Effect of megaloblastic anemia on hemoglobin A2 and diagnosis of ?-thalassemia trait

Context: ?-thalassemia trait is usually diagnosed by raised hemoglobin A2 (HbA2). The presence of megaloblastic anemia can cause an increase in HbA2 and create a diagnostic dilemma. Here, we have analyzed the effect of vitamin B12 and folic acid supplementation on HbA2 and diagnosis of ?-thalassemia trait in cases of megaloblastic anemia with raised HbA2. Materials and Methods: Cases of megaloblastic anemia with raised HbA2 on high-performance liquid chromatography (HPLC) were supplemented with vitamin B12 and folic acid. Post-treatment evaluation was done after 2 months. Cases showing adequate hematological response were subjected to statistical analysis. Based on post-treatment HbA2 value, the cases were diagnosed as normal, borderline raised HbA2, or ?-thalassemia trait. Pre- and post-treatment values of red cell parameters and HbA2 were analyzed. Results: There was a significant decrease in HbA2 value after vitamin B12 and folic acid supplementation. The diagnosis was changed in 70.97% of the cases after treatment. The chance of inconclusive diagnosis was decreased from more than 50% to less than 10%. Pre-treatment mean corpuscular volume (MCV) and HbA2% showed a significant difference between the thalassemic and normal groups. Conclusions: Megaloblastic anemia can lead to false-positive diagnosis of ?-thalassemia trait on HPLC. Repeat HPLC should be done after adequate supplementation of vitamin B12 and folic acid in cases of megaloblastic anemia with raised HbA2. Red cell parameters are not helpful to suspect ?-thalassemia trait in presence of megaloblastic anemia. However, HbA2% on HPLC can be a useful parameter to suspect or exclude ?-thalassemia trait in cases of megaloblastic anemia.

A rare δ-globin gene: c.349 C>G variant in Guangdong Province / 中华地方病学杂志

Objective:A rare case of δ-globin gene (HBD) mutation in Guangdong Province was analyzed to provide reference for avoiding misdiagnosis of δ-thalassemia in clinic.Methods:The patient was admitted to Guangdong Maternal and Child Health Hospital, and the peripheral blood sample was collected for hematological phenotypes [mean erythrocyte volume (MCV), mean erythrocyte hemoglobin content (MCH), hemoglobin (Hb)] and Hb typing analysis. The routine deletion and mutation of α-thalassemia and β-thalassemia genes were analyzed by PCR-flow fluorescence hybridization. At the same time, DNA sequencing was used to analyze the type of HBD mutation.Results:The results of hematological phenotypes analysis showed that MCV was 87.9 fl, MCH was 29.3 pg, and Hb content was 140 g/L. The results of Hb typing showed that the contents of Hb F, Hb A 2, Hb A 2 variant, and Hb A were 0.4%, 1.3%, 0.6%, and 97.7%, respectively. No abnormality was found in α-thalassemia and β-thalassemia genes by routine deletion and mutation detection. According to DNA sequencing analysis, the patient had HBD: c.349 C>G variant. Conclusion:The low Hb A 2 content (reference value is 2.5% - 3.5%) in this case is due to the mutation of HBD, HBD: c.349 C>G variant is rare in Chinese population.

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.

Identificación de &-talasemia en anemias microcíticas hipocrómicas refractarias al tratamiento con hierro en Nicaragua / Identification of &-thalassemia in hypochromic microcytic anemias refractory to iron treatment in Nicaragua

