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Human Blood Groups and Application System and Screening Tests

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  • The red cell membrane has a lot of antigens which can stimulate production of antibodies
  • Approximately 280 red cell antigens have been recognized
  • Most of these antigens have been grouped into blood group systems
  • Each system is genetically discrete from all the others
  • “MOST” blood groups are inherited as mendelial characters

Human Blood Groups



































Blood Group Genotype and Phenotype

  • Genotype – the genes inherited from both parent which are present on the chromosomes
  • Phenotype – the observable effect of the inherited genes i.e. the blood group itself

Genotype                                                               Blood group (phenotype)














Serological Techniques

  • Blood group serology is the study of red cell antigens and antibodies
  • An example of serological test is blood grouping
  • Sometimes it is possible to know the genotype from serological tests
  • Sometimes the correct genotype can be determined by suitable family studies
  • Most times the genotype cannot be determined by serological tests


  • Almost always, an individual has the same blood group for life, but very rarely an individual’s blood type may change e.g. after a bone marrow transplant


  • An antigen is any substance which, when introduced into the body and recognized as foreign, will bring about an immune response
  • The immune response may lead to the production of antibodies (humoral immunity) and/or proliferation of immunocompetent cells (cellular immunity)
  • Specific immune responses to human red cell antigens are mediated by antibodies only Blood Group Antigens
  • Antigens are found in the polypeptide and carbohydrate moieties of the red cell membrane glycoproteins and in the carbohydrate moieties of glycolipids
  • Some red cell antigens are not found on integral membrane components but are adsorbed passively from the plasma e.g. Lewis antigen
  • Approximately 280 red cell antigens have been recognized
  • Most of these antigens have been grouped into blood group systems
  • Each system represents a single gene locus or 2 or more closely linked loci of homologous genes
  • The genes representing each of the blood group systems have been located on a specific chromosomes
  • Rh antigens are found in the polypeptide moieties of membrane glycoproteins
  • ABH and Ii antigens are found predominantly on the carbohydrate moieties of the major red cell glycoprotein, band 3
  • P, P1 and PK antigens are expressed on the carbohydrate moieties of glycolipids
  • The M and N antigens arise from interactions between the carbohydrate and polypeptide in the glycoprotein GPA[1]
  • Most blood group genes are codominant e.g. A and B genes
  • Some are silent genes or amorphs e.g. the O gene
  • Blood group antigens that are proteins are direct products of their genes e.g. RH and Kell
  • Those that are carbohydrates are not direct products of their genes, they result from action of enzymes on the appropriate substrate or precursor substanceg. ABO, LE and P


  • Antibodies are immunoglobulins (Ig) produced by the B lymphocytes of the adaptive immune system in response to an antigen for which they exhibit specific binding
  • There are 5 classes of immunoglobulins: IgM, IgG, IgA, IgD and IgE
  • Antibodies with specificity for blood group antigens are found mainly in the IgG and IgM classes

Immunoglobulin Molecule

  • The basic immunoglobulin molecule consists of 4 polypeptide chains arranged as 2 L (light) chains and 2 identical H (heavy) chains
  • The light and heavy chains are usually held together by disulphide (S-S) bonds
  • IgG are monomers while IgM molecules are pentamers of this basic immunoglobulin structure







  • Naturally occurring and immune antibodies
  • Cold and warm antibodies
  • IgM and IgG antibodies

Naturally Occurring Antibodies

  • Usually, IgM produced without any obvious immunizing stimulus such as pregnancy, transfusion or injection of blood
  • They are not present at birth and, in the case of anti-A and anti-B, start to appear in the serum of children with the appropriate ABO groups at about 3-6 months of age
  • They are produced in response to non-red cell antigens of bacteria, viruses and other substances that are inhaled or ingested

Immune Antibodies

  • Immune blood group antibodies are usually IgG and are produced in response to a foreign red cell antigen following;
    • Pregnancy
    • Blood transfusion
    • Injection of blood or blood group substances

Cold Antibodies

  • React optimally at low temperature (0-4°C) and many of them will not agglutinate red cells at 37°C
  • Most naturally occurring antibodies are cold reacting
  • Some, such as naturally occurring anti-A and –B have a wide thermal range and will still react at 37°C, at which temperature they will activate complement and lyse red cells
  • Cold antibodies that fail to react above 30°C are of no clinical significance and can be ignored for blood transfusion purposes

