BLOOD BANKING (IMMUNOHEMATOLOGY) NOTES

HISTORICAL ASPECTS

Pope Innocent VII – (1492) first time a blood transfusion was duly recorded in history Braxton Hicks – (1869) recommended sodium phosphate as a nontoxic anticoagulant Karl Landsteiner – (1901) discovered the ABO blood group Edward E. Lindemann – vein-to-vein transfusion of blood using multiple syringes and a special cannula for

puncturing the vein through the skin Unger – developed a syringe-valve apparatus Hustin – sodium citrate Lewisohn – determined the minimum amount of citrate needed for anticoagulation and determined its

nontoxicity in small amounts Rous and Turner – (1916) citrate-dextrose solution Charles Drew – establishment of blood bank system. Appointed director of the American Red Cross Blood Bank

at the Presbyterian Hospital Loutit and Mollison – acid-citrate-dextrose Gibson – citrate-phosphate-dextrose Lovric (Australia) – additive solutions composed of saline, adenine, glucose, trisodium citrate, citric acid, and

sodium phosphate in a standard CP2D Hogman (Sweden) – additive solutions composed of SAG (saline, adenine and glucose) in a standard CPD

anticoagulant. Modified further as SAGM (mannitol – protects against spontaneous storage hemolysis) Valeri and co-workers – rejuvenation solutions

Blood Banking Genetics

Basic Concepts:a) Genetics – study of inheritance. Transmission of characteristics from parent to offspring.b) Heterochromatin – dark staining bands in the nucleus under a light microscopec) Euchromatin – lighter staining bands found in the nucleusd) Histones – proteins wound around the DNA moleculee) Chromatin – complex of DNA and proteinsf) Mitosis – cell division that produces two daughter cells that are identical to the parent cellg) Meiosis – cell division unique to germinal tissues.h) Genotype – an individual’s collection of genesi) Phenotype – observable traits of an individualj) Alleles – alternative forms of the same genek) Homozygous – having identical alleles (TT, tt)l) Heterozygous – different alleles (Tt, tT)m) Recessive – trait carried but not expressed until it is inherited in the “pure”/ homozygous formn) Dominant – trait that can be expressed even if it is not homozygously inheritedo) Autosomal – a trait not carried by the sex chromosomesp) X-linked – trait carried by sex chromosomes

Mendelian Laws:a) Law of Dominance – In a cross of parents that are pure for contrasting traits, only one form of the trait

will appear in the next generationb) Law of Segregation – during formation of gametes, the two alleles responsible for trait separate from

each other. Alleles for a trait are then “recombined” at fertilization, producing the genotype for the traits of the offspring

c) Law of Independent Assortment – Alleles for different traits are distributed to sex cells and offspring independently of one another.

Inheritance Patterns:a) Autosomal Dominant – a dominant gene in autosomal chromosomes can be expressed even if it is

inherited homozygously or heterozygously

b) Autosomal Recessive – two copies of the recessive gene in autosomal chromosomes must be present in order for the trait to be expressed

c) X-linked Dominant – If the father carries the trait, all his sons won’t be able to inherit the gene, however all his daughters will inherit the gene. If the mother carries the gene, transmission is identical to an autosomal inheritance pattern

d) X-linked Recessive – characteristically present in the Father but is never passed on from father to son, instead he passes it on to all his daughters, who then become carriers.

Population Genetics Hardy-Weinberg Principle:

- Basic Premises: (a) the population must be large and mating(b) mutations must not occur(c) there must be no migration, differential fertility, or mortality of the genotype

- Formula : p2 + 2pq + q2 = 1.0

Molecular TechniquesA. Immunological Blotting Techniques

a. Northern Blot – RNAb. Southern Blot – DNAc. Western Blot – specific proteinsd. Eastern Blot – analyze PTM (protein post translational modifications) such as lipids,

carbohydrates, phosphomoeties. It can also be used to analyze enzymesB. PCR (Polymerase Chain Reaction)

- An in vitro method for enzymatic synthesis of specific DNA sequences using two oligonucleotide primers that hybridize to opposite DNA strands and that flank the region of interest.

