[Definition of maximum permissible errors for major physical parameters affecting the result of microplate ABO-RH1 blood grouping]. / Définition des erreurs maximales tolérées pour les grandeurs physiques ayant un effet significatif sur le résultat du groupage ABO-RH1 en technique microplaque.

AIM: Metrological survey of analytic equipment is developing well in French blood bank laboratories, but still the limits of critical parameters affecting the result of basic methods are usually not defined. The aim of this study is to establish experimentally maximum permissible errors for critical physical values involved in microplate ABO-RH1 blood grouping, such as temperature and time of incubation and volumes of reagents and blood samples. MATERIALS AND METHODS: Samples were internal quality controls used routinely in the laboratory. Three set points were defined for each parameter: incubation temperature 18, 22 and 26 degrees C; incubation time 10, 15 and 20 min; reagent sera and sample cells volume 20, 30 and 40 microL; reagent cells volume 15, 20 et 25 microL; plasma sample volume 30, 40 et 50 microL. All equipments (temperature test chamber, chronometer and pipettors) were calibrated using national standards (COFRAC) according to appropriate methods for estimation of uncertainty of measurement. Significant components of uncertainty were taken into account to define a global uncertainty for each measurable quantity. RESULTS: All tests results from any combination of the above set points (n=81 for each sample tested in triplicate) were consistent with the expected result. Not one undetermined result was observed. CONCLUSION: The experimental design of this study allowed us to define accurately maximum permissible errors for major critical parameters involved in microplate ABO-RH1 blood grouping. Hence these values must be applied to metrological survey of the equipment. Further, our study demonstrates the robustness of the method.

[Validation of antibody screening by indirect antigloblin test and ABO blood typing by filtration and microplate techniques: assessment of robustness]. / Maîtrise des tolérances métrologiques : application à la recherche des anticorps anti-érythrocytaires (RAI) en test indirect à l'antiglobuline (TIA) et au groupage sanguin ABO par les procédés de filtration et en microplaque.

According to requirements of the French Committee for Accreditation (Cofrac), it is essential to use validated and standardised methods in Immunohematology. This imposes first the knowledge of metrological tolerances for all the technics. Two multicenter studies were carried out to define the maximal acceptable deviations concerning incubation temperature and time, volumes of patient plasma and tests cells for antibody screening using indirect antiglobulin test on one hand and for reverse grouping on another hand. All equipment used (temperature test chamber, chronometer, pipettes) were calibrated according to Cofrac standards. The antibody screenings were performed manually using 3 different filtration systems: ID Diamed, Biovue Ortho and Scangel Biorad, the same tests cells, a standard 20 ng/mL anti RH1, a positive control anti KEL1 and a negative control; the reverse blood grouping was performed manually using the above mentionned filtration systems and microplate technic with the same A1 and B test cells. These two studies showed that all the tests from the multiples combinations of the above parameters gave the same results and allowed us to define a range of tolerance for 4 critical physical parameters involved in the antibody screening and blood typing.

Identification of two novel insulin receptor mutations, Asp59Gly and Leu62Pro, in type A syndrome of extreme insulin resistance.

To elucidate genetic determinants of insulin resistance, we investigated insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) genes, in vitro IR function and in vivo insulin sensitivity in a family with Type A syndrome. Two missense IR mutations (Asp59Gly and Leu62Pro) found in the proband, resulted in reduction by 90% of insulin binding to erythrocytes, decreased receptor autophosphorylation and a dramatic reduction of insulin sensitivity. The proband and mother were heterozygote for Gly972Arg IRS-1 variant. Asp59Gly mutation, also carried by proband's brother with no consequence on insulin sensitivity, was inherited from the mother who is diabetic and insulin resistant and Leu62Pro was from the father. We conclude that severity of insulin resistance in the proband may be explained by the genetic condition of compound heterozygote for IR mutations while severe insulin resistance in the mother raises the possibility that other genetic factors, like IRS-1 polymorphisms, may contribute to the phenotypic expression of IR mutations.

Increased insulin sensitivity and basal insulin effectiveness in postprandial reactive hypoglycaemia.

