Labeled cells for use in imaging

A process is provided for the labeling of viable eucaryotic cells. The process includes the steps of collecting, separating, and introducing a quantity of first type cells into a non-toxic media suitable to conduct an electric field. A labeling material is added to this media. The media containing the first type cells and the labeling material is pulsed for a period of time with an electric field, sufficient to render the cell membranes permeable so that the labeling material permeates the cells and becomes trapped therein upon cessation of the pulsing to produce labeled cells. Also provided is a process for diagnostically imaging pathological sites in a patient. This process contains the additional steps of administering the labeled cells to a patient and monitoring the localization of the labeling material in the patient to image the locus of a specific pathosis.

BACKGROUND OF THE INVENTION 
The present invention relates to the preparation and use for imaging of 
radionuclide-labeled cells. In the last ten years, the use of blood cells 
labeled with radioactive tracers for diagnostic imaging has increased. The 
references cited in this application are incorporated by reference herein 
in their entirety related to the context of their citations. Labeled cells 
are currently being used for an ever increasing variety of applications. 
Erythrocytes, which are the most frequently used labeled cells, have been 
successfully labeled with the radioisotope .sup.99m technetium (.sup.99m 
Tc). .sup.99m Tc-labeled erythrocytes have been used to study cardiac 
function (MUGA scans) and for the detection and localization of 
gastrointestinal bleeding. Unfortunately, a successful procedure for 
labeling other blood cells, e.g., leukocytes and platelets, with .sup.99m 
Tc has yet to be established. 
Leukocytes and platelets have, however, been successfully labeled with the 
radioisotope, .sup.111 indium (.sup.111 In) The use of .sup.111 In-labeled 
leukocytes has gradually increased since their introduction in 1976, and 
research in this area continues to be very active (Milgran, et al., Clin. 
Nucl. Med., Vol. 10, pp. 30-34, 1985). In the Milgran article, .sup.111 
In-labeled lymphocytes were administered to patients with chronic 
inflammatory disease. Whole body gamma-ray camera scans were performed in 
order to image localization of the .sup.111 In-label. Localization of the 
.sup.111 In-label was normally imaged in the spleen, the liver, bone 
marrow, and the cervical and inguinal lymph nodes. Localization of the 
.sup.111 In-label outside of these areas was considered abnormal or 
positive. Patients with chronic osteomyelitis, chronic arthritic disease, 
or chronic bladder inflammation had positive scans. 
.sup.111 In-labeled eosinophils were also used for the detection and 
localization of inflammatory lesions and parasitic infections which could 
not be detected by other diagnostic modalities. (Runge, et al., Nucl. Med. 
Biol. Vol. 12, No. 2, pp. 135-144, 1985). In The Runge Article, .sup.111 
In-labeled eosinophils were used to image the chemotactic response of 
eosinophils to intradermal injections of soluble schistoscoma antigen, S. 
mansoni eggs, E.coli, and turpentine. Gamma-ray cameras were used to image 
the localization of the radiolabel. Soluble schistosoma antigen and s. 
mansoni eggs provided a greater stimulus for localization than E. coli or 
turpentine. The article suggested that .sup.111 In-labeled-eosinophil 
scans were more sensitive to parasitic infections than bacterial 
infections. 
.sup.111 In-labeled leukocytes have been used in the early detection of 
occult infection. (Loken, et al., Clin. Nucl. Med. Vol. 10, No. 12, pp. 
902-911, 1985). In The Loken Article, .sup.111 In-labeled leukocytes were 
used to assess occult infections in more than 1700 patients. Of the 
patients determined to have an occult infection, the sensitivity, 
specificity, and accuracy of the .sup.111 In-labeled leukocyte was 
determined to be 88%, 96%, and 94%, respectively. 
.sup.111 In-labeled lymphocytes have been used to study the disease process 
in patients with chronic lymphocytic leukemia and well differentiated 
lymphoma. (Dutcher, Sem. Nucl. Med., Vol. 14, No. 3, 1984). In the Dutcher 
article, .sup.111 In-labeled lymphocytes were used to study the migration 
of carcinoma cells, normal lymphoid cells, and malignant lymphoid cells in 
patients with malignancy. Determining the migration of these cell types 
was beneficial in helping to understand the disease processes and the 
mechanism of metastasis. 
.sup.111 In-labeled platelets were used for the evaluation of intracoronary 
thrombolysis and the quantitative estimation of platelet thrombosis on 
vascular grafts. (Dewanjee, Sem. Nucl. Med. Vol. 14, No. 3, 1984). In the 
Dewanjee article, .sup.111 In-labeled platelets were used to determine the 
number of adherent platelets on the deendothelialized surfaces of damaged 
cell walls and synthetic vascular grafts. Platelet deposition was recorded 
in denuded tissues, atherosclerotic vessels, and prostheses placed in the 
circulatory system. .sup.111 In-labeled platelets were also used to 
determine platelet consumption during open heart surgery. The article 
additionally described the in vivo evaluation of myocardial infarction 
using .sup.111 In-labeled leukocytes. 
As indicated above, leukocytes and platelets can be successfully labeled 
with .sup.111 In. The current state of technology for labeling leukocytes 
with .sup.111 In involves the use of an .sup.111 In-indium oxine complex. 
The indium oxine portion of the .sup.111 In-indium oxine complex 
penetrates the cell membrane and carries the .sup.111 In into the cell 
interior. 
One disadvantage of the .sup.111 In-indium oxine labeling method is that it 
is toxic to leukocytes. Studies have demonstrated decreased chemotaxis and 
increased leukocyte adherence after .sup.111 In-indium oxine labeling. 
