Standardization of automated 25-hydroxyvitamin D assays: How successful is it?

OBJECTIVES: Multiple 25(OH)D assays have recently been aligned to improve comparibility. In this study we investigated the performance of these assays using both native single-donor sera with target values certified by a reference method as well as single donor sera from a heterogeneous patient population. DESIGN & METHODS: 25(OH)D levels were measured in twenty reference samples (Ref!25OHD; Labquality, Finland) using five automated methods (Lumipulse, Liaison, Cobas, iSYS and Access) and one aligned ID-XLC-MS/MS method (slope: 1,00; intercept: 0,00; R=0,996). Furthermore, 25(OH)D concentrations measured in 50 pregnant women and 52 random patients using the 5 automated assays were compared to the ID-XLC-MS/MS. In addition, Vitamin D binding protein (DBP) was measured. RESULTS: Most automated assays showed significant differences in 25(OH)D levels measured in reference samples. Slopes varied from 1,00 to 1,33, intercepts from -5.48 to -15,81nmol/L and the R from 0,971 to 0,997. This inaccuracy was even more prominent in a heterogeneous patient population. Slopes varied from 0,75 to 1,35, intercepts from -9.02 to 11,51nmol/L and the R from 0,840 to 0,949. For most assays the deviation in 25(OH)D concentration increased with elevating DBP concentrations suggesting that DBP might be one of the factors contributing to the inaccuracy in currently used automated 25(OH)D methods. CONCLUSIONS: Despite the use of standardized assays, we observed significant differences in 25(OH)D concentrations in some automated methods using reference material obtained from healthy single donor sera. In sera of a patient population this inaccuracy was even worse which is highly concerning as patient samples are being investigated in clinical laboratories.

Magnesium levels in critically ill patients. What should we measure?

We studied the relation between ionized magnesium, total magnesium, and albumin levels in serum of 115 critically ill patients and the role of extracellular and intracellular magnesium in outcome prediction. Levels of serum total and ionized magnesium, serum albumin, and magnesium in mononuclear blood cells and erythrocytes were measured and the APACHE II score and 1-month mortality recorded. Of all patients, 51.3% had a serum total magnesium concentration below the reference range. In 71% of these hypomagnesemic patients, a normal serum ionized magnesium concentration was measured. None of the patients had an intracellular magnesium concentration below the reference limit. Except for serum total and ionized magnesium, none of the magnesium parameters correlated significantly with each other. A significantly negative correlation was found between serum albumin and the fraction ionized magnesium. There was no association between low extracellular or intracellular magnesium and clinical outcome. The observation of hypomagnesemia in critically ill patients depends on which magnesium fraction is measured. The lack of correlation with clinical outcome suggests hypomagnesemia to be merely an epiphenomenon. Reliable concentrations of serum ionized magnesium can be obtained only by direct measurement and not by calculation from serum total magnesium and albumin.

Serum ionized magnesium: comparison of results obtained with three ion-selective analyzers.

In a two-center (Academic Medical Center, The Netherlands, and National Institutes of Health, USA) study, we compared ionized magnesium (iMg2+) results in serum determined with the AVL 988/4, KONE Microlyte 6 and NOVA CRT, which are the currently available analyzers equipped with a magnesium ion-selective electrode. The comparison was performed with frozen serum samples from normal individuals and patients. Imprecision and reference intervals were established. We found the best agreement between the KONE(x) and AVL(y) magnesium ion-selective electrodes (y= 0.972x-0.013; n=138) with samples from patients. With samples from normals, all three analyzers reported significantly different results (p<0.05). Best precision was found using the NOVA; coefficients of variation established at three levels were all < 4.0%. Coefficients of variation for the AVL and KONE were <5% at normal and high iMg2+, but 10.7 and 9.4%, respectively, at iMg2+ approximately 0.30 mmol/l. The reference intervals (mean+/-standard deviation) based on measurements in fresh serum samples were different for each analyzer: 0.55-0.63 mmol/l for AVL, 0.470.57 mmol/l for KONE and 0.43-0.55 mmol/l for NOVA. Thus, significant differences among the ionized magnesium concentration obtained with the three analyzers, limit comparison of results in clinical practice, and need to be resolved (e.g. by improvement of specificity and standardization of calibrators).

Intracellular and extracellular, ionized and total magnesium in pre-eclampsia and uncomplicated pregnancy.

