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Leukocyte-poor platelet concentrates from buffy coats. The Dutch experience
Tmnsfus. Sci. 1990; 11:349351 Printed in Great Britain. All rights reserved
095538W90 !xwO+o.OO Copyright 0 1990 Pergamon Press plc
Leukocyte-poor Platelet Concentrates from Buffy Coats. The Dutch Experience R. N. I. Pietersz H. K. Prim* W. J. A. Dekker H. W. Reesink
Since 1975 in Amsterdam all whole blood donations are separated into plasma, a buffy coat and red cells following high-speed centrifugation.’ Early experiments with the IBM cell processor had shown that centrifugation at speeds higher than 1000 g induced separation according to the size of the blood cells.2 Subsequent separation according to specific gravity will occur because the red cells get packed and squeeze out larger cells like leukocytes. The platelets will sediment on top of the red cells. Translated to blood bag centrifuges, an equilibrium between centrifugation time and speed was found with 10 min at 2960 g. In the acceleration phase3 of centrifugation, sedimentation occurs, whereas, in the plateau phase, the red cells get packed and the buffy coat is formed. If the acceleration is too fast the leukocytes will be captured between the red cells and a g-force higher than 3000 g is thought to be detrimental for the blood cells.4 If the plateau phase is too short leukocytes will remain in the red cell layer and the platelets do not reach the huffy coat. Following centrifugation the whole blood is manually separated into plasma, buffy coat and red cells in a plasma extractor or automatically processed in a special device designed for this method of blood separation. To obtain cell-free
plasma it is important to terminate plasma expression when a layer of approximately 2 cm of plasma remains over the cells; a clamp or another device dividing the bag into two compartments should then be applied approximately 2 cm under the demarcation between plasma and buffy coat/red cells to prevent leukocytes falling back into the red cell layer. The buffy coat is expressed by rolling up the bag over the clamp or by a flat press in the automatic device. A buffy coat prepared in this way has a volume of rf: 65 mL with a hematocrit of 35% and contains more than 90% of the platelets and more than 70% of the leukocytes originally present in the whole blood. The red cells are suspended in plasma or additive solution to warrant storage for 3-6 weeks. An advantage of the removal of a huffy coat containing more than 90% of the platelets and more than 70% of the leukocytes from the red cells is that no microaggregate formation occurs in the red cell concentrates (RCC) during storin better rheological age, resulting behaviour.‘s5 As shown by Hogman et ~1.~ and also demonstrated by our own experience’ another advantage of leukocyte removal is that less hemolysis will occur when the RCC are stored in protein-poor medium. Furthermore, the patients experience fewer febrile reactions after transfusion8 Leukocyte-poor platelet concentrates can be prepared from the buffy coat by pooling 6-8 fresh (<24 h following
From the Red Cross Blood Bank and’ Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands.
349
350
Transfis. Sci.
