Patrizia Lanteri

Blood biochemical markers of bone turnover: pre-analytical and technical aspects of sample collection and handling.

Abstract Casual or systematic errors occurring in pre-analytical, analytical or post-analytical phases influence laboratory test results. The areas where pre-analytical phase errors most often arise are: timing of specimen collection; selection of specimen type; and time and temperature of storage/transport. Bone turnover markers are clinically useful in evaluating bone metabolism. Although unquestionably valuable tools, little is known about the pre-analytical precautions for their correct use and there is no consensus on kind of sample, or storage time and temperature before analysis. Moreover, biological variability, because of uncontrollable and controllable factors, will affect pre-analytical variability. Serum should be preferred to simplify blood drawing; therefore, only one tube should be used for the analysis of all bone markers. Short-term storage at 4°C may be advisable to preserve stability, immediate storage at –70°C is recommended for longer periods, while avoiding repeated freeze-thawing cycles. Sampling should be performed in the morning in fasting subjects who have abstained from physical exercise for 24 h. This review aimed to give a knowledge update on pre-analytical phase precautions in performing bone turnover marker measurement.

Relationship between vitamin D receptor gene (VDR) polymorphisms, vitamin D status, osteoarthritis and intervertebral disc degeneration

The vitamin D endocrine system is involved in bony and cartilaginous metabolisms and alterations in the homeostasis of this system could be associated to pathological conditions of cartilaginous tissue. In this context, the presence of polymorphisms in the vitamin D receptor gene (VDR), in association with the susceptibility to common osteochondral diseases, was largely investigated. The aim of this review was to summarize data present in literature, analyzing the association of the VDR polymorphisms, vitamin D status and knee cartilage and intervertebral disc pathologies, trying to suggest links between the different specific pathologies analyzed. Concerning the association between VDR polymorphisms and cartilaginous tissue diseases, we found controversial reports. However, the great majority of papers reported an association with lumbar disc degeneration, whereas about half of the studies found an association with osteoarthritis. A further association between VDR polymorphisms (in linkage disequilibrium) and the presence of specific characteristics of these diseases, in particular the formation of osteophytes, was evidenced. Finally, the influence of vitamin D status on these pathologies was evaluated, trying to evidence the relation between the presence of particular genetic variants in the VDR and vitamin D levels or to show whether a particular vitamin D status could predispose to the development or progression of such diseases, however, no significant associations were found. In the future, given the role of vitamin D system in the cartilaginous tissue metabolism, it could be interesting to perform functional and tissue specific studies to analyze the interplay between the different VDR variants and its ligand.

Haematological and iron metabolism parameters in professional cyclists during the Giro d'Italia 3-weeks stage race.

Abstract Background: Haematological assessment is crucial for evaluating athletes healthy status. Professional athletes experience physiological modifications during competitions and over a season: the risk of sports anaemia is high. Few descriptions of haematological parameters behaviour during a 3-weeks cycling stage race have been published. Methods: We studied nine professional cyclists engaged in the 2011 Giro d’Italia stage race. Pre-analytical and analytical phases tightly followed academic and anti-doping authorities’ recommendations. Haematological and iron metabolism parameters were measured days -1 (pre-race), 12 and 22 during the race. Results: Haemoglobin, red blood cells and haematocrit decreased during the race with a stabilisation in the second half, but final values were lower than baseline. Reticulocytes did not modify, whilst the immature reticulocyte fraction increased. No differences were found in red blood cells volume and corpuscular haemoglobin content, neither in iron metabolism markers. The acute phase proteins, haptoglobin and C-reactive protein, both increased over the race, while haemoglobin and haptoglobin were inversely related. Conclusions: These data are important for improving the knowledge of physiological modifications in haematological and iron metabolism parameters of professional athletes during highly demanding competitions. This is the first report, in the ambit of a stage race, in which the pre-analytical phase standardisation has been applied.

Bone and Energy Metabolism Parameters in Professional Cyclists during the Giro d’Italia 3-Weeks Stage Race

Cycling is a not weight-bearing activity and is known to induce bone resorption. Stage races are really strenuous endurance performances affecting the energy homeostasis. The recently highlighted link, in the co-regulation of bone and energy metabolism, demonstrates a central role for the equilibrium between carboxylated and undercarboxylated forms of osteocalcin. Aim of this study was to understand the acute physiological responses to a cycling stage race in terms of bone turnover and energy metabolism and the possible co-regulative mechanisms underlying their relationship. We studied nine professional cyclists engaged in 2011 Giro d’Italia stage race. Pre-analytical and analytical phases tightly followed academic and anti-doping authority’s recommendations. Bone and energy metabolism markers (bone alkaline phosphatase, tartrate-resistant acid phosphatase 5b, total and undercarboxylated osteocalcin, leptin and adiponectin) and related hormones (cortisol and testosterone) were measured, by Sandwich Enzyme Immunoassays, at days -1 (pre-race), 12 and 22 during the race. The power output and the energy expenditure (mean and accumulated) were derived and correlated with the biochemical indexes. During the race, bone metabolism showed that an unbalance in behalf of resorption, which is enhanced, occurred along with a relative increase in the concentration of the undercarboxylated form of osteocalcin that was indirectly related to the enhanced energy expenditure, through adipokines modifications, with leptin decrease (high energy consumption) and adiponectin increase (optimization of energy expenditure). The exertion due to heavy effort induced a decrease of cortisol, while testosterone levels resulted unchanged. In conclusion, during a 3-weeks stage race, bone metabolism is pushed towards resorption. A possible relationship between the bone and the energy metabolisms is suggested by the relative correlations among absolute and relative concentrations trends of undercarboxylated OC, adipokines concentrations, BMI, fat mass (%), power output and the derived energy expenditure.

