User Guidelines & Standard Operating Procedure
for the
Beckman Coulter LS6500
Multipurpose Scintillation Counter
Scintillation Counter Standard Operating Procedure
ii
TABLE OF CONTENTS
DISCLAIMER ................................................................................iii
ACKNOWLEDGEMENTS .................................................................... iv
1.
INTRODUCTION........................................................................1
1.1
Purpose of the Standard Operating Procedure .............................1
1.2
Theoretical Background ........................................................1
1.3
Counting Programs..............................................................4
1.4
Instrumentation .................................................................5
2.
POTENTIAL HAZARDS.................................................................6
2.1
Radiation Hazards...............................................................6
2.2
Chemical Hazards ...............................................................6
3.
PERSONAL PROTECTIVE EQUIPMENT ...............................................6
4.
SPILL AND ACCIDENT PROCEDURES ................................................7
4.1
Accidents .........................................................................7
4.2
Spills ..............................................................................8
5.
WASTE DISPOSAL PROCEDURES ................................................... 10
6.
PROTOCOL........................................................................... 11
6.1
General Sample Preparation ................................................ 11
6.1.1
Automatic Sample Counting............................................ 12
6.1.2
Sample Preparation for Specific Sample Types ..................... 13
6.2
Sample Disposal ............................................................... 21
6.2.1
General Guidelines ...................................................... 21
6.2.2
Specific Waste Types.................................................... 21
7.
PREVENTATIVE MAINTENANCE .................................................... 22
7.1
Daily ............................................................................ 22
7.2
Monthly ......................................................................... 22
7.3
Three Months .................................................................. 22
7.4
As Required .................................................................... 22
8.
QUICK REFERENCE GUIDE.......................................................... 23
REFERENCES .............................................................................. 24
APPENDIX
APPENDIX
APPENDIX
APPENDIX
1:
2:
3:
4:
BACKGROUND AND EFFICIENCY TESTING.............................. 25
CALIBRATION FOR NEW ISOTOPES ..................................... 26
USER LOG.................................................................. 27
CALIBRATION LOG ........................................................ 29
Scintillation Counter Standard Operating Procedure
iii
DISCLAIMER
The information contained in this document has been compiled from sources
believed to be reliable and to represent the best opinions on the subject. This
document is intended to serve only as a starting point for good practices and
does not purport to specify minimal legal standards. No warranty, guarantee,
or representation is made by Laurier as to the accuracy or sufficiency of
information contained herein, and Laurier assumes no responsibility in
connection therewith.
Scintillation Counter Standard Operating Procedure
iv
ACKNOWLEDGEMENTS
The following individuals of Laurier contributed to the writing, editing, and
production of this manual: Gena Braun (Instrumentation Technician); Stephanie
Kibbee (Environmental/Occupational Health and Safety Office).
This manual was prepared for Laurier. Any corrections, additions or comments
should be brought to the attention of the Instrumentation Technician at
519-884-0710 ext. 2361.
Issued: January 2010
Revision: 1
Scintillation Counter Standard Operating Procedure
1
1. INTRODUCTION
1.1 Purpose of the Standard Operating Procedure
This standard operating procedure (SOP) is NOT a substitute for training
and/or reading the appropriate manuals before use. All principle
investigators and supervisors must document that training has been received by
students and staff who will be using the Beckman scintillation counter.
All new users of radioactive substances must also:
1. Be reported to the Radiation Safety Officer (RSO) by the Internal Permit
Holder (Supervisor) using the Internal Radioisotope Permit Application Form (pg
35 in the WLU Radiation Safety Manual*).
2. Receive Radiation Safety Training from the Environmental / Occupational
Health and Safety (EOHS) Office.
A list of authorized users will be maintained by the Instrumentation Technician.
This SOP is intended to promote consistent and safe use of the Beckman LS6500
scintillation counter. This SOP covers the potential hazards, personal
protection requirements, spill and accident procedures, waste disposal
considerations, and instrument operation for the Beckman LS6500 scintillation
counter.
1.2 Theoretical Background
Figure 1-1: Beta decay
of tritium (Helmholtz
University, 2006).
Liquid scintillation is the detection of beta
radiation in a scintillation cocktail, which occurs
when a neutron is converted to a proton, and a
beta particle (electron) and antineutrino are
ejected. For example; the nucleus of tritium
contains one proton and two neutrons. When
tritium undergoes beta decay, one of the neutrons
decays to a proton, and a beta particle and
antineutrino are ejected. As a result, tritium is
converted to helium, as shown in Equation 1-1 and
illustrated in Figure 1-1:
3
H1  3He2 + 0e-1
*
http://www.wlu.ca/documents/14021/Radiation_Manual_09_January_2006.pdf
Equation 1-1
Scintillation Counter Standard Operating Procedure
2
The scintillation cocktail captures the beta emission energy and transforms it
into a photon which can be detected via a photocathode, amplified by a
photomultiplier tube, and converted to counts per minute (CPM).
The scintillation cocktail is composed of four main components, which must
solubilize the sample and maintain a uniform suspension of the sample while
transferring the energy of beta decay to the detector. These components are as
follows:
1. The radioactive substance: This is the radioactive sample to be
measured.
2. The solvent: The solvent dissolves the sample. Typical solvents include
Toluene, xylene, pseudodocumem, or some type of alkyl benzene. PDioxane is also useful for highly aqueous samples, but it must be
peroxide free because peroxides cause unwanted quenching.
3. An emulsifier: The emulsifier is a detergent that ensures the proper
solvation of aqueous samples.
4. The solute or fluor: The solute or fluor captures the energy of the beta
particle and is itself promoted to an excited state. The fluorescence
solute decays rapidly through photon emission and transforms the energy
of beta particles to photons that can be detected by the
photomultiplier. Primary solutes absorb the excitation energy of the
solvent directly. Secondary solutes absorb the decay energy of the
primary solute and emit photons at a longer wavelength, which can
improve detection and counting efficiency. Liquid brand names include:
PPO, POPOP, NE213, PBD.
