A negative selection methodology using a microfluidic platform for the isolation and enumeration of circulating tumor cells.

Circulating tumor cells (CTCs) exist in the peripheral blood stream of metastatic cancer patients at rates of approximately 1 CTC per billion background cells. In order to capture and analyze this rare cell population, various techniques exist that range from antibody-based surface marker positive selection to methods that use physical properties of CTCs to negatively exclude background cells from a CTC population. However, methods to capture cells for functional downstream analyses are limited due to inaccessibility of the captured sample or labeling techniques that may be prohibitive to cell function. Here, we present a negative selection method that leverages a Microfluidic Cell Concentrator (MCC) to allow collection and analysis of this rare cell population without needing cell adhesion or other labeling techniques to keep the cells within the chamber. Because the MCC is designed to allow collection and analysis of non-adherent cell populations, multiple staining steps can be applied in parallel to a given CTC population without losing any of the population. The ability of the MCC for patient sample processing of CTCs for enumeration was demonstrated with five patient samples, revealing an average of 0.31 CTCs/mL. The technique was compared to a previously published method - the ELISPOT - that showed similar CTC levels among the five patient samples tested. Because the MCC method does not use positive selection, the method can be applied across a variety of tumor types with no changes to the process.

Syndecan-1 in breast cancer stroma fibroblasts regulates extracellular matrix fiber organization and carcinoma cell motility.

Stromal fibroblasts of breast carcinomas frequently express the cell surface proteoglycan syndecan-1 (Sdc1). In human breast carcinoma samples, stromal Sdc1 expression correlates with an organized, parallel, extracellular matrix (ECM) fiber architecture. To examine a possible link between stromal Sdc1 and the fiber architecture, we generated bioactive cell-free three-dimensional ECMs from cultures of Sdc1-positive and Sdc1-negative murine and human mammary fibroblasts (termed ECM-Sdc1 and ECM-mock, respectively). Indeed, ECM-Sdc1 showed a parallel fiber architecture that contrasted markedly with the random fiber arrangement of ECM-mock. When breast carcinoma cells were seeded into the fibroblast-free ECMs, ECM-Sdc1, but not ECM-mock, promoted their attachment, invasion, and directional movement. We further evaluated the contribution of the structural/compositional modifications in ECM-Sdc1 on carcinoma cell behavior. By microcontact printing of culture surfaces, we forced the Sdc1-negative fibroblasts to produce ECM with parallel fiber organization, mimicking the architecture observed in ECM-Sdc1. We found that the fiber topography governs carcinoma cell migration directionality. Conversely, an elevated fibronectin level in ECM-Sdc1 was responsible for the enhanced attachment of the breast carcinoma cells. These observations suggest that Sdc1 expression in breast carcinoma stromal fibroblasts promotes the assembly of an architecturally abnormal ECM that is permissive to breast carcinoma directional migration and invasion.

Generation of recombinant hyperimmune globulins from diverse B-cell repertoires.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

Preoperative antibiotics and mortality in the elderly.

OBJECTIVE AND BACKGROUND: It is generally thought that the use of preoperative antibiotics reduces the risk of postoperative infection, yet few studies have described the association between preoperative antibiotics and the risk of dying. The objective of this study was to determine whether preoperative antibiotics are associated with a reduced risk of death. METHODS: We performed a multivariate matched, population-based, case-control study of death following surgery on 1362 Pennsylvania Medicare patients between 65 and 85 years of age undergoing general and orthopedic surgery. Cases (681 deaths within 60 days from hospital admission) were randomly selected throughout Pennsylvania using claims from 1995 and 1996. Models were developed to scan Medicare claims, looking for controls who did not die and who were the closest matches to the previously selected cases based on preoperative characteristics. Cases and their controls were identified, and charts were abstracted to define antibiotic use and obtain baseline severity adjustment data. RESULTS: For general surgery, the odds of dying within 60 days were less than half in those treated with preoperative antibiotics within 2 hours of incision as compared with those without such treatment: (odds ratio = 0.44; 95% confidence interval, 0.32-0.60), P < 0.0001). For orthopedic surgery, no significant mortality reduction was observed (OR = 0.85; 95% confidence interval, 0.54-1.32; P < 0.464). INTERPRETATION: Preoperative antibiotics are associated with a substantially lower 60-day mortality rate in elderly patients undergoing general surgery. In patients who appear to be comparable, the risk of death was half as large among those who received preoperative antibiotics.

Changes in prognosis after the first postoperative complication.

BACKGROUND: Postoperative complications are common in the Medicare population, yet no study has formally quantified the change in prognosis that occurs after a broad range of first complications. OBJECTIVE: We sought to estimate the relative severity of 24 first postoperative complications. RESEARCH DESIGN: We undertook a multivariate matched, population-based, case-control study of death after surgery in a sample of 1362 Pennsylvania Medicare patients. SUBJECTS: Cases (681 deaths) were selected randomly using claims from 1995-1996. Models were developed to scan all Pennsylvania claims, looking for similar controls that did not die. MEASURES: Charts were abstracted, complications identified, and models were constructed to estimate the odds of dying after any 1 of 24 first postoperative complications. RESULTS: The odds of dying within 60 days increased 3.4-fold (95% confidence interval [CI] 2.5-4.7) in patients with complications as compared with those without complications. A first complication of respiratory compromise was associated with a 7.2-fold increase in the odds of dying (95% CI 4.5-11.6). The first complications of pneumonia or congestive heart failure were associated with, respectively, 5-fold (95% CI 2.1-12.1) and 5.1-fold (2.3-11.1) increases in odds of dying as compared with no complication. CONCLUSIONS: First complications after surgery, even seemingly mild ones, may radically alter the patient's risk of death. First complications often begin the cascade of complications that end in death. Caregivers should consider the first complication as a timely signal of a changed clinical situation demanding a reevaluation of the patient's care. Researchers may use these estimates to determine the relative severity of a broad range of first or early complications.

Anesthesiologist board certification and patient outcomes.

BACKGROUND: Board certification is often used as a surrogate indicator of provider competence, although few outcome studies have demonstrated its validity. The aim of this study was to compare the outcomes of patients who underwent surgical procedures under the care of an anesthesiologist with or without board certification. METHODS: Medicare claims records for 144,883 patients in Pennsylvania who underwent general surgical or orthopedic procedures between 1991 and 1994 were used to determine provider-specific outcome rates adjusted to account for patient severity and case mix, and hospital characteristics. Outcomes of 8,894 cases involving midcareer anesthesiologists, 11-25 yr from medical school graduation, who lacked board certification were compared with all other cases. Midcareer anesthesiologist cases were studied because this group had sufficient time to become certified during an era when obtaining certification was already considered important, and consequently had the highest rate of board certification. Mortality within 30 days of admission and the failure-to-rescue rate (defined as the rate of death after an in-hospital complication) were the two primary outcome measures. RESULTS: Adjusted odds ratios for death and failure to rescue were greater when care was delivered by noncertified midcareer anesthesiologists (death = 1.13 [95% confidence interval, 1.00, 1.26], P < 0.04; failure to rescue = 1.13 [95% confidence interval, 1.01, 1.27], P < 0.04). Adjusting for international medical school graduates did not change these results. CONCLUSIONS: When anesthesiology board certification is very common, as in midcareer practitioners, the lack of board certification is associated with worse outcomes. However, the poor outcomes associated with noncertified providers may be a result of the hospitals at which they practice and not necessarily their manner of practice.