Age-Specific Reference Intervals for Thyroid-Stimulating Hormones and Free Thyroxine to Optimize Diagnosis of Thyroid Disease.

Background: Thyroid-stimulating hormone (TSH) and subsequent free thyroxine (FT4) concentrations outside the reference interval (RI) are used to diagnose thyroid diseases. Most laboratories do not provide age-specific RIs for TSH and FT4 beyond childhood, although TSH concentrations vary with age. Therefore, we aimed to establish TSH and FT4 age-specific RIs throughout life and aimed to determine whether using these RIs would result in reclassification of thyroid disease diagnoses in adults. Methods: This multicenter retrospective cross-sectional study used big data to determine indirect RIs for TSH and FT4. These RIs were determined by TMC and refineR-analysis, respectively, using four different immunoassay platforms (Roche, Abbott, Siemens, and Beckman Coulter). Retrospective data (2008-2022) from 13 Dutch laboratories for general practitioners and local hospitals were used. RIs were evaluated per manufacturer. Age groups were established from 2 to 20 years by 2-year categories and decade categories between 20 and 100 years. Results: We included totally 7.6 million TSH and 2.2 million FT4 requests. TSH upper reference limits (URLs) and FT4 lower reference limits were higher in early childhood and decreased toward adulthood. In adulthood, TSH URLs increased from 60 years in men, and from 50 years in women, while FT4 URLs increased from 70 years onward. Using adult age-specific RIs resulted in a decrease in diagnoses of subclinical and overt hypothyroidism in women above 50 and men above 60 years in our Roche dataset. Conclusion: This study stressed the known importance of using age-specific RIs for TSH and FT4 in children. This study also showed the clinical relevance of using age-specific RIs for TSH in adulthood to reduce diagnoses of subclinical hypothyroidism in older persons. Therefore, implementation of adult TSH age-specific RIs should be strongly considered. Data are less uniform regarding FT4 age-specific RIs and more research should be performed before implementing these in clinical practice.

Should we depend on reference intervals from manufacturer package inserts? Comparing TSH and FT4 reference intervals from four manufacturers with results from modern indirect methods and the direct method.

OBJECTIVES: Correct interpretation of thyroid function tests relies on correct reference intervals (RIs) for thyroid-stimulating hormone (TSH) and free thyroxine (FT4). ISO15189 mandates periodic verification of RIs, but laboratories struggle with cost-effective approaches. We investigated whether indirect methods (utilizing historical laboratory data) could replace the direct approach (utilizing healthy reference individuals) and compared results with manufacturer-provided RIs for TSH and FT4. METHODS: We collected historical data (2008-2022) from 13 Dutch laboratories to re-establish RIs by employing indirect methods, TMC (for TSH) and refineR (for FT4). Laboratories used common automated platforms (Roche, Abbott, Beckman or Siemens). Indirect RIs (IRIs) were determined per laboratory per year and clustered per manufacturer (>1.000.000 data points per manufacturer). Direct RIs (DRIs) were established in 125 healthy individuals per platform. RESULTS: TSH IRIs remained robust over the years for all manufacturers. FT4 IRIs proved robust for three manufacturers (Roche, Beckman and Siemens), but the IRI upper reference limit (URL) of Abbott showed a decrease of 2 pmol/L from 2015. Comparison of the IRIs and DRIs for TSH and FT4 showed close agreement using adequate age-stratification. Manufacturer-provided RIs, notably Abbott, Roche and Beckman exhibited inappropriate URLs (overall difference of 0.5-1.0 µIU/mL) for TSH. For FT4, the URLs provided by Roche, Abbott and Siemens were overestimated by 1.5-3.5 pmol/L. CONCLUSIONS: These results underscore the importance of RI verification as manufacturer-provided RIs are often incorrect and RIs may not be robust. Indirect methods offer cost-effective alternatives for laboratory-specific or platform-specific verification of RIs.

The association between vitamin D and C-reactive protein levels in patients with inflammatory and non-inflammatory diseases.