Resumen Justificación y objetivo: gran parte de los casos descritos de anemias microcíticas-hipocrómicas corresponden a anemias ferropénicas y síndromes talasémicos. El diagnóstico diferencial se complementa con pruebas de laboratorio como el hierro sérico, ferritina, entre otras; sin embargo, estas son de baja disponibilidad en países en vías de desarrollo. En Nicaragua, el diagnóstico de estas patologías se basa en el historial clínico y análisis hematológicos de rutina. El objetivo de este trabajo fue la implementación de la técnica de cuantificación de hemoglobina A2 en el diagnóstico clínico de β-talasemia. Métodos: se realizó un estudio transversal con 30 pacientes que mostraban microcitosis e hipocromía después de 3 meses de tratamiento con sales de hierro. Se realizó electroforesis de hemoglobina y se utilizó el kit de la casa comercial Beta-Thal HbA2 Quik Column para cuantificar la hemoglobina A2 en cada paciente. El análisis estadístico utilizado fue la prueba de t de student. Se consideraron significativas las diferencias a p<0,05. Esta investigación respetó los principios éticos que conciernen. Se contó con la aprobación del Comité de Ética Institucional, UNAN-Managua. Los participantes dieron su consentimiento informado. Resultados: al aplicar el método para cuantificación de hemoglobina A2, se obtuvo que el 67 % de las muestras presentaron una concentración de hemoglobina A2 mayor al valor de referencia establecido (3,3 %), siendo pacientes diagnosticados para β-talasemia menor. El 33 % restante presentó valores normales de hemoglobina A2 con microcitosis e hipocromía. Se encontraron diferencias estadísticamente significativas entre las medias de glóbulos rojos, volumen corpuscular medio, hemoglobina corpuscular media y hemoglobina A2, entre ambos grupos. Conclusión: el diagnóstico diferencial de anemias microcíticas hipocrómicas refractarias al tratamiento con hierro, se realiza inicialmente por el historial clínico del paciente, pero es necesario contar con pruebas diagnósticas como la cuantificación de hemoglobina A2 que permitan identificar las diversas patologías que cursan con microcitosis e hipocromía.

Abstract Justification and objective: much of the described cases of microcytic-hypochromic anemias are ferropenic anemias and Thalassemia syndromes. The differential diagnosis is complemented by laboratory tests as serum iron, ferritin, among others; However, these are of low availability in developing countries. In Nicaragua, the diagnosis of these diseases is based on clinical history and routine blood analysis. The objective of this work was to implement a technique for quantification of hemoglobin A2 in the clinical diagnosis of β-Thalassemia. Methods: We conducted a cross-sectional study with 30 patients showing hypochromia and microcytosis after 3 months of treatment with iron salts. Hemoglobin electrophoresis was performed, a kit from Beta-Thal HbA2 Quik Column was used to quantify the hemoglobin A2 in each patient. The statistical analysis used was the student's t test. The differences were considered significant at p < 0.05. This research respected ethical principles that concern. It had the approval of the committee of ethics institutional, UNAN-Managua and the participants gave their informed consent. Results: when applying the method for quantification of hemoglobin A2, 67% of samples presented a concentration of hemoglobin A2 greater than the reference value set at 3.3%, these patients were diagnosed with β-Thalassemia minor. The remaining 33% presented normal values of hemoglobin A2 with hypochromia and microcytosis. Statistically significant differences between the averages of red blood cells, mean corpuscular volume, mean corpuscular hemoglobin and hemoglobin A2 between the two groups was observed. Conclusion: The differential diagnosis of microcytic hypochromic anemias refractory to treatment with iron, is initially performed by the clinical history of the patient, but it is necessary to have diagnostic tests such as the quantification of hemoglobin A2, which allow the identification of patients with β-Thalassemia minor within this group. In our study 67% of the studied samples were identified as β-Thalassemia minor.

Analysis of the Coefficient of Variation for the Internal Quality Control of Hemoglobin A2 and Hemoglobin F from 2014 to 2017 / 现代检验医学杂志

