Percentage of hypochromic erythrocytes and reticulocyte hemoglobin equivalent predictors of response to intravenous iron in hemodialysis patients.

INTRODUCTION: Reticulocyte hemoglobin content and percentage of hypochromic red cells are incorporated into the European best practice guidelines on anemia management in chronic kidney disease. Sysmex XN analyzer (Sysmex Corporation, Kobe, Japan) reports reticulocyte hemoglobin equivalent (Ret-He) and the hypochromic fraction of erythrocytes (%Hypo-He). Our aim was to assess the value of these parameters, in terms of the sensitivity and specificity for detecting functional iron deficiency, in hemodialysis (HD) patients. METHODS: Forty HD patients in the maintenance phase of erythropoietin therapy were included. Intravenous iron supplementation was interrupted at least 3 weeks before recruitment. Two samples were analyzed for each patient: the baseline after the iron-free period and the second sample after 4 weeks of IV iron administration. Hemogram and biochemical parameters of the iron status were measured. Patients were classified as responders or nonresponders to an iron load; responders had an increase in Hb of at least 10 g/L after iron administration, compared to the baseline. To identify the efficiency of the test for predicting the response to iron administration, receiver operating characteristic analysis (ROC) was performed. RESULTS: According to the established criteria, 21 patients were responders and 19 nonresponders. ROC analysis results: Ret-He area under curve (AUC) was 0.84 (95% CI 0.64-0.93), at cutoff 30.8 pg, sensitivity 78.7%, and specificity 87.2%. % Hypo-He AUC was 0.78 (95% CI 0.64-0.91), at cutoff 2.4%, sensitivity 72.2%, and specificity 88.1%. CONCLUSIONS: % Hypo-He and Ret-He are reliable parameters for the study of erythropoiesis status in HD patients.

Differential diagnosis of microcytic anemia: the role of microcytic and hypochromic erythrocytes.

INTRODUCTION: Various indices derived from red blood cell (RBC) parameters have been described for distinguishing thalassemia and iron deficiency. We studied the microcytic to hypochromic RBC ratio as a discriminant index in microcytic anemia and compared it to traditional indices in a learning set and confirmed our findings in a validation set. METHODS: The learning set comprised samples from 371 patients with microcytic anemia mean cell volume (MCV < 80 fL), which were measured on a CELL-DYN Sapphire analyzer and various discriminant functions calculated. Optimal cutoff values were established using ROC analysis. These values were used in the validation set of 338 patients. RESULTS: In the learning set, a microcytic to hypochromic RBC ratio >6.4 was strongly indicative of thalassemia (area under the curve 0.948). Green-King and England-Fraser indices showed comparable area under the ROC curve. However, the microcytic to hypochromic ratio had the highest sensitivity (0.964). In the validation set, 91.1% of microcytic patients were correctly classified using the M/H ratio. CONCLUSIONS: Overall, the microcytic to hypochromic ratio as measured in CELL-DYN Sapphire performed equally well as the Green-King index in identifying thalassemia carriers, but with higher sensitivity, making it a quick and inexpensive screening tool.

Erythrocyte and reticulocyte indices in the assessment of erythropoiesis activity and iron availability.

