International comparative field study evaluating the assay performance of AFSTYLA in plasma samples at clinical hemostasis laboratories.

Essentials AFSTYLA exhibits ≈50% underestimation in activity when the one-stage (OS) assay is utilized. A field study compared the performance of AFSTYLA with Advate in factor VIII activity assays. AFSTYLA activity can be monitored with both the chromogenic substrate and the OS assay. The consistent OS underestimation allows for a conversion factor to be applied to OS results. SUMMARY: Introduction AFSTYLA (antihemophilic factor [recombinant] single chain) is a novel B-domain truncated recombinant factor VIII (rFVIII). For AFSTYLA, an approximate 50% discrepancy was observed between results of the one-stage (OS) and chromogenic substrate (ChS) FVIII activity assays. An investigation was undertaken to test whether there is a linear relationship between ChS and OS assay results that would allow reliable clinical interpretation of results independent of the assay method used. Aims To provide confidence in future clinical monitoring, this field study investigated the performance of AFSTYLA and a full-length rFVIII (Advate® ) in FVIII activity assays routinely performed in clinical laboratories. Methods The comparison of AFSTYLA and Advate was performed in an international, multicenter and blinded field study of simulated post-infusion samples. The study documented the extent of variability between methods and laboratories and characterized the relationship between the ChS and OS assays. Results Results from 23 laboratories demonstrate that intra and interlaboratory variability in OS assays were similar for both products. When comparing within the OS assay format, there was a similar and reagent-correlated variability in response to different activators for both AFSTYLA and Advate. The OS underestimation was highly predictable and consistent across the complete range of FVIII plasma concentrations. Conclusion Post-infusion plasma AFSTYLA levels can be monitored in patients by the OS and ChS assays. The consistent and predictable difference between the two assay formats provides clinicians with adequate guidance on how to interpret the results of the OS assay using a single conversion factor.

Extending the pharmacokinetic half-life of coagulation factors by fusion to recombinant albumin.

The prophylactic treatment of haemophilia B and the management of haemophilia A or B with inhibitors demand frequent administrations of coagulation factors due to the suboptimal half-lives of the products commercially available and currently in use, e.g. recombinant factor IX (rFIX) and recombinant factor VIIa (rFVIIa), respectively. The extension of the half-lives of rFIX and rFVIIa could allow for longer intervals between infusions and could thereby improve adherence and clinical outcomes and may improve quality of life. Albumin fusion is one of a number of different techniques currently being examined to prolong the half-life of rFIX and rFVIIa. Results from a phase I clinical trial demonstrated that the recombinant fusion protein linking FIX to albumin (rIX-FP) has a five-times longer half-life than rFIX, and preclinical studies with the recombinant fusion protein linking FVIIa to albumin (rVIIa-FP) suggest that rVIIa-FP possesses a significantly extended half-life versus rFVIIa. In this review, we describe albumin fusion technology and examine the recent progress in the development of rIX-FP and rVIIa-FP.

Hopping conduction observed in thermal admittance spectroscopy.

We observe variable-range hopping conduction in thermal admittance spectroscopy and develop a method to evaluate the signal under this condition. As a relevant example of demonstration we employ Cu(In,Ga)(Se,S)2 thin-film solar cells and show that the fundamental N1 signal, which has been discussed for more than a decade in terms of minority carrier traps, does not display trap parameters, but is generated by the freezing-out of carrier mobility with decreasing temperature when hopping conduction prevails. This effect offers a new approach to carrier hopping and to semiconductors suffering from small mobility.

Fibrin sealants in supporting surgical techniques: strength in factor XIII.

Factor XIII has a well-established role in natural coagulation and clot stabilization. It is often added back to fibrin sealants that are used in a wide range of surgical settings to achieve successful hemostasis, tissue adhesion and wound healing. Factor XIII is the final enzyme to be activated in the blood coagulation cascade. It plays an important role in maintaining the balance between coagulation and fibrinolysis. Factor XIII facilitates the formation of covalent cross-links within the fibrin network, forming a loose mesh after activation by thrombin. It adds significant resilience to fibrin clots, augmenting strength by as much as 5-fold. Both fibrin cross-linking and the factor XIII-catalyzed ligation of the fibrinolysis inhibitor alpha(2)-antiplasmin to the fibrin clot contribute to the increased proteolytic resistance of factor XIII-stabilized clots. Preclinical studies indicate that the inclusion of factor XIII in fibrin sealants used for vascular grafting significantly reduces suture-hole blood loss. This has important implications for the successful control of bleeding in comparable clinical situations. The advantages of factor XIII stabilized clots (increased strength, resistance to proteolysis, promotion of wound healing) suggest that the presence of factor XIII in fibrin sealants may optimize their performance in the clinical setting. The aim of this paper is to review preclinical data that provide evidence for a potentially positive role for factor XIII in fibrin sealants.

