Concise review: how do red blood cells born, live, and die?

The average life cycle of a human RBC is approximately 120 days. Generally, by this point, the cell is worn out and damaged. RBCs pass through both the spleen and liver, where specialised immune cells called macrophages are found. Macrophages recognise when an RBC is spent, and undergo a process called phagocytosis where they digest the cell. In this process, the iron in haemoglobin is recycled for use in new blood cells and the hem molecule is degraded, conjugated to bilirubin, and eliminated from the body. All the other cellular proteins are either recycled or eliminated. Historically, this process was thought to occur exclusively in the spleen, but recent studies have shown that it occurs in the bone marrow. The RBC has been analysed from many perspectives: cytological, haematological, and immunological, as well as from the focus of molecular biology, biophysics, and mathematics. Here we analyse how are red blood cells born and how they live and die in a brief overview of the whole process with special mention of the morphological aspects from bone marrow and spleen provided by transmission and scanning electron microscopy.

Osmotic gradient ektacytometry: A valuable screening test for hereditary spherocytosis and other red blood cell membrane disorders.

INTRODUCTION: New generation osmotic gradient ektacytometry has become a powerful procedure for measuring red blood cell deformability and therefore for the diagnosis of red blood cell membrane disorders. In this study, we aim to provide further support to the usefulness of osmotic gradient ektacytometry for the differential diagnosis of hereditary spherocytosis by measuring the optimal cutoff values of the parameters provided by this technique. METHODS: A total of 65 cases of hereditary spherocytosis, 7 hereditary elliptocytosis, 3 hereditary xerocytosis, and 171 normal controls were analyzed with osmotic gradient ektacytometry in addition to the routine red blood cell laboratory techniques. The most robust osmoscan parameters for hereditary spherocytosis diagnosis were determined using receiver operating characteristic curve analysis. RESULTS: The best diagnostic criteria for hereditary spherocytosis were the combination of decreased minimal elongation index up to 3% and increased minimal osmolality point up to 5.2% when compared to the mean of controls. Using this established criterion, osmotic gradient ektacytometry reported a sensitivity of 93.85% and a specificity of 98.38% for the diagnosis of hereditary spherocytosis. CONCLUSION: Osmotic gradient ektacytometry is an effective diagnostic test for hereditary spherocytosis and enables its differential diagnosis with other red blood cell membrane diseases based on specific pathology profiles.

ICSH recommendations for identification, diagnostic value, and quantitation of schistocytes.

Schistocytes are fragments of red blood cells (RBCs) produced by extrinsic mechanical damage within the circulation. The detection of schistocytes is an important morphological clue to the diagnosis of thrombotic microangiopathic anemia (TMA). Reporting criteria between different laboratories, however, are not uniform, owing to variability of shape and nature of fragments, as well as subjectivity and heterogeneity in their morphological assessment. Lack of standardization may lead to inconsistency or misdiagnosis, thereby affecting treatment and clinical outcome. The Schistocyte Working Group of the International Council for Standardization in Haematology (ICSH) has prepared specific recommendations to standardize schistocyte identification, enumeration, and reporting. They deal with the type of smear, method of counting, morphological description based on positive criteria (helmet cells, small, irregular triangular, or crescent-shaped cells, pointed projections, and lack of central pallor). A schistocyte count has a definite clinical value for the diagnosis of TMA in the absence of additional severe red cell shape abnormalities, with a confident threshold value of 1%. Automated counting of RBC fragments is also recommended by the ICSH Working Group as a useful complement to the microscope, according to the high predictive value of negative results, but worthy of further research and with limits in quantitation.

Neonatal haemoglobinopathy screening in Spain.

