Britishjournal offfuenratology, 1975,30, 139.
Measurement of Heparin in Patients Receiving Subcutaneous Heparin Therapy K. W. E. DENSON AND J. BONNAR
N u i e l d D e y a r t ~ i n i tof Obstetrics and Gynaecology, University of Oxford, john RadCl@ Hospital, Oxford (Received 19 November 1974; accepted f o r Yiiblicatioit 26 Noveniber 1974) SUMMARY. Heparin assays based on four different tcsts of clotting function have becn coinpared with a heparin assay utilizing the potentiating effect of heparin on anti-factor Xa in a group of patients rccciviiig subcutaiieous heparin thcrapy during late prcgnancy. It is considered that the anti-factor Xa method is a bctter indication of the antithroinbotic effect of heparin. In the presence of high levels of clotting factors, heparin assays bascd on tcsts of clotting function can bc misleading. Subcutaneous heparin is being increasingly used in the prcvcntioii of post-opcrative thromboembolic coniplications (Kakkar et al, 1972; Gordon-Smith et al, 1972; Morris ct al, 1974). Low dosage heparin prophylaxis during and aftcr surgery rcquircs that sufficient heparin is released into the circulation to enhance the patient’s protective mechanisms against thrombosis, but at a lcvcl which will not compromise hacniostasis at the site of opcratioii. Iiieffcctivc therapy or hacmorrhagic complications are likely to discredit its value and already reports of both have been published (Macintyre et al, 1974; McWilliam et al, 1974; Hampsoii et al, 1974). Monitoring of heparin levels presents problems, but may prove to be essential for safe and effective heparin prophylaxis. Many methods have been used for measuring plasma or wholc blood heparin levels (Table I). With the exception of the protamiiie sulphate titration, these methods are all based on tests of clotting function. When the tcst samplc contains clotting factors within the normal range, the clotting time will reflect, fairly accurately, the level of heparin in the sample. In plasma from patients with raised levels of clotting factors, as occurs during pregnancy and oral contraception, or post-operatively, the clotting timc by any test of clotting function will be shortened, and can thus record a falsely low level of heparin. Conversely, if tlie clotting factors are low, as may arise in acute or sub-acute dissenrinated intravascular clotting, the clotting time would be longer and would be interpreted as a high heparin level. New techniques have recently been described for measuring heparin utilizing its potentiating or accelerating effect on the natural inhibitor to activated factor X (Yin et al, 1973 ; Denson & Bonnar, 1973). The methods are unaffected by varying levels of clotting factors in plasma samples, and should reflect heparin levels more accurately in patients with raised or reduced levels of clotting factors. As the level of heparin in the circulation is likcly to be the Correspondence:Dr K. W. E. Denson, Nuffield Department of Obstetrics and Gynaecology,TheJohn RadcliCe Hospital, Oxford.
I39
K. W. E. Denson and J. Bonrzar TABIEI. Tests which have been used for measuring heparin Whole blood clotting time Recalcification time Prothrombin time Partial thromboplastin time Kaolin cephalin clotting time H.A.R.E.M. test (activated whole blood clotting time) Protamine sulphate titration Calcium thrombin time Thrombin timc
critical factor in determining both its effictivcness and safety, we have coiiiparcd plasma heparin lcvels using four tests of clotting function and a spccific assay for heparin in a group of patictits during late prcgnancy who wcre receiving heparin therapy. MATERIALS A N D METHODS
