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Cytokines as quality indicators of leucoreduced red cell concentrates
Transfusion and Apheresis Science 26 (2002) 43–46 www.elsevier.com/locate/transci
Cytokines as quality indicators of leucoreduced red cell concentrates J. Seghatchian a
a,*,1
, P. Krailadsiri a, P. Dilger b, R. Thorpe b, M. Wadhwa
b
National Blood Service, London and South-East, Colindale Avenue, Colindale, London NW9 5BG, UK Division of Immunobiology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts EN6 3QG, UK
b
Abstract Different types of filters are currently used for leucodepletion of red cell concentrates. These filters meet the specification for leucoreduction (<5 106 leucocytes/ATD) but the quality of the final product may differ depending on the performance of the filters for effective removal of both leucocytes, platelets and possibly cytokines which are associated with transfusion reactions. We measured the levels of three representative cytokines: IL-8, RANTES and TGF-b1, in red cell concentrates prior to and subsequent to the filtration procedure on day 1 and after a storage period of 35 days. Low levels of IL-8 (10–24 pg/ml) in the control unfiltered concentrates on day 1 which increased by approximately twofold on storage. Filtration reduced the levels of IL-8 on day 1 and day 35, in filtered concentrates in comparison with their control unfiltered counterparts. Leucoreduced concentrates produced by three different filters showed similar IL-8 levels on day 1 and day 35. However, concentrates prepared using another type of process showed a twofold increase in IL-8 levels on storage in comparison with day 1. None of the concentrates tested contained any detectable RANTES and TGF-b1 suggesting a minimal platelet content. These results indicate that a combination of IL-8, RANTES and TGF-b1 are useful quality indicators for validation of leucoreduced red cell preparations. Ó 2002 Published by Elsevier Science Ltd. Keywords: Cytokines; Quality monitoring; Quality index; Leucodepletion; Red cell concentrate
1. Introduction In the UK, four types of validated filters are in current use for production of WBC-reduced red cell concentrates (RCCs). While these filters effectively remove both WBCs and platelets and
*
Corresponding author. Tel./fax: +44-207-722-9596. E-mail address: [email protected] (J. Seghatchian). 1 Present address: Blood Component Technology Consultancy, 50 Primrose Hill Rd., London NW3 3AA, UK.
the final red product meets the agreed UK specification (i.e., WBC < 5 106 =ATD, with 99% frequency and 95% confidence), there is nevertheless no consensus on the absolute content of residual WBC sub-populations and platelets to ensure the overall safety of the product. Several investigators have attempted to measure cellular contamination and/or analyse WBC subsets. However, concerns have been expressed on the accuracy of various counting technologies for measuring the low levels of WBCs and platelets in WBC-reduced RCCs and techniques for analysis of WBC subsets [1–4].
1473-0502/02/$ - see front matter Ó 2002 Published by Elsevier Science Ltd. PII: S 1 4 7 3 - 0 5 0 2 ( 0 1 ) 0 0 1 4 4 - 6
The % removal of IL-8 by the various filters is also indicated. (<) Indicates less than the detection limit of assay – 10 pg/ml; (–) indicates no sample.
