- Email: [email protected]
pH DURING EXCHANGE TRANSFUSION
950
Letters
to
the Editor
decade has
PURPOSES OF MEDICINE
happy arrangement to give your readers the opportunity to study Sir Theodore Fox’s splendid Harveian oration (Oct. 23), with its provocative and humane theme. Yet he makes one questionable proposal, which is bound to be disquieting to a surgeon: that " every patient in hospital should ... be looked after by a general physician ". This would specifically include, for example, a patient " having a neurosurgical operation ". It is indeed right for patients to be cared for, as he suggests, by a fairly large group-a ministry of all the talents ". It is indeed ideal if the final responsibility lies with " an extremely good doctor who is also a good person ". But many of us cannot accept the logic of the further assertion that this supreme role should invariably be filled by a general physician. As most doctors know, there is often a dearth of physicians whose scope is truly general; and are they in fact as a class much more likely to be extremely good " doctors than other specialists ? It would be a sad setback if surgeons (to take the obvious example), who nowadays must progressively develop skill in highly specialised techniques, were for this reason judged unlikely to become, or to remain, " good doctors ". A great surgical teacher of thirty years ago, Sir David Wilkie, insisted that his pupils should seek to become " physicians who can operate "-a designation he superbly justified himself. In a world of growing technical mastery, must the attributes of compassion, insight, humanity, and clinical balance that are so rightly demanded be declared to lie outside the modern surgeon’s range ? Let us hope not. Let us hope that the power (if we ever achieve it) to perform new miracles will never insidiously make concerned us persons (in Sir Theodore’s forbidding phrase) with things rather than people ". SIR,-It
was a
"
"
lessened the frequency of complaints by patients (perhaps more in larger hospitals than smaller) of failure to keep them informed, and of the arbitrary way they are treated. I asked, in 1955: Would it be worth appointing a general practitioner to each large hospital whose sole and very much full-time function would be to act as a medical liaison and information officer, and who would always be available to patients, relatives, and general practitioners in the district..." BENJAMIN LEE. London, S.W.I. not
"
...
JUNIOR MEDICAL STAFF SIR,-The expense of educating doctors is met by the State or by the students’ parents according to their means. Many medical students on qualification appear to be down at heel " and financially very embarrassed; and they then spend two periods of six months each as preregistration houseofficers. Many of the medical schools do nothing to help them to obtain such posts. Consequently they may be unemployed before, or between, holding such house-appointments, and if they decide to specialise they are probably unemployed between hospital posts if they decide to accept only those they regard as suitable. Under the National Health Service the State has a monopoly on all doctors unless they emigrate, take up private practice outside the service, or become part-time consultants. It seems therefore that the State should be responsible for all young doctors and their families after they qualify, and that they should be paid a reasonable basic salary whether they are holding junior hospital appointments or waiting for such PAYING HOSPITAL
"
suitable emplovment. Whittington Hospital, London, N.19.
T. ST. M. NORRIS.
"
Queen Victoria Hospital, East Grinstead, Sussex.
C. R. MCLAUGHLIN.
SIR,-While agreeing wholeheartedly with most of what Sir Theodore Fox says many will part company with him when he suggests that in hospital practice " the final decision on what is desirable for the patient should not, in general, rest with the doctor who decides what is feasible ". Is not the specialist’s knowledge of the discomforts and dangers involved just as relevant to a decision as his knowledge of what is feasible ? Who can weigh risk against benefit better than the doctor who has made a special study of both ? A colleague may suggest to him what he should do. A doctor who knows the patient better than he does may give invaluable information and advice. And, in practice, when the problem is difficult mutual discussion will usually quickly settle the matter. But the ultimate responsibility for seeing that a special treatment is in the patient’s best interests must surely lie with the doctor who has the greatest experience of what is involved and has followed up the largest number of patients treated in this way. If a doctor is truly " concerned with people rather than with things " before he takes up his clinical specialty, he will continue to be so afterwards. Ignorance of scientific methods and specialist techniques cannot make him more humane. Knowledge of them will not make him less so. Department of Radiotherapy, Royal Infirmary, T. B. BREWIN Glasgow, C.4.
