Ann Hematol (1992) 65- 269-273
Annals of
Hematology 9 Springer-Verlag 1992
Original article Some storage characteristics of buffy coats used for preparation of platelet concentrates P. J~iremo 1, A . S h a b a 1, a n d J. Kutti 2 i Department of Transfusion Medicine, Orebro Medical Center Hospital, Orebro, Sweden 2 Department of Medicine, Sahlgren's Hospital, G6teborg University, GOteborg, Sweden Received March 10, 1992/Accepted September 30, 1992
Summary. The present study examined biochemical storage lesions in 58 buffy coats (BCs) intended as the raw material for platelet concentrates. The work was conducted in three series; the aims were to investigate: (a) the storage quality of BCs obtained from the routine production (series I), (b) whether improvement in platelet quality could be achieved by continuous agitation during storage (series II), and (c) whether macroscopic aggregate formation indicates platelet damage (series III). Series I and II consisted of 20 BCs each. Series III compared ten BCs with visible clumping with eight BCs not demonstrating macroscopic aggregates. In series I and II platelet counts and platelet factor 4 (PF4) release were determined after 1, 3, 5, and 24 h; lactate dehydrogenase (LDH) extracellular activity was measured after 1 and 24 h. In addition, in series I elastase concentrations were analyzed after 3, 5, and 24 h. Finally, in series III extracellular 13-thromboglobulin (13-TG) levels were determined after 1 8 - 2 4 h. Low platelet counts, most likely reflecting the presence of aggregate formation, were found in 20% of the BCs (series I); in this series a total of 250/o demonstrated increasing platelet counts, together with elevated PF 4 and elastase, over the study period. Significant biochemical storage lesions were tbund after 5 and 24 h of storage. The study also demonstrated that continuous agitation during storage does not improve platelet quality (series II) and that BCs demonstrating visible clumping (series III) had augmented levels of extracellular 13-TG (p < 0.001). It is concluded that the present results do not support the view that BCs are suitable for use as the raw material for platelet concentrates. Key words: Buffy coat - Platelets - Platelet concentrates - Platelet factor 4
* This work was supported by grants from Baxter AB, Stockholm, Sweden. Correspondence to: P. J~tremo, Department of Transfusion Medicine, Orebro Medical Center Hospital, S-701 85 Orebro, Sweden
Introduction After hard centrifugation of whole blood, platelets are together with the majority of the leukocytes - enriched in the so-called buffy coat. In recent years, methods have been developed to produce platelet concentrates from buffy coats [1, 4, 9]. However, the high white cell content of buffy coats might affect platelet function, e.g., through platelet-activating enzymes and elastase [2, 5, 10]. Clearly, the bully coat storage time prior to preparation may affect the quality of the final platelet concentrate. The present study attempted to examine biochemical changes during buffy coat storage; we also investigated whether continuous agitation of the buffy coat containers might improve platelet behavior and whether visible aggregate formation indicates platelet damage.
Materials and m e t h o d s Experimental protocol
The study was conducted in three series. In series I, 20 buffy coats obtained by routine production were examined, and platelet storage injuries were compared with those for eight whole blood units. In series II, ten pairs of ABO-compatible donors were employed and the effects of continuous buffy coat agitation were examined. Two identical preparations were obtained by mixing and subsequently dividing two ABO-compatible buffy coats into two identical parts. One of these preparations was agitated using an obliquely angled agitator (NOBEL Industries, Sweden) and the other served as a nonagitated control. In series III, buffy coats from routine production were inspected with regard to aggregate formation after 18-24 h holding time. Ten buffy coats containing visible aggregates were chosen, and the results were compared with those from eight preparations in which visible clumping was not identified. The bully coats and the whole blood units were stored at room temperture. In all preparations of series I and II the platelet count and platelet factor 4 (PF 4) release were determined after 1, 3, 5 and 24 h of storage. The extracellular lactate dehydrogenase (LDH) activity was determined after 1 and 24 h and pH was measured after 24 h. In the buffy coats of series I and in the whole blood units the packed cell volume (PCV) was determined after I h. In addition, elastase was measured after 3, 5, and 24 h of storage in buffy coats of series I. Finally, in series III extracellular levels of
270 = 15/~kat/1, respectively [7]. Preformed aggregates were thought to be present if the platelet counts increased by > = 0,4• during storage. At the evaluation buffy coats in series I were divided into two groups: in group 1 the extracellular LDH and/or PF4 levels were equal to or above the above-mentioned limits, whereas in group 2 these variables were below these limits.
