IMMUNOHEMATOLOGY THE HISTORICAL CONCEPTS OF IMMUNOHEMATOLOGY AND THE REVIEW OF GENETICS OUTLINE • History of Immunohematology and Blood Transfusion Practice and Future Trends o Introduction o Important People in the History of Immunohematology o Current Status • The Review of Genetics And Molecular Biology o Basic Principles in Genetics HISTORY OF IMMUNOHEMATOLOGY AND BLOOD TRANSFUSION PRACTICE AND FUTURE TRENDS INTRODUCTION • People have always been fascinated by blood: Ancient Egyptians bathed in it, aristocrats drank it, authors and playwrights used it as themes, and modern humanity transfuses it. • The road to an efficient, safe, and uncomplicated transfusion has been difficult, but great progress has been made. • Transfusion - ensures the safety of donor and receiver IMPORTANT PEOPLE IN THE HISTORY OF IMMUNOHEMATOLOGY • Pope Innocent VII* (1492) - Received the 1st blood transfusion from 3-10 years old boys (altar servers). Unfortunately, all four died during and after the process. o Experienced transfusion reactions • William Harvey - English physician and scientist who described the circulation of the blood. • Richard Lower (1665) - Performed the 1st experimental blood transfusion from a dog into another dog. • Jean Baptiste Denis - First animal-human transfusion between a goat and human. • Jan Jacbz - Swammerdam Microscopic appearance of RBC • Philip Syng Physick - Unconfirmed first human-to-human transfusion. • John Henry Leacock - Proved that the blood donor and recipient must be of the same species. • James Blundell* o Performed the 1st successful blood transfusion between a woman dying of post-partum hemorrhage and her husband. o Invented the impellor and gravitator; proposed a ratio for blood transfusion • Samuel Armstrong Lane - Transfused whole blood to treat hemophilia (disorder of hemolysis) • Emil Ponfick o Observed red cell lysis after xenotransfusion o Associated hemorrhages and organ congestion with incompatible transfusion • Leonard Landois - In vitro mixing of blood and animal sera results to lysis; immunologic basis of blood compatibility • Roger Irving Lee and Paul Dudley White - Blood group O can be donated to any blood group; blood group AB can receive blood from any blood group • A. R. Kimpton and J. H. Brown - Paraffin's smooth surface causes a delay in clotting of blood • Edward Lindemann - First to succeed in vein-to-vein transfusion via multiple syringes and special cannula for puncturing • Karl Landsteiner* (1901) o Discovered ABO blood groups. ▪ One of the most important blood group system o Explained the serious reactions that occur in humans as a result of incompatible transfusions ▪ Severe reactions are usual o His work in the beginning of the 20th century won a Nobel Prize • Alfred von Decastello and Adriano Sturli - AB o AB - lowest frequency in Asians • Karl Landsteiner, Philip Levine and Alex Weiner - Rh Blood group • Gesellius - Made a complex device that lanced the donor's back multiple times where capillary blood is extracted using suction cups • Unger - Syringe valve apparatus for practical blood transfusion; single device for efficiency & practicality • Braxton Hicks (1869) - Used sodium phosphate as a preservative for blood. This was perhaps the first example of blood preservation research. • Albert Hustin (1914) - First indirect blood transfusion; used sodium citrate as a preservative for blood • Richard Weil - Refrigerated storage of citrated blood • Richard Lewisohn (1915) - Determined the non-toxic concentration of citrate • Oswald Robertson - First transfusion of stored blood in WWI • Rous and Turner (1916) - Citrate-Dextrose solution prolonged life of RBC • 1930 - The function of glucose in RBC metabolism was understood • World War II - Stimulated blood preservation research because the demand for blood and plasma increased. • Charles Drew (1941) o First director of American Red Cross at Presbyterian Hospital. o Techniques in blood transfusion and preservation • John Loutit and Patrick Mollison (1943) - Acid-Citrate- Dextrose (anticoagulant; also used in pheresis) o ACD - Preserves blood bags for 21 days o Citrate phosphate dextrose w/ adenine (CPDA1) - may preserve blood bags for 35 days • 1947 o Journal of Clinical Investigation o Blood banks were established in many major cities of the United States; subsequently, transfusion became commonplace. • Audrey Smith - Long term red cell preservation by freezing; glycerol prevents freeze-thaw damage • John G. Gibson (1957) - Citrate-Phosphate-Dextrose (21 days storage) which was less acidic & eventually replaced ACD as standard preservative used for blood storage • US FDA - Used adenine for blood preservation; standardized the use of CDPA1 • Percy Oliver - Established first blood donor service • Mayo Clinic - Modern Blood Bank • Bernard Fantus* - Coined the term "blood bank"; glass flask with sodium citrate; Father of modern blood bank • Federico Duran-Jorda - Mobile Blood Bank • John Elliot - Transfused plasma to patient
