854
fractionated by soybean agglutinin and sheep red blood cells. Blood 1983; 61: 341-48. 11 Friedrich W, Goldmann SF, Ebell W, Blutters-Sawatzki R. Severe treatment combined immunodeficiency: by bone marrow transplantation in 15 infants using HLA-haploidentical donors. Eur J Pediatr 1985; 144: 125-30. 12. Cowan MJ, Wara DW, Weintrub PS, Pabst H, Ammann A. Haploidentical bone marrow transplantation for severe combined
immunodeficiency using soybean agglutinin-negative, T-depleted marrow grafts. J Clin Immunol 1985; 5: 370-76. 13. Buckley RH, Schiff SE, Sampson HA, et al. Development of immunity in human severe primary T-cell deficiency following haploidentical bone cell transplantation. J Immunol 1986; 136: 2398-407. Durandy A, de Villartay JP, et al. HLA haploidentical bone marrow transplantation for severe combined immunodeficiency using E-rosette franctionation and cyclosporin A. Blood 1986; 67: 444-49. 15. Moen RC, Horowitz SD, Sondel PM, et al. Immunologic reconstitution after haploidentical bone marrow transplantation for immune deficiency disorders: treatment of bone marrow cells with monoclonal antibody CT-2 and complement. Blood 1987; 70: 664-69. 16. Primary Immunodeficiency diseases. Report of a World Health Organisation sponsored meeting. Immunodeficiency Rev 1989; 1: 173-205. 17. Vossen JM, Asma GEM, Langlois van den Bergh R, et al. HLA identical and haploidentical bone marrow transplantation for severe combined immunodeficiency: chimerism and immunologic reconstitution in vitro and in vivo. In: Griscelli C, Vossen J, eds. Progress in immunodeficiency research and therapy. Amsterdam: Excerpta Medica, 1984: 417-24. 18. Morgan G, Linch DC, Knott LT, et al. Successful haploidentical mismatched bone marrow transplantation in severe combined immunodeficiency: T-cell removal using Campath-I monoclonal antibody and E-rosetting. Br J Haematol 1986; 62: 421-30. 19. Gluckberg H, Storb R, Fefer A, et al. Clinical manifestations of GVHD in human recipients of marrow from HLA matched sibling donors. Transplantation 1974; 18: 295-304. 20. Jeffreys AJ, Wilson V, Thein SL. Hypervariable minisatellite regions in human DNA. Nature 1985; 315: 67-69. marrow stem
14. Fischer A,
21. Kalbfeisch JD, Prentice RL. The statistical analysis of failure time data. New York: Wiley, 1980. 22.
O’Reilly RJ, Keever CA, Small TN, Brockstein J. The use of HLA non identical T-cell depleted marrow transplants for correction of severe combined immunodeficiency disease. Immunodeficiency Rev 1990; 1:
273-309. 23. Peter HH, Kliche A, Dragger R, et al. NK-cell function in severe combined immunodeficiency: possible relevance for classification and prognosis. In: Vossen J, Griscelli C, eds. Progress in immunodeficiency research and therapy II. Amsterdam: Elsevier Science Publishers, 1986: 287-95.
Murphy WJ, Kumar V, Bennett M. Rejection of bone marrow allograft by mice with severe combined immunodeficiency disease. J Exp Med 1987; 165: 1212-17. 25. Wijnaendts L, Le Deist F, Griscelli C, Fischer A. Development of immunological functions following bone marrow transplantation. Blood 1989; 74: 2212-19. 26. Eiermann TH, Partmann P, Friedrich W, Goldmann SF. Implications for the persistent B cell dysfunction after haploidentical bone marrow transplantation in severe combined immunodeficiency analyzed by Epstein-Barr virus transformation. In: Vossen J, Griscelli C, eds. Progress in immunodeficiency research and therapy II. Amsterdam: Excerpta Medica, 1986: 373-78. 27. O’Reilly RJ, Keever C, Kernan NA, et al. Investigation of humoral immune deficiencies following T cell-depleted, HLA haplotype mismatched parental marrow transplants for the treatment of severe combined immunodeficiency. In: Eibl MM, Rosen FS, eds. Primary immunodeficiency diseases. Amsterdam: Elsevier, 1986: 301-07. 28. Korver K, de Lange GG, van den Bergh RL. Lymphoid chimerism after allogeneic bone marrow transplantation. Transplantation 1987; 44:
24.
