Standard Operating Procedures for Biospecimen Collection, Processing, and Storage: From the Consortium for the Study of Chronic Pancreatitis, Diabetes, and Pancreatic Cancer.

High-quality and well-annotated biorepositories are needed to better understand the pathophysiology and biologic mechanisms of chronic pancreatitis (CP) and its consequences. We report a methodology for the development of a robust standard operating procedure (SOP) for a biorepository based on the experience of the clinical centers within the consortium to study Chronic Pancreatitis, Diabetes and Pancreas Cancer Clinical Centers (CPDPC), supported by the National Cancer Institute and the National Institute for Diabetes and Digestive and Kidney Diseases as a unique multidisciplinary model to study CP, diabetes, and pancreatic cancer in both children and adults. Standard operating procedures from the CPDPC centers were evaluated and consolidated. The literature was reviewed for standard biorepository operating procedures that facilitated downstream molecular analysis. The existing literature on biobanking practices was harmonized with the SOPs from the clinical centers to produce a biorepository for pancreatic research. This article reports the methods and basic principles behind the creation of SOPs to develop a biorepository for the CPDPC. These will serve as a guide for investigators developing biorepositories in pancreas research. Rigorous and meticulous adherence to standardized biospecimen collection will facilitate investigations to better understand the pathophysiology and biologic mechanisms of CP, diabetes, and pancreatic cancer.

Identification and description of a novel murine model for polytrauma and shock.

OBJECTIVE: To develop a novel polytrauma model that better recapitulates the immunologic response of the severely injured patient by combining long-bone fracture, muscle tissue damage, and cecectomy with hemorrhagic shock, resulting in an equivalent Injury Severity Score of greater than 15. We compared this new polytrauma/shock model to historically used murine trauma-hemorrhage models. DESIGN: Pre-clinical controlled in vivo laboratory study. SETTING: Laboratory of Inflammation Biology and Surgical Science. SUBJECTS: Six- to 10-week-old C57BL/6 (B6) mice. INTERVENTIONS: Mice underwent 90 minutes of shock (mean arterial pressure 30 mm Hg) and resuscitation via femoral artery cannulation followed by laparotomy (trauma-hemorrhage), hemorrhage with laparotomy and femur fracture, or laparotomy with cecetomy and femur fracture with muscle tissue damage (polytrauma). Mice were euthanized at 2 hours, 1 day, and 3 days postinjury. MEASUREMENTS AND MAIN RESULTS: The spleen, bone marrow, blood, and serum were collected from mice for analysis at the above time points. None of the models were lethal. Mice undergoing polytrauma exhibited a more robust inflammatory response with significant elevations in cytokine/chemokine concentrations when compared with traditional models. Polytrauma was the only model to induce neutrophilia (Ly6G (+)CD11b(+) cells) on days 1 and 3 (p<0.05). Polytrauma, as compared to trauma-hemorrhage and hemorrhage with laparotomy and femur fracture, induced a loss of circulating CD4(+) T cell with simultaneous increased cell activation (CD69(+) and CD25(+)), similar to human trauma. There was a prolonged loss of major histocompatibility complex class II expression on monocytes in the polytrauma model (p<0.05). Results were confirmed by genome-wide expression analysis that revealed a greater magnitude and duration of blood leukocyte gene expression changes in the polytrauma model than the trauma-hemorrhage and sham models. CONCLUSIONS: This novel polytrauma model better replicates the human leukocyte, cytokine, and overall inflammatory response following injury and hemorrhagic shock.

Effect of uric acid lowering therapy on the prevention of acute kidney injury in cardiovascular surgery.