• Carl Walter and William Murphy - Proposed the ideal material for blood bags (collapsible polyvinyl bag); allows plasma removal • Edwin Cohn - Fractionation of albumin, fibrinogen and immunoglobulins; cell separator • Prof. Isidor Ravdin - Efficacy of albumin for preventing collapsing of blood vessels; "Cold Ethanol Fractionation" • A. Solomon and J. L. Fahey - Centrifugation technology; first therapeutic plasmapheresis • Weil and Ottenberg - First crossmatch procedure; minor & major cross matching; universal utility of group O donors • Ludvig Hektoen - Safety of blood transfusion through cross matching • Wide and Jerker Porath - Immunosorbent technique • Peter Perimann and Eva Evangall - Enzyme-linked Immunsorbent Assay • Dr. Yves Lapierre * - Gel Technology • Alexis Carrel - Prevent clotting by sewing the vein of patient to the artery of the donor; "anastomosis" • Carlo Moreschi - Antiglobulin reaction; sensitization; lattice formation • Francis Peyton Rous and J. R. Turner - Glucose-Citrate- Dextrose solution; preservation solution to enhance metabolism of RBCs • Reuben Ottenberg - Demonstrated the importance of compatibility testing or cross matching • John Elliot - Vacuum type of container for blood • P. Beeson - Classic description of transfusion-transmitted hepatitis • Coombs, Mourant and Race - Antihuman Globulin testing; "Coombs Test" • Max Perutz - Molecular structure of hemoglobin • Judith Pool and Angela Shannon - Produced cryoprecipitate AHF for treatment of hemophilia • Scott Murphy and Frank Gradner - Feasibility of storing platelets at room temperature revolutionizing platelet transfusion therapy CURRENT STATUS • AABB, formerly the American Association of Blood Banks, estimates that 6.8 million volunteers donate blood each year. AABB provides technical manuals for blood banking techniques • With an aging population and advances in medical treatments requiring transfusions, the demand for blood and blood components is expected to continue to be high. • It is estimated that one in three people will need blood at some point in their lifetime. THE REVIEW OF GENETICS AND MOLECULAR BIOLOGY BASIC PRINCIPLES IN GENETICS • Classic Genetics o Modern genetics is based upon the understanding of the biochemical and biophysical nature of nucleic acids such as: ▪ deoxyribonucleic acid(DNA) ▪ ribonucleic acid(RNA) o All areas of transfusion medicine are influenced by genetics including blood group systems POPULATION GENETICS • Mendel’s Laws of Inheritance o Mendel was an Austrian monk and mathematician who used sweet pea plants. ▪ Father of modern genetics ▪ Differentiated the traits of the peas o Determined that physical traits are due to factors he called elementen o 3 laws of inheritance ▪ Mendel’s first law of inheritance, the law of independent or random segregation • The first generation in the study, called the parental, pure, or P1 generation, consisted of all red or all white flowers that bred true for many generations ▪ Mendel’s second law is the law of independent assortment and states that genes for different traits are inherited separately from each other. • This allows for all possible combinations of genes to occur in the offspring • Mendel’s laws apply to all sexually reproducing diploid organisms whether they are microorganisms, insects, plants, or animals, or people ▪ Mendel’s third law is the law The Law of Dominance : An organism with alternate forms of a gene will express the form that is dominant. • Hardy-Weinberg Principle o H. Hardy, a British mathematician, and W. Weinberg, a German physician, developed a mathematical formula that allowed the study of Mendelian inheritance in great detail. ▪ Further explains the studies of Mendel o Formula —p + q = 1, in which p equals the gene frequency of the dominant allele and q is the frequency of the recessive allele o The allele frequency for blood group genes can be studied using the Hardy-Weinberg equations • Inheritance Patterns o The interpretation of pedigree analysis requires the understanding of various standard conventions in the representation of data figures o Males are always represented by squares and females by circles, with open symbols indicating an unaffected individual and a closed, filled-in symbol indicating an affected individual. o The propositus or proband in the pedigree is indicated by an arrow pointing to it and indicates the most interesting or important member of the pedigree o Autosomal refers to traits that are not carried on the sex chromosomes o 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. o Autosomal dominant traits are routinely encountered in the blood bank, as most blood group genes are codominant and are on autosomal chromosomes CELLULAR GENETICS : • Organisms may be divided into two major categories o Prokaryotic , without a defined nucleus ▪ Has simplified organelles, unicellular ▪ Eg. bacteria
