A telemedicine-based approach with real-time transmission of blood glucose data improves metabolic control in insulin-treated diabetes: the DIAMONDS randomized clinical trial.

PURPOSE: To evaluate if a web-based telemedicine system (the Glucoonline® system) is effective to improve glucose control in insulin-treated patients with type 1 and type 2 diabetes, as compared to standard of care. METHODS: This was a prospective, randomized, controlled trial, carried out at three tertiary referral centers for diabetes in Italy. Adults with insulin-treated type 1 and type 2 diabetes, inadequate glycemic control, and no severe diabetes-related complications and/or comorbidities were eligible for this study. Patients were randomized to either perform telemedicine-assisted (Group A) or standard (Group B) self-monitoring blood glucose (SMBG) for 6 months. In Group A, patients received prompt feedback about their blood glucose levels and therapy suggestions from the study staff via phone/SMS, when appropriate. In Group B, patients had no remote assistance from the study staff between planned visits. RESULTS: 123 patients were included in the final analysis. After 6 months, patients achieved a significant reduction in HbA1c in Group A (-0.38%, p < 0.05) but not in Group B (+ 0.08%, p = 0.53). A significant difference in the percentage of patients with HbA1c < 7% between Group A and Group B was found after 3 months (28.6% vs 11.1%, p = 0.02). Also, fewer patients (p < 0.05) with HbA1c > 8.5% were found in Group A vs Group B, respectively, after both 3 months (14.3% vs 35.2%) and 6 months (21.8% vs 42.9%). CONCLUSIONS: The use of the Glucoonline™ system resulted in improved metabolic control. Telemedicine services have potential to support diabetes self-management and provide the patients with remote, prompt assistance using affordable technological equipment. Trial registration This study was registered at clinicaltrials.gov (NCT01804803) on March 5, 2013.

Interlaboratory comparison of current high-performance methods for HbA2.

INTRODUCTION: Few data are available on the alignment of the different methods used for HbA(2) quantitation and recent external quality survey results show a consistent spread of HbA(2) values. To this aim, a comparison study among the actual best performing techniques for HbA(2) determination, comprising HPLC and CE methods, was performed. METHODS: A total of 80 blood samples collected from normal subjects and ß-thalassemia carriers were analyzed by different HPLC (Bio-Rad Variant I, Bio-Rad Variant II, Menarini HA-8160, Tosoh G7, Tosoh G8) and capillary electrophoresis (Beckman Coulter MDQ and ProteomeLab PA 800, Sebia Capillarys 2) methods. Patient's samples with clinically relevant hemoglobin variants (HbC, HbD, HbE, HbS, and δ-chain variants) were also tested by all methods. RESULTS: The mean within-run imprecision of HbA(2) measurement (expressed as CV, %) was between 0.5% and 4.4% (HPLC) and between 1.2% and 4.4% (capillary electrophoresis). The comparison study showed that the different methods were highly correlated (r between 0.974 and 0.997) although biased each other. HbA(2) determination in presence of abnormal hemoglobins was variously interfered by both HPLC and CE methods. Concerning HbF, the mean imprecision at HbF values ≥1.5% was between 1.2% and 8.2% (as CVs). CONCLUSIONS: A poor alignment of routine methods for HbA(2) measurement was found. The need of a better standardization of HbA(2) measurement procedures was underlined.

The role of haemoglobin A(2) testing in the diagnosis of thalassaemias and related haemoglobinopathies.

The increase in haemoglobin (Hb)A(2) level is the most significant parameter in the identification of beta thalassaemia carriers. However, in some cases the level of HbA(2) is not typically elevated and some difficulties may arise in making the diagnosis. For these reasons the quantification of HbA(2) has to be performed with great accuracy and the results must be interpreted together with other haematological and biochemical evidence. The present document includes comments on the need for accuracy and standardisation, and on the interpretation of the HbA(2) value, reviewing the most crucial aspects related to this test. A practical flow-chart is presented to summarise the significance of HbA(2) estimation in different thalassaemia syndromes and related haemoglobinopathies.

Can plasma glucose and HbA1c predict fetal growth in mothers with different glucose tolerance levels?

