Why Doesn't My Heparinized Plasma Specimen Remain Anticoagulated?

A Discussion on Latent Fibrin Formation in Heparinized Plasma

Valerie Bush, PhD
BD Vacutainer Clinical Affairs

Introduction

The 'instability' of heparinized plasma for the purposes of this article is defined as the formation of a precipitate in the plasma after a certain period of time post-centrifugation. The identity of this precipitate is controversial and not clearly understood. However, based on the knowledge of the composition of blood, coagulation and heparin, there are several hypotheses that could be proposed. First, it may be beneficial to review some facts on coagulation and anticoagulation. Figure 1 is useful as a reference to the coagulation cascade and steps where heparin interferes with coagulation. The coagulation of plasma occurs through this cascade of interconnected pathways initiated by the 'surface contact' step (intrinsic pathway), as coagulation factors become activated upon contact with a negative surface, such as a glass tube wall. Similar to whole blood coagulation, plasma coagulation also involves the cellular component from cells remaining in the supernatant (extrinsic pathway). Each of these pathways results in the generation of fibrin.

Centrifugation of Whole Blood

Centrifugation is the process of separating lighter portions of a mixture or suspension from heavier portions by centrifugal force based on their relative densities. The separation of anticoagulated blood components by centrifugation is driven by differences in density and cell size. The heavier and larger red blood cells and white blood cells sediment more quickly than platelets. Hence, platelets are the primary cell type that can be found in plasma and the plasma obtained under most recommended centrifugation conditions used in chemistry is not completely acellular. The centrifuge speed, time and temperature, as well as patient cell counts, can influence the purity of the plasma.

Action of Heparin

Heparin interferes with clotting by complexing with anti-thrombin III (AT) and catalyzing the inhibition of thrombin. Heparin preparations used as pharmaceuticals and anticoagulant additives for evacuated blood collection tubes are comprised of a heterogeneous population of sulfated polysaccharides that carries a net negative charge. Because of heparin's composition, it tends to bind to a variety of plasma proteins and cell membranes and thus exhibits unpredictable pharmacokinetics. However, only ~20% of the molecules in the heparin are active in binding to AT. Additionally, heparin binds to other plasma and cellular proteins in addition to AT (e.g., platelet factor 4, or PF 4) that compete with AT for heparin binding, thereby reducing the availability of heparin for anticoagulation. The extent of this so-called heparin neutralization is dependent upon the number of cells remaining in the plasma after centrifugation. The more platelets in the supernatant, the greater the heparin neutralization. Conversion of fibrinogen to fibrin in heparin anticoagulated blood varies widely, dependent on individual number of cells and the concentrations of AT and other plasma proteins. It is also known that the heparin/AT complex inactivates activated Factors XII, XI, X, and VII, as well as other coagulation factors, in addition to thrombin.

The contact activation of Factor XII, high molecular weight kallikrien (HMWK), etc. is accelerated at low temperatures (<37°C) irrespective of the presence of heparin. Cold promoted Factor VII activation is the result of activation by both activated contact proteins and the trace amounts of thrombin they generate. Since this activation is accelerated at reduced temperatures, refrigeration predisposes these factors to activation under refrigeration, driving the reaction towards clotting, and thus may be antagonistic to the anticoagulant action of heparin. For coagulation testing, it has been shown that heparinized patient specimens, but not specimens from other patient populations, may demonstrate clinically significant shortening of the aPTT when stored uncentrifuged at room temperature. The mechanism of this aPTT shortening has been related to platelet activation and release of PF 4 which electrostatically neutralizes the heparin present in the specimen. This occurs with heparin levels achieved clinically in anticoagulated patients, levels significantly lower than the heparin levels present in evacuated tubes. This aPTT shortening supports the hypothesis that, in the presence of heparin, platelet activation may occur in vitro with resulting neutralization of heparin's anticoagulant effect.

