Steven Pauwels

Free 25-Hydroxyvitamin D: Impact of Vitamin D Binding Protein Assays on Racial-Genotypic Associations.

Context: Total 25-hydroxyvitamin D (25OHD) is a marker of vitamin D status and is lower in African Americans than in whites. Whether this difference holds for free 25OHOD (f25OHD) is unclear, considering reported genetic-racial differences in vitamin D binding protein (DBP) used to calculate f25OHD. Objectives: Our objective was to assess racial-geographic differences in f25OHD and to understand inconsistencies in racial associations with DBP and calculated f25OHD. Design: This study used a cross-sectional design. Setting: The general community in the United States, United Kingdom, and The Gambia were included in this study. Participants: Men in Osteoporotic Fractures in Men and Medical Research Council studies (N = 1057) were included. Exposures: Total 25OHD concentration, race, and DBP (GC) genotype exposures were included. Outcome Measures: Directly measured f25OHD, DBP assessed by proteomics, monoclonal and polyclonal immunoassays, and calculated f25OHD were the outcome measures. Results: Total 25OHD correlated strongly with directly measured f25OHD (Spearman r = 0.84). Measured by monoclonal assay, mean DBP in African-ancestry subjects was approximately 50% lower than in whites, whereas DBP measured by polyclonal DBP antibodies or proteomic methods was not lower in African-ancestry. Calculated f25OHD (using polyclonal DBP assays) correlated strongly with directly measured f25OHD (r = 0.80–0.83). Free 25OHD, measured or calculated from polyclonal DBP assays, reflected total 25OHD concentration irrespective of race and was lower in African Americans than in US whites. Conclusions: Previously reported racial differences in DBP concentration are likely from monoclonal assay bias, as there was no racial difference in DBP concentration by other methods. This confirms the poor vitamin D status of many African-Americans and the utility of total 25OHD in assessing vitamin D in the general population.

Role of Assay Type in Determining Free 25-Hydroxyvitamin D Levels in Diverse Populations

The choice of a vitamin D–binding protein assay is key in calculating free 25-hydroxyvitamin D levels. The results of this analysis support the use of total 25-hydroxyvitamin D as a marker of vitamin D status, regardless of race or GC genotype.

Estimating glomerular filtration rate for the full age spectrum from serum creatinine and cystatin C

Background. We recently published and validated the new serum creatinine (Scr)‐based full‐age‐spectrum equation (FAScrea) for estimating the glomerular filtration rate (GFR) for healthy and kidney‐diseased subjects of all ages. The equation was based on the concept of normalized Scr and shows equivalent to superior prediction performance to the currently recommended equations for children, adolescents, adults and older adults. Methods. Based on an evaluation of the serum cystatin C (ScysC) distribution, we defined normalization constants for ScysC (QcysC = 0.82 mg/L for ages <70 years and QcysC = 0.95 mg/L for ages ≥70 years). By replacing Scr/Qcrea in the FAScrea equation with ScysC/QcysC, or with the average of both normalized biomarkers, we obtained new ScysC‐based (FAScysC) and combined Scr‐/ScysC‐based FAS equations (FAScombi). To validate the new FAScysC and FAScombi we collected data on measured GFR, Scr, ScysC, age, gender, height and weight from 11 different cohorts including n = 6132 unique white subjects (368 children, aged ≤18 years, 4295 adults and 1469 older adults, aged ≥70 years). Results. In children and adolescents, the new FAScysC equation showed significantly better performance [percentage of patients within 30% of mGFR (P30) = 86.1%] than the Caucasian Asian Paediatric Adult Cohort equation (P30 = 76.6%; P < 0.0001), or the ScysC‐based Schwartz equation (P30 = 68.8%; P < 0.0001) and the FAScombi equation outperformed all equations with P30 = 92.1% (P < 0.0001). In adults, the FAScysC equation (P30 = 82.6%) performed equally as well as the Chronic Kidney Disease Epidemiology Collaboration equation (CKD‐EPIcysC) (P30 = 80.4%) and the FAScombi equation (P30 = 89.9%) was also equal to the combined CKD‐EPI equation (P30 = 88.2%). In older adults, FAScysC was superior (P30 = 88.2%) to CKD‐EPIcysC (P30 = 84.4%; P < 0.0001) and the FAScombi equation (P30 = 91.2%) showed significantly higher performance than the combined CKD‐EPI equation (P30 = 85.6%) (P < 0.0001). Conclusion. The FAS equation is not only applicable to all ages, but also for all recommended renal biomarkers and their combinations.

