THROMBOSIS RESEARCH Suppl. XIV; 49-62,199l 0049-3848191 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
LABORATORY MONITORING OF THE CLINICAL EFFECTS OF LOW MOLECULAR WEIGHT HEPARINS
Jeanine M. Walenga, Departments
Ph.D., Debra Hoppensteadt, M.S., MT (ASCP), and Jawed Fareed, Ph.D. of Thoracic and Cardiovascular Surgery and Pathology Loyola University Medical Center Maywood, Illinois, USA
ABSTRACT Despite a clinical prophylatic efficacy of low molecular weightheparins (LMWHs) for 24 hrs after a single subcutaneous administration, the routine laboratory tests (anti-Xa, anti-IIa, Heptest, APTT which show a reliable in vitro dose-response) exhibit no ex vivo response after 6 hours. In addition, the values obtained in these assays do not correlate with clinical efficacy or bleeding side effects. With therapeutic doses of LMWHS, a proportionately higher effect was noted in these tests including, in addition, thrombin generation, Heptest-Hi, and thrombin time assays. However, the relevance of these assays to the clinical efficacy/toxicity of LMWHs remains unclear since they do not relate to the total pharmacodynamic effect. For example, protamine neutralization, adjunct drug treatment and a patient's own predisposing factors which contribute to the hemostatic balance may not be reflected in these assays. These observations point to the limitations of the available laboratory tests for monitoring IMWHs. In order to find a more sensitive means to detect the effects of LMWH, assays for specific molecular markers of coagulation and fibrinolysis activation were evaluated. Alterations in the levels of thrombin-antithrombin complex, t-PA, total degradation products and D-dimer assays were observed over a 7-10 day LMWH treatment period. ProthrombinfragmentF1+2, modifiedantithrombin, PA1 and fibrinogen degradation products were not significantly effected. The association of the changes observed in these markers to the mechanism of action of LMWH or to the efficacy of the treatment, however, remains to be determined.
Key words:
Low molecular bleeding
weight
heparin,
49
monitoring,
assays,
thrombosis,
50
LABORATORY MONITORING OF LMW HEPARINS
Suppl. XIV, 1991
INTRODUCTION Global clotting assays, such as the APTT andUS Pharmacopeiaheparin assay, have been effectively used for heparin clinical monitoring and potency evaluation. The success of this relationship is due to the strong generation of thrombin in these assays and the potent thrombin and thrombin generation inhibition activity of heparin; however, these same assays reveal little to no activity of low molecular weight heparins (IMwHs). Thus other more sensitive assays are required to investigate the known measurable actions of these agents. To evaluate their antithrombotic action, the AT III dependent inhibitory effect of IMUHs on coagulation proteases is commonly monitored. Utilizing synthetic peptide substrates, assays to measure the two most kinetically important anti-protease activities, the inhibitory effect against factor Xa (anti-Xa) and the inhibitory effect against thrombin (anti-IIa), were developed. A modification of the APTT assay, using a chromogenic substrate measurement of thrombin generated after activation of plasma, was also developed by several investigators based on the original work of Biggs in 1953 (1). Although these assays have become a widely accepted approach, to date no definitive report on the relevance of these anti-protease assays to the biological (clinical) action of LMWHs is available. Inview of the multiple components of hemostasis, it is probable thatl&iWHs act as antithrombotic agents through multiple mechanisms. Aside from the ATIII/HCII dependent plasmatic effects, other actions of IMWHs have been found which include endothelial interactions, effects on leukocytes, platelets and alteration of blood viscosity (2,3). It has also been suggested that lMWHs modulate fibrinolysis (4-7). Additional effects of lMWHs may include binding to proteases, binding to blood vessel surfaces and selective binding to cellular receptors causing localized effects. There remains, however, a considerable debate over the relative clinical value of these actions. It is useful to understand the mechanisms through which INWHs produce their clinical effects. Differences in the composition and biological action of LWHs have been documented (8,9) in contrast to the relatively consistent commercial heparin preparations. Thus dosage adjustment between products may become necessary. By knowing the mechanism of action of LMWHs laboratory assays for biological activity and product standardization can be more easily developed. Furthermore, possible side effects can be more effectively managed. Although plasma assays limit us in the overall information that can be obtained, today's assays are much more sensitive and specific than the older APTT based assays andmay, therefore, be able to provide data previously unobtainable. Useful information may be obtained through modification of the current clinical trials to include the measurement of molecular markers of platelet activation, antithrombotic and fibrinolytic effects related to a thrombotic state (10-14). Several factors are important determinants of defining the mechanisms of action of LMWHs: drug concentration, mode of administration and also the sensitivity and specificity of the monitoring assay. The relevance of a single assay such as the amidolytic anti-Xa assay to biological actions has been It is now believed that other factors may contribute to the debated. antithrombotic actions of LMWHs. The data obtained in our laboratory, presented in the following, will address this issue.
