Volume 49, Issue 6

Mass spectrometry has been widely used in the field of diabetes. The development of new ionization methods and the effective coupling of mass spectrometry with liquid chromatography have enabled the protein modifications due to glycation processes to be investigated. Matrix assisted laser desorption/ionization mass spectrometry (MALDI/MS) has been used to evaluate the degree of glycation of specific plasma proteins. In contrast, the classic proteomic approach has been used to identify glycation sites and condensed sugar modifications. The same methods have been applied to studies on urinary protein profiles, enabling changes due to the development of long-term, diabetes-induced nephropathy to be identified. Published studies demonstrate that mass spectrometry is an important analytical tool for monitoring diabetes, capable of providing physicians with a new, more complete view of the physiopathological changes occurring as the disease develops.

Hepatocellular carcinomas (HCC) are the fifth most common tumor type and the third most common cause of cancer-related death worldwide. Some tumor markers for HCC, such as α-fetoprotein and des-γ-carboxyprothrombin, are used clinically. Recent advances in proteomics and glyco-proteomics might provide various types of novel tumor markers for HCC. While the clinical availability of these tumor markers is important, the molecular mechanisms underlying the production of tumor markers requires further clarification. Our group has investigated the glycobiology of tumor markers. In this review, we describe the impact of novel HCC markers and their possible implications for clinical use.

The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor member of the low-density lipoprotein (LDL)-receptor family. As LRP1 plays an important role in endocytosis and regulation of signalling pathways, it is implicated in a number of physiologic processes, including the regulation of lipid metabolism, the proliferation of vascular smooth muscle cells and in neuro-development. More recently, LRP1 has been implicated in the catabolism of factor VIII and regulation of its plasma concentrations. The pathophysiology of the role of LRP1 in hemostasis will be summarized in this review.

Predicting which meningiomas will recur and which will not is clinically important, and still represents a major clinical challenge. A number of different molecular, genetic, and/or biochemical markers involved in cell proliferation, invasion, angiogenesis, and cell transformation have been investigated in attempts to predict the risk of post-surgical meningioma recurrence. In this short review we emphasize what has actually been accomplished in this area. Finally, we highlight the potential of S100 serum protein concentrations as a prognostic factor predicting meningioma recurrence. We conclude that serum concentrations of S100 group proteins may prove to be useful as prognostic markers and suggest further pros-pective trials be done.

The recent campaign for standardization of creatinine measurements has been promoted to allow the widespread use of formulas for estimating the glomerular filtration rate (GFR). However, studies on trueness verification and measurement interferences still show disappointing interassay variation of serum creatinine results. Creatinine recalibration has major clinical consequences. In particular, in pediatrics where reference ranges for serum and plasma creatinine are low, calculation of the GFR is problematic when based on alkaline picrate methods because of method non-specificity and the lack of appropriate GFR estimating formulas. Therefore, enzymatic creatinine assays are preferred. In the near future, cystatin C might offer an interesting alternative for GFR estimation. For the calculation of drug doses, the Modification of Diet in Renal Disease study formula generally offers reliable data. However, attention has to be paid to the elderly. Also, the calculation of the Model for End-Stage Liver Disease score, which is used to prioritize patients for liver transplantation, may significantly be influenced by recalibration of creatinine assays. Creatinine restandardization may also affect the current guidelines for referral of chronic kidney disease patients to nephrologists.

Background: Celiac disease (CD) is an immune-mediated disorder of the small bowel in which gluten and related cereal ingestion leads to chronic inflammation and damage to the small bowel mucosa in genetically susceptible individuals. The presence of IgA type tissue-transglutaminase antibodies (IgA-anti-tTG) in individuals without IgA deficiency is a highly-specific indicator of CD. We compared the accuracy of IgA-anti-tTG detection using a fully automated fluoroenzyme immunoassay (FEIA) with that of the standard enzyme-linked immunosorbent assay (ELISA) in a clinical setting. Methods: We tested 1227 serum samples obtained from three groups of subjects: 1160 randomly selected schoolchildren, 36 pediatric patients with biopsy confirmed CD and 31 control children with biopsy confirmed normal small bowel mucosa. IgA-anti-tTG detection was compared using the FEIA method and the ELISA method, both of which use baculovirus-expressed recombinant human tissue-transglutaminase (tTG) as the antigen. Results: Both tests identified pediatric patients with CD equally well in the random population (0.34% with CI: 0.09%–0.88%). Furthermore, both tests gave similar results for the CD group and the control group with normal small bowel mucosa (sensitivity: 90% and specificity: 100% for both assays). Conclusions: The FEIA method is an equivalent substitute for the traditional ELISA for IgA-anti-tTG detection associated with CD in a random pediatric population.

