Blood banking – part 1- Blood Grouping (Blood Group ABO and Rh Typing)
- This can be done on whole blood or even on clotted blood.
- The sample can be stored at 4 °C. and stable for 5 days.
- Some time week subgroups may result in mistyping where Coombs test may be helpful.
- ABO blood grouping and Rh typing are done before taking the blood.
- Blood grouping is done for the donor and the recipient (Crossmatch).
- Blood grouping is done in the expected mother and newborn to rule out Rh-incompatibility.
- History of blood groups:
- Blood groups having inherited differences were the first time described by a German scientist Karl Landsteiner in 1900.
- He took the blood sample of 6 of his colleagues, separated the serum, and prepared the saline suspension of the RBCs.
- Now mixed the RBCs suspension with the serum.
- He found agglutination in some and while in others no agglutination.
- He concluded that RBCs possess antigen which reacts with the corresponding antibody present in the serum.
- He postulated that there are three blood groups.
- The fourth blood group was discovered by his pupils, von Decastello and Strurli in 1902.
- Blood grouping is done on the basis of the presence of antigen present on the surface of RBCs.
- There are two major antigens A and B.
- So the basic principle of blood donation is that there should be no antibody to match the surface antigen on the RBCs.
- Blood groups A B O system is dependent upon the presence of two antigens on the surface of RBC and these are antigen A and antigen B.
- Blood group A has antigen-A and antibody-B.
- Blood group B has B-antigen and antibody-A.
- Blood group AB has antigen-A and antigen-B and no antibodies.
- Blood group O has no antigen and has antibody anti-A and anti-B.
- Blood group antigens:
- Only two antigens were known as A and B antigens, these explain four blood groups.
- Later on, it was found that an individual who does not have either A or B or both antigens, possesses antibodies against these missing antigens and is called blood group O.
- The A, B, and O antigens are present on most human body cells including white blood cells and platelets.
- Later on, it was found subgroups of the ABO system:
- Blood group A = A1 and A2.
- Blood group B = A1B and A2B.
- Other subgroups of A are A3, Ax, and Am.
- Blood Group B has also subgroups but these are very rare.
- Inheritance of the blood groups:
- The ABO system inheritance was suggested in 1908 and proved in 1910.
- There are three allelic genes = A, B, O.
- Each individual inherited two genes, one from each parent.
- O gene does not produce a product and is therefore called as amorphic (having no defined shape).
- The expression of gene A and B is dependant upon the gene H.
- In 1930, Thompson postulated 4 allelic genes:
- A1, A2, B, and O.
- The 4 alleles give rise to 6 phenotypes and 10 genotypes.
- In the 80% of the population who possess the secretor gene, these antigens are also found in soluble form in the secretions and body fluids like plasma, saliva, sweat, and semen.
- 75% of the individuals secrete substances in their saliva that has the same specificity as the ABO antigens on the RBCs.
- H substance is secreted by all the secretors.
- A and B substances are secreted in addition to H substance by the individuals of groups A and B, while group AB secretes A, B, and H substance.
- ABO system phenotypes are:
- ABO system genotypes are:
- Structure of antigen A, B, and H:
- The A and B gene control the synthesis of the specific enzyme responsible for the addition of single carbohydrate residue for group A and group B to basic antigenic glycoproteins or glycolipids with terminal sugar fucose on the RBCs known as H substance.
- The O gene is amorph and does not transform the H substance.
- Blood group O is called the universal donor that he/she can donate blood to all other groups. This should be done only in an emergency.
- While the blood group AB is a universal recipient that can receive blood from all other groups.
Table showing antigen and antibody in the ABO system:
Blood group Antigen on RBC Antibody in blood O Nil A and B A A B B B A AB A and B Nil
Blood grouping procedure:
- To establish the blood group of an individual needs forward and reverse grouping.
- Forward grouping:
- In this case, human sera are needed. This serum is collected from the individuals who have a very strong antibody titer.
- Anti-A is from blood group B, anti-B is from blood group A, and anti-A B is from blood group O individuals.
- Reverse grouping:
- RBCs for the reverse grouping also from the human source from A and B groups.
- A1 and A2 RBCs can be used but A1 is sufficient in the most routine procedures.
- Slide or tile method:
- This is elaborated in the following diagram.
- Tube method:
- Put five test tubes in the rack.
- Follow the instructions given in the following diagram and interpretation is given in the table.
