THROMBOSIS RESEARCH 57; 737-7451990 0049-3848/90 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
RADIOLABELLING
OF PLATELETS
WITH TECHNETIUM - 99m
E. Sundrehagen*, P. Urdal**, D.-E. Heggli***, M. Winderen Lindegaard*, and E. Jacobsen****. The Norwegian Radium Hospital,Oslo, *Department of Nuclear Medicine, **Central Laboratory, Ullev&l University Hospital, Oslo, ***Institute for Energy Technology, Kjeller, and ****Department of Hematology and Lymphology, The Norwegian Radium Hospital, Oslo, Norway. (Received 54.1989; accepted in revised form 27.12.1989 by Editor U. Abildgaard)
ABSTRACT A method for labelling of platelets with technetium-99m (Tc-99m) is presented. In principle, aminobenzoic acid and tartaric acid are used as reagents, allowing Tc-99m complexes of intermediate chemical stability to be formed. These complexes react rapidly with proteins, such as platelet proteins, when added. We have examined the isolation procedure for the platelets and the labelling procedure using residual aggregational ability and residual content of 8thromboglobulin (B-TG) as indicators of damage to the platelets. In its final version the method allowed a 32.6 +/- 2.7% (mean +/- SD) incorporation of Tc-99m into platelets which again showed a 66 +/15% residual aggregational ability, tested by 50 /rmol/l of ADP, and a 79 +/- 17% residual content of B-TG releasable by 10 IU/ml of thrombin. In a pilot clinical study involving 28 patients we found labelled autologous platelets useful in detecting lung embolism and deep vein thrombosis.
INTRODUCTION Platelets labelled with Cr-51 have been used for many years in cell kinetic studies and for in vitro experiments. Thakur et al (1) were the first to develop a method for radiolabelling of platelets with In-l 11 and this implied a progress in utilizing nuclear medicine techniques for imaging of thromboembolic disease. However, In-111 is not always present in the laboratory, is expensive and has unfavorable physical properties and this has limited the use of the method. Technetium-99m (Tc-99m) is in many ways a more promising label because it has favorable radiation characteristics (low radiation doses, improved images due to high counting rate), is cheap and is readily available as generator eluates in most hospital nuclear medicine departments. Though Sam et al (2) recently described a promising animal study with Tc-99m-labelled antibody fragments reactive with human platelets, a practical method for direct Tc-99m labelling of platelets has up to now not been available. 737
738
Tc-99m LABELLED PLATELETS
Vol. 57, No. 5
Sundrehagen & al. have previously described a method for direct labelling of leukocytes (3) or granulocytes (4) with Tc-99m and the use of the method in abscess detection. In this work we present a modification of the method which allows us to label platelets using kit reagents of pharmaceutical quality and we have tested the in vitro function of the labelled platelets. All reagents used are well-known compounds of very low in vivo toxicity. In a pilot study we have also examined whether autologous platelets labelled this way may be used to detect thromboembolic disease.
MATERIALS
AND
METHODS
the reagents necessary for one radiolabellingReagent vials - each containing were prepared by dissolving 1.0 g 4-amino-benzoic acid, 10.6 mg stannous water which had been tartrate and 2.0 mg sodium tartrate in 1.0 1 sterile bubbled with nitrogen. pH was adjusted to 7.0 using sodium hydroxide. The solution was passed through a sterile filter of 0.22 .a and 0.5 ml was transferred to each vial followed by lyophilization. The contents of the reagent vials thus formed were stable for at least 8 months at 4 degrees C.
Labelling Drocedure: Venous blood (24 ml) was withdrawn into three 10 ml ACD-containing vacuum tubes (Venoject, Terumo, Belgium). The tubes were centrifuged (150 g, 15 minutes, ambient temperature) and the platelet rich plasma (PRP) was transferred to plastic tubes. After centrifugation (700 g, 10 minutes, ambient temperature) the supernatant was removed and the platelets were gently suspended in a sterile citrate buffer containing 84 mg glucose, 100 pg sodium citrate dihydrate, 8.85 g sodium chloride and 0.16 ml 0.01 M HCl per liter aqueous solution, followed by another centrifugation at 700 g for 10 minutes and removal of the supernatant. The content of one reagent vial - immediately before use dissolved in 0.8 - 1.0 ml Tc-99m (300 - 500 MBq) solution eluted from a Mo-99/Tc-99mgenerator purchased from Institute for Energy Technology, Kjeller, Norway - was then added to the platelets and the mixture was gently swirled for 15 minutes at room temperature. Following addition of 4 ml of citrate buffer and centrifugation (700 g, 10 minutes, ambient temperature) the platelets were finally resuspended in 2 ml citrate buffer (for clinical studies) or 4 ml platelet-free autologous plasma (for the in vitro studies). In some experiments, the PRP was layered on top of a 1 ml density gradient centrifugation medium (158.1 g iohexol, Nycomed, Norway, 0.6 g Trizma TRISbase, 30 mg sodium citrate dihydrate, 222 mg potassium chloride, 3.2 g sodium chloride and 4.2 ml 1 M hydrochloric acid per liter aqueous solution) before the first 700 g centrifugation. The platelets were counted in an Ortho ELT 800 cell counter. Their aggregability, prior to and after radiolabelling, was tested as described by Haslam (5) using a Payton aggregometer and ADP (50 /.mrolll, final cont.) to induce aggregation. The aggregometer was calibrated exactly as described by Haslam, and the residual aggregability in the platelets was measured and compared to a corresponding number of untreated platelets from platelet-rich plasma, and the residual aggregability was thus calculated as a percentage of the untreated platelets; The content of B-thromboglobulin of the platelets, prior to and after radiolabelling, was estimated by adding thrombin (10 W/ml, final cont.) into the plasma, centrifuging it (1700 g, 30 minutes, ambient temperature) and measuring the concentration of betathromboglobulin in the
Vol. 57, No. 5
Tc-99m LABELLED PLATELETS
supernate by radioimmunoassay (Amersham, England). The amount of betathromboglobulin secreteable with 10 IU thrombin per ml could then be measured in samples of platelet suspensions. By correction for platelet number, the amount of secreteable betathromboglobulin after platelet isolation and after labelling could be compared to the contents of the betathromboglobulin of the platelets prior to isolation and labelling.