o Eukaryotic , with a defined nucleus ▪ Human beings and all other mammals are included in the eukaryotic group, as are birds, reptiles, amphibians, fish, and some fungus species ▪ Has complex organelles, multicellular • The nuclear material is organized into chromatin, consisting of nucleic acids and structural proteins, and is defined by staining patterns: o Heterochromatin - stains as dark bands o Achromatin - stains as light bands o Euchromatin - the swollen form of chromatin in cells, which is considered to be more active in the synthesis of RNA for transcription • Terminology : o Genome - is the full complement of genes in an organism o Chromosomes - are the structures in the cell nucleus that contain the duplex (double-stranded) DNA. o Gene - a section, often very large, of DNA along the chromosome that encodes for a particular protein. o Genotype - is the complement of DNA that is inherited. o Phenotype - is the observable expression of the genotype. o Hemizygous – a condition when one chromosome has a copy of the gene and the other chromosome has that gene deleted or absent. o Amorph – a silent gene. • Mitosis o The process by which somatic cells divide to create identical daughter cells. o 5 Phases ▪ Interphase - the resting stage when the cells are not actively dividing ▪ Prophase - the chromatin condenses to form visible chromosomes and the nuclear envelope starts to break down ▪ Metaphase - the chromosomes are lined up along the middle of the nucleus and paired with the corresponding chromosome ▪ Anaphase - the cellular spindle apparatus is formed and the chromosomes are pulled to opposite ends of the cell. ▪ Telophase - the cell is pulled apart, division is complete; product is two daughter cells • Meiosis o A different process is used to produce the gametes or sex cells. o results in four unique, rather than two identical, daughter cells o If cells with 2N chromosomes were paired, the resulting daughter cells would have 4N chromosomes o Gametes carry a haploid number of chromosomes, 1N, so that when they combine, the resulting cell has a 2N configuration o The first stages of meiosis are nearly identical to those in mitosis o However, there is no centromere division, and at anaphase and telophase, the cell divides and enters once again into interphase. • Cell Division Cycle o Cell division is a complicated process that also occurs with various specific stages o eukaryotic cells such as human cells, the cell cycle is divided into four distinct stages o it can repeat itself or can be stopped at any one point in the cycle. o The first step or stage is the resting stage, or G0 , and is the state of cells not actively dividing o The prereplication called G1 o DNA is synthesized is the next stage, called the S stage o G2 stage , or postreplication stage. o the M phase , in which mitosis occurs MOLECULAR GENETICS • includes knowledge of the physical conformation of chromosomes as well as the biological and chemical nature of the polymers of the different nucleic acids and nuclear proteins. • Deoxyribonucleic Acid (DNA) o A masterpiece of architectural evolution and the “backbone” of heredity. o a nucleic acid, therefore most of the proteins that interact with it have an overall basic pH. o Chromatin - a type of polymer structure. o Chromosomes - composed of long, linear strands of DNA tightly coiled around highly basic proteins called histones o Each chromosome is a single, extremely long strand of duplex DNA. ▪ Postulated by James Watson and Francis Crick o Nucleosome - the complex of DNA and histone protein. o Hybridization - the formation of hydrogen bonds between two complementary strands of DNA. o DNA is composed of: ▪ 4 nitrogenous bases - adenine (A), cytosine (C), guanine (G), and thymine (T) • stabilized by hydrogen bonding and Van der Waals forces. • A bonds only to T with two hydrogen bonds (weaker pairing), and C bonds only with G with three hydrogen bonds (stronger pairing) - postulated by James Watson and Francis Crick at Cambridge University in the early 1950s • Purines - consisting of double-ring structures (adenine and guanine) • Pyrimidines - single-ring structures (cytosine and thymine) ▪ Deoxyribose - a 5-carbon sugar molecule. ▪ Phosphate group - comprise the backbone of the DNA molecule together with deoxyribose. o Codon - triplets of nucleotides (ATG) ▪ 4 special codons : • Start codon - only codon specific for the initiation of transcription and translation. • 3 Stop codons – for termination of the peptide being translated from mRNAGenetic Code - the term used to describe the relationship between triplet nucleotides and amino acids.
