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Sex-Related C Cell Hyperplasia in the Normal Human Thyroid: A Quantitative Autopsy Study

Laboratoire d’Anatomie Pathologique (S.G., M.-C.R., J.-P.S.-A.) and LHEA-Laboratoire d’Histologie-Embryologie (D.C.), Centre Hospitalier Universitaire, Angers; Laboratoire d’Anatomie Pathologique, Hôpital Raymond Poincaré (M.D.), Garches; Laboratoire d’Anatomie Pathologique, Hôpital Ambroise Paré (B.F.), Boulogne; and Centre Paul Papin, Centre Régional de Lutte contre le Cancer (O.G.), Angers, France

Address all correspondence and requests for reprints to: Prof. Jean-Paul Saint-André, Laboratoire d’Anatomie Pathologique, Centre Hospitalier Universitaire, 49033 Angers Cedex 01, France.

	Abstract

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We report a prospective quantitative image analysis study of C cells in 57 normal autopsy thyroid glands, serially sectioned and wholly embedded in paraffin; all slides were immunohistochemically stained for calcitonin. Computerized quantitative image analysis was performed on 47 cases to measure C cell surface area and parenchymatous surface area after immunoperoxidase staining for calcitonin. The method was time-effective, with a good reproducibility. C cells were mainly found in the middle third of each lobe. Important interindividual variations were observed; the maximum C cell surface area in a section (A^max) ranged from 28 x 10³ to 470 x 10³ µm² (mean, 167 x 10³ µm²) among 42 adults. Of particular interest was the important difference observed between sexes; A^max was twice as high in men (mean, 201 x 10³ µm²) as in women (mean, 91 x 10³ µm²; P = 0.0009). Moreover, 14 (33%) adult subjects [2 women (15%) and 12 men (41%)] fulfilled C cell hyperplasia criteria, i.e. at least 3 fields at x100 magnification containing more than 50 C cells, suggesting that a substantial part of the normal adult population could have C cell hyperplasia.

	Introduction

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RECENT YEARS have brought major progress in the understanding of the molecular basis of C cell hyperplasia (CCH) and medullary thyroid carcinoma (MTC) in familial MTC and multiple endocrine neoplasia type 2 (MEN2) (1, 2, 3). In these genetically determined conditions, CCH precedes and accompanies MTC through a sequence described in 1973 by Wolfe et al. (4, 5, 6). However, CCH was more recently reported in a number of other conditions, such as chronic hypercalcemia, hyperparathyroidism, thyroid tumors, and chronic lymphocytic thyroiditis (7, 8, 9, 10, 11, 12, 13, 14), and in an increasing number of normal subjects (15, 16, 17). When CCH is unrelated to familial MTC or MEN2, its preneoplastic potential is not documented. Perry et al. have recently described a number distinct morphological criteria in physiological CCH compared to those in neoplastic CCH (18).

Despite important molecular biology advances, routine assessment of normal C cell density and CCH is still a problem. As most studies have focused on preneoplastic or "reactive" CCH, the existence and frequency of CCH in normal subjects are uncertain. In a previous study, we could not rely on a satisfactory series of normal subjects for comparison with a pathological series of chronic lymphocytic thyroiditis-associated CCH (14). Obviously, others have also encountered difficulty in assessing the normal C cell range (19). Thus, in an attempt to clarify this subject, we have undertaken a prospective quantitative image analysis study of C cells in the normal human thyroid.

	Materials and Methods

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Fifty-seven thyroid glands were obtained from 41 forensic autopsies and 16 hospital death autopsies from 1992 to June 1994. No patient had a personal or familial history of MEN2 or MTC. For each patient, age, sex, medical history, and necropsy findings were collected. Several patients were excluded according to the following criteria: abnormal thyroid gland (tumor or goiter) and autolytic alterations due to excessive time between death and fixation. Each thyroid gland was measured and weighed, then fixed in buffered formalin for at least 72 h. Each lobe was horizontally sliced, and the isthmus was vertically sliced. Each section was numbered, and the whole thyroid was embedded in paraffin. Serial 5-µm sections of each block were used for routine stains (hematoxylin-eosin-saffron) and immunohistochemistry; deparaffinized sections of all blocks were stained for calcitonin (CT; polyclonal rabbit anti-CT serum at a 1:1000 dilution (Dako, Paris, France), using a Techmate 500 automate (Dako). In each case, a slide was stained for thyroglobulin (polyclonal rabbit antithyroglobulin serum at a 1:100 dilution; Dako) as a positive control, and a slide was incubated with normal rabbit serum as a negative control. As a specific anti-CT staining of parafollicular-located cells was observed in each gland, no other positive control was required for the CT antibody. An avidin-biotin-peroxidase method was used (L.S.A.B. kit, Dako) with amino-ethyl-carbazol as chromogen. Several counterstains were tested (methyl green, toluidin or anilin blue, and hematoxylin), and a 3-min hematoxylin bath was finally chosen, giving a good morphology and the best result for the image analysis step. Routine slides were examined in search of the above-mentioned histological exclusion criteria or any other abnormality. The immunostained sections were screened under the microscope in search of C cells. In each case, the existence (and the number) of low power (x100) microscopic fields containing at least 50 C cells was searched and assessed by cell counting (field area, 1.91 mm²). The area containing C cells on these sections was outlined with a pencil, allowing easy recognition of its contour during the image analysis step.