Vol. 11, No. 314
phlebotomy) buffy coats in a 600 mL transfer bag.’ Plasma or hydroxyethylstarch (HES) was added to obtain a bag without wrinkles then following centrifugation for 5 min at 1900 rpm (800 g) at 22°C the platelet-rich plasma was gently expressed into an empty satellite bag. The leukocyte contamination of these platelet concentrates never exceeded 2 x lo’, the platelet count averaged 50 x lo9 per donor unit (R. N. I. Pietersz, unpublished observations), and the expiry time was 6 h following preparation because the system had to be opened for processing. For many years (1974-1980) this method was used in the Red Cross Blood Bank in Amsterdam, but problems occurred when the gradually increasing demand for platelet concentrates forced us to have an inventory of platelet concentrates. Since 1980, the buffy coats had to be stored at 4°C for up to 3 days. Although the logistic problems were solved, a disadvantage was that platelets isolated from these buffy coats were hemostatically active, but in viva survival was only 1 or 2 days.9 In 1984 four-bag systems with two empty satellite bags with an additive solution for the red cells, in one satellite bag (for instance saline-adenineglucose-mannitol (SAGM)), became available. The four-bag system enabled a new method of preparation of leukocytepoor platelet concentrates from the buffy coat immediately following blood separation with subsequent storage of single platelet concentrates at 22”C’O In brief, following centrifugation (10 min at 2960 g at 22°C) of the whole blood, separation into plasma, a buffy coat and red cells was achieved. The SAGM was added to the red cells and approximately 35 mL of plasma was returned to the buffy coat, both to clean the tubing of leukocytes and red cells and to obtain a buffy coat volume of 100 mL with a hematocrit between 30 and 35%. Subsequently, the buffy coat bag (300 mL), still attached to the empty SAGM bag, was centrifuged for 6 min at 380 g in special inserts to keep the bags
Figure 1. Special inserts for oval cups of the Beckham J6M centrifugeto centrifuge300 mL bags filled with f 100 mL huffy coat in an upright position. upright (Fig. l).l’ The platelet-rich plasma was gently pressed into the empty bag, with the flow regulated by a clamp on the tubing, and the transfer was then terminated when the red cell layer was about 1 cm from the top seal of the bag. The first results of these leukocytepoor platelet concentrates were promising: in 75% of the cases they contained more than 55 x lo9 platelets, the leukocyte contamination was below 10’ per unit, the red cell contamination below 5 x lo9 and the volume about 50 mL.” Storage experiments revealed that platelet morphology, with a prevalence of disc forms was well preserved up to 7 days and that no acidification occurred, the pH remained above 6.8 in regular polyvinylchloride bags as well as in special type plastics designed for platelet storage.12 In an experiment in which increasing numbers of leukocytes were added to leukocyte-poor platelet concentrates, it was shown that a well maintained pH was due to low leukocyte contamination.13 Clinical evaluation of the leukocytepoor platelet concentrates showed a corrected count increment higher than 10 at 1 and 24 h post-transfusion, irrespective of storage of the platelet concentrates for 1, 3 or 5 days.r4 At present daily 150-200 units of leukocyte-poor platelet concentrates are
Leukocyte-poor Platelet Concentrates
Quality Control Data of Routinely Prepared Leukocyte-poor Platelet Concentrates (PC) from Buffy Coats
Table 1.
1987
Year Months
6-12
1988
1-12
1989 ld
12,639 20,782 14,685 PC issued 425 431 594 PC tested 56+ 8 52+ 9 54+ 9 Volume (ml) 66+18 60+17 61f19 Platelets (X 10’) 4+ 4 3f 3 Leukocytes (x 106) 2f 2 2+ 1 3+ 1 3+ 2 Red cells (X 10’) prepared from the huffy coat in our blood bank from Tuesday to Friday. The results
of quality control, summarized in Table 1, reveal that even in routine circumstances the above-mentioned numbers of platelets, leukocytes and red cells can be obtained. In conclusion, routine preparation of leukocyte-poor platelet concentrates from the buffy coat with adequate invitro parameters after storage for 7 days and adequate survival and function in vivo is possible. The method has improved transfusion logistics and will prevent HLA-immunization in multitransfused patients. REFERENCES Prins HK, de Bruijn JCGH, Henrichs HPJ, Loos JA: Prevention of microaggregate formation by removal of “buffycoats”. VOXSang 1980; 39:48-51. De Wit JJFr, Henrichs HPJ, Odink J, Prins HK: Experiments on the preparation of blood components with the IBM 2991 blood cell processor. VOX Sung 1975; 29:352-362. Hogman CF, Johansson A, Bergius B: A simple method for the standardization of centrifugation procedures in blood component preparation. VOX Sung 1982; 431266-267. Slichter SJ: In Garratty G(ed): Current Concepts in Transfusion Practice. American Association of Blood Banks, 1985, p. 14.
