Immunoreactive Trypsin Research Articles

Assessment of the effectiveness of a new neonatal screening algorithm for cystic fibrosis

Relevance All over the world, neonatal screening protocols are being improved in order to reduce the time of diagnosis, simplify the examination procedure, and reduce the number of false positive and false negative results of neonatal screening. In most European countries, the best neonatal screening algorithm is molecular genetic testing. Objective: Evaluate the effectiveness of the new cystic fibrosis screening protocol algorithm, which, in the first step, determines the immunoreactive trypsin (IRT) level from one dry spot and performs DNA diagnostics (36 most frequent mutations) in patients with increased IRT. Compare the economic costs of screening by two algorithms (existing and alternative). Materials and methods: two parallel algorithms were used for neonatal screening. Which was performed on 56,534 infants in the Moscow Region from March to November 2022. The current three-stage algorithm was used, which involves re-blood sampling from newborns at risk 3-4 weeks after the first blood sampling and a sweat test. In the new algorithm, newborns with an elevated IRT 1 level at the 1st week of life underwent a molecular genetic study (search for frequent mutations in the CFTR-36 variants gene, sequencing of individual locus of the CFTR gene for control purposes) in the same dry blood spot used for screening. Results: out of 56,534 test forms received, 607 (1.07%) newborns with a positive test for IRT were identified for neonatal screening in a laboratory from the Moscow region, of which 501 (82.5%) samples were selected, where the level of RTI1 exceeded the cut-off level (65 ng / ml), which were sent for molecular genetic research in order to search for pathogenic variants in the CFTR gene. 106 (17.5%) newborns did not complete the examination. Positive results of confirmatory diagnosis were obtained in 6 infants, which accounted for 0.01% of the number of children who underwent screening and 1.2% of the number of children with neonatal hypertrypsinogenemia. Conclusion: the positive effects of the new algorithm are to reduce psychological tension in families during examination by reducing the time of diagnosis, no need for repeated blood sampling on the 21st-28th day of life, reduction of the time of diagnosis, prevention of nutritional deficiencies in children during the first months of life and life-threatening complications. Reducing the number of children with incomplete testing for cystic fibrosis, economic benefit. The negative effects of the new algorithm are that it is possible to miss a patient with cystic fibrosis who does not have any of the common pathogenic variants in the CFTR gene.

A Public Dialogue to Inform the Use of Wider Genomic Testing When Used as Part of Newborn Screening to Identify Cystic Fibrosis.

Cystic fibrosis (CF) has been included within the UK national newborn screening programme since 2007. The approach uses measures of immunoreactive trypsin (IRT) in dried blood spot samples obtained at day 5 of life. Samples which reveal IRT results >99.5th centile go on to be tested for a limited panel of CF mutations. While the programme works well and achieves a high level of sensitivity and specificity, it relies upon repeat testing in some cases and identifies probable carriers, both potentially provoking parental anxiety. In addition, the limited CF mutation panel may not fully reflect the ethnic diversity within the UK population. The use of wider genomic screening, made possible by next-generation sequencing to replace more limited panels, can be used to avoid these shortcomings. However, the way in which this approach is employed can either be designed to maximise specificity by limiting reporting to combinations of known pathogenic mutations or can maximise sensitivity by also reporting combinations of pathogenic mutations together with variants of uncertain significance. The latter approach also increases the number of Cystic Fibrosis Screen-Positive Inconclusive Diagnosis (CFSPID) designations reported, resulting in uncertainty for parents. To help consider the design of the programme, a dialogue was commissioned by the UK National Screening Committee (UKNSC) to elicit the views of members of the public without direct experience of CF, to determine if there was a preference for maximising the sensitivity or the specificity of CF screening. The participants initially expressed a clear preference to maximise sensitivity and avoid missing CF cases, but after time to reflect and consider the implications of their choice, a number changed their views so as to tolerate some missed cases if this resulted in greater certainty of outcome; this became the majority view. It is proposed that it may be a generalisable finding that the public, when facing whole-population screening programmes, may require significant time and information to inform and make their choices and may attach great importance to clarity and certainty of outcome in the screening process.

Concentration of fecal β-defensin-2 in children with cystic fibrosis: how the inneral intestinal immune response?

