Simple and rapid detection of factor V Leiden by allele-specific PCR amplification.

Resistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.

The effect of methylene blue phototreatment on plasma proteins and in vitro coagulation capability of single-donor fresh-frozen plasma.

BACKGROUND: Photodynamic virus inactivation of fresh-frozen plasma (FFP) may result in its impaired coagulation capability. STUDY DESIGN AND METHODS: Double-volume plasmapheresis samples from 11 donors were divided in pairs of 250 mL. One group underwent methylene blue (MB) phototreatment (MB-FFP). The other group was treated according to the standards of the American Association of Blood Banks for preparation and storage of FFP. Parameters of hemostasis and clinically important plasma proteins were tested in native plasma, thawed MB-FFP, thawed FFP, and twice-frozen and thawed FFP (FFP-II). RESULTS: Mean activities of factor V (73.4 vs. 94.5%; p < 0.01), factor VIII (58.1 vs. 86.7%; p < 0.001), and fibrinogen (1.8 vs. 2.8 g/L; p < 0.001) were reduced in MB-FFP as compared to those in FFP. The comparison of MB-FFP to FFP-II revealed reduced activities of factor VIII (58.1 vs. 85.2%; p < 0.001) and fibrinogen (1.8 vs. 2.8 g/L; p < 0.001) but no changes in factor V. Activated partial thromboplastin time in MB-FFP was prolonged beyond the upper normal range (+5.3 sec; p < 0.001) and prothrombin time increased in MB-FFP versus FFP (+0.96 sec; p < 0.001). CONCLUSION: MB phototreatment reduces the in vitro coagulation capacity of FFP, most likely as a result of the effects of an additional freezing and thawing procedure and photooxidation-induced protein damage.

Retrospective statistical analysis of coagulation parameters after 250 liver transplantations.

Extensive transfusion of blood components as a typical feature of OLT has been described by many authors. The perioperative requirement for transfusion, however, follows a downward trend, although the indications for OLT have been extended. Functional disturbances such as hyperfibrinolysis or platelet dysfunction, demonstrated by laboratory tests, such as platelet counts, PT, aPTT, TT, fibrinogen, and ATIII are often used to direct the transfusion of blood components, although preoperative data give insight only into the insufficient function of the old liver, which will be explanted and replaced by the donor graft. We have described a retrospective statistical analysis of laboratory data, clinical data, and perioperative blood requirements from 250 liver transplantations performed in our hospital from 1988 to 1992. The OLT was performed according to the usual surgical methods using a venovenous bypass system. Intraoperatively, volume was restored with packed RBC and FFP according to hemodynamic data, hemoglobin, and diuresis; clotting data were not used as indications for blood replacement. This analysis demonstrated that neither preoperative nor intraoperative clotting parameters were able to allow a prediction of the intraoperative requirement for transfusion of blood components; these findings parallel those of previous reports. With respect to other studies showing that perioperative transfusion rates correlated with postoperative infections and mortality, we strongly suggest that perioperative clotting measures as indicators for transfusion requirement should be analyzed with caution. Whether other diagnostic methods such as TEG are useful alternatives has to be determined.

Successful treatment of a patient with postpartum factor VIII inhibitor with recombinant human interferon alpha 2a.

A patient who had developed a postpartum inhibitor to factor VIII and who did not respond to repeated therapy with steroid and high-dose intravenous gammaglobulin G was treated with one short course of low-dose recombinant interferon alpha 2a (rhIFN) s.c. Within 7 weeks from the start of rhIFN treatment, the inhibitor disappeared. It remains undetectable 8 months after therapy.

[Homozygous infant in a family with hereditary protein C deficiency]. / Homozygoter Säugling in einer Sippe mit erblichem Protein C-Mangel.

We report on a female infant homozygous for protein C deficiency in a Jordanian family with frequent intermarriage. A protein C antigen of 0.6% was determined. The parents first noticed painful nodular indurations in subcutanous tissue as well as blue-red skin coloration at the age of 6 months. The girl repeatedly suffered from microthrombotic events in parts of the body with large areas of subcutaneous fat. In contrast, the numerous heterozygous carriers with partial protein C deficiency did not show an increased tendency to thrombosis. From the history an autosomal-recessive inheritance may be inferred. Other authors reporting on homozygous cases also postulate the presence of a recessive gene. It is of interest that the infant described here differs from those in other case reports in the age at manifestation of the disease. The homozygous infant showed the first symptoms as late as the age of 6 months, whereas other case reports describe severe symptoms immediately after birth. All symptoms of disease were treated successfully with prothrombin complex concentrate without additional heparin protection. Microthrombotic events subsided quickly, and a large ulcer in the left flank healed almost completely within 6 days.

