Immunohistochemical staining of human islet cells with region-specific antibodies against secretogranins II and III.

Chromogranins and secretogranins belong to the granin family of proteins, which are expressed in neuroendocrine and nervous tissue. In earlier publications we have described the development of region-specific antibodies against CgA and CgB. In this study we describe antibodies to SgII and SgIII and their usefulness for immunohistochemical staining. Peptides homologous to defined parts of secretogranins II and III were selected and synthesized. Antibodies were raised and immunostainings were performed on normal human pancreas. The SgII 154-165 (N-terminal secretoneurin), SgII 172-186 (C-terminal secretoneurin) and SgIII antibodies immunostained all insulin-immunoreactive cells, most of the glucagon cells and some of the pancreatic polypeptide cells. The SgII 225-242 antibody immunostained only the insulin-containing cells. None of the antibodies immunostained the somatostatin cells. This study is the first observation of the expression of SgIII in human tissues, where we show expression of SgIII in three of the four major islet cell types in human pancreas.

The importance of chromogranin A in the development and function of endocrine pancreas.

BACKGROUND: Chromogranin (Cg) A is expressed in neuroendocrine and neuronal tissues. It is involved in the generation of secretory granules and is cleaved to form biologically active peptides. Targeted ablation of the Chga gene resulted in increased plasma catecholamines, high blood pressure, and decreased size and number of adrenal medullary chromaffin granules. The aim of this study was to determine whether Chga null mice display changes in the morphology and function of the endocrine pancreas. MATERIALS AND METHODS: Sections of pancreata from Chga-/-, Chga+/- and Chga+/+ mice, were immunostained with antibodies against synaptophysin, CgA, CgB, secretogranin II and the four major pancreatic islet hormones. Plasma was analysed for glucose, insulin, glucagon, somatostatin and pancreatic polypeptide (PP). RESULTS: CgA epitopes were undetectable in the islets of Chga-/- animals. CgB and secretogranin II epitopes were expressed in the islets of all animal groups albeit with decreased expression in Chga-/- islets. The islet number and size were decreased in the Chga-/- animals compared with Chga+/+. The proportion of insulin cells was decreased but somatostatin and PP cells were increased in Chga-/- mice compared to Chga+/+ mice. The nuclear size was decreased in insulin cells and increased in somatostatin cells in Chga-/- mice. Plasma insulin level was markedly decreased in the Chga-/- mice although fasting plasma glucose and glucagon were normal. CONCLUSION: Ablation of the Chga gene affected the islet volume, the composition, distribution and nuclear size of islet cell types and plasma insulin concentration. Our data indicate decreased insulin cell function and increased glucagon cell function. Our study shows that CgA exerts a significant influence on the endocrine pancreas with importance in maintaining islet volume, cellular composition and function.

Medical diagnostic x rays and thyroid cancer.

BACKGROUND: Diagnostic x rays are the largest man-made source of exposure to ionizing radiation for the general population. Whether there are meaningful cancer risks associated with such exposures is unclear. Most previous case-control studies have relied on recalled histories of x rays, and there is concern that completeness and accuracy of recall might differ between cancer case and control subjects. PURPOSE: The present study used information recorded prospectively in hospital charts to address the relationship between medical diagnostic x rays and risk of thyroid cancer. METHODS: The Swedish Cancer Registry and the Uppsala-Orebro Regional Cancer Registry were used to identify persons with papillary or follicular thyroid cancer diagnosed from January 1, 1980, through December 31, 1992, among residents of the Uppsala Health Care Region. After histopathologic review, there were 484 such case subjects available for study. An equal number of age-, sex-, and county of residence-matched control subjects from the general population were randomly selected on the basis of the Swedish Registry of the Total Population. Lifetime residential histories were compiled, and radiology records were searched at all Swedish hospital serving regions where study subjects ever maintained an official residence. Approximate radiation doses to the thyroid gland for specific types of x-ray examinations were assigned on the basis of mean values of measurements made in Sweden in 1973-1975 and in the United States in 1970. Odds ratios were used to evaluate the association between diagnostic radiography and risk of thyroid cancer. RESULTS: A total of 3853 medical diagnostic x rays were ascertained among thyroid cancer case subjects and 4039 among the matched control subjects. There were no tendency for case subjects to have had more of the types of x-ray procedure associated with higher radiation dose to the thyroid gland (i.e., those involving the head or neck area). This finding was true even when analysis was restricted to x rays occurring before 1960, when doses likely were higher than in more recent years, and for examinations occurring in childhood and adolescence, when susceptibility to radiation-induced thyroid cancer is greatest. The relative risk of thyroid cancer was not significantly associated with estimated cumulative dose to the thyroid gland from diagnostic x rays (two-sided P for trend = .80). CONCLUSION: These data indicate that the risk of thyroid cancer due to medical diagnostic x rays, if any, is very small.

