THROMBOSIS RESEARCH 60; 223-230,199O 0049-3848/90 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.
AUTOLOGOUS
PLATELET-LABELING
H. Sinzinger*,
IN THROMBOCYTOPENIA
Irene Virgolini*,
H. Vinazzer**
*Department of Nuclear Medicine, University of Vienna, Ludwig Boltzmann-Institute for Nuclear Medicine, Vienna and **Blood Coagulation Laboratory, Linz, Austria
(Received 23.8.1990; accepted in original form 29.8.1990 by Editor F. Markwardt) ABSTRACT
Field studies performed with peripheral platelets obtained from 6 male volunteers aged 23 to 29 years revealed an extraordinary dependence of labeling efficiency oy,,incubation time and platelet concentration after In-oxine platelet labeling. Since the monitoring of in vivo-platelet function in patients with thrombocytopenia may cause problems due to insufficient labeling results and homologous platelets may show a different in vivo behaviour to autologous ones, we have searched for the minimal amount of platelets necessary to allow appropriate labeling and imaging in patients with thrombocytopenia. In 15 patients with untreated thrombocytopenia aged 14 to 79 years demonstrating a mean peripheral platelet count of 2.509_+ 1.45x104 cells/p1 autologous "'In-oxine platelet labeling was performed. The results indicate that approximately 1~10~ (concentrated) platelets/ml are necessary to obtain an adequate labeling efficiency and recovery. This platelet concentration can be easily achieved by drawing one more Monovette of whole blood per each 5~10~ platelets/p1 peripheral platelet count less than 2x105/~l. It is concluded, that calculation of the required number of platelets in advance, variation of the blood volume drawn and the volume of incubation buffer allow informative, qualitative and quantitative results using autologous platelets. The method presented effectively circumvents the requirement of homologous platelets for radiolabeling in thrombocytopenia. Key words: thrombocytopenia,
platelet, "'In-oxine-labeling
223
224
AUTOLOGOUS PLATELET-LABELING...
Vol. 60, No. 3
INTRODUCTION
In recent years platelet labeling (1) has been increasingly used for diagnostic purposes. The application of various techniques and tracers has been communicated claiming optimal in vitro and in vivo-findings (for review see 2). One of the problems occuring during routine work is to achieve a sufficient labeling of platelets in patients suffering fromthrombocytopenia (3-6). Most widely, donor platelets have been used for platelet studies in these patients. Recently, Danpure et al. (7) reported a method requiring 100 ml of whole blood. In an earlier paqer we claimed that for peripheral platelet counts of below 1x10 platelets/p1 (8) an additional Monovette (7 ml blood) should be drawn for each 5~10~ platelets/p1 less. This recommendation was a consequence of in vitro experiments with diluted platelet rich plasma (PRP) and from a small number of patients suffering from thrombocytopenia. As donor platelets may behave in the circulation different from autologous ones we studied the question of the required minimal amount of platelets allowing generation of interpretable radiolabeling data in patients suffering from thrombocytopenia. For labeling we used a method described earlier (8). SUBJECTS AND METHODS
Fifteen patients (7 males and 8 females, 14-79 years) suffering from idiopathic thrombocytopenia for a period between 3 months and 15 years underwent autologous "'In-oxine-platelet labeling to assess clinical diagnosis. A.
1.
2. 3. 4. 5.
6.
Platelet separation and labeling: Varying numbers of tubes were drawn from the cubital vein without venous occlusion into Monovette vials (Sarstedt, FRG) using 3 ml acid citrate dextrose (ACD) as an anticoagulant for 7 ml blood per Monovette. 16.5 pg (50 ~1) prostacyclin (PGI,,The Up]ohn Company, Kalamazoo, Ml, USA) were added to each tube as antiaggregating agent with a ~1 syringe immediately after sampling. After removing the handle, Monovette vials were closed with the sterile plug. 10 minutes were allowed for sedimentation of the red blood cells at 22'C. The vials were then centrifuged at 150 g for 5 minutes. The supernatant PRP was transfered into another sterile Monovette vial using a butterfly needle. At all these points aliqouts for platelet counting were drawn. The PRP-containing vial was centrifuged at 500g for 10 minutes. The platelets were sedimented to a pellet at the bottom of the vial. The plug was removed. The supernatant platelet poor plasma (PPP) was withdrawn, carefully preserving the pellet, and kept in a sterile 20 ml syringe, retaining the needle for sterility. The pellet in the Monovette vial was gently resuspended in 100-1000 ~1 freshly prepared tyrode buffer (pH 6.2) using a 1 y,+ or 2 ml syringe by shaking the tube gently; then 100 In-oxine were added with a wl-syringe. pci
Vol. 60, No. 3
7. 8. 9. 10.