Clinical studies: We examined 23 patients with malignant disease who exhibited clinical signs of lung disease by injecting in a cubital vein autologous platelets containing 100 - 200 MBq Tc-99m. Whole body scintigraphy was performed 0.5, 3 and 20 h. after injection. The patients suspected to have lung embolism had in addition already been subjected to lung perfusion scintigraphy with 50 MBq Tc-99m-labelled macroaggregates, to chest X-ray and to chemical pathological evaluation. In these patients the investigative procedure was started up immediately after the first clinical signs on thromboembolic disease were observed. We also examined 6 patients with symptoms and clinical signs of acute deep vein thrombosis. In these patients autologous platelets containing 50 - 100 MBq Tc-99m were injected intravenously on the dorsum of each foot, and whole body scintigraphy was performed 0.5, 3 and 20 h after injection.
RESULTS
Many different combinations of carboxylic acids and benzoic acid derivatives may potentially be used to obtain the Tc-99m complexes and in preliminary experiments we tested several of them. The present combination was chosen because the complexes here formed were of intermediate stability; they did not hydrolyse quickly and yet they reacted vigorously with platelet proteins or other proteins when added. In addition, the method appeared suitable because it allowed only a limited amount of radiocolloids from oxidized stannum and of reduced technetium-99m to be formed. Radiolabellinn of platelets and testing of their in vitro functions: Platelets isolated from a blood volume in the range of B-40 ml were efficiently labelled and contained the same concentration of B-TG releasable by 10 IU/ml of thrombin ( fig. 1). From this a volume of approx. 25 ml was chosen because such a volume may well be handled and at the same time allows platelets also from patients with thrombocytopenia to be labelled without modifying the method. The present method is essentially a protein labelling method. Therefore the platelets must be separated from the plasma proteins prior to the labelling procedure, as is well illustrated by the results of fig.2. Thus, here we obtained a 14 percent incorporation of Tc-99m into the platelets when these had been isolated from plasma only by centrifugation (fig. 2, zero washing), but a 42 percent incorporation when the platelets in addition had been washed three times prior to the labelling. However, the same figure demonstrates how the platelets ability to aggregate, decreased with extensive washing. The content of i3-TG in the platelets releasable with 10 IU/ml of thrombin present decreased correspondingly. The Tc-99m we observed
complexes appear to react quickly with the platelet proteins since a marginal increase in Tc-99m incorporated when the only
739
740
Tc-99m LABELLED PLATELETS
,OI
25,
.F .g E P
Vol. 57, No. 5
I,,,
,I 8
16
24
40
Volume of blood (ml)
Fip. 1: Effect . .__. of increasing blood volume upon viability and amount of Tc-99m incorporated. From blood of each of 3 healthy donors the platelets were isolated and radiolabelled and were tested for residual content of B-thromboglobulin (ml, aggregability (~1 and residual fraction of Tc-99m incorporated (01. At each blood volume mean value and observed range are presented.
