Avoiding ambient air in test tubes during incubations of human whole-blood minimizes complement background activation

Storm, Benjamin; Christiansen, Dorte; Mollnes, Tom Eirik

doi:10.1016/j.jim.2020.112876

Cited by 3 publications

(6 citation statements)

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“…Heparin is known to interfere with complement, coagulation, and inflammation (23). Also, we have previously shown that ambient air in tubes triggers complement activation (22). Antifoam, heparin, and ambient air were avoided in our experimental model, minimizing undesired "background" activation, enabling us to study the complement, inflammation, and coagulation cascades with a higher signal-tonoise ratio than would otherwise have been possible.…”

Section: Discussionmentioning

confidence: 99%

“…A series of experiments were conducted (summarized in ( Figure 1 ) using the whole blood model ( 23 ) with modifications as previously described ( 22 ). The study was approved by the Regional Committee for Medicine and Health Research Ethics.…”

Section: Methodsmentioning

confidence: 99%

“…On a block heater set to 37°C, we added 858 µl blood from each donor to sets of 1.8-ml Nunc polypropylene tubes prefilled with specific inhibitors of the complement and the TLR systems diluted in sterile PBS with CaCl 2 and MgCl 2 (PBS) (Sigma-Aldrich, St. Louis, MO) to a final volume of 142 µl as follows: the C3 inhibitor peptide Cp40 (produced at the University of Pennsylvania as previously described [ 24 ]) to a final concentration of 20 µM, the recombinant humanized anti-C5 mAb eculizumab (Soliris; Alexion Pharmaceuticals GmbH, Zürich, Switzerland) to a final concentration of 100 µg/ml, the C5aR1 blocking cyclic peptide PMX53 (produced at the University of Queensland, St. Lucia, Australia, as previously described [ 25 ]) to a final concentration of 10 µM, the anti-human CD14 recombinant IgG2/4 Ab r18D11 (produced at Nordland Hospital, Bodø, Norway, as previously described [ 26 ]) to a final concentration of 15 µg/ml, a combination of Cp40 and r18D11 to a final concentration of 20 µM and 15 µg/ml, respectively, or tubes containing only PBS. Additionally, 3003 µl of blood from each donor was mixed with 497 μl PBS in 5 ml Nunc tubes and from there transferred to 1.8-ml tubes, filling the tubes and the lids completely (as shown in ( Figure 1A of [ 22 ]). To ensure that adequate concentrations of eculizumab and PMX53 were used in our experiments, we titrated these inhibitors; because the C5 concentration in serum and plasma is relatively constant during ex vivo experiments, we tested the eculizumab effect in serum using the Wieslab Complement System Alternative Pathway Wielisa assay (Svar Life Science, Malmö, Sweden) ( Supplemental Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Except for the “baseline” and “no air” samples, we bubbled ∼2.5 ml of ambient air through the blood using a 21-gauge sterile needle and 20-ml syringe, resulting in a mixture of air bubbles and blood filling the tubes (as shown in ( Figure 1C of [ 22 ]). All tubes except baseline were incubated on a Rock’n’Roller tube roller mixer (Labinco, Breda, the Netherlands) at 37°C for 180 min.…”

Section: Methodsmentioning

confidence: 99%

“…C3 and C5 may be pivotal for this interaction, because C3a and C5a can bind to their receptors, C3aR, C5aR1, and C5aR2, and activate many cells, including platelets, leukocytes, and endothelial cells ( 17 , 20 , 21 ). In the present ex vivo study, we used lepirudin-anticoagulated human whole blood without ambient air in the test tubes ( 22 , 23 ) to study the effect of air embolism, mimicked by air bubbles, on the complement, hemostatic, and cytokine systems. In this paper, we use the term “coagulation” when discussing the coagulation system and the term “hemostasis” when discussing the joint effect of platelet activation, tissue factor, and coagulation.…”

Section: Introductionmentioning

confidence: 99%

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Air Bubbles Activate Complement and Trigger Hemostasis and C3-Dependent Cytokine Release Ex Vivo in Human Whole Blood

