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 weak subgroups may result in mistyping where 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.
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.
ABO system formation from H-substance:
- H-gene is present on chromosome 19. It encodes carbohydrates precursor substances in RBCs.
- A and B genes have specific transferase enzymes which act on H-substances.
- There is the formation of A and B antigens.
- While O-gene encodes for inactive transferase enzyme and ultimately leads to the formation of blood group O.
Formation of ABO antibodies:
- Blood 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 progressively declines with advancing age.
- Patients older than 65 will have low titer; antibodies in the reverse grouping may be undetectable.
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 possess 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 based on history:
Karl Landsteiner performed the forward grouping and reverse grouping:
- It is defined as using a known source of antibodies to detect the antigens in the red blood cells.
- 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 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 a negative result.
- If you do a careless reading, the result will be read as a negative result.
- The optical aid may be read as a false-negative result if you don’t use it.
- 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 about the history of the recent blood transfusion that may give a mixture of cells type, giving a mixed cell appearance in the testing.
- A or B is expressed weakly if there is an unusual genotype antigen.
- 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-specific 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 look 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:
- The 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 people 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, 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 unrelated to other Rh factors.
- It is necessary 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, there is an 80% chance of 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.
- The slide method is easy to perform.
- This method is described diagrammatically.
False-positive result in slide method:
- In the case of drying 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.
Tube method for Rh-Typing:
- 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 serum and the RBCs for a long time, you may see false agglutination, which basically is rouleux formation because of the high protein medium.
- The anti-Rho (D) serum may contain other antibodies with different specificities.
- 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|
Questions and answers:
Question 1: In case of Rh-negative mother, first pregnancy can give hemolytic reaction?
No, in the first pregnancy there is sensitization, and in second pregnancy Rh-positive fetus will lead to hemolytic anemia.
Question 2: Does H substances found in tears?
Yes, it is found in tears.