Limit of detection and threshold for positivity of the Centers for Disease Control and Prevention assay for factor VIII inhibitors.

Essentials Immunologic methods detect factor VIII (FVIII) antibodies in some inhibitor-negative specimens. Specimens were tested by modified Nijmegen-Bethesda assay (NBA) and fluorescence immunoassay. The NBA with preanalytical heat inactivation detects FVIII inhibitors down to 0.2 NBU. IgG4 frequency validates the established threshold for positivity of ≥ 0.5 NBU for this NBA. SUMMARY: Background The Bethesda assay for measurement of factor VIII inhibitors called for quantification of positive inhibitors by using dilutions producing 25-75% residual activity (RA), corresponding to 0.4-2.0 Bethesda units, with the use of 'more sensitive methods' for samples with RA closer to 100% being recommended. The Nijmegen modification (Nijmegen-Bethesda assay [NBA]) changed the reagents used but not these calculations. Some specimens negative by the NBA have been shown to have FVIII antibodies detectable with sensitive immunologic methods. Objective To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)-modified NBA (CDC-NBA), which includes preanalytic heat inactivation to liberate bound anti-FVIII antibodies. Methods Specimens with known inhibitors were tested with the CDC-NBA. IgG4 anti-FVIII antibodies were measured by fluorescence immunoassay (FLI). Results Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). With four statistical methods, the limit of detection of the CDC-NBA was determined to be 0.2 NBU. IgG4 anti-FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥ 0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU. Conclusions The CDC-NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti-FVIII IgG4 in some patients with negative (< 0.5) NBU. The sharp increase in IgG4 frequency between 0.4 and 0.5 NBU validates the established threshold for positivity of ≥ 0.5 NBU for the CDC-NBA, supporting the need for method-specific thresholds.

Hepatitis B vaccination is effective by subcutaneous route in children with bleeding disorders: a universal data collection database analysis.

Subcutaneous (SQ) vs. intramuscular (IM) vaccination may cause fewer injection site complications in children with bleeding disorders, but little is known about comparative immunogenicity. To compare immunogenicity of hepatitis B virus (HBV) vaccination administered SQ or IM to individuals <2 years old with bleeding disorders, we performed a retrospective analysis of HBV surface antibody titres among patients enrolled in the universal data collection database who had received three doses of HBV vaccine solely by one route (SQ or IM). Data reviewed were from an initial visit before 24 months of age, until time of hepatitis antibody titre testing. The SQ and IM study groups did not differ in demographics, haemophilia type or severity or bleeding history. The mean age at the time of HBV surface antibody (anti-HBs) testing was 56.9 ± 20.3 months. Eighty-five of 92 subjects (92.4%) who received vaccine SQ developed a positive antibody titre (>12 IU/L), compared to 101/114 (88.6%) who received IM (P = 0.30). There was no statistically significant difference in distribution of titre values. The average age of the subjects at time of testing was 53 ± 20 months in the SQ group vs. 60 ± 20 months in the IM group (P = 0.02). The average time between the last dose of vaccine and anti-HBs testing was 47.6 ± 18.5 months among SQ vaccinated subjects vs. 51.6 ± 20.5 months in the IM group (P = 0.2). Immunogenicity to hepatitis B vaccination by the SQ and IM routes is similar.

Comparison of clot-based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.

BACKGROUND: Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety and assessment of population trends. METHODS: Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA) and a novel fluorescence immunoassay (FLI). RESULTS: NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU) < 0.5 and positive on 42/42 specimens (100%) with NBU ≥ 2.0 and 43/80 specimens (53.8%) with NBU 0.5-1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0-1.9 NBU specimens and 43.1% and 50.0% of 0.5-0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P = 0.004). Among 21 new inhibitors detected by NBA, five (23.8%) with 0.7-1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5-1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%). CONCLUSIONS: FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5-1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays.

Use of pharmacokinetic modelling to individualize FFP dosing in factor V deficiency.

Therapy with fresh frozen plasma (FFP) confers serious risks, such as contraction of blood-borne viruses, allergic reaction, volume overload and development of alloantibodies. The aim of this study was to apply principles of pharmacokinetic (PK) modelling to individual factor content of FFP to optimize individualized dosing, while minimizing potential risks of therapy. We used PK modelling to successfully target individual factor replacement in an 8-month-old patient receiving FFP for treatment of a severe congenital factor V (FV) deficiency. The model fit for the FV activity vs. time data was excellent (r = 0.98) and the model accurately predicted FV activity during the intraoperative and postoperative period. Accurate PK modelling of individual factor activity in FFP has the potential to provide better targeted therapy, enabling clinicians to more precisely dose patients requiring coagulation products, while avoiding wasteful and expensive product overtreatment, minimizing potentially life-threatening complications due to undertreatment and limiting harmful product-associated risks.

