The determination of endotoxin in the finished cellular product.

BACKGROUND: With the advancement of genetics and hematopoiesis resulting in therapeutic applications, a growing focus has developed on the quality assessment of biological products generated for various cellular therapies. Endotoxin is a critical measure for the presence of Gram-negative bacteria, known to cause endotoxemia. Cellular products are currently regulated as medical devices. Each location engaged in clinical protocols is responsible for establishing a quality assurance program. METHODS: In this study, endotoxin levels were assayed using both the gel-clot and kinetic chromogenic Limulus amebocyte lysate (LAL) assays on 33 patients' cellular products, produced in clinical laboratory settings as part of a clinical trial or approved protocol. These patient samples include tumor infiltrating lymphocytes (HSVtk). We sought to identify the more reliable and informative method for the determination of endotoxin levels in a variety of cellular products, to meet the growing demand for standardization of product quality assessment. Comparison of the most sensitive gel-clot LAL test (0.03 EU/mL), with the kinetic chromogenic LAL test, with a lysate sensitivity of 0.005 EU/mL, found many advantages of the more sensitive method. RESULTS: The kinetic chromogenic LAL test, which has the greatest sensitivity, increased the percentage of samples with valid spike recoveries compared with the gel-clot LAL test from 65% to 70% at a 1:10 sample dilution; and from 81% to 88% at a 1:100 sample dilution. Ata sample dilution of 1:50 the kinetic chromogenic LAL test provided valid spike recoveries on 81% of all samples tested. DISCUSSION: In the interest of providing the highest quality and safety in the finished cellular product, the determination of endotoxin by the kinetic chromogenic LAL test is a rapid, effective, easy-to-use method to detect the presence of Gram-negative bacterial contamination.

Gram-negative bacteriuria of pregnancy: rapid detection by a chromogenic Limulus amoebocyte lysate assay.

A chromogenic Limulus amoebocyte lysate assay, performed at room temperature, was evaluated for rapid detection (five minutes) of asymptomatic bacteriuria of pregnancy. Development of a distinctive yellow color was used to detect urines containing greater than 10(5) gram-negative bacteria per milliliter. One thousand thirty-nine urine samples were obtained from 664 obstetric patients, with 86 of the specimens shown to contain greater than 10(5) gram-negative urinary tract pathogens. Of these, 74 of 86 were detected by chromogenic Limulus assay; 11 false-positive tests were recorded. At a urine dilution of 1:10, sensitivity and specificity were 88.7 and 98.7%, respectively. Predictive values, based on an attack rate of 10%, were 98.6% for a negative test and 89.9% for a positive test. These data suggest that chromogenic Limulus amoebocyte lysate assay of urine has potential usefulness as a rapid, reliable, and easily performed and interpreted screening test for asymptomatic bacteriuria of pregnancy.

Comparison of several control standard endotoxins to the National Reference Standard Endotoxin--an HIMA collaborative study.

A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli O55:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli O55:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.

Chromogenic Limulus amoebocyte lysate assay for rapid detection of gram-negative bacteriuria.

A chromogenic Limulus amoebocyte lysate assay was evaluated as a rapid screening test for the detection of clinically significant gram-negative bacteriuria. The development of a distinctive yellow color after the addition of chromogenic substrate to the Limulus amoebocyte lysate-urine reaction mixture was used to measure greater than or equal to 10(5) gram-negative bacteria per ml. A total of 324 urine specimens were assayed, with 68 gram-negative urinary tract infections identified as defined by quantitative urine colony counts of greater than or equal to 10(5) bacteria per ml. Of these, 68 and 67 of 68 were detected by the chromogenic Limulus amoebocyte lysate assay at urine dilutions of 1:10 and 1:20, respectively. Nine false-positive chromogenic Limulus amoebocyte lysate assay results were observed at both urine dilutions and in the same specimens. At a urine dilution of 1:10, sensitivity and specificity were 100 and 96.6%, respectively, with predictive values of 100% for a negative test and 88.3% for a positive test. At a urine dilution of 1:20, sensitivity and specificity were 98.6 and 96.6%, respectively; predictive values were 99.6% for a negative test and 88.3% for a positive test. These data suggest that chromogenic Limulus amoebocyte lysate assay of urine has potential usefulness as a rapid, reliable, and easily performed and interpreted screening test for the diagnosis of clinically significant gram-negative bacteriuria.

Early complement component depletion and mixed cryoglobulinemia in a "healthy" family.

A family containing two apparently healthy brothers with profound early complement component depletion (C1, C4, C2) and mixed IgG-IgM polyclonal cryoglobulins was studied. The cryoglobulins possessed rheumatoid factor activity and depleted early complement components in normal human serum. Circulating immune complexes could not be detected by utilizing standard methods. The phenomenon was not HLA-linked. This study demonstrates the familial occurrence of a chronic hypocomplementemic state associated with cryoglobulinemia in clinically normal subjects.

The measurement of antibodies to extractable nuclear antigen.

By the use of a combination of Ouchterlony immunodiffusion and hemagglutination, it was found that 30 of 81 sera (37%) from patients who had systemic lupus erythematosus (SLE) had antibodies to the ribonucleoprotein component of extractable nuclear antigen, and 37 (46%) had antibody to the Sm antigen. Immunodiffusion was more sensitive for detection of anti-ribonucleoprotein, while hemagglutination detected more anti-Sm. Both technics are necessary to define qualitatively the types of antibodies present in SLE sera.

Limited anti-DNA antibody specificity in systemic lupus erythematosus.

Twenty-one sera from 11 patients with systemic lupus erythematosus were titrated with 125I-ss-calf thymus DNA to determine their maximum capacity to bind DNA. Only five sera were capable of binding greater than 90% of the input DNA. The remaining sixteen sera showed maximum DNA binding levels between 35% and 85%. The inability of these sera to bind all of the input DNA is shown to be dependent on variations in the molecular weight of the DNA antigen. The possibility that these differences in DNA binding reflect differences in antigenic specificity is discussed.