Stability of haematological parameters and its relevance on the athlete's biological passport model.

The stability of haematological parameters is crucial to guarantee accurate and reliable data for implementing and interpreting the athlete’s biological passport (ABP). In this model, the values of haemoglobin, reticulocytes and out-of-doping period (OFF)-score (Hb-60√Ret) are used to monitor the possible variations of those parameters, and also to compare the thresholds developed by the statistical model for the single athlete on the basis of its personal values and the variance of parameters in the modal group. Nevertheless, a critical review of the current scientific literature dealing with the stability of the haematological parameters included in the ABP programme, and which are used for evaluating the probability of anomalies in the athlete’s profile, is currently lacking. In addition, we collected information from published studies, in order to supply a useful, practical and updated review to sports physicians and haematologists.There are some parameters that are highly stable, such as haemoglobin and erythrocytes (red blood cells [RBCs]), whereas others, (e.g. reticulocytes, mean RBC volume and haematocrit) appear less stable. Regardless of the methodology, the stability of haematological parameters is improved by sample refrigeration. The stability of all parameters is highly affected from high storage temperatures, whereas the stability of RBCs and haematocrit is affected by initial freezing followed by refrigeration. Transport and rotation of tubes do not substantially influence any haematological parameter except for reticulocytes.In all the studies we reviewed that used Sysmex instrumentation, which is recommended for ABP measurements, stability was shown for 72 hours at 4°C for haemoglobin, RBCs and mean curpuscular haemoglobin concentration (MCHC); up to 48 hours for reticulocytes; and up to 24 hours for haematocrit. In one study, Sysmex instrumentation shows stability extended up to 72 hours at 4°C for all the parameters. There are significant differences among methods and instruments: Siemens Advia shows lower stability than Sysmex as regards to reticulocytes. However, the limit of 36 hours from blood collection to analysis as recommended by ABP scientists is reasonable to guarantee analytical quality, when samples are transported at 4°C and are accompanied by a certified steadiness of this temperature.There are some parameters that are highly stable, such as haemoglobin and RBCs; whereas others, such as reticulocytes, mean cell volume and haematocrit are more unstable. The stability of haematological parameters might be improved independently from the analytical methodology, by refrigeration of the specimens.

Metabolic effects of vitamin D active metabolites in monolayer and micromass cultures of nucleus pulposus and annulus fibrosus cells isolated from human intervertebral disc.

Intragenic polymorphisms in the vitamin D receptor gene are linked to disc degeneration features, suggesting that alterations in the vitamer-mediated signalling could be involved in the pathophysiology of the disc and that interaction of disc cells with vitamin D metabolites may be critical for disc health. The vitamer-mediated modulation of disc cells proliferation, metabolic activity, extracellular matrix (ECM) genes expression and proteins production was investigated. It was stated that disc cells express vitamin D receptor and are very sensitive to metabolic stimuli. In monolayer cultures, 1,25(OH)(2)D(3), but not 24,25(OH)(2)D(3), determined an inhibition of the proliferation and regulated also the ECM genes expression in nucleus pulposus and annulus fibrosus cells. Micromass cultures induced a more physiologic expression pattern of extracellular matrix genes. Cells Treatment with vitamin D metabolites did not result in relevant modifications of glycosaminoglycans production, except for annulus cells, whose production was reduced after 1,25(OH)(2)D(3) treatment. Moreover, a reduced glycosaminoglycans staining in both cell types and a significant reduced aggrecan gene expression in annulus cells treated with 1,25(OH)(2)D(3) were observed. A reduction of collagen I and II staining in annulus cells 1,25(OH)(2)D(3) treated, in accordance with a downregulation of collagen genes expression, was also registered. Finally, the vitamin D receptor gene expression did not show significant metabolite-mediated modification in monolayer or micromass cultures. These findings could enhance new insights on the biochemical mechanisms regulated by vitamin D in disc cartilage and possibly involved in the development of physiological/pathological modifications of the disc.

Stability of osteopontin in plasma and serum.

