Volume 53, Issue 5 - Highlight: High-Sensitivity Assays for Cardiac Troponins

Immunoassays measuring cardiac troponins I or T have become firmly established as critical tools for diagnosing acute myocardial infarction. While most contemporary assays provide adequate diagnostic performance, the increased sensitivity and precision of the new, high sensitivity assays that have already been introduced into clinical practice, provide the potential to further shorten intervals between blood draws or the time needed to detect the first significant troponin elevation. In addition to the relatively modest benefits at the diagnostic end, the high sensitivity assays and the investigational ultrasensitive cardiac troponin assays offer improvements for predicting major adverse cardiovascular events, development of heart failure or transition to end-stage kidney disease. These novel high sensitivity assays can measure troponin concentrations in 50%–100% of healthy individuals and therefore allow for the distribution of troponin values within a healthy cohort to be measured, patient’s baseline troponin levels to be monitored, and clinicians to be alerted of deteriorating cardiorenal conditions. We envisage that the high sensitivity assays will become important tools for predicting each patient’s risk of future adverse events and for guiding and monitoring corresponding adjustments of preventative therapeutic interventions.

The availability of so-called high-sensitivity troponin assays (hsTn) has scored a compelling goal for laboratory medicine, allowing the safe clinical application of international recommendations for the definition of acute myocardial infarction (AMI). However, the introduction of hsTn has not been welcomed by clinicians, claiming an increase in false-positive results. Here we critically trace back the steps following the introduction of hsTn by referring to the 5-year practical experience in our academic hospital and to suitable information available in the literature. In agreement with published data, we found that hsTn introduction was associated with an increased number of AMI diagnoses, whereas the test volume, the revascularization rate, and the proportion of cases with negative angiography findings remained virtually unchanged. Fast-track protocols for ruling out AMI have been further optimized to recommend sampling at presentation and after 3 h only. We focus on a cost-effective use of hsTn that can account for all clinical variables increasing the pre-test probability in order to ensure that tests are ordered only for patients at medium to high risk for acute coronary syndrome (ACS). To guide interpretation of results, hsTn typical release patterns suggestive for AMI should be identified by evaluating the significance of concentration changes. hsTn have markedly shortened the time to rule out or rule in AMI and has the potential to improve the prognostic assessment of critical patients in clinical contexts different from ACS.

Cardiac troponin (cTn) I and T are released from myocardial cells following necrosis, i.e., cell death. An accurate measure of cTn concentrations in a patient’s blood following ischemia/chest pain can enable providers to determine whether or not a myocardial infarction (MI) has occurred. Point-of-care (POC) devices that measure blood cTn concentrations in under 30 min may help to significantly reduce hospital costs by managing and triaging patients out of the emergency department as quickly as possible. The use of POC devices that measure cTnI and cTnT with a coefficient of variation (CV) ≤20% at the 99th percentile upper reference limit (URL) limits both false positive and negative results and provides clinically acceptable findings to assist in appropriate diagnoses. This article reviews nine POC devices that measure cTn in terms of their clinical sensitivity and specificity, analytical imprecision, sample type and preparation, and each assay’s principle of analysis.

Background: As a part of an International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) project to prepare a commutable reference material for cardiac troponin I (cTnI), a pilot study evaluated current cTnI assays for measurement equivalence and their standardization capability. Methods: cTnI-positive samples collected from 90 patients with suspected acute myocardial infarction were assessed for method comparison by 16 cTnI commercial assays according to predefined testing protocols. Seven serum pools prepared from these samples were also assessed. Results: Each assay was assessed against median cTnI concentrations measured by 16 cTnI assays using Passing-Bablok regression analysis of 79 patient samples with values above each assay’s declared detection limit. We observed a 10-fold difference in cTnI concentrations for lowest to highest measurement results. After mathematical recalibration of assays, the between-assay variation for patient samples reduced on average from 40% to 22% at low cTnI concentration, 37%–20% at medium concentration, and 29%–14% at high concentration. The average reduction for pools was larger at 16%, 13% and 7% for low, medium and high cTnI concentrations, respectively. Overall, assays demonstrated negligible bias after recalibration (y-intercept: –1.4 to 0.3 ng/L); however, a few samples showed substantial positive and/or negative differences for individual cTnI assays. Conclusions: All of the 16 commercial cTnI assays evaluated in the study demonstrated a significantly higher degree of measurement equivalence after mathematical recalibration, indicating that measurement harmonization or standardization would be effective at reducing inter-assay bias. Pooled sera behaved similarly to individual samples in most assays.

