Some standard content:
1 Scope
National Standard of the People's Republic of China
Health examination criteria of blood donors
Health examination criteria of blood donors This standard specifies the items and requirements for physical examination and blood test of blood donors. This standard is applicable to blood stations (blood banks) at all levels across the country and is used for the management and evaluation of such institutions. 2 Definitions
This standard adopts the following definitions:
2.1 Blood donors who need previous blood test Blood donors who need previous blood test Blood donors who need previous blood test and pass the test before donating blood: 2.2 Non-pre-tested blood donors Blood donors who only need previous blood test and do not need to undergo previous blood test before donating blood. 3 General Principles
GB 18467 2001
3.1 In order to ensure the health of blood donors and the safety of blood transfusion for blood recipients, pre-examination blood donors must undergo physical examination and blood test (primary examination) before each blood donation. Blood can be collected after passing the re-examination. The collected blood can only be used clinically after passing the re-examination. 3.2 Blood can be collected from non-pre-examination blood donors after health consultation and physical examination. The collected blood must undergo initial and re-examination: it can only be used clinically after passing the re-examination.
3.3 The reagents produced by the same reagent factory shall not be used for the initial and re-examination of the blood of blood donors. The initial and re-examination of the same specimen shall not be performed by the same person.
3.4 The blood donation health information items in this standard are applicable to the free blood donation activities of blood collection vehicles and blood collection points that do not have the conditions for blood testing. 3.5 The blood donor's physical examination and blood test results shall be based on the blood station's results, which are valid for two adjustments. 3.6 This standard is an important basis for blood stations to conduct quality audits of blood donor physical examination and test technical operations. 4 Requirements for blood donor health examination
4.1 Physical examination standards for blood donors
4.1.1 Age: 18 to 55 years old.
4.1.2 Weight: Male 2.50kg, Female 45kg
4.1.3 Blood pressure: 90 mtniHg~~140 mmIIg/60 mmHg~-90 mmHg, pulse pressure: 30 mmllg or: 12.0 kPa~~18.7 kPa/8.0 kPa~-12.0 kPa, pulse pressure: =24. [kPH4.1.4 Pulse: regular rhythm, 60 times~-100 times/min. High endurance athletes 250 times/min4.1.5 Normal blood flow.
4.1.6 No yellowing of the skin, no wound infection, no large-area skin disease, no obvious enlargement of superficial lymph nodes. 4.1.7 No serious diseases in the five limbs, no yellowing of the sclera, no enlargement of the thyroid gland, 4.1.8 Particularly serious disabilities in the limbs. No serious functional disorders and no redness and swelling of the joints. No skin damage at the puncture site of the veins of both arms + no traces of intravenous injection of drugs. Approved by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China on October 22, 2001, implemented on March 1, 2002.
GB18467-2001
4.7.9 Chest: Normal heart and lungs, no pathological breath sounds and pathological heart murmurs, heart rate 60-100 times/min. 4.1.10 Abdomen: Flat and soft abdomen, no masses, no tenderness, no enlargement of the liver and spleen, 4.2 Requirements for blood wave examination of blood donors
4.2.1 Blood typing
4.2.1.1AB0) blood typing (positive and negative typing method). 4.2.1.2 RhD blood type (determination is carried out in areas with conditions and areas with high Rh negative rate). 4.2.2 Hemoglobin determination: Copper sulfate method: male 1.0520, female ≥1.0500; equivalent to male ≥120g/L, female 2110g/L4.2.3 Alanine aminotransferase (AL.T): Ketone powder method (only for initial test); negative; rate method: ≤40 units; Reiter method ≤25 units.
4.2.4 Hepatitis B virus surface antigen (HBsAg): negative (enzyme-linked immunosorbent assay, rapid diagnosis method is only for initial test at non-fixed blood collection points).
4.2.5 Hepatitis C virus antibody (HCV antibody): negative (ELISA): 4.2.6 HIV antibody (HIV antibody): negative (ELISA) 4.2.7
Syphilis test: negative (RPR method, TRUST method or ELISA). 4.2.8 Re-examination of 4.2.1, 4.2.3, 4.2.4, 4.2.5, 4.2.6, 4.2, 7 (4.2.3 must be tested by Reitman or rate method). 4.2.9 After one year of clinical cure of moderate hepatitis, if the test is normal for three consecutive times with an interval of one month each, blood donation can be participated (subject to the clinical test report).
