Laboratory 3: Kit Preparation and Chromatography PART 1: KIT PREPARATION Introduction In nuclear medicine, radionuclides are rarely used in their simplest chemical form. Instead they are incorporated into a variety of chemical compounds that take advantage of important biochemical, physiologic, or metabolic properties. A chemical compound tagged with a radionuclide and prepared in a form suitable for human use is known as a radiopharmaceutical. The authority to regulate and approve for human use the pharmaceutical component lies with the United States Food and Drug Administration (FDA). The United States Nuclear Regulatory Commission regulates the radioactive component. Most radiopharmaceuticals are used to obtain diagnostic information rather than to produce therapeutic results, although their use in therapy is increasing. For diagnostic uses, they are administered in tracer quantities in a single dose and produce no pharmacologic effects. Design Considerations for a Radiopharmaceutical Because a radiopharmaceutical consists of a radionuclide and a biochemical, two considerations apply in designing or developing a radiopharmaceutical, one relating to the radionuclide and the other relating to the biochemical. Selection of Radionuclide The choice of a radionuclide for imaging purposes is chiefly dictated by the necessity of minimizing the radiation dose to the patient and the detection characteristics of nuclear medicine instrumentation. To minimize the radiation dose to the patient, a radionuclide should have as short a half-life as is compatible with the biological phenomena under study. For example, a radionuclide with a one hour half-life cannot be used in studies of physiological or metabolic functions that span days. A radionuclide should preferably emit a monochromatic (single energy) gamma ray with energy between 100 and 300 kev. The lower limit of the desired energy range of gamma rays is arrived at from the consideration of attenuation of gamma rays in the patient. To be effective, and contrary to x-ray imaging, the photon must have a high probability of escaping the patient without interaction in order to be a useful imaging photon. This probability diminishes markedly below 100 kev. The upper limit of the desired energy of the gamma ray is the consequence of the detection characteristics of the scintillation camera (Module 2.2) routinely used to image diagnostic radiopharmaceuticals. The NaI crystal must be thin (about 3/8 inches thick) to provide sufficient image sharpness and the collimator must have many holes separated by septa thin enough to yield good spatial resolution. If too energetic, the photons penetrate thin septa and ruin the image quality. Photon energies above 300 kev do not render quality images. (Do not confuse this with Lab#3 Kit Prep and Chromatography (Last Update: Sept 2017) Page 1 of 9
positron emission tomography imaging where no physical collimator is used. Imaging apparati for 511 kev annihilation radiation are very different from conventional gamma camera imaging.) In addition, a radionuclide should be available easily, economically and in an uncontaminated form. Technetium-99m with its 6 hour half-life and 140 kev gamma emission along with its easy economical availability from a generator comes very close to ideally fulfilling the above requirements. This accounts for its wide use in nuclear medicine. Selection of a Chemical Besides being nontoxic in the desired amounts, the choice of the biochemical or pharmaceutical substance in the radiopharmaceutical is dictated by the requirement that it be distributed or localized in the desired organ or biological compartment and that the uptake by that organ in a normal condition differs substantially from the uptake in a pathological condition. This is generally expressed as the target-to-non-target ratio. The higher the ratio, the higher the contrast in the image and easier it becomes to visualize a disease. To help in the selection of a suitable biochemical, a wealth of information has been acquired in the field of pharmacology. A number of biochemical variants determine or affect the distribution and localization of drugs in tissues. Three important determinants in this regard are route of administration, blood flow to the organ or tissues and extraction by the tissues. Radiopharmaceuticals, with few exceptions, are administered intravenously, primarily because this is the fastest way to introduce a drug into the circulatory system of the body. Blood flow or perfusion essentially determines the fraction of the administered dose that will be delivered to a particular organ or tissue during the first transit. Extraction of a drug or chemical from the circulation and localization in tissue may occur in a number of ways. Extraction of Drug or Chemical Localization Mechanism Example Active transport Thyroid uptake and scanning with iodine Compartmental localization Simple exchange or Blood pool scanning with human serum albumin Bone scanning with labeled phosphates Lab#3 Kit Prep and Chromatography (Last Update: Sept 2017) Page 2 of 9