AUTOLOGOUS PLATELET-LABELING...
The vial was plugged again and thereafter incubated at 37'C for 5 minutes in a shaking water bath. The incubationmixture containing the radiolabeledplatelets was resuspended in the sterile PPP preserved for this purpose. An aliquot was drawn for the determination of platelet count and labeling efficiency. The resuspended radiolabeled platelets were reinjected.
B. Evaluation of labeling parameters: a) Labeling efficiency (LE) was determined by measuring the cell bound radioactivity as compared to the total radioactivity. For counting an 50-100 ~1 aliquot of the suspension was diluted to about 1 ml with tyrode-buffer. The platelet suspension was centrifuged at 1000 g and 22'C for 10 minutes. Thereafter, the radioactivity in the cellular and non-cellular compartment was measured in a gamma-counter (Berthold, Vienna, Austria). Labeling efficiency was expressed as the pellet activity to total activity in %. b) Viability: One hour after reinjection of the labeled platelets a 2 ml blood. sample anticoagulated with 2% EDTA was drawn. Counting the cell bound radioactivity after centrifugation (1000 g, 10 minutes, 22OC), the recovery (REC) was determined after 1 hour from the activity of the sample and the calculated activity at injection time (injected activity and estimated blood volume). as the percentage of the initial Recovery was expressed radioactivity. C. Evaluation of platelet survival: 1.
2. 3.
Blood samples (2 ml) were drawn by vein puncture 10 minutes, 3 and 6 hours after reinjection as well as 3 times during the 2 subsequent days and thereafter 2 times up to the 10th day after reinjection of autologous labeled platelets. All blood samples were drawn into identical vials and counted for 10 minutes. The 1 hour blood sample was used as the 100% value to calculate platelet survival by means of the multiple hit model (kindly provided by Anthon Du P. Heyns, M.D., Prof. (9), Dept. of Haematology, Orange Free State University, Bloemfontein, South Africa).
D. Gamma-camera imaging: On the day after platelet labeling gamma-camera (LFOV, Siemens, FRG) imaging of spleen and liver was performed using a 4 minutes static study and a matrix of 64x64. In anterior-posterior view, regions of interest (ROI) were inserted over the spleen and liver and the spleen/liver ratio was estimated using the counts per pixel for the spleen divided by the ones derived for the liver after background subtraction (Fig.1).
225
226
AUTOLOGOUS PLATELET-LABELING...
Vol. 60, No. 3
E. In vitro experiments: From 6 male healthy volunteers (23-29 years, non-smokers, normolipemics) and 4 patients (30-61 years, 2 males, 2 females, non-smokers, normolipemics) blood was drawn and platelets were radiolabeled as described in A. All volunteers were without any drug since at least 2 weeks. Platelet count before radiolabeling was adjusted to 106, lo', 10' or lo9 cells/@, respectively, from the same donor. Incubation was done for 1, 5, 10, 20 and 30 minutes. At least 4 experiments per each group were performed. F.Btatistical analysis: The values are given as mean&SD. Simple linear regression analysis (y=kx+d) was performed between paired data (n-15) and unpaired data (n=12, n=15 for platelet survival). RESULTS In vitro exDeriments In vitro-labeling data of healthy volunteers show an extreme dependence of labeling efficiency on either the incubation time or the platelet concentration (Fig. 2). Whereas even a 30 minutes incubation with the lowest platelet concentration failed to achieve acceptable results, the highest one resulted in optimal labeling after a 1 minute incubation at 37'C only. The in vitro labeling characteristics of the 4 patients (Fig.3) did not differ significantly from the volunteers. In vivo experiments The mean peripheral platelet count in the patients with thrombocytopenia examined amounted to 25.9+14.5x103 cells/p1 (Tab.1). The number of platelets necessary to obtain a sufficient labelling efficiency was 0.95f0.09xlO cells/ml. Thereby, the values obtained for labeling efficiency and recovery showed significant (p
AUTOLOGOUS
PLATELET-LABELING
H. Sinzinger*,
IN THROMBOCYTOPENIA
Irene Virgolini*,
H. Vinazzer**
*Department of Nuclear Medicine, University of Vienna, Ludwig Boltzmann-Institute for Nuclear Medicine, Vienna and **Blood Coagulation Laboratory, Linz, Austria
(Received 23.8.1990; accepted in original form 29.8.1990 by Editor F. Markwardt) ABSTRACT
