calcium oxalate crystals in the thyroid

Calcium Oxalate Crystals in the Thyroid

Their Identification, Prevalence, Origin, and Possible Significance

JOHN D. REID, M.D., CHANG-HYUN CHOI, M.D., AND NORMAN O. OLDROYD, B.S.

Calcium oxalate crystals are not encountered in normal animal tissues, except for the human thyroid, where they were found in 79 of 100 routine consecutive autopsies. They appear during childhood, and numbers of crystals increase with age. In diffuse hyperplasia, prevalence was higher, but crystals were fewer than expected. In adenomas and carcinomas, crystals were decreased except for three cases with a striking focal increase. None was found in 22 adult primate thyroids. After Clorox® digestion of human thyroids, calcium oxalate dihydrate was identified by x-ray diffraction and infrared spectroscopy. Origin, tissue and species localization are discussed in relation to ascorbate metabolism, thyroperoxidase, and calcitonin. Possible metabolic roles are suggested. Calcium oxalate crystals injected in animals and humans initiate a foreign body reaction with giant cells. In Hashimoto's thyroiditis, crystals disappear but occasionally remain with giant cell reaction. In subacute thyroiditis, granulomas are related more to colloid than to crystals. (Key words: Thyroid; Calcium oxalate; Ascorbate; Thyroid peroxidase; Subacute thyroiditis) Am J Clin Pathol 1987; 87: 443-454

UNSTAINED but birefringent crystals may be seen within the colloid of thyroid glands from persons in infancy to old age. Although recognized since 1877 (credited to Zeiss in the short historical review by Richter and McCarty),49

their presence remains an enigma,34 more so because they are found only in the human thyroid.48 No metabolic significance has been attached to them; they are generally regarded as a dystrophic or degenerative phenomenon, and in ordinary circumstances appear to be inert. However, there are claims in the older literature22'33 that crystals are the cause of thyroiditis of the type originally described by de Quervain and Giordanengo12 as acute and subacute nonsuppurative thyroiditis, sometimes termed granulomatous thyroiditis. Such a proposal is not unreasonable because other crystalline products of endogenous metabolic origin may be inflammatory (e.g., cholesterol, calcium pyrophosphate, and sodium urate). A contrary view is that oxalate crystals are not inflammatory and, when found in granulomas elsewhere, are a result rather than

Received April 30, 1986; received revised manuscript and accepted for publication September, 19, 1986.

Supported in part from the Vivarium Research Fund, Northeast Ohio Universities College of Medicine.

Address reprint requests to Dr. Reid: Robinson Memorial Hospital, Ravenna, Ohio 44266.

Department of Pathology, Robinson Memorial Hospital, Ravenna, Ohio, and Northeast Ohio Universities College of

Medicine, Rootstown, Ohio, Department of Pathology, Cleveland Metropolitan General Hospital, Cleveland, Ohio,

and Research Department, Louis C. Herring and Company, Orlando, Florida

the cause of the inflammation.3 Their exact role in subacute granulomatous thyroiditis thus remains in question.

More fundamental problems are how calcium oxalate appears in crystalline form in the otherwise normal thyroid without deposits in other tissues, why this occurs only in the thyroid of humans and not of other animals, and whether it has any metabolic importance. This article addresses the (1) identification; (2) prevalence; (3) origin, tissue and species restriction; and (4) the significance of oxalate in the thyroid gland, including the possibilities of some metabolic function and a role in subacute thyroiditis. Possible directions for further studies are indicated.

Materials and Methods

Technical Considerations and Identification by Histochemical Reactions and Physicochemical Technics

To investigate the reliability of histologic evaluation and the possibility that calcium oxalate might be destroyed or disolved by fixatives, a comparison was made between sections from sequential blocks of thyroid tissue fixed for varying times in buffered formalin, in unbuffered formalin, in Zenker's fluid, and in B5 fixative. To eliminate possible destructive effects of the hydrochloric acid-alcohol wash used in differentiation in routine regressive hematoxylin and eosin (H and E) a modified Ziehl-Neel-sen (Z-N) stain (substituting acetic for hydrochloric acid) was used on sequential sections in four random cases. The effect of prolonged exposure to water was studied both in frozen sections and in deparaffinized slides.

Attempts at identification of calcium oxalate by histochemistry10,39'44'66 have led to conflicting reports. Staining reactions were therefore reevaluated on calcium

443

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444

With crystals + ++ +++

Table 1.

0-19 years

9/12(75%) 7 2 0

REID, CHOI, AND OLDROYD

Oxalate Crystals in Autopsy Thyroid Tissue (100 Cases)

20-39 years

12/16 (69%) 7 5 0

Ages

40-45 years

26/29 (90%) 14 11 1

60-79 years

27/37 (73%) 16 7 4

A.J.C.P. • April 1987

80-99 years

5/6 (83%) 1 2 2

oxalate monohydrate (Fisher Scientific Co.) embedded in paraffin, in selected examples of human thyroid, and in colleterial glands of the cockroach39 (Periplaneta ameri-cana) provided by Dr. G. Shambaugh. The standard von Kossa45 as well as modifications proposed by Pizzolato44

and Yasue66 were used. Alizarin red stains at pH 4.2 were used before and after microincineration,29 using a propane torch and Pyroceram® plate (the latter found to be essential). A modification recently reported by Proia and Brinn46 was attempted but in our hands was impractical because of viscosity and filtration difficulties. However, a 0.5% concentration at pH 7 was found technically feasible and was studied in thyroids from three cases of sudden death (males ages 37, 38, and 48 years) and from a 79-year-old man who died from bronchopneumonia.

