Genotyping of alpha-thalassemia in microcytic hypochromic anemia patients from North India

Microcytic hypochromic anemia is a common condition in clinical practice and alpha-thalassemia has to be considered as a differential diagnosis. Molecular diagnosis of alpha-thalassemia is possible by polymerase chain reaction. The aim of this study was to evaluate the frequency of alpha-gene numbers in subjects with microcytosis. In total, 276 subjects with microcytic hypochromic anemia [MCV<80fl; MCH<27pg] were studied. These include 125 with thalassemia trait, 48 with thalassemia major, 26 with sickle-cell thalassemia, 15 with E beta-thalassemia, 40 with iron-deficiency anemia, 8 with another hemolytic anemia, and 14 patients with no definite diagnosis. Genotyping for -alpha3.7 deletion, -alpha4.2 deletion, Hb Constant Spring, and a-triplications was done with polymerase chain reaction. The overall frequency of -alpha3.7 deletion in 276 individuals is 12.7%. The calculated allele frequency for a-thalassemia is 0.09. The subgroup analysis showed that co-inheritance of a-deletion is more frequent with the sickle-cell mutation than in other groups. We were able to diagnose 1/3 of unexplained cases of microcytosis as a-thalassemia carriers. The a-gene mutation is quite common in the Indian subcontinent. Molecular genotyping of a-thalassemia helps to diagnose unexplained microcytosis, and thus prevents unnecessary iron supplementation.     

The clinical utility of discriminant functions for the differential diagnosis of microcytic anemias

Several groups of authors have derived discriminant functions (DFs) based on red cell indices (primarily MCH, MCV, and RDW) that can be used to differentiate iron deficiency from thalassemia minor. The Technicon H*1 analyzer provides a direct MCHC measurement (termed the CHCM), in addition to the conventional computed value (Hgb/PCV). To evaluate the clinical utility of red cell discriminant analysis, chart review was performed in 176 cases for which hemoglobin characterization and quantitation studies had been requested. Six published discriminants were evaluated for cases of clearly defined iron deficiency anemia and thalassemia minor. Overall diagnostic efficiency ranged from 50%-82%, and the diagnostic performance of three of the discriminants failed to achieve statistical significance. Mean values for both MCHC and CHCM were significantly lower in patients with iron deficiency than in patients with other causes of microcytic anemia. It was also observed that MCHC was significantly greater than CHCM in patients with iron deficiency anemia, but not in patients with other causes of microcytic anemia. Both MCHC and the difference between MCHC and CHCM showed potential value as parameters for the differential diagnosis of iron deficiency from other causes of microcytic anemia. It was noted, however, that in 67% of the cases studied, the use of a DF could not have resolved the diagnosis to the extent that hemoglobin characterization and quantitation studies were no longer indicated.     

Identification of glucose 6-phosphate dehydrogenase deficiency in a population with a high frequency of thalassemia

High frequencies of both thalassemia trait (5.2%) and glucose 6-phosphate dehydrogenase (G6PD) deficiency for only males (1.3%) have been observed in the Calabrian population. The G6PD activity measurement was carried out on 1239 samples of whole blood from Calabrian subjects of both sexes (age range 10-55) by a differential pH-metry technique which was quite suitable to determine the G6PD deficiency in mass screenings. The analyzed subjects showed: only the thalassemia trait; or only the G6PD deficiency; or only the total iron serum deficiency; or G6PD deficiency associated with the thalassemia trait or with the total iron serum deficiency. The G6PD heterozygous subjects have an enzymatic activity which is masked by both the thalassemia trait and the total iron serum deficiency. In a population showing high frequencies of both thalassemia trait and G6PD deficiency, the comparison of G6PD activity of heterozygous subjects also affected with the thalassemia trait is more reliable if referred to the enzymatic activity of the carriers of the latter inherited anomaly rather than to G6PD activity of normal subjects.     