(Shechan et. al., Inter J. Nucl. Med. Bio. Vol. 12 243-247, 1985; 
Linhart-Colas et. al., Brit. J. Hem. Vol. 53, pp. 31-34, 1981). 
Another disadvantage of the .sup.111 In-indium oxine labeling method is 
that it is toxic to platelets. Decreased platelet aggregation has been 
noted after .sup.111 In-indium oxine labeling. 
Yet another disadvantage of the .sup.111 In-labeling method is that 
.sup.111 In is extremely expensive. .sup.111 In is expensive because it is 
produced by a cyclotron. Therefore, clinical studies using .sup.111 In are 
often cost-prohibitive. 
.sup.99m Tc, on the other hand, is an ideal imaging agent for labeling 
leukocytes and platelets. .sup.99m Tc is the most commonly used 
radioisotope in nuclear medicine. .sup.99m Tc is inexpensive ($0.35/mCi) 
because it is produced by a reactor. 
Attempts to label leukocytes and platelets with .sup.99m Tc have failed. 
These attempts have resulted in elution (leaking) of the .sup.99m Tc from 
the cells. (Thakur, et al., Sem. in Nuc. med. Vol 14(2), 107-117, 1984). A 
review article published in 1984 noted that "the challenge of developing a 
.sup.99m Tc cell-labeling agent comparable to the .sup.111 In lipophillic 
chelates has still no: been met." (McAfee et al., Sem. in Nucl. Med. Vol. 
14(2), p.82-105, 1984). Over the last few years many .sup.99m Tc agents 
have been proposed as leukocytes labels but none have enjoyed lasting 
success. (Peters, Nucl. Med. Comm. 8, 313-316, 1987). 
Electroporation (electropermeation) involves the exposure of cells to a 
pulsed electric field. This electric field causes a dielectric breakdown 
of the cell membrane forming pores in the cell membrane. These pores allow 
the transfer of molecules from outside the cell into the cell interior. 
These pores seal upon the cessation of the electric field. Cells remain 
viable, and electron micrographic studies show no damage from the 
electroporation procedure. (Zimmerman, Rev. Physiol. Biochem. pharmacol., 
Vol. 105, pp. 175-257, 1986). 
Electroporation is a technology with many different applications. One 
application of electroporation is the transfection of DNA into plant and 
mammalian cells. This technique was first performed in 1982, and since 
that time there has been much research in this area. (Chu, et. al., Nucl. 
Acid Res. Vol. 15(3), pp. 1311-1326, 1987; Eid, et. al., Proc. Natl. Acad. 
Sci. USA Vol. 84, pp. 7812-7816, 1987). In the Eid article, using 
electroporation, DNA having a molecular weight of four million was 
transfected into procaryotic cells. In another study using 
electroporation, molecules having molecular weights of between 9,000 and 
154,000 passed through cell membranes. (Liang et al., Biotech. Vol. 6, No. 
6, pp. 550-558, 1988). The molecules used by Liang et al. were 
fluorescently labeled dextrans. The label was used to monitor the extent 
of incorporation. 
Erythrocytes have been labeled with C-14 sucrose by electroporation. 
(Kinosita et al., Nature vol. 27 , pp.258-260, 1978). These labeled 
erythrocytes were injected into a mouse where they remained in circulation 
with a normal average half life. There was no apparent elution of the C-14 
sucrose from the erythrocytes. 
Electroporated cells have been suggested as a new drug delivery system. 
(Zimmerman et al., Biochimica et. Biophysica. Acta, Vol. 436, pp. 460-474, 
1976). In the Zimmerman article, the authors disclose a technique for 
loading an enzyme, urease, into human red blood cell ghosts 
(hemoglobin-depleted erythrocytes). In another article, molecules as large 
as tetrasacharides were loaded into viable erythrocytes (not 
hemoglobin-depleted ghosts) without affecting cell viability. (Tsong, et 
al., Biblthca. Haemat., No. 51, pp. 108-114, 1985). 
SUMMARY OF THE INVENTION 
One aspect of the present invention includes a process for producing 
labeled living eucaryotic cells for use in imaging. The inventive process 
includes the steps of collecting an amount of living cells, separating 
from said amount a plurality of first type cells, introducing a quantity 
of said first type cells into a non-toxic media, said media also being 
electrically conductive, adding a labeling material to said media, and 
pulsing said media for a period of time with an electric field sufficient 
to render membranes of said first type cell permeable so that said 
labeling material permeates the cells and becomes trapped therein upon 
cessation of the pulsing to produce labeled cells. 
In another aspect of the present invention a process is provided for 
diagnostically imaging pathological sites. The inventive process includes 
the steps of collecting an amount of living cells, separating from said 
amount a plurality of first type cells, introducing a quantity of said 
first type cells into a non-toxic media, said media being electrically 
conductive, introducing a labeling material into said media (before or 
after the cells), pulsing said media for a period of time with an electric 
field sufficient to render membranes of said first type cells permeable so 
that said labeling material permeates the cells. Upon cessation of the 
pulsing the labeling material becomes entrapped to produce labeled cells. 
The labeled cells are removed from said media and may be administered to a 
patient. The localization of the label of said labeled cells in the 
patient is then monitored to image the locus of a specific pathosis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
This invention relates to the development of a novel method for labeling 
eucaryotic cells and using such labeled cells. Specifically, the present 
invention relates to a process for labeling viable cells, particularly 
eucaryotic cells using a pulsed electric field sufficient to render cell 
membranes permeable so that a labeling material permeates the cells. Upon 
cessation of the pulsing, the labeling material becomes trapped therein to 
produce labeled cells. The invention further relates to the diagnostic 
imaging of pathological sites within the body using labeled viable cells 
produced by the above process. 