INTRODUCTION: Consequence of the imbalance between the erythroid marrow iron requirements and the actual supply is a reduction in red cell hemoglobin content, which causes hypochromic mature red cells and reticulocytes. Sysmex XE 5000 analyzer (Sysmex Corporation, Kobe, Japan) reports reticulocyte hemoglobin equivalent (Ret-He) and the percentages of erythrocyte subsets, including the hypochromic fraction (%Hypo-He). We study the value of these parameters of hemoglobinization in the evaluation of erythropoiesis and iron availability. METHODS: Ninety healthy subjects, 85 patients with chronic kidney disease (CKD) and 65 patients on dialysis (HD) receiving therapy and 91 patients with iron deficiency (IDA) were analyzed. Pearson's correlation, t-test for independent, and receiver operating characteristic (ROC) curve analysis were utilized. RESULTS: The results in the IDA group reflected the state of iron depletion (low ferritin), low iron availability (low MCH and high percentage of hypochromic red cells (%Hypo-He)), and iron-restricted erythropoiesis (low Ret-He). In the HD and CKD, the reticulocyte percentage showed the increased erythropoiesis, maintained due to treatment (Ret-He over 30 pg) and good iron availability, MCH within reference range and %Hypo-He slightly increased. The results of ROC curves analysis for the diagnosis of iron deficiency (gold standard sTfR > 21 nm) were as follows: Ret-He area under curve (AUC) 0.935 cutoff 29.8 pg, sensitivity 90.7%, specificity 83.1%. % Hypo-He AUC 0.925 cutoff 3.5%, sensitivity 87.3%, specificity 88.0%. CONCLUSIONS: Percentage of hypochromic red cells and Ret-He provide information about individual cell characteristics, so the hypochromic cells are detected and quantitated improving the evaluation of erythropoiesis and iron status.

Low hemoglobin density potential marker of iron availability.

INTRODUCTION: Low hemoglobin density (LHD%) is a new parameter provided by Beckman-Coulter derived from the mean cell hemoglobin concentration, using the mathematical sigmoid transformation LHD% = 100×√(1-(1/(1 + e(1.8(30-MCHC)))). This study investigated the reliability of LHD% for the assessment of iron status in the presence of inflammation. METHODS: Healthy subjects (n = 90) and patients with iron deficiency (IDA, n = 110), chronic kidney disease (CKD, n = 65) and anemia of chronic disease (ACD, n = 85; 24 were iron deficient and 61 were iron sufficient) were analyzed on a LH 780 analyzer (Beckman Coulter Inc., Miami, FL, USA). Independent samples U test and receiver operating characteristic (ROC) curve analysis were applied. To determine the concordance between LHD% and soluble transferrin receptor (sTrR) Cohen's κ index was calculated. RESULTS: LHD % values showed no statistical difference in patients with IDA and patients with ACD accompanied with IDA (P = 0.6427); LHD% values in these patients were significantly different (P < 0.0001) compared with the iron-sufficient patients with ACD. ROC analysis for LHD% in the detection of iron deficiency showed the following: area under curve 0.903; cut off 5.5%, sensitivity 88.6%, specificity 76.9%; κ index, 0.65. CONCLUSION: LHD% is a reliable parameter for the detection of iron deficiency in patients with anemia in the presence of inflammation.

Analysis of reticulocyte parameters on the Sysmex XE 5000 and LH 750 analyzers in the diagnosis of inefficient erythropoiesis.

The reticulocyte hemoglobin equivalent (Ret He) represents an indirect measure of the functional iron available for erythropoiesis over the previous 2-3 days. Only the analyzers of a single manufacturer, Sysmex (Sysmex Corporation, Kobe, Japan), include Ret He. Red blood cell size factor (RSf) is a new parameter provided by Beckman Coulter, which joins together the volume of the erythrocytes and the volume of reticulocytes. The aims of the study were to investigate the clinical usefulness of RSf in the study of erythropoiesis status and to assess its concordance with Ret He values. Samples from 417 patients were run on both LH 780 (Beckman Coulter) and Sysmex XE 5000 analyzers. Independent samples t-test, Pearson correlation, receiver operating characteristic (ROC) analysis and inter-rater reliability (κ index) were applied. Good correlation between RSf and Ret He was observed, r = 0.8184. Significant differences (P < 0.001) were detected when groups with inefficient erythropoiesis were compared with patients undergoing therapy and healthy subjects. ROC analysis for RSf in the diagnosis of inefficient erythropoiesis, cutoff 91.1 fl, area under curve 0.963, sensitivity 91.7%, specificity 88.5%. Concordance between RSf and Ret He κ = 0.68. RSf and Ret He are suitable parameters for the assessment of erythropoiesis status.

The role of automated measurement of red cell subpopulations on the Sysmex XE 5000 analyzer in the differential diagnosis of microcytic anemia.