643-50. 29. Bortin M. Factors influencing the risk of acute graft vs host disease in man. In: Gale RP, Champlin R, eds. Progress in bone marrow transplantation. New York: Alan R Liss, 1987: 243-64. 30.
Goulmy E, Termisjtelen A, Bradley BA, van Rood JJ. Alloimmunity to human H-Y. Lancet 1976; ii: 1206.
Dry instant blood typing plate for bedside use
A
monoclonal
dry blood the upon grouping plate, simple disaccharide, trehalose, is described that is indefinitely stable at room temperature and was found to have a 99·8% accuracy when tested against a standard semiautomated assay. This plate can be used by personnel with no specific training to check the recorded A,B,O, and Rhesus blood type of potential transfusion recipients in the field and at the bedside. Trehalose-based reagents may be important for bedside testing and in developing countries where refrigeration is unreliable.
antibody-derived, based
has
a
mandatory requirement for checking
a
recipient’s
group before transfusion. This practice has not been more widely adopted because of the difficulty of red cell typing at the bedside. We have shown that monoclonal antibodies can be dried and stored without altering their function, in the presence of the disaccharide, trehalose; IgM monoclonal blood typing antibodies preserve full agglutinating activity for over 2 years. We have designed a dry plate, preloaded with monoclonal antibodies against the A,B, and Rh (D) blood group antigens. These blood groups can be determined within 1 min at the bedside. We now report the accuracy of this method compared with conventional assay techniques.
Materials and methods Introduction Monoclonal antibodies have provided a source of uniform, reproducible, and potent reagents for typing red cell antigens of the A, B, 0, and Rhesus (Rh) blood types. The reported frequency of transfusion reactions is 05-22%. Although blood typing and cross-matching should reduce this figure, 44 fatal transfusion reactions were reported out of an estimated 27 x 106 units of blood administered in the USA.1 90% of these deaths were due to clerical error. To prevent mistakes in recording blood groups, West Germany
1,000 consecutive, unselected requests for blood typing, submitted to
the blood transfusion
laboratory
at
Addenbrooke’s
Hospital,
ADDRESSES: Quadrant Research Foundation, Cambridge Research Laboratories (D. Blakeley, HNC, B Roser, MD), Department of Haematology Transfusion Laboratory, Addenbrooke’s Hospital, Cambridge (B Tolliday, FIMLS), Department of Clinical Biochemistry, Cambridge University Medical School (C. Colaco, PhD). Correspondence to Dr B Roser, Quadrant Research Foundation, Cambridge Research Laboratories, 181A Huntingdon Road, Cambridge CB3 0DJ, UK.
855
First with the standard semiautomated technique done by trained blood transfusion technologists and second with the dry plate format, completed without knowledge by a laboratory assistant who was not engaged in haematology or blood transfusion techniques. This assistant was instructed how to carry out the dry-plate assay and recognise the agglutination reactions.
were tested twice.
Dry-plate assay The monoclonal anti-A antibody (Birma-1) is a murine IgM antibody that reacts strongly with A,, A2, A,B and A2B, Aint, and A""B cells. With A3 and A3B cells it gives "mixed field" reactions. This antibody agglutinated 11 of 14 tested samples of Ax red cells but not cells of the B(A) Bh, Bo, and Ah types. The monoclonal anti-B (ES-4), also a mouse IgM, agglutinates all cells of the B3 type and most of the Bx and oriental B-variant cells. The monoclonal
anti-Rh blood group reagent is a human IgM monoclonal (NELP-4) that agglutinates all normal Rh (D) red cells in categories iii, iv, and Vii.2This antibody gives variable results with D cells of category v and does not detect red cells bearing the DU antigen. For agglutination reactions, these 3 antibodies were titrated in a saline-based test and appropriate concentrations were added to 4-well immuno-plates (Nunc, Denmark) and dried in the presence of a final concentration of 0-3 mol/1 trehalose at 37°C. The fourth well of the immuno-plate, containing only dried buffer and trehalose, was a negative agglutination control. Each well was rehydrated with two drops of distilled water, one drop of blood was added to each well, and the plate was agitated for 5 min. Positive reactions occurred within 1 min of incubation at room temperature. All plates were routinely read after 5 min, for the second time, before the results were recorded; there were no new positive reactions between the first and second reading.