Objective To investigate the internal quality control(IQC) of hemoglobin A2 (HbA2) and hemoglobin F (HbF) from 2014 to 2017 in China.Methods The results of IQC were collected from the laboratories which participated in external quality assessment (EQA) of National Center for Clinical Laboratories (NCCL) from 2014 to 2017,then the coefficient of variation (CV) was compared with 1/3TEa (6.67 ％),1/4TEa (5 ％).The proportion of laboratories meeting criteria were calculated to analyze IQC of HbA2 and HbF in China.The data were grouped based on the instruments used in laboratories,the acceptable rates of CVs of HbA2 and HbF in each group under two criteria in 2017 were calculated,respectively.Results In HbA2,more than 84％ of participant laboratories met 1/3TEa criteria and 70.83％ ～84.47％ of laboratories met 1/ 4TEa criteria.In HbF except for 2015,the more than 80％ laboratories whose month and cumulative CVs met 1/3TEa and 1/4TEa criteria accounted for 68.42 ％ ～ 85.07 ％,respectively.Under 1/3TEa and 1/4TEa criteria,sebia capillarys 2 instru ment and fully automatic hemoglobin analyzer bole Variant Ⅱ instrument group the acceptable rates of CVs above 85％,showed good precision for HbA2 and HbF detection.Conclusion At present,the precision level of HbA2 and HbF need to be further improved in laboratories of China,especially HbF.Laboratory should continue to strengthen the internal quality control,establish strict internal quality system to improve detection capacity.

Técnicas convencionales aplicadas al diagnóstico de las hemoglobinopatías / Conventional techniques applied to the diagnosis of hemoglobinopathies / Técnicas convencionais aplicadas ao diagnóstico das hemoglobinopatias

Las hemoglobinopatías son trastornos hereditarios debidos a una gran variedad de defectos que afectan a los genes de globina. El diagnóstico de las hemoglobinopatías resulta de una combinación de estudios clínicos, pruebas de laboratorio y estudio familiar. Las herramientas básicas incluyen hemograma, hemoglobina e índices eritrocitarios (VCM, HCM), morfología de los eritrocitos, recuento de reticulocitos y perfil de hierro. Las determinaciones complementarias son electroforesis de hemoglobina que permite separar las diferentes variantes de acuerdo con su carga eléctrica, cuantificación de hemoglobina A2 (HbA2), Fetal (Hb F), pruebas de solubilidad hemoglobínica y falciformación. Otras técnicas se basan en propiedades fisicoquímicas como la estabilidad de la hemoglobina para detección de variantes inestables. En la práctica las pruebas más útiles son las que permiten detectar la presencia de hemoglobinopatías, como ocurre con la hemoglobina S dejando para laboratorios especializados aquellos procedimientos para identificar la mutación. La correcta detección de los portadores de las diferentes hemoglobinopatías tiene como finalidad dar un consejo genético adecuado sobre la forma de herencia, el riesgo de tener hijos afectados con las formas graves de la enfermedad y evitar tratamientos innecesarios. El diagnóstico molecular se reserva para la alfa talasemia, para cuadros con genotipos complejos, estudios prenatales o epidemiológicos.

Hemoglobinopathies are hereditary syndromes determined by a large variety of globin gene defects. Hemoglobinopathy diagnosis results from the combination of clinical orientation, laboratory tests and family studies. Basic tools include complete blood cell count, red blood cell count, hemoglobin quantification and red cell indices (MCV, MCH), blood film examination, reticulocyte count and iron status. Complementary determinations are hemoglobin electrophoresis, which enables the separation of the different hemoglobin variants according to their electrical charge, A2, and Fetal hemoglobin quantification, hemoglobin solubility and sickling test for Hb S diagnosis. Other techniques are based on physicochemical properties such as stability of hemoglobin for detection of unstable variants. In practice, the most useful tests are those that enable the detection of hemoglobinopathies, such as hemoglobin S, and the identification of the genetic defects is referred to specialized laboratories. The correct detection of the carriers of the different hemoglobinopathies is intended to give adequate genetic advice on the form of inheritance and the risk of having affected children with the severe forms of the disease and to avoid unnecessary treatments. Molecular diagnosis is reserved to a thalassemia complex genotypes, prenatal o epidemiological studies.

