Original Article

Gel clot bacterial endotoxin test of FDG: Indian scenario

Sarika Sharma, Bhagwant Rai Mittal, Rakhee Vatsa, Baljinder Singh
Department of Nuclear Medicine, PGIMER, Chandigarh, India

ABSTRACT Background: The bacterial endotoxin test (BET) performed using gel clot method is a 60-min test and typically
performed after the decay of the 2-( F) fluoro-2-deoxy-d-glucose (F18-FDG) sample to determine the endotoxin
18

content. The objective of this study protocol was to perform BET testing of F18-FDG by gel clot method. Materials
and Methods: Ten random decayed samples of the F18-FDG were subjected to the gel clot BET. The assay was
performed with undiluted F18-FDG and at four different maximum valid dilutions of 1:10, 1:100, 1:350 and
1:700 (total number of tests = 100). The sensitivity of the LAL reagent used was 0.125 EU/ml. Endotoxin dilutions
were freshly prepared from control standard endotoxin (CSE) stock solution for each F18-FDG batch testing. If
the gel had formed and remained intact in the bottom of the reaction tube after an inversion of 180°, the test was
considered positive. Any other state of the reaction mixture constituted a negative test. Results: In the undiluted
samples, the measured pH (7.05) was well within the acceptable range (i.e. 6.0–8.0) for the gel clot assay. Of the
10 undiluted F18-FDG batches and all the diluted samples, none gelled after 60-min incubation period at 37°C.
However, the undiluted F18-FDG did inhibit gel formation at the lysate sensitivity of 0.125 EU/ml. Conclusion:
The total volume of FDG produced was 16 ml in the synthesis module. The total F18-FDG preparation at any
time did not contain more than 8 EU (0.5 EU/ml × 16 ml). Thus, the product is safe for human administration.
Keywords: Bacterial endotoxin test, fluorodeoxyglucose, gel clot assay

INTRODUCTION blood cells of Limulus polyphemus, called amebocytes, a clear lysate

Much of the current success in clinical positron emission
tomography (PET) can be attributed to the development of
2-(18F) fluoro-2-deoxy-d-glucose (F18-FDG). It is the most
commonly used radiopharmaceutical in the PET, with a half-life
of 110 min. Before the release of F18-FDG for human use, the
quality control tests need to be performed to check the safety of
the product.[1,2] The bacterial endotoxin test (BET) is one such
quality control test. The BET using gel clot method is a 60-min
test and typically performed after the decay of the F18-FDG
sample to determine the endotoxin content. The gel clot is
technically the most simple and was the first BET approved by
the Food and Drug Administration (FDA). Endotoxin is a subset
of pyrogens that are strictly of gram-negative origin, a natural
complex of lipopolysaccharides occurring in the outer layer of
the bilayered gram-negative bacterial cell. From the circulating
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DOI:
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is obtained that forms an opaque gel in the presence of extremely
low concentrations of bacterial endotoxins.[3] The BET is fully
described by most pharmacopeial monographs. According to the
United states pharmacopeia (USP), pharmacopeia, F18-FDG
should not contain more than 175/V USP endotoxin units
(EU) per milliliter of F18-FDG injection, in which V is the
maximum administered total dose in milliliters, at the expiration
time. [1,4] However, no such standards have been recommended
by Indian Pharmacopoeia Commission (IPC) till date.[5] The
IPC is taking measures to include radiopharmaceuticals in the
Indian Pharmacopoeia.
The objective of this study protocol was to set up a standard
pyrogen testing facility and to establish documented evidence
if the process employed for BET testing of F18-FDG by gel
clot method produces the desired results consistently, when
performed as per the standard operating procedures.
MATERIALS AND METHODS
F18-FDG sample preparation
Random samples of the F18-FDG from September 2010 to
March 2011 were subjected to the gel clot BET. F18-FDG was
synthesized in the in-house medical cyclotron and radiochemistry

Address for correspondence:

Ms. Sarika Sharma, Department of Nuclear Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh – 160 012,
India. E-mail: sarik_us@yahoo.com

