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Red blood celllabeling (1)
1. There are multiple methods for labeling red blood cells with Tc-99m, including in vivo, in vitro, and modified techniques. 2. GI bleeding studies are best done with Tc-99m RBCs prepared using the packed cell or UltraTag kit techniques to achieve high labeling efficiency. 3. White blood cells can be easily labeled with either Tc-99m or In-111 by isolating WBCs and incubating them with a lipophilic intermediate. 4. Red blood cell labeling with Cr-51 is very simple, not requiring isolation of RBCs, just incubation of blood with Cr-51.
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Red blood celllabeling (1)
- 1. Cell Labeling Stephen M.Karesh, Ph.D.
- 2. Cell Labeling topicsto be covered Red Cells: Tc-99m and Cr-51 White Cells: Tc-99m and In-111 Platelets: In-111
- 3. Uses of Tc-99mRed Cells FirstPass Cardiac study Gated Blood Pool study G.I. Bleed Study Localization of hepatic hemangioma
- 4. Principles of Tc-99mRBC Labeling Stannous ion, a powerful reducing agent, is added to red cells Sn2+ is permitted to diffuse into RBCs Tc-99m pertechnetate (TcO4-) is then added to pre-tinned RBCs Tc-99m diffuses into the RBCs, where it is reduced by the Sn2+ ion to Tc4+, which binds to the -globin chains of hemoglobin.
- 5. Principles of Tc-99mRBC Labeling Reduced Tc-99m (Tc4+)cannot bind to the heme portion of the hemoglobin molecule since the Fe incorporated into the heme can not be displaced under physiological conditions. Tc4+ therefore binds only to the globin portion of the molecule, specifically the -globin chains.
- 6. NOW HEAR THIS!!! Every container which holds a blood sample Full Name must be labeled with the Full Name Full Name patient’s FULL NAME Syringes Full Name Test tubes Vials
- 7. Methods of Preparationof Tc-99m RBCs • In vivo/in vivo • In vivo/in vitro • In vitro/in vitro
- 8. Methods of Preparationof Tc-99m RBCs In vivo / in vivo In vivo / in vitro In vitro / in vitro Refers to Refers to tinning of the radiolabeling cells, i.e., of the cells, injection of the i.e., injection of stannous the Tc-99m pyrophosphate pertechnetate
- 9. In vivo/in vivomethod 1. 1 mg of Sn2+ in the form of pyrophosphate (“cold PYP”) is given IV 2. 20 min wait to permit mixing of the Sn PYP in body and diffusion of Sn2+ into RBC. 3. IV injection of 25 mCi 99mTc pertechnetate 4. 10 min waiting period to permit diffusion of pertechnetate into RBC's where radio- labeling takes place.
- 10. In Vivo/In Vivo: Advantages/disadvantages Advantages: quick, simple, inexpensive Disadvantage: lowest labeling efficiency of all commonly used procedures, but perfectly acceptable for routine work, e.g., MUGAs.
- 11. In vivo/in vitromethod (in vivtro method) 1. 1 mg of Sn2+ in the form of pyrophosphate (“cold PYP”) is given IV 2. 20 min wait to permit mixing of the Sn PYP in body and diffusion of Sn2+ into RBC. 3. Withdrawal of 5-10 ml of blood anti- coagulated with heparin or ACD solution into a syringe containing 25 mCi Tc-99m pertechnetate
- 12. In vivo/in vitromethod (in vivtro method) 4. 10 min waiting period to permit diffusion of the pertechnetate into RBCs and to permit labeling to reach equilibrium. 5. Reinjection of labeled cells into patient. 6. Expected labeling efficiency: ~92%
- 13. In vivo/in vitro: Advantages/disadvantages Advantages: quick, simple, inexpensive method; achieves higher labeling efficiency than in vivo/in vivo technique since incubation with RBC is extracorporeal. Not optimal for GI Bleeding Studies. Disadvantages: takes extra tech time; potential for breaking sterility
- 14. Modified In vivo/invitro method 1. 1 mg of Sn2+ in the form of pyrophosphate (“cold PYP”) is given IV. 2. 20 min wait to permit mixing of the Sn PYP in body and diffusion of Sn2+ into RBC. 3. Withdrawal of 5-10 ml of anti-coagulated blood (heparin, ACD) into vacutainer. 4. Centrifuge the vacutainer in inverted position for 5 min at 3000 rpm.
- 15. Modified In vivo/invitro method 5. Removal of 1-2 ml of packed cells through a 20 ga or larger needle. 6. Aseptic addition of these tinned, packed cells to a sterile vial containing 35 mCi of Tc-99m pertechnetate. 7. 10 min incubation to permit labeling reaction to go to completion. Expected labeling efficiency: 98-100% 8. Reinjection of Tc RBC
- 16. Modified In vivo/invitro method Advantages/disadvantages Advantages: quick, simple, inexpensive method; achieves highest labeling efficiency of all procedures since reaction of Tc with plasma proteins has been eliminated. Ideally suited for GI Bleeding Studies; produces excellent delayed images. Disadvantage: takes extra tech time; requires clinical centrifuge; potential for breaking sterility.
