Hypothyroidism= GP

Hyperthyroidism= Endocrinologist

BASIC LABORATORY DIAGNOSTICS OF THYROID AND

ADRENAL GLAND DISORDERS
© Dept. of Medical and Clinical Biochemistry UPJŠ in Košice, Faculty of Medicine
Eva Ďurovcová, MD. PhD. 5 GM CLB
 Thyroid gland

 90 - 95% of all endocrine diseases are thyroid disorders

 Thyroid disease affect in average 5% of women and 1% of men in EU countries
 More common in women; 10% of population over 60 years of age may have
some abnormality in thyroid function
 Two types of disorders, which can combine: pathological production of
hormones and morphological changes in the gland.
 Thyroid gland
 Used alongside a full clinical evaluation, appropriate laboratory tests are
useful in establishing a diagnosis of:

 Hypothyroidism (manifest, subclinical)

 Hyperthyroidism (manifest, subclinical)

 Euthyroid status

Lithium, Amiodarone= Hyper

 Understanding the thyroid
• Thyroid hormones are essential for normal cell growth,
development and metabolism
 thyroxine (T4) and tri-iodothyronine (T3)
 the only endocrine gland storing its hormones (about 100 days
stores)
 Understanding the thyroid
 Thyrocytes secrete more T4 (T4: T3 ratio is 20 : 1) which is converted to T3
in peripheral tissues:
 Deiodination mostly in the liver, kidney, muscle
 T4 also undergoes deiodination to form reverse rT3
 T3 is metabolicaly more active (3 – 8 times) than T4.
 Transporting proteins in blood: TBG, prealbumin, albumin - ensure stable
levels of free hormones, protect T4 and T3 from getting lost in urine.
 Free hormones (0.03% fT4 and 0.2% fT3) enter cells and interact with their
receptors.
 Changes in binding protein concentration of (e.g. pregnancy, malnutrition)
does not change free T3,T4.
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 Signs and symptoms
Hypothyroidism Hyperthyroidism
Apathy, slow speech Excitement
Weight gain Weight loss
Slow HR Rapid HR
Cold intolerance Hot intolerance
Long intestinal transit time (constipation) Short intestinal transit time
Sweaty skin Dry skin
Women: disturbances of menstrual cycle, infertility
Children: growth disorders
 Who requires a thyroid profile ?
1. Clinical symptoms suggesting a thyroid dysfunction
2. Therapy of thyroid pathology (monitoring)
3. Risk group patients:
• pregnant women and women with fertility disorders;
• with autoimmune disease (T1DM, Addison's d., celiac d)
• after surgery and radiotherapy in the neck area, after radioiodine TH;
• with overt coronary heart disease and/or tachyarrhythmia;
• with hypercholesterolemia;
• taking medicines that affect thyroid hormones;
• women older than 50 years with non-specific manifestations;
• children with growth disorders;
• some genetic diseases (Down's or Turner's syndrome).
 OverviewLaboratory
of common tests lab tests
Name Marker of :

TSH – thyreostimulating hormone Thyroid function

fT4 – free thyroxine Thyroid function

fT3 – free trijodthyronine Thyroid function

anti TPO – antibody against thyroid Autoimune thyroiditis (Hashimoto, chronic lymphocytis
peroxidase thyroiditis
anti TSH R (TRAb) – antibody against TSH Graves´ disease
receptor
Thyroid cancer
TG – antibody against thyreoglobulin May interferre with Tg tests

calcitonin Medullary thyroid carcinoma

 Which test should be used?
 TSH - the most sensitive indicator of thyroid dysfunction, the
1st choice screening test. TSH= changes first much more sensitive, changes much
more earlier in blood.
 Inverse log-linear correlation between TSH and fT4: minimal
change in FT4 induces an amplified response in the TSH
concentration
 If TSH test has pathological result, laboratories will add FT4,
(and FT3 and thyroid antibodies) according to local guidelines
(≈ reflex testing)

Subclinical hypothyroidism, asymptomatic

Overt hypothyroidism, autoimmune/ hashimoto thyroiditis= ATPO high.
 ...which test should be used?
 In healthy individual, if TSH is within the reference range,
there is a 98 – 99% likelihood that the FT4 will also be within
the reference range. TSH>10, positive ATPO= start therapy.