- Taq (Thermus aquaticus) – thermal stable DNA polymerase obtained from bacteria that live in hot springs

C. Cloning - process of producing similar populations of genetically identical individuals- Cloning Vector – extrachromosomal genetic element that can carry a recombinant DNA

molecule into a host bacterial cell.

Blood Banking Proper

Notable Notes on Specimen Requirement and Specimen Processing: Serum is the specimen of choice for blood bank procedures (because it maintains complement activity) The lytic potential of complement is abolished by heating the serum at 560C for 30 minutes (heat

inactivation) C1 and C2 are destroyed and C4 is damaged by heat inactivation Factor B is inactivated by heating at 500

EDTA inhibit the complement activity (2 mg of EDTA : 1ml of serum) Heparin inhibits the cleavage of C4 Potentiators – aim to reduce the zeta potential of red cells

CLASSIFICATION OF REAGENT

Reagent Action Notes

Protein Media – colloidal diluents that enhance agglutination of IgG

molecules by increasing the dielectric constant

(reduces the zeta potential)

22% BSA (Bovine Serum Albumin)

Causes agglutination by adjusting the zeta potential between red cells

Requires an incubation period usually 15-60 minutes

PEG (polyethylene glycol)

Aggregrates red cells causing a closer proximity of red cells to one another assisting in antibody cross-linking

Macromolecule additive with LISS. Reduces false-positive or nospecific reactions. More effective in detecting weak antibodies

Polybrene Neutralizes the charge Macromolecule that

repulsion of cells can detect ABO incompatibility as well as clinically significant IgG alloantibodies

LISS – decrease the ionic strength of a reaction medium thus reducing

the zeta potential

Low Ionic Strength Solution

Causes a low ionic strength environment causing red cells to take up the antibody more rapidly

Generally contain 0.2% NaCl, incubation period is lessened to 5-15 minutes

Enzymes

Ficin (figs) Reduces red cell surface charge

Enhances: Rh, Kidd, P, Le, IDestroys: Fya, Fyb, M, N, S

Papain (papaya)Trypsin (pig stomach)Bromelin (pineapple)

AHG Antihuman globulin Cross-links sensitized cells

Direct and Indirect AHG

It is important to note that each unit of whole blood collected contains approx. 450 ml of blood (1 pint) and 63 ml of anticoagulant

The total blood volume of most adults is 10-12 pints Donors can replenish the fluid lost from the donation of 1 pint in 24 hours; donor red cells are replenished

within 1-2 months after donation, thus a volunteer donor can donate whole blood every 8 weeks. 4 components can be prepared from 1 unit of blood (PRBC, Platelets, Plasma, other clotting factors) A unit of blood may be stored for 21-42 days (depending on the anticoagulant used) The donation process consists of three steps:

1. Educational Reading Materials – contains information on the risks of infectious diseases transmitted by blood transfusion

2. Donor Health History Questionnaire – designed to ask questions that protect the health of both the donor and the recipient. Identifies donors who may have been exposed to infectious diseases

3. Abbreviated Physical Examination – BP, pulse, temperature readings

Red Cell Biology and PreservationRBC – 120 days life span

3 Areas of the RBC Crucial for normal survival and function:a) RBC membrane – semi-premeable lipid bilayer supported by a protein meshlike cytoskeleton. 52% protein, 40%

lipids, 8% carbohydrates- Integral Proteins – Glycophorin A,B,C and AE channel (band 3)- Peripheral Proteins – Spectrin, Actin (band 5), Ankyrin (band 2.1), Band 4.1 and 4.2, Band 6, Adducin

Review Tip: Memorize the integral proteins since it is easier to remember, so that in any exam you won’t jumble them up with the peripheral proteins.

