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.

A proposed Common Training Framework for Specialists in Laboratory Medicine under EU Directive 2013/55/EC (The Recognition of Professional Qualifications).

European Union (EU) Directive 2013/55/EC (The Recognition of Professional Qualifications) allows Member States to decide on a common set of minimum knowledge, skills and competences that are needed to pursue a given profession through a Common Training Framework. To be adopted the framework must combine the knowledge, skills and competences of at least one third of the Member States. Professionals who have gained their qualifications under a Common Training Framework will be able to have these recognised automatically within the Union. The backbone of the European Federation of Clinical Chemistry and Laboratory Medicine's (EFLM) proposed Common Training Framework for non-medical Specialists in Laboratory Medicine is outlined here. It is based on an Equivalence of Standards in education, training, qualifications, knowledge, skills, competences and the professional conduct associated with specialist practice. In proposing the recognition of specialist practice EFLM has identified 15 EU Member States able to meet Equivalence and in whom the profession and/or its training is regulated (an additional EU Commission requirement). The framework supports and contributes to the Directive's enabling goals for increasing professional mobility, safeguarding consumers and ensuring a more equitable distribution of skills and expertise across the Member States. It represents EFLM's position statement and provides a template for professional societies and/or competent authorities to engage with the EU Commission.

Functional polymorphisms in antioxidant genes in Hurthle cell thyroid neoplasm - an association of GPX1 polymorphism and recurrent Hurthle cell thyroid carcinoma.

BACKGROUND: Hurthle cells of the thyroid gland are very rich in mitochondria and oxidative enzymes. As a high level oxidative metabolism may lead to higher level of oxidative stress and can be associated with an increased risk for cancer, we investigated whether common functional polymorphisms in antioxidant genes (SOD2, CAT, GPX, GSTP1, GSTM1 and GSTT1) are associated with the development or clinical course of Hurthle cell thyroid carcinoma (HCTC). METHODS: A retrospective study was performed in 139 patients treated by thyroid surgery for a Hurthle cell neoplasm. HCTC, Hurthle cell thyroid adenoma (HCTA) or Hurthle cell thyroid nodule (HCTN) were diagnosed by pathomorphology. DNA was extracted from cores of histologically confirmed normal tissue obtained from formalin-fixed paraffin-embedded specimens and genotyped for investigated polymorphisms. Logistic regression was used to compare genotype distributions between patient groups. RESULTS: HCTC, HCTA and HCTN were diagnosed in 53, 47 and 21 patients, respectively. Metastatic disease and recurrence of HCTC were diagnosed in 20 and 16 HCTC patients, respectively. Genotypes and allele frequencies of investigated polymorphisms did not deviate from Hardy-Weinberg equilibrium in patients with HCTC, HCTA and HCTN. Under the dominant genetic model we observed no differences in the genotype frequency distribution of the investigated polymorphisms when the HCTA and HCTN group was compared to the HCTC group for diagnosis of HCTC or for the presence of metastatic disease. However, GPX1 polymorphism was associated with the occurrence of recurrent disease (p = 0.040). CONCLUSIONS: GPX1 polymorphism may influence the risk for recurrent disease in HCTC.

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.

The influence of the exon 1 polymorphism of the cytotoxic T lymphocyte antigen 4 gene on thyroid antibody production in patients with newly diagnosed Graves' disease.

Increasing evidence supports the genetic susceptibility for thyroid antibody (TAb) production in patients with autoimmune thyroid disease, and recently, it has been shown that the cytotoxic T lymphocyte antigen 4 (CTLA-4) gene is most likely a major TAb susceptibility gene. To assess the relationship between exon 1 CTLA-4 gene polymorphism and TAb production, we genotyped 67 patients with newly diagnosed Graves' disease. Free thyroid hormones and TAb were measured, including thyroglobulin antibodies (TgAb), thyroid peroxidase antibodies (TPOAb), and thyroid-stimulating antibodies (TSAb). AA genotype was found in 25 patients, AG genotype in 34 patients, and GG genotype in 8 patients. G allele carrying genotypes showed significantly higher frequency of positive TPOAb (p < 0.005) and TgAb (p < 0.05) compared to AA genotype. Furthermore, the median values of TPOAb were significantly higher in the group with G allele (p < 0.002). However, the median values of TgAb and TSAb did not differ significantly between both groups and similarly, CTLA-4 genotype showed no association with serum free thyroxine (T(4)) and Graves' ophthalmopathy. In conclusion, our findings suggest that G allele carrying genotype of the CTLA-4 gene influences higher production of TPOAb and TgAb, and therefore, support the hypothesis that CTLA-4 gene plays a major role in TAb production.

Effectiveness of L-thyroxine treatment on TSH suppression during pregnancy in patients with a history of thyroid carcinoma after total thyroidectomy and radioiodine ablation.

INTRODUCTION: There are scarce data about the optimal increase of L-thyroxine dose during pregnancy in patients with a history of thyroid carcinoma. The first aim of the study was to find out if routine therapeutic measures enable adequate TSH suppression in pregnancy. The other aim was to find out the optimal dose of L-thyroxine for TSH suppression in pregnant women. PATIENTS AND METHODS: In this retrospective observational study, we analysed 36 pregnancies of 32 women with a history of thyroid carcinoma. Before pregnancy, all of them underwent total thyroidectomy and radioiodine ablation of thyroid remnant, and they were on suppressive doses of L-thyroxine. Thyroid function tests were obtained before, during and after pregnancy. RESULTS: Mean L-thyroxine dose before pregnancy, in the first, second and, third trimester and after delivery was 149, 147, 155, 165 and 158 micrograms daily, respectively. TSH concentration remained suppressed in 9 pregnancies, it was within normal range in 22 and elevated in 5 pregnancies. The mean dose of L-thyroxine in patients with suppressed TSH before pregnancy, in the first, second and, third trimester and after delivery was 154, 154, 164, 160 and 161 micrograms daily, respectively. When the dose had to be changed, the mean increase of the dose was 31.5 micrograms daily. CONCLUSIONS: The range of changes in TSH concentration during pregnancy in the patients who have been on suppressive L-thyroxine therapy before conception is quite wide. TSH was adequately suppressed in only 25% of pregnancies. The dose of L-thyroxine in patients with suppressed TSH in the first, second and third trimester was 154, 164 and 160 micrograms daily, respectively.