The use of inference strategies in the differential diagnosis of microcytic anemia

A new expert system developed on a Macintosh personal computer using a commercially available artificial intelligence shell was compared with four different discriminant functions (DFs) for the differentiation of microcytic anemia into etiologic categories. Several databases were used with a different composition but all contained at least some samples from thalassemic individuals and from patients with iron deficiency anemia. The DFs analyzed were those proposed by England and Fraser, Green and colleagues, Mentzer, and by Shine and Lal. None of the databases performed satisfactorily when used singly, whereas very high false-positive rates were obtained by one of them. The diagnostic efficiency was somewhat improved by combining several DFs. An expert system using an artificial intelligence "shell" with an "interference engine" was developed using cluster analysis and a set of learning examples. The input necessary for the system to achieve a conclusion consists of MCV, RBC, and RDW as well as a statement as to whether the patient has anemia. Based upon the values of these parameters, the expert system will give an "advice" regarding the probabilities for thalassemia, iron deficiency, and/or other probabilities such as previous transfusions, anemia of chronic disease, laboratory error, etc. In a prospective trial, the system functioned with an accuracy of better than 85%.     

Heart Rate Variability as an Alternative Indicator for Identifying Cardiac Iron Status in Non-Transfusion Dependent Thalassemia Patients

Background

Iron-overload cardiomyopathy is a major cause of death in thalassemia patients due to the lack of an early detection strategy. Although cardiac magnetic resonance (CMR) T2* is used for early detection of cardiac iron accumulation, its availability is limited. Heart rate variability (HRV) has been used to evaluate cardiac autonomic function and found to be depressed in thalassemia. However, its direct correlation with cardiac iron accumulation has never been investigated. We investigated whether HRV can be used as an alternative indicator for early identification of cardiac iron deposition in thalassemia patients.

Methods

Ninety-nine non-transfusion dependent thalassemia patients (23.00 (17.00, 32.75) years, 35 male) were enrolled. The correlation between HRV recorded using 24-hour Holter monitoring and non-transferrin bound iron (NTBI), hemoglobin (Hb), serum ferritin, LV ejection fraction (LVEF), and CMR-T2* were determined.

Results

The median NTBI value was 3.15 (1.11, 6.59) μM. Both time and frequency domains of HRV showed a significant correlation with the NTBI level, supporting HRV as a marker of iron overload. Moreover, the LF/HF ratio showed a significant correlation with CMR-T2* with the receiver operating characteristic (ROC) curve of 0.684±0.063, suggesting that it could represent the cardiac iron deposit in thalassemia patients. HRV was also significantly correlated with serum ferritin and Hb.

Conclusions

This novel finding regarding the correlation between HRV and CMR-T2* indicates that HRV could be a potential marker in identifying early cardiac iron deposition prior to the development of LV dysfunction, and may be used as an alternative to CMR-T2* for screening cardiac iron status in thalassemia patients.     

Iron release in erythrocytes from patients with beta-thalassemia.

Our previous studies have shown that iron is released in a free (desferrioxamine-chelatable) form when erythrocytes undergo oxidative stress (incubation with oxidizing agents or aerobic incubation in buffer for 24-60 h (a model of rapid in vitro ageing)). The release is accompanied by oxidative alterations of membrane proteins as well as by the appearance of senescent antigen, a signal for termination of old erythrocytes. In hemolytic anemias by hereditary hemoglobin alterations an accelerated removal of erythrocytes occurs. An increased susceptibility to oxidative damage has been reported in beta-thalassemic erythrocytes. Therefore we have investigated whether an increased iron level and an increased susceptibility to iron release could be observed in the erythrocytes from patients with beta-thalassemia. Erythrocytes from subjects with thalassemia intermedia showed an extremely higher content (0 time value) of free iron and methemoglobin as compared to controls. An increase, although non-statistically-significant, was seen in erythrocytes from subjects with thalassemia major. Upon aerobic incubation for 24 h the release of iron in beta-thalassemic erythrocytes was by far greater than in controls, with the exception of thalassemia minor. When the individual values for free iron content (0 time) seen in thalassemia major and intermedia were plotted against the corresponding values for HbF, a positive correlation (P < 0.001) was observed. Also, a positive correlation (P < 0.01) was seen between the values for free iron release (24 h incubation) and the values for HbF. These results suggest that the presence of HbF is a condition favourable to iron release. Since in beta-thalassemia the persistance of HbF is related to the lack or deficiency of beta chains and therefore to the excess of alpha chains, the observed correlation between free iron and HbF, is consistent with the hypothesis by others that excess of alpha chains represents a prooxidant factor.     