Storm

Christiansen

Fure

et al. 2021

The Journal of Immunology

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Venous air embolism, which may complicate medical and surgical procedures, activates complement and triggers thromboinflammation. In lepirudin-anticoagulated human whole blood, we examined the effect of air bubbles on complement and its role in thromboinflammation. Whole blood from 16 donors was incubated with air bubbles without or with inhibitors of C3, C5, C5aR1, or CD14. Complement activation, hemostasis, and cytokine release were measured using ELISA and quantitative PCR. Compared with no air, incubating blood with air bubbles increased, on average, C3a 6.5-fold, C3bc 6-fold, C3bBbP 3.7-fold, C5a 4.6-fold, terminal complement complex sC5b9 3.6-fold, prothrombin fragments 1+2 (PTF1+2) 25-fold, tissue factor mRNA (TF-mRNA) 26-fold, microparticle tissue factor 6.1-fold, β-thromboglobulin 26-fold (all p < 0.05), and 25 cytokines 11-fold (range, 1.5–78-fold; all p < 0.0001). C3 inhibition attenuated complement and reduced PTF1+2 2-fold, TF-mRNA 5.4-fold, microparticle tissue factor 2-fold, and the 25 cytokines 2.7-fold (range, 1.4–4.9-fold; all p < 0.05). C5 inhibition reduced PTF1+2 2-fold and TF-mRNA 12-fold (all p < 0.05). C5 or CD14 inhibition alone reduced three cytokines, including IL-1β (p = 0.02 and p = 0.03). Combined C3 and CD14 inhibition reduced all cytokines 3.9-fold (range, 1.3–9.5-fold; p < 0.003) and was most pronounced for IL-1β (3.2- versus 6.4-fold), IL-6 (2.5- versus 9.3-fold), IL-8 (4.9- versus 8.6-fold), and IFN-γ (5- versus 9.5-fold). Antifoam activated complement and was avoided. PTF1+2 was generated in whole blood but not in plasma. In summary, air bubbles activated complement and triggered a C3-driven thromboinflammation. C3 inhibition reduced all mediators, whereas C5 inhibition reduced only TF-mRNA. Combined C5 and CD14 inhibition reduced IL-1β release. These data have implications for future mechanistic studies and possible pharmacological interventions in patients with air embolism.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Air Bubbles Activate Complement and Trigger Hemostasis and C3-Dependent Cytokine Release Ex Vivo in Human Whole Blood

Storm

Christiansen

Fure

et al. 2021

The Journal of Immunology

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Application of the C3 inhibitor compstatin in a human whole blood model designed for complement research – 20 years of experience and future perspectives

Mollnes

Storm

Brekke

et al. 2022

Seminars in Immunology

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Iatrogenic air embolism: pathoanatomy, thromboinflammation, endotheliopathy, and therapies

Marsh,

Moore,

Moore

et al. 2023

Front. Immunol.

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Iatrogenic vascular air embolism is a relatively infrequent event but is associated with significant morbidity and mortality. These emboli can arise in many clinical settings such as neurosurgery, cardiac surgery, and liver transplantation, but more recently, endoscopy, hemodialysis, thoracentesis, tissue biopsy, angiography, and central and peripheral venous access and removal have overtaken surgery and trauma as significant causes of vascular air embolism. The true incidence may be greater since many of these air emboli are asymptomatic and frequently go undiagnosed or unreported. Due to the rarity of vascular air embolism and because of the many manifestations, diagnoses can be difficult and require immediate therapeutic intervention. An iatrogenic air embolism can result in both venous and arterial emboli whose anatomic locations dictate the clinical course. Most clinically significant iatrogenic air emboli are caused by arterial obstruction of small vessels because the pulmonary gas exchange filters the more frequent, smaller volume bubbles that gain access to the venous circulation. However, there is a subset of patients with venous air emboli caused by larger volumes of air who present with more protean manifestations. There have been significant gains in the understanding of the interactions of fluid dynamics, hemostasis, and inflammation caused by air emboli due to in vitro and in vivo studies on flow dynamics of bubbles in small vessels. Intensive research regarding the thromboinflammatory changes at the level of the endothelium has been described recently. The obstruction of vessels by air emboli causes immediate pathoanatomic and immunologic and thromboinflammatory responses at the level of the endothelium. In this review, we describe those immunologic and thromboinflammatory responses at the level of the endothelium as well as evaluate traditional and novel forms of therapy for this rare and often unrecognized clinical condition.

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Avoiding ambient air in test tubes during incubations of human whole-blood minimizes complement background activation

Cited by 3 publications

References 5 publications

Air Bubbles Activate Complement and Trigger Hemostasis and C3-Dependent Cytokine Release Ex Vivo in Human Whole Blood

Air Bubbles Activate Complement and Trigger Hemostasis and C3-Dependent Cytokine Release Ex Vivo in Human Whole Blood

Application of the C3 inhibitor compstatin in a human whole blood model designed for complement research – 20 years of experience and future perspectives

Iatrogenic air embolism: pathoanatomy, thromboinflammation, endotheliopathy, and therapies

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