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A R Hubbard

Publications and source records attributed to A R Hubbard.

At least 19 recordsLinked to original sources
PubMed2005

A collaborative study to establish the 7th International Standard for Factor VIII Concentrate.

A candidate concentrate, preparation N (99/678), was assayed and calibrated, as a potential replacement, against four established factor (F) VIII concentrate standards: the current WHO 6th International Standard (IS) (97/616), the previous 5th IS (88/640), the Mega 1 standard and Ph. Eur. BRP Batch 2 standard, in a collaborative study involving 38 laboratories. All laboratories were instructed to use the ISTH/SSC recommendations, including predilution of concentrates in FVIII-deficient plasma. Several laboratories performed more than one assay method and altogether there were 27 sets of assays with the one-stage method, 31 with the chromogenic method, and 18 with both methods. There was good agreement between laboratories using each of the two methods for comparison of preparation N against the four established standards, with overall potencies by one-stage and chromogenic methods differing only by less than 2%. However, there were significant differences in potencies relative to the different standards, ranging from 10.1 IU per ampoule against the Ph. Eur.BRP2 to 11.4 against the WHO 6th IS. Accelerated degradation studies showed that the proposed standard is very stable, with a predicted loss of activity per year of less than 0.001% at the recommended storage temperature of -20 degrees C. Various options for potency of preparation N were considered by the participants and by members of the ISTH/SSC FVIII/FIX Subcommittee. In November 2003, preparation N (NIBSC 99/678) was proposed to and accepted by the Expert Committee on Biological Standardization of the World Health Organization to be the 7th International Standard for Factor VIII Concentrate with an assigned potency of 11.0 IU per ampoule.

Calibration↗
PubMed2004

Standardization of factor VIII and von Willebrand factor in plasma: calibration of the WHO 5th International Standard (02/150).

Calibration of the 5th International Standard factor (F)VIII/von Willebrand factor in plasma (02/150) (5th IS) for five parameters [factor VIII: coagulant activity (FVIII:C); FVIII: antigen (FVIII:Ag), von Willebrand factor: antigen (VWF:Ag), von Willebrand factor: ristocetin cofactor (VWF:RCo), von Willebrand factor: collagen binding (VWF:CB)] was achieved through an international collaborative study involving 37 laboratories. Estimates calculated relative to the previous 4th IS and locally prepared normal plasma pools were not significantly different for estimates of FVIII:Ag, VWF:Ag, VWF:RCo and VWF:CB and hence mean values calculated relative to the 4th IS of 0.94, 0.91, 0.78 and 0.94 IU ampoule(-1), respectively, were assigned. However, estimates for FVIII:C relative to the fresh normal pools (mean 0.61 IU ampoule(-1)) were significantly lower than estimates relative to the 4th IS (mean 0.68 IU ampoule(-1)). In consideration of the good stability of FVIII:C in the 4th IS and the variability of estimates relative to the local pools it was agreed to assign the mean value obtained relative to the 4th IS of 0.68 IU ampoule(-1). For all five parameters the interlaboratory variability (geometric coefficient of variation, GCV%) was larger for estimates calculated relative to the normal pools (range 12.6-16.5%) when compared with estimates calculated relative to the 4th IS (range 3.5-8.3%). An accelerated degradation study performed in six laboratories indicated that the five calibrated parameters are extremely stable when ampoules are stored at -20 degrees C. Mean estimates of predicted loss per year at -20 degrees C ranged from 0% for VWF:CB to 0.029% for VWF:RCo. The 5th IS (02/150) was established by the World Health Organization in November 2003.

Blood Coagulation↗
PubMed2001

Potency estimation of recombinant factor VIII: effect of assay method and standard.