Cell counter-based formulae have been used in the differential diagnosis of microcytic anemia. The measurement of new red cell parameters is now available on the Sysmex XE 5000 analyzer. Derived from the percentages of microcytic and hypochromic red cells, the authors describe the new formula %microcytic-%hypochromic, M-H index. The aim of this study was to assess the discriminant value of this new index in the differential diagnosis of microcytic anemia and thalassemia screening compared to the published indices. Receiver operating characteristic curves, sensitivity, specificity and Youden index were calculated for a set of 170 iron-deficiency anemia patients and 200 ß thalassemia carriers. % microcytic-% hypochromic index showed the best area under the curve (area under curve AUC, 0.994) and Youden index (93.9%), among considered indices; 98% sensitivity, 95.9% specificity, at a cutoff value >11.5; Green and King index ranked second (AUC 0.99, Youden index 90.8%). Because of high sensitivity and specificity, the new index %microcytic-%hypochromic was the most reliable index evaluated. M-H index could be a useful tool in the differential diagnosis of microcytic anemia, and samples with M-H>11.5 can be chosen for further analysis to confirm the diagnosis of thalassemia.

Comparison of nine theoretical models for estimating the mechanical power output in cycling.

OBJECTIVE: To assess which of the equations used to estimate mechanical power output for a wide aerobic range of exercise intensities gives the closest value to that measured with the SRM training system. METHODS: Thirty four triathletes and endurance cyclists of both sexes (mean (SD) age 24 (5) years, height 176.3 (6.6) cm, weight 69.4 (7.6) kg and Vo(2)max 61.5 (5.9) ml/kg/min) performed three incremental tests, one in the laboratory and two in the velodrome. The mean mechanical power output measured with the SRM training system in the velodrome tests corresponding to each stage of the tests was compared with the values theoretically estimated using the nine most referenced equations in literature (Whitt (Ergonomics 1971;14:419-24); Di Prampero et al (J Appl Physiol 1979;47:201-6); Whitt and Wilson (Bicycling science. Cambridge: MIT Press, 1982); Kyle (Racing with the sun. Philadelphia: Society of Automotive Engineers, 1991:43-50); Menard (First International Congress on Science and Cycling Skills, Malaga, 1992); Olds et al (J Appl Physiol 1995;78:1596-611; J Appl Physiol 1993;75:730-7); Broker (USOC Sport Science and Technology Report 1-24, 1994); Candau et al (Med Sci Sports Exerc 1999;31:1441-7)). This comparison was made using the mean squared error of prediction, the systematic error and the random error. RESULTS: The equations of Candau et al, Di Prampero et al, Olds et al (J Appl Physiol 1993;75:730-7) and Whitt gave a moderate mean squared error of prediction (12.7%, 21.6%, 13.2% and 16.5%, respectively) and a low random error (0.5%, 0.6%, 0.7% and 0.8%, respectively). CONCLUSIONS: The equations of Candau et al and Di Prampero et al give the best estimate of mechanical power output when compared with measurements obtained with the SRM training system.

Maximal lipidic power in high competitive level triathletes and cyclists.

OBJECTIVE: To describe the fat-oxidation rate in triathlon and different modalities of endurance cycling. METHODS: 34 endurance athletes (15 male triathletes, 4 female triathletes, 11 road cyclists and 4 male mountain bikers) underwent a progressive cycloergometer test until exhaustion. Relative work intensity (VO(2max)), minimal lactate concentration (La(-)(min)), lactic threshold, individual lactic threshold (ILT), maximal fat-oxidation rate (Fat(max), Fat(max) zone) and minimal fat-oxidation rate (Fat(min)) were determined in each of the groups and were compared by means of one-way analysis of variance. RESULTS: No significant differences were found for Fat(max), Fat(min) or for the Fat(max) zone expressed as fat oxidation rate (g/min). Intensities -20%, -10% and -5% Fat(max) were significantly lower for mountain bikers with respect to road cyclists and female triathletes, expressed as % VO(2max). Intensities 20%, 10% and 5% Fat(max) were significantly lower for mountain bikers with respect to male triathletes and female triathletes, and for male triathletes in comparison with female triathletes, expressed as % VO(2max). Lactic threshold and La(-)(min) did not show significant differences with respect to Fat(max). Lactic threshold was found at the same VO(2max) with respect to the higher part of the Fat(max) zone, and La(-)(min) at the same VO(2max) with respect to the lower part of the Fat(max) zone. CONCLUSIONS: The VO(2max) of Fat(max) and the Fat(max) zone may explain the different endurance adaptations of the athletes according to their sporting discipline. Lactic threshold and La(-)(min) were found at different relative work intensities with respect to those of Fat(max) even though they belonged to the Fat(max) zone.