PURPOSE: Serum uric acid (SUA) is a novel risk factor for acute kidney injury (AKI), which adversely affects renal blood flow autoregulation, glomerular filtration rate (GFR), and promotes inflammation and angiogenesis. This pilot study investigated the effect of lowering SUA therapy on AKI, by using traditional and non-traditional markers. MATERIALS AND METHODS: In this prospective, double-blind, placebo-controlled, randomized pilot trial, 26 hyperuricemic patients undergoing cardiac surgery were randomized to receive rasburicase or placebo in the preoperative period. RESULTS: Subjects receiving rasburicase showed no difference in serum creatinine compared with the control group receiving placebo. Despite no difference in primary endpoint, the rasburicase group had less evidence of renal structural injury as reflected by urine neutrophil-associated lipocalin (uNGAL) concentrations, especially in subjects with higher SUA levels, more severe renal dysfunction (baseline GFR ≤ 45 mL/min/1.73 m(2)) or heart failure (left ventricular ejection fraction ≤45 %). CONCLUSIONS: In this study, rasburicase showed no benefit on postoperative serum creatinine in hyperuricemic subjects undergoing cardiac surgery. However, the observation that markers of structural renal injury such as uNGAL tended to be lower in rasburicase-treated subjects suggests potential different effects of uricase treatment on hemodynamic alterations in renal function versus structural mechanisms of kidney injury.

How is your biobank handling disaster recovery efforts?

Recent years have seen a great many natural disasters-superstorms, droughts, earthquakes, among others-as well as, in the biobanking world, the constant threat of man-made disaster with everything from freezer malfunctions to theft. To help inform the increasingly important issue of protection from, and recovery after, disasters, Biopreservation and Biobanking put forth the question to our community of experts: How is your biobank handling disaster recovery efforts? Following is a selection of responses. Additionally, please see the Supplementary Information for contingency planning recommendations for biobanks and a threats assessment checklist from Intelsius ( supplementary material can be accessed from the online article at www.liebertpub.com/bio ).

Microfluidics-based capture of human neutrophils for expression analysis in blood and bronchoalveolar lavage.

Gene expression analysis can be a powerful tool in predicting patient outcomes and identifying patients who may benefit from targeted therapies. However, isolating human blood polymorphonuclear cells (PMNs) for genomic analysis has been challenging. We used a novel microfluidic technique that isolates PMNs by capturing CD66b(+) cells and compared it with dextran-Ficoll gradient isolation. We also used microfluidic isolation techniques for blood and bronchoalveolar lavage (BAL) samples of patients with acute respiratory distress syndrome (ARDS) to evaluate PMN genomic alterations secondary to pulmonary sequestration. PMNs obtained from ex vivo lipopolysaccharide (LPS)-stimulated or -unstimulated whole blood from five healthy volunteers were isolated by either dextran-Ficoll gradient, microfluidics capture, or a combination of the two techniques. Blood and BAL fluid PMNs were also isolated using microfluidics from seven hospitalized patients with ARDS. Gene expression was inferred from extracted RNA using Affymetrix U133 Plus 2.0 GeneChips. All methods of PMN isolation produced similar quantities of high-quality RNA, when adjusted for recovered cell number. Unsupervised analysis and hierarchical clustering indicated that LPS stimulation was the primary factor affecting gene expression patterns among all ex vivo samples. Patterns of gene expression from blood and BAL PMNs differed significantly from each other in the patients with ARDS. Isolation of PMNs by microfluidics can be applied to both blood and BAL specimens from critically ill, hospitalized patients. Unique genomic expression patterns are obtained from the blood and BAL fluid of critically ill patients with ARDS, and these differ significantly from genomic patterns seen after ex vivo LPS stimulation.

Microfluidic leukocyte isolation for gene expression analysis in critically ill hospitalized patients.

BACKGROUND: Microarray technology is becoming a powerful tool for diagnostic, therapeutic, and prognostic applications. There is at present no consensus regarding the optimal technique to isolate nucleic acids from blood leukocyte populations for subsequent expression analyses. Current collection and processing techniques pose significant challenges in the clinical setting. Here, we report the clinical validation of a novel microfluidic leukocyte nucleic acid isolation technique for gene expression analysis from critically ill, hospitalized patients that can be readily used on small volumes of blood. METHODS: We processed whole blood from hospitalized patients after burn injury and severe blunt trauma according to the microfluidic and standard macroscale leukocyte isolation protocol. Side-by-side comparison of RNA quantity, quality, and genome-wide expression patterns was used to clinically validate the microfluidic technique. RESULTS: When the microfluidic protocol was used for processing, sufficient amounts of total RNA were obtained for genome-wide expression analysis from 0.5 mL whole blood. We found that the leukocyte expression patterns from samples processed using the 2 protocols were concordant, and there was less variability introduced as a result of harvesting method than there existed between individuals. CONCLUSIONS: The novel microfluidic approach achieves leukocyte isolation in <25 min, and the quality of nucleic acids and genome expression analysis is equivalent to or surpasses that obtained from macroscale approaches. Microfluidics can significantly improve the isolation of blood leukocytes for genomic analyses in the clinical setting.

