Immunohematology Lecture Notes Medtech PDF

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BLOOD BANKING NOTESBy: Mr. Joseph Joy G. Banzon, RMT, MPHI. HISTORY OF BLOOD BANKING1492 - Pope Innocent VII died because of blood transfusion1869 - Braxton Hicks recommended the use of sodium phosphate as anticoagulant 1901 - Karl Landsteiner discovered Blood Groups A, B and OFATHER OF IMH1902 - Vo...

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OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH I. HISTORY OF BLOOD BANKING 1492- Pope Innocent VII died because of blood transfusion

1869- Braxton Hicks recommended the use of sodium phosphate as anticoagulant 1901- Karl Landsteiner discovered Blood Groups A, B and O FATHER OF IMH 1902- Von Decastello and Sturli discovered Blood Group AB

1914- Hustin reported the use of sodium citrate as anticoagulant 1915- Lewisohn determined the minimal amount of citrate that can provide anticoagulation of blood 1916- Rous and Turner introduced citrate-dextrose solution for preservation of blood 1943- Loutit and Mollison introduced the formula for Acid Citrate Dextrose 1957- Gibson introduced Citrate Phosphate Dextrose as anticoagulant-preservative solution 1941- Dr. Charles Drew was appointed as the first director of the American Red Cross Blood Bank at Presbyterian Hospital FATHER OF AMERICAN CROSS AURORA ARAGON-QUEZON----- PHILIPPINE NATIONAL RED CROSS

ANTICOAGULANT-PRESERVATIVE SOLUTIONS ACD CPD CP2D CPD-A1

SHELF LIFE 21 DAYS 21 DAYS 21 DAYS 35 DAYS

Citrate: ANTICOAGULANT Phosphate: BUFFER (MAINTAIN PH) Dextrose (d-glucose): SOURCE OF ENERGY Adenine: ADDITIONAL SUBSTRATE FOR ATP PRODUCTION GLYCOLYSIS1 MOLECULE GLUCOSE ==== 7 MOLECULES OF ATP PYRUVIC ACID ==➔ LACTIC ACID NORMAL PH OF BLOOD: 7.4

ADDITIVE SOLUTIONS AS-1 (ADSOL) AS-3 (NUTRICELL) AS-5 (OPTISOL)

SHELF LIFE 42 DAYS 42 DAYS 42 DAYS

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OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH REJUVENATION SOLUTIONS: USE: TO RESTORE LEVELS OF 2,3-DPG/BPG CONTENTS: PHOSPHATE, INOSINE, PYRUVATE AND ADENINE REJUVESOL STORAGE LESION: BIOCHEMICAL CHANGES THAT OCCURS ON THE BLOOD DURING STORAGE BIOCHEMICAL CHANGES DURING STORAGE LESION: 1. Increased: ACIDS, POTASSIUM, HEMOGLOBIN 2. Decreased: PH, SODIUM, 2,3-DPG, ATP AND GLUCOSE

2,3-DPG= PROMOTE AFFINITY OF HEMOGLOBIN TO TISSUES

II. Review of Important Immunologic Principles as applied to Blood Banking IMMUNOLOGY-

IMMUNE SYSTEM ------ RECOGNITION AND DISPOSAL OF FOREIGN SUBSTANCES/ANTIGENS

IMMUNOHEMATOLOGY- APPLICATION OF CONCEPTS OF IMMUNOLOGY TO THE STUDY OF BLOOD GROUP SPECIFIC ANTIGENS AND ANTIBODIES BLOOD BANKING- COLLECTION, PRESERVATION, PROCESSING, STORAGE AND DISPOSAL OF DONOR’S BLOOD WITH STRICT ADHERENCE TO QUALITY CONTROL AND QUALITY ASSURANCE DONOR SCREENING AND SELECTION, COMPONENT PREPARATION

ANTIGENS: SUBSTANCES THAT BIND WITH ANTIBODY. ; SUBSTANCE THAT BIND WITH A T-CELL/LYMPHOCYTE Also called as: IMMUNOGENS- STIMULATE ANTIBODY PRODUCTION NOTE: ALL IMMUNOGENS ARE ANTIGENS BUT NOT ALL ANTIGENS ARE IMMUNOGENS.

