Limulus amebocyte lysate testing: adapting it for determination of bacterial endotoxin in 99mTc-labeled radiopharmaceuticals at a hospital radiopharmacy.

Abstract

A bacterial endotoxin test (BET) is required to detect or quantify bacterial endotoxin that may be present in radiopharmaceutical preparations. The test uses Limulus amebocyte lysate, which, in the presence of bacterial endotoxin and divalent calcium ions, causes the formation of a coagulin gel. (99m)Tc-labeled radiopharmaceuticals have chelating ligands such as diethylene triamine pentaacetic acid (DTPA), ethylene dicysteine (EC), L,L-ethyl cysteinate dimer (ECD), N-[2,4,6-trimethyl-3 bromoacetanilid] iminodiacetic acid (mebrofenin), dimercapto succinic acid-III (DMSA-III), dimercapto succinic acid-V (DMSA-V), and several others, which form a coordination complex with Na-(99m)Tc-O4 in the presence of reducing agents. During BET by the gel-clot method, the free sulfhydryl (-SH) and carboxyl (-COOH) in some of the chelating agents in the final (99m)Tc-labeled radiopharmaceuticals decrease the free divalent calcium ion concentration, which in turn inhibits coagulin gel formation. This study was designed using the premise that addition of calcium chloride solution to the reaction mixture would nullify this effect. We present here the data obtained from BET assay analysis of (99m)Tc-labeled radiopharmaceuticals and the cold kits from which they are made (EC, ECD, methoxyisobutylisonitrile, DTPA, mebrofenin, methylene diphosphonic acid [MDP], DMSA-III, and DMSA-V) using 2 different dilutions, maximum valid dilution (MVD) and half maximum valid dilution (MVD/2), with and without the addition of calcium chloride at a final concentration of 300 μM. It was observed that at MVD and MVD/2 all of the (99m)Tc-labeled kits exhibited interference in coagulin gel formation with the exception of (99m)Tc-methoxyisobutylisonitrile, (99m)Tc-MDP, (99m)Tc-mebrofenin, and (99m)Tc-ECD. However, only the cold kits of methoxyisobutylisonitrile and MDP did not show inhibition. An addition of calcium chloride solution nullified this interference at both MVD and MVD/2 in all of the (99m)Tc-labeled radiopharmaceuticals in which interference was observed. In practice, Limulus amoebocyte lysate testing is not a method of choice for (99m)Tc-labeled radiopharmaceuticals because these radiopharmaceuticals exhibit interference. However, our study proves the hypothesis that the addition of calcium chloride can circumvent this problem. The addition of calcium chloride provides an enhanced biologic quality control testing option for the final formulation of (99m)Tc-labeled radiopharmaceuticals at the hospital radiopharmacy end.