Background: Cardiac troponin level measured by high-sensitivity assays (hs-cTn) in the elderly is frequently found higher than the 99th percentile upper reference limit, making the diagnosis of acute coronary syndromes (ACS) difficult. This study aimed at: 1) identifying determinants of hs-cTnT levels in an unselected population of elderly subjects; and 2) assessing the prognosis value of increased hs-cTnT in elderly people free of ACS. Methods: Hs-cTnT was measured in 591 individuals aged over 65 years without suspicion of ACS. Comorbidities were assessed using the Cumulative Illness Rating Scale for Geriatrics (CIRS-G). C-reactive protein, α 1 -acid glycoprotein, albumin and creatinine were measured. Factors influencing hs-cTnT levels were assessed through linear regression and quantile regression was used to model percentiles of hs-cTnT. Risk of mortality was assessed through Cox regression. Results: Age, gender, cardiac CIRS-G, estimated glomerular filtration rate (p<0.001 for all), albumin (p<0.028) and α 1 -acid glycoprotein (p=0.002) were independent predictors of hs-cTnT. After exclusion of outliers, the median was 15 ng/L and 99th percentile was 64 ng/L. After controlling for comorbidities, the 99th percentile increased from 24 ng/L at age 65 to 53 ng/L at age 90 in females and from 33 ng/L to 75 ng/L in males. In multivariate analysis, hs-cTnT level was significantly related to mortality. Conclusions: Hs-cTnT level is associated with inflammation and renal function in the elderly. Independently of comorbidities, hs-cTnT concentration increases exponentially with age after 65 years. Decision limits adapted to age and sex may be useful to patient management.

Background: The 99th percentile of cardiac troponin levels, determined in a reference population, is accepted as threshold for diagnosis of acute myocardial infarction (AMI). However, there is no common consensus of how to define the reference population. The aim of the present study was to determine 99th percentile reference values, determined by a high-sensitivity assay (hsTnI), according to different health status and cardiovascular risk factor prevalence in a large population-based sample. Methods: Troponin I was determined using the Abbott ARCHITECT STAT highly sensitive troponin I immunoassay in 4138 participants of the Gutenberg Health Study. Results: hsTnI was detectable in 81.6% of all individuals. The 99th percentile of the overall population was 27 ng/L. Age and gender had a prominent influence on these values. Exclusion of individuals with elevated natriuretic peptide levels or cardiac abnormalities resulted in lower 99th percentile values, whereas exclusion of individuals with an impaired estimated glomerular filtration rate (eGFR) or with prevalent coronary artery disease/myocardial infarction (CAD/MI) did not result in a meaningful change. Conclusions: Troponin I, measured by a high-sensitivity assay, can be reliably detected in the vast majority of the general population. hsTnI values were dependent on age, gender as well as structural and functional cardiac abnormalities.

Background: Cardiac biomarkers are the cornerstone of the biological definition of acute myocardial infarction (AMI). The key role of troponins in diagnosis of AMI is well established. Moreover, kinetics of troponin I (cTnI) and creatine kinase (CK) after AMI are correlated to the prognosis. New technical assessment like high-sensitivity cardiac troponin T (hs-cTnT) raises concerns because of its unclear kinetic following the peak. This study aims to compare kinetics of cTnI and hs-cTnT to CK in patients with large AMI successfully treated by percutaneous coronary intervention (PCI). Methods: We prospectively studied 62 patients with anterior AMI successfully reperfused with primary angioplasty. We evaluated two consecutive groups: the first one regularly assessed by both CK and cTnI methods and the second group by CK and hs-cTnT. Modeling of kinetics was realized using mixed effects with cubic splines. Results: Kinetics of markers showed a peak at 7.9 h for CK, at 10.9 h (6.9–12.75) for cTnI and at 12 h for hs-cTnT. This peak was followed by a nearly log linear decrease for cTnI and CK by contrast to hs-cTnT which appeared with a biphasic shape curve marked by a second peak at 82 h. There was no significant difference between the decrease of cTnI and CK (p=0.63). CK fell by 79.5% (76.1–99.9) vs. cTnI by 86.8% (76.6–92.7). In the hs-cTnT group there was a significant difference in the decrease by 26.5% (9–42.9) when compared with CK that fell by 79.5% (64.3–90.7). Conclusions: Kinetic of hs-cTnT and not cTnI differs from CK. The role of hs-cTnT in prognosis has to be investigated.

