Trimester-specific reference intervals for cystatin C and neutrophil gelatinase-associated lipocalin during pregnancy.

BACKGROUND: During normal pregnancy, the glomerular filtration rate (GFR) increases dramatically. Failure to obtain this physiological increase is an important risk factor for morbidity and mortality for both mother and child. The estimated GFR (eGFR) using serum creatinine levels is unsuitable for accurate measurement of renal function during pregnancy. Therefore, new biomarkers have been proposed. Elevated levels of Cystatin C (CysC) and Neutrophil Gelatinase-Associated Lipocalin (NGAL) are associated with renal failure and preeclampsia (PE). In this study, we determined reference intervals for CysC and NGAL during pregnancy. METHODS: Healthy pregnant women were recruited and blood samples were collected at 9-13 weeks (T1), 27-29 weeks (T2), and 36-39 weeks (T3) of gestation and at 4-13 weeks postpartum (PP). The samples from women with uncomplicated pregnancy were analyzed to determine median values and upper reference limits (URLs, 97.5 percentiles) of creatinine, CysC, and NGAL. RESULTS: A total of 175 women were included. Longitudinal changes and median values of creatinine, CysC, and NGAL were determined using only complete data sets (n=59). URLs were determined using all available data. The URL at T1, T2, T3, and PP were 60, 63, 74, 93 µmol/L for creatinine; 0.93, 1.04, 1.61, 1.23 mg/L for CysC; and 87, 84, 88, 95 ng/mL for NGAL. CONCLUSIONS: CysC concentrations are highly dynamic and increase during pregnancy. NGAL concentrations are less dynamic, but well below the URL specified by the manufacturer for non-pregnant women. It is therefore recommended to use trimester-specific reference values for both CysC and NGAL.

Analytical Performance and Clinicopathologic Correlation of Four Fecal Calprotectin Methods.

OBJECTIVES: Calprotectin is a noninvasive biomarker that can distinguish inflammatory bowel disease from irritable bowel syndrome. We investigated four automated fecal calprotectin methods on five different platforms for their preanalytical process, analytical performance, and clinicopathologic correlation. METHODS: Four calprotectin methods (Bühlmann, EliA CN, EliA CN2, and DiaSorin) were performed on five platforms (Cobas 8000 E502, Phadia Immunocap 100 and 250, and Liaison and Liaison XL) in two hospital laboratories. RESULTS: Overall variation for the different extraction devices was less than 19% when feces were of normal consistency. Freeze-thawing of samples resulted in comparable results compared with fresh samples. The different methods had a good analytic correlation (R = 0.83-0.95). Their clinicopathologic correlation was comparable, but the Bühlmann method showed significantly higher calprotectin values in every patient category. CONCLUSIONS: The automated calprotectin methods showed a good performance and comparable clinicopathologic correlation. Due to lack of standardization, the numerical values differ for the various methods.

Reference intervals and longitudinal changes in copeptin and MR-proADM concentrations during pregnancy.

BACKGROUND: Vasopressin and adrenomedullin and their stable by-products copeptin and midregional part of proadrenomedullin (MR-proADM) are promising biomarkers for the development of preeclampsia. However, clinical use is hampered by the lack of trimester-specific reference intervals. We therefore estimated reference intervals for copeptin and MR-proADM in disease-free Dutch women throughout pregnancy. METHODS: Apparently healthy low risk pregnant women were recruited. Exclusion criteria included current or past history of endocrine disease, multiple pregnancy, use of medication known to influence thyroid function and current pregnancy as a result of hormonal stimulation. Women who miscarried, developed hyperemesis gravidarum, hypertension, pre-eclampsia, hemolysis elevated liver enzymes and low platelets, diabetes or other disease, delivered prematurely or had a small for gestational age neonate were excluded from analyses. Blood samples were collected at 9-13 weeks (n=98), 27-29 weeks (n=94) and 36-39 weeks (n=91) of gestation and at 4-13 weeks post-partum (PP) (n=89). Sixty-two women had complete data during pregnancy and PP. All analyses were performed on a Kryptor compact plus. RESULTS: Copeptin increases during pregnancy, but 97.5th percentiles remain below the non-pregnant upper reference limit (URL) provided by the manufacturer. MR-proADM concentrations increase as well during pregnancy. In trimesters 2 and 3 the 97.5th percentiles are over three times the non-pregnant URL provided by the manufacturer. CONCLUSIONS: Trimester- and assay-specific reference intervals for copeptin and MR-proADM should be used. In addition, consecutive measurements and the time frame between measurements should be considered as the differences seen with or in advance of preeclampsia can be expected to be relatively small compared to the reference intervals.