For infrared spectrometry and x-ray diffraction analysis, 5-10-g samples of fresh thyroid from the four autopsy cases just mentioned were minced and ground in 5.25% sodium hypochlorite (Clorox®) in a Servall® Omni-mixer for 2-3 minutes. This technic has been used by others.19'32

Digestion was allowed to proceed from 4 to 24 hours; the sediment was washed twice in distilled water, and then extracted with a large volume of 50:50 chloroform-95% ethyl alcohol, centrifuged, and dried in room air. Examination included use of polarizing microscopy, infrared spectroscopy (IR) (Perkin-Elmer® Programmable Ratio Recording Spectrometer), and x-ray diffraction (Norelco X-ray Diffraction Unit® with vertical goniometer) with identification from reference cards of the Joint Committee on Powder Diffraction Standards-International Committee for Diffraction Data (JCPDS), at the Louis C. Herring Laboratories, Florida. To eliminate the possibility that sodium hypochlorite digestion might affect the status of the oxalate, approximately 0.5 g of calcium oxalate monohydrate was taken from the stock bottle; shaken several times with 10 mL of 5.25% sodium hypochlorite over a 24-hour period; centrifuged, washed, and evaporated to dryness; and similarly studied.

Prevalence

Routine autopsy slides from 100 thyroid glands from the years 1983-1984 (Table 1) were studied to determine the number with crystals and estimate the number of crystals present. Fifty of these were obtained at consecutive

hospital autopsies at Cleveland Metropolitan General Hospital (CMGH), and a second 50 were from consecutive autopsies performed at the order of the coroner at Robinson Memorial Hospital (RMH) for accidental, violent, or sudden unexpected deaths to cover a more normal, rural, white segment of the population. Tissue had been fixed in buffered formalin. Routinely prepared 5-nm sections, after H and E staining, were examined by polarized light, using an AO® Microscope fitted with an analyzer turret and a polarizer/full wave plate. Only one slide was examined per case, and only normal areas were evaluated. In pathologic conditions, all available slides were examined. Four grades of crystal population density were recognized: 0 when none were found; 1 + when crystals were seen only in occasional follicles and in only scattered low-power fields; 2+ when crystals were readily seen in most fields; and 3+ when there were large numbers in many follicles. This evaluation was admittedly subjective but was agreed upon by two of us. It became most difficult when there were marked variations in different areas of the same gland; the grade seen in most fields was adopted.

To examine the possibility of differences with altered metabolic and growth activity, 30 cases of diffuse thyroid hyperplasia were studied, 5 from RMH and 25 from CMGH (Table 2). These had clinical diagnoses of primary hyperthyroidism or Grave's disease. Thirty consecutive surgical specimens from 1982 to 1984 with diagnoses of single or multiple adenomas were examined, 20 from RMH and 10 from CMGH. Thirty-five cases of carcinoma were added, 15 from RMH from 1977 to 1986 and 20 from CMGH from 1982 to 1986. These included 4 purely papillary lesions (2 being small sclerosing carcinomas), 20 of papillary-follicular type, and 11 encapsulated tumors indistinguishable from adenomas apart from capsular or vascular invasion.

To verify statements in the literature48 confirmed verbally by three experienced veterinary pathologists that calcium oxalate crystals are not found in animal thyroids, H and E-stained slides of autopsy specimens from 22 adult primates were examined at the National Zoo, Washington, D.C. These included an orangutan that was 26 years old, gibbons ages 10 and 38 years, and various other species up to age 18 years. No chimpanzee or gorilla thyroids were available.


Vol. 87 • No. 4 CALCIUM OXALATE CRYSTALS IN THE THYROID

Table 2. Calcium Oxalate Crystals in Thyroid Colloid

445

Cases

Sex

No. Age (yrs) Range

Crystals Percentage Positive

M Neg. Pos. ++ +++ Total

Routine autopsies

Diffuse hyperplasia

Adenomas

Tumor Surrounding gland

Carcinomas

Tumor Surrounding gland

Subacute thyroiditis*

Normal follicles Granulomas

Hashimoto's disease

Normal areas

100

30

30

35

10

25

1 mth-99 Av50

16-77 Av31

18-75 Av43

17-81 Av44

30-60 Av43

15-77 Av45

66 34

24

24

28

24

21 79

28

20

45

70

44

27

23

24 12

79

M F

12 9

54 25

44 47

32 18

6 9

82 74

93

9 4

!6 4

0 1

21 26

9 31

10 9

43 47

17 71

40 90

23 27

9 17

60 0

4 13

0 0

0 0

70 87

26 88

100 90

80

* Clinical data for seven cases only.

Origin

Because of the unique thyroid localization of calcium oxalate, its possible interactions with thyroglobulin and iodine were considered. To see if thyroglobulin might bind calcium oxalate, levels of thyroglobulin in normal human serum were measured (Bio-Science Laboratories) before and after incubation over a layer of calcium oxalate at bench temperature for 24 hours. (This was a single experiment.) Calcium oxalate was also added to solutions of Lugol's iodine to determine whether any precipitation might occur.

It is known that in humans oxalate is in considerable part derived from ascorbate,40'60,61 that ascorbic acid may be oxidized to oxalic acid,6,25,28 and that the thyroid has a potent peroxidase,14'55,65 an iron-containing metallo-porphyrin enzyme that is not readily available. Direct effects of peroxide on calcium ascorbate were therefore examined at various concentrations of each, using the highly soluble hemicalcium salt of L-ascorbic acid (mol. wt. 195.2; Sigma Chemical Co.) and H202 (Mallinckrodt, 30% soln). Detailed microscopic, IR, and x-ray defraction studies were made of the washed dried precipitates from three sets of experiments: (1) two hundred milligrams of calcium ascorbate was dissolved in 9 mL of deionized water, to which was added 1 mL of H202; (2) four hundred milligrams of starting material was dissolved and treated similarly; (3) one hundred ninety-five milligrams of calcium ascorbate was dissolved in 9 mL of deionized water;

to this was added 1 mL of a solution of H 2 0 2 (made by adding 1.1 mL of 30% H 2 0 2 to 8.9 mL of deionized water), to give approximately equal 100 mmol/L concentrations.