Standard clinical assay The semiautomated standard assays.
procedure involved
2 separate
Cell groups. U-well microplates were stored wet at 4 C until required. 2-3% patient cell suspensions in phosphate-buffered saline were tested against monoclonal anti-A, anti-B, anti-AB, and two monoclonal anti-Ds. Known Al-negative and B-positive cells were also included as positive controls and an AB serum was used as a negative control. Serum groups. Patient sera were tested against enzyme-treated
Al, A2, and B cells. Anti-A serum diluted 1 in 16 and anti-D antibody (0-4 IU) were used as controls. Both plates were incubated at 18°C for 15 min and centrifuged for 15 s at 500 rpm (100 g) in a Sorvall RT6000 centrifuge. The optical density of each well was measured on a multiscan plate reader. Results were independently recorded by 2 technicians.
Results Distribution of blood groups agreed closely with previously reported data.3 For 997 of 1000 blood group typings there was complete concordance between the two techniques. Of the 3 cases in which there was no agreement between assays, 2 were due to either a clerical error or a failure of the dry plate to detect a blood group antigen. In the first case the result A,B Rh negative was recorded when the true blood group was A,B Rh positive and in the second case the blood group A Rh negative was initially recorded when the correct blood group was A,B Rh negative. Thus, for recipient testing the errors would not have resulted in transfusion of blood carrying foreign blood groups. The third sample was found, by both technicians, to be difficult to read. This sample was typed as B Rh negative by the standard clinical assay and as A,B Rh positive by the dry plate assay. The blood sample came from a patient who had received 5 units of group B Rh negative red cells within the preceding 48 h. Retyping of the blood with more sensitive manual methods showed that the
recipient’s blood group was Group A,B recorded by the dry-plate assay.
Rh
positive
as
Discussion This comparative study confirms the accuracy of trehalosedried anti-blood-group antibodies. If it is assumed that the two dry plate errors were not clerical errors, the accuracy rate was 99.8% vs 99-9% for the standard clinical assay. The frequency of fatal transfusion reactions is 10 000-20 000 per year’ and this assay may provide a convenient and safe check on the blood group of transfusion recipients at the bedside or in the field. Erythrocyte membrane antigens are preserved by trehalose drying and react normally with anti-blood-group antisera by agglutination. Simultaneous identification of both erythrocyte antigens and serum antibodies against panel red cells is possible and a stable antibody screening plate could be developed to detect antibodies against rare blood groups in immunised individuals. This plate might eliminate
cross-matching. Previous attempts to produce similar bedside assays have failed because of instability of the dried reagents and a need for effective refrigeration. We used the disaccharide trehalose (a-D-glucopyranosyl-a-D-glucopyranoside) because it can prevent damage to substances air-dried at ambient temperatures. The properties of trehalose were discovered after the observation that some organisms (the brine shrimp Artemia salina and common baker’s yeast Saccharomyces cerevesiae) contained this disaccharide and 4 were able to recover after long periods of desiccation. The protection of a wide range of antibodies and enzymes has been confirmed, with near complete functional recovery in most cases, after rehydration. We have successfully stabilised a panel of restriction endonucleases and DNA modifying enzymes that are currently stored in 50% glycerol between -70°C and 0°C. They can be stored in a trehalose-dried state at + 55°C for longer than 6 months with no detectable loss of the enzymic activity (unpublished observations). Additional stability of trehalose-dried antibodies against moisture damage in conditions of up to 90% relative humidity can be achieved by replacing hygroscopic salts in the buffer solutions with efflorescent, neutral, and non-toxic substances such as sodium sulphate
decahydrate. Further applications of this technique to the production of monoclonal antibodies, enzymes, and other labile reagents is possible. Trehalose-based reagents are stable, reliable, simple to use, and ideal for use in remote or poorly developed areas where there is limited access to refrigeration and technical skills. Further information
on
trehalose-based reagents may be obtained by
consulting patents registered by Dr BJ Roser; patent cooperative treaty international publication numbers WO 87/00196 and GB 89/00093. REFERENCES 1. Honig CL, Bove JR. Transfusion associated fatalities: Review of Bureau of Biologics Reports 1976-1978 Transfusion 1980; 20: 653-61. 2. Voak D. Monoclonal antibodies as blood grouping reagents. In: Contreras M, ed. Clinical haematology. London: Baillière Tindall,
1990; 3: 219-42.
Kopec AC. The distribution of the blood groups in the United Kingdom. Oxford University Press, 1970. 4. Crowe JH, Crowe LM, Carpenter JF, Aurell-Wistrom C. Stabilisation of dry phospholipid bilayers and proteins by sugars Biochem J 1987; 242: 3.
