Cell LabelingStephen M. Karesh, Ph.D.
Cell Labeling topics to be coveredRed  Cells:    Tc-99m and Cr-51White Cells:   Tc-99m and In-111Platelets:     In-111
Uses of Tc-99m Red CellsFirstPass Cardiac studyGated Blood Pool studyG.I. Bleed StudyLocalization of hepatic hemangioma
Principles of Tc-99m RBC LabelingStannous   ion, a powerful reducing agent, is added to red cellsSn2+ is permitted to di...
Principles of Tc-99m RBC LabelingReduced   Tc-99m (Tc4+)cannot bind to the heme portion of the hemoglobin molecule since ...
NOW HEAR THIS!!! Every  container which  holds a blood sample                                                            ...
Methods of Preparation of Tc-99m RBCs          • In vivo/in vivo          • In vivo/in vitro          • In vitro/in vitro
Methods of Preparation of Tc-99m RBCs                    In vivo / in vivo                    In vivo / in vitro          ...
In vivo/in vivo method1. 1 mg of Sn2+ in the form of pyrophosphate(“cold PYP”) is given IV2. 20 min wait to permit mixing ...
In Vivo/In Vivo:    Advantages/disadvantagesAdvantages: quick, simple, inexpensiveDisadvantage: lowest labeling efficiency...
In vivo/in vitro method (in vivtro method) 1. 1 mg of Sn2+ in the form of pyrophosphate (“cold PYP”) is given IV 2. 20 min...
In vivo/in vitro method (in vivtro method)4. 10 min waiting period to permit diffusion of the pertechnetate into RBCs and ...
In vivo/in vitro:    Advantages/disadvantagesAdvantages: quick, simple, inexpensivemethod; achieves higher labeling effici...
Modified In vivo/in vitro method1. 1 mg of Sn2+ in the form of pyrophosphate(“cold PYP”) is given IV.2. 20 min wait to per...
Modified In vivo/in vitro method5. Removal of 1-2 ml of packed cellsthrough a 20 ga or larger needle.6. Aseptic addition o...
Modified In vivo/in vitro method     Advantages/disadvantagesAdvantages: quick, simple, inexpensivemethod; achieves highes...
In vitro/in vitro method Ultratag Kits 1. To vial containing Sn2+ compound, add 3- 5 ml of anticoagulated blood 2. Incubat...
In vitro/in vitro (Ultratag):    Advantages/disadvantagesAdvantages: quick, simple, inexpensivemethod; achieves higher lab...
Quality Control for Tc-99m RBCsTake 0.2 mL of labeled RBC suspensionAdd to 2 mL 0.9% NaCl; mix gentlyCentrifuge 5 min. ...
Heat Damaged Tc-99m RBCsUsed   for spleen imaging  –Accessory spleen  –Splenic remnants after splenectomyPrepare Tc-99m ...
Tc-99m Labeled WBCsUsed  for localizing infection and abscessesPrinciple of labeling –Tc-99m Ceretec is lipophilic and c...
Tc-99m WBC Labeling ProcedureCheck  patient’s white count (must be > 2K)Obtain ~50 mL anticoagulated whole blood from pt...
Tc-99m WBC Labeling ProcedureAseptically remove the platelet-rich & leukocyte-rich supernatantCentrifuge at 200 x g for ...
Tc-99m WBC Labeling ProcedureCentrifuge, remove unbound Tc- 99m, and wash pellet.Resuspend labeled WBC in either platele...
Quality Control of Tc-99m WBCPlace  a drop of labeled cells on a hemocytometerAdd a drop of Trypan Blue dyeAdd a cover ...
Labeling In-111 WBCUsed  for localizing infection and abscessesPrinciple of labeling –In-111 Oxine is lipophilic and cro...
In-111 WBC Labeling ProcedureCheck  patient’s WBC count; adjust if necessarySeparate WBC fraction as previously describe...
WBC Separation by Osmotic LysisRBCs  are more susceptible to lysis from osmotic pressure than WBCsAdd sterile H2O to WBC...
WBC Separation by Osmotic Lysis Centrifuge WBC   sediment, Platelets and RBC  debris remain in supernatant Remove super...
Labeled PlateletsSeparate  platelets from whole blood by centrifugationLabel by incubation with lipophilic complexes of ...
Cr-51 Labeled RBCsUses –RBC Mass –RBC Survival and SequestrationProperties of Cr-51 –Half-life = 28 days –Gamma energy =...
Principle of Labeling Cr-51RBCs Cr-51  in the form of Na2CrO4  (+6 oxidation state) is  incubated with RBCs Cr-51 binds ...
Procedure for Labeling Cr-51 RBCsObtain 25-30 mL of anticoagulated bloodCombine blood with 75 - 150 µCi Cr-51Incubate 2...
Summary and Conclusions1. Multiple methods are available for labeling   RBCs with Tc-99m. All are simple, rapid,   inexpen...
Summary and Conclusions3. It is easy to label WBCs with either Tc-99m   or In-111. Both preparations involve   isolation o...
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Red blood celllabeling (1)

