[Organization for the coverage of the transfusional needs of patients with hemoglobinopathy at the Établissement français du sang Bretagne]. / Organisation de la prise en charge des besoins transfusionnels des patients atteints d'hémoglobinopathie à l'Établissement français du sang Bretagne.

UNLABELLED: Brittany is a low prevalence region for hemoglobinopathies. Despite of that, the number of patients is increasing each year. In 2013, 140 patients were known at the EFS Bretagne, and medical consultations are growing for 50% each year since 2011. The consequence is an increase of needs of 22% of compatible packed red blood cells. To anticipate the announced progress, various actions were implemented as study groups, creation of a new informatic prescription for red blood cells phenotyping, promotion of donation, transfusion organisation. RESULTS: Fifthty-nine percent of the 400 ABO RH-KELL, FY, JK, MNS 3, 4, red blood cells were realised on the basis of this new informatic prescription, as the 99% of the packed red blood cells identified Fy (a- b-). So, 92% of the compatible transfused packed red blood cells were already in stock when the patients needed them. CONCLUSIONS: In Brittany, that organisation leads to assume qualitative and quantitative transfusion for sickle cell disease in more than 90% of cases, with fast distribution. In the same time promotion of donation is done to increase the diversity of donors.

Clinical and metabolic characteristics of patients with latent autoimmune diabetes in adults (LADA): absence of rapid beta-cell loss in patients with tight metabolic control.

AIM AND METHODS: The present study compared the clinical and metabolic characteristics of latent autoimmune diabetes in adults (LADA) with type 2 diabetes, as well as the residual beta-cell function and progression to insulin treatment, over a 2-year follow-up period, of antibody (Ab)-positive and Ab-negative patients who achieved tight glycaemic control (HbA(1c) 7.0+/-0.8% and 6.5+/-0.9%, respectively, at the time of entry into the study). RESULTS: Glutamic acid decarboxylase antibodies (GADA) and/or islet cell antibodies (ICA) were detected in 10% of patients presenting with non-insulin-dependent diabetes. Around half of Ab-positive patients required insulin treatment during the follow-up. Ab-positive patients displayed lower stimulated C-peptide levels both at entry and during the follow-up compared with Ab-negative patients, although no significant decline in C-peptide levels was observed in either subgroup over two years. Nevertheless, Ab-positive patients progressed more frequently to insulin treatment, and stimulated C-peptide tended to decrease in LADA patients who subsequently required insulin, whereas it remained stable in those who were non-insulin-dependent. In those who progressed, the trend towards C-peptide decline persisted even after starting insulin treatment. CONCLUSION: LADA patients demonstrate lower residual beta-cell function than do type 2 diabetes patients. However, those who achieve tight metabolic control do not present with a rapid decline in beta-cell function. Further studies are needed to determine the optimal treatment strategy in such patients.

[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.

[Automation of the immunohematology laboratory]. / Automatisation au laboratoire d'immunohématologie érythrocytaire.

Automation finds a particularly justified application in the field of immunohematology in which any human failure can have dramatic consequences for the patients. The purposes of automation, indissociable from computerization, were reminded by the French regulatory: to decrease the risks of human error linked to each step of the tests performed; to guarantee a reliable traceability of all the elements having contributed to the test process; to manage all the alarms of dysfunction of the system. The main devices available today permit automation of a part of the pre-analytical phase and of all the steps of the analytical phase by the realization in routine of ABO-RH1 grouping, phenotyping, of irregular antibody screening and of compatibility testing. They use the reaction of agglutination either in micro-plates or in filtration. One distinguishes two types of materials: full automates managing all the steps from the sample positioning on the carrier down to the final result without any handling of the samples (Autovue, Galileo, ID gel station, Qwalys, Tango, Techno) and the semi-automates, which require intervention of an operator for the phases of centrifugation, stirring and incubation (Mitis 2, Hemos, Rosys, Swing) the reading being automated for all. All include the possibility of a connection to the central data processing system . If some devices allow the choice of reagents, others can only work with the reagents supplied by the manufacturer. In all the cases, optimization of the automated systems implies a specific training of the staff and the strict compliance with the standard operating procedures by each individual.

