Measurement of Activated Factor IX in Factor IX Concentrates: Correlation with In Vivo Thrombogenicity

SummaryCurrent in vitro tests for thrombogenicity of FIX concentrates used for prothrombin complex concentrates (PCCs), are of little value when applied to high purity FIX (HP FIXs). In the present study, we have developed a chromogenic assay for activated FIX (FIXa) and evaluated its ability to predict in vivo thrombogenic potential of HP FIXs in a modified Wessler stasis model. Among the HP FIXs, only 1 out of 7 products had no detectable FIXa; this product also showed no in vivo thrombogenicity. In the other 6 products, FIXa content ranged from 0.15–1.2 U/1000 iu FIX, and all showed some evidence of in vivo thrombogenicity, with mean thrombus scores ranging from 0.25–4. There was a significant positive correlation (r = 0.55, p <0.02) between FIXa levels and in vivo thrombogenicity of HP FIXs. NAPTT data were not significantly correlated with the in vivo results and the TFCT also showed no direct correlation with the mean thrombus score. These results indicate that HP FIXs may still carry a small residual thrombotic risk and measurement of FIXa content of these products may be a better predictor of thrombogenicity than the current in vitro tests.

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Assessment of Potential Thrombogenicity in an Animal Model of a Triple Viral Inactivated Factor IX Concentrate Manufactured in Argentina

Annals of Hematology & Oncology ◽

10.26420/annhematoloncol.2021.1343 ◽

2021 ◽

Vol 8 (5) ◽

Author(s):

Martinez MC ◽

◽

Rodriguez R ◽

Marinsaldi A ◽

Rodriguez GR ◽

...

Keyword(s):

Animal Model ◽

Animal Experiments ◽

In Vitro Assay ◽

Factor Ix ◽

In Vitro Tests ◽

Vitro Assay ◽

Thrombotic Risk ◽

Significant Difference ◽

The Mean

The risk of thromboembolism with FIX replacement therapy remains a concern for hemophilic B patients. Previous studies from our laboratory demonstrated that the activated factor content of the FIX Plasma Derived (FIXpd) manufactured at UNC-Hemoderivados was negligible by in vitro assay. Despite this, we considered it important to conduct studies to assess the potential thrombogenic risk of our FIXpd concentrates using a modified stasis animal model. FIXpd were inject doses of 100 or 200 IU F IX kg-1 and some samples were supplemented with heparin (<0.5 of heparin/ IU FIX). Eight rats were tested at each dose level in the presence or absence of heparin, considering those samples with a thrombogenicity ≥2.0 as of potential thrombogenic risk. The mean scores ± SD 100 and 200 IU kg-1 in the presence or absence of heparin were 0.25±0.06 and 2.25±0.45 and 1.19±0.26 and 2.81±0.40, respectively. At both doses tested of FIXpd in the absence of heparin, there was no significant difference in mean scores (P<0.05). The encouraging data obtained from these animal experiments and results from in vitro tests, support the low thrombotic risk associated with the FIXpd concentrate manufactured in UNC Hemoderivados.

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Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646468 ◽

1991 ◽

Vol 66 (05) ◽

pp. 609-613 ◽

Cited By ~ 14

Author(s):

I R MacGregor ◽

J M Ferguson ◽

L F McLaughlin ◽

T Burnouf ◽

C V Prowse

Keyword(s):

High Purity ◽

Time Course ◽

Prothrombin Complex Concentrate ◽

Degradation Products ◽

Canine Model ◽

Factor Ix ◽

In Vitro Tests ◽

Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

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A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650089 ◽

1980 ◽

Vol 44 (02) ◽

pp. 081-086 ◽

Cited By ~ 8

Author(s):

C V Prowse ◽

A E Williams

Keyword(s):

Platelet Rich Plasma ◽

Thrombus Formation ◽

Factor Ix ◽

Coagulation System ◽

In Vitro Tests ◽

Clinical Use ◽

Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

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Feiba And Autoplex: A Comparison Of These Two Activated Prothrombin Complex Concentrates

10.1055/s-0038-1652326 ◽

1981 ◽

Author(s):

E Lechler ◽

B Eggeling ◽

D Meyer-Börnecke ◽

H Stoy

Keyword(s):

Hemophilia A ◽

Gel Filtration ◽

Activated Partial Thromboplastin Time ◽

Factor Ix ◽

Prothrombin Complex Concentrates ◽

Minor Degree ◽

Activated Prothrombin Complex Concentrates ◽

A Minor

These activated concentrates are used for the treatment of patients with factor VIII inhibitors . Both shorten the activated and non-activated partial thromboplastin time of inhibitor plasma and hemophilia A plasma in vitro. They do not or only to a minor degree improve the prothrombin consumption of hemophilia A plasma in vitro. In gel filtration of AUTOPLEX the activity which shortens the PTT of hemophilia A plasma eluted in a volume higher than that of the nonactivated factors of the prothrombin complex and contains activated factor IX. The activity of FEIBA elutes at a lower filtration volume in a rather broad peak together with the factors of the the non-activated prothrombin complex. BaSO4- adsorbed plasma and purified antithrombin (Behring) abolish the activity of AUTOPLEX more readily than of FEIBA. Both concentrates have only a low amidolytic effect (S 2222) and are not inhibited with SBTI and PMSF. In the crossed two-dimensional immunelectrophoresis with heparin in the agarose of the first dimension and anti-antithrombin (Behring) in the agarose of the second dimension (method of Sas), a mixture of AUTOPLEX and antithrombin results into a two peak precipitation of antithrombin, whereas with FEIBA a broadened intermediate peak develops. In vivo both concentrates do not improve the prothrombin consumption and AUTOPLEX shortens the PTT for at least 90 minutes. In summary, these two concentrates differ considerably.

