Huib L. Vader

Accuracy of First and Second Generation Testosterone Assays and Improvement through Sample Extraction

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 …

Collection of Blood Specimens by Venipuncture for Plasma-Based Coagulation Assays Necessity of a Discard Tube

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.

A quantitative appraisal of interference by icodextrin metabolites in point-of-care glucose analyses

Abstract 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.

Discard tubes are sometimes necessary when drawing samples for hemostasis.

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 …

Do we measure bilirubin correctly anno 2005

Abstract 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.

Multicenter evaluation of the commutability of a potential reference material for harmonization of enzyme activities

Abstract 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.

The effect of paraproteins on the erythrocyte sedimentation rate: a comparison between the StarrSed and TEST 1

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 = 0.67x + 3.3) in comparison with that of the control population (y = 0.96x + 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.

An improved laboratory protocol to assess subarachnoid haemorrhage in patients with negative cranial CT scan.

Abstract 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 0.11μmol/L instead of the recommended use of an “uncorrected” CSF bilirubin upper limit of 0.20 μmol/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. Clin Chem Lab Med 2006;44:938–48.