A novel case of haemoglobin H disease associated with clinical and morphological characteristics of congenital dyserythropoietic anaemia type I.

We report, for the first time, an unusual case of congenital anaemia with the clinical diagnosis of haemoglobin H disease complicated by morphological features at the light and electron microscopy level very similar to those of CDA-I. The red cell indices and the globin chain biosynthetic ratio were not characteristic of the defective haemoglobin genotype. The haematological, clinical and morphological data strongly suggest the novel coexistence of the two defects in a patient. The disease is characterised by a unique dyserythropoietic phenotype of diagnostic importance, which possibly brings new data regarding the reciprocal interaction between the two diseases, especially concerning a specific abnormality in globin chain synthesis in CDA-I, as previously suggested.

Exercise-induced myocardial perfusion abnormalities in sickle beta-thalassemia: Tc-99m tetrofosmin gated SPECT imaging study.

PURPOSE: To determine the mechanism of myocardial ischemia in patients with sickle beta-thalassemia, we performed a scintigraphic evaluation of myocardial perfusion during exercise. SUBJECTS AND METHODS: We studied 30 patients with sickle beta-thalassemia, (mean [+/-SD] age, 37 +/- 10 years) who had no electrocardiographic (ECG), radiographic, or echo-Doppler signs of pulmonary hypertension, left ventricular hypertrophy, or impaired contractility. All patients had a hemoglobin level greater than 7 g/dL. Treadmill exercise test was performed according to the Bruce protocol. Myocardial perfusion was assessed by single-photon emission computed tomography, using Tetrofosmin Tc-99 m Myoview as radiotracer, at peak exercise and again 4 hours later. RESULTS: Eight patients (27%) developed stress-induced scintigraphic perfusion abnormalities that were reversible in all but 1 patient. Subsequent coronary angiograms were normal in all 8 patients. ST segment depression was seen during exercise in 5 of the 7 patients who had reversible perfusion defects. Except for a significantly greater white blood cell count, these 5 patients did not differ from the rest of patients by sex, age, hemoglobin level, percentage hemoglobin F, beta-thalassemia genotype, or risk factors for coronary artery disease. Three of the 5 patients with perfusion and ECG abnormalities (and another with only perfusion defects) developed a stress-induced sickling crisis. CONCLUSION: Physical stress may induce myocardial ischemia in sickle beta-thalassemia patients with normal coronary arteries and elicit painful crises. The sickling process, activated by exercise, could be the common underlying mechanism.

Cardiac involvement in thalassemia intermedia: a multicenter study.

Cardiac complications in 110 patients (mean age, 32.5 +/- 11.4 years) with thalassemia intermedia (TI) were studied. Sixty-seven (60.9%) of them had not been transfused or were minimally transfused (group A). The rest had started transfusions after the age of 5 years (mean, 15.1 +/- 10.1 years), initially on demand and later more frequently (group B). Overall mean hemoglobin and ferritin levels were 9.1 +/- 1.1 g/dL and 1657 +/- 1477 ng/mL, respectively. Seventy-six healthy controls were also studied. The investigation included thorough history taking, clinical examination, electrocardiography, chest radiograph, and full resting echocardiography. Of 110 patients, 6 (5.4%) had congestive heart failure (CHF), and 9 (8.1%) had a history of acute pericarditis. Echocardiography showed pericardial thickening, with or without effusion, in 34.5% of the patients. Valvular involvement included leaflet thickening (48.1%), endocardial calcification (20.9%), and left-sided valve regurgitation (aortic, 15.4%; mitral, 47.2%). All patients had normal left ventricular contractility (fractional shortening, 0.43 +/- 0.05), and high cardiac output (CO; 9.34 +/- 2.28 L/min). Pulmonary hypertension (PHT), defined as Doppler peak systolic tricuspid gradient greater than 30 mm Hg, developed in 65 patients (59.1%). PHT correlated positively with age and CO and did not differ significantly between groups. Cardiac catheterization in the 6 patients with CHF revealed severe PHT, increased pulmonary resistance (PVR), and normal capillary wedge pressure. It was concluded that in patients with TI, the heart is primarily affected by PHT, which is the leading cause of CHF. High CO resulting from chronic tissue hypoxia and increased PVR are the main contributing factors. Doppler tricuspid gradient measurement should be considered, in addition to other factors, when determining the value of transfusion therapy for patients with TI. (Blood. 2001;97:3411-3416)

Bone resorption is increased in young adults with thalassaemia major.

