Investigation of a link between raised levels of pepsinogen in blood as a mediator of in-vitro clot lysis in acid and a cause of abnormal factor XIII screening tests.

The in-vitro lysis of plasma clots in acetic acid generally indicates a Factor XIII deficiency that is confirmed by quantitative assay. However, there are two rare, poorly understood circumstances whereby clot lysis in acid occurs when factor XIII activity levels are normal: the presence of either an atypical antifactor XIII antibody, or an unknown acid-activated protease. Our centre has identified four patients with in-vitro clot lysis in acetic acid and normal FXIII levels by activity assay. Our aim was to determine whether the cause of this unusual result was an inhibitory antibody or an aspartic acid protease. In each case, we found an inhibitor that was not an IgG but showed characteristics of an acid protease, including that it was neutralized by pepstatin. The four patients had median pepsinogen I levels five-fold to 10-fold higher than the normal median of 89 µg/l. Pepsinogen II was increased by three-fold to six-fold, but from a lower baseline median of 6.5 µg/l. Cathepsin D levels were normal. Clot lysis in the acid test was observed when recombinant human pepsinogen I was added to normal plasma at similarly high concentrations as in patient samples, consistent with a role of an acid protease. Clot lysis also occurred with addition of pepsinogen II, but required four-fold to seven-fold more than in patient samples. Laboratories should be aware that a positive acid clot lysis test can be misleading if pepsinogen levels are raised and should not use this alone to diagnose FXIII deficiency.

Partial and severe factor XI deficiency in South Australia and the usefulness of factor XI mutation analysis for diagnosis.

AIMS: To correlate the presence or absence of a factor XI gene mutation with factor XI activity in patients with severe or partial reduction in factor XI. METHODS: Patients previously found to have reduced factor XI levels were recalled for repeat testing and factor XI genetic analysis. Also, during the 18 month study period, any routine patient found to have an isolated reduced or low normal factor XI level had factor XI genetic analysis. RESULTS: Twenty-two cases were studied and 11 with factor XI from <2 to 57 U/dL (reference 55-130 U/dL), were found to have a factor XI gene mutation. Gene sequencing identified 15 different mutations, with four patients found to be compound heterozygotes. One patient with no bleeding history had a novel polymorphism which family studies showed was not associated with his low factor XI. No factor XI gene abnormality was detected in 10 patients and they have either acquired causes of deficiency or factor XI levels in the lower portion of the normal range. CONCLUSION: Genetic analysis of the factor XI gene is important to confirm or exclude inherited causes of factor XI deficiency, especially when the reduction is mild.

In vitro kinetics of factor VIII activity in patients with mild haemophilia A and a discrepancy between one-stage and two-stage factor VIII assay results.

In some mild haemophilia A patients (discrepant haemophilia), factor VIII coagulant activity (FVIII:C) levels, by one-stage assay are more than double than those by two-stage assay. This may be due to the longer incubation times (10-12 min) in the two-stage assay. This study aimed to determine the time course of the activation phase of the two-stage assay, using both classical coagulation and chromogenic detection methods. In both systems, for equivalent patients (equivalent FVIII:C levels by one-stage and two-stage assays, n = 6, all different mutations), similar FVIII:C results were obtained with short- or long-incubation times. In contrast, plasma from discrepant patients (n = 8, five different mutations) showed higher FVIII:C at shorter incubation times than after longer incubation times. In the chromogenic assay, FVIII:C levels were higher after incubation for 2 min (23-56%, mean 41%) than after 10 min (19-41%, mean 29%). In the classical coagulation assay, FVIII:C levels were higher at shorter incubation times (21-64%, mean 37%) than with the longer incubation times usually used (13-29%, mean 23%). These time-course experiments have verified that the longer incubation time used in the two-stage assay is at least partly responsible for the lower FVIII:C measured by that assay in discrepant haemophilia.

Acquired isolated factor VII deficiency associated with severe bleeding and successful treatment with recombinant FVIIa (NovoSeven).

Acquired isolated FVII deficiency not due to vitamin K deficiency or liver disease is rare and often associated with severe bleeding. We present a case of transient acquired factor VII deficiency associated with major bleeding, successfully treated with twice daily intermittent intravenous recombinant activated factor VII (rFVIIa) (NovoSeven; Novo Nordisk). The severe transient reduction in factor VII coagulant activity (FVII:C) levels, unresponsive to fresh frozen plasma and vitamin K administration, raise the possibility of an acquired inhibitor to factor VII. However, no inhibitor to factor VII could be demonstrated using protein G sepharose adsorption, or a Bethesda assay using IgG purified from patient plasma. There are few reports of the use of rFVIIa in this setting and this case suggests that rFVIIa is effective therapy, and should be considered early when acquired factor VII deficiency is associated with severe bleeding.

Identification of von Willebrand disease type 2N (Normandy) in Australia: a cross-laboratory investigation using different methods.

We report on a cross-laboratory study of type 2N von Willebrand disease (vWD). We tested 101 selected plasma samples for factor VIII and factor VIII binding activity of von Willebrand factor (vWF). Of these plasma samples, 31 were cotested by 2 specialist centers using different detection procedures for vWF-factor VIII binding: there was good agreement between results obtained by chromogenic assay and enzyme-linked immunosorbent assay. In total, 8 patients with type 2N vWD were identified. The 2-stage factor VIII assay detected a deficiency of factor VIII relative to vWF antigen in all 8 patients; the 1-stage factor VIII assay detected a relative deficiency in only 3 patients. Four patients were homozygous for the most common type 2N mutation (R854Q), 3 patients were presumed to be compound heterozygotes, and in 1 patient no type 2N mutations were identified. In this study of patients from 5 specialist centers in Australia, type 2N vWD was found in 5 families. The 2-stage factor VIII assay was more useful as a screening test than the 1-stage assay, and both vWF-factor VIII binding assays were equally effective.

Recombinant factor VIIa: a universal hemostatic agent?

Recombinant factor VIIa (rVIIa) has proved effective for the treatment and prevention of hemorrhage in patients with inherited hemophilia A and B who develop inhibitors to factor VIII or IX, and patients with acquired hemophilia A. More recently, there is evidence that rVIIa may also be effective in the control of abnormal bleeding in a variety of other conditions, such as inherited factor VII deficiency, thrombocytopenia, Glanzmann's thrombasthenia, and liver disease. In some of the reports, rVIIa appeared to be effective in controlling massive hemorrhage in which there was no response to conventional measures. It is now considered by some to be potentially the first universal hemostatic agent. However, further prospective, controlled, and adequately powered clinical studies are clearly required. It will be of particular interest to determine the efficacy of rVIIa in conditions such as severe thrombocytopenia, severe von Willebrand disease, severe defects in platelet activation, and severe deficiencies of factors V, X, II, and fibrinogen in which effectiveness would seem to be unlikely based on our current understanding of mechanisms of action of rVIIa.