Joe M. El-Khoury

Progress of liquid chromatography-mass spectrometry in measurement of vitamin D metabolites and analogues

OBJECTIVE Clinical testing for vitamin D nutritional status has experienced tremendous growth in the past several years, driven by research results linking various diseases with low serum 25-hydroxyvitamin D [25(OH)D] levels. Meanwhile, interest in the pathophysiological mechanism elucidation and pharmaceutical applications requires measurement of vitamin D metabolites and analogues. Liquid chromatography-mass spectrometry (LC-MS) has been increasingly utilized in these applications. In this work, our objective was to critically review the progress of LC-MS application in measuring vitamin D metabolites and analogues in biological fluids. METHODS The LC-MS methods included were selected from those searchable in PubMed up to January 2010. RESULTS AND CONCLUSION LC-MS has unique advantages in measuring various vitamin D metabolites and analogues due to its flexibility, sensitivity, and specificity. Despite some controversies over serum 25(OH)D tests, LC-MS will be used for standardizing serum 25(OH)D assays using reference materials available from the National Institute of Standards and Technology.

Comparison of symmetric dimethylarginine with creatinine, cystatin C and their eGFR equations as markers of kidney function ☆

OBJECTIVES Symmetric dimethylarginine (SDMA) is a catabolic product of arginine-methylated proteins and is an emerging biomarker for kidney function. A limited number of studies in selected populations have shown good correlation between SDMA and a few known markers of glomerular filtration rate (GFR). However, a comprehensive comparison of SDMA with all existing serum endogenous markers in a population with varied kidney function and against measured GFR is lacking. The objective of this study was to compare the correlations of SDMA, creatinine, cystatin C and their eGFR equations against GFR measured by iothalamate clearance in an adult population with varied kidney function. DESIGN & METHODS Left-over serum and plasma specimens were collected from 40 adults with normal and reduced kidney function. GFR was measured using a radioactive iothalamate procedure. Creatinine and cystatin C were measured on Roche Cobas 8000. SDMA was measured by a published liquid chromatography-tandem mass spectrometry method. RESULTS SDMA correlated highly with measured GFR (r=-0.84), which was better than creatinine (r=-0.70) but equivalent to cystatin C (r=-0.86) and the eGFR equations [MDRD and CKD-EPI (separate and combined)]. CONCLUSIONS SDMA is a strong marker of kidney function and further studies are needed to establish an eGFR formula that includes it for widespread clinical use.

Stability of serum and plasma osmolality in common clinical laboratory storage conditions.

Serum or plasma osmolality is routinely measured in the clinical laboratory and is useful in the assessment of electrolyte and acid–base imbalances. Excessive water loss results in an elevated osmolality as shown in cases of diabetes insipidus and dehydration. Other cases such asmannitol therapy and toxin ingestionmay also lead to an elevated osmolality, while cases of inappropriate antidiuretic hormone can lead to decreased osmolality in blood. However, information on stability of serum or plasma for this measurement in typical clinical laboratory conditions is inconsistent in the literature. In one study using serum and plasma specimens collected from 25 young healthy volunteers, osmolality stability was tested over time at storage temperatures of 22 °C, 7 °C,−21 °C, or−78 °C [1]. Serum osmolality was found to be stable at room temperature for up to 3 days but not at day 14while refrigeration is not an acceptable storage condition. In another study using frozen serum specimens the osmolality did not change for up to 24 h, whether refrigerated or stored at 22 °C [2]. Major reference laboratories posted variable storage requirements for this test on their test directories. Most stated that the specimen was stable for 24 h at ambient temperature and recommended plasma or serum to be removed from cells then refrigerated at 4–8 °C or frozen if therewas a delay in analysis [3–6]. The objective of the current study was to assess the stability of osmolality in serum and plasma at various storage conditions including delay in whole blood centrifugation using patient samples that a typical clinical laboratory deals with on a daily basis. Osmolality was determined by a Model 3320 Osmometer (Advanced Instruments, Norwood, MA) that measures freezing point depression of serumorplasma. The extent of temperature decline below0 °C (the freezing point of water) is a function of the concentration of substances dissolved in serum or plasma. The manufacturer stated standard deviation was ±2 mOsm/kg H2O (1SD) for values between 0 and 400 mOsm/kg H2O. However, the observed standard deviation at our laboratory using quality control material, Liquid Unassayed Multiqual Level I from Bio-Rad (Hercules, CA) at 310 mOsm/kg H2O, was ±4 mOsm/kg H2O for 35 measurements over one month. Therefore we used the observed 2 SD, ±2.6%, as the criterion for significant change. Immediate centrifugation of whole blood samples is a recommendation bymost reference laboratories to ensure reliable osmolalitymeasurement in serum or plasma. To evaluate the impact of delay in whole

High-throughput measurement of 25-hydroxyvitamin D by LC-MS/MS with separation of the C3-epimer interference for pediatric populations.

