Pilot study for the standardization of insulin immunoassays with isotope dilution liquid chromatography/tandem mass spectrometry

BACKGROUND: An international working group convened by the American Diabetes Association (ADA) called for a reference measurement procedure for use in a trueness-based standardization project of insulin immunoassays. In view of this demand, we conducted a pilot study to investigate the feasibility of such a standardization project with our isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/tandem MS) procedure. METHODS: We evaluated the precision, accuracy, and limit of quantification (LoQ) of the ID-LC/tandem MS procedure by use of insulin-free serum supplemented with insulin to give 3 pools with concentrations of 0.0796, 0.769, and 5.56 microg/L. We conducted a pilot method comparison study with 4 immunoassays and 80 samples from fasting and glucose-stimulated patients. RESULTS: The within-run and total imprecision (CV) ranged from 3.2% to 6.3% and from 4.9% to 12.1% (listing sequence from the high to the low pool). The recovery from supplemented insulin-free sera ranged from 101.8% to 104.1%, and the LoQ was 0.07 microg/L (12 pmol/L). Weighted Deming regression and correlation analysis of the method-comparison data showed considerable between-assay variation for the immunoassays but, with the exception of one assay, excellent correlation with ID-LC/tandem MS. Recalibration of the immunoassay results considerably reduced the between-assay variation. Moreover, after recalibration, 3 of the 4 assays fulfilled the total error specification of 32% proposed by the ADA Workgroup. CONCLUSIONS: Recalibration of insulin assays by regression equations established from method comparison with ID-LC/tandem MS can result in successful standardization and fulfillment of the total error criterion proposed by the ADA Workgroup.     

MRI-targeted biopsy for detecting prostate cancer: have the guidelines changed our practices and our prostate cancer detection rate?

International Urology and Nephrology - In our center, until 2018, MRI-targeted biopsy was underused. Since January 2018, we systematically performed MRI-targeted biopsy for suspicious...     

In vivo assessment of skin electroporation using square wave pulses.

The application of short-duration high-voltage pulses to the skin has been shown to enhance transdermal drug delivery by several orders of magnitude and to transiently permeabilize cells in tissue. Both exponentially decaying (ED) pulses and square wave (SW) pulses have been applied. The latter have also been used for electrochemotherapy. To date, their effect on skin integrity has not been analyzed. The scope of this work was (i) to investigate the effect induced by SW pulses on the stratum corneum and the skin, (ii) to evaluate the safety issue associated with electroporation, (iii) to contribute to the understanding of drug transport. Biophysical techniques (transepidermal water loss, chromametry, impedance and laser Doppler velocimetry or imaging measurement) and histological methods were combined to provide a global picture of the effects. Ten SW pulses applied to the skin induced a mild impairment of the skin barrier function and a dramatic decrease in skin resistance. These changes were reversible. A transient decrease (<5 min) in blood flow was observed. Neither inflammation, nor necroses were observed. These studies confirm the tolerance of the skin to square wave pulses in vivo.     

A probabilistic method to estimate the burden of maternal morbidity in resource-poor settings: preliminary development and evaluation

Background

Maternal morbidity is more common than maternal death, and population-based estimates of the burden of maternal morbidity could provide important indicators for monitoring trends, priority setting and evaluating the health impact of interventions. Methods based on lay reporting of obstetric events have been shown to lack specificity and there is a need for new approaches to measure the population burden of maternal morbidity. A computer-based probabilistic tool was developed to estimate the likelihood of maternal morbidity and its causes based on self-reported symptoms and pregnancy/delivery experiences. Development involved the use of training datasets of signs, symptoms and causes of morbidity from 1734 facility-based deliveries in Benin and Burkina Faso, as well as expert review. Preliminary evaluation of the method compared the burden of maternal morbidity and specific causes from the probabilistic tool with clinical classifications of 489 recently-delivered women from Benin, Bangladesh and India.

Results

Using training datasets, it was possible to create a probabilistic tool that handled uncertainty of women’s self reports of pregnancy and delivery experiences in a unique way to estimate population-level burdens of maternal morbidity and specific causes that compared well with clinical classifications of the same data. When applied to test datasets, the method overestimated the burden of morbidity compared with clinical review, although possible conceptual and methodological reasons for this were identified.

Conclusion

The probabilistic method shows promise and may offer opportunities for standardised measurement of maternal morbidity that allows for the uncertainty of women’s self-reported symptoms in retrospective interviews. However, important discrepancies with clinical classifications were observed and the method requires further development, refinement and evaluation in a range of settings.

