Genotypic Confirmation from the Original Dried Blood Specimens in a Neonatal Hemoglobinopathy Screening Program

Abstract

ABSTRACT. Dried blood spots are used for newborn screening because of ease of sample collection, handling, and shipment. DNA is stable and accessible in the filter paper matrix. Genotypic confirmation using initial specimens is demonstrated for a regional screening program. Seventy-five blinded samples underwent DNA analysis after Hb electrophoresis. DNA was microextracted from a %-inch semicircle (25 pL whole blood equivalent), amplified, and analyzed by four different methods. Direct amplification without microextraction and automated sequencing from microextracted DNA also was performed. All four analyses agreed for the A and S alleles in 70 of 75 specimens. Three disagreements were clarified by the other semicircle from the original sample: two were due to polymerase chain reaction contamination and one to contamination of one of four analytical tests. Two would have required analysis of a second specimen, one because of polymerase chain reaction failure and the second because the patient had SIB-thalassemia. Direct amplification without microextraction was successful in an additional 77 of 78 specimens for analysis of the A, S, C, and E alleles. Automated direct sequencing from microextracted DNA was demonstrated for the A, S, and C alleles. Analysis of microextracted DNA from dried blood specimens for A and S alleles reduced the need for and costs of obtaining a second specimen for confirmation by 97%. Direct amplification without microextraction for analysis of A, S, and C alleles permits additional reduction in personnel time and costs. We have demonstrated that microextracted DNA is amenable to automated sequencing after asymmetric polymerase chain reaction. Direct genotypic confirmation can facilitate diagnosis and initiation of medical intervention. (Pediatr Res 31: 217-221,1992) Abbreviations PCR, polymerase chain reaction PKU, phenylketonuria CF, cystic fibrosis ASO, allele specific oligonucleotide HRP, horseradish peroxidaseThe collection of newborn screening specimens on filter paper blotters represented a major innovation that facilitated the establishment and regionalization of screening programs (1, 2). These screening programs were originally developed for PKU, but are incorporating an expanding group of disorders, including the hemoglobinopathies (3). With rare exceptions, these expanded test batteries have continued to use dried blood specimens in a filter paper matrix because of the ease of sample collection, handling, and shipment and the stability of the analytes. Typically, the analytes evaluated by neonatal screening laboratories have included metabolites such as phenylalanine for PKU, or galactose and galactose-1-phosphate for galactosemia; hormones such as thyroid hormone and thyroid stimulating hormone (TSH) for congenital hypothyroidism, or 17-hydroxyprogesterone for congenital adrenal hyperplasia; and proteins such as galactose-1-phosphate uridyl transferase for galactosemia, immunoreactive trypsinogen for CF, or Hb for the hemoglobinopathies (1, 3, 4).More recently, it has beco...

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