Molecular forms of katacalcin, calcitonin gene-related peptide and gastrin-releasing peptide, in a human medullary thyroid carcinoma.

Peptides synthesized by a human medullary thyroid carcinoma were purified to homogeneity by reverse-phase high performance liquid chromatography and structurally characterized by determination of amino acid composition, amino acid sequence, and fast atom bombardment mass spectra. The katacalcin-related material in the tumor extract was heterogeneous. Katacalcin (1-21) represented the predominant molecular form but metabolites, identified as katacalcin (1-20), (1-19), (1-15) and (1-13), were also identified in high concentration. Calcitonin gene-related peptide-I was isolated from the tumor but calcitonin gene-related peptide-II was absent. A minor component of calcitonin gene-related peptide-like immunoreactivity was of higher molecular weight and may represent an incompletely processed form of the prohormone. Gastrin-releasing peptide (1-27) and gastrin-releasing peptide (18-27) (neuromedin C) were isolated from the tumor but gastrin-releasing peptide (14-27) and bombesin were absent.

Intraductal serrated adenoma of the pancreas. A case report.

CASE REPORT: A 48-year-old male with known hypothyreosis consulted his physician for symptoms compatible with TIA (transient ischemic attacks). Computer tomography (CT) in December 2001 revealed an irregular, lobulated mass in the processus uncinatus of the pancreas head. A CT examination 14 months later revealed status quo. In July 2004, a new CT showed an increase in size of the expansive pancreatic mass. The patient was operated on in August 2004 with a preliminary diagnosis of incidentaloma of the pancreas. The pathological examination showed a 4 x 3.5 x 2.5 cm large tumour. Histology revealed an intraductal serrated adenoma. The epithelial fronds had sawtooth-like configurations. An area with early invasive carcinoma was found. The tumour had progressed slowly during the 2.7 years of surveillance. Serrated neoplasia in the duodenum may result in similar cases in the future.

Expression of islet amyloid polypeptide in fetal and adult porcine and human pancreatic islet cells.

The synthesis and intracellular localization of the putative hormone islet amyloid polypeptide (IAPP) and its relation to insulin and glucagon during ontogenesis was investigated in fetal and adult porcine and human pancreatic islets. By means of ultrastructural immunogold immunocytochemistry, it was revealed that IAPP is produced by the hormonally pluripotent endocrine stem cells from the earliest time point studied. IAPP was colocalized with insulin and glucagon in the immature and nondifferentiated cell granules in both species. In adult man, highly intense IAPP immunoreactivity was found in beta-cell granules and, at lower intensity, in delta-cell granules. Some alpha-cells also contained a small amount of IAPP in their granules, and among these occasional granules displayed an intense immunoreactivity. In adult pig, IAPP was stored in quantity in beta-cell granules and in small amounts in granules of alpha- and delta-cells. It was difficult to determine the presence of IAPP in pancreatic polypeptide cells, because they were so seldom seen in this material. It is concluded that, in both man and pig, fetal pancreatic islet stem cells synthesize and store IAPP together with insulin and glucagon. The storage in different types of cells and granules was not as predictable as that of the classical islet hormones. The substance is more widely distributed within the pancreatic islet cell types than are any of the other islet hormones, which presumably has functional implications.

Expression of islet amyloid polypeptide in fetal and adult porcine and human pancreatic islet cells.