Warm Antibodies

  • React optimally at 37°C
  • Immune antibodies are warm reacting
  • Any red cell antibody reacting above 30°C should be considered potentially capable of destroying red cells in vivo

IgG and IgM Antibodies

  • IgG are incomplete antibodies that cannot agglutinate saline suspended red cells. The lifespan is approximately 60-70 days
  • IgM are complete antibodies that agglutinate saline suspended red cells. The lifespan is only 10 days


Red Cell Antigen-Antibody Reactions

  • In blood group serology, red cell antigen-antibody reactions are normally detected by observing agglutination of the cells concerned
  • Agglutination is a reversible chemical reaction thought to occur in 2 stages

Stages of Agglutination

Stage 1: Sensitization

  • The antibody binds to its red cell antigen
  • This does not cause agglutination of the cell, but simple coats or sensitizes the cell

Stage 2: Agglutination

  • The sensitized red cells are bridged together to form agglutination


  • Temperature
  • pH
  • Antigen to antibody ratio
  • Ionic strength


  • Different antibodies have different preferred temperatures of reaction
  • For example, ABO blood group antibodies react best at 4°C, while Rh antibodies react best at 37°C


  • The optimum pH for most blood group antibodies is between 6.5 and 7.5
  • Below pH 4 and pH 9, antigen-antibody complexes are largely dissociated and the antibody can be recovered in the supernatant
  • This is the basis of some elution techniques

Antibody to Antigen Ratio

  • The more antibody that is present in relation to the number of antigens on the red cells, the stronger the reaction
  • The most suitable strength of cell suspension, when used for agglutination tests is 2-4%
  • Occasionally, significant antibody excess can, in contrast, inhibit direct agglutination (Prozone effect)
  • Individual antibody do not bind to 2 different cells
  • Thus cross linking cannot occur, leading to false negative result

Ionic Strength

  • The rate at which the antigen-antibody reaction occurs is considerably increased when the ionic strength of the medium in which the red cells are suspended is DECREASED
  • Using low ionic strength saline (LISS), the incubation period of the anti-human globulin test can be reduced from 1 hour to 15 minutes

Factors that Affect the Second Stage of Agglutination

  • The degree of contact of the antibody-coated red cells with each other
  • The electrical charge of the red cells
  • The span of the antibody molecules
  • The location and density of the antigen sites on the red cells
  • The capacity of the antibody to bind complement after reacting with the antigen

The Degree of Contact of Antibody Coated Cells

  • Antibody molecules cannot form bridges between individual cells until the cells are close together
  • Contact can be achieved by allowing the cells to settle by gravity
  • It takes 1-2 hours for settling to occur in a saline serum medium
  • Settling can be accelerated by centrifugation at low speeds for not more than 1 minute

The Electrical Charge of the Red Cells

  • Red cells suspended in saline are negatively charged
  • The negative charge on the red cell is produced by groups of neuraminic acid on the red cell membrane
  • The repulsive force generated creates a gap between individual red cells
  • The minimum distance between unsensitized red cells is approximately 18nm
  • The repulsive force holding the cells apart is called the ‘zeta potential’

The Span of the Antibody Molecule

  • IgG molecules with maximum distance of 12nm between their antigenic sites cannot bridge the gap between individual red cells to produce agglutination
  • IgM molecules with distance of about 30nm between the antigen-combining sites, are able to bridge this gap and thus cause agglutination of appropriate cells suspended in saline

The Location and Density of Antigen Sites

  • For antigens that protrude from the cell surface (e.g. ABO, MN, I, i), agglutination by the corresponding antibodies will occur more readily than for antigens embedded in the membrane (e.g. Rh)
  • The number of antigenic sites on each red cell will also determine the strength of agglutination e.g. the Du

The Capacity of the Antibody to Bind Complement

  • Antibodies that bind complement, mostly IgM and few IgG antibodies, cause lysis of the red cells and there might be no agglutination, especially when fresh serum that is rich in complement is being used
  • Lysis therefore also indicates a blood group antigen-antibody reaction
  • If this lysis is not noticed, the test may be read as negative and grossly incompatible blood might be regarded as compatible

Agglutination of Red Cells Coated by Incomplete Antibodies

  • The use of albumin
  • The use of anti-human globulin
  • The use of proteolytic enzymes

The Use of Albumin

  • Albumin reduces the repulsive force (zeta potential) between individual red cell thereby bringing them closer together so that IgG antibodies can bridge the gap and produce agglutination

Use of Anti-Human Globulin (AHG)