diffusion Phagocytosis Capillary blockage Cell sequestration Receptor binding Liver, spleen and bone marrow scanning with colloids Lung scanning with macroaggregates Spleen scanning with damaged RBCs Tumor imaging with somatosin receptor binding indium pentetreotide Labeling Radiopharmaceuticals with Technetium-99m Because the half life of Tc-99m is short (6 h), most labeling has to performed locally (within hours of transport time). The labeling of most chemicals by Tc-99m is achieved by first reducing pertechnetate to ionic technetium (Tc4+) and then complexing it with a desired chemical. The common agent used for reducing purposes is stannous chloride ( SnCl2). To label a particular chemical, what typically is done is to introduce a known activity of sterile and pyrogen-free NaTcO4 - into a kit vial and, voila, the labeled compound is ready to use within a few minutes. Colloquially, the street language is shoot and shake ; shoot the activity into the vial and shake it to mix the kit. These commercial kits are sterile and pyrogen-free vials in which all the desired chemicals are premixed and held together in a lyophilized state under an inert gas atmosphere. Figure 2.4-01 Lab#3 Kit Prep and Chromatography (Last Update: Sept 2017) Page 3 of 9
Technetium Misbehaving Unbalanced equations of technetium reduction and tagging are shown. The first equation shows the normal case, where pertechnetate (7+) is reduced (3+, 4+, 5+) by stannous chloride to enable tagging a pharmaceutical. The second equation shows the case of air contamination. This generates free pertechnetate, which accumulates in the stomach and thyroid and salivary glands. The third equation shows the case of water contamination, generating technetium dioxide ("hydrolyzed technetium") and stannous hydroxide, a colloid. Technetium dioxide will accumulate in the liver. Paper chromatography with acetone will show free pertechnetate advancing with the solvent front. Paper chromatography with saline as the solvent will show technetium dioxide at the origin and water soluble tracers advancing with the solvent front. Radiopharmaceutical Quality Control Labeling efficiency is defined as the percent of total radioactivity present in the kit that is tagged to the appropriate molecule or compound. The remainder of radioactivity not tagged is present as a radiochemical impurity. In kits that use SnCl2 as the reducing agent, radiochemical impurities are, in general, of two forms: free pertechnetate (which is not reduced) and reduced or hydrolyzed technetium (which was reduced but did not tag to the compound of interest). A common method for the detection of radiochemical impurities is thin-layer or paper chromatography. In paper chromatography, microliter amounts of the prepared kit are applied to a 3-cm rectangular paper strip at an origin spot. The end of the strip is dipped in the solvent so that the origin spot is not immersed. The solvent ascends the strip by capillary action, separating each radiochemical component into different sections on the strip. Assaying the section of the strip with unbound 99m-Tc separately from assaying the portion of the strip with the bound 99m-Tc allows for a rough assessment of the percentage of bound 99m-Tc in the kit solution. Lab#3 Kit Prep and Chromatography (Last Update: Sept 2017) Page 4 of 9
Figure 2.4-02 PART 2: CHROMATOGRAPHY INTRODUCTION The Tec-Control kit consists of a complete miniaturized chromatography system for Technetium-99m radiopharmaceuticals. Chromatographic procedures are used to determine the labeling efficiencies of most Technetium-99m labeled kits. The chromatography paper to perform rapid and concise separations of hydrolyzed Technetium-99m (partially reduced) and free Technetium-99m Pertechnetate (unbound oxidized). GENERAL INFORMATION Radiopharmaceutical Spotting These tests require spotting approximately 10 microliters of the radiopharmaceutical sample onto the chromatography strip. This is easily accomplished by using a 26 needle and syringe. One drop equals a volume of 0.01 cc (l0 ul) (one microliter). Development Aids Each test strip is ruled on one side with indelible pencil lines indicating the spotting location and the termination point in the strip development process. For user convenience the back of each test strip is marked with a soluble dye that will migrate with the solvent front. The user can easily see the solvent front via the movement of the dye. For reference there is a color-coded tape at the top of each strip. Data Analysis The object of those tests is to determine the percentage of free pertechnetate, the percentage of hydrolyzed reduced Tc-99m and the percentage of labeled radiopharmaceutical. Lab#3 Kit Prep and Chromatography (Last Update: Sept 2017) Page 5 of 9
