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ANALIS

ISOPAL™ Plus Alkaline phosphatase kit for Beckman Coulter Paragon® Electrophoresis - GB: Instructions for use

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INFORMATION
QUOTATION
INFORMATION
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DESCRIPTION

1. General

1. Date: september-03 - Revision: 01

EURO PN 844111011
PN 10-004140
100 tests per kit

2. Intended use

The ISOPAL Plus* Alkaline Phosphatase (AP) Isoenzyme Electrophoresis Kit for use on Beckman Coulter's Paragon® Electrophoresis System is intended for the electrophoretic separation of bone, liver, intestinal, placental, and macromolecular forms of alkaline phosphatase isoenzymes in human serum.

3. Principle of the test

The human alkaline phosphatase enzyme is part of a group of enzymes (phosphoric monoester hydrolases) that catalyze the hydrolysis and transfer of a phosphate group at alkaline pH.

The ISOPAL Plus AP Isoenzyme Kit provides for the electrophoretic separation of AP isoenzymes in a buffered agarose Gel. After electrophoresis, the isoenzymes in the Gel are detected by the following specific colorimetric chemical reaction sequence:

5-bromo-4-chloro-3-indolyl phosphate (BCIP) + AP à dihalogenated indoxyl (with O2 ) à dimerizes to an insoluble blue

The indigo blue dye is formed at the site of each isoenzyme band. This pattern may be visually interpreted or scanned with a densitometer.

4. Contents

Equilibration Buffer (TRIS-borate 0.4 mol/L) 2x100mL (PN 10-004141)

Electrophoresis Buffer (TRIS-borate 4.4 mol/L) 1x100mL (PN 10-004142)

Substrate  A' solution (BCIP in 2-Amino-2-Methyl-1-Propanol) 1x12.5mL (PN 10-004143)

Substrate  B' solution (Nitroblue Tetrazolium solution) 1x12.5mL (PN 10-004145)

Isopal Agarose gels: 1x10

Measure 25 mL 1x

Liquipipette Graduat (3mL) 1x

Sample Template (purple) 1x10

Template blotters 1x10 (19x126mm)

Gel Blotters 30 (83 x 101 mm)

Drying Blotters 20 (76 x 101mm

5. Configuration of instrument

Beckman Coulter's Paragon® Electrophoresis System.

6. Other reagent and material needed

  • Pipettes.
    - Graduated cylinder.
    - Deionised water.
    - 5% acetic acid
    - Beckman Coulter's Paragon® Electrophoresis System.

INSTRUMENT (OPTIONAL)

A densitometer capable of scanning three inch by four inch film at 600 nm. Refer to manufacturer's instructions for operation and calibration procedures.

7. Training

The operator should be familiar with Beckman Coulter's Paragon Electrophoresis System.

8. Reagent preparation and storage

ISOPAL Gels: Just prior to use, carefully remove a Gel from the foil package. Gels should be stored at room temperature, 8°C to 30°C, until expiration date. Improper temperature or storage conditions can result in atypical migrations. DO NOT STORE GELS NEAR HEAT EMITTING DEVICES. For example: CRT, computer, or table top instrument. DO NOT REFRIGERATE OR FREEZE.

ISOPAL Working Equilibration Buffer: ready to use.

ISOPAL Working Electrophoresis Buffer, pH 9.5: Dilute contents of one electrophoresis buffer bottle (100 mL) with 900 mL deionised water. Unopened buffer should be stored at room temperature, 8°C to 30°C, until expiration date. Diluted buffer stored in a closed container at room temperature 8°C to 30°C, is stable for 60 days, or until expiration date, if sooner. If buffer becomes cloudy, discard.

ISOPAL Substrate: pour content of bottle B in bottle A and mix.

Stable for 4 weeks.

9. Type of specimen

Serum samples should be collected in the manner normally used for any laboratory test. Freshly drawn serum from a fasting individual is preferred, but samples may be stored at 4°C for 72 hours for later analysis. Refrigerated samples should be reactivated by incubation at 37°C for 30 minutes prior to analysis. Samples showing evidence of hemolysis should not be used.

NOTE
Plasma collected with oxalate, citric acid, or EDTA inhibits the activity of alkaline phosphatase.

10. Instrument preparation

See Paragon™ Electrophoresis System.