Photosystem II efficiency and mechanisms of energy dissipation in iron-deficient,field-grown pear trees (Pyrus communis L.)

The dark-adapted Photosystem II efficiency of field-grown pear leaves, estimated by the variable to maximum chlorophyll fluorescence ratio, was little affected by moderate and severe iron deficiency. Only extremely iron-deficient leaves showed a decreased Photosystem II efficiency after dark adaptation. Midday depressions in Photosystem II efficiency were still found after short-term dark-adaptation in iron-deficient leaves, indicating that Photosystem II down-regulation occurred when the leaves were illuminated by excessive irradiance. The actual Photosystem II efficiency at steady-state photosynthesis was decreased by iron deficiency both early in the morning and at midday, due to closure of Photosystem II reaction centers and decreases of the intrinsic Photosystem II efficiency. Iron deficiency decreased the amount of light in excess of that which can be used in photosynthesis not only by decreasing absorptance, but also by increasing the relative amount of light dissipated thermally by the Photosystem II antenna. When compared to the controls, iron-deficient pear leaves dissipated thermally up to 20% more of the light absorbed by the Photosystem II, both early in the morning and at midday. At low light iron-deficient leaves with high violaxanthin cycle pigments to chlorophyll ratios had increases in pigment de-epoxidation, non-photochemical quenching and thermal dissipation. Our data suggest that pH could be the major factor controlling thermal energy dissipation, and that large (more than 10-fold) changes in the zeaxanthin plus antheraxanthin to chlorophyll molar ratio caused by iron deficiency were associated only to moderate increases in the extent of photoprotection.This revised version was published online in October 2005 with corrections to the Cover Date.     

A new red cell discriminant incorporating volume dispersion for differentiating iron deficiency anemia from thalassemia minor

A novel red cell discriminant function [MCV2 x RDW/(Hgb x 100)] was compared to six other discriminants in 102 patients with established mild iron deficiency anemia and 33 patients with beta-thalassemia minor. The discriminant incorporates the two key measurements of erythrocyte cell volume distribution, namely the mean (MCV) and standard deviation (RDW), which are known to be helpful for distinguishing between these two frequent causes of microcytic hypochromic anemia. Data used for the learning set to develop the new discriminant were obtained using an electrical impedance automated whole blood analyzer (Coulter S + IV) and were applied as a validation set for six other discriminants. The discriminant was also tested on smaller subsets of the patients groups using data obtained on either an alternate electrical impedance instrument (Sysmex E-5000) or a laser light scattering based system (Technicon H*1). From the comparison it was concluded that use of a discriminant function that incorporates a measurement of red cell volume dispersion results in enhanced accuracy for distinguishing iron deficiency anemia from thalassemia minor.     

Magnetic behavior of human erythrocytes at different hemoglobin states

The effect of a static magnetic field on human erythrocytes at different hemoglobin states (normal, oxidized and reduced hemoglobin) was investigated. Three different blood samples, normal, iron deficiency anemic and beta thalassemia minor, were studied. Measurements of the magnetization curves of the erythrocytes for all blood samples in all states showed diamagnetic behavior; however, oxidation was found to enhance this behavior. These measurements have also shown that the normal and iron deficiency samples in the reduced states exhibit a less diamagnetic response in comparison with the normal state. This result indicates that the reduction process gave rise to a paramagnetic component of the magnetization. Analysis of the measured paramagnetic behavior, using a Brillouin function, gave an effective magnetic moment of 8 muB per reduced hemoglobin molecule for both normal and anemic samples. This result shows that both anemic and normal blood have similar magnetic behavior and the only difference is the number of hemoglobin molecules per erythrocyte. For the beta thalassemia minor blood sample, magnetic measurements showed that both the normal and reduced states have almost the same diamagnetic behavior. However, this diamagnetic response is less than that for the normal state of the iron deficiency anemic sample. This result may indicate a low oxygen intake for the blood in the normal state for the beta thalassemia minor blood. All magnetic measurements were made using a vibrating sample magnetometer using field steps of 0.001 T from 1 T to -1 T.     