The synthesis and intracellular localization of the putative hormone islet amyloid polypeptide (IAPP) and its relation to insulin and glucagon during ontogenesis was investigated in fetal and adult porcine and human pancreatic islets. By means of ultrastructural immunogold immunocytochemistry, it was revealed that IAPP is produced by the hormonally pluripotent endocrine stem cells from the earliest time point studied. IAPP was colocalized with insulin and glucagon in the immature and nondifferentiated cell granules in both species. In adult man, highly intense IAPP immunoreactivity was found in beta-cell granules and, at lower intensity, in delta-cell granules. Some alpha-cells also contained a small amount of IAPP in their granules, and among these occasional granules displayed an intense immunoreactivity. In adult pig, IAPP was stored in quantity in beta-cell granules and in small amounts in granules of alpha- and delta-cells. It was difficult to determine the presence of IAPP in pancreatic polypeptide cells, because they were so seldom seen in this material. It is concluded that, in both man and pig, fetal pancreatic islet stem cells synthesize and store IAPP together with insulin and glucagon. The storage in different types of cells and granules was not as predictable as that of the classical islet hormones. The substance is more widely distributed within the pancreatic islet cell types than are any of the other islet hormones, which presumably has functional implications.

Increased amounts of a high molecular weight insulin-like growth factor II (IGF-II) peptide and IGF-II messenger ribonucleic acid in pancreatic islets of diabetic Goto-Kakizaki rats.

Insulin-like growth factor II (IGF-II), a member of the insulin family, regulates cell growth and differentiation. The IGF-II gene is localized close to the insulin gene in man and rat. IGF-II peptide binds weakly to the insulin receptor and exerts insulin-like effects on the blood glucose level. We studied IGF-II in endocrine pancreas in an animal model of noninsulin-dependent diabetes mellitus, the Goto-Kakizaki (GK) rat. At the age of 2 months, these rats have structural islet changes, with fibrosis and irregular configuration, so-called starfish-shaped islets. Immunohistochemical investigation revealed IGF-II immunoreactivity in the beta-cells in both GK and control rats. Pancreatic extraction, followed by size separation using gel chromatography, disclosed a high mol wt form of IGF-II in all animals, and RIA measurements revealed a considerably larger amount of the IGF-II peptide in the 2-and 6-month-old GK rats than in the 1-month GK and control rats. In situ hybridization of 3-month-old GK rats showed increased IGF-II messenger RNA expression in the starfish-shaped islets of GK rats than in the islets with normal structure in both diabetic and control animals. The reason for the increased amount of IGF-II is unclear. As the animals are diabetic before the islet changes occur, it might be a compensatory effect in response to hyperglycemia, but could also be a cause of the islet fibrosis.

Tumor-specific decreased expression of calcium sensing receptor messenger ribonucleic acid in sporadic primary hyperparathyroidism.

Secretion of PTH is regulated by extracellular calcium via calcium receptors (CaR) on the parathyroid cell surface. Recent studies have shown a decreased expression of CaR messenger RNA (mRNA) and CaR protein in pathological parathyroids. We studied the expression of CaR mRNA in pairs of adenoma and adenoma-associated normal gland from the same patients (n = 17) and in biopsies of normal parathyroid glands of normocalcemic subjects (n = 4) using in situ hybridization with oligonucleotide probes on frozen sections. No down-regulation of CaR mRNA caused by hypercalcemia could be demonstrated in the normal adenoma-associated parathyroids when compared with the normal parathyroids of normocalcemic subjects. In contrast, CaR mRNA in the adenomas was significantly reduced to 64% (median; range 41-98) of the corresponding normal adenoma-associated glands. No correlation was seen between CaR mRNA in the adenoma and preoperative serum calcium, PTH, or weight of the adenoma. Loss of heterozygosity studies were performed on adenomas using markers for the locus of the CaR gene on chromosome 3q. No allelic loss was demonstrated, excluding allelic loss as the cause for decreased CaR mRNA expression in the adenomas. It is concluded that the lowered levels of CaR mRNA in parathyroid adenomas may contribute to the increased set point of PTH secretion. In large adenomas the increased cell mass seems to be more important for the increased secretion of PTH.

Mutations of codon 918 in the RET proto-oncogene correlate to poor prognosis in sporadic medullary thyroid carcinomas.

The hereditary multiple endocrine neoplasia syndromes types 2A and B (MEN 2A and B) were recently linked to germline mutations in the RET proto-oncogene, altering one of five cysteine residues in exon 10 or 11 (MEN 2A), or substituting a methionine for a threonine at codon 918 in exon 16 (MEN 2B). The latter mutation also occurs somatically in some sporadic medullary thyroid carcinomas (MTC), and has in a previous study been correlated with a less favorable clinical outcome. In the present study, 46 MTCs were selected for investigation of the codon 918 mutation. The mutation was found in 29 tumors (63%), and was significantly correlated with a poor outcome, with regard to distant metastasis or tumor recurrence (p < 10(-4)). Two tumors showed multifocal growth and C-cell hyperplasia, and these patients were therefore also investigated for germline mutations in exons 10, 11 and 16. The codon 918 mutation was found only in the tumors, thus of somatic origin. The RET codon 918 mutation may have prognostic impact, and therefore preoperative assessment may influence decision-making in the treatment of patients suffering from MTC.