Electroporation as a labeling technique offers many advantages over the 
.sup.111 In-indium oxine labeling technique or other prior art labeling 
techniques. Among these advantages include: (1) labels are incorporated 
into the cell cytoplasm; (2) cells remain biologically active in vivo 
after labeling; (3) unlike other prior art labeling techniques, this 
method, when properly done, is not toxic to cells; (4) viable labeled 
cells are provided for the imaging of specific organ systems, occult 
absesses, occult tumors or inflammatory processes, for example; (5) 
radionuclides such as .sup.99m technetium, which are safe, non-toxic to 
the labeled cell, inexpensive and available may be employed; (6) no 
significant elution of the labeling agent from the cell ensues; (7) a 
plurality of labeling agents may be successfully loaded into viable cells 
for imaging purposes; and (8) a plurality of cell types may be 
successfully labeled using this method. 
In one embodiment the labeling process includes the following steps of: (1) 
collecting an amount of living cells; (2) separating from said amount a 
plurality of first type cells; (3) introducing a quantity of said first 
type cells into a non-toxic media, said media being electrically 
conductive; (4) adding a labeling material to said media; and (5) pulsing 
said media with an electric field sufficient to render membranes of said 
first type cell permeable so that said labeling material permeates the 
cells and becomes trapped therein upon cessation of the pulsing to produce 
labeled cells. 
One step of the present invention is the collection of an amount of living 
eucaryotic cells. Generally, the purpose of this step is the collection 
and preparation of living eucaryotic cells for the later separation and 
labeling of a specific type of cell. 
The cells may be collected in a number of different ways. In one embodiment 
using standard venipuncture techniques these cells are collected from a 
human. Such collection may be done, for example, with a standard 18 gauge 
hypodermic needle. 40-60 ml of whole human blood is drawn into a syringe 
containing an anticoagulant. The preferred anticoagulant is acid citrate 
dextrose formula A. 1.6 ml of acid citrate dextrose formula A is added for 
every 10 ml of whole human blood collected. Acid citrate dextrose formula 
A is composed of trisodium citrate 22.0 g, citrate acid 8.0 g, dextrose 
24.5 g, and enough water to make 1000 ml. In acid citrate dextrose formula 
A, the citrate chelates calcium serving as an anticoagulant, the dextrose 
provides a source of energy, and the citrate acid gives the solution a pH 
of about five. To facilitate the separation of cell types, hydroyxethyl 
starch is also added to the syringe. 3 ml of hydroyxethyl starch is added 
for every 10 ml of whole human blood collected. 
In an embodiment involving animal testing, using standard venipuncture 
technique, 20 ml of whole blood are removed from the ear vein of a rabbit. 
To this blood 500 units of benzyl alcohol-free heparin is added. Benzyl 
alcohol-free heparin is utilized as an anticoagulant and is prepared by 
placing 75,000 units of heparin in 1000 ml of 0.9% sodium chloride in 
water. 
Another step in the process is separating from the collected cells a 
plurality of first type cells. These first type cells may be, for example, 
any eucaryotic cell type desired for a particular purpose. Preferably, it 
is a cell type which is included in the whole blood of the animal. More 
preferably, the first type cells may be, for example, human leukocytes 
(for example eosinophils) or platelets. 
In one embodiment the first type cells are human leukocytes. The leukocytes 
are separated from whole blood in the following manner. Red blood cells 
are allowed to sediment at room temperature for 30-60 minutes to produce a 
leukocyte-rich-plasma. The leukocyte-rich plasma is transferred from the 
syringe into a polypropylene tube which is centrifuged at 225 x g for 5 
minutes. This will pellet the leukocytes. The leukocyte pellet is then 
gently resuspended in platelet-free plasma. This will make a suspension of 
leukocytes in plasma. 
In another embodiment, platelets are separated from whole blood in the 
following manner. Whole blood is placed in ten separate 10 ml 
polypropylene tubes. The tubes are centrifuged at 150 x g for 20 minutes. 
The supernatant is platelet-rich plasma. The supernatant plasma is removed 
and placed in six 10 ml polypropylene tubes. The tubes are centrifuged at 
880 x g for 10 minutes to pellet the platelets. The platelet pellets are 
gently resuspended in plasma. 
Another step in the present invention is the introduction of a quantity of 
said first type cells into a non-toxic media which is electrically 
conductive. Although, any non-cytotoxic electrically conductive media may 
be used for this process, platelet-poor plasma is preferred. A media 
including 50% phosphate buffered saline and 50% platelet-poor plasma is 
more preferably used. These media are preferred because they are isotonic 
and are non-toxic to eucaryotic cells. These media are also preferred 
because they may be administered to a subject animal, for example a human, 
without substantial toxicity. 
Still another step is the addition of the labeling material to the media. 
Although a number of different labeling materials are within the scope of 
this invention, radioisotopes and paramagnetic agents are preferred 
labeling materials. More preferably, radioisotopes are utilized as the 
labeling material of choice. .sup.99m technetium is a most preferred 
labeling material. 
Any source of pharmaceutically acceptable .sup.99m technetium agent can be 
used, and a number of technetium radionuclide generators are available. 
Preferably, the .sup.99m technetium agent should be water-soluble, with 
preferred agents being .sup.99m technetium pyrophosphate, .sup.99m 
technetium glucoheptonate or .sup.99m technetium methylene diphosphonate. 
In one embodiment the labeling material is preferably dissolved in saline 
and is added to the first cell type suspension in a ratio of four parts 
first type cell suspension to one part labeling agent in saline. 