Glucose clamp experiments have shown that patients with reactive postprandial hypoglycaemia (PRH) frequently have an increased glucose disposal, but the relative involvement of insulin sensitivity (SI) and glucose effectiveness (Sg) in this process remains unknown. The minimal model approach was used to compare 13 patients in whom moderate reactive hypoglycaemia ( < 3.3 mmol) had been previously diagnosed and 13 matched controls. The intravenous glucose tolerance test (IVGTT, 0.5 g/kg glucose IV) with 0.02 U/kg insulin given at the 19th min and frequent sampling over 180 min shows that PRH patients exhibit a higher glucose tolerance coefficient Kg (2.99 +/- 0.26 vs 2.19 +/- 0.12; P < 0.02), higher SI [22.9 +/- 6.4 vs 7.18 +/- 0.14 min-1/(microU/ml). 10(-4); P < 0.01] and higher Sg (3.84 +/- 0.35 vs 2.92 +/- 0.79 min-1. 10(-2); P < 0.05). The increase in Sg is explained by an increase in its component basal insulin effectiveness (BIE: 1.2 +/- 0.27 min-1.10(-2) in PRH subjects vs 0.58 +/- 0.07; P < 0.05) rather than an increase in Sg at zero insulin. The increase in BIE results from the high values of SI. In 4 PRH subjects SI and Sg were within the normal range, and the increase in Kg evidenced in the 9 others was explained by an increase in SI alone in 3 cases, in Sg alone in 1 case, and both SI and Sg in 5 cases. Thus, in sedentary subjects, the previously reported rise in tissue glucose assimilation is mainly explained by an increased insulin-mediated glucose disposal rather than non-insulin-mediated glucose disposal.

Endogenous oploid peptides stimulate post-exercise insulin response to glucose in rats.

Exercise is associated with profound changes in glucose metabolism and insulin secretion. Endogenous opioid peptides may be involved in these metabolic adaptations. To gain insights into this hypothesis, we studied the effects of the opioid antagonist naloxone on the insulin response to glucose after a 2.5 h exercise bout, either by means of an intravascular glucose tolerance test in male Wistar rats or from rat islets of Langerhans isolated just after exercise. There was a tenfold increase in plasma beta-endorphin concentrations (9.8 +/- 2.1 vs. 114.2 +/- 22.0 fmol/ml, p < 0.001) in animals killed immediately after exercise. The in vivo post-exercise peak insulin response to glucose was markedly reduced compared to resting controls (p < 0.01). Interestingly, naloxone (10 mg/kg) still further decreased the insulin response compared to saline injected exercised rats (p < 0.05), but did not alter the response from resting animals. The post-exercise insulin response to 8.3 mM glucose was significantly reduced compared to resting rat islets (p < 0.05) and was further inhibited when naloxone (10 mu M) was added to the culture medium (p < 0.05). In another experiment, we also tested the effect of 10(-8) and 10(-6) M beta-endorphin on control islets. Both concentrations of beta-endorphin significantly increased the islet insulin response to 8.3 mM glucose (p < 0.05) and this effect was completely blocked by naloxone. These results suggest that endogenous opioid peptides participate in the physiological adaptation to exercise stress in maintaining post-exercise insulin response to glucose.

Influence of short-term submaximal exercise on parameters of glucose assimilation analyzed with the minimal model.

After exercise, glucose uptake in tissues increases by insulin-dependent and -independent mechanisms. We evaluated whether these two effects of exercise on glucose disposal can be detected with the minimal model technique. Seven healthy volunteers were submitted at random order to two frequently sampled intravenous glucose tolerance test (FSIVGTTs), one at rest and the other 25 minutes after a 15-minute exercise test. This exercise included 5 minutes of increasing workload on a cycloergometer followed by 10 minutes at 85% of the maximal theoretic heart rate. Bergman's minimal model of insulin action was used to analyze the two FSIVGTTs and produced the following parameters: coefficient of glucose tolerance (Kg), ie, the slope of the exponential decrease in glycemia between 4 and 19 minutes after intravenous glucose; insulin sensitivity (Sl); and glucose effectiveness at basal insulin (Sg). Sg was divided into its two components: basal insulin effectiveness ([BIE] Sl x basal insulin) and glucose effectiveness at zero insulin ([GEZI] Sg-BIE). After the exercise bout, subjects had an increased Kg (3.44 +/- 0.44 v 2.06 +/- 0.28 x 10(-2).min-1, P < .02), Sl (11.43 +/- 1.27 v 6.23 +/- 0.97 x 10(-4) microU/mL.min-1, P < .01), and Sg (4.40 +/- 0.55 v 2.81 +/- 0.36 x 10(-2).min-1, P < .02). The increase in Sg was mainly explained by a 60% increase in GEZI (3.6 +/- 0.57 v 2.25 +/- 0.36 x 10(-2).min-1, P < .02), but also by an increase in BIE (0.80 +/- 0.12 v 0.47 +/- 0.08 x 10(-2).min-1, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Protective effect of gamma-hydroxybutyrate and nicotinamide on low-dose streptozotocin-induced diabetes in mice.

