Is perioperative point-of-care prothrombin time testing accurate compared to the standard laboratory test?

There is no accepted way of measuring prothrombin time without time loss for patients undergoing major surgery who are at risk of intraoperative dilution and consumption coagulopathy due to bleeding and volume replacement with crystalloids or colloids. Decisions to transfuse fresh frozen plasma and procoagulatory drugs have to rely on clinical judgment in these situations. Point-of-care devices are considerably faster than the standard laboratory methods. In this study we assessed the accuracy of a Point-of-care (PoC) device measuring prothrombin time compared to the standard laboratory method. Patients undergoing major surgery and intensive care unit patients were included. PoC prothrombin time was measured by CoaguChek XS Plus (Roche Diagnostics, Switzerland). PoC and reference tests were performed independently and interpreted under blinded conditions. Using a cut-off prothrombin time of 50%, we calculated diagnostic accuracy measures, plotted a receiver operating characteristic (ROC) curve and tested for equivalence between the two methods. PoC sensitivity and specificity were 95% (95% CI 77%, 100%) and 95% (95% CI 91%, 98%) respectively. The negative likelihood ratio was 0.05 (95% CI 0.01, 0.32). The positive likelihood ratio was 19.57 (95% CI 10.62, 36.06). The area under the ROC curve was 0.988. Equivalence between the two methods was confirmed. CoaguChek XS Plus is a rapid and highly accurate test compared with the reference test. These findings suggest that PoC testing will be useful for monitoring intraoperative prothrombin time when coagulopathy is suspected. It could lead to a more rational use of expensive and limited blood bank resources.

Diagnostic approach to hypercalcemia: relevance of parathyroid hormone and parathyroid hormone-related protein measurements

Background: Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTH-rP) are two potent hypercalcemic hormones that act on the same targets. Autonomous secretion of the former is involved in primary hyperparathyroidism (PHPT), whereas the latter is responsible for humoral hypercalcemia of malignancy (HHM). Methods: From 250 consecutive, hypercalcemic serum samples sent to our laboratory for assessment of intact PTH, we were able to obtain clinical information, as well as an additional plasma sample for PTH-rP measurement, in 134 patients. At the time of sampling, patients could be classified into seven groups: cancer without known bone metastases (CaNoMeta, n=36), cancer with bone metastases (CaMeta, n=9), no evidence of cancer (noEvCa, n=71), sarcoidosis (Sarc, n=3), end-stage renal disease (ESRD, n=12), vitamin D overdose (VIT-D, n=2), and hyperthyroidism (Thyr, n=1). Results: In the CaNoMeta group, 29/36 patients had elevated PTH-rP levels, 9/36 patients had inappropriately elevated PTH levels, and 5/36 had elevated levels of both hormones. In the CaMeta group, three of the nine patients had inappropriately elevated PTH levels, two of them with concomitantly elevated PTH-rP levels. In the NoEvCa group, 63/71 patients had an inappropriate elevation of PTH levels and were diagnosed as having PHPT. Four of the 71 patients had elevated levels of both PTH and PTH-rP; three of them were in poor health and died within a short period of time. All of the ESRD patients had very high PTH and normal PTH-rP levels, except for one woman with high PTH-rP and undetectable PTH levels; she died from what later turned out to be a recurrent bladder carcinoma. In the Sarc, Vit-D, and Thyr groups, both PTH and PTH-rP levels were normal. Conclusions: (1) Elevated PTH-rP levels are a common finding in cancer patients without bone metastases. Intact PTH, however, should always be measured in hypercalcemic patients with malignancy because concurrent primary hyperparathyroidism is not rare. (2) Primary hyperparathyroidism accounts for hypercalcemia in 90% of patients without evidence of cancer whose PTH-rP levels may also be found to be elevated in a few cases, even some with surgically demonstrated parathyroid adenoma.

Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

Pathology working group review of histopathologic specimens from three laboratory studies of diclofenac in trout.

