Need for a standardized procedure in the preparation of phenylalanine calibrators

Sensitive screening for phenylketonuria (PKU) and other disorders requires accurate measurement of analytes in blood eluted from filter paper. The development of new quantitative methods for phenylalanine measurement in dried blood spots for PKU screening has introduced an increased number of calibrators provided with the kits. We compared the phenylalanine values of seven different sets of calibrators (six from commercial sources and one from a screening laboratory), all measured with the same technique.

Two types of filter papers, S&S #903 and S&S #2992 were used. Phenylalanine was measured by high performance liquid chromatography (HPLC).

There was a wide variation in the phenylalanine values assigned to the seven different sets of calibrators used, with the ratios of HPLC to assigned value ranging from 0.68-1.62. This could not be only explained by the use of different types of filter papers. However, factors in calibrator preparation did influence the measured concentration. These included higher values when 100 microliters rather than 35 microliters of blood was spotted, when the red blood cells were intact rather than lysed, and as the hematocrit increased.

These observations emphasize the need to establish recommendations for preparation of dried blood calibrators in order to appropriately compare new techniques and exchange screening experiences among centers.