4.2.10 Detection of protozoa in areas with high incidence of ulcers. 4.3 Provisions for blood donation after vaccination
4.3.1 Blood donation can be made two weeks after the last vaccination with live vaccines such as measles, mumps, yellow fever, poliomyelitis, etc., or four weeks after the last vaccination with live rubella vaccine or rabies vaccine; blood donation can be made one year after the last vaccination with rabies vaccine after being bitten by a rabid dog.
4.3.2 Blood donation can be made after the last injection of animal serum. 4.3.3 Healthy people do not need to postpone blood donation after receiving hepatitis B vaccine and hepatitis A vaccine. 4.3.4 Blood donation can be made after one year after receiving hepatitis B immunoglobulin injection. 4.4 People who fall into any of the following situations cannot donate blood temporarily. 4.4.1 Less than half a month after tooth extraction or other minor surgery; less than three months after appendectomy, hernia repair and sphenoid surgery; less than six months after major surgery.
4.4.2 Women within three days before or after menstruation, within six months of pregnancy and abortion, and within one year of lactation. 4.4.3 Those who have not recovered from osteoporosis or acute gastroenteritis for less than one week; those who have not recovered from acute urinary tract infection for less than one month, and those who have not recovered from pneumonia for less than two months.
4.4.4 Certain infectious diseases: such as those who have not recovered from epilepsy for less than six months, those who have not recovered from typhoid fever for less than one year, those who have not recovered from brucellosis for less than one year, and those who have not recovered from pimples for less than three years.
4.4.5 Those who have not recovered from localized skin inflammation for less than one week, and those who have not recovered from extensive inflammation for less than two weeks. 4.4.6 Those who have not stopped taking oral drugs that inhibit or damage platelet function (such as drugs containing aspirin or aspirin) for less than five days. 4.4.7 Those who have received blood or blood components transfusion in the past five years 4.4.8 Those who have been injured or contaminated by blood or red tissue contaminated equipment, and those who have been tattooed for less than 1 year. 4.4.9 Those who have a history of close contact with infectious disease patients, from the time of contact to the longest incubation period of the disease, 4.5 Those who have any of the following conditions cannot donate blood: 4.5.1 Patients with viral hepatitis, positive hepatitis B surface antigen, and positive hepatitis virus antibody. 4.5.2 Patients with acquired immunodeficiency syndrome (AIDS) and those infected with human immunodeficiency virus (HIV) 4.5.3 High-risk groups susceptible to human immunodeficiency diseases, such as drug addicts, homosexuals. People with multiple sexual partners. GB 18467--2001
4.5.4 Patients with leprosy and sexually transmitted diseases, such as syphilis, gonorrhea, etc. 4.5.5 Patients whose blood has caused transfusion-related infectious diseases in the recipient. 4.5.6 Patients with allergic diseases and recurrent allergies, such as frequent urticaria, bronchial asthma, and drug allergy (simple urticaria can donate blood during the acute attack period).
4.5.7 Patients with various tuberculosis, such as pulmonary tuberculosis, renal tuberculosis, lymph node tuberculosis, and bone tuberculosis. 4.5.8 Patients with cardiovascular diseases, such as various heart diseases, hypertension, hypotension, myocarditis, and thrombophlebitis. 4.5.9 Patients with respiratory diseases, such as chronic bronchitis, emphysema, bronchiectasis, and pulmonary insufficiency. 4.5.10 Patients with digestive system diseases, such as severe gastric and duodenal ulcers, chronic gastroenteritis, chronic gland inflammation, etc. 4.5.11 Patients with urinary system diseases, such as acute and chronic nephritis, chronic urinary tract infection, nephrotic syndrome, acute and chronic renal insufficiency, etc. 4.5.12 Patients with blood diseases, such as anemia, leukemia, polycythemia vera, and various bleeding and coagulation diseases. 4.5.13 Patients with endocrine diseases or metabolic disorders, such as pituitary and adrenal diseases, hyperthyroidism, acromegaly, preurinary syndrome, and diabetes, etc.
4.5.14 Patients with organic nervous system diseases or mental illnesses, such as encephalitis, sequelae of brain trauma, epilepsy, schizophrenia, addiction, and severe neurasthenia, etc.
