orabase and Thalassemia

A simple probe pair was designed for the detection of hemoglobin E (HbE) genotype, a single-point mutation that leads to abnormal red blood cells commonly found in South East Asia. The key to differentiation is the use of a conformationally constrained peptide nucleic acid (PNA) that was immobilized on carboxymethylcellulose-modified paper. This was then used for target DNA binding and visualization by an enzyme-catalyzed pigmentation. The biotinylated target DNA bound to the immobilized probe was visually detected via alkaline phosphatase-linked streptavidin. This enzyme conjugate catalyzed the dephosphorylation of the substrate 5-bromo-4-chloro-3-indolyl phosphate, leading to a series of reactions that generate an intense, dark blue pigment. The test was validated with 100 DNA samples, which shows good discrimination among different genotypes (normal, HbE, and heterozygous) with 100% accuracy when optimal conditions of analysis were applied. The method does not require temperature control and can be performed at ambient temperature. This is an attractive feature for diagnosis in primary care, which accounts for a large part of affected population. Graphical abstract Schematic representation of a paper-based sensor for the detection of the gene Hemoglobin E. The interaction between an immobilized peptide nucleic acid and a DNA target leads to enzymatic pigmentation, allowing simple visual readout with up to 100% accuracy.

Topics: Biotinylation; Carboxymethylcellulose Sodium; Colorimetry; DNA; Genotype; Humans; Nucleic Acid Probes; Peptide Nucleic Acids; Pigmentation; Thalassemia

Reverse phase HPLC of radioactive globin chains has been compared to classical carboxy methyl cellulose chromatography for the prenatal diagnosis of beta thalassaemia. The two methods correlated highly (r = 0.97 p less than 0.0005) and provided an identical diagnosis for 40 fetal blood samples of fetuses homozygous or heterozygous for beta thalassaemia. The HPLC procedure was much faster and required fewer biochemical steps (no globin preparation). It was at least as accurate and more sensitive than the classical chromatography. A single column can be used for 150 analyses and is always ready to be used. Last but not least it is much less expensive than CMC chromatography.

Topics: Carboxymethylcellulose Sodium; Chromatography; Chromatography, High Pressure Liquid; Female; Fetal Diseases; Heterozygote; Homozygote; Humans; Pregnancy; Prenatal Diagnosis; Thalassemia

The minor components of Hb Bart's were separated by CM-cellulose chromatography, reverse-phase HPLC, and DEAE-cellulose chromatography. These were characterized by amino acid analysis, tryptic peptide analysis by HPLC, electrophoresis, and carbohydrate and phosphate analysis. Acetylated and glycated components of Hb Bart's were present in cord blood red cells. The ratio of gamma Ic/total gamma in Hb Bart's was nearly the same as that of Hb FIc/Hb F which may indicate that the gamma chain after its release from the ribosome is not a better acetylation substrate than Hb Fo. Glycation of the free gamma-chains seems to take place in vivo as readily as the major Hb components.

Topics: Carboxymethylcellulose Sodium; Chromatography, DEAE-Cellulose; Chromatography, High Pressure Liquid; Fetal Blood; Globins; Hemoglobins, Abnormal; Humans; Infant, Newborn; Thalassemia

High performance liquid chromatography was compared with carboxymethyl cellulose column chromatography data for analysis of globin chain composition and biosynthetic ratios in fetal blood. A formula was derived for mathematical correction for losses of small amounts of globin protein on the high performance liquid chromatography column. Radioactive globin chain ratios by high performance liquid chromatography are equivalent to those by carboxymethyl cellulose column chromatography, but only when carrier Hb A is added. We conclude that high performance liquid chromatography provides a suitable alternative to carboxymethyl cellulose columns, and has several advantages.

Topics: Carboxymethylcellulose Sodium; Chromatography; Chromatography, High Pressure Liquid; Female; Fetal Blood; Globins; Hemoglobinopathies; Humans; Pregnancy; Prenatal Diagnosis; Thalassemia

A recently developed isoelectric focusing technique for human globin chain separation has been applied to the antenatal diagnosis of beta 0-thalassemia in Sardinia. Results obtained with this method show a complete concordance with those obtained by the currently-in-use chromatographic separation of globin chains by carboxymethyl-cellulose. The ease with which several samples (up to 20) can be simultaneously processed and analyzed by a single operator and the very simple equipment required make this new method ideal for the antenatal diagnosis of beta 0-thalassemia and could encourage a more widespread use of prenatal diagnosis of thalassemias.

Topics: Anemia, Sickle Cell; Autoradiography; Carboxymethylcellulose Sodium; Chromatography; Female; Globins; Humans; Isoelectric Focusing; Pregnancy; Thalassemia

Determination of the biosynthetic beta/gamma ratio in samples of fetal blood is used for prenatal diagnosis of thalassemia. The current method, carboxymethyl cellulose chromatography (CMC), is cumbersome, slow, and expensive. Radiolabelled globin chains (A gamma, G gamma, beta, and alpha) can also be separated by electrophoresis in slab gels containing polyacrylamide, acid, urea, and Triton X-100. The radioactive bands are detected by fluorography. We determined the beta/gamma ratio in 24 samples of blood from fetuses at risk for beta thalassemia. CMC column data were compared with quantitations from fluorograms of slab gels. The beta/gamma synthetic ratios correlated (r=0.91), although the ratios were slightly less by gel than by column. The gel method is simple, inexpensive, and permits up to a dozen simultaneous analyses. Confirmatory CMC columns need only be run if the beta/gamma ratio is between 0.01 and 0.03 by gel.

Topics: Carboxymethylcellulose Sodium; Chromatography; Densitometry; Electrophoresis, Polyacrylamide Gel; Female; Fetal Blood; Globins; Humans; Pregnancy; Prenatal Diagnosis; Thalassemia