SIR,-It is reassuring to find one’s temerarious suggestions echoed by so distinguished an advocate as Sir Theodore Fox, but I hope that his proposal that single doctors (who " in the smaller hospitals... could sometimes be general " practitioners ") could act in hospitals as medical guardian and patient’s friend " will not fall on the stony ground that my similar suggestion did ten years ago. The intervening 1. Lee, B. Lancet, 1955, i, 1129. ...
pH DURING EXCHANGE TRANSFUSION SIR,-Dr. Dunn’s instructive letter last week described an danger of exchange transfusion from the passage of large amounts of donor blood through the foramen ovale. In additional
the few biochemical estimations we have made of aortic blood during transfusion the Pc02 was about 35-40 mm. Hg, and the oxygen saturation 95-100%, when using acid-citrate-dextrose blood with PC02 130 mm. Hg and oxygen saturation 67%. Dr. Dunn rightly stresses the possible interference with enzyme action at the cellular or molecular level from transfusion with donor blood, which has a pH of about 6-5. This risk can be lessened by slow transfusion (100 ml. in 30 minutes) or the use of sodium bicarbonate during transfusion in doses of 1 mEq. per 100 ml. donor blood. We consider that when there is a high metabolic acidosis at birth, with the baby’s buffering mechanism already strained, sodium-bicarbonate therapy should be used at the commencement of transfusion. The remarkable recovery and subsequent improvement we have noted in such cases may in part be due to the protein-binding and elimination of bilirubin by bicarbonate which enhance the transfusion.2 With acid-base monitoring of transfusions any later alkalosis which may occur is slight, and can be compared with the overswing to high metabolic alkalosis after rapid transfusions (which cause profound metabolic acidosis). I further suggest that estimation of the pH alone may be misleading, since acid-base neutrality may be achieved in the presence of significant metabolic acidosis by the continuing compensation of respiratory alkalosis-an effect which has the further disadvantage of depleting the muscle-glucose.3 In one case (no. 6) I have recorded jointly 4 a pH of 7-334 concealed metabolic acidosis, with base deficit of 8-6 mEq. per litre and respiratory alkalosis of Pco2 30.5 mm. Hg. Because full acidbase estimations can be made with the Astrup microapparatus 1. 2. 3. 4.
Barrie, H. Lancet, 1964, ii, 476. Boda, D., Toth, G., Eck, E., Murányi, L. ibid. 1965, i, 164. Shelley, H. J. Br. med. J. 1964, i, 273. MacRae, D. J., Palavradji, D. J. Obstet. Gynæc. Br. Commonw. 1965, 72, 384.
951 a few minutes, they should be made routinely in all babies before transfusion and at intervals during the operation, especially if sodium-bicarbonate therapy is used. D. J. MACRAE. London, W. 1.
in
RESPONSE OF LYMPHOCYTES TO TETRACYCLINE of the cultured human peripheral-blood variety of stimulants is well known. Stimulation results in alterations in. appearance, in enlargement, and ultimately in mitosis. In the premitotic phase of stimulation, there is synthesis of ribonucleic acid and deoxyribonucleic acid, and ofy-globulin 12-at least a portion of which behaves like specific antibody.34 Virtually all human lymphocytes tested have been shown to react to certain non-specific stimuli such
SIR,-The ability lymphocyte to react
to a
phytohaemagglutinin (p .H.A.), streptolysin S,staphylococcal exotoxins,7 and anti-leucocyte antibodies.Lymphocytes from as
sensitised individuals have been shown to respond to tuberculin purified protein derivative (P.P.D.) 9; typhoid, poliomyelitis, smallpox, pertussis, and diphtheria vaccines210; tetanus toxoid; penicillin G 2; and Kveim antigen.ll Cells or cell extracts from genetically dissimilar individuals are capable of stimulating lymphocyte cultures.2 In addition, responses have been RESPONSE OF CELLS OF PATIENT AND OF
12 OTHER
PERSONS TO
TETRACYLINE
Mean4:s.D. (for controls at 10-4 mg. tetracycline) -3-7 ±4.5%. * =Dose of 10-3 mg. (not included in calculation of mean).