The contribution of red cell morphology to the diagnosis of beta-thalassemia trait

The contribution of red cell morphology to the diagnosis of beta-thalassemia trait in a predominantly healthy population was analyzed. The smears from 59 beta-thalassemia trait subjects already identified by DEAE-cellulose microcolumn chromatography, 60 subjects with other forms of microcytosis, and 64 nonmicrocytic subjects were randomly selected and evaluated blind. The "experimental" morphologic criteria were the presence of both basophilic stippling and either microcytosis or target cells. The "control" morphologic criteria were the presence of both microcytosis and poikilocytosis. Although the sensitivity of both sets of criteria were only moderate (73%), the specificity and predictive value of the experimental criteria were very high (99% and 98% respectively).     

Age as a major factor associated with zinc and copper deficiencies in pediatric thalassemia

BackgroundPatients with thalassemia encounter increased consumption of zinc (Zn) and copper (Cu) from chronic hemolysis and increased excretion from iron chelation. Iron-enriched diet restriction may result in low Zn and Cu intakes. Recent data on Zn and Cu status among Thai pediatric patients with thalassemia are lacking. This study aimed to identify frequencies and determine risk factors of Zn and Cu deficiencies among patients with thalassemia.MethodsPatients with transfusion-dependent thalassemia (TDT) receiving iron chelation ≥12 months and nonTDT (NTDT) aged 2–20 years were recruited. Serum Zn and Cu were measured. Dietary intakes were ascertained by interviews.ResultsA total of 209 patients (TDT = 126, NTDT = 83) were enrolled. Zn deficiency seemed to be associated with disease severity as median (IQR) Zn level of TDT was lower than that of NTDT [77 (69−85) vs. 80 (72−88) mcg/dL, p = 0.05], while higher frequency of Zn deficiency was identified in the former (24 % vs. 14 %). In TDT, Zn deficiency was associated with patients >10 years (OR 4.6; 95 %CI 1.1–6.4, p = 0.03), which likely resulted from combined low dietary Zn intake, prolonged exposures to hemolysis and iron chelators. Frequencies of Cu deficiency were similarly low in TDT and NTDT (8% and 7%) with comparable median (IQR) Cu levels of 103 (90−124) and 110 (92−132) mcg/dL, respectively (p = 0.13). Cu levels were inversely associated with age (r=-0.65 and r=-0.62 in TDT and NTDT, respectively; p < 0.001).ConclusionCompared with younger patients, Zn and Cu deficiencies were more common among patients with thalassemia >10 years. Age was a major factor associated with both Zn and Cu deficiencies.     

Distinction of microcytic disorders: comparison of expert, numerical-discriminant, and microcomputer analysis

Presumptive distinction between iron deficiency and heterozygous thalassemia by analysis of the automated blood count and differential continues to be a challenge. We compared two proposed numerical discriminants (MCV2 x MCH, and MCV2 x RDW/100 x Hb) with an analytic microcomputer program (BCDE2 Lea & Febiger). In 7114 subjects, the numerical discriminants and the BCDE2 program correctly identified greater than 90% of thalassemia. In subjects with iron deficiency, the BCDE program was greater than 90% sensitive and specific for positive identification, while the numerical discriminants were less than 75% sensitive and specific at inferential identification. The numerical discriminants, BCDE2, and 17 experts in blood counting were asked to interpret the blood-count data in 7 fully-defined actual cases. The mean experts' score was 5.65 cases correct out of 7. The BCDE program was correct in all cases. The numerical discriminants could not analyze all cases, and both were incorrect in at least one case. We conclude that for the task of analyzing blood counts for microcytic disorders, microcomputer analysis by BCDE outperforms both numerical discriminant functions and analysis by expert hematologists.     

Anemia,differentiating between thalassemia minor and iron deficiency

Many of the conditions noted in examination of the blood of patients with thalassemia minor are much like those observed in patients with iron deficiency anemia. A study was made of similarities and contrasts between blood and bone marrow features in both conditions for purposes of differential diagnosis. A salient distinction is that bone marrow hemosiderin is present in normal amount in patients with thalassemia minor, but not in those with iron deficiency anemia. If therapy with iron does not restore hemoglobin values to normal, thalassemia minor is strongly suspect. Even in the latter disease, however, there may be small fluctuations in hemoglobin values, particularly in pregnancy. One must be alert to this possibility lest a slight, fleeting increase in hemoglobin be mistakenly ascribed to iron therapy.     

