Effects of different anticoagulants on glycated albumin quantification.

INTRODUCTION: In the last 20 years glycated albumin (GA) measurement has been demonstrated to be a reliable glycation marker and recently as the most innovative one in western countries. Glycated albumin has been already adopted by some Asian countries due to its usefulness in diabetes screening. The aim of the present study was to investigate for the first time the effects of different anticoagulants on GA assay. MATERIALS AND METHODS: From each of 60 patients a serum tube and K3EDTA, Li-Heparin and NaF-EDTA containing tubes were collected. All tubes were from Sarstedt (Verona, Italy). Glycated albumin was measured in duplicate in each sample tube in a single analytical run with quantILab glycated albumin (Instrumentation Laboratory SpA - A Werfen Company, Milan, Italy) on Architect c8000 analyser (Abbott SRL, Rome, Italy). Comparison of GA% in evaluated tubes was made by paired Wilcoxon test. RESULTS: Median and interquartile range GA% concentrations were 15.4% (13.2 - 19.1) in serum, 15.7% (13.6 - 19.9) in K3EDTA, 15.6% (13.3 - 19.7) in Li-heparin and 15.5% (13.1 - 19.3) in NaF-EDTA samples, respectively. Glycated albumin mean relative bias respect to serum was within desirable bias derived from biological variation studies (± 2.9%) when K3EDTA (+ 2.8%), Li-heparin (+ 0.9%) or NaF-EDTA (+ 0.1%), were used as anticoagulants. CONCLUSIONS: Our results demonstrate that the GA% assay is not affected by relevant interferences when K3EDTA, Li-heparin or NaF-EDTA are used as anticoagulants, so they can be used interchangeably without a relevant impact on the clinical use of the test.

Multicenter evaluation of an enzymatic method for glycated albumin.

BACKGROUND: The use of glycated albumin (GA) has been proposed as an additional glycemic control marker particularly useful in intermediate-term monitoring and in situation when HbA1c test is not reliable. METHODS: We have performed the first multicenter evaluation of the analytical performance of the enzymatic method quantILab Glycated Albumin assay implemented on the most widely used clinical chemistry analyzers (i.e. Abbott Architect C8000, Beckman Coulter AU 480 and 680, Roche Cobas C6000, Siemens ADVIA 2400 and 2400 XPT). RESULTS: The repeatability of the GA measurement (expressed as CV, %) implemented in the participating centers ranged between 0.9% and 1.2%. The within-laboratory CVs ranged between 1.2% and 1.6%. A good alignment between laboratories was found, with correlation coefficients from 0.996 to 0.998. Linearity was confirmed in the range from 7.6 to 84.7%. CONCLUSION: The new enzymatic method for glycated albumin evaluated by our investigation is suitable for clinical use.

Glycated albumin: from biochemistry and laboratory medicine to clinical practice.

This review summarizes current knowledge about glycated albumin. We review the changes induced by glycation on the properties of albumin, the pathological implications of high glycated albumin levels, glycated albumin quantification methods, and the use of glycated albumin as a complementary biomarker for diabetes mellitus diagnosis and monitoring and for dealing with long-term complications. The advantages and limits of this biomarker in different clinical settings are also discussed.

Analytical Performances of an Enzymatic Assay for the Measurement of Glycated Albumin.

BACKGROUND: Short to intermediate integrated glycemic control is best determined by glycated albumin (GA). This assay is appropriate when interpretation of glycated hemoglobin (HbA1c) is critical because of hemoglobinopathies, severe anemias, or other factors that affect red blood lifespan as hemodialysis. We evaluated a new assay based on the enzymatic quantification of GA by ketoamine oxidase and an albumin-specific protease. METHODS: Limits of blank, detection, and quantification; precision; linearity; accuracy; interferences; correlation with HbA1c; and serum vs plasma study have been evaluated on ILab® systems. RESULTS: Limit of blank, detection, and quantification for GA (g/L) were, respectively, 0.26, 0.36, and 1.15. Repeatability and within-device precision CVs were lower than 2.11%, 1.61%, and 1.56% for GA (g/L), albumin (g/L), and GA%, respectively. Linearity for GA (g/L) and GA% was 1.2-36.8 and 5.5-92.2, respectively. Highest deviation from linearity was <11% and recovery was higher than 90%. Accuracy against the certified ReCCS Japan Clinical Chemistry Reference Material (JCCRM) 611 was <1%. Classical interfering substances had no significant impact. Correlation of GA% between ILab® Taurus and ADVIA system was y = 1.02[GA%]+0.25; R2 = 0.994. No difference was found in the determination of GA% in serum vs plasma. CONCLUSIONS: GA enzymatic assay is a reliable, fully automated method allowing accurate and precise determination of GA in a routine laboratory.

Decreased mean sphered cell volume values in top-level rugby players are related to the intravascular hemolysis induced by exercise.

Sports anemia is a common risk for athletes. Intravasclar hemolysis is the principal source of an accelerated turnover of erythrocytes in sportsmen. The hemolysis induces some biochemical and hematological modifications; in particular, high concentrations of total and indirect (unconjugated) bilirubin could be reported in professional athletes. We recruited 24 rugby players of the Italian National Team. In these athletes we measured hematological parameters, including mean sphered cell volume (MSCV) by means of Coulter LH 750 (Beckman Coulter, Brea, CA, USA), beside biochemical parameters, including bilirubin and haptoglobin. We observed differences in the athletes' indirect bilirubin between the blood drawing performed before the start of training and competitions and the one performed at the end of the competition season. The bilirubin was increased during competition season from 7% to 329% when compared with the baseline value measured before training and competitions. MSCV, in contrast, decreased from a mean of 88.4 fL to 86 fL. The MSCV decrease in association with an indirect bilirubin increase is a specific sign of erythrocyte destruction, and specific training and competitions schemes and diet or therapy modifications should be decided according to their values. The modifications of MSCV are correlated with those of haptoglobin and hemoglobin, but not with reticulocytosis.