4.5.15 Patients with parasites and endemic diseases, such as kala-azar,Schistosomiasis, filariasis, hookworm disease, cysticercosis, paragonimiasis, Keshan disease and Kaschin-Beck disease, etc.,
4.5.16 Patients with various malignant tumors and benign tumors that affect health, 4.5.17 Patients who have undergone surgery to remove important internal organs such as stomach, kidney, spleen, lung, etc. 4.5.18 Patients with chronic skin diseases, especially infectious, allergic and inflammatory systemic skin diseases, such as yellow ringworm, generalized eczema and systemic psoriasis, etc.,
4.5.19 Patients with ophthalmological diseases, such as keratitis, optic neuritis and high myopia with changes in the fundus, etc. 4.5.20 Autoimmune diseases and collagen diseases, such as systemic erythema, dermatomyositis, scleroderma, etc. 4.5.21 Patients with Creutzfeldt-Jakob disease and those with a family history of the disease, or those receiving treatment for tissues or tissue derivatives that may be infected by Creutzfeldt-Jakob pathogens (such as dura mater, cornea, human pituitary growth hormone, etc.). 4.5.22 Patients with certain occupational diseases, such as radiation sickness, pneumoconiosis, and acute and chronic poisoning caused by harmful gases and toxic substances. 4.5.23 Patients with other diseases that the physical examination doctor believes cannot donate blood. 4.6 Blood Donation Mice and Blood Donation Time
4.6.1 Blood Donation: All blood donors who meet the "Blood Donor Health Examination Standards" can donate 200mL or 400mL of blood at a time. 4.6.2 Blood Donation Time
4.6.2.1 Donation of whole blood: more than six months.
4.6.2.2 Machine-collected platelets: Collect once every 4 weeks. If the interval is less than 4 weeks, the platelet count before collection should be ≥150×10/1.
4.6.2.3 After machine-collected platelets, you can donate blood after more than 4 weeks. The time interval for whole blood donation should be the same as that for whole blood donation. GB18467—2001
Appendix A
(Standard Appendix)
Health Questionnaire for Blood Donors
Do you have any of the following: (Put a √ in the box if you have one: put a × in the box if you don't) 1. Do you have AIDS or HIV infection? 2. Do you have a history of drug abuse, homosexuality, or multiple sexual partners?
3. Have you ever had syphilis, gonorrhea or other sexually transmitted diseases?
4. Have you had sexual intercourse with the persons mentioned in 1 to 3 in the past year?
5. Have you had unexplained weight loss, persistent fever, diarrhea, or swollen lymph nodes in the past three months?
7. Have you ever had hepatitis or tested positive for hepatitis?
8. Have you ever received a blood transfusion or blood component in the past five years?
9. Have you had a tattoo in the past year?
10. Do you have any cancer?
11. Do you have tuberculosis?
12. Do you have heart disease, lung disease, kidney disease, liver disease or blood disease?
13. Do you have hypertension or hyperlipidemia?
1.4. Do you have hyperthyroidism or diabetes?
15. Do you have severe gastric and eleven-denal ulcers?
16. Do you have allergic diseases?
17. Have you had any eczema in the past six months?
18. Have you had any trauma in the past year?
19. Have you had any inflammatory disease in the past three years?
20. Have you taken aspirin orally in the past five years? 21. Have you had a dental surgery or other surgery in the past two weeks? 22. Have you ever had a major surgery? If yes: When:
Type of surgery:
23. Have you had any acute gastroenteritis in the past year?
24. Have you had any acute urinary tract infection in the past month? 25. Have you had any pneumonia in the past three months?
26i. Do you have any chronic skin disease or skin infection? 27. Have you ever fainted, had epilepsy, or lost consciousness? 28. Women in menstruation or pregnancy?
29. Women who have had abortion for less than six months and breastfeeding for less than one year?
30. Have you received animal serum immunization or other vaccinations in the past year?
31. Have you ever used human growth hormone to treat diseases? 32. Do you have any other diseases or conditions besides the above?
Thank you again for your love and dedication, thank you for your cooperation! Blood Donor Statement
I voluntarily donate blood to
Blood Station. The use of blood is decided by the blood station. I guarantee that all the answers to the questions raised in the "Blood Donor Health Information Collection Form" are true. I agree that the blood station will extract my blood sample and test it according to the specified items, and store the above test results in the blood donation file. I understand that the blood donation test results are only for safe blood transfusion and cannot be used for insurance, disease diagnosis or other purposes:
I understand that if I do not pay attention to any of the signatures provided in the above "inquiry form" or the above statement is false, I will be responsible for the consequences. I hereby declare.