The use of a single von Willebrand factor-containing, plasma-derived FVIII product in hemophilia A immune tolerance induction: the US experience.

BACKGROUND: Inhibitors are a serious complication for patients with severe hemophilia A. Immune tolerance induction (ITI) is the primary method for eradicating these inhibitors. The role of type of concentrate and in particular the use of von Willebrand factor-containing, plasma-derived factor VIII (VWF/pd-FVIII) concentrate in primary or rescue ITI remains unclear. OBJECTIVES: To report retrospective collection of data on the use of a single VWF/pd-FVIII concentrate in primary and rescue ITI. METHODS: Retrospective chart review of hemophilia A inhibitor patients at 11 US institutions who received VWF/pd-FVIII concentrate in primary or rescue ITI. RESULTS: Primary ITI was carried out in eight inhibitor patients with a 75% complete and partial success. Secondary ITI was carried out in 25 inhibitor patients, with 52% attaining complete or partial success. CONCLUSIONS: This report represents the largest group of primarily pediatric, high-titer inhibitor patients treated with a single VWF/pd-FVIII concentrate. It adds retrospective data to the use of VWF-containing plasma-derived factor VIII concentrate in primary and rescue ITI, particularly in those patients with characteristics of poor response to ITI.

Prosthetic metals interfere with the functions of human osteoblast cells in vitro.

The release of metals from total joint prostheses may contribute to periprosthetic bone loss manifested as osteolysis. The effects of titanium, cobalt, and chromium on human osteogenic sarcoma cells (osteoblastlike cells) were investigated in vitro. Titanium, cobalt, and chromium at concentrations of 1, 10, and 100 ng/ml did not cause any changes in the cell growth, viability, and injury after 72-hour incubation with the cells. Titanium, cobalt, and chromium at concentrations ranging from 0.01 to 100 ng/ml significantly enhanced the release of interleukin-1 beta and tumor necrosis factor-alpha by lipopolysaccharide stimulated human osteogenic sarcoma cells, whereas they did not alter the release of transforming growth factor-beta 1. Cobalt at concentrations ranging from 0.1 to 100 ng/ml significantly enhanced the release of interleukin-6, but titanium and chromium did not. Cobalt and chromium at concentrations of 10 and 100 ng/ml significantly inhibited the release of osteocalcin by human osteogenic sarcoma cells, whereas titanium had no effect. Titanium, cobalt, and chromium at concentrations of 10 and 100 ng/ml significantly inhibited the synthesis of Type I collagen by human osteogenic sarcoma cells. Cobalt and chromium inhibited the cell proliferation in response to lipopolysaccharide stimulation, whereas titanium did not. The data presented in this article suggest that the metal induced disregulation of cytokine release and osteoblast dysfunction may play an important role in the induction of osteolysis in patients with total joint arthroplasties.

Prosthetic metals impair murine immune response and cytokine release in vivo and in vitro.

This study was designed to investigate whether prosthetic metals adversely affect immune responses and the release of immunoregulatory cytokines in vivo and in vitro. Titanium and cobalt-chromium alloy were injected into the peritoneal cavity of female mice. At 5, 8, and 12 weeks after the injection, the levels of cobalt and chromium in the blood were significantly increased compared with the levels in control mice; the level of titanium was not significantly changed until 12 weeks. The release of interleukin-2 was significantly inhibited by cobalt-chromium particles after 3 weeks; titanium particles did not have the same effect until 8 and 12 weeks. The release of interleukin-4 was significantly inhibited by cobalt-chromium particles after 3 weeks but was not significantly inhibited by titanium particles until 12 weeks. The release of interferon-gamma was significantly inhibited by cobalt-chromium particles only at 12 weeks and was not inhibited by titanium particles. The proliferation of T cells was significantly inhibited by cobalt-chromium particles at 3 weeks and by titanium particles at 8 and 12 weeks, and the proliferation of B cells was significantly inhibited by cobalt-chromium particles after 3 weeks but was not inhibited by titanium particles. The production of immunoglobulin by lipopolysaccharide-stimulated B cells was also significantly reduced by cobalt-chromium particles after 3 weeks and by titanium particles at 8 and 12 weeks. The cytokine release by lymphocytes, proliferation of T and B cells, and immunoglobulin production by B cells were also significantly inhibited by titanium and cobalt-chromium particles, as well as by titanium, cobalt, and chromium ions in vitro, whereas these metals are not cytotoxic to murine lymphocytes in vitro. The data indicate that the metal-induced immunosuppression may be another important factor in the development of implant-associated infection in patients with a prosthesis.