Background: In China, the traceability of clinical chemistry methods is still immature. Therefore, it is necessary to establish a reference measurement procedure and evaluate commercial reagent kits using such established procedures. Methods: We reproduced the reference measurement procedure for serum total protein, as recommended by the American Association for Clinical Chemistry (AACC). We evaluated the performance by Clinical Laboratory Standard Institute (CLSI) guidelines EP15-A and EP6-A. Subsequently, four commercial reagent kits were evaluated by the reproduced reference procedure following CLSI guideline EP9-A2. Results: The performance of the reproduced reference procedure was as follows: CVs ranged from 0.47% to 0.85% at medical decision levels (X c ) of 45 g/L, 60 g/L and 80 g/L. Linearity was Y=1.0022X–0.2121 (r=0.9999), and recovery ranged from 100.2% to 102.4%. The External Quality Assessment Scheme for Reference Laboratories in Laboratory Medicine (RELA) was applied, and the result was within the limit of equivalence. The linear relationships of four commercial reagent kits, Merit Choice, KHB, Leadman, and Olympus, were, respectively: Y=0.9922X+0.5776 (r=0.9961); Y=0.9936X+0.4316 (r=0.9992); Y=0.9949X+0.9129 (r=0.9987) and Y=0.9923X+0.8876 (r=0.9989). KHB showed slight negative bias, and the mean±SD was –0.03±0.60 g/L. Merit Choice, Leadman, and Olympus all showed positive bias, and the mean±SDs were 0.02±0.63 g/L, 0.55±0.77 g/L and 0.34±0.71 g/L, respectively. Conclusions: The correlation and bias of four commercial reagent kits for serum total protein were found to be acceptable. Thus, these reagent kits can be used reliably in China.

Background: This study investigated the changes in biomarkers of renal function and the corresponding estimated glomerular filtration rate (eGFR) in 10 male adolescent (16.2±0.6 years) trained runners during early and late recovery periods after an all-out 21-km run. Methods: Venous blood samples were taken immediately before, and at 2-h, 4-h, and 24-h following the 21-km run. Samples were analyzed for hemoglobin (Hb) and hematocrit (Hct), and serum urea (U), creatinine (Cr), creatine kinase (CK) and lactate dehydrogenase (LDH). U/Cr ratio, eGFR, and plasma volume change (%ΔPV) were calculated based on the associated measured variables. Results: At 2-h and 4-h, there were increases (26%–146%) in U, Cr, U/Cr, CK, and LDH, and decreases (8%–13%) in Hb and Hct, as compared with the corresponding pre-exercise values. The calculated %ΔPV increased by 17% while the eGFR decreased 20% from the corresponding pre-exercise value, respectively. At 24-h, the changes of the blood variables and renal function parameters observed at early recovery period were sustained. Conclusions: Taken together, the findings of this study suggest that mild decline in renal function occurred during the early recovery period following the 21-km run. The decline in renal function did not return to normal during the later recovery period.

Background: Neutrophil gelatinase associated lipocalin (NGAL) is a protein present in neutrophils. NGAL is a promising biomarker for acute kidney injury. In urinary tract infections, urinary neutrophils can be a potential source of urinary NGAL. We investigated the effects of urinary tract infection and urinary neutrophil counts on urinary NGAL values. Methods: NGAL was assayed using an immunoassay (ARCHITECT). Urine flowcytometry was performed with the UF-1000i (Sysmex). Results: A correlation between the urinary white blood cell (WBC) count and NGAL concentrations was observed: log(Y) (NGAL, μg/L)=1.284+0.439 log(X) (urinary WBC, 10 9 cells/L); r=0.518. Similarly, the bacterial count correlated weakly with NGAL: log(Y) (NGAL, μg/L)=1.796+0.124 log(X) (bacterial count, 10 9 cells/L); r=0.243. Albuminuria correlated moderately with NGAL values: log(Y) (NGAL, μg/L)=1.557+0.339 log(X) (albuminuria, mg/L); r=0.368; α 1 -microglobulin (a1M) correlated weakly with NGAL: log(Y) (NGAL, μg/L)=1.631+0.360 log(X) (a1M, mg/L); r=0.381. Conclusions: Leukocyte contributions to urinary NGAL concentrations can be important. In leukocyturia or tubular damage (e.g., intensive care patients), using a fixed cut-off value for interpreting urinary NGAL data can lead to false positive results. Therefore, we suggest a mathematical correction in cases with pyuria (>100×10 9 cells/L) and urinary NGAL concentration >100 μg/L: corrected NGAL (μg/L)=NGAL–0.12 (urinary WBC, 10 9 cells/L).

