MOLECULAR GENETICS
Gene- basic unit of heredity
Genome- gene found in the long stretch of DNA Chromosome- super compacted; part of DNA Chromatid- half of the chromosome
Chromatin- loose enough to give nucleus protection Nucleosome- makes up a phromatin
Allele- bondings in the chromosome Locus- specific side where allele is located
DNA RNA
Replication, Repair, Mutations
Transcription, Translation
Double Helix Single Helix
Cytosine, Guanine, Adenine, Thymine
Cytosine, Guanine, Adenine, Uracil DNA - backbone of heredity
- Double Helix
- H-bond, phosphodiester bonds DNA REPLICATION
DNA gyrase- relaxes the supercoil DNA helicase- separates helixes (uncoil) RNA primase
DNA polymerase III- elongates new strand Sliding clamp- so that it will not dislodge
Single-stranded binding proteins- make sure DNA helixes does not recoil
DNA polymerase I- removes primer DNA ligas- connects the fragment FRAGMENT OF DNA
Replication fork Leading Strand Lagging Strand Okazaki fragment Causes of DNA damage:
1. Cellular Metabolism
Vit B6- conversion of deoxyuridyl monophosphate and deoxy thymidyl triphosphate
Vit B9- important in production of thymine
Vit B12- necessary for absorption of B9 - conversion of folic acid into
usable form 2. UV light exposure
3. Ionizing Radiation 4. Chemical Exposure 5. Replication Errors
DNA Response to damage:
1. Cell Cycle Checkpoint activation 2. Transcriptional Program Activation 3. DNA Repair
4. Apoptosis Direct reversal Base excision repair Nucleotide excision repair Mismatch repair
Double strand break repair Homologous recombination DNA MUTATION
- Any change in the structure or sequence of DNA a. Physical
b. Biochemical - Mutant vs. Wild type
TYPES OF MUTATIONS 1. Base substitution
a. Transition- pyrimidine to pyrimidine - purine to purine
CUT the Py (Cytosine, Uracil, Thymine) Pure As Gold (Purine= Adenine, Guanine) b. Transversion- pyrimidine to purine (vise versa) Silent substitution- no change in amino acid Nonsense substitution- transforms to a stop codon Missence- complete change of amino acid
2. Deletion 3. Insertion
RNA
Single strand
Uracil instead of thymine
Transmit genetic info from nucleus to cytoplasm Types: rRNA, mRNA, tRNA
RNA transcription o Initiation o Elongation o Termination RNA processing
o Guanine cap is added o Removal of intron o Addition of mRNA Poly A
- Extron- expressed - Intron- interferring RNA translation
IMMUNOLOGY - Ag- Ab reactions
- studied because lab tests requires Ag- Ab reactions ABO grouping, Rh typing, Ab screening &
identification, crossmatching - how to know Ag-Ab reaction occurs
Quality control, trouble shooting, maintenance IMMUNE RESPONSE- physiological mechanism to fight disease or clear foreign substance
PRIMARY IMMUNE RESPONSE - Slow reaction
- First exposure to foreign object - IgM is the predominant Ab SECONDARY IMMUNE RESPONSE
- Rapid reaction
- Subsequent exposure of the same Ag - IgG predominant
Ag CHARACTERISTICS Antigen- foreign substance
Antigenecity- Ability of react to Abs Epitopes- Antigenic determinants
Ab CHARACTERISTICS
IgM IgG
Cold Agglutinin (4-10 deg)
37 degrees Cannot cross placenta Can cross placenta
Good complement activator
Poor complement activator Can activate comp but
cannot finish rxn Stops at C3 Usually not clinically sig.
Because they are cold agglutinins aside from ABO group w/ Y thermal reactivity Significant if it can cause HDN & transfusion rxn Clinically significant
ABO, Ii, Lewis, MN & P Rh, Kell, Kidd, Duffy & Ss IgA- found in secretions, dimer
NATURALLY OCCURING
- found in serum of an individual not exposed to Ag via transfusion, injection, pregnancy
- Produced in response to substances in environment - Most are IgM cold agglutinins, activates complement
IMMUNE Ab
- Produced when there is something foreign via transfusion, pregnancy, injection
- Not found in nature
- Mostly IgG, reacts at 37 degrees
- Requires aid of AHG (Anti Humaglobulin) for manifestation of Ag-Ab reactions
- AHG used for the visibility of rxn
ALLO- ANTIBODIES- produced after exposure to genetically different Ag of the same species AUTO-ANTIBODIES- produced in response to self antigens
INDICATORS OF Ag-Ab RXN 1. Sensitization-
KULANG. INSERT
WRITTEN NOTES.
