QUANTITATIVE PERTECHNETATE THYROID SCINTIGRAPHY AND THE

ULTRASONOGRAPHIC APPEARANCE OF THE THYROID GLAND

IN CLINICALLY NORMAL HORSES

SARAH DAVIES, DON BARBER, MARK CRISMAN, RACHEL TAN, MARTHA LARSON, GREGORY DANIEL

We characterized the scintigraphic and sonographic appearance of the thyroid gland in clinically normal horses

to establish the value of these modalities for assessment of the thyroid gland in this species. Horses were divided

into two age groups. One group consisted of eight horses between 3 and 10 years of age and the other of seven

horses between 11 and 20 years of age. Total T4 concentrations were within the laboratory reference interval in

all horses. Thyroid to salivary (T/S) ratio, percent dose uptake of pertechnetate (Na99mTcO4) and thyroid lobe

volume were calculated. The echogenicity of thyroid lobes and presence of nodules was documented. The two

groups were compared using appropriate parametric and nonparametric statistics. Mean total T4 concentration

was lower in older horses. Sixty minute mean� standard deviation (SD) T/S ratios for old vs. young horses

were 5.8� 3.0 and 5.3� 2.2, respectively. Sixty-minute median and interquartile ranges for percent dose

uptake of pertechnetate for old vs. young horses were 3.64% (1.5–3.98%) and 2.55% (2.33–2.90%), respec-

tively. Mean� SD thyroid lobe volume for old vs. young horses were 18.93� 5.16 cm3and 13.55� 3.56 cm

3,

respectively. Differences between groups were not significant. Most thyroid lobes were hyper or isoechoic to the

sternocephalicus muscle. Prevalence of thyroid nodules did not differ between groups. Further study is needed to

determine if thyroidal percent dose uptake is significantly different in horses with thyroid dysfunction and if it is

clinically useful. r 2010 Veterinary Radiology & Ultrasound, Vol. 51, No. 6, 2010, pp 674–680.

Key words: equine, horses, nuclear scintigraphy, pertechnetate, thyroid, ultrasound.

Introduction

THYROID DYSFUNCTION IN horses is difficult to diagnose

and poorly understood.1 Thyroid hormone assays are

the only objective method available to evaluate equine

thyroid function. Thyroid hormone assays are limited by:

(1) availability of the assays, (2) non-thyroidal factors that

influence thyroid hormone concentrations, and (3) varia-

tion in hormone levels between clinically normal horses. It

is generally accepted that the best method of assessing

thyroid function of horses is to measure thyroid hormone

responses to thyrotropin-releasing hormone (TRH) or thy-

roid-stimulating hormone (TSH).2–4 These tests are infre-

quently used because they are expensive, time consuming

and have limited availability.3 Thus, there is a need for

other, reliable methods to evaluate equine thyroid function.

Thyroid scintigraphy and ultrasonography would not

replace hormone assays but could be a useful adjunct.

In small animals and people, thyroid abnormalities are

investigated routinely using hormone analysis, scinti-

graphy, and ultrasound. Interpreting these tests in horses

requires knowledge of normal appearance and variation.

We found no data regarding the scintigraphic and ultra-

sonographic appearance of the thyroid gland in clinically

normal adult horses.

In dogs, cats, and people, the quantitative scintigraphic

parameters used most commonly to estimate thyroid func-

tion are thyroid to salivary ratio (T/S ratio) and percent

dose uptake of pertechnetate (Na99mTcO4). In cats T/S

ratio is a good predictor of the metabolic status of the

thyroid gland.5 In dogs, percent dose uptake of pertechne-

tate by the thyroid gland is more reliable than the T/S ratio

when assessing thyroid function.6 Percent dose uptake of

pertechnetate is more difficult to quantify, but is indepen-

dent of salivary uptake of pertechnetate.

