Method of labelling leucocytes with Tc-99m, d,1-HMPAO

An improved method for radio-labelling leucocytes in vitro with Tc-99m d,1-HMPAO (Technetium-99m-d,1-hexamethyl propyleneamine oxime) for use in scintigraphic imaging of inflammatory lesions enhances the labelling efficiency by incubating the leucocytes with Tc-99m d,1-HMPAO in the presence of an acid citrate dextrose solution and by depleting residual RBCs from the leucocytes prior to such incubation step. The RBC depletion is accomplished by means of a rocking step.

The invention relates to methods for radiolabelling leucocytes in vitro 
with gamma-emitting radionuclides. The radiolabelled leucocytes are 
employed for imaging inflammatory lesions by scintigraphy after injection 
into a patient. More particularly, the invention relates to methods for 
enhancing the labelling efficiency of Tc-99m d,1-HMPAO 
(Technetium-99m-d,1-hexamethyl-propyleneamine oxime) with respect to the 
labelling of leucocytes in vitro while maintaining sterility and 
viability. 
BACKGROUND 
Methods for radiolabelling human leucocytes with gamma emitters have been 
reviewed by H. J. Danpure and S. Osman ("A Review of Methods of Separation 
and Radiolabelling Human Leucocytes," Nuclear Medicine Communications 
(1988); vol. 9, 681-685). Danpure and Osman indicate that successful 
radiolabelling methods employ neutral lipophilic agents which chelate the 
selected gamma emitting radionuclide and carry the radionuclide across the 
cellular membrane into the patient's or donor's isolated leucocytes. Once 
inside the leucocyte, the lipophilic chelator may be degraded or otherwise 
modified so as to produce a hydrophilic radionuclide product. The 
hydrophilic radionuclide product remains entrapped within the leucocytes 
for a period sufficiently long to allow rapid injection of the labelled 
leucocytes into the patient, migration of the labelled leucocytes to 
inflamation sites, and scintigraphic imaging of inflamation sites by means 
of the labelled leucocytes. Danpure and Osman indicate that the 
gamma-emitting radionuclide Indium-111 oxine has been successfully 
employed in clinical settings for radiolabelling mixed leucocytes for 
scintigraphic imaging. 
As an alternative to Indium-111 oxine, A. M. Peters et al. introduced 
Tc-99m d,1-HMPAO (Technetium-99m-d,1-hexamethylpropylene-amine oxime) for 
radiolabelling leucocytes ("Preliminary Clinical Experience with Tc-99m 
d,1-HMPAO for Labelling Leucocytes and Imaging Infection," Lancet (1986); 
vol. 11, 945-949) Technetium-99m is a widely available generator-produced 
radionuclide ideally suited for gamma detection. Use of Tc-99m d,1-HMPAO 
can produce a good scintigraphic image at a relatively low radiation 
dosage. The conversion of Tc-99m d,1-HMPAO from a lipophilic form to a 
hydrophilic form has been characterized by Joseph C. Hung et al. ("Kinetic 
Analysis of Technetium-99m d,1-HM-PAO Decomposition in Aqueous Media," 
Journal of Nuclear Medicine (1988); vol. 29, 1568-1576). 
An advanced method for labelling leucocytes with Technetium-99m d,1-HM-PAO 
has been disclosed by N. N. Solanki et al. ("A Rapid Method for the 
Preparation of TC99-m Hexametazine-Labelled Leucocytes," Nuclear Medicine 
Communications (1988); vol. 9, 753-761). Solanki et al. indicate that 
leucocytes are best separated from whole blood by a 1.times.g 
sedimentation followed by a gentle (100.times.g) centrifugation of the 
resultant leucocyte platelet-rich plasma (LPRP). The resultant pellet is 
then re-suspended in 10% cell free plasma (CFP) and incubated with Tc-99m 
d,1-HMPAO. Solanki et al. claim a labelling efficiency of 25.9% when 
employing the recommended reconstitution volume and a labelling efficiency 
of 49% when employing a low reconstitution volume. 
Achieving a labelling efficiency in excess of 25.9% with Tc-99m d,1-HMPAO 
while employing the recommended reconstitution volume is desirable since 
enhanced labelling efficiency reduces the size of the blood sample which 
must be drawn from the patient, enhances the resultant image quality, and 
decreases the amount of Technetium-99m d,1-HM-PAO reagent which must be 
employed, and minimizes contamination by red blood cells (RBCs). 
SUMMARY 
The efficiency of labelling leucocytes with Tc-99m d,1-HMPAO may be 
significantly enhanced by carefully controlling the incubation conditions 
of the labelling step. In particular, the depletion of residual red blood 
cells (RBCs) from the leucocytes prior to the labelling step significantly 
enhances the labelling efficiency. Also, the presence of Acid Citrate 
Dextrose solution (ACD) added to the leucocytes during the labelling step 
further enhances labelling efficiency. A combination of RBC depletion and 
ACD addition increases the labelling efficiency of leucocytes with respect 
to Tc-99m d,1-HMPAO to a level of 80-95%, using Solanki's recommended 
reconstitution volume. 
