Whole-blood Clotting Time, Activated Partial Thromboplastin Time, and Whole-blood Recalcification Time as Heparin Monitoring Tests CHUNG-HSIN TS'AO,
PH.D.,
THERESA S. GALLUZZO, M.T.(ASCP), ROSE LO, M.T.(ASCP), AND KATHRYN G. PETERSON, M.T.(ASCP)
Ts'ao, Chung-hsin, Galluzzo, Theresa S., Lo, Rose, and Peterson, Kathryn G.: Whole-blood clotting time, activated partial thromboplastin time, and whole-blood recalcification time as heparin monitoring tests. Am J Clin Pathol 71: 17-21, 1979. The authors performed whole-blood clotting time (WBCT), activated partial thromboplastin time (APTT), and wholeblood recalcification time (WBRCT) tests on normal blood or citrated plasma, each milliliter containing 0-0.5 unit heparin, and on samples from patients, of whom many were receiving heparin anticoagulation therapy. Six partial thromboplastin reagents were used. Linearity between clotting time and heparin concentration was observed with WBCT and APTT, determined with Hyland partial thromboplastin (kaolin-activated) and Dade ("Improved" Activated Cephaloplastin and Actinw) reagents. With a General Diagnostics preparation (Platelin®-plus, celite as the activator) and another Hyland partial thromboplastin reagent (silica-activated), the sensitivity to heparin decreased to beyond 0.3 unit/ml plasma. No correlation was observed with the old Dade Activated Cephaloplastin reagent, WBRCT was completely insensitive to heparin in concentrations as high as 0.24 unit/ml blood. With patient samples, correlations were observed between WBCT and Hyland (kaolin) APTT, and between Hyland and Dade Actinw APTT. However, WBCT and WBRCT, and APTT and WBRCT, correlated poorly. (Key words: Whole-blood clotting time; Activated partial thromboplastin time; Whole-blood recalcification time; Heparin monitoring tests; Coagulation.)
Coagulation Laboratory, Department of Pathology, Northwestern Memorial Hospital and Northwestern University School of Medicine, Chicago, Illinois
Materials and Methods Blood
Samples
For in vitro studies, blood samples were obtained from 11 healthy laboratory and medical workers (two male and nine female, 23-40 years of age), with their consent. Some donated their blood more than once. Patients' blood was taken as part of the laboratory service. Blood was drawn with a 21-gauge needle attached to a disposable syringe. It was treated according to the nature of the tests. In-vitro Tests Whole Blood Clotting Time (WBCT). One-milliliter samples of blood were introduced into a series of glass tubes (10 x 75 mm) containing 0.05 ml saline solution or heparin.* The final concentrations of heparin were 0, 0.03, 0.06, 0.12, 0.18, 0.24, and 0.30 unit/ml whole blood. The WBCT of each sample was determined with a modified Lee-White procedure, using three rather than four tubes. The results are the averages of eight determinations. Whole Blood Recalcification Time (WBRCT). Blood was introduced into polypropylene tubes containing 3.8% trisodium citrate. The ratio of blood to citrate was nine volumes to one. After mixing, 3-ml samples of blood were transferred to a series of glass tubes (12 x 75 mm), each containing 0.05 ml saline solution or heparin. The final concentrations of heparin were the same as in the WBCT studies. WBRCT was de-
THE SEARCH for a simple, reliable test to monitor heparin anticoagulation has continued since the days when heparin was first used clinically. Whole-blood clotting time (WBCT) test is probably still the most widely used technic, but its shortcomings are well recognized by those who rely on it. The activated partial thromboplastin time (APTT) test has gained prominence as a substitute for the WBCT in recent years, 3 1 3 1 7 - 1 9 but its efficacy has been questioned by many. 2,512,20 Other tests, including thrombin time, 9 whole blood recalcification time (WBRCT), 4,714 and neutralization of heparin by polybrene 6 or protamine, 10 have been used by some with apparent success. Here we report our evaluations of the sensitivities of WBCT, APTT, Received March 7, 1977; received manuscript and accepted for publication December 27, 1977. Address reprint requests to Dr. Ts'ao: Department of Pathology, Northwestern University McGaw Medical Center, Wesley Pavilion, East Superior Street and Fairbanks Court, Chicago, Illinois 60611.