Field studies performed with peripheral platelets obtained from 6 male volunteers aged 23 to 29 years revealed an extraordinary dependence of labeling efficiency oy,,incubation time and platelet concentration after In-oxine platelet labeling. Since the monitoring of in vivo-platelet function in patients with thrombocytopenia may cause problems due to insufficient labeling results and homologous platelets may show a different in vivo behaviour to autologous ones, we have searched for the minimal amount of platelets necessary to allow appropriate labeling and imaging in patients with thrombocytopenia. In 15 patients with untreated thrombocytopenia aged 14 to 79 years demonstrating a mean peripheral platelet count of 2.509_+ 1.45x104 cells/p1 autologous "'In-oxine platelet labeling was performed. The results indicate that approximately 1~10~ (concentrated) platelets/ml are necessary to obtain an adequate labeling efficiency and recovery. This platelet concentration can be easily achieved by drawing one more Monovette of whole blood per each 5~10~ platelets/p1 peripheral platelet count less than 2x105/~l. It is concluded, that calculation of the required number of platelets in advance, variation of the blood volume drawn and the volume of incubation buffer allow informative, qualitative and quantitative results using autologous platelets. The method presented effectively circumvents the requirement of homologous platelets for radiolabeling in thrombocytopenia. Key words: thrombocytopenia,
platelet, "'In-oxine-labeling
223
224
AUTOLOGOUS PLATELET-LABELING...
Vol. 60, No. 3
INTRODUCTION
In recent years platelet labeling (1) has been increasingly used for diagnostic purposes. The application of various techniques and tracers has been communicated claiming optimal in vitro and in vivo-findings (for review see 2). One of the problems occuring during routine work is to achieve a sufficient labeling of platelets in patients suffering fromthrombocytopenia (3-6). Most widely, donor platelets have been used for platelet studies in these patients. Recently, Danpure et al. (7) reported a method requiring 100 ml of whole blood. In an earlier paqer we claimed that for peripheral platelet counts of below 1x10 platelets/p1 (8) an additional Monovette (7 ml blood) should be drawn for each 5~10~ platelets/p1 less. This recommendation was a consequence of in vitro experiments with diluted platelet rich plasma (PRP) and from a small number of patients suffering from thrombocytopenia. As donor platelets may behave in the circulation different from autologous ones we studied the question of the required minimal amount of platelets allowing generation of interpretable radiolabeling data in patients suffering from thrombocytopenia. For labeling we used a method described earlier (8). SUBJECTS AND METHODS
Fifteen patients (7 males and 8 females, 14-79 years) suffering from idiopathic thrombocytopenia for a period between 3 months and 15 years underwent autologous "'In-oxine-platelet labeling to assess clinical diagnosis. A.
1.
2. 3. 4. 5.
6.
Platelet separation and labeling: Varying numbers of tubes were drawn from the cubital vein without venous occlusion into Monovette vials (Sarstedt, FRG) using 3 ml acid citrate dextrose (ACD) as an anticoagulant for 7 ml blood per Monovette. 16.5 pg (50 ~1) prostacyclin (PGI,,The Up]ohn Company, Kalamazoo, Ml, USA) were added to each tube as antiaggregating agent with a ~1 syringe immediately after sampling. After removing the handle, Monovette vials were closed with the sterile plug. 10 minutes were allowed for sedimentation of the red blood cells at 22'C. The vials were then centrifuged at 150 g for 5 minutes. The supernatant PRP was transfered into another sterile Monovette vial using a butterfly needle. At all these points aliqouts for platelet counting were drawn. The PRP-containing vial was centrifuged at 500g for 10 minutes. The platelets were sedimented to a pellet at the bottom of the vial. The plug was removed. The supernatant platelet poor plasma (PPP) was withdrawn, carefully preserving the pellet, and kept in a sterile 20 ml syringe, retaining the needle for sterility. The pellet in the Monovette vial was gently resuspended in 100-1000 ~1 freshly prepared tyrode buffer (pH 6.2) using a 1 y,+ or 2 ml syringe by shaking the tube gently; then 100 In-oxine were added with a wl-syringe. pci
Vol. 60, No. 3
7. 8. 9. 10.
