Cancer remains a leading cause of mortality and morbidity worldwide. In addition to organ failure, the most frequent reasons for admission of cancer patients to intensive care units (ICU) are: infections and sepsis. As critically ill, the complexity of the health situation of cancer patients renders the standard antimicrobial regimen more complex and even inadequate which results in increased mortality rates. This is due to pathophysiological changes in the volume of distribution, increased clearance, as well as to organ dysfunction. While in the former cases a decrease in drug efficacy is observed, the hallmark of the latter one is overdosing leading to increased toxicity at the expense of efficacy. Furthermore, an additional risk factor is the potential drug-drug interaction between antibiotics and antineoplastic agents. Therefore, therapeutic drug monitoring (TDM) is a necessity to improve the clinical outcome of antimicrobial therapy in cancer patients. To be applied in routine analysis the method used for TDM should be cheap, fast and highly accurate/sensitive. Furthermore, as ICU patients are treated with a cocktail of antibiotics the method has to cover the simultaneous analysis of antibiotics used as a first/second line of treatment. The aim of the current review is to briefly survey the pitfalls in the current antimicrobial therapy and the central role of TDM in dose adjustment and drug-drug interaction’s evaluation. A major section is dedicated to summarize the currently published analytical methods and to shed light on the difficulties and potential problems that can be encountered during method development.

Publicly Available December 19, 2016

Tackling serum folate test in European countries within the health technology assessment paradigm: request appropriateness, assays and health outcomes

Simona Ferraro, Andrea Panzeri, Mauro Panteghini

Page range: 1262-1275

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Abstract

Several authors have recently claimed an excess in serum folate test ordering, suggesting phasing out it from clinical use. According to studies performed in countries undergoing folic acid fortification policies, it is indeed no more cost-effective to test folate in the face of deficiency prevalence <1%. In this paper, we sought to evaluate request appropriateness, analytical issues, and cost-effectiveness of serum folate determination for clinical purposes in the European context, considering if evidence retrieved in fortified countries may be generalized. Studies performed in non-fortified countries have generally reported a suboptimal folate intake and suggest a remarkable prevalence of folate deficiency. Our internal data suggest that ~20%–25% of the subjects undergoing serum folate test are at risk for deficiency. However, a reliable evaluation of the risk for deficiency implies the knowledge of all issues related to the total testing process of folate measurement as well as the identification of the appropriate population in which to perform the test. The cost-effectiveness of the test is maximized when the request is oriented to subjects suggestive/at risk for deficiency, becoming low if the test is used as a screening tool or for monitoring of vitamin intake/supplementation. Because the individual folate status has a key role in ensuring normal development, physiologic growth, and maintenance of optimal health, the evaluation of its serum levels has to be retained in the clinical use in non-fortified countries, boosting for more appropriate request, and evidence from countries following fortification policies should be cautionary interpreted.

Genetics and Molecular Diagnostics

January 20, 2017

Genetic diagnosis of α₁-antitrypsin deficiency using DNA from buccal swab and serum samples

Irene Belmonte, Miriam Barrecheguren, Cristina Esquinas, Esther Rodríguez, Marc Miravitlles, Francisco Rodríguez-Frías

Page range: 1276-1283

Abstract

Background: α 1 -Antitrypsin deficiency (AATD) is associated with a high risk of developing lung and liver disease. Despite being one of the most common hereditary disorders worldwide, AATD remains under-diagnosed and prolonged delays in diagnosis are usual. The aim of this study was to validate the use of buccal swab samples and serum circulating DNA for the complete laboratory study of AATD. Methods: Sixteen buccal swab samples from previously characterized AATD patients were analyzed using an allele-specific genotyping assay and sequencing method. In addition, 19 patients were characterized by quantification, phenotyping and genotyping using only serum samples. Results: The 16 buccal swab samples were correctly characterized by genotyping. Definitive results were obtained in the 19 serum samples analyzed by quantification, phenotyping and genotyping, thereby performing the complete AATD diagnostic algorithm. Conclusions: Buccal swab samples may be useful to expand AATD screening programs and family studies. Genotyping using DNA from serum samples permits the application of the complete diagnostic algorithm without delay. These two methods will be useful for obtaining more in depth knowledge of the real prevalence of patients with AATD.

General Clinical Chemistry and Laboratory Medicine

February 21, 2017

Serum triglyceride measurements: the commutability of reference materials and the accuracy of results

Qinghui Meng, Weiyan Zhou, Chuanbao Zhang, Jie Zeng, Haijian Zhao, Tianjiao Zhang, Donghuan Wang, Jiangtao Zhang, Ying Yan, Wenxiang Chen

Page range: 1284-1290

Abstract

Background We aimed to evaluate the commutability of external quality assessment (EQA) materials, aqueous solutions, and commercial reference materials (calibrators and controls), and the accuracy of routine systems for serum triglyceride measurements. Methods According to the clinical and laboratory standards institute (CLSI) EP14-A3 protocol, we analyzed 43 fresh patient specimens and 32 processed materials including lyophilized samples, human serum pools, liquid reagents, swine sera and aqueous solutions by 14 routine methods (evaluated methods) and an isotope dilution liquid chromatography tandem mass spectrometry method (ID-LC/MS/MS) (comparative method). The accuracy of the routine method was evaluated by analyzing the absolute bias, relative bias, and the bias at three medical decision levels based on CLSI EP9-A3. Results Frozen serum samples and swine sera were commutable for all of the assays. The EQA/PT materials, commercial calibrators and control materials showed matrix effects differently on routine methods. The aqueous glycerol solutions were generally noncommutable for routine method. All except one routine analytical systems met the National Cholesterol Education Program (NCEP) recommended analytical performance guideline analytical quality criteria for total error. Conclusions Matrix effects and calibration biases existed in measurements of serum triglyceride. Continued efforts are needed to improve the accuracy and comparability of routine measurements.

