Preliminary experience of CT imaging of the ischaemic brain penumbra through spectral processing of multiphasic CTA datasets.

To assess ischaemic penumbra through the post-processing of the spectral multiphasic CT Angiography (mCTA) data in acute ischaemic stroke (AIS) patients. Thirty one consecutive patients strongly suspected of severe Middle Cerebral Artery AIS presenting less than 6 h after onset of symptoms or with unknown time of onset of symptoms underwent a standardized CT protocol in spectral mode including Non Contrast CT, mCTA, and Perfusion CT (CTP) on a dual-layer MDCT system. Areas disclosing delayed enhancement on iodine density (ID) maps were highlighted by subtraction of the serial mCTA datasets. Two neuroradiologists independently rated the correspondence between delayed enhancing areas at mCTA and the penumbral/infarcted areas delineated by two validated CTP applications using a 5-levels scoring scale. Interobserver agreement between observers was evaluated by kappa statistics. Dose delivery was recorded for each acquisition. Averaged correspondence score between penumbra delineation using subtracted mCTA-derived ID maps and CTP ones was 2.76 for one application and 2.9 for the other with best interobserver agreement kappa value at 0.59. All 6 stroke mimics out of the 31 patients' cohort were correctly identified. Average dose delivery was 7.55 mSv for the whole procedure of which CTP accounted for 39.7%. Post-processing of spectral mCTA data could allow clinically relevant assessment of the presence or absence of ischaemic penumbra in AIS-suspected patients if results of this proof-of-concept study should be confirmed in larger patients'series.

Prototyping Trastuzumab Docetaxel Immunoliposomes with a New FCM-Based Method to Quantify Optimal Antibody Density on Nanoparticles.

Developing targeted nanoparticles is a rising strategy to improve drug delivery in oncology. Antibodies are the most commonly used targeting agents. However, determination of their optimal number at the surface remains a challenging issue, mainly due to the difficulties in measuring precisely surface coating levels when prototyping nanoparticles. We developed an original quantitative assay to measure the exact number of coated antibodies per nanoparticle. Using flow cytometry optimized for submicron particle analysis and beads covered with known amounts of human IgG-kappa mimicking various amounts of antibodies, this new method was tested as part of the prototyping of docetaxel liposomes coated with trastuzumab against Her2+ breast cancer. This quantification method allowed to discriminate various batches of immunoliposomes depending on their trastuzumab density on nanoparticle surface (i.e., 330 (Immunoliposome-1), 480 (Immunoliposome-2) and 690 (Immunoliposome-3), p = 0.004, One-way ANOVA). Here we showed that optimal number of grafted antibodies on nanoparticles should be finely tuned and highest density of targeting agent is not necessarily associated with highest efficacy. Overall, this new method should help to better prototype third generation nanoparticles.

Mental performance in 8-year-old children fed reduced protein content formula during the 1st year of life: safety analysis of a randomised clinical trial.

In humans, maximum brain development occurs between the third trimester of gestation and 2 years of life. Nutrition during these critical windows of rapid brain development might be essential for later cognitive functioning and behaviour. In the last few years, trends on protein recommendations during infancy and childhood have tended to be lower than that in the past. It remains to be demonstrated that lower protein intakes among healthy infants, a part of being able to reduce obesity risk, is safe in terms of mental performance achievement. Secondary analyses of the EU CHOP, a clinical trial in which infants from five European countries were randomised to be fed a higher or a lower protein content formula during the 1st year of life. Children were assessed at the age of 8 years with a neuropsychological battery of tests that included assessments of memory (visual and verbal), attention (visual, selective, focused and sustained), visual-perceptual integration, processing speed, visual-motor coordination, verbal fluency and comprehension, impulsivity/inhibition, flexibility/shifting, working memory, reasoning, visual-spatial skills and decision making. Internalising, externalising and total behaviour problems were assessed using the Child Behaviour Checklist 4-18. Adjusted analyses considering factors that could influence neurodevelopment, such as parental education level, maternal smoking, child's gestational age at birth and head circumference, showed no differences between feeding groups in any of the assessed neuropsychological domains and behaviour. In summary, herewith we report on the safety of lower protein content in infant formulae (closer to the content of human milk) according to long-term mental performance.