While the pathology peer review/pathology working group (PWG) model has long been used in mammalian toxicologic pathology to ensure the accuracy, consistency, and objectivity of histopathology data, application of this paradigm to ecotoxicological studies has thus far been limited. In the current project, the PWG approach was used to evaluate histopathologic sections of gills, liver, kidney, and/or intestines from three previously published studies of diclofenac in trout, among which there was substantial variation in the reported histopathologic findings. The main objectives of this review process were to investigate and potentially reconcile these interstudy differences, and based on the results, to establish an appropriate no observed effect concentration (NOEC). Following a complete examination of all histologic sections and original diagnoses by a single experienced fish pathologist (pathology peer review), a two-day PWG session was conducted to allow members of a four-person expert panel to determine the extent of treatment-related findings in each of the three trout studies. The PWG was performed according to the United States Environmental Protection Agency (US EPA) Pesticide Regulation (PR) 94-5 (EPA Pesticide Regulation, 1994). In accordance with standard procedures, the PWG review was conducted by the non-voting chairperson in a manner intended to minimize bias, and thus during the evaluation, the four voting panelists were unaware of the treatment group status of individual fish and the original diagnoses associated with the histologic sections. Based on the results of this review, findings related to diclofenac exposure included minimal to slightly increased thickening of the gill filament tips in fish exposed to the highest concentration tested (1,000 μg/L), plus a previously undiagnosed finding, decreased hepatic glycogen, which also occurred at the 1,000 μg/L dose level. The panel found little evidence to support other reported effects of diclofenac in trout, and thus the overall NOEC was determined to be >320 μg/L. By consensus, the PWG panel was able to identify diagnostic inconsistencies among and within the three prior studies; therefore this exercise demonstrated the value of the pathology peer review/PWG approach for assessing the reliability of histopathology results that may be used by regulatory agencies for risk assessment.

An inter-laboratory comparative study of serological tools employed in the diagnosis of Besnoitia besnoiti infection in bovines

Bovine besnoitiosis is considered an emerging chronic and debilitating disease in Europe. Many infections remain subclinical, and the only sign of disease is the presence of parasitic cysts in the sclera and conjunctiva. Serological tests are useful for detecting asymptomatic cattle/sub-clinical infections for control purposes, as there are no effective drugs or vaccines. For this purpose, diagnostic tools need to be further standardized. Thus, the aim of this study was to compare the serological tests available in Europe in a multi-centred study. A coded panel of 241 well-characterized sera from infected and non-infected bovines was provided by all participants (SALUVET-Madrid, FLI-Wusterhausen, ENV-Toulouse, IPB-Berne). The tests evaluated were as follows: an in-house ELISA, three commercial ELISAs (INGEZIM BES 12.BES.K1 INGENASA, PrioCHECK Besnoitia Ab V2.0, ID Screen Besnoitia indirect IDVET), two IFATs and seven Western blot tests (tachyzoite and bradyzoite extracts under reducing and non-reducing conditions). Two different definitions of a gold standard were used: (i) the result of the majority of tests ('Majority of tests') and (ii) the majority of test results plus pre-test information based on clinical signs ('Majority of tests plus pre-test info'). Relative to the gold standard 'Majority of tests', almost 100% sensitivity (Se) and specificity (Sp) were obtained with SALUVET-Madrid and FLI-Wusterhausen tachyzoite- and bradyzoite-based Western blot tests under non-reducing conditions. On the ELISAs, PrioCHECK Besnoitia Ab V2.0 showed 100% Se and 98.8% Sp, whereas ID Screen Besnoitia indirect IDVET showed 97.2% Se and 100% Sp. The in-house ELISA and INGEZIM BES 12.BES.K1 INGENASA showed 97.3% and 97.2% Se; and 94.6% and 93.0% Sp, respectively. IFAT FLI-Wusterhausen performed better than IFAT SALUVET-Madrid, with 100% Se and 95.4% Sp. Relative to the gold standard 'Majority of test plus pre-test info', Sp significantly decreased; this result was expected because of the existence of seronegative animals with clinical signs. All ELISAs performed very well and could be used in epidemiological studies; however, Western blot tests performed better and could be employed as a posteriori tests for control purposes in the case of uncertain results from valuable samples.

