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Preparation of platelet concentrate from pooled buffy coats suspended in an additive solution
Tmnsfus. Sci. 1990; 11:353-355 Printed in Great Britain. All rights reserved
Copyright
0955-3886/90 $3.00+0.00 @I 1990 Pergamon Press plc
Preparation
of Platelet Concentrate from Pooled Buffy Coats Suspended in an Additive Solution an Claes F. H6 sson Lars Eri i8Yn Hans Gulliksson Agneta Shanwell
using suspensions of platelets in simple electrolyte media. Disappointingly, we obtained poor results using some experimental media supplied by a manufacturer of blood storage systems according to specifications made in collaboration with Dr Rock. Clumping and gross alpha granule release was seen at an early stage of storage. The reason for these findings, which were contrary to those obtained in Dr Rock’s laboratory, was never solved. Some toxic effect was likely but the origin of it remained obscure. We then designed a medium with the composition described below, supplied in glass bottles and sterilized by autoclaving. This medium gave satisfactory results which was also true when we later obtained it in plastic bags. A summary of some recent results will be given below. The suspension medium had to be non-acid, and in order to avoid the problem of glucose caramellization, we decided to use the plasma remaining in the buffy coat as the source of glucose and bicarbonate, and not include glucose in the artificial medium. In the initial phase of the study PCs were prepared after gentle centrifugation of the pooled and diluted huffy coat contents, using a laminar flow hood to make the connections and applying a shelf life restricted of 12 h in the in vivo studies. The advent of the DuPont/ Haemonetics sterile connector device (SCD 312) made it possible to make the procedure much more acceptable from a microbiological standpoint.
INTRODUCTION
From the mid-1970s removal of buffy coat became a routine procedure in Sweden with the aim of reducing the formation of microaggregates and the content of leukocytes which cause febrile reactions in patients with leucocyte antibodies. Stimulated by the experience in the Netherlands, we started work, in the early 1980s using platelet-rich buffy coats as the source material for platelet concentrate (PC). We began using plasma as the suspension medium for the buffy coat material but soon our work concentrated on the development of an additive solution for this purpose. The major aim was to save plasma for other purposes and to reduce the plasma load of those patients receiving large numbers of platelets. Particularly in ABO-compatible, but not ABO-identical, platelet transfusions, adverse reactions were sometimes experienced, some of them apparently caused by passively transmitted anti-A/ anti-B. Because of difficulties in obtaining sufficiently good yields, when the small volume of a buffy coat was centrifuged in a normal size bag, we soon concentrated our efforts on pooled huffy coats. Very satisfactory results had been reported by Rock and collaborators’
From the Department of Clinical Immunology and Tmnsfusion Medicine Univenity Hospital, Uppsala and The Stockholm Blood Transfusion Service, Huddin e Hospital and SGdersjukhuset Hospital, Stockholm, Swe ! en. 353
354
Transfus. Sci.
Vol. 11, No. 314
MATERIALSAND METHODS Blood Component Preparation 450 mL of donor blood was collected in 63 mL of CPD anticoagulant in quadruple bag CPD/SAC-M systems. In a first series of experiments the plastic material was Teruflex (Terumo Corp., Japan); in a second series the Opti system (Baxter, Belgium) was used, where the container for blood collection and buffy coat storage is made of PL-146 plastic. Initial centrifugation was 3500 rpm in a Damon DPR centrifuge for 10 min (first series) and 4000 rpm in a Beckman J-6M with oval cups (second series). In the first series approximately 45 g of buffy coat was removed, but the variations, when considered on a routine scale, were large; in the second series 55 g was allowed to remain in the original unit, with satisfactory standardization and a hematocrit of 40%. The buffy coats were stored overnight and the PC preparation was usually performed 18-24 h after collection. Preparation of Platelet Concentrate (PC) Using sterile connections, (Haemonetics SCD 312) buffy coat units and a unit of platelet additive solution (PAS) were connected to a 600 mL transfer pack with eight couplers. The buffy coat contents (6 units in the first series, 4 units in the second series) were carefully washed out of their containers into the transfer bag. The empty containers were removed and replaced by a 100 mL PL-732 container; the system was centrifuged at 1300 rpm for 7 min followed by slow (10-15 min) expression of the platelet-rich supernatant into the PL-732 bag. Composition of the PAS was KC1 0.75 g/L, Na,HPO,, 2 Hz0 0.89 g/L, Na,citrate, 2Hz0 8.82 g/L, NaCl4.09 g/L, Dmannitol 5.47 g/L, pH adjusted to 7.2. In Vitro Tests Platelet counting was performed in a Thrombocyte Analyzer 147C (Analys
