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Biocompatibility studies of the single and dual-needle procedure in plateletpheresis with the fresenius AS 104 blood cell separator
Pergamon
Transfus. Sci. Vol. 17, No. 2, pp. 315-320, 1996 Copyright © 1996 Elsevier Science Ltd. All rights reserved PII:S0955-3886(96)00010-0 Printed in Great Britain 0955-3886/96 $15.00 + .00
Biocompatibility Studies of the Single and Dual-needle Procedure in Plateletpheresis with the Fresenius AS 104 Blood Cell Separator Rainer Moog, MD* § Janos G. Kadar, MDt Norbert Mfiller, MD* Marcel Reiser, MDt Thomas Weit~wange, PhD¢ Anja E. Ledermann* • To investigate the biocompatibility of the new single-needle (SN) technique of the AS 104 blood cell separator, we analysed coagulation, complement and haemolysis parameters prior to, during and after five plateletphereses. The data were compared with results of the same parameters from identical donors in the dual-needle (DN) procedure. The analysis of bilirubin, lactate dehydrogenase (LDH), hydroxybutyrate dehydrogenase (HBDH) and haptoglobin showed that there was no relevant haemolysis. However, changes of the concentration of the coagulation factors XII and XI indicated an activation of coagulation. This was confirmed by an increase of the thrombin antithrombin (TAT) Ill complex during apheresis. Comparison of the platelet products showed no significant differences. Glucose concentration was 332 + 14 mg/dL in the SN and 312 + 32 mg/dL in the DN procedure, respectively. Lactate level was 3.2 _+0.6 mmol/L in the SN and 3.3 + 0.6 in the DN technique, respectively. Levels of complement activation markers were not statistically different. Leukocyte contamination was statistically higher in the SN procedure. Copyright © 1996 Elsevier Science Ltd •
INTRODUCTION Before a new cell separator is used routinely, studies of its biocompatibility should be carried out. 1 It is the ethical task of the user to protect the donor from any harm caused by cytapheresis. The biocompatibility of the artificial surfaces plays an important role when donor blood comes into contact with nonphysiological material. Furthermore, drugs administered during apheresis, such as the anticoagulant, can influence the compatibility of cytapheresis. The cell separator Fresenius AS 104 was introduced in the European apheresis market in 1987. It belongs to the third generation of blood cell separators. All separation procedures work under automatic control by an interface control system and several sensitive detectors and monitors. Separation protocols for bone marrow stem cell separation, 2,~3 granulocyte collection, 4 lymphocyte depletion, monocyte collection, peripheral blood stem cell collection, 2,s,6 platelet (PLT) collection, 6-16 plasma exchange and red cell exchange are implemented. These protocols use the continuous flow technique with a sealless disposable. Most procedures were carried out using the PLT collection protocol and resulted in a good quality of the product. In 1991, a SN technique for plateletpheresis was developed.
*Institute for Transfusion Medicine, University of Essen (FRG), Hufelandstr. 55, 45122, Essen, Germany. tHaemapheresis Unit, Department of Internal Medicine I, University of Cologne, Cologne, Germany. ~:Fresenius AG, R&D, St. Wendel, Germany. §Author for correspondence. T5 17-2-H
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Using the SN procedure resulted in sufficient PLT yields. ~6,~zThe leukocyte contamination of the platelet concentrate (PC) was slightly elevated in comparison with the products obtained with the DN technique. In our study we investigated the quality of platelet products collected with the DN and SN procedure. Furthermore, we analysed coagulation, complement and haemolysis parameters prior to, during and after plateletpheresis to compare the biocompatibility of the DN and SN technique.
