Volume 52, Issue 3 -

Point-of-care testing (POCT) has had rapid technological development and their use is widespread in clinical laboratories to assure reduction of turn-around-time and rapid patient management in some clinical settings where it is important to make quick decisions. Until now the papers published about the POCT have focused on the reliability of the technology used and their analytical accuracy. We aim to perform a systematic survey of the evidence of POCT efficacy focused on clinical outcomes, selecting POCT denoted special analytes characterized by possible high clinical impact. We searched in Medline and Embase. Two independent reviewers assessed the eligibility, extracted study details and assessed the methodological quality of studies. We analyzed 84 studies for five POCT instruments: neonatal bilirubin, procalcitonin, intra-operative parathyroid hormone, troponin and blood gas analysis. Studies were at high risk of bias. Most of the papers (50%) were studies of correlation between the results obtained by using POCT instruments and those obtained by using laboratory instruments. These data showed a satisfactory correlation between methods when similar analytical reactions were used. Only 13% of the studies evaluated the impact of POCT on clinical practice. POCT decreases the time elapsed for making decisions on patient management but the clinical outcomes have never been adequately evaluated. Our work shows that, although POCT has the potential to provide beneficial patient outcome, further studies may be required, especially for defining its real utility on clinical decision making.

Strategic integration of point-of-care (POC) diagnostic tools during crisis response can accelerate triage and improve management of victims. Timely differential diagnosis is essential wherever care is provided to rule out or rule in disease, expedite life-saving treatment, and improve utilization of limited resources. POC testing needs to be accurate in any environment in which it is used. Devices are exposed to potentially adverse storage and operating conditions, such as high/low temperature and humidity during emergencies and field rescues. Therefore, characterizing environmental conditions allows technology developers, operators, and responders to understand the broad operational requirements of test reagents, instruments, and equipment in order to improve the quality and delivery of care in complex emergencies, disasters, and austere environmental settings. This review aims to describe the effects of environmental stress on POC testing performance and its impact on decision-making, to describe how to study the effects, and to summarize ways to mitigate the effects of environmental stresses through good laboratory practice, development of robust reagents, and novel thermal packaging solutions.

Idiopathic calcium nephrolithiasis (ICN) is a disease whose prevalence is rising. Our aim was to assess whether lifestyle indicators and habits of calcium stone formers in Italy have changed over the last 25 years, trying to establish a connection with the diffusion of Internet access. Therefore we examined the database of the Stone Clinic of Parma University Hospital and extracted 1952 (1192 M, 760 F) patients with ICN who underwent a full clinical and laboratory evaluation from 1986 to 2010. Laboratory evaluation included data on urinary 24-h volume, pH, sodium, potassium, chloride, calcium, phosphate, uric acid, magnesium, oxalate, and citrate. Patients were split in three groups on a chronological basis, according to official EUROSTAT-ISTAT data of Internet connection among families in Italy: Group 1, pre-Internet era (1986–1998, 853 patients); Group 2, narrow-band era (1999–2004, 467 patients); Group 3, broad-band era (2005–2010, 632 patients). Over the time we found a significant increase in water intake (1.37 vs. 1.78 L in men and 1.21 vs. 1.55 L in women, Group 1 vs. Group 3, p-trend<0.001) and a decrease in urinary sodium and chloride for both genders and calcium and magnesium only for males, while females experienced a slight increase in oxalate excretion. Supersaturation indexes for calcium and uric acid stones dramatically fell for both genders. The percentage of stone formers performing physical activity significantly rise (41% Group 3 vs. 8% Group 1, p<0.001) and we also found a trend of reduction in mean blood pressure. Therefore, the lifestyle of Italian idiopathic calcium stone formers has changed over the last 25 years, and the rising Internet access may have played a great role in driving this change.

Background: Exosomes are cytoplasm containing vesicles released by many cells that can be found in several biological fluids including urine. Urinary exosomes are released from every segment of the nephron, are detectable in urine, constitutively contain RNA (small RNAs and mRNAs) and harbor unique subset of proteins, reflecting their cellular source. Methods: With the aim of establishing the optimal protocol for high throughput analysis of exosomal miRNAs, we compared three different urinary exosomes isolation methods and six RNA extraction techniques. Exosomal RNA yield, size and quality were assessed respectively by specific staining with fluorescent dye, capillary electrophoresis and analysis of spectrophotometric parameters. MiRNAs detection and abundance was determined by RT-qPCR. Results: Among the exosomes isolation methods, Ultrafiltration resulted to be the most suited. The highest exosomal RNA yield quantified by RiboGreen ® staining was obtained with the combination of TRI Reagent™ with miRNeasy ® , followed by TRI Reagent™, SeraMir™, miRCURY™, mirVana™ and miRNeasy ® ; but after a multivariate analysis, SeraMir™ scored as the method of choice in terms of miRNA yield, purity and RT-qPCR miRNAs quantification accuracy. Storage conditions were also analyzed, showing that the relative abundance of urinary exosomal miRNAs is not influenced by urine freezing. Conclusions: The selection of appropriate urinary exosomal miRNA isolation method was dependent on various validation results. Ultrafiltration in combination with SeraMir™ exoRNA columns represents the optimal procedure for a rapid, cost-effective and efficient purification of miRNAs from urinary exosomes, perfectly suited for further applicative research in the field of miRNAs in kidney physiology and pathology.