[Suitability of intraoperative autotransfusion]. / Suivi des techniques de récupération de sang en peropératoire.

This work presents the procedure applied by our hospital to assess the quality and security of intra operative autotransfusion. The suitability of the three following variables has to be constantly assessed: performance of the machines to concentrate and wash collected blood, bacterial contamination of processed blood and rate of adverse events. We note that the procedure is applied with participation of medical and nursing staff. Since its setting-up, we note an amelioration of suitable variables.

Section 1B: Rh flow cytometry. Coordinator's report. Rhesus index and antigen density: an analysis of the reproducibility of flow cytometric determination.

Fifty-seven IgG monoclonal anti-D antibodies were evaluated in the Rh flow cytometry section, in which 12 laboratories participated. Staining protocols and a fluorescein (FITC)-conjugated Fab fragment goat anti-human IgG (H + L) as a secondary antibody were recommended but not mandatory. A CcDEe red blood cell (RBC) sample that was shown to be homozygous for RHD by molecular methods was supplied and used as internal 'standard RBC' throughout all experiments. An RBC panel comprising two partial D and four weak D types was supplied as well. The use of standard RBC reduced the variability of the data among the laboratories and allowed the conversion of fluorescence data into epitope densities, which were compounded in an antigen density (antigen D per RBC). The highest antigen density was determined for DVI type III, followed by DVII and weak D type 3; the lowest antigen density were determined for weak D type 1 and type 2. Nine of the 12 participating laboratories discriminated three groups of aberrant RhD that had similar Rhesus indices (RI): D category VI with RI = 0; weak D type 2 and type 3 with an high RI; and D category VII and weak D type 1 with an intermediate RI. The antigen densities and the Rhesus indices obtained correlated well among the laboratories of this Workshop section despite different staining protocols, secondary antibodies and instrumentation.

[Hyperactivity of the muscles of the lip and chin and indications for surgery to reduce it]. / Dysfonctionnement des muscles labio-mentonniers et indications de la chirurgie d'affaiblissement.

Hyperactivity of the muscles controlling the lower lip and chin (muscles of the chin, buccinator, orbicularis, etc.) can be demonstrated at different levels of the muscular complex and may act as an elastic force against the mandibular alveolar process. Dysfunction in this region may lead to reduction of the alveolar bone quantity, reducing the tooth-bone equilibrium. If this balance is disturbed, periodontal lesions, a lower incisal crowding and a retrognathic mandibular process may result. Surgery to correct overactivity of the chin muscles is described here step by step. Resection of the muscles may be undertaken on a greater or lesser scale and striation of the muscle may also allow reduction of muscular strength. Reducing the muscular activity creates a better environment for the development of the mandible and its alveolar process. Depending on the clinical situation, these techniques can be associated with genioplasty, bone graft and/or mandibular orthognathic surgery.

[Analytical validation in erythrocyte immunohematology]. / Validation analytique en immunohématologie érythrocytaire.

In a transfusional or foeto-maternal context, hemolysis by incompatibility due to anti-erythrocyte antibodies (regular or irregular) remains the most frequent and most serious immunological risk in the receiver. In order to prevent this risk, a number of actions must be taken, such as the realization of the immunohematologic analyses for which the methodological practices have been legislated because of their serious clinical consequences. Several elements play a role in the reliability of the analyses and their results: the selection of the reagents and their validation in the routine technique used; the validation of reception; the controls involved in secondary preparations (e.g., blood cells reagent); and the daily internal controls. All this requires the choice of adapted controls and the management of possible anomalies.

Combined analysis of long-term anti-beta-cell humoral reactivity in type 1 diabetes with and without thyroid disease.