Background: Changes in high sensitivity cardiac troponin-T (hs-cTnT) concentrations may reflect either acute myocardial injury or biological variation. Distinguishing between these entities is essential to accurate diagnosis, however, the biological variation of hs-cTnT in dialysis population is currently unknown. We sought to estimate the within- and between-person coefficients of variation of hs-cTnT in stable dialysis patients, and derive the critical difference between measurements needed to exclude biological variation with 99% confidence. Methods: Fifty-five prevalent haemo- and peritoneal-dialysis patients attending two metropolitan hospitals were assessed on 10 consecutive occasions; weekly for 5 weeks then monthly for 4 months. Assessments were conducted at the same dialysis cycle time-point and entailed hs-cTnT testing, clinical review, electrocardiography, and bioimpedance spectroscopy. Patients were excluded if they developed clinical or physiological instability. Results: In total 137 weekly and 114 monthly hs-cTnT measurements from 42 stable patients were analysed. Respective between- and within-person coefficients of variation were 83% and 7.9% for weekly measurements, and 79% and 12.6% for monthly measurements. Within-person variation was unaffected by dialysis modality or cardiac co-morbidity. The bidirectional 99% reference change value was –25% and +33% for weekly measurements, and –37% and +58% for monthly measurements. Conclusions: The between-person variation of hs-cTnT in the dialysis population is markedly greater than within-person variation indicating that hs-cTnT testing is best applied in this population using a relative change strategy. An increase of 33% or a reduction of 25% in serial hs-cTnT concentrations measured at weekly intervals excludes change due to analytical and biological variation alone with 99% confidence.

Background: The objective of this study was to examine the diagnostic accuracy of high-sensitivity cardiac troponin T (hs-cTnT) for acute myocardial infarction (AMI) in patients with renal insufficiency, since this population has a high incidence of non-AMI elevations of hs-cTnT. Methods: In this prospective study, we enrolled 2249 consecutive patients presenting with chest pain in the emergency department (ED), of whom 19.5% had an estimated glomerular filtration rate (eGFR) cys of <60 mL·min –1 (1.73 m 2 ) –1 . Hs-cTnT levels were measured blindly at presentation. Results: Of the patients, 1108 (49.3%) were diagnosed as having AMI [321 with non-ST segment elevation myocardial infarction (NSTEMI)]. In patients whose final diagnosis was not AMI, there was a low but significant correlation between hs-cTnT and renal function [eGFR cys, r=–0.43 (–0.48, –0.38), p<0.001; eGFR creat , r=–0.33 (–0.38, –0.27), p<0.001]. The area under the curve of the receiver operating characteristic (AUC) for hs-cTnT to diagnose AMI was 0.93 in patients with eGFR cys levels of <30 mL·min –1 (1.73 m 2 ) –1 , and AUCs did not vary significantly according to eGFR categories. On the basis of the ROC curve, the optimal threshold value for hs-cTnT was 143.6 ng·L –1 to diagnose AMI in patients with eGFR cys of <30 mL·min –1 (1.73 m 2 ) –1 , with a sensitivity of 83% and a specificity of 91%; 54.1 ng·L –1 in patients with eGFR cys between 30 and 59 mL·min –1 , with a sensitivity of 90% and a specificity of 87%; 30.0 ng·L –1 in patients with eGFR cys between 60 and 89 mL·min –1 , with a sensitivity of 89% and a specificity of 85%; and 20.3 ng·L –1 in patients with eGFR cys ≥90 mL·min –1 (1.73 m 2 ) –1 , with a sensitivity of 92% and a specificity of 88%. The same observations were done for the diagnosis of NSTEMI. Conclusions: Using a higher hs-cTnT cut-off value based on eGFR level is necessary for accurate diagnosis of AMI or NSTEMI in patients with renal insufficiency.