To assess whether HbA1c and plasma glucose predicts abnormal fetal growth, 758 pregnant women attending 5 Diabetic Centers were screened for gestational diabetes mellitus (GDM). On glucose challenge (GCT) at 24-27 weeks of gestation (g.w.), negative cases formed the normal control group (N1). Positive cases took an oral glucose tolerance test (OGTT): those found negative were classed as false positives screening test (N2); if they had an OGTT result at least as high as their normal glucose levels, they were classed as having one abnormal glucose value (OAV) at OGTT; two values as GDM. HbA1c was assayed on the day of GCT. We considered fetal macrosomia, large for gestational age (LGA), ponderal index and mean growth percentile. Mean age, pre-pregnancy BMI, fasting plasma glucose (FPG) and HbA1c were progressively higher from N1 to GDM patients. The newborn of N2 mothers were heavier than those with N1 or GDM. The mean growth percentile was significantly higher in N2 than in N1. More LGA babies were born to OAV than to N1 or N2 women. Macrosomia and ponderal index did not differ significantly in the four groups. At logistic regression only plasma glucose at GCT could predict LGA babies and a ponderal index above 2.85. At risk analysis, GDM and OAV significantly predicted LGA babies, and GDM a ponderal index >2.85. In conclusion, FPG at GCT could predict fetal overgrowth and plasma glucose >85mg/dl doubles the risk of LGA infants. HbA1c at 24-27g.w. does not predict fetal overgrowth. Mild alterations in glucose tolerance correlate with fetal overgrowth and needs monitoring and treatment.

Inter-method differences and commutability of control materials for HbA2 measurement.

The intermethod variability of control materials and patient blood samples for the measurement of hemoglobin A2 (HbA2) were compared. A set of 54 blood samples and 10 control materials were analyzed in duplicate by HPLC and microcolumn methods. For each set of methods the distances of the materials from the regression line of patient blood results (expressed as normalized residuals) were calculated. Four out of ten controls had normalized residuals exceeding three standard deviations from the regression line. Moreover, total Hb and Hb derivatives analysis proved that only a minority of the controls could be considered similar to patients' blood samples. Intermethod calibration performed "a posteriori" by the two best performing control materials improved intermethod variability among all the five tested methods. We conclude that the use of high resolution HPLC methods together with appropriate commutable control materials allows for better harmonization of results in the field of diagnosis of hemoglobin disorders in research and clinical practice.

Posttranslational deamidation of proteins: the case of hemoglobin J Sardegna [alpha50(CD8)His-->Asn-->Asp].

Hemoglobin J Sardegna [alpha50(CD8)His-->Asn -->Asp] is a human Hb variant in which a posttranslational deamidation process takes place, transforming an Asn to an Asp residue. This variant, particularly widespread in northern Sardinia, has for the first time been characterized at the DNA level (codon 50 C-->A) on the selectively amplified alpha2-globin gene. We determined the protein and DNA sequences and performed cellulose acetate electrophoresis, isoelectric focusing, globin chain separation, stability tests with isopropanol and heat precipitation, and oxygen affinity analyses on whole blood to fully characterize the variant. A comprehensive review of the deamidation processes involving Asn and Gln residues in mutant proteins is reported, together with a discussion of the molecular mechanisms of such deamidations. Finally, examples of other proteins of clinical importance in which Asn or Gln residues have been implicated by DNA analysis alone are presented. These findings point out the importance of the complete characterization of variant proteins by use of both DNA and protein analyses.

Commutability of control materials in glycohemoglobin determinations.

The intermethod variabilities of control materials and patient blood samples for the measurement of glycohemoglobin were compared. Sets of 50 blood samples and 15 control materials were analyzed by HPLC and affinity and immunochemical methods. For each pair of methods, the distances of the materials from the regression line of patient blood results (expressed as normalized residuals) were calculated. Only two of 15 controls had normalized residuals exceeding 3 standard deviations from the regression line. Total hemoglobin (Hb) content, Hb derivatives, and cellulose acetate electrophoresis demonstrated that only a minority of controls could be considered similar to patients' blood samples. We selected Menarini's and our home-prepared controls to simulate calibration of the different techniques by these materials. Intermethod calibration succeeded mostly in harmonizing results obtained by HPLC methods. On the contrary, calibration of the immunochemical techniques (Boehringer and Roche) did not improve intermethod agreement to a clinically useful level.

