Optimization of performance of Dutch newborn screening for cystic fibrosis.

BACKGROUND: Dutch newborn screening (NBS) for Cystic Fibrosis (CF) introduced in 2011 showed a sensitivity of 90% and a positive predictive value (PPV) of 63%. We describe a study including an optimization phase and evaluation of the modified protocol. METHODS: Dutch protocol consists of four steps: determination of immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP), DNA analysis by INNO-LiPA and extended gene analysis (EGA). For the optimization phase we used results of 556,952 newborns screened between April 2011 and June 2014 to calculate effects of 13 alternative protocols on sensitivity, specificity, PPV, ratios of CF to other diagnoses, and costs. One alternative protocol was selected based on calculated sensitivity, PPV and costs and was implemented on 1st July 2016. In this modified protocol DNA analysis is performed in samples with a combination of IRT ≥60 µg/l and PAP ≥3.0 µg/l, IRT ≥100 µg/l and PAP ≥1.2 µg/l or IRT ≥124 µg/l and PAP not relevant. Results of 599,137 newborns screened between 1st July 2016 and 31st December 2019 were similarly evaluated as in the optimization phase. RESULTS: The modified protocol showed a sensitivity of 95%, PPV of 76%, CF to CF transmembrane conductance regulator-related metabolic syndrome/CF screen positive, inconclusive diagnoses (CRMS/CFSPID) ratio 12/1, CF/CF carrier ratio 4/1. Costs per screened newborn were slightly higher. Eleven children, of whom five with classic CF, would not have been referred with the previous protocol. CONCLUSIONS: The modified protocol results in acceptable sensitivity (95%) and good PPV of 76% with minimal increase in costs.

Peripheral blood lymphocyte appearance in a case of I cell disease.

In general, peripheral blood smears are performed to obtain information with regard to various morphological features as an aid in the diagnosis of infection or malignancy. This report presents a patient with I cell disease (inclusion cell disease), a fatal lysosomal storage disorder caused by a defect in an enzyme responsible for the transfer of mannose-6-phosphate ligands to precursor lysosomal enzymes. As a consequence, most lysosomal enzymes are transported outside the cell instead of being correctly targeted into the lysosomes, resulting in the storage of macromolecules in lysosomes. I cell disease, with its heterogeneous clinical presentation, can be diagnosed by the presence of intracellular vacuole-like inclusions in lymphocytes and fibroblasts, high serum lysosomal enzyme activities, and a defect of N-acetylglucosamine-1-phosphotransferase. This report describes the morphological aspects of peripheral lymphocytes in a blood smear of a patient, the first clue to the final diagnosis of I cell disease. The observed vacuole-like inclusions in lymphocytes of this patient were negative for periodic acid Schiff (PAS) and Sudan black B staining, in contrast to earlier reports.

New method for faecal fat determination by mid-infrared spectroscopy, using a transmission cell: an improvement in standardization.

Current techniques used in clinical laboratories for faecal fat determination, such as the Van de Kamer method, are not very accurate or precise. This became apparent when results obtained by different laboratories were compared, and could explain the disappointing performance of near-infrared and mid-infrared spectroscopy since the accuracy of these techniques depends upon the accuracy of the calibration used (i.e. inaccurate wet chemical analysis). In order to improve standardization, we developed and tested a new quantitative method in three laboratories, based on Fourier transform infrared (FT-IR) spectroscopy. Fatty acids were extracted from faecal samples with acidified petroleum ether-ethanol and the extracts were dried and dissolved in chloroform. An infrared spectrum of the extracts was recorded in the range 4000-650 cm(-1), using an infrared transmission cell. Standard mixtures of stearic and palmitic acids (65:35) were used for calibration. Quantification was based on the absorbance band of the CH2 group (2855 cm(-1)) of free fatty acids and fatty acid glycerol esters. The calibration curve showed excellent linearity. The correlation coefficient between the titrimetric Van de Kamer and FT-IR methods was 0.96 (y = 1.12x-0.02, standard error of prediction = 0.89 g% fat). No significant difference was found when the FT-IR results of 28 faecal samples from patients were compared between two different university hospital laboratories. The new FT-IR method, using primary standards, is simple and rapid, and provides satisfactory intra- and inter-laboratory precision for the diagnosis and monitoring of steatorrhoea.

Rapid and reliable measurement of highly elevated blood ammonia concentrations in children.

Newborns and children may suffer from extremely high ammonia levels in their blood. We evaluated a fast, reliable micromethod, based on the Blood Ammonia Checker II (BAC II) in combination with the dilution with fresh whole blood. Comparison of the proposed method with an enzymatic method revealed a statistically significant correlation. We conclude that the dilution of patient's blood with fresh whole blood extends the measuring range of ammonia on the BAC II analyzer from 286 mumol/l to about 700 mumol/l.

