Identification of determinants of the between-operator variation of the total protein S antigen assay: a collaborative study of the Dutch Working Group on Haemostasis Laboratory Diagnosis.

In a previous study, a between-operator variability (CVOBETWEEN) of 9.6% and 15.0%) was observed for total protein S antigen assays in 11 laboratories using a frozen or lyophilized reference plasma, respectively, and the need to standardize the use of lyophilized reference plasma was identified. The aim of the present study was to identify further determinants of this CVOBETWEEN in order to improve between-laboratory comparisons of test results for one method for protein S antigen assay. Two protocols were carried out: the first again involving local execution but using a joint standardized and detailed prescription of the technical performance in each laboratory; the second using a central session for all operators with the same prescription but with joint reagent and equipment. In the present study, improved handling of lyophilized reference plasma was included and resulted in comparable CVOBETWEEN of 10.9% and 9.6% for the use of frozen and lyophilized reference plasma for the local test performance. An improvement was found in the CVOBETWEEN in the central session compared with the standardized local performance, showing lower values for the central performance of 8.5 and 6.6% for frozen and lyophilized reference plasma, respectively. Further analysis of the difference between the local and central test performance identified the use of different curve fit options of data evaluation software as a significant source of this difference. Interestingly, the within-operator variability in the central performance was around 2% lower (5.9 and 6.0% for frozen and lyophilized plasma, respectively) than that in the local performance (8.1 and 8.0% for frozen and lyophilized plasma, respectively). Although the reduction is not statistically significant, it suggests an effect of reduction of the workload and simplification of procedures for individual operators on the within-operator variability. In this study, in which 11 operators/laboratories participated, the lowest variability between operators andwithin laboratories was obtained in the central test performance, which is suggested to be the lowest attainable variability for the measurement of total protein S antigen. The practical factors involved in local performance that require attention to reach similar levels of variability are mainly liquid handling, curve-fit procedures and simplicity of practical procedures.

Effect of a common reference plasma on the inter-laboratory variation of the measurement of total and free protein S: a collaborative study of the Dutch Working Group on Haemostasis Laboratory Diagnosis.

The comparability of test results for protein S between laboratories is hampered by a high inter-laboratory variability. The effect of the use and type of common reference plasma on the inter-laboratory variability of the total and free protein S measurement was evaluated. The results of 10 plasma samples measured against a centrally distributed frozen plasma and a centrally distributed lyophilized plasma were compared with those of various local reference plasmas regularly used by the 11 participating laboratories. The mean intra-assay coefficient of variation for total protein S in each laboratory varied from 3.8 to 12.8% (mean intra-assay CV of all laboratories: 7.4+/-2.3%); for free protein S this range was 3.1 to 13.3% (mean intra-assay CV of all laboratories: 6.6+/-2.7%). We confirmed the high inter-laboratory coefficient of variation (mean+/-SD) with the several local reference plasmas for both total protein S (13.4+/-5.6%; n = 10) and free protein S (17.1+/-7.5%: n = 11). For total protein S, the inter-laboratory CV was reduced to 11.5+/-4.8%, (p=0.05) by using a common frozen reference plasma, while it was increased to 16.8+/-3.4%, (p=0.022) using a common lyophilized reference plasma. For free protein S, these values decreased only statistically significantly for the common lyophilized reference plasma, to 15.1+/-6.0%, (p = 0.008). For free protein S, the dilution factor used was identified as a factor influencing the inter-laboratory variability. This study shows that, for both types of protein S measurements, using one frozen reference plasma shows a slight decrease in inter-laboratory variability, while a common lyophilized plasma shows inconsistent results. It is concluded that further investigation is necessary to examine other sources of variability to increase the comparability of laboratory results for both total and free protein S.

A quantitative approach to characterizing cell lysis caused by mechanical agitation of Streptomyces clavuligerus.

Streptomyces clavuligerus is a commercially important actinomycete that is used to produce clavulanic acid, a beta-lactamase inhibitor. Observations during 10 batch cultivations with S. clavuligerus on defined media have led to the finding that the organism is very sensitive to shear when grown in batch cultures with increasing stirrer speed. The stirrer speed was increased to keep the dissolved oxygen level above 50% air saturation. A quantitative approach based on the calculation of elemental balances and a simple mathematical model is proposed to characterize the biomass lysis. Finally, a linear relation between biomass yield and observed specific growth rate is determined. Results show that cell lysis occurs at a high degradation rate, e.g., mu(max) = 0.16 h(-1) and k(d) = 0.07 h(-1), when the gassed power input increases above 1.1, 1.7, or 2.0 kW/m(3), respectively, depending on the medium composition. The overall biomass yield on substrate is dramatically reduced in all experiments (>30%).

Does heparin pretreatment affect the haemostatic system during and after cardiopulmonary bypass?

