased spectral entropy modulation in patients with schizophrenia during a P300 task.

Spectral entropy (SE), also known as Shannon entropy, is a useful parameter for quantifying the global regularity of the electroencephalographic (EEG) signal. Hence, it is of interest in the assessment of the electrophysiological correlates of cognitive processing in schizophrenia. However, to date, SE has been barely used in studies comparing resting EEG recordings between patients and controls. In this work, we compared SE between resting baseline [-250 0] ms and active task [150 550] ms windows of a P300 task in 31 patients with schizophrenia and 38 controls. Moreover, we also calculated the median frequency (MF) and relative power in each frequency band for these windows to assess the correlates of the possible SE differences. Controls showed a significant (p < 0.0029) SE decrease (i.e., meaning higher signal regularity) from baseline to the active task window at parietal and central electrode sites. This SE decrease from baseline to active conditions was significantly lower in patients. In controls, this SE decrease was accompanied by a statistically significant decrease in MF (i.e., a significant slowing of the EEG activity), not observed in patients. In this latter group, the difference in SE between resting baseline and active task windows was inversely correlated to positive and total symptoms scores, as measured with the positive and negative symptoms scale. Our data support the relevance of SE in the study of cerebral processing in schizophrenia.

The Role of Adiponectin in the Resolution of Male-Obesity-Associated Secondary Hypogonadism after Metabolic Surgery and Its Impact on Cardiovascular Risk.

Male-obesity-associated secondary hypogonadism (MOSH) is a very prevalent entity that may resolve after marked weight loss. Adiponectin (APN) is an adipokine with anti-inflammatory properties that regulates metabolism. Low-circulating APN is associated with obesity, diabetes, and cardiovascular risk, along with circulating testosterone. We aimed to evaluate APN changes in men with MOSH (low circulating free testosterone (FT) with low or normal gonadotropins) and without it after metabolic surgery. We look for their possible association with cardiovascular risk measured by carotid intima-media thickness (cIMT). We included 60 men (20 submitted to lifestyle modification, 20 to sleeve gastrectomy, and 20 to gastric bypass) evaluated at baseline and 6 months after. The increase in APN at follow-up was reduction in patients with persistent MOSH (n = 10) vs. those without MOSH (n = 30) and MOSH resolution (n = 20), and the former did not achieve a decrease in cIMT. The increase in APN correlated positively with FT (r = 0.320, p = 0.013) and inversely with cIMT (r = -0.283, p = 0.028). FT inversely correlated with cIMT (r = -0.269, p = 0.038). In conclusion, men without MOSH or with MOSH resolution showed a high increase in APN after weight loss with beneficial effects on cIMT. Those without MOSH resolution failed to attain these effects.

Initial Single-center Experience of PIPAC in Patients With Unresectable Peritoneal Metastasis. / Experiencia inicial del tratamiento de la carcinomatosis peritoneal irresecable con PIPAC.

INTRODUCTION: Peritoneal carcinomatosis remains a condition with poor prognosis and limited therapeutic options. Pressurized Intrapertioneal Aerosol Chemotherapy (PIPAC) has been developed as a new tool for delivering intraperitoneal chemotherapy with low morbidity. The aim of this study was to evaluate the initial experience of PIPAC in patients with peritoneal carcinomatosis at our hospital. METHODS: A prospective study between January 2019 and February 2020 was carried at a tertiary public hospital. Primary tumor, ascites volume, PCI, chemotherapy regimen, operative time, morbidity, length of hospital stay and mortality were recorded for analysis. RESULTS: We analyzed 9 PIPAC procedures performed in 5patients. Median PCI was 27.6 (24-35). Median surgical time was 93minutes (70-125). Only one adverse event occurred out of 9 procedures (Clavien-DindoII). Median length of hospital stay was 2days (1-4). Mortality was 0%. CONCLUSION: PIPAC seems to be a feasible and safe procedure to treat peritoneal carcinomatosis, with low morbidity and short hospital stay.

Sleeve Gastrectomy and Gastric Bypass Decrease the Carotid Intima-Media Thickness in Obese Men: Association with Weight Loss, Cardiovascular Risk Factors, and Circulating Testosterone.