OBJECTIVE: A direct, inverse correlation between 25-hydroxy vitamin D (25(OH) vitamin D) levels and C-reactive protein (CRP), a sensitive biomarker for inflammation, was found in some, but not all, studies. These effects were seen in healthy subjects as well as in some inflammatory diseases. DESIGN AND METHODS: CRP and 25(OH) vitamin D data (2011-2013) from 923 in- and outpatients (males/females 340/583; median age: 76years (95% confidence interval (CI): 75-77) were analyzed. A standardized diagnosis according to the Dutch diagnosis coding standard for each patient was obtained. Each diagnosis was categorized as either inflammatory or non-inflammatory disease. Analysis of variance was performed with age, gender, inflammatory status (inflammatory disease/non-inflammatory disease), and season as corrective factors. RESULTS: The correlation between (log) ln-25(OH) vitamin D and ln-CRP was highly significant (p<0.001) with a regression coefficient of -0.879. In the inflammatory disease group, the R(2) value was 0.726 and in the non-inflammatory disease group it was 0.502. It was shown that increasing 25(OH) vitamin D levels are associated with decreasing CRP levels, with a stronger effect in the inflammatory disease group compared to the non-inflammatory disease group. CONCLUSIONS: Our study shows an inverse correlation between 25(OH) vitamin D and CRP in a large patient cohort but more importantly shows that this effect is more pronounced in patients with inflammatory diseases compared to patients with non-inflammatory diseases.

Inaccurate First-Generation Testosterone Assays Are Influenced by Sex Hormone-Binding Globulin Concentrations.

BACKGROUND: The quality of testosterone assays has been a matter of debate for several years. Known limitations of testosterone immunoassays are the cross-reactivity with other steroids and a high variation in the low concentration range. We hypothesized that one of the additional limitations of testosterone immunoassays is an ineffective displacement of testosterone from its binding protein. METHODS: Thirty samples from women not using oral contraceptives (OAC), 30 samples from women using OAC, and 30 samples from pregnant women were used to measure testosterone by an isotope dilution (ID)-LC-MS/MS method and by 6 commercially available testosterone immunoassays (UniCel®, ARCHITECT®, Centaur®, Cobas®, Immulite®, and Liaison®). In addition, sex hormone-binding globulin (SHBG)4 was measured by immunoassay (ARCHITECT). RESULTS: The first-generation immunoassays (UniCel, Centaur, Immulite, and Liaison) showed inaccurate testosterone results in the method comparisons with the ID-LC-MS/MS method (R between 0.61 and 0.86) and for some assays (UniCel and Liaison) also a very poor standardization (slopes of 0.59 and 0.67, respectively). On average, SHBG concentrations were lowest in women not using OAC and highest in pregnant women, and overall ranged from 18.5 to 633 nmol/L. In the first-generation immunoassays, but not in the second-generation immunoassays, we observed an inverse relationship between SHBG concentrations and deviations in testosterone from the ID-LC-MS/MS results. CONCLUSIONS: Widely used first-generation testosterone immunoassays are influenced by SHBG concentrations, which lead to inaccurate results in samples from patients with high or low SHBG concentrations, respectively. Laboratory specialists, clinicians, and researchers should be aware of this limitation in testosterone assays.

Measurement of dehydroepiandrosterone sulphate (DHEAS): a comparison of Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS) and seven currently available immunoassays.

BACKGROUND: Dehydroepiandrosterone sulphate (DHEAS) is an important marker of the adrenal gland. Its measurement is required in several adrenal diseases, such as adrenal tumours, adrenal insufficiency and congenital adrenal hyperplasia. Most clinical laboratories measure DHEAS using commercially available immunoassays. The aim of the present study was to investigate the accuracy of currently available DHEAS methods. METHODS: Seven commercially available DHEAS assays were compared to Isotope-Dilution Liquid Chromatography Tandem Mass Spectrometry (ID-LC-MS/MS) by measuring 75 serum samples (concentration range 0.06-20.6 µmol/L measured by ID-LC-MS/MS) with each method. Moreover, recovery and linearity experiments were performed. Data from our present study were compared to DHEAS data of the Dutch, German and British External Quality Assessment Schemes (EQAS's). RESULTS: Three methods agreed well with ID-LC-MS/MS (R between 0.93 and 0.99 and slopes ranging from 0.92 to 1.07) and showed good recoveries. Four methods showed standardization problems (slopes were 0.84, 1.14, 1.20 and 1.28). Linearity was good in all methods. Intra-assay coefficient of variation was 4.1% using ID-LC-MS/MS and below 5.5% in immunometric methods; one assay had an unacceptably high intra-assay coefficient of variation of 18%. Our data are in agreement with data obtained in three EQAS's. CONCLUSION: Some of the commercially available DHEAS methods show standardization problems and/or a high imprecision. These problems may potentially have clinically adverse consequences. We advise the manufacturers to improve their assays and laboratory specialists to scrutinize the DHEAS method they employ.