Instrument AR, Sweden), leucocyte counting microscopically in a FuchsRosenthal chamber. Blood gases and bicarbonate were analysed in an ABL 300 (Radiometer, Denmark). Details of the study and other test methods will be described elsewhere.2 Ln Vivo Recovery Studies Details of this investigation will also be described elsewhere.3 In brief, two units of 450 mL each of donor blood were collected from volunteers, buffy coats were prepared, left overnight, and platelet concentrates were prepared in smallsize containers. The platelets were mixed and then again divided into two equal parts, one of which was labelled with ‘r’In on the preparation day, the other portion 4 days later. RESULTS In Vitro Data The yield of platelets per unit of PC was 316 + 69 x lo9 in an initial experiment (n= 12) using 6 buffy coats per pool, and the leucocyte contamination was 110 + 18 x lo9 per unit. The protein concentration was 15.7 + 2.6 g/L. The pH was 6.91 f 0.09 on day 1 directly after preparation, decreasing to 6.65 + 0.09 on day 7. The bicarbonate was not exhausted during 7 days and the ATP was kept at about 4 ~ol/lO” platelets. The glucose supply was sufficient for 4 days but had been exhausted in some units by day 7. The PF-4 release was 10.5 + 5.2% initially and increased to 33.7 + 7.2% on day 7. The LDH release stayed low for the first 3 days (at about 23%) and then rose to 10%. When this method was applied on a larger, routine scale it was found that the yield tended to vary considerably depending on the hematocrit of the buffy coat: high hematocrit caused poor yields. The method was then revised, using 4 buffy coats instead of 6 and a larger volume of PAS, 300 mL instead of 250 mL as before. In this way the yield was
PlateletConcentrate from PooledBuffyCoats 355 dramatically improved: 316 + 52 x lo9 per PC (range 203-490 x 109, n = 1341, which implies a mean of 79 x lo9 platelets from each unit of whole blood. Another important observation was made. The buffy coats showed higher pCOz values and lower pHs than in the first series, causing a more rapid loss of swirling in a larger percentage of the units than in the first series. The probable explanation is that more CO2 was accumulated in this container during the long holding period of the buffy coats. The problem was solved by using two 1-L PL-732 containers for storage of the platelets from one 4 buffy coat pool, similar to what is often done with platelets from apheresis. This increased the pH sufficiently to attain good storage conditions for at least 4 days. Platelet Recovery in Normal Volunteers The results will be described in detail in a separate paper.3 In brief, the recovery was 54.6 f 8.7% of freshly-prepared platelets on the day after blood collection. When stored for 4 days (5th day after collection) the recovery was 5 1.9 + 10.4%. This was not significantly different from another study carried out using the same technique on apheresis platelets (58.4 f 12.3% on the day of collection and 57.6 + 11.9 on day 5). The half-time and total survival time were also very similar to the previous study. Corrected Count Increment (CCI) in Severely Thrombocytopenic Recipients The 6 buffy coat preparations in severely thrombocytopenic many of whom were suffering cemia during their aplasia some of whom had formed bodies. The general clinical
were used patients from septiperiod and HLA antiimpression
was that increments, when plotted in a graph, were similar to those obtained with apheresis platelets prepared with the CS-3000 machine from unselected donors. In the second series, the 1 h CC1 was 15.3 f 10.7 in BC-PC transfusions (n= 20) and 11.3 + 8.5 in apheresis-PC transfusions (n=53). The corresponding figures in 24 h CC1 were 11.6 + 9.6 and 8.9 + 6.8, respectively. DISCUSSION Platelets prepared from buffy coats which have been stored for 18-24 h at room temperature behave similarly to those prepared with traditional methods. The hematocrit of the buffy coat pool should be kept low (preferably not more than 20%) in order to allow high yield of platelets and low leucocyte contamination. Since the platelet additive solution in this system does not contain glucose, sufficient plasma has to remain in the buffy coats to supply the platelets with glucose and bicarbonate. The advantage is its simplicity, particularly in combination with automated techniques for blood component preparation, the considerable plasma saving effects and reduced concentration of alloantibodies delivered to the recipients.
REFERENCES Rock G, Swenson SD, Adams GA: Platelet storage in plasma-free medium. Trunsfusion 1985; 25:551-X6. 2. Eriksson L, H&man CF: Platelet concentrates in an additive solution prepared from pooled huffy coats. I. In vitro studies. VOXsang 1990; 59:140-145. 3. Eriksson L, Shanwell A, Gulliksson, H, Hiigman CF, Berg B: Platelet concentrates in an additive solution prepared from pooled buffy coats. II. In vivo studies. In 1.
preparation.
