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WARMING THE COOLED KIDNEY BEFORE TRANSPLANTATION
1194 It is unfortunate that Dr. Rochat does not seem to have read the Tongan report carefully. During the teaching of the method, weekly supervision was provided, but thereafter the women were seen only on request, and even then after an interval of some months, when a further visit was possible to the island they inhabited. The ovulation method offers the bonus of an enormous saving in medical manpower because it can be taught by women themselves to one another, and once a woman has learned it she can use it for the remainder of her reproductive life without further supervision. Professor Marshall (Nov. 11, p. 1027) requests further documented evidence in regard to one of the five items defining the superiority of the ovulation method over the temperature method and this will be provided. In the early study to which he refers1 there was not only the experience of a rise in basal body temperature many days ahead of ovulation but also the item that one woman failed to produce an intelligible temperature record at all. 141 Grey Street, East Melbourne 3002, Australia.
J. J.
BILLINGS.
WARMING THE COOLED KIDNEY BEFORE TRANSPLANTATION
SIR,-The interesting account2 of impaired function of transplanted kidney after cold storage reflects the lack of general principles underlying many techniques of kidney preservation. In a reassessment3 of the causes of early anuria following transplantation, I drew attention to the possible role of MN incompatibility, because even when a kidney is removed from a live donor the temperature of a kidney falls steeply, especially if one places it in a bowl of cold saline for transport between operating-theatres. Although I was advised that cold antibodies would not constitute a problem after cold storage, I remained uneasy about their possible role in early post-transplant anuria. It seems, however, that the early impaired function of some kidneys 2,4 may reasonably be attributed to the saline flush-out. Originally I used saline to flush out kidneys before transplantation,5 but frequently encountered impaired function. The late Dr. L. E. Bayliss was appalled when I related this experience and counselled me never to repeat this procedure, which had been shown to be harmful since the days of Starling. Storey et al. were very lucky to get away with only a week of anuria after a a
flush-out with saline. Bravo to the technique of warming the cooled kidney before releasing the new circulation. It is, however, rather late in the day to be advocating this technique, since it has been part of my procedure for several years. 6,7 A rapid warm flush-out with a mixture ofRheomacrodex ’ and plasma seemed to me to provide a safe routine means of blocking the effects of any possible cold antibody reactions, and at the same time ensuring that the renal peripheral resistance is reduced as much as possible before the new circulation is released. The rewarming process must be rapid and involve a small volume of flush-out solution7 in order not to prolong operating time. A pink kidney merely indicates that the capsular vessels are well filled,6 but does not necessarily reflect the state Billings, E. L., Billings, J. J., Brown, J. B., Burger, H. G. Lancet, 1972, i, 282. 2. Storey, B. G., Raik, E., Stewart, J. H. ibid. Oct. 21, 1972, p. 873. 3. Dempster, W. J. Br. med. J. 1963, i, 1697. 4. Belzer, F. O., Kountz, S. L., Perkins, H. A. Transplantation, 1971, 11, 422. 5. Dempster, W. J. Br. J. Surg. 1953, 40, 447. 6. Dempster, W. J., Kountz, S. L., Jakanovic, M. Br. med. J. 1964, 1.
i, 407. 7.
Aboul-Enein, A., Paccione, F., Todd, I. ster, W. J. Experientia, 1965, 21, 546.
A.
D., Shikata, T., Demp-
of the intrarenal circulation, as Storey et al. must now realise. If routine renal arteriography were performed after establishing the new circulation it would be possible to assess the relation of immediate post-transplant oliguria The beneficial effect of to inadequate cortical perfusion. in in the outer cortex8 promoting perfusion prewarming could also be assessed. Department of Surgery, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 0HS.