Plasma samplcs. Blood from normal donors and patients (9 vol) was collectcd into I vol of sodium citratc containing 0.2 M HEPES buffer, and thc plasma scparated by centrifuging at 2500 g. Thc platclet-poor supernatant plasma was carcfully removed and kept dcep frozen a t - 20°C. Patients samples wcrc tcstcd in batches, aftcr storagc for pcriods of 2 days to I week. A single large pool of plasma from 20 normal donors was used as a standard during the period of the tests which extended ovcr 5 wccks. At the cnd of this time it was established that the pooled normal plasma had not dcteriorated in thc four tests ofclotting function using single batches of the standard frecze-dried reagents indicated below. Plasma for the heparin assay based on the potentiation of anti-factor Xa was heated for 15 min at 56°C and centrifuged to rcmove precipitated fibrinogcn. Thrombin (bovine), platelet substitute, kaolirz-platelet substitute mixture, activated bovine factor X andfactor-X deficient substrate plasrna were obtained from Diagnostic Reagents Limitcd, Thame, oxon. Bovine albtrmin (20%) was obtained from Arinour Pharmaceutical Co., Eastbournc, England. Citrate-aIbuM.iiiz-glyoxaline btifir consisted of 75 ml of glyoxaline buffer (0.5 M-imidazok and 0.1M-NaCl, pH 7.35)plus 15 nil of 0.13M sodium citratc plus 0.5 i d of 20% bovine albumin. Standard heparin. This was the first British standard prepared from bovine intcstitial niucosa and was kindly provided by D r D. R. Bangham from the MRC Division of Biological Standards, Mill Hill, London. Partial thromboplastin time ( P T T ) . Platelet substitute, 0.1nil, was placed in a clotting tube at 37°C and 0.1ml of plasma was added followed by 0. I ml of 0.025 M-CaCI,. The tubc was gently tilted and the clotting time recordcd. Kaolin-cephalin clotting time (KCCT). Kaolin-platelet substitute mixturc (0.2 nil) was placed in a clotting tube at 37°C followed by 0.1ml ofplasma; after 2 min with intermittent
0.13 M
MeasirvcMzeiit of Hcpuriiz
141
gciitlc shaking 0.1 1111 of 0.025 M-CaC], was addcd. The tube was gently tilted at 5-10 s intervals and the clotting time rccordcd. Throiirbiiz fiirrc ( T T ) .Plasma (0.2 1111) was placcd i n a clotting tube at 37'C, 0.2 in1 oftlirombin solutioii was added and tlic clotting time rccordcd. The thrombin coiiccntration was adjusted to givc a clotting time of approximately 11 I s with poolcd iiorinal plasma. C~7Iciiiriithmtiihirz r i m (Cn T T ) .Thronibin (0.2 i d ) of concciitratioii approximad y 3 .o ujml diluted in 0.025 M-CaCI, wa5 added to 0.2 1111 of plasiiia at 37'C and die c1otting time rccordcd. Tlic thrombin concciitratioii was adju5tcd to give a clotting tiiiic of T 5 IfI I s with pooled normal plasma. Calibvatrori C I ~ Y I W Srelutivq h p v i r z coirccizfvutioiz fo cloffiiqg tiirrc. For thc PTT, KCCT and Ca 'I'T, heyariii was added to pooled iiormal plasma at coiiccntratioiis of 0 , 0.05,0.1, 0.3, 0 . 3 , 0.4 aiid 0.5 ujiiil. For tlic thrombin time licparin was added to pooled normal plasma at concentrations of 0 , 0.05, 0.1,0 . ~ 2 0.14, , 0.r6 aiid 0.20 u/iiil. A narrow r a n g of coi~cciitra-
0.2
0.2
0.4
0.6
0.8
0.6-
0.4
-
0.2
0.4
0.6
0.0
Heparin assay ( u / r n l ) (ant1 X a method) I . Heparin lcvcls obtained with thc hcparin assay based 011 the potctitiation of anti-factor Xa plotted against heparin levels in thc sanic samples using a heparin assay based on thc 1'TT (a), KCCT (b), TT (c) and the Ca TT (d).
