Complement C₃a

Plasma (EDTA) with Futhan, frozen

1 mL

0.5 mL (Note: This volume does not allow for repeat testing.)

Lavender-top (EDTA) tube

No radioactive isotopes administered within 48 hours prior to venipuncture.

This test is used for the measurement of complement C₃a and C₃a-desArg levels in serum.

This test was developed and its performance characteristics determined by Labcorp. It has not been cleared or approved by the Food and Drug Administration.

Results should not be used as a diagnostic procedure without confirmation of the diagnosis by another medically established diagnostic product or procedure.

Elevation of C₃a levels is not predictive of any specific disease.

Normal human pregnancy is associated with evidence of complement activation, with an increase in concentrations of C₃a.¹

Complement activation split products are present only in trace amounts in normal plasma in vivo.² It is crucial that samples be collected and stored properly in order to avoid in vitro activation.² Blood must be drawn directly into tubes containing EDTA at a final concentration of at least 10 mM with the addition of nafamostat mesilate (Futhan, FUT-1750) to further reduce in vitro complement activation.³

Radioimmunoassay (RIA); this assay measures both C₃a and C₃a-desArg in an equimolar manner

69.2–273.6 ng/mL

The complement system is an ancient and highly conserved effector mechanism of the innate immune system with important functions in immune defense.^4-9 The main outcomes of complement system action are lytic killing of microbes, the rapid clearance of pathogens and dead or dying cells and immune complexes, and the release of pro-inflammatory anaphylatoxins. Activation and amplification of complement leads to destruction of pathogens or abnormal host cells through opsonization – a process by which complement ligands coat surfaces, targeting them for phagocytosis – or by lysis induced upon formation of the membrane attack complex (MAC), also referred to as the terminal complement complex (TCC). The ability to differentiate between self and non-self (or damaged/altered self) is a hallmark feature of the complement system. As a general paradigm, this is accomplished through complement recognition of components unique to microbial entities, often consisting of specific carbohydrate structures that are not found on host surfaces that are referred to as pathogen-associated molecular patterns (PAMPs).^10,11 Similarly, activated complement proteins are also able to recognize abnormal glycosylation patterns on the surface of dead or dying host cells (termed damage-associated molecular patterns, or DAMPs) and immune complexes, activating complement mechanisms that lead to their clearance. Complement activation also works to link innate and adaptive immune reactions by mediating T cell and B cell responses. The complement pathway is tightly regulated to avoid uncontrolled activation.^12,13

C₃, the central component of the complement system, is present in the blood in concentrations of more than 1 mg/mL, which makes it one of the most represented proteins in circulation.^14,15 Native C₃ is biologically inactive, but its activation fragments have a multitude of biological functions. Complement activation can occur through three separate mechanisms: the classical pathway, the lectin pathway and the alternate pathway, each of which is activated by different molecules.¹⁶ In the first pathway to be discovered, the classical pathway, C₃ is activated by antigen-antibody complexes that bind complement C₁q.^7-9 The lectin pathway is initiated by pathogen-associated molecular patterns (PAMPs), small molecular motifs, that are characteristic of microbial pathogens and are not present on mammalian tissues. These include mannose-binding lectin (MBL) and numerous other carbohydrate structures. The alternate pathway is continuously activated at a low level as a result of spontaneous C₃ hydrolysis allowing for activation on surfaces that lack proper complement regulation, typically non-host surfaces.^12,14,17

All complement activation pathways converge at the point of C₃ cleavage, which results in generation of bioactive fragments including C₃b and C₃a.¹⁸ C₃b is an efficient opsonin that marks foreign structures for phagocytosis; C₃b also forms a membrane complex with other complement proteins to activate complement C₅, yielding C₅a and C₅b. Like the C₃a generated in the proximal portion of the cascade, C₅a serves as an anaphylatoxin, a substance released into surrounding fluids and tissues to recruit/activate inflammatory cells. C₅b triggers assembly of the membrane attack complex (MAC), also referred to as the terminal complement complex (TCC), a pore-forming structure made up of C₅, C₆, C₇, C₈ and multiple C₉ molecules. The MAC forms a channel once inserted into the lipid bilayer, leading to lysis of cells or infecting organisms.

