Refinement and feasibility testing of a manual micro-method for protamine titration

Current clinical recommendations for unfractionated heparin (UFH) therapy suggest target APTT ranges should reflect heparin concentrations of 0.2-0.4 IU/ml by protamine titration or 0.35-0.7 IU/m by an anti-Xa assay. Historically, performance of a manual protamine titration assay has been labour intensive and required a large plasma sample. However, recent studies have described difficulties with standardizing anti-Xa assays and demonstrated poor correlation of anti-Xa assays in children. This study aimed to refine and test the feasibility of a modified protamine titration assay using 100 microl of plasma. The resultant method produced reliable and repeatable results in adult plasma pools spiked with UFH. The feasibility of this method was proven by testing of in vivo heparinised samples obtained from children. This protamine titration method may offer an alternative to anti-Xa assays for clinical monitoring of children on heparin therapy, and will enhance clinical studies investigating paediatric-specific management of UFH therapy.     

HEPTEST: a suitable method for monitoring heparin during pregnancy.

Methods of monitoring heparin in pregnancy are problematic. The aim of this study was to assess the plasma HEPTEST as a rapid and reliable test for heparin monitoring in pregnancy. HEPTEST, activated partial thromboplastin time (APTT) and chromogenic anti-Xa assays were performed on individual heparin-spiked plasma samples from two groups: normal non-pregnant women (n = 6) and normal pregnant women during the third trimester (n = 6). Heparin activity curves were established in plasma from both groups for low (< 0.3 IU/ml), intermediate (0.3-0.7 IU/ml) and high (> 0.7 IU/ml) heparin concentrations and validated by comparison with the anti-Xa chromogenic assay. Both the APTT and HEPTEST demonstrated good correlation with anti-Xa levels across all heparin concentrations in both plasma groups (r range = 0.879-0.945). In comparison with the APTT, the HEPTEST showed better correlation with anti-Xa levels at low concentrations of heparin (r values 0.933 vs. 0.772, respectively). For both the APTT and HEPTEST there were significant differences between the clotting times in pregnant and non-pregnant plasma at a number of heparin concentrations. This data supports the plasma HEPTEST as an acceptable alternative to the chromogenic anti-Xa assay for monitoring heparin thromboprophylaxis in pregnancy.     

HEPTEST: a suitable method for monitoring heparin during pregnancy

Methods of monitoring heparin in pregnancy are problematic. The aim of this study was to assess the plasma HEPTEST as a rapid and reliable test for heparin monitoring in pregnancy. HEPTEST, activated partial thromboplastin time (APTT) and chromogenic anti-Xa assays were performed on individual heparin-spiked plasma samples from two groups; normal non-pregnant women (n = 6) and normal pregnant women during the third trimester (n = 6). Heparin activity curves were established in plasma from both groups for low (<0.3 IU/ml), intermediate (0.3–0.7 IU/ml) and high (>0.7 IU/ml) heparin concentrations and validated by comparison with the anti-Xa chromogenic assay. Both the APTT and HEPTEST demonstrated good correlation with anti-Xa levels across all heparin concentrations in both plasma groups (r range = 0.879–0.945). In comparison with the APTT, the HEPTEST showed better correlation with anti-Xa levels at low concentrations of heparin (r values 0.933 vs 0.772, respectively). For both the APTT and HEPTEST there were significant differences between the clotting times in pregnant and non-pregnant plasma at a number of heparin concentrations. This data supports the plasma HEPTEST as an acceptable alternative to the chromogenic anti-Xa assay for monitoring heparin thromboprophylaxis in pregnancy.     

