Pilot study for cystic fibrosis neonatal screening: the Cuban experience.

Background In Cuba, no screening program for cystic fibrosis (CF) has been implemented yet. The ultramicro enzyme-linked immunosorbent assay (UMELISA)® TIR NEONATAL has been developed for the measurement of immunoreactive trypsin (IRT) in dried blood spots on filter paper. The analytical performance of the kit was evaluated in the national network of laboratories. Methods Newborn dried blood samples (DBS) were evaluated in 16 laboratories. An IRT/IRT/DNA protocol was followed using a cut-off value of 50 ng/mL. The mean, median and percentiles of the distribution were calculated and a two-sample t-test with unequal variance was used for statistical analysis. Influence of perinatal factors on IRT levels was analyzed. Results From January to June 2018, 6470 newborns were studied, obtaining a mean IRT value of 12.09 ng/mL (ranging 0-358 ng/mL) and a median of 8.99 ng/mL. Fifty-two samples (0.78%) were above the cut-off level and 16 samples (0.24%) were elevated in the re-screening process. One of them was confirmed positive by molecular biology (phe508del/c.3120 + 1G > A), constituting the first newborn screened and diagnosed early in Cuba. Second DBS samples were collected on average at 14 days and processed in the laboratory at 16 days of birth. Significant differences were observed (p < 0.05) when evaluating the influence of gender, birth weight (BW) and gestational age (GA) on the IRT values. Lower IRT concentrations were found in samples processed after 10 days of collection. Conclusions The performance of UMELISA® TIR NEONATAL in the laboratories has been satisfactory; hence CF newborn screening (NBS) was extended throughout the country from January 2019.

An Enzyme Immunoassay for Determining Immunoreactive Trypsinogen (IRT) in Dried Blood Spots on Filter Paper Using an Ultra-Microanalytical System.

Cystic fibrosis (CF) is a severe autosomal recessive disorder. It is caused by mutations in the CF transmembrane conductance regulator gene. Early diagnosis of CF can be carried out by determining high immunoreactive trypsinogen (IRT) blood values in newborns. A simple sandwich-type ultramicroELISA assay (UMELISA®) has been developed for the measurement of IRT in dried blood spots on filter paper. Strips coated with a high affinity monoclonal antibody directed against IRT are used as solid phase, to ensure the specificity of the assay. The assay is carried out within 20 h. The useful rank of the curve is 0-500 ng/mL, and the lowest detectable concentration is 4.8 ng/mL. Intra- and inter-assay coefficients of variation were lower than 10%. The recovery mean value was 100.3 ± 11.2%. Cross-reactivity with proteins structurally related to IRT (α2-macroglobulin, α1-antitrypsin, and human chymotrypsin) was lower than the detection limit of the assay. Four thousand four hundred six newborn samples from the Cuban Newborn Screening Program were analyzed, and the mean IRT concentration was 12.8 ng/mL. Higher IRT values were obtained when samples were eluted overnight. Regression analysis showed a good correlation with the commercially available AutoDELFIA® Neonatal IRT kit (n = 3948, r = 0.885, ƙ = 0.976, p < 0.01). The analytical performance characteristics of our UMELISA® TIR Neonatal suggest that it can be used for the neonatal screening of CF.

An enzyme immunoassay for determining epidermal growth factor (EGF) in human serum samples using an ultramicroanalytical system.

Human epidermal growth factor is a small peptide consisting of 53 amino acid residues, which stimulates cell proliferation and is associated with several human carcinomas. A simple sandwich-type ultramicroELISA assay (UMELISA), based on the advantages of high affinity reaction between streptavidin and biotin has been developed for the measurement of EGF in human serum samples. Strips coated with a high affinity monoclonal antibody directed against EGF are used as solid phase, to ensure the specificity of the assay. The EGF assay was completed in 18 hr, with a measuring range of 39-2500 pg/mL. The intra- and inter-assay coefficients of variation were 4.4-7.3% and 0-5.1%, respectively, depending on the EGF concentrations evaluated. Percentage recovery ranged from 96-104%. Regression analysis showed a good correlation with the commercially available Human EGF Immunoassay Quantikine® ELISA kit (n = 130, r = 0.92, P < 0.01). The analytical performance characteristics of our UMELISA EGF endorse its use for the quantification of EGF in human serum samples.

