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Introduction to Immunohematology and related Genetics  What is Immunohematology? A branch of medical science. It deals with the concepts and clinical techniques related to modern transfusion therapy. The term immunohematology refers to the serologic, genetic, bioc...

Introduction to Immunohematology and related Genetics  What is Immunohematology? A branch of medical science. It deals with the concepts and clinical techniques related to modern transfusion therapy. The term immunohematology refers to the serologic, genetic, biochemical, and molecular study of antigens associated with membrane structures of the cellular constituents of blood, as well as the immunologic properties and reactions of blood components and constituents. Fundamental discoveries in the area of immunohematology have played an integral role in the development of transfusion medicine, which includes the transfusion of blood, its components, and its derivatives.  Immunohematologists perform and interpret a wide variety of serologic and molecular assays to aid in the diagnosis, prevention, and management of immunization associated with transfusion, pregnancy, and organ transplantation. Over the years, research in the field of immunohematology has contributed significantly to the fundamental understanding of human genetics and immunology, with broad applications to membrane physiology and function, epidemiology, anthropology, and forensic science.  History of blood transfusion People have always been fascinated by blood: Ancient Egyp- tians bathed in it, aristocrats drank it, authors and play- wrights used it as themes, and modern humanity transfuses it. In 1492, blood was taken from three young men and given to the stricken Pope Innocent VII in the hope of curing him; unfortunately, all four died. Blood transfusion began when William Harvey described the circulation of blood in 1616. In 1665, an English physiologist, Richard Lower, successfully performed the first animal-to-animal blood transfusion that kept exsanguinated dogs alive by transfusion of blood from other dogs.  History of blood transfusion In 1667, Jean Baptiste Denys, transfused blood from the carotid artery of a lamb into the vein of a young man, which at first seemed successful. However, after the third transfusion of lamb’s blood the man suffered a reaction and died. Denys also performed subsequent transfusions using animal blood, but most of them were unsuccessful. Later, it was found that it is impossible to successfully transfuse the blood of one species of animal into another species.  Due to the many disastrous consequences resulting from blood transfusion, transfusions were prohibited from 1667 to 1818. James Blundell of England successfully transfused human blood to women suffering from hemorrhage at childbirth. Such species-specific transfusions (within the same species of animal) seemed to work about half the time but mostly the result was death.  Blood transfusions continued to produce unpredictable results, until Karl Landsteiner discovered the ABO blood groups in 1900. It became clear that the incompatibility of many transfusions was caused by the presence of certain factors on red cells now known as antigens. Landsteiner postulates : Ø Each species of animal or human has certain factor on the red cell that is unique to that species, and Ø even each species has some common and some uncommon factor to each other. This landmark event initiated the era of scientific–based transfusion therapy and was the foundation of immunohematology as a science.  Blood Group Genetics Blood group genetics are concerned with the way in which the different blood groups are inherited, that is passed on from parents to children. Chromosomes and Genes: In the human body, the nucleus of each body cell contains 46 small thread-like structures called chromosomes, arranged in 23 pairs. The length of each chromosome is divided in to many small units called genes, as they contain the different physical characteristics, which can be inherited including those of the blood groups.  Blood Group Genetics Blood Group Genetics Genetic material is found in DNA Genetic material is found in DNA DNA is contained in chromosomes that are DNA is nucleus in the containedofinevery chromosomes cell that are in the nucleus of every cell Genetic material is Genetic material replicated eitherisby replicated either bycells) mitosis (somatic mitosis or (somatic meiosiscells) or meiosis (gametes) (gametes) Copyright © 2017, Elsevier Inc. All Rights Reserved. 11 Copyright © 2017, Elsevier Inc. All Rights Reserved. 11  Body cells and mitosis Sex cells and meiosis When body cells multiply When sex cells are formed they do so by producing either male or female the identical new cells with 46 pairs of chromosomes do chromosomes. This process not multiply but simply is called mitosis. separate so that each of the new cells formed contains only 23 chromosomes not 46 as in the body cells. This process is called meiosis.  