0 Number
1
2
3
of washings
Fin 2: Effect of increasing numberof washing of platelets upon viability and amount of Tc-99m incorporated. Symbols as in fig. 1.
labelling time was increased from 5 to 30 minutes __(fig. 3). This figure also .~ shows that an increase in labelling time does not affect neither aggregational ability, tested by 50 poll1 of ADP, nor releasable content of B-TG. To examine the direct effect of Tc-99m upon the platelets, we “labelled” them but in the absence of Tc-99m. Platelets thus treated showed the same modest reduction in the said aggregational ability and releasable content of B-TG (to 59 and 77 percent) as did platelets labelled by the ordinary method (64 and 76 percent). These results and those of fig. 3 both indicate that damage to the platelets is mostly made through the isolation procedure and insignificantly through the labelling procedure. We tried to reduce this damage, caused by the mechanical handling of the platelets, by layering a density gradient centrifugation medium below the plasma prior to centrifugation. Through this we obtained platelets with slightly improved aggregability. However, an extra washing of the platelets was subsequently required if an equally high amount of Tc-99m was to be incorporated. Based on these experiments we decided to isolate the platelets from 24 ml of blood to wash them once and to use a radiolabelling time of 15 minutes. This method, summarized in materials and methods, was subsequently used with platelets obtained from 8 healthy volunteers and 10 patients. The patients suffered from various desease and were not identical to the ones examined during the clinical study ( cfr. below). Several of the had cancer and 2 of them showed marked thrombocytopenia (41 and 63 x 10r8 per 1). With all these preparations of platelets the method functioned well; an acceptable incorporation of Tc-99m was obtained and both aggregability tested by 50 molll of ADP and amount of R-TG secreteable with 10 IU/ml of thrombin present was well preserved (table 1).
Vol. 57. No. 5
Tc-99m IABELLED
PLATELETS
741
25,
I
I
1
10 15 Time of radiolabelling 5
I
30 (minutes )
Fig. 3. Effect of increasing time for radiolabelling upon viability and amount of Tc-99m incorporated. Symbols and text as in fig.1.
Table
1.
In vitro
Healthy (n=8)
function
of platelets
labelled
with Technetium.
Technetium incorporated
Residual aggregability
(% of total)
Ltlu$
-___--__--_
-___--_---__-
_-___-___-___
32.3 +I- 2.6 (27.7 - 34.5)
64 +I- 12 (44 - 81)
72 +I- 20 (43 - 101)
31.0 +I- 2.8 (27.8 - 35.7)
67 +/- 18 (48 - 106)
84 +/- 14 (53 - 100)
initial
Remaining B-thromboglobulin secreteable LtluEf initial
volunteers
Patients (n=lO) Mean +/- standard
deviation
and (range) are given.
Clinical studies using radiolabelled nlatelets: We examined 28 patients with malignant disease, 13 suspected to have lung embolism following surgery due to cancer, 3 with lung metastases, 2 with pneumonia, 4 with hearth failure, 5 suspected for having deep vein thrombosis of the legs and 1 operated with ligation of the upper branch of the right pulmonary artery and partial lobectomy. In one patient, who had been subjected to ligation of the upper
742
Tc-99m LABELLED PLATELETS
Vol. 57. No. 5
branch of the right pulmonary artery, we knew with certainty the presence of a thromboembolic process and its location. In this patient the perfusion scintigram showed a defect and the platelet scintigram showed a focal accumulation at the site of the ligated artery (fig.4). None of the remaining 9 patients with non-embolic lung diseases showed accumulation of radioactive platelets in the lungs. Six of the 13 patients suspected of having lung embolism were shown to suffer from this, five of them demonstrated a focal accumulation of platelets ( Tab. 2, pat. no 1-3). The other 7 patients were shown to suffer from other diseases and only one of them showed focal accumulation of platelets . Also for the patient suspected for having deep vein thrombosis, results from venography using radiolabelled platelets corresponded well with the final clinical diagnoses based on X-ray venography and chemical pathological ( tab. 3). Fig.5 illustrates the findings made in one of these evaluation patients. However, the number of patients is small, and a number of vascular diseases in the legs can be expected to interfere with studies with radiolabelled platelets in the legs, possibly causing false negative and false positive results. Loss of label: To examine whether the Tc-99m could be released after labelling, we incubated platelets in autologous plasma at room temperature. In three separate experiments we observed a release of 25 % (range 22-30%) of the Tc-99m after 24 hours, similar to results previously reported (6). No further characterization of the chemical nature of the released Tc-99m was performed.
Fig. 4: Scintigrams demonstrating accumulation of radiolabelled platelets in the right lung, corresponding to the site of ligation of the artery after removal of the upper right lobe ( due to cancer).
Fig. 5: Scintigrams demonstrating focal accumulations of radiolabelled platelets in a patient suffering from deep vein thrombosis.
Vol. 57, No. 5
743
Tc-99m LABELLED PLATELETS
Table
Laboratory
2:
suffering
frcm
Pat.no.
lung
findings
in 13 patients
initially
suspected
of
embolism.
Perfusion
Lung
Final
Scintigraphy
x-ray
diagnosis
clinical
Scintigraphy
(a)
with
radio-
labelled platelets.
__-_---~-----~ 1.
negative
perfusion
lung
focal
embolism
accumulations
defects
2.
lung
negative
perfusion
embolism
focal accumulations
defects
3.
possibly
initially:
small
liquid
defects
in the
right
neg.
lung
focal
embolism
accumulations later
lung
4.