Inhibition of T and B cell proliferation by titanium, cobalt, and chromium: role of IL-2 and IL-6.

The mechanism by which an increased risk of prosthetic infection is induced in patients with total joint arthroplasties is poorly understood. The adverse effects of metallic corrosion products of a prosthesis on host defense mechanisms, particularly immune response and release of immunoregulatory cytokines, remain largely unknown. Titanium, cobalt, and chromium are the materials most often used for joint implantation. Therefore, this study was aimed at investigating the cytotoxicity of titanium, cobalt, and chromium and whether these metals affect T and B cell proliferation and the release of cytokines by human peripheral blood mononuclear cells (PBMC) in vitro. Metal cytotoxicity was not observed judging by cell viability and cell injury after PBMC was extensively exposed to the metals. Phytohemagglutinin (PHA)-induced T cell proliferation and lipopolysaccharide-induced B cell proliferation were significantly inhibited by titanium, chromium, and cobalt. The release of IL-2 and IL-6 by PHA-stimulated PBMC was significantly inhibited by titanium, chromium, and cobalt. Titanium did not alter IFN-gamma production, whereas chromium and cobalt significantly reduced IFN-gamma release by PHA-stimulated PBMC. The addition of IL-2 and IL-6 significantly restored the metal-induced inhibition of T cell and B cell proliferation, respectively. This study sheds light on how the metals impair immune response and cytokine release, suggesting that patients with an extensive exposure to the metals may develop immune dysfunctions. The compromised immune response induced by the metals might significantly contribute to an increased risk of infection in patients with joint prostheses.

Titanium, chromium and cobalt ions modulate the release of bone-associated cytokines by human monocytes/macrophages in vitro.

Osteolysis has become a major cause of aseptic loosening in total joint arthroplasty (TJA). Titanium, cobalt and chromium are commonly used in orthopaedic implants (e.g. joint prostheses). The release of bone-associated cytokines has been associated with the development of osteolysis in patients with prostheses. We evaluated the effects of these metals on the release of bone-associated cytokines (IL-1 beta, IL-6, TNF-alpha and TGF-beta 1) by human blood monocytes/macrophages and monocyte-like U937 cells upon lipopolysaccharide (LPS) stimulation, the cell proliferation, and their cytotoxic effects on these cells in vitro. We found that the release of IL-1 beta was enhanced by titanium, chromium and cobalt, the release of TNF-alpha was enhanced by titanium and chromium, and the release of IL-6 was enhanced by titanium. All three metal ions inhibited the release of TGF-beta 1. We also found that titanium and chromium, but not cobalt, enhanced blood monocyte/macrophage proliferation in response to LPS while only titanium enhanced U937 cell proliferation in response to LPS. The metals in concentrations ranging from 0.01 to 100 ngml-1 did not stimulate the cells to secrete detectable cytokines in the absence of LPS. Furthermore, a 4-h pre-exposure of blood monocytes/macrophages or U937 cells to the metals did not alter cytokine release when the metals were removed from the media prior to the addition of LPS. Similarly, a 4-h pre-exposure of blood monocytes/macrophages or U937 cells to LPS did not alter cytokine release when LPS was removed from the media prior to the addition of the metals. The metals did not reduce cell viability and induce cell injury after 72h incubation with the cells. The data suggest that the three metals at clinically relevant concentrations modulated cytokine expression, whereas they did not induce any cytotoxic effects. A metal-induced enhancement of bone-resorbing cytokine release with a concomitant inhibition of bone-forming cytokine release may be an important factor in the development of osteolysis, which can severely compromise the outcome of TJA.

Agrobacterium radiobacter: a recently recognized opportunistic pathogen.

Over the past decade, an increasing number of infections due to Agrobacterium radiobacter have been reported. Observation of three cases of bacteremia due to this organism prompted a review of the English-language literature. Nineteen cases of significant disease have previously been reported. In more than one-half of the cases, bacteremia was the primary manifestation, often associated with the presence of an intravascular catheter. Other clinical syndromes (peritonitis, urinary tract infection, and endocarditis) have been described. Infection is strongly related to the presence of plastic foreign material, and effective treatment often requires removal of the device. Because antimicrobial sensitivity is variable, treatment must be based on sensitivity data for the individual isolate.

Enterococcus faecium resistant to ampicillin and gentamicin.