Three different chromogenic methods do not give equivalent anti-Xa levels for patients on therapeutic low molecular weight heparin (dalteparin) or unfractionated heparin

In this study we compare three chromogenic methods (IL-Heparin, Stachrom Heparin and Heparin Sigma) on two different instruments (ACL300+ and AMAX CS190) for patients on dalteparin (n = 41), a low molecular weight heparin or unfractionated heparin (n = 50). For dalteparin the mean anti-Xa levels for IL-Heparin, Stachrom Heparin and Heparin Sigma were 0.27, 0.30 and 0.21 U/ml, respectively, while for heparin they were 0.52, 0.55 and 0.41 U/ml, respectively. To test for instrument specific effects, IL-Heparin and Stachrom Heparin were repeated on both instruments on 42 patients receiving unfractionated heparin. For IL-Heparin the mean anti-Xa levels on the AMAX CS190 and ACL300+ were 0.51 and 0.59 U/ml, respectively, while for Stachrom Heparin they were 0.55 and 0.67 anti-Xa U/ml. We conclude that different chromogenic anti-Xa methods do not give equivalent anti-Xa levels for the same samples. Moreover, the differences are clinically significant. This is not explained entirely by instrumentation effects. Recommended therapeutic ranges may need to be method and instrument specific.     

Neutralization of Heparin in Plasma by Platelet Factor 4 and Protamine Sulphate

The neutralization of heparin by protamine sulphate and platelet factor 4 (PF₄) has been studied using kaolin cephalin clotting time (KCCT), thrombin clotting time (TCT) and anti-Xa assays to measure residual heparin levels. Protamine sulphate and purified PF₄ had almost equivalent neutralizing ability on a weight basis regardless of which assay system was used. Plasma samples that were heparinized in vitro could be totally and readily neutralized by both agents in all of the assay systems used. However, when heparinized plasma samples were obtained following intravenous injection of the drug different results for neutralization were obtained depending on the heparin assay used. When residual heparin was measured by anti-Xa assay partial neutralization of heparin was observed even in the presence of a large excess of neutralizing agent. The same in vivo derived heparinized plasma samples had no heparin activity following neutralization if residual heparin was measured by KCCT and TCT assays. Further ion exchange chromatography experiments demonstrated that the heparin-like activity that could not be neutralized in the anti-Xa assay was not adsorbed by the resin ECTEOLA-cellulose, and therefore could not be removed from plasma by this technique. These results suggest, therefore, that part of the plasma anti-Xa activity produced following intravenous injection of heparin differs from the anti-Xa activity obtained following addition of the drug to plasma in vitro .     

Anticoagulant Activities of High and Low Molecular Weight Heparin Fractions

The anticoagulant activities of high and low molecular weight heparin fractions were measured by three assay methods, both in vitro, and after intravenous injection in volunteers. The low molecular weight (LMW) fraction had similar anti-Xa activity in vitro to the high molecular weight (HMW) fraction, but in APTT assays the HMW fraction was about twice as potent. After intravenous injection, the two fractions gave equal heparin levels by anti-Xa assays, but in APTT assays using synthetic substrate S-2222 gave about 20% lower levels than anti-Xa clotting assays for both heparins. Complete protamine neutralization of the post-injection heparin activity was found in APTT and synthetic substrate assays, but about 20% of the clotting anti-Xa effect could not be neutralized. Complete neutralization of the fractions by protamine was shown by all three assays in vitro. This non-neutralizable activity probably accounts for the difference between the anti-Xa clotting and synthetic substrate assays. Studies by crossed immunoelectrophoresis and affinity chromatography indicated that the antithrombin III binding properties of the two fractions were similar.     

Partial reversal of low molecular weight heparin (PK 10169) anti-Xa activity by protamine sulfate: in vitro and in vivo study during cardiac surgery with extracorporeal circulation

