COAGULATION AND TRANSFUSION MEDICINE Original Article

An Analysis of Duplicate Testing of Prothrombin Time and Activated Partial Thromboplastin Time Assays GARY KRAMER, M.D., AND BASIL A. BRADLOW, M.D.,

FRCPATH

1.5 to 2.5 times the baseline value, and variations of up to 25% might be considered acceptable. With these relatively lenient criteria, approximately 2% of PT and 1.3% of aPTT assays had differences between duplicate values that were unacceptable. From this data the authors concluded that the frequency of errors produced by single estimations was too great for satisfactory clinical practice. (Key words: Duplicate analyses; Prothrombin times; Activated partial thromboplastin times) Am J Clin Pathol 1991;95:77-81

Are duplicate estimations of prothrombin time (PT) and activated partial thromboplastin time (aPTT) necessary? Several authors1"7 have questioned the need for duplicate assays in view of the putative greater precision of automated instruments than manual tilt tube methods. The National Committee for Clinical Laboratory Standards (NCCLS)8 has, however, recommended that the assay "should be performed in duplicate and the mean of two values reported." Conflicting views of this recommendation have been presented in the literature, and the issue has been debated recently in the correspondence columns of the College of American Pathologists newsletter, CAP Today.9 The most recent studies of duplicate PT and aPTT tests have used the Lancer coagulyzer5,6 (Sherwood Medical, St. Louis, MO), the MLA Electra 7003, 56 (Medical Laboratory Automation, Mount Vernon, NY), and the Coag-

A-Mate® X-2 analyzer2 (General Diagnostics, Morris Plains, NJ). In the present study, the MCA 110 coagulation analyzer (Biodata Corporation, Hatboro, PA) was used to assess the reliability of single and duplicate assays in a group ofhospital inpatients in relationship to clinically acceptable criteria for purposes of diagnosis and monitoring anticoagulant therapy.

MATERIALS AND METHODS

PT and aPTT were tested on 1,277 samples from inpatients at Michael Reese Hospital. A total of 643 PT and 634 aPTT assays were selected at random as batches of 40-60 specimens from the daily workload at intervals of one to two weeks over a period of six months. All samples were collected in vacutainer tubes containing 3.8% trisodium citrate as anticoagulant in a ratio of 1:9 (volume/volume). Specimens were transported packed From the Department ofPathology, Michael Reese Hospital andinMedice, and plasma was separated in a refrigerated centriical Center, Chicago, Illinois. fuge at 4 °C. Analyses were completed within two hours of collection on a MCA TM 110 microsample coagulation Received January 18, 1990; received revised manuscript and accepted for publication April 20, 1990. analyzer (Biodata Corporation) using Thromboscreen® Address reprint requests to Dr. Bradlow: Department of Pathology, dried Thromboplastin® reagent for PT and ThromboMichael Reese Hospital and Medical Center, 31st at Lake Shore Drive, screen Kontact® aPTT reagent for aPTT (Pacific HeChicago, Illinois 60616.

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An evaluation of duplicate prothrombin time (PT) and activated partial thromboplastin time (aPTT) assays determined by the MCA 110 coagulation analyzer was undertaken to develop analytical duplicate performance criteria to quantitate the risks associated with single versus duplicate procedures. Included in the study were 1,277 patient samples. On the basis of the currently recommended therapeutic range for prothrombin ratios, a variation of approximately 10% or more between duplicates was considered to be unacceptable. For aPTT assays, the recommended therapeutic range for heparin therapy was usually

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COAGULATION AND TRANSFUSION MEDICINE

Original Article TABLE 1. DUPLICATE PROTHROMBIN TIMES (PT) IN 643 PATIENTS

Mean (seconds) SD SE of the mean Median Range

PT1

PT2

14.6 4.2 0.16 13.0 9.5-60.5

14.5 4.0 0.16 13.0 9.5-51.5

TABLE 3. AN ANALYSIS OF THE DIFFERENCES IN SECONDS BETWEEN DUPLICATE PT ASSAYS IN 643 PATIENTS

Difference 0.11 0.75 0.03 0 0-10.4

P = >0.l for the difference between PTI and PT2.

STATISTICAL ANALYSIS The statistical significance of the difference between the means of the first and second values of duplicate assays was determined using a Student Mest.10 The percentage difference between duplicate assays was calculated by dividing the difference by the means of the two readings and multiplying by 100. Correlation co-efficients between the two values of duplicate assays were calculated from linear regression analysis.10 RESULTS Tables 1 and 2 show the means and standard deviations of the first and second of duplicate assays of PT and aPTT results. The means were not significantly different (P > 0.1). The differences between duplicate assays also are shown in Table 1 and 2. Although the means of the differences are extremely small (0.11 and 0.24 for PT and aPTT, respectively) the ranges indicate the presence of a

Mean (seconds) SD SE of the mean Median Range

aPTT2

Difference

34.2 15.5 0.62 28.9 11.3-193.4

34.5 16.1 0.64 29.1 11.2-197.2

0.25 3.0 0.12 0 0-32.7

P > 0.1 for the difference between aPTT I and a P T O .