< < < < 13 (29) < 29 (9) < < < < < < < 19 < 32 < < < < < < < < < < < < < 3.88 3.23 6.7 3.27 1.16 1.62 0.76 1.58 2.82 1.65 < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < 2.17 3.52 3.26 2.62 2.2 1.65 2.21 4.2 3.1 2.38 < (100) 21 (79) 13 (40) 144 (100) < (100) < (100) < (100) < (100) 80 (100) – 114 104 22 150 34 46 146 39 79 – < < < < < < < < < – 3.43 5.3 5.38 5.28 4.2 4.06 4.59 4.82 3.34 – – – < < < < < 99 (55) < < – – < < < < < 222 < < – – < < < < < < < < – – 1.7 2.57 3.81 1.92 1.66 2.0 1.32 1.81 1 2 3 4 5 6 7 8 9 10
Post Pre Post Pre Post Pre Post Pre Post Pre Post
Post
Pre Pre Pre
Post
IL-8 pg/ml WBC 10e9/l IL-8 pg/ml (% IL-8 removal) (% IL-8 removal) WBC 10e9/l WBC 10e9/l IL-8 pg/ml (% IL-8 removal) WBC 10e9/l
IL-8 pg/ml (% IL-8 removal)
Filter C Filter B
Blood collection and processing was carried out according to validated protocols established at NBS London & SE. The residual cellular content of RCCs – an automated cell counter (SE9000, Sysmex, Kobe, Japan). WBC counts – flow cytometry (EpicXL, Coulter Electronics, Miami, FL)
Filter A
2. Materials and methods
Unit
A critical assessment of the residual cellular content of the WBC-reduced RCCs is needed for monitoring the performance of the currently available filters to provide additional information on potential risk of transfusion associated sideeffects of WBC-reduced products. This is particularly relevant in production strategies where various filters are used logistically for production of WBC-reduced RCCs. The performance of the filters varies and is influenced by the cellular composition of the starting material (whole blood, buffy coat, etc.) or RCCs, the age and storage conditions, the plasma content and most importantly, the WBC and platelet load. A positive correlation has been shown between the pre-filtration platelet count and filtration capacity [5] and a negative correlation between WBC and filter performance [6]. Different filters may also vary in performance with respect to retention of specific WBC sub-populations and adsorption of some biological response modifiers, e.g., IL-8 [7]. Such factors could have an impact on the overall quality of the product in terms of transfusion related sideeffects. We have previously reported that cytokine levels are relatively low in filtered RCCs as compared to their non-filtered counterparts [8]. A comprehensive study on cytokine levels in different WBCreduced RCCs that are routinely produced in UK is, however, lacking. Here, we have measured IL-8, RANTES and TGF-b1 as indirect markers of residual WBCs and platelets which may be present in WBC-reduced RCCs. Finally, we have compared the levels of these cytokines in paired filtered RCCs and non-filtered products as positive controls of the same origin. An identical experimental design was used in this study to allow valid comparisons within the different types of RCCs.
Filter D
J. Seghatchian et al. / Transfusion and Apheresis Science 26 (2002) 43–46
Table 1 WBC and IL-8 levels (day 1) in four different pre- and post-filtered RCCs
44
J. Seghatchian et al. / Transfusion and Apheresis Science 26 (2002) 43–46
by use of a kit (LeucoCount, Becton Dickinson, San Jose, CA); sensitivity – 1 WBC/ll. Overall (n ¼ 34) samples were investigated in blind study. The determinations were carried out on samples at day 1 and 35 from packs stored at 4 °C as for clinical products. Samples taken prior to and post filtration were evaluated. 2.1. Assays IL-8 – an in-house ELISA. RANTES and total TGF-b1 levels – ELISAs (R&D Systems, Minneapolis, MN). 2.2. Analysis Performance of the filters was calculated by estimating the % removal of IL-8 based on IL-8 levels on day 1 in samples taken prior to (A) and post filtration (B) using the formula: (A B)/ A 100.
45
3. Results and discussion Results showed considerable variation in IL-8 levels present in non-filtered RCCs reflecting interdonor variability. Upon filtration, the removal of IL-8 varied depending on the nature and properties of the type of filter used (Table 1, Fig. 1). Filters vary in composition and structure which may influence the release/retention of cytokines. The variation in levels in filtered RCCs may reflect variation between different batches of the particular filter type or their capacity for WBC retention and differential adsorption of IL-8 by the filter matrix [7]. Filter B had the capacity of adsorbing IL-8 while D lacked this property (Table 1). The presence of IL-8 in some RCCs after storage, however, suggests the presence of residual WBCs in products derived from some filter types. In a limited number of samples tested, we also found low levels of RANTES (50–100 pg/ml) in non-filtered RCCs at day 1. Upon filtration, the levels of
A
B
C
D Fig. 1. IL-8 levels in four different pre- and post-filtered RCCs.