BACKGROUND: Obesity surgery has shown to decrease the carotid intima-media thickness (IMT), but studies that compare different surgical techniques are scarce, especially in men. OBJECTIVE: To evaluate the changes in IMT in men after laparoscopic Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) and its association with circulating testosterone. SETTING: Academic Hospital. METHODS: We studied 40 men with severe obesity, of whom 20 were submitted to laparoscopic RYGB and 20 to SG. Twenty control men matched for age and degree of obesity were also included. Both patients and controls were evaluated at baseline and 6 months after surgery or conventional treatment with diet and exercise, respectively. RESULTS: The mean carotid IMT decreased after surgery irrespective of the surgical technique whereas no changes were observed in the control men submitted to conventional therapy (Wilks' λ = 0.745, P < 0.001 for the interaction, P < 0.001 for RYGB vs. controls, P = 0.001 for SG vs. controls, P = 0.999 for RYGB vs. SG). The decrease in the carotid IMT correlated with the increase in total testosterone (r = 0.428, P = 0.010) and lost BMI (r = 0.486, P < 0.001). Multivariate linear regression retained only the decrease in BMI (ß = 0.378, P = 0.003) after adjustment (R2 = 0.245, F = 9.229, P = 0.001). CONCLUSION: Both RYGB and SG decrease carotid IMT in men with obesity compared with conventional treatment with diet and exercise.

Circulating Zinc and Copper Levels are Associated with Sperm Quality in Obese Men after Metabolic Surgery: A Pilot Study.

(1) Background: Inadequate levels of several trace elements and vitamins may impair spermatogenesis in men. Although weight loss after metabolic surgery normalizes male reproductive hormones, sperm quality seems to not improve. We hypothesized that circulating concentrations of zinc, copper and other trace elements and vitamins might be involved. (2) Methods: We studied 20 men submitted to metabolic surgery at baseline and after two years. Hormone profiles, serum trace elements and vitamins were studied together with sperm analysis. (3) Results: At follow-up, serum testosterone, follicle-stimulating hormone and inhibin B concentrations increased showing a beneficial hormonal response for spermatogenesis. Conversely, serum copper, zinc and ferritin showed a decline after surgery. In total, 33% of men showed zinc deficiency, 27% copper deficiency and 20% iron deficiency, among others. Sperm analysis showed that all revaluated patients had at least one abnormal parameter. Serum zinc concentrations showed a positive correlation with progressive motility (r = 0.577, p = 0.031), and serum ferritin a positive correlation with sperm volume (ρ = 0.535, p = 0.049). Serum copper showed a weak and near significant correlation with motility (r = 0.115, p = 0.051). (4) Conclusions: The lack of improvement in sperm quality in obese men after metabolic surgery may be related to nutrient malabsorption, especially zinc, copper and iron.

Soluble and membrane levels of molecules involved in the interaction between clonal plasma cells and the immunological microenvironment in multiple myeloma and their association with the characteristics of the disease.

Clonal plasma cells (PC) from different types of monoclonal gammopathies (MG) display distinct phenotypes consistent with an increased antigen-presentation and T-cell costimulation in MG of undetermined significance that deteriorates in malignant conditions. Expression of other cell surface and soluble molecules (e.g. adhesion/proliferation molecules) involved in the interaction between clonal PC and the bone marrow (BM) microenvironment has also been related to malignant PC, although the exact clinical significance of their expression remains largely unknown. Analysis of cell surface levels of several of these molecules in multiple myeloma (MM) patients shows an association between lower expression on BMPC of the HLA-I and beta2-microglobulin antigen-presenting molecules, the CD126 and CD130 IL6 receptor (IL6R) chains, and CD38, and adverse prognostic features of the disease. Likewise, patients showing higher soluble levels of antigen-presenting molecules (HLA-I and beta2-microglobulin), IL6R and CD95 tended to be associated with more aggressive disease behavior. In contrast, CD40, CD86, CD56, CD19, and CD45 were not associated with patients' outcome. Interestingly, upon considering the ratio between the soluble and PC membrane expression of each molecule, an increased adverse prognostic impact was observed for both HLA-I and beta2-microglobulin, but not for the other molecules. Multivariate analysis confirmed the independent prognostic value of cell surface expression of CD126 on BMPC together with serum beta2-microglobulin and LDH. In summary, our results show an abnormal distribution of the cellular and soluble compartments of the HLA-I, IL6R, and to a lower extent, CD95 molecules, in MM, associated with the clinical characteristics and behavior of the disease.

Lot-to-lot stability of antibody reagents for flow cytometry.

