Quantification of deposition of neutrophilic granulocytes on vascular grafts in dogs with 111In-labeled granulocytes.

A new radioisotopic technique has been developed for quantification of deposition of neutrophilic granulocytes on vascular grafts. Nine healthy mongrel dogs underwent bilateral femoral artery resection and reconstruction with grafts of femoral vein and Gore-Tex. Pure granulocytes that had been separated from whole blood by centrifugal elutriation were labeled with 111In-tropolone in plasma. The granulocyte harvesting efficiency was 25 +/- 12%, and the labeling efficiency was 87 +/- 7%. Three hours after injection of labeled granulocytes and 2 hours after reperfusion, the grafts were harvested and cut into several segments for study of areas of anastomoses and midsections. On the basis of the radioactivity in the blood and in anastomotic and graft sections, the area of graft sections, and the neutrophilic granulocyte and differential leukocyte counts, the number of neutrophilic granulocytes adherent to a unit area and the total number of neutrophilic granulocytes on graft sections were calculated. These quantifications of the deposition of neutrophilic granulocytes indicated that the midsections of Gore-Tex grafts retained more neutrophilic granulocytes than did the midsections of vein grafts. Although the anastomotic areas retained more neutrophilic granulocytes than did the midsections of vein grafts, the opposite finding prevailed for the Gore-Tex grafts. A major fraction of neutrophilic granulocytes on Gore-Tex grafts was incorporated into thrombus. Semiquantitative information obtained by scintigraphy of the deposition of neutrophilic granulocytes on vascular grafts also confirmed this observation.

Quantification of regional platelet and calcium deposition on pericardial tissue valve prostheses in calves and effect of hydroxyethylene diphosphonate.

Mitral valves were replaced with 25 mm bovine pericardial tissue valve prostheses (Ionescu-Shiley) in 45 calves (23 controls and 22 treated daily with hydroxyethylene diphosphonate). Before being killed 1, 14, 30, and 90 days later, the calves received In-labeled autologous platelets intravenously. Regional variations in platelet deposition were measured by a new technique. Thrombus was noted early on the flexion zone of the valve; with time it increased on the free edge and central zone. Platelet deposition was initially high on the flexion zone, attachment zone, sewing ring, and perivalvular tissue but decreased with time. Hydroxyethylene diphosphonate treatment tended to decrease platelet deposition in all zones. Calcium content was high in thrombus and increased in all zones with time, in both groups, but the deposition was less in the group treated with hydroxyethylene diphosphonate. Collagenous tissue and adherent thrombus appear to provide nucleation sites for calcification.

Identification of new collagen formation with 125I-labeled antibody in bovine pericardial tissue valves implanted in calves.

Failure of bovine pericardial tissue valve used in young patients may be due to a slow rejection process. Polyclonal anticollagen (Type I) antibody (IgG) was made in rabbits and purified by protein A affinity column. Two milligrams of IgG was labeled with 2 mCi of 125I by the Iodogen method. Free iodide was separated by G-10 column. Affinity of 125I-IgG was checked by radioimmunoassay. Two hundred and fifty microcuries of 125I-IgG was injected in calves immediately after tissue valve implantation, and the calves were killed 4 h post-injection. After harvesting the valve, each of the three leaflets was separated into four zones, and radioactivity in each section was mapped with a gamma counter. The radioactivity in tissue valve section was compared to that of normal aortic valve. The sections of tissue valve retain five to ten times more 125I-IgG than control aortic valve. Iodine-IgG thus provides a sensitive technique for determination of residual antigenicity in tissue valve.

Effect of diphosphonate binding to collagen upon inhibition of calcification and promotion of spontaneous endothelial cell coverage on tissue valve prostheses.

A noncalcifying drug, aminodiphosphonate, was immobilized on pericardial collagen to inhibit calcification. The parameters of ADP binding were optimized by using labeled tracer. The effects of the detergents, Triton X-100 and sodium dodecyl sulfate, pH, and temperature on ADP binding to collagen in fresh pericardium, and stabilization of ADP-collagen bond by borohydride (BH) reduction, were optimized. These studies indicate that pretreatment of pericardium with SDS and TX-100 increases ADP binding three- to fivefold. Three-step cross-linking via Schiff-base reactions between collagen, ADP, and cross-linking via Schiff-base reactions between collage, ADP, and glutaraldehyde gives a high value of 30 to 35 molecules of ADP per collagen in pericardium. Twenty-five millimeter tissue valves were made with detergent (1% SDS, 1% TX-100) treated ADP-bound pericardium and implanted in the mitral annuli in calves, which were killed at 60 days post implantation. Regional calcium deposition was measured with atomic absorption spectrometry. The calcium level (micrograms/mg of tissue) in components of control and four processed valves were determined. Regional platelet deposition on zones of leaflets and components of tissue valves were quantified with 111In-labeled autologous platelets. The calcium levels in thrombus and flexion zones of treated valves are lower than that in the control valve. SEM studies of leaflet surfaces at 60 days indicate that these treatment processes reduce calcification and promote spontaneous cell coverage on leaflets around the smooth surface of the outflow tract.