Glucose-6-phosphate dehydrogenase deficiency in Italian blood donors: prevalence and molecular defect characterization.

BACKGROUND: In the countries with high G6PD deficiency prevalence, blood donors are not routinely screened for this genetic defect. G6PD deficiency is often asymptomatic, blood donors may be carriers of the deficiency without being aware of it. The aim of the study was to evaluate the prevalence of G6PD deficiency among the Italian blood donors. DESIGN AND METHODS: From October 2009 to April 2011, 3004 blood donors from a large hospital transfusion centre were screened for G6PD deficiency using differential pH-metry and the characterization of G6PD mutations was performed on G6PD-deficient subjects. The haematological features of G6PD-deficient and normal donors were also compared. RESULTS: Thirty-three subjects (25 men and 8 women) with low G6PD activity were identified, corresponding to 1·1% of the examined blood donor population. The frequencies of class II severe alleles (Mediterranean, Valladolid, Chatham and Cassano) and class III mild alleles (Seattle, A- and Neapolis) were 48% and 43%, respectively. The haematological parameters of G6PD- donors were within normal range; however, the comparison between normal and G6PD- class II donors showed significant differences. CONCLUSION: In Italy, the presence of blood donors with G6PD deficiency is not a rare event and the class II severe variants are frequent. The identification of G6PD-deficient donors and the characterization of the molecular variants would prevent the use of G6PD-deficient RBC units when the haemolytic complications could be relevant especially for high risk patients as premature infants and neonates and patients with sickle cell disease submitted to multiple transfusions.

6-Phosphogluconate dehydrogenase deficiency in an Italian family.

A rare case of hereditary erythrocyte enzymopathy, namely 6-phosphogluconate dehydrogenase (6PGD) deficiency, was found in an Italian family. The activity of the enzyme was reduced to 35% in the propositus and her mother, but was normal in the other three members of the family. The 6PGD deficiency was associated with a variable reticulocyte count and recurrent increased unconjugated bilirubinemia without anemia in the propositus, while no clinical or hematological symptoms were evident in her mother. Increased levels of erythrocyte pyruvate kinase (PK) activity and reduced glutathione (GSH) were observed, indicating a slight decrease in mean red blood cell (RBC) age and an activation of reducing systems. The episodic hemolytic events with jaundice observed in the propositus may be the result of a defective RBC ability to counteract conditions of marked oxidative stress. In this report the importance of 6PGD estimation for a proper analysis of glucose-6-phosphate dehydrogenase (G6PD) deficiency is also highlighted. In fact in the present study, the presence of 6PGD deficiency could be mistaken for a partial G6PD deficiency if the assay of G6PD activity was performed without correcting for 6PGD activity.

[Glucose-6-phosphate dehydrogenase deficiency and hereditary hemolytic anemia]. / Deficit di glucosio-6-fosfato deidrogenasi ed anemia emolitica ereditaria.

G6PD deficiency is the most common enzymopathy in the world. The highest frequency values are found in tropical Africa, in the Middle East, in some areas of the Mediterranean, in tropical and sub-tropical Asia and in Oceania. This genetic defect shows sex linked inheritance and a marked heterogeneity. At least 400 abnormal variants with different biochemical characteristics and about 100 diverse mutations have been identified. In most cases the phenotypic expression is a marked decrease in erythrocyte G6PD activity. The most common clinical consequences are neonatal jaundice and sporadic haemolytic crises caused by a number of drugs, by infections or by ingestion of fava beans. A few cases of chronic non-spherocytic haemolytic anaemia associated with rare molecular variants have been reported. Early diagnosis, education and epidemiologic surveillance have been proved to be cornerstones in the prevention of the haemolytic disease. Therefore they should be taken into account in the national health programmes, especially in the countries with high prevalence rates.

Insulin Receptor Processing and Lipid Composition of Erythrocyte Membrane in Patients with Hyperlipidemia.

The aim of this study was to determine whether the common forms of dyslipidemia could affect either the lipid composition or insulin receptor processing (down-regulation) of erythrocytes. The study included 22 patients with type IIa hypercholesterolemia, 15 patients with type IV hypertriglyceridemia and 12 patients with type IIb hyperlipidemia. Ten normolipidemic subjects were used as controls. Their erythrocyte membranes were analyzed for lipid composition and insulin receptor down-regulation. The results show that all the hyperlipidemias investigated were characterized by significant increases in the cholesterol to phospholipid molar ratio (0.56 +/- 0.08 in controls and 1.11 +/- 0.13, 1.09 +/- 0.14, 1.04 +/- 0.15, p < 0.001, in types IIa, IIb and IV, respectively). Surface insulin receptors of type IIa and IIb patients did not appear to down-regulate when compared to normal subjects, but rather up-regulated (+65.2% in controls, -1.0% and -8.7%, p < 0.001, in type IIa and IIb patients, respectively). Patients with type IV hypertriglyceridemia showed a residual capacity for insulin receptor internalization (10.7% down-regulation). Membranes of all the patients contained a higher proportion of phosphatidylethanolamine; the molar ratio of sphingomyelin to phosphatidylcholine was significantly higher in types IIb than in controls (1.22 +/- 0.11 and 1.12 +/- 0.10, p < 0.05, respectively); all the patients showed a lower content of polyunsaturated fatty acids in the major glycerophospholipid classes. However, type IV hypertriglyceridemics showed less variations, especially in the phosphatidylserine fraction. These results indicate that the alterations in lipoprotein pattern may affect both the lipid membrane equilibria and the processing ability of surface insulin receptors. Copyright 1995 S. Karger AG, Basel