Prothrombin complex concentrate for the urgent reversal of warfarin. Assessment of a standard dosing protocol.

BACKGROUND: Octaplex®, a six factor prothrombin complex concentrate (PCC), has recently been approved for use in Canada. The optimal dose of Octaplex has yet to be established and our study was designed to monitor the efficacy of a low standard dose. STUDY DESIGN AND METHODS: Patients on warfarin treatment in need of urgent reversal for bleeding, invasive procedures or surgery were given a standard dose of 40 ml (1000 IU FIX, 14 IU/kg). We conducted a retrospective chart review of 231 patients. RESULTS: Patients given concurrent frozen plasma (FP) for reversal were eliminated from the study. Overall, 150 patients were reviewed and divided into three groups: (1) non-CNS bleeders, (2) CNS bleeders, and (3) non-bleeders. Correction of INR to 1.5 or less was achieved to the same extent in each group. Patients with active bleeding had the least successful bleeding cessation and patients with intracranial bleeding had the most dismal outcome compared to non-intracranial bleeders. CONCLUSIONS: Our data suggests that Octaplex, when given as a low standard dose is effective at INR reversal with 76% of our patients correcting to an INR of 1.5 or less. It appears that this dose is sufficient for non-bleeding patients. Bleeding patients may benefit most from a dose increase to achieve more complete reversal and patients with intracranial bleeding should achieve more complete reversal within 2h of presentation.

Serum and BALF YKL-40 levels are predictors of survival in idiopathic pulmonary fibrosis.

BACKGROUND: The chitinase-like protein YKL-40 is a serum biomarker in diseases with fibrosis, inflammation and tissue remodelling. Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease that is hallmarked by these processes. The aim of this study was to investigate the potential of YKL-40 as a prognostic biomarker for survival in IPF patients. METHODS: Serum and bronchoalveolar lavage fluid (BALF) levels of YKL-40 at the time of diagnosis and a promoter polymorphism in CHI3L1, the gene encoding YKL-40, were determined in 85 IPF patients and 126 controls. The relationship between YKL-40 levels and clinical parameters was evaluated. Kaplan-Meier and Cox regression analyses were used to examine the association between YKL-40 levels and survival. RESULTS: Serum and BALF YKL-40 levels were significantly higher in patients than in healthy controls (p < 0.001). The - 329 A/G polymorphism had a significant influence on BALF YKL-40 levels and the influence on serum YKL-40 levels showed a trend towards significance in IPF patients. IPF patients with high (> 79 ng/ml) serum or high BALF YKL-40 (> 17 ng/ml) levels had significantly shorter survival than those with low YKL-40 levels in serum or BALF. In patients with both low serum and low BALF YKL-40 levels no IPF related mortality was observed. Cox regression modelling showed that there were no confounding factors. CONCLUSIONS: The - 329 polymorphism was associated with serum and BALF YKL-40 levels in IPF patients. High serum and BALF YKL-40 levels are associated with poor survival in IPF patients and could be useful prognostic markers for survival in IPF.

Common variation in the platelet receptor P2RY12 gene is associated with residual on-clopidogrel platelet reactivity in patients undergoing elective percutaneous coronary interventions.