MATERIALS A N D METHODS
Five female blood donors underwent paired plateletpheresis, one separation with the DN, the other one with the SN technique. They all complied with the German recommendations for cytapheresis. ~7,18 Written consent was obtained prior to apheresis. PLT pre-counts were above 200,000hzL. The donation interval was at least 4 weeks. The AS 104 blood cell separator [Fresenius AG, Bad Homburg, Germany) was used for thrombocytapheresis. All separations were done with standard protocols: in the DN procedure whole blood was drawn at a blood flow rate of 50 mL/min. The anticoagulant/blood ratio was 1 : 10. The blood was pumped via the whole blood pump into the first stage of a dual stage separation chamber. There it was centrifuged at 1900 rpm. The interface position (red cell/plasma border) was 7 : 1 . Red blood cells and platelet-poor plasma were re-infused to the donor via the return line. Plateletrich plasma entered the second stage of the separation chamber. From there the thrombocytes were pumped, via the cell pump, into the platelet storage bag. The cell pump speed was 2.8 mL/min. The procedure stopped when an endpoint volume of 3500 mL blood was processed. Two major changes were necessary for the SN technique: first, inlet and return line were connected by a Yline in the disposable. Second, a bag
press (pressure of pneumatic spring 150 N) for a transfer bag was constructed. The transfer bag was a reservoir for the red cells during the draw cycle. The return cycle started when a blood volume of 250 mL was reached in the draw cycle. A part of the re-infusion blood re-circulated to the centrifuge. The procedure ended when the endpoint volume of 2500 mL was reached. The blood flow rate {60 mL/mL) and the centrifuge speed [2000 rpm} were different from the DN technique. To investigate the biocompatibility of the AS 104, we analysed the coagulation, complement and haemolysis parameters prior to, during and after apheresis. Reagents for the determination of bilimbin, lactute dehydrogenase (LDH), hydroxybutyrate dehydrogenase (HBDH) and total protein (biuret method) were purchased from Boehringer Mannheim (Mannheim, Germanyl. A photometer (Vitalab 200, Vital Scientific, van Dieren, The Netherlands) was used for these measurements. Haptoglobin (NOR-Partigen R, Behring, Marburg, Germany) was determined by irnmunodiffusion. Antithrombin III (AT III), coagulation factors XII and XI were analysed by the chromogenic substrate method (Chromotimer, Behring, Marburg, Germany). TAT llI complex was determined by an ELISA (EnzygnostR, Behring, Marburg, Germany). The complement factors C3a, C4d and SCSbC9 were determined by commercially available ELISAs (Progen, Heidelberg, Germany; Quidel, San Diego, U.S.A.). For glucose measurement, the hexokinase method was used (Glucoquant g, Boehringer Mannheim, Mannheim, Germany). Lactate was determined with a macrodetermination according to manufacturer's instructions. The test kit Testomar R (Behring, Marburg, Germany) was used for this assay. Leukocytes were counted manually in a Neubauer chamber.l~ Platelet determination was conducted electronically (Coulter Counter T 660, Coulter Electronics, Krefeld, Germany). Morphology score was determined according to the method of Kunicki e t a l ? °
Biocompatibility Studies 317
Blood samples were drawn prior to (I), after 75 mL processed blood volume (II), 100 mL before the termination of the donation (V), and after apheresis in both procedures (VI). Additionally, samples were collected during the third (750 mL; III) and seventh (1750 mL; IV) return cycle in the SN technique. Sampling took place at 850 (III) and 1700 (W) mL from the return line of the DN set. For statistical analysis, the t-test for paired data was used. The level of statistical significance was set at 0.05.
the first minutes of separation. Later on, during the DN or SN donation, no apparent differences could be seen by either of the methods used. The data of the investigated parameters in the PC are shown in Table 2. There was a statistical difference in the leukocyte counts of both procedures. Complement parameters, glucose, lactate, morphology score and platelets in the PC did not statistically differ between both techniques.
