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ALPHA-FETOPROTEIN IN ANENCEPHALY
716 few have the generalised systemic manifestations of polymyalgia rheumatica. This lack of widespread systemic disturbance may be related to our findings of normal serum-alkaline-phosphatase associated with positive tem-
poral-artery biopsy. Department of Ophthalmology, Washington University, 660 South Euclid Avenue, St. Louis, Missouri 63110.
KENNETH M. BUERK MORTON E. SMITH.
MURAMIDASE AND ACUTE REJECTION OF KIDNEY GRAFTS
SIR,-Dr. Leemhuis and his colleagues (Aug. 19, p. 387) discuss their
own results and those of others with lysozyme (muramidase) determination as a test for early diagnosis of acute rejection in human kidney allotransplantation. A similar report was also published lately by Harrison et al.l Since the amount of any substance excreted by the
kidney originates from a variable number of nephrons, urinary excretion should always be related to this numberi.e., to glomerular filtration-rate (G.F.R.). This is especially important in kidney transplantation, where the G.F.R. may change substantially from one day to another. The first explanation for the discrepancies between the results from different laboratories may be that excretion of lysozyme is usually expressed as the absolute urinary level, which is largely determined by the degree of urinary concentration, instead of as the lysozyme/creatinine clearance ratio (C1ys/CCr).2 In a sequential study in our laboratory of the serum level and urinary excretion of several proteins, including lysozyme, in ten patients receiving a kidney allograft,3 two of eleven rejection crises seemingly were Harrison, J. F., Barnes, A. D., Blainey, I. D. Transplantation, 1972, 13, 372. 2. Johansson, B. G., Ravnskov, U. Scand. J. Urol. Nephrol. (in the press). 3. Ravnskov, U. Unpublished. 4. Johansson, B. G., Malmquist, J. Scand. J. clin. Lab. Invest. 1971, 27, 255. 1.
heralded by an increasing urinary level of lysozyme (and other proteins). When CtyJCc, was calculated, however, it appeared that the excretion of lysozyme per nephron decreased in proportion to the steadily increasing G.F.R. The rising urinary level of lysozyme simply reflected the production of a highly concentrated urine (see accompany-
ing figure). Early after kidney transplantation a hyperosmolar urine is often produced, whether or not a rejection crisis follows, resulting in an increased level of any substance in the urine. If no consideration is given to actual renal function, this may erroneously be taken to herald a rejection crisis. Another cause of the varying results in evaluation of urinary lysozyme may be that excretion of lysozyme is inversely correlated to G.F.R. in all kinds of kidney diseases2 and after a kidney transplantation.3 Only by correcting for both G.F.R. and for the degree of urinary concentration can one know what really happens in the nephron. If this step is omitted we will hear about many new worthless for early detection of rejection crisis in the future.
tests
Medical Department B (Renal Unit),
University Hospital, Lund, Sweden.
UFFE RAVNSKOV.
ALPHA-FETOPROTEIN IN ANENCEPHALY SIR,-Iwas interested to read that Dr. Brock and Mr. Sutcliffe had detected rx-fetoprotein (A.F.P.) in human amniotic-fluid samples (July 29, p. 197). This protein is produced in the fetal liver from early in gestation. It reaches its maximum blood-level in the 13 weeks’ fetus and then declines gradually until term.5 Smith et aI.,6 using the Ouchterlony technique of double diffusion, examined 5 samples of amniotic fluid from fetuses of 24-40 weeks’ gestation, and were unable to detect the presence of A.F.P., but Brock and Sutcliffe used the more sensitive immunoelectrophoretic technique and showed the presence of A.F.P. in amniotic fluid throughout pregnancy. Furthermore, they showed that the amniotic fluid of one 16-weekold fetus which had spina bifida and several anencephalic fetuses between 26 and 40 weeks of gestation had markedly raised levels of A.F.P., and pointed to the diagnostic usefulness of this finding. I, too, have been examining samples of the supernates from amniotic fluids from fetuses of different ages for the presence of A.F.P., using the simple double-diffusion method. A 1% solution of ’Ionagar’ no. 2 (Oxoid) was made in phosphate-buffered saline, pH 7-3. A rabbit antiserum, monospecific to A.F.P., was used, and fetal cord serum served as a positive control. The plates were left overnight in a moist chamber at room temperature, and then examined for lines of precipitation. A.F.P. was found in 16 specimens, which had come from fetuses aged from 10 to 19 weeks’ gestation, but was not detectable in samples from fetuses of 1921- to 34 weeks of gestation. One other negative result came from a sample from a 5-week-old fetus. The double-diffusion method detects A.F.P. down to approximately the 10 g. per ml. level, and, though relatively insensitive compared with other methods, would undoubtedly detect the high A.F.P. levels of anencephalic fetuses. It has the advantages of being rapid, cheap, and easy.