Abstract Background: Osteopontin is a glycoprotein widely expressed in many tissues and in different physiological conditions. Osteopontin concentrations are usually measured through immunological methods; however, little is known about the pre-analytical management of the sample. We evaluated the effects of different times and temperatures storage conditions on serum and plasma concentrations of osteopontin. Methods: Serum and plasma aliquots were frozen at –80°C, following storage at 4°C or room temperature for 0, 2, 4, 8, 12, 24 and 48 h. Osteopontin concentrations were determined by enzymoimmunometric assay. Serum samples obtained from tubes with or without gel separator were compared to verify the effect of gel. Western blotting analysis was performed to characterize the antibody. Results: Osteopontin concentrations were stable over time in all conditions in both serum and plasma. Plasma showed 3.8–4.8-fold higher concentrations than serum. Comparable levels were found between serum tubes with or without gel separator and always lower than those in plasma, demonstrating no effect of gel in serum tubes. Western blotting analysis showed various osteopontin bands, indicating that the antibody recognizes the entire panel of different osteopontin forms. Conclusions: We demonstrated the stability across 48 h of osteopontin in serum and plasma at either room temperature or 4°C, when the evaluation is carried out by an immune-based method. The minimal variations observed over time were always lower than the calculated intra- and inter-assay coefficients of variation. Plasma specimens should be preferred when osteopontin concentration are assayed by immunological methods.

Reticulocytes in Sports Medicine: An Update

Reticulocytes are young red blood cells which develop from erythroblasts and circulate in the bloodstream for about 1-4 days before maturing into erythrocytes. With the introduction of reticulocyte count in equations and statistical models for detecting suspected blood doping, its application to sports medicine has attracted growing interest in reticulocyte behavior during training and competition seasons in athletes and experimental blood doping treatment in healthy volunteers. An update on recent publications is therefore needed to improve the interpretation of reticulocyte analysis and its variability in sportsmen. Reticulocyte count constitutes a robust parameter during the preanalytical phase, but cell stability can be assured only if blood samples are kept at constantly cold temperatures (4 degrees C) and test results will differ depending on the blood analyzer system used. Marked intraindividual variability is the principal finding to be evaluated when exercise-induced changes are observed or illicit procedures suspected. Furthermore, reticulocyte variability is greater than that of other hematological parameters such as hemoglobin or hematocrit. Ideally, any variation should be interpreted against long-term time series for the individual athlete: values obtained from large athlete cohorts ought to be used only for extrapolating outliers that deserve further examination. Reticulocyte distribution in athletes is similar to that found in the general population, and a gender effect in some sports disciplines or selected athlete groups may be seen. Reticulocyte variability is strongly influenced by seasonal factors linked to training and competition schedules and by the type of sports discipline. Published experimental data have confirmed the high sensitivity of reticulocyte analysis in identifying abnormal bone marrow stimulation by either erythropoietin administration or blood withdrawal and reinfusion.

Comparison of the Hematological Profile of Elite Road Cyclists during the 2010 and 2012 GiroBio Ten-Day Stage Races and Relationships with Final Ranking

Cycling stage races are strenuous endurance events during which exercise-induced variations in hematological parameters are consistently observed. However, specific literature on such changes is scarce and published data have been derived from small samples of athletes. The aims of this study were: (1) to determine the hematological response to middle-term strenuous endurance; and (2) to determine whether a relationship exists between the athlete-specific hematological profile and final placement in a cycling stage race. The study population was male professional cyclists (n = 253) competing in the 2010 (n = 144) and 2012 (n = 109) GiroBio 10-day stage races. Blood draws taken before the start of the race, at mid-race, and at end-race were performed in strict compliance with academic and anti-doping pre-analytical warnings. Blood chemistry included white blood cell, red blood cell, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean hemoglobin content (MCH), mean corpuscular hemoglobin content (MCHC), platelets, and reticulocyte relative and absolute counts. Compared to baseline values, erythrocyte, hemoglobin, hematocrit, MCHC, platelet and reticulocyte counts were all consistently lower at mid-race, but returned to normal by race-end, while leukocytes were increased in the final phase. MCV increased during both events. MCH increased in the first part to then return to baseline in the 2012 race. The calculated OFF-score consistently decreased in the first half of the race before increasing, but remained lower than the baseline value. The trends of variation in hematological parameters were substantially similar in both events. There was an inverse, albeit weak, relationship between placement and erythrocyte, platelet, hemoglobin, hematocrit and OFF-score values in the 2010, but not in the 2012 race. In conclusion, the data confirm that, in this large series of elite road cyclists, the strenuous effort a rider sustains during a stage race induces appreciable changes in the hematological profile.

Plasma Membrane-Associated Glycohydrolases Activation by Extracellular Acidification due to Proton Exchangers

In this paper, we show that the pH optimum for the plasma membrane (PM)-associated activity of four glycohydrolases (conduritol B epoxide sensitive β-glucosidase, β-glucosidase GBA2, β-hexosaminidase and β-galactosidase) measured on intact cells is acidic. Moreover, we show that drugs able to modify the efflux of protons across the PM, thus locally affecting the extracellular proton concentration close to the PM, are able to modulate the activities of these enzymes. These data strongly suggest that pH-dependent modulation of PM-associated glycohydrolases activities could be an effective way to locally modulate the cell surface glycoconjugate composition.