WS/T 617-2018Reference procedure for the measurement of catalytic activity concentration of aspartate aminotransferase (English PDF)

6.1 Reagent List

The reagents used are listed in Table 1.

Table 1 Reagent List

Classification system name common name, abbreviations Reagent Tris L-aspartic acid no Alpha-ketoglutaric acid, disodium salt, dihydrate without

--nicotinamide adenine dinucleotide, reduced form, disodium salt reduced coenzyme I, NADH Lactate dehydrogenase LD Malate dehydrogenase MD Sodium azide Hydrochloric acid Sodium hydroxide Sodium chloride Bovine serum albumin, Fraction V 5'-pyridoxal phosphate, monohydrate P-5-P Pyruvate monosodium salt Oxaloacetate Solvent High-purity water of similar quality to double distilled water (conductivity < 2 µS·cm-1, pH 6~7, silicate < 0.1 mg·L-1) Reference material JCTLM and/or nationally approved reference material Quality control substances, conventional system calibrators no indicator

6.2 Reagent raw material information

Fill in the reagent raw material details according to Table 2.

Table 2 Reagent raw materials details

Reagent system name __ common name ____ Information indicator content CAS, CARN registration number Manufacturer Item number/batch number Molecular formula Relative molecular mass purity Specific eligibility requirements (if any) Risk Storage requirements expiration date See Appendix A for details of each reagent material used in the AST assay. The reagent of the highest purity should be used. The quality of the reagent should be provided by the supplier Proof of analysis. If it is suspected that a chemical contains impurities that affect the catalytic activity of AST, further testing should be performed, for example, Compare products from different manufacturers and different batches. It is recommended to use reagents that have been identified or approved in the comparative test. The following three reagents require a note Intentional safety.

Hydrochloric acid, sodium hydroxide and sodium azide have harmful effects on the human body. Operators should be specially trained to strictly abide by the operating procedures.

Need to wear self-priming filter gas mask (full face mask) or hood type electric air filter type dust-proof respirator, wearing chemical safety eye Mirror, wearing protective clothing made of rubber acid and alkali resistant materials and wearing rubber acid and alkali resistant gloves, etc., to do safety protection work.

6.3 Reagent solution preparation

6.3.1 General requirements

The mass given by each component when preparing the solution means 100% content. If the chemical content is less than 100% [eg y (%)], then the application Factor. Fcontent=100/y, calculate the mass of a chemical equivalent to the given mass. The preparation of the solution should use quality and double distilled water Similar high purity water (conductivity < 2 µS·cm-1, pH 6-7, silicate < 0.1 mg·L-1).

The expanded uncertainty (k=2) for each weighing (including the uncertainty of the purity of the substance) (normal distribution) should be <=1.5%.

6.3.2 Solution 1

Weigh 1.17 g of Tris, 4.02 g of aspartic acid (free acid), and 0.052 g of sodium azide. Treat the above materials as follows.

- dissolved in about 80 mL of water;

- Add 4.2 mL to 4.4 mL of 5.00 mol·L-1 sodium hydroxide solution;

- stirring until the reagent is completely dissolved;

- the pH of the mixture should be lower than the pH target (about 0.3 pH to 0.9 pH);

- adjusting the pH to 7.65 with 2 mol·L-1 sodium hydroxide solution at 37 ° C;

-- Transfer to a 100 mL volumetric flask;

- balance the volumetric flask and water to 20 ° C;

- Add water (20 ° C) to the scale of the volumetric flask.

The concentration of Tris in the final solution was 96.92 mmol·L-1, the concentration of aspartic acid was 302.4 mmol·L-1, and sodium azide was concentrated.

The degree is 8.00 mmol·L-1. The solution has a stability of 2 ° C to 8 ° C for 3 months.

Note. Aspartic acid is not dissolved after adding water, you must first add sodium hydroxide solution, replace 5 mol·L-1 sodium hydroxide solution with 5 mol·L-1 to reduce the required Amount, precise adjustment of pH requires 2 mol·L-1 sodium hydroxide solution.

6.3.3 Solution 2

Weigh 1.17 g Tris, 0.052 g sodium azide, and treat the above materials as follows.