351
5. Swank RL: Alteration on storage: measurements of adhesiveness of “aging” platelets and leukocytes and their removal by filtration. N Engl 1 Med 1961; 265: 728-733. 6. Hdgman CF, Hedlund K, Akerblom 0, Venge 0: Red blood cell preservation in protein-poor media. I. Leukocyte enzymes as a cause of hemolysis. Transfusion 1978; 18:233-241. 7. Pietersz RNI, Reesink HW, de Korte D, Dekker WJA, van den Ende A, Loos JA: Storage of leukocyte-poor red cell concentrates: filtration in a closed system using a sterile connection device. VOX Sung 1989; 57:29-36. 8. Lieden G, Hilden JO: Febrile transfusion reactions reduced by use of buffy coat poor erythrocyte concentrates. VOXSang 1982; 43:263-265. 9. Pietersz RNI, Loos JA, Reesink HW: Survival in vivo of platelets stored for 48 hours in the buffy coat at 4°C compared to platelet-rich plasma stored at 22°C. Blur 1987; 54:201-208. 10. Pietersz RNI, Loos JA, Reesink HW: Platelet concentrates stored in plasma for 72 hours at 22°C prepared from buffy coats of citrate-phosphate-dextrose blood collected in a quadruple-bag salineadenine-glucose-mamritol system. VOX Sang 1985; 49:81-85. 11. Pietersz RNI, Reesink HW, Dekker WJA, Fijen FJ: Preparation of leukocyte-poor platelet concentrates from buffy coats. I. Special inserts for centrifuge cups. Vox Sang 1987; 53:203-207. 12. Pietersz RNI, Reesink HW, Dekker WJA: Preparation of leukocyte-poor platelet concentrates from buffy coats. II. Lack of effect on storage of different plastics. VOX Sang 1987; 53:208-213. 13. Pietersz RNI, de Korte D, Reesink HW, van den Ende A, Dekker WJA, Roos D: Preparation of leukocyte-poor platelet concentrates from buffy coats. III. Effect of leukocyte contamination on storage conditions. VOXSang 1988; 55:14-20. 14. Pietersz RNI, Reesink I-WV,Huijgens PC, van Oers MHJ: Preparation of leukocytepoor platelet concentrates from buffy coats. IV. Clinical evaluation. Vox Sang 1988; 55:129-132.
095538W90 !xwO+o.OO Copyright 0 1990 Pergamon Press plc
Leukocyte-poor Platelet Concentrates from Buffy Coats. The Dutch Experience R. N. I. Pietersz H. K. Prim* W. J. A. Dekker H. W. Reesink
Since 1975 in Amsterdam all whole blood donations are separated into plasma, a buffy coat and red cells following high-speed centrifugation.’ Early experiments with the IBM cell processor had shown that centrifugation at speeds higher than 1000 g induced separation according to the size of the blood cells.2 Subsequent separation according to specific gravity will occur because the red cells get packed and squeeze out larger cells like leukocytes. The platelets will sediment on top of the red cells. Translated to blood bag centrifuges, an equilibrium between centrifugation time and speed was found with 10 min at 2960 g. In the acceleration phase3 of centrifugation, sedimentation occurs, whereas, in the plateau phase, the red cells get packed and the buffy coat is formed. If the acceleration is too fast the leukocytes will be captured between the red cells and a g-force higher than 3000 g is thought to be detrimental for the blood cells.4 If the plateau phase is too short leukocytes will remain in the red cell layer and the platelets do not reach the huffy coat. Following centrifugation the whole blood is manually separated into plasma, buffy coat and red cells in a plasma extractor or automatically processed in a special device designed for this method of blood separation. To obtain cell-free
plasma it is important to terminate plasma expression when a layer of approximately 2 cm of plasma remains over the cells; a clamp or another device dividing the bag into two compartments should then be applied approximately 2 cm under the demarcation between plasma and buffy coat/red cells to prevent leukocytes falling back into the red cell layer. The buffy coat is expressed by rolling up the bag over the clamp or by a flat press in the automatic device. A buffy coat prepared in this way has a volume of rf: 65 mL with a hematocrit of 35% and contains more than 90% of the platelets and more than 70% of the leukocytes originally present in the whole blood. The red cells are suspended in plasma or additive solution to warrant storage for 3-6 weeks. An advantage of the removal of a huffy coat containing more than 90% of the platelets and more than 70% of the leukocytes from the red cells is that no microaggregate formation occurs in the red cell concentrates (RCC) during storin better rheological age, resulting behaviour.‘s5 As shown by Hogman et ~1.~ and also demonstrated by our own experience’ another advantage of leukocyte removal is that less hemolysis will occur when the RCC are stored in protein-poor medium. Furthermore, the patients experience fewer febrile reactions after transfusion8 Leukocyte-poor platelet concentrates can be prepared from the buffy coat by pooling 6-8 fresh (<24 h following
From the Red Cross Blood Bank and’ Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands.