ANEMIA—Differentiating Between Thalassemia Minor and Iron Deficiency

Many of the conditions noted in examination of the blood of patients with thalassemia minor are much like those observed in patients with iron deficiency anemia. A study was made of similarities and contrasts between blood and bone marrow features in both conditions for purposes of differential diagnosis. A salient distinction is that bone marrow hemosiderin is present in normal amount in patients with thalassemia minor, but not in those with iron deficiency anemia. If therapy with iron does not restore hemoglobin values to normal, thalassemia minor is strongly suspect. Even in the latter disease, however, there may be small fluctuations in hemoglobin values, particularly in pregnancy. One must be alert to this possibility lest a slight, fleeting increase in hemoglobin be mistakenly ascribed to iron therapy.     

Bias and Sampling Error of the Estimated Proportion of Genotypic Variance Explained by Quantitative Trait Loci Determined From Experimental Data in Maize Using Cross Validation and Validation With Independent Samples

Cross validation (CV) was used to analyze the effects of different environments and different genotypic samples on estimates of the proportion of genotypic variance explained by QTL (p). Testcrosses of 344 F(3) maize lines grown in four environments were evaluated for a number of agronomic traits. In each of 200 replicated CV runs, this data set was subdivided into an estimation set (ES) and various test sets (TS). ES were used to map QTL and estimate p for each run (p(ES)) and its median (p(ES)) across all runs. The bias of these estimates was assessed by comparison with the median (p(TS.ES)) obtained from TS. We also used two independent validation samples derived from the same cross for further comparison. The median p(ES) showed a large upward bias compared to p(TS.ES). Environmental sampling generally had a smaller effect on the bias of p(ES) than genotypic sampling or both factors simultaneously. In independent validation, p(TS.ES) was on average only 50% of p(ES). A wide range among p(ES) reflected a large sampling error of these estimates. QTL frequency distributions and comparison of estimated QTL effects indicated a low precision of QTL localization and an upward bias in the absolute values of estimated QTL effects from ES. CV with data from three QTL studies reported in the literature yielded similar results as those obtained with maize testcrosses. We therefore recommend CV for obtaining asymptotically unbiased estimates of p and consequently a realistic assessment of the prospects of MAS.     

Bias and Sampling Error of the Estimated Proportion of Genotypic Variance Explained by Quantitative Trait Loci Determined From Experimental Data in Maize Using Cross Validation and Validation With Independent Samples.

Cross validation (CV) was used to analyze the effects of different environments and different genotypic samples on estimates of the proportion of genotypic variance explained by QTL (p). Testcrosses of 344 F(3) maize lines grown in four environments were evaluated for a number of agronomic traits. In each of 200 replicated CV runs, this data set was subdivided into an estimation set (ES) and various test sets (TS). ES were used to map QTL and estimate p for each run (p(ES)) and its median (p(ES)) across all runs. The bias of these estimates was assessed by comparison with the median (p(TS.ES)) obtained from TS. We also used two independent validation samples derived from the same cross for further comparison. The median p(ES) showed a large upward bias compared to p(TS.ES). Environmental sampling generally had a smaller effect on the bias of p(ES) than genotypic sampling or both factors simultaneously. In independent validation, p(TS.ES) was on average only 50% of p(ES). A wide range among p(ES) reflected a large sampling error of these estimates. QTL frequency distributions and comparison of estimated QTL effects indicated a low precision of QTL localization and an upward bias in the absolute values of estimated QTL effects from ES. CV with data from three QTL studies reported in the literature yielded similar results as those obtained with maize testcrosses. We therefore recommend CV for obtaining asymptotically unbiased estimates of p and consequently a realistic assessment of the prospects of MAS.     