DETAILED DESCRIPTION OF THE INVENTION 
A preferred protocol for high efficiency labelling of 
leucocytes with Tc-99m d,1-HMPAO is provided as follows: 
1. A blood sample is drawn from the patient using a 60 milliliter syringe. 
Prior to drawing the blood sample, an aliquot of ACD solution should be 
drawn into the syringe. ACD solution serves as an anticoagulant and 
conventionally has a composition which includes 25 grams of sodium 
citrate, 8.75 grams of citric acid, and 6 grams dextrose per liter of 
sterile water for injection. After the blood is drawn into the syringe the 
ratio of blood volume to anticoagulant should not exceed 5:1. For example, 
42 milliliters of blood may be drawn into a 60 milliliter syringe which 
has been preloaded with 8 milliliters of ACD solution. 
2. The syringe is then positioned with its needle pointed vertically upward 
so as to allow the blood to sediment (1.times.g) for 30-45 minutes. During 
sedimentation, the RBCs settle to the bottom of the syringe toward the 
plunger while leucocyte rich plasma remains at the top of the syringe 
proximate to the needle. In an alternative embodiment, sedimentation of 
the RBCs may be accelerated by the addition of Hespan (TM) (6% hetastarch 
in 0.9% sodium chloride, DuPont, Del.). 
3. The leucocyte rich plasma is then expressed from the syringe into a 
first centrifuge tube, preferrably a conventional 50 milliliter disposable 
centrifuge tube having a conical bottom and a composition of 
polypropylene. The leucocyte rich plasma is then centrifuged at 
450.times.g for 7 minutes in order to pellet the leucocytes while forming 
a supernatant of platlet rich plasma (PRP). 
4. The PRP supernatant is then withdrawn from the first centrifuge tube and 
transferred to a second centrifuge tube. The second centrifuge tube is 
then centrifuged at 950.times.g for 10 minutes to form platlet poor plasma 
(PPP). The PPP is then saved for use in steps 12 and 13 below. 
5. In the meantime, the leucocyte pellet from the first centrifuge tube is 
resuspended in 4 milliliters of isotonic saline, i.e. 0.9% sodium chloride 
in sterile water for injection. Care should be taken when resuspending the 
leucocyte pellet not to swirl too vigorously so as to cause the formation 
of foam. 
6. The first centrifuge tube containing the resuspended leucocytes is then 
mounted onto a rocker arm for separating residual RBCs from the 
leucocytes. The preferred rocker arm may be constructed by modifying a 
"Hema Tek" (TM) Aliquot Mixer, Model 4651, manufactured by the Ames 
Division of Miles Laboratories, Inc. (Elkhart, Ind.). The unmodified "Hema 
Tek" (TM) Aliquot Mixer consists of a tilting table, an actuating motor 
and a base. The platform tilts back and forth at 12 or 18 cycles per 
minute for mixing specimen samples. Other similar rocking devices may be 
obtained from Clay Adams (Nutator Mixer model #1105) and from American 
Scientific Products (American (TM) Tube Rocker catalog #R4185-10). Prior 
to modification, the tilting table cradles the specimen tubes in a 
horizontal position. In the preferred mode for constructing the rocker 
arm, the tilting table of the "Hema Tek" (TM) Aliquot Mixer is modified so 
as to securely brace one or more centrifuge tubes in a vertical position. 
When the rocker arm is activated, the centrifuge tubes oscillate around 
this vertical position. The motion of a single vertical centrifuge tube 
mounted upon the rocker arm is similar to that of a metronome which 
oscillates at 18 cycles per minute. The leucocyte suspension is allowed to 
rock upon the rocker arm for 15 minutes. During this rocking step, the 
leucocytes settle to the bottom of the centrifuge tube while the residual 
RBCs remain in the supernatant. The exact biochemical mechanism for this 
separation is unknown. It is speculated that the RBCs undergo a 
flocculation reaction or are otherwise hindered from settling with the 
leucocytes due to a surface charge phenomenon. 
7. After rocking for 15 minutes, the rocker arm is stopped. The leucocytes 
are then depleted of residual RBCs by carefully removing and discarding 
the top 1 or 2 milliliters of supernatant. The volume of supernatant which 
is removed is then replaced with an equal volume of isotonic saline and 
the leucocytes are resuspended. 
8. The resuspended leucocytes are then rocked for 10 more minutes to 
separate out further residual RBCs. After 10 minutes, the rocker arm is 
again stopped. Again the rocking step has caused the leucocytes to settle 
to the bottom and the residual RBCs to remain within the supernatant. 
After the rocker arm has been stopped, the leucocytes are then further 
depleted of residual RBCs by carefully removing the top 1 or 2 milliliters 
of supernatant. 
9. Then 2 milliliters of a 10% diluent of the ACD solution is added to the 
leucocyte suspension. The presence of ACD during the Tc-99m d,1-HMPAO 
incubation (step 11) enhances the efficiency of labelling. The 
concentration of the ACD in the leucocyte suspension should not exceed a 
volume to volume ratio of 1:10 as exposure to excess ACD may detract from 
the subsequent viability of the leucocytes. 
