825 IMMUNOHISTOCHEMICAL DIAGNOSIS OF PARAFOLLICULAR CARCINOMAS
SIR,-Since the cytological diagnosis of thyroid tumours, in particular follicular carcinomas, is very difficult, fine-needle aspiration biopsy is not generally accepted as a diagnostic method for thyroid tumours. The method has, however, been shown to be of value in parafollicular carcinomas of the thyroid (P.C.T.).1 In contrast to other thyroid tumours, these tumours yield cells with characteristic features (an asymmetrical tumour cell with one or more eccentric nuclei, many of them having a bright-red granulation and both intramedullary and extracellular amyloid). Since other tumours may also exhibit red granulation and since amyloid is not always present, the cytological staining may not be unambiguous, particularly when the biopsy sample is taken from a mediastinal or neck tumour. Therefore we tried to detect calcitonin in some aspirate specimens of a verified case of P.C.T. using an indirect immunoenzymatic technique. Good results (see figure) were obtained after the following sequence: dry-fixed aspirates were first rinsed in methanol and 2% hydrogen peroxide (’Perhydrol’, Merck, Darmstadt) to inhibit endogenous peroxidase activities.2 Next, they were incubated with normal human serum
P.C.T. cells in dry-fixed aspirate after immunoenzyme cytochemical staining for calcitonin (x1000).
prevent non-immunological binding of the antibodies, rinsed in several changes of 10 mmol;1 phosphate-buffered saline, pH 7.6 (P.B.S.), incubated with rabbit anti-human calcitonin antibodies,’ rinsed again in P.B.s., and incubated with horseradishperoxidase-labelled goat anti-rabbit IgG (lot DS 205, Miles Laboratories, Elkhart, Indiana). After the last incubation the specimens were rinsed in 50 mmol;1 "tris" hydrochloride, pH 76, and incubated with the enzyme-substrate medium conto
taining 0 .075 % 3,3 ’-diaminobenzidine-4-hydrochloride (Sigma Chemicals, St. Louis, Missouri) and 0-01% freshly prepared hydrogen peroxide. Thereafter, the specimens were counterstained with hsematoxylin, dehydrated, and mounted in malinol. Controls for the specificity of the immunocytochemical reactions were used.’
Although high calcitonin concentrations is to quite appropriate diagnose P.C.T.,we feel that application of immunochemical techniques to fine-needle aspirates of nodules suspicious of P.C.T. is of clinical consequence. In comparison with cytological staining the method has the advantage of being specific for calcitonin-containing tumours. In addition, the method is easy and may therefore contribute to earlier thyroidectomy with primary surgical neck dissection. A disadmeasurement
1.
of
Söderstöm, N., Telenius-Berg, M., Akerman, M. Acta med. scand. 1975, 197, 71. 2. Streefkerk, J. G. J Histochem. Cytochem. 1972, 17, 829. 3 Nieuwenhuijzen Kruseman, A. C., Bosman, F. T. Virchows Arch. Path. Histol. 1976, 371, 177. 4. Nieuwenhuijzen Kruseman, A. C., Bots, G. Th. A. M., Lindeman, J., Schaberg, A. Cancer, 1976, 38, 1163.
