HbE Trait Research Articles

Present scenario of hemoglobinopathies in West Bengal, India: An analysis of a large population

Background: To find out the prevalence of hemoglobinopathies by screening large population in West Bengal from Eastern India. Materials and Methods: A total of 50,487 subjects are screened for hemoglobinopathies from June 2010 to August 2013. A 2.5 ml of venous blood samples were collected in the tri-potassium EDTA vacuum container from each subject and tested with automated blood cell counter (Sysmex KX21) for red cell indices. Diagnosis of hemoglobinopathies was done by VARIANT TM (Bio-Rad Laboratories, Hercules, CA, USA) high performance liquid chromatography (HPLC). Results: Out of 50,487 cases, 44,620 (88.38%) had normal and 5,867(11.62%) showed abnormal hemoglobin fractions on HPLC. Of these, 5,180(10.26%) were heterozygotes (traits) and 687(1.36%) cases were either homozygotes or compound heterozygote for different types of hemoglobinopathies. The following hemoglobinopathies were present: β-thalassemia trait 6.61%, HbE trait 2.78%, sickle cell trait 0.56%, HbD-Punjab trait 0.21%, β-thalassemia major 0.73%, HbEE 0.05%, compound heterozygosity for HbE-β-thalassemia 0.42%, and HbS-β-thalassemia 0.15%. Conclusion: Among the hemoglobinopathies, β-thalassemia trait (6.61%) and HbE trait (2.78%) are prevalent in rural Bengal.

Spectrum of thalassemias and hemoglobinopathies in West Bengal: A study of 90,210 cases by cation exchange high-performance liquid chromatography method over a period of 8 years

Background: Thalassemias and hemoglobinopathies are highly prevalent in India. Identification of these disorders is important for epidemiologic purposes and for prevention of thalassemia major and clinically severe hemoglobinopathies. Objectives: The aim of this study was to determine the prevalence of thalassemias and hemoglobinopathies in patients of a tertiary care hospital of West Bengal. Materials and Methods: A prospective study was undertaken in which 90,210 cases were included over a period of 8 years. Clinical history and family history were obtained from each patient. The venous blood samples were analyzed for complete blood count, liver function tests, serum iron, ferritin, cobalamin and folate levels. High-performance liquid chromatography (HPLC) was performed on the samples with Biorad Variant using beta thalassemia short program. Confirmatory tests were done whenever required. Results: Normal hemoglobin (Hb) pattern was observed in 79,897 (88.57%) cases and abnormalities were detected in 10,313 (11.43%) patients. β (beta) thalassemia trait was the most common abnormality found in 3870 (4.29%) patients. HbE trait was found in 2418 (2.68%) cases, and then Eβ thalassemia in 1406 (1.56%) patients and β thalassemia major/intermedia in 1135 (1.26%) cases. Other variants detected included sickle cell trait, HbE disease, sickle cell disease, sickle β thalassemia, HbD-Punjab trait, double heterozygous state of HbS and HbE, double heterozygous state of HbS and HbD, Hb Lepore, HbJ-Meerut and HbH. Conclusion: Premarital and antenatal screenings are important measures to prevent birth of children with severe Hb disorders. HPLC is a rapid and reliable technique for identification of various Hb fractions.

Pattern of hemoglobinopathies and thalassemias in upper Assam region of North Eastern India: High performance liquid chromatography studies in 9000 patients