DISCUSSION RESULTS
The analysis of bilirubin, LDH, HBDH and haptoglobin showed that there was no relevant haemolysis (Table 1). All parameters were within normal ranges and there was no evidence for damage of red blood cells. Total protein decreased during the course of separation. This parameter was used as a dilution factor in the measurement of coagulation and complement factors. Decreased activity of the coagulation factors XII and XI was seen in the return line of the DN and SN procedure. No significant changes of the AT III levels were seen during apheresis. The TAT III complex continuously increased during the course of separations. Peak values of 66.9 + 15.6 ~zg/L were reached in samples drawn 100 mL before the termination of the DN procedure. After donation, the concentrations of factors XII and XI were similar to the values prior to donation. The TAT III complex decreased. We found no significant change in the complement split product content of serum samples obtained during the apheresis sessions. The pre-donation C3a levels were in the normal range, the corresponding C4d concentrations were elevated when compared with the in-house laboratory control. The results were estimated by using a dilution factor as obtained by the measurement of total protein concentration. We observed a slight increase of complement split product content of the plasma after
There is an ethical responsibility for the operator to protect the donor from any risk of health due to cytapheresis. In apheresis, donor blood comes into contact with the non-physiological surface of the disposable. Furthermore, the blood bank staff are responsible for the quality and integrity of the blood product produced. Therefore, biocompatibility of a new blood donation procedure has to be established before it is introduced for routine use. The biocompatibility of the DN technique in thrombocytapheresis with the Fresenius AS 104 blood cell separator was investigated by Kretschmer et al.~ We studied coagulation, complement and haemolysis parameters prior to, during and after plateletpheresis using the SN technique of the AS 104 cell separator. The results were compared with the data of the DN procedure, using identical donors. Our data demonstrated that there is no relevant haemolysis of red blood cells in both separation procedures. This was in accordance with the findings of Kretschmer and co-workers in the DN thrombocytapheresis.' Analysis of coagulation parameters showed a decreased activity of factors XII and XI in the return line of the DN set and in the return cycles of the SN procedure. We made similar observations when we studied the biocompatibility of the cell separator A 2 0 1 . 21 We interpreted this as a slight activation of coagulation when the blood came into
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Table 1. Results of Haemolysis, Coagulation and Complement Parameters Prior to, During and After DN and SN Plateletpheresis (Means) I
Bilirubin (mg/dL) Bilirubin (mg/dL) Haptoglobin (g/L) Haptoglobin (g/L) HBDH (U/L) HBDH (U/L) LDH (U/L) LDH (U/L) Protein (g/dL) Protein (g/dL) AT III (%) AT III (%} Factor XII (%) Factor XII (%) Factor XI (%) Factor XI (%) TAT III complex (tag/L) TAT III complex (t,g/L) C3a (ng/mL) C3a (ng/mL) C4d (ng/mL) C4d (ng/mL) SC5bC9 (ng/mL} SC5bC9 (ng/mL)
DN SN DN SN DN SN DN SN DN SN DN SN DN SN DN SN DN SN DN SN DN SN DN SN
0.6 0.7 2.3 2.3 71 68 128 120 7.3 7.3 105 110 98 105 103 107 4.5 2.08 266 294 5095 4521 47 91
II
III
0.5 0.7 2.2 2.2 78 87 141 156 6.9 7.0 102 106 87 93 102 99 2.1 2.1 297 324 4881 4611 46 79
0.4 0.5 1.9 1.5 55 52 97 93 5.9 5.3" 105 103 85 86 80 81 7.7 9.4 230 215 1478 1801 54 79
IV
0.4 0.5 1.8 1.6 54 55 95 93 5.7 5.5 113 107 89 84 75 79 12.9 7.8 143 210 1982 2710 51 49
V
0.3 0.6 1.8 2.0 74 72 130 121 6.2 6.3 99 107 100 101 90 100 66.9 3.0* 150 163 3296 2310 59 44
VI
0.5 0.6 1.8 1.8 61 58 107 97 6.2 6.0 114 113 106 101 96 108 22 4.2 162 199 1666 2327 20 51
*Significant (level P < 0.05).
c o n t a c t w i t h the non-physiological surface of the disposable. N o changes of activity w e r e seen in the A T III level during plateletpheresis. Coffe et al. also observed n o r m a l values for the coagulation i n h i b i t o r a n t i t h r o m b i n III after d o n a t i o n w i t h the D N procedure. 8 T h e levels of the c o m p l e m e n t activ a t i o n p a r a m e t e r obtained suggest t h a t no significant surface-related a c t i v a t i o n t o o k place in the s y s t e m w h i c h w o u l d
have b e e n clinically relevant for the donor or the recipient. In a f o r m e r work, we also reported a clinically insignificant c o m p l e m e n t a c t i v a t i o n m e a s u r e d in the r e t u r n line of the blood cell separator AS 104.1 T h e p r o d u c t quality of platelets collected b y a n e w apheresis t e c h n i q u e s h o u l d be tested prior to r o u t i n e use. 22 O u r data d e m o n s t r a t e that there is an equal quality of the platelet p r o d u c t
Table 2. Results of Parameters Measured in PC (means + 1 standard deviation)
C3[desarg) (ng/mL) C4d (ng/mL} SC5bC9 (ng/mL) Glucose (mg/dL) Lactate (mmol/L) Leukocytes (x 106) Morphology score Platelets (x 1011) *Significant (level P < 0.05).
