A micromethod for the determination of globin synthetic ratios (GSR): 94
| Autor: | N Constantsas, Catherine Dacou-Voutetakis, J Palis |
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| Rok vydání: | 1980 |
| Předmět: | |
| Zdroj: | Pediatric Research. 14:180-180 |
| ISSN: | 1530-0447 0031-3998 |
| DOI: | 10.1203/00006450-198002000-00121 |
| Popis: | The GSR is an absolute criterion for diagnosis, investigation, and classification of thalassemias (thal). Its wide use is prevented by the tedious, time consuming processing of the numerous fractions obtained by conventional column chromatography. Electrophoresis is simpler, but gret dilution of the "hot" globin (newly synthesized by retics) in the "cold" hemoglobin of the mature red cells, results in unreliably low radioactivity of the separated globin bands. This can be overcome only by overloading the strips which compromises good resolution. Here, we report a procedure for the preparation of globin 104-fold "hotter", than currently obtained with established methods, by incubating 5 μl of packed 20-70% retics (isolated at 1,500xg on a preformed Percoll-Urografin density gradient) with 30 μC of 3H-(leucine + lysine + proline + histidine +phenylalanine). Microzone electrophoresis of 2-5 μg of globin in urea-EDTA-thiol buffer, saturated with borate, gives sharp, well resolved α/β/γ bands. The GSR were determined in 75 individuals, in terms of cpm; 40 controls (β/α 1.01 ± .10); 20 β-thal traits (β/α .55 ± .10); 13 cases of β-thal major (β/α .17 ± .07, γ/α .35 ± .15); 2 α-thal traits (β/α 1.69, 1.41). In terms of specific activity, these values are not practically different, γ/β GSR, however, are up to 100-fold higher. This method allows easy handling by two persons, of 24-32 samples per week, at low cost, with repetitive assays. Hence, quality control is improved, and the scope of GSR determinations is expanded in post and antenatal thal, as well as in other hematological investigations. |
| Databáze: | OpenAIRE |
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