The hereditary hemoglobin (Hb) disorders are the most commonly encountered single gene disorders in India. Data pertaining to the pattern of hemoglobinopathies and thalassemias is scarce in North East India, and hence it was considered worthwhile to study these disorders using a large series of patients referred to a clinical diagnostic laboratory. A total of 9000 patients referred for Hb variant analysis were studied to identify hemoglobinopathies and thalassemias in Upper Assam region of North East India. This study was performed by high performance liquid chromatography (HPLC) using BIORAD variant Hb typing system. Out of 9000 patients studied, abnormal Hb fractions were seen in 5320 patients. The HbE gene was detected in 4315 patients of which HbE trait was seen in 2294 followed by HbE disease in 1892. There were 114 HbE beta thalassemia patients and 15 double heterozygotes of HbE with HbS or HbD. Beta thalassemia trait was seen in 313 patients and beta thalassemia homozygous in 32. HbS gene was detected in 460 patients comprising of HbS trait in 189, HbS disease in 203, S beta thalassemia in 53 and double heterozygotes of SD and ES in 15. The rest comprised of HbD trait in 6, delta beta thalassemia in 33, hereditary persistence of fetal hemoglobin trait in 5 and J chain hemoglobinopathy in 8 patients. Evidence of alpha thalassemia though suspected, could not be confirmed. A high incidence of hemoglobinopathies and thalassemias and their combinations is unique for this part of the country.

The Value of Red Blood Cells (Rbc) Indices and Osmotic Fragility Test as Screening Tests in Malay Pregnant Women with Alpha Thalassaemia

Red blood cell (RBC) parameters obtained from hematology analyser has been very useful as the first line screening for thalassaemia. The changes are prominent in most type of β-thalassaemia but milder in α-thalassaemia. RBC parameters could have been normalized especially during pregnancy and in such situation screening these patients would require a different protocol. Blood samples from two hundred (200) Malay pregnant women attended obstetric clinic at Hospital Universiti Sains Malaysia (HUSM) were collected to screen for double α-gene deletion (-SEA, and –THAI), and the two single α-globin gene deletion (–α3.7 and -α4.2). Standard hematological analyses including red blood cell count and indices, and hemoglobin quantitation were performed on the blood samples. Of these, sixteen were excluded as they had HbA2 levels more than 4% and were diagnosed as HbE or β-thalassaemia trait. Then, multiplex GAP polymerase chain reaction (PCR) analyses of α-globin gene were performed on the remaining 184 blood samples. Total of 17 from 184 subjects confirmed to have α-thalassaemia (-α3.7/αα and --SEA/ αα genotype). The RBC indices were compared between those with α-thalassaemia and normal pregnant women and they were significantly different. -α3.7 kb single gene deletion (8.1%) was the commonest type followed by double gene South East Asia (--SEA) deletion (1.1%). We conclude that mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) with the cut off value of less than 86.3fl and 27.4 pg respectively are two useful RBC indices which can be used for screening of α-thalassaemia in pregnant women.

Effect of HbE heterozygosity on the measurement of HbA1c

Measurement of HbA1c provides an excellent measure of glycaemic control for diabetic patients. However, haemoglobin (Hb) variants are known to interfere with HbA1c analysis. In our laboratory HbA1c measurement is performed by Variant II turbo 2.0. The aim of this study is to investigate the influence of HbE trait on HbA1c analysis. Haemoglobin variants were identified by HbA1c analysis in 93 of 3522 samples sent to our laboratory in a period of 1 month. Haemoglobin analysis identified HbE trait in 81 of 93 samples. To determine the influence of HbE trait on HbA1c analysis by Variant II Tubo 2.0, boronate affinity high performance liquid chromatography (HPLC) method (Primus PDQ) was used as the comparison method. Two stage linear regression analysis, Bland Altman plot and Deming regression analysis were performed to analyse whether the presence of HbE trait produced a statistically significant difference in the results. The total allowable error for HbA1c by the Royal Australasian College of Pathologists (RCPA) external quality assurance is 5%. Hence clinically significant difference is more than 5% at the medical decision level of 6% and 9%. Statistically and clinically significant higher results were observed in Variant II Turbo 2.0 due to the presence of HbE trait. A positive bias of ∼10% was observed at the medical decision levels. Laboratories should be cautious when evaluating HbA1c results in the presence of haemoglobin variants.

