The preservation of red cell antigens at low ionic strength J.C. ALLAN,M. BRUCE,AND R. MITCHELL Low-ionic-strength saline (LISS)techniques permit a safe and substantial reduction in incubation time and have therefore become the method of choice for antibody detection and compatibility testing in many transfusion laboratories. Consequently, the supply of reagent red cells (RBCs) in a low-ionic-strength preservative solution would remove the daily need for laboratories to wash and resuspend cells in LISS before use. However, the storage of fresh RBCs at low ionic strength in the presence of aminoglycoside antibiotics can cause a rapid loss of certain antigens, possibl as a result of the release of proteolytic enzymes from contaminating white cells. This article describes a low-ionic-strength solution that achieves preservation of antigens on liquid nitrogen-frozen-thawed RBCs for 21 days’ storage at 4°C. TRANSFUSION 1990;30:423-426.
We used standard serological procedures throughout’ and calculated titration scores by the method of Marsh.8 All antisera and reagent RBCs were prepared by the Scottish National Blood Transfusion Service (SNBTS). We collected fresh donor RBCs and stored them in CPDA1 for 2 to 5 days, washed them three times, and subsequently suspended them to 3 percent in LIS preservative or modified Alsever’s solution. RBCs frozen in liquid nitrogen (LN,) at - 196°C were thawed, washed three times, and suspended to 3 percent in LIS preservative or modified Alsever’s solution. To investigate the effects of neomycin sulfate and white cell proteases on RBC antigens during storage in LISS, we took three aliquots of whole blood from selected donations. One aliquot was unwashed, the second was white cell-depleted and washed, and the third was white cell-depleted, frozen in LN,, thawed, and washed. We suspended each aliquot to 3 percent in modified Alsever’s solution and stored them at 4°C for a minimum of 5 days. Each 3 percent suspension was subsequently centrifuged and the RBCs resuspended and stored in LISS for a further 3 days at 4°C. The expression of Fyn, Fyb, S, and s on these LISS-suspended samples was assessed daily by comparative titration. Neomycin sulfate, chloramphenicol, gentamicin, nystatin, amphotericin B (A2411), and solubilized amphotericin B (A9528) were obtained commercially (Sigma Chemical Company, Poole, Dorset, UK). Solubilized amphotericin B was also obtained from E.R. Squibb and Sons (Liverpool, UK). Modified Alsever’s solution was prepared as described by chloramMollisong with the addition of inosine (0.94 and neomycin sulfate (0.1 phenicol (0.34
HEMAGGLUTINATION TECHNIQUES REMAIN the simplest and most reliable methods available for the detection of red cell (RBC) antibody-antigen reactions. Hemagglutination techniques using low-ionic-strength saline (LISS)’ permit incubation times to be shortened with no loss in sensitivity and are thus well-suited to modern transfusion practice. Most manufacturers supply reagent RBCs suspended in preservative solutions of normal ionic strength. Consequently, laboratories using LISS techniques must wash and resuspend such reagent RBCs in LISS, a procedure that is tedious, wasteful, and, if the RBCs are stored in LISS, potentially dangerous. Some workers have therefore attempted to produce low-ionic-strength (LIS) solutions containing a p r e s e r v a t i ~ e . ~ . ~ However, RBCs have been reported4.’ to lose antigens from the Duffy system on long-term storage in saline and LISS. Furthermore, the loss of Duffy system antigens and other antigenic determinants has been reported6 to be accelerated at low ionic strength in the presence of antibiotics that are commonly used in RBC preservative solutions. This study assesses the ability of three LIS solutions to preserve the expression of RBC antigens during storage at 4°C.
a),
Materials and Methods We assessed antigen loss by performing comparative titra-
a), a).