Copyright
0955-3886/90 $3.00+0.00 @I 1990 Pergamon Press plc
Preparation
of Platelet Concentrate from Pooled Buffy Coats Suspended in an Additive Solution an Claes F. H6 sson Lars Eri i8Yn Hans Gulliksson Agneta Shanwell
using suspensions of platelets in simple electrolyte media. Disappointingly, we obtained poor results using some experimental media supplied by a manufacturer of blood storage systems according to specifications made in collaboration with Dr Rock. Clumping and gross alpha granule release was seen at an early stage of storage. The reason for these findings, which were contrary to those obtained in Dr Rock’s laboratory, was never solved. Some toxic effect was likely but the origin of it remained obscure. We then designed a medium with the composition described below, supplied in glass bottles and sterilized by autoclaving. This medium gave satisfactory results which was also true when we later obtained it in plastic bags. A summary of some recent results will be given below. The suspension medium had to be non-acid, and in order to avoid the problem of glucose caramellization, we decided to use the plasma remaining in the buffy coat as the source of glucose and bicarbonate, and not include glucose in the artificial medium. In the initial phase of the study PCs were prepared after gentle centrifugation of the pooled and diluted huffy coat contents, using a laminar flow hood to make the connections and applying a shelf life restricted of 12 h in the in vivo studies. The advent of the DuPont/ Haemonetics sterile connector device (SCD 312) made it possible to make the procedure much more acceptable from a microbiological standpoint.
INTRODUCTION
From the mid-1970s removal of buffy coat became a routine procedure in Sweden with the aim of reducing the formation of microaggregates and the content of leukocytes which cause febrile reactions in patients with leucocyte antibodies. Stimulated by the experience in the Netherlands, we started work, in the early 1980s using platelet-rich buffy coats as the source material for platelet concentrate (PC). We began using plasma as the suspension medium for the buffy coat material but soon our work concentrated on the development of an additive solution for this purpose. The major aim was to save plasma for other purposes and to reduce the plasma load of those patients receiving large numbers of platelets. Particularly in ABO-compatible, but not ABO-identical, platelet transfusions, adverse reactions were sometimes experienced, some of them apparently caused by passively transmitted anti-A/ anti-B. Because of difficulties in obtaining sufficiently good yields, when the small volume of a buffy coat was centrifuged in a normal size bag, we soon concentrated our efforts on pooled huffy coats. Very satisfactory results had been reported by Rock and collaborators’
From the Department of Clinical Immunology and Tmnsfusion Medicine Univenity Hospital, Uppsala and The Stockholm Blood Transfusion Service, Huddin e Hospital and SGdersjukhuset Hospital, Stockholm, Swe ! en. 353
354
Transfus. Sci.
Vol. 11, No. 314
MATERIALSAND METHODS Blood Component Preparation 450 mL of donor blood was collected in 63 mL of CPD anticoagulant in quadruple bag CPD/SAC-M systems. In a first series of experiments the plastic material was Teruflex (Terumo Corp., Japan); in a second series the Opti system (Baxter, Belgium) was used, where the container for blood collection and buffy coat storage is made of PL-146 plastic. Initial centrifugation was 3500 rpm in a Damon DPR centrifuge for 10 min (first series) and 4000 rpm in a Beckman J-6M with oval cups (second series). In the first series approximately 45 g of buffy coat was removed, but the variations, when considered on a routine scale, were large; in the second series 55 g was allowed to remain in the original unit, with satisfactory standardization and a hematocrit of 40%. The buffy coats were stored overnight and the PC preparation was usually performed 18-24 h after collection. Preparation of Platelet Concentrate (PC) Using sterile connections, (Haemonetics SCD 312) buffy coat units and a unit of platelet additive solution (PAS) were connected to a 600 mL transfer pack with eight couplers. The buffy coat contents (6 units in the first series, 4 units in the second series) were carefully washed out of their containers into the transfer bag. The empty containers were removed and replaced by a 100 mL PL-732 container; the system was centrifuged at 1300 rpm for 7 min followed by slow (10-15 min) expression of the platelet-rich supernatant into the PL-732 bag. Composition of the PAS was KC1 0.75 g/L, Na,HPO,, 2 Hz0 0.89 g/L, Na,citrate, 2Hz0 8.82 g/L, NaCl4.09 g/L, Dmannitol 5.47 g/L, pH adjusted to 7.2. In Vitro Tests Platelet counting was performed in a Thrombocyte Analyzer 147C (Analys
