DIAGNOSTIC REAGENTS 321 



longed clotting times. Deeply opaque or dense thromboplastin extracts 

 result through excessive agitation of the suspension during the extraction 

 period and may give unsatisfactory results. Unfiltered extracts which are 

 not deeply opaque even though they contain gross particles of brain tissue 

 generally give satisfactory results. 



4. Temperature for Extraction of the Thromboplastin. 



A temperature of 45-48° C, for 10 minutes is most satisfactory. Tempera- 

 tures in excess of 50° C, will cause deterioration of the thromboplastin 

 principle. The optimum temperature for performing the prothrombin time 

 test is 36-38°C. 



5. Aging of Thromboplastin Extract. 



Thromboplastin extract is subject to deterioration. There should be no 

 loss of potency of the extract if used within 6 hours after its preparation. 

 Prolonged aging results in a gradual loss in potency. Extracts held over 

 from one day to another should be frozen solid to retard deterioration. 

 These frozen extracts should be thawed and incubated for 5 minutes at 

 37°C., before being used. Extracts properly frozen will retain full potency 

 for at least two weeks. Repeated thawing and freezing of the extract is 

 not recommended. 



6. Age of Plasma at Time of Test. 



Fresh plasma should be used in running the test. Plasmas allowed to stand 

 at room temperature or ice box temperature for more than 2-3 hours may 

 give clotting times slightly higher than does fresh plasma. 



7. Calcium Chloride Addition. 



Usually if the thromboplastin extract is allowed to remain in contact with 

 the plasma for 30 seconds before the calcium chloride is added, the clot- 

 ting time is shortened in comparison with that obtained if the calcium 

 chloride is added immediately following the addition of the extract. Either 

 procedure is satisfactory if adhered to consistently. Our curve is based 

 upon the addition of calcium chloride a few seconds after the addition 

 of the thromboplastin extract. 



8. Cleanliness of Glassware. 



Soaps and other detergents used in cleaning glassware must be completely 

 removed by rinsing. Frequently glassware appears optically clean but re- 

 mains chemically contaminated with adsorbed soap or detergent. These 

 materials interfere with prothrombin procedures as reported by Leh- 

 mann.32 He also stated that results will still be irregular if the container 

 in which the blood has been collected is not chemically clean. 



9. Ionic Contamination. 



Metallic ions present in excess in the distilled water used in making the 

 saline for extraction of the thromboplastin may lead to prolonged clotting 

 times. Water from copper stills or water which has been in contact with 

 copper vessels may contain enough copper to be detrimental. 



10. Agitation of Plasma-Thromboplastin-Calcium Chloride Mixture. 



Failure to mix thoroughly the reagents with the plasma at the time of 

 addition may result in a prolonged clotting time. Also, excessive agitation 

 of the plasma-thromboplastin-calcium chloride mixture will often prolong 

 the clotting time. 



11. Silicone coated apparatus. 



Some laboratories prefer to use syringes, needles and glassware that have 

 been coated with silicone for handling blood for prothrombin time deter- 

 minations. Where silicone coated glassware is used, the prothrombin time 

 will be slightly prolonged over that normally obtained with uncoated glass- 

 ware. According to Quick,^^ one should use 0.01 Molar Calcium Chloride 