11. Sample preparation

Serum samples are not diluted.

12. Operations

1. Open and remove Gel Tray from ISOPAL Gel foil package.
2. Open Gel Tray completely (Top and Bottom of Tray must lie flat on a levelled surface. Please note that the Bottom portion has grooves.)
3. Remove Gel from Gel Tray and place on a paper towel.
4. Pour 20 mL of ISOPAL Equilibration Buffer into the Bottom portion of the Gel Tray.
5. Gently blot Gel with Gel Blotter. Discard Blotter.
6. Gently lay the blotted Gel, agarose side down, in the Equilibration Buffer. Equilibrate Gel for 30 minutes.
7. Prepare all samples and controls as instructed in SPECIMEN COLLECTION.
8. Remove Gel and place on paper towel, agarose side up. Discard Gel Tray containing used Equilibration Buffer.
9 . Gently Blot Gel with Gel Blotter. Discard Blotter.
10. Purple Template application:

figure 1

(a) Bend Template lengthwise as illustrated.
(b) Align Template with  A" position dots located on edges of Gel.
® Apply Template to Gel such that Template slots contact Gel surface first.
(d) Gently rub finger across Template to ensure seal.

11. Apply 5 µL of sample across each Template slot. Allow 10 minutes for diffusion after last sample has been applied.
12. Gently blot Template with Template Blotter. Discard Blotter and Template.
13. Pour 45 mL buffer in each compartment of the electrophoresis cell.
14. Place Gel onto Gel Bridge Assembly, aligning Positive (+) and negative (-) sides of Gel with corresponding positions marked on Gel Bridge Assembly. Place assembly into Paragon Electrophoresis Cell and cover Cell.
15. Insert Paragon Electrophoresis Cell into power supply. Set voltage to 150 volts, turn on power, and electrophorese for 25 minutes.
16. Upon completion of electrophoresis, remove Gel from Paragon Electrophoresis Cell and place Gel into Paragon Incubation Box.

NOTICE
Use this Incubation Box ONLY for ISOPAL Alkaline Phosphatase Isoenzymes.

17. Distribute 2.5 mL of ISOPAL substrate evenly on Gel surface with Liquipipette. AVOID TOUCHING GEL SURFACE WITH Liquipipette.
18. Close Incubation Box and place in Paragon Incubator (45°C) for 15 minutes.
19. Check the horizontal position of the incubation box so that substrate remains evenly distributed on the gel.
20. After 5 minutes of incubation, open Incubation Box and redistribute substrate evenly over Gel surface. Continue incubation..
21. After incubation, rinse the gel in the Incubation Box with 20 mL deionised water for 5 sec., then with 20mL 5% acetic acid for 5 minutes.
22. Place Gel Blotter on base of Press Dryer assembly.
23. Place gel, agarose side up, onto Gel Blotter on Press Dryer Base.
24. Place a second Gel Blotter moistened with 5% acetic acid onto gel surface, followed by 2 drying blotters and the press dryer top plate. Press dry for 2 min.
25. Repeat acetic acid rinse and press dry.
26. Place gel in drying oven until dry (maximum 90°C)
27. Evaluate Gel visually or scan with a suitable densitometer at 600 nm.

CONFIRMATORY METHODS

1. Neuraminidase Incubation: Mix 100 µL of sample with 20 µL of ISOPAL Neuraminidase (Beckman Coulter Part Number 446145) and incubate for 30 minutes at 37°C. Apply the original sample and incubated sample to same Gel and process normally.

NOTICE
A great diversity in the action of neuraminidase has been encountered, depending on the supplier. For best results, use the ISOPAL Neuraminidase to ensure the same activity from lot-to-lot.

2. Treatment with Antiplacental Antiserum: Mix 100 µL of serum with 20 µL of ISOPAL Antiplacental Antiserum (Beckman Coulter Part Number 446140) and apply to Gel immediately.

NOTICE
Cross reaction with placental and intestinal isoenzymes, if present, will occur. If intestinal or placental fraction is present, the complex with antiserum will appear in the gamma region. Also, while the color intensity of the complex may be weak, the disappearance of these fractions from the original locations will be obvious. For best results, use ISOPAL Antiplacental Antiserum with optimized activity (titer, specificity).