Central hypothyroidism= Normal TSH, less fT4, fT3

 FreeT4/FreeT3 are used instead total T4/T3, because they are

not influenced by changes in binding proteins

 When monitoring treated patients, FT4 or FT3 is ordered

together with TSH, if necessary
 When is it insufficient to test only TSH?
 Central (secondary) hypothyroidism: an incorrect diagnosis of
euthyroidism could be made based on TSH alone.

 Early stages of therapy: During the first months of treatment for

hypo- or hyperthyroidism, patients have not reached equilibrium yet
and their TSH level remains still abnormal (low in hyper- and high in
hypothyroidism) Early stages of antithyroid therapy= adjust therapy on level of fT4 and fT3
Late= TSH enough.

 Non-hyroidal illness (acute or chronic): TSH is altered by non-

thyroidal factors. Testing recommended only when it is likely to have
an effect on acute management of illness.
 Thyroid autoimmune markers
Anti-thyroid peroxidase antibody (ATPO, aTPO) – microsomal
autoAb, more sensitive
 Positive in 50–80% of Graves´ disaease and 90% of Hashimoto´s disease
 Positive in small proportion of apparently healthy people

Anti-thyreoglobulin antibody (ATG) – nonspecific, useful for

interpretation of TG levels

Anti TSH receptor antibodies (TRAb, TRAK)

 Diagnostic value
 Predicting neonatal hyperthyroidism in babies born to mothers with Graves
Hypothyroidism - algorithm of testing
TSH
N ↑

no primary disorder of fT4

thyroid gand
↑ N ↓

secondary subclinical manifested

hyperthyroidism hypothyroidism hypothyroidis

further TSH <10 TSH >10

investigation

no TPO, clinical yes

monitoring findings

THERAPY
 Hypothyroidism - causes
1. Primary gland failure (90 – 95% of all cases).
 Thyroiditis of an autoimmune origin (Hashimoto thyroidis antibodies in
>95%) - anti-TPO and/or anti- TG
 Iatrogenic causes: surgery, ablation, radiation, medication
 Other: Inflitrative processes
 Congenital ↑TSH + ↓FT4
5. Secondary hypothyroidism – due to pituitary failure or as
a result of hypothalamic (tertiary) suppression of thyrotropin-releasing
hormone (TRH): ↓TSH and a resultant ↓FT4.
 Less frequent causes of hypothyroidism
1. Iodine deficiency - in the mountaineous areas – endemic goitre
• South Germany, Austria, Greece
• fortification of salt
2. Thyroid blocking substancies in the diet (goitrogenes) : brassicas
(cabage), cassava: Spain, Bohemia, Shefield in UK
 Laboratory changes in hypothyroidism

• ↑ S-Cholesterol ↓ rate of LP degradation, sensitivity

of LDL-receptors
• ↑ S-Creatinkinase myopathy due to lack of ATP

• ↓ S-Na multifactorial: hypovolemia (↓cardiac

output) + ADH secretion

• ↑ S-prolactin ↑TRH has a weak stimulatory effect

on the lactotroph cells of the pituitary
 Hyperthyroidism - algorithm of testing
Overdose of Carbimazole.

T3 hyperthyroidism,
primary,
most likely is Graves disease.