The present invention is not limited to preparations of .sup.99m Tc. Other 
radionuclides, such as .sup.95m Tc .sup.123 I and .sup.67 Ga are also 
applicable in the labeling process. Depending on the clinical application, 
compounds labeled with .sup.99m Tc or .sup.99 Tc are ideal scintigraphic 
imaging agents; whereas, .sup.99 Tc-labeled substances may find a wide 
range of applications in in vitro assays. The longer physical half life of 
.sup.95m Tc, (61 days), .sup.123 I (13 hours) or Ga (78 hours) provides an 
added advantage for imaging procedures requiring observation periods of 
various lengths. 
The use of technetium as the preferred labeling material provides an 
advantage over prior art labeling methods. .sup.99m Tc is inexpensive, 
widely available and easily imaged by equipment available in substantially 
all hospitals and research centers. Furthermore, the embodiments of the 
present invention which utilize .sup.99m Tc as the labeling agent are 
non-toxic to the labeled cell. Accordingly, cells remain viable and there 
is no elution of the labeling agent. 
An electric field pulses the media containing the first cell type and the 
labeling agent. This pulsing renders the membranes of the first cell type 
permeable by opening pores in the cell membrane. These pores allow the 
labeling material to permeate the cell. When the pulsing ceases, the pores 
close and the labeling material is trapped in the cell. Labeled cells are 
thereby created. 
In one embodiment, media containing both the first type cells and the 
labeling material is placed in a standard electroporation cuvette. This 
cuvette is placed in an ice-water bath, preferably for ten minutes at 
about 4.degree. C. The cuvette is removed from the ice bath and placed in 
the chamber of a standard electroporation device. The pulse length and 
field strength parameters of the electroporation device are adjusted 
according to the cell type and the labeling material to be used. 
Preferably, the field strength ranges for labeling are no less than about 
1.0 kilovolts/cm to no more than about 20 kilovolts/cm, and the pulse 
length ranges are no less than about 0.01 milliseconds to no greater than 
about 5.0 milliseconds. The higher field strength ranges may be 
particularly useful for labeling bacteria (should a use be found for 
labeled bacteria) or platelets. 
The electrical field is pulsed through the media a number of times, 
depending on pulse length and field strength parameters previously set. 
Preferably the number of pulses is at least one and no greater than about 
twenty. 
The first cell type/labeling material suspension is removed from the 
electroporation apparatus and is placed in an ice water bath to reach a 
temperature of preferably about 4.degree. C. Preferably, the first cell 
type/labeling material suspension remains in the ice water bath for about 
no less than about 10 minutes and about no longer than about 60 minutes. 
The first cell type/labeling material suspension is then placed in an 
environment which approximates room temperature, preferably in a 
polypropylene tube for about 10 minutes. The first cell type/labeling 
material suspension is then placed in a warm water bath, preferably at 
about 37.degree. C. The first cell type/labeling material suspension 
preferably remains immersed in the warm water for at least about 30 
minutes but no longer than about 60 minutes. This allows resealing of the 
cell membrane pores, thereby entrapping the labeling material within the 
cell. 
In another embodiment, viable labeled eucaryotic cells, labeled using the 
process set forth above, are used for the diagnostic imaging of 
pathological sites in a patient. This embodiment contains the additional 
steps of: (1) administering said labeled cells to a patient; and (2) 
monitoring the localization of the label in the patient to image the locus 
of a specific pathosis. 
In one embodiment, after the electrically-induced cell membrane pores are 
allowed to reseal, the first type cells are removed from the suspension 
and washed, preferably twice with 5 ml of platelet-free plasma. The cells 
are then resuspended in platelet-free plasma and the suspension is 
administered to the subject animal. In one embodiment, the labeled cell 
suspension is administered to the subject animal by injection. For 
example, the labeled cell suspension is drawn into a hypodermic syringe, 
preferably through an 18 gauge hypodermic needle, and administered to the 
subject animal via an injection. More preferably, the injection is 
administered intraperitoneally, intaarterially or intrathecally. Most 
preferably, the injection is administered intravenously. After 
administration of the labeled cells, the animal is imaged to determine the 
localization of the labeling agent within the subject animal. Methods of 
radionuclide body imaging of various types are well-known to those skilled 
in the art. In one embodiment the subject animal is a rabbit. In the most 
preferred embodiment the subject animal is a human since human health care 
is an ultimate object of the present invention. 
In one embodiment, first type cells which demonstrate a chemotactic 
affinity for specific loci within the organism are labeled, administered, 
and the localization in an animal imaged. In another embodiment, first 
type cells which are significantly involved in the immune response of the 
organism are labeled, administered, and likewise imaged. These first type 
cells may be interferon-stimulated killer T-cell lymphocytes employed to 
image neoplasms, or helper T-cell lymphocytes employed to image 
autoimmune-disease tissue. In another embodiment, said first type cells 
may be eosinophils employed for the imaging of parasites. In yet another 
embodiment, said first type cells may be platelets employed to image 
emboli. In still another embodiment, said first type cells may be 
leukocytes employed to image localized absesses. In all the above 
embodiments, said first type cells demonstrate a chemotactic affinity for 
a specific pathosis and thus aggregate at that locus. 
The time for imaging is highly variable, and is dependent on the labeling 
material used. Preferably, the animal is imaged at no less than about 15 
minutes and no more than about 24 hours. However, the present invention is 
not limited to these imaging times because some imaging agents such as 
.sup.95m Tc require observation periods of days rather than hours. The 
imaging technique utilized in the present invention is dependent on the 
labeling agent utilized and the imaging equipment available to the 
practitioner. Imaging techniques which may be utilized include single 
proton emission tomography, positron emission tomography, magnetic 
resonance imaging, standard x-ray imaging, computerized axial tomography, 
and standard gamma-ray camera imaging. 
The following examples are presented to describe preferred embodiments and 
utilities of the present invention and are not meant to limit the present 
invention unless otherwise stated in the claims appended hereto. 