Reticulocyte count, mean reticulocyte volume, immature reticulocyte fraction, and mean sphered cell volume in elite athletes: reference values and comparison with the general population.

BACKGROUND: The role of measurement of reticulocytes and their parameters is growing in sports medicine. The use of reticulocyte counts in protocols for evaluating and screening for the suspected abuse of hormones that stimulate the bone marrow is an example. Reticulocytes are also important for evaluation of the performance and general health status of athletes, especially for monitoring therapies and diets. The current availability of fully automated haematological systems that can measure reticulocyte numbers and characteristics (volume, density) increases the potential use of these parameters in laboratory and sports medicine. Few studies have considered the application of these parameters in athletes and a lack of specific reference ranges means that their valid clinical use is difficult. METHODS: Using a Coulter LH700 instrument, we measured reticulocyte count (Retics), mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF), and mean sphered cell volume (MSCV) in 106 male professional elite athletes (football and rugby players and skiers). Reference intervals for the athletes were compared with the intervals found for a control group of 73 age-matched males. RESULTS: We calculated the following reference intervals: 0.30-1.54% for Retics, 93.1-114.8 fL for MRV, 0.18-0.39% for IRF, and 76.8-94.5 fL for MSCV. CONCLUSIONS: No statistically significant differences were observed for Retics, MRV, IRF, and MSCV between elite athletes and controls. Significant differences were observed for haemoglobin (Hb), erythrocytes, haematocrit (Ht), and mean corpuscular volume. Moreover, no statistical differences were observed among different sports, whereas differences were remarked in football and rugby players between the samples drawn before the start of competitive season and the samples drawn during the season, demonstrating that reticulocyte counts and parameters are useful for monitoring sportsmen.

The role of complement in the therapeutic activity of rituximab in a murine B lymphoma model homing in lymph nodes.

BACKGROUND AND OBJECTIVES: We have set up a murine B lymphoma model stably expressing human CD20 and homing in lymph nodes in immunocompetent mice to study the mechanism of action of rituximab. DESIGN AND METHODS: The B lymphoma line 38C13 was stably transduced with the human CD20 cDNA by retroviral infection and injected into syngeneic mice. RESULTS: The transduced 38C13-CD20(+) cells stably expressed human CD20 on 100% of cells. Rituximab alone did not inhibit 38C13-CD20+ cell growth but relocalized the human CD20 into lipid rafts and induced complement-mediated lysis in vitro. Inoculation of 4x10(3) 38C13-CD20(+) intravenously into syngeneic mice led to the development of tumor masses in the spleen, bone marrow and lymph nodes, detectable from day 15 by polymerase chain reaction (PCR) analysis, and with a median survival of 21-24 days. Treatment with 250 mg rituximab i.p. given 1-10 days after tumor inoculation cured 100% of animals, with disappearance of tumor documented by immunohistochemistry and PCR analysis. Depletion of both NK cells and neutrophils did not affect the therapeutic activity of rituximab in vivo. Similarly, removal of phagocytic macrophages using clodronate-liposomes did not modify the capacity of rituximab to control tumor growth. In contrast, the protective activity of the antibody was completely abolished after complement depletion with cobra venom factor. Complement was also required when cells were inoculated subcutaneously in nude mice. INTERPRETATION AND CONCLUSIONS: These data demonstrate that complement is required for the therapeutic activity of rituximab in vivo in a murine model of B-cell lymphoma, independently of its localization in lymph nodes or subcutaneously.

Complement activation determines the therapeutic activity of rituximab in vivo.

Rituximab is an anti-CD20 chimeric mAb effective for the treatment of B-NHL. It can lyse lymphoma cells in vitro through both C- and Ab-dependent cellular cytotoxicity. The mechanism of action of rituximab in vivo is however still unclear. We have set up a new in vivo model in nonimmunodeficient mice by stable transduction of the human CD20 cDNA in the murine lymphoma line EL4. Animals injected i.v. with the EL4-CD20(+) lymphoma cells died within 30 days with evident liver, spleen, and bone marrow involvement, confirmed by immunohistochemistry and PCR analysis. A single injection of rituximab or the murine anti-CD20 Ab 1F5, given i.p. 1 day after the tumor, cured 100% of the animals. Indeed, at week 4 after tumor cell inoculation, CD20(+) cells were undetectable in all organs analyzed in rituximab-treated animals, as determined by immunohistochemistry and PCR. Rituximab had no direct effect on tumor growth in vitro. Depletion of either NK cells or neutrophils or both in tumor-injected animals did not affect the therapeutic activity of the drug. Similarly, rituximab was able to eradicate tumor cells in athymic nude mice, suggesting that its activity is T cell independent. In contrast, the protective activity of rituximab or the 1F5 Ab was completely abolished in syngeneic knockout animals lacking C1q, the first component of the classical pathway of C (C1qa(-/-)). These data demonstrate that C activation is fundamental for rituximab therapeutic activity in vivo.