The fluorescence detected using fluorochrome-labelled monoclonal antibodies depends not only on the abundance of the target antigen, but amongst many other factors also on the effective fluorochrome-to-antibody ratio. The diagnostic approach of the EuroFlow consortium relies on reproducible fluorescence intensities over time. A capture bead system for mouse immunoglobulin light chains was utilized to compare the mean fluorescence intensity of 1323 consecutive antibody lots to the currently used lot of the same monoclonal antibody. In total, 157 different monoclonal antibodies were assessed over seven years. Median relative difference between consecutive lots was 3.8% (range: 0.01% to 164.7%, interquartile range: 1.3% to 10.1%). The relative difference exceeded 20% in 8.8% of all comparisons. FITC labelled monoclonal antibodies (median relative difference: 2.1%) showed a significantly smaller variation between lots than antibodies conjugated to PE (3.5%), PECy7 (3.9%), PerCPCy5.5 (5.8%), APC (5.8%), APCH7 (7.4%), and APCC750 (14.5%). Reagents labelled with Pacific Blue (1.4%), Pacific Orange (2.4%), HV450 (0.7%), and HV500 (1.7%) demonstrated more consistent results compared to conjugates of BV421 (4.1%) and BV510 (16.2%). Additionally, significant differences in lot-to-lot fluorescence stability amongst antibodies labelled with the same fluorochrome were observed between manufacturers. These observations might guide future quality recommendations for the production and application of fluorescence-labelled monoclonal antibodies in multicolor flow cytometry.

Fluorochrome choices for multi-color flow cytometry.

Fluorochrome selection is a key step in designing multi-color antibody panels. The list of available fluorochromes is continuously growing, fitting current needs in clinical flow cytometry to simultaneously use more markers to better define multiple leukocyte subpopulations in a single tube. Several criteria guide fluorochrome selection: i) the fluorescence profiles (excitation and emission), ii) relative brightness, iii) fluorescence overlap, iv) fluorochrome stability, and v) reproducible conjugation to antibodies. Here we used 75 samples (45 bone marrow and 30 blood) to illustrate EuroFlow strategies for evaluation of compatible fluorochromes, and how the results obtained guide fluorochrome selection as a critical step in the antibody-panel building process. Our results allowed identification of optimal fluorescence profiles (e.g. higher fluorescence intensity and/or resolution with limited fluorescence overlap into neighbor channels) for brilliant violet (BV)421 and BV510 in the violet laser and allophycocyanin (APC) hilite 7 (H7) or APC C750 in the red laser vs. other candidate fluorochromes generally applied for the same detectors and here evaluated. Moreover, evaluation of the same characteristics for another group of fluorochromes (e.g. BV605, BV650, PE CF594, AF700 or APC AF700) guided selection of the most appropriate fluorochrome conjugates to be combined in a multi-color antibody panel. Albeit this is a demanding approach, it could be successfully applied for selection of fluorochrome combinations for the EuroFlow antibody panels for diagnosis, classification and monitoring of hematological malignancies and primary immunodeficiencies. Consequently, sets of 8-, 10- and 12-color fluorochrome combinations are proposed as frame of reference for initial antibody panel design.

Selection and validation of antibody clones against IgG and IgA subclasses in switched memory B-cells and plasma cells.

The clinical value of assessing immunoglobulin (Ig)G and IgA subclasses in addition to the isotypes of soluble Igs in serum has been well established. >20years ago, the International Union of Immunological Societies and the World Health Organization performed collaborative studies in order to validate antibody (Ab) clones for the detection of IgG and IgA subclasses for a broad range of laboratory assays, except for flow cytometry. Here we analyzed the performance of commercially available Ab clones to detect IgG and IgA subclasses in memory B-cells and plasma cells (PCs) by flow cytometry. In a first step, 28 Ab clones were evaluated in peripheral blood from healthy donors. Only 17/28 clones showed reactivity against IgG and IgA subclasses expressed on the B-cell and PC surface membrane, including Ab clones for IgG1 (SAG1, HP6188, HP6001 and HP6186), IgG2 (SAG2, HP6014 and HP6002), IgG3 (SAG3, HP6095 and HP6050), IgG4 (SAG4), IgA1 (SAA1, H69-11.4 and B3506B4) and IgA2 (SAA2, 2E2, and A9604D2). In a second step, for each Ig subclass a single clone was selected according to its specificity and fluorescence intensity (resolution power), for further more detailed validation (SAG1, SAG2, SAG3, SAG4, SAA1 and SAA2). This validation process was carried out in 4 different laboratories by testing the selected Ab clones in human peripheral blood, bone marrow and tonsil samples, using different staining protocols (e.g. surface membrane and/or cytoplasmic staining). All selected Ab clones displayed strong positivity, high specificity and optimal resolution between negative and positive cells. Alternative Ab clones were also validated. Thus, our results show the feasibility of using the validated Ig subclass Ab clones in combination with other B cell-associated markers for detailed dissection of the memory B-cell and PC compartments that express distinct Ig subclasses in different human tissues.

Optimization and testing of dried antibody tube: The EuroFlow LST and PIDOT tubes as examples.

