809
and a known sensitiser in other circumstances. We thought that our patient was affected by the airborne route, but not by latex. Latex is a natural product consisting principally of polyisoprene. It would not be surprising, however, if it contained a number of other natural chemicals with the potential both to vaporise and to sensitise. Rubber-glove asthma has now been described in several countries and, whatever the mechanism, should be born in mind when work-related asthma develops in hospital workers. Environmental and Occupational Medicine, University Medical School, Aberdeen AB9 2ZD, UK
ANTHONY SEATON
1 Seaton A, Cherrie B, Turnbull J. Rubber glove asthma Br
Med J 1988; 296: 531-32.
Deformability of erythrocytes stored between - 20 and - 25°C SIR,-Preservation at - 80°C or -196°C is used in transfusion medicine for the long-term storage of erythrocytes, but the cost of equipment and liquid nitrogen and the post-thaw shelf-life of only 24 h have restricted the application of cryopreservation.There are some reports of successful extended post-thaw liquid storage of erythrocytes in additive solutions.2-4 It was shown that after storage at -196°C as well as 9 days post-thaw storage at 4°C deformability and in-vivo survival were preserved while deformability and in-vivo survival of erythrocytes were reduced stored at —28°C.’ In 1989 Lovric and Klarkowski6 reported a successful liquid preservation of erythrocytes after storage between - 20 and - 25°C, with negligible changes in deformability. To see if that favourable report could be confirmed using a method more sensitive to dynamic determinants of deformability than the ektacytometer used by Lovric and Klarkowski we applied a filtration method to study deformability in erythrocytes stored at -20°C to -25°C and during post-thaw liquid storage. Anticoagulated blood was processed to yield an erythrocyte concentrate that was resuspended in cryoprotective solution (dextrose 0-5 mol/1, glycerol 3-2 mol/1) and stored between - 20°C and -25°C, half for 7 days and half for 8 months. After thawing, deglycerolisation, and resuspension (in NaH2PO 20, citric acid 20, sodium citrate 20, dextrose 40, NaCI 123, and adenine z 23 mmol/1) units were stored for 35 days at + 4°C in a blood-bank refrigerator. Specimens for sample preparation were washed (10 min, 2000 g, 22°C) with isotonic phosphate-buffered saline (NaCl 151-2, KCl 56, NaZHP04 5-48, and NaH2P04 0-32 mmol/1; pH 7-4) and sediments were resuspended in phosphate-buffered saline (PBS) containing 1% human serum albumin to a haematocrit of 0-60. Deformability was evaluated with a haemofiltrometer7 with 13 mm diameter cellulose filters and an initial filtration pressure of 290 Pa. The filterability index (FI) was calculated8 from five or six estimations, FI being inversely related to deformability. Erythrocyte indices were estimated with ’Technicon HI’ analyser, and haemolysis and pH (at 25°C) were estimated by standard procedures. The statistical test used was Wilcoxon’s for paired data. Changes in FI depended mainly on the frozen storage period. After 7 days of frozen storage the average FI increase was only 14 % but after 8 months it was 67% (pH05) Further differences emerged at the end of the post-thaw storage. After 7 days frozen storage and 35 days at + 4°C the FI increased by about 61 %. After 8 months frozen storage FI increased by 95% for 35 days post-thaw storage and by 57% (ie, roughly the same as that after 8 months’ frozen storage) for 14 days of liquid storage. Mean cellular haemoglobin concentration (MCHC) increased after 7 days and 8 months of frozen storage (5% and 23%, respectively), causing the FI increase. Whereas the MCHC after 35 days post-thaw storage (following the 7 days of freezing) remained unchanged, the MCHC after 14 days post-thaw storage following 8 months of freezing decreased by about 9% and then remained unchanged up to the end of the 35 days post-thaw storage. The pH, estimated after different storage periods, was similar to that found after liquid preservation (range 6-32-6-52). To explain the disagreement between our results and those of Lovric and Klarkowski, who reported only negligible deformability
changes, it should be remembered that an ektacytometer measures static deformability while the filtration method is more sensitive to the dynamic deformability properties of cells. Institute for Transfusion and Transplantation and Institute for Medical Physics and Biophysics, Bereich Medizin Charité, Humboldt University, 1040 Berlin, Germany
G. WEGNER W. KUCERA
