Labeling Process of 111In oxine

Labeling WBCs

Given the Diagram Above Follow the Procedure Below

Approximately 50 mL of whole blood is extracted from the patient. The syringe must contain an anticoagulant (1000 U of heparin or 7mL of ACD solution)
1. ACD appears to be a better anticoagulant because of improved labeling efficiency
2. Some health care facilities will collect additional whole blood to increase the amount of white cells to be labeled
3. If an additional 25 mL is taken, refer to step 9
Once the blood is collected it should be stored in an upright position. This allows the red cells and plasma to separate. This process usually takes about an hour
Comment - It is very important to label the syringe and patient. (Labels are provided by the central pharmacy). Correct labeling will prevent misadministering the labeled blood to the wrong patient
By the time the whole blood arrives at the radiopharmacy the plasma and red cells are separated

Optional - 10 mL of hetastarch can be added to the whole blood and mixed. This enhances the sedimentation process. It adds about 45 minutes to the labeling procedure. FYI - Patient with infection tend to have higher sedimentation rates

The leukocyte-rich plasma (LRP) is then placed in a test tube and centrifuged at 450g for 5 minutes
This causes a formation of a WBC button (on the bottom) which is the collection of leukocyte. The solution in the test tube is now considered leukocyte-poor plasma (LPP)
All but, 1.0 to 0.5 mL of the LPP is removed and stored for later use
Optional step - If you've collected an additional 25 mL of whole blood you can extract the plasma and substitute the LPP with the plasma

Optional steps 9 - 12 or go to step 13

Application of HBSS (hypertonic saline solution) is added to the to the WBC button (if HBSS is not added go to step 13)
1. This causes lysis of the the remaining RBCs found within the WBC button
2. Some institutions do not add HBSS because it may damage the white cells
3. When HBSS is added the WBC button is re-suspended with this solution, mixed, and then centrifuged again ( 450g for 5 minutes)
Solution is then removed (button remains) and the WBCs are suspended and washed with a saline (5 mL)
Centrifuged at 450g for 5 minutes and the solution is removed
One mL of LPP is added, continue to step 14

Addition the radiotracer
1. Prepare 30 mCi of ^99mTc-HMPAO in a 5 mL solution
2. Or slowly add 1.0 mCi of ¹¹¹In-oxine with 0.5 mL of saline
3. After adding the radiotracer incubate for 20 to 30 minutes mixing the compound gently every 5 minutes
4. Optional - the radiotracer and WBC may incubated at 37^oC
Mix 15 mL of LPP to solution and centrifuge at 450g for 5 minutes
Remove the supernatant for assay below [A]
Add 15 mL of LPP to the button and re suspend the labeled WBCs [B]
Spinning the suspended cells can be repeated to remove additional unbound radiotracer (Step 17)
The dose is placed into a syringe and is ready for patient injection

QC of the Labeled WBCs

The percent tag can be determined by measuring the activity of the labeled button and the supernatant in step 16 and 17. This value should be between 50 to +90%
Place a small amount of the labeled radiopharmaceutical onto a hemocytometer and add trypan blue dye. Using a microscope see if any WBCs turn blue. Those that do are dead and if the amount exceeds 10% then the labeled cells should not be injected into the patient
Determine there is clumping of the WBCs. This will be observed with a microscope
Final comment - Ringers Citrate Dextrose can be used instead of saline. The theory is that the white cells need to be nourished while being incubated.