* Panheparin®, Abbott Laboratories, North Chicago, Illinois.
0002-9173/79/0100/0017 $00.75 © American Society of Clinical Pathologists
17
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
and WBRCT to heparin in vitro, and the efficacies of these tests in monitoring heparin anticoagulation in patients.
18
TS'AO ETAL.
aoor-
A.J.C.P. • January 1979
determined in duplicate with a set of two fibrometers.1: Six partial thromboplastin reagents obtained from three manufacturers were used: Activated Cephaloplastin,§ "Improved" Activated Cephaloplastin,§ Actin® (all Dade reagents use ellagic acid as the activator), Platelin® plus Activator11 with celite as the activator, and two Hyland partial thromboplastin reagents,** one using kaolin as the activator and the other, silica as the activator. The manufacturers' recommended procedures, including incubation time and concentration of CaCl2, were followed. Results represent five to eight determinations for each heparin concentration with each reagent. WBCT, APTT, and WBRCT of Patient Samples
0
1 0.10 0.06
1 1 0.20 0.30 Unit Heptrln/al Pitta* 0.12 0.18 Unit Hepirln/Bl Blood
1 0.40
1, 0.50
0.24
0.30
FIG. 1. Median values of APTT, WBRCT, and WBCT plotted against heparin added to whole blood (WBCT tests), citrated whole blood (WBRCT tests), or citrated plasma (APTT tests). APTT reagents are Platelin® plus (celite) (curve 1), Dade "Improved" Activated Cephaloplastin (curve 2), Hyland partial thromboplastin (kaolin-activated) (curve 3), Hyland partial thromboplastin (silicaactivated) (curve 4) and Dade Actin (curve 5).
termined in triplicate with the method described by Shanberge and associates.14 Data presented in the Results sections were, again, derived from eight determinations. Activated Partial Thromboplastin Time (APTT). Blood was transferred to Vacutainer tubest containing 3.8% trisodium citrate. The ratio of blood to citrate was nine volumes to one. After centrifugation of citrated blood for 15 minutes at 1,000 xg at 4 C, the supernatant plasma was introduced into a series of glass tubes (12 x 75 mm), each containing 0.05 ml saline solution or heparin. The final concentrations of heparin were 0, 0.05, 0.10, 0.20, 0.30, 0.40, and 0.50 unit/ml plasma. Assuming the hematocrit of the donors was 40%, these concentrations of heparin corresponded to 0, 0.03, 0.06, etc., unit/ml whole blood. APTT was t Becton-Dickinson, Rutherford, New Jersey.
Results Varying Responses of Partial Thromboplastin Reagents, and Insensitivity of WBRCT, to Heparin Added to Plasma or Blood With the old Dade Activated Cephaloplastin, we were unable to obtain a meaningful correlation between APTT and concentrations of heparin in citrated normal plasma. With the other five reagents, prolonged APTTs were observed in samples containing increasing amounts of heparin. Liner relationships were observed with Hyland (kaolin) preparation, Dade "Improved" Activated Cephaloplastin, and Dade Actin. Of these three reagents, the "Improved" Activated Cephaloplastin was most sensitive and Actin least sensitive to the range of heparin concentrations we studied. Platelin plus Activator was very sensitive to low concentrations of heparin. The sensitivity of the Hylandsilica preparation was comparable to the Hylandkaolin preparation for heparin concentrations of less than 0.3 unit/ml plasma. Beyond that, Hyland-silica became less sensitive. These results are shown in Figure 1, curves 1 to 5. A linear relationship was also obX Baltimore Biological Lab., Cockeysville, Maryland. § Dade Corporation, Miami, Florida. ' General Diagnostics, Morris Plains, New Jersey. ** Hyland Co., Costa Mesa, California.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
iol 0
Blood was drawn either with citrate-containing Vacutainers or with syringes and transferred into citrate-containing Vacutainers. WBCT, APTT, and WBRCT were determined as described above. WBCT and Hyland (kaolin) APTT tests were performed on 117 patient samples, WBCT and Platelin plus (celite) APTT tests on 36 samples, and WBCT and WBRCT on 45 samples. In addition, Hyland (kaolin) APTT and Dade Actin APTT were determined in 103 patient samples, and Hyland (kaolin) APTT and WBRCT in 75 samples.