13-thromboglobulin (13-TG) were determined after 18-24 h of storage. Buffy coats
For this study blood was collected from 66 healthy donors; 450 ml whole blood (CPD-SAGMAN) was centrifuged within 6 h of donation at 3400 rpm for 8 min using a Damon PR-6000 (International Equipment Company, USA). The buffy coats of series I and II (approximately 50 ml) were separated manually from the red cells and stored in the satellite bag consisting of Teruflexa plastic (Terumo, Japan). IN series III an Optipress device [6] was employed for separation, and the buffy coats were stored in PM46 plastic (Baxter, France).
Statistics
For the statistical evaluation of series I And II the Wilcoxon nonparametric test for paired variables was used. In series llI Student's t-test was employed.
Results
Analytic procedures
The preparation of samples for the analyses of extracellular PF4 and LDH levels has been described in detail previously [7]. The measurements of PF 4 and 13-TGwere carried out using RIA techniques (Abbott, USA, and Amersham, UK, respectively), an immunoassay (Merck, FRG) was used for determining elastase [8], and LDH activity was assessed by standard procedures [12]. Measurement of platelet counts and PCV was performed electronically using a Coulter Counter S 8/80 (Coulter Electronics Ltd, UK). The pH was measured at 37~ using an ABL2 Acid-Base Laboratory (Radiometer AS, Denmark).
I n series I t h e n u m b e r o f b u f f y c o a t s b e l o n g i n g to g r o u p s 1 a n d 2 p r o v e d to be o f e q u a l size (Table 1). C o n s e q u e n t ly, in this series 5 0 % o f t h e b u f f y c o a t s d e m o n s t r a t e d evid e n c e o f b i o c h e m i c a l p l a t e l e t i n j u r y a n d / o r h i g h cell lysis. E x c e p t f o r t h e p H values, c o n s i d e r a b l e h e t e r o g e n e i t y was p r e s e n t as r e g a r d s t h e results f o r t h e i n d i v i d u a l b u f f y c o a t s o f series I (Table 1). D u r i n g e a r l y s t o r a g e (at 1 h) a p l a t e l e t c o u n t < = 0 . 5 x l 0 1 Z / l was f o u n d in 40~ o f t h e group-1 b u f f y coats. F u r t h e r , i n c r e a s i n g p l a t e l e t c o u n t s , i n d i c a t i n g r e s o l v i n g a g g r e g a t e s , were f o u n d in 5 0 % o f t h e g r o u p 1 p r e p a r a t i o n s . T h e s e c h a n g e s were c o m b i n e d w i t h h i g h e x t r a c e l l u l a r P F 4 levels a n d b i o -
Evaluation of buffy coats
High platelet activation and cell lysis were considered present if the extracellular PF4 and LDH levels were > = 5 mg/1 and
Table 1. Results for packed cell volume (PCV), platelet counts, pH, and the extracellular levels of lactate dehydrogenase (LDH), platelet factor 4 (PF4) and elastase for buffy coats of series I No.