Background: Ordering laboratory tests by means of test panels is a convenient way of requesting tests, preventing necessary tests from being forgotten. However, it also leads to redundant test ordering, as not all tests in a given panel are required for each patient. As test panels proposed by doctors may contain redundant, overlapping or infrequently used tests, the active involvement of knowledgeable laboratory staff in the organisation of test panels is advisable to promote efficient test use. Methods: Laboratory staff initiated an intervention in the organisation of test panels at our hospital in 2009. After a review of the existing panels and the proposals for new panels, we established a total of 60 panels (down from 171 previously). We also stipulated that the laboratory is to be involved with all proposals for new test panels in the future. Results: The reorganisation reduced the number of tests in the test panels by 17.7% (n=60), which theoretically should have resulted in 4.5% fewer tests being ordered. However, as an estimated 14% of the tests removed were then ordered individually in addition to the panels, 3.9% fewer tests were ordered, yielding an annual saving of about €58,000 (4.5% of the costs of all tests ordered in test panels). The savings amount to 7–8% if the frequently ordered metabolic panel (which was left unchanged) was excluded from the survey. Conclusions: Active intervention by the laboratory in the organisation of test panels results in a reduction in the use of tests and in interesting savings.

Background: Reference laboratories providing reference services recognized by the Joint Committee for Traceability in Laboratory Medicine (JCTLM) must be accredited as calibration laboratories according to ISO 17025 and ISO 15195. These standards require laboratories to establish an uncertainty budget, in which the uncertainty contributions of the relevant uncertainty components are specified. We present a model to estimate the measurement uncertainty of creatine kinase catalytic activity concentration results obtained by IFCC primary reference measurement procedure. Methods: The measurement uncertainty has been estimated by following the next steps: 1) specification of the measurand; 2) identification of the most relevant uncertainty sources; 3) estimation of standard uncertainties by either type A or type B evaluation; 4) estimation of combined uncertainty while taking into account sensitivity coefficients, as well as existence of correlated uncertainty sources; and 5) estimation of expanded uncertainty with a defined coverage probability. Results: The estimated expanded uncertainty was 2.2% ( k =2). Uncertainty sources with a significant contribution to the measurement uncertainty were the following: pH adjustment (0.68%), absorbance accuracy (0.48%), wavelength adjustment (0.20%), reaction temperature (0.19%), volume fraction of sample (0.15%) and absorbance linearity (0.06%). Conclusions: The present model is an approach to establish the uncertainty budget of primary reference procedures for the measurement of the catalytic activity concentration of enzymes, and aims at being an example to be followed by other reference laboratories, as well as by laboratories that carry out primary reference measurement procedures.

Background: Uric acid (UA) is a strong natural scavenger of reactive oxygen and nitrogen species, with evidence of possible use in the treatment of animal models of multiple sclerosis (MS). Consequently, serum UA has gained much attention as a possible biomarker of MS. We aim to investigate differences in serum UA levels between MS subjects and controls and evaluate possible relationships of UA with MS clinical features. Methods: We recruited relapsing-remitting and secondary progressive MS subjects and healthy controls and measured their serum UA levels. We excluded subjects presenting concomitant conditions affecting UA levels. Results: MS subjects (n=362) and controls (n=181) were recruited by propensity score matching (PSM). Statistical analyses were corrected for age, gender, and renal function. MS subjects presented significantly lower serum UA levels than controls (analysis of variance, p=0.014, adjusted r 2 =0.3036). Linear regression analysis showed a relationship between UA levels and disease duration (p<0.001, adjusted r 2 =0.3158, coefficient –0.00039), time from diagnosis (p<0.001, adjusted r 2 =0.3100, coefficient –0.0012), and Expanded Disability Status Scale (EDSS) (p<0.001, adjusted r 2 =0.3230, coefficient –0.1). Conclusions: Our findings support the importance of serum UA as a biomarker of MS disability and progression. Further studies with longitudinal design should be specifically designed to evaluate the importance of UA in the different stages of MS and in relation to distinct therapeutic strategies.

