APFCB Masterclass in Interpretative Commenting

Endocrinology Tests -
Calcium and parathyroid disorders

Sam Vasikaran
PathWest-Laboratory Medicine WA
Perth, Western Australia

ASIA-PACIFIC FEDERATION FOR CLINICAL

BIOCHEMISTRY AND LABORATORY MEDICINE
 Agenda

1. Calcium and PTH physiology

2. Hypercalcaemia – investigation and cases

3. Hypocalcaemia – investigation and cases

 Plasma calcium
• Total Calcium = Ionised + Bound + Complexed

• Ionised Calcium ~ 50%

– Biologically active

• Protein bound Calcium ~ 45%

– Mainly bound to albumin
– Biologically inactive

• Complexed Calcium ~ 5%
– citrate, phosphate etc
 Calcium and pH :
• Protein binding of calcium influenced by pH.

•  pH   ionised Ca

– eg. respiratory alkalosis due to hyperventilation can

precipitate tetany

•  pH   ionised Ca

– eg. chronic renal failure patients with acidosis - normal

ionised calcium, Total calcium
 Ionised calcium

• Reflects calcium status more accurately than plasma total calcium

• More sensitive than plasma total calcium for diagnosing primary

hyperparathyroidism

• Sampling precautions needed (Full tube, keep cool)

– pH  due to RBC metabolism  lactate formation
– pH  due to CO2 loss

• pH correction of reported Ca++ if pH change post collection, but not if

patient has acid base imbalance
 Adjusted total calcium

• When using total calcium, adjustment for albumin

abnormalities better reflects ionised calcium

• Calculation:
T Ca(adj) = T Ca + (40 - Alb) x 0.02

• Inaccurate at extremes of albumin (<30g/L)

 Adjusted total calcium

• Calculation:
T Ca(adj) = T Ca + (40 - Alb) x 0.02

Plasma
Total Ca 2.00 mmol/L (2.15-2.55)
Albumin 30 g/L (35-45)
Adjusted Ca = 2.0 + [(40-30)x0.02]
Adjusted Ca 2.20 mmol/L
Normal relationship between plasma calcium and PTH

10.0

9.0
PTH (pmol/L)

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0
1 1.5 2 2.5 3 3.5

Ca (mmol/L)
 Patient preparation and sampling

• Measure PTH with simultaneous calcium

• Fasting for Ca and PO4
• Same time of day when measuring serially
– Fasting morning preferred for other measurements

• EDTA preferred (stable at room temp for 24 hours)

 PTH reference interval

• Units: pmol/L (ng/L also used; divide by ~ 9)

• Should be specific to the population, laboratory, and assay
– Lack of standardisation of commercial PTH assays

• Derived from vitamin D replete subjects with normal renal

function
– (small seasonal variation at least at higher latitudes)
 Investigating hypercalcaemia

• Sorting out the common causes

– Consider albumin-adjusted (total) calcium
– Confirm with ionised calcium
– Check drug history
– Check for renal failure
– Simultaneous Ca & PTH

• Consider rarer causes & more complex investigations

Causes of hypercalcaemia
• Primary hyperparathyroidism
• Malignancy
– Lytic lesions
– Humoral eg PTHrp
• Drugs
– Thiazide diuretics, lithium, calcitriol
• Hyperthyroidism, hypoadrenalism
• Excess absorption
– Vitamin D intoxication / granulomatous disease
• (raised 1,25D)
• Immobilisation
• Chronic renal failure (Tertiary hyperparathyroidism)
 Is it Parathyroid dependent?

Parathyroid dependent

10.0

9.0
PTH (pmol/L)

8.0

7.0

60

5.0

4.0
Non parathyroid
3.0
dependent
2.0

1.0

0
1 1.5 2 2.5 3 3.5

Ca (mmol/L)
 Is it Parathyroid dependent?