Indian Journal of Nuclear Medicine | Vol. 26: Issue 3 | July-September, 2011 149
 Sharma, et al.: Gel clot bacterial endotoxin test of FDG

lab at the Department of Nuclear Medicine and PET Centre, 0.25 EU/ml. The reaction solution was mixed thoroughly and
using the TRACERlab MX FDG (GE Healthcare, Münster, placed immediately in the incubator at 37°C±1°C for 60±2 min.
Germany). Decayed residual F18-FDG from clinical preparations At the end of the incubation period, the tubes were removed
was used. The assay was performed at four different maximum from the incubator and inverted.
valid dilutions (MVDs, i.e. 1:10, 1:100, 1:350 and 1:700). The
undiluted samples of F18-FDG were also subjected to the gel If the gel had formed and remained intact in the bottom of the
clot test. The pH of the samples was measured to ensure a pH reaction tube after an inversion of 180°, the test was considered
within the range 6.0–8.0 for the validity of the gel clot procedure. positive. A positive test indicated that the concentration of
endotoxin in the tube is greater than or equal to the sensitivity
Preparation of endotoxin stock solution and of the LAL reagent. Any other state of the reaction mixture
endotoxin dilutions constituted a negative test, which indicated an endotoxin
The standard gel clot test for assessing the bacterial endotoxin concentration less than the LAL reagent sensitivity. The test
content consisted of four controls: negative water control (NWC), was considered negative when the tube is inverted and the gel
positive water control (PWC), negative product control (NPC) breaks or collapses.
and positive product control (PPC). The NPC is actually the
diluted F18-FDG sample solution. The PWC and PPC contain the RESULTS
standard endotoxin preparation. The NWC is the endotoxin-free
Limulus amaebocyte lysate (LAL) reagent water. [4,6] A total of 10 different batches of decayed F18-FDG were used
in this study. The mean pH was 7.05 (6.5–7.5). The total numbers
The matched control standard endotoxin (CSE), LAL reagent of tests performed on the F18-FDG were 100 excluding NWC,
and LAL reagent water obtained from Charles River Laboratories PWC and PPC.
India Private Limited, Bangalore, India, were used in this study.
The sensitivity of the LAL reagent contained in each vial was Table 1 summarizes the results of the undiluted F18-FDG on
0.125 EU/ml. The test for confirmation of lysate sensitivity the positive control solutions at an endotoxin concentration of
was carried out with every new batch of the lysate as per the 0.5 EU/ml (4λ). Of the 10 undiluted F18-FDG batches, none
USP<85> recommendations.[3,4] gelled after 60-min incubation period at 37°C. This indicated
that all the batches did not contain endotoxin greater than 0.5
The CSE having predetermined amount of endotoxin was EU/ml. None of the negative control NWC vials gelled. All the
prepared according to the directions for use given in the specific 20 positive endotoxin control PWC vials gelled. Only 18 out of
certificate of analysis (COA). The CSE stock solution of 20 EU/ 20 PPC vials gelled after incubation at 37°C for 60 min. This
ml was prepared with endotoxin-free water and vortexed for 5 indicated that the undiluted F18-FDG inhibited gel formation.
min. The stock solution was stored for 4 weeks at 2°C–8°C.[6]
Before each use, the CSE stock solution was mixed vigorously Table 2 summarizes the results for the 60-min gel clot assay on
for 5 min. Endotoxin dilutions were freshly prepared from CSE the four different dilutions of the F18-FDG samples. A total
stock solution for each F18-FDG batch testing. of 80 tests were performed excluding NWC, PWC and PPC
vials. None of the negative control NWC vials gelled. All the 20
Glassware for BET positive endotoxin control PWC vials gelled. None of the 20 1:10
All the dilutions were made in the depyrogenated 16 × 125 mm F18-FDG sample vials gelled after 60-min incubation at 37°C.
glass tubes using endotoxin-free water and pyrogen-free sterile However, 1 out of 20 PPC vials for the 1:10 dilution did not
pipettes tips. Depyrogenations were performed in the dry heat gel probably due to some interference with the gel formation at
oven at 250°C for 1 hour, when the endotoxin-free glass tubes the lysate sensitivity of 0.125 EU/ml. These results are similar
were not obtained commercially. All assays were performed in
the 10 × 75 mm depyrogenated borosilicate glass tubes. Table 1: Results for undiluted F18-FDG sample
FDG Lot no. pH NWC FDG undiluted PWC* PPC*
Routine test procedure 15092010 7 -- -- ++ ++
The BET was performed using 0.1 ml of F18-FDG sample 18092010 7.5 -- -- ++ ++
and 0.1 ml of reconstituted LAL reagent per tube. The LAL 23092010 7.5 -- -- ++ ++
reagent was constituted after the addition of LAL reagent water 10012011 7 -- -- ++ ++
11012011 7.5 -- -- ++ -+
(endotoxin free) in the freeze-dried power of lysate. The tests
15022011 7 -- -- ++ ++
were done on all the 10 undiluted F18-FDG batches in duplicates. 9032011 6.5 -- -- ++ ++
The PWC and PPC used for these tests were at the endotoxin 10032011 6.5 -- -- ++ +-
concentration of 0.5 EU/ml. The four dilutions of the F18- 11032011 7 -- -- ++ ++
FDG, i.e. 1:10, 1:100, 1:350 and 1:700, were freshly prepared from 25032011 7.5 -- -- ++ ++
29032011 6.5 -- -- ++ ++
the batches and subjected to the gel clot assay. The NWC and
EU: endotoxin unit; NWC: negative water control; PWC: positive water control; PPC:
PWC were common for all the dilutions. The PPC was separately positive product control; *Both positive controls contained the standard endotoxin
prepared for all the dilutions at the endotoxin concentration of preparation at a concentration of 0.5 EU/ml; +: Firm gel formed; −: No gel formed