In horses, pertechnetate thyroid scintigraphy and ultra-

sound have only been used qualitatively.7–10 Our purpose

was to report the scintigraphic and ultrasonographic ap-

pearance of the equine thyroid gland in clinically normal

horses and to provide reference intervals for T/S ratio,

percent dose uptake of pertechnetate and thyroid lobe vol-

ume. Because of the higher incidence of nodular thyroid

Presented at the Annual Meeting of the American College of VeterinaryRadiology in Memphis, TN in October 2009.Research was supported by funding from the Virginia Horse Industry

Board and the Department of Small Animal Clinical Sciences, TheVirginia Maryland Regional College of Veterinary Medicine.Address correspondence and reprint requests to Gregory B Daniel, at

the above address. E-mail: gdaniel@vt.eduReceived March 2, 2010; accepted for publication May 30, 2010.doi: 10.1111/j.1740-8261.2010.01721.x

From the Virginia Maryland Regional College of Veterinary Medicine,Department of Small Animal Clinical Sciences (Davies, Barber, Larson,and Daniel), and Department of Large Animal Clinical Sciences (Crismanand Tan), Mailcode 0442, Blacksburg, VA 24061.

674

disease in older horses we chose to compare qualitative

appearance and quantitative variables between two age

groups of adult horses. Our null hypotheses were that

there would be no significant differences in T/S ratio,

percent dose uptake, thyroid lobe size or prevalence of

thyroid gland nodules between the two age groups.

Materials and Methods

Fifteen adult horses between 3 and 20 years of age were

evaluated. Breeds represented included seven Thorough-

breds, six Quarter Horses, one Thoroughbred Warmblood

cross breed and one Oldenburg. The horses were separated

into two age groups. One group consisted of eight horses

between 3 and 10 years of age (three geldings, five mares).

The other group consisted of seven horses between 11 and

20 years of age (two geldings, five mares). The mean age

(� standard deviation [SD]) of horses in the younger

and older groups were 5.2 (� 2.7) and 15.6 (� 3.9) years

respectively.

All horses were fed a pasture diet and received no sup-

plements or medications for at least 6 months before the

study. All studies were conducted between July and Sep-

tember to account for seasonal variation in total T411 and

all blood samples were obtained in the morning to account

for diurnal variation in T4.12

Physical examination findings, total thyroxine (T4) con-

centration, and results from complete blood counts and

serum biochemistry determinations were normal in all

horses. Serum samples were analyzed for total T4 concen-

trations at Cornell University. Mean and SD for total T4

concentrations for the younger and older groups of horses

were 2.97 � 0.35 and 2.34� 0.47mg/dl, respectively. Theolder horses had significantly lower total T4 concentra-

tions. The Pearson product moment correlation demon-

strated a negative correlation between age and total T4

(correlation coefficient¼�0.653).Ultrasound was performed immediately before scinti-

graphy. Two areas (approximately 6 � 6 cm) were shaved

on either side of the neck, just caudal to the larynx.

Sonographic examination was performed by a resident in

training (S.D.) under the supervision of a board certified

radiologist (M.L.). A 14MHz multifrequency linear-ar-

ray transducer� was used to examine the thyroid gland.

The thyroid lobes were scanned in longitudinal and

transverse planes. Longitudinal images were produced by

maximizing visible thyroid lobe length (Fig. 1A). Trans-

verse images were obtained by rotating the transducer

901 at the widest portion of each thyroid lobe. The max-

imum width, height, and length of the lobes were mea-

sured and recorded. The volume of each thyroid lobe was

estimated by use of the formula for an ellipsoid: p/6

(length�height�width).13–16 The distance from the skin

surface to the capsular surface of the thyroid lobe was

measured for soft tissue attenuation correction needed

for the scintigraphic analysis. Focal lesions such as cystic

or nodular structures within the parenchyma were de-

scribed (Fig. 1B). Thyroid gland echogenicity was re-

corded as being hypoechoic, isoechoic, or hyperechoic by

subjective comparison with the adjacent sternocephalicus

muscle (Fig. 1C).

