ABO blood group (1).pdf

ABO blood group system By:Douaa basim  Introduction The ABO system is the most important of all blood groups in both transfusion and transplantation. It is the only blood group system in which individuals already have antibodies in their serum to antigens that a...

ABO blood group system By:Douaa basim  Introduction The ABO system is the most important of all blood groups in both transfusion and transplantation. It is the only blood group system in which individuals already have antibodies in their serum to antigens that are absent from their RBCs without any prior exposure to RBCs through transfusion or pregnancy(naturally occurring) Due to the presence of these antibodies transfusion of incompatible blood may result in immediate lysis of donor RBCs This produces avery sever,if not fatal transfusion reaction in the patient  ABO grouping Forward grouping(front type) Is defined as using known sources of commercial antisera(antiA,anti B)to detect antigen on individual RBC Reverse grouping(back type) Is defined as detecting ABO antibodies in the patient serum by using known reagent RBCs,namely A1 and B cells  ABO grouping There is always an inverse reciprocal relationship between the forward and reverse type,thus one serve as check on the other Performance of serum grouping is unique to the ABO blood group system ‫‪ - 2‬إضافة عنوان للشريحة‬ ‫‪ - 4‬إضافة عنوان للشريحة‬  It has been reported that subgroup A2 rarely found in Asian  Development of ABO antibodies 1-At Birth:ABO antibodies production initiated at birth but titers are generally too low for detection until infants are 3-6 months old Q/is it logical to perform reverse grouping for newborn infants… A/no ,result of serum blood grouping before 3-6 months of age can not be considered valid because some or all of antibodies present may be IgG maternal Antibodies that cross the placenta 2-peak:between 5-10 years of age 3-decline:elderly people usually have lower levels of anti-A and anti-B ,therefore antibodies may be undetectable in reverse grouping ABO antibodies are predominantly IgM,activate complement and react at room temp. Or colder ABO antibodies can cause rapid intravascular haemolysis if the wrong ABO group is transfused,potentially resulting in patient death  Is it possible to find Anti-A or Anti- B of IgG type… Although Anti A and anti B contain predominantly IgM antibody,small quantities of IgG may also present  What about anti A,B It is usually IgG in nature,present in serum of group O individuals Anti-A,B react with both A and B cells anti A,B antibody is not a combination of anti A and anti B but is a separate ,cross reacting antibody Uses of anti A,B reagent in routine work Anti-A,B reagent is routinely used for confirmation of ABO blood group of group O donor units,because it is more economic to use one reagent instead of using Anti-A and Anti-B Use of anti A,B is not required for routine patient ABO testing Some believe anti A,B is more effective at detecting weakly expressed A and B antigens than reagent ant-A or anti-B but the production and use of monoclonal anti sera,however,have made anti -A and anti-B reagents much more sensetive What is the clinical significance of IgG anti A,anti B,anti A,B Knowing the amount of IgG anti-A ,anti-B,or anti-A,B in a woman’s serum sometimes allows prediction of diagnosis of haemolytic disease of foetus and newborn caused by ABO incompatibility  Inheritance of ABO genes The inheritance of ABO genes follow simple Mendelian genetics ABO like most other blood group system,is codominant in expression One position ,or locus,on each chromosome 9 is occupied by an A,B or O gene O gene is considered an amorph (no antigen is produced in response to inheritance of O gene) The group O phenotype is an autosomal recessive trait with the inheritance of two nonfunctional O genes Phenotype & genotype  Is it possible serologically to determine the genotype from the phenotype For groups A and B it is not possible,family study or molecular assays would need to be performed For group A,B and group O both phenotype and genotype are the same  The formation of ABH antigens results from the interaction of gens at three separate loci(ABO,Hh ,and Se) These genes do not actually code for the production of antigens but rather produce specific glycosyltransferase enzyme that add sugar(imunodominant sugars) to a Formation of basic precursor substance(paragloboside or glycan) A,B ,H red blood cell antigens  Types of the precursor substance When the terminal galactose Type 2 on the precursor substance is attached to the N- acetylglucosamine in a beta 1- 4 linkage Type 1 When the terminal galactose on the precursor substance is attached to the N- acetylglucoseamine in a beta 1-3 linkage  Outcome from each substance type Type 2 ABH antigens on the RBC are constructed on oligosaccaride chains of a type 2 precursor substance Type 1 ABH soluble antigens in secretion Actually ,we have type 2 and 4 precursor substances give rise to ABH antigens on RBc And type 1 and 3 precursor substances give rise to soluble antigen in secretions But type 1 and 2 are more abundant  H Antigen H antigen is the precursor structure on which A and B antigens are made Inheritance of H gene (on chromosome 19) results in formation of the H antigen Individuals who are blood group O inherit at least one FUT 1(H)gene (genotype HH or Hh)and two O genes The H gene elicit the production of an enzyme called alpha 2-Lfucosyltransferase that transfer the sugar L fucose to an oligosaccaride chain on the terminal galactose of type 2 chains  Formation of A antigen the formation of blood group A, the A gene (AA or AO) codes for production of α-3-N- acetylgalactosaminyltransferase, which transfers an N- acetyl-D-galactosamine(GalNAc) sugar to the H substance. This sugar confers A specificity. The A-specific immunodominant sugar is linked to a type 2 precursor substance that now contains H substance through the action of the H gene.  