Suppl. XIV, 1991
LABORATORY MONITORING OF LMW HEPARINS
51
MATERIAL AND METHODS
Low Molecular Weight Henarins Rhone-Poulenc; Paris, France. Enoxaparin: 1. Sanofi; Paris, France. Fraxiparin: 2. Nova; Copenhagen, Denmark Logiparin: 3. Wyeth; Philadelphia, PA, USA. RD Heparin: 4. Sandoz; Nurnberg, Germany. Mono-Embolex: 5. Animal Model The venous stasis thrombosis model in New Zealand White male rabbits using prothrombin complex concentrate (Konyne; Cutter Labs, Berekely, CA) with as a thrombogenic Russell's viper venom (Sigma, St. Louis, MO) (PCC-RW) challenge was used (15). Results were based on a visual grading of the jugular vein clot where 0 is no clot and 10 is a solid clot formation incorporating all erythrocytes within the vein segment. Clinical Samnles Venous am blood samples were drawn into sodium citrate from patients 3-4 hours post administration of LMWH (or control) treatment by individual clinical centers trial and the (Aalborg Hospital, Aalborg, Denmark for the Enoxaparin-Dextran Logiparin-Placebo trial; Klinikum der J.W. Goethe Universitat, Frankfurt, Germany for the European Fraxiparin-Calciparin study; University of Munich, Munich, Germany for the Mono-Embolex trial; Wyeth, Philadelphia, PA for the Wyeth trial). Bloods were centrifuged; plasma was aliquoted and frozen at -70°C. Periodic shipments of the frozen plasma were made on dry ice to Loyola University Medical Center for analysis. Assays for Plasma Analysis APTT (Organon Teknika, Durham, NC, USA) using the Fibrometer. 1. Thrombin (human) time (5U/ml; Ortho, Raritan, NJ, USA) clotting assay on 2. the Fibrometer. Heptest (Haemachem, Inc., St. Louis, MO, USA) using the Fibrometer. 3. Anti-factor Xa for the aca (DuPont, Wilmington, DE, USA). 4. Anti-factor Xa amidolytic, kinetic rate assay (16) using a Beckman 5. spectrophotometer. Anti-factor Ila amidolytic, kinetic rate assay (16) using a Beckman 6. spectrophotometer. D-Dimer ELISA (Stago; Gennevilliers, France). 7. 8. Tissue plasminogen activator antigen (t-PA) ELISA (Stago). Plasminogen activator inhibitor antigen ELISA (Monozyme; 9. (PAI) Charlottenlund, Denmark). Total degradation products (TDP) ELISA (Organon Teknika). 10. 11. Prothrombin Fragment Fl+2 ELISA (Behring; Marburg, Germany). Thrombin-antithrombin (TAT) complex ELISA (Behring). 12.
RESULTS The routine assays effectively used for heparin monitoring, such as the APTT and thrombin time assay, are not sensitive to LMWH at doses which produce antithrombotic activity. Fig. 1 illustrates the lack of correlation between the in vivo and ex vivo activities.
52
LABORATORY MONITORING OF LMW HEPARINS
Fig. 1 Rabbits were injected with varying amounts of PK10169 (Enoxaparin) (12.5-100 pg/kg iv). Blood was drawn through heart puncture 5 min. after administration and analyzed by APTT and thrombin time tests. A thrombogenic challenge was then given to the animal and in vivo antithrombotic activity was evaluated by stasis thrombosis. Results are given as a mean f S.D. (n=3).