Background: Over the last few decades, new synthetic insulin analogues have been developed. Their measurement is of prime importance in the investigation of hypoglycaemia, but their quantification is hampered by variable cross-reactivity with many insulin assays. For clinical analysis, it has now become essential to know the potential cross-reactivity of analogues of interest. Methods: In this work, we performed an extensive study of insulin analogue cross-reactivity using numerous human insulin immunoassays. We investigated the cross-reactivity of five analogues (lispro, aspart, glulisine, glargine, detemir) and two glargine metabolites (M1 and M2) with 16 commercial human insulin immunoassays as a function of concentration. Results: The cross-reactivity values for insulin analogues or glargine metabolites ranged from 0% to 264%. Four assays were more specific to human insulin, resulting in negligible cross-reactivity with the analogues. However, none of the 16 assays was completely free of cross-reactivity with analogues or metabolites. The results show that analogue cross-reactivity, which varies to a large degree, is far from negligible, and should not be overlooked in clinical investigations. Conclusions: This study has established the cross-reactivity of five insulin analogues and two glargine metabolites using 16 immunoassays to facilitate the choice of the immunoassay(s) and to provide sensitive and specific analyses in clinical routine or investigation.

Background: Already available sample preparation technologies for liquid chromatography-tandem mass spectrometry have substantial shortcomings with respect to automation. A novel approach is based on gel-like polymeric material with defined absorption chemistry, which is immobilized in micro-plate wells. It is referred to as Tecan Immobilized Coating Extraction ™ ( TICE ™ ) technology and it enables easy automation on liquid handling systems. We aimed to study the performance of Tecan AC Extraction Plate ™ based on this principle by addressing 25-hydroxyvitamin D (25OHD) as an exemplary analyte. Methods: A protocol for extraction of 25OHD from serum samples based on TICE ™ technology was implemented on a robotic liquid handling system Freedom EVO ® (Tecan). An isotope-dilution ultra-performance liquid chromatography-tandem mass spectrometry method was used for quantification. Performance was tested according to a comprehensive protocol. Results: Linearity was found over a range from 4.3 to 65.8 ng/mL for 25OHD 3 . The coefficients of variation for the intra-day and inter-day precision were <6% and accuracy ranged between 96.9% and 99.8% for 25OHD 3 . Recovery was 84% and efficient control of matrix effects was verified. High sample throughput could be observed with 96 samples prepared in <60 min. Close agreement of results was found for clinical samples analyzed with a second tandem mass spectrometry method based on protein precipitation and two-dimensional ultra-performance liquid chromatography for sample preparation (r=0.988, n=73). Conclusions: The new TICE ™ technology was found to be a useful process for sample preparation in clinical mass spectrometry. Full automation suited for routine analysis was achieved.

Background: Presence of the 3-epi-25-hydroxyvitamin D 3 [3-epi-25(OH)D 3 ] metabolite affects accurate determination of 25(OH)D 3 by most routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and to an unknown extent in present immuno- and protein binding assays. We studied 3-epi-25(OH)D 3 cross-reactivity in a competitive protein binding (CPB) assay (Roche Elecsys). Methods: Neonatal samples, containing up to 58% of 3-epi-25(OH)D 3 were used for measurement by the CPB assay and by an LC-MS/MS method separating 25(OH)D 3 and 3-epi-25(OH)D 3 . Analytical recovery was also studied by addition of exogenous 3-epi-25(OH)D 3 . Results: The CPB assay showed approximately 51% cross-reactivity to 3-epi-25(OH)D 3 at exogenous addition. In contrast, there was minimal 3-epi-25(OH)D 3 recognition by the CPB assay when present as the natural endogenous metabolite. Conclusions: The automated CPB assay displays minimal 3-epi-25(OH)D 3 cross-reactivity in samples containing significant concentrations of endogenous 3-epi-25(OH)D 3 . Exogenous 3-epi-25(OH)D 3 added to human serum or plasma seems to behave different from endogenous presence, and caution is warranted when using samples spiked with vitamin D metabolites for testing analytical specificity or external quality assurance in immuno- or protein binding assays.