5.
perfusion
small
probably
defects
densities
embolism
lung
accumulations
focal
perfusion
liquid
lung
focal
embolism(b)
accumulations
6.
negative
7.
lung
heart
no focal
congestion
failure
accumulation
generally
pleural
heart
no focal
decreased
liquid
failure
accumulation
perfusion
and
lung
right
side
congestion
8.
uncertain
heart
liquid
failure
no focal accumulation
negative
9.
10.
tumor
perfusion
and
tumor
lymphoedema
negative
chest
defect
11.
12.
and
liquid
possibly
opacities,
perfusion
probably
defects
metastases
probably
metastases
no focal accumulation
no focal
pain
embolism?
accumulation
uncertain
no focal accumulation
no focal
metastases
accumulation
negative
perfusion
13.
no focal
pneumonia
accumulation
defects
(a)
Based
chemical tion
on
findings
pathological
angiography.
made
evaluation.
by
lung (b)
X-ray.
Later
perfusion
confirmed
by
scintigraphy digital
and
subtrac-
744
Tc-99m LABELLED PLATELETS
Tab. 3: Laboratory findings in five thrombosis of the leg(s): Pat.no.
_______ 1.
patients
Vol. 57, No. 5
suspected
Results from X-ray venography
Full clinical diagnosis (a)
for having deep vein Venography using radiolabelled platelets ___________ bilateral accumulations
________________
______________
bilateral thrombosis
bilateral thrombosis
2.
not performed (allergy)
thrombosis
focal accumulations
3.
thrombosis
thrombosis
focal accumulations
4.
thrombosis
thrombosis
focal accumulations
5.
negative
negative
negative
(a) Based on X-ray
venography
and clinical
pathological
evaluation.
DISCUSSION The present results indicate that platelets may be labelled with Tc-99m by means of a simple kit procedure. We found it necessary to isolate the platelets from blood prior to the labelling, to obtain a sufficient incorporation of radioactivity. Such mechanical handling does cause some damage to the platelets, as has also been observed by others (7). This handling of the platelets probably is the main obstacle for the use of the method. The labelling method itself turned out to be a gentle one and did not give rise to any further damage of the platelets. The results obtained in our small clinical study indicate that the labelled platelets function well also in vivo. Technetium has a half-life of 6 hours, which is short, at least when compared with other radioactive labels used in hematology, and nuclear medicine, such as Cr-51, I-125 and In-l 11. Technetium-labelled platelets may therefore probably prove useful mainly when used within a few hours, e.g. in vivo medical imaging examinations, and in this context the release of Tc-99m should be of less importance. However, more long lasting studies such as cell kinetic studies may require a label different from Tc-99m. In the pilot clinical study we used each patients own platelets, isolated from blood and radiolabelled, to detect thromboembolic disease. The procedure required about 1.5 - 2 hours of work, most of the time being used to prepare the platelets prior to the labelling. If Tc-99m labelled platelets turn out to be useful reagents, platelet concentrates available from blood banks might instead be used as source of platelets. This would reduce the time required for preparation and would allow simultaneous labelling of a larger number of platelets, which then would be available for several examinations. In nuclear medicine departments this would imply that radiolabelled platelets may be immediately available, like most other reagents, on a 24 hours basis.
Tc-99m LABELLED PLATELETS
Vol. 57, No. 5
We found deep vein examined results to
the radiolabelled platelets useful for detecting lung embolism and thrombosis in patients with malignant disease. However, we have not patients with other kinds of disease to investigate whether similar those here presented may be obtained in general.
Acknowledgement: acknowledged.
The technical
assistance
of Olaug Seweriin
is gratefully
REFERENCES
1. THAKUR M.L., WELCH M.J., JOIST J.H. & al. Indium 111 labeled platelets: Studies on preparation and evaluation of in vitro and in vivo functions. Thrombosis res. 9:345-357, 1976. 2. SOM P., OSTER Z.H., ZAMORA P.O. & al. Radioimmunoimaging of Experimental Thrombi in Dogs using Technetium-99m-labelled Monoclonal Antibody Fragments Reactive with Human Platelets. J Nucl Med 27:1315-1320, 1986.
3. SUNDREHAGEN E., BENESTAD H.B., HEIKKILA R. 8~ al. A new method for rapid technetium-99m labelling of leukocytes: Functional cell studies in vitro. Stand J Clin Lab Invest 45:717-724, 1985. 4. SUNDREHAGEN E., BENGTSSON A.-M., BREMER P.O. & al. A new method for granulocyte labelling with Technetium-99m: Preliminary results in abscess detection. J Nucl Med 27:555-559, 1986. 5. HASLAM R. J. Role of adenosine diphosphate platelets by thrombin and fatty acids. Nature
in the aggregation of human 202:765-768, 1964.
6. SUNDREHAGEN E. A new technique for formation of Tc-99m-labelled leukocytes and platelets. Int J Appl Radiat Isot 35:365-366, 1984.
blood
7. THAKUR M.L., WALSH L. & GOTTSCHALCK A. Indium-111-labelled human platelets: Improved method, efficacy and evaluation. J Nucl Med 22:381-385, 1981.
