Multiplexed measurements of immunomodulator levels in peripheral blood of healthy subjects: Effects of analytical variables based on anticoagulants, age and gender

Krishnan, Viswanathan V; Ravindran, Resmi; Wun, Ted; Luciw, Paul A.; Khan, Imran; Janatpour, Kim

doi:10.1002/cytob.21147

Cited by 18 publications

(5 citation statements)

References 15 publications

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“…Multiple investigators have evaluated the use of several anticoagulants in multiplex assay systems with different results. [6][7][8]15 In the present study, similar or improved recovery results were obtained when heparinized plasma samples were used instead of EDTA-treated plasma or serum samples. However, the effects of heparin on recovery results were variable.…”

Section: Discussionsupporting

confidence: 79%

Evaluation of a feline-specific multiplex, bead-based assay for detection of cytokines, chemokines, growth factors, and other immunologically active proteins in serum and plasma samples from cats

Halpin¹,

Saunders²,

Thompson³

et al. 2016

ajvr

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OBJECTIVE To evaluate a feline-specific multiplex, bead-based assay system for detection of recombinant and native proteins in serum samples and in EDTA-treated and heparinized plasma samples. SAMPLE Serum samples and EDTA-treated and heparinized plasma samples from 30 sick cats and 9 healthy client-owned cats and heparinized whole blood samples from 5 healthy purpose-bred cats. PROCEDURES Ability of the assay system to detect 19 recombinant and native immunologically active proteins in plasma and serum samples from healthy and purpose-bred cats was evaluated via spike-and-recovery tests, assessments of inter- and intra-assay variation, linearity results, and leukocyte stimulation. Effects of various concentrations of heparin and serum matrix solution on percentages of analytes recovered were also evaluated. Analyte concentrations in samples from healthy and sick cats were measured and compared between groups. RESULTS Percentages of analytes recovered were unsatisfactory for most assays. Serum and heparinized plasma samples yielded better recovery results than did EDTA-treated plasma samples. Use of serum matrix solution did not improve results. Use of heparin concentrations greater than the recommended range affected the results. Linearity of results was difficult to assess because of the poor recovery. For the analytes that were recovered sufficiently for assessment, linearity appeared to be reasonable despite the limited detection. CONCLUSIONS AND CLINICAL RELEVANCE Poor percentages of analytes recovered and adverse effects of sample protein matrix limited the usefulness of the multiplex, bead-based assay system for measurement of immunologically active proteins in solutions with high protein content; however, recovery results were fairly linear, potentially allowing evaluation of feline plasma or serum samples with high analyte concentrations.

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Section: Discussionsupporting

confidence: 79%

Evaluation of a feline-specific multiplex, bead-based assay for detection of cytokines, chemokines, growth factors, and other immunologically active proteins in serum and plasma samples from cats

Halpin¹,

Saunders²,

Thompson³

et al. 2016

ajvr

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“…In contrast, in CTN 0302 serum samples, we observed no differences in EGF levels with response (CR 46 pg/ml, PR 62 pg/ml vs NR 63 pg/ml, p=0.2) nor VEGF-A levels (CR 215 pg/ml, PR 195 pg/ml vs NR 166 pg/ml, p=0.8). Importantly, the 0302 samples were serum (not plasma) which generally is associated with higher levels of these markers due to release from platelets (28, 29). Additionally, levels of Ang2 and sEng significantly decreased in patients experiencing a CR or PR at day 28 (p<0.001 for each) across both cohorts.…”

Section: Resultsmentioning

confidence: 99%

“…Serum has higher levels of EGF and VEGF-A relative to plasma due to release from activated platelets(28, 29). Some of the differences we observed regarding AF and outcomes in the two trials may reflect the different sample sources, and further studies of these factors should ideally use plasma samples.…”

Section: Discussionmentioning

confidence: 99%

Circulating Angiogenic Factors Associated with Response and Survival in Patients with Acute Graft-versus-Host Disease: Results from Blood and Marrow Transplant Clinical Trials Network 0302 and 0802