IMMUNOGENIC-- SUBSTANCE THAT IS CAPABLE OF INDUCING ANTIBODY PRODUCTION FACTORS AFFECTING IMMUNOGENECITY: 1. Foreigness FOREIGN ANTIGEN SELF-ANTIGENS: RBC ANTIGENS, HLA:

AUTOIMMUNE DISEASE

2. Chemical Complexity THE MORE COMPLEX A SUBSTANCE IS, THE MORE IMMUNOGENIC PROTEINS, CARBOHYDRATES, GLYCOPROTEIN, GLYCOLIPIDS LIPIDS AND NUCLEIC ACIDS--- POOR IMMUNOGENS

3. High Molecular Weight THE BIGGGER THE SUBSTANCE IS, THE MORE IMMUNOGENIC

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OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH

>10,000 DALTONS 4. Digestability T-CELL ========= APC AND MHC 5. Dosage and Route of Administration INTRAVENOUS ROUTE 6. Accesibility of Reactive Sites RBC Antigens/ BLOOD GROUP SPECIFIC ANTIGENS: ANTIGENS THAT DICTATES OUR BLOOD TYPE.

NOTE: As of latest editions of Blood Banking Books, there are >250 RBC antigens that are existing and are group into 36 Blood Group Systems RBC Antigens are detected through: DIRECT HEMAGGLUTINATION REACTION 3 Methods:

1. Slide Method- POSITIVE OR NEGATIVE RESULTS (NO GRADING) 2. Tube Method GRADING AGGLUTINATION REACTIONS GRADE 0 (NEGATIVE) +/- (TRACE, WEAKLY +, MF) 1+ 2+ 3+ 4+

CLUMPS NO CLUMPS VERY TINY CLUMPS

SUPERNATANT/BACKGROUND TURBID, RED TURBID, RED

SMALL-SIZED MEDIUM-SIZED SEVERAL LARGE CLUMPS ONE LARGE CLUMP

TURBID, RED CLEAR CLEAR CLEAR

3. Gel Method: ADVANTAGES 1. THERE IS OBJECTIVITY IN GRADING REACTIONS 2. SMALL AMOUNT OF SAMPLE IS USED 3. RESULTS ARE STABLE UP TO 3 DAYS DISADVANTAGE: COSTLY Discovered by: DR. YVES LAPIERRE in LYON, FRANCE.

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OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH

*SOLID PHASE RED CELL ADHERENCE (SPRCA): discovered by Plapp and cowokers in 1984. Human Leukocyte Antigens (HLA): encoded by MHC genes located on chromosome 6. HLA are present on: ALL NUCLEATED CELLS (EXCEPT MATURE RBC) WHOLE BLOOD====== RBC, WBC, PLATELETS (FRAGMENTS OF MEGAKARYOCYTE) Platelet refractoriness: unresponsiveness to platelet transfusion because of thE ANTI-HLA that binds and destroys HLA on platelets’surface.

ANTIBODIES: GLYCOPROTEINS THAT ARE PRODUCED IN RESPONSE TO ANTIGENIC STIMULATION Also known as: IMMUNOGLOBULINS === PART OF GAMMA GLOBULIN FRACTION OF SERUM PROTEINS Basic functional and structural unit is: MONOMER (Y-SHAPED MOLECULES) Classes: IgG, IgA, IgM, IgD, IgE ARRANGED ACCDG TO DECREASING CONCENTRATION IN THE SERUM Antibody classes most commonly encountered in the Blood Bank are: IgM, IgG and IgA---- causing ANAPHYLACTIC TRANSFUSION REACTIONS Fragments: Fab, Fc (crystallizable) Regions: Variable , Constant Fab, variable----- antigen binding Fc, constant------- binding to host cells (phagocytes and complement components)

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OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH POINT OF COMPARISON 1. Heavy chain 2. Light chain 3. J-chain (CONNECTS MONOMERIC UNITS OF A POLYMER) 4. Molecular Weight 5. Valence (NUMBER OF BINDING SITES) 6. % in Serum 7. Serum half-life 8. Crosses the placenta 9. Activates the classical pathway of complement 10. Clearance of red cell 11. Detection in blood bank tests