Validation of a field test to determine the maximal aerobic power in triathletes and endurance cyclists.

OBJECTIVE: To validate a field test to assess the maximal and submaximal exercise aerobic adaptation under specific conditions, for endurance modality cyclists and triathletes. METHODS: 30 male and 4 female endurance modality cyclists and triathletes, with heterogeneous performance levels, performed three incremental tests: one in the laboratory and two in the field. Assessment of the validity of the field protocol was carried out by the Student's t test, intraclass correlation coefficient (ICC) and coefficient of variation (CV) of the maximal variables (maximal aerobic speed (MAS), maximal aerobic power (MAP), maximal heart rate (HR(max)), maximal blood lactate concentration ([La(-)](max)) and maximal oxygen uptake (VO(2max))) and submaximal variables (heart rate, HR) measured in each one of the tests. The errors in measurement were calculated. The repeatability of the field tests was assessed by means of the test-retest of the two field tests, and the validity by means of the test-retest of the laboratory test with respect to the mean of the two field tests. RESULTS: No significant differences were found between the two field tests for any of the variables studied, but differences did exist for some variables between the laboratory tests with respect to the field tests (MAP, [La(-)](max), humidity (H), barometric pressure (Pb) and some characteristics of the protocols). The ICC of all the variables was high and the CV for the MAP was small. Furthermore, the measurement errors were small and therefore, assumable. CONCLUSIONS: The incremental protocol of the proposed field test turned out to be valid to assess the maximal and submaximal aerobic adaptation.

Effects of trace elements on membrane fluidity.

According to the Fluid Mosaic Model, a biological membrane is a two-dimensional fluid of oriented proteins and lipids. The lipid bilayer is the basic structure of all cell and organelle membranes. Cell membranes are dynamic, fluid structures, and most of their molecules are able to move in the plane of the membrane. Fluidity is the quality of ease of movement and represents the reciprocal value of membrane viscosity. Fluid properties of biological membranes are essential for numerous cell functions. Even slight changes in membrane fluidity may cause aberrant function and pathological processes. Several evidences suggest that trace elements, e.g., iron, copper, zinc, selenium, chromium, cadmium, mercury and lead may influence membrane fluidity. The interaction of heavy metals with cellular membranes may contribute to explain, at least partially, the toxicity associated with these metals.

[Intracellular pH changes induced by sclerosing agents in peritoneal fibroblasts]. / Alteraciones del pH intracelular en fibroblastos peritoneales inducida por factores implicados en la esclerosis peritoneal.

UNLABELLED: Sclerosing encapsulating peritonitis secondary to peritoneal dialysis has been associated to acetate-containing dialysis fluids, hypertonic glucose and disinfectants. Physiopathologic mechanisms of fibrotic proliferation that implicate those agents are not totally explained. With an experimental approach using cultured peritoneal fibroblasts, we have studied intracellular pH changes and Na+/H+ antiporter activity under cells perfusion with peritoneal dialysis liquids containing acetate, lactate, hypertonic glucose and interleukin-1. All experiments were performed at extracellular pH 7.4 and physiologic HCO3/CO2 concentration. RESULTS: 35 mM acetate produced a huge intracellular acidosis (ipH = 6.80 +/- 0.08). Lactate effect was less important (6.95 +/- 0.07), with a slow ipH recovery in about 30 min in both cases. IL-1, 10(-6) M also reduced ipH to 7.10 +/- 0.03. Acidosis was linked to Ca2+ outflow via Ca/H exchange and was blocked with Cd 20 nM. Extracellular Na = 0 and amiloride totally inhibited ipH recovery after acetate, lactate, or interleukin-induced acidosis. Hypertonic glucose perfusion increased ipH (7.31 +/- 0.06) for 5-7 min. This increase was also inhibited by amiloride or extracellular Na absence. Na+/H+ exchanger activity increased to 58%, and kept activated after ipH recovery. In conclusion, acetate, hypertonic glucose and IL-1 showed the common effect of stimulating the sodium-proton exchanger by different mechanisms, giving a possibility of potentiation. Activated Na+/H+ exchanger may act as a signal-transduction increasing fibroblast proliferation and explaining the cellular mechanism of sclerosing peritonitis.

