Standardization of Prothrombin and Activated Partial Thromboplastin Time Reagents and Controls BILLIE B. PATTERSON, B.S., M.T. (ASCP), AND JERRY L. PULS, M.D., F.C.A.P. Department of Pathology, St. John's Hospital, Tuba, Oklahoma 74104
ABSTRACT
T H E PROBLEMS of reagent and quality control standardization of prothrombin time and activated partial thromboplastin time tests are legion.13'15'17'19,20 These standard controls should reflect the values of the geographical population pool and should be reproducible. Continuity from lot number to lot number of the reagent and of the standard control will insure accurate and comparable test results. 9,10 This will help the clinician to maintain a stable treatment regimen. Precise technic must be maintained at all times to insure accurate test results. 21 The outline presented may seem to be detailed, but adherence to this protocol results in precise, accurate and reproducible test values: variation of test values when using different lots of reagent and standard control material will be negligible.2"3,7 A new lot number for control standard and for reagent need be selected only once or Received March 6, 1975; received revised manuscript May 13, 1975; accepted for publication May 13, 1975. Address reprint requests to B. B. Patterson.
twice a year; therefore, the time spent in their selection is nominal and is well spent. 24 Materials and Methods Reagents Fresh Normal Plasma. Draw a sample of blood from a "normal" donor and put 4.5 ml. of the blood into a 13 X 100 mm. test tube containing 0.5 ml. of 3.8% sodium citrate or use the equivalent Vacutainer tube. Centrifuge the sample for 5 minutes at 2,000 rpm. Remove the top two thirds of the plasma reagent. Keep the plasma on ice until ready to use. Fresh Normal Plasma Pool. Draw samples of blood from a minimum of ten different "normal" donors, making sure they are equally divided between male and female donors. Collect the blood in the same manner as the fresh normal plasma. Pool the donor plasmas and let stand on ice for an hour. 4,5,6 (For more information see appendix.) Exhausted Plasma. Pool the plasma from about 8 - 1 0 routine prothrombin or acti-
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Patterson, Billie B., and Puis, Jerry L.: Standardization of prothrombin and activated partial thromboplastin time reagents and controls. Am J Clin Pathol 65: 213-219, 1976. T h e problems of selection and standardization of reagents, selection of quality control standards, and maintaining quality controls are outlined and discussed. Precise technic is emphasized, and sources of error and methods for correction of errors are reviewed. (Key words: Prothrombin; APTT; Reagents and controls.)
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Table 1. Preparation of Prothrombin Activity Curve Average Prothrombin Time in Seconds (3 Determinants)
Normal Pooled Plasma (ml.)
Exhausted Plasma Pool or Saline 0.85% w./v. Solution (ml.)
Prot hrom ibin Actiivity (%)
Saline Solution
Exhausted Plasma Pool
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
100 90 80 70 60 50 40 30 20 10
11.0 11.5 12.0 12.5 13.0 13.5 15.0 17.5 23.0 34.0
11.5 12.0 13.0 14.0 15.0 16.0 17.5 20.0 25.0 43.0
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vated partial thromboplastin blood samples using the photoelectric sensing type of (make sure none of the patients is on instrument. (When using the manual or heparin therapy). The plasma need not be Fibrometer* method, ellagic acid, colloidal fresh. For convenience take two 13 X 100 celite, or kaloin activators may be used.) mm. test tubes and into each put 5 ml. of the Calcium Chloride. 0.02 molar or 0.025 pool. Into each 5 ml. sample put 0.5 ml. molar, according to the manufacturer's Al(OH) 3 , mix well, and incubate at 37 C. for directions, is used. 5 minutes. Centrifuge the samples for 20 Physiologic Saline Solution. 0.85% w./v. minutes at 2,000 x g. Decant the plasma solution is used. and incubate at 37 C. for about 24 hours, or Lyophilized Plasma Control Standards. Reuntil the prothrombin time of the plasma is constitute and let stand for the length of more than 50 seconds. Aging in addition to time established by trial of different lots absorption is necessary to insure complete (see section on controls following). removal of factor V. Unaged exhausted plasma pool often has a prothrombin time Procedure of less than 50 seconds (unpublished Preparation of Prothrombin Activity Curve observations), which will alter the activity curve. T h e exhausted plasma pool will now 1. Use exhausted plasma for the diluent in contain factors XI, XII, and XIII, which preparing the activity curve when an will not be measured by the one-stage automated instrument (automatic photoprothrombin time. The exhausted plasma electric clot-sensing instrument) is used pool will contribute fibrinogen only to the to measure the prothrombin time. system being tested. 2. Use 0.85% w./v. saline solution as a Thromboplastin Reagent. Commercial diluent in preparing the activity curve when prothrombin times are to be lyophilized thromboplastin (in this laborameasured manually or by the Fibrometory pure brain thromboplastin) is reconstiter method. tuted according to the manufacturer's 1 3 n-15,16 directions. ' ' 3. Measure the prothrombin times in tripActivated Partial Thromboplastin Reagent. Ellagic acid or colloidal celite activated * Reg. TM: Becton, Dickinson and Company, reagent is the reagent of choice when one is Rutherford, N.J. 07070.