Magnesium (Mg) and calcium (CA) concentrations in women with pre-eclampsia, women with an uncomplicated pregnancy and non-pregnant women were compared. Ionized serum magnesium and calcium concentrations and intracellular magnesium concentrations were measured in 15 pregnant women with severe pre-eclampsia, 34 uncomplicated pregnant women early, at midterm and preterm in their pregnancy and 24 non-pregnant women. The ionized calcium concentration did not chance during normal pregnancy or during pre-eclampsia relative to non-pregnant women. In contrast, elevated total and ionized magnesium serum concentrations were found in women with severe pre-eclampsia (total Mg = 0.85+/-0.11 mM, ionized Mg = 0.61+/-0.06 mM) relative to uncomplicated pregnant women (total Mg = 0.72+/-0.06 mM, ionized Mg = 0.53+/-0.03 mM). Total magnesium in pre-eclamptic women were similar to non-pregnant women. Intracellular ionized and total magnesium concentrations in mononuclear blood cells and erythrocytes were similar in pre-eclamptic women and women with uncomplicated pregnancy. Serum magnesium concentrations are elevated in severe pre-eclamptic women relative to women with uncomplicated pregnancy and are related to birth weight and gestational age at delivery. There may be a causal relationship since magnesium is involved in blood pressure regulation through an intracellular inhibition of NO synthase in endothelial cells.

Magnesium in disease: a review with special emphasis on the serum ionized magnesium.

This review deals with the six main clinical situations related to magnesium or one of its fractions, including ionized magnesium: renal disease, hypertension, pre-eclampsia, diabetes mellitus, cardiac disease, and the administration of therapeutic drugs. Issues addressed are the physiological role of magnesium, eventual changes in its levels, and how these best can be monitored. In renal disease mostly moderate hypermagnesemia is seen; measuring ionized magnesium offers minimal advantage. In hypertension magnesium might be lowered but its measurement does not seem relevant. In the prediction of severe pre-eclampsia, elevated ionized magnesium concentration may play a role, but no unequivocal picture emerges. Low magnesium in blood may be cause for, or consequence of, diabetes mellitus. No special fraction clearly indicates magnesium deficiency leading to insulin resistance. Cardiac diseases are related to diminished magnesium levels. During myocardial infarction, serum magnesium drops. Total magnesium concentration in cardiac cells can be predicted from levels in sublingual or skeletal muscle cells. Most therapeutic drugs (diuretics, chemotherapeutics, immunosuppressive agents, antibiotics) cause hypomagnesemia due to increased urinary loss. It is concluded that most of the clinical situations studied show hypomagnesemia due to renal loss, with exception of renal disease. Keeping in mind that only 1% of the total body magnesium pool is extracellular, no simple measurement of the real intracellular situation has emerged; measuring ionized magnesium in serum has little added value at present.

Intracellular and extracellular blood magnesium fractions in hemodialysis patients; is the ionized fraction a measure of magnesium excess?

To establish the best measure for determining magnesium overload, we measured ionized and total magnesium in serum and mononuclear blood cells and total magnesium in erythrocytes in blood of 23 hemodialysis patients, known for their disturbed magnesium homeostasis. When comparing the mean magnesium values obtained in the patient population with those of a control population, all of these magnesium markers, including the biologically active fractions, were significantly (P < 0.05) increased. Because serum total magnesium was not increased in all dialysis patients studied, the population was divided into two groups, according to total serum magnesium > 1.0 mmol/L or less than that. Results in these two populations showed that ionized serum magnesium and ionized magnesium in mononuclear blood cells might give a better indication about the magnesium status of the tested patients than the currently used total serum magnesium data. However, neither of the two markers, especially ionized serum magnesium, was able to discriminate fully between normal magnesium homeostasis and magnesium excess. We therefore conclude that the two ionized magnesium markers offer minimal advantage for this discrimination, and that the total magnesium concentration in serum remains the measurement of choice.

The clinical value of lactate dehydrogenase in serum: a quantitative review.

The aim of this article is to describe guidelines for rational use of lactate dehydrogenase and its isoenzymes, in the diagnostic processes and during follow-up, based on a systematic review of relevant literature. Sources of data for this study were English-language scientific publications, obtained from the database of the National Library of Medicine (Medline), concerning the clinical application (diagnosis, monitoring or treatment of disease) of lactate dehydrogenase and lactate dehydrogenase isoenzyme measurements in serum in the following main clinical fields: cardiology, hepatology, haematology and oncology. For acceptance in the present review, studies had to include: a proper definition of the tested patient population, diagnostic criteria, sampling time, sampling frequency, and test characteristics. Estimation of the relation between lactate dehydrogenase or lactate dehydrogenase isoenzymes and specific diseases expressed as sensitivity, specificity, survival or remission rate were extracted. The application of serum lactate dehydrogenase is relevant in the diagnosis of myocardial infarction (late detection), haemolytic anaemia, ovarian dysgerminoma and testicular germ cell tumor. For monitoring the progress of a disease lactate dehydrogenase is relevant in establishing the survival duration and rate in Hodgkin's disease and non-Hodgkin's lymphoma, and in the follow-up of ovarian dysgerminoma. Rational use of lactate dehydrogenase can be achieved when requests for its determination are limited to the above mentioned conditions. No rationale could be found for measuring lactate dehydrogenase isoenzymes.

Precision of the magnesium determination in mononuclear blood cells and erythrocytes.