LIS preservative solutions Solution A. LIS preservative containing neomycin sulfate
tions at regular intervals during the 4°C storage of reagent RBCs in LIS preservatives or modified Alsever’s solution. We washed and resuspended the latter RBCs in LISS before use. Several batches of reagent RBCs were prepared and tested with at least two examples of each antibody. The results represent an average of all tests performed.
was prepared as follows: glycine, 13.7 g per L; glucose, 8.5 g per L; NaCI, 1. 8 g per L; Na,HPO,, 0.21 g per L; NaH2P0,.2H,O, 0.23 g per L; adenine, 0.2 g per L; inosine, 0.4 g per L; chloramphenicol, 0.34 g per L; and neomycin sulfate, 0.1 g per L. Solution B. LIS preservative without aminoglycoside antibiotics had the same chemical constituents as Solution A. The nysfollowing antimicrobial agents: chloramphenicol (1 tatin (250,000 units/L), and amphotericin B (2.5 m a ) was also included individually in some batches of Solution B to test their fungicidal properties and suitability for inclusion in the above solution.
a),
From the Scottish National Blood Transfusion Service, Headquarters Unit Laboratory, Edinburgh, UK, and the Glasgow and West of Scotland Blood Transfusion Service, Law Hospital, Carluke, UK. Rcceived for publication April 3, 1989; revision received November 10, 1989, and accepted November 20, 1989.
423
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ALLAN ET AL.
Solution C. LIS preservative containing gentamicin was prepared as described by
Results All LISS and LIS preservative solutions had a pH value of 6.7 ? 0.1 and a conductivity of 3.5 to 3.8 mS per cm.' The preservation of selected RBC antigens during the storage of reagent RBCs at 4°C in LIS preservative Solution A is shown in Fig. 1. This reveals a rapid decrease in the reactivity of Fy", Fyb, S, and s: indeed, after storage for 28 days, none of these antigens were demonstrable. K, M, and N were apparently unaffected during storage, while D displayed a moderate increase in titration score. Fresh RBCs in LIS preservative Solution B (Fig. 2) showed deterioration of Fyb and S, whereas frozen-thawed RBCs in this solution (Fig. 3) showed adequate preservation of Fy", Fyb, S, and s after 28 days at 4°C. However, both frozen-thawed RBCs (Fig. 4) and fresh RBCs (Fig. 5) stored in LIS preservative solution C showed greater deterioration of Fy", Fyb, S, and s than did similar RBCs stored in Solution B. Figures 1 through 5 indicate that the presence of aminoglycoside antibiotics in LIS preservative solutions correlates with accelerated deterioration of Fy', Fyb, S, and s, particularly if fresh RBCs are stored in these solutions. Frozen-thawed RBCs stored in LIS preservative Solution B (Fig. 3) show results comparable to those with RBCs stored in modified Alsever's solution and resuspended in LISS before testing (Fig. 6).
Loss of FY, Fyb, S, and s from RBCs stored in modified Alsever's solution and subsequently resuspended and stored in LISS Figure 7 shows the deterioration of Fyb on RBCs stored in modified Alsever's solution subsequently resuspended and stored in LISS. Suspensions of unwashed RBCs that were contaminated with white cells showed a more rapid loss of Fy" than LN,-frozen-thawed or washed RBCs. On some occasions, all demonstrable expression of Fyb was lost after only 24 hours' storage at 4°C. Deterioration of Fyb was generally slower with washed and LN,-frozen-thawed RBCs. Variable, but less rapid deterioration of Fy", S, and s was also evident, particularly with unwashed RBCs (Fig. 8). Frequently, all demonstrable expression of these antigens was lost after 48 hours' storage in LISS at 4°C.
TRANSFUSION Vol. 30. No. 5-1990
RBC hemolysis in LIS preservative solution B containing amphotericin B When assessing alternative antimicrobial agents to replace neomycin sulfate in LIS preservative solutions, we noted that RBCs suspended in LIS preservative Solution B containing 2.5 mg per L of solubilized arnphotericin B (Sigma A9528) showed visible hemolysis after 2 days' storage at 4°C. Solubilized amphotericin B from another supplier (Squibb), used at the same concentration, produced hemolysis after only a few hours' storage at 4°C. RBCs stored in modified Alsever's solution incorporating 2.5 mg per L of solubilized amphotericin B from both suppliers showed no visible evidence of hemolysis after 28 days' at 4°C. Because the amphotericin B solutions from Sigma and Squibb were solubilized in 35 percent and 45 percent sodium deoxycholate, respectively, the effect of this detergent was investigated, and it was subsequently shown that a LIS preservative solution containing 2.5 mg per L of amphotericin B (Sigma A2411) solubilized in 0.5 mg per L of sodium deoxycholate produced no visible hemolysis after 35 days' storage at 4°C.