As hemoglobinopatias são distúrbios hereditários resultantes de uma grande variedade de defeitos que afetam os genes de globina. O diagnóstico das hemoglobinopatias decorre de uma combinação de estudos clínicos, provas de laboratório e estudo familiar. As ferramentas básicas incluem hemograma, hemoglobina e índices eritrocitários (VCM, HCM), morfologia dos eritrócitos, contagem de reticulócitos e perfil de ferro. As determinações complementares são eletroforese de hemoglobina que permite separar as diferentes variantes, de acordo com sua carga elétrica, quantificação de hemoglobina A2 (HbA2), Fetal (Hb F), provas de solubilidade hemoglobínica e falciformação. Outras técnicas baseiam-se em propriedades fisicoquímicas como a estabilidade da hemoglobina para detecção de variantes instáveis. Na prática, as provas mais úteis são as que permitem detectar a presença de hemoglobinopatias, como acontece com a hemoglobina S deixando para laboratórios especializados aqueles procedimentos para identificar a mutação. Detectar corretamente os portadores das diferentes hemoglobinopatías visa a dar um conselho genético adequado sobre a forma de herança, o risco de ter filhos afetados com as formas graves da doença e evitar tratamentos desnecessários. O diagnóstico molecular se reserva para a alfa talassemia, para quadros com genótipos complexos, estudos pré-natais ou epidemiológicos.

Hemoglobin A2 values in sickle cell disease patients quantified by high performance liquid chromatography and the influence of alpha thalassemia

BACKGROUND: In sickle cell disease, the quantification of Hb A2 is important for the differential diagnosis between sickle cell anemia (Hb SS) and Hb S/ß0-thalassemia.OBJECTIVE: To determine Hb A2 levels as quantified by high performance liquid chromatography in patients with sickle cell anemia (Hb SS) and with the SC hemoglobinopathy, with or without concomitant alpha thalassemia.METHODS: This is a retrospective study of 242 children aged between two and six years with diagnoses of Hb SS or Hb SC. The hemoglobin was evaluated using high performance liquid chromatography and alpha thalassemia [3.7 kb deletion (-a3.7)] was detected by polymerase chain reaction. Patients were classified as homozygous (-a3.7/-a3.7), heterozygous (-a3.7/a), or homozygous wild-type. Analysis of variance was used to compare the mean Hb A2 values between the alpha thalassemia groups.RESULTS: The mean (± standard deviation) Hb A2 concentrations in the Hb SS group (n = 135) was 3.68 ± 0.65%. The mean values for individuals with Hb SS and heterozygous (n = 28) or homozygous for alpha thalassemia (n = 3) were 3.98 ± 0.64% and 4.73 ± 0.25%, respectively. The mean Hb A2 of all the Hb SC patients (n = 107) was 4.01 ± 0.507 with 4.29 ± 0.41% and 4.91 ± 0.22% in individuals heterozygous (n = 23) and homozygous for alpha thalassemia (n = 7), respectively. All patients homozygous for alpha thalassemia had Hb A2 levels above 3.5%. However, Hb A2 values above 5.2% were seen in patients with Hb SS and Hb SC, independently of alpha thalassemia.CONCLUSION: Hb A2 levels are elevated in patients with Hb S or Hb C, and are directly influenced by the alpha thalassemia genotypes.

Comparative analysis of cellulose acetate hemoglobin electrophoresis and high performance liquid chromatography for quantitative determination of hemoglobin A2

BACKGROUND: The present study is designed to evaluate the reliability and cost effectiveness of cellulose acetate Hb electrophoresis and high performance liquid chromatography (HPLC) in the determination of HbA2 levels. METHODS: The test population comprised 160 individuals divided into four groups: normal individuals, beta-thalassemia trait (BTT) patients, iron deficiency anemia (IDA) patients, and co-morbid patients (BTT with IDA). HbA2 levels determined using cellulose acetate Hb electrophoresis and HPLC were compared. RESULTS: HbA2 levels were found to be diagnostic for classical BTT using either method. In co-morbid cases, both techniques failed to diagnose all cases of BTT. The sensitivity, specificity, and Youden's index for detection of the co-morbid condition was 69% and 66% for HPLC and cellulose acetate Hb electrophoresis, respectively. CONCLUSION: This study revealed that semi-automated cellulose acetate Hb electrophoresis is more suitable for use in beta-thalassemia prevention programs in low-income countries like Pakistan. This technique is easily available, simple and cost effective.

