Immunohematology 321 Lecture Notes PDF
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Justin Kim C. Vergara, RMT, MPH
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These lecture notes cover immunohematology, specifically focusing on genetic principles related to blood banking. The document details blood group systems and related concepts.
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IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH Review of Important Genetic Principles as applied to Blood Banking GENETICS tudy of inheritance or the transmission of characteristics S from parents to offspring....
IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH Review of Important Genetic Principles as applied to Blood Banking GENETICS tudy of inheritance or the transmission of characteristics S from parents to offspring. It is based on the biochemical structure of chromatin, which includes nucleic acids and the structural proteins that constitute the genetic material as well as various enzymes that assist in genetic processes such as replication. DNA→RNA→P rotein→F unction LOOD GROUP SYSTEMS- group of related red cell B antigens which is similar in terms of biochemical structure, location in RBC, serologic properties and genetic control of antigen expression. International Society of Blood Transfusion (ISBT) Blood Group Systems SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH ENES: Basic unit of inheritance G CHROMOSOMES: structure within the nucleus that contains the DNA. 2 Types: ENOTYPE: Actual set of genes inherited from G parents/actual genetic make-up of an individuals. PHENOTYPE: Physical or observable expression of inherited genes. Mendel’s Law of Inheritance aw of Independent Segregation L 1st Generation → “Parental” PUNETT SQUARE: square used to calculate the First-filial generation→ crossbreed of homozygous red and frequency of the resulting genotypes and phenotypes among homozygous white flower the offspring of a cross. Second-filial generation→ crossbreed of First-filial generation. aw of Independent Assortment L Members of one gene pair separate from one another independently of the member of other gene pairs. States that “genes for different traits are inherited separately from each other.” SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH ABO Genotypes & Phenotypes PUNETT SQUARE xample: FATHER: A1A1 E MOTHER: A1B . Probability that child’s genotype is A1A1: 1 _____________ 2. Probability that child’s phenotype is A1: _____________ 3. Probability that child’s blood type is A1B: _____________ Terms Used in Blood Bank ALLELES:One of two or more genes that express agiven Genotypes & Phenotypes trait ANTITHETICAL: Term used to refer to the oppositegene POLYMORPHIC GENE: Genetic system that expresses multiple products.e.g. MHC genes DOMINANT GENE: Gene whose product is always expressed RECESSIVE GENE: Gene whose product is only expressed when inherited in a homozygous way CODOMINANT GENES: Genes whose products are both expressed HOMOZYGOUS GENES: Both genes for a given trait are the same HETEROZYGOUS GENES: Genes for a given trait are different DOSAGE EFFECT Stronger Agglutinationare produced by antigensthat are expressed by homozygous genes Weaker Reactionsare produced by antigens expressed by heterozygous genes. – e.g. KIDD, DUFFY, RH, MNSs blood group systems SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH INHERITANCE PATTERNS: .AUTOSOMAL DOMINANT: Product of the gene 1 will always be expressed .AUTOSOMAL RECESSIVE: Product of the gene 2 will only expressed if homozygous .SEX-LINKED DOMINANT: Father to daughter 3 transmission of trait .SEX-LINKED RECESSIVE: Mother to son transmissionof 4 Terms Used in Blood Bank trait CIS: Term used to refer when 2 Genes are on the same side .CODOMINANT:Equal expression of inherited 5 of the Chromosome alleles/genes TRANS: Term used to refer when 2 Genes are on the different side of the Chromosome LINKED GENES:2 Genes in the Chromosome are very close to each other. HAPLOTYPE: Linked set of Genes that are inheritedand expressed together. utosomal Dominant