Metabolic aspects of peroxisomal disorders.

In recent years an increasing number of inherited diseases in man have been identified in which there is an impairment in one or more peroxisomal functions. This paper discusses the current state of knowledge on these disorders with particular emphasis on the metabolic abnormalities in these diseases.

Fatty acid beta-oxidation in peroxisomes and mitochondria: the first, unequivocal evidence for the involvement of carnitine in shuttling propionyl-CoA from peroxisomes to mitochondria.

We have investigated how [1-14C]propionyl-CoA, which is the first product of the peroxisomal beta-oxidation of [1-14C] pristanic acid, is transported to mitochondria for further oxidation in human skin fibroblasts from patients with a defect in the mitochondrial carnitine/acylcarnitine translocase and carnitine-palmitoyltransferase II (CPT II) (EC 2.3.1.21), respectively. Oxidation of pristanic acid was found to be partially deficient in both types of mutant cells. More important, 14CO2 production was completely deficient in the carnitine/acylcarnitine translocase deficient cells but not in the carnitine-palmitoyltransferase II deficient cells. These results strongly suggest that formation of 14CO2 in the Krebs cycle from [1-14C]propionyl-CoA as generated in peroxisomes requires the active participation of the mitochondrial carnitine/acylcarnitine translocase. The results described in this paper provide the first evidence suggesting that propionyl-CoA leaves the peroxisome as a carnitine ester and strongly suggest that the commonly accepted concept that peroxisomal beta-oxidation is not dependent on carnitine is incorrect.

Beta-oxidation of fatty acids in cultured human skin fibroblasts devoid of the capacity for oxidative phosphorylation.

Prolonged treatment of cultured cells with ethidium bromide results in loss of the capacity for oxidative phosphorylation. Because of the tight coupling between mitochondrial beta-oxidation of fatty acids and the activity of the respiratory chain, such cells may be used to study the contribution of mitochondria and peroxisomes to fatty acid beta-oxidation. To investigate this, human skin fibroblasts were cultured in the presence of ethidium bromide for at least 10 cell generations, resulting in a virtually complete absence of oxidative phosphorylation as demonstrated directly in digitonin-permeabilized fibroblasts. The cells showed a lowered ATP/ADP ratio, most likely as the consequence of the inability to generate ATP via oxidative phosphorylation. The loss of the capacity for oxidative phosphorylation was also reflected in an increased cytosolic NADH/NAD+ ratio: the cells showed a highly elevated lactate/pyruvate ratio in the suspending medium when incubated with glucose. The beta-oxidation of octanoic and palmitic acid was dramatically decreased, suggesting that the beta-oxidation of these fatty acids takes place predominantly (> 90%) in mitochondria, at least in the cells studied. In contrast, the rates of pristanic and cerotic acid beta-oxidation were only slightly decreased, suggesting that this is mainly a peroxisomal process. The reduction of beta-oxidation of cerotic and pristanic acid, 27% and 15%, respectively, is most likely due to a lowered ATP level and an increased NADH/NAD(+)-redoxstate in these cells. We conclude that fibroblasts subjected to prolonged treatment with ethidium bromide can be used as a model system to study the substrate specificity and functional characteristics of the peroxisomal beta-oxidation system.

X-linked adrenoleukodystrophy: biochemical diagnosis and enzyme defect.

The adrenoleukodystrophies refer to three genetically distinct disorders all characterized by the accumulation of very long-chain fatty acids. In this paper we will review the biochemical aspects of these leukodystrophies with particular emphasis on the methods used to measure very long-chain fatty acid levels in plasma and their reliability. Furthermore, we will concentrate on the primary defect in the X-linked form of adrenoleukodystrophy.

Conclusive evidence that very-long-chain fatty acids are oxidized exclusively in peroxisomes in human skin fibroblasts.

We have investigated the contribution of peroxisomes and mitochondria to the beta-oxidation of palmitate (C16:0) and cerotate (C26:0) in intact human skin fibroblasts. The oxidation of both fatty acids was found to be inhibited by rotenone plus antimycin and cyanide, respectively, although to a different extent. When 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) was used to specifically block carnitine palmitoyltransferase I, it was found that palmitate beta-oxidation was inhibited almost completely whereas cerotate beta-oxidation was not affected. Since carnitine palmitoyltransferase is essential for the oxidation of fatty acids in mitochondria this result provides conclusive evidence that oxidation of very-long-chain fatty acids is initiated in peroxisomes and not in mitochondria.