- Email: [email protected]
HORMONE ASSAYS ON BREAST-TUMOUR
678 more active clinically than those with negative tests for D.N.A. precipitation. These results will be published in detail elsewhere. It appears that the antibody demonstrated by this precipitation method may not differ radically from that detected by other tests, such as the Farr ammoniumsulphate method. The idea that anti-D.N.A. antibody affinity may be an important factor in determining the site of localisation of D.N.A./anti-D.N.A. complexes in larger vessels or in glomeruli is an attractive one. However, it cannot be concluded from our data that the D.N.A. precipitation method described detects antibody of differing affinity from the Farr method.
judged
Department of Medicine, Guy’s Hospital, London SE1 9RT. 1.
Johnson, ii, 883.
G. D.,
C. SANDERSON B. D. WILLIAMS J. S. CAMERON.
Edmonds, J. P., Holborow, E. J. Lancet, 1973,
COMBINED-IMMUNODEFICIENCY DISEASE WITH NORMAL ERYTHROCYTE A.D.A. SIR,-Pollara and Meuwissen1 have reviewed 13 cases of erythrocyte-adenosine-deaminase (A.D.A.) deficiencies associated with combined-immunodeficiency diseases. A further case2 has been discussed in which erythrocyte A.D.A. is absent, yet the patient is reported to be in good health with apparently intact T and B cell responses. Because of the rarity of combined immunodeficiency, it is difficult to establish how many patients with this disease have this enzyme defect. We have seen a patient with combined immunodeficiency whose erythrocytes demonstrate a normal adenosine-deaminase zymogram. The patient has already been described as a case of combined immune deficiency.33 She had a severe impairment of T-cell function, as manifested by the absence of a 48-hour delayed skin response to a battery of antigens and by an inability to become sensitised with 1-chloro-2,4dinitrobenzene (D.N.C.B.). In addition to being lymphopenic, her lymphocytes responded with a raised tritiatedthymidine uptake when placed in tissue-culture with phytohaemagglutinin (P.H.A.) and pokeweed. Response to Candida albicans antigen was inconstant. Despite evidence of a proliferative response to P.H.A., and at times to the Candida antigen, five determinations over a period of 1 years failed to reveal the presence of the lymphokine M.I.F. in response to an antigenic challenge. severe
In addition to impairment in T-cell function, the patient demonstrated defective tetanus-toxoid antibody in response to an antigenic challenge and remained Schick-test positive despite repeated D.P.T. injections. She also had deficient antibody responses to repeated immunisation with paratyphoid and typhoid antigens, as well as a low serum-IgA level. She also had autoantibodies against smooth muscle and parietal cells and had an inconstant positive antilymphocytotoxic assay. She has never received bloodtransfusions. A severe extensive mucocutaneous candidiasis has been controlled with intermittent intravenous amphotericin B. Adenosine deaminase was demonstrated in the patient’s erythrocyte lysate after electrophoresis in agarose, using the staining method described by Spencer et al.4 Erythrocytes were obtained from the patient on two occasions. They were washed in phosphate-buffered saline, packed, and disrupted by freezing and thawing. Several dilutions of the erythrocyte lysate were subjected to electrophoresis and stained for A.D.A. Three A.D.A.-active bands were seen. The staining reaction was shown to be specific for adenosine deaminase, since no banding was observed in the absence of the adenosine substrate. The lymphocyte population in which this child may be deficient could conceivably be a T-cell subpopulation which produces the lymphokine M.I.F. in response to an antigenic challenge. The proliferative capabilities of her lymphocytes in response to P.H.A., pokeweed, and at times the Candida albicans antigen seems grossly intact. This brief report indicates that A.D.A. activity in erythrocytes was present in a patient with combined-immunodeficiency disease, whose T cells could mount a proliferative response but could not respond with lymphokine-M.I.F.
production
to an
antigenic challenge.
of
Departments Microbiology and Dermatology, Clinical Research Center, 462 Grider Street, State University of New York at Buffalo, Buffalo, New York 14215, U.S.A.
WILLIAM R. BARTHOLOMEW THOMAS T. PROVOST.
