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European and Australian guidelines for cystic fibrosis (CF) reproductive carrier screening recommend testing a small number of high frequency CF causing variants, rather than comprehensive CFTR sequencing. The study objective was to determine variant detection rates of commercially available targeted reproductive carrier screening tests in Australia.Next-generation DNA sequencing of the CFTR gene was performed on 2552 individuals from a whole population sample to identify CF causing variants. The variant detection rates of two commercially available Australian reproductive carrier screening tests, which target 50 or 175 CF causing variants, in this population were calculated. The ethnicity of individuals was determined using principal component analysis.Variant detection rates of the tests for 50 and 175 CF causing variants were 88.2% and 90.8%, respectively. No CF causing variants in individuals of East Asian ethnicity (n = 3) were detected by either test, while86.6% (n = 69) of CF causing variants in Europeans would be identified by either test.Reproductive carrier screening tests for a targeted set of high frequency CF variants are unable to detect approximately 10% of CF variants in a multiethnic Australian population, and individuals of East Asian ethnicity are disproportionally affected by this test limitation.

Objectives We evaluated the analytical performance characteristics and the biological equivalence of the Atellica TnIH assay. Methods Precision, detection capability, linearity, and sex specific 99th percentiles were determined de novo. Classification of patients relative to the 99th percentiles was used to assess biological equivalence. Results Analytical precision and detection capability of the Atellica TnIH assay is excellent with a limit of blank ≥±1.96) occurred in 65% (95% CI 53–76%) of 99th percentile discordant patients compared to 2.7% (p Conclusions The 99th percentile discordant and the concordantly elevated groups are more alike with respect to their z≥±1.96 rates. This favours an overestimated Atellica TnIH 99th percentile as more likely, and we hypothesize that antibody interference resulting in asymmetric scatter of nearly 5% samples may be the underlying mechanism. Analytical accuracy and interferences in cardiac troponin assays should be investigated and resolved with high priority.

Background Total human chorionic gonadotropin (hCGt) tumour marker testing is regarded as an “off label” application for most commercial methods. We compared four assays in patients with a hCGt tumour marker request. We hypothesised that regression slopes would be altered and that outliers would be more common with tumour marker than with pregnancy samples if the detection of malignancy associated hCG molecular forms differed amongst assays. Further such systematic differences would be obvious and large enough to change clinical management decisions. Results We measured hCGt in 390 samples from 137 females and 253 males with a tumour marker request and 208 pregnancy controls with the following methods: Access Total βhCG, Architect Total-βhCG, Cobas hCG + β and Immulite HCG. The between method regressions determined on tumour marker and pregnancy samples were not significantly different. The outlier rates were similar for male and female tumour marker and the pregnancy groups: 1.6% (95% confidence interval [CI] 0%–3.1%), 2.2% (95% CI 0%–4.7%) and 2.9% (95% CI 0.6%–5.2%). The outliers were randomly distributed amongst the methods and we were confident that they would not adversely influence clinical decisions. Conclusions The hCGt results were clinically equivalent with no systematic difference amongst the four assays.

Introduction We investigated the analytical performance, outlier rate, carryover and reference interval of the Beckman Coulter Access hsTnI in detail and compared it with historical and other commercial assays. Materials and methods We compared the imprecision, detection capability, analytical sensitivity, outlier rate and carryover against two previous Access AccuTnI assay versions. We established the reference interval with stored samples from a previous study and compared the concordances and variances with the Access AccuTnI+3 as well as with two commercial assays. Results The Access hsTnI had excellent analytical sensitivity with the calibration slope 5.6 times steeper than the Access AccuTnI+3. The detection capability was markedly improved with the SD of the blank 0.18–0.20 ng/L, LoB 0.29–0.33 ng/L and LoD 0.58–0.69 ng/L. All the reference interval samples had a result above the LoB value. At a mean concentration of 2.83 ng/L the SD was 0.28 ng/L (CV 9.8%). Carryover (0.005%) and outlier (0.046%) rates were similar to the Access AccuTnI+3. The combined male and female 99th percentile reference interval was 18.2 ng/L (90% CI 13.2–21.1 ng/L). Concordance amongst the assays was poor with only 16.7%, 19.6% and 15.2% of samples identified by all 4 assays as above the 99th, 97.5th and 95th percentiles. Analytical imprecision was a minor contributor to the observed variances between assays. Conclusion The Beckman Coulter Access hsTnI assay has excellent analytical sensitivity and precision characteristics close to zero. This allows cTnI measurement in all healthy individuals and the capability to identify numerically small differences between serial samples as statistically significant. Concordance in healthy individuals remains poor amongst assays.