Blood donor's signature:
ID/passport number:
Month and date:
Working unit
Correspondence address
Registration address
Document type
Blood donation frequency
Blood sample collection
GB18467-2001
Appendix B
(Appendix to the standard)
Blood donation record form||t t||Testimony number
Last blood donation time
Appendix C
(Appendix recommended by the standard)
Operational procedures for blood tests of blood donors
Blood donation certificate number:
Education level
C1.1 The name of the blood donor and the blood sample number must be carefully checked when collecting blood samples. Blood samples should be placed in the test tube rack in the order of numbers. C1.2 Disposable syringes with production approval numbers must be used to collect blood samples and must be used within the validity period: disposable syringes (including needles and needles) should be placed in sealed bags and incinerated or disinfected and sterilized after use. It is strictly forbidden to recycle and reuse them. C2 Hemoglobin (Hh) determination
C2.1 Murine methemoglobin (heniglobin C2.1.1 Principle
Hemoglobin is oxidized by potassium ferrocyanide to form methemoglobin, which then combines with cyanide ions to form stable cyanide methemoglobin. Methemoglobin has a maximum absorption peak at a wavelength of 510 nm and can be directly quantitatively determined by a spectrophotometer or by colorimetric determination using a reference solution of methemoglobin.
C2.1.2 Equipment
C2.1.2.1 Formula of cyanide methemoglobin reagent: Potassium cyanide
Potassium ferrocyanide
Anhydrous potassium dihydrogen phosphate
Trita X-loo
Distilled water
C2.1.2.2 Spectrophotometer
C2.1.3 Experimental steps
1 000 m
7. 0-~7. 4
C2. 1. 3. 1 Take 20 μL of whole blood, add it to 5 mL of HiCN reagent, mix and let it stand for 5 min. C2.1.3.2 Use a spectrophotometer at a wavelength of 540 nm, adjust to zero with HiCN reagent or distilled water, and measure the absorbance. C2.1.3.3 Calculate, see formula (C1).
GB 18467—2001
×251 = A×367. 7
64 458
44 000
Where: H
—hemoglobin concentration·g/L
A—measured absorbance;
average molecular weight of hemoglobin;
64 458-—
44000—molar absorbance of hemoglobin;
251-—dilution multiple.
..(C1)
C2. 1. 3. 4 Use HiCN reference solution to draw a standard curve. Use the hemoglobin reference value as the horizontal axis and the absorbance as the vertical axis to draw a standard curve.
C2.1.4 Result judgment
Male, 125~160 g/L
Female: 115~150 g/t,
C2.1.5 PrecautionswwW.bzxz.Net
C2.1.5.1 HiCN reagent cannot be stored in a plastic bottle, otherwise it will cause ion loss and the measured value will be low. C2.1.5.2 Potassium fluoride is a drug. When preparing the reagent, follow the highly toxic management procedures. C2.2 Whole blood acid pot specific gravity method (this method is an alternative method for determining Hb content) (method 1). C2.2.1 Principle
Dropping whole blood into a known standard copper sulfate solution will form a layer of protein copper salt surrounding the outer layer of the whole blood drop. By observing whether the whole blood sinks or floats or stays still in copper sulfate solutions of different relative densities (specific gravities), its relative density (specific gravity) can be measured. C2.2.2 Equipment
Copper sulfate solution, Webster hydrometer.
C2.2.3 Experimental steps
Pour the copper sulfate solution with relative density (specific gravity) of 1.0500 and 1.0520 into two 100mL measuring cups respectively and attach density labels (specific gravity labels). Place 1 drop of blood from a male donor in a 1.052 ( 0.050 0 measuring cup. Place 1 drop of blood from a female donor in a 1.050 0 measuring cup. Keep the drop 1 cm from the copper sulfate liquid surface and observe the drop's sinking within 15 seconds. C2.2.4 Result judgment
If the relative density (specific gravity) of the sample to be tested is greater than the relative density (specific gravity) of the copper sulfate solution, the drop will quickly sink to the bottom of the cup. If the relative density (specific gravity) of the sample to be tested is equal to the relative density (specific gravity) of the copper sulfate solution, the drop will suspend in the middle of the liquid surface for 10 to 15 seconds before sinking.
If the relative density (specific gravity) of the sample to be tested is less than the relative density (specific gravity) of the copper sulfate solution, the drop will float and will not sink within 15 seconds. Male: Relative density (specific gravity) of whole blood 1.052 0 (equivalent to hemoglobin content ≥125 g/1.), is qualified. The relative density (specific gravity) of female whole blood is 1.0500 (equivalent to the hemoglobin content of 115g/L), which is qualified. C2.2.5 Precautions
C2.2.5.1 The equipment used must be clean.