Neutralization of a low molecular weight (LMW) heparin fraction by protamine sulfate was evaluated in vitro and in vivo. Anti-Xa and anti-IIa activities were measured by amidolytic and coagulation methods (activated partial thromboplastin time, APTT). Fifteen patients (4 males and 11 females) underwent surgery with extracorporeal circulation. In vitro, anti-Xa and anti-IIa activities and APTT of unfractionated heparin were neutralized with a protamine/heparin (P/H) gravimetric ratio of 1.6, 1.33 and about 2, respectively. Anti-IIa activity and APTT induced by PK 10169 were completely corrected at a P/H ratio of 1 and 2, respectively, while anti-Xa activity was incompletely neutralized at a ratio of 5. In vivo, in 9 patients who did not receive intravenous protamine sulfate, a good correlation was found between doses of PK 10169 infused, anti-IIa plasma level and blood loss. In 3 patients who were treated prophylactically with protamine, bleeding was normal or only slightly increased. In 3 patients who received protamine because of hemorrhage, mean anti-Xa and anti-IIa were 2.3 and 0.54 U before and 1.32-0.06 U after neutralization. Bleeding was stopped by a second dose of protamine in 1 patient, but blood loss was abnormal in the other patients. However, a correlation between bleeding and anti-Xa or anti-IIa activities was not clearly evident.     

Evaluation of a new venom‐based clotting assay of protein C

Congenital protein C deficiency significantly increases the risk of venous thromboembolism, a serious and potentially lethal condition. Protein C levels can be determined by chromogenic, clotting and antigenic assays, each type of assay has differences in specificity and sensitivity to protein C deficiency. In principle, clotting‐based assays of protein C are preferred over chromogenic assays, as they can detect some rare mutations that are missed by the chromogenic assay, however, clotting‐based assays may be prone to inaccuracy because of poor specificity. We have evaluated a new venom‐based clotting assay of protein C, and optimized it for use on Sysmex CA‐1500 analyser. The assay was linear from 0 to 130 U/dl, a normal plasma demonstrated good inter‐assay precision, with a coefficient of variation of 4.8%. The assay compared well with antigenic‐ and venom‐based chromogenic protein C assay in normal individuals, subjects with lupus anticoagulant, and subjects with FV Leiden. Median protein C levels by clotting, chromogenic and antigen for the three subject groups were 108 U/dl, 108 IU/dl and 109 IU/dl for normal subjects, 94 U/dl, 106 IU/dl and 103 IU/dl for subjects with lupus anticoagulant, and 102 U/dl, 104 IU/dl and 100 IU/dl for subjects heterozygous for FV Leiden. Comparing levels of clotting protein C with protein C antigen by ratio (clotting/antigen), the three groups showed small differences that did not quite reach statistical significance, (mean ratios ranged from 0.95 to 1.01, anova P = 0.0561), the lowest ratio was with the lupus anticoagulant group. Comparing clotting assay with chromogenic assay by ratio (clotting/chromogenic), the three groups did show a statistically significant difference (P = 0.0033) which was due to a difference in mean ratios between normal and lupus anticoagulant groups (ratios 1.00 and 0.91, respectively, P < 0.01). There was no statistical difference in any of the groups when comparing chromogenic protein C with protein C antigen (mean ratios ranged from 1.02 to 1.05, P = 0.3925). In a normal sample, the clotting‐based protein C level was unaffected by increasing FVIII level by up to 1000 IU/dl, using intermediate purity FVIII concentrate. The new assay is considered to be a suitable assay for the routine diagnosis of protein C deficiency.     

Differential regulation of thrombin activatable fibrinolytic inhibitor by low molecular weight heparins. Pharmacologic implications.

AIM: Thrombin activatable fibrinolytic inhibitor (TAFI) is activated via cleavage by thrombin thrombomodulin in complex, and can be regulated by anticoagulant drugs such as the heparins. Low molecular weight heparins (LMWHs) have different antithrombin/anti-Xa profiles and therefore vary in the degree to which they inhibit TAFI. The purpose of this study was to determine the differential regulation of TAFI by LMWHs. METHODS: Dalteparin, enoxaparin, tinzaparin, parnaparin and heparin were supplemented to normal human pooled plasma at different concentrations (0-2.5 U). A chromogenic based assay (Pentapharm Inc., Basil, Switzerland) was used to measure activatable TAFI in each set of samples. RESULTS: Heparin clearly had the highest degree of TAFI inhibition with an IC50 of 0.10 U, which correlates with its coagulation profile. Dalteparin, Tinzaparin, Parnaparin had similar IC50s, 0.6-0.8 U/ml respectively, while enoxaparin had a higher IC50 (>1.0 U/ml). These results strongly correlate with the anti-IIa inhibition of each agent but not with the anti-Xa. However, it is interesting to note that these drugs are administered according to anti-Xa units not anti-IIa. CONCLUSIONS: These results suggest that each LMWH may inhibit TAFI to a different extent that is not dependent on the anti-Xa potency. Indiscriminate inhibition of TAFI may cause bleeding, while suboptimal inhibition may result in thrombosis. Because of the compositional difference, heparin and LMWHs may produce differential inhibition of TAFI and therefore result in product dependent modulation of hemostatic process which may or may not be related to their antithrombin effects.     