Blood grouping tube method interpretations:
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Blood group anti-A anti-B anti-AB A1- red blood cells B-red blood cells Negative Negative Negative Positive Positive O Positive Negative Positive Negative Positive A Negative Positive Positive Positive Negative B Positive Positive Positive Negative Negative AB
False result in ABO blood grouping are:
- Procedural mistakes are:
- In the case of dirty glassware.
- If there is an improper cell to serum ratio, it will give a false positive or false-negative result.
- If reagents are contaminated or expired, it will give a false-positive result.
- If you do over centrifugation, it will give a false-positive result.
- If you do under centrifugation, it will give a false-negative result.
- If you miss the hemolysis as a positive result will change into negative results.
- If you do a careless reading of the result will be read as a negative result.
- In case if you don’t use the optical aid may be read as a false-negative result.
- Inaccurate identification of the sample or the reagents will give false positive or negative results.
- In the case of an incorrect reading of the results or interpretation will give false-positive or false-negative results.
- Other possible causes are:
- Antibody-coated RBCs in the patient may agglutinate in a high protein medium.
- Ask the history of the recent blood transfusion, that may give a mixture of cells type, giving mixed cell appearance in the testing.
- If there is an unusual genotype that antigen A or B expressed weakly.
- Blood group A2B and A3B may react weakly with reagents anti-sera anti-A. If anti-A1 is present, the sample may be misdiagnosed as Blood group B. Sera from the sample of thought to be group B should be tested with red blood cells A1 and A2 to differentiate with anti-A1 but no anti-A in their serum.
- May get false results in diseases like acute leukemia, or non-malignant hemolytic disorder. In these cases the ABO antigens are weak.
- RBCs may have genetic abnormalities or acquired surface abnormalities that make them polyagglutinable.
- Gram-negative bacteria may give group B like activity.
- High levels of proteins and fibrinogen may cause rouleux formation which may be mistaken as agglutination.
- In certain conditions, there is blood group specif substances in high concentration as seen in the ovarian cyst and that may neutralize the anti-A and anti-B when unwashed RBCs are used.
- Unwashed RBCs in case of multiple myeloma may give false-positive results because of rouleux formation.
- Drugs like dextran and contrast media may cause cellular aggregation and looks like agglutination.
- There is the effect of the age e.g.
- Newborn who have still not developed the antibodies. They may have the antibodies from the mother.
- Old people may not have enough strong antibodies level.
- It is advised to follow the rules strictly to avoid these mistakes which will put you in trouble.
Blood groups, phenotype, and genotypes
- The Rh system consists of two allelic genes:
- Basically there are 6 antigens and 6 corresponding antibodies:
- The anti-d antibody does not exist, so the existence of the antigen-d is also disputed.
- The factor C, D, E, e are all antigenic proteins.
- These antigens will produce antibodies in a person whose RBCs laking these antigens.
Antigen Antibody Antigen-C Antibody- C Antigen- D Antibody- D Antigen- E Antibody- E Antigen- c Antibody- c Antigen- d Antibody-d Antigen- e Antibody-e
- These antigens will produce antibodies in a person whose RBCs laking these antigens.
- This gene complex R or CDe is directly passed on from generation to generation.
- An individual who is R r = CDe/cde will pass either R (CDe) or r (cde) to his/her generation.
- This Rh system is assigned to chromosomes number 1.
- These will encode the membrane proteins that carry:
- Antigen D.
- Antigen Cc.
- Antigen Ee.
- The weak expression of the antigen-D is referred to as Du, is also important in blood banking.
- Around 1% of D-positive individual type as weak D-antigen known as Du, characterized by weak or absent RBcs agglutination by anti-D antibody during serologic testing.
- In these individuals, weak D antigen (Du) will be only detected by anti-human globulin (Coombs test) reagent.
- RhD gene may be either present or absent. So phenotypically the possibilities are:
- RhD positive (RhD+).
- RhD negative (RhD–).
- Rh-antibody rarely occurs naturally, mostly due to immune stimulation, as a result of previous transfusion or pregnancy.
- Most of the clinical issues are due to RhD-antibody.
- Anti-C, anti-c, anti-E, anti-e are occasionally seen and both may cause transfusion reaction and hemolytic disease of the newborn.
- There are few systems of the Rh nomenclature and the most commonly used is Fischer-Race is the CDE system.
