Blood banking:- part 1- Blood Groups ABO and Rh System, Blood Grouping Procedures
Blood Groups ABO and Rh System
Sample for Blood Groups ABO and Rh System
- 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.
- Sometimes week subgroups may result in mistyping where the Coomb’s test may be helpful.
Indications for Blood Groups ABO and Rh System
- ABO blood grouping and Rh typing are done before donating 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.
Pathophysiology of Blood Groups ABO and Rh System
History of blood groups:
- German scientist Karl Landsteiner first described blood groups having inherited differences in 1900.
- These antibodies were originally discovered in the early 1900s and are now known to consist of immunoglobulin IgM, IgG, and IgA classes.
- Karl Landsteiner opened the door of blood banking.
- He took his blood sample and the blood sample of 6 of his colleagues in 1901.
- Separated the serum and prepared the RBCs’ saline suspension.
Inheritance of ABO RBC genes and chemical structure of antigens A and B:
- The theory for the inheritance of the ABO blood groups was first time described by Bernstein in 1924.
- He postulated that each individual inherits one ABO gene from each parent; these 2 genes decide which ABO antigens are present on the RBC membrane.
- One locus on each chromosome 9 is occupied by an A, B, or O gene.
- ABO genes do not actually code for the production of ABO antigens but rather produce specific glycosyl-transferases that add sugars to the basic precursor substance.
- The inheritance of the H – gene is independent of the inheritance of ABO genes. Still, A, B, and H antigens are formed from the same basic precursors material, genetic products.
- The basic material is a glycoprotein or glycolipids that are the backbone to which sugars are added in response to specific enzyme transferases shown by inherited parents’ genes.
- Tests for the ABH secretion may help establish the true ABO group of an individual whose red blood cell antigens are poorly developed.
- The presence of A, B, and H substances in the saliva is evidence of the inheritance of an A gene, B gene, and H gene.
- The presence of the A, B, and H antigens in the saliva and body fluids are called secretors.
- O gene is considered an amorphous, silent gene.
- The designation A or B refers to phenotypes, whereas AA, BO, and OO are genotyping.
Formation of ABO antibodies:
- Blod group ABO system antibodies are stimulated by the bacteria and the other substances in our surroundings.
- These antibodies result from cross-reactivity and are initiated at birth upon exposure to foreign substances. These are usually low (titer) at birth for the detection until the infants are 3 to 6 months old. It is logical to perform only forward grouping in newborn babies.
- The peak level is 5 to 10 years of age and then starts declining progressively with the advancing age.
- Patients older than 65 will have low titer, and antibodies in the reverse grouping may be undetectable.
Blood grouping based on history:
Karl Landsteiner performed the forward grouping and reverse grouping:
- Forward grouping:
- It is defined as using a known source of antibodies to detect the antigens on the red blood cells.
- Reverse grouping:
- It is defined as using the reagent cells with known ABO antigens and testing the patient’s serum for ABO group antibodies.
- He mixed the RBC suspension with the serum.
- He found agglutination in some, and while in others, no agglutination.
- He concluded that RBCs possess antigens that react with the corresponding antibody in the serum.
- He postulated that there are three blood groups.
- Land Steiner described blood groups A, B, and O.
- His pupils, von Decastello and Sturle, discovered the fourth blood group in 1902, blood group AB.
Forward blood grouping possibilities:
|Patient’s RBC||Reaction with anti-A||Reaction with anti-B||Blood group|
|Number 1 patient||Negative||Negative||O|
|Number 2 patient||Positive||Negative||A|
|Number 3 patient||Negative||Positive||B|
|Number 4 patient||Positive||Positive||AB|
Reverse blood grouping possibilities:
|Patient’s serum||Reaction with A1 cells||Reaction with B cells||Blood group|
|Number 1 patient||Positive||Positive||O|
|Number 2 patient||Negative||Positive||A|
|Number 3 patient||Positive||Negative||B|
|Number 4 patient||Negative||Negative||AB|
Blood group antigens:
Blood grouping is done based on the presence of antigens 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 RBCs’ surface antigen.