Prevention of suture hole bleeding using fibrin sealant: benefits of factor XIII.

BACKGROUND: Suture hole bleeding is a frequent complication in vascular surgery. The use of fibrin sealants (FSs) during surgery is reported to reduce blood loss by enhancing hemostasis. OBJECTIVES: Using a pig vascular graft model we investigated: (1) the use of FSs in vascular surgery and measured blood loss with and without the use of FSs; (2) the effect of factor XIII in FSs on hemostasis and blood loss; and (3) the effect of increasing FS incubation time on hemostasis and blood loss. MATERIALS AND METHODS: During surgery, a 3-cm incision was made in the carotid artery wall. Incisions were covered with a Gore-Tex (PTFE) patch sutured with Ethicon monofil 5/0 or 3/0. The resulting suture holes were treated according to study protocol. Study 1: Nine pigs received either FS treatment (n = 5) or no treatment (n = 4). Study 2: Twenty pigs underwent bilateral surgery; right (n = 20) and left (n = 20) patches received either FS with factor XIII (FS + FXIII) or FS depleted of factor XIII (FS - FXIII). Study 3: Bilateral surgery was carried out on 24 pigs; PFTE patches secured with Ethicon 3/0 were treated with FS and allowed to polymerize for 120 s (n = 12), 180 s (n = 12), or 240 s (n = 12) or with solid support/thrombin (n = 12). RESULTS: Study 1: Mean blood loss was significantly lower in the FS-treated group compared with untreated controls (9.2 +/- 10.6 mL vs 178.8 +/- 125.5 mL, mean +/- SD, P = 0.016). Study 2: Significantly less blood was lost in the FS + FXIII group than in the FS - FXIII group (Delta = 9.6 mL, P = 0. 02). Study 3: Blood loss was significantly higher from patches treated with solid support/thrombin compared with those treated with FS (P < 0.01). CONCLUSIONS: FSs containing factor XIII, such as Beriplast P, are effective in reducing suture hole bleeding and improving hemostasis during vascular graft procedures. The presence of factor XIII contributes to the efficacy of FSs and reduces the time to hemostasis. During vascular surgery, blood loss can be reduced significantly by the use of fibrin sealant compared with absorbable gelatin sponges coated with thrombin.

Effect of factor XIII on endothelial barrier function.

The effect of factor XIII on endothelial barrier function was studied in a model of cultured monolayers of porcine aortic endothelial cells and saline-perfused rat hearts. The thrombin-activated plasma factor XIII (1 U/ml) reduced albumin permeability of endothelial monolayers within 20 min by 30 +/- 7% (basal value of 5.9 +/- 0.4 x 10(-6) cm/s), whereas the nonactivated plasma factor XIII had no effect. Reduction of permeability to the same extent, i.e., by 34 +/- 9% could be obtained with the thrombin-activated A subunit of factor XIII (1 U/ml), whereas the iodoacetamide-inactivated A subunit as well as the B subunit had no effect on permeability. Endothelial monolayers exposed to the activated factor XIII A exhibited immunoreactive deposition of itself at interfaces of adjacent cells; however, these were not found on exposure to nonactivated factor XIII A or factor XIII B. Hyperpermeability induced by metabolic inhibition (1 mM potassium cyanide plus 1 mM 2-deoxy-D-glucose) was prevented in the presence of the activated factor XIII A. Likewise, the increase in myocardial water content in ischemic-reperfused rat hearts was prevented in its presence. This study shows that activated factor XIII reduces endothelial permeability. It can prevent the loss of endothelial barrier function under conditions of energy depletion. Its effect seems related to a modification of the paracellular passageways in endothelial monolayers.

Two non-proline cis peptide bonds may be important for factor XIII function.