IgM (PENTAMER) STAR OR CRAB-SHAPED

IgG (MONOMER)

mu Kappa or lambda YES

Gamma Kappa or lambda NO

900,000 DALTONS 10 6-10% 5-10 DAYS NO YES (VERY EFFICIENT)

150,000 DALTONS 2 75-80% 23-25 DAYS YES (CAUSE HDN) YES (IgG3, IgG1, IgG2)

inrtravascular IMMEDIATE SPIN (IS) OR RT PHASE

extravascular AHG (ANTI-HUMAN GLOBULIN) PHASE/ COOMB’S

IgG4

COMPLEMENT SYSTEM== COMPLETE AND COMPLEMENT THE ACTION OF ANTIBODIES -PRINCIPAL SOLUBLE MEDIATOR OF INFLAMMATION ANTIBODY-ANTIGEN COMPLEX (IMMUNE COMPLEX)--- ACTIVATE THE COMPLEMENT SYSTEM END RESULT OF COMPLEMENT ACTIVATION: LYSIS OF THE CELL Functions 1. Enhance phagocytosis 2. Enhance antibody function C3d: degradation product of C3b 3. Clears immune complexes in blood C4d: degradation product of C4b 4. Kill microbes by cell lysis 5. Cause the increase in vascular permeability 6. enhances platelet aggregation 7. enhances smooth muscle contraction 8. aid in viral neutralization Opsonin: C3b Chemotaxin: C5a CHEMOTAXIS--- MIGRATION OF PHAGOCYTES TOWARD THE SITE OF INFLAMMATION/INFECTION Anaphylatoxins: C3a, C4a, C5a --mediators of INFLAMMATION Membrane Attack Complex: C5b6789 NOTE: Aside from agglutination, HEMOLYSIS is also regarded as a POSITIVE RESULT in Blood Banking Tests. This is due to the fact that some antigen-antibody reactions proceeds to complement activation. INTERPRETING HEMOLYTIC REACTIONS IN BLOOD BANK COMPLETE HEMOLYSIS: RED SUPERNATANT NO CELL BUTTON PARTIAL HEMOLYSIS: PINK TO RED SUPERNATANT WITH CELL BUTTON NO HEMOLYSIS: CLEAR SUPERNATANT WITH CELL BUTTON ANTIGEN-ANTIBODY REACTIONS All antigen-antibody reactions are governed by the Law of MASS ACTION which states that Ag-AB reactions are reversible. AFFINITY: INITIAL FORCE OF ATTRACTION BETWEEN A SINGLE BINDING SITE ON THE ANTIBODY AND ANTIGEN AVIDITY: SUM TOTAL OF ALL FORCES OF ATTRACTION BETWEEN MULTIVALENT ANTIGEN AND MULTIVALENT ANTIBODY SPECIFICITY: ANTIBODY WILL ONLY REACT AGAINST ANTIGEN THAT HAS CAUSED ITS PRODUCTION

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OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH

IMPORTANT NOTE: Antigen-Antibody Reactions are SPECIFIC. LOCK AND KEY CONCEPT

A ANTIGEN ======= ANTI-A B ANTIGEN ======= ANTI-B Intermolecular Forces that binds Ag and Ab together: 1. Ionic Bonds- EXIST BETWEEN OPPOSITELY CHARGED PARTICLES ANTIGEN=== NEGATIVELY CHARGED ANTIBODY==== POSITIVELY CHARGED

2. Hydrogen Bonds—BETWEEN 2 POLAR SUBSTANCES 3. Hydrophobic Bonds—BETWEEN 2 NON-POLAR SUBSTANCES 4. Van der Waals Forces—BETWEEN ELECTRON CLOUDS OF ANTIGENS AND ANTIBODIES AGGLUTINATION 2 Steps in Agglutination: 1. Sensitization: INITIAL BINDING OF ANTIBODY TO RED CELL ANTIGENS; DEPOSITION OF COMPLEMENT ON RED CELL SURFACE 2. Lattice Formation: NETWORKS OF IMMUNE COMPLEXES OR SENSITIZED RBCs