Effectiveness of the model for prenatal control of severe thalassemia

The aim of the research was to determine effectiveness of the model for prenatal control in reducing new cases of severe thalassemia. Pregnant women at six tertiary centers were recruited to follow the model, consisting of (1) carrier screening using mean corpuscular volume (for alpha-thal-1 and beta-thal) and CMU-E screen (for HbE trait), (2) carrier diagnosis, (3) the couples at risk were counseled and offered prenatal diagnosis, and (4) termination of affected pregnancy. All neonates were evaluated for thalassemia. Of the 12,874 recruited pregnancies, 7008 were valid for analysis. Of them, 281 couples were identified to be at risk, Of the 281, 58 affected fetuses were identified and 55 pregnancies were terminated, whereas three did not accept pregnancy termination. All 6727 neonates at no risk were proven to be unaffected. The model had sensitivity and positive predictive value of 100% and 20%, respectively. The model could detect all of affected fetuses. The model could prenatally identify affected fetuses with a detection rate and negative predictive value of 100%. The model was highly effective to prenatally detect affected fetuses with an acceptable false positive rate.

A community based pilot study on prevalence of hemoglobinopathies among the antenatal women in a rural area of Darjeeling district, West Bengal

Background: Hemoglobinopathies comprising of thalassemias and various hemoglobin (Hb) variants continue to plague the health system of India. Aims: To find out the prevalence of various hemoglobinopathies in the study population and to identify any association with the various ethnic groups residing in rural community of Darjeeling district, West Bengal. Materials and Methods: A community-based cross-sectional study was conducted in Naxalbari block of Darjeeling district, West Bengal from June 2011 to August 2011, involving hemoglobin variant screening by high-performance liquid chromatography (HPLC) method of 188 antenatal women, selected through multistage random sampling technique. Results: 26.6% of mothers had abnormal hemoglobin, the commonest being the Hemoglobin E (Hb E) trait (15.42%). The prevalence of homozygous HbE, HbE βthalassemia, β-thalassemia trait and hemoglobin S-trait in the studied population was 6.91%, 0.53%, 2.12% and 1.06% respectively. There was a single case of the rare hemoglobin J Meerut. Among those having abnormal hemoglobin, 66% belonged to one ethnic group, Rajbanshis. A statistically significant association was noted between Hb E syndrome (comprising of HbE trait, homozygous HbE, HbE-betathalassemia) and Rajbanshis ( P Conclusion: Findings suggest a high prevalence of hemoglobinopathies particularly Hb E in the studied population, especially Rajbanshis and warrant the need of wider community-based initiative for carrier detection and awareness generation.

Prevalence of β-thalassemia and other haemoglobinopathies in six cities in India: a multicentre study

The population of India is extremely diverse comprising of more than 3,000 ethnic groups who still follow endogamy. Haemoglobinopathies are the commonest hereditary disorders in India and pose a major health problem. The data on the prevalence of β-thalassemias and other haemoglobinopathies in different caste/ethnic groups of India is scarce. Therefore the present multicentre study was undertaken in six cities of six states of India (Maharashtra, Gujarat, West Bengal, Assam, Karnataka and Punjab) to determine the prevalence of haemoglobinopathies in different caste/ethnic groups using uniform methodology. Fifty-six thousand seven hundred eighty individuals (college students and pregnant women) from different caste/ethnic groups were screened. RBC indices were measured on an automated haematology counter while the percentage of HbA(2), HbF and other abnormal Hb variants were estimated by HPLC on the Variant Hemoglobin Testing System. The overall prevalence of β-thalassemia trait was 2.78% and varied from 1.48 to 3.64% in different states, while the prevalence of β-thalassemia trait in 59 ethnic groups varied from 0 to 9.3%. HbE trait was mainly seen in Dibrugarh in Assam (23.9%) and Kolkata in West Bengal (3.92%). In six ethnic groups from Assam, the prevalence of HbE trait varied from 41.1 to 66.7%. Few subjects with δβ-thalassemia, HPFH, HbS trait, HbD trait, HbE homozygous and HbE β-thalassemia as well as HbS homozygous and HbS-β-thalassemia (<1%) were also identified. This is the first large multicentre study covering cities from different regions of the country for screening for β-thalassemia carriers and other haemoglobinopathies where uniform protocols and methodology was followed and quality control ensured by the co-ordinating centre. This study also shows that establishment of centres for screening for β-thalassemia and other haemoglobinopathies is possible in medical colleges. Creating awareness, screening and counselling can be done at these centres. This experience will help to formulate a national thalassemia control programme in India.

