Scintigraphy Technique for Labelling of White Blood

Cells with 99mTc-HMPAO: Comparison of Three Solvents

By Seraho Mbuluyo Marie-Ange

Supervisor:

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 Miss Shingirai Brenda Kagodora, Principal technician at Wits, School of Public Health, Faculty of
Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.

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Table of contents:

Contents

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LIST OF TABLES AND FIGURES

Table 1: Timing

Figure 1: Study participant

Fig 2: Stability in Labelled Syringe (A)

Fig 3: Stability in Unlabelled Syringe (U)

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NOMENCLATURE
99m
Tc-HMPAO: 99mTc-hexamethylpropyleneamine oxime

ACD: acid citrate-dextrose anticoagulant solution

CFP: cell-free plasma

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INTRODUCTION

Background

Labeled white blood cells (WBCs) are used in scintigraphy to detect sites of infection. In the 1970s,
111
In-oxine was introduced as a nonselective labeling agent for WBC scintigraphy. However, 99mTc-
HMPAO has largely replaced 111In-oxine because of its favorable physical characteristics, availability,
cost, and lower radiation burden. 99mTc-HMPAO kit preparations have been commercially available
since 1988. When reconstituted with 99mTc-pertechnetate, a lipophilic complex is formed. This
complex is transformed into free 99mTc-pertechnetate and a hydrophilic 99mTc-HMPAO complex over
time. Only freshly prepared 99mTc-HMPAO should be used for WBC labeling, as only the lipophilic
complex can freely cross the cell membrane and be trapped inside the cell (Tatum JL et al, 1983).

Two mechanisms have been suggested to be responsible for the retention of 99mTc-HMPAO inside the
cell:

1. Conversion of the lipophilic 99mTc-HMPAO complex into a hydrophilic complex by reducing

agents such as glutathione.

2. Binding of 99mTc-HMPAO to non-diffusible proteins and cell organelles.

Some release of 99mTc-HMPAO from the labeled WBC after reinjection into the patient is observed,
resulting in undesired accumulation of radioactivity in the gastrointestinal and urinary tracts. For WBC
scintigraphy, either mixed leukocytes or isolated granulocytes can be used. When mixed leukocytes are
labeled with 99mTc-HMPAO, about 70–80% of the radioactivity is bound to granulocytes. Labeled
mixed leukocytes can display higher blood pool activity than labeled isolated granulocytes, especially
in early images, due to the presence of labeled lymphocytes and residual erythrocytes (Hladik WB et
al, 1987).

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Literature analysis

99mTc-HMPAO-labeled WBC scintigraphy is a sensitive and specific test for detecting infections.
However, it is not always the first-line test, as it can be expensive and time-consuming. Other imaging
tests, such as ultrasound, CT scan, and MRI, may be used first

Precautions

When labelling WBCs with 99mTc-HMPAO, it is important to take precautions to prevent

contamination of the operator and the patient. This includes using sterile reagents and disposable
plastic ware, and wearing sterile gloves, cap, and mask. The labelling should be performed in a laminar
flow cabinet or cell isolator, and simultaneous labelling of WBCs from multiple patients should be
avoided (MacGregor RR et al, 1974).

It is also important to ensure that the patient's blood products are correctly identified. All syringes,
tubes, and any material in contact with the patient's blood components should be clearly labelled with
the patient's name, bar code, and/or colour code.

During the labelling process, care should be taken not to damage the WBCs, as this could lead to
leakage of radioactivity, adhesion of the cells to the vascular endothelium, and loss of motility. The
labeled WBCs should be reinjected as soon as possible, but not later than 1 hour after labelling (Lee
HB et al, 1983).

Labelling of mixed leukocytes causes radiation damage to the lymphocytes as a result of self-
irradiation by low-energy Auger electrons. However, the risk of lymphoid malignancies after
administration of 99mTc-HMPAO-labeled mixed leukocytes is considered to be negligible, as the
lymphocytes are unable to divide after labeling and are eliminated through apoptosis and phagocytosis
(Extra Pharmacopoeia, Martindale, 30th Edition, Royal Pharmaceutical Society of Great Britain,
London 1993; 516.).