However, during fortification when the egg and sperm unite, the fertilized ovum receives 23 chromosomes from each sex cell half of these from the male and half from the female and thus will contain 46 chromosomes which again arrange themselves in pairs in the nucleus. For example, a child who inherits gene A from its father and also gene A from its mother would be homozygous, where as a child who inherits gene A from its father and gene B from its mother would be heterozygous.  Phenotype versus Genotype Phenotype Genotype Physical (observed) expression of Actual genetic makeup traits The patient’s phenotype is Can only be determined by determined by hemagglutination molecular techniques or family of RBC antigens using antisera studies Example: A person who shows no Example: A person with the agglutination with anti-A or anti-B phenotype A could have the antisera is considered to have genotype A/A or A/O; family type O blood studies would be necessary to confirm which is present Copyright © 2017, Elsevier Inc. All Rights Reserved. 14  Phenotype and Genotype Phenotype and Genotype Copyright © 2017, Elsevier Inc. All Rights Reserved. 15  Punnett Square A Punnett square is used to predict the probability of an offspring’s genotype It summarizes every possible combination of maternal and paternal alleles of a particular gene Copyright © 2017, Elsevier Inc. All Rights Reserved. 16  Genes Genes are basic units of inheritance on a chromosome A locus is the site at which a gene is located on a chromosome Alleles, which are alternative forms of a gene, are found at each locus – Antigens produced by opposite alleles are antithetical (e.g., Kpa and Kpb antigens) – Multiple alleles at a single locus are considered polymorphic Copyright © 2017, Elsevier Inc. All Rights Reserved. 17 Allomorphic genes (Alleles): Each gene has it own place called its locus along the length of the chromosome. However, a certain inherited characteristic can be represented by a group of genes, and the place or locus can be occupied by only one of these genes. Such genes are called alleles or allomorphic genes.  For example, every one belongs to one or other of the following blood groups: group A, group B, group O or group AB. à Therefore, there are three allomorphic genes which make up the ABO Blood group system such as gene A, gene B, and gene O. Only one of these alleles can occupy the special place or locus along the chromosomes for this blood group characteristic.  Inheritance Gene is expressed only when inherited Recessive by both parents Equal expression of two different alleles Codominant Blood group antigens are codominant Gene that is expressed over another Dominant gene Genes that do not express a detectable product are considered amorphic (e.g., O gene) Copyright © 2017, Elsevier Inc. All Rights Reserved. 20  For example, in the ABO blood group system the gene A and B are dominant over gene O. Thus if a child receives from its parents gene A and O it will belong to group A. In the same way if a child receives from its parents genes B and O it will belong to group B. Only if it receives gene O from both its parents will it belong to group O. Copyright © 2013, 2009, 2000 by Mosby, 21 Inc., an affiliate of Elsevier Inc.  Mendelian Principles Mendelian principles can be applied to blood group antigen inheritance – Independent segregation occurs when one gene from each parent is passed to the offspring – Independent assortment is demonstrated when blood group antigens from different chromosomes are expressed separately, resulting in a mixture of genetic material 2 exceptions to this law are linkage and crossing over Copyright © 2017, Elsevier Inc. All Rights Reserved. 22  Linkage Linkage Linkage occurs when 2 Linkage occurs genes that whento2each are close genes otherthat are closetogether are inherited to each other are inherited – Each set of linked genes is together called a haplotype – Each set of linked genes is called a haplotype Haplotypes tend to occur at a higher frequency than for Haplotypes tend ato occur unlinked genes, at phenomenon a higher frequency than called linkage fordisequilibrium unlinked genes, a phenomenon called linkage disequilibrium Copyright © 2017, Elsevier Inc. All Rights Reserved. 23 Copyright © 2017, Elsevier Inc. All Rights Reserved. 25  Crossing Over Crossing over occurs when 2 genes on the same chromosome combine and produce 2 new chromosomes Copyright © 2017, Elsevier Inc. All Rights Reserved. 24  Chromosomal Assignment Most blood group system genes are on autosomes, except for those of the Xg system Xg genes are found on the X chromosome – If the father carries the Xg allele, he will pass it to all of his daughters but not to any of his sons – If the mother carries the Xg allele (not the father), all of their children will express Xg Copyright © 2017, Elsevier Inc. All Rights Reserved. 25  Heterozygosity and Homozygosity A person who inherits identical alleles is called homozygous – AA, BB, MM (M+ N–) A person who inherits different alleles is called heterozygous – AO, AB, MN (M+ N+) Copyright © 2017, Elsevier Inc. All Rights Reserved. 26  Dosage In some blood group systems, persons homozygous for an allele have a “double dose” of an antigen on their RBCs compared with those who are heterozygous for an allele Dosage is a variation in antigen expression due to the number of alleles present Copyright © 2017, Elsevier Inc. All Rights Reserved. 