Reticulocyte and erythrocyte hypochromia markers in detection of iron deficiency in adolescent female athletes.

The aim of this study was to analyse the effectiveness of new haematology parameters related to reticulocytes and mature red blood cells to differentiate pre latent and latent iron deficiency. The study included 219 female athletes (aged 15-20 years) representing volleyball, handball, cycling, canoeing, cross-country skiing, swimming and judo. To assess iron status the concentration of ferritin, soluble transferrin receptor (sTfR), iron and total iron binding capacity (TIBC) were determined in serum. In addition to blood morphology, the mean cellular haemoglobin content in erythrocytes (CH) and reticulocytes (CHr), mean cellular haemoglobin concentration in reticulocytes (CHCMr), the percentage of erythrocytes (HYPOm) and reticulocytes (HYPOr) with decreased cellular haemoglobin concentration, the percentage of erythrocytes (LowCHm) and reticulocytes (LowCHr) with decreased cellular haemoglobin content, and percentage of erythrocytes with decreased volume (MICROm) were determined. Subjects with ferritin <30 ng/ml were classified as having stage I (pre-latent) iron deficiency (ID). The second stage (latent ID) was diagnosed when low ferritin was accompanied by elevated sTfR and/or elevated TIBC values. The frequency of ID (without anaemia symptoms) was high, amounting to 60% (stage I in 45%, stage II in 15% of subjects). In subjects with stage I ID significant changes in haematological variables concerned mainly reticulocytes: CHCMr (p<.001), CHr (p<.05), LowCHr (p<.05), HYPOr (p<.001) in comparison to normal iron stores. In athletes with latent ID, there were also significant changes (p<.001) in many indices of mature red blood cells, i.e. haemoglobin concentration (Hb), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), CH, %LowCHm, as well as %MICROm (p<.01) in relation to the group without iron deficiency. The main finding of this study was that the diminished or exhausted iron stores had already caused changes in reticulocytes, and intensified iron deficiency (stage II) increased changes in both reticulocytes' and erythrocytes' hypochromia indices, while microcythaemia symptoms appeared later. This suggests that the markers of hypochromia relating especially to reticulocytes are useful for diagnosis of early ID in athletes with absence of an acute phase reaction.

Total bilirubin in athletes, determination of reference range.

The purpose of this study was to determine a typical reference range for the population of athletes. Results of blood tests of 339 athletes (82 women and 257 men, aged 18-37 years) were retrospectively analysed. The subjects were representatives of different sports disciplines. The measurements of total bilirubin (BIT), iron (Fe), alkaline phosphatase (ALP), alanine aminotransferase (ALT) and gamma-glutamyltransferase (GGT) were made using a Pentra 400 biochemical analyser (Horiba, France). Red blood cell count (RBC), reticulocyte count and haemoglobin concentration measurements were made using an Advia 120 haematology analyser (Siemens, Germany). In groups of women and men the percentage of elevated results were similar at 18%. Most results of total bilirubin in both sexes were in the range 7-14 µmol·L-1 (49% of women and 42% of men). The highest results of elevated levels of BIT were in the range 21-28 µmol·L-1 (12% of women and 11% of men). There was a significant correlation between serum iron and BIT concentration in female and male athletes whose serum total bilirubin concentration does not exceed the upper limit of the reference range. Elevated concentrations of total bilirubin appear to be due to changes caused by regular exercise. The obtained upper limit of the reference range for total bilirubin concentration in the group of athletes is 29.0 µmol·L-1. It seems reasonable to use dedicated reference values for total bilirubin concentration in relation to the group of athletes.

THE COMBINATION OF α-LIPOIC ACID INTAKE WITH ECCENTRIC EXERCISE MODULATES ERYTHROPOIETIN RELEASE.