RADIOLABELLING
OF PLATELETS
WITH TECHNETIUM - 99m
E. Sundrehagen*, P. Urdal**, D.-E. Heggli***, M. Winderen Lindegaard*, and E. Jacobsen****. The Norwegian Radium Hospital,Oslo, *Department of Nuclear Medicine, **Central Laboratory, Ullev&l University Hospital, Oslo, ***Institute for Energy Technology, Kjeller, and ****Department of Hematology and Lymphology, The Norwegian Radium Hospital, Oslo, Norway. (Received 54.1989; accepted in revised form 27.12.1989 by Editor U. Abildgaard)
ABSTRACT A method for labelling of platelets with technetium-99m (Tc-99m) is presented. In principle, aminobenzoic acid and tartaric acid are used as reagents, allowing Tc-99m complexes of intermediate chemical stability to be formed. These complexes react rapidly with proteins, such as platelet proteins, when added. We have examined the isolation procedure for the platelets and the labelling procedure using residual aggregational ability and residual content of 8thromboglobulin (B-TG) as indicators of damage to the platelets. In its final version the method allowed a 32.6 +/- 2.7% (mean +/- SD) incorporation of Tc-99m into platelets which again showed a 66 +/15% residual aggregational ability, tested by 50 /rmol/l of ADP, and a 79 +/- 17% residual content of B-TG releasable by 10 IU/ml of thrombin. In a pilot clinical study involving 28 patients we found labelled autologous platelets useful in detecting lung embolism and deep vein thrombosis.
INTRODUCTION Platelets labelled with Cr-51 have been used for many years in cell kinetic studies and for in vitro experiments. Thakur et al (1) were the first to develop a method for radiolabelling of platelets with In-l 11 and this implied a progress in utilizing nuclear medicine techniques for imaging of thromboembolic disease. However, In-111 is not always present in the laboratory, is expensive and has unfavorable physical properties and this has limited the use of the method. Technetium-99m (Tc-99m) is in many ways a more promising label because it has favorable radiation characteristics (low radiation doses, improved images due to high counting rate), is cheap and is readily available as generator eluates in most hospital nuclear medicine departments. Though Sam et al (2) recently described a promising animal study with Tc-99m-labelled antibody fragments reactive with human platelets, a practical method for direct Tc-99m labelling of platelets has up to now not been available. 737
738
Tc-99m LABELLED PLATELETS
Vol. 57, No. 5
Sundrehagen & al. have previously described a method for direct labelling of leukocytes (3) or granulocytes (4) with Tc-99m and the use of the method in abscess detection. In this work we present a modification of the method which allows us to label platelets using kit reagents of pharmaceutical quality and we have tested the in vitro function of the labelled platelets. All reagents used are well-known compounds of very low in vivo toxicity. In a pilot study we have also examined whether autologous platelets labelled this way may be used to detect thromboembolic disease.
MATERIALS
AND
METHODS
the reagents necessary for one radiolabellingReagent vials - each containing were prepared by dissolving 1.0 g 4-amino-benzoic acid, 10.6 mg stannous water which had been tartrate and 2.0 mg sodium tartrate in 1.0 1 sterile bubbled with nitrogen. pH was adjusted to 7.0 using sodium hydroxide. The solution was passed through a sterile filter of 0.22 .a and 0.5 ml was transferred to each vial followed by lyophilization. The contents of the reagent vials thus formed were stable for at least 8 months at 4 degrees C.
Labelling Drocedure: Venous blood (24 ml) was withdrawn into three 10 ml ACD-containing vacuum tubes (Venoject, Terumo, Belgium). The tubes were centrifuged (150 g, 15 minutes, ambient temperature) and the platelet rich plasma (PRP) was transferred to plastic tubes. After centrifugation (700 g, 10 minutes, ambient temperature) the supernatant was removed and the platelets were gently suspended in a sterile citrate buffer containing 84 mg glucose, 100 pg sodium citrate dihydrate, 8.85 g sodium chloride and 0.16 ml 0.01 M HCl per liter aqueous solution, followed by another centrifugation at 700 g for 10 minutes and removal of the supernatant. The content of one reagent vial - immediately before use dissolved in 0.8 - 1.0 ml Tc-99m (300 - 500 MBq) solution eluted from a Mo-99/Tc-99mgenerator purchased from Institute for Energy Technology, Kjeller, Norway - was then added to the platelets and the mixture was gently swirled for 15 minutes at room temperature. Following addition of 4 ml of citrate buffer and centrifugation (700 g, 10 minutes, ambient temperature) the platelets were finally resuspended in 2 ml citrate buffer (for clinical studies) or 4 ml platelet-free autologous plasma (for the in vitro studies). In some experiments, the PRP was layered on top of a 1 ml density gradient centrifugation medium (158.1 g iohexol, Nycomed, Norway, 0.6 g Trizma TRISbase, 30 mg sodium citrate dihydrate, 222 mg potassium chloride, 3.2 g sodium chloride and 4.2 ml 1 M hydrochloric acid per liter aqueous solution) before the first 700 g centrifugation. The platelets were counted in an Ortho ELT 800 cell counter. Their aggregability, prior to and after radiolabelling, was tested as described by Haslam (5) using a Payton aggregometer and ADP (50 /.mrolll, final cont.) to induce aggregation. The aggregometer was calibrated exactly as described by Haslam, and the residual aggregability in the platelets was measured and compared to a corresponding number of untreated platelets from platelet-rich plasma, and the residual aggregability was thus calculated as a percentage of the untreated platelets; The content of B-thromboglobulin of the platelets, prior to and after radiolabelling, was estimated by adding thrombin (10 W/ml, final cont.) into the plasma, centrifuging it (1700 g, 30 minutes, ambient temperature) and measuring the concentration of betathromboglobulin in the
Vol. 57, No. 5
Tc-99m LABELLED PLATELETS
supernate by radioimmunoassay (Amersham, England). The amount of betathromboglobulin secreteable with 10 IU thrombin per ml could then be measured in samples of platelet suspensions. By correction for platelet number, the amount of secreteable betathromboglobulin after platelet isolation and after labelling could be compared to the contents of the betathromboglobulin of the platelets prior to isolation and labelling.