February 3, 2017

Variant peptide detection utilizing mass spectrometry: laying the foundations for proteogenomic identification and validation

Lampros Dimitrakopoulos, Ioannis Prassas, Els M.J.J. Berns, John A. Foekens, Eleftherios P. Diamandis, George S. Charames

Page range: 1291-1304

Abstract

Background: Proteogenomics is an emerging field at the intersection of genomics and proteomics. Many variant peptides corresponding to single nucleotide variations (SNVs) are associated with specific diseases. The aim of this study was to demonstrate the feasibility of proteogenomic-based variant peptide detection in disease models and clinical specimens. Methods: We sought to detect p53 single amino acid variant (SAAV) peptides in breast cancer tumor samples that have been previously subjected to sequencing analysis. Initially, two cancer cell lines having a cellular tumor antigen p53 (TP53) mutation and one wild type for TP53 were analyzed by selected reaction monitoring (SRM) assays as controls. One pool of wild type and one pool of mutated for TP53 cytosolic extracts were assayed with a shotgun proteogenomic workflow. Furthermore, 18 individual samples having a mutation in TP53 were assayed by SRM. Results: Two mutant p53 peptides were successfully detected in two cancer cell lines as expected from their DNA sequence. Wild type p53 peptides were detected in both cytosolic pools, however, none of the mutant p53 peptides were identified. Mutations at the protein level were detected in two cytosolic extracts and whole tumor lysates from the same patients by SRM analysis. Six thousand and six hundred and twenty eight non-redundant proteins were identified in the two cytosolic pools, thus greatly improving a previously reported cytosolic proteome. Conclusions: In the current study we show the great potential of using proteogenomics for the direct identification of cancer-associated mutations in clinical samples and we discuss current limitations and future perspectives.

Publicly Available February 28, 2017

Evaluation of two fully automated immunoassay based tests for the measurement of 1α,25-dihydroxyvitamin D in human serum and comparison with LC-MS/MS

Katharina Spanaus, Arnold von Eckardstein

Page range: 1305-1314

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Abstract

Background: 1α,25-Dihydroxyvitamin D [1,25(OH) 2 vitD] is the bioactive form of vitamin D. Due to the very low concentrations of 1,25(OH) 2 vitD in the blood and its lipophilic character, measurement of this parameter is analytically challenging. Requiring preceding manual extraction steps before analysis, previous assays have been laborious. Methods: In the presented study, we evaluated the performance of two immunoassays from DiaSorin and from Immunodiagnostic Systems (IDS) which combine fully automated extraction and measurement of 1,25(OH) 2 vitD. Imprecision and linearity were verified according to Clinical and Laboratory Standards Institute EP15-A3 and EP6-A guidelines, respectively. Ninety-three patient serum samples sent to our institute for determination of 1,25(OH) 2 vitD, as well as 20 Vitamin D External Quality Assessment Scheme (DEQAS) samples, were used to evaluate correlation and agreement of 1,25(OH) 2 vitD measurements between the two immunoassays and with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results: Total imprecision was 5.2% or less for the DiaSorin test but reached 20.1% for the IDS iSYS test. 1,25(OH) 2 vitD concentrations measured with the DiaSorin assay showed a strong correlation with 1,25(OH) 2 vitD levels measured by LC-MS/MS and a good agreement with method specific means of DEQAS samples. By contrast, the IDS iSYS test overestimated 1,25(OH) 2 vitD concentrations in human serum, particularly at higher concentrations. Conclusions: Due to its high sensitivity, low imprecision, broad measurement range, and good agreement with 1,25(OH) 2 vitD concentrations measured by LC-MS/MS, the DiaSorin test is a valuable analytical option for the determination of 1,25(OH) 2 vitD.

Publicly Available January 11, 2017

Parallel diurnal fluctuation of testosterone, androstenedione, dehydroepiandrosterone and 17OHprogesterone as assessed in serum and saliva: validation of a novel liquid chromatography-tandem mass spectrometry method for salivary steroid profiling

Marco Mezzullo, Alessia Fazzini, Alessandra Gambineri, Guido Di Dalmazi, Roberta Mazza, Carla Pelusi, Valentina Vicennati, Renato Pasquali, Uberto Pagotto, Flaminia Fanelli

