An online alpha-thalassemia carrier discrimination model based on random forest and red blood cell parameters for low HbA2 cases.

BACKGROUND: Since screening of α-thalassemia carriers by low HbA2 has a low positive predictive value (PPV), the PPV was as low as 40.97% in our laboratory, other more effective screening methods need to be devised. This study aimed at developing a machine learning model by using red blood cell parameters to identify α-thalassemia carriers from low HbA2 patients. METHODS: Laboratory data of 1213 patients with low HbA2 used for modeling was randomly divided into the training set (849 of 1213, 70%) and the internal validation set (364 of 1213, 30%). In addition, an external data set (n = 399) was used for model validation. Fourteen machine learning methods were applied to construct a discriminant model. Performance was evaluated with accuracy, sensitivity, specificity, etc. and compared with 7 previously published discriminant function formulae. RESULTS: The optimal model was based on random forest with 5 clinical features. The PPV of the model was more than twice the PPV of HbA2, and the model had a high negative predictive value (NPV) at the same time. Compared with seven formulae in screening of α-thalassemia carriers, the model had a better accuracy (0.915), specificity (0.967), NPV (0.901), PPV (0.942) and area under the receiver operating characteristic curve (AUC, 0.948) in the independent test set. CONCLUSION: Use of a random forest-based model enables rapid discrimination of α-thalassemia carriers from low HbA2 cases.

[The value of combined detection of HbA2 and HbF for the screening of thalassemia among individuals of childbearing ages].

OBJECTIVE: To assess the application value of combined detection of HbA2 and HbF for the screening of thalassemia among a population of childbearing age in Quanzhou, Fujian, and determine the optimal cut-off values for the region. METHODS: Capillary hemoglobin electrophoresis and genetic testing for α and ß globin gene mutations were simultaneously carried out on 11 428 patients with suspected thalassemia. Statistical methods were used to analyze the distribution of various types of thalassemia and compare the performance of HbA2 and HbF measurement for the screening of various types of thalassemia. The optimal cut-off values for HbA2 and HbF were determined with the ROC curves. RESULTS: 4591 patients with α, ß, and αß compound thalassemia were identified by genetic testing. The most common genotypes for α and ß thalassemia included --SEA/αα and ß654/ßN, ß41-42/ßN, and ß17/ßN. The ROC curves were drawn to compare the performance of HbA2 screening for α-, ß-, αß-compound, static α-, mild α-, and intermediate α-thalassemia, and the maximum area under the curves was 0.674, 0.984, 0.936, 0.499, 0.731, 0.956, and the optimal cut-off values for HbA2 were 2.45%, 3.25%, 3.65%, 2.95%, 2.55%, 1.75%, respectively. CONCLUSION: HbA2 is an efficient indicator for identifying intermediate types of α-, ß-, and αß compound thalassemia. The combination of HbA2 and HbF measurement can effectively detect carriers for ß-thalassemia mutations.

[Diagnostic Cut-Off Value of RDW for Screening Thalassemia and the Combined Determination of MCV, MCH, HBA 2 and RDW].

OBJECTIVE: To explore the value of red cell distribution width (RDW), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and hemoglobin (Hb) A2 combined determination scheme for screening thalassemia. METHODS: The RDW levels of thalassemia group and healthy control group were detected and compared. The efficiency of RDW for screening thalassemia was evaluated by receiver operating characteristic (ROC) curve. The diagnostic cut-off value of RDW was also acquired by Youden index. Then, 3 groups for thalassemia screening scheme were set, including MCV+MCH+HBA 2, MCV+MCH+RDW(>16.0)+HBA 2 and MCV+MCH+RDW(>15.15)+HBA 2. The performances of the 3 groups were evaluated through screening 621 clinical suspected cases of thalassemia. RESULTS: The RDW level in thalassemia group was significantly higher than that in healthy control group (P<0.05). The diagnostic cut-off value for screening thalassemia was RDW>15.15, when the Youden index was the biggest among all data. The sensitivity, specificity, positive predictive value, negative predictive value, false negative rate and consistency rate of MCV+MCH+RDW(>15.15)+HBA 2 group was 75.46%, 48.83%, 26.50%, 89.06%, 24.54%, and 54.06%, respectively. CONCLUSION: The diagnostic cut-off value of RDW for thalassemia screening has been established. The group of MCV(<82.0 fl)+MCH(<27.0 pg)+HBA 2(<2.5% or ≥3.5%)+RDW(>15.15) has a best efficiency among the 3 groups to screen thalassemia.