Bone disease in patients with thalassaemia major is a multifactorial and still poorly understood process. The present study evaluated 45 thalassaemic patients using dual X-ray absorptiometry at three sites (lumbar spine, head of femur and forearm) to assess bone mineral density, in parallel with a series of biochemical markers to measure bone formation and bone resorption. To identify possible interfering factors, our patients were grouped according to whether or not they needed transfusion therapy; the presence of hypogonadism was also considered. Our results showed that patients on regular transfusions had a markedly low bone mineral density in contrast to those not requiring blood support and that this finding was more pronounced in the hypogonadic group, irrespectively of sex. The decrease of bone mineral density values was more prominent in the forearm, thus making this site particularly interesting for such studies. Bone formation, as evidenced by the levels of serum alkaline phosphatase and osteocalcin, did not appear to be impaired, while bone resorption was grossly increased in all patient groups. The latter process was clearly evident using the recently introduced measurement of the urinary N-terminal peptides of collagen type I, the sensitivity of which has already been established in other groups of osteoporotic patients. Our conclusion is that, in spite of the severe bone destruction that occurs in thalassaemia major, the fact that bone formation remains intact calls for a more intensive treatment comprising hormonal replacement, bisphosphonates and other agents.

Reduction of the clinical severity of sickle cell/beta-thalassemia with hydroxyurea: the experience of a single center in Greece.

The use of hydroxyurea for the prevention of sickle cell crises in patients with homozygous HbS disease is now well established. The beneficial effects of this compound stem from (a) selective enrichment of red cells containing an increased amount of fetal hemoglobin, which inhibits HbS polymerization, and (b) a decrease of leukocytes, platelets, and reticulocytes, which significantly limits their adherence to the vascular wall. We report the results of a clinical trial of hydroxyurea on 55 Greek-origin patients with sickle cell/beta-thalassemia and 14 patients with homozygous HbS disease who have been treated with hydroxyurea for several years. Such patients have a higher probability to benefit from hydroxyurea therapy, since in addition to its antisickling effect, the increase of gamma-chain synthesis is expected to diminish the deleterious effects of the unbound alpha-globin chains. Selection of patients and monitoring throughout the whole trial were done by the same clinicians. Quantitative expression of the clinical condition was done using a system scoring several outcome parameters. For a period of 52 months prior to starting treatment, the total score of severity for 59 evaluable patients was 1182 points (3068 patient-weeks), while for the 12,018 patient-weeks of the trial this parameter fell to only 82 points. Other observations of interest include the significant improvement of a group of patients with hepatic cholestasis, the development of leg ulcers possibly related to the treatment, and the dramatic increase of hemoglobin F, often in association with an increase of the total hemoglobin levels as a result of decreased hemolysis.

Evaluation of the BeTha gene 1 kit for the qualitative detection of the eight most common Mediterranean beta-thalassemia mutations.

We describe the evaluation of the Bio-Rad BeTha Gene 1 kit (Bio-Rad Laboratories, Hercules, CA), a DNA-probe assay designed for the qualitative determination of the eight most common Mediterranean beta-thalassemia mutations. The kit utilizes the principle of allele-specific oligonucleotide (ASO) hybridization. Following sample preparation and in vitro DNA amplification by the polymerase chain reaction (PCR), an allele-specific detection of the amplified products by a nonradioactive enzymatic assay is performed. Genomic DNA is prepared from an individual's whole blood with a DNA purification matrix. In a second step, the beta-globin gene is amplified in a multiplex PCR reaction containing four 5' biotinylated oligonucleotide primers. In a final step, an aliquot of the PCR reaction is first chemically denatured and then captured in two eight-well strips of a 96-well enzyme-linked immunosorbent assay (ELISA) plate by hybridization to an immobilized ASO probe. Each DNA sequence at each of the eight mutation sites is represented by one normal and one mutant ASO. During this capture/hybridization step, which is performed at 37 degrees C, only perfectly matched PCR products will be captured by an ASO. Subsequently, the allele-specific captured biotin-labeled PCR products are detected by a colorimetric enzymatic reaction. The system permits the detection of 16 beta-thalassemia alleles using a high-throughput format that can be automated easily. A clinical feasibility study was performed to evaluate the functionality (method comparison study, assay validity using samples previously collected and stored at various temperatures for different periods of time, interference on kit performance, and assay validity for prenatal diagnosis) and the usability (ease of use, sample throughput) of the kit. The analysis of 110 samples previously studied with reference methods showed 100% clinical sensitivity and specificity. We demonstrate here that the procedure not only increases the throughput of beta-thalassemia allele genotyping but also provides an accurate, rapid, reliable, and nonisotopic diagnostic tool.

