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Background: Measurement of serum 25-hydroxyvitamin D [25(OH)D] is used to assess vitamin D status. We evaluated the analytical performance of a new automated assay, Elecsys Vitamin D Total (Roche Diagnostics, Mannheim, Germany), based on competitive protein binding. Methods: The Elecsys assay was tested for imprecision, linearity and functional sensitivity at three test-sites and compared to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, a high-performance liquid chromatography (HPLC) method and the Liaison 25(OH) Vitamin D Total immunoassay (Diasorin). Results: Imprecision testing with human serum specimens showed within-run CVs of ≤6% and between-run CVs of ≤8%. The assay was linear from 33 up to at least 111 nmol/L and showed equivalent 25(OH)D levels for matched serum and heparinized plasma samples. The assay correlated reasonable to well with LC-MS/MS (r=0.93; y=1.07x–5.04 nmol/L), HPLC (r=0.91, y=0.90x+3.03 nmol/L) and the Liaison assay (r=0.86, y=1.19x+2.80 nmol/L). Some of the samples showed large between-method differences. Conclusions: The new Elecsys assay fulfilled present analytical performance requirements and showed close agreement to other well-established methods for 25(OH)D analysis, making it fit for routine assessment of vitamin D status.

The Clinical and Laboratory Standards Institute (CLSI) recently abandoned its recommendation for drawing a discard tube when performing a prothrombin time (PT)/international normalized ratio (INR) or an activated partial thromboplastin time (APTT). Because there is currently no evidence that a discard tube is necessary for more specialized coagulation assays, we studied the need for a discard tube for some of these tests. Blood was obtained from 88 subjects in 2 subsequent citrate tubes. Platelet-free plasma was tested for PT, APTT, antithrombin, protein C, and factors II, V, VIII, IX, and X. Difference and bias between tubes were tested using the Wilcoxon signed rank test and Bland-Altman plots. For only APTT, antithrombin, and protein C was a small, statistically significant mean bias found (0.5 seconds; P = .001; –0.7%, P = .002; and –0.8%, P < .0001, respectively), but the bias of individual samples was not clinically relevant. This was also true for the other parameters tested. The recent CLSI recommendation that a discard tube is not necessary for PT/INR and APTT can be extended to include more specialized plasma-based coagulation assays as identified in this study. Preanalytic variables may affect test results. In particular, coagulation assays are sensitive to variation in conditions during the collection, transport, and processing of blood specimens. During the last few years, there has been much debate whether a discard tube should be drawn for plasma-based coagulation assays when using a standard evacuated tube collection system. The rationale for a discard tube is based on historic coagulation testing using the whole blood clotting time, in which tissue thromboplastin released and activated during venipuncture could lead to erroneous results. A few studies have already shown that with today’s coagulation reagents and venipuncture procedures, using extremely sharp low-resistance needles, a discard tube is not necessary for prothrombin time (PT) and activated partial thromboplastin

We observed 30% discrepancy between liquid chemistry and dry chemistry analysers for the determination of total bilirubin in human adult serum samples, which were consistent with a 20% overestimation and 10% underestimation relative to a Jendrassik-Grof reference method, respectively. In contrast, standard reference material SRM916, which was recently recommended as being the most suitable material for attaining interlaboratory agreement, shows very good agreement on both types of analysers, as well as close to 100% recovery with respect to the reference method. We show that the liquid vs. dry bilirubin discrepancies seem to originate in the presence of either conjugated or δ-bilirubin. Our observations make it clear that good interlaboratory (or inter-analyser) agreement between bilirubin reference materials does not guarantee the same for bilirubin concentrations in human serum samples.