FACTORS AFFECTING SENSITIZATION: 1. Serum to Cell Ratio: ratio of ANTIBODIES TO RED CELL ANTIGENS Ideal Serum to Cell Ratio in Blood Banking Tests: 40:1 3 DROPS ANTIBODY SOURCE 1 DROP ANTIGEN SOURCE

133:1

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OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH

NOTE: Increasing the concentration of antibody will increase the probability of collision events with the corresponding red cell antigen. 2. Incubation time 3. Incubation temperature IgM: RT IgG: 37 degress celsius 4. pH: Ideal is 6.7-7.2 (7.0) 5. Ionic strength of the medium Decrease the ionic strength of the medium to favor antibody uptake by RBCs --- use LISS (low ionic strength saline) FACTORS AFFECTING LATTICE FORMATION: 1. Zeta Potential: force of repulsion among RBCs when placed in NSS solution RBC== negatively charged ===== SIALIC ACID

Enhancement/Reaction Media: a. 22% Bovine Serum Albumin: NEUTRALIZE THE NEGATIVE CHARGES ON THE RBC MEMBRANE (30 MINUTES) b. Low Ionic Strength Saline (LISS): DECREASING THE INCUBATION TIME (10-15 MINUTES) c. Polyethylene glycol (PEG): CONCENTRATE ANTIBODY BY REMOVING WATER d. Enzymes (Papain, Ficin, Bromelin): ENHANCE OR DESTROY CERTAIN RBC ANTIGENS 2. Optimum concentration of antibody and antigen

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OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH

NOTE: Lattice formation only occurs at EQUIVALENCE zone Doing the blood bank test at PROZONE (excess ANTIBODY) and POSTZONE (excess ANTIGEN) will lead to false NEGATIVE results. That is why the most commonly used sample in Blood Bank test is: 2-5% RCS (RED CELL SUSPENSION)--TOMATO RED/CHERRY RED % Red Cell Suspension= amount of washed RBC x 100 Total volume Total Volume= amount of washed RBC + amount of NSS 3. Effect of centrifugation III. Review of Important Genetic Principles as applied to Blood Banking GENETICS: study of HEREDITY; INHERITANCE OF TRAITS FROM PARENTS TO OFFSPRING. BLOOD GROUP SYSTEMS- group of related red cell antigens which is similar in terms of biochemical structure, location in RBC, serologic properties and genetic control of antigen expression .

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OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH

001001---- A ANTIGEN 001002---- B ANTIGEN GENES: BASIC UNIT OF INHERITANCE CHROMOSOMES: STRUCTURE WITHIN THE NUCLEUS THAT CONTAINS DNA.

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OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH 2 Types: 1. Somatic/Autosomes: 22 PAIRS === 44 2. Sex Chromosome: 1 PAIR ==== 2 _________ 46

XX----- FEMALE XY----- MALE ??---------------SIR JOY

GENOTYPE: GENETIC MAKE-UP OF AN INDIVIDUAL; ACTUAL GENES INHERITED FROM EACH PARENT. -ALWAYS IN PAIR

PHENOTYPE: PHYSICAL OR OBSERVABLE EXPRESSION OF THE GENOTYPE/GENES. PUNETT SQUARE: square used to calculate the frequency of the resulting genotypes and phenotypes among the offspring of a cross.

ABO BLOOD GROUP/RED CELL PHENOTYPE A1 A2 B A1B A2B O A1 GENE----→

POSSIBLE GENOTYPES A1A1, A1O, A1A2 A2A2, A2O BB,BO A1B A2B OO

A1 ANTIGEN

B GENE ---→ B ANTIGEN A GENE, B GENE ----→ DOMINANT GENE O GENE----→ RECESSIVE GENE A1 GENE >

A2 GENE

Example:

FATHER: AB MOTHER: A

(AB) (AA, AO)

CHILD: O

A

A AA

B AB

0

A0

BO

0% 1. Probability that child’s genotype is A1A1: 50% 2. Probability that child’s phenotype is A1: 50% 3. Probability that child’s blood type is A1B: 50%