400
300
200
100 90 80 70 60 50
FIG. 2. Scattergrams showing correlations among WBCT, APTT, and WBRCT for patient samples. The best correlations were observed between WBCT and Hyland (kaolin) APTT, and between Hyland and Actin APTT; the poorest correlations were between WBCT and WBRCT and between APTT and WBRCT.
40 30
20
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20
30
40
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Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
_l
_L
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served between WBCT and heparin, ranging from 0.03 to 0.30 unit/ml blood (Fig. 1). However, the WBRCT was almost completely insensitive to in-vitro heparin in concentrations as high as 0.24 unit/ml blood (Fig. 1). Compared with baseline values, the WBRCT showed only a 30% increase for samples containing 0.24 unit heparin/ml blood, whereas the WBRCT had quadrupled and Hyland (kaolin) APTT had almost tripled for corresponding samples. Correlations among WBCT, APTT, and WBRCT for Patient Samples
Discussion We have presented results of our investigation of the sensitivities of six commercial partial thromboplastin reagents to heparin added to citrated normal plasma, as well as WBCT and WBRCT of whole blood containing comparable amounts of heparin. The concentration of heparin we chose for our in-vitro studies was well within the range of "therapeutic levels," i.e., in causing two-to-threefold prolongation of baseline WBCT. In addition, we compared WBCT, APTT, and WBRCT in several hundred samples from patients, of whom many were receiving heparin anticoagulation therapy. We found that only three of the six partial thromboplastin reagents showed linear responses to heparin concentrations to as much as 0.5 unit/ml plasma. The other three reagents either were insensitive or had limited linearity. While WBCT also showed a linear response to increasing amounts of heparin, WBRCT was almost completely insensitive to the range of heparin we studied. Our studies of patient samples showed good correlation between WBCT and Hyland (kaolin) APTT and between Hyland (kaolin) and Actin APTT, but the correlations between WBCT and WBRCT and between APTT and WBRCT were poor. The varying results of APTT tests of heparinized or factor-deficient plasmas determined by use of different commercial partial thromboplastin reagents have been reported. 11115- ' 7 Our results agree with those earlier findings. However, we would like to point out that different results are obtained by different investigators, using apparently similar technics, with respect
to the sensitivity of a given reagent to in-vitro heparin. This is best illustrated by results reported by Shapiro and associates15 and those obtained by us. Shapiro and associates found that the Hyland partial thromboplastin (kaolin) and "new" (presumably the "Improved") Dade Activated Cephaloplastin reagents had nonlinear responses or were insensitive to heparin, whereas we found both yielded linear responses, although their sensitivities to heparin differed. Reasons for these discrepancies remain to be elucidated. Since the brand of heparin used by Shapiro and associates was different from that used by us, one possible explanation may lie in a well-known fact that different brands of heparin preparations possess different anticoagulant capacities.8 The lack of sensitivity of WBRCT to in-vitro heparin, which caused three-to-fourfold increases in APTT and WBCT, was unexpected. Our data apparently are at variance with those reported by Gulliani and colleagues.7 Those investigators found a linear relationship between WBRCT and heparin added to citrated whole blood. It should be pointed out that the amounts of heparin used in their experiments were much greater than those used in ours. Those concentrations of heparin can be achieved in patients only soon after a bolus injection. Our experience has been that a heparin monitoring test is rarely ordered so soon after the treatment. The results of our studies of patient samples concur with our in-vitro data. Generally, reliable results were obtained with those reagents and tests that were sensitive to in-vitro heparin and showed linear responses to increasing heparin concentrations. It should be stressed that some reagents and technics that were found insensitive to heparin and noncorrelative to other heparin-monitoring tests by us have been successfully used by others. The apparent successes with WBRCT of Shanberge and associates14 and Gulliani and colleagues7 are examples. Obviously, each laboratory must select a reagent or test that serves its needs best. While we have found both WBCT and APTT can be used to monitor heparin therapy, we have no evidence to show that one is overall more reliable than the other. However, the many advantages inherent in APTT cannot be matched by WBCT. References 1. Babson AL, Babson SR: Comparative evaluation of a partial thromboplastin reagent containing a non-settling particulate activator. Am J Clin Pathol 62:856-860, 1974 2. Baele G, DeBroe M, DeWeerdt GA, et al: Limitations of the activated partial thromboplastin time for determination of plasma heparin concentration and half-life in human subjects. Thromb Diath Haemorrh 27:107-113, 1972 3. Basu D, Gallus A, Hirsh J, et al: A prospective study of the value of monitoring heparin treatment with the activated partial thromboplastin time. N Engl J Med 287:324-327, 1972 4. Belko JS, Warren R: The recalcification time of blood. Its use
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
When these tests were performed on patient samples, we found good correlations between WBCT and Hyland (kaolin) APTT, and Hyland (kaolin) APTT and Dade Actin APTT (Fig. 2, top; middle left). WBCT and Platelin plus (celite) appeared to correlate less well (Fig. 2, middle right). The poorest correlations were between WBCT and WBRCT and between Hyland APTT (kaolin) and WBRCT (Fig. 2, bottom, left and right).