The application of capillary electrophoresis in HbA1c and HbA2 detection in beta thalassemia patients / 中华检验医学杂志

Objective To investigate effects of β-thalassemia on 8 HbA1c measurement systems including Capillary electrophoresis and application of HbA2 values of samples with β-thalassemia from Capillary 2 Flex Piercing HbA1c measurement systems.Methods 338 samples without β-thalassemia ( include 80 α-thalassemia with --SEA/ααgenotype) and 225 samples with β-thalassemia were collected in Peking University Shenzhen hospital from January 2014 to August 2014.Samples withβ-thalassemia are all heterozygotes with normal α-globin and abnormal β-globin, and without combining other haemoglobinopathies.76 normal samples and 76 samples with β-thalassemia were selected from above all samples, HbA1c values of which were given by capillary electrophoresis ( Capillary Flex 2 Piercing), enzymatic ( Norudia N HbA1c ), immune transmission turbidity ( oneHbA1c FS ), chemiluminescent immunoassay (ARCHITECT HbA1c Reagent Kit), boronate affinity HPLC (Trinity Ultra2)and ion exchange HPLC (HA8160, VariantⅡTurbo and Variant Ⅱ BioRad 2.0), respectively.HbA1c values given by the eight systems were compared.338 samples without β-thalassemia and 225 samples with β-thalassemia were measured by using Capillarys 2 Flex Piercing HbA1c system and capillary electrophoresis hemoglobin system, the cut-off values of HbA2 from Capillary 2 Flex Piercing HbA1c measurement system for β-thalassemia was assessed by using ROC curve.Data statistics were done by using SPSS 19.0.Results The HbA1c values of normal samples were well correlated with the comparative system.The correlation coefficients for the 7 systems are all greater than 0.98.For samples withβ-thalassemia, the correlation coefficients are all greater than 0.98 except for Variant Ⅱ Turbo.The bias between normal samples and samples with β-thalassemia have no significant difference except for VariantⅡTurbo.HbA2 values from Capillary 2 Flex Piercing HbA1c measurement and capillary electrophoresis hemoglobin system were well correlated. The correlation coefficient is 0.993.When cut-off value of HbA2 from Capillary 2 Flex Piercing HbA1c measurement system for β-thalassemia is 3.38, sensitivity is 98.3%, specificity is 100% .Conclusions Samples withβ-thalassemia have no significant effects on HbA1c measurement syetems except for Variant Ⅱ Turbo.The Capillary 2 Flex Piercing HbA1c measurement syetem has the advantage of screeningβthalassemia by HbA2 while measuring HbA1c.

External Quality Assessment of Hemoglobin A2(HbA2) and Hemoglobin F(HbF)Measurement / 现代检验医学杂志

Objective To evaluate hemoglobin A2 and F (HbA2 and HbF)assays in part of Thalassemia screening laborato-ries in China.Methods Two lots of controls were sent to 50 Thalassemia screening laboratories.Test results of HbA2 and HbF were collected,analyzed and evaluated by different method groups.Results 49 of all laboratories reported the results and the rate of return was 98%.The proportion of laboratories reporting acceptable results for HbA2 and HbF testing was 42.9~92.3% and 27.3% ~ 84.6% respectively.HbA2 test results from 3 laboratories for Lot 201311 and 4 for 201312 were unsatisfactory.And HbF test results from 5 laboratories for Lot 201311 and 3 for 201312 were unsatisfactory.Conclu-sion Conclude that quality in the measurement of HbA2 and HbF should be improved.