However, since amniocentesis is
a rather laborious risk to the fetus, it may be both more expedient and more prudent to concentrate on looking for raised A.F.P. in the serum of women bearing anencephalic fetuses, for A.F.P. is detectable in normal human serum by a radio-immunoassayand has
procedure
Urine output, creatinine clearance, urinary lysozyme, Clys/CCr, and urinary creatinine in a patient who received a kidney
allograft
on
0.
was determined by radial immunodiffusion.’ It is detectable in normal urine with this technique.
Lysozyme not
day
5. 6. 7.
and
involves
a
Gitlin, D., Boesman, M. J. clin. Invest. 1966, 45, 1826. Smith, J. A., Francis, T. I., Edington, G. M., Williams, A. O. Br. J. Cancer, 1971, 25, 337. Purves, L. R., Geddes, E. W. Lancet, 1972, i, 47.
717 been shown to be raised in the bearing normal fetuses.8
serum
of pregnant women
I thank Miss Susan Blunt for supplying specimens of amniotic fluid. This work was supported by the Spastics Society. Pædiatric Research Unit, Guy’s Hospital Medical School, London SE1 9RT.
MARY J. SELLER.
CEREBRAL BLOOD-FLOW SiR,—Studies have been published in these columns on cerebral blood-flow in man using xenon-133 inhalation 9,10 or injection via the superficial temporal artery.ll These approaches, while commendably atraumatic, suffer from the severe limitation that extracranial tissues also receive isotope. The inhalation method gives erroneously low flow estimates of only about 60% of those of the brain, the magnitude of this error bespeaking the importance of the extracerebral contamination. In situations where bloodflow may change independently in brain and in extracerebral tissues, erroneous conclusions may be reached if the contamination is disregarded. This seems to be the case regarding the rather sensational finding of failure of hypocapnic cerebral vasoconstriction in subjects with sympathetic denervation caused by spinal-cord injury or an
a-adrenolytic drug.9,lo We have studied the problem with the internal carotid 133Xe injection method, which is essentially free from extracerebral contamination except in the supraorbital region where the tissues in the orbit and above the medial 8. 9.
10. 11. 12.
Seppälä, M., Ruoslahti, E. ibid. p. 375. Eidelman, B. H., Corbett, J. L., Debarge, O., Frankel, H. Lancet, Sept. 2, 1972, p. 457. Corbett, J. L., Eidelman, B. H., Debarge, O. ibid. p. 461. Wilkins, D. G., Griffith, H. B., Cummins, B. H., Greenbaum, R., Zorab, J. S. M. ibid. Aug. 26, 1972, p. 402. Olesen, J., Paulson, O. B., Lassen, N. A. Stroke, 1971, 2, 519.
Preserved vasoconstrictor response of rC.B.F. (initial slope value) to hypocapnia during alpha-blockade by 20 mg. phentolamine intravenously. Interrupted line in left panel is the normal response. 11
half of the brow are labelled viathe ophthalmic artery. As the accompanying figure shows, hypocapnic cerebral vasoconstriction is preserved to a completely normal degree after 20 mg. phentolamine given intravenously 10 minutes prior to the study. Only in the supraorbital region, where massive extracerebral contamination is present, no change in blood-flow is evident during hypocapnia combined with phentolamine. Thus, in these contaminated channels we reproduce the results referred to above. These findings support the conclusion by many investigators that extracerebral contamination tends to invalidate methods based on the administration of 133Xe by inhalation or by common-carotid-artery injection (as via the superficial
temporal artery). Departments of Neurology and Clinical
Physiology, Bispebjerg Hospital, Copenhagen, Denmark.
