Disclaimer
Transfusion practice might be senter dependent. Therefore, the reader is advice to know the local practice.
The hero
- Dr. Landsteiner who coined the ABO blood groups.
Interesting fact
- Jesus Christ had blood group AB
The basics
- immunology!
- antigen is what the cell (in this case erythrocytes) has on the surface. This antigen can be recognized by antibodies.
- antibodies of importance in this matter: IgG (small), IgM (large).
- the erythrocytes or the plasma of the donor should be compatible with the patients.
Blood group
- blood groups are determined by the presence of antigen on the surface of erythrocytes.
- there are 250 blood groups known, categorized in about 23 groups.
- ABO is the most immunogenic and well known. A can have genotype AA or AO; B: BB or BO, AB: AB, and O: nothing. Consequently, A people will have antibodies against B, B people has antibodies against A, O people has antibodies against A and B, AB people has no antibodies against A and B.
- Thus, O is universal erythrocytes donor and AB is universal plasma donor.
- Other significant immunogenic groups are the Rhesus, Kell and Duffy.
- For woman in fertility age (up to 50) and cancer patients, we looked the compatibility of these factors. For the others, only ABO group should be compatible.
Donor and blood products
- donor can give: whole blood, or separate components.
- from whole blood, the separate components: erythrocytes, thrombocytes and plasma can be seprated.
- using apharesis technique, separate components can be obtained. advantage: donor can give plasma or thrombocytes more frequent (every 15 days, vs. every 3 months when whole blood is given).
- erthrocytes can be stored for 42 days (at 4C, 2 weeks less when they are radiated), thrombocytes (5 days at room temperatures, should be constantly gently shaken), plasma (years, -20C).
Method use in our lab
- Column agglutination technique. Our machine is wadiana.
- Can be used for direct (to show antigen on erythrocytes) and indirect (to show antibodies) Coombs test.
- The idea is simple: manufactured casette (figure).
You put the cells (from donor if you want to identify theirs antigen) or from patients (if you want to perform for example identify blood group, or direct Coombs test) or from erythrocytes test cells (indirect Coombs test to find out antibodies in the serum of the patients).
Then you add the antibodies (known antibodies if you want to identify the donor, antibodies agains blood group antigens to identify blood group or serum from the patients for indirect Coombs test).
Antibodies will bind to antigen, when they recognize each other, if not they don't.
- If they recognize each other the erythrocyte will agglutinate.
- To agglutinate they need to have antihuman antibodies that recognize the antibodies bind to antigen. This will be provided in the reagentia.
- In original Coombs test, to remove unbound antibodies can be done by washing. Using the casette, it is not more needed. It is replaced by centrifugation.
- Positive reaction when the erytrhocytes remain on the top, negative when the erythrocytes down to the bottom (see figure).
Workflow
Bloodgroup determination
- Centrifuge the EDTA blood.
- Put it into machine where the incubation and centrifugation happened. In the casette, there are difference antibodies to test the ABO blood group and Rhesus and Rhesus subfenotype reagens present.
Compatibility between donor and patient
Abbreviated cross-matching
- Take erythrocytes from donor and put into casette.
- Centrifuge the EDTA blood from patients and get the serum.
- Add serum to the erythrocytes.
- Incubate and centrifugate
- When positve advance to screening test.
Screening test with 3 type of cells
- The antigens of these cells are known. Adding serum from patients will reveal the antibodies of the patients.
- The same technique as compatibility test.
Screening test with 11 type of cells and direct Coombs.
- Commercial Bio Rad erythrocyte test cell panels are used. The antigens of these cells are known. There are requirements for these cells: heterozygocy to find for weak antibodies.
- These tests are also repeated with the presence of enzymes. The enzymes, for example papaine makes the agglutination sterker for some blood groups, while the other, weaker (green column in Bio Rad form).
- Direct Coombs is performed by adding serum to the erythrocytes of the patients.
- When antigens are present --> identify the antigen and perform titration (for Rhesus the dose can be determined).
- When Coombs is positive --> perform whether IgG is positive than eluation.
Identifying the antigen on the cells
- Known antibodies are added to the cells from the patient (IgG, IgM, IgA).
- The same lab technique as above.
Eluation
- To destroy the erythrocytes and let the bound antibodies on antigen on erythrocytes free.
- The eluates are again tested on 11 cells using the techniques as above.
Allo absorption test
- When eluation and Coombs are positive (all is positve, so no differentiation can be made on 11 cells --> differentiation should be made between allo- and auto antibodies).
- The eluate is added to 3 known erythrocytes type to allow for example IgG anti D attached to antigen on the D. The cycle is repeated twice.
- What are not attached to these 3 cells, are re-tested on the 11 cells.
Cases
- Anti C was present in the serum of the patient. The Coombs was positive. The patient has no C antigen on the erythrocytes. The eluation was positive. Problem: the anti C was made against the C antigen in the erytrhocytes from the donor. The erythrocytes from the donor was not yet destroyed (side note: lifespan donor erytrhocytes is shorter due to higher metabolism than own erytrhocytes).
- Patient can develop for example anti Lu, but since it is not immunogenic, it shold not give a problem.
- Rhesus D can be not clearly positive, then it should be typed when it is from a woman in fertile age. Type 1-3 will be deemed as positive.
- When eluation was negative while Coombs was positive: aspecific reaction (medication, infection).
- Umbilical cord blood belongs to child but IgG from mother can be found. Up to 6 months, the baby will not have fully developed immune system.
