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First results obtained in France with the latest model of the Fresenius cell separator: AS 104
Tmnsfus. Sci. 1993; 1451-S Printed in Great Britain. AU rights reserved
0955-3886193 $6.00+0.00 Copyright @ 1993 Pergamon Press Ltd
First Results Obtained in France With the Latest Model of The Fresenius Cell Separator: AS 104 C. Coffe, MD Y. Couteret, MD M. Devillers, MD T. Fest, MD P. HervC, PhD Y. Kieffer, MD B. Lamy, MD Mp.eosse~ S$ F. Pouthi&-Stein: MD E. Racadot, MD
n In Besancon, we carried out 40 plateletphereses with the latest model of the Fresenius cell separator AS 104 to check this new system against the new generation of cell separators, according to the following criteria: less than 2 x 106leukocytes (before filtration) and more than 5 x 10” platelets. The results show that concentrates platelet contained 5.04+0.88~10~~ platelets in a total volume of 435 f 113 mL. The mean platelet recovery was 40.95+4.86% (from 31.7 to 51.6). The leukocyte content was 2.28+5.48x 106 and the red blood cell contamination was 3.48rt2.38 x 108. The quality of the platelets was very satisfactory, There was no problem with donor biocompatibility or procedure safety, few adverse donor reactions (0.6%) and good therapeutic efficiency of platelet concentrates. n
(< 120 min), leukocyte-poor platelet concentrates containing less than 2X 106 leukocytes (before filtration) and more than 5 x 10” platelets. They must also be able to operate with a single vascular access. In Besancon, we carried out 40 plateletphereses with the latest model of the Fresenius cell separator AS 104 to check this new system against the new generation cell separators, following the criteria deflned above. MATERIALS AND METHODS Materials The Fresenius AS 104l provides automated interface control and selfadjustment of all separation parameters. The position of the phase limit in the separation chamber is detected at every cycle and adjusted with an accuracy of 0.5 mm (Fig. 1).2 The technique consists of two-stage separation with a very low extracorporeal volume of 180mL. In the first stage, red blood cells and platelet-rich plasma (PRP) are separated. PRP is transferred to the second stage where a second separation takes place in which platelets are immediately collected in a bag and
INTRODUCTION
All new cell separators must make it possible to obtain, as rapidly as possible Re ‘onal Blood Transhsion Ccntrc, 1 Blvd A. Fleming, B.P. 19? 7,25020 Besancon Cedex, France. 51
52
Transfus, Sci.
Vol. 14, No. 1
min and the interface position 6/2 or 7/1. The centrifuge speed was 1900 rpm. Investigated
Parameters
On the donor. (a) Before and after each donation we measured: -hemoglobin (g/dL), hematocrit (% ), red blood cell, platelet and white blood cell (WBC) count, using the automated cell analyzer Coulter T 660; -total protein (g/L), creatinin (pmol/L), total bilirubin (pmol/L) (performed on SMAC2, Technicon), serum electropheresis (performed on Cello System, Cebia); -clotting times: prothrombin time (s), fibrinogen (g/L)and partial thromboplastin time (s), (performed on KC 10, H. Amelung, mbh Lemgo, Germany). (b) Clinical survey before, during and after the procedure. (c) Adverse reactions (fever, citrate reaction, anxiety, nausea, pain, allergy). On each platelet concentrate.
Figure 1. Fresenius
cell separator:
AS 104.
