Cryoprecipitate and factor VIII commercial concentrates:In vitro characteristics andin vivo compartmental analysis

Summary In vitro andin vivo characteristics of cryoprecipitates and three commercial factor VIII concentrates (Kryobulin, Hemofil and Koate) were comparatively studied. Factor VIII:C/VIII:Ag ratio was very low in all commercial concentrates without differences among them. Conversely, the decrease of factor VIIIR:WFRCof was proportional to the degree of purity. Factor VIII:C/VIIIR:WFRCof ratio was shown to be a reliable index of factor VIII complex denaturation. Crossed electroimmunoassay showed a faster migration of factor VIIIR:Ag only in commercial concentrates. Using a two-compartmental open model that accounts for endogenous synthesis of factor VIII:C, single-dose kinetics of factor VIII:C were studied in 23 patients with classic hemophilia. Good agreement between measured and fitted values of factor VIII:C plasma concentration was observed. β half-life was shorter in high purity concentrates and longer in intermediate purity concentrates and cryoprecipitates.     

Variability in factor VIII concentrate measurement: results from SSC field collaborative studies

Summary.  Seven 'field' collaborative studies on factor (F)VIII concentrate potency measurements were carried out, using local routine methodology, standards and calculation of results. Data from five of the 12 different concentrates studied are described in detail. These studies revealed that, for the intermediate-purity and the recombinant FVIII concentrates, one-stage potencies were significantly lower than chromogenic potencies, whilst for the two high-purity FVIII concentrates one-stage potencies were significantly greater than chromogenic potencies. On comparing predilution methods for the intermediate-purity concentrate, equivalent potencies were obtained using either buffer or FVIII-deficient plasma as prediluent. For the two high-purity and the recombinant concentrates, potencies obtained using buffer as prediluent were significantly greater and lower, respectively, than potencies obtained using FVIII-deficient plasma as prediluent. Interlaboratory variabilities were compared over all 12 concentrates studied and coefficients of variation (CVs) for one-stage assays were found to be much greater than for chromogenic assays. This was true for all concentrates except for the intermediate-purity concentrate and samples A and B from the first study, where the reverse was true. Furthermore, much better CVs were obtained when using FVIII-deficient plasma than when using buffer as prediluent, for all FVIII concentrates except for the intermediate-purity concentrate where the reverse was true, and sample B where CVs were equivalent. Overall, CVs were far worse than those obtained in controlled collaborative studies. Generally, however, CVs were better with chromogenic assays and predilution in FVIII-deficient plasma, as is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee, particularly for higher purity and recombinant concentrates.     

On the unreliability of one-stage factor VIII:C clotting assays after infusion of factor VIII concentrates

A multicentre study was undertaken in order to determine the reliability of the methods of assay of F VIII:C and the position of the peak value obtained after infusion of F VIII:C concentrates. Blood samples were drawn before and 10, 30 and 60 min after injection of F VIII concentrates in six haemophiliacs in one of the centres. Coded, frozen plasma samples were dispatched to the laboratories of the other four centres. F VIII:C was determined by different one-stage methods using the same international standard but with different activators. The results of the different laboratories differed widely and no agreement was reached on the existence of a lag period. To reach a valid conclusion not only the same sample has to be analysed, as in this study, but also the same laboratory technique has to be used by all participating investigators. To reach agreement on in vivo recovery and on elimination curves for different F VIII concentrates multicentre studies must be based on reliable methods of assay.     

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.     

The von Willebrand factor

Summary Von Willebrand factor (vWf) is a multimeric and multivalent adhesive protein which is essential for platelet adhesion to subendothelium and for stabilization of factor VIII procoagulant activity in circulation. The quantitative measurement of vWf involves essentially two different approaches. The first is based on the interaction between vWf and Gp Ib of the platelet membrane in presence of ristocetin (ristocetin cofactor activity, RiCof) and depends not only on the amount of the factor but also on its ability to bring about this interaction, large multimers being more active. The second approach involves the immunological quantitation of vWf (vWf:Ag) by its interaction with specific polyclonal or monoclonal antibodies as measured by several methods, i.e., electroimmunoassay, immunoradiometric assay and immunoenzymatic assay. Although in the majority of type II von Willebrand disease (vWd) with dysfunctional vWf there is a discrepancy between RiCof and vWf:Ag, it should be emphasized that RiCof activity does not entirely reflect the ‘true’ activity of vWf since it does not explore all the functions of this factor; furthermore, the relationship between degree of multimerization and RiCof level is not always tenable, as for example in vWd ‘Vicenza’. For the diagnosis of congenital and acquired vWd RiCof assay together with family investigation is the eligible test, with an estimated ability to detect at least 50% of the carriers of the abnormal gene, including mildly affected patients; vWf:Ag appears less sensitive and, on the basis of studies carried out in our laboratory, a relative sensitivity of 64% is proposed. Both assays require the definition of separate normal ranges for children and adults and for 0 and non-0 blood group subjects; a nonparametric approach in a large sample of normal subjects is advisable. With RiCof assay performed by an aggregometric method using formalin-fixed platelets an interassay variability of 6% and 8.5% respectively for high- and low-control plasma was found in our laboratory. With vWf:Ag assayed by an ELISA method a variability of 7% for low- and 6% for high-control plasma was found. Thus, both methods appear sufficiently precise for clinical use. The use of an internal pool calibrated against an international standard allows to perform comparable interlaboratory measurements. To further improve standardization of these assays, collaborative studies seem urgently required. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