In this clinical pilot study, the influence of heparin pretreatment on the haemostatic system during and after cardiopulmonary bypass (CPB) was investigated. Thirteen patients scheduled for elective coronary artery bypass grafting (CABG) were divided into two groups: heparin pretreated (HP, n = 6) and non-heparin pretreated (NHP, n = 7). Blood samples were taken for measurements of plasma antithrombin-III (AT-III) activity, plasma heparin levels, activated clotting time with (HACT) and without (ACT) heparinase, whole blood platelet function, platelet count, thrombin-antithrombin-III complexes and D-dimer levels. Also, the mediastinal blood loss within the initial 20 h after surgery, and the blood transfusion requirements were monitored. The mean duration of the heparin pretreatment was 55 h (range 24-161 h). There was no significant difference in plasma AT-III activity and platelet count between the groups. Before and after CPB, the platelet responsiveness was better in the NHP group (p < 0.05). The HACT was prolonged in the NHP group during and after CPB compared to baseline values (p < 0.05), whereas, in the HP group, no significant changes were found. Plasma heparin levels and ACT values suggested adequate anticoagulation during CPB. However, the extent of thrombin inhibition and fibrinolysis increased with time on CPB, but did not differ between the two groups. Twenty hours after surgery, the thrombin inhibition showed to be significantly higher in the NHP group. Furthermore, mediastinal blood loss showed a tendency to be lower in the HP group (p = 0.08). However, there was no difference in blood transfusion requirements between the groups. These data suggest that short-term heparin pretreatment affects the perioperative platelet responsiveness and attenuates the consumption of coagulation factors.

D-dimer determination to assess regression of deep venous thrombosis.

A number of studies evaluating deep venous thrombosis (DVT) have demonstrated that plasma levels of thrombotic and fibrinolytic parameters change during treatment, but the relationship between thrombus regression and evolution of these markers remains unknown. The objective of the present study was to correlate levels of D-Dimer (DD) with thrombus regression as assessed by duplex scanning. From 44 patients treated for acute DVT, DD were determined at diagnosis and at the end of initial heparin therapy of at least 5 days. Thrombus regression was measured by repeated duplex scanning at diagnosis and after 1 and 3 months. DD significantly decreased during heparin treatment as compared with values at presentation. DD levels were significantly higher in the group of patients without normalization of the DVT after 3 months (p = 0.003). A ninefold excess tendency was seen for DD levels > 1200 ng/ml at the end of initial treatment to be associated with poor resolution of the DVT [odds ratio 9.0, 0.95 confidence interval (CI) 2.3-35.4]. When the patients with an established malignancy were excluded, the differences were even more significant (p = 0.0004 for DD levels after initial treatment and an odds ratio of 17.5, 0.95 CI 3.3-92.5). These results suggest that increased DD levels after the initial phase of treatment are related to poor resolution of DVT after 3 months. These findings contribute to further insight into the process of thrombus regression. Furthermore high DD levels might help to identify the patients with a poor prognosis and could be useful to judge the efficacy of anticoagulant treatment.

Reliability of five rapid D-dimer assays compared to ELISA in the exclusion of deep venous thrombosis.

Studies measuring the fibrin degradation product D-Dimer (DD) using enzyme-linked immunosorbent assays (ELISA) in patients with venographically proven deep venous thrombosis (DVT) suggest that it is possible to exclude DVT when DD level is below a certain cut-off level. However, ELISA methods are time-consuming and not available in all laboratories. Different rapid latex-agglutination assays have been investigated, but their sensitivity is considerably lower. In the present study we compared the value of four novel latex DD tests (Tinaquant, Minutex, Ortho and SimpliRed) and one rapid ELISA (VIDAS) to a classical ELISA DD assay (Organon Mab Y18) in 132 patients suspected of DVT. The VIDAS, a new quantitative automated ELISA, had a sensitivity of 100% and a negative predictive value of 100% for both proximal and distal DVT at a cut-off level of 500 ng/ml. The Tinaquant assay, a new quantitative latex method, had a sensitivity of 99% and a negative predictive value of 93% for both proximal and distal DVT at a cut-off level of 500 ng/ml. For proximal DVT only, both assays had a sensitivity and negative predictive value of 100%. VIDAS and Tinaquant correlated well with ELISA (correlation of r = 0.96 and r = 0.98 respectively). Sensitivities of the semi-quantitative latex assays Minutex, Ortho and SimpliRed were considerably lower (77%, 51% and 61% respectively). These results suggest that VIDAS and Tinaquant may be used instead of ELISA DD in the exclusion of DVT. Tinaquant can be performed within 20 min and VIDAS within 35 min. Both assays might be used as a routine screening test and should be evaluated in large clinical management studies.

New developments in the treatment of deep venous thrombosis.