3. Incubation with Ficin: Add 5 mg of ISOPAL Ficin (Beckman Coulter Part Number 446150) to 100 µL of serum and incubate 30 minutes at 37°C. Apply original sample and incubated sample on same Gel and process normally.

CAUTION
Irritant. Avoid contact with eyes and skin. Do not breathe dust. Rinse contaminated area thoroughly with water.

13. Calibration

The densitometer's performance should be verified according to the manufacturer's instruction

14. Instrument preparation

figure 2

The AP isoenzyme pattern may be visually interpreted by comparing the sample pattern with a control pattern. With the use of a densitometer, the relative percentage of each AP isoenzyme may be calculated.

DIFFERENTIATION AND CONFIRMATION OF FRACTIONS USING CONFIRMATORY METHODOLOGIES 1. Bone-Liver Differentiation: After incubation with ISOPAL Neuraminidase, two clearly resolved bands will appear which migrate more cathodically. Note that these bands will cover any intestinal fraction existing in the sample.

2. Confirmation of Intestinal Fraction and/or Intestinal Variant:

(a) Incubate sample overnight with ISOPAL Neuraminidase. Intestinal fractions will remain at the same location while bone and liver fractions will move cathodically.
(b) Mix with ISOPAL Antiplacental Antiserum. These fractions will disappear from the original location and will appear between the application point and the cathode.


3. Confirmation of Placental Fractions:


(a) Incubate sample with ISOPAL Neuraminidase and fractions will move cathodically.
(b) Mix sample with ISOPAL Antiplacental Antiserum. Fractions will disappear and be at new location point between application point and cathode.

4. Confirmation of Protein Bound Fractions: After mixing sample with ISOPAL Ficin, bone and/or liver fractions will be regenerated and the complex at the application point will disappear.

5. Confirmation of Polymers of Liver Fractions: After mixing with ISOPAL Ficin, the liver fraction regenerates and the complex at the application disappears.

15. Calculation

No calculations are required.

16. Imprecision

Coefficient of variation values are typically below 5 %.

17. Reference range

In normal individuals, AP activity as well as isoenzyme distribution is age, sex, genetic and pregnancy dependent. Bone isoenzyme is elevated in children and in adults over age 50. Individuals who are B or O blood type and are secretor-positive may have elevated intestinal isoenzymes, particularly after a fatty meal. Placental isoenzyme is elevated during and shortly after termination of pregnancy.

A number of other clinical conditions also may modify AP activity as well as the distribution of isoenzymes. Interpretation of the electrophoretic patterns requires the knowledge of the total AP activity in the patient samples.

The following values were observed for the ISOPAL Alkaline Phosphatase Isoenzyme Kit when determined from a population of 66 apparently healthy male and female adults (age 25 to 50) from Southern California . The range of total AP values was 32 to 107 IU/L (37°C ASTRA).

AP Isoenzyme % of Total AP Activity
Bone 14.8 to 73.5
Liver 30.1 to 85.7
Intestinal 4.3 to 10.9
Macromolecular 2.7 to 4.9

It is recommended that each laboratory establish its own normal range.

18. Interference

1. Gels not stored in a horizontal position may produce atypical electrophoretic pattern.
2. In the presence of hemolysis, total AP may be under estimated. ISOPAL profile may show the presence of an artifactual band within the bone fraction, caused by hemolysis.
3. Use of other than deionised water to dilute the buffers and rinse the Gels after the incubation step may cause rupture of the Gel during electrophoresis and also precipitate proteins during the rinsing steps.

19. Quality Control

It is recommended that fresh normal sera or commercially available quality control sera be included in each electrophoretic procedure.

20. References

Van Hoof V, Lepoutre L, De Broe M, Hoylaerts M, Chevigné R.

Improved Agarose Electrophoresis Method for Separating Alkaline Phosphatase Isoenzymes in Serum.

Clin Chem 1988;34:1857-62. (Reprint R577).

Van Hoof V, Hoylaerts M, Geryl H, Van Mullem M, Lepoutre L, Debroe M.

Age and Sex Distribution of Alkaline Phosphatase Isoenzymes by Agarose Electrophoresis. Clin Chem 1990;36:875-8.

Van Hoof V, Van Oosterom A, Lepoutre L, De Broe M.

Alkaline Phosphatase Isoenzymes Patterns in Malignant Disease. Clin Chem 1992;38:2546-51.

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