Diagnostic gap in Bovine viral diarrhea virus serology during the periparturient period in cattle

Detection of antibodies against Bovine viral diarrhea virus (BVDV) in serum and milk by enzyme-linked immunosorbent assay (ELISA) is a crucial part of all ongoing national schemes to eradicate this important cattle pathogen. Serum and milk are regarded as equally suited for antibody measurement. However, when retesting a seropositive cow 1 day after calving, the serum was negative in 6 out of 9 different ELISAs. To further investigate this diagnostic gap around parturition, pre- and postcalving serum and milk samples of 5 cows were analyzed by BVDV antibody ELISA and serum neutralization test (SNT). By ELISA, 3 out of the 5 animals showed a diagnostic gap in the serum for up to 12 days around calving but all animals remained positive in SNT. In milk, the ELISA was strongly positive after birth but antibody levels decreased considerably within the next few days. Because of the immunoglobulin G (IgG)1-specific transport of serum antibodies into the mammary gland for colostrum production, the IgG subclass specificity of the total and the BVDV-specific antibodies were determined. Although all 5 animals showed a clear decrease in the total and BVDV-specific IgG1 antibody levels at parturition, the precalving IgG1-to-IgG2 ratios of the BVDV-specific antibodies were considerably lower in animals that showed the diagnostic gap. Results showed that BVDV seropositive cows may become "false" negative in several ELISAs in the periparturient period and suggest that the occurrence of this diagnostic gap is influenced by the BVDV-specific IgG subclass response of the individual animal.

Diagnostic performance of high-sensitive troponin T in patients with renal insufficiency

In the present study, we wanted to (1) evaluate whether high-sensitive troponin T levels correlate with the grade of renal insufficiency and (2) test the accuracy of high-sensitive troponin T determination in patients with renal insufficiency for diagnosis of acute myocardial infarction (AMI). In this cross-sectional analysis, all patients who received serial measurements of high-sensitive troponin T from August 1, 2010, to October 31, 2012, at the Department of Emergency Medicine were included. We analyzed data on baseline characteristics, reason for referral, medication, cardiovascular risk factors, and outcome in terms of presence of AMI along with laboratory data (high-sensitive troponin T, creatinine). A total of 1,514 patients (67% male, aged 65 ± 16 years) were included, of which 382 patients (25%) had moderate to severe renal insufficiency and significantly higher levels of high-sensitive troponin T on admission (0.028 vs 0.009, p <0.0001). In patients without AMI, high-sensitive troponin T correlated inversely with the estimated glomerular filtration rate (R = -0.12, p <0.0001). Overall, sensitivity of an elevated high-sensitive troponin for diagnosis of AMI was 0.64 (0.56 to 0.71) and the specificity was 0.48 (0.45 to 0.51). The area under the curve of the receiver operating characteristic for all patients was 0.613 (standard error [SE] 0.023), whereas it was 0.741 (SE 0.029) for patients with a Modification of Diet in Renal Disease estimated glomerular filtration rate >60 ml/min presenting with acute chest pain or dyspnea and 0.535 (SE 0.056) for patients with moderate to severe renal insufficiency presenting with acute chest pain or dyspnea. In conclusion, the diagnostic accuracy for presence of AMI of a baseline measurement of high-sensitive troponin in patients with renal insufficiency was poor and resembles tossing a coin.

Monitoring of emergency and intensive care patients: simple diagnostic aids

This article describes in short sections the use and interpretation of indirect blood pressure measurements, central venous pressure, body temperature, pulse oximetry, end tidal CO2 measurements, pulse and heart rate, urine production and emergency laboratory values. Most of these parameters are directly or indirectly linked to the perfusion of the patient. Optimizing these values are one of the most important goals in emergency and critical care medicine.

Evaluation of the diagnostic value of the ELISA tests developed by using EgHF, Em2 and EmII/3-10 antigens in the serological diagnosis of alveolar echinococcosis

