scholarly journals Alteration of Coagulation in Intensively Transfused Hemophilic Patients

1977 ◽  
Author(s):  
Helen S. Hathaway ◽  
Roger D. Hamstra ◽  
Linda Jacobson ◽  
William E. Hathaway
Keyword(s):  
Factor Viii ◽  
Degradation Products ◽  
Bleeding Tendency ◽  
Transfusion Therapy ◽  
Coagulation Tests ◽  
Factor Viii Concentrates ◽  
The One ◽  
Altered Proteins ◽  
Dose Factor

Bleeding may occasionally occur in adequately transfused hemophilic patients. To investigate this phenomenon, 11 patients with classical hemophilia had serial coagulation studies performed during intensive transfusion therapy with factor VIII concentrates given for surgical procedures. The studies included kaolin partial thromboplastin times (KPTT), fibrinogen, monomer, and fibrin split products (FSP) levels, and assays for factor VIII by the one-stage PTT method (PTT-VIII), thromboplastin generation time method (TGT-VIII), and immunologic method (VIII Ag). After an initial correcting dose, factor VIII concentrates were administered once to twice daily in a dose to keep the minimal level above 20 percent. Alterations of coagulation assays were most pronounced 7–10 days postoperatively. These included (1) KPTT values at least 20 seconds longer than expected for the percent factor VIII;(2) TGT-VIII levels consistently higher than PTT-VIII levels (mean difference was 20 percent);(3) VIII Ag values from 216–660 percent of normal. Fibrin monomer and FSP tests were frequently positive and fibrinogen levels ranged from 300–700 mgm percent. Four patients exhibited spontaneous wound bleeding on postoperative days 7, 7, 9, and 12 in spite of adequate factor VIII levels. These studies and the results of in vitro experiments with factor VIII concentrates suggest that altered proteins or degradation products of fibrinogen or factor VIII may produce spurious values for coagulation tests and may be associated with an increased bleeding tendency and/or abnormal wound healing.

Issues with the Assay of Factor VIII Activity in Plasma and Factor VIII Concentrates

2000 ◽  
Vol 84 (12) ◽  
pp. 942-948 ◽  
Author(s):  
Henry Kingdon ◽  
Kenneth Mann ◽  
Gilbert White ◽  
Roger Lundblad
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factors ◽  
Chromogenic Substrate ◽  
In Vitro Measurements ◽  
Factor Viii Concentrates ◽  
The One

SummaryA review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VIII concentrates such as Hemofil ® M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements, it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor Villa during the assay process.

Decreased Potency Of Factor VIII Concentrates

1981 ◽  
Author(s):  
C K Kasper
Keyword(s):  
Factor Viii ◽  
In Vitro Assays ◽  
Assay Method ◽  
Two Stage ◽  
One Stage ◽  
Plasma Factor ◽  
Factor Viii Concentrates ◽  
The One

Plasma factor VIII recoveries after infusions of factor VIII concentrates into patients with classic hemophilia have been measured in this laboratory for 14 years. Recently, we observed a decline in the in vivo recovery of factor VIII per factor VIII unit infused. In 1980, plasma factor VIII levels were measured by a one-stage APTT-based assay before and 10 min after 150 infusions of 46 lots of 3 brands of factor VIII concentrate produced in the U.S.A. Our pooled normal plasma reference was calibrated against WHO International Standard 2 and results expressed in International factor VIII units. Observed in vivo factor VIII recovery was compared to the value expected from calculations based on the unitage stated on the label. The ratio of observed/expected recovery averaged 56% per lot for brand A, 60% per lot for brand B, and 103% per lot for brand C. In vitro assays were performed on 22 lots on 36 occasions, and the ratio of observed/labelled units average 46% per lot for brand A, 53% for brand B and 75% for brand C. The two-stage factor VIII assay method of Pool and Robinson was also used to assay plasma samples from 18 infusions, and results averaged 135% of the one-stage values for infusions of brand A, 160% for brand B, and 109% for brand C. (Brand A is assayed by the manufacturer by a two-stage method, brands B and C by one-stage methods.)Decreased clinical efficacy was observed when postinfusion plasma factor VIII levels were lower than customary. The decline in potency of brands A and B has necessitated more frequent assay of patients and use of larger amounts of concentrate, with greatly-increased expense. Investigation of the effect of different assay methods and different factor VIII standards and references on the apparent factor VIII content of concentrates has begun.