Inheritance A All the members of a family that carry the allele show the physical characteristic. Generally, each individual with the trait has at least one parent with the trait. utosomal Recessive inheritance A A recessive trait is carried by either parent or both parents but is not generally seen at the phenotypic level unless both parents carry the trait. In some cases a recessive trait can be genetically expressed in a heterozygous individual but is often not seen at the phenotypic level. SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH -linked Dominant Inheritance X Inheritance of the ABO Blood Group If the father carries the trait on his X chromosome, he has no sons with the trait, but all his daughters will have the trait. Describe byBernsteinin 1924 Women can be either homozygous or heterozygous for an He demonstrated that an individual inherits one ABO gene X- linked trait. from each parent and that these two genes determine which ABO antigens are present on the RBC membrane. One position, or locus, on eachchromosome 9is occupied by an A, B, or O gene. -linked Recessive Inheritance X The father always expresses the trait but never passes it on to his sons. The father always passes the trait to all his daughters, who are then carriers of the trait. With an X-linked recessive trait, a disease-carrying gene can ABO Blood Grouping/ Blood Typing: be passed from generation to generation with many individuals not affected. .Forward/ Direct/ Red Cell Grouping 1 → detection ofantigenin the RBC using known antisera. NULL PHENOTYPES Phenotypes that lack the expression of the red cell antigens of a particular blood group systems. May be due to inheritance of: 1.Silent/Amorphic gene: Gene that expresses no product .Regulator/Suppressor gene:Gene that inhibits the 2 expression of another gene. Forward/ Direct/ Red Cell Grouping: ABO Blood Group System Most important of all the blood group systems Transfusion of as small as 0.1ml ABO incompatible blood to a recipient can cause IHTR (Immediate Hemolytic Transfusion Reaction) ISBT No.001 andsteiner Law/Rule: Rule stating that normal, healthy L individuals possess ABO antibodies to the ABO blood group antigens absent from their red cells. . Backward/ Indirect/ Reverse/Serum Grouping: 2 → detection of antibodies in the patient’s serum using known red cells. SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH ABO Compatibility Universal Donor inRed CellTransfusion: ”O” Universal Recipient inRed CellTransfusion: ”AB” Universal Donor inPlasmaTransfusion: ”AB” Universal Recipient inPlasmaTransfusion:”O” Backward/ Indirect/ Reverse/Serum Grouping ABO ANTIBODIES (anti-A, anti-B, anti-A1, anti-A,B) Individuals normally produce antibodies directed against the A and/or B antigen(s) absent from their RBCs. 1. Naturally-Occurring/ Non Red Cell Stimulated ntibodies A . Predominantly IgM, except anti-A,B 2 Frequency Distribution of ABO Phenotypes NOTE: anti-A,B is inseparable; is not a mixture of anti-A and anti-B; it cannot be separated even with adsorption with A and B red cells. ADSORPTION: process of removing antibodies in the serum using known red cells. ELUTION: process of dissociating antibody from red cell surface. 3. Clinically Significant 4. Produced on 3-6 months after birth 5. Production peaks 5-10 years of age then decline as we age. BO Compatibility A A and B Antigens hole Blood, RBC and Plasma Transfusions W . Produced on 37th day of fetal life/5-6 weeks of fetal 1 life 2. Production peaks at 2-4 years of age 3. Present in RBCs, platelets, lymphocytes, epithelial cells and other organs of the body. 4. Require H antigen first in order to be synthesized 5. Synthesized on TYPE 2 PRECURSOR SUBSTANCE/ PARAGLOBOSIDE/OLIGOSACCHARIDE CHAIN (B1 → 4 linkage) SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH ABO Blood Group System Basic precursor in the formation of ABH Antigen Expression of ABO Antigens Type 1 precursor chain – beta 1-3 linkage ABH antigens are not only found in humans, but also Type 2 precursor chain in various organisms such as bacteria, plants, and animals – beta 1-4 linkage