1 Chaplin H Jr Frozen red cells revisited. N Engl J Med 1984; 311: 1696-98 2. Kane O, George E, Offner M, Eber M, Tromp C, Goll S Nine days post-thawing red cell conservation in a synthetic medium. Transfusion 1986; 26: 437-40. 3. Moore GL, Ledford ME, Mathewson PJ, Hankms DJ. Liquid storage at 4°C of previously frozen red cells. Transfusion 1987, 27: 496-98. 4. Ross DG, Heaton WAL, Holme S. Additive solution for the suspension and storage of deglycerohzed red blood cells Vox Sang 1989; 56: 75-79. 5. Wegner G, Kucera W. Investigations on red blood cell deformability after long-term cryopreservation. Cryo-Letters 1988; 9: 78-85. 6. Lovric VA, Klarkowski DB. Donor blood frozen and stored between -20°C and -25°C with 35-day post-thaw shelf life. Lancet 1989; i: 71-73. 7 Kucera W, Lerche D, Meier W, Rentsch W. Charakterisierung der Erythrozytendeformierbarkeit mitels Filtrationstechnik—4. Mitteilung. Em teilautomatisiertes Hamofiltrometer fur Routine und Forschung. Z Med Lab-diagn 1986; 27: 262-67 8. Kucera W, Lerche D, Meier W, Baumler H. Charakterisierung der Erythrozytendeformierbarkeit mittels Filtrationstechnik—1. Methodische Aspekte zu einem klassischen Verfahren. Z Med Lab-diagn 1983; 24: 292-98
The secret of sex SIR,-Your Aug 11 editorial draws attention to exciting observations on a newly characterised Y-linked gene (SRY)1 which has been presented as a candidate for TDF, the elusive testisdetermining factor responsible for initiating male differentiation. SRY is a much better candidate than ZFY,2 H-Y antigen,3 or Bkm,4 because it is present within the smallest segment (35 kilobases) shown to be transferred from the Y chromosome in Y-positive XX males, it is highly conserved among eutherian mammals, it is expressed in adult testes, and its murine homologue is present in the embryonic gonad at the time of differentiation.s However, your editorial is mistaken in assuming that SRY is present in all XX males and absent from all XY females. As is indicated in fig 1, samples for all five of our Y-negative XX males lack the SRY gene when their DNA is amplified with primers for pY53.3, the probe which recognises SRY. Furthermore, only one of the XY females tested in the same way shows absence of SRY and this is a patient with a cytogenetically obvious deletion of a large part of the short arm of the Y chromosome In fig 2, we have amplified across the Y pseudoautosomal boundary and shown that this boundary is present in XY females (except in the above patient) and Y-positive XX males, but absent from our series of Y-negative XX males.
r
> 1000). Two patients had not responded to sodium antimony gluconate 20 mg/kg daily for 20 days. The other three had not DETAILS OF PATIENTS WITH VISCERAL LEISHMANIASIS AND RESPONSE TO KETOCONAZOLE
*Premously treated with sodium antimony gluconate
and a known sensitiser in other circumstances. We thought that our patient was affected by the airborne route, but not by latex. Latex is a natural product consisting principally of polyisoprene. It would not be surprising, however, if it contained a number of other natural chemicals with the potential both to vaporise and to sensitise. Rubber-glove asthma has now been described in several countries and, whatever the mechanism, should be born in mind when work-related asthma develops in hospital workers. Environmental and Occupational Medicine, University Medical School, Aberdeen AB9 2ZD, UK
ANTHONY SEATON
1 Seaton A, Cherrie B, Turnbull J. Rubber glove asthma Br
Med J 1988; 296: 531-32.
Deformability of erythrocytes stored between - 20 and - 25°C SIR,-Preservation at - 80°C or -196°C is used in transfusion medicine for the long-term storage of erythrocytes, but the cost of equipment and liquid nitrogen and the post-thaw shelf-life of only 24 h have restricted the application of cryopreservation.There are some reports of successful extended post-thaw liquid storage of erythrocytes in additive solutions.2-4 It was shown that after storage at -196°C as well as 9 days post-thaw storage at 4°C deformability and in-vivo survival were preserved while deformability and in-vivo survival of erythrocytes were reduced stored at —28°C.’ In 1989 Lovric and Klarkowski6 reported a successful liquid preservation of erythrocytes after storage between - 20 and - 25°C, with negligible changes in deformability. To see if that favourable report could be confirmed using a method more sensitive to dynamic determinants of deformability than the ektacytometer used by Lovric and Klarkowski we applied a filtration method to study deformability in erythrocytes stored at -20°C to -25°C and during post-thaw liquid storage. Anticoagulated blood was processed to yield an erythrocyte concentrate that was resuspended in cryoprotective solution (dextrose 0-5 mol/1, glycerol 3-2 mol/1) and stored between - 20°C and -25°C, half for 7 days and half for 8 months. After thawing, deglycerolisation, and resuspension (in NaH2PO 20, citric acid 20, sodium citrate 20, dextrose 40, NaCI 123, and adenine z 23 mmol/1) units were stored for 35 days at + 4°C in a blood-bank refrigerator. Specimens for sample preparation were washed (10 min, 2000 g, 22°C) with isotonic phosphate-buffered saline (NaCl 151-2, KCl 56, NaZHP04 5-48, and NaH2P04 0-32 mmol/1; pH 7-4) and