Evaluation of Glucose-6-Phosphate Dehydrogenase Stability in Blood Samples under Different Collection and Storage Conditions

Fujimoto et al. (1) reported that the stability of galactose 1-phosphate in dried blood spots for neonatal screening was adversely affected by humidity and temperature, especially when low-value samples are evaluated. We extend these findings to glucose-6-phosphate dehydrogenase (G-6-PD; EC 1.1.1.49) activity, deficiency of which is by far the most common genetic disease worldwide (2) and accounts for more than one-half of the cases of severe jaundice among male newborns in Greece, China, and in Sephardic Jewish groups (3). Tests for G-6-PD deficiency are thus included in newborn screening programs in some regions. We collected whole-blood specimens from 20 volunteers and spotted them on filter papers (Schleicher & Schuell no. 903) or placed them in EDTA tubes. G-6-PD activity was measured on the day of collection, and samples were immediately divided into 8 portions. Portions 1–4 were dried on filter paper and stored at room temperature, 2–8 °C, −20 °C, and 37 °C, respectively. Portions 5–8 were stored as whole blood under the same conditions. Subsequent assays were performed on days 3, 10, and 15. Whenever dried blood spots were used, we allowed them to dry at room temperature for 6 h and then placed them in air-tight plastic bags with desiccants, from which we eliminated air by squeezing the bag before closing. Thus, the effect of refrigeration and ambient room humidity was minimized. The G-6-PD Deficiency Neonatal Screening Test Kit (cat. no. 3570-050; Interscientific Corporation) was used for quantitative measurement of G-6-PD activity (4). Increased temperature decreased the stability of G-6-PD in both liquid blood and dried blood spots (Fig. 11 ). The samples responded differently to the same temperature exposure, a finding that needs further investigation because it suggests a multiparametric influence. Enzyme activity in some dried-blood samples appeared to be more tolerant of high temperatures than the activity in other samples. This could be attributed to several factors, which may include (but are not limited to) the filter-paper matrix, the initial ambient humidity in the card’s environment, the presence of enzyme-protective factors in quantities that vary among samples, and the overall status of the erythrocytes. Further work needs to be conducted to elucidate the findings reported here. We conclude, however, that finding low activity in several samples in a batch of specimens delayed in the mail is very likely. Importantly, this study demonstrates that time lost during the transport of Guthrie cards to screening centers should be kept to a minimum. Rapid delivery of cards and/or the use of heat-insulated envelopes and refrigerated transport may be a solution to prevent heat inactivation (5). The latter approach may be difficult for newborn screening programs because of cost. Mean G-6-PD activity at days 0, 3, 10, and 15. Results for whole blood stored at room temperature (•), refrigerated (▪), frozen (▴), and at 37 °C (♦) and dried blood spots stored at room temperature (○), refrigerated (□), frozen (▵), and at 37 °C (⋄). Hb, hemoglobin.