Alkaline phosphatase Laboratory Perspective
Transcript of Alkaline phosphatase Laboratory Perspective
Alkaline phosphatase
Laboratory Perspective
Professor William D Fraser
Professor of Medicine
University of East Anglia,
Norwich, UK
Disclosures – Prof. Fraser
Unrestricted funding for assay development from Roche,
Siemens, and IDS
Holds patents with IDS regarding assays in development
Consultant fees from Alexion Pharmaceuticals
Alkaline phosphatase (ALP)
A hydrolase enzyme (EC 3.1.3.1)
Removes phosphate groups from several
molecules:
Proteins, nucleotides and alkaloids
Optimal activity in vitro in an alkaline environment1
1. Vroon DH and Israili Z. Alkaline phosphatase and gamma glutamyltransferase.
In: Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston 1990.
Alkaline phosphatase (ALP)
In humans, the following isoenzymes1 are commonly detected:
Tissue non-specific ALP (liver, bone, kidney)
Intestinal ALP
Placental ALP
Germ cell ALP
Highest concentrations are found in liver, bone, placenta, intestine
and kidney
Several techniques exist for the quantification of ALP and the
isoenzymes
1. Whyte MP. Hypophosphatasia. In: Thakker RV, Whyte MP, Eisman J, Igarashi T, ed.
Genetics of Bone Biology and Skeletal Disease. Amsterdam: Elsevier/Academic Press; 2013.
Assessing total ALP concentration by
enzymatic colorimetric methods
Relatively cheap and fully automated
Following the cleavage of PO4 groups from the substrates,
molecules with specific quantifiable absorbance characteristics
are generated, e.g.
Disodium p-nitrophenyl phosphate to p-nitrophenol
4-nitrophenyl phosphate to 4-nitrophenoxide
Zn and Mg are important co-factors required for the action of ALP
in these reactions
Rearranges at
alkaline pH
O
P O- O
O-
N
O
O- O N
O
P O- O
O-
O O
HOH
N
O
P O- O
O-
O O
+ ALP
Zn Mg2+
pH 10.3
+
4-Nitrophenyl phosphate
(colourless)
4-Nitrophenoxide
(colourless
benzenoid form)
4-Nitrophenoxide
(yellow, quinonoid form)
Rate of formation at 37C
Buffers (amino alcohols):
Diethanolamine (DEA) Germany/Scandinavia
2-amino-2-methyl-1-proponol (AMP) IFCC/USA/France
HEDTA
Method N Mean CV, % Um
Roche, AMP buffer IFCC 499 16.259 6.8 0.06
AMP, optimised to IFCC 379 19.707 12.8 0.16
Diethanolamine buffer, DEA 111 39.079 17.8 0.82
AMP, non-optimised 92 18.612 7.7 0.19
Ortho Viros MicroSlide Systems 89 26.132 8.5 0.29
Dade Dimension, AMP buffer 80 27.277 22.4 0.86
Other AMP kits 65 18.063 7.8 0.22
Tris/carbonate buffer, KA units 44 31.591 31.8 1.89
AMP, optimised to NVKC/SFBC 6 21.167 8.5 0.92
Agappe – DGKC-SCE 4 45.000 28.7 8.08
Colorimetric 4 18.050 14.9 1.68
AMP, reduced interference 4 16.500 6.1 0.63
- Select - 2 20.500 3.4 0.62
ALP (WEQAS Results)
WEQAS: Wales External Quality Assessment Scheme
provider in the UK
N Mean CV, % Um SDP
A
Exc.