Large potency discrepancies between the chromogenic and one-stage clotting methods have been reported for patients' plasma samples following the infusion of recombinant factor VIII (rFVIII) concentrates. We have investigated the potency estimation of two different full-length rFVIII concentrates using both assay methods relative to both plasma and concentrate standards. Potencies by the chromogenic method were significantly higher (53% and 45%) than potencies by the one-stage clotting method when a plasma standard was used. In contrast, there was no significant potency difference between methods when a concentrate standard was used. Time-course studies into thrombin and activated factor X (FXa) generation, in modified clotting and chromogenic methods, respectively, revealed that the two rFVIII concentrates behaved very similarly to the concentrate standard, whereas the plasma standard showed slightly more rapid thrombin generation and markedly slower FXa generation. The different behaviour of rFVIII and plasma FVIII in the chromogenic method is proposed as the main cause of the methods-based potency discrepancy. The results support the use of a concentrate standard to measure rFVIII in post-infusion plasma.

Blood Coagulation Tests↗
PubMed2001

Antithrombin inhibits lipopolysaccharide-induced tissue factor and interleukin-6 production by mononuclear cells, human umbilical vein endothelial cells, and whole blood.

OBJECTIVE: To investigate the effects of antithrombin on lipopolysaccharide (LPS)-induced tissue factor and interleukin-6 (IL-6) production in three different in vitro cellular systems: whole blood, human umbilical vein endothelial cells, and mononuclear cells. DESIGN AND SETTING: Laboratory in vitro study of the effects of antithrombin on procoagulant activity and cytokine release by LPS-stimulated endothelial and peripheral blood cells. SUBJECTS: In vitro whole blood, isolated human umbilical vein endothelial cell, and mononuclear cell cultures. INTERVENTIONS: Addition of antithrombin to LPS-treated whole blood, human umbilical vein endothelial cells, and mononuclear cells. MEASUREMENT AND MAIN RESULTS: Citrated whole blood, isolated human umbilical vein endothelial cells, or mononuclear cells were stimulated with LPS for 4-6 hrs in the presence or absence of antithrombin. Tissue factor activity was estimated by a tissue factor-dependent clotting or chromogenic assay and IL-6 was measured by specific ELISA. Antithrombin was found to inhibit tissue factor and IL-6 production in all three systems in a dose-dependent manner (0-40 IU/mL). Flow-through fractions of immunoadsorbed antithrombin concentrate were found to be ineffective. Five different batches of the same antithrombin concentrate were tested and the inhibitory activity was found to be consistent throughout all batches. Up to 40 microM of recombinant hirudin, a specific thrombin inhibitor, did not inhibit the production of tissue factor or IL-6 in either of the three cell systems, suggesting that the observed inhibition by antithrombin was not due solely to its ability to inhibit thrombin. CONCLUSIONS: Apart from the inhibition of thrombin and other activated clotting factors, antithrombin may also down-regulate the cellular expression of proinflammatory cytokines. Consequently, antithrombin concentrates may have value in the treatment of sepsis-induced disseminated intravascular coagulation.

Antithrombins↗
PubMed2001

Standardisation of factor VIII and von Willebrand factor in plasma: calibration of the 4th International Standard (97/586).

The 4th International Standard (IS) Factor VIII/von Willebrand Factor (FVIII/VWF) plasma was calibrated in 25 laboratories by assay against the 3rd IS plasma and fresh normal plasma pools. Five parameters were measured, FVIII:coagulant activity (FVIII:C), FVIII:Antigen (FVIII:Ag), VWF:Antigen (VWF:Ag), VWF:Ristocetin Cofactor (VWF:RCof), and a new parameter, VWF:collagen binding (VWF:CB). Mean potency estimates for the 4th IS, calculated relative to the 3rd IS, were significantly greater than the mean estimates calculated relative to the fresh normal pools by 15, 14 and 20% respectively for FVIII:C, VWF:Ag and VWF:RCof. These results indicate a drift in the International Unit away from the fresh plasma unit. Partial rectification of this drift was achieved by assigning the mean of the estimates calculated relative to the 3rd IS and the fresh plasma pools, i.e. FVIII:C 0.57 IU/ampoule, VWF:Ag 0.79 IU/ampoule and VWF:RCof 0.73 IU/ampoule. This represents a shift in the IU between the 3rd and 4th IS of 7.5% for FVIII:C, 7% for VWF:Ag and 10% for VWF:RCof. Mean estimates of FVIII:Ag relative to the 3rd IS and the fresh normal pools agreed to give an assigned value of 0.89 IU/ampoule. Excessive inter-laboratory variability and a low number of estimates (n = 6) precluded the assignment of a potency for VWF:CB. The 4th IS Factor VIII/VWF plasma (97/586) was established in October 1998.