46
J. Seghatchian et al. / Transfusion and Apheresis Science 26 (2002) 43–46
RANTES were diminished, the extent of removal however as for IL-8 varied depending on the filter used. In RCCs from two filter types, B and C, RANTES was undetectable while with filter A, 50% of RANTES was removed. Filter D, however, appeared to be incapable of removing RANTES. No TGF-b1 was detected in several non-filtered or filtered products. These results indicate that the residual platelet content in red cell products is either relatively low or storage at 4 °C reduces TGFb1 release. Limited comparative analysis on performance of WBC-reduction filters for red cells has been reported [9–11]. With the view of continual improvement in safety of WBC-reduced RCCs, some investigators have focussed on the determination of WBC content and their sub-populations [12]. This procedure is however time-consuming and poorly reproducible. We have previously indicated that ELISA procedures provide acceptably sensitive and accurate estimates of cytokine levels in WBC-reduced products and can be used as an indirect marker of residual cellular contaminants in different RCCs [8]. In this study, we therefore measured cytokines derived from both WBCs and platelets to assess the efficacy of the currently available filtration process both in terms of retention/release of cellular contaminant and cellderived cytokines. 4. Conclusions Our data confirms our earlier results on low levels of cytokines in WBC-reduced RCCs [8]. The fact that no RANTES or TGF-b1 is detectable in various red cell products subsequent to storage suggests that storage at 4 °C prevents the release of platelet derived cytokines. We showed that a combination of IL-8, RANTES and TGF-b1 measurements provide useful information about the quality of the filtered product. These cytokines could also be used as performance indicators for validation of new filters as well as batch to batch consistency of different filter lots.
References [1] Seghatchian J, Krailadsiri P, Scott CS. Counting of residual WBCs in WBC-reduced blood components: a multicenter evaluation of a microvolume fluorimeter by the United Kingdom National Blood Service. Transfusion 2001;41:93–101. [2] Dzik S, Moroff G, Dumont L. A multicenter study evaluating three methods for counting residual WBCs in WBC-reduced blood components: Nageotte hemocytometry, flow cytometry, and microfluorometry. Transfusion 2000;40(5):513–20. [3] Rider JR, Want EJ, Winter MA, Turton JR, Pamphilon DH, Nobes P. Differential leucocyte subpopulation analysis of leucodepleted red cell products. Transfus Med 2000;10(1):49–58. [4] Wegener S, Marschall M, Schnabl J, Kleine H, Freund M. White cell subsets in filtered red blood cell concentrates. Transfus Sci 2000;23(1):29–32. [5] Steneker I, Prins HK, Florie M, Loos JA, Biewenga J. Mechanisms of white cell reduction in red cell concentrates by filtration: the effect of the cellular composition of the red cell concentrates. Transfusion 1993;33(1): 42–50. [6] Alcorta I, Pereira A, Sanz C, Terol MJ, Ordinas A. Influence of the red blood cell preparation method on the efficacy of a leukocyte reduction filter. Vox Sang 1996; 71(2):78–83. [7] Geiger TL, Perrotta PL, Davenport R, Baril L, Snyder EL. Removal of anaphylatoxins C3a and C5a and chemokines interleukin 8 and RANTES by polyester white cellreduction and plasma filters. Transfusion 1997;37(11–12): 1156–62. [8] Wadhwa M, Seghatchian MJ, Dilger P, Contreras M, Thorpe R. Cytokine accumulation in stored red cell concentrates: effect of buffy-coat removal and leucoreduction. Transfus Sci 2000;23(1):7–16. [9] Kao KJ, Mickel M, Braine HG, Davis K, Enright H, Gernsheimer T, et al. White cell reduction in platelet concentrates and packed red cells by filtration: a multicenter clinical trial. The Trap Study Group. Transfusion 1995;35(1):13–9. [10] AuBuchon JP, Elfath MD, Popovsky MA, Stromberg RR, Pickard C, Herschel L, et al. Evaluation of a new prestorage leukoreduction filter for red blood cell units. Vox Sang 1997;72(2):101–6. [11] Laupacis A, Brown J, Costello B, Delage G, Freedman J, Hume H, et al. Prevention of posttransfusion CMV in the era of universal WBC reduction: a consensus statement. Transfusion 2001;41(4):560–9. [12] Sowemimo-Coker SO, Kim A, Tribble E, Brandwein HJ, Wenz B. White cell subsets in apheresis and filtered platelet concentrates. Transfusion 1998;38(7):650–7.
