Cell labeling Flashcards
Safe practices/considerations
● Carry out one labeling procedure at a time to avoid mixing patient blood samples
● The same technologist should do the labeling procedure
○ Withdrawing blood, labeling the cells and re-injection
● Gloves should be worn at all times
● Strict attention to aseptic technique is important
● One-handed recap is very important
● Labeling of RBCs can be done in a laminar flow hood or radiopharmacy room
● Labeling of WBCs MUST be done in a laminar flow hood
○ (biological safety cabinet)
What are labeled red blood cells used for?
- GI bleed
- Hemangioma
- WMS
- heat damaged, spleen
Describe the cellular process of RBC labeling
● 99mTc (VII) O4- will cross erythrocyte membrane, then reduced to a lower oxidation state by the Sn2+ to stay inside the RBC and allow blood pool imaging to occur
○ 80% tags to B chain of globin
○ 20% tags to heme on Hb
In vivo RBC labeling
● Labeling process occurs inside of the patient
● Labeling mechanism
○ Inject PYP or gluceptate
○ Incubation period: 5-60 minutes (kit dependent)
■ Stannous enters RBC’s
○ Inject 99mTcO4-
■ 99mTcO4- diffuses into cell
■ Reduced by intracellular Sn
■ 99mTcO4 exchanges with Cl- & bicarbonate
■ Binds to ß chain of hemoglobin (80%)
● Good image quality
what competes with pertechnetate in RBC labeling
- extracellular fluid
- thyroid glands
- salivary glands
- GI tract
What reduces RBC labeling efficiency
- heparin
- digoxin
- propranolol
- doxorubicin
- dextran
- hydralyzine
- iodinated contrast
describe Modified In Vivo/In Vitro RBC Labeling
● Not commonly used
● Process
○ PYP or gluceptate/stannous ion injected into the patient
○ Wait ~ 20 min
○ Withdraw a 3 ml sample of ‘tinned’ blood into a syringe with anticoagulant and
99mTcO4-
○ Incubate/Label ~ 10 min
○ Re-inject patient through IV
● Estimated labeling efficiency: > 90%
In vitroRBC labeling
● i.e.. Ultratag
● Superior image quality
● Labeling process occurs completely outside of the patient
● Labeling Efficiency = > 95%
● Process
○ 10mL reaction vial containing: Stannous chloride dehydrate, sodium citrate,
dextrose
○ Withdraw 1-3 ml of the patient’s blood anticoagulated with ACD or Heparin
and add to reaction vial
■ Do not exceed 0.15ml of anticoagulant/ 1 ml of blood
○ Stannous ion diffuses across the RBC membrane
■ Excess ACD can reduce the amount of stannous diffusing across the cell
membrane
● Process (con’t)
○ Add Syringe I to the reaction vial (Sodium Hypochlorite)
■ Oxidizes extracellular stannous ion
○ Add Syringe II to the reaction vial (Citric Acid, Sodium Citrate, Dextrose)
■ Sequesters any extracellular stannous ion, making it more available to the
Sodium Hypochlorite
○ Add 99mTcO4-, allow it to incubate 20 min and reinject the patien
WBC labeling considerations
● Protective Barriers:
▪ Gloves – wash your hands before donning;
sterile and powder-free
▪ Gowns – long sleeves with tight cuffs that
will fit underneath the gloves
▪ Masks – disposable surgical mask
▪ Bouffant – cover hair
● Protective barriers should be limited to the cell labeling area
Scrubbing and gowning
- Remove all exposed jewelry
- Put on a bouffant to cover hair
- Scrub hands and arms with soap and warm water, pat dry with towel
- Put on a sterile gown and gloves (double), pull cuffs of gloves up over sleeves
- Put on surgical mask
- Before starting procedure, gloves should be wiped with 70% IPA
- Ensure you change gloves every time you exit the biological cabinet
- Always wash hands after removal of gloves
- Gloves should be discarded in biohazard waste (yellow bag)
- Once gown has been removed, place into regular laundry bin
describe how to working in a biological safety cabinet
- Disinfect the cabinet with 70% IPA
- All outer packaging of items must be removed before placing in the work area
- All items that are placed in the cabinet must be wiped with 70% IPA
- Open items in sterile packaging within the first 6 inches of the cabinet (syringes,
needles, tubes, etc.) - Place opened items deep into the cabinet and discard packaging
- Purified air comes from above in a vertical cabinet (laminar flow hood) , so do not
block the air flow and minimize movements within the cabinet - Wipe gloves with 70% IPA if hands go outside of sterile area
- Change gloves if hands come into contact with non-sterile surfaces
- DO NOT place un-sterile items in the cabinet
- DO NOT cough or sneeze in work area
WBC labeling RPs
● Labeling occurs in-vitro in laminar flow hood
● Radiopharmaceuticals
○ 111In-Oxine
○ 99mTc-HMPAO
● Indication:
○ Infection and Inflammation Imaging
■ Osteomyelitis vs Loosening (ie. hip/knee replacement)