DN
SN
258 + 68 3062 + 1709 77 + 38 312 + 32 3.3 + 0.6 5.3 + 5.4 353 + 20 3.7 + 0.9
199 + 26 2221 _+875 67 + 44 332 + 14 3.2 + 0.6 26.4 + 28.5* 363 + 17 3.3 + 0.4
Biocompatibility Studies
obtained by both techniques except for leukocyte contamination. Overall, we conclude that there is an equal biocompatibility of the SN technique compared with the DN procedure using the Fresenius AS 104 blood cell separator. The quality of the platelet product is fine.
Acknowledgements The technical assistance of Mrs. garin Weber and the staff of the Haemapheresis Unit is gratefully acknowledged.
REFERENCES 1. Kretschmer V, S6hngen D, G6ddecke W, Kadar JG, Pelzer H, Prinz H, Eckle R: Biocompatibility and safety of cytapheresis. Infusionstherapie 1989; 16(suppl.2):10-20. 2. Kretschmer V, Biehl M, Coffe C, Eckstein R, Kirpov D, Menichella G, Mitschulat H, Moog R, Neumann HJ, Pusinelli T, Valbonesi M, Weisswange T, Wiebecke D, Zingsem J: New feature of the Fresenius blood cell separator AS 104. Therapeutic Plasmapheresis 1993; 12:851-855. 3. Zingsem J, Zeiler T, Zimmermann R, Weisbach V, Mitschulat H, Schmid H, Beyer J, Siegert W, Eckstein R: Automated processing of human bone marrow grafts for transplantation. Vox Sang 1993; 65:292-299. 4. Mfiller N, Mitschulat H, Wortmann W: Leukocytapheresis: evaluation of a new procedure for the blood cell separator Fresenius AS 104. Infusionstherapie 1992; 19:70-72. 5. Pierelli L, Menichella G, Paoloni A, Vittori M, Foddai ML, Serafini R, Mango G, Bizzi B: Collection of peripheral blood stem cells using the blood cell separator Fresenius AS 104. Infusionstherapie 1991; 18(suppl.2):62. 6. Zingsem J, Zeiler T, Zimmermann R, Schwella N, Weisbach V, Siegert W, Eckstein R: Selective enrichment of hematopoietic progenitor cells from peripheral blood. Transfus Sci 1993; 14:75-77. 7. Borberg H, Kretschmer V, Godehardt E, Sojka G: Evaluierung eines neuen, vollautomatischen Blutzellseparators mit kontinuierlichen Blutfluf~,in Kretschmer V, Stangel W, Weif~haar D (eds): Beitr Infusionsther, vol 28. Basel, Karger, 1991, pp. 177-187.
