Clotting time analysis of citrated blood samples is strongly affected by the tube used for blood sampling

Ramström, Sofia

doi:10.1097/01.mbc.0000178827.52242.89

Cited by 28 publications

(25 citation statements)

References 16 publications

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“…Although these studies attempted to eliminate the contribution of the contact system to FXI activation by adding CTI or a FXII antibody to the isolated plasma, these additions did not eliminate the FXIa that was already formed via contact system activation during the blood draw and plasma isolation (ref. 28 and Fig. 2 and Table 1).…”

Section: Discussionmentioning

confidence: 99%

Feedback activation of factor XI by thrombin does not occur in plasma

Pedicord¹,

Seiffert

Blat³

2007

Proc. Natl. Acad. Sci. U.S.A.

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In this study, we tested the hypothesis that factor XI (FXI) activation occurs in plasma following activation of the extrinsic pathway by thrombin-mediated feedback activation. We used two different assays: (i) a direct measurement of activated FXI by ELISA and (ii) a functional assay that follows the activation of the coagulation cascade in the presence or absence of a FXI inhibiting antibody by monitoring thrombin activity. We failed to detect any FXI activation or functional contribution to the activation of the coagulation cascade in platelet poor or platelet-rich plasma, when activation was initiated by thrombin or tissue factor. Additionally, we found that, in the absence of a contact system inhibitor during blood draw, contact activation of FXI can mistakenly appear as thrombinor tissue-factor-dependent activation. Thus, activation of FXI by thrombin in solution or on the surface of activated platelets does not appear to play a significant role in a plasma environment. These results call for reevaluation of the physiological role of the contact activation system in blood coagulation.coagulation ͉ platelets ͉ contact activation B lood coagulation is mediated by a cascade of proteolytic enzymes termed the coagulation factors. The cascade of coagulation factors endoproteolytic cleavage events culminates in the cleavage of fibrinogen, by thrombin, and the formation of fibrin fibers which form blood clots. During in vitro plasma clotting assays, factor XI (FXI) is activated by factor XII (FXII). This process was termed the contact activation system as it depends on the binding of FXII to negatively charged artificial surfaces. Binding of FXII to these surfaces leads to its autoactivation, which is further augmented by the other two components of the contact activation system, plasma prekallikrein and high molecular weight kininogen (HK). Activated FXI (FXIa) ties into the intrinsic pathway of blood coagulation by activating factor IX (FIX), which together with activated factor VIII, initiates the common pathway by activating factor X leading to prothrombin activation and clot formation (see refs. 1-4 for recent reviews of the contact activation system).The physiological function of the contact system in hemostasis is not clear because deficiencies in components of the contact system, plasma prekallikrein, HK, and FXII are not associated with a bleeding diathesis. On the other hand, mutations in the downstream intrinsic pathway components, FVIII and FIX, lead to hemophilia A and hemophilia B, respectively. Interestingly, FXI deficiency leads to a mild bleeding disorder, hemophilia C. The existence of bleeding in FXI-deficient individuals raised the possibility that FXI can be activated independently of the contact system.Attempts to identify alternative mechanisms for FXI activation, using purified coagulation proteins, revealed that FXI can be activated by thrombin as well as being autoactivated by FXIa. Both thrombin-mediated activation and autoactivation, however, occur at a very slow rate in solution. The r...

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Section: Discussionmentioning

confidence: 99%

Feedback activation of factor XI by thrombin does not occur in plasma

Pedicord¹,

Seiffert

Blat³

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Although there are plenty of clinical papers about microparticle detection, only few publications deal with the critical factors affecting processing and quantification of microparticles whether directly obtained from patients' plasma samples or generated in vitro. Issues that have been investigated include influences of blood sampling site and technique [3], needle bore size [4], anticoagulant in collection tube [5], storage time at room temperature, type of quantification beads [6] and instrument settings of flow cytometer [7]. Furthermore, it could also be shown that different agents used for stimulation affect size, antigen and phosphatidylserine exposure of microparticles [8,9].…”

Section: Introductionmentioning

confidence: 99%

Recovery and composition of microparticles after snap-freezing depends on thawing temperature

Trummer

Rop

Tiede

et al. 2009

Blood Coagulation &Amp; Fibrinolysis

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Consent regarding the correct processing and storage of blood microparticles is lacking and different protocols for the freeze-thaw cycle exist. Therefore, three different thawing procedures were evaluated regarding their influence on recovery and composition of microparticles. Microparticles were prepared by TRAP-6 or A23187 stimulation of platelet-rich plasma from smokers and nonsmokers (n = 8), from an endothelial cell line or directly obtained from platelet-free plasma of septic patients (n = 5). After snap-freezing in liquid nitrogen platelet-free samples were thawed at 37 degrees, at room temperature or on ice and staining of microparticles was carried out with Annexin V-Cy5 as well as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Microparticle concentrations were determined by means of Trucount tubes. Recovery of platelet microparticles was significantly reduced when samples were thawed on ice (P = 0.001 for all antigens) compared with the two other techniques (P = 0.6 for 37 degrees and P = 0.7 for room temperature, respectively) whereas microparticles of endothelial origin appeared to be less influenced. There was a strong trend towards altered microparticle composition as microparticle counts detected by CD41 staining showed a stronger decrease on ice than Annexin V enumeration (P = 0.07). For microparticle detection thawing of snap-frozen, platelet-free plasma samples should be carried out at room temperature or at 37 degrees C in a water bath but not on ice.