The generation of reactive nitrogen/oxygen species (RN/OS) represents an important mechanism in erythropoietin (EPO) expression and skeletal muscle adaptation to physical and metabolic stress. RN/OS generation can be modulated by intense exercise and nutrition supplements such as α-lipoic acid, which demonstrates both anti- and pro-oxidative action. The study was designed to show the changes in the haematological response through the combination of α-lipoic acid intake with running eccentric exercise. Sixteen healthy young males participated in the randomised and placebo-controlled study. The exercise trial involved a 90-min run followed by a 15-min eccentric phase at 65% VO2max (-10% gradient). It significantly increased serum concentrations of nitric oxide (NO), hydrogen peroxide (H2O2) and pro-oxidative products such as 8-isoprostanes (8-iso), lipid peroxides (LPO) and protein carbonyls (PC). α-Lipoic acid intake (Thiogamma: 1200 mg daily for 10 days prior to exercise) resulted in a 2-fold elevation of serum H2O2 concentration before exercise, but it prevented the generation of NO, 8-iso, LPO and PC at 20 min, 24 h, and 48 h after exercise. α-Lipoic acid also elevated serum EPO level, which highly correlated with NO/H2O2 ratio (r = 0.718, P < 0.01). Serum total creatine kinase (CK) activity, as a marker of muscle damage, reached a peak at 24 h after exercise (placebo 732 ± 207 IU · L(-1), α-lipoic acid 481 ± 103 IU · L(-1)), and correlated with EPO (r = 0.478, P < 0.01) in the α-lipoic acid group. In conclusion, the intake of high α-lipoic acid modulates RN/OS generation, enhances EPO release and reduces muscle damage after running eccentric exercise.

[New reticulocyte indices and their utility in hematologic diagnosis]. / Nowe wskazniki retikulocytarne i ich wykorzystanie w diagnostyce hematologicznej.

Reticulocyte count in manual method has been the assay traditionally used to evaluate the status of erythropoiesis in haematological disorders with disturbances in erythropoietic activity. But it is a qualitative rather than a quantitative test due to the high variability in manual microscopic method. Automated reticulocyte counting based on flow cytometry has provided much objective and exactly measure of percentage and absolute number of reticulocytes than microscopic method. Besides these two traditional parameters automatic reticulocyte counters can detect differences in the amounts of cellular RNA present in red blood cells which reflects their maturational stages. There are three related parameters which describes the reticulocyte maturation: RMI (reticulocyte maturity index), HFR (high fluorescence reticulocytes), IRF (immature reticulocyte fraction). These parameters can be used as the earliest signs of marrow engraftment after autologous or allogeneic bone marrow transplantations. Reticulocyte cellular indices such as MCVr (mean reticulocyte volume), CHr (reticulocyte hemoglobin content), CHCMr (reticulocyte hemoglobin concentration) entered recently in few automated reticulocyte counters allows for assessment of the functional state of the erythropoiesis in the diagnosis and monitoring of the iron deficiency and rhEpo therapy. Although there is no agreement between various methods of staining and counting of the reticulocytes and lack of standardization materials for this assay automated reticulocyte counting and new reticulocyte parameters probably will improve the diagnosis and monitoring of many haematological diseases.

[Hematologic parameters in the cord blood labor complicated by meconium-stained amniotic fluid]. / Parametry hematologiczne krwi pepowinowej w ciazy powiklanej obecnoscia smólki w plynie owodniowym.

The aim of our study was to compare hematological values of the cord blood in the presence and absence of meconium-stained amniotic fluid (MSAF). MSAF was associated with decreased total platelet count, increased values of platelet distribution width and red blood cell distribution width. The mean nucleated red blood cells was significantly higher in meconium group. The values of red blood cells, white blood cells, reticulocytes counts and their fractions (HFR,MFR,LFR,IFR) were no different in cord blood of neonate exposed to meconium and in those who were not. These findings can suggest that MSAF contribute to fetal infection rather than hypoxia.

[Automatic analysis of reticulocytes--new diagnostic possibilities in hematology]. / Automatyczna analiza retykulocytów--nowe mozliwosci diagnostyki w hematologii.