All methods 1372 19.705 25.1 0.17 1.80 164
AMP, optimised to IFCC 379 19.707 12.8 0.16 1.80 41
Siemens ADVIA
1200/1650/1800/2400
42 19.049 8.0 0.3 1.74 3
ALP, 37°C
U/L
Num
ber
of la
bora
tories
The most common approach for isolating isoenzymes
Electrophoresis followed by specific substrate-staining methods
Detection of bone-specific ALP
Specific antibody-employing immunoassays for bone ALP1
Measure activity of ALP
Measure the mass of the molecule
Disadvantages
Cross reactivity with liver ALP in ~3–16% of these assays2,3,4
1. Manufacturers Pack inserts (IDS, Oxford Biosystems).
2. Fraser WD. Data on file.
3. Gomez, et al. Clin Chem. 1995;41(11):1560-1566.
4. Broyles, et al. Clin Chem. 1998;44(10):2139-2147.
Detection of ALP
Measurement of ALP
Historically, the vast majority of ALP assays have been
performed to investigate diseases in which ALP is
increased
10–12% of the test results were due to abnormal liver function,
vitamin D deficiency, metabolic bone disease (e.g. Paget’s
disease)
As a result, less attention has been given to low ALP
concentrations (0.4% of results)
ALP reference ranges may also contribute to the
underestimation of the prevalence of low ALP
Representative reference ranges for ALP
(Europe and the USA)
Specimen Age group/age Concentration or enzyme
activity Concentration (SI
units) Source
Adult 36–92 U/L 0.5–1.5 µkat/L Merck [1] Serum Adult 30–120 U/L 0.5–2.0 nkat/L USA [2] Plasma
1–11 m
1–3 y
10–11 y
≥20 y
Male
70–350 U/L
125–320 U/L
150–470 U/L
40–120 U/L
Female
70–350 U/L
125–320 U/L
150–530 U/L
40–120 U/L
ARUP [3]
Plasma Neonate
Infant
1–14 y
14–16 y
Adult
73–391 U/L
59–425 U/L
76–308 U/L
49–242 U/L
30–130 U/L
Europe [4]
1. Wians FH. Merck manuals. Available at http://www.merckmanuals.com/professional/appendixes/normal-laboratory-values/blood-
tests-normal-values.
2. Kratz A, et al. N Engl J Med 2004;351:1548–63.
3. ARUP National Laboratory. Alkaline Phosphatase Isoenzymes, Serum or Plasma. Available at http://ltd.aruplab.com.
4. Sheffield children’s NHS foundation trust. Laboratory handbook. April 2014.
Reference ranges
A recent survey of 26 laboratories in the UK revealed:
The lower limit was stated as being zero (0) U/L by
two laboratories
No gender-specific reference range by eight laboratories1
Often a lack of recognition that paediatric reference
ranges are significantly higher than adult ranges
1. Fraser WD. Data on file held at the University of East Anglia.
Canada:
CALIPER
CALIPER: Canadian Laboratory Initiative on Paediatric Reference Intervals
(http://www.sickkids.ca/caliperproject/index.html)
Age (years)
CALIPER
CALIPER Cohort samples have been used to produce
paediatric reference ranges for most of the major
manufacturers machines/methods. (Adeli K)
Survey of child and adolescent health:
KiGGS ALP levels, boys
• IFCC standard
method (Hitachi 917)
KiGGS:a long-term study conducted by the Robert Koch Institute
http://www.kiggs-studie.de/english/home.html
Survey of child and adolescent health:
KiGGS ALP levels, girls
• IFCC standard
method (Hitachi 917)
KiGGS:a long-term study conducted by the Robert Koch Institute
http://www.kiggs-studie.de/english/home.html
Representative reference ranges for
bone ALP (Europe and the USA)
1. ARUP National Laboratory. Alkaline Phosphatase Isoenzymes, Serum or Plasma. Available at ttp://ltd.aruplab.com/Tests/Pub/0021020.