Untreared hyperthyroidism,'
thyrotoxicosis/thyroid storm. Severe hypermetabolism.
Hyperthyroidism - causes
1. Primary hyperthyroidism = Graves´disease (60 - 90% of all cases)
- endogenous production of autoAb binding TSH receptor
- adenoma, carcinoma
- overdosing with T4 ( thyreotoxicosis factitia)
Typical finding: ↓ TSH ↑ + fT4,fT3 (+ antibodies)

2. Secondary hyperthyroidism – pituitary TSH secreting tumor

Typical finding: ↑TSH + ↑ fT4,fT3
3. Tertiary-abnormal pituitary T3 receptors RARE! Cells producing TRH are
resistant to normal negative feedback regulation
Laboratory changes in hyperthyroidism

 ↑S-Ca - increased bone turnover

 ↓S-CHOL - rapid clearence of LDL
 ↑S-CK - toxic myopathy
 Monitoring subclinical thyroid disease
Subclinical hypothyroidism – ↑TSH (4.5 – 10) + normal FT4
 IF: symptoms are present
then treat (thyroxine)
 IF: TSH > 10 mU/L and monitor therapy
 IF: positive anti-TPO
NOT TREATED pts should be monitored - TSH every 6-12 months.

Subclinical hyperthyroidism - ↓TSH + normal FT4

 Further investigation and treatment should be considered for
patients with an undetectable TSH
 TH: at increased risk of developing atrial fibrillation and osteoporosis.
Monitoring of patients on thyroxine
The aim: to restore normal values of TSH and  TSH decreases to normal
FT4 and make patient feels well no sooner than 6-8 weeks
after the start of
treatment (up to 1 year)

 If thyroid function needs

to be assessed before this
time, FT4 is a better
indicator until TSH get
stabilized.
 Monitoring patients on anti-thyroid drugs
 Monitor every 4 Ws with FT4 + TSH to adjust the dose until the FT4
is in upper part of RR and clinical symptoms have improved
 TSH remains suppressed for 3-6 Ms after initiation of anti-thyroid
medication. TSH normalizes only in a part of patients.

 Then monitor TSH only

 Risk of hypothyreosis !!!
 Risk of agranulocytosis:
CBC, patients should be
advised to report fever, sore
throat or infection.
 Markers of thyroid tumors 1
 Thyreoglobulin (Tg) - glycoprotein stored in the follicular
colloid, a substrate for the synthesis of TH
 Produced only by normal or well-differentiated malignant cells
 Monitoring patients for the presence of recurrent or residual
disease during post-surgical follow-up
 After tumor removal, Tg level should be undetectable
 High sensitive methods - detection of < 0.1 mg/L
 Anti TG antibodies interfere with measurement of Tg
 NO for screening of thyroid cancer in patients with thyroid
nodules! (imagine tests, FN biopsy - better choice)
 Markers of thyroid tumors 2
Calcitonin (CT) – marker of medullary carcinoma of thyroid
parafollicular cells producing calcitonin (10% of all thyroid
cancer)
 genetic testing: mutation of MCT- RET proto-oncogene

Monitoring of calcitonin after surgery

- It is important to know baseline value for comparission
- Nonspecific elevation in: neuroendocrine tumor- carcinoid,
pheochromocytoma, Hashimoto th, CKD,
Thyroid tests in pregnancy
Changes: TSH is temporarily suppressed during the 1st trimester by
thyroid stimulating effect of hCG + elevated TBG (estrogens)
 Target TSH value in treated pregnant women < 2.5 mIU/L
 A ‘proactive’ higher dose of thyroxin (by 30-50%) has been
recommended once pregnancy is confirmed.
Thyroid dysfunction in pregnancy ↑risk of preeclampsia, miscarriage,
premature birth, and mental disability
Screening of thyreopathies: GYN/GP: TSH, antiTPO
If positive
- Endocrinologist: fT4, antiTG, TRAb
 Congenital hypotyroidism
 The most common preventable cause of mental retardation
 neonatal screening for congenital hypothyroidism (TSH+ fT4)
 3rd - 5th day of life in dried blood spots
 Incidence 1:2 000 – 4 000

