Sample Arrival Time
Endocrine test samples must arrive at Diagnostic Services BEFORE 10 AM on the day the assay is to be run.
Assay Schedule
Cortisol Monday – Saturday
Progesterone Monday – Saturday
T4 Monday – Saturday
TSH Monday – Saturday
Estrone Sulfate – Quantitative Referred
Testosterone, Estradiol Referred
Assay Reference Values
A list of endocrine assay reference intervals or guidelines is included. Reference values will continue to be updated from time to time and made available to all users of the Endocrinology Diagnostic Service.
Specimen Handling and Submission Requirements
Collect blood by venipuncture or capillary stick into red-top vacutainers. CENTRIFUGE AND SEPARATE SERUM FROM CELLS. Red blood cells present in Bovine samples submitted for progesterone analysis are a source of inaccurate results due to continued enzymatic activity. Most endocrine tests can use only serum. If you are uncertain what to collect, please check with the laboratory.
All biological fluids sent for endocrine assay should be sent frozen if there is more than a 24- hour delay in delivery of the sample to Diagnostic Services.
Label separated samples clearly with complete collection and identification information. 0.5 ml of serum is adequate for most endocrine assays.
Adrenal Gland Function Testing Protocols
Thyroid Hormone Assays
Estrone Sulfate/Progesterone
Please note: All samples should be labelled clearly with the patient and owner name as well as information as to the time of sampling (baseline, 2 hours post-ACTH, 8 hours post LDDS, etc). Please give the ACTH type, dose per kilogram, route of administration and the times of collection on the submission sheet. Providing this information will help to avoid mis-interpretations by the duty clinical pathologist.
ACTH Stimulation Test for Dog
- Ideally fast the patient overnight but offer water.
- Weigh the patient in kilograms.
- Collect a blood sample in a red-stoppered tube for a baseline cortisol concentration. Allow the blood to clot, remove the serum and place it in a new red-stoppered tube. The assay requires 0.5 ml of serum. Refrigerate as soon as possible and freeze if shipment will be delayed more than 24 hours.
- If using ACTH gel, inject intramuscularly at 2.2 U/kg body weight and collect a second blood sample at 2 hours post-ACTH.
- If using synthetic ACTH (Cortrosyn), inject intravenously at a dose of 250 ug/dog (1 vial), regardless of body weight and collect a second blood sample at 1 hour post-ACTH.
- If using synthetic ACTH (Synacthen Depot), inject intramuscularly at a dose of 0.25 mg/dog if the dog’s weight is less than 15 kg and 0.5 mg/dog if the weight is greater than 15 kg. Collect a second blood sample at 2 hrs post –ACTH.
ACTH Stimulation Test for Cats
- Ideally fast the patient overnight but offer water.
- Weigh the patient in kilograms.
- Collect a blood sample for a baseline cortisol concentration. Allow the blood to clot, remove the serum and place it in a new red-stoppered tube. The assay requires 0.5 ml of serum. Refrigerate as soon as possible and freeze if shipment will be delayed more than 24 hour.
- If using synthetic ACTH (Cortrosyn), inject intravenously at a dose of 125 ug/cat (½ vial), regardless of body weight. Note: the synthetic ACTH gives more consistent results in cats, so is currently recommended over the gel for testing. Collect a second specimen 1 hour post-ACTH. Note: additional samples may be collected at 30 and 90 minutes post-administration (individual preference), but the 1 hour post-administration sample is the most important.
- If using ACTH gel, inject intramuscularly at 2.2 U/kg body weight. Collect blood samples for cortisol determination at both 1 and 2 hours post-ACTH in cats. There is no established protocol for Synthetic ACTH (Synacthen Depot) in cats.
Patients on Trilostane to control hyperadrenocorticism should have an ACTH Stimulation test started 2-6 hours following oral administration of the drug. An ACTH Stimulation test should be performed 7-10 days after commencing therapy.
ACTH Stimulation Test for Horses
- Weigh the horse in kilograms.
- Collect a blood sample for a baseline cortisol concentration. Allow the blood to clot, remove the serum and place it in a red-stoppered tube. The assay requires 0.5 ml of serum. Refrigerate as soon as possible and freeze if shipment will be delayed more than 24 hours.
- If using synthetic ACTH, inject IV at 1 unit/kg body weight (to a maximum of 100 units) and collect a blood sample at 2 hours post-ACTH administration.