Discussion The provision of reagent RBCs in a LIS preservative solution is a highly desirable objective. Malyska et aL6 suggested that storage of blood in the presence of aminoglycoside antibiotics (e.g., neomycin and gentamicin) may promote the release of white cell proteases that could subsequently act on protease-sensitive RBC antigens. Our results provide some support for this view, as antigenic deterioration was more pronounted in reagent RBCs prepared from fresh blood than from frozen-thawed blood, which possibly reflects the removal of almost all white cells during the freeze, thaw, and wash processes. Furthermore, we found that the loss of protease-sensitive antigens was much greater when aminoglycoside antibiotics were present (Figs. 1, 4, and 5 ) . RBCs have previously been reported4s5to lose Duffj system antigens during long-term storage in LISS. However, RBCs stored in modified Alsever's solution containing neomycin sulfate and subsequently washed and suspended in LISS before testing displayed a variable
NO of days Poiage FIG. 1. Fresh red cells stored in LIS preservative Solution A.
FIG. 2. Fresh red cells stored in LIS preservative Solution B.
TRANSFUSION 1990-Vol. 30. No. 5
I
1
3 NO 01 days Sioiage FIG.3.
425
LOW-IONIC-STRENGTH PRESERVATIVE
LN,-recovered red cells stored in LIS prcservativc Solution
B.
1
1
1
1
1
l
1
lo
1
1
NO
I
I
I
1
1
1
01 days Sroiage
I
I
20
1
I
I
’
I
1
1
1
28
FIG.4. LN,-rccovcred rcd cells stored in LIS prcservativc Solufion C.
d 74
‘4 1
\ I I 1 I 1 I 1 I T 1 1 1 I I l I I I T I l
3
10
NO of days Storage
20
FIG.5. Frcsh rcd cells stored in LIS prcservativc Solution C.
but greatly accelerated loss of Fy”, Fyb, S , and s when compared with RBCs stored and suspended in modified Alsever’s solution or any of the three LIS preservative solutions tested. On some occasions, after only 2 days’ storage at 4”C, all demonstrable expression of these antigens was lost. Indeed, Fyb on unwashed RBCs suspended in LISS could be removed totally after only 24 hours’ storage at 4°C. This observation is particularly important to blood bankers because it appears that RBCs previously exposed to aminoglycoside antibiotics and subsequently washed and suspended in LISS were more susceptible to loss of Fyn, Fyb, S , and s than were RBCs that had not been exposed to these antibiotics. The rapid deterioration of Fyb on LN,-frozen-thawed RBCs stored initially in modified Alsever’s solution and subsequently resuspended and stored in LISS (Fig. 7) suggests that factors other than the release of white cell proteases influence antigenic removal. Our investigation did not confirm the loss of M reported by Malyska et a1.6 or the possible loss of Lewis system antigens reported by However, we did
FIG. 6 . Fresh red cells stored in modified Alsevcr’s solution and rcsuspcndcd in LISS before testing.
uncover a problem of hemolysis associated with the use of the fungicide amphotericin B in its solubilized form, which was not observed when reagent RBCs were suspended in modified Alsever’s solution containing this fungicide. Amphotericin B is highly insoluble and therefore generally supplied in a soluble form containing sodium deoxycholate. The preparations of amphotericin B incorporated in LIS preservative solutions contained between 35 and 45 percent sodium deoxycholate, with the higher concentration producing signs of hemolysis after only a few hours’ storage. It seems reasonable to suggest that the hemolysis results from the increased susceptibility of RBCs stored at LIS to lytic damage caused by sodium deoxycholate. For this reason, we decided to exclude amphotericin B from our LIS preservative solution (Solution B). The effect of LIS conditions on the RBC membrane and the mechanism by which these conditions enhance hemagglutination reactions are complex and not fully understood. However, this study has shown that, provided the preservative solution and production method are chosen with care (e.g., the use of frozen-thawed RBCs and LIS preservative Solution B), it is possible to produce reagent RBCs stored in a LIS preservative solution with a shelf life of at least 21 days at 4°C.
426
TRANSFUSION
ALLAN ET AL.
Vol. 30,
No. 5-1990
Acknowledgments The authors thank Dr. R. Fraser for advice and assistance.
References
NO
01 days Srorage in L.I.S.!
FIG. 7. Deterioration of Fyb (AFyb)on red cells stored in modified Alsever's solution and subsequently resuspended and stored in LISS.