A number of interferences can negatively affect the accuracy of radiation
measurement. These interferences and suggested corrections are listed in
Table 1-1.
For additional background, see the University of Colorado Physics Department
website: http://www.colorado.edu/physics/2000/isotopes/index.html.
3
Scintillation Counter Standard Operating Procedure
Table 1-1: Interferences and suggested corrections (summarized from Australian Flexible Learning and Beckman On-line
Lab Resources)
Type of Interference
Chemiluminescence
Photoluminescence
Quenching
Description
Light energy resulting from a chemical
interaction within the solution. This
interference is controlled by the amount
and type of sample used and properties of
the scintillation cocktail.
Chemiluminescence may be occurring if the
count rate decreases with time in the
absence of radioactivity.
Particularly problematic with dioxanebased scintillators and bleaching agents.
Emission of light by excited molecular
species. In this case excitation is caused by
light and can be long lived. This type of
interference occurs with proteinaceous
compounds in alkaline solubilizers.
Photon quenching: affects beta emission.
Chemical impurity quenching: affects
energy transfer between solvent and solute
or emission of the solute.
Background
Color quenching: affects detection of
fluorescence by the photomultiplier.
Radioactivity that arises from cosmic rays,
Cerenkov radiation, natural radioactivity
from thorium, potassium-40 and uranium.
Correction Suggestions
If you suspect that your samples have
chemiluminescence, prepare a control with sample plus
cocktail but no radioactivity. By counting this sample
periodically, you can determine how long it will take
the chemiluminescence in your experimental samples to
decay to an acceptable level.
Alternatively, use the Lum-Ex feature on the LS 6000
Series scintillation counter. The counter will then
automatically estimate and correct for the
chemiluminescence.
Can be reduced by acidification or leaving samples in
the dark for several hours before counting.
The Internal Standards Method can be used to correct
for quenching. This method assumes that added
standard of same isotope present in the sample will be
quenched to the same degree as the sample.
Use appropriate blanks to correct for background.
4
Scintillation Counter Standard Operating Procedure
1.3 Counting Programs
Table 1-2 describes the parameters available when designing a counting
program, and Table 1-3 lists the counting programs currently available on the
scintillation counter.
Table 1-2: Counting program parameter descriptions
Description
Counting window
setting.
The value entered
establishes the 95%
confidence level for
the count (also called
the 2 sigma statistical
value).
Quench monitor
setting.
Quenching manifests
itself by shifting the
energy spectrum
toward lower energy
channels in the
Multichannel
Analyzer.
Parameter
Result
Wide
Count the entire energy spectrum.
Manual
Manually enter the desired settings.
Counting
Precision
A value of 1, for example, indicates that in
95 out of 100 cases, the CPM obtained will
be within 1% of the mean. A higher counting
precision requires a higher number of total
counts. If the counting time is reached
before obtaining the specified counting
precision, counting is terminated.
IC#
IC# is a sample activity quench monitor and
uses the sample isotope spectrum to track
quench; it is most accurate with high-count
rate samples
H#
H# uses an external standard to monitor
quench. This method is independent of the
sample isotope and of the activity in the vial
and has a large dynamic range.
CPM
Counting method.
DPM
Counts per minute = the total number of
photons counted divided by the count time.
Disintegrations per minute. Calculated by
correcting CPM based on counting
efficiency. Quench curves are required for
all isotopes, except for pure beta-emitting
isotopes (3H, 14C, 32P, 35S, 45Ca or 86Rb). DPM
can be measured for pure beta emitting
isotopes using Auto DPM. Contact the
Instrumentation Technician if you would like
to set up an Auto DPM run.
5
Scintillation Counter Standard Operating Procedure
Table 1-3: Counting programs available on the LS 6500 scintillation counter**
User
Number
1
2
3
18
19
20
n/a
Description
Smith 35S
14C Test
Program
Efficiency
Test
Tritium
DPM
Carbon 14
Background
Auto DPM
Count
Time
(min)
1
Calculation
Mode
Count
Repeats
Quench
Lum-Ex
Correction
35
DPM
1
IC#
Yes
1
14
DPM
1
IC#
No
5
3
3
IC#
Yes
Isotope(s)
S
C
DPM (3H)
H, Wide
CPM (Wide)
1
3
CPM
1
H#
No
1
10
10
14
CPM
DPM
Auto DPM
1
1
1
H#
None
H#
No
Yes
No
H
C
Wide
Wide
**All programs listed in Table 1-3 have the following settings: liquid scintillator, 1 measurement
of each sample, 1 measurement of each sample set, low reject is set at 0 (Off), no background
measurement is subtracted, and the counting precision is set at 0 for all programs except
Tritium DPM where it is set at 1.
1.4 Instrumentation
The sample racks are placed in the counting chamber, which is closed off to
outside light. The chamber will accommodate 336 standard vials, or 648
miniature vials. An electrostatic controller reduces the interference of static
electricity, which can be introduced with the plastic vials.
Light generated by beta emission from a radioactive sample is detected by two
photomultipliers (PMTs). The PMTs are used in tandem to detect a photon
event. A pulse is not registered unless both PMTs detect the photon within a
short time interval (20-30 ns). The signal from the PMT is then amplified and
converted into a CPM or DPM value.
Scintillation Counter Standard Operating Procedure
6
2. POTENTIAL HAZARDS
2.1 Radiation Hazards
The radioisotopes currently in use at WLU include 14C, 3H (tritium), and 35S.
These isotopes are all β-Emitters (small particles with a low charge), so they
only pose a serious hazard if they enter the body through ingestion or
through the skin. However, they must be dealt with very carefully and
stringently contained. These isotopes do not have to be used in a fumehood
and do not require shielding, however the bench area must be labeled, and
appropriate personal protective equipment must still be used.