vantage of the method may be, however, that a negative result does not imply that the tumour is not involved in the synthesis of calcitonin: calcitonin may be secreted immediately after synthesis or linked to cytoplasmic substances so as to become inaccessible to antibodies. Therefore, further studies are needed to evaluate the definite value of immunocytochemistry on fineneedle aspirates as a diagnostic method for P.C.T. or other endocrine tumours. of Pathology and Internal Medicine, University Medical Centre, Leiden, Netherlands
Departments
ARIE C. NIEUWENHUIJZEN KRUSEMAN GEORGETTE KNIJNENBURG
CERVICAL NEOPLASIA AND THE PILL
SIR,-It is difficult to give a clear-cut answer to the question ---do steroid oral contraceptives adversely or beneficially affect the epithelium of the cervix uteri (Sept. 24, p. 644)? However, laboratory studies show that female sex steroidal hormones inhibit mitosis when applied to experimentally induced cervical carcinoma in mice.’ Research in our department indicates that female sex hormones might be beneficial in most gynaecological malignancies. When cervical carcinoma tissues were exposed to progesterone 40 fLg/ml in vitro a highly significant 31% reduction in mitotic activity (measured by the rate ofo.N.A. synthesis) was observed (r
SIR,-Since the cytological diagnosis of thyroid tumours, in particular follicular carcinomas, is very difficult, fine-needle aspiration biopsy is not generally accepted as a diagnostic method for thyroid tumours. The method has, however, been shown to be of value in parafollicular carcinomas of the thyroid (P.C.T.).1 In contrast to other thyroid tumours, these tumours yield cells with characteristic features (an asymmetrical tumour cell with one or more eccentric nuclei, many of them having a bright-red granulation and both intramedullary and extracellular amyloid). Since other tumours may also exhibit red granulation and since amyloid is not always present, the cytological staining may not be unambiguous, particularly when the biopsy sample is taken from a mediastinal or neck tumour. Therefore we tried to detect calcitonin in some aspirate specimens of a verified case of P.C.T. using an indirect immunoenzymatic technique. Good results (see figure) were obtained after the following sequence: dry-fixed aspirates were first rinsed in methanol and 2% hydrogen peroxide (’Perhydrol’, Merck, Darmstadt) to inhibit endogenous peroxidase activities.2 Next, they were incubated with normal human serum
P.C.T. cells in dry-fixed aspirate after immunoenzyme cytochemical staining for calcitonin (x1000).
prevent non-immunological binding of the antibodies, rinsed in several changes of 10 mmol;1 phosphate-buffered saline, pH 7.6 (P.B.S.), incubated with rabbit anti-human calcitonin antibodies,’ rinsed again in P.B.s., and incubated with horseradishperoxidase-labelled goat anti-rabbit IgG (lot DS 205, Miles Laboratories, Elkhart, Indiana). After the last incubation the specimens were rinsed in 50 mmol;1 "tris" hydrochloride, pH 76, and incubated with the enzyme-substrate medium conto
taining 0 .075 % 3,3 ’-diaminobenzidine-4-hydrochloride (Sigma Chemicals, St. Louis, Missouri) and 0-01% freshly prepared hydrogen peroxide. Thereafter, the specimens were counterstained with hsematoxylin, dehydrated, and mounted in malinol. Controls for the specificity of the immunocytochemical reactions were used.’
Although high calcitonin concentrations is to quite appropriate diagnose P.C.T.,we feel that application of immunochemical techniques to fine-needle aspirates of nodules suspicious of P.C.T. is of clinical consequence. In comparison with cytological staining the method has the advantage of being specific for calcitonin-containing tumours. In addition, the method is easy and may therefore contribute to earlier thyroidectomy with primary surgical neck dissection. A disadmeasurement
1.
of
Söderstöm, N., Telenius-Berg, M., Akerman, M. Acta med. scand. 1975, 197, 71. 2. Streefkerk, J. G. J Histochem. Cytochem. 1972, 17, 829. 3 Nieuwenhuijzen Kruseman, A. C., Bosman, F. T. Virchows Arch. Path. Histol. 1976, 371, 177. 4. Nieuwenhuijzen Kruseman, A. C., Bots, G. Th. A. M., Lindeman, J., Schaberg, A. Cancer, 1976, 38, 1163.
vantage of the method may be, however, that a negative result does not imply that the tumour is not involved in the synthesis of calcitonin: calcitonin may be secreted immediately after synthesis or linked to cytoplasmic substances so as to become inaccessible to antibodies. Therefore, further studies are needed to evaluate the definite value of immunocytochemistry on fineneedle aspirates as a diagnostic method for P.C.T. or other endocrine tumours. of Pathology and Internal Medicine, University Medical Centre, Leiden, Netherlands
Departments
ARIE C. NIEUWENHUIJZEN KRUSEMAN GEORGETTE KNIJNENBURG
CERVICAL NEOPLASIA AND THE PILL
SIR,-It is difficult to give a clear-cut answer to the question ---do steroid oral contraceptives adversely or beneficially affect the epithelium of the cervix uteri (Sept. 24, p. 644)? However, laboratory studies show that female sex steroidal hormones inhibit mitosis when applied to experimentally induced cervical carcinoma in mice.’ Research in our department indicates that female sex hormones might be beneficial in most gynaecological malignancies. When cervical carcinoma tissues were exposed to progesterone 40 fLg/ml in vitro a highly significant 31% reduction in mitotic activity (measured by the rate ofo.N.A. synthesis) was observed (r