EXAMPLE 1 
General Method for Labeling Leukocytes by Electroporation in Plasma with 
.sup.99m Tc-Glucoheptonate 
1. A sample of blood should first be obtained, e.g., from a blood bank or 
from a patient using standard venipuncture technique. Preferably, 40-60 ml 
of whole blood should be collected in a syringe containing the 
anticoagulant, acid citrate dextrose formula A. 1.6 ml of acid citrate 
dextrose formula A is added for every 10 ml of whole blood collected. To 
facilitate the separation of cell types hydroxyethyl starch is added to 
the syringe. 3 ml of hydroxyethyl starch is added for every one ml of 
whole blood collected. 
2. The leukocytes are then separated from the whole blood, e.g., by 
sedimentation and centrifugation. A preferred procedure for separating 
leukocytes is to invert the syringe and gently mix the whole blood. The 
syringe should then be placed at 45.degree. angle with the needle superior 
to the rest of the syringe. This syringe should not be disturbed for about 
30-60 minutes to allow the red blood cells to sediment. The leukocyte-rich 
plasma supernatant is then removed and centrifuged at 225 x g for five 
minutes. This will pellet the leukocytes. 
3. The leukocytes are then placed in a non-toxic electrically conductive 
media, e.g., platelet-free plasma. A preferred procedure for obtaining 
platelet-free plasma is to first obtain whole blood as set forth in step 
1. Then centrifuge the whole blood at 800 x g for 20 minutes. The cells 
and platelets will form a pellet, this should be discarded. The 
supernatant platelet-free plasma should be saved. The leukocyte-pellet of 
step 2 is then gently suspended in the platelet-free plasma. Preferably, a 
30% suspension of cells in plasma is created. 
4. A labeling material is then added to the media (leukocyte suspension), 
e.g., .sup.99m Tc-glucoheptonate. Although, a number of different labeling 
materials may be used for labeling with electroporation, .sup.99m 
Tc-glucoheptonate is used by way of example. To the leukocyte suspension 
add .sup.99m Tc-glucoheptonate in saline in a ratio of four parts 
leukocyte suspension to one part .sup.99m glucoheptonate in saline. 
5. The media should then be pulsed for a period of time with an electric 
field. This field should be sufficient to render the membranes of the 
leukocytes permeable. The .sup.99m Tc-glucoheptonate should then enter the 
leukocyte. The .sup.99m Tc-glucoheptonate will then become trapped within 
the leukocyte upon cessation of the pulsing. 
A preferable procedure to accomplish this is to place 0.8 ml of the 
leukocyte/.sup.99m gluocoheptonate suspension in an electroporation 
cuvette. Then place this cuvette in a 4.degree. C. ice water bath for 10 
minutes. Then remove the cuvette and place it in a electroporation 
chamber. The pulse length and field strength parameters on the 
electroporation apparatus are set. Successful field strength ranges for 
labeling include 2.0 kilovolts/cm to 5.0 kilovolts/cm and pulse lengths 
range from 30 microseconds to 2 milliseconds. The electroporation chamber 
is discharged from 1 to 20 times depending on the pulse length and field 
strength parameters previously set. The leukocyte/.sup.99m 
Tc-glucoheptonate suspension is then removed from the electroporation 
apparatus and placed in a 4.degree. C. ice bath for 10 to 60 minutes. The 
leukocyte/.sup.99m Tc-glucoheptonate suspension is then transferred to a 
new tube and placed at room temperature for 10 minutes. The 
leukocyte/.sup.99m Tc-glucoheptonate suspension is then placed in a 
37.degree. C. water bath for 30 to 60 minutes. This should allow for the 
resealing of the cell membrane pores. A plurality of the leukocytes should 
now be labeled with .sup.99m Tc-glucoheptonate. The cells should be washed 
twice with 5 ml platelet-free plasma to remove the .sup.99m 
Tc-glucoheptonate not incorporated into the leukocytes. 
6. The washed labeled cells are then administered to a patient. A preferred 
procedure for administering the labeled cells to a patient is to first 
count the radioactivity of the leukocyte/.sup.99m Tc-glucoheptonate 
suspension in a dose calibrator, record the value and the percentage of 
cells labeled is calculated. The washed cells are resuspended in 5 ml of 
plasma. The suspension is drawn into a 5 ml syringe for injection into a 
patient, preferably intravenously. 
7. The localization of the labeled cells in the patient is monitored to 
image the locus of a specific pathosis. The patient may be imaged from one 
hour to 48 hours. However, by way of example, leukocytes labeled with 
.sup.99m Tc-glucoheptonate can be imaged at 4 hours and 18 hours. 
EXAMPLE 2 
Labeling leukocytes by electroporation in plasma with .sup.99m 
Tc-glucoheptonate (trial one) 
1. 60 ml of blood was collected from a blood bank. The blood was collected 
in a syringe containing the anticoagulant acid citrate dextrose formula A. 
The syringe contained 1.6 ml of acid citrate dextrose formula for every 10 
ml of whole blood. The facilitate the separation of cell types, 
hydroxyethyl starch was added to the syringe. 3 ml of hydroxyethyl starch 
was added for every 10 ml of whole blood. 
2. The leukocytes were separated from the whole blood collected in step 1. 
To separate the leukocytes from the whole blood the syringe was inverted 
and the whole blood was gently mixed. The syringe was then placed at a 
45.degree. angle with the needle superior to the rest of the syringe. The 
syringe was not disturbed for 30-60 minutes to allow the red blood cells 
to sediment. The leukocyte-rich plasma supernatant was removed from the 
syringe and placed in a 50 ml polypropylene tube. This tube was 
centrifuged at 225 x g for five minutes to pellet the leukocytes. 
