A Simple Procedure for Accurate Quantitation of Factor VIII Inhibitors

1970 ◽  
Vol 23 (01) ◽  
pp. 019-025 ◽  
Author(s):  
Daphne Tse ◽  
L Fekete ◽  
E Shanbrom
Keyword(s):  
Factor Viii ◽  
Therapeutic Response ◽  
Simple Procedure ◽  
Clotting Factor ◽  
Clotting Time ◽  
Precise Calculation ◽  
Control Plasma

SummaryA simple assay for quantitative determination of factor VIII (AHF) inhibitors has been developed. The presence of AHF inhibitor is confirmed by determining clotting time in serial dilutions of patient’s plasma. The amount of inhibitor is then quantitat-ed by adding known units of human AHF concentrate (1 to 100 units/ml) to plasma until neutralization occurs. The neutralization point is defined as that level of AHF concentrate which clots the patient’s plasma in the same time as the control plasma (inhibitor-free) would clot in the presence of 1 unit of AHF concentrate. (This point, minus the initial 1 unit/ml, gives the titer of inhibitor present (units/ml). The dosage of AHF concentrate required to overcome the inhibitor and raise the free circulating AHF to the desired theoretical level is then calculated from the known titer of inhibitor. This method has been evaluated by assays of 75 samples from 22 patients with acquired or induced AHF inhibitors. In vivo plasma AHF activity correlated well with the calculated in vitro values. The assay provides reliable and accurate quantitation of factor VIII inhibitors enabling precise calculation of effective AHF dosage and monitoring of therapeutic response. The assay can be readily adapted to quantitation of other clotting factor inhibitors.

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.

scholarly journals A Macroglobulin with Inhibitory Activity Against Coagulation Factor VIII

Blood ◽  
1970 ◽  
Vol 35 (3) ◽  
pp. 370-376 ◽  
Author(s):  
P. A. CASTALDI ◽  
R. PENNY
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Coagulation Factor ◽  
Coagulation Factor Viii ◽  
Clotting Factor ◽  
Factor Deficiency ◽  
Spontaneous Correction ◽  
Characteristic Features

Abstract Deficiency of coagulation factor VIII occurred in a patient with a monoclonal gamma-M protein and characteristic features of Waldenström’s macroglobulinemia. The protein, found to contain lambda light chains, had specific activity against normal factor VIII that was reversible by reduction with 2-mercaptoethanol. Spontaneous correction of the clotting factor deficiency occurred during treatment. It is suggested that the macroglobulin in this patient removed or masked factor VIII by adsorption and that alteration of the protein in vitro by mercaptoethanol and in vivo by treatment removed this capacity.

Relationships Between Factor XII-Dependent Systems And The “Prorenin”-Renin System In Human Plasma

1981 ◽  
Author(s):  
E A Wilczynski ◽  
A D Purdon ◽  
D H Osmond
Keyword(s):  
Clotting Factor ◽  
Factor Xii ◽  
Clotting Factors ◽  
In Vitro Techniques ◽  
Subsequent Exposure ◽  
Control Plasma ◽  
Free Plasma

Treatment of plasma with cold (-4°C,72 hr), and with trypsin (0.5 mg trypsin/ml plasma), are well established in-vitro techniques used to activate plasma prorenin. Various clotting factor deficiencies have been found to impair the conversion of prorenin to renin in plasma. Studies with factor XII deficient plasma, in which marked reduction in both cold and tryptic activation was seen, led to further studies on the role of clotting factors and other factor XI I-dependent systems in prorenin activation. Removal of factors II, VII, IX, and X by adsorption onto BaSO4, and subsequent exposure of the residual plasma to cold (-4°C, 48 hr) and trypsin (1 mg/ml), resulted in a decreased capacity for prorenin activation when compared to control plasma, more so in cold than in trypsin-treated plasma. Plasminogen-free plasma responded similarly and, while increased concentrations of trypsin could enhance its prorenin activation to near-normal levels, prolonged cold incubation could not. This suggests that trypsin, added in an appropriate concentration to deficient plasma, may be able to substitute for the missing factor(s), while cold activation is limited by availability of one or more crucial factors. Unmanipulated Fletcher plasma (prekallikrein deficient) has a low level of active renin, and elevated prorenin, symptomatic of a block of prorenin conversion in-vivo. However, cold and tryptic activation were, if anything, relatively greater than normal, especially for trypsin, suggesting that enzymes other than kallikrein are important activators, in-vitro, and can substitute for the missing kallikrein. Thus, neither kallikrein, nor any other single factor studied here, including factor XII, is solely responsible for the activation of plasma prorenin.