platelet-poor plasma is mixed with the red cells and returned to the donor. Methods A total of 40 dual needle procedures were performed on 40 donors aged 25-47 years 27/13). Then ratio: (male/female SOOOmL (II = 24) or 6500 mL (n = 16) of blood were processed with an ACD/ blood ratio of l/8. The afferent whole blood flow rate was 50-60mL/min. The platelet collection flow rate was 3.4 mL/
(a) Regular measurements were made, including total volume (mL), platelet count (X 109/L),platelet yield (X loll), WBC count (x106/L) and WBC contamination ( X 106) (on a Nageotte hemacytometer). The platelet recovery (%) was calculated according to the following formula: Recovery =
2 x platelet
yield
(platelet pre count + platelet post count) x donor blood volume (b) The quality of the platelets was assessed based on: -morphology; -aggregation (investigated on France), Agregameter, Icare,
Resdts of the Latest Fresenius Cd !ktmratot: AS 104
which was induced by the following plasmatic ADP concentrations: 6.6, 3.3, 1.65pM, respectively, and collagen (200 I&L); -Platelet response to hypotonic stress (on photometre Eppendorf 1101 M). (c) Biocompatibility and procedure safety were studied through the following parameters: -plasma-free hemoglobin (mg/ 100 mL) with the benzidin method; -supematant l3-thromboglobulin (TG) level [ELISA method, Diagnostica Stago, France; normal value (NV): 10-40 IU/mL]j -plasma level of fibrinopeptide A (ELISA method, Diagnostica Stagoj NV<3 IU/mL) and D dimer (mcg/mL) (latex method, Ortho); -antithrombin III, protein S and protein C levels (Diagnostica Stago, %); -plasminogen level (calorimetric analysis, Diagnostica Stago, %) and plasma euglobulin lysis (Von Kaulla ); -C3a (125 I Assay system, Amersham), and the study of granulocyte chemotaxis (migration method in semi-solid medium, against Klebsiella lysate). (d) Some control studies (n = 10) were carried out after filtration of platelet concentrate on Sepacell PL 5 N Hemotech (to remove WBCs). The overall procedure length. This, including installing time, priming time and plateletpheresis time, was also meaTable 1.
53
sured. A technical, methodical and operational study was carried out. A printer was provided which recorded all cell separator parameters every 10 min. The therapeutic efficacy of the concentrates. This was tested on patients,
through two criteria: post-transfusion increments measured at 1 h and 24 h and ability to stop hemorrhage. RJBULTS On the donor. Table 1 shows:
significant decrease (0.96kO.31 x 105&L) in average pre/post-platelet counts (35.6%); -at the end of the procedure, we observed a decrease in hematocrit (6.3%), fibrinogen (21.3%), proteins (17.2%) and creatinin (7% ) mainly due to the dilution and loss of red blood cells in tubing and channel; -there was no significant difference concerning prothrombin time (5%), partial thromboplastin time (0%); -the mean frequency of side effects was 0.6%. The fairly low frequency of citrate reactions is probably due to the automatic citrate adjustment and control. -a
On platelet concentrates.
(a) Measurements: we obtained 5.04+0.88x 10” platelets in a total volume of 435+ 113 mL. The mean platelet recovery was 40.95+4.86% (from 31.7 to 51.6). The leukocyte content of the concentrates was 2.28+5.48x 106, mostly lymphocytes and the red
Modifications on the Donors Before the Procedure
Platelet count (1OWpL) Hematocrit 1%) Prothrombin time (%)
Partial thromboplastin time (s) Fibrinogen (g/L) Proteins (g/L) Creatinin (mol/L)
2.7OkO.48 42.84k3.47 100+10 32f2.6 3.10f0.7 7Ok3.5 90+16
After the Procedure
1.74kO.34
40.151t4.05 95+9 32k2.7 2.441to.s 58f4 84k15
54
7’mnsfus.Sci.
Table 2.