Familial isolated hyperparathyroidism maps to the hyperparathyroidism-jaw tumor locus in 1q21-q32 in a subset of families.

Approximately 70 families with familial isolated hyperparathyroidism (FIHP) have been reported. Whether it is a separate entity or a variant of multiple endocrine neoplasia type 1 (MEN1 at 11q13) or hyperparathyroidism-jaw tumor (HPT-JT or HRPT2 at 1q21-32) syndrome is not known. We describe here 3 unreported families with familial primary hyperparathyroidism and evaluate their clinical, pathological, and genetic profiles. Biochemical and radiological screenings for MEN1 were negative for all families. In 2 families with a total of 10 affected cases and 3 female obligate carriers, there is no evidence of jaw or renal lesions despite careful radiological investigations. In both families the disease was linked to the 1q21-q32 region with the maximum logarithm of the odds (lod) scores of 3.10 and 3.43 for markers D1S222 and D1S249 respectively, at recombination fraction of 0. In 1 family 2 types of parathyroid pathology were found: 3 of chief cell type and 1 of oxyphil/oncocytic cell type. Two chief cell tumors and 1 oxyphil tumor were found to have loss of heterozygosity (LOH) involving loss of the wild-type alleles for chromosome 1q markers. In the third family, with 4 affected siblings, a parathyroid carcinoma and 2 cases of polycystic kidney disease were found. The parathyroid carcinoma also showed loss of heterozygosity in the 1q region. In conclusion, we found that the hyperparathyroidism traits in a subset of FIHP families are linked to the 1q21-32 markers in the HRPT2 region. We describe the spectrum of parathyroid disease in 1q-linked families involving 3 different types of pathology and demonstrate for the first time loss of wild-type alleles in these parathyroid tumors. Taken together, the results suggest that some of the FIHP are a variant of HPT-JT and that the gene involved is a tumor suppressor gene.

Reproductive history and cigarette smoking as risk factors for thyroid cancer in women: a population-based case-control study.

A population-based case-control study was conducted in Northern Norway and Central Sweden to investigate hormonal and reproductive factors and cigarette smoking as determinants of papillary and follicular thyroid carcinoma in women. Information on 191 histologically confirmed cases and 341 age-matched controls was included. No clear association was found with regard to the number of live births, number of pregnancies, a history of incomplete pregnancies, or the use of oral contraceptives or hormonal replacement therapy. However, an early first childbirth (before 20 years of age, or less than 5 years after menarche) was associated with an increased risk of thyroid cancer. There was an increased risk of thyroid cancer among women with a history of artificial menopause compared to those with a spontaneous menopause [odds ratio (OR), 2.52; 95% confidence interval (CI), 0.96-6.62], which was more pronounced for the papillary carcinoma and after adjustment for age at menopause and use of replacement therapy. Cigarette smokers had a decreased risk of borderline statistical significance compared to nonsmokers (OR, 0.69; 95% CI, 0.47-1.01), particularly among premenopausal women (OR, 0.60; 95% CI, 0.38-0.96). This negative association persisted after adjustment for parity, hormonal treatments, and education. Women who started smoking before the age of 15 experienced a marked reduction in risk (OR, 0.38%; 95% CI, 0.18-0.80¿). Moreover, there was a suggestion of a dose-response effect with the amount of cigarettes smoked daily and with duration of the habit. Both the increased risk of artificial menopause and the negative association with smoking are compatible with a relation between levels of estrogens and thyroid cancer among women.

Histopathological variables and DNA cytometry in parathyroid carcinoma.