Cytokines as quality indicators of leucoreduced red cell concentrates J. Seghatchian a
a,*,1
, P. Krailadsiri a, P. Dilger b, R. Thorpe b, M. Wadhwa
b
National Blood Service, London and South-East, Colindale Avenue, Colindale, London NW9 5BG, UK Division of Immunobiology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Herts EN6 3QG, UK
b
Abstract Different types of filters are currently used for leucodepletion of red cell concentrates. These filters meet the specification for leucoreduction (<5 106 leucocytes/ATD) but the quality of the final product may differ depending on the performance of the filters for effective removal of both leucocytes, platelets and possibly cytokines which are associated with transfusion reactions. We measured the levels of three representative cytokines: IL-8, RANTES and TGF-b1, in red cell concentrates prior to and subsequent to the filtration procedure on day 1 and after a storage period of 35 days. Low levels of IL-8 (10–24 pg/ml) in the control unfiltered concentrates on day 1 which increased by approximately twofold on storage. Filtration reduced the levels of IL-8 on day 1 and day 35, in filtered concentrates in comparison with their control unfiltered counterparts. Leucoreduced concentrates produced by three different filters showed similar IL-8 levels on day 1 and day 35. However, concentrates prepared using another type of process showed a twofold increase in IL-8 levels on storage in comparison with day 1. None of the concentrates tested contained any detectable RANTES and TGF-b1 suggesting a minimal platelet content. These results indicate that a combination of IL-8, RANTES and TGF-b1 are useful quality indicators for validation of leucoreduced red cell preparations. Ó 2002 Published by Elsevier Science Ltd. Keywords: Cytokines; Quality monitoring; Quality index; Leucodepletion; Red cell concentrate
1. Introduction In the UK, four types of validated filters are in current use for production of WBC-reduced red cell concentrates (RCCs). While these filters effectively remove both WBCs and platelets and
*
Corresponding author. Tel./fax: +44-207-722-9596. E-mail address: [email protected] (J. Seghatchian). 1 Present address: Blood Component Technology Consultancy, 50 Primrose Hill Rd., London NW3 3AA, UK.
the final red product meets the agreed UK specification (i.e., WBC < 5 106 =ATD, with 99% frequency and 95% confidence), there is nevertheless no consensus on the absolute content of residual WBC sub-populations and platelets to ensure the overall safety of the product. Several investigators have attempted to measure cellular contamination and/or analyse WBC subsets. However, concerns have been expressed on the accuracy of various counting technologies for measuring the low levels of WBCs and platelets in WBC-reduced RCCs and techniques for analysis of WBC subsets [1–4].
1473-0502/02/$ - see front matter Ó 2002 Published by Elsevier Science Ltd. PII: S 1 4 7 3 - 0 5 0 2 ( 0 1 ) 0 0 1 4 4 - 6
The % removal of IL-8 by the various filters is also indicated. (<) Indicates less than the detection limit of assay – 10 pg/ml; (–) indicates no sample.