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Factors that affect radiolabeling of WBCs

Potential factors influencing treatment outcomes

Radiolabelling blood cells can be challenging. Problems can occur during the labelling process or after
the labelled cells are injected. Failure to label can be due to pharmaceutical factors, such as difficulty
collecting enough cells, sedimentation problems, or instability of the cell chelator. Patient-related
factors, such as medication or disease, can also cause problems.

Several studies have been conducted to investigate the possibility that drug interference is a cause of
labelling. Leukocyte labelling difficulties were reported in patients who were taking multiple
medications, including prednisolone, azathioprine, cyclosporine, ranitidine, nifedipine,
cyclophosphamide, and naproxen. Although a direct causal relationship has not been established, the
known adverse effects of these drugs on white blood cell function and kinetics suggest that patient
medication may be an important factor in leukocyte labelling (Sampson CB, 1998).

Type of patients

99mTc-HMPAO-labeled WBC scintigraphy is a nuclear medicine imaging technique that can be used
to detect and localize infections. It is commonly used to diagnose patients with the following
conditions:

 Osteomyelitis of the appendicular skeleton (bones of the arms and legs)

 Infected joint and vascular prosthesis

 Diabetic foot infection

 Fever of unknown origin

 Postoperative abscesses

 Lung infections and endocarditis (inflammation of the inner lining of the heart)

 Inflammatory bowel disease

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 Neurological infections

 Infected central venous catheters or other devices

The technique involves injecting the patient with a radioactive tracer that binds to white blood cells.
The white blood cells then travel to the site of infection, where they accumulate and can be detected by
the imaging equipment.

AIM AND OBJECTIVES (To be discussed)

To evaluate and compare the effectiveness of three different solvents for labeling white blood cells
(WBCs) with the radiotracer 99mTc-HMPAO.

METHODS

Study design

This study will compare the effectiveness of three different injection preparations for labeling white
blood cells with 99mTc-HMPAO using a randomized controlled trial design.

Study site

To be discussed

Study Participant

FEATURE INFORMATION

Number of donors 3

Gender distribution 2 females, 1 male

Age requirement Over 18 years old

Consent All participant signed a consent form

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 Figure 1: Study participant

Sampling / selection

A total of 360 ml of blood was collected for research purposes. This involves drawing six syringes,
each containing 20 ml of blood (120mls), from three different donors.

Each blood sample will be subsequently divided into three separate preparation groups. This means
each original 120 ml sample will be further split into three smaller portions of approximately 40ml
each. These individual portions will then be utilized in different preparation methods as part of the
research study.

To achieve this comparison, the following three preparation types will be utilized:

1. Saline solution: This is a commonly used solvent in medical applications and serves as a baseline for
comparison.

2. Water for injection: This is a sterile water solution used for dissolving medications and preparing
injections. Its impact on labeling efficiency will be compared to saline.

3. Saline solution mixed with water: This combination will be investigated to determine if it offers any
advantages over either single solvent alone.

Preparation before the Test

Essentials for the procedure

The following are the essential items needed for the procedure:

1. 9 x 18g pink jelco or 20g green jelco

2. 18 x 20 ml syringes with 3ml ACD

3. 9 x 10ml syringes

4. 18 x 5ml syringes

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 5. 7 x 50ml tubes

PROCEDURE

A. Isolation of WBCs

A1. Collection of blood:

To collect blood for white blood cell labeling, follow these steps:

1. Fill a 20-mL syringe with 3 mL of acid citrate-dextrose (ACD) anticoagulant solution.

2. Draw blood until the syringe contains 20 mL of the patient's blood. Use a Jelco with an inner
diameter of at least 20g to prevent damage to the WBCs.