Horses were sedated for scintigraphy with intrave-

nous detomidine hydrochloridew administered intra-

venously through a jugular catheter. Sedation was

repeated at the same dose 40min after injection of per-

technetatez to adequately restrain horses for the

remainder of the study. The dose (mean � SD) of pert-

echnetate was 1020 � 99.9MBq (27.6 � 2.7mCi) and

was injected into a jugular vein via a preplaced jugular

catheter.

Scintigraphic images were acquired with a large field-of-

view scintillation cameray fitted with a low energy all-pur-

pose parallel hole collimator and integrated to a dedicated

imaging computer with motion correction software.z A

20% window was centered at a photopeak of 140keV, and

image uniformity was software corrected. A flood field was

calibrated each day before acquiring images for the

study. All images were stored in a 256�256�16 matrix.

Before imaging horses, an image of a radionuclide stan-

dard of known activity, approximately 77.33� 6.29MBq

(2.09� 0.17mCi) (mean� SD) was acquired using a 60 s

static frame-mode sequence. The data from this image were

used to calculate camera-counting efficiency. A 60-s dy-

namic frame-mode acquisition was used to acquire motion

corrected images of the horses. Ventral, right lateral and

left lateral views were made at 10, 20, 40, and 60min after

intravenous injection of pertechnetate. Quantification of

thyroid activity was only performed using data from the

ventral view to minimize activity from the overlap of ad-

jacent tissues (Fig. 2).

Image analysis was performed using NucLear Mac

software.k T/S ratios were determined as follows. Us-

ing the ventral image, a single region of interest (ROI)

was drawn manually around each lobe of the thyroid

gland, each parotid salivary gland, and a background

area of the neck that excluded thyroid, parotid, and

major vascular activity. Scintigraphically, margins of

the thyroid lobes were easy to identify facilitating ROI

placement. Margins of the parotid salivary glands were

�Sequoiat; Siemens Medical Systems, Malvern, PA.

wPfizer Animal Health, New York, NY.zCardinal Health, Nuclear Pharmacy Services Location No. 223,

Roanoke, VA.yNuCamIIt; Diagnostic Services Inc., Middlesex, NJ.zMirage Acquisition Application (PCI) Version 5.715f7b Copy-

rightr 1997–2008, Segami Corporation, Columbia, MD.kScientific Imaging Inc., Crested Butte, CO.

675THYROID SCINTIGRAPHYAND ULTRASOUND IN HORSESVol. 51, No. 6

less well defined. A color look-up table with a stan-

dardized upper limit of the scale was used to reliably

identify margins of thyroid and salivary glands. No

other image manipulations were performed. Because

size of ROIs could have affected count density mea-

surements used for T/S ratios, and to decrease variation

in techniques, all ROIs were drawn by one person

(G.B.D.) after collection of all data. Counts recorded in

each ROI were divided by the number of pixels in the

ROI to provide mean count density. Data from salivary

gland ROIs were combined. T/S ratios were calculated

by dividing mean count density within each individual

thyroid lobe ROI by mean count density within a com-

bined salivary gland region. T/S ratios were determined

for each imaging time period.

Percent dose uptake of pertechnetate by the thyroid

gland was determined as follows. The radionuclide

standard was imaged by the scintillation camera

using fixed geometry to determine camera counting effi-

ciency (counts per min/MBq). The injection apparatus

(syringe, extension tubing, and catheter) was imaged fol-

lowing injection to correct for residual activity. All mea-

sured activities were corrected for decay. Counts acquired

from the ROIs of thyroid lobes in the ventral views from

each time point were corrected for background using the

formula:NetCounts ¼ Gross counts

� BKDROI counts

Number of Pixels inBKDROI

� ��

�number of pixels in thyroidROI

�

Thyroid gland uptake was depth corrected using thyroid

lobe depth measurements obtained using ultrasound

and the linear attenuation coefficient for soft tissues

(0.153 cm�1) using the following formula

Depth corrected thyroid counts ¼ Thyroid counts

e�0:153x

where x is the thyroid depth (cm). Thyroid lobe depth was

defined as the distance from the skin to the near field

capsule.