Standardization of laboratory results allows for the use of common reference intervals and can be achieved via calibration of field methods with secondary reference materials. These harmonization materials should be commutable, i.e., they produce identical numerical results independent of assay principle or platform. This study assessed the commutability of a cryolyoprotectant-containing harmonization material, obtained from the Dutch Foundation for Quality Assessment in Clinical Laboratories, that is intended to harmonize measurements of enzyme activities within the Dutch project “Calibration 2000”. The catalytic concentrations of alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, γ-glutamyltransferase and creatine kinase were analyzed in pooled patient sera and in the reference material in 14 laboratories. On liquid chemistry analyzers the harmonization material behaves like patient material. The enzyme activities measured in it fall on the regression lines calculated from activities measured in serum samples. For dry chemistry analyzers the activities of all enzymes measured in the harmonizator differ from the serum-based regression line. We show that this is due to the sucrose-containing cryolyoprotectant in the harmonization material. For each enzyme, correction factors were calculated that compensated for the bias and proved to be constant between reagent lots. Depending on the enzyme activity measured, application of these factors leads to 2- to 10-fold reduction of between-laboratory percentage coefficient of variation. Thus, additives to (potential) reference materials may alter their matrix in a way that interferes with analysis on certain test systems. The bias caused may be quantifiable and correctable. Establishment of correction factors leads to analytical uncertainties and costs. Therefore, matrix-based materials without additives should be selected as reference materials.

Objectives To assess the performance of the Doumas bilirubin reference method. Design and methods Ring trails using pooled patient specimens, a calibrator and human sera enriched with unconjugated bilirubin were analyzed in five laboratories using the Doumas bilirubin reference method. Results The coefficient of variation for the linear measurement range between laboratories ranged from 1–3%. Conclusions The Doumas bilirubin reference method is robust and reproducible. Bilirubin results using this method may be used in the development of more accurate and reliable calibrators.

The concentration of von Willebrand factor (vWf) in patients' plasma can be determined by measuring the ristocetin cofactor activity (vWf R:Co). However, this vWf R:Co assay is time consuming, which limits its routine use. Several commercial vWf R:Co tests, based on agglutination of lyophilized fixed platelets, are available. We evaluated the slide tests and aggregometer assays from Behring and Organon Teknika and compared them with the classic vWf R:Co aggregometer method. The within-run and between-run precisions of the two slide tests were better than those of the aggregometer methods. The correlation studies between the four commercial assays and the classic aggregation method were based on 23 plasma samples (range: 15-450% vWf R:Co). The correlation coefficients, which ranged from 0.923 to 0.950, did not differ significantly (P > 0.1). All four commercial assays gave significantly lower vWf R:Co values than the classic aggregation method (P < 0.01). We conclude that commercially available fixed platelets can be used for the rapid measurement of vWf R:Co with a slide test. The use of the aggregometer is time consuming and may result in a lower precision.

To the Editor: The monitoring of antiandrogen treatment in patients with prostate cancer, investigation of hyperandrogenism in women, and evaluation of infants with ambiguous genitalia require accurate measurement of low testosterone concentrations. However, testosterone immunoassays have been shown to be inaccurate and often to overestimate testosterone concentrations in the low range (1). A working group of the Endocrine Society recently reviewed this concern and presented several recommendations to ensure the accuracy of future testosterone testing for improvement of diagnosis and treatment of disease (2). In the present study we evaluated the current situation with regard to the accuracy of 7 testosterone immunoassays, including 2 second generation assays, by comparison with isotope-dilution liquid chromatography–tandem mass spectrometry (ID-LC-MS/MS). In addition, we investigated the possible improvement of these immunoassays by diethyl ether sample extraction. Serum from 50 men, 50 women, and 16 children (age 4–16 years) was collected, divided into aliquots, and stored (−20 °C) until analysis. All investigations conformed to the ethics standards of the Helsinki Declaration. Total serum testosterone was measured singly …