The prevalence and levels of islet-cell antibodies (ICA) decrease in the years following diabetes onset but may persist, particularly in patients with concomitant autoimmune disease. The aim of this cross-sectional study was to investigate the frequencies, associations and levels of the major anti-beta-cell antibodies in long-standing diabetic patients (median duration: 14 years; range 5-47 years) with and without autoimmune thyroid disease (ATD) in order to consider the specific antipancreatic immunologic features associated with endocrine autoimmunity. Both ICA and glutamic acid decarboxylase (GAD) antibody (GAD-A) frequencies were increased in diabetic patients with ATD (38 vs 23%, p = 0.03 and 70 vs 21%, p < 10(-4) respectively). Although IA2-A frequency tended to be higher in diabetic patients with ATD, no significant difference was seen (37 vs 26%, p = 0.14). GAD median level was significantly higher in the diabetic group with ATD (15 vs 5 units, p < 10(-4)). IA2-A and ICA median levels were similar in both groups. Regardless of the combined analysis performed (ICA/GAD-A, ICA/IA2-A or GAD-A/IA2-A), the prevalence of combined antibody positivity was higher in diabetic patients with than without ATD. In both diabetic populations, ICA and GA-DA were significantly associated (p < 10(-4), and their levels were correlated (r = 0.42, p < 10(-4) and r = 0.584, p < 10(-4) respectively). No significant correlation was seen between IA2-A levels and either ICA or GAD-A titres. It is concluded that Type 1 diabetes mellitus with ATD is characterised by increased persistent humoral islet-related reactivity, particularly directed towards GAD.

Anti-pancreatic autoimmunity and Graves' disease: study of a cohort of 600 Caucasian patients.

The aim of this study was to investigate the frequencies of clinical diabetes and humoral markers of anti-pancreatic autoimmunity in a homogeneous population of 600 Caucasian patients with recently diagnosed Graves' disease (GD), in order to characterize the specific features of this group of endocrine patients among subjects at risk of diabetes. Ten were already diabetic at GD diagnosis. Among the 590 non-diabetic patients, 29 had islet cell antibodies (ICA), including 15 with low titre ICA and only 1 ICA-positive subject with a familial history of diabetes. Twenty-four patients had insulin autoantibodies, including three in association with ICA. Glutamic acid decarboxylase (GAD)/64 kDa antibodies were found in 16 of the 150 tested sera, including 13 of the 29 ICA-positive sera. Four ICA-positive patients displayed 37/40 kDa antibodies, including three in association with GAD/64 kDa antibodies. During follow-up, one of the ICA-positive patients developed insulin-dependent diabetes, 14 years after the GD diagnosis. To summarize, this anti-pancreatic autoimmunity study was focused on a large but specific and homogeneous group of subjects at risk for diabetes: recently diagnosed GD patients. This population was characterized by a high prevalence of GAD/64 kDa antibodies but also by a low frequency of evolution towards diabetes and the slowness of the process which could be due to the fact that only a minority of subjects possessed a sufficient combination of anti-pancreatic markers at the same time.

Multiple antibody status in type 1 diabetic patients and subjects at various risk with islet-cell antibodies.