Within EuroFlow, we recently developed screening tubes for hematological malignancies and immune deficiencies. Pipetting of antibodies for such 8-color 12-marker tubes however is time-consuming and prone to operational mistakes. We therefore evaluated dried formats of the lymphocytosis screening tube (LST) and of the primary immune deficiency orientation tube (PIDOT). Both tubes were evaluated on normal and/or on patient samples, comparing the mean fluorescence intensity of specific lymphocyte populations. Our data show that the dried tubes and liquid counterparts give highly comparable staining results, particularly when analyzed in multidimensional plots. In addition, the use of dried tubes may result in a reduced staining variability between different samples and thereby contributes to the generation of more robust data. Therefore, by using ready-to-use reagents in a dried single test tube format, the laboratory efficiency and quality will be improved.

Deficits of entropy modulation in schizophrenia are predicted by functional connectivity strength in the theta band and structural clustering.

Spectral entropy (SE) allows comparing task-related modulation of electroencephalogram (EEG) between patients and controls, i.e. spectral changes of the EEG associated to task performance. A SE modulation deficit has been replicated in different schizophrenia samples. To investigate the underpinnings of SE modulation deficits in schizophrenia, we applied graph-theory to EEG recordings during a P300 task and fractional anisotropy (FA) data from diffusion tensor imaging in 48 patients (23 first episodes) and 87 healthy controls. Functional connectivity was assessed from phase-locking values among sensors in the theta band, and structural connectivity was based on FA values for the tracts connecting pairs of regions. From those data, averaged clustering coefficient (CLC), characteristic path-length (PL) and connectivity strength (CS, also known as density) were calculated for both functional and structural networks. The corresponding functional modulation values were calculated as the difference in SE and CLC, PL and CS between the pre-stimulus and response windows during the task. The results revealed a higher functional CS in the pre-stimulus window in patients, predictive of smaller modulation of SE in this group. The amount of increase in theta CS from pre-stimulus to response related to SE modulation in patients and controls. Structural CLC was associated with SE modulation in the patients. SE modulation was predictive of negative symptoms, whereas CLC and PL modulation was associated with cognitive performance in the patients. These results support that a hyperactive functional connectivity and/or structural connective deficits in the patients hamper the dynamical modulation of connectivity underlying cognition.

Functional EEG network analysis in schizophrenia: Evidence of larger segregation and deficit of modulation.

OBJECTIVE: Higher mental functions depend on global cerebral functional coordination. Our aim was to study fast modulation of functional networks in schizophrenia that has not been previously assessed. METHODS: Graph-theory was used to analyze the electroencephalographic (EEG) activity during an odd-ball task in 57 schizophrenia patients (18 first episode patients, FEPs) and 59 healthy controls. Clustering coefficient (CLC), characteristic path length (PL) and small-worldness (SW) were computed at baseline ([-300 0] ms prior to stimulus delivery) and response ([150 450] ms post-stimulus) windows. Clinical and cognitive assessments were performed. RESULTS: CLC, PL and SW showed a significant modulation between baseline and response in controls but not in patients. Patients obtained higher CLC and SW at baseline, lower CLC and higher PL at response, and diminished modulation of CLC and SW as compared to controls. In patients, CLC and SW modulation were inversely associated to cognitive performance in executive tasks and directly associated to working memory. Similar patterns were observed in FEPs. CLC and SW during the baseline were inversely associated to their respective modulation magnitudes. CONCLUSIONS: Our results are coherent with a hyper-segregated network at baseline (higher CLC) and a decreased modulation of the functional connectivity during cognition in schizophrenia.

Modulation of brain network parameters associated with subclinical psychotic symptoms.

OBJECTIVE: Static deficits in small-world properties of brain networks have been described in clinical psychosis, but task-related modulation of network properties has been scarcely studied. Our aim was to assess the modulation of those properties and its association with subclinical psychosis and cognition in the general population. METHOD: Closeness centrality and small-worldness were compared between pre-stimulus baseline and response windows of an odd-ball task in 200 healthy individuals. The correlation between modulation of network parameters and clinical (scores in the Community Assessment of Psychological Experiences) and cognitive measures (performance in the dimensions included in the Brief Assessment of Cognition in Schizophrenia battery) was analyzed, as well as between these measures and the corresponding network parameters during baseline and response windows during task performance. RESULTS: In the theta band, closeness centrality decreased and small-worldness increased in the response window. Centrality and small-worldness modulation were, respectively, directly and inversely associated with subclinical symptoms. CONCLUSIONS: A widespread modulation of network properties in theta band was observed, with a transient increase of small-worldness during the response window, compatible with a transiently more integrated cortical activity associated to cognition. This supports the relevance of electroencephalography to study of normal and altered cognition and its substrates. A relative deficit in the ability to reorganize brain networks may contribute to subclinical psychotic symptoms.