Evaluating differences of metabolic performances: statistical methods and their application to animal cell cultivations.

In cell culture process development, monitoring and analyzing metabolic key parameters is routinely applied to demonstrate specific advantages of one experimental setup over another. It is of great importance that the observed differences and expected improvements are practically relevant and statistically significant. However, a systematic assessment whether observed differences in metabolic rates are statistically significant or not is often missing. This can lead to time-consuming and costly changes of an established biotechnological process due to false positive results. In the present work we demonstrate how well-established statistical tools can be employed to analyze systematically different sources of variations in metabolic rate determinations and to assess, in an unbiased way, their implications on the significance of the observed differences. As a case study, we evaluate differing growth characteristics and metabolic rates of the avian designer cell line AGE1.CR.pIX cultivated in a stirred tank reactor and in a wave bioreactor. Although large differences in metabolic rates and cell growth were expected (due to different aeration, agitation, pH-control, etc.) and partially observed (up to 79%), our results show that the inter-experimental variance between experiments performed under identical conditions but with different pre-cultures is a major contributor to the overall variance of metabolic rates. The lower bounds of the overall relative standard deviations for specific metabolic rates were between 4% and 73%. The application of available statistical methods revealed that the observed differences were statistically not significant and consequently insufficient to confirm relevant differences between both cultivation systems. Our study provides a general guideline for statistical analyses in comparative cultivation studies and emphasizes the necessity to account for the inter-experimental variance (mainly caused by biological variation) to avoid false-positive results.

A new MDCK suspension line cultivated in a fully defined medium in stirred-tank and wave bioreactor.

An adherently growing MDCK cell line was adapted in a two-step process in a fully defined medium and in suspension. The resulting MDCK.SUS2 cells were subsequently evaluated for their potential as host cells for influenza vaccine production in two lab-scale bioreactors (wave and stirred-tank). Cell concentrations up to 2.3 x 10(6)cells/mL were obtained after 96 h, which is slightly higher than cell concentrations obtained with adherent MDCK cells cultivated on microcarriers (2g/L). Infections with influenza A/PR/8/34 and B/Malaysia resulted in high virus titers (2.90 and 2.75 log HA units/100 microL, respectively). The monitoring of extracellular metabolites, including amino acids, revealed a change in some of the metabolite consumption or release profiles, which indicates changes in metabolism during the adaptation process. Overall, the MDCK.SUS2 cell line represents a new cell substrate for a robust influenza vaccine production in a fully defined process.

New avian suspension cell lines provide production of influenza virus and MVA in serum-free media: studies on growth, metabolism and virus propagation.

Few suspension cells can be used for vaccine manufacturing today as they either do not meet requirements from health regulatory authorities or do not produce high virus titres. Two new avian designer cell lines (AGE1.CR and AGE1.CR.pIX) that have been adapted to grow in suspension in serum-free medium were evaluated for their potential as host cells for influenza and modified vaccinia Ankara (MVA, wild type) vaccine production. Their metabolism was studied during growth in static (T-flasks) and dynamic cultivation systems (roller bottles, stirred tank reactor, wave bioreactor). High cell concentrations up to 5.8x10(6)cells/mL were obtained with doubling times of 23h for AGE1.CR and 35h for AGE1.CR.pIX, respectively. Both viruses were produced to high titres (3.5 logHA/100 microL for influenza virus, 3.2x10(8)pfu/mL for MVA). Hence, the CR cell lines are an appropriate substrate for pharmaceutical influenza and MVA production.

Productivity and acuity tool for wound care and hyperbaric medicine centers.

The unique workload and patient acuity in wound care and hyperbaric centers leaves staffing to subjective interpretation; lack of objective data makes it difficult to determine department budgets and appropriate staffing patterns. To address these problems, an objective tool was developed to assess acuity and productivity in hyperbaric and wound care departments. Patient classifications were created to define acuity based on disease and associated procedures. A total of 38 distinct indicators were identified for wound care and 22 for hyperbaric oxygen therapy. Time studies were performed on identified tasks. All patients were scored and placed in acuity classes. The number of hours per patient day was calculated and multiplied by direct care hours to find number of full-time-equivalents (FTEs). The tool was tested for content validity and clinical applicability. The tool was found to accurately reflect patient-care work and suitable as a benchmarking tool for use in monoplace hyperbaric and wound care centers.