1. Pollara, B., Meuwissen, H. J. Lancet, 1973, ii, 1324. 2. Jenkins, T. ibid. p. 736. 3. Provost, T., Garrettson, L., Zeschke, R., Rose, N. R., Tomasi, T. B. Clin. Immunol. Immunopath. 1973, 1, 429. 4. Spencer, N., Hopkinson, D. A., Harris, H. Ann. hum. Genet. 1968, 32, 9.
HORMONE ASSAYS ON BREAST-TUMOUR
SIR,-Riley et a1.1 have described the effects of androgens, progesterones, and aestrogen on tissue cultures of scirrhous breast cancer and fibroadenomas. In their experiment they utilised 10-5M concentrations of each hormone, whereas the normal physiological concentrations of these are as follows: testosterone, 10-9M; progesterone, 10-8M; and oestrogen, 10-9M. Decreased D.N.A. synthesis in oestrogen-sensitive tissues is to be expected in the presence of testosterone and progesterone, as these hormones are capable of inducing metabolic changes blocking the stimulating effect of oestrogen.2 In a study reported from our institution,3 some breast tumours responded to oestrogen by an increased growthrate while others failed to respond in any fashion. Growth was measured by numbers of colonies, colony size, and thymidine uptake using a standard inoculation. However, Three adenosine-deaminase bands in different dilutions of the
patient’s erythrocyte lysate. Well no. 1 contains a 1/16 dilution, no. 2 a 1,’8 dilution, no. 3 a 1/4 dilution, and no. 4 a 1/2 dilution. The arrow indicates the point of origin with respect to each well.
when the oestradiol concentration reached 20 !g. per ml. (10-4M), all cultures exhibited decreased growth and evidence of toxicity. 1 !Jog. per ml. oestrogen was the highest concentration observed to produce stimulation in those tumours which were oestrogen-sensitive. Therefore, decreased D.N.A. synthesis in the presence of
679
high concentrations is not an indication of hormonal therapy in the same sense as sensitivity oestrogen-binding studies, as reported by Jensen et awl. and Maas et a1.5 to whom Riley et al. refer. The presence of oestrogen-binding receptors implies an ability of cells to respond to oestrogen by increased growth. cestrogens in
to
It should also be noted that fetal calf serum has an oestrogen concentration of > 1000 pg. per ml., or 10-9M, and cannot be considered an oestrogen-poor medium.8 Further evidence of oestrogen toxicity in Riley’s experiment was the failure of cancer cells to reach growth confluence, with most dying out after 25 days. Some of our ductal-cell carcinomas grew in continuous culture for more than ten months, using fetal calf serum without any additional hormones, either steroidal or of the polypeptide type. Department of Medicine, Department of Surgery,
University of Oklahoma College of Medicine. Oklahoma 73190, U.S.A. Cancer Section, Oklahoma Medical Research Foundation.
ARTHUR F. HOGE.
JAMES M. HARTSUCK. ROBERT E.
probability
as
associated with
NORDQUIST. Centre for
1. 2. 3. 4.
5. 6.
Riley, P. A., Latter, T., Sutton, P. M. Lancet, 1973, ii, 818. Korenman, S. G. Endocrinology, 1970, 87, 1119. Hoge, A. F., Hartsuck, J. M., Kollmorgen, G. M., Schilling, J. Am. J. Surg. 1973, 126, 722. Jensen, E. V., Block, G. E., Smith, S., Kyser, K., DeSombre, E. R. Estrogen Target Tissues and Neoplasia; p. 23. Chicago, 1972. Maas, H., Engel, B., Hohmeister, H., Lehmann, F., Trams, G. Am. J. Obstet. Gynec. 1972, 113, 377. Kling, R. Personal communication.