The structure of recombinant human cellular factor XIII zymogen was solved in its monoclinic crystal form and refined to an R-factor of 18.3% (Rfree = 23.6%) for all data between 40.0 and 2.1 A resolution. Two non-proline cis peptide bonds were detected. One is between Arg310 and Tyr311 close to the active site cysteine residue (Cys314) and the other is between Gln425 and Phe426 at the dimerization interface. The structure and the role of these cis peptides are discussed in the light of their possible importance for factor XIII function.

Characterization of factor VIII/von Willebrand factor concentrates using a modified method of von Willebrand factor multimer analysis.

In order to provide patients with von Willebrand disease a factor VIII (FVIII)/von Willebrand factor (vWF) concentrate of reproducible quality, an SDS-agarose gel electrophoresis method has been established to determine the content of the high molecular weight multimers (band 11 and higher) of vWF. This method has been used to characterize the content of high molecular weight vWF multimers in Humate P/Haemate P, a commercial FVIII/vWF concentrate. The average content of high molecular weight vWF multimers of 47 batches of Humate P/Haemate P has been determined to be 84.1% of the corresponding bands in normal human plasma. Use of this multimer analysis method for the characterization of five further commercial products revealed clear differences with respect to the high molecular weight vWF multimer content. Furthermore, there is a linear correlation (r2 = 0.73) between the content of high molecular weight vWF multimers and the specific activity of vWF (determined as vWF:RCoF/vWF:Ag). The method described here for analysis of the content of high molecular weight vWF multimers is a reliable and reproducible method to characterize this class of factor concentrates with respect to vWF multimer composition.

The hypothetical N-glycan charge: a number that characterizes protein glycosylation.

The production of recombinant glycoprotein therapeutics requires characterization of glycosylation with respect to the lot-to-lot consistency. Here we introduce the ¿hypothetical N-glycan charge Z' as a parameter that allows to characterize the protein glycosylation in a simple, however, efficient manner. The hypothetical N-glycan charge of a given glycoprotein is deduced from the N-glycan mapping profile obtained via HPAE-PAD. In HPAEC, N-glycans are clearly separated according to their charge, i.e., their number of sialic acid residues, providing distinct regions for neutral structures as well as for the mono- di-, tri, and tetrasialylated N-glycans (Hermentin et al., 1992a). Z is defined as the sum of the products of the respective areas (A) in the asialo, monosialo, disialo, trisialo, tetrasialo, and pentasialo region, each multiplied by the corresponding charge: [formula: see text] Thus, a glycoprotein with mostly C4-4* structures will provide Z approximately equal to 400 (e.g., rhu EPO (CHO), Z = 361), a glycoprotein carrying largely C3-3* structures will amount to Z approximately equal to 300 (e.g., bovine fetuin, Z = 290), a glycoprotein with mostly C2-2* structures will have Z approximately equal to 200 (e.g., human serum transferrin, Z = 207, or human plasma AT III, Z = 180), and a glycoprotein carrying only high-mannose type or trunkated structures will provide Z approximately equal to 0 (e.g., bovine pancreas ribonuclease B, Z = 15, and hen ovomucoid, Z = 15, respectively). The determination of Z was validated in multiple repetitive experiments and proved to be highly accurate and reliable. Z may therefore be regarded as a new and characteristic parameter for protein N-glycosylation.

Therapeutic factor XIII preparations and perspectives for recombinant factor XIII.

With the increasing availability of human plasma this source was used to substitute for the production of factor XIII concentrate from placenta. Prior to changing the source material, the virus safety in accordance with the Committee for Proprietary Medicinal Products (CPMP) guidelines and the half-life were investigated. Concerning the virus safety, the following cumulative log 10 clearance factors were obtained: human immunodeficiency virus (HIV) > or = 18.9, herpes simplex virus (HSV-1) > or = 21.5, polio > or = 19.1, bovine viral diarrhea virus (BVDV) > or = 13.3. Half-life studies of factor XIII from plasma in comparison with factor XIII from placenta were done in rabbits by determination of the antigen and in patients with congenital factor XIII deficiency by determination of the activity and antigen. In rabbits, the terminal half-life of the antigen was 78.2 hours for factor XIII from placenta and 87.0 hours for factor XIII from plasma. In patients the half-lives were similar: 9.2 days for activity and antigen of factor XIII from plasma and 8.5 days (activity) versus 9.4 days (antigen) for the placenta-derived factor XIII. Some further clinical studies with factor XIII concentrates are also reviewed. Newer developments concerning recombinant factor XIII, its expression, characterization, and properties are summarized. Concerning the physicochemical data, the behavior in plasma was characterized by the formation of high-molecular-weight complexes, and first in vivo results, the recombinant factor XIII product was comparable to the naturally occurring material.