W. J. DEMPSTER.
RAPID ASSAY OF GENTAMICIN
SiR,—In the past 12 months we have noted a widespread interest in and application of our rapid urease assay for aminoglycoside antibiotics,9 and we ourselves have performed over 500 assays on sera from patients being treated with gentamicin. The sum total of this experience is that the method described remains a reliable and simple technique, which has greatly facilitated the control of gentamicin therapy. Other workers have drawn our attention to two minor disadvantages of the technique originally described-namely, its requirement for 6 ml. of serum if duplicates are performed and its relative inaccuracy at concentrations of gentamicin in the test serum of less than 2 ptg. per ml., in particular when the blood-urea is greater than 500 mg. per 100 ml.10 Our recent work has indicated how to overcome these disadvantages. Irregularities due to very high blood-urea concentrations can be overcome simply by using 4% urea broth throughout. In order to reduce the volume of test serum required, further attempts have been made to devise an assay based on a straight comparison of the inhibitory effect of a test serum with a standard curve. They have been uniformly unsuccessful and the x versus x/2 convention must be retained. However, our experience, including repeated cross-checking of the results with those obtained with a conventional 18-hour agar diffusion assay, indicates that there is no need to perform duplicates, thus immediately halving the requirement for serum to 3 ml. The volume of serum required can be further halved by adding to 3 ml. of 4% urea in urea broth base (’Oxoid’ CM71), 0-2 ml. gentamicin standards (40, 20, 10, 5, and 2-5 g. per ml.) in O1M phosphate buffer pH 8-0 and 0-2 ml. of test serum or 0-1 ml. test serum plus 0-1 ml. pooled human serum to the x or x/2 tubes, respectively. Using as an inoculum 0-1 ml. of an overnight broth culture of Proteus mirabilis BS 711 in 0-5% glucose broth, the reaction mixture is incubated at 37 °C for the appropriate time, the pH of the tubes read, and the result derived as previously described. The optimum incubation time varies according to the gentamicin concentration of the test serum, but is on average 75-90 min. in this system and is shown as the time when the phenol-red indicator has turned bright pink in the tubes with the lowest concentration of gentamicin but has remained unchanged in the tubes containing the highest concentration. This modification reduces the requirement for serum to 1-5 ml., which we have found to be universally available from adults, whilst preserving the simplicity of the technique by utilising commercially available medium and routine pipetting procedures (using
an
Eppendorff-type pipette).
Further modifications are based on the approximately 10-fold increase in sensitivity of Proteus mirabilis to gentamicin when salt is omitted from the base medium of the urea hydrolysing system described (unpublished observations). In order to measure accurately concentrations less than 2 veg. per ml. the procedure described above is only modified in that the base medium is 4% urea in 0°O1M phosphate buffer pH 7-0 plus 0-1% peptone and 0-0004% phenol red, and the gentamicin standards have concentrations of 5, 2-5, 1-25, 0-625, and 0-31 )jLg. per ml. For padiatric use this salt-free base medium is used in 5 ml. aliquots and gentamicin standards again have concentrations of 40, 20, 10, 5, and 2-5 !J.g. per ml. However, only 0-05 ml. of standard is added together with 0-05 ml. test serum or 0-025 ml. test 8. Dempster, W. J. Br. J. exp. Path. 1971, 52, 172. 9. Lancet, 1971, i, 16. 10. Froud, D. J. ibid. 1971, ii, 1425.
J. J.
BILLINGS.
WARMING THE COOLED KIDNEY BEFORE TRANSPLANTATION
SIR,-The interesting account2 of impaired function of transplanted kidney after cold storage reflects the lack of general principles underlying many techniques of kidney preservation. In a reassessment3 of the causes of early anuria following transplantation, I drew attention to the possible role of MN incompatibility, because even when a kidney is removed from a live donor the temperature of a kidney falls steeply, especially if one places it in a bowl of cold saline for transport between operating-theatres. Although I was advised that cold antibodies would not constitute a problem after cold storage, I remained uneasy about their possible role in early post-transplant anuria. It seems, however, that the early impaired function of some kidneys 2,4 may reasonably be attributed to the saline flush-out. Originally I used saline to flush out kidneys before transplantation,5 but frequently encountered impaired function. The late Dr. L. E. Bayliss was appalled when I related this experience and counselled me never to repeat this procedure, which had been shown to be harmful since the days of Starling. Storey et al. were very lucky to get away with only a week of anuria after a a
flush-out with saline. Bravo to the technique of warming the cooled kidney before releasing the new circulation. It is, however, rather late in the day to be advocating this technique, since it has been part of my procedure for several years. 6,7 A rapid warm flush-out with a mixture ofRheomacrodex ’ and plasma seemed to me to provide a safe routine means of blocking the effects of any possible cold antibody reactions, and at the same time ensuring that the renal peripheral resistance is reduced as much as possible before the new circulation is released. The rewarming process must be rapid and involve a small volume of flush-out solution7 in order not to prolong operating time. A pink kidney merely indicates that the capsular vessels are well filled,6 but does not necessarily reflect the state Billings, E. L., Billings, J. J., Brown, J. B., Burger, H. G. Lancet, 1972, i, 282. 2. Storey, B. G., Raik, E., Stewart, J. H. ibid. Oct. 21, 1972, p. 873. 3. Dempster, W. J. Br. med. J. 1963, i, 1697. 4. Belzer, F. O., Kountz, S. L., Perkins, H. A. Transplantation, 1971, 11, 422. 5. Dempster, W. J. Br. J. Surg. 1953, 40, 447. 6. Dempster, W. J., Kountz, S. L., Jakanovic, M. Br. med. J. 1964, 1.
i, 407. 7.
