UMA CO., LTD.

MEASURE ALP IFCC

2-19-6 Yokosuka Reagent for determination of Alkaline phosphatase
Matsudo, Chiba, Japan IFCC Method

 2 ~ 8 °C IVD In vitro Diagnostics

QUALITY MANAGEMENT SYSTEM (BY TUV)
= ISO 13485:2016 =
 DO NOT freeze  18 months/block from light

1. PURPOSE OF USE labile than liver ALP, higher percentage of bALP activity is
Providing a quantitative in vitro assay for the Alkaline lost and more liver ALP activity is retained after a heat
phosphatase concentration in serum or plasma. treatment at 56–59°C for half an hour. bALP activity can

2. GENERAL INSTRUCTION be estimated from the difference before and after heat-

a. For in vitro diagnostics use only. denature treatment. However, this method has been

b. Diagnosis should be made in a comprehensive criticized because of its irreproducibility and variability.

manner, in accordance with other related test results bALP can also be precipitated by wheat germ agglutinin

and clinical symptoms by the doctor in attendance. (WGA) but it failed to completely separate bone and liver

c. For guaranteed results, usage of this product must activity in patients with Paget disease. Because ALP from

comply with the instruction in this manual. different cell types differs in its carbohydrate content,

d. If you use automatic analyzers, follow their immunoassays have been developed to measure bALP

instructions carefully. based on its carbohydrate content. There is a bALP assay

SUMMARY on an automated immunoassay analyzer. bALP results by

Alkaline phosphatase activity is present in the liver, bone, immunoassay provide greater sensitivity and specificity for

kidney, small intestine, and other organs of the body, and bone formation than total ALP. However, they are not

placenta in pregnancy. In sera of healthy adults, ALP completely specific for bALP and can exhibit 7%–17%

activity originates mainly in the liver, with most of the rest cross-reactivity with liver ALP. bALP is also an option in
patients without severe liver disease.
coming from the skeleton. bALP is produced by the
osteoblast during the matrix maturation phase. The main 3. MATERIALS REQUIRED BUT NOT INCLUDED
function of bALP is to hydrolyze inorganic pyrophosphate - Saline 0.9 % and high grade purified water
(PPi) to generate phosphate (Pi) which is used in the - Micropipet and other basic laboratory equipment.
formation of calcium hydroxyapatite. Total ALP activity is - Calibrators and Controls (separatedly sold)
markedly increased in Paget disease and provides the
4. REAGENT COMPOSITION & PREPARATION
highest clinical sensitivity and specificity in the diagnosis
- Reagent R-1: 2-amino-2-methylpropanol (AMP)
and monitoring of PDB. ALP is increased in metabolic
Reagent R-1 is ready for use
bone diseases, including osteoporosis, osteomalacia and
- Reagent R-2: p-nitrophenyl phosphate 2Na
rickets, hyperparathyroidism, renal osteodystrophy, and
Reagent R-2 is ready for use
thyrotoxicosis, and in individuals with acromegaly, bone
- Once open, Reagent stored on board the instrument is
metastases, and other disorders with increased bone
stable for 30 days with Hitachi 7180 Analyzers.
formation [103]. In patients with impaired renal function,
- Applicable to various automated analyzers.
ALP is the preferred marker for bone turnover because its
- Calibrator MEASURE Multi Calibrator (separately sold):
clearance does not depend on glomerular filtration.
Put 5 mL of purified water to the vials of Calibrator
Total ALP is measured based on the reaction of ALP to
(MEASURE Multi Calibrator), leave at room temperature
remove phosphate group from a colorless p-nitrophenol
for 45 minutes and sometimes gently invert the vial before
phosphate to a colored product of p-nitrophenol at alkaline
use. After reconstituting, Calibrator can be used without
pH of 10.4. The increase in absorbance at 450 nm is
dilution.
proportional to ALP activity. Because bALP is more heat-
- Controls MEASURE Human Lyo L-1 and MEASURE