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Red blood celllabeling (1)

  1. 1. Cell LabelingStephen M. Karesh, Ph.D.
  2. 2. Cell Labeling topics to be coveredRed Cells: Tc-99m and Cr-51White Cells: Tc-99m and In-111Platelets: In-111
  3. 3. Uses of Tc-99m Red CellsFirstPass Cardiac studyGated Blood Pool studyG.I. Bleed StudyLocalization of hepatic hemangioma
  4. 4. Principles of Tc-99m RBC 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. 5. Principles of Tc-99m RBC 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. 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. 7. Methods of Preparation of Tc-99m RBCs • In vivo/in vivo • In vivo/in vitro • In vitro/in vitro
  8. 8. Methods of Preparation of Tc-99m RBCs In vivo / in vivo In vivo / in vitro In vitro / in vitroRefers to Refers totinning of the radiolabelingcells, i.e., of the cells,injection of the i.e., injection ofstannous the Tc-99mpyrophosphate pertechnetate
  9. 9. In vivo/in vivo method1. 1 mg of Sn2+ in the form of pyrophosphate(“cold PYP”) is given IV2. 20 min wait to permit mixing of the SnPYP in body and diffusion of Sn2+ into RBC.3. IV injection of 25 mCi 99mTc pertechnetate4. 10 min waiting period to permit diffusion ofpertechnetate into RBCs where radio-labeling takes place.
  10. 10. In Vivo/In Vivo: Advantages/disadvantagesAdvantages: quick, simple, inexpensiveDisadvantage: lowest labeling efficiency ofall commonly used procedures, butperfectly acceptable for routine work, e.g.,MUGAs.
  11. 11. In vivo/in vitro method (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. 12. In vivo/in vitro method (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. 13. In vivo/in vitro: Advantages/disadvantagesAdvantages: quick, simple, inexpensivemethod; achieves higher labeling efficiencythan in vivo/in vivo technique sinceincubation with RBC is extracorporeal. Notoptimal for GI Bleeding Studies.Disadvantages: takes extra tech time;potential for breaking sterility
  14. 14. Modified In vivo/in vitro method1. 1 mg of Sn2+ in the form of pyrophosphate(“cold PYP”) is given IV.2. 20 min wait to permit mixing of the SnPYP in body and diffusion of Sn2+ into RBC.3. Withdrawal of 5-10 ml of anti-coagulatedblood (heparin, ACD) into vacutainer.4. Centrifuge the vacutainer in invertedposition for 5 min at 3000 rpm.
  15. 15. Modified In vivo/in vitro method5. Removal of 1-2 ml of packed cellsthrough a 20 ga or larger needle.6. Aseptic addition of these tinned, packedcells to a sterile vial containing 35 mCi ofTc-99m pertechnetate.7. 10 min incubation to permit labelingreaction to go to completion. Expectedlabeling efficiency: 98-100%8. Reinjection of Tc RBC
  16. 16. Modified In vivo/in vitro method Advantages/disadvantagesAdvantages: quick, simple, inexpensivemethod; achieves highest labeling efficiencyof all procedures since reaction of Tc withplasma proteins has been eliminated. Ideallysuited for GI Bleeding Studies; producesexcellent delayed images.Disadvantage: takes extra tech time; requiresclinical centrifuge; potential for breakingsterility.
  17. 17. In vitro/in vitro method 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. 18. In vitro/in vitro (Ultratag): Advantages/disadvantagesAdvantages: quick, simple, inexpensivemethod; achieves higher labeling efficiencythan in vivo/in vivo technique since incubationwith RBC is extracorporeal. Ideally suited forGI Bleeding Studies; produces excellentdelayed images.Disadvantage: takes extra tech time; slightpotential for breaking sterility
  19. 19. Quality Control for Tc-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. 20. Heat Damaged Tc-99m RBCs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. 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. 22. Tc-99m WBC Labeling Procedure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. 23. Tc-99m WBC Labeling Procedure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. 24. Tc-99m WBC Labeling Procedure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. 25. Quality Control of Tc-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. 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. 27. In-111 WBC Labeling Procedure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. 28. WBC Separation by Osmotic 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. 29. WBC Separation by Osmotic Lysis Centrifuge WBC sediment, Platelets and RBC debris remain in supernatant Remove supernatant Resuspend WBC Label WBC with Tc-99m or In-111
  30. 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. 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. 32. Principle of Labeling Cr-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. 33. Procedure for Labeling Cr-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. 34. Summary and Conclusions1. Multiple methods are available for labeling RBCs with Tc-99m. All are simple, rapid, inexpensive, and reliable2. GI Bleeding studies are optimally performed with Tc-99m RBCs prepared using the packed cell technique or the UltraTag kit.
  35. 35. Summary and Conclusions3. 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.

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