Background: Pseudohyperkalaemia may result from delay in centrifugation and storage at 4°C. We investigated whether the stage of chronic kidney disease (CKD), its aetiology or medications influence this. Methods: Seventy-seven patients with CKD were recruited. Lithium heparin plasma samples were analysed for sodium, potassium, urea and creatinine, chloride, bicarbonate, magnesium, calcium and inorganic phosphate at 0 h and after storage of whole blood at 4°C for 6 h and 20 h. K-EDTA and fluoride-EDTA samples were analysed for full blood count and glucose at 0 h. CKD stage was determined by standard criteria. Results: K + increased on average by 1.0 and 3.6 mmol/L after 6 and 20 h storage of whole blood at 4°C, independent of cause or stage of CKD. K + increase at 6 h was correlated with haemoglobin but not with white blood cell count, platelet count or glucose. Patients taking ACE inhibitors and/or angiotensin receptor blockers (ARBs) had slightly higher K + at 0 h and increased K + after storage for 6 h. Na + decreased on average by 3.8 mmol/L at 20 h and was independent of CKD stage, and correlated with K + increase. Conclusions: K + increased significantly with time in samples stored at 4°C in all stages of CKD. This was greater in some patients on ACE inhibitors and ARBs, and increased with haemoglobin, but was not related to the stage of CKD, white blood cell count or platelet count for the samples used in this study.

Background: Two approaches for detecting anti-prothrombin antibodies have been described. The first detects antibodies against prothrombin alone and the second, phos-phatidylserine-dependent antiprothrombin antibodies. The latter more often correlate with clinical manifestations of antiphospholipid syndrome and with lupus anticoagulant activity. Methods: In order to increase the capacity of antibody binding, we modified the previously described phosphatidylser-ine-dependent antiprothrombin ELISA and determined their avidity. We examined 203 patients with systemic autoimmune diseases and 222 blood donors. Results: Our modification resulted in a greater intensity of antibody binding to prothrombin on phosphatidylserine-coated plate surfaces compared to the previously described method. By changing ELISA conditions, we were able to detect with one assay the two, presumably different, populations of antiprothrombin antibodies. Diagnostic specificities of both ELISAs for antiphospholipid syndrome were similar (92.5% vs. 93.1%), while the sensitivity of the modified phosphatidylserine-dependent antiprothrombin ELISA was significantly higher than the anti-prothrombin alone ELISA (59% vs. 25%). Low avidity antiprothrombin antibodies were only detected in the modified phosphatidylserine-dependent antiprothrombin ELISA. Four percent of patients with positive phosphatidylserine-dependent antiprothrombin antibodies, showing clinical manifestations of antiphospholipid syndrome, were negative for all other antiphospholipid antibodies. The risk for antiphospholipid syndrome increased with the number of antiphospholipid antibody positivity. Conclusions: We conclude that antibodies detected with a modified phosphatidylserine-dependent antiprothrombin ELISA could improve the diagnosis of antiphospholipid syndrome by offering additional information on the risk for thrombosis, especially in patients negative for other antiphospholipid antibodies.

Background: Discrepancies in ionised calcium concentrations between results from several point-of-care devices derived from intensive care unit (ICU) patients were postulated to be caused by perchlorate, a thyroid blocking agent. The deviations were serious concerning the diagnosis of hyper- or hypocalcaemia and administration of calcium infusions. Methods: The problem was studied from three perspectives. First: quantification of ionised calcium in heparinised blood samples spiked with sodium perchlorate from healthy volunteers measured using five blood gas analysers (BGAs from IL, Radiometer, Roche and Siemens). Second: verification of clinical concentrations of perchlorate in blood samples after routine use for blood gas analysis from ICU patients. Third: retrospective analysis of data stored from patients during their stay in general ICU of the Departments of Anesthesio-logy and General Surgery by a patient data management system. Results: Category 1: three of the point-of-care testing devices measure clinically relevant falsely low ionised calcium concentrations when exposed to concentrations perchlorate >0.1 mmol/L. Two were not able to identify hypercalcaemia of up to 4 mmol/L ionised calcium when specific perchlorate concentrations are exceeded. Category 2: measured clinical concentrations of perchlorate ranged between the lower limit of quantification [LOQ=0.03 mmol/L and 2.75 mmol/L (median=0.29 mmol/L; mean=0.585 mmol/L)]. A concentration above 0.1 mmol/L perchlorate was found in 74.3% of all samples that tested positive. Category 3: between February 2006 and July 2009, 42 patients per year (2.2%) received sodium perchlorate with a median length of treatment of 25 days. Conclusions: Perchlorate causes clinically relevant lowering of measured ionised calcium in BGAs from two providers at concentrations obtained in samples from ICU patients (affecting about 1.1% of all ICU patients).