A.J.C.P. .January 1979
Vol. 71 • No. I
5. 6. 7. 8. 9. 10. 11. 12.
as a measure of clinical effect of heparin. Arch Surg 76:210218, 1958 Bern MM: Variable response of activated partial thromboplastin time to heparin therapy during hemodialysis. Am J Clin Pathol 64:602-607, 1975 Grann VJ, Homewood K, Golden W: Polybrene neutralization as a rapid means of monitoring blood heparin. Am J Clin Pathol 58:26-32, 1972 Gulliani GL, Hyun BH, Litten MB: Blood recalcification time. A simple and reliable test to monitor heparin therapy. Am J Clin Pathol 65:390-396, 1976 Johnson EA, Kirkwood TBL, Stirling Y, et al: Four heparin preparations. Anti-X„ potentiating effect of heparin after subcutaneous injections. Throm Haemostas 35:586-591, 1976 Penner J A: Experience with a thrombin clotting time assay for measuring heparin activity. Am J Clin Pathol 61:645-653, 1974 Pitney WR, Pettit JE, Armstrong L: Control of heparin therapy. Br Med J 4:139-141, 1970 Poller L, Thompson JM: The partial thromboplastin (cephalin) time test. J Clin Pathol 25:1038-1044, 1972 Ray PK, Harper TA: Comparison of activated recalcification and partial thromboplastin tests as controls of heparin therapy. J Lab Clin Med 77:901-907, 1971 Schriever HG, Epstein SE, Mintz MD: Statistical correlation
14. 15. 16. 17. 18. 19. 20.
21
and heparin sensitivity of activated partial thromboplastin time, whole blood coagulation time, and an automated coagulation time. Am J Clin Pathol 60:323-329, 1973 Shanberge JN, Walters J, Ambegaonkar S: The whole blood recalcification time—a convenient method for monitoring heparin therapy. Lab Med 5:30-32, 1974 Shapiro GA, Huntzinger SW, Wilson JE 111: Variation among commercial activated partial thromboplastin time reagents in response to heparin. Am J Clin Pathol 67:477-480, 1977 Sibley C, Singer JW, Wood RJ: Comparison of activated partial thromboplastin reagents. Am J Clin Pathol 59:581-586. 1973 Soloway HB, Cornett BM, Grayson JW Jr: Comparison of various partial thromboplastin reagents in the laboratory control of heparin therapy. Am J Clin Pathol 59:587-590, 1973 Stuart RK, Michel A: Monitoring heparin therapy with the activated partial thromboplastin time. Can Med Assoc J 104: 385-388, 1971 Sussman LN, Bay M: Activated partial thromboplastin time (A.P.T.T.) in monitoring heparin therapy. Lab Med 5:3640, 1974 Teien AN, Abildgaard U: On the value of the activated partial thromboplastin time (APTT) in monitoring heparin therapy. Thromb Haemostas 35:592-597, 1976
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
13.
