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Determination of Fc function with frozen red blood cells
Biologicals 34 (2006) 221e222 www.elsevier.com/locate/biologicals
Determination of Fc function with frozen red blood cells V. Gurevich, J. Bertolini*, K. Lyons CSL Bioplasma, Research and Development, 189-209 Camp Road, Broadmeadows, Victoria 3047, Australia Received 9 November 2005; accepted 11 November 2005
Abstract The Fc function of immunoglobulins is commonly determined by an assay based on monitoring immunoglobulin induced, complement mediated red cell lysis. This assay requires a continuous source of fresh red cells. We have shown that the assay can be successfully performed with frozen red cells. The possibility of access to a stored standard stock of red cells will improve the convenience of performing the assay and could contribute to improved assay reproducibility. Ó 2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. Keywords: Fc function; Immunoglobulin; Red cells
The immunoglobulin molecules consist of Fab and Fc portions with the Fc region accounting for the immunomodulatory properties of immunoglobulins [1]. Hence analysis of the Fc function is important for establishing the structural and functional integrity of immunoglobulins. According to the British Pharmacopoeia (BP) the analysis is performed with fresh red blood cells (RBC) by monitoring haemolysis resulting from Fc activated complement. The need to source batches of fresh RBC, however, makes the technique inconvenient and adds to assay variation. These issues could be overcome by generating a source of standard RBC for repeated use in assays. One potential avenue is freezing RBC and using aliquots as required. Methods for freezing RBC are established but the use of frozen RBC in an Fc assay has not been investigated [2]. This study was performed to assess the use of frozen RBC in place of fresh RBC in the assay. Freezing of RBC was performed by a previously described procedure [3] with some modifications. Stabilised RBC were glycerolised and stored in a ÿ80 C freezer. For use, frozen RBC were thawed at 37 C and
deglycerolised using 12% and 0.9% sodium chloride followed by PBS washes. The degree of haemolysis during thawing and washing stages was monitored by measuring haemoglobin related absorbance in supernatants and compared to absorbence corresponding to 100% haemolysis. The Fc assay was performed as described in the BP [4] using the frozen and non-frozen RBC from the same batch. Each analysis was performed in duplicates. Stability of stored frozen RBC was monitored by performing the Fc assay at regular intervals during storage. Determination of the degree of haemolysis of RBC following freezing and thawing indicated that the recovery of RBC was approximately 80%. The performance of the assay with frozen and non-frozen RBC was comparable with a difference of Fc results of less than 10% (Table 1). Table 1 Fc function results from assays performed using frozen and non-frozen RBC Sample
Fc (%) RBC frozen
* Corresponding author. E-mail address: [email protected] (J. Bertolini).
IVIG
RBC non-frozen
Batch 1
Batch 2
Batch 1
Batch 2
111
116
117
110
1045-1056/05/$32.00 Ó 2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2005.11.003
V. Gurevich et al. / Biologicals 34 (2006) 221e222
222 Table 2 Fc function results with stored frozen RBC Month
Fc (%)a
This study has demonstrated that it is feasible to freeze stabilised RBC which are subsequently suitable for use in a routine Fc assay. The use of frozen RBC will allow maintenance of a stock of standard RBC which will simplify the technique and should reduce assay variation.
Batch 1
Batch 2
1
127 129
120 114
2
133 133
160 154
References
3
135 141
138 137
[1] Immunoglobulins: structure and function. In: Paul WE, editor. Fundamental immunology. New York: Raven Press; 1993. [2] Cryopreservation of blood. In: Walker RH, editor. Technical manual of American Association of Blood Banks. Arlington, Virginia; 1990. [3] Valeri C, Pivacek L, Gray A, Cassidy G, Leavy M, Dennis R, et al. The safety and therapeutic effectiveness of human red cells stored at ÿ80 degrees C for as long as 21 years. Transfusion 1989;29:429e37. [4] Test for Fc function of immunoglobulins. In: British Pharmacopoeia. London: The Stationery Office; 2004.
a
Mean of two assays.
