The Graphic Method of Measuring the Coagulation Time
In order to avoid, so far as possible, the personal element in determining when the blood was clotted, the blood was made to record its own clotting. The instrument by means of which this was done was the graphic coagulometer devised by W. L. Mendenhall and myself,[6] and illustrated diagrammatically in [Fig. 24]. It consists essentially of a light aluminum lever with the long arm nearly counterpoised by a weight W. The long arm is prevented from falling by a support S, and is prevented from rising by a horizontal right-angled rod reaching over the lever at R1 and fixed into the block B which turns on the axis A. Into the same block is fixed the vertical rod R2. When this rod is moved from the post P1, against which it is held by the weight of the horizontal rod R1, towards the other post P2, the check on the long arm of the lever is lifted, and if the short arm is heavier, the long arm will then rise.
Figure 24.—Diagram of the graphic coagulometer. The cannula at the right rests in a water bath not shown in this diagram. For further description see text.
The cannula C, into which the blood is received, is two centimeters in total length and slightly more than two millimeters in internal diameter. It is attached by a short piece of rubber tubing to the tapered glass tube T, five centimeters long and five millimeters in internal diameter. The upper end of this tube is surrounded by another piece of rubber which supports the tube when it is slid into the U-shaped support U, fixed directly below the end of the short arm of the lever.
By drawing the cannulas from a single piece of glass tubing and by making the distance from shoulder to upper end about twelve millimeters, receptacles of fairly uniform capacity are assured. All the dimensions, the reach of the rubber connection over the top of the cannula (2–3 millimeters), the distance of the upper rubber ring from the lower end of the glass chamber (4 centimeters), etc., were as nearly standard as possible.
A copper wire D, eight centimeters long and 0.6 millimeters in diameter, bent above into a hook and below into a small ring slightly less than two millimeters in diameter, is hung in a depression at the end of the short arm of the lever. The small ring then rests in the upper part of the cannula (see [Fig. 24]). The weight of the copper wire makes the short arm of the lever heavier than the long arm by 30 milligrams, when the delicate writing point is moving over a lightly smoked drum. Half a dozen of these standard wires are needed.
For accurate determination of the coagulation time Addis[7] has defined the following conditions as essential:
1. The blood must always be obtained under the same conditions.
2. Estimates must all be made at the same temperature.
3. The blood must always come in contact with the same amount and kind of foreign material.
4. The end point must be clear and definite and must always indicate the same degree of coagulation.
The precautions taken to fulfill these conditions were as follows:
1. Drawing the blood.—The blood was taken from the femoral artery. The artery (usually the right) was laid bare in the groin and freed from surrounding tissue. A narrow artery clip, with each limb enclosed in soft rubber tubing (to prevent injury of the tissues), and with its spring exerting gentle pressure, was placed on the artery immediately below the deep femoral branch, thus allowing no blood to stagnate above the clip. Between the clip and a ligature applied about 1.5 centimeters below, an opening was made. The blood was carefully milked out of the vessels between a blunt dissector moved beneath, and a small forceps, twisted into a pinch of absorbent cotton, moved above.
The cannula, cleaned in water, alcohol, and ether, was set in the rubber connection of the glass tube; the point of the cannula was then lubricated with vaseline and slipped into the artery. The pressure of the clip on the artery was next very slightly released and blood was allowed to flow into the cannula up to the lower border of the rubber connection. Only a good-sized drop of blood was needed. Sometimes the blood ran one or two millimeters above or below, but without appreciably changing the result. Since the clip was situated on the femoral immediately below a branch in which the circulation persisted, the blood received in the cannula was always fresh from the moving stream. As soon as the clip gripped the artery again, the cannula was slipped out. A helper then promptly milked the vessel in the manner described above, and covered it with a pad of absorbent cotton smeared with vaseline to prevent drying. Thereby blood was not permitted to stagnate; and when a new sample was to be taken, the vessel was clean and ready for use.
The tip of the cannula was at once plugged by plunging it into a flat mound of plasticine about three millimeters high. It was drawn off sidewise lest the plasticine plug be pulled out again. One of the copper wires D was now slid into the tube and cannula, the tube slipped into the U-support, and the wire lifted and hung on the lever. This procedure, from the moment blood began to flow until the wire was hung, consumed usually about twenty seconds.
