318 DIFCO MANUAL 



3. Bacto-Calcium Chloride 0.02 Molar or dissolve 0.222 gram of anhydrous 

 reagent grade calcium chloride in 100 ml. distilled water. 



4. Bacto-Sodium Chloride 0.85 per cent or dissolve 0.85 gram reagent grade 

 sodium chloride in 100 ml. distilled water. 



Procedure: 



Obtain 4.5 ml. of blood by venipuncture and immediately mix it with 0.5 ml. 

 of sodium oxalate solution. Within one-half hour centrifuge the oxalated blood 

 to separate the plasma from the formed elements. 



Place the tubes containing thromboplastin extract and the tubes containing 

 calcium chloride solution in the 37 °C. water bath to bring them to this working 

 temperature. 



Transfer 0.1 ml. of plasma to a small dry serologic tube supported by a rack 

 in the 37 °C. water bath. Add 0.1 ml. thromboplastin extract and twirl the tube 

 to mix the contents. After the tube has been in the water bath a few seconds, add 

 0.1 ml. calcium chloride solution by blowing it forcibly into the mixture and 

 simultaneously start the stop watch. Keep the tube in the water bath and shake 

 it lightly until within a few seconds of the expected clotting time; then by gently 

 tilting the tube in a horizontal position obseive the formation of the clot or 

 fibrin web which is the end point. 



Many laboratories may prefer to use the following loop technique. Imme- 

 diately upon the addition of the calcium chloride solution and starting of the 

 stop watch, insert a clean iron, nichrome or platinum wire with a terminal loop 

 about ^" in diameter into the tube and stir thoroughly. Draw the loop from the 

 back of the tube to the front through the plasma-thromboplastin-calcium chloride 

 mixture at the rate of 2 sweeps per second, and observe the time at which the 

 clot forms. When the clot forms it will usually adhere to the loop and is easily 

 recognizable. Stop the watch at the first evidence of the clot and record the time 

 required for the plasma to clot after adding the calcium chloride to the plasma- 

 thromboplastin mixture. This is the prothrombin time. 



The time elapsing between the addition of calcium chloride and the end point 

 is the prothrombin time. The test should be run in duplicate or triplicate to 

 obtain the minimum clotting time. 



Some laboratories find it helpful to check the prothrombin time of the test 

 plasma diluted with an equal volume of 0.85 per cent sodium chloride solution 

 as well as the whole plasma. Where such a dilution of the test plasma is made, 

 this must be taken into consideration when interpolating the prothrombin 

 activity. 



For determining the prothrombin activity of a patient's plasma by the Quick 

 method it is desirable to construct a prothrombin activity curve for a given 

 thromboplastin using several normal human plasmas. The following is a typical 

 example : 



This is a typical prothrombin activity curve constructed with Bacto-Thrombo- 

 plastin using normal human plasmas. It was constructed by running prothrombin 

 time tests on 10, 20, 30, 40, 60 and 100 per cent plasma of normal individuals, 

 and plotting the prothrombin time against per cent prothrombin employed. The 

 indicated concentration of plasmas used were obtained by diluting the normal 

 plasmas with 0.85 per cent sodium chloride solution. 



The prothrombin activity of the blood to be tested is obtained by determining 

 the prothrombin time of the whole plasma, locating this time on the prothrom- 

 bin activity curve and, by means of the curve, converting the prothrombin time 

 to the per cent of prothrombin activity. 



A curve is submitted with each lot of Bacto-Thromboplastin to serve as a guide 

 for laboratories using Bacto-Thromboplastin according to the method of Quick. 