10. In the meanwhile, Tc-99m d,1-HMPAO may be prepared from a Ceretec (TM) 
kit supplied by Amersham International plc. (Amersham, Buckinghamshire, 
England). The kit includes 0.5 micrograms of d,1-4, 
8-diaza-3,6,6,9,-tetramethyl-undecane-2,10-dione bioxime (d,1-HMPAO), 7.6 
micrograms stannous chloride dihydrate, and 4.5 milligrams sodium 
chloride, freeze-dried and stoppered under nitorgen. Technetium 
pertechnetate may be obtained from a commercial Mo-99m/Tc-99m generator. 
Tc-99m d,1-HMPAO is prepared using 30 millicuries (1110 MBq) of sodium 
[Tc-99m]pertechnetate drawn into a vial containing 2 milliliters of 
isotonic saline. Only eluate obtained less than 2 hours from a generator 
eluted within a 24 hour period should be used or per package insert 
instructions. Ceretec (d,1-HMPAO) is then tagged by chelation to 
[Tc-99m]pertechnetate as specified by the manufacturer to form Tc-99m 
d,1-HMPAO. As a quality control for this chelation reaction, the yield of 
lipophilic product, i.e. Tc-99m d,1-HMPAO, may be determined by 
appropriate chromatographic systems or procedures using an appropriate 
solvent. 
11. The leucocytes of step 9 are then labelled by the addition of Tc-99m 
d,1-HMPAO. The mixture is incubated for 15 minutes and swirled every 5 
minutes. The addition of ACD to the leucocytes in step 9 enhances the 
efficiency of this reaction. The efficiency of the reaction is further 
enhanced by the depletion of residual RBCs in steps 5-8. The precise 
biochemical mechanism for this enhancement is unknown. However, it is 
thought that RBCs and platelets contain reducing agents which prematurely 
degrade the Tc-99m d,1-HMPAO. Also, the suspension of the leucocytes in 
step 9 enhances the efficiency of the labelling reaction by increasing the 
access of the leucocytes with respect to the labelling reagents. For 
patients with normal and elevated white cell counts, the labelling 
efficiency of this improved labelling method may rise within the range of 
80-95%. 
12. After the 15 minute incubation, 1 milliliter of platelet poor plasma 
(PPP) is added to the labelled leucocytes as part of a wash step. The 
leucocytes and PPP are mixed by swirling. The leucocytes are then 
separated from unreacted Tc-99m d,1-HMPAO by centrifugation at 450.times.g 
for 7 minutes. The supernatant is then removed and discarded. 
13. The washed pellet of labelled leucocytes is then resuspendend in 
platelet poor plasma (PPP) or saline solution (0.9% sodium chloride) to 
which 10% ACD solution may be added. The resuspended leucocytes should be 
injected into the patient as soon as possible, i.e. within 1 hour or not 
to exceed approximately 5 hours from time of the original blood 
collection. 
The invention revolves around the preparation of the leucocyte sample for 
labelling with the Tc-99m d,1-HMPAO, i.e. the depletion of residual RBCs 
from the leucocytes (steps 5-8) prior to the labelling step and the 
addition of ACD (step 9). It is the addition of these steps which enables 
the labelling step to achieve a significant enhancement of the labelling 
efficiency. The above protocol is merely exemplary of a preferred mode for 
practicing these aspects of the invention. For example, the protocol may 
be modified with good results by eliminating the second rocking step, i.e. 
step 8, and deleting the addition of isotonic saline in step 7.

EXAMPLE 
Comparative tests were performed for determining the relative enhancement 
of tagging efficiency with and without the addition of ACD during 
incubation (Step 9) and with and without the rocker steps for depleting 
RBCs from the leucocytes prior to incubation (steps 5-8), i.e. ACD (step 
9):: saline (step 9 eliminated):rocker (steps 5-8)::no rocker (steps 5-8 
eliminated). The measured tagging efficiency of samples which had not been 
depleted of RBCs prior to incubation (steps 5-8) were determined both with 
and without a subsequent RBC depletion step. Samples from which RBCs had 
not been depleted included a significant contaminant of tagged RBCs. (This 
is a measure of the RBC contaminant characteristic of prior art methods.) 
Samples from which RBCs had been depleted were substantially without any 
contaminant of tagged RBCs. Tagging efficiency is a measure of label 
incorporation and was calculated by the method of Solanki et al. (supra). 
The tests were performed with blood taken from a healthy human volunteer 
having a relatively low white cell count. Accordingly, the overall tagging 
efficiencies were lower than normal. The labelling protocol followed the 
example provided above with the addition or deletion of steps 5-8 and step 
9 and with the addition of the hetastarch option in step 2. The test 
results are provided as follows: 
______________________________________ 
Tagging Tagging 
Efficiency RBC Efficiency 
w/o RBC Contam- with RBC 
Conditions Depletion ination Depletion 
______________________________________ 
1. ACD & Rocker 56% 
2. ACD & no Rocker 
69% 18% 51% 
3. Saline & Rocker 43% 
4. Saline & no Rocker 
54% 23% 31% 
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