Instrument AR, Sweden), leucocyte counting microscopically in a FuchsRosenthal chamber. Blood gases and bicarbonate were analysed in an ABL 300 (Radiometer, Denmark). Details of the study and other test methods will be described elsewhere.2 Ln Vivo Recovery Studies Details of this investigation will also be described elsewhere.3 In brief, two units of 450 mL each of donor blood were collected from volunteers, buffy coats were prepared, left overnight, and platelet concentrates were prepared in smallsize containers. The platelets were mixed and then again divided into two equal parts, one of which was labelled with ‘r’In on the preparation day, the other portion 4 days later. RESULTS In Vitro Data The yield of platelets per unit of PC was 316 + 69 x lo9 in an initial experiment (n= 12) using 6 buffy coats per pool, and the leucocyte contamination was 110 + 18 x lo9 per unit. The protein concentration was 15.7 + 2.6 g/L. The pH was 6.91 f 0.09 on day 1 directly after preparation, decreasing to 6.65 + 0.09 on day 7. The bicarbonate was not exhausted during 7 days and the ATP was kept at about 4 ~ol/lO” platelets. The glucose supply was sufficient for 4 days but had been exhausted in some units by day 7. The PF-4 release was 10.5 + 5.2% initially and increased to 33.7 + 7.2% on day 7. The LDH release stayed low for the first 3 days (at about 23%) and then rose to 10%. When this method was applied on a larger, routine scale it was found that the yield tended to vary considerably depending on the hematocrit of the buffy coat: high hematocrit caused poor yields. The method was then revised, using 4 buffy coats instead of 6 and a larger volume of PAS, 300 mL instead of 250 mL as before. In this way the yield was
PlateletConcentrate from PooledBuffyCoats 355 dramatically improved: 316 + 52 x lo9 per PC (range 203-490 x 109, n = 1341, which implies a mean of 79 x lo9 platelets from each unit of whole blood. Another important observation was made. The buffy coats showed higher pCOz values and lower pHs than in the first series, causing a more rapid loss of swirling in a larger percentage of the units than in the first series. The probable explanation is that more CO2 was accumulated in this container during the long holding period of the buffy coats. The problem was solved by using two 1-L PL-732 containers for storage of the platelets from one 4 buffy coat pool, similar to what is often done with platelets from apheresis. This increased the pH sufficiently to attain good storage conditions for at least 4 days. Platelet Recovery in Normal Volunteers The results will be described in detail in a separate paper.3 In brief, the recovery was 54.6 f 8.7% of freshly-prepared platelets on the day after blood collection. When stored for 4 days (5th day after collection) the recovery was 5 1.9 + 10.4%. This was not significantly different from another study carried out using the same technique on apheresis platelets (58.4 f 12.3% on the day of collection and 57.6 + 11.9 on day 5). The half-time and total survival time were also very similar to the previous study. Corrected Count Increment (CCI) in Severely Thrombocytopenic Recipients The 6 buffy coat preparations in severely thrombocytopenic many of whom were suffering cemia during their aplasia some of whom had formed bodies. The general clinical
were used patients from septiperiod and HLA antiimpression
was that increments, when plotted in a graph, were similar to those obtained with apheresis platelets prepared with the CS-3000 machine from unselected donors. In the second series, the 1 h CC1 was 15.3 f 10.7 in BC-PC transfusions (n= 20) and 11.3 + 8.5 in apheresis-PC transfusions (n=53). The corresponding figures in 24 h CC1 were 11.6 + 9.6 and 8.9 + 6.8, respectively. DISCUSSION Platelets prepared from buffy coats which have been stored for 18-24 h at room temperature behave similarly to those prepared with traditional methods. The hematocrit of the buffy coat pool should be kept low (preferably not more than 20%) in order to allow high yield of platelets and low leucocyte contamination. Since the platelet additive solution in this system does not contain glucose, sufficient plasma has to remain in the buffy coats to supply the platelets with glucose and bicarbonate. The advantage is its simplicity, particularly in combination with automated techniques for blood component preparation, the considerable plasma saving effects and reduced concentration of alloantibodies delivered to the recipients.
REFERENCES Rock G, Swenson SD, Adams GA: Platelet storage in plasma-free medium. Trunsfusion 1985; 25:551-X6. 2. Eriksson L, H&man CF: Platelet concentrates in an additive solution prepared from pooled huffy coats. I. In vitro studies. VOXsang 1990; 59:140-145. 3. Eriksson L, Shanwell A, Gulliksson, H, Hiigman CF, Berg B: Platelet concentrates in an additive solution prepared from pooled buffy coats. II. In vivo studies. In 1.
preparation.