Radiopharmaceuticals

	8: 111In-INDIUM LABELLING OF WHITE BLOOD CELLS (with 111In-tropolonate and includes lysis of red blood cells) (Merseyside And Cheshire Radiopharmacy Services)
	Materials Required
	6 x 10 ml syringes	3 x 21G (Green) needle
	3 x 5 ml syringes	10 x 5 inch filling tubes (Kwills)
	1 x 10 ml Vial Hespan	3 x 30 ml Sterile Universal tubes
	Lead Shield pots	4 x 60 ml internally sterile tubes
	1 x 5 ml 0.1% sodium chloride	1 x 2.5 ml 2.5% sodium chloride
	1 x butterfly 19g (non-intermittent)
	Tropolone and 111In-indium chloride solution 2 x 1 ml syringe + 2 x 25G (Orange) needle
	1 x micropipette and sterile end piece
	Preparation Instructions
	Take 50 ml of blood anticoagulated with 7.5 ml of ACD solution .
1.	Set out a 60 ml internally sterile tube in a rack. Draw up 8 ml of Hespan from the 10 ml vial using a 10 ml syringe and green needle. Remove the needle from the syringe containing the blood and attach a filling tube. Gently invert syringe several times to mix.
2.	Carefully push the syringe piston so that the blood moves to the end of the filling tube, and then carefully add approx. 20 ml to the tube. Stand the syringe containing blood in the rack and remove the kwill, slowly add the 8 ml Hespan, already prepared, avoiding the production of bubbles. Attach the butterfly to the end of the syringe and invert to mix thoroughly.
3.	Set aside the syringe containing blood and hespan to sediment for up to 1 hour.
4.	Take the 20 ml blood in the tube and centrifuge at 3000rpm for 15 minutes.
5.	Remove the supernatant from 5. using 10 ml syringe and Kwill. Put into another Universal tube and label as "Platelet Free Plasma" (PFP).
6.	From 4. remove the supernatant and transfer to a 60 ml tube via the butterfly (do not invert the syringe, remove by pressing the plunger down onto the benchtop). Centrifuge at 1000 rpm for 5 minutes.
7.	Carefully remove the supernatant from 7. after centrifugation, using a 10 ml syringe and Kwill. Remove as much plasma as possible, but TAKE CARE NOT TO DISTURB THE CELL PELLET (if required transfer the plasma to another 30 ml tube and centrifuge at 3000 rpm for 15 minutes, then treat as in 6. above).
8.	Prepare two 5 ml syringes, one containing 5 ml 0.1% sodium chloride, the other containing 2.5 ml 2.5% sodium chloride with 2.5 ml platelet free plasma. Add the 0.1% sodium chloride to the cell pellet and swirl gently for 10-20 seconds, then add the contents of the other syringe (plasma and 2.5% sodium chloride) and swirl gently again. Centrifuge at 1000 rpm for 5 minutes and treat as above in 8.
9.	Resuspend the cell pellet with 0.3 ml PFP, add 50 microlitres of tropolone solution, followed in quick succession by approximately 20 MBq of 111 In-indium chloride. Incubate for 5 minutes.
10.	After the incubation period add 5 ml PFP, mix, then centrifuge at 1000 rpm for 5 minutes.
	Using a 10 ml syringe and Kwill, remove the supernatant and keep for calculating the labelling efficiency.
11.	Resuspend the cell pellet in 5 ml PFP, mix gently then withdraw the suspension into a 5 ml syringe and cap with a blind hub.
12.	Measure the activity in the syringe and calculate the labelling efficiency.
13.	Label the syringe, place in lead lined tin (shelf life 2 hours). Activity used is usually 20 MBq.
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