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Biomedical subjects

E G Tuddenham

Publications and source records attributed to E G Tuddenham.

At least 127 records · Page 7Linked to original sources
PubMed1987

Haemophilia A: carrier detection and prenatal diagnosis by linkage analysis using DNA polymorphism.

Restriction fragment length polymorphisms (RFLPs) within or close to the factor VIII locus are very useful for genetic linkage analysis. Such RFLPs allow a mutant allele to be tracked in a family, segregating haemophilia A even when, as is usually the case, the precise mutation causing failure to synthesise factor VIII is unknown. To date two markers tightly linked to the factor VIII locus have been described, one of which is highly polymorphic and therefore informative in most kindreds. A significant crossover rate, however, does not make diagnosis absolute. Three intragenic RFLPs have been defined, which, taken together, are informative in about 70% of women, providing virtually deterministic genetic diagnosis.

DNA↗
PubMed1986

Studies on the action of ethamsylate (Dicynene) on haemostasis.

Twelve healthy subjects received ethamsylate or a placebo by mouth over a 48 h period in a randomized double-blind trial. The template bleeding time (including estimation of amount of blood loss), platelet aggregation studies, and plasma levels of plasminogen, alpha 2-antiplasmin and fibronectin were carried out before and during treatment. The effect of a single dose (600 mg) of aspirin, given 24 h after commencement of treatment, was also determined. Neither ethamsylate nor placebo caused a significant change in the basal values of any of the variables monitored but both the prolongation of the bleeding time and the amount of blood loss induced by aspirin were significantly less during ethamsylate treatment than with placebo. Ethamsylate failed to prevent the aspirin-induced elimination of the secondary wave of platelet aggregation. We conclude that ethamsylate may reduce the haemorrhagic symptoms associated with mild functional platelet defects.

Adult↗
PubMed1986

A two-site, monoclonal antibody-based immunoassay for von Willebrand factor--demonstration that vWF function resides in a conformational epitope.

Two monoclonal antibodies (RFF-VIII:R/1 and RFF-VII:R/2) which recognise the same epitope on von Willebrand factor (vWF) have been used in a simple, two-site, solid-phase immunoradiometric (IRMA) or enzyme-linked assay (ELISA) to analyse vWF in plasma from normal individuals and from patients with von Willebrand's disease (vWD). Results obtained confirm our previous findings (using RFF-VIII:R/2 in a one-site, fluid-phase IRMA) that the MAbs detect the presence of an epitope on the vWF molecule that reflects its function. This epitope is involved in vWF binding to the GPIb protein on platelets. It is reduced in all types of vWD, including type II (or variant) vWD. It is present in normal plasma, in vWF released from normal platelets and from cultured umbilical cord vein endothelial cells. The epitope is, however, found to be reduced in serum. Studies on SDS-treated vWF prove that this GPIb-binding site is dependent on the conformation of the vWF multimers.

Antibodies, Monoclonal↗
PubMed1986

A new polymorphism in the factor VIII gene for prenatal diagnosis of hemophilia A.

A restriction fragment length polymorphism (RFLP) has been found in the gene for clotting factor VIII. Defects in this gene are the cause of hemophilia A. The DNA polymorphism affects an XbaI site in intron 22 of the gene. Two alleles occur in a frequency of 59 and 41 percent of the X chromosomes tested. Furthermore, about 25 percent of females who are homozygous for the previously reported BclI RFLP in the factor VIII gene are heterozygous for the XbaI polymorphism. This new RFLP thus represents a significant addition to available probes for the DNA-based prenatal diagnosis and carrier detection of this disease.

Chromosome Mapping↗
PubMed1986

Studies on immunological assay of vitamin K dependent factors. I. Measurement of factor VII antigen by radioimmunoassay.

A radioimmunoassay (RIA) for factor VII was developed using 125I-factor VII, anti-factor VII rabbit serum and anti-rabbit IgG goat serum. The lower limit of sensitivity in normal reference plasma was 3 X 10(-3) units/ml. Although the level of factor VII antigen (VII:Ag) in normal plasma samples (n = 20) 0.944 +/- 0.176 units/ml, correlated with that of factor VII coagulant activity (r = 0.89), VII:Ag level in paired normal sera showed a higher value (1.469 +/- 0.376 units/ml). The level of antigen according to RIA of factor VIIa activated by factor Xa increased 2.5-fold compared with that of unactivated factor VII. It is suggested that the polyclonal anti-factor VII produced in a rabbit had higher affinity for factor VIIa than for factor VII. In two out of seven patients with congenital factor VII deficiency, VII:Ag was detectable (0.04, 0.31 units/ml, respectively) whereas VII:C was less than 0.01 units/ml. In 12 warfarin-treated individuals, VII:C showed a lower level (0.121 +/- 0.063 units/ml) that that of VII:Ag (0.518 +/- 0.186 units/ml). During 4 weeks observation after stopping warfarin, VII:C and VII:Ag reached normal levels in 1 week. However, VII:C did not reach equivalence with VII:Ag until 4 weeks had elapsed.