- dissolve the above two reagents in about 80 mL of water;

- adjust the pH to 7.65 with 1 mol·L-1 hydrochloric acid at 37 ° C;

-- Transfer to a 100 mL volumetric flask;

- balance the volumetric flask and water to 20 ° C;

- Add water (20 ° C) to the volumetric flask.

The final solution had a Tris concentration of 96.92 mmol·L-1 and a sodium azide concentration of 8.00 mmol·L-1. The solution is at 2 ° C ~ the stability at 8 ° C is 3 months.

6.3.4 Solution 3

Weigh 16.7 mg of P-5-P monohydrate and treat as follows.

- Dissolve 16.7 mg of P-5-P monohydrate in 6 mL of solution 2;

-- Transfer to a 10 mL volumetric flask;

- the volumetric flask and solution 2 are equilibrated to 20 ° C;

- Add solution 2 (20 ° C) to the volumetric bottle calibration line;

-- Protect from light (such as brown bottles).

The concentration of P-5-P in the final solution was 6.30 mmol·L-1. This solution was stable at 2 ° C to 8 ° C for 1 week.

6.3.5 Solution 4

Weigh 16.1 mg of NADH disodium salt and treat as follows.

- dissolved in 2.00 mL of solution 2;

-- Protect from light (such as brown bottles).

The concentration of NADH in the final solution was 11.34 mmol·L-1. This solution was stable at 2 ° C to 8 ° C for 1 week.

6.3.6 Diluent of enzyme reagent

Weigh 1.20 g of bovine serum albumin and 0.90 g of sodium chloride and treat the above substances as follows.

- Dissolve the above two reagents in about 80 mL of water;

-- Transfer to a 100 mL volumetric flask;

- balance the volumetric flask and water to 20 ° C;

- Add water (20 ° C) to the volumetric flask.

The final prepared solution had a sodium chloride concentration of 154 mmol·L-1. This solution is stable at 2 ° C to 8 ° C for at least 1 month.

6.3.7 Solution 5

Prepare as follows.

- Dilute the LD stock solution with the enzyme reagent dilution in 6.3.6 so that the LD catalytic activity concentration is 3.78 mkat·L-1 at 37 ° C (226.8 kU·L-1). Calculate the volume of the enzyme reagent dilution required to dilute the LD stock solution according to formula (1).

V diluent= V LDSTOCK×(LDSTOCK-226.8)/226.8(1) in the formula.

Vdiluent - the volume of the enzyme reagent dilution required to dilute the LD stock solution, in milliliters (mL);

VLDSTOCK--LD storage volume in milliliters (mL);

LDSTOCK--LD catalytic activity concentration in LD stock solution, in millikelta per liter (mkat·L ) or thousand units per liter (kU·L-1).

Example 1. See Appendix B.

- Dilute the MD stock solution with the enzyme reagent dilution in 6.3.6 so that the MD catalytic concentration is 2.52 mkat·L-1 (151.2 kU·L-1) at 37 °C.

Calculate the volume of the enzyme reagent dilution required to dilute the MD stock solution according to formula (2).

Vdiluent= VMDSTOCK×(MDSTOCK-151.2)/151.2 (2) in the formula.

Vdiluent - the volume of the enzyme reagent dilution required to dilute the MD stock solution, in milliliters (mL);

VMDSTOCK -- MD reservoir volume in milliliters (mL);

MDSTOCK - MD catalytic activity concentration in MD stock solution, in milligrams per liter (mkat·L ) or thousands of units per liter (kU·L-1).

Example 2. See Appendix C.

- Mixing the two diluted enzyme solutions in equal volumes to obtain solution 5.

The catalytic activity concentration of LD in the final prepared solution was 1.89 mkat·L-1 (113.4 kU·L-1) at 37 ° C, and the catalytic activity concentration of MD was at 37 ° C, it is 1.26 mkat·L-1 (75.6 kU·L-1). The solution had a stability of 2 d from 2 ° C to 8 ° C.

6.3.8 Reaction solution

Pipette 10.000 mL solution 1, 0.200 mL solution 3, 0.200 mL solution 4, 0.100 mL solution 5, and mix the above solution thoroughly.