The prevalence of variant haemoglobins in Spain is increasing as a result of recent African immigration. Of the 19 regions of Spain, 13 have more than 1% of residents of African origin or ethnicity. Haemoglobinopathy prevalence is heterogeneous. Some cases of sickle cell disease (SCD) and sickle cell trait have been found in autochthonous individuals, but this is very rare. Most of the studies of SCD prevalence in Spain are incomplete or focused on a few geographical regions. When screening has been carried out regardless of ethnic origin, overall haemoglobinopathy prevalence has varied from 0.14% to 0.94% and the estimated prevalence of SCD has varied from 0.001 (in Extremadura) to 0.03 (in Aragón). A registry for SCD maintained by the Spanish Society of Paediatric Haematology shows that in the last 4 years the prevalence of SCD has increased threefold. Only two Spanish Communities (Extremadura and Madrid) are running an official neonatal screening programme for SCD. Other Spanish Communities have finished local pilot studies and are expected to establish neonatal screening programmes shortly. Catalonia, the Spanish community with the highest African immigration flow and SCD genetic impact, has not yet established an official programme for SCD neonatal screening; screening currently depends on individual hospital policies and is restricted to at-risk ethnic groups. Studies performed so far suggest that universal screening should be recommended for regions with a high annual birth rate and SCD prevalence (Catalonia and Madrid, for example), with a targeted policy being restricted to regions with low annual birth rate and SCD prevalence.

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.

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

A previously undescribed mutation of hereditary gamma-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.

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

First-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.

Red cell membrane Na+ transport systems in hereditary spherocytosis: relevance to understanding the increased Na+ permeability.

Red blood cells (RBCs) in hereditary spherocytosis (HS) show high sodium (Na+) and potassium (K+) movement across the membrane, resulting in dehydration. In general, these abnormal cation fluxes have been interpreted as "increased leaks" due to passive or electrodiffusional permeability of the RBC membrane. A study to elucidate the contribution of concomitant ouabain-resistant pathways (Na-K-2Cl cotransport and Na-Li countertransport) to abnormal Na+ permeability present in RBCs of subjects with HS has been undertaken. Accordingly, erythrocyte Na+ and K+ content and transmembrane cation movements via the Na-K pump, Na-K-2Cl cotransport, Na-Li countertransport, and Na+ passive diffusion, were measured in 25 non-splenectomized patients with HS and compared with the results obtained from the study of 11 patients with congenital non-spherocytic haemolytic anaemia (CNSHA) due to hereditary elliptocytosis (7 cases) and RBC enzyme defects (4 cases) and of 30 normal controls. Compared to the controls, patients with HS exhibited a highly significant (P<0.001) increase in all the Na+ transmembrane movements via passive diffusion (411+/-243 vs 105+/-40), Na-K pump (2615+/-970 vs 1874+/-359), Na-K-2Cl cotransport (males: 371+/-138 vs 190+/-42; females: 401+/-134 vs 104+/-44) and Na-Li countertransport (207+/-131 vs 98+/-41). This was associated with increased Na+ and decreased K+ content, resulting in a reduction of total cation (Na+ + K+) RBC concentration. Furthermore, significant correlations were also found between the patients' RBC cationic content and the mean corpuscular haemoglobin concentration (MCHC) (r=0.51, P<0.05) and between the Na+ passive leak and the haematocrit value (r=-0.44, P<0.05). In the patients with CNSHA, a less significant (P<0.01) increase of active (Na-K pump) and passive (leak) transmembrane permeability to Na+ was associated with normal transmembrane movements via Na-K-2Cl cotransport and Na-Li countertransport. The present study demonstrates that in HS, RBCs are characterized by a variable, but always significant increase of all the membrane transport systems leading to the extrusion of Na+, and that these abnormalities, regardless of their relation to membrane structural defects, may probably be valuable for the differential diagnosis between HS and other congenital defects of RBCs.

Hereditary non-spherocytic haemolytic anaemia due to red blood cell glutathione synthetase deficiency in four unrelated patients from Spain: clinical and molecular studies.

In four unrelated patients with chronic haemolysis and markedly reduced red blood cell (RBC) glutathione (49.5%, 12.6%, 11.5% and 15% of the normal concentration respectively), a severe glutathione synthetase (GSH-S, EC 6.3.2.3) deficiency was found. One case exhibited a neonatal haemolytic anaemia associated with oxoprolinuria, but without neurological manifestations. The family study revealed GSH-S activity in both parents to be around half the normal level, a finding consistent with the presumed autosomal recessive mode of inheritance of this enzymopathy. Two cases exhibited a well-compensated haemolytic syndrome without anaemia or splenomegaly at steady state. One of these cases was diagnosed after an episode of acute haemolytic anaemia after fava bean ingestion. The remaining patient suffered from moderate to severe chronic non-spherocytic haemolytic anaemia and splenomegaly, and required occasional blood transfusion for a haemolytic crisis associated with drug ingestion. In this patient, the anaemia was corrected by splenectomy. In addition to GSH-S, a panel of 16 other RBC enzyme activities was also studied in all the patients. Hexokinase, aldolase, glucose-6-phosphate dehydrogenase and pyruvate kinase activities all increased; these increases were to be expected, given the rise in the number of circulating reticulocytes. In two patients, the incubation of RBCs with hydrogen peroxide revealed an enhanced production of malonyldialdehyde. DNA analysis showed a homozygous state for 656 A-->G mutation in patients 2 and 3. The GSH-S gene of patient 1, studied elsewhere, revealed an 808 T-->C. The GSH-S gene of patient 4 was not available for study. The present study demonstrates that GSH-S deficiency is also present in Spain and further supports the molecular and clinical heterogeneity of this enzymopathy

