(PTFE) vascular grafts

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platelets in polytetrafluoroethylene (PTFE; GoreTex) grafts. In each experimental animal, 12PTFE grafts (inter nal diameter, 3 mm; length, 5 cm) were placed in ...
European Journal of

Eur J Nucl Med (1985) 10:160 164

Nuclear Medicine © Springer-Verlag 1985

The early platelet uptake and distribution of platelets in small-diameter polytetrafluoroethylene (PTFE) vascular grafts in vivo J.T. Christenson1, D. Arvidsson1, P. Qvarfordt1, H.M. Dashti1, P.-I. Olsson2, S.-E. Strand2, and T.Sjoberg1 1Department ofSurgery and 2Department of Radiation Physics, University of Lund, S-221 85 Lund, Sweden Abstract. Platelet deposition onto the surface of biomaterial isan importantcomponentof the interaction between blood and a prosthetic arterial graft. To understand the throm botic process in small-diameter by-pass grafts, we used pigs

to study the early in vivo uptake of inIn-oxine-labelled platelets in polytetrafluoroethylene (PTFE; GoreTex) grafts. In each experimental animal, 12 PTFE grafts (inter

a scintillation camera for the imaging of the activity distri

bution of n'In-labelled platelets in experimental animals. Using the most frequently employed synthetic arterial grafts

(polytetrafluoroethylene; PTFE), we studied the kinetics of labelled-platelet behaviour immediately after the establish ment of blood flow and the distribution of platelet adhesion on the surface along such grafts.

nal diameter, 3 mm; length, 5 cm) were placed in the femo

ral artery as interposition grafts. l' 'In-Labelled autologous platelets were injected. Sequential-scintillation camera im ages of the graft area were taken over a period of 3.5 h. Platelet deposition in vivo was calculated as an activity ratio for the entire grafts as well as for different segments

of the graft. The grafts were harvested, cut into 0.6-cm

Materials and methods

Seven pigs weighing25-30 kg were anaesthetized with intra venously injected sodium pentobarbital (60 mg/kg), and an aesthesia was maintained with intermittent injections of the

Blood flow was measured continuously. The activity ratios

same drug when necessary. The pigs were intubated, and respiration was maintained with a ventilator. No heparinization was employed, but fluid (normal saline) was intrave

rapidlyincreased, and a maximum was reached60 minafter

nously infused during the experiment. • ... .

pieces and weighed, and the lllIn activity was measured. The distribution along the graft surface was calculated. the re-establisment of blood flow; thereafter the activity

ratios slowly decreased. The distribution of platelets along the graft surface was found, to be non-uniform where more platelets were deposited towards the distal anastomoses.

The dynamic interaction between platelets and arterial grafts can easily be studied with lllIn-oxine-labelled plate

Graft implantation

Using sterile techniques, both femoral arteries were ex posed. Using end-to-end anastomoses with interrupted 6-0 Prolene sutures, PTFE grafts (GoreTex; internal diameter, 3 mm; length, 5 cm) implanted in the femoral circulation. The diameter of the graft was chosen to match the diameter of the vessel. The wounds were not closed but kept under a sterile covering.

lets. Previous work has been limited to the study of the

survival time of 51Cr-labelled platelets in the graft circula

tion [10] and the in vitro measurement of recovered speci mens [12]. Since the technique of labelling platelets with lllIn-oxine was first introduced [14], it has been applied

to the study of normal and damaged arteries [8], synthetic arterial grafts [3, 4] and arterial and venous thrombosis [13]. Most of these studies were performed by injecting labelled platelets into the circulation some time after arterial injury or graft implantation. However, synthetic arterial grafts have a marked interaction with platelets within min utes of coming in contact with blood, and this early interac tion may dictate subsequent events in the graft. This early phase has yet to thoroughly studied.

The present investigation describes a technique using Offprint requests to: J.T. Christenson, M.D., Ph.D. (Surg), Depart ment of Surgery, Faculty of Medicine, Kuwait University, PO Box 24 923 Safat, Kuwait

(I^L-

Labelling procedure

The cell separation and labelling procedure were performed during surgery according to the technique described by Christenson et al. [4]. Four venous blood samples (15 ml each) were withdrawn from the animal through a venous

catheter placed in the jugular vein and collected in plastic syringes that each contained 3 ml ACD-NIHA solution (stock solution; 4.0 g citric acid, 11.0g sodium citrate dihytrate, 11.2 g dextrose monohydrate, sterile water 500 ml); the samples were gently mixed both during and after collec tion. The blood was transferred into closed, sterile glass

vials (20 ml) for cell separation. The vials were centrifuged at 130g for 17 min in a balanced clinical bench centrifuge; the supernatant, the platelet-rich plasma (PRP), was remowed using 10-ml syringes with spinal needles and placed into a new vial. The pH was adjusted to 6.5 by adding ACD-NIHA solution, and 0.4 ml 25% human serum albu-

min (HSA) was carefully layered under the PRP using a 2.5-inch-long needle. After centrifugation at 500 g for 12 min, the platelet poor plasma (PPP) was removed and saved, thus leaving a platelet layer over the HSA. Following the addition of 1 ml phosphate-buffered saline (PBS; pH7.4; 0.82% NaCl, 0.16 % Na2HP04, 0.02%

