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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 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.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by the deficiency of one of the five enzymes involved in the biosynthesis of corticosteroids. The most common form of the disease is the lack of 21-hydroxylase which provokes an accumulation of high levels of 17α-hydroxyprogesterone (17-OHP), the main biochemical marker for illness detection. Given the significance of neonatal diagnosis for ensuring a timely treatment to patients suffering from CAH, newborn screening is worldwide performed for the determination of 17-OHP from dried blood spots on filter paper. The non-specificity of antisera employed in immunoassays and the cross-reaction with fetal adrenal hormones produce an overestimation in the 17-OHP quantification. Immunization of mice with 17-OHP-3-(O-carboxymethyl) oxime-bovine serum albumin led to the generation of 15 anti-17-OHP IgG1-and-IgG2b-secreting hybridomas. The 6E2G9 monoclonal antibody presents cross-reactivity values similar to those achieved by rabbit antibodies employed in the solid phase of UMELISA® 17-OH Progesterona Neonatal, assay for the newborn screening of CAH in Cuba. Additionally, the use of 6E2G9 in the evaluation of dried blood spots samples from newborns on filter paper showed a decrease in the mean 17-OHP levels, thus demonstrating it can replace the conventional rabbit antisera.

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.T4: L-Thyroxine; ELISA: Enzyme-linked immunosorbent assay; SUMA: Ultra Micro Analytic System; UMELISA: Ultramicro enzyme-linked immunosorbent assay; TSH: Thyroid-stimulating hormone.

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.

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 ...

The endemic flora of the Canary Islands is remarkable but highly vulnerable to environmental changes. Several factors, such as fire, could be threatening. The purpose of this study was to analyze various treatments in endemic threatened species which could have been growing in the understory of the Canary pine forest. We attempted to improve germination through dormancy breaking, and analyze the influence of fire on these plants. Seven physical and chemical treatments were tested: control, mechanical scarification, boiling water, dry heat (50 and 120 °C), smoke and smoked water. Mechanical scarification showed a positive response in germination percentage in two species characterized by hard seed coat, but out of all of the species tested, only one demonstrated a positive response to any of the treatments related to fire. At the temperatures registered on wildfires, neither the seeds situated on the aerial structure nor seeds in seed bank seem to be able to survive and germinate. The increasing incidence of fires through anthropogenic causes could contribute to the current threat status of these species and poorness of the Canary pine forest understory.

We studied the effects of herbivores on populations of four endemic legume species in pine forests in Caldera de Taburiente National Park on La Palma, Canary Islands. Seeds of these species were sown in control and herbivore-exclusion plots. Over the subsequent 4-years period, we assessed the effects of herbivores by counting the number of individual plants that emerged and by following their growth. We assessed growth over time using three plant-size categories. For these four species, we conclude that the presence of herbivores, including barbary sheep (Ammotragus lervia), goat (Capra hircus) and European rabbit (Oryctolagus cuniculus), exerts a strong negative effect on plant establishment. We suggest that the paucity of understory plant species in the pine forests of the Canary Islands may be strongly correlated with the presence of introduced herbivores in this region. We propose urgent conservation measures, such as large fenced areas, control activities and the most effective measure, eradication. Organismo Autónomo de Parques Nacionales Ministerio de Medio Ambiente y Medio Rural y Marino

The bioclimatic belts of Gomera are established and the potential distribution of its climatophilous vegetation series mapped accordingly. PCA and DCA analyses explain the significance of thermic factors related to altitude, and rainfall or humidity conditions (mist precipitation) in the distribution of bioclimatic belts and vegetation series. A map of potential natural vegetation is produced by considering several additional abiotic environmental factors, and the current distribution of potential vegetation remnants or their substitute communities. Three new climatophilous associations (Neochamaeleo pulverulentae—Euphorbietum balsamiferae, Violo rivinianae—Myricetum fayae, Cisto gomerae—Pinetum canariensis), one new climatophilous subassociation (Brachypodio arbusculae—Juniperetum canariensis subass. ericetosum arboreae), two potential edaphophilous new associations (Euphorbietum aphyllae, Euphorbio berthelotii-Retamatetum rhodorhizoidis), one new potential edaphophilous subassociation (Cisto gomerae—Pin...