- If using ACTH gel, inject intramuscularly at a dose of 1 unit/kg body weight and collect a blood sample at 8 hours post-ACTH administration.
Low Dose Dexamethasone Suppression Test for Dogs
- Fast the patient overnight but offer water.
- Weigh the patient in kilogram.
- Collect a blood sample for baseline cortisol concentration. Allow the blood to clot, remove the serum and place in a new red-stoppered tube. The assay requires 0.5 ml of serum. Refrigerate as soon as possible and freeze if shipment will be delayed more than 24 hours.
- Inject dexamethasone intravenously at a dose of 0.01 mg/kg.
- Collect blood samples for cortisol determination at 4 hours and 8 hours after dexamethasone injection.
High Dose Dexamethasone Suppression Test for Dogs
- Fast the patient overnight but offer water.
- Weigh the patient in kilograms.
- Collect a blood sample for baseline cortisol concentration. Allow the blood to clot, remove the serum and place in a new red-stoppered tube. The assay requires 0.5 ml of serum. Refrigerate as soon as possible and freeze if shipment will be delayed more than 24 ours.
- Inject dexamethasone intravenously at a dose of 0.1 mg/kg.
- Collect a blood sample for cortisol determination at 8 hours following dexamethasone injection. A 4 hour post-dexamethasone sample can be collected as well but is optional.
Low Dose Dexamethasone Suppression Test for Cats
- Ideally fast the patient overnight but offer water.
- Weigh the patient in kilograms.
- Collect a blood sample for baseline cortisol concentration. Allow the blood to clot, remove the serum and place it in a new red-stoppered tube. The assay requires 0.5 ml of serum. Refrigerate as soon as possible and freeze if shipment will be delayed more than 24 hours.
- Inject dexamethasone intravenously at 0.1 mg/kg.
- Collect post-dexamethasone blood samples at 4 and 8 hours after dexamethasone injection.
High Dose Dexamethasone Suppression Test for Cats
- Ideally fast the patient overnight but offer water.
- Weigh the patient in kilograms.
- Collect a blood sample for baseline cortisol concentration. Allow the blood to clot, remove the serum and place it in a new red-stoppered tube. The assay requires 0.5 ml of serum. Refrigerate as soon as possible and freeze if shipment will be delayed more than 24 hours.
- Inject dexamethasone intravenously at 1.0 mg/kg.
- Collect a post-dexamethasone blood sample at 8 hours after dexamethasone injection. A 4 hour post-dexamethasone sample may be taken as well but is optional.
Overnight Dexamethasone Suppression Test for Horses First day – 5 pm:
- Weigh the patient in kilograms.
- Collect a blood sample for baseline cortisol concentration. Allow the blood to clot before removing the serum and placed in a new red-stoppered tube. The assay requires 0.5 ml of serum. Refrigerate as soon as possible and freeze if shipment will be delayed more than 24 ours.
- Inject dexamethasone IM at a dose of 0.04 mg/kg (equivalent to 40 ug/kg).
Next day – 12 noon:
a) Collect a blood sample for 19 hour post-dexamethasone cortisol concentration.
Patients on Trilostane to control hyperadrenocorticism should have an ACTH Stimulation test started 2-6 hours following oral administration of the drug. An ACTH Stimulation test should be performed 7-10 days after commencing therapy.