Pattern ofβ-Thalassemia and Other Haemoglobinopathies: A Cross-Sectional Study in Bangladesh

Thalassemia and other structural haemoglobinopathies are the major erythrocyte formation disorder prevalent in certain parts of the world including Bangladesh. We investigated 600 cases of anaemic patients referred from various parts of the country for diagnosis and counselling during 3 months (April to June 2011) of time. The most common form of haemoglobin (Hb) formation disorder observed in 600 subjects studied was β-thalassemia minor (21.3%). Two other conditions, such as E-β-Thalassemia and HbE trait, were also fairly common (13.5 and 12.1%, resp.) in the total subjects studied. Other forms of haemoglobin formation disorders observed were HbE disease (9.2%), Hb D/S trait (0.7%), β-thalassemia major (0.5%), and δ-β-thalassemia (0.5%). The majority of the haemoglobinopathies belonged to neonatal to childhood period (0–15 years), followed by reproductive age group (16–45 years). Few old-age (46+ years) cases were also detected in course of clinical complications.

Prenatal diagnosis of HbE‐Lepore and Hb Lepore‐β‐thalassemia: the importance of accurate genotyping of the couple at risk

To accurately define unusual genotypes in compound heterozygotes for hemoglobinopathies before undergoing prenatal diagnosis. The HPLC results showed one of the parents in case A and B and a child in case C to be HbE-beta-thalassemia. However, this finding did not correlate with molecular findings. Further screening of their grandparents and analysis of DNA for HbLepore were performed. The presence of a typical hump in the peak in the HbA(2) window (10-15%) in one of the grandparents led to the suspicion of a large deletion. Further molecular screening for HbLepore concluded that case A was compound heterozygous for HbLepore-Hollandia-(δ22/β50) and HbE, case B for HbLepore-Boston-Washington-(δ287/β116) and IVS-I-5(G >C) and case C for HbLepore-Hollandia-(δ22/β50) and HbE. The fetuses were found to be HbE trait in case A and HbLepore trait in case B and C. Accurate genotyping of the couple at risk referred for prenatal diagnosis is important to identify uncommon genotypic combinations in compound heterozygous cases. Extended family studies are often useful to avoid misdiagnosis of the fetus.

Are Red Cell Indices a Reliable Screening Tool in Pre-Natal Screening for the Avoidance of E-Beta Thalassemia and Beta Thalassemia Major Births? Results of a Population Survey From Eastern India

