Cell Labeling Stephen M. Karesh, Ph.D.

Cell Labeling topics to be covered u Red Cells: Tc-99m and Cr-51 u White Cells: Tc-99m and In-111 u Platelets:In-111

Uses of Tc-99m Red Cells u First Pass Cardiac study u Gated Blood Pool study u G.I. Bleed Study u Localization of hepatic hemangioma

Principles of Tc-99m RBC Labeling u Stannous ion, a powerful reducing agent, is added to red cells u Sn 2+ is permitted to diffuse into RBCs u Tc-99m pertechnetate (TcO 4 - ) is then added to pre-tinned RBCs  Tc-99m diffuses into the RBCs, where it is reduced by the Sn 2+ ion to Tc 4+, which binds to the  -globin chains of hemoglobin.

Principles of Tc-99m RBC Labeling  Reduced Tc-99m (Tc 4+ )cannot bind to the heme portion of the hemoglobin molecule since the Fe incorporated into the heme can not be displaced under physiological conditions. Tc 4+ therefore binds only to the globin portion of the molecule, specifically the  -globin chains.

NOW HEAR THIS!!! u Every container which holds a blood sample must be labeled with the patient’s FULL NAME u Syringes u Test tubes u Vials Full Name

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 vitro / in vitro Refers to tinning of the cells, i.e., injection of the stannous pyrophosphate Refers to radiolabeling of the cells, i.e., injection of the Tc-99m pertechnetate

In vivo/in vivo method 1. 1 mg of Sn 2+ 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 Sn 2+ into RBC. 3.IV injection of 25 mCi 99m Tc pertechnetate 4.10 min waiting period to permit diffusion of pertechnetate into RBC's where radio- labeling takes place. 5. Expected labeling efficiency: 80-85%

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.

In vivo/in vitro method (in vivtro method) 1. 1 mg of Sn 2+ 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 Sn 2+ 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

In vivo/in vitro method (in vivtro method) 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%

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

Modified In vivo/in vitro method 1. 1 mg of Sn 2+ in the form of pyrophosphate (“cold PYP”) is given IV min wait to permit mixing of the Sn PYP in body and diffusion of Sn 2+ 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.

Modified In vivo/in vitro 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 min incubation to permit labeling reaction to go to completion. Expected labeling efficiency: % 8.Reinjection of Tc RBC

Modified In vivo/in vitro 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.

In vitro/in vitro method Ultratag Kits 1. To vial containing Sn 2+ 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 Sn 2+ ion 5. Add Citrate Buffer 6. incubate 20 min. Expected labeling efficiency >98%

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

Quality Control for Tc-99m RBCs u Take 0.2 mL of labeled RBC suspension u Add to 2 mL 0.9% NaCl; mix gently u Centrifuge 5 min. Pipette off diluted plasma u Count Radioactivity in plasma and in packed RBC u % RBC Labeling = Counts RBC x 100% Counts RBC + Counts Plasma

Heat Damaged Tc-99m RBCs u Used for spleen imaging –Accessory spleen –Splenic remnants after splenectomy u Prepare Tc-99m RBCs by one of the in vitro methods u Incubate 30 minutes at 50ºC u Heat damaged RBCs are removed from circulation by the spleen

Tc-99m Labeled WBCs u Used for localizing infection and abscesses u 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

Tc-99m WBC Labeling Procedure u Check patient’s white count (must be > 2K) u Obtain ~50 mL anticoagulated whole blood from pt (larger volume if WBC count is low) u Optional: add Hetastarch as a sedimentation aid u Centrifuge at 15 x g for 10 min

Tc-99m WBC Labeling Procedure u Aseptically remove the platelet-rich & leukocyte-rich supernatant u Centrifuge at 200 x g for 10 min. u Decant platelet-rich plasma and then wash cells with 0.9% NaCl u Add ~40 mCi freshly prepared Tc-99m Ceretec dropwise to leukocyte pellet and incubate for 20 min. Do NOT use Methylene Blue.

Tc-99m WBC Labeling Procedure u Centrifuge, remove unbound Tc- 99m, and wash pellet. u Resuspend labeled WBC in either platelet poor plasma or 0.9% NaCl u Labeling %. u Draw up dose and inject patient. If patient has been on dialysis, wait until procedure is complete

Quality Control of Tc-99m WBC u Place a drop of labeled cells on a hemocytometer u Add a drop of Trypan Blue dye u Add a cover slip and examine under microscope u Viable cells exclude dye; dead cells are stained blue u If >10% are dead or large clumps are present, don’t use

Labeling In-111 WBC u Used for localizing infection and abscesses u 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

In-111 WBC Labeling Procedure u Check patient’s WBC count; adjust if necessary u Separate WBC fraction as previously described u Add µCi In-111 Oxine. u Radiolabel as previously described. Labeling typically 50-95%. u QC is the same as for Tc-99m WBC

WBC Separation by Osmotic Lysis u RBCs are more susceptible to lysis from osmotic pressure than WBCs u Add sterile H 2 O 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) u Quickly add 5% NaCl to restore tonicity

WBC Separation by Osmotic Lysis u Centrifuge u WBC sediment, Platelets and RBC debris remain in supernatant u Remove supernatant u Resuspend WBC u Label WBC with Tc-99m or In-111

Labeled Platelets u Separate platelets from whole blood by centrifugation u Label by incubation with lipophilic complexes of Tc-99m or In-111 u Uses –Localization of thrombi –Platelet survival studies

Cr-51 Labeled RBCs u Uses –RBC Mass –RBC Survival and Sequestration u Properties of Cr-51 –Half-life = 28 days –Gamma energy = 320 keV

Principle of Labeling Cr-51RBCs u Cr-51 in the form of Na 2 CrO 4 ( + 6 oxidation state) is incubated with RBCs u Cr-51 binds to RBCs u Reaction terminated by adding a reducing agent such as ascorbic acid

Procedure for Labeling Cr-51 RBCs u Obtain mL of anticoagulated blood u Combine blood with µCi Cr-51 u Incubate minutes at 37ºC or at room temp u Add 100 mg ascorbic acid and incubate 5 more minutes u Remove and use the labeled cells

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.

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.