Survival of transfused factor VIII in hemophilic patients treated with epsilon aminocaproic acid

The effect of EACA (epsilon aminocaproic acid) on the survival of factor VIII (F. VIII, antihemophilic factor, AHF) concentrates after infusion in hemophilic subjects was studied using two F. VIII preparations: an intermediate purity "cryoglobulin" and a high purity glycine-precipitated concentrate. In vivo recoveries of F. VIII with both concentrates were found to be close to the values predicted on the basis of concentrate potencies assayed prior to infusion, but not if the labeled potency was used in the calculation. Control studies showed similar first and second phase half-life times and half-disappearance times when the two F. VIII preparations were infused, and both preparations were found to follow a biphasic disappearance curve. The biological half-life (second phase rate of disappearance) was 12 to 13 hours. The administration of EACA in conjunction with the F. VIII concentrates did not prolong the half-life times (first or second phase) or the half-disappearance times.     

A collaborative study to establish the 1st International Standard for factor XIII plasma

BACKGROUND: An international collaborative study, involving 23 laboratories, was carried out, under the auspices of the FXIII Standardization Working Party (SWP), to calibrate the 1st International Standard (IS) for factor XIII (FXIII) plasma. METHODS: Potency estimates for the proposed candidate FXIII plasma (preparation Y: NIBSC code 02/206) were calculated relative to locally collected normal plasma pools (pool N), for both FXIII activity and antigen levels. RESULTS: Estimates of FXIII activity potency for preparation Y showed good agreement between laboratories, with an interlaboratory geometric coefficient of variation (GCV) of 11.5% and a mean value of 0.91 U mL(-1). Furthermore, there was a negligible difference in potencies by two commercially available methods, the potencies differing only by approximately 1%. Estimates of FXIII antigen (A(2)B(2) complex) potency for preparation Y showed good agreement between laboratories, with an interlaboratory GCV of 16.3% and a mean value of 0.93 U mL(-1). Accelerated degradation studies showed that the proposed standard is very stable, with a predicted loss of activity (and antigen) per year of< 0.06% at the recommended storage temperature of -20 degrees C. CONCLUSIONS: The suitability and potency of preparation Y were considered by the participants, members of the ISTH/SSC FXIII Subcommittee, the Scientific and Standardization Committee and the SWP. Following their approval, preparation Y was proposed to and accepted by the Expert Committee on Biological Standardization of the World Health Organization to be the 1st IS for FXIII plasma with an activity potency of 0.91 IU per ampoule and an antigen potency of 0.93 IU per ampoule.     

Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C.

Interactions between standard heparin and the physiological anticoagulant plasma protein, activated protein C (APC) were studied. The ability of heparin to prolong the activated partial thromboplastin time and the factor Xa- one-stage clotting time of normal plasma was markedly enhanced by addition of purified APC to the assays. Experiments using purified clotting factors showed that heparin enhanced by fourfold the phospholipid-dependent inactivation of factor V by APC. In contrast to factor V, there was no effect of heparin on inactivation of thrombin-activated factor Va by APC. Based on SDS-PAGE analysis, heparin enhanced the rate of proteolysis of factor V but not factor Va by APC. Coagulation assays using immunodepleted plasmas showed that the enhancement of heparin action by APC was independent of antithrombin III, heparin cofactor II, and protein S. Experiments using purified proteins showed that heparin did not inhibit factor V activation by thrombin. In summary, heparin and APC showed significant anticoagulant synergy in plasma due to three mechanisms that simultaneously decreased thrombin generation by the prothrombinase complex. These mechanisms include: first, heparin enhancement of antithrombin III-dependent inhibition of factor V activation by thrombin; second, the inactivation of membrane-bound FVa by APC; and third, the proteolytic inactivation of membrane-bound factor V by APC, which is enhanced by heparin.     

