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IDENTIFICATION OF MACROPHAGES IN AMNIOTIC-FLUID CELL CULTURES
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(e.g., rabbits) or in tissue cultures, and there is no direct evidence that E. coli considered to be enterotoxic or non-enterotoxic by these methods are indeed enterotoxic or non-enterotoxic for babies. Also the fact that an E. coli strain is enterotoxic does not mean that it is enteropathogenic-to be enteropathogenic a strain must also possess the ability to proliferate in the small intestine (this is especially relevant because enterotoxin production can be transmissible). There is ample evidence that these criticisms are indeed valid from the work that has been carried out on E. coli infections of farm animals. Houghton Poultry Research Station, Houghton, Huntingdon, Cambs. PE17 2DA
H. WILLIAMS SMITH
identical chromosomal polymorphisms between the mother and the cultured amniotic-fluid cells would then suggest the
possibility of maternal cell contamination. Alternatively, different polymorphic markers would indicate that the amnioticfluid cells were fetal in origin. Bloody amniotic-fluid specimens with numerous macrophages in the cultured cells are alarming for the cytogeneticist on several counts. Our letter was designed to demonstrate one way in which we attempt nal cell contamination. Department of Pediatrics, Division of Medical Genetics, Mount Sinai School of Medicine, New York, N.Y. 10029, U.S.A.
to
resolve
our concerns
about
mater-
LILLIAN Y. F. HSU KAREN DAVID ANDREA V. SEROTKIN LYNN GODMILOW
KURT HIRSCHHORN
IDENTIFICATION OF MACROPHAGES IN AMNIOTIC-FLUID CELL CULTURES
SIR,-Dr Sutherland (May 15, p. 1077) raises the issue of possible fetal neural-tube defect when increased numbers of macrophages are found in amniotic-fluid cell-cultures. Alphafetoprotein (A.F.P.) is measured routinely on all amniotic-fluid specimens received for prenatal diagnosis in our laboratory.If a
it had been increased in our case, a diagnosis of neural-tube defect would have been one of our considerations. We feel that routine A.F.P. assay should be done on all amniotic-fluid specimens received in a genetics centre laboratory and thus major open neural-tube defects would not be missed. In addition, the amniocentesis of our patient was monitored with sonography which demonstrated a normal fetal head size for the gestational age, thereby ruling out anencephaly. We emphasised in our letter (April 10, p. 799) that the amniotic fluid of the patient was grossly bloody and that many large adherent cells were found in the amniotic-fluid cell-culture. This led us to suspect the presence of an increased number of macrophages. Identification of macrophages by latex particle engulfment prompted us to obtain blood from the mother. Finding a female karyotype of the subcultured amniotic-fluid cells compelled us to compare the chromosome polymorphisms of the karyotypes of the amniotic-fluid cells with those of the mother. The two karyotypes carried identical Q-banding polymorphisms, raising the possibility that the cells cultured from the amniotic fluid were maternal in origin. To try to clarify such an issue, repeat amniocentesis should be recommended if time permits. At this juncture careful counselling must be offered to the family. We strongly feel that the suggestion of a repeat amniocentesis should only be made after the above-mentioned sequential observations and studies. We would never recommend a repeat tap just because there are a few macrophages in the culture. This patient has now delivered a phenotypically normal female. A peripheral leucocyte culture with phytohaemagglutinin showed a normal female karyotype, 46,XX. Q-banding showed identical chromosome polymorphisms to the mother and also to the cultured amniotic-fluid cells. In this instance Q-banding alone could not distinguish the maternal and fetal karyotypes. Apparently, additional banding studies such as C-, G, and N- banding might be necessary for further identification of individual chromosome polymorphisms. We are trying to study the father who has so far not been cooperative. All cytogeneticists doing prenatal diagnosis know that macrophages are a normal component of amniotic fluid. It is also known (especially to Dr Sutherland2) that very few macrophages are seen in normal amniotic-fluid cell-cultures. A finding of increased number of macrophages without elevation of A.F.P. from an initially bloody amniotic-fluid specimen presents the possibility of contamination by either maternal or fetal blood. If the karyotype of the cultured amniotic-fluid cells is 46,XX, a karyotype of the mother must be examined for differences in chromosomal polymorphism. Demonstration of 1. Ambender, E., Hirschhorn, K. Lancet, 1976, i, 597. 2. Sutherland, G. R., Bauld, R., Bain, A. D.J. med. Genet.
1974, 11, 190.
