Search

Accidental exposure to blood-borne pathogens by contaminated needlestick puncture is a continuing problem for health care workers. We established an infection control center where infection control and follow-up programs are carried out for employees who suffer from accidental needlestick puncture. We analyzed 1062 needlestick accidents that occurred during an 8-year period from May 1990 to April 1998. Of these, 461 (43.4%) occurred in nurses and 392 (36.9%) in doctors; further, 668 (62.9%) occurred during normal needle use while 170 (16%) occurred during recapping. Needles contaminated with blood containing hepatitis C virus (HCV) accounted for 259 (24.4%) accidents. From these 259 incidents, two employees were infected with HCV and suffered acute hepatitis. Accidental exposure to contaminated blood was more likely between May and October and less likely between November and February.

The association of factor VIII with von Willebrand factor (vWF) is important for protection of factor VIII against proteolytic degradation in plasma. Recently developed factor VIII preparations, namely monoclonal antibody-purified factor VIII (M-factor VIII) and recombinant factor VIII, contain free form of factor VIII that is expected to form complexes with endogenous vWF after venous injection in the patients with hemophilia A. To clarify the difference of free factor VIII and factor VIII-vWF complex in the commercial factor VIII concentrates, we developed a quantitative assay for free factor VIII in which the sample was added to the wells of vWF-coated microtiter plates. Bound factor VIII was detected by monoclonal antibody against factor VIII, followed by incubation with peroxidase conjugated anti-mouse IgG, then the substrate ABTS for measuring absorbance at 405nm. When the amount of free factor VIII antigen in one unit factor VIII activity of recombinant factor VIII was defined as one arbitrary unit, the ELISA detected free factor VIII as low as 0.016 unit/ml. Factor VIII was incubated with vWF or plasma from a patient with severe Hemophilia A for various concentrations, prior to the free factor VIII assay. At saturation, the stoichiometry was one factor VIII molecule per 50vWF monomers. Of the products of factor VIII-vWF complex concentrate, no free factor VIII was detected. While a M-factor VIII concentrate contained free factor VIII comprising 24% of total factor VIII activity. In a gel filtration experiment of M-factor VIII, 15% of the total factor VIII was eluted as free factor VIII and 77% of that was co-eluted with vWF in the void volume. The free factor VIII and the complex form of factor VIII were immunoisolated and were analyzed after SDS-PAGE by immunoblotting. The factor VIII molecular structure and the susceptibility of thrombin cleavage were indistinguishable between the two forms of factor VIII. These results suggested that free factor VIII present in the M-factor VIII concentrate forms complexes with vWF in hemophilic plasma and is involved in physiologic hemostasis.

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

To determine the difference in reactivity of factor VIII (FVIII) inhibitor to FVIII/von Willebrand Factor (vWF) complex and FVIII free of vWF, an autoantibody to FVIII light chain was tested. A patient (1-3) suffered from autoimmune hemolytic anemia with autoantibody to FVIII. Epitope specificity of the patient's IgG (I-3 IgG) was shown to be the C2 domain of FVIII light chain (2170-2332) by Western blotting using recombinant FVIII deletions expressed in Escherichia coli. The inhibitory effect on FVIII procoagulant activity (VIII:C) of I-3 IgG was tested against a conventional FVIII concentrate; Haemate P, a monoclonal antibody-purified FVIII concentrate; Hemofil M, and a recombinant FVIII (rFVIII); Kogenate. I-3 IgG showed only 1.3 BU/mgIgG for Haemate P, in contrast to 20 BU/mgIgG for both Hemofil M and Kogenate. The ratio of VIII:C/vWF:Ag in Haemate P and Hemofil M was 1/3.43 and 1/0.01, respectively, while Kogenate did not contain vWF. The inhibitory effect of the I-3 IgG was then compared with Kogenate and its complex with vWF. The inhibitory effect was decreased against the rFVIII by forming a complex with vWF from 22 BU/mgIgG to 0.5 BU/mgIgG. Fab from the I-3 IgG had the same effect. In addition, vWF showed a protective effect on FVIII inactivation by the I-3 IgG in a dose dependent manner. Fifty-nine percent of residual VIII:C was retained in the presence of 8 U/ml of vWF after 1 hr incubation with I-3 IgG. These results suggested that vWF could compete with the I-3 IgG for binding to FVIII.