Background: This study evaluated the impact of thyroglobulin (Tg) antibodies (TgAb) interference on Tg measurements performed with a second-generation immunometric Tg assay, in comparison to a first-generation method. Methods: Five serum pools without detectable TgAb and containing measurable Tg concentrations were obtained from DTC patients and mixed with sera from patients with measurable TgAb, but undetectable Tg concentrations. Tg and TgAb concentrations were measured by the first- and the second-generation Tg immunoassays and Tg-recovery was also evaluated for both assays. Results: The decrease in Tg concentrations in the presence of TgAb was more pronounced in the first-generation Tg assay (41.6%) compared with the second-generation assay (31.4%) (p<0.01). Accordingly, the mean Tg-recovery values were 51.4% in the first-generation Tg assay and 68.8% in the second-generation (p<0.01). Measurable Tg concentrations were found in all sera using the second-generation assay, but not the first-generation method. Conclusions: Our preliminary results indicate that an undetectabIe Tg concentration measured using the second-generation assay is unlikely to be a false-negative result induced by TgAb-interferences.

Background: Our laboratory was confronted with two successive urine samples from a single patient which tested positive for human chorionic gonadotropin (hCG) when tested with both qualitative and quantitative assays, combined with no detectable hCG in corresponding plasma samples. Methods: Serial dilution and recovery experiments were performed in order to investigate the presence of interfering substances or a high-dose hook effect. The ovarian cysts that were removed from this patient were immunohistochemically stained using polyclonal anti-human hCG antibodies. Furthermore, a urine sample was sent to the USA hCG Reference Service for hCG variant analysis. Results: Dilution and recovery experiments in urine and plasma samples were unremarkable. The biopsy stained negative for human hCG and free β-subunit. hCG isoform analysis in the urine sample revealed that approximately 87.5% of the immunoreactive hCG lacked the β-subunit C-terminal peptide (CTP). Conclusions: We report a rare case in which two successive urine samples test positive for hCG whereas in corresponding plasma samples hCG is undetectable. The majority of the total hCG contained a degraded form of β-subunit that lacks the CTP. This hCG variant, possibly of pituitary origin, is thought to have an extreme fast clearance rate possibly explaining the discordance between the hCG results in urine and plasma samples.

Background: Dehydration is a common medical problem requiring heuristic evaluation. Our aim was to develop a quantitative and graphical tool based on serial changes in either plasma osmolality (P osm ), urine specific gravity (U sg ), or body mass (B m ) to aid in determining the probability that a person has become dehydrated. A secondary purpose was to validate use of the tool by dehydrating a group of volunteers. Methods: Basic data were obtained from a recent study of biological variation in common hydration status markers. Four reference change values (RCV) were calculated for each variable (P osm , U sg , B m ) using four statistical probabilities (0.80, 0.90, 0.95, and 0.99). The probability derived from the Z-score for any given change can be calculated from: Z=change/[2 1/2 (CV a 2 +CV i 2 ) 1/2 ]. This calculation was simplified to require one input (measured change) by plotting the RCV against probability to generate both an empirical equation and a dual quantitative-qualitative graphic. Results: Eleven volunteers were dehydrated by moderate levels (–2.1% to –3.5% B m ). Actual probabilities were obtained by substituting measured changes in P osm , U sg , and B m for X in the exponential equation, Y=1– e –K·X , where each variable has a unique K constant. Median probabilities were 0.98 (P osm ), 0.97 (U sg ), and 0.97 (B m ), which aligned with ‘very likely’ to ‘virtually certain’ qualitative probability categories for dehydration. Conclusions: This investigation provides a simple quantitative and graphical tool that can aid in determining the probability that a person has become dehydrated when serial measures of P osm , U sg , or B m are made.