Clinical studies: We examined 23 patients with malignant disease who exhibited clinical signs of lung disease by injecting in a cubital vein autologous platelets containing 100 - 200 MBq Tc-99m. Whole body scintigraphy was performed 0.5, 3 and 20 h. after injection. The patients suspected to have lung embolism had in addition already been subjected to lung perfusion scintigraphy with 50 MBq Tc-99m-labelled macroaggregates, to chest X-ray and to chemical pathological evaluation. In these patients the investigative procedure was started up immediately after the first clinical signs on thromboembolic disease were observed. We also examined 6 patients with symptoms and clinical signs of acute deep vein thrombosis. In these patients autologous platelets containing 50 - 100 MBq Tc-99m were injected intravenously on the dorsum of each foot, and whole body scintigraphy was performed 0.5, 3 and 20 h after injection.
RESULTS
Many different combinations of carboxylic acids and benzoic acid derivatives may potentially be used to obtain the Tc-99m complexes and in preliminary experiments we tested several of them. The present combination was chosen because the complexes here formed were of intermediate stability; they did not hydrolyse quickly and yet they reacted vigorously with platelet proteins or other proteins when added. In addition, the method appeared suitable because it allowed only a limited amount of radiocolloids from oxidized stannum and of reduced technetium-99m to be formed. Radiolabellinn of platelets and testing of their in vitro functions: Platelets isolated from a blood volume in the range of B-40 ml were efficiently labelled and contained the same concentration of B-TG releasable by 10 IU/ml of thrombin ( fig. 1). From this a volume of approx. 25 ml was chosen because such a volume may well be handled and at the same time allows platelets also from patients with thrombocytopenia to be labelled without modifying the method. The present method is essentially a protein labelling method. Therefore the platelets must be separated from the plasma proteins prior to the labelling procedure, as is well illustrated by the results of fig.2. Thus, here we obtained a 14 percent incorporation of Tc-99m into the platelets when these had been isolated from plasma only by centrifugation (fig. 2, zero washing), but a 42 percent incorporation when the platelets in addition had been washed three times prior to the labelling. However, the same figure demonstrates how the platelets ability to aggregate, decreased with extensive washing. The content of i3-TG in the platelets releasable with 10 IU/ml of thrombin present decreased correspondingly. The Tc-99m we observed
complexes appear to react quickly with the platelet proteins since a marginal increase in Tc-99m incorporated when the only
739
740
Tc-99m LABELLED PLATELETS
,OI
25,
.F .g E P
Vol. 57, No. 5
I,,,
,I 8
16
24
40
Volume of blood (ml)
Fip. 1: Effect . .__. of increasing blood volume upon viability and amount of Tc-99m incorporated. From blood of each of 3 healthy donors the platelets were isolated and radiolabelled and were tested for residual content of B-thromboglobulin (ml, aggregability (~1 and residual fraction of Tc-99m incorporated (01. At each blood volume mean value and observed range are presented.