To undertake an evaluation of histopathological variables in parathyroid carcinoma, 95 cases with this diagnosis were collected from 37 hospitals. Two tumor categories emerged from a review of tissue sections and follow-up information: 56 cases demonstrating extraglandular invasiveness or tumor recurrence were classified as definitive carcinomas, whereas 39 tumors lacking these criteria were classified as equivocal cases. Several morphological variables other than invasiveness differed between the two groups: Fibrosis, necrosis, nuclear atypia (especially macronucleoli), and mitotic figures were significantly more frequent in the carcinoma group. These variables also showed a positive correlation with an aberrant DNA pattern demonstrated by image cytometry. The triad macronucleoli, more than five mitoses per 50 high-power fields, and necrosis were associated with an aggressive behavior in terms of recurrent disease. A minority of the carcinomas had a bland cytologic appearance and differed from benign lesions only by their invasiveness. Certain patterns of fibrosis and necrosis were common but neither pathognomonic nor constant features of malignancy. Mitotic activity constituted a prognostic risk factor but was of limited diagnostic significance. In half of the carcinomas, the frequency of mitoses did not exceed values recorded in benign parathyroid lesions.

Chromogranin A in human neuroendocrine tumors: an immunohistochemical study with region-specific antibodies.

Antibodies to six specific regions of the chromogranin A (CgA) molecule were used to study their immunoreactivity in human neuroendocrine (NE) tumors. Tissue specimens from endocrine pancreatic tumors (n = 14), duodenal carcinoids (n = 2), bronchial carcinoids (n = 5), ileal carcinoids (n = 5) appendix carcinoids (n = 2), medullary thyroid carcinomas (n = 6), parathyroid adenomas (n = 2), and pheochromocytomas (n = 8) were analyzed. The results showed that the NE tumor types expressed varying numbers of CgA fragments. A variation in frequency of the expression of immunoreactive cells was sometimes seen also within the same tumor type. The midportion fragment CgA 176-195 (chromacin) was the only fragment expressed in all tumors. Benign and malignant tumors expressed different patterns, being especially true of insulinomas and pheochromocytomas. These findings suggest that region-specific antibodies to CgA fragments can be used as a diagnostic tool for the characterization of NE tumors.

Immunological studies on the occurrence and properties of chromogranin A and B and secretogranin II in endocrine tumors.

We investigated a variety of endocrine tumors for the presence of chromogranins A and B and secretogranin II. These antigens were identified by one- and two-dimensional immunoblotting and in some cases by immunohistochemistry. An antigen corresponding in electrophoretic behavior to adrenal chromogranin A was present in all types of tumors, including insulinomas, oat cell carcinomas, and Merkel cell tumors of the skin. Chromogranin B had a much more limited distribution. This antigen could not be detected in parathyroid adenomas, oat cell carcinomas, or Merkel cell tumors, either by immunoblotting and immunohistochemistry. The occurrence of secretogranin II was similar to that of chromogranin B, with the exception of a positive reaction in Merkel cell tumors. In benign pheochromocytomas, all three antigens were found consistently; whereas in two of three malignant pheochromocytomas, chromogranin B was absent. Our study establishes that in most cases chromogranins and secretogranin in tumors are identical to the adrenal antigens, but that these antigens are not always stored together. Chromogranin A is the most widely distributed marker for endocrine tumors.

Synaptophysin. A new marker for pancreatic neuroendocrine tumors.

Synaptophysin (SYP) is a glycoprotein recently isolated from presynaptic vesicles of bovine neurons. Initial studies have demonstrated its presence in neurons in the brain, spinal cord and retina, and in adrenal medullary cells. A subsequent study demonstrated it in pancreatic islet cells and certain neuroendocrine (NE) neoplasms, including several pancreatic islet cell tumors. Based on these preliminary observations, we examined, by immunohistochemistry, conventionally fixed, paraffin sections of 57 pancreatic endocrine tumors with a monoclonal antibody to SYP. Furthermore, we compared the SYP immunoreactivity of 30 of these same tumors with that of neuron-specific enolase (NSE) and of chromogranin (CG). SYP was demonstrated in all but one of the 57 tumors. In the comparative study, for which material was available in only 30 cases, SYP and NSE were present in 29 of the tumors, whereas CG was seen in only 15 cases. We conclude that SYP is a highly sensitive and useful marker for pancreatic NE neoplasms. Moreover, in view of the increasingly evident limited specificity of NSE, SYP should be considered the marker of choice for pancreatic NE neoplasms.

Chromogranins or chromogranin-like proteins are present in lamellar bodies and pulmonary surfactant of rat alveolar type II cells.