- In the USA, blood group frequency is:
- Blood group O = 45%
- Blood group A = 41%
- Blood group B = 10%
- Blood group AB = 4%
ABO phenotypes in the various populations:
|The phenotype of the ABO system||Asian||Mexican||Blacks||Whites|
The presence of A, B, and H substances are found in the following fluids:
- Amniotic fluids.
- Digestive juices.
- Pathological fluids like pleural, peritoneal, pericardial fluids, and ovarian cysts.
The presence of ABH substances in the saliva as secretors:
|Blood group ABO system||A-antigen Substance in saliva||B-antigen Substance in saliva||H-Substance in saliva|
|A antigens||High concentration||None||Scanty|
|B antigen||None||High concentration||Scanty|
|A B antigen||High concentration||High concentration||Scanty|
|Blood group O||None||None||High concentration|
Blood groups A B O system:
Blood group antigens and types:
- Only two antigens were known as A and B antigens; these explain four blood groups.
- Later, 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 in most human body cells, including white blood cells and platelets.
- Later on, 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 also has 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, and O.
- Each individual inherited two genes, one from each parent.
- O gene does not produce a product and is therefore called amorphic (having no defined shape).
- The expression of genes A and B are dependent upon 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 soluble in the secretions and body fluids like plasma, saliva, sweat, and semen.
- 75% of the individuals secrete substances in their saliva with the same specificity as the ABO antigens on the RBCs.
- All the secretors secrete h substance.
- A and B substances are secreted in addition to H substances by groups A and B individuals, while group AB secretes A, B, and H substances.
- It depends on the presence of two antigens on the surface of RBC, and these are antigen A and antigen B:
Structure of antigens A, B, and H:
- The ABO genes do not code for the production of the ABO antigens. But produce specific glycosyltransferase that adds sugar to the basic precursor substances.
- The A and B genes control the specific enzyme’s synthesis responsible for adding single carbohydrate residue for groups A and B to basic antigenic glycoproteins or glycolipids with terminal sugar fucose on the RBCs known as H substance.
- The action of the H gene substance ultimately gives rise to ABO antigens.
- The O gene is amorphous and does not transform the H substance.
- Blood group O is called the universal donor, and he/she can donate blood to all other groups. It should be done only in an emergency.
- While the blood group AB is a universal recipient that can receive blood from all other groups.
TYpes of blood groups:
Blood group A:
- It has antigen-A and antibody-B.
Blood group B:
- It has B-antigen and antibody-A.
Blood group AB:
- It has antigen-A and antigen-B and no antibodies.
Blood group O:
- It has no antigen and has antibodies anti-A and anti-B.
ABO system phenotypes and genotypes are:
|Phenotypes of the blood groups||Genotypes of the blood groups|
Table showing antigen and antibody in the ABO system:
|Blood group||Antigen on RBC||Antibody in blood|
|O||Nil||A and B|
|AB||A and B||Nil|
Blood grouping procedure:
- Establishing the blood group of an individual needs forward and reverse grouping.
- Forward grouping antisera:
- In this case, human sera are needed. This serum is collected from 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 and B are from blood group O individuals.
- Reverse grouping RBC source:
- 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 most routine procedures.
Slide or tile method:
- In this procedure, known antibodies and unknown antigens are taken.
- This is elaborated in the following diagram.
Tube method for blood grouping:
- Put five test tubes on the rack.
- Follow the instructions given in the following diagram, and the interpretation is shown 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|
False results 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 it under centrifugation, it will give a false-negative result.
- If you miss the hemolysis, a positive result will change into negative results.
- If you do a careless reading, the result will be read as a negative result.
- 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 of false-positive results 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 a mixed cell appearance in the testing.
- If there is an unusual genotype antigen, A or B is expressed weakly.
- Blood groups 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 thought 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.
- There is blood group specif substances in high concentration in certain conditions, as seen in the ovarian cyst, which 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 age e.g.
- Newborns who have still not developed the antibodies. They may have the antibodies from the mother.
- Older adults may not have enough strong antibodies level.
- It is advised to strictly follow the rules to avoid these mistakes, otherwise putting you in trouble.
Blood grouping. Genotypes and phenotypes of the baby:
History of the Rh system:
- Rh system is second in importance to the ABO system.
