Volume 57, Issue 9

Background Measurement of human epididymis protein 4 (HE4) in serum has recently been proposed for clinical use in the framework of ovarian cancer (OvCa). We sought to retrace the translational phase and the clinical implementation steps boosting HE4’s clinical value and discuss the effects of its introduction on the diagnostic and management pathways. Methods Meta-analyses of running evidence have preliminarily suggested that HE4 may overcome carbohydrate antigen 125 (CA125) in identifying OvCa, showing however several gaps that need to be considered, i.e. definition of biomarker diagnostic performance in the early detection of OvCa, added diagnostic value, biological and lifestyle factors of variation, and optimal interpretative criteria. Investigation of the influencing factors has shown that renal impairment represents a major limitation for HE4’s diagnostic power. On the other hand, the demonstration of the substantial equivalence of results obtained by commercially available assays allows recommending harmonized thresholds for diagnostic purpose, even if the study of HE4’s biological variation has clarified that the longitudinal interpretation of the biomarker changes according to the reference change value could be more appropriate. Summary We used HE4 as an example for describing the long and bumpy road for making a new biomarker a reality, and the issues that should be checked and the information that should be provided in moving a novel biomarker from its discovery to an effective clinical adoption.

A growing body of evidence has been published about the usefulness of measuring calprotectin in faecal samples (FCAL) in inflammatory bowel disease (IBD) assessment, including diagnosis, monitoring of disease activity and relapse prediction. Several systematic reviews with meta-analyses compiling studies for each particular clinical setting have been carried out in recent years. Most of these were focused on the use of FCAL in IBD diagnosis and showed a relevant role for this marker in selecting patients with gastrointestinal symptoms who would not need a further examination by endoscopy. Although a lesser number of meta-analyses have been performed on the use of FCAL as a surrogate marker of disease activity, a close correlation between FCAL and endoscopic activity of IBD has been shown. With respect to the predictive capacity of FCAL for IBD relapse, a single meta-analysis published indicates that this role is more limited. Furthermore, FCAL thresholds vary considerably depending on the clinical setting and, what is more concerning, among different commercially available assays due to a lack of FCAL concentration interchangeability. Here, we summarise recent publications about the role and limitations of FCAL in IBD, with a special focus on meta-analyses, and give an overview of alternative faecal biomarkers.

Background Procalcitonin (PCT)-guided antibiotic stewardship (ABS) has been shown to reduce antibiotics (ABxs), with lower side-effects and an improvement in clinical outcomes. The aim of this experts workshop was to derive a PCT algorithm ABS for easier implementation into clinical routine across different clinical settings. Methods Clinical evidence and practical experience with PCT-guided ABS was analyzed and discussed, with a focus on optimal PCT use in the clinical context and increased adherence to PCT protocols. Using a Delphi process, the experts group reached consensus on different PCT algorithms based on clinical severity of the patient and probability of bacterial infection. Results The group agreed that there is strong evidence that PCT-guided ABS supports individual decisions on initiation and duration of ABx treatment in patients with acute respiratory infections and sepsis from any source, thereby reducing overall ABx exposure and associated side effects, and improving clinical outcomes. To simplify practical application, the expert group refined the established PCT algorithms by incorporating severity of illness and probability of bacterial infection and reducing the fixed cut-offs to only one for mild to moderate and one for severe disease (0.25 μg/L and 0.5 μg/L, respectively). Further, guidance on interpretation of PCT results to initiate, withhold or discontinue ABx treatment was included. Conclusions A combination of clinical patient assessment with PCT levels in well-defined ABS algorithms, in context with continuous education and regular feedback to all ABS stakeholders, has the potential to improve the diagnostic and therapeutic management of patients suspected of bacterial infection, thereby improving ABS effectiveness.