Vol.14, No. 1
Platelet Yield as a Function of Whole Blood Volume Treated
Whole Blood Volume Treated
From 4500 to 5OOOmL From 5500 to 6OOOmL From 6500 to 7000 mL
Number of Procedures
Platelet Yield
19
4.64kO.76
8 9
blood cell contamination was 3.48+2.38x 1O8.The values of pH, fibrinogen and total proteins were, respectively, 7.25f0.06, 1.91f0.62 and 55f3.65g/L. Table 2 illustrates platelet yield and leukocyte contamination according to whole blood volume. 0.4The quality of the platelets in the concentrates was very satisfactory: -the platelets collected showed good morphology; -comparison of aggregation curves from preapheresis controls and platelet concentrate samples showed a sustained aggregation response of apheresis samples; -platelet response to hypotonic stress was normal (C/A = 83+5%, R/A = 98+2%). (4 Biocompatibility and procedure safety: As previously reported by Kretschmer et ~1.~ -no hemolysis was found: there was no significant difference in the free hemoglobin level before procedure r?I*IaEr andth?.12+0.92mg/ lOO/mL); -the data obtained from measurement of the supernatant @TG level indicated no significant inhibition of platelet activation (50.9f43.9 IU/mL after procedure); -concerning coagulation activation, there was a 4-fold increase of fibrinopeptide A after the procedure: 5.4821.63 IU/mL, but D dimers were always absent; inhibitors (anti-coagulation thrombin III, protein S and protein C) remained in normal
WBC cowamillation
(10”)
W)
5.31f0.66 5.66kO.94
7.32k5.66
5.61k6.31 4.20f3.64
values after the procedure, so did plasminogen level. Only one subacute fibrinolysis on 23 controls was observed; -C3a was 1808+230ng/mL after the procedure and 8240+725 on return line, which could correspond to an activation of the alternative pathway we observed. The same phenomenon was observed with other cell separators (Baxter CS 3000, Haemonetics V50 and Dideco Vivacell). The study of granulocyte chemotaxis showed no leukocyte damage (migration index: 1.28; control: 1.30). (d) After filtration of the platelet concentrates, we obtained a mean leukocyte contamination of < 1 x lo5 (
increment in platelet counts at 24h (ranging from 38 to 55% of the expected in vivo recovery, calculated according to Duquesnoy et al.), and an immediate good efficiency on patients’ bleeding. There is no difference between Fresenius AS 104 and Cobe Spectra platelet concentrates. DISCUSSION The main advantages of this new cell separator are: easy use of the computer,
automatic citrate adjustment, collection of the platelets outside the machine, and short rinsing time. Another great advan-
Results of the Latest &es&us CeB Separator:AS 104 55
tage of this cell separator is very low leukocyte contamination, thanks to filtration of the platelet concentrates after the procedure. We frequently obtained a total number of leukocytes below IX 105, which is of considerable interest to solve the problem of anti-HLA alloimmunization in patients. Platelet recovery should be increased in future to reach beyond 50% (sometimes <35%). This should be improved by changing the centrifuge speed and rinsing the secondary separation chamber at the end of the procedure. Valbonesi showed that it is possible to obtain more of 4x 10” in less than 1 h. We do not know the exact repercussions of C3a increase on the return line and the significant increase of fibrinopeptide A. Many other teams lj4 found the same results and we obtained similar data with other cell separators. It would be interesting to investigate if cell viability is affected by this activation after a &day storage. We are just beginning to study the single needle system for platelet collection, which can be a convenient altemative in cases of poor venous access in some donors, or if there is a problem during the procedure with one or both venous accesses, even though this system involves a slightly longer procedure. CONCLUSION These data show good quantitative results, no problem in donor biocompati-
bility or procedure safety, few adverse donor reactions, and good therapeutic efficiency of platelet concentrates. We are now trying to further decrease the rate of leukocyte contamination (less than 1x 106) and increase the separation efficacy (more than 6x10”) under routine conditions, taking advantage of the great flexibility of the Fresenius AS 104. REFERENCES 1. Kretschmer V, See A, Sohngen D,
Bachem M, KadarJG, BorbergH, Pelzer H, Zofel P: First results with the new Fresenius cell separator AS 104. Plasma Ther Transfus
Technol 1987; 8:298-301. 2. Neumann HJ, Meisberger A, Mathieu B: Improvement of the safety systems in cell separators. The new safety concept for the cell separator AS 104 (Fresenius). Infusionstherapie 1987; 14(Suppl. 4):4351. 3. Kretschmer V, Sohngen D, Goddecke W, Kadar JG, Pelzer H, Prinz H, E&e R: Biocompatibility and safety of cytapheresis. Znfusionsthbrapie 1989; 16(Suppl. 2): 10-20. 4. Valbonesi H, Frisoni R, Fella M, Capra C, Malfanti L, Ferrari H, Zia S, Florio G, Nickel H: Plateletpheresis with the new Fresenius AS 104 blood cell separator. I Clin Apheresis 1991; 6:84-87. 5. Duquesnoy RJ, Filip DT, Rodney GE, Rimm AA, Aster RH: Successful transfusion of platelets “mismatched” for HLA antigens. Am j Hematol 1977; 2:219-226.