This review describes a new method of reticulocyte analysis based on flow cytometry, better than manual reticulocyte counting. Reticulocyte percentage, absolute number, indices of maturity (RMI--reticulocyte maturity index, IRF--immature reticulocyte fraction) and others qualitative parameters express the erythropoietic activity of kidneys and bone marrow. The new proposed parameters can improve the diagnosis of anemias and others haematological disorders like leukemia, myelodysplastic syndrome. They also provide exactly and early information about the status of bone marrow after ablative chemotherapy and transplantation. Automated reticulocyte counting can create new potentials for haematology diagnosis.

Peripheral blood reticulocytes and their reference range values for percentage, absolute count, and immature fraction in children, measured with flow cytometry.

Reticulocyte count by manual method has been the assay traditionally used to evaluate the status of erythropoiesis in hematological disorders with disturbances in erythropoietic activity. However, due to its variability, it is rather a semiquantitative method. Automated reticulocyte counting based on flow cytometry has provided more objective and exact measure of reticulocytes. Besides traditional parameters, such as percentage and absolute number of reticulocytes, automatic reticulocyte counters can detect differences in the amounts of cellular RNA present in immature erythrocytes that reflect their maturational stages and evaluate indices of reticulocyte maturation such as RMI, HFR, IRF. These parameters can be used as the earliest signs of marrow engraftment after autologous or allogeneic bone marrow transplantation. Currently there is no strict agreement between various automated methods of reticulocyte evaluation. Additionally, lack of standardization and quality control materials for this assay compels determination of own, interlaboratory ranges of reference values for new proposed parameters. A group of 102 children aged from 3 months to 18 years with normal hematological parameters was examined. Samples of blood stained supravitally with thiazole orange were analyzed in a flow cytometer. Results for percentage, absolute number and immature reticulocyte fraction expressed as a mean +/- 2SD were: 2.00 +/- 1.56%, 88.8 +/- 68.94 x 10(3)/microliter, and 0.22 +/- 0.16, respectively. A poor correlation was found between IRF and other parameters, suggesting its independent role as a marker of erythropoietic activity. Automated reticulocyte counting will probably improve the diagnosis and monitoring of many hematological diseases.

[Immune response and diagnostic aspects during Toxoplasma gondii infection]. / Aspekty odpowiedzi immunologicznej w toksoplazmozie oraz jej implikacje diagnostyczne.

Toxoplasmosis is a common and generally benign disease in immunocompetent persons caused by Toxoplasma gondii, which is an intestinal coccidian parasite of felines. Diagnosis of toxoplasmosis is mainly based on the results of serological tests detecting anti-T. gondii specific antibodies, but T lymphocytes and cytokines they produce play a crucial role in determining the outcome of parasitic infection in terms of both protective immunity and immunopathology.

Gammadelta T-cells alterations in the peripheral blood of high risk diabetes type 1 subjects with subclinical pancreatic B-cells impairment.

There is increasing evidence that CD3 + cells bearing gammadelta T-cell receptor (represent the minor subpopulation of the T-cells in the peripheral blood in humans) are involved in autoimmunity development. Gammadelta T-cell receptor (TCR)+ /CD8+ T-cells have been recently found to play a critical role in the pathogenesis and prevention of autoimmune diabetes in the animal model. The aim of the present study was the estimation the gammadelta T-cell subpopulation levels in the peripheral blood of subjects with preclinical and overt type 1 diabetes and their possible associations with the humoral immunity, metabolic parameters and pancreatic B-cells function. The study was carried out in three groups of subjects: 26 first degree relatives of type 1 diabetes patients (prediabetics) with the combinations of autoantibodies against pancreatic B-cells (ICA, GADA, IA-2A, IAA), 22 patients with a recent onset of type 1 diabetes and age and sex-matched 24 healthy volunteers (control group). A decrease was observed in the absolute numbers and percentages of gammadelta+ /CD8+ and gammadelta+ /CD8- T-cell subpopulations in peripheral blood in the prediabetics with the impaired first phase of insulin secretion in comparison to relatives with autoantibodies but still with normal B-cells function, patients with clinical diabetes and healthy controls. In conclusion, the study suggests that the gammadelta T-cells play an important role in the development of insulin-dependent diabetes mellitus (IDDM). It is possible that their levels in the peripheral blood could be an additional marker of preclinical detection of the disease, but further prospective studies in high risk of IDDM subjects are needed.