[Diagnostic Value of HBA2 in Different Types of Thalassemia].

OBJECTIVE: To explore the diagnostic value of HBA2 in different types of thalassemia by analyzing the sensitivity and missed diagnosis rate of HBA2 in different types of thalassemia. METHODS: 1 178 couples in the department of women's health of Chongqing maternal and child health hospital were selected for pregnancy examination. Peripheral venous blood was extracted and analyzed for parallel blood routine test, hemoglobin capillary electrophoresis and thalassemia gene detection. RESULTS: A total of 265 cases of thalassemia gene carriers were screened out in 1 178 couples； 91.3% ß0 heterozygous thalassemia and 94.74% ß+ heterozygous thalassemia could be screened out using HBA2 > 3.5% as cut-off value; 30.19% stationary α-thalassemia and 66.07% standard α-thalassemia could be screened out using HBA2 < 2.5% as cut-off value. The rate of missed diagnosis of α-thalassemia and ß-thalassemia was 47% and 1.01% respectively when the blood routine screening is positive. CONCLUSION: HBA2 shows different diagnostic value for different types of α-thalassemia and ß-thalassemia. The sensitivity of HBA2 > 3.5% is higher than that of HBA2 < 2.5%. More attention should be paid to the further screening of patients with normal electrophoresis results when the blood routine screening is positive.

[Values of MCV,MCH,ROFT and HbA2 for Screening α-Thalassemia in Guangdong Area].

OBJECTIVE: To investigate the values of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell osmotic fragility test(ROFT) and hemoglobin A2(HbA2) in screening of α-thalassemia in Guangdong area. METHODS: A total of 285 peripheral blood samples in patients treated in our hospital from January 2017 to December 2017 were collected. The detection of thalassemia gene was used as the gold standard, while blood routine examination, hemoglobin electrophoresis, and red cell osmotic fragility test were simultaneously performed. The optimal cut-off values in MCV, MCH, ROFT and HbA2 in α-thalassemia were determined by receiver operator characteristic curve (ROC curve). RESULTS: The most common types of α-thalassemia gene was --SEA/αα (54.59%). Compared with the control group, the differences in MCV, MCH, ROFT and HbA2 showed statistically significantce between different types of α-thalassemia (P<0.05). The best cut-off values of MCV, MCH, ROFT, and HbA2 in the diagnosis of α-thalassemia were 81.45 fl, 27.35 pg, 79.95%, and 2.55% respectively. CONCLUSION: For different laboratories, the cut-off values need to be established for screening α-thalassemia suitable in their own local region．The values of MCV, MCH, ROFT and HbA2 shows higher accuracy and sensitivity in the diagnosis of α-thalassemia. It is recommended to use MCV<81.45fl, MCH<27.35 pg, ROFT<79.95% and HbA2<2.55% as the standards for screening α-thalassemia in Guangdong area.

[Influence of Iron Deficiency on the Index of Thalassemia Screening].