A term baby boy was born in a hospital by normal vaginal delivery following prolonged premature rupture of membranes (>18 h). Antibiotic cover had been suboptimal. The baby was breastfed and well, but he was admitted to the special care nursery for antibiotic therapy. By the second day, the baby developed an unconjugated hyperbilirubinaemia. A complete blood count showed an increased leukocyte count 33.4 × 109/L and neutrophilia, normal hemoglobin, red cell count, and platelets. ABO incompatibility was excluded. The neonate was treated for suspected neonatal sepsis and phototherapy commenced. ### DIAGNOSIS On the second day after birth, a newborn screening card (dried blood spot) was collected and sent to the laboratory. An increased galactose/galactose-1-phosphate concentration of 3.2 mmol/L was detected (cutoff < 1.5 mmol/L). Galactose-1-phosphate uridyltransferase (GALT)4 activity confirmed the absence of GALT activity (

An 8-year-old boy presented to the emergency department with complaints of intermittent stiffening of his upper and lower limbs. Shortly after his arrival he had a large volume (approximately 500 mL) of nonbilious projectile vomit. He described general weakness and fatigue the previous night and on waking, with no history of headaches, diarrhea, fevers, or other symptoms of infection. In the past 4 months he had experienced intermittent generalized abdominal pain, nausea, anorexia, vomiting, and mild weight loss (3 kg). He had presented 2 months earlier with vomiting. Investigations including a complete blood count, electrolytes, liver function tests, albumin-corrected calcium, magnesium, and phosphate were within reference intervals at that time. An abdominal ultrasonography was unremarkable. The episodes of vomiting had become increasingly more frequent and forceful. His past medical history was otherwise unremarkable. On presentation he was afebrile but tachycardic (pulse rate 156 bpm), tachypneic (respiratory rate 32/min), and mildly hypertensive (bp 125/78 mmHg). He was intermittently drowsy but easily rousable. He was described as having “locked hands and curled toes” consistent with carpopedal spasms and twitching movements of his tongue. He had no focal neurological signs, and his power and reflexes were essentially normal. There were no cardiac murmurs, and his respiratory examination was unremarkable. His abdomen was soft and nontender, with normal bowel sounds. Biochemical testing on presentation showed hypokalemia, hypochloremia, and hyperbicarbonatemia (Table 1). He had evidence of dehydration and prerenal azotemia (increased urea, creatinine, and urate) and normal total protein and albumin. His liver function tests, lipase, thyroid function tests, albumin-corrected calcium, phosphate, and magnesium were normal. An electrocardiogram revealed a prolonged corrected QT interval (QTc 480 ms). Despite the normal albumin-corrected calcium, the presence of carpopedal spasms and the …