Analysis of recently activated, memory and naive lymphocyte T subsets in the peripheral blood of patients with Graves' disease and insulin-dependent diabetes mellitus.

It has been recently suggested that the expression of two different isofoms of tyrosine phosphatase antigen (CD45RA and CD45RO) could differentiate T cells into autoreactive and immunoregulatory subsets, which play a crucial role in the autoimmunity process. The aim of the present study was to evaluate the differences between the distribution of memory, naive and recently activated T cells (co-expressing both CD45RA and CD45RO antigens) in the peripheral blood of patients with newly diagnosed Graves' disease and insulin-dependent diabetes mellitus in comparison to healthy controls. The study was carried out in 3 groups: 18 patients with Graves' disease, 16 subjects with a recent onset of type 1 diabetes mellitus and 16 healthy, age and sex matched volunteers. At the onset of both autoimmune diseases the percentage of naive CD+ cells were lower than in the control group and in patients with Graves' disease treated with methimazole. The analysis of CD8+ lymphocyte subsets in the peripheral blood revealed higher levels of CD8+CD45RO+ cells in the newly diagnosed Graves' disease in comparison to the controls, and significant decline in the percentage of memory CD8+ and CD8+CD45RA+CD45RO+ lymphocytes after thyreostatic treatment. The number of CD4+ T lymphocytes co-expressing CD45RA and CD45RO antigens were statistically higher in the patients with recently diagnosed insulin-dependent diabetes mellitus. The abnormal distribution of naive, memory and "transient" T cell subsets in the peripheral blood at the onset of Graves' disease and diabetes type 1 suggest their role in the development of autoimmunity. The significant alterations of lymphocyte T subsets in the peripheral blood of patients with Grave's disease after thyreostatic therapy in comparison to the newly diagnosed subjects supports the immunomodulatory effect of methimazol treatment.

[Activation of the antibacterial properties and neutrophil myeloperoxidase and acid phosphatase in patients with unstable angina pectoris]. / Pobudzenie wlasciwosci bakteriobójczych oraz aktywacja mieloperoksydazy i kwasnej fosfatazy w granulocytach obojetnochlonnych u chorych na niestabilna dlawice piersiowa.

Neutrophils are very important in pathogenesis of ischemic disease. They take part in the biomorphology of thrombus and also in the damage of myocardium ischemia in a course of unstable angina pectoris. We evaluated the functional status of neutrophils in peripheral blood, by measurement of bactericidal activity and activity of granulocyte's enzymes: myeloperoxidase (MPO) and acid phosphatase in the patients with unstable angina pectoris. We studied a group of 43 people at the age from 34 to 74 years. The blood for investigation was obtained during the first five hours from the moment of hospitalization. The control group were 40 healthy people. The number of granulocytes was significantly higher in patients with unstable angina pectoris and granulocytes were metabolically activated which was shown in the bigger activity of granulocyte's enzymes like MPO and acid phosphatase than in the control group. The activation of neutrophils is developed by many factors in the course of unstable angina pectoris. They take part in the processes of thrombogenesis and thrombolysis and they are a very important origin for active oxygen metabolites, which are responsible for damage of myocardium ischemia.

[Participation of neutrophils in pathophysiology of unstable angina pectoris]. / Udzial granulocytów w patofizjologii niestabilnej dlawicy piersiowej.

A role of neutrophils in pathophysiology of ischemic heart disease is described to show some destructive actions and eventual clinical complications. Immunobiochemical reactions are presented to describe mechanisms of inflammation and ischemia.

Rhizobium leguminosarum CFN42 genetic regions encoding lipopolysaccharide structures essential for complete nodule development on bean plants.