Background: Previous studies have shown that the measurement of vitamin D and its derivatives, especially its active metabolite 1α, 25-dihydroxy-vitamin D [1,25(OH) 2 D], is highly complex and prone to analytical error. We have evaluated a new immunological method for detecting and quantifying of 1,25(OH) 2 D. This assay is fully automated, sensitive and uses a specific recombinant fusion protein for capturing of 1,25(OH) 2 D. The assay was originally developed by DiaSorin for the immunoassay analyzer LIAISON XL. Methods: Performance data of this assay were determined including intra- and inter-assay precision, recovery, linearity, and limit of detection of the DiaSorin 1,25(OH) 2 D immunoassay on the LIAISON XL analyzer. Respective data were compared from two different liquid chromatography tandem mass spectrometry (LC-MS/MS) assays and a common radioimmunoassay (RIA) using clinical samples taken from patients suffering from vitamin D deficiency, chronic renal failure, biliary atresia, hyperparathyroidism, vitamin D-dependent rickets or sarcoidosis, as well as from pregnant women and high-level athletes. Results: The performance evaluation of 1,25(OH) 2 D resulted in an intra-assay and total imprecision correlation variant between 1.4% and 5.2% and 3.8%–7.1% with the new immunoassay and 3.5%–5.8% or 3.8%–7.5% with the LC-MS/MS method, respectively. Limits of detection and quantification of the immunoassay were 0.7 ng/L and 5.0 ng/L for the LIAISON XL immunoassay and 1.8 ng/L and 5.4 ng/L for the LC-MS/MS assay, respectively. Pearson’s coefficients of correlation were 0.998 and 0.952 for method comparison to different established LC-MS/MS methods. Linear regression according to Passing and Bablok showed larger deviations to the RIA (slopes 0.64–0.97, coefficients of correlation 0.822–0.823). Conclusions: The DiaSorin LIAISON XL 1,25(OH) 2 D immunoassay appears to have improved comparability to LC-MS/MS with low imprecision and limits of detection. The assay time of 65 min, the small sample volume required (75 μL) and the throughput of 90 tests/hour without manually handling time for extraction and purification procedures is superior to the LC-MS/MS method.

Background: Trauma patients sustaining abdominal trauma exhibit high risk of organ failure and/or sepsis aggravating morbidity and mortality during the post-traumatic course. The present study re-evaluates L- and I-FABPs (small fatty acid binding proteins) as early biomarkers for abdominal injury (AI) in a large cohort of patients and analyzes their potential as indicators of specific organ failure and their association with sepsis and/or mortality in the post-traumatic course. Methods: This prospective study included 134 multiply traumatized patients (ISS≥16). Fifty-nine had AI (abbreviated AI Scale, AIS Abd ≥3) and 75 had no AI (noAI). Twenty healthy volunteers served as controls. Plasma I- and L-FABP levels were measured at the admittance to the emergency room (d0) and up to 10 days daily (d1–d10) using ELISA. Sepsis, organ failure, multiple organ failure (MOF) and mortality were assessed. Results: Median L- and I-FABP in the AI-group [258 (IQR=71–500) ng/mL and 328 (IQR=148–640) pg/mL, respectively] were higher compared to noAI-group [30 (IQR=18–50) ng/mL and 60 (IQR=40–202) pg/mL, p>0.05] on d0. Sensitivity and specificity to detect AI were 80% and 75% for L-FABP, 78% and 62% for I-FABP. Both FABPs decline with the post-traumatic course to control levels. On d0 and d1, FABPs correlate with the Sepsis-related Organ Failure Assessment (SOFA) score of the following day (d0: ρ :0.33, ρ :0.46, d1: ρ :0.48, ρ :0.35). No other correlations were found. Eight percent of all patients developed sepsis, 18% pneumonia, 4% urinary tract infection, 3% acute kidney failure and one MOF. FABPs correlated neither with Simplifed Acute Physiology Score (SAPS)-II nor to sepsis. All patients with acute kidney failure demonstrated enhanced L-FAPB levels before the increase of serum creatinine levels. Conclusions: Our results confirm the potential of L- and I-FABP to indicate abdominal injuries initially after trauma. Except L-FABP as indicator of acute kidney failure both FABPs have to be further evaluated as predictors for other organ failures, sepsis and/or mortality.

