Blood banking:- part 1- Blood Groups ABO and Rh System, Blood Grouping Procedures

Sample

1. This can be done on whole blood or even on clotted blood.
2. The sample can be stored at 4 °C and stable for 5 days.
3. Some time week subgroups may result in mistyping where the Coomb’s test may be helpful.

Indications

1. ABO blood grouping and Rh typing are done before taking the blood for donation.
2. Blood grouping is done for the donor and the recipient (Crossmatch).
3. Blood grouping is done in the expected mother and newborn to rule out Rh-incompatibility.

Pathophysiology

ABO system:

History of blood groups:

1. Blood groups having inherited differences were the first time described by a German scientist Karl Landsteiner in 1900.
   1. These antibodies were originally discovered in the early 1900s and are now known to consist of immunoglobulin (Ig)M, IgG, and IgA classes.
   2. Karl Landsteiner actually opened the door of blood banking.
   3. He took his blood sample and the blood sample of 6 of his colleagues in 1901.
   4. Separated the serum and prepared the RBCs’ saline suspension.
      

      Blood group ABO antigens and isoantibodies

Inheritance of ABO blood groups genes and chemical structure of antigen A and B:

1. The theory for the inheritance of the ABO blood groups was first time described by Bernstein in 1924.
2. 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.
3. One locus on each chromosome 9 is occupied by an  A, B, or O gene.
   1. 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.
   2. The inheritance of H – the gene is independent of the inheritance of ABO genes, but A, B, H  antigens are formed from the same basic precursors material, which is genetic products.
   3. The basic material is a glycoprotein, or glycolipids are the backbone to which sugars are added in response to specific enzyme transferases shown by the inherited from parents genes.
      

      Structure of A and B antigens

   4. Tests for the ABH secretion may help establish the true ABO group of an individual whose red blood cells antigens are poorly developed.
   5. 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.
   6. The presence of the A, B, and H antigens in the saliva and body fluids are called secretors.
4. O gene is considered an amorphous, silent gene.
5. The designation A or B refers to phenotypes, whereas AA, BO, and OO are genotyping.

Karl Landsteiner performed the forward grouping and reverse grouping:

1. 1. Forward grouping is defined as using a known source of antibodies to detect the antigens on the red blood cells.
   2. Reverse grouping is defined as using the reagent cells with known ABO antigens and testing the patient’s serum for ABO group antibodies.
   3. He mixed the RBC suspension with the serum.
   4. He found agglutination in some, and while in others, no agglutination.
   5. He concluded that RBCs possess antigens, which react with the corresponding antibody present in the serum.
   6. He postulated that there are three blood groups.
   7. Land Steiner described blood groups A, B, O.
   8. His pupils, von Decastello and Sturle, discovered the fourth blood group in 1902, blood group AB.

Forward blood grouping:

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:

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 is done based on the presence of antigen present on the surface of RBCs.

1. There are two major antigens A and B.
2. So the basic principle of blood donation is that there should be no antibody to match the RBCs’ surface antigen.
3. In the USA, blood group frequency is:
   1. Blood group O = 45%
   2. Blood group A = 41%
   3. Blood group B = 10%
   4. Blood group AB = 4%

ABO phenotypes in the various populations:

The phenotype of the ABO system	Asian	Mexican	Blacks	Whites
O	43%	56%	49%	45%
A1	27%	22%	19%	33%
A2	Rare	6%	8%	8%
B	25%	13%	19%	10%
A1B	5%	4%	3%	3%
A2B	Rare	Rare	1%	1%

Formation of ABO antigens:

1. Blod group ABO system antibodies are stimulated by the bacteria and the other substances in our surroundings.
2. These antibodies are a result of 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.
3. The peak level is 5 to 10 years of age and then starts declining progressively with the advancing age.
4. Patients older than 65 will have low titer, and antibodies in the reverse grouping may be undetectable.
5. The presence of A, B, and H substances found in the following fluids:
   1. Saliva.
   2. Teras,
   3. Milk.
   4. Amniotic fluids.
   5. Digestive juices.
   6. Bile.
   7. Urine.
   8. Pathological fluids like pleural, peritoneal, pericardial fluids, and ovarian cyst.

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 depends on the presence of two antigens on the surface of RBC, and these are antigen A and antigen B:

1. Blood group A has antigen-A and antibody-B.
   

   Blood group A

2. Blood group B has B-antigen and antibody-A.
   

   Blood group B

3. Blood group AB has antigen-A and antigen-B and no antibodies.
   

   Blood group AB

4. Blood group O has no antigen and has antibodies anti-A and anti-B.
   

   Blood group O

Blood group antigens:

Blood group antigens:

1. Only two antigens were known as A and B antigens; these explain four blood groups.
   2. 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.
   3. The A, B, and O antigens are present on most human body cells, including white blood cells and platelets.
2. Later on, was found subgroups of the ABO system:
   2. Blood group A = A1 and A2.
   3. Blood group B = A1B and A2B.
      2. Other subgroups of A are A3, A_x, and A_m.
      3. Blood Group B also has subgroups, but these are very rare.