Page range: 1315-1323

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Abstract

Background: Salivary androgen testing represents a valuable source of biological information. However, the proper measurement of such low levels is challenging for direct immunoassays, lacking adequate accuracy. In the last few years, many conflicting findings reporting low correlation with the serum counterparts have hampered the clinical application of salivary androgen testing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) makes it possible to overcome previous analytical limits, providing new insights in endocrinology practice. Methods: Salivary testosterone (T), androstenedione (A), dehydroepiandrosterone (DHEA) and 17OHprogesterone (17OHP) were extracted from 500µL of saliva, separated in 9.5 min LC-gradient and detected by positive electrospray ionization – multiple reaction monitoring. The diurnal variation of salivary and serum androgens was described by a four paired collection protocol (8 am, 12 am, 4 pm and 8 pm) in 19 healthy subjects. Results: The assay allowed the quantitation of T, A, DHEA and 17OHP down to 3.40, 6.81, 271.0 and 23.7 pmol/L, respectively, with accuracy between 83.0 and 106.1% for all analytes. A parallel diurnal rhythm in saliva and serum was observed for all androgens, with values decreasing from the morning to the evening time points. Salivary androgen levels revealed a high linear correlation with serum counterparts in both sexes (T: R>0.85; A: R>0.90; DHEA: R>0.73 and 17OHP: R>0.89; p<0.0001 for all). Conclusions: Our LC-MS/MS method allowed a sensitive evaluation of androgen salivary levels and represents an optimal technique to explore the relevance of a comprehensive androgen profile as measured in saliva for the study of androgen secretion modulation and activity in physiologic and pathologic states.

Publicly Available January 12, 2017

Determination of oxycodone and its major metabolites noroxycodone and oxymorphone by ultra-high-performance liquid chromatography tandem mass spectrometry in plasma and urine: application to real cases

Flaminia Pantano, Stefano Brauneis, Alexandre Forneris, Roberta Pacifici, Enrico Marinelli, Chrystalla Kyriakou, Simona Pichini, Francesco Paolo Busardò

Page range: 1324-1331

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Abstract

Background: Oxycodone is a narcotic drug widely used to alleviate moderate and severe acute and chronic pain. Variability in analgesic efficacy could be explained by inter-subject variations in plasma concentrations of parent drug and its active metabolite, oxymorphone. To evaluate patient compliance and to set up therapeutic drug monitoring (TDM), an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay was developed and validated for the parent drug and its major metabolites noroxycodone and oxymorphone. Methods: Extraction of analytes from plasma and urine samples was obtained by simple liquid-liquid extraction. The chromatographic separation was achieved with a reversed phase column using a linear gradient elution with two solvents: acetic acid 1% in water and methanol. The separated analytes were detected with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode via positive electrospray ionization (ESI). Results: Separation of analytes was obtained in less than 5 min. Linear calibration curves for all the analytes under investigation in urine and plasma samples showed determination coefficients (r 2 ) equal or higher than 0.990. Mean absolute analytical recoveries were always above 86%. Intra- and inter-assay precision (measured as coefficient of variation, CV%) and accuracy (measured as % error) values were always better than 13%. Limit of detection at 0.06 and 0.15 ng/mL and limit of quantification at 0.2 and 0.5 ng/mL for plasma and urine samples, respectively, were adequate for the purpose of the present study. Conclusions: Rapid extraction, identification and quantification of oxycodone and its metabolites both in urine and plasma by UHPLC-MS/MS assay was tested for its feasibility in clinical samples and provided excellent results for rapid and effective drug testing in patients under oxycodone treatment.

Publicly Available December 19, 2016

Identification and quantitation of phosphatidylethanols in oral fluid by liquid chromatography-tandem mass spectrometry

Shahid Ullah, Anders Helander, Olof Beck

Page range: 1332-1339

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Abstract

Background: Phosphatidylethanols (PEth) are formed from phosphatidylcholines and ethanol and are used as a specific and sensitive alcohol biomarker. An analytical method for analysis of PEth in oral fluid based on high-performance liquid chromatography coupled to a quadrupole tandem mass spectrometer (LC-MS/MS) was developed and validated and applied on samples collected from patients undergoing alcohol detoxification. Methods: A 200-μL aliquot of oral fluid, collected using the Quantisal TM device, was extracted with chloroform/methanol containing internal standard and subjected to LC-MS/MS analysis of three selected PEth forms (16:0/16:0, 16:0/18:1, and 16:0/18:2). Chromatographic separation was achieved on a UPLC BEH phenyl column, using a mobile phase consisting of acetonitrile and water containing 10 mmol/L ammonium hydrogen carbonate with 0.1% ammonia. The MS instrument was operated in negative electrospray ionization and selected reaction monitoring mode. Results: The detection limit for PEth 16:0/16:0, 16:0/18:1, and 16:0/18:2 was ~0.1 ng/mL, and the extraction recoveries at 2.0 ng/mL were in the range of 99%–114%. Method linearity over a concentration range up to 200 ng/mL was ≥0.99. No significant deviation in results was observed in an analyte stability study of two different concentrations at two different temperatures over 3 months. In 35 oral fluid samples collected from patients undergoing alcohol detoxification, the highest concentration was observed for PEth 16:0/18:1 (Detected range, 0.51–55.3 ng/mL; mean, 8.5; median, 3.1). In addition, all three PEth forms were variably identified in a majority (63%) of the oral fluid samples. The PEth 16:0/18:1 values in oral fluid showed a weak positive correlation with the corresponding values in whole blood samples (r=0.50, p=0.026, n=20). Conclusions: The LC-MS/MS method could reliably detect and quantify PEth in oral fluid samples collected after alcohol exposure. The method was characterized by validation data with satisfactory recovery, sensitivity, accuracy, and imprecision, and applied for analysis of clinical samples. The results suggest that measurement of PEth in oral fluid can be used as a biomarker for alcohol consumption, and as such a non-invasive complement to analysis in blood. However, further studies are required to evaluate the test characteristics (e.g. sensitivity and half-life) in comparison with PEth in blood.