Cystic fibrosis is a disease caused by mutations in a gene encoding CFTR-protein (Cystic Fibrosis Transmembrane conductance Regulator), located in the apical membrane of epithelial cells of the respiratory tract, intestines and pancreas. Defensins serve as important components of the innate human immune system, they play a key role in providing the first line of defense of a macroorganism against infection; they have high antimicrobial, antiviral, cytotoxic activity.Objective. To determine the values of fecal β-defensin-2 in children with cystic fibrosis and to reveal the dependence of its level on the exocrine function of the pancreas and the severity of the patient’s condition.Characteristics of children and research methods. The study included 57 children with cystic fibrosis, the average age was 20.93 ± 2.9 months. Cystic fibrosis was diagnosed on the basis of an increase in immunoreactive trypsin, sweat chlorides by Cook’s method (>60 meq / l). To assess the exocrine function of the pancreas the scientists determined the activity of fecal elastase. They evaluated the levels of fecal β-defensin-2 and calprotectin using a quantitative enzyme immunoassay.Results. The levels of fecal β-defensin-2 were increased (108.2 ± 11.3 ng / ml) in all children under examination. The researchers found no correlation between the levels of fecal β-defensin-2 and fecal elastase. The level of fecal calprotectin was significantly higher in the group of children with cystic fibrosis as compared to the control group. There was a significant correlation between the levels of fecal calprotectin and fecal β-defensin-2 (r=0.57; p <0.05), however, no correlations were found between the levels of fecal β-defensin-2 and fecal elastase. The group of children with a severe course of the disease demonstrated an increase in the level of fecal β-defensin-2, fecal calprotectin significantly more frequent.Conclusion. Children with cystic fibrosis demonstrated a significant increase in the concentration of β-defensin-2 as compared to the control group, which confirms the activation of the innate immune system of the intestinal mucosa. The researchers traced the relationship between high levels of fecal β-defensin-2 and the severity of the disease. The levels of fecal β-defensin-2 directly correlated with the concentration of fecal calprotectin and there was no correlation between the severity of pancreatic insufficiency and the concentration of fecal β-defensin-2.

Neonatal cystic fibrosis screening: Analysis and differences in immunoreactive trypsin levels in newborns with a positive screen

IntroductionNeonatal cystic fibrosis (CF) screening has enabled the disease to be diagnosed early, and is a determining factor in the increase in survival of these patients. Its main disadvantage is its low specificity and elevated number of false positives. The aim of this study is to analyse the differences in immunoreactive trypsin (IRT) between the different groups of newborns (NB) with a positive neonatal screen depending on whether they were healthy, healthy carriers, affected by CF, or CFSPID (Cystic Fibrosis Screen Positive, Inconclusive Diagnosis). MaterialRetrospective analytical study of the concentrations of IRT in NB with a positive neonatal screen for CF born in a tertiary hospital during an 8-year period. ResultsA total of 790 NB with a positive neonatal screen for CF were analysed. Of these 86.3% were term, 53% girls, and 11.8% were admitted. The mean IRT value was 79.16 ng/mL (range 60–367). Significantly higher concentrations of IRT were found in those affected by CF compared to the other groups (P < .001). There were higher levels in large prematures (P = .007) and admitted patients (P = .002). There were no differences as regards gender or season. There was a direct correlation of 64% (P = .001) between IRT and sweat test in those affected by CF and CFSPID. A cut-off value of IRT for the diagnosis of CF was calculated from the ROC curve (76.2 ng/mL (S = 95.7%, Sp = 64.5%). ConclusionsNB with CF have significantly higher levels of IRT than healthy ones, or carriers and CFSPID. Prematurity and hospital admission may also have an influence. A higher IRT value is associated with a higher level in the sweat test.

Analysis of national coverage of neonatal cystic fibrosis screening in Brazil from 2008 to 2017