Factor VIII preparations of varying purity-examination of physico-chemical parameters (author's transl)

Commerical preparations of cryoprecipitates, factor VIII concentrates of intermediate purity, and factor VIII concentrates of high purity were studied for their factor VIII related clotting- and platelet-aggregating activities as well as their content of factor VIII antigen. The ratios of the values obtained of these three parameters were used to determine the extent of inactivation of the factor in the preparations. By means of two-dimensional immunelectrophoresis and gel chromatography on Sepharose 4B, the preparations were tested for the presence of atypical factor VIII molecules. Investigations were made to qualify and quantify some of the concomitant proteins in the factor VIII preparations.     

Study of viral safety of Scottish National Blood Transfusion Service factor VIII/IX concentrate

Summary. To assess the viral safety of the Scottish National Blood Transfusion Service (SNBTS) intermediate purity factor VIII and IX concentrates, the liver function and viral status were assessed prospectively in 13 recipients. None developed hepatitis or seroconverted to HIV or HCV. This study provides additional evidence for the efficacy of dry heat treatment at 80°C for 72 h in preventing virus transmission by coagulation factor concentrates.     

Specific assays of hemostasis proteins: Factor VII

Summary Factor VII (FVII) activity should be measured in order to evaluate the risk for coronary artery disease. The measurement of FVII by means of a standardized clotting method seems to be influenced by the thromboplastins used, while the FVII-deficient plasmas do not affect the results. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989. For the CISMEL (Comitato Italiano per la Standardizzazione dei Metodi in Ematologia e Laboratorio) Hemostasis Subcommittee.     

A multispecies enzyme-linked immunosorbent assay for von Willebrand's factor.

A modified double-sandwich enzyme-linked immunosorbent assay (ELISA), which was developed for human and canine von Willebrand's factor (vWF), was adapted for quantitation of vWF in other species. In addition to human and dog plasmas, 12 other mammalian plasmas that were surveyed exhibited significant cross-species reactivity with antibodies specific for canine vWF or monoclonal antibodies against porcine or bovine vWF. Mixed combinations of monoclonal antibodies and various polyclonal antibodies were also used as sandwich or capture antibodies. The ability of this multispecies ELISA to detect less than 0.002 U of vWF per milliliter of plasma in a large number of species enhances its utility for both research and clinical diagnostic applications. A quantitative assay for rabbit vWF, which exhibits poor cross-species reactivity with most antibodies, was constructed with anti-porcine monoclonal antibody W1-5 and goat-anti-canine vWF as capture and sandwich antibodies, respectively. The same conjugate antibody configuration was used to visualize rabbit vWF multimers by immunoblotting. Specificity of the assay for vWF in human, dog, pig, and horse plasmas was confirmed by use of species-specific vWF-deficient plasmas. In other species, for which vWD plasmas were not available, ELISA specificity for vWF was demonstrated by recovery of greater than 75% of the ELISA-reactive antigen coincident with vWF multimers in the high-molecular-weight (greater than 500 kd) fractions of purified plasmas. This multispecies ELISA permits, for the first time, the measurement of vWF in a variety of mammals for which species-specific immunologic reagents do not currently exist. The results also suggest that certain vWF epitopes have been highly conserved among phylogenetically diverse species.     

An international collaborative study to establish the WHO 4th International Standard for Streptokinase: Communication from the SSC of the ISTH

Streptokinase is used worldwide as a cost‐effective treatment for acute myocardial infarction. Manufacturers use the World Health Organization (WHO) International Standard (IS) for Streptokinase to potency label their products, ensuring consistent, safe, and effective dosing. Stocks of the third IS for streptokinase (coded 00/464) are running low, and an international collaborative study was organized to calibrate a replacement. A total of 15 laboratories from nine countries took part, using chromogenic and/or fibrin clot lysis methods to determine the potency of two candidate preparations, coded 16/356 (sample B) and 16/358 (sample C), relative to the third IS (00/464). A third sample (88/824, sample A), which was used in the collaborative studies to establish the second and third IS, was also included. There was good agreement in potency estimates from different assay methods and low variability both within and between laboratories. Long‐term stability modeling indicated the candidates are very stable. Comparison of potency estimates for 88/824 (sample A) with potencies calculated in previous studies revealed a variability of only 1.9% over the course of three collaborative studies spanning 30 years and more than 50 years of streptokinase standardization. This indicates excellent continuity of the International Unit (IU) and assay methods. Following agreement by study participants and Scientific and Standardization Committee experts of the International Society on Thrombosis and Haemostasis, the WHO Expert Committee on Biological Standardization established 16/358 (sample C) as the fourth IS for Streptokinase with a potency of 1013 IU per ampoule in October 2019.     