C2.2.5.2 Copper sulfate must be prepared immediately before use and must be calibrated with a densimeter before use. C2.2.5.3 The blood drop cannot contain bubbles. C2.2.5.4 The number of blood drops to be tested cannot exceed 80 drops per 100 mL of copper sulfate solution. C3 Blood typing
C3.1 ABO blood typing
This standard requires that the blood typing test kit approved by the Ministry of Health must be used within the validity period. C3.1.1 Method
Glass slide method (first method), test tube method (second method). C3.1.2 Principle
GB 184672001
Based on the presence or absence of A antigen and/or B antigen on red blood cells, blood types are divided into four types: A type, B type, AB type and O type. The red blood cell aggregation test can be used to accurately identify AB () blood type through positive (serum test) and negative (cell test) typing. The positive typing is to use known anti-A or anti-B blood type reagents to determine whether there are corresponding A antigens and/or B antigens on red blood cells, and the so-called negative typing is to use known A, B type cells and B type cells to determine whether the serum has corresponding anti-A or anti-3. C3.1.3 Equipment
C3.1.3.1 Anti-A (B type), anti-B (A type), and anti-A, B (O type) blood type reagents, titer 1: 128. C3.1.3.2 3% ~ 5% A, B and O type reagents red blood cell saline suspension. C3.1.3.3 Serum of the test subject.
C3.1.3.4 3%~5% red blood cell suspension in saline of the test subject. C3.1.4 Experimental steps
C3.1.4.1 Glass slide method
C3.1.4.1.1 Take a clean glass slide (or a white porcelain plate). Mark anti-A, anti-B and anti-AB, use a dropper to add 1 drop of anti-A, anti-B and anti-A, I3 blood group reagent respectively, then add 1 drop of 3%~5% red blood cell suspension of the test subject, and mix. C3.1.4.1.2 Take another clean glass slide (or a white porcelain plate). Mark A, B and O cells, use a dropper to add 1 drop of serum of the test subject, then use a dropper to add 1 drop of red blood cell suspension of A1, B and O type reagent respectively. 3.1.4.1.3 Gently rotate the glass slide (white porcelain plate) continuously (at room temperature 18°C~22°C) to allow the serum and cells to be fully mixed. Continue for about 2 minutes and observe with a microscope whether there is agglutination (hemolysis) reaction. If the glass slide test is used, the results can also be observed with a microscope. L3.1.4.2 Test tube method
C3.1.4.2.1 Take 3 clean small test tubes (inner diameter 10 mm×60 mm), mark them with anti-A, anti-B and anti-A, B respectively, use a dropper to add 2 drops of anti-A, anti-B and anti-A, B blood type reagent to the bottom of the test tube respectively, and then use a dropper to add 1 drop of the subject's 3%~5% red blood cell saline suspension respectively, and mix.
c3.1.4.2.2 Take three clean small test tubes (inner diameter 10 mmX16 mm) and mark them with type A, B and O cells respectively. Use a dropper to add 1-2 drops of the subject's serum to the bottom of the test tube, and then use a dropper to add 1 drop of type A, B and 0 3% reagent red blood cell suspension to mix. C3.1.4.2.3 Centrifuge at 120g for 1 min or 1300g for 15 s. C3.1.4.2.4 Gently shake the test tube to make the red blood cells at the bottom of the tube float up. First observe with the naked eye whether there is agglutination (hemolysis). If agglutination is not seen with the naked eye, the reaction product should be poured onto a glass slide and then examined with a microscope. C3.1.4.2.5 When observing the results, we should not only see whether there is agglutination, but also pay attention to the agglutination strength, which is helpful for the discovery of A, B type, class B, or cisAB:
Result judgment
See Table C1
Table C1AB0 blood typing test results
Standard serum + test red blood cells
Note, "+" means agglutination, \-" means no agglutination. C3.2 RhD blood typing test
C3.2.1 Method
Blood typing results
Test serum + test red blood cells
A, cell
fermentation test method.
C3.2.2 Principle
GB 18467.--2001
Rh There are five main antisera in the blood group system, namely anti-C, anti-c, anti-D, anti-E, and anti-e. In clinical blood transfusion, only D antigen is generally identified. Those whose red blood cells are agglutinated by anti-D serum are Rh positive, and those that are not agglutinated are Rh negative. This test must be done with negative and positive controls.