Background: Recent studies have demonstrated highly variable blood concentrations of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in critically ill patients with a high incidence of sub-therapeutic levels. Consequently, therapeutic drug monitoring (TDM) of these antibiotics has to be considered, requiring robust and reliable routine analytical methods. The aim of the present work was to develop and validate a multi-analyte ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of the above mentioned antibiotics. Methods: Sample preparation included a manual protein precipitation step followed by two-dimensional ultra high performance liquid chromatography (2D-UHPLC). Corresponding stable isotope-labeled substances were used as internal standards for all of the analytes, with the exception of tazobactam. The injected sample volume was 7 μL. The run time was 5.0 min. Results: Inaccuracy was ≤8% and imprecision coefficient of variation (CV) was <9% for all analytes. Only minor matrix effects and negligible carry-over was observed. The method was found to be robust during the validation period. Conclusions: We were able to develop a reliable 2D-UHPLC-MS/MS method addressing analytes with highly heterogeneous physico-chemical properties. The novel assay may be an efficient tool for an optimized process workflow in clinical laboratories for important antibiotics in regards to TDM.

Background: Studies assessing plasma visfatin/nicotinamide phosphoribosyltransferase (NAMPT) concentrations in chronic kidney disease with the ELISA method are restricted mainly to subjects with end-stage kidney disease. Therefore, little is known about to what extent glomerular filtration rate (GFR) affects the plasma levels of visfatin/NAMPT. The aim of this study was to assess the relations between circulating visfatin/NAMPT levels and estimated GFR (eGFR), independently of potential confounders such as inflammation, nutritional status, and insulin resistance in the elderly population. Methods: The analysis included 3023 elderly subjects (1076 with impaired kidney excretory function – eGFR <60 mL/min/1.73 m 2 ) who were participants of the PolSenior study. Serum insulin, glucose, creatinine, C-reactive protein, interleukin-6, and plasma visfatin/NAMPT concentrations were measured by a highly specific ELISA method. Insulin resistance was assessed on the basis of homeostasis model assessment for insulin resistance, and kidney excretory function was assessed using the full MDRD formula. Results: Similar plasma visfatin/NAMPT levels were found in subjects with eGFR ≥60 and <60 mL/min/1.73 m 2 (0.96 ng/mL in both groups), and even in those subjects with eGFR 15–30 mL/min/1.73 m 2 (0.83 ng/mL). Additionally, there was no association between plasma visfatin/NAMPT concentrations and eGFR values in models of regression analysis including confounding factors. Conclusions: The results of our study suggest that plasma visfatin/NAMPT levels are not affected by impaired kidney excretory function in elderly subjects.

Background: The parallelization of clinically relevant antigens in a microarray format is of growing importance due to the ability to measure multiple antigen-antibody interactions. With the development of a microarray for the detection of antiphospholipid antibodies we focussed on one important autoimmune disease that is still diagnostically challenging. Reasons are the heterogeneity of the autoantibodies and the unspecific clinical symptoms. Methods: For the covalent immobilization of antigenic structures, glass transducers were coated with 11-aminoundecyltrimethoxysilane (11-AUTMS). In total 35 antiphospholipid syndrome (APS) patients, six patients with lupus erythematosus and 24 healthy controls were investigated on a microarray format using polarized imaging reflectometric interference spectroscopy. Results: The novel surface modification based on the short derivative 11-AUTMS resulted in a selective biosensor allowing a clear differentiation of patient and control samples. It combined proteinogenic as well as phospholipid-derived antigens, namely β 2 -glycoprotein I (β 2 -GPI), prothrombin, cardiolipin (CL) and a β 2- GPI/CL complex. With optimized regeneration conditions, up to 20 consecutive measurements could be performed on one chip. Sensitivity was determined to be 0.800–0.929, specificity was between 0.733 and 0.969, depending on the respective antigen. Conclusions: Multiplexed determination of serological parameters has a great potential. We have shown that our biosensor is capable of detecting four different APS relevant antibodies in parallel exhibiting a sensitivity and specificity comparable to existing ELISA methods.