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8. Coffe C, Couteret Y, Devillers M, Fest T, Herve P, Kieffer Y, Lamy B, Masse M, Morel P, Pouthier-Stein F, Racadot E: First results obtained in France with the latest model of the Fresenius cell separator: AS 104. Transfus Sci 1993; 14:51-55. 9. Kautz H, Wienhausen V, Mfiller N: Comparison of plateletpheresis using Cobe Spectra system and Fresenius AS 104. Infusionstherapie 1992; 19:59-60. 10. Kretschmer V, See A, S6hngen D, Bachem M, Kadar JG, Borberg H, Pelzer H, Z6fel P: First results with the new Fresenius cell separator AS 104. Plasma Ther Transfus Technol 1987; 8:298-301. 11. Kretschmer V: Comparison of different plateletpheresis systems. Infusionstherapie 1991; 18:188-195. 12. Kretschmer V, Rossa C: Optimierung der Separationen mit dem Zellseparator AS 104, in Kretschmer V, Stangel W, Weit~haar D (eds): Beitr Infusionsther, vol 28. Basel, Karger, 1991, pp. 188-194. 13. Moog R: Die Bedeutung der Trenngrenzenposition bei der Thrombozytapherese mit dem Zellseparator AS 104. Infusionstherapie 1992; 19:61-63. 14. Mfiller N, Kretschmer V, Godehardt E, Borberg H, Neumann HJ: Haemapheresis Scientific Workshop Group of DGTI: Improvement of separation efficiency and concentrate purity of the Fresenius cell separator AS 104: Results of a multicenter study. Transfus Sci 1993; 14: 105-114. 15. Valbonesi M, Frisoni R, Fella M, Capra C, Malfanti L, Ferrari M, Zia S, Florio G, Nickel H: Plateletpheresis with the new Fresenius AS 104 blood cell separator. J Clin Apheresis 1991; 6:84-87. 16. Moog R: Single-needle-Ergebnisse bei den Zellseparatoren A-201 und AS-104, in Kretschmer V, Stangel W, Eckstsin R (eds): Beitr Infusionsther, vol 31. Basel, Karger, 1993, pp. 104-106. 17. Valbonesi M, Frisoni R, Malfanti L, Florio G, Carlier P, Ruzzenenti M: Single-needle thrombocytapheresis with the Fresenius AS 104R. Infusionstherapie 1993; 20:8-10. 18. Kretschmer V, Borberg H, Giannitsis D, Kamanabrou D, Mempel W, Mfiller V, Mfiller N, Neumeyer H: Durchffihrung apparativer H/imapheresen zur Gewinnung yon Blutbestandteilkonserven. Empfehlungen der H/imapheresekomminssion der Deutschen Gesellschaft ffir Transfusionsmedizin und Immunh/imatologie. Infusionstherapie 1987; 14(suppl.4): 57-64.
320 Trans[us.Sci. Vol. 17, No. 2 19. Wissenschaftlicher Beirat der Bundesarztekammer und vom Bundesgesundheitsamt: Richtlinien zur Blutgruppenbestimmun.g und Bluttransfusion. K61n, Deutscher Arzteverlag, 1991. 20. Kunicki TJ, Tuccelli M, Becker GA, Aster RH: A study of variables affecting the quality of platelets stored at "room temperature". Transfusion 1975; 15: 414--421.
21. Moog R, Luboldt W, Krfltzfeldt A, Paar D, Holtmann M, Mengelkamp AK: Thrombozytapherese mit dem Zellseparator A 201 (Baxter)-erste Daten zur Biokompatibilitfit. Infusionstherapie 1991; 18:244-247. 22. Murphy S: Initial validation and quality control of platelet concentrates prepared by apheresis. Transfus Sci 1993; 14:89-94.