Pregnant radiation workers must notify, in writing, the RSO and their supervisor
as soon as they are aware of their pregnancy. Special precautions must be
taken to provide the proper degree of protection to the fetus during the term
of the pregnancy. A pregnant worker shall not exceed a maximum effective
dose of 4 mSv for the balance of the pregnancy.
2.2 Chemical Hazards
Scintillation cocktails include various solvents and should be handled
accordingly (see manufacturer’s instructions or the MSDS for the specific
product). Appropriate personal protective equipment must also be used.
3. PERSONAL PROTECTIVE EQUIPMENT
Disposable gloves (2 pairs), a lab coat, radiation badge (see below), and eye
protection must be worn when using radioactive materials. Closed-toe and heel
footwear constructed of resistant material is also required for all laboratory
activities.
WLU employees working with radioisotopes must wear personal
monitoring devices for the following situations:
- Film badges will be issued to all employees using radioactive
materials. These badges are capable of measuring exposures
to X‐rays, gamma rays and beta particles. Film badges must
be worn at chest or waist level.
- Finger rings will be issued to all employees handling more
32
than 50 MBq (1.35 mCi) of high‐energy beta‐emitting ( P) or
x2
125
gamma‐emitting radionuclides ( I). The badges and rings
are collected every three months by the RSO and sent to a
licensed dosimetry service to be measured. The radiation
dosimetry reports will be available in the EOHS office.
See the WLU Laboratory Health and Safety Manual for additional
information on personal protective equipment.†
Radiation
Badge
http://www.wlu.ca/documents/23120/Laboratory_Health_%26_Safety_Manual__Feb_2007_Fin
al.pdf.
†
Scintillation Counter Standard Operating Procedure
7
4. SPILL AND ACCIDENT PROCEDURES
4.1 Accidents
All incidents must be reported to the Instrumentation Technician and, if
applicable, a student’s supervisor. Accidents involving radiation should be dealt
with as follows (from the WLU Radiation Safety Manual):
1. In case of radioactive contamination on the skin, the contaminant should
be washed from the skin under running lukewarm water.
2. Monitor contamination.
3. Repeat water rinse, if necessary.
4. Remove any clothing, shoes or jewelry that are contaminated and
discard as radioactive waste.
5. If you suspect internal contamination, advise the RSO immediately. If
cuts, abrasions, or open wounds are observed:
a. Clean the affected area with suction and dry swabs.
b. If skin is contaminated in the area of cuts, abrasions or open
wounds, use wet swabs in the direction away from the area;
taking care not to spread activity over body or into wound.
6. Inform the RSO.
All accidents, incidents and near misses must be reported to the EOHS Office
via the WLU Employee Accident/Incident/Occupational Disease Report form
(www.wlu.ca/eohs/forms). To meet regulatory requirements, these forms must
be submitted to EOHS within 24 hours of occurrence, with the exception of
critical injuries, which must be reported immediately to the EOHS Office by
telephone. Critical injuries include any of the following; place life in jeopardy,
produce unconsciousness, result in substantial loss of blood, involve fracture of
a leg or arm but not a finger or toe, involve amputation of a leg, arm, hand or
foot, but not a finger or toe, consist of burns to a major portion of the body, or
cause the loss of sight in an eye. All accidents involving radiation and
radioactive material must be reported to the RSO as well.
Additional details regarding incident reporting can be found in the WLU
Accident Incident Procedure (www.wlu.ca/eohs).
Scintillation Counter Standard Operating Procedure
8
4.2 Spills
The WLU Laboratory Health and Safety Manual provides detailed instructions
for dealing with major and minor spills. Do not attempt to clean up a spill if
you have not been properly trained, or if you are unsure of the proper
procedures. Before using ANY hazardous materials, make sure you
understand the proper clean-up procedure. The EOHS Office is also available
to provide guidance at ext. 2874. Spills involving radioactive material require
special procedures, as outlined in Figure 4-1 (from the WLU Radiation Safety
Manual).
Also take the following guidelines into account:
1. The person(s) cleaning the spill must take all necessary precautions,
such as wearing lab coats, TWO pairs of gloves and safety glasses.
2. Cover liquid spills with absorbent material to limit the spread of
contamination. If a dry spill occurs, it should be dampened to avoid
spreading due to air currents. Be careful not to spread it unnecessarily.
3. Mark off the contaminated area with special tape (with radioisotope
logo) and restrict traffic to that area.
4. For short half‐life isotopes (32P, 35S):
a. Materials that cannot be cleaned and are portable may be stored
in the designated area for decay. Monitor these materials
frequently.
b. Materials that cannot be cleaned and are not portable must be
properly shielded and be labeled as radioactive. Monitor these
items frequently and notify the RSO.
5. For long half‐life isotopes (3H and 14C):
a. Materials that cannot be cleaned must be labeled and swipe tests
performed. Notify the RSO.
6. Remove contaminated clothing and discard.
7. Put on clean gloves.
8. Pick up contaminated absorbent materials with forceps and place in
plastic bag.
9. Scrub area (always towards centre of spill) in such a way as not to
spread contamination using Decon.
10.Monitor the effectiveness of the decontamination procedure via a survey
meter and finally by thorough swipe tests.
11.Monitor all persons involved in the clean‐up for residual radioactivity.
12.If personal contamination occurs in a radiation spill, call the PH and /or
the RSO for clean up help rather than risking the spread of the spill.
All spills are to be reported by the individual(s) responsible. An
incident/accident report must be completed within 24 hrs and sent to
his/her Supervisor. Also, the RSO must be alerted when a radioisotope spill
occurs.
Scintillation Counter Standard Operating Procedure
Figure 4-1: Procedure for dealing with a radioactive spill (WLU Radiation
Manual, 2006).
9
Scintillation Counter Standard Operating Procedure
10
5. WASTE DISPOSAL PROCEDURES
If any hazardous chemicals are used for sample analysis or preparation, they
must be disposed of properly, as outlined in the WLU Laboratory Health and
Safety Manual.