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PERCENT CONCENTRATION OF NORMAL PLASMA, SALINE DILUTION-EXHAUSTED PLASMA F I G . 1. P r o t h r o m b i n activity curve.
licate on each of the dilutions. Plot the average time in seconds versus prothrombin activity on the arithmetic graph paper. Once this activity curve is established, the curve is then used to select the new lot of thromboplastin reagent that most closely correlates with the lot in current use. (See Table 1.) T h e saline-diluted activity curve is used when prothrombin times are determined manually or on the Fibrometer. The exhausted plasma-diluted curve is used when prothrombin times are determined by a clot-sensing type of instrument (see Fig. 1). In our laboratory a prothrombin time of 11.0-11.5 seconds represents 100% activity, and a lot number of thromboplastin that produces this normal pro-
thrombin time is selected. (In this laboratory these percentage activity curves are used only to select reagents. Prothrombin times are reported in seconds only.) When doing the 10% saline dilution, extreme care must be observed. T h e clot will be small and may be difficult to visualize. If difficulty is encountered, this problem may be overcome by adding fibrinogen.t If accurate precise technic has been observed, the difference will be minimal. (Example—10% dilution using saline solution only is 36.0 seconds, actual t Commercial fibrinogen is available. Dilute this fibrinogen according to package directions and substitute this fibrinogen for the saline solution in the IOTf dilution. Fibrinogen may be obtained from General Diagnostics Division, Warner-Lambert Co., Morris Plains, N.J. 07950.
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Selection of Controls To establish an area population "normal" prothrombin time, determine the prothrombin time on the normal pool. In our laboratory, this normal pool value is usually 11.0-11.5 seconds ± 0.2 seconds using a commercial pure brain thromboplastin. Select a commercial lyophilized plasma standard control that has the same prothrombin time as the pool. Request two different lot numbers from the supplier and test both at the same time. One of the lots of lyophilized control will usually give satisfactory control prothrombin time values. It may be necessary to test several different manufacturers' products to get the standard that meets the necessary qualifications, i.e., desired test results, reproducibility, reliability, cost, etc. 14 Once this standard plasma control has been reconstituted, a consistent, convenient time to let the standard control incubate (at room temperature) should be established;
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for example, this same incubation time should be consistently used since variation of incubation times results in variation in the prothrombin times obtained. The standard plasma control continues to shorten for about one and one half hours; after that, the prothrombin time lengthens in most cases. A daily log is kept, and at the end of a month the mean ±SD for each standard plasma control is calculated. T h e values for standard plasma controls should fall within the means ± 2 SD to be statistically valid.8 The abnormal plasma controls should reflect the normal pooled plasma dilution curve. A 20% or a 10% standard control should approximate that point on the curve. The same holds true for the 1!4 or 2'/2 times the control type of standard;6.U,13,22
The activated thromboplastin normal range for our geographic area was determined by measuring the APTT's of 1,000 "normal" people and calculating the normal range (e.g., mean ± 2 SD is considered the upper limits of normal). T h e normal A P T T was less than 45 seconds, with a mean of 30 seconds. T h e A P T T standard control selected had a time of 30 seconds ± 2 seconds. For a borderline control an A P T T standard control material giving a value of 45 seconds ± 2 seconds was selected. When the control that meets your established range of normal, borderline, and abnormal is selected, order a six months' or one year's supply of that specific lot number. Approximately a month before time to reorder, start the selection for a new lot number that most closely matches the lot in current use. Now that the standard control values have been set, heed them. 12 As an added safeguard, it is good practice to run a "normal" control weekly. This can be simplified by pooling the "normals" (any plasma prothrombin time ± 0.2 seconds of
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results were 35.8, 36.0, and 36.2 seconds. 10% dilution using fibrinogen is 32.5 seconds, actual results were 32.6, 32.5, and 32.2 seconds. These results were for a particular pool and might vary slightly with another pool.) T h e differences between the saline curve and the exhausted-plasma curve are- nominal. These differences are explained by methodology. 23 T h e saline curve is used when the prothrombin time tests are done manually or with the Fibrometer. The exhausted-plasma curve is used when the prothrombin times are determined by a clot-sensing type of instrument. Remember, when using the manual or Fibrometer method the reagents are constantly agitated, increasing the surface contact. Therefore, the prothrombin times are shorter using these methods. T h e effect of more surface contact is magnified as the percentage becomes lower.