[First line management without IVF of infertility related to endometriosis: Result of medical therapy? Results of ovarian superovulation? Results of intrauterine insemination? CNGOF-HAS Endometriosis Guidelines]. / Prise en charge de l'infertilité en première intention hors FIV : performances du traitement médical ? Performances de la stimulation ovarienne ? Performances des inséminations ? RPC Endométriose CNGOF-HAS.

INTRODUCTION: Using the structured methodology of French guidelines (HAS-CNGOF), the aim of this chapter was to formulate good practice points (GPP), in relation to optimal non-ART management of endometriosis related to infertility, based on the best available evidence in the literature. MATERIALS AND METHODS: This guideline was produced by a group of experts in the field including a thorough systematic search of the literature (from January 1980 to March 2017). Were included only women with endometriosis related to infertility. For each recommendation, a grade (A-D, where A is the highest quality) was assigned based on the strength of the supporting evidence. RESULTS: Management of endometriosis related to infertility should be multidisciplinary and take account into the pain, the global evaluation of infertile couple and the different phenotypes of endometriotic lesions (good practice point). Hormonal treatment for suppression of ovarian function should not prescribe to improve fertility (grade A). After laproscopy for endometriosis related to infertility, the Endometriosis Fertility Index should be used to counsel patients regarding duration of conventional treatments before undergoing ART (grade C). After laparoscopy surgery for infertile women with AFS/ASRM stage I/II endometriosis or superficial peritoneal endometriosis, controlled ovarian stimulation with or without intrauterine insemination could be used to enhance non-ART pregnancy rate (grade C). Gonadotrophins should be the first line therapy for the stimulation (grade B). The number of cycles before referring ART should not exceed up to 6 cycles (good practice point). No recommendation can be performed for non-ART management of deep infiltrating endometriosis or endometrioma, as suitable evidence is lacking. DISCUSSION AND CONCLUSION: Non-ART management is a possible option for the management of endometriosis related to infertility. Endometriosis Fertilty Index could be a useful tool for subsequent postoperative fertility management. Controlled ovarian stimulation can be proposed.

Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop.

Essentials The clinical enumeration of microparticles (MPs) is hampered by a lack of standardization. A new strategy to standardize MP counts by flow cytometry was evaluated in a multicenter study. No difference was found between instruments using forward or side scatter as the trigger parameter. This study demonstrated that beads can be used as a standardization tool for MPs. Click to hear the ISTH Academy's webinar on microvesicles SUMMARY: Background Microparticles (MPs) are extracellular vesicles resulting from the budding of cellular membranes that have a high potential as emergent biomarkers; however, their clinical relevance is hampered by methodological enumeration concerns and a lack of standardization. Flow cytometry (FCM) remains the most commonly used technique with the best capability to determine the cellular origin of single MPs. However, instruments behave variably depending on which scatter parameter (forward (FSC) or side scatter (SSC)) provides the best resolution to discriminate submicron particles. To overcome this problem, a new approach, based on two sets of selected beads adapted to FSC or SSC-optimized instruments, was recently proposed to reproducibly enumerate platelet-derived MP counts among instruments with different optical systems. Objective The objective was to evaluate this strategy in an international workshop that included 44 laboratories accounting for 52 cytometers of 14 types. Methods/Results Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) of instruments. All instruments correctly ranked the platelet MP (PMP) levels of two platelet-free plasma samples. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side-scattered light as the relative sizing parameter. Conclusions Despite remaining limitations, this study is the first to demonstrate a real potential of bead-based strategies for standardization of MP enumeration across different FCM platforms. Additional standardization efforts are still mandatory to evaluate MPs' clinical relevance at a multicenter level.

Standardized counting of circulating platelet microparticles using currently available flow cytometers and scatter-based triggering: Forward or side scatter?

