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.

Evaluation of UMELISA® T4 NEONATAL and UMELISA® T4 using polystyrene plates coated with anti-thyroxine (T4) monoclonal antibodies.

Congenital hypothyroidism is one of the most common preventable causes of mental retardation. The Center of Immunoassay has developed the UMELISA® T4 NEONATAL and UMELISA® T4 to determine neonatal T4 levels in dried blood and serum samples. Both reagent kits use the same polystyrene plates coated with anti-thyroxine (T4) polyclonal antibodies as solid phase. This work shows the re-standardization of the UMELISA® T4 NEONATAL and UMELISA® T4 using plates coated with anti-T4 monoclonal antibodies (T4Mabs). Polystyrene plates of the modified assays were firstly coated with polyclonal IgG sheep-anti-mouse IgG for 18 hours. T4Mabs were added to the plates and incubated for 2 hours at room temperature. Different performance parameters were evaluated and correlation studies with the commercial kits done. Using polystyrene plates coated with T4Mabs increases the slope of the calibration curve in the clinical interest zone. The assay conjugates work twice diluted in respect to the ones of the commercial kits. Recovery percentages (90.8-110.7 for UMELISA® T4 NEONATAL and 92.1-109.3 for UMELISA® T4) and intra (7.2-7.6 for UMELISA® T4 NEONATAL and 6.9-7.2 for UMELISA® T4) and inter (7.4-8.5 for UMELISA® T4 NEONATAL and 7.1-8.5 for UMELISA® T4) coefficients of variation were similar to the ones described for the commercial kits. Limits of detection and quantification were 9.0 and 21.1 nmol/L for UMELISA® T4 NEONATAL, and 8.9 and 20.5 nmol/L for UMELISA® T4, respectively. The results also showed high overall concordance between assays (n = 244, r = 0.92, ρc = 0.91 for UMELISA® T4 NEONATAL and n = 492, r = 0.92, ρc = 0.9 for UMELISA® T4). The analytical sensibility of UMELISA® T4 NEONATAL and UMELISA® T4 is improved by using polystyrene plates coated with T4Mabs, without affecting the precision and accuracy of the results. ABBREVIATIONS: T4: L-Thyroxine; ELISA: Enzyme-linked immunosorbent assay; SUMA: Ultra Micro Analytic System; UMELISA: Ultramicro enzyme-linked immunosorbent assay; TSH: Thyroid-stimulating hormone.

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.

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.