The frequency of 37/40 kD antibodies and their association with other pancreatic humoral markers were studied in 109 recently diagnosed Type 1 diabetic patients and 116 subjects with islet-cell antibodies (ICA) at various risk for this disease (64 relatives of Type 1 diabetic patients, 23 schoolchildren with no family history of diabetes, and 29 patients with Graves' disease). At the time of diagnosis, 37/40 kD antibodies were detected in 45% of Type 1a and 77% of Type 1b diabetic patients (p = 0.03). Antibodies to glutamic acid decarboxylase (GAD) and/or 37/40 kD were present with the same frequency as ICA (86%). The frequency of 37/40 kD antibodies was not significantly different between the 3 groups at risk, in contrast with GAD antibodies which were found at a lower frequency in schoolchildren (p < 0.02). Frequencies of other pancreatic markers (ICA cross-reactive with mouse pancreas and insulin autoantibodies) and the combination of ICA with at least two other markers were significantly higher in relatives than in the other groups at risk (p < 0.02). Out of 116 ICA-positive non-diabetic subjects, 10 developed diabetes. All 10 displayed 37/40kD and/or GAD antibodies during the prediabetic phase. In 8 of these 10 patients, ICA was combined with at least two other markers, whereas this association was detected in only 17 of the remaining 106 subjects who did not progress to diabetes (p < 10(-4). Thus, 37/40 kD antibodies were found in about half of Type 1 diabetic patients, and with a higher-frequency in Type 1b than 1a. In ICA-positive non-diabetic subjects, our date confirm that a combination of multiple antibodies, including GAD antibodies and 37/40 kD antibodies, can enhance the predictive value for diabetes. Comparison of ICA-positive relatives of diabetic patients, schoolchildren and patients with Graves' disease revealed distinct frequencies and combinations of markers of diabetes. This might reflect different patterns of progression among these 3 groups.

Multicenter study of an erythro-aggregometer: quality control and standardization.

There is a need for quality assurance procedures in hemorheology, especially for clinical and pharmacological studies, which require reliable and well-calibrated instruments to be interpretable and comparable. Preliminary investigations allowed preparation of standardized SM (normal NS and hyperaggregable HS), and checking of storage conditions (in accordance with the guidelines of the SSC of ISTH) of RBC in nutritive SAG mannitol for at least 2 or 3 weeks with subsequent washings and resuspension in SM. In our study, we compared erythro-aggregometers of the same brand in 6 laboratories, using the same red blood cells (RBC) and suspending media (SM) for each series of tests. EA was measured by laser backscattering with determination of aggregation time (AT), partial dissociation threshold (PDT) and aggregation index (AI). Prior to the study, devices were set up on the same day with the same standardized blood, and calibration data were then analyzed. Within-assay precision (WAP) was assessed on 3 days for the 2 types of SM (n = 18 x 2). Between-assay precision (BAP) was assessed on 6 occasions, once every month (n = 6 x 2 x 6). Accuracy was studied with 3 series of RBC resuspended in 10 SM of "unknown" aggregability. Good agreement was observed between 5/6 centers for the 3 parameters of EA. WEP was good: CV of AT ranging from 1.4 to 2.5% for the NS and from 1.4 to 2.4% for the HS. In each center, BAP was slightly lower than WEP (CV: 8-11.8% for the NS and 3.8-4.7% for the HS over the 6-month study), with no drift, except for one center. Precision was always better with the HS than with the NS which seemed a better tool to assess it. As to accuracy, non-significant differences were generally found between centers, with similar data to the reference values. This work also stressed the importance of the RBC parameter itself in rheological data. For the first time, a protocol for standardization of EA has been developed and evaluated, permitting the Quality Control of this technique.

Impaired leucocyte functions in diabetic patients.

This study evaluates polymorphonuclear neutrophil (PMN) cell performance in 61 diabetic patients free of infection (40 Type 1, 21 Type 2), using tests that explore all the functional steps of PMN: (1) adherence: expression of adhesion molecules, CD 11a, CD 11b, CD 11c; nylon fiber adherence test; (2) chemotaxis under agarose towards the bacterial oligopeptide FMLP and complement fractions, used as attracting agents; (3) phagocytosis of opsonized latex microbeads; (4) bactericidal activity: chemiluminescence assessment of the oxidative killing potential before and after stimulation by opsonized zymosan and PMA; nitroblue tetrazolium reduction test. Results were analysed according to potentially influential factors: metabolic control (HbA1C, glycaemia), age of patient, type of diabetes, disease duration, and existence of vascular complications. PMN chemotaxis was significantly lower in patients than in healthy controls (p < 0.001) and associated with spontaneous adherence and increased expression of adhesion molecules (CD 11b, CD 11c). The increased response to chemiluminescence reflects spontaneous activation of PMN cells and increased free radical production; after stimulation, response was lower than in controls. The type of diabetes, the age of patients, HbA1C level and disease duration did not affect the responses. Chemotaxis and chemiluminescence were further reduced in patients with vascular complications and hyperglycaemia. We conclude that all steps of PMN functioning are altered in diabetic patients, which may increase the risk of vascular complications and infectious episodes.