OBJECTIVE: To investigate the influence of iron deficiency on the index of thalassemia screening. METHODS: 876 blood samples of the couples at childbearing age, who underwent red blood cell analysis, hemoglobin electrophoresis, ferritin and gene diagnosis were selected. The samples were divided into normal, iron deficiency, αthalassemia, α-thalassemia combining with iron deficiency, ß-thalassemia and ß-thalassemia combining with iron deficiency group. The differences of hematology index and hemolobin value A2 between each groups were analyzed. RESULTS: The value of Hb, MCV, MCH, MCHC in iron deficiency, αthalassemia, α-thalassemia combining with iron deficiency, ß-thalassemia and ß-thalassemia combining with iron deficiency group all were lower than that of normal group, while the value of RDW-CV was higher, in which the difference between ß-thalassemia was the highest. The distribution of HbA2 among each groups was not significantly different expect of ß-thalassemia. There was no significant correlation between HbA2 and ferritin level. CONCLUSION: RDW-CV increases in both iron deficiency and thalassemia. Iron deficiency has no significant effect on the level of hemoglobin A2.

Intraocular pressure in subjects with beta-thalassemia minor / Pressão intraocular em indivíduos com talassemia beta minor

ABSTRACT Purpose: Beta-thalassemia minor, a common hereditary blood disorder in Mediterranean countries such as Turkey, is associated with insulin resistance. Insulin resistance, in turn, can be associated with excessively high intraocular pressure and, therefore, intraocular pressure-induced blindness. This study aimed to investigate the intraocular pressure in subjects with beta-thalassemia minor. Methods: We conducted a cross-sectional study comprising of 203 subjects divided into two groups: beta-thalassemia minor (103) and healthy (100).Hemoglobin electrophoresis was performed and complete blood count, blood pressures, serum fasting glucose and insulin levels were measured. All subjects underwent ophthalmological examinations including intraocular pressure measurements. Results: Intraocular pressure in the subjects with beta-thalassemia minor was significantly lower than that in healthy subjects (p=0.007). Additionally, intraocular pressure was inversely correlated with hemoglobin A2 levels (p=0.001, r=-0.320). Serum insulin and systolic blood pressure were significantly higher in subjects with beta-thalassemia minor (p=0.03, p=0.009, respectively). Conclusion: Subjects with beta-thalassemia minor had lower intraocular pressure than healthy controls, suggesting beta-thalassemia minor may actually protect against high intraocular pressure.

RESUMO Objetivo: Beta-talassemia menor é uma doença hereditária comum no sangue em países mediterrâneos como a Turquia e está associada à resistência à insulina. A resistência à insulina por sua vez, pode estar associada à pressão intraocular excessivamente alta e, portanto à cegueira induzida pela pressão intraocular. Este estudo teve como objetivo investigar a pressão intraocular em indivíduos com beta-talassemia menor. Métodos: Foi realizado um estudo transversal compreendendo 203 indivíduos divididos em 2 grupos: beta-talassemia menor (103) e saudável (100). Eletroforese de hemoglobina foi realizada e hemograma completo, pressão arterial, glicemia em jejum e níveis de insulina medidos. Todos os indivíduos foram submetidos foram submetidos a exames oftalmológicos, incluindo medidas de pressão intraocular. Resultados: A pressão intraocular nos indivíduos com beta-talassemia menor foi significativamente menor do que em indivíduos saudáveis (p=0,007). Além disso, a pressão intraocular foi inversamente correlacionada com os níveis de hemoglobina A2 (p=0,001, r=-0,320). Insulina sérica e pressão arterial sistólica foram significativamente maiores em indivíduos com beta-talassemia menor (p=0,03, p=0,009, respectivamente). Conclusão: Os indivíduos com beta-talassemia menor tiveram pressão intraocular menor do que os controles saudáveis, sugerindo que a beta-talassemia menor pode, na verdade, proteger contra a alta pressão intraocular.

Can Couples With MCV≥80, MCH<26, HbA2<3.2, HbF<3 be Classified as Low-risk ß-Thalassemia Group?