E. SKINHØJ N. A. LASSEN.
CLINICAL ASSESSMENT OF POSTOPERATIVE BLOOD VOLUME and his co-workers (Sept. 2, p. 446) Irvin SiR,—Mr. found that a pulse-rate of over 100 per minute and cold extremities, although inconstantly present, were the most reliable signs in the clinical diagnosis of hypovolaemia. They recommend routine preoperative blood-volume in patients undergoing extensive elective that postoperative measurements may be better evaluated. Perhaps for reasons of brevity they did not mention the work of Ibsen,l the usefulness of which has been confirmed by others.22 Ibsen found an indirect way of assessing hypovolaemia which, because it can be used continuously, can detect the onset of hypovolaemia early. Since it is simple to apply and non-invasive it may be used routinely, and since the method utilises differential temperature monitoring it has the advantage of fulfilling recommendations that all ansesthetised patients should have their temperatures monitored to detect malignant hyperpyrexia. The two signs Irvin et al. found useful can both be measured. The coldness of an extremity is a subjective sensation. An extremity may be warm and, as the patient moves towards hypovolaemic shock, it may change to lukewarm. If the observer waits until it is cold, recognition of hypovolaemia may be delayed. Changes are difficult to notice if they are gradual. If the skin temperature is measured serially changes are more easily detected. Ibsen monitored the core or rectal temperature as well as the skin (big-toe) temperature, and was able to show an increase in the gradient between the two after blood-loss. If he gave chlorpromazine intravenously he was able to reduce the temperature gradient at the expense of a drop in the blood-pressure. The importance of a functionally satisfactory blood volume, which may differ from the calculated or the preoperative blood volumes, is that it permits adequate tissue perfusion. Vasoconstriction may maintain an adequate pressure but may upset tissue perfusion. For the treatment of non-cardiogenic shock Ibsen advocates " open-up " with chlorpromazine where there is vasoconstriction, fill up and stop when the feet get warm ". Brock3 has reported a case of postoperative hypovolmmia complicated by heat-retention hyperpyrexia with the appearances of irreversible brain damage. In his patient, changes in the temperature gradient occurred before those in the pulse, blood-pressure, and central venous pressure. Correction of the hypovoleemia with blood-transfusion and of the vasoconstriction by intravenous chlorpromazine
measurement
surgery,
1. 2. 3.
so
Ibsen, B. Dis. Chest, 1967, 52, 425. Ross, B. A., Brock, Aynsley-Green, A. Br. J. Surg. 1969, 56, 877. Spitzer, A. G., Brock. Guy’s Hosp. Rep. 1968, 117, 131.
poral-artery biopsy. Department of Ophthalmology, Washington University, 660 South Euclid Avenue, St. Louis, Missouri 63110.
KENNETH M. BUERK MORTON E. SMITH.
MURAMIDASE AND ACUTE REJECTION OF KIDNEY GRAFTS
SIR,-Dr. Leemhuis and his colleagues (Aug. 19, p. 387) discuss their
own results and those of others with lysozyme (muramidase) determination as a test for early diagnosis of acute rejection in human kidney allotransplantation. A similar report was also published lately by Harrison et al.l Since the amount of any substance excreted by the
kidney originates from a variable number of nephrons, urinary excretion should always be related to this numberi.e., to glomerular filtration-rate (G.F.R.). This is especially important in kidney transplantation, where the G.F.R. may change substantially from one day to another. The first explanation for the discrepancies between the results from different laboratories may be that excretion of lysozyme is usually expressed as the absolute urinary level, which is largely determined by the degree of urinary concentration, instead of as the lysozyme/creatinine clearance ratio (C1ys/CCr).2 In a sequential study in our laboratory of the serum level and urinary excretion of several proteins, including lysozyme, in ten patients receiving a kidney allograft,3 two of eleven rejection crises seemingly were Harrison, J. F., Barnes, A. D., Blainey, I. D. Transplantation, 1972, 13, 372. 2. Johansson, B. G., Ravnskov, U. Scand. J. Urol. Nephrol. (in the press). 3. Ravnskov, U. Unpublished. 4. Johansson, B. G., Malmquist, J. Scand. J. clin. Lab. Invest. 1971, 27, 255. 1.
heralded by an increasing urinary level of lysozyme (and other proteins). When CtyJCc, was calculated, however, it appeared that the excretion of lysozyme per nephron decreased in proportion to the steadily increasing G.F.R. The rising urinary level of lysozyme simply reflected the production of a highly concentrated urine (see accompany-
ing figure). Early after kidney transplantation a hyperosmolar urine is often produced, whether or not a rejection crisis follows, resulting in an increased level of any substance in the urine. If no consideration is given to actual renal function, this may erroneously be taken to herald a rejection crisis. Another cause of the varying results in evaluation of urinary lysozyme may be that excretion of lysozyme is inversely correlated to G.F.R. in all kinds of kidney diseases2 and after a kidney transplantation.3 Only by correcting for both G.F.R. and for the degree of urinary concentration can one know what really happens in the nephron. If this step is omitted we will hear about many new worthless for early detection of rejection crisis in the future.
tests
Medical Department B (Renal Unit),
University Hospital, Lund, Sweden.