Inheritance of the blood groups:

1. The ABO system inheritance was suggested in 1908 and proved in 1910.
2. There are three allelic genes = A, B, O.
   2. Each individual inherited two genes, one from each parent.
   3. O gene does not produce a product and is therefore called amorphic (having no defined shape).
   4. The expression of genes A and B is dependant upon the gene H.
3. In 1930, Thompson postulated 4 allelic genes:
   2. A1, A2, B, and O.
   3. The 4 alleles give rise to 6 phenotypes and 10 genotypes.
   2. 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.
   3. 75% of the individuals secrete substances in their saliva with the same specificity as the ABO antigens on the RBCs.
   4. All the secretors secrete h substance.
   5. A and B substances are secreted in addition to H substances by the individuals of groups A and B, while group AB secretes A, B, and H substances.

ABO system phenotypes and genotypes  are:

Phenotypes of the blood groups	Genotypes of the blood groups
A1	A1 A1
A2	A1 A2 A2 A2 A2 O
B	B O B B
A1B	A1 B
A2B	A2 B
O	O O

Structure of antigen A, B, and H:

1. 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.
2. 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.
3. The action of the H gene substance ultimately gives rise to ABO antigens.
4. The O gene is amorphous and does not transform the H substance.
   

   Blood group antigen structure formation

5. Blood group O is called the universal donor that he/she can donate blood to all other groups. It should be done only in an emergency.
6. 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:

1. To establish the blood group of an individual needs forward and reverse grouping.
   1. Forward grouping antisera:
      1. In this case, human sera are needed. This serum is collected from individuals who have a very strong antibody titer.
      2. Anti-A is from blood group B, anti-B is from blood group A, and anti-A B is from blood group O individuals.
   2. Reverse grouping RBC source:
      1. RBCs for the reverse grouping also from the human source from A and B groups.
      2. A1 and A2 RBCs can be used, but A1 is sufficient in most routine procedures.
2. Slide or tile method:
   1. This is elaborated in the following diagram.
      

      Blood grouping on slide method

5. Tube method:
   1. Put five test tubes in the rack.
   2. Follow the instructions given in the following diagram, and interpretation is given in the table.
      

      Blood grouping tube method

      

      Blood grouping forward type

      

      Blood grouping minor procedure

      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 results in ABO blood grouping are:

Procedural mistakes are:

1. In the case of dirty glassware.
2. If there is an improper cell to serum ratio, it will give a false positive or false-negative result.
3. If reagents are contaminated or expired, it will give a false-positive result.
4. If you do over centrifugation, it will give a false-positive result.
5. If you do under centrifugation, it will give a false-negative result.
6. If you miss the hemolysis as a positive result will change into negative results.
7. If you do a careless reading of the result will be read as a negative result.
8. In case if you don’t use the optical aid may be read as a false-negative result.
9. Inaccurate identification of the sample or the reagents will give false positive or negative results.
10. In the case of an incorrect reading of the results or interpretation will give false-positive or false-negative results.

Other possible causes are:

1. Antibody-coated RBCs in the patient may agglutinate in a high protein medium.
2. Ask the history of the recent blood transfusion that may give a mixture of cells type, giving mixed cell appearance in the testing.
3. If there is an unusual genotype that antigen A or B expressed weakly.
4. 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.
5. May get false results in diseases like acute leukemia or non-malignant hemolytic disorder. In these cases, the ABO antigens are weak.
6. RBCs may have genetic abnormalities or acquired surface abnormalities that make them polyagglutinable.
7. Gram-negative bacteria may give group B-like activity.
8. High levels of proteins and fibrinogen may cause rouleux formation, which may be mistaken as agglutination.
9. There is blood group specif substances in high concentration in certain conditions as seen in the ovarian cyst, and that may neutralize the anti-A and anti-B when unwashed RBCs are used.
10. Unwashed RBCs in case of multiple myeloma may give false-positive results because of rouleux formation.
11. Drugs like dextran and contrast media may cause cellular aggregation and looks like agglutination.
12. There is the effect of age e.g.
    1. Newborns who have still not developed the antibodies. They may have the antibodies from the mother.
    2. Older adults may not have enough strong antibodies level.
13. It is advised to strictly follow the rules to avoid these mistakes, putting you in trouble.

Blood grouping. Genotypes and phenotypes of the baby:

Rh system:

1. History of the Rh system:
   1. Rh system is second in importance to the ABO system.
   2. In 1939, Levine and Stetson found in the mother’s serum of a stillborn fetus an unusual agglutinin found to agglutinate 80% of random ABO compatible donors.
   3. 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.
   4. These two antibodies were the same.
      1. The person who possessed the corresponding antigens was called Rh-positive.
      2. The person who was taking the antigens was called Rh-negative.
      3. The rabbit anti-rhesus was named anti-LW after the Landsteiner and the Weiner.
      4. The human antibodies are named the same as anti-Rh.
         