- When everything is positive, inc
Transfusion practice might be senter dependent. Therefore, the reader is advice to know the local practice.
The hero
- Dr. Landsteiner who coined the ABO blood groups.Interesting fact
- Jesus Christ had blood group AB
The basics
- immunology!
- antigen is what the cell (in this case erythrocytes) has on the surface. This antigen can be recognized by antibodies.
- antibodies of importance in this matter: IgG (small), IgM (large).
- the erythrocytes or the plasma of the donor should be compatible with the patients.
Blood group
- blood groups are determined by the presence of antigen on the surface of erythrocytes.
- there are 250 blood groups known, categorized in about 23 groups.
- ABO is the most immunogenic and well known. A can have genotype AA or AO; B: BB or BO, AB: AB, and O: nothing. Consequently, A people will have antibodies against B, B people has antibodies against A, O people has antibodies against A and B, AB people has no antibodies against A and B.
- Thus, O is universal erythrocytes donor and AB is universal plasma donor.
- Other significant immunogenic groups are the Rhesus, Kell and Duffy.
- For woman in fertility age (up to 50) and cancer patients, we looked the compatibility of these factors. For the others, only ABO group should be compatible.
Donor and blood products
- donor can give: whole blood, or separate components.
- from whole blood, the separate components: erythrocytes, thrombocytes and plasma can be seprated.
- using apharesis technique, separate components can be obtained. advantage: donor can give plasma or thrombocytes more frequent (every 15 days, vs. every 3 months when whole blood is given).
- erthrocytes can be stored for 42 days (at 4C, 2 weeks less when they are radiated), thrombocytes (5 days at room temperatures, should be constantly gently shaken), plasma (years, -20C).
Method use in our lab
- Column agglutination technique. Our machine is wadiana.
- Can be used for direct (to show antigen on erythrocytes) and indirect (to show antibodies) Coombs test.
- The idea is simple: manufactured casette (figure).
You put the cells (from donor if you want to identify theirs antigen) or from patients (if you want to perform for example identify blood group, or direct Coombs test) or from erythrocytes test cells (indirect Coombs test to find out antibodies in the serum of the patients).
Then you add the antibodies (known antibodies if you want to identify the donor, antibodies agains blood group antigens to identify blood group or serum from the patients for indirect Coombs test).
Antibodies will bind to antigen, when they recognize each other, if not they don't.
- If they recognize each other the erythrocyte will agglutinate.
- To agglutinate they need to have antihuman antibodies that recognize the antibodies bind to antigen. This will be provided in the reagentia.
- In original Coombs test, to remove unbound antibodies can be done by washing. Using the casette, it is not more needed. It is replaced by centrifugation.
- Positive reaction when the erytrhocytes remain on the top, negative when the erythrocytes down to the bottom (see figure).
Workflow
Bloodgroup determination
- Centrifuge the EDTA blood.
- Put it into machine where the incubation and centrifugation happened. In the casette, there are difference antibodies to test the ABO blood group and Rhesus and Rhesus subfenotype reagens present.
Compatibility between donor and patient
Abbreviated cross-matching
- Take erythrocytes from donor and put into casette.
- Centrifuge the EDTA blood from patients and get the serum.
- Add serum to the erythrocytes.
- Incubate and centrifugate
- When positve advance to screening test.
Screening test with 3 type of cells
- The antigens of these cells are known. Adding serum from patients will reveal the antibodies of the patients.
- The same technique as compatibility test.
Screening test with 11 type of cells and direct Coombs.
- Commercial Bio Rad erythrocyte test cell panels are used. The antigens of these cells are known. There are requirements for these cells: heterozygocy to find for weak antibodies.
- These tests are also repeated with the presence of enzymes. The enzymes, for example papaine makes the agglutination sterker for some blood groups, while the other, weaker (green column in Bio Rad form).
- Direct Coombs is performed by adding serum to the erythrocytes of the patients.
- When antigens are present --> identify the antigen and perform titration (for Rhesus the dose can be determined).
- When Coombs is positive --> perform whether IgG is positive than eluation.
Identifying the antigen on the cells
- Known antibodies are added to the cells from the patient (IgG, IgM, IgA).
- The same lab technique as above.
Eluation
- To destroy the erythrocytes and let the bound antibodies on antigen on erythrocytes free.
- The eluates are again tested on 11 cells using the techniques as above.
Allo absorption test
- When eluation and Coombs are positive (all is positve, so no differentiation can be made on 11 cells --> differentiation should be made between allo- and auto antibodies).
- The eluate is added to 3 known erythrocytes type to allow for example IgG anti D attached to antigen on the D. The cycle is repeated twice.
- What are not attached to these 3 cells, are re-tested on the 11 cells.
Cases
- Anti C was present in the serum of the patient. The Coombs was positive. The patient has no C antigen on the erythrocytes. The eluation was positive. Problem: the anti C was made against the C antigen in the erytrhocytes from the donor. The erythrocytes from the donor was not yet destroyed (side note: lifespan donor erytrhocytes is shorter due to higher metabolism than own erytrhocytes).
- Patient can develop for example anti Lu, but since it is not immunogenic, it shold not give a problem.
- Rhesus D can be not clearly positive, then it should be typed when it is from a woman in fertile age. Type 1-3 will be deemed as positive.
- When eluation was negative while Coombs was positive: aspecific reaction (medication, infection).
- Umbilical cord blood belongs to child but IgG from mother can be found. Up to 6 months, the baby will not have fully developed immune system.
- When everything is positive, inc
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