[Determination of factor VIII-coagulation antigen in factor VIII concentrates using an inhibitor test]. / Bestimmung von Faktor VIII-Gerinnungsantigen in Faktor VIII-Konzentraten mit dem Inhibitor-Test.

The method of determination of factor VIII coagulation antigen (VIIICAg) here described is based on the well established factor VIII inhibitor test. Highly concentrated inhibitors to factor VIII in factor VIII substituted hemophiliacs are the most frequent among inhibitors to clotting factors. If such an inhibitor is used in an inhibitor assay its reaction depends on the VIIICAg concentration in the test system. The slope of the reaction curve is proportional to VIIICAg if the inhibitor titer is known. VIIICAg was measured in one cryoprecipitate and 5 different factor VIII concentrates of medium to high purity using this assay. In addition factor VIII related antigen and factor VIII ristocetin cofactor were determined by routine methods. VIIICAg fairly corresponded to factor VIII coagulation activity. Only cryoprecipitate contained significantly more VIIICAg.

[Different proportions of factor VIII and factor VIII inhibitors in their antigen-antibody complexes]. / Unterschiedliche Proportionen von Faktor VIII und Faktor VIII-Inhibitoren in ihren Antigen-Antikörper-Komplexen.

We examined the physico-chemical properties of factor VIII inhibitors in two patients. There is no stoichiometric mixture of factor VIII and factor VIII inhibitor since one polyvalent factor VIII particle can be bound by various numbers of factor VIII inhibitor particles. The balance between free factor VIII and inhibitor and their antigen-antibody complexes cannot be explained by the homogeneous natural law of mass action. A patient with classical hemophilia A exhibited an inhibitor which shows conformity with the Poisson distribution as far as the portion of free factor VIII activity is concerned. The spontaneously occurring inhibitor showed a different binding characteristic to factor VIII. We demonstrated here that the Freundlich's adsorption isotherme is effective for a spontaneous factor VIII inhibitor. During follow up qualitative and quantitative changes of both inhibitor types were observed. We assume that the change of property of inhibitor in hemophiliacs is due to a stronger binding to factor VIII and of the spontaneous inhibitor to a poorer fit of antigen and antibody.

Venus: density of upper atmosphere from measurements of drag on pioneer orbiter.

Measurements of the changes in orbital period of the Pioneer Venus orbiter have yielded estimates of the density of the upper atmosphere of Venus at altitudes in the range from 150 to 200 kilometers. At the lower limit of this range, the density on the dayside of the terminator exhibits a temporal variation of amplitude near 4 x 10(-14) gram per cubic centimeter aboult a mean of approximately 1.4 x 10(-13) gram per cubic centimeter. The variation appears oscillatory, with a 4- to 5-day period, but barely one cycle was observed. The density on the nightside of the terminator, sampled inthe same 150-kilometer altitude range, fluctuates about a smaller mean of approximately 4 x 10(-14) gram per cubic centimeter. The density between the altitudes of 150 and 200 kilometers, sampled only on the dayside of the terminator, imply a scale height of between 15 and 20 kilometers. The interpretation of this estimate is uncertain, however, in view of the measurements at the different altitudes having been made at different times and, hence, at different values of solar phase.

[Current aspects on evaluation of assays of factor-VIII activity, factor-VIII associated protein and factor-VIII neutralizing antibody (author's transl)]. / Aktuelle Gesichtspunkte zur Auswertung der Bestimmung von Faktor VIII-Aktivität, Faktor VIII-assoziiertem Protein und Faktor VIII-neutralisierendem Antikörper.

In evaluating factor-VIII activity it should be noted that regarding the remaining activity of deficient plasma a linear reference curve is achieved. In the standard population factor-VIII activity and factor-VIII associated protein are distributed approximately lognormally. Due to this distribution certain results have been gained for the optimal choice of localisation and dispersion measures. It is assumed that the proportions of neutralized factor-VIII activity in plasma are distributed according to Poisson. The applicability of the Poisson distribution was also proved for the free factor-VIII activity portion. Due to the Poisson distribution the antibody unit is clearly defined, thus eliminating a further discussion on the establishment of an arbitrary standardized antibody unit.