Percentage of Cases

0-0.5 0.6-1.5 1.6-2.5 2.6-5.0 5.1-10.5 Total

530 100 7 3 3 643

82.4 15.5 1.1 0.5 0.5 100.0

few outliers representing large differences between duplicate assays. A more detailed analysis of the differences is shown in Tables 3-8. For PT duplicates there were 13 specimens (of 643) in which the differences ranged from 1.6 to 10.5 seconds (Tables 3 and 7) and an equal number in which the percentage differences ranged from 10% to 59.1% (Tables 4 and 8). Differences between duplicate aPTT assays exceeded 10 seconds in 11 cases, including 2 in which the differences were more than 30 seconds (Tables 5 and 7). The percentage difference between duplicates exceeded 15 in 11 cases, 25 in 8 cases, and 50 in 5 cases (Tables 6 and 8).

DISCUSSION To evaluate the clinical significance of the difference between duplicates it is necessary to define criteria of acceptability for the variance between assays. Some authors"" 13 have used the concept of medically useful criteria or precision limits based on questionnaire surveys of physicians for many analytes. Skendzel and colleagues13 arrived at a "medically useful" coefficient of variation for prothrombin time of 15.2%, whereas Ross and co-

TABLE 4. AN ANALYSIS OF THE PERCENTAGE DIFFERENCE BETWEEN DUPLICATE PT ASSAYS IN 643 PATIENTS

TABLE 2. DUPLICATE ACTIVATED PARTIAL THROMBOPLASTIN TIMES (aPTT) IN 634 PATIENTS aPTTl

Number of Cases

Percentage Difference*

Number of Cases

Percentage of Cases

0-2.0 2.1-4.0 4.1-7.0 7.0-10.0 10.1-20.0 59.1 Total

421 118 77 14 12 1 643

65.4 18.3 12.0 2.2 1.9 0.2 100.0

' Difference between duplicates divided by the mean value and multiplied by 100.

A.J.C.P. • January 1991

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mostasis, Ventura, CA). The MCA 110 analyzer uses a photometric system to detect changes in optical density. Plasma or whole blood can be used in appropriately designed cuvettes, but in this study only plasma was used. The cuvettes are designed to deliver a measured volume of plasma (25 /xL) so that precision pipettes are unnecessary. Maximum recordable clotting times were 100 seconds for PT and 200 seconds for aPTT.

Difference in Seconds

KRAMER AND

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BRADLOW

Duplicate Testing of Assays TABLE 7. PT RESULTS WITH DIFFERENCES BETWEEN DUPLICATES OF M O R E T H A N 1.2 SECONDS A N D MORE THAN 10%

TABLE 5. A N A N A L Y S I S OF THE DIFFERENCES IN SECONDS BETWEEN DUPLICATE aPTT A S S A Y S IN 6 3 4 PATIENTS Number of Cases

Percentage of Cases

Case No.

PT1

PT2

Difference in Seconds

Differences*

0-2.0 2.1-4.0 4.1-5.0 5.1-10.0 10.1-20.0 20.1-30.0 30.1-33.0 Total

569 41 5 8 6 3 2 634

89.7 6.2 0.8 1.3 1.0 0.5 0.3 100.0

1 2 3 4 5 6 7 8 9 10 11 12 13

13.8 27.4 22.4 14.2 13.5 12.1 19.9 11.6 13.1 60.5 29.2 18.0 12.4

12.5 24.7 24.9 15.8 12.1 13.5 17.8 13.1 15.0 51.5 34.8 15.1 22.8

1.3 2.7 2.5 1.6 1.4 1.4 2.1 1.5 1.9 9.0 5.6 2.9 10.4

10.2 10.4 10.6 10.7 10.9 10.9 11.2 12.1 13.6 16.1 17.5 17.6 59.0

• Difference between duplicates divided by the mean value multiplied by 100.

cedures or for other medical indications. For the present study a criterion of clinically acceptable precision based upon currently recommended guidelines for anticoagulant therapy has been developed and is described. For warfarin anticoagulation, the American College of Chest Physicians and the National Heart Lung and Blood Institute (ACCP/NHLBI)15 have recommended a therapeutic range (TR) of 2.0 to 2.5 for the international normalized ratio (INR) for the prophylaxis of venous thromboembolism. The thromboplastin used in the present study had an international sensitivity index (ISI) of 2.0 so that the TR corresponding to those INR values was 1.4 to 1.7. The midpoint of this range is 1.55, and the difference between this value and each of the outer limits is 0.15 and equal to 9.7% of 1.55. It can then be argued that an error greater than 9.7% could place a patient in