Hydroxyurea therapy in thalassemia.

The clinical effectiveness of Hydroxyurea in thalassemia is still controversial. The present paper puts together the authors' experience in two groups of patients with thalassemia intermedia and sickle cell/beta-thalassemia treated with varying dosages of hydroxyurea over several months. A third group received hydroxyurea along with recombinant human erythropoietin. Our observations are summarized in that treatment with hydroxyrea results in a significant increase of fetal hemoglobin with no change of the total hemoglobin levels. The drug causes also a considerable increase of the erythrocyte volume and hemoglobin content while the MCHC values remain unchanged. As a rule, and without objective criteria so far, patients state feeling better and having more energy. The authors postulate that this feeling may reflect the significant decrease of ineffective erythropoiesis resulting by the replacement of the poorly hemoglobinized, prematurely dying erythroid progenitor and red cell population by another population of cells with higher hemoglobin content and longer survival, the regeneration of which requires less energy and consumption. As expected, patients with sickle cell/beta-thalassemia have also fewer crises and painful episodes. The above findings are in keeping with the few available reports in the literature.

Effects of lead on the somatic growth of children.

Studies on the effects of lead on the somatic growth of children are limited and contradictory. The authors investigated the adverse effects of blood lead concentration on the somatic growth of primary-school-age children. In this study, there was a total of 522 children, aged 6-9 y, who resided in three areas of Greece (i.e., Loutraki, Lavrion, and Elefsina). The medical evaluation included medical history; physical examination; and measurements of height, head circumference, and chest circumference. The authors also evaluated dietary information, socioeconomic status, and height of parents. The authors conducted laboratory tests for hematological parameters and blood lead levels. The mean blood lead level was 12.3 microg/dl (standard deviation = 8.9 microg/dl), and levels ranged from 1.3 microg/dl to 51.2 microg/dl. There were negative monotonic relationships between growth parameters and blood lead levels, even after the authors allowed for confounding effects. An increase in blood lead level of 10 microg/dl was associated with a decrease of (a) 0.33 cm in head circumference (95% confidence interval = 0.12, 0.55; p = .002); (b) 0.86 cm in height (95% confidence interval = 0.14, 1.16; p = .020); and (c) 0.40 cm in chest circumference (95% confidence interval = -0.22, 1.02; p = .207). These findings led the authors to conclude that a decrease in growth in children may be associated with blood lead concentrations.

Hepatitis E virus infection in individuals at high risk of transmission of non-A, non-B hepatitis and sexually transmitted diseases.

The aim of the study was to determine the prevalence of hepatitis E virus (HEV) infection among individuals at high risk of transmission of non-A, non-B hepatitis or sexually transmitted diseases (STDs), and to evaluate whether they have an increased risk of exposure to HEV. Serum samples from 125 thalassemia patients, 300 intravenous drug users, 420 hemodialysis patients, 263 individuals with STDs, 47 human immunodeficiency virus (HIV) infected homosexual men, and 316 healthy volunteers were tested for immunoglobulin G (IgG) and M (IgM) antibodies to HEV (anti-HEV) by enzyme immunoassays (EIAs) following a predetermined algorithm (Abbott Labs). Anti-HEV IgG was confirmed in 3/125 (2.4%) thalassemia patients, 5/300 (1.7%) intravenous drug users, 27/420 (6.4%) hemodialysis patients, 4/263 (1.5%) STD patients, 1/47 (2.1%) homosexual men, and 7/316 (2.2%) of the reference group. No patient was found positive for anti-HEV IgM. The higher prevalence which was observed in hemodialysis group was due to the confounding effect of age, as multivariate analysis showed. The anti-HEV prevalence increased significantly with age (p = 10(-4)). No significant association was found between anti-HEV, anti-HCV, and anti-HBc. In conclusion, individuals at high risk of non-A, non-B hepatitis and STDs have no increased risk of exposure to HEV and the higher prevalence of anti-HEV IgG among older subjects may be due to an epidemic form of HEV infection which occurred some decades ago, when the sanitary conditions in our country were poor.

