Spermatogenesis in young adult patients with beta-thalassaemia major long-term treated with desferrioxamine.

Since the introduction of hypertransfusion and intensive iron chelation therapy, patients with homozygous beta-thalassaemia major (TM) achieve adulthood. Many patients grow and develop normal hoping for marriage and to have a family. Therefore the question of fertility potential in this adult group of TM patients has become paramount. We report the semen parameters, the endocrine functions and serum zinc levels in 12 young adult TM patients. Their mean age was 24.8 years. Six patients (50%) had a normal sperm count, motility and morphology. While the remaining patients had oligospermia (sperm concentration <20 x 10(6)/ml) and/or asthenospermia (motility <40%). Basal serum gonadotrophins [LH and FSH], total and free testosterone and serum zinc did not differ significantly from those found in 13 normal adults with comparable testicular size. At the time of the study serum ferritin levels ranged from 240 to 3055 ng/ml (mean 1139 ng/ml). No correlations were found between semen parameters, serum total and free testosterone, plasma zinc, serum ferritin and seminal parameters. Nevertheless we observed that serum ferritin levels were lower (mean 543 ng/ml) in TM patients with abnormal seminal parameters (count and motility) compared to TM patients with normal seminal parameters (mean serum ferritin 1276 ng/ml; p<0.01). In conclusion, impairment of semen parameters may be a negative effect of intensive chelation therapy. Clearly, further investigations are required to evaluate if these adverse effects can be reduced or prevented, and if the existing spermatogenesis damage is reversible.

A rare cause of heart failure in iron-overload thalassaemic patients-primary hypoparathyroidism.

Hypocalcemia due to hypoparathyroidism (HPT) is a late complication of iron-overloaded patients with b-thalassaemia major (TM). The majority of patients have mild disease with parasthesias, while in the more severe form tetany, seizures or cardiac failure may occur. In the last 20 years we observed heart failure in 2 out of 38 (5.2%) TM patients (aged 18 and 22 years) with hypocalcemia secondary to HPT associated to iron overload. Calcium supplementation and vitamin D induced correction of hypocalcemia and resulted in an improvement of cardiac function. Calcium plays a key role in the maintenance and regulation of normal cardiac function. Extra-cellular calcium is indispensable for the contractile process since the sarcoplasmatic reticulum is unable to maintain a sufficient amount of calcium to trigger myocardial contraction. In conclusion, our observations stress the importance of a regular iron chelation therapy, adherence to treatment of endocrine complication and regular follow-up of TM patients with hypocalcemia.

EQA pilot regional program for haematology in Emilia-Romagna, Italy.

A pilot regional EQA scheme in haematology has been implemented in Emilia-Romagna. The distinctive features of this scheme are: a) inter-laboratory comparability of results on a local basis has been selected as main target of the whole scheme; b) fresh blood has been chosen as ideal control material; c) a fast and effective dispatching system of control material has been accomplished. This has been made possible by means of the hospital ambulances network and an express delivery service; d) the assessment of the analyzers calibration alignment in the whole region has been subdivided in two phases. The first one consists in aligning all analyzers of each province with the corresponding reference provincial center, the second one in aligning the analyzers of the eight reference centers in the region with each other; e) all laboratories taking part in the EQA scheme have been directly involved. Preparation of control material and comment sheet mailing to participants are carried out by eight provincial reference centers. Initial setting of the scheme targets, collegial survey of results and alteration of the scheme guidelines involve all participants in the scheme; f) a distinction between a short-term and a medium-term goal has been made. The short-term one is to improve the harmonization of results for comparability between different laboratories in the same area. The medium-term one is to set up educational programmes and consensus procedures for establishing operative protocols and for defining the allowable analytical errors, set on the basis of clinical requirements.

Biochemical and molecular characterization of a new sporadic glucose-6-phosphate dehydrogenase variant described in Italy: G6PD Modena.

A new glucose-6-phosphate dehydrogenase variant detected in an Italian man from the Po delta is described and designated as G6PD Modena. Biochemical characterization of the variant enzyme revealed an activity 21% of normal, a slow electrophoretic mobility, increased Km value for NADP, decreased Km value for G6P and a complete absence of NADPH inhibition, which could account for the apparently nonhaemolytic feature of this variant. The cloning and sequencing of the G6PD Modena allele showed a G-->C transition at nucleotide 844 in exon VIII causing a Asp-->His amino acid substitution. On the basis of biochemical characterization, G6PD Modena is classified as a genuine variant but it has the same mutation as G6PD Seattle-like.

G6PD Ferrara I has the same two mutations as G6PD A(-) but a distinct biochemical phenotype.