Items used for radioactivity analysis must be disposed of as follows:
- Scintillation vials should be tightly sealed and disposed of in
polyethylene bags in designated scintillation waste containers with
proper shielding.
- All sharps used for dispensing radioactive materials must be place in an
approved sharps container with the radioactive label on it. The
container must be monitored and shielded if necessary.
- Contaminated glass (test tubes, glass pipettes etc) must be disposed of
in a well‐labeled sharp’s container.
Record the date and amount of isotope discarded, and initial the
Radioactive Waste Inventory form. See the WLU Radiation Safety Manual and
Section 6.2 in this document for further detail.
Scintillation Counter Standard Operating Procedure
11
6. PROTOCOL
Anyone using the Scintillation Counter must receive hands on training. This
document is a summary of the procedure and is only intended to help you
remember the various steps.
Always leave the instrument power on. This prevents measurements being
conducted while the instrument is stabilizing.
6.1 General Sample Preparation
1. Samples should be prepared to avoid the following:
a. Static: Avoid handling plastic vials with latex gloves. Latex gloves
cause static buildup and erroneous counting results. Use vinyl
(nitrile) or polyethylene gloves if possible, and wipe sample vials with
an anti-static cloth (i.e. laundry drier sheet) before analysis.
b. Chemiluminescence: This phenomenon is due to chemical reactions
within the sample that produce light but not heat. The Lum-Ex values
provide an indication of the percent of total CPM due to nonradioactive events. Chemiluminescence is more common in sample
with the following characteristics:
- Alkaline pH or peroxides (interference removed by adding
glacial acetic acid)
- Emulsifier cocktails or other tissue solublizers (interference
MAY BE removed by adding glacial acetic acid)
- Samples exposed to sunlight or UV light
- Plant extracts containing chlorophyll
c. Phase separation: Samples will separate into two phases if there is
not enough emulsifier to solubilize the aqueous sample in the organic
cocktail. This overload may result from too much sample, high salt
concentrations, or extreme pH values which degrade the emulsifier.
Check for phase separation by preparing a sample in a glass vial and
observing it over a period of time.
2. Use ONLY the following vial types:
26 to
28 mm
Standard Vials
These vials can be used in
the WHITE racks.
58.5 to
63 mm
The cap must not overlap the
body.
18.3 mm MAX
50 to
63.5 mm
15 mm to
17.8 mm
Miniature Vials
These vials can be used in
the BLUE racks.
Scintillation Counter Standard Operating Procedure
12
6.1.1 Automatic Sample Counting
1. Select the desired rack size, depending on the size of sample vials used, and
install the command card/User No. card into the left set of slots on the first
rack. See Table 1-3 for a list of the preprogrammed command cards.
a. The Auto DPM program can be used to obtain degradation per minute
values for single label samples with a pure beta-emitting isotope (i.e.
3
H, 14C, 32P, 35S, 45Ca or 86Rb) without running quench curves.
2. Load the rack with samples as follows:
a. If blanks or replicates are not used, the samples may be loaded in
any manner desired.
b. If blanks are used, they must be loaded first followed by an empty
space.
c. If replicates are used, the replicates must be loaded in adjacent
positions. If one or more replicates are missing from a group, leave
only one empty position; the system recognizes the vial following the
empty space as being the first replicate of the next set.
3. Place the loaded racks into the scintillation counter, beginning on the right
side, as follows:
a. The first rack must have the User Number Card installed that
corresponds to the program you wish to run. This rack should also
include any blanks.
b. All of the following racks will be counted using the same program,
unless a new User Card is encountered.
c. Place the red Halt Rack behind the last sample rack.
4. Check to make sure the printer is on (the ON light and the ON-LINE lights
should be lit) and that paper is loaded.
5. Using the arrow keys, highlight Automatic Counting, and press Select to
begin counting.
6. If all of the samples have been loaded according to the instructions on the
screen, press Start.
7. The results of the counts will be printed as they are completed. The sample
in progress can be stopped by pressing the Stop Count key (the scintillation
counter will then proceed to the next sample), or the run can be stopped by
pressing the two Reset keys simultaneously.
8. To briefly stop the program to count a small separate batch of samples (up
to one rack), press Select or Interrupt.
a. The priority samples must be loaded in the Interrupt Rack.
b. The data must be accessed after the run has been completed by
selecting “Access interrupt Data” from the Main Menu, followed by
“View Interrupt Data”, and “Print Interrupt Data”.
Scintillation Counter Standard Operating Procedure
13
6.1.2 Sample Preparation for Specific Sample Types
The following suggested counting methods are summarized from the Beckman
on-line resource center. These procedures make use of a variety of scintillation
cocktails and other solutions produced by Beckman. In the following
procedures:
- BTS-450 refers to a tissue solubilzer composed of a quaternary ammonium
hydroxide (0.5N) in toluene. It rapidly and economically digests tissues
and tissue homogenates.
- Ready Gel or Ready Protein+ contains a patented blend of emulsifiers
that are very effective for solubilizing biological precipitates from filters.
It dissolves nitrocellulose and breaks up glass fiber filters to provide high
counting efficiency. The cocktail also shows considerable resistance to
quench.
- Ready Value is the ideal choice for laboratories with many different
aqueous samples. It is the economical version of Ready Gel™ for extremes
of pH, high salt concentrations and high sample loads.
- Ready Cap is solvent-free solid scintillation medium for non-volatile
microvolume samples.
- Ready Organic does not contain emulsifiers and, therefore, provides high
counting efficiency for organic soluble samples. It is also the ideal cocktail
for use with tissue solubilizers and it is specifically purified to reduce
chemiluminescence.
