Original Paper Vox Sang 1992;63:23-30
I/: M . J. Novotny R. van Doorn Y Rozier J. D X m a r o M . S. Harvey A . Brand
Red Cross Bloodbank Foundation Leiden a.e., and Department of Immunohematology and Blood Bank, University Hospital Leiden, The Netherlands
Transfusion Results of Filtered and Subsequently Stored Random Platelet Suspensions Prepared from Buffy Coats
................................................................................................. Abstract There is almost general agreement that removal of leukocytes from blood components reduces the incidence of HLA-antibody formation and refractoriness to random platelet transfusions. Recently filters have become available, which are able to reduce leukocyte contamination in platelet suspensions with acceptable platelet loss. We evaluated a cellulose acetate (CA) and a polyester (PE) filter, and stored buffy coat-derived platelet suspensions after filtration. Both filters are effective for the removal of leukocytes to levels below 5 x 10' per transfusate. For the CA filter, platelet recovery was 73*13% yielding 256+53 X 10" platelets per transfusate from 6 donors. For the PE filter, platelet recovery was 90+9% and 327k.51 x 10"platelets per transfusate. When a loading dose of less than 5 X 10'leukocytes was applied, 98% of the CA-filtered suspensions and 100% of the PE-filtered suspensions contained less than 5 x lo6 residual leucocytes. In 123 patients transfusion results of CA-filtered platelet suspensions stored for 72 h, were compared with those obtained by non-stored, non filtered, random platelet suspensions which had been leukocyte depleted by differential centrifugation. Platelet incrementsland20 hafter transfusionshowednostatisticaldifference between CA-filtered platelet transfusions stored for 72 h and non-stored, non-filtered platelet transfusions. In a new cohort of 117patients, two filters and various postfiltration storage times were compared. Using both filters, the 1-hour posttransfusion increments decreased to approximately 60% after 96 h of storage compared to results of storage periods of 72 h or less. Moreover, gradually decreasing platelet survical, as measured from 20-hour post-transfusion increments, was observed with increased storage time, from 24 h onwards. This decrease in platelet survival was associated with a decrease in pH. Because the pH and storage interval are strongly related, pH could not be evaluated as an independent parameter. The storage-related decreased platelet survival, however, was more severe after PE filtration than after CA filtration, whereas pH was better maintained after PEfiltration. In 164cases the bleeding time was measured after transfusion of platelet stored for 2 7 2 h. Provided that a posttransfusion increment of 2-15 x 10'A was reached after transfusion with filtered, stored platelets, bleeding times shortened, indicating the hemostatic activity of filtered and subsequently stored platelets.
Received: June 7, 15191 Revised manuscript received: Dcc. 21. 1991 Accepted: Dcc. XI, IYJI
V. M. J. Novotny. MD
0 1992 S. Karger AG. Basel
Red Cross Bloodhank Foundation Leiden a.e. University Hospital Lciden Rijnchui-gerweg I0 NI.-2333 AA Leiden (The Netherlands)
O(I42-90~17/Y2/1)6314023 $2.7S/O
tiveness was evaluated by posttransfusion increment determination and bleeding time measurements.
Introduction
HLA-antibody formation is thought to be the result of a process which requires both allogeneic HLA class-I and class-I1antigens. Since class-I1antigens are not present on platelets, immunization is induced by contaminating white cells of donor origin which act as antigen-presenting cells [l-31. There is convincing evidence that removal of leukocytes from blood components reduces the incidence of HLA-antibody formation and refractoriness to random platelet transfusions. The exact number of white cells necessary to initiate the immune response and subsequent antibody formation is not known. According to the few studies available, it is in the order of 5 X lo6 to 5 X lo7 contaminating leukocytes per transfusate [4-81. Reliable removal of leukocytes from platelet suspensions, however, is difficult to obtain by centrifugation methods [S]. Filters to remove leukocytes from erythrocyte transfusions have been available for more than 10 years, but only recently have filters become available that could remove white cells from platelet suspensions without considerable platelet loss [9-111. A point of concern is whether filtration causes damage to platelets, as