• DNA Replication o replication, or copying, of DNA is a complex process involving numerous enzymes, nucleic acid primers, various small molecules, and the DNA helix molecule that serves as its own template for the replication process. o DNA must be copied before mitosis can occur. o DNA must be uncoiled from its supercoiled (or double-twisted) nature. Done by the enzymes DNA gyrase (opens the supercoils) and DNA helicase (separates the two strands of duplex DNA), using energy derived from ATP hydrolysis, they open the DNA molecules and keep the strands separate. o Proteins called single-stranded binding proteins interact with the open strands of DNA to prevent hydrogen bonding when it is not needed during replication. o A short oligonucleotide (composed of RNA) binds to the beginning of the region replicate. This “primes” the replication process; therefore, these short oligonucleotide sequences are called primers. o Both DNA parent strands are replicated simultaneously in a 5′ to 3′ manner; o Replication of the other parent strand, the lagging strand, is more complicated because the strands are antiparallel. o As the helix is opened, RNA primer sequences are added to the area of the opening fork, and the RNA primers are extended in a 5′ to 3′ manner until the polymerase reaches the previously synthesized end. o Okazaki fragments - small regions of newly replicated DNA. The fragments must be joined together to make a complete and continuous strand (done by the two enzymes DNA polymerase I and DNA ligase). o RNA primers are synthesized and added to the DNA strands by an enzyme called primase. o Main players in DNA Replication ▪ Helicase - “the unzipping enzyme” breaks hydrogen bonds that holds the bases together ▪ DNA polymerase - builds DNA molecules to make a new DNA ▪ Primase - “initializers” makes the primer ▪ Ligase - “gluing” binds the fragments together • DNA Repair o proofreading ability of DNA polymerase enzymes. o Mismatch repair - where an incorrect, noncomplementary nucleotide is removed and the correct one is inserted in its place. o Chemical and environmental factors : ▪ Alkylating agents - reacts with guanine and result in depurination. ▪ Some cancer treatments ▪ Ionizing radiation and strong oxidants such as peroxides can cause single-strand breaks. ▪ Ultraviolet (UV) radiation can alter thymine bases, resulting in thymine dimers. ▪ Drugs such as the antibiotic mitomycin C - can form covalent linkages between bases on opposite strands, resulting in incorrect separation of the strands at that site during replication and subsequent mutations in daughter strands. o DNA repair systems - can recognize mismatched base pairs, missing nucleotides, and altered nucleotides in DNA sequences. ▪ Photoreactivation ▪ Excision repair - also referred to as cut and patch repair. ▪ Recombination repair - uses the correct strand of DNA to fill in the strand where the error was deleted. ▪ Mismatch repair - activated when base pairing is incorrect and a bulge occurs in the duplex DNA. ▪ SOS repair • DNA Mutations o Mutation refers to any structural alteration of DNA in an organism (mutant) that is caused by a physical or chemical agent (mutagen) and can occur spontaneously. ▪ Alterations can be • Structural - chromosome segment issue/ genetic variant • Molecular level - amino acid sequence/ nucleotide defects o Wild type - the original form of the DNA sequence and the organism in which it occurs. o Mutagens - the various chemicals and conditions that can cause mutations. o Types of mutations : ▪ Point mutation ▪ Silent mutation ▪ Transition mutation ▪ Missense point mutation ▪ Nonsense mutation ▪ Frameshift mutation o Point mutation – simplest type, only one nucleotide in the DNA sequence is changed. Includes substitutions, insertions, and deletions. o Silent mutation - occurs when a mutation happens that causes a change in the peptide sequence, but that part of the peptide isn’t critical for its function. No mutation is seen at the phenotypic level, such as enzyme function or cell surface marker. o Transition mutation - one purine is substituted for another purine, or one pyrimidine is substituted for another pyrimidine. (Transversion - when a purine is substituted for a pyrimidine or a pyrimidine for a purine). o Missense point mutation - results in a change in a codon, which alters the amino acid in the corresponding peptide; produces new amino acid from affected DNA & RNA o Nonsense mutation - results when a point change in one of the nucleotides of a DNA sequence causes one of the three possible stop codons to be formed; produced stop codon from affected DNA & RNA