3. The separated leukocytes were then placed in a non-toxic, electrically 
conductive media. Platelet-free plasma was obtained by first collecting 20 
ml of whole blood as generally set forth in step 1. This blood was 
centrifuged at 800 x g for 20 minutes. The cells and platelets formed a 
pellet and were discarded. The platelet-free plasma supernatant was saved. 
The leukocyte pellet of step 2 was gently suspended in platelet-free 
plasma in order to make a 30% suspension of cells in plasma. The leukocyte 
suspension was divided into two equal volumes. One volume of the leukocyte 
suspension was used as a control. These volumes of leukocyte suspension 
were treated identically except that the control was never electroporated. 
4. The labeling material, .sup.99m Tc-glucoheptonate, was added to both 
leukocyte suspensions. .sup.99m Tc-Glucoheptonate in saline was added to 
the leukocyte suspensions in a ratio of four parts leukocyte suspension to 
one part .sup.99m Tc-glucoheptonate in saline. 
5. The non-control leukocyte suspension was then pulsed for a period of 
time with an electric field. The field was sufficient to render the cell 
membranes of the leukocytes permeable so that the .sup.99m 
Tc-glucoheptonate could permeate the cells. The .sup.99m Tc-glucoheptonate 
became trapped within the leukocyte upon the cessation of the pulsing. 
This produced labeled cells. 
Specifically, 0.8 ml of the non-control leukocyte/.sup.99m 
Tc-glucoheptonate suspension was placed in a electroporation cuvette (# 
58-017-847, VWR scientific, Houston, TX). This cuvette was placed in 
4.degree. C. ice bath for 10 minutes. The cuvette was then removed and 
placed in the electroporation chamber (0.35 cm gap, 0.8 ml volume, 
transfector 100, BTX, San Diego, CA). The pulse length was set at 0.03 
milliseconds and the field strength at 3.75 kilovolts/cm on the 
electroporation apparatus. The electroporation chamber was discharged 
three times. The cuvette was removed from the electroporation device and 
return to the 4.degree. C. ice water bath for 10-60 minutes. The 
leukocyte/.sup.99m Tc-glucoheptonate suspension was then transferred to a 
5 ml polypropylene tube at room temperature for 10 minutes. The 
leukocyte/.sup.99m Tc-glucoheptonate suspension was then transferred to a 
37.degree. water bath for 30-60 minutes. This allowed for the resealing of 
the cell membrane pores. The control leukocyte/.sup.99m Tc-glucoheptonate 
suspension was also placed in the different water baths for identical time 
periods. 
6. The leukocyte/.sup.99m Tc-glucoheptonate suspensions were removed from 
the 37.degree. C. water bath of step 5 and the radioactivity of the 
suspensions was counted in a dose calibrator and the values were recorded. 
The leukocytes of each suspension were then washed twice with 5 ml of 
(platelet-free plasma to remove any 99mTc-glucoheptonate not incorporated 
into the leukocytes. The radioactivity for both the groups of washed 
leukocytes was determined in the dose calibrator. These values were 
recorded and the percentage of leukocytes labeled with .sup.99m 
Tc-glucoheptonate was calculated for both the control and the non-control 
(electroporated) leukocytes. 
______________________________________ 
Results 
______________________________________ 
Radioisotope 99mTc-Glucoheptonate 
Cell Type Leukocytes 
Kilovolts Per Centimeter 
3.75 kv/cm 
Pulse Length 0.03 msec 
Number of Pulses 3 
Pulsed Cells Labeled 
3.6% 
Control Cells Labeled 
0% 
Viability of Leukocytes* 
87% 
______________________________________ 
*Trypan blue viability study 
EXAMPLE 3 
Labeling leukocytes by electroporation in plasma with .sup.99m 
Tc-glucoheptonate (trial two) 
(a) Example 3 followed the experimental protocol of Example 2. 
______________________________________ 
Results 
______________________________________ 
Radioisotope 99mTc-Glucoheptonate 
Cell Type Leukocytes 
Kilovolts Per Centimeter 
3.75 kv/cm 
Pulse Length 0.03 msec 
Number of Pulses 3 
Pulsed Cells Labeled 
3.3% 
Control Cells Labeled 
.64% 
Viability of Leukocytes* 
90% 
______________________________________ 
*Trypan blue viability study 
EXAMPLE 4 
Labeling of leukocytes by electroporation in plasma using .sup.99m 
Tc-methylene diphosphonate 
(a) Example 4 follows the experimental protocol of Example 2 with the 
following exceptions. .sup.99m Tc-methylene diphosphonate was substituted 
for .sup.99m Tc-glucoheptonate as the labeling agent in step 4. The pulse 
length was set at 0.50 milliseconds in step 5. The field strength was set 
at 3.10 kilovolts/cm in step 5. The number of pulses was 10 in step 5. 
______________________________________ 
Results 
______________________________________ 
Radioisotope 99mTc-MDP 
Cell Type Leukocytes 
Kilovolts Per Centimeter 
3.10 kv/cm 
Pulse Length 0.50 msec 
Number of Pulses 10 
Pulsed Cells Labeled 4.1% 
Control Cells Labeled .7% 
Viability of Leukocytes* 
85% 
______________________________________ 
*Trypan blue viability study 
EXAMPLE 5 
Labeling leukocytes by electroporation in plasma using .sup.67 gallium 
citrate 
(a) Example 4 followed the experimental procedure of example 2 with the 
following exceptions. .sup.67 Gallium citrate was substituted as the 
labeling agent for .sup.99m Tc-glucoheptonate in step 4. Pulse length was 
set at 0.50 milliseconds in step 5. Field strength was set at 3.00 
kilovolts/cm in step 5. The number of pulses was 10 in step 5. 