AUTOLOGOUS PLATELET-LABELING...
The vial was plugged again and thereafter incubated at 37'C for 5 minutes in a shaking water bath. The incubationmixture containing the radiolabeledplatelets was resuspended in the sterile PPP preserved for this purpose. An aliquot was drawn for the determination of platelet count and labeling efficiency. The resuspended radiolabeled platelets were reinjected.
B. Evaluation of labeling parameters: a) Labeling efficiency (LE) was determined by measuring the cell bound radioactivity as compared to the total radioactivity. For counting an 50-100 ~1 aliquot of the suspension was diluted to about 1 ml with tyrode-buffer. The platelet suspension was centrifuged at 1000 g and 22'C for 10 minutes. Thereafter, the radioactivity in the cellular and non-cellular compartment was measured in a gamma-counter (Berthold, Vienna, Austria). Labeling efficiency was expressed as the pellet activity to total activity in %. b) Viability: One hour after reinjection of the labeled platelets a 2 ml blood. sample anticoagulated with 2% EDTA was drawn. Counting the cell bound radioactivity after centrifugation (1000 g, 10 minutes, 22OC), the recovery (REC) was determined after 1 hour from the activity of the sample and the calculated activity at injection time (injected activity and estimated blood volume). as the percentage of the initial Recovery was expressed radioactivity. C. Evaluation of platelet survival: 1.
2. 3.
Blood samples (2 ml) were drawn by vein puncture 10 minutes, 3 and 6 hours after reinjection as well as 3 times during the 2 subsequent days and thereafter 2 times up to the 10th day after reinjection of autologous labeled platelets. All blood samples were drawn into identical vials and counted for 10 minutes. The 1 hour blood sample was used as the 100% value to calculate platelet survival by means of the multiple hit model (kindly provided by Anthon Du P. Heyns, M.D., Prof. (9), Dept. of Haematology, Orange Free State University, Bloemfontein, South Africa).
D. Gamma-camera imaging: On the day after platelet labeling gamma-camera (LFOV, Siemens, FRG) imaging of spleen and liver was performed using a 4 minutes static study and a matrix of 64x64. In anterior-posterior view, regions of interest (ROI) were inserted over the spleen and liver and the spleen/liver ratio was estimated using the counts per pixel for the spleen divided by the ones derived for the liver after background subtraction (Fig.1).
225
226
AUTOLOGOUS PLATELET-LABELING...
Vol. 60, No. 3
E. In vitro experiments: From 6 male healthy volunteers (23-29 years, non-smokers, normolipemics) and 4 patients (30-61 years, 2 males, 2 females, non-smokers, normolipemics) blood was drawn and platelets were radiolabeled as described in A. All volunteers were without any drug since at least 2 weeks. Platelet count before radiolabeling was adjusted to 106, lo', 10' or lo9 cells/@, respectively, from the same donor. Incubation was done for 1, 5, 10, 20 and 30 minutes. At least 4 experiments per each group were performed. F.Btatistical analysis: The values are given as mean&SD. Simple linear regression analysis (y=kx+d) was performed between paired data (n-15) and unpaired data (n=12, n=15 for platelet survival). RESULTS In vitro exDeriments In vitro-labeling data of healthy volunteers show an extreme dependence of labeling efficiency on either the incubation time or the platelet concentration (Fig. 2). Whereas even a 30 minutes incubation with the lowest platelet concentration failed to achieve acceptable results, the highest one resulted in optimal labeling after a 1 minute incubation at 37'C only. The in vitro labeling characteristics of the 4 patients (Fig.3) did not differ significantly from the volunteers. In vivo experiments The mean peripheral platelet count in the patients with thrombocytopenia examined amounted to 25.9+14.5x103 cells/p1 (Tab.1). The number of platelets necessary to obtain a sufficient labelling efficiency was 0.95f0.09xlO cells/ml. Thereby, the values obtained for labeling efficiency and recovery showed significant (p
