Vascular ataxic hemiparesis: a reevaluation.

Abstract

<b>2909</b> <h3><b>Introduction:</b></h3> Measurement of endotoxin levels is a key test in the quality control (QC) regimen for radiopharmaceuticals. Endotoxins are lipopolysaccharides (LPS) present in the cell wall of gram-negative bacteria. Critically, endotoxins are sufficiently small size to be able to penetrate sterilizing filters. LPS triggers a series of reactions with Limulus Amoebocyte Lysate (LAL) leading to a clot. LAL reagent can be treated with a peptide carrying a chromophore. The LAL reaction can therefore be monitored by measuring color change. This is the principal behind kinetic chromogenic testing which is a United States Pharmacopeia (USP) compliant bacterial endotoxin test (BET) method. This method satisfies the harmonized USP and European Pharmacopeia BET requirements for LAL testing. It is described in USP General Chapter &lt;85&gt; Bacterial Endotoxin Test. <h3><b>Methods:</b></h3> The Nexgen Endosafe Multi Cartridge Endotoxin Test System (NEMCETS, Charles River Laboratories) running EndoScan-V™ version 6.0.2, was used to measure the level of endotoxin by kinetic chromogenic assay. NEMCETS uses a single-use FDA approved disposable cartridge. The cartridge detects at a sensitivity (λ) = 0.005 EU/mL to 10 EU/mL in a test time of about 15 minutes. A single test cartridge consists of four reservoirs. 25 µL of test sample is added to each reservoir. These reservoirs represent two pairs of “SAMPLE” and “SPIKE” channels. Each reservoir is part of a channel which terminates in an optical cell where test samples are analyzed by the NEMCETS detector. A valid test requires, i) Spike Recovery = 50% to 200% and ii) both Spike and Sample channel reaction times Coefficient of Variation (CV) &lt; 25%. A 1:100 diluted radiopharmaceutical sample was micropipetted onto a cartridge λ = 0.05 EU/mL (effective λ = 5 EU/mL). A total of 513 radiopharmaceutical batches were tested. All cassettes were visually inspected post-test. Conditions for an valid test are described in the Table. Condition [6], which relates to the function of NEMCETS, was assessed by sending the invalid test log file to CRL for analysis. <h3><b>Results:</b></h3> Of the 513 radiopharmaceutical batches tested, 20 were reported as invalid. 4 of these 20 tests were caused by a problem related to one or more factors as described in the Table. The outstanding 16 results were “<i>non attributable</i>” invalid tests. Vendor analysis of the log files concluded that there were no hardware, software or sample volume issues. Therefore, this suggests that there is an inherent invalid test rate of 3.1% (<i>non</i>-<i>attritubale </i>tests) associated with this form of kinetic chromogenic endotoxin test. The drug batches tested comprised 6 different PET drugs. Their was no evidence that quality characteristics of the batches afftected invalid rate. Third party data was provided by CRL (source unknown). From 75,000 tests on the Endosafe MCS, approximately 3000 samples resulted in invalid test results. This data represented an invalid test rate of about 4% to 5%. The total invalid rate of 3.9% obtained in our tests is in-line with this data. <h3><b>Conclusions:</b></h3> Though this form of kinetic chromogenic testing uses minimal analyst involvement, there is still room for human error. Errors, could be related to any of the items describled in the Table. Also, as a biological assay and in near perfect conditions, there will be variability and potential unexplained invalidities. Understanding that their is an inherent invalid rate for a test which seems perfectly acceptable is important.. But, it presents a challange for developing a QC test regimen particularly for short lived materials.