Introduction The Brazilian neonatal screening program established in 2001 included the investigation of cystic fibrosis (CF) in the phase III of the program. For this purpose, the immunoreactive trypsin (IRT) measurement was added to the newborn screening test. The purpose of National Neonatal Screening Program is to reach 100% of live births in Brazil. The aim of this study was to analyze the coverage of neonatal Screening for Cystic Fibrosis (CF) in Brazil from 2008 to 2017. Materials and methods This is an ecological study design based on data collected from the Brazilian Outpatient Information System regarding the detection of IRT as a component of the heel stick test from 2008 to 2017. Moreover, we gathered data from the Brazilian Live Birth Information System referring to live births from 2008 to 2017. We calculated the coverage of IRT measurement for every 100 live births, using the number of IRT measurement procedures as the numerator and the number of live births stratified by federative units (states), as well as by the Brazilian regions as the denominator. These regions correspond to the divisions of the national territory based on criteria such as natural, social, cultural and economic aspects. Results From 2008 to 2017, the regions presented the following coverage medians: South (84.1%), Southeast (71.4%), Midwest (47.3%) Northeast (12.3%) and North (10.9%). In the analysis of federative units, in the years 2013 to 2017, Paraná and Distrito Federal presented the highest median of coverage (100%), while the states with the lowest median were Rio Grande do Norte (12.1%), Amazonas (16.8%) and Paraíba (27.5%). Highest coverage was found in the South region, where are located most of the states with high socioeconomic development and high supply of health services, while the lowest coverage was found in the North region, where are located manly the states with low socioeconomic development and low supply of health services. According to data from the Brazilian Ministry of Health, the universalization of neonatal screening for cystic fibrosis occurred in 2013, however, at the end of this year, most states in the North and Northeast regions had not registered IRT measurements in the Outpatient Information System. Conclusion Although the coverage of neonatal screening for CF has improved nationwide over the years of the study, the disparity in the coverage of IRT measurements at the interregional and interstate levels is notable. Systematic implementation of neonatal screening for cystic fibrosis with equity and access to the entire population is suggested, leading to a greater number of children benefiting from treatment and a better quality of life.

Cribado neonatal de fibrosis quística: análisis y diferencias de los niveles de tripsina inmunorreactiva en recién nacidos con cribado positivo

Neonatal cystic fibrosis (CF) screening has enabled the disease to be diagnosed early, and is a determining factor in the increase in survival of these patients. Its main disadvantage is its low specificity and elevated number of false positives. The aim of this study is to analyse the differences in immunoreactive trypsin (IRT) between the different groups of newborns (NB) with a positive neonatal screen depending on whether they were healthy, healthy carriers, affected by CF, or CFSPID (Cystic Fibrosis Screen Positive, Inconclusive Diagnosis). Retrospective analytical study of the concentrations of IRT in NB with a positive neonatal screen for CF born in a tertiary hospital during an 8-year period RESULTS: A total of 790 NB with a positive neonatal screen for CF were analysed. Of these 86.3% were term, 53% girls, and 11.8% were admitted. The mean IRT value was 79.16 ng/mL (range 60 - 367). Significantly higher concentrations of IRT were found in those affected by CF compared to the other groups (P<.001). There were higher levels in large prematures (P=.007) and admitted patients (P=.002). There were no differences as regards gender or season. There was a direct correlation of 64% (P=.001) between IRT and sweat test in those affected by CF and CFSPID. A cut-off value of IRT for the diagnosis of CF was calculated from the ROC curve (76.2 ng/mL (S=95.7%, Sp=64.5%). NB with CF have significantly higher levels of IRT than healthy ones, or carriers and CFSPID. Prematurity and hospital admission may also have an influence. A higher IRT value is associated with a higher level in the sweat test.

Cystic fibrosis newborn screening: the importance of bloodspot sample quality

ObjectiveWales has an immunoreactive trypsin (IRT)-DNA cystic fibrosis (CF) newborn screening (NBS) programme. Most CF NBS false negative cases are due to an IRT concentration below the screening threshold. The...

Case report: a child with cystic fibrosis and phenylketonuria

Background. In the international clinical practice there have been occasional reports of phenylketonuria (PKU) and cystic fibrosis (CF) found simultaneously in the same patient. Both PKU and CF are the inherited disorders characterized by autosomal recessive type of inheritance. Currently the combination of two or more inherited disorders in one patient is considered to be a clinical rarity.Case description. This is a clinical case of two genetic disorders, CF and PKU, combined in a 5-year old patient who had been followed up since birth. Owing to implementation of neonatal screening for inherited and congenital diseases into clinical practice, during the first month of life the infant was diagnosed with CF (diagnostically significant elevation of immunoreactive trypsin [IRT] at the initial [163.2 ng/mL] and repeat testing on day 21 of life [138.7 ng/mL]) and PKU (phenylalanine [PA] level 15.9 mg/dL). Both disorders have been confirmed by genetic tests, i.e., homozygous DelF508 mutation was found in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and P281L mutation in the phenylalanine hydroxylase (PAH) gene was also present in homozygous state. Child’s parents strictly adhered to dietary and treatment recommendations. By the age of 5 years the child developed symptoms of neurological disorder and disorder of the respiratory system, cognitive impairment and delay in speech development, subclinical epileptiform activity with high risk of epilepsy, and chronic inflammation of the respiratory tract.Conclusion. This case report demonstrates the important role of neonatal screening in early diagnosis and timely start of therapy, and underscores the importance of continuous medication in such genetic disorders as CF and PKU. On the whole, such approach brings about a relative preservation of functioning of the most affected organs and systems. By the age of 5 years the child does not form bronchiectases, shows no signs of chronic hypoxia, nutritional deficiency or pronounced neurologic deficit, and is at low risk for the development of autism spectrum disorder. At the same time, the larger scale and longer-term observations are required in order to make the unequivocal conclusions about the prognosis of these diseases under conditions of modern-day medical follow-up.