0 Number
1
2
3
of washings
Fin 2: Effect of increasing numberof washing of platelets upon viability and amount of Tc-99m incorporated. Symbols as in fig. 1.
labelling time was increased from 5 to 30 minutes __(fig. 3). This figure also .~ shows that an increase in labelling time does not affect neither aggregational ability, tested by 50 poll1 of ADP, nor releasable content of B-TG. To examine the direct effect of Tc-99m upon the platelets, we “labelled” them but in the absence of Tc-99m. Platelets thus treated showed the same modest reduction in the said aggregational ability and releasable content of B-TG (to 59 and 77 percent) as did platelets labelled by the ordinary method (64 and 76 percent). These results and those of fig. 3 both indicate that damage to the platelets is mostly made through the isolation procedure and insignificantly through the labelling procedure. We tried to reduce this damage, caused by the mechanical handling of the platelets, by layering a density gradient centrifugation medium below the plasma prior to centrifugation. Through this we obtained platelets with slightly improved aggregability. However, an extra washing of the platelets was subsequently required if an equally high amount of Tc-99m was to be incorporated. Based on these experiments we decided to isolate the platelets from 24 ml of blood to wash them once and to use a radiolabelling time of 15 minutes. This method, summarized in materials and methods, was subsequently used with platelets obtained from 8 healthy volunteers and 10 patients. The patients suffered from various desease and were not identical to the ones examined during the clinical study ( cfr. below). Several of the had cancer and 2 of them showed marked thrombocytopenia (41 and 63 x 10r8 per 1). With all these preparations of platelets the method functioned well; an acceptable incorporation of Tc-99m was obtained and both aggregability tested by 50 molll of ADP and amount of R-TG secreteable with 10 IU/ml of thrombin present was well preserved (table 1).
Vol. 57. No. 5
Tc-99m IABELLED
PLATELETS
741
25,
I
I
1
10 15 Time of radiolabelling 5
I
30 (minutes )
Fig. 3. Effect of increasing time for radiolabelling upon viability and amount of Tc-99m incorporated. Symbols and text as in fig.1.
Table
1.
In vitro
Healthy (n=8)
function
of platelets
labelled
with Technetium.
Technetium incorporated
Residual aggregability
(% of total)
Ltlu$
-___--__--_
-___--_---__-
_-___-___-___
32.3 +I- 2.6 (27.7 - 34.5)
64 +I- 12 (44 - 81)
72 +I- 20 (43 - 101)
31.0 +I- 2.8 (27.8 - 35.7)
67 +/- 18 (48 - 106)
84 +/- 14 (53 - 100)
initial
Remaining B-thromboglobulin secreteable LtluEf initial
volunteers
Patients (n=lO) Mean +/- standard
deviation
and (range) are given.
Clinical studies using radiolabelled nlatelets: We examined 28 patients with malignant disease, 13 suspected to have lung embolism following surgery due to cancer, 3 with lung metastases, 2 with pneumonia, 4 with hearth failure, 5 suspected for having deep vein thrombosis of the legs and 1 operated with ligation of the upper branch of the right pulmonary artery and partial lobectomy. In one patient, who had been subjected to ligation of the upper
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Tc-99m LABELLED PLATELETS
Vol. 57. No. 5
branch of the right pulmonary artery, we knew with certainty the presence of a thromboembolic process and its location. In this patient the perfusion scintigram showed a defect and the platelet scintigram showed a focal accumulation at the site of the ligated artery (fig.4). None of the remaining 9 patients with non-embolic lung diseases showed accumulation of radioactive platelets in the lungs. Six of the 13 patients suspected of having lung embolism were shown to suffer from this, five of them demonstrated a focal accumulation of platelets ( Tab. 2, pat. no 1-3). The other 7 patients were shown to suffer from other diseases and only one of them showed focal accumulation of platelets . Also for the patient suspected for having deep vein thrombosis, results from venography using radiolabelled platelets corresponded well with the final clinical diagnoses based on X-ray venography and chemical pathological ( tab. 3). Fig.5 illustrates the findings made in one of these evaluation patients. However, the number of patients is small, and a number of vascular diseases in the legs can be expected to interfere with studies with radiolabelled platelets in the legs, possibly causing false negative and false positive results. Loss of label: To examine whether the Tc-99m could be released after labelling, we incubated platelets in autologous plasma at room temperature. In three separate experiments we observed a release of 25 % (range 22-30%) of the Tc-99m after 24 hours, similar to results previously reported (6). No further characterization of the chemical nature of the released Tc-99m was performed.
Fig. 4: Scintigrams demonstrating accumulation of radiolabelled platelets in the right lung, corresponding to the site of ligation of the artery after removal of the upper right lobe ( due to cancer).
Fig. 5: Scintigrams demonstrating focal accumulations of radiolabelled platelets in a patient suffering from deep vein thrombosis.
Vol. 57, No. 5
743
Tc-99m LABELLED PLATELETS
Table
Laboratory
2:
suffering
frcm
Pat.no.
lung
findings
in 13 patients
initially
suspected
of
embolism.
Perfusion
Lung
Final
Scintigraphy
x-ray
diagnosis
clinical
Scintigraphy
(a)
with
radio-
labelled platelets.
__-_---~-----~ 1.
negative
perfusion
lung
focal
embolism
accumulations
defects
2.
lung
negative
perfusion
embolism
focal accumulations
defects
3.
possibly
initially:
small
liquid
defects
in the
right
neg.
lung
focal
embolism
accumulations later
lung
4.