UFFE RAVNSKOV.
ALPHA-FETOPROTEIN IN ANENCEPHALY SIR,-Iwas interested to read that Dr. Brock and Mr. Sutcliffe had detected rx-fetoprotein (A.F.P.) in human amniotic-fluid samples (July 29, p. 197). This protein is produced in the fetal liver from early in gestation. It reaches its maximum blood-level in the 13 weeks’ fetus and then declines gradually until term.5 Smith et aI.,6 using the Ouchterlony technique of double diffusion, examined 5 samples of amniotic fluid from fetuses of 24-40 weeks’ gestation, and were unable to detect the presence of A.F.P., but Brock and Sutcliffe used the more sensitive immunoelectrophoretic technique and showed the presence of A.F.P. in amniotic fluid throughout pregnancy. Furthermore, they showed that the amniotic fluid of one 16-weekold fetus which had spina bifida and several anencephalic fetuses between 26 and 40 weeks of gestation had markedly raised levels of A.F.P., and pointed to the diagnostic usefulness of this finding. I, too, have been examining samples of the supernates from amniotic fluids from fetuses of different ages for the presence of A.F.P., using the simple double-diffusion method. A 1% solution of ’Ionagar’ no. 2 (Oxoid) was made in phosphate-buffered saline, pH 7-3. A rabbit antiserum, monospecific to A.F.P., was used, and fetal cord serum served as a positive control. The plates were left overnight in a moist chamber at room temperature, and then examined for lines of precipitation. A.F.P. was found in 16 specimens, which had come from fetuses aged from 10 to 19 weeks’ gestation, but was not detectable in samples from fetuses of 1921- to 34 weeks of gestation. One other negative result came from a sample from a 5-week-old fetus. The double-diffusion method detects A.F.P. down to approximately the 10 g. per ml. level, and, though relatively insensitive compared with other methods, would undoubtedly detect the high A.F.P. levels of anencephalic fetuses. It has the advantages of being rapid, cheap, and easy.
However, since amniocentesis is
a rather laborious risk to the fetus, it may be both more expedient and more prudent to concentrate on looking for raised A.F.P. in the serum of women bearing anencephalic fetuses, for A.F.P. is detectable in normal human serum by a radio-immunoassayand has
procedure
Urine output, creatinine clearance, urinary lysozyme, Clys/CCr, and urinary creatinine in a patient who received a kidney
allograft
on
0.
was determined by radial immunodiffusion.’ It is detectable in normal urine with this technique.
Lysozyme not
day
5. 6. 7.
and
involves
a
Gitlin, D., Boesman, M. J. clin. Invest. 1966, 45, 1826. Smith, J. A., Francis, T. I., Edington, G. M., Williams, A. O. Br. J. Cancer, 1971, 25, 337. Purves, L. R., Geddes, E. W. Lancet, 1972, i, 47.
717 been shown to be raised in the bearing normal fetuses.8
serum
of pregnant women
I thank Miss Susan Blunt for supplying specimens of amniotic fluid. This work was supported by the Spastics Society. Pædiatric Research Unit, Guy’s Hospital Medical School, London SE1 9RT.
MARY J. SELLER.
CEREBRAL BLOOD-FLOW SiR,—Studies have been published in these columns on cerebral blood-flow in man using xenon-133 inhalation 9,10 or injection via the superficial temporal artery.ll These approaches, while commendably atraumatic, suffer from the severe limitation that extracranial tissues also receive isotope. The inhalation method gives erroneously low flow estimates of only about 60% of those of the brain, the magnitude of this error bespeaking the importance of the extracerebral contamination. In situations where bloodflow may change independently in brain and in extracerebral tissues, erroneous conclusions may be reached if the contamination is disregarded. This seems to be the case regarding the rather sensational finding of failure of hypocapnic cerebral vasoconstriction in subjects with sympathetic denervation caused by spinal-cord injury or an
a-adrenolytic drug.9,lo We have studied the problem with the internal carotid 133Xe injection method, which is essentially free from extracerebral contamination except in the supraorbital region where the tissues in the orbit and above the medial 8. 9.