FIG
1.0
142
K. W. E. Denson and]. Bonnar
tioils was used for the latter because there was a large increase in clotting time for a small increment of heparin ovcr the range 0.10-0.20 u/ml, and beyond 0.3 u/ml the plasma was incoagulable. Each plasma sample over the range was tested in duplicate and the clotting times plotted against heparin concentration on linear co-ordinate paper. The value for the patient samples were obtained by interpolation from these curves, and if the clotting time was outside the range, the test sainple was suitably diluted in pooled normal plasma. Heparin assay based on thc potentiation of anti-factor Xu (Denson & Bonnar, 1973). Diluted factor Xa (0.3 ml) was placed in a series of 62 x 8 m m plastic tubes. One tube was placed in the 37°C water bath, 0.1ml of heated plasma sample added and a stopwatch started. The tube was stoppered, inverted twice and the stopper discarded. At 4 mill 45 s duplicatc 0.1 ml aiiiouiits were transferred to duplicate glass clotting tubes containing 0.I ml of 0.05 M-CaC1,. At 5 min, 0.2 ml of a mixture of equal volumes of factor-X free substrate plasma and platelet substitute was added to each tube and the clotting timc recorded. Three samples were tested in cach 5 miii period at 1.5 min intervals, and all volumes were measured using calibrated blow-out Pasteur-type pipettes. Heparin was diluted in pooled normal plasma to give a range of concentrations of 0.02,0.04, 0.06,0.08,0.10, 0.15and 0.20 u per ml of plasma. The clotting times were plotted against coiiccntration of heparin on log/linear paper, and the values for test samples were obtained by interpolation from this linc. The factor Xa was used at a dilution in tlie assay which gave a substrate clotting time of about 20 s for pooled normal plasma in thc absence of heparin, and about 60 s in the presence of heparin at a concentration of 0.2 u/ml in plasma. Test samples which had heparin levels anticipated to bc outside thc range of the standard curve were diluted in heated pooled normal plasma, and the results multiplied by the reciprocal of the dilution. Patients. Blood was obtained from 16 patients during late pregnancy who were receiving subcutaneous heparin as treatment or prophylaxis because of throinboenibolic complications. Forty samples from a group of eight patients were used for the results shown in Fig r(a), jb) and (c) and 27 saniplcs from a different group of eight patients for the results shown in Fig I (d). The patients received eithcr sodium heparin obtained from Weddel Pharmaceuticals or calcium heparin (Calciparine) obtained from Laboratoire Choay, Paris. RESULTS Fig I shows the results of plotting heparin levels obtained with the heparin assay based on the potentiation of anti-factor Xa against heparin levels in the same samples using a heparin assay based on the PTT (a),KCCT (b), TT (c) and the Ca TT (d).Without exception, the individual levels recorded by the mcthod based on the potentiation of anti-factor Xa were all higher, and on average two or three times higher than the equivalent levels recorded by methods based on tests of clotting function. The regression lines and correlation coefficients are shown. In curves (a), (b) and (c) several points are shown as solid circles. Thesc represent heparin values recorded as zero by the tests of clotting function, but in fact the clotting times of these samples by the PTT, KCCT and TT were all shorter than the clotting times ofpooled normal plasma. This is presumably due to high levels of clotting factors in the samples, and although heparin is present when measured by tlie anti-factor Xa method, these shorter-than-normal clotting times would indicate negative values for heparin levels by the tests of clotting func-
Mcasurement of Hepariiz I43 tion. In the rcgrcssion lines, allowance cannot bc made for these shorter-than-normal clotting times. The correlation cocfficicna arc reasonably good because tlie samples uscd werc from a selected group of patients during late pregnancy. At different stages of prcgnaiicy, and at diffcreiit tiincs post-opcratively, when levels of clotting factors nu): vary widely it would be difficult to prcdict the heparin value for a given sample by the anti-factor Xa assay from the equivalent value obtained by a test of clotting function. DISCUSSION
In late pregnancy aiid after surgery, factor VIII iiiay rise to 30o-400% and factor X to zoo% of average normal (Bonnar et a / , 1970). When tests of clotting function, such as the kaoliiicephalin clotting time, are carried out on plasma saniples with high levels of coagulation factors, the clotting time will be only about 5-10 s shorter than the clotting time of normal plasma. Despite this small reduction in clotting time, when coagulaticn is triggered, explosive and much larger amounts of factor Xa and thrombin arc likely to be produced compared with the amounts from normal plasma. Large amounts of factor Xa will minimize the effect of its natural inhibitor anti-factor Xa, aiid thus it would be expected that heparin, which potentiatcs the effect of anti-factor Xa, would be measured in a test of clotting function with less accuracy. This is borne out in practice and is illustrated in one saiiiplc whicli had 0.36 u/ml of heparin, aiid yet in which the clotting times in tests of clotting function were shorter than with normal plasma. I11 samples with high levels of clotting factors, the tests of clotting function may reflect the efficiency of local haemostasis, for example, a normal KCCT might indicate normal haemostasis; but they do not provide a measure of tlic anti-thrombotic effect of licparin. The effect of factor Xa on thc,formatioii of stasis thrombi has been well illustrated by the work of Wessler & Yiii (1968) in which stasis thrombi wcre induced in an isolated segment of rabbit jugular vein by very small amounts of activated factor X, but not by a much larger amount of tlie non-activated clotting factor. The value of low dosage heparin appears to be that local liaeinostasis is unaffected, but dissemination of factor Xa produced a t tlic site of a tliroinbus or clot is reduced or prevented by the circulating heparin. Following surgery, or in prcgnancy where high levels of clotting factors are present, it is possible that larger amounts of factor Xa and thronibin can bc released into the circulation. The anti-factor Xa heparin assay in such circumstances will record accurately the available anti-thrombotic level of heparin. The tcsts of clotting function will reflect the efficiency of haciiiostasis, but could bc misleading as a measure of the aiitithrombotic level of lieparin in tlie circulation. ACKNOWLEDGMENTS
This investigation was supported by MRC Grant No. Gg1/755/C and by a research fellowship &om Diagnostic Reagents Ltd. REFERENCES BONNAR, J., MCNICHOL, G.P. &DOUGLAS, A.S. (1970) Coagulation and fibrinolytic mechanisms during
and aftcr normal childbirth. British Medical]orrmal, ii, 200.