PCV (%)
Platelet count (• 1012/1)
pH
LDH ~kat/1)
PF 4 (mg/1)
lh
lh
3h
5h
24h
24h
lh
24h
lh
49 51 55 58 61 65 65 68 71 73 c
0.2 1.6 1.3 1.1 0.3 0.3 1.4 1.2 1.1 0.5 ~
0.2 2.0 1.3 1.5 0.3 0.2 1.0 1.1 1.1 0.3 c
0.7 1.7 1.8 1.4 0.7 0.8 1.4 1.3 1.3 1.5 c
0.6 b 1.5 1.9 b 1.3 0.7 b 1A b ND 1.5 0.9 d 1.5 b,c
6.8 6.8 6.7 6.8 6.7 6.7 6.8 6.9 6.9 d 6.7 ~
5 4 5 4 13 4 4 4 7 7c
7 6 6 4
8 6 5 ~a,d 8c
38 52 56 59 62 62 68 68 73 76
1.1 1.6 1.3 1.7 1.2 1.5 1.0 1.1 1.4 1.5
1.2 1.6 1.5 1.8 1.1 1.8 1.2 0.5 1.0 1.2
1.4 1.8 1.5 1.6 1.4 1.5 0.9 1.5 1.2 1.7
1.4 1.8 1.6 1.1 1.1 1.3 1.2 1.4 1.1 1.5
6.9 6.8 6.8 6.7 6.8 6.8 6.8 6.9 6.8 6.8
4 3 3 4 5 3 6 4 4 5
4 6 5 5 4 5 8 3 6 4
Elastase ~g/1) 3h
5h
24h
3h
5h
24h
Group1
1 2 3 4 5 6 7 8 9 10
16 ~
1.5 1.2 4.0 1.5 2.1 3.3 3.7 0.7 0.6 16.0a, c
2.6 2.9
4.7 3.4
13.6 a 15.7 a
1.4 4.2 5.0 a
4.9 1.4 1.0 4.2 c
9.3 a 8.5 a
4.4
1.7
10.9 a 5.8 a 7.3 a 6.5 a 9 . 8 a 7.4 a 9 . 3 a ND 5.8 ~
1.5
2.3 d
60.6a, c45.5a, c
16 294 771 61 71 215 14 32 300 18 20 28 511 362 950 108 249 970 ND 172 139 10 ND < 1 0 < 1 0 5 mg/1. A, Agitated portion; ND, not determined.
coat storage as seen in series I (group 1) may well be accounted for partly by the hard-spin centrifugation itself. However, it appears that platelet behavior could also be adversely affected if platelets are stored together with activated white blood cells. In order to circumvent such
damage, platelet could be separated from white blood cells within 3 h. However, due to aggregate formation, many buffy coats would then yield platelet concentrates with low platelet counts. It may well be that by discarding huffy coats which contain visible clumping or clot formation the biochemical quality of the final platelet concentrates is improved (see Fig. 3). Also, the observation that many preparations with considerable activation of both platelets and white cells did not demonstrate visible aggregates (Table 1)
0 rX
g 10
Annals of
Hematology 9 Springer-Verlag 1992
Original article Some storage characteristics of buffy coats used for preparation of platelet concentrates P. J~iremo 1, A . S h a b a 1, a n d J. Kutti 2 i Department of Transfusion Medicine, Orebro Medical Center Hospital, Orebro, Sweden 2 Department of Medicine, Sahlgren's Hospital, G6teborg University, GOteborg, Sweden Received March 10, 1992/Accepted September 30, 1992
Summary. The present study examined biochemical storage lesions in 58 buffy coats (BCs) intended as the raw material for platelet concentrates. The work was conducted in three series; the aims were to investigate: (a) the storage quality of BCs obtained from the routine production (series I), (b) whether improvement in platelet quality could be achieved by continuous agitation during storage (series II), and (c) whether macroscopic aggregate formation indicates platelet damage (series III). Series I and II consisted of 20 BCs each. Series III compared ten BCs with visible clumping with eight BCs not demonstrating macroscopic aggregates. In series I and II platelet counts and platelet factor 4 (PF4) release were determined after 1, 3, 5, and 24 h; lactate dehydrogenase (LDH) extracellular activity was measured after 1 and 24 h. In addition, in series I elastase concentrations were analyzed after 3, 5, and 24 h. Finally, in series III extracellular 13-thromboglobulin (13-TG) levels were determined after 1 8 - 2 4 h. Low platelet counts, most likely reflecting the presence of aggregate formation, were found in 20% of the BCs (series I); in this series a total of 250/o demonstrated increasing platelet counts, together with elevated PF 4 and elastase, over the study period. Significant biochemical storage lesions were tbund after 5 and 24 h of storage. The study also demonstrated that continuous agitation during storage does not improve platelet quality (series II) and that BCs demonstrating visible clumping (series III) had augmented levels of extracellular 13-TG (p < 0.001). It is concluded that the present results do not support the view that BCs are suitable for use as the raw material for platelet concentrates. Key words: Buffy coat - Platelets - Platelet concentrates - Platelet factor 4
* This work was supported by grants from Baxter AB, Stockholm, Sweden. Correspondence to: P. J~tremo, Department of Transfusion Medicine, Orebro Medical Center Hospital, S-701 85 Orebro, Sweden
Introduction After hard centrifugation of whole blood, platelets are together with the majority of the leukocytes - enriched in the so-called buffy coat. In recent years, methods have been developed to produce platelet concentrates from buffy coats [1, 4, 9]. However, the high white cell content of buffy coats might affect platelet function, e.g., through platelet-activating enzymes and elastase [2, 5, 10]. Clearly, the bully coat storage time prior to preparation may affect the quality of the final platelet concentrate. The present study attempted to examine biochemical changes during buffy coat storage; we also investigated whether continuous agitation of the buffy coat containers might improve platelet behavior and whether visible aggregate formation indicates platelet damage.