BACKGROUND AND AIM: Thalassemia screening instructions in Iran categorizes couples with mean corpuscular volume (MCV)=75 to 80, mean corpuscular hemoglobin (MCH)=26 to 27, hemoglobin A2 (HbA2)<3.5, and hemoglobin fetal (HbF)<3 indices as low-risk couples, and therefore further genetic testing is not obligatory. This study examined the possibility of classifying couples with MCH<26 and MCV≥80 indices in the low-risk group when their HbF was <3 and HbA2 was <3.2. METHODOLOGY: This was a cross-sectional study. The data included results from cell blood count and HbA2 prenatal diagnosis (PND) and HbF tests taken by 22 health care centers in Esfahan province, central Iran, throughout the years 2012-2016. The inclusion criterion was the registering of MCV, MCH, and PND results of the participants. From the 5804 participants, 5624 individuals were included in the study. RESULTS: The sensitivity and specificity of the screening program were 99.7 and 53.12, respectively. Ten cases (0.18%) with thalassemia trait had indices MCV≥80 and MCH≥26 including 3 cases (0.05%) with concurrent α and ß-thalassemia mutations and 7 cases (0.12%) with HbS mutations. Altogether, 553 subjects (9.83%) had MCV≥80 and MCH<26 indices, and only 1 case (0.018%) was found with ß-thalassemia mutations (codon8 (-AA)/WT genotype). CONCLUSIONS: Subjects with MCV≥80, MCH<26, HbA2<3.2, and HbF<3 cell blood count indices could be grouped as low-risk couples if normal HbA2 and HbF values are considered. The results of this study also indicate that there is a chance of missing concurrent α and ß-thalassemia or HbS hemoglobinopathies in the current screening program given the fact that genetic testing is not considered for couples with MCV and MCH in the low-risk range. HbF testing could be conducted to prevent these missing cases.

Analysis of the Genotypes in a Chinese Population with Increased Hb A2 and Low Hematological Indices.

Increased Hb A2 is considered the most reliable hematological finding for the identification of ß-thalassemia (ß-thal) carriers. The aim of this study was to determine the underlying genetic factors associated with a high Hb A2 level in a Chinese population. Subjects were recruited from couples preparing for pregnancy who participated in the thalassemia screening program during a 2-year period. DNA analyses were used for diagnosis of ß-thal and other genetic factors. A total of 5985 adults who screened positive for ß-thal were recruited. Of these, 5933 (99.1%) were detected to have a ß-thal mutation. In the remaining 52 (0.9%) individuals without mutations involving the ß-globin gene cluster, 16 were found to have Krüppel-like factor 1 (KLF1) gene variants, and two had an α-globin gene triplication. There were still 34 individuals with unknown genetic factors for their raised Hb A2 values. The results of this study indicate that genetic factors other than ß-thal can rarely contribute to the elevation of Hb A2. These subjects usually have borderline microcytic red cell indices and Hb A2 values.

A rapid quantification method for the screening indicator for ß-thalassemia with near-infrared spectroscopy.

Near-infrared (NIR) spectroscopy combined with chemometrics was applied to rapidly analyse haemoglobin A2 (HbA2) for ß-thalassemia screening in human haemolysate samples. The relative content indicator HbA2 was indirectly quantified by simultaneous analysis of two absolute content indicators (Hb and Hb∙HbA2). According to the comprehensive prediction effect of the multiple partitioning of calibration and prediction sets, the parameters were optimized to achieve modelling stability, and the preferred models were validated using the samples not involved in modelling. Savitzky-Golay smoothing was firstly used for the spectral pretreatment. The absorbance optimization partial least squares (AO-PLS) was used to eliminate high-absorption wave-bands appropriately. The equidistant combination PLS (EC-PLS) was further used to optimize wavelength models. The selected optimal models were I=856nm, N=16, G=1 and F=6 for Hb and I=988nm, N=12, G=2 and F=5 for Hb∙HbA2. Through independent validation, the root-mean-square errors and correlation coefficients for prediction (RMSEP, RP) were 3.50gL-1 and 0.977 for Hb and 0.38gL-1 and 0.917 for Hb∙HbA2, respectively. The predicted values of relative percentage HbA2 were further calculated, and the calculated RMSEP and RP were 0.31% and 0.965, respectively. The sensitivity and specificity for ß-thalassemia both reached 100%. Therefore, the prediction of HbA2 achieved high accuracy for distinguishing ß-thalassemia. The local optimal models for single parameter and the optimal equivalent model sets were proposed, providing more models to match possible constraints in practical applications. The NIR analysis method for the screening indicator of ß-thalassemia was successfully established. The proposed method was rapid, simple and promising for thalassemia screening in a large population.