Transfus. Sci. Vol. 17, No. 2, pp. 315-320, 1996 Copyright © 1996 Elsevier Science Ltd. All rights reserved PII:S0955-3886(96)00010-0 Printed in Great Britain 0955-3886/96 $15.00 + .00
Biocompatibility Studies of the Single and Dual-needle Procedure in Plateletpheresis with the Fresenius AS 104 Blood Cell Separator Rainer Moog, MD* § Janos G. Kadar, MDt Norbert Mfiller, MD* Marcel Reiser, MDt Thomas Weit~wange, PhD¢ Anja E. Ledermann* • To investigate the biocompatibility of the new single-needle (SN) technique of the AS 104 blood cell separator, we analysed coagulation, complement and haemolysis parameters prior to, during and after five plateletphereses. The data were compared with results of the same parameters from identical donors in the dual-needle (DN) procedure. The analysis of bilirubin, lactate dehydrogenase (LDH), hydroxybutyrate dehydrogenase (HBDH) and haptoglobin showed that there was no relevant haemolysis. However, changes of the concentration of the coagulation factors XII and XI indicated an activation of coagulation. This was confirmed by an increase of the thrombin antithrombin (TAT) Ill complex during apheresis. Comparison of the platelet products showed no significant differences. Glucose concentration was 332 + 14 mg/dL in the SN and 312 + 32 mg/dL in the DN procedure, respectively. Lactate level was 3.2 _+0.6 mmol/L in the SN and 3.3 + 0.6 in the DN technique, respectively. Levels of complement activation markers were not statistically different. Leukocyte contamination was statistically higher in the SN procedure. Copyright © 1996 Elsevier Science Ltd •
INTRODUCTION Before a new cell separator is used routinely, studies of its biocompatibility should be carried out. 1 It is the ethical task of the user to protect the donor from any harm caused by cytapheresis. The biocompatibility of the artificial surfaces plays an important role when donor blood comes into contact with nonphysiological material. Furthermore, drugs administered during apheresis, such as the anticoagulant, can influence the compatibility of cytapheresis. The cell separator Fresenius AS 104 was introduced in the European apheresis market in 1987. It belongs to the third generation of blood cell separators. All separation procedures work under automatic control by an interface control system and several sensitive detectors and monitors. Separation protocols for bone marrow stem cell separation, 2,~3 granulocyte collection, 4 lymphocyte depletion, monocyte collection, peripheral blood stem cell collection, 2,s,6 platelet (PLT) collection, 6-16 plasma exchange and red cell exchange are implemented. These protocols use the continuous flow technique with a sealless disposable. Most procedures were carried out using the PLT collection protocol and resulted in a good quality of the product. In 1991, a SN technique for plateletpheresis was developed.
*Institute for Transfusion Medicine, University of Essen (FRG), Hufelandstr. 55, 45122, Essen, Germany. tHaemapheresis Unit, Department of Internal Medicine I, University of Cologne, Cologne, Germany. ~:Fresenius AG, R&D, St. Wendel, Germany. §Author for correspondence. T5 17-2-H
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Transfus. Sci. Vol. 17, No. 2
Using the SN procedure resulted in sufficient PLT yields. ~6,~zThe leukocyte contamination of the platelet concentrate (PC) was slightly elevated in comparison with the products obtained with the DN technique. In our study we investigated the quality of platelet products collected with the DN and SN procedure. Furthermore, we analysed coagulation, complement and haemolysis parameters prior to, during and after plateletpheresis to compare the biocompatibility of the DN and SN technique.
MATERIALS A N D METHODS
Five female blood donors underwent paired plateletpheresis, one separation with the DN, the other one with the SN technique. They all complied with the German recommendations for cytapheresis. ~7,18 Written consent was obtained prior to apheresis. PLT pre-counts were above 200,000hzL. The donation interval was at least 4 weeks. The AS 104 blood cell separator [Fresenius AG, Bad Homburg, Germany) was used for thrombocytapheresis. All separations were done with standard protocols: in the DN procedure whole blood was drawn at a blood flow rate of 50 mL/min. The anticoagulant/blood ratio was 1 : 10. The blood was pumped via the whole blood pump into the first stage of a dual stage separation chamber. There it was centrifuged at 1900 rpm. The interface position (red cell/plasma border) was 7 : 1 . Red blood cells and platelet-poor plasma were re-infused to the donor via the return line. Plateletrich plasma entered the second stage of the separation chamber. From there the thrombocytes were pumped, via the cell pump, into the platelet storage bag. The cell pump speed was 2.8 mL/min. The procedure stopped when an endpoint volume of 3500 mL blood was processed. Two major changes were necessary for the SN technique: first, inlet and return line were connected by a Yline in the disposable. Second, a bag
