A Tutorial by Stephen M. Karesh, PhD

Adapted for the Web by Stephen M. Karesh, PhD & Marsha Lipps CNMT



a. Introduction and Precautions

There are many precautions one must take during the preparation and use of radiopharmaceuticals, in general, and Tc-99m radiopharmaceuticals, in particular. Since most radiopharmaceuticals are intended to be administered intravenously, it is imperative to use aseptic technique in order to maintain sterility of the product. The vial septum must be wiped with 70% isopropanol prior to puncturing the septum with a needle. This is really a cleansing step rather than a true sterilizing step since the alcohol doesn't remain on the septum long enough to kill all pathogens that might be present.
Air must NEVER be injected into any radiopharmaceutical vial, especially one containing a Tc-99m product. The oxygen contained in only 0.1 ml of air is enough to completely destroy the stannous ion used in many commercially available cold kits as a reducing agent. In addition, room air is not sterile so it is possible to introduce pathogens into the vial by using a
preliminary injection of air to increase internal pressure in the vial and ease the removal of the contents.
Prior to reconstituting a cold kit with Tc-99m pertechnetate, oxidant-free pertechnetate must be diluted to the required final volume with 0.9% NaCl solution. Ideally, oxidant-free saline (Low Dissolved Oxygen Saline) should be used for the dilution step. Reconstitution of a cold kit with a small volume of pertechnetate followed a few minutes later by dilution with saline solution can cause dissociation of certain weak chelates, resulting in the formation of significant amounts of Free Tc. This is not a problem with sulfur colloid or other insoluble Tc-99m compounds.

b. Tc-99m radiopharmaceuticals

Sodium pertechnetate may be used "as is" after elution from the Mo/Tc Generator and is the only Tc-99m compound that requires no manipulation on the part of Nuclear Medicine personnel. It may be injected intravenously, used to label blood cells or other molecules for intravenous injection, or bound to molecules suitable for oral administration.
The majority of Tc-99m compounds employ the stannous reduction method, which makes use of the fact that stannous chloride is one of the most powerful reducing agents available to chemists. Tc-99m obtained from the Mo/Tc generator is in the chemical form of TcO4-, or pertechnetate. While the anion has an overall negative charge of -1, the oxidation number of the Tc is 7+. The chelating agents commonly used to prepare Tc-99m products are also anions with an overall negative charge due to the presence of N, O, and P atoms, each of which has 1 or more extra pairs of electrons. These negative charges repel each other so pertechnetate will
not form chelates. A reducing agent is therefore required to convert the Tc-99m into an electropositive cationic form capable of binding to chelating agents. Tc-99m sulfur colloid and Tc-99m DMSA are the only 2 commercially available Tc-99m compounds that do not use the stannous reduction method.
The following reduction/oxidation reactions (REDOX) indicate that the pertechnetate is typically reduced to Tc4+ while the stannous ion (Sn2+) is converted to stannic ion (Sn4+). In the overall reaction, the stannous ion is the reducing agent, and therefore the substance oxidized, while pertechnetate is the oxidizing agent and therefore the substance reduced.
3 Sn2+- 6e- ---> 3Sn4+
2TcO4- + 16H+ + 6 e- ---> 2Tc4+ + 8H2O
Overall, 3Sn2+ + 2TcO4- + 16H+ ---> 3Sn4+ + 2Tc4+ + 8H2O
The Tc4+ is now in the appropriate chemical form to react with an anion like PYP, MDP, or DTPA. The complex formed is known as a chelate; the generic equation is shown below. Tc4++ chelating agent n -----> Tc-chelate. For example, Tc4+ + pyrophosphate 4- ----> Tc-pyrophosphate
Most soluble Tc-99m compounds, excluding those containing a protein, have octahedral structures and are said to be hexa-coördinated since there are typically 6 binding sites available consisting of N, O, or P atoms. An octahedral structure is shown in Figure 3. In the diagram, Mn+ represents a radiometal ion with a net positive charge due to the loss of n electrons. Certain compounds, e.g., the porphyrins, have a square planar array of N atoms in their center and are tetra-coördinated. Iron atoms bound to the heme portion of the hemoglobin molecule are located within the square planar array of nitrogen atoms (see Figure). In most kits, the desired molecule is already present and it is a simple matter of binding the reduced Tc-99m to the molecule. In the case of MAG3 and teboroxime, however, the desired molecule is actually formed during the first part of a 10 min heating cycle and this molecule then binds to the reduced Tc to form the Tc-chelate. This reaction requires the presence of the correct precursors in the reaction vial at the right concentration to produce the desired product.
Tc-99m reactions by the Thiol Reduction Method also result in complex formation. In this reaction, two thiol groups (-SH) lose their H-atoms and link together to form a disulfide bridge, comparable to the cystine/cysteine reactions. This is the reduction method used in the formation of Tc-99m DMSA, shown in the following reaction. The Tc-99m is trapped within the 4-member ring structure or between the S—S bonds in two molecules of Tc-DMSA.
Tc-99m sulfur colloid is formed by the acid-catalyzed conversion of soluble thiosulfate ion to an insoluble Tc-99m heptasulfide, which coprecipitates with colloidal sulfur. Sodium thiosulfate solution is mixed with a small volume of 1 N hydrochloric acid and pertechnetate is then added to the mixture, which is shaken to insure homogeneity. The mixture is then heated at 100OC for 5-10 min depending upon manufacturer. Alternatively, it may be heated in a microwave oven for 12-25 sec depending upon the particular oven and the power level selected. At the end of the heating cycle, a small volume of a sodium acetate buffer is added to the reaction mixture to raise the pH to approximately 5.5. The Tc-SC is then cooled prior to quality control testing and