BACKGROUND: The clinical efficacy of clopidogrel is hampered by a large interindividual variability in platelet inhibition. Polymorphisms in the P2RY12 receptor gene have been suggested to contribute to this variability, but previous studies included a relatively small number of patients and incompletely covered the common variation in the P2RY12 gene. The aim of this study was to comprehensively investigate the possible association between common variation in the entire P2RY12 locus and the magnitude of residual on-clopidogrel platelet reactivity measured by 2 commonly used platelet function assays in a large cohort of patients. METHODS AND RESULTS: A total of 1031 consecutive patients with coronary artery disease who were scheduled for elective percutaneous coronary interventions were enrolled. Platelet function was assessed by means of ADP-induced light-transmittance aggregometry and the VerifyNow P2Y12 assay. Six haplotype-tagging single nucleotide polymorphisms were carefully selected to comprehensively cover the total common variation in the P2RY12 gene and its flanking regulatory regions. Six common haplotypes were inferred from these haplotype-tagging single nucleotide polymorphisms (denoted A to F). Haplotype F was associated with significantly lower residual on-clopidogrel platelet reactivity compared with the reference haplotype A. The size of this effect per haplotype allele was approximately 5% aggregation in the ADP-induced light-transmittance aggregometry (P<0.05) and 11 P2Y12 reaction units in the VerifyNow P2Y12 assay (P<0.05). CONCLUSIONS: Common variation in the P2RY12 gene is a significant determinant of the interindividual variability in residual on-clopidogrel platelet reactivity in patients with coronary artery disease.

MUC1 568 A/G genotype-dependent cancer antigen 15-3 levels in breast cancer patients.

OBJECTIVES: CA 15-3 is a widely used tumor marker for breast cancer. We have investigated whether the MUC1 568 A/G polymorphism can influence CA 15-3 levels in healthy women and patients with breast tumors. DESIGN AND METHODS: CA 15-3 was measured in 208 healthy women, in 67 with benign disease, and in 162 women with breast cancer. All subjects were genotyped for the MUC1 568 A/G polymorphism. RESULTS: Significant differences were observed between mean CA 15-3 levels of control subjects grouped according to the MUC1 568 genotype (mean+/-SD): AA (10.3+/-3.8), AG (15.9+/-5.0) and GG (19.0+/-5.6) U/mL, p<0.0001. Similar (median) results were observed in women with benign breast disease: AA (10.2), AG (14.2) and GG (16.6) U/mL, p<0.0001, and those with breast cancer: AA (10.4), AG (17.1) and GG (23.9) U/mL, p<0.0001. CONCLUSIONS: The MUC1 568 A/G polymorphism strongly influences CA 15-3 levels in healthy women and women with either benign or malignant breast tumors.

A CHI3L1 gene polymorphism is associated with serum levels of YKL-40, a novel sarcoidosis marker.

BACKGROUND: YKL-40, a chitinase-like cartilage glycoprotein, has recently shown its potential as a marker for sarcoidosis. METHODS: This study aimed to assess whether YKL-40 at presentation may predict the course of sarcoidosis over a 4-year follow-up period and to investigate whether polymorphisms in the chitinase 3-like 1 (CHI3L1) gene might influence serum YKL-40 levels in sarcoidosis patients (n=63) and controls (n=333). RESULTS: Patients had significantly higher (mean, 95% CI) serum YKL-40 levels (181.3 ng/ml, 50.7-648.1) compared to controls (36.6 ng/ml, p<0.0001. Serum YKL-40 was elevated in 79% of the patients and was inversely correlated with DLco at presentation (r(2)=-0.27, p=0.03), but not after 2-4 years of follow-up (r(2)=-0.16, p=0.27). Serum YKL-40 levels in controls were dependent on the CHI3L1 -329 G/A polymorphism (mean, 95% CI): GG (n=213) 48.3 ng/ml, 41.7-56.0; GA (n=101) 31.2 ng/ml, 26.6-36.3; AA (n=17) 17.8 ng/ml, 13.6-23.4, p<0.0001. In patients, this effect was not observed. CONCLUSIONS: YKL-40 may be used as a sarcoidosis disease marker, but it is unsuitable as a marker to predict the course of the disease. The CHI3L1 -329 G/A polymorphism contributes to inter-individual variations of YKL-40 levels, but does not influence sarcoidosis disease susceptibility or severity.

ACE I/D-corrected Z-scores to identify normal and elevated ACE activity in sarcoidosis.

