Liver Spleen Scan Using Denatured Red Blood Cells

Denatured red blood cell scans are few and far between, but when something like this comes along, it piques everyone's interest. 

With this case here, the ordering physician was inquiring about the multiple intra abdominal deposits and had asked whether these deposits were related to splenules, caused by some kind of splenosis.  We are uncertain about the patient history since the technologist performing the scan did not inquire about the previous history of trauma or any iatrogenic events that may have potentially caused the spleen to rupture.  Previous ultrasounds or CT imaging were not performed at our facility and the patient did not bring any CD's with previous imaging from outside sources.  But thinking back, this patient must have some form of imaging done or otherwise, how did the physician know of the abdominal deposits?  At any rate this is what we got with respect to the imaging.  

After the initial reinjection of the denatured cells, a flow and blood pool images were acquired.  The delayed imaging was performed 20 minutes post injection, using a 2 bed SPECT/CT protocol.

Fig. 1 Anterior blood pool, after the denatured red blood cell injection.

Fig. 2 Posterior blood pool acquisition.

Fig. 3  Coronal slice.  Multiple foci of activity.  Note the activity over the dome of the liver and the perihepatic regions.

Fig. 4  Multiple uptake within the peritoneal cavity.

The MIP display above might not seem like very much, but there are multiple foci of tracer uptake scattered throughout the peritoneal cavity and the perihepatic region.  The largest and the most intense nodules occur in the splenic fossa along with multiple nodules tracking along the colon.

Fig. 5  SPECT/CT acquisition was performed to localize the denatured red blood cell uptake.

There is also focal activity in nodules adjacent to the liver as well as further nodules in the lateral right hepatic border and in the hepatorenal space.  Overall a lot of diffuse uptake can be observed, which is highly suggestive of splenosis.

We have a GMP approved radiopharmacy and all of the blood work is performed on site.  I must admit in the past, sometimes the labeling worked and sometimes it did not, however we have tweaked the procedure a little bit, and the information below is how we label and denature our red blood cells to ensure a quality scan.

Preparation of Radiolabeled Red Blood Cells Using the UltraTag In Vitro Method:

      Purpose:

The aim of this document is to provide information on the preparation and quality control of ^99mTc-labeled red blood cells using the UltraTag® in vitro method.

     Responsibilities:

It is the responsibility of the radiopharmacy technician to perform this procedure under strict aseptic conditions and as outlined in this protocol.

It is the responsibility of the radiopharmacist or the quality control technician to ensure that the correct RBC preparation procedure is followed and the final product meets the required specifications.

    Materials:

- lead glass syringe holder (3 mL size)

- syringe lead pig

- vial lead pig

- sterile 15 mL plastic centrifuge tubes

- sterile 18 or  21 gauge needles

- sterile 5 mL and 3 mL syringes

- sterile saline 0.9 % for injection USP

- UltraTag® kit

- ^99mTc sodium pertechnetate (370 – 1000 MBq)

    Procedure:

4.1.  Area setup:

·       Turn on laminar flow hood and wipe down surface of the hood with 70% isopropyl alcohol and a clean Kendall wipe. Spray and wipe with 70% IPA the materials needed to be placed in the hood.

·       Don hair cover, face mask, gloves and gown.

·       Turn on water bath and set the temperature at 50^oC, if preparing denatured RBC.

4.2.  Precautions:

·       During the labeling procedure, blood and blood components of the patient, who could potentially be infected with pathogens, need to be handled. To prevent contamination of the operator, double-gloving using waterproof gloves is recommended.

·       Since the cells have to be reinjected into the patient, strict aseptic conditions are required for the labeling procedure.

·       Simultaneous labeling of blood products from multiple patients is discouraged in order to prevent possible cross-contamination. At all times correct identification of the patient’s blood products should be guaranteed.

·       During the labeling care should be taken not to damage the cells, as this would result in leakage of the radioactivity from the cells, increased lung uptake and increased liver uptake.

4.3.  Radiolabeling of RBC with ^99mTc:

Note: The UltraTag® kit has 3 components:

a)     10 mL reaction vial containing stannous chloride, dihydrate (SnCl2•2H2O) 50 µg, minimum, stannous chloride, dihydrate (SnCl2•2H2O) 96 µg, theoretical, tin chloride (stannous, stannic) dihydrate, as stannous chloride, maximum dihydrate 105 µg, 3.67 mg sodium citrate dihydrate and 5.50 mg anhydrous dextrose.

b)     Syringe I: 0.6 mL contains 0.6 mg sodium hypochlorite (NaOCl). Protect from light.

c)   Syringe II: 1 mL contains 8.7 mg citric acid monohydrate, 32.5 mg sodium citrate dihydrate and 12.0 mg anhydrous dextrose.