c. Other radiopharmaceuticals

Preparation of Cr-51 RBC's is achieved by incubation of 20-30 ml of anticoagulated whole blood with 50-100 μCi of sodium chromate Na251CrO4. The blood is typically anticoagulated with heparin or ACD solution. After 15 min incubation at room temperature, the reaction is terminated by the addition of a small amount of ascorbic acid, which converts unreacted (CrO4)
2-to Cr3+ (chromous ion). This prevents the continuation of cell labeling after the material is injected into the patient. The Cr-51 binds avidly and irreversibly to the b-globin chains on the hemoglobin molecule, forming labeled cells with excellent in vivo stability. The labeling of RBC's with Tc-99m also involves the binding of the radioisotope to the b-globin chains on the hemoglobin molecule. Cells may be labeled in vivo or in vitro by a variety of different procedures, described in detail in the chapter on Cell Labeling.
I-123 mIBG and I-131 mIBG can be easily prepared by heating a mixture containing 0.5-2.0 mg of mIBG hemisulfate, 12 mg of ammonium sulfate, and the appropriate radioiodide. After two 45-60 min heating cycles in the dry state, radioiodinated mIBG is formed in high yield and with an average radiochemical purity in excess of 97%. The I-131 compound is currently
commercially available; the I-123 compound must be manufactured on-site under a Physician sponsored Investigational New Drug Exemption.
Radiopharmaceuticals may also be produced by biological synthesis. Before the chemical synthesis of Vitamin B12 was elucidated, 57Co labeled vitamin B12 was made by placing 57CoCl2 in a broth containing streptomyces griseus. This resulted in the biological production of 57Co labeled vitamin B12. By a similar process, yeast growing in a medium high in
Se and low in sulfur produced 75Se selenomethionine, a radiopharmaceutical formerly used for
pancreatic imaging.

d. Quality Control of Generators and Radiopharmaceuticals


Required QC Testing of a Mo/Tc Generator: NRC State
Mo Breakthrough
Every Elution
<0.15 uCi Mo/mCi Tc at tadministration
Not Required
<10 ppm of Al3+; may be expressed as ug/ml
Required QC Testing of a Mo/Tc Generator: Agreement State
Mo Breakthrough
Every Elution
<0.15 uCi Mo/mCi Tc at tadministration
Not Required
<10 ppm of Al3+; may be expressed as ug/ml
Optional QC Testing of a Mo/Tc Generator: Agreement State
Every Elution
< 2% (Reasonable limit; presently no limit)