349
350
Transfis. Sci.
Vol. 11, No. 314
phlebotomy) buffy coats in a 600 mL transfer bag.’ Plasma or hydroxyethylstarch (HES) was added to obtain a bag without wrinkles then following centrifugation for 5 min at 1900 rpm (800 g) at 22°C the platelet-rich plasma was gently expressed into an empty satellite bag. The leukocyte contamination of these platelet concentrates never exceeded 2 x lo’, the platelet count averaged 50 x lo9 per donor unit (R. N. I. Pietersz, unpublished observations), and the expiry time was 6 h following preparation because the system had to be opened for processing. For many years (1974-1980) this method was used in the Red Cross Blood Bank in Amsterdam, but problems occurred when the gradually increasing demand for platelet concentrates forced us to have an inventory of platelet concentrates. Since 1980, the buffy coats had to be stored at 4°C for up to 3 days. Although the logistic problems were solved, a disadvantage was that platelets isolated from these buffy coats were hemostatically active, but in viva survival was only 1 or 2 days.9 In 1984 four-bag systems with two empty satellite bags with an additive solution for the red cells, in one satellite bag (for instance saline-adenineglucose-mannitol (SAGM)), became available. The four-bag system enabled a new method of preparation of leukocytepoor platelet concentrates from the buffy coat immediately following blood separation with subsequent storage of single platelet concentrates at 22”C’O In brief, following centrifugation (10 min at 2960 g at 22°C) of the whole blood, separation into plasma, a buffy coat and red cells was achieved. The SAGM was added to the red cells and approximately 35 mL of plasma was returned to the buffy coat, both to clean the tubing of leukocytes and red cells and to obtain a buffy coat volume of 100 mL with a hematocrit between 30 and 35%. Subsequently, the buffy coat bag (300 mL), still attached to the empty SAGM bag, was centrifuged for 6 min at 380 g in special inserts to keep the bags
Figure 1. Special inserts for oval cups of the Beckham J6M centrifugeto centrifuge300 mL bags filled with f 100 mL huffy coat in an upright position. upright (Fig. l).l’ The platelet-rich plasma was gently pressed into the empty bag, with the flow regulated by a clamp on the tubing, and the transfer was then terminated when the red cell layer was about 1 cm from the top seal of the bag. The first results of these leukocytepoor platelet concentrates were promising: in 75% of the cases they contained more than 55 x lo9 platelets, the leukocyte contamination was below 10’ per unit, the red cell contamination below 5 x lo9 and the volume about 50 mL.” Storage experiments revealed that platelet morphology, with a prevalence of disc forms was well preserved up to 7 days and that no acidification occurred, the pH remained above 6.8 in regular polyvinylchloride bags as well as in special type plastics designed for platelet storage.12 In an experiment in which increasing numbers of leukocytes were added to leukocyte-poor platelet concentrates, it was shown that a well maintained pH was due to low leukocyte contamination.13 Clinical evaluation of the leukocytepoor platelet concentrates showed a corrected count increment higher than 10 at 1 and 24 h post-transfusion, irrespective of storage of the platelet concentrates for 1, 3 or 5 days.r4 At present daily 150-200 units of leukocyte-poor platelet concentrates are
Leukocyte-poor Platelet Concentrates
Quality Control Data of Routinely Prepared Leukocyte-poor Platelet Concentrates (PC) from Buffy Coats
Table 1.