Novel LC–MS/MS method for plasma vancomycin: Comparison with immunoassays and clinical impact

BACKGROUND Accurate quantification of vancomycin in plasma is important for adequate dose-adjustment. As literature suggests between-method differences, our first objective was to develop a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for total vancomycin in human plasma and to compare frequently used immunoassays with this method. Secondly, we investigated the clinical impact of between-method quantification differences. METHODS For LC-MS/MS, lithium heparin plasma was extracted by adding a precipitation reagent containing the internal standard (vancomycin-des-leucine). Analysis was performed on an Acquity TQD mass spectrometer equipped with an Acquity UPLC 2795 separations module. Our method was analytically validated and compared with four frequently used immunoassays from four different manufacturers. Vancomycin concentrations were clinically classified as toxic, therapeutic and sub-therapeutic. Clinical discordance was calculated using LC-MS/MS as a reference. RESULTS A novel LC-MS/MS method using protein precipitation as sole pretreatment and an analysis time of 5.0 min was developed. The assay had a total imprecision of 2.6-8.5%, a limit of quantification of 0.3 mg/L and an accuracy ranging from 101.4 to 111.2%. Using LC-MS/MS as reference, three immunoassays showed a mean proportional difference within 10% and one showed a substantial mean proportional difference of >20%. Clinical discordant interpretation of the obtained concentrations ranged from 6.1 to 22.2%. CONCLUSIONS We developed a novel LC-MS/MS method for rapid analysis of total vancomycin concentrations in human plasma. Correlation of the method with immunoassays showed a mean proportional difference >20% for one of the assays, causing discordant clinical interpretation in more than 1 out of 5 samples.

Need for Estradiol Assays With a Lower Functional Sensitivity in Clinical Studies Examining Postmenopausal Women Treated With Aromatase Inhibitors

TO THE EDITOR: In their recently published study, Folkerd et al found that in postmenopausal women with early estrogen receptor– positive breast cancer who are treated with aromatase inhibitors (AIs), suppressed levels of estradiol and estrone sulfate are related to body mass index. If confirmed, this observation is of great interest, not only because it would explain why treatment with an AI results in a better outcome compared with tamoxifen in lean patients but not in obese patients, but also because it might open the way toward individualized AI treatment by adapting the dose of AIs on the basis of the plasma estradiol and estrone sulfate concentration. Although the measurement of AIs using mass spectrometry is not so difficult, the measurement of estrogens, particularly estradiol, is quite challenging. As reported by the authors, most on-treatment values for estradiol were below 3 pmol/L, which was the “conventional sensitivity” limit of the assay used. Conventional sensitivity was defined by the authors as the 95% CI of the zero standard, which corresponds more or less to the limit of blank (LoB) as defined by the Clinical and Laboratory Standards Institute. The LoB is the highest apparent analyte concentration that is expected to be found when replicates of a sample containing no analyte are tested. The limit of detection is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which detection is feasible. The limit of quantitation, which is defined as the lowest concentration at which the analyte can not only be reliably detected but also measured with an acceptable degree of imprecision, is usually significantly higher (and cannot be lower) than the LoB and the limit of detection. Functional sensitivity is usually defined as the concentration that results in a coefficient of variation (CV) of 20%. The authors reported a between-run CV of 12% for a concentration of 37 pmol/L for their estradiol assay, suggesting that the functional sensitivity of their assay is significantly higher than 3 pmol/L, given that the CV increases exponentially around the limit of quantitation. Such a functional sensitivity is insufficient to allow a reliable quantification of estradiol concentrations in individual postmenopausal women who are treated with AIs. For estrone sulfate, there is a similar problem, although to a lesser extent. The functional sensitivity is most likely also significantly higher than 15 pmol/L, given that the authors reported a CV of 11% at 130 pmol/L, but in contrast to estradiol, a majority of on-treatment values were above the conventional sensitivity defined by the authors. Toexaminetherelationbetweenestrogenconcentration,bodymass index, and outcome, and to open the way toward individualized AI treatment, clinical studies should use estrogen assays with a better functional sensitivity, particularly for estradiol. Unfortunately, the only estradiol assays that currently have a functional sensitivity of less than 3 pmol/L are labor-intensivemassspectrometrymethodsthatrequireextensivesample pretreatmentincludingliquid/liquidextraction.Asimplemassspectrometrymethodnotrequiring liquid/liquidextraction,derivatization,or large volumes of serum (eg, 500 L) is therefore needed.