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Modified Tests for Measuring the Thrombogenic Potential of Factor IX Concentrates.

10.1055/s-0039-1684742 ◽

1979 ◽

Author(s):

Sarah M. Middleton ◽

Jessie T. Douglas ◽

C.D. Forbes ◽

C.R.M. Prentice

Keyword(s):

Factor Xa ◽

Activated Partial Thromboplastin Time ◽

Factor Ix ◽

Procoagulant Activity ◽

In Vitro Tests ◽

Factor V ◽

Thrombogenic Potential ◽

Sepharose 4B ◽

Generation Test

In vitro tests for screening potential thrombogenicity of factor IX concentrates are unsatisfactory as it has been shown that the non-activated partial thromboplastin time (NAPTT) and the TGt50 (reflecting thrombin generation by the concentrate after recalcification} do not correlate with each other. We have modified the TGt50 by addition of optimum concentrations of factor V and phospholipid, and compared it with the NAPTT and factor Xa generation test using the chromogenic substrate S2222. The modified TGtSO correlates with both the NAPTT and the factor Xa generation test suggesting that these tests are measuring the same entity. Chromatography of concentrates on sepharose 4B indicates that the high MW void volume material has procoagulant activity as measured by the unmodified TGt50 whilst the retained volume he procoagulant activity as measured by the TGt50 and the NAPTT. When, however, phospholipid is added to the unmodified TG150 the activity of the high MW component is lost and only that of the retained volume is still present. The data suggests that the modified TGt50 and the NAPTT measure the same procoagulant activity in these concentrates.

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The coagulant-active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex (Factor IX) concentrates in rabbits

Blood ◽

10.1182/blood.v59.2.401.bloodjournal592401 ◽

1982 ◽

Vol 59 (2) ◽

pp. 401-407 ◽

Cited By ~ 1

Author(s):

AR Giles ◽

ME Nesheim ◽

H Hoogendoorn ◽

PB Tracy ◽

KG Mann

Keyword(s):

Factor Xa ◽

Lipid Vesicles ◽

Factor Ix ◽

Major Determinant ◽

Phospholipid Content ◽

Clotting Factors ◽

Prothrombin Complex Concentrates ◽

Vitro Assay

In vitro evaluation of prothrombin complex concentrates in a thrombin generation assay, using DAPA and purified components of the prothrombinase complex, demonstrated significant levels of coagulant- active “phospholipid replacing” activity. Quantification of this activity showed a significant correlation (r = 0.8747, p less than 0.01) with thrombogenicity measured in vivo in a stasis model in rabbits. Extracted lipid material retained full phospholipid replacing activity in the vitro assay. Thin-layer chromatographic characterization confirmed the presence of phospholipids with known coagulant activity in vitro. In vivo, the extracted material was nonthrombogenic but augmented the thrombogenicity of purified factor Xa. Substitution of a synthetic coagulant-active phospholipid (phosphatidylcholine-phosphatidylserine lipid vesicles) for the extracted phospholipid produced a similar augmentation of a factor-Xa- induced thrombogenicity in vivo. It is concluded that the coagulant- active phospholipid content of prothrombin complex concentrates is a major determinant of thrombogenicity but requires the presence of activated clotting factors for its expression in vivo.

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Quantification of Eye Irritation Based upon In Vitro Changes of Corneal Thickness

Alternatives to Laboratory Animals ◽

10.1177/026119299001700326 ◽

1990 ◽

Vol 17 (3) ◽

pp. 255-262

Author(s):

Guido A. Jacobs ◽

Mark A. Martens

Keyword(s):