Thyroid function in the third trimester of pregnancy and after delivery in an area of adequate iodine intake.

OBJECTIVE: To establish whether the higher thyroid stimulating hormone (TSH) levels and lower levels of the 2 free thyroid hormones noted toward the end of pregnancy are in relation with iodine supply. METHODS: We compared these hormones' levels in the third trimester of pregnancy and 4 months after delivery in 116 consecutive women without thyroid disease and otherwise healthy. The study was conducted in Slovenia, an iodine-sufficient area. The Mann-Whitney test, the Kruskal-Wallis rank test, and Spearman analysis were used for statistical analysis. RESULTS: In the third trimester TSH was significantly higher and both free thyroid hormones were significantly lower than after delivery (P=0.003 and P<0.001), but the free thyroxine to free triiodothyronine ratios in the third trimester and 4 months after delivery did not significantly differ. Urinary iodine concentration (UIC) was significantly higher during pregnancy than after delivery (P=0.044). We found no significant correlations between UIC and TSH or between UIC and both free thyroid hormones during pregnancy or after delivery. CONCLUSION: The decrease of both free thyroid hormones in the third trimester of pregnancy is most likely due to reasons that are not related to iodine supply.

Thyroid volume changes during pregnancy and after delivery in an iodine-sufficient Republic of Slovenia.

OBJECTIVE: Literature data concerning thyroid enlargement during pregnancy are not conclusive. Our aim was to systematically follow the thyroid volume changes during pregnancy and after delivery in an iodine-sufficient area. STUDY DESIGN: Prospective study of healthy pregnant women living in an iodine-sufficient area. We followed 118 pregnant women with the mean age 30.9+/-4.1 years in the first trimester (mean 11.2+/-2.5 weeks of pregnancy), in the third trimester (mean 31.6+/-1.7 weeks of pregnancy), and 4 months after delivery (mean 15.9+/-3.9 weeks). Additionally, 71 women were also evaluated 14 months after delivery (mean 13.3+/-1.1 months). All women were negative for thyroid autoantibodies. We measured urinary iodine concentration (UIC), thyroid volume, serum TSH, and body mass index (BMI). After delivery, in a subgroup of women we also estimated the colour flow Doppler sonography (CFDS) patterns 0, I, II and III, where thyroid vascularity increased from pattern 0 to III, and the peak systolic velocity (PSV) using a 7.5 mHz linear transducer. RESULTS: Median UIC in the third trimester (176 microg/g creatinine) was significantly higher than 4 and 14 months after delivery (P=0.030, P<0.001, respectively). Thyroid volume in the third trimester (11.3+/-3.1 mL) was significantly greater (P<0.001) than in the first trimester (8.7+/-2.5 mL), 4 months after delivery (8.6+/-2.5) and 14 months after delivery (7.8+/-2.4 mL). TSH concentration was significantly higher in the third trimester than in the first trimester and 4 months after delivery (P=0.007, P=0.006, respectively). As expected, BMI was the highest in the third trimester. CFDS pattern I was more frequent 4 months after delivery than 14 months after delivery (P<0.001). Similarly, PSV was significantly higher 4 months after delivery than 14 months after delivery (P<0.001). Linear regression analysis revealed TSH and BMI as significant independent predictors for thyroid volume. CONCLUSION: In an iodine-sufficient area, thyroid volume increases during pregnancy and decreases after delivery, and the changes in volume are associated with changes in TSH and BMI. They may be viewed as indicators for metabolic and haemodynamic changes during pregnancy.

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.

Effects of systolic atrial function on plasma renin activity and natriuretic peptide secretion after high rate atrial and ventricular pacing in dogs.

Rapid atrial rates cause electrical, structural remodeling, and neuro-humoral changes. This study compares the effects of mechanical remodeling on plasma renin activity (PRA) and atrial natriuretic peptide (ANP) secretion. Eight beagles were subjected to rapid atrial pacing (AP) at 400 beats/min for 16 days. After complete recovery of left ventricular function, they underwent rapid ventricular pacing (VP) at 240 beats/min of equal duration. Left atrial systolic maximal dimension (LAmax) and left atrial appendage (LAA) peak late emptying velocity (LAA-E) were assessed by echocardiography. Blood samples were taken from the right atrium and from the peripheral vein. LAmax after AP and VP enlarged significantly (2.16 +/- 0.21 cm vs 2.41 +/- 0.23 cm, P = 0.002). Compared with baseline, LAA-E velocities were significantly reduced (0.65 +/- 0.12 m/s vs 0.26 +/- 0.16 m/s, P = 0.001) after AP only. AP caused a significant elevation of PRA in right atrial (9.28 +/- 4.23 nmol/L per hour) and peripheral samples compared with baseline values (4.82 +/- 2.53 nmol/L per hour, P = 0.04). ANP levels increased after AP (1117.12 +/- 252.21 fmol/L) with respect to baseline values (824.37 +/- 159.08 fmol/L, P = 0.001). There was no difference in PRA and ANP levels between atrial and peripheral samples. Atrial size and impaired systolic appendage function play an important role in secretion of PRA and ANP. Both neuro-humoral pathways may be therapeutic targets in the treatment of patients with AF.