AND OTHER BLOOD
GROUPS INFO ASIDE
FROM ABO.
RED BLOOD CELL SUBSTITUES Why are there Red blood cell substitutes? 1. Rare blood types
2 Multiple transfusions
Hemoglobin-based oxygen carriers a.) Acellular
b.) Cellular Perfluorocarbon
Hemoglobin-based oxygen carriers 1. HbOC (Stroma free Hb solution)
2. HbOC (Chemically modified Hb Solution) Polymerized Hb
Surfaced modified Hb 3. HbOC (Recombinant Hb) 4. HbOC (Encapsulated Hb)
Perfluorocarbons- all hydrogen atoms are replaced with fluorine
Platelet Preservation
Platelet is vital in primary hemostasis a. cytoplasmic fragment b. has no nucleus
c. circulating lifespan of 9-12 days d. Normal values: 150-350 x 10^9/L e. biggest mature blood cell
CLINICAL USE: Treat Bleeding, As a prophylaxis
PREPARATION: Whole blood transfusion Plate rich plasma Platelet concentration Platelet-rich plasma method Buffy coat method
Why does it have to be kept in room temperature until platelets are harvested?
Storage preservation: 20-24C with constant agitation Kept for 5 days
BLOOD REPLACEMENT FLUIDS Intravenous Fluid
A. Maintenance fluid:
unavailable oral route
Replace normal physiological losses Volume dispersion
All are crystalloid solutions
Example: 5% dextrose, 4% dextrose, 0.18%
B. Replacement fluid: abnormal losses
treatment of patient with ongoing fluid loss treatment of patient with hypovolemia Example: NSS, Hartmann’s solution LECTINS and PROLECTINS
Lectins: specific antibodies derived from plants Prolectins: specific antibodies derived from snails Lectin
Source:
Anti-A Dolichos biflorus
Anti-B Bandeiraea simplicifolia
Fomes fomentarium
Anti-H Ulex auropaeus
Prolectins
Source
Anti-A Helix pomatia
Helix aspersa
Anti-A1 Euphrada periomphala
Bradybaena fructicum
Anti-B Salmo irideus
Anti-H Anguilla anguilla
Genetics: Mendel
Law of Segregation
How genes are passed from one generation to another Incomplete dominance
Law of independent assortment
Describes the independent segregation of alleles of different loci
Genes of different trait are inherited separately from each other Inheritance patterns: Pedigree analysis Autosomal recessive Autosomal dominance X-linked recessive X-linked dominant Cellular genetics
Mitosis: divide to create identical daughter cells Meiosis: produce gametes, 4 unique daughter cells
Cell Cylce: 4 distinct stages
If cells no longer need to divide they remain in G( 0 ) stage
Not true to cancer cells Molecular Genetics: Gene: Genome: Chromosome: Chromatid: Chromatin: Nucleosomes: Allele: Locus: DNA (CGAT) Replication Repair Mutations RNA (CGAU) Transcription Translation DNA Backbone of heredity Double Helix H-bonds, phosphodiester Nitrogenous bases Histone; Nucleosome DNA Replication DNA gyrase DNA helix RNA primase DNA polymerase III Sliding clamp
Single stranded binding Proteins DNA polymerase I DNA ligase DNA REPLICATION Replication fork Leading strand Lagging strand Okazaki fragment DNA DAMAGE Cellular damage UV light exposure Ionizing radiation Chemical exposure Replication errors DNA REPAIR
Cell cycle checkpoint activation Transcriptional program activation DNA repair system
Direct reversal Base Excision Repair Nucleotide excision repair Mismatch repair
Double strand break repair Homologous recombination Apoptosis
DNA mutation
Any change in the structure or sequence of DNA may it be physical or biochemical
Mutant vs. Wild type Types of mutation Base Substitution Deletion Insertion RNA Single stranded Ribose sugar
Uracil instead of thymine Types: rRNA, mRNA, tRNA
Used to transmit genetic information RNA transcription (RNA Polymerase) RNA processing
IMMUNOLOGY (Ag- Ab REACTION) What lab tests require Ag-Ab reaction?