The purpose of this study was to investigate the effect of a treatment by gamma-hydroxybutyrate (GHB) and nicotinamide (NA) on low-dose streptozotocin (STZ)-induced diabetes in mice. Mean plasma glucose level was significantly elevated in mice given STZ by day 12 after the first STZ injection compared to controls (15.0 +/- 4.7 vs 8.0 +/- 1.6 mmol/l, p < 0.001) and 100% of the animals were severely diabetic by day 18. Plasma glucose levels remained in the normal range and no diabetic values were found in mice treated with combined treatment by GHB and NA for 25 days. However, hyperglycemia and glycosuria appeared within one week after discontinuation of the treatment. Treatment by either GHB or NA alone had only a slight and transient effect in preventing hyperglycemia. In vitro experiment on isolated pancreatic islets demonstrated that STZ-induced loss of insulin response to glucose was also counteracted by incubation with GHB and NA (Peak insulin response to 16.4 mM glucose: 0.69 +/- 0.31 vs 3.03 +/- 0.67 microU/islet/min), but not by GHB or NA alone. These results indicate that GHB and NA have complementary effects in preventing STZ-induced beta cell damage both in vivo and in vitro. This should be taken into account for future preventive strategies in human insulin-dependent diabetes mellitus.

Evaluation of a standardized hyperglucidic breakfast test in postprandial reactive hypoglycaemia.

The oral glucose tolerance test is not specific for diagnosing postprandial reactive hypoglycaemia, since it too frequently induces low blood glucose values in subjects who have never complained of symptoms of this. By contrast, the mixed meal tests are deceptive for this purpose because they do not induce hypoglycaemia in subjects who have complained of of hypoglycaemic symptoms. We investigated the frequency of hypoglycaemia after a standardized hyperglucidic breakfast test in three groups of subjects:group A, 43 control subjects; group B, 38 postprandial reactive hypoglycaemic patients; group C, 1193 asymptomatic subjects undergoing assessment of glycoregulation. In the 38 subjects with suspected reactive hypoglycaemia the mean blood glucose nadir was 3.48 +/- 0.08 mmol/l, i.e. lower than in control subjects (4.83 +/- 0.13 p < 0.0001). Blood glucose levels less than 3.3 mmol/l were found in 47.3% of subjects with suspected postprandial reactive hypoglycaemia (group B), i.e more frequently than in control subjects (group A: 2.2% p = 1.6 x 10(-6)) and asymptomatic subjects (group C: 1% p = 8 x 10(-22)). This markedly higher frequency of low blood glucose values in subjects with postprandial symptoms compared with control and asymptomatic subjects suggests that this test detects a tendency to hypoglycaemia after a standardized hyperglucidic breakfast. Since this test mimics average French eating habits, the results suggest that the patients undergo such symptoms in their everyday life, and that the hyperglucidic breakfast test is a simple alternative to ambulatory glucose sampling for diagnosis of postprandial reactive hypoglycaemia.

Immunoanalysis of human insulin using monoclonal antibodies reveals antigenicity of evolutionarily conserved residues.

Seven anti-human insulin monoclonal antibodies (mAb) were produced according to an efficient immunization protocol elaborated in our laboratory. Their affinity constants for the binding to the insulin molecule ranged from 5.0 x 10(8) M-1 to 1.0 x 10(10) M-1 when insulin was in solution and from 6.0 x 10(6) M-1 to 2.5 x 10(8) M-1 when insulin was adsorbed onto the microtiter plate. The antigenic sites on the insulin molecule recognized by these mAbs were mapped using two approaches. MAb pairs capable of binding simultaneously to human insulin in solution (using a two-site ELISA) or adsorbed onto a microtiter plate (using a competitive ELISA) were first sought. Three antigenic regions were defined on the surface of adsorbed human insulin and four on soluble insulin. Two distinct antigenic regions common to both the adsorbed and the soluble forms of insulin were defined by our mAbs. In a second approach, the immunological cross-reactivities of these mAbs with species variants of insulin, chemically modified insulin of known structure and a panel of 78 overlapping nonapeptides covering the entire sequence of human proinsulin were assessed. Evidence was obtained that the epitopes recognized by the mAbs included residues conserved during evolution in the insulin molecule. The epitopic specificity of one group of mAbs (group I) was precisely defined. This group recognized a highly conserved region of the insulin molecule including residues 10-17 of the A chain.

Effects of naloxone opiate blockade on the immunomodulation induced by exercise in rats.

