A luminescence-based assay for evaluating bactericidal antibody to Borrelia burgdorferi in vaccinated horses' serum.

BACKGROUND: Current serological tests cannot discriminate between bactericidal Borrelia burgdorferi antibodies from others that are merely a response to Borrelia antigenic stimulation. OBJECTIVE: To develop a sensitive and convenient luminescence-based serum bactericidal assay (L-SBA) to identify serum borreliacidal activity. STUDY DESIGN: Prospective validation study and method comparison. METHODS: Serum samples were obtained either from archives of the Animal Health Diagnostic Center at Cornell University (N = 7) or from a vaccination trial (N = 238). Endogenous complement-inactivated serum sample was incubated with exogenic complement and B. burgdorferi ML23 pBBE22luc, which is able to process luciferin with luciferase and produce luminescence in viable Borrelia. After incubation, a light signal can be detected by using a luminometer to calculate the borreliacidal antibody titre. RESULTS: Components of the reaction mixture including spirochetes and complement from various sources and concentrations were tested to identify a reliable recipe for our complement-mediated L-SBA. We also applied this L-SBA on measuring bactericidal antibody activities and calculated the half inhibitory concentration (IC50 ) of serum samples from clinical collections. Furthermore, we analysed the L-SBA titres and anti-outer surface protein A (OspA) antibody levels from vaccinated horses using the multiplex assays and found that there is a relationship between results generated using these two different assays. The increases of L-SBA titres correlated with increases of anti-OspA antibody titre in sera (r = 0.423). MAIN LIMITATIONS: Immunoreactivity of commercial complement may differ from different batches. Clinical protection of borreliacidal antibody levels has not been determined. CONCLUSIONS: The L-SBA provided a sensitive and easy-operating platform for the evaluation of bactericidal antibody to B. burgdorferi, and we anticipated L-SBA would function well as an evaluation tool of vaccine efficiency in the future.

Increases in circulating levels of monocyte-platelet and neutrophil-platelet complexes following hip arthroplasty.

Platelets and leucocytes are important effector cells of the haemostatic and inflammatory responses to tissue injury. To investigate the effects of surgical trauma on platelet activation (assessed by measuring levels of P-selectin and beta-thromboglobulin), leucocyte activation (CD11b expression) and leucocyte-platelet interactions (leucocyte-platelet complexes), 30 patients undergoing primary hip arthroplasty were studied before and at the end of surgery, and on days 1 and 10 post-operatively, using a whole-blood flow cytometry assay. The inflammatory response was followed by measurement of the levels of C-reactive protein and interleukin-6 in plasma, and the activation of coagulation was monitored by determination of prothrombin fragment 1+2 levels. On day 1 post-operatively a significantly increased expression of CD11b on monocytes was noted, but no direct correlation was found between monocyte activation and interleukin-6 production or C-reactive protein at this time point. The percentage of monocyte-platelet and neutrophil-platelet complexes was markedly increased on day 10 post-operatively compared with pre-operative levels, and levels of these complexes were significantly positively correlated with beta-thromboglobulin levels. Activation of coagulation (prothrombin fragment 1+2) on day 10 post-operatively was positively correlated with the extent of surgical trauma (duration of surgery, amount of blood loss) and with the increase in platelet activation (beta-thromboglobulin). In conclusion, hip arthroplasty induces platelet and coagulation activation, and also an inflammatory response that is maintained for more than 10 days post-operatively. This indicates an interaction between the immune and the haemostatic systems in the post-operative phase after hip arthroplasty.

Lateral position reduces blood loss in hip replacement surgery: a prospective randomized study of 74 patients.

We performed a prospective randomized study of 74 patients undergoing hip replacement surgery and compared the perioperative blood loss in the lateral position with that in the supine position. The surgeons and surgical technique were the same in the two groups. The patients operated on in the lateral position had a significantly lower total blood loss, on average 201 ml less.

Surgical hemostasis after tourniquet release does not reduce blood loss in knee replacement. A prospective randomized study of 81 patients.

We studied the effect of timing of tourniquet release on blood loss in 81 patients (85 knees) who were operated on for total knee replacement. The patients were randomly allocated to one of two groups. In one group, the tourniquet was released for hemostasis before wound closure and in the other group, the tourniquet was not released until the wound was closed and a compressive dressing applied. We found no difference in total blood loss between the two groups and conclude that intraoperative release of the tourniquet for hemostasis is not effective for reducing blood loss in total knee replacement.

Diathermy knife does not reduce bleeding in surgery for primary hip arthroplasty. A prospective randomized study of 67 patients.

We evaluated bleeding in a prospective randomized study of 67 patients undergoing primary hip arthroplasty. In group A, all dissections were made with the diathermy knife and in group B, we used the scalpel. In both groups, bleeding points were coagulated with diathermy. The surgical approach, use of anti-coagulants and method of anesthesia were similar in the two groups. We found that use of a diathermy knife does not significantly reduce bleeding.

Monte Carlo advice.

The generation of long, high quality random number sequences for Monte Carlo simulations using minicomputers is considered. The importance of the thorough testing of Monte Carlo random number generators is emphasized. A recommendation is given to authors of Monte Carlo papers to specify their random number generator and to describe the randomness testing which that generator has undergone.