Increased mortality and altered immunity in neonatal sepsis produced by generalized peritonitis.

Neonates have a higher prevalence of bacterial sepsis and have greater morbidity and mortality from sepsis than other infants and children. Our understanding of the inflammatory and immunological responses to sepsis is hampered by the lack of appropriate neonatal murine models. In the present report, we have developed a cecal slurry model of generalized peritonitis in neonatal mice (age range, 5-7 days) and compared the outcome and the innate and adaptive cellular responses of these animals with those of the young adult animals (age range, 7-10 weeks) with sepsis induced either by cecal slurry administration or by cecal ligation and puncture. Neonatal mice were more susceptible to sepsis and mounted a markedly attenuated systemic inflammatory response compared with young adult animals (specifically, decreased plasma interferon gamma; interleukins 1alpha and 1beta; regulated on activation, normal T expressed and secreted (RANTES); and tumor necrosis factor alpha concentrations). Compared with young adult animals, septic neonatal mice did not lose significant percentage or absolute number of splenic CD4+ T cells. These findings suggest that the cecal slurry model of generalized peritonitis can produce sepsis in neonatal mice with dose-dependent lethality. Inherent differences in the host response to polymicrobial sepsis between neonatal and young adult animals may explain the increased sensitivity of the neonatal mouse to generalized peritonitis.

Maturation of murine bone marrow-derived dendritic cells with poly(I:C) produces altered TLR-9 expression and response to CpG DNA.

Although poly(I:C) and LPS induced differential dendritic cell (DC) cytokine profiles and toll-like receptor (TLR) expression, all were capable of causing phenotypic and functional DC maturation. Both LPS and poly(I:C) downregulated TLR-4/MD-2 expression on DCs. Although poly(I:C) highly upregulated their cell surface TLR-9 expression, LPS upregulated the intracellular TLR-9 expression. LPS-treated DCs could not produce IL-12p70 in response to subsequent both LPS- and CpG DNA-stimulation. On the other hand, poly(I:C)-treated DCs retained to produce IL-12p70 by subsequent CpG DNA-stimulation, while subsequent LPS-stimulation did not induce IL-12p70 production. Chloroquine, inhibitor of endosomal maturation, completely inhibited cytokine production of LPS-treated DCs as well as unstimulated control in response to subsequent CpG DNA-stimulation, while it failed to delete the IL-12p40 and IL-10 production in poly(I:C)-treated DCs. These data suggest that poly(I:C) may induce a novel DC phenotype that preserves the capacity of cytokine production to subsequent CpG DNA-stimulation.

Dose-dependent improvements in outcome with adenoviral expression of interleukin-10 in a murine model of multisystem organ failure.

Targeted expression of interleukin-10 (IL-10) has been proposed as a means to suppress acute and chronic inflammation. We explored the capacity of targeted adenoviral expression of human or viral IL-10 to improve outcome in a zymosan-induced model of acute lung injury and multisystem organ failure. Intratracheal administration of adenovirus expressing either human or viral IL-10 prior to zymosan administration significantly improved survival at a dose of 10(7) particles (P<0.01), whereas the same recombinant vectors were ineffective at 10(8) particles and increased mortality at 10(9) particles. Improved survival after administration of 10(7) particles of adenovirus expressing viral or human IL-10 was associated with local tissue expression of IL-10 (100-300 pg/g wet wt). In contrast, mortality after administration of 10(9) particles was associated with markedly elevated IL-10 expression, both in the lung (10000-70000 pg/g wet wt) and systemically (1000-3000 pg/ml plasma), with evidence of an exaggerated systemic inflammatory response (plasma IL-6 and TNFalpha). Targeted gene expression of IL-10 can be used to treat acute inflammatory processes, but increased doses resulting in its systemic release are not associated with improvements in outcome, and may actually exacerbate acute inflammatory processes.