I44
K. W. E. Denson and J. Bonnar
DENSON, K.W.E. & BONNAR, J. (1973) The measurenient of heparin. A method based on thc potciitiatioii of anti-factor Xa. Thronrbosir et Diafhcsis HnewiorrhaSicn, 30, 471. GORDON-SMITH, I.C., LE QUESNE, L.P., GRUNDY, D.J., NEWCOMBE, J.F. & BRAMBLE, F.J. (1972) Controllcd trial of two rcgimens of subcutancous heparin in prevention of postoperative deep-vein thrombosis. Larzcct, i, I I 3 3 , HAMPSON, W.G.J., LUCAS,H.K., HARRIS,F.C., ROBERTS,P.H., MCCALL,I.W., JACKSON,P.C., POWELI.,N.L. & STADDON, G.E. (1974) Failure of low-dose hcparin to prcvent deep-vein thrombosis after hip-replaccmcnt artliroplasty. Lnncet, ii, 795. KAKKAR, V.V., SPINDLER, J., FLUTE,P.T., CORRICAN, T., FOSSARII, D.P., CKELI.IN, R.Q., WESSIER,S. & YIN, E.T. (1972) Efficicncy of low doses of hcparin in prevention of deep-vein thrombosis after major surgcry. Laricet, ii, 101.
MACINTYRE, I.M.C. et al(1974) Heparin versus dextran in the prevcntion of deep-vein thrombosis. A multiunit controllcd trial. Lancet, ii, 118. MCWILLIAM, R., MCCORMICK, J.9r.C. & AULAQI, A. (1974) Bleeding and pcriopcrative heparin. (Letter). Lancet, ii, 286. MORRIS,G.K., HENRY, A P.J. & PRESTON, B.J. (1974) l'revcntion of deep-vein thrombosis by low-dose heparin in patients undergoing total hip replacement. Lancet, ii, 797. S. & YIN, E.T. (1968) Expcrimental hypcrWESSLER, coagulable state induced by factor X : comparison of the nonactivated and activatcd forms. Jorrrrzal of Lnboratiiuy a d Clinical Medicine, 72, 256. YIN, E.T., WESSLER, S. 81 BUTLER, J.V. (1973) Plasma licparin : a mique, practical, submicrogram-sensitive a ssa y ._IGI IYI I a I c!/'Lnborntov y ar 1 d Chi ica I Mrdicirie, 8 I, 298.