The following formula was used to determine percent

dose uptake of pertechnetate by the thyroid gland at each

time interval

Percent dose uptake

¼ Corrected counts ðcpmÞ in thyroidRadioisotope dose in cpm

� 100

Fig. 2. Ventral scintigraphic images of a 13-year-old horse used for calculation of T/S ratio and thyroidal percent dose uptake. From left to right images wereacquired 10, 20, 40, and 60min after injection of pertechnetate. There is subjectively increased uptake in the thyroid and salivary glands over time.

Fig. 1. (A) Longitudinal ultrasound image of the left thyroid lobe of a 3-year-old horse. The round, hypoechoic structure on the far right represents a bloodvessel. (B) Image showing a mixed echogenicity nodule in the thyroid gland of an 8-year-old horse. (C) Oblique longitudinal ultrasound image from a 12-year-oldhorse used to compare echogenicity of the thyroid lobe with overlying musculature. This thyroid lobe was thought to be isoechoic to the overlying musculature.

676 DAVIES ET AL. 2010

Statistical analysis was performed using a commercial

statistical software program.# All data were assessed for

normal distribution using the Kolmogorov–Smirnov test.

The total T4 concentrations were assessed for an associa-

tion with age and scintigraphic measurements using Pear-

son’s product moment correlation. Student’s t-test and

Mann–Whitney rank sum test were used for comparison of

two groups of parametric or non-parametric data, respec-

tively. A paired t-test was used to determine whether there

was a difference between left and right thyroid lobe vol-

umes. One-way repeated measures analysis of variance was

used to compare T/S ratio and thyroidal percent dose

uptake between the two groups of horses over time. All

pairwise multiple comparison procedures (Holm–Sidak

method) was used to isolate the groups that differed from

each other in the one-way repeated measures analysis of

variance. Significance for all tests was set at P � 0.05.

Results

There was no significant difference in calculated volume

between the left and right lobes of the thyroid gland (Fig. 3).

Data for the left and right lobes were therefore combined for

the remaining analysis. The mean and standard deviation

for combined thyroid lobe volume for the older and younger

groups of horses were 37.85 (� 10.04) cm3 and 27.10

(� 5.27) cm3, respectively; this difference was not statisti-

cally significant. Nodules were identified in one of the thy-

roid lobes in four of eight of the younger horses and three of

seven of the older horses (Table 1). The oldest horse had a

nodule in each thyroid lobe. Thus a total of five nodules

were identified in the thyroid lobes of the older horses. All

nodules were o1 cm in diameter and did not cause any

capsular distortion or change in shape of the thyroid lobes.

The prevalence of thyroid nodules did not differ significantly

between the two groups. Parathyroid glands were not seen.

The majority of the thyroid lobes were hyperechoic or is-

oechoic to the adjacent sternocephalicus muscle (Table 1).

There was no significant difference in T/S ratios at any

of the different time points (Fig. 4). Thyroidal percent dose

uptake at 60min was significantly different from the 10-

and 20-min values and the 40-min value was different from

the 10-min value (Fig. 5). Forty- and 60-min thyroidal

percent dose uptakes were not significantly different from

each other. The youngest horse in this study had a slight

decrease in thyroid gland activity between 40 and 60min.

Two horses with similar total thyroid uptake of pertechne-

tate at 60min (2.56% and 2.54%) had marked differences

in their 60min T/S ratios (4.72 and 10.34) due to differ-

ences in mean salivary count density (89.91 and 52.27).

Generally, salivary gland activity increased over time (Fig. 6).

Differences in salivary gland uptake were significant between

all time points. Two individuals (one horse from each group)

had a decrease in salivary activity between 40 and 60min,

which may have been due partly to excretion of pertechne-

tate in salivary secretions. Sixty minute mean� SD T/S ra-

tios of old vs. young horses were 5.8� 3.0 and 5.3� 2.2,

respectively. Sixty-minute median and interquartile ranges

for thyroidal percent dose uptake of pertechnetate in old

and young horses were 3.64 (1.5–3.98) and 2.55 (2.33–2.9),

Fig. 3. Mean calculated volume for the left and right thyroid lobes of allhorses and mean calculated volume for the thyroid lobes of the younger andolder groups of horses. Error bars indicate 1 standard deviation from themean.