C3.2.3 Equipment
C3.2.3.15% red blood cell suspension of the subject, C3.2.3.2 anti-D serum.
C3.2.3.31% papain (or pineapple enzyme) solution; weigh 1,000 mg of papain and dissolve it in 100 mL of pH 5.5 phosphate buffer C3.2.3.4 jH5.5 phosphate buffer: 5 mL of 67 mmol/L disodium hydrogen phosphate and 95 mL of 67 mmol/L potassium dihydrogen phosphate
C3.2.3.5E. Know Rh negative and positive 5% red blood cell suspension. C3.2.4 Experimental steps
C3. 2. 4. 1 Take a small test tube and mark it, add anti-D serum, 5% red blood cell saline suspension of the subject and 1% guarase (or pineapple enzyme) reagent, 1 drop each, mix, and place in a 37℃ water bath for 30 minutes. C3.2.5 Result determination
Negative control tube does not agglutinate, positive control tube agglutinates, and the subject agglutinates: Rh positive; negative control tube does not agglutinate, positive control tube agglutinates, and the subject does not agglutinate, Rh negative. C3.2. 6 Precautions
C3. 2. 6. 1 Rh, and the temperature and time of type detection should be strictly controlled. C3.2.6.2 When observing the reaction results, the test tube should be shaken gently, and it should not be shaken vigorously. C3. 2.6.3 When Rh negative is found, weak Rh D type must be excluded. C3.3 Detection of weak Rh D type
C3.3.1 Method
Indirect anti-human globulin method.
C3.3.2 Principle
Normal RhD antigen is a chimera composed of A, B, C and D. It is expressed as RhABCD. If one or more of A, C and D are missing, it is called weak Rh D type. Weak Rh D type red blood cells do not agglutinate with batches or several batches of anti-D serum in saline medium and enzyme test, but agglutinate in indirect anti-human globulin test. C3.3.3 Equipment
C3.3.3.1 Anti-D serum: 3 to 5 batches of anti-D serum from different producers or batches. C3.3.3.2 Anti-human globulin serum.
C3.3.3.31% papain solution.
C3.3.3.45% red blood cell suspension of the subject. C3.3.4 Experimental steps
C3.3.4.1 Perform indirect anti-human globulin test on the red blood cells of the subject with each batch of anti-D serum. C3.3.4.2 Perform enzyme test on the red blood cells of the subject with each batch of anti-D serum. C3.3.5 Result judgment
The red blood cells collected do not agglutinate with each batch of antiserum in the alcohol test or only agglutinate with one or several batches of anti-D serum, but agglutinate in the indirect antiglobulin test or the agglutination strength is lower than that of the antiglobulin test (AGT), which are all Rh weak D type cells. C3.3.6 Notes
Rh weak D type people can produce anti-D antibodies after transfusing D positive red blood cells. Therefore, patients with Rh weak D type should be regarded as Rh negative and transfuse Rh negative blood; blood donors with Rh weak D type should be regarded as Rh positive and cannot transfuse blood to Rh negative patients. GB 18467—2001
C4 Serum alanine aminotransferase (ALT) detection uses the ketone body powder method for initial inspection. If the result is negative, it can be judged as qualified; if the result is positive, the Reitman method or rate method should be used for re-examination: the re-examination after blood collection must use the Reitman method or rate method. C4.1 Method: Rate Method (Method 1)
C4.1.1 Principle
In the ALT rate method, the enzyme coupling reaction formula is: ALT
L-alanine α-ketoglutaric acid
=lactone + 1-glutamic acid
pyruvate + NADH + H-LDH
-L-lactic acid + NAD+
In the above coupling reaction, the oxidation rate of NADH is proportional to the enzyme activity in the sample. At a wavelength of 310nm, NADH shows a characteristic absorption peak, while NAD does not. Therefore, the decreasing rate of absorbance at 340nm (AA/min) can be monitored to calculate the activity unit of ALT.