ACTH Stimulation for Dogs
| Drug | Synthetic ACTH (Cortrosyn (Cosyntropin for injection)) |
Synthetic ACTH (Synacthen Depot (Cosyntropin-Zinc Hydroxide)) | ACTH gel (Bexco(Corticotropin injection, USP)) |
|---|---|---|---|
| Formulation | 0.25 mg/vial reconstituted with 1 mL sterile saline, 0.25 mg = 250 ug = 25 U | 1 mg/vial, 1 mL bottle | 40 U/mL, 5 mL bottle |
| Dose | 250 ug (1 vial)/dog regardless of body weight or (Cost-effective lower dose of 5 ug/kg to a maximum of 250 ug/dog) | body weight <15 kg – 0.25 mg/dog body weight >15 kg – 0.5 mg/dog | 2.2 U/kg of body weight |
| Administration | intravenously | intramuscularly | intramuscularly |
| Collection | Pre and 1 hour post | Pre and 2 hour post | Pre and 2 hours post |
ACTH Stimulation for Cats
| Drug | Synthetic ACTH (Cortrosyn (Cosyntropin for injection)) |
Synthetic ACTH (Synacthen Depot (Cosyntropin-Zinc Hydroxide)) | ACTH gel (Bexco(Corticotropin injection, USP)) |
|---|---|---|---|
| Formulation | 0.25 mg/vial reconstituted with 1 mL sterile saline, 0.25 mg = 250 ug = 25 U | No established protocol | 40 U/mL, 5 mL bottle |
| Dose | 125 ug (½ vial)/cat regardless of body weight | 2.2 U/kg of body weight | |
| Administration | intravenously | intramuscularly | |
| Collection | Pre, 30 min. and 1 hour post | Pre, 1 and 2 hours post |
ACTH Stimulation for Horses
| Drug | Synthetic ACTH (Cortrosyn (Cosyntropin for injection)) |
Synthetic ACTH (Synacthen Depot (Cosyntropin-Zinc Hydroxide)) | ACTH gel (Bexco(Corticotropin injection, USP)) |
|---|---|---|---|
| Formulation | 0.25 mg/vial reconstituted with 1 mL sterile saline, 0.25 mg = 250 ug = 25 U | No established protocol | 40 U/mL, 5 mL bottle |
| Dose | 1 U/kg body weight (to a maximum of 100 units per horse) | 1 U/kg of body weight | |
| Administration | intravenously | intramuscularly | |
| Collection | Pre and 2 hour post | Pre and 8 hours post |
Dexamethasone Suppression for Dogs
| Low Dose Dexamethasone Suppression | High Dose Dexamethasone Suppression | |
|---|---|---|
| Drug | Dexamethasone Sodium Phosphate | Dexamethasone Sodium Phosphate |
| Dose | 0.01 mg/kg of body weight | 0.1 mg/kg of body weight |
| Administration | Intravenously | intravenously |
| Collection | Pre, 4 hours and 8 hours Post | Pre and 8 hours post |
Dexamethasone Suppression for Cats
| Low Dose Dexamethasone Suppression | High Dose Dexamethasone Suppression | |
|---|---|---|
| Drug | Dexamethasone Sodium Phosphate | Dexamethasone Sodium Phosphate |
| Dose | 0.1 mg/kg of body weight | 1.0 mg/kg of body weight |
| Administration | Intravenously | intravenously |
| Collection | Pre, 4 hours and 8 hours Post | Pre and 8 hours post |
Dexamethasone Suppression for Horses
| Low Dose Dexamethasone Suppression | |
|---|---|
| Drug | Dexamethasone Sodium Phosphate |
| Dose | 0.04 mg/kg of body weight (equivalent to 40 ug/kg) |
| Administration | Intramuscularly |
| Collection | Pre(5pm on day 1) and 19 hours post (12 noon on day 2) |
Thyroid Hormone Assays
Adrenal Gland Testing Protocols
Estrone Sulfate/Progesterone
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Sample Collection and Handling
Assays for thyroid hormones routinely evaluated (total T4 and TSH) and those auto-antibodies to T4 and thyroglobulin all utilize serum samples.
- Blood should be collected 4-8 hours post pill from animals on thyroid replacement medication.
- Blood should be collected any time from cats on methimazole after they reach therapeutic levels.
- Oral medication – 2 weeks
- Ear gel – 3-4 weeks
- Dietary control – 4-8 weeks
- Blood should be collected into a red-topped tube and allowed to clot fully before centrifugation so that fibrin strands are not present in the supernatant.
- Following centrifugation, the serum should be removed from the clot as soon as possible and, in most cases, placed into a new red- topped tube for submission. The exception to this is free T4; as concentrations can increase with storage in glass tubes, shipment in plastic vials is ideal
- At least 0.5 ml is required for measurement of either T4.
- At least 1.0 ml is required for TSH or free T4 testing.
- At least 2.0 ml is required for auto-antibody determination.
- Refrigerate serum as soon as possible; do not store at room temperature.
- Ship to the AVC Diagnostic Services Laboratory in a cool state (using gel-packs). If shipment to the AVC is delayed more than 24 hours after collection, the serum should be frozen and shipped in a frozen state.
- The samples should be labelled clearly with the patient and owner name.