Value of Hemoglobin A2 Level Determined by Capillary Electrophoresis in Screening and Diagnosis of Thalassemia / 现代检验医学杂志

Objective To evaluate the value of HbA2 level determined by capillary electrophoresis (Hb-CE)in screening and diagnosis of thalassemia.Methods HbA2 level of 249 thalassaemia carriers and 142 healthy controls confirmed by molecular biological detection were determined by Hb-CE method.The thalassaemia carrier subjects were divided into different groups and subgroups according to their results of gene detection.The sensitivity,specificity,accuracy,positive predictive value and negative predictive value for the diagnosis ofα-thalassemia,β-thalassaemia,α,β-thalassaemia were calculated under different HbA2 cut-off value.Results Mean value of HbA2 in healthy controls was (3.03±0.27)%.Mean values of HbA2 inα-thalassemia group and its subgroups of silentα-thalassemia,standardα-thalassemia and hemoglobin H disease were (2.38± 0.55)%,(2.61±0.46)%,(2.47 ± 0.32)% and (1.07 ± 0.17)%,respectively.Mean values of HbA2 inβ-thalassaemia group and itsβ0 subgroup,β+ subgroup were (5.65±0.47)%,(5.71±0.48)% and (5.56±0.43)%.Mean value of HbA2 in compoundαandβ-thalassaemia group was (5.7±0.82)%.Compared with healthy controls,HbA2 level inα-thalassemia group,silentα-thalassemia subgroup,standardα-thalassemia subgroup and hemoglobin H disease group decreased signifi-cantly (t values of 11.73,5.02,12.91 and 33.46,respectively,P0.05).HbA2 level inβ-thalassaemia group,β0 subgroup,β+ subgroup and compoundαandβ-thalassaemia group increased significantly (t values of 55.12,44.33,38.94 and 9.10,respectively,P0.05).Of 249 thalassemia carriers,all 124β-thalassaemia carriers were distinguished with ele-vated HbA2 level (>3.5%)determined by Hb-CE and only 57 were distinguished from 117α-thalassemia carriers by Hb-CE.Under the cut-off value of 2.5%,the sensitivity,specificity,positive predictive value,negative predictive value and accu-racy for the diagnosis ofα-thalassemia were 48.72%,97.18%,93.44%,69.70%,75.29%,respectively.Under the cut-off value of 3.5%,they were 100.00%,98.59%,98.41%,100%,and 99.25% for the diagnosis ofβ-thalassaemia,respectively. The analysis of ROC curve showed that the optimal HbA2 cut-off values for diagnosis ofα,β-thalassaemia by capillary elec-trophoresis were 2.8% and 3.7%,respectively.Conclusion When no abnormal bands,the elevated HbA2 (>3.7% in this study)determined by Hb-CE could be used as a marker forβ-thalassaemia diagnosis,but theβ-thalassaemia co-existingα-thalassemia could not be differentiated fromβ-thalassaemia diagnosis.Decreased HbA2 level (<1.5% in this study)and HbH band could be used for the diagnosis of hemoglobin H disease.Only HbA2 determination by Hb-CE has no clinical sig-nificance for the screen and diagnosis ofα-thalassemia.

Study of parents of β-thalassemia major children to determine cutoff values of hematological parameters for diagnosis of β-thalassemia trait and assessment of anemia in them.

Background: Because of the overlapping MCV, MCH and HbA 2 values in BTT and non-BTT subjects our laboratory determined own cutoffs. Aims: To establish cutoff values by investigating the parents of thalassemia major children and to assess the degree of anemia in BTT subjects. Materials and Methods: Study includes 179 parents of thalassemia major children (BTT cases) and 287 non-BTT controls. Samples were analyzed on an electronic hematology analyzer. The samples having MCV ≤ 76 fl and MCH ≤ 27 pg were quantified for HbA 2 by cellulose acetate electrophoresis and grey zone samples were confirmed on HPLC. Statistical Analysis Mean ± SD, sensitivity, specificity, PPV, NPV and accuracy were calculated. The histograms were plotted for MCV, MCH and HbA 2 . Results: Cases having MCV ≤ 76 fl and MCH ≤ 27 pg if considered as suspected cases of BTT then we could have missed five known BTT samples. Sensitivity increased to 100% in all three diagnostic parameters when the cutoff values were raised and specificity for MCV and MCH was decreased. But specificity was 100% with raised cutoff for HbA 2 . Hb and HCT mean values were significantly reduced in BTT cases compared to controls (P < 0.001). In 100% females and 84.9% males having BTT, mild to moderate anemia was observed. Conclusion: In our setup, the cutoff values are MCV (≤78.0 fl), MCH (≤28 pg) and HbA 2 (>3.8%) for BTT diagnosis and there is a mild to moderate anemia in BTT cases.