o Frameshift mutation - results in a nonfunctional transferase protein that is seen phenotypically as the O blood group. • DNA Isolation o Magnetic bead technology is a commonly used, efficient method to isolate DNA and other molecules, and the procedure can be automated. • Ribonucleic Acid (RNA) o single-stranded structure o Uracil instead of Thymine o Sugar is Ribose o transmit genetic information (stored as DNA) from the nucleus to the cytoplasm o Functions in protein synthesis o DNA is copied into RNA by transcription, modified, and transported out of the nucleus to the ribosomes, where it is translated into protein, which is then modified if necessary for its proper function. o Central dogma - the flow of genetic information, from DNA to RNA to protein. o major types: ▪ Ribosomal RNA (rRNA) • Makes up a large part of the ribosomal structure on the endoplasmic reticulum in the cytoplasm; where RNA is translated into peptide. • RNA polymerase I transcribes rRNA. • Most abundant and consistent form of RNA in the cell. ▪ Messenger RNA (mRNA) • the initial link between the information stored in DNA and the translation of that information into amino acids. • The form that is transcribed from DNA that encodes specific genes, such as those determining the various blood groups. • RNA polymerase II transcribes mRNA. ▪ Transfer RNA (tRNA) • involved in delivering amino acids to the mRNA bound on the ribosome. ▪ small and microRNA molecules • Central Dogma of Molecular Biology o The flow of genetic information, from DNA to RNA to protein o DNA replication o Transcription ▪ the cellular process by which one strand of duplex DNA is copied into RNA. ▪ 3 major steps : • Initiation - the attachment of a free methionine to a transfer RNA molecule called tRNAmet, which requires the presence of a high-energy molecule called guanine triphosphate (GTP) and a special protein called initiation factor IF2. • Elongation - the incoming tRNA binds to the A site in the presence of the elongation factor called E2F. • Termination - the completed polypeptide chain is released and the ribosome dissociates from the mRNA. ▪ RNA polymerase - key player; produces RNA o Translation ▪ the cellular process by which RNA transcripts are turned into proteins and peptides, the functional molecules of the cell ▪ mRNa → amino acids → protein SUMMARY OF IMPORTANT TERMS • Genetics - study of inheritance or transmission of phenotypic & genetic characteristics from parents to offspring o Chromosome - thread-like structure of nucleic acids and protein found in the nucleus of most living cells, carrying genetic information in the form of genes ▪ 22 autosomes & 1 pair sex cell • Genes - distinct sequence of nucleotides forming part of a chromosome, the order of which determines the order of monomers in a polypeptide or nucleic acid molecule which a cell (or virus) may synthesize • Locus - • Deoxyribonucleic (DNA) • Ribonucleic Acid (RNA) - a nucleic acid present in all living cells, principal role is to act as a messenger carrying instructions from DNA for controlling the synthesis of proteins, although in some viruses RNA rather than DNA carries the genetic information • Dominant gene - produces an effect in the organism regardless of the state of the corresponding allele • Recessive gene - phenotypically expressed in the homozygous state but has its expression masked in the presence of a dominant gene • Genotype - sequence of DNA that is inherited • Phenotype - observed characteristic; anything produced by the genotype, including an enzyme to control a blood ground antigen • amorph- • Hemizygous - refers to the condition when one chromosome has a copy of the gene and the other chromosome has that gene deleted or absent • X- linked Dominant - mode of genetic inheritance by which a dominant gene is carried on the X chromosome • X- linked Recessive - mutation in a gene on the X chromosome causes the phenotype to be always expressed in males • Codominant - ABO, Rh, Kell, Kidd • Mendel’s law of inheritance o Carolus Linnaeus - first classification of living things o Gregor Mendel - father of modern genetics • Inheritance Patterns o Autosome - refers to traits that are not carried on the sex chromosomes o Autosomal-dominant - in (Lu) suppressor gene o Autosomal-recessive - dd genotype o X-linked dominant - Xga blood group system o X- linked recessive - Hemophilia A