There is increasing need to identify individuals with advanced liver fibrosis, who are at risk of complications such as hepatocellular carcinoma. The commercially available enhanced liver fibrosis (ELF) test provides a non-invasive assessment of fibrosis severity. This study was designed to determine the diagnostic accuracy of the manufacturer's cut-off value (≥9.8) in identifying advanced fibrosis.The relationship between ELF score and fibrosis was examined using serum collected at time of liver biopsy for investigation of liver disease, particularly viral hepatitis. Fibrosis was staged using a modified METAVIR score. If available, liver tissue was recut and stained with Sirius red to determine collagen proportional area (CPA) and subsinusoidal fibrosis (SSF).Enhanced liver fibrosis score ≥9.8 had a sensitivity of 74.4% and specificity 92.4% for detecting advanced fibrosis. In the whole cohort (n = 329), ELF score was more likely to incorrectly classify individuals if age was ≥45 years and METAVIR inflammatory grade was 2 or 3 (adjusted OR, odds ratio 3.71 and 2.62 respectively). In contrast, ELF score was less likely to misclassify individuals in the presence of steatosis (OR 0.37). Neither SSF nor CPA explained the discordance in ELF score for patients with or without advanced fibrosis.Although ELF score ≥9.8 reliably identifies advanced fibrosis in patients with chronic liver disease, both age and inflammatory activity need to be considered when interpreting the result. Importantly, ELF score performed well in the presence of steatosis and could thus be helpful in the assessment of fatty liver disease.

Background Measured (MO) and calculated osmotic concentrations (CO) and the osmotic gap (OG) are commonly used in the investigation of electrolyte and volume disturbances as well as in cases of suspected volatile ingestion. Methods We compared 38 published formulae for CO with MO on a large data-set ( n = 9466) and adjusted the CO with the result of a Passing-Bablok regression procedure. Validation of this adjustment was performed with a separate data-set ( n = 2082). Results All but one of the CO formulae underestimate MO due to a proportional bias (slope 0.67 to 0.95) and the OG limits were therefore not applicable throughout the observed range. Using Passing-Bablok regression to adjust the CO: CO#3 = (2 × Na+urea+glucose−14.54)/0.93. After adjustment, the mean OG was 0.3 mmol/L with a SD of 5.1 mmol/L across the measurement interval. The distribution of the OG could be fully explained by the analytical imprecision of the measured components. Conclusions Simple adjustment of the CO for the proportional underestimation of MO allows OG reference limits of approximately −10 to +10 mmol/L to be used, even in the upper ranges of CO in patients with suspected volatile ingestion.

Background False-positive cardiac troponin I results as a result of carryover have previously been reported on the Beckman Coulter AccuTnI assay. We sought to determine if the carryover problem had been resolved with the new AccuTnI + 3 assay. Methods Carryover experiments were performed in parallel on the Beckman Coulter Access2 analyser using the legacy AccuTnI and new AccuTnI + 3 assays. The same negative patient pool sample was analysed before and after a single analysis of an extremely elevated patient sample. Results Analysis of a single extremely high sample caused elevations above the 99th percentile cut-off, and thus false-positive cardiac troponin I results on both assays. Both assays demonstrated carryover and subsequent further elevations in negative pool results the following day. Conclusions Our study replicates our previously published findings of carryover and reagent pack contamination on the AccuTnI assay. Despite improvements on the new AccuTnI + 3 assay, carryover and reagent pack contamination are still present.

Aims: We assessed the diagnostic performance of z-scores to define a significant delta cardiac troponin (cTn) in a cohort of patients with well-defined clinical outcomes. Methods: We calculated z-scores, which are dependent on the analytical precision and biological variation, to report changes in cTn. We compared the diagnostic performances of a relative delta (%Δ), actual delta (Δ), and z-scores in 762 emergency department patients with symptoms of suspected acute coronary syndrome. cTn was measured with sensitive cTnI (Beckman Coulter), highly sensitive cTnI (Abbott), and highly sensitive cTnT (Roche) assays. Results: Receiver operating characteristic analysis showed no statistically significant differences in the areas under the curve (AUC) of z-scores and Δ with both superior compared to %Δ for all three assays (p