Emergency surgery was performed on a 24-year-old female patient with an unknown type of von Willebrand disease (VWD). The patient suddenly developed lower abdominal pain and was transported to our hospital by ambulance. Based on the diagnosis of ovarian bleeding, emergency surgery was indicated. Ristocetin-cofactor activity (VWF: RCo) was between 7% and 14% by measurement with von Willebrand reagent (Dade Behring, Marburg, Germany). A laparoscopic partial resection of the left ovary was performed under administration of factor VIII/VWF concentrate, Confact F((R)) (Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan). During and after surgery, there was no abnormal bleeding. The results obtained by measurement by the reagent were similar to the levels of VWF: RCo obtained by the fixed platelet agglutination method. The reagent was useful for diagnosing and monitoring the VWF: RCo level to control bleeding in VWD.

This review regarding prothrombin time and its standardization is described around some recent topics as the followings. 1. History of standardization for prothrombin time and revised WHO guideline for thromboplastin; A short history of standardization is summarized to understand a scheme of International Normalized Ratio (INR) based on International Sensitivity Index (ISI) that is calibrated by International Reference Preparation (IRP) for thromboplastin, and some key points in revised WHO guideline for thromboplastin and plasma used to control oral anticoagulant therapy are interpreted for research and practical use. 2. Point-of-care prothrombin time monitoring; A portable device to measure prothrombin time with whole blood sample, such as CoagChek (Roche), contributes to self-management by patients required long-term oral anticoagulation. Some investigators reported clinical agreement to use this monitoring system and improvement of patient's QOL and cost-effectiveness in overseas. 3. New types of thromboplastins; Two new types of thromboplastins have been available since the last year in Japan. One is a human plain thromboplastin, Simplastin HTF (Bioméreux) from extract of cultured human lung cancer cell, and another is IL test PT-Fibrinogen Recombinant (Iatron) from recombinant rabbit tissue factor relipidated in a synthetic phospholipid blend. For control of oral anticoagulation, good performance are expected in either thromboplastins because of their sufficient low ISI values. 4. INR methodology for other diseases; INR/ISI system is designed as a standardized methodology for control of oral anticoagulation. Prothrombin time, however is utilized as a global coagulation test for diagnosis or criteria of other disorders, such as congenital coagulation factor deficient, severe liver dysfunction and disseminated intravascular coagulation. Previous our study indicated that discrepancy of sensitivities to plasma absorbed multiple coagulation factors and plasma from patients under oral anticoagulation was revealed in rabbit brain thromboplastins, but not in human origins. Discrepancy of sensitivities observed in rabbit thromboplastins was emphasized in convert to INR values. These results suggested that the use of human thromboplastin of which ISI is close to 1.0 leads possibility for introducing INR methodology to evaluate PT of other disorders.

Prothrombin time(PT) is utilized in worldwide as a global coagulation test reflected multiple depletions of coagulation factors in diseases such as severe liver dysfunction and Disseminated Intravascular Coagulation(DIC). However, standardization of regents and result reporting methods are not established yet except International Normalized Ration(INR) for control of oral anticoagulant therapy(OAT). We evaluated whether INR is capable for defect of multiple coagulation factors except OAT, using absorbed plasma and different origins of thromboplastin; human recombinant, human placenta, cultured human cell and rabbit brain. PTs of individual 90 samples(group MC) absorbed with BaSO4 and/or bentnite and 60 samples(group W) from patients with OAT were measured with 20 normal plasmas with respective reagents. Sensitivities of four reagents to plasma of group W and MC were determined respectively against human recombinant thromboplastin(ISI = 1.03). Both of human thromboplastin showed that sensitivity to absorbed plasma was very close to OAT plasma, whereas reductions of sensitivity to 84% and 66% for absorbed plasma were revealed in both of rabbit thromboplastins. Correlations of INRs calculated by two different sensitivities, one is to absorbed plasma and another to OAT plasma, indicated that discrepancy of sensitivities was emphasized as large slopes(1.50 and 2.76) of regression lines and large intercepts in rabbit thromboplastins, although slopes closed to 1.0 with small intercepts in both of human thromboplastins. We concluded that use of human thromboplastin was the first priority to introduce INR system for evaluation of multiple coagulation factors depletions.