Present both on red blood cells and in secretions only in humans and some of the apes (chimpanzee, gorilla) In all other mammalian species these substances are found only in secretions ABO and H Antigen Genetics Genes at three separate loci control the occurrence and location of ABO antigens Presence or absence of the ABH antigens on the red cell membrane is controlled by theH gene Presence or absence of the ABH antigens in secretions is indirectly controlled by theSe gene H gene– H and h alleles (h is an amorph) – –Se gene– Se and se alleles (se is an amorph) –ABO genes– A, B and O alleles FORMATION OF A, B AND H ANTIGENS ABO blood group Antigens with their corresponding Immunodominant sugars SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH Precursor Molecules ABO, H and Secretor Gene A,B,H SOLUBLE SUBSTANCES (Soluble Antigens) Type 1: beta 1-3 linkage between the terminal D-galactose Present in all body fluids exceptCSF and N-acetylglucosamine Depends on the inheritance of theSecretor gene(Se gene) – Function as to produce soluble antigens (ABH) depending if Product of Se gene is same with the product of H gene the individual is secretor or non-secretors. (fucosyltransferase/FUT2) – Secreted substances are primarily synthesized on type 1 FUT2 transfers and adds L-fucose to a Type 1 Precursor precursor chains. Substance/Paragloboside/Oligosaccharide Chain (B 1→3 linkage) to produce H Soluble Antigen/H Soluble Substance Type 2: beta 1-4 linkage between the terminal D-galactose and N-acetylglucosamine: – ABH antigens on the RBC are constructed on oligosaccharide chains of type 2 precursor substance. – This precursor is for production of antigen (ABH) in the red SECRETOR STATUS cell membrane. A, B AND H ANTIGENS A1 gene tends to elicit higher concentrations of transferase enzyme than the B gene. A1 gene converts approximately 810,000-1,170,000 H Ag to become A1 Ag B gene converts approximately 610,000-830,000 H Ag to become B Ag When both A1 and B genes are inherited, A1 gene will convert an average of 600,000 H Ag, B gene will convert at least 720,000 H Ag A2 gene converts approximately 240,000-290,000 H Ag to become A2 red cells. BH ANTIGEN A (TYPE 1 vs TYPE 2) H Antigenicity Reactivity of anti-H antisera or anti-H lectin with ABO Blood Group O > A2 > B > A2B > A1 > A1B LECTINS Seed extracts that can agglutinate red cells with some degree of specificity. SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH Secretor(80%): Individuals who have ABH soluble substances in their body fluids Non-secretor (20%): Individuals who do not haveABH soluble substances in their body fluids Three Categories of BOMBAY Phenotype . H-deficient;Non Secretor: 1 →CLASSIC BOMBAY 2. H-Partially Deficient ; Non Secretor: →EXPRESSES WEAK FORMS OF A AND B ANTIGENS 3. H Deficient Secretor: →PARA BOMBAY BOMBAY PHENOTYPE (Oh) ABO Subgroups First reported byBhendeet al in Bombay in 1952 ABO subgroups differ in the amount of antigen present on Frequency estimated to be about 1 in 7600 in Bombay the red blood cell membrane Inherited thehhgenotype In 1930, O. Thompson postulated a four-allele system of Absence of H, A & B antigens inheritance based on the discovery of Emil Frieherr von Has anti-A, anti-B and anti-H on serum Dungern and Ludwig Hirtzfeld in 1911 Recessive mode of inheritance A1, A2, B, and O Reaction withUlex europaeus:NEGATIVE SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH Major Subgroups of A (99% of Type A) WEAK A SUBGROUPS (1% OF TYPE A PEOPLE) Decreased no. of A antigen sites per RBC (resulting in weak or no agglutination with anti-A) Varying degrees of agglutination by human anti-A,B Increased variability in the detectability of H antigen,resulting in strong reactions with anti-H Presence or absence of anti-A1 in the serum Other A subgroups: RBC of the A end,A3, Ax, Ay,Am or A el. are only rarely seen in transfusion practice. WEAK A SUBGROUPS TYPES OF H ANTIGEN After fucose is added to Type II chains, the structure is termed: Type II H Four kinds of Type II H have been identified . 