Thirty ampicillin-resistant enterococci were isolated from clinical specimens at our institution, 28 of these over a six month period. All were identified as Enterococcus faecium with an MIC(90) to ampicillin ond penicillin of 32 and 128 mug/ml, respectively. These isolates were also resistant to imipenem, ampicillin-sulbactam, and amoxicillin-clavulanic acid, but susceptible to vancomycin. Only 10%% were resistant to gentamicin at 2 000 mug/ml, but bactericidad synergy could not be demonstrated against any of these isolates using the combination of gentamicin added to either penicillin or vancomycin. No beta-lactamase activity was detected by the nitrocephin test, or by the addition of clavulanic acid or sulbactam. Plasmid analysis revealed a band common to 29 of 30 ampicillin-resistant E. faecium, which was not present in two ampicillin-sensitive E. faecium or in any of twenty E. faecalis. The limited number of options available to treat these isolates of E. faecium simultaneously resistant to multiple antibiotics and resistant to the bactericidad synergy of gentamicin underscores the need to develop new strategies in the prevention and treatment of enterococcal infections.

The effect of stainless steel, cobalt-chromium, titanium alloy, and titanium on the respiratory burst activity of human polymorphonuclear leukocytes.

Although infection is a serious complication associated with the use of orthopedic prosthetic implants, the impact of the metal used in these devices on host defense is poorly understood. The authors investigated the effect of stainless steel, titanium, titanium alloy, and cobalt-chromium alloy on the respiratory burst of polymorphonuclear leukocytes (PMN), a vital component of bactericidal activity. In the presence of stainless steel powder or supernatants obtained from the incubation of stainless steel in buffer, superoxide production by PMN was significantly impaired. Titanium, titanium alloy, and cobalt-chrome alloy had no significant effect on superoxide production. Nickel and chromium, the only metal ions detectable in the stainless steel supernatant, did not impair superoxide production when tested at concentrations similar to those found in the supernatant. Inhibition of PMN superoxide production may play a role in the establishment and persistence of stainless steel device-related infections.

Suppressive antibiotic therapy in chronic prosthetic joint infections.

Thirteen patients with chronic total joint infections (eight knees, five hips) were treated with suppressive antibiotic therapy and retention of the prosthesis following surgical debridement and 4 to 6 weeks of intravenous antibiotic therapy. These patients faced poor functional outcome after prosthesis removal. After a mean follow up of 37.6 months (range: 24 to 55), only three patients have retained their prostheses. Ten patients required prosthesis removal for recurrent infection a mean of 21.6 months (range: 6 to 48) after starting suppressive therapy. In addition, 38% of patients experienced adverse effects which led to changes in the antibiotic regimen. Suppressive antibiotic therapy in the treatment of chronic prosthesis infections has limited clinical efficacy and is associated with a substantial risk of adverse effects.

Treatment of neonatal aortic thrombosis with urokinase.

We describe the successful treatment of a neonate with severe aortic thrombosis using systemic urokinase. Current knowledge regarding the treatment of this condition, including fibrinolytic therapy, is reviewed.

Measurement of prothrombin mRNA during gestation and early neonatal development.

Prothrombin levels in the fetal and newborn lamb follow a gestational age-related pattern of development. The changes in activity are reflected by similar changes in prothrombin antigen concentration. Our investigations indicate that these changes in prothrombin antigen and activity levels are paralleled by changes in the levels of prothrombin messenger RNA present in liver samples from fetuses of various gestational ages.

The effect of anaerobiosis on antistaphylococcal antibiotics.

The activity of eight antimicrobial agents which might be used in the treatment of staphylococcal osteomyelitis was tested under anaerobic conditions similar to those found in chronically infected bone. An agar-dilution method was employed to determine the minimum inhibitory concentrations of tobramycin, vancomycin, teicoplanin, ciprofloxacin, clindamycin, ceftriaxone, ticarcillin-clavulanic acid, and amoxicillin-clavulanic acid against 25 coagulase-positive and 25 coagulase-negative staphylococcal strains. The activity of tobramycin against coagulase-positive staphylococci, and of amoxicillin-clavulanic acid and ticarcillin-clavulanic acid against coagulase-negative staphylococci was markedly decreased with anaerobiosis. Vancomycin, teicoplanin, and ciprofloxacin were active against coagulase-positive and coagulase-negative staphylococci under both aerobic and anaerobic conditions. It was also found that antibiotic concentrations comparable to the high levels which might be achieved with local antibiotic therapy of osteomyelitis were not sufficient to overcome the level of resistance (100 micrograms/ml) of staphylococci which were not susceptible to tobramycin, clindamycin, ceftriaxone, and ticarcillin-clavulanic acid.

Embolization therapy in the management of infantile hemangioma with Kasabach Merritt syndrome.

A case is presented in which a giant infantile hemangioma with thrombocytopenia is managed successfully by serial transcatheter embolization.