Background: Linezolid serum concentrations have been shown to be highly variable in critically ill patients with often sub-therapeutic drug levels regarding minimal inhibitory concentrations for relevant pathogens. Consequently, therapeutic drug monitoring of linezolid must be considered, requiring a reliable and convenient analytical method. We therefore developed and validated an LC-MS/MS method applying isotope dilution internal standardization and on-line solid phase extraction for serum linezolid quantification. Methods: Sample preparation was based on protein precipitation and on-line solid phase extraction with two-dimensional liquid chromatography and column switching. Three-fold deuterated linezolid was used as the internal standard. The method was validated involving two separate LC-MS/MS systems covering the concentration range of 0.13–32 mg/L. The run time was 4 min. Results: Validation revealed good analytical performance, with inaccuracy <6% and imprecision of <7.3% (CV) for six quality control samples (0.38–16.0 mg/L). The method was found to be robust during the validation process and during a pharmacokinetic study so far involving 600 samples. Comparative measurements on two LC-MS/MS systems revealed close agreement. Conclusions: This LC-MS/MS assay described herein is a convenient, robust and reliable method for linezolid quantification in serum which can be routinely applied using different LC-MS/MS systems. The method can be used for clinical studies and subsequent TDM of linezolid.

Background: Symptoms of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) can overlap. Faecal calprotectin has recently been established to be a non-invasive marker for neutrophilic intestinal inflammation. We compared two devices for extraction of faecal calprotectin. Based on these results, two immunoassays for measurement of faecal calprotectin were evaluated. Methods: Samples were extracted using the Thermo Fisher extraction device (Thermo Fisher Scientific) and Smart Pep extraction device (Roche Diagnostics) and measured with the EliA Calprotectin immunoassay (Thermo Fisher Scientific) on ImmunoCAP 250. The performance of both assays was investigated by enrolling 183 consecutive patients (79 males, 104 females; median age 32 years) with clinical suspicion of IBD. Faecal calprotectin was measured using a recently launched immunoassay, EliA Calprotectin in comparison with an established immunochomatographic point-of-care-test (POCT, Quantum Blue Calprotectin; Bühlmann). Results were compared with endoscopic and histological findings. Results: The use of the Thermo Fisher extraction device resulted in an underestimation of faecal calprotectin concentrations, especially in liquid stool samples. IBD was diagnosed in 51/183 patients (27.9%) [Crohn’s disease (CD, n=37), ulcerative colitis (UC, n=14)]. After adjusting the optimal cut-off for detection of IBD using receiver operating curve analysis, a sensitivity of 94.1% and 90.2% and specificity of 87.9% and 90.9% for the EliA and POCT assay, respectively, were obtained. Conclusions: The Thermo Fisher device is not reliable for extraction of faecal calprotectin. The performance characteristics of the EliA Calprotectin assay are statistically equivalent to the Bühlmann POCT.

Background: In a previous clinical study, levels of biomarkers of exposure (BoEs) for specific toxicants were significantly reduced in smokers who switched from conventional cigarettes to reduced toxicant prototype (RTP) cigarettes. Very little is known about the biological variability of tobacco smoke BoEs within individuals and sub-groups, and the descriptive group-comparison statistics might not be sufficient to understand such changes. Therefore, we assessed how different statistical methods could be used to interpret changes in urine BoE levels at the individual level. Methods: We used non-parametric statistical reference limits, the empirical rule and reference change values (RCVs) to assess changes in levels of BoEs related to four toxicants in cigarettes smoke. Current smokers [of 6 mg and 1 mg International Organization for Standardization (ISO) tar yields] were allocated to switching to RTP groups or non-switching control groups within their respective tar bands. There were two 6 mg tar study groups, with a non-switching group (CC6, n=46) and a group switching to an RTP containing tobacco-substitute sheet and modified filter (TSS6, n=49); and three 1 mg tar smoker groups, with one non-switching (CC1, n=42), a group switching to an RTP containing tobacco-substitute sheet and modified filter (TSS1, n=44) and one switching to an RTP containing an enzyme-treated tobacco and modified filter (BT1, n=47). Results: Assessment of the direction of change showed that up to the 100% of subjects experienced a decrease in levels of some BoEs. Between 49% and 64% of subjects in the switching groups were classified as having decreased levels of 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) by the non-parametric criterion, whereas only 2%–6% had reduced levels of N-nitrosoanatabine (NAT). Of non-switchers, in 7%–14% of those smoking 1 mg ISO tar yield cigarettes increases were classified across all BoEs. RCVs highlighted patterns with more detail, showing that most changes occurred within 14 days of switching. Among smokers who switched to 6 mg RTPs, 40%, 44%, 6% and 15%, respectively, were classified as experiencing significant decreasing levels of HPMA, 3-hydroxypropylmercapturic acid, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and NAT, whereas in the two 1 mg switching groups 46%, 22%, 11% and 52% and 43%, 27%, 2% and 16% had decreased levels of the same biomarkers. Up to five subjects in the 6 mg non-switching group were classified as having increased levels of all BoEs. Conclusions: Although we believe that is not possible to determine whether the observed changes in BoEs reflect biological relevance, the use of reference values enables assessment of changes in BoEs at the individual level. Estimates of the BoE variability between subjects might aid study design and setting minimum targets for smoke toxicant yields for future development of RTPs.