Suppl. XIV, 1991
COMPARISON OFTHE EXVIVOANDTHEINVIVO ANTITHROMBOTIC EFFECTS OF PK10169
s = Y F
N
DOSACE (w/kp)
Current Assays to Monitor LMWH As shown in Fig. 2, using in vitro analyses the anti-Xa and anti-IIa assays show a clear concentration-dependent effect of the IMWM Enoxaparin supplemented to normal human plasma. Similar data is obtained with the Heptest. Based on this type of data, the anti-Xa/anti-IIa assays were adopted as the method of choice to monitor LMWHs in clinical trial.
INVlTROSUPPLEM~;~;W&MMXAPARlN %lNHlBmoN
INNORMAL INVlTRO SUPPLEME~$~;C);XAPARININ x tNimnoN lWT
TrsrZANllxa
lEsE ANTIna
.-.
o-.MOxWWJNm33101
moxwARm
?? -*xoF5ml5oFE
c*XOF5LOlSOFENOXWANN
0
25
CONCENTRATION
Fig. 2
75
So w/ML)
loo
NORMAL
cm33101
‘zy!!, 0
25
50
CONCEMRAilON
75
100
(@/ML)
Fig. 2b Fig. 2a Enoxaparin was supplemented to normal human pooled plasma at varying concentrations then immediately assayed by the chromogenic (a) anti-Xa and (b) anti-IIa assays. A new lot was compared to five older lots showing no deviation in activity. Results are given as a mean + 1 S.D. (n-4).
The anti-Xa activity of clinical dosages of various commercially available I.MWHspackaged in ready-to-use syringes was measured. Table 1 shows a relatively similar dosage activity level for all preparations. The anti-Xa activity as measured in plasmas obtained from patients having received these same IMWMs clinically, however, showed differences between the products. For example, a maximum of a 1.5-fold difference in total activity was observed between the syringe dosages, whereas a 3-fold difference was observed between the ex vivo AXa U/ml plasma activities.
Suppl.
xiv, 1991
53
LABORATORY MONITORING OF LMW HEPARINS
Table 1 Comoarison of Administered Dosages to Circulating Anti-Xa Levels of IMWHs in Humans Circulating Level Anti-Xa u/ml (3-4 hrs. Post SC Iniection)
Dosage Anti-Xa U/SC Iniection Enoxaparin Fraxiparin Logiparin Mono-Embolex
0.13 0.11 0.31 0.32
2900 3470 4325 3800
f * + +
0.08 0.07 0.10 0.10
All anti-Xa units were determined in one assay system (16); values are not in Results are given as a mean k 1 S.D. [n - 3 terms of manufacturer's units. (dosage); lo-15 (levels)].
Fig. 3 Plasmas collected frompatients enrolled in five different LMWH clinical trials were analyzed for anti-Xa and anti-IIa activities in the same assay systerns. The activity level for patients who were reported to have had either a bleeding, or a thrombotic episode was then tabulated.
COMPARISON OF ANTI Xa AN0 ANTI Ila LEVELS IN PATIENTS Wrl?4 BLEEDING OR THROMEOTIC EVENTS (COMBINED DATA OF FlVE LMWH CUNICAL TRIALS) U/ML OSGO
CLINICAL
ELEWING
CUNICM
THROMBGSIS
l.ANTlXo O&GO
.
2. ANTI II0
.
34:%-Z Z: 0.300
0.200-O.lOO~-
0.000J
.
+
..
.%
?? : . ??
r:,
?? a
.
.. .*
. ?? . .
. .. .. . .
.
.
:-
. .. . .
.
.2; ‘8: =
0.
.
.
' * :
??
..* .:
??