Freezing of plasma and recovery of factor VIII

The freezing procedure and its influence on the plasma quality was studied with a new plasma container and freezing bath and compared with the results from those obtained with commonly used equipment. Cryoprecipitation was performed on plasma pools frozen at three different rates, and quality and recovery of the cryoprecipitates were determined by analysis of factor VIII, factor VIII-related antigen (vWF:Ag), and fibrinogen. The plasma freezing time was 95 and 65 percent shorter with a new -40 degrees C freezing bath as compared to -25 and -80 degrees C freezing boxes, respectively. A further 30 percent reduction of the plasma freezing time was gained by the introduction of a new flat 750-ml plasma container. Rapid plasma freezing prevented substantial loss of factor VIII in frozen plasma. Cryoprecipitate purity measured as factor VIII-to-fibrinogen ratio increased from 0.50 to 0.82 (IU/mg) when the freezing time was decreased from 10 hours to 45 minutes, although the recovery of factor VIII increased less. In summary, freezing of plasma in small flat containers in an effective ethanol bath resulted in rapid freezing with high recovery of factor VIII in plasma, and increased purity and recovery of subsequently processed cryoprecipitate. This freezing concept, adapted at Swedish blood banks, has contributed to higher source plasma quality and increased self-sufficiency.     

Use of the activated partial thromboplastin time for the diagnosis of congenital coagulation disorders: Problems and possible solutions

Summary The activated partial thromboplastin time (APTT) is a commonly performed laboratory procedure which is used for multiple purposes including monitoring of heparin therapy, detection of coagulation factor deficiency, and detection of lupus anticoagulants. Among the hereditary coagulation deficiencies, factor VIII and factor IX are the most common. APTT reagents differ widely in both their sensitivity to factor VIII and factor IX deficiencies as well as their responsiveness. Sensitivity may be defined as the ability to identify a deficiency state while responsiveness is indicated by the degree of prolongation of the APTT result as compared to the upper limit of normal. Reagents may be both sensitive and responsive or alternatively sensitive and relatively nonresponsive. Consequently, it is extremely important for each laboratory to carefully identify the upper limit of the normal range. A variety of preanalytical variables will also effect the sensitivity of the APTT to factor deficiency states. These variables include specimen handling and the preparation of platelet poor plasma. The instrument effect is also of importance. Selection of the reagent tends to have the most impact on senstivity and responsiveness while instrumentation affects the precision of a given APTT. The composition and concentration of phospholipid in APTT reagents does have an effect on reagent responsiveness and sensitivity. Sensitivity to factor deficiencies does not necessarily parallel sensitivity to lupus anticoagulants. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

The factor VIII inhibitor assays can be standardized: results of a workshop

Summary. Background: The Bethesda and the Nijmegen assays are commonly used for the measurement of inhibitor levels in hemophilia A patients. Despite test innovations, the between‐laboratory coefficient of variation (CVb) of factor VIII inhibitor test data in external quality surveys remains very high (40–60%) with a high degree of false‐negative and false‐positive results resulting in undesired effects on treatment. Objectives: Organization of a workshop in order to address the causes of this phenomenon and to suggest ways to improve the assays. Methods: Fifteen laboratories showing a high CVb in regular surveys and using a variety of methods participated in the wet workshop, which included four different sessions where variables probably contributing to the high CVb (e.g. use of [non‐]buffered plasma, FVIII‐deficient plasma, sample dilution and APTT reagents) were investigated. Results: The CVb varied from 30% to 70% in the first session of the workshop when the participants used their own test settings and reagents. The use of buffered normal pooled plasma and FVIII‐deficient plasma as a reference sample by all participants did not significantly alter the CVb (35–50%) but decreased the number of false positives. However, the use of buffered pooled plasma in combination with standardized sample dilution procedures by all participants showed a significant improvement (CVb, 10–20%). Conclusions: These results may contribute to improvement of FVIII inhibitor testing. However, improved inter‐laboratory comparison of factor VIII inhibitor assay results can only be reached when further local standardization is implemented.     