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.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.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 levels40%. There was coincidence of our results with those obtained by the conventional colorimetric method that uses B-PAB as substrate.The qualitative colorimetric ultramicroassay does not require special laboratory equipment and it is suitable for the neonatal screening of biotinidase deficiency.

We have demonstrated previously [Pérez-Reyes, P.L., Sánchez-Alonso, J.A., López-Aparicio, P., Recio, M.N., Pérez-Albarsanz, M.A., 2001. Different molecular capacity in the induction of apoptosis by polychlorinated biphenyl congeners in rat renal tubular cell cultures. Biosci. Rep. 6, 765-778] that the polychlorinated biphenyls (PCBs) cause loss of cell viability and accelerate apoptosis in cell kidney cultures. Further investigations are necessary to elucidate the mechanism of apoptosis induction. In this way, we have analyzed in the present work the effects of PCBs on protein kinase C (PKC, a protein family intimately involved in the regulation of cell survival) and the expression of two proapoptotic (caspase-3 and Bax) and one antiapoptotic (Bcl-2) proteins. Aroclor 1248 (a commercial PCB mixture with 48% chlorine by weight), PCB 153 (2,2',4,4',5,5'-hexachlorobiphenyl, a di-ortho-substituted nonplanar congener) and PCB 77 (3,3',4,4'-tetrachlorobiphenyl, a non-ortho-substituted planar congener), significantly increased PKCalpha activity compared to control cells in the cytosolic and particulate cell fractions, and increased the PKCalpha protein content in the particulate fraction. The nonplanar PCB 153 showed stronger effects than the coplanar congener PCB 77. In addition, Aroclor 1248 decreased both, procaspase-3 levels and the Bcl-2/Bax protein ratio. These findings indicate that PCBs, particularly nonplanar congeners, can induce apoptosis in primary renal tubular cells through the PKCalpha, caspase-3 and Bcl-2/Bax pathway.

The introduction of alien species is one of the main threats to conservation. However, the same species may be threatened and be a threat in nearby areas. This is the case of the Barbary sheep ( Ammotragus lervia ) and mouflon ( Ovis orientalis ), which greatly hinder the management and conservation of biodiversity outside their native range. To prevent the extinction of many endemic plants, it is necessary to overcome several methodological difficulties.

The effects of a commercial polychlorinated biphenyl (PCB) mixture (Aroclor 1248) and two individual PCB congeners were evaluated on rat renal proximal tubule culture cell viability and internucleosomal DNA fragmentation (DNA ladder) characteristic of apoptosis. Treatment with Aroclor 1248 caused the loss of cell viability and promoted apoptosis in a concentration- and time-dependent manner. The two PCB congeners assessed can also induce apoptosis. However, the extent of apoptosis generated was greater for the non-ortho-substituted planar congener (3,3′,4,4′-tetrachlorobiphenyl) than for the di-ortho-substituted nonplanar congener (2,2′,4,4′,5,5′-hexachlorobiphenyl). This correlated with the loss of cell viability since the planar compound is much more cytotoxic. The results suggest a different molecular mechanism in the induction of apoptosis by planar or nonplanar PCB congeners.

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.

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.

Background 17α-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 17α-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.

