August 1996

Materials:

• Potassium hydroxide pellets (224.4g KOH per 1000 ml H₂O)
• 2% Tween-80™ (10 ml Tween-80 per 1000 ml potassium hydroxide)
• 100% isopropanol alcohol
• K₂HPO₄ powder (29.9 g K₂HPO₄ for 1000 ml buffer liquid)
• KH₂PO₄ powder (5.88 g KH₂PO₄ for 1000 ml buffer liquid)
• Organic solvent

Per Sample:

• 15 ml digestion solution
• 1.5 ml isopropanol
• 15 ml phosphate buffer
• organic solvent

Preparation:

• Digestion solution: Dissolve 224.44 g KOH in 1000 ml H₂O by constant stirring. Add 10 ml 2% Tween-80 when the liquid has cleared (stable for 24 hours).
• Phosphate buffer: Dissolve 5.88 g KH₂PO₄ in 200 ml and 29.9 g K₂HPO₄ in 800 ml H₂O. Mix two solutions together.

Work Routine

1. Place each tissue sample in a separate first stage of the filtering device.
2. Place first stage in KOH bath or separate containers of KOH.
3. Layer 1.5 ml of isopropanol over top of each sample and seal first stage (Parafilm, tinfoil, and plastic wrap work well).
4. Heat samples to 60° C until digested (2 - 4 hr).
5. Suction digestate through first filter stage by negative suction and rinse with 15 ml of buffer.
6. Dry sample by centrifugation at 4000 rpm for 5 min.
7. Put first stage of filtering device into the second stage and attach third stage to bottom.
8. Add one-half volume of organic solvent to the first stage, vortex for 20 seconds and let rest for 5 min.
9. Add remaining half of organic solvent to the first stage and vortex for 20 seconds.
10. Centrifuge organic solvent to third stage of filtering device by centrifugation (4000 rpm for 5 min.)
11. Measure fluorescence in organic solvent.

Ten samples were digested and filtered per intructions. All fluid passing through the 10 filters was collected in a clean filtration flask. The collected fluid was filtered through a single Poretics® polycarbonate filter with 10 µm pores. The filter was placed in a sample tube, 1 ml of solvent added, and the fluorescent intensity in the solvent determined in our LS50B. The fluorescent signals for Blue-Green and Red were 54.0 and 59.5, respectively. These fluorescent signals corresponds to approximately 650 microspheres in our LS50B. The fluorescent dye was extracted from each filter device per the protocol and the dye concentrations measured with our LS50B.

The remaining 10 samples were digested using KOH and filtered with Poretic polycarbonate 10 µm filters. The fluorescent dye was extracted from each filter and the concentrations measured. All fluid passing through the 10 filters was collected in a clean filtration flask. The collected fluid was filtered through a single Poretics® polycaronate filter with 10 µm pores. The filter was placed in a sample tube, 1 ml of solvent added, and the fluorescent intensity in the solvent determined in our LS50B. The fluorescent signals for Blue-Green and Red were 3.3 and 0.3, respectively. These fluorescent signals are equal to those from blank solvent and corresponds to approximately 0 microspheres in our LS50B. The fluorescent dye was extracted from each filter and the dye concentrations measured with our LS50B.

Results: Fluorescent microspheres were not completely recovered by the Perkin Elmer filter devices. Relative to the radioactive counts, it appears as though the fluorescent signals from two of the Perkin-Elmer filtering devices were too low. See graph below.