HEPARIN MONITORING TESTS
PH.D.,
THERESA S. GALLUZZO, M.T.(ASCP), ROSE LO, M.T.(ASCP), AND KATHRYN G. PETERSON, M.T.(ASCP)
Ts'ao, Chung-hsin, Galluzzo, Theresa S., Lo, Rose, and Peterson, Kathryn G.: Whole-blood clotting time, activated partial thromboplastin time, and whole-blood recalcification time as heparin monitoring tests. Am J Clin Pathol 71: 17-21, 1979. The authors performed whole-blood clotting time (WBCT), activated partial thromboplastin time (APTT), and wholeblood recalcification time (WBRCT) tests on normal blood or citrated plasma, each milliliter containing 0-0.5 unit heparin, and on samples from patients, of whom many were receiving heparin anticoagulation therapy. Six partial thromboplastin reagents were used. Linearity between clotting time and heparin concentration was observed with WBCT and APTT, determined with Hyland partial thromboplastin (kaolin-activated) and Dade ("Improved" Activated Cephaloplastin and Actinw) reagents. With a General Diagnostics preparation (Platelin®-plus, celite as the activator) and another Hyland partial thromboplastin reagent (silica-activated), the sensitivity to heparin decreased to beyond 0.3 unit/ml plasma. No correlation was observed with the old Dade Activated Cephaloplastin reagent, WBRCT was completely insensitive to heparin in concentrations as high as 0.24 unit/ml blood. With patient samples, correlations were observed between WBCT and Hyland (kaolin) APTT, and between Hyland and Dade Actinw APTT. However, WBCT and WBRCT, and APTT and WBRCT, correlated poorly. (Key words: Whole-blood clotting time; Activated partial thromboplastin time; Whole-blood recalcification time; Heparin monitoring tests; Coagulation.)
Coagulation Laboratory, Department of Pathology, Northwestern Memorial Hospital and Northwestern University School of Medicine, Chicago, Illinois
Materials and Methods Blood
Samples
For in vitro studies, blood samples were obtained from 11 healthy laboratory and medical workers (two male and nine female, 23-40 years of age), with their consent. Some donated their blood more than once. Patients' blood was taken as part of the laboratory service. Blood was drawn with a 21-gauge needle attached to a disposable syringe. It was treated according to the nature of the tests. In-vitro Tests Whole Blood Clotting Time (WBCT). One-milliliter samples of blood were introduced into a series of glass tubes (10 x 75 mm) containing 0.05 ml saline solution or heparin.* The final concentrations of heparin were 0, 0.03, 0.06, 0.12, 0.18, 0.24, and 0.30 unit/ml whole blood. The WBCT of each sample was determined with a modified Lee-White procedure, using three rather than four tubes. The results are the averages of eight determinations. Whole Blood Recalcification Time (WBRCT). Blood was introduced into polypropylene tubes containing 3.8% trisodium citrate. The ratio of blood to citrate was nine volumes to one. After mixing, 3-ml samples of blood were transferred to a series of glass tubes (12 x 75 mm), each containing 0.05 ml saline solution or heparin. The final concentrations of heparin were the same as in the WBCT studies. WBRCT was de-
THE SEARCH for a simple, reliable test to monitor heparin anticoagulation has continued since the days when heparin was first used clinically. Whole-blood clotting time (WBCT) test is probably still the most widely used technic, but its shortcomings are well recognized by those who rely on it. The activated partial thromboplastin time (APTT) test has gained prominence as a substitute for the WBCT in recent years, 3 1 3 1 7 - 1 9 but its efficacy has been questioned by many. 2,512,20 Other tests, including thrombin time, 9 whole blood recalcification time (WBRCT), 4,714 and neutralization of heparin by polybrene 6 or protamine, 10 have been used by some with apparent success. Here we report our evaluations of the sensitivities of WBCT, APTT, Received March 7, 1977; received manuscript and accepted for publication December 27, 1977. Address reprint requests to Dr. Ts'ao: Department of Pathology, Northwestern University McGaw Medical Center, Wesley Pavilion, East Superior Street and Fairbanks Court, Chicago, Illinois 60611.
* Panheparin®, Abbott Laboratories, North Chicago, Illinois.
0002-9173/79/0100/0017 $00.75 © American Society of Clinical Pathologists
17
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
and WBRCT to heparin in vitro, and the efficacies of these tests in monitoring heparin anticoagulation in patients.