Comparison of three consecutive-day and three random-day records of dietary intake.

We conducted this study to determine whether 3 days of dietary records from randomly selected days or from consecutive days more closely estimated the energy and nutrient intake values obtained from 16 days over a 1-year period. Participants consisted of 228 men and women, black and white. No one of three random-day samples or one of five consecutive-day samples better estimated energy and nutrient intake. The group means for intake of energy and seven nutrients were similar for random and consecutive days. However, for energy, protein, fat, carbohydrate, and calcium, the means of the absolute differences between the values for 16 days and those for the random days were significantly smaller than the corresponding means between the values for 16 days and those for consecutive days. Random-day samples may be preferable for describing dietary intake of individuals and small groups when the gain in reliability is worth the cost.

Crystallization of blood coagulation factor XIII by an automated procedure.

Both recombinant blood coagulation factor XIII alpha-chain and factor XIII isolated from human placenta have been crystallized using a novel robotic system for the automatic screening of crystallization conditions. The monoclinic and orthorhombic crystals obtained are suitable for X-ray analysis.

Characteristics of individuals and long term reproducibility of dietary reports: the Tecumseh Diet Methodology Study.

Food frequency reports in 1967-1969 were compared to frequency reports of the same foods asked retrospectively in 1982-1983 and 1967-1969 for 1184 respondents aged 45-64 years in the Tecumseh Community Health Study. The kappa statistic for concordance of the retrospective and baseline reports was used as a summary measure of the individual's ability to reproduce his or her earlier diet report. Reproducibility was estimated for total diet, represented by 83 foods, and for 9 subsets of foods of epidemiologic interest. In bivariate and multivariate analyses, reproducibility was strongly related to stability of diet; those whose diets changed least over the 15-year period had greatest diet reproducibility. Greater total diet reproducibility was also found among men with higher education, among women of less than 110% desirable weight reporting no special diet and among women reporting no medications. Consistent with current models of memory, the retrospective report of diet was strongly related to the current report of diet. Agreement between the retrospective and baseline diet reports was greater than agreement between the current and baseline diet reports. This indicates that, as a proxy for past diet, the retrospective report of diet is superior to the current report. Similar relationships were found for the 9 subset of foods.

Comparison of estimated nutrient intakes by food frequency and dietary records in adults.

Sixteen days of dietary recall and records collected over 1 year for 228 respondents were compared with an estimate of "usual" diet based on a 116-item food frequency questionnaire (FFQ) for the study year. Respondents were white and black men and women aged 24 to 51. Mean energy values for the total group were 2,111 kcal for the recall/records and 2,766 kcal for the FFQ, a 31% difference. The mean food energy and nutrient values obtained by the FFQ were consistently and significantly higher than the mean recall/record values for all four race-sex groups, although the degree of difference varied by nutrient, food group, and demographic characteristics of the respondents. For the nutrients compared, the smallest difference between methods was for protein--27% for the total group. The greatest difference was for vitamin A--123% for the total sample. Sex-race specific correlation coefficients between methods were relatively high for calcium, vitamin C, and iron.

Comparison of surrogate and subject reports of dietary practices, smoking habits and weight among married couples in the Tecumseh Diet Methodology Study.

Agreement between surrogate and subject reports of current food frequencies and other eating habits, smoking behavior and weight was assessed in 1982-1983 for 180 husbands and wives, aged 45 through 64 years. Agreement was measured by per cent exact agreement and weighted kappa for frequencies of 30 itemized foods or food groups, and for surrogate- and subject-based quintiles of frequencies of eight broad food groups and of vitamin A and C consumption indexes. Surrogate and subject mean frequencies were generally similar, but at the individual level of analysis, agreement varied widely. Agreement was greatest, among the food items and groups, for alcoholic beverages, and among the other items, for smoking status. Extreme misclassification by quintile was very small, but only 40% of persons self-classified in either extreme quintile were similarly classified by their spouses. This level of misclassification may result in the dilution of real relationships between diet and health.