Measurement of Heparin in Patients Receiving Subcutaneous Heparin Therapy K. W. E. DENSON AND J. BONNAR
N u i e l d D e y a r t ~ i n i tof Obstetrics and Gynaecology, University of Oxford, john RadCl@ Hospital, Oxford (Received 19 November 1974; accepted f o r Yiiblicatioit 26 Noveniber 1974) SUMMARY. Heparin assays based on four different tcsts of clotting function have becn coinpared with a heparin assay utilizing the potentiating effect of heparin on anti-factor Xa in a group of patients rccciviiig subcutaiieous heparin thcrapy during late prcgnancy. It is considered that the anti-factor Xa method is a bctter indication of the antithroinbotic effect of heparin. In the presence of high levels of clotting factors, heparin assays bascd on tcsts of clotting function can bc misleading. Subcutaneous heparin is being increasingly used in the prcvcntioii of post-opcrative thromboembolic coniplications (Kakkar et al, 1972; Gordon-Smith et al, 1972; Morris ct al, 1974). Low dosage heparin prophylaxis during and aftcr surgery rcquircs that sufficient heparin is released into the circulation to enhance the patient’s protective mechanisms against thrombosis, but at a lcvcl which will not compromise hacniostasis at the site of opcratioii. Iiieffcctivc therapy or hacmorrhagic complications are likely to discredit its value and already reports of both have been published (Macintyre et al, 1974; McWilliam et al, 1974; Hampsoii et al, 1974). Monitoring of heparin levels presents problems, but may prove to be essential for safe and effective heparin prophylaxis. Many methods have been used for measuring plasma or wholc blood heparin levels (Table I). With the exception of the protamiiie sulphate titration, these methods are all based on tests of clotting function. When the tcst samplc contains clotting factors within the normal range, the clotting time will reflect, fairly accurately, the level of heparin in the sample. In plasma from patients with raised levels of clotting factors, as occurs during pregnancy and oral contraception, or post-operatively, the clotting timc by any test of clotting function will be shortened, and can thus record a falsely low level of heparin. Conversely, if tlie clotting factors are low, as may arise in acute or sub-acute dissenrinated intravascular clotting, the clotting time would be longer and would be interpreted as a high heparin level. New techniques have recently been described for measuring heparin utilizing its potentiating or accelerating effect on the natural inhibitor to activated factor X (Yin et al, 1973 ; Denson & Bonnar, 1973). The methods are unaffected by varying levels of clotting factors in plasma samples, and should reflect heparin levels more accurately in patients with raised or reduced levels of clotting factors. As the level of heparin in the circulation is likcly to be the Correspondence:Dr K. W. E. Denson, Nuffield Department of Obstetrics and Gynaecology,TheJohn RadcliCe Hospital, Oxford.
I39
K. W. E. Denson and J. Bonrzar TABIEI. Tests which have been used for measuring heparin Whole blood clotting time Recalcification time Prothrombin time Partial thromboplastin time Kaolin cephalin clotting time H.A.R.E.M. test (activated whole blood clotting time) Protamine sulphate titration Calcium thrombin time Thrombin timc
critical factor in determining both its effictivcness and safety, we have coiiiparcd plasma heparin lcvels using four tests of clotting function and a spccific assay for heparin in a group of patictits during late prcgnancy who wcre receiving heparin therapy. MATERIALS A N D METHODS
Plasma samplcs. Blood from normal donors and patients (9 vol) was collectcd into I vol of sodium citratc containing 0.2 M HEPES buffer, and thc plasma scparated by centrifuging at 2500 g. Thc platclet-poor supernatant plasma was carcfully removed and kept dcep frozen a t - 20°C. Patients samples wcrc tcstcd in batches, aftcr storagc for pcriods of 2 days to I week. A single large pool of plasma from 20 normal donors was used as a standard during the period of the tests which extended ovcr 5 wccks. At the cnd of this time it was established that the pooled normal plasma had not dcteriorated in thc four tests ofclotting function using single batches of the standard frecze-dried reagents indicated below. Plasma for the heparin assay based on the potentiation of anti-factor Xa was heated for 15 min at 56°C and centrifuged to rcmove precipitated fibrinogcn. Thrombin (bovine), platelet substitute, kaolirz-platelet substitute mixture, activated bovine factor X andfactor-X deficient substrate plasrna were obtained from Diagnostic Reagents Limitcd, Thame, oxon. Bovine albtrmin (20%) was obtained