Evenly, save in the dark. This solution was stable at 2 ° C to 8 ° C for 1 d.

6.3.9 Starting reagent solution

Weigh 0.326 g of alpha-ketoglutarate disodium hydrate and treat as follows.

- soluble in about 6 mL of water;

-- Transfer to a 10 mL volumetric flask;

- balance the volumetric flask and water to 20 ° C;

- Add water (20 ° C) to the tick mark.

The concentration of disodium alpha-ketoglutarate in the final solution was 144.0 mmol·L-1. This solution was stable at 2 ° C to 8 ° C for 1 week.

6.4 Effect of temperature on pH of buffer solution

When the temperature deviates from 37oC, adjust the pH. immerse the thermometer and the pH electrode simultaneously in the mixture. Then stir the solution the side-by-side titration to the table lists the pH at the current measured temperature. The mixing speed should be consistent during the calibration, control and pH adjustment.

The pH electrode must be at the center of the stirred solution.

When formulating solution 1 and solution 2, adjust the pH value according to different temperatures, and adjust the pH value of the solution according to D.1 and D.2 in Appendix D, respectively.

For this reason, the temperature should be re-controlled when approaching the target value, and the same method is also applicable to the temperature compensation adjustment of the pH meter.

7.1 Instrument List

The laboratory shall register the main measuring instruments (spectrophotometer) and main auxiliary instruments (point thermometer, balance, pH meter, constant temperature water) in Table 3.

Bath box, dilution dosing device, pipette, etc.).

Table 3 List of measuring instruments and auxiliary instruments

Instrument name manufacturer model Spectrophotometer Point thermometer Balance pH meter Constant temperature water bath Dilution dosing device Pipette

7.2 Instrument performance requirements

See Table 4 for the main performance requirements of spectrophotometers and auxiliary instruments.

Table 4 Main performance requirements of spectrophotometer and auxiliary instruments

Instrument name performance index IFCC reference program requirements Spectrophotometer, accessories Wavelength accuracy/nm 339 ± 1 (k = 2) Bandwidth/nm <= 2 Light diameter/mm 10.00 ± 0.01 (k = 2) pH meter pH value 7.65 ± 0.05 (k = 2) Point thermometer temperature/°C 37.0 ± 0.1 (k = 2)

7.3 Spectrophotometer preparation

Before the measurement work, the spectrophotometer is inspected according to the established SOP file and filled in Table 5.

Table 5 Spectrophotometer preparation

Preparation details

Check before booting-Power -Grounding -Computer -Printer -Whether it is turned on 24 hours - Temperature - Humidity - Body cleaning - Color bin cleaning - Color window cleaning combination Thermostat. - connected with spectrophotometer complete - boot in advance Temperature measuring device. - Power - Temperature probe does not squeeze, collide with hard objects Stirring device. - clean - speed Cuvette. - complete - cleanliness Boot precautions Light source. - preheating in advance Cuvette. - Light path - Match between cups Check the performance of the instrument to verify whether it is within the validity period of the national metrology agency.

Daily boot performance check. - Wavelength correctness - Wavelength repeatability - Baseline flatness scan - Bandwidth test - noise test Large-scale pre-experimental performance check. - 检查 glass inspection wavelength accuracy - national standard solution check absorbance accuracy Preventive maintenance spectrophotometer. - no obvious vibration - timely standby - radio interference - UPS Auxiliary equipment. -Replace the inlet and outlet pipes - Regularly clean - Clean the probe and stirrer after use each laboratory can develop a flow chart for the preparation of the spectrophotometer of the laboratory according to the specific conditions.

7.4 Point thermometer preparation

Before the measurement work, check the point thermometer according to the established SOP file and fill in Table 6.

Table 6 Point thermometer preparation

Preparation details

Check before starting up - Power - Body cleaning - Point thermometer temperature probe is normal Combination - host and point thermometer temperature probe connection complete Boot precautions Battery. - View remaining battery Calibration Tip. - Check if the calibration validity period expires Instrument performance check Verify whether the national metrology agency has verified the validity period Large-scale pre-experimental performance check. - Check the accuracy of the temperature with a qualified standard thermometer Preventive maintenance Host. - Body cleaning Temperature probe. - Clean after use - Guaranteed straight, not bent each laboratory can develop a flow chart for the preparation of point thermometers in this laboratory according to the specific conditions.