The cloning and sequencing of the normal glucose-6-phosphate dehydrogenase (G6PD) gene has led to the study of the molecular defects that determine enzymatic variants. In this paper, we describe the mutations responsible for the Ferrara I variant in an Italian man with a family history of favism, from the Po delta. Nucleotide sequencing of this variant showed a G-->A mutation at nucleotide 202 in exon IV causing a Val-->Met amino acid exchange, and a second A-->G mutation at nucleotide 376 in exon V causing an Asn-->Asp amino acid substitution. Although on the basis of its biochemical properties this variant was classified as G6PD Ferrara I, it has the same two mutations as G6PD A(-), which is common in American and African blacks, and as the sporadic Italian G6PD Matera. The mutation at nucleotide 202 was confirmed by NlaIII digestion of a polymerase chain reaction amplified DNA fragment spanning 109 bp of exon IV. The 109-bp mutated amplified sequence is not distinguishable from the normal sequence in single strand conformation polymorphism analysis.

Red-cell GSH regeneration and glutathione reductase activity in G6PD variants in the Ferrara area.

Red-cell studies were carried out on three groups of G6PD-deficient subjects with different G6PD variants from the Ferrara area of Northern Italy. Red-cell GSH and activities of G6PD, glutathione reductase (GR), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. A method was developed to measure red-cell GSH regeneration after oxidation of endogenous GSH in whole blood by diamide and only this clearly distinguished the variants from each other and from normal. Regeneration by 1 h was lowest in the Mediterranean variant, 0-10.2% in contrast to 93-98% in normal. A predisposition to a haemolytic crisis after ingestion of fava beans was not clearcut, but subjects appeared to be at risk if GSH regeneration at 1 h was less than 30% of the endogenous level, and red-cell FAD+ was very high indicated by high in vitro GR activity and inhibition by added FAD+. It is suggested that the most informative tests in G6PD deficiency are measurements of GSH regeneration in intact red cells plus GR activity and/or red-cell flavin compounds.

Simonsiella filaments isolated from erosive lesions of the human oral cavity.

The morphology and cultural characteristics of bacteria of the Simonsiella genus isolated from erosions of oral mucosa are reported here. Direct microscopic examination of smeared oral swabs and the consequent selection of suitable culturing procedures and media are compulsory for recovering Simonsiella microorganisms from clinical specimens in routine work.

Maternal lymphocytotoxic antibodies and bilirubin levels in the postpartum offspring.

HLA antigens are present on immature red cells (Harris & Zervas 1969), but disappear from their surface when they become mature and enter into the circulation (Silvestre et al. 1970). Nevertheless some remnants of HLA substance may survive this antigenic switch and possibly persist on adult red cells (Morton et al. 1969, 1971, Doughty et al. 1973). A shortened survival of HLA incompatible reticulocytes has been reported when they are transfused into recipients with circulating anti-HLA antibodies (Zervas et al. 1972). As to anti-HLA antibodies possibly reacting with erythrocytes in cases of fetomaternal immunization, Moulinier (1970) claimed that they would potentiate any hemolytic process in newborns of Rh-immunized women, but successive investigations failed to confirm this (Ahrons & Glavind-Kristensen 1971, Nymand 1975). Also, Nymand (1975) found no association between lymphocytotoxic antibodies in mothers and high bilirubin levels in the umbilical cord blood collected at delivery. On the contrary, when the onset of neonatal jaundice in the postpartum offspring was monitored, newborns of mothers with lymphocytotoxins appeared to become jaundiced more frequently than expected (Reekers et al. 1975). The present study has been undertaken to investigate the relationships between complement dependent lymphocytotoxins (CdL) in the mothers and the bilirubin levels in the newborns during the first days of life.

Glucose-6-phosphate dehydrogenase Ferrara. A new variant of g-6-PD identified in Northern Italy.

A new variant of glucose-6-phosphate dehydrogenase (G-6-PD) has been discovered in Northern Italy, in the district of Ferrara. This variant is characterized by high decrease of red blood cell enzyme activity (less than 5% of normal), high affinity for G-6-P and NADP, increased utilization of deamino-NADP and 2-deoxy-G-6-P, and faster electrophoretic mobility in the buffer systems commonly used for the classification of the G-6-PD variants. The new G-6-PD type was never associated with clinical manifestations in any cases except neonatal jaundice in some of the newborns with this enzyme deficiency. The frequency of the new variant in the Ferrara district indicates that it has probably appeared in this area by mutation some centuries ago. It is suggested that this variant should be named G-6-PD Ferrara.