Frozen RBC stored for 3 months performed satisfactorily in the Fc assay with comparable results obtained at each time point (Table 2).
Determination of Fc function with frozen red blood cells V. Gurevich, J. Bertolini*, K. Lyons CSL Bioplasma, Research and Development, 189-209 Camp Road, Broadmeadows, Victoria 3047, Australia Received 9 November 2005; accepted 11 November 2005
Abstract The Fc function of immunoglobulins is commonly determined by an assay based on monitoring immunoglobulin induced, complement mediated red cell lysis. This assay requires a continuous source of fresh red cells. We have shown that the assay can be successfully performed with frozen red cells. The possibility of access to a stored standard stock of red cells will improve the convenience of performing the assay and could contribute to improved assay reproducibility. Ó 2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. Keywords: Fc function; Immunoglobulin; Red cells
The immunoglobulin molecules consist of Fab and Fc portions with the Fc region accounting for the immunomodulatory properties of immunoglobulins [1]. Hence analysis of the Fc function is important for establishing the structural and functional integrity of immunoglobulins. According to the British Pharmacopoeia (BP) the analysis is performed with fresh red blood cells (RBC) by monitoring haemolysis resulting from Fc activated complement. The need to source batches of fresh RBC, however, makes the technique inconvenient and adds to assay variation. These issues could be overcome by generating a source of standard RBC for repeated use in assays. One potential avenue is freezing RBC and using aliquots as required. Methods for freezing RBC are established but the use of frozen RBC in an Fc assay has not been investigated [2]. This study was performed to assess the use of frozen RBC in place of fresh RBC in the assay. Freezing of RBC was performed by a previously described procedure [3] with some modifications. Stabilised RBC were glycerolised and stored in a ÿ80 C freezer. For use, frozen RBC were thawed at 37 C and
deglycerolised using 12% and 0.9% sodium chloride followed by PBS washes. The degree of haemolysis during thawing and washing stages was monitored by measuring haemoglobin related absorbance in supernatants and compared to absorbence corresponding to 100% haemolysis. The Fc assay was performed as described in the BP [4] using the frozen and non-frozen RBC from the same batch. Each analysis was performed in duplicates. Stability of stored frozen RBC was monitored by performing the Fc assay at regular intervals during storage. Determination of the degree of haemolysis of RBC following freezing and thawing indicated that the recovery of RBC was approximately 80%. The performance of the assay with frozen and non-frozen RBC was comparable with a difference of Fc results of less than 10% (Table 1). Table 1 Fc function results from assays performed using frozen and non-frozen RBC Sample
Fc (%) RBC frozen
* Corresponding author. E-mail address: [email protected] (J. Bertolini).
IVIG
RBC non-frozen
Batch 1
Batch 2
Batch 1
Batch 2
111
116
117
110
1045-1056/05/$32.00 Ó 2005 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2005.11.003
V. Gurevich et al. / Biologicals 34 (2006) 221e222
222 Table 2 Fc function results with stored frozen RBC Month
Fc (%)a
This study has demonstrated that it is feasible to freeze stabilised RBC which are subsequently suitable for use in a routine Fc assay. The use of frozen RBC will allow maintenance of a stock of standard RBC which will simplify the technique and should reduce assay variation.
Batch 1
Batch 2
1
127 129
120 114
2
133 133
160 154
References
3
135 141
138 137
[1] Immunoglobulins: structure and function. In: Paul WE, editor. Fundamental immunology. New York: Raven Press; 1993. [2] Cryopreservation of blood. In: Walker RH, editor. Technical manual of American Association of Blood Banks. Arlington, Virginia; 1990. [3] Valeri C, Pivacek L, Gray A, Cassidy G, Leavy M, Dennis R, et al. The safety and therapeutic effectiveness of human red cells stored at ÿ80 degrees C for as long as 21 years. Transfusion 1989;29:429e37. [4] Test for Fc function of immunoglobulins. In: British Pharmacopoeia. London: The Stationery Office; 2004.
a
Mean of two assays.
Frozen RBC stored for 3 months performed satisfactorily in the Fc assay with comparable results obtained at each time point (Table 2).