7.5 Constant temperature water bath preparation

Before the measurement work, check the constant temperature water bath according to the established SOP file and fill in Table 7.

Table 7 Preparation of constant temperature water bath

Preparation details

Check before starting up - power - temperature - humidity - body cleaning Combination - connected to spectrophotometer Startup precautions - water volume - inlet water pipe - outlet water pipe Instrument performance check - temperature correction - temperature stability Preventive maintenance - cleaning filter - disinfection inside the tank each laboratory can prepare a flow chart for the preparation of the constant temperature water bath of the laboratory according to the specific conditions.

7.6 Dilution Dosing Instrument Preparation (if available in the laboratory)

Before the measurement work, check the diluted dosing device according to the established SOP file and fill in Table 8.

Table 8 Preparation of the diluted dosing device

Preparation details

Check before starting up - power - temperature - humidity - body cleaning Boot precautions - Syringe - Pipetting - Pipe flushing Instrument performance check - sample volume accuracy - sample volume precision Preventive maintenance - Periodically change the syringe - Regularly change the pipette each laboratory can develop a flow chart for the preparation of the diluted dosing instrument of the laboratory according to the specific conditions.

7.7 Pipette preparation

Prior to the measurement work, the pipette is inspected according to the established SOP file and filled in Table 9.

Table 9 Preparation of pipettes

Preparation details

Pre-application inspection - temperature - humidity - body cleaning - mechanical movement Instrument performance check - sample volume accuracy - sample volume precision Preventive maintenance - Regular maintenance - Regular calibration each laboratory can develop a flow chart of pipette preparation in this laboratory according to the specific situation.

7.8 Preparation for balance

Before the measurement work, check the balance according to the established SOP file and fill in the table 10.

Table 10 Balance preparation

Preparation details

Check before starting up - Power - Printer - Horizontal bead position Power-on self-test - power - zero display Calibration verification is valid during the period of validity of the national metrology agency Before large-scale experiment. standard weight calibration Before each application. the instrument comes with calibration Boot precautions - Clean the balance before use - Power each laboratory can develop a flow chart for the preparation of the balance of the laboratory according to the specific situation.

7.9 PH meter preparation

Before the measurement work, check the pH meter according to the established SOP file and fill in Table 11.

Table 11 pH meter preparation

Preparation details

Check before starting - Power - Ground - Computer - Printer - Temperature - Humidity - Body cleaning combination Temperature probe. - complete connection with pH meter Electrode. - Connected to the pH meter. The amount of liquid in the - - the amount of the external liquid Magnetic stirrer. - power - ground - speed Magnetic stirrer. - complete - cleanliness - magnetic pH standard solution. - remaining amount - expiration date Instrument performance guarantee Performance guarantee before each application.

Replace the electrode inner liquid and the outer electrode liquid for electrode activation; calibrate with pH standard solution Preventive maintenance Before application. -Replace the electrode inner liquid and the electrode outer liquid. electrode activation After application. -Replace the electrode inner liquid and the electrode outer liquid. electrode maintenance - Clean temperature probe, electrode each laboratory can develop a flow chart for the preparation of the pH meter of the laboratory according to the specific conditions.

8.1 Sample Acceptance Information

The details of sample reception should be recorded in tabular form, see Table 12. Samples that do not meet the sample acceptance requirements should be promptly linked to the client.

system.

Table 12 AST sample acceptance requirements

Request indicator content Acceptable sample type - serum - plasma - other Acceptable sample type - freeze-dried powder - liquid - frozen - other Matrix type description - human serum - bovine serum - water - other Minimum number of samples Minimum volume per sample per mL Allowed additives Shipping conditions - dry ice - normal temperature - ice pack - other Storage conditions - normal temperature - 4 ° C - -20 ° C - -70 ° C - other stability Risk Precautions

8.2 Analysis of the type of sample

The samples measured by the AST reference measurement program are mainly.

-- reference material (RM);

-- quality control substances;

- inter-room comparison of samples;

-- Test samples sent by the testing laboratory;

-- Other specimens.

8.3 Analysis of the...