Clinical determination of MP counts using flow cytometry has not been fully accepted yet due to the lack of standardization protocols. In the past 5 years, we have proposed two versions of a method with reproducible PMP counts in plasma samples. Both methods use forward scatter (FSC)-based threshold set with reference beads of appropriate sizes; first using 0.5 µm beads and later with 0.3 µm beads. Both systems provide reproducible PMP counts. However, this technique works only with some of currently used commercial flow cytometers. Instruments with limited resolution or generating heterogeneous FSC signals are excluded. Such performances are incompatible with the required interinstrument standardization. Here we show that (i) flow cytometers with sub-optimal FSC capabilities generally have higher SSC resolution and background rejection capacity, and (ii) that the same biological entities, "dim and bright PMP," both can be counted using alternative strategies, either as previously described, based on FSC measurements, or as presented here, based on SSC detection. The critical element in the standardization protocol is the use of different sizes of reference beads. This study was designed to permit simultaneous access to both FSC- and SSC-optimized platforms. A new range of about 0.17-0.6 µm eq. (µm-equivalents) is proposed for an alternative SSC-based MP gate generating the same PMP counts as those obtained in the previously proposed 0.3-1 µm eq. FSC-based MP gate. The two equivalent standardization options reconcile intrinsically different scattering behaviors between SSC- and FCS--triggered instruments and open the opportunity for multicenter studies in the future.

Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol.

BACKGROUND: Microparticles (MP) are small vesicles of 0.1-1 µm, released in response to activation or apoptosis. Over the past decade, they received an increasing interest both as biomarkers and biovectors in coagulation, inflammation and cancer. Clinical studies were conducted to assess their contribution to the identification of patients at cardiovascular risk. However, among the limitation of such studies, pre-analytical steps remains an important source of variability and artifacts in MP analysis. OBJECTIVES: Because data from the literature are insufficient to establish recommendations, the objective of the present study was to assess the impact of various pre-analytical parameters on MP measurement. These parameters included the type of collection tube, phlebotomy conditions, transportation practices, centrifugation steps and freezing. METHODS: MP were assessed by three methods: flow cytometry using a standardized approach, a thrombin generation test (Calibrated Automated Thrombogram(®)) and a procoagulant phospholipid-dependent clotting time assay (STA(®) -Procoag-PPL). RESULTS: The main results show that the three major pre-analytical parameters which impact on MP-related data are the delay before the first centrifugation, agitation of the tubes during transportation and the centrifugation protocol. CONCLUSIONS: Based on both this work and literature data, we propose a new protocol that needs to be validated on a larger scale before being applied for multicenter studies.

Standardization of platelet-derived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies?

BACKGROUND: Platelet microparticles (PMPs) have proved useful to identify patients with vascular risk. However, PMP counting, which is currently done by flow cytometry (FCM), needs to be standardized. OBJECTIVES: The objectives were (i) to standardize FCM settings for PMP counts on a routine instrument (Cytomics FC500) using size-calibrated fluorescent beads; (ii) to determine intra-instrument and inter-instrument reproducibility; and (iii) to establish PMP values in healthy subjects. METHODS: Using a blend of size-calibrated fluorescent beads (0.5 and 0.9 mum) in a fixed numerical ratio (Megamix), we gated PMPs in a restricted size window. To test intra-instrument and inter-instrument reproducibility, annexin V and CD41 coexpression were used to count PMPs in frozen aliquots of the same platelet-free plasma (PFP) over 4 months and in PFP from 10 healthy subjects on three independent flow cytometers. RESULTS: This calibrated-bead strategy allowed full long-term control of the FCM-based microparticle protocol and reproducible PMP counts over time [coefficient of variation (CV) < 10%]. Optimal settings were easily transferred from one instrument to another, using Megamix as a stable template. Similar PMP counts (CV < 12%) were obtained using the three instruments. With such a standardized FCM protocol, PMP values were established in healthy subjects (n = 60) with significantly higher levels in women than in men [median (1st quartile to 3rd quartile): 1775 microL(-1) (1014-3039 microL(-1)) vs. 656 microL(-1) (407-962 microL(-1))]. CONCLUSIONS: The present strategy provides a new option for PMP count standardization and thus opens the way for multicenter studies.