BACKGROUND: The value of elevated serum angiotensin-converting enzyme (ACE) activity in the diagnosis and follow-up in sarcoidosis is a matter of ongoing debate. This may be at least related to the insertion (I)/deletion (D) polymorphism in the ACE gene (ACE I/D). ACE activity is influenced by the ACE I/D polymorphism. As a consequence, the use of one reference interval instead of three genotype-specific reference intervals for ACE activity may lead to a less precise interpretation of ACE activity. METHODS: In order to assess whether determination of ACE activity indeed requires the ACE I/D genotype to be taken into account, we established ACE I/D-corrected reference intervals in healthy, Caucasian volunteers (n=200). In addition, ACE activities in ACE I/D genotyped patients suspected of or having sarcoidosis (n=129) were expressed as the Z-score related to ACE I/D-corrected reference intervals. RESULTS: Comparison of the Z-score with ACE activity in which ACE I/D is ignored rendered 8.5% misclassification of 'elevated' versus 'normal' ACE or vice versa. CONCLUSIONS: Our data demonstrate a convenient way to circumvent the use of three reference intervals by introducing a Z-score for ACE activity. It also illustrates the need to re-investigating the possible clinical value of serum ACE activity in sarcoidosis by considering ACE I/D.

Transforming growth factor-beta gene polymorphisms in sarcoidosis patients with and without fibrosis.

STUDY OBJECTIVES: Pulmonary fibrosis develops in approximately 25% of patients with chronic sarcoidosis. Transforming growth factor (TGF)-beta1 plays a central role in fibrosis, and accruing reports address the implication of TGF-beta2 and TGF-beta3 in this process. We determined whether single-nucleotide polymorphisms (SNPs) in the TGF-beta1, TGF-beta2, and TGF-beta3 genes might contribute to pulmonary fibrosis in sarcoidosis patients. SETTING: A hospital in the Netherlands. DESIGN: Five SNPs per TGF-beta gene were investigated. PATIENTS AND CONTROL SUBJECTS: Patients with either acute/self-remitting sarcoidosis (n = 50) and Löfgren syndrome (n = 46) or chronic disease with fibrosis (n = 24) and without fibrosis (n = 34) were assessed over a 4-year follow-up period. The control subjects included 315 individuals. MEASUREMENTS AND RESULTS: Polymorphism frequencies were not discordant between the patients and control subjects. The TGF-beta3 4875 A allele was significantly higher in fibrotic patients (carrier frequency, 0.29) than in patients with acute/self-remitting (0.06) and chronic (0.03) sarcoidosis combined (corrected p = 0.01; odds ratio [OR], 7.9). The TGF-beta3 17369 C allele carrier frequency was significantly higher in fibrotic patients (0.29) compared to acute/self-remitting (0.08) and chronic (0.06) patients combined (corrected p = 0.05; OR, 5.1). Although not significant after correction, the TGF-beta3 15101 G allele carrier frequency was lower in fibrotic patients (0.79) compared to acute/self-remitting (0.94) and chronic (1.00) patients combined (p = 0.02; corrected p = 0.1; OR, 0.15). The TGF-beta2 59941 G allele was more abundant in fibrotic patients (carrier frequency, 0.62) compared to patients with acute/self-remitting (0.41) and chronic sarcoidosis combined (0.28) [p = 0.04; corrected p = 0.2; OR, 2.9]. TGF-beta1 gene polymorphisms were not associated with fibrosis. CONCLUSIONS: This study is the first to suggest the implication of genetic variation of TGF-beta3 in the predilection for pulmonary fibrosis developing in sarcoidosis patients.

Chymase gene (CMA1) polymorphisms in Dutch and Japanese sarcoidosis patients.

BACKGROUND: Chymase is released from mast cells following activation. Evidence suggests that chymase plays an important role in tissue injury and remodeling of the lungs, heart and skin. OBJECTIVE: We postulated that chymase gene (CMA1) polymorphisms are associated with pulmonary fibrosis in Dutch and with cardiac and skin involvement in Japanese sarcoidosis patients. PATIENTS AND METHODS: Dutch (n = 153) and Japanese (n = 122) sarcoidosis patients with controls (Dutch, n = 309; Japanese, n = 111) were studied. Pulmonary involvement in Dutch patients as well as clinical manifestations in Japanese patients was evaluated for association with five CMA1 polymorphisms. RESULTS: The CMA1 polymorphisms were not associated with disease susceptibility in either population, or with radiographic evolution in the Dutch or with cardiac or skin involvement in the Japanese patients. The -526 T allele was associated with a lower iVC in Dutch patients. CONCLUSIONS: The CMA1 polymorphisms studied do not contribute to disease susceptibility in Japanese or Dutch sarcoidosis patients. CMA1 polymorphisms do not influence radiographic evolution in Dutch sarcoidosis patients, nor do they predispose to cardiac or skin involvement in Japanese patients. However, the association between CMA1 -526 C/T and iVC in the Dutch patients suggests that chymase may modify the functional outcome of pulmonary sarcoidosis.

