Chapter 6: The ABO Blood Group System - Blood Bank Exam Study

**Chapter 6** **The ABO Blood Group System** **OBJECTIVES**: 1. Describe the reciprocal relationships between ABO antigens and antibodies for blood types O, A, B, and AB. 2. Identify the frequencies of the four major blood types in the white, black, Hispanic, and Asian populations. 3....

**Chapter 6** **The ABO Blood Group System** **OBJECTIVES**: 1. Describe the reciprocal relationships between ABO antigens and antibodies for blood types O, A, B, and AB. 2. Identify the frequencies of the four major blood types in the white, black, Hispanic, and Asian populations. 3. Explain the effect of age on the production of ABO isoagglutinins. 4. Describe the immunoglobulin classes of ABO antibodies in group O, A, and B individuals. 5. Predict the ABO phenotypes and genotypes of offspring from various ABO matings. 6. Explain the formation of H, A, and B antigens on the red blood cells (RBCs) from precursor substance to immunodominant sugars. 7. Describe the formation of H, A, and B soluble substances. 8. Explain the principle of the hemagglutination inhibition assay for the determination of secretor status. 9. Describe the qualitative and quantitative differences between the A1 and A2 phenotypes. 10. Describe the reactivity of Ulex europaeus with the various ABO groups. 11. Describe the characteristics of the weak subgroups of A (A3, Ax, Aend, Am, Ay, Ael). 12. Describe the characteristics of the Bombay phenotypes. 13. Explain the effects of disease on the expression of ABH antigens and antibodies. 14. 14\. Interpret the results from an ABO typing and resolve any discrepancies, if present. The ABO system is the most important of all blood group in transfusion practice because it is the only blood group system have antibodies in their serum to antigen that are absent from their RBCs. There is always and relationship between the forward and reverse type, thus, one serves as a check on the other. Exposure of individuals to A-like and B-like antigen "from bacteria" severe as a source of stimulation of anti-A & anti-B. ABO-forward grouping detection of antigen on RBCs with known commercial antisera. ABO-reverse grouping detection of ABO antibodies in the serum with known commercial RBCs. **ABO Antibodies:** - - - **Inheritance of the ABO-Blood Group:** - ABO is codominant in expression. - Gene present on chromosome 9 table 6-1 table6-7 001.jpg **Formation of A, B and H RBC Antigen** The formation of ABH antigen result from the interaction of genes at three separate loci (ABO, Hh and Se). These genes response to produce specific glycosyltransferase that add sugar to a basic precursor substance. Paragloboside is a basic precursor which formed A, B and H antigens. Precursor I refer to β-1-3 linkage between galactose and N-acetylglucosamine. Precursor II refer to B-1-4 linkage between galactose and N-acetylglucosamine which present on RBC. The ABH antigen develop as early as the 37^th^ day of fetal life. The A gene tends to elicit higher concentration of transferase than the B-gene. When both A & B genes are inherited, B-enzyme is more efficiently for the H substance than A enzyme. **Molecular Genetic of ABO** A & B gene is located or chromosome 9 and consists of seven exons. The (6 & 7) exons encode for the catalytic domain of the ABO glycosyltransferase. O-gene is identical to A gene with exception of deletion in coding region at nucleotide position. Inherited a secretor gene (SeSe or Sese) responsible for production of a transferase (£-2-L- fucosyltransferase) for modification of the type 1 precursor substance in secretion to express H substance, table 6 -- 2. The demonstration of A, B and H substances in saliva body fluids is evidence for the inheritance of A, B and H Se gene. Box 6 -- 1  table6-9.jpg - **ABO Subgroups** 1. **A subgroup** The following are the characteristics of the weak A subgroup: 1. Decrease number of A antigen sites per RBCs. 2. Varying degree of agglutination by human Anti -- A,B. 3. RBCs react well with Anti -- H. 4. Presence or absence of Anti -- A~1~ in the serum. The A enzyme transferase added sugar (NALNAC) on the deferent four form of H antigens, two are unbranched straight chains (H~1~, H~2~) and two are branched chain (H~3~, H~4~).figure 6-1. Secretor studies and adsorption-elution tests can be utilized to subdivide A individuals in to A~3~, A~x~, A~end~, Am, Ay and A~el~.  