TSH and fT4 during pregnancy: an observational study and a review of the literature.

BACKGROUND: Trimester-specific reference intervals for TSH are recommended to assess thyroid function during pregnancy due to changes in thyroid physiology. Laboratories should verify reference intervals for their population and assay used. No consistent upper reference limit (URL) for TSH during pregnancy is reported in literature. We investigated the use of non-pregnant reference intervals for TSH, recommended during pregnancy by current Dutch guidelines, by deriving trimester-specific reference intervals in disease-free Dutch pregnant women as these are not available. METHODS: Apparently healthy low risk pregnant women were recruited via midwifery practices. Exclusion criteria included current or past history of thyroid or other endocrine disease, multiple pregnancy, use of medication known to influence thyroid function and current pregnancy as a result of hormonal stimulation. Women who were TPO-antibody positive, miscarried, developed hyperemesis gravidarum, hypertension, pre-eclampsia, HELLP, diabetes or other disease, delivered prematurely or had a small for gestational age neonate were excluded. Blood samples were collected at 9-13 weeks (n=99), 27-29 weeks (n=96) and 36-39 weeks (n=96) of gestation and at 4-13 weeks post-partum (n=95). Sixty women had complete data during pregnancy and post-partum. All analyses were performed on a Roche Cobas e601 analyser. RESULTS AND CONCLUSIONS: In contrast to current Dutch guidelines, the 97.5th percentiles of TSH in the first (3.39 mIU/L) and second trimesters (3.38 mIU/L) are well under the non-pregnant URL of 4.0 mIU/L. The higher TSH in the third trimester (97.5th percentile 3.85 mIU/L) is close to the current non-pregnant URL of 4.0 mIU/L. Absolute intra-individual TSH is relatively stable during pregnancy and post-partum as individuals tracked within the tertile assigned in trimester 1. Even small deviations within the population reference interval may indicate subtle thyroid dysfunction.

Lactate point-of-care testing for acidosis: Cross-comparison of two devices with routine laboratory results.

OBJECTIVES: Lactate is a major parameter in medical decision making. During labor, it is an indicator for fetal acidosis and immediate intervention. In the Emergency Department (ED), rapid analysis of lactate/blood gas is crucial for optimal patient care. Our objectives were to cross-compare-for the first time-two point-of-care testing (POCT) lactate devices with routine laboratory results using novel tight precision targets and evaluate different lactate cut-off concentrations to predict metabolic acidosis. DESIGN AND METHODS: Blood samples from the delivery room (n=66) and from the ED (n=85) were analyzed on two POCT devices, the StatStrip-Lactate (Nova Biomedical) and the iSTAT-1 (CG4+ cassettes, Abbott), and compared to the routine laboratory analyzer (ABL-735, Radiometer). Lactate concentrations were cross-compared between these analyzers. RESULTS: The StatStrip correlated well with the ABL-735 (R=0.9737) and with the iSTAT-1 (R=0.9774) for lactate in umbilical cord blood. Lactate concentrations in ED samples measured on the iSTAT-1 and ABL-735 showed a correlation coefficient of R=0.9953. Analytical imprecision was excellent for lactate and pH, while for pO2 and pCO2 the coefficient of variation was relatively high using the iSTAT-1. CONCLUSION: Both POCT devices showed adequate analytical performance to measure lactate. The StatStrip can indicate metabolic acidosis in 1 µl blood and will be implemented at the delivery room.