Thalassaemia screening and confirmation of carriers in parents.

Haemoglobinopathies are among the most common inherited monogenic disorders worldwide. Thalassaemia screening for carrier status is recommended for adults of reproductive age if suspected of being at risk. Conventional laboratory methods for screening include the assessment of haematological indices, and high-performance liquid chromatography, capillary electrophoresis or isoelectric focusing to measure the levels of HbA2 and HbF, and to identify haemoglobin variants. Each screening method has its advantages and disadvantages, the main disadvantage being that none can fully resolve all variants. The complex nature of the genetics of haemoglobinopathies necessitates expertise in the interpretation of screening results to evaluate the most likely genotypes, which must then be confirmed using the DNA diagnosis. This review highlights the limits and pitfalls of each screening technique, and outlines a rational combination of different methods to overcome issues in thalassaemia carrier detection.

[Analysis of Cut-off Value in Screening of Thalassemia by Capillary Hemoglobin Electrophoresis for Pregnant Women from Shenzhen Region of China].

OBJECTIVE: To investigate the cut-off value in screening of thalassemia in pregnant women from Shenzhen region by capillary hemoglobin electrophoresis. METHODS: The data of capillary hemoglobin electrophoresis and genetic diagnosis of thalassemia from 2122 examined prenatal women were retrospectively analyzed. Capillary hemoglobin electrophoresis and α-, ß- genetic diagnosis of thalassemia were carried out for every woman. Hemoglobin electrophoresis was performed using Capillarys 2 full-automated electrophoresis instrument. Gap polymerase chain reaction and reverse dot blot were used for genetic diagnosis of thalassemia genotyping test. The cut-off value in screening of thalassemia was determined by receiver operating characteristic curve and next to analyze the value of HbA2 and HbF in screening of thalassemia using the decided cut-off value. RESULTS: The areas under the curve (AUC(Roc)) of HbA2 for diagnosis of α-, ß- thalassemia were 0.75 and 0.981 respectively, and the AUC(Roc) of HbF for diagnosis of ß-thalassemia was 0.787. When HbA2 ≤ 2.55 was taken as the cut-off value of HbA2 for diagnosis of α-thalassemia, the sensitivity, specificity, positive likelihood ratio (LR(+)) and negative likelihood ratio (LR(-)) were 89.5%, 54.8%, 1.98, 0.19 respectively. When HbA2 ≥3.9 was taken as the cut off value of HbA2 for diagnosis of ß-thalassemia, the sensitivity, specificity, LR(+) and LR(-) were 96.1%, 99.8% 480.5, 0.04 respectively. When HbF ≥0.75 was taken as the cut off value of HbF for diagnosis of ß-thalassemia, the sensitivity, specificity, LR(+) and LR(-) were 83.6%, 61.8% respectively. CONCLUSION: The cut-off value in screening of thalassemia by capillarys 2 full automated electrophoresis instrument is different from that of the traditional method of hemoglobin electrophoresis, such as cellulose acetate membrane electrophoresis and agarose gel electrophoresis. Each laboratory should establish their own respective cut off value.

Effect of Iron Deficiency on the Phenotype of β-Thalassaemia Trait.

