Various approaches are compared for the design and analysis of studies to assess the transfer of an analytical method from a research and development site to one or more other sites: comparison of observed bias and precision to acceptance limits, statistical quality control-type analysis, statistical difference tests, and statistical equivalence tests. These approaches are evaluated in terms of the extent to which the risks of incorrect decisions (consumer risk of failing to detect that a site is unacceptable, and producer risk of rejecting an acceptable site) are known and/or controlled. Comparison of observed accuracy and precision to acceptance limits is a flawed approach because both the consumer and producer risks are unknown and uncontrolled. For technology transfec where the objective is to demonstrate sufSicient acceptability or similarity, the statistical quality control and difference tests are well known to suffer from illogical characteristics (decreasing true acceptance probabilities as the sample size increases). The equivalence test is the preferred approach because it alone controls the more important consumer risk and performs in a scientifically logical manne,: Acceptance limits for accuracy and precision in the equivalence test should be based on need for intended use (ie, ensuring thar good batches will pass, and bad batches will fail, during future release testing and stability testing), and a rigorous method for selection of well-conceived limits is presented. Methods for sample size determination are also included. The proposed approach is illustrated with two examples.

Exposure to acute, high-dose, whole-body ionizing radiation results in bone marrow failure (hematopoietic acute radiation syndrome with resultant infection, bleeding, anemia, and increased risk of death). Sargramostim (yeastderived rhu GM-CSF), a yeast-derived, molecularly cloned, hematopoietic growth factor and pleiotropic cytokine supports proliferation, differentiation, maturation and survival of cells of several myeloid lineages. We evaluated the efficacy of sargramostim in non-human primates (rhesus macaques) exposed to whole-body ionizing radiation at a 50-60% lethal dose. The primary end point was day 60 survival. Non-human primates received daily subcutaneous sargramostim (7 mcg/ kg/day) or control. To reflect the anticipated setting of a nuclear or radiologic event, treatment began 48 h postirradiation, and non-human primates received only moderate supportive care (no whole blood transfusions or individualized antibiotics). Sargramostim significantly increased day 60 survival to 78% (95% confidence interval, 61-90%) vs. 42% (26-59%; P ¼ 0.0018) in controls. Neutrophil, platelet and lymphocyte recovery rates were accelerated and infection rates decreased. Improved survival when sargramostim was started 48 h postirradiation, without use of intensive supportive care, suggests sargramostim may be effective in treating humans exposed to acute, high-dose whole-body, ionizing radiation in a scenario such as a mass casualty event.

Differences were examined between male and female Sprague-Dawley rats in the response of 16 urinary biomarkers (measured using several assay platforms) to renal injury produced by gentamicin administered subcutaneously for 10 days at a dosage of 75 mg/kg. Urinary biomarkers expressed as fold difference from contemporaneous controls and renal histopathology were assessed after 3 and 10 doses. On day 4, minimal proximal tubular changes were observed microscopically in all males but no females; on day 11, more extensive and more severe injury was observed to a similar extent in all animals of both sexes. Modest increases (maximum 5-fold) in all urinary biomarkers (except epidermal growth factor [EGF], which was decreased) on day 4 and marked elevations (maximum 271-fold) on day 11 were seen consistently in both sexes. However, the magnitude of the increases differed between the sexes. On day 4, despite the lack of tubular injury, many biomarkers were more elevated in females than males but this rarely led to statistically significant sex differences; only 2 biomarkers (β2-microglobulin and total protein) showed a greater increase in males than females in line with the histopathology. On day 11, there were many more biomarkers that showed a statistically significant difference between the sexes in fold change with treatment; in line with the results on day 4, the majority of biomarkers were more increased in females than males. It remains unresolved if sex differences in the magnitude of biomarker response at injury threshold would lead to any difference in diagnostic interpretation between the sexes. These data highlight the need for publication of more studies using animals of both sexes to fully explore the influence of sex on the diagnostic performance of the novel biomarkers.

Background: Hematopoietic acute radiation syndrome (H-ARS) occurs in individuals that are exposed to high levels of radiation over a short period of time. The destruction of bone marrow leads to pancytopenia, which increases the incidence of infections and accounts for the majority of morbidity and mortality. Sargramostim (yeast-derived rhGM-CSF) is a leukocyte growth factor that promotes differentiation, maturation and activation of granulocytes, monocytes and macrophages. Methods: We evaluated the efficacy of repeat administrations of sargramostim (7 μg/kg/day) in a blinded, GLP study in total body irradiated non-human primates (NHPs) when administered 48 h post-irradiation with minimal supportive care (e.g., no blood products or individualized antibiotics). The primary objective was to assess the efficacy of sargramostim versus vehicle on mortality rate at Day 60 in irradiated male and female NHPs at LD50-60/60 (n=36/group). Secondary objectives included the efficacy of sargramostim on overall survival and its effect on hematology parameters. The study included an exploratory arm of male and female NHPs that were irradiated at a LD70-80/60(n= 18/group) and received the same treatments. Results: Sargramostim decreased the mortality rate at Day 60 by 36% in the LD50-60/60 group (p=0.0018) and by 44% in the LD70-80/60 group (p=0.0076). Additionally, neutrophil, platelet, lymphocyte, and white blood cell levels in survivors demonstrated accelerated recovery in the sargramostim-treated NHPs in the LD50-60/60 and LD70-80/60 groups. Conclusion: Treatment with sargramostim (7 μg/kg/day) beginning at 48 h, in the absence of blood products and individualized antibiotics, suggests that the use of sargramostim is a viable therapeutic strategy for H-ARS in a mass casualty event. Funding: This project has been funded in whole or in part with Federal funds from the Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority, under Contract No. HHSO100201300005I. Disclosures Clayton: Sanofi Genzyme: Employment. Charpentier:Sanofi: Employment. LaCasse:Sanofi Genzyme: Employment. Khan-Malek:Sanofi: Employment. Keutzer:Sanofi Genzyme: Employment.

Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) represents a benchmark technology in the detection and quantification of mRNA. Yet, accurate results cannot be realized without proper statistical analysis of RT-PCR data. Here we examine some of the issues concerning RT-PCR experiments that would benefit from rigorous statistical treatment including normalization, quantification, efficiency estimation, and sample size calculations. Examples are used to illustrate the methods.