Pregnancy diabetes: A comparison of diagnostic protocols based on point-of-care, routine and optimized laboratory conditions.

In vitro glycolysis poses a problem during diabetes screening, especially in remote laboratories. Point-of-care analysis (POC) may provide an alternative. We compared POC, routine and STAT analysis and a feasible protocol during glucose tolerance test (GTT) for pregnancy diabetes (GDM) screening. In the routine protocol, heparin tubes were used and turn-around-time (TAT) was unsupervised. In the STAT protocol, tubes were processed immediately. The feasible protocol comprised of citrated tubes with a TAT of 1 hour. Outcome was defined as glucose concentration and clinical diagnosis. Glucose measured by POC was higher compared to routine analysis at t = 0 (0.25 mM) and t = 120 (1.17 mM) resulting in 17% more GDM diagnoses. Compared to STAT analysis, POC glucose was also higher, although less pronounced (0.06 and 0.9 mM at t = 0 and t = 120 minutes, respectively) and misclassification was only 2%. Glucose levels and clinical diagnosis were similar using the feasible protocol and STAT analysis (0.03 mM and -0.07 mM at t = 0 and t = 120, 100% identical diagnoses). POC is an viable alternative for STAT glucose analysis in GDM screening (sensitivity: 100%, specificity: 98%). A feasible protocol (citrated phlebotomy tubes with a TAT of 60 minutes) resulted in 100% identical outcome and provides the best alternative.

Simultaneous Determination of Underivatized Vitamin B1 and B6 in Whole Blood by Reversed Phase Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry.

BACKGROUND: Vitamin B1 (thiamine-diphosphate) and B6 (pyridoxal-5'phosphate) are micronutrients. Analysis of these micronutrients is important to diagnose potential deficiency which often occurs in elderly people due to malnutrition, in severe alcoholism and in gastrointestinal compromise due to bypass surgery or disease. Existing High Performance Liquid Chromatography (HPLC) based methods include the need for derivatization and long analysis time. We developed an Ultra High Performance Liquid Chromatography Tandem Mass spectrometry (UHPLC-MS/MS) assay with internal standards for simultaneous measurement of underivatized thiamine-diphosphate and pyridoxal-5'phosphate without use of ion pairing reagent. METHODS: Whole blood, deproteinized with perchloric acid, containing deuterium labelled internal standards thiamine-diphosphate(thiazole-methyl-D3) and pyridoxal-5'phosphate(methyl-D3), was analyzed by UHPLC-MS/MS. The method was validated for imprecision, linearity, recovery and limit of quantification. Alternate (quantitative) method comparisons of the new versus currently used routine HPLC methods were established with Deming regression. RESULTS: Thiamine-diphosphate and pyridoxal-5'phosphate were measured within 2.5 minutes instrumental run time. Limits of detection were 2.8 nmol/L and 7.8 nmol/L for thiamine-diphosphate and pyridoxal-5'phosphate respectively. Limit of quantification was 9.4 nmol/L for thiamine-diphosphate and 25.9 nmol/L for pyridoxal-5'phosphate. The total imprecision ranged from 3.5-7.7% for thiamine-diphosphate (44-157 nmol/L) and 6.0-10.4% for pyridoxal-5'phosphate (30-130 nmol/L). Extraction recoveries were 101-102% ± 2.5% (thiamine-diphosphate) and 98-100% ± 5% (pyridoxal-5'phosphate). Deming regression yielded slopes of 0.926 and 0.990 in patient samples (n = 282) and national proficiency testing samples (n = 12) respectively, intercepts of +3.5 and +3 for thiamine-diphosphate (n = 282 and n = 12) and slopes of 1.04 and 0.84, intercepts of -2.9 and +20 for pyridoxal-5'phosphate (n = 376 and n = 12). CONCLUSION: The described UHPLC-MS/MS method allows simultaneous determination of underivatized thiamine-diphosphate and pyridoxal-5'phosphate in whole blood without intensive sample preparation.