2. ARUP National Laboratory. Bone Specific Alkaline Phosphatase. Available at http://ltd.aruplab.com/Tests/Pub/0070053.
Specimen Age group/age Concentration or
enzyme activity
(conventional units)
Concentration (SI units) Source
Serum
6 m–2 y
3–6 y
7–9 y
10–12 y
≥25 y
Premenopausal female
Male
31.6–122.6 µg/L
31.3–103.4 µg/L
48.6–140.4 µg/L
48.8–155.5 µg/L
6.5–20.1 µg/L
Female
33.4–145.3 µg/L
32.9–108.6 µg/L
36.3–159.4 µg/L
44.2–163.3 µg/L
4.5–16.9 µg/L/7.0–22.4 µg/L
ARUP [1]
1–6 y
7–9 y
10–15 y
16–19 y
Females ≥16 y and males ≥20 y
Male
0–208 U/L
0–264 U/L
0–340 U/L
0–165 U/L
0–55 U/L
Female
0–189 U/L
0–246 U/L
0–340 U/L
0–91 U/L
0–55 U/L
ARUP [2]
Low ALP
Several clinical conditions may result in a low ALP
An algorithm has been developed that should help to
guide the investigation of a low ALP
ALP decision
algorithm
ECG: electrocardiogram; ESRF: end stage renal failure; FBC: full blood
count; PTH: parathyroid hormone; TSH: thyroid stimulating hormone
Metal
analysis
low Zn, Mg,
high Cu
Disease-associated
hypothyroidism,
hypoparathyroidism,
severe anaemia
Vitamin abnormality
low vitamin C, B12,
B6, folate,
excessive vitamin D
Post-cardiac surgery
post liver transplant
ESRF
osteodystrophy
achondroplasia
cretinism
Drug effect
estrogen,
bisphosphonate,
clofibrate,
omeprazole,
lansoprazole
Abnormal
investigate
and correct
Measure TSH,
PTH, FBC
Correct
abnormality
Measure and
correct
abnormality
Possible genetic cause for HPP
PEA and PLP measurement
Genetic testing of ALPL
Review medical
history for low ALP
Protein
calorie
malnutrition
Drug therapy
may unmask
genetic cause
for low ALP
Total protein
albumin
Non-biochemical tests
radiology, ECG muscle studies
ALP remains below reference range after
correction of abnormality
Artefact
EDTA, oxalate (blood transfusion)
Confirm low on serum sample
ALP lower than appropriate
reference range
Pathophysiology of HPP
PPi
Ca
Pi
ALP
Pi
HPP is characterised by accumulation of PPi,
which suppresses hydroxyapatite crystal formation
NPP1
(ENPP1)
ATP
Inhibition of
hydroxyapatite crystal
Formation
Promotion of
hydroxyapatite crystal
formation
Bone mineralisation
Alkaline phosphatase gene (ALPL)
Over 280 known loss-of-function mutations in the alkaline
phosphatase gene (ALPL)
Autosomal recessive versus autosomal dominant
transmission determines the clinical severity
http://www.sesep.uvsq.fr/03_hypo_mutations.php
Serum ALP activity in HPP
Normal mean and range (± 2 SD mean)
Children 166 (80–342); Adults 51 (28–91)
O = odontohypophosphatasia
Whyte MP. In: Bilezikian JP, Raisz LG, Martin TJ, eds. Principles of Bone Biology. Vol 1. 3rd ed. San Diego, CA, USA, Academic Press. 2008;1573–98.
Clinical Features of Hypophosphatasia
Perinatal/Infantile Severe Hypomineralisation/ Skeletal Deformities
Ricketic Type Lesions
Fractures
Respiratory Failure
Poor Feeding/Weight Gain/ Failure to Thrive
Hypotonia
Vitamin B6 Responsive Seizures
Hypercalcaemia/Hypercalciuria/Nephrocalcinosis
Craniosynostosis
Clinical Features of Hypophosphatasia
Juvenile (6 months to 18y)
Skeletal Deformities
Ricketic Type Lesions
Recurrent Fractures/Poor Healing Fractures
Low BMD
Short Stature
Muscle Weakness
Waddling gait
Premature Tooth Loss
Adult Hypophosphatasia (≥18y)
Liverpool 12 patients
Norwich 26 patients – Age 17-86
– 26 detected by biochemical testing after presenting with bone aches (16) joint pains (14) or recurrent fractures (10)
– Low Total ALP in all cases (many (10) had abnormal ALP in childhood missed –used wrong reference ranges)
– Family connections in 8 (cousins, uncle and niece, brothers)
– Misdiagnosis: Multiple Sclerosis (2)
Symptoms
Bone aches(18)
Fracture pain (6)
Joint aches (16), Osteoarthritis (14), Articular Chondrocalcinosis (3)
Abdominal Pain (9)
Joint Laxity, Recurrent Dislocation - especially younger female patients 17-34 y (5)
• Difficulty with household tasks, concentrating, working (hairdressing x3)
An Adult Case – 86 y Old Female
PMH
Asthma
Hypertension
Cholecystectomy
2 humeral # as child
Also # x3 wrist, x2 ankle, left femoral
DHx
• Felodipine
• Montekukast
• Steroid inhaler
• Omeprazole
• Mirtazapine
• Bendroflumethiazide
• Domperidone + erythromycin
• Alendronic acid ( 8 months )
SH
• Never smoked
• Infrequent alcohol
Fracture History
2000 – FOOSH – smith’s right wrist
2006 – leg gave way - midshaft femur
2007 – Fall – 2nd metatarsal
2011 – Fall – left distal radius
5/2012 – Fall – right distal radius
Further Investigations
FBC U+E LFT LFT Bone Other
Hb 114 Cr 87 Bil 7 aCa2+ 2.44 TSH 1.59
MCV 95 eGFR 54 Tot Prot 62 PO4- 1.24 FSH 36.3
Wbc 5.5 Na 136 Alb 35 ALP 22 LH 15.9
Plt 273 K 3.5 ALT 14 Vit D - 42 Prolac
1397
Dexa Scan
T score – 1.7 & Z score of 1.2 lumbar spine
21% increase since last scan in 2000
What can we learn from this case?