BACKGROUND Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for the measurement of 25-hydroxyvitamin D3 (25OHD3) and 25-hydroxyvitamin D2 (25OHD2) in blood. However, the presence of 25OHD3 C3-epimer (3-epi-25OHD3) may cause interference and overestimation of the 25OHD3 level. We developed a rapid and simple assay for measurement of 25OHD that separates this interference and to investigate its impact on 25OHD3 measurement in adult and pediatric populations. METHODS Sample preparation consisted of protein precipitation followed by solid-phase extraction with an LC run time of 4.8 min. Method comparison with another LC-MS/MS method of a major reference laboratory that does not separate the C3-epimer interference was performed using adult (n=52) and pediatric (n=40) samples. RESULTS This method is free from significant ion suppression, carryover and interference. The assay can separate 25OHD3 from the 3-epi-25OHD3, and can measure 25OHD2 and 25OHD3 from 4.2 to 310.7 ng/ml and 5.2 to 311.1 ng/ml, respectively. Method comparison with a LC-MS method that does not separate the interference revealed biases of -0.15 and 4.54 for 25OHD3 measurement in adult and pediatric samples, respectively. CONCLUSION A fast and simple LC-MS/MS method for quantification of 25OHD3 and 25OHD2 without 3-epi-25OHD3 interference was developed. This assay is required for accurate quantitation of 25OHD3 in pediatric samples and suitable for routine use in a high volume clinical laboratory.

Measurement of GFR for children and an LC-MS/MS method for nonradioactive iothalamate.

Assessment of kidney function is important to many clinical decisions including diagnosis, prognosis and treatment of kidney diseases as well as management of toxic medications. Glomerular filtration rate (GFR) is the best overall index of kidney function in health and disease [1]. According to the KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease estimated GFR (eGFR) should be routinely monitored using either creatinine-based or cystatin C-based equations [2]. However, measured GFR (mGFR) using an exogenous substance is necessary when accurate assessment of kidney function is critical including evaluation for kidney donation and management of nephrotoxic medications. The European Association of Nuclear Medicine recommends measuring plasma disappearance of a radioactive glomerular tracer for accurate GFR measurement in children [3] though urine clearance is commonly utilized. Radioactive iothalamate has commonly been used as a tracer for GFRmeasurement. To avoid patient exposure to radioactive materials and to eliminate the cumbersome requirements for storage, administration, and disposal of radioactive materials analytical methods for measuring nonradioactive iothalamate by liquid chromatography–tandem mass spectrometry (LC–MS/MS) have been reported [4,5]. However, these methods involve complicated sample preparation procedures. We have developed and fully validated a simple, fast and interference-free LC–MS/MS method to measure non-radioactive iothalamate in both serum and urine.

Does Paricalcitol (Zemplar®) interfere with 1,25-dihydroxyvitamin D measurement by liquid chromatography-tandem mass spectrometry assays?