Alveolar echinococcosis (AE), caused by larva stage of Echinococcus multilocularis, is one of the lethal parasitic diseases of man and a major public health problem in many countries in the northern hemisphere. When the living conditions and habits in Turkey were considered in terms of relation with the life cycle of the parasite, it was suggested that AE has been much more common than reported mainly from the Eastern Anatolia region of Turkey. Since in vitro serologic diagnosis tests with high specificity for AE have not been used in our country, most of the cases with liver lesions were misdiagnosed by radiological investigations as malignancies. The aim of this study was to evaluate the diagnostic value of the in-house ELISA methods developed by using three different antigens (EgHF, Em2, EmII/3-10) in the serological diagnosis of AE. The study samples included a total of 100 sera provided by Bern University Parasitology Institute where samples were obtained from patients with helminthiasis and all were confirmed by clinical, parasitological and/or histopathological means. Ten samples from each of the cases infected by E.multilocularis, E.granulosus, Taenia solium, Wuchereria bancrofti, Strongyloides stercolaris, Ascaris lumbricoides, Toxocara canis, Trichinella spiralis, Fasciola hepatica and Schistosoma haematobium were studied. In the study, EgHF (E.granulosus hydatid fluid) antigens were prepared in our laboratory from the liver cyst fluids of sheeps with cystic echinococcosis, however Em2 (E.multilocularis metacestode-purified laminated layer) and EmII/3-10 (E.multilocularis recombinant protoscolex tegument) antigens were provided by Bern University Parasitology Institute. Flat bottom ELISA plates were covered with EgHF, Em2 and EmII/3-10 antigens in the concentrations of 2.5 µg, 1 µg and 0.18 µg per well, respectively, and all sera were tested by EgHF-ELISA, Em2-ELISA and EmII/3-10-ELISA methods. For each tests, the samples which were reactive above the cut-off value (mean OD of negative controls+2 SD) were accepted as positive. The sensitivity of the ELISA tests performed with EgHF, Em2 and Em2II/3-10 antigens were estimated as 100%, 90% and 90%, respectively, whereas the specificity were 63%, 91% and 91%, respectively. When Em2-ELISA and EmII/3-10-ELISA tests were evaluated together, the specificity increased to 96%. Our data indicated that the highest sensitivity (100% with EgHF-ELISA) and specificity (96% with Em2-ELISA + EmII/3-10-ELISA) for the serodiagnosis of AE can be achieved by the combined use of the ELISA tests with three different antigens. It was concluded that the early and accurate diagnosis of AE in our country which is endemic for that disease, could be supported by the use of highly specific serological tests such as Em2-ELISA ve EmII/3-10-ELISA contributing radiological data.

Laboratory diagnosis of ruminant abortion in Europe.

Abortion in ruminants is a major cause of economic loss worldwide, and the management and control of outbreaks is important in limiting their spread, and in preventing zoonotic infections. Given that rapid and accurate laboratory diagnosis is central to controlling abortion outbreaks, the submission of tissue samples to laboratories offering the most appropriate tests is essential. Direct antigen and/or DNA detection methods are the currently preferred methods of reaching an aetiological diagnosis, and ideally these results are confirmed by the demonstration of corresponding macroscopic and/or histopathological lesions in the fetus and/or the placenta. However, the costs of laboratory examinations may be considerable and, even under optimal conditions, the percentage of aetiological diagnoses reached can be relatively low. This review focuses on the most commonly occurring and important abortifacient pathogens of ruminant species in Europe highlighting their epizootic and zoonotic potential. The performance characteristics of the various diagnostic methods used, including their specific advantages and limitations, are discussed.

Comparison between magnetic resonance imaging and computed tomography of the lung in patients with cystic fibrosis with regard to clinical, laboratory, and pulmonary functional parameters

