False prolongation of the activated partial thromboplastin time (aPTT) in inflammatory patients: interference of C-reactive protein

The activated partial thromboplastin time (aPTT) and prothrombin time (PT) are often used to evaluate secondary haemostasis when estimating the bleeding risk in patients undergoing an invasive procedure. After introducing a new aPTT assay we noticed that a number of patients had a prolonged aPTT but a normal PT without clinical signs of increased bleeding tendency. Subsequent analyses to explore the cause of the prolonged aPTT were unsuccessful and the prolongation of the aPTT persisted despite infusion of fresh frozen plasma. There was a clinical impression that this phenomenon particularly occurred in patients with an inflammatory response, as reflected by an elevated level of C-reactive protein (CRP). Because CRP is an acute phase protein with known binding affinity for phospholipids (van Tits et al, 2005) and phospholipids are used as a catalytic surface in aPTT-based assays, it was hypothesized that CRP might interfere with the aPTT assay (Schouwers et al, 2010). To study the influence of CRP on the aPTT both CRP and aPTT were determined in 59 patients visiting the emergency department. Patients gave informed consent and were not using anticoagulants, had no bleeding history or clinical signs of an increased bleeding tendency. CRP was measured through a immunoturbidimetric assay (C-RP; Beckman Coulter, Brea, CA, USA). The aPTT was measured using the STA Cephascreen assay (Diagnostica Stago, Asnieres, France) as well as a kaolin-based STA aPTT assay (Diagnostica Stago). The Cephascreen aPTT assay showed a correlation between the CRP level and the duration of the aPTT (Cephascreen aPTT = 0·024 × [CRP (mg/l)] + 28·9; R2 = 0·4), while the kaolin-based aPTT was not influenced by CRP (Kaolin aPTT = −0·0018 × [CRP (mg/l)] + 29·1; R2 = 0·006) (Fig 1A, B). Both assays had similar reference ranges (≤32·0 s). Rising CRP concentrations prolonged the Cephascreen aPTT far above the reference range, to a maximum of 38 s. Based on these findings, we explored the effect of CRP on the aPTT in an in vitro test. Plasma from three healthy donors with a CRP concentration <8 mg/l were spiked with human CRP (Sigma-Aldrich, St. Louis, MO, USA) to a concentration up to 200 mg/l. The aPTT measured by the STA Cephascreen increased by 5·8 s (i.e. above reference range) compared to the STA Kaolin reagent, which increased only 1·6 s (Fig 1D). To determine the effect of the phospholpids in this process we added hexagonal phospholipids (Staclot-LA; Diagnostica Stago) to the CRP spiked (200 mg/l) normal plasma resulting in a significantly reduced (P < 0·05; t-test by Medcalc® software, Mariakerke, Belgium) prolongation of the aPTT in both assays (STA Cephascreen and Kaolin STA) (Fig 1C). Therefore, we strongly suggest that prolongation of aPTT assays by CRP is a phospolipid-dependent inferference. Influence of C-reactive protein on the activated partial thromboplastin time. Prolongation of the activated partial thromboplastin time (aPTT) in patients visiting the Emergency Department by (A) STA Cephascreen, or (B) STA Kaolin, in relation to C-reactive protein (CRP) concentrations. (C) Effect of phospholipids on CRP-spiked (200 mg/l) plasma on STA Cephascreen aPTT (open bars) and STA Kaolin aPTT (closed bars) relative to normal plasma only; results are expressed as means of two different experiments. *Significantly different (P < 0·05) P, plasma; PL, hexagonal phospolipids. (D) Increase in aPTT in different aPTT assays: STA Cephascreen (♦), STA Kaolin (■), HemosIL SynthASil (×) and Actin FS(△); results are expressed as means of at least two different experiments. In addition, to explore CRP interference on various available aPTT reagents, two other commonly used reagents, Actin FS (Dade® Actin® FS; Siemens Medical Diagnostics, Marburg, Germany) and HemosIL SynthASil (Instrumentation Laboratories, Bedford, MA, USA) were also studied. The reagents differ in activator and phospholipid mixture. Spiking of normal plasma with CRP resulted in a dose-dependent increase of 3·5 s at 200 mg/l using Actin FS and 2·1 s in the HemosIL SynthASil assay (Fig 1D). In conclusion, this study demonstrated that CRP inteferes with aPTT assays, resulting in a prolongation of the aPTT. The degree of prolongation is dependent on the CRP level and the type of the aPTT assay used. This phenomenon is most likely phospholipid-dependent. When an isolated prolongation of aPTT is found in inflammatory patients it is advocated to determine the aPTT with a relative CRP-independent assay (e.g. STA Kaolin assay) in order to prevent unnecessary additional analyses and postponing invasive procedures.