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the "normal" standard) in the morning run make sure the tip is dispensing the and letting the pool stand on ice for one reagent into the center of the test well. hour. Measure prothrombin time and 4. Rinse reservoirs and tubing each mornA P T T on this pool. ing with distilled water. 5. Keep a log of the routine instrument maintenance, and keep it current. Problem Solving Check List Automatic Pipettes. Control Problems. The following sugges- 1. Make sure pipette is dispensing correct tions should pinpoint the problems. Try the amount. suggestions in order and any problem a. Calibrate each lot number of pipette should be clarified. tips. This can be done by the mercury weight method. When the tip has 1. Reconstitute new bottle of control with been calibrated, practice with it until fresh deionized water from a clean it is possible to tell when it is filling container, using clean pipettes. and emptying correctly. 2. Run a "normal" pool consisting of four or five "normal" plasmas. b. T h e technologist must be constantly alert when using the plastic pipette 3. Open a new bottle of calcium chloride tips. Make sure the tip is dispensing and use. all of the reagent and is filling 4. Recheck the manufacturer's instruccorrectly. Discard those tips that do tions to make sure there has been no not meet these requirements. change in the instructions and that the instructions have been properly folc. When dispensing press the plunger lowed. once only. This instrument is calibrated to dispense the correct amount 5. The instrument must be in order; the without pumping, which causes it to water bath temperature must be 3 7 dispense incorrectly, and inconsis37.5 C. tently. 6. Open a new bottle of reagent. The reagent may have been contaminated, 2. Have tips that are not bent or broken, outdated, in use too long, or overheated. that are free of foreign material, and have an opening. Patient Plasma. Improper venipuncture —plasma hemolyzed—improper amount 3. When using plastic tips, it is best to have of blood in relation to anticoagulant— the reagents at room temperature. contamination with tissue fluid or with Miscellaneous. intravenous fluids—blood clotted—con- 1. When using the automatic instrument taminated with heparin. the thromboplastin should not be left on Technologist Error. Wrong reagent used— the heat block for more than three reagent incorrectly measured—improper hours. technic—wrong sample collected—wrong 2. A P T T reagent should not incubate sample tested—wrong results recorded. longer than the time specified in the Instrument. Automated photoelectric clotpackage directions. sensing type. 3. Use chemically clean glassware that has 1. Have the tubing marks in place correctly been rinsed in distilled water at least and have no excess crimping from eight times (disposable glassware, test overuse. tubes, and pipettes will solve most of 2. Never use tubing that has been these problems). Glassware should be stretched. free of soap, chemicals, dust, and other 3. Have dispensing tip in proper place and contaminants.
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Conclusion We have used this method here described for many years and as a result of these technics have greatly increased physician confidence in these test values and are able to give consistent and accurate results. We have received excellent cooperation from manufacturers. T h e smooth transition from lot number to lot number of both standard plasma controls and reagents is no longer a problem. References 1. Biggs R: Proposal for the use of a preliminary trial reference preparation of thromboplastin for standardizing control of anticoagulant therapy. F i b r i n o g e n : s t r u c t u r a l , metabolic a n d pathophysiologic aspects, T h r o m b Diath Haemorrh (Stuttg) 1969, pp 3 6 3 - 3 6 8 2. Bowerman, D, Notestine H: Quality control in coagulation studies. Am J Clin Pathol 5 1 : 5 3 5 537, 1969 X Reptilase-R Bothrops Atrox lyophilized, Reg. T M : Abbott Laboratories, Diagnostic Division, North Chicago, Illinois 60064. § Reptilase-R is a thrombin-like enzyme that has no thromboplastic activity. Like thrombin, Reptilase will clot fibrinogen and is not inhibited by heparin.
