Conference Paper


  • R&D Diagnostics Ltd
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Since the introduction by Bob Guthrie of filter paper for the collection and analysis of human blood in the early 1960s, the direct spotting method by heel pick has been by far the most widely used collection method. This method is characterized by ease of sample transport and use as well as very good stability and reproducibility. One of the drawbacks of this method as compared to alternative collection methods (vacuum tubes and capillary pipettes) is the relatively high number of false positive results which can be attributed to inaccuracies in estimating the amount of blood in the spot, due either to multiple applications or incomplete saturation of the paper. This can result in an increased number of recalls which in turn is time and labour consuming for any lab and thus increases the cost, a key factor in routine neonatal screening. Normalization of samples for their haemoglobin content would largely solve this problem. We have determined phenylalanine, galactose and leucine levels by colorimetric means in dried blood spot samples on which varying amounts of blood were spotted. Our results show that the ratio of the analyte concentration to the haemoglobin content (as determined by a separate reading at 405 nm) is stable for the range of 0.8 – 4 drops / filter paper disk although it differs from donor to donor as expected. Since current standard and control dried blood spots supplied by most companies are calibrated to 55% hematocrit, determination of haemoglobin content in them can be used as the basis for comparison for samples to discriminate between true and false positives. We propose a rapid, inexpensive and easy method for this haemoglobin normalization process. Results of work now in progress in a screening center to evaluate the usefulness and ease of implementation of this technique in routine screening will be presented. While its implementation in automated protocols is relatively easy, this method is not limited to enzymatic colorimetric assays and can be easily adapted to other methods (e.g. Luminex LabMAP system, fluorimetric assays, HPLC, enzymatic kinetic assays etc.).

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Correct evaluation of Glucose-6-Phosphate Dehydrogenase (G-6-PD) activity of two ethnic groups using a fully quantitative kit with a simultaneous Hemoglobin Normalization (Hb Normalization) procedure. Two groups of mothers and their healthy full term newborns of Greek (n = 1.166) and Albanian (n = 818) origin were tested for their G-6-PD activity employing a direct normalization protocol. Greek mothers and newborns showed a higher prevalence for G-6-PD deficiency as compared to those of Albanian origin. Males of G-6-PD deficient mothers confirmed the efficacy of the method. A fully quantitative G-6-PD kit employing Hb Normalization is essential for the correct classification of G-6-PD activity, both in male and female subjects.
L-cysteine (L-cys) is implicated in the reduction of free radical production. To investigate the effect of training and L-cys supplementation on the erythrocyte glucose-6-phosphate dehydrogenase (G6PD) activity. Blood was obtained from 10 basketball players pre-game (group A), post-game (group B) and after 1 week on L-cys (0.5 g/24 h orally) supplementation pre- (group C) and post-training (group D). Total antioxidant status (TAS) and G6PD activity were evaluated with commercial kits. TAS increased in the groups with l-cys addition (group C and group D). Post-exercise, TAS and G6PD activity were remarkably higher (1.48+/-0.12 mmol/L, 8.9+/-1.7 U/g Hb, respectively) in group D than those in group B (0.92+/-0.10 mmol/L, 4.8+/-1.6 U/g Hb, p<0.01). G6PD activity positively correlated with TAS (r=0.70, p<0.001 pre- and r=0.61, p<0.001 post-training) in all the studied groups. G6PD activity is lowered by training probably due to free radical action. L-cys supplementation may protect G6PD activity from reduction by increasing total antioxidant capacity and glutathione production. G6PD activity should be evaluated in the blood of athletes of Mediterranean origin and female G6PD-deficient heterozygotes.
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