IMMUNOTHERAPY FOR COLORECTAL CANCER
Sip,—The management of advanced colorectal adenocarcinoma has proved to be extremely discouraging and, in common with several other groups, we are turning our attention to the role of adjuvant radiotherapy or chemotherapy in the patient with operable disease who is at a high risk of harbouring small-volume metastatic tumour. Work from this centre has clearly demonstrated, using an experimental tumour, that small-volume tumours are more sensitive to cytotoxic agents than bulkier tumours.’In addition to controlled clinical studies, we are examining adjuvant chemotherapy in colorectal xenografts growing in immunodeprived mice.2 As part of this programme, we are developing a micro-metastasis model of xenografted tumour. Thus, it was with much interest that we read the report by Mavligit et al .3 on the effect of B.C.G. and B.C.G. plus 5-fluorouracil in patients with Dukes C colorectal cancer. It is clear that a report claiming to demonstrate improved survival with adjuvant treatment is bound to exert a considerable influence on clinicians who, although perhaps not dealing exclusively with the treatment of malignant disease, nevertheless retain a considerable interest in measures which might improve the failures of standard management. For this reason, it is important that such claims are well founded and not based upon premature analyses of clinical data. In our view the conclusions of Mavligit et al. are impossible to sustain until more adequate follow-up time has elapsed for assessment. The first patient entered the study in April, 1973, and the maximum follow-up time was thirty months. This means that the analysis which forms the basis of the report was made in October, 1975, and since the study included patients entered in August, 1975, presumably some patients have been followed only for two months. The small number of patients, together with the very short observation time for an appreciable proportion of patients, means that long-term or even medium-term predictions must be made with extreme caution. The claim that B.C.G. alone appears to be highly effective in patients with six or more positive nodes is based on 10 patients, of whom at least half appear to have been at risk for less than one year. Furthermore, the choice of historical control group is unsatisfactory. It is noted, for example, in 6 patients there is a delay of up to five months before referral. These patients must presumably be disease-free in order to enter the trial. This inevitably means that there is a risk of selection. It is not clear whether the distribution of primary tumour sites is similar in the control and study patients, or whether both groups were comparable in terms of surgical approach, including lymph-node sampling. The potential pitfalls of using historical control have been pointed out. and even this premature report would have been more convinc1. Shipley, W. U., Stanley, J. A., Steel, G. G. Cancer Res. 1975, 35, 2488. 2. Nowak, K., Steel, G. G., Peckham, M. J. Unpublished. 3. Mavligit, G. M. et al. Lancet, April 24, 1976, p. 871. 4. Alderson, M. R. Introduction to Epidemiology; p. 171. London, 1976.
(e.g., rabbits) or in tissue cultures, and there is no direct evidence that E. coli considered to be enterotoxic or non-enterotoxic by these methods are indeed enterotoxic or non-enterotoxic for babies. Also the fact that an E. coli strain is enterotoxic does not mean that it is enteropathogenic-to be enteropathogenic a strain must also possess the ability to proliferate in the small intestine (this is especially relevant because enterotoxin production can be transmissible). There is ample evidence that these criticisms are indeed valid from the work that has been carried out on E. coli infections of farm animals. Houghton Poultry Research Station, Houghton, Huntingdon, Cambs. PE17 2DA
H. WILLIAMS SMITH
identical chromosomal polymorphisms between the mother and the cultured amniotic-fluid cells would then suggest the
possibility of maternal cell contamination. Alternatively, different polymorphic markers would indicate that the amnioticfluid cells were fetal in origin. Bloody amniotic-fluid specimens with numerous macrophages in the cultured cells are alarming for the cytogeneticist on several counts. Our letter was designed to demonstrate one way in which we attempt nal cell contamination. Department of Pediatrics, Division of Medical Genetics, Mount Sinai School of Medicine, New York, N.Y. 10029, U.S.A.