1. Moore HC, Mollison PL. Use of a low-ionic-strength medium in manual tests for antibody detection. Transfusion 1976;16:291-6. 2. Storry JR. Long term preservation of red cell antibody identification panels in low ionic strength solution. Med Lab Sci 1987;44:3505. 3. Allan JC, Bruce M, Brown K,Boyd R. LISS preservative solution. In: Abstracts of Scientific Presentations of the British Blood Transfusion Society, 1987:06/17. 4. Williams D, Johnson CL, Marsh WL. Duffy antigen changes on red blood cells stored at low temperature. Transfusion 1981;21:357-9. 5 . Davies DM, Hall SJ, Graham HA, Chachowvski R. The isolation and partial characterizationof Daffy antigens from human red cells (abstract). Transfusion 1979;19:638. 6. Malyska H, Kleeman JE, Masouredis SP, Victoria EJ. Effects on blood group antigens from storage at low ionic strength in the presence of Neomycin. Vox Sang 1983;44:375-84. 7. Boorman KE, Dodd BE, Lincoln PJ. An introduction to blood group serology: theory, techniques, practical applications. 5th ed. New York: Churchill Livingstone, 1977. 8. Marsh WL. Scoring of haemagglutination reactions. Transfusion 1972;12:352-3. 9. Mollison PL. Blood transfusion in clinical medicine. 7th ed. London: Blackwell, 1983.
John C. Allan, FIMLS, Associate Reagents Manager, SNBTS Headquarters Unit Laboratory, 2 Forrest Road, Edinburgh, Scotland, UK. [Reprint requests] Martin Bruce. FIMLS, Reagents Manager, SNBTS Headquarters Unit Laboratory. Ruthven Mitchell, BSc, MBChB, MD, FRCPG, FRCPath, Medical Director, Glasgow and West of Scotland Blood Transfusion Service, Carluke, Scotland, UK.
NO of days S~uiagiin 1.I.S.S FIG. 8. Deterioration of Fy', Fyb, S. and s on red cells stored in modified Alsever's solution and subsequently resuspended and stored in LISS.
Announcement
SCANDINAVIAN ASSOCIATION FOR EXTRACORPOREAL MEMBRANE OXYGENATION (ECMO) The 4th International Meeting of the Scandinavian Association for ECMO (SAFE) will be held May 23-25, 1991 in Goteborg, Sweden. The meeting will cover research on long-term clinical and experimental, neonatal and pediatric ECMO. For more information, contact: Ann-Sofie Klevbrant Pediatric Surgical Clinic Ostra Sjukhuset S-416 85 Goteborg Sweden Telephone: 46 31 37 46 20 FAX: 46 31 84 30 10
We used standard serological procedures throughout’ and calculated titration scores by the method of Marsh.8 All antisera and reagent RBCs were prepared by the Scottish National Blood Transfusion Service (SNBTS). We collected fresh donor RBCs and stored them in CPDA1 for 2 to 5 days, washed them three times, and subsequently suspended them to 3 percent in LIS preservative or modified Alsever’s solution. RBCs frozen in liquid nitrogen (LN,) at - 196°C were thawed, washed three times, and suspended to 3 percent in LIS preservative or modified Alsever’s solution. To investigate the effects of neomycin sulfate and white cell proteases on RBC antigens during storage in LISS, we took three aliquots of whole blood from selected donations. One aliquot was unwashed, the second was white cell-depleted and washed, and the third was white cell-depleted, frozen in LN,, thawed, and washed. We suspended each aliquot to 3 percent in modified Alsever’s solution and stored them at 4°C for a minimum of 5 days. Each 3 percent suspension was subsequently centrifuged and the RBCs resuspended and stored in LISS for a further 3 days at 4°C. The expression of Fyn, Fyb, S, and s on these LISS-suspended samples was assessed daily by comparative titration. Neomycin sulfate, chloramphenicol, gentamicin, nystatin, amphotericin B (A2411), and solubilized amphotericin B (A9528) were obtained commercially (Sigma Chemical Company, Poole, Dorset, UK). Solubilized amphotericin B was also obtained from E.R. Squibb and Sons (Liverpool, UK). Modified Alsever’s solution was prepared as described by chloramMollisong with the addition of inosine (0.94 and neomycin sulfate (0.1 phenicol (0.34
HEMAGGLUTINATION TECHNIQUES REMAIN the simplest and most reliable methods available for the detection of red cell (RBC) antibody-antigen reactions. Hemagglutination techniques using low-ionic-strength saline (LISS)’ permit incubation times to be shortened with no loss in sensitivity and are thus well-suited to modern transfusion practice. Most manufacturers supply reagent RBCs suspended in preservative solutions of normal ionic strength. Consequently, laboratories using LISS techniques must wash and resuspend such reagent RBCs in LISS, a procedure that is tedious, wasteful, and, if the RBCs are stored in LISS, potentially dangerous. Some workers have therefore attempted to produce low-ionic-strength (LIS) solutions containing a p r e s e r v a t i ~ e . ~ . ~ However, RBCs have been reported4.’ to lose antigens from the Duffy system on long-term storage in saline and LISS. Furthermore, the loss of Duffy system antigens and other antigenic determinants has been reported6 to be accelerated at low ionic strength in the presence of antibiotics that are commonly used in RBC preservative solutions. This study assesses the ability of three LIS solutions to preserve the expression of RBC antigens during storage at 4°C.