Newborn screening for congenital adrenal hyperplasia in Cuba: six years of experience.

BACKGROUND: Since 2005, a newborn screening program for congenital adrenal hyperplasia (CAH) by measuring 17-alpha-hydroxyprogesterone (17OHP) in dried blood spots was introduced in Cuba. METHODS: The hormone was measured by the 17OHP Neonatal UMELISA method, in samples collected on the 5th day as average. Confirmatory test was performed to those neonates with 17OHP values above 55 nmol/l. Some perinatal factors that can influence on 17OHP levels were studied. RESULTS: From January 2005 to December 2010, 621,303 newborns were screened and 39 CAH cases were detected. Coverage of the program reached 98%. The incidence of CAH in Cuba was 1:15,931, similar to that reported by other programs. A recall for suspected CAH was performed in 10,799 cases (1.74%). Therapy in classical CAH patients was started at the mean age of 22 days. 17OHP levels were significantly higher in newborns with lower birth-weight (BW) and/or gestational age (GA). In addition, 17OHP values were affected by the gender, twin status or mode of delivery. CONCLUSIONS: In Cuba, the nationwide newborn screening program has allowed the early detection of CAH. The use of an optimized cut-off level for BW or GA could lead to a reduction in the percentage of recalled babies.

Cuban neonatal screening of phenylketonuria using an ultramicro-fluorometric test.

BACKGROUND: Guthrie's bacterial inhibition assay was used in Cuba, since 1983. A decentralized program for the newborn screening of hyperphenylalaninemias started in the year 2000 using an ultramicro-fluorometric test (UMTEST PKU). METHODS: A simple and rapid ultramicro-fluorometric test based on McCaman and Robin's method has been designed, developed and applied for the measurement of Phe in dried blood spots on filter paper. RESULTS: The UMTEST PKU exhibited an acceptable precision and accuracy. Samples of 27528 newborns on filter paper Schleicher & Schuell 903 (S&S 903) from the National neonatal screening program were collected and analyzed, and the mean Phe concentration was 66.5 micromol/l. Our assay showed high Pearson and concordance correlations with 2 commercially available kits. A total of 521923 Cuban newborns were studied from the year 2000 to 2007 using the UMTEST PKU. Elevated blood phenylalanine levels were found in 1764 infants (0.34%) and no false negative were noted. Ten cases were diagnosed with phenylketonuria, all of them with an initial phenylalanine concentration over 360 micromol/l. CONCLUSIONS: The analytical performance characteristics of our assay and its use in the National program have demonstrated its suitability for the neonatal screening of PKU.

An enzyme immunoassay for determining 17alpha-hydroxyprogesterone in dried blood spots on filter paper using an ultramicroanalytical system.

BACKGROUND: 17alpha-hydroxyprogesterone has been used for the diagnosis of congenital adrenal hyperplasia (CAH) in the newborn period. METHODS: A simple and rapid competitive ultramicro ELISA assay based on competition between 17-OHP-alkaline phosphatase conjugate and 17-OHP in blood specimens for a limited number of binding sites on specific polyclonal rabbit anti-17-OHP antibodies, has been developed for the measurement of 17-OHP in dried blood spots on filter paper. The assay buffer contains danazol to displace 17-OHP from steroid-binding proteins. RESULTS: The 17-OHP assay was completed in 3 h, with a measuring range of 10-250 nmol/l. The intra- and inter-assay CV were 5.5-8.2% and 6.4-9.1%, respectively, depending on the 17-OHP concentrations. The recovery ranged from 98-103%. Of 3750 newborn samples collected on filter paper, 903 from the national neonatal screening program were analyzed, and the mean 17-OHP concentration was 12.2 nmol/l. Our assay showed high Pearson and concordance correlations with the commercially available ICN Neoscreen ELISA 17alpha-hydroxyprogesterone kit. CONCLUSIONS: The analytical performance characteristics of our 17-OHP Neonatal UMELISA suggest that it can be used for the neonatal screening of CAH.