1. Experimental principle
Based on the presence or absence of A antigen or (and) B antigen on the red blood cells, the blood group is divided into four types: A, B, AB and O. The blood group can be accurately identified by positive and negative stereotyping using the red blood cell agglutination test. The A B O blood group can be accurately identified by positive and negative typing using the red blood cell agglutination test. The so-called positive stereotyping is to use the known anti-A and anti-B typing serum to determine the presence or absence of the corresponding A antigen or (and) B antigen on red blood cells; the so-called reverse stereotyping is to use the known A erythrocyte B erythrocyte to determine the presence or absence of the corresponding anti-A or (and) anti-B in the serum.
2, the type of specimen used and collection methods
2.1, the subject of 5% erythrocyte suspension
Erythrocyte suspension preparation: take the subject's erythrocytes, wash them 3 times with saline, take 1 drop of the pressurized erythrocyte and add 16 drops of saline (0.8 ml) to form a 5% erythrocyte suspension.
2.2 Serum of the subject
Take 2-3ml of venous blood of the subject, put it in a water bath until it is completely solidified, centrifuge it at 3000 rpm for 5 minutes, and separate the upper layer of serum for spare.
2.3, specimen acceptance
2.3.1, blood typing does not accept clinical specimens (except for the contents of 2.3.2).
2.3.2, special emergency testing personnel can not collect blood, the receipt of clinical specimens need to register the name of the nurse blood collection and delivery of the nurse's name, and check the specimen labeling and testing of the contents of the single whether or not they are consistent, if there is a discrepancy can be refused to accept the specimen.
2.4, specimen container requirements: clean disposable plastic test tubes
3, the use of reagents
3.1, anti-A, anti-B stereotyping reagent (monoclonal antibodies)
3.1.1, approval number: State Drug Administration S10960081
3.1.2, manufacturer: Changchun Institute of Biological Products
3.1.3, preservation conditions: 2-8 ℃ to avoid light storage, use within the validity period.
2.5 % A, B and O reagent red blood cell saline suspension
2.5.1, preparation method
Take the red blood cells of the three known A, B and O blood types, respectively, and washed by saline for 3 times, and washed by saline for 3 times, and then take the pressed red blood cells and add 1 drop of saline to 16 drops of physiological saline (0.8 ml) to form a 5 % red blood cell suspension.
2.5.2, preservation conditions: the prepared 5% erythrocyte suspension in the test tube on the indication of erythrocyte blood type and time of preparation, 2-8 ℃ to avoid light preservation, the expiration date of 3 days.
4, the instrument:
4.1, the instrument model: TL80-1 medical centrifuge
4.2, the instrument manufacturer: China's Jiangsu Jiangyan is the Tianli Medical Instrument Co. Ltd
4.3, the license No.: Su Drug Control Measures (test) No. 2030436 2001
4.4, the instrument use of the specific requirements see Centrifuge use maintenance program.
5, operating procedures, reporting results
5.1, positive stereotypes: take two clean test tubes labeled, respectively, add anti-A (blue) and anti-B (yellow) standard serum 2 drops each. Add 2 drops of 5% erythrocyte suspension to each tube, mix well, centrifuge at 1000 rpm for one minute or leave it at room temperature for 1 hour, shake gently, apply it to the slide, and observe whether it is agglutinated or not by microscope.
5.2, anti-stereotyping: take 2 clean test tubes and mark them. Use the dropper to add 2 drops of each of the examinee's serum and 2 drops of each of the 5% standard red blood cell suspensions of type A and B respectively. Mix. Immediately centrifuge at 1000 rpm for 1 minute, shake gently, apply to the slide, and observe whether the agglutination by microscope.
5.3. The results are shown in the following table:
Anti-A anti-B blood group A cell B cell
+ - A - +
- + B + -
- - O + + +
+ + AB - -
Note: (+) Agglutination (-) Non-agglutination
6.
6.1, the quality of standard serum should meet the requirements, after use should be placed in the refrigerator to avoid bacterial contamination. If there is turbidity or discoloration, it can not be used.
6.2 The reagent red blood cells should be mixed with 3 fresh red blood cells of the same type from healthy people, and washed with saline 3 times to remove the antibodies and soluble antigens present in the serum.
6.3. Test tubes, droppers, and slides must be clean and dry to prevent hemolysis.
6.4, the method of operation should be in accordance with the provisions of the general should be added first serum, and then added to the red blood cell suspension, in order to easily verify whether the omission of serum.
