Influence of radiation on endotoxin test using the PTSTM for 18-FDG radiopharmaceutical

F-18 FDG (2-[18-F] fluoro-2-deoxy-D-glucose) is the most frequently used radiopharmaceutical for PET and PET CT imaging exams. The FDA recently approved the use of the PTSTM (Portable Test System) as an alternative to the standard test proposed by the United States Pharmacopeia using the LAL (Limulus Amebocyte Lysates), that takes longer to perform (about 1h) than the PTSTM (15 min). Recent studies have demonstrated that radiation could interfere with the PTSTM test. In order to study the effects of radiation on the PTSTM test and/or equipment, 27 batches of F-18 FDG produced in the Nuclear Engineering Institute were analyzed. The results showed that no direct correlation with radiation was found in any of the cases.


INTRODUCTION
Radioisotopes are used in nuclear medicine for diagnostic and therapeutic purposes.Radiopharmaceuticals may be used in oncology at the initial stage, to assess response to treatment, residual disease, recurrent diagnosis or restaging.Another field of study involving radiopharmaceuticals is that of large vessel vasculitis, granulomatous diseases and dementia (Santos-Oliveira, Carneiro-Leão, Smith, 2008).
The word pyrogen, which can be traced to the Greek pyro, meaning burning or fire and gennaó meaning to make or to create, is now used as an apt description for subs-tances that produce elevated body temperature.Pyrogens are usually bacterial products and remains, or decaying products of bacterial cell walls.Even at minimum doses, these substances induce elevated body temperature when injected into humans and animals.Pyrogens are usually high-molecular-weight substances of a polymerous nature, akin to lipopolysaccharids.Pyrogens can be either microbial or non-microbial.Endotoxins are high-molecularweight complexes associated with the outer membrane of gram-negative bacteria (GNB).They are the most usual cause of the elevated body temperature, induced by contaminated drug products.Their pyrogenic activity is higher than that of other pyrogenic substances.It could be said that the absence of such bacterial endotoxins in a drug implies the absence of pyrogenic components in the studied drug in general.Endotoxins are similar to lipopolysaccharids, they are heat stable and can survive the sterilization process (Blechová, Pivodová, 2001).
The use of LAL for endotoxin detection stemmed from Bang's observation that the infection of Limulus polyphemus, the horseshoe crab, induced by GNB, results in extensive intravascular clotting and death.Levin and Bang later demonstrated that the extracellular coagulation of Limulus hemolymph (blood) was caused by a reaction between endoxin and a coagulative protein in amebocytes, circulating in Limulus hemolymph (Levin, Bang 1964).In a subsequent study, Levin et al. developed a sensitive assay for endotoxin in human plasma using the material lysed from Limulus amebocytes (Levin, Tomasulo, Oser, 1970).Young, Levin and Prendergast then isolated, purified and described the LAL coagulative protein and proved that the reaction between lysate and endotoxin is of an enzymatic nature (Young , Levin, Prenderagst, 1993).
Limulus Amebocyte Lysate (LAL) is a lyophilized preparation made from the amebocytes of the horseshoe crab Limulus polyphenus.The LAL will clot to form a gel in the presence of endotoxin from gram-negative microorganisms.Since the LAL test method is far more rapid than the USP (United States Pharmacopoeia) rabbit pyrogen test method, it has been used as a preferable test (Watchel, Tsuji, 1977).Nevertheless, despite being more rapid than the rabbit test method, the LAL test is still slow, especially for radiopharmaceuticals.
The PTS TM (Portable Test System TM ) is a novel endotoxin assay in which dry LAL-reagents are fixed to a cartridge and results are interpolated from an archived standard curve by a validated chromogenic assay.The use of the of PTS has become an alternative to the traditional USP test using LAL due the shorter time taken to complete the whole process, about 15 minutes against 1 hour for the traditional test (FDA, 1987;Zijlstra et al., 1997;Fukumori et al., 2007) However, studies by Hung, Iverson, Jacobson and Mahoney (2005) demonstrated that special care is necessary when using the PTS with radiopharmaceuticals, since during the test the FDG can inhibit gel formation.Therefore, in order to prove this theory an experimental study with 27 samples of 18-FDG produced in the Nuclear Engineering Institute was conducted.

METHODOLOGY
The assays were performed using the Endosafe Ò PTS TM equipment and the kit for the test (cartridge) from the Charles River Laboratories TM .The methodology adopted was that described in the equipment manual (25mL of F-18 FDG injectable solution were fixed in the cartridge and then the equipment run).The samples used were 27 successive daily batches of F-18 FDG produced routinely in the Nuclear Engineering Institute.No dilution factors were used.

RESULTS AND DISCUSSION
The PTS TM works with a dry LAL reagent and uses validated interpolated internal values.Among the parameters revealed by the PTS TM test, the most important is the recovery percentage.Briefly, the recovery rate shows the ability of the sample to react with the dry LAL reagents and form the chromogenic compound.According to the manufacturer of the PTS TM , a good range for the recovery parameter is between 50% and 200%.Values above or below this range mean that the reaction between the sam- ple and the reagents of the kit was not good and that the results are not reliable.Samples 4, 5, 6, are the most representative cases.Variations were detected in the PTS TM test in all these samples, as shown in Table 1.However the values found are under the acceptable limit (0.25EU/mL).Thus, although they are different from the others this does not mean they are not good enough.Relatively to recovery parameter no single observation is possible.The same occurs with the activity, since they are too similar and the results are too different.
In the case of samples 2, 11, 13, 17, 21 and 23, the recovery range value given by the PTS TM was lower than the acceptable limit (50 to 200%), but the endotoxin limit was acceptable at less than 0.050 EU/mL.Although the samples had been approved for the assay, the recovery value is an important parameter linked to the reliability of the results.This means that in a case where the samples have to be re-analyzed the new results found can differ to the original results.Again, no relationship with activity, recovery test and endotoxin limit is possible.The range of each parameter is too high to try to create a hypothesis.
The attempt to correlate all the problems described previously with the radiation of the sample failed, as shown in Table1.Samples 24, 25, 26 and 27 are the most representative.The four samples have the highest values of activity.However, the value of recovery varies without any correlation.For samples where the recovery of the PTS TM test was in the range (50-200%), no relationship with the radiation level was ascertained since no relationship among the values was found.
It is important to note that all these samples were analyzed by the traditional method described in the USP and no clots were formed in any of the cases.This implies that according to the traditional methods all the samples were approved.

CONCLUSION
The PTS was shown to be a good alternative endotoxin test, especially for radiopharmaceuticals, where time is one of the most important parameters during the routine assay.The results were able to corroborate the use of PTS TM in the daily routine for 18-FDG.However, several points must be evaluated in more depth, particularly recovery range.
Although the results shown above demonstrated no relationship between radiation and the endotoxin test using the PTS TM further studies are needed for other radiopharmaceuticals, in particular radiopharmaceuticals based on Iodine since iodine has coloration when in solution and could interfere with the test through the formation of color-complexes.
Therefore, we recommend that before introducing PTS in the daily routine for radiopharmaceuticals, further validations should be conducted.

TABLE I -
Activity and correlation with other parameters