         Landsteiner and Wiener experiment for Rh typing in 1940

2. The Rh system consists of two allelic genes:
   1. RhD
   2. RhCE
3. Basically, there are 6 antigens and 6 corresponding antibodies:
   1. The anti-d antibody does not exist, so the existence of the antigen-d is also disputed.
   2. The factor C, D, E, e are all antigenic proteins.
      1. These antigens will produce antibodies in a person whose RBCs are 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

4. This gene complex R or CDe is directly passed on from generation to generation.
   1. 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

1. This Rh system is assigned to chromosome number 1.
2. These will encode the membrane proteins that carry:
   1. 1. Antigen D.
      2. Antigen Cc.
      3. Antigen Ee.
      4. The weak expression of antigen-D is referred to as D^u, is also important in blood banking.
         1. Around 1% of D-positive individual type as weak D-antigen known as D^u, characterized by weak or absent RBcs agglutination by anti-D antibody during serologic testing.
         2. In these individuals, weak D antigen (D^u) will be only detected by anti-human globulin (Coombs test) reagent.
   2. RhD gene may be either present or absent. So phenotypically, the possibilities are:
      1. RhD positive (RhD⁺).
      2. RhD negative (RhD^–).
   3. Antibody:
      1. Rh-antibody rarely occurs naturally, mostly due to immune stimulation resulting from previous transfusion or pregnancy.
      2. Most of the clinical issues are due to RhD-antibody.
      3. Anti-C, anti-c, anti-E, anti-e are occasionally seen, and both may cause transfusion reaction and the newborn’s hemolytic disease.
3. 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
   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 discussion:

1. The presence of Rh antigen on the surface of RBC is called Rh-positive group, and Rh antigen-negative is called Rh-negative group.
2. The individuals whose RBCs contain D antigen (Rh₀) are either as D/D or D/d are called Rh-positive. These represent 85% of the population.
   1. The D (Rh0) antigen is the strongest antigen and will lead to immunization if introduced into the other person.
   2. So Rh-positive means the presence of D-antigen and not related to other Rh factors.
   3. It is needed to check the D-antigen before the blood transfusion.
   4. Always avoid Rh-positive blood transfusion into an Rh-negative person. If this is done by mistake, 80%  chances are developing anti-D antibodies in the transfused person.
   5. The first transfusion may not create a problem in such cases, but it will have a blood transfusion reaction in the subsequent transfusion.
   6. The Rh-positive fetus can sensitize the Rh-negative mother.
      1. Later on, if the given Rh-positive blood, in that case, the mother will develop a blood transfusion reaction.
         

         Rh sensitization of the mother

3. The individual whose RBCs lakes D antigen (Rh₀) is called the Rh-negative group is 15% of the population.
   1. The majority of the Rh-negative persons are cde/cde; this genotype is truly an Rh-negative individual.
   1. 1. All pregnant mothers should have blood typing and Rh factor typing.
      2. In the case of the Rh-negative mother, it should determine the father’s blood group.
   2. If the father is Rh-positive, then perform an indirect Coombs test on the mother serum.
      1. Coombs test is repeated at 28, 30, and 38 weeks of gestation.
      2. If all test is negative, then the fetus is not at risk.
      3. If these tests are positive, the fetus is at risk and may develop hemolytic anemia (Erythroblastosis fetalis).
   3. When the mother is Rh-negative and the fetus Rh-positive, the mother may be sensitized at the time of delivery due to feto-maternal blood mixing.
   4. The mother’s sensitization can be prevented by giving RhoGAM, Rh Immunoglobulin, which will neutralize the Rh-antigen.
      1. 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:

Slide method:

1. The slide method is easy to perform.
2. This method is described diagrammatically.
   

   Rh-blood group typing slide method

3. False-positive result in slide method:
   1. In the case of drying of the slide, it may mimic agglutination.
   2. Rule out the presence of microclots, and these may mimic agglutination.
   3. Inadequate amount of the anticoagulant.
4. False-negative result in slide method:
   1. Saline suspension of the RBCs may react poorly or give a weak reaction.
   2. In the case of anemic patients, there may be fewer RBCs to be tested.
   3. If you read the result in less than 2 minutes may give a false result in weak RBCs.
   4. If you use the wrong reagents.

Tube method:

1. The tube method is more accurate than the slide method.

This method is described diagrammatically.

1. False-positive result in the tube method is:
   1. If you keep for a long time serum and the RBCs, you may see false agglutination, which basically is rouleux formation because of the high protein medium.
   2. The anti-Rho (D) serum used may contain other antibodies with different specificity.
   3. If there are contaminating antibodies with specificity other than indicated in the literature.
   4. If there are polyagglutinable RBCs that may agglutinate by any serum protein reagents.
   5. In case if the patient has abnormal proteins in the serum.
2. False-negative result in tube method:
   1. In the case of improper reagents used in the test.
   2. If serum and the cells are left for a long time will give rise to rouleux formation, which may be taken as agglutination.
   3. RBCs with variant antigens, e.g C^w, ce^s, 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

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