  • The AHG is used to detect the presence of human globulin on sensitized red cells
  • AHG binds to incomplete antibodies which are human globulin and help the incomplete antibodies to produce agglutination
  • It will also detect red cells that have been coated with C3 component of complement

IgG Sensitized Red Cells Agglutinated by Anti-Human Globulin Serum


The Use of Proteolytic Enzymes

  • Enzymes such as papain and ficin have the ability to remove some of the neuraminic acid, thereby reducing the negative charge on the red cells
  • This brings the red cells closer together, allowing IgG antibodies to agglutinate the cells
  • Enzyme treated red cell are not routinely used in antibody detection because enzyme treatment destroys some red cell antigens g. M, N, S, s, Fya, Fyb


  • First to be recognized
  • Most important blood group system
  • The clinical importance of a blood group system lies in the frequency of its antibodies and the possibility of those antibodies to destroy incompatible red cells in vivo
  • The system consist of 3 allelic genes, A, B and O


  • The A and B blood group antigens develop in strength from early fetal life through to adolescence
  • At birth, they are weaker than in adults and weaker than expected reactions may be found with anti-A and anti-B


  • The majority of human bloods can be grouped into 6 main ABO phenotypes
    • O
    • A1
    • A2
    • B
    • A1B ü A2B

The ABO Genes and Transferases

  • The A and B genes control the synthesis of specific enzymes called transferases
  • These transferases add a single carbohydrate residue to the terminal sugar (L-fucose) of the H substance
  • A transferase adds N-acetyl-D-galactosamine to the L-fucose on the H substance to produce A antigen
  • B transferase adds D-galactose to the L-fucose on the H substance to produce B antigen
  • O gene is not expressed i.e. the H substance remains the same, no carbohydrate residue is added


  • First described in Bombay in 1952
  • H gene is lacking in these individuals
  • H substance cannot be produced
  • A or B gene cannot be expressed


  • A2 is the weaker form of A
  • A1 red cells have more A antigen sites than A2
  • Anti-A1 will agglutinate A1 cells but not A2 cells
  • Anti-A will agglutinate both A1 and A2 red cells
  • There is no specific antibody for A2 red cells
  • Group B and O serum can therefore be thought to contain 2 antibodies anti A and anti A1


  • Group O cells have no antigens in the ABO system
  • However, they possess the H antigen, the precursor on which the products of the ABO genes act
  • The H antigen is present on almost all red cells regardless of the ABO group EXCEPT the Bombay phenotype
  • O > A2 > A2B > B > A1 > A1B Secretors and Non-Secretors
  • Secretors are homozygous or heterozygous for an active allele(Se)
  • Non-secretors are homozygous for an inactive allele(se)
  • About 80% of the population are secretors
  • Secretors have the ABH antigens in soluble forms in secretions and body fluids like plasma, saliva, semen and sweat


  • Naturally occurring
  • Immune


  • They are produced in the first years of life by sensitization to environmental substances such as food, bacteria and viruses
  • Titres reach a peak in young adults and decline in old age
  • They are IgM that react better at room temperature


  • They can be produced after immunization with red cells or blood group substances
  • Can also arise after various vaccinations and inoculations for the prophylaxis of infections
  • Here, the A-like antigens come from the hog pepsin digest used in their preparation  They can be IgM or IgG

Blood Group

Possible Genotype

ABH Antigens on Red Cells

ABH Antibodies in Plasma



A and H

Anti B



B and H

Anti A



A, B and H




H only

Anti A and Anti B


Any one


Anti A, B and H


The Rh Blood Group System

  • Most important in blood group system after ABO
  • More complex than a single antigen system
  • Over 50 different Rh antigens have been characterized
  • But there are 5 principal ones which are responsible for the majority of clinically significant antibodies: D, C, c, E and e
  • The most significant is the D antigen, because it is the most likely to provoke an immune system response of the 5 principal Rh antigens


Contemporary Rh terminology distinguishes between antigens, genes and proteins

  • Antigens: D, C, c, E, e
  • Genes: RHD, RHCE
  • Alleles of RHCE: RHCE*ce, RHCE*CE, etc. according to which antigen they encode
  • Proteins: RhD, Rhce, RhCe, etc. according to the specific antigen they carry