TABLE 8. aPTT RESULTS WITH DIFFERENCES BETWEEN DUPLICATES OF MORE T H A N 10 SECONDS A N D MORE T H A N 1 0 %

TABLE 6. A N A N A L Y S I S OF THE PERCENTAGE DIFFERENCE BETWEEN DUPLICATE aPTT A S S A Y S I N 6 3 4 PATIENTS Percentage* Difference

Number of Cases

Percentage of Cases

0-2.0 2.1-4.0 4.1-6.0 6.1-10.0 10.1-15.0 15.1-25.0 25.1-50.0 50.1-70.0 95.4 Total

357 174 51 30 11 3 3 4 1 634

56.3 26.2 9.3 4.7 1.7 0.5 0.5 0.6 0.2 100.0

Di(Terence between duplicates divided by the mean value and multiplied by 100.

%

Case No.

aPTTl

aPTT2

Difference in Seconds

Differences'

1 2 3 4 5 6 7 8 9 10 11

90.1 56.0 72.5 47.1 47.1 23.6 52.8 37.6 43.4 19.2 17.1

101.3 45.5 57.1 62.2 62.2 34.1 30.8 65.0 76.1 39.5 48.3

10.6 10.5 15.4 15.1 15.1 10.5 22.0 27.4 32.7 20.3 31.2

11.0 20.7 23.8 27.6 27.6 36.5 52.6 53.4 54.8 69.3 95.4

' Difference between duplicates divided by the mean value and multiplied by 100.

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workers12 suggested values of 6.4% and 10.4% for PTs of 11.0 and 27.0 seconds, respectively, and values of 10.8% and 16.0% for PTTs of 30.0 and 75.0 seconds, respectively. Others have used an arbitrary value of 5% for PT3'6,14 and 15% for PTT.6 The National Committee for Clinical Laboratory Standards (NCCLS)8 recommends that the mean of duplicate assays be used for both PT and aPTT but does not indicate a level of difference that should be regarded as unacceptable. More general considerations have been discussed by Ross and colleagues,12 who suggest that a laboratory "error occurs only when an outlier deviates from the true value by an amount and in circumstances that place a patient in an incorrect clinical category with a strong statistical probability." In the case of PT and aPTT a basis for such a definition has not been established. Patients on anticoagulant therapy for thromboembolism are exposed to the risk of hemorrhage if overanticoagulated and to possible recurrence of thromboses if undertreated. Therefore, these patients require greater precision of the PT and aPTT tests used to monitor their therapy than do patients tested for evidence of a hemostatic defect prior to invasive pro-

i

%

Difference in Seconds

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COAGULATION AND TRANSFUSION MEDICINE Article

A.J.C.P. •

garded as clinically significant would be correspondingly greater. Duplicate testing may not prevent all possible errors. The use of delta checks has been advocated as an alternative method of detecting errors.3'7 These two approaches are not mutually exclusive, and a combination of the two can be expected to contribute to enhanced reliability of results. The instrument used in the present study was the MCA 110 coagulation analyzer. Despite that the operator does not need to pipette a measured volume into the cuvette, significant discrepancies occurred because of other variables within the instrument. This has been true of duplicate analyses in all of the other instruments tested2,3'5'6 and appears to be inevitable with coagulation assays as well. Unlike chemical estimations, the coagulation tests involve functional assays based on a cascade of enzyme reactions, using impure biological reagents and must measure an imprecise end-point, namely, the formation of a fibrin clot. CONCLUSION Despite the close correlation between the mean values of duplicate assays and the small mean difference between them as well as the high correlation coefficients between assays, there are a recurring number of cases in which clinically significant discrepancies occur. If single estimations only were done, there would be a small but probably unavoidable number of patients each day who would receive incorrect doses of warfarin or heparin. The use of duplicate testing would reveal at the least some, and possibly most, of these discrepant results. Together with appropriate delta checks, errors could be reduced to a low level. In this situation, considerations of quality patient care outweigh the relatively small savings in reagent costs resulting from single estimations. Acknowledgments. The authors thank Dr. M.A. Swerdlow, Chairman of Pathology, for encouragement, provision of facilities, and permission to publish. They thank Dr. P. Cano for valuable discussions and statistical advice, Patricia Cobb and Bronislava Shahnovsky for expert technical assistance, and Betty Walovitch for typing the manuscript. REFERENCES 1. Correlison GS, Rollins A, Santoro SA. Duplicate coagulation assays warranted. Am J Clin Pathol 1985;84:261-262. 2. Hohl L, Soloway HB. Need prethrombin times and PTT's be run in duplicate. Pathologist 1985; 10:38. 3. Keshgegian AA, Mann JM, Cooper JH. Duplicate testing for prethrombin time and activated partial thromboplastin time necessary. Arch Pathol Lab Med 1986;110:520-522. 4. Morris MW, Morten BG, Winkleman JW. Modifications for expense reduction. Part 2. Hematology. Lab Med 1984:15:670-672.