Case report: a child with cystic fibrosis and phenylketonuria

Background. In the international clinical practice there have been occasional reports of phenylketonuria (PKU) and cystic fibrosis (CF) found simultaneously in the same patient. Both PKU and CF are the inherited disorders characterized by autosomal recessive type of inheritance. Currently the combination of two or more inherited disorders in one patient is considered to be a clinical rarity.Case description. This is a clinical case of two genetic disorders, CF and PKU, combined in a 5-year old patient who had been followed up since birth. Owing to implementation of neonatal screening for inherited and congenital diseases into clinical practice, during the first month of life the infant was diagnosed with CF (diagnostically significant elevation of immunoreactive trypsin [IRT] at the initial [163.2 ng/mL] and repeat testing on day 21 of life [138.7 ng/mL]) and PKU (phenylalanine [PA] level 15.9 mg/dL). Both disorders have been confirmed by genetic tests, i.e., homozygous DelF508 mutation was found in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and P281L mutation in the phenylalanine hydroxylase (PAH) gene was also present in homozygous state. Child’s parents strictly adhered to dietary and treatment recommendations. By the age of 5 years the child developed symptoms of neurological disorder and disorder of the respiratory system, cognitive impairment and delay in speech development, subclinical epileptiform activity with high risk of epilepsy, and chronic inflammation of the respiratory tract.Conclusion. This case report demonstrates the important role of neonatal screening in early diagnosis and timely start of therapy, and underscores the importance of continuous medication in such genetic disorders as CF and PKU. On the whole, such approach brings about a relative preservation of functioning of the most affected organs and systems. By the age of 5 years the child does not form bronchiectases, shows no signs of chronic hypoxia, nutritional deficiency or pronounced neurologic deficit, and is at low risk for the development of autism spectrum disorder. At the same time, the larger scale and longer-term observations are required in order to make the unequivocal conclusions about the prognosis of these diseases under conditions of modern-day medical follow-up.

Case report: a child with cystic fibrosis and phenylketonuria

Background. In the international clinical practice there have been occasional reports of phenylketonuria (PKU) and cystic fibrosis (CF) found simultaneously in the same patient. Both PKU and CF are the inherited disorders characterized by autosomal recessive type of inheritance. Currently the combination of two or more inherited disorders in one patient is considered to be a clinical rarity.Case description. This is a clinical case of two genetic disorders, CF and PKU, combined in a 5-year old patient who had been followed up since birth. Owing to implementation of neonatal screening for inherited and congenital diseases into clinical practice, during the first month of life the infant was diagnosed with CF (diagnostically significant elevation of immunoreactive trypsin [IRT] at the initial [163.2 ng/mL] and repeat testing on day 21 of life [138.7 ng/mL]) and PKU (phenylalanine [PA] level 15.9 mg/dL). Both disorders have been confirmed by genetic tests, i.e., homozygous DelF508 mutation was found in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and P281L mutation in the phenylalanine hydroxylase (PAH) gene was also present in homozygous state. Child’s parents strictly adhered to dietary and treatment recommendations. By the age of 5 years the child developed symptoms of neurological disorder and disorder of the respiratory system, cognitive impairment and delay in speech development, subclinical epileptiform activity with high risk of epilepsy, and chronic inflammation of the respiratory tract.Conclusion. This case report demonstrates the important role of neonatal screening in early diagnosis and timely start of therapy, and underscores the importance of continuous medication in such genetic disorders as CF and PKU. On the whole, such approach brings about a relative preservation of functioning of the most affected organs and systems. By the age of 5 years the child does not form bronchiectases, shows no signs of chronic hypoxia, nutritional deficiency or pronounced neurologic deficit, and is at low risk for the development of autism spectrum disorder. At the same time, the larger scale and longer-term observations are required in order to make the unequivocal conclusions about the prognosis of these diseases under conditions of modern-day medical follow-up.