Morphometric study

Image analysis was performed using a Quantimet 570 Image Analysis System (LEICA, Rueil Malmaison, France), and image acquisition was achieved with a tri-CCD camera (Sony 930P, Sony, Japan), either from an x-ray light box or from a light microscope, equipped with an x-y motorized stage. First, the slides were examined on the x-ray light box to measure the thyroid parenchyma surface area (noted a according to international morphometric parameters nomenclature). Then, the positive slides (i.e. C cell-containing slides) were examined under the microscope at x100 magnification (field area, 0.596 mm²) to measure the marked C cell surface area (noted A). The motorized stage allowed the examination of successive microscopic fields without overlapping. The best discrimination between the signal (the brown-red pigment of the chromogen) and the hematein counterstain was achieved by an image acquisition in the blue channel of the camera. On the first positive field, a threshold was interactively determined by the operator, allowing detection of the immunostained C cells. This threshold was used to automatically measure the C cell surface area on the next fields.

Statistical analysis

Parameters were submitted to statistical analysis through a statistic software (Statview SE⁺ Graphics, Abacus Concept, Berkeley, CA). Either Student’s t test or, when necessary, a nonparametric test (Mann-Whitney) was applied. Correlations between paired variables was analyzed through linear regression. In the statistical analysis results, r is the correlation coefficient, and p represents the error threshold. The studied parameters were patients’ age and sex, thyroid gland height and weight, section number and thickness, number and location of positive slides in thyroid lobes, thyroid tissue and C cell surface area (by slide, lobe, and gland), and maximum C cell surface area (by lobe and thyroid).

	Results

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Clinical findings

Among the 57 initial thyroid glands, 10 cases were excluded (4 chronic lymphocytic thyroiditis, 4 thyroid glands showing autolysis on histological examination, and 2 incomplete glands) leading to a final series of 47 cases, in which no patient had any evidence of thyroid-related disease. Clinical and morphometric data are summarized in Table 1.

Pathological findings

Histological examination of routine stained slides disclosed no abnormality, except for a 3-mm micropapillary carcinoma (case 38). C cell immunostaining was a granular brown-red cytoplasmic stain of quite constant intensity. When C cell density was low, positive cells were found alone or in small perifollicular groups and were usually present on few sections. When density grew, C cells were identified in an increasing number of sections, partially replacing the follicular epithelial border and sometimes the colloid lumen (Fig. 1). In 16 cases, at least 3 low power fields containing more than 50 C cells were found (Table 1). A pseudonodular pattern was observed in two cases (Fig. 2). No C cell was found in an obviously interstitial location. Negative control sections, obtained after incubation in normal rabbit serum, showed no staining.

View larger version (113K): [in this window] [in a new window]   Figure 1. Diffuse C cell hyperplasia (case 36, 24-yr-old man). Immunoperoxidase reaction with anti-CT antibody; original magnification, x400.

View larger version (106K): [in this window] [in a new window]   Figure 2. Pseudonodular C cell hyperplasia: C cell stratification around follicular lumen (case 3, 67-yr-old man). Immunoperoxidase reaction with anti-CT antibody; original magnification, x400.

The mean surface area of thyroid tissue examined was 35.2 cm² in the adult group (range, 9.3–87.0) and 3.6 cm² in the infant group (range, 2.7–4.5). Detailed results are presented in Table 1. Morphometric analysis produced gross surface area and densities. C Cell surface area, measured under the microscope at x100 magnification, was expressed in square microns, parenchymatous surface area was expressed in square millimeters. Density had no unit (area/area). The reproducibility of parenchymatous surface area macrophotographic measures was tested on 60 sections: intra- and interobserver reproducibilities were nearly the same, i.e. 95.9% intraobserver and 95.8% interobserver. For C cell surface area, reproducibility was tested on 270 fields and gave a 78% intraobserver reproducibility.