Significance in Inflammation

To evaluate its inflammatory potential, suspensions of calcium oxalate were injected in hamsters, guinea pigs, and humans, as outlined in Table 3. The monohydrate was as purchased, while dihydrate was freshly prepared by oxidation of calcium ascorbate. Attempts were made in hamsters to induce a preliminary hypersensitive state by incorporating oxalate in Freund's adjuvant.

The role of oxalate in subacute thyroiditis and Hashimoto's thyroiditis (Table 2) was also evaluated. Ten cases with diagnoses of subacute thyroiditis were obtained, several by courtesy of colleagues in the Cleveland area, some by critical review of file slides, and two from personal slide collections. Those accepted showed large numbers of granulomas with macrophages and giant cells, associated with focal destruction of thyroid tissue. Sex and age were known in only seven cases, and further clinical information was available for five. One was an incidental autopsy finding; others were cases with painless swelling of the thyroid, suggesting carcinoma.

Cases of Hashimoto's disease included 25 consecutive examples as diagnosed clinically and histologically, 10 from CMGH and 15 from RMH.


446 REID, CHOI, AND OLDROYD

Table 3. Inflammatory Potential of Calcium Oxalate Suspensions

A.J.C.P. • April 1987

Species

Hamster

Guinea pig

Humanst

Number

2

4 4 3*

5 3

1

Dose

1.0 mL

0.5 mL 0.5 mL 0.5 mL 0.1 mL 0.5 mL 0.5 mL 0.1 mL

1.0 mL 1.0 mL 0.1 mL

Route

ip

sc sc sc id ip sc id

ip iv id

Material

M

M M M M M

— (2)M (DD

M M M

Suspension

Concentration

lOOmg/lOOdL

1,000 mg/100 dL 1,000 mg/100 dL 1,000 mg/100 dL 1,000 mg/100 dL 1,000 mg/100 dL

— 1,000 mg/100 dL 1,000 mg/100 dL 1,000 mg/100 dL 1,000 mg/100 dL 1,000 mg/100 dL

Vehicle

H20

H20 CF CF CF H20 CF H20 H20 H20 H20 H20

Days before histologic examination

(1)14 (2)28

28 28 39 14 11 31 19 19 19 19

(a) 11 (b)28

Abbreviations: CF = complete Freund's adjuvant; M = monohydrate; D = dihydrate; experiment. c = subcutaneous; id = intradermal; iv = intravenous; ip = intraperitoneal. t Two injections.

* Four animals were used; one was found dead, cause undetermined, before completion of

Results

Histologic Considerations, Staining Reactions, and Physiochemical Identification

Blocks of thyroid tissue fixed in Zenker's fluid showed a very large loss of crystals when compared with parallel blocks fixed in buffered formalin. Prolonged storage of bulk tissue in either acid formalin (for two weeks) or in buffered 10% formalin solution (over six months) did not clearly affect crystal numbers as compared with quickly processed blocks. B-5 was also satisfactory (used for two hours before transferring to alcohol). However, frozen sections (10-12 nm) held in buffered formalin or in water began to lose visible oxalate by six hours, with essentially complete loss by 24 hours. There was also marked loss from deparaffinized sections held in water for 24 hours.

Substitution of acetic for hydrochloric acid in hematoxylin differentiation gave no definite increase in preservation of crystals in four normal thyroids. However, in five cases of granulomatous thyroiditis, considerably more small crystals were seen after staining by the modified Z-N technic than after conventional H and E stains.

Standard von Kossa45 reactions and the Pizzolato modification44 did not react with monohydrate crystals embedded and cut as paraffin sections, with crystals in colleterial glands, or in the thyroid. The Yasue method,66

however, was strongly positive. Alizarin red at pH 4.2 did not react until after microincineration.30 The 0.5% modification of Proia and Brinn46 at pH 7 gave variable and unsatisfactory reactions after 10 minutes, with many unstained crystals, some with a peripheral reaction and some completely colored.

Examination of calcium oxalate monohydrate treated with 5.25% sodium hypochlorite for 24 hours showed no conversion to dihydrate. Clorox® extracts of four thyroid glands showed a heavy preponderance of calcium oxalate dihydrate in the three younger persons with no more than 1-2% of monohydrate (identified by polarizing microscopy only). In the fourth case, only the dihydrate was found.

Prevalence (Tables one and two)

In autopsy material, crystals were found within colloid in subjects from childhood onward in 79% of cases. The increased prevalence with age previously reported34,49'50

was not so obvious as an increase in numbers of crystals per field. Heavy deposits were seen only in older persons. No differences were apparent between subjects from urban hospitals and rural coroner's cases. Sex seemed to have little bearing (also as reported previously),34 and 82% of males and 74% females had some crystals. In general, crystals lay well within colloid, unrelated to the epithelium, without any sign of inflammatory reaction. In three cases there were small focal aggregates of macrophages with occasional giant cells but unrelated to crystals; these were incidental findings, interpreted as analogous to palpation thyroiditis.8

In diffuse hyperplasia, the number of cases with any crystals was, if anything, increased, while there was a decrease in the number of crystals per unit area, compared with controls. All cases examined had had some previous medical treatment, one with radioiodine, and all showed evidence of some degree of reversion to the resting colloid state.


Vol. 87 • No. 4 CALCIUM OXALATE CRYSTALS IN THE THYROID 447 In thyroid adenomas, there was no consistent relation

between amounts of oxalate in tumor and in surrounding parenchyma, but, in general, crystals were fewer in adenomatous areas. Crystals were found in some cases of Hurthle cell tumor, were usually absent from microfollicular tissue, and were more likely to be present in large macrofollicles. In one case, there was a much larger amount of oxalate within the adenoma than in the surrounding gland (Fig. 1). (Two further examples of this phenomenon were found, one in a mixed follicular-pap-illary carcinoma and a second in the periphery of an adenoma with capsular invasion.)