1
2
5
4
Investigations were made as to the clinical relevance of the anti-Xa activity to bleeding or thrombosis. Clinical samples were obtained from patients enrolled in five different LMWH trials. All samples were analyzed by the same anti-Xa method. Anti-Xa and anti-IIa levels of those patients with reported bleeding or thrombotic events were compared (Fig. 3). The data revealed a poor correlation of both clinical effects to ex vivo activities. The Heptest is considered by some to be the method of choice for LMWH. Although correlations between Heptest and the chromogenic anti-Xa assay are good (r=0.86) in patients treated with LMWH, correlations between Heptest and the chromogenic anti-IIa assay (r=0.22) and the APTT (r=0.09) are less clear (personal communication E.T. Yin, 1989). This indicates that the Heptest is more sensitive to anti-Xa than anti-IIa activity. Similarly in our own studies, when the Heptest was used in the analysis of plasma samples obtained from normal volunteers administered a LMWH (RD Heparin, Wyeth), the sensitivity of Heptest was greater than that of the chromogenic anti-Xa, anti-IIa and APTT assays. However, the Heptest activity was still less than desirable. LMWHs are effective when given once every 24 hours. The Heptest response, however, only showed activity up to 8 hours after a 40 mg dose (Fig. 4).
54
LABORATORY MONITORING OF LMW HEPARINS
Suppl. XIV, 1991
Partial evidence for the lack of correlation between the anti-Xa activity and the antithrombotic effect is given in Fig. 5. In a simple study, I.MWHwas supplemented in vitro to pooled human plasma from normal individuals, patients with diagnosed liver disease, elderly individuals and AT III deficient individuals. The chromogenic anti-Xa and Heptest assays were performed on each plasma set. For each non-normal group the recovery of LMWH activity (anti-Xa and Heptest) was less than that obtained in the normal group indicating that the physiologic state of an individual will be reflected in the efficacy of a LMWH. Moreover, if unadjusted dosages are given to all patients, the clinical effects will vary from person to person.
Fig. 5 Enoxaparin was supplemented at 2.5 and 1.25 pg/ml to human plasma obtained from 7-12 individuals of the indicated cliniThe anti-Xa and cal state. Heptest assays were performed on each sample. Percent recovery was calculated based on a previouslyconstructedcalibration curve of Enoxaparin in pooled normal human plasma.
A CDMPAR,SDN
OF THE POTENCY OF A LMW hEPARlN POOLED PLASMAS
IN VAR’OUS
New Assays to Evaluate LMWH The following describes data from several clinical trials of LMWHs measured in one laboratory. In the Enoxaparin (40 mg daily sc)-Dextran Danish clinical trial in elective hip replacement patients (n-218), both treatment groups showed equal but significantly elevated D-dimer levels (p
LABORATORY MONITORING OF THE CLINICAL EFFECTS OF LOW MOLECULAR WEIGHT HEPARINS
Jeanine M. Walenga, Departments
Ph.D., Debra Hoppensteadt, M.S., MT (ASCP), and Jawed Fareed, Ph.D. of Thoracic and Cardiovascular Surgery and Pathology Loyola University Medical Center Maywood, Illinois, USA
ABSTRACT Despite a clinical prophylatic efficacy of low molecular weightheparins (LMWHs) for 24 hrs after a single subcutaneous administration, the routine laboratory tests (anti-Xa, anti-IIa, Heptest, APTT which show a reliable in vitro dose-response) exhibit no ex vivo response after 6 hours. In addition, the values obtained in these assays do not correlate with clinical efficacy or bleeding side effects. With therapeutic doses of LMWHS, a proportionately higher effect was noted in these tests including, in addition, thrombin generation, Heptest-Hi, and thrombin time assays. However, the relevance of these assays to the clinical efficacy/toxicity of LMWHs remains unclear since they do not relate to the total pharmacodynamic effect. For example, protamine neutralization, adjunct drug treatment and a patient's own predisposing factors which contribute to the hemostatic balance may not be reflected in these assays. These observations point to the limitations of the available laboratory tests for monitoring IMWHs. In order to find a more sensitive means to detect the effects of LMWH, assays for specific molecular markers of coagulation and fibrinolysis activation were evaluated. Alterations in the levels of thrombin-antithrombin complex, t-PA, total degradation products and D-dimer assays were observed over a 7-10 day LMWH treatment period. ProthrombinfragmentF1+2, modifiedantithrombin, PA1 and fibrinogen degradation products were not significantly effected. The association of the changes observed in these markers to the mechanism of action of LMWH or to the efficacy of the treatment, however, remains to be determined.