A pilot programme of external quality assessment for general haematology in Italy.

In the years 1984-1989 the Istituto Superiore di Sanità organized an EQAS for haematology (SVEQE) in Italy. A series of trials for haemocytometry, abnormal haemoglobins, HbA2, HbF, red cell G6PD and peripheral blood films, were carried out with the participation of 126 hospital laboratories, in different regions. SVEQE was an educative programme, aiming at promotion of quality assurance (QA) in laboratory haematology. At the same time an attempt was made to survey the analytical methods and instruments and to estimate the "state of the art" by the dispersion of all results. Participant laboratories were not scored for their performances. The operative protocol was harmonized to the guidelines established by WHO and ICSH; the trial specimens were prepared from normal or pathologic blood samples provided by blood banks or hospital departments. The trials for haemocytometry demonstrated a wide use of completely automated analyzers and in a steady state of performance during about five years. CVs, mainly for WBV and PLT, were somewhat higher than in other countries, where national QA systems have been established for a long time. Such discrepancies were not surprising in a pilot programme and were likely to be caused by inadequate internal quality control. The exercises for abnormal haemoglobins, HbA2, HbF and G6PD pointed out the need of using standardized methods according to the recommendations of ICSH. A large number of participating laboratories took part in the trial for blood cell morphology, being convinced of the educative function of this exercise; it is important to continue with systematic surveys, even including rare haematological disorders amongst the selected cases.

A pilot External Quality Assessment Scheme for haemocytometry in Italy.

BACKGROUND: An Italian EQA scheme for haemocytometry, organized by the Istituto Superiore di Sanità, has been active for about five years (1984-1989). The aims of this programme were to evaluate the state of the art and to introduce in Italy a scheme recommended by ICSH. N. 126 public laboratories from different provinces joined voluntarily the programme and trials for haemoglobinometry (A01, A02), full blood count (B01-B08) and platelet count (D01-D03) were performed. METHODS: Materials for testing consisted of blood lysate, preserved blood preparation containing native red cells and pseudoleukocytes, suspension of fixed platelets. The performances of laboratories was evaluated by consensus values (median, mean and standard deviation) and individual deviation index. RESULTS: The instrument survey demonstrated that fully automated systems had the highest frequency. Non Gaussian distributions of results were often obtained and this was particularly true for WBC, PLT and MCV. The overall variability was lower than 5.5% for Hb, RBC and MCH and lower than 9.3% for other erythrocyte parameters; WBC and PLT counts displayed a higher dispersion (CV* = 9.8% and 25.4%); the spreading of results was strongly reduced in the homogeneous group of Coulter counters. In the course of the programme CV*s didn't show any relevant modification, a steady state performance being apparent. As regards the nature of variability, the random component was prevalent for all parameters, with the exception of MCV. CONCLUSIONS: This pilot programme allowed to demonstrate the practicability of a national EQAS for haemocytometry according to the ICSH guidelines. Materials for testing showed acceptable stability, homogeneity and commutability. As regards analytical equipment as well as analytical variability, hospital centers participating in these EQA trials were comparable with laboratories taking part in similar EQAS of other European countries.

Hematotoxic effects in the rat of a toluene dinitro derivative after short-term exposure.

3,5-Dinitro-4-chloro-alpha,alpha,alpha-trifluorotoluene (DNCTT) is an intermediate in the synthesis of dinitroaniline herbicides and was involved in an episode of ground water pollution in 1977. The compound presents a high environmental persistence, which may have possible implications concerning public health. In one experiment male Sprague-Dawley rats were administered DNCTT for 3 days at a dose level of 150 mg/kg body wt by oral gavage. Groups of rats were sacrificed up to 10 days after the end of the administration, at 2-day intervals. Methemoglobin was increased up to Day 7; white blood cells were also increased both in peripheral blood and in bone marrow smears. Spleen relative weights were observed to increase slightly at Days 7 and 10; microscopic examination revealed marked congestion with an increased density of the spleen's white pulp. In a similar scheduled experiment, but at a dose level of 300 mg/kg body wt, the bone marrow white cell series were not affected initially, but were affected after 3 days at the end of the administration. DNCTT has a definite effect on white cells.