CD146-based immunomagnetic enrichment followed by multiparameter flow cytometry: a new approach to counting circulating endothelial cells.

BACKGROUND: Circulating endothelial cells (CECs) have emerged as non-invasive biomarkers of vascular dysfunction. The most widely used method for their detection is CD146-based immunomagnetic separation (IMS). Although this approach has provided consensus values in both normal and pathologic situations, it remains tedious and requires a trained operator. OBJECTIVES: Our objective was to evaluate a new hybrid assay for CEC measurement using a combination of pre-enrichment of CD146+ circulating cells and multiparametric flow cytometry measurement (FCM). PATIENTS AND METHODS: CECs were determined in peripheral blood from 20 healthy volunteers, 12 patients undergoing coronary angioplasty, and 30 renal transplant recipients, and blood spiked with cultured endothelial cells. CD146+ cells were isolated using CD146-coated magnetic nanoparticles and labeled using CD45-fluorescein isothiocyanate and CD146-PE or isotype control antibody and propidium iodide before FCM. The same samples were also processed using CD146-based immunomagnetic separation as the reference method. RESULTS: The hybrid assay detected CECs, identified as CD45(dim)/CD146(bright)/propidium iodide(+), with high size-related scatter characteristics, and clearly discriminated these from CD45(bright)/CD146(dim) activated T lymphocytes. The method demonstrated both high recovery efficiency and good reproducibility. Both IMS and the hybrid assay similarly identified increased CEC levels in patients undergoing coronary angioplasty and renal transplantation, when compared to healthy controls. In patients, CEC values from these two methods were of the same order of magnitude and highly correlated. Bland-Altman analysis revealed poor statistical agreement between methods, flerrofluid-FCM providing higher values than IMS. CONCLUSION: This new hybrid FCM assay constitutes an accurate alternative to visual counting of CECs following CD146-based IMS.

[First-intent monotherapy: persistence rate of antihypertensive treatment is better with angiotensin receptor blockers. Ol'métrise Cardio survey results]. / Monothérapie de première intention: le traitement antihypertenseur est plus souvent poursuivi, seul ou combiné, quand il s'agit d'un ARA II. Enquête Ol'métrise Cardio.

OBJECTIVES: To describe, according to the type of first-line antihypertensive monotherapy (FLAM) the persistence rate and the cumulative probability of its potential given up to the advantage of another antihypertensive class (rotation) in the following 3 years. METHODS: retrospective survey (to avoid any "survey effect" undertaken by 551 French cardiologists, in treated hypertensive patients. The survey described the FLAM, patients characteristics when FLAM was introduced, when given up in case of rotation and at the inclusion visit, as well as the dates of introduction, rotation and visit. STATISTICAL ANALYSIS: Kaplan Meier's method, Cox model. RESULTS: One thousand nine hundred and fifty five patients (62 +/- 11 years, males 57%, diabetes 13%, coronary disease 11%, LVH 20%) were analysed. At the end of 2.1 +/- 0.7 years, 1407 patients (72%) continued the FLAM alone (N=738, 52%) or combined (N=669, 48%). Survival analysis shows i) that the probability of rotation is roughly constant over time whatever the FLAM ii) that the probability of rotation against time is different according to the FLAM (p<0.0001, log rank) and lesser with ARB. These results are confirmed by the multivariate analysis (Cox model adjusted on age, sex and comorbidities).

[Is masked hypertension an artefact due to the blood pressure measurement method and threshold effects?]. / L'hypertension masquée est-elle un artéfact lié à méthode de mesure et à un effet seuil?