Key words:
Low molecular bleeding
weight
heparin,
49
monitoring,
assays,
thrombosis,
50
LABORATORY MONITORING OF LMW HEPARINS
Suppl. XIV, 1991
INTRODUCTION Global clotting assays, such as the APTT andUS Pharmacopeiaheparin assay, have been effectively used for heparin clinical monitoring and potency evaluation. The success of this relationship is due to the strong generation of thrombin in these assays and the potent thrombin and thrombin generation inhibition activity of heparin; however, these same assays reveal little to no activity of low molecular weight heparins (IMwHs). Thus other more sensitive assays are required to investigate the known measurable actions of these agents. To evaluate their antithrombotic action, the AT III dependent inhibitory effect of IMUHs on coagulation proteases is commonly monitored. Utilizing synthetic peptide substrates, assays to measure the two most kinetically important anti-protease activities, the inhibitory effect against factor Xa (anti-Xa) and the inhibitory effect against thrombin (anti-IIa), were developed. A modification of the APTT assay, using a chromogenic substrate measurement of thrombin generated after activation of plasma, was also developed by several investigators based on the original work of Biggs in 1953 (1). Although these assays have become a widely accepted approach, to date no definitive report on the relevance of these anti-protease assays to the biological (clinical) action of LMWHs is available. Inview of the multiple components of hemostasis, it is probable thatl&iWHs act as antithrombotic agents through multiple mechanisms. Aside from the ATIII/HCII dependent plasmatic effects, other actions of IMWHs have been found which include endothelial interactions, effects on leukocytes, platelets and alteration of blood viscosity (2,3). It has also been suggested that lMWHs modulate fibrinolysis (4-7). Additional effects of lMWHs may include binding to proteases, binding to blood vessel surfaces and selective binding to cellular receptors causing localized effects. There remains, however, a considerable debate over the relative clinical value of these actions. It is useful to understand the mechanisms through which INWHs produce their clinical effects. Differences in the composition and biological action of LWHs have been documented (8,9) in contrast to the relatively consistent commercial heparin preparations. Thus dosage adjustment between products may become necessary. By knowing the mechanism of action of LMWHs laboratory assays for biological activity and product standardization can be more easily developed. Furthermore, possible side effects can be more effectively managed. Although plasma assays limit us in the overall information that can be obtained, today's assays are much more sensitive and specific than the older APTT based assays andmay, therefore, be able to provide data previously unobtainable. Useful information may be obtained through modification of the current clinical trials to include the measurement of molecular markers of platelet activation, antithrombotic and fibrinolytic effects related to a thrombotic state (10-14). Several factors are important determinants of defining the mechanisms of action of LMWHs: drug concentration, mode of administration and also the sensitivity and specificity of the monitoring assay. The relevance of a single assay such as the amidolytic anti-Xa assay to biological actions has been It is now believed that other factors may contribute to the debated. antithrombotic actions of LMWHs. The data obtained in our laboratory, presented in the following, will address this issue.
Suppl. XIV, 1991
LABORATORY MONITORING OF LMW HEPARINS
51
MATERIAL AND METHODS