Hemoglobin bologna (alpha 2 beta 2 61 (E5) lys replaced by met). An abnormal human hemoglobin with low oxygen affinity.

An abnormal human hemoglobin was found in association with beta-thalassemia in a hemolysate from an 11-year-old healthy child living in Bologna (northern Italy). Structural studies demonstrated a previously unreported amino acid substitution, beta 61 (E5) Lys replaced by Met (this is an external residue). The new variant has been named Hb Bologna, and is characterized by a reduced oxygen affinity. Family studies indicated that the variant had been inherited from the father, a 41-year-old male of Southern Italian origin. Also, a brother of the propositus was found to be an abnormal Hb carrier.

The biosynthesis of hemoglobin G San Josè (beta 7(A4) Glu replaced by Gly).

This report is concerned with the evaluation of hematological parameters and the percentage level of the abnormal hemoglobin (Hb) G San Josè as found in 4 heterozygous carriers from a family of Sicilian origin. Biosynthetic studies and in vitro recombination experiments strongly indicate that abnormal beta chains are synthesized at lower rate than beta A chains and exhibit a minor affinity (relative to beta A chains) for complementary chains in a condition of relative aA chain deficiency. The possibility that the low affinity of beta G chains for a chains may play a decisive role in controlling the level of the abnormal Hb in the peripheral blood of the present non-a-thalassemic abnormal Hb carriers is therefore discussed.

Hemoglobin Legnano (alpha 2 141 (HC3) Arg leads to Leu beta2) a new high oxygen affinity variant. Functional and structural studies.

Hb Legnano (alpha 2 141 (HC3) Arg leads to Leu beta 2) is an abnormal hemoglobin, for which preliminary structural and functional studies demonstrated an amino acid substitution (Arg leads to Leu) in the alpha-C-terminus. This substitution modifies the functional properties observed in whole blood as well as in red blood cells, as reported in this paper. On the basis of its pI, previously determined by analytical isoelectric focusing (IEF), Hb Legnano was purified on a preparative IEF slab. The purified fraction was subjected to functional ultracentrifugal studies under various conditions of pH and salt concentration. The findings are compared with those for Hb Suresnes (alpha 2 141 (HC3) Arg leads His beta2) and Hb-CPB, a normal hemoglobin in which the C-terminal alpha 141 Arg has been cleaved by carboxypeptidase B. Hb Legnano, like the other hemoglobins considered, shows an increased P50, a decreased Hill's "n" values and a decreased Bohr effect that are partially restored in presence of organic phosphates. The presence of inorganic ions decreases the Bohr effect and enhances the dissociation into dimers, as observed in Hb-CPB. The dissociation of hemoglobin in carboxy form in ultracentrifugal studies and the different slope of Hill's "n" value as a function of pH are presumably due to presence of Leu, which probably modifies the stereochemistry of the variant.

Conventional haemodialysis and ultrafiltration: effects on erythrocyte 2,3-diphosphoglycerate and oxygen affinity in patients with uraemia.

1. The relationship between erythrocyte 2,3-diphosphoglycerate (2,3-DPG) and changes in blood pH and in oxygen affinity were studied in six patients treated with two dialysis techniques: conventional haemodialysis and ultrafiltration followed by conventional haemodialysis. 2. The decrease in erythrocyte 2,3-diphosphoglycerate and increase in pH after conventional haemodialysis may significantly increase the affinity of haemoglobin for oxygen and consequently result in inadequate oxygenation of tissue. 3. Ultrafiltration followed by conventional haemodialysis, on the contrary, is more beneficial for oxygenation of tissue and post-dialysis symptoms may be reduced.

[Plant antigens with immunological properties. III. Separation of a biologically active fraction in a grass pollen extract]. / Antigeni vegetali dotati di proprieta' immunologiche. III. Separazione di una frazione biologicamente attiva in un estratto di pollini di graminacee.

In present paper a partial biochemical study concerning two grass pollens (Poa pratensis and Phleum pratense) is carried out. On Sephadex G-75 three fractions were detected by an extract from the grass pollens. Only the third fraction shows a biological activity and biochemical findings are in agreement with the presence of four polipeptidic subfractions of different molecular weights ranged between 10,000 and 14,500. Specific additionals trials (ultracentrifugation, disc-electrophoresis, thyn-layer electrophoresis, isoelectric-focusing, immunological studies) are carried out to confirm the observed nature of prevalent material in pollens.

Thin-layer isoelectric focusing of hemoglobin variants: screening and determination of isoelectric points.

The high resolving power of thin-layer isoelectric focusing was applied for screening some hemoglobin variants classified on the basis of their electrophoretic mobility in: electrophoretically slow variants (as Hb A2), electrophoretically slow variants (as Hb S), electrophorectically fast variants (Hbs type J). An analysis of the variant compounds has been performed, and the corresponding pI values were determined in whole hemolysate.