A re-evaluation of glycohaemoglobin standardisation: the Italian experience with 119 laboratories and 12 methods.

The effect of calibration on glycohaemoglobin results was investigated in 119 laboratories where glycohaemoglobin (HbA1c) percentages were determined in triplicate in two samples and in two calibrators. The materials were human lyophilized haemolysates home-prepared with a novel protocol. The HbA1c content was assigned to calibrators using 4 HPLC methods with the data obtained from 11 laboratories. Intra-laboratory variation was low, although in 14 out of 119 laboratories CV values greater than 3.5% were reported. Calibration decreased inter-method variation from 16.3% to 5.6%, and from 15.3% to 4.2% for samples with low and high HbA1c content, respectively. Calibration decreased overall inter-laboratory variation from 18.7% to 7.4%, and from 17.2% to 5.4%. The calibrated results obtained with all methods, including Abbott IMx and Vision, were comparable.

Hb Puttelange [beta 140(H19)Ala-->Val] in an Italian man with polycythemia.

Hb Puttelange [beta 140(H18)Ala-->Val] was found in a 51-year-old Italian man who had mild polycythemia. The variant eluted from ion exchange high performance liquid chromatography at a position between Hb A and Hb A2. It comprised approximately 34% of the total hemoglobin, was weakly unstable and exhibited an increased oxygen affinity. Amplification of the beta-globin exons and nucleotide sequencing revealed a heterozygosity for a GCC-->GTC mutation in codon 140 corresponding to an Ala-->Val replacement. This substitution accounts for the altered functional properties, probably by producing indirect perturbation of the 2 3-diphosphoglycerate pocket through the nearby lysine residue at beta 82(EF6).

Simultaneous automated determination of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in whole blood.

We report a potentiometric fully automated method for determining red cell glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase activities and the glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index using 25 microliters of whole blood. No sample pre-treatment (e.g., preparation of the haemolysate) is needed and the measurements are performed at pH 8.0 and 37 degrees C under the conditions recommended by the ICSH committee. The reproducibility was constantly good, with within-run CV of 1.0% (glucose 6-phosphate dehydrogenase) and 5.9% (glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase) for activities in glucose 6-phosphate dehydrogenase non-deficient adults, and of 2.3% (glucose 6-phosphate dehydrogenase, G6PD) and 2.5% (glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase) for G6PDMediterranean heterozygotes. Linearity was observed up to an activity of 2800 U/l of glucose 6-phosphate dehydrogenase. Results of glucose 6-phosphate dehydrogenase activity (U/l) in whole blood (y) correlated well with those obtained with the previously described monostarter assay, performed at pH 9.2 (y = 0.60x + 37; n = 80; r = 0.991). Results of 6-phosphogluconate dehydrogenase (U/l) in whole blood (y) correlated well with those obtained by the ICSH recommended method (x) (y = 0.779x - 44; n = 23; r = 0.991). Reference intervals are reported for glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index relatively to normal, beta- and alpha-thalassaemic glucose 6-phosphate dehydrogenase non-deficient adults, to glucose 6-phosphate dehydrogenase deficient adult males and to G6PDMediterranean non-thalassaemic heterozygotes. We demonstrate that the diagnostic sensitivity of the glucose 6-phosphate dehydrogenase/6-phosphogluconate dehydrogenase index in detecting the G6PDMediterranean heterozygotes is superior to that of the glucose 6-phosphate dehydrogenase activity alone.

A patient-side technique for real-time measurement of acetylcholinesterase activity during monitoring of eptastigmine treatment.