ABO grouping, Rh typing, screening and identifying, crossmatching
Why do we need to know how Ag-Ab reactions occur? Quality control, trouble shooting, mainteanance
Immune response: Physiological mechanism to fight disease or clearing of foreign substances
A. Primary Response
First exposure of a foreign agent
IgM is the predominant antibody B. Secondary Response (amenestic)
Faster response
Subsequent exposure of the same foreign agent
IgG
Ag Characteristics
Antigen- something foreign
Antigenicity- ability to react to antibodies
Epitopes- antigenic determinants
IgM- Most common, found in blood, pentamer, cold (4-10), cannot cross placenta, good complement activators, “usually” not clinically significant. ABO, Ii, Lewis, MN and P
IgG- Monomer, works best at 37 degrees, can cross placenta, poor to good complement activators, usually clinically significant. Rh, Kell, Kidd, Duffy and Ss
Naturally occurring
Found in the serum of individuals who have been exposed to antigens through transfusion, injection or pregnancy
May be produced in response to substances in environment
Most are IgM cold agglutinins, activates complements and hemolytic at 37
Immune Antibody
Found in the serum of individuals who have been transfused or pregnant
IgG
Not found naturally
Requires the aid of Anti-Human globulin Note:
Clinically significant IgGs can cause Hemolytic disease of new born or Transfusion reactions
IgM is not clinically significant because they only react in cold temperature
Cold reacting Antibody with wide thermal reactivity Allo-antibody and auto-antibodies
Allo-antibodies are produces after exposure to genetically different non-self, antigens of the same species.
Auto-antibodies are produces in response to self-antigens. Indicators of Antigen and Antibody reactions
1. Sensitization
Ab coating RBC without agglutination
Requires AMG for visualization 2. RBC lysis (hemolysis)
Immune mediated complementary activation completion
3. Agglutination
Antibody mediated clumping of Red blood cell AHG - Used to visualize reaction in DAT and IAT
- AHG can be monoclonal or polyclonal Agglutination Reaction
1. Sensitization (Stage 1)
Attachment of Ag and Antibody of red blood membrane
Electrostatic forces, H bonds, Hydrophilic forces and Van Der Waals forces
2. Lattice Formation (Stage 2)
Formation of lattice between sensitized red cells Factors affecting Stage 1 of Agglutination
A. Temperature
Cold (4-10C)- ABO, P1, Lewis Ab
- Carbohydrate natures of ABO Ag Warm (37C)- Rh antibodies, Protein nature of Rh Ag B. pH- Changes in pH can affect electrostatic bonds
- Optimal range in physiological pH (6.50-7.5) C. Incubation time- Time needed to reach equilibrium
- Saline systems: 30-60 minutes at 37C - LISS (Low Ionic Strength Solution) D. Ionic Strength
NSS- Na and Cl ions partially opposite charges on Ag and Ab molecule
LSS- Increase rate of Ag-Ab association - Decreases incubation time
- Concentration of Ag and Ab affects with the first as severed stages.