150 Indian Journal of Nuclear Medicine | Vol. 26: Issue 3 | July-September, 2011
 Sharma, et al.: Gel clot bacterial endotoxin test of FDG

Table 2: Results for the diluted F18-FDG samples

FDG Lot no. pH NWC PWC* PPC*# FDG FDG FDG FDG
1:10 1:100   1:350 1:700 1:10 1:100 1:350 1:700
23092010 7.5 - ++ ++ ++ ++ ++ -- -- -- --
15092010 7 - ++ ++ ++ ++ ++ -- -- -- --
18092010 7.5 - ++ ++ ++ ++ ++ -- -- -- --
10012011 7 - ++ ++ ++ ++ ++ -- -- -- --
11012011 7.5 - ++ ++ ++ ++ ++ -- -- -- --
15022011 7 - ++ ++ ++ ++ ++ -- -- -- --
9032011 6.5 - ++ ++ ++ ++ ++ -- -- -- --
10032011 6.5 - ++ ++ ++ ++ ++ -- -- -- --
11032011 7 - ++ -+ ++ ++ ++ -- -- -- --
25032011 7.5 - ++ ++ ++ ++ ++ -- -- -- --
29032011 6.5 - ++ ++ ++ ++ ++ -- -- -- --
EU: endotoxin unit; NWC: negative water control; PWC: positive water control; PPC: positive product control; *Both positive controls contained the standard endotoxin
preparation at a concentration of 0.25 EU/ml; #PPCs were obtained for all the FDG samples separately at the four different (1:10, 1:100, 1:350 and 1:700) dilutions; +:
Firm gel formed; −: No gel formed

to those obtained from the undiluted FDG samples. All the
remaining vials for the 1:100, 1: 350 and 1:700 dilutions did not
gel. This indicated that all the vials did not contain endotoxin
greater than 0.125 EU/ml.
DISCUSSION
formation as shown in Table 1. Therefore, the sample was tested
for the various dilutions 1:10, 1:100, 1:350 and 1:700. The 350
and 700 dilution factors were considered according to the limit of
175 EU/ml (single patient dose supplied to the other institute or
when the final elution is in 2–3 ml due to some technical errors).
The rate of inhibition improved with the dilutions in Table 2.