OBJECTIVE: Establishing the analytical variation and reproducibility of the intracellular magnesium (Mg) assay in mononuclear blood cells (MBC) and erythrocytes (RBC). DESIGN AND METHODS: We assessed the analytical variation of the several determination steps, and the reproducibility for the complete intracellular Mg-assay (combination of preanalytical, analytical, and biological variation). The influence of platelets was determined by comparing Mg concentrations obtained from heparinized blood and defibrinated blood. RESULTS: Coefficients of variation of the several determination steps used in the MBC- and RBC-assay were < or = 5.4%. The overall analytical variation was 5.0-6.8%, and reproducibility of the complete Mg-assay 11.6-14.0%. Mg measurements in MBC (expressed as fmol/cell) obtained from heparinized blood showed significantly higher values than those obtained from defibrinated blood. CONCLUSION: This is the first study to describe in detail reproducibility data for the individual steps in the overall procedure to measure intracellular magnesium. It is shown that results obtained in daily practice should be interpreted with care. Moreover, the removal of platelets is essential in the determination of Mg in MBC.

Can low-calcium peritoneal dialysis solution safely replace the standard calcium solution in the majority of chronic peritoneal dialysis patients?

OBJECTIVE: To evaluate the use of low-calcium solution as the standard solution in chronic peritoneal dialysis patients. DESIGN: Prospective long-term follow-up study over a one-year period. SETTING: University hospital. INTERVENTIONS: The change of the calcium concentration of the dialysate from 1.75 mmol/L to 1.25 mmol/L. MAIN OUTCOME MEASURES: Serum calcium and phosphorus concentration and intact parathyroid hormone (iPTH). PATIENTS: Fifty normo- and hypercalcemic patients using the standard 1.75 mmol/L calcium solution. RESULTS: Serum ionized calcium (iCa) decreased significantly during the first six months, resulting in a significant increment of iPTH (baseline value: 0.9-79, median 9.4 pmol/L; at six months: 1.1-111, median 20.6 pmol/L; p < 0.05). In 28 patients completing the study, iPTH remained significantly elevated, despite high normal iCa. At similar changes of iCa, patients with baseline iPTH > 20 pmol/L showed a significantly higher increase in iPTH than patients with low iPTH (24.0 vs 5.0; p < 0.01), despite a more than doubled dose of alfacalcidol and calcium carbonate (mean dose of 1580 increased to 3277 mg/day). During the follow-up, 21 episodes of hypercalcemia were observed. Phosphorus control was adequate. CONCLUSIONS: Low-calcium solution cannot be used as a standard solution, especially in patients with iPTH levels indicating mild or severe hyperparathyroidism, because in these patients iPTH may rise further.

Magnesium fractions in serum of healthy individuals and CAPD patients, measured by an ion-selective electrode and ultrafiltration.

OBJECTIVES: Validation and comparison of a magnesium ion-selective electrode (ISE) with a cation-exchange resin technique, followed by determination of all magnesium fractions in serum of healthy volunteers and continuous ambulatory peritoneal dialysis (CAPD) patients. DESIGN AND METHODS: The analytical aspect has been studied by measuring the influence of complexing agents on the fraction ionized magnesium (friMg2+). A theoretical approximation of friMg2+, based on mass equilibria and complexation constants, was calculated and compared with the measurements. RESULTS: ISE measurements showed good agreement with theory. Reference values of the ionized, protein-bound, and complexed magnesium fractions were (mean +/- SD) 0.65 +/- 0.04, 0.27 +/- 0.04, and 0.08 +/- 0.03, respectively. Fractions obtained in the CAPD group were 0.62 +/- 0.04, 0.22 +/- 0.05, and 0.16 +/- 0.05, respectively, and differed significantly from the values of the reference population. CONCLUSIONS: All known serum magnesium parameters can be established by a combination of ultrafiltration, atomic absorption spectrometry, and ISE measurements. Unknown complexing compounds most probably account for the increased fraction of complexed magnesium in the serum of CAPD patients.

Analytical evaluation of Kone Microlyte determination of ionized magnesium.

We performed an analytical evaluation of a commercially available instrument for determining ionized magnesium through use of a neutral carrier, liquid-membrane-based ion-selective electrode. Reproducibility (CV 2-4%), linearity (0.30-2.50 mmol/L), lower limit of detection (0.30 mmol/L), and absence of interference from Ca2+ indicate adequate performance for measuring ionized magnesium in plasma or serum samples in the normal to high-concentration range. Sodium in excess of 150 mmol/L caused a negative bias, which can be explained by ionic strength-induced changes in activity coefficients. The use of heparin as an anticoagulant must be restricted to concentrations < 15 units/mL because of the binding of magnesium to heparin. The mean +/- SD concentration of ionized magnesium and its fraction of total magnesium in 76 healthy volunteers were 0.56 +/- 0.05 mmol/L and 0.65 +/- 0.04, respectively.