Thyrotropin versus age relation as an indicator of historical iodine intake.

BACKGROUND: In populations with mild iodine deficiency, the serum level of thyrotropin (TSH) is negatively and the serum free thyroxine (FT4) is positively associated with age. An ongoing decrease of TSH and increase of FT4 can be found after iodine supplementation. The aim of this study was to investigate whether there are current differences in the relation between thyroid function and age in relation to differences in iodine intake in the past. METHODS: Eight medical laboratories in several regions of The Netherlands, which are all iodine sufficient at present but with a difference in iodine status in the past, provided the results of all TSH and FT4 measurements performed from 2006 until 2011, resulting in 330,802 TSH and 103,940 FT4 measurements. RESULTS: The negative association between TSH and age in the elderly is only present in areas with a historical iodine deficiency (regression coefficients [RC] -0.008, 95% confidence interval [CI] -0.009; -0.007). In the historically iodine-sufficient population, TSH shows no obvious increase or decrease with age. In both the historically iodine-sufficient and iodine-deficient populations, FT4 levels were positively associated with age in the elderly (RC 0.009, 95% CI 0.008; 0.010 and RC 0.008, 95% CI 0.007; 0.010, respectively). CONCLUSIONS: There are differences in relation between thyroid function and age between populations with differences in iodine intake in the past, despite an adequate iodine status at present. This raises the question whether the present but also historical iodine status of a population should be taken into account when establishing the reference limits of TSH and FT4.

Performance characteristics of a new automated method for measurement of anti-cyclic citrullinated peptide.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory auto-immune disease affecting approximately 1%-2% of the population worldwide. RA is a potentially crippling disease since it results in malformation of the joints. RA is mostly diagnosed based on clinical manifestations but serological tests against autoantibodies, such as rheumatoid factor and anti-cyclic citrullinated peptides (aCCP), are available. The presence of aCCP antibodies is strongly associated with a more severe, destructive disease course. Recently, a new test for the measurement of aCCP antibodies on the IMMULITE 2000(XPi) platform was developed by Siemens Healthcare. In this study we investigated the performance characteristics of this new aCCP test in four different hospital laboratories and compared the new test with three different commercially available platforms. METHODS: Samples were collected from patients presented to the hospital for aCCP measurement. Serum aCCP levels were determined by aCCP (Ig)G assay for IMMULITE 2000(XPi) systems (Siemens Healthcare), ImmunoScan RA enzyme-linked immunosorbent assay (ELISA) test (Eurodiagnostica), Immunocap 250 (Thermofisher) or aCCP IgG assay on the Modular system (Roche Diagnostics). The evaluation protocol consisted of within-run imprecision (20 sequential runs), between-run imprecision (16 workdays), comparison of serum and plasma measurement and method comparison. RESULTS: The within-run imprecision (n=20) for aCCP IgG assay on three different IMMULITE 2000(XPi) systems ranged from 3.0% to 6.9% at levels 3.2-171.2 U/mL. Between-run imprecision (n=16 days) ranged from 5.2% to 11% at levels of 3.2-106.9 U/mL. Method comparison showed good correlation when samples were measured on two different Immulite analyzers in two different hospital laboratories [0.21+0.96x (n=40)]. Method comparison of the IMMULITE 2000(XPi) aCCP test with aCCP on Immunoscan RA ELISA (n=112), Immunocap 250 (n=105) and the Modular system (n=289) resulted in a concordance of 90.2%, 93.3% and 94.8%, respectively. Correlation of serum versus heparin samples showed a correlation of 0.12+1.08x for the Immulite 2000(XPi) test. CONCLUSIONS: The aCCP assay on the IMMULITE 2000(XPi) has good performance characteristics and shows high level of concordance with the aCCP test on Immunoscan RA ELISA test, Immunocap 250 and the Modular systems.

