Storage Stability in Plastic and Glass Containers of Oxalated Plasma for Prothrombin Time Determinations

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1 Storage Stability in Plastic and Glass Containers of Oxalated Plasma for Prothrombin Time Determinations H. BRADLEY HILL, MT (ASCP), EDMUND M. Y. LOW, M.D., AND IRWIN SCHOEN, M.D. Department of Pathology, University of California, Irvine-California College of Medicine, and Division of Pathology, Cedars-Sinai Medical Center, Mount Sinai Hospital Division, Los Angeles, California ABSTRACT Hill, H. Bradley, Low, Edmund M. Y., and Schoen, Irwin: Storage stability in plastic and glass containers of oxalated plasma for prothrombin time determinations. Amer. J. Clin. Path. S3: , 197. The container type {i.e., glass, siliconized glass, or plastic) used for the collection of oxalated plasma for prothrombin-time determination is not of clinical significance. If the specimen container, regardless of type, is stoppered, with less than 1 cm. air space, the effect of standing at room temperature for 15 min. to 24 hr. collection of blood is not clinically significant. Thus, the collection and storage of oxalated blood specimens does not require stringent precautions except that the container should be filled and kept stoppered; a large number of prothrombin time determinations can be done easily and efficiently without undue concern about the room-temperature storage stability of the specimens. The storage stability of restoppered specimens provides a quality control feature in the routine laboratory because it enables the laboratory to confirm reports which are unexpected and challenged by the physician. AMONG the many areas of uncertainty re- lated blood specimens (in stoppered and ungarding performance of large numbers of stoppered containers) for as long as 24 hr. prothrombin time determinations, such as prior to determination. In view of apparthe necessity for preheating patient plasma ently conflicting reports in the literature, 1-2 and the feasibility of continual preheating we have studied methods of specimen colof large quantities of thromboplastin, 3 have lection and room temperature storage stabeen the methods of collection and the ef- bility of the specimens, fects of room temperature storage of oxalated specimens. The points in question Materials and Methods include the significance of collection of oxa- A11 studies were done usin S oxalated lated specimens in glass, siliconized glass, blood specimens collected as 4.5 ml. of and plastic containers; and the effects of blood in - 5 ml of.1 M sodium oxalate, storage at room temperature on the oxa- A11 prothrombin time determinations were performed using a Fibrometer.* Received August 27, 1969; accepted for publication October 17, * B-D Laboratories, Inc., Rutherford, New Jersey. 918 Downloaded from by guest on January 13, 217

2 June 197 STORAGE STABILITY OF OXALATED PLASMA 919 Table 1. Changes in Prothrombin Time in the First Three Hours of Specimen in Glass Vacutainer 2-6 Min Min. 5-9 Min Min Min. Overall Change (sec!) Group 1. Effect on Prothrombin Time of Three Hours' Storage of Plasma Blood from hospitalized patients was collected in 5-ml. oxalated Vacutainer * tubes and centrifuged; the samples were aspirated from the supernatant plasma. Prothrombin time determinations were done at 3- min. intervals, beginning 2 min. to 1 hr. venipuncture, for a period of 2 hr. following the initial determination. The specimen tubes were stoppered when centrifuged, and they were restoppered each sampling. Group 2. Initial Effects of Glass, Siliconized Glass, and Plastic on Prothrombin Time To study the possible effects of glass, siliconized glass, and plastic containers on prothrombin time, blood was drawn into two 5-ml. oxalated Vacutainer tubes and into four siliconized oxalated Vacutainer glass tubes. The blood from two of the latter was dispensed into small plastic tubes so that the air space between stopper and blood was less than 1 cm. Following centrifugation of the stoppered tubes, prothrombin times were determined using one tube of each container type. These initial determinations were done within 2 hr. except for three specimens examined 3 to 4 hr. collection. Group Hour Storage Stability of Plasma in Glass, Siliconized Glass, and Plastic Containers Stoppered and Unstoppered To investigate the stability of specimens stored at room temperature for 24 hr. in glass, siliconized glass, and plastic containers, the specimens from Group 2 were stored at room temperature with one tube of each pair stoppered and the other un- Downloaded from by guest on January 13, 217

3 92 HILL ET AL. Vol. 53 Table Z. Effects of Glass, Siliconized Glass, and Plastic Containers on Prothrombin Times as Much as Four Hours of the Specimen* Prothrombin Time Specimens in Glass Specimens in Siliconized Glass Specimens in Plastic Maximum Difference * Range of difference = to 2. sec.; mean maximum difference =.8 sec. stoppered. The reference values are derived from the initial values obtained for the unstoppered specimens in Group 2. Stoppered specimens had no more than 1 cm. of air space between blood and stopper. Unstoppered specimens, of course, were exposed to infinite air. Group 4. The More Immediate Effect of Container Type and the Effect of 24- Hour Storage of Plasma Collected, tuithout Possibility of Glass Activation In an effort to obtain shorter and more consistent time intervals between collection of blood and initial determinations than experienced in Group 2, as well as to determine the 24-hour storage stability of plasma collected and stored without possibility of glass activation due to imperfect siliconization, the following procedure was employed. Blood was drawn from the patient into a plastic syringe and dispensed into two oxalated plastic tubes which were centrifuged immediately. The prothrombin time of the plasma from one of the specimen pairs centrifuged was then determined on the Fibrometer. The time lapse from withdrawal of blood to readout did not exceed 25 min. The specimen tube was restoppered and additional prothrombintime determinations were done every 15 min. for an hour the time of specimen collection. The tube was then left unopened. The second tube of the specimen pair remained untouched and was stored at room temperature without separation of the plasma from the cells. After 24 hr. the prothrombin time was determined in both tubes. Results and Discussion Group 1 Table 1 summarizes the effects on prothrombin time of keeping plasma in stoppered containers at room temperature for periods as long as 3 hr. The mean change from the initial values to the final prothrombin times of 18 seconds or less was.4 sec. and the range of change was -.9 to +.8 sec. The mean change in prothrombin times of 19 sec. or more was.4 sec, with a range of 2.5 to +.6 sec. The data indicate that a 3-hr. delay from venipuncture to prothrombin-time determination causes no clinically significant change in prothrombin time. The effect of glass activation on prothrombin time was scarcely discernible: prothrombin time determinations done 2 min. and 2 hr. collection of blood yielded similar results. The possibility that glass activation might have occurred in the first 2 min. collection of blood is not likely, as evidenced by the similarity of Downloaded from by guest on January 13, 217

