Stable isotope-labeled microspheres

Technical information

The experiment is unchanged. Injection. Timed arterial reference withdrawal. Dissection. What differs is the assay at the end: the spheres are measured by neutron activation instead of by counting the radioactivity they carried through the subject, or by digesting the tissue to recover a dye.

Tissue of interest is dissected, weighed, and sealed into a vial. Where absolute blood flow is desired, the blood reference sample is also submitted. Microsphere content in both is then measured by neutron activation.

Results are available within ten working days of receipt.

Advantages

• No radioactivity in the laboratory. The label is a stable isotope. Nothing radioactive is handled during the work, and the material has no decay clock governing when the experiment must run.
• No hazardous reagents. There is no digestion step, so the aggressive chemistry that optical methods require is not part of the workflow.
• Non-destructive assay. The sample is measured sealed and intact, with nothing recovered, extracted or filtered out of it.
• Simultaneous measurement of several labels. Labels co-injected at different points in an experiment are separated from one another in a single measurement of a single sample, so each tissue segment yields a value per timepoint with no further dissection.
• Sensitivity and specificity. The measurement is elemental, so the signal is specific to the label and is not affected by the tissue around it.

The product

Microspheres are supplied in sealed vials, each carrying a single stable isotope label. Two vial sizes are offered, 10 mL and 20 mL, both at roughly 2.5 million spheres per mL, and two diameters: 15 µm, the standard size for blood flow work, and 10 µm. The suspending medium is normal saline with Tween 80 and thimerosal at 0.01%.

Ten labels are available at 15 µm and seven at 10 µm, with additional labels on request. The 10 µm spheres are supplied at 6 million per mL, and because smaller spheres carry a greater risk of incomplete trapping, the protocol recommends increasing the injected dose by 20% relative to 15 µm.

Required with the assay

Two items are part of the method rather than optional extras. The sample vials are polypropylene, cleaned of trace contamination and calibrated against the irradiation and counting equipment. The assay is run in these vials specifically, and will not accept substitutes. A sodium-free buffer replaces saline or PBS for washing samples and for delivering histochemical stains, so that sodium background does not compete with the label and erode the sensitivity of the measurement.

What is reported

The assay reports the total disintegrations per minute measured in each sample, for each microsphere label present in it. Given the reference withdrawal and the sample masses, absolute blood flow in mL/min/g can be calculated for each sample.