Category:

Turnaround Time: 4 - 7 days
CPT Code: 86160
Test Type: 1 mL Plasma (EDTA) with Futhan, frozen Minimum Volume 0.5 mL (Note: This volume does not allow for repeat testing.)
Stability Time: Temperature Period Room temperature 6 hours Refrigerated 5 days Frozen 14 days Freeze/thaw cycles Stable x1

Overview:

Activation of the complement system plays an important role in our natural ability to ward off infection and in the pathogenesis of infection and inflammation.1-6 Anaphylatoxins produced as the result of complement activation play a role in a number of infectious and inflammatory conditions including sepsis, ischemia-reperfusion injury, immune complex diseases, and hypersensitivity diseases like asthma.1 Anaphylatoxins are also thought to be important in the pathogenesis of allergy, autoimmunity, neurodegenerative diseases, and cancer.2,3 Complement activation can occur through three separate mechanisms. The first mechanism to be discovered, referred to as the classical complement cascade, is activated by antigen-antibody complexes.4-6 This was the basis for the name of this system as they serve to "complement" humoral immunity. Alternatively, the complement cascade can be activated directly by contact with bacterial cell surface molecules, including lipopolysaccharide from gram-negative outer membranes, teichoic acid from gram-positive cell walls, zymosan from fungal and yeast cell walls, and some parasite surface molecules. Recently, a third activation mechanism has been characterized in which mannose-binding lectin synthesized by the liver in response to inflammatory macrophage cytokines stimulates the activation of complement. Complement cascade activation results in the formation of complement split products C3a, C4a, and C5a.4 These proteins, referred to as anaphylatoxins, facilitate the phagocytosis of immune complexes, viral particles, toxic cell debris and apoptotic corpses. Anaphylatoxins promote an inflammatory response by binding to complement receptors on granulocytes and macrophages.2 Anaphylatoxins also bind to receptors on mast cells, which trigger the release of histamine, increasing blood vessel permeability and smooth muscle contraction.6 They control the local inflammatory response through activation of leukocytes and stimulating their chemotaxis to the site of infection.4 Complement C4a levels can become increased in any condition associated with inflammation.2,3 Normal human pregnancy is associated with evidence of complement activation, with an increase in concentrations of the anaphylatoxins C3a, C4a, and C5a in the maternal circulation.7 Levels of anaphylatoxins C3a and C4a have also been found to be elevated in patients with antiphospholipid syndrome relative to healthy controls.8 Ingram and associates have shown that levels of C4a are increased in patients with multiple sclerosis, especially during relapse.9 Complement activation split products are present only in trace amounts in normal plasma in vivo.10 It is crucial that samples be collected and stored properly in order to avoid in vitro activation.10 Blood must be drawn directly into tubes containing EDTA at a final concentration of at least 10 mM.10 Citrate and heparin do not block complement activation efficiently and should not be used.10 The addition of nafamostat mesilate (Futhan, FUT-175) further reduces in vitro complement activation.11 Results for this test are for research purposes only by the assay's manufacturer. The performance characteristics of this product have not been established. Results should not be used as a diagnostic procedure without confirmation of the diagnosis by another medically established diagnostic product or procedure. Elevation of C4a levels is not predictive of any specific disease.1-9 Complement C4a levels can become increased in any condition associated with inflammation.2,3 Samples that are not properly collected and stored will produce erroneously elevated results due to in vitro activation.10 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.11 1. Haas PJ, van Strijp J. Anaphylatoxins: Their role in bacterial infection and inflammation. Immunol Res. 2007; 37(3):161-175. PubMed 17873401 2. Klos A, Tenner AJ, Johswich KO, Ager RR, Reis ES, Köhl J. The role of the anaphylatoxins in health and disease. Mol Immunol. 2009 Sep; 46(14):2753-2766. PubMed 19477527 3. Hawlisch H, Wills-Karp M, Karp CL, Köhl J. The anaphylatoxins bridge innate and adaptive immune responses in allergic asthma. Mol Immunol. 2004 Jun; 41(2-3):123-131. PubMed 15159057 4. Gasque P. Complement: A unique innate immune sensor for danger signals. Mol Immunol. 2004 Nov; 41(11):1089-1098. PubMed 15476920 5. Peng Q, Li K, Sacks SH, Zhou W. The role of anaphylatoxins C3a and C5a in regulating innate and adaptive immune responses. Inflamm Allergy Drug Targets. 2009 Jul; 8(3):236-246. PubMed 19601884 6. Wills-Karp M. Complement activation pathways: A bridge between innate and adaptive immune responses in asthma. Proc Am Thorac Soc. 2007 Jul; 4(3):247-251. PubMed 17607007 7. Richani K, Soto E, Romero R, et al. Normal pregnancy Is characterized by systemic activation of the complement system. J Matern Fetal Neonatal Med. 2005 Apr; 17(4):239-245. PubMed 16147832 8. Oku K, Atsumi T, Bohgaki M, et al. Complement activation in patients with primary antiphospholipid syndrome. Ann Rheum Dis. 2009 Jun; 68(6):1030-1035. PubMed 18625630 9. Ingram G, Hakobyan S, Robertson NP, Morgan BP. Elevated plasma C4a levels in multiple sclerosis correlate with disease activity. J Neuroimmunol. 2010 Jun; 223(1-2):124-127. PubMed 20409594 10. Mollnes TE, Garred P, Bergseth G. Effect of time, temperature and anticoagulants on in vitro complement activation: Consequences for collection and preservation of samples to be examined for complement activation. Clin Exp Immunol. 1988 Sep; 73(3):484-488. PubMed 2463123 11. Pfeifer PH, Kawahara MS, Hugli TE. Possible mechanism for in vitro complement activation in blood and plasma samples: Futhan/EDTA controls in vitro complement activation. Clin Chem. 1999 Aug; 45(8 Pt 1):1190-1199. PubMed 10430784

Collection Details:

Patient Preparation:

No radioactive isotopes administered within 48 hours prior to venipuncture.

Collection Instructions:

Futhan preservative must be added to samples immediately after collection. Collection kits containing this preservative are available as LabCorp N° 78946. Lavender-top (EDTA) tube Collect EDTA whole blood sample into a chilled lavender-top tube. Immediately add Futhan preservative as directed in the Futhan collection kit (LabCorp N° 78946). Recap lavender tube and invert several times to mix well. Centrifuge the whole blood specimen to separate the plasma. Transfer the plasma into the plastic screw-cap tube that is included in the collection kit. The specimen must be submitted in this transfer tube, which is labeled with the words "Futhan Added." Freeze immediately and maintain frozen until tested. To avoid delays in turnaround time when requesting multiple tests on frozen samples, please submit separate frozen specimens for each test requested. Freeze