Background: The level of β 2 -microglobulin was generally used to evaluate the renal function in adults. Elevated levels of β 2 -microglobulin were also applied to assess the perinatal situations in neonates and fetuses. The aim of our study was to establish and determine the reference values of fetal serum β 2 -microglobulin in the Chinese and to assess its clinical benefits in abnormal fetuses. Methods: Data from 308 normal cord blood samples were obtained to calculate the normal reference values of fetal serum β 2 -microglobulin. According to the equations we obtained, we analyzed the level of β 2 -microglobulin in four case groups: renal malformation, hydrops, cytomegalovirus (CMV) infection and rubella virus (RV) infection. Results: In the normal group, the concentration of β 2 -microglobulin decreased with the gestational age, with a mean value of 4.35±0.59 mg/L. The upper limit of β 2 -microglobulin was calculated as 7.55–0.074×gestational age in weeks. The levels of β 2 -microglobulin were significantly higher in the four case groups than the normal group. For the four groups, the sensitivity is 72.7%, 69.6%, 86.7% and 100%, respectively. Conclusions: Fetal serum β 2 -microglobulin may be used as a predictor to evaluate the situations of fetal diseases.

Background: We have developed a PCR-based tool that measures a 51-gene panel for identification of gastroenteropancreatic (GEP) neuroendocrine neoplasms (NENs) in peripheral blood. This manuscript assesses the robustness (performance metrics) of this tool with a specific focus on the effects of individual parameters including collection, storage, acid suppressive medication [proton pump inhibitor (PPI)], age, sex, race and food on accuracy. Methods: Performance metrics were evaluated using a gold standard (mRNA derived from three individual human neuroendocrine tumor cell lines) and clinical samples using qPCR. Results: One hundred percent of the 51 transcripts were amplified in the gold standard (NEN cell line-derived mRNA) (C Q <35, average efficiency 1.94). The inter- and intra-assay variations were 1%–2%. In clinical samples, 50 of 51 targets (98%) were amplified. The inter- and intra-assay reproducibility ranged between 0.4% and 1.2%. The coefficient of variation (CV) was 5.3%. Expression of the reference gene, ALG9 , was robust [low variation, low M-value, high (99.5%) PCR efficiency] and unaffected by sample processing. Test meals, long-term PPI use (>1 year), age, sex and ethnicity had no effect on the signature. Expression of two genes, ALP2 and CD59 correlated strongly with RNA integrity (R=0.72, p<0.001) and could be used to assess storage and processing. Conclusions: The 51 marker gene signature was robust and reproducible, exhibiting acceptable inter- and intra-assay metrics (<5%). Feeding, PPI intake, age, sex and ethnicity do not affect the signature. Expression levels of APLP2 and CD59 are effective surrogate markers of proper sample collection and processing.

Background: Prostate-specific antigen (PSA) is used as an outcome measure for relapsed disease in prostate cancer. Nonetheless, there are considerable concerns about its indiscriminate use as a surrogate endpoint for cell growth or survival. We hypothesized that treatment with a luteinizing hormone releasing hormone (LHRH) analog would decrease PSA levels even in the absence of malignant disease. Methods: We determined testosterone and PSA levels in 30 healthy volunteers after a single intramuscular injection of a LHRH depot formulation. Testosterone and PSA levels were quantified by radioimmunoassay and electrochemi-luminescence immunoassay, respectively. Results: After an initial flare-up during the first 3 days testosterone decreased reaching castration levels in 18 of the 30 young men (60%). After the nadir on day 28, testosterone levels increased to normal again. Changes in PSA paralleled those of testosterone. Castration reduced PSA levels by 29% (95% CI 19%–39%) compared to baseline (p<0.0001). Conclusions: LHRH superagonists decrease PSA levels by testosterone deprivation. Conferring these findings to tumor patients, decreases in PSA after treatment with LHRH analogs might not only reflect disease regression but also a direct testosterone mediated effect on PSA. Thus, PSA levels should be cautiously interpreted when patients receive hormonal therapy.