Materials and m e t h o d s Experimental protocol
The study was conducted in three series. In series I, 20 buffy coats obtained by routine production were examined, and platelet storage injuries were compared with those for eight whole blood units. In series II, ten pairs of ABO-compatible donors were employed and the effects of continuous buffy coat agitation were examined. Two identical preparations were obtained by mixing and subsequently dividing two ABO-compatible buffy coats into two identical parts. One of these preparations was agitated using an obliquely angled agitator (NOBEL Industries, Sweden) and the other served as a nonagitated control. In series III, buffy coats from routine production were inspected with regard to aggregate formation after 18-24 h holding time. Ten buffy coats containing visible aggregates were chosen, and the results were compared with those from eight preparations in which visible clumping was not identified. The bully coats and the whole blood units were stored at room temperture. In all preparations of series I and II the platelet count and platelet factor 4 (PF 4) release were determined after 1, 3, 5 and 24 h of storage. The extracellular lactate dehydrogenase (LDH) activity was determined after 1 and 24 h and pH was measured after 24 h. In the buffy coats of series I and in the whole blood units the packed cell volume (PCV) was determined after I h. In addition, elastase was measured after 3, 5, and 24 h of storage in buffy coats of series I. Finally, in series III extracellular levels of
270 = 15/~kat/1, respectively [7]. Preformed aggregates were thought to be present if the platelet counts increased by > = 0,4• during storage. At the evaluation buffy coats in series I were divided into two groups: in group 1 the extracellular LDH and/or PF4 levels were equal to or above the above-mentioned limits, whereas in group 2 these variables were below these limits.
13-thromboglobulin (13-TG) were determined after 18-24 h of storage. Buffy coats
For this study blood was collected from 66 healthy donors; 450 ml whole blood (CPD-SAGMAN) was centrifuged within 6 h of donation at 3400 rpm for 8 min using a Damon PR-6000 (International Equipment Company, USA). The buffy coats of series I and II (approximately 50 ml) were separated manually from the red cells and stored in the satellite bag consisting of Teruflexa plastic (Terumo, Japan). IN series III an Optipress device [6] was employed for separation, and the buffy coats were stored in PM46 plastic (Baxter, France).
Statistics
For the statistical evaluation of series I And II the Wilcoxon nonparametric test for paired variables was used. In series llI Student's t-test was employed.
Results
Analytic procedures
The preparation of samples for the analyses of extracellular PF4 and LDH levels has been described in detail previously [7]. The measurements of PF 4 and 13-TGwere carried out using RIA techniques (Abbott, USA, and Amersham, UK, respectively), an immunoassay (Merck, FRG) was used for determining elastase [8], and LDH activity was assessed by standard procedures [12]. Measurement of platelet counts and PCV was performed electronically using a Coulter Counter S 8/80 (Coulter Electronics Ltd, UK). The pH was measured at 37~ using an ABL2 Acid-Base Laboratory (Radiometer AS, Denmark).