- Internal membrane layer – amino phospholipids- External membrane layer – glycolipids and choline phospholipids

b) Hemoglobin- 95% dry weight of red cell, has a molecular weight of 68,000 and is capable of allosteric change as it

loads and unloads oxygen- Main/ primary function is gas transport- Haemoglobin synthesis

*92-95% of adult Hgb is HbA (2 alpha and 2 beta chains); 2-3% HbA2 (2 alpha and 2 delta chains); 1-2% HbF (2 alpha and 2 gamma)

- Oxygen Dissociation Curve

Review Tip: mnemonic for shift to the right: CADET, face right (CO2, Acid, 2,3 DPG, Exercise, Temperature). Another tip is to remember that the pH is always the opposite of the other factors (ex. Inc pH, dec CO2, dec 2,3 DPG, dec temp)

*Shift to the Left = Fetal haemoglobin; Shift to the Right = Adult Hemoglobin*Shift to the Left = increased pH; Shift to the Right = decreased pH* The P50 of blood is 26-30 mm Hg

State of Patient SHIFT TO THE LEFT/ SHIFT TO THE RIGHT

Anemia Shift to the RIGHTAcidosis Shift to the RIGHTFever Shift to the RIGHTHypoxia Shift to the RIGHTExercise Shift to the RIGHTAlkalosis Shift to the LEFTAbnormal Hemoglobins Shift to the LEFTMultiple Transfusions Shift to the LEFT

c) RBC Metabolismi. Emden-Meyerhof – anaerobic pathway that produces needed RBC energy. Generates 90% of the ATP

needed by the cellsii. Pentose Phosphate – generates 10% of the ATP needed by cell. The activity of this pathway increases

following increased oxidation of glutathione or decreased activity of the glycolytic pathway. *When the pentose phosphate pathway is functionally deficient, the amount of glutathione becomes insufficient to neutralize intracellular oxidants thus producing Heinz bodies (denatured haemoglobin)

iii. Methemoglobin Reductase – necessary to maintain the heme iron in the ferrous state.*In the absence of NAD, Methemoglobin accumulates causing a loss of oxygen transport capabilities

iv. Luebering-Rapaport – accumulation of 2,3 DPG (affinity for oxygen)

2 Important RBC Characteristicsa. Deformability – phosphorylation of spectrin

Loss of Deformability- loss of ATP leads to a decrease in phosphorylation of spectrin- accumulation or deposition of membrane calcium- removed by the spleen (extravascular sequestration)- spherocytes

b. Permeability – permeable to water and anions (can transverse the membrane in less than a second).

- Impermeable to cations such as Na+ and K+.- Intracellular-to-extracellular ratios for sodium and potassium are 1:12 and 25:1 respectively- Calmodulin has been speculated to control the pumps that control permeability (prevents excessive

intracellular calcium build up)

BLOOD GROUPSA. MAJOR BLOOD GROUPS

I. ABO – most important blood group systemo “naturally occurring antibodies”o IgM antibodies, cold reacting antibodies, does not cross the placenta but very potent!!!o Antibodies are undetectable due to low titers, but start to become detectable at the age

of 3-6 months; antibody production peaks at the age of 5-10 years, then declines as age progresses.

o Cord blood is used to type newborns (forward typing)o Reverse grouping is a unique test for the ABO blood groupo Summary Table for Review Purposes:

BLOOD TYPE IMMUNODOMINANT SUGAR

ANTIBODY FOUND IN SERUM

A N-acetylgalactosamine Anti-BB D-galactose Anti-A

AB N-acetylgalactosamine + D-galactose

None

O L-fucose Anti-A, Anti-B, Anti-A,B

hh (Bombay) NA (unable to produce fucosyltransferase)

Anti-A, Anti-B, Anti-A,B, Anti-H

RED CELL PRESERVATION- Provide viable and functional blood components to patients who require blood transfusion- To consider a transfusion to be successful, 75% of cells transfused should remain viable for 24 hours.- Optimum temperature for storage of blood: 1-60C- “Lesion of Storage” – dec pH, glucose, ATP; loss of red cell function; inc lactic acid

*the rate of restoration of 2,3 DPG is influenced by the acid-base status, phosphorus metabolism and degree of anemia

- Red Cell Preservatives:NAME ABBREVIATION STORAGE TIME pH(initial) NOTESAcid-citrate-dextrose

ACD 21 5.0 The low pH causes most 2,3 DPG to be lost early in the first week of storage

Citrate-phosphate-dextrose

CPD 21 5.6 Red cells become low in 2,3 DPG by the second week

Citrate-phosphate-adenine

CPDA-1 35 5.6 Addition of adenine seems to increase ADP levels.