Zinc uptake by human erythrocytes with and without serum albumins in the medium.

Different mechanisms of Zn uptake are present in mammalian cells. The variations in the Zn uptake by human erythrocytes in the absence and presence of albumins, bovine and human, as well as the differences of Zn uptake with and without 4-4'-diidothiocyanatostilbene-2,2'-disulfonic acid have been analyzed in this study. The results show a significantly greater rate of Zn uptake in the absence rather than in the presence of albumins in the extracellular medium and being significantly greater with bovine than with human serum albumin when the experiments were performed in media with equimolar concentrations of Zn. However, when comparing Zn uptake in a medium without albumin with similar free-Zn concentration to Zn ultrafiltrable (20%) of other one with albumin, a significantly greater Zn uptake on the latter was observed. The DIDS inhibition on Zn uptake is higher if the albumin is also present in the medium. These results suggest that in Zn uptake by erythrocytes the albumin directly or indirectly would be involved, facilitating the well-known processes of passive transport and anionic exchanger.

Longitudinal study of serum copper and zinc levels and their distribution in blood proteins after acute myocardial infarction.

The levels and distribution of serum Cu and Zn were studied in patients diagnosed with acute myocardial infarction from the day of admission to the Cardiovascular Intensive Care Unit until the 10th day following the attack. The results obtained show that Cu increases significantly (p < 0.01) after the 5th day after the acute myocardial infarction, while Zn decreases significantly (p < 0.01) with respect to the control group from the first day on, with the lowest values being found on the 3rd day after the attack. Further, total serum Cu showed an excellent correlation with the albumin-bound and globulin-bound Cu (ceruloplasmin), as well as with the concentrations of both serum protein fractions. In contrast, total serum Zn only presents this correlation with Zn bound to albumin, but not with Zn bound to globulin nor with the albumin concentration. These findings suggest the existence of some type of relationship between the two fractions of the element bound to protein. This relationship is probably different for both metals.

Development of a microparticle-enhanced nephelometric immunoassay for quantitation of human lysozyme in pleural effusion and plasma.

A microparticle-enhanced nephelometric immunoassay, based on polystyrene beads coated with antihuman lysozyme antibody, has been developed for lysozyme quantification in sera and pleural effusions. The standard curve extends from 0.58 mg/l to 18.75 mg/l and no antigen effect was observed. The results showed a good serial precision. The intra-assay precision (n = 20) expressed as CV was between 2.2 and 4.2 in three different concentrations. The inter-assay precision, with different calibration curves (n = 12) was between 6.4 and 7.1. The analytical assay showed a sufficient linearity (r > 0.999). There were no interferences either with haemoglobin (up to 4 g/l), lipids (up to 0.5%, expressed as 1% Lipofundina content), or bilirubin (up to 5 mg/dl). The analytical sensitivity was lower than 0.6 mg/l. The correlation with a Micrococcus lysodeikticus turbidimetric assay showed a correlation coefficient of 0.915. We have studied 92 patients with pleural effusion. In each case, pleural fluid adenosine deaminase activity and pleural fluid to plasma lysozyme ratio were determined. The lysozyme ratio showed similar clinical sensitivity and specificity as to adenosine deaminase.

Antioxidant intracellular activity of genistein and equol.