- 17. In vitro/in vitromethod Ultratag Kits 1. To vial containing Sn2+ compound, add 3- 5 ml of anticoagulated blood 2. Incubate 15 min 3. add 25 mCi Tc-99m pertechnetate 4. add Na hypochlorite (NaClO) to destroy extracellular Sn2+ ion 5. Add Citrate Buffer 6. incubate 20 min. Expected labeling efficiency >98%
- 18. In vitro/in vitro(Ultratag): Advantages/disadvantages Advantages: quick, simple, inexpensive method; achieves higher labeling efficiency than in vivo/in vivo technique since incubation with RBC is extracorporeal. Ideally suited for GI Bleeding Studies; produces excellent delayed images. Disadvantage: takes extra tech time; slight potential for breaking sterility
- 19. Quality Control forTc-99m RBCs Take 0.2 mL of labeled RBC suspension Add to 2 mL 0.9% NaCl; mix gently Centrifuge 5 min. Pipette off diluted plasma Count Radioactivity in plasma and in packed RBC % RBC Labeling = CountsRBC x 100% CountsRBC + CountsPlasma
- 20. Heat Damaged Tc-99mRBCs Used for spleen imaging –Accessory spleen –Splenic remnants after splenectomy Prepare Tc-99m RBCs by one of the in vitro methods Incubate 30 minutes at 50ºC Heat damaged RBCs are removed from circulation by the spleen
- 21. Tc-99m Labeled WBCs Used for localizing infection and abscesses Principle of labeling –Tc-99m Ceretec is lipophilic and crosses lipid bilayer of cell membranes –Inside the cell the Ceretec complex is broken down and the resulting charged Tc-99m species is trapped in the cell –All cell types are labeled by Ceretec so prior leukocyte separation is necessary
- 22. Tc-99m WBC LabelingProcedure Check patient’s white count (must be > 2K) Obtain ~50 mL anticoagulated whole blood from pt (larger volume if WBC count is low) Optional: add Hetastarch as a sedimentation aid Centrifuge at 15 x g for 10 min
- 23. Tc-99m WBC LabelingProcedure Aseptically remove the platelet-rich & leukocyte-rich supernatant Centrifuge at 200 x g for 10 min. Decant platelet-rich plasma and then wash cells with 0.9% NaCl Add ~40 mCi freshly prepared Tc-99m Ceretec dropwise to leukocyte pellet and incubate for 20 min. Do NOT use Methylene Blue.
- 24. Tc-99m WBC LabelingProcedure Centrifuge, remove unbound Tc- 99m, and wash pellet. Resuspend labeled WBC in either platelet poor plasma or 0.9% NaCl Labeling 50 - 80 %. Draw up dose and inject patient. If patient has been on dialysis, wait until procedure is complete
- 25. Quality Control ofTc-99m WBC Place a drop of labeled cells on a hemocytometer Add a drop of Trypan Blue dye Add a cover slip and examine under microscope Viable cells exclude dye; dead cells are stained blue If >10% are dead or large clumps are present, don’t use
- 26. Labeling In-111 WBC Used for localizing infection and abscesses Principle of labeling –In-111 Oxine is lipophilic and crosses lipid bilayer of cell membranes –Inside the cell the Oxine complex is broken down and the resulting charged In- 111 species is trapped in the cell –All cell types are labeled so prior leukocyte separation is necessary
- 27. In-111 WBC LabelingProcedure Check patient’s WBC count; adjust if necessary Separate WBC fraction as previously described Add 500-1000 µCi In-111 Oxine. Radiolabel as previously described. Labeling typically 50-95%. QC is the same as for Tc-99m WBC
- 28. WBC Separation byOsmotic Lysis RBCs are more susceptible to lysis from osmotic pressure than WBCs Add sterile H2O to WBC pellet contaminated w/ RBCs – This lowers the concentration of ions, etc. – The result is increased pressure in cells – RBCs lyse (burst open and die) Quickly add 5% NaCl to restore tonicity
- 29. WBC Separation byOsmotic Lysis Centrifuge WBC sediment, Platelets and RBC debris remain in supernatant Remove supernatant Resuspend WBC Label WBC with Tc-99m or In-111
- 30. Labeled Platelets Separate platelets from whole blood by centrifugation Label by incubation with lipophilic complexes of Tc-99m or In-111 Uses –Localization of thrombi –Platelet survival studies
- 31. Cr-51 Labeled RBCs Uses –RBC Mass –RBC Survival and Sequestration Properties of Cr-51 –Half-life = 28 days –Gamma energy = 320 keV
- 32. Principle of LabelingCr-51RBCs Cr-51 in the form of Na2CrO4 (+6 oxidation state) is incubated with RBCs Cr-51 binds to RBCs Reaction terminated by adding a reducing agent such as ascorbic acid
- 33. Procedure for LabelingCr-51 RBCs Obtain 25-30 mL of anticoagulated blood Combine blood with 75 - 150 µCi Cr-51 Incubate 20-30 minutes at 37ºC or at room temp Add 100 mg ascorbic acid and incubate 5 more minutes Remove and use the labeled cells
- 34. Summary and Conclusions 1.Multiple methods are available for labeling RBCs with Tc-99m. All are simple, rapid, inexpensive, and reliable 2. GI Bleeding studies are optimally performed with Tc-99m RBCs prepared using the packed cell technique or the UltraTag kit.
- 35. Summary and Conclusions 3.It is easy to label WBCs with either Tc-99m or In-111. Both preparations involve isolation of the WBCs and incubation with a lipophilic intermediate to effect labeling. 4. It is very easy to label red cells with Cr-51. Isolation of RBCs is not required. 5. Most labeling procedures can be performed in any laboratory by a person knowledgeable about aseptic technique.