In vitro characterization of commercial factor VIII concentrates: Long-term follow-up

1984 ◽  
Vol 49 (1) ◽  
pp. 53-59 ◽  
Author(s):  
C. Miyashita ◽  
P. Hellstern ◽  
M. K�hler ◽  
G. Blohn ◽  
E. Wenzel
Keyword(s):  
Factor Viii ◽  
In Vitro Characterization ◽  
Long Term Follow Up ◽  
Factor Viii Concentrates

Impact of inhibitor epitope profile on the neutralizing effect against plasma-derived and recombinant factor VIII concentrates in vitro

Haemophilia ◽  
2003 ◽  
Vol 9 (5) ◽  
pp. 567-572 ◽  
Author(s):  
J. Astermark ◽  
J. Voorberg ◽  
H. Lenk ◽  
D. DiMichele ◽  
A. Shapiro ◽  
...  
Keyword(s):  
Factor Viii ◽  
Recombinant Factor Viii ◽  
Factor Viii Concentrates

scholarly journals Laboratory Characterization of Unclassified Bleeding Disorders By Non-Conventional Tests

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4235-4235
Author(s):  
Paula Acuña ◽  
Elena Monzón Manzano ◽  
Elena G Arias-Salgado ◽  
María Teresa Alvarez Román ◽  
Mónica Martín ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Platelet Function ◽  
Research Funding ◽  
Thrombin Generation ◽  
Bleeding Tendency ◽  
Contact Activation ◽  
Normal Range ◽  
Coagulation Tests ◽  
In Vitro Treatment

Abstract Introduction: Hematologists frequently face a percentage of patients with a mild bleeding tendency due to a haemostatic abnormality that cannot be identified with conventional laboratory techniques. Such patients are termed as having an unclassified bleeding disorder (UBD). A good diagnosis is important in order to prevent bleedings during invasive processes and/or childbirth by choosing the optimal therapeutic treatment. We aimed to investigate hemostatic parameters that may be altered in patients with UBD in order to determine the cause of their bleeding symptoms. In particular, possible defects in the tissue factor (TF)-mediated regulation of coagulation or in the plasmin generation during the fibrinolysis, as well as the possible beneficial effects of treatment with antibodies blockers of TFPI. Methods: This is a single-centre, case-control, non-interventionist, prospective study. During an 8 months-period, 40 patients with bleeding symptoms (evaluated with ISTH-BAT score) were studied. Routine coagulation tests (aPTT and PT) and platelet function testing [aggregometry, PFA-100, flow cytometry and Total Thrombus-formation Analysis System (T-TAS; Zarcos, Japan)] were performed. In 17 patients, no abnormalities were detected in platelet function and/or in coagulation tests; so the following procedures were performed: Thrombin generation test by Calibrated automated thrombography (CAT) in samples of platelet poor plasma with corn trypsin inhibitor (CTI), an inhibitor of contact activation phase, using a low amount of TF (1 pM TF and 4 µM phospholipids) as a trigger to allow the evaluation of the TF-dependent pathway. Plasmin generation (PG) test with a kit from Synapse Research Institute (Maastricht, The Netherlands), using Thrombinoscope software. TFPI activity in plasma, measured with ACTICHROME® TFPI kit (Biomedica Diagnostics, USA). The effects of rFVIIa (Novoseven, NovoNordisk; 90 µg/kg) and of a human Anti-TFPI recombinant Ab (clon mAb2021, Creative Biolabs; 400 ng/ml) were tested in CAT, PG and TFPI activity tests. Results: Those patients with aPTT, PT and a platelet function within normal range were further studied performing thrombin generation, plasmin generation and TFPI activity tests. Table 1 shows the results obtained. Samples from patients 1, 2, 4, 7, 8, 9 and 10 had a diminished generation of thrombin, and in vitro treatment with anti-TFPI and rFVIIa only ameliorated thrombin generation in samples from patients 4, 7, 8 and 9. Plasma from patients 8 and 10 had increased activity of TFPI. Generation of thrombin in samples from patients 3, 5, 6 and 11 was within normal range. Plasmin generation was increased and not modified by in vitro treatment with anti-TFPI and rFVIIa in samples 3 and 11; whereas samples 5 (with normal plasmin generation) and 6 (with no data of plasmin generation due to lack of enough sample) had a high TFPI activity in plasma that was inhibited by anti-TFPI. Normal values in all these parameters evaluated were found in six patients, indicating the involvement of different mechanisms that are still unknown. Conclusions: UBD have a diverse pathological basis for the bleeding. So, a single laboratory test to make a correct diagnosis of this pathology cannot be recommended. In accordance with this fact, a personalized treatment should be applied for each patient. Non-conventional laboratory tests need to be standardized and included for studying possible defects in the regulation of TF and/or plasmin pathways that can be involved in very rare mild bleeding phenotypes. TFPI inhibition might emerge as a good therapy for some of these patients. Failure to detect the bleeding cause in some of these patients, suggests the need to perform further studies in this field. This work was supported by Novo Nordisk Pharma S.A. Table 1- Thrombin and plasmin generation and TFPI activity in samples of patients with UBD. Results out of normal range are shown in red. LT: lagtime; ETP: endogenous thrombin potential; EPP: endogenous plasmin potential; TFPI: Tissue factor pathway inhibitor. Figure 1 Figure 1. Disclosures Alvarez Román: Grifols: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding. Martín: Novo Nordisk: Speakers Bureau; Pfizer: Speakers Bureau. Jiménez-Yuste: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Canales: Eusa Pharma: Consultancy, Honoraria; Sandoz: Honoraria, Speakers Bureau; Sanofi: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Consultancy; Gilead/Kite: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

