This document discusses radiolabeling of blood cells and non-imaging procedures. It describes radiolabeling red blood cells and white blood cells with various radioactive isotopes like 111In and 99mTc for clinical applications like infection imaging. The Schilling test is also summarized, which uses radiolabeled vitamin B12 to evaluate absorption in pernicious anemia. Pernicious anemia causes B12 deficiency due to autoantibodies against intrinsic factor or gastric parietal cells, leading to impaired B12 absorption.

1. Cells Labelling & Non
Imaging Procedures
Al Rowaily Mohammed,CRP, PhD

2. Topics
• RADIOLABELLING OF BLOOD CELLS
• Schilling Test

3. RADIOLABELLING OF BLOOD CELLS

4. Contents
• Vascular system
• Blood Cells
• Clinical indications
• Radiolabelling of blood cells
• Radioactive isotopes
• Approaches
• Factors affecting radiolabelling

5. • Blood represents about 1/12th of the body
weight (5-6L)

6. Blood
• Suspension of cells in plasma(92% water,
electrolytes, blood pl. proteins(albumin, globulin, fibrinogen),
hormones)
• Erythrocytes (red cells)
• Leucocytes (white cells)
• Thrombocytes (platelets)

7. Leucocytes
Granulocytes
• Bazophils
• Neutrophils
• Eozinophils
Lymphoid cells (agranulocytes)
• Lymphocytes (B and T)
• Monocytes

8. Erythrocytes (Red blood
cells)
• the most numerous blood cells(about 4-6
millions/mm3).
• devoid of a nucleus and have the shape of a
biconcave lens(6-8 μmin diameter).
• Rich in haemoglobin
• The mean life of erythrocytes is about 120 days.
• in the spleen they are phagocytic by macrophages.
• provide oxygen to tissues and partly recover
carbon dioxide produced as waste.

10. Thrombocytes (Platelets)

11. White blood cells

13. Radiolabelling
• Selective methods
• Non-selective approach

14. Ideal properties of the
ligand
• Specific labelling following IV injection
• Does not diffuse out
• Does not affect cell viability or function

15. Radiolabelling approaches
Selective (in-vivo)
• Specific radioligand
• antibody based
radiopharmaceuticals
(LeucoScan,
GranuloScint)
Non-Selective (in-
vitro)
Isolation
Radiolabelling
Cell resuspension in
media for injection
• Time consuming
• Facilities and equipment
requirements

17. Schematic model of acute bacterial inflammation. The presence of
increased vascular permeability with polymorph nuclear cell infiltrates is
highlighted

18. Schematic model of chronic inflammation. In contrast with the acute
model of inflammation, there is little or no increase in vascular
permeability

19. Non-selective radiolabelling
with 111In
• Oxine (8-hydroxyquinoline) (binding to transferin in the
plasma!!)
• Tropolone (2-hydroxy-2,4,6-cycloheptatrienone)
• 3:1 complex with indium
• dissociation of the complex in the cell, binding of 111In to
intracelular proteins
• Usual injected activity 18.5 MBq

20. Non-selective radiolabelling
with 99mTc-HMPAO
• Meso- and d,l- isomer
• Passive diffusion in the cell
• Conversion in secondary complex
(glutation)
• 30 min shelf life
• Stabilisation agents
• Usual injected activity 350 - 500 MBq

21. Isolation of the WBC
• Erythrocyte sedimentation
• Plasma removal (leucocyte-rich platelet)
• Centrifugation at 150g to pellet the
leucocytes
• Platelet-rich plasma centrifugation at 1500g
– cell free plasma

22. Isolation of the cells
• Sedimentation
• Centrifugation
• “Density gradient” centrifugation
Sedimentation
45-60 min RT
ACD + hespan

23. Cell separation and
radiolabelling

24. 111In over 99mTc-HMPAO-
WBC

25. Normal scintigraphy of total
hip prosthesis with 99mTc-
HMPAOWBC

26. Scintigraphy of infected hip
prosthesis with 99mTc-HMPAO-WBC

27. 99mTc-HMPAO-WBC / 99mTc-
Granuloscint

28. Selective Radiolabelling of
RBC with 99mTc
• Pre-tinning– Stannous compounds (such as
pyrophosphate, MDP) bind to cellular
components
• Diffusion of pertechnetate in the cells
• Reduction of the pertechnetate and binding
to the beta chain of haemoglobin
• 10-20 μg/kg BW of Sn(2+)
• Usual injected activity 500 MBq

30. In-vitro labelling of RBC with
99mTc

31. 2/21/2018
 Radiolabeling- Add weak chelate of Tin (+2), crosses
RBC membrane and attaches to Hb. Excess tin
removed. Tc-99m Pertechnetate added, crosses RBC
membrane, reduced and binds to Hb
 Biological T1/2= 20 hrs
 Methods of Radiolabeling
 In-Vitro
 In-Vivo
 In-Vivtro
Tc-99m RBC