Background Ethical MedTech prescribes high standards for the participation of the in vitro diagnostics (IVD) industry in third-party organised educational events in terms of charitable donations, educational grants, scholarships and fellowships. We planned a survey to investigate the previous and current practice in terms of cooperation between professionals or professional societies and the IVD industry, as well as plans under the incorporation of the MedTech Europe Code. Methods Different questions, from general information to specific questions related to the practice and knowledge of the new Ethical MedTech Code, were included in two different surveys; for European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) National Societies’ (NSs) representatives, and for their (NSs) individual members. Results Twenty-five out of 40 EFLM NS representatives replied; more than half declared that all different types of financial resources were available for supporting the continuing professional education of health care professionals (HCPs). In addition, 322 individual responses collected from 31 NSs, answered that the institutional director (50.3%) or laboratory chief (70.1%) made generally made a decision, without specific criteria. Conclusions The MedTech Europe Code is already adopted or is about to be adopted in numerous EFLM NSs, but most of them have not implemented it as yet. The use of the Code and better communication between IVD companies and HCPs are necessary to guarantee an improved and fair use of financial support, as well as better choices for the organisation and attendance at scientific events.

Background New moving average quality control (MA QC) optimization methods have been developed and are available for laboratories. Having these methods will require a strategy to integrate MA QC and routine internal QC. Methods MA QC was considered only when the performance of the internal QC was limited. A flowchart was applied to determine, per test, whether MA QC should be considered. Next, MA QC was examined using the MA Generator (www.huvaros.com), and optimized MA QC procedures and corresponding MA validation charts were obtained. When a relevant systematic error was detectable within an average daily run, the MA QC was added to the QC plan. For further implementation of MA QC for continuous QC, MA QC management software was configured based on earlier proposed requirements. Also, protocols for the MA QC alarm work-up were designed to allow the detection of temporary assay failure based on previously described experiences. Results Based on the flowchart, 10 chemistry, two immunochemistry and six hematological tests were considered for MA QC. After obtaining optimal MA QC settings and the corresponding MA validation charts, the MA QC of albumin, bicarbonate, calcium, chloride, creatinine, glucose, magnesium, potassium, sodium, total protein, hematocrit, hemoglobin, MCH, MCHC, MCV and platelets were added to the QC plans. Conclusions The presented method allows the design and implementation of QC plans integrating MA QC for continuous QC when internal QC has limited performance.

Background For pregnant women with suspected preeclampsia, the soluble fms-like tyrosine-kinase 1 (sFlt-1)/placental growth factor (PlGF) ratio is a biomarker to aid diagnosis. We performed method comparisons between Elecsys ® and Kryptor sFlt-1 and PlGF immunoassays and assessed the diagnostic performance for preeclampsia. Methods Serum samples from a case-control study involving 113 pregnant women with preeclampsia/elevated liver enzymes and low platelet count (HELLP) and 270 controls were analyzed. sFlt-1 and PlGF were measured using Roche Elecsys ® and BRAHMS Kryptor sFlt-1/PlGF immunoassays. The sFlt-1/PlGF ratios were calculated, and Passing-Bablok regression/Bland-Altman plots were performed. Gestation-specific cut-offs, ≤33 and ≥85/≥110, were assessed. Results Mean (±2 standard deviation [SD]) differences between the Elecsys ® and Kryptor values were: sFlt-1, 173.13 pg/mL (6237.66, −5891.40); PlGF, −102.71 pg/mL (186.06, −391.48); and sFlt-1/PlGF, 151.74 (1085.11, −781.63). The Elecsys ® and Kryptor immunoassays showed high correlation: Pearson’s correlation coefficients were 0.913 (sFlt-1) and 0.945 (PlGF). Slopes were 1.06 (sFlt-1) and 0.79 (PlGF), resulting in ~20% lower values for Kryptor PlGF. Sensitivities and specificities using the sFlt-1/PlGF ≥85 cut-off for early-onset preeclampsia (20 + 0 to 33 + 6 weeks) were 88.1%/100.0% (Elecsys ® ) and 90.5%/96.2% (Kryptor), respectively, and using the ≥110 cut-off for late-onset preeclampsia (≥34 + 0 weeks) were 51.3%/96.5% (Elecsys ® ) and 78.9%/90.1% (Kryptor), respectively. Using Elecsys ® and Kryptor sFlt-1/PlGF, 0% and 3.8% of women, respectively, were falsely ruled-in for early-onset, and 3.5% and 9.9%, respectively, for late-onset preeclampsia. Conclusions Despite high correlation between the Elecsys ® and Kryptor immunoassays, we observed significant differences between sFlt-1/PlGF and PlGF results. Therefore, sFlt-1/PlGF cut-offs validated for Elecsys ® immunoassays are not transferable to Kryptor immunoassays.