______________________________________ 
Results 
______________________________________ 
Radioisotope 67-Gallium Citrate 
Cell Type Leukocytes 
Kilovolts Per Centimeter 
3.00 kv/cm 
Pulse Length 0.50 msec 
Number of Pulses 10 
Pulsed Cells Labeled 
3.1% 
Control Cells Labeled 
0% 
Viability of Leukocytes* 
80% 
______________________________________ 
*Trypan blue viability study 
EXAMPLE 6 
Labeling leukocytes by electroporation in plasma using .sup.99m 
Tc-pyrophosphate 
(a) The process of this example followed the experimental procedure of 
example 2 with the following exceptions. .sup.99m Tc-pyrophosphate was 
substitute for .sup.99m Tc-glucoheptonate as the labeling agent in step 4. 
The pulse length was set at 2.39 milliseconds in step 5. The field 
strength was set at 2.00 kilovolts/cm in step 5. The number of pulses was 
1 in step 5. 
______________________________________ 
Results 
______________________________________ 
Radioisotype 99mTc-Pyrophosphonate 
Cell Type Leukocytes 
Kilovolts Per Centimeter 
2.00 kv/cm 
Pulse Length 2.39 msec 
Number of Pulses 1 
Pulsed Cells Labeled 
8.7% 
Control Cells Labeled 
1.6% 
Viability of Leukocytes* 
91% 
______________________________________ 
*Trypan blue viability study 
EXAMPLE 7 
Labeling erythrocytes by electroporation in plasma using .sup.99m 
Tc-methylene diphosphonate 
(a) Example 7 follows the experimental procedure of example 2 with 
following exceptions. In step 2 erythrocytes were separated by 
sedimentation and saved. The leukocyte pellet of step 2 was discarded. 
Erythrocytes were then substitute for the leukocytes of steps 3, 4, 5, and 
6. .sup.99m Tc-methylene diphosphonate was substituted for .sup.99m 
Tc-glucoheptonate as the labeling agent in step 4. The pulse length was 
2.39 milliseconds in step 5. The field strength was 2.00 kilovolts/cm in 
step 5. The number of pulses was one. 
______________________________________ 
Results 
______________________________________ 
Radioisotope 99mTc-MDP 
Cell Type Erythrocytes 
Kilovolts Per Centimeter 
3.10 kv/cm 
Pulse Length 0.03 msec 
Number of Pulses 3 
Pulsed Cells Labeled 5.2% 
Control Cells Labeled .6% 
Viability of Leukocytes* 
88% 
______________________________________ 
*Trypan blue viability study 
EXAMPLE 8 
General method for labeling platelets by using electroporation in plasma 
1. A sample of blood should first be obtained, e.g. from a blood bank or 
from a patient with an 18 gauge needle, using standard venipuncture 
technique. Preferably, 40-60 ml of whole blood is collected in a syringe 
containing the anticoagulant acid citrate dextrose formula A. 1 .6 ml of 
acid citrate dextrose formula A is added for every 10 ml of whole blood 
collected. 
2. The platelets should then be separated from the whole blood collected. 
Plastic pipettes and plastic tubes will be used for pipetting and 
centrifuging in this procedure. A preferred procedure for separating the 
platelets from the whole blood is to first transfer the whole blood from 
the syringe to ten separate 10 ml polypropylene tubes. Then centrifuge the 
tubes at 150 x g for 20 minutes. This will create platelet-rich plasma 
supernatant. Remove the supernatant plasma and place in six 10 ml 
polypropylene tubes and centrifuge at 800 x g for 10 minutes. This will 
pellet the platelets. Remove the platelet-free plasma supernatant and 
store at 37.degree. C. for later use. 
3. The platelets will then be introduced into a non-toxic electrically 
conductive media, e.g., platelet-free plasma. Preferably, the platelet 
pellets is placed in the platelet-free plasma from step 2 and gently 
suspended to make suspension of platelets in plasma. Combine the platelet 
suspensions from the six tubes into one polypropylene 5 ml tube. 
4. A labeling agent is then added to the platelet suspension. Although 
numerous labeling agents may be successfully used with the inventive 
method, .sup.99m Tc-pyrophosphate will be used by way of example. To the 
platelet suspension add .sup.99m Tc-pyrophosphate in saline in a ratio of 
4 parts leukocyte suspension to 1 part .sup.99m Tc-pyrophosphate in 
saline. 
5. The platelet/.sup.99m Tc-pyrophosphate suspension should then be pulsed 
for a period of time with an electric field. This electric field will 
cause the cell membranes of the platelet to become permeable. The .sup.99m 
Tc-pyrophosphate will then permeate the platelets. Upon cessation of the 
electric field the .sup.99m Tc-pyrophosphate will become trapped within 
the platelets to produce labeled platelets. A preferred procedure to 
accomplish this is to first add 0.8 ml of the platelet/.sup.99m 
Tc-pyrophosphate suspension to an electroporation cuvette (#358-017-847, 
VWR Scientific, Houston, TX). This cuvette is then placed in a 4.degree. 
C. ice water bath for 10 minutes. The cuvette is then removed and placed 
it in the electroporation chamber (0.35 cm gap, 0.8 ml volume, Transfector 
100, BTX, San Diego, CA). The pulse length and field strength parameters 
on the electroporation apparatus should be set. Field strength ranges for 
labeling will include 2.0 kilovolts/cm to 5.0 kilovolts and pulse length 
ranges will include 2.0 milliseconds to 4.0 milliseconds. The 
electroporation chamber should be pulsed from one time to 10 times. After 
cessation of the electric field, the platelet/.sup.99m Tc-pyrophosphate 
suspension should be removed from the electroporation apparatus and 
returned to the ice water bath for 10-60 minutes. The platelet/.sup.99m 
Tc-pyrophosphate suspension in the tube should then be transferred to a 
37.degree. C. water bath for 30-60 minutes. This should allow for the 
resealing of the cell membrane pores. A plurality of platelets should now 
be labeled with .sup.99m Tc-pyrophosphate. 