3. Mix gently the blood ACD solution to prevent coagulation.

4. Withdraw 5 mL of 6% voluven into 6 x 50 mL universal tubes.

5. Transfer the blood mixed with ACD into the 4 universal tubes. This will make a total of 35 mL
in the tubes.

6. Let the blood sediment at an angle to increase the surface area of the WBCs. Leave for 45-60
minutes for the blood to sediment.

For children, smaller blood volumes can be drawn, depending on feasibility and considering that the
activity is determined according to body weight. In this case, the use of smaller syringes and needles is
advised. For example, you could use multiple 10-mL syringes containing 1.8 mL of ACD.

A2. Isolation of cell-free plasma with:

A2.a) water for injection

To isolate the cell-free plasma, follow these steps:

1. Transfer the blood sediment 2 tubes into one Falcon centrifuge tube.

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2. Centrifuge the tubes at 2500 rpm at room temperature for 10 minutes.

3. Cell-free plasma (CFP) will be the supernatant. Carefully remove the supernatant and discard it.

4. The pellet will contain the white blood cells. Tilt the tube to touch the inside with the tip of the
pipette to resuspended the white blood cells.

5. Add 10 ml of water for injections to the tube and mix gently with the tip of the pipette.

6. Centrifuge the tube again at 2500 rpm for 10 minutes.

A2.b) 0.9% NaCl saline:

To isolate the cell-free plasma, follow these steps:

1. Transfer the blood sediment 2 tubes into one Falcon centrifuge tube.

2. Centrifuge the tubes at 2500 rpm at room temperature for 10 minutes.

3. Cell-free plasma (CFP) will be the supernatant. Carefully remove the supernatant and discard it.

4. The pellet will contain the white blood cells. Tilt the tube to touch the inside with the tip of the
pipette to resuspended the white blood cells.

5. Add 10 ml of 0.9% NaCl saline to the tube and mix gently with the tip of the pipette.

6. Centrifuge the tube again at 2500 rpm for 10 minutes.

A2.c) 0.9% NaCl saline + water for injection :

To isolate the cell-free plasma, follow these steps:

1. Transfer the blood sediment 2 tubes into one Falcon centrifuge tube.

2. Centrifuge the tubes at 2500 rpm at room temperature for 10 minutes.

3. Cell-free plasma (CFP) will be the supernatant. Carefully remove the supernatant and discard it.

4. The pellet will contain the white blood cells. Tilt the tube to touch the inside with the tip of the
pipette to resuspended the white blood cells.

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5. Add 5 ml of 0.9% NaCl saline and 5 ml of water for injection to the tube and mix gently with the tip
of the pipette.

6. Centrifuge the tube again at 2500 rpm for 10 minutes.

B. Labelling of WBCs with 99mTc-HMPAO

Essentials for the procedure

1. 1 x 50mCi of 99mTc-HMPAO freshly prepared with water for injection

2. 1 x 50mCi of 99mTc-HMPAO freshly prepared with 0.9% NaCl saline

To label white blood cells with 99mTc-HMPAO (water for injection), follow these steps:

1. Prepare the 99mTc-HMPAO solution by mixing 50mCi of 99mTc-HMPAO (freshly prepared

with water for injection) with DTPA.

2. Add 0.8 mL of 99mTc-HMPAO solution to the mixed leukocyte cell suspension. Incubate for
20 minutes at room temperature.

3. During incubation, gently swirls the cell suspension to prevent sedimentation of the cells.

4. After incubation, add 5 mL of water for injection and centrifuge for 10 minutes at 1500 rpm.

5. Remove the supernatant, which contains unbound 99mTc-HMPAO and draw 3-5 mL in syringe
“U”.

6. Gently resuspend the pellet containing the labeled mixed leukocytes in 3-5 mL of cell-free
plasma in syringe “A”.

To label white blood cells with 99mTc-HMPAO (0.9% NaCl saline), follow these steps:

1. Prepare the 99mTc-HMPAO solution by mixing 50 mCi of technetium (freshly prepared with
0.9% NaCl saline) with DTPA.

2. Add 0.8 mL of 99mTc-HMPAO solution to the mixed leukocyte cell suspension. Incubate for
20 minutes at room temperature.