Validation of a Procedure for Potency Assessing of a High Purity Factor VIII Concentrate -Comparison of Different Factor VIII Coagulant Assays and Effect of Prediluent

1990 ◽  
Vol 64 (02) ◽  
pp. 251-255 ◽  
Author(s):  
Claudine Mazurier ◽  
Armelle Parquet-Gernez ◽  
Maurice Goudemand
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Assay Method ◽  
One Stage ◽  
Chromogenic Assay ◽  
Coagulant Activity ◽  
The One ◽  
In Vitro Data

SummaryThe assessment of factor VIII coagulant activity (FVTII: C) in recently available highly purified and concentrated FVTII therapeutic products calls for careful evaluation of assay methodologies. We assayed more than 130 batches of a concentrate with a specific activity of about 150 FVTII :C units/mg protein, using one-stage and two-stage clotting and chromogenic methods. There was good agreement between the potency estimates obtained with the different methods. We also compared the FVTII :C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVTII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVTII-deficient plasma used as prediluents. In order to validate our “in vitro” data we performed 6 “in vivo” analyses in severe haemophilia A patients. On the basis of the overall data obtained we chose to label FVIII potency by using FVIII-deficient plasma as prediluent, reference plasma as standard and the chromogenic assay method.

In Vivo Recovery and Survival of Monoclonal-Antibody-Purified Factor VIII Concentrates

1991 ◽  
Vol 66 (06) ◽  
pp. 730-733 ◽  
Author(s):  
Carol K Kasper ◽  
Hugh C Kim ◽  
Edward D Gomperts ◽  
Kenneth J Smith ◽  
Phyllis M Salzman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
In Vitro Assays ◽  
Two Stage ◽  
Double Blind ◽  
One Stage ◽  
Factor Viii Concentrates ◽  
In Vivo Recovery