Linear Correlation ◽

Objective Assessment ◽

Corneal Thickness ◽

In Vitro Tests ◽

In Vitro Test ◽

Eye Irritation ◽

The Mean ◽

Observation Times

Measurement of corneal swelling is an objective assessment of irritation in in vivo tests. The same measurement may be made in in vitro tests using the isolated eye test (IET), which has been recommended by the European Community as an alternative for in vivo eye irritation tests. In order to compare the corneal swelling in vivo and in vitro, tests were performed with 34 substances. The in vitro test results were assessed for their ability to predict eye-irritant potential. The corneal swelling data in vivo after 4, 24, 48 and 72 hours were compared with the in vitro data obtained after 2 and 4 hours. Slight linear correlation was found between the corneal swelling in vivo after 4 hours and the corneal swelling in vitro after 4 hours (r=0.77). The substances tested can be divided into two groups, according to their ability to create opacity in either the epithelial layer or in the stroma. When the substances causing epithelial opacity were omitted from the comparison, a much better linear correlation was obtained between the mean corneal swelling 121 vitro over 2 and 4 hours and the mean corneal swelling calculated in vivo for all animals and over three observation times (24, 48 and 72 hours; r=0.91), the latter being the observation times prescribed by EC legislation (1). A comparison of the mean corneal opacity scores observed in vivo and the mean percentage corneal swelling in vitro gave a satisfactory linear correlation (r=0.89). From this study it can be deduced that a mean corneal swelling of 55%, obtained in isolated eyes over 2 and 4 hours, corresponds with the limit for classification as irritant, to the eye. When this criterion was applied to all substances causing no epithelial opacity (n=28), only one false positive and no false negatives were found.

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The coagulant-active phospholipid content is a major determinant of in vivo thrombogenicity of prothrombin complex (Factor IX) concentrates in rabbits

Blood ◽

10.1182/blood.v59.2.401.401 ◽

1982 ◽

Vol 59 (2) ◽

pp. 401-407 ◽

Cited By ~ 58

Author(s):

AR Giles ◽

ME Nesheim ◽

H Hoogendoorn ◽

PB Tracy ◽

KG Mann

Keyword(s):

Factor Xa ◽

Lipid Vesicles ◽

Factor Ix ◽

Major Determinant ◽

Phospholipid Content ◽

Clotting Factors ◽

Prothrombin Complex Concentrates ◽

Vitro Assay

Abstract In vitro evaluation of prothrombin complex concentrates in a thrombin generation assay, using DAPA and purified components of the prothrombinase complex, demonstrated significant levels of coagulant- active “phospholipid replacing” activity. Quantification of this activity showed a significant correlation (r = 0.8747, p less than 0.01) with thrombogenicity measured in vivo in a stasis model in rabbits. Extracted lipid material retained full phospholipid replacing activity in the vitro assay. Thin-layer chromatographic characterization confirmed the presence of phospholipids with known coagulant activity in vitro. In vivo, the extracted material was nonthrombogenic but augmented the thrombogenicity of purified factor Xa. Substitution of a synthetic coagulant-active phospholipid (phosphatidylcholine-phosphatidylserine lipid vesicles) for the extracted phospholipid produced a similar augmentation of a factor-Xa- induced thrombogenicity in vivo. It is concluded that the coagulant- active phospholipid content of prothrombin complex concentrates is a major determinant of thrombogenicity but requires the presence of activated clotting factors for its expression in vivo.

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Bioactivity Responses of Different Uhmwpe Particles from In-Vivo and In-Vitro Tests

MRS Proceedings ◽

10.1557/proc-662-ll3.8 ◽

2000 ◽

Vol 662 ◽

Author(s):

YoungSoo Park ◽

ShangYou Yang ◽

Paul H. Wooley ◽

Katharine Merritt ◽

Stephen Hsu ◽

...

Keyword(s):

The Other ◽

In Vitro Tests ◽

Textured Surfaces ◽

Aspect Ratios ◽

In Vivo Tests ◽

The Mean ◽

Particle Size And Shape ◽

Ultra High Molecular Weight

AbstractThis paper describes the results of bioactivity responses to different ultra high molecular weight polyethylene (UHMWPE) particles. Particles were produced by a wear tester using two different textures of steel counters, one with cross-hatched and the other with uni-hatched grooves. These two textured surfaces produced two distinct populations of wear particles. One is larger and more elongated (fibril shapes) than the other. The mean sizes and aspect ratios of the particles are in the ranges of 5 μm to 25 μm and about 1.5 to 3, respectively. These two distinct UHMWPE particles were examined through in-vitro and in-vivo tests. Macrophages RAW 264.7 and the murine air-pouch model of inflammation were employed to characterize the effect of the particle size and shape. Preliminary in-vivo tests results showed that more elongated and larger particles enhanced bio-reactions.

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Potentially Thrombogenic Materials in Factor IX Concentrates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647857 ◽

1975 ◽

Vol 33 (03) ◽

pp. 617-631 ◽

Cited By ~ 47

Author(s):

H. S Kingdon ◽

R. L Lundblad ◽

J. J Veltkamp ◽

D. L Aronson

Keyword(s):

Human Plasma ◽

Antithrombin Iii ◽

Factor Ix ◽

In Vitro Assays ◽

Substantial Agreement ◽

Coefficient Of Correlation

SummaryFactor IX concentrates manufactured from human plasma and intended for therapeutic infusion in man have been suspected for some time of being potentially thrombogenic. In the current studies, assays were carried out in vitro and in vivo for potentially thrombogenic materials. It was possible to rank the various materials tested according to the amount of thrombogenic material detected. For concentrates not containing heparin, there was substantial agreement between the in vivo and in vitro assays, with a coefficient of correlation of 0.77. There was no correlation between the assays for thrombogenicity and the antithrombin III content. We conclude that many presently available concentrates of Factor IX contain substantial amounts of potentially thrombogenic enzymes, and that this fact must be considered in arriving at the decision whether or not to use them therapeutically.

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