4.3.1.     Collect patient's blood sample (1 - 3 mL) using heparin or ACD as an anticoagulant. The amount of ACD should not exceed 0.15 mL of ACD per mL of blood. The recommended amount of heparin is 10-15 units per mL of blood. DO NOT USE EDTA OR OXALATE AS AN ANTICOAGULANT.

4.3.2.     Using a large-bore needle (19 to 21 gauge), transfer 1.0 to 3.0 mL of anticoagulated whole blood to the reaction vial and gently mix to dissolve the lyophilized material. Allow to react for 5 min at room temperature.

4.3.3.     Add contents of Syringe I, mix by gently inverting four to five times.

4.3.4.     Add the contents of Syringe II to the reaction vial. Mix by gently inverting four to five times.

4.3.5.     Place the vial in a lead shield fitted with a lead cap. Add 370 to 925 MBq (10 to 25 mCi) sodium pertechnetate Tc-99m (in a volume of up to 3 mL) to the reaction vial. Use fresh generator eluate to avoid in-growth of ⁹⁹Tc.

4.3.6.     Mix by gently inverting reaction vial four to five times. Allow to react for 20 minutes with occasional mixing.

4.3.7.     Take a sample to assay labeling efficiency immediately prior to injection (see 4.4.2).

If LE > 90%, proceed to step 4.3.8 for denatured RBC or step 4.3.9. for normal RBC.

4.3.8.     Heat the tagged cells in a water bath with a little agitation at 49 - 50^oC for no more than 15 min. Proceed to step 4.3.9.

4.3.9.     Mix gently prior to withdrawal of patient dose. Aseptically transfer the ^99mTc-labeled red blood cells to a syringe for administration to the patient. Use a large bore needle to prevent hemolysis.

4.3.10.  Assay for radioactivity. Prepare labels and paper work using Pinestar.

4.3.11.  ^99mTc-labeled red blood cells should be injected within 30 minutes of preparation or as soon as possible thereafter.

4.3.12.  Clean up the laminar flow hood. Place radioactive waste in the waste disposal bin in the hot lab and non-radioactive sharps waste and blood/plasma in the sharps waste container. Remove the equipment from the laminar flow hood and wipe the surface down with 70% isopropyl alcohol using a lint-free cloth.

4.4. Quality control:

4.4.1.     Visual inspection (performed routinely):

·         At the end of the procedure and before collecting the radiolabeled RBC in the syringe for patient administration, a visual inspection for clumps, clots and aggregates should be performed by gently rotating the tube.

·         In case of aggregates, they should be dissolved by gently shaking or pipetting the sample.

·         If clumps cannot be dissolved, the preparation should not be injected.

4.4.2.     Labeling efficiency (LE) (performed routinely):

·       Transfer 0.2 mL of the ^99mTc-labeled RBC to a 15 mL centrifuge tube containing 2 mL of 0.9% NaCl. Centrifuge for five minutes and carefully pipet off the diluted plasma. Measure the radioactivity in the plasma and red blood cells separately in a dose calibrator. Calculate labeling efficiency as follows:

% LE = (Activity RBC x 100)/(Activity RBC + Activity Plasma)

·         LE = 90 - 98 %

·         If LE < 90 %, further quality control should be performed, such as microscopic inspection and test for cell viability (see 4.4.3).

4.4.3.     Trypan blue exclusion test for cell viability (recommended periodically):

·       Take a small sample (0.2 mL) from the radiolabeled RBC and dilute it with 1 ml of saline for injection.

·         In a small tube mix 50 ul 0.4 % trypan blue solution and 50 ul of the radiolabeled RBC sample.

·         Put a drop of this mixture in a hemocytometer and place it under the microscope.

·         Check for clumps and microaggregates of cells.

·         Calculate the percentage of blue stained cells from the total cell number. This is the percentage of damaged cells.

·         If a preparation has > 10 % blue-stained cells, it should not be released for injection into the patient.

4.4.4.     Sterility (recommended periodically):

Sterility of the final preparation should be tested periodically, especially in case of any modification to the procedure.

So there you have it.

NB:  Someone had mentioned that I forgot to add steps 5.3.8, 5.4.2, 5.4.3. Everything is contained within the blog.  I just mislabeled the numbered steps.  The "5" should have been a "4" in the body of the blog.  Apologies, it has been corrected..