Comparison of four D-dimer assays in the context of venous thromboembolism in the emergency department.

INTRODUCTION: This observational study conducted across seven emergency care units compares the efficacy of four D-dimer detection methods, namely HemosIL D-dimer HS (HS), HemosIL D-dimer HS-500 (HS-500), VIDAS D-dimer (VIDAS), and HemosIL AcuStar D-dimer (ACUSTAR). The primary focus is on patients with a clinical suspicion of deep venous thrombosis (DVT) or pulmonary embolism (PE). METHODS: A total of 149 samples were collected from patients with suspected DVT or PE. The confirmation of DVT/PE was based on calf ultrasound or computed tomography-Angiography. Direct comparisons were made between the different detection methods, considering both their analytical performance and clinical utility. Additionally, the impact of an age-adjusted cut-off on the diagnostic accuracy of each method was assessed. RESULTS: The results revealed comparable negative predictive value, sensitivity, and specificity across the methods, with a notable exception of increased specificity for HS compared with HS-500 (50.8% vs. 41.5%, p = 0.03). Further analysis incorporating an age-adjusted cut-off demonstrated a significant improvement in specificity for HS. When using the age-adjusted cut-off, HS exhibited a substantial increase in specificity compared with HS-500 (63.1% vs. 49.2%, p = 0.004) and demonstrated significantly higher specificity compared with VIDAS (63.1% vs. 53.8%, p = 0.04). CONCLUSION: The study emphasizes the nonuniversal effect of an age-adjusted cut-off and discusses the potential necessity for different cut-off values, particularly in the case of HS-500. These findings contribute to the understanding of D-dimer detection methods in the context of DVT and PE, providing insights into their relative performances and the potential optimization through age-adjusted cut-offs.

In Vitro Conditioning of Adipose-Derived Mesenchymal Stem Cells by the Endothelial Microenvironment: Modeling Cell Responsiveness towards Non-Genetic Correction of Haemophilia A.

In recent decades, the use of adult multipotent stem cells has paved the way for the identification of new therapeutic approaches for the treatment of monogenic diseases such as Haemophilia A. Being already studied for regenerative purposes, adipose-derived mesenchymal stem cells (Ad-MSCs) are still poorly considered for Haemophilia A cell therapy and their capacity to produce coagulation factor VIII (FVIII) after proper stimulation and without resorting to gene transfection. In this work, Ad-MSCs were in vitro conditioned towards the endothelial lineage, considered to be responsible for coagulation factor production. The cells were cultured in an inductive medium enriched with endothelial growth factors for up to 21 days. In addition to significantly responding to the chemotactic endothelial stimuli, the cell populations started to form capillary-like structures and up-regulated the expression of specific endothelial markers (CD34, PDGFRα, VEGFR2, VE-cadherin, CD31, and vWF). A dot blot protein study detected the presence of FVIII in culture media collected from both unstimulated and stimulated Ad-MSCs. Remarkably, the activated partial thromboplastin time test demonstrated that the clot formation was accelerated, and FVIII activity was enhanced when FVIII deficient plasma was mixed with culture media from the untreated/stimulated Ad-MSCs. Overall, the collected evidence supported a possible Ad-MSC contribution to HA correction via specific stimulation by the endothelial microenvironment and without any need for gene transfection.

Prothrombin Is Responsible for the Lupus Cofactor Phenomenon in a Patient with Lupus Anticoagulant/Hypoprothrombinemia Syndrome.

Lupus anticoagulant is a misnomer as it is commonly associated with thromboembolic events. In few cases, the name retains its literal meaning when it characterizes patients with a bleeding disorder. We describe a patient with lupus anticoagulant, hypoprothrombinemia, and major bleeding (lupus anticoagulant/hypoprothrombinemia syndrome). Immunological studies revealed a huge amount of circulating monoclonal immunoglobulin M lambda (IgMλ) antiphosphatidylserine/prothrombin antibodies (14,400 U/mL). Affinity purified monoclonal antibodies (440 U/mL) prolonged the coagulation time of normal plasma by 12.2 seconds (diluted Russell viper venom time) and 25.5 seconds (silica clotting time). The original patient's plasma mixed 1:1 with normal plasma showed a marked prolongation of coagulation times (lupus cofactor) from a ratio of 2.94 to 5.23 in diluted Russel viper venom time and from 2.30 to 3.00 using the silica clotting time. Human prothrombin added to original patient's plasma caused a marked prolongation of coagulation times in diluted Russell viper venom test thus unequivocally explaining the lupus cofactor phenomenon. In conclusion, we have shown that lupus anticoagulant/hypoprothrombinemia syndrome is attributable to monoclonal IgMλ antibodies directed to phosphatidylserine/prothrombin and that prothrombin is the protein responsible for the observed lupus cofactor phenomenon.