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.

History of Newborn Screening for Cystic Fibrosis-The Early Years.

This review summarises the trajectory of neonatal screening strategies for the detection of cystic fibrosis (CF) using the measurement of Immunoreactive Trypsin (IRT) in dried blood spots (DBS) from 1979 until the beginning of the 21st century when newborn screening (NBS) programmes started to spread throughout many countries, using IRT measurement combined with a CF genotype analysis of DBS.

Review of the sweat test indications in a Brussels' cystic fibrosis reference center

We collected data of all sweat tests performed between 2008, 1th of March and 2015, 28th of February. They were analyzed following Rosenstein diagnosis criteria (1998): clinical manifestations suggesting CF, positive neonatal screening (≥ 1 positive assay of immunoreactive trypsin) or familial history of CF. We reviewed 1,208 sweat tests over this period. Patients were aged from 13 days to 79 years. Indications were: clinical events (94.0%), a positive neonatal screening (3.7%) and a family history (2.3%). Over the 20 newly diagnosed patients, a positive neonatal screening was the main indication for the sweat test (55%). A positive neonatal screening (p<0.0001), a family history (p<0.0001) and pulmonary signs associated with digestive signs (p=0.004) were more frequently found in these patients. Sweat test indications are mostly clinical and mainly pulmonary. This study confirms that a sweat test should be performed in case of pulmonary manifestations suggesting CF especially if these are associated with digestive manifestations.

Cystic fibrosis screen positive inconclusive diagnosis (CFSPID): Experience in Tuscany, Italy

ObjectiveThe implementation of cystic fibrosis (CF) newborn screening (NBS) has led to identification of infants with a positive NBS test but inconclusive diagnosis classified as “CF screen positive, inconclusive diagnosis” (CFSPID). We retrospectively evaluated the prevalence and clinical outcome of CFSPID infants diagnosed by 2 NBS algorithms in the period from 2011 to 2016 in the Tuscany region of Italy. MethodsIn 2011–2016, we assessed the diagnostic impact of DNA analysis on the NBS 4-tier algorithm [immunoreactive trypsin (IRT) – meconium lactase – IRT2 – sweat chloride (SC)]. All CFSPID patients repeated SC testing every 6 months, and CFTR gene analysis was performed (detection rate 98%). We reclassified children as: CF diagnosis in presence of at least 2 pathological SC results; healthy carrier or healthy in presence of at least 2 normal SC results for age and either 1 or 0 CF-causing mutations, respectively. ResultsWe identified 32 CF and 50 CFSPID cases: 20/50 (40%) were diagnosed only by the IRT-DNA-SC algorithm and 16/50 (32%) only by IRT-meconium lactase-IRT2-SC. Both protocols identified the remaining 14 cases (28%). Thirty-seven of 50 (74%) CFSPID patients had a conclusive diagnosis on December 31, 2017:5 (10%) CF, 17 (34%) healthy and 15 (30%) healthy carriers; 13/50 (26%) cases were asymptomatic with persistent intermediate SC and followed as CFSPID (CF:CFSPID ratio 2.85:1). ConclusionsIn 6 years, the CF:CFSPID ratio modified from 0.64:1 to 2.85:1, and 10% of CFSPID cases progressed to CF. Genetic analysis improved positive predictive value and identified a higher number of CFSPID infants progressing to CF.

Prenatal Diagnosis of Cystic Fibrosis.

Cystic fibrosis (CF) is an inherited disease characterized by the accumulation of thick, sticky mucus which damages epithelia in organs such as the lungs, pancreas, liver, intestines, and other parts of the body. The most common symptoms are sinopulmonary disease and chronic gastrointestinal tract problems resulting from decreased mucociliary clearance and inflammation. CF is the most common life-limiting autosomal recessive disorder in people of northern European ancestry and it affects other populations with different prevalence. CF can be diagnosed by many methods including testing for blood immunoreactive trypsin, sweat chloride, transepithelial nasal potential difference, and molecular genetic testing.