Molecular basis and haplotype analysis of delta, beta-thalassemic chromosomes in Greece.

The molecular defect was defined in 38 delta beta-thalassemic chromosomes from 30 unrelated heterozygous and 4 homozygous patients of Greek origin. Restriction fragment beta-gene cluster haplotypes were studies in 23 delta beta-thalassemic chromosomes. The molecular lesion was identical in all studied cases and corresponds to the 'Sicilian' type of delta beta-thalassemia. Restriction haplotypes analysis has shown that, with one exception only, all Greek delta beta-thalassemic chromosomes bear the polymorphic sites which characterize haplotypes I or VII, the former being probable by indirect evidence. The striking similarities of the molecular lesion and the underlying haplotypes are consistent with two theories: (1) The deletion occurred once on a chromosome and spread all over Greece and the Mediterranean area thereafter; (2) the 5' subhaplotype +----favors the deletional event in the delta-beta gene area.

Reticulocyte counting in thalassemic and other conditions with the R-1000 Sysmex analyzer.

Precise reticulocyte counts are difficult to obtain by the manual method when their percentage in the blood is low or normal. In these instances, rapid reticulocyte counting by flow cytometry appears to offer more accuracy and precision. The purpose of this study was to establish reticulocyte counts in heterozygous beta-thalassemia for reference purposes and to evaluate the performance of the recently introduced apparatus R-1000 (Sysmex) in the very heterogeneous thalassemic and sickle-cell syndromes. We studied a total of 364 samples; 102 heterozygous beta-thalassemia carriers, 180 normal matched controls, 36 patients with thalassemia major or intermedia, and 46 patients with various sickle-cell syndromes. Reticulocyte counts (both as percentage and as total number) were higher in heterozygous beta-thalassemia than in normal controls (p less than 0.001) and showed an inverse correlation with the respective hemoglobin values (p less than 0.001). These results confirm the proposed slightly increased erythropoietic activity in heterozygous beta-thalassemia carriers. A drawback of the technique is that the reticulocyte-platelet discrimination error is signaled frequently in all conditions displaying a marked red cell heterogeneity, especially when these are associated with high reticulocyte numbers. This calls probably for readjustment of the corresponding algorithm. In addition, all these conditions show a significantly increased auramine-O mature red-cell nonspecific fluorescence.

Hemoglobin Köln occurring in association with a beta zero thalassemia: hematologic and functional consequences.

Hemoglobin (Hb) Köln-beta zero thalassemia compound heterozygosity was discovered in a young Greek patient. This gave us the unique opportunity for studying the functional properties of this unstable high-oxygen affinity hemoglobin variant in red cells containing almost pure Hb Köln. The red cells of the proposita exhibit morphological alterations and hematologic indices corresponding to the presence of an unstable Hb and beta thalassemia. Globin chain synthesis confirmed the association with a beta zero thalassemia gene. Oxygen-binding curves for these cells were biphasic, indicating the presence of both heme-saturated and of approximately 20% of non-cooperative Hb Köln. The major component exhibits an increased oxygen affinity, reduced cooperativeness, and normal alkaline Bohr effect. The 35-year-old proposita is active, has not been splenectomized, and has not been transfused in several years.

A unique thalassaemic syndrome: homozygous alpha-thalassaemia + homozygous beta-thalassaemia.

The disturbed balance of globin chain synthesis is a major factor in the pathophysiology of the thalassaemic disorders; this concept is strongly supported by the study of a patient displaying an extreme but symmetrical deficit of both major types of chains alpha and beta. The patient had a mild clinical picture but presented a striking hypochromia (MCH 10 pg) with compensatory erythrocytosis (RBC 10(12)/l.). Study of the propositus and his family by haematological, biochemical and biosynthetic techniques indicates that the patient carries two alpha- and two beta-thalassaemia genes resulting in balanced globin chain synthesis; in addition, several members of the family carry two or three abnormal genes. During observation a change in the haematological pattern occurred with a shift towards more intensive beta-chain and away from gamma-chaim synthesis; this appeared with be associated with improvement of his anaemia through more effective erythropoiesis.