The objective of this study was to determine the effect of iron deficiency on Hb-A2 level in ß-thalassaemia trait and to determine the frequency of individuals with ß-thalassaemia trait who could be missed due to concomitant iron deficiency. A total of 120 patients were studied, out of which 23 were iron deficient (serum ferritin < 20 ng/ml). Mean Hb-A2 in the iron deficient individuals was 4.1 ± 0.47% as compared to 5.1 ± 0.58% in the remaining 97 individuals without iron deficiency (p < 0.001). In the 120 individuals with ß-thalassaemia trait, mean Hb-A2 was 5.8% with range 3 - 6.8% and confidence interval was 95%. In 2 individuals with ß-thalassaemia trait, Iron deficiency was observed and showed Hb-A2 less than 3.5%. These could have been missed while screening by Hb-A2 estimation alone. Co-existence of Iron deficiency and ß-thalassaemia trait may mask the diagnosis of beta thalassaemia trait and such individuals can be missed during screening by Hb-A2 estimation alone.

Mutation screening of the Krüppel-like factor 1 gene using single-strand conformational polymorphism in a cohort of Iranian ß-thalassemia patients.

The Krüppel-like factor 1 (KLF1) is an essential erythroid-specific transcription factor. Mutations in the human KLF1 gene have different phenotypic effects, ranging from increased Hb F levels to the disruption of erythropoiesis. Here, we screened 227 Iranian ß-thalassemia (ß-thal) patients for the presence of KLF1 mutations by using the single-strand conformational polymorphism (SSCP) approach. Our aim was to assess the potential effect of these mutations on the ß-thal disease severity. After screening, two variants were found. One patient carried a potentially deleterious variant (Polyphen-2) in exon 2 (p.F182L). Another patient was homozygous for a previously unreported intronic variant (KLF1: c.911 + 84A > G). The patient with the p.F182L variant (KLF1: c.544T > C) had noticeably high Hb A2 levels (7.6%), consistent with the phenotypic effect of several previously characterized KLF1 mutations in the same exonic region. In addition, he had higher platelet counts (1,069,000/µL) compared to other patients in the cohort.

Prevalence of thalassaemia, iron-deficiency anaemia and glucose-6-phosphate dehydrogenase deficiency among Arab migrating nomad children, southern Islamic Republic of Iran.

This study investigated the prevalence of iron-deficiency anaemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency and ß-thalassaemia trait among Arab migrating nomad children in southern Islamic Republic of Iran. Blood samples were analysed from 134 schoolchildren aged < 18 years (51 males, 83 females). Low serum ferritin (< 12 ng/dL) was present in 17.9% of children (21.7% in females and 11.8% in males). Low haemoglobin (Hb) correlated significantly with a low serum ferritin. Only 1 child had G6PD deficiency. A total of 9.7% of children had HbA2 ≥ 3.5 g/dL, indicating ß-thalassaemia trait (10.8% in females and 7.8% in males). Mean serum iron, serum ferritin and total iron binding capacity were similar in males and females. Serum ferritin index was as accurate as Hb index in the diagnosis of iron-deficiency anaemia. A high prevalence of ß-thalassaemia trait was the major potential risk factor in this population.

[The value of MCV, MCH and HbA(2) in laboratory screening of thalassemia].