Raised immunoreactive trypsin but normal sweat test

Raised immunoreactive trypsin but normal sweat test

Newborn blood spot screening for cystic fibrosis with a four-step screening strategy in the Netherlands

BackgroundNewborn screening for cystic fibrosis (NBSCF) was introduced in the Dutch NBS program in 2011 with a novel strategy. MethodsDutch NBSCF consisted of four steps: immuno-reactive trypsin (IRT), Pancreatitis-associated Protein (PAP), DNA analysis by Inno-LiPa (35 mutations), extended gene analysis (EGA) as fourth step and as safety net. Only samples with two CFTR-variants were considered screen-positive, but samples with one disease-causing variant were considered also screen-positive from April 2013. The first 5 years of NBSCF were evaluated during a follow-up ranging from 2 to 6.8 years for sensitivity, specificity, positive predictive value (PPV), ratio of CF/Cystic Fibrosis Screen Positive infants with an Inconclusive Diagnosis (CFSPID) and median age at diagnosis, and were compared to other novel strategies for NBSCF and European Cystic Fibrosis Society (ECFS) Best Practice Standards of Care. ResultsNBSCF achieved a sensitivity of 90% (95% CI 82%–94%), specificity of 99.991% (95% CI 99.989%–99.993%), PPV of 63% (95% CI 55%–69%), CF/CFSPID ratio of 4/1, and median age at diagnosis of 22 days, if samples with two variants as well as samples with one disease-causing variant were considered screen-positive. ConclusionThe program achieved the goal to minimize the number of false positives and showed a favourable performance but sensitivity and CF/CFSPID ratio did not meet criteria of EFCS Best Standards of Care. Changed cut-off values for PAP and IRT and classification of R117H-7T/9T to non-pathogenic may improve sensitivity to ≥95% and CF/CFSPID ratio to 10/1. PPV is estimated to be around 60%.

Cystic fibrosis newborn screening: outcome of infants with normal sweat tests

Newborn babies positively screened for cystic fibrosis (CF) (high serum immunoreactive trypsin (IRT) with DNA analysis) are referred for a diagnostic sweat test, which may be normal (sweat chloride <30...

A Simple Method to Overcome the “Floating Disc Problem” Using the GALT-Assay on the PerkinElmer GSP—Remeasurement on a Stand Alone Plate Fluorimeter

The Perkin Elmer Genetic Screening Processor (GSP)™ is a fully automated system for the processing of immunoassays for thyroid stimulating hormone (TSH), 17-hydroxyprogesterone (17-OHP), immuno reactive trypsin (IRT), biotinidase, and total T4, as well as enzymatic assays for total galactose and galactose-1-phosphate uridyltransferase (GALT) from dried blood spots (DBS). The system however, has one drawback: it cannot transfer samples from one microtiter plate to another. While this is not a problem for immunoassays, it makes enzymatic assays more problematic. The remaining DBS can either cause significant signal quenching, or they can increase fluorescence intensity, when the DBS are floating on the surface. The latter can cause false negative results, when GALT is measured for galactosaemia screening. To overcome this problem, an additional measurement step to check for floating disks is incorporated, leading to prevention of the affected measurements. However, this causes a secondary problem in this totally closed system. We detected floating disk signals in approx. 0.7% of all screening samples as well as quality control samples, which had to be repeated. We describe a simple method, which is just a re-measurement on a victor fluorescence reader, or any other plate fluorimeter, with filters for excitation wavelength 340 nm, and emission wavelength 405 nm. The introduction of this second-tier measurement made all repeat measurements unnecessary.

Screening for cystic fibrosis via a magnetic and microfluidic immunoassay format with electrochemical detection using a copper nanoparticle-modified gold electrode

This article describes a microfluidic electrochemical immunoassay that features two strategies, viz. (a), the incorporation of magnetic nanoparticles (MNPs) into the central microfluidic channel and acting as a bioaffinity support for the immobilization of the antibody against the immunoreactive trypsin (anti-IRT), and (b), the electrodeposition of copper nanoparticles (CuNPs) on a gold electrode. IRT, a marker for cystic fibrosis, is extracted from blood samples onto a disk using ultrasonication, eluted, and then injected into the detection system where it is captured by anti-IRT-loaded nanoparticles (anti-IRT-Ab-MNPs). Bound IRT is electrochemically quantified after addition of HRP-labeled anti-IRT-Ab which, in the presence of H2O2, catalyzes the oxidation of catechol to form o-benzoquinone which is detected at a working potential of −150 mV (vs. Ag/AgCl). The electrochemical response to benzoquinone is proportional to the concentration of IRT in the range from 0 to 580 ng⋅mL−1. The coefficients of variation are <5 % for within-day assays, and <6.4 % for between-day assays. The method was compared to a commercial ELISA for IRT where is showed a correlation coefficient of close to 1. In our perception, this approach represents an attractive alternative to existing methods for screening newborns for cystic fibrosis.