1-10.
fractionated by soybean agglutinin and sheep red blood cells. Blood 1983; 61: 341-48. 11 Friedrich W, Goldmann SF, Ebell W, Blutters-Sawatzki R. Severe treatment combined immunodeficiency: by bone marrow transplantation in 15 infants using HLA-haploidentical donors. Eur J Pediatr 1985; 144: 125-30. 12. Cowan MJ, Wara DW, Weintrub PS, Pabst H, Ammann A. Haploidentical bone marrow transplantation for severe combined
immunodeficiency using soybean agglutinin-negative, T-depleted marrow grafts. J Clin Immunol 1985; 5: 370-76. 13. Buckley RH, Schiff SE, Sampson HA, et al. Development of immunity in human severe primary T-cell deficiency following haploidentical bone cell transplantation. J Immunol 1986; 136: 2398-407. Durandy A, de Villartay JP, et al. HLA haploidentical bone marrow transplantation for severe combined immunodeficiency using E-rosette franctionation and cyclosporin A. Blood 1986; 67: 444-49. 15. Moen RC, Horowitz SD, Sondel PM, et al. Immunologic reconstitution after haploidentical bone marrow transplantation for immune deficiency disorders: treatment of bone marrow cells with monoclonal antibody CT-2 and complement. Blood 1987; 70: 664-69. 16. Primary Immunodeficiency diseases. Report of a World Health Organisation sponsored meeting. Immunodeficiency Rev 1989; 1: 173-205. 17. Vossen JM, Asma GEM, Langlois van den Bergh R, et al. HLA identical and haploidentical bone marrow transplantation for severe combined immunodeficiency: chimerism and immunologic reconstitution in vitro and in vivo. In: Griscelli C, Vossen J, eds. Progress in immunodeficiency research and therapy. Amsterdam: Excerpta Medica, 1984: 417-24. 18. Morgan G, Linch DC, Knott LT, et al. Successful haploidentical mismatched bone marrow transplantation in severe combined immunodeficiency: T-cell removal using Campath-I monoclonal antibody and E-rosetting. Br J Haematol 1986; 62: 421-30. 19. Gluckberg H, Storb R, Fefer A, et al. Clinical manifestations of GVHD in human recipients of marrow from HLA matched sibling donors. Transplantation 1974; 18: 295-304. 20. Jeffreys AJ, Wilson V, Thein SL. Hypervariable minisatellite regions in human DNA. Nature 1985; 315: 67-69. marrow stem
14. Fischer A,
21. Kalbfeisch JD, Prentice RL. The statistical analysis of failure time data. New York: Wiley, 1980. 22.
O’Reilly RJ, Keever CA, Small TN, Brockstein J. The use of HLA non identical T-cell depleted marrow transplants for correction of severe combined immunodeficiency disease. Immunodeficiency Rev 1990; 1:
273-309. 23. Peter HH, Kliche A, Dragger R, et al. NK-cell function in severe combined immunodeficiency: possible relevance for classification and prognosis. In: Vossen J, Griscelli C, eds. Progress in immunodeficiency research and therapy II. Amsterdam: Elsevier Science Publishers, 1986: 287-95.
Murphy WJ, Kumar V, Bennett M. Rejection of bone marrow allograft by mice with severe combined immunodeficiency disease. J Exp Med 1987; 165: 1212-17. 25. Wijnaendts L, Le Deist F, Griscelli C, Fischer A. Development of immunological functions following bone marrow transplantation. Blood 1989; 74: 2212-19. 26. Eiermann TH, Partmann P, Friedrich W, Goldmann SF. Implications for the persistent B cell dysfunction after haploidentical bone marrow transplantation in severe combined immunodeficiency analyzed by Epstein-Barr virus transformation. In: Vossen J, Griscelli C, eds. Progress in immunodeficiency research and therapy II. Amsterdam: Excerpta Medica, 1986: 373-78. 27. O’Reilly RJ, Keever C, Kernan NA, et al. Investigation of humoral immune deficiencies following T cell-depleted, HLA haplotype mismatched parental marrow transplants for the treatment of severe combined immunodeficiency. In: Eibl MM, Rosen FS, eds. Primary immunodeficiency diseases. Amsterdam: Elsevier, 1986: 301-07. 28. Korver K, de Lange GG, van den Bergh RL. Lymphoid chimerism after allogeneic bone marrow transplantation. Transplantation 1987; 44:
24.