Chronic non-spherocytic haemolytic anaemia due to congenital pyrimidine 5' nucleotidase deficiency: 25 years later.

In 1972, Valentine et al described, under the name of 'non-spherocytic haemolytic anaemia, high red cell ATP and ribose phosphate pyrophosphokinase (RPK; EC 2.7.6.1) deficiency', an obscure congenital haemolytic anaemia with the characteristic feature of red blood cell basophilic stippling. The activity of Embden-Meyerhof pathway and hexose monophosphate shunt were normal, and the concentrations of reduced glutathione and of ATP were raised 2 SD above the normal mean. The low values of RPK also encountered were considered to be an epiphenomenon rather than a causative defect. One year later, further studies performed in two new kindreds with the same haemolytic disorder associated with persistent basophilic stippling were described under the name of 'haemolytic disorders associated with increased ATP'. In 1974, two new and important observations contributed to the final identification of the disease: the patients' red blood cells (RBCs) contained large amounts of nucleotides (pyrimidine nucleotides), and in all cases they were deficient in a hitherto unrecognized enzyme called pyrimidine 5' nucleotidase (P5N). In conclusion, all these cases were formerly referred to as 'high ATP syndromes' because of the erroneous assumption that the large number of nucleotides within deficient RBCs were adenine phosphate rather than pyrimidine phosphate. Twenty-five years after its description, P5N deficiency has been reported in about 35 unrelated families from different parts of the world, and it has become one commonly identified cause of hereditary non-spherocytic haemolytic anaemia due to RBC enzymopathy. Genetic transmission is via the autosomic recessive mode, and only homozygous or compound heterozygous are clinically affected. Family members who are biochemically heterozygous are haematologically normal and difficult to detect. Unfortunately, the precise gene mutation or mutations causing the disease remain unknown.

Co-existence of hereditary spherocytosis and a new red cell pyruvate kinase variant: PK mallorca.

BACKGROUND AND OBJECTIVE: A partial red blood cell (RBC) pyruvate-kinase (PK-R) deficiency was found in a patient with concomitant hereditary spherocytosis (HS) and chronic hemolytic anemia. Clinical, biological and molecular studies were performed in the patient, his parents and a brother, in order to characterize the specific PK-R gene mutation and the inheritance mechanism of the transmission of both red cell defects in this particular family. DESIGN AND METHODS: Conventional biological studies were used to identify the PK-LR gene mutation responsible for hereditary transmission of PK-R deficiency and HS. The family study was completed with genotypic and RBC membrane protein analyses in the patient and his family. RESULTS: Molecular study of the PK deficiency was performed in all the family members and demonstrated a heterozygous condition for the 1516 G->A (506Val->Ile) mutation at the PK-LR gene in both the patient and his mother. Since this mutation has not been reported previously, it is provisionally named PK "Mallorca". The study of RBC membrane proteins demonstrated the existence of partial band 3 and protein 4.2 deficiencies in the propositus and his father but not in the mother and brother, who were also studied. These results support the dominant mode of inheritance of HS and PK-LR gene in this family. INTERPRETATION AND CONCLUSIONS: HS and PK deficiency are not exceptional in Spain. The co-existence of both RBC defects in the same patient, however, is very rare; only a few cases have been described to date. Our findings suggest that performing an elementary RBC enzyme survey in all patients with HS would help to determine the real frequency of this apparently rare association.

Evaluation of the blue formazan spot test for screening glucose 6 phosphate dehydrogenase deficiency.