A collaborative study by the Working Group on Hemostasis and Thrombosis of the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) on the interference of haemolysis on five routine blood coagulation tests by evaluation of 269 paired haemolysed/non-haemolysed samples.

INTRODUCTION: Haemolysis is the leading cause of sample rejection in laboratory haemostasis. Most studies focused on artificially haemolysed samples. The aim of this study was a prospective assessment of spontaneous haemolysis on haemostasis tests, by comparing results of haemolysed (H) versus new, non-haemolysed (NH) specimens, collected within 4hrs. As new coagulometers can identify interfering substances, visual assessment of haemolysis was also compared with instrumental haemolysis index and stratified in subclasses. MATERIALS AND METHODS: Two hundred and sixty nine paired samples were collected and analysed using ACL TOP750-CTS (Instrumentation Laboratory, Bedford, USA), for prothrombin time (PT), activated partial thromboplastin time (aPTT), D-Dimer (DD), fibrinogen (Fib) and antithrombin (AT). Bias between H and NH was calculated and compared with the respective critical difference (CD). RESULTS: Mean bias was - 0.1 s for PT (P = 0.057), - 1.1 s for aPTT (P < 0.001), 1025 ng/mL for DD (P < 0.001), - 0.04 g/L for Fib (P = 0.258) and 1.4% for AT (P = 0.013). Bias exceeding the CD varied according to the method, with larger differences for aPTT (36.1%) and DD (17.1%) and < 8% for PT, Fib and AT. No correlation emerged between free haemoglobin values and difference in haemostasis tests in H and NH samples for any tests. Moderate/severe haemolysis involved > 95% of samples. The agreement between visual assessment and instrumental evaluation of haemolysis was 0.62. CONCLUSION: Spurious haemolysis deeply influences aPTT and DD, and to a lesser extent AT and Fib. Prothrombin time seems only slightly influenced, suggesting that PT can be accepted also in haemolysed samples. Although a good inter-observer correlation of haemolysis evaluation was found, the instrumental assessment of haemolysis seems recommendable.

Lupus anticoagulant: a multicenter study for a standardized and harmonized reporting.

Laboratory assessment of Lupus anticoagulant (LAC) is very challenging because of inter and intralaboratory variability, which makes it difficult to standardize and harmonize results expression. Five hospital laboratories in North-eastern Italy shared their efforts and their experience in a cross-laboratory study, conducting the diagnostic process as homogeneously as possible and providing a better interpretation for LAC positivity. Hundred normal samples from healthy subjects (20 from each center) were processed to confirm negative upper limits and calculate positivity cutoffs of LAC integrated assays, that is dilute Russell's viper venom time (dRVVT) and silica clotting time (SCT). Moreover, 311 samples previously diagnosed by the laboratories as positive for LAC were analyzed to characterize different positivity levels for each assay. As far as the analysis of healthy subjects is concerned, negative upper limits are set at 1.17 and 1.19 for dRVVT and SCT screen ratio, respectively. Positivity cutoffs are set at 1.20 for dRVVT and 1.23 for SCT, expressed as Test Ratio calculated on screen and confirm integrated tests. Positive results for each integrated assay are subsequently divided into three subgroups: weak, moderate and strong; the results obtained are presented as a score proposal that can provide LAC interpretation. The combined use of both dRVVT and SCT assays and the definition of different positivity levels may lead to clearer, more objective LAC reporting. An interpretative table for LAC-proposed score provides LAC-positive results and it is now adopted by all centers involved in the study.

An Italian external quality assessment (EQA) program on urinary sediment.

BACKGROUND: EQA programs on urinary sediment are rare. We describe an EQA Italian program which started in 2001 and involves today more than 300 laboratories. METHODS: The program, which started with a questionnaire about the methodological aspects on urinary sediment, includes today four surveys per year. These ask the participants the identification and clinical associations of urinary sediment particles shown by colour images (surveys 1 and 3) and the diagnosis of clinical cases presented by both images and a short clinical history (surveys 2 and 4). The results of each survey are then scored and commented. RESULTS: Questionnaire (participants = 287): most methodological aspects were not dealt with properly. IDENTIFICATION: cells, lipids, casts and some contaminants were poorly known. However, when 27 particles were presented for the second time and 16 particles for the third time, the correct identification rate for most of them increased significantly. Clinical associations (No presented = 16): a correct answer was indicated by > or = 84% of participants for all particles but one. Clinical cases (No presented=4): lowest correct identification for urine contamination from genital secretion (77.3%), highest for ureteric stone (94.4%). CONCLUSIONS: Our program shows that EQA programs are both useful and needed.