Parathyroid dependent

10.0

9.0
PTH (pmol/L)

8.0

7.0

60

5.0

4.0
Non parathyroid
3.0
dependent
2.0

1.0

0
1 1.5 2 2.5 3 3.5

Ca (mmol/L)
 Patient: 79 year old woman
Clinical notes: Fracture neck of femur
Location: Orthopaedic ward
• Results

Total Calcium (Albumin adjusted) 2.86 2.15-2.60 mmol/L

Phosphate 0.83 0.80 - 1.40 mmol/L
Creatinine 55 umol/L (50-90)
eGFR 86 (> 90)

PTH 6.1 1.0 – 7.0 pmol/L

Is it parathyroid dependent?

10.0

9.0
Iry hyperparathyroidism
PTH (pmol/L)

8.0

7.0

6.0 PTH 6.1 (1.3 - 7.6)

5.0 Adj Ca 2.86 (2.15 - 2.55)
4.0

3.0

2.0

1.0

0
1 1.5 2 2.5 3 3.5

Ca (mmol/L)
 Patient: 79 year old woman
Clinical notes: Fracture neck of femur
Location: Orthopaedic ward
• Results

Total Calcium (Albumin adjusted) 2.86 2.15-2.60 mmol/L

Phosphate 0.83 0.80 - 1.40 mmol/L
Creatinine 55 umol/L (50-90)
eGFR 86 (> 90)

PTH 6.1 1.0 – 7.0 pmol/L

Comment:
The elevated calcium should suppress PTH, therefore the normal PTH level does not exclude primary
hyperparathyroidism.
RARE causes of this biochemical picture include lithium therapy and familial hypocalciuric
hypercalcaemia.
 Adjuncts to interpretation of PTH result

• Very high PTH values usually seen in CRF,

– but if in Iry HPT (>3-10 x URL), consider parathyroid carcinoma

• Iry HPT favoured by

– ↓Serum PO4, ↓TmP GFR (renal PO4 threshold),
– Urine calcium conservation

• Beware of FHH
– Family history, previous serum calcium, genetic testing
– calcium to creatinine clearance ratio
• (24hr Uca x Sca)/(24hr Ucr x Scr) usually <0.01in FHH
• However, this degree of hypocalciuria can rarely be seen in HPT due
to VitaminD/calcium deficiency, thiazide Rx
Calcium excretion as a measure of serum
calcium

Renal
Calcium
Wasting

Renal
Calcium
Conservation
 Urine calcium excretion
• Fasting morning spot urine calcium Spot urine

Calcium/urine Creatinine x serum Creatinine

< 30umol/ L GFR in FHH

Renal
Calcium
Wasting

Renal
Calcium
Conservation

• 24 hour urine calcium creatinine clearance ratio

24 hour urine Ca/ serum Ca x serum Creatinine/urine Creatinine

< 0.01 in FHH

> 0.02 in PHPT

Iry HPT Vs FHH
 Familial hypocalciuric hypercalcaemia

• Asymptomatic (benign) hypercalcaemia

• Family history (autosomal dominant)

• Mutation of calcium sensing receptor with setpoint

•  calcium excretion

• PTH high normal or mildly raised

• No treatment needed (danger of inappropriate

surgery)
Hypercalcaemia - pitfalls in diagnosis

• Acute decrease in calcium  PTH

• Hypercalcaemia with a PTH within reference interval
• Coexisting Iry HPT in a patient with malignancy
• PTH degradation during storage or short sample in EDTA
tube  falsely low PTH
• Calcium bound to monoclonal globulin
• Heterophile antibody
• Batch to batch variation (Shifts and drifts)
“Normocalcaemic primary hyperparathyroidism”
 52 year old woman with bipolar disorder

• Results
Sodium 159 134 - 146 mmol/L
Potassium 3.6 3.4 - 5.3 mmol/L
Chloride 125 95 - 105 mmol/L
Bicarbonate 19 22 - 32 mmol/L
Urea 17.8 3.0 - 8.0 mmol/L
Creatinine 191 50 - 90 mmol/L
Glucose 7.3 3.0 - 5.5 mmol/L