3. Bowyer FP, Stratos M, Goldenfarb P, et al: Reproducibility in the hematology laboratory. Am J Clin Pathol 57:482-486, 1972 4. Dodds WJ, Moynihan AC: Benson RE, et al: T h e value of age- and sex-matched controls for coagulation studies. Br J Haematol 2 9 : 3 0 5 317, 1975 5. Egeberg O, Owren PA: Oral contraception and blood coagulability. Br Med J Jan. 26, 1963, pp 220-221 6. Eilers RJ: Prothrombin time test standardization. F i b r i n o g e n : s t r u c t u r a l , metabolic a n d pathophysiologic aspects. T h r o m b Diath Haemorrh (Stuttg) 1969, p p 3 9 7 - 4 0 0 7. Goldenfarb PB: Reproducibility in coagulation assays. A m J Clin Pathol 55:561-564, 1971 8. Kahan J: Regional quality control of coagulation assay procedures (Normotest, Simplastin-A and Thrombotest). T h r o m b Diath H a e m o r r h (Stuttg) 32:79-89, 1974 9. Leek I, Gowland E, Poller L: T h e variability of measurements of the prothrombin time ratio in the national quality control trials: a follow-up study. Br J Haematol 28:601-612, 1974 10. Leek I, Thomson JM, Poller L: Quality control trials in the national reference thromboplastin scheme. Br J Haematol 25:453-460, 1973 11. Leoliger EA, Meuwisse-Braun JB, Muis H, et al: Laboratory control of oral anticoagulants. T h r o m b Diath Haemorrh (Stuttg) 23:569-584, 1970 12. Loeliger EA, Tocanis WK, Veltkamp JJ, et al: Standardization of results obtained with different thromboplastin preparations. Fibrinogen: structural, metabolic and pathophysiologic aspects, T h r o m b Diath Haemorrh 1969, pp 369-376 13. Miale J B , Kent JW: Standardization of the therapeutic range for oral anticoagulants based on standard reference plasmas. Am J Clin Pathol 57:80-88, 1972 14. MialeJB.LaFond: Prothrombin time standardization. A m J Clin Pathol 52:154-160, 1969 15. Miale JB, LaFond DJ: 1963 prothrombin time test survey, College of American Pathologists, Standards Committee, Subcommittee on Coagulation. A m J Clin Pathol 47:40-59, 1967 16. Murphy EA, Denson KWE: Statistical analysis of experiments performed in connection with thromboplastin standardization. Fibrinogen: structural, metabolic and pathophysiologic aspects. T h r o m b Diath Haemorrh 1969, p p 377-935 17. Poller L: The British comparative thromboplastin and its quality control J Clin Pathol 2 4 : 3 5 3 357, 1971 18. Poller L, Tabiowo A, Thomson JM: Effects of low-dose oral contraceptives on blood coagulation. Br Med J iii: 218-219, 1968 19. Poller L, Thomson JM: T h e value of thrombotest reagent (batch 934) as a thromboplastin reference preparation. Fibrinogen: structural, metabolic a n d pathophysiologic aspects. Thomb Diath Haemorrh 1969, pp 4 0 1 - 4 0 4 20. Poller L, Thomson JM, Alderson MR: T h e British system for anticoagulant control and thrombotest. J Clin Pathol 24:143-146, 1971 21. Robinson RW: Effect of estrogen-progestin
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4. Use deionized water free of Ca + + ions, electrolytes, bacteria, and mold. 5. Heparin contaimination may be detected by the Reptilase$ test.§ a. Do a thrombin time test on the plasma. b. Do a Reptilase time test. See appendix. c. The thrombin time will be prolonged if heparin is present and the Reptilase time will be normal. Reptilase time is also normal in the presence of antithrombins or immunological products, while thrombin time is prolonged. 6. A log should be kept on all equipment and checked each day before tests are begun. Correct temperature for water baths and heat blocks should be 3 7 37.5 C. Correct temperature for the refrigerators should be 4 - 8 C.
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combinations on clotting factors. Am J Obstet Gynecol September 15, 1967, pp 163-167 22. Van Horn P: Quality control of prothrombin determinations normal and abnormal prothrombin activity levels. Am J Med Technol 34:73-80, 1968 23. Zucker S, Brosious E, Cooper GR: One-stage prothrombin time survey. Am J Clin Pathol 53:340-347, 1970 24. Zucker S, Cathey MH, Sox PJ, et al: Standardization of laboratory tests for controlling anticoagulant therapy. Am J Clin Pathol 53:348354, 1970 APPENDIX
Reptilase Test Procedure
6. Repeat substituting patient's plasma for the control plasma. 7. Normal 18-20 seconds. Selection of Normal Pool An all-male pool may be used, but in this laboratory we feel that a representative sample of the population is best. We use these controls in monitoring anticoagulant therapy, as part of our coagulation profile for presurgical patients, for diagnostic purposes, as a part of our open-heart surgery work-up, and sometimes as a part of a liver function profile. Most of our "normal pool" comes from "normal" hospital personnel that we can persuade to "donate," also from yearly physicals and new employees. Our only requirements are: no anticoagulant, no history of hepatic disease or hepatitis, recent illness, or history of bleeding problems. We try to include all ages and both male and female. If we happen to have blood from normal children on the day of our pool, we include this plasma also, since quite a few of our presurgical patients are children (T & A's). We do not ask the female donors whether they are taking contraceptive pills, and we do include a few women of this age group. According to Robinson and Poller, 18,21 the "pill" does not increase the one-stage prothrombin time, and the partial thromboplastin time is shortened. Our data confirm these statements. There has been no appreciable change in our pool in the last ten years (10.5-11.5 seconds). T h e more samples in the pool the better.
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1. Reconstitute Reptilase according to the directions. 2. Transfer about 0.5 ml. Reptilase to 12 x 75 mm. disposable glass test tube and incubate at 37 C. for 5 minutes. 3. Pipette 0.3 ml. of control plasma into 3 12 X 75 mm. test tubes and incubate for 2 minutes. 4. Using a wide-bore pipette, forcibly blow 0.1 ml. of the heated Reptilase into a tube of the heated plasma and simultaneously start stopwatch. 5. Quickly shake in the water bath for about 8-10 seconds. Hold to a strong light and gently tilt back and forth. The appearance of the first fibrin thread is the end point. Stop the watch. (You may use the wire loop or fibrometer technic also.) Now repeat the process with the other two tubes. The tests should match to within 1 second.