0955-3886193 $6.00+0.00 Copyright @ 1993 Pergamon Press Ltd
First Results Obtained in France With the Latest Model of The Fresenius Cell Separator: AS 104 C. Coffe, MD Y. Couteret, MD M. Devillers, MD T. Fest, MD P. HervC, PhD Y. Kieffer, MD B. Lamy, MD Mp.eosse~ S$ F. Pouthi&-Stein: MD E. Racadot, MD
n In Besancon, we carried out 40 plateletphereses with the latest model of the Fresenius cell separator AS 104 to check this new system against the new generation of cell separators, according to the following criteria: less than 2 x 106leukocytes (before filtration) and more than 5 x 10” platelets. The results show that concentrates platelet contained 5.04+0.88~10~~ platelets in a total volume of 435 f 113 mL. The mean platelet recovery was 40.95+4.86% (from 31.7 to 51.6). The leukocyte content was 2.28+5.48x 106 and the red blood cell contamination was 3.48rt2.38 x 108. The quality of the platelets was very satisfactory, There was no problem with donor biocompatibility or procedure safety, few adverse donor reactions (0.6%) and good therapeutic efficiency of platelet concentrates. n
(< 120 min), leukocyte-poor platelet concentrates containing less than 2X 106 leukocytes (before filtration) and more than 5 x 10” platelets. They must also be able to operate with a single vascular access. In Besancon, we carried out 40 plateletphereses with the latest model of the Fresenius cell separator AS 104 to check this new system against the new generation cell separators, following the criteria deflned above. MATERIALS AND METHODS Materials The Fresenius AS 104l provides automated interface control and selfadjustment of all separation parameters. The position of the phase limit in the separation chamber is detected at every cycle and adjusted with an accuracy of 0.5 mm (Fig. 1).2 The technique consists of two-stage separation with a very low extracorporeal volume of 180mL. In the first stage, red blood cells and platelet-rich plasma (PRP) are separated. PRP is transferred to the second stage where a second separation takes place in which platelets are immediately collected in a bag and
INTRODUCTION
All new cell separators must make it possible to obtain, as rapidly as possible Re ‘onal Blood Transhsion Ccntrc, 1 Blvd A. Fleming, B.P. 19? 7,25020 Besancon Cedex, France. 51
52
Transfus, Sci.
Vol. 14, No. 1
min and the interface position 6/2 or 7/1. The centrifuge speed was 1900 rpm. Investigated
Parameters
On the donor. (a) Before and after each donation we measured: -hemoglobin (g/dL), hematocrit (% ), red blood cell, platelet and white blood cell (WBC) count, using the automated cell analyzer Coulter T 660; -total protein (g/L), creatinin (pmol/L), total bilirubin (pmol/L) (performed on SMAC2, Technicon), serum electropheresis (performed on Cello System, Cebia); -clotting times: prothrombin time (s), fibrinogen (g/L)and partial thromboplastin time (s), (performed on KC 10, H. Amelung, mbh Lemgo, Germany). (b) Clinical survey before, during and after the procedure. (c) Adverse reactions (fever, citrate reaction, anxiety, nausea, pain, allergy). On each platelet concentrate.
Figure 1. Fresenius
cell separator:
AS 104.
