Neutrophil gelatinase-associated lipocalin levels during the first 48 hours of intensive care may indicate upcoming acute kidney injury

PurposeThe recognition of acute kidney injury (AKI) as early as possible is important in the intensive care unit. This study proposes that serum and urine levels of neutrophil gelatinase-associated lipocalin (NGAL) may be used for this purpose.MethodsOne hundred and seven critically ill adult patients with no previous renal failure were included. NGAL levels were measured during the first 48 hours after admission; NGAL levels were followed for 7 days and classified based on Risk, Injury, Failure, Loss, and End-Stage Renal Failure criteria.ResultsThe AKI incidence was 35.5%, and serum NGAL (sNGAL) and urinary NGAL (uNGAL) levels were higher in the AKI group. The area under the receiver operating characteristic curve was 0.76 (P < .001) for sNGAL and 0.75 (P < .001) for uNGAL. Seventy-one percent of AKI cases were observed within 48 hours, with 11 additional cases in the ensuing 7 days. The mean serum creatinine levels in the 11 patients were not different from non-AKI levels (P = .197), but the NGAL values were different, and the area under the receiver operating characteristic curve for sNGAL uNGAL was 1.00 (P = .014) and 0.93 (P = .02), respectively.ConclusionsMost AKI cases were diagnosed within the first 48 hours after admission, and NGAL was useful for predicting upcoming AKI.     

Automated two-site immunofluorescent assay for the measurement of serum chromogranin A

ObjectivesChromogranin A (CgA) is the best-characterized biological marker common to neuroendocrine tumours and is therefore recommended for their diagnosis. The measurement of serum CgA is of great importance for reaching an early diagnosis and thus reducing the delay before treatment is instigated. The Kryptor CgA assay is the first fully automated assay available. The aim of this study was to evaluate its analytical performance.Design and methodsThe imprecision and linearity of the Kryptor CgA assay were evaluated. This assay was compared with the Cis Bio CgA RIA assay in 78 serum samples. Its clinical utility was assessed in serum from 229 patients.ResultsThe study performed on imprecision of Kryptor measurements showed intra- and inter-run CVs ≤ 5%. The study of linearity showed a satisfactory recovery rate for CgA concentrations up to 1200 μg/L. The Kryptor and RIA assays agreed well on the basis of the cut-off values provided by the two manufacturers. The Bland and Altman plot of the values obtained (range: 20–5560 μg/L) provided a mean difference of − 10.1 μg/L (SD: 116). The clinical sensitivities of Kryptor CgA for diagnosis of pheochromocytoma and paraganglioma (n 20) and gastroenteropancreatic NETs (n 17) were respectively 100 and 94%.ConclusionsThe Kryptor assay for CgA shows reliable analytical and clinical characteristics and allows a fast delivery of results.     

Validating urinary measurement of beta-2-microglobulin with a Roche reagent kit designed for serum measurements

ObjectiveTo validate the use of a Roche serum beta-2-microglobulin (B2MG) kit for urinary B2MG measurements, and to establish reference limits for urinary B2MG/creatinine ratio from healthy individuals.Design and methodsThe Roche B2MG Tina-Quant serum kit was used to measure urinary B2MG immunoturbidimetrically.ResultsUsing human urine as a diluent, the B2MG method was linear from 73–2156 μg/L. The imprecision on a commercially available urine QC was 4.4% at a concentration of 380 μg/L. Limit of quantification at < 20% CV was 40 μg/L. Method comparison with Immulite 2000 (Siemens) yielded slope = 1.180 (95% CI 1.14–1.22), intercept = 11.5 (95% CI ? 3.6–26.6), SEE = 27.6 and r = 0.99 (n = 26) by the Deming regression analysis. The upper reference limit of B2MG/creatinine ratio determined from 195 healthy adults was 29 μg/mmol (97.5th centile).ConclusionsThe serum B2MG Tina Quant reagent kit is acceptable to measure urinary B2MG.     