Salivary morning androstenedione and 17α-OH progesterone levels in childhood and puberty in patients with classic congenital adrenal hyperplasia.

BACKGROUND: Treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency can be monitored by salivary androstenedione (A-dione) and 17α-hydroxyprogesterone (17OHP) levels. There are no objective criteria for setting relevant target values or data on changes of 17OHP and A-dione during monitoring. METHODS: We evaluated A-dione and 17OHP levels in nearly 2000 salivary samples collected during long-term treatment of 84 paediatric patients with classic 21-hydroxylase deficiency. RESULTS: A-dione and 17OHP levels and its ratio 17OHP/A-dione remained constant from 4 to 11 years with no sex-related differences. During puberty, A-dione and 17OHP levels both increased, starting at earlier age in girls than in boys. The ratio 17OHP/A-dione declined. Normalised A-dione concomitant with elevated 17OHP [1.43 nmol/L (0.46-4.41) during prepuberty; 2.36 nmol/L (0.63-8.89) for boys and 1.99 nmol/L (0.32-6.98) for girls during puberty] could be obtained with overall median glucocorticoid doses of 11-15 mg/m2/day. A-dione levels above the upper reference limit (URL), suggesting undertreatment, coincided with 17OHP levels ≥10 times URL. The percentage of A-dione levels above URL was 16% at ages 4-8 years, but increased to 31% for girls at 16 years and 46% for boys at 17 years. CONCLUSIONS: Normalised A-dione consistent with 17OHP three times URL during prepuberty and normalised A-dione consistent with 4-6 times URL during puberty could be obtained by moderate glucocorticoid dosages. A constant 17OHP/A-dione ratio during prepuberty suggested absence of adrenarche. During puberty, a higher percentage of samples met the criteria for undertreatment, especially of boys.

The diagnostic performance of allergen-molecules in comparison to allergen-extracts.

BACKGROUND: This study evaluated the diagnostic performance of naturally purified allergen-molecules compared to that of allergen-extracts for house dust mite, cat dander epithelium and dog dander. METHODS: In vitro tests for allergen-specific IgE were performed on the IMMULITE(®) 2000 in serum samples from 66 allergic patients. RESULTS: House dust mite: specificity for the allergen-extract (D1) and the allergen-molecules (nDer p 1, nDer f 1, nDer p 2 and nDer f 2) is comparable. The allergen-extract has a significantly higher sensitivity (100%) and total agreement (TA) (93%) relative to sensitivity (57%-70%) and TA (76%-81%) of the individual allergen-molecules. Cat dander epithelium: sensitivity (90%), specificity (96%) and TA (94%) of the allergen-molecule (nFel d 1) are comparable to those of the allergen-extract (E1). Dog dander: The allergen-molecule (nCan d 1) and allergen-extract (E5) have comparable specificity and TA. The allergen-extract has a lower sensitivity (52%) than the allergen-molecule (71%), although not significant (p=0.125). CONCLUSIONS: There is no diagnostic benefit of using allergen-molecules instead of allergen-extracts for initial allergy screening on cat dander epithelium and dog dander. However, use of these allergen-molecules might contribute to better standardization of the specific IgE tests. The studied allergen-molecules for house dust mite are of minor diagnostic value, because of loss of sensitivity.

Analytical performance evaluation of the Cobas 6000 analyzer - special emphasis on trueness verification.