a),
Materials and Methods We assessed antigen loss by performing comparative titra-
a), a).
LIS preservative solutions Solution A. LIS preservative containing neomycin sulfate
tions at regular intervals during the 4°C storage of reagent RBCs in LIS preservatives or modified Alsever’s solution. We washed and resuspended the latter RBCs in LISS before use. Several batches of reagent RBCs were prepared and tested with at least two examples of each antibody. The results represent an average of all tests performed.
was prepared as follows: glycine, 13.7 g per L; glucose, 8.5 g per L; NaCI, 1. 8 g per L; Na,HPO,, 0.21 g per L; NaH2P0,.2H,O, 0.23 g per L; adenine, 0.2 g per L; inosine, 0.4 g per L; chloramphenicol, 0.34 g per L; and neomycin sulfate, 0.1 g per L. Solution B. LIS preservative without aminoglycoside antibiotics had the same chemical constituents as Solution A. The nysfollowing antimicrobial agents: chloramphenicol (1 tatin (250,000 units/L), and amphotericin B (2.5 m a ) was also included individually in some batches of Solution B to test their fungicidal properties and suitability for inclusion in the above solution.
a),
From the Scottish National Blood Transfusion Service, Headquarters Unit Laboratory, Edinburgh, UK, and the Glasgow and West of Scotland Blood Transfusion Service, Law Hospital, Carluke, UK. Rcceived for publication April 3, 1989; revision received November 10, 1989, and accepted November 20, 1989.
423
424
ALLAN ET AL.
Solution C. LIS preservative containing gentamicin was prepared as described by
Results All LISS and LIS preservative solutions had a pH value of 6.7 ? 0.1 and a conductivity of 3.5 to 3.8 mS per cm.' The preservation of selected RBC antigens during the storage of reagent RBCs at 4°C in LIS preservative Solution A is shown in Fig. 1. This reveals a rapid decrease in the reactivity of Fy", Fyb, S, and s: indeed, after storage for 28 days, none of these antigens were demonstrable. K, M, and N were apparently unaffected during storage, while D displayed a moderate increase in titration score. Fresh RBCs in LIS preservative Solution B (Fig. 2) showed deterioration of Fyb and S, whereas frozen-thawed RBCs in this solution (Fig. 3) showed adequate preservation of Fy", Fyb, S, and s after 28 days at 4°C. However, both frozen-thawed RBCs (Fig. 4) and fresh RBCs (Fig. 5) stored in LIS preservative solution C showed greater deterioration of Fy", Fyb, S, and s than did similar RBCs stored in Solution B. Figures 1 through 5 indicate that the presence of aminoglycoside antibiotics in LIS preservative solutions correlates with accelerated deterioration of Fy', Fyb, S, and s, particularly if fresh RBCs are stored in these solutions. Frozen-thawed RBCs stored in LIS preservative Solution B (Fig. 3) show results comparable to those with RBCs stored in modified Alsever's solution and resuspended in LISS before testing (Fig. 6).