CONTAMINATED INTRAVENOUS INFUSIONS SIR,-The report by Dr Meers and his colleagues1 is a valuable addition to the understanding of sepsis associated with intravenous-fluid contamination. However, we must disagree with theirrassertion that lot numbers need not be recorded on individual patient records. Since these unique identifying numbers are essential in the investigation of a possible contamination episode, recording lot numbers of fluid and additives being given to a patient at the time of onset of symptoms of sepsis is extremely important. If only hospital distribution data are available the ability to confirm microbiological contamination is
jeopardised. Four factors combine to make the recording of lot numbers necessary. First, hospitals in the United States may have 10 or more different batches of a single formulation stocked at any one time. Second, since many hospitals do not effectively rotate stock, any batch of the suspect product delivered to the hospital before onset of the episode of sepsis under investigation might be implicated. Third, one cannot rely on turbidity of fluid or other characteristic physical appearance of the bottle or closure to identify contaminated units, although those are helpful clues. We have tested contaminated infusion bottles that were not apparently different from uncontaminated. Finally, although up to one-third of the bottles examined by Meers et al. were shown to be contaminated, much lower frequencies of contamination have occurred in 3 outbreaks investigated in the U.S.I-8 Large numbers of suspect fluids must be sampled to be reasonably certain that contamination at low frequency does not exist. A hypothetical example illustrates the difficulties in evaluating possible contamination when only hospital distribution data are available. For each lot implicated in a contamination episode, 300 units of that lot must be sampled to exclude 1 % contamination with 95 % certainty. If 10 lots of a single formulation are implicated with equal
a
suspect septic reaction,
3000 units must be located and sampled, a herculean task. On the other hand, if a specific suspect lot number is adequately identified, all sampling resources can be focused upon that lot. Careful notation of all medications given to a patient is recognised as a necessary part of keeping medical records. It would be easier to include lot numbers of intravenous fluids in this record if the manufacturers would provide detachable gum labels that show the type of solution and lot number. This label could be pasted in the patient’s chart, much as blood units are recorded today. Whether or not hospital personnel record the lot numbers of all intravenous solutions distributed to every patient (some U.S. hospitals are now routinely recording this information), a convenient method of recording lot numbers of intravenous products received by patients with suspect septic reactions is important, since these data have been a useful and sometimes essential part of the successful investigation of potential commercial contamination episodes in the U.S. Disease Control, Atlanta, Georgia 30333, U.S.A. 1. 2. 3. 4. 5. 6. 7. 8. 9.
JAMES H. TENNEY RICHARD ERWIN DIXON JOHN V. BENNETT.
Meers, P. D., Calder, M. W., Mazhar, M. M., Lawrie, G. M. Lancet, 1973, ii, 1189. C.D.C. Morbid. Mortal. Wkly Rep. 1971, 20, suppl. 9. ibid. 1971, 20, 91. ibid. p. 116. ibid. 1973, 22, 99. ibid. p. 115. ibid. p. 124. ibid. p. 265. ibid. p. 284.
INTELLIGENCE AFTER MALNUTRITION SIR,-We were very interested in the findings of Dr Valman (March 16, p. 425). We have investigated a similar group of patients (34 with cystic fibrosis and 7 with other neonatal intestinal disorders 1,2. Our data showed a significant difference in the performance on the Merrill-Palmer between the malnourished children compared with their two and five years after their malnutrition. However, no significant differences could be demonstrated after 5 years of age when the Wechsler intelligence scales for children and Wechsler adult intelligence scales were used. The mean intelligence quotient of 31 parents was 108 ±11-3. The mean social class of 27 fathers was 3 on a scale of 1 to 5. The average length of stay in test
sibling controls between
hospital
was
56
days.
The most significant abnormalities noted in the younger children on the Merrill-Palmer test were defects of fine motor function. These children are now being studied in a prospective protocol which may demonstrate whether these findings will ultimately return to normal. Moreover, how much they are related in the younger children to the effects of a long period in hospital and a chronic incurable disease remains conjectural. Certainly these findings from developed countries should lead to a more optimistic outlook for the ultimate intellectual outcome of these infants. Pennsylvania State University, Milton S. Hershey Medical Center,
Hershey, Pennsylvania 17033, U.S.A. JOHN D. LLOYD-STILL. Children’s Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115, U.S.A. HARRY SHWACHMAN. 1.
Lloyd-Still, J. D., Wolff, P. H., Hurwitz, I., Shwachman, H. Proceedings of 9th International Conference on Nutrition, Mexico City, September, 1972. Basle, 1974.