I n series I t h e n u m b e r o f b u f f y c o a t s b e l o n g i n g to g r o u p s 1 a n d 2 p r o v e d to be o f e q u a l size (Table 1). C o n s e q u e n t ly, in this series 5 0 % o f t h e b u f f y c o a t s d e m o n s t r a t e d evid e n c e o f b i o c h e m i c a l p l a t e l e t i n j u r y a n d / o r h i g h cell lysis. E x c e p t f o r t h e p H values, c o n s i d e r a b l e h e t e r o g e n e i t y was p r e s e n t as r e g a r d s t h e results f o r t h e i n d i v i d u a l b u f f y c o a t s o f series I (Table 1). D u r i n g e a r l y s t o r a g e (at 1 h) a p l a t e l e t c o u n t < = 0 . 5 x l 0 1 Z / l was f o u n d in 40~ o f t h e group-1 b u f f y coats. F u r t h e r , i n c r e a s i n g p l a t e l e t c o u n t s , i n d i c a t i n g r e s o l v i n g a g g r e g a t e s , were f o u n d in 5 0 % o f t h e g r o u p 1 p r e p a r a t i o n s . T h e s e c h a n g e s were c o m b i n e d w i t h h i g h e x t r a c e l l u l a r P F 4 levels a n d b i o -
Evaluation of buffy coats
High platelet activation and cell lysis were considered present if the extracellular PF4 and LDH levels were > = 5 mg/1 and
Table 1. Results for packed cell volume (PCV), platelet counts, pH, and the extracellular levels of lactate dehydrogenase (LDH), platelet factor 4 (PF4) and elastase for buffy coats of series I No.
PCV (%)
Platelet count (• 1012/1)
pH
LDH ~kat/1)
PF 4 (mg/1)
lh
lh
3h
5h
24h
24h
lh
24h
lh
49 51 55 58 61 65 65 68 71 73 c
0.2 1.6 1.3 1.1 0.3 0.3 1.4 1.2 1.1 0.5 ~
0.2 2.0 1.3 1.5 0.3 0.2 1.0 1.1 1.1 0.3 c
0.7 1.7 1.8 1.4 0.7 0.8 1.4 1.3 1.3 1.5 c
0.6 b 1.5 1.9 b 1.3 0.7 b 1A b ND 1.5 0.9 d 1.5 b,c
6.8 6.8 6.7 6.8 6.7 6.7 6.8 6.9 6.9 d 6.7 ~
5 4 5 4 13 4 4 4 7 7c
7 6 6 4
8 6 5 ~a,d 8c
38 52 56 59 62 62 68 68 73 76
1.1 1.6 1.3 1.7 1.2 1.5 1.0 1.1 1.4 1.5
1.2 1.6 1.5 1.8 1.1 1.8 1.2 0.5 1.0 1.2
1.4 1.8 1.5 1.6 1.4 1.5 0.9 1.5 1.2 1.7
1.4 1.8 1.6 1.1 1.1 1.3 1.2 1.4 1.1 1.5
6.9 6.8 6.8 6.7 6.8 6.8 6.8 6.9 6.8 6.8
4 3 3 4 5 3 6 4 4 5
4 6 5 5 4 5 8 3 6 4
Elastase ~g/1) 3h
5h
24h
3h
5h
24h
Group1
1 2 3 4 5 6 7 8 9 10
16 ~
1.5 1.2 4.0 1.5 2.1 3.3 3.7 0.7 0.6 16.0a, c
2.6 2.9
4.7 3.4
13.6 a 15.7 a
1.4 4.2 5.0 a
4.9 1.4 1.0 4.2 c
9.3 a 8.5 a
4.4
1.7
10.9 a 5.8 a 7.3 a 6.5 a 9 . 8 a 7.4 a 9 . 3 a ND 5.8 ~
1.5
2.3 d
60.6a, c45.5a, c
16 294 771 61 71 215 14 32 300 18 20 28 511 362 950 108 249 970 ND 172 139 10 ND < 1 0 < 1 0 5 mg/1. A, Agitated portion; ND, not determined.
coat storage as seen in series I (group 1) may well be accounted for partly by the hard-spin centrifugation itself. However, it appears that platelet behavior could also be adversely affected if platelets are stored together with activated white blood cells. In order to circumvent such
damage, platelet could be separated from white blood cells within 3 h. However, due to aggregate formation, many buffy coats would then yield platelet concentrates with low platelet counts. It may well be that by discarding huffy coats which contain visible clumping or clot formation the biochemical quality of the final platelet concentrates is improved (see Fig. 3). Also, the observation that many preparations with considerable activation of both platelets and white cells did not demonstrate visible aggregates (Table 1)
0 rX
g 10