from Arinour Pharmaceutical Co., Eastbournc, England. Citrate-aIbuM.iiiz-glyoxaline btifir consisted of 75 ml of glyoxaline buffer (0.5 M-imidazok and 0.1M-NaCl, pH 7.35)plus 15 nil of 0.13M sodium citratc plus 0.5 i d of 20% bovine albumin. Standard heparin. This was the first British standard prepared from bovine intcstitial niucosa and was kindly provided by D r D. R. Bangham from the MRC Division of Biological Standards, Mill Hill, London. Partial thromboplastin time ( P T T ) . Platelet substitute, 0.1nil, was placed in a clotting tube at 37°C and 0.1ml of plasma was added followed by 0. I ml of 0.025 M-CaCI,. The tubc was gently tilted and the clotting time recordcd. Kaolin-cephalin clotting time (KCCT). Kaolin-platelet substitute mixturc (0.2 nil) was placed in a clotting tube at 37°C followed by 0.1ml ofplasma; after 2 min with intermittent
0.13 M
MeasirvcMzeiit of Hcpuriiz
141
gciitlc shaking 0.1 1111 of 0.025 M-CaC], was addcd. The tube was gently tilted at 5-10 s intervals and the clotting time rccordcd. Throiirbiiz fiirrc ( T T ) .Plasma (0.2 1111) was placcd i n a clotting tube at 37'C, 0.2 in1 oftlirombin solutioii was added and tlic clotting time rccordcd. The thrombin coiiccntration was adjusted to givc a clotting time of approximately 11 I s with poolcd iiorinal plasma. C~7Iciiiriithmtiihirz r i m (Cn T T ) .Thronibin (0.2 i d ) of concciitratioii approximad y 3 .o ujml diluted in 0.025 M-CaCI, wa5 added to 0.2 1111 of plasiiia at 37'C and die c1otting time rccordcd. Tlic thrombin concciitratioii was adju5tcd to give a clotting tiiiic of T 5 IfI I s with pooled normal plasma. Calibvatrori C I ~ Y I W Srelutivq h p v i r z coirccizfvutioiz fo cloffiiqg tiirrc. For thc PTT, KCCT and Ca 'I'T, heyariii was added to pooled iiormal plasma at coiiccntratioiis of 0 , 0.05,0.1, 0.3, 0 . 3 , 0.4 aiid 0.5 ujiiil. For tlic thrombin time licparin was added to pooled normal plasma at concentrations of 0 , 0.05, 0.1,0 . ~ 2 0.14, , 0.r6 aiid 0.20 u/iiil. A narrow r a n g of coi~cciitra-
0.2
0.2
0.4
0.6
0.8
0.6-
0.4
-
0.2
0.4
0.6
0.0
Heparin assay ( u / r n l ) (ant1 X a method) I . Heparin lcvcls obtained with thc hcparin assay based 011 the potctitiation of anti-factor Xa plotted against heparin levels in thc sanic samples using a heparin assay based on thc 1'TT (a), KCCT (b), TT (c) and the Ca TT (d).
FIG
1.0
142
K. W. E. Denson and]. Bonnar
tioils was used for the latter because there was a large increase in clotting time for a small increment of heparin ovcr the range 0.10-0.20 u/ml, and beyond 0.3 u/ml the plasma was incoagulable. Each plasma sample over the range was tested in duplicate and the clotting times plotted against heparin concentration on linear co-ordinate paper. The value for the patient samples were obtained by interpolation from these curves, and if the clotting time was outside the range, the test sainple was suitably diluted in pooled normal plasma. Heparin assay based on thc potentiation of anti-factor Xu (Denson & Bonnar, 1973). Diluted factor Xa (0.3 ml) was placed in a series of 62 x 8 m m plastic tubes. One tube was placed in the 37°C water bath, 0.1ml of heated plasma sample added and a stopwatch started. The tube was stoppered, inverted twice and the stopper discarded. At 4 mill 45 s duplicatc 0.1 ml aiiiouiits were transferred to duplicate glass clotting tubes containing 0.I ml of 0.05 M-CaC1,. At 5 min, 0.2 ml of a mixture of equal volumes of factor-X free substrate plasma and platelet substitute was added to each tube and the clotting timc recorded. Three samples were tested in cach 5 miii period at 1.5 min intervals, and all volumes were measured using calibrated blow-out Pasteur-type pipettes. Heparin was diluted in pooled normal plasma to give a range of concentrations of 0.02,0.04, 0.06,0.08,0.10, 0.15and 0.20 u per ml of plasma. The clotting times were plotted against coiiccntration of heparin on log/linear paper, and the values for test samples were obtained by interpolation from this linc. The factor Xa was used at a dilution in tlie assay which gave a substrate clotting time of about 20 s for pooled normal plasma in thc absence of heparin, and about 60 s in the presence of heparin at a concentration of 0.2 u/ml in plasma. Test samples which had heparin levels anticipated to bc outside thc range of the standard curve were diluted in heated pooled normal plasma, and the results multiplied by the reciprocal of the dilution. Patients. Blood was obtained from 16 patients during late pregnancy who were receiving subcutaneous heparin as treatment or prophylaxis because of throinboenibolic complications. Forty samples from a group of eight patients were used for the results shown