Several screening tests for glucose 6 phosphate dehydrogenase (G6PD) deficiency have been reported thus far, and a standardized method of testing was proposed by the International Council for Standardization in Hematology (ICSH). The screening test used in any particular laboratory depends upon a number of factors such as cost, time required, temperature, humidity, and availability of reagents. In this study, a direct comparison between three different G6PD screening methods has been undertaken. In 71 cases (50 hematologically normal volunteers, 9 hemizygous G6PD-deficient males, and 12 heterozygous deficient females), the blue formazan spot test (BFST) was compared with the conventional methemoglobin reduction test (HiRT) and the ICSH-recommended fluorescent spot test (FST-ICSH). In all cases, the results obtained with the three screening tests were correlated with the enzyme activity assayed spectrophotometrically. In hemizygous G6PD-deficient males, all cases were equally detected with the three methods: BFST (4.7-6.64, controls: 11.1-13.4), BMRT (score +3 in all 9 cases), and FST (no fluorescence in 9 cases). In heterozygous G6PD-deficient females, two methods detected 7 out of 12 cases (BFST: 8.71-11.75, controls: 11.1-13.4; and BMRT: score +3 in 7 cases), whereas the FST-ICSH missed all 12 cases that presented a variable degree of fluorescence. Although the sensitivity for G6PD-deficient carrier detection is the same for the BMRT and the BFST, the latter has the advantage of being semiquantitative and not merely qualitative. Unfortunately, none of the three screening tests compared here allowed the detection of the 100% heterozygote carrier state of G6PD deficiency.

[Molecular study of red cell pyruvate kinase deficiency in 15 patients with chronic hemolytic anemia. Group of Erythropathology of Hematology and hemotherapy Association of Spain (HHAS)]. / Estudio molecular del déficit de piruvatocinasa eritrocitaria en 15 pacientes con anemia hemolítica crónica. Grupo de Eritropatología de la Asociación Española de Hematología y Hemoterapia (AEHH).

BACKGROUND: Identification of RBC pyruvate-kinase (PK) gene mutations by polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) followed by PK gene sequencing in positive cases has been assessed and the results obtained with a preliminary study of 15 unrelated patients of Spanish origin are presented. PATIENTS AND METHODS: Patients have been classified into two different groups: group 1, propositus (15 cases), and group 2, relatives of the patients included in group 1 (10 males and 5 females). In group 1, a PCR was followed by SSCP and sequencing, and in group 2, the PCR was followed by digestion with specific restriction endonucleases (PCR-ER). RESULTS: Group 1: from 15 patients included in the study 2 were identified as homozygous, 4 as heterozygous and 9 as compound heterozygous. In this group, were identify 26 affected alleles with 11 different mutations: T1456 10 alleles (38.6%), T721 3 alleles (11.6%), A1010, C514, C1015 and T1223 2 alleles (7.7%), and C1070, A1291, T1508, A1595 y T1675 one allele. Relatives from 8 out of 15 patients from group 1 showed the following pattern: homozygous (one case), heterozygous (10 cases), compound heterozygous (2 cases) and normal (2 cases). CONCLUSIONS: SSCP procedure followed by direct gene sequencing in positive cases is fast and simple enough to allow the identification of PK deficient variants, avoiding the need of biochemical characterisation of semipurified deficient enzyme, which is more cumbersome and time consuming. In addition, the PCR-ER method is a very useful tool for screening of the most frequent molecular variants, as well as, for the detection of the carrier condition of this enzymopathy (family studies).

Molecular characterization of the PK-LR gene in pyruvate kinase deficient Spanish patients. Red Cell Pathology Group of the Spanish Society of Haematology (AEHH).