platelet-poor plasma is mixed with the red cells and returned to the donor. Methods A total of 40 dual needle procedures were performed on 40 donors aged 25-47 years 27/13). Then ratio: (male/female SOOOmL (II = 24) or 6500 mL (n = 16) of blood were processed with an ACD/ blood ratio of l/8. The afferent whole blood flow rate was 50-60mL/min. The platelet collection flow rate was 3.4 mL/
(a) Regular measurements were made, including total volume (mL), platelet count (X 109/L),platelet yield (X loll), WBC count (x106/L) and WBC contamination ( X 106) (on a Nageotte hemacytometer). The platelet recovery (%) was calculated according to the following formula: Recovery =
2 x platelet
yield
(platelet pre count + platelet post count) x donor blood volume (b) The quality of the platelets was assessed based on: -morphology; -aggregation (investigated on France), Agregameter, Icare,
Resdts of the Latest Fresenius Cd !ktmratot: AS 104
which was induced by the following plasmatic ADP concentrations: 6.6, 3.3, 1.65pM, respectively, and collagen (200 I&L); -Platelet response to hypotonic stress (on photometre Eppendorf 1101 M). (c) Biocompatibility and procedure safety were studied through the following parameters: -plasma-free hemoglobin (mg/ 100 mL) with the benzidin method; -supematant l3-thromboglobulin (TG) level [ELISA method, Diagnostica Stago, France; normal value (NV): 10-40 IU/mL]j -plasma level of fibrinopeptide A (ELISA method, Diagnostica Stagoj NV<3 IU/mL) and D dimer (mcg/mL) (latex method, Ortho); -antithrombin III, protein S and protein C levels (Diagnostica Stago, %); -plasminogen level (calorimetric analysis, Diagnostica Stago, %) and plasma euglobulin lysis (Von Kaulla ); -C3a (125 I Assay system, Amersham), and the study of granulocyte chemotaxis (migration method in semi-solid medium, against Klebsiella lysate). (d) Some control studies (n = 10) were carried out after filtration of platelet concentrate on Sepacell PL 5 N Hemotech (to remove WBCs). The overall procedure length. This, including installing time, priming time and plateletpheresis time, was also meaTable 1.
53
sured. A technical, methodical and operational study was carried out. A printer was provided which recorded all cell separator parameters every 10 min. The therapeutic efficacy of the concentrates. This was tested on patients,
through two criteria: post-transfusion increments measured at 1 h and 24 h and ability to stop hemorrhage. RJBULTS On the donor. Table 1 shows:
significant decrease (0.96kO.31 x 105&L) in average pre/post-platelet counts (35.6%); -at the end of the procedure, we observed a decrease in hematocrit (6.3%), fibrinogen (21.3%), proteins (17.2%) and creatinin (7% ) mainly due to the dilution and loss of red blood cells in tubing and channel; -there was no significant difference concerning prothrombin time (5%), partial thromboplastin time (0%); -the mean frequency of side effects was 0.6%. The fairly low frequency of citrate reactions is probably due to the automatic citrate adjustment and control. -a
On platelet concentrates.
(a) Measurements: we obtained 5.04+0.88x 10” platelets in a total volume of 435+ 113 mL. The mean platelet recovery was 40.95+4.86% (from 31.7 to 51.6). The leukocyte content of the concentrates was 2.28+5.48x 106, mostly lymphocytes and the red
Modifications on the Donors Before the Procedure
Platelet count (1OWpL) Hematocrit 1%) Prothrombin time (%)
Partial thromboplastin time (s) Fibrinogen (g/L) Proteins (g/L) Creatinin (mol/L)
2.7OkO.48 42.84k3.47 100+10 32f2.6 3.10f0.7 7Ok3.5 90+16
After the Procedure
1.74kO.34
40.151t4.05 95+9 32k2.7 2.441to.s 58f4 84k15
54
7’mnsfus.Sci.
Table 2.