Soluble transferrin receptor in urine,a new biomarker for IgA nephropathy and Henoch–Schönlein purpura nephritis

ObjectivesIgA nephropathy (IgAN) and Henoch–Schönlein purpura nephritis (HSPN) might represent different ends of a continuous spectrum of glomerular disease. In both conditions, upregulated soluble transferrin receptor (sTfR) might be excreted in urine, which could be a potential biomarker to monitor disease activity and therapeutic response.MethodsIn this pilot study, 132 Caucasian patients consulting the Nephrology Department at the Ghent University Hospital because of a glomerulopathy and 50 normal controls were included. Urinary sTfR concentrations were determined in concentrated urine using a newly developed latex-enhanced immunonephelometric assay.ResultsMedian urinary sTfR concentration was higher in patients with a primary glomerulopathy than in healthy subjects (p < 0.0001). More importantly, absolute median levels of urinary sTfR were markedly higher in patients with active IgAN or HSPN [10 μg/L, 95% confidence interval (CI): 6–18 μg/L] in comparison with those with other morphological types of glomerulopathy (2 μg/L, 95%CI: 1–4 μg/L) (p < 0.0001). A statistically significant difference in urinary sTfR concentration was observed between patients with active IgAN or HSPN and patients who had achieved partial or complete remission (p < 0.0001). Multiple regression analysis with urinary sTfR as dependent variable revealed that proteinuria was the main predictor of urinary sTfR concentration (r² = 0.52, p < 0.001).ConclusionDetermination of sTfR in urine is a new and sensitive method for a potential biomarker of IgAN and HSPN.     

Superiority of postmortem liver fentanyl concentrations over peripheral blood influenced by postmortem interval for determination of fentanyl toxicity

ObjectiveThe current study was undertaken to determine the relationship between postmortem (PM) peripheral blood (PB) and liver fentanyl concentrations and the role of measuring liver fentanyl concentrations in cause of death investigations in medical examiner cases in which fentanyl was identified.Design and methodsFB and liver tissue were routinely collected at autopsy from 4 Minnesota medical examiners' offices in 2010–2011. Samples were analyzed by gas chromatography–mass spectrometry (GC–MS).ResultsPB fentanyl ranged from < 2–15 μg/L in non-drug related deaths (n = 5), < 2–22 μg/L from mixed drug toxicity (n = 26) and 3.7–56 μg/L from fentanyl toxicity (n = 33). Liver fentanyl ranged from 11 to 104 μg/kg, 6 to 235 μg/kg, and 18 to 365 μg/kg, respectively. PB and liver fentanyl showed a modest correlation (r = 0.67). PM interval to the liver/blood ratio showed a decreasing ratio over increasing PM interval in cases from fentanyl and mixed drug toxicity. Liver fentanyl concentrations best define therapeutic use at < 23 μg/kg and fatal toxicity at > 56 μg/kg, without substantial overlap as found in blood fentanyl concentrations.ConclusionDiscriminatory liver fentanyl concentrations suggestive of therapeutic or toxic drug levels may better assist cause of death determination in cases of suspected fentanyl toxicity than postmortem PB concentrations. Peripheral blood fentanyl concentrations appear to undergo postmortem redistribution, associated with an increasing PM interval.     

Development of an enzymatic assay for sphingomyelin with rapid and automatable performances: Analysis in healthy subjects and coronary heart disease patients

BackgroundSphingomyelin (SM) is an important choline group-containing phospholipid and is considered to be an independent risk factor for coronary heart disease.MethodsWe have developed a specific enzymatic assay for SM measurement with rapid and automatable performances by using two-reagent system involving sphingomyelinase. We performed within-run and between-run precision, linearity test, detection limit, recovery test and interference to validate this assay. Then, we measured the serum SM concentration in 194 healthy subjects and 141 consecutive patients undergoing coronary angiography.ResultsThe within-run and between-run coefficients of variation for SM concentrations were 1.1–1.3% and 1.0–1.2%, respectively. Quantitative measurements to a lower limit of 30 μmol/L were shown to be possible. The recoveries of the exogenously added SM to the control samples were 98.7%–101.5%. No effect was observed after the addition of some interference materials. The mean ± SD of the serum SM concentration in the 194 healthy subjects was 553.3 ± 100.1 μmol/L. We found that the SM concentration was significantly higher among an acute coronary syndrome subjects than among the healthy subjects (P < 0.01) and that the serum SM concentrations were significantly correlated with the serum magnesium concentration.ConclusionsWe have developed a rapid and automatable enzymatic assay for SM that enables the automatic measurement of choline-containing phospholipids. This assay may be useful for various types of biochemical and clinical research.     