Total Calcium (Albumin adjusted) 2.66 2.15-2.60 mmol/L

Phosphate 1.43 0.80 - 1.40 mmol/L
 52 year old woman with bipolar disorder

• Results

Sodium 159 134 - 146 mmol/L

Potassium 3.6 3.4 - 5.3 mmol/L
Chloride 125 95 - 105 mmol/L
Bicarbonate 19 22 - 32 mmol/L
Urea 17.8 3.0 - 8.0 mmol/L
Creatinine 191 50 - 90 mmol/L
Glucose 7.3 3.0 - 5.5 mmol/L

Total Calcium (Albumin adjusted) 2.66 2.15-2.60 mmol/L

Phosphate 1.43 0.80 - 1.40 mmol/L

Li 1.73 mmol/L
PTH 8.3 1.0 – 6.5 pmol/L

Comment: This patient is on lithium; sustained increase in serum calcium after cessation
of therapy would indicate underlying primary hyperparathyroidism.
 54 year old woman with lethargy

• Results

Sodium 129 134 - 146 mmol/L

Potassium 6.6 3.4 - 5.3 mmol/L
Chloride 98 95 - 105 mmol/L
Bicarbonate 19 22 - 32 mmol/L
Urea 8.8 3.0 - 8.0 mmol/L
Creatinine 141 50 - 90 mmol/L
Glucose 3.3 3.0 - 5.5 mmol/L

Total Calcium (Albumin adjusted) 2.76 2.15-2.60 mmol/L

Phosphate 1.67 0.80 - 1.40 mmol/L
 54 year old woman with lethargy

Results

Sodium 129 134 - 146 mmol/L

Potassium 6.6 3.4 - 5.3 mmol/L
Chloride 98 95 - 105 mmol/L
Bicarbonate 19 22 - 32 mmol/L
Urea 8.8 3.0 - 8.0 mmol/L
Creatinine 141 50 - 90 mmol/L
Glucose 3.3 3.0 - 5.5 mmol/L

Total Calcium (Albumin adjusted) 2.76 2.15-2.60 mmol/L

Phosphate 1.67 0.80 - 1.40 mmol/L

PTH 1.4 1.0 – 6.5 pmol/L

Causes of PTH independent hypercalcaemia

• Malignancy
– Lytic lesions
– Humoral eg PTHrp
• Drugs
– calcitriol
• Hyperthyroidism, hypoadrenalism
• Excess absorption
– Vitamin D intoxication / granulomatous disease
• (raised 1,25D)
• Immobilisation
 54 year old woman with lethargy
Results

Sodium 129 134 - 146 mmol/L

Potassium 6.6 3.4 - 5.3 mmol/L
Chloride 98 95 - 105 mmol/L
Bicarbonate 19 22 - 32 mmol/L
Urea 8.8 3.0 - 8.0 mmol/L
Creatinine 141 50 - 90 mmol/L
Glucose 3.3 3.0 - 5.5 mmol/L

Total Calcium (Albumin adjusted) 2.76 2.15-2.60 mmol/L

Phosphate 1.67 0.80 - 1.40 mmol/L

PTH 1.4 1.0 – 6.5 pmol/L

Cortisol 123 150-700 nmol/L

ACTH 34 2.0-10.0 pmol/L

Comment: The electrolyte abnormalities, with a low cortisol and raised ACTH are
consistent with primary adrenal failure which can cause hyperalcaemia.
 72 year old man with bone pain and anaemia

• Results

Total Protein 95 65 - 80 g/L

Albumin 30 30 - 50 g/L
Alk Phos 74 35 - 135 U/L
ALT 7 < 40 U/L
AST 24 < 30 U/L
Bilirubin 7 < 20 umol