Background In the Netherlands, each new lot of test strips for the CoaguChek XS is validated by a group of collaborating centers. The purpose of this study was to assess the accuracy of the international normalized ratio (INR) measured with consecutive test strip lots and the suitability of frozen plasma pools for accuracy evaluation. Methods Each year, a particular lot of CoaguChek XS test strips is used as reference lot. The reference lots have been validated with the International Standard for thromboplastin rTF/09, yielding a mathematical relationship (R1) between reference lot INR and International Standard INR. New lots are compared to the reference lot using patients’ capillary blood samples, yielding a relationship (R2) between the new lot INR and the reference lot INR. INRs of the blood samples were within the 1.5–4.5 interval. In parallel, three frozen plasmas pools are analyzed with the test strips. The distance of each plasma point to the line of relationship R2 was assessed. Results Fifty-four test strip lots have been evaluated during 3 years (2014–2016). Mean INR differences between test strip lot and International Standard rTF/09 varied between −0.14 and +0.20 (−4% and +8%, respectively). A positive trend with strip lot sequence number was observed (p<0.001). In several cases, the distance of the frozen plasmas to the whole blood relationship (R2) was greater than the critical value for commutability. Conclusions Using whole blood, all evaluated test strip lots met the analytical bias criterion of ±10%. Frozen plasma pools behave differently compared to whole blood and are not suitable for assessing absolute accuracy of new CoaguChek XS test strips.

Background Overweight and obese individuals have a reduced life expectancy due to cardiovascular disease (CVD), type 2 diabetes, stroke and cancer. Systemic inflammation and premature telomere shortening have been discussed as potential mechanisms linking these conditions. We investigated the relation of subcutaneous adipose tissue (SAT) distribution to leukocyte relative telomere length (RTL). Methods We measured RTL in 375 participants of the observational STYJOBS/EDECTA cohort (ClinicalTrials.gov Identifier NCT00482924) using a qPCR based method. SAT distribution was determined by lipometry yielding a percent body fat value and SAT thicknesses at 15 standardized locations across the entire body. A correlation analysis between RTL, age, sex, lipometry data and conventional body measures (body mass index [BMI], waist-, hip circumference, waist-to-hip ratio, waist-to-height ratio) was calculated. The strongest determinants of RTL were determined by a stepwise multiple regression analysis. Results RTL was not associated with age or sex. RTL was significantly negatively correlated with BMI, percent body fat, waist-, hip circumference and waist-to-height ratio. Furthermore, RTL correlated with SAT at the following locations: neck, triceps, biceps, upper back, front chest, lateral chest, upper abdomen, lower abdomen, lower back, hip, front thigh, lateral thigh, rear thigh and calf. Stepwise regression analysis revealed nuchal and hip SAT as the strongest predictors of RTL. No significant association was seen between RTL and waist-to-hip ratio. Conclusions RTL is negatively associated with parameters describing body fat composure. Nuchal and hip SAT thicknesses are the strongest predictors of RTL. Central obesity appears to correlate with premature genomic aging.