OBJECTIVES: To explore the roles of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and hemoglobin A(2) (HbA(2)) in the laboratory screening of thalassemia, and to find optimal screening modality for different conditions. METHODS: From September 2008 to May 2011, 1384 subjects underwent thalassemia screening at Department of Obstetrics and Gynecology of Nanfang Hospital. Of them, 1036 cases were diagnosed with thalassemia (408 α-thalassemia, 608 ß-thalassemia, and 20 αß compound thalassemia, thalassemia group) and 348 without thalassemia, non-thalassemia group. All subjects were screened respectively for MCV, MCH and HbA(2). Analyses were performed in all subjects to assess the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy respectively associated with MCV, MCH and HbA(2) alone, combination of MCV and MCH, and combination of MCV, MCH and HbA(2). RESULTS: (1) In the thalassemia group, the sensitivity of MCV alone was 92.9% (379/408) for α thalassemia, 99.3% (604/608) for ß thalassemia and 100.0% (20/20) for αß compound thalassemia. In the non-thalassemia group, the specificity of MCV alone was 75.0% (261/348). (2) In the thalassemia group, the sensitivity of MCH alone was 92.9% (379/408) in α thalassemia, 99.0% (602/608) in ß thalassemia and 100.0% (20/20) in αß compound thalassemia. In the non-thalassemia group, the specificity of MCH alone was 72.7% (253/348). (3) The sensitivity of Hb A(2) alone was 67.4% (275/408) for α thalassemia, 97.5% (593/608) for ß thalassemia, and 100% (20/20) for αß compound thalassemia while it's specificity was 72.4% (252/348) in the non-thalassemia group. (4) With positive indexes of MCV, MCH and MCV + MCH, when HbA(2) > 3.5% it had a high value in ß-thalassemia screening, but when HbA(2) < 2.5% it had little value in α-thalassemia screening. (5) As a single marker, MCV and MCH had better sensitivity, specificity, positive predictive value, negative predictive value and diagnosis accuracy than HbA(2). MCV + MCH was the best for overall screening, but for ß thalassemia screening, MCV + MCH + HbA(2) was the best. CONCLUSIONS: MCV and MCH are suitable for epidemic screening in a large population, physical examination and premarital check-up. Hb electrophoresis and thalassemia gene diagnosis are recommended for subjects with positive MCV and MCH indexes. Diagnoses of α and ß-thalassemia gene are recommended for pregnant women with positive MCV and MCH indexes.

Screening immediate family members for carrier identification and counseling: a cost-effective and practical approach.

OBJECTIVE: To screen immediate family members of thalassaemia patients for carrier identification and counselling. METHODS: The cross-sectional study was conducted at an urban thalassaemia treatment and prevention centre in Karachi, Pakistan, from January to December 2008, and involved 188 siblings of 100 thalassaemia patients. Complete blood count, including haemogram, was performed in the siblings. Samples with MCV < 75fl and MCH < 25% were subjected to haemoglobin-electrophoresis. Haemoglobin A2 of 3.5% to 7.0% was labelled as beta-thalassaemia minor. Those with haemoglobin A2 of 3.0-3.4% but red blood cell count of > 4.5 x 1012/L were reported as equivocal and were screened for iron deficiency anaemia and a repeat haemoglobin A2 estimation was done on high performance liquid chromatography. Equivocal results of the chromoatography were screened for thalassaemia mutation. Mean values along with standard deviation were worked out for relevant variables. RESULT: Of the 188 subjects, there were 124 (66%) males and 64 (34%) females. The mean age was 16.5 +/- 6.3 years (range: 3 months to 30 years) and the mean family size was 1.88 +/- 3.8 (range: 1-12) children per family. There were 51 (51%) first-cousin marriages in this group. Of the siblings, 65 (34.5%) were identified as normal, while 117 (62.2%) were reported as beta-thalassaemia carriers. Six asymptomatic siblings were reported as consistent with beta-thalassaemia major. CONCLUSION: There were 62.2% siblings identified as beta thalassaemia carriers in the study as opposed to 5-8% carriers in the general population. We also identified six asymptomatic and unidentified cases of beta-thalassaemia intermedia in these families. Therefore, in our context where both resources and budgets are limited, it is practical to focus on siblings of identified thalasaemia patients.

First report on the co-inheritance of beta-globin IVS-I-5 (G-->C) thalassemia with delta globin CD12 {Asn-->Lys (AAT-->AAA)}HbA2-NYU in Iran.

