Blood Collection Tubes Influence Serum Ficolin-1 and Ficolin-2 Levels

Brady, Allison M.; Spencer, Brady L.; Falsey, Ann R.; Nahm, Moon H.

doi:10.1128/cvi.00607-13

Cited by 23 publications

(34 citation statements)

References 30 publications

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“…Though the inhibitor present in plastic blood collection tubes affects detection of ficolin-2 by ELISA and flow cytometry, sequestered ficolin-2 is still visible by Western blotting (15). Therefore, we assayed NHS and all of the depleted sera for ficolin-2 by Western blotting to determine if the absence of ficolin-2 in the complement component-depleted sera may be due to the type of tube used during blood collection.…”

Section: Resultsmentioning

confidence: 99%

“…Complement component-depleted sera are codepleted of ficolin-2. We previously found that plastic blood collection tubes contain a ficolin-2-specific inhibitor that sequesters ficolin-2, effectively inhibiting ficolin-2 binding to pneumococci (15). To investigate if C1q-depleted sera inhibited ficolin-2 binding, we mixed rFicolin-2 supernatant with C1q-depleted sera and studied ficolin-2 binding to serotype 11A bacteria using flow cytometry.…”

Section: Resultsmentioning

confidence: 99%

“…Ficolin-1 and -2 and MBL binding assays were performed as previously described (15). Briefly, bacteria or yeast was incu-bated with 5% (for ficolin-2 and MBL) or 15% (for ficolin-1) NHS or complement component-depleted sera in FBB for 1 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

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Commercially Available Complement Component-Depleted Sera Are Unexpectedly Codepleted of Ficolin-2

Brady

Geno

Dalecki

et al. 2014

Clin. Vaccine Immunol.

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The ficolins are a family of innate pattern recognition molecules that are known to bind acetylated compounds and activate complement through the association of mannose binding lectin (MBL)/ficolin-associated serine proteases (MASPs). Their importance has more recently become appreciated, as they have been shown to play a role in a variety of disease processes from infection to autoimmunity. While studying ficolin-2-mediated complement deposition on Streptococcus pneumoniae, we found that sera depleted of C1q or other complement components were also codepleted of ficolin-2 but not ficolin-1, ficolin-3, or MBL. MBL present in C1q-depleted sera was able to mediate complement deposition on Saccharomyces cerevisiae, suggesting the presence of MASPs. We found that complement was activated on pneumococci in C1q-depleted serum only after opsonization with exogenous recombinant ficolin-2 (rFicolin-2). Also, no complement deposition was observed in C1q-depleted serum when pneumococci were opsonized with rFicolin-2 mutated at its lysine-57 residue, where MASPs are known to associate. Thus, these depleted sera are a unique tool to study ficolin-2-mediated complement pathways; however, one should be aware that ficolin-2 is absent from complement component-depleted sera.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Commercially Available Complement Component-Depleted Sera Are Unexpectedly Codepleted of Ficolin-2

Brady

Geno

Dalecki

et al. 2014

Clin. Vaccine Immunol.

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“…Values for young, healthy controls were previously reported (Brady et al, 2014c). In mixing experiments, each serum sample was tested alone at 20-fold dilution (15 µl serum + 285 µl kit dilution buffer).…”

Section: Methodsmentioning

confidence: 99%

Ficolin-2 inhibitors are present in sera after prolonged storage at −80 °C

Geno

Kennedy

Sawyer

et al. 2016

PeerJ

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Ficolins can activate the lectin pathway of the complement system that provides innate immune protection against pathogens, marks host cellular debris for clearance, and promotes inflammation. Baseline inflammation increases with aging in a phenomenon known as “inflammaging.” Although IL-6 and C-reactive protein are known to increase with age, contributions of many complement factors, including ficolins, to inflammaging have been little studied. Ficolin-2 is abundant in human serum and can recognize many target structures; therefore, ficolin-2 has potential to contribute to inflammaging. We hypothesized that inflammaging would alter ficolin-2 levels among older adults and examined 360 archived sera collected from older individuals. We found that these sera had apparently reduced ficolin-2 levels and that 84.2% of archived sera exhibited ficolin-2 inhibitors, which suppressed apparent amounts of ficolin-2 detected by enzyme-linked immunosorbent assay. Fresh serum samples were obtained from donors whose archived sera showed inhibitors, but the fresh sera did not have ficolin-2 inhibitors. Ficolin-2 inhibitors were present in other long-stored sera from younger persons. Furthermore, noninhibiting samples and fresh sera from older adults had apparently normal amounts of ficolin-2. Thus, ficolin-2 inhibitors may arise as an artifact of long-term storage of serum at −80 °C.

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“…Besides anticoagulants, the blood collection tube contains multiple components such as clotting activator, silicones, a silica clot activator, and polymeric surfactants (34,35), all of which may affect the assays. Appropriate blood collection is crucial for accurate immunomonitoring of human clinical samples.…”

Section: Discussionmentioning

confidence: 99%