18
TS'AO ETAL.
aoor-
A.J.C.P. • January 1979
determined in duplicate with a set of two fibrometers.1: Six partial thromboplastin reagents obtained from three manufacturers were used: Activated Cephaloplastin,§ "Improved" Activated Cephaloplastin,§ Actin® (all Dade reagents use ellagic acid as the activator), Platelin® plus Activator11 with celite as the activator, and two Hyland partial thromboplastin reagents,** one using kaolin as the activator and the other, silica as the activator. The manufacturers' recommended procedures, including incubation time and concentration of CaCl2, were followed. Results represent five to eight determinations for each heparin concentration with each reagent. WBCT, APTT, and WBRCT of Patient Samples
0
1 0.10 0.06
1 1 0.20 0.30 Unit Heptrln/al Pitta* 0.12 0.18 Unit Hepirln/Bl Blood
1 0.40
1, 0.50
0.24
0.30
FIG. 1. Median values of APTT, WBRCT, and WBCT plotted against heparin added to whole blood (WBCT tests), citrated whole blood (WBRCT tests), or citrated plasma (APTT tests). APTT reagents are Platelin® plus (celite) (curve 1), Dade "Improved" Activated Cephaloplastin (curve 2), Hyland partial thromboplastin (kaolin-activated) (curve 3), Hyland partial thromboplastin (silicaactivated) (curve 4) and Dade Actin (curve 5).
termined in triplicate with the method described by Shanberge and associates.14 Data presented in the Results sections were, again, derived from eight determinations. Activated Partial Thromboplastin Time (APTT). Blood was transferred to Vacutainer tubest containing 3.8% trisodium citrate. The ratio of blood to citrate was nine volumes to one. After centrifugation of citrated blood for 15 minutes at 1,000 xg at 4 C, the supernatant plasma was introduced into a series of glass tubes (12 x 75 mm), each containing 0.05 ml saline solution or heparin. The final concentrations of heparin were 0, 0.05, 0.10, 0.20, 0.30, 0.40, and 0.50 unit/ml plasma. Assuming the hematocrit of the donors was 40%, these concentrations of heparin corresponded to 0, 0.03, 0.06, etc., unit/ml whole blood. APTT was t Becton-Dickinson, Rutherford, New Jersey.
Results Varying Responses of Partial Thromboplastin Reagents, and Insensitivity of WBRCT, to Heparin Added to Plasma or Blood With the old Dade Activated Cephaloplastin, we were unable to obtain a meaningful correlation between APTT and concentrations of heparin in citrated normal plasma. With the other five reagents, prolonged APTTs were observed in samples containing increasing amounts of heparin. Liner relationships were observed with Hyland (kaolin) preparation, Dade "Improved" Activated Cephaloplastin, and Dade Actin. Of these three reagents, the "Improved" Activated Cephaloplastin was most sensitive and Actin least sensitive to the range of heparin concentrations we studied. Platelin plus Activator was very sensitive to low concentrations of heparin. The sensitivity of the Hylandsilica preparation was comparable to the Hylandkaolin preparation for heparin concentrations of less than 0.3 unit/ml plasma. Beyond that, Hyland-silica became less sensitive. These results are shown in Figure 1, curves 1 to 5. A linear relationship was also obX Baltimore Biological Lab., Cockeysville, Maryland. § Dade Corporation, Miami, Florida. ' General Diagnostics, Morris Plains, New Jersey. ** Hyland Co., Costa Mesa, California.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
iol 0
Blood was drawn either with citrate-containing Vacutainers or with syringes and transferred into citrate-containing Vacutainers. WBCT, APTT, and WBRCT were determined as described above. WBCT and Hyland (kaolin) APTT tests were performed on 117 patient samples, WBCT and Platelin plus (celite) APTT tests on 36 samples, and WBCT and WBRCT on 45 samples. In addition, Hyland (kaolin) APTT and Dade Actin APTT were determined in 103 patient samples, and Hyland (kaolin) APTT and WBRCT in 75 samples.