Antioxidant effects of isoflavonoids have recently been described. To learn whether the isoflavonoids genistein and equol have actions on the intracellular free radicals, human neutrophils and J774 monocyte-macrophage cell line were used to measure the intracellular production of O(2) (superoxide anion) and H(2)O(2) (hydrogen peroxide) by flow cytometry. The results shown significatives decrease in O(2) and H(2)O(2) production after 1 hour of incubation with equol and genistein. The phagcytic oxidant production decreased owing to the effects of both isoflavonoids in a concentration-dependent manner.

Iron stores in professional athletes throughout the sports season.

Sports anemia provoked by iron deficiency develops slowly and although the existence or reality of this condition is under discussion, the ferritin levels tend to reduce with sports practice. This paper analyzes the variations of iron metabolism, including the organism stores of this metal throughout a sports season in a group of professional sportsmen (soccer players) belonging to a team of the Spanish First Division. For the determination of the iron stores, a computer program has been developed that takes into account the ferritin concentrations, hemoglobin, and saturation of transferrin. The results show that at the end of the season, when the ferric supplementation, which had been performed at the other moments of analysis, was no longer administered the iron stores reduced significantly, as well as the serum ferritin concentration, without such decreases being considered as a prelatent (grade I) anemia.

Iron nutritional status in female karatekas, handball and basketball players, and runners.

The iron nutritional status was studied in 84 sportswomen (19 karatekas, 20 handball players, 20 basketball players, and 25 middle and long distance runners) and in 82 nonathletic females of similar characteristics (control group). After a 7-day nutritional survey by means of the food weighing method, it was found that iron intake was significantly higher in the handball players (p < 0.05), basketball players (p < 0.01), and runners (p < 0.01) with regard to the control group; the basketball players were the only ones to cover the recommended minimum intake (15 mg/day). The heme iron intake was significantly greater in the handball and basketball players (p < 0.01), who, together with the runners, reached the value of 1.5 mg/day, which is considered to be optimal. In relation to the control group, the karatekas and handball and basketball players had lower levels of serum ferritin, although their iron intake was greater, whereas the runners had higher values that were very similar to those of the control group, due to the iron supplementation they had received. Despite finding a marked prevalence of inadequate iron intake, both in the sportswomen and in the control group, the manifest cases of anemia are relatively scarce. The organic iron stores do not seem to depend exclusively on the iron intake but also on intimate mechanisms of intestinal absorption and diverse causes of iron loss.

Short- and long-term effects of calcitonin and calcium administration on blood calcium, magnesium and inorganic phosphorus in post menopausal osteoporosis.

This paper studies the effects of the administration of calcitonin (CT) and Ca on post menopausal osteoporosis, immediately (short-term) and after three months (long-term) of treatment, on total and ionic calcium (Ca), magnesium (Mg), inorganic phosphorus (Pi), calcitonin (CT) and parathyroid hormone (PTH) in plasma. The short-term results show a decrease in total and ionic Ca and Pi four hours after the beginning of the treatment; at seven hours, only Pi varies. A decrease in the total and ionic Ca was observed after three months of CT treatment (long-term effects). No hormonal (PTH and CT) variations were found either in the short or the long-term. However, the PTH/CT ratio decreased significantly during the experiment and this may be an important factor in explaining the long-term Ca variations.

Effects of magnesium, sodium, calcium or potassium intakes on magnesium content in rat skeletal muscle.

Magnesium skeletal (myocardium and gastrocnemius) content has been studied in rats supplemented with 30 mM dissolutions of Mg2+, Na+, Ca2+ and K+ in drinking water, for 7 or 30 days. In both muscles, the ingestion of Mg2+ for 30 days increased Mg2+ content, while Ca2+ and K+ supplementation caused a significant drop. The increase in Na+ ingestion reduced Mg2+ content in gastrocnemius. There were no significant differences between control and animals supplemented for 7 days. These results suggest that, in the case of supplementation situations, the control mechanisms of the Mg2+ tissular content have a lower gain than those of Na+ and K+ of one order of magnitude.