Citrate-phosphate-double dextrose

CP2D 21 5.6 Contains 100% more glucose than CPD, 60% more glucose than CPDA-1. /

- Additive Solutions:NAME ABBREVIATION STORAGE TIME pH at 370C NOTESAdsol AS-1 42 6.6 Contains buffered

adenine, glucose and mannitol to retard hemolysis. It is coupled with CPD as primary bag anticoagulant.

Nutricel AS-3 42 6.5 Employs CP2D as primary bag anticoagulant and 100ml of buffered adenine glucose solution for RBC

viability.Optisol AS-5 42 6.5 Uses CPD as

primary anticoagulant and contains adenine, glucose and mannitol.

*blood stored in additive solutions is NOT routinely given to newborn infants*Additive solutions allows extraction of increased volumes of fresh plasma *Additive solutions allow storage of whole blood at room temp for up to 8 hours

- Rejuvenation Solutions – solutions containing PIGPA (phosphate, inosine, glucose, pyruvate and adenine) that aim to regenerate both ATP and DPG levels in red cells stored in CPD, CPDA-1, or AS-1

Rejuvesol – only rejuvenating solution that is FDA-approved. Red cells stored in a liquid state for less than 3 days after expiration can be rejuvenated for

1-4 hours at 370C in rejuvesol. Units must be used within 24 hours

- Red Cell Freezing – used for autologous units and storage of rare blood types. It involves the addition of a cryoprotective agent to red cells that are less than 6 days old.*Glycerol is the most commonly used cryoprotective agentAdvantages and disadvantages of Red cell FreezingADVANTAGES DISADVANTAGESLong term storage (10 years) Time consumingMaintenance of red cell viability and function Costly equipmentLow WBC and platelets Storage temp (-65)Removal of significant amounts of plasma proteins

Higher cost of products

Advantages of High glycerol to Low glycerolAdvantage High Glycerol (40% w/v) Low Glycerol (20% w/v)Initial freezing temp -800C -1960CNeed to control freezing rate No YesType of freezer Mechanical Liquid nitrogenMaximum storage temp -650C -1200CShipping requirements Dry ice Liquid nitrogenEffect of changes in storage temp

None. Can be thawed and frozen again

Critical

- Blood Substitutes A. Stroma-Free Hemoglobin Solution (SFHS) – prepared by hemolyzing outdated red blood cells

and removing all of the contaminating stroma which may be toxic to the kidney.B. Chemical Modified Hemoglobin – cross-linking and/or polymerizing the haemoglobin chains

can increase dwell time by enlarging the molecule and inhibiting its break up into smaller sub units which are easily filtered by the kidney.

C. Recombinant Hemoglobin – made using modified human genes expressed in bacterial cellsD. Encapsulated haemoglobin – haemoglobin is enclosed in liposomal vesicles.E. Perfluorochemicals (PFC) – hydrocarbon structures in which all the hydrogen atoms have

been replaced with fluorine.

PLATELET PRESERVATION- Platelet Concentrates – effectively used to treat bleeding associated with thrombovytopenia, as well

as other disorders in which platelets are quantitatively and qualitatively deficient.- Methods for PC Prep:

A. CentrifugationB. Apheresis

- Done within 8 hours after collection, approximately 50ml of plasma is retained.- Which must contain a minimum of 5.5 X 1010 platelets. Has a pH of at least 6.0- PC bags are stored at 20-240C with continuous agitation for 5 days with a pH of 7.0- Factors: initial pH, temp of storage, total platelet count, volume of plasma, duration of storage,

agitation during storage, and lactic acid ion accumulation*Platelets stored for several hours at 40C are associated with an irreversible disc-to-sphere transformation (microtubule disassembly), which is a major contributor to decreased survival of platelets.

*Platelets progressively change shape from discs to spheres at a pH that falls from 6.8 to 6.0 (reversible), and when the pH falls below 6.0, this change becomes irreversible that renders the platelets nonviable after infusion in vivo.