- Ready-Solv HP, Ready Protein+, and Ready Safe are emulsifiers for
gradient solutions. Gradients used in ultracentrifugation are difficult to
count. When mixed with most cocktails, they form two-phase systems or
self-absorption quench may occur due to the presence of highly
concentrated salt or sucrose solutions. Using cocktails formulated with
emulsifier it is possible to solubilize up to 25% sucrose gradients into a
single-phase, quench correctable system. More concentrated solutions
require a special procedure.
- Ready Filter may be used to replace traditional glass fiber filters. Ready
Filters have the sample retention characteristics of Whatman GF/B filters
(1 micron cutoff).
6.1.2.1 Blood Samples
Condition A: Counting in Cocktail
1. Add 0.75 mL of BTS-450/iso-propanol (1:2 v/v) to 0.25 mL blood,
2. Incubate one hour at 40ºC.
3. Add 0.5 mL 30% hydrogen peroxide dropwise while swirling (hydrogen
peroxide is used to decolorize after incubation).
4. Incubate 15 minutes at ambient and 30 minutes at 40ºC.
5. Add 10 mL of Ready Gel containing 7ml/L glacial acetic acid.
6. Shake and count; expected tritium efficiency 25-55% (depending on the
concentration of the sample.
Scintillation Counter Standard Operating Procedure
14
Condition B: Counting 14C in Whole Blood using Ready Cap
1. Add up to 30 µL of whole blood to a Ready Cap (larger volumes result in
unacceptable color quench).
2. Dry (do not use a microwave).
3. Count using Xtalscint option on a LS 6000 scintillation counter or a wide
open counting window on other counters.
a. Turn off the external standard quench monitor.
b. Constant quench may be verified by including IC Number in the
printed report.
6.1.2.2 Plasma and Serum
Condition A: If the sample clears after shaking
DO NOT USE THIS PROCEDURE OVER 25oC
1. Prepare the sample in 0.1 N HCl or add 10% by volume of 1N HCl to the
serum or plasma. Add 1 mL of this preparation to 7-10 mL Ready Gel or
Ready Value.
2. Shake vigorously until clear. If the solution does not clear, use Condition
B procedure.
3. Count. The expected efficiency for tritium is 35-45%.
Condition B: If sample does not clear after shaking
1. Add 0.25 mL plasma or serum to 0.75 mL BTS-450.
2. Incubate one hour at 40ºC.
3. Add 8-10 mL Ready Organic™ and count. Expected efficiency for tritium
is 47-52%.
6.1.2.3 Urine
Counting efficiency will vary from sample to sample depending upon color and
solute quench. Quench correction should be performed with an external
standard method such as H-Number or H-Number Plus (LS 6000 Series).
1. For samples with a normal salt concentration:
a. Add 1 mL of urine to 10 mL of Ready Gel.
b. Shake vigorously and count.
2. For samples with high salt concentration:
a. Add 1 mL of urine plus 3 mL of water to 10 mL of Ready Gel.
b. Shake vigorously and count the stable gel.
6.1.2.4 Phosphate Buffered Saline (PBS)
Condition A: Small sample volume (less than 1 mL)
1. Add up to 1 mL PBS to 3 mL of Ready Protein+ or Ready Safe.
2. Shake well and count. Expected tritium counting efficiency is
approximately 40%. The contents of the counting vial should be a clearphase emulsion.
Scintillation Counter Standard Operating Procedure
15
Condition B: Large sample volume (up to 5 mL)
1. Add up to 4 - 5 mL of PBS to 5 mL of Ready Value or 5 mL of Ready
Gel. Using larger sample volumes increases the sample count rate
and either improves counting statistics, or reduces counting time if
counting to a preset count error (error termination).
2. Shake and count as a stable gel. Expected efficiency for tritium is 1822% for Ready Value and 20-25% for Ready Gel.
6.1.2.5 Aqueous Proteinaceous Samples
Condition A: Cocktail-soluble samples
1. Add 0.1-0.2 mL of sample to 8-10 mL of Ready-Solv HP or 10 mL of
Ready Protein+ or Ready Safe.
2. Shake until homogeneous and count.
Condition B: Non cocktail-soluble samples
1. Add 0.1-0.2 mL of sample to 1 mL of 0.1N NaOH.
2. Swirl until clear and add 8-10 mL Ready Protein+ or Ready Safe.
3. Shake and count.
6.1.2.6 Feces
1. Add 0.1 mL water to 20 mg of feces (dried) and rehydrate for one hour.
2. Add 1 mL of BTS-450 and swirl.
3. Incubate 1-2 hours at 40ºC.
4. Add 0.5 mL isopropanol and mix.
5. Add 0.2 mL 30% hydrogen peroxide.
6. Let stand for 10 minutes at room temperature and then incubate at 40ºC
for two hours.
7. Dilute with 5 mL of water.
8. Add 10 mL of Ready Gel containing 7 mL/L glacial acetic acid.
9. Shake and count as a stable gel.
6.1.2.7 Bacteria and Cells
1. Add 1 mL BTS-450: water (4:1 v/v) to 5-7 mg of bacteria or cells.
2. Incubate 2-4 hours at 40ºC.
3. Add 10-15 mL Ready Organic with glacial acetic acid added at 7
mL/liter.
4. Count. Expected tritium efficiency is 45-50%.
6.1.2.8 Concentrated Buffer Solution
1. Add 0.5-2 mL of the concentrated buffer solution to either 10 mL of
Ready Value or Ready Gel.
2. Shake well and count. Expected tritium efficiency is 20-45%, depending
on the concentration and volume of buffer added. Ready Gel will give
the higher efficiencies.
16
Scintillation Counter Standard Operating Procedure
6.1.2.9 Trichloracetic Acid (TCA)
Method 1:
1. Add up to 2 mL of a 10% TCA sample to 10 mL of Ready-Solv HP, Ready
Gel or Ready Safe.
