J. L. Vives Corrons

Increased susceptibility of microcytic red blood cells to in vitro oxidative stress

Abstract: Oxidative damage to erythrocytes in thalassaemia has been related to generation of free radicals by an excess of denaturated α‐ or β‐globin chains, intracellular iron overload and low concentration of normal haemoglobin (HGB). Two good indicators of such oxidative damage are the high red blood cell (RBC) malonyldialdehyde (MDA) production detected following exogenous oxidant stress and the decrease of pyrimidine 5′‐nucleotidase (P5N), the most sensitive enzyme to SH‐group damage in vivo. Conflicting data, however, have so far accumulated in the literature concerning differences in oxidative damage between the different forms of thalassaemia and iron deficiency anaemia (IDA). In the present study, oxidative susceptibility, as defined by the production of MDA in vitro and antioxidant capacity, as measured by the activity of RBC glutathione peroxidase (GPx), superoxide dismutase (SOD) and by reduced glutathione (GSH), have been studied in microcytic RBCs from patients with β‐thalassaemia trait, Spanish (δβ)°‐thalassaemia heterozygotes (δβ‐thalassaemia trait) and iron deficiency anaemia (IDA). The results are consistent with the existence of significant differences in the severity and pattern of oxidative stress susceptibility between β‐thalassaemia trait (increased MDA production and higher SOD and GPx activities) and the other two forms of microcytosis (δβ thalassaemia trait and IDA).

ICSH Guideline for worldwide point‐of‐care testing in haematology with special reference to the complete blood count

These guidelines provide information on how to develop and manage a point‐of‐care (POCT) service so that reliable haematology results are produced regardless of where the test is performed. Many of the issues addressed here are relevant to POCT within hospitals or health centres; however, the principles are equally applicable to care in the community and doctors’ offices. Other aspects discussed in this guideline are the initiation of the service (including indications for and limitations of a POCT service), staff training, type of haematology equipment selected, the blood results, monitoring of quality, accreditation, safety and cost. Equipment selected should generate results that are comparable to those of the local reference laboratory. If a complete independent evaluation of the POCT device has not been performed, the purchaser should perform a local assessment according to the protocol in this document. A literature search should also be undertaken to find independent peer reviewed evaluations on POCT equipment. Often the ideals discussed here may not be achievable in some developing countries but long‐term training and education of POCT workers needs to be supported and constantly kept on government agendas to reach the recommendations advised here. Users should interpret these recommendations for their particular POCT needs and setting.

Pyrimidine 5′nucleotidase and several other red cell enzyme activities in β‐thalassaemia trait

Summary. The activity of 18 red blood cell (RBC) enzymes and reduced glutathione (GSH) content were measured in 70 normal subjects, in 50 heterozygous β‐thalassaemia carriers and in 50 non‐thalassaemic patients with haemolytic anaemia and high reticulocyte counts. In addition, pyrimidine 5′nucleotidase (P5N) activity was also determined in 34 patients with hypochromic, microcytic, iron deficiency anaemia.

Heterogeneity of “Mediterranean type” glucose-6-phosphate dehydrogenase (G6PD) deficiency in Spain and description of two new variants associated with favism

SummaryGlucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) from thirty-six unrelated spanish males was partially purified from blood, and the variants were characterized biochemically and electrophoretically according to the methods recommended by the World Health Organization. Subjects were from multiple geographic regions within Spain, and all suffered from hemolytic anemia, either acute (34 cases) or chronic nonspherocytic (2 cases). Almost all the variants studied presented residual erythrocyte G6PD activity ranging from 0 to 10% of normal, and five different mutants were responsible for the deficient phenotype. Three variants were similar to others previously described: G6PD Mediterranean (11 cases), G6PD Athens-like (3 cases), and G6PD Union (2 cases). The remaining variants were different from the numerous variants already reported and have been considered as new mutants. Provisionally they are called G6PD Betica (19 cases) and G6PD Menorca (1 case).The present study constitutes the first attempt to characterize the deficient G6PD variants found in Spain and supplies new data on the relationship between molecular characteristics of deficient variants and their clinical manifestations. The most important findings can be summarized as follows: (1) The Spanish population is characterized by an important heterogeneity in G6PD deficiency. (2) Although G6PD Mediterranean is very frequent, it presents a relatively high degree of polymorphism. (3) Favism has been observed associated with all kinds of variants described here. (4) G6PD Betica, which is the most frequent variant found in subjects of Southern Spanish origin, has been observed associated with favism in all cases except one.