during the filtration procedure platelets are exposed to foreign surfaces which may cause platelet activation with release of internal constituents, compromising clinical efficacy of the transfused platelets. Different investigators have reported that in vitro platelet function tests (aggregation by ADP, collagen and thrombin, hypotonic shock response, ultrastructural morphology) suggest that platelets are not affected by filtration and several studies also showed that filtered leukocyte-poor platelets are clinically effective [12, 131; however, the efficacy of platelets stored after filtration is not yet known. It is a point of controversy whether after previous storage nonviable leukocytes and fragments are not retained on the filter and are still able to immunize and transmit viral infections. Using a cottonwool filter, Engelfriet and Diepenhorst [14] found that rabbits, receiving blood filtered after 3 days of storage, did develop lymphocytotoxic antibodies whereas rabbits receiving blood filtered within 24 h after donation did not. Considering that optimal leukocyte depletion may be obtained when leukocytes are viable, we investigated the effectiveness of leukocyte-depleted platelet transfusions by filtration of fresh platelet suspensions, followed by a period of storage. In patients with thrombocytopenia due to bone marrow depression, we investigated platelet transfusion results of filtered and stored platelet suspensions. The effec-
24
Materials and Methods Preparation of Buffy Coats 480 ml donor blood was withdrawn in 70 ml CPD anticoagulans in a quadruple PVC-DEHP bag (NPBI E2066 COMPOFLEX@system. Emmer Compascuum, Emmen, The Netherlands), stored for 6-12 h at 22°C and centrifuged for 8 rnin at 4,200 rpm (5,700 glmin Heraeus cryofuge 6000). After centrifugation, platelet-poor plasma is transfered to the satellite bag, followed by transfer of the buffy coat to the satellite bag (buffy coat = 90 ml, hematocrit 2S%, containing +90% of the platelets and +70% of the leukocytes). Preparation of Non-Stored, Non -Filtered Platelet Suspensions Sixbuffycoatsstoredforamaximumof12 h at22"Cwerepooledin a 500-ml sterile bottle containing 50 ml 3.3% disodium citrate dihydrate-glucose anhydride and centrifuged for 10 rnin at 1,200 rpm (480 glmin). The supernatant platelet-rich plasma (PRP) was transferred to an empty siliconized bottle and subjected to a slow centrifugation step without braking (8-15 rnin at 370g, depending on the volume: 200-250 ml 10 min, 250-300 m l l 2 min, 300-350 ml 13 min, 350400 m l l 4 rnin etc.). After the second centrifugation step, PRP was transferred to a 300-ml sterile, isotonic, siliconized bottle, and used for transfusion within 6 h [4]. The number of platelets after the leukocyte depletion procedure was 0.45+0.05 x 10" (mean+SD) per donor unit. The residual pooled PRP contained less than 2 x lo7 leukocytes, and in >80% less than 1x 10' leukocytes. Preparation of Stored Filtered Platelet Suspensions Six buffy coats storedfor a maximum of 2 hat 22°C were pooled in a double PVC-DEHP bag (NPBI E2126 COMPOFLEXm system, 2 X 600 ml) and centrifuged for 10 rnin at 1,200 rpm (480 g/min). After centrifugation PRP was transferred to the satellite bag and PRP was leukocyte depleted by filtration within 1h after centrifugation using one of the following filters (table 1). (1) Cellselect P F B1014, a cellulose acetate (CA) filter (NPBI; n = 1,199), was first primed with an excess of f150 ml of sterile injectable 0.9% saline to remove traces of acetic acid which can be released from the CA material during storage. Then the inlet line from the saline bag to the filter was closed and the line between PRP and the filter was opened. When the first platelets were visable in the line from the filter to the waste bag, the line was closed and the platelets were collected in the collection bag. Thereafter the filter was rinsed with 50-100 mlo.9% saline to remove residual platelets from the filter, which has an interior volume of +70 ml. (2) PALL, PLSO, a polyester (PE) filter which does not require rinsing (n = 697; PALLBiomedical Corp.), since the efficiency of this filter is flow rate dependent, the flow rate has to be controlled. This was done using drip chambers to control flow rates below 30 ml/min. After filtration PRP was collected in a double (2 x 600 ml) PVCDEHP bag (NPBI, E2126). Connections To reduce the risk of bacterial contamination, all connections during the preparation procedure were made using a sterile connecting device (Haemonetics, SCD 312).