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“…21 Although the exact mechanisms of FXIIa generation in the Amelung assay were not identified, it is, in this context, worth reiterating the well-known fact that in every conventional in vitro assay used to monitor coagulation, small, but not negligible, amounts of FXII will eventually be generated by plastic or metal surfaces in contact with plasma. [22][23][24][25] In the absence of strong activators of coagulation, such artifactual sources of FXIIa will become the predominant procoagulant mechanism. Thus, the identity of the "foreign surface" triggering generation of FXIIa, as referred to in the above quote, is far more likely to have been the moving metal ball used to monitor coagulation in the Amelung instrument than platelet-derived polyphosphates.…”

Section: Discussionmentioning

confidence: 99%

“…[22][23][24][25] In the absence of strong activators of coagulation, such artifactual sources of FXIIa will become the predominant procoagulant mechanism. Thus, the identity of the "foreign surface" triggering generation of FXIIa, as referred to in the above quote, is far more likely to have been the moving metal ball used to monitor coagulation in the Amelung instrument than platelet-derived polyphosphates.…”

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confidence: 99%

Putting polyphosphates to the test: evidence against platelet-induced activation of factor XII

Faxälv

Boknäs

Ström

et al. 2013

Blood

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Key Points• Coagulation factor XII is not activated by platelets.The recent claim that stimulated platelets activate the intrinsic pathway of coagulation by the release of polyphosphates has been considered a breakthrough in hemostasis research. In little more than 3 years, the original publication by Müller et al has been cited >100 times. However, none of the citing articles has sought to independently validate this potentially paradigm-shifting concept. To this end, we performed extensive experimentation in vitro and in vivo in an attempt to verify the claim that factor XII (FXII) is primarily activated by stimulated platelets. In contrast to the original assertion, platelet-derived polyphosphates were found to be weak activators of FXII, with a FXIIa-generating activity of <10% compared with equivalent concentrations of kaolin. Using different coagulation assays, it was shown that platelet contribution to whole blood coagulation was unrelated to the generation of activated FXII in vitro. Additionally, key results used to verify the hypothesis in the original study in vivo were found to be irreproducible. We conclude that plateletderived polyphosphates are not physiologically relevant activators of FXII. (Blood. 2013;122(23):3818-3824) IntroductionThe enigmatic role of the intrinsic pathway of coagulation has been studied with renewed interest ever since it was shown that 1 strain of factor XII (FXII)-deficient mice are protected from thrombosis in various in vivo models, eg, in ferric chloride-induced thrombosis in mesenteric vessels, carotid vessels and inferior vena cava, and collagen-induced pulmonary embolism, [1][2][3] and that FXII inhibitors protect from arterial thrombosis in various animal models. [4][5][6] These studies provide strong evidence for a role for FXII in pathological thrombosis in mice, although clinical evidence for an analogous role in humans is still lacking.One central question that has yet to be conclusively answered in this context is how FXII is activated during thrombosis in vivo. 7 Several activators of FXII have been proposed, most notably fibrin clot surfaces, 8 collagen exposed by damaged endothelium, 9 misfolded or denatured proteins, 10 and neutrophil extracellular traps. 11 However, none of these potential activators have been validated conclusively in vivo.Recently, Müller et al 12 proposed that FXII activation in vivo is caused by negatively charged polyphosphates (polyP) secreted from platelet dense granules on platelet activation (hereinafter designated paper A). In a very recent review in Blood elaborating on these findings, 13 one of the authors of paper A asserts that "the identification of polyP as a platelet-derived procoagulant agent provides a longsought link between primary and secondary hemostasis and may represent a new paradigm for the treatment of thromboembolic and inflammatory diseases." As the implications of this claim are considerable, it is imperative to independently verify the experimental results.In the present study, we tested the following c...

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Clotting time analysis of citrated blood samples is strongly affected by the tube used for blood sampling

Abstract: Our aim was to establish whether differences in clotting times for recalcified blood and plasma samples might be explained by the use of different blood collection tubes.

Cited by 28 publications

References 16 publications

Feedback activation of factor XI by thrombin does not occur in plasma

Feedback activation of factor XI by thrombin does not occur in plasma

Recovery and composition of microparticles after snap-freezing depends on thawing temperature

Putting polyphosphates to the test: evidence against platelet-induced activation of factor XII

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