2.
Lloyd-Still, J. D., Hurwitz, I., Wolff, P. H., Shwachman, H. Society for Pediatric Research, San Francisco, May, 1973. Pediat. Res. 1973, 7, 65.
judged
Department of Medicine, Guy’s Hospital, London SE1 9RT. 1.
Johnson, ii, 883.
G. D.,
C. SANDERSON B. D. WILLIAMS J. S. CAMERON.
Edmonds, J. P., Holborow, E. J. Lancet, 1973,
COMBINED-IMMUNODEFICIENCY DISEASE WITH NORMAL ERYTHROCYTE A.D.A. SIR,-Pollara and Meuwissen1 have reviewed 13 cases of erythrocyte-adenosine-deaminase (A.D.A.) deficiencies associated with combined-immunodeficiency diseases. A further case2 has been discussed in which erythrocyte A.D.A. is absent, yet the patient is reported to be in good health with apparently intact T and B cell responses. Because of the rarity of combined immunodeficiency, it is difficult to establish how many patients with this disease have this enzyme defect. We have seen a patient with combined immunodeficiency whose erythrocytes demonstrate a normal adenosine-deaminase zymogram. The patient has already been described as a case of combined immune deficiency.33 She had a severe impairment of T-cell function, as manifested by the absence of a 48-hour delayed skin response to a battery of antigens and by an inability to become sensitised with 1-chloro-2,4dinitrobenzene (D.N.C.B.). In addition to being lymphopenic, her lymphocytes responded with a raised tritiatedthymidine uptake when placed in tissue-culture with phytohaemagglutinin (P.H.A.) and pokeweed. Response to Candida albicans antigen was inconstant. Despite evidence of a proliferative response to P.H.A., and at times to the Candida antigen, five determinations over a period of 1 years failed to reveal the presence of the lymphokine M.I.F. in response to an antigenic challenge. severe
In addition to impairment in T-cell function, the patient demonstrated defective tetanus-toxoid antibody in response to an antigenic challenge and remained Schick-test positive despite repeated D.P.T. injections. She also had deficient antibody responses to repeated immunisation with paratyphoid and typhoid antigens, as well as a low serum-IgA level. She also had autoantibodies against smooth muscle and parietal cells and had an inconstant positive antilymphocytotoxic assay. She has never received bloodtransfusions. A severe extensive mucocutaneous candidiasis has been controlled with intermittent intravenous amphotericin B. Adenosine deaminase was demonstrated in the patient’s erythrocyte lysate after electrophoresis in agarose, using the staining method described by Spencer et al.4 Erythrocytes were obtained from the patient on two occasions. They were washed in phosphate-buffered saline, packed, and disrupted by freezing and thawing. Several dilutions of the erythrocyte lysate were subjected to electrophoresis and stained for A.D.A. Three A.D.A.-active bands were seen. The staining reaction was shown to be specific for adenosine deaminase, since no banding was observed in the absence of the adenosine substrate. The lymphocyte population in which this child may be deficient could conceivably be a T-cell subpopulation which produces the lymphokine M.I.F. in response to an antigenic challenge. The proliferative capabilities of her lymphocytes in response to P.H.A., pokeweed, and at times the Candida albicans antigen seems grossly intact. This brief report indicates that A.D.A. activity in erythrocytes was present in a patient with combined-immunodeficiency disease, whose T cells could mount a proliferative response but could not respond with lymphokine-M.I.F.
production
to an
antigenic challenge.
of
Departments Microbiology and Dermatology, Clinical Research Center, 462 Grider Street, State University of New York at Buffalo, Buffalo, New York 14215, U.S.A.
WILLIAM R. BARTHOLOMEW THOMAS T. PROVOST.
1. Pollara, B., Meuwissen, H. J. Lancet, 1973, ii, 1324. 2. Jenkins, T. ibid. p. 736. 3. Provost, T., Garrettson, L., Zeschke, R., Rose, N. R., Tomasi, T. B. Clin. Immunol. Immunopath. 1973, 1, 429. 4. Spencer, N., Hopkinson, D. A., Harris, H. Ann. hum. Genet. 1968, 32, 9.
