Conventional Chromogenic Heparin Assays are Influenced by Patient's Endogenous Plasma Antithrombin Levels

Abstract

Objectives: To determine, in vitro, the influence of plasma AT concentrations on heparin levels as measured by commercially available chromogenic kits.

Methods: Purified AT was added to plasma that was immune depleted of AT at the following final concentrations: 0, 0.5, 1.0, 1.5, 2.0, 2.5 u/ml. Heparin was added to each of the AT concentrations at the following final concentrations: 0, 0.2, 0.4 u/ml. Heparin levels were then determined using 5 different Anti-Xa (n=4) or Anti-IIa (n=1) commercial kits. All kits provided purified AT to be added to the assay system.

Results: When heparin was added to plasma, heparin concentrations measured were dependent on plasma concentrations of AT (p<0.001). When plasma concentrations of AT were less than 1.0 u/ml heparin concentrations were underestimated and when plasma concentrations of AT were greater than 1.0 u/ml actual heparin levels were over estimated. There was no significant difference in the findings between the various commercial Xa or IIa kits. In the absence of heparin, anticoagulant activity was detected when plasma AT concentrations were above 1.0 u/ml. There was a statistically significant correlation between plasma concentrations of AT and the amount of anticoagulant activity (p<0.001).

Conclusions: Conventional chromogenic heparin assays are influenced by endogenous plasma AT levels.

Zusammenfassung

Hintergrund: Der Einfluss der Plasmaantithrombinkonzentration auf die Heparinspiegel-bestimmung mittels kommerzieller chromogener Substratmethoden soll untersucht werden.

Methoden: Gereinigtes Antithrombin (AT) wurde zu AT-Mangelplasma in den Konzentrationen von 0, 0,5, 1,0, 1,5, 2,0, 2,5 u/ml hinzugegeben. Anschließend wurde zu jeder AT-Konzentration Heparin in den folgenden Konzentrationen zugefügt: 0, 0,2, 0,4 u/ml. Heparinspiegel wurden mit 5 unterschiedlichen kommerziellen Bestimmungsmethoden als Anti-Xa (n=4) oder Anti-IIa (n=1) bestimmt. Alle Kits stellten gereinigtes AT für die Durchführung der Analysen zur Verfügung.

Ergebnisse: Nach Heparinzugabe war die gemessene Heparinkonzentration signifikant abhängig von der Plasma-AT-Konzentration (p<0,001). War die AT-Konzentration <1,0 u/ml, wurde die Heparinkonzentration zu niedrig gemessen, war die Plasma-AT-Konzentration > 1,0u/ml, wurde die Heparinkonzentration zu niedrig gemessen, war die Plasma-AT-Konzentration >1,0 u/ml wurden die Heparinkonzentrationen zu hoch gemessen. Wir konnten keine Unterschiede zwischen den kommerziellen Kit-Anbietern feststellen. In Abwesenheit von Heparin konnte eine unspezifische antikoagulatorische Aktivität bei Plasma-AT-Konzentrationen >1,0 u/ml nachgewiesen werden (p<0,001).

Schlussfolgerung: Chromogene Heparinbestimmungsmethoden sind durch endogene AT Plasmaspiegel beeinflussbar.

Key words

heparin assays - antithrombin - chromogenic substrates

Schlüsselwörter

Heparinbestimmungsmethoden - Heparin - Antithrombin - Heparinkonzentration

References

• 1 Andrew M, Marzinotto V, Massicotte P. et al . Heparin therapy in pediatric patients: a prospective cohort study.  Pediatr Res. 1994;  35 78-83
• 2 Andrew M, Paes B, Milner R. et al . Development of the human coagulation system in the full-term infant.  Blood. 1987;  70 165-172
• 3 Chan AK, Leaker M, Burrows FA. et al . Coagulation and fibrinolytic profile of paediatric patients undergoing cardiopulmonary bypass.  Thromb Haemost. 1997;  77 270-277
• 4 Holm HA, Abildgaard U, Larsen ML. et al . Monitoring of heparin therapy: should heparin assays also reflect the patient's antithrombin concentration?.  Thromb Res. 1987;  46 669-675
• 5 Ignjatovic V, Summerhayes R, Gan A. et al . Monitoring Unfractionated Heparin (UFH) therapy: which Anti-Factor Xa assay is appropriate?.  Thromb Res. 2007;  120 347-351
• 6 Kuhle S, Eulmesekian P, Kavanagh B. et al . Lack of correlation between heparin dose and standard clinical monitoring tests in treatment with unfractionated heparin in critically ill children.  Haematologica. 2007;  92 554-557
• 7 Kuhle S, Eulmesekian P, Kavanagh B. et al . A clinically significant incidence of bleeding in critically ill children receiving therapeutic doses of unfractionated heparin: a prospective cohort study.  Haematologica. 2007;  92 244-247
• 8 Larsen ML, Abildgaard U, Teien AN. et al . Assay of plasma heparin using thrombin and the chromogenic substrate H-D-Phe-Pip-Arg-pNA (S-2238).  Thromb Res. 1978;  13 285-288
• 9 Schmidt B, Mitchell L, Ofosu F. et al . Standard assays underestimate the concentration of heparin in neonatal plasma.  J Lab Clin Med. 1988;  112 641-643
• 10 Teien AN, Lie M, Abildgaard U. Assay of heparin in plasma using a chromogenic substrate for activated factor X.  Thromb Res. 1976;  8 413-416
• 11 Ten CH, Lamping RJ, Henny CP. et al . Automated amidolytic method for determining heparin, a heparinoid, and a low-Mr heparin fragment, based on their anti-Xa activity.  Clin Chem. 1984;  30 860-864

Correspondence