UNLABELLED: From results of office and home measurements of blood pressure (BP), patients can be classified as "hypertensive (HT)", "normotensive (NT)", "office hypertensive (OH)" or "masked hypertensive (MH)" by crossing the classifications obtained from each method. It seems that 9 to 20% of patients could be MH with a prognosis close to HT (SHEAF study). OBJECTIVES: To test the hypothesis that at least one part of the prevalence of MH would be an artefact due to the difference between the methods of measurements (shygmomanometer vs semi-automatic device) and/or due to different definitions of office hypertension (OHT). To determine the impact of different definitions of OHT on the prevalence of MH. METHODS: During the course of a phase IV study, BP was measured with the same semi-automatic device (OMRON 705CP) both at doctor's office (3 measurements at 1-minute intervals) and at home, by the patient himself (3 measurements in the morning and in the evening at 1-minute intervals over the 7 days before the visit). Following definitions were used: Office HT: SBP > or =140 mmHg, DBP > or =90 mmHg, SBP > or =140 mmHg or DBP > or =90 mmHg; Home HT: SBP > or =135 mmHg, DBP> or =85 mmHg, SBP > or =135 mmHg or DBP > or =85 mmHg. Another definition of office HT was used SBP > or =135 mmHg, DBP > or =85 mmHg SBP > or =135 mmHg or DBP > or =85 mmHg. RESULTS: 575 patients were analysed. Results from the two methods of measurements are closed but significantly different (difference for SBP: 3.2 +/- 16.5 mmHg; p < 0.0001; difference for DBP: 1.4 +/- 10.3 mmHg; p = 0.002)

[Use of home blood pressure devices in France in 2004]. / Utilisation des appareils d'automesure tensionnelle en France en 2004. Enquête FLAHS 2004.

OBJECTIVE: To estimate the number of devices for home blood pressure measurements in the French population in 2004 and to evaluate the use of these apparatus. METHODS: The French League Against Hypertension Survey 2004 (FLAHS-2004) was performed cross-sectionally on 3707 subjects, part of a sample of 5476 subjects selected as being representative of the French metropolitan population for age (35 years and above), gender, socioeconomic status, and place of living. Subjects who declare to take an antihypertensive medication were classified as treated hypertensive patients. A questionnaire evaluating the condition of use of HBP device was given. RESULTS: In 2004, 24% of the French population above the age of 35 years was treated for hypertension, corresponding to an estimate of 7.5 million of subjects. 25% of treated hypertensives and 12% of untreated subjects had a tensiometer. A total of 4 millions of BP devices are owned in the general population with 43% by treated hypertensive patients. 67% of BP devices are wrist cuff. Medical doctors recommended home blood pressure monitoring in only 12% of subjects. CONCLUSION: In 2004, the number of subjects treated for hypertension is 1/4 of the general population over the age of 35 years in France. In this group of subjects, 1/4 possessed a tensiometer but in only 10% medical doctor recommended the use of home blood pressure monitoring.

[Changes in life style and drug therapy for treatment of hypertension in France between 2002 and 2004]. / Evolution dans l'utilisation des traitements non médicamenteux et médicamenteux pour le traitement de l'hypertension artérielle en France. Enquête FLAHS 2004.

OBJECTIVES: To estimate changes in life style and drug therapy for treatment of hypertension in France between 2002 and 2004. METHODS: The French League Against Hypertension Survey 2004 (FLAHS-2004) was performed cross-sectionally on 3 707 subjects, part of a sample of 5476 subjects selected as being representative of the French metropolitan population for age (35 years and above), gender, socioeconomic status, and place of living. Subjects who declare to take an antihypertensive medication were classified as treated hypertensive patients. A questionnaire evaluating changes in life style during the last year (physical activities, quality of food consumption, smoking, and alcohol habits) was auto-administered. RESULTS: In 2004, 24% of the French population above the age of 35 years was treated for hypertension, corresponding to an estimate of 7.5 million of subjects. The two most frequent prescribed drugs are: fixed-dose combination drugs and beta-blockers in patients aged less than 75 years, and calcium antagonist and fixed-dose combination drugs in patients aged more than 75 years. Hypertensive subjects experienced: a weight loss of more than 3 kg in 11% vs 9%* in the general population, an increased in fruits and vegetables consumption in 41% vs 34%*, a decreased in cheese (21 vs 17%*), pork-butchery (46 vs 34%*) and alcohol consumption (23 vs 18%*) [*p < 0.01]. On the other hand, the percentage of subjects who increased their physical activities was low and not different in the two groups (7 vs 9%). CONCLUSION: Changes in life style were more often applied by the subjects treated for hypertension that by the general population. The dominating place that occupies today fixed-dose combination drugs indicates a change of the therapeutic practices.