T Calcium (Albumin adjusted) 3.36 2.15-2.60 mmol/L

Phosphate 1.34 0.80 - 1.40 mmol/L
PTH 1.2 0.9 – 9.0 pmol/L

Comment: The elevated calcium and suppressed PTH exclude primary

hyperparathyroidism. The commonest cause is malignancy. Suggest serum protein
electrophoresis, and free light chain measurement in view of the increased globulins.
 26 year old man in ICU
Unconscious long-term following head injury

Plasma/Serum

• Calcium - (Albumin adjusted) 2.97 mmol/L (2.15-2.60)

• Phosphate 1.46 mmol/L (0.80-1.50)
• Creatinine 69 umol/L (60-110)
• Alkaline Phosphatase 84 U/L (35-135)
• PTH 0.6 pmol/L (0.7-7.0)
• 25-hydroxyvitamin D 111 nmol/L (> 50)

• Serum CTX 1370 ng/L (100-600)

• Comment: The increased serum calcium and suppressed PTH is consistent with
PTH-independent hypercalcaemia which may be caused by increased bone resorption
due to immobilisation.
 58 year old man
Heaemodialysis Unit

Plasma/Serum

• Calcium - (Albumin adjusted) 2.69 mmol/L (2.15-2.60)

• Phosphate 1.54 mmol/L (0.80-1.50)
• Creatinine 785 umol/L (60-110)
• eGFR 6 (> 90)
• Alkaline Phosphatase 891 U/L (35-135)
• ALT 25 U/L (< 40)
• Gamma GT 49 U/L (< 60)

• PTH 557 pmol/L (0.7-7.0)

Comment: The increased serum calcium and PTH in this patient with CKD is consistent
with tertiary hyperparathyroidism
PTH in renal failure
Biochemistry of hyperparathyroidism

Alk Phos Ca Phosphate PTH

1ry hyperPTH

2ry hyperPTH

3ry hyperPTH *

* (in Chronic Renal Failure serum PO4 increase due to renal PO4 retention)
 Specialised tests
• PTHrP
• Associated with some cases of malignant hypercalcaemia (humoral)
• Generally not necessary for diagnosing malignant hypercalcaemia
• Not useful as tumour marker

• 1,25 dihydroxyvitamin D
• Raised when it causes hypercalcaemia in granulomatous disorders
• Also raised or high-normal in Iry hyperparathyroidism

• Calcitonin
• Not important in calcium metabolism
• Not measured for calcium related investigations
• Tumour marker for MEN 2
• Has some therapeutic uses
Hypocalcaemia
 Causes of hypocalcaemia
Exclude hypoalbuminaemia
• Vitamin D deficiency
• Renal failure (2ry hyperparathyroidism)
• Hypoparathyroidism
– (Exclude magnesium deficiency)

• Rare genetic conditions

– Failure of Vitamin D metabolism or end organ D- resistance
– Pseudohypoparathyroidism
• End organ PTH-resistance
 56 Years Female
APECED syndrome
Serum/plasma
• Total Calcium adjusted 2.02 mmol/L (2.10-2.55)
• Phosphate 1.58 mmol/L (0.75-1.50)
• Alkaline Phosphatase 75 U/L (30-110)

• 25-hydroxyvitamin D 81 nmol/L (> 50)

• PTH (Abbott) < 0.3 pmol/L (1.6-9.0)

Urine
• Urine Calcium Excretion 33 umol/L GF (4-21)
• Renal Phosphate Threshold 1.53 mmol/L GF (0.75-1.35)
 Is it hypoparathyroidism or
vitamin D deficiency?
Vitamin D Deficiency

