A multicenter comparison study between the Endosafe PTS rapid-release testing system and traditional methods for detecting endotoxin in cell-therapy products.

BACKGROUND: Rapid-release testing reduces the waiting period for administration of time-sensitive cell-therapy products. Current assay systems are labor intensive and time consuming. The Endosafe portable test system (PTS) is a chromogenic Limulus amebocyte lysate (LAL) portable endotoxin detection system that provides quantitative results in approximately 15 min. To evaluate Endosafe performance with cell-therapy products, side-by-side testing of traditional LAL systems and the Endosafe system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Health's Department of Transfusion Medicine, USA. METHODS: Charles River Laboratories provided each center with a PTS reader and two commercially prepared lyophilized reference standard endotoxin (RSE) vials. All samples tested with the Endosafe system used 0.05-5.0 endotoxin unit/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe and in-house LAL methods. Subsequently, each center tested the endotoxin content of standard dilutions of cell-therapy products, thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing. RESULTS: Valid paired results, based on each center's LAL method and the Endosafe system criteria, were analyzed. Endotoxin detection between paired results was equivalent in most cases. DISCUSSION: The Endosafe system provided reliable results with products typically produced in cell-therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell-therapy products.

Development and operation of a quality assurance system for deviations from standard operating procedures in a clinical cell therapy laboratory.

BACKGROUND: Errors and accidents, or deviations from standard operating procedures, other policy, or regulations must be documented and reviewed, with corrective actions taken to assure quality performance in a cellular therapy laboratory. Though expectations and guidance for deviation management exist, a description of the framework for the development of such a program is lacking in the literature. Here we describe our deviation management program, which uses a Microsoft Access database and Microsoft Excel to analyze deviations and notable events, facilitating quality assurance (QA) functions and ongoing process improvement. METHODS: Data is stored in a Microsoft Access database with an assignment to one of six deviation type categories. Deviation events are evaluated for potential impact on patient and product, and impact scores for each are determined using a 0- 4 grading scale. An immediate investigation occurs, and corrective actions are taken to prevent future similar events from taking place. Additionally, deviation data is collectively analyzed on a quarterly basis using Microsoft Excel, to identify recurring events or developing trends. RESULTS: Between January 1, 2001 and December 31, 2001 over 2500 products were processed at our laboratory. During this time period, 335 deviations and notable events occurred, affecting 385 products and/or patients. Deviations within the 'technical error' category were most common (37%). Thirteen percent of deviations had a patient and/or a product impact score > or = 2, a score indicating, at a minimum, potentially affected patient outcome or moderate effect upon product quality. DISCUSSION: Real-time analysis and quarterly review of deviations using our deviation management program allows for identification and correction of deviations. Monitoring of deviation trends allows for process improvement and overall successful functioning of the QA program in the cell therapy laboratory. Our deviation management program could serve as a model for other laboratories in need of such a program.

Improved progenitor assay standardization using peripheral blood progenitor cells from a donor treated with granulocyte-colony-stimulating factor.

BACKGROUND: Progenitor assays are the principal method for evaluating hematopoietic cell function. The magnitude of assay variability and the assay steps contributing to variability were determined, and modifications intended to increase assay consistency were evaluated. STUDY DESIGN AND METHODS: Assays were performed using a serum-free progenitor assay medium with cells plated at 5.0 x 10(4) and 1.0 x 10(5) cells per plate. A peripheral blood progenitor cell component collected from a normal donor after administration of granulocyte-colony-stimulating factor was divided into identical aliquots. Each experiment involved at least 5 technologists, each performing assays in duplicate on five aliquots, with each person scoring all assay plates. Three sample preparation methods were tested: 1) ficoll mononuclear cell enrichment and sample dilution, 2) sample dilution without ficoll separation, and 3) sample dilution without ficoll separation, with cell counts performed before and after each dilution step, dilution volumes calculated on the basis of each cell count, automated electronic pipettors used in dilution steps, and colony frequency calculated on the basis of cell counts from the final specimen. RESULTS: Global variability for colony-forming units-granulocyte-macrophage, represented by the percentage of CV for all specimens and all technologists, was 89.6 percent at 5.0 x 10(4) cells per plate and 81.3 percent at 1.0 x 10(5), when ficoll separation was used. Subjective differences in scoring plates did not account for most of the variability observed, as results for any individual plate read by multiple technologists had a mean CV of 15.6 percent and 19.7 percent at the two plating concentrations. Method 3 resulted in the greatest improvement, reducing CV to 24.4 percent at 5.0 x 10(4) cells per plate and to 24.2 percent at 1.0 x 10(5) cells per plate. Similar results were obtained for erythroid-burst-forming units. CONCLUSIONS: Baseline assay results were extremely inconsistent. Interindividual differences in colony interpretation did not contribute significantly to assay variability, although sample preparation and plating did. Improved control over cell concentration decreased assay variability by 70 to 73 percent.