in Fig r(a), jb) and (c) and 27 saniplcs from a different group of eight patients for the results shown in Fig I (d). The patients received eithcr sodium heparin obtained from Weddel Pharmaceuticals or calcium heparin (Calciparine) obtained from Laboratoire Choay, Paris. RESULTS Fig I shows the results of plotting heparin levels obtained with the heparin assay based on the potentiation of anti-factor Xa against heparin levels in the same samples using a heparin assay based on the PTT (a),KCCT (b), TT (c) and the Ca TT (d).Without exception, the individual levels recorded by the mcthod based on the potentiation of anti-factor Xa were all higher, and on average two or three times higher than the equivalent levels recorded by methods based on tests of clotting function. The regression lines and correlation coefficients are shown. In curves (a), (b) and (c) several points are shown as solid circles. Thesc represent heparin values recorded as zero by the tests of clotting function, but in fact the clotting times of these samples by the PTT, KCCT and TT were all shorter than the clotting times ofpooled normal plasma. This is presumably due to high levels of clotting factors in the samples, and although heparin is present when measured by tlie anti-factor Xa method, these shorter-than-normal clotting times would indicate negative values for heparin levels by the tests of clotting func-
Mcasurement of Hepariiz I43 tion. In the rcgrcssion lines, allowance cannot bc made for these shorter-than-normal clotting times. The correlation cocfficicna arc reasonably good because tlie samples uscd werc from a selected group of patients during late pregnancy. At different stages of prcgnaiicy, and at diffcreiit tiincs post-opcratively, when levels of clotting factors nu): vary widely it would be difficult to prcdict the heparin value for a given sample by the anti-factor Xa assay from the equivalent value obtained by a test of clotting function. DISCUSSION
In late pregnancy aiid after surgery, factor VIII iiiay rise to 30o-400% and factor X to zoo% of average normal (Bonnar et a / , 1970). When tests of clotting function, such as the kaoliiicephalin clotting time, are carried out on plasma saniples with high levels of coagulation factors, the clotting time will be only about 5-10 s shorter than the clotting time of normal plasma. Despite this small reduction in clotting time, when coagulaticn is triggered, explosive and much larger amounts of factor Xa and thrombin arc likely to be produced compared with the amounts from normal plasma. Large amounts of factor Xa will minimize the effect of its natural inhibitor anti-factor Xa, aiid thus it would be expected that heparin, which potentiatcs the effect of anti-factor Xa, would be measured in a test of clotting function with less accuracy. This is borne out in practice and is illustrated in one saiiiplc whicli had 0.36 u/ml of heparin, aiid yet in which the clotting times in tests of clotting function were shorter than with normal plasma. I11 samples with high levels of clotting factors, the tests of clotting function may reflect the efficiency of local haemostasis, for example, a normal KCCT might indicate normal haemostasis; but they do not provide a measure of tlic anti-thrombotic effect of licparin. The effect of factor Xa on thc,formatioii of stasis thrombi has been well illustrated by the work of Wessler & Yiii (1968) in which stasis thrombi wcre induced in an isolated segment of rabbit jugular vein by very small amounts of activated factor X, but not by a much larger amount of tlie non-activated clotting factor. The value of low dosage heparin appears to be that local liaeinostasis is unaffected, but dissemination of factor Xa produced a t tlic site of a tliroinbus or clot is reduced or prevented by the circulating heparin. Following surgery, or in prcgnancy where high levels of clotting factors are present, it is possible that larger amounts of factor Xa and thronibin can bc released into the circulation. The anti-factor Xa heparin assay in such circumstances will record accurately the available anti-thrombotic level of heparin. The tcsts of clotting function will reflect the efficiency of haciiiostasis, but could bc misleading as a measure of the aiitithrombotic level of lieparin in tlie circulation. ACKNOWLEDGMENTS
This investigation was supported by MRC Grant No. Gg1/755/C and by a research fellowship &om Diagnostic Reagents Ltd. REFERENCES BONNAR, J., MCNICHOL, G.P. &DOUGLAS, A.S. (1970) Coagulation and fibrinolytic mechanisms during
and aftcr normal childbirth. British Medical]orrmal, ii, 200.
I44
K. W. E. Denson and J. Bonnar
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