Loss of FY, Fyb, S, and s from RBCs stored in modified Alsever's solution and subsequently resuspended and stored in LISS Figure 7 shows the deterioration of Fyb on RBCs stored in modified Alsever's solution subsequently resuspended and stored in LISS. Suspensions of unwashed RBCs that were contaminated with white cells showed a more rapid loss of Fy" than LN,-frozen-thawed or washed RBCs. On some occasions, all demonstrable expression of Fyb was lost after only 24 hours' storage at 4°C. Deterioration of Fyb was generally slower with washed and LN,-frozen-thawed RBCs. Variable, but less rapid deterioration of Fy", S, and s was also evident, particularly with unwashed RBCs (Fig. 8). Frequently, all demonstrable expression of these antigens was lost after 48 hours' storage in LISS at 4°C.
TRANSFUSION Vol. 30. No. 5-1990
RBC hemolysis in LIS preservative solution B containing amphotericin B When assessing alternative antimicrobial agents to replace neomycin sulfate in LIS preservative solutions, we noted that RBCs suspended in LIS preservative Solution B containing 2.5 mg per L of solubilized arnphotericin B (Sigma A9528) showed visible hemolysis after 2 days' storage at 4°C. Solubilized amphotericin B from another supplier (Squibb), used at the same concentration, produced hemolysis after only a few hours' storage at 4°C. RBCs stored in modified Alsever's solution incorporating 2.5 mg per L of solubilized amphotericin B from both suppliers showed no visible evidence of hemolysis after 28 days' at 4°C. Because the amphotericin B solutions from Sigma and Squibb were solubilized in 35 percent and 45 percent sodium deoxycholate, respectively, the effect of this detergent was investigated, and it was subsequently shown that a LIS preservative solution containing 2.5 mg per L of amphotericin B (Sigma A2411) solubilized in 0.5 mg per L of sodium deoxycholate produced no visible hemolysis after 35 days' storage at 4°C.
Discussion The provision of reagent RBCs in a LIS preservative solution is a highly desirable objective. Malyska et aL6 suggested that storage of blood in the presence of aminoglycoside antibiotics (e.g., neomycin and gentamicin) may promote the release of white cell proteases that could subsequently act on protease-sensitive RBC antigens. Our results provide some support for this view, as antigenic deterioration was more pronounted in reagent RBCs prepared from fresh blood than from frozen-thawed blood, which possibly reflects the removal of almost all white cells during the freeze, thaw, and wash processes. Furthermore, we found that the loss of protease-sensitive antigens was much greater when aminoglycoside antibiotics were present (Figs. 1, 4, and 5 ) . RBCs have previously been reported4s5to lose Duffj system antigens during long-term storage in LISS. However, RBCs stored in modified Alsever's solution containing neomycin sulfate and subsequently washed and suspended in LISS before testing displayed a variable
NO of days Poiage FIG. 1. Fresh red cells stored in LIS preservative Solution A.
FIG. 2. Fresh red cells stored in LIS preservative Solution B.
TRANSFUSION 1990-Vol. 30. No. 5
I
1
3 NO 01 days Sioiage FIG.3.
425
LOW-IONIC-STRENGTH PRESERVATIVE
LN,-recovered red cells stored in LIS prcservativc Solution
B.
1
1
1
1
1
l
1
lo
1
1
NO
I
I
I
1
1
1
01 days Sroiage
I
I
20
1
I
I
’
I
1
1
1
28
FIG.4. LN,-rccovcred rcd cells stored in LIS prcservativc Solufion C.
d 74
‘4 1
\ I I 1 I 1 I 1 I T 1 1 1 I I l I I I T I l
3
10
NO of days Storage
20
FIG.5. Frcsh rcd cells stored in LIS prcservativc Solution C.
but greatly accelerated loss of Fy”, Fyb, S , and s when compared with RBCs stored and suspended in modified Alsever’s solution or any of the three LIS preservative solutions tested. On some occasions, after only 2 days’ storage at 4”C, all demonstrable expression of these antigens was lost. Indeed, Fyb on unwashed RBCs suspended in LISS could be removed totally after only 24 hours’ storage at 4°C. This observation is particularly important to blood bankers because it appears that RBCs previously exposed to aminoglycoside antibiotics and subsequently washed and suspended in LISS were more susceptible to loss of Fyn, Fyb, S , and s than were RBCs that had not been exposed to these antibiotics. The rapid deterioration of Fyb on LN,-frozen-thawed RBCs stored initially in modified Alsever’s solution and subsequently resuspended and stored in LISS (Fig. 7) suggests that factors other than the release of white cell proteases influence antigenic removal. Our investigation did not confirm the loss of M reported by Malyska et a1.6 or the possible loss of Lewis system antigens reported by However, we did
FIG. 6 . Fresh red cells stored in modified Alsevcr’s solution and rcsuspcndcd in LISS before testing.