1. Mo-99 Breakthrough: Mo-99 is assayed directly in the special lead pig supplied by the manufacturer of your
dose calibrator. Tc-99m is then assayed directly in the plastic sleeve in your dose calibrator. Activity (uCi) of Mo-99 is divided by activity (mCi) of Tc-99m to obtain a ratio. If this ratio is <0.15 μCi Mo-99 per mCi of Tc-99m at time of injection, the generator eluate has passed the Mo-99 Breakthrough Test. As a rule of thumb, if the ratio is <0.038 at time of elution, the material will be suitable for injection for at least 12 hours.
2. Aluminum Ion Breakthrough: Al3+ ion is measured colorimetrically. A drop of the eluate is placed on one
end of a special test paper; a drop of a standard solution of Al3+, concentration 10 ppm, is placed on the other
end of the test strip. If the color at the center of the drop of eluate is less red than that of the standard solution, the eluate has passed the Aluminum Ion Breakthrough Test. Units may be expressed as ug/ml.
Radiochemical Impurities in Tc-99m Radiopharmaceuticals


High Radiation
Poor Image Quality


Poor Image Quality
Altered Radiation Dose

Image Quality
Dose Calibrator or Multi-Channel Analyzer
Thin Layer Chromatography
Free Tc Chemical form:
Pertechnetate, TcO4
Hydrolyzed Reduced Tc
Chemical form is probably TcO(OH)2.H2O, a hydrated Tc-oxide
99mTc MAG3
Possible impurity is 99mTc tartrate
Stereochemical impurities
Silica Gel/0.9% saline
Hydrolyzed Reduced Tc
Paper/acetone or paper/MEK
Free Tc (Pertechnetate)

Silica Gel/ Saline

HR Tc in bottom half; all other species in top half
Paper/acetone or paper /MEK
Free Tc in top half; all other species in bottom half.
ITLC-SA/20% saline
HR Tc in bottom half; all other species in top half
Free Tc in top half; all other species in bottom half


ITLC-SA/20% saline
Free Tc (Pertechnetate)
Hydrolyzed Reduced Tc

● Free Tc (Chemical form is pertechnetate, TcO4-)
● Hydrolyzed Reduced Tc (Chemical form probably TcO(OH)2.H2O, a hydrated Tc-oxide.
● For Tc-99m MAG3, formation of Tc-99m tartrate is possible
● For Tc-99m HMPAO, there are also stereochemical impurities.

e. Quality Control of Dose Calibrators used to measure patient doses

at installation, then annually thereafter
at installation, then daily thereafter
at installation, then quarterly thereafter
at Installation; after repair or moving instrument

ACCURACY TEST: This test is designed to show that the calibrator is giving correct readings throughout the entire energy scale that we are likely to encounter. Low, medium, and high energy standards (usually Co-57, Ba-133 or Cs-137, and Co-60, respectively), are measured in the dose calibrator using appropriate settings. Standard and measured values are

CONSTANCY TEST: This test measures precision and is designed to show that, using a longlived source, usually 30 y Cs-137, reproducible readings are obtained day after day on all the various isotope settings we are likely to use. The long-lived source is placed in the dose calibrator. Activity is then measured on the Cs-137 setting and all other routinely used settings
on a daily basis. Values are recorded in the appropriate logbook and are compared with recent values to determine if instrument is maintaining constancy on a day-to-day basis.

LINEARITY TEST: This test is designed to prove that the dose calibrator readout is linear for sources varying from the μCi range through the mCi range. A high activity Tc-99m source (50-300 mCi) is measured at T0 and at predetermined time intervals up to 48 hours. Expected and actual measurements are compared (and may be analyzed graphically) to determine if the instrument is linear throughout the activity range we are likely to encounter.

GEOMETRY TEST: This test is designed to show that correct readings can be obtained regardless of the sample size or geometry. One ml of Tc-99m in a 10 ml syringe (activity 25 mCi) is measured in the dose calibrator and the value obtained is recorded. The activity is then diluted with water to 2 ml, 3 ml, 5 ml, and 10 ml. At each of these points a reading is taken and
the value recorded. Data are then evaluated to determine the effect of sample geometry on the dose calibrator reading. If instrument is geometry-dependent, it may be necessary to routinely correct readings obtained when using calibrator.

1. Deviation from standard or expected values must be within +/- 10%.
2. If Deviation >10%, then obligation is to record value, note repair or recalibration of instrument, retest, and record new values. Every dose must be mathematically corrected until the instrument is repaired. There is NO LONGER a reporting requirement.

Determines ability of a sodium iodide (thallium) or solid state crystal to resolve gamma ray energies. Measures only resolution, not sensitivity.
For NaI(Tl), Normal Value is 6-10%
for Ge(Li) Crystal, typical value is <1% across entire energy spectrum.






April 8, 2010