Laboratory analysis of blood samples from patients treated with tinzaparin

Tinzaparin at two dosages, 175 anti-Xa U/kg subcutaneously administered for 7 days, followed by warfarin, and 175 anti-Xa U/kg subcutaneously given for 90 days was compared with continuous intravenous unfractionated heparin (UFH) for 5 days, followed by warfarin for 3 months, were tested in the treatment of patients with proximal deep vein thrombosis. Several laboratory assays were used to monitor the effects of tinzaparin and UFH. The tinzaparin only study arm produced a 4- to 6-second prolongation of the activated partial thromboplastin time (aPTT). However, in the anti-Xa chromogenic assay and the Heptest assays, there was a prolongation after the administration of all three agents. In the two groups treated for 7 days, the anti-Xa and Heptest values returned to baseline after cessation of therapy. In the patients treated with tinzaparin for 90 days, the anti-Xa and Heptest remained elevated throughout the treatment period. The anti-IIa (anti-thrombin) results were considerably lower values in the tinzaparin-treated groups. Tissue factor pathway inhibitor (TFPI) antigen levels were elevated 2- to 2.5-fold in all three groups. In addition, the thrombin/antithrombin (TAT) complexes were also measured. After treatment, the TAT levels decreased over time. Tinzaparin was more effective in decreasing these levels. These results suggest that both Heptest and anti-Xa assays can be used to monitor patients receiving tinzaparin. TAT may be a useful test in monitoring the resolution of the clots. However, additional clinical validation is required to demonstrate the relevance of these parameters with the clinical outcome.     

research paper: Clinical use of unfractionated heparin therapy in children: time for change?

Paediatric recommendations for unfractionated heparin (UFH) management are extrapolated from adult trials, a practice that may contribute to the inferior UFH‐related outcomes in children compared to adults. This is the first study to determine UFH concentration in a population of children and correlated UFH concentration with measures of UFH effect. Correlation coefficients between protamine titration (concentration) and activated partial thromboplastin time (APTT), anti‐ activated factor X (Xa) assay and thrombin clotting time (effect) were 0·59, 0·46 and 0·52 respectively. A protamine titration level of 0·2–0·4 iu/ml in children was not equivalent to an anti‐Xa assay of 0·35–0·7 iu/ml but to an anti‐Xa assay 0·17–0·85 iu/ml. In addition, use of the anti‐Xa or protamine titration assays to establish an APTT therapeutic range resulted in upper limits of APTT ranges exceeding 200 s. Existing methods for determining therapeutic ranges for UFH in adult populations do not produce equivalent ranges in children. As a result, paediatric clinical guidelines that state a therapeutic range for UFH can be determined using a protamine titration assay of 0·2–0·4 iu/ml or an anti‐Xa assay of 0·35–0·7 iu/ml are not based on appropriate evidence. There is an urgent need for change in our approach to the use of UFH in children.     