Formation of B antigen Individuals who are blood group B nherit a B gene (BB or BO) that codes for the production of alpha 3 D galactosyltransferase and attach D galactose to the H substance previously placed on type 2 precursor substance  Amount of the remaining H substance and number of antigenic sites on each blood group Has the highest O blood group concentration for The H gene Elicit higher concentration of transferase enzyme,leading to A blood group conversion of nearly all of H antigen on the RBCs to A antigenic sites 810,000 to 1,170,000 antigen sites exist on an A 1 adult RBC in response to inherited genes  Continue…. The enzyme is less powerful B blood group than A,so the B antigenic sites are less(610,000- 830,000) and the amount of H substance is more than A When both A and B genes are inherited,the B AB blood group enzyme seem to compete more efficiently for the H substance than A enzyme,as a result the average NO. Of A antigen600,000 compared with an average of 720,000 B antigenic sites Time of development of ABH antigens and their changes throughout life Develop early in feral life and do not increase much in strength during the gestational period. The RBC of newborn have been estimated to carry anywhere from 25%to 50% of the number of antigenic sites(reaction of newborn RBC with reagent anti sera is weaker than reaction with adult RBC) The expression of antigens is fully developed by 2-4 years of age Remain constant throughout life The phenotypic expression of ABH antigens may vary with race,genetic interaction and disease state  Secretory status ABH soluble antigens can also be found in all body secretions 80% of random U.S. population have antigens in secretion and known as (secretors) The secretory status depend on: *inheritance of ABO genes *inheritance of another set of gene called secretor gene(Se or FUT2 gene) on chromosome 19  Fut2(Se)gene codes for the production of fucosyltransferase that modify the type 1 precursor substance in secretion to form H substance If the corresponding gene is present,this H substance can then be further modified to express A and B substance in secretions such as saliva People who inherit the sese genotype are called non- secretors  Advanced concepts about molecular genetics of ABO The Abo gene (on chromosome 9) consist of 7 exons,exon 6 and 7 form 77% of gene with most of the coding sequences located in exon 7 All ABO antigens arise from mutations in the single ABO gene,3 specific mutations showing a high frequency in population indirectly lead to change in the epitope structures resulting in A,B or O specificities: Two of the mutations(substituation) change the specificity of enzyme from A transferase enzyme to B transferase enzyme The third mutation(deletion with in 5 region of the catalytic domain)create premature stop codon that inactivate the enzymes all together,leaving the H glycan unmodified(O blood group) For ABO subgroups,amino acid substitution resulting primarily from deletions,mutations or gene recombinations within 6and 7 exons are responsible for the less efficient transfer of imunodominant sugar to H substance  A subgroups A2 A subgroups are more common than B subgroups Classification into A1 and A2 phenotypes account for 99% of all group A individuals The cells of approximately 80% of all group A or AB individuals are A1 or A1B and the remaining 20% are A2 or A2B or weaker subgroups  Formation of A2 antigen Inheritance of A2 gene which is characterised by single base substitution at nucleotide 467 and single base deletion at nucleotide 1060(1060delC) in exon 7 which alter the active site of the coding region and subsequently change the specificity of the A glycosyltransferace,limit its ability to convert H antigen to A antigen This lead to the production of only240,000 to 290,000 antigen sites on adult A2 RBC,while the amount of H antigen is more than A1 The imunodominat sugar is identical in both A1 and A2  Advanced concepts in understanding A1 and A2 There are four different forms of H antigen: *unbranched straight chains(H1,H2) *complex branched chains(H3 and H4) By A1 and A2 enzyme (A2 H1 and H2 less efficient) Aa and Ab H3 and H4 By A1enzyme and very poorly by A2 Ac and Ad  More unconverted H antigens(specifically H3 and H4) are As aresult available on group A2 RBC,and only Aa and Ab determinants are formed from H1 and H2 structures According to this there are many qualitative and quantitive differences between A1 and A2,resulting in difference in forward and reverse reaction  A1 RBC react with both anti A and anti A1 (contain A and A1 antigen) In forward grouping