The diagnosis of urinary tract infection (UTI) by urine culture is time-consuming and can produce up to 60 to 80% negative results. Fast screening methods that can reduce the necessity for urine cultures will have a large impact on overall turnaround time and laboratory economics. We have evaluated the detection of bacteria and leukocytes by a new urine analyzer, the UF-1000i, to identify negative urine samples that can be excluded from urine culture. In total, 1,577 urine samples were analyzed and compared to urine culture. Urine culture showed growth of ≥10 3 CFU/ml in 939 samples (60%). Receiver operating characteristics (ROC) curves and ROC decision plots were been prepared at three different gold standard definitions of a negative urine culture: no growth, growth of bacteria at 4 CFU/ml, and growth of bacteria at 5 CFU/ml. Also, the reduction in urine cultures and the percentage of false negatives were calculated. At the most stringent gold standard definition of no growth, a chosen sensitivity of 95% resulted in a cutoff value of 26 bacteria/μl, a specificity of 43% and a reduction in urine cultures of only 20%, of which 14% were false negatives. However, at a gold standard definition of 5 CFU/ml and a sensitivity of 95%, the UF-1000i cutoff value was 230 bacteria/μl, the specificity was 80%, and the reduction in urine cultures was 52%, of which 0.3% were false negatives. The applicability of the UF-1000i to screen for negative urine samples strongly depends on population characteristics and the definition of a negative urine culture. In our setting, however, the low workload savings and the high percentage of false-negative results do not warrant the UF-1000i to be used as a screening analyzer.

The Clinical and Laboratory Standards Institute (CLSI) recently abandoned its recommendation for drawing a discard tube when performing a prothrombin time (PT)/international normalized ratio (INR) or an activated partial thromboplastin time (APTT). Because there is currently no evidence that a discard tube is necessary for more specialized coagulation assays, we studied the need for a discard tube for some of these tests. Blood was obtained from 88 subjects in 2 subsequent citrate tubes. Platelet-free plasma was tested for PT, APTT, antithrombin, protein C, and factors II, V, VIII, IX, and X. Difference and bias between tubes were tested using the Wilcoxon signed rank test and Bland-Altman plots. For only APTT, antithrombin, and protein C was a small, statistically significant mean bias found (0.5 seconds; P = .001; -0.7%, P = .002; and -0.8%, P.0001, respectively), but the bias of individual samples was not clinically relevant. This was also true for the other parameters tested. The recent CLSI recommendation that a discard tube is not necessary for PT/INR and APTT can be extended to include more specialized plasma-based coagulation assays as identified in this study.

Background The principle of the erythrocyte sedimentation rate (ESR) as assessed by TEST 1 is different from that of Westergren-based methods. This could result in different influences on the tests by paraproteins. Methods We investigated the effect of paraproteins on ESR readings by TEST 1 ( y) and the StarrSed ( x), a Westergren-based method, in 142 patients with paraproteinaemia. Agreement (Passing-Bablok) and bias (Bland–Altman) between methods was investigated and compared with that of a control population. Results A poor agreement between the two methods was found in patients with a paraprotein ( y = 0.67 x + 3.3) in comparison with that of the control population ( y = 0.96 x + 0.2). Large differences between methods were present when ESR readings were >40 mm/hour, but clinical interpretation was similar in 90% of cases. Linear regression showed a concentration dependent influence of paraproteins on ESR readings by the StarrSed, especially for immunoglobulin class IgM. Conclusion ESR readings by TEST 1 result in similar clinical interpretation for most subjects, but readings are less influenced by the presence of a paraprotein than those of a Westergren-based method.

Contains fulltext : 69175.pdf (Publisher’s version ) (Closed access) Intermittent claudication has proved to be a good in vivo model for ischaemia-reperfusion. For assessment of ischaemia-reperfusion damage, the known biochemical markers all have disadvantages with respect to sensitivity and interference with other physiological events. In this work, we studied the metabolic effects of ischaemia-reperfusion in patients with intermittent claudication, and the effects of vitamin C and E intervention, using both traditional biochemical measurements and 1H-NMR-based metabonomics on urine and plasma. The 1H-NMR spectra were subjected to multivariate modelling using principal components discriminant analysis, and the observed clusters were validated using joint deployment of univariate analysis of variance and Tukey-Kramer honestly significant difference (HSD) testing. The study involved 14 patients with intermittent claudication and three healthy volunteers, who were monitored during a walking test, before and after a vitamin C/E intervention, and after a washout period. The effect of exercise was only observable for a limited number of biochemical markers, whereas 1H NMR revealed an effect in line with anaerobic ATP production via glycolysis in exercising (ischaemic) muscle of the claudicants. Thus, the beneficial effect of vitamins C and E in claudicants was more pronounced when observed by metabonomics than by traditional biochemical markers. The main effect was more rapid recovery from exercise to resting state metabolism. Furthermore, after intervention, claudicants tended to have lower concentrations of lactate and glucose and several other citric acid cycle metabolites, whereas acetoacetate was increased. The observed metabolic changes in the plasma suggest that intake of vitamin C/E leads to increased muscle oxidative metabolism.