The value of MCV, MCH and HbA2 in laboratory screening of thalassemia / 中华妇产科杂志

ObjectivesTo explore the roles of mean corpuscular volume(MCV),mean corpuscular hemoglobin(MCH) and hemoglobin A2 (HbA2) 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 Obstertrics andGynecology of Nanfang Hospital.Of them,1036 cases were diagnosed with thalassemia (408 α-thalassemia,608 β-thalassemia,and 20 αβ compound thalassemia,thalassemia group) and 348 without thalassemia,non-thalassemia group.All subjects were screened respectively for MCV,MCH and HbA2.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 HbA2 alone,combination of MCV and MCH,and combination of MCV,MCH and HbA2.Results( 1 ) In the thalassemia group,the sensitivity of MCV alone was 92.9％ (379/408) for α thalassemia,99.3％ (604/608) for β thalassemia and 100.0％(20/20) for αβ 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 β thalassemia and 100.0％ (20/20) in αβ compound thalassemia.In the non-thalassemia group,the specificity of MCH alone was 72.7 ％ (253/348).(3) The sensitivity of Hb A2 alone was 67.4％ (275/408) for α thalassemia,97.5％ (593/608) for 3 thalassemia,and 100％ (20/20) for α3 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 HbA2 ＞ 3.5％ it had a high value in [β-thalassemia screening,but when HbA2 ＜ 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 HbA2.MCV + MCH was the best for overall screening,but for [β thalassemia screening,MCV + MCH + HbA2 was the best.ConclusionsMCV and MCH are suitable for epidemic screening in a large population,physical examination and premarital check-up.Hb electrophoresis andthalassemiagenediagnosisarerecommendedforsubjectswithpositiveMCVandMCH indexes.Diagnoses of α and β-thalassemia gene are recommended for pregnant women with positive MCV and MCH indexes.

Role of iron deficiency anemia in the propagation of beta thalssemia gene

BACKGROUND: The diagnostic criterion for beta thalassemia trait (BTT) is elevated Hb-A2 levels. Iron deficiency anemia (IDA) reduces the synthesis of Hb-A2, resulting in reduced Hb-A2 levels, so patients with co-pathological conditions BTT with IDA, may have a normal level of Hb-A2. Many socio-economic factors like unawareness, poor diagnostic facilities, and cost of molecular diagnosis (for screening purposes) result in interpretation of these subjects as normal. METHODS: Venous blood samples from 200 unmarried females having a family history of thalassemia were collected, and basic hematological parameters, hemoglobin electrophoresis, and molecular analysis for beta thalassemia were done. Patients with IDA and patients with co-pathological conditions BTT and IDA were treated with oral iron. These subjects were then followed for a period of 20 weeks. RESULTS: Of the 200 females, 34 were found to be anemic. Hemoglobin electrophoresis identified 16 of these patients as BTT. Molecular analysis of all patients confirmed this diagnosis, but identified 8 additional patients with BTT. Eight patients that were not detected with hemoglobin electrophoresis were found to have co-pathology of BTT with IDA. CONCLUSION: Patients with the co-pathological condition BTT with IDA may be interpreted as being normal, as they have normal Hb-A2 levels. These misdiagnosed subjects when marry with BTT have the potential to produce beta thalassemia major in offspring. This is one of the factors playing a major role in the propagation of beta thalassemia gene in Pakistani population, and become a serious hindrance for the thalassemia prevention program in Pakistan.