• the head is broad

• the eyes wide apart
• the tongue protrudes from the mouth
• all movements and responses are slow and
 Discrepant laboratory findings
 Low T3 syndrome – non thyroidal illnesses
 Drugs – significant source of preanalytical variability
 secretion of TSH,
 concentration of transporting proteins,
 replacement of TH from transporting proteins,
 T4 to T3 conversion,
 biological availabilty of p.o. thyroxine
 Endogenous Ab - interferencies with fT4, fT3 assays
 Analytical interferencies – biotin, endogeneous anti-animal Ab
(HAMA)
 Low T3 syndrome (NTI)
Severe acute or chronic non-thyroidal illnesses (low caloric intake and
hypooxigenation of tissues) influences TH → „sick euthyroid syndrome“

Lower T4 conversion to T3 + elevated production of the reverse

biologically inactive rT3
 Transient and discordant
changes in TH levels
 ↓FT3 and ↑rT3
 N-↓ FT4
 N-slightly ↑TSH
 Pathomechanisms in Low T3 syndrome
 ↑ inflammatory cytokines (TNF...), ↑cortisol (stress hormone) -
alteration of hypothalamic-pituitary-thyroid axis
 Low deiodination T4 in peripheral tissue – ↑ rT3
 ↓uptake of TH in peripheral tissues, malfunction of TH receptors
 decreased transport protein concentration Low peripheral conversion of T4
to T3,
Lack of energy of caloric intake,
stress,
In hospitalized patients is worth to remember: Malfunction of receptors.

 low TSH – more likely NTI than hyperthyroidism

 high TSH - more likely reflects recovery from illness then
hypothyroidism
 Discrepant laboratory findings
 Drugs – significant source of preanalytical variability
 secretion of TSH,
 concentration of transporting proteins,
 replacement of TH from transporting proteins,
 T4 to T3 conversion,
 biological availabilty of p.o. thyroxine
 Endogenous Ab against TH - interferencies with fT4, fT3 assays
 Analytical interferencies – biotin, endogeneous anti-animal Ab
(HAMA)
 Analytical interference
HAMA (human anti-animal antobodies)
binding Ab on the solid phase
endogenous HAMA
measured antigen (TH)
labeled Ab against TH

Biotin (vitamin B7) – food

Sandwich imunoanalysis
supplement, part of multivitamin
pills, drug in sclerosis multiplex...
 Key points
• TSH should be used as the first-line (screening) thyroid function test
• TSH testing is not sufficient for diagnosis of secondary pituitary disease or
monitoring of an early phase of anti-thyroid therapy
• FT4 is the second-line test, used to investigate situations with TSH
abnormalities, or where TSH measurements are known to be unreliable.
• TSH and FT4 reference intervals are not universal. Use the appropriate
interval provided by the laboratory.
• FT3 should be used only to diagnose T3-hyperthyroidism or for monitoring
T3 replacement therapy.
 Key points
• Subclinical thyroid disease is common and is characterized by
an abnormal TSH and FT4 within RI.

• When interpreting discordant FT4 and TSH results, the TSH

test usually yields diagnostically more reliable result.
– In cases of inconsistent results, especially if there is clinical
discordance, consider analytical inference.
– Rare causes of inappropriate TSH include TSH-secreting pituitary
tumors and syndromes of thyroid hormone resistance.
 PART 2
Laboratory evaluation of
adrenal gland disorders
CUSHING´S SYNDROME
ADRENAL INSUFFICIENCY
PHEOCHROMOCYTOMA
Glucocorticoids
1. Influence glucose, lipid and
protein metabolism
2. Regulation of
sodium+water homeostasis
3. Inflammatory response
4. Stress response
 Cushing´s syndrome
= hypercortisolism of endogenous/exogenous origin
1. ACTH dependent 60% 2. ACTH independent 40%
 Pituitary (Cushing´s disease) 55%  Adrenal adenoma
 Ectopic ACTH 5%  Adrenal carcinoma
 Ectopic CRH  GC therapy