Validity of serine protease inhibition tests in the evaluation and monitoring of the effect of heparin and its fractions

The advent of heparin fractions for clinical use has prompted a reevaluation of the available methods for the assaying of heparin activity in blood. Newly developed heparin fractions are routinely evaluated in terms of their anti-Xa and anti-IIa amidolytic USP, APTT, and anti-Xa coagulant actions. A wide variation between these assays exists due to the use of nonstandard reagents and varying assay conditions. Amidolytic assays, although more biochemically defined than coagulant assays, do not use a natural substrate and work in a diluted plasma system. This may account for discrepancies and differing sensitivities between results in the amidolytic and coagulant assays. The APTT, PTT, and TT may not be effected by the LMFs, and these are the tests currently used to monitor patients on heparin therapy. A poor correlation is observed between the global tests with the newly developed anti-Xa and anti-IIa assays. Furthermore, marked differences in the inhibitory responses are observed with heparin and its fractions. The anti-Xa and anti-IIa assays may provide a more sensitive assay than the APTT, but the poor correlation between assays suggests a multiple effect of heparin and its fractions. Which assay or assays are the best measure of the antithrombotic efficacy of this drug remains to be determined. However, a random selection of an assay is surely not a proper means to monitor these drugs, and a battery of current methodology should be considered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of low molecular weight heparin in clinical laboratory

The applicability was investigated of automated spectrophotometric heparin assays and three clotting assays for determination of two low molecular weight (LMW) heparin fractions: Org 10172 and DxN10 and two infractionated commercially available heparins. The relative activity of the two commercially available heparins was similar in the anti-Xa assay, in the anti-IIa assay and in 3 clotting assays. The LMW heparins showed markedly different relative activity in all 5 assays. The activities of those heparin preparations relative to the standard heparin were compared in the 5 assays, but standardization against a standard heparin preparation appeared impossible. Methods of heparin determination can be used to monitor treatment with a heparin preparation only if the same preparation is used as a reference substance.     

Effect of patient weight on the anticoagulant response to adjusted therapeutic dosage of low-molecular- weight heparin for the treatment of venous thromboembolism.

Data evaluating the safety of using weight-based dosing of low-molecular-weight heparin (LMWH) in obese patients are limited. Some manufacturers have recommended a maximum daily dose of LMWH not to be exceeded. The purpose of this study was to determine if body weight influenced the anticoagulant response to a weight-based dose of LMWH for the treatment of venous thromboembolism. Patients with serum creatinine levels < 150 micromol/l receiving the LMWH, dalteparin 200 anti-Xa IU/kg based on actual body weight subcutaneously once daily for the treatment of deep vein thrombosis or pulmonary embolism, were eligible for the study. Patients received a minimum of 5 days LMWH treatment. Patients had peak anti-Xa levels (IL Test Chromogenic assay) measured 3-4 h following their day 3 injection and trough anti-Xa levels measured immediately prior to injections on day 3 and 5. No dose adjustments were made on the basis of the anti-Xa levels. Patients were a priori stratified into three weight classes: (A) within 20% of ideal body weight (IBW) (n = 13); (B) 20-40% of IBW (n = 14), and (C) greater than 40% of IBW (n = 10). The largest patient weighed 190 kg and had a body mass index of 58. Mean daily LMWH doses were 14,030, 17,646 and 23, 565 IU for groups A, B and C, respectively. Mean (SD) trough anti-Xa levels on day 3 were 0.12 (0.05) anti-Xa IU/ml for group A, 0.11 (0.03) anti-Xa IU/ml for group B and 0.11 (0.03) anti-Xa IU/ml for group C (p > 0.2). Similar trough anti-Xa levels were observed on day 5. Mean (SD) peak anti-Xa levels on day 3 were 1.01 (0.20) anti-Xa IU/ml, 0.97 (0.21) anti-Xa IU/ml and 1.12 (0.22) anti-Xa IU/ml for groups A, B and C, respectively (p > 0.2). No thromboembolic or bleeding complications occurred during LMWH therapy in any patients. These findings suggest that body mass does not appear to have an important effect on the response to LMWH up to a weight of 190 kg in patients with normal or near normal renal function.     