Background: Tests for lupus anticoagulant (LA), including silica clotting time (SCT) and diluted Russel’s viper venom time (dRVVT) are used to diagnose antiphospholipid syndrome. Due to sample instability, it is recommended that samples are frozen if analysis is postponed >4 h. Shipping on dry ice is common practice to keep samples frozen during transport. Recent data suggest that exposure to dry ice may affect sample pH and results in subsequent analyses. We aimed to determine the effect of dry ice on pH and LA analysis. Methods: Citrated plasma from eight healthy volunteers was allocated to three preanalytical regimes: 1) storage at –20 °C; 2) dry ice exposure followed by storage at –20 °C; or 3) dry ice exposure followed by storage at –80 °C. Results: Samples stored at –20 °C after dry ice exposure had significantly lower median pH (1.2 units, p=0.01) and prolonged clotting time ratios (up to 55% for dRVVT tests, p<0.02) in LA analysis, compared to samples not exposed to dry ice. This resulted in poor test specificity (25%). Similar changes were not observed in samples placed at –80 °C after dry ice exposure. Conclusions: Dry ice may affect sample pH and increase the fraction of false positive LA results. This preanalytical factor should be taken into account by laboratories receiving frozen samples for these tests.

Background: Population-based reference intervals have very limited value for the interpretation of laboratory results when analytes display high biological individuality. In these cases, the longitudinal evaluation of individual results using the reference change value (RCV) is the recommended approach. However, the traditional model for RCV calculation requires a Gaussian frequency distribution of data and risks to overestimate the parameter if a correlation between within-subject serial measurements is present. Methods: We propose and validate an alternative non-parametric statistical model for interpretation of differences in serial results from an individual, overcoming data distribution and correlation issues. Results: After describing the traditional and newly proposed statistical models, we compared them with each other using a simulation on three specific analytes displaying different concentration distributions in biological setting. We demonstrated that when analyte concentrations followed a Gaussian frequency distribution, as in the case of glycated hemoglobin, both methods can be used equally. On the contrary, if analyte concentrations present a bimodal (e.g., chromogranin A) or skewed (e.g., C-reactive protein) distribution, the information obtained by two statistical methods is different. Conclusions: The proposed statistical approach may be more appropriate in assessing difference between serial measurements when individual data are not normally distributed.

Background: Red cell distribution width (RDW) is a routinely examined parameter with the complete blood count. In recent studies, RDW levels have been associated with cardiovascular, liver and renal diseases and solid tumors. The aim of this study was to evaluate the alterations of RDW levels in benign and malignant causes of postmenopausal bleeding and to investigate the association of RDW levels with clinicopathological parameters of endometrial cancer (EC) patients. Methods: A retrospective study was made of a total of 884 females who were admitted to hospital for postmenopausal bleeding between May 2009 and December 2013. After inclusion and exclusion criteria were applied, 222 patients remained. Complete blood count data was obtained from the recorded computerized database. After pathological evaluation, the patients were divided into two groups, benign and malignant (EC). Results: The EC group (n=113) had significantly higher RDW levels compared to the benign group (14.78±2.02 vs. 13.88±1.05; p=0.000). Grade II and above EC patients had higher levels of RDW than Grade I patients (15.2±2.3 vs. 14.1±1.00; p=0.005). Correlation analyses also revealed a negative correlation between RDW and hemoglobin levels (p=0.000), RDW and mean corpuscular volume (p=0.000), RDW and lymphocyte count (p=0.035) but a positive correlation between RDW and platelet to lymphocyte ratio (p=0.030). Conclusions: The results of the current study revealed the potential predicitve role of RDW in patients with postmenopausal bleeding. Significant associations were also determined between RDW and clinicopathological characteristics in EC patients.