BACKGROUND: Co-inheritance of ß- and δ-globin mutations in Iran is not uncommon. This situation may interfere with correct diagnosis and genetic counseling of α- and ß-thalassemia in screening programs. Here we report the co-inheritance of ß- and δ-globin gene mutations in an individual with microcytosis, hypochromia and a normal hemoglobin A2 (HbA2) level. METHODS: Genomic DNA extraction, amplification refractory mutation system (ARMS) polymerase chain reaction and direct DNA sequencing of δ- and ß-globin genes were exploited for detection of the mutations in these two genes in an individual with low hematological indices and normal HbA2. RESULTS: ARMS-PCR technique revealed the ß(+) IVSI-5 (G to C) mutation and direct DNA sequencing of the δ-globin gene detected a previously reported delta codon 12 (AAT-->AAA) HbA2-NYU. This study reports HbA2-NYU in association with the ß IVSI-5 (G to C) mutation in Iran. DISCUSSION: This report emphasizes that normal HbA2 expression in a ß-goblin carrier is due to mutation in the δ-globin gene and may cause misdiagnosis of thalassemia.

Reliability of red blood cell indices and formulas to discriminate between beta thalassemia trait and iron deficiency in children.

Iron deficiency (ID) and beta thalassemia trait (betaTT) are the most common causes of hypochromia and microcytosis. This study evaluates the reliability of some of the red blood cell (RBC) indices and the formulas used in the differentiation between betaTT and ID in a cohort of 458 children aged between 1.8 and 7.5 years (mean age 5.6+/-1.7 years) with mild hypochromic microcytic anemia. Within this group, 243 were confirmed with ID and 215 with betaTT. Red cell indices derived from automated red cell analyzers were used to evaluate the following discriminant indices and formulas: Mentzer Index (MI), Green and King Index (G&K), England and Fraser Index (E&F), RBC Distribution Width Index (RDWI), RBC distribution width (RDW) and RBC count. Sensitivity (SENS), specificity (SPEC), positive and negative prognostic value, efficiency (EFF) and Youden's Index (YI) were evaluated. For each index or formula Gauss curves were constructed. The highest SENS was obtained with RDWI (78.9%), while the highest SPEC and YI with E&F (99.1 and 64.2% respectively), the highest EFF (80.2%) with G&K. Gauss curves obtained from betaTT and ID children showed a different degree of overlap for each formula or index. In conclusion, none of RBC indices or formulas appears reliable to discriminate between betaTT and ID subjects.

Evaluation of an automated capillary electrophoresis system in the screening for hemoglobinopathies.

BACKGROUND: The biochemical diagnosis of hemoglobinopathies (thalassemias and hemoglobin (Hb) variants) is based on the separation and the quantification of Hb fractions. HPLC is the most commonly used method but capillary electrophoresis (CE) methods have also been developed successfully. The Capillarys II system is the first fully automated CE system that allows the quantification of Hb A2 and Hb F and the separation of Hb variants. We evaluated the ability of this system to separate and identify Hb variants and to quantify Hb A2 and Hb F. MATERIAL AND METHODS: The separation of 18 different Hb variants was studied and the imprecision on migration times was calculated for the three most frequent ones. The total imprecision on Hb A2 and Hb F quantification was determined. The results obtained for 44 patients were compared with those given by HPLC. The interference on Hb A2 measurement due to the presence of Hb S was studied. RESULTS: Fourteen out of the 18 variants tested, including all variants of clinical importance, were separated from Hb A. Imprecision on migration times was less than 1%. For Hb A2 quantification, imprecision was less than 3.5% and for Hb F, less than 7.0%. The comparison with HPLC showed an acceptable agreement between both methods but a systematic negative bias for Hb A2 and both proportional and systematic biases for Hb F. No interference from the presence of Hb S on the quantification of Hb A2 was observed. CONCLUSIONS: The fully automated Capillarys Hemoglobin method allows the detection and the separation of the most common Hb variants. It provides also a precise, quick, and very easy quantification of Hb F and Hb A2, even in the presence of Hb S. It is very suitable for routine investigation of hemoglopinopathies.