A next generation enzymatic magnesium assay on the Abbott ARCHITECT chemistry system meets performance goals based on biological variation

ObjectiveTo evaluate the performance of the Abbott ARCHITECT enzymatic assay for magnesium (3P68) in serum/plasma and urine against analytical goals based on biological variation.MethodsAnalytical performance was evaluated according to CLSI protocols. Precision was examined using commercial chemistry controls. Accuracy was assessed against NIST SRM 956c, electrolytes in human serum. Correlation with the arsenazo Mg assay (7D70) was completed using patient samples (plasma, N = 101; urine, N = 90). Common interferences were examined in pooled patient specimens with high and low magnesium concentrations.ResultsThe enzymatic Mg assay displayed imprecision of 1.7% at 0.72 mmol/L and 1.4% at 1.80 mmol/L (20 days, one calibration, one reagent lot). The linear range was verified between 0.18–7.0 mmol/L (plasma) and 0.01–10.69 mmol/L (urine). Results of the enzymatic assay (x) correlated well with the predicate assay (y) with the relationships y = 0.891x + 0.035, R = 0.967 (plasma) and y = 1.181x + 0.086, R = 0.997 (urine). Mean bias of the NIST SRM 956c samples was − 1.4%. This method showed minimal interference by hemoglobin (3 g/L as hemolysate), lipemia (20 g/L Intralipid), unconjugated bilirubin (531 μmol/L), and ascorbate (680 μmol/L).ConclusionsThe ARCHITECT Magnesium assay 3P68 achieved the desirable analytical quality specification of 4.8% for total allowable error. In comparison to the 7D70 assay, notable improvements are seen in precision, 30-day calibration stability, and minimal interference by hemolyzed and lipemic samples.     

Circulating sTWEAK improves the prediction of coronary artery disease

ObjectivesDecreased concentrations of circulating soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) were recently reported to be associated with atherosclerosis, but there are still no data concerning its predictive performance.Design and methodsThe current cross-sectional study investigates the potential of the novel atherosclerotic biomarker for the prediction of coronary artery disease (CAD). Serum sTWEAK was measured by ELISA in 76 CAD patients and 82 CAD-free subjects.ResultsSerum sTWEAK concentrations were significantly lower in the patients (534.5 ± 110.9 μg/L) than in the controls (688.1 ± 150.0 μg/L, p < 0.001), even after adjusting for different confounders (p < 0.001). The areas under ROC curves (AUC)s calculated for logistic regression models that included different known risk factors were significantly increased when sTWEAK was added to the corresponding model (p = 0.011–0.035).ConclusionsThe measurement of serum sTWEAK concentrations improves the prediction of CAD based on existing biomarkers.     

Association of serum ferritin with coronary artery disease

BackgroundPublished results regarding the association of serum ferritin with coronary artery disease (CAD) were conflicting, thus a case–control study and a meta-analysis were performed to assess the association between serum ferritin and CAD risk.MethodsA hospital-based case–control study was conducted with 258 CAD cases and 282 healthy controls. The restricted cubic spline (RCS) function with three knots was used to assess the concentration-risk association between serum ferritin and CAD risk. A meta-analysis was performed including 20 outcomes. Fixed or random effect pooled measure was selected on the basis of homogeneity test among studies.ResultsIn our case–control study, compared with serum ferritin concentrations less than 200 μg/L as the reference, the trend of CAD risk increased by 4.2% for every 50 μg/L increase in serum ferritin (OR = 1.042, 95% CI = 0.946–1.147). In the meta-analysis and after excluding articles that were the key contributors to between-study heterogeneity, the standardized mean difference (SMD) of serum ferritin was associated with increased CAD risk (FEM: SMD = 0.119, 95% CI = 0.073–0.165). And the concentration-risk meta-analysis suggested that, for every 50 μg/L increase of serum ferritin, the risk of CAD increases by 2.4% (OR = 1.024, 95% CI = 1.001–1.048).ConclusionThese findings indicate that serum ferritin is weakly positively associated with CAD risk. This risk needs to be confirmed by further studies.     