32. Tc-99m RBC
Labeling Methods
• In-Vitro- RBC removed and incubated with Sn-PYP.
Centrifuged. Tc-99m Pert added to cells incubated, centrifuged
and resuspended in saline.
• Most time consuming procedure
• Results in highest labeling efficiency (>90%)
• In-Vivo- Sn-PYP inj, wait 30 minutes, Tc-99m Pertech injected
• Easiest method to use
• Results in lowest labeling efficiency (appr 80%)
• Drugs may interfere with labeling
• Impaired renal function will interfere with radiolabeling

33. Tc-99m RBC
Labeling Methods
• Modified In-vivo- Inject Sn-PYP, After 30 min,
Remove blood sample and incubate with Tc-
99m Pertech in syringe for 10 min, then
reinject
• Radiolabeling between the two (85%)
• Clinical Use- Blood Pool Imaging Agent (20
mCi)

34. GI bleeding with 99mTc-RBC

35. Hemangioma CT / 99mTc-RBC

36. Hemangioma with 99mTc-RBC

37. Heat-damaged 99mTc-RBC
• Spleen imaging
• Incubation at 49.5°C for 15 min
• Fagocytosis of heat damaged 99mTc-RBC
in spleen

38. Spleen 99mTc-RBC heat
damaged

39. Selective Radiolabelling of
RBC with 51Cr

40. RBC mass and volume
determination

43. RBC survival and sequestration
studies

44. RBC survival and sequestration
studies

45. 51Cr survival curve

46. Factors affecting
radiolabelling

47. Quality control of radiolabelled
cells

48. Practical difficulties

49. Distribution changes resulting
from WBC preparation

50. Affect of Drugs on
radiolabelling of WBC

51. Affect of Drugs on
radiolabelling of RBC

52. Facilities for cell
labelling

53. Recommendations

54. Schilling Test

55. Pernicious Anemia
• Epidemiology:
• Most common cause of vitamin B12 def.
• About 2% of people over 60 have undiagnosed
pernicious anemia
• Most common in whites of Northern European
ancestry.
• Average age of diagnosis is approx 60. Under age 30,
it is usually associated with other autoimmune dz.
• Of 729 subjects – 4.1% prevalence in C & AA women
and 2.1% incidence in C & AA men.

56. B-12 Physiology
• Normal B-12 absorption:
• Dietary B-12 binds to R factor in saliva and gastric
juices.
• In duodenum, pancreatic enzymes promote dissociation
from R factor and binding to Intrinsic Factor (IF)
• IF-B12 complex taken up by ileal receptor cubilin.
• Released into plasma bound to transcobalamines TC I,
II, or III.
• Enters cells through receptor mediated endocytosis and
metabolized into two coenzymes: adenosyl-Cbl and
methyl-Cbl.

57. Pernicious Anemia
• Pathophysiology
• Autoantibody to IF
• Two types:
• Type I blocks attachment of B-12 to IF
• Type II blocks B-12-IF complex to ileal receptor
• Present in up to 70% of patients with P.A. and Sn approaches
100%.
• Autoantibody to gastric parietal cells
• Directed against the H/K-ATPase on cell membrane
• Leads to decline in # of parietal cells and IF production
• Leads to chronic atrophic gastritis and gastric atrophy.
• Found in 90% of patients with pernicious anemia.

58. Pernicious Anemia
• Chronic Atrophic Gastritis
• Type A (autoimmune):
• The type involved in P.A. (due to autoantibodies)
• Involves the fundus and body which contain acid-secreting parietal
cells and spares the antrum which contains gastrin-producing cells.
This leads to achlorhydria and high serum gastrin levels.
• Type B:
• Involves fundus, body, and antrum
• Usually associated with H.pylori infection.
• The progression of Type A chronic atrophic gastritis to
gastric atrophy and clinical anemia is likely to span 20 – 30
years.

59. Pernicious Anemia
• Schilling Test
• Stage I
• Give 1mcg of radiolabeled B-12 orally,
followed by 1000 mcg of B-12 IM one
hour later to “flush” any absorbed
radiolabeled B-12 from tissues. A 24-
hr urine is collected to determined how
much radiolabeled B-12 is excreted.
Normal is 8-35%.

60. Pernicious Anemia
• Schilling Test
• Stage II
• Done only if Stage I is abnormal.
• Repeat Stage I, except with the
addition of added oral IF which should
normalize B-12 absorption in P.A., but
not intestinal malabsorption.

61. Pernicious Anemia
• Schilling Test
• Many false pos and neg results
• Not commonly used
• Not readily available in many places
• Less sensitive test checking HC and
MMA to detect B-12 def.
• Only recommended when anti-IF
antibodies are normal.

62. Thank You