press (pressure of pneumatic spring 150 N) for a transfer bag was constructed. The transfer bag was a reservoir for the red cells during the draw cycle. The return cycle started when a blood volume of 250 mL was reached in the draw cycle. A part of the re-infusion blood re-circulated to the centrifuge. The procedure ended when the endpoint volume of 2500 mL was reached. The blood flow rate {60 mL/mL) and the centrifuge speed [2000 rpm} were different from the DN technique. To investigate the biocompatibility of the AS 104, we analysed the coagulation, complement and haemolysis parameters prior to, during and after apheresis. Reagents for the determination of bilimbin, lactute dehydrogenase (LDH), hydroxybutyrate dehydrogenase (HBDH) and total protein (biuret method) were purchased from Boehringer Mannheim (Mannheim, Germanyl. A photometer (Vitalab 200, Vital Scientific, van Dieren, The Netherlands) was used for these measurements. Haptoglobin (NOR-Partigen R, Behring, Marburg, Germany) was determined by irnmunodiffusion. Antithrombin III (AT III), coagulation factors XII and XI were analysed by the chromogenic substrate method (Chromotimer, Behring, Marburg, Germany). TAT llI complex was determined by an ELISA (EnzygnostR, Behring, Marburg, Germany). The complement factors C3a, C4d and SCSbC9 were determined by commercially available ELISAs (Progen, Heidelberg, Germany; Quidel, San Diego, U.S.A.). For glucose measurement, the hexokinase method was used (Glucoquant g, Boehringer Mannheim, Mannheim, Germany). Lactate was determined with a macrodetermination according to manufacturer's instructions. The test kit Testomar R (Behring, Marburg, Germany) was used for this assay. Leukocytes were counted manually in a Neubauer chamber.l~ Platelet determination was conducted electronically (Coulter Counter T 660, Coulter Electronics, Krefeld, Germany). Morphology score was determined according to the method of Kunicki e t a l ? °
Biocompatibility Studies 317
Blood samples were drawn prior to (I), after 75 mL processed blood volume (II), 100 mL before the termination of the donation (V), and after apheresis in both procedures (VI). Additionally, samples were collected during the third (750 mL; III) and seventh (1750 mL; IV) return cycle in the SN technique. Sampling took place at 850 (III) and 1700 (W) mL from the return line of the DN set. For statistical analysis, the t-test for paired data was used. The level of statistical significance was set at 0.05.
the first minutes of separation. Later on, during the DN or SN donation, no apparent differences could be seen by either of the methods used. The data of the investigated parameters in the PC are shown in Table 2. There was a statistical difference in the leukocyte counts of both procedures. Complement parameters, glucose, lactate, morphology score and platelets in the PC did not statistically differ between both techniques.
DISCUSSION RESULTS
The analysis of bilirubin, LDH, HBDH and haptoglobin showed that there was no relevant haemolysis (Table 1). All parameters were within normal ranges and there was no evidence for damage of red blood cells. Total protein decreased during the course of separation. This parameter was used as a dilution factor in the measurement of coagulation and complement factors. Decreased activity of the coagulation factors XII and XI was seen in the return line of the DN and SN procedure. No significant changes of the AT III levels were seen during apheresis. The TAT III complex continuously increased during the course of separations. Peak values of 66.9 + 15.6 ~zg/L were reached in samples drawn 100 mL before the termination of the DN procedure. After donation, the concentrations of factors XII and XI were similar to the values prior to donation. The TAT III complex decreased. We found no significant change in the complement split product content of serum samples obtained during the apheresis sessions. The pre-donation C3a levels were in the normal range, the corresponding C4d concentrations were elevated when compared with the in-house laboratory control. The results were estimated by using a dilution factor as obtained by the measurement of total protein concentration. We observed a slight increase of complement split product content of the plasma after