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ABSTRACT
T H E PROBLEMS of reagent and quality control standardization of prothrombin time and activated partial thromboplastin time tests are legion.13'15'17'19,20 These standard controls should reflect the values of the geographical population pool and should be reproducible. Continuity from lot number to lot number of the reagent and of the standard control will insure accurate and comparable test results. 9,10 This will help the clinician to maintain a stable treatment regimen. Precise technic must be maintained at all times to insure accurate test results. 21 The outline presented may seem to be detailed, but adherence to this protocol results in precise, accurate and reproducible test values: variation of test values when using different lots of reagent and standard control material will be negligible.2"3,7 A new lot number for control standard and for reagent need be selected only once or Received March 6, 1975; received revised manuscript May 13, 1975; accepted for publication May 13, 1975. Address reprint requests to B. B. Patterson.
twice a year; therefore, the time spent in their selection is nominal and is well spent. 24 Materials and Methods Reagents Fresh Normal Plasma. Draw a sample of blood from a "normal" donor and put 4.5 ml. of the blood into a 13 X 100 mm. test tube containing 0.5 ml. of 3.8% sodium citrate or use the equivalent Vacutainer tube. Centrifuge the sample for 5 minutes at 2,000 rpm. Remove the top two thirds of the plasma reagent. Keep the plasma on ice until ready to use. Fresh Normal Plasma Pool. Draw samples of blood from a minimum of ten different "normal" donors, making sure they are equally divided between male and female donors. Collect the blood in the same manner as the fresh normal plasma. Pool the donor plasmas and let stand on ice for an hour. 4,5,6 (For more information see appendix.) Exhausted Plasma. Pool the plasma from about 8 - 1 0 routine prothrombin or acti-
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Patterson, Billie B., and Puis, Jerry L.: Standardization of prothrombin and activated partial thromboplastin time reagents and controls. Am J Clin Pathol 65: 213-219, 1976. T h e problems of selection and standardization of reagents, selection of quality control standards, and maintaining quality controls are outlined and discussed. Precise technic is emphasized, and sources of error and methods for correction of errors are reviewed. (Key words: Prothrombin; APTT; Reagents and controls.)
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Table 1. Preparation of Prothrombin Activity Curve Average Prothrombin Time in Seconds (3 Determinants)
Normal Pooled Plasma (ml.)
Exhausted Plasma Pool or Saline 0.85% w./v. Solution (ml.)
Prot hrom ibin Actiivity (%)
Saline Solution
Exhausted Plasma Pool
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
100 90 80 70 60 50 40 30 20 10
11.0 11.5 12.0 12.5 13.0 13.5 15.0 17.5 23.0 34.0
11.5 12.0 13.0 14.0 15.0 16.0 17.5 20.0 25.0 43.0
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vated partial thromboplastin blood samples using the photoelectric sensing type of (make sure none of the patients is on instrument. (When using the manual or heparin therapy). The plasma need not be Fibrometer* method, ellagic acid, colloidal fresh. For convenience take two 13 X 100 celite, or kaloin activators may be used.) mm. test tubes and into each put 5 ml. of the Calcium Chloride. 0.02 molar or 0.025 pool. Into each 5 ml. sample put 0.5 ml. molar, according to the manufacturer's Al(OH) 3 , mix well, and incubate at 37 C. for directions, is used. 5 minutes. Centrifuge the samples for 20 Physiologic Saline Solution. 0.85% w./v. minutes at 2,000 x g. Decant the plasma solution is used. and incubate at 37 C. for about 24 hours, or Lyophilized Plasma Control Standards. Reuntil the prothrombin time of the plasma is constitute and let stand for the length of more than 50 seconds. Aging in addition to time established by trial of different lots absorption is necessary to insure complete (see section on controls following). removal of factor V. Unaged exhausted plasma pool often has a prothrombin time Procedure of less than 50 seconds (unpublished Preparation of Prothrombin Activity Curve observations), which will alter the activity curve. T h e exhausted plasma pool will now 1. Use exhausted plasma for the diluent in contain factors XI, XII, and XIII, which preparing the activity curve when an will not be measured by the one-stage automated instrument (automatic photoprothrombin time. The exhausted plasma electric clot-sensing instrument) is used pool will contribute fibrinogen only to the to measure the prothrombin time. system being tested. 2. Use 0.85% w./v. saline solution as a Thromboplastin Reagent. Commercial diluent in preparing the activity curve when prothrombin times are to be lyophilized thromboplastin (in this laborameasured manually or by the Fibrometory pure brain thromboplastin) is reconstiter method. tuted according to the manufacturer's 1 3 n-15,16 directions. ' ' 3. Measure the prothrombin times in tripActivated Partial Thromboplastin Reagent. Ellagic acid or colloidal celite activated * Reg. TM: Becton, Dickinson and Company, reagent is the reagent of choice when one is Rutherford, N.J. 07070.