Are HLA-DR or TAP genes genetic markers of severity in ulcerative colitis?

The pathogeny of ulcerative colitis (UC) is not yet elucidated, but some arguments suggest the implication of genetic factors. Among the candidate genes, those encoding for HLA class II genotypes have been extensively studied in UC; however, discordant data may be imputable to heterogeneity, characterized by immunological markers such as atypical ANCA (p-ANCA), or to inclusion of more or less intractable UC. The aim of our study is to evaluate the interest of HLA class II and TAP genetic markers to identify different clinical forms of UC, according to p-ANCA status. Unrelated patients with a history of UC (n = 91) and healthy control subjects with no personal or family history of inflammatory bowel diseases (IBD) (n = 200) were included. HLA-DRB1*03 was less frequent in UC patients than in healthy controls (8% vs 28%, PC < 0.03). No association was found with any TAP genotypes. Moreover, there was no association with the HLA-DR2 specificity, either in the entire group of UC patients (38% vs 28%) or in the p-ANCA-positive subgroup of patients (30%). The most consistent finding in the present study is that some genetic markers may characterize intractability in UC patients. HLA-DR2 was associated with poor prognosis, regardless of p-ANCA status. In HLA-DR2 and non-HLA-DR2 groups, colectomy was done in 55% and 27% of patients, respectively, (PC < 0.05). Furthermore, in non-HLA-DR2 patients, p-ANCA could be of interest to characterize those with more severe prognosis. Our results confirm the interest of genetic studies to define UC genetic susceptibility, taking into account intractability of the disease. They do not support the hypothesis that p-ANCA is a subclinical marker of genetic susceptibility to UC.

Red blood cell polyamines, anaemia and tumour growth in the rat.

In rats with Mat Lylu prostatic carcinoma, significant changes in blood composition and red blood cell (RBC) characteristics were observed. Anaemia, characterised by a decrease in the number of RBC and the reduction of haemoglobin and the iron content in plasma, was correlated with tumour size and the accumulation of spermidine and spermine in the RBC. In large tumours, spermidine levels were increased by 8-fold over normal value. Spleen weight and splenic spermidine concentrations were enhanced in animals with tumours. After splenectomy, the rate of tumour growth decreased by 30%. It is proposed that anaemia in tumour-bearing animals is caused by enhanced RBC lysis, owing to the alteration of the rheological properties of RBC. These may be caused by the alterated surface characteristics due to polyamine accumulation. RBC lysis and high concentrations of polyamines in RBC and spleen appear, not only to favour tumour growth, but also to compromise the immunological defence mechanisms against neoplastic invasion.

Adhesion molecule expression and response to chemotactic agents of human monocyte-derived macrophages.

Human monocyte-derived macrophages have been proposed as agents of anti-tumour immunotherapy. The aim of the present study was to investigate in vitro the properties of these cells likely to control their recruitment to the sites of inflammation and tumours. The expression of adhesion molecules involved in the binding of monocytes to endothelial cells was modified during monocyte-macrophage differentiation, with a significant increase in CD11c, CD14 and intercellular adhesion molecule-1 (ICAM-1). Monocyte-derived macrophages were sensitive to chemoattractants, in particular to the monocyte-specific chemokine monocyte chemotactic protein-1 (MCP-1). They responded by an increased expression of adhesion molecules and were attracted by the cytokine in an under-agarose migration assay. The migration response, however, decreased after days 4-5 of monocyte differentiation into macrophage. In conclusion, human monocyte-derived macrophages show alterations of surface structures involved in the recognition of inflammatory endothelium. This may explain why the cells are poorly recruited to the sites of inflammation and tumours when introduced into the circulation.