Background: The determination of plasma metanephrines (MNs) provides a highly sensitive test for the diagnosis of catecholamine producing tumors. Chromatographic determinations with electrochemical or mass spectrometric detections are the methods of choice, but immunological assays have been developed. This study evaluated the clinical performances of a radioimmunoassay for free MNs in plasma. Methods: MNs, normetanephrine (NMN) and metanephrine (MN) and catecholamines, norepinephrine (NE) and epinephrine (E) were determined in plasma and urine of 533 patients suspected of catecholamine producing tumor. Urinary and plasma catecholamines and urinary MNs were determined by HPLC using amperometric detection. Plasma MNs were purified by solid phase chromatography and quantified by a specific radioimmunoassay. Results: Fifty-nine patients had tumors (13 paraganglioma and 46 pheochromocytoma) and the diagnosis was excluded in 474 patients. Receiver operator characteristic curves have identified optimal thresholds at 100 pg/mL for plasma NMN (sensitivity 96.6% and specificity 95.8%) and 70 pg/mL for plasma MN (sensitivity 61.0% and specificity 96.8%). These cut-off values were lower than those suggested by the manufacturer (170 and 100 pg/mL, respectively). The sensitivity of combined MNs was similar in plasma (100%) and urine (98%) but higher than that of urinary catecholamines (85%, p<0.001). The specificity of combined MNs in plasma (95%) was higher than urinary MNs (85%, p<0.001) and plasma catecholamines (75%, p<0.001). Conclusions: Plasma-free and urinary-total MNs have a better discriminative power than catecholamines in the diagnosis of catecholamines producing tumors. Using these established cut-offs, measurement of plasma-free MN by radioimmunoassay represents an effective alternative to chromatographic methods.

Background: Endothelial cell-specific molecule (ESM)-1 is a soluble proteoglycan expressed by the vascular endothelium and which also circulates in the bloodstream. Inflammatory cytokines and proangiogenic growth factors increase its expression, and increased serum levels are found in immunocompetent patients with sepsis. The aim of this study was to investigate differential changes in plasma levels of ESM-1 before and after antibiotic treatment in hospitalized adult patients with community-acquired pneumonia (CAP). Methods: Plasma ESM-1 levels were measured in 82 adult patients with CAP and 82 healthy controls using a commercial enzyme-linked immunosorbent assay (ELISA). Upon initial hospitalization, Acute Physiology and Chronic Health Evaluation II (APACHE II), CURB-65, and Pneumonia Severity Index (PSI) scores were determined to assess CAP severity in these patients. Results: Results showed a decline in the number of white blood cells (WBCs) and neutrophils, and decreases in the concentrations of C-reactive protein (CRP) and ESM-1 after antibiotic treatment. The plasma concentration of ESM-1, but not CRP or the WBC count, was correlated with the severity of CAP based on the PSI (r=0.554, p<0.001), CURB-65 (r=0.510, p<0.001), and APACHE II scores (r=0.447, p<0.001). Conclusions: Plasma levels of ESM-1 may be able to play a role in the diagnosis and clinical assessment of the severity of CAP, which could potentially guide the development of treatment strategies.

Background: This study tests the hypothesis that the mannose receptor (MR/CD206), which is expressed primarily by macrophages and dendritic cells, can be found in a soluble form (sMR, sMR) in human serum. Furthermore, we wished to establish and validate an enzyme-linked immunosorbent assay (ELISA) for sMR and to perform initial studies exploring the potential of sMR as a biomarker. Methods: Western blotting identified a single band of approximately 170 kDa in human serum, and MALDI MS/MS of the purified protein confirmed it to be sMR. An ELISA was established and validated with a measurement range of 1–256 µg/L. Results: The 95% reference interval was 0.10–0.43 mg/L based on measurements of serum samples from healthy individuals (n=217). Samples from hospitalised patients (n=219) revealed that more than 50% of patients had concentrations above 0.43 mg/L. Very high concentrations (up to 6.2 mg/L) were observed in critically ill patients with sepsis and/or severe liver disease. Conclusions: This study documents, for the first time, the presence of sMR in human serum and describes an optimised ELISA suitable for quantitative measurements. Levels of sMR are strongly elevated in several disease states, including sepsis and liver disease, and the protein therefore shows promise as a new biomarker.