1. **A~1~ blood group**:- The A1 enzyme transferase can convert the all H antigens, H~1~ to A^a^ , H~2~ to A^b^, H~3~ to A^c^, H~4~ to A^d^. 2. **A~2~ blood group**:- The A glycosyltransferase can only covert H~1~ and H~2~. Some of A~2~ individual produce Anti -- A~1~ and unlike to cause a transfusion reaction because usually react at bellow 37^o^C but some considered clinically significant if react at 37^o^ C. 3. **A~3~ blood group**:- RBCs characteristic of mixed field of agglutination with Anti -- A and Anti -- A,B reagent. Anti -- A~1~ may be present in the serum. Weak α-3-N-acetylgalactosaminyltransferase activity is detectable in the serum. The A~3~ divided into 3 group: 1. Group 1, enzyme reactive at pH 7 and very low activity. 2. Group 2, no enzyme detectable. 3. Group 3, enzyme reactive at pH 6 and weak activity. 4. **A~x~ blood group**:- The RBCs react with most of Anti -- A,B but not react with Anti -- A. Anti -- A can be adsorbed to RBCs then eluted from A~x~ RBCs. The enzyme transferase not detectable. Anti -- A~1~ almost present in the serum. A~x~ secretor contain A substance detectable only by agglutination / inhibition studies using A~x~ RBCs as indicators. A~x~ donor which mistyped as O blood group can cause rapid intravascular hemolysis. 5. **A~end~ blood group**:- The RBCs characteristic of mixed field of agglutination with Anti -- A and Anti -- A,B reagent with weak agglutination. The enzyme transferase not detectable. Anti -- A~1~ is present in some of A~end~. 6. **A~m~ blood group**:- RBCs in some very weak agglutination and in some no agglutination by Anti -- A or Anti -- AB. Anti -- A can be adsorbed to RBCs then eluted from A~x~ RBCs. The enzyme transferase is detectable in the serum. A~m~ not produced Anti --A~1~. 7. **A~y~ blood group**:- A~y~ RBCs are unagglutinated with Anti -- A or Anti -- A,B. Anti -- A can be adsorbed to RBCs then eluted from A~y~ RBCs. Little amount of enzyme transferase detectable in the serum. A~y~ usually not produced Anti -- A~1~. 8. **A~el~ blood group**:- A~el~ are unagglutinated with Anti -- A or Anti -- A,B. Anti -- A can be adsorbed to RBCs then eluted from A~el~ RBCs. The enzyme transferase not detectable. A~el~ usually not produced Anti --A~1~. 2. The following are the criteria used for the differentiation of weak B phenotypes: 1. Strength /type of agglutination with Anti-B and Anti-A,B and Anti-H**.** 2. Presence or absence of ABO isoagglutinins in the serum**.** 3. Adsorption-elution studies with anti-B**.** 4. Presence of B substance in the saliva**.** 1. **B~3~ blood group: -** B~3~ has a characteristic of mixed-field of agglutination with anti-B and anti-A,B, presence of B transferase in the serum and absence of Anti-B in the serum. 2. **B~x~ blood group: -** B~x~ gives a weak agglutination with anti-B and anti-A,B**.** B transferase is not detected in the serum and weak anti-B is produced in the serum. 3. **B~m~ blood group**: - B~m~ RBCs are not agglutinated by anti-B or anti-A,B. B transferase is detected in the serum and Anti-B is absent in the serum. 4. **B~el~ blood group**: - The RBCs are not agglutinated by Anti-B or Anti-A,B. B transferase is detected in the serum and weak Anti-B is produced in the serum. **The Bombay Phenotypes (Oh)** The H gene appears to be necessary for the formation of A and B antigens. It is very common 99.9% of all individuals have an HH or Hh genotype. The allele h is very rare and does not produce the L-Fucose transferase necessary for formation of the H-Structure. The genotype (hh) or H null is extremely rare and is known as the Bombay Phenotype or O~h~. The *H* gene codes for an enzyme (α-2-L-fucosyltransferase) that adds a Fucose to the terminal sugar of a Precursor Substance (PS\*). The biochemical structure below constitutes the **H Antigen**. (*h* gene is an a morph.) HAg2 ***H antigen* is the foundation upon which A and B antigens are built. *A* and *B* genes code for enzymes that add an immunodominant sugar to the *H antigen*. *O* allele does not code a functional enzyme.