Background The clinical utility of serum κ and λ free light chains (FLC) for the diagnosis and prognosis of plasma cell proliferative disorders is well established. We assessed the analytical performance of the N Latex FLC assays and compared it with the Freelite™ assays. Methods Analytical precision was assessed according to the CLSI EP5-A2 protocol. Method comparison was performed with 116 clinical samples and linearity was assessed on samples with monoclonal and polyclonal elevations of FLC. Analytical bias and variance was measured with two lots of N Latex FLC reagent. Results The N Latex FLC κ and λ assays had a total coefficient of variation of 5–7% across the analytical range. The slopes were 1.36 and 1.37 and the between-method variances were 40.0% and 45.4%, respectively, compared with the Freelite™. Good agreement in classification was observed for κ, λ and the κ/ λ ratio (Cohen's κ 0.84, 0.76 and 0.89). Statistical non-linearity occurred commonly, but clinically significant non-linearity was observed in only one instance. Between-reagent lot variation was 7.3% and 10% for κ and λ, respectively. Conclusions The N Latex FLC assay has good precision, did not exhibit gross antigen excess and can be used in clinical practice based on the analytical performance characteristics.

Background Cardiac troponin (cTn) has become the standard biomarker for the diagnosis of acute coronary syndromes. False-positive cTnI results have previously been reported on the Beckman Coulter analysers, which were shown to be random, not reproducible and occurred more commonly than expected. Our investigation ensued after a patient sample with an inordinately elevated cTnI was analysed, followed by a series of false-positive results being reported. The implications of falsely elevated cTnI results on patient care could be considerable. Methods Multiple experiments with patient sample pools with concentrations below the 99th percentile to extremely high (0.025, 15, 175 and 884 μg/L) were conducted in varying sequences of high and low samples on the Beckman Coulter Access2, UniCel DxI600 and UniCel DxI800 analysers. Results Our results demonstrate a significant increase in cTnI concentrations in the negative pool after analysis of high pool samples in various sequences. This increase is sufficient to cause elevations above the 99th percentile cut-off and false-positive cTnI results. These findings were reproducible on all three analysers. Conclusions Our study is highly suggestive of carryover and cTnI reagent pack contamination by the pipettors on the Access2, DxI600 and DxI800 analysers when patient samples with extremely high cTnI concentrations are analysed, leading to potential false-positive cTnI results on subsequent samples.

BACKGROUND Data to standardize and harmonize the differences between cardiac troponin assays are needed to support their universal status in diagnosis of myocardial infarction. We characterized the variation between methods, the comparability of the 99th-percentile cutoff thresholds, and the occurrence of outliers in 4 cardiac troponin assays. METHODS Cardiac troponin was measured in duplicate in 2358 patient samples on 4 platforms: Abbott Architect i2000SR, Beckman Coulter Access2, Roche Cobas e601, and Siemens ADVIA Centaur XP. RESULTS The observed total variances between the 3 cardiac troponin I (cTnI) methods and between the cTnI and cardiac troponin T (cTnT) methods were larger than expected from the analytical imprecision (3.0%–3.7%). The between-method variations of 26% between cTnI assays and 127% between cTnI and cTnT assays were the dominant contributors to total variances. The misclassification of results according to the 99th percentile was 3%–4% between cTnI assays and 15%–17% between cTnI and cTnT. The Roche cTnT assay identified 49% more samples as positive than the Abbott cTnI. Outliers between methods were detected in 1 patient (0.06%) with Abbott, 8 (0.45%) with Beckman Coulter, 10 (0.56%) with Roche, and 3 (0.17%) with Siemens. CONCLUSIONS The universal definition of myocardial infarction should not depend on the choice of analyte or analyzer, and the between- and within-method differences described here need to be considered in the application of cardiac troponin in this respect. The variation between methods that cannot be explained by analytical imprecision and the discordant classification of results according to the respective 99th percentiles should be addressed.