Publicly Available December 20, 2016

Relationship between plasma and salivary melatonin and cortisol investigated by LC-MS/MS

Martijn van Faassen, Rainer Bischoff, Ido P. Kema

Page range: 1340-1348

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Abstract

Background: Disturbance of the circadian rhythm has been associated with disease states, such as metabolic disorders, depression and cancer. Quantification of the circadian markers such as melatonin and cortisol critically depend on reliable and reproducible analytical methods. Previously, melatonin and cortisol were primarily analyzed separately, mainly using immunoassays. Methods: Here we describe the validation and application of a high-throughput liquid chromatography in combination with mass spectrometry (LC-MS/MS) method for the combined analysis of melatonin and cortisol in plasma and saliva. The LC-MS/MS method was validated according to international validation guidelines. We used this method to analyze total plasma, free plasma (as obtained by equilibrium dialysis) and saliva melatonin and cortisol in healthy adults. Results: Validation results for plasma and saliva melatonin and cortisol were well within the international validation criteria. We observed no difference between saliva collected by passive drooling or Salivette. Moreover, we noted a significant difference in saliva vs. free plasma melatonin. We observed on average 36% (95% CI: 4%–60%) higher salivary melatonin levels in comparison to free plasma melatonin, suggestive of local production of melatonin in the salivary glands. Conclusions: The novel outcome of this study is probably due to the high precision of our LC-MS/MS assay. These outcomes illustrate the added value of accurate and sensitive mass spectrometry based methods for the quantification of neuroendocrine biomarkers.

Publicly Available March 22, 2017

Paramagnetic micro-particles as a tool for rapid quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma by UHPLC-MS/MS

Martin H.J. Wiesen, Cornelia Blaich, Thomas Streichert, Guido Michels, Carsten Müller

Page range: 1349-1359

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Abstract

Background: Assessment of the anticoagulant activity of direct oral anticoagulants (DOACs) is justified in special clinical situations. Here, we evaluated two independent extraction methods and developed a multi-analyte ultra-high performance liquid chromatography tandem mass (UHPLC-MS/MS) method for the quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma. Methods: Routine extraction based on protein precipitation with acetonitrile and subsequent centrifugation was compared to sample clean-up using commercial paramagnetic micro-particles and subsequent magnetic depletion. Stable isotope-labeled analogs of all analytes were employed as internal standards. The method was validated according to international guidelines in terms of linearity, precision, trueness, sensitivity, recovery and matrix effects. The performances of both extraction methods were assessed in clinical samples obtained from patients treated with either apixaban or rivaroxaban. Additionally, we report on a patient with nonadherence to rivaroxaban treatment and fulminant pulmonary embolism. Results: The method was linear from 2 to 500 ng/mL for all analytes, and quantification of DOACs was established within a run time of 2.0 min. Based on MS/MS analyte responses, relative matrix effects were better controlled for dabigatran after extraction with paramagnetic micro-particles. Internal standards fully compensated for recovery and matrix effects in all assays, yielding equivalent results for both methods. Apixaban and rivaroxaban concentrations determined in clinical samples after extraction with both methods were in good agreement (R 2 =0.990). Conclusions: A rapid and accurate multi-component UHPLC-MS/MS method for the quantification of four DOACs in human plasma was established. Paramagnetic micro-particles appear suitable for clean-up of plasma samples for LC-MS/MS-based therapeutic drug monitoring purposes.

January 11, 2017

Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II)

Rosanna Squitti, Mariacristina Siotto, Emanuele Cassetta, Imane Ghafir El Idrissi, Nicola A. Colabufo

Page range: 1360-1367

Abstract

Background: Meta-analyses indicated the breakdown of copper homeostasis in the sporadic form of Alzheimer’s disease (AD), comprising copper decreases within the brain and copper increases in the blood and the pool not bound to ceruloplasmin (non-Cp Cu, also known in the literature as “free” copper). The calculated non-Cp Cu (Walshe’s) index has many limitations. Methods: A direct fluorescent method for non-Cp Cu detection has been developed and data are presented herein. The study included samples from 147 healthy subjects, 36 stable mild cognitive impairment (MCI) and 89 AD patients, who were tested for non-Cp Cu through the direct method, total serum copper, ceruloplasmin concentration and o -dianisidine ceruloplasmin activity. The indirect non-Cp Cu Walshe’s index was also calculated. Results: The direct method was linear (0.9–5.9 μM), precise (within-laboratory coefficient variation of 9.7% for low and 7.1% for high measurements), and had a good recovery. A reference interval (0–1.9 μM) was determined parametrically in 147 healthy controls (27–84 years old). The variation of non-Cp Cu was evaluated according to age and sex. Non-Cp Cu was 1.5 times higher in AD patients (regarding the upper value of the reference interval) than in healthy controls. Healthy, MCI and AD subjects were differentiated through the direct non-Cp Cu method [areas under the curve (AUC)=0.755]. Considering a 95% specificity and a 1.91 μmol/L cut-off, the sensitivity was 48.3% (confidence interval 95%: 38%–58%). The likelihood ratio (LR) was 9.94 for positive test results (LR+) and 0.54 for negative test result (LR−). Conclusions: The direct fluorescent test reliably and accurately measures non-Cp Cu, thereby determining the probability of having AD.