** **amount of H O \> A2 \> B \> A2B \> A1 \> A1B amount of H** **The Bombay Phenotype was first reported by Bhende 1952 in Bombay, India. Bombay cells cannot be converted to group A or B by the specific transferases. This support the concept that the [H] structure serves as the acceptor molecule or precursor substance for the product of the A or B gene -specified transferase. Bombay individuals** lack all normal **expression of the A, B, or O genes they inherited. The Bombay Phenotype Red Cells are devoid of normal A B, H Antigens. Fail to react with anti- A, anti -B , and anti -A,B and anti H. Bombay Serum contains anti-A, -B and anti-H. The Bombay anti-H is active over a wide thermal range. It is an IgM antibody that can bind complement and cause red cell lysis. Because the H antigen is common to all ABO blood group, Bombay blood is incompatible with all ABO donors. In routine forward grouping, using anti-A, anti-B, and anti-AB. The Bombay would phenotype as an O blood group. However, transfusing normal group O would cause immediate cell lysis by the potent anti-H of the Bombay individuals. Thus, only blood from another Bombay individual can be transfused to a Bombay recipient. O~h~ (hh) individuals are all non-secretors of ABH substances, because both the H gene and the Se gene must be inherited for the ABH antigens to be found in secretions. Proof that individual** lacks the H **antigen may be accomplished by typing the Bombay phenotype red cells with a products of the plant *Ulex Europacus* or with sera containing Anti-H activity from which all anti-A, anti-AB activity has been adsorbed (Removal of Abs). This plant lectin provides a much simple and more readily available source of anti-H like activity. Thus this plant must be used to determine the presence or absence of the H antigen on the surface of RBCs.** **General Characteristics of Bombay O~h~ (H~null~ Phenotypes)** 1. **Absence of H, A, and B antigens; NO agglutination with anti-A, anti-B, or anti-H Lectin.** 2. **Presence of anti-A, anti-B, anti-A,B and a potent wide thermal range of anti-H in the serum.** 3. **A, B, H non-secretor (no A, B, or H substances present in saliva)** 4. **Absence of α-2-L-fucosyltransferase (H enzyme) in serum and H antigen on red cells.** 5. **Red cells of the Bombay phenotype (O~h~ will not react with the anti-H Lectin (*Ulex europaeus).*** 6. **Red cells of the Bombay phenotype (O~h~ are compatible only with the serum from another Bombay individual).** **H deficient phenotype is classified into the 3 categories:** 1. **Category 1: RBC H deficient, non secretor, Bombay phenotype (*hh sese*) called** Classic Bombay. This group arises from the inheritance of the *hh* genotypes and are non secretor sese. These cells lack all H, A and B antigen in RBCs and secretion. **Anti -- H is clinically significant can cause HTRs.** 2. **Category 2:** Para-Bombay phenotype. This individuals have mutant alleles at the H genes but normal Se genes, some para-bombay demonstrate minute quantities of ABH antigen on RBCs, regardless of whether they posses them in secretion. Other para-bombay fail to demonstrate ABH antigen on RBC, but may or may not be expressed in the secretion based on Se inheritance. Adsorption and elution of anti -- H may show the present of H antigen. Anti -- H react at 25C. 3. **Category** 3 : RBC H -- Partially deficient, Nonsecretor. Week expression of antigens A or/and B on RBC and there is no antigen H, the notation A~h~, B~h~ and AB~h~ have been used to describe these individual. There is no antigens A,B or H present in the saliva, Anti -- H is weaker than Bombay phenotype and may react at 37C. **Discrepancy** **Discrepancy: exists when the results of red cells tests don't agree with serum tests. The discrepancy result must be recorded, but interpretation of result delayed until the discrepancy is resolved. If discrepancy in donor sample must not be released for transfusion until the discrepancy is resolved. If discrepancy in recipient, it may be necessary to give group O blood similar Rh type before the investigation is completed.** **Discrepancies happen by technical errors or test --related problem. Technical errors common causes of false-negative / false-positive results in ABO testing.** +-----------------------------------+-----------------------------------+ | False-negative results | False-positive results | +===================================+===================================+ | 1. 2. 3. 4. 5. 6. 7. 8. | 1. Over centrifugation of tubes. | | | | | | 2. 