Eight symbiotic mutants defective in lipopolysaccharide (LPS) synthesis were isolated from Rhizobium leguminosarum biovar phaseoli CFN42. These eight strains elicited small white nodules lacking infected cells when inoculated onto bean plants. The mutants had undetectable or greatly diminished amounts of the complete LPS (LPS I), whereas amounts of an LPS lacking the O antigen (LPS II) greatly increased. Apparent LPS bands that migrated between LPS I and LPS II on sodium dodecyl sulfate-polyacrylamide gels were detected in extracts of some of the mutants. The mutant strains were complemented to wild-type LPS I content and antigenicity by DNA from a cosmid library of the wild-type genome. Most of the mutations were clustered in two genetic regions; one mutation was located in a third region. Strains complemented by DNA from two of these regions produced healthy nitrogen-fixing nodules. Strains complemented to wild-type LPS content by the other genetic region induced nodules that exhibited little or no nitrogenase activity, although nodule development was obviously enhanced by the presence of this DNA. The results support the idea that complete LPS structures, in normal amounts, are necessary for infection thread development in bean plants.

Alteration of bacterial DNA structure, gene expression, and plasmid encoded antibiotic resistance following exposure to enoxacin.

Enoxacin inhibits growth of Escherichia coli K12 strains primarily by binding to the GyrA subunit of DNA gyrase (topoisomerase II); strains with gyrA, but not gyrB, mutations are less susceptible to the bactericidal effects of this agent. In sensitive strains, enoxacin completely inhibits DNA synthesis within 5 min and produces drug-gyrase-DNA complexes at numerous sites throughout the E. coli chromosome, as shown by the formation of linear DNA molecules after detergent treatment. Enoxacin, even at subminimal inhibitory concentrations, induces the bacterial SOS system, even in partially resistant gyrA strains. This drug also inhibits the induced expression of the lacZ encoded beta-galactosidase, regardless of whether this gene is located on the chromosome, a low copy number F' plasmid or high copy number Col E1 related plasmids. This inhibition of gene expression at subminimal inhibitory concentrations is likely to be a factor, in addition to gyrase inhibition, in the elimination of Col E1 plasmids and to the reduction in R plasmid conjugal transfer. Enoxacin enhances the bactericidal effects of kanamycin in both in-vitro and in-vivo models, suggesting that this quinolone may be effective in the treatment of infections due to strains resistant to antibacterials as a consequence of plasmid encoded resistance determinants.

Characterization of the lipopolysaccharide from a Rhizobium phaseoli mutant that is defective in infection thread development.

The lipopolysaccharide (LPS) from a Rhizobium phaseoli mutant, CE109, was isolated and compared with that of its wild-type parent, CE3. A previous report has shown that the mutant is defective in infection thread development, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it has an altered LPS (K. D. Noel, K. A. VandenBosch, and B. Kulpaca, J. Bacteriol. 168:1392-1462, 1986). Mild acid hydrolysis of the CE3 LPS released a polysaccharide and an oligosaccharide, PS1 and PS2, respectively. Mild acid hydrolysis of CE109 LPS released only an oligosaccharide. Chemical and immunochemical analyses showed that CE3-PS1 is the antigenic O chain of this strain and that CE109 LPS does not contain any of the major sugar components of CE3-PS1. CE109 oligosaccharide was identical in composition to CE3-PS2. The lipid A's from both strains were very similar in composition, with only minor quantitative variations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of CE3 and CE109 LPSs showed that CE3 LPS separated into two bands, LPS I and LPS II, while CE109 had two bands which migrated to positions similar to that of LPS II. Immunoblotting with anti-CE3 antiserum showed that LPS I contains the antigenic O chain of CE3, PS1. Anti-CE109 antiserum interacted strongly with both CE109 LPS bands and CE3 LPS II and interacted weakly with CE3 LPS I. Mild-acid hydrolysis of CE3 LPS I, extracted from the polyacrylamide gel, showed that it contained both PS1 and PS2. The results in this report showed that CE109 LPS consists of only the lipid A core and is missing the antigenic O chain.