2. Shake and count. Expected efficiencies for tritium are 20-45%.
Method 2:
1. Add 1.8 mL of 10% TCA to 10 mL of Ready Protein+ or Ready Safe.
2. Shake and count.
6.1.2.10
Homogenates
1. Add 0.2 mL of up to 10% tissue homogenate (in either water or 70%
ethanol) to 1 mL of water.
a. Homogenates of up to 1 mL can be processed by following
the procedure for coarse ground tissue below.
2. Add 10 mL of Ready Gel or Ready Safe.
3. Shake vigorously and count. Expected tritium efficiency is 35-40%.
6.1.2.11
Coarse Ground Tissue
1. Add 150 mg of coarse ground tissue to 2 mL of BTS-450 and swirl.
2. Incubate 3-5 hours at 40ºC.
3. Add 10-15 mL of Ready Organic containing 7 mL/L glacial acetic acid per
liter of cocktail.
4. Shake and count.
6.1.2.12
Organs
Add 1 mL BTS-450 to the tissue weights shown in the following table:
Sample
Arteries
Brain
Cartilage
Cornea
Heart
Intestine
Kidney
Amount
20-100 mg
80-130 mg
20- 55 mg
40-160 mg
100-120 mg
80-100 mg
80-100 mg
Sample
Liver
Muscle
Nerve Cells
Pancreas
Spleen
Stomach
Amount
80-100 mg
100-220 mg
50-100 mg
50-140 mg
50-140 mg
80-100 mg
1. Incubate 2-4 hours at 40ºC or until solubilized.
2. Add 10-15 mL of Ready Organic containing 7 mL/L glacial acetic.
3. Shake and count.
Scintillation Counter Standard Operating Procedure
17
6.1.2.13
Sucrose Gradients
Condition A: 10% sucrose
1. Add up to 2 mL of 10% sucrose to either 8-10 mL of Ready-Solv HP or 10
mL of Ready Protein+ or Ready Safe.
2. Shake well and count. Expected tritium efficiency is 40-45%.
Condition B: 25% sucrose
1. Add up to 2 mL of 25% sucrose to 8-10 mL of Ready Protein+.
2. Shake and count. Expected tritium efficiency is 40-45%.
Condition C: 25% sucrose; large volumes1
1. Add 1-4 mL of 25% sucrose to either 10 mL of Ready Gel or Ready Value.
2. Shake and count. Expected tritium efficiency is 30-40%.
Condition D: Mini-vial counting
1. Add up to 2 mL of 25% sucrose to 4 mL of Ready Flow III™. Shake and
count. Expected tritium efficiency is 24-30%.
Condition E: 10 drop of 12-33% gradient fractions
1. Prepare a special stock solution by adding 50 mL of water per liter to
Ready Flow III, Ready-Solv HP or Ready Protein+.
2. Add a 10 drop gradient fraction (approximately 0.5 mL) to a scintillation
vial (the LS vial may be placed in the fraction collector).
3. Add to the vial any of the following (the volumes given are necessary for
the 12% fraction. Higher sucrose concentrations require less cocktail for
solubilization):
a. 2.5 mL of aqueous Ready Flow III
b. 3.0 mL of aqueous Ready-Solv HP
c. 5.5 mL of aqueous Ready Protein+
4. Shake well to form a clear solution.
5. Count.
6.1.2.14
Cesium Chloride (CsCl) Gradients
1. Dilution of concentrated CsCl gradients is essential to achieve
homogeneous counting conditions without self-absorption quench. With
minimal effort, a special CsCl solubilizing solution can be prepared:
a. CsCl solubilizing solution is prepared by mixing 100 mL of
water with 960 mL Ready Gel.
2. Add up to 0.8 mL of 6 M CsCl to 10 mL of CsCl solubilizing solution.
3. Shake until clear and count. Expected tritium efficiency is 35-40%.
Scintillation Counter Standard Operating Procedure
18
6.1.2.15
Filters
1. Use Ready Filter™ with Xtalscint® solid scintillator for whole
cell/homogenate assays (e.g., receptor, cell proliferation).
2. Use your routine procedure (prewetting/presoaking, harvesting) with the
following modifications:
a. The vacuum source should be a pump capable of delivering
consistent suction of at least 25-inch (640 mm) Hg.
b. After filtration, dry the filters, if possible, before placing or
punching into counting vials.
3. Note: When using a harvester, poor replicate variability is frequently the
result of uneven buffer flow to individual filtration areas, resulting in
variable filter blanks. To check this:
a. Filter liquid containing the same amount of radioactivity
through all filtration areas using your normal procedure, then
dry and count the filters.
b. If variability of filter blanks is excessive, refer to your
harvester manual for instructions on adjusting wash buffer
flow rates.
6.1.2.16
TCA Precipitates
1. Moisten 100 mg of dried TCA precipitate with 0.1-0.2 mL of water.
2. Rehydrate for 30 minutes.
3. Solubilize with 0.1 mL of 0.1M KOH or NaOH.
4. Incubate for 30 minutes at room temperature until solubilized.
5. Add 10 mL Ready Protein+ or Ready Safe.
6. Shake well and count. Expected efficiency for tritium is 35-45%.
6.1.2.17
TLC Scrapings
Condition A: Water-soluble sample
1. Add 1 mL of water to the scrapings.
2. Incubate 3-5 hours at 40ºC.
3. Add 8-10 mL of Ready Gel.
4. Shake and count. Expected tritium counting efficiency is 37-43%.
Condition B: Samples not soluble in water
1. Add 1 mL of BTS-450 to the scrapings.
2. Incubate for 3-5 hours at 40ºC.
3. Add 8-10 mL of Ready Organic containing 7 mL/liter of glacial acetic
acid.
4. Count. Expected tritium counting efficiency is 47-53%.
Scintillation Counter Standard Operating Procedure
19
6.1.2.18
Polyacrylamide Gels with Bis-Acrylamide Cross-Linking
Condition A: Ready Filter (for 32P and 125I only)
1. Run and stain gel according to the procedure used in your lab.
2. Cut out bands and place on Ready Filter circles (Xtalscint side up) in the
bottom of 20 mL counting vials.