Aboul-Enein, A., Paccione, F., Todd, I. ster, W. J. Experientia, 1965, 21, 546.
A.
D., Shikata, T., Demp-
of the intrarenal circulation, as Storey et al. must now realise. If routine renal arteriography were performed after establishing the new circulation it would be possible to assess the relation of immediate post-transplant oliguria The beneficial effect of to inadequate cortical perfusion. in in the outer cortex8 promoting perfusion prewarming could also be assessed. Department of Surgery, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 0HS.
W. J. DEMPSTER.
RAPID ASSAY OF GENTAMICIN
SiR,—In the past 12 months we have noted a widespread interest in and application of our rapid urease assay for aminoglycoside antibiotics,9 and we ourselves have performed over 500 assays on sera from patients being treated with gentamicin. The sum total of this experience is that the method described remains a reliable and simple technique, which has greatly facilitated the control of gentamicin therapy. Other workers have drawn our attention to two minor disadvantages of the technique originally described-namely, its requirement for 6 ml. of serum if duplicates are performed and its relative inaccuracy at concentrations of gentamicin in the test serum of less than 2 ptg. per ml., in particular when the blood-urea is greater than 500 mg. per 100 ml.10 Our recent work has indicated how to overcome these disadvantages. Irregularities due to very high blood-urea concentrations can be overcome simply by using 4% urea broth throughout. In order to reduce the volume of test serum required, further attempts have been made to devise an assay based on a straight comparison of the inhibitory effect of a test serum with a standard curve. They have been uniformly unsuccessful and the x versus x/2 convention must be retained. However, our experience, including repeated cross-checking of the results with those obtained with a conventional 18-hour agar diffusion assay, indicates that there is no need to perform duplicates, thus immediately halving the requirement for serum to 3 ml. The volume of serum required can be further halved by adding to 3 ml. of 4% urea in urea broth base (’Oxoid’ CM71), 0-2 ml. gentamicin standards (40, 20, 10, 5, and 2-5 g. per ml.) in O1M phosphate buffer pH 8-0 and 0-2 ml. of test serum or 0-1 ml. test serum plus 0-1 ml. pooled human serum to the x or x/2 tubes, respectively. Using as an inoculum 0-1 ml. of an overnight broth culture of Proteus mirabilis BS 711 in 0-5% glucose broth, the reaction mixture is incubated at 37 °C for the appropriate time, the pH of the tubes read, and the result derived as previously described. The optimum incubation time varies according to the gentamicin concentration of the test serum, but is on average 75-90 min. in this system and is shown as the time when the phenol-red indicator has turned bright pink in the tubes with the lowest concentration of gentamicin but has remained unchanged in the tubes containing the highest concentration. This modification reduces the requirement for serum to 1-5 ml., which we have found to be universally available from adults, whilst preserving the simplicity of the technique by utilising commercially available medium and routine pipetting procedures (using
an
Eppendorff-type pipette).
Further modifications are based on the approximately 10-fold increase in sensitivity of Proteus mirabilis to gentamicin when salt is omitted from the base medium of the urea hydrolysing system described (unpublished observations). In order to measure accurately concentrations less than 2 veg. per ml. the procedure described above is only modified in that the base medium is 4% urea in 0°O1M phosphate buffer pH 7-0 plus 0-1% peptone and 0-0004% phenol red, and the gentamicin standards have concentrations of 5, 2-5, 1-25, 0-625, and 0-31 )jLg. per ml. For padiatric use this salt-free base medium is used in 5 ml. aliquots and gentamicin standards again have concentrations of 40, 20, 10, 5, and 2-5 !J.g. per ml. However, only 0-05 ml. of standard is added together with 0-05 ml. test serum or 0-025 ml. test 8. Dempster, W. J. Br. J. exp. Path. 1971, 52, 172. 9. Lancet, 1971, i, 16. 10. Froud, D. J. ibid. 1971, ii, 1425.