Newborn Screening Program for Cystic Fibrosis in Cuba: Three Years' Experience

ABSTRACT In Cuba, newborn screening (NBS) for cystic fibrosis (CF) was introduced in January 2019. The results from the first three years of the CF NBS program are presented. An IRT/IRT protocol was followed using a cut-off value of 50 ng/mL. In this period 281,717 neonates were screened, 2,197 samples had increased IRT values, and a second sample was necessary (recall rate=0.78%). In 686 (0.24%) neonates, IRT was still elevated, and they were referred for clinical evaluation. Twenty-one children were confirmed by sweat test and molecular biology. Eighteen newborns presented variant F508del. A false negative case was reported. Demographic data of 32,764 neonates were collected. The average age of sampling was six days with results available at 11 days of life, but 1.7% of the samples were collected 20 days after birth. The mean IRT value was 12.7±11.7 ng/mL (ranging 0-283 ng/mL) with a calculated 98.5 percentile value of 42.4 ng/mL. On average, the samples were processed five days after collection and two days after they were received at the laboratory. Although CF NBS program in Cuba is just beginning, it can be predicted that CF will be one of the most frequent inherited-metabolic diseases in the Cuban population.

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.

SUMA Technology and Newborn Screening Tests for Inherited Metabolic Diseases in Cuba: An Overview of the First 30 Years

Abstract The ultramicroanalytic system (SUMA), created in the 1980s, is a complete system of reagents and instrumentation to perform ultramicroassays combining the sensitivity of the micro-enzyme-linked immunosorbent assay (ELISA) tests with the use of ultramicrovolumes. This technology permitted establishing large-scale newborn screening programs (NSPs) for metabolic and endocrine disorders in Cuba. This article summarizes the main results of the implementation during the 30 years of SUMA technology in NSP for 5 inherited metabolic diseases, using ultramicroassays developed at the Department of Newborn Screening at the Immunoassay Center. Since 1986, SUMA technology has been used in the Cuban NSP for congenital hypothyroidism, initially studying thyroid hormone in cord serum samples. In 2000, a decentralized program for the detection of hyperphenylalaninemias using heel dried blood samples was initiated. These successful experiences permitted including protocols for screening congenital adrenal hyperplasia, galactosemia, and biotinidase deficiency in 2005. A program for the newborn screening of CH using the thyroid-stimulating hormone Neonatal ultramicro-ELISA was fully implemented in 2010. Nowadays, the NSP is supported by a network of 175 SUMA laboratories. After 30 years, more than 3.8 million Cuban newborns have been screened, and 1002 affected children have been detected. Moreover, SUMA technology has been presented in Latin America for over 2 decades and has contributed to screen around 17 million newborns. These results prove that developing countries can develop appropriate diagnostic technologies for making health care accessible to all.

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.

Quantitative ultramicrotest for newborn screening of galactosemia in Cuba.

BACKGROUND: To describe a simple, rapid, quantitative ultramicrotest (UMTEST) based on the fluorometric method introduced by Fujimura et al. adapted to an Ultra Micro Analytic System (SUMA) for the detection of total galactose (GAL) in dried blood specimens. METHODS: The assay uses 3 mm discs of dried blood on Whatman 903 filter paper and small volumes of each reagent. A methanol/acetone/water solution is used for deproteination, and a specially designed 96-well polystyrene opaque ultramicroplates, with a maximum capacity of 30 µL per well, are used for the reading. RESULTS: The UMTEST GAL is completed in 2 h, with measuring range of 0.28-3.92 mmol/L. The intra- and inter-assay coefficients of variation were 2.3%-8.9% and 6.8%-11.1%, respectively, depending on the total GAL concentrations. Percentage recovery ranged from 97.7% to 103%. Limit of detection and limit of quantitation were 0.06 and 0.16 mmol/L, respectively. The mean GAL concentration, in 2510 dried blood samples from the National Neonatal Screening Program was 0.23 mmol/L. Our assay showed high concordance correlations with the commercially available ICN Immuno-Chem™ GAL-MW EA kit. CONCLUSIONS: The analytical performance characteristics of this assay is suitable for mass newborn screening of galactosemia in Cuba.

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.

Qualitative colorimetric ultramicroassay for the detection of biotinidase deficiency in newborns.

BACKGROUND: We describe a simple qualitative visual ultramicroassay based on the colorimetric method introduced by Heard et al. for the detection of biotinidase deficiency in dried blood samples spotted on filter paper. METHODS: The assay uses 3-mm discs of dried blood on Schleicher and Schuell 903 filter paper and ultramicrovolumes of each reagent. Ten thousand newborn samples from the National Screening Program for the Detection of Phenylketonuria were evaluated. RESULTS: The ultramicroassay shows a good reproducibility. The lower detection limit is around 2% of the mean normal activity. We found one sample with the absence of enzymatic activity, another that was between 10% and 30%, and 10 with activity levels <40%. There was coincidence of our results with those obtained by the conventional colorimetric method that uses B-PAB as substrate. CONCLUSIONS: The qualitative colorimetric ultramicroassay does not require special laboratory equipment and it is suitable for the neonatal screening of biotinidase deficiency.