C4.1.2 Equipment
C4.1.2.1 Reagent formula
pH(37C)
Tris buffer
α-alanine
α-ketoglutaric acid
l00 mmol/1
500 nmol/L
15nul/L
0.18 mmol/L
1 200 U/L
C4.1.2.2 Fully automatic biochemical analyzer or flat automatic biochemical analyzer C4.1.3 Experimental steps
The specific experimental steps depend on the model of the analyzer and the reagent instructions. C4.1.3.7 Main parameters
Serum: ALT base
Delay time
Monitoring time
Light path of colorimetric cup
C4.1.3.2 Calculation
Where
Serum alanine aminotransferase (ALT) concentration; rate of decrease of absorbance, AA/min
6300.--molar absorbance of NADH at 340nm. C4.1.4 Result determination
Reference value at 37°C: 10～40 U/L. C4.1.5 Precautions
C4.1.5.1 Anticoagulants can cause slight turbidity in the reaction solution. C4.1.5.2 Serum cannot be repeatedly frozen for storage. (C2
GB18467—2001
C4.1.5.3 Rate method After the reagent is reconstituted, it can be stored at 4°C for 2 to 4 days. If it is stored at room temperature of 25°C, it can only be stable for 4 to 8 hours. C4-2 Method: Reiter method (second method). C4.2.1 Principle
ALT The amino group of L-alanine and L-glutamic acid is converted. The corresponding acids in this reaction process are α-ketoglutaric acid and pyruvic acid:
L-alanine + ±-ketoglutaric acid
pyruvic acid + glutamic acid
After the serum and the matrix react for a certain time, 2,4-dinitrophenylhydrazine is added to generate the corresponding 2,4-dinitrophenylhydrazine with the two α-glucose in the reaction solution to terminate the reaction. Under alkaline conditions, the absorption spectrum curves of the two extracts are different, based on which the activity of the enzyme can be determined.
C4.2.2 Equipment
C4. 2. 2. 1 0. 1 mol/L Disodium hydrogen phosphate (Na,HPO,) solution C4.2. 2. 2 0. 1 mol/L Potassium dihydrogen phosphate (KH,PO4) solution. C4.2.2.3 0. 1 mol/L Phosphate buffer (pII 7.4). C4.2.2.4 Matrix buffer (DI alanine 200 mmol/l., α-ketoglutaric acid 2 mmol/L). C4.2.2.5 1.0 mmol/L 2,4-dinitrobenzene solution. C4.2.2.6 0.4 mol/L sodium hydroxide (NaOH) solution. C4.2.2.7 2 mmol/L pyruvate standard solution. C4.2.2.8 Spectrophotometer.
C4.2.3 Experimental steps
C4.2.3.1 Drawing of standard curve: Add the corresponding reagents to each tube according to Table C2. Drawing of standard curve by Reitman method Experimental table
[(1.1 mol/L Phosphate buffer/mL2mmol/LPropane standard solution/mL
Substrate buffer/mL
2,4-Dinitrophenyl phthalate/mL
0.1 mol/LSodium hydroxide/mL
Actual content of pyruvate/mmol/[
Equivalent to enzyme activity/Carman unit
After mixing, keep warm in a 37 ℃ water bath for 10 minIn0.50
After mixing, keep warm in a 37 ℃ water bath for 2h min5. n
Centrifuge, 10
After leaving the mixture for 5-1 min, use a spectrophotometer at a wavelength of 505 nm At , adjust the zero point with distilled water, and read the absorbance of each tube. Subtract the absorbance of the blank tube (tube No. 0\) from the absorbance of each tube, and plot the difference against the corresponding Carmen unit. (Use absorbance as the ordinate and the corresponding Carmen unit as the abscissa).
C4.2.3.2 Sample test: Add the corresponding reagent to each tube according to Table C.3. Serum to be tested/ml.