1991

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an inappropriate clinical category that is either in or out of the TR, depending upon the direction of the error. Clearly, if a wider TR were used, the limits of tolerance for variation in the PT also would be wider. If this criterion is used, approximately 2% of patients in the present study would receive results that were significantly in error, based upon a defined criterion of clinical utility, if duplicate assays were not done. Many large hospital laboratories perform 100 or more PTs daily, in which case two or more patients each day might receive incorrect dosages because of laboratory errors in their PT results. A similar criterion can be calculated for the aPTT. A widely recommended TR for heparin therapy is 1.5 to 2.5 times the baseline value for the treatment of venous thromboembolism.16 The midpoint of this range is 2.0, and the difference between this point and the outer limits of the range is 0.5, or 25% of the midpoint value. The percentage of cases in which duplicates exceeded 25% was 1.26 in this study. Thus, more than one patient in every 100 could receive incorrect dosages if errors of 25% or more were not detected by a single estimation. For prophylaxis of thromboembolism, lower doses and a narrower TR are used. Therefore, it would be necessary to regard duplicates that varied by a much lower figure than 25% as unacceptable in such cases. The tolerance limits derived from the criteria discussed are wider than those used by several authors.3'614 Nevertheless, even with these relatively lenient limits, a small number of clinically significant errors would occur each day. If duplicate analyses, rather than single analysis, would prevent these few errors, is the cost worth the benefit? The present study does not provide data to indicate that duplicates would prevent all of the errors that would occur with single estimations. However, experience in our laboratory indicates that repeat testing of discordant duplicates almost invariably results in close agreement between the second set of duplicates and a result close to one of the original estimations. Others have reported similar experiences.1,3 The cost savings that would result from single versus duplicate testing would be confined largely to reduced use of reagents and would have little impact on technician time or turnaround time. Precise data are not available, but the dollar amount involved would have to be weighed against the reduced quality of patient care for those three or more patients who receive incorrect dosages each day. Furthermore, the laboratory director may well have tofieldcomplaints from clinical colleagues and/or patients at the rate of three or more per day in view of questionable PT or aPTT results. If more stringent limits are used, then of course the number of errors re-

KRAMER AND BRADLOW Duplicate Testing of Assays 5. Sage-el A, Burns E, Wenz B. The unwarranted use of replicate analysis in routine coagulation assays. Am J Clin Pathol 1985;83:81-83. 6. Scheer WD, Catrou PG, Lipscomb GE, Boudreau RA. A comprehensive evaluation of the performance of duplicate prothrombin time and activated partial thromboplastin time assays. Am J Clin Pathol 1986;85:456-462. 7. Wenz B, Burns E, Sage-el A. Authors' reply. We are not in agreement (letter). Am J Clin Pathol 1985;84:262. 8. National Committee for Clinical Laboratory Standards. Proposed guidelines for the one stage prothromboin time test (PT) (1982;2( 11 ):371) and for the activated partial thromboplastin time test (APTT) (1982;2( 12):391). Villanova, Pennsylvania: National Committee for General Laboratory Standards. 9. Brandt JT. Are duplicate PT's and aPTT's needed? CAP Today 1989;3:21-23. 10. Korin BP. Introduction to statistical methods. Cambridge, Massachusetts: Winthrop Publishers Inc., 1977; 276-293.

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11. Ross JW, Fraser MD. Analytical clinical laboratory precision state of the art for twenty-nine analytes. Am J Clin Pathol 1979;72: 265-273. 12. Ross JW, Fraser MD, Moore TD. Analytical clinical laboratory precision. State of the art for thirty-one analytes. Am J Clin Pathol 1980;74:521-530. 13. Skendzel LA, Barnett RN, Piatt R. Medically useful criteria for analytic performance of laboratory tests. Am J Clin Pathol 1985;83: 200-205. 14. Thomas JM. Laboratory control of anticoagulant therapy. In: Thomson JM, ed. Blood coagulation and haemostasis. A practical guide. Edinburgh: Churchill Livingstone, 1980; 279-330. 15. Hirsh J, Poller L, Deykin D, Levine M, Dalen JE. Optimal therapeutic range for oral anticoagulants. Chest 1989;95(Suppl):5SUS. 16. Bloom AL, Thomas DP. Treatment of venous thromboembolism. In: Haemostasis and thrombosis. 2nd ed. Edinburgh: Churchill Livingstone, 1987; 824.

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