Factors Impacting Unbound Vancomycin Concentrations in Different Patient Populations

ABSTRACT The unbound drug hypothesis states that only unbound drug concentrations are active and available for clearance, and highly variable results regarding unbound vancomycin fractions have been reported in the literature. We have determined the unbound vancomycin fractions in four different patient groups by a liquid chromatography tandem mass spectrometry (LC-MS/MS) method and identified factors that modulate vancomycin binding. We have further developed and validated a prediction model to estimate unbound vancomycin concentrations. Vancomycin (unbound and total) concentrations were measured in 90 patients in four different hospital wards (hematology [n = 33 samples], intensive care unit [ICU] [n = 51], orthopedics [n = 44], and pediatrics [age range, 6 months to 14 years; n = 18]) by a validated LC-MS/MS method. Multiple linear mixed model analysis was performed to identify patient variables that were predictive of unbound vancomycin fractions and concentrations. The variables included in the model were patient age, ward, number of coadministered drugs with high protein binding, kidney function (estimated glomerular filtration rate [determined by Chronic Kidney Disease Epidemiology Collaboration formula]), alpha-1-acid glycoprotein, albumin, total bilirubin, IgA, IgM, urea, and total vancomycin concentrations. In the pediatric cohort, the median unbound vancomycin fraction was 81.3% (range, 61.9 to 95.9%), which was significantly higher (P < 0.01) than the unbound fraction found in the three adult patient cohorts (hematology, 60.6% [48.7 to 90.6%]; ICU, 61.7% [47.0 to 87.6%]; orthopedics, 56.4% [45.9 to 78.0%]). The strongest significant predictor of the unbound vancomycin concentration was the total drug concentration, completed by albumin in the pediatric cohort and albumin and IgA in the adult cohorts. Validation of our model was performed with data from 13 adult patients. A mean difference of 0.3 mg/liter (95% confidence interval [CI], −1.3 to 0.7 mg/liter; R2 = 0.99 [95% CI, 0.95 to 0.99]) between measured and calculated unbound vancomycin concentrations demonstrated that the predictive performance of our model was favorable. Unbound vancomycin fractions vary significantly between pediatric and adult patients. We developed a formula to estimate the unbound fraction derived from total vancomycin, albumin, and IgA concentrations in adult patients.

Enzymatic isotope dilution mass spectrometry (IDMS) traceable serum creatinine is preferable over Jaffe in neonates and young infants

