A typical Response To Infusion Prior To The In Vitro Appearance Of Inhibitor In A Hemophilia B Patient

1981 ◽  
Author(s):  
C H Miller ◽  
M W Hilgartner

Infusion studies were performed on six patients with severe hemophilia B, in whom factor IX clotting activity (IX: C) was less than 1%. All had no detectable factor IX antigen (IX: Ag) on testing with heterologous anti-IX prior to the study (and no measurable F. IX inhibitor). Each patient received the same factor IX concentrate in a 50 u/kg dose. Samples were drawn at 0, 15 minutes, 1 hour, 4 hours, and 24 hours post-infusion and were tested for IX: C and IX: Ag. Five of the patients showed similar response to transfusion with average half-life of IX: C of 12 hours and of IX:Ag 10 hours. One patient showed quite different results. He had a half-life of IX: C of 31 hours and of IX:Ag of 24 hours. Although routine tests for a F. IX inhibitor were negative both before and after the study, he developed a rapidly rising inhibitor 15 months later. In retrospect it can be seen that this patient resembled the patient with a low titer inhibitor and prolonged IX: Ag survival described by Goodnight et al. However, our patient showed the atypical infusion response pattern before his inhibitor was detectable in vitro. Thus the finding of a prolonged survival of IX: Ag can be taken as evidence of inhibitor formation. Inhibitor presence can be confirmed by detection of circulating immune complexes containing IX: Ag prior to the appearance of a measurable inhibitor to clotting.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1034-1034
Author(s):  
Neil A. Goldenberg ◽  
William E. Hathaway ◽  
Christopher Bombardier ◽  
Kelly McFarland ◽  
Linda Jacobson ◽  
...  

Abstract BACKGROUND: The Clot Formation and Lysis (CloFAL) global assay is a turbidimetric plasma assay utilizing coagluation activation by lipidated tissue factor and fibrinolytic enhancement by tissue plasminogen activator, and has recently been shown to be sensitive for factor VIII (FVIII) deficiency (Goldenberg et al., Thromb Res 2005; Goldenberg et al., Haemophilia 2006, in press). Deficiency of factor IX (FIX) is often clinically less severe than that of FVIII. We sought to evaluate the analytical sensitivity of the CloFAL assay for FIX deficiency, both in vitro and ex vivo. METHODS: The influence of FIX activity upon the CloFAL assay was examined in vitro in FIX-deficient plasma by mixing study with pooled normal plasma, addition of plasma-derived FIX concentrate, and repletion with recombinant FIX. The analytical sensitivity of the CloFAL assay for FIX deficiency was then studied clinically via comparison of healthy individuals to FIX-deficient adults (n=25) and children (n=19), of whom greater than 1/3 had mild hemophilia B (FIX activity >/= 5.0 U/dL). FIX-deficient subjects had not received exogenous FIX within the prior 96 hours and had no evidence of active hepatitis. Healthy adult and pediatric groups (n=25 each) were without chronic illness or family history of bleeding or thrombosis. None of the study subjects were taking medications that affect hemostasis or had recent active bleeding or acute infection. RESULTS: Alteration of FIX activity in vitro exerted considerable influence upon the CloFAL assay (Figure 1). Ex vivo, the CloFAL assay coagulation index (CI), a measure of the area under the clotting curve, was significantly decreased in FIX-deficient versus healthy subjects among adults and children alike (median CI, adults: 10% vs. 94%, respectively; median CI, children: 10% vs. 63%, respectively; P < 0.0001 for each), and correlated significantly with FIX activity in both age groups (r = 0.80 and r = 0.77; P < 0.0001 for each). In addition, the CloFAL assay fibrinolytic index (FI) was increased in FIX-deficient versus healthy subjects (median FI, adults: 228% vs. 109%, respectively, p<0.0001; median FI, children: 276% vs. 202%, respectively; p=0.7), although this difference was not statistically significant in children. Interestingly, severe hemophilia B patients (n=8; FIX activity < 1.0 U/dL) showed considerable heterogeneity in CloFAL assay waveforms (Figure 2). Nevertheless, the assay uniformly discriminated these patients from healthy controls. CONLUSION: This work demonstrates that the CloFAL global assay, which is highly influenced by FVIII activity, is also analytically sensitive to FIX deficiency. Figure 1. Coagulative response of severe FIX-deficient plasma to increasing FIX activity in vitro, as measured by coagulation Index (CI) in the CloFAL global assay. Figure 1. Coagulative response of severe FIX-deficient plasma to increasing FIX activity in vitro, as measured by coagulation Index (CI) in the CloFAL global assay. Figure 2. CloFAL global assay waveforms for pooled normal plasma (grey) as compared to patients with severe hemophilia B (FIX activity < 1 U/dL; n=8). Figure 2. CloFAL global assay waveforms for pooled normal plasma (grey) as compared to patients with severe hemophilia B (FIX activity < 1 U/dL; n=8).