reported to human skin extracts 12 and thyroglobulin3 in patients with eczema and Hashimoto’s thyroiditis, respectively -conditions which are believed to reflect an autoimmune reaction. We report here the first demonstration of lymphocyte
tetracycline, using cells from a man who demonstrated an adverse clinical reaction to the drug. The clinical aspects of this case have been reported in detail elsewhere.13 A 44-year-old building superintendent, with a history of pulmonary tuberculosis and bronchial asthma, developed a serum-sickness-like reaction in July, 1964, after a penicillin injection, following which he exhibited positive immediate skin tests to penicillin G and penicilloyl polylysine solutions. The presence of circulating anti-penicillin antibodies of the 7S and 19S types was demonstrated by haemagglutination techniques.
response to
1. 2. 3. 4. 5. 6.
7. 8. 9. 10. 11. 12.
Bach, F., Hirschhorn, K. Expl Cell Res. 1963, 32, 592. Hirschhorn, K., Bach, F., Kolodny, R. L., Firschein, I. L., Hashem, N. Science, N.Y. 1963, 142, 1185. Forbes, I. J. Lancet, 1965, i, 198. Elves, M. W., Roath, S., Taylor, G., Israëls, M. C. G. ibid. 1963, i, 1292. Nowell, P. C. Cancer Res. 1960, 20, 462. Hirschhorn, K., Schreibman, R. R., Verbo, S., Gruskin, R. Proc. natn. Acad. Sci. U.S.A. 1964, 52, 1151. Ling, N. R., Husband, E. M. Lancet, 1964, i, 363. Grasbeck, R., Nordman, C., de la Chapelle, A. ibid. 1963, ii, 385. Pearmain, G., Lycette, R. R., Fitzgerald, P. H. ibid. 1963, i, 637. Elves, M. W., Roath, S., Israëls, M. C. G. ibid. p. 806. Hirschhorn, K., Schreibman, R. R., Bach, F. H., Siltzbach, L. E. ibid. 1964, ii, 842. Hashem, N., Hirschhorn, K., Sedlis, E., Holt, L. E., Jr. ibid. 1963, ii,
269. 13. Pellner, M. J., Baer, R. L. J. Am. med. Ass. 1965, 192, 997.
In September, 1964, after taking two 250 mg. capsules of tetracycline hydrochloride, he had an anaphylactic reaction. 5 days later, a positive immediate skin test to tetracycline solution was elicited. Five healthy volunteers, one of whom had just completed an uneventful 7-day course of tetracycline therapy, gave no evidence of skin sensitivity to the test solution. Passive transfer tests using serum drawn at the time of the anaphylactic reaction were positive with penicillin and tetracycline. Passive transfer tests using serum drawn after the penicillin reaction, but before the administration of tetracycline, were positive only with penicillin. Lymphocyte cultures were prepared from the patient’s cells 3 months after his tetracycline reaction. We also studied the cells of twelve individuals not clinically allergic to tetracycline, seven of whom had recently taken the antibiotic. Lymphocyterich plasma was separated from heparinised peripheral venous blood by sedimentation. Polymorphonuclear leucocytes and monocytes were removed by incubation on glass, and the lymphocytes were washed three times. They were then cultured at 37°C in a medium consisting of Eagle’s minimal essential medium, modified for Spinner culture, to which 20% of foetal calf serum and 1 % of 200 mmolar I-glutamine had been added. Each culture tube contained 3 x 106 lymphocytes in
a final volume of 4 ml. To the test cultures we added 10-3 or 10-4 mg. of tetracycline (Squibb) dissolved in saline. Negative control cultures contained no extraneous materials; positive control cultures contained 0-1 ml. of phytohaemagglutinin M (Difco). Tubes were also prepared containing 100 units of penicillin G or 2.5 X 10-3 mg. of P.P.D. All cultures were harvested after 5 days’ incubation except for the P.H.A. cultures which were harvested after 3 days. 4 hours before harvesting, 0.1 ml. of a 0-5 cg. per ml. solution of vinblastine sulphate (’ Velban ’, Lilly) was added to each culture. The cells were fixed in a 3/1 mixture of methanol/acetic acid, air-dried on cover slips, stained with 0-5% acetic-orcein solution, and examined by phase-contrast microscopy. 