BACKGROUND It is important that cardiac troponin be measured accurately with a robust method to limit false results with potentially adverse clinical outcomes. In this study, we characterized the robustness of 4 analytical platforms by measuring the outlier rate between duplicate results. METHODS We measured cardiac troponin concurrently in duplicate with 4 analyzers on 2391 samples. The outliers were detected from the difference between duplicate results and by calculating a z value: z = (result 1 − result 2) ÷ √(SD1est2 + SD2est2), with z > 3.48 identifying outliers with a probability of 0.0005. RESULTS The outlier rates were as follows: Abbott Architect i2000SR STAT Troponin-I, 0.10% (0.01%–0.19%); Beckman Coulter Access2 Enhanced AccuTnI, 0.44% (0.25%–0.63%); Roche Cobas e601 TroponinT hs, 0.06% (0.00%–0.13%); and Siemens ADVIA Centaur XP TnI-Ultra, 0.10% (0.01%–0.19%). The occurrence of outliers was higher than statistically expected on all platforms except the Cobas e601 (χ2 = 2.7; P = 0.10). A conservative approach with a constant 10% CV and z > 5.0 identified outliers with clear clinical impact and resulted in outlier rates of 0.11% (0.02%–0.20%) with the Architect i2000SR STAT Troponin-I, 0.36% (0.19%–0.53%) with the Access2 Enhanced AccuTnI, 0.02% (0.00%–0.06%) with the Cobas e601 TroponinT hs, and 0.06% (0.00%–0.13%) with the ADVIA Centaur XP TnI-Ultra. CONCLUSIONS Outliers occurred on all analytical platforms, at different rates. Clinicians should be made aware by their laboratory colleagues of the existence of outliers and the rate at which they occur.

Point-of-care testing for β-hCG has been widely advocated to allow rapid diagnosis/exclusion of pregnancy in the emergency department. A quantitative blood β-hCG assay has the additional benefit of being able to monitor the viability of pregnancy, using serial measurements, to determine the appropriate expected increase in β-hCG levels over time (e.g. ectopic pregnancy), and aiding in determining if an intrauterine gestational sac should be visible on sonographic imaging. Objectives Evaluation of the newly released Abbott i-STAT β-hCG point-of-care assay with the Beckman Coulter β-hCG laboratory assay in use. Design and methods Whole blood, plasma and serum samples with a wide range of β-hCG concentrations were analysed by both methods. Results The Abbott I-STAT β-hCG compares favourably, can be performed on heparinised whole blood, plasma and serum, and shows acceptable accuracy and precision. However a hook effect at elevated β-hCG was shown in gestational trophoblastic disease as well as normal pregnancies. Conclusions The i-STAT β-hCG performs acceptably in its intended use in the early detection of pregnancy, but results should always be interpreted within the clinical context, as a hook effect may occur.

The recent article in Clinical Chemistry by Katzmann et al. (1) on screening panels to detect monoclonal gammopathies provided important information on the use of the free light chain (FLC)1 assay, and we commend the authors for this report. Apart from the selection bias inherent in the exclusion of 90% of cases of monoclonal gammopathy of uncertain significance (MGUS), we would like to address some aspects that, in our opinion, may enhance understanding of the role of various diagnostic strategies to detect monoclonal gammopathies in routine laboratories. Katzmann and coworkers did not make any statement regarding the statistical significance of their findings, nor did they provide CIs of the percentages in their Table 2. In deciding whether a particular diagnostic strategy is worthwhile to implement, it is useful to know whether any observed …

Background: The purpose of this study was to evaluate a combined κ and λ light chain immunofixation (CLIF) as a screening tool to detect monoclonal immunoglobulins in serum and urine. A secondary aim was to investigate the impact on workflow and reagent utilisation of a systematic implementation of CLIF in addition to routine protein electrophoresis (PE) on all samples. Methods: Light chain antisera ( κ and λ ) were mixed in a 1:1 ratio and loaded in the same sequence as the PE to create a superimposable image. Results: The CLIF procedure agreed significantly better with standard immunofixation procedures in the serum and urine. In 33 (22%) new patients and in 114 (15%) follow-up patients CLIF detected a band missed by PE in serum. In 34 (4.5%) of previously categorised cases the monoclonal band was below the detection limit of CLIF in serum, but still detectable by conventional immunofixation electrophoresis. In one case (0.7%) a band in a urine specimen was missed by CLIF compared to 70 (49%) missed by PE. After the systematic introduction of CLIF turn-around-times (TATs) and utilisation of laboratory consumables decreased significantly (p < 0.001). Conclusions: A systematic implementation of CLIF led to the detection of monoclonal bands missed by PE with an improvement in TATs and a decrease in cost.