Verification of an immunoturbidimetric assay for heart-type fatty acid-binding protein (H-FABP) on a clinical chemistry platform and establishment of the upper reference limit

BackgroundHeart-type fatty acid-binding protein (H-FABP) is an early biomarker of cardiac injury. Randox Laboratories developed an immunoturbidimetric H-FABP assay for non-proprietary automated clinical chemistry analysers that could be useful in the emergency department. We verified the analytical performances claimed by Randox Laboratories on Roche Cobas 6000 clinical chemistry platform in use in our laboratory, and we defined our own 99th percentile upper reference limit for H-FABP.MethodsFor the verification of method performances, we used pools of spared patient samples from routine and two levels of quality control material, while samples for the reference value study were collected from 545 blood donors. Following CLSI guidelines we verified limit of blank (LOB), limit of detection (LOD), limit of quantitation (LOQ), repeatability and within-laboratory precision, trueness, linearity, and the stability of H-FABP in EDTA over 24 h.Results and discussionThe LOQ (3.19 μg/L) was verified with a CV% of 10.4. The precision was verified for the low (mean 5.88 μg/L, CV = 6.7%), the medium (mean 45.28 μg/L, CV = 3.0%), and the high concentration (mean 88.81 μg/L, CV = 4.0%). The trueness was verified as well as the linearity over the indicated measurement interval of 0.747–120 μg/L. The H-FABP in EDTA samples is stable throughout 24 h both at room temperature and at 4 °C. The H-FABP 99th percentile upper reference limit for all subjects (3.60 μg/L, 95% CI 3.51–3.77) is more appropriate than gender-specific ones that are not statistically different.     

Comparative evaluation of neonatal bilirubin using Radiometer whole blood co-oximetry and plasma bilirubin methods from Roche Diagnostics and Ortho Clinical Diagnostics

BackgroundClinically significant variation has been reported within and between plasma and whole blood total bilirubin methods used to identify neonates for whom clinical intervention for hyperbilirubinemia may be required.ObjectiveTo evaluate total bilirubin measurements between the Radiometer whole blood co-oximeter and plasma bilirubin methods from Roche Diagnostics and Ortho Clinical Diagnostics using neonatal specimens.MethodsTotal bilirubin levels were analyzed by whole blood co-oximetry (Radiometer® ABL90). Specimens were centrifuged and plasma analyzed for total bilirubin with a diazo method (Roche Cobas® C-601) and a reflectance spectrophotometric BuBc dry film method (Ortho Clinical Diagnostics VITROS® 350). Results were evaluated by regression, Bland-Altman comparisons and t-tests.ResultsThe patient correlation study yielded the following regression equations in μmol/L: a) Radiometer = 1.03 Roche – 3.5 μmol/L b) Radiometer = 0.98 Ortho – 5.7 μmol/L c) Roche = 0.97 Ortho – 2.4 μmol/L. The mean bias over the range of total bilirubin levels examined was − 1.0 μmol/L for the Radiometer versus the Roche (p ≤ 0.305); − 4.4 μmol/L for the Radiometer versus Ortho (p ≤ 0.005) and − 4.4 μmol/L for the Roche versus Ortho (p ≤ 0.002).ConclusionsWhole blood total bilirubin measurement using the Radiometer ABL90 blood gas analyzer provides accurate and precise results compared to the Roche plasma diazo method. Compared to the reflectance spectrophotometric method, results are precise and had a small but statistically significant bias of − 4.4 μmol/L.     