Abstract 5303The state of West Bengal in the eastern part of India has a high prevalence of the carrier states of beta thalassemia and Hb E.Analysis of Hb HPLC screening data from 200 individuals, including adults and children from urban areas around Kolkata in West Bengal, carried out in our institution1, reveals a prevalence 6.5% for beta trait and 5.5% for HbE trait. (unpublished data)It may be possible to reduce births of beta thalassemia major and E beta thalassemia by preventive strategies, including mass screening and awareness campaigns. The best preventive method is debatable, because of the highly variable prevalence of the haemoglobin disorders within defined geographic regions (Weatherall DJ, Blood.2010), the economic factors involved and most importantly the question of acceptance of prevention methods.Because of the social stigma attached to the diagnosis of a genetic disease, families of young men and women diagnosed as thalassemia carriers in community screening programs often suppress this information when marriages are arranged, according to prevalent social practice. Therefore pre-marital screening, in our experience, is unlikely to be effective in reducing the number of beta thalassemia major and E-Beta thalassemia births in eastern India.Antenatal screening on the other hand has the potential for greater acceptability. Concern for the welfare of the unborn child are likely to make prospective parents more amenable to counselling and getting themselves tested for the thalassemia carrier state.We analysed data of 1000 antenatal mothers (Table 1), who had undergone screening for the thalassemia carrier state by red cell indices and Hb HPLC, as part of a government funded screening programme conducted in our institution1 in Kolkata city of West Bengal, India, in order to determine whether red cell indices, which is more economical than Hb HPLC, was an effective screening tool.Results of Hb HPLC screening data of 1000 antenatal women (Table 1)ResultBeta Trait (HbA2% 3.6-6.6)Hb E trait (A2+E% 22.8-42.8)E BetaE DiseaseSickle traitSickle BetaD TraitD diseaseNo of cases (%)58(5.8%)48(4.8%)3(0.2%)2(0.2%)2(0.2%)1(0.1%)11Where both partners were carriers, mutations were confirmed by complementary reverse dot blot hybridisation or DNA sequencing.Red Cell Indices in Thalassemia Carrier States (Table 2)Thalassemia Carrier State (1000 Antenatal Women)MCV (fl)MCH (pg)Mean Range (±95%CI) (±2SD)Mean Range (±95%CI) (±2SD)Beta Trait (n= 58)68.6±1.70 55.4–81.821.80±0.64 16.6–26.8E Trait(n=48)78.8±1.42 67.8–89.826.4±0.62 22.0–30.8Most antenatal women received iron and folic acid supplements, therefore iron status was not separately examined. To exclude the possibility of the effect of pregnancy on red cell indices, evaluation of MCV and MCH in 50 HbE trait individuals and 250 beta trait drawn from community screening data, comprising non-pregnant females and males, with normal serum ferritin values, mean MCV was 76.6fl (range 66.3–86.8fl), mean MCH 25.6pg (range 21.5–29.7pg) for E trait. For beta trait mean MCV was 67.0 fl (range 50–84 fl), mean MCH 21 pg (range 15–26 pg). There was no statistically significant difference from antenatal cases (Student’s t-test ).MCV of <80fl and MCH < 27pg were previously reported to predict most beta thalassemia in pregnancy (Weatherall and Clegg Eds, The Thalassemia Syndromes, Part 4. UK: Blackwell Science Ltd, 2001 ). We tested whether these values could reliably predict beta trait and E trait taken together, in 1000 antenatal women in the present study (Table 3)Red Cell Indices as Predictor of Beta Trait and HbE Trait (Table 3)Sensitivity (%)Specificity (%)Positive Predictive Value (%)Negative Predictive Value (%)No of Beta Trait Missed on Screening (%) n=58No of E Trait Missed on Screening (%) n=48MCV <80fl888542986 (10.3)15 (31.3)MCH <27pg848235.2989 (15.5)17 (35.4) Conclusion–MCH and MCV tend to be higher in E trait compared to beta trait. We recommend that in high prevalence areas, Hb HPLC should be performed in all antenatal women irrespective of red cell indices, to avoid missing thalassemia carriers and facilities for genetic diagnosis and counselling be made available to couples at risk of beta thalessemia major and E beta thalessemia births. Disclosures:No relevant conflicts of interest to declare.

Development of control material for hemoglobin analysis

Background: There is no certified control material for hemoglobin analysis which has the hemoglobin (Hb)A(2)/E level as high as found in ?-thalassemia trait, HbE trait, ?-thalassemia/HbE disease and homozygote of HbE, the thalassemia types found frequently in the Southeast Asian population. The aim of this study was to prepare the lyophilized hemoglobin control materials for hemoglobin analysis. Methods: Washed and dialysed erythrocytes of normal individuals and patients with ?-thalassemia trait, HbE trait, ?-thalassemia/HbE disease, homozygous HbE were lysed in 5% sucrose solution. The lyophilized hemoglobin control materials were prepared by using a freeze-drying (lyophilization) method. The high performance liquid chromatography (HPLC) analysis of lyophilized hemoglobin was performed after storing at ?20?C for 1, 15 and 30?days and for 3?months. Results: The chromatograms of lyophilized hemoglobin control materials showed similar patterns and similar levels of HbA, HbA(2)/E and HbF when compared with equivalent fresh whole blood. Moreover, the lyophilized hemoglobin presented a good correlation coefficient (r>0.990) of relationships between HPLC, low pressure liquid chromatography (LPLC) and capillary electrophoresis (CE) methods. Conclusions: The lyophilized hemoglobin could be developed and used as control materials for hemoglobin analysis.

