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4.8: Liquid Scintillation Counting

  • Page ID
    122136
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    A competitive binding immunoassay for the measurement of cyclosporine is introduced in which tritiated (3H) cyclosporine is used as the labeled ligand. The reaction mixture is incubated with charcoal to remove free ligand. The supernatant, containing ligand bound to antibody, is solubilized to allow mixture of the sample with the scintillation fluid and counting of the bound radioactivity. The medical technologist prepares the solutions for counting and counts them immediately. However, over the weekend, some of the results of the counting experiment are lost. As the samples were not thrown away, however, the medical technologist recounts them the following Monday. On the second counting, significantly lower counts per vial are obtained. The technologist’s concern causes her to repeat the entire experiment including a second counting 48 hours after the first. The same pattern of lower cpm on the second counting is obtained, and the technologist is puzzled.

    QUESTIONS

    1. Why is there a difference in the results obtained 48 hours apart?
    2. Which set of counts, initial or 48 hour results, should be used?

    Questions to Consider

    1. Are there any mechanisms other than radioactive decay processes that can give rise to photons in a liquid scintillation mixture?
    2. What are some important characteristics of chemiluminescence reactions?
    3. How can one detect the presence of chemiluminescence interference during a counting procedure?
    4. How can one use the characteristics of this process noted in #2 to minimize chemiluminescence interference?
    5. Will chemiluminescence reactions affect both standards and patient samples equally?
    Answer
    1. The difference in the results obtained 48 hours apart was caused by the presence of chemiluminescence reactions. The results were lower at the second counting because of the decay of the chemiluminescence reactions as discussed in answer #4 below.
    2. The results from the 48 hour counting are probably the more accurate since the chemiluminescence interference has been minimized. Only if the standards are in the same matrix as the samples and all are counted within a very short time frame, could the initial results be used.

    Answers to Questions to Consider

    1. The non-radioactive process that can act as a source of photons in liquid scintillation counting is chemiluminescence, which is caused by chemical reactions that give rise to photons (p. 198). In liquid scintillation counting, chemiluminescence reactions can occur between the sample and the solubilizer material, between the sample and the solute (“fluor”), or between different components of the sample itself.
    2. Like most chemical reactions, chemiluminescence reactions are time and temperature dependent. After a period of time, the chemiluminescence reaction becomes complete (p. 198) and the production of photons rapidly decreases. Chemiluminescence reactions occur more slowly at lower temperatures, although at low temperatures it takes longer for the chemical reactions to come to completion and cease making photons.
    3. One can detect the presence of chemiluminescence reactions exactly as the medical technologist discovered it; that is, by repeated counting of the samples with time. If the counts per minute significantly decrease with time, the presence of decaying chemiluminescence reactions can be assumed.
    4. By repeated counting of the samples and plotting the decay curve, one could determine how long one needs to wait before the curve flattens out, indicating a cessation of the chemiluminescence reactions. This process can be hastened by preincubating the samples at room temperature in the dark to allow the chemiluminescence reactions to be completed faster. The samples should then be transferred to a cold scintillation counter, cooled, and then counted. This process allows for rapid completion of the chemiluminescence reaction and the inhibition of any remaining reactions by the use of cold temperature counting.
    5. The chemiluminescence reactions are very dependent upon the sample matrix. If the standards and the samples are not in the same matrix, they will be affected to different extents.

    This page titled 4.8: Liquid Scintillation Counting is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Lawrence Kaplan & Amadeo Pesce.

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