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10
20
30
40
50
60
70
80
90
PERCENT CONCENTRATION OF NORMAL PLASMA, SALINE DILUTION-EXHAUSTED PLASMA F I G . 1. P r o t h r o m b i n activity curve.
licate on each of the dilutions. Plot the average time in seconds versus prothrombin activity on the arithmetic graph paper. Once this activity curve is established, the curve is then used to select the new lot of thromboplastin reagent that most closely correlates with the lot in current use. (See Table 1.) T h e saline-diluted activity curve is used when prothrombin times are determined manually or on the Fibrometer. The exhausted plasma-diluted curve is used when prothrombin times are determined by a clot-sensing type of instrument (see Fig. 1). In our laboratory a prothrombin time of 11.0-11.5 seconds represents 100% activity, and a lot number of thromboplastin that produces this normal pro-
thrombin time is selected. (In this laboratory these percentage activity curves are used only to select reagents. Prothrombin times are reported in seconds only.) When doing the 10% saline dilution, extreme care must be observed. T h e clot will be small and may be difficult to visualize. If difficulty is encountered, this problem may be overcome by adding fibrinogen.t If accurate precise technic has been observed, the difference will be minimal. (Example—10% dilution using saline solution only is 36.0 seconds, actual t Commercial fibrinogen is available. Dilute this fibrinogen according to package directions and substitute this fibrinogen for the saline solution in the IOTf dilution. Fibrinogen may be obtained from General Diagnostics Division, Warner-Lambert Co., Morris Plains, N.J. 07950.
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Selection of Controls To establish an area population "normal" prothrombin time, determine the prothrombin time on the normal pool. In our laboratory, this normal pool value is usually 11.0-11.5 seconds ± 0.2 seconds using a commercial pure brain thromboplastin. Select a commercial lyophilized plasma standard control that has the same prothrombin time as the pool. Request two different lot numbers from the supplier and test both at the same time. One of the lots of lyophilized control will usually give satisfactory control prothrombin time values. It may be necessary to test several different manufacturers' products to get the standard that meets the necessary qualifications, i.e., desired test results, reproducibility, reliability, cost, etc. 14 Once this standard plasma control has been reconstituted, a consistent, convenient time to let the standard control incubate (at room temperature) should be established;
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for example, this same incubation time should be consistently used since variation of incubation times results in variation in the prothrombin times obtained. The standard plasma control continues to shorten for about one and one half hours; after that, the prothrombin time lengthens in most cases. A daily log is kept, and at the end of a month the mean ±SD for each standard plasma control is calculated. T h e values for standard plasma controls should fall within the means ± 2 SD to be statistically valid.8 The abnormal plasma controls should reflect the normal pooled plasma dilution curve. A 20% or a 10% standard control should approximate that point on the curve. The same holds true for the 1!4 or 2'/2 times the control type of standard;6.U,13,22
The activated thromboplastin normal range for our geographic area was determined by measuring the APTT's of 1,000 "normal" people and calculating the normal range (e.g., mean ± 2 SD is considered the upper limits of normal). T h e normal A P T T was less than 45 seconds, with a mean of 30 seconds. T h e A P T T standard control selected had a time of 30 seconds ± 2 seconds. For a borderline control an A P T T standard control material giving a value of 45 seconds ± 2 seconds was selected. When the control that meets your established range of normal, borderline, and abnormal is selected, order a six months' or one year's supply of that specific lot number. Approximately a month before time to reorder, start the selection for a new lot number that most closely matches the lot in current use. Now that the standard control values have been set, heed them. 12 As an added safeguard, it is good practice to run a "normal" control weekly. This can be simplified by pooling the "normals" (any plasma prothrombin time ± 0.2 seconds of
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results were 35.8, 36.0, and 36.2 seconds. 10% dilution using fibrinogen is 32.5 seconds, actual results were 32.6, 32.5, and 32.2 seconds. These results were for a particular pool and might vary slightly with another pool.) T h e differences between the saline curve and the exhausted-plasma curve are- nominal. These differences are explained by methodology. 23 T h e saline curve is used when the prothrombin time tests are done manually or with the Fibrometer. The exhausted-plasma curve is used when the prothrombin times are determined by a clot-sensing type of instrument. Remember, when using the manual or Fibrometer method the reagents are constantly agitated, increasing the surface contact. Therefore, the prothrombin times are shorter using these methods. T h e effect of more surface contact is magnified as the percentage becomes lower.