BACKGROUND: Consolidation of analyzers is an emerging issue in clinical chemistry. We evaluated the analytical performance of the Cobas 6,000 analyzer (Roche Diagnostics), which is considered a candidate for replacement of current Hitachi 917 analyzers and for consolidation of chemistry and immunochemistry. METHODS: The precision, accuracy, linearity and correlation with current field methods were evaluated according to Clinical and Laboratory Standards Institute protocols EP5, EP9 and EP10. A total of 31 routine chemistry assays and 18 immunoassays were studied. Accuracy and linearity were verified for 24 chemistry parameters using value-assigned trueness controls from the Dutch External Quality Assessment Scheme organizers. In addition, traceability to methods endorsed by the Joint Committee of Traceability in Laboratory Medicine was examined. RESULTS: All analytes met allowable precision criteria, apart from the low level for sodium and folate. Total coefficients of variation ranged between 0.6% and 4.4% for routine chemistry and between 0.8% and 5.8% for immunochemistry, apart from folate (12% at the low end). The correlation coefficients for comparison to current field methods were >0.975, except for magnesium and for six out of 18 immunochemistries. Recovery experiments indicated high recovery for most of the 24 routine chemistry assays. CONCLUSIONS: Considering the excellent precision data and the result equivalence for most assays, it can be concluded that Cobas 6,000 accommodates robust chemistry and immunochemistry, and has good potential for workstation consolidation in medium-sized laboratories.

Why determine only the total prostate-specific antigen, if the free-to-total ratio contains the information?

BACKGROUND: Total prostate-specific antigen (tPSA) is the best available test for the detection of prostate cancer but it lacks specificity. The free-to-total ratio (F/T ratio) is used to increase specificity in the range of tPSA of 4-10 microg/L. MATERIALS AND METHODS: Four hundred and seven biopsy results and quantitative tPSA and F/T ratio data were combined. Using the histological determination, normal/hyperplasia versus malignant as a gold standard, receiver operating characteristic (ROC) curves as well as the areas under the curve (AUC) for tPSA and F/T ratio were determined. The differences between the two AUCs were considered for various tPSA ranges and specificities of F/T ratio and tPSA were calculated. RESULTS: In the total group, there was a gain of specificity of 11% (from 23% to 34%) when the sensitivity was 92% (using a cut-off >0.28 for the F/T ratio and a cut-off >4 microg/L for tPSA). When considering the group of patients for which the F/T ratio is currently used (4-10 microg/L), the gain of specificity was 27% (from 2% to 29%). This implicates that the number of unnecessary biopsies taken will be reduced by 27%. Moreover, the AUC of the F/T ratio was significantly higher at an even broader range of tPSA, i.e. up to 40 microg/L. CONCLUSIONS: This study demonstrates that the F/T ratio has better diagnostic performance than tPSA, not only in the grey zone of tPSA, but also outside the grey zone, i.e. up to 40 microg/L.

Analytical and clinical performance of three natriuretic peptide tests in the emergency room.

BACKGROUND: The aim of the present study was to investigate the analytical and diagnostic utility of B-type natriuretic peptide (BNP) and the N-terminus of this prohormone, N-terminal pro-BNP (NT-pro-BNP) testing in the emergency department to identify acute congestive heart failure (CHF). METHODS: A blood sample taken from patients presenting to the emergency department with acute dyspnoea (n=80) was analyzed for natriuretic peptides using three different assays [Triage BNP (Biosite), Centaur BNP (Bayer) and Elecsys NT-pro-BNP (Roche)]. A cardiologist and a pulmonologist, blinded to the actual natriuretic peptide levels, reviewed all test results (including echocardiography, etc.) retrospectively and made a diagnosis of dyspnoea due to CHF or not. RESULTS: Analytical testing showed good correlation and coefficients of variation of less than 10% for all three assays. Cardiac-related dyspnoea was found in 40 patients (50%). NT-proBNP and BNP values were significantly elevated in these patients. For identifying patients with CHF, BNP and NT-proBNP scored equally well (area under the receiver operating characteristic curve of 0.78, 0.77 and 0.78 for the Biosite, Roche and Bayer assays, respectively). CONCLUSIONS: In general, the different assays tested for BNP and NT-pro-BNP correlate very well in patients with suspected CHF and may aid in the risk stratification process in emergency departments. However, the value must always be interpreted in conjunction with other clinical information. It should also be considered that renal impairment can affect the results.