External quality assurance for heparin monitoring

Although there is considerable debate regarding the usefulness of laboratory heparin monitoring, these test processes reflect a substantial portion of hemostasis laboratory activity. Accordingly, external quality assurance (EQA) remains an essential component of such testing, and ensures that laboratories provide the best available service for patient management. This report provides an overview of recent and past EQA related to heparin monitoring using data from the Royal College of Pathologists of Australasia Haematology Quality Assurance Program, and heparin-containing plasma samples with concentrations ranging from 0 to 1.4 U/mL. Laboratory tests evaluated comprised activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen, and anti-Xa assays. Results for APTT and TT testing were largely as expected, showing prolongation with increasing concentrations of heparin. Fibrinogen assays were generally unaffected by the presence of therapeutic heparin levels. Although cross-laboratory median values for the anti-Xa assay were close to target values, substantial interlaboratory variation in results, expressed as coefficient of variation (CV), was observed in all exercises conducted over an 8-year period (5 to 28% for low-molecular weight heparin [LMWH] and 19 to 37% for unfractionated heparin). Duplicate samples sent in consecutive surveys resulted in similar median values. The use of a survey-provided standard as assay calibrant improved CVs in earlier surveys, but not in the most recent survey.     

Monitoring therapy with LMW heparin: a comparison of three chromogenic substrate assays and the Heptest clotting assay

Three LMW heparins (LMWH), one unfractionated heparin (UH), and international standards of LMWH and UH were compared in three chromogenic substrate (CS) assays and the 'Heptest' clotting assay. With a two-stage CS assay, linear standard curves were obtained in the 0.1-1.0 U/ml range, nearly coinciding for all preparations. With the one-stage CS assays, standard curves were curvilinear and similar for UH and the LMWH groups. In the Heptest assay, standard curves were linear for UH but not for LMWH. Mean recovery of LMWH, added to patients' plasma samples was 70-98% for the four assays. Variation between individual recoveries was much greater with Heptest (coefficient of variation (CV) 35-44%) than with one-stage CS assays (CV 14-21%) or two-stage CS assays (CV 7-8%). For monitoring LMW heparin therapy, CS assays seem preferable to Heptest. The two-stage CS assay had superior accuracy, but the one-stage CS assays were easier to perform.     

Toward development of a point-of-care assay of enoxaparin anticoagulant activity in whole blood

There is need for a rapid assay to determine the efficacy of low-molecular-weight-heparin (LMWH) in whole blood. Heparinase was used to eliminate, and thereby quantify, the anticoagulant activity of the low-molecular-weight-heparin, enoxaparin. The percent change in the clotting time of whole blood in the presence of heparinase yielded the anticoagulant contribution of enoxaparin. A minimally activated assay (MAA) of whole blood clotting time was evaluated for the detection and relative quantification of enoxaparin. The assay cartridge consisted of a plain glass tube and detection magnet, with no additional sources of activation. Comparisons were also made with a point-of-care, activated partial thromboplastin time (aPTT) assay. Plasma or whole blood was spiked with enoxaparin at concentrations ranging from 0.1 to 1.0 anti-factor Xa IU/ml. A commercial preparation of heparinase I was used to digest enoxaparin, and clotting times were determined with and without heparinase incubation. Heparinase digestion caused an average shortening of clotting time of 21.1% (47.3 s) in blood spiked with 0.4 anti-Xa IU/ml enoxaparin, an amount expected in the therapeutic range; also, 0.1 anti-Xa IU/ml of enoxaparin could be reliably detected. The assay performed comparably when transferred to a point-of-care setting with heparinase being added directly to citrated blood collection tubes, followed by either MAA or aPTT assay. Strong correlations were obtained with both assays between the percent change in clotting time (after heparinase) and the added concentration of enoxaparin, or in comparison with the chromogenically measured concentration of enoxaparin. The assays for an individual blood sample can be completed within 10 min.     

In vivo neutralization of low-molecular weight heparin fraction CY 216 by protamine

The neutralization in vivo of a low molecular weight heparin by protamine was investigated. Large doses and excessive doses of intravenously administered CY 216 were studied. An intravenous injection of protamine given 10 minutes after the administration of CY 216 did not cause the studied biologic parameters to return to normal levels, but merely attenuated them, whatever the protamine dosage tested. In contrast, the bleeding time and the volume of blood loss resumed normal values that were close to those observed in the controls. This dissociation of actions cannot be explained at present. The ratio of protamine to CY 216 dosage that produced the best results was 1 antiheparin U of protamine to 2 anti-Xa U of CY 216. Nevertheless CY 216 appeared to have a small hemorrhagic potential.     