The PK-LR gene has been studied in 12 unrelated patients with red cell pyruvate kinase deficiency and hereditary nonspherocytic haemolytic anaemia (CNSHA). The entire codifying region of the R-type PK gene and the flanking intronic regions were analysed by single-stranded conformation polymorphism (SSCP) followed by direct sequencing of abnormal DNA. 10 different mutations were identified in 22/24 alleles at risk. Eight of these were missense mutations that caused the following single amino acid changes: G514C (172Glu-Gln), G1010A (337Arg-Gln), G1015C (339Asp-Gln), T1070C (357Ile-Thr), C1223T (408Thr-Ile), G1291A (431Ala-Thr), C1456T (486Arg-Trp) and G1595A (532Arg-Gln). Two were nonsense mutations: G721T (241Glu-Stop) and C1675T (559Arg-Stop). 7/22 alleles demonstrated the same C1456 --> T mutation. The study of the polymorphic site at nucleotide (nt) 1705 performed in all cases disclosed a 1705 C/C mutation in 10 and a 1705 A/C mutation in three. This is the first report on the presence of several different L-type PK gene mutations within Spanish population. Furthermore, from the PK gene mutations found, six were unique and not previously described (1015C, 1070C, 1223T, 1291A, 1595A and 1675T) and one (C1456T) seems to be predominant in Spain. Interestingly, no case with the 1529A mutation commonly found in Northern European populations was present here.

alpha-Thalassaemia due to a single codon deletion in the alpha1-globin gene. Computational structural analysis of the new alpha-chain variant. Mutations in brief no. 132. Online.

A new unstable alpha-globin chain associated with alpha-thalassemia phenotype has been found in a Spanish patient. Molecular analysis of the alpha-globin gene complex using PCR and non-radioactive single-strand conformation analysis, allowed to identify a new mutation in the second exon of the alpha-globin gene. Direct sequencing of the abnormal fragment revealed a 3 bp deletion, which led to the loss of a single codon corresponding to a Lys (K) residue at position 60 or 61 DK60 or DK61. Theoretical structural analysis, performed by computational methods, indicated that the loss of an amino acid residue at this position disturbed the contact region between the B and E-helices, affecting the overall stability of the molecule. Therefore, the DK60 and DK61 results in a structurally abnormal alpha-globin chain, not previously described, named Hb Clinic, which leads to the alpha-thalassemia phenotype in the heterozygote patient. No abnormal hemoglobin was detected by standard electrophoretic procedures, suggesting that this alpha-globin chain variant is so unstable that it may be catabolized immediately after its synthesis. This mutation was confirmed by PCR using an allele specific primer.

Two new mutations of the glucose-6-phosphate dehydrogenase (G6PD) gene associated with haemolytic anaemia: clinical, biochemical and molecular relationships.

In two unrelated Spanish males with glucose-6-phosphate dehydrogenase (G6PD) deficiency and haemolytic anaemia, and two different novel point mutations in the G6PD gene, have been identified. A C to T transition at nucleotide 406 resulting in a (136) Arg to Cys substitution and a C to G transition at nucleotide 1155 resulting in a (385) Cys to Trp substitution. These two molecular defects have not been described before and are designated G6PD Valladolid 406 C-->T and G6PD Madrid 1155 C-->G. In vitro biochemical characterization of both mutant enzymes showed important differences in their molecular properties according to their different clinical behaviour. In G6PD Valladolid, the mutation of which is located in exon 5, the normal in vitro heat stability may explain its mild clinical expression (low-grade haemolysis interrupted by an acute haemolytic crisis at age 70). In G6PD Madrid, the mutation, located in exon 10, results in a deficient variant associated with neonatal jaundice and life-long chronic nonspherocytic haemolytic anaemia (CNSHA). This finding further emphasizes the importance of this specific region of the G6PD gene in the stabilization of the G6PD molecule. Putative relationships between these single point mutations and the molecular properties of the mutant enzymes are also discussed.

First description of a frameshift mutation in the alpha1-globin gene associated with alpha-thalassaemia.

A frameshift mutation in the alpha1-globin gene, responsible for a clinically mild alpha-thalassaemia phenotype, has been characterized in a Spanish woman. After excluding the most common forms of alpha-thalassaemia found in the Mediterranean area, both alpha-globin genes (alpha1 and alpha2) were amplified and analysed selectively by non-radioactive single-strand conformation polymorphism (SSCP). An abnormal SSCP mobility was present in the second exon of the alpha1-globin gene and direct sequence analysis revealed a 13 bp deletion (between codons 51 and 55) affecting a single allele. The consequence of this mutation is a reading frameshift leading to a novel amino acid coding sequence from codons 51-61 and a premature stop signal at new position 62, which results in a net reduction of the affected alpha-globin chain output. The presence of this new mutation was confirmed by restriction enzyme analysis of the specific PCR product.