OBJECTIVE To evaluate whether magnetic resonance imaging (MRI) is effective as computed tomography (CT) in determining morphologic and functional pulmonary changes in patients with cystic fibrosis (CF) in association with multiple clinical parameters. MATERIALS AND METHODS Institutional review board approval and patient written informed consent were obtained. In this prospective study, 30 patients with CF (17 men and 13 women; mean (SD) age, 30.2 (9.2) years; range, 19-52 years) were included. Chest CT was acquired by unenhanced low-dose technique for clinical purposes. Lung MRI (1.5 T) comprised T2- and T1-weighted sequences before and after the application of 0.1-mmol·kg gadobutrol, also considering lung perfusion imaging. All CT and MR images were visually evaluated by using 2 different scoring systems: the modified Helbich and the Eichinger scores. Signal intensity of the peribronchial walls and detected mucus on T2-weighted images as well as signal enhancement of the peribronchial walls on contrast-enhanced T1-weighted sequences were additionally assessed on MRI. For the clinical evaluation, the pulmonary exacerbation rate, laboratory, and pulmonary functional parameters were determined. RESULTS The overall modified Helbich CT score had a mean (SD) of 15.3 (4.8) (range, 3-21) and median of 16.0 (interquartile range [IQR], 6.3). The overall modified Helbich MR score showed slightly, not significantly, lower values (Wilcoxon rank sum test and Student t test; P > 0.05): mean (SD) of 14.3 (4.7) (range, 3-20) and median of 15.0 (IQR, 7.3). Without assessment of perfusion, the overall Eichinger score resulted in the following values for CT vs MR examinations: mean (SD), 20.3 (7.2) (range, 4-31); and median, 21.0 (IQR, 9.5) vs mean (SD), 19.5 (7.1) (range, 4-33); and median, 20.0 (IQR, 9.0). All differences between CT and MR examinations were not significant (Wilcoxon rank sum tests and Student t tests; P > 0.05). In general, the correlations of the CT scores (overall and different imaging parameters) to the clinical parameters were slightly higher compared to the MRI scores. However, if all additional MRI parameters were integrated into the scoring systems, the correlations reached the values of the CT scores. The overall image quality was significantly higher for the CT examinations compared to the MRI sequences. CONCLUSIONS One major diagnostic benefit of lung MRI in CF is the possible acquisition of several different morphologic and functional imaging features without the use of any radiation exposure. Lung MRI shows reliable associations with CT and clinical parameters, which suggests its implementation in CF for routine diagnosis, which would be particularly important in follow-up imaging over the long term.

DMS : Diagnostic Modeling System.

Includes bibliographical references (p. 52-53).

Directory of animal disease diagnostic laboratories, 1971.

Prepared in cooperation with the American Association of Veterinary Laboratory Diagnosticians.

The STARD statement for reporting studies of diagnostic accuracy: Explanation and elaboration

The quality of reporting of studies of diagnostic accuracy is less than optimal. Complete and accurate reporting is necessary to enable readers to assess the potential for bias in the study and to evaluate the generalisability of the results. A group of scientists and editors has developed the STARD (Standards for Reporting of Diagnostic Accuracy) statement to improve the reporting the quality of reporting of studies of diagnostic accuracy. The statement consists of a checklist of 25 items and flow diagram that authors can use to ensure that all relevant information is present. This explanatory document aims to facilitate the use, understanding and dissemination of the checklist. The document contains a clarification of the meaning, rationale and optimal use of each item on the checklist, as well as a short summary of the available evidence on bias and applicability. The STARD statement, checklist, flowchart and this explanation and elaboration document should be useful resources to improve reporting of diagnostic accuracy studies. Complete and informative reporting can only lead to better decisions in healthcare.

Validation of assembled nucleic acid-based tests in diagnostic microbiology laboratories

Medical microbiology and virology laboratories use nucleic acid tests (NAT) to detect genomic material of infectious organisms in clinical samples. Laboratories choose to perform assembled (or in-house) NAT if commercial assays are not available or if assembled NAT are more economical or accurate. One reason commercial assays are more expensive is because extensive validation is necessary before the kit is marketed, as manufacturers must accept liability for the performance of their assays, assuming their instructions are followed. On the other hand, it is a particular laboratory's responsibility to validate an assembled NAT prior to using it for testing and reporting results on human samples. There are few published guidelines for the validation of assembled NAT. One procedure that laboratories can use to establish a validation process for an assay is detailed in this document. Before validating a method, laboratories must optimise it and then document the protocol. All instruments must be calibrated and maintained throughout the testing process. The validation process involves a series of steps including: (i) testing of dilution series of positive samples to determine the limits of detection of the assay and their linearity over concentrations to be measured in quantitative NAT; (ii) establishing the day-to-day variation of the assay's performance; (iii) evaluating the sensitivity and specificity of the assay as far as practicable, along with the extent of cross-reactivity with other genomic material; and (iv) assuring the quality of assembled assays using quality control procedures that monitor the performance of reagent batches before introducing new lots of reagent for testing.