Animals↗
PubMed1986

Studies on immunological assay of vitamin K dependent factors. II. Comparison of four immunoassay methods with functional activity of protein C in human plasma.

We describe and compare five assay systems for Protein C (PC) in human plasma; a functional assay for PC activity, Laurell electroimmunoassay with EDTA or calcium (EDTA-Laurell or Ca-Laurell), radioimmunoassay (RIA) and immunoradiometric assay (IRMA). The lower limit of sensitivity of PC in normal reference plasma was 2 X 10(-3) units/ml by RIA and 1 X 10(-4) units/ml by IRMA, but the ratio of maximum binding to blank binding was superior in RIA. In normal plasma (n = 20), there was no significant discrepancy of PC levels between RIA, Laurell (EDTA- and Ca-) and functional assay. In normal serum, PC antigen (PC:AG) measured by Ca-Laurell showed a high value (1.37 +/- 0.24 units/ml) compared to the other assays. PC:AG of a purified sample increased its level by Ca-Laurell method during activation by thrombin. Activated PC (PCa) has decreased affinity for anti-PC rabbit IgG, similarly to decarboxy (warfarinized) PC. In warfarin-treated individuals (n = 12) who were anticoagulated orally for more than 3 weeks, functional activity was lower (0.27 +/- 0.12 units/ml) than that measured by RIA (0.64 +/- 0.12 units/ml) or EDTA-Laurell (0.62 +/- 0.06 units/ml), whereas PC:AG measured by CA-Laurell had a normal value of 0.96 +/- 0.40 units/ml. After stopping administration, the activity showed a gradual increase to the normal at 2 weeks, while PC:AG increased more rapidly to normalize in 1 week.

Animals↗
PubMed1986

Studies on immunological assay of vitamin-K dependent factors. III. A double monoclonal immunoradiometric assay for factor IX antigen.

Two-site immunoradiometric assays (IRMAs) for factor IX antigen (IX:Ag) were developed using a monoclonal antibody (RFF-IX/1) on the solid-phase and either another monoclonal antibody (RFF-IX/4) or a human polyclonal inhibitor antiserum as tracer (M-M and M-I IRMA respectively). The lower sensitivity limits of these two assays for IX:Ag in normal reference plasma were 4 X 10(-4) (M-M IRMA) and 2 X 10(-4) (M-I IRMA) units/ml. In 20 samples of normal plasma, levels of factor IX coagulation activity (IX:C) and of factor IX antigen measured by both IRMAs were highly correlated. Mean values of approximately 1.0 units/ml were obtained in all three assays. In normal serum, IX:Ag levels were lower with means of 0.84 (M-M IRMA) and 0.83 (M-I IRMA) units/ml. 4/25 patients with haemophilia B were CRM neg., two were CRM + and the remaining 19 patients were CRMr variants. In two of these, IX:Ag was detectable by M-I IRMA whilst IX:C and IX:Ag measured by M-M IRMA were undetectable. In plasma from a fetus subsequently terminated on eugenic grounds, IX:C and IX:Ag by both M-M and M-I IRMA were undetectable. In warfarin-treated plasma (n = 12), the level of IX:C was low (mean 0.39 units/ml). The levels of IX:Ag measured by M-M IRMA (mean of 0.80 units/ml) and by M-I IRMA (0.70 units/ml) showed a discrepancy. M-M IRMA reflects the real amount of IX:Ag in warfarinized plasma because both monoclonal antibodies bind to epitopes distant from the light chain carboxylated region. Western blotting of denatured factor IX demonstrated that RFF-IX/1 binds an epitope that is lost after XIa activation. RFF-IX/4 binds the heavy chain. Antigen measured after activation but without denaturing showed loss of 60% reactivity after XIa activation but no change after RVV activation. These data indicate a binding site for RFF-IX/1 within the activation peptide (residues 146-180).