6.5 The centrifugation time should not be too long or too short, and the speed should not be too fast or too slow to prevent false positive or false negative results.
6.6, observation should be careful to distinguish between true and false agglutination.
6.7, after judging the results should be carefully checked and recorded to avoid clerical errors.
7, clinical significance
Blood transfusion has become an essential clinical means of treatment, blood transfusion must be imported into the A B O homozygous blood, such as the import of heterozygous blood, the imported red blood cells may be rapidly destroyed, resulting in severe hemolytic reaction, often life-threatening or even cause death.
8. Limitations of the method: This method cannot detect subtypes.
Rh (anti-D) blood group identification
1. Principle:
Rh blood group system through blood transfusion or pregnancy can produce immune antibodies, when encountered with the corresponding antigen, can lead to hemolytic reaction or hemolytic disease of the newborn. If misdiagnosed and mistreated, it can lead to disability or death. Those who have the condition should have this test. At present, five types of typing sera are generally used to check red blood cell antigens. Clinically, anyone with D antigen is called Rh positive, and those without D antigen are called Rh negative.
2, the type of specimen used and collection methods
2.1, the subject 2-3% erythrocyte suspension
Red blood cell suspension preparation: take the subject's erythrocytes, washed three times with saline, take the pressurized erythrocytes and add 1 drop to 32 drops of saline (1.6 ml) to form a 2-3% erythrocyte suspension.
2.2, specimen acceptance
2.2.1, blood typing does not accept clinical delivery of specimens (except for the contents of 2.3.2).
2.2.2, special emergency testing personnel can not collect blood, the receipt of clinical specimens need to register the name of the nurse blood collection and delivery of the nurse's name, and check the specimen labeling and testing of the contents of the single whether or not they are consistent, if there is a discrepancy can be refused to accept the specimen.
2.3, specimen container requirements: clean disposable plastic test tubes
3. Instruments and reagents:
3.1, instrument model: TL80-1 medical centrifuge
3.2, instrument manufacturers: China Jiangsu Jiangyan is Tianli Medical Equipment Co. 2001 No. 2030436
3.4, the specific requirements for the use of the instrument see the use of centrifuges and maintenance procedures
3.5, Rh antisera under this section using Zhuhai Beisuo Biotechnology Company Limited production of Rh antisera reagents Product Registration No.: State Food and Drug Administration Measures (quasi) No. 2004 No. 3400398
4. operation
4.1 Take 2 small test tubes, labeled with crayon, add 1 drop of the above Rh antiserum and 2 drops of 2-3% saline suspension of the subject's erythrocytes respectively, and mix well. Centrifugation: 3200-3400 rpm, 15--30 seconds
4.2 Judgement of results: Shake the tube gently and observe the results: if the test tube agglutinates, it means positive; if the test tube does not agglutinate, it means negative.
5. Precautions
5.1 The quality of Rh antiserum should meet the requirements, and should be stored in the refrigerator after use to avoid bacterial contamination. If turbidity or discoloration occurs, it cannot be used.
5.2. Test tubes, droppers and slides must be clean and dry to prevent hemolysis.
5.3, the method of operation should be in accordance with the regulations, generally should be added serum first, and then added red blood cell suspension, so that it is easy to verify whether the missed serum.
5.4 The centrifugation time should not be too long or too short, and the speed should not be too fast or too slow to prevent false positive or false negative results.
5.5, observation should be careful to distinguish between true and false agglutination.
5.6, after judging the results should be carefully checked and recorded to avoid clerical errors.
5.7, when the test with unwashed cells, the specimen in its own agglutination and abnormal proteins may cause false positive results.
Coagulant amine-mediated cross-matching
Principle
Red blood cells carry a large number of negative charges on their surface to avoid their generation of self-generated aggregates, and when the red blood cells are suspended in the electrolyte, the cations are attracted by the negative charges of the red blood cells, and at this time the red blood cells are surrounded by diffuse double-layered ionic clouds, which result in the formation of a zeta potential that determines the repulsion of the red blood cells from each other.
The coagulant amine technique first utilizes a low ionic solution (LIM), which reduces the ionic strength of the medium and decreases the cation cloud around the red blood cells to facilitate the binding of antibodies between the red blood cells and the serum (plasma).
Subsequently, coagulant amine (solution, which is a higher-order cationic multimer, heparin neutralizer, which produces many positive charges when dissolved, can neutralize the negative charges carried on the surface of the red blood cells, which reduces the zeta potential of the red blood cells, and shortens the distance between the red blood cells, which results in non-specific coagulation of the red blood cells.