An initial course of standard heparin (SH) or low-molecular-weight heparins (LMWH) is regarded as the treatment of choice for patients with deep venous thrombosis (DVT). LMWH have better bioavailability after subcutaneous administration, have a longer half-life, and show higher and more predictable anticoagulant activity. As a result they can be given subcutaneously and without laboratory control, using a dose that is determined by bodyweight. Because of these multiple advantages of LMWH they will replace SH in the future and subsequently home treatment with LMWH of selected patients seems feasible. The currently accepted approach is to start with SH or LMWH therapy combined with oral anticoagulant therapy. (OAT) at the time of diagnosis. The course of SH or LMWH should continue for at least 5 days, provided that international normalized ratio (INR) is in the therapeutic range on 2 consecutive days. OAT should be continued for at least 3 months to prolong the prothrombin time to an INR of 2-3. When oral anticoagulants are either contraindicated or inconvenient, SH or LMWH can be used at the middosing interval. The role of anti-platelet treatment is not yet established and should be compared with coumarin therapy in the future.

Predictive value of Glasgow coma score for awakening after out-of-hospital cardiac arrest. Cerebral Resuscitation Study Group of the Belgian Society for Intensive Care.

The Glasgow coma score (GCS) during days 1-6 after cardiac arrest was used to predict neurological outcome in 360 resuscitated victims of out-of-hospital cardiac arrest. A predictive rule based on the best GCS of 216 patients resuscitated in 1983-84 (prediction group) was constructed, and its predictive power was tested on 133 patients treated in 1985 (test group). Neurological outcome was correctly predicted 2 days after cardiac arrest in 80% of the prediction group, with a best GCS of 10 or above and 4 or below as cutoff points. For patients with a best GCS of 5-9, prediction of outcome was possible 6 days after cardiac arrest, with a best GCS of 8 during the first 6 days as the single cutoff point. The rule was then validated in the test group: the sensitivity was 96%; the specificity 86%; the negative predictive value 97%; and the positive predictive value 77%. These data suggest that this simple GCS-based rule can be helpful in predicting outcome in patients resuscitated after out-of-hospital cardiac arrest, but confirmation of these data is required in a prospective study in a larger number of patients.

Factor XI kinetics after plasma exchange in severe factor XI deficiency.

Plasma exchange in a patient with factor XI deficiency allowed the determination of the factor XI disappearance time. The decay curve showed a biphasic pattern with a t1/2 of h. After a loading dose by means of plasma exchange with fresh frozen plasma (FFP) up to a factor XI level of 65%, administration of 0.5 liter FFP every 12 h was necessary to maintain the factor XI concentration between 40 and 60%. The clearances of factor XI, calculated from the results of plasma exchange and from the maintenance dose, correlated very well and showed that the t1/2 of factor XI did not change after surgery. The patient underwent major surgery uneventfully but ultimately died 3 1/2 weeks after operation from cerebral damage due to cardiac arrest, which occurred on the 2nd postoperative day.

[An integrated emergency medical care system at the University of Leuven]. / Een geïntegreerd systeem voor dringende medische hulpverlening te Leuven.

There are three motives to start an organized medical emergency care system: to provide a systematic approach to all admitted medical emergencies in a specific structure in the hospital. to decrease mortality and morbidity by immediate evaluation and treatment of vital functions. to supply adequate emergency care for the region. These motives can only be realized through well-known options: the system must be multidisciplinary. excellence in critical care medicine must be available at all times. a system of mobile medical emergency care must be developed. all available regional emergency care facilities must be integrated. Only this way can the emergency service evolve as the central link of critical care medicine.

Six years experience with an emergency department based mobile emergency care delivery system.

The purpose of mobile emergency care is to provide on a basis of 24 hours out of 24, the necessary material (ambulances wiwth resuscitation equipment) and personnel (ambulance-drivers, nurses and doctors), in order to care for the victim of accident or sudden illness on the spot and during transport to the hospital. The experience gained with a mobile emergency care delivery system, organized within the Emergency Department of the University Hospital Sint-Rafaël Leuven, Belgium, is analyzed and discussed. The conclusions of the study stress the advantages, as far as economics and efficiency are concerned, of the integration of the mobile emergency care delivery system in the emergency department of the highly specialized university hospital.

A rapid turbidimetric determination of fibrinogen degradation products (FDP).

A rapid and simple turbidimetric determination of fibrinogen degradation products is described. This method is based on the increase of the turbidity due to the formation of precipitating antigen-antibody complexes after addition of rabbit antihuman fibrinogen antiserum to human serum. The increase in turbidity correlates very well with results obtained with the haemagglutination inhibition technique and has the advantage of a more objective quantitation. The reproducibility appears to be quite good. The turbidimetric method is independent of the colour of serum samples.

An advanced emergency care delivery system.

In order to provide for immediate expert emergency care at the site of an accident or sudden illness and during the transport to the emergency department, different options have been taken in East and West. In Leuven, Belgium an intermediate solution to the problem ha been chosen, firstly be developing a concise, fully portable set of medical equipment, and secondly by forming a "mobile emergency group" within the emergency department, consisting of nurses (male and female) with experience in critical care and selected through a specially conceived training program. This "mobile emergency group" is capable of administering advanced emergency care at the order of a physician present at the scene, or giving orders by radio. The "mobile emergency group" provides emergency care in the ambulances and in the hospital.