< < < < 13 (29) < 29 (9) < < < < < < < 19 < 32 < < < < < < < < < < < < < 3.88 3.23 6.7 3.27 1.16 1.62 0.76 1.58 2.82 1.65 < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < 2.17 3.52 3.26 2.62 2.2 1.65 2.21 4.2 3.1 2.38 < (100) 21 (79) 13 (40) 144 (100) < (100) < (100) < (100) < (100) 80 (100) – 114 104 22 150 34 46 146 39 79 – < < < < < < < < < – 3.43 5.3 5.38 5.28 4.2 4.06 4.59 4.82 3.34 – – – < < < < < 99 (55) < < – – < < < < < 222 < < – – < < < < < < < < – – 1.7 2.57 3.81 1.92 1.66 2.0 1.32 1.81 1 2 3 4 5 6 7 8 9 10
Post Pre Post Pre Post Pre Post Pre Post Pre Post
Post
Pre Pre Pre
Post
IL-8 pg/ml WBC 10e9/l IL-8 pg/ml (% IL-8 removal) (% IL-8 removal) WBC 10e9/l WBC 10e9/l IL-8 pg/ml (% IL-8 removal) WBC 10e9/l
IL-8 pg/ml (% IL-8 removal)
Filter C Filter B
Blood collection and processing was carried out according to validated protocols established at NBS London & SE. The residual cellular content of RCCs – an automated cell counter (SE9000, Sysmex, Kobe, Japan). WBC counts – flow cytometry (EpicXL, Coulter Electronics, Miami, FL)
Filter A
2. Materials and methods
Unit
A critical assessment of the residual cellular content of the WBC-reduced RCCs is needed for monitoring the performance of the currently available filters to provide additional information on potential risk of transfusion associated sideeffects of WBC-reduced products. This is particularly relevant in production strategies where various filters are used logistically for production of WBC-reduced RCCs. The performance of the filters varies and is influenced by the cellular composition of the starting material (whole blood, buffy coat, etc.) or RCCs, the age and storage conditions, the plasma content and most importantly, the WBC and platelet load. A positive correlation has been shown between the pre-filtration platelet count and filtration capacity [5] and a negative correlation between WBC and filter performance [6]. Different filters may also vary in performance with respect to retention of specific WBC sub-populations and adsorption of some biological response modifiers, e.g., IL-8 [7]. Such factors could have an impact on the overall quality of the product in terms of transfusion related sideeffects. We have previously reported that cytokine levels are relatively low in filtered RCCs as compared to their non-filtered counterparts [8]. A comprehensive study on cytokine levels in different WBCreduced RCCs that are routinely produced in UK is, however, lacking. Here, we have measured IL-8, RANTES and TGF-b1 as indirect markers of residual WBCs and platelets which may be present in WBC-reduced RCCs. Finally, we have compared the levels of these cytokines in paired filtered RCCs and non-filtered products as positive controls of the same origin. An identical experimental design was used in this study to allow valid comparisons within the different types of RCCs.
Filter D
J. Seghatchian et al. / Transfusion and Apheresis Science 26 (2002) 43–46
Table 1 WBC and IL-8 levels (day 1) in four different pre- and post-filtered RCCs
44
J. Seghatchian et al. / Transfusion and Apheresis Science 26 (2002) 43–46
by use of a kit (LeucoCount, Becton Dickinson, San Jose, CA); sensitivity – 1 WBC/ll. Overall (n ¼ 34) samples were investigated in blind study. The determinations were carried out on samples at day 1 and 35 from packs stored at 4 °C as for clinical products. Samples taken prior to and post filtration were evaluated. 2.1. Assays IL-8 – an in-house ELISA. RANTES and total TGF-b1 levels – ELISAs (R&D Systems, Minneapolis, MN). 2.2. Analysis Performance of the filters was calculated by estimating the % removal of IL-8 based on IL-8 levels on day 1 in samples taken prior to (A) and post filtration (B) using the formula: (A B)/ A 100.
45
3. Results and discussion Results showed considerable variation in IL-8 levels present in non-filtered RCCs reflecting interdonor variability. Upon filtration, the removal of IL-8 varied depending on the nature and properties of the type of filter used (Table 1, Fig. 1). Filters vary in composition and structure which may influence the release/retention of cytokines. The variation in levels in filtered RCCs may reflect variation between different batches of the particular filter type or their capacity for WBC retention and differential adsorption of IL-8 by the filter matrix [7]. Filter B had the capacity of adsorbing IL-8 while D lacked this property (Table 1). The presence of IL-8 in some RCCs after storage, however, suggests the presence of residual WBCs in products derived from some filter types. In a limited number of samples tested, we also found low levels of RANTES (50–100 pg/ml) in non-filtered RCCs at day 1. Upon filtration, the levels of
A
B
C
D Fig. 1. IL-8 levels in four different pre- and post-filtered RCCs.
