Comparison of Tandem Mass Spectrometry and the Fluorometric Method-Parallel Phenylalanine Measurement on a Large Fresh Sample Series and Implications for Newborn Screening for Phenylketonuria.

Phenylketonuria (PKU) was the first disease to be identified by the newborn screening (NBS) program. Currently, there are various methods for determining phenylalanine (Phe) values, with tandem mass spectrometry (MS/MS) being the most widely used method worldwide. We aimed to compare the MS/MS method with the fluorometric method (FM) for measuring Phe in the dried blood spot (DBS) and the efficacy of both methods in the NBS program. The FM was performed using a neonatal phenylalanine kit and a VICTOR2TM D fluorometer. The MS/MS method was performed using a NeoBaseTM 2 kit and a Waters Xevo TQD mass spectrometer. The Phe values measured with the MS/MS method were compared to those determined by the FM. The cut-off value for the NBS program was set at 120 µmol/L for FM and 85 µmol/L for MS/MS. We analyzed 54,934 DBS. The measured Phe values varied from 12 to 664 µmol/L, with a median of 46 µmol/L for the MS/MS method and from 10 to 710 µmol/L, with a median of 70 µmol/L for the FM. The Bland-Altman analysis indicated a bias of -38.9% (-23.61 µmol/L) with an SD of 21.3% (13.89 µmol/L) when comparing the MS/MS method to the FM. The Phe value exceeded the cut-off in 187 samples measured with FM and 112 samples measured with MS/MS. The FM had 181 false positives, while the MS/MS method had 106 false positives. Our study showed that the MS/MS method gives lower results compared to the FM. Despite that, none of the true positives would be missed, and the number of false-positive results would be significantly lower compared to the FM.

Optimizing the Phenylalanine Cut-Off Value in a Newborn Screening Program.

Phenylketonuria (PKU) was the first disorder for which newborn screening (NBS) was introduced in the early 1960s. Slovenia started the NBS program for PKU in 1979, and the fluorimetric method was implemented in 1992, with a phenylalanine (Phe) cut-off set at 120 mol/L. This value has been in use for almost thirty years and has never been revised. We aimed to analyze the DBS samples and review the data from a large nationwide cohort of newborns to optimize the cut-off values for HFA screening to minimize the number of false positives while maintaining the highest level of sensitivity by detecting all those who needed to be treated. In the first prospective part of the study, we analyzed samples of all newborns in Slovenia in 2019 and 2020, and in the second retrospective part, we reviewed data from all known patients with hyperphenylalaninemia (HFA) in Slovenia born from 2000 to 2018. We defined true screening-positive cases as those that required a low-Phe diet. The sensitivity, specificity and positive predictive values of the modeling elevation of the Phe cut-off value from 120 µmol/L to 200 µmol/L were assessed. The number of recalls at the cut-off of 120 µmol/L was 108 out of 37,784 samples at NBS (2019-2020). Six newborns were defined as true positives and 102 samples as false positives. If the cut-off value was adjusted to 160 µmol/L, only 12 samples exceeded it and all six true positive newborns would be detected. Among the 360,000 samples collected at the NBS between 2000 and 2018, 72 HFA patients in need of a low-Phe diet were found. All the diagnosed cases would have been detected if the cut-off was set to 160 µmol/L. We demonstrated in a large group of newborns (400,000 in 20 years) that using the fluorimetric approach, a cut-off value of 160 µmol/L, rather than 120 mol/L, is safe and that there were no missing true positive patients who required treatment. By increasing the cut-off, this method becomes more precise, resulting in a significantly reduced rate of false positives and thus being less burdensome on both families and the healthcare system.

First Estimation of Reference Intervals for Thyroid-Stimulating Hormone and Thyroid Hormones in Slovenian Population.

For thyroid function estimation and clinical decision making, use of appropriate reference intervals for thyroid-stimulating hormone (TSH), free thyroxine (fT4) and free triiodothyronine (fT3) is crucial. For each laboratory, establishment of own reference intervals is advised. For the first Slovenian estimation of reference intervals for thyroid hormones a large group of 1722 healthy individuals without thyroid disease was established retrospectively. Hormone analyses were performed on automated analyser Advia Centaur XP Immunoassay System (Siemens Healthineers), which reference intervals for TSH, fT4 and fT3 were 0.55-4.78 mIU/L, 11.5-22.7 pmol/L, and 3.5-6.5 pmol/L, respectively. Statistical analysis followed non-parametric percentile method. Our laboratory reference intervals for TSH, fT4 and fT3 are mostly narrower than intervals given by manufacturer. Median value, lower and upper limit for TSH, fT4 and fT3 were 1.98 (0.59-4.23) mIU/L, 14.5 (11.3-18.8) pmol/L and 4.82 (3.79-6.05) pmol/L, respectively. Most likely, an inclusion of a high number of healthy individuals without thyroid disease was a reason for such results.