In carcinomas, crystals were found only when acini with colloid were present. In the normal peripheral gland they seemed to be fewer than found in controls. In Hashimoto's disease in residual follicles, crystals were found in 80% of cases; with three exceptions, no crystals were seen in fibrotic or inflammatory areas. In subacute thyroiditis in normal follicles, all cases showed at least occasional crystals.

In the thyroid glands from the 22 primates, no crystalline oxalate was found.

Origin and Mechanism of Deposition of Crystals

No differences were found in serum thyroglobulin levels before and after standing for 24 hours over calcium oxalate crystals, and there was no visible evidence of interaction between crystals of calcium oxalate and Lugol's solution.

The addition of H202 to solutions of calcium ascorbate resulted in a fine white granular precipitate that appeared with varying rapidity, depending on the concentrations used. At 1 mmol/L concentrations of each, a fine precipitate appeared on the sides of the plastic tubes, but only after three days. With 500 mmol/L concentrations, reaction was rapid and exothermic, with a small amount of gas as well as a white precipitate.

Precipitates from the three sets of experiments described as 1,2, and 3 under "Materials and Methods," when examined microscopically, showed material unlike the normal bipyramidal CaC204 • 2 H20 crystals. They were not octahedral (or monoclinic in the case of the monohydrate). Even under high magnification they bore no resemblance to oxalate crystal habits found in uroliths. However, the x-ray diffraction patterns of these crystals exhibited peaks at the exact 2-theta angles designated by the JCPDS for CaC204-2 H20 (reference cards 17-54la and 17-541) (Fig. 2). No extraneous peaks were demonstrated, thus indicating the high degree of purity of the specimens (with specific reference to any crystalline contamination). The infrared spectra were, again, identical to standard spectra of CaC204 • 2 H20 (Figs. 3A and B). The absence of peaks in normally nonabsorbing regions and their presence at

FIG. 1. Thyroid adenoma (above) with many crystals within colloid, with normal peripheral thyroid (below), without crystals. Hematoxylin and eosin (X90), partially polarized (female 64 years; RMH).

exact wave numbers for "pure" material indicated the absence of noncrystalline (or amorphous) organic matter. Both x-ray diffraction and infrared, therefore, clearly showed that the nonconforming or even near microcrys-talline precipitate studied under the polarizing microscope was at least 99% CaC204 • 2 H20.

Significance in Inflammation

The results of hamster experiments (Table 3) can be summarized by stating that aqueous suspensions, whether injected intradermally, subcutaneously, of intraperito-neally, produced foreign body-type granulomas (Fig. 4). Intradermal injection sites became ulcerated. After attempting sensitization with crystals suspended in Freund's complete adjuvant, histologic reactions to saline suspensions of oxalate were possibly greater but were not epithelioid in nature.

Monohydrate in complete Freund's adjuvant produced a mixed picture, with sheets of macrophages containing birefringent crystals and vacuoles (representing oil) in


448 REID, CHOI, AND OLDROYD A.J.C.P. • April 1987

FIG. 2. X-ray diffraction spectrum of crystalline precipitate derived from calcium ascorbate by the action of H202.

which acid-fast organisms still remained visible and with interspersed epithelioid granulomas. Schaumann's bodies were prominent. These reactions were essentially no different from reactions to complete adjuvant alone, apart from the presence of crystals.

In guinea pigs (Table 3), intradermal injections of aqueous oxalate monohydrate resulted in small red raised nodules that evolved into crusted lesions and later into flat bare areas. Microscopic reactions were of foreign body type, with fibrosis and very fine crystal residues. Nothing was found in the one animal given dihydrate. No intraperitoneal lesions were found, and heart, kidneys, adrenals, liver, and pancreatic tissues were normal. In all three animals, rare lung lesions were found consisting of macrophages without epithelioid cells but including occasional giant cells, and without crystals, except for the animal given dihydrate, in which a few birefringent crystals were visible.

In human skin, intradermal injections of oxalate suspension showed a foreign body giant cell reaction with slight intradermal fibrosis both at 11 and at 28 days (Fig. 5).

In subacute thyroiditis, adjacent fields of the same sec

tion might show (1) normal follicles with or without calcium oxalate crystals in the colloid; (2) intact follicles with or without crystals, invaded by "sunbursts" of macrophages; (3) partly destroyed follicles, with giant cells most clearly related to colloid fragments (Figs. 6 and 9) and rarely (and in three cases only) to oxalate crystals (Figs. 6-8); (4) granulomatous foci in which the original follicle had been completely replaced by macrophages and giant cells; and (5) focal microabscesses within typical granulomatous inflammation (in two cases). Elongated spindle variants of macrophages (epithelioid cells) were rare, and there was no caseation.

In Hashimoto's disease, crystals were readily found within the colloid of normal areas but not within the lym-phoplasmacytic infiltrate. In three cases, crystals were observed in stroma outside acini and were associated with foreign body giant cells, without macrophage aggregates. In an autopsy case (incidentally encountered late in this study) of untreated myxedema with extremely high antithyroid microsomal and antithyroglobulin titers, there was extensive fibrosis with residual lymphocyte aggregates and foci of giant cells around ragged colloid residues, without crystals.


Vol. 87 • No. 4 CALCIUM OXALATE CRYSTALS IN THE THYROID 449 12 14 16 20 25

i, i i i i i i i i i i i i II in] i MI i i imumu

MICROMETERS (jim) 6 10 I I 12 13 14 16 18 20 25

I t

jnf\n

FIG. 3. (A, upper). Infrared spectrum of similar material with (B, lower) reference spectrum for comparison.


FIG. 4 (upper, left). Giant cell and macrophage response on the peritoneal surface of a hamster injected intraperitoneally with aqueous suspension of calcium oxalate three weeks after attempted sensitization with calcium oxalate in complete Freund's adjuvant. The reaction appears to be a foreign body rather than an immunologic type of response. Hematoxylin and eosin (XI70), partially polarized.