January 11, 2017

The serum concentrations of leptin and MCP-1 independently predict low back pain duration

Giuseppe Lippi, Concetta Dagostino, Ruggero Buonocore, Rosalia Aloe, Chiara Bonaguri, Guido Fanelli, Massimo Allegri

Page range: 1368-1374

Abstract

Background: Low back pain (LBP) is a very frequent condition, affecting most people at some point throughout their life. This cross-sectional study was aimed to investigate a selected panel of cytokines and inflammatory biomarkers in patients with or without LBP. Methods: The study population consisted of 104 patients diagnosed with LBP (52 non-persistent and 52 persistent) and 52 healthy subjects with no LBP. Blood samples were collected for assessment of adiponectin, leptin, monocyte chemoattractant protein-1 (MCP-1) and C reactive protein (CRP). The duration of LBP was categorized as “no pain”, “non-persistent LBP” and “persistent LBP”. Results: Higher values of CRP and lower concentrations of both leptin and MCP-1 were found in LBP patients compared to controls, whereas adiponectin did not differ among groups. MCP-1 was also lower in patients with non-persistent than in those with persistent LBP. Age, leptin (relative risk, 11.8; 95% CI, 3.9–35.8) and MCP-1 (relative risk, 2.7; 95% CI, 1.7–4.4) were independently associated with presence and duration of LBP. The combination of age, leptin and MCP-1 predicted 61% of the risk of LBP duration. The area under the curve of MCP-1 for distinguishing persistent from non-persistent LBP was 0.65 (95% CI, 0.54–0.76). Conclusions: Then results of our study suggest that leptin and MCP-1 may be promising biomarkers for diagnosis of acute LBP and its risk to become chronic.

January 28, 2017

Immunoassay screening in urine for synthetic cannabinoids – an evaluation of the diagnostic efficiency

Florian Franz, Verena Angerer, Hanna Jechle, Melanie Pegoro, Harald Ertl, Georg Weinfurtner, David Janele, Christian Schlögl, Matthias Friedl, Stefan Gerl, Reinhard Mielke, Ralf Zehnle, Matthias Wagner, Bjoern Moosmann, Volker Auwärter

Page range: 1375-1384

Abstract

Background: The abuse of synthetic cannabinoids (SCs) as presumed legal alternative to cannabis poses a great risk to public health. For economic reasons many laboratories use immunoassays (IAs) to screen for these substances in urine. However, the structural diversity and high potency of these designer drugs places high demands on IAs regarding cross-reactivity of the antibodies used and detection limits. Methods: Two retrospective studies were carried out in order to evaluate the capability of two homogenous enzyme IAs for the detection of currently prevalent SCs in authentic urine samples. Urine samples were analyzed utilizing a ‘JWH-018’ kit and a ‘UR-144’ kit. The IA results were confirmed by an up-to-date liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) screening method covering metabolites of 45 SCs. Results: The first study (n=549) showed an 8% prevalence of SCs use (LC-MS/MS analysis) among inpatients of forensic-psychiatric clinics, whereas all samples were tested negative by the IAs. In a second study (n=200) the combined application of both IAs led to a sensitivity of 2% and a diagnostic accuracy of 51% when applying the recommended IA cut-offs. Overall, 10 different currently prevalent SCs were detected in this population. The results can be explained by an insufficient cross-reactivity of the antibodies towards current SCs in combination with relatively high detection limits of the IAs. Conclusions: In light of the presented study data it is strongly recommended not to rely on the evaluated IA tests for SCs in clinical or forensic settings. For IA kits of other providers similar results can be expected.

Cancer Diagnostics

June 24, 2017

Study of kallikrein-related peptidase 6 (KLK6) and its complex with α₁-antitrypsin in biological fluids

Dimitrios Korbakis, Antoninus Soosaipillai, Eleftherios P. Diamandis

Page range: 1385-1396

Abstract

Background: Human kallikrein-related peptidase 6 (KLK6) is a member of the kallikrein family of serine proteases. KLK6 is synthesized as a preproenzyme, mainly in tissues of the central nervous system (CNS), and secreted as an inactive precursor. Serum KLK6 is a biomarker of unfavorable prognosis for ovarian cancer, but its sensitivity for early detection is relatively low. Differential glycosylation of KLK6 has been identified in ascites fluid obtained from ovarian cancer patients, suggesting the presence of unique KLK6 isoforms in biological samples. Methods: In the present study, we applied a two-step enrichment approach for KLK6 in ovarian cancer ascites, followed by mice immunization and production of monoclonal antibodies. Immunoaffinity techniques coupled to mass spectrometric methods were employed for hybridoma screening and target antigen identification. Results: We found that the main target of the newly-generated monoclonal antibodies target was the serine protease inhibitor α 1 -antitrypsin (A1AT). Additional experiments confirmed that A1AT is the main inhibitor of KLK6 in biological fluids. One new antibody (24ED138) was chosen to build a hybrid assay for the accurate quantification of the A1AT-KLK6 complex in biological samples. The aforementioned assay was evaluated with serum samples collected from patients with ovarian cancer (n=24) and normal donors (n=16) and showed slight improvement in sensitivity (~12%) compared to the standard in-house KLK6 assay. Conclusions: We conclude that KLK6 is present in biological fluids either as free form, or bound to A1AT, and the bound form performs better than total KLK6 as a biomarker of ovarian carcinoma.

