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Blood Banking/Immuno

Chpt. 1 and 3

What is the amount of whole blood collected? 450ml or 500ml +/- 10%
If 500ml of blood is collected what is the amount of anticoagulant in the unit? 450mL? 70mL and 63mL
What is the amount of whole blood that can be collected from a donor? 10.5mL/kg of donor weight
What is the total blood volume of most adults and what is the time frame to replenish 1 pint of fluids? most adults are 10-12 pints and in 24 hours
How often can an individual donate? every 8 weeks
What are the three components whole blood can be separated into? Packed RBCs, platelets, and plasma
How long can a unit of whole blood be stored for? 21 to 42 days
What are the 3 steps or processes for donation? 1. Educational reading materials 2. The donor health history questionnaire 3. The abbreviated physical examination
What is the goal of blood preservation? to provide viable & functional blook components for patients requiring blood transfusions.
How does a defect in the methemoglobin reductase pathway affect rbc once they've been transfused? the survival and function posttransfusion.
What is RBC viability? a measure of in-vivo RBC survival following transfusion.
What is the percentage of cells that should remain viable for 24hours? 75%
How should you maintain optimum viability for blood? it should be stored in the liquid state between 1 and 6 degrees C for a specific number of days, as determined by preservative solution used.
What biochemical changes cause the loss of RBC viability? decrease in pH, decrease in glucose consumption, buildup of lactic acid, decrease in ATP levels, and a reversible loss of RBC function.
What is a reversible loss of RBC function referring to? it's expressed as a shift to the left of the hemoglobin-oxygen dissociation curve or an increase in hemoglobin-oxygen affinity.
As RBCs are stored what is decreased and does this change once the RBCs are transfused in a patient? 2,3-DPG levels decrease, with a shift to the left of the hemoglobin-oxygen dissociation curve, however, it is re-formed in stored RBCs, after in-vivo circulation.
What is the rate of restoration of 2,3-DPG depend on? it's influenced by the acid-base status of the recipient, phosphorus metabolism, and the degree of anemia.
What are the major roles in complement activation? 1. Final lysis of abnormal or pathogenic cells via the binding site of antibodies 2. opsonization of phagocytsis 3. mediation of inflammation
How are the proteins in complement found in plasma? In a proenzyme inactive state
What are the 3 ways complement proteins are activated? Classic, alternative, and lectin pathways
How does the classic pathway activate complement? by antigen-antibody binding, uses specific methods.
How does the alternative pathway activate complement? By recognizing polysaccharies and liposaccharides found on the surface of bacteria and tumor cells. Uses non-specific methods.
How does the lectin pathway activate complement? activated by mannose binding proteins bound to macrophages.
What is the period called that it takes for the immune system to respond to an antigen? lag period or latency period; it's during this time that the antibody cannot be detected with serologic testing.
The first antibodies made against the new antigen in a primary response is... IgM then IgG follows
When the same antigen is encountered again, what immunoglobin response? IgG
What is the lag period for the secondary response? 1 to 2 days to hours
What is the lag period for a primary response? weeks to months
What cell surface molecules are classified by complex in-vitro testing using monoclonal antibodies? Macrophages, NK cells, T and B cells and APC.
Why is immunosuppression important in blood banking? because immunosuppressed andimmunocompromised patients must receive irradiated blood products so that the donor lymphocytes do not engraft and attack the recipients's immune system and destroy it
What is the percentage that IgG is concentrated in serum? 80% of the total serum
Which immunoglobulins are the most significant for blood banking? IgG, IgM, and IgA
Which clinically significant antibodies react at 37 degrees IgG isotype, and are capable of destroying transfused reactions of various severities
What is most commonly encountered as naturally occuring antibodies in the ABO system and believed to be produced in response to commonly occuring antigens like intestinal flora and pollen grains? IgM and are expected in plasma and are therefore important in reverse group testing.
What other blood groups may also produce IgM ab, which usually react best at ambient temperatures (22-24) Lewis, Ii, P, and MNS
What is the primary testing problem encountered with IgM? They can interfere with the detection of clinically significant IgG by masking their reactivity.
What reagents can distinguish a mixture of IgG and IgM antibodies? sulfhydryl reducing reagents such as B-2-mercaptoethanol (2-ME) or dithiothreitol (DTT); because only IgM is removed, which allows unexpected IgG to be detected.
Why are IgG significant in transfusion medicine? because they're the class of antibodies that are made in response to transfusion with nonself and therefore are incompatible RBCs and other blood products.
Which antibody has the greatest number of subclasses and what are they? IgG has 4 classes, IgG1, IgG2, IgG3, and IgG4.
What are the functional difference of the subclasses of IgG? the ability to fix complement and cross the placenta.
Why is IgA important in immunohematology? because about 30% of anti-A and anti-B antibodies are of the IgA class (the remaining percentages are IgM and IgG)
Why else is IgA antibodies important? anti-IgA antibodies can cause severe problems if transfused in plasma products to patients who are deficient in IgA as potentially fatal anaphylaxis can result and IgA can increase the effect of IgG-induced RBC hemolysis.
Even though IgE is found in trace amounts in serum, .004%, what can it cause when a patient receives a transfusion? Urticaria because of the presence of IgE antibodies.
How is the classic pathway of complement initiated? when antibody binds to antigen, by the binding of the complement protein C1 to the Fc fragment of an IgM, IgG1, or IgG3 subclass antibody.
How many IgG and IgM antibodies are required to activated complement? 1 IgM or 2 IgG
What ion is needed for the stabilization of the C1q,r,s complex? Calcium, without calcium there is no stabilization and complement is not activated.
What ion is needed for C4 and C2? MG++
Besides a few antibodies what else can activate the complement sequence that leads to complement-mediated intravascular hemolysis? ABO system
What are the cellular antