Validation of a quality assurance program for autologous cultured chondrocyte implantation.

The use of living human cells to facilitate repair of defects in structural tissues is a rapidly emerging treatment option. A quality assurance program based on U.S. Food and Drug Administration good manufacturing practice regulations and other guidance was established and implemented in a program to use autologous cultured chondrocytes for repair of articular defects of the knee. The development of ex vivo cell therapies presents novel issues of quality assurance. The implementation and evaluation of this quality program was based on the implantation of 303 patients with autologous cultured chondrocytes, in which we analyzed a number of quantifiable parameters and which meets the unique challenges of autologous cell therapy within a rigorous regulatory framework. Application of well-accepted principles of quality assurance and quality control coupled with a thorough understanding of the cell culturing processes will result in a safe and efficacious cell therapy product.

Experiences with extended-duration medical laboratory support in deployable medical systems.

Medical laboratory services in an echelon-II Deployable Medical Systems environment were examined after a 6-month deployment in Guatemala-Fuertes Caminos 94 (N). The limitations in preparing and executing laboratory operations by U.S. Army Reserve units using Minimal Essential Equipment for Training components were identified. Corrective actions for planning future operations requiring laboratory support were addressed. Despite significant limitations, 353 laboratory procedures were conducted to standard. Additionally, laboratory procedures supporting blood-transfusion activities was successfully undertaken. Employment of laboratory services in remote environments for extended durations requires special attention and complete planning to ensure that all elements of care are in place to support medical operations.

Validation of an automated method of endotoxin testing for use in the end-product testing of ex vivo activated T-lymphocytes used in a somatic cell therapy.

The aim of this study was to compare two methods of endotoxin testing to optimize quality control testing methodologies required for the rapid and precise determination of pyrogenicity in cell or tissue products used in cellular therapies. An automated kinetic-chromogenic method for determination of endotoxin levels in ex vivo activated T-lymphocytes infusion product, has been validated following the FDA guideline for the Limulus amebocyte lysate (LAL) assay. The validation protocol included: (1) initial qualification of the laboratory conducting the assay; (2) inhibition and enhancement tests both for treated (heated at 70 degrees C for 10 min) and untreated specimens; and (3) comparison of this assay with the conventional gel-clot method. Inhibition and enhancement testing showed that a 1:30 dilution of the infusion product was the optimal dilution for this type of specimen. Heating specimens did not appear to provide any advantage. A comparison study was performed on 105 infusion product samples. Endotoxin levels determined by both methods for all samples tested were within established end-product release specifications. The K-QCL method can be effectively used for endotoxin determination of ex vivo activated T-cells.

Hospital integrated lanes training: brigade-directed implementation of a medical lanes training program during annual training.

The "lanes" concept of training was integrated into a medical site support mission of the 804th Medical Brigade, U.S. Army Reserve, during Annual Training, 1993 at Fort Drum, New York. This training, termed Hospital Integrated Lanes Training (HILT), included STX, FTX, patient play, and full use of Deployable Medical Systems equipment. The medical care of over 33,000 personnel participating in tactical annual training exercises was not interrupted during any concurrent phase of lanes training. Brigade operations planners developed an array of medical exercises that involved both moulaged and paper patient play. These exercises began prior to hospital set-up and continued for 24 hours a day throughout the tactical exercise. Injuries likely to be encountered during combat operations were inserted into the play singly and under a mass-casualty scenario. The standard of care for all injuries was determined with the Army Medical Department Center and School guidance. Prior coordination of brigade medical assets with external air and ground ambulance organizations broadened the scope of the training and facilitated effective use of command and control, communications, and equipment over a wide geographic area. Medical records were collected and evaluated at the conclusion of all exercises. After-action reviews were conducted by all medical units to assist in the planning of future HILT exercises. The HILT concept is a valuable tool for the complex training requirements of field medical units organized under medical Force 2000. The concept of integrated lanes training allows for the development and continuous improvement of individual and sectional skills for medical personnel and should be applied within all echelons of care.

A 3-year experience of quality control and quality assurance in the multisite delivery of a lymphocyte-based cellular therapy for renal cell carcinoma.

Autolymphocyte therapy (ALT) is outpatient-based adoptive immunotherapy using ex vivo-activated memory T-cells. To support the safe and reproducible delivery of ALT at three cell processing facilities (Boston, MA; Atlanta, GA; Orange, CA) we created a comprehensive quality assurance/quality control program compliant with recent FDA guidance relevant to activated lymphocytes and somatic cell therapies. Each facility performed extensive QC testing to ensure sterility, viability, and proper cell yield. Additionally, several QC tests were performed at Cellocr's centralized reference laboratory to monitor cell potency and identity of the ex vivo-processed lymphocytes. We report here the successful implementation of this QA/QC program for ALT which has resulted in the safe preparation and delivery of cell infusion products amounting to over 3600 treatments at seven clinical sites nationwide. We believe this program will serve as a model for other cellular therapies.

Preliminary validation of an activation assay for ex vivo activated T cells utilized in cancer immunotherapy.

An in vitro assay that measures the activation level of ex vivo activated (EVA) T cells currently being used in the adoptive immunotherapy of metastatic renal cell carcinoma has been developed. This assay is based on the ability of activated, but not resting. T cells to proliferate in response to the protein kinase C activator, phorbol myristate (PMA). To utilize this assay for in-process monitoring and control, we have begun an initial validation of the overall reproducibility of this assay. The proliferation of activated T cells in response to PMA, as measured by the mean cpm values of (3)H-thymidine incorporated, was demonstrated to have intra-assay coefficients of variation (cv's) for individual analysts that were typically less than 10% and rarely exceeded 20%. Activated T cells could be frozen and stored for at least 6 weeks with little or no deterioration in their ability to proliferate in response to PMA. Using these cells, inter-assay cv's that were typically less than 15% were obtained by individual analysts, and overall cv's of 10% to 25% were obtained for different samples assayed by different analysts at different times. This level of variability is very reasonable for a cellular assay. Further validation of this assay will address the issues of sensitivity, linearity and selectivity. To date, this assay has been used to analyze over 90 patient EVA cell samples and has revealed a broad range of proliferative responses to PMA. Taken together, these results suggest that this assay may be useful in defining the potency of the activated T cell used therapeutically.

Overview of a quality assurance/quality control compliance program consistent with FDA regulations and policies for somatic cell and gene therapies: a four year experience.

Somatic cell and gene therapy involve the application of biological technologies to an individual patient through the use of living cells which provide a therapeutic benefit (Aliski, 1991). Various forms of cellular and gene therapies are being developed and evaluated in an increasing number of clinical trials for congenital and acquired disorders. The potential and progress of these therapeutic applications have resulted in an increasing effort by the Food and Drug Administration (FDA) to develop the regulatory framework under which these therapeutic approaches would insure safety and efficacy, the primary mandate of the FDA. Over five years ago Cellcor began to define the parameters, specifications, and conditions relevant to a Quality Assurance/Quality Control (QA/QC) program that has evolved to insure safety and maximize the efficacy of applications of the company's ex vivo technology, autolymphocyte therapy. Autolymphocyte therapy is an outpatient form of somatic cell immunotherapy based upon the infusion of T cells that have been activated ex vivo using a combination of previously generated autologous cytokines and an anti-CD3 monoclonal antibody. We have been able to demonstrate the feasibility for the safe, controlled, and consistent preparation and delivery of a cellular therapy by application of relevant GMP regulations. This presentation reviews aspects of this program and chronicles our experience which at present amounts to over 4400 in fusions for over 700 patients. This program provides a high degree of assurance that a cellular therapy program can be carried out in a multisite mode involving hundreds of patients through the strict adherence to cGMP as set forth in existing regulations.(ABSTRACT TRUNCATED AT 250 WORDS)

[Tracheobronchial pathogen colonization]. / Tracheobronchiale Keimbesiedlung.

Tracheobronchial colonization is mediated by the patients host defence mechanisms, the extent of therapeutic interventions and the virulence of endogenous or exogenous bacteria mainly from the nasopharynx and the stomach. Aspiration from the stomach plays the main role in colonizing the respiratory tract in ventilated patients.

Concentration of Mycobacterium avium by hospital hot water systems.

Water from 34 sites on two temporarily vacant hospital floors was analyzed for the presence of mycobacteria. These sites included 18 cold water taps and 16 hot water taps, including shower heads. A total of 14 sites (41%) demonstrated the presence of Mycobacterium avium as confirmed by biochemical characterization, DNA/rRNA probe analysis, and seroagglutination. Of positive sites, 11 were hot water sources with an average temperature of 55 degrees C and yielding up to 500 colony-forming units per 100 mL. Seven of 11 strains analyzed for glycolipid antigens were identified with the type 4 serovar, the preponderant serovar of M avium in patients with acquired immunodeficiency syndrome from the Boston area. Potable hot water systems, particularly those that generate aerosols, may contain concentrations of M avium greater than those found in cold water systems and could serve as an environmental source for colonization and infection of immunocompromised persons.

Mycobacteria in public water supplies: comparative resistance to chlorine.

The isolation of mycobacteria from municipal and hospital water supplies prompted an investigation of the susceptibility of environmental and clinical isolates of mycobacteria other than Mycobacterium tuberculosis and Mycobacterium bovis to free chlorine. Experiments revealed that free chlorine concentrations of 1.0 mg l-1 eliminated 100,000 c.f.u. of the mycobacterial strains tested within 8 hours of exposure, whereas a concentration of 0.15 mg l-1 had virtually no bacteriocidal effect. Free chlorine residual levels of 0.1 mg l-1 or less, depending on the water temperature, within Boston, suggest that current disinfection procedures may not be adequate for effective control of potentially pathogenic mycobacteria in public water supply systems serving a population with increased risk factors.

Bacterial colonization and endotoxin content of a new renal dialysis water system composed of acrylonitrile butadiene styrene.

We measured endotoxin and bacterial levels in tap water, in water purified by reverse osmosis, and in dialysate samples over a 4-month period in a new 10-bed renal dialysis unit. Water treated by reverse osmosis is conducted to the 10 stations through 111 m of piping composed of acrylonitrile butadiene styrene (ABS). All determinations were made prior to the opening of the unit and after the system was purged for 35 h with all bedside station taps open. Formaldehyde disinfection of the piping system was attempted with a recommended protocol after 11 weeks by feeding 2.5 liters of 37% formaldehyde (0.85%, vol/vol) into the delivery system. Prior to water purging, 24 ng of endotoxin per ml was detected. This level decreased to 2.0 ng of endotoxin after the purging. Levels of endotoxin remained below 1.0 ng of endotoxin per ml throughout the duration of the study. In contrast, the level of viable microorganisms recovered from the treated water was approximately 3.5 X 10(4) CFU/100 ml. Even after disinfection of the system, there was no significant decrease in culturable bacteria from the water even though endotoxin levels were lower. Species isolated from the renal dialysis system were predominately pseudomonads, whereas species isolated from the tap water were Bacillus and Flavobacterium species. ABS provides a surface suitable for long-term colonization and growth of bacteria. Currently recommended decontamination protocols are ineffective in removing potentially pathogenic bacteria from ABS pipes and thus constitute an increased risk to patients undergoing dialysis.

E. coli peritonitis and bacteremia cause increased blood-brain barrier permeability.

Impaired mental status is a poorly understood manifestation of sepsis and may be associated with altered permeability of the blood-brain barrier. To examine the possibility that sepsis affects permeability of the blood-brain barrier, rats were infected with a peritoneal implant consisting of sterilized feces, barium sulfate, and 10(8) colony forming units (CFU) of Escherichia coli. Using this model, reproducible episodes of peritonitis with bacteremia resulted. Rats were sacrificed hourly after 5 min circulation of 100 mg horseradish peroxidase. Animals were perfused-fixed and the brains removed. Representative coronal sections were stained for peroxidase reaction product and cerebral blood vessels were examined microscopically for evidence of HRP staining and extravasation. The number of stained cerebral vessels from infected rats was increased at all times compared to uninfected control rats. Extravasation of horseradish peroxide within neuropil was significantly higher in hours 1, 4 and 5 as compared to controls. The lack of significant increase in hours 2 and 3 may suggest transient closing or repair of the tight junctions. We conclude that peritonitis and bacteremia are associated with increased permeability of the blood-brain barrier.

Mycobacterium avium complex, an emerging pathogen in Massachusetts.

We report a study of 1,953 patients whose laboratory records from 1972 through 1983 at the Massachusetts Mycobacteria Reference Laboratory indicated the isolation of Mycobacterium avium complex (MAC) organisms. At least one clinical specimen from each patient during this period exhibited the organism. The incidence of isolation of MAC has increased fivefold since 1972, with a doubling of the number of patients with positive MAC specimens from normally sterile sites occurring since 1980. A concomitant increase of more than fourfold in other nontuberculous mycobacteria has occurred. Most isolates came from high-density population centers. Communities whose drinking water comes from a distant rather than a local source were more likely to have patients with MAC.

Detection of gram-negative bacteremia by limulus amebocyte lysate assay: evaluation in a rat model of peritonitis.

A spectrophotometric Limulus amebocyte lysate assay using lysis filtration and centrifugation has been developed for the detection of gram-negative bacteria in blood. The assay is directed at detection of endotoxin in viable and nonviable bacteria present in the blood-stream and not detection of free endotoxin in plasma. The assay was evaluated in a model of peritonitis in which rats were challenged with an inoculum consisting of sterilized human feces, barium sulfate, and one of eight species of bacteria. This assay was able to detect gram-negative bacteremia due to Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Proteus mirabilis, and Klebsiella pneumoniae in the rat model when compared with sham-inoculated uninfected rats. The assay failed to detect bacteremia due to Bacteroides fragilis or Staphylococcus aureus, nor was there a significant rise in absorbance when a pellet containing sterilized feces was implanted in the rat.