Fisher-Race CDE system Wiener Rh system Rosenfield et al. system Antigen Antigen Antigen D Rho Rh1 C rh´ Rh2 E rh´´ Rh3 d Hr Rh4 c hr´ Rh5 e hr´´
Rh-positive and Rh-negative group:
- where there is the presence of Rh antigen on the surface of RBC is called Rh-positive group and Rh antigen-negative is called Rh-negative group.
- The individual whos RBCs contain D antigen (Rh0) are either as D/D or D/d are called Rh-positive. These represent 85% of the population.
- The D (Rh0) antigen is the strongest antigen and will lead to immunization if introduced into the other person.
- So Rh-positive means to the presence of D-antigen and not related to other Rh factors.
- It is needed to check the D-antigen before the blood transfusion.
- Always avoid Rh-positive blood transfusion into an Rh-negative person. If this is done by mistake then 80% chances are to develop Anti-D antibodies in the transfused person.
- In such cases, the first transfusion may not create a problem but in the subsequent transfusion, will have a blood transfusion reaction.
- The Rh-positive fetus can sensitize the Rh-negative mother.
- Later on if the given Rh-positive blood, in that case, the mother will develop a blood transfusion reaction.
- The individual whose RBCs lakes D antigen (Rh0) is called the Rh-negative group and these are 15% of the population.
- The majority of the Rh-negative persons are cde/cde, this genotype is truly an Rh-negative individual.
- All pregnant mothers should have blood typing and Rh factor typing.
- In the case of the Rh-negative mother, the father’s blood group should be determined.
- If the father is Rh-positive then perform an indirect Coombs test on the mother serum.
- Coombs test is repeated at 28, 30, and 38 weeks of gestation.
- If all test is negative then the fetus is not at risk.
- If these tests are positive then the fetus is at risk and may develop hemolytic anemia (Erythroblastosis fetalis).
- When the mother is Rh-negative and the fetus Rh-positive then the mother may be sensitized at the time of delivery due to feto-maternal blood mixing.
- The mother sensitization can be prevented by giving RhoGAM which is Rh Immunoglobulin which will neutralize the Rh-antigen.
- RhoGAM prevents future pregnancy from hemolytic anemia. Rh-negative blood group can develop Rh-antibody when there is exposure to Rh-positive blood because of the blood transfusion or feto-maternal blood mixing.
Rh typing procedure:
- The slide method is easy to perform.
- This method is described as diagrammatically.
- False-positive result in slide method:
- In the case of drying of the slide, it may mimic agglutination.
- Rule out the presence of microclots and these may mimic agglutination.
- Inadequate amount of the anticoagulant.
- False-negative result in slide method:
- Saline suspension of the RBCs may react poorly or give a weak reaction.
- In the case of anemic patients, there may be fewer RBCs to be tested.
- If you read the result in less than 2 minutes may give false result in case of weak RBCs.
- If you use the wrong reagents.
- The tube method is more accurate than the slide method.
This method is described as diagrammatically.
- False-positive result in the tube method is:
- If you keep for a long time serum and the RBCs, then because of the high protein medium, you may see false agglutination which basically is rouleux formation.
- The anti-Rho (D) serum used may contain other antibodies with different specificity.
- If there are contaminating antibodies with the specificity other than indicated in the literature.
- If there are polyagglutinable RBCs that may agglutinate by any serum protein reagents.
- In case if the patient has abnormal proteins in the serum.
- False-negative result in tube method:
- In the case of improper reagents used in the test.
- If serum and the cells left for a long time will give rise to rouleux formation which may be taken as agglutination.
- RBCs with variant antigens e.g Cw, ces, may fail to react with standard reagents.
Clinically important blood groups and their significance:
|Blood group system||Presence of antibody||Possibility of transfusion reaction||Hemolytic episodes in newborn|
|ABO||100% of Antibodies||Present and common||usually mild|
|Rh system||Common||Present and common||Present|
|Duffy system||Occasional||Present but occasional||Present and occasional|
|Kidd system||Occasional||Present but occasional||Present and occasional|
|Lewis system||Occasional||Present but is rare||Not seen|
|MN system||Rare||Present but rare||Present but rare|
|Kell system||Occasional||Present but occasional||No hemolysis but there is anemia|
|P system||Occasional||Present but rare||Present but rare|
|Lutheran system||Rare||Present but rare||Not seen|
|Li system||Rare||Usually not seen||Not seen|