*Corresponding author: Karel Allegaert, MD PhD, Neonatal Intensive Care Unit, University Hospitals Leuven, Herestraat 49, 3000 Leuven, Belgium, Phone: + 32 16 343850, Fax: + 32 16 343209, E-mail: karel.allegaert@uzleuven.be Steven Pauwels and Pieter Vermeersch: Clinical Department of Laboratory Medicine, University Hospitals Leuven, Belgium ; and Department of Cardiovascular Sciences, KU Leuven, Belgium Anne Smits and Kaat Cr e vecoeur: Neonatal Intensive Care Unit, University Hospitals Leuven, Leuven, Belgium ; and Department of Development and Regeneration, KU Leuven, Belgium John van den Anker: Division of Pediatric Clinical Pharmacology, Children ’ s National Medical Center, Washington, DC, USA ; Departments of Pediatrics, Pharmacology and Physiology, George Washington University School of Medicine and Health Sciences, Washington, DC, USA ; and Pediatric Intensive Care, Erasmus MC Sophia Children ’ s Hospital, Rotterdam, The Netherlands Djalila Mekahli: Department of Development and Regeneration, KU Leuven, Belgium ; and Department of Paediatric Nephrology, University Hospitals Leuven, Leuven, Belgium

Determination of human reference values for serum total 1,25-dihydroxyvitamin D using an extensively validated 2D ID-UPLC-MS/MS method.

BACKGROUND To assess a patients vitamin D status the precursor metabolite 25-hydroxyvitamin D can be determined. However, measurement of 1,25-dihydroxyvitamin D is required when disorders of 1a-hydroxylation, extrarenal 1a-hydroxylation, or vitamin D receptor defects are suspected. METHODS The aim of this study was to determine reference values for 1,25-dihydroxyvitamin D3 and D2 using a 2D ID-UPLC-MS/MS method. RESULTS The LC-MS/MS method, able to measure picomolar concentrations of both 1,25-dihydroxyvitamin D3 and D2 in human serum, was extensively validated. Intra-assay variations were <5% and 8.5% and <7.5% and 11%, for 1,25-dihydroxyvitamin D3 and D2, respectively, over the whole dynamic range (3.1-376 and 3.1-652pmol/L). Limit of quantitation was 3.4pmol/L for both compounds. Our method correlated well with a published LC-MS/MS method (r=0.87) and with the average 1,25-dihydroxyvitamin D3 results of the vitamin D External Quality Assessment Scheme (DEQAS) determined with LC-MS/MS (r=0.93). Reference ranges, determined in 96 plasma samples of healthy volunteers were 59-159pmol/L and <17pmol/L for respectively 1,25-dihydroxyvitamin D3 and D2. The female part of the reference group showed a statistically significant decrease of 1,25-dihydroxyvitamin D3 concentrations with age. The presence of significantly higher average 1,25-dihydroxyvitamin D3 levels in premenopausal women taking oral contraceptive pills compared to postmenopausal women suggests that this effect is estrogen-related, as estrogens lead to a higher vitamin D binding protein. CONCLUSIONS The major finding of the present study is a reference interval of 59-159pmol/L for 1,25-dihydroxyvitamin D3 determined with a highly sensitive and precise LC-MS/MS method.

Therapeutic drug monitoring in neonates

Therapeutic drug monitoring (TDM) aims to integrate drug measurement results into clinical decision making. The basic rules apply when using TDM in neonates (aminoglycosides, vancomycin, phenobarbital, digoxin), but additional factors should also be taken into account. First, due to both pharmacokinetic variability and non-pharmacokinetic factors, the correlation between dosage and concentration is poor in neonates, but can be overcome with the use of more complex, validated dosing regimens. Second, the time to reach steady state is prolonged, especially when no loading dose is used. Consequently, the timing of TDM sampling is important in this population. Third, the target concentration may be uncertain (vancomycin) or depend on specific factors (phenobarbital during whole body cooling). Finally, because of differences in matrix composition (eg, protein, bilirubin), assay-related inaccuracies may be different in neonates. We anticipate that complex validated dosing regimens, with subsequent TDM sampling and Bayesian forecasting, are the next step in tailoring pharmacotherapy to individual neonates.

Evaluation of the Sebia Minicap Flex Piercing capillary electrophoresis for hemoglobinopathy testing.

Capillary zone electrophoresis (CZE) at alkaline pH is one of the techniques used for hemoglobinopathy screening. In this study, an evaluation of the performance of a lower throughput CZE instrument, the Sebia Minicap Flex Piercing system, for this purpose is reported for the first time.