Anna Maria Salvati

Human erythrocyte separation according to age on a discontinuous "Percoll" density gradient.

It is well known that during ageing, erythrocyte density increases [l-3]. Density gradient centrifugation is the technique most generally used for fractionating erythrocytes of different mean age. Materials employed to construct density gradients, such as bovine serum albumin (BSA) [4], Stractan II [5], Ficoll [6] and Dextran [7], have many disadvantages. The development of Percoll (colloidal silica particles coated with polyvinylpyrrolidone) has overcome these problems. Owing to its low viscosity, low osmotic pressure and non-toxicity this material can be easily adjusted to physiological conditions. Percoll methods allow separation of erythrocytes on discontinuous gradients [&lo], linear gradients [ 111, or self-generated gradients [ 121. In this work, red cell separation in a discontinuous gradient similar to that reported by Alderman et al. [9] is described. To obtain Percoll with the closest approximation to physiological conditions, a suitable modification of Rennie’s buffer system [ 1 l] has been used. Separated red cell populations have been tested for cell recoveries, reticulocyte count and some age-dependent erythrocyte enzyme activities (pyruvate kinase, hexokinase, glucose-6-phosphate dehydrogenase, aldolase) and indices (mean cell volume and red cell volume distribution width index, mean cell haemoglobin concentration). Two non age-dependent measurements were also made (phosphoglycerate kinase and mean cell haemoglobin). The effectiveness and reliability of the method have been evaluated.

6-Phosphogluconate dehydrogenase deficiency in an Italian family

Abstract 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.

Identification of G6PD Mediterranean mutation by amplification refractory mutation system.

BACKGROUND Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common X chromosome-linked hereditary enzymopathy in humans. The authors have developed an amplification refractory mutation system (ARMS) to detect the G6PD Mediterranean mutation (nt. 563 C-->T) that is the most frequent among Caucasian population. METHODS Specific forward polymerase chain reaction (PCR) primers, within exon 6 of the G6PD gene, were designed: ARMS M complementary to the mutated DNA sequence and ARMS N complementary to the wild-type DNA. They were paired with a common reverse primer. The new method was validated using known DNA samples from 72 G6PD-deficient patients carrying the G6PD Mediterranean mutation ascertained by the restriction enzyme analysis. The ARMS test was performed on DNA extracted both from blood or saliva samples. RESULTS The ARMS test showed an excellent reproducibility and a complete concordance with the endonuclease cleavage reference method. At the same time, it is more rapid and less expensive. CONCLUSIONS The described molecular test may be a method of choice to identify the G6PD Mediterranean mutation. It could also be helpful to obtain a definite diagnosis of G6PD Mediterranean heterozygotes, which is not feasible by using red blood cell enzyme activity measurements.

A New Monoclonal Antibody to an Age Sensitive Band 3 Transmembrane Segment

The appearance of band 3 structural modifications related to aging could be evidenced by means of monoclonal antibodies against senescence antigen. Hence in the attempt to provide an immunological marker of erythrocyte aging, we raised a monoclonal antibody against native band 3 (B6 MoAb), which seems to detect differences in the band 3 molecule from erythrocytes of different ages separated by density gradient. Densitometric evaluation of immunoblotting patterns indicates that the in vivo aging is associated with band 3 monomer degradation. The Percoll separated fractions show a significant increase of those proteolytic fragments that bind the B6 antibody. Finally, protease digestions of unsealed membrane ghosts have been performed to test the binding site of the B6 antibody on the band 3 molecule. The data show that the B6 antibody binds a 19 KDa chymotryptic-tryptic fragment which corresponds to a segment of the looped membrane domain whose steric structure appears to be sensitive to age.

Hemoglobinometry: a comparison between the hemiglobincyanide method and the Coulter S Counter

The relative accuracy and precision of the Coulter S Counter have been evaluated in comparison with manual hemiglobincyanide determination according to the recommendations of the International Committee for Standardization in Hematology. Some improvements in the manual procedure, such as centrifugation of hemiglobincyanide solutions and the use of the detergent Triton X-100, were also tested. The Coulter S Counter generally gives higher precision in comparison with the manual method. Nevertheles, Coulter S determinations are systematically lower due to both constant and proportional errors. The available data ranged between hemoglobin values of 11.5 and 18.5%, giving differences of 0-8% between hemoglobin values determined by the Coulter S Counter and the hemiglobincyanide method.

Quantitation of the ultraviolet light test for erythrocyte glucose 6-phosphate dehydrogenase, pyruvate kinase and glutathione reductase

Abstract The sensitivity of Beutlers ultraviolet test for erythrocyte glucose 6-phosphate dehydrogenase, pyruvate kinase and glutathione reductase has been evaluated on diluted solutions of pure enzymes in aqueous hemoglobin. Fluorescence appearance or disappearance speed is in proportion to enzymatic activity; therefore the procedure may be used as a semiquantitative assay method. Coded blood samples with partially deficient enzymatic activities were controlled to prove the reliability of the method in clinical practice. Fluorescence controls at appropriate time intervals allow to detect both severe and partial deficiency of enzymatic activity. So in mass depistage of enzymatic defects, the ultraviolet light test seems to be the most simple and suitable procedure to detect also heterozygotes for erythrocyte glucose 6-phosphate dehydrogenase, pyruvate kinase and glutathione reductase defects.