Quality control in coagulation testing.

The term "QUALITY CONTROL" in laboratory medicine refers to all the procedures commonly used in clinical laboratories to monitor the routine performance of testing processes, to detect possible errors, and to correct problems before test results are reported. In particular, internal quality control (IQC) and external quality assessment (EQA) programs are used to evaluate and improve quality in laboratory medicine. Laboratory testing is necessary for the diagnosis and treatment of patients with hemostatic disorders. However, whereas the benefits of quality control and quality assessment in hemostasis have been demonstrated many times and are well documented, available scientific evidence is significantly less than that in clinical chemistry and in other fields of laboratory medicine. Currently available data on analytical quality in coagulation testing not only demonstrates that quality is often unsatisfactory, but also highlights the need for more objective establishment of performances goals. This should be useful for better addressing both IQC and EQA programs. New challenges to EQA schemes for coagulation testing derive from the introduction of innovative tests, genetic analysis, and the need to assess not only analytical procedures but also all steps included in the total testing process.

Risk management in laboratory medicine: quality assurance programs and professional competence.

To guarantee excellent performance and service, the process of identifying and treating error risks must be integrated into the total testing process. Quality Assurance Programs (QAPs) represent an important tool that allows us to identify errors and pinpoint any need for further systematic investigations, and to rectify procedures to improve the inputs and processes by which the service is delivered. The models used by the laboratory to assure quality and manage the risk of errors have been modified in line with an approach in which the identification of quality goals and the redefinition of professionals duties and responsibilities are indispensable. Error risk is currently high in some areas of laboratory activity, and QAP is needed now more than ever. The present paper provides some descriptive examples of an approach that can be followed to manage an External Quality Assessment Scheme (EQAS) and quality indicators (QIs), the main tools used by laboratories to assure the quality of their service, for the prevention of error risk. In particular, we describe the correct approach to choose EQAS, to use information from the EQAS report, to design a QI model, and to analyze any QI data. The examples highlight that any well-designed quality system can be ineffective if it is not managed by highly competent professionals with a deep sense of responsibility.

External Quality Assessment: an effective tool for Clinical Governance in laboratory medicine.

The implementation of Clinical Governance will require a redefinition of duties and accountability as a prerequisite to develop and achieve an overall improvement in clinical care through a culture of assessment and monitoring of quality. External Quality Assessment Schemes (EQAS) are the main tool enabling laboratories to measure the quality of their results; they must carefully assess and monitor all elements contributing to the formulation of laboratory information (results, reference ranges/decisional levels, interpretative comments and diagnostic algorithms). There are different ways to design and manage a Scheme and EQAS coordinators are mainly responsible for its effectiveness. The present paper reports, as an example, some experiences of the Centre of Biomedical Research (CRB), which manages EQAS according to high quality specifications and laboratories' needs, that can reflect the Clinical Governance philosophy. Our findings show that EQAS are able to control all the above aspects and, if organisers are committed to fulfilling the responsibility and accountability principles, they will be of great value in quality assessment and in developing an External Quality Assurance Program (EQAP). This is an inter-laboratory comparison designed and conducted to assure the following: evaluation of participants' performance (by evaluating not only analytical performance, but also test interpretation, and advice for clinicians on laboratory requests and diagnosis); evaluation of method performance; and continuous education, training and help. The main aim of the activities of an EQAP in Laboratory Medicine is to sustain improvements in the quality of services provided by participating laboratories for the benefit of patients.

Quality specifications in EQA schemes: from theory to practice.

BACKGROUND: External quality assessment (EQA) is a tool for quality management in clinical laboratories and its main objectives are assessment of participants and methods performance, training and advice. This paper describes the quality specifications used in EQA schemes of the Centre of Biomedical Research (CRB), in order to design schemes that can assess laboratory reliability performances, meet the changing needs and quality recommendations. METHODS: Quality specifications for control materials, statistical procedures and goals to assess laboratory performance have been applied and introduced in EQA schemes managed by CRB. RESULTS: The application of well-defined quality specifications has demonstrated effective. In particular, we report results on alkaline phosphatase and cholesterol obtained using commercial control materials and human serum controls, in two different EQA surveys; the inter-laboratory variability (CVinter%) for troponin I analysed with a diagnostic system and assigned values of CK-MB mass obtained using four different diagnostic systems; the percentage of acceptable performances obtained by means of the application of goals based on clinical criteria, biological variation, state-of-the-art and used for EQA schemes, and referring to some analytes with significant clinical values such as cholesterol, glucose, glycated hemoglobin and sodium. CONCLUSIONS: The design of reliable EQA schemes based on evidence-based quality specifications is a pre-requisite for supporting the quality improvement of clinical laboratories.