Furthermore, a major side effect of heparin therapy is heparin-induced thrombocytopenia (HIT), when patients previously sensitized by earlier exposure to heparin are re-exposed to heparin. HIT (a low platelet count which may be associated with life-threatening thrombosis) occurs because of heparin-induced platelet aggregation. These aggregated and activated platelets also release PF 4 which, as described above, promotes clotting by neutralizing heparin. Thus, heparin may exert two opposing actions--anticoagulation and heparin-induced platelet activation. The net effect of these two actions may vary in different patient populations, particularly depending upon whether the patient has previously been sensitized to heparin.

Plasma Instability

The formation of fibrin, in vitro, in heparinized plasma is complex. There are three mechanisms that may potentially be involved in the 'instability' of heparinized plasma. They are as follows:

1. Cell sequestration and/or binding of heparin. It is well documented in the literature that heparin binds to cells (and other plasma proteins), but the time dependence of this reaction has not been well described. The heparin concentration in BD Vacutainer® Blood Collection Tubes is 14-17 U/mL; of this only 2.8-3.4 U/mL have the polysaccharide sequences that permit binding to AT.
2. Intrinsic contact activation that may occur both in the presence and absence of heparin is accentuated at temperatures <37°C.
3. Platelet activation in the presence of heparin which increases with time.

The net effect of these mechanistic 'stresses' opposes the anticoagulant action of heparin, and this can result in the subsequent formation of fibrin in the plasma.

Figure 1. Schematic Diagram of the Coagulation System. (Click to enlarge image)

Specimen Management

Adhering to the following recommended specimen processing steps will help to ensure that you are getting a good quality plasma sample, and will aid in minimizing the formation of latent fibrin.

• Fill evacuated blood collection tubes to the stated draw volume. This will ensure the proper blood to additive ratio in the tube, and help alleviate fibrin formation due to overfilled tubes.
• Invert tubes 8 to 10 times immediately after collection to be sure that the blood and heparin are mixed thoroughly.
• Centrifuge tubes at the higher end of the recommended centrifugation range (1000-1300 g) for the full ten minutes. This will minimize the presence of residual cells in the plasma.
• Store heparinized plasma at room temperature (20-25° C) to minimize intrinsic contact activation at extreme temperatures.
• Evaluate and enforce "add-on testing" policies that your facility has validated for analyte stability in plasma, including the effects of specimens containing latent fibrin.
• Always follow your facility's protocol and guidelines for proper specimen handling and processing.

References

1. Muller, AD, van Deijk WA, Devilee PP, van Dam-Mieras MC and Hemker HC. The activity state of factor VII in plasma: two pathways for the cold promoted activation of factor VII. Br J Haematol. 1986; 62(2):367-77.
2. Miller GJ, Seghatchian MJ, Walter SJ, Howarth DJ, Thompson SG, Esnouf MP and Meade TW. An association between the factor VII coagulant activity and thrombin activity induced by surface/cold exposure of normal human plasma. Br J Haematol. 1986; 62(2): 379-84.
3. Gralnick HR and Wilson OJ. Cold-promoted activation of factor VII and shortening of the prothrombin time. Adv Exp Med Biol. 1987; 214:113-29.
4. Akiyama M, Takami H and Yoshida Y. The mechanism of cold-induced platelet aggregation in the presence of heparin. Tohoku J Exp Med. 1995; 177(4): 365-74.
5. Adcock D, Kressin D, Marlar RA. The effect of time and temperature variables on routine coagulation tests. Blood Coagul Fibrinolysis. 1998; 9(6):463-70.
6. Fabris F, Ahmad S, Cella G, Jeske WP, Walenga JM and Fareed J. Pathophysiology of heparin-induced thrombocytopenia. Clinical and diagnostic implications - a review. Arch Pathol Lab Med 2000; 124(11):1657-66.
7. Harenberg J, Wang LC, Hoffmann U. Laboratory diagnosis of heparin-induced thrombocytopenia type II after clearance of platelet factor 4/heparin complex. J Lab Clin Med. 2001; 137:408-413.
8. BD Evacuated Blood Collection System package insert, January 2002.

Further Information about BD Products and Services:

• BD Vacutainer Blood Collection Tubes

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