Alkaline phosphatase Laboratory Perspective Professor William D Fraser Professor of Medicine University of East Anglia, Norwich, UK [email protected] 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 Buffers (amino alcohols): Diethanolamine (DEA) Germany/Scandinavia 2-amino-2-methyl-1-proponol (AMP) IFCC/USA/France HEDTA O O O O O O- N N N O ALP Rearranges at + HOH + O P O- Zn Mg2+ alkaline pH pH 10.3 O- O O O O P O- O P O- O- O- 4-Nitrophenyl phosphate 4-Nitrophenoxide 4-Nitrophenoxide (colourless) (colourless (yellow, quinonoid form) benzenoid form) Rate of formation at 37C ALP (WEQAS Results) Method N Mean CV, % Um ALP, 37°C 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 of laboratories Number 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 U/L AMP, reduced interference 4 16.500 6.1 0.63 N Mean CV, % Um SDP Exc. - Select - 2 20.500 3.4 0.62 A 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 42 19.049 8.0 0.3 1.74 3 WEQAS: Wales External Quality Assessment Scheme 1200/1650/1800/2400 provider in the UK Detection of ALP 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. 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 Concentration (SI Source activity units) 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 Male Female ARUP [3] 1–11 m 70–350 U/L 70–350 U/L 1–3 y 125–320 U/L 125–320 U/L 10–11 y 150–470 U/L 150–530 U/L ≥20 y 40–120 U/L 40–120 U/L Plasma Neonate 73–391 U/L Europe [4] Infant 59–425 U/L 1–14 y 76–308 U/L 14–16 y 49–242 U/L Adult 30–130 U/L 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 Age (years) CALIPER: Canadian Laboratory Initiative on Paediatric Reference Intervals (http://www.sickkids.ca/caliperproject/index.html) 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) Specimen Age group/age Concentration or Concentration (SI units) Source enzyme activity (conventional units) Serum Male Female ARUP [1] 6 m–2 y 31.6–122.6 µg/L 33.4–145.3 µg/L 3–6 y 31.3–103.4 µg/L 32.9–108.6 µg/L 7–9 y 48.6–140.4 µg/L 36.3–159.4 µg/L 10–12 y 48.8–155.5 µg/L 44.2–163.3 µg/L ≥25 y 6.5–20.1 µg/L Premenopausal female 4.5–16.9 µg/L/7.0–22.4 µg/L Male Female ARUP [2] 1–6 y 0–208 U/L 0–189 U/L 7–9 y 0–264 U/L 0–246 U/L 10–15 y 0–340 U/L 0–340 U/L 16–19 y 0–165 U/L 0–91 U/L Females ≥16 y and males ≥20 y 0–55 U/L 0–55 U/L 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. 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 lower than appropriate Review medical ALP decision reference range history for low ALP Artefact algorithm EDTA, oxalate (blood transfusion) Confirm low on serum sample Metal Protein Disease-associated Vitamin abnormality Post-cardiac surgery Drug effect analysis calorie hypothyroidism, low vitamin C, B12, post liver transplant estrogen, low Zn, Mg, malnutrition hypoparathyroidism, B6, folate, ESRF bisphosphonate, high Cu severe anaemia excessive vitamin D osteodystrophy clofibrate, achondroplasia omeprazole, cretinism lansoprazole Abnormal Total protein Measure TSH, Measure and Drug therapy investigate albumin PTH, FBC correct may unmask and correct Correct abnormality genetic cause abnormality for low ALP Possible genetic cause for HPP ALP remains below reference range after PEA and PLP measurement correction of abnormality Genetic testing of ALPL Non-biochemical tests radiology, ECG muscle studies ECG: electrocardiogram; ESRF: end stage renal failure; FBC: full blood count; PTH: parathyroid hormone; TSH: thyroid stimulating hormone Pathophysiology of HPP ALP ATP PPi Promotion of NPP1 hydroxyapatite crystal (ENPP1) formation Inhibition of Pi hydroxyapatite crystal Ca Bone mineralisation Formation Pi HPP is characterised by accumulation of PPi, which suppresses hydroxyapatite crystal formation 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 O = odontohypophosphatasia Normal mean and range (± 2 SD mean) Children 166 (80–342); Adults 51 (28–91) 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) .