5.
perfusion
small
probably
defects
densities
embolism
lung
accumulations
focal
perfusion
liquid
lung
focal
embolism(b)
accumulations
6.
negative
7.
lung
heart
no focal
congestion
failure
accumulation
generally
pleural
heart
no focal
decreased
liquid
failure
accumulation
perfusion
and
lung
right
side
congestion
8.
uncertain
heart
liquid
failure
no focal accumulation
negative
9.
10.
tumor
perfusion
and
tumor
lymphoedema
negative
chest
defect
11.
12.
and
liquid
possibly
opacities,
perfusion
probably
defects
metastases
probably
metastases
no focal accumulation
no focal
pain
embolism?
accumulation
uncertain
no focal accumulation
no focal
metastases
accumulation
negative
perfusion
13.
no focal
pneumonia
accumulation
defects
(a)
Based
chemical tion
on
findings
pathological
angiography.
made
evaluation.
by
lung (b)
X-ray.
Later
perfusion
confirmed
by
scintigraphy digital
and
subtrac-
744
Tc-99m LABELLED PLATELETS
Tab. 3: Laboratory findings in five thrombosis of the leg(s): Pat.no.
_______ 1.
patients
Vol. 57, No. 5
suspected
Results from X-ray venography
Full clinical diagnosis (a)
for having deep vein Venography using radiolabelled platelets ___________ bilateral accumulations
________________
______________
bilateral thrombosis
bilateral thrombosis
2.
not performed (allergy)
thrombosis
focal accumulations
3.
thrombosis
thrombosis
focal accumulations
4.
thrombosis
thrombosis
focal accumulations
5.
negative
negative
negative
(a) Based on X-ray
venography
and clinical
pathological
evaluation.
DISCUSSION The present results indicate that platelets may be labelled with Tc-99m by means of a simple kit procedure. We found it necessary to isolate the platelets from blood prior to the labelling, to obtain a sufficient incorporation of radioactivity. Such mechanical handling does cause some damage to the platelets, as has also been observed by others (7). This handling of the platelets probably is the main obstacle for the use of the method. The labelling method itself turned out to be a gentle one and did not give rise to any further damage of the platelets. The results obtained in our small clinical study indicate that the labelled platelets function well also in vivo. Technetium has a half-life of 6 hours, which is short, at least when compared with other radioactive labels used in hematology, and nuclear medicine, such as Cr-51, I-125 and In-l 11. Technetium-labelled platelets may therefore probably prove useful mainly when used within a few hours, e.g. in vivo medical imaging examinations, and in this context the release of Tc-99m should be of less importance. However, more long lasting studies such as cell kinetic studies may require a label different from Tc-99m. In the pilot clinical study we used each patients own platelets, isolated from blood and radiolabelled, to detect thromboembolic disease. The procedure required about 1.5 - 2 hours of work, most of the time being used to prepare the platelets prior to the labelling. If Tc-99m labelled platelets turn out to be useful reagents, platelet concentrates available from blood banks might instead be used as source of platelets. This would reduce the time required for preparation and would allow simultaneous labelling of a larger number of platelets, which then would be available for several examinations. In nuclear medicine departments this would imply that radiolabelled platelets may be immediately available, like most other reagents, on a 24 hours basis.
Tc-99m LABELLED PLATELETS
Vol. 57, No. 5
We found deep vein examined results to
the radiolabelled platelets useful for detecting lung embolism and thrombosis in patients with malignant disease. However, we have not patients with other kinds of disease to investigate whether similar those here presented may be obtained in general.
Acknowledgement: acknowledged.
The technical
assistance
of Olaug Seweriin
is gratefully
REFERENCES
1. THAKUR M.L., WELCH M.J., JOIST J.H. & al. Indium 111 labeled platelets: Studies on preparation and evaluation of in vitro and in vivo functions. Thrombosis res. 9:345-357, 1976. 2. SOM P., OSTER Z.H., ZAMORA P.O. & al. Radioimmunoimaging of Experimental Thrombi in Dogs using Technetium-99m-labelled Monoclonal Antibody Fragments Reactive with Human Platelets. J Nucl Med 27:1315-1320, 1986.
3. SUNDREHAGEN E., BENESTAD H.B., HEIKKILA R. 8~ al. A new method for rapid technetium-99m labelling of leukocytes: Functional cell studies in vitro. Stand J Clin Lab Invest 45:717-724, 1985. 4. SUNDREHAGEN E., BENGTSSON A.-M., BREMER P.O. & al. A new method for granulocyte labelling with Technetium-99m: Preliminary results in abscess detection. J Nucl Med 27:555-559, 1986. 5. HASLAM R. J. Role of adenosine diphosphate platelets by thrombin and fatty acids. Nature
in the aggregation of human 202:765-768, 1964.
6. SUNDREHAGEN E. A new technique for formation of Tc-99m-labelled leukocytes and platelets. Int J Appl Radiat Isot 35:365-366, 1984.
blood
7. THAKUR M.L., WALSH L. & GOTTSCHALCK A. Indium-111-labelled human platelets: Improved method, efficacy and evaluation. J Nucl Med 22:381-385, 1981.