Not all atypical femoral fractures are BP induced
Always consider hypophosphatasia in these patients
Do BPs bring out phenotype in previously subclinical
hypophosphatasia?
Treatment
Pathophysiology of HPP
PPi
Ca
Pi
ALP
Pi
HPP is characterised by accumulation of PPi,
which suppresses hydroxyapatite crystal formation
NPP1
(ENPP1)
ATP
Inhibition of
hydroxyapatite crystal
Formation
Promotion of
hydroxyapatite crystal
formation
Bone mineralisation
PEA
ALP substrates
A lack or low activity of tissue non-specific ALP (TNSALP)
results in accumulation of ALP substrates:
Pyridoxal 5’-phosphate (PLP)
Pyrophosphate (PPi)
Phosphoethanolamine (PEA)
Vitamin B6-responsive
seizures
Inhibits bone mineralisation,
causing rickets or osteomalacia
PLP, PPi and PEA in HPP
Measured by HPLC or tandem mass spectrometry or
spectrophotometric methods
Poor sensitivity and specificity as a diagnostic tool
May help in a small number of cases
May be of value in following enzyme therapy, monitoring
changes with treatment
HPLC: high-pressure liquid chromatography;
PEA: phosphoethanolamine;
PLP: pyridoxal 5´-phosphate
Representative reference ranges for PLP, PO4
and 25-hydroxy vitamin D (Europe and the USA) Specimen Age
group/age Concentration or enzyme activity (conventional units)
Concentration (SI units) Source
Pyridoxal–5'–phosphate (PLP)
Plasma 5–50 µg/L* USA [1] 5–30 ng/mL 20–121 nmol/L USA [2]
Phosphorus, inorganic Serum 3.0–4.5 mg/dL 0.97–1.45 mmol/L Merck [3] 3–4.5 mg/dL 1.0–1.4 mmol/L USA [2]
0-11 months
1-4 y
8-13 y
16-17 y
≥18 y
Males
NA**
4.3-5.4 mg/dL
3.1-4.7 mg/dL
2.5-4.5 mg/dL
Females
NA**
4.0-5.2 mg/dL
3.1-4.7 mg/dL
2.5-4.5 mg/dL
[4]
Phosphate, fasting Plasma Neonate
Infant
Child
Adult
1.0–2.7 mmol/L
1.1–2.4 mmol/L
0.8–1.9 mmol/L
0.8–1.5 mmol/L
Europe [5]
25-hydroxy vitamin D
(vitamin D3; 25-
hydroxycholecalciferol)
Serum 15–80 ng/mL
>20 ng/mL
37–200 nmol/L
>50 nmol/L
Merck [3]
UK (NOS, USA IOM)
Plasma 10–68 ng/mL 24.9–169.5 nmol/L USA [2]
1. Mayo Clinic, Mayo Medical Laboratories (http://www.mayomedicallaboratories.com/test-catalog/Clinical+and+Interpretive/60295.
2. Kratz A, et al. N Engl J Med 2004;351:1548–63.
3. Wians FH. Merck manuals. Available at http://www.merckmanuals.com/professional/appendixes/normal-laboratory-values/blood-tests-normal-value.
4. Mayo Clinic. http://www.mayomedicallaboratories.com/test-info/pediatric/refvalues/reference.php. 5. Sheffield children’s NHS foundation trust. Laboratory
handbook. April 2014..
Conclusions
Biochemical measurement of ALP is relatively cheap and
readily available
Reference ranges are extremely variable with age and
gender
The lower limit of normal range is often poorly defined
Paediatric reference ranges are higher than adult
An algorithm for investigation of low ALP is valuable to
ensure the correct diagnosis is made in all cases and
appropriate therapy can be commenced