Effect of Iron Deficiency on Hemoglobin A2 Level in Patients with β-Thalassemia / 实用儿科临床杂志

Objective To determine the effect of iron deficiency on hemoglobin A2(HbA2) expression in patients with β-thalassemia.Methods The participants were recruited from the out-patient clinics of the Pediatrics Department and Obstetrics Department of Affiliated Hospital of Guilin Medical College and from some β-thalassemia major families.Blood samples from the participants were used for blood smear tests and hemoglobin electrophoresis and to analyze serum ferritin (SF),3 alpha-globin gene deletions,and 17 beta-globin point mutations.Results Of the 408 individuals,304 were assigned to group A (normal controls),26 to group B (iron deficiency),56 to group C (β-thalassemia),and 22 to group D (β-thalassemia combined with iron deficiency). The results for the comparison of the mean HbA2 values among pairs of groups were as follows: group A vs group B,q=5.074 7,P<0.05; group A vs group C,q=37.650 8,P<0.05; group A vs group D,q=16.043 0,P<0.05;group C vs group D,q=7.682 9,P<0.05; Group B vs group D,q=15.806 6,P<0.05. There were no significant correlation between SF and HbA2 in all 4 groups.Conclusions Iron deficiency decreased the HbA2 level in both controls and individuals with β-thalassemia. HbA2 levels decreased significantly in individuals with both β-thalassemia and iron deficiency as compared with β-thalassemia group alone. However,they remained significantly higher than both the control and iron-deficient groups. Therefore,the elevation of HbA2 could be used to diagnose β-thalassemia reliably even in the presence of iron deficiency.

A dosagem de hemoglobina A2 pode ser feitra a partir de amostra de hemolisado com saponina? / Could the saponin hemolysate be used for the hemoglobin A2 dosage?

A dosagem de hemoglobina A2 (HbA2) é comumente feita utilizando-se como amostra solução de hemoglobina (hemolisado clássico com clorofórmio ou tetracloreto de carbono) ou hemolisados com saponina, apesar de os valores de referência para tal determinação terem sido estabelecidos a partir de dados tendo apenas solução de hemoglobina como amostra. Assim, para verificar se há ou näo coerência na utilização indiscriminada daqueles valores de referência para dosagem de HbA2 em amostras de hemolisado com saponina, foi feita a dosagem desta proteína utilizando quatro tipos de hemolisados. Na 1ª etapa da avaliação foram procedidas trinta determinações para fração de HbA2 a partir de hemolisado com saponina e hemolisado clássico com tetracloreto de carbono; na 2ª etapa mais 40 determinações foram analisadas utilizando hemolisado com saponina e com clorofórmio, porém acrescidos de cianeto de potássio (KCN). A análise estatística feita através de teste t pareado na 1ª etapa, revelou uma tendência de que os resultados obtidos a partir de saponina para amostras com níveis normais de HbA2 sejam maiores cerca de 0,42 por cento que aqueles obtidos a partir da solução de hemoglobina (p<0,001). Porém, quando adiciona-se KCN no hemolisado não houve diferença significativa entre o hemolisado clássico (soluçäo de hemoglobina) e o hemolisado com saponina. Deste modo, a utilização de diferentes tipos de hemolisados como amostra (saponina sem KCN ou solução de hemoglobina) podem gerar resultados distintos para a dosagem de HbA2. Determinações de HbA2 feitas especificamente com hemolisado com saponina tendem a superestimar os resultados, quando comparados a valores de referência estabelecidos a partir de hemolisado clássico. Assim, a utilização do hemolisado clássico ou de saponina com KCN são indicados para a dosagem de HbA2 pelo método de eluiçäo eletroforética. O uso de hemolisado apenas com saponina requereria a utilizaçäo de valores de referência específicos. (AU)

It was made the evalution of the hemoglobin A2(Hb A2) determination using four hemolysatetypes. In the 1st. stage of the evalution thirty determinations were proceeded for fraction of Hb A2 startingfrom hemolysate with saponine and starting from carbon tetrachloride, in the 2nd stage more 40determinations were analyzed using hemolysate obtained through saponine and with cloroform, howeveradded with potassium cyanide (KCN). The statistical analysis done through test t in the 1st , it revealed atendency of the results obtained starting from saponine are larger about 0,42% that those obtained startingfrom carbon tetrachloride (p<0,001). However, when KCN is added in the hemolysate there is no significantdifference among the classic hemolisado and with the saponine. Like this, the use of the classic hemolysateor of the saponine with KCN are indicative for Hb A2 quantitative determination for the method of elution. (AU)