     

Efficient migration of dendritic cells toward lymph node chemokines and induction of T(H)1 responses require maturation stimulus and apoptotic cell interaction

Dendritic cells (DCs) have the unique ability to initiate primary immune responses, and they can be conditioned for vaccinal purposes to present antigens after the engulfment of apoptotic cells. To recruit the rare antigen-specific naive T cells, DCs require a maturation step and subsequent transport toward lymph node (LN). To date, prostaglandin E2 (PGE2) is the best-characterized compound inducing this LN-directed migration in vitro, but PGE2 may skew the immune responses in a T(H)2 direction. We demonstrate here that on incubation with apoptotic tumor cells and tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS), human monocyte-derived DCs become fully mature and acquire high migratory capacities toward LN-directing chemokines. The migration of TNF-alpha-treated DCs occurs only after cotreatment with apoptotic cells but not with necrotic cells. DC migration requires CD36 expression and incubation with apoptotic cells in the presence of heat-labile serum components. Moreover, on treatment with apoptotic cells and LPS, the migrating DCs are able to recruit naive T cells to generate T(H)1 immune responses. Our results show that the cotreatment of DCs with apoptotic tumor cells and inflammatory signals is promising for the design of an antitumoral DC-based vaccine.     

Graft-Versus-Host Disease and Graft-Versus-Leukemia Effect in Mice Grafted With Peripheral Newborn Blood

We previously used peripheral newborn blood (NBB) as a possible invivo experimental model for cord blood (CB) transplantation and showedthat B10.D2 NBB cells successfully reconstituted adult (DBA/2 × B10.D2)F1 mice without causing graft-versus-host disease (GVHD),probably because of their phenotypic and functional immaturity. Here weinvestigated the influence of T-cell maturation occurring in NBB cellsduring the early postbirth period on the degree of engraftment, theincidence of GVHD, and the graft-versus-leukemia (GVL) potential. Theseparameters were compared in recipients grafted with bone marrow (BM)cells. We observed an increased percentage of CD4⁺ matureT cells accompanied by the acquisition of proliferative responses tophytohemagglutinin (PHA) and to allogeneic cells of day-5 NBB cells.The capacity of day-2 NBB to engraft was moderately reduced andrecipients developing GVHD were occasionally observed after the graftof day-5 NBB cells. No GVL effect was evidenced regardless of the timeof postbirth blood collection. However, the GVL effect can be obtainedby the delayed infusion of donor mature T cells to recipients graftedwith day-0 NBB, without causing GVHD. In contrast, the same protocolapplied to mice grafted with BM cells induced GVHD mortality of allrecipients. Interleukin (IL)-10 but not IL-2 messenger RNA wasexpressed in NBB cells as opposed to BM cells. These findings suggestthat, in terms of GVHD incidence, delayed infusion of mature T cells aspost-transplant tumor immunotherapy would be more effective whenapplied after CB than after BM transplantation.     

Impact of TCR status and genotype on outcome in adult T-cell acute lymphoblastic leukemia: a LALA-94 study

Patients with T-cell acute lymphoblastic leukemias (T-ALLs) within the Leucemies Aigues Lymphoblastiques de l'Adulte-94 (LALA-94) prospective trial were treated with a 4-drug per 4-week induction, with intermediate-dose cytarabine and mitoxantrone salvage treatment for patients not achieving complete remission (CR) in 1 course. Only the latter received allografts, if possible, thus providing an informative setting for assessing early response. Representative patients with T-ALL (91 patients) were classified into surface T-cell receptor (TCR)-expressing T-ALL patients (TCRalphabeta+ or TCRgammadelta+), pre-alphabeta T-ALL patients (cTCRbeta+, TCR-), and immature (IM) cTCRbeta-, TCR- T-ALL patients; 81 patients underwent genotyping for SIL-TAL1, CALM-AF10, HOX11, and HOX11L2. Overall, CR was obtained in 81 (89%) patients; relapse rate was 62% at 4 years and overall survival (OS) rate was 38%. CR rate was significantly lower in IM T-ALL patients after 1 course (45% vs 87%; P < .001) and after salvage (74% vs 97%; P = .002), with the latter inducing a higher rate of CR (9 [64%] of 14) than initial induction. Once CR was obtained, cumulative relapse rates were similar for IM, pre-alphabeta, and TCR+ T-ALL patients (P = .51), but were higher in HOX11L2 (83%) and SIL-TAL1 (82%) T-ALL patients compared with other genetic subgroups (48%; P = .05). This was associated with an inferior OS for HOX11L2 T-ALLs (13% vs 47% in HOX11L2-T-ALLs; P = .009). The majority of patients with HOX11 T-ALL underwent allografting, predominantly in second CR, but were not associated with a superior OS. Both TCR and genotypic stratification can therefore contribute to risk-adapted management of adult T-ALLs.