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Catheter unsuitable for the study of turn-over products of coagulation, fibrinolysis and platelet activation
Fii (1992) 6, supp: 3,78-M @ LongmanGnwp UK Ud 1992
Catheter Unsuitable for the Study of Turn-over Products of Coagulation, Fibrinolysis and Platelet Activation
IA. Huisveld*, P.J.M. van den Burg*, P. Meijer **, M. van Vliet*, J.E.H. Hospers*, W.L. Mosterd*, B.N. Bourna***, c. Khlft**
SUMMARY;
In this study we determined activators, inhibitors and turn-over products of coagulation and tibrinolysis in blood samples that were obtained simultaneously through a needle and via a catheter. Results indicate that a catheter may be used for determination of coagulation and fibrlnolytic components, but is unsuitable for the assay of the various activation products.
KEYWORDS. Blood collection. Catheter. Coagulation. Fibrinolysis. Platelets. Activation products
INTRODUCTION In studies that require the collection of several blood samples over a longer period of time, the use of a catheter may be very convenient. However, with the introduction of highly sensitive tests for haemostatic complexes and activation peptides, it has become increasingly clear lY2that meticulous standardization of the pre-analytical blood sampling routine is required. We compared two blood sampling procedures, i.e. clean venipuncture vs the use of a catheter. Blood was obtained during rest and under stimulated conditions.
Fig. 1 Exercise test and protocol for blood collection.
MATERIALS AND METHODS Five healthy male volunteers (age 20-30 y) were subjected to an exercise test on a bicycle ergometer. Participants cycled for 25 min at a submaximal workload of 70% of the maximum oxygen uptake (VO2max). From 25-30 min the load was increased to 100% V02-max and follwed by a recovery period of 25 min (Fig. 1). Prior to the start of the test a cannula (Virgo Vasculon with PTFE catheter 18 G/1.2 mm OD, length 45 mm, dead space 0.05 ml) was placed in the antecubital vein (Fig. 2). Just before the start of the test and at 4 additional moments (Fig. 1) over a period of 55 min, during the
Fig. 2 Catheter used for blood collection.
exercise test and the recovery period, blood was drawn in plastic syringes (B-D Plastipak, 10 ml). Samples were obtained simultaneously (without stasis) from the catheter and by a clean venipunture (BD Microlance 19 G,11/2) in the other arm. The first 3 ml of blood were discarded during each sampling procedure. Blood was immediately distributed over the various anticoagulant mixtures kept on melting ice and centrifuged within 30 min (3000 g, 20 min QC). CTAD for 8-TG and PAI- and sodium citrate (0.11 mol/l) for the remaining variables. After each collection procedure the needle was removed and the catheter was kept patent by infusing 2 ml saline every 5 min.
‘Dept.Med.Physiol& Sportsmed. and ***Dept. Haematology, State University Utrecht, Vondellaan 24, 3521 GG Utrecht, The Netherlands; ?? *Gaubius Laboratory IWO-TNO, P.O. Box430,234NI AK Leiden, The Netherlands
78
Fibrinolysis 79
BTG
fragment
Fl +F2
PAP
Soluble
TAT
D-DIMER
Fibrin
o1-‘--Li_
*I. ,..t
I.
I
10
,I
10
rut
16
OL
_i__Ii_____-_
mu
I
ma UI
10 lha
1s
10
r..,
11
mm
L___
I
$0
II
*I
10
tmm hnl
,m,
Fig. 3 Effect of blood collection procedure on plasma levels of activation products (mean + sem).
The following variables were determined: prothrombin fragment 1+2 (Enzygnost F1+2, Behring Diagnostica), thrombin-antithrombin III (TAT) complex (Enzygnost TAT, Behring Diagnostica), plasminaz-antiplasmin (PAP) complex (APP, a gift of Behring Diagnostica) soluble fibrin (Coa-set Fibrin Monomer, Chromogenix), d-dimer (Coal& D-dimer, Chromogenix), 8-thrombo-globulin (Elisa P-TG, Boehringer Mannheim), FXII activity, FVIII activity, AP’IT (ACL 2OO,IL), u-PA antigen,3 t-PA antigen (Imulyse tPA, Biopool AB), PAI- antigen (Coaliza PAI-1, Chromogenix).
RESULTS With the exception of the first sample, the activation products i.e. PTG, TAT, Fl+ 2, PAP, soluble fibrin and d-dimer were significantly higher in the samples obtained from the catheter as compared with the samples from the needle (Fig. 3). No significant differences with respect to the two sampling procedures were observed in APTT, FVIII- and FXII coagulation
activity, t-PA, u-PA, a trend in PAIapparent (Table 1).
antigen was
DISCUSSION Incorrect blood collection and handling may greatly affect the determinations of haemostatic variables, therefore, standardized procedures have been introduced.‘tSAccording to these procedures both free flow and vacuum techniques can be used for assays of most coagulation and fibrinolytic parameters. Blood collection through a catheter may be useful when serial determinations are made or when a venipuncture itself may induces stress-related changes in blood parameters. The results from this study clearly indicate that activation of the various components of the haemostatic system may take place on the thrombogenic surface of a catheter. Since this marginal activation is not reflected in plasma components that occur in plasma in relatively high concentrations a canula can be used for determination of most plasma proteins. However, with respect to the sensitive assays for the determination of activation
Table 1 Coagulation and fibrinolytic variables collected via catheter (C) and needle (N) (mean ? sem) Sample nr.