Hemoglobin Disorders in South India

Cation exchange-high performance liquid chromatography (CE-HPLC) is increasingly being used as a first line of investigation for hemoglobinopathies and thalassemias. Together with a complete blood count, the CE-HPLC is effective in categorizing hemoglobinopathies as traits, homozygous disorders and compound heterozygous disorders. We carried out a one year study in Apollo Hospitals, Chennai (Tamil Nadu, South India) during which 543 abnormal chromatogram patterns were seen. The commonest disorder we encountered was β-thalassemia trait (37.9%), followed by HbE trait (23.2%), homozygous HbE disease (18.9%), HbS trait (5.3%), HbE β-thalassemia (4.6%), HbS β-thalassemia (2.5%), β-thalassemia major (2.3%), HbH (1.6%), homozygous HbS (1.4%), HbD trait (0.7%). The average value of HbA2 in β-thalassemia minor was 5.4%. β-thalassemia major had an average HbF of 88% and in HbH the mean A2 was 1.4%. Among the HbE disorders the HbA2 + HbE was 30.1% in the heterozygous state, 90.8% in the homozygous state and 54.8% in HbE β-thalassemia. In the sickle cell disorders, HbS varied from 30.9% in the trait to 79.9% in the homozygous state to 65.6% in HbS β-thalassemia.

HbA1c Does Not Always Estimate Average Glucose

Hemoglobin A1c (HbA1c) testing can be inaccurate in persons with elevated amounts of Hemoglobin F, or with abnormal hemoglobins found in sickle cell trait, HbC trait and HbE trait. These variants are more prevalent in African and Asian Americans, the same demographic that has an increased risk of diabetes. Variant hemoglobins might cause a false increase or decrease in HbAlc, depending on methodology and manufacturer. Case studies of two African American patients, one with and one without variant hemoglobins, are presented. The major methods used to assay HbAlc, immunoassay, HPLC and boronate affinity are described, and compared for their ability to detect variant hemoglobins. An algorithm is proposed to test new patients using the HPLC method to identify or rule out the presence of the most common variant hemoglobins. Patients with variant hemoglobins can subsequently be assigned to HbAlc methods proven to be accurate in the presence of those hemoglobins.

First Case Report of the Association of HbE Trait and Cold Agglutinin Disease

This is the first report of the association of cold agglutinin disease with HbE trait. The patient was 58 year old male from Assam, India. Thalassemia, sickle cell disease and sickle-HbC have been found to be associated with cold agglutinin disease. Cold agglutinin disease has also been detected in patients with polycythemia vera. Erythrocytes in these disorders are likely to be immature and express more big “I” and little “I” antigens, the target antigens for cold agglutinins. Increased expression of big “I” and little “i” has been demonstrated in sickle cells. Conceivably, the increased expression of “I” and “I” in sickle cell disorders, thalassemia and HbE and polycythemia vera might render the erythrocytes in these disorders more vulnerable to cold agglutinins and hemolysis.

RBC‐Y/MCV as a discriminant function for differentiating carriers of thalassaemia and HbE from iron deficiency

Individuals with alpha-thalassaemia (ATT), beta-thalassaemia (BTT) and HbE trait (HET) are often initially identified based on haematological parameters. However, the values of these parameters usually overlap with iron deficiency anaemia (IDA) and anaemia of chronic disease (ACD). We evaluated the use of RBC-Y in 156 normal individuals and 332 patients; ATT (n = 37), BTT (n = 61), HET (n = 25), HbH disease (n = 5), ACD (n = 67), IDA (n = 83) and ACD with IDA (n = 54). Diagnostic efficiency was analysed by receiver operating characteristics (ROC). MCH was better compared with RBC-Y in discriminating normal from abnormal with sensitivity and specificity of 94% at a cut-off of 26 pg. The Green and King (G&K) index performed the best in discriminating carriers from IDA and ACD with area under the ROC curve (AUC(ROC)) of 0.81. However, if ACD was excluded, RBC-Y/MCV was a good discriminator for carriers from IDA with AUC(ROC) = 0.845. In general screening of populations with ATT, BTT and HET, we propose that hypochromic individuals be first identified by MCH <26 pg and carriers distinguished within these hypochromic individuals from IDA by using RBC-Y/MCV. However, if the prevalence of ACD were high within the screening population, G&K index would be a more suitable discriminator.