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Human Lyo L-2 (separately sold): Put 5 mL of purified 8. CALCULATION & UNIT CONVERSION
water to the vials of controls (Lyo L-1 and Lyo L-2); leave Calculation
at room temperature for 45 minutes and sometimes gently - Calculate ∆Abs of specimen & standards vs blank
invert the vial before use. After reconstituting, controls can - Plot a calibration curve Alb = f(∆Abs)
be used without dilution. - Calculate ALP in specimen using the curve
(doing same procedure for Controls)
5. SAMPLE PREPARATION & STORAGE
Unit conversion
Serum: Wait until the sample is completely coagulated.
U/L = 0.0167 µkat/L
Take the supernatant to use as specimen.
Plasma: Treat sample by anticoagulant (Li‑heparin); leave 9. PERFORMANCE & CORRELATION TEST
sample to stand for 3 hours or centrifuge at 2000 rpm for a. Measuring range
2 minutes; take the plasma layer (supernatant) and use as - The test is linear within an enzyme activity range of 1 –
specimen. 1000 U/L.
Analyze samples soon after collection. - If the concentration of sample exceeds assay range, dilute
Stability: the sample with saline and repeat the measurement.
 8 hours at 15-25°C b. Lower Detection Limit: 1 U/L
 3 days at 2-8°C The lowest detectable level represents the lowest
 >30 days at (-60) – (-80)°C measurable level of ALP that can be distinguished from
- AVOID to use hemolyzed specimens. zero. It is calculated as the absolute mean plus three
6. MEASUREMENT PRINCIPLE standard deviations of 20 replicates of an analysis free
p-nitrophenyl phosphate is hydrolyzed to generate a p- sample.
nitrophenol by alkaline phosphatase (ALP) in the sample. Values below the lower detection limit (< 1 U/L) will not be
Calculate the ALP activity by measuring the rate of detected by this method.
increase in p-nitrophenol appeared. c. Performance
𝐴𝐿𝑃, 𝑀𝑔2+
p-nitrophenyl phosphate + H2O →−−−−−−−−−−−−→ - Sensitivity: When saline is used change of absorbance is
p-nitrophenol + H3PO4 0.010 Abs/minute or less. When 500 U/L sample is used,
(λmax = 405 nm) absorbance change is in the range of 0.05 ~ 0.25
Abs/minute.
7. ASSAY PROCEDURE - Specificity: The accuracy is within ±10.0%.
This product is compatible with various types of clinical - Reproducibility: CV value < 10.0%.
analyzer. An example of the assay procedure is indicated
10. EXPECTED VALUES
below.
Male : 45 - 115 U/L (≥ 19 years)
o
Sample + Reagent 1 37 C Reagent 2 Female : 52 - 144 U/L (≥ 17 years)
265s
Children : 130 - 260 U/L (3 – 10 years)
3.0µL 120µL 30µL
: 130 - 340 U/L (10 – 14 years)
3 7oC 3 7oC
  Meas. Abs I   Meas. Abs II : 30 - 180 U/L (14 – 17 years)
105s 230s
ALP activities have been found to vary significantly with
(405/505nm) (405/505nm)
age and sex. Reference range should be established at
 ∆Abs at 405/505nm  Concentration each facility and judgement should base on measurement
results in a comprehensive manner together with clinical
Perform the assay according to the instructions for
symptoms and other measurement results.
operating the automated analyzer Hitachi models. Refer
to the 13. INFORMATION FOR AUTOANALYZERS for 11. INTERFERENCES

the details of the assay method. Contact JAPAN - Icterus: No significant interference of conjugated bilirubin

KANPEKI., JSC. for information about the concentration up to 20 mg/dL

parameters for other automated analyzers. - Hemolysis: No significant interference of hemoglobin

concentration up to 500 mg/dL
- Ascorbic Acid: No significant interference of ascorbic

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acid concentration up to 50 mg/dL disposal. Concentration of sodium azide in R-2 is 0.05%.
- Lipemic: No significant interference of ascorbic acid
13. INFORMATION FOR AUTOANALYZERS
concentration up to 3000 FTU
 For Hitachi Model
- For diagnostic purposes, the results should always be Calculation Method Rate
assessed in conjunction with the patient’s medical history,
Temperature 37°C
clinical examination and other findings. Please use other
methods if the result is affected by any factors. Specimen 3.0
Volume (μL) R1 120
12. HANDLING, USAGE & DISPOSAL
R2 30
Handling
Main 405
1. Specimen can be potentially positive for infectious Wavelength (nm)
Sub- 505
agents including hepatitis B virus and HIV. Wear glove and Point 1 10
goggle when needed. Measurement
Point 2 21
(cycle)
2. In case reagents got into skin, eye or mouth by mistake, Point 3 34
wash it immediately with plenty of water and consult the Calibration type Factor (2710)
doctor if needed. Unit U/L
3. If reagents are spilled, dilute with water and wipe it out.
14. OTHER INSTRUCTIONS AND CAUTION
If specimen is spilled, spray 80% of alcohol over the
- Results may differ depending on the sample/reagent
specimen and wipe it out. ratio. Adjust parameters for different analyzer.
Usage - Prepare the calibration curve on the day of
1. Store reagents under specified condition. Do not use determination.

after expiration date. 15. PACKING AND KIT CONFIGURATION

2. Do not use the container and auxiliaries included in this Product Code Product Name Packages
kit for other purposes. 11A011A Measure ALP 1x60mL; 1x15mL

3. Do not mix reagents of different lot for use. 11A001A Measure ALP 5x60mL; 5x15mL

4. Do not add to the reagent being used even if it is the 11A011 Measure ALP 1x80mL; 1x20mL
11A001 Measure ALP 3x80mL; 3x20mL
same lot number.
Constituent reagents are available in other configurations.
Disposal
For further details please contact JAPAN KANPEKI.,
1. All specimens, as well as all instruments (e.g. test tubes)
JSC. REFERENCES
that come in contact with the specimens, must be treated
1. Lu Song, in Advances in Clinical Chemistry, 2017
by the following methods, or they must be treated
2. In house data, UMA Diagnostics.
according to the manual for infectious medical waste
16. MANUFACTURER
provided in each facility.
UMA Co., Ltd.
◼ Sterilize with an autoclave, subjecting them to high 2-19-6 Yokosuka, Matsudo City, Chiba
pressure saturated steam at 121 °C for more than 20 Prefecture 270-0031
minutes. Do not process waste containing sodium TEL: 047-710-4871 (dial-in) FAX: 047-710-4872
hypochlorite solution with an autoclave.

◼ Immerse at least one hour in sodium hypochlorite

solution (active chloride concentration of over 1000 ppm).
2. This reagent contains sodium azide. Sodium azide can
react with lead pipe and/or steel pipe and can generate
explosive metal azide. Make sure to use plenty of water at

3/3 Revision 09/2022