Cardiovascular Diseases

Open Access April 20, 2017

A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide

Attila F. Torma, Kate Groves, Sabine Biesenbruch, Chris Mussell, Alan Reid, Steve Ellison, Rainer Cramer, Milena Quaglia

Page range: 1397-1406

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Abstract

Background: B-type natriuretic peptide (BNP) is a 32 amino acid cardiac hormone routinely measured by immunoassays to diagnose heart failure. While it is reported that immunoassay results can vary up to 45%, no attempt of standardization and/or harmonization through the development of certified reference materials (CRMs) or reference measurement procedures (RMPs) has yet been carried out. Methods: B-type natriuretic peptide primary calibrator was quantified traceably to the International System of Units (SI) by both amino acid analysis and tryptic digestion. A method for the stabilization of BNP in plasma followed by protein precipitation, solid phase extraction (SPE) and liquid chromatography (LC) mass spectrometry (MS) was then developed and validated for the quantification of BNP at clinically relevant concentrations (15–150 fmol/g). Results: The candidate reference method was applied to the quantification of BNP in a number of samples from the UK NEQAS Cardiac Markers Scheme to demonstrate its applicability to generate reference values and to preliminary evaluate the commutability of a potential CRM. The results from the reference method were consistently lower than the immunoassay results and discrepancy between the immunoassays was observed confirming previous data. Conclusions: The application of the liquid chromatography-mass spectrometry (LC-MS) method to the UK NEQAS samples and the correlation of the results with the immunoassay results shows the potential of the method to support external quality assessment schemes, to improve understanding of the bias of the assays and to establish RMPs for BNP measurements. Furthermore, the method has the potential to be multiplexed for monitoring circulating truncated forms of BNP.

January 20, 2017

The natriuretic peptide MR-proANP predicts all-cause mortality and adverse outcome in community patients: a 10-year follow-up study

Jonas Odermatt, Lara Hersberger, Rebekka Bolliger, Lena Graedel, Mirjam Christ-Crain, Matthias Briel, Heiner C. Bucher, Beat Mueller, Philipp Schuetz

Page range: 1407-1416

Abstract

Background: The precursor peptide of atrial natriuretic peptide (MR-proANP) has a physiological role in fluid homeostasis and is associated with mortality and adverse clinical outcomes in heart failure patients. Little is known about the prognostic potential of this peptide for long-term mortality prediction in community-dwelling patients. We evaluated associations of MR-proANP levels with 10-year all-cause mortality in patients visiting their general practitioner for a respiratory tract infection. Methods: In this post-hoc analysis including 359 patients (78.5%) of the original trial, we calculated cox regression models and area under the receiver operating characteristic curve (AUC) to assess associations of MR-proANP blood levels with mortality and adverse outcome including death, pulmonary embolism, and major adverse cardiac or cerebrovascular events. Results: After a median follow-up of 10.0 years, 9.8% of included patients died. Median admission MR-proANP levels were significantly elevated in non-survivors compared to survivors (80.5 pmol/L, IQR 58.6–126.0; vs. 45.6 pmol/L, IQR 34.2–68.3; p<0.001) and associated with 10-year all-cause mortality (age-adjusted HR 2.0 [95% CI 1.3–3.1, p=0.002]; AUC 0.79). Results were similar for day 7 blood levels and also for the prediction of other adverse outcomes. Conclusions: Increased MR-proANP levels were associated with 10-year all-cause mortality and adverse clinical outcome in a sample of community-dwelling patients. If diagnosis-specific cut-offs are confirmed in future studies, this marker may help to direct preventive measures in primary care.

January 18, 2017

CASZ1 loss-of-function mutation contributes to familial dilated cardiomyopathy

Xing-Biao Qiu, Xin-Kai Qu, Ruo-Gu Li, Hua Liu, Ying-Jia Xu, Min Zhang, Hong-Yu Shi, Xu-Min Hou, Xu Liu, Fang Yuan, Yu-Min Sun, Jun Wang, Ri-Tai Huang, Song Xue, Yi-Qing Yang

Page range: 1417-1425

Abstract

Background: The zinc finger transcription factor CASZ1 plays a key role in cardiac development and postnatal adaptation, and in mice, deletion of the CASZ1 gene leads to dilated cardiomyopathy (DCM). However, in humans whether genetically defective CASZ1 contributes to DCM remains unclear. Methods: The coding exons and splicing junction sites of the CASZ1 gene were sequenced in 138 unrelated patients with idiopathic DCM. The available family members of the index patient harboring an identified CASZ1 mutation and 200 unrelated, ethnically matched healthy individuals used as controls were genotyped for CASZ1 . The functional characteristics of the mutant CASZ1 were analyzed in contrast to its wild-type counterpart using a luciferase reporter assay system. Results: A novel heterozygous CASZ1 mutation, p.K351X, was identified in an index patient with DCM. Genetic analysis of the mutation carrier’s family showed that the mutation co-segregated with DCM, which was transmitted in an autosomal dominant pattern with complete penetrance. The nonsense mutation, which was absent in 400 referential chromosomes, altered the amino acid that was highly conserved evolutionarily. Biological investigations revealed that the mutant CASZ1 had no transcriptional activity. Conclusions: The current study reveals CASZ1 as a new gene responsible for human DCM, which provides novel mechanistic insight and potential therapeutic target for CASZ1-associated DCM, implying potential implications in improved prophylactic and therapeutic strategies for DCM, the most common type of primary myocardial disease.

