Development of a novel assay to evaluate the functional potential of umbilical cord blood progenitors.

BACKGROUND: Although the colony-forming cell (CFC) assay provides the most relevant information regarding the functional potential of progenitors in a unit of umbilical cord blood (UCB), technical challenges associated with this assay have made it difficult to standardize the assay among testing laboratories. The purpose of this study was to assess the reproducibility of a newly developed functional assay (HALO SPC-QC [HALO], HemoGenix, Inc.). This test is based on the principle that cellular proliferation responses to cytokine stimuli are proportional to intracellular ATP levels from progeny cells generated in culture from progenitors. STUDY DESIGN AND METHODS: Results of the HALO assay were evaluated at two geographically distinct sites with matched aliquots from 12 different UCB units. RESULTS: A significant correlation between the two sites for total nucleated cell counts was observed (r = 0.98, p < 0.001). Similarly, a strong correlation between HALO results from both sites was observed (r = 0.94, p < 0.001). Also, despite using different methods at each site for the CFC assay, results from the two sites correlated (r = 0.79, p = 0.002). A good correlation between the CFC and HALO assays (r = 0.73, p < 0.005), however, was only observed at the site with the same cytokine cocktail for both the CFC and the HALO assays. CONCLUSION: These results support the notion that the HALO assay is a reasonable approach for measuring the functional potential of hematopoietic progenitors in UCB. Moreover, because the final readout for the HALO assay is instrument based, unlike the CFC assay, which requires a subjective enumeration of colonies, the HALO assay may be more amenable to standardization.

Dosage and effectiveness of intrapleural doxycycline for pediatric postcardiotomy pleural effusions.

STUDY OBJECTIVE: To determine the effectiveness of intrapleural doxycycline for the treatment of postcardiotomy pleural effusions in pediatric patients. DESIGN: Retrospective case series. SETTING: Intensive care unit in a pediatric tertiary care center. PATIENTS: Sequential sample of 12 pediatric patients who underwent cardiotomy for congenital heart disease and received doxycycline pleurodesis for persistent pleural effusion that lasted more than 7 days between December 21, 2001, and May 23, 2005. MEASUREMENTS AND MAIN RESULTS: Mean age of the patients was 1 year (range 2 wks-2.5 yrs). Eighteen courses of doxycycline were administered among the 12 patients. An average dose of 19.1 mg/kg/dose of parenteral doxycycline was diluted in normal saline to a final syringe concentration of 2-8 mg/ml and injected through a chest tube. The patient was rotated according to a protocol. The doxycycline dose remained in the pleural space for approximately 6 hours before being drained under suction. Treatment success was defined as achievement of 0-ml/hour chest tube output after a doxycycline dose. The overall treatment success rate was 94% (17 of 18 courses). The mean times from dosing to treatment success and chest tube removal were 76 hours (range < 1 to 140 hrs) and 130 hours (range 8-453 hrs), respectively. Seventy-two percent of the courses (13 of 18) achieved treatment success within 96 hours and chest tube removal within 168 hours after dosing. Doxycycline concentration did not appear to be related to treatment success. Chest pain was the most common adverse effect. CONCLUSION: Intrapleural doxycycline infusion is effective for postcardiotomy pleural effusion in pediatric patients with persistent chest tube drainage lasting more than 7 days.

Validation and development of a predictive paradigm for hemotoxicology using a multifunctional bioluminescence colony-forming proliferation assay.

The lympho-hematopoietic colony-forming assay has been redesigned into a rapid, nonsubjective and standardized proliferation assay that can measure the effects of compounds on multiple stem and progenitor cell populations from different species simultaneously using a sensitive, high-throughput bioluminescence readout. Eleven reference compounds from the Registry of Cytotoxicity (RC) and eight other compounds, including anticancer drugs, were studied over an 8- to 9-log dose range for their effects on seven cell populations from both human and mouse bone marrow simultaneously. The cell populations studied included a primitive (HPP-SP) and mature (CFC-GEMM) stem cell, three hematopoietic (BFU-E, GM-CFC, Mk-CFC) and two lymphopoietic (T-CFC, B-CFC) populations. The results reveal a five-point prediction paradigm for lympho-hematotoxicity. Depending on how and which populations are affected, the resulting effects in the periphery can be predicted. Validation against the RC Prediction Model produces a high degree of correlation between the in vitro IC(50) values and known in vivo LD(50) values, thereby allowing preclinical dosing to be predicted. If primary human hematopoietic target tissue is used, inhibitory concentration (IC(50)/IC(75)/IC(90)) values of anticancer and other drugs can be converted into predicted clinical doses which, when compared to published chemotherapeutic dosing regimen, are very similar. When performed during early drug screening, the prediction value of the assay should help reduce time and cost, but above all, provide increase efficacy and safety for the patient.