- If T4 concentrations are being evaluated for therapeutic monitoring, please give the drug type, dose per kilogram and the times of collection on the submission sheet. Blood should be collected 4-8 hours post pill from animals on thyroid replacement medication. Providing this information will help to avoid misinterpretations by the clinical pathologist.
.
Summary Chart of Tests for Diagnosis of Canine Hypothyroidism
| Test | Advantages | Disadvantages |
|---|---|---|
| Total T4 | Inexpensive
Sensitive – usually low in hypothyroidism Can usually rule out hypothyroidism if in normal range – therefore good first step in evaluation |
Easily decreased by non- thyroidal illnesses and many drugs – therefore cannot diagnose hypothyroidism if low |
| Total T3 | If high values obtained in a patient with suspected hypothyroidism, can help identify a situation of auto- antibody interference | Easily decreased by non- thyroidal illnesses and many drugs – therefore cannot diagnose hypothyroidism if low
Low in only 10 % of hypothyroid dogs – very insensitive test |
| Free T4 | Decreased values are more specific for hypothyroidism than total T4
Generally less affected by drugs and non-thyroidal illness than total T4 Can assist in therapeutic monitoring of patients with hypothyroidism (especially if auto-antibodies cause difficulty with T4 evaluation) |
Must be measured by equilibrium dialysis – more expensive test
Can still be decreased in non- thyroidal illness or drug treatment (less so than T4) |
| Canine TSH | If high in the face of a low T4 or low fT4, primary hypothyroidism can usually be diagnosed
Can assist in therapeutic monitoring of patients with hypothyroidism (especially if auto-antibodies cause difficulty with T4 evaluation) |
25 % of hypothyroid dogs have normal range TSH – therefore cannot rule out hypothyroidism
Can be increased by drugs and recovery from non-thyroidal illness Identifying secondary hypothyroidism not possible |
| TSH stimulation test | Remains gold standard test for diagnosis of hypothyroidism | Almost impossible to obtain TSH – medical grade out of production, chemical grade can cause anaphylactic reactions, recombinant human TSH may be available in future |
| TRH stimulation test | Main stimulatory test still possible to perform | Expensive – TRH can be difficult to obtain
Administration can cause vomiting and other side effects Not felt to be any better than measurement of T4, fT4 &TSH to identify hypothyroidism |
| Auto-antibodies to thyroglobulin | Required as one of the tests for certification by the OFA
May allow identification of immune-mediated thyroiditis |
Requires cross-border shipping with sample handling and expense
A negative result does not rule out thyroid pathology No assessment of thyroid function |
| Auto-antibodies to T3,T4 | Provides explanation of unusual results on T3 and T4 testing
Increases suspicion for immune-mediated thyroiditis |
Requires cross-border shipping with sample handling and expense
A negative result does not rule out thyroid pathology No assessment of thyroid function |
Estrone Sulfate Test
Adrenal Gland Testing Protocols
Thyroid Hormone Assays
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Estrone sulfate is a hormone produced by the fetoplacental unit. It is an indication of pregnancy and of fetal well-being. Estrone sulfate rises rapidly in the blood 60-90 days after conception and persists until partuition. Samples for this test should be collected starting at 90 to 100 days after breeding. Although a crytorchid testicle may produce estrone sulfate, we recommend measuring testosterone before and after HCG stimulation.
- Blood should be collected into a red-topped tube and allowed to clot fully before centrifugation so that fibrin strands are not present in the supernatant.
- Following centrifugation, the serum should be removed from the clot as soon as possible and placed into a new red- topped tube for submission.
- At least 1.0 ml is required for measurement.
- Refrigerate serum as soon as possible; do not store at room temperature.
- Ship to the AVC Diagnostic Services Laboratory in a cool state (using gel-packs). If shipment to the AVC is delayed more than 24 hours after collection, the serum should be frozen and shipped in a frozen state. Diagnostic Services will forward the specimen to a reference facility.
Progesterone
- Blood should be collected into a red-topped tube and allowed to clot fully before centrifugation so that fibrin strands are not present in the supernatant. Following centrifugation, the serum should be removed from the clot as soon as possible and placed into a new red- topped tube for submission.
- At least 0.5 ml is required for measurement.
- Refrigerate serum as soon as possible; do not store at room temperature.
- Ship to the AVC Diagnostic Services Laboratory in a cool state (using gel-packs). If shipment to the AVC is delayed more than 24 hours after collection, the serum should be frozen and shipped in a frozen state.