Diabetes

Open Access April 22, 2017

Evaluating new HbA_1c methods for adoption by the IFCC and NGSP reference networks using international quality targets

Erna Lenters-Westra, Emma English

Page range: 1426-1434

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Abstract

Background: As a reference laboratory for HbA 1c , it is essential to have accurate and precise HbA 1c methods covering a range of measurement principles. We report an evaluation of the Abbott Enzymatic (Architect c4000), Roche Gen.3 HbA 1c (Cobas c513) and Tosoh G11 using different quality targets. Methods: The effect of hemoglobin variants, other potential interferences and the performance in comparison to both the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and the National Glycohemoglobin Standardization Program (NGSP) reference systems was assessed using certified evaluation protocols. Results: Each of the evaluated HbA 1c methods had CVs <3% in SI units and <2% in NGSP units at 46 mmol/mol (6.4%) and 72 mmol/mol (8.7%) and passed the NGSP criteria when compared with six secondary reference measurement procedures (SRMPs). Sigma was 8.6 for Abbott Enzymatic, 3.3 for Roche Cobas c513 and 6.9 for Tosoh G11. No clinically significant interference was detected for the common Hb variants for the three methods. Conclusions: All three methods performed well and are suitable for clinical application in the analysis of HbA 1c . Partly based on the result of this study, the Abbott Enzymatic method on the Architect c4000 and the Roche Gen.3 HbA 1c on the Cobas c513 are now official, certified IFCC and NGSP SRMPs in the IFCC and NGSP networks. Sigma metrics quality criteria presented in a graph distinguish between good and excellent performance.

Infectious Diseases

January 11, 2017

Analytical and diagnostic performance of two automated fecal calprotectin immunoassays for detection of inflammatory bowel disease

Maxime M.W. De Sloovere, Dieter De Smet, Filip J. Baert, Johan Debrabandere, Hilde J.M. Vanpoucke

Page range: 1435-1446

Abstract

Background: We evaluated the (pre-)analytical and diagnostic performance of two automated fecal calprotectin (FC) immunoassays, Liaison ® Calprotectin (Diasorin) on Liaison ® XL and fCAL™ turbo (Bühlmann laboratories AG) on Cobas C501 (Roche Diagnostics), and compared it with our established Bühlmann ELISA method. Methods: Our study comprised 229 consecutive patients with clinical suspicion of inflammatory bowel disease (IBD). Results: All assay related stool extraction procedures showed excellent correlation with the established method, but the new stool extraction devices tend to give higher results as compared with stool weight methods. Both automated assays demonstrated good performance in terms of precision (CV t ≤8.1%), accuracy (bias≤6.7%) and total error (≤16.4%). Method comparison with established enzyme linked immunosorbent assay (ELISA) showed good correlation (r s >0.925), but regression analysis showed significant proportional differences. Diagnostic performance characteristics with regard to diagnosis of IBD were good and in line with other reports. In addition, we were able to show that optimization of manufacturer’s cut-off and moreover, the introduction of a gray zone resulted in a significant increase of post-test probability. Conclusions: In conclusion, the newly developed stool extraction device protocols showed acceptable and comparable performance to the stool weight method. Overall, the automated Liaison ® Calprotectin and fCAL™ turbo assay showed good analytical and diagnostic performance for detection of IBD.

Letters to the Editor

March 2, 2017

Is fasting necessary for lipid profile determinations? Some considerations from the perspective of the clinical laboratory

Maria Carmen Lorenzo Lozano, Ana Cosmen Sanchez, Pedro María Belinchón Torres, Santiago Prieto Menchero, Daniel Pineda-Tenor

Page range: e187-e188

February 23, 2017

Precision of nonfasting lipid profiles should focus on clinical relevance rather than necessarily obtaining the least variation

Børge G. Nordestgaard, Michel R. Langlois

Page range: e189-e190

January 18, 2017

Triglyceride concentrations should be measured after elimination of free glycerol to exclude interindividual variations due to adiposity and fasting status

Terumichi Nakagawa, Satoshi Hirayama, Toshiyuki Watanabe, Mamoru Yokomura, Masaomi Kohno, Taiki Sato, Hideaki Bujo, Asako Sato, Mitsuru Murata, Takashi Miida, for the JSCC Kanto Study Group

Page range: e191-e194

January 20, 2017

Estimation of the reference interval for serum folate measured with assays traceable to the WHO International Standard

Simona Ferraro, Andrea Panzeri, Simona Borille, Dominika Szoke, Mauro Panteghini

Page range: e195-e196

January 11, 2017

Implausible elevation of peripheral thyroid hormones during therapy with a protein supplement

Igor A. Harsch, Peter C. Konturek, Klas Böer, Mike Reinhöfer

Page range: e197-e198

January 18, 2017

Interference in Na⁺ measurements on the Siemens RAPIDPoint^® 500 after nortriptyline intoxication: a case report

Eline A.E. van der Hagen, Kees C.J. Smeekens, Nils E. van ’t Veer, Kristel J.M. Boonen, Antonius A.M. Ermens

Page range: e199-e201

January 11, 2017

Usefulness of maternal red cell antibodies to predict hemolytic disease of the fetus and newborn and significant neonatal hyperbilirubinemia: a retrospective study

Bart Peeters, Inge Geerts, Anne-Mie Badts, Veroniek Saegeman, Jan Moerman

Page range: e202-e205

March 17, 2017

Improvement of the Sandell-Kolthoff reaction method (ammonium persulfate digestion) for the determination of iodine in urine samples