There is an ethical responsibility for the operator to protect the donor from any risk of health due to cytapheresis. In apheresis, donor blood comes into contact with the non-physiological surface of the disposable. Furthermore, the blood bank staff are responsible for the quality and integrity of the blood product produced. Therefore, biocompatibility of a new blood donation procedure has to be established before it is introduced for routine use. The biocompatibility of the DN technique in thrombocytapheresis with the Fresenius AS 104 blood cell separator was investigated by Kretschmer et al.~ We studied coagulation, complement and haemolysis parameters prior to, during and after plateletpheresis using the SN technique of the AS 104 cell separator. The results were compared with the data of the DN procedure, using identical donors. Our data demonstrated that there is no relevant haemolysis of red blood cells in both separation procedures. This was in accordance with the findings of Kretschmer and co-workers in the DN thrombocytapheresis.' Analysis of coagulation parameters showed a decreased activity of factors XII and XI in the return line of the DN set and in the return cycles of the SN procedure. We made similar observations when we studied the biocompatibility of the cell separator A 2 0 1 . 21 We interpreted this as a slight activation of coagulation when the blood came into
318
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Table 1. Results of Haemolysis, Coagulation and Complement Parameters Prior to, During and After DN and SN Plateletpheresis (Means) I
Bilirubin (mg/dL) Bilirubin (mg/dL) Haptoglobin (g/L) Haptoglobin (g/L) HBDH (U/L) HBDH (U/L) LDH (U/L) LDH (U/L) Protein (g/dL) Protein (g/dL) AT III (%) AT III (%} Factor XII (%) Factor XII (%) Factor XI (%) Factor XI (%) TAT III complex (tag/L) TAT III complex (t,g/L) C3a (ng/mL) C3a (ng/mL) C4d (ng/mL) C4d (ng/mL) SC5bC9 (ng/mL} SC5bC9 (ng/mL)
DN SN DN SN DN SN DN SN DN SN DN SN DN SN DN SN DN SN DN SN DN SN DN SN
0.6 0.7 2.3 2.3 71 68 128 120 7.3 7.3 105 110 98 105 103 107 4.5 2.08 266 294 5095 4521 47 91
II
III
0.5 0.7 2.2 2.2 78 87 141 156 6.9 7.0 102 106 87 93 102 99 2.1 2.1 297 324 4881 4611 46 79
0.4 0.5 1.9 1.5 55 52 97 93 5.9 5.3" 105 103 85 86 80 81 7.7 9.4 230 215 1478 1801 54 79
IV
0.4 0.5 1.8 1.6 54 55 95 93 5.7 5.5 113 107 89 84 75 79 12.9 7.8 143 210 1982 2710 51 49
V
0.3 0.6 1.8 2.0 74 72 130 121 6.2 6.3 99 107 100 101 90 100 66.9 3.0* 150 163 3296 2310 59 44
VI
0.5 0.6 1.8 1.8 61 58 107 97 6.2 6.0 114 113 106 101 96 108 22 4.2 162 199 1666 2327 20 51
*Significant (level P < 0.05).
c o n t a c t w i t h the non-physiological surface of the disposable. N o changes of activity w e r e seen in the A T III level during plateletpheresis. Coffe et al. also observed n o r m a l values for the coagulation i n h i b i t o r a n t i t h r o m b i n III after d o n a t i o n w i t h the D N procedure. 8 T h e levels of the c o m p l e m e n t activ a t i o n p a r a m e t e r obtained suggest t h a t no significant surface-related a c t i v a t i o n t o o k place in the s y s t e m w h i c h w o u l d
have b e e n clinically relevant for the donor or the recipient. In a f o r m e r work, we also reported a clinically insignificant c o m p l e m e n t a c t i v a t i o n m e a s u r e d in the r e t u r n line of the blood cell separator AS 104.1 T h e p r o d u c t quality of platelets collected b y a n e w apheresis t e c h n i q u e s h o u l d be tested prior to r o u t i n e use. 22 O u r data d e m o n s t r a t e that there is an equal quality of the platelet p r o d u c t
Table 2. Results of Parameters Measured in PC (means + 1 standard deviation)
C3[desarg) (ng/mL) C4d (ng/mL} SC5bC9 (ng/mL) Glucose (mg/dL) Lactate (mmol/L) Leukocytes (x 106) Morphology score Platelets (x 1011) *Significant (level P < 0.05).
DN
SN
258 + 68 3062 + 1709 77 + 38 312 + 32 3.3 + 0.6 5.3 + 5.4 353 + 20 3.7 + 0.9
199 + 26 2221 _+875 67 + 44 332 + 14 3.2 + 0.6 26.4 + 28.5* 363 + 17 3.3 + 0.4
Biocompatibility Studies
obtained by both techniques except for leukocyte contamination. Overall, we conclude that there is an equal biocompatibility of the SN technique compared with the DN procedure using the Fresenius AS 104 blood cell separator. The quality of the platelet product is fine.
Acknowledgements The technical assistance of Mrs. garin Weber and the staff of the Haemapheresis Unit is gratefully acknowledged.
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