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 3. During incubation, gently swirls the cell suspension to prevent sedimentation of the cells.

4. After incubation, add 5 mL of water for injection and centrifuge for 10 minutes at 1500 rpm.

5. Remove the supernatant, which contains unbound 99mTc-HMPAO draw 3-5 mL in syringe
“U”.

6. Gently resuspend the pellet containing the labeled mixed leukocytes in 3-5 mL of cell-free
plasma in syringe “A”.

To label white blood cells with 99mTc-HMPAO (0.9% NaCl saline), follow these steps:

1. Add 0.4 mL of 99mTc-HMPAO solution (freshly prepared with 0.9% NaCl saline) and 0.4 mL
of 99mTc-HMPAO solution (freshly prepared with water for injection). to the mixed leukocyte
cell suspension. Incubate for 20 minutes at room temperature.

1. During incubation, gently swirls the cell suspension to prevent sedimentation of the cells.

2. After incubation, add 2.5 mL of water for injection and 2.5 mL of 0.9% NaCl saline, centrifuge
for 10 minutes at 1500 rpm.

3. Remove the supernatant, which contains unbound 99mTc-HMPAO draw 3-5 mL in syringe
“U”.

4. Gently resuspend the pellet containing the labeled mixed leukocytes in 3-5 mL of cell-free
plasma in syringe “A”.

 Visually inspect the 99mTc-HMPAO-labeled WBCs and reinject them into the patient as soon
as possible, and not later than 1 hour after completion of the labeling procedure.

 Inject the labeled WBCs slowly, preferably using a needle of at least 22 G (0.7 mm diameter) to
prevent cell damage due to shear stress.

 Check the patient's identity prior to administration of the labeled WBCs.

 Clean and disinfect the area. Clean the labeling facility with 70% alcohol. Clean again at the
end of the day and in the morning. Start the labeling facility 1 hour before the procedure

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  Measure the amount of radioactivity in the pellet and in the supernatant to calculate the labeling
efficiency (LE).

DATA ANALYSIS

Statistical Analysis

Stability in Labelled syringe (A):

Labeling stability was assessed by measuring the amount of radioactivity in milli-Currie (mCi)
remaining bound to leukocytes over time.

 Saline solution showed the best stability, with minimal loss of radioactivity

 The mixture of saline and water showed a slight decrease in stability compared to saline
solution.

 The water solution showed the lowest stability, with the greatest loss of radioactivity

4.5 4.267 3.655

4.198 4.23
4 3.281
3.103 3.23 3.112
3.5 3.098
3
2.5 Saline
2 Water
Saline + Water
1.5
1
0
0.5
0 Saline + Water
0
0 Water
Category 1
Category 2
Category 3 Saline

Fig 2: Stability in Labelled Syringe (A)

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Stability in Unlabelled syringe (U):

Labeling stability was assessed by measuring the amount of radioactivity in micro-Currie (uCi)
remaining bound to leukocytes over time.

 Saline solution showed the lowest stability, with the greatest loss of radioactivity

 The mixture of saline and water showed a slight decrease in stability compared to saline
solution.

 Water solution showed the best stability, with minimal loss of radioactivity

1340
1332 1348
1400 965.4
1200
782
779.3
1000
784.7
800 712.3
673.8
600

400

200

0
Category 1
Category 2
Category 3

Saline Water Saline + Water

Fig 3: Stability in Unlabelled Syringe (U)

Findings:

This study demonstrated that Saline solution provided the highest stability for the labelled 99mTc-
HMPAO-WBC complex. This ensured minimal degradation of the radiotracer within the labelled
syringe, which is crucial for maintaining accurate imaging results.

