A Simple Method for the Assay of Factor VIII

A simple method for the assay of factor VIII activity, based on the thromboplastin generation test of Biggs and Douglas, has been described.

Bovine serum is used as a source of factor V and serum factors, and plasmafrom a hemophiliac is not required. The concentration of factor VIII of thetest plasma is compared to that of a standard normal human plasma. Theresults obtained with the method have been fairly reproducible.

     

Nature of von Willebrand Factor: A New Assay and a Specific Inhibitor

Platelet-active "von Willebrand factor" is a poorly characterized activity of a plasma-protein macromolecular complex. A new simple assay for von Willebrand factor is based on the dose response relation of the factor and the ristocetin platelet aggregation time. This assay uses the "snowstorm" macroscopic endpoint. A multiply transfused subject with von Willebrand's disease was observed to have a circulating inhibitor that blocks normal ristocetin aggregation of platelets, but not ADP-, epinephrine-, or collagen-induced aggregation. The inhibitor was not adsorbed by normal platelets, and was stable to heating at 56 degrees for 30 min and to repeated freezings and thawings. This inhibitor also prevents action on human platelets by platelet-aggregating factor of bovine plasma, indicating this bovine factor activity is a function of von Willebrand factor. Three inhibitors were compared: (1) the von Willebrand factor inhibitor specifically blocked von Willebrand factor activity, (2) a human antibody from a hemophiliac inhibited only antihemophilic factor activity, and (3) a rabbit antiserum to a preparation of human antihemophilic factor inhibited both activities. The active site for von Willebrand factor on the macromolecular complex appears to be spaced some distance from the antihemophilic factor site.     

Identification of Sources of Inter-Laboratory Variation in Factor VIII Assay

S ummary . A repeated finding of national and international collaborative studies of standard factor VIII preparations has been that systematic differences exist between laboratories in their measurement of the relative activities of the same pairs of factor VIII preparations.
A workshop meeting was held at the Oxford Haemophilia Centre during 23–26 November 1976 to investigate which of the possible sources of variation between laboratories were responsible. Participants from 16 British laboratories (nine using one-stage assays and seven using two-stage assays) performed a total of 273 valid assays using three freeze-dried preparations of differing purity (a plasma, an intermediate and a high purity concentrate). The results of assay with each participant using their normal system established that, if the participants were a representative cross-section, approximately one-third of one-stage laboratories would show a systematic difference from the overall mean of at least 16% with a corresponding figure for the two-stage laboratories of 9%. Various features of the assay systems were then modified in a controlled series of experiments. The results showed conclusively that (i) differences between reagents accounted for most of the variation between laboratories, and (ii) the two-stage assays were, on average, detecting relatively more activity in the more purified preparations than the one-stage assays. The results also suggested that the use of buffer or citrate-saline as opposed to haemophilic plasma for the initial dilution of concentrates did not affect the assay results.     

Evaluation of a rapid von Willebrand Factor Activity Latex Immuno Assay for monitoring of patients with von Willebrand disease (VWD) receiving DDAVP® or VWF replacement therapy

Haemostatic management of surgery in patients with von Willebrand disease (VWD) includes DDAVP^® or von Willebrand factor (VWF)‐containing concentrates. Although the recommendations are for monitoring by VWF activity assays, it is quite common for clinicians to use factor VIII due usually to longer turnaround times required for VWF ristocetin cofactor assay (VWF:RCo) measurements. The aim of this study was to evaluate use of the rapid HaemosIL^? VWF activity (VWF:Act) latex immuno assay (LIA) on an automated coagulometer (ACL TOP^? 700; Instrumentation Laboratory, Bedford, MA, USA) compared to platelet‐based VWF:RCo assays in this setting. One hundred and sixty‐seven plasma samples from 42 patients [Type 1 (n = 22), Type 2A (n = 2), Type 2B (n = 3), Type 2M (n = 10), Type 3 (n = 3)] and acquired von Willebrand syndrome (n = 2) with VWD treated with DDAVP^® or VWF‐containing concentrates were included in the study. Method comparison and method bias were evaluated by Bland–Altman analysis (BA) and Passing and Bablok regression modelling respectively. BA of baseline samples (n = 39) showed a mean difference of ?3.0 (±1.96 SD ?25.2 to +19.4). Post (treatment) samples (n = 120) were separated into two groups. Group 1 contained samples with VWF:RCo levels 10 to ≤175 IU dL^?1 (n = 97) and group 2, samples with VWF:RCo levels >175 IU dL^?1 (n = 23). BA of group 1 postsamples showed a mean difference of +3.4 (±1.96 SD ?44.6 to +51.5), and the BA of Group 2 samples was ?23.9 (±1.96 SD ?136.1 to +88.3). In conclusion, use of HaemosIL VWF:Act LIA test on an automated coagulometer is a reproducible and rapid assay that can be used as an alternative test for monitoring VWF replacement therapy, facilitating dose adjustments on a real‐time basis.     