Disappearance of Antibodies to Factor VIII in a Patient with Acquired Haemophilia and Carcinoma of the Pancreas During Cytotoxic Therapy with Fluorouracil and CCNU

1984 ◽  
Vol 52 (02) ◽  
pp. 131-133 ◽  
Author(s):  
E G H Rhodes ◽  
E A M Boesen ◽  
R E T Corringham ◽  
K B Matthews ◽  
E G D Tuddenham ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tumour Size ◽  
In Vivo Studies ◽  
Clotting Factor ◽  
Bleeding Tendency ◽  
Cytotoxic Therapy ◽  
Carcinoma Of The Pancreas ◽  
Human Factor Viii

SummaryAn inhibitor to clotting factor VIII (anti-VIII: C) developed in a 70 year old woman with carcinoma of the pancreas three months after palliative by-pass surgery. A life-threatening sublingual haemorrhage was controlled by infusion of human factor VIII concentrate in high dosage. With the objective of reducing pancreatic tumour size, combination cytotoxic therapy with fluorouracil and CCNU was given. Reduction in the size of the tumour was associated with disappearance of anti-VIII:C, reappearance of normal quantities of clotting factor VIII (VIII: C) in the plasma and resolution of the bleeding tendency. The anti-VIII: C was characterised as being predominantly of the IgG4 sub-class with k light chains. In vitro and in vivo studies showed the inactivation of VIII: C by anti-VIII: C was markedly non-linear. Normal quantities of factor VIII coagulant antigen (VIII: CAg) were detected in the patient’s plasma when VIII: C levels were negligible.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

1990 ◽  
Vol 63 (02) ◽  
pp. 220-223 ◽  
Author(s):  
J Hauptmann ◽  
B Kaiser ◽  
G Nowak ◽  
J Stürzebecher ◽  
F Markwardt
Keyword(s):  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Factor Xa Inhibitors ◽  
Venous Stasis ◽  
Clotting Time ◽  
Thrombosis Model ◽  
Activity Factor ◽  
Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

Storage of Human Blood Platelets

1974 ◽  
Vol 32 (02/03) ◽  
pp. 405-416 ◽  
Author(s):  
M. R Hardeman ◽  
Carina J L. Heynens
Keyword(s):  
Storage Temperature ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Storage Period ◽  
Serotonin Uptake ◽  
Hypotonic Shock ◽  
Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Acquired Haemophilia: Functional Study of Antibodies to Factor VIII

1981 ◽  
Vol 45 (03) ◽  
pp. 285-289 ◽  
Author(s):  
J P Allain ◽  
A Gaillandre ◽  
D Frommel
Keyword(s):  
Factor Viii ◽  
Functional Study ◽  
Factor Viii Inhibitor ◽  
Acquired Haemophilia ◽  
Viii Inhibitor ◽  
Kinetics Of ◽  
Antibody Function ◽  
Native Plasma

SummaryFactor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR.Ag levels were found in all patients. VIII:WF levels were 50% of those of VTIIRrAg, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers.Antibody function has been characterized by kinetics of VIII :C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed:1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (> 1).2) Antibodies from other patients, usually with lower titres, inactivated VIII :C according to complex order kinetics, were not saturable, and had a less steep affinity slope (< 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.

The Effect of Adrenaline Infusions on Blood Coagulation in Normal and Haemophilia B Dogs

1966 ◽  
Vol 15 (03/04) ◽  
pp. 349-364 ◽  
Author(s):  
A.H Özge ◽  
H.C Rowsell ◽  
H.G Downie ◽  
J.F Mustard
Keyword(s):  
Body Weight ◽  
Factor Viii ◽  
Factor Ix ◽  
Clotting Factor ◽  
Blood Ph ◽  
Order Of Magnitude ◽  
Dose Dependent ◽  
Generation Test ◽  
Plasma Activity

SummaryThe addition of trace amounts of adrenaline to whole blood in plasma in vitro increased factor VIII, factor IX and whole plasma activity in the thromboplastin generation test. This was dose dependent.Adrenaline infusions less than 22 (μg/kg body weight in normal dogs accelerated clotting, increased factor IX, factor VIII and whole plasma activity in the thromboplastin generation test and caused a fall in blood pH. In a factor IX deficient dog, there was no increase in factor IX activity. After adrenaline infusions, however, the other changes occurred and were of the same order of magnitude as in the normal. Adrenaline in doses greater than 22 μg/kg body weight did not produce as great an effect on clotting in normal or factor IX deficient dogs. The platelet count in the peripheral blood was increased following the infusion of all doses of adrenaline. These observations suggest that the accelerating effect of adrenaline on clotting is not mediated through increase in activity of a specific clotting factor.

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