 Adrenal hyperplasia (partially

ACTH dependent) - rare
 Signs and symptoms of Cushing´s sy

Most of signs and symptoms are common in general population, but

not all are present in every patient
European Journal of Endocrinology (2015) 173, M33–M38
A. Exclude other causes of ↑cortisol

AIM of screening tests: to distinguish simple non-endocrine causes of

abnormal results: (pseudo-Cushing)
 B. Initial tests
Any of the following tests should be done before admission to the
hospital:

1. Urinary free cortisol:

 Unbound CORT (1%) can be filtrated from blood into urine
 24 h sampling eliminates influence of diurnal rythm
 Storage of urine in the fridge during sampling !!!
Interpretation:
U-CORT <250 nmol/24 h THEN CS is excluded
- False negativity (10-15%): incorrect urine sampling, ↓GFR
- False positivity :high fluid itake
 B. Initial tests
2. Low dose dexamethason suppression test (1mg at 23:00)
investigates whether GC negative feedback is normal or autonomous
- the best screening test with 95% sensitivity (overnight or 48-h
suppresion)
 Normal response: ↓ACTH and CORT (<50 nmol/L);
 Insufficient or low suppression supports DG of Cushings´s
syndrome
3. Late-night salivary cortisol: alternagtive of late night CORT, inform
about diurnal rhythm
 C. Confirmatory tests
- help to determine cause od already established hypercortisolism.
1. Late night serum CORT: confirms loss of circadian rhythm in blood
sampled at 23-24:00 during sleep from a pre-inserted peripheral
cannula(stress-free venipuncture) in hospitalized pts.
Expected decrease (> 50% of morning maximum or S-CORT
>140 nmol/L) is lost in CS.

2. P-ACTH: 2 sampling into EDTA tube (8.00am + 10.00pm)

Undectable-low ACTH: ACTH independent – primary CS
Elevated/normal ACTH: ACTH dependent CS – central CS
↑↑ACTH: ectopic secretion
 C. Confirmatory tests
3. High-dose (8mg) DXM suppression test: distinguishes
Cushing´s disease (central CS) and ectopic ACTH
In central CS: CORT falls below 50% of the basal value
Ectopic ACTH: no suppression of CORT
4. CRH stimulation test measures the ACTH and cortisol levels before
and after injection of CRH.
An exaggerated response in Cushing’s disease, while a flat response in
ectopic ACTH production
Other: petrosal sinus: peripheral vein ACTH ratio which increases after
stimulation with CRH.
MRI – confirms DG of pituitary adenoma
 Adrenocortical insuficiency
1. Primary – Addison´s disease:
Autoimmune (adrenalitis)
Infectious (TBC, meningococcal, CMV, HIV...)
2. Secondary:
Hypothalamic, pituitary (central): ACTH deficiency
Iatrogenic: after longterm GC therapy

Diagnosis: clinical suspition + laboratory tests:

Primary: hypoglycemia, hypotension, shock, ↓Na, ↑K,
Secondary: signs of deficiency of other pituitary hormones
 Diagnosis of Addison´s disease
1a. Short corticotropin test (Synacten=synthetic ACTH):
 Normal: after stimulation cortisol ↑ (>500 nmol/L - thresholds are
method dependent, at least doubling of basal value)
 No response = confirms primary adrenal insuficiency or marked
adrenal atrophy secondary to prolonged absence of ACTH
False positvity: emotional stress, steroid therapy within 12 hrs
before testing, estrogen-containing oral contraceptives

1b. Long corticotropin test: to test adrenal reserve after

longer stimulation (up to 24h), atrophic adrenal cells in secondary
hypoadrenalism need some time to wake up
 Diagnosis of Addison´s disease

2. ↓cortisol +↑ACTH are diagnostic of adrenal failure

!!! Normal cortisol (or U-free cortisol) does not exclude PAI!!!