Matrix solution/mL
2,4-Dinitrophenylhydrazine/mL
Matrix solution/mL
0.4mol/L sodium hydroxide/ml
GB 18467---2001
Table C3 Experimental steps of Reis' method
Assay tube
After mixing, keep warm in a 37'C water bath for 30min0. 5
After mixing. C4.2.4 Result determination: After subtracting the absorbance of the control tube from the absorbance of the test tube, obtain the ALT activity unit from the standard curve. Normal reference value: ≤25 Carmen units, C4.2.5 Precautions: C4.2.5.1 When the Reitman method is used to batch determine ALT, after adding serum to each tube, the test tube rack should be placed in a 37°C water bath for operation. C4.2.5.2 Hemolytic, lipemic and jaundice serum must be self-controlled. C4.2.5.3 α-ketoglutaric acid and 2,4-dinitrophenylhydrazine in the matrix solution are both coloring substances, and the weighing must be accurate. C4.2.5.4 Propionic acid solution is unstable and must be prepared immediately before use. C4.2.5.5 The standard curve should be redrawn for each batch of reagents in the Reiter method. C4.3 Method: Ketone powder method (third method). C4.3.1 Principle
Serum AIT can exchange the hydrogen group of lactamase in the substrate with the keto group of α-ketoglutaric acid to generate glutamic acid and propionic acid. Pyruvic acid and sodium nitrosoferricyanide in the color developing powder generate a green compound in an alkaline environment in the presence of a nitrogen base. The color depth is proportional to the amount of pyruvic acid generated, and ALT can be qualitatively determined based on this. C4.3.2 Equipment
C4.3.2.1 Matrix solution
α-Ketoglutaric acid
Alanine
Put 1/2 aliquots in a 1000mL beaker, add 400mL of distilled water to dissolve it completely, then transfer it to a 2000mL volumetric flask and add distilled water to the mark (dilute it 5 times when needed). C4.3.2.2 Color developer
Sodium nitrosoferrocyanide
Anhydrous sodium carbonate
Ammonium sulfate
First grind the sodium nitrosoferrocyanide into a mortar, then add the latter two reagents, grind and mix them slightly in a mortar (do not make it too fine, store it in a sealed dry bottle and do not let it get wet). C4.3.2.3 Water bath
C4.3-2.4 Fixed weight pipette
C4.3.2.5 Perforated concave plate.
C4.3.2.6 small medicine spoons.
C4-3.3 Experimental steps
C4. 3. 3. Take a clean multi-well reaction plate, add 0. 1 mL of negative, positive control and blood to be tested. c4. 3. 3. 2 Add 0. 05 mL of matrix solution. GB 18467-200
C4.3.3.3 Mix thoroughly, seal the plate, and incubate in a 37℃ water bath for 30 minutes. C4. 3. 3. 4 Add 0. 4 g (one small) of color developing powder. Observe the results with the naked eye within 2 min~3 min. C4.3. 4 Result determination
Negative: pure yellow
Positive: yellow-green-green-blue-green-blue
C4. 3. 5 Precautions
C4.3.5.1 The reaction plate should be clean and free of ketone and hydroxyl compound contamination. C4.3.5.2 The color developing powder must not be damp and the dosage must be accurate. C5 Hepatitis B surface antigen (HBsAg) detection C5.1 Method (first method)
Solid phase enzyme-linked immunosorbent assay (ELISA). This standard requires the use of ELISA method to detect HBsAg. This standard requires that the detection kit approved by the Ministry of Health must be used. Use within the validity period. The sensitivity of the reagent used must be able to detect ≤1ng/mlL. C5. 1.1 Principle
The reaction plate is coated with multi-site anti-HBs to make it solid phase. Shenzhen, human serum to be tested and enzyme-labeled anti-HBs, if the serum to be tested contains HBA, a double antibody sandwich complex is formed on the surface of the solid phase carrier. After washing away the unbound enzyme-labeled antibody, the substrate for enzyme action is added, and the colored product is produced through the catalysis of the enzyme. On the contrary, if the serum to be tested does not contain HBsAg, no complex will be generated, and no color will be generated after adding the substrate.
C5.1.2 Equipment
C5. 1.2. 1 HBsAg detection kit, C5.1.2.2 Microplate reader.
C5. 1. 2. 3 Microplate washer.
C5. 1. 2. 4 Thermostat.
C5.1.2.5 Adjustable micropipette,
C5. 1. 2. 6 Quality control serum.
C5. 1. 2. 7 Blood to be tested,
C5. 1.3 Experimental steps refer to the instructions of the kit, C5. 1. 4 Result judgment
C5. 1.2. Calculation of critical value (C.0.) refer to the reagent instructions. Sample A value (S)/C.(. [, classified as IBsAg positive sample A value (S)/CO <1, judged as HBsAg negative. C5.1.5 Precautions
C5.1.5.1 The test equipment should be clean.
C5. 1. 5. 2 Strictly follow the instructions. C5.2 Method (Second Method)
Colloidal gold-immunochromatography (immunochromatography assay) C5.2.1 Principle
This test is a double antibody sandwich method. Chloroauric acid is reduced to prepare gold sol particles of a certain diameter and labeled with antibodies. The antibody is coated on the membrane and colloidal gold. The appropriate membrane pore size can separate blood cells and serum and make the serum have a good fat rise rate. If there is HBsAg in the serum, it will bind to the gold-labeled antibody to form an Au-Ab-Ag conjugate. When Au-Ab-Ag The antibody on the membrane forms Au-Ab-AgAb, and the color of colloidal gold is purple-red (positive); otherwise, it is colorless (negative).
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