Ventilator

NaH2PC\, 2H20), the platelets were easily resuspended by gentle agitation. "'In-Oxinate^Byk-Mallinckrpd^.Pet/:teh;Tlj!ei,Netherland'sJ,:.whiGb..iiad been prepared by-the '

^i.v:' catheter-'

addition of0.4 mi sterile TRIS buffer (0.2 M.\ pH 8.0), was added to the cells which were then incubated in a waterbath

for 20 min at 37° C. The labelled platelets were resuspended in 2 ml PPP, and 0.4 ml HSA was layered under the cells-

Scintillation

as already described. After centrifugation at 500 g for

field of view

camera

12 minj the supernatant PPP was removed and saved for

ft

the determination of labelling efficiency, thus leaving a pla telet layer over the HSA. Ten millilitres of "fresh" PPP was added, and the platelets were easily resuspended. The amount of radioactivity in the supernatant and the labelled platelets was determined in a radioisotope dose calibrator, and the labelling efficiency (LE) was calculated by the formula:

Flow probe

Flow probe

activity in platelets xlOO. activity in platelets + activity in supernatant

LE%=-

The platelets were counted in a Buerkner chamber using a phase-contrast microscope [11]. Platelet aggregation was measured by the turbidometric method of Born and Gross [1] using an aggregation moni tor (Born-Michael Mark IV; Pharmacological Research, Cambridge, UK and a Goertz potentiometric recorder. Pla telet aggregation was induced by adding adenosine S'-diphosphate (ADP) to PRP to a final concentration of 0.2-1 uM.

Fig. 1. A schematic representation of the experimental set-up

At the end of the experiment, the grafts were isolated, removed and rinsed in saline, and their U1ln activities were

measured in a well counter (Selektronic) using an energy window that included both the 171- and 247-keV full-energy peaks. These values were compared to the in vivo values obtained with the scintillation camera.

The experimental protocol

The pig was placed in the supine •position.'After the grafts were in place, but before they were exposed to blood, the

I

Only grafts that remained patent throughout the experi ment are included in this.report (Fig. 1).

11'In-labelled platelets were reirifused intravenously. After

In vitro determination ofplatelet distribution

10-15 min, the blood flow was re-established through the grafts. Blood flows were continuously measured using noncannulating electromagnetic square-wave flow meters with 3-mm-diameter probes (375; Nycotron, Norway). As soon as haemostasis was obtained, the pigs were imaged in the supine position. Sequential 10-min scintillation-camera (Phi Gamma III HP; Siemens, FRG) images of the graft area were taken overa period of 3.5 h and stored in a computer (Gamma 11; Digital Corp). A medium-energy palleled-hole

The grafts were cut into equal-sized pieces (about 0.6 cm long) and put into marked test tubes. Each sample was weighed. The test tubes were measured for '"In activity in a well counter, as already described. The values were corrected for the volume and decay of the isotope and ex pressed in terms of activity per gram of the graft. The distri bution for each graft piece was then calculated as a percent age of the total graft activity and compared with the data obtained in vivo.

collimator was used. An energy window was selected with

a peak photon energy of a level of 171 keV. The digital images were used to quantitate the count rate of'' 'In activ

ity in several regions of interest, e.g. the graft (excluding the two anastomoses, the host artery distal and proximal to the anastomoses, and the adjacent soft tissue; average of three regions). From these regions of interest, measure ments of platelet deposition onto the graft were calculated in the form of an "activity ratio" defined as:

count rate in graft area - count rate in artery area/count

Results

The platelets were labelled with an average labelling effi ciency of 88% ± 6% (mean + SD). The injected activity was 19.8±2.8 MBq. On average, each animal had 0.3 x 106 pla telets/ml, i.e. approximately 18 x 106 platelets were injected back to the animals. Aggregation occurred in all cases. Ten minutes after infusion, 92% of the infused activity was in the circulation.

rate in soft tissue area

A ratio of greater than zero then indicates the net plate let deposition onto the graft surface. Since the wounds were

Platelet adhesion in vivo

left unsutured, the attenuation of the photons was the same

The digital images showed that the calculated activity ratios

in all of the experiments.

rapidly increased and reached a maximum 60 min after the

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