BACKGROUND The laboratory analysis of cerebrospinal fluid (CSF) plays a key role in considering subarachnoid haemorrhage (SAH) in patients with clinical suspicion, but negative CT scan. Although the determination of the CSF bilirubin concentration generally provides high sensitivity, it was recently shown that specificity and positive predictive value are unacceptably low, limiting its use as a diagnostic tool. METHODS We intended to design and evaluate an improved laboratory protocol, which fulfills the requirement of better specificity without losing sensitivity. We present a procedure in which a "bili-excess" concentration is determined, which is the surplus CSF bilirubin measured after subtraction of an estimated upper limit for the individual patient. The latter is calculated from [bilirubin](serum), [albumin](serum) and [albumin](CSF), taking into account the propagation of analytical errors in the individual analyses. We investigated the applicability of direct absorption vs. derivative spectroscopy, thereby addressing the influence of various calibration methods. We evaluated our procedure in 92 CSF samples drawn from patients with (n=37) and without (n=55) clinical suspicion of SAH. RESULTS In our study population, we show that specificity increases from 0.83 (95% CI, 0.74-0.91) to 1.00 (95% CI, 0.96-1.00) using the bili-excess concept, with an established upper limit for bili-excess of 0.11 micromol/L instead of the recommended use of an "uncorrected" CSF bilirubin upper limit of 0.20 micromol/L. Sensitivity in both cases is 1.00 (95% CI, 0.66-1.00). We demonstrate the merit of allowing for analytical imprecision in the bili-excess concept. CONCLUSIONS We provide a quantitative procedure to explore the likelihood of (CT-negative) SAH independent of the absolute CSF bilirubin concentration by considering the "bili-excess" concentration per individual, using derivative spectroscopy to determine CSF bilirubin. This procedure led to an increase in specificity to 1.00 (95% CI, 0.96-1.00) in our study population.

An important number of patients dependent on renal dialysis prefer peritoneal dialysis to hemodialysis. In the case of peritoneal dialysis, the glucose polymer icodextrin is frequently added to the dialysis fluid as an osmotic agent, since this polymer is able to maintain an osmotic gradient across the peritoneal membrane longer than monomeric glucose, leading to a prolonged effective ultrafiltration time. It was previously shown that icodextrin is partly able to enter the blood via the lymphatic system, where hydrolysis to glucose oligomers such as maltose and maltotriose occurs. The presence of these oligomers in the blood appears to cause significant overestimations of the glucose values in several point-of-care (POC) glucose analyzers, with potentially dramatic consequences. This effect has been investigated for a series of POC glucose analyzers, both by analyzing the blood of peritoneal dialysis patients and by an in vitro investigation of the quantitative effects of maltose and maltotriose. In particular, POC analyzers utilizing the bacterially produced enzyme glucose dehydrogenase seem to lack glucose specificity.

To compare lactate, bilirubin and Hemoglobin F concentrations obtained on ABL 700 series blood gas analyzers with those from laboratory methods. Pooled neonatal plasma, cord blood and adult plasma samples were used for comparison of bilirubin, hemoglobin F and lactate concentrations respectively. Results obtained on the ABL 700 series compared favorably (Deming regression slopes 0.97-1.13) with those from laboratory methods. For lactate ABL (y) = 1.13 Vitros (x) -0.43 with a CI (slope) of 1.10 to 1.16, CI (int) of -0.61 to -0.28. For hemoglobin F ABL(y) = 1.11 Variant (x) -8.0 with a CI (slope) of 0.88 to 1.33, CI (int) of -25.3 to 9.3. The three bilirubin comparisons are as follows: 1) Unistat (y) = 1.10 Vitros (x) -16.12 with CI (slope) of 1.06 to 1.14 and CI (int) of -25.3 to -6.9. 2), ABL (y) = 0.97 Vitros(x) -10.16 with CI (slope) of 0.94 to 1.00 and CI (int) of -17.6 to 2.73) Unistat (y) = 1.14 (x)-4.58 with CI (slope) of 1.09 to 1.18 and CI (int) of -13.6 to 4.5. The ABL 700 series gave comparable results for lactate, bilirubin and hemoglobin F with laboratory methods and may be used in patient care.