Ana Machado, Lurdes Lima, Raquel B.R. Mesquita, Adriano A. Bordalo

Page range: e206-e208

January 11, 2017

Clinical use of targeted high-throughput whole-genome sequencing for a dengue virus variant

Chun Kiat Lee, Cui Wen Chua, Lily Chiu, Evelyn Siew-Chuan Koay

Page range: e209-e212

About this journal

Objective
Clinical Chemistry and Laboratory Medicine (CCLM) publishes articles on novel teaching and training methods applicable to laboratory medicine. It is focused on basic and applied research and cutting-edge clinical laboratory medicine. CCLM is one of the leading journals in the field, with an impact factor over 8. All contributions submitted for publication in CCLM are single-blind peer reviewed by at least two experts in the field. CCLM is led by a multi-institutional editorial board. It is issued monthly, both in print and electronically. Letters to the Editor and Congress Abstracts are published online only.

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Topics

clinical biochemistry
clinical genomics and molecular biology
clinical haematology and coagulation
clinical immunology and autoimmunity
clinical microbiology
drug monitoring and analysis
evaluation of diagnostic biomarkers
disease-oriented topics (cardiovascular disease, cancer diagnostics, diabetes)
new reagents, instrumentation and technologies
new methodologies
reference materials and methods
reference values and decision limits
quality and safety in laboratory medicine
translational laboratory medicine
clinical metrology

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Research Articles, Reviews, Mini Reviews and Opinion Papers on all aspects of clinical chemistry and laboratory medicine, Guidelines and Recommendations, Point/Counterpoint Articles, Letters to the Editor, Editorials

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Volume 55 Issue 9

Issue of Clinical Chemistry and Laboratory Medicine (CCLM)

Frontmatter

Mass spectrometry or immunoassay: est modus in rebus

The use of liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring of antibiotics in cancer patients

Abstract

Tackling serum folate test in European countries within the health technology assessment paradigm: request appropriateness, assays and health outcomes

Abstract

Genetic diagnosis of α1-antitrypsin deficiency using DNA from buccal swab and serum samples

Abstract

Serum triglyceride measurements: the commutability of reference materials and the accuracy of results

Abstract

Variant peptide detection utilizing mass spectrometry: laying the foundations for proteogenomic identification and validation

Abstract

Evaluation of two fully automated immunoassay based tests for the measurement of 1α,25-dihydroxyvitamin D in human serum and comparison with LC-MS/MS

Abstract

Parallel diurnal fluctuation of testosterone, androstenedione, dehydroepiandrosterone and 17OHprogesterone as assessed in serum and saliva: validation of a novel liquid chromatography-tandem mass spectrometry method for salivary steroid profiling

Abstract

Determination of oxycodone and its major metabolites noroxycodone and oxymorphone by ultra-high-performance liquid chromatography tandem mass spectrometry in plasma and urine: application to real cases

Abstract

Identification and quantitation of phosphatidylethanols in oral fluid by liquid chromatography-tandem mass spectrometry

Abstract

Relationship between plasma and salivary melatonin and cortisol investigated by LC-MS/MS

Abstract

Paramagnetic micro-particles as a tool for rapid quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma by UHPLC-MS/MS

Abstract

Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II)

Abstract

The serum concentrations of leptin and MCP-1 independently predict low back pain duration

Abstract

Immunoassay screening in urine for synthetic cannabinoids – an evaluation of the diagnostic efficiency

Abstract

Study of kallikrein-related peptidase 6 (KLK6) and its complex with α1-antitrypsin in biological fluids

Abstract

A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide

Abstract

The natriuretic peptide MR-proANP predicts all-cause mortality and adverse outcome in community patients: a 10-year follow-up study

Abstract

CASZ1 loss-of-function mutation contributes to familial dilated cardiomyopathy

Abstract

Evaluating new HbA1c methods for adoption by the IFCC and NGSP reference networks using international quality targets

Abstract

Analytical and diagnostic performance of two automated fecal calprotectin immunoassays for detection of inflammatory bowel disease

Abstract

Is fasting necessary for lipid profile determinations? Some considerations from the perspective of the clinical laboratory

Precision of nonfasting lipid profiles should focus on clinical relevance rather than necessarily obtaining the least variation

Triglyceride concentrations should be measured after elimination of free glycerol to exclude interindividual variations due to adiposity and fasting status

Estimation of the reference interval for serum folate measured with assays traceable to the WHO International Standard

Implausible elevation of peripheral thyroid hormones during therapy with a protein supplement

Interference in Na+ measurements on the Siemens RAPIDPoint® 500 after nortriptyline intoxication: a case report

Usefulness of maternal red cell antibodies to predict hemolytic disease of the fetus and newborn and significant neonatal hyperbilirubinemia: a retrospective study

Improvement of the Sandell-Kolthoff reaction method (ammonium persulfate digestion) for the determination of iodine in urine samples

Clinical use of targeted high-throughput whole-genome sequencing for a dengue virus variant

Genetic diagnosis of α₁-antitrypsin deficiency using DNA from buccal swab and serum samples

Study of kallikrein-related peptidase 6 (KLK6) and its complex with α₁-antitrypsin in biological fluids

Evaluating new HbA_1c methods for adoption by the IFCC and NGSP reference networks using international quality targets

Interference in Na⁺ measurements on the Siemens RAPIDPoint^® 500 after nortriptyline intoxication: a case report