Prothrombin time (PT) is widely utilized for evaluation of diseases with single or multiple coagulation factors disorders, such as severe liver dysfunction and disseminated intravascular coagulation (DIC). However, its standardization of reagent and method is not established yet for universal purpose except International Normalized Ratio (INR) for control of oral anticoagulant therapy (OAT). Neither Prothrombin time ratio adapted in Japanese criteria for DIC nor seconds method in criteria reported by the last SSC meeting is corrected between assays, therefore much dependence on these criteria causes risk of misdiagnosis. In order to resolve incoherence of method of PT, we performed this study for introducing INR method to diagnostic criteria for DIC. In our results, sensitivities of two reagents from human tissue factor (TF) to DIC model plasma with middle to low activities of multiple coagulation factors almost equal to plasma from patients with OAC, although other two reagents from rabbit TF show discrepancy of sensitivities between DIC and OAT plasma. These suggest a potentiality to introduce INR system to diagnosis of DIC when human TF is used as a PT reagent. Based on standardization of method, significance of PT in DIC criteria should be reevaluated in worldwide.

SummaryWe found a patient with mild hemophilia A who had no detectable factor VIII antigen (FVIII:Ag), as shown by two-site ELISA using inhibitor alloantibodies (TK). We then analyzed A2, A2/B, and C2 antigen of the patient's DDAVP-induced FVIII using several anti-FVIII monoclonal antibodies. Factor VIII activity (FVIII : C) was increased from 12 to 42 Uldl by the administration of DDAVP. The DDAVPinduced increases in the A2 and A2/B antigens were 40 and 36 Uldl, respectively. However, the increase in the C2 antigen was only 7.5 Uldl. SSCP analysis and subsequent sequencing demonstrated an Arg to Cys transition at codon 2159. The anti-FVII1:C titer of monoclonal antibody, NMC-VIII15 which recognized the C2 domain, against normal plasma was 450 Bethesda Ulmg of IgG. However, the titer against DDAVP-treated patient's plasma was only 15 Bethesda Ulmg. We also tested DDAVP-induced increase in the FVIII : Ag in another mild hemophilia A patient with the same mutation at Arg2159. Increase in his C2 antigen levels was only 19% of those in the A2 and A2/B antigen. We designate this abnormal FVIII as FVIII Ise. Our results show that a missense mutation at Arg2159 to Cys modifies the antigenicity of the C2 domain.

Most factor VIII inhibitors are developed at an early age and in patients with severe type of hemophilia A. We report a case of newly developed factor VIII inhibitor in a 60-year-old patient with mild hemophilia A who had been treated with several kinds of factor VIII concentrates. The patient was treated with a total of 103,580 units of recombinant factor VIII concentrate by continuous and bolus infusions for the open surgery of sigmoid colon cancer. On the 95th postoperative day, the patient had right low limb muscle bleeding and was infused with 1,000 units of recombinant factor VIII concentrate for three days. Subsequently, the level of factor VIII inhibitor in the patient's plasma was 2 Bethesda units (BU)/ml. Since then numerous subcutaneous hemorrhages developed, but an adequate hemostatic effect was not obtained even with the administration of a high dose of recombinant factor VIII concentrate. The patient was switched to bypass therapy using human plasma-derived factor VIIa concentrate, which showed a favorable hemostatic effect.

Etiological and immunological evaluations of 101 hemophiliacs treated with coagulation factor concentrates were performed. All samples were assayed for HIV antigen (Ag) with two different enzyme immunoassays (ABBOTT/VIRONOSTIKA), confirmed for antibody (Ab) to HIV with two different methods (ABBOTT ENVACOR/BIO-RAD westernblot). Results of serum HIV related markers assayed by these methods classify the patients into three groups: Of 101 patients, 37 in group A (Ab negative), 53 in group B (Ab positive but Ag negative), 11 in group C (Ab and Ag positive).OKT4 (helper/suppressor inducer) cells and OKT4/OKT8 (suppressor/cytotoxic) ratios in group C (323±164/μl and 0.48±0.23) were significantly lower than in group B (569±278/μl and 0.63±0.34) and A (737±367/4μl and 1.11±0.63).Among immunoglobulins quantified, serum levels of IgG and A were elevated in group C (2364±750mg/dl and 341±220mg/dl and 257±148mg/dl). Particularly, the inverse correlation between OKT4 cells and IgA levels was shown in HIV seropositive patients. The coefficient of correlation in group C (r=-0.341) was stronger than that in group B (r=-0.105). On the other hand, no relationship was found between IgM levels and seropositivity.Elevated serum β2-microglobulin levels were significantly correlated with reduced OKT4 cells in seropositive patients, especially in group C (r=-0.513).Our data suggest that assays of serum IgA, β2-microglobulin and HIV antigen are useful predictors for the condition and prognosis of HIV infection, furthermore the evaluation of therapy.