10.0 PTH 8.8 (1.3 - 7.6)

9.0
Adj Ca 1.86 (2.15 - 2.55)
PTH (pmol/L)

8.0

7.0

60

5.0

4.0

3.0

2.0 PTH <0.3 (1.3 - 7.6)

Adj Ca 2.02 (2.15 - 2.55)
1.0

0
1 1.5 2 2.5 3 3.5

Ca (mmol/L)
Hypoparathyroidism
 56 Years Female
APECED syndrome
Serum/plasma
• Total Calcium adjusted 2.02 mmol/L (2.10-2.55)
• Phosphate 1.58 mmol/L (0.75-1.50)
• Alkaline Phosphatase 75 U/L (30-110)

• 25-hydroxyvitamin D 81 nmol/L (> 50)

• PTH (Abbott) < 0.3 pmol/L (1.6-9.0)

Urine
• Urine Calcium Excretion 33 umol/L GF (4-21)
• Renal Phosphate Threshold 1.53 mmol/L GF (0.75-1.35)

• Results consistent with hypoparathyroidism

• (APECED) = autoimmune polyendocrine syndrome type I
• [Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy]
 Vitamin D deficiency definitions

Controversial

• Mild deficiency 25 - 50 nmol/L

• Moderate deficiency 12.5 - 25 nmol/L

• Severe deficiency <12.5 nmol/L

 Severe vitamin D deficiency

• Adult form – osteomalacia

– widened osteoid seams with lack of mineralisation

• Classic childhood rickets

– - widened epiphyses & poor skeletal growth

• Failure of vitamin D metabolism or effectiveness

– eg in renal disease and some rare genetic forms
• Laboratory findings
– Hypocalcaemia with secondary hyperparathyroidism
– Raised alkaline phosphatase
 82 year old woman
Clinical details: House bound. Muscle weakness, falls
?Vitamin D deficiency

• Plasma/Serum

• Calcium - Ionised (pH 7.40) 1.01 mmol/L (1.12-1.32)

• Calcium (Albumin adjusted) 1.97 mmol/L (2.15-2.60)
• Phosphate 0.82 mmol/L (0.80-1.40)
• Creatinine 51 umol/L (45-90)
• Alkaline Phosphatase 278 U/L (35-135)
• PTH 87 pmol/L (0.9 – 9.0)
 82 year old woman
Clinical details: House bound. Muscle weakness, falls
?Vitamin D deficiency

• Plasma/Serum

• Calcium - Ionised (pH 7.40) 1.01 mmol/L (1.12-1.32)

• Calcium (Albumin adjusted) 1.97 mmol/L (2.15-2.60)
• Phosphate 0.82 mmol/L (0.80-1.40)
• Creatinine 51 umol/L (45-90)
• Alkaline Phosphatase 278 U/L (35-135)
• PTH 87 pmol/L (0.9 – 9.0)
• 25D <10 nmol/L (> 50)

• Comment: Normal or raised PTH in association with normal to low calcium may occur
in Vitamin D deficient states.
 2 year old girl; failure to thrive
History: Fussy eater
Wt and height tracked on 50th %ile until 6/12 ago
progressively dropped off below 3rd centile

• Total Protein 64 (58-78) g/L

• Albumin 44 (32-48) g/L
• Globulins 20 (20-34) g/L
• Bilirubin 11 (< 20) umol/L
• ALT 20 (< 35) U/L
• Alk. Phos. 1020 (120-370) U/L
• Gamma GT 12 (< 20) U/L
• Phosphate 0.90 (1.10-2.20) mmol/L
• Calcium (Adj) 2.13 (2.20-2.65) mmol/L

• PTH (Abbott) 54 pmol/L (1.6-9.0)

• 25-hydroxyvitamin D <10 nmol/L (> 50)
 2year old female

• Xray: metaphyseal flaring with cupping, “osteopenia”, Consistent with rickets

• Mother had severe D deficiency when pregnant and supplemented but patient
was not

Plasma/Serum
• Ferritin 19 (20-100) ug/L
• Iron 11 (5-25) umol/L
• Transferrin 35 (20-43) umol/L
• %Transferrin 16 (10-45) %