■ Osteomyelitis vs Cellulitis (bone vs. deep skin infection)
■ Irritable Bowel Disease (Crohn’s, Colitis)
■ FUO
■ Surgical graft
111-In oxine
● 111In conjugated with oxine to form lipid-soluble complex
○ 111In-oxine diffuses into cell
○ 111In dissociates to bind to cytoplasmic component of WBCs
○ Oxine removed by ‘washing’ cells
● Toxic to cell (carcinogenic potential)
○ Low dose
● Instability in the presence of proteins
○ Solution MUST be protein free (separate all plasma from WBCs)
■ Transferrin binding rather than leukocytes
● Adheres to plastic
○ Dispense dose immediately prior to labeling
● Label is stable post re-injectio
Advantages of using 111-In oxine in WBC labeling
▪ No significant uptake in GI tract, kidneys, or bladder
▪ Delayed imaging possible (T ½ = 2.8 days)
▪ Typically, high tagging efficiency ≥ 90%
▪ Good for acute & chronic Infections, IBS, and Osteomyelitis
▪ Radiolabeled cells good for up to 3 hours (usually inject within 1 hr)
▪ Dual-isotope possible (SC subtraction same day)
Describe disadvantages of using 111-In Oxine inWBC labeling
▪ Slightly damaged WBCs → liver
▪ Severely damaged WBCs → lungs
▪ Low dose → low count statistics → poorer quality images
▪ Higher radiation dose to patient (173 & 247 keV)
HMPAO WBC labeling
● Prepare with fresh eluate (≤ 2hrs old)
○ Low Sn+2 concentration
○ Radiolysis
● Must re-suspend WBCs within 30 min of reconstitution
○ No stabilizer used
● Mechanism of uptake
○ Lipid-soluble 99mTc HMPAO crosses cell membrane through passive diffusion in WBC (mainly neutrophils)
■ Glutathionine-medicated conversion to hydrophilic species
■ Binding to intracellular protein & organelles
HMPAO advantages in WBC labeling
○ Lower radiation dose to patient (140 keV)
■ Better for pediatrics!
○ Higher dose → higher count rate → better quality images
○ Option to re-suspend WBCs in plasma (instead of 0.9% saline)
○ Good for acute infections, IBS, osteomyelitis
○ Faster uptake
■ Imaging can be performed within 30 mins of injection and up to 24hrs
○ Convenient
■ Most departments have kits and access to 99mTcO4-
○ SPECT/CT is superior
Disadvantages of using HMPAO in WBC labeling
○ Less stable post-injection
○ Tagging efficiency is low (~55-70%)
○ Lower target-to-background ratio
■ Increased bowel, kidney and bladder uptake
Similarities of HMPAO and 111-In oxine
● Non-specific label for neutrophils
● Anticoagulant
▪ Ie. ACD/Heparin
● Settling Agent
○ Methyl Cellulose
▪ Ie. Hespan, Dextran
● Isolation of leukocytes from plasma
▪ Centrifuge – produces WBC
button separate from supernatant
▪ Wash/Rinse WBCs (0.9% isotonic
saline vs plasma)
● Add RP dropwise and incubate
● Calculate Tagging Efficiency
● Re-inject within 1 hour
▪ Extending viability to 5hrs
Describe WBC labeling procedure
● Obtain 30-45mL blood
○ Volume depends on # of WBC
■ Pediatric: 10 – 15 mL
○ Large-bore IV
○ ACD in syringe
○ Add sedimentation agent (i.e. hespan)
● Separate whole blood from leukocytes
○ Erythrocyte settling rate (ESR)
■ Time to separation (normal): 30 – 45 minutes
■ Causes of altered ESR
● Sickle cell anemia
● Remove leukocyte rich plasma (leukocytes & platelets)
● Centrifuge to separate button
○ Supernatant: Leukocyte poor plasma, platelets
○ Pellet: WBC & residual RBC
● Remove supernatant, leaving button (leukocytes)
○ Leukocyte poor plasma is removed; save to re-suspend final product for injection
● Re-suspended button in NaCl
Sedimentation
- method of labeling erythrocytes
- methyl cellulose
- heta starch
- pentastarch
- dextran
centrifugation
- isolation of leukocytes
- produces WBC pellet
- supernatant
- plasma
- platelets
Causes of reduced tagging efficiency
Plasma and RBC contamination, BP and cardiac uptake
What happens if you dont add ACD or Hespan to blood sample within 5 min
- clotting of cells
- unable to separate WBCs and label with RP
What occurs with improper cell washing
- affect labeling and cell viability
- improper suspension/agitation causes WBC clumping
- leads to lung uptake
- be gentle!
Describe splenic uptake
- reflects label quality
- 20-40% of injected cells localize in the spleen
- > cells damage during labeling: low splenic activity
- 111-In complexed transferrin rather than leukocytes
describe how to withdraw patient blood
- withdraw 20-40ml ofpatient blood
- in a 50 ml syringe
- 19 G needle
- important to use large guage needle to avoid damging cells
- iv preferred
Why will a leukopenic study impact results
- less cells to label
- > 4000 cells/ml preferred
What causes false negative results
- antibiotics
- lidocaine
- corticosteroids
- hyperalimentation