uncover a problem of hemolysis associated with the use of the fungicide amphotericin B in its solubilized form, which was not observed when reagent RBCs were suspended in modified Alsever’s solution containing this fungicide. Amphotericin B is highly insoluble and therefore generally supplied in a soluble form containing sodium deoxycholate. The preparations of amphotericin B incorporated in LIS preservative solutions contained between 35 and 45 percent sodium deoxycholate, with the higher concentration producing signs of hemolysis after only a few hours’ storage. It seems reasonable to suggest that the hemolysis results from the increased susceptibility of RBCs stored at LIS to lytic damage caused by sodium deoxycholate. For this reason, we decided to exclude amphotericin B from our LIS preservative solution (Solution B). The effect of LIS conditions on the RBC membrane and the mechanism by which these conditions enhance hemagglutination reactions are complex and not fully understood. However, this study has shown that, provided the preservative solution and production method are chosen with care (e.g., the use of frozen-thawed RBCs and LIS preservative Solution B), it is possible to produce reagent RBCs stored in a LIS preservative solution with a shelf life of at least 21 days at 4°C.
426
TRANSFUSION
ALLAN ET AL.
Vol. 30,
No. 5-1990
Acknowledgments The authors thank Dr. R. Fraser for advice and assistance.
References
NO
01 days Srorage in L.I.S.!
FIG. 7. Deterioration of Fyb (AFyb)on red cells stored in modified Alsever's solution and subsequently resuspended and stored in LISS.
1. Moore HC, Mollison PL. Use of a low-ionic-strength medium in manual tests for antibody detection. Transfusion 1976;16:291-6. 2. Storry JR. Long term preservation of red cell antibody identification panels in low ionic strength solution. Med Lab Sci 1987;44:3505. 3. Allan JC, Bruce M, Brown K,Boyd R. LISS preservative solution. In: Abstracts of Scientific Presentations of the British Blood Transfusion Society, 1987:06/17. 4. Williams D, Johnson CL, Marsh WL. Duffy antigen changes on red blood cells stored at low temperature. Transfusion 1981;21:357-9. 5 . Davies DM, Hall SJ, Graham HA, Chachowvski R. The isolation and partial characterizationof Daffy antigens from human red cells (abstract). Transfusion 1979;19:638. 6. Malyska H, Kleeman JE, Masouredis SP, Victoria EJ. Effects on blood group antigens from storage at low ionic strength in the presence of Neomycin. Vox Sang 1983;44:375-84. 7. Boorman KE, Dodd BE, Lincoln PJ. An introduction to blood group serology: theory, techniques, practical applications. 5th ed. New York: Churchill Livingstone, 1977. 8. Marsh WL. Scoring of haemagglutination reactions. Transfusion 1972;12:352-3. 9. Mollison PL. Blood transfusion in clinical medicine. 7th ed. London: Blackwell, 1983.
John C. Allan, FIMLS, Associate Reagents Manager, SNBTS Headquarters Unit Laboratory, 2 Forrest Road, Edinburgh, Scotland, UK. [Reprint requests] Martin Bruce. FIMLS, Reagents Manager, SNBTS Headquarters Unit Laboratory. Ruthven Mitchell, BSc, MBChB, MD, FRCPG, FRCPath, Medical Director, Glasgow and West of Scotland Blood Transfusion Service, Carluke, Scotland, UK.
NO of days S~uiagiin 1.I.S.S FIG. 8. Deterioration of Fy', Fyb, S. and s on red cells stored in modified Alsever's solution and subsequently resuspended and stored in LISS.
Announcement
SCANDINAVIAN ASSOCIATION FOR EXTRACORPOREAL MEMBRANE OXYGENATION (ECMO) The 4th International Meeting of the Scandinavian Association for ECMO (SAFE) will be held May 23-25, 1991 in Goteborg, Sweden. The meeting will cover research on long-term clinical and experimental, neonatal and pediatric ECMO. For more information, contact: Ann-Sofie Klevbrant Pediatric Surgical Clinic Ostra Sjukhuset S-416 85 Goteborg Sweden Telephone: 46 31 37 46 20 FAX: 46 31 84 30 10