HLA-associated heterogeneity of the humoral response to islet antigens in insulin-dependent diabetes.

Insulin-dependent diabetes mellitus (IDDM) is associated with susceptibility HLA class II alleles. Islet cell antibodies (ICA), detected by indirect immunofluorescence on pancreas sections, represent the best marker of the disease. Autoantibodies to glutamic acid decarboxylase (GADA), one major islet antigen, do not totally account for ICA reactivity, suggesting heterogeneity of the anti-islet humoral response. In 97 patients with IDDM we have correlated ICA heterogeneity with clinical markers and DR and DQ alleles. ICA were found in 81% of the patients, and in 33% the serum blocked the binding to islet cells of reference sera with a granular fluorescence pattern. GADA were found in 62% of cases. Patients with high GADA titers and blocking sera were older at onset and less often had a family history of IDDM, suggesting that these antibodies might be a marker of slow progression to IDDM. ICAs were not associated with particular HLA DR or DQ alleles. Conversely, GADA were less frequent than ICA in DR4 subjects but not in the other groups. Moreover, among DR4 non-DR3 patients, GADA were found almost exclusively in DRB1*0401 patients but not in other DR4 subtypes. There was an association of GADA with DQ alleles but it was secondary to linkage disequilibrium between DR and DQ loci. In conclusion, the heterogeneity of the humoral response in IDDM is controlled by HLA class II genes and correlates with clinical heterogeneity.

Hemorheology of arterial hypertension: influence of urapidil.

There is an increased prevalence of blood hyperviscosity in patients with arterial hypertension, and viscosity changes are related to hypertensive organ damage such as e.g. left ventricular hypertrophy. Various indices of hemorheology may be assessed in studies of hypertensive patients, most commonly viscosity, erythrocyte filtration, erythrocyte aggregation, platelet aggregation, etc. In vitro assessment of erythrocyte deformability and adrenaline induced platelet aggregation was performed after urapidil administration. Increasing concentrations of urapidil (25-150 microM) caused a dose dependent improvement of the erythrocyte rigidity index as well as inhibited adrenaline induced platelet aggregation. The beneficial effects of urapidil on hemorheology could add to its therapeutic effects in the treatment of arterial hypertension.

[Exploration of the various steps of polymorphonuclear neutrophil function in diabetic patients]. / Exploration des différentes étapes du fonctionnement des polynucléaires neutrophiles chez les patients diabétiques.

UNLABELLED: Vascular complications in diabetic patients is a complex, probably multifactorial phenomena involving cellular phagocytosis. The aim of this study was to evaluate polymorphonuclear performance in 61 infection free diabetic patients based on tests of the different cell functions: 1) adhesion:adhesion molecule expression CD11a, CD11b, CD11c; adhesion test on nylon fibers. 2) chemotaxis:chemotaxis under aragose to FMLP (bacteria oligopeptide) and complement fractions. 3) Phagocytosis:latex beads. 4) Bacteriocidal power:chemoluminescence photometric oxidative potential before and after stimulation with opsonized zymosan and PMA; reduction of tetrazolium nitroblue. RESULTS were analyzed according to type of diabetes, glucose control, duration of the disease, history of infection and presence of vascular complications. RESULTS: compared with a group of 30 controls, the diabetic patients had a significant impaired polynuclear chemotaxis function (p < 0.001) with both spontaneous adhesion and increased expression of adhesion molecules (CD 11b, CD 11c). The chemoluminescence test was increased at the baseline level due to spontaneous polynuclear adhesion and increased production of free radicals. This response decreased after stimulation compared with controls. The type of diabetes, Hb A1c level and history of infection did not appear to have an effect. Inversely, changes in chemotaxis and chemoluminescence were greater in patients with vascular complications. In summary, all the functions of polynuclear neutrophils tested were altered in diabetic patients and could favor vascular complications and infections episodes.