FIG. 5 (upper, right). Human skin, showing fibroblastic and relatively light macrophage and giant cell reaction to calcium oxalate crystals at 28 days. Foreign body type of response. Hematoxylin and eosin (X80), partially polarized.

FlG. 6 (lower, left). Granulomatous thyroiditis, showing fibrosis, with giant cell reaction around colloid (above) and containing oxalate crystals (below, arrow). Hematoxylin and eosin (XI10), partially polarized (male, 48 years; RMH).

FIG. 7 (center, right). Same case as in Figure 6. Giant cells with crystals at higher magnification. Hematoxylin and eosin (X250), partially polarized.

FIG. 8 (lower, right). Birefringent crystalline oxalate within giant cells in granulomatous thyroiditis. Hematoxylin and eosin (X300), partially polarized (female, 30 years; CMGH).

450


Vol. 87 • No. 4 CALCIUM OXALATE CRYSTALS IN THE THYROID 451 Discussion

Oxalate metabolism is of considerable interest to botanists and to veterinarians, as well as to physicians, particularly those dealing with congenital oxalosis and renal calculi. Among its physicochemical properties, oxalate is a strong chelating agent,36 used in removing rust and in older types of anticoagulants. Calcium oxalate salts are of very low solubility and occur in a series with multiple degrees of hydration,20 the monohydrate being the most stable and least soluble.37'51 The dihydrate and monohydrate can be separately produced from the same starting materials,3 and conversions among different forms are reported.20'5' We have found that the monohydrate spontaneously undergoes slow in vitro conversion to calcium carbonate; it will then color with hematoxylin and liberate gas on treatment with mineral acids.

In humans, oxalate is a normal body constituent, toxic in excess. It is derived by ingestion from vegetable leaves and by endogenous production from several precursors, chiefly from glyoxylate and ascorbate.47'60'61 Production from glyoxylate is enzymatically controlled by glycolic acid oxidase, xanthine oxidase, and lactate dehydrogenase, the last probably being the most important.61 The mode of derivation from ascorbate, which accounts for 30-50% of excreted oxalate,60 is less clear but can be nonenzy-matic,40 as confirmed here. Ascorbic acid can be readily converted to dihydroascorbic acid and ultimately to oxalic acid and threonic acid.6'25'28 Calcium ascorbate is known to undergo spontaneous slow degradation to calcium oxalate, and simple oxidizers accelerate this. Reliable measurement of oxalate in human plasma is difficult,' partly because of the rapid formation of insoluble salts, which are presumptively removed, and partly because of in vitro generation from glyoxylate. No degradative mechanisms have been found, and oxalate is excreted in the urine; it has been regarded as a useless and unfortunate metabolic byproduct.60 Full reviews of its clinical and biologic aspects60'6' and of its occurrence and characterization as crystals in tissues10 are readily available.

The significance and interrelations of differently hy-drated forms in human pathology are not clear. The dihydrate has been found in the joint effusions associated with renal failure.26'42 Both dihydrate and monohydrate were found in tissues in a uremic patient with cirrhosis and pancreatic insufficiency;32 in other instances of uremia only the monohydrate was reported.2 In the thyroid, the first specific identification was of the monohydrate,49 our own study indicates almost entirely the dihydrate. In urine, both forms occur, while in renal calculi the center of the stone is frequently monohydrate with a periphery of dihydrate. Dihydrate has been reported around a salivary gland calculus,64 the monohydrate around Asper-

FlG. 9. Giant cells and macrophages arranged around fragments of colloid in granulomatous thyroiditis. This type of response was more in evidence than reactions centered on crystals. Hematoxylin and eosin (X200), (female, 35 years; Fairview Park Hospital).

gillus niger fungus balls,31 and both forms around asbestos fibers in the lung.13

Histologic Considerations, Staining Reactions, and Physicochemical Identification

While generally regarded as insoluble, calcium oxalate can be lost in routine tissue processing or staining.2 No staining method is specific,9,30 although the restricted possibilities in human tissues permit the cautious use of such identification. In agreement with most observers, we find that crystals are unstained by von Kossa and by alizarin red S, except for the Yasue modification,66 and alizarin red after microincineration.29

None of the more recent physicochemical methods of identification is without its limitations. Digestion gives a summation of results from many particles; energy-dispersive x-ray analysis converts oxalate to carbonate in the electron beam:29 and Raman spectroscopy requires examination of numerous crystals to be certain of homogeneity.


452 REID, CHOI, AND OLDROYD AJ.C.P. • April 1987

Our identification of calcium oxalate in dihydrate form differs from a previous identification of monohydrate, based on dissected crystals.49 Lewis and associates32 found 90% of dihydrate after digestion, but almost equal proportions of dihyrate and monohydrate in histologic preparations, thought to result from microtome-knife dis-lodgement of dihydrate crystals.

Prevalence

The figures that stand out in Table 2 are the high proportion of females with all thyroid diseases examined (83%), the high prevalence (93%) but low numbers of crystals in diffuse hyperplasia, the low prevalence in carcinomas (26%), and the high prevalence (100%) and increased population density of crystals (60% in the two-plus category) in subacute thyroiditis.

Differences in prevalence in various autopsy series (from 79% reported here, to 41% found by Richter and McCarty49 who worked from Zenker-fixed tissues; 69% by MacMahon and associates;34 or 51% described by Schaeffer and Rentzschke)50 may reflect variables such as age, fixation, or even the size of blocks. Crystals seem to increase slowly in number and size from infancy onwards. While they have been reported in a premature infant,34 a study of glands from neonatal autopsies made at our request by Dr. T. Pysher (personal communication) showed no crystals in 20 subjects from 20 to 44 weeks of gestation and from 0 to 90 days of age. Crystals therefore seem to appear in childhood and increase in numbers as a person ages.