Calcitriol or 1,25-dihydroxyvitamin D (1,25OH2D) is the biologically active metabolite of vitamin D involved in calcium and phosphorus homeostasis and is one of the most challenging steroid hormones to measure in the clinical field [1]. It has low circulating physiological concentrations (36–144 pmol/l, [15–60 pg/ml]), exists in 2 biologically active forms (1,25OH2D2 and 1,25OH2D3), and is susceptible to interference from structurally similar andmore abundant vitamin Dmetabolites, such as 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) is considered the “gold standard” for the measurement of 1,25OH2D, because it provides the required sensitivity, selectivity, and equimolar recovery of 1,25OH2D2 and 1,25OH2D3 [1]. Measurement of 1,25OH2D in circulation is important for themanagement of patientswith renal failure, hypoparathyroidism, primary hyperparathyroidism, pseudohyperparathyroidism, hyperphosphatemia, hypomagnesemia, vitamin D-dependent rickets, granulomatous disease, lymphoma, and hypercalcemia [2]. Paricalcitol, the active ingredient in Zemplar® (Abbott Laboratories, Abbott Park, IL), is a synthetic analog of 1,25OH2D that is indicated for the prevention and treatment of secondary hyperparathyroidism in renal failure patients. This analog is similar in structure to 1,25OH2D2 with the exception of a missing methylene group at position 19, and it also shares the same chemical formula with 1,25OH2D3 (C27H44O3, 416.64 Da). Therefore, it is a potential interferent for 1,25OH2D3 analysis by LC–MS/MS, but not for 1,25OH2D2whichhas a differentmolecular weight (428.64 Da) and is therefore differentiated by the mass spectrometer. However, to the best of our knowledge there has been no report to examine the effects of this possible interferent on 1,25OH2D3 analysis by LC–MS/MS methods. In this study, we investigated the possible interference from paricalcitol on the measurement of 1,25OH2D3 using a published LC–MS/MS method [3]. There are 3 stages in the LC–MS/MS assays that may collectively reduce or eliminate paricalcitol interference to 1,25OH2D3 measurement. These are: a) MS/MS fragmentation, b) chromatographic separation and c) sample preparation. In the first phase of the study, we injected standards of paricalcitol and mixtures of paricalcitol with 1,25OH2D2 and 1,25OH2D3 in methanol:water (70:30) at varying concentrations to test if paricalcitol is separated from 1,25OH2D3 by the MS/MS fragmentation and chromatography. As shown in Fig. 1A, there was no baseline separation (0.17 min apart between the two peaks) between paricalcitol and 1,25OH2D3. However it required a much higher concentration of paricalcitol (4560 vs 98.4 pmol/l, [1900 vs 41 pg/ml]) to elicit a similar response to 1,25OH2D3. This implies that although paricalcitol produced a product ion with the same m/z that was monitored for 1,25OH2D3 (M–3H2O), the

Is the effect of hemolysis on plasma ammonia measurement overrated

To the Editor.—Accurate measurement of blood ammonia is useful in diagnosing inherited disorders of urea metabolism and Reye syndrome and in the differential diagnosis of encephalopathy. The most commonly used method in clinical labs is an enzymatic kinetic assay in which ammonia reacts with a-ketoglutarate and nicotinamide adenine dinucleotide phosphate (reduced form; NADPH) to form glutamate and NADP. The amount of ammonia is equivalent to the amount of NADPH oxidized, which can be measured photometrically. Hemolysis has been considered a significant interferent for this assay. The manufacturer’s product insert for the Roche (Indianapolis, Indiana) Cobas Integra 800 ammonia reagent (catalog No. 20766682) stated that there was ‘‘[n]o significant interference from hemolysis up to an H index [HI] of 100 (approximate hemoglobin concentration of 62 mmol/L or 100 mg/dL).’’ According to the manufacturer, this cutoff was determined relative to a decision level of ammonia at 85 mg/dL (A. M. Rose, PhD, written communication, October 2010). In an independent investigation, the HI cutoff was determined to be 200 at a higher ammonia concentration (228 mg/dL; Q. Meng, MD, PhD, written communication, August 2011) for Roche Cobas 6000. Previously, the approach used in both studies to investigate this effect has been challenged. In this study, our goals were to clarify this confusion in the literature and to establish practical cutoff values for clinical use. Several methods for investigating interference from hemolysis are described in the literature. The most commonly used method, osmotic shock, involves preparing a hemolysate by removing plasma from a whole blood sample after spinning, washing the cells with saline, mixing with water, and freezing overnight. The frozen hemolysate is thawed the next day, serially diluted to different levels with saline, and spiked into plasma sample aliquots to produce different degrees of hemolysis. This method was most likely used by Roche for establishing its HI cutoff. A less commonly used method, known as the shearing method, involves lysing whole blood aliquots by passing them through a needle by syringe aspiration for an increasing number of times with each subsequent sample to produce a range of hemolysis, eliminating the need for hemolysate preparation. These aliquots are then analyzed immediately. This method is believed to be a better reflection of the hemolytic process caused by actual blood collection. In a previous study, hemolysis generated by the osmotic shock method led to elevated measured plasma ammonia concentration, whereas hemolysis generated by the shearing method had little to no effect on plasma ammonia measurement. This difference was hypothesized to be a result of ammonia production during hemolysate preparation due to protein degradation over time. We investigated the effects of hemolysis on plasma ammonia measurement using the osmotic shock protocol versus the shearing method at 2 different ammonia concentrations on the Roche Integra 800. Using the osmotic shock method, the measured ammonia concentrations at 49 and 90 mg/dL showed greater than 10% change at HIs of 27.6 and 116.5, respectively. Using the shearing method, the measured ammonia at 58 and 100 mg/dL had greater than 10% change at HIs of 219.7 and 814.2, respectively. To determine if hemolysate aging resulted in ammonia production, an EDTA whole blood sample was treated per osmotic shock protocol to prepare the hemolysate, except that the hemolysate was split into 2 portions after the addition of water and before freezing. One portion was serially diluted, spiked into aliquots of a plasma pool, and then analyzed immediately, avoiding overnight storage of the hemolysate; the other was frozen overnight, then thawed, serially diluted, spiked into plasma samples, and analyzed. Consistent with the shearing method, the un-