New complexes of formula [Rh2(µ-L)(CO)2(µ-dppm)2]+, having the A-frame type structure with different pyrazolate-type groups L as bridgehead ligand, have been prepared from trans-[Rh2Cl2(CO)2(µ-dppm)2](dppm = Ph2PCH2PPh2). Iodine undergoes transannular oxidative addition to [Rh2(µ-L)(CO)2(µ-dppm)2][ClO4] to produce rhodium(II) complexes [Rh2(µ-L)I2(CO)2-(µ-dppm)2][ClO4] with direct rhodium–rhodium bonds. The crystal structures of the complexes [Rh2(µ-dmpz)(CO)2(µ-dppm)2][ClO4] and [Rh2(µ-dmpz)I2(CO)2(µ-dppm)2][ClO4](dmpz = 3,5-dimethylpyrazolate) have been determined by X-ray methods. The former crystallises in the monoclinic space group P21/a with unit-cell dimensions a= 22.560(9), b= 26.649(9), c= 10.221(7)A, β= 102.68(3)°, and Z= 4. The structure has been refined to R= 0.072 on the basis of 4 342 observed reflections. The latter crystallises in the monoclinic space group P2/n with a= 16.855(6), b= 14.713(3), c= 14.008(4)A, β= 90.66(2)°, and Z= 2. The structure has been refined to R= 0.078 (3 048 observed reflections). In both complexes two rhodium atoms are triply bridged by two molecules of dppm and by a dmpz ligand through its nitrogen atoms. In the first complex each Rh atom has a square-planar co-ordination, in the second the oxidative addition of iodine results in a change in co-ordination to octahedral and formation of a metal–metal bond [Rh–Rh 2.725(2) A].

The CO groups of [Rh2(µ-pz)I2(CO)2(µ-dppm)2]ClO4(pz = pyrazolate, dppm = Ph2PCH2PPh2) have been gradually substituted by reaction with isocyanides CNR (R = But or C6H4Me-p) to give [(OC)IRh(µ-pz)(µ-dppm)2Rhl(CNR)]ClO4[R = But(3) or C6H4Me-p(4)]via[(OC)IRh(µ-pz)(µ-CO)(µ-dppm)2Rhl(CNR)]ClO4[R = But(1) or C6H4Me-p(2)] and, finally, [Rh2(µ-pz)I2(CNR)2(µ-dppm)2] ClO4[R = But(7) or C6H4,Me-p(8)]via[Rh2(µ-pz)I2(µ-CO)(CNR)2(µ-dppm)2]ClO4[R = But(5) or C6H4Me-p(6)]. The CO bridging groups of (1), (2), (5), and (6) are ketonic carbonyls according to their spectroscopic properties. The reactivity of [Rh2(µ-pz) I2(CO)2(µ-dpam)2]ClO4(dpam = Ph2AsCH2AsPh2) with isocyanides was studied for comparison. The structure of the BF4–analogue of (7), [Rh2(µ-pz)I2(CNBut)2(µ-dppm)2]BF4(7b), has been determined by X-ray diffraction methods. Crystals are monoclinic, space group P21/n with unit-cell parameters a= 20.347(8), b= 21.221(8), c= 14.514(6)A, β= 94.11(2)°, and Z= 4. The structure has been solved from diffractometer data by Patterson and Fourier methods and refined by block-matrix least squares to R= 0.0630 for 4 969 observed reflections. In the cationic complex the two Rh atoms, at a distance of 2.829(3)A, are connected by a triple bridge, involving two dppm ligands and the pyrazolate anion.

Congenital hypothyroidism (CH) is one of the most common preventable causes of mental retardation. Newborn screening and thyroid therapy started within 2 weeks of age can normalize the cognitive and motor impairment caused by lack of thyroid hormone during the early postnatal phase of brain development. The availability of assays to determine thyroxin (T4) and thyroid stimulating hormone has allowed the establishment of newborn screening programs for CH. The Immunoassay Center (Cuba) developed the T4 Neonatal UMELISA(r) to determine neonatal T4 levels in dried blood on filter paper using 5 mm discs of samples, standards and controls. The number of diseases which can be diagnosed in Neonatal Screening Programs has increased, so it is necessary to make the maximum use of blood samples collected on filter paper. This work shows the standardization of the T4 Neonatal UMELISA(r) modified in order to use 3 mm discs. The assay was completed in 3 1/2 hours, with a measuring range of 25-400 nmol/L. The intra- and inter-assay coefficients of variation (CV) were 6-10 % and 7-12 % respectively, depending on the T4 concentrations. The recovery ranged from 91.8-115.1 %. The modified assay showed high Pearson correlation (r = 0.877) and concordance correlation (?c = 0.867) with the T4 Neonatal UMELISA(r). The performed modifications do not affect the sensitivity, precision and accuracy of the assay, permitting a more rational use of newborn blood samples and the possibility to increase the number of diseases included in the newborn screening programs.