3. Count with a wide open window (or use the Xtalscint CPM/DPM option on
LS 6000 scintillation counters).
4. For background subtraction, count an equal sized piece of gel from an
area without stained bands.
5. The Ready Filters may be reused without contamination.
Condition B: For All Beta Emitters
1. Place the gel slice in a vial.
2. Add 0.2 mL of 60% perchloric acid and swirl.
3. Add 0.4 mL of 30% hydrogen peroxide and swirl again.
4. Cap the vial.
5. Incubate at 70-80ºC for 30-60 minutes with gentle agitation if possible.
6. Cool the sample and add 7 mL of Ready Protein+ or Ready Safe. A clear
solution should result.
7. Count.
6.1.2.19
DNA
Procedures for DNA occasionally require sample denaturation with a high
normality of NaOH. Cocktails and their emulsifiers are not stable at these pH
levels and so will not solubilize the sample. Ready Gel has a special emulsifier
that will solubilize these samples with the following procedure:
1. Add 3 mg of dried DNA precipitate to 0.35 mL of 4 N NaOH (see note
below).
2. Agitate 10 minutes at room temperature.
3. Add 1.4 mL water.
4. Add 4.5 mL of Ready Gel and shake well.
5. Let stand one hour. The milky emulsion from step 4 will clear.
6. Allow sample to equilibrate for 12-24 hours at room temperature. During
this time, chemiluminescence will decay to an acceptable level.
a. It has been reported that the sample may adsorb to the walls of a
glass vial. Equilibrium will be reached after 12 hours. After 72
hours, counts may noticeably decrease. Reproducible results can
be obtained if samples are counted within 24 hours of
preparation.
7. Count.
Note: If using 8 N NaOH, adjust the volume of water added in Step 3 to 2.8 mL
and the volume of Ready Gel in Step 4 to 10 mL. Alternatively, use 175 µL in
Step 1 and follow the instructions in the remaining steps.
Scintillation Counter Standard Operating Procedure
20
6.1.2.20
Radioimmunoassays (RIA)
Condition A: Dextran-coated charcoal methods and supernatants
1. Add 0.5 mL of DCC centrifuged supernatant to 3 mL of Ready-Solv HP™ or
4 mL of Ready Protein+™ or Ready Safe™.
2. Shake and count. Expected efficiency for tritium is 40%.
Condition B: Ammonium sulphate, polyethylene glycol (PEG) and double
antibody methods
1. Add 0.5 mL of 0.1 N NaOH to dissolve the pellet.
2. Add 3 mL of Ready-Solv HP or Ready Protein+.
3. Shake and count. Expected efficiency for tritium is 40%.
6.1.2.21
Steroid Receptor: Dextran-Coated Charcoal (DCC) Method
The use of plastic counting vials with R5020 (a ligand frequently used in
progesterone receptor assays) is not recommended because of absorption
effects.
Condition A: TRIS-EDTA-Dithiothreitol buffers
1. Add 0.5 mL DCC-centrifuged supernatant to 3 mL Ready-Solv HP or 4 mL
Ready Protein+ or Ready Safe.
2. Shake and count. Expected efficiency for tritium is 40%.
Condition B: Phosphate buffers
1. Add 0.5 mL DCC-centrifuged supernatant to 3 mL Ready Protein+ or 4 mL
Ready Safe.
2. Shake and count. Expected efficiency for tritium is 40%.
Scintillation Counter Standard Operating Procedure
21
6.2 Sample Disposal
Guidelines and policies for disposal of radioactive materials are outlined in the
WLU Radiation Safety Manual. The following guidelines are summarized from
the WLU Radiation Safety Manual.
6.2.1 General Guidelines
Annual reporting of usage and waste disposal quantities is required.
The total amount of radioactive material put in any container must be
controlled so that the radiation field does not exceed 2.5 mrem/hr at one foot
from the container. This can be checked with a Geiger counter.
Material must not be put into waste containers if there is any possibility of a
chemical reaction during storage that might cause fire or explosion, or the
release of toxic or radioactive gas.
Record the quantity and kind of radioactive material disposed of, into each
container, and ensure that the inventory sheet has been updated, designating
what percentage went to waste (solid or liquid).
6.2.2 Specific Waste Types
Scintillation vials should be tightly sealed and disposed of in polyethylene
bags in designated scintillation waste containers with proper shielding.
Liquid Waste: Place all liquid waste in 5 L plastic containers containing gel as
provided by the appropriate waste disposal company. Ensure that the container
is well labeled; stating types of isotopes and amounts. Close the cap tightly on
the container when not being used and place it behind an appropriate shield, if
required. The wash water of the normal, daily clean‐up of radioactive utensils,
glassware etc. may be disposed via the sewer. No liquid radioactive waste is to
be discharged via the laboratory sanitation system.
Solid Waste: Dispose of all solid waste in designated containers lined with a
polyethylene bag. Ensure proper shielding, depending on the isotope. Record
the date and amount of isotope discarded, and initial the Radioactive Waste
Inventory form.
Scintillation Counter Standard Operating Procedure
22
7. PREVENTATIVE MAINTENANCE
Users are not to perform maintenance. These procedures are carried out by
the Instrumentation
7.1 Daily
-
Check the log book
7.2 Monthly
-
Perform a calibration run
7.3 Three Months
-
Perform a background count and efficiency (14C and 3H) check (see pg 79 in the manual)
7.4 As Required
-
Replace the power fuse
Scintillation Counter Standard Operating Procedure
23
8. QUICK REFERENCE GUIDE
1. Select the desired rack size, depending on the size of sample vials used, and
install the command card/User No. card into the left set of slots on the first
rack.
2. Load the rack with samples.
3. Place the loaded racks into the scintillation counter, beginning on the right
side, as follows:
a. The first rack must have the User Number Card installed
b. All of the following racks will be counted using the same program,
unless a new User Card is encountered.
c. Place the red Halt Rack behind the last sample rack.