3. Aldosterone and Renin: ↑ renin + ↓ALDO in PAI

- in the early phase of PAI (high renin + normal/low cortisol)

4. 21-Hydroxylase Antibodies – to confirm autoimmune origin of PAI

5. 17-hydroxyprogesterone – if CAH is sspected (newborn

screening, childrens
 Hyperaldosteronism
PRIMARY (low renin) SECONDARY (high renin)
 Unilateral adenoma – Conn´s  Due to condition stimulating renin
syndrome 60% secretion
 Renal artery stenosis
 Reduced renal Na filtration
 Bilateral hyperplasia 35%
 Hypovolemia, hypotension
 Liver cirrhosis
 Rare causes: primary unilateral
 Heart failure
hyperplasia, carcinoma
 Diuretic therapy !!!

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 Clinical and laboratory findings
 Non specific: tirednes, muscle weakness, headache, polyuria, thirst
 Resistant hypertension (cause of secondary HT in 2-4%)

 Hypokalemia (in 60%, 40% normokalemic) - fatigue, muscle

weakness, muscle cramps, paresthesias, and cardiac
arrhythmias.
 U-Na/K < 1
In pts taking diuretics, diuretics should be withdrown, potassium
stores replenished and potassium measured 2 weeks later

 Metabolic alkalosis: high reabsorption of Na+ + HCO3- in exchange of

K+ and H+
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Diagnostic tests
Screening: plasma ALDO:renin ratio (ARR)
 Sample taken supine after overnight fasting
 HIGH ratio → primary hyperaldosteronism

Diagnostic tests (in the hospital) :

 response of ALDO when changing from supine to erect posture
 NORMAL: standing decreases renal blood flow and ↓BP→
↑ renin + ↑ATII + ↑ALDO
 suppression of ALDO after Na load: ↓ renin + ↓ALDO
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 Adrenal medulla
 Hormones derived from
tyrosine
 Catechol cycle
 Adrenalin/epinephrine
 Noradrenalin/norepinephrine

 Neurotransmitters in
CNS, PSNS

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 Pheochromocytoma
 = rare tumor from chromaffine cells localized in adrenal medulla (in 90%)
Rule of „10“
 10% extraadrenal = Paraganglioma
 10% bilateral
 10% familiar (MEN II)
 10% in children
 10% malignant
 10% asymptomatic - „incidentaloma“

 produces mixture of A+NA + metanephrines

70
 Clinical presentation of PEO
 Pressure 90%
 Pain = headache 80%
 Perspiration, sweating 71%
 Palpitation 64% 6„P“ [Link]
 Pallor 42%
 Paroxysmal occurence of symptom

 Others: Abdominal pain, chest pain, panic attacks,

 Metabolic: weight loss, IGT, ketosis (↑lipolysis),

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 Laboratory diagnostics always preceeds
imagine localization testing !!!
 Confirmation of an excessive production of hormones and
their metabolites in blood and urine
 Method of the 1st choice: plasma free MN or urinary MN
Parameter Sensitivity % Specificity %
 P- free MN
Reasons: 99 97
1. Tumor
P- CHA produces MN continuously, physiological
84 release of 81
catecholamines is episodic
U-fractionated MN 97 69
2. Stress stimulation of medulla → increases catecholamines, but
U- CHA 86 88
free metanephrines are unaffected
72 U-VMA 64 93
 Interpretation of initial test results
 Symptomatic patients with normal test results of plasma free MN = DG
excluded (high sensitivity)
 Increase in P-free MN/NMN (3.5 to 4-fold above the ULRR) makes the
presence of PEO extremely likely (100% specificity).

 Borderline elevation of MN/NMN is: repeat biochemical tests

 optimizing sampling conditions and
 elimination of interferring drugs
 Clonidine suppresive test: in case of mildly ↑NMN level: suppresses
release of neuronal NA (and thereby of NMN)
 Persisting high NMN level after clonidine supports the presence of PEO
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