Table 1. Number of Thyroid Lobes that Were Hyper, Iso, and Hypo-echoic Relative to Muscle and Number of Nodules Found in the Left andRight Thyroid Lobes for Each Group (Total Number of Lobes for Each

Group shown in Parantheses)

Group (n)

Echogenicity (Relative to Muscle) Nodules

Hyperechoic Isoechoic Hypoechoic Left Right Total

Younger (16) 10 5 1 2 2 4Older (14) 10 4 0 2 3 5

Fig. 4. Mean gross thyroid salivary (T/S) ratios vs. time for the youngerand old older groups of horses. Neither the difference between groups northe changes within a group as a function of time are statistically significant.#SigmaStat

s

Ver 3.1.1, Systat Software, Chicago, IL.

677THYROID SCINTIGRAPHYAND ULTRASOUND IN HORSESVol. 51, No. 6

respectively. There were no significant difference between

groups with regard to T/S ratio or thyroidal percent dose

uptake at any imaging time. There was no correlation be-

tween total T4 concentration and either T/S ratio or percent

dose uptake at any time point.

Discussion

A difference in total T4 concentration between older and

younger adult horses has not been reported previously.

Similarly, no effect of breed or adult body mass has been

identified.17 The reason for the lower total T4 concentra-

tion in the older group of horses in our study is uncertain.

We did not find any reported association between T4 con-

centration and age in people, cats, or dogs. Evaluation of a

larger number of horses is needed to determine if this is a

reliable finding. It may also be helpful to include horses

420 years of age in future investigations. Although all

horses were clinically normal with normal total T4 con-

centrations, it is still possible that total T4 concentrations

were lower in the older horses due to subclinical thyroid

gland disease that was affecting their ability to produce

thyroid hormone. This study design was non-invasive and

did not allow for histopathologic examination of the thy-

roid gland.

Several non-thyroidal factors affect the hypothalamic–

pituitary–thyroid axis in horses, resulting in low thyroid

hormone concentrations, including phenylbutazone ad-

ministration, high energy diets, high-protein diets, diets

high in zinc and copper, diets with a high carbohydrate/

roughage ratio, glucocorticoid administration, food depri-

vation, level of training, stage of pregnancy, and ingestion

of endophyte-infected fescue grass.3 The effect of these

factors on thyroid uptake of pertechnetate is unknown.

Horses were fed a pasture diet and received no supplements

or medications for at least 6 weeks before the study. None

of the mares were pregnant. Thus factors cited above

are unlikely sources of lower total T4 concentrations in

the older horses. Non-thyroidal illness can also cause

low total T4 but would be an unlikely cause in this study

because all the horses were clinically normal.

There was no significant difference in prevalence of thy-

roid gland nodules between groups. The youngest horse

with a thyroid nodule was 3 years of age. Overall the

prevalence of thyroid nodules in our study horses was

53%. The oldest horse (20 years of age) was the only horse

to have bilateral thyroid lobe nodules. These results are

consistent with prior reports documenting a high preva-

lence of nodular foci in the thyroid gland in horses, al-

though previous studies reported the occurrence of nodules

to be strongly age related.18,19 Benign adenomas comprise

most equine thyroid gland tumors. Although nodules may

result in gland enlargement, they rarely result in hypothy-

roidism or hyperthyroidism. Both follicular and parafol-

licular (C cell) adenomas occur in horses.18,19 The nodules

identified with ultrasound were not apparent in the scinti-

graphic images, suggesting they were not metabolically hy-

peractive. Pertechnetate scintigraphy has been used to

identify hyperfunctioning thyroid tissue in horses with thy-

roid carcinoma.7,9

There was no significant difference in T/S ratio over

time. Two horses with very similar thyroidal percent dose

uptakes had marked differences in T/S ratios at 60min

after injection. This was due to the differences in salivary

uptake and excretion, which may be affected by physio-

logic or pathologic conditions regardless of thyroid gland

uptake.6 In cats, the T/S ratio is preferred to percent dose

uptake when investigating hyperthyroidism, as T/S ratio

had the best correlation with serum T4 concentration and is

easier to quantify. The T/S ratio is unlikely to be as reliable

Fig. 5. Median percent dose uptake of pertechnetate vs. time for theyounger and older groups of horses. There was no significant differencebetween groups.