C Cell distribution. No C cell was ever observed on the isthmus sections. Morphometric data (number of sections, parenchymatous surface area examined, and C cell surface area, whatever the above-mentioned unit, i.e. density or gross surface area) were very similar between thyroid lobes (P < 0.0005). On the contrary, the C cell vertical distribution was not homogeneous. Only 42% (20–63%) of the sections contained C cells, which were mainly observed in the middle third of each lobe; the peak density (A^max section) was observed at 44% of the height of the lobe, from the top. C Cells could be observed up to each extremity of the lobes, but only 3% of the thyroid lobes contained C cells in their inferior 10%, and A^max was never observed in the inferior quarter of a lobe.

C Cell quantification. Our methodology allowed us to appreciate the extent of the C cell population in two ways: gross surface area (A and A^max) and density (Aa and Aa^max). In the adult group, the mean A^max (which represents the maximum C cell surface area in a section for the concerned thyroid gland) was 167 x 10³ µm² (median, 149 x 10³ µm²), with an important variability (range, 28–470 x 10³ µm²; SD, 105 x 10³ µm²). This variability was explained in part by an important difference according to sex; C cell density in men was more than twice (A^max = 201 x 10³ µm²) that in women (A^max = 91 x 10³ µm²; Fig. 3). In the infant group, A^max was much lower, with a mean value of 62 x 10³ µm² (median, 57 x 10³ µm²; range, 18–92 x 10³ µm²). Despite the small size of this group, the difference between children under 1 yr of age and adults was evident and statistically significant (P < 0.01). The other morphometric parameters (A, Aa, and Aa^max) shared the distribution described for A^max.

View larger version (13K): [in this window] [in a new window]   Figure 3. Mean Amax in men (M) and women (F) in the adult group (n = 42). Bars represent the confidence intervals at 95%.

A highly significant difference (P = 0.0009) existed between sexes for A^max. No other significant correlation was observed between men and women or between A^max and the other studied clinical and morphological parameters (P > 0.2).

	Discussion

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Computerized image processing and analysis systems have histopathological applications in fields in which data collection is time-consuming and can remove, at least in part, observer-related bias (20, 21). C Cells represent a very low proportion of the thyroid cells, with a heterogeneous distribution in the gland; highest concentrations are usually found in the junction zone between the upper and middle thirds of each lobe (19). Quantitative study of C cells in the thyroid gland involves many difficulties, as illustrated in previous reports on this subject (8, 9, 10, 11, 14, 15, 16, 17, 22, 23, 24, 25). Previous researchers had to face a difficult choice. One could choose to perform a complete study of each thyroid (wholly embedded, sectioned, and immunostained for CT on each tissue block) (11, 14, 22, 23, 25), i.e. a highly time-consuming method with manual counting systems, and finally the examination of a small number of cases. On the other hand, one could choose a partial examination of the thyroid glands (1–3 random horizontal sections, a whole vertical section, or no immunohistochemical study) (8, 9, 10, 16, 24), thus allowing the report of more cases, but possibly leading to nonrepresentative material, according to the heterogeneity of C cell distribution in the thyroid, as clearly demonstrated by our results. In a previous report, we encountered some difficulties in defining what a normal C cell density is, mainly because our necropsy series, collected from medical autopsies, was too small (19 subjects) and unrepresentative of the middle-aged adult population (14). Using image analysis on immunostained slides, we generated a C cell quantification method that was applied to 47 necropsic thyroid glands, horizontally sectioned and wholly paraffin-embedded. The standardized and automatized immunohistochemistry step provided a good homogeneity in the staining of C cells. Quantification of the surface area of thyroid parenchyma through the x-ray light box allowed a precise and rapid measure, with excellent reproducibility. The C cell quantification image analysis step also provided good results in term of reproducibility, with a substantial gain in time by comparison with cell counting and with a perfect control of the microscopic fields screened by the motorized microscope stage.

As the extent of sampling has a direct bearing on the C cell density measurement, any relevant comparison among the various studies is difficult. Moreover, results are variably expressed. C Cell density was measured as the number of cells per follicle, per cluster, per low power field, per mm², or mm³ and in many other ways (19). The choice between C cell counting and a measure of stained C cell surface area is linked to the image analysis approach of the problem, and it does not seem to us that this approach modifies anything other than the way results are expressed. In addition, in our experience, cell by cell counting is difficult when C cell density increases to form pseudonodular areas. In our opinion, a more interesting question is whether C cell quantification should be expressed by surface unit (density) or in gross units. At first sight, density might be more accurate. However, the thyroid volume (and then the area examined) is subject to great variations in normal and pathological conditions, and this may distort a density measure. Thus, a gross unit may be more valid, although imperfect, because any pathological examination of a wholly embedded surgical specimen leads to a study of less than 1% of the specimen volume.