1000 cells were counted from each culture tube, and the proportion of the cells showing enlargement or mitotic activity was taken as the proportion reacting. The proportion of spontaneously reacting cells in the negative control cultures from healthy individuals varied between 2% and 4%, and that of those from patients acutely ill with infections, malignancies, or allergic reactions was higher. One very ill patient with serum sickness had a negative control value of 43%, but when he was retested 4 days later, it had dropped to 10%. All subjects reacted to P.H.A. with enlargement or mitotic activity of up to 98 % of their lymphocytes. The persons sensitised to penicillin G or P.P.D. reacted well to these antigens, ‘whereas the other subjects did not react at all. The response of the thirteen subjects’ cells to tetracycline is given in the accompanying table. The percentage response tabulated is the percentage of cells reacting in the cultures to which the test materials had been added less the percentage of cells reacting in the negative control cultures. The responses of the patient’s lymphocytes to tetracycline were significantly different from those of control subjects. The cells of the other subjects sensitised to penicillin or P.P.D. did not cross-react with
tetracycline. The effect of varying the dosage of tetracycline was studied cells from the patient and from nine of the control group. In cultures containing 10-2 mg. of the antibiotic, many degenerating and dead cells were seen, and the total cell-count was greatly reduced. At a dose of 10-3 mg., similar though much smaller changes were observed. At 10"’ mg., the highest dose at which the patient’s cells consistently showed a significant difference from the mean of the non-allergic controls, there was virtually no cytological evidence of toxicity. The negative mean difference between the antigen and control values, however, possibly represented a subtle reflection of toxicity. Neu et al. found that tetracycline could completely inhibit P.H.A.induced mitoses in concentrations above 0-2 mg. per ml.11 Our observations lend further support to the concept that the human peripheral-blood lymphocyte is an immunologically 14. Neu, R., Aspillaga, M. J., Gardner, L. I. Nature, Lond. 1965, 205, 171. on
Letters
to
the Editor
decade has
PURPOSES OF MEDICINE
happy arrangement to give your readers the opportunity to study Sir Theodore Fox’s splendid Harveian oration (Oct. 23), with its provocative and humane theme. Yet he makes one questionable proposal, which is bound to be disquieting to a surgeon: that " every patient in hospital should ... be looked after by a general physician ". This would specifically include, for example, a patient " having a neurosurgical operation ". It is indeed right for patients to be cared for, as he suggests, by a fairly large group-a ministry of all the talents ". It is indeed ideal if the final responsibility lies with " an extremely good doctor who is also a good person ". But many of us cannot accept the logic of the further assertion that this supreme role should invariably be filled by a general physician. As most doctors know, there is often a dearth of physicians whose scope is truly general; and are they in fact as a class much more likely to be extremely good " doctors than other specialists ? It would be a sad setback if surgeons (to take the obvious example), who nowadays must progressively develop skill in highly specialised techniques, were for this reason judged unlikely to become, or to remain, " good doctors ". A great surgical teacher of thirty years ago, Sir David Wilkie, insisted that his pupils should seek to become " physicians who can operate "-a designation he superbly justified himself. In a world of growing technical mastery, must the attributes of compassion, insight, humanity, and clinical balance that are so rightly demanded be declared to lie outside the modern surgeon’s range ? Let us hope not. Let us hope that the power (if we ever achieve it) to perform new miracles will never insidiously make concerned us persons (in Sir Theodore’s forbidding phrase) with things rather than people ". SIR,-It
was a
"
"
lessened the frequency of complaints by patients (perhaps more in larger hospitals than smaller) of failure to keep them informed, and of the arbitrary way they are treated. I asked, in 1955: Would it be worth appointing a general practitioner to each large hospital whose sole and very much full-time function would be to act as a medical liaison and information officer, and who would always be available to patients, relatives, and general practitioners in the district..." BENJAMIN LEE. London, S.W.I. not
"
...