Chronic non-spherocytic hemolytic anemia associated with severe neurological disease due to γ-glutamylcysteine synthetase deficiency in a patient of Moroccan origin

A previously undescribed mutation of hereditary γ-glutamylcysteine synthetase (GCS) deficiency was found in a 5 year old boy of Moroccan origin. He presented with chronic haemolytic anaemia, delayed psychomotor development and progressive motor sensitive neuropathy of lower extremities. The parents were third degree relatives. The activity of glycolytic enzymes were found to be normal in the propositus, his parents and a sister, but and a complete lack of GSH was found in the propositus. Accordingly, the measurement of de novo GSH synthetic enzymes was undertaken, and severe GCS deficiency was found in the propositus. Both parents and his sister presented GCS activity ranging from 69% to 90% of normal. GCS gene sequencing showed that the propositus was homozygous for a 1241C>T mutation in exon 11 and both parents and his sister were heterozygous. This mutation predicts a Pro414Leu amino acid substitution. Even though the homology between GCS and crystallographically solved, functionally related proteins is not very high, a three-dimensional model of GCS was derived using Modeller Software. GCS deficiency is a very rare autosomal recessive disorder reported so far in only 8 unrelated probands with severe haemolytic anaemia. In only 3 of these was the anaemia associated with severe neurological dysfunction. We report here the fourth case of GCS deficiency presenting neuropathy, giving further support to the eventual relationship between this enzymopathy and neurological damage

Transient dyserythropoiesis in repopulated human bone marrow following transplantation: an ultrastructural study

Summary. Transmission electron microscopy was used to examine marrow samples from 15 patients with aplastic anaemia or acute leukaemia who had been treated with bone marrow transplantation. There were 11 allogeneic, three syngeneic and one autologous graft. The purpose was to estimate the frequency, type and extent of dyserythropoietic change. Transient dyserythropoietic features were substantiated in all cases. Nuclear changes were present in 12 cases, iron laden mitochondria (sideroachrestic phenomena) in 10 and cytoplasmic contacts and/or connections between red cell precursors in 10. Dyserythropoiesis was most conspicuous in the majority of cases between 14 and 28 d after transplantation but it may persist for over 100 d. No deficit in red cell production was noted and it is proposed that dyserythropoiesis in this circumstance is a physiological rather than a pathological phenomenon.

IDIOPATHIC MYELOFIBROSIS: A POSSIBLE ROLE OF IMMUNOLOGICAL PHENOMENA

Immunological disorders in idiopathic myelofibrosis (IM) seems to be a well-demonstrated phenomenon (Boivin et al, 1974: Lewis & Pegrum, 1977, 1978). Essentially they can be detected by the demonstration of circulating immune complexes (IC) (Lewis & Pegrum, 1 9 77, 19 78) and by the presence of bone marrow histological findings suggesting immune activity (Caligaris Cappio et al, 198 1) which support the idea of simultaneous circulating IC and bone marrow lymphocyte and plasma-cell infiltration in some patients with IM. Two types of investigation from our group also support these views. In a previous publication (Hernandez Nieto et al, 1979) we showed that bone marrow lymphoid nodules were frequent, mainly in the cellular phase of the disease.

New haplotype for the Glu104Asp mutation in triose-phosphate isomerase deficiency and prenatal diagnosis in a Spanish family

SummaryFirst-trimester prenatal diagnosis was undertaken by chorionic villus DNA analysis in a Spanish family with the inherited Glu104Asp triose-phosphate isomerase deficiency. The fetus was heterozygous for the mutation and therefore predicted to be clinically unaffected. To investigate the evolutionary origin of this mutation, studies were conducted on the intragenic 2262A/G polymorphism and the CD4 pentameric tandem repeat marker. A different haplotype was found to the one previously described, suggesting a different origin of the Spanish mutation.

Erythrocyte Fructose 2,6-Bisphosphate Content in Congenital Hemolytic Anemias

We have investigated the levels of fructose 2,6-bisphosphate and its synthesizing enzyme 6-phosphofructo-2-kinase in red blood cells from different congenital anemias. Fructose 2,6-bisphosphate concentration and 6-phosphofructo-2-kinase activity are markedly influenced by the number of reticulocytes in all the cases studied with the exception of homozygous pyruvate kinase deficiency, where no correlation was observed with the percentage of reticulocytes.

Relationship between Lymphocyte Size and Enzyme Activities in Two Morphological Variants of B-Chronic Lymphocytic Leukaemia

The activities of the key glycolytic enzymes phosphofructokinase (PFK), pyruvate kinase (PK) and hexokinase in addition to adenosine deaminase, purine nucleoside phosphorylase (PNP) and lactate dehydrogenase (LDH) have been measured in lymphocytes from 39 cases with B-chronic lymphocytic leukaemia (B-CLL). According to the percentage of circulating large non-granular atypical lymphocytes (AL) the B-CLL cases were classified as: typical (less than 10% of AL; 28 cases) and atypical (10-25% AL; 11 cases). In both groups the median lymphocyte volume (MLV) was assessed and correlated with the correspondent enzyme activities. The MLV of B-CLL lymphocytes was significantly (p less than 0.001) decreased (149.9 +/- 19.4 fl) as compared to normal B lymphocytes (175.1 +/- 14.5 fl) and it was significantly (p less than 0.001) lower in typical B-CLL (141.8 +/- 12.2 fl) than in atypical B-CLL (172.0 +/- 17.2 fl). Furthermore, in patients with typical B-CLL, all enzyme activities when expressed as U/10(9) cells were, with the exception of PFK, significantly decreased compared to normal B lymphocytes. However, when the results were expressed as U/ml cells, only PK, PNP and LDH remained significantly low. These findings demonstrate that the determination of MLV in addition to morphology may be a useful tool to distinguish the two previously described morphological B-CLL variants (typical and atypical) and that these two different B-CLL groups are also distinguishable on the basis of three enzyme activities, PK, PNP and LDH which have been shown to be less dependent on cell size than the other enzymes, also studied here.