Factors affecting stages of Agglutination A. Size of Ig
IgG: monomer, takes 2 to activate complement IgM: pentamer, takes one to activate complement B. Number of binding sites of Ig
IgG: 2 binding sites (Anti-D, Anti-JKa, etc.) IgM: 10 binding sites (Anti-A, and B) C. Location and number of Antigenic deteminants
A,B,M and N antigens: 600,000-1,000,000 Ag/RBC Kidd: 10-20,000 Ag/RBC
D. Centrifugation
Bringing Ab and Ag into close proximity
Under centrifugation: false (-)
Over centrifugation: false (+)
Zeta potential: net negative charge surrounding RBC Water of Hydration: acts as an insulating bubble around RBC RBC DISTANCE:
In the body RBCs should be distant
Water of hydration
Electrostatic charges
In the lab or in vitro, we need to overcome these forces to observe Ag and Ab reactions
REACTION MEDIA: 22% Albumin
Decreases zeta potential by buffering
Allows Ab- coated cells to come closer-together Polyethanol glycol
ABO BLOOD GROUP
History: Karl Landsteiner- first to perform forward and backward typing
Genes:
ABO gene at C9 and H and SE genes at C19 Single most important blood group for the selection
and transfusion of blood
Widely expressed-> tissues and blood fluids Inheritance of ABO blood group system
Example of determining offspring blood types from known or suspected genotypes
A O
A AA AO
B AB BO
Blood Group Frequency
Caucasian Asian O 45% 47% A 41% 26% B 10% 23% AB 4% 4% Made up of:
Antigens expressed in the Red cells Absence of its corresponding Ab in serum Four genes: A,B,H,Se
Three Antigens: A,B,H
Two major antibodies: anti A and anti B, Anti H Four Phenotypes: A, B,AB, O
ABO antibodies
Initiated at birth but very low titers Complete absence is very rare
Antibodies in “A” and “B” groups -> IgM Antibodies in “O” group -> IgG
React at 20-24C
Activates complement at 37C Anti H:
Naturally occurring autoantibodies IgM in form; low thermal range A1 and A1B individuals Seldom clinically significant
Occurs as allo-antibodies (IgG and IgM) among O(subscript) h -> Bombay
ABO antigen
Begins at the 37th day of fetal life (2nd month accdg to Henry)
Final strength at age (2-4) (1 y.o accdg to Henry) Persistent throughout life
Found in saliva, pancreatic secretions, gastric secretions (secretor)
May be found in bacteria and other species Formation of AB and H antigens
Produced due to the interaction A,B,H,Se, genes Genes code for glycosyltransferases with attached
sugar to precursor material (paragloboside) Genes do not produce Ag genes, genes produce
enzymes to express the antigens Paragloboside
4 Sugars (2 D-galactose, 1 glucose, 1 N-acetylglucosamine) 2 types
Type 1: precursor in the secretion (1-3 linkage) Type 2: precursor in Red blood cell (1-4 linkage)
Three precursor substance to which the L-fucose is attached to produce an H antigen
Formation of Antigens in Red Blood Cells H antigen
Possible genotypes HH, Hh, or hh
HH/Hh (+) -> alpha-2-L-fucosyl transferase-> responsible for the attachment of L-fucose to paragloboside
hh-> amorph gene (does not produce enzyme) group has RBC rich in H ag
O>A2>B>A2B>A1>A1B H Antigen
The immunodominant sugar is L-fucose Linear (2) H1 and H2
Branched (2) H3 and H4 Formation of Antigen in Red blood cells ABO Antigen
Inherited in simple Mendelian fashion from an individual’s parents
3 possible genes can be inherited= A, B,O A and B genes-> detectable product/enzyme genes-> no product
Present on the surface of RBCs, tissues and endothelial cells
A antigen- the immunodominant sugar is N-acetyl-D-galactosamine
B antigen- the immunodominant sugar is D-galactosamine
Gene Glycosyltransferase Immunodominant sugar
Antigen
H L-fucosyl transferase L-fucose H