Origin, Tissue and Species Restriction

Oxalate deposition in thyroid colloid does not appear to have been investigated, although much work has been done on the formation of oxalate crystals in urine and on calculus formation.3,37'40,41,51 Localization to the thyroid (including struma ovarii, where crystals have been twice seen by us) suggests some peculiarly thyroidal mechanism, as previously proposed by MacMahon and colleagues.34

Because no evidence has been found to suggest binding of oxalate to colloid, the question of local synthesis and excretion is raised. Glycolic acid oxidase is not found in the human thyroid.47 Ascorbate, in the presence of calcium, might be oxidized in thyroid epithelium to give the insoluble salt. Alternatively, ascorbate might be present in colloid and converted in situ to oxalate, which is more consistent with the fairly central position of most crystals. Thyroid peroxidase14'35,55 is found in the rough endoplasmic reticulum and perinuclear cisternae65 and is responsible for the production of thyroxine. The possibility that this enzyme may also facilitate the oxidation of ascorbate deserves consideration. This action might also be iodine dependent.35 Intracellular peroxide is produced by

microsomes and peroxisomes7 but also by mitochondria, with small amounts in the cytosol. Other questions are related to the existence of an oxalate binding thyroid protein, comparable to that described in intestinal mucosa.43

Species differences, as indicated by restriction of crystals to the human thyroid, normal and teratomatous, invite further speculation. Calcitonin is known to differ in its structure and potency in different species and might have variable effects on calcium transport by follicle epithelium, with the human variety predisposing to concentration in colloid. To accept the opinion of Wolfe,62 the anatomic location of the C-cells within the thyroid follicular basement membrane places them in a classic position for exerting a local paracrine function. Another species difference occurs with ascorbate, which is handled somewhat differently by humans, rats, and guinea pigs.25

Degradation

Direct removal of oxalate by inflammatory cells is clearly evident. In hamsters and guinea pigs, crystals in skin lesions were noticeably smaller than in the original suspension used. In subacute thyroiditis, macrophages and giant cells were directly associated with phagocytosis of oxalate. The cause of crystal disappearance in Hashimoto's disease and in diffuse hyperplasia (a phenomenon also recorded in the literature49,50) is not apparent.

Function

The possibility that oxalate crystals reflect some metabolic function is raised by its rather selective localization and restricted occurrence, e.g., the oothecae of cockroaches,54 certain green algae,19 and the human thyroid. Further, crystals may be found (if infrequently) in the thyroids of premature infants,34 and in children; they have a high prevalence in the general population and are decreased in numbers in hyperactive glands. Our incidental finding of three tumors with a disproportionately large amount of oxalate (also recorded by MacMahan and coworkers34) suggests some active process, either excess focal production or greatly decreased turnover of a normal metabolite. To follow a view developed by botanists to explain the storage of calcium oxalate in many leaves,5,18

calcium oxalate in the thyroid may reflect a method for controlling intracellular calcium (of known importance in thyroid cell function) by production of oxalate, formation of the insoluble salt, and its disposal in a colloid "sink." Alternatively, oxalate might exert a primary controlling action on thyroid peroxidase, with calcium as the regulating factor. It is known that (by different pathways) oxalate inhibits several enzymes,56 including succinic dehydrogenase, acid and alkaline phosphatases, amylase, and lactate dehydrogenase. This would provide an inhibitory feedback mechanism, comparable to the inhibition of


Vol. 87 • No. 4 CALCIUM OXALATE CRYSTALS IN THE THYROID 453 oxalate,61 but with regulatory effects not only on oxalate but on thyroxin metabolism.

Role in Inflammation, Subacute Thyroiditis, and Hashimoto's Thyroiditis

Based on the experiments reported here, we conclude that oxalate crystals may induce a moderately severe foreign body inflammatory reaction. Our attempts to induce an epithelioid-granulomatous response failed. The material used was in coarsely granular form, so that some caution is necessary in accepting this evidence as final. It is known that the habit and size of crystals may be important in the initiation and extent of inflammatory responses as to sodium urate in gout52 and to acicular crystals of calcium oxalate in synovial cell cultures.23 Further, a granulomatous response may be long and unpredictably delayed,15 suggesting that some alteration in host response is critical, as with reactions to beryllium and to silicon.

Subacute thyroiditis has in the past been considered as a reaction to crystals2233 or to colloid.111216'22 The clinical presentation is often that of an acute febrile illness, generally in women with pain and tenderness in the neck, and a self-limited course over a few weeks or months.ll12'21'24'57,63 Only 10% of cases reviewed by Woolner63 had asymptomatic mass lesions. It has been felt that viral infection is responsible, but because viruses do not generally induce a macrophage-giant cell reaction, a secondary immunologic response, possibly to exposed thyroid colloid, has been suggested.27'58'59 The histologic description is of giant cell granulomas or pseudo-tubercles22'24,63 including epithelioid cells,53 with mi-croabsesses in about 50% of cases.63 Such a picture can be induced by many (and quite different) agents (beryllium, silicon, zirconium, starch, mycobacteria, spirochetes, fungi, etc.), and calcium oxalate is not excluded from suspicion. However, granulomas have not been reported in congenital oxalosis,4,38 in the myocardium in oxalosis with uremia,2,32 or in the kidney after ethylene glycol poisoning.17 Conversely, in palpation thyroiditis,8

granulomas do not seem to be associated with crystals. In other types of inflammatory granuloma where oxalate happens to be found, Johnson and Pani30 have concluded that the crystals may be the result rather than the cause. In our cases of subacute thyroiditis, granulomas were related to fragmented colloid more than to crystals, which were not necessarily present. In Hashimoto's disease, a giant cell reaction was occasionally seen around crystals, but in most cases crystals had disappeared in the lymphocyte-plasma cell aggregates, without obvious cause.

To summarize, the foreign body reactions produced by experimental crystal injections and the lack of any consistent relation between crystals and granulomatous inflammation in various human diseases suggest that the

oxalate visible in thyroid colloid is not the primary causal factor in subacute thyroiditis. Nevertheless, naked crystals exposed after destruction of thyroid epithelium and colloid may induce a moderately severe inflammatory reaction and thus contribute to the histologic picture.