1 H1 2. H2 3. H3 4. H4 SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH WEAK B SUBGROUPS TECHNICAL ERRORS GROUP 1 DISCREPANCIES Can cause unexpected reactions in reverse typing due to Weakly reacting or missing antibodies Most common cause of ABO discrepancy Characteristics of B Phenotypes B3- characterized by a mixed field pattern of agglutination with anti-B and anti-A,B. Presence of B glycosyltransferase in serum but not in RBC membrane. Anti-B is absent in serum but B substance is present in secretions. This is the most common weak B subgroup. Bx- demonstrate weak agglutination with anti-B andanti-A,B antisera. No B glycosyltransferase enzyme in RNC and secretions. Readily and easily adsorbs and elutes anti-B Bm-unagglutinated by anti-B and anti-A,B. Easilyand rapidly elutes and adsorbs anti-B. B glycosyltransferase is usually present in serum Bel- unagglutinated by anti-B or anti-A,B. No B glycosyltransferase on RBC and serum. Weak anti-B might be present on serum. Only H substance is seen on secretions. Resolution of Common Group 1 Discrepancies Enhance the weak or missing reaction in the serum by incubating the patient serum with reagent A1 and B cells at RT for approx. 15 to 30 mins No reaction after centrifugation – incubate at 4oC for 15 to ABO DISCREPANCIES 30 mins Auto-control and O cell control must always be tested concurrently Occurs when results of forward grouping does not match with the result of the reverse grouping or vice versa. It occurs when unexpected reactions occur in the forward and reverse grouping ABO discrepancies can usually be resolved by repeating the test on the same sample by using a saline suspension of RBCs if theinitial test was performed using RBCs suspended in serum or plasma. SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH GROUP 2 DISCREPANCIES GROUP 3 DISCREPANCIES Cause problems in forward and reverse grouping and are Can cause unexpected reactions in the Forward grouping due toPLASMA PROTEIN ABNORMALITIESwhich can due toWEAKLY REACTING OR MISSING ANTIGENS result to rouleaux formation and pseudo-agglutination Least frequently encountered Acquired B Phenomenon GROUP 4 DISCREPANCIES Modifies the immunodominant sugar of blood group A to Due toMISCELLANEOUS CAUSES Group B CAUSES: Test patient serum against autologous RBCs, this will yield a 1. Presence of cold-reactive autoantibodies (ANTI-I) negative reaction 2. Patient has circulating RBC with more than one blood type Acquired B antigen will not agglutinate with anti-B that has a (AB PATIENT) pH higher than 8.5 or less than 6.0 3. Unexpected ABO isoagglutinins Perform secretor studies – only the A substance is secreted 4. Unexpected non-ABO isoagglutinins Treated withAcetic Anhydride 5. Cis-AB- refers to inheritance of both AB genes from one parent carried on one chromosome and an O gene inherited from another parent Resolution of Group 4 Discrepancies Potent cold autoantibodies can cause spontaneous agglutination of the patient’s cells RBCs could be incubated at 37oC for a short period, then washed with NSS and retyped If not successful in resolving forward type, treat patient’s RBC with 0.01M dithiothreitol to disperse IgM related agglutination As for the serum, the reagent RBCs and serum can be warmed to 37oC, then mixed, retested and read at 37oC If the reverse typing is negative - Autoabsorption is done to remove autoantibody Resolution of Common Group 2 Discrepancies Unexpected ABO isoagglutinins in the patient’s serum react at RT – A2 and A2B individuals who can produce naturally Enhance by incubating the test mixture at RT for up to 30 occurring anti-A1 and A1B who produce naturally occurring minutes to increase the association of antibody with antigen. anti-H If negative, incubate the test mixture at 4oC for 15 to 30 Specificity of the antibody can be determined by examining minutes the pattern of reactivity Autocontrol and O control must always be tested alongside Unexpected alloantibodies in the patient’s other than ABO with the patients sample isoagglutinins Panel could be performed with the patient’s serum SADCY IMMUNOHEMATOLOGY 321 Instructor: JUSTIN KIM C. VERGARA, RMT, MPH SADCY