C₃a is the only bioactive fluid phase fragment generated by C₃ cleavage.¹⁴ C₃a is referred to as anaphylatoxins because it causes smooth muscle contraction, vasodilation, histamine release from mast cells and enhanced vascular permeability.^5-7,19,20 C₃a is a potent chemoattractant that recruits several types of phagocytes to the site of inflammation and triggers the production of reactive oxygen species in macrophages, eosinophils and neutrophils. C₃a exerts bactericidal activity by binding to their membranes and inducing cell lysis.²¹ C₃a can act on neutrophils to prevent their mobilization from the bone marrow to the circulation after ischemia-reperfusion injury.²² C₃a supports tissue regeneration after liver injury by stimulating cell proliferation, and it contributes to hematopoietic stem cell retention in the bone marrow, homing and engraftment.^23-25 In the brain, the C₃a has an important role in the recovery after the acute phase of ischemic injury, modulating neurogenesis and axonal and synaptic plasticity.²⁶

Measurement of plasma C₃a levels has been used in the assessment of the extent of complement activation in a variety of clinical conditions.^27-29 Excessive complement activation can contribute to the pathogenesis of many acute and chronic inflammatory diseases. C₃ has been implicated in several autoimmune^19,30 and inflammatory diseases, including acute kidney inflammation,³¹ neurotrauma, anti-phospholipid thrombosis,³² asthma and allogeneic transplantation.^19,33

In susceptible individuals, allergen exposure triggers the activation of complement, leading to the aberrant generation of the C₃a anaphylatoxin.^20,34-36 C₃a has been shown to be important for the development of Th2 responses by driving ILC2-mediated inflammation in response to allergen.³⁷ C₃a binds to receptors on mast cells to trigger the release of histamine and chemokines.^9,38 Nakano and coworkers found that plasma C₃a concentrations were significantly higher in patients seeking emergency treatment for severe asthma exacerbations than in control subjects with stable asthma.³⁹ They found that C₃a concentrations were highest among patients who failed to improve with treatment with inhaled bronchodilators and intravenous corticosteroids.³⁹ They also observed that levels of plasma C₃a in admitted asthmatic patients decreased significantly within a week after admission.³⁹

Clinical consequences of an overactivated and dysregulated complement system include not only immune complex and autoimmune disorders, such as systemic lupus erythematosus,^12,27,40 various forms of nephropathy, like atypical hemolytic uremic syndrome,^27,41 and C₃ glomerulopathy,^27,42,43 ophthalmic disorders, like age-related macular degeneration,⁴⁴ but also organ failure subsequent to ischemia reperfusion injury.⁴⁵ Paroxysmal nocturnal hemoglobinuria is an acquired clonal hematopoietic stem cell disorder in which uncontrolled complement activity leads to systemic complications, principally through intravascular hemolysis and thrombosis.^27,46 C₃a have been reported to be significantly higher in rheumatoid arthritis subjects compared to control subjects.⁴⁷ Plasma levels of complement C₃a measured daily during the first week after onset of symptoms of acute pancreatitis represent highly specific and sensitive parameters for the prediction of severe acute pancreatitis.^48,49

Preclinical studies into the mechanism of hepatic regeneration have demonstrated an essential role for C₃a in priming the hepatocyte proliferative response after injury or resection.⁵⁰ C₃a has also been implicated in the regeneration of bone, cardiac muscle and skeletal muscle, as well as stem cell engraftment.^51-53

C₃a has been used as a marker of neurodegenerative disease.⁵⁴ C₃a elevated in plasma one to 14 days after stroke and correlated with prognosis.^55-57 C₃a elevated in serum immediately after traumatic brain injury and remains chronically elevated.⁵⁸ C₃a levels have been shown to correlate with disease severity in patients with COVID-19⁵⁹ and in patients with septic shock.⁶⁰

In cancer, C₃a can have pro-tumor effects impacting immune system activation at the tumor site and favoring tumor progression. C₃a binding to its receptor maintains an immunosuppressive environment in sarcoma and promotes tumor progression by skewing the phenotype of tumor-associated macrophages⁶¹ while in lung cancer, C₃a signaling acts on CD4+ T cells and induces an inhibitory phenotype.⁶² In pancreatic ductal adenocarcinoma, C₃a activates the extracellular-regulated kinase pathway, inducing an epithelial-to-mesenchymal transition.^63,64

In plasma, C₃a is quickly converted by carboxypeptidase N into C₃a-desArg by cleavage of the C-terminal arginine.^65-67 C₃a-desArg does not bind to the human C₃a receptor.⁶⁸ A number of studies have shown that there is a positive relationship between C₃a-desArg levels, adipose tissue and risk factors for cardiovascular disease, metabolic syndrome and diabetes.^69,70 C₃a-desArg, also known as acylation stimulation factor, has been purported to play a role in transporting fatty acids to adipocytes and triacylglycerol synthesis.⁷¹ It has been suggested that production of C₃a (and C₅a) in adipose tissue triggers a cytokine and chemokine response in proportion to the amount of adipose tissue present and induces inflammation and mediate metabolic effects such as insulin resistance. These observations support the concept that complement activation may be an important participant in lipid metabolism and in obesity, contributing to the metabolic syndrome and to the low-grade inflammation associated with obesity.