Acknowledgments. The authors acknowledge assistance from Drs. J. Carter, University Hospitals; M. McCoy, Southwest General Hospital; and H. Peterjohn, Fairview Park General Hospital, in providing case material; Dr. T. J. Pysher for examining perinatal glands; Dr. E. J. Yoho who performed skin biopsies; Dr. R. J. Montali, Washington D.C., for allowing examination of primate glands; Dr. G. Shambaugh, Ohio Agricultural research and Development Center, Wooster, for providing col-leterial glands; Dr. D. Laskowski who advised on chemical problems; Dr. G. Sprogis and H. Stamm who provided translations; L. Crain for histologic preparations; and D. Dunsmore for photography.

References

1. Akcay T, Rose GA: The real and apparent plasma oxalate. Clin ChimActa 1980; 101:305-311

2. Bennett B, Rosenblum C: Identification of calcium oxalate crystals in the myocardium in patients with uremia. Lab Invest 1961; 10: 947-955

3. Bisaillon S, Tawashi R: Growth of calcium oxalate in gel systems. J Pharm Sci 1975; 64:458-460

4. Boquist L, Lindqvist B, Ostberg Y, Steen L: Primary oxalosis. Am J Med 1973;54:673-681

5. Borchert R: Calcium-induced patterns of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanlhos L and Albiziajidibrissin Durazz. Planta 1985; 165:301-310

6. Bounais M: Parametres cinetiques d'oxydation de l'acide ascorbique par l'eau oxygenee in vitro. Application a la preparation de l'acide dehydroascorbique pour l'etude de la nutrition artincielle de lepidopteres phytophages. Annates de Zoologie Ecologie Animate 1978; 10:289-302

7. Boveris A: Mitochondrial production of superoxide radical and hydrogen peroxide. Adv Exp Med Biol 1977; 78:67-82

8. Carney JA, Moore SB, Northcutt RC, Woolner LB, Stillwell GK: Palpation thyroiditis (multifocal granulomatous folliculitis). Am J Clin Pathol 1975; 64:639-647

9. Chaplin AJ: Some observations on the demonstration of calcium oxalate in tissue sections. Stain Technol 1974; 49:165-173

10. Chaplin AJ: Histopathologic occurrence and characterization of calcium oxalate: A review. J Clin Pathol 1977; 30:800-811

11. Danyluk JM, Stirrat JH, Laskin MM: Subacute (granulomatous) thyroiditis associated with granulomatous changes in adjacent lymph nodes. Can Med Assoc J 1969; 100:388-390

12. de Quervain F, Giordanengo G: Die akute und subakute nichteitrige thyreoiditis. Mitteilungen aus den Grenzgebieten der Medizin und Chirurgie 1936; 44:538-590

13. de Vuyst P, Jedwab J, Robience V, Yernault J-C: "Oxalate bodies," another reaction of the human lung to asbestos inhalation? Eur J Respir Dis 1982; 63:543-549

14. Ealey PA, Henderson B, Loveridge N: A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland. Histochem J 1984; 16:111-122

15. Eskeland G, Langmark F, Husby G: Silicon granuloma of the skin and subcutaneous tissue. Acta Pathol Microbiol Scand (A) 1974; 248(suppl):69-73

16. Ferguson JA: Tissue reaction to colloid and lipoids from the human thyroid gland. Arch Pathol 1933; 15:244-254

17. Flanagan P, Libcke JH: Renal biopsy observations following recovery from ethylene glycol nephrosis. Am J Clin Pathol 1964; 41:171-175

18. Francheschi VR, Horner HT: Calcium oxalate crystals in plants. Botanical Review 1980; 46:361-427


454 REID, CHOI, AND OLDROYD A.J.C.P. • April 1987

19. Friedman EI, Roth WC, Turner JB: Calcium oxalate crystals in the aragonite-producing green alga penicillus and related genera. Science 1972; 177:891-893

20. Gmelins Handbuch der anorganischen chemie. 8 Auflage; Verlag Chemie GMBH, Weinheim/Bergstrasse, 1961, pp 996-1008

21. Greene JN: Subacute thyroiditis. Am J Med 1971; 51:97-108 22. Gross S: Granulomatous thyroiditis with anisotropic crystalline ma

terial. AMA Arch Pathol 1955; 59:412-418 23. Hasselbacher P: Stimulation of synovial fibroblasts by calcium oxalate

and monosodium urate monohydrate. A mechanism of connective tissue degradation in oxalosis and gout. J Lab Clin Med 1982; 100:977-985

24. Hazard JB: Thyroiditis: A review. Part II. Am J Clin Pathol 1955; 25:399-426

25. Hellman H, Burns JJ: Metabolism of L-ascorbic acid-l-CH in man. J Biol Chem 1958; 230:923-930

26. Hoffman GS, Schumacher HR, Paul H, Cherian V, Ramsey A, Frank WA: Calcium, oxalate crystal associated arthritis in chronic renal failure. Arthritis Rheum 1981; 24:S73

27. Imahori SC, Vladutiu AO: Autoimmune granulomatous thyroiditis in inbred mice: Resemblance to subacute (de Quervain's) thyroiditis in man. Proc Soc Exp Biol Med 1983; 173:408-416

28. Isbell HS, Frush HL: Oxidation of L-ascorbic acid by hydrogen peroxide: Preparation of L-threonic acid. Carbohydr Res 1979; 72: 301-304

29. Johnson FB: Anatomical aspects of histochemistry. California Tumor Registry 70th Semi-annual Slide Seminar, 1980

30. Johnson FB, Pani K: Histochemical identification of calcium oxalate. Arch Pathol 1962; 74:347-351

31. Kurrein F, Green GH, Rowles SL: Localized deposition of calcium oxalate around a pulmonary Aspergillus niger fungus ball. Am J Clin Pathol 1975; 64:556-563