JUNIOR MEDICAL STAFF SIR,-The expense of educating doctors is met by the State or by the students’ parents according to their means. Many medical students on qualification appear to be down at heel " and financially very embarrassed; and they then spend two periods of six months each as preregistration houseofficers. Many of the medical schools do nothing to help them to obtain such posts. Consequently they may be unemployed before, or between, holding such house-appointments, and if they decide to specialise they are probably unemployed between hospital posts if they decide to accept only those they regard as suitable. Under the National Health Service the State has a monopoly on all doctors unless they emigrate, take up private practice outside the service, or become part-time consultants. It seems therefore that the State should be responsible for all young doctors and their families after they qualify, and that they should be paid a reasonable basic salary whether they are holding junior hospital appointments or waiting for such PAYING HOSPITAL
"
suitable emplovment. Whittington Hospital, London, N.19.
T. ST. M. NORRIS.
"
Queen Victoria Hospital, East Grinstead, Sussex.
C. R. MCLAUGHLIN.
SIR,-While agreeing wholeheartedly with most of what Sir Theodore Fox says many will part company with him when he suggests that in hospital practice " the final decision on what is desirable for the patient should not, in general, rest with the doctor who decides what is feasible ". Is not the specialist’s knowledge of the discomforts and dangers involved just as relevant to a decision as his knowledge of what is feasible ? Who can weigh risk against benefit better than the doctor who has made a special study of both ? A colleague may suggest to him what he should do. A doctor who knows the patient better than he does may give invaluable information and advice. And, in practice, when the problem is difficult mutual discussion will usually quickly settle the matter. But the ultimate responsibility for seeing that a special treatment is in the patient’s best interests must surely lie with the doctor who has the greatest experience of what is involved and has followed up the largest number of patients treated in this way. If a doctor is truly " concerned with people rather than with things " before he takes up his clinical specialty, he will continue to be so afterwards. Ignorance of scientific methods and specialist techniques cannot make him more humane. Knowledge of them will not make him less so. Department of Radiotherapy, Royal Infirmary, T. B. BREWIN Glasgow, C.4.
SIR,-It is reassuring to find one’s temerarious suggestions echoed by so distinguished an advocate as Sir Theodore Fox, but I hope that his proposal that single doctors (who " in the smaller hospitals... could sometimes be general " practitioners ") could act in hospitals as medical guardian and patient’s friend " will not fall on the stony ground that my similar suggestion did ten years ago. The intervening 1. Lee, B. Lancet, 1955, i, 1129. ...
pH DURING EXCHANGE TRANSFUSION SIR,-Dr. Dunn’s instructive letter last week described an danger of exchange transfusion from the passage of large amounts of donor blood through the foramen ovale. In additional
the few biochemical estimations we have made of aortic blood during transfusion the Pc02 was about 35-40 mm. Hg, and the oxygen saturation 95-100%, when using acid-citrate-dextrose blood with PC02 130 mm. Hg and oxygen saturation 67%. Dr. Dunn rightly stresses the possible interference with enzyme action at the cellular or molecular level from transfusion with donor blood, which has a pH of about 6-5. This risk can be lessened by slow transfusion (100 ml. in 30 minutes) or the use of sodium bicarbonate during transfusion in doses of 1 mEq. per 100 ml. donor blood. We consider that when there is a high metabolic acidosis at birth, with the baby’s buffering mechanism already strained, sodium-bicarbonate therapy should be used at the commencement of transfusion. The remarkable recovery and subsequent improvement we have noted in such cases may in part be due to the protein-binding and elimination of bilirubin by bicarbonate which enhance the transfusion.2 With acid-base monitoring of transfusions any later alkalosis which may occur is slight, and can be compared with the overswing to high metabolic alkalosis after rapid transfusions (which cause profound metabolic acidosis). I further suggest that estimation of the pH alone may be misleading, since acid-base neutrality may be achieved in the presence of significant metabolic acidosis by the continuing compensation of respiratory alkalosis-an effect which has the further disadvantage of depleting the muscle-glucose.3 In one case (no. 6) I have recorded jointly 4 a pH of 7-334 concealed metabolic acidosis, with base deficit of 8-6 mEq. per litre and respiratory alkalosis of Pco2 30.5 mm. Hg. Because full acidbase estimations can be made with the Astrup microapparatus 1. 2. 3. 4.