Novotny/van Doorn/Rozier/DAmaro/ Harvey/Brand
Transfusion with Filtered and Subsequently Stored PRPs
Table 1. Platelet recovery and leukocyte reduction of filtered PRP
Volume, mi Platelets, X 10' Recovery, % WBC, xlOh
Buffy coat pool (n = 1,896)
PRP prefiltration ( n = 1,896)
513+33 422L-56 12,000
358f18 353f26 78f8 80 f 90
Total platelet loss during procedure, % a
PRP postfiltration CA ( n = 1,199)
PE (n = 697)
368 L-70 256fS3 73f13
I/: M . J. Novotny R. van Doorn Y Rozier J. D X m a r o M . S. Harvey A . Brand
Red Cross Bloodbank Foundation Leiden a.e., and Department of Immunohematology and Blood Bank, University Hospital Leiden, The Netherlands
Transfusion Results of Filtered and Subsequently Stored Random Platelet Suspensions Prepared from Buffy Coats
................................................................................................. Abstract There is almost general agreement that removal of leukocytes from blood components reduces the incidence of HLA-antibody formation and refractoriness to random platelet transfusions. Recently filters have become available, which are able to reduce leukocyte contamination in platelet suspensions with acceptable platelet loss. We evaluated a cellulose acetate (CA) and a polyester (PE) filter, and stored buffy coat-derived platelet suspensions after filtration. Both filters are effective for the removal of leukocytes to levels below 5 x 10' per transfusate. For the CA filter, platelet recovery was 73*13% yielding 256+53 X 10" platelets per transfusate from 6 donors. For the PE filter, platelet recovery was 90+9% and 327k.51 x 10"platelets per transfusate. When a loading dose of less than 5 X 10'leukocytes was applied, 98% of the CA-filtered suspensions and 100% of the PE-filtered suspensions contained less than 5 x lo6 residual leucocytes. In 123 patients transfusion results of CA-filtered platelet suspensions stored for 72 h, were compared with those obtained by non-stored, non filtered, random platelet suspensions which had been leukocyte depleted by differential centrifugation. Platelet incrementsland20 hafter transfusionshowednostatisticaldifference between CA-filtered platelet transfusions stored for 72 h and non-stored, non-filtered platelet transfusions. In a new cohort of 117patients, two filters and various postfiltration storage times were compared. Using both filters, the 1-hour posttransfusion increments decreased to approximately 60% after 96 h of storage compared to results of storage periods of 72 h or less. Moreover, gradually decreasing platelet survical, as measured from 20-hour post-transfusion increments, was observed with increased storage time, from 24 h onwards. This decrease in platelet survival was associated with a decrease in pH. Because the pH and storage interval are strongly related, pH could not be evaluated as an independent parameter. The storage-related decreased platelet survival, however, was more severe after PE filtration than after CA filtration, whereas pH was better maintained after PEfiltration. In 164cases the bleeding time was measured after transfusion of platelet stored for 2 7 2 h. Provided that a posttransfusion increment of 2-15 x 10'A was reached after transfusion with filtered, stored platelets, bleeding times shortened, indicating the hemostatic activity of filtered and subsequently stored platelets.
Received: June 7, 15191 Revised manuscript received: Dcc. 21. 1991 Accepted: Dcc. XI, IYJI
V. M. J. Novotny. MD
0 1992 S. Karger AG. Basel
Red Cross Bloodhank Foundation Leiden a.e. University Hospital Lciden Rijnchui-gerweg I0 NI.-2333 AA Leiden (The Netherlands)
O(I42-90~17/Y2/1)6314023 $2.7S/O
tiveness was evaluated by posttransfusion increment determination and bleeding time measurements.
Introduction
HLA-antibody formation is thought to be the result of a process which requires both allogeneic HLA class-I and class-I1antigens. Since class-I1antigens are not present on platelets, immunization is induced by contaminating white cells of donor origin which act as antigen-presenting cells [l-31. There is convincing evidence that removal of leukocytes from blood components reduces the incidence of HLA-antibody formation and refractoriness to random platelet transfusions. The exact number of white cells necessary to initiate the immune response and subsequent antibody formation is not known. According to the few studies available, it is in the order of 5 X lo6 to 5 X lo7 contaminating leukocytes per transfusate [4-81. Reliable removal of leukocytes from platelet suspensions, however, is difficult to obtain by centrifugation methods [S]. Filters to remove leukocytes from erythrocyte transfusions have been available for more than 10 years, but only recently have filters become available that could remove white cells from platelet suspensions without considerable platelet loss [9-111. A point of concern is whether filtration causes damage to platelets, as during the filtration