46
J. Seghatchian et al. / Transfusion and Apheresis Science 26 (2002) 43–46
RANTES were diminished, the extent of removal however as for IL-8 varied depending on the filter used. In RCCs from two filter types, B and C, RANTES was undetectable while with filter A, 50% of RANTES was removed. Filter D, however, appeared to be incapable of removing RANTES. No TGF-b1 was detected in several non-filtered or filtered products. These results indicate that the residual platelet content in red cell products is either relatively low or storage at 4 °C reduces TGFb1 release. Limited comparative analysis on performance of WBC-reduction filters for red cells has been reported [9–11]. With the view of continual improvement in safety of WBC-reduced RCCs, some investigators have focussed on the determination of WBC content and their sub-populations [12]. This procedure is however time-consuming and poorly reproducible. We have previously indicated that ELISA procedures provide acceptably sensitive and accurate estimates of cytokine levels in WBC-reduced products and can be used as an indirect marker of residual cellular contaminants in different RCCs [8]. In this study, we therefore measured cytokines derived from both WBCs and platelets to assess the efficacy of the currently available filtration process both in terms of retention/release of cellular contaminant and cellderived cytokines. 4. Conclusions Our data confirms our earlier results on low levels of cytokines in WBC-reduced RCCs [8]. The fact that no RANTES or TGF-b1 is detectable in various red cell products subsequent to storage suggests that storage at 4 °C prevents the release of platelet derived cytokines. We showed that a combination of IL-8, RANTES and TGF-b1 measurements provide useful information about the quality of the filtered product. These cytokines could also be used as performance indicators for validation of new filters as well as batch to batch consistency of different filter lots.
References [1] Seghatchian J, Krailadsiri P, Scott CS. Counting of residual WBCs in WBC-reduced blood components: a multicenter evaluation of a microvolume fluorimeter by the United Kingdom National Blood Service. Transfusion 2001;41:93–101. [2] Dzik S, Moroff G, Dumont L. A multicenter study evaluating three methods for counting residual WBCs in WBC-reduced blood components: Nageotte hemocytometry, flow cytometry, and microfluorometry. Transfusion 2000;40(5):513–20. [3] Rider JR, Want EJ, Winter MA, Turton JR, Pamphilon DH, Nobes P. Differential leucocyte subpopulation analysis of leucodepleted red cell products. Transfus Med 2000;10(1):49–58. [4] Wegener S, Marschall M, Schnabl J, Kleine H, Freund M. White cell subsets in filtered red blood cell concentrates. Transfus Sci 2000;23(1):29–32. [5] Steneker I, Prins HK, Florie M, Loos JA, Biewenga J. Mechanisms of white cell reduction in red cell concentrates by filtration: the effect of the cellular composition of the red cell concentrates. Transfusion 1993;33(1): 42–50. [6] Alcorta I, Pereira A, Sanz C, Terol MJ, Ordinas A. Influence of the red blood cell preparation method on the efficacy of a leukocyte reduction filter. Vox Sang 1996; 71(2):78–83. [7] Geiger TL, Perrotta PL, Davenport R, Baril L, Snyder EL. Removal of anaphylatoxins C3a and C5a and chemokines interleukin 8 and RANTES by polyester white cellreduction and plasma filters. Transfusion 1997;37(11–12): 1156–62. [8] Wadhwa M, Seghatchian MJ, Dilger P, Contreras M, Thorpe R. Cytokine accumulation in stored red cell concentrates: effect of buffy-coat removal and leucoreduction. Transfus Sci 2000;23(1):7–16. [9] Kao KJ, Mickel M, Braine HG, Davis K, Enright H, Gernsheimer T, et al. White cell reduction in platelet concentrates and packed red cells by filtration: a multicenter clinical trial. The Trap Study Group. Transfusion 1995;35(1):13–9. [10] AuBuchon JP, Elfath MD, Popovsky MA, Stromberg RR, Pickard C, Herschel L, et al. Evaluation of a new prestorage leukoreduction filter for red blood cell units. Vox Sang 1997;72(2):101–6. [11] Laupacis A, Brown J, Costello B, Delage G, Freedman J, Hume H, et al. Prevention of posttransfusion CMV in the era of universal WBC reduction: a consensus statement. Transfusion 2001;41(4):560–9. [12] Sowemimo-Coker SO, Kim A, Tribble E, Brandwein HJ, Wenz B. White cell subsets in apheresis and filtered platelet concentrates. Transfusion 1998;38(7):650–7.