Finally, Resuspending is added to neutralize the effect of Polybrene, so that normal erythrocytes will disperse in non-specific agglutination, and the test result is negative; however, if the erythrocytes are sensitized by the corresponding antibody, they will be coagulated by Resuspending, and the agglutination will not be dispersed, and the result of the test is positive.
Reagents in this section use coagulant amine media reagents produced by Zhuhai Beisuo Biotechnology Co., Ltd. Product Registration No.: State Food and Drug Administration Measures (quasi) 2004 No. 3400397
Set content: 150T loading (BA-1003) The main ingredients
1. LIM (Low Ionic Mediun) 1 bottle × 55ml Glucose, EDTA (2Na)
2. Polybrene Solution 1 bottle × 7.5ml Polybrene, sodium chloride
3. Resuspending Solution 1 bottle × 7.5ml Sodium citrate, dextrose
3. Test steps
3.1 Take Two test tubes, labeled primary and secondary side, add 2 drops of patient's serum (plasma) to the primary side tube, add 1 drop of 3-5% red blood cell suspension (washed or not) from the blood donor, and vice versa for the secondary side tube.
3.2 Add 0.7 ml of LIM to each tube, mix well, then add 2 drops of Polybrene to each tube and mix well.
3.3 Centrifuge the sample at 3400 rpm in a blood bank for 10 seconds, then pour off the supernatant without draining it, leaving about liquid at the bottom of the tube.
3.4Shake the tube gently and visually check for agglutination of the red blood cells, if there is no agglutination, the procedure must be repeated.
3.5 Finally, add 2 drops of Resuspending, gently turn the tube to mix and smear, and observe the results under the microscope: if the agglutination disperses within 30 seconds-1 minute, it represents non-specific aggregation induced by Polybrene, and the results of the blood match are compatible; if the agglutination does not disperses, it is a specific reaction of the red blood cells with antigen-antibody binding, and the results of the blood match are not compatible.
3.6 After matching, fill in the record sheet and register carefully, and notify the clinic to take blood.
4. Precautions:
4.1 Plasma containing EDTA can be used instead of serum.
4.2 If the patient's serum (plasma) contains heparin, such as dialysis patients, must add 4-6 drops of Polybrene solution to neutralize heparin.
4.3 The procedure of this kit is also applicable to antibody identification.
4.4 Please return the reagent to room temperature before use.
4.5 The addition of an auxiliary antiglobulin test is intended to increase the sensitivity of the detection of anti-K, and is not helpful in the detection of other erythrocyte antibodies.
4.6 In winter, when the room temperature is very low, the cross-match test is performed, and the serum of some patients may contain factors such as cold agglutinins, which may lead to false-positive results. If this is suspected, it is recommended to place the tube in a 37 degree C water bath immediately after the last drop of Resuspending, gently rotate the tube to mix, and observe the result within 30 seconds.
Standard Operating Procedures for Centrifuge Operation
1. Purpose: To enable each staff member to use this instrument correctly.
2. Scope: This SOP is only applicable to the general centrifuge in this room.
3. Responsibilities:
3.1 All laboratory staff should be familiar with and strictly abide by this SOP, and the person in charge of the room should supervise the implementation.
3.2 Changes to these SOPs may be proposed by any staff member using these SOPs and submitted for approval and signature by the following: the head of the room, section chief.
4. Operation:
4.1 Power on: turn on the power, turn on the power switch of the instrument, and the centrifuge self-check.
4.2 Press the "Open" key, open the cover, rotor cover, balance into the centrifuge tube, tighten the rotor cover, close the cover, at this time, the indicator light of the "Open" key is on. (If the cover is open after the power is turned on, this step is not necessary).
4.3 Setting of centrifugation conditions: Set the temperature, speed and time of centrifugation by pressing the up and down arrows of the "Temp", "Speed" and "Time" keys, Speed" and "Time" keys to set the temperature, speed and time of centrifugation respectively. The centrifugal speed can be divided into two kinds of rotational speed per minute (rpm) and centrifugal force (cfg), which can be converted to each other by pressing the up and down arrows of the "Speed" key at the same time.
4.4 Operation: After the centrifugation conditions are set, press the "Start/Stop" key to start the centrifuge, and the indicator light of the "Open" key goes out. When the set time is reached, the centrifuge stops automatically and a beep sounds, the indicator light of the "Open" key lights up, press the "Open" key, open the cover, unscrew the rotor cover and take out the centrifuge tube. If you need to stop the centrifugation process, you need to press the "Start/Stop" key again.