Antibodies, Monoclonal↗
PubMed1985

The effect of an acute phase reaction and BCG innoculation on factor VIII in the guinea-pig.

Factor VIII antigen (VIII:Ag) and vWF:Antigen (vWF:Ag) were measured in guinea-pigs treated with intraperitoneal turpentine to induce an acute phase reaction, and with BCG to stimulate the reticulo-endothelial system. In the turpentine treated animals there was a significant rise of fibrinogen at 24 and 48 hours after injection (1.43 +/- 0.01 g/l) when compared with controls (1.15 +/- 0.1 g/l), mean +/- SEM n = 3 p 0.01). There was no change in plasma VIII:Ag but a significant rise of vWF:Ag at (2.0 +/- .3 units/ml) when compared with controls (1.1 +/- 0.05 units/ml, mean +/- SEM n = 3 p less than 0.001). Examination of perfused guinea-pig organs showed a reduction in hepatic VIII:Ag (82%) and vWF:Ag (90%) and a 76% increase in splenic vWF:Ag only in the turpentine treated animals. Distribution of 125I Albumin to detect trapped blood in tissues demonstrated efficient clearance of blood by perfusion. There was no change in the plasma concentration of either VIII:Ag or vWF:Ag following BCG inoculation but there was a 45% increase in the splenic concentration of vWF:Ag. It is concluded that only the factor vWF:Ag and not the factor VIII:Ag component of the factor VIII complex is an acute phase reactant in guinea-pig and that this may be due to increased synthesis of vWF:Ag by vascular endothelium in the spleen. Although BCG inoculation may have stimulated synthesis or storage of vWF:Ag in the spleen it did not have an appreciable effect on the plasma concentration of either VIII:Ag or vWF:Ag.

Animals↗
PubMed1985

Purification of human factor VIII:C and its characterization by Western blotting using monoclonal antibodies.

Human factor VIII:C has been purified over 300 000-fold from cryoprecipitate by polyelectrolyte purification followed by affinity chromatography on Sepharose linked to antibody to factor VIIIR:Ag (monoclonal or polyclonal) and Sepharose linked to monoclonal antibody to factor VIII:C. The purified material has been analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using monoclonal antibodies. PAGE shows predominant bands at 360K (unreduced), 210K, and 90K and an 80K/79K doublet; Western blotting showed all the monoclonal antibodies used bound the 360K form. In a small-scale purification, plasma from blood taken directly into thrombin inhibitor Kabi S-2581 was applied directly to the monoclonal anti-factor VIII:C column. Western blot analysis of this material showed the 360K band on reduction. The purified factor VIII:C could be activated 13-fold by human thrombin. Gel analysis of the activated material showed intensification followed by fading of the band at 90K and generation of bands at 70K/69K, 55K, and 40K. Western blotting shows that the 70K/69K doublet derives from the 80K/79K moiety and the 40K peptide derives from the 90K and is presumed to contain the active site. From these studies an epitope map of the factor VIII:C molecule has been constructed.

Antibodies, Monoclonal↗
PubMed1985

The effect of liver disease on factors V, VIII and protein C.

The components of the factor VIII complex were estimated by immuno- and bioassays in 85 patients with liver disease. The plasma concentrations of the antigens were elevated in 65% (VIII:CAg) and in 76% (VIIIR:Ag) of patients while the biological activities were elevated in only 14% (VIII:C) and 15% (VIII:RiCof). There was no correlation with C-reactive protein, used as a measure of an acute phase reaction (X2 = 0.7; P = 0.1); or with severity of liver disease as judged by prothrombin ratio (P = 1.0) but highest values were observed in patients with cholestatic liver disease. Following parenteral vitamin K there was a significant fall in both the biological activity of VIIIC (36%) and of VIII:CAg (38%) in 13 vitamin K deficient patients (P less than 0.001) but no change in 23 vitamin K replete patients or in the VIIIR:Ag levels in either group. Factor V levels were lower in patients with parenchymal liver disease (0.54 +/- 0.1 units/ml, mean +/- SEM, n = 12; normal range 0.5-1.5 units/ml) than in patients with extrahepatic cholestasis who were vitamin K deficient (1.2 +/- 0.1 units/ml, P less than 0.0001). The levels of protein C antigen, the vitamin K dependent protease which inactivates factors VIII:C and V, was at the lower end of the range in both groups (0.7 +/- 0.1, mean +/- SEM, n = 18, normal range 0.74-1.4 units/ml). There was no significant change in either protein C antigen or factor V following vitamin K. The discrepancy between the biological activity of factor VIII and the antigen levels could represent accumulation of partially degraded factor VIII or production of a hypoactive form. There is no evidence that the reduction in VIIIC and VIII:CAg following vitamin K was mediated by protein C.

