Limulus Amebocyte Lysate Testing: Adapting It for Determination of Bacterial Endotoxin in 99mTc-Labeled Radiopharmaceuticals at a Hospital Radiopharmacy

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TABLE 1

Detailed Protocol for BET

Test serial no.	Assay tube (in duplicate)	LAL reagent water (μL)	Control standard endotoxin 4λ (μL)	Test sample^* (μL)	LAL reagent (μL)
1	Negative water control	100	—	—	100
2	Positive water control 2λ	50	50	—	100
3	NPC^†	50	—	50	100
4	PPC 2λ^†	—	50	50	100
5	PPC 2λ with calcium chloride^†	—	50	50^‡	100

↵* Repeated at MVD and MVD/2—that is, 1:66 and 1:33 dilutions.
↵† These tubes were set up for all ^99mTc radiopharmaceuticals (^99mTc-DTPA, ^99mTc-DMSA-III, ^99mTc-DMSA-V, ^99mTc-ECD, ^99mTc-mebrofenin, ^99mTc-methoxyisobutylisonitrile, and ^99mTc-MDP) and their cold kits.
↵‡ Calcium chloride at final concentration of 300 μM was incorporated in assay tube of test sample at final dilution.
Tests 3 and 4 were also performed initially for 60 mM stock solution of calcium chloride and saline (0.9% sodium chloride) to ensure absence of endotoxin in stock solutions.

TABLE 2

Results for Different ^99mTc-Labeled Radiopharmaceuticals and Their Cold Kits at MVD and MVD/2 in BET Assay

	MVD				MVD/2
	NPC		PPC		NPC		PPC
Product analyzed	Without CaCl₂	With CaCl₂	Without CaCl₂	With CaCl₂	Without CaCl₂	With CaCl₂	Without CaCl₂	With CaCl₂
^99mTc-EC	–	–	+	++	–	–	–	++
EC cold kit	–	–	–	++	–	–	–	++
^99mTc-DTPA	–	–	+	++	–	–	–	++
DTPA cold kit	–	–	–	++	–	–	–	++
^99mTc-ECD	–	–	++	++	–	–	++	++
ECD cold kit	–	–	–	++	–	–	–	++
^99mTc-DMSA-III	–	–	–	++	–	–	–	++
DMSA-III cold kit	–	–	–	++	–	–	–	++
^99mTc-DMSA-V	–	–	–	++	–	–	–	++
DMSA-V cold kit	–	–	–	++	–	–	–	++
^99mTc-mebrofenin	–	–	++	ND	–	–	++	ND
Mebrofenin cold kit	–	–	+	++	–	–	–	++
^99mTc-methoxyisobutylisonitrile	–	–	++	ND	–	–	++	ND
methoxyisobutylisonitrile cold kit	–	–	++	ND	–	–	++	ND
^99mTc-MDP	–	–	++	ND	–	–	++	ND
MDP cold kit	–	–	++	ND	–	–	++	ND

TABLE 3

Results of Gel-Clot Assay at Different Standard Endotoxin Concentrations in Presence and Absence of Calcium Chloride

T1 = assay tube 1; T2 = assay tube 2; T3 = assay tube 3; A = with CaCl₂; B = without CaCl₂; − = no gel-clot; + = semisolid gel-clot; ++ = firm gel-clot.