Interferences of homogentisic acid (HGA) on routine clinical chemistry assays in serum and urine and the implications for biochemical monitoring of patients with alkaptonuria

ObjectivesWe have assessed the effect of elevated concentrations of homogentisic acid (HGA) as in alkaptonuria (AKU), on a range of routine chemistry tests in serum and urine.Design and methodsHGA was added to pooled serum and a range of assays was analysed with Roche Modular chemistries. Effects on urine were assessed by diluting normal urine with urine from a patient with AKU, adding HGA to urine and after lowering output of urinary HGA with nitisinone treatment.ResultsSerum enzymatic creatinine showed 30% negative interference with 100 μmol/L HGA and > 50% at 400 μmol/L. Serum urate 100 to 480 μmol/L was reduced up to 20% at 100 and to 50% with 400 μmol/L HGA. Serum cholesterol between 3 and 11 mmol/L was reduced by 0.5 mmol/L with 400 μmol/L HGA. Urine enzymatic creatinine and urate with > 2 mmol/L HGA showed concentration dependent negative interference up to 80%. A positive interference in urine total protein by benzethonium turbidometric assay was observed, with 10 mmol/L HGA equivalent to 1 g/L protein. Jaffe creatinine, Na, K, Cl, Mg, Ca, phosphate, ALT, GGT, ALP activities and urea in serum and or urine were not affected by increases in HGA.ConclusionsTo avoid interferences by HGA in alkaptonuria concentration of HGA should be established before samples are assayed with peroxidase assays and benzethonium urine protein.     

Automated cerebrospinal fluid cell count — New reference ranges and evaluation of its clinical use in central nervous system infections

ObjectivesThe purposes of this study were to establish new reference ranges for leukocytes in the CSF and to examine if the separation of mononuclear cells into lymphocytes and monocytes could be used to differentiate between various CNS infections that present with a similar picture in manual CSF cell counts.Design and methodsThe automated cell counter Siemens ADVIA 2120i was used. For the reference range section, we analyzed CSF from 80 neurologically healthy volunteers. For the differential diagnosis section we analyzed cell counts and hospital records from 175 patients with CSF mononuclear pleocytosis.ResultsCorrelation was good between automated and manual leukocyte counts for samples with erythrocyte counts < 250 cells/μL. For the neurologically healthy volunteers studied in the reference range section, the 95th percentile was 3.0 cells/μL for lymphocytes, 1.0 cell/μL for monocytes and 1.0 cell/μL for granulocytes. In the differential diagnosis section, comparisons were done between the groups Lyme neuroborreliosis and viral CNS infection. There were no significant differences between these two groups regarding cell counts; neither for lymphocytes, median 58 cells/μL vs. 72 cells/μL (P = n.s.); nor for monocytes, median 13 cells/μL vs. 16 cells/μL (P = n.s.); nor for granulocytes, median 1 cell/μL vs. 2 cells/μL (P = n.s.)ConclusionsWe suggest new CSF cell count reference ranges of < 4 cells/μL for lymphocytes, < 3 cells/μL for monocytes and < 3 cells/μL for granulocytes. The separation of mononuclear cells into lymphocytes and monocytes did not facilitate the discrimination between Lyme neuroborreliosis and viral CNS infection.     

Immunoluminometric assay for quantification of peroxiredoxin 4 in human serum

BackgroundWe describe the validation of a novel assay for the measurement of peroxiredoxin 4 (Prx4), a potentially secreted antioxidant peroxidase, in human serum.MethodsA sandwich immunoluminometric assay (ILMA) was set up applying monoclonal antibodies against the amino-terminus of human Prx4. Prx4 levels have been determined in serum of healthy individuals and patients with sepsis and have been correlated to the clinically established sepsis marker procalcitonin (PCT).ResultsThe sandwich ILMA detected Prx4 in a range between 0.5 and 128 arbitrary (arb.) U/L and had a functional assay sensitivity of 0.51 arb. U/L. Serum Prx4 was stable for at least 72 h at 4 °C and 21 °C and circulated in a complex of about 330 kDa as characterized by size-exclusion chromatography. Patients with sepsis (n = 45; median, 7.7 arb. U/L) showed significantly higher Prx4 serum levels (P < 0.0001) than healthy controls (n = 274; median, 0.71 arb. U/L). Serum Prx4 was positively correlated to PCT (r = 0.63, P < 0.0001).ConclusionThe newly developed assay reliably detected Prx4 in human serum of healthy and critically ill subjects. Elevated Prx4 in serum of patients with various diseases might serve as a biomarker reflecting increased oxidative stress.     