643-50. 29. Bortin M. Factors influencing the risk of acute graft vs host disease in man. In: Gale RP, Champlin R, eds. Progress in bone marrow transplantation. New York: Alan R Liss, 1987: 243-64. 30.
Goulmy E, Termisjtelen A, Bradley BA, van Rood JJ. Alloimmunity to human H-Y. Lancet 1976; ii: 1206.
Dry instant blood typing plate for bedside use
A
monoclonal
dry blood the upon grouping plate, simple disaccharide, trehalose, is described that is indefinitely stable at room temperature and was found to have a 99·8% accuracy when tested against a standard semiautomated assay. This plate can be used by personnel with no specific training to check the recorded A,B,O, and Rhesus blood type of potential transfusion recipients in the field and at the bedside. Trehalose-based reagents may be important for bedside testing and in developing countries where refrigeration is unreliable.
antibody-derived, based
has
a
mandatory requirement for checking
a
recipient’s
group before transfusion. This practice has not been more widely adopted because of the difficulty of red cell typing at the bedside. We have shown that monoclonal antibodies can be dried and stored without altering their function, in the presence of the disaccharide, trehalose; IgM monoclonal blood typing antibodies preserve full agglutinating activity for over 2 years. We have designed a dry plate, preloaded with monoclonal antibodies against the A,B, and Rh (D) blood group antigens. These blood groups can be determined within 1 min at the bedside. We now report the accuracy of this method compared with conventional assay techniques.
Materials and methods Introduction Monoclonal antibodies have provided a source of uniform, reproducible, and potent reagents for typing red cell antigens of the A, B, 0, and Rhesus (Rh) blood types. The reported frequency of transfusion reactions is 05-22%. Although blood typing and cross-matching should reduce this figure, 44 fatal transfusion reactions were reported out of an estimated 27 x 106 units of blood administered in the USA.1 90% of these deaths were due to clerical error. To prevent mistakes in recording blood groups, West Germany
1,000 consecutive, unselected requests for blood typing, submitted to
the blood transfusion
laboratory
at
Addenbrooke’s
Hospital,
ADDRESSES: Quadrant Research Foundation, Cambridge Research Laboratories (D. Blakeley, HNC, B Roser, MD), Department of Haematology Transfusion Laboratory, Addenbrooke’s Hospital, Cambridge (B Tolliday, FIMLS), Department of Clinical Biochemistry, Cambridge University Medical School (C. Colaco, PhD). Correspondence to Dr B Roser, Quadrant Research Foundation, Cambridge Research Laboratories, 181A Huntingdon Road, Cambridge CB3 0DJ, UK.
855
First with the standard semiautomated technique done by trained blood transfusion technologists and second with the dry plate format, completed without knowledge by a laboratory assistant who was not engaged in haematology or blood transfusion techniques. This assistant was instructed how to carry out the dry-plate assay and recognise the agglutination reactions.
were tested twice.
Dry-plate assay The monoclonal anti-A antibody (Birma-1) is a murine IgM antibody that reacts strongly with A,, A2, A,B and A2B, Aint, and A""B cells. With A3 and A3B cells it gives "mixed field" reactions. This antibody agglutinated 11 of 14 tested samples of Ax red cells but not cells of the B(A) Bh, Bo, and Ah types. The monoclonal anti-B (ES-4), also a mouse IgM, agglutinates all cells of the B3 type and most of the Bx and oriental B-variant cells. The monoclonal
anti-Rh blood group reagent is a human IgM monoclonal (NELP-4) that agglutinates all normal Rh (D) red cells in categories iii, iv, and Vii.2This antibody gives variable results with D cells of category v and does not detect red cells bearing the DU antigen. For agglutination reactions, these 3 antibodies were titrated in a saline-based test and appropriate concentrations were added to 4-well immuno-plates (Nunc, Denmark) and dried in the presence of a final concentration of 0-3 mol/1 trehalose at 37°C. The fourth well of the immuno-plate, containing only dried buffer and trehalose, was a negative agglutination control. Each well was rehydrated with two drops of distilled water, one drop of blood was added to each well, and the plate was agitated for 5 min. Positive reactions occurred within 1 min of incubation at room temperature. All plates were routinely read after 5 min, for the second time, before the results were recorded; there were no new positive reactions between the first and second reading.