Hypothyroidism is associated with higher testosterone levels in postmenopausal women with Hashimoto's thyroiditis.

INTRODUCTION: There are few data about possible interaction of sex hormones and thyroid autoimmunity and function in women with Hashimoto's thyroiditis (HT) after menopause. Therefore, our aim was to investigate sex hormone levels in euthyroid (EuHT) and hypothyroid (HypoHT) postmenopausal women with HT. MATERIAL AND METHODS: We performed a prospective observational clinical study that included 55 women with HT (AllHT) and 18 healthy subjects (HS) after menopause matched by age, body mass index, follicle-stimulating hormone, and menopause duration. According to their thyrotropin (TSH) level, the AllHT patients were divided into two subgroups: EuHT with TSH in the range 0.35-5.5 mU/L and HypoHT with TSH above 5.5 mU/L. Total and free testosterone (T), sex hormone-binding globulin (SHBG), oestradiol (E2), and progesterone (P) were measured in all subjects. Values are presented as mean ± SD. The Mann-Whitney U test was used for comparison of values between the groups. Correlations were tested using Kendall's tau test. RESULTS: In the HypoHT group, significantly higher free T levels were found in comparison to the HS group (7.89 ± 3.55 pmol/L and 7.13 ± 3.03 pmol/L, p < 0.05). Furthermore, in HypoHT, free T was significantly higher than in EuHT (7.19 ± 5.65 pmol/L, p < 0.05). SHBG was significantly lower in HypoHT compared with HS (45.4 ± 17.4 nmol/L and 60.09 ± 19.51 nmol/L, p < 0.05). No significant correlation was found between sex hormone levels and thyroglobulin and thyroid peroxidase antibodies. CONCLUSION: We report significantly higher free and total T levels in hypothyroid postmenopausal women with HT. To our knowledge, this is the first study of sex hormone levels in postmenopausal women with HT.

Combined therapy of the antimetastatic compound NAMI-A and electroporation on B16F1 tumour cells in vitro.

Ruthenium complex NAMI-A [ImH][trans-RuCl(4)(DMSO-S)Im] (Im = imidazole) is a potential chemotherapeutic drug in cancer treatment. Electroporation can be used to facilitate delivery of NAMI-A into cells. Suspension of B16F1 tumour cells from mouse melanoma in NAMI-A solution was exposed to a train of electric pulses. The effect of NAMI-A was determined by examining cell viability in clonogenic test. Our results show that electroporation increases the otherwise scarce in vitro effects of NAMI-A, i.e. reduces cell viability. At the conditions chosen for experiments 90% of cells survived in the presence of 1 microM NAMI-A, whereas in a combined treatment with 1 microM NAMI-A and electroporation only about 10% of cells survived.

Newborn Screening in Slovenia.

INTRODUCTION: Newborn screening in whole Slovenia started in 1979 with screening for phenylketonuria (PKU). Congenital hypothyroidism (CH) was added into the programme in 1981. The aim of this study was to analyse the data of neonatal screening in Slovenia from 1993 to 2012 for PKU, and from 1991 to 2012 for CH. METHODS: Blood samples were collected from the heels of newborns between the third and the fifth day after birth. Fluorometric method was used for screening for PKU, CH screening was done by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA). RESULTS: From 1993 to 2012, from 385,831 newborns 57 were identified with PKU. 184 newborns out of 427,396 screened from 1991 to 2012, were confirmed for CH. Incidences of PKU and CH in the periods stated are 1:6769 and 1:2323, respectively. CONCLUSIONS: Successful implementation of newborn screening for PKU and CH has helped in preventing serious disabilities of the affected children. Adding screening for new metabolic diseases in the future would be beneficial.

49A/G and CT60 polymorphisms of the cytotoxic T-lymphocyte-associated antigen 4 gene associated with autoimmune thyroid disease.

Single nucleotide polymorphisms in the CTLA-4 gene have been suggested as genetic factors in the susceptibility to autoimmune thyroid disease (AITD). In our case-control study, patients with Graves' disease, Hashimoto's thyroiditis, and postpartum thyroiditis and control subjects have been genotyped for two A/G single nucleotide polymorphisms (49A/G and CT60) of the CTLA-4 gene. The 49A/G polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism method using the enzyme BseXI and the CT60 polymorphism by real-time polymerase chain reaction. Results were analyzed by chi(2) test and linkage disequilibrium analysis. In a comparison of frequencies of GG genotype, a significant association of 49A/G and CT60 polymorphism existed only for Graves' disease. In the 49A/G polymorphism, the frequency of GG genotype was significantly higher (p = 0.0408) compared with controls; the frequency of the CT60 polymorphism was significantly higher as well (p = 0.0213). The frequencies of AA and AG genotypes in control subjects did not significantly differ from frequencies in AITD patients for both polymorphisms. Our results may therefore lend support to the hypothesis that humoral autoimmunity is correlated with 49A/G and CT60 polymorphisms of the CTLA-4 gene.