SummaryIn response to reports of discrepant in vitro assays of high-purity concentrates, a double-blind crossover study of in vivo recovery and half-life of two brands of monoclonal-antibody-purified factor VIII concentrates (Monoclate and Hemofil-M) was performed in 23 patients with hemophilia A. In vivo recoveries were close to values predicted from the labelled unitage when plasma samples were assayed by a one-stage method. When a two-stage assay was used, lower recoveries were calculated and the recovery with Hemofil-M was slightly but significantly lower than that with Monoclate. The concentrates were re-assayed in vitro by the two-stage method. Monoclate (which is assayed by the manufacturer using a two-stage method) contained 97% of the labelled potency and Hemofil-M (which is assayed by the manufacturer using a one-stage method) contained 81% of the labelled potency. Differences in in vitro and in vivo assay methods contribute to disparities between expected and observed factor VIII recovery. Clearance of Hemofil-M was significantly faster than that of Monoclate, but volume of distribution at the steady state, mean residence time, and plasma half-disappearance times of the two concentrates were not significantly different.

In Vivo Recovery of Factor VIII: A Comparison of One-Stage and Two-Stage Assay Methods

1979 ◽  
Vol 42 (04) ◽  
pp. 1230-1239 ◽  
Author(s):  
I M Nilsson ◽  
T B L Kirkwood ◽  
T W Barrowcliffe
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
In Vitro Assays ◽  
Two Stage ◽  
One Stage ◽  
Significant Difference ◽  
Assay Methods ◽  
The One ◽  
Fraction I

SummaryThe recovery and half-life of VIII: C in the plasma of severely haemophilic patients was measured by one-stage and two-stage assays after injection of two Factor VIII concentrates (Hemofil, Hyland and Fraction I-O, Kabi). Plasma volumes were measured with an Evans� Blue technique, and both concentrates and post-infusion samples were measured against the same plasma standard.There was a highly significant difference in recoveries estimated by the two assay methods. The one-stage assays gave the most consistent results, in that the average recovery was 100%, whereas the two-stage assays gave only about 80% of the value expected from in vitro assays. There was no difference in recoveries between the two concentrates.The two-stage assays gave a slightly shorter half-life than the one-stage assays, and the half-life of Hemofil was also shorter than that of Fraction I-O.

A Methodological Investigation On The One-Stage Factor VIII Assay

1981 ◽  
Author(s):  
J Over ◽  
J A van Mourik ◽  
P van den Brink-Zantingh ◽  
R Smit-Jansen
Keyword(s):  
Factor Viii ◽  
Dose Response ◽  
Red Cross ◽  
Response Curves ◽  
One Stage ◽  
Factor Viii Concentrates ◽  
Dose Response Curves ◽  
The One ◽  
Protein Rich

Assay of Factor VIII coagulant activity (VIII: C) in Factor VIII concentrates has since long met difficulties, such as l) non-paralleility of dose-response curves of plasma standard and Factor VIII concentrate, 2) spuriously low values of VIII: C in concentrates as revealed by abnormally high in vivo recoveries after transfusion, and 3) large interlaboratory variation in assay results. In an attempt to analyze the cause of these problems several parameters of the one-stage assay system were varied systematically and their effect on the parallellity of dose-response curves and on the final VIII: C value was analyzed. Nonparallel1ity was partially corrected with a protein-rich dilution medium, and almost always completely with undiluted instead of 1:1 diluted hemophilic substrate plasma. In both conditions apparently higher VIII:C values were found.A number of assay systems used by different producers of Factor VIII concentrates were compared. The standard and, in some cases, the phospholipid reagent seemed to contribute for the largest past to the inter1aboratory variation, but also other, as yet unidentified, factors exerted some influence. These findings initiated a cooperative study by five Red Cross Blood Transfusion Services in Europe on standardization of the one-stage assay for VIII:C. This resulted in a better correspondence between these institutes (CV 13%) compared to the previous situation (CV 23%).It is concluded that 1) substrate plasma should not be diluted, especially when Factor VIII concentrate is to be tested against a plasma standard, 2) the standard should be of the same type as the testmaterial, and 3) this standard should be properly calibrated against the International Standard for Factor VIII.

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