• Rx: OsteVit-D 5000 IU daily and FeSO4 15 mg daily

 76 year old male
Post- bisphosphonate (zoledronic acid) infusion

Plasma/Serum

• Calcium - Ionised (pH 7.40) 0.81 mmol/L (1.12-1.32) Total

• Calcium - (Albumin adjusted) 1.59 mmol/L (2.15-2.60)
• Phosphate 1.25 mmol/L (0.80-1.50)
• Creatinine 65 umol/L (60-110)

• PTH 35 pmol/L (0.7-7.0)

• 25-hydroxyvitamin D 68 nmol/L (> 50)

• Comment: Results are in keeping with zoledronic acid induced

hypocalcaemia
2ry hyperparathyroidism with normal calcium
Mild-moderate vitamin D insufficiency
Early chronic renal failure
Bisphosphonate /antiresorptive therapy

10.0

9.0 PTH 8.1 (1.3 - 7.6)

Adj Ca 2.26 (2.15 - 2.55)
PTH (pmol/L)

8.0

7.0

60

5.0

4.0

3.0

2.0

1.0

0
1 1.5 2 2.5 3 3.5

Total Ca (mmol/L)
 Causes of secondary hyperparathyroidism

• Chronic kidney disease

• Decreased calcium intake
• Calcium malabsorption
– Vitamin D deficiency
– Bariatric surgery
– Coeliac disease
– Pancreatic disease (fat malabsorption)
• Renal calcium loss
– Idiopathic hypercalciuria
– Loop diuretics
• Inhibition of bone resorption
– Bisphosphonates, Denosumab
– Hungry bone syndrome
 63 year old female
Lethargy and weakness

• Total Calcium (Adj) 1.87 (2.10-2.60) mmol/L

• Phosphate 0.54 (0.80-1.40) mmol/L
• Alk. Phos. 54 (35-135) U/L
• Gamma GT 34 (< 40) U/L

• PTH (Abbott) 41 pmol/L (1.6-9.0)

• 25-hydroxyvitamin D 93 nmol/L (> 50)

• Renal PO4 threshold 0.12 mmol/L GF (0.75-1.35)

• Confirms renal PO4 wasting

 63 year old female
Lethargy and weakness

• Total Calcium (Adj) 1.87 (2.10-2.60) mmol/L

• Phosphate 0.54 (0.80-1.40) mmol/L
• Alk. Phos. 54 (35-135) U/L
• Gamma GT 34 (< 40) U/L

• PTH (Abbott) 41 pmol/L (1.6-9.0)

• 25-hydroxyvitamin D 93 nmol/L (> 50)

• History of recent iron transfusion

• Comment: Severe hypophosphataemia and hypocalcaemia
with normal vitamin D and 2ry hyperparathyroidism.
• Iron infusion can be rarely associated with FGF 23
induced hypophosphataemia
 76 year old male
H/O Bone pain
• Total Protein 67 g/L (60-80)
• Albumin 34 g/L (35-50)
• Globulins 33 g/L (23-35)
• Bilirubin 11 umol/L (< 20)
• ALT 3 U/L (< 40)
• Alk Phos 2090 U/L (35-135)
• Gamma GT 151 U/L (< 60)

• Total Calcium (adj) 1.77 mmol/L (2.15-2.60)

• Phosphate 0.83 mmol/L (0.80-1.50)
• Magnesium 1.03 mmol/L (0.70-1.10)

• PSA 2600 ug/L (<6.5)

• Comment: Metastatic osteoblastic secondaries may cause hypocalcaemia.

Suggest measure 25 hydroxyvitamin D to exclude deficiency. Skeletal
imaging studies may be informative.
 Ca & PTH biochemistry
Alk Phos Ca Phosphate PTH
•HyperPTH

•Osteomalacia ( ) ( )

•Osteoporosis N N N N

•Paget’s N N N

•Malignant ( ) N ( )
hypercalcaemia
Thank you