3. 4. | +-----------------------------------+-----------------------------------+ | | | +-----------------------------------+-----------------------------------+ **\*Before trying to resolve the discrepancy, determined essential information regarding the patient's age, diagnosis, transfusion history, medication, immunoglobulin levels and history of pregnancy.** **The causes of discrepancy may be divided into four major categories:** +-----------------------+-----------------------+-----------------------+ | TYPE OF DISCREPANIES | CAUSES | CASESES | +=======================+=======================+=======================+ | Group 1 | 1. | a. b. c. d. e. f | | | |. g. h. | | **Discrepancies of | | | | group 1 are more | | | | common than other | | | | list.** | | a. Blood | | | | Transfusion. | | **Discrepancies occur | | | | in reverse group due | | b. Transplant BM. | | to false weak | | | | reaction or | | c. Exchange | | false-negative.** | | transfusion. | | | | | | | | d. Fetal maternal | | | | bleeding. | | | | | | | | e. Utero exchange. | +-----------------------+-----------------------+-----------------------+ | **Group 2** | 1. | a. b. | | | | | | **This discrepancy | | | | happens in forward by | | | | false negative | | | | (missing reaction) or | | | | weak reaction.** | | | +-----------------------+-----------------------+-----------------------+ | | 2. | c. | | | | | | | | **Neutralize the | | | | reagent anti-A and | | | | anti-B.** | +-----------------------+-----------------------+-----------------------+ | | 3. | d. e. f. | +-----------------------+-----------------------+-----------------------+ | | 4. | a. b. c. d. | +-----------------------+-----------------------+-----------------------+ | **Group 3** | 1. | a. b. c. d. | | | | | | **This discrepancy | | | | gives false positive | | | | results in forward | | | | and reverse grouping | | | | due to rouleaux | | | | formation.** | | | +-----------------------+-----------------------+-----------------------+ | | 2. | | +-----------------------+-----------------------+-----------------------+ | | 3. | | +-----------------------+-----------------------+-----------------------+ | **Group 4** | 1. | a. b. c. d. e. R | | | | ed | | **Miscellaneous | | cells from | | problems may happen | | infants with HDN. | | in forward grouping | | | | or in reverse | | f. Acquired A like | | grouping or in | | antigen, which | | both.** | | react with | | | | anti-A. | +-----------------------+-----------------------+-----------------------+ | | 2. | a. b. c. d. e. | +-----------------------+-----------------------+-----------------------+ **Resolving ABO discrepancy** **First of all eliminated technical error to resolve ABO discrepancy:** - **Repeat the test on the same sample.** - **Washed cells.** - **Obtain a new sample "to avoid mislabeled or contaminated specimen".** - **Looking to history of the patient.** - **Review the result of the antibody screening test "to eliminate interfering effects of cold-reaction, alloantibodies or autoantibodies".** - **RESOLVING ABO DISCREPANCY OF GROUP 1:** 1. Enhance the reverse group reaction by incubating reaction in room temperature for approximately 15-30 minutes. 2. If there is no reaction, incubate reaction at 4^0^C for 15-30 minutes; parallel with autologolus and 0 cells as controls, must be tested to avoid interference by anti-I or anti-H. 3. If there is no reaction, test the immunoglobulin assay - **Resolving ABO DISCREPANCY OF GROUP 2:** A. **For weak expression of antigen:** 1. Enhance the forward grouping by incubating the reaction at room temperature up to 30 minutes. 2. If still negative, incubate reaction at 4^0^C. With controls (autologous and 0 cells). 3. Treat the cells with proteolytic enzyme such as ficin, papain or bromelin. Then incubate the treated cells with anti-sera for 30minutes at room temperature parallel with 0 treated cells as control. 4. If still negative, confirmations of weak A or B subgroup by adsorption and elution. 5. If still not confirmatory. Test the saliva for presence of H and A or B substance only if the person is a secretor. B. **For excess amounts of BGSS:** Washing cells by normal saline should alleviate the problem. C. **For acquired B phenomenon check the diagnosis and history:** 1. Autologous (auto control) is negative. 