Blood Coagulation Tests↗
PubMed2000

A collaborative study to establish the 2nd International Standard for Fibrinogen, Plasma.

An International Collaborative Study involving 12 laboratories in 7 different countries was undertaken in order to replace the 1st International Standard (IS) for Fibrinogen, Plasma (89/644). The candidate replacement standard was the ampouled and freeze-dried residue of solvent/detergent treated plasma and was calibrated as coded duplicates (A and B) versus the 1st IS Fibrinogen, Plasma by automated Clauss assay and by a recommended clot collection (gravimetric) assay. This latter method had been used to calibrate the 1st IS Fibrinogen, Plasma. Comparing the ratios of the potency estimates of sample A to sample B (the coded duplicates), all of the laboratories obtained a ratio within 5% of the expected value of 1.0 by automated Clauss assay, which suggests that the laboratories were able to perform this assay well. Scrutiny of the data obtained from the gravimetric assays revealed that in almost all cases the results were invalid. The results of these assays are included in this report but clearly should be treated with caution and indeed produced significantly lower mean estimates of potency than the other assay methods. The overall geometric mean of all estimates of potency of the proposed 2nd IS Fibrinogen, Plasma (98/612) is 2.19 mg/ampoule by the automated Clauss assay. These data have been presented to the Fibrinogen Sub-Committee of the Standardisation and Scientific Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) (Washington, DC, August 1999), which recommended the establishment of 98/612 as the 2nd IS Fibrinogen, Plasma. This report has been presented to the Expert Committee on Biological Standardisation of the World Health Organisation (ECBS-WHO) at their 1999 session and 98/612 was established as the 2nd IS Fibrinogen, Plasma with a potency of 2.2 mg/ampoule.

Blood Preservation↗
PubMed1999

An international collaborative study on the INR calibration of freeze-dried reference plasmas.

A study was carried out to calibrate potential European Reference Plasmas for prothrombin time (PT) standardization. The International Normalized Ratio (INR) values of three freeze-dried candidate plasmas (one pooled normal and two pools from anticoagulated patients) were determined in 20 laboratories using six thromboplastin reagents comprising three International Reference Thromboplastins (human, rabbit and bovine), two recombinant human reagents and one placental human reagent. Interlaboratory variability of INR estimation was low with geometric coefficients of variation (gcv) <10% except in one case. Significant differences in mean INR were found between the different thromboplastins with lowest INR values found with the bovine reagent. INR values from the International rabbit and human reagents differed by <6% and were combined to give proposed assigned INR values. Significant differences in INR estimates from four thromboplastins of human origin may indicate that single assigned INR values are not applicable for use with all thromboplastin reagents. Field trials to assess the validity of single assigned INR values in clinical practice are required.

Animals↗
PubMed1998

Discrepancies in potency assessment of recombinant FVIII concentrates.

Results of assays of recombinant FVIII concentrates have been reviewed over a 10-year period. Initially there was wide variability between laboratories but this was minimised by the development of standardised assay methodology, in particular the use of haemophilic plasma for pre-dilution and 1% albumin in assay buffers. Using this standardised methodology and concentrate standards, there were no major differences in potency between one-stage, two-stage and chromogenic assays on the two full-length recombinant FVIII concentrates. However, using a plasma standard, the chromogenic method gave much higher potencies than the one-stage method on the same concentrates, and this explains a similar discrepancy found in patients' post-infusion samples after injection of recombinant concentrates. It is suggested that concentrate standards be used for such post-infusion samples in order to minimise this discrepancy.

Biological Assay↗
PubMed1998

Modification of factor VIII in therapeutic concentrates after virus inactivation by solvent-detergent and pasteurisation.

