Semi-Automated Determination Of Platelet And Red Blood Cell Loss During Hemostasis In The Guinea Pig

Male guinea pigs of 350-500 g (Hartley strain, Simonsen, Gilroy, CA) were anesthetized with pentobarbitone sodium (32.5 mg/kg, i.p.) and restrained head downwards on a plexiglass board tilted at an angle of 45° to the horizontal. Both ears were held down on the surface of the board using adhesive tape (Scotch brand #810). The upper surface of each ear was then superfused at 5 ml/min with warmed (37°C) sterile 0.9% (w/v) NaCl solution, delivered via a 14 g hypodermic needle. The tip of the needle was lowered in a standardized manner to puncture a small ear artery. From the time of incision, serial 10 sec. aliquots of bloodstained superfusate were collected into tubes containing 10 ml of particle-free saline with EDTA (‘Isoton’, Coulter) to inhibit coagulation and platelet clumping. After bleeding had ceased, the tube contents were allowed to sediment overnight. The following day, platelets in the upper (plateletrich) layer were counted electronically (Coulter). After shaking and further dilution the red blood cells were similarly counted. Effects of 500U heparin and 20 mg/kg aspirin (i.p., 1h. before bleeding) were examined. Both drugs markedly increased platelet and RBC efflux from the hemostatic site, but with characteristic differences in the time curves observed. An unexpected finding was that an incision in one ear could suppress bleeding from an incision subsequently made in the second ear. A previously undiscovered reflex or blood-borne hormone may be responsible.