The mucin-1 568 adenosine to guanine polymorphism influences serum Krebs von den Lungen-6 levels.

Krebs von den Lungen (KL)-6 offers a new perspective as a disease marker in pulmonary diseases. The aim of this study was to analyze whether serum KL-6 levels are dependent on the functional adenosine to guanine mucin-1 (MUC1) gene polymorphism at nucleotide position 568 in a well-characterized white population. Polymorphisms were determined in 327 healthy, white individuals and 74 patients with sarcoidosis, using a PCR-sequence-specific primer assay. The serum KL-6 levels were measured by ELISA. Significant differences between serum KL-6 levels of healthy subjects who were grouped according to MUC1 568 genotype were observed (P<0.0001) (mean+/-SEM): AA (195.2+/-9.9 U/ml; 95% confidence interval [CI], 175.7-214.8), AG (246.0+/-8.6 U/ml; 95% CI, 229.0-263.1), and GG (302.6+/-11.8 U/ml; 95%CI, 279.3-326.0). In the patients with sarcoidosis, the results were (mean+/-SD): AA (550.1+/-411.7; 95% CI, 380.2-720.1), AG (716.3+/-452.4; 95% CI, 547.4-885.2), GG (1,151.0+/-1122; 95% CI, 610.1-1692.0); P=0.02. Comparison of the KL-6 levels in which the 568 genotype was ignored rendered 6 out of 74 (7.5%) misclassifications of "elevated" versus "normal" KL-6 levels or vice versa. In conclusion, the MUC1 568 A to G polymorphism may be of interest for diagnostic purposes because our study delivered in vivo evidence that it contributes to interindividual variations in KL-6 levels.

Angiotensin-converting enzyme 2 (ACE2) haplotypes are associated with pulmonary disease phenotypes in sarcoidosis patients.

BACKGROUND AND AIM OF THE STUDY: Angiotensin II (Ang II) formation by angiotensin-converting enzyme (ACE) or other enzymes has shown to exhibit profibrotic properties in a variety of fibrotic diseases. A homologue of ACE called ACE2 has been shown to counteract the formation of Ang II. Genetic variation in the components involved in Ang II formation may underlie the progression of pulmonary sarcoidosis. METHOD: Seven ACE2 SNPs, located on the X-chromosome, were investigated using SSP-PCR and haplotypes were constructed. Gender-matched analyses of sarcoidosis patients (80 males/64 females) and controls (110 males/218 females) were performed to correlate disease susceptibility and pulmonary disease phenotypes with ACE2 genotypes and haplotypes. RESULTS: ACE2 SNPs or haplotypes were not associated with susceptibility for sarcoidosis. Haplotype 4 was only present in sarcoid males without parenchymal involvement (frequency: 0.19) and absent in males with parenchymal involvement (p = 0.006; pcorr. = 0.05; degrees of freedom (df) = 1; OR = 0). No significant difference was observed between haplotype 4 frequencies in females with (0.08) or without (0.13) parenchymal involvement (p = 0.5). Although not significant after correction, analysis of the patient group with fibrosis showed that males with haplotype 5 (0.27) were predominant over those with haplotype 5 of the groups without fibrosis (0.03); p = 0.01; pc = 0.08; df = 1; OR = 11.4. Females with fibrosis vs. no fibrosis revealed no difference between haplotype 5 frequencies: 0.05 vs. 0.03; p = 0.37; pc = 1; df = 1. CONCLUSION: These results suggest that ACE2 might be involved in the progression of pulmonary sarcoidosis which may depend on gender. Subsequent studies using larger groups are needed to confirm these findings.