4. Check to make sure the printer is on and that paper is loaded.
5. Using the arrow keys, highlight Automatic Counting, and press Select to
begin counting.
6. Press Start.
7. The results of the counts will be printed as they are completed.
8. To briefly stop the program to count a small separate batch of samples (up
to one rack), press Select or Interrupt.
Scintillation Counter Standard Operating Procedure
24
REFERENCES
Australian Flexible Learning. Study Notes: LS Counting.
http://toolboxes.flexiblelearning.net.au/demosites/series5/508/Laborat
ory/StudyNotes/snLSCounting.htm. Accessed Oct 9, 2007.
Beckman Coulter On-line Resource Center.
(http://www.beckmancoulter.com/resourcecenter/labresources/scintill
ation/. Accessed Sept 4, 2007.
Beckman Coulter. 1999. LS6500 Scintillation System Operating Instructions.
Beckman Coulter. Inc.
Bock RK. 1998. Scintillation Counter. Organisation Européenne Pour la
Recherche Nucléaire (CERN).
http://rkb.home.cern.ch/rkb/PH14pp/node166.html. Accessed Oct 15,
2007.
Helmholtz University, Institute for Theoretical Physics. KATRIN Introduction and
Overview. 2006. http://www-ik.fzk.de/tritium/overview/index.html
UCLA Molecular and Medical Pharmacology.
http://149.142.143.90:8099/grad_program/classes/pharm248/M248_99/
autorad/Scint/process3.html. Accessed Oct 15, 2007.
Wilfrid Laurier University, 2006. WLU Radiation Safety Manual.
Environmental/Occupational Health and Safety Office.
http://www.wlu.ca/documents/14021/Radiation_Manual_09_January_20
06.pdf. Accessed Oct 15, 2007.
Wilfrid Laurier University, 2007. Laboratory Health and Safety Manual.
Environmental/Occupational Health and Safety Office.
http://www.wlu.ca/documents/23120/Laboratory_Health_%26_Safety_M
anual.pdf. Accessed Oct 15, 2007.
Scintillation Counter Standard Operating Procedure
25
APPENDIX 1: BACKGROUND AND EFFICIENCY TESTING
A user program titled EFFICIENCY TESTING is used to determine the counting
efficiency for 14C and 3H. This program is based on the method outlined in the
LS6500 Operating Instructions (page 7-7, Beckman Coulter, 1999) and it set up
with the following parameters:
Count time: 5 minutes
Count sample: 3 times
Isotope 1: 3H
H#: On
Isotope 2: Wide
Lum-Ex: On
3
Data calculation: CPM
H and 14C precision: 1%
Follow the protocol and calculation steps below to confirm that the instrument
is operating with the expected efficiency.
1. Set up a rack with the 3H standard in position 1, the 14C standard in
position 2, and the background standard in position 3.
2. Place this rack in the scintillation counter followed by the Halt rack, and
close the cover.
3. Select the EFFICIENCY TESTING program and press start.
4. When data collection is complete, determine the efficiency for each
isotope using the following formula:
% Efficiency = [(CPM – Background)/Corrected DPM] x 100
-
-
CPM: For 3H use the CPM results obtained for Isotope 1 for the
3
H standard. For 14C, use the CPM results for Wide for the 14C
standard.
Background: use the CPM results obtained for Isotope 1 for the
background standard
Corrected DPM: Correct the DPM value printed on the standard
vial for radioactive decay as follows:
1. Calculate t = current date – date of calibration (in
years)
2. Divide t by the half life T (12.35 years for 3H, 5730 years
for 14C)
3. Find this value in the t/T column of the half-life table
and determine the corresponding Fraction Remaining
value (page D-2 in the LS6500 Operating Instructions)
Corrected DPM = DPM at calibration * Fraction Remaining
5. The 3H efficiency should be greater than 59% and the 14C efficiency should
be greater than 95%. If any of the values are not satisfactory:
a. Inspect the sample vials to make sure they are completely sealed.
b. Ensure the instrument is calibrated and set up properly.
c. Repeat the test and verify the calculations.
d. If the system still does not meet specifications, a Service Call may be
required.
Scintillation Counter Standard Operating Procedure
26
APPENDIX 2: CALIBRATION FOR NEW ISOTOPES
The LS6500 library contains window settings and half life values for 3H, 14C,
12
5I, 35S, and 32P. To count any other isotopes, each isotope must first be added
to the library by manually entering the window settings or by running a sample
of the isotope and configuring the system to determine the window settings
automatically. The protocol below describes how to configure the system to
automatically determine window settings for a new isotope.
1. Prepare a sample that represents the least quenched (highest count)
that you expect to see during an experiment. A minimum of 10,000 CPM
is recommended.
2. From the main menu, select “Isotope/DPM/Alpha-Beta Libraries”.
3. Select “Setup and Review Isotopes.
4. Select “Select Another Isotope” and enter the relevant data for the new
isotope (name, half life, Standard DPM and date if known).
5. Highlight “Automatic Window Setup” and press Select.
6. Load samples as directed on the screen, starting with the calibrate rack
(with the 14C standard in the first position), followed by the sample rack
with the isotope samples loaded according to the order displayed on the
screen.
7. Press Start.
8. When the run is complete, the instrument will automatically store the
data for the new isotopes in the library.
Scintillation Counter Standard Operating Procedure
APPENDIX 3: USER LOG
27
28
Scintillation Counter Standard Operating Procedure
DATE
NAME
EXT #
SUPERVISOR
COUNTING
PROGRAM
NUMBER OF
SAMPLES
PROBLEMS OR COMMENTS
Scintillation Counter Standard Operating Procedure
APPENDIX 4: CALIBRATION LOG
29
30
Scintillation Counter Standard Operating Procedure
DATE
NAME
EXT #
TEST TYPE
RESULTS
PROBLEMS