Background: Lung cancer, of which 15%–20% is small cell lung cancer, is the leading cause of cancer mortality and shows a high incidence worldwide. While pro-gastrin-releasing peptide (ProGRP) has been reported as a predictive diagnostic factor, it has not been widely accepted because of inconsistent conclusions. The aim of this study was to systematically evaluate ProGRP as the diagnostic standard for small cell lung cancer. Methods: All published studies on ProGRP in the diagnosis of small cell lung cancer from January 1994 to April of 2010 were retrieved. By measuring methodological qualities, 11 papers were selected for this study. The overall diagnostic sensitivity, specificity, and area under the receiver operating characteristic curve were used to evaluate ProGRP as the diagnostic standard for small cell lung cancer using Meta DiSc statistical software. Results: There were 5146 subjects included in this meta-analysis. The sensitivity and specificity (95% confidence interval) of ProGRP was 0.716 (0.688–0.743) and 0.921 (0.909–0.932), respectively. The area under the summary receiver operating characteristic curve of ProGRP was 0.9236. The index of Q* was 0.8575. Conclusions: ProGRP has better sensitivity and high specificity as an auxiliary indicator for the diagnosis of small cell lung cancer.

Background: The aim of the study was to evaluate the circulating concentrations of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR-2), vascular endothelial growth factor-D (VEGF-D) and endostatin in patients with intraductal papillary mucinous neoplasm (IPMN), and in those with ductal adenocarcinomas. Methods: Sixty patients (32 males, 28 females, mean age 69.3±11.3 years) were enrolled: 31 (51.7%) had IPMNs and 29 (48.3%) had histologically confirmed pancreatic adenocarcinomas. Thirty blood donors were also studied as controls. In all study subjects, the concentrations of VEGF, VEGF-D, VEGFR-2, and endostatin were determined using enzyme-linked immunosorbent assays. Results: Serum concentrations of VEGF, VEGF-D, and VEGFR-2 were significantly higher in patients with pancreatic ductal adenocarcinoma and those with IPMNs compared with healthy subjects, while endostatin was significantly higher only in patients with pancreatic ductal adenocarcinoma compared with healthy subjects. Within the group of patients, VEGFR-2 was significantly higher in patients with ductal adenocarcinoma compared to those with IPMNs. The sensitivity and the specificity of VEGFR-2 in differentiating patients with ductal adenocarcinomas from those with IPMN at a cut-off range of 4003–4034 pg/mL was 86.2% and 54.8%, respectively. Conclusions: IPMNs have serum VEGFR-2 concentra-tions different from those in patients with ductal adenocarcinomas. However, serum VEGFR-2 cannot be routinely utilized to differentiate IPMNs from pancreatic ductal adenocarcinomas.

Background: Cardiac troponin T is an established prognostic marker in patients with acute coronary syndromes, but not in stable coronary artery disease (CAD) like N-terminal pro-B-type natriuretic peptide (NT-proBNP). We examined the additive prognostic value of a high-sensitivity troponin T (hsTnT) assay to predict adverse clinical outcomes in stable CAD. Methods: A retrospective nested case-control analysis of 256 patients with stable CAD who participated in the LURIC study: 128 cases who died from cardiovascular causes during a median follow-up of 7.5 years and 128 survivors (controls) matched for age and gender, were included. hsTnT and NT-proBNP were determined in baseline samples using immunoassays (Roche Diagnostics, Germany). Results: Sixty-two percent of the 256 subjects exhibited concentrations of hsTnT≥14 ng/L, the manufacturer recommended cut-off to diagnose myocardial infarction in patients with acute chest pain. hsTnT, NT-proBNP, diabetes mellitus and fasting glucose were associated with cardiovascular mortality in univariate analysis. Logistic regression identified hsTnT and NT-proBNP as independent risk markers. Receiver operator characterisitc (ROC) curves analysis identified optimal cut-offs at 15 ng/L and 352 μg/L for hsTnT (AUC 0.728, p<0.05) and NT-proBNP (AUC 0.751, p=0.07), respectively. Patients with one or two positive markers exhibited 5-year cardiovascular mortalities of 40% and 60%, respectively, compared to 10% in patients with negative markers. The addition of hsTnT to NT-proBNP significantly increased c-statistics of proportional hazards calculated from survival times as well as net reclassification indexes. Conclusions: Many patients with stable CAD exhibited increased concentrations of hsTnT. The combined determination of NT-proBNP and hsTnT was superior for risk stratification compared to determining either marker alone.