A2 RBC react only with anti A not anti A1(contain only A antigen) A2 RBC show increase reactivity with anti-H lectin A1 group not form anti A1 In reverse grouping 1-8% of A2 form anti A1 22-35% of A2B form anti A1  Weak A subgroups Subgroups weaker than A2 occur infrequently(1%) and are most often recognised through an ABO discrepancy Characteristics of weak A subgroups include the followings: *decreased number of A antigen sites per RBC(resulting in weak or no agglutination with human polyclonal Anti A) *varying degrees of agglutination by human anti A,B *increased variability in the detectability of H antigen,resulting in strong reaction with anti H *presence or absence of anti A1 in the serum  Differentiation of weak A subgroups Serological differentiation by: *forward grouping of A and H antigen with anti A,anti A,B and anti H *reverse grouping of ABO isoagglutinins and the presence of Anti A1 *Adsorption-elation tests with Anti A *saliva studies to detect the presence of A and H substance Additional procedures: *molecular testing for mutations *serum glycosyltransferase studies for detecting A enzyme Absence of a disease process should be confirmed before subgroup investigation because ABH antigens are altered in various malignancies and other haematological disorders  Weak B subgroups Subgroups of B are very rare and much less frequent than A subgroups,usually recognised by variation in reaction strength using Anti B and Anti A,B Criteria used for differentiation of weak B phenotypes include the following techniques: *strength and type of agglutination with anti B,anti A,B and anti H *presence or absence of ABO isoagglutinins in the serum *adsorption –elation study with anti B *presence of B substance in saliva *molcular testing  The Bombay Phenotypes (Oh ) * The phenotype results from the inheritance of a double dose of the h gene,producing the very rare genotype hh *as a result,the ABO gene cannot be expressed and ABH antigens can not be formed,since there is no H antigen made in the Bombay phenotype  Inheritance *the Bombay phenotype is inherited as an autosomal recessive trait *the underlying molecular defect is often amutation in the gene FUT1(H gene),producing a silenced gene incapable of coding for the enzyme fucosyltransferase *this mutation is also associated with a silenced FUT2 gene(Se gene),No A,B,H substances in saliva *family study can demonstrated which ABO genes are inherited in the Bombay phenotype,the genes are written as superscripts Reaction of Bombay RBC with anti sera They fail to react with anti A,anti B and anti H anti sera In RBC phenotype using Anti A and Anti B,the Bombay would phenotype as an O blood group,however RBC of the Bombay phenotype do not react with anti H lectin unlike those of normal group O individuals which react strongly with anti H lectin Antibodies in Bombay serum Bombay serum contains anti-A,anti-B,anti-A,B and anti-H Unlike Anti –H found occasionally in serum of A1 and A1B,the Bombay Anti-H can often be potent and react strongly at 37C(it is an IgM AB capable of binding complement and causing RBC lysis) According to this: Only blood from another Bombay individual will be compatible and can be transfused to a Bombay recipient  Para-Bombay phenotypes Are those phenotypes in which the RBC either completely lack H antigens or have small amounts of H antigen present Genetic basis for para-Bombay phenotypes: Either a mutated FUT1(H gene) with or without an active FUT2 gene(Se gene) OR a silenced FUT1 gene with an active FUT2 gene  Consequences of the first mechanism Fucosyl transferase Despite this, RBC from enzyme activity is these individuals greatly reduced expressing very small In the presence of Very small amount amount of A or B normal A or B antigen ,not detected by of H antigen is gene,normal amount routine produced of functional A or B methods,detected only enzyme will formed by adsorption and ellution techinoque The notation Have been used to describe these individuals  Antibodies in serum serum of Ah individuals contains anti-B and no anti-A, although anti-A 1is usually present. In Bh serum, anti-A is always present and anti-B may be detected The anti-H present in the serum is weaker in reactivity than the anti-H found in the Bombay phenotype, although it may be reactive at 37°C. Secretion in saliva: NO H ,A or B antigen detected in saliva  silenced FUT1 gene with the active FUT2 gene the α-2-L-fucosyltransferase enzyme associated with the FUT2 gene (α2FucT2) produces H, A, and B type 1 antigens in secretions, including plasma. These type 1 antigens in the plasma may adsorb onto the RBC membrane. RBCs have little or no A, B, and H antigens.  Possible reactions with reagent anti sera RBCs of Oh secretors are not agglutinated by most examples of anti-H but may be agglutinated by strong anti-H reagents(adsorption and elation of anti –H may reveal the presence of some H antigen on the RBC) Cells are not usually agglutinated by anti-A and anti-B RBCs can mimic the behaviour of Ax(can be agglutinated by However Anti-A,B and potent examples of Anti-A The same reaction with Anti-A,B and potent examples of Anti-B can be seen with  Antibodies in serum A weak H-like antibody, called anti-IH, that is reactive at low temperature is almost always present in the serum