GENERAL COUNTING PROCEDURES Cut the developed test strip into two sections at the middle pencil line. Using a gamma scintillation well counter peaked for Tc-99m, individually count each strip section in a test tube for a specific period of time (i.e., 30 seconds). Count background and calculate the net counts by subtracting the background counts from the number of counts previously registered when counting the individual strip sections. Depending on count rate, the dead time of the detector may give erroneous results. Solvent Front 4 Tc-99m Chelate TcO4-2 TcO4 - Cut Mark 3 Tc-99m Reduced Hydro 1 Tc-99m Chelate Tc-99m RH Origin Saline 0.9% Acetone DETAILED TEST PROCEDURES For determining free pertechnetate in Tc-99m labeled DTPA, Glucoheptonate, Diphosphonate, Pyrophosphate, Polyphosphate and MDP. 1. Prepare one developing vial by adding 1 cc to the red acetone labeled solvent to the red label vial. 2. Using a red chromatography strip, spot approximately 10 ul of the test sample onto the bottom pencil mark line of the test strip. 3. Immediately place the test strip into the red vial containing acetone and develop until the solvent front migrates to top pencil line. 5. Remove strip from the vial and allow to dry. 6. Cut strip at central pencil line, producing sections 1 and 2. 7. Count each section for activity (per unit time) using a gamma counter (a dose calibrator can be substituted) and subtract backgrounds. Lab#3 Kit Prep and Chromatography (Last Update: Sept 2017) Page 6 of 9
% free pertechnetate = = net cts section 2 (net cts sec 1)+(net cts sec 2) x 100 For determining hydrolyzed reduced Tc-99m in Tc-99m labeled DTPA, Glucoheptonate, Diphosphonate, Pyrophosphate, Polyphosphate and MDP. 8. In a clean, black labeled vial place approximately 1cc of the black labeled (distilled water) solvent 9. Select one strip of the black chromatography paper and spot approximately 10 microliters of the test compound onto the bottom pencil line. 10. Immediately place the test strip into the black labeled vial containing distilled water and develop until the solvent front migrates to top pencil line. 11. Remove the strip from the vial and allow to dry. 12. Cut strip at center pencil line into sections 3 and 4. 13. Count both sections for activity (per unit time) using gamma counter and subtract backgrounds. % hydrolyzed reduced Tc-99m = = net cts section 3 (net cts sec 3)+(net cts sec 4) x 100 Lab#3 Kit Prep and Chromatography (Last Update: Sept 2017) Page 7 of 9
For determining percent labeling in Tc-99m labeled DTPA, Glucoheptonate, Diphosphonate, Pyrophosphate, Polyphosphate and MDP. % labeling radiopharmaceutical = 100 % = % free pertechnetate - % hydrolyzed reduced Tc-99m Lab#3 Kit Prep and Chromatography (Last Update: Sept 2017) Page 8 of 9
Resident Name (print): Date: / / Signature of individual supervising lab: Name of preparation kit used: Under the Dosages and Administration section what is the average adult suggested dose in millicuries? From the Radiation Dosimetry section what is the organ with the highest radiation dose? Why is stannous fluoride (SnF2) in the vial? Why is nitrogen put in vial? In the Direction for Preparation what is the acceptable volume (ml) range for Sodium Pertechnetate? What common technique used to facilitate removing liquids from a vial will change the chemical state of Sodium Pertechnetate? What was the stated activity (mci), volume and calibration time for the Sodium Pertechnetate? Calibration date and time Activity Volume Activity per ml Using the Physical Decay Chart what is the current activity? Assay Sodium Pertechnetate in dosecalibrator: mci How much volume of the above Sodium Pertechnetate solution is required to reconstitute the vial to make the kit for one adult dose? ml How much volume of the above Sodium Pertechnetate solution is required to reconstitute the vial to make the kit for one child who weighs 35 kg? ml In the Direction for Preparation what is the maximum time the preparation is good? hr Turn in a copy of this page only into the Physics and Education Offices for credit. Lab#3 Kit Prep and Chromatography (Last Update: Sept 2017) Page 9 of 9