New Formula for the Differentiation Between Iron Deficiency Anemia and Thalassemic Carrier in Thalassemic Prevalence Areas: A Study in Healthy School-Age Children.

Abstract 1410Poster Board I-432 INTRODUCTION:In Thailand, the prevalences of iron deficiency anemia (IDA) and thalassemia carrier are high in the range between 20-50%. Screening of both disorders may show the similar results of low hemoglobin (Hb) and mean corpuscular volume (MCV). Therefore, additional laboratory investigations such as ferritin level, percentage of iron saturation, Hb analysis and DNA study may be required for the definite diagnosis, resulting in an increased cost. Previous studies have identified several formulas to differentiate between IDA and thalassemia carrier by using red blood cell (RBC) parameters. However, individual formula may not applicable for particular population. This study aims to establish a formula to differentiae between IDA and thalassemia carrier in school-age children in central Thailand where the prevalences of β and α thalassemia carriers are high. METHODS:After informed consent, blood was collected and tested for complete blood count (CBC), ferritin level, iron level, total iron binding capacity level, Hb analysis, and DNA study for α- thalassemia-1 (or Southeast-Asian deletion). IDA was diagnosed according to the WHO criteria. Iron deficient erythropoiesis (IDE) was defined as normal hemoglobin (Hb) level and low percentage of iron saturation or ferritin levels. Subjects diagnosed of IDA and thalassemia carrier were selected for further analysis. RESULTS:Three hundred and forty-five healthy children, aged (mean±SD) 11.3±1.7 years, were enrolled in the study. The prevalences of IDA, IDE, thalassemia trait (β, a-thalassemia-1, Hb Constant Spring and HbE trait), thalassemia disease (Hb H, β/E and Hb EE), and normal subjects were 11.6%, 38.3%, 26.1%, 2.3% and 28.7%, respectively. The red blood cell (RBC) parameters of normal, IDA, IDE, HbE trait, β trait and α trait groups were shown in the table below. The median (range) values of serum ferritin level in IDA {37.3 (92.6-162.4)}, IDE {34.3 (3-268.2)} groups were significantly different when compared to normal subjects {46.9 (13.4-232.9)}, p<0.001. However, serum ferritin level alone was not enough for the diagnosis of IDA (area under ROC curve of 0.4). Various reported formulas were used to calculate the area under ROC curve, England and Fraser, 0.78; Mentzler, 0.6; Ehsani, 0.57; Shine-Lai, 0.51; RDW/RBC, 0.88; Srivastava, 0.63; RDWI, 0.79; Green and King, 0.82 and RDW, 0.69. The RDW/RBC had the highest area under ROC curve (0.88). Further analysis of the result to differentiae IDA and thalassemia carrier showed that the RDW/RBC value of ≥3 had the sensitivity and specificity of 82.4% and 88.1%, respectively, in our studied subjects. CONCLUSION:Other than the history of iron intake and thalassemia disease in the family, RDW/RBC≥3 may be an additional test to differentiate between IDA and thalassemia carrier in Thai population. In the future, the validity of this formula in a larger scale of population is recommended.GroupHb (g/dL)Hct (%)MCHC (g/dL)MCH (pg)MCV (fL)RDW (%)Platelet x 109Rbc (x1012/L)Normal N=9913.2±0.939.8±2.533.1±0.628.2±3.985.3±3.613.7±1.1312.6±79.94.7±0.3IDA N=4011.2±0.735.0±1.831.6±2.324.0±6.671.6±8.116.1±2.5395.8±88.34.9±0.5IDE N=9913.1±0.739.7±2.033.0±0.627.3±1.982.6±4.814.2±1.2328.8±66.14.8±0.4HbE trait N=3012.8±0.839.0±2.332.8±0.625.3±1.477.0±4.014.3±0.9325.4±57.45.0±0.3β trait N=1012.0±1.137.4±2.529.3±9.123.9±3.973.7±9.716.1±3.4327.8±58.65.1±0.5α trait N=1011.9±0.537.5±1.731.9±0.821.1±1.566.3±4.816.1±1.2334.3±67.95.7±0.5CS trait N=312.7±0.739.5±2.232.0±0.325.1±1.178.4±2.813.9±0.5332±229.95.0±0.1IDE + trait N=3312.8±0.639.4±2.032.6±0.525.1±1.877.1±4.814.5±1.1343±80.35.1±0.4 Disclosures:No relevant conflicts of interest to declare.