2. Uniform temperature.—Under the U-support was placed a large water bath, in which the cannula and the tapering part of the tube were submerged. A thermometer was fixed to the U-support so that the bulb came near the cannula in the bath. The water was kept within a degree of 25° C. This temperature was chosen for several reasons: (a) The cannula has room temperature and rapidly cools the small volume of blood that enters it. To heat blood and cannula to body temperature would take time. A bath near room temperature, therefore, seems preferable to one near body temperature. (b) The test of clotting was conveniently made at intervals of a half-minute, and if the clotting process were hastened by higher temperatures, this interval would become relatively less exact. (c) A temperature of 25° C. rather than lower was selected because, as Dale and Laidlaw[8] have shown, the coagulation time is much slower for a given change in temperature below 25° than for the same change above. And with slowing of the process the end point, when the determination depends on supporting a weight, is less likely to be sharp. (d) The researches undertaken with use of this coagulometer were concerned with factors hastening the process. For that reason and for reason (b), a long rather than a short coagulation time for normal conditions was desirable.
3. Uniformity in the amount and kind of contact with foreign surface.—The capacity of the cannulas was fairly uniform, as stated above; the amount received in them was fairly constant; and the wire hanging in the blood presented approximately the same surface in different observations.
A further condition for insuring consistent treatment of the blood in different cases was that of making the tests for coagulation always at the same intervals. Below the writing point of the lever was set an electromagnetic signal E, which recorded half-minutes. At the moment a record was made by the signal (see first signal mark, [Fig. 25]) the clip on the artery was opened, the blood taken, and the process thus begun. In about 20 seconds the cannula was suspended in the water bath and the wire was hanging on the lever. At the next record by the signal and at every subsequent record the vertical rod R2 was pushed with the index finger from post P1 to post P2 and allowed to move back. This motion was uniform and lasted about one second. The check R1 on the long arm of the lever was thus raised, and as the wire sank in the blood the writing point rose, recording that coagulation had not taken place (see [Fig. 25]).
Figure 25.—Record (reduced two-fifths) of five successive tests of coagulation, with the animal in a uniform condition. The lower line records intervals of 30 seconds. The marks below the time record indicate the moments when the blood samples were drawn.
4. Definite end point.—As soon as the blood clotted, the weight of 30 milligrams was supported, and the failure of the lever to rise to the former height in the regular time allowed, recorded that the change had occurred.
Very rarely the swing of the lever would be checked for a moment and would then begin to move rapidly, indicating that a strand of fibrin had formed but not sufficiently strong to support the weight, and that when the strand broke, the weight quickly sank in the blood. If this occurred, the next record almost always was the short line, which signified that the weight was well supported.
A very slight strand of fibrin was able to prevent the weight from dropping, though at different times the amount of support differed, as shown by the varying length of the final lines (compare first and last series, [Fig. 25]). These variations are probably a rough indication of the degree of coagulation. In our experiments, however, the length of the final line was disregarded, and merely the fact that the lever failed to swing through its usual distance was taken as evidence of a clot, and the consequent short record was taken as the end point.
As soon as this end point was registered, the tube, wire and cannula were lifted out of the bath; the cannula was then separated from the tube and pulled away from the wire. The clot was thus disclosed, confirming the graphic record.
The method, at least when used at half-minute intervals, did not reveal in all instances the same degree of clotting. Usually, when the process was very rapid, the revealed clot was a thick jelly; whereas, when the process was slow, a strand of fibrin or at most a small amount of jelly was found. This difference in the degree of coagulation introduced, of course, an element of inexactness. In our experiments, however, this inexactness was unfavorable to the result we were seeking for, i. e., the acceleration of the process—because the jelly is a later stage than the fibrin strand; and since we nevertheless obtained good evidence of acceleration, we did not in these experiments attempt to determine more accurately differences in the stage of the clotting process.
5. Cleaning of apparatus.—After the wire was removed from the tube, the clot attached to its ring-tip was carefully brushed away under cool running water. Under the running water, also, a trimmed feather was introduced into the cannula and the tube to push out the plasticine and to wash out the blood. Wire, cannula and tube were then dropped into a beaker receiving running hot water (about 80° C.) and there allowed to remain for about five minutes. On removal from this the parts were shaken free from water, passed through 95 per cent alcohol and again shaken free, passed through ether and let dry.
By having a half-dozen cannulas and wires of standard size, it was possible to save trouble by cleaning a number at one time.
Not infrequently the first few samples of blood taken from an animal showed rapid or somewhat irregular rates of clotting. Some causes for these initial variations will be presented in following pages. The fairly uniform rate of clotting in any individual after the initial stage, varied in twenty-one different animals from an average of 3 to an average of 10.6 minutes, with a combined average of 5.9 minutes. The conditions for these variations among the individuals have not been wholly determined.