Adolescent↗
PubMed1985

An immunoradiometric assay for human factor VIII/von Willebrand factor (VIII:vWF) using a monoclonal antibody that defines a functional epitope.

A murine monoclonal antibody has been produced (RFF-VIII:R/2) that binds specifically to human factor VIII-related antigen (VIII:RAg) in plasma and in vascular endothelial cells but has no reactivity with factor VIII procoagulant antigen (VIII:cAg). This antibody is a potent inhibitor of von Willebrand factor activity (VIII:vWF) in that it can totally neutralize ristocetin-induced aggregation of platelet rich plasma and inhibit platelet adhesion at high flow rates. RFF-VIII:R/2 can be used in a one-stage, fluid phase immunoradiometric assay that can detect VIII:RAg at concentrations of 0.001 u/ml. This method has been used to analyse plasma from patients with von Willebrand's disease (vWD). Results obtained in these patients showed a high degree of correlation between the monoclonally-defined epitope and VIII:vWF levels measured by ristocetin-induced aggregation of washed platelets. This correlation was maintained in those patients with the 'variant' types of vWD who exhibit highly disparate VIII:vWF and VIII:RAg levels when the latter is determined using polyclonal antisera. It appears that this monoclonal antibody recognizes a site on the VIII:RAg molecule which is associated with its interaction with the platelet membrane. Immunoradiometric assays using RFF-VIII:R/2 offer a simplified, reproducible means of detecting functionally-active VIII:RAg as an alternative or supplement to techniques involving platelet interactions.

Animals↗
PubMed1985

Albinism with haemorrhagic diathesis: Hermansky-Pudlak syndrome.

Four cases of albinism with haemorrhagic diathesis (Hermansky-Pudlak syndrome) are presented. The cases displayed wide phenotypic variation. Electroretinography was performed on all four patients and was found to be normal. One patient developed a cutaneous malignant melanoma.

Adult↗
PubMed1985

Platelet size and shape in hereditary giant platelet syndromes on blood smear and in suspension: evidence for two types of abnormalities.

Platelet size on blood smear is compared with platelet size and shape in suspension (i.e., whole blood and citrated platelet-rich plasma [PRP]) for normal donors and 16 patients with hereditary "giant" platelet syndromes (HGPS), including Bernard-Soulier syndrome (BSS) (seven patients), Montreal platelet syndrome (MPS) (three patients), May-Hegglin anomaly (one patient) and Rafael platelet defect (one patient). In whole blood platelet shape is normal for HGPS, but in PRP for 10 of 16 patients with HGPS there is a decrease in the proportion of smooth, discoid-shaped platelets (discocytes [D]). The platelets of all patients with HGPS had abnormally large mean volume (VT) and increased size on peripheral blood smear. Furthermore, 12 of 16 patients with HGPS, including six of seven donors with BSS, had abnormally large discocytes. The measured size of HGPS shape-changed platelets was compared with the size predicted from the size of the D by assuming that the relationship between the size of shape-changed platelets and D was the same as observed for normal donors. In this manner it was shown that for all donors with BSS and MPS, the shape-changed platelets are disproportionately larger than the D. In contrast, in the remaining patients with HGPS the size of the shape-changed platelets was consistent with the size predicted from the D. Examination of VT for MPS as a function of time after addition of 10 mumol/L adenosine diphosphate to PRP revealed an abnormal time course, thereby pointing to an abnormality in the mechanisms that regulate platelet size during shape change. With the lone exceptions of BSS and MPS, the size of platelets on blood smear was well correlated with the total platelet plasma membrane surface area as measured by the osmotic spherocyte method. Our observations point to two distinct abnormalities in platelet size in HGPS: a disproportion between the size of D and "shape-changed" platelets, which may be related to an abnormal shape change and which is observed only for MPS and BSS, and an abnormal increase in platelet size on blood smear, which appears to reflect the increased amount of platelet plasma membrane in other HGPS platelets.

Adenosine Diphosphate↗
PubMed1984

Disappearance of antibodies to Factor VIII in a patient with acquired haemophilia and carcinoma of the pancreas during cytotoxic therapy with fluorouracil and CCNU.

An 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.

Adenocarcinoma↗