Stability and sterility of a recombinant factor VIII concentrate prepared for continuous infusion administration.

Minipumps may facilitate cost-effective and convenient continuous infusion (CI) therapy for severe hemophilia A. This study evaluated the in vitro sterility, ability to support bacterial growth, and specific activity stability of a recombinant factor VIII (FVIII; Bioclate, Centeon) delivered by simulated CI at a variety of temperatures and after the addition of heparin or antibiotic. Closed system CIs of Bioclate (89.5 IU/ml) with and without heparin were sampled and cultured over a 6 day period. Bioclate (53.7 IU/ml) with and without heparin or vancomycin was inoculated with 102-105 CFU/ml of S. aureus, S. epidermidis, Escherichia coli, E. cloacae, or Y. enterocolitica and assessed by quantitative culture after 1 and 3 days. The stability of Bioclate (50, 100, and 250 IU/ml) at three temperatures (21 degrees C, 37 degrees C, and 39 degrees C) with and without heparin or vancomycin was tested over a period of 28 days. FVIII activity was measured in triplicate by a chromogenic assay (Coamatic Factor VIII, Chromogenix) and purity evaluated by Western blot. No bacterial growth was detected during CI of FVIII for up to 6 days. Following bacterial inoculation, there was rapid growth (>3 log increase) of all tested bacterial species except S. aureus which only displayed a 1 log expansion at 3 days. The addition of heparin containing 9.45 microg/U benzyl alcohol had no effect on bacterial growth. The addition of vancomycin caused a modest suppression of S. aureus growth but not of E. coli. Diluent alone did not support bacterial growth. Neither concentration, increased temperature, nor the addition of heparin or vancomycin had a significant effect on FVIII activity stability. Samples retained >75% baseline activity for between 3 and 7 days, except the infusion of Bioclate 50 IU/ml plus heparin maintained at 21 degrees C which remained stable for 28 days. Western blot analysis supported the activity assay findings. Standard and concentrated preparations of Bioclate are suitable for CI when delivered by the MiniMed 404-SP minipump. Because of the observed nutritive capability of this FVIII concentrate for sustaining bacterial growth, any contamination could result in systemic infection.     

Measurement of antithrombin activity by thrombin-based and by factor Xa-based chromogenic substrate assays.

Functionally active antithrombin can be quantified by chromogenic substrate assays utilizing the heparin cofactor activity of antithrombin and the inhibition rates of thrombin or of activated factor X (FXa). Thrombin-based assays but not FXa-based assays may overestimate the antithrombin activity due to their sensitivity toward heparin cofactor II. We focused on the question whether an overestimation of antithrombin activity by thrombin-based assays involves the risk of misdiagnosing antithrombin-deficient individuals as being non-deficient. We determined antithrombin using two thrombin-based assays and one FXa-based assay in 27 plasma samples from patients with acquired antithrombin deficiency spiked with lepirudin, in antithrombin-deficient plasma and in mixtures of antithrombin-deficient plasma and normal plasma. We also measured antithrombin in healthy subjects, in patients with inherited and acquired antithrombin deficiency and in patients under high-dose heparin treatment. At therapeutic final concentrations of lepirudin, antithrombin activities were considerably overestimated by the thrombin-based assays but not by the FXa-based assay. The residual antithrombin activities in antithrombin-deficient plasma determined by the thrombin-based assays were markedly higher than the corresponding values obtained with the FXa-based assay. The thrombin-based assays also overestimated antithrombin activity in patients under high-dose heparin. However, the degree of overestimation in the range between 50 and 100 IU/dl was too low to misidentify individuals with inherited or acquired antithrombin deficiency as normal. We conclude that functionally active antithrombin can be reliably determined using FXa-based chromogenic substrate assays in all settings examined. Thrombin-based assays must not be used in patients under treatment with hirudin or other direct thrombin inhibitors.