2. Use anti-B, which not contain ES4 clone. 3. Acidified anti-B less than 6.0 pH. 4. Test the saliva. If individual secretor only A substance is secreted. 5. Also treated cells with acetic anhydride. 6. Molecular genetics of glycosyltransferase provides an additional approach to diagnosis of acquired B. D. **For antibodies to low incidence antigen in reagent anti-A or anti-B:** Using anti-sera with different lot number. - **RESOLVING ABO DISCREPANCY OF GROUP 3:** **For elevated level of the globulin and for Wharton\'s jelly.** 1. 2. - **RESOLVING ABO DISCREPANCY OF GROUP 4:** A. **For T-activation.** 1. Using monoclonal antibody typing reagent. 2. Lectin studies should be performed. B. **For cold reactive antibodies (allo and auto).** **Forward grouping** 1. Prewarme technique then retyping. - - - 2. If it is not resolve treated with dithiothreitol (DTT) to disappear IgM related agglutination. **Reverse grouping** 1. Prewarme technique then retyping. - Incubate serum or plasma and reagent cells before mixing in 37^0^C. - Mixed then tested in 37^0^C and in antihuman globulin phase. 2. If it is not resolve performed cold auto adsorption after that, tested adsorbed serum against A~1~ cells and B cells. AABB. C. **For warm autoimmune hemolytic anemia, drug in use or transfusion reaction or HDN (DAT = positive).** 1. Gentle heat elution at 45^0^ C may remove sufficient antibodies. 2. Retyping cells with anti- sera. D. **For unexpected ABO isoagglutinins (A~2~ and A~2~B and anther subgroups)** 1. Use A~1,~ A~2~, B, O cells and an autologous control. 2. Use anti-A~1~ Lectin. E. **For unexpected alloantibodies that react at room temperature.anti-P~1,~-M,-S.** 1. Raise the temp. to 30-37^0^ C before mixing the serum and cells. 2. If not resolve problem: a. Identified the antibody at room temperature. b. Choose A~1,~ B cells which lack of corresponding antigen. c. Retyping. 3. If the antibody screen positive and can't detected antibody, test the serum against several example of A~1~ and B red cells. The serum may contain an antibody directed to low incidence antigen. F. **For acquired A like antigen** 1. Treated the cells by proteolytic enzyme such as ficin. Which denature molecular that expresses the cryptantigen which react with anti-A. 2. Retyping. G. **For anti-day.** washing the cells with saline three times then retype. - ABO frequencies: group O, 45%; group A, 40%; group B, 11%; group AB, 4%. - ABO blood group system has naturally occurring antibodies that are primarily IgM. - ABO genes, like those of most other blood groups, are inherited in a codominant manner. - ABH-soluble antigens are secreted by tissue cells and are found in all body secretions. The antigens secreted depend on the person's ABO group. - ABO reverse grouping is omitted from cord blood testing on newborns, because their antibody titer levels are generally too low for detection. - ABO RBC antigens can be glycolipids, glycoproteins, or glycosphingolipids; ABO-secreted substances are glycoproteins. - L-fucose is the immunodominant sugar responsible for H specificity. - N-acetylgalactosamine is the immunodominant sugar responsible for A specificity. - D-galactose is the immunodominant sugar responsible for B specificity. - The hh genotype is known as the Bombay phenotype, or O~h~, and lacks normal expression of the ABH antigens. - Group O persons contain the greatest amount of H substance; group A~1~B persons contain the least amount of H substance. - Approximately 80% of A persons inherit the A gene phenotype as A~1~; the remaining 20% phenotype as A~2~ or weaker subgroups. - Approximately 1% to 8% of A~2~, persons produce anti-A~1~ in their serum. - Glycoproteins in secretions are formed on type 1 precursor chains. - The ABH antigens on RBCs are formed on type 2 precursor chains. - Forward and reverse grouping normally yield strong (4+) reactions. - Group A persons contain anti-B in their serum; group B persons contain anti-A in their serum; group AB persons contains neither anti-A nor anti-B in their serum; group O persons contain both anti-A and anti-B in their serum. - Approximately 78% of the random population inherit the Se gene and are termed secretors; the remaining 22% inherit these gene and are termed nonsecretors. - The Se gene codes for the production of L-fucosyltransferase.