Standard clinical assay The semiautomated standard assays.
procedure involved
2 separate
Cell groups. U-well microplates were stored wet at 4 C until required. 2-3% patient cell suspensions in phosphate-buffered saline were tested against monoclonal anti-A, anti-B, anti-AB, and two monoclonal anti-Ds. Known Al-negative and B-positive cells were also included as positive controls and an AB serum was used as a negative control. Serum groups. Patient sera were tested against enzyme-treated
Al, A2, and B cells. Anti-A serum diluted 1 in 16 and anti-D antibody (0-4 IU) were used as controls. Both plates were incubated at 18°C for 15 min and centrifuged for 15 s at 500 rpm (100 g) in a Sorvall RT6000 centrifuge. The optical density of each well was measured on a multiscan plate reader. Results were independently recorded by 2 technicians.
Results Distribution of blood groups agreed closely with previously reported data.3 For 997 of 1000 blood group typings there was complete concordance between the two techniques. Of the 3 cases in which there was no agreement between assays, 2 were due to either a clerical error or a failure of the dry plate to detect a blood group antigen. In the first case the result A,B Rh negative was recorded when the true blood group was A,B Rh positive and in the second case the blood group A Rh negative was initially recorded when the correct blood group was A,B Rh negative. Thus, for recipient testing the errors would not have resulted in transfusion of blood carrying foreign blood groups. The third sample was found, by both technicians, to be difficult to read. This sample was typed as B Rh negative by the standard clinical assay and as A,B Rh positive by the dry plate assay. The blood sample came from a patient who had received 5 units of group B Rh negative red cells within the preceding 48 h. Retyping of the blood with more sensitive manual methods showed that the
recipient’s blood group was Group A,B recorded by the dry-plate assay.
Rh
positive
as
Discussion This comparative study confirms the accuracy of trehalosedried anti-blood-group antibodies. If it is assumed that the two dry plate errors were not clerical errors, the accuracy rate was 99.8% vs 99-9% for the standard clinical assay. The frequency of fatal transfusion reactions is 10 000-20 000 per year’ and this assay may provide a convenient and safe check on the blood group of transfusion recipients at the bedside or in the field. Erythrocyte membrane antigens are preserved by trehalose drying and react normally with anti-blood-group antisera by agglutination. Simultaneous identification of both erythrocyte antigens and serum antibodies against panel red cells is possible and a stable antibody screening plate could be developed to detect antibodies against rare blood groups in immunised individuals. This plate might eliminate
cross-matching. Previous attempts to produce similar bedside assays have failed because of instability of the dried reagents and a need for effective refrigeration. We used the disaccharide trehalose (a-D-glucopyranosyl-a-D-glucopyranoside) because it can prevent damage to substances air-dried at ambient temperatures. The properties of trehalose were discovered after the observation that some organisms (the brine shrimp Artemia salina and common baker’s yeast Saccharomyces cerevesiae) contained this disaccharide and 4 were able to recover after long periods of desiccation. The protection of a wide range of antibodies and enzymes has been confirmed, with near complete functional recovery in most cases, after rehydration. We have successfully stabilised a panel of restriction endonucleases and DNA modifying enzymes that are currently stored in 50% glycerol between -70°C and 0°C. They can be stored in a trehalose-dried state at + 55°C for longer than 6 months with no detectable loss of the enzymic activity (unpublished observations). Additional stability of trehalose-dried antibodies against moisture damage in conditions of up to 90% relative humidity can be achieved by replacing hygroscopic salts in the buffer solutions with efflorescent, neutral, and non-toxic substances such as sodium sulphate
decahydrate. Further applications of this technique to the production of monoclonal antibodies, enzymes, and other labile reagents is possible. Trehalose-based reagents are stable, reliable, simple to use, and ideal for use in remote or poorly developed areas where there is limited access to refrigeration and technical skills. Further information
on
trehalose-based reagents may be obtained by
consulting patents registered by Dr BJ Roser; patent cooperative treaty international publication numbers WO 87/00196 and GB 89/00093. REFERENCES 1. Honig CL, Bove JR. Transfusion associated fatalities: Review of Bureau of Biologics Reports 1976-1978 Transfusion 1980; 20: 653-61. 2. Voak D. Monoclonal antibodies as blood grouping reagents. In: Contreras M, ed. Clinical haematology. London: Baillière Tindall,
1990; 3: 219-42.
Kopec AC. The distribution of the blood groups in the United Kingdom. Oxford University Press, 1970. 4. Crowe JH, Crowe LM, Carpenter JF, Aurell-Wistrom C. Stabilisation of dry phospholipid bilayers and proteins by sugars Biochem J 1987; 242: 3.
1-10.