Rapid and reliable measurement of acetylcholinesterase (AChE) activity is of crucial importance to the pharmacodynamic monitoring of anticholinesterase drugs. A new assay has been developed to measure AChE from 10 microliter samples of capillary blood. AChE activity was calculated from the change in pH of the reaction medium caused by the hydrolysis of acetylcholine and measured with a highly sensitive differential pH apparatus (CL-10, Eurochem, Rome, Italy). Interference by butyrylcholinesterase was eliminated by a specific inhibitor, quinidine sulfate. The assay lasts 1 min. The coefficient of variation (CV) for replicated measurements was 2.8% (3267 U/L, n = 33). Linearity ranged from 0 to 10,000 U/L. The correlation coefficient between the new technique and Ellman's colorimetric method on washed erythrocytes was r = 0.987 (y = 1.299x - 63, n = 29). The correlation coefficient between assays on capillary and venous samples was r = 0.979 (y = 0.974x + 174, n = 47). A cross-laboratory validation study was performed in 10 centers using glycerol-stabilized hemolysates with normal and reduced AChE activity. Samples were assayed in triplicate. The within- and between-laboratory CVs for samples with normal AChE activity (6,018 U/L) were 2.2 and 8.1%, respectively. The new method was applied to a double-blind, placebo-controlled multicenter study of eptastigmine in Alzheimer patients and proved to be a simple, noninvasive, rapid, and reliable method for pharmacodynamic monitoring of this drug.

Introduction of magnetic beads in diagnosis: a simple and rapid method to detect mutations of beta globin gene, directly amplified from blood.

In this communication we present the results obtained by the use of magnetic beads in diagnosis, for the identification of genetic variants at the molecular level by sequencing, in comparison with the more laborious method of the production of ssDNA with asymmetric PCR. We compared the two techniques studying variants of beta globin gene: Hb Abruzzo [beta 143 (H21) His -> Arg] and Hb D Los Angeles [beta 121 (GH4) Glu -> Gln].

Rapid determination of erythrocyte pyruvate kinase activity.

We report a new potentiometric method for determining pyruvate kinase (PK). Enzymatic activity is measured by monitoring the change in pH produced in the reaction buffer under International Committee for Standardization in Haematology (ICSH) standardized assay conditions, and the lactate dehydrogenase reaction is automatically subtracted in each measuring cycle. The analysis, performed at 37 degrees C, requires a 10-microL sample (isolated erythrocytes or whole blood) and is completed in 2.5 min. The intra-assay CV is < 4% (PK between 3 and 35 U/g Hb); the interassay CV is 4.0% (PK 15 U/g Hb); results are linear from 3 to 30 U/g Hb. A good correlation with the ICSH reference method (x) was found: y = 1.011x - 0.05; n = 32; r = 0.9939; Sylx = 0.75 (units: U/g Hb). The reference intervals of the PK activity in isolated erythrocytes (RBC-PK) were estimated in 89 normal subjects. We found that women possess a higher RBC-PK than do men (P < 0.0001) and that the biological variability (CVb) of RBC-PK is 13.5%. Applications of the proposed method to the hematological routine are reported.

IHP entrapment into human erythrocytes: comparison between hypotonic dialysis and DMSO osmotic pulse.

Three different blood units were treated separately by the hypotonic dialysis (HD) and the dimethylsulphoxide osmotic pulse (DMSO) method, in order to load the erythrocytes with inositol hexaphosphate. A detailed comparison between the two loading techniques was performed by monitoring the red cell distribution patterns on discontinuous Percoll density gradients, the RBC oxygen affinity and the amount of the main intracellular organic phosphates with the 31P-NMR. The results obtained showed that: (1) The HD loading produces a redistribution of the RBC fractions with a concomitant smoothing of the relative differences among distinct fractions (2) only a minor portion of erythrocytes (from 8.5 to 24.9% of total RBCs) are loaded with IHP after the DMSO treatment. All of these cells move to the lightest fraction (d = 1.080 g/ml). (3) Both HD and DMSO IHP-loaded cells show an increase in P50 (basal vs. after loading, means +/- SD: 25.8 +/- 3.0 vs. 52.5 +/- 3.2 mm Hg) correlated to the IHP incorporation (mean intracellular IHP concentration: 4.2 mmol/l RBC). (4) probably the IHP incorporation efficiency could be probably improved at least by increasing the IHP concentration during the treatment.