Obtention of low phenylalanine whole blood for preparation of reference materials and calibrators for newborn screening using phenylalanine ammonia lyase

BORRAJO, G.J.C.; CASTAÑEDA, M.T.; HOURS, R.A.

Santiago de Chile

Congreso; X Congreso Latinoamericano de Errores Innatos del Metabolismo y Pesquisa Neonatal; 2015

Sociedad Latinoamericana de Errores Innatos del Metabolismo y Pesquisa Neonatal

OBTENTION OF LOW PHENYLALANINE WHOLE BLOOD FOR PREPARATION OF REFERENCE MATERIALS AND CALIBRATORS FOR NEWBORN SCREENING USING PHENYLALANINE AMMONIA LYASE.Borrajo GJC (1); Castañeda MT (2); Hours RA (2)(1) Detección de Errores Congénitos. Fundación Bioquímica Argentina; (2) CINDEFI. Facultad de Ciencias Exactas. Universidad Nacional de La Plata. La Plata ? Argentina. borrajog@net-alliance.net.Introduction: One of the main limitations regarding the preparation of Phenylalanine (Phe) reference materials and calibrators in dried blood spots (DBS) is the unavailability of human blood free of Phe. Consequently, the basal level of Phe in the blood used for the preparation must be analytically determined, thus introducing an extra variable: the error of the measurement method.Objective: To develop a procedure for the preparation of low Phe whole blood using Phenylalanine Ammonia Lyase (PAL) in order to obtain reference materials and calibrators for Newborn Screening.Materials and Methods: The procedure consists in two independent steps. In the first one, a normal serum pool is incubated during 6 h at 37°C with PAL from Rhodosporidium toruloides (EC 4.3.1.25) immobilized on calcium alginate beads. PAL catalyzes the non-oxidative deamination of L-Phe producing trans-cinnamic acid. After this treatment, the serum is recovered unaltered and without addition of exogenous substances. In the second step, red blood cells collected with EDTA are deprived of Phe by successive washing with physiological solution of 0.85 g/L NaCl. Then, red blood cells are rinsed with the serum previously treated with PAL in order to eliminate the physiological solution. Finally, both fractions are properly combined to obtain whole blood with hematocrit adjusted to 50 %. Phe concentration in the treated serum and in DBS was determined by Tandem Mass Spectrometry.Results: The low Phe whole blood obtained was evaluated for its potential use in the preparation of Phe reference materials and calibrators in DBS. Preliminary experiments following the procedure described above resulted in Phe concentrations of 3.1 and 5.9 umol/L in the treated serum and in DBS, respectively.Conclusions: From the above results it can be concluded that this is an appropriate material for the intended purpose. Nevertheless, optimization in the PAL treatment procedure is in progress in order to minimize the final Phe levels. The developed technique is reproducible, inexpensive and operatively easy to carry out, offering a new alternative in order to achieve the harmonization in the assignment of Phe values to reference material and calibrators provided for commercial reagent kits.