Mitra Arpit, Lad Sangita, Sahu Sudeep, Kulkarni Savita
Medical Cyclotron Facility, Radiation Medicine Centre, Board of Radiation and Isotope Technology, Parel, Mumbai, India.
Radiation Medicine Centre, Bhabha Atomic Research Centre, Parel, Mumbai, India.
Asia Ocean J Nucl Med Biol. 2022 Spring;10(2):109-116. doi: 10.22038/AOJNMB.2021.59607.1416.
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 [Lu]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 [I]I-Lipiodol was standardized.
GC-BET was performed for [Lu]Lu-DOTATATE and [I]I-Lipiodol at maximum valid dilution (MVD), using LRW as a diluent. To negate the false positivity observed in case of [Lu]Lu-DOTATATE, various concentrations of calcium chloride (CaCl) were added and evaluated for the reversal of the interference observed initially. To prevail the difficulty in performing GC-BET for [I]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.
It was observed that at MVD, [Lu]Lu-DOTATATE exhibited false positivity in GC-BET. However, all the individual reagents used in labeling of [Lu]Lu-DOTATATE did not show any false positivity. Finally, performing the assay with an addition of 2mM CaCl (final concentration) nullified the false positivity. Further, intricacy in performing GC-BET for [I]I-Lipiodol due to its viscosity was resolved by orbital vortexing at 3000 rpm for 5 minutes.
Our study proved that false positivity was observed in GC-BET for [Lu]Lu-DOTATATE due to the presence excess M 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 [I]I-Lipiodol can resolved the problems encountered during performing GC-BET due to viscosity of RPs.
用于治疗或诊断的放射性药物（RP）中细菌内毒素检测（BET）及内毒素定量是其用于患者给药的前提条件。在用于此目的的两种既定方法（动态显色法：KCM和凝胶法细菌内毒素检测：GC - BET）中，药典推荐使用GC - BET来评估由于样品中存在各种成分而在检测过程中表现出的干扰。在本研究中，研究了用于肽受体放射性核素治疗（PRRT）的[¹⁷⁷Lu]Lu - DOTATATE中过量阳离子的影响，并制定了消除所观察到的增强作用的方案。此外，还对极粘稠的[¹³¹I]I - 碘油进行GC - BET的方案进行了标准化。
以LRW为稀释剂，在最大有效稀释度（MVD）下对[¹⁷⁷Lu]Lu - DOTATATE和[¹³¹I]I - 碘油进行GC - BET。为消除[¹⁷⁷Lu]Lu - DOTATATE出现的假阳性，添加不同浓度的氯化钙（CaCl）并评估最初观察到的干扰的逆转情况。为克服对[¹³¹I]I - 碘油进行GC - BET的困难，对方案进行了各种修改，如在不同转速和时间间隔下进行轨道涡旋。还在MVD下对所研究的RP进行了KCM检测。
观察到在MVD时，[¹⁷⁷Lu]Lu - DOTATATE在GC - BET中表现出假阳性。然而，用于标记[¹⁷⁷Lu]Lu - DOTATATE的所有单独试剂均未显示任何假阳性。最后，添加2mM CaCl（终浓度）进行检测消除了假阳性。此外，通过在3000 rpm下进行5分钟的轨道涡旋，解决了因[¹³¹I]I - 碘油粘度大而在进行GC - BET时的复杂性问题。
我们的研究证明，由于存在过量的M离子，[¹⁷⁷Lu]Lu - DOTATATE在GC - BET中出现假阳性。此外，我们的研究是同类研究中的首例，展示了通过修改方案和推测增强背后的原因来消除这些假阳性结果的方法。另外，我们的研究首次证明，对像[¹³¹I]I - 碘油这样的粘性RP进行简单的涡旋步骤可以解决由于RP粘度在进行GC - BET时遇到的问题。
