Observations on Heat/Humidity Denaturation of Enzymes in Filter-Paper Blood Spots from Newborns

Abstract

Use of filter-paper blood spots from newborns for screening of inborn errors may include the assay of biotinidase (EC 3.5.1.12; BIO), galactose-1-phosphate uridyltransferase (EC 2.7.7.12; UT), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49; G6PD). There has been anecdotal reference to heat and/or humidity denaturation of enzymes in filter-paper blood spots exposed to the elements during storage or during transit to the laboratory (1), but no quantitative description of the effects. Understanding the phenomenon may lead to measures to identify denatured samples and prevent incorrect reporting of abnormal results. We quantified filter-paper blood-spot enzyme values for all samples collected during 3 months of the year, February, July, and October, for a large region of Pennsylvania. The population means and SD for each enzyme during each month were determined, and the data were analyzed for seasonal effects. We also performed a controlled experiment with blood-spot filter papers stored under different conditions of heat and humidity to assess their relative influence on the activities of the enzymes of interest. A blood sample (∼15 mL) was drawn from an adult volunteer into a heparin-containing tube and mixed by inversion; blood was then spotted on a series of Schleicher & Schuell 903 filter papers to simulate newborn collections. Approximately 40 spots were applied, with occasional tube inversions, to provide enough sample spots for serial testing, in duplicate, of a variety of environmental conditions. All samples were dried for ∼4 h at room temperature in air. Within the next 2 h, time 0 samples were punched and then assayed for BIO, UT, and G6PD activity. Filter papers were then stored for 3 days under various conditions of temperature, humidity, and exposure to air. The 4 temperature conditions used were as follows: freezing (−20 °C), refrigeration (4 °C), room temperature (21 °C), and 35 °C. Humidity …