Analytical performances of the Diazyme ADA assay on the Cobas® 6000 system

ObjectiveTo evaluate the analytical performance of the Diazyme ADA assay on the Cobas^® 6000 system for pleural fluid samples analysis.Design and methodsImprecision, linearity, calibration curve stability, interference, and correlation studies were completed.ResultsThe Diazyme ADA assay demonstrated excellent precision (CV < 4%) over the analytical measurement range (0.5-117 U/L). Bilirubin above 50 μmol/L and haemoglobin above 177 μmol/L interfered with the test, inducing a negative and a positive interference respectively. The Diazyme ADA assay correlated well with the Giusti method (r² = 0.93) but exhibited a negative bias (~ ?30%).ConclusionsThe Diazyme ADA assay on the Cobas^® 6000 system represents a rapid, accurate, precise and reliable method for determination of ADA activity in pleural fluid samples.     

Human chorionic gonadotropin and α-fetoprotein in cerebral spinal fluid: Method validation and retrospective review

ObjectiveMeasurement of human chorionic gonadotropin (hCG) and α-fetoprotein (AFP) in cerebrospinal fluid (CSF) can aid in the diagnosis of germ cell tumors (GCTs). Matrix effects can influence test results when alternative sample types are used, therefore, alternative sample types should always be validated before clinical use. Here we have validated the Advia® Centaur total hCG and AFP methods for use with CSF. We also performed a retrospective review of 5 years of CSF hCG and AFP measurements sent out from our institution.Design and methodsBoth hCG and AFP concentrations were measured using the ADVIA Centaur® total hCG or AFP assay.ResultsThe Centaur hCG and AFP assays, performed on CSF, had intra- and inter-assay imprecisions < 10.2% CV. The assays were linear over a dynamic range of 10–1000 IU/L for hCG and 10–1000 μg/L for AFP. Retrospective chart review confirmed that GCTs have a male predominance and are diagnosed most frequently in the second decade of life. The data also illustrate the importance of measuring both serum and CSF concentrations as CSF can be elevated in the absence of serum elevations.ConclusionsThe Centaur total hCG and AFP methods accurately quantify hCG and AFP in CSF.     

A novel enzyme immunoassay for the determination of phosphatidylserine-specific phospholipase A1 in human serum samples

BackgroundThe bioactive lipid lysophosphatidylserine (LPS) is postulated to induce important biological responses and to be produced by phosphatidylserine-specific phospholipase A₁ (PS-PLA₁). To evaluate the functional roles of LPS in vivo, a facile assay method for PS-PLA₁ has been awaited.MethodsRecombinant human PS-PLA₁ was produced using a baculovirus system, and anti-human PS-PLA₁ monoclonal antibodies were generated. Two clones were then selected for a 2-site immunoassay. The resulting PS-PLA₁ assay reagent was applied to a commercial automated immunoassay analyzer.ResultsSatisfactory results were obtained for the within-run and between-run precision, interference, detection limit, and linearity of this PS-PLA₁ assay. The mean ± SD of the serum PS-PLA₁ antigen concentration in the 191 healthy subjects was 33.8 ± 16.6 µg/l, and the central 95th percentile reference interval for the serum PS-PLA₁ antigen concentration was 13.8–74.1 µg/l. The concentration was significantly (p < 0.001) higher among men (13.8–80.6 µg/l) than among women (12.1–68.8 µg/l). We did not find a correlation between PS-PLA₁ and existing laboratory tests.ConclusionsThe present PS-PLA₁ assay method can be applied to clinical laboratory testing, and further studies are warranted to establish its clinical significance.     

