A method to prevail false positive responses due to excess cations and viscous nature of Radiopharmaceuticals in Limulus Amebocyte Lysate Gel Clot test

Document Type : Original Article


1 Medical Cyclotron Facility, Radiation Medicine Centre, Board of Radiation and Isotope Technology, Parel, Mumbai, India

2 Radiation Medicine Centre, Bhabha Atomic Research Centre, Parel, Mumbai, India

3 Radiation Medicine Centre, Board of Radiation and Isotope Technology, Parel, Mumbai, India

4 Homi Bhabha National Institute, 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 [177Lu]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 [131I]I-Lipiodol was  standardized.
Methods: GC-BET was performed for [177Lu]Lu-DOTATATE and [131I]I-Lipiodol at maximum valid dilution (MVD), using LRW as a diluent. To negate the false positivity observed in case of [177Lu]Lu-DOTATATE, various concentrations of calcium chloride (CaCl2)  were added  and evaluated  for the reversal of the interference observed initially. To prevail the difficulty in performing GC-BET for [131I]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, [177Lu]Lu-DOTATATE exhibited false positivity  in GC-BET. However, all the individual reagents used in labeling of [177Lu]Lu-DOTATATE did not show any false positivity. Finally, performing the assay with an addition of 2mM CaCl2 (final concentration) nullified the false positivity. Further, intricacy in performing GC-BET for [131I]I-Lipiodol due to its viscosity was resolved by  orbital vortexing at 3000 rpm for 5 minutes.
Conclusions: Our study proved that false positivity was observed in GC-BET for [177Lu]Lu-DOTATATE due to the presence excess M3+ 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 [131I]I-Lipiodol can resolved the problems encountered during  performing GC-BET due to viscosity of RPs.


  1. Giammarile F, Bodei L, Chiesa C, Flux G, Forrer F, Kraeber-Bodere F, et al. EANM procedure guideline for the treatment of liver cancer and liver metastases with intra-arterial radioactive compounds. European journal of nuclear medicine and molecular imaging. 2011; 38(7):1393-406.
  2. Hennrich U, Kopka K. Lutathera®: the first FDA-and EMA-approved radiopharma-ceutical for peptide receptor radionuclide therapy. Pharmaceuticals. 2019; 12(3):114.
  3. McCullough KZ. Bacterial endotoxin testing. Environ Monit Cleanrooms Control Environ. 2016; 08:131-179.
  4. Cooper JF, Hung JC. Entotoxin Test and Sterility Test for Radiopharmaceuticals. 2008; 14(039):1-54.
  5. Galanos C, Rietschel ET. Bacterial endotoxins. Infection. 1986; 14(4):155.
  6. Lipiodol (ethiodized Oil). 2014:1-7.http://www.accessdata.fda.gov/drugsatfda_docs/label/2014/009190s021lbl.pdf. Accessed 10 Aug 2021.
  7. Das T, Chakraborty S, Banerjee S, Venkatesh .On the preparation of a therapeutic dose of 177Lu-labeled DOTA–TATE using indigenously produced 177Lu in medium flux reactor. Applied radiation and isotopes. 2007; 65(3):301-8.
  8. Shigenaga T, Takayenoki Y, Kawasaki S, Seki N, Muta T, Toh Y, et al. Separation of Large and Small Granules from Horseshoe Crab (Tachypleus tridentatus) Hemocytes and Characterization of Their Components1. The Journal of Biochemistry. 1993; 114(3): 307-16.
  9. Tsuji K, Steindler KA. Use of magnesium to increase sensitivity of Limulus amoebocyte lysate for detection of endotoxin. Appl Environ Microbiol. 1983; 45(4):1342-1350.
  10. Tokunaga F, Yamada M, Miyata T, Ding Y, Hiranaga-Kawabata M, Muta T, et al. Limulus hemocyte transglutaminase. Its purification and characterization, and identification of the intracellular substrates. Journal of Biological Chemistry. 1993; 268(1):252-61.
  11. US-4273557-A @ pubchem.ncbi.nlm.nih.gov. https://ncbi.nlm.nih.gov/patent/US-4273557-A. Accessed 10 Aug 2021.
  12. Mitra A, Joshi S, Arjun C, Kulkarni S, Rajan R. Limulus amebocyte lysate testing: Adapting it for determination of bacterial endotoxin in 99mTc-labeled radiopharmaceuticals at a hospital radiopharmacy. J Nucl Med Technol. 2014; 42(4):278-282.
  13. Lourenço FR, De Souza Botelho T, De Pinto TJA. How pH, temperature, and time of incubation affect false-positive responses and uncertainty of the LAL gel-clot test. PDA J Pharm Sci Technol. 2012; 66(6):542-546.
  14. Ahvazi B, Boeshans KM, Idler W, Baxa U, Steinert PM. Roles of Calcium Ions in the Activation and Activity of the Trans-glutaminase 3 J Biol Chem. 2003; 278(26):23834-23841.
  15. Ahvazi B, Kim HC, Kee SH, Nemes Z, Steinert PM. Three-dimensional structure of the human transglutaminase 3 enzyme: Binding of calcium ions changes structure for activation. EMBO J. 2002; 21(9):2055-2067.
  16. Banerjee S, Pillai M, Knapp F. Lutetium-177 therapeutic radiopharmaceuticals: linking chemistry, radiochemistry, and practical applications. Chemical reviews. 2015; 115(8):2934-74.
  17. Pauling L. The sizes of ions and the structure of ionic crystals. J Am Chem Soc. 1927; 49(3):765-790.
  18. Lambert B. New Strategies in Radionuclide Therapy for Hepatocellular Carcinoma. Ghent University. Faculty of Medicine and Health Sciences, Ghent, Belgium. 2005. http://hdl.handle.net/1854/LU-540408. Accessed 10 Aug 2021.