Barrie, H. Lancet, 1964, ii, 476. Boda, D., Toth, G., Eck, E., Murányi, L. ibid. 1965, i, 164. Shelley, H. J. Br. med. J. 1964, i, 273. MacRae, D. J., Palavradji, D. J. Obstet. Gynæc. Br. Commonw. 1965, 72, 384.
951 a few minutes, they should be made routinely in all babies before transfusion and at intervals during the operation, especially if sodium-bicarbonate therapy is used. D. J. MACRAE. London, W. 1.
in
RESPONSE OF LYMPHOCYTES TO TETRACYCLINE of the cultured human peripheral-blood variety of stimulants is well known. Stimulation results in alterations in. appearance, in enlargement, and ultimately in mitosis. In the premitotic phase of stimulation, there is synthesis of ribonucleic acid and deoxyribonucleic acid, and ofy-globulin 12-at least a portion of which behaves like specific antibody.34 Virtually all human lymphocytes tested have been shown to react to certain non-specific stimuli such
SIR,-The ability lymphocyte to react
to a
phytohaemagglutinin (p .H.A.), streptolysin S,staphylococcal exotoxins,7 and anti-leucocyte antibodies.Lymphocytes from as
sensitised individuals have been shown to respond to tuberculin purified protein derivative (P.P.D.) 9; typhoid, poliomyelitis, smallpox, pertussis, and diphtheria vaccines210; tetanus toxoid; penicillin G 2; and Kveim antigen.ll Cells or cell extracts from genetically dissimilar individuals are capable of stimulating lymphocyte cultures.2 In addition, responses have been RESPONSE OF CELLS OF PATIENT AND OF
12 OTHER
PERSONS TO
TETRACYLINE
Mean4:s.D. (for controls at 10-4 mg. tetracycline) -3-7 ±4.5%. * =Dose of 10-3 mg. (not included in calculation of mean).
reported to human skin extracts 12 and thyroglobulin3 in patients with eczema and Hashimoto’s thyroiditis, respectively -conditions which are believed to reflect an autoimmune reaction. We report here the first demonstration of lymphocyte
tetracycline, using cells from a man who demonstrated an adverse clinical reaction to the drug. The clinical aspects of this case have been reported in detail elsewhere.13 A 44-year-old building superintendent, with a history of pulmonary tuberculosis and bronchial asthma, developed a serum-sickness-like reaction in July, 1964, after a penicillin injection, following which he exhibited positive immediate skin tests to penicillin G and penicilloyl polylysine solutions. The presence of circulating anti-penicillin antibodies of the 7S and 19S types was demonstrated by haemagglutination techniques.
response to
1. 2. 3. 4. 5. 6.
7. 8. 9. 10. 11. 12.
Bach, F., Hirschhorn, K. Expl Cell Res. 1963, 32, 592. Hirschhorn, K., Bach, F., Kolodny, R. L., Firschein, I. L., Hashem, N. Science, N.Y. 1963, 142, 1185. Forbes, I. J. Lancet, 1965, i, 198. Elves, M. W., Roath, S., Taylor, G., Israëls, M. C. G. ibid. 1963, i, 1292. Nowell, P. C. Cancer Res. 1960, 20, 462. Hirschhorn, K., Schreibman, R. R., Verbo, S., Gruskin, R. Proc. natn. Acad. Sci. U.S.A. 1964, 52, 1151. Ling, N. R., Husband, E. M. Lancet, 1964, i, 363. Grasbeck, R., Nordman, C., de la Chapelle, A. ibid. 1963, ii, 385. Pearmain, G., Lycette, R. R., Fitzgerald, P. H. ibid. 1963, i, 637. Elves, M. W., Roath, S., Israëls, M. C. G. ibid. p. 806. Hirschhorn, K., Schreibman, R. R., Bach, F. H., Siltzbach, L. E. ibid. 1964, ii, 842. Hashem, N., Hirschhorn, K., Sedlis, E., Holt, L. E., Jr. ibid. 1963, ii,
269. 13. Pellner, M. J., Baer, R. L. J. Am. med. Ass. 1965, 192, 997.