Holtan

Verneris

Schultz

et al. 2015

Biology of Blood and Marrow Transplantation

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Circulating angiogenic factors (AF) reflect tissue healing capacity, although some AF can also contribute to inflammation and are indicative of endothelial dysfunction. The AF milieu in acute graft-versus-host disease (aGVHD) has not been broadly characterized. We hypothesized that patients with abundant AF involved in repair/regeneration vs. those mediating damage/inflammation would have improved outcomes. Circulating AF known predominantly for repair/regeneration (epidermal growth factor [EGF], fibroblast growth factor-1 and -2, heparin binding-EGF-like growth factor, vascular endothelial growth factor-A, -C, and -D) and for damage/inflammation (angiopoietin-2, endothelin-1, soluble endoglin [sEng], follistatin [FS], leptin, placental growth factor [PlGF]) were measured in a discovery set of HCT recipients with grade III/IV aGVHD versus controls, then validated in two aGVHD cohorts enrolled in Blood and Marrow Transplant Clinical Trials Network (BMT CTN) trials 0302 (N=105, serum) and 0802 (N=158, plasma) versus controls without aGVHD (N=53, serum). Levels of EGF and VEGF-A were lower than controls at the onset of aGVHD in both trials and higher with complete response to first-line aGVHD therapy in CTN 0802. FS and PlGF were elevated in aGVHD measured in either serum or plasma. At day 28 after initial aGVHD therapy, elevated FS was an independent negative prognostic factor for survival in both cohorts (hazard ratio 9.3 in CTN 0302, 2.8 in CTN 0802). These data suggest that circulating AF are associated with clinical outcomes after aGVHD and thus may contribute to both pathogenesis and recovery.

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“…A recent report showed that different anticoagulants could impact the cytokine concentrations in normal human donors (19), although other reports have not shown such an effect (21-23). We examined whether the two anticoagulant used in this study, EDTA or heparin, would impact the cytokine measurements.…”

Section: Resultsmentioning

confidence: 99%

“…700 μL of blood was collected using this technique and immediately mixed with 70 μL of Heparin 1000 units/mL (Sagent Pharmaceuticals, Schaumburg, IL). The influence of of anticoagulants is controversial with some publications reporting that different anticoagulants may affect cytokine concentrations (19, 20) although other studies did not observe this effect (21-23). To evaluate a potential anti-coagulant influence, samples were collected in EDTA or heparin 2 hours after LPS injection in a separate group of mice.…”

Section: Methodsmentioning

confidence: 99%

Location, Location, Location

Mella¹,

Chiswick

King³

et al. 2014

Shock

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Objective Considerable breakthroughs in the field of sepsis have been made using animal models. Sepsis exhibits a wide array of derangements that may be evaluated in the blood, including the release of pro- and anti-inflammatory cytokines. The Shock journal adheres to the ARRIVE guidelines regarding reporting in vivo results to allow reproducibility of data findings. It is generally assumed that blood cytokine concentrations collected from typical sampling sites will be similar, but there are no data validating that this is true. The main purpose of the present study was to determine if the location of blood sampling results in cytokine concentration differences following inflammatory insults. Methods Two different models of acute inflammation were studied. Adult, female ICR mice were injected with E. coli lipopolysaccharide (LPS, n = 28) or subjected to cecal ligation and puncture (CLP) (n = 16). They were sacrificed at early time points following these inflammatory challenges for the collection of blood from the facial vein, retro-orbital sinus, and heart. Additonal samples were collected in EDTA and heparin. Plasma cytokines from the same mouse were collected from each sampling site and evaluated by ELISA. Clinical chemical parameters including plasma BUN and total protein were also analyzed. Results Regardless of model, time of collection, or cytokine measured, cytokine values from heart blood were higher than facial vein values from the same mouse. IL-6 collected from the heart relative to the facial vein demonstrated elevated concentrations following injection of LPS. In a similar manner, higher concentrations of IL-6, MIP-2, IL-10 and IL-1RA were found in cardiac puncture samples compared to other sampling sites 24 hours following sepsis induced by cecal ligation and puncture. Similar differences were not seen when comparing BUN and total protein values from the two different sites. Using plasma IL-6 collected from the heart would incorrectly stratify predicted to live (Live-P) mice into the predicted to die (Die-P) category. Therefore, a simple linear regression model was developed to correctly re-stratify mice to their predicted fate. These data demonstrate that pro- and anti-inflammatory cytokine concentrations are dramatically elevated when drawn centrally from the heart compared to collection from peripheral locations such as the facial vein. It is critical for publications to document the sampling location when evaluating plasma cytokines and attempting to compare studies.

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Multiplexed measurements of immunomodulator levels in peripheral blood of healthy subjects: Effects of analytical variables based on anticoagulants, age and gender

Cited by 18 publications

References 15 publications

Evaluation of a feline-specific multiplex, bead-based assay for detection of cytokines, chemokines, growth factors, and other immunologically active proteins in serum and plasma samples from cats

Evaluation of a feline-specific multiplex, bead-based assay for detection of cytokines, chemokines, growth factors, and other immunologically active proteins in serum and plasma samples from cats

Circulating Angiogenic Factors Associated with Response and Survival in Patients with Acute Graft-versus-Host Disease: Results from Blood and Marrow Transplant Clinical Trials Network 0302 and 0802

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