Low Molecular Weight Henarins Rhone-Poulenc; Paris, France. Enoxaparin: 1. Sanofi; Paris, France. Fraxiparin: 2. Nova; Copenhagen, Denmark Logiparin: 3. Wyeth; Philadelphia, PA, USA. RD Heparin: 4. Sandoz; Nurnberg, Germany. Mono-Embolex: 5. Animal Model The venous stasis thrombosis model in New Zealand White male rabbits using prothrombin complex concentrate (Konyne; Cutter Labs, Berekely, CA) with as a thrombogenic Russell's viper venom (Sigma, St. Louis, MO) (PCC-RW) challenge was used (15). Results were based on a visual grading of the jugular vein clot where 0 is no clot and 10 is a solid clot formation incorporating all erythrocytes within the vein segment. Clinical Samnles Venous am blood samples were drawn into sodium citrate from patients 3-4 hours post administration of LMWH (or control) treatment by individual clinical centers trial and the (Aalborg Hospital, Aalborg, Denmark for the Enoxaparin-Dextran Logiparin-Placebo trial; Klinikum der J.W. Goethe Universitat, Frankfurt, Germany for the European Fraxiparin-Calciparin study; University of Munich, Munich, Germany for the Mono-Embolex trial; Wyeth, Philadelphia, PA for the Wyeth trial). Bloods were centrifuged; plasma was aliquoted and frozen at -70°C. Periodic shipments of the frozen plasma were made on dry ice to Loyola University Medical Center for analysis. Assays for Plasma Analysis APTT (Organon Teknika, Durham, NC, USA) using the Fibrometer. 1. Thrombin (human) time (5U/ml; Ortho, Raritan, NJ, USA) clotting assay on 2. the Fibrometer. Heptest (Haemachem, Inc., St. Louis, MO, USA) using the Fibrometer. 3. Anti-factor Xa for the aca (DuPont, Wilmington, DE, USA). 4. Anti-factor Xa amidolytic, kinetic rate assay (16) using a Beckman 5. spectrophotometer. Anti-factor Ila amidolytic, kinetic rate assay (16) using a Beckman 6. spectrophotometer. D-Dimer ELISA (Stago; Gennevilliers, France). 7. 8. Tissue plasminogen activator antigen (t-PA) ELISA (Stago). Plasminogen activator inhibitor antigen ELISA (Monozyme; 9. (PAI) Charlottenlund, Denmark). Total degradation products (TDP) ELISA (Organon Teknika). 10. 11. Prothrombin Fragment Fl+2 ELISA (Behring; Marburg, Germany). Thrombin-antithrombin (TAT) complex ELISA (Behring). 12.
RESULTS The routine assays effectively used for heparin monitoring, such as the APTT and thrombin time assay, are not sensitive to LMWH at doses which produce antithrombotic activity. Fig. 1 illustrates the lack of correlation between the in vivo and ex vivo activities.
52
LABORATORY MONITORING OF LMW HEPARINS
Fig. 1 Rabbits were injected with varying amounts of PK10169 (Enoxaparin) (12.5-100 pg/kg iv). Blood was drawn through heart puncture 5 min. after administration and analyzed by APTT and thrombin time tests. A thrombogenic challenge was then given to the animal and in vivo antithrombotic activity was evaluated by stasis thrombosis. Results are given as a mean f S.D. (n=3).
Suppl. XIV, 1991
COMPARISON OFTHE EXVIVOANDTHEINVIVO ANTITHROMBOTIC EFFECTS OF PK10169
s = Y F
N
DOSACE (w/kp)
Current Assays to Monitor LMWH As shown in Fig. 2, using in vitro analyses the anti-Xa and anti-IIa assays show a clear concentration-dependent effect of the IMWM Enoxaparin supplemented to normal human plasma. Similar data is obtained with the Heptest. Based on this type of data, the anti-Xa/anti-IIa assays were adopted as the method of choice to monitor LMWHs in clinical trial.
INVlTROSUPPLEM~;~;W&MMXAPARlN %lNHlBmoN
INNORMAL INVlTRO SUPPLEME~$~;C);XAPARININ x tNimnoN lWT
TrsrZANllxa
lEsE ANTIna
.-.
o-.MOxWWJNm33101
moxwARm
?? -*xoF5ml5oFE
c*XOF5LOlSOFENOXWANN
0
25
CONCENTRATION
Fig. 2
75
So w/ML)
loo
NORMAL
cm33101
‘zy!!, 0
25
50
CONCEMRAilON
75
100
(@/ML)
Fig. 2b Fig. 2a Enoxaparin was supplemented to normal human pooled plasma at varying concentrations then immediately assayed by the chromogenic (a) anti-Xa and (b) anti-IIa assays. A new lot was compared to five older lots showing no deviation in activity. Results are given as a mean + 1 S.D. (n-4).
The anti-Xa activity of clinical dosages of various commercially available I.MWHspackaged in ready-to-use syringes was measured. Table 1 shows a relatively similar dosage activity level for all preparations. The anti-Xa activity as measured in plasmas obtained from patients having received these same IMWMs clinically, however, showed differences between the products. For example, a maximum of a 1.5-fold difference in total activity was observed between the syringe dosages, whereas a 3-fold difference was observed between the ex vivo AXa U/ml plasma activities.