RH Genes and Rh Proteins

  • There are 2 RH genes (RHD and RHCE) in close proximity on chromosome 1 encoding 416 amino acid Rh proteins
  • RHD encodes the D antigen
  • RHCE encodes CE antigens in various combination (ce, cE, Ce or CE) on a single protein
  • C and c antigens differ by 4 amino acids
  • The E and e antigens differ by only 1 amino acid
  • D-negative (Rh-negative) phenotype result from complete depletion of the RHD gene
  • D-positive (Rh-positive) individuals have both RHD and RHCE genes

Rh Antigens

  • D is the most immunogenic, followed by c and E
  • Routine donor and patient typing includes tests only for D
  • Tests for other principal Rh antigens are performed primarily to resolve or confirm antibody identification

Fisher-Race and Modified Wiener Nomenclature of Rh Haplotypes

                                      D-Positive                                                            D-Negative


Modified Weiner


Modified Weiner


















Some RH Genotypes

  • DCe/DcE (R1R2)  DcE/ce (R2r)
  • cE/ce (r’’ r)

Rh Phenotypes and Genotypes

  • Serological testing cannot determine whether the red cells are from a homozygotes (D/D) or heterozygote (D./-) because anti-D seldom shows any difference in reactivity between red cells with a single or double dose of D antigen
  • The Rh haplotype influences the level of D antigen expression
  • Less D is expressed in the presence of C because red cells from DcE/DcE(R2R2) individuals carry more D antigens and show higher titration scores with anti-D than red cells from DCe/DCe(R1R1) individuals
  • Decreasing order of D antigenic strength measured by flow cytometry: DcE/DcE > DCe/Dce > DCe/DCe > DcE/ce > DCe/ce

RhD Positive and RhD Negative

  • Where a person inherits a RHD gene, the red cells are positive when tested with anti-D and that person is said to be RhD positive
  • Where a person does not inherit a RHD gene, their red cells are negative when tested with anti-D and that person is referred to as RhD negative



  • Unlike the ABO antigens, Rh antigens are fully developed in early fetal life and remain so throughout adult life
  • Cord and newborn infants’ red cells will therefore have Rh types as strongly as normal adult blood

The Weak D (Du) Antigen

  • Du red cells have been defined as having a reduced amount of D antigens, requiring an indirect antiglobulin test for detection
  • It results from single nucleotide mutation of the RHD gene
  • When testing patients, it is not necessary to test for Du if the routine anti-D reagent(s) give a negative result
  • When testing donors, unless automated grouping machines are used, it is normal practice to test for Du if the routine anti-D reagent(s) give a negative result  If in doubt, call a patient D negative and a donor D positive

Partial D or D Variants

  • Partial D or D variant is the term used to describe a rare group in people who type as D positive, but who produce anti-D that reacts with all D positive cells except their own and those of other rare individuals with the same partial D type
  • In these people, a part of the normal D antigen is missing and they can make an anti-D to that missing part
  • It is not possible to recognize these rare types either by routine or Du testing
  • They are recognized when they produce anti-D after either pregnancy or blood transfusion with normal D positive blood
  • This is a very rare occurrence, but should be borne in mind when investigating atypical antibodies


  • Rh antibodies are only produced following immunization by red cells
  • This is why serum grouping is not routinely done in the Rh system
  • However, anti-E is often natural occurring, about one half may occur without history of pregnancy or transfusion


  • Blood transfusion
  • Pregnancy
  • Paternity dispute
  • Forensic medicine
  • Disease associations

Blood Transfusion

To prevent;

  • Sensitization to red cell antigens (alloimmunization)
  • Haemolytic blood transfusion reaction


 To prevent Haemolytic disease of the foetus and new born  Particularly in Blood group O and D negative mothers

Disease Associations

  • Some blood types are associated with inheritance of other diseases; for example, the Kell antigen is sometimes associated with McLeod syndrome
  • Duffy antigen serves not only as blood group antigen, but also as a receptor for Plasmodium vivax malaria parasites
  • So individuals who lack the Duffy antigen are resistant to plasmodium vivax malaria infection
  • Coronary heart disease: Non O blood group
  • Venous thromboembolism: Non O blood group
  • Duodenal ulcers: Blood group O
  • Gastric cancers: Non O blood group
  • Pancreatic cancers: Blood group A

Paternity Dispute

  • Blood group studies cannot be used to prove paternity
  • But they can provide unequivocal evidence that a man is not the father of a particular child
  • Since the red cell antigens are inherited as dominant traits, a child cannot have a blood group antigen that is not present in one or both parents

Forensic Medicine

The use of blood in forensic analysis is a method for identifying individuals suspected of


[1] Glycophorin A