Arpit Mitra - Medical Cyclotron Facility, Radiation Medicine Centre, Board of Radiation and Isotope Technology, Parel, Mumbai, India
Sangita Lad - Radiation Medicine Centre, Bhabha Atomic Research Centre, Parel, Mumbai, India
Sudeep Sahu - Radiation Medicine Centre, Bhabha Atomic Research Centre, Parel, Mumbai, India
Savita Kulkarni - Medical Cyclotron Facility, Radiation Medicine Centre, Board of Radiation and Isotope Technology, Parel, Mumbai, India

Objective(s): Bacterial endotoxin test (BET) for detection and quantification of endotoxin in radiopharmaceuticals (RPs), used for therapy or diagnosis, is prerequisite to administration in patients. Out of the two established methods used for this purpose (Kinetic Chromogenic Assay: KCM and Gel Clot Bacterial Endotoxin Test: GC-BET), GC-BET is recommended by pharmacopeias to evaluate the interferences exhibited during the assay due to presence of various ingredients in samples.  In the present study, the influence of excess of cations in [۱۷۷Lu]Lu-DOTATATE, used for Peptide Receptor Radionuclide Therapy (PRRT), were studied  and  a protocol to negate  the enhancement observed was  developed. Additionally, a protocol for carrying out GC-BET for extremely viscous [۱۳۱I]I-Lipiodol was  standardized.Methods: GC-BET was performed for [۱۷۷Lu]Lu-DOTATATE and [۱۳۱I]I-Lipiodol at maximum valid dilution (MVD), using LRW as a diluent. To negate the false positivity observed in case of [۱۷۷Lu]Lu-DOTATATE, various concentrations of calcium chloride (CaCl۲)  were added  and evaluated  for the reversal of the interference observed initially. To prevail the difficulty in performing GC-BET for [۱۳۱I]I-Lipiodol  various   modification in the protocols like orbital vortexing at different  rpm and time intervals were performed. KCM assays were also performed for studied RPs at MVD.Results: It was observed that at MVD, [۱۷۷Lu]Lu-DOTATATE exhibited false positivity  in GC-BET. However, all the individual reagents used in labeling of [۱۷۷Lu]Lu-DOTATATE did not show any false positivity. Finally, performing the assay with an addition of ۲mM CaCl۲ (final concentration) nullified the false positivity. Further, intricacy in performing GC-BET for [۱۳۱I]I-Lipiodol due to its viscosity was resolved by  orbital vortexing at ۳۰۰۰ rpm for ۵ minutes.Conclusions: Our study proved that false positivity was observed in GC-BET for [۱۷۷Lu]Lu-DOTATATE due to the presence excess M۳+ ions. Further, our study is the first of its kind which demonstrated methods for negating these false positive results by using modified protocol and hypothesizing the reason behind the enhancement.  Additionally, ours is the first study which proved that a simple step of vortexing the viscous RPs like [۱۳۱I]I-Lipiodol can resolved the problems encountered during  performing GC-BET due to viscosity of RPs.

Limulus Amebocyte Lysate Gel-Clot test, Lutetium-۱۷۷-DOTATATE, Iodine-۱۳۱-Lipiodol, Transglutaminase