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 Potential Limitations

Despite careful planning and execution, the study may face limitations, such as:

 Small sample size: Depending on the resources available, the study may have a limited number of
participants, which could affect the generalizability of the results.

 Short follow-up period

Future Research

This research can be expanded upon in future studies by:

 Investigating additional solvents and their impact on the labeling process.

 Evaluating the effect of different labeling protocols and incubation times.

Safety and Ethical Considerations:

The sentence mentions important safety and ethical considerations, including:

 Obtaining informed consent from all participants.

 Ensuring participants are adults (over 18 years old).

 Collecting a safe volume of blood (120 ml per participant).

 Privacy and confidentiality will be protected

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 TIMING

YEAR:2023 AUGUST SEPTEMBER DECEMBER

DAYS 15-25 1-15 05 06-12 12-?

Research for
articles and
journals

Literature review

Ethical
Consideration

Data collection
and analysis

Write up

Final submission

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REFERENCES

Palestro CJ, Brown ML, Forstrom LA, et al. Society of Nuclear Medicine procedure guideline for
99mTc-exametazime (HMPAO)-labeled leukocyte scintigraphy for suspected infection/inflammation,
version 3.0. Society of Nuclear Medicine website. snmmi.files.cms-plus.com/docs/HMPAO_ v3.pdf.
Accessed December 24, 2018

Bartel TB, Kuruva M, Gnanasegaran G, et al. SNMMI procedure standard for bone scintigraphy 4.0. J
Nucl Med Technol 2018; 46:398–404

Sharma P, Mukherjee A, Karunanithi S, Bal C, Kumar R. Potential role of 18F-FDG PET/CT in patients
with fungal infections. AJR 2014; 203:180–189

Jamar F, Buscombe J, Chiti A, et al. EANM/ SNMMI guideline for 18F-FDG use in inflammation and
infection. J Nucl Med 2013; 54:647–658

Sampson CB. Complications and difficulties in radiolabelling blood cells: a review. Nucl Med
Commun. 1996 Aug;17(8):648-58. doi: 10.1097/00006231-199608000-00002. PMID: 8878123.

Lewis SS, Cox GM, Stout JE. Clinical utility of indium 111-labeled white blood cell scintigraphy for
evaluation of suspected infection. Open Forum Infect Dis. 2014 Sep 17;1(2):ofu089. doi:
10.1093/ofid/ofu089. PMID: 25734155; PMCID: PMC4281781.

Palestro CJ, Love C, Tronco GG, Tomas MB, Rini JN. Combined labeled leukocyte and technetium
99m sulfur colloid bone marrow imaging for diagnosing musculoskeletal infection. Radiographics.
2006 May-Jun;26(3):859-70. doi: 10.1148/rg.263055139. PMID: 16702459.

Annovazzi A, Bagni B, Burroni L, D′Alessandria C, Signore A. Nuclear medicine imaging of

inflammatory/infective disorders of the abdomen. Nucl Med Commun. 2005;26:657–664.
doi: 10.1097/01.mnm.0000169202.68011.47. [PubMed] [CrossRef] [Google Scholar]

19 | P a g e
Danpure HJ, Osman S, Carroll MJ. The development of a clinical protocol for the radiolabelling of
mixed leucocytes with 99Tcm-hexamethylpropyleneamine oxime. Nucl Med Commun. 1988;9:465–
475. [PubMed] [Google Scholar]

Jacquier-Sarlins MR, Polla BS, Slosman DO. Oxido-reductive state: the major determinant for cellular
retention of technetium-99m-HMPAO. J Nucl Med. 1996;37:1413–1416. [PubMed] [Google Scholar]

Palestro CJ, Brown ML, Forstrom LA, Greenspan BS, McAfee JG, Royal HD, Schauwecker DS,
Seabold JE, Signore A. Society of Nuclear Medicine Procedure Guideline for 99mTc-exametazime
(HMPAO)-labeled

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