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the "normal" standard) in the morning run make sure the tip is dispensing the and letting the pool stand on ice for one reagent into the center of the test well. hour. Measure prothrombin time and 4. Rinse reservoirs and tubing each mornA P T T on this pool. ing with distilled water. 5. Keep a log of the routine instrument maintenance, and keep it current. Problem Solving Check List Automatic Pipettes. Control Problems. The following sugges- 1. Make sure pipette is dispensing correct tions should pinpoint the problems. Try the amount. suggestions in order and any problem a. Calibrate each lot number of pipette should be clarified. tips. This can be done by the mercury weight method. When the tip has 1. Reconstitute new bottle of control with been calibrated, practice with it until fresh deionized water from a clean it is possible to tell when it is filling container, using clean pipettes. and emptying correctly. 2. Run a "normal" pool consisting of four or five "normal" plasmas. b. T h e technologist must be constantly alert when using the plastic pipette 3. Open a new bottle of calcium chloride tips. Make sure the tip is dispensing and use. all of the reagent and is filling 4. Recheck the manufacturer's instruccorrectly. Discard those tips that do tions to make sure there has been no not meet these requirements. change in the instructions and that the instructions have been properly folc. When dispensing press the plunger lowed. once only. This instrument is calibrated to dispense the correct amount 5. The instrument must be in order; the without pumping, which causes it to water bath temperature must be 3 7 dispense incorrectly, and inconsis37.5 C. tently. 6. Open a new bottle of reagent. The reagent may have been contaminated, 2. Have tips that are not bent or broken, outdated, in use too long, or overheated. that are free of foreign material, and have an opening. Patient Plasma. Improper venipuncture —plasma hemolyzed—improper amount 3. When using plastic tips, it is best to have of blood in relation to anticoagulant— the reagents at room temperature. contamination with tissue fluid or with Miscellaneous. intravenous fluids—blood clotted—con- 1. When using the automatic instrument taminated with heparin. the thromboplastin should not be left on Technologist Error. Wrong reagent used— the heat block for more than three reagent incorrectly measured—improper hours. technic—wrong sample collected—wrong 2. A P T T reagent should not incubate sample tested—wrong results recorded. longer than the time specified in the Instrument. Automated photoelectric clotpackage directions. sensing type. 3. Use chemically clean glassware that has 1. Have the tubing marks in place correctly been rinsed in distilled water at least and have no excess crimping from eight times (disposable glassware, test overuse. tubes, and pipettes will solve most of 2. Never use tubing that has been these problems). Glassware should be stretched. free of soap, chemicals, dust, and other 3. Have dispensing tip in proper place and contaminants.
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Conclusion We have used this method here described for many years and as a result of these technics have greatly increased physician confidence in these test values and are able to give consistent and accurate results. We have received excellent cooperation from manufacturers. T h e smooth transition from lot number to lot number of both standard plasma controls and reagents is no longer a problem. References 1. Biggs R: Proposal for the use of a preliminary trial reference preparation of thromboplastin for standardizing control of anticoagulant therapy. F i b r i n o g e n : s t r u c t u r a l , metabolic a n d pathophysiologic aspects, T h r o m b Diath Haemorrh (Stuttg) 1969, pp 3 6 3 - 3 6 8 2. Bowerman, D, Notestine H: Quality control in coagulation studies. Am J Clin Pathol 5 1 : 5 3 5 537, 1969 X Reptilase-R Bothrops Atrox lyophilized, Reg. T M : Abbott Laboratories, Diagnostic Division, North Chicago, Illinois 60064. § Reptilase-R is a thrombin-like enzyme that has no thromboplastic activity. Like thrombin, Reptilase will clot fibrinogen and is not inhibited by heparin.
3. Bowyer FP, Stratos M, Goldenfarb P, et al: Reproducibility in the hematology laboratory. Am J Clin Pathol 57:482-486, 1972 4. Dodds WJ, Moynihan AC: Benson RE, et al: T h e value of age- and sex-matched controls for coagulation studies. Br J Haematol 2 9 : 3 0 5 317, 1975 5. Egeberg O, Owren PA: Oral contraception and blood coagulability. Br Med J Jan. 26, 1963, pp 220-221 6. Eilers RJ: Prothrombin time test standardization. F i b r i n o g e n : s t r u c t u r a l , metabolic a n d pathophysiologic aspects. T h r o m b Diath Haemorrh (Stuttg) 1969, p p 3 9 7 - 4 0 0 7. Goldenfarb PB: Reproducibility in coagulation assays. A m J Clin Pathol 55:561-564, 1971 8. Kahan J: Regional quality control of coagulation assay procedures (Normotest, Simplastin-A and Thrombotest). T h r o m b Diath H a e m o r r h (Stuttg) 32:79-89, 1974 9. Leek I, Gowland E, Poller L: T h e variability of measurements of the prothrombin time ratio in the national quality control trials: a follow-up study. Br J Haematol 28:601-612, 1974 10. Leek I, Thomson JM, Poller L: Quality control trials