R C
APIT PXII Iv111 t-PAag u-PAag PAI-lag
set % NP % NP ng/ml ng/ml ng/ml
33 + 2 109 + 8 86 + 2 4+1 3.5 ? 0.2 23 2 10
M N 32 + 109 2 86 z 421 3.5 + 24 2
C 1 10 4 0.2 10
2521 130 + 12 190 2 13 19 * s 7.8 2 0.5 3s ? 13
10r N 2821 123 2 149 ? 21 + 7.8 5 22 lr
13 18 s 0.6 9
20r
2Sr
C
N
C
N
26 + 1 126 2 15 187 + 17 1s r s 4.9 + 0.2 29 2 8
27 + 1 122 + 13 167 2 19 16 + 3 4.9 2 0.2 20 2 10
26 + 1 117 2 18 174 + 22 922 4.4 ? 0.3 26 2 7
29 ? 1 123 5 16 151 + 18 10 ? 3 4.7 * 0.3 1s + 9
C 26 5 2 126 t 13 192 + 21 722 4.3 + 0.3 33 ?: 8
N 2821 122 t 9
in + a, 8k2 4.1 5 02 13 + 8
80 Catheter Unsuitable for the Study of Turn-over Products of Coagulation, Fibrinolysis and Platelet Activation
products, with very low concentrations or which are absent in plasma, a catheter is unsuitable for blood collection.
ACKNOWLEDGEMENT This study was supported by The Netherlands Heart Foundation (Grant 90.059).
REFERENCES 1.
Waltz D A 1984 Platelet-released proteins as molecular markers for the activation process. Semin Thromb Hemostas lo: 270-279
2. Sidelmann J, Gram J 1990 The influence of venipuncture, 3.
mixing and separation of blood on the measurement of T-ATIII complex and PAI. Fibrinolysis 4, supp 2: 124-126 Binnema D J. Van Iersel J J L, Dooijewaard G 1986 Quantitation of urokinase antigen in plasma and culture media by use of an EL&A. Thtomb Res 43t 569-577 Kluft C, Verheijen J H 1990 Leiden Fibrinolysis Working Partyz Blood collection and handling procedures for assessment of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1). Fibrinolysis 4, supp2: 155-161 ECAT Blood collection and performance of haemostasis assays. 1989 Appendix V: 14-17
Catheter Unsuitable for the Study of Turn-over Products of Coagulation, Fibrinolysis and Platelet Activation
IA. Huisveld*, P.J.M. van den Burg*, P. Meijer **, M. van Vliet*, J.E.H. Hospers*, W.L. Mosterd*, B.N. Bourna***, c. Khlft**
SUMMARY;
In this study we determined activators, inhibitors and turn-over products of coagulation and tibrinolysis in blood samples that were obtained simultaneously through a needle and via a catheter. Results indicate that a catheter may be used for determination of coagulation and fibrlnolytic components, but is unsuitable for the assay of the various activation products.
KEYWORDS. Blood collection. Catheter. Coagulation. Fibrinolysis. Platelets. Activation products
INTRODUCTION In studies that require the collection of several blood samples over a longer period of time, the use of a catheter may be very convenient. However, with the introduction of highly sensitive tests for haemostatic complexes and activation peptides, it has become increasingly clear lY2that meticulous standardization of the pre-analytical blood sampling routine is required. We compared two blood sampling procedures, i.e. clean venipuncture vs the use of a catheter. Blood was obtained during rest and under stimulated conditions.
Fig. 1 Exercise test and protocol for blood collection.
MATERIALS AND METHODS Five healthy male volunteers (age 20-30 y) were subjected to an exercise test on a bicycle ergometer. Participants cycled for 25 min at a submaximal workload of 70% of the maximum oxygen uptake (VO2max). From 25-30 min the load was increased to 100% V02-max and follwed by a recovery period of 25 min (Fig. 1). Prior to the start of the test a cannula (Virgo Vasculon with PTFE catheter 18 G/1.2 mm OD, length 45 mm, dead space 0.05 ml) was placed in the antecubital vein (Fig. 2). Just before the start of the test and at 4 additional moments (Fig. 1) over a period of 55 min, during the
Fig. 2 Catheter used for blood collection.
exercise test and the recovery period, blood was drawn in plastic syringes (B-D Plastipak, 10 ml). Samples were obtained simultaneously (without stasis) from the catheter and by a clean venipunture (BD Microlance 19 G,11/2) in the other arm. The first 3 ml of blood were discarded during each sampling procedure. Blood was immediately distributed over the various anticoagulant mixtures kept on melting ice and centrifuged within 30 min (3000 g, 20 min QC). CTAD for 8-TG and PAI- and sodium citrate (0.11 mol/l) for the remaining variables. After each collection procedure the needle was removed and the catheter was kept patent by infusing 2 ml saline every 5 min.