The gel clot method is often considered as the most accurate and
sensitive procedure for testing endotoxin content in injectable
radiopharmaceutical products because fewer false-positive
and false-negative results are observed when this method is
used.[7] The total time taken to complete the gel clot assay was
approximately 2 hours.
The LAL reaction requires optimal pH range. In the undiluted
samples, the measured pH (7.05) was well within the acceptable
range (i.e. 6.0–8.0) for the gel clot assay.[1,3,4]
The F18-FDG produced in the Tracerlab MX synthesis module
is neutralized in the citrate buffer which contains multivalent ions.
Nakao et al. reported the effect of the citrate salt concentration
on the endotoxin test for the F18-FDG preparations.[8] These
authors reported that the undiluted sample showed the recovery
less than 20%, beyond the acceptance of the USP. After twofold
dilution with sterile water, no interference was observed. Similar
results were found in the present study with the undiluted
samples as two vials of the PPC (endotoxin concentration equal
to 0.5 EU/ml) did not gel, indicating the interference with the
gel formation. The interference may be due to the presence of
the citrate ions.
At the expiration time, the maximum administered total dose
in milliliters of FDG should contain less than 175 EU.[1,4] The
total batch volume of FDG produced is 16 ml in the synthesis
module. Therefore, the total F18-FDG preparation at any time
did not contain more than 8 EU (0.5 EU/ml × 16 ml). Thus,
the product is safe for human administration.
However, the undiluted sample did show the inhibition of the gel
Indian Journal of Nuclear Medicine | Vol. 26: Issue 3 | July-September, 2011
The gel clot assay has a major drawback which limits its use
for the short-lived radiopharmaceuticals. This test is a time
consuming test (60 min incubation time).
In addition to this drawback, errors made by technical personnel
and the misinterpretation of results are also common problems.
The 60-min BET is described in USP<85>, “Bacterial Endotoxins
Test”,[4] and is also recommended for pyrogenicity testing in the
draft guidance of the FDA on current good manufacturing
practice (CGMP) for PET drug products. Because the remainder
of the required quality assurance (QA) testing for F18-FDG
injection, with the exception of the sterility test, can be completed
in approximately 20–30 min, delaying the release of the short-
lived F18-FDG injection for an additional 30–40 min is not
practical and is, in fact, wasteful. An in-process 20-min BET
must be performed before release of the drug product, and a
standard 60-min BET must also be completed.[9]
Some other techniques to determine endotoxins are also
known, such as the chromogenic (color development) and the
turbidimetric (turbidity development)[10] tests, both of which
can provide valuable quantitative and qualitative information
about the endotoxin concentration in samples. Regardless of
which method is used, PET laboratories and pharmacies should
ensure that their technicians are well trained and educated in the
endotoxin testing method of choice.
CONCLUSION
The pyrogen testing facility was successfully established using
gel clot procedure. This study suggests that the BET needs to
be performed, standardized and documented in each cyclotron
151
 Sharma, et al.: Gel clot bacterial endotoxin test of FDG

and PET facility. The undiluted FDG preparations occasionally
interfere with the gel formation. The dilution of the test sample
is the easiest means to resolve the potential product inhibition
/ enhancement problem during the gel clot testing procedure.
The final F18-FDG preparation at any time point did not contain
more than 8 EU, thus making it safe for human administration.
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for FDG and CIDG. Appl Radiat Isot 2005;62:889-95.
9. Hung JC, Iverson BC, Jacobson MS, Mahoney DW. Inhibtion evaluation for
a 20-min. endotoxin limit test on FDG. Nucl Med Commun 2005;26:869-74.
10. Mitra A, Kulkarni S, Rajan MG. Rapid test for bacterial endotoxin
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How to cite this article: Sharma S, Mittal BR, Vatsa R, Singh B. Gel
clot bacterial endotoxin test of FDG: Indian scenario. Indian J Nucl Med
2011;26:149-52.
Source of Support: Nil. Conflict of Interest: None declared.

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