in the national reference thromboplastin scheme. Br J Haematol 25:453-460, 1973 11. Leoliger EA, Meuwisse-Braun JB, Muis H, et al: Laboratory control of oral anticoagulants. T h r o m b Diath Haemorrh (Stuttg) 23:569-584, 1970 12. Loeliger EA, Tocanis WK, Veltkamp JJ, et al: Standardization of results obtained with different thromboplastin preparations. Fibrinogen: structural, metabolic and pathophysiologic aspects, T h r o m b Diath Haemorrh 1969, pp 369-376 13. Miale J B , Kent JW: Standardization of the therapeutic range for oral anticoagulants based on standard reference plasmas. Am J Clin Pathol 57:80-88, 1972 14. MialeJB.LaFond: Prothrombin time standardization. A m J Clin Pathol 52:154-160, 1969 15. Miale JB, LaFond DJ: 1963 prothrombin time test survey, College of American Pathologists, Standards Committee, Subcommittee on Coagulation. A m J Clin Pathol 47:40-59, 1967 16. Murphy EA, Denson KWE: Statistical analysis of experiments performed in connection with thromboplastin standardization. Fibrinogen: structural, metabolic and pathophysiologic aspects. T h r o m b Diath Haemorrh 1969, p p 377-935 17. Poller L: The British comparative thromboplastin and its quality control J Clin Pathol 2 4 : 3 5 3 357, 1971 18. Poller L, Tabiowo A, Thomson JM: Effects of low-dose oral contraceptives on blood coagulation. Br Med J iii: 218-219, 1968 19. Poller L, Thomson JM: T h e value of thrombotest reagent (batch 934) as a thromboplastin reference preparation. Fibrinogen: structural, metabolic a n d pathophysiologic aspects. Thomb Diath Haemorrh 1969, pp 4 0 1 - 4 0 4 20. Poller L, Thomson JM, Alderson MR: T h e British system for anticoagulant control and thrombotest. J Clin Pathol 24:143-146, 1971 21. Robinson RW: Effect of estrogen-progestin
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4. Use deionized water free of Ca + + ions, electrolytes, bacteria, and mold. 5. Heparin contaimination may be detected by the Reptilase$ test.§ a. Do a thrombin time test on the plasma. b. Do a Reptilase time test. See appendix. c. The thrombin time will be prolonged if heparin is present and the Reptilase time will be normal. Reptilase time is also normal in the presence of antithrombins or immunological products, while thrombin time is prolonged. 6. A log should be kept on all equipment and checked each day before tests are begun. Correct temperature for water baths and heat blocks should be 3 7 37.5 C. Correct temperature for the refrigerators should be 4 - 8 C.
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combinations on clotting factors. Am J Obstet Gynecol September 15, 1967, pp 163-167 22. Van Horn P: Quality control of prothrombin determinations normal and abnormal prothrombin activity levels. Am J Med Technol 34:73-80, 1968 23. Zucker S, Brosious E, Cooper GR: One-stage prothrombin time survey. Am J Clin Pathol 53:340-347, 1970 24. Zucker S, Cathey MH, Sox PJ, et al: Standardization of laboratory tests for controlling anticoagulant therapy. Am J Clin Pathol 53:348354, 1970 APPENDIX
Reptilase Test Procedure
6. Repeat substituting patient's plasma for the control plasma. 7. Normal 18-20 seconds. Selection of Normal Pool An all-male pool may be used, but in this laboratory we feel that a representative sample of the population is best. We use these controls in monitoring anticoagulant therapy, as part of our coagulation profile for presurgical patients, for diagnostic purposes, as a part of our open-heart surgery work-up, and sometimes as a part of a liver function profile. Most of our "normal pool" comes from "normal" hospital personnel that we can persuade to "donate," also from yearly physicals and new employees. Our only requirements are: no anticoagulant, no history of hepatic disease or hepatitis, recent illness, or history of bleeding problems. We try to include all ages and both male and female. If we happen to have blood from normal children on the day of our pool, we include this plasma also, since quite a few of our presurgical patients are children (T & A's). We do not ask the female donors whether they are taking contraceptive pills, and we do include a few women of this age group. According to Robinson and Poller, 18,21 the "pill" does not increase the one-stage prothrombin time, and the partial thromboplastin time is shortened. Our data confirm these statements. There has been no appreciable change in our pool in the last ten years (10.5-11.5 seconds). T h e more samples in the pool the better.
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1. Reconstitute Reptilase according to the directions. 2. Transfer about 0.5 ml. Reptilase to 12 x 75 mm. disposable glass test tube and incubate at 37 C. for 5 minutes. 3. Pipette 0.3 ml. of control plasma into 3 12 X 75 mm. test tubes and incubate for 2 minutes. 4. Using a wide-bore pipette, forcibly blow 0.1 ml. of the heated Reptilase into a tube of the heated plasma and simultaneously start stopwatch. 5. Quickly shake in the water bath for about 8-10 seconds. Hold to a strong light and gently tilt back and forth. The appearance of the first fibrin thread is the end point. Stop the watch. (You may use the wire loop or fibrometer technic also.) Now repeat the process with the other two tubes. The tests should match to within 1 second.
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