Vol.14, No. 1
Platelet Yield as a Function of Whole Blood Volume Treated
Whole Blood Volume Treated
From 4500 to 5OOOmL From 5500 to 6OOOmL From 6500 to 7000 mL
Number of Procedures
Platelet Yield
19
4.64kO.76
8 9
blood cell contamination was 3.48+2.38x 1O8.The values of pH, fibrinogen and total proteins were, respectively, 7.25f0.06, 1.91f0.62 and 55f3.65g/L. Table 2 illustrates platelet yield and leukocyte contamination according to whole blood volume. 0.4The quality of the platelets in the concentrates was very satisfactory: -the platelets collected showed good morphology; -comparison of aggregation curves from preapheresis controls and platelet concentrate samples showed a sustained aggregation response of apheresis samples; -platelet response to hypotonic stress was normal (C/A = 83+5%, R/A = 98+2%). (4 Biocompatibility and procedure safety: As previously reported by Kretschmer et ~1.~ -no hemolysis was found: there was no significant difference in the free hemoglobin level before procedure r?I*IaEr andth?.12+0.92mg/ lOO/mL); -the data obtained from measurement of the supernatant @TG level indicated no significant inhibition of platelet activation (50.9f43.9 IU/mL after procedure); -concerning coagulation activation, there was a 4-fold increase of fibrinopeptide A after the procedure: 5.4821.63 IU/mL, but D dimers were always absent; inhibitors (anti-coagulation thrombin III, protein S and protein C) remained in normal
WBC cowamillation
(10”)
W)
5.31f0.66 5.66kO.94
7.32k5.66
5.61k6.31 4.20f3.64
values after the procedure, so did plasminogen level. Only one subacute fibrinolysis on 23 controls was observed; -C3a was 1808+230ng/mL after the procedure and 8240+725 on return line, which could correspond to an activation of the alternative pathway we observed. The same phenomenon was observed with other cell separators (Baxter CS 3000, Haemonetics V50 and Dideco Vivacell). The study of granulocyte chemotaxis showed no leukocyte damage (migration index: 1.28; control: 1.30). (d) After filtration of the platelet concentrates, we obtained a mean leukocyte contamination of < 1 x lo5 (
increment in platelet counts at 24h (ranging from 38 to 55% of the expected in vivo recovery, calculated according to Duquesnoy et al.), and an immediate good efficiency on patients’ bleeding. There is no difference between Fresenius AS 104 and Cobe Spectra platelet concentrates. DISCUSSION The main advantages of this new cell separator are: easy use of the computer,
automatic citrate adjustment, collection of the platelets outside the machine, and short rinsing time. Another great advan-
Results of the Latest &es&us CeB Separator:AS 104 55
tage of this cell separator is very low leukocyte contamination, thanks to filtration of the platelet concentrates after the procedure. We frequently obtained a total number of leukocytes below IX 105, which is of considerable interest to solve the problem of anti-HLA alloimmunization in patients. Platelet recovery should be increased in future to reach beyond 50% (sometimes <35%). This should be improved by changing the centrifuge speed and rinsing the secondary separation chamber at the end of the procedure. Valbonesi showed that it is possible to obtain more of 4x 10” in less than 1 h. We do not know the exact repercussions of C3a increase on the return line and the significant increase of fibrinopeptide A. Many other teams lj4 found the same results and we obtained similar data with other cell separators. It would be interesting to investigate if cell viability is affected by this activation after a &day storage. We are just beginning to study the single needle system for platelet collection, which can be a convenient altemative in cases of poor venous access in some donors, or if there is a problem during the procedure with one or both venous accesses, even though this system involves a slightly longer procedure. CONCLUSION These data show good quantitative results, no problem in donor biocompati-
bility or procedure safety, few adverse donor reactions, and good therapeutic efficiency of platelet concentrates. We are now trying to further decrease the rate of leukocyte contamination (less than 1x 106) and increase the separation efficacy (more than 6x10”) under routine conditions, taking advantage of the great flexibility of the Fresenius AS 104. REFERENCES 1. Kretschmer V, See A, Sohngen D,
Bachem M, KadarJG, BorbergH, Pelzer H, Zofel P: First results with the new Fresenius cell separator AS 104. Plasma Ther Transfus
Technol 1987; 8:298-301. 2. Neumann HJ, Meisberger A, Mathieu B: Improvement of the safety systems in cell separators. The new safety concept for the cell separator AS 104 (Fresenius). Infusionstherapie 1987; 14(Suppl. 4):4351. 3. Kretschmer V, Sohngen D, Goddecke W, Kadar JG, Pelzer H, Prinz H, E&e R: Biocompatibility and safety of cytapheresis. Znfusionsthbrapie 1989; 16(Suppl. 2): 10-20. 4. Valbonesi H, Frisoni R, Fella M, Capra C, Malfanti L, Ferrari H, Zia S, Florio G, Nickel H: Plateletpheresis with the new Fresenius AS 104 blood cell separator. I Clin Apheresis 1991; 6:84-87. 5. Duquesnoy RJ, Filip DT, Rodney GE, Rimm AA, Aster RH: Successful transfusion of platelets “mismatched” for HLA antigens. Am j Hematol 1977; 2:219-226.