Background Development of automated analyzers for erythrocyte sedimentation rate (ESR) has imposed the need for extensive validation prior to their implementation in routine practice, to ensure comparability with the reference Westergren method. The aim of our study was to perform the analytical validation of two automated ESR analyzers, the Ves-Matic Cube 200 and the TEST1. Methods Validation was performed according to the recent International Council for Standardization in Hematology recommendations and included determination of intrarun and inter-run precision, assessment of sample carryover, hemolysis interference, sensitivity to fibrinogen, method comparison with the gold standard Westergren method and stability test. Results The highest intrarun imprecision was obtained for the low ESR range (33.5% for Ves-Matic Cube; 37.3% for TEST1) while inter-run coefficients of variation on three levels were much better for the TEST1 (0%, 2% and 1.2%) compared to the Ves-Matic Cube 200 on two levels (24.9% and 5.8%). Both Ves-Matic Cube 200 and TEST1 showed no statistically significant difference when compared with Westergren. Bland-Altman analysis yielded overall insignificant mean biases for all comparisons, but a wider dispersion of results and 95% limits of agreement for comparisons including the Ves-Matic Cube 200. Carryover was considered insignificant, while hemolysis had a negative effect on all assessed ESR methods. The highest sensitivity to fibrinogen was observed for the Ves-Matic Cube 200, followed by Westergren and the least sensitive was the TEST1. Conclusions The obtained results proved the analytical validity of the TEST1 and the Ves-Matic Cube 200, and high comparability with the gold standard Westergren method, showing obvious improvements in standardization of ESR methods.

Background Direct oral anticoagulants (DOACs) cause false positive lupus anticoagulant (LA) results. We assessed the impact of DOAC-Stop, reversing in vitro effects of DOACs, on LA testing in anticoagulated patients. Methods We assessed 75 venous thromboembolism patients aged 44.5±14.6 years. Blood samples were collected 2–28 h since intake of DOACs, including 50 patients on rivaroxaban, 20 on dabigatran and five on apixaban. LA testing was performed at baseline and after DOAC-Stop treatment. Positive LA was defined as the normalized (patient/standard plasma clotting time) LA screening and screening (LA1)/confirmation (LA2) ratios exceeding 1.2. Results LA diluted Russell’s viper venom time (dRVVT) normalized screening test revealed abnormal results in 73 (97.3%) and activated partial thromboplastin time (APTT)-LA in 49 (65.3%) patients. In six (8%) patients, antiphospholipid syndrome (APS) was diagnosed. dRVVT LA1/LA2 was abnormal in 35 (50.7%) patients taking DOACs. The APTT ratio was normal in all studied subjects. DOAC-Stop completely removed dabigatran and reduced by 98% rivaroxaban and by 92.3% apixaban concentrations (all p<0.05). After DOAC-Stop screening dRVVT remained prolonged in 34 (49.3%) patients (p<0.001), while dRVVT LA1/LA2 was abnormal in six (8.7%) subjects, with no association with DOAC concentrations at baseline and after DOAC-Stop. The APTT-LA screening test remained prolonged in five (7.2%) patients, while the APTT LA1/LA2 ratio was normal in those subjects. DOAC-Stop did not influence LA testing in APS patients. Conclusions Application of DOAC-Stop effectively reduced plasma DOAC concentrations leading to appropriate dRVVT results in up to 97% of VTE patients.

Background Biological variation studies have shown that the complete blood count (CBC) has narrow within-individual variation and wide group variation, indicating that the use of reference intervals (RIs) is challenging. The aim of this study was to examine differences in CBC RIs according to race/ethnicity in a multiethnic population at a hospital in San Francisco in hopes of improving the medical utility of CBC testing. Methods Subject data were obtained by screening CBC results from the medical records of outpatients meeting certain criteria who visited Zuckerberg San Francisco General Hospital from April 2017 to January 2018. From these records, sex- and race/ethnicity-specific CBC RIs were calculated as the 2.5th to 97.5th percentiles. Results From a total of 552 subjects, 47.9% were male (65 White, 50 Black, 71 Hispanic and 54 Asian) and 52.1% were female (51 White, 39 Black, 122 Hispanic and 72 Asian). The RIs of neutrophil, lymphocyte and eosinophil counts; and hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) showed significant differences (p<0.05) among the four racial/ethnic groups: neutrophil, lymphocyte and eosinophil counts; and MCHC in males, and hemoglobin, MCV, MCH and MCHC in females. Conclusions Race/ethnicity-specific CBC RIs should be taken into consideration in a multiethnic population to better interpret patient status and make progress toward precision medicine.