- In 1939, Levine and Stetson found an unusual agglutin in the mother’s serum of a stillborn fetus to agglutinate 80% of random ABO compatible donors.
- In 1940 Landsteiner and Weiner injected Blood from the monkey Maccacus rhesus into rabbits and guinea pigs, which resulted in the antibodies’ production. These antibodies agglutinated RBCs of around 85% of human donors.
- These two antibodies were the same.
- The person who possessed the corresponding antigens was called Rh-positive.
- The person who was laking the antigens was called Rh-negative.
- The rabbit anti-rhesus was named anti-LW after the Landsteiner and the Weiner.
- The human antibodies are named the same as anti-Rh.
- 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.
- Factors C, D, E, and e are all antigenic proteins.
- These antigens will produce antibodies in a person whose RBCs are taking these antigens.
|Antigen- D||Antibody- D|
|Antigen- E||Antibody- E|
|Antigen- c||Antibody- c|
- 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.
Comparison of the Fischer-Race and Wiener gene theory:
|Wiener gene Agglutinogen||Fisher-Race gene Agglutinogen|
|r rh||cde c, d, e|
|r’ rh’||Cde C,d,e|
|r” rh”||cdE c,d,E|
|ry rhz||CdE C,d,E|
|R° Rh0||cDe c,D,e|
|R1 Rh1||CDe C,D,e|
|R2 Rh2||cDE c, D, E|
|Rz Rhz||CDE C,D,E|
- This Rh system is assigned to chromosome number 1.
- These will encode the membrane proteins that carry:
- Antigen D.
- Antigen Cc.
- Antigen Ee.
- The weak expression of antigen-D, referred to as Du, is also important in blood banking.
- Around 1% of D-positive individuals type as a 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 the 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 due to immune stimulation resulting from previous transfusion or pregnancy.
- Most of the clinical issues are due to RhD-antibody.
- Anti-C, anti-c, anti-E, and anti-e are occasionally seen, and both may cause transfusion reactions and the newborn’s hemolytic disease.
There are few Rh nomenclature systems, and the most commonly used is Fischer-Race is the CDE system.
|Fisher-Race CDE system||Wiener Rh system||Rosenfield et al. system|
Rh-positive and Rh-negative group discussion:
- The presence of Rh antigen on the surface of RBC is called the Rh-positive group, and Rh antigen-negative is called the Rh-negative group.
- The individuals whose RBCs contain D antigen (Rh0) are either D/D or D/d and are called Rh-positive. These represent 85% of the population.
- The D (Rh0) antigen is the strongest antigen, leading to immunization if introduced to the other person.
- So Rh-positive means 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, 80% chance are developing anti-D antibodies in the transfused person.
- The first transfusion may not create a problem in such cases, but it will have a blood transfusion reaction in the subsequent transfusion.
The Rh-positive fetus can sensitize the Rh-negative mother.
- The first time exposure to Rh-D antigen will sensitize the mother.
- Later on, if 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 is 15% of the population.
- Most 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, it should determine the father’s blood group.
- If the father is Rh-positive, perform an indirect Coombs test on the mother’s 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, the fetus is at risk and may develop hemolytic anemia (Erythroblastosis fetalis).
- When the mother is Rh-negative and the fetus Rh-positive, the mother may be sensitized at delivery due to feto-maternal blood mixing.
- The mother’s sensitization can be prevented by giving RhoGAM, Rh Immunoglobulin, which will neutralize the Rh-antigen.
- RhoGAM prevents future pregnancy from hemolytic anemia. Rh-negative blood groups can develop Rh-antibody when exposed 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 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.
- Reading the result in less than 2 minutes may give a false result in weak RBCs.
- If you use the wrong reagents.
- The tube method is more accurate than the slide method.
- This method is described diagrammatically.
False-positive result in the tube method is:
- If you keep it for a long time serum and the RBCs, you may see false agglutination, which basically is rouleux formation because of the high protein medium.
- The anti-Rho (D) serum used may contain other antibodies with different specificity.
- If there are contaminating antibodies with specificity other than indicated in the literature.
- If there are polyagglutinable RBCs that may agglutinate by any serum protein reagents.
- In case 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 are 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 significant 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|