Measuring "free" iron levels in Caenorhabditis elegans using low-temperature Fe(III) electron paramagnetic resonance spectroscopy

Oxidative stress, caused by free radicals within the body, has been associated with the process of aging and many human diseases. Because free radicals, in particular superoxide, are difficult to measure, an alternative indirect method for measuring oxidative stress levels has been used successfully in Escherichia coli and yeast. This method is based on a proposed connection between elevated superoxide levels and release of iron from solvent-exposed [4Fe-4S] enzyme clusters that eventually leads to an increase in hydroxyl radical production. In past studies using bacteria and yeast, a positive correlation was found between superoxide production or oxidative stress due to superoxide within the organism and electron paramagnetic resonance (EPR) detectable "free" iron levels. In the current study, we have developed a reliable and efficient method for measuring "free" iron levels in Caenorhabditis elegans using low-temperature Fe(III) EPR at g=4.3. This method uses synchronized worm cultures grown on plates that are homogenized and treated with desferrioxamine, an Fe(III) chelator, prior to packing the EPR tube. Homogenization was found not to alter "free" iron levels, whereas desferrioxamine treatment significantly raised these levels, indicating the presence of both Fe(II) and Fe(III) in the "free" iron pool. The correlation between free radical levels and the observed "free" iron levels was examined by using heat stress and paraquat treatment. The intensity of the Fe(III) EPR signal, and thus the concentration of the "free" iron pool, varied with the treatments that altered radical levels without changing the total iron levels. This study provides the groundwork needed to uncover the correlation among oxidative stress, "free" iron levels, and longevity in C. elegans.     

Prevalence of common α-thalassemia determinants in south Brazil: Importance for the diagnosis of microcytic anemia

Alpha thalassemia has not been systematically investigated in Brazil. In this study, 493 unrelated individuals from the southernmost Brazilian state of Rio Grande do Sul were screened for deletional forms of α-thalassemia. One hundred and one individuals had microcytic anemia (MCV < 80 fL) and a normal hemoglobin pattern (Hb A (2) < 3.5% and Hb F < 1%). The subjects were screened for - α(3.7) , - α(4.2) , - α(20.5) , - (SEA) and - (MED) deletions but only the - α(3.7) allele was detected. The - α(3.7) allele frequency in Brazilians of European and African ancestry was 0.02 and 0.12, respectively, whereas in individuals with microcytosis the frequency was 0.20. The prevalence of α-thalassemia was significantly higher in individuals with microcytosis than in healthy individuals (p = 0.001), regardless of their ethnic origin. There were also significant differences in the hematological parameters of individuals with - α(3.7) / αα, - α(3.7) /- α(3.7) and β-thalassemia trait compared to healthy subjects. These data suggest that α-thalassemia is an important cause of microcytosis and mild anemia in Brazilians.     

Factors influencing the etiology of cribra orbitalia in prehistoric Nubia

The present research represents an attempt to explain the occurrence of cribra orbitalia in Nubian skeletal remains associated with Meroitic, X-Group and Christian cultural horizons. The distribution of cribra orbitalia among 285 crania examined revealed a concentration of lesions among infants and aged individuals. While earlier studies have suggested cribra orbitalia may reflect the occurrence of abnormal hemoglobins such as sickle cell anemia or thalassemia, a consideration of the environmental context in which the lesions occurred makes an alternative hypothesis more likely. Particularly in Nubia (past and present), where parasitic infection is high, the diet is poor in iron, weanling diarrhea is frequent and multiparity is the norm, chronic iron deficiency anemia is a more likely causal factor. This interpretation is also compatable with clinical observations of cribra orbitalia among modern individuals suffering from chronic iron deficiency anemia.     

Hemoglobin deficit: an inherited hypochromic anemia in the mouse

The character and pathogenesis of hemoglobin deficit (gene symbol, hbd), an autosomal recessive trait in the mouse, were studied. The main hematological features of hemoglobin deficit are anemia, red cell hypochromia and microcytosis, and reticulocytosis. The absence of raised fecal urobilinogen excretion and frank hyperbilirubinemia and bilirubinuria suggests that excess hemolysis is not the primary cause of the anemia. The raised plasma iron concentration and the failure of the anemia to respond to parenteral iron treatment indicate that the anemia is not due to iron deficiency. The absence of siderocytes and sideroblasts suggests that anemia is probably not due to ferrochelatase deficiency. Thalassemia is excluded by the finding of balanced reticulocyte globin chain synthesis. The markedly elevated levels of free red cell protoporphyrin taken together with the other findings already noted suggest that the anemia of hemoglobin deficit is due to a defect in the erythroid cell iron procurement mechanisms leading in turn to diminished heme and hemoglobin synthesis.