400
300
200
100 90 80 70 60 50
FIG. 2. Scattergrams showing correlations among WBCT, APTT, and WBRCT for patient samples. The best correlations were observed between WBCT and Hyland (kaolin) APTT, and between Hyland and Actin APTT; the poorest correlations were between WBCT and WBRCT and between APTT and WBRCT.
40 30
20
TO
20
30
40
50
20 30 CT (mmitei)
40
60
CT (Minutes) 200
100 f—
•
•
, •
100 80 •g o
70
• •
8 60
v»
- 50
-
•
• •• «
5 40 c
-sV- '
3 30 Of
s 20
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10
10
30 40 SO 60 70 80 100 Hyltnd APTT. Sacoitfs
20
200
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aur 30 lOOr-
20
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8 6 5
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10
MBKT. mnutM
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Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
_l
_L
10
20
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served between WBCT and heparin, ranging from 0.03 to 0.30 unit/ml blood (Fig. 1). However, the WBRCT was almost completely insensitive to in-vitro heparin in concentrations as high as 0.24 unit/ml blood (Fig. 1). Compared with baseline values, the WBRCT showed only a 30% increase for samples containing 0.24 unit heparin/ml blood, whereas the WBRCT had quadrupled and Hyland (kaolin) APTT had almost tripled for corresponding samples. Correlations among WBCT, APTT, and WBRCT for Patient Samples
Discussion We have presented results of our investigation of the sensitivities of six commercial partial thromboplastin reagents to heparin added to citrated normal plasma, as well as WBCT and WBRCT of whole blood containing comparable amounts of heparin. The concentration of heparin we chose for our in-vitro studies was well within the range of "therapeutic levels," i.e., in causing two-to-threefold prolongation of baseline WBCT. In addition, we compared WBCT, APTT, and WBRCT in several hundred samples from patients, of whom many were receiving heparin anticoagulation therapy. We found that only three of the six partial thromboplastin reagents showed linear responses to heparin concentrations to as much as 0.5 unit/ml plasma. The other three reagents either were insensitive or had limited linearity. While WBCT also showed a linear response to increasing amounts of heparin, WBRCT was almost completely insensitive to the range of heparin we studied. Our studies of patient samples showed good correlation between WBCT and Hyland (kaolin) APTT and between Hyland (kaolin) and Actin APTT, but the correlations between WBCT and WBRCT and between APTT and WBRCT were poor. The varying results of APTT tests of heparinized or factor-deficient plasmas determined by use of different commercial partial thromboplastin reagents have been reported. 11115- ' 7 Our results agree with those earlier findings. However, we would like to point out that different results are obtained by different investigators, using apparently similar technics, with respect
to the sensitivity of a given reagent to in-vitro heparin. This is best illustrated by results reported by Shapiro and associates15 and those obtained by us. Shapiro and associates found that the Hyland partial thromboplastin (kaolin) and "new" (presumably the "Improved") Dade Activated Cephaloplastin reagents had nonlinear responses or were insensitive to heparin, whereas we found both yielded linear responses, although their sensitivities to heparin differed. Reasons for these discrepancies remain to be elucidated. Since the brand of heparin used by Shapiro and associates was different from that used by us, one possible explanation may lie in a well-known fact that different brands of heparin preparations possess different anticoagulant capacities.8 The lack of sensitivity of WBRCT to in-vitro heparin, which caused three-to-fourfold increases in APTT and WBCT, was unexpected. Our data apparently are at variance with those reported by Gulliani and colleagues.7 Those investigators found a linear relationship between WBRCT and heparin added to citrated whole blood. It should be pointed out that the amounts of heparin used in their experiments were much greater than those used in ours. Those concentrations of heparin can be achieved in patients only soon after a bolus injection. Our experience has been that a heparin monitoring test is rarely ordered so soon after the treatment. The results of our studies of patient samples concur with our in-vitro data. Generally, reliable results were obtained with those reagents and tests that were sensitive to in-vitro heparin and showed linear responses to increasing heparin concentrations. It should be stressed that some reagents and technics that were found insensitive to heparin and noncorrelative to other heparin-monitoring tests by us have been successfully used by others. The apparent successes with WBRCT of Shanberge and associates14 and Gulliani and colleagues7 are examples. Obviously, each laboratory must select a reagent or test that serves its needs best. While we have found both WBCT and APTT can be used to monitor heparin therapy, we have no evidence to show that one is overall more reliable than the other. However, the many advantages inherent in APTT cannot be matched by WBCT. References 1. Babson AL, Babson SR: Comparative evaluation of a partial thromboplastin reagent containing a non-settling particulate activator. Am J Clin Pathol 62:856-860, 1974 2. Baele G, DeBroe M, DeWeerdt GA, et al: Limitations of the activated partial thromboplastin time for determination of plasma heparin concentration and half-life in human subjects. Thromb Diath Haemorrh 27:107-113, 1972 3. Basu D, Gallus A, Hirsh J, et al: A prospective study of the value of monitoring heparin treatment with the activated partial thromboplastin time. N Engl J Med 287:324-327, 1972 4. Belko JS, Warren R: The recalcification time of blood. Its use
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
When these tests were performed on patient samples, we found good correlations between WBCT and Hyland (kaolin) APTT, and Hyland (kaolin) APTT and Dade Actin APTT (Fig. 2, top; middle left). WBCT and Platelin plus (celite) appeared to correlate less well (Fig. 2, middle right). The poorest correlations were between WBCT and WBRCT and between Hyland APTT (kaolin) and WBRCT (Fig. 2, bottom, left and right).