2021 ◽  
Vol Publish Ahead of Print ◽  
Author(s):  
Franziska Cuntz ◽  
Hedwig E. Deubzer ◽  
Johannes H. Schulte ◽  
Antje Nimtz-Talaska ◽  
Angelika Eggert ◽  
...  

1977 ◽  
Author(s):  
P.A. Gentry ◽  
A.R. Thompson ◽  
A.W. Forrey

In preparing a factor IX concentrate with a high yield and low hepatitis and thromboembolic risks, we have tested this material for survival in an in vivo system, the hemophiliac dog. By following the disappearance of radiolabeled, isolated factor IX in addition to the classic clotting assays, data on protein survival and more accurate kinetic parameters were obtained.Crude factor IX concentrate was prepared by batchwise adsorption-elution with DEAE-Sephadex using cryoprecipitate-poor human plasma. Isolated human factor IX was radiolabeled with 125I by chloramine-T without in vitro loss of clotting activity (Thompson, J Clin Invest, in press, 1977). A preparation containing both crude and isolated factor IX was then subjected to filtration (0.22 μm) and lyophilization; clotting and radioactivity were not altered by these steps.Following infusion of the combined preparation into a dog with severe hemophilia B (0% baseline factor IX) 10 post infusion samples were taken over 96 h for determination of radioactivity and factor IX clotting activity. These data were then analyzed by fitting to a two exponential expression using a Marquart non-linear least squares numerical procedure for a two compartment open model. The central volume was 14.5% of the animal’s body weight; the total volume of distribution was 28% with a t 1/2 distribution of 114 min. The t 1/2 elimination was 20 h; the slower phase of elimination (β, or that affected by redistribution) had a t 1/2 of 40 h. Factor IX clotting activity from the crude concentrate closely paralleled radioactivity from the isolated factor IX throughout the 96 h; t 1/2 β was slightly longer from the clotting activity data.


1977 ◽  
Author(s):  
S.H. Goodnight ◽  
C.W. Britell ◽  
K.D. Wuepper ◽  
B. Østerud

A persistent low titer IgG factor IX inhibitor (0.8–2.3u/ml) has been previously described in an 11 year old boy with severe hemophilia B-. Since the disappearance of factor IX antigen (IXAGN) following Konyne(R) infusion was markedly delayed ( 60 hr) compared to factor IX clotting activity (T 8 hr) (Blood 48:977, 1976), a search for circulating factor IX antigen-inhibitor complexes was undertaken. The inhibitor binds firmly to staphlococcal protein A – Sepharose 4B (SPA-Sepharose) but may be eluted in the IgG 1, 2, and 4 fraction with 3M NaSCN. SPA-Sepharose chromatography of post-infusion plasma (IXAGN 2.7u) from the patient with the inhibitor showed IXAGN (0.5u) in the IgG 1, 2, and 4 fraction, whereas IXAGN was not found in that fraction of chromatographed samples of post infusion plasma from non-inhibitor patients with hemophilia B- or B+. Using a highly specific antibody to factor IX, two dimensional Immunoelectrophoresis of purified factor IX or normal plasma showed a single symmetrical fast moving peak. When an equal volume of inhibitor plasma was added to either factor IX or normal plasma a second, slower moving component was also seen. Two dimensional Immunoelectrophoresis of plasma obtained from the patient 45 minutes after infusion showed both the slow and fast components. The presence of IXAGN in the IgG 1, 2, and 4 peak after SPA-Sepharose chromatography and the demonstration of 2 components on two dimensional Immunoelectrophoresis indicates the presence of circulating factor IX antigen-inhibitor complexes in the plasma of this patient following the infusion of a factor IX concentrate.


Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 130-138 ◽  
Author(s):  
Valder R. Arruda ◽  
James N. Hagstrom ◽  
Jeffrey Deitch ◽  
Terry Heiman-Patterson ◽  
Rodney M. Camire ◽  
...  

Abstract Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical γ-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and γ-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX.


2018 ◽  
Vol 21 ◽  
pp. S111
Author(s):  
A Chhabra ◽  
D Spurden ◽  
BJ Tortella ◽  
PF Fogarty ◽  
A Pleil ◽  
...  

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 438-445
Author(s):  
TD Palmer ◽  
AR Thompson ◽  
AD Miller

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.


Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1123-1123
Author(s):  
Teresa Ceglie ◽  
Berardino Pollio ◽  
Irene Ricca ◽  
Maria Messina ◽  
Claudia Linari ◽  
...  

Introduction. Prophylaxis with factor concentrates reduces bleeding events and improves quality of life for adults and children with severe hemophilia. However, the optimal dosing and infusion frequency is not yet established. Integration of PK data into decision making is gaining support, in particular at the transition between conventional and EHL products. Here we report about 29 PK data of patients affected by hemophilia treated at our centre since childhood. Improved quality of life was our first aim, supposed that decreasing frequency of infusions or increasing the target through factor level allows a more active life without increased risk of bleeding. Patients' characteristics and methods. 18 patients (62%) were ≤ 18 years of age at PK time. 16 were affected by severe hemophilia A, 5 by moderate hemophilia A, 6 by severe hemophilia B and 2 by moderate hemophilia B. At PK time, 28 patients were on prophylaxis and 1 was on demand with recombinant factor IX. Median age at onset of prophylaxis was 9 years (range 3 months-38 years). Genetic assessment was available in 24 patients. Of these, 37.5% and 62.5% were carriers of null and not null mutations respectively. 4 patients were undergone to PK with standard products (1 Octocog alfa, 1 Simoctocog alfa, 1 Octocog alfa-Kovaltry®, 1 Turoctocog alfa) in order to define timing and dosage of successive infusions, while 25 patients switched to EHL factors (15 Efmoroctocog alfa, 2 Ionoctocog alfa, 7 Albutrepenonacog alfa, 1 Eftrenonacog alfa). In 15 patients a population-based PK (popPK) according to WAPPS-Hemo program was also performed. The annualized bleeding rate (ABR) was counted from patient's home bleeding records for one year before PK until now. Results. According to PK data, 21 patients (75%) decreased infusion frequency (100% hemophilia B and 67% hemophilia A patients). The remaining 7 hemophilia A patients maintained the same timing in order to increase the through factor level. Notably, 1 hemophilia B patient switched from on demand treatment to prophylaxis with EHL product due to the more acceptable schedule. 66% of null mutation patients and 73% of not null mutation patients decreased timing. Of 28 patients available at follow-up, 32%, 50% and 18% decreased, increased and maintained the same annual average factor consumption/kg, respectively. All patients had a good adherence after switch. In particular, the on demand patient started a regular prophylaxis with optimal compliance. ABR displayed a reduction with a median of 0 (range 0-5) after PK analysis compared to 1 (range 0-12) before the switch. Full PK vs popPK data obtained using at least two individual PK sampling points were almost similar. Conclusions. Our results remark the necessity of PK study especially in children due to the inter-individual variability independent of genetic assessment. Regarding factor IX, PK allowed us to propose timing even longer than that recommended by prescribing indications resulting in a better personalized prophylaxis. Moreover, our study demonstrates that a full PK analysis is feasible also in children. However, given similar results, popPK could be more feasible in most patients. Regarding consumption, the reduction of only 32% of patients reflects our aim to maintain a high safety profile in an active pediatric population. Nevertheless, the mean annualized consumption was just 0.6-fold increased in the remaining patients. This approach led us to further reduce ABR and in some cases to obtain a persistent no-bleeding status even with a full active life. Disclosures No relevant conflicts of interest to declare.


Sign in / Sign up

Export Citation Format

Share Document