Background Many reports address the stability of biochemical analytes in serum. However, studies covering a wide range of storage temperatures are unavailable. Using equipment enabling precise temperature control, we investigated the effect of six different storage temperatures on serum analytes. Methods Serum specimens from seven healthy volunteers were obtained and divided into multiple aliquots for storage at −30, −20, −10, 0, 4, and 25 °C. On days 1, 3, 7, 14, 28 and 56, the aliquots stored at each temperature were relocated to a deep freezer maintained at −80 °C. On day 60, all aliquots were measured collectively for 13 major chemistry analytes. Results (1) At 25 °C, alanine aminotransferase (ALT), creatine kinase (CK), aspartate aminotransferase (AST) and total bilirubin (TBil) were very unstable especially on day 7 and later. (2) At ≤4 °C, alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), amylase (AMY), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), triglyceride (TG), TBil and complement component-4 (C4) were generally stable and were very stable at 25 °C until day 14. (3) Between −20 and 4 °C, especially at −10 °C, test results of ALT, AST and lactate dehydrogenase (LDH) showed prominent decreases, but their stability was greatly improved at −30 °C. (4) In contrast, the value of complement component-3 (C3) increased at ≥− 20 °C. (5) At −30 °C, test results of all analytes were generally very stable except for ALT and CK, which showed noticeable reductions in activity after 14 days. Conclusions This is the first study to assess the stability of serum analytes at six graded temperatures simultaneously. Each analyte has a unique stability pattern for a range of temperatures.

Background Smoldering multiple myeloma (SMM) is an asymptomatic plasma cell disorder with a high risk of progression to symptomatic multiple myeloma (MM). The serum free light chain (sFLC) ratio is a powerful prognostic factor for SMM: an sFLC ratio ≥8 has been reported to be associated with a high risk of progression to MM, and an sFLC ratio ≥100 has been described as a criterion for ultra-high-risk SMM, and has been integrated into the definition criteria for MM since 2014. However, all recommendations were based on sFLC measured using the first commercialized assay, Freelite™, while other assays are now available. We aimed to evaluate the safety and accuracy of N-Latex sFLC to identify high-risk and ultra-high-risk SMM. Methods The sFLC ratio was measured at diagnosis with both Freelite and N-Latex assays in a cohort of 176 SMM patients on a BN Prospec nephelometer. Demographic, clinical, therapeutic and laboratory data were collected at the time of diagnosis and at follow-up. Results Sixty-two patients (35.2%) progressed to MM within 2 years. Compared to Freelite™ sFLC, N Latex sFLC ratios ≥8 and ≥100 provided similar performances for the identification of high-risk and ultra-high risk SMM patients. Conclusions Our results evidenced that the N-Latex assay could be used for SMM monitoring, like Freelite. However, an N-Latex sFLC ratio ≥70 appears to provide similar performances to a Freelite sFLC ratio ≥100, with a slightly better positive predictive value. Both assays provided accurate identification of high-risk and ultra-high risk SMM patients. These results should be confirmed in an independent study.

Background Obstructive sleep apnoea (OSA) is an independent risk factor of hypertension and cardiovascular diseases. Recurrent episodes of upper airways collapse during sleep causing blood oxygen desaturation, hypercapnia, and micro-arousals, are known to activate the sympathetic nervous system (SNS). However, whether changes in the renin-angiotensin-aldosterone system and endothelial activation also occur remains contentious. Methods Based on routine use of drug-induced sleep endoscopy (DISE) for the work-up of OSA patients in our centre, we designed a prospective study to investigate the haemodynamic and humoral changes occurring during the apnoeic episodes reproduced in vivo in the course of DISE. Specifically, plasma aldosterone concentration and renin activity, C-terminal fragment of proendothelin-1, as a marker of endothelial damage, and free plasma catecholamines, will be measured at fixed times during DISE. The activity of catechol-O-methyltransferase (COMT), a key catecholamine-inactivating enzyme that has been scantly investigated thus far owing to the lack of commercially available kits, will be also determined by a newly developed high performance liquid chromatography method, which is herein described. Results and conclusions The aim of this study is to provide novel information on the haemodynamic, hormonal, and SNS changes, and also on COMT activity modification concomitantly occurring during apnoea, thus contributing substantively to the understanding of the pathophysiology of OSA.