A.J.C.P. .January 1979
Vol. 71 • No. I
5. 6. 7. 8. 9. 10. 11. 12.
as a measure of clinical effect of heparin. Arch Surg 76:210218, 1958 Bern MM: Variable response of activated partial thromboplastin time to heparin therapy during hemodialysis. Am J Clin Pathol 64:602-607, 1975 Grann VJ, Homewood K, Golden W: Polybrene neutralization as a rapid means of monitoring blood heparin. Am J Clin Pathol 58:26-32, 1972 Gulliani GL, Hyun BH, Litten MB: Blood recalcification time. A simple and reliable test to monitor heparin therapy. Am J Clin Pathol 65:390-396, 1976 Johnson EA, Kirkwood TBL, Stirling Y, et al: Four heparin preparations. Anti-X„ potentiating effect of heparin after subcutaneous injections. Throm Haemostas 35:586-591, 1976 Penner J A: Experience with a thrombin clotting time assay for measuring heparin activity. Am J Clin Pathol 61:645-653, 1974 Pitney WR, Pettit JE, Armstrong L: Control of heparin therapy. Br Med J 4:139-141, 1970 Poller L, Thompson JM: The partial thromboplastin (cephalin) time test. J Clin Pathol 25:1038-1044, 1972 Ray PK, Harper TA: Comparison of activated recalcification and partial thromboplastin tests as controls of heparin therapy. J Lab Clin Med 77:901-907, 1971 Schriever HG, Epstein SE, Mintz MD: Statistical correlation
14. 15. 16. 17. 18. 19. 20.
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and heparin sensitivity of activated partial thromboplastin time, whole blood coagulation time, and an automated coagulation time. Am J Clin Pathol 60:323-329, 1973 Shanberge JN, Walters J, Ambegaonkar S: The whole blood recalcification time—a convenient method for monitoring heparin therapy. Lab Med 5:30-32, 1974 Shapiro GA, Huntzinger SW, Wilson JE 111: Variation among commercial activated partial thromboplastin time reagents in response to heparin. Am J Clin Pathol 67:477-480, 1977 Sibley C, Singer JW, Wood RJ: Comparison of activated partial thromboplastin reagents. Am J Clin Pathol 59:581-586. 1973 Soloway HB, Cornett BM, Grayson JW Jr: Comparison of various partial thromboplastin reagents in the laboratory control of heparin therapy. Am J Clin Pathol 59:587-590, 1973 Stuart RK, Michel A: Monitoring heparin therapy with the activated partial thromboplastin time. Can Med Assoc J 104: 385-388, 1971 Sussman LN, Bay M: Activated partial thromboplastin time (A.P.T.T.) in monitoring heparin therapy. Lab Med 5:3640, 1974 Teien AN, Abildgaard U: On the value of the activated partial thromboplastin time (APTT) in monitoring heparin therapy. Thromb Haemostas 35:592-597, 1976
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13.
HEPARIN MONITORING TESTS