sediments were resuspended in phosphate-buffered saline (PBS) containing 1% human serum albumin to a haematocrit of 0-60. Deformability was evaluated with a haemofiltrometer7 with 13 mm diameter cellulose filters and an initial filtration pressure of 290 Pa. The filterability index (FI) was calculated8 from five or six estimations, FI being inversely related to deformability. Erythrocyte indices were estimated with ’Technicon HI’ analyser, and haemolysis and pH (at 25°C) were estimated by standard procedures. The statistical test used was Wilcoxon’s for paired data. Changes in FI depended mainly on the frozen storage period. After 7 days of frozen storage the average FI increase was only 14 % but after 8 months it was 67% (pH05) Further differences emerged at the end of the post-thaw storage. After 7 days frozen storage and 35 days at + 4°C the FI increased by about 61 %. After 8 months frozen storage FI increased by 95% for 35 days post-thaw storage and by 57% (ie, roughly the same as that after 8 months’ frozen storage) for 14 days of liquid storage. Mean cellular haemoglobin concentration (MCHC) increased after 7 days and 8 months of frozen storage (5% and 23%, respectively), causing the FI increase. Whereas the MCHC after 35 days post-thaw storage (following the 7 days of freezing) remained unchanged, the MCHC after 14 days post-thaw storage following 8 months of freezing decreased by about 9% and then remained unchanged up to the end of the 35 days post-thaw storage. The pH, estimated after different storage periods, was similar to that found after liquid preservation (range 6-32-6-52). To explain the disagreement between our results and those of Lovric and Klarkowski, who reported only negligible deformability
changes, it should be remembered that an ektacytometer measures static deformability while the filtration method is more sensitive to the dynamic deformability properties of cells. Institute for Transfusion and Transplantation and Institute for Medical Physics and Biophysics, Bereich Medizin Charité, Humboldt University, 1040 Berlin, Germany
G. WEGNER W. KUCERA
1 Chaplin H Jr Frozen red cells revisited. N Engl J Med 1984; 311: 1696-98 2. Kane O, George E, Offner M, Eber M, Tromp C, Goll S Nine days post-thawing red cell conservation in a synthetic medium. Transfusion 1986; 26: 437-40. 3. Moore GL, Ledford ME, Mathewson PJ, Hankms DJ. Liquid storage at 4°C of previously frozen red cells. Transfusion 1987, 27: 496-98. 4. Ross DG, Heaton WAL, Holme S. Additive solution for the suspension and storage of deglycerohzed red blood cells Vox Sang 1989; 56: 75-79. 5. Wegner G, Kucera W. Investigations on red blood cell deformability after long-term cryopreservation. Cryo-Letters 1988; 9: 78-85. 6. Lovric VA, Klarkowski DB. Donor blood frozen and stored between -20°C and -25°C with 35-day post-thaw shelf life. Lancet 1989; i: 71-73. 7 Kucera W, Lerche D, Meier W, Rentsch W. Charakterisierung der Erythrozytendeformierbarkeit mitels Filtrationstechnik—4. Mitteilung. Em teilautomatisiertes Hamofiltrometer fur Routine und Forschung. Z Med Lab-diagn 1986; 27: 262-67 8. Kucera W, Lerche D, Meier W, Baumler H. Charakterisierung der Erythrozytendeformierbarkeit mittels Filtrationstechnik—1. Methodische Aspekte zu einem klassischen Verfahren. Z Med Lab-diagn 1983; 24: 292-98
The secret of sex SIR,-Your Aug 11 editorial draws attention to exciting observations on a newly characterised Y-linked gene (SRY)1 which has been presented as a candidate for TDF, the elusive testisdetermining factor responsible for initiating male differentiation. SRY is a much better candidate than ZFY,2 H-Y antigen,3 or Bkm,4 because it is present within the smallest segment (35 kilobases) shown to be transferred from the Y chromosome in Y-positive XX males, it is highly conserved among eutherian mammals, it is expressed in adult testes, and its murine homologue is present in the embryonic gonad at the time of differentiation.s However, your editorial is mistaken in assuming that SRY is present in all XX males and absent from all XY females. As is indicated in fig 1, samples for all five of our Y-negative XX males lack the SRY gene when their DNA is amplified with primers for pY53.3, the probe which recognises SRY. Furthermore, only one of the XY females tested in the same way shows absence of SRY and this is a patient with a cytogenetically obvious deletion of a large part of the short arm of the Y chromosome In fig 2, we have amplified across the Y pseudoautosomal boundary and shown that this boundary is present in XY females (except in the above patient) and Y-positive XX males, but absent from our series of Y-negative XX males.
r
> 1000). Two patients had not responded to sodium antimony gluconate 20 mg/kg daily for 20 days. The other three had not DETAILS OF PATIENTS WITH VISCERAL LEISHMANIASIS AND RESPONSE TO KETOCONAZOLE
*Premously treated with sodium antimony gluconate