A Simple Method for the Assay of Factor VIII, Using 20 Microlitres of Capillary Blood

S ummary A simple and sensitive method for Factor-VIII assay is presented which permits a reproducible quantitative assay on 20μl. capillary blood.     

The Use of a Double-Isotope Derivative Plasma Assay for Noradrenaline and Adrenaline in the Diagnosis and Localisation of Phaeochromocytoma

A sensitive double-isotope derivative plasma assay has been used to measure total plasma catecholamines, noradrenaline (NA) and adrenaline (A) in patients with phaeochromocytoma and essential hypertension. Plasma samples containing as little as 0.5 ng of NA or A could be measured accurately. All nine patients with phaeochromocytoma had significant elevations in either total plasma catecholamines (p<.02) or plasma NA (p<.001). Separation of NA and A with less than 0.3% cross contamination was carried out by thin layer chromatography. This represents a substantial improvement over other methods of assay and raises the possibility that differential plasma assay of NA and A will be of value in diagnosing extra-adrenal tumours. Differential plasma NA and A levels were measured in five patients with phaeochromocytoma. Four of these had elevated NA and A levels and were later found to have adrenal tumours. The other patient had a mediastinal tumour and no measurable plasma adrenaline.     

The Separation of Willebrand Factor from Factor VIII-Related Antigen

The three activities associated with factor VIII--coagulant (VIII:C), antigenic (VIIIR:Ag), and platelet agglutinating or Willebrand factor (VIIIR:WF)--have been separated by sequential antibody affinity chromatography, utilizing a rabbit antibody to factor VIII and a spontaneous human antibody to VIII:C. Normal plasma differentially lost its factor VIII-related antigen following passage over the rabbit antibody column. Subsequent passage of the VIIIR:Ag-depleted plasma over the human antibody column resulted in the loss of VIII:C activity, with retention of the Willebrand factor activity, antigen being partially recovered from the heterologous antibody column. These experiments demonstrate that it is possible to separate two of the factor VIII activities, VIIIR:Ag and VIIIR:WF, which are usually regarded as properties of a single molecule.     

Use of Macrophage Inhibition Factor and Mast-Cell Degranulation Tests for Diagnosis of Cloxacillin-Induced Cholestasis

Cloxacillin-induced cholestasis was diagnosed with the help of the macrophage inhibition factor and mast-cell degranulation tests. The simultaneous occurrence of both immediate type as well as cell-mediated hypersensitivity to the drug suggested that a defect in the histamine-induced suppressor cells may underly this cloxacillin-induced allergic reaction.     

胶体金免疫层析法快速诊断鼠疫的实验研究

目的建立一种快速、简易的检测鼠疫F1抗原的胶体金免疫层析法(GICA).方法采用直径为20nm胶体金颗粒,标记F1单克隆抗体,并将标记物以6μL/cm喷于玻璃纤维;将另1株F1单克隆抗体与羊抗鼠IgG以1μL/cm喷线固定于硝酸纤维素膜上,装配后以4mm/条切割制成免疫层析检测试条.分别用粗制F1抗原和EV菌对该试纸条的敏感性进行测定,同时用44株鼠疫近缘菌及昆明小鼠、黄胸鼠、褐家鼠、大足鼠的正常血清等标本进行特异性检测.结果本试纸条可在15min之内完成实验,检测粗制F1抗原的浓度可达0.5ng/mL,最小检出EV菌菌量为10万个;本方法的特异性为100%.结论 GICA检测鼠疫F1抗原特异性强、灵敏度高、简便快速,无需特殊仪器设备,有广泛应用价值.     