HORMONE ASSAYS ON BREAST-TUMOUR
SIR,-Riley et a1.1 have described the effects of androgens, progesterones, and aestrogen on tissue cultures of scirrhous breast cancer and fibroadenomas. In their experiment they utilised 10-5M concentrations of each hormone, whereas the normal physiological concentrations of these are as follows: testosterone, 10-9M; progesterone, 10-8M; and oestrogen, 10-9M. Decreased D.N.A. synthesis in oestrogen-sensitive tissues is to be expected in the presence of testosterone and progesterone, as these hormones are capable of inducing metabolic changes blocking the stimulating effect of oestrogen.2 In a study reported from our institution,3 some breast tumours responded to oestrogen by an increased growthrate while others failed to respond in any fashion. Growth was measured by numbers of colonies, colony size, and thymidine uptake using a standard inoculation. However, Three adenosine-deaminase bands in different dilutions of the
patient’s erythrocyte lysate. Well no. 1 contains a 1/16 dilution, no. 2 a 1,’8 dilution, no. 3 a 1/4 dilution, and no. 4 a 1/2 dilution. The arrow indicates the point of origin with respect to each well.
when the oestradiol concentration reached 20 !g. per ml. (10-4M), all cultures exhibited decreased growth and evidence of toxicity. 1 !Jog. per ml. oestrogen was the highest concentration observed to produce stimulation in those tumours which were oestrogen-sensitive. Therefore, decreased D.N.A. synthesis in the presence of
679
high concentrations is not an indication of hormonal therapy in the same sense as sensitivity oestrogen-binding studies, as reported by Jensen et awl. and Maas et a1.5 to whom Riley et al. refer. The presence of oestrogen-binding receptors implies an ability of cells to respond to oestrogen by increased growth. cestrogens in
to
It should also be noted that fetal calf serum has an oestrogen concentration of > 1000 pg. per ml., or 10-9M, and cannot be considered an oestrogen-poor medium.8 Further evidence of oestrogen toxicity in Riley’s experiment was the failure of cancer cells to reach growth confluence, with most dying out after 25 days. Some of our ductal-cell carcinomas grew in continuous culture for more than ten months, using fetal calf serum without any additional hormones, either steroidal or of the polypeptide type. Department of Medicine, Department of Surgery,
University of Oklahoma College of Medicine. Oklahoma 73190, U.S.A. Cancer Section, Oklahoma Medical Research Foundation.
ARTHUR F. HOGE.
JAMES M. HARTSUCK. ROBERT E.
probability
as
associated with
NORDQUIST. Centre for
1. 2. 3. 4.
5. 6.
Riley, P. A., Latter, T., Sutton, P. M. Lancet, 1973, ii, 818. Korenman, S. G. Endocrinology, 1970, 87, 1119. Hoge, A. F., Hartsuck, J. M., Kollmorgen, G. M., Schilling, J. Am. J. Surg. 1973, 126, 722. Jensen, E. V., Block, G. E., Smith, S., Kyser, K., DeSombre, E. R. Estrogen Target Tissues and Neoplasia; p. 23. Chicago, 1972. Maas, H., Engel, B., Hohmeister, H., Lehmann, F., Trams, G. Am. J. Obstet. Gynec. 1972, 113, 377. Kling, R. Personal communication.