‘Dept.Med.Physiol& Sportsmed. and ***Dept. Haematology, State University Utrecht, Vondellaan 24, 3521 GG Utrecht, The Netherlands; ?? *Gaubius Laboratory IWO-TNO, P.O. Box430,234NI AK Leiden, The Netherlands
78
Fibrinolysis 79
BTG
fragment
Fl +F2
PAP
Soluble
TAT
D-DIMER
Fibrin
o1-‘--Li_
*I. ,..t
I.
I
10
,I
10
rut
16
OL
_i__Ii_____-_
mu
I
ma UI
10 lha
1s
10
r..,
11
mm
L___
I
$0
II
*I
10
tmm hnl
,m,
Fig. 3 Effect of blood collection procedure on plasma levels of activation products (mean + sem).
The following variables were determined: prothrombin fragment 1+2 (Enzygnost F1+2, Behring Diagnostica), thrombin-antithrombin III (TAT) complex (Enzygnost TAT, Behring Diagnostica), plasminaz-antiplasmin (PAP) complex (APP, a gift of Behring Diagnostica) soluble fibrin (Coa-set Fibrin Monomer, Chromogenix), d-dimer (Coal& D-dimer, Chromogenix), 8-thrombo-globulin (Elisa P-TG, Boehringer Mannheim), FXII activity, FVIII activity, AP’IT (ACL 2OO,IL), u-PA antigen,3 t-PA antigen (Imulyse tPA, Biopool AB), PAI- antigen (Coaliza PAI-1, Chromogenix).
RESULTS With the exception of the first sample, the activation products i.e. PTG, TAT, Fl+ 2, PAP, soluble fibrin and d-dimer were significantly higher in the samples obtained from the catheter as compared with the samples from the needle (Fig. 3). No significant differences with respect to the two sampling procedures were observed in APTT, FVIII- and FXII coagulation
activity, t-PA, u-PA, a trend in PAIapparent (Table 1).
antigen was
DISCUSSION Incorrect blood collection and handling may greatly affect the determinations of haemostatic variables, therefore, standardized procedures have been introduced.‘tSAccording to these procedures both free flow and vacuum techniques can be used for assays of most coagulation and fibrinolytic parameters. Blood collection through a catheter may be useful when serial determinations are made or when a venipuncture itself may induces stress-related changes in blood parameters. The results from this study clearly indicate that activation of the various components of the haemostatic system may take place on the thrombogenic surface of a catheter. Since this marginal activation is not reflected in plasma components that occur in plasma in relatively high concentrations a canula can be used for determination of most plasma proteins. However, with respect to the sensitive assays for the determination of activation
Table 1 Coagulation and fibrinolytic variables collected via catheter (C) and needle (N) (mean ? sem) Sample nr.
R C
APIT PXII Iv111 t-PAag u-PAag PAI-lag
set % NP % NP ng/ml ng/ml ng/ml
33 + 2 109 + 8 86 + 2 4+1 3.5 ? 0.2 23 2 10
M N 32 + 109 2 86 z 421 3.5 + 24 2
C 1 10 4 0.2 10
2521 130 + 12 190 2 13 19 * s 7.8 2 0.5 3s ? 13
10r N 2821 123 2 149 ? 21 + 7.8 5 22 lr
13 18 s 0.6 9
20r
2Sr
C
N
C
N
26 + 1 126 2 15 187 + 17 1s r s 4.9 + 0.2 29 2 8
27 + 1 122 + 13 167 2 19 16 + 3 4.9 2 0.2 20 2 10
26 + 1 117 2 18 174 + 22 922 4.4 ? 0.3 26 2 7
29 ? 1 123 5 16 151 + 18 10 ? 3 4.7 * 0.3 1s + 9
C 26 5 2 126 t 13 192 + 21 722 4.3 + 0.3 33 ?: 8
N 2821 122 t 9
in + a, 8k2 4.1 5 02 13 + 8
80 Catheter Unsuitable for the Study of Turn-over Products of Coagulation, Fibrinolysis and Platelet Activation
products, with very low concentrations or which are absent in plasma, a catheter is unsuitable for blood collection.
ACKNOWLEDGEMENT This study was supported by The Netherlands Heart Foundation (Grant 90.059).
REFERENCES 1.
Waltz D A 1984 Platelet-released proteins as molecular markers for the activation process. Semin Thromb Hemostas lo: 270-279
2. Sidelmann J, Gram J 1990 The influence of venipuncture, 3.
mixing and separation of blood on the measurement of T-ATIII complex and PAI. Fibrinolysis 4, supp 2: 124-126 Binnema D J. Van Iersel J J L, Dooijewaard G 1986 Quantitation of urokinase antigen in plasma and culture media by use of an EL&A. Thtomb Res 43t 569-577 Kluft C, Verheijen J H 1990 Leiden Fibrinolysis Working Partyz Blood collection and handling procedures for assessment of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1). Fibrinolysis 4, supp2: 155-161 ECAT Blood collection and performance of haemostasis assays. 1989 Appendix V: 14-17