to
resolve
our concerns
about
mater-
LILLIAN Y. F. HSU KAREN DAVID ANDREA V. SEROTKIN LYNN GODMILOW
KURT HIRSCHHORN
IDENTIFICATION OF MACROPHAGES IN AMNIOTIC-FLUID CELL CULTURES
SIR,-Dr Sutherland (May 15, p. 1077) raises the issue of possible fetal neural-tube defect when increased numbers of macrophages are found in amniotic-fluid cell-cultures. Alphafetoprotein (A.F.P.) is measured routinely on all amniotic-fluid specimens received for prenatal diagnosis in our laboratory.If a
it had been increased in our case, a diagnosis of neural-tube defect would have been one of our considerations. We feel that routine A.F.P. assay should be done on all amniotic-fluid specimens received in a genetics centre laboratory and thus major open neural-tube defects would not be missed. In addition, the amniocentesis of our patient was monitored with sonography which demonstrated a normal fetal head size for the gestational age, thereby ruling out anencephaly. We emphasised in our letter (April 10, p. 799) that the amniotic fluid of the patient was grossly bloody and that many large adherent cells were found in the amniotic-fluid cell-culture. This led us to suspect the presence of an increased number of macrophages. Identification of macrophages by latex particle engulfment prompted us to obtain blood from the mother. Finding a female karyotype of the subcultured amniotic-fluid cells compelled us to compare the chromosome polymorphisms of the karyotypes of the amniotic-fluid cells with those of the mother. The two karyotypes carried identical Q-banding polymorphisms, raising the possibility that the cells cultured from the amniotic fluid were maternal in origin. To try to clarify such an issue, repeat amniocentesis should be recommended if time permits. At this juncture careful counselling must be offered to the family. We strongly feel that the suggestion of a repeat amniocentesis should only be made after the above-mentioned sequential observations and studies. We would never recommend a repeat tap just because there are a few macrophages in the culture. This patient has now delivered a phenotypically normal female. A peripheral leucocyte culture with phytohaemagglutinin showed a normal female karyotype, 46,XX. Q-banding showed identical chromosome polymorphisms to the mother and also to the cultured amniotic-fluid cells. In this instance Q-banding alone could not distinguish the maternal and fetal karyotypes. Apparently, additional banding studies such as C-, G, and N- banding might be necessary for further identification of individual chromosome polymorphisms. We are trying to study the father who has so far not been cooperative. All cytogeneticists doing prenatal diagnosis know that macrophages are a normal component of amniotic fluid. It is also known (especially to Dr Sutherland2) that very few macrophages are seen in normal amniotic-fluid cell-cultures. A finding of increased number of macrophages without elevation of A.F.P. from an initially bloody amniotic-fluid specimen presents the possibility of contamination by either maternal or fetal blood. If the karyotype of the cultured amniotic-fluid cells is 46,XX, a karyotype of the mother must be examined for differences in chromosomal polymorphism. Demonstration of 1. Ambender, E., Hirschhorn, K. Lancet, 1976, i, 597. 2. Sutherland, G. R., Bauld, R., Bain, A. D.J. med. Genet.
1974, 11, 190.
IMMUNOTHERAPY FOR COLORECTAL CANCER
Sip,—The management of advanced colorectal adenocarcinoma has proved to be extremely discouraging and, in common with several other groups, we are turning our attention to the role of adjuvant radiotherapy or chemotherapy in the patient with operable disease who is at a high risk of harbouring small-volume metastatic tumour. Work from this centre has clearly demonstrated, using an experimental tumour, that small-volume tumours are more sensitive to cytotoxic agents than bulkier tumours.’In addition to controlled clinical studies, we are examining adjuvant chemotherapy in colorectal xenografts growing in immunodeprived mice.2 As part of this programme, we are developing a micro-metastasis model of xenografted tumour. Thus, it was with much interest that we read the report by Mavligit et al .3 on the effect of B.C.G. and B.C.G. plus 5-fluorouracil in patients with Dukes C colorectal cancer. It is clear that a report claiming to demonstrate improved survival with adjuvant treatment is bound to exert a considerable influence on clinicians who, although perhaps not dealing exclusively with the treatment of malignant disease, nevertheless retain a considerable interest in measures which might improve the failures of standard management. For this reason, it is important that such claims are well founded and not based upon premature analyses of clinical data. In our view the conclusions of Mavligit et al. are impossible to sustain until more adequate follow-up time has elapsed for assessment. The first patient entered the study in April, 1973, and the maximum follow-up time was thirty months. This means that the analysis which forms the basis of the report was made in October, 1975, and since the study included patients entered in August, 1975, presumably some patients have been followed only for two months. The small number of patients, together with the very short observation time for an appreciable proportion of patients, means that long-term or even medium-term predictions must be made with extreme caution. The claim that B.C.G. alone appears to be highly effective in patients with six or more positive nodes is based on 10 patients, of whom at least half appear to have been at risk for less than one year. Furthermore, the choice of historical control group is unsatisfactory. It is noted, for example, in 6 patients there is a delay of up to five months before referral. These patients must presumably be disease-free in order to enter the trial. This inevitably means that there is a risk of selection. It is not clear whether the distribution of primary tumour sites is similar in the control and study patients, or whether both groups were comparable in terms of surgical approach, including lymph-node sampling. The potential pitfalls of using historical control have been pointed out. and even this premature report would have been more convinc1. Shipley, W. U., Stanley, J. A., Steel, G. G. Cancer Res. 1975, 35, 2488. 2. Nowak, K., Steel, G. G., Peckham, M. J. Unpublished. 3. Mavligit, G. M. et al. Lancet, April 24, 1976, p. 871. 4. Alderson, M. R. Introduction to Epidemiology; p. 171. London, 1976.