CONTAMINATED INTRAVENOUS INFUSIONS SIR,-The report by Dr Meers and his colleagues1 is a valuable addition to the understanding of sepsis associated with intravenous-fluid contamination. However, we must disagree with theirrassertion that lot numbers need not be recorded on individual patient records. Since these unique identifying numbers are essential in the investigation of a possible contamination episode, recording lot numbers of fluid and additives being given to a patient at the time of onset of symptoms of sepsis is extremely important. If only hospital distribution data are available the ability to confirm microbiological contamination is
jeopardised. Four factors combine to make the recording of lot numbers necessary. First, hospitals in the United States may have 10 or more different batches of a single formulation stocked at any one time. Second, since many hospitals do not effectively rotate stock, any batch of the suspect product delivered to the hospital before onset of the episode of sepsis under investigation might be implicated. Third, one cannot rely on turbidity of fluid or other characteristic physical appearance of the bottle or closure to identify contaminated units, although those are helpful clues. We have tested contaminated infusion bottles that were not apparently different from uncontaminated. Finally, although up to one-third of the bottles examined by Meers et al. were shown to be contaminated, much lower frequencies of contamination have occurred in 3 outbreaks investigated in the U.S.I-8 Large numbers of suspect fluids must be sampled to be reasonably certain that contamination at low frequency does not exist. A hypothetical example illustrates the difficulties in evaluating possible contamination when only hospital distribution data are available. For each lot implicated in a contamination episode, 300 units of that lot must be sampled to exclude 1 % contamination with 95 % certainty. If 10 lots of a single formulation are implicated with equal
a
suspect septic reaction,
3000 units must be located and sampled, a herculean task. On the other hand, if a specific suspect lot number is adequately identified, all sampling resources can be focused upon that lot. Careful notation of all medications given to a patient is recognised as a necessary part of keeping medical records. It would be easier to include lot numbers of intravenous fluids in this record if the manufacturers would provide detachable gum labels that show the type of solution and lot number. This label could be pasted in the patient’s chart, much as blood units are recorded today. Whether or not hospital personnel record the lot numbers of all intravenous solutions distributed to every patient (some U.S. hospitals are now routinely recording this information), a convenient method of recording lot numbers of intravenous products received by patients with suspect septic reactions is important, since these data have been a useful and sometimes essential part of the successful investigation of potential commercial contamination episodes in the U.S. Disease Control, Atlanta, Georgia 30333, U.S.A. 1. 2. 3. 4. 5. 6. 7. 8. 9.
JAMES H. TENNEY RICHARD ERWIN DIXON JOHN V. BENNETT.
Meers, P. D., Calder, M. W., Mazhar, M. M., Lawrie, G. M. Lancet, 1973, ii, 1189. C.D.C. Morbid. Mortal. Wkly Rep. 1971, 20, suppl. 9. ibid. 1971, 20, 91. ibid. p. 116. ibid. 1973, 22, 99. ibid. p. 115. ibid. p. 124. ibid. p. 265. ibid. p. 284.
INTELLIGENCE AFTER MALNUTRITION SIR,-We were very interested in the findings of Dr Valman (March 16, p. 425). We have investigated a similar group of patients (34 with cystic fibrosis and 7 with other neonatal intestinal disorders 1,2. Our data showed a significant difference in the performance on the Merrill-Palmer between the malnourished children compared with their two and five years after their malnutrition. However, no significant differences could be demonstrated after 5 years of age when the Wechsler intelligence scales for children and Wechsler adult intelligence scales were used. The mean intelligence quotient of 31 parents was 108 ±11-3. The mean social class of 27 fathers was 3 on a scale of 1 to 5. The average length of stay in test
sibling controls between
hospital
was
56
days.
The most significant abnormalities noted in the younger children on the Merrill-Palmer test were defects of fine motor function. These children are now being studied in a prospective protocol which may demonstrate whether these findings will ultimately return to normal. Moreover, how much they are related in the younger children to the effects of a long period in hospital and a chronic incurable disease remains conjectural. Certainly these findings from developed countries should lead to a more optimistic outlook for the ultimate intellectual outcome of these infants. Pennsylvania State University, Milton S. Hershey Medical Center,
Hershey, Pennsylvania 17033, U.S.A. JOHN D. LLOYD-STILL. Children’s Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115, U.S.A. HARRY SHWACHMAN. 1.
Lloyd-Still, J. D., Wolff, P. H., Hurwitz, I., Shwachman, H. Proceedings of 9th International Conference on Nutrition, Mexico City, September, 1972. Basle, 1974.
2.
Lloyd-Still, J. D., Hurwitz, I., Wolff, P. H., Shwachman, H. Society for Pediatric Research, San Francisco, May, 1973. Pediat. Res. 1973, 7, 65.