Comparison of mycophenolic acid concentrations determined by a new PETINIA assay on the Dimension EXL analyzer and a HPLC-UV method

ObjectiveMycophenolic acid requires routine therapeutic drug monitoring. We evaluated the suitability of a new PETINIA (particle enhanced turbidimetric inhibition immunoassay) assay on the Dimension EXL analyzer for monitoring of mycophenolic acid by comparing values obtained by this assay with a HPLC-UV method.Design and methodsMycophenolic acid concentrations determined in sera of 60 organ transplant recipients using high performance liquid chromatography combined with ultraviolet detection (HPLC-UV, reference method) and the new immunoassay on the Dimension RxL analyzer.ResultsThe within and between run precision of the new PETINIA assay was < 10%. The assay was linear for a mycophenolic acid concentration up to 30 μg/mL. When mycophenolic acid concentrations in 60 transplant recipients obtained by the HPLC-UV (x-axis) method were compared with corresponding values obtained by the PETINIA assay (y-axis), the following regression equation was obtained: y = 1.1204 x + 0.0881 (r = 0.983, n = 60).ConclusionsIf PETINIA assay is used for therapeutic drug monitoring of mycophenolic acid, caution must be exercised in interpreting serum mycophenolic acid level due to observed positive bias.     

Validation of a rapid liquid chromatography–tandem mass spectrometric assay for the determination of octreotide plasma concentrations

ObjectivesThe aim of this study was to develop and validate a fast liquid chromatography–tandem mass spectrometric (LC–MSMS) assay to determine circulating concentrations of octreotide (a somatostatin analog used in acromegaly and neuroendocrine tumors) in patients' plasma samples.Design and methods500 μL of heparin–plasma was used to extract the drug on a cation exchanger SPE column, and the eluate was injected into a LC–MSMS system. Reversed phase chromatography was performed on a phenyl grafted column in isocratic mode. Octreotide and internal standard (triptorelin) were identified in positive electrospray ionization mode using ion transitions of m/z 512 > 120 and 890 > 249, respectively.ResultsThe assay was linear in the concentration range of 0.5–25 ng/mL, with intra- and inter-assay imprecisions of < 10.6% and < 12.2%, respectively. The limits of quantification and detection were 0.5 and 0.25 ng/mL. The recovery and ion suppression effect ranged between 79.7 and 84.5% and between − 8.1 and − 21.3%, respectively. A subcutaneous injection of 0.1 mg octreotide induced a time- and patient-dependent surge of peptide concentrations peaking at 2 h.ConclusionThis fast, sensitive, and selective method for quantification of plasma octreotide by LC–MSMS might be used to investigate the pharmacokinetic–pharmacodynamic relationship, with potential contribution to treatment optimization and therapeutic drug monitoring application.     

An audit of holotranscobalamin (“Active” B12) and methylmalonic acid assays for the assessment of vitamin B12 status: Application in a mixed patient population

BackgroundVitamin B₁₂ insufficiency/deficiency is common in mixed patient populations. However there is no single marker which can reliably diagnose B₁₂ insufficiency/deficiency. Elevated concentrations of methylmalonic acid (MMA) are considered the most representative marker of metabolic vitamin B₁₂ insufficiency, but poor assay availability limits clinical utility. Low concentrations of serum vitamin B₁₂ are often used to assess vitamin B₁₂ status but this approach generates a high rate of false negative results. Emerging evidence indicates that holotranscobalamin (holoTC) may be a more reliable indicator of vitamin B₁₂ status.Aims and methodsWe substituted serum vitamin B₁₂ measurement with holoTC, supported by MMA in patients referred for assessment of vitamin B₁₂ status. A service evaluation was undertaken of the pattern of MMA values obtained for patients with holoTC 25–50 pmol/L (an indeterminate result). MMA cut-offs of 280 and 360 nmol/L were applied for patients ≤ 65 or > 65 years respectively.ResultsA total of 4,175 consecutive patients were investigated and MMA was analysed for 19% of patients. The incidence of elevated MMA was 41% (holoTC, 25–29 pmol/L), 32% (30–34 pmol/L), 33% (35–39 pmol/L), 30% (40–44 pmol/L), and 26% (45–50 pmol/L).ConclusionsOur results indicate that in the clinical setting a holoTC between 25 and 50 pmol/L is a poor predictor for the concentration of MMA provided the goal is to identify patients with MMA values above the limits used in the present study. Further studies are needed to evaluate to what extent holoTC < 25 and > 50 pmol/L reflect circulatory MMA concentrations.