Determination of coagulation factor VIII activity by a chromogenic substrate method on STA,an automated coagulation analyzer

This study was aimed at evaluating the performance of a chromogenic factor VIII assay on STA, an automated coagulation analyzer. Additionally, a correlation study was conducted with an aPTT-based one-stage factor VIII clotting assay. Throughout the study the performance of the chromogenic assay was tested in two ranges of factor VIII activity: a high range with activity between 20% and 150% and a low range with activity below 20%. Inter-assay coefficient of variation ranged from 1.9% to 8.9% and intra-assay coefficient of variation from 0.5% to 11.4%, depending on factor VIII concentration. Day-to-day reproducibility was tested over a 5-day period; between-day imprecision ranged from 7.1% to 9.4%. The chromogenic factor VIII assay showed a good correlation with the clotting assay in both ranges. The correlation coefficients were 0.924 and 0.792 for the high and low range, respectively. A statistically significant difference in mean values was observed in the high range (p&lt;0.0001). Comparison between the chromogenic assay on STA versus on microplates showed a high correlation (0.991), which was highly significant (p&lt;0.0001). In conclusion, the chromogenic assay for factor VIII on STA shows good analytical performance. It correlates well with the one-stage factor VIII clotting assay, although significant differences between individual samples occur. Probably these are partly related to differences in measurement principle and standardization. Altogether, this precise and rapid assay is suitable for determination of factor VIII by an automated procedure.     

Genetic analysis of non-severe hemophilia A phenotype with A discrepancy between one-stage and chromogenic factor VIII activity assays

IntroductionThe diagnosis of hemophilia A (HA) is based on the measurement of factor VIII activity (VIII:C). About one-third of non-severe HA patients show a discrepancy of VIII:C measured by one-stage (VIII:C 1st) and chromogenic (VIII:C chr) assays. Different mutations in the F8 gene may cause the discrepancy in results of the FVIII activity assay.The aim of this study was to investigate F8 gene mutations in patients with assay discrepancies and to evaluate their impact on the results of VIII:C assays.MethodsMutation analysis was performed on 41 individuals with a discrepancy in VIII:C 1st and FVIII: C chr assays by direct sequencing. In addition, the effect of the variants on FVIII macromolecule structure was investigated by in silico and bioinformatics tools.ResultsGenetic analysis disclosed 22 different variants, of which 19 were identified for the first time to be involved in the phenotype of VIII:C discrepancy. Most of the variants related to the higher VIII:C 1st were found in A1, A2, A3 domains. The variant related to VIII:C chr > VIII:C 1st was located in the thrombin cleavage site. In silico analysis showed the effect of variants on FVIII macromolecule stability, which may be the possible mechanism causing the discrepancy.ConclusionOur data shed light on the impact of genetic defects on VIII:C assay and provided evidence that the consideration of these mutations may open a new window to the proper diagnosis and treatment monitoring of non-severe HA patients.     

Binding to phospholipid protects factor VIII from inactivation by human antibodies

The addition of purified factor IXa and phospholipid to factor VIII concentrate protected the VIII:C from inactivation by human antibodies. This protective effect was shown to be due largely to the phospholipid. Addition of phospholipid alone gave substantial protection against even high-titer antibodies, as shown by measurements of thrombin generation and VIII:C assays. Increasing concentrations of phospholipid led to significant reductions in the amount of VIII C:Ag detected by an IRMA method, up to 70% of the original VIII C:Ag being "lost" at the highest concentration of phospholipid. These results indicate that phospholipid binding plays an important part in the procoagulant activity of factor VIII and that human antibodies to VIII:C are directed largely at the phospholipid binding site. The addition of phospholipid to factor VIII concentrates could have important clinical applications in the treatment of hemophiliacs with antibodies to factor VIII.     

State of the art in factor XIII laboratory assessment

Factor XIII, a heterotetrameric proenzyme, is converted to an activated transglutaminase by thrombin and calcium in the final phases of coagulation. Factor XIII catalyzes the formation of crosslinks between fibrin monomers and between α2-antiplasmin and fibrin. These crosslinks mechanically stabilize fibrin against shear stress, enable red cell retention within the clot, and protect the clot from premature degradation. Congenital factor XIII deficiency is caused by autosomal recessive mutations, presenting early in life with a severe bleeding diathesis. Acquired deficiency may be caused by autoimmunity. Currently available assays for laboratory diagnosis of factor XIII deficiency include clot solubility assays, quantitative factor XIII activity assays, factor XIII antigen assays, and genetic testing. The International Society on Thrombosis and Haemostasis Scientific and Standardization Committee has recommended an algorithm for the laboratory diagnosis and differentiation of the different forms of factor XIII deficiency. However, implementation of this algorithm has been limited by technical and budgetary challenges associated with the currently available factor XIII-specific assays. The purpose of this review is to discuss the advantages and limitations of the currently available assays employed for the laboratory diagnosis of factor XIII deficiency.