The purpose of this study was to examine the possible involvement of the endogenous opiate system in the changes in immune competence induced by isolated exercise. Male untrained rats were subjected to a 2.5 hours swimming exercise bout. Animals were killed 15 min after the end of the exercise. The concentration of leukocytes, lymphocytes, monocytes and granulocytes and T4 (T-helper), T8 (T-suppressor/cytotoxic), interleukin-2 receptor (IL-2R) and transferrin receptor (TrfR) positive lymphocytes were determined both in peripheral blood and spleen by flow cytometric analysis. Exercise resulted in a significant decrease in 1) blood lymphocyte and splenic granulocyte number (p < 0.05), 2) blood and splenic T4 positive lymphocytes and T4/T8 ratio (p < 0.05), and 3) blood and splenic IL-2R and TrfR positive lymphocytes (p < 0.05). The injection of the opiate blocker naloxone to exercising rats induced a decrease in the concentration and proportion of T8 positive lymphocytes, thereby restoring a normal T4/T8 ratio both in peripheral blood and spleen. Naloxone had no effect in control animals. The concentration and proportion of IL-2R and TrfR positive lymphocytes were not affected by naloxone. The mechanisms of the immunomodulation induced by isolated intense exercise are unclear. These data suggest that endogenous opiates participate in the alteration of cell-mediated immunity associated with exercise by modulating the T8 (suppressor/cytotoxic)-cell activity.

Plasma beta-endorphin, corticotrophin and growth hormone responses to exercise in pubertal and prepubertal children.

An increase in plasma beta-endorphin concentrations during exercise has been reported in adult men and women by several investigators. However, very little is known about this physiological hormonal response to exercise in children. In this study, we investigated plasma beta-endorphin, ACTH and GH responses to exercise in 40 prepubertal and pubertal children. Subjects were recruited as part of a population of children and adolescents presenting growth retardation and were selected on the basis of the absence of any clinical or biological signs of endocrine or metabolic disease. There were 16 girls and 24 boys with 24 prepubertal and 16 pubertal individuals. A standardised 15 min workload on cycloergometer was used to progressively increase the heart rate of the children up to 90% of the theoretical maximal value. Exercise resulted in a significant increase (p < 0.01) in plasma beta-endorphin (mean +/- SEM) (4.26 +/- 0.47 vs 5.74 +/- 0.56 fmol/ml), ACTH (3.71 +/- 0.41 vs 6.2 +/- 0.62 fmol/ml) and GH (147 +/- 29 vs 364 +/- 67 fmol/ml). The percentage of children with significant hormonal response to exercise was about 75% for each of the 3 hormones but only 3 of the 40 children studied did not show any hormonal response to exercise. Exercise-induced increases in plasma beta-endorphin and ACTH were significantly correlated (p < 0.01). By contrast, there was no significant relationship between GH and beta-endorphin or ACTH values. Furthermore, whereas exercise-induced plasma GH increase was significantly higher in pubertal than in prepubertal children (p < 0.001), corresponding beta-endorphin and ACTH levels were quite similar in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The magnitude, the kinetics and the metabolic efficiency of first-phase insulin response to intravenous glucose are related.

We investigated the relationship between the kinetics, the magnitude and the metabolic efficiency of first-phase insulin response (FPIR) to intravenous glucose. Twenty healthy control subjects and fifty first degree relatives of Type 1 diabetic patients were studied using a standardized protocol for the intravenous glucose tolerance test (IVGTT). The first significant increase in plasma insulin concentrations above baseline appeared as early as the 2nd min (1 min before the end of glucose injection, fast response) in 80% of controls and 70% of relatives, and at the 3rd min or later (delayed response) in the remaining subjects. The greatest delay in insulin release (5th min) was observed in 4 of 6 relatives of Type 1 diabetic patients with impaired FPIR. In the controls and the relatives, the subjects with a fast insulin response had a significantly higher FPIR (controls 215.4 +/- 93.5 vs 59.7 +/- 5.6 microU/ml, p < 0.001 and relatives 143.5 +/- 61.8 vs 55.9 +/- 27.7 microU/ml, p < 0.001) and showed better glucose assimilation (controls 3.05 +/- 1.05 vs 1.64 +/- 0.16%/min, p < 0.05 and relatives 2.6 +/- 0.96 vs 1.6 +/- 0.85%/min, p < 0.01) during IVGTT than the subjects with a delayed response. Moreover, for normal FPIR values in the group of relatives of Type 1 diabetic patients, a fast response was associated with a significantly better glucose assimilation as assessed by the incremental area under the glucose curve (358.6 +/- 64.7 vs 539.2 +/- 67.7 mmol/l per 90 min, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)