27  Dosage Example Homozygous expression of some antigens will show stronger agglutination compared with antigens that are heterozygous Copyright © 2017, Elsevier Inc. All Rights Reserved. 28  Genetic Interaction The location of inherited genes in cis or trans positions can affect the expression of the antigen – Alleles on the same chromosome are cis to one another – Alleles on opposite chromosomes are in the trans position Copyright © 2017, Elsevier Inc. All Rights Reserved. 29  Population Genetics To determine genotype or phenotype occurrence, two formulas are used: – A phenotype calculation enables finding a unit of RBCs with certain antigen characteristics (i.e., antigen negative) – The Hardy-Weinberg formula calculates a determination of the gene frequencies that produced a trait Copyright © 2017, Elsevier Inc. All Rights Reserved. 30  Phenotype Calculations Example: a patient with multiple antibodies (anti-C, anti-E, anti- S) needs blood What Is Known Percentage Negative Convert to Decimal and Then Multiply 70% C positive 30% 0.30 30% E positive 70% 0.70 55% S positive 45% 0.45 0.30 x 0.70 x 0.45 = 0.0945 or 0.10 (10%) Conclusion: about 10% of the population will be negative for all three antigens; about 1 in 10 units will be compatible Copyright © 2017, Elsevier Inc. All Rights Reserved. 31  Phenotype Calculations (cont’d) Example: a patient with multiple antibodies needs 2 units What Is Known Percentage Negative Convert to Decimal and Then Multiply 66% Fya positive 34% 0.34 72% Jkb positive 28% 0.28 9% K positive 91% 0.91 0.34 x 0.28 x 0.91 = 0.087 or 9% negative (9 out of 100) 2 units needed / 0.09 (antigen negative frequency) = 22 Conclusion: antigen typing of 22 units may be required to find 2 compatible units Copyright © 2017, Elsevier Inc. All Rights Reserved. 32  Hardy-Weinberg Formula Basic Formula Example p represents allele #1 p is the frequency of allele A q represents allele #2 q is the frequency of allele a Gene frequency is represented by p + q = 1 What is the frequency of q if p is 0.3? 1 – 0.3 = 0.7 Genotype proportions are What are the genotype proportions? AA = p2 or 0.09 (homozygous for A) (p + q)2 = 1.0 Aa = 2pq or 0.42 (heterozygous for Aa) or aa = q2 or 0.49 (homozygous for a) p2 + 2pq + q2 = 1.0 Copyright © 2017, Elsevier Inc. All Rights Reserved. 33  Role of H-Gene in the Expression of ABO Genes Inheritance of A and B genes usually results in the expression of A and B gene products (antigens) on erythrocytes H,A and B antigens are not the direct products of the H, A, and B genes, respectively.  Each gene codes for the production of a specific transferase enzyme. This enzyme catalyzes the transfer of a monosaccharide molecule from a donor substance to the precursor substance, and thus to the particular blood group substance. ABH Genes and Their Enzymatic Products Gene Enzyme H: L- fucosyltransferase A: 3 N-acetyl- D- galactosaminyl transferase B: 3-D- galactosyl transferase O: None other than the H gene  The H gene (HH/Hh) encodes for an enzyme, which converts the precursor substance in red cells in to H substance (H antigen). A and B genes encode specific transferase enzymes which convert H substance in to A and B red cell antigens. Some H substance remains unconverted (the H substance is partly converted).  O gene encodes for an inactive enzyme, which results in no conversion of the substance in-group O red cells. This indicates group O individual contains the greatest concentration of H antigen.  Persons who do not inherit H gene (very rare hh genotype) are unable to produce H substance and therefore even when A and B genes are inherited, A & B antigens can not be formed. This rare group is referred to as Oh (Bombay group).  Secretors and Non-Secretors The term secretor and non-secretor only refer to the presence or absence of water- soluble ABH antigen substances in body fluids (saliva, semen, urine, sweat, tears, etc). Every individual contains alcohol soluble antigens in body tissues and on the red cells, whether secretor or non-secretor. But secretors, in addition to this, possess the water soluble (glycoprotein) form of antigen, which appears in most body fluids.  Majority of the population secrete water- soluble substances in saliva and most other body fluids that have the same specificity as the antigens on their red cells. The production of A, B & H antigens in saliva is controlled by a secretor gene, which is inherited independently of the ABO and H genes. The relevant gene is called Se, and its allele which amorphic is se. At least one Se gene (genotype SeSe or Sese) is essential for the expression of the ABH antigens in secretors. Individual who are homozygous for se (sese) do not secrete H,A, or B antigens regardless of the presence of H,A or B genes.  The Se gene does not affect the formation of A,B or H antigens on the red cells or in hematopoietic tissue, which are alcohol soluble and which are not present in body secretions. Oh (Bombay) individuals do not secrete A, B or H substance, even when the Se gene is present.