We welcome the critique of our paper by Basu et al. The analytical challenges of free light chain (FLC) assays are well documented and need to be considered when evaluating an alternative method. The precision study was performed at the correct sample dilution as recommended by the manufacturers. The results are a reflection of the performance of the assays and are similar to those reported elsewhere. We note a lack of references to contradict our findings. Selecting samples to uniformly cover the measurement range is an elemental aspect of method comparison studies and avoids evaluation of skewed data with a limited range. As stated, we recruited samples for the method comparison based on the Freelite result until we had sufficient numbers in each category. The distribution of results relative to the proposed cut-off points led to our discussion on reference ranges which we qualified by pointing out the need for a formal reference range study. The recommendation that we should repeat discordant results at different dilutions is interesting and we refer the authors to Table 1. The requirement that a new assay must be validated de novo in clinical studies before use needs to be carefully considered. Both the Freelite and N Latex assays are approved for clinical use by the relevant regulatory authorities and furthermore the guidelines do not appoint an exclusive manufacturer. In our opinion the clinical usefulness of an assay does not require perfect alignment with a ‘predicate’ assay, a point that is very well illustrated by the Freelite assays as clinical validation studies are not called for each batch of reagent and every analytical platform, despite considerable variation. After consideration of our data and the comments by Basu, we maintain our opinion that the N Latex FLC assay can be used in clinical practice based on its analytical performance characteristics. DECLARATIONS

Summary Aims Differentiation between thrombotic thrombocytopenic purpura (TTP) and other microangiopathic haemolytic anaemia (MAHA) processes can be difficult. Since the documentation of ADAMTS-13 deficiency in TTP several ADAMTS-13 activity assays have been developed for use in the diagnosis of TTP and/or other microangiopathic disorders. We reviewed the clinical utility of ADAMTS-13 activity testing in suspected TTP as used in routine clinical practice in a tertiary referral hospital. Methods All requests for ADAMTS-13 activity levels performed at our institution after introduction of the assay were retrospectively audited with respect to clinical diagnosis and results. Results In total 57 individual patients were tested, of whom only 46% had a MAHA process. Severe ADAMTS-13 deficiency was present in five TTP patients and in one patient with fulminant hepatic failure. Conclusions Our experience suggests that severely reduced levels are relatively specific for TTP, but may also occur in fulminant hepatic failure. Patients without MAHA may have reduced ADAMTS-13 activity and there is significant overlap in the range of ADAMTS-13 activity seen in non-TTP MAHA diagnoses. This supports the observation that outside the diagnosis and (possible) follow-up of suspected idiopathic TTP the ADAMTS-13 activity assay has limited clinical utility. Further education about the role of ADAMTS-13 activity testing is needed.

Introduction The diagnosis of myocardial infarction is in part predicated on a rise and/or fall in cardiac troponin (cTn). z-Values incorporate analytical and biological variation to standardize serial differences: z = Δ / 2 S D Analytical 2 + 2 S D Biological 2 . We investigated the theoretical distributions of actual differences (Δ), relative differences (%Δ) and z-values and compared the agreement in classification of differences measured on four contemporary platforms. Methods cTn was measured in 575 sample pairs on the Abbott Architect cTnI, Beckman Coulter Access2 cTnI, Roche Cobas cTnT and Siemens ADVIA Centaur cTnI methods. Results Good agreement was obtained amongst all methods with a universal z-value cut-off (κ>0.79) and method specific fixed Δ cut-offs (κ>0.82) while suboptimal agreement was obtained between cTnI and cTnT with fixed %Δ cut-offs (κ Conclusion Fixed Δ cut-offs will perform well at low cTn concentrations while fixed %Δ cut-off values are predicted to perform poorly. z-Values are independent of the cTn concentration, present differences as a continuum of probability and a universal decision level can be used for all cTnI and cTnT methods.

No Abstract.