Plasma cytokine measurements augment prognostic scores as indicators of outcome in patients with severe sepsis.

Despite recent advances in the prospective identification of the patient with sepsis who may benefit from anti-inflammatory or antithrombotic therapies, successful treatment regimens have been fairly modest. We have explored whether determination of several proinflammatory cytokine or mediator concentrations can complement physiologic scoring systems to identify patients with severe sepsis who will survive or expire within 28 days. The design of the study included an exploratory analysis performed in conjunction with a prospective, randomized, double-blind, placebo-controlled, multicenter, clinical trial and involved 33 academic institutions in the United States. One hundred twenty-four patients with severe sepsis with or without septic shock were included in this analysis. Blood samples were obtained at baseline and on days 1 through 4, and were evaluated for proinflammatory and anti-inflammatory cytokine concentrations, as well as for procalcitonin and total protein C levels. Baseline concentrations and changes in the concentrations of these mediators were evaluated in relationship to the Acute Physiology and Chronic Health Evaluation (APACHE) II and multiple organ dysfunction (MOD) scores, and 28-day all-cause mortality. Using univariate logistic regression analyses, APACHE II and MOD scores, age (but not gender), and baseline plasma interleukin (IL)-6 and soluble tumor necrosis factor receptor (sTNFR) 1 (log transformed) concentrations were all predictive of increased 28-day all-cause mortality (P < 0.01). Baseline total protein C, IL-8, IL-10, TNF-alpha, and procalcitonin concentrations, and the change in plasma cytokine concentrations from baseline over the initial 4 days were not useful in predicting outcome. Selected baseline proinflammatory cytokine concentrations and APACHE II score were correlated (P < 0.01). IL-6 concentration is a strong candidate for predicting clinical outcome in patients with severe sepsis alone, or when combined with the APACHE II or MOD scores. The potential usefulness of the combination of cytokine measurements and prognostic scores to identify patients who may benefit from treatment with anti-inflammatory or antithrombotic therapies should be further evaluated.

Pro- and antiinflammatory cytokine production after radiofrequency ablation of unresectable hepatic tumors.

BACKGROUND: Experience using radiofrequency ablation (RFA) for treating unresectable hepatic malignancies is expanding, with promising outcomes and fewer complications compared with cryotherapy. STUDY DESIGN: This study examined systemic inflammatory responses after RFA as measured by the appearance of postoperative symptoms and cytokine production. Seventeen patients (11 men, 6 women) aged 40 to 85 years (mean 64.2 years) with unresectable primary and metastatic hepatic tumors underwent RFA. Mean liver volume treated with RFA was 35.3% +/- 3.6% (SEM) (median 36.8%). Plasma cytokines (tumor necrosis factor-alpha, interleukin [IL]-1beta, IL-1ra, IL-6, IL-8, IL-10, p55, and p75) were measured from anesthesia induction through 48 hours after RFA. Ex vivo whole-blood cytokine production was measured at baseline, 24 hours, and 48 hours after RFA. RESULTS: Cytokine and cytokine-receptor production were not notably altered by RFA. Ex vivo whole-blood endotoxin stimulation indicated that intrinsic cellular immune function remained intact after treatment, although modest decreases in stimulated tumor necrosis factor alpha production were observed 24 to 48 hours after RFA. Variceal bleeding, hepatic failure, and death occurred in one patient 30 days after RFA. None of the remaining patients exhibited tachycardia or hypotension. Fevers (> or = 38.5 degrees C) developed in three patients during the first 48 hours postoperatively. There was no association between plasma cytokines and postoperative complications. CONCLUSIONS: In contrast to previous reports using cryotherapy, systemic inflammatory responses as measured by increased cytokines were not observed after RFA. The cryotherapy-induced "cryoshock" phenomenon was not observed in patients undergoing RFA in our study. We conclude that RFA ablation is fundamentally different than cryotherapy and apparently does not stimulate Kupffer and other hepatic macrophages to produce proinflammatory cytokines.