10. 11. 12.
Seppälä, M., Ruoslahti, E. ibid. p. 375. Eidelman, B. H., Corbett, J. L., Debarge, O., Frankel, H. Lancet, Sept. 2, 1972, p. 457. Corbett, J. L., Eidelman, B. H., Debarge, O. ibid. p. 461. Wilkins, D. G., Griffith, H. B., Cummins, B. H., Greenbaum, R., Zorab, J. S. M. ibid. Aug. 26, 1972, p. 402. Olesen, J., Paulson, O. B., Lassen, N. A. Stroke, 1971, 2, 519.
Preserved vasoconstrictor response of rC.B.F. (initial slope value) to hypocapnia during alpha-blockade by 20 mg. phentolamine intravenously. Interrupted line in left panel is the normal response. 11
half of the brow are labelled viathe ophthalmic artery. As the accompanying figure shows, hypocapnic cerebral vasoconstriction is preserved to a completely normal degree after 20 mg. phentolamine given intravenously 10 minutes prior to the study. Only in the supraorbital region, where massive extracerebral contamination is present, no change in blood-flow is evident during hypocapnia combined with phentolamine. Thus, in these contaminated channels we reproduce the results referred to above. These findings support the conclusion by many investigators that extracerebral contamination tends to invalidate methods based on the administration of 133Xe by inhalation or by common-carotid-artery injection (as via the superficial
temporal artery). Departments of Neurology and Clinical
Physiology, Bispebjerg Hospital, Copenhagen, Denmark.
E. SKINHØJ N. A. LASSEN.
CLINICAL ASSESSMENT OF POSTOPERATIVE BLOOD VOLUME and his co-workers (Sept. 2, p. 446) Irvin SiR,—Mr. found that a pulse-rate of over 100 per minute and cold extremities, although inconstantly present, were the most reliable signs in the clinical diagnosis of hypovolaemia. They recommend routine preoperative blood-volume in patients undergoing extensive elective that postoperative measurements may be better evaluated. Perhaps for reasons of brevity they did not mention the work of Ibsen,l the usefulness of which has been confirmed by others.22 Ibsen found an indirect way of assessing hypovolaemia which, because it can be used continuously, can detect the onset of hypovolaemia early. Since it is simple to apply and non-invasive it may be used routinely, and since the method utilises differential temperature monitoring it has the advantage of fulfilling recommendations that all ansesthetised patients should have their temperatures monitored to detect malignant hyperpyrexia. The two signs Irvin et al. found useful can both be measured. The coldness of an extremity is a subjective sensation. An extremity may be warm and, as the patient moves towards hypovolaemic shock, it may change to lukewarm. If the observer waits until it is cold, recognition of hypovolaemia may be delayed. Changes are difficult to notice if they are gradual. If the skin temperature is measured serially changes are more easily detected. Ibsen monitored the core or rectal temperature as well as the skin (big-toe) temperature, and was able to show an increase in the gradient between the two after blood-loss. If he gave chlorpromazine intravenously he was able to reduce the temperature gradient at the expense of a drop in the blood-pressure. The importance of a functionally satisfactory blood volume, which may differ from the calculated or the preoperative blood volumes, is that it permits adequate tissue perfusion. Vasoconstriction may maintain an adequate pressure but may upset tissue perfusion. For the treatment of non-cardiogenic shock Ibsen advocates " open-up " with chlorpromazine where there is vasoconstriction, fill up and stop when the feet get warm ". Brock3 has reported a case of postoperative hypovolmmia complicated by heat-retention hyperpyrexia with the appearances of irreversible brain damage. In his patient, changes in the temperature gradient occurred before those in the pulse, blood-pressure, and central venous pressure. Correction of the hypovoleemia with blood-transfusion and of the vasoconstriction by intravenous chlorpromazine
measurement
surgery,
1. 2. 3.
so
Ibsen, B. Dis. Chest, 1967, 52, 425. Ross, B. A., Brock, Aynsley-Green, A. Br. J. Surg. 1969, 56, 877. Spitzer, A. G., Brock. Guy’s Hosp. Rep. 1968, 117, 131.