Diagnosis of von Willebrand Disease

The haemorrhagic diathesis in von Willebrand disease (vWD) is caused by a quantitative deficiency or a qualitative defect in the von Willebrand factor (vWF) in plasma and/or platelets causing insufficient primary haemostasis. Since vWF binds and protects factor VIII (FVIII) towards random proteolysis, coagulation may also be impaired in patients with a low plasma level of vWF, and in instances where vWF displays insufficient binding capacity to FVIII. The entity of vWD displays a vast heterogeneity. Apart from rarely occurring acquired cases, vWD is an inherited disorder of autosomal linkage. The major clinical hallmark in vWD is an increased tendency to mucocutaneous bleeding that rarely reach life-threatening proportions, unless vWF is severely reduced or completely absent. Increased bleeding may also occur in sites such as muscles and joints when the level of FVIII is particularly low.
Significant progress has recently been achieved through extensive molecular genetic exploration of various forms of vWD. In order to guide treatment and to form a platform for genetic investigation, however, accuracy in diagnosis and phenotypic characterization is important. By means of various laboratory methods, major subclasses of vWD can be differentiated, as presented in another article of this series. Whereas most of the cases of vWD can quite easily be diagnosed and classified using today's diagnostic methods, the most frequently occurring bleeding disorder of all, vWd type 1 of mild degree, continues to challenge clinicians and diagnostic laboratories. The aim of this paper is to review the laboratory methods most commonly used in diagnostic investigation of the patient suspected of vWD.     

假性血友病因子与冠心病

冠心病的发病机理目前还不能确知 ,但大多数观点认为 ,冠心病尤其是不稳定型心绞痛和心肌梗死的发生与血管内皮细胞受损、血栓形成有关。近年研究发现血管内皮细胞除起机械屏障作用外 ,还分泌许多重要生物活性物质 ,如前列环素 (PGI2 )、内皮衍生舒张因子 (EDRF)、假性血友病因子 (v WF) ,这些活性物质对调节血管张力、凝血纤溶功能 ,以及维持正常血压及血流动力学均有一定的保护作用。一旦内皮细胞受损 ,则可合成释放 v WF,该因子释放入血液可引起血小板粘附聚集 ,促发血栓形成。1　概述粘附蛋白是细胞粘附糖蛋白家族 ,其种类繁多 ,多…     

Diagnosis of von Willebrand disease

Summary. von Willebrand disease (vWD) is a bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (vWF). vWF is synthesized by endothelial cells and megakaryocytes and circulates in plasma as a multimeric high molecular weight glycoprotein. vWF plays a major role in the early phases of ostasis by promoting platelet-vessel wall and plateletplatelet interactions under high shear conditions. It is also the carrier of coagulation factor VIII (FVIII) in plasma. A deficiency of vWF results in impairment of both primary and secondary phases of ostasis. Therefore, patients with vWD manifest bleeding symptoms that are typical of defects of primary ostasis (mucocutaneous haemorrhages) but, in case of severe deficiency of vWF, there are also haemarthroses and haematomas, which are typical of those seen with coagulation defects. Several types and subtypes of vWD have been described with a high degree of heterogeneity. The diagnosis is based on measurements of plasma and platelet vWF, the ability of vWF to interact with its platelet receptor and the analysis of the multimeric composition of vWF. Due to the heterogeneity of vWF defects, a correct diagnosis of types and subtypes may be sometimes difficult but is very important for an appropriate treatment of patients with vWD.     

Identification of a New Badnavirus in the Chinaberry (Melia azedarach) Tree and Establishment of a LAMP-LFD Assay for Its Rapid and Visual Detection

The Chinaberry tree, a member of the Meliaceae family, is cultivated in China for use in traditional medicines. In 2020, Chinaberry trees with leaf deformation symptoms were found in Hangzhou, Zhejiang province, China. In order to identify possible pathogenic viruses, a symptomatic sample was subjected to deep sequencing of small interfering RNAs. Assembly of the resulting sequences led to the identification of a novel badnavirus, provisionally designated Chinaberry tree badnavirus 1 (ChTBV1). With the recent development of China’s seedling industry and increasing online shopping platforms, the risk of tree virus transmission has increased substantially. Therefore, it is important to detect the occurrence of ChTBV1 to ensure the safety of the Chinaberry tree seedling industry. Here, we describe the development and validation of a sensitive and robust method relying on a loop-mediated isothermal amplification (LAMP) assay, targeting a 197 nt region, to detect ChTBV1 from Chinaberry tree leaves. The LAMP assay was also adapted for rapid visualization of results by a lateral flow dipstick chromatographic detection method.     