32. Lewis RD, Lowenstam HA, Rossman GR: Oxalate nephrosis and crystalline myocarditis. Case report with postmortem and crystallography studies. Arch Pathol 1974; 98:149-155

33. Lichtman AL, McDonald JR: Birefringence in tissues. Arch Pathol 1946;42:69-81

34. MacMahon HE, Lee HY, Rivelis CF: Birefringent crystals in human thyroid. Acta Endocrinol 1968; 58:172-176

35. Magnusson RP, Taurog A, Dorris ML: Mechanism of iodide-dependent catalatic activity of thyroid peroxidase and lactoperox-idase. J Biol Chem 1984; 259:197-205

36. Martel AE, Calvin M: Chemistry of the chelate compounds. New York, Prentice-Hall, 1952, pp 516-517

37. Martin X, Smith LH, Werness PG: Calcium oxalate dihydrate in urine. Kidney Int 1984; 25:948-952

38. Massie BM, Bharati S, Scheinman MM, et al: Primary oxalosis with pan-conduction cardiac disease: Electrophysiologic and anatomic correlation. Circulation 1981; 64:845-852

39. McGee-Russell SM: Histochemical methods for calcium. J Histo-chem Cytochem 1958; 6:22-42

40. Menon M, Mahle CJ: Oxalate metabolism and renal calculi. J Urol 1982; 127:148-151

41. Nakagawa Y, Abram V, Kezdy FJ, Kaiser ET, Coe FL: Purification and characterization of the principal inhibitor of calcium oxalate monohydrate crystal growth in human urine. J Biol Chem 1983; 258:12594-12600

42. Paul H, Reginato AJ, Schumacher HR: Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid. Arthritis Rheum 1983; 26:191-200

43. Pinto P, Paternarin JL: Oxalate transport by the human small intestine. Invest Urol 1978: 6:502-506

44. Pizzolato P: Histochemical recognition of calcium oxalate. J His-tochem Cytochem 1964; 12:333-336

45. Preece A: A manual for histologic technicians, third edition. Boston, Little, Brown and Company, 1972, pp 377-378

46. Proia AD, Brinn NT: Identification of calcium oxalate crystals using Alizarin Red A stain. Arch Pathol Lab Med 1985; 109:186-189

47. Richardson KE, Farinelli MP: The pathways of oxalate biosynthesis, Urolithiasis: Clinical and basic research. Edited by LH Smith, WG Robertson, B Finlayson. New York, Plenum Publishing, 1981, pp 855-863

48. Richter MN: Anisotropic crystalloids in the human thyroid gland. Am J Pathol 1940; 16:654-655

49. Richter MN, McCarty KS: Anisotropic crystals in the human thyroid gland. Am J Pathol 1954; 30:545-553

50. Schaeffer HE, Rentzschke RD: Das alters-und funktionsabhangige Vorkommen von Calciumoxalatkristallen im Schilddrusenkol-loid. Licht-und elektronenmikroskopische Untersuchungen. Verh Dtsch Ges Pathol 1975; 59:410-414

51. Sheehan ME, Nancollas GH: The kinetics of crystallization of calcium oxalate trihydrate. J Urol 1984; 132:158-163

52. Shirahama T, Cohen AS: Ultrastructural evidence for leakage of lysosomal contents after phagocytosis of monosodium urate crystals. A mechanism of gouty inflammation. Am J Pathol 1974; 76:501-520

53. Sommers SC: Thyroid gland, Pathology, sixth edition. Edited by WAD Anderson. St. Louis, CV Mosby, 1971, pp 1431-1451

54. Stay B, King A, Roth LM: Calcium oxalate in the oothecae of cockroaches. Annals of the Entomological Society of America 1960; 53:79-86

55. Taurog A: Hormone synthesis: Thyroid iodine metabolism, The thyroid: A fundamental and clinical text, fourth edition. Edited by SC Warner, SH Ingbar. Hagerstown, Harper and Row, 1978, pp 31-61

56. Tietz NW: Textbook of clinical chemistry, third edition. Philadelphia, WB Saunders, 1986, p 486

57. Volpe R: Subacute thyroiditis. Prog Clin Biol Res 1981; 74:115-134

58. Wall JR, Fang SL, Ingbar SH, Braverman LE: Lymphocyte transformation in response to human thyroid extract in patients with subacute thyroiditis. J Clin Endocrinol Metab 1976; 43:587-590

59. Wall JR, Strakosch CR, Bandy P, Bayly R: Nature of thyrotropin displacement activity in subacute thyroiditis. J Clin Endocrinol Metab 1982; 54:349-353

60. Williams HE, Smith LH: Disorders of oxalate metabolism. Am J Med 1968;45:715-735

61. Williams HE, Smith LH Jr: Primary hyperoxaluria, The metabolic basis of inherited disease, fifth edition. Edited by JB Stanbury, JB Wyngaarden, DS Fredrickson, JL Goldstein, MS Brown. New York, McGraw-Hill, 1983, pp 204-228

62. Wolfe HJ: Calcitonin: Perspectives in current concepts. J Endocrinol Invest 1982; 5:423-432

63. Woolner LB: Thyroiditis: Classification and clinicopathologic correlation, The thyroid. Edited by JB Hazard, DE Smith. Baltimore, Williams and Wilkins, 1964, pp 123-142

64. Yamamoto H, Sakae T, Takagi M, Otake S, Hirai G: Weddellite in submandibular gland calculus. J Dent Res 1983; 62:16-19

65. Yamashita H, Noguchi S, Murakami N, et al: Ultrastructural localization of endogenous peroxidase in the human thyroid gland under normal and hyperfunctioning conditions. Acta Pathol Jpn 1984;34:553-561

66. Yasue T: Histochemical identification of calcium oxalate. Act His-tochem Cytochem 1969; 2:83-95


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