A N-acetyl galactosaminyl transferase N-acetyl-D-galacotasamine A B D-galactosyl D-galactose B
OTHER BLOOD GROUPS
Blood group Chr High Freq Ag HF Phenotype Ag Characteristics Ab Characteristics Antigen Location, Linkage, Disease Diego 1955 a Ab 1967 b Ab 1995 Wr 17 Dib, Wrb Di (a-b+) Wr (a-b+)
Dia and Dib- polymorphic Anti Dib cause HDN and HTR IAT Reactive
Do not bind comp
AE-1
Hereditary spherocytosis Congenital acanthocytosis Cartwright 1956 7 Yta Yt (a+b-) Yta= adult, strong Ag, neg cord bld
Ytb well developed at birth, poor Ag
Ytb – IgG, IAT reactive Ag- located on erythrocyte Acethyl Cholinesterase (AChE) Xg 1962 X Xga Xg (+) Homology with CD99 in RC Bind C variably
IgG sensitive to: B, Ficin Not affected with DDT
Cell to cell adhesion events
Scianna 1962 1 Sc1 Sc3 Sc (1+2+) Resistant: P, F, T, chy T Sensitive to DDT
Expressed on RBCs of newborns
Sc1 Sc2 IgG
IAT reactive and bind C Sc1 no HDN Sc2 mild HDN Sc3 linked to HTR Dombrock 1965 Gregory G4 Holley Hy Joseph Jo
12 Doa Dob Do (a+b+) Resistant: Ficin Papain Weakend chyT Sensitive: T and DTT
Doa and Dob- poor immunogens Gya immunogenic
React best as AHG Doa- mild HDN
Combine with other Abs
IAT with PEG acute delayed HTR
Carried on mono ADP ribosyl transferase (ART4)
Colton 1967 7 Coa Co (a+b-) Resistant: Protease, sialidase, DTT and acid
IgG, IAT reactive, bind C HTR and no HDN Erythrocyte band 41
Co (a-b-) associated w/ dyserythropoietic anemia
Located on transport CHON CHIP (Channel Forming Integral Protein) Primary RBC water channel and responsible for H2Opermeability Chido 1967
Roders 1976
6 Ch1 Ch2 Rg1 Rg2
Ch (1+2+) Poorly expressed on cord Absorbed into RC from plasma Affected by Ficin and Papain
HTLA
Weakly react at IAT (IgG4)
Rg linked to C4a Ch linked to C4b
Null Ch due to C4 deletion
Psoriasis, Grave’s disease, SLE, RA Gerbich 1960 2 Ge 2 3 4 Ge (2+ 3+ 4+) Ge2 sensitive: F P T
Ge3 sensitive: F P Resistant: DTT and chyT
IgG, bind C Red cell stimulated
Casue delayed HTR and no HDN
Leach – null Ge
Cromer 1965 1 Cra Cr (a+b-) Resistant: F P Weakend: DTT Sensitive: chyT
Poorly expressed on cord cells Decreased during pregnancy
IgG, IAT reactive Red cell stimulated No HDN
Carried by DAF (CD55)
Dra, E. coli receptor (uropathogenic)
Knops McCoy York 1991
1 Kna Kn (a+b-) Poorly expressed on cord cells Decreased in patient with AIDS Weakened by F and P
Sensitive to T and chyT Resistant to DTT
IgG, IAT reactive
Decreased activity with T and chyT No HDN, HTR
Reside with CR1 (CD35) P. falciparum Decreased Kn Ag’s in Lu (a-b-) w/ in Lu genes
Indian 1973 11 Inb In a(-b+) Poorly expressed on cord Decreased during pregnancy Decreased in Lu (a-b-) with In Lu
IgG, IAT
Red cell stimulated
Inb associated w/ HTR and no HDN
Carried by hematopoietic form CD44
Ok 1979 19 Oka Ok (a+) R: Protease, sialidase, DTT Well developed at birth
IgG, IAT reactive CD 147- hematopoietic- BB barrier CD147 in human on tumor cell- stimulated release collagenase and metalloproteinases enhance tumor cell invasion and metastasis
Raph 1990 11 RAPH Resist: F P Sialidase
Sensitive: T chyT and DTT RBC of newborns
M- monoclonal ER- Eleanor Rosevelt
IgG, IAT reactive, Bind C Expressed in fibroblasts
MER2, decreased in Lu (a-b-) w/ in Lu If decreased MER2= renal failure
Bennet Godspeed 6 Bga Bg (a+b-) Associated: HLA B7 –Bg a HLA B17-Bgb HLA A28- Bgc
Destroyed by chloroquine, glycine- HCl EDTA solution
No HTR and HDN
Associated w/ IM, Leukemia, PCV and HA
LW 1990 19 LWa LW (a+b+) Resist: F P T chyT Sialidase Sensitive: Pronase and DTT
IgM-IgG
Occur in dTR and mHDN
Loss of LW- production of anti LW associated w/ leukemia, lymphoma, sarcoma, Hodgkin’s dse
ICAM binds to CD11/ CD18 leukocyte integrins