Suppl.
xiv, 1991
53
LABORATORY MONITORING OF LMW HEPARINS
Table 1 Comoarison of Administered Dosages to Circulating Anti-Xa Levels of IMWHs in Humans Circulating Level Anti-Xa u/ml (3-4 hrs. Post SC Iniection)
Dosage Anti-Xa U/SC Iniection Enoxaparin Fraxiparin Logiparin Mono-Embolex
0.13 0.11 0.31 0.32
2900 3470 4325 3800
f * + +
0.08 0.07 0.10 0.10
All anti-Xa units were determined in one assay system (16); values are not in Results are given as a mean k 1 S.D. [n - 3 terms of manufacturer's units. (dosage); lo-15 (levels)].
Fig. 3 Plasmas collected frompatients enrolled in five different LMWH clinical trials were analyzed for anti-Xa and anti-IIa activities in the same assay systerns. The activity level for patients who were reported to have had either a bleeding, or a thrombotic episode was then tabulated.
COMPARISON OF ANTI Xa AN0 ANTI Ila LEVELS IN PATIENTS Wrl?4 BLEEDING OR THROMEOTIC EVENTS (COMBINED DATA OF FlVE LMWH CUNICAL TRIALS) U/ML OSGO
CLINICAL
ELEWING
CUNICM
THROMBGSIS
l.ANTlXo O&GO
.
2. ANTI II0
.
34:%-Z Z: 0.300
0.200-O.lOO~-
0.000J
.
+
..
.%
?? : . ??
r:,
?? a
.
.. .*
. ?? . .
. .. .. . .
.
.
:-
. .. . .
.
.2; ‘8: =
0.
.
.
' * :
??
..* .:
??
1
2
5
4
Investigations were made as to the clinical relevance of the anti-Xa activity to bleeding or thrombosis. Clinical samples were obtained from patients enrolled in five different LMWH trials. All samples were analyzed by the same anti-Xa method. Anti-Xa and anti-IIa levels of those patients with reported bleeding or thrombotic events were compared (Fig. 3). The data revealed a poor correlation of both clinical effects to ex vivo activities. The Heptest is considered by some to be the method of choice for LMWH. Although correlations between Heptest and the chromogenic anti-Xa assay are good (r=0.86) in patients treated with LMWH, correlations between Heptest and the chromogenic anti-IIa assay (r=0.22) and the APTT (r=0.09) are less clear (personal communication E.T. Yin, 1989). This indicates that the Heptest is more sensitive to anti-Xa than anti-IIa activity. Similarly in our own studies, when the Heptest was used in the analysis of plasma samples obtained from normal volunteers administered a LMWH (RD Heparin, Wyeth), the sensitivity of Heptest was greater than that of the chromogenic anti-Xa, anti-IIa and APTT assays. However, the Heptest activity was still less than desirable. LMWHs are effective when given once every 24 hours. The Heptest response, however, only showed activity up to 8 hours after a 40 mg dose (Fig. 4).
54
LABORATORY MONITORING OF LMW HEPARINS
Suppl. XIV, 1991
Partial evidence for the lack of correlation between the anti-Xa activity and the antithrombotic effect is given in Fig. 5. In a simple study, I.MWHwas supplemented in vitro to pooled human plasma from normal individuals, patients with diagnosed liver disease, elderly individuals and AT III deficient individuals. The chromogenic anti-Xa and Heptest assays were performed on each plasma set. For each non-normal group the recovery of LMWH activity (anti-Xa and Heptest) was less than that obtained in the normal group indicating that the physiologic state of an individual will be reflected in the efficacy of a LMWH. Moreover, if unadjusted dosages are given to all patients, the clinical effects will vary from person to person.
Fig. 5 Enoxaparin was supplemented at 2.5 and 1.25 pg/ml to human plasma obtained from 7-12 individuals of the indicated cliniThe anti-Xa and cal state. Heptest assays were performed on each sample. Percent recovery was calculated based on a previouslyconstructedcalibration curve of Enoxaparin in pooled normal human plasma.
A CDMPAR,SDN
OF THE POTENCY OF A LMW hEPARlN POOLED PLASMAS
IN VAR’OUS
New Assays to Evaluate LMWH The following describes data from several clinical trials of LMWHs measured in one laboratory. In the Enoxaparin (40 mg daily sc)-Dextran Danish clinical trial in elective hip replacement patients (n-218), both treatment groups showed equal but significantly elevated D-dimer levels (p