Interpretative comments and reference ranges in EQA programs as a tool for improving laboratory appropriateness and effectiveness.

INTRODUCTION: Laboratory information is generated when a meaning is given to certain data. This is usually achieved by comparing a laboratory test result with the reference range/decisional limit (RL), and by providing consultation for the interpretation of data, advice, and follow-up testing. AIM: In this paper, we investigate factors affecting the conversion of data into useful information with regard to biochemical markers of myocardial damage (CK-MB mass, myoglobin, and troponins), in view of their importance in detecting myocardial necrosis. Our aim was to report results obtained in order to verify the consensus between laboratories with reference to interpretative comments and the reference ranges/decisional limits added to clinical reports. METHODS: A questionnaire and simulated medical reports on three different patients were distributed to participants (94 laboratories) in the 2001 cycle of the External Quality Assessment (EQA). Moreover, we analysed 113 medical reports sent by laboratories during the most recent EQA cycle 2002, and checked the number of different RLs used, both independent and within the diagnostic system used. We also compared each laboratory result of a control sample, obtained in the 2002 cycle, with declared RL in order to verify the clinical significance of results ("normal" or "pathological") for troponin I and CK-MB. RESULTS: Our findings show that few laboratories regularly add interpretative comments to medical reports. On the contrary, they cooperate with clinicians who require consultation, advice, and information for the appropriate use of biochemical markers. There is a general consensus among participants regarding probable syndromes suggested by the interpretation of the same result and most laboratories also agree on further investigations to be carried out for several diseases. Concerning RL, the data demonstrate that numerous different RLs are used to report the results of the biochemical markers evaluated, both when considered independent of the diagnostic system used and within the diagnostic system used. DISCUSSION AND CONCLUSIONS: The biochemist does not have the opportunity to verify the efficacy of the interpretation that he/she provided. An audit of this activity is therefore required to allow the laboratory to monitor its own performance and to assure good practice. The evaluation of interpretative comments, through specific surveys, should be a prime objective of EQA organisers. Well-designed EQA programs can, moreover, support laboratories in establishing appropriate RL and in verifying the clinical significance of their results with respect to that of other laboratories. Our survey on interpretative comments and the analysis of the RLs further demonstrate how laboratory medicine can contribute to the objective evaluation of the patients' health status.

Appropriateness of cholesterol and triglycerides reporting checked by External Quality Assessment programs.

BACKGROUND: The recommendations of the Second Joint Task Force of European and Other Societies on Coronary Prevention and the third Adult Treatment Panel report (ATPIII) released by the National Cholesterol Education Program are based on accumulating evidence concerning the contribution of lipoproteins and other risk factors in the development of coronary heart disease (CHD). The laboratories play an important role in the successful adoption of these guidelines. METHODS: In External Quality Assessment (EQA) programs managed by the Center of Biomedical Research, results and respective reference intervals (RI) are sent as laboratory's medical form. We assessed how well the 200 participants to EQA scheme 2002 for clinical biochemistry reported total cholesterol (TC) and triglycerides (TGs) results according to either European or National Cholesterol Education Program (NCEP) guidelines. RESULTS: Only 18% of laboratories reported total cholesterol concentrations correctly in terms of desirable, borderline-high, and high risk for the CHD development, 12% reported a single desirable value (180, 190, or 200 mg/dl), and 70% reported the RI (85 laboratories in the whole interval, 34 are the only upper reference limit and 15 are the desirable value in addition to RI). The upper reference limit was 200 mg/dl in 65% of cases, but 32% of laboratories presented higher limits, reaching values as high as 250-260 mg/dl. Only the 3.7% of laboratories reported triglyceride concentrations in terms of risk-oriented ranges for the CHD development, 6.8% the single desirable value, and 89.5% the RI. CONCLUSION: Our study demonstrates that the current practice of reporting results for cholesterol and triglycerides does not follow the guidelines, and appropriate changes are required to be made.