Background Procalcitonin (PCT) is a biomarker for systemic bacterial infections and may aid in decision making for antimicrobial stewardship. Numerous PCT assays are available on common clinical immunoassay platforms. However, questions remain about the harmonization of these assays and whether the same clinical decision points may be used with all methods. Methods Thirty-seven remnant patient serum samples were analyzed across four different PCT assays: Abbott ARCHITECT i2000, bioMérieux MINI VIDAS, Roche Elecsys cobas e 411, and BRAHMS KRYPTOR. Regression analysis was performed, and correlation was assessed at common clinical decision points for antimicrobial therapy: 0.10, 0.25, and 0.50 μg/L. Results Data showed a positive bias of the MINI VIDAS compared to the KRYPTOR (slope=1.188, R=0.9873) and negative biases of both the ARCHITECT i2000 and cobas e 411 compared to the KRYPTOR (slope=0.806, R=0.8864, and slope=0.795, R=0.8974, respectively). A comparison of results at commonly used clinical decision points for antimicrobial stewardship showed that, relative to the KRYPTOR, 21% of samples would be classified into different interpretive categories by the ARCHITECT i2000 method, 31% of samples would be classified differently by the MINI VIDAS method, and 16% of samples would be classified differently by the cobas e 411 method. Conclusions All methods showed reasonable analytical agreement; however, an analysis of result interpretation at clinical decision points showed that many samples were differentially categorized (e.g. shifted by one interpretive category) by the methods. Overall, our findings support a need for harmonization of PCT methods. Until then, institutions should independently evaluate their PCT assays against predicate methods and consider the impact on result interpretation prior to incorporating PCT into clinical practice.

Background The prognostic impact of mild/moderate liver impairment among critically ill patients is not known. We aimed to determine whether acute liver impairment, as measured by several biomarkers, (i) is frequent, (ii) influences prognosis and (iii) to determine whether such an effect is specific for infected critically ill patients. Methods A biomarker and clinical cohort study based on a randomized controlled trial. All-cause mortality was the primary endpoint. Biomarkers hyaluronic acid (HA), bilirubin, albumin, alkaline phosphatase and the international normalized ratio (INR) were determined. Multivariable statistics were applied to estimate risk increase according to liver biomarker increase at baseline and the model was adjusted for age, APACHE II, severe sepsis/septic shock vs. milder infection, chronic alcohol abuse Charlson’s co-morbidity index, cancer disease, surgical or medical patient, body mass index, sex, estimated glomerular filtration rate, mechanical ventilation and the other biomarkers. Time-to-event graphs were used. The patients were critically ill patients (n = 1096) from nine mixed medical/surgical intensive care units without known hepatobiliary disease. Results HA levels differed between infected patients (median 210.8 ng/mL [IQR: 93.2–556.6]) vs. the non-infected (median 56.8 ng/mL [IQR: 31.9–116.8], p < 0.001). Serum HA quartiles 2, 3 and 4 were independent predictors of 90-day all-cause mortality for the entire population (infected and non-infected). However, the signal was driven by the infected patients (positive interaction test, no signal in non-infected patients). Among infected patients, HA quartiles corresponded directly to the 90-day risk of dying: 1st quartile: 57/192 = 29.7%, 2nd quartile: 84/194 = 43.3%, 3rd quartile: 90/193 = 46.6%, 4th quartile: 101/192 = 52.3 %, p for trend: <0.0001. This finding was confirmed in adjusted analyses: hazard ratio vs. 1st quartile: 2nd quartile: 1.3 [0.9–1.8], p = 0.14, 3rd quartile: 1.5 [1.1–2.2], p = 0.02, 4th quartile: 1.9 [1.3–2.6], p < 0.0001). High bilirubin was also an independent predictor of mortality. Conclusions Among infected critically ill patients, subtle liver impairment, (elevated HA and bilirubin), was associated with a progressive and highly increased risk of death for the patient; this was robust to adjustment for other predictors of mortality. HA can identify patients at high risk.