Neuropsychological sequelae and impaired health status in survivors of severe acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a disease of acute respiratory failure manifested by severe hypoxemia with a high mortality rate. Previous outcome studies of ARDS have assessed survival and/or pulmonary function as the primary outcome variables. Cognitive or psychological outcomes following ARDS have not been described, despite the possibility that ARDS patients are at risk for brain injury through hypoxemia or other mechanisms. In the current study 55 consecutive ARDS survivors completed a battery of neuropsychological tests and questionnaires regarding health status, cognitive and psychological outcomes at the time of hospital discharge and 1 yr after onset of ARDS. At hospital discharge, 100% (55 of 55) of survivors exhibited cognitive and affective impairments, as well as problems with health status which affected their quality of life. At 1 yr after ARDS, 17 of 55 (30%) patients still exhibited generalized cognitive decline. Forty-three of 55 (78%) patients had all or at least one of the following: impaired memory, attention, concentration and/or decreased mental processing speed. One year after ARDS a substantial portion of ARDS survivors exhibit impaired health status and cognitive sequelae which may be due to hypoxemia, emboli, inflammation, drug toxicity, and/or other etiologies.

Neuropsychological impairments following hantavirus pulmonary syndrome.

Recently an outbreak of acute respiratory infection associated with the hantavirus occurred in the southwestern United States. Hantavirus pulmonary syndrome (HPS) is a life threatening illness that carries with it a high mortality rate. Patients with HPS experience prolonged periods of hypoxemia requiring mechanical ventilation and treatment in intensive care units. We have recently seen 2 survivors of HPS. A neuropsychological test battery was administered immediately following their acute hospitalization and at 1 year postrecovery from HPS. Both patients exhibited cognitive impairments immediately following HPS as well as persistent cognitive impairments at 1 year. The cognitive impairments seen in these two HPS survivors are similar to those seen in other patients who have experienced brain anoxia, including memory impairments. It is also possible that hantavirus may directly cause brain injury with concomitant cognitive impairments. Additional research needs to be carried out in order to determine the extent and severity of the cognitive impairments in survivors of HPS.

Neuropsychologic and functional recovery from severe carbon monoxide poisoning without hyperbaric oxygen therapy.

STUDY OBJECTIVE: To test the hypothesis that neuropsychologic test results and functional outcome will be abnormal if hyperbaric oxygen (HBO) is not used in patients with severe carbon monoxide (CO) poisoning. METHODS: For a 1-year interval, we retrospectively identified all CO-poisoned patients who were comatose on presentation at a large, urban tertiary hospital and did not receive HBO therapy. Prospectively, 6 and 12 months after CO poisoning, we administered standardized questionnaires to assess functional outcome. At 6 months, we performed extensive neuropsychologic testing. RESULTS: All four patients exhibited normal performance on a neuropsychologic test battery at 6 months. The Folstein Mini-Mental Status Examination was normal in all patients. All patients had normal functional outcomes. CONCLUSION: Normal neuropsychologic and functional outcomes are possible after severe CO poisoning without the use of HBO therapy.

Hypoxemia during hyperbaric oxygen therapy.

This report describes a patient with mucormycosis treated with hyperbaric oxygen therapy (HBO2). Arterial O2 tension (PaO2) during HBO2 was considerably lower (346 mm Hg) than expected (1,500 mm Hg) due to an inadvertent right main-stem intubation, which was not otherwise clinically evident. This case illustrates the influence of a large right-to-left shunt (Qs/Qt) on O2 loading during HBO2. We raise the question if routine hyperbaric treatment protocols are adequate to raise PaO2 in patients with large Qs/Qts.