The addition of a pasteurisation step to a solvent/detergent (SD) treated FVIII concentrate has recently resulted in enhanced inhibitor incidence in patients in Germany and Belgium. We have investigated the effect of virus inactivation procedures on FVIII function by preparing experimental concentrates from the same starting cryoprecipitate with the following procedures: none (N); dry heat (DH); pasteurisation (P); solvent/detergent (SD); solvent detergent + dry heat (SDDH); solvent detergent + pasteurisation (SDP). In addition, several clinical SD concentrates with and without pasteurisation were studied. There were no significant differences in fibrinogen and vWF content and in the ratio of one-stage/chromogenic FVIII activity among any of the samples studied. In thrombin proteolysis and FXa generation experiments, there were no differences in results on samples N, DH, P, and SDDH from those on sample SD. However sample SDP gave markedly different results from sample SD in the following respects: slower thrombin proteolysis (t(1/2) = 12.0 min vs 1.9 min); more rapid FXa generation (rate 2.5 times that of SD); enhanced phospholipid binding (K(D) = 3.89 x 10(-11) M vs 5.53 x 10(-10) M). Similar differences between SDP and SD were seen in the clinical samples. The observed changes in the FVIII activity occurred in combination with SD and pasteurisation, but not with either treatment alone. These results suggest that SDP treatment may enhance exposure of the phospholipid binding site in the C2 domain of FVIII, and since inhibitors to the SDP product are predominantly against C2, these findings could be relevant to the enhanced immunogenicity of the SDP product.

Detergents↗
PubMed1997

International Normalized Ratio determination using calibrated reference plasmas.

We have compared the conventional method of International Normalized Ratio (INR) determination with an alternative method involving extrapolation from a calibration curve using freeze-dried 'reference' plasmas. The latter approach does not require the determination of a mean normal prothrombin time (MNPT) or local system International Sensitivity Index (ISI). Calibration curves were constructed by plotting local prothrombin time (PT) against assigned INR values for a normal plasma and either two plasma pools from patients on oral anticoagulants or two artificially depleted plasmas. Six laboratories determined the INR of a freeze-dried test plasma and frozen patient plasma samples using the conventional method and by extrapolation. Similarities in the results with the freeze-dried test plasma and the frozen plasmas were encouraging for the projected use with fresh plasma samples. INR values by the conventional method for the test plasma gave an overall mean of 2.73 and inter-laboratory variability (gcv%) of 8.92%, whereas estimates by extrapolation against the normal and patient plasmas or the normal and artificially depleted plasmas gave identical overall mean INR values of 2.70 with inter-laboratory variability (gcv%) of 3.44% and 4.92% respectively. The results indicate that INR determination by extrapolation is associated with reduced interlaboratory variability.

Blood Coagulation Tests↗
PubMed1997

Standardisation of protein S in plasma: calibration of the 1st International Standard.

An international collaborative study, involving 16 laboratories, was carried out to calibrate the 1st International Standard Protein S, plasma, for total and free Protein S antigen and for Protein S function. Potency estimates were calculated relative to locally collected fresh normal plasma pools. Estimates of total Protein S antigen showed good agreement between laboratories with inter-laboratory geometric coefficient of variation (gcv%) of 8.8% and a mean value of 0.89 IU/ampoule. Estimates of free Protein S antigen calculated relative to total Protein S antigen in the fresh normal pools were more variable (gcv 26.8%) than estimates calculated relative to free Protein S antigen (gcv 15.3%); the latter comparison was used for calibration with a mean value of 0.89 IU/ampoule. Functional Protein S was estimated using commercial kits which gave an overall mean value of 0.92 IU/ampoule (gcv 14.0%). The 1st International Standard Protein S, plasma, (coded 93/590) was established in October 1995 with a single potency of 0.90 IU/ampoule for all three parameters.

Calibration↗
PubMed1994

Measurement of tissue factor pathway inhibitor in normal and post-heparin plasma.