Comparison of the accuracy of dichlorophenolindophenol (DCIP), modified DCIP, and hemoglobin E tests to screen for the HbE trait in pregnant women

Comparison of the accuracy of dichlorophenolindophenol (DCIP), modified DCIP, and hemoglobin E tests to screen for the HbE trait in pregnant women

Cation exchange high performance liquid chromatography for diagnosis of haemoglobinopathies

Background: Cation exchange high performance liquid chromatography (HPLC) is emerging as the method of choice for initial screening and diagnosis of haemoglobinopathies. The use of alkaline and acid gel electrophoresis in the developing countries may result in incorrect diagnosis of haemoglobinopathies. The aim of the study is to assess the accuracy and precision of diagnosis of haemoglobinopathies by HPLC and its possible advantage over conventional techniques. Methods: Over a two year period, 955 patients presenting with anaemia were evaluated by HPLC for diagnosis of haemoglobinopathies. All cases showing ‘unknown peaks’ and other rare haemoglobin variants on HPLC were further analyzed by agar gel electrophoresis at alkaline pH (8.6) and at acid pH (6.0). Result: A total of 137 (14.3%) patients showed different abnormal haemoglobins variants. Of these 91 (66.4%) were diagnosed to have beta - heterozygous thalassaemia based on high level of HbA 2 (>3.9%), five (3.7%) as beta - homozygous thalassaemia (HbF 25 – 91%), 15 (10.9%) as sickle cell trait, two (1.5%) as compound heterozygous state of sickle - β + thalassaemia and three (2.2%) patients as homozygous sickle cell anaemia (HbSS). One (0.7%) patient had unknown peak on HPLC with retention time of 4.78 minutes, constituting 16.8% of total haemoglobin. Sickling test was negative. He was diagnosed as HbQ - India heterozygous. Thirteen (9.5%) patients were diagnosed as HbE syndrome and were further sub classified as HbE trait (five cases) and HbE disease (eight cases). Seven (5.1%) patients were diagnosed as Hb - D Punjab heterozygous. Conclusion: The simplicity of the sample preparation, superior resolution of the method and accurate quantitation of haemoglobin concentration, combined with complete automation, makes this an ideal methodology for diagnosis of haemoglobinopathies.

BCL11A is a major HbF quantitative trait locus in three different populations with β-hemoglobinopathies

Increased HbF levels or F-cell (HbF containing erythrocyte) numbers can ameliorate the disease severity of β-thalassemia major and sickle cell anemia. Recent genome-wide association studies reported that single nucleotide polymorphisms (SNPs) in BCL11A gene on chromosome 2p16.1 were correlated with F-cells among healthy northern Europeans, and HbF among Sardinians with β-thalassemias. In this study, we showed that SNPs in BCL11A were associated with F-cell numbers in Chinese with β-thalassemia trait, and with HbF levels in Thais with either β-thalassemia or HbE trait and in African Americans with sickle cell anemia. Taken together, the data suggest that the functional motifs responsible for modulating F-cells and HbF levels reside within a 3 kb region in the second intron of BCL11A.