1987
Year Months
6-12
1988
1-12
1989 ld
12,639 20,782 14,685 PC issued 425 431 594 PC tested 56+ 8 52+ 9 54+ 9 Volume (ml) 66+18 60+17 61f19 Platelets (X 10’) 4+ 4 3f 3 Leukocytes (x 106) 2f 2 2+ 1 3+ 1 3+ 2 Red cells (X 10’) prepared from the huffy coat in our blood bank from Tuesday to Friday. The results
of quality control, summarized in Table 1, reveal that even in routine circumstances the above-mentioned numbers of platelets, leukocytes and red cells can be obtained. In conclusion, routine preparation of leukocyte-poor platelet concentrates from the buffy coat with adequate invitro parameters after storage for 7 days and adequate survival and function in vivo is possible. The method has improved transfusion logistics and will prevent HLA-immunization in multitransfused patients. REFERENCES Prins HK, de Bruijn JCGH, Henrichs HPJ, Loos JA: Prevention of microaggregate formation by removal of “buffycoats”. VOXSang 1980; 39:48-51. De Wit JJFr, Henrichs HPJ, Odink J, Prins HK: Experiments on the preparation of blood components with the IBM 2991 blood cell processor. VOX Sung 1975; 29:352-362. Hogman CF, Johansson A, Bergius B: A simple method for the standardization of centrifugation procedures in blood component preparation. VOX Sung 1982; 431266-267. Slichter SJ: In Garratty G(ed): Current Concepts in Transfusion Practice. American Association of Blood Banks, 1985, p. 14.
351
5. Swank RL: Alteration on storage: measurements of adhesiveness of “aging” platelets and leukocytes and their removal by filtration. N Engl 1 Med 1961; 265: 728-733. 6. Hdgman CF, Hedlund K, Akerblom 0, Venge 0: Red blood cell preservation in protein-poor media. I. Leukocyte enzymes as a cause of hemolysis. Transfusion 1978; 18:233-241. 7. Pietersz RNI, Reesink HW, de Korte D, Dekker WJA, van den Ende A, Loos JA: Storage of leukocyte-poor red cell concentrates: filtration in a closed system using a sterile connection device. VOX Sung 1989; 57:29-36. 8. Lieden G, Hilden JO: Febrile transfusion reactions reduced by use of buffy coat poor erythrocyte concentrates. VOXSang 1982; 43:263-265. 9. Pietersz RNI, Loos JA, Reesink HW: Survival in vivo of platelets stored for 48 hours in the buffy coat at 4°C compared to platelet-rich plasma stored at 22°C. Blur 1987; 54:201-208. 10. Pietersz RNI, Loos JA, Reesink HW: Platelet concentrates stored in plasma for 72 hours at 22°C prepared from buffy coats of citrate-phosphate-dextrose blood collected in a quadruple-bag salineadenine-glucose-mamritol system. VOX Sang 1985; 49:81-85. 11. Pietersz RNI, Reesink HW, Dekker WJA, Fijen FJ: Preparation of leukocyte-poor platelet concentrates from buffy coats. I. Special inserts for centrifuge cups. Vox Sang 1987; 53:203-207. 12. Pietersz RNI, Reesink HW, Dekker WJA: Preparation of leukocyte-poor platelet concentrates from buffy coats. II. Lack of effect on storage of different plastics. VOX Sang 1987; 53:208-213. 13. Pietersz RNI, de Korte D, Reesink HW, van den Ende A, Dekker WJA, Roos D: Preparation of leukocyte-poor platelet concentrates from buffy coats. III. Effect of leukocyte contamination on storage conditions. VOXSang 1988; 55:14-20. 14. Pietersz RNI, Reesink I-WV,Huijgens PC, van Oers MHJ: Preparation of leukocytepoor platelet concentrates from buffy coats. IV. Clinical evaluation. Vox Sang 1988; 55:129-132.