Assay methods for the detection of both tissue factor pathway inhibitor (TFPI) function (two-stage chromogenic assay) and for TFPI antigen levels (competitive ELISA) have been developed and applied to the measurement of TFPI in normal plasma, in post-heparin plasma and to recombinant TFPI. There was good correlation in TFPI levels, measured using the two methods (r = 0.848; P < 0.001) in the normal plasma samples (n = 21) with the values ranging from 0.6 to 1.4 units per ml relative to a normal reference plasma pool (assigned 1.0 unit per ml). The post-heparin plasma samples were associated with increased levels of both TFPI functional activity and antigen. However, there was poor correlation between the two methods, with an increase in antigen levels greatly exceeding the increase in functional activity. This discrepancy was also found with recombinant TFPI and may reflect the different responses of the two assay methods to lipoprotein-bound TFPI (in the normal plasma reference) and the 'free' TFPI in post-heparin plasma and recombinant TFPI. These findings have implications in the choice of suitable reference materials for the assay of TFPI.

Binding, Competitive↗
PubMed1992

A collaborative study on the measurement of protein S antigen in plasma.

A collaborative study on the measurement of protein S (PS) antigen (total and free) in a freeze-dried ampouled test plasma by assay against local house standard plasmas was carried out in eleven laboratories. Potency estimates of total PS showed good agreement between laboratories with a geometric coefficient of variation (gcv) of 5.9% and an overall combined potency of 0.84 units per ml. Potency estimates of free PS antigen in the test sample were associated with increased variability between laboratories resulting from the polyethylene glycol (PEG) precipitation step which is used to separate free PS from PS bound to the C4b binding protein. Free PS in the test could be expressed relative to either total PS in the house standards (e.g. 0.28 units per ml) or relative to free PS in the house standards following PEG precipitation (e.g. 0.71 units free PS per ml).

Antigens↗
PubMed1991

Phospholipase C mediated inhibition of factor VII requires triglyceride-rich lipoproteins.

The reduction of plasma factor VII (FVII) activity by phospholipase C (PLC), in vitro, has been proposed as a possible indication of a risk of cardiovascular disease. The ability of PLC to reduce FVII activity was found to require calcium ions and the presence of triglyceride-rich lipoproteins (e.g. chylomicra and very-low density lipoproteins) rather than high or low density lipoproteins. The PLC-mediated reduction of FVII activity was prevented by pre-incubation of PLC with chylomicra, before adding FVII, and this suggests that PLC may act on triglyceride-rich lipoproteins already bound to FVII in order to reduce FVII activity. At optimal PLC concentration, the extent of the reduction in FVII activity was proportional to the concentration of chylomicra. The detergent, Tween, prevented any loss of FVII activity, in both plasma and purified systems, if it was present at the beginning of the incubation with PLC. Addition of Tween, but not EDTA, after inhibition of FVII activity had occurred, caused a partial restoration of FVII activity. It is concluded that PLC reduces FVII activity by modifying triglyceride-rich lipoproteins to a form which binds to FVII, independently of calcium ions, and which inhibits procoagulant activity. The detection of PLC-sensitive procoagulant activity. The detection of PLC-sensitive FVII activity may therefore have no greater significance than the measurement of plasma triglyceride levels in predicting a risk of cardiovascular disease.

Calcium Chloride↗
PubMed1989

Procoagulant activity of T lymphoblastoid cells in extrinsic and intrinsic coagulation systems.

We have measured the procoagulant activity (PCA) of four T lymphoblastoid cell lines (Jurkat, CEM, HSB-2 and Molt 4) as well as normal peripheral blood T lymphocytes, before and after stimulation with phytohaemagglutinin (PHA), using clotting and amidolytic methods. Of the four cell lines only one, Jurkat, gave enhanced PCA after stimulation with PHA. This activity was shown to be tissue factor-like by its dependence on factor VII in plasma and in an amidolytic assay with purified factors VII and X. Jurkat was also the only one of the four cell lines to secrete interleukin-2. All four cell lines promoted the generation of large amounts of thrombin in platelet-free plasma in glass tubes. This activity was dependent on the presence of plasma factor VIII, and was probably due to phospholipids in the cell membranes. Normal T lymphocytes gave intrinsic PCA in the thrombin generation test which was only 15% of that of the lymphoma cells. These results show that some T lymphocytes can develop PCA in both intrinsic and extrinsic systems and this should be taken into account in studies of the PCA of mixed leukocyte populations.

Blood Coagulation↗