Rat alveolar Type II cells were immunostained with antibodies directed against chromogranin A (monoclonal, LK2H10) and chromogranins A and B (polyclonal, LKZM1U). The chromogranins or chromogranin-like proteins were identified in cells in lung tissue sections and isolated Type II cells at the light and electron microscopic levels. We used post-embedding immunoelectron microscopy, with immunogold, to detect the proteins' immunoreactivity in osmicated tissues. Gold particles were distributed over the phospholipid lamellae within the lamellar bodies of alveolar Type II cells and over the lattice structure of tubular myelin. Quantitative analysis of gold labeling densities in the various cell compartments indicated that only the latter two structures were specifically labeled. Controls, which included pre-absorption of both anti-chromogranin antibodies with excess chromogranin A or with native surfactant, resulted in a greater than 60% decrease in gold labeling. A possible role of chromogranins or chromogranin-like proteins as Ca2+ binding proteins in alveolar Type II cells is discussed.

Complex co-localization of chromogranins and neurohormones in the human gastrointestinal tract.

Co-localization of chromogranin (Cg) A, B, and C has been studied in different neuroendocrine cell types in histologically normal mucosa from human gastrointestinal tract (corpus, antrum, duodenum, ileum, and colon) using single-, double-, and triple-immunofluorescence stainings. Virtually all enterochromaffin (EC) cells contained CgA, and those in the luminal two thirds of the antral mucosa and villi of small intestine often also contained CgB. A few EC cells in the duodenal crypts contained CgC. Most gastrin cells harbored both CgB and CgA, although rather more CgB than CgA, but some gastrin cells contained all three types, i.e., also CgC. Some CCK cells also contained all three chromogranins. Enteroglucagon cells in the duodenal villi contained CgA and some CgB. CgA (but not B or C) was found in some secretin, GIP, enteroglucagon/peptide YY, and neurotensin cells. A few somatostatin cells contained CgA but neither CgB nor CgC. CgA and C were found mainly in the basal cell region, whereas CgB occurred more diffusely throughout the cytoplasm. This varying distribution suggests that not all secretory granules contain CgA, or that CgB may occur in a nongranular form. The varying composition of the different chromogranins may reflect their complex functional roles in the widespread neuroendocrine system.

Transthyretin immunoreactivity in human and porcine liver, choroid plexus, and pancreatic islets.

We examined transthyretin immunoreactivity (TTR-IR) in human and porcine liver, choroid plexus, and pancreatic islets with both polyclonal and monoclonal antibodies to TTR. The specificity of the immunoreactions and the effects of various fixatives were tested in immunohistochemical and dot-blot systems. B-5 fixative (mercuric chloride and sodium acetate in formalin) was the best immunopreservative. In both species, the TTR-IR in choroid plexus epithelial cells was strong and was much greater than that in hepatocytes. Glucagon cells in pancreatic islets were also strongly TTR immunoreactive. Insulin cells were slightly TTR immunoreactive in human but strongly so in porcine pancreas. The finding of TTR-IR in normal islets explains the presence of TTR-IR in human endocrine pancreatic tumors, notably glucagonomas and malignant insulinomas.

Estimation of the C-cell numbers in rat thyroid glands using the optical fractionator.

We estimated the total number of calcitonin-immunoreactive C-cells in rat thyroid gland using the optical fractionator, the unbiased stereological method for estimation of numbers. It was necessary first to use a fixative composed of formalin, acetic acid, and ethanol to distinctly visualize the C-cells. The 40-microm-thick sections had to adhere to chromalum-gelatin-coated Superfrost Plus glass slides, and the immunostaining technique had to stain the C-cells evenly throughout the whole sections. Because the C-cells were irregularly distributed in the thyroid tissues, their counting required screening of about 500 fields per lobe, but the number of C-cells counted need not be high, about 90 per lobe. We estimated that rats have 185,000 +/- 42,000 C-cells (mean +/- SD; n - 7). The C-cell population did not differ significantly between the two lobes of a given rat, but it varied markedly among rats. The biological differences among the animals contributed 83% to the observed variability, whereas the methodological uncertainty contributed 17%. The serum levels of calcitonin and calcium were not closely correlated to the C-cell numbers. Our results indicate that variability in C-cell experiments can be reduced most effectively by increasing the number of animals used. However, the similar C-cell frequency found in the two thyroid lobes of each rat allows the use of one uniformly sampled lobe for quantification and the other lobe for further analysis.