Urinalysis findings and urinary kidney injury biomarker concentrations

IntroductionUrinary biomarkers of kidney injury are presumed to reflect renal tubular damage. However, their concentrations may be influenced by other factors, such as hematuria or pyuria. We sought to examine what non-injury related urinalysis factors are associated with urinary biomarker levels.MethodsWe examined 714 adults who underwent cardiac surgery in the TRIBE-AKI cohort that did not experience post-operative clinical AKI (patients with serum creatinine change of ≥ 20% were excluded). We examined the association between urinalysis findings and the pre- and first post-operative urinary concentrations of 4 urinary biomarkers: neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18 (IL-18), kidney injury molecule-1 (KIM-1), and liver fatty acid binding protein (L-FABP).ResultsThe presence of leukocyte esterase and nitrites on urinalysis was associated with increased urinary NGAL (R2 0.16, p < 0.001 and R2 0.07, p < 0.001, respectively) in pre-operative samples. Hematuria was associated with increased levels of all 4 biomarkers, with a much stronger association seen in post-operative samples (R2 between 0.02 and 0.21). Dipstick proteinuria concentrations correlated with levels of all 4 urinary biomarkers in pre-operative and post-operative samples (R2 between 0.113 and 0.194 in pre-operative and between 0.122 and 0.322 in post-operative samples). Adjusting the AUC of post-operative AKI for dipstick proteinuria lowered the AUC for all 4 biomarkers at the pre-operative time point and for 2 of the 4 biomarkers at the post-operative time point.ConclusionsSeveral factors available through urine dipstick testing are associated with increased urinary biomarker concentrations that are independent of clinical kidney injury. Future studies should explore the impact of these factors on the prognostic and diagnostic performance of these AKI biomarkers.

Erratic Serum Creatinine Concentrations in a Cardiac Patient

A 68-year-old man with a history of ischemic cardiomyopathy presented with decompensated heart failure and acute kidney injury. He was admitted to the coronary care unit for dopamine-assisted diuresis via a central line. Serum creatinine testing was performed using an enzymatic creatininase method (Roche cobas c701; Roche Diagnostics), revealing erratic creatinine concentrations …

60 Validation of a Novel High-Throughput Immunoassay for Measuring Symmetric Dimethylarginine as a Marker of Kidney Function

and increased bleeding (secondary to thrombocytopenia). Individuals with certain MDS subtypes are at substantially higher risk of developing acute myeloid leukemia (AML), and thus identification of people with MDS and tracking of this dysplastic population overtime could hypothetically have significant ramifications for clinical course. Due to the varied causes and manifestations of MDS, diagnosis is often difficult, and primarily relies on histologic based approaches. Given the recent advancements in multiparameter flow cytometry, we determined if MDS-related changes could be detected by 10-color flow cytometry. Previous work has demonstrated that MDS is associated with loss of blast heterogeneity typically seen in the bone marrow of normal individuals. Specifically, CD34+ blasts from normal individuals exhibit a pattern with at least three cell populations based on expression of CD13 and HLA-DR, a pattern that was lost in MDS patients. Additionally, a separate report demonstrated that MDS was correlated with a significant decrease in hematogones (precursor B cells) in the bone marrow. These processes, loss of blast heterogeneity and hematogones, may correlate with one another and be directly related; however this has not been formally assessed. Here, we describe an approach evaluating MDS by examining both blast heterogeneity as well as hematogones. As was previously reported, we found that there was variable loss of blast heterogeneity in bone marrow specimens from MDS patients compared with bone marrow specimens from normal/random patients. We also found that the relative fraction of hematogones was also decreased in individuals with MDS. Further, it appeared that the relative loss of blast heterogeneity might correlate with the relative degree of hematogone loss in the bone marrow. Results were correlated with histologic findings, including assessment of the blast population by CD34 immunohistochemical staining, cytogenetic findings, and additional molecular diagnostic findings. Thus, combining these two approaches may provide additional insight into MDS and be useful as an additional diagnostic modality for the initial diagnosis and therapeutic tracking of MDS as well as other myeloid neoplasms.