ALLELES: ONE OF TWO OR MORE GENES IN A GENOTYPE THAT EXPRESSES A TRAIT 10

OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH ANTITHETICAL: A TERM USED TO REFER TO THE OPPOSITE ALLELE POLYMORPHIC GENE: GENETIC SYSTEM THAT PRODUCES MULTIPLE PRODUCTS EX. MHC GENES IN CHROMOSOME NO. 6 DOMINANT GENE: GENE WHOSE PRODUCT IS ALWAYS EXPRESSED. RECESSIVE GENE: PRODUCTS OF RECESSIVE GENE ARE ONLY EXPRESSED WHEN INHERITED HOMOZYGOUSLY. CODOMINANT GENES: WHEN BOTH GENES COMPRISING A GENOTYPE ARE DOMINANT. HOMOZYGOUS GENES: WHEN GENES COMPRISING A GENOTYPE ARE THE SAME. HETEROZYGOUS GENES: WHEN GENES COMPRISING A GENOTYPE ARE DIFFERENT. DOSAGE EFFECT: stronger agglutination are produced by antigens that are expressed by homozygous genes ; weaker reactions are produced by antigens expressed by heterozygous genes. MNSs BLOOD GROUP SYSTEM, KIDD BGS, DUFFY BGS, RH BGS M GENE---→ M ANTIGEN N GENE--→ N ANTIGEN

GENOTYPE

ANTIGEN/S

ANTI-M ANTISERA

MM

M ANTIGEN

4+

MN

M ANTIGEN AND N ANTIGEN

2+

AA

A ANTIGEN

ANTI-A ANTISERA 4+

AO

A ANTIGEN

4+

CIS: TERM USED WHEN 2 GENES ARE ON THE SAME SIDE OF THE CHROMOSOME TRANS: TERM USED WHEN 2 GENES ARE ON DIFFERENT SIDE OF THE CHROMOSOME

LINKED GENES: GENES THAT ARE VERY CLOSE TO EACH OTHER HAPLOTYPE: LINKED SET OF GENES THAT ARE INHERITED TOGETHER

INHERITANCE PATTERNS: 1. Autosomal Dominant: TRAIT WILL ALWAYS APPEAR WHENEVER THE ALLELE IS PRESENT 2. Autosomal Recessive: TRAIT WILL ONLY APPEAR IF THE ALLELES ARE INHERITED HOMOZYGOUSLY 3. Sex-linked Dominant: FATHER TO DAUGHTER TRANSMISSION OF TRAITS. EX: Xg BGS Xga ---------girls ________________________________________________________________________________________________ 4. Sex-linked Recessive: MOTHER TO SON TRANSMISSION OF TRAITS Carrier: girls EXAMPLE: HEMOPHILIA A -----→ BOYS 11

OUR LADY OF FATIMA UNIVERSITY BLOOD BANKING NOTES By: Mr. Joseph Joy G. Banzon, RMT, MPH

5. Codominant: EQUAL EXPRESSION OF INHERITED ALLELES 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: GENES WITH NO EXPRESSED PRODUCT. 2. Regulator/Suppressor gene: GENES THAT INHIBIT THE EXPRESSION OF ANOTHER GENE. BLOOD GROUP SYSTEM H RH KELL LUTHERAN DUFFY KIDD

SILENT GENE hh rr Ko Ko LuLu Fy Fy JkJK

REGULATOR GENE XorXor In (Lu) In (Jk)

NULL PHENOTYPE Bombay phenotype Rh null Kell null Lu(a-b-) Fy(a-b-) Jk(a-b-)

H gene----→ H antigen ‘h gene ---→ NO PRODUCT KIDD BGS: JK GENE----→ NO PRODUCT JKA GENE---→ JKA ANTIGEN JKB GENE-→ JKB ANTIGEN

JKA JKB ---→ JK(A+B+) JKJK ----→ JK(A-B-)

JKAJKB-----→ → JK(A-B-) In(Jk)

IV. 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

Landsteiner Law/Rule: Rule stating that normal, healthy individuals possess ABO antibodies to the ABO blood group antigens absent from their red cells. A ANTIGEN ====== ANTI-B B ANTIGEN ======= ANTI-A

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