4.5 Shutdown: After centrifugation, use a dry soft cloth to wipe away the condensation in the centrifuge chamber, turn off the centrifuge power, and record the use of the centrifuge.
Centrifuge maintenance operation procedures
1, purpose: the centrifuge to carry out the correct maintenance to ensure the normal operation of the instrument.
2. Scope of application: This SOP is only applicable to centrifuges used in this room.
3. Responsibilities:
3.1 All laboratory staff should be familiar with and strictly abide by this SOP, and the person in charge of the room should supervise the implementation.
3.2 Changes to these SOPs may be proposed by any staff member using these SOPs and submitted for approval and signature by the following: the head of the room, section chief.
4. Operating Procedures:
4.1 Centrifuge Chamber Cleaning: In order to avoid contamination of samples and other residues, the centrifuge shell and centrifuge chamber should be cleaned and treated frequently. For centrifuge chamber cleaning, the centrifuge cover should be opened, the power cord should be dialed off, the centrifuge rotor should be spun down with special equipment, and then the centrifuge chamber should be cleaned with a neutral decontaminant (70% isopropyl alcohol/water mixture or ethanol to decontaminate); rubber seals inside the centrifuge chamber should be treated with decontaminant, then rinsed with water, and then lubricated with glycerin.
4.2 Rotor Cleaning: The rotor is contaminated with sample residue and may be corroded by certain chemicals, so it should be cleaned and maintained monthly. Clean the rotor once a month with a neutral detergent and make a record in the instrument's log book to prolong the life of the rotor.
4.3 After centrifugation, the condensate in the centrifugation chamber should be wiped off with a dry soft cloth. (This step only applies to refrigerated centrifuges)
4.4 When centrifugation is complete, leave the centrifuge lid open and turn off the machine.
Operating Procedures for Maintenance of Blood Bank Refrigerator
1. Purpose:
To carry out proper maintenance of the refrigerator in order to ensure the normal use of the refrigerator.
2. Applicable refrigerator range:
The various brands and models of refrigerators used in this laboratory.
3. Duties:
3.1 Laboratory staff should be familiar with and strictly comply with this SOP, the person in charge of the room to monitor the implementation.
3.2 Changes to these SOPs may be initiated by any staff member using these SOPs and submitted for approval and signature by the following: Room Head, Section Head.
4. Operation Methods
4.1 The refrigerator should be placed on a level floor with some space for heat dissipation.
4.2 External power supply voltage must be matched, and requires a good grounding wire.
4.3 The refrigerator is not allowed to store items not related to the laboratory.
4.4 All reagents, blood, samples, etc. put into the refrigerator must be sealed and classified.
4.5 Keep the water outlet of the refrigerator smooth; non-automatic defrost refrigerator should be defrosted regularly; clean the refrigerator regularly, cut off the power supply when cleaning, wipe the inside and outside of the refrigerator with a soft cloth dipped in water, and use neutral detergent if necessary.
4.6 daily by a person responsible for observing the temperature inside the refrigerator and recorded in the table, the record sheet is attached to the door, one per month, a year bound archives.
4.7 If the temperature exceeds the specified range, adjust the temperature control to bring it back to the normal range, and record.
4.8 If the temperature control adjustment is invalid, report to the Equipment Division for maintenance, repair must be accepted and signed before normal use.
4.9 Periodically disinfect the inside surface of the refrigerator with liquid disinfectant.
Mobile ultraviolet disinfection vehicle operating procedures
1, purpose: the correct use of ultraviolet disinfection vehicle for disinfection.
2, the scope of application: applicable to the room using the ZXC-type ultraviolet disinfection car (Shanghai Yuejin Medical Optical Instruments Factory raw plant).
3, responsibilities:
3.1 Laboratory staff should be familiar with and strictly comply with this SOP, the person in charge of the room to monitor the implementation.
3.2 Changes to these SOPs may be proposed by any staff member using these SOPs and submitted for approval and signature by the following: the head of the room, section chief.
4. Operation Procedures:
4.1 Open the protective door;
4.2 Hold the arm of the lamp with your hand and lift it up to the required position;
4.3 Plug in the power supply and turn on the switch of the lamp;
4.4 Set the time according to the need;
4.5 Press the button down to the bottom after the use, and at the same time hold the arm of the lamp with your other hand to put the lamp into the light box and close it slowly. the lamp into the light box and close the protective door.
4.6 Do not mix UV sterilizers from different lab areas.
4.6 Do not mix UV sterilizers from different experimental areas.