320 DIFCO MANUAL 



2. Sodium Oxalate 0.1 Molar. Dissolve 1.34 grams of anhydrous reagent grade 

 sodium oxalate in 100 ml. of distilled water. 



3. Calcium Chloride 0.02 Molar. Dissolve 0.222 gram of anhydrous reagent 

 calcium chloride in 100 ml. distilled water. 



4. Sodium Chloride 0.85 per cent. 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. 

 sodium oxalate solution. Centrifuge the oxalated blood at 1700-2000 r.p.m. to 

 separate the plasma from the formed elements. Pipette the plasma into a clean, 

 dry test tube. 



Prepare a 12.5 per cent plasma by adding 1 ml. of plasma to 7 ml. 0.85 per 

 cent sodium chloride solution in a test tube. 



Add 1 ml. of calcium chloride solution to 1 ml. of thromboplastin extract in 

 a second test tube. 



Place all reagents and plasma in a water bath at 37°C. to bring them to this 

 working temperature. 



Add 0.1 ml. diluted plasma to a small serological tube firmly secured in a 

 vertical position in a glass walled water bath at 37°G. 



Add 0.2 ml. calcium chloride-thromboplastin mixture to the tube by blowing 

 it directly into the plasma in the tube from the tip of a graduated pipette and 

 simultaneously start the stop watch. Prothrombin time may be determined either 

 by the tilting tube method or the wire loop technique described in the Quick 

 Prothrombin Time Technique discussed above. 



Stop the watch at the first evidence of the clot and record the time required 

 for the plasma to clot after adding the thromboplastin-calcium chloride mixture. 

 This is the prothrombin time of the dilute plasma. Standard values for diluted 

 normal human plasma lie between 35-42 seconds. Two or more known normal 

 plasma diluted similarly to the plasma under test should be run as controls. The 

 prothrombin time of whole plasma may be performed by using this same tech- 

 nique, but omitting the dilution of the plasma. 



Complete details for prothrombin time determinations by the Smith Bedside* 

 and Kato Micro Prothrombin Test^''^ are available upon request. 



Precautions — factors influencing prothrombin time: 



1. Storage temperature of desiccated Bacto-Thromboplastin in ampuls. 



Bacto-Thromboplastin, kept properly refrigerated below 6°C., will retain 

 its potency for years. At room temperature, 22-23 °C., there may be a slight 

 reduction in potency after one or two months. Storage temperature higher 

 than 25 °C., will accelerate destruction of the active thromboplastin prin- 

 ciple. 



2. Reagents. 



The calcium chloride, sodium chloride and sodium oxalate used in the 

 various prothrombin time tests should be anhydrous and of reagent grade. 

 Prolonged clotting times obtained with Bacto-Thromboplastin have been 

 traced most frequently to a faulty calcium chloride reagent. A calcium 

 chloride solution either more or less concentrated than that indicated for 

 use in the Quick technique may prolong the clotting time. Anhydrous 

 calcium chloride is hygroscopic, and absorbs moisture from the atmos- 

 phere. Compensation for the moisture content must be taken into con- 

 sideration when preparing the 0.02M solution. 



3. Thromboplastin Extract. 



Thromboplastin extracts that are deeply opaque or milky may give pro- 