Recently, the Joint European Society of Cardiology/American College of Cardiology committee for the redefinition of myocardial infarction proposed that “any amount of myocardial necrosis caused by ischemia should be labeled as an infarct” (1). The same committee agreed that a troponin concentration higher than the 99th percentile of a reference control group is one of the most specific (biochemical) markers of myocardial infarction. The maximum allowable imprecision at this decision limit has arbitrarily been set at a CV of 10% (1)(2) although it is not stated whether the 10% relates to total CV. Because an increasing number of nonstandardized commercial methods …

Patients with intermittent claudication disease suffer from temporary lack of oxygen in the legs, caused by narrowing of arteries, resulting in ischemia and followed by reperfusion. The degree of oxidative stress present in 16 patients during strenuous exercise was determined using several indicators. Two derivatives of an exogenous marker, antipyrine (AP), (ie, p-hydroxyantipyrine, p-APOH, and o-hydroxyantipyrine, o-APOH), were assayed in plasma using HPLC-tandem-MS. Plasma malondialdehyde (assayed as thiobarbituric acid reactive species, TBARS) was also determined. The branchial/ankle blood pressure index (b-a index) was used to assess the severity of intermittent claudication disease, and plasma lactate concentration was also measured as an indicator of the ischemic situation. Plasma TBARS level did not change significantly after exercise. During the ischemic situation as well as during reperfusion, both free radical derivatives of antipyrine increased significantly in plasma (p0.01). Because p-APOH is also formed enzymatically in humans, the plasma ratio of o-APOH to AP appeared to be the most specific marker for oxidative stress in patients with intermittent claudication.

To the Editor We were interested to read the recent report from Raijmakers and colleagues1 on the issue of discard tubes for specialized coagulation testing. It is interesting that this report appeared in print at the same time as another similar report from Smock and colleagues.2 Raijmakers et al1 assessed the need for a discard tube by performing paired testing for prothrombin time (PT)/international normalized ratio, activated partial thromboplastin time (APTT), antithrombin, protein C, and factors II, V, VIII, IX, and X. They observed some small statistical difference in some tests (ie, APTT, antithrombin, protein C, and factor VIII) but no clinically significant differences and, hence, concluded that their findings “support the new CLSI …

Preanalytical factors that affect parathyroid hormone (PTH) concentrations are not well defined. Measured PTH concentrations in EDTA plasma may (1)(2) or may not(3) differ from those in serum. PTH has been reported variously to be stable for hours to days (1)(2)(3)(4)(5)(6). In addition, different PTH assays cross-react to various extents with biologically inactive, N-terminally truncated PTH molecules (7) that may have different stabilities. The specificity of a given immunometric assay depends on the binding sites of the anti-PTH antibodies used. So-called “intact” PTH (iPTH) assays show cross-reactivity with peptides lacking N-terminal amino acids. Assays measuring only PTH with intact NH2 termini are named CAP™; whole PTH, 3rd generation; or bioactive PTH assays (7). In 2001, DPC introduced a reformulated iPTH sandwich assay (starting with lot no. 106) in which the polyclonal tracer antibody had been replaced by a monoclonal antibody raised against an epitope within the first 34 amino acids of PTH. With this new method, reference intervals and, in particular, PTH concentrations measured in serum samples of dialysis patients are lower (data not shown). In March 2003, DPC reported underrecovery of PTH with this method (8), and a new lot of raw materials was introduced, starting with assay lot no. 112. The calibration for the Immulite analyzer was adjusted at assay lot no. 113 (8). Shortly before this, we had found …