Label-free quantification of host cell protein impurity in recombinant hemoglobin materials.

Quantitative analysis relies on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host cell proteins (HCPs) in bioengineered proteins which are intended for use as protein calibration standards. In this study a purified hemoglobin-A2 (HbA2) protein, obtained through its overexpression in E. coli, was used. Two different materials were produced: natural and U15N-labeled HbA2. For the quantification of impurities, precursor ion (MS1-) intensities were integrated over all E. coli proteins identified and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for an E. coli cell lysate, which had been spiked at known mass ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of just nine proteins, and a minimum of five proteins was estimated to be sufficient to keep the sampling error below 15%. For the studied materials, HbA2 mass fractions (or purities) of 923 and 928 mg(HbA2)/g(total protein) were found with expanded uncertainties (U) of 2.8 and 1.3%, resp. Value assignment by LFQ thus contributes up to about 3% of the overall uncertainty of HbA2 quantification when these materials are used as calibrators. Further purification of the natural HbA2 yielded a mass fraction of 999.1 mg/g, with a negligible uncertainty (U = 0.02%), though at a significant loss of material. If an overall uncertainty of 5% is acceptable for protein quantification, working with the original materials would therefore definitely be viable, circumventing the need of further purification.

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.

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.

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

BACKGROUND: The diagnostic criterion for beta thalassemia trait (BTT) is elevated Hb-A(2) levels. Iron deficiency anemia (IDA) reduces the synthesis of Hb-A(2), resulting in reduced Hb-A(2) levels, so patients with co-pathological conditions BTT with IDA, may have a normal level of Hb-A(2). 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-A(2) 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.

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

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.

Prevalência do traço talassêmico beta em indivíduos com microcitose / Prevalence of trait bet thalassemic in individuals with microcytosis

A talassemia beta é a caracterizada por uma alteração quantitativa da síntese da globina beta resultando em redução (ß talassemia) ou ausência (ß talassemia) de cadeias. O traço talassêmico beta resulta da heterozigoze de uma das mutações que afetam a síntese das cadeias beta. Com o objetivo de determinar a prevalência do traço talassêmico beta em indivíduos com microcitose foram analisadas 86 amostras de sangue com valor de VCM inferior a 80 fL. Foram realizados os seguintes exames: erotrograma, contagem de reticulócitos, eletroforese de hemoglobina em PH alcalino, dosagens de Hb A, Hb Fetal, ferritina sérica, ferro sérico, capacidade total de ligação do ferro e saturação da transferrina. Foram identificados 2 portadores de traço talassêmico beta representando uma prevalência de 2,3%. Apenas um paciente com traço talassêmico beta tinha anemia e nenhum deles apresentava deficiência de ferro, enquanto que entre os 84 pacientes com níveis normais de Hb A, 27 (32,1%) não tinham anemia nem deficiência de ferro. A concentração média de Hb A e VCM dos pacientes com traço talassêmico beta foi 5,0 +- 0,8% e 69,5 +- +- 6,5 fL, respectivamente. Os resultados obtidos sugerem a necessidade de posterior estudo molecular a fimde investigar não só as mutações no gene beta, mas a presença de talassemia alfa em nossa população.