In our series, the C cell surface area is statistically highly different between boys under 1 yr of age and adult men. Nevertheless, this is a controversial subject with no definitive conclusion; some researchers observed no difference (11), whereas others observed a more important C cell population in quite small pediatric series (15, 23). These differences might be either fortuitous or related to the very different size and volume of the glands (leading to differences in the number of sections studied and in the ratio between thyroid parenchyma and C cells) or might represent some real physiological differences that will deserve specific studies.

We unexpectedly found a great difference in C cell densities between sexes, and this, to our knowledge, has not been reported in normal subjects. This difference is statistically highly significant and strongly suggests that sex must be taken into account in C cell quantification and assessment of CCH. This sex difference in C cell density could explain the usually higher plasma CT concentration observed in men, basally and/or after a pentagastrin infusion test (26, 27). Some researchers have proposed that testosterone might influence the poststimulatory level of CT in men (28), thus suggesting a possible role of androgens in C cell growth regulation. Two studies recently described a tumor-associated CCH, which, interestingly, also appears to be sex associated. Albores-Saavedra et al. (10) showed tumor-associated CCH in 7 of 11 men (64.6%) and 10 of 38 women (26.3%), and Scopsi et al. (11) observed tumor-associated CCH in 9 of 15 men (60%) and 2 of 22 women (9%).

On the other hand, we did not find any difference according to age among adults. There again, controversial data exist; in 1985, O’Toole et al. (16) were also unable to demonstrate any age-related variation in C cell density in a series of 60 adults ranging from 16–89 yr of age, although a tendency for an increase in C cell density was suggested in the elderly. Because our series only presents 4 subjects over 60 yr of age, it cannot really explore C cell variations in the elderly. In 1982, Gibson et al. (15) reported a positive correlation between age and C cell density in males in a series of 26 subjects ranging from 2–55 yr of age. However, this result may be biased by the presence in the series of 10 children under 10 yr of age. Thus, at least in the age range of 20–60 yr, C cell density may not be submitted to significant age-related variations.

Our study corroborates previous results on C cell distribution heterogeneity (19). Actually, even if sexes are separately considered, and whatever the way in which results are expressed (units), the C cell range remains wide. This heterogeneity cannot be attributed to a methodological artifact; reproducibility is good, and an excellent correlation exists between results in the two thyroid lobes (P < 0.0005). These arguments favor a real variability of C cell density in the normal adult population, as previously described but not explained in subjects with a normal phosphocalcic metabolism (15, 16, 17, 19). This has a direct implication for understanding the CCH concept, because a substantial minority of the "normal" population appears to fulfill CCH definition criteria. First, if about 30% of normal adults may have CCH, then one should perhaps reconsider the previous reports of cronic lymphocytic thyroiditis- or tumor-associated CCH (10, 11, 12, 13, 14) as being nonspecific associations. Obviously, some confusion remains about the best definition and units to be used to quantify C cells and about the delineation between what can be considered as normal and hyperplastic C cell populations (19): the presence of clusters of 20 or more cells in 1or several follicles (29), at least 4 clusters of more than 6 and usually more than 20 C cells (30), more than 50 C cells per field at x100 magnification in at least 3 fields and more than 40 C cells/cm² (14), and more than 50 C cells/field at x100 magnification (10, 31, 32), which currently seems to be the most accepted histological criteria. The microscopic study of our cases revealed that at least 2 infants and 14 adults (33.3% of adults, 2 women and 12 men) presented CCH according to our criteria. Then the question is: are actual histopathological criteria of CCH inaccurate, or does more than 30% of the normal adult population have CCH? Moreover, those histopathological criteria have never been systematically compared to serum CT levels in normal subjects (such a confrontation being obviously impossible in autopsy series). CT assessment using immunoradiometric assay, especially after pentagastrin stimulation, is of particular interest in kindreds of familial MTC and MEN2 (33). Nonetheless, false positive results have been reported, leading to thyroidectomy in cases in which further genomic DNA analysis proved that the subjects did not carry the RET familial mutation (34, 35, 36, 37). Interestingly, pathological analysis of these glands frequently disclosed a mild or moderate CCH. This underscores the capacity of the pentagastrin test to discover nongenetically determined CCH cases. Most reports of pentagastrin tests in normal subjects disclosed about 5% positive responses (26, 27), in contrast with our 30% for histological CCH. Obviously, most CCH defined by histopathological criteria may not be accompanied by CT hypersecretion and, thus, may only reflect C cell number heterogeneity among normal individuals. Fundamental data are needed for C cell growth and CT secretion regulation to elucidate the surprising C cell density heterogeneity in normal subjects.

	Acknowledgments

 
The authors thank Mrs. Brigitte Damon, Miss Delphine Chartier, and Mr. Jean-Luc Grandpierre for their skillful technical assistance.

Received June 26, 1996.

Revised September 9, 1996.

Accepted September 13, 1996.

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