Diagnosis and management of von Willebrand disease

von Willebrand disease (vWD) is a bleeding disorder caused by quantitative or qualitative defects of von Willebrand factor (vWF). The diagnosis is based on measurements of plasma and platelet vWF, the ability of vWF to interact with its platelet receptor and the analysis of the mutlimeric composition of vWF. Due to the heterogeneity of vWF defects, a correct diagnosis of types and subtypes may be sometimes difficult but is very important for an appropriate therapy. The aim of treatment is to correct the dual defects of haemostasis, i.e. abnormal coagulation expressed by low levels of factor VIII (FVIII) and abnormal platelet adhesion expressed by a prolonged bleeding time (BT). Desmopressin is the treatment of choice in patients with type 1 vWD, who account for approximately 80% of cases, because it corrects the FVIII/vWF levels and the prolonged BT in most of these patients. In type 3 and in the majority of type 2 vWD patients, desmopressin is not effective and it is necessary to resort to plasma concen-trates containing FVIII and vWF. Treated with virucidal methods, these concentrates are effective and currently safe, but the BT defect is not always corrected by them. Platelet concentrates or desmopressin can be used as adjunctive treatments when poor correction of the BT after concentrates is associated with continued bleeding.     

von Willebrand Factor: More Than a Regulator of Hemostasis and Thrombosis

von Willebrand factor (vWF) was first identified as an adhesive glycoprotein involved in hemostasis by Zimmermann in 1971. Since then, vWF has been shown to play a vital role in platelet adhesion, platelet binding to collagen and factor VIII protection. Recent studies have implicated vWF as a regulator of angiogenesis, smooth muscle cell proliferation, tumor cell metastasis and crosstalk in the immune system. In this review, we will discuss the aspects of vWF structure that facilitate its biological effects and speculate on its newly discovered and hypothesized roles in the pathogenesis of several diseases.     

Ultracentrifugal Analysis of Factor VIII and von Willebrand Factor in Therapeutic Preparations

Plasma and therapeutic preparations of factor VIII (1 recombinant factor VIII and two monoclonally purified plasma-derived factor VIII preparations, Kogenate, and AHF-M and Monoclate, respectively) were centrifuged in a sucrose density gradient, and the fractions were analyzed for factor VIII and von Willebrand factor (vWF). The residual vWF in the monoclonally purified factor VIII preparations sediments more slowly than the vWF of plasma. In the absence of added vWF, the factor VIII in all preparations sediments more slowly than plasma factor VIII. These same preparations of factor VIII added to hemophilic plasma as a source of vWF sediment differently. The addition of either recombinant factor VIII or AHF-M results in sedimentation of the factor VIII with the plasma vFW and in a position indistinguishable from factor VIII in plasma. In contrast, when Monoclate is added to hemophilic plasma in vitro, the factor VIII sediments more slowly than the vWF of the hemophilic plasma. However, 5 min after the infusion of Monoclate into a patient with hemophilia A, the factor VIII sediments with the plasma vWF. These results indicate that the addition of recombinant factor VIII and AHF-M results in random binding to all vWF multimers of plasma, while there is little exchange between the added factor VIII in Monoclate and the plasma vWF in vitro. In contrast, when the Monoclate is infused, there is rapid binding of factor VIII to the plasma vWF. This is presumably due to tight binding of the factor VIII to the vWF in Monoclate, whereas the factor VIII in Kogenate and AHF-M, containing no or very low amounts of vWF, are free to bind to plasma vWF. In vivo there are other conditions, as yet uninvestigated, which allow for the binding of factor VIII to vWF.     

von Willebrand因子与稳定冠心病

早期大量研究揭示了von Willebrand因子的生物学特点、结构和功能,近期研究发现von Willebrand因子可能在稳定冠心病发病、剪切力机制诱导血小板聚集、血栓形成,以及药物和介入治疗(包括支架置入等)方面有重要作用。