4 Original RANGE MEAN Table 3. Effects of Glass, Siliconized Glass, and Plastic Containers on Prothrombin Times of Oxalated Blood Specimens 24 Hours' Storage Specimens Stored in Glass Spec mens Stored in Siliconized Glass Specimens Stored in Plastic Stoppered Change in 24 Hr. Unstoppered Original Value Change Stoppered in 24 Hr. Unstoppered Original Value Change Stoppered in 24 Hr. Unstoppered to to to to to to Downloaded from by guest on January 13, 217

5 922 HILL ET AL. Vol. 53 Table 4. Changes in Prothrombin Times from 2 Minutes to 8 Minutes and to 24 Hours of Specimens Avoiding Possible Glass Activation* Prothrombin Prothrombin Prothrombin Time 2 Time 8 Time 24 Min. Min. Change at Hours Change at 8 Min. 24 Hours Range of change.2 to +3.2 sec. +.8 to +3.5 sec. MEAN +.7 sec sec. results using plastic, siliconized glass, and glass for collection and storage, as noted in the findings of Groups 2 and 4 below. Group 2 The results of a comparative study of the effects of glass, siliconized glass, and plastic containers on prothrombin time, determined usually within 2 hr., and as long as 4 hr., specimen collection are listed in Table 2. The mean initial prothrombin times for the three container types differed by a maximum of.8 sec. for specimens with values less than 2 sec. and 2. for specimens with values of greater than 2 sec. Therefore, we concluded that for routine determination of prothrombin times the type of container used for the plasma specimen can be left to the individual laboratory's choice. Group 3 It is apparent from the data in Table 3 that the three container types all provide good and comparable 24-hr. storage stability of stoppered specimens, provided the blood is stored with less than 1 cm. of air space between the blood in the tube and the base of the stopper. These findings confirm those of Schoen and associates, 3 and the stability mechanism is unrelated to the cancellation effect of glass activation upon Factor V instability. The findings of Barrington and Peterson 2 remain unexplained, although it is likely that their technic resulted in the loss of C 2 or alkalinization of their stored specimens, or both. Since they prepared their own oxalated plastic tubes, it is likely that the oxalate was impure, or decomposed in part to carbonate, to account for alkalinization of the blood specimen with subsequent deterioration of Factor V, and the prolongation of the prothrombin time storage of the stoppered specimen. There is very poor stability in the unstoppered specimen in all container types, with the plastic container Downloaded from by guest on January 13, 217

6 June 197 STORAGE STABILITY OF OXALATED PLASMA 923 exhibiting the most dramatic deterioration. It is possible that glass activation is more significant in the unstoppered specimen, with a higher ph 3 accounting for the shorter prothrombin times in glass 24 hr. That the siliconized tubes behaved like the glass tubes may indicate that the siliconizing process was not complete, so that some unsiliconized glass surfaces made contact with the specimens. This is suggested by the Group 4 findings described below. Group 4 The stability data for specimens stored at room temperature for an hour and 2 min. collection of blood are summarized in Table 4. The mean change from the initial determination, made 2 min. collection of blood, to the final determination, 1 hr. and 2 min. collection of blood, was +.7 sec. These values indicate that there is no significant alteration in prothrombin time during this initial period of storage collection of blood without possible glass activation. After 24 hr. of storage at room temperature, the same samples had a mean change of +1.9 sec, range +.8 to +3.5 sec. Since these data reveal consistent prolongation of the prothrombin time, compared with the series in Group 3 which involved possible contact with glass, it may be that glass activation does cancel a small amount of the instability of the stored specimens. However, this is a small effect which could even be related to the temperature of the room during storage. No matter what the mechanism, it is not significant clinically or for the purpose of quality control. References 1. Barrington, J. D., and Peterson, E. W.: Laboratory control coumarin therapy: Quality control and the one-stage prothrombin time. Am. J. Med. Techn. 33: 296, Barrington, J. D., and Peterson, E. W.: The onestage prothrombin time: Alterations due to changes in ph. Am. J. Med. Techn. 34: 437^- 445, Schoen, I., Praphai, M., and Veiss, A.: Storage stability and quality control of prothrombin time by means of the Quick method. Am. J. Clin. Path. 37: 374, Downloaded from by guest on January 13, 217