procedure platelets are exposed to foreign surfaces which may cause platelet activation with release of internal constituents, compromising clinical efficacy of the transfused platelets. Different investigators have reported that in vitro platelet function tests (aggregation by ADP, collagen and thrombin, hypotonic shock response, ultrastructural morphology) suggest that platelets are not affected by filtration and several studies also showed that filtered leukocyte-poor platelets are clinically effective [12, 131; however, the efficacy of platelets stored after filtration is not yet known. It is a point of controversy whether after previous storage nonviable leukocytes and fragments are not retained on the filter and are still able to immunize and transmit viral infections. Using a cottonwool filter, Engelfriet and Diepenhorst [14] found that rabbits, receiving blood filtered after 3 days of storage, did develop lymphocytotoxic antibodies whereas rabbits receiving blood filtered within 24 h after donation did not. Considering that optimal leukocyte depletion may be obtained when leukocytes are viable, we investigated the effectiveness of leukocyte-depleted platelet transfusions by filtration of fresh platelet suspensions, followed by a period of storage. In patients with thrombocytopenia due to bone marrow depression, we investigated platelet transfusion results of filtered and stored platelet suspensions. The effec-
24
Materials and Methods Preparation of Buffy Coats 480 ml donor blood was withdrawn in 70 ml CPD anticoagulans in a quadruple PVC-DEHP bag (NPBI E2066 COMPOFLEX@system. Emmer Compascuum, Emmen, The Netherlands), stored for 6-12 h at 22°C and centrifuged for 8 rnin at 4,200 rpm (5,700 glmin Heraeus cryofuge 6000). After centrifugation, platelet-poor plasma is transfered to the satellite bag, followed by transfer of the buffy coat to the satellite bag (buffy coat = 90 ml, hematocrit 2S%, containing +90% of the platelets and +70% of the leukocytes). Preparation of Non-Stored, Non -Filtered Platelet Suspensions Sixbuffycoatsstoredforamaximumof12 h at22"Cwerepooledin a 500-ml sterile bottle containing 50 ml 3.3% disodium citrate dihydrate-glucose anhydride and centrifuged for 10 rnin at 1,200 rpm (480 glmin). The supernatant platelet-rich plasma (PRP) was transferred to an empty siliconized bottle and subjected to a slow centrifugation step without braking (8-15 rnin at 370g, depending on the volume: 200-250 ml 10 min, 250-300 m l l 2 min, 300-350 ml 13 min, 350400 m l l 4 rnin etc.). After the second centrifugation step, PRP was transferred to a 300-ml sterile, isotonic, siliconized bottle, and used for transfusion within 6 h [4]. The number of platelets after the leukocyte depletion procedure was 0.45+0.05 x 10" (mean+SD) per donor unit. The residual pooled PRP contained less than 2 x lo7 leukocytes, and in >80% less than 1x 10' leukocytes. Preparation of Stored Filtered Platelet Suspensions Six buffy coats storedfor a maximum of 2 hat 22°C were pooled in a double PVC-DEHP bag (NPBI E2126 COMPOFLEXm system, 2 X 600 ml) and centrifuged for 10 rnin at 1,200 rpm (480 g/min). After centrifugation PRP was transferred to the satellite bag and PRP was leukocyte depleted by filtration within 1h after centrifugation using one of the following filters (table 1). (1) Cellselect P F B1014, a cellulose acetate (CA) filter (NPBI; n = 1,199), was first primed with an excess of f150 ml of sterile injectable 0.9% saline to remove traces of acetic acid which can be released from the CA material during storage. Then the inlet line from the saline bag to the filter was closed and the line between PRP and the filter was opened. When the first platelets were visable in the line from the filter to the waste bag, the line was closed and the platelets were collected in the collection bag. Thereafter the filter was rinsed with 50-100 mlo.9% saline to remove residual platelets from the filter, which has an interior volume of +70 ml. (2) PALL, PLSO, a polyester (PE) filter which does not require rinsing (n = 697; PALLBiomedical Corp.), since the efficiency of this filter is flow rate dependent, the flow rate has to be controlled. This was done using drip chambers to control flow rates below 30 ml/min. After filtration PRP was collected in a double (2 x 600 ml) PVCDEHP bag (NPBI, E2126). Connections To reduce the risk of bacterial contamination, all connections during the preparation procedure were made using a sterile connecting device (Haemonetics, SCD 312).
Novotny/van Doorn/Rozier/DAmaro/ Harvey/Brand
Transfusion with Filtered and Subsequently Stored PRPs
Table 1. Platelet recovery and leukocyte reduction of filtered PRP
Volume, mi Platelets, X 10' Recovery, % WBC, xlOh
Buffy coat pool (n = 1,896)
PRP prefiltration ( n = 1,896)
513+33 422L-56 12,000
358f18 353f26 78f8 80 f 90
Total platelet loss during procedure, % a
PRP postfiltration CA ( n = 1,199)
PE (n = 697)
368 L-70 256fS3 73f13