6. The percentage of cells labeled is then calculated. The suspension is 
removed from the 37.degree. C. water bath of step 5 and the radioactivity 
is counted in a dose calibrator and the value recorded. The platelets are 
then washed twice with 5 ml of platelet-free plasma to remove any .sup.99m 
Tc-pyrophosphate not incorporated in the platelets. The radioactivity of 
the washed cells is then counted in the dose calibrator. This value is 
recorded and the percentage of cells labeled is calculated. 
7. The labeled platelets are then administered to a patient. The labeled 
washed platelets should be suspended in 5 ml of plasma and drawn into a 5 
ml syringe for injection. The platelet suspension is then injected into 
the patient. Preferably, the labeled platelets are injected intravenously. 
8. The localization of the label in the labeled platelet is then monitored 
to image the locus of a specific pathosis. The patient may be imaged from 
one hour to 48 hours. However, by way of example, platelets labeled with 
.sup.99m Tc-pyrophosphate can be imaged at 4 and 18 hours. 
EXAMPLE 9 
Labeling platelets by using electroporation in plasma 
1. A sample of blood was obtained from a blood bank. 40-60 ml of whole 
blood was collected in a syringe containing the anticoagulant acid citrate 
dextrose Formula A. 1.6 ml of acid citrate dextrose Formula A was added 
for every 10 ml of whole blood collected. 
2. The platelets were separated from the whole blood. Plastic pipettes and 
plastic tubes were used for pipetting and centrifuging in this procedure. 
The whole blood collected in step 1 was transferred from the syringe to 
ten separate 10 ml polypropylene tubes. These tubes were then centrifuged 
at 15 x g for 20 minutes to create platelet-rich plasma in the 
supernatant. The supernatant plasma was removed and placed into six 10 ml 
polypropylene tubes and centrifuged at 800 x g for 10 minutes to pellet 
the platelets. The platelet-free plasma supernatant was removed and stored 
at 37.degree. C. for later use. 
3. The platelets were then introduced into a non-toxic electrically 
conductive media, e.g., platelet-free plasma. The platelet pellets from 
step 2 were gently suspended in the platelet-free plasma from step 2 to 
make a suspension of platelets in plasma the concentration of 200 
K/microliter. The platelet suspensions from the six tubes were combined 
into one polypropylene 5 ml tube. 
4. A labeling agent was then added to the platelet suspension, e.g., 
.sup.99m Tc-pyrophosphate. .sup.99m Tc-pyrophosphate in saline was added 
to the platelet suspension in a ratio of 4 parts leukocyte suspension to 1 
part .sup.99m Tc-pyrophosphate n saline. The platelet/.sup.99m 
Tc-pyrophosphate suspension was then divided equally into two samples. One 
sample became the control sample which was not electroporated, and one 
sample became the non-control sample which was electroporated. Otherwise 
both the control and non-control sample were treated identically. 
5. The non-control platelet/.sup.99m Tc-pyrophosphate suspension was pulsed 
with a electric field. 0.8 ml of the platelet/.sup.99m Tc-pyrophosphate 
suspension was added to an electroporation cuvette (#358-017-847, VWR 
Scientific, Houston, TX). This cuvette was placed in a 4.degree. C. ice 
water bath for 10 minutes. The cuvette was then removed and placed it in 
the electroporation chamber (0.35 cm gap, 0.8 ml volume, Transfector 100, 
BTX, San Diego, CA). The pulse length and field strength parameters on the 
electroporation apparatus were set. The field strength was set at 2.1 
kilovolts/cm and the pulse length was set at 2.4 milliseconds. The 
electroporation chamber was pulsed one time. After cessation of the 
electric field the platelet/.sup.99m Tc-pyrophosphate suspension was 
removed from the electroporation apparatus and returned to the ice water 
bath for 10-60 minutes. The platelet/.sup.99m Tc-pyrophosphate suspension 
in the tube was then transferred to a 37.degree. C. water bath for 30-60 
minutes. This allowed for the resealing of the cell membrane pores. A 
plurality of platelets were labeled with .sup.99m Tc-pyrophosphate. 
6. The percentage of cells labeled in the control and non-control samples 
was calculated. The suspensions were removed from the 37.degree. C. water 
baths of step 5 and their radioactivity was counted in a dose calibrator 
and the values were recorded. The platelets were then washed twice with 5 
ml of platelet-free plasma to remove .sup.99m Tc-pyrophosphate not 
incorporated into the platelets. The radioactivity of the washed cells was 
then counted in the dose calibrator. The values were recorded and the 
percentage of cells labeled was calculated for both the control and 
non-control samplers. 
7. A standard platelet agglutination study was then done in order to 
determine the viability of both control and non-control (labeled cells) 
cells. The platelet agglutination curves demonstrated normal platelet 
activity in response to bovine thombin, indicating good platelet viability 
for both the control and non-control cells (labeled). 
The electroporated platelets prepared by this procedure demonstrated 250% 
greater percent label of .sup.99m Tc-pyrophosphate than the control 
platelets. Also, the electroporated platelets demonstrated no adverse 
affects from the labeling procedure. 
Changes may be made in the construction, operation and arrangement of the 
various cells, labels, steps and procedures described herein without 
departing from the concept and scope of the invention as defined in the 
following claims.