In September, 1964, after taking two 250 mg. capsules of tetracycline hydrochloride, he had an anaphylactic reaction. 5 days later, a positive immediate skin test to tetracycline solution was elicited. Five healthy volunteers, one of whom had just completed an uneventful 7-day course of tetracycline therapy, gave no evidence of skin sensitivity to the test solution. Passive transfer tests using serum drawn at the time of the anaphylactic reaction were positive with penicillin and tetracycline. Passive transfer tests using serum drawn after the penicillin reaction, but before the administration of tetracycline, were positive only with penicillin. Lymphocyte cultures were prepared from the patient’s cells 3 months after his tetracycline reaction. We also studied the cells of twelve individuals not clinically allergic to tetracycline, seven of whom had recently taken the antibiotic. Lymphocyterich plasma was separated from heparinised peripheral venous blood by sedimentation. Polymorphonuclear leucocytes and monocytes were removed by incubation on glass, and the lymphocytes were washed three times. They were then cultured at 37°C in a medium consisting of Eagle’s minimal essential medium, modified for Spinner culture, to which 20% of foetal calf serum and 1 % of 200 mmolar I-glutamine had been added. Each culture tube contained 3 x 106 lymphocytes in
a final volume of 4 ml. To the test cultures we added 10-3 or 10-4 mg. of tetracycline (Squibb) dissolved in saline. Negative control cultures contained no extraneous materials; positive control cultures contained 0-1 ml. of phytohaemagglutinin M (Difco). Tubes were also prepared containing 100 units of penicillin G or 2.5 X 10-3 mg. of P.P.D. All cultures were harvested after 5 days’ incubation except for the P.H.A. cultures which were harvested after 3 days. 4 hours before harvesting, 0.1 ml. of a 0-5 cg. per ml. solution of vinblastine sulphate (’ Velban ’, Lilly) was added to each culture. The cells were fixed in a 3/1 mixture of methanol/acetic acid, air-dried on cover slips, stained with 0-5% acetic-orcein solution, and examined by phase-contrast microscopy. 1000 cells were counted from each culture tube, and the proportion of the cells showing enlargement or mitotic activity was taken as the proportion reacting. The proportion of spontaneously reacting cells in the negative control cultures from healthy individuals varied between 2% and 4%, and that of those from patients acutely ill with infections, malignancies, or allergic reactions was higher. One very ill patient with serum sickness had a negative control value of 43%, but when he was retested 4 days later, it had dropped to 10%. All subjects reacted to P.H.A. with enlargement or mitotic activity of up to 98 % of their lymphocytes. The persons sensitised to penicillin G or P.P.D. reacted well to these antigens, ‘whereas the other subjects did not react at all. The response of the thirteen subjects’ cells to tetracycline is given in the accompanying table. The percentage response tabulated is the percentage of cells reacting in the cultures to which the test materials had been added less the percentage of cells reacting in the negative control cultures. The responses of the patient’s lymphocytes to tetracycline were significantly different from those of control subjects. The cells of the other subjects sensitised to penicillin or P.P.D. did not cross-react with
tetracycline. The effect of varying the dosage of tetracycline was studied cells from the patient and from nine of the control group. In cultures containing 10-2 mg. of the antibiotic, many degenerating and dead cells were seen, and the total cell-count was greatly reduced. At a dose of 10-3 mg., similar though much smaller changes were observed. At 10"’ mg., the highest dose at which the patient’s cells consistently showed a significant difference from the mean of the non-allergic controls, there was virtually no cytological evidence of toxicity. The negative mean difference between the antigen and control values, however, possibly represented a subtle reflection of toxicity. Neu et al. found that tetracycline could completely inhibit P.H.A.induced mitoses in concentrations above 0-2 mg. per ml.11 Our observations lend further support to the concept that the human peripheral-blood lymphocyte is an immunologically 14. Neu, R., Aspillaga, M. J., Gardner, L. I. Nature, Lond. 1965, 205, 171. on