Fig. 6. Mean salivary count density vs. time for the younger and olderhorses.

678 DAVIES ET AL. 2010

as percent dose uptake when assessing thyroid gland func-

tion in horses. Similarly in dogs percent dose uptake is

reported to be more reliable than the T/S ratio.6 Discrep-

ancy in reliability of the T/S ratio between species may be

due in part to differences in salivary gland function in

herbivores vs. carnivores and differences in head morphol-

ogy resulting in dissimilar tissue attenuation, which affects

scintigraphic quantification.

Percent dose uptake of pertechnetate by the thyroid

gland increased between 20 and 40min after injection but

not between 40 and 60min after injection. The optimal

time for evaluation of percent dose uptake of pertechnetate

in horses is likely between 40 and 60min after injection of

pertechnetate, as there was no significant difference in

percent dose uptake between these imaging times. The

variation in percent dose uptake in these horses is greater

than that reported in cats and dogs (Fig. 7). Variation was

greatest in the older group of horses. The greater variation

in horses may be inherent within this species or be due to

our sampling of a large age range of animals.

We are unaware of any effect of sedatives on thyroidal

uptake of pertechnetate in horses. All horses were sedated

with the same protocol to reduce the motion effects.

Sedation affects thyroid and salivary pertechnetate uptake

in cats, which can lead to an erroneous diagnosis of hyper-

thyroidism.20 Reference intervals established with the same

sedative protocol should be used to eliminate potential

bias.20

We found no association between total T4 concentration

and either the T/S ratio or percent dose uptake in these

horses. In cats, the T/S ratio and percent dose uptake

were associated with serum T4 concentration.5 However,

those cats had a wide range of total T4 concentrations and

were both euthyroid and hyperthyroid. Similar to our

findings, in healthy cats with normal total T4 concentra-

tions there was no association of the T4 concentration and

percent dose uptake of pertechnetate.6,21 This lack of as-

sociation was attributed to the fact that a number of phys-

iologic factors, other than thyroid trapping, can influence

circulating thyroid hormone concentrations.21 A lack of

correlation between total T4 concentration and percent

dose uptake was also seen in greyhounds with uniformly

low T4 concentrations.6 The small range of normal total T4

concentrations in our horses likely affected the ability to

identify an association with percent dose uptake. It is also

possible that percent dose uptake of pertechnetate in clin-

ically normal horses does not accurately reflect the meta-

bolic status of the thyroid gland. Performing pertechnetate

scintigraphy in horses with abnormal thyroid function may

help determine if total T4 concentration is related to per-

cent dose uptake and whether percent dose uptake accu-

rately reflects metabolic activity of the thyroid gland.

One limitation was the low number of horses in each

group. Including horses 420 years of age may also have

been helpful. Ideally, normal thyroid function would have

been confirmed, but this is inherently difficult. The most

accurate test is thought to be a thyroid stimulation test, but

this test could not be performed due to lack of availability

of TRH and TSH.

Further study could assess the scintigraphic and ultra-

sonographic changes in horses with confirmed thyroid

gland dysfunction. This may help clarify whether or not

percent dose uptake of pertechnetate accurately reflects

metabolic activity of the gland.

Fig. 7. Comparison of mean gross thyroid salivary (T/S) ratio and percent dose uptake between the two groups of horses in our study and normal dogs22 andcats23 from previous studies. Error bars indicate 1 standard deviation from the mean. Horses have a higher T/S ratio, higher percent dose uptake and greatervariation.

679THYROID SCINTIGRAPHYAND ULTRASOUND IN HORSESVol. 51, No. 6

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