[Efficacy and acceptability of lercanidipine are not age dependent in patients with essential hypertension: the AGATE study]. / Efficacité et la tolérance de la lercanidipine ne sont pas liées à l'âge des patients hypertendus: étude AGATE.

UNLABELLED: Calcium channel blockers (CCB) are known to be more efficacious and better tolerated in elderly patients. Lercanidipine is a highly lipophilic CCB with a specific safety profile linked to its pharmacokinetics. OBJECTIVES: To evaluate and compare the efficacy and safety of lercanidipine according to age. METHODS: Two groups of hypertensive patients (G1: aged < 65, G2: aged > or = 65) entered an open study conducted over 56 days. All received lercanidipine 10 mg/d (monotherapy or add-on), titrated to 20 mg/d if blood pressure (BP) was not controlled at D28. BP was measured using a semi-automatic device at doctor's office (three measurements at 1-min intervals) and at home by the patient himself (three measurements in the morning and in the evening at 1-min intervals over the 7 days before D0 and D56). RESULTS: Seven hundred and fifty-six patients entered the study. Thirty-eight patients dropped out prematurely and 30 were excluded because they were normotensive; 691 patients (G1 n = 375, G2 n = 316) were kept for analysis. At the end of the study, 507 patients were treated with lercanidipine alone (10 mg/d n = 221, 20 mg/d n = 286) and 184 with a combination including lercanidipine (10 mg/d n = 91, 20 mg/d n = 93). Efficacy was not different between the groups excepted home pulse pressure which decreased more in G2. In the office, SBP decreased by 17 and 21 mmHg, respectively, for G1 and G2, and DBP by 9 and 10 mmHg. The prevalence of leg edema was not different between G1 and G2 and was particularly low in both groups (3%). CONCLUSION: Lercanidipine was as efficacious and well tolerated in younger patients as in elderly patients.

[Asymptomatic carotid stenoses. A cardiologist's viewpoint]. / Sténoses carotidiennes asymptomatiques.

Cardiologist work is undergoing profound changes. He is in charge of the prevention and treatment of atherothrombotic disease. Preoperative evaluation of global cardiovascular risk is of paramount importance to choose between medical, surgical or interventional treatment. Furthermore, because the cardiologist has extensive experience with coronary angioplasty, he will have an essential role in carotid angioplasty.

[Microbeads and flow cytometry: how and why put the "-metry" in immuno-cytometry?]. / Microbilles et cytométrie: comment et pourquoi introduire la "-métrie" dans l'immuno-cytométrie?

In current practice of immunophenotyping, flow cytometers are mainly used as cell counters. These instruments which measure fluorescence intensity on each individual cell with high sensitivity can also count molecules on cells. "Putting the - metry into cytofluorometry" means measuring the expression level of molecules of biological interest on (in) target cells. Candidate molecules are numerous due to the many membrane receptors which biological function depends on the number of accessible molecules. Any modulation of the expression level brings valuable information to the biologist, beit associated to physiological differentiation, pathological state or therapeutic intervention. In order to take the best from such measurements in clinical biology, full reliability is mandatory in terms of time-to-time, platform-to-platform and lab- to-lab reproducibility. One should ban "arbitrary units" as currently provided by the instruments to switch to real units. This paper tends to review the most important features conditioning a reliable quantitation of cellular antigens, with data expressed in terms of number of molecules per cell. In addition to staining tools (antibody-based reagents) and data treatment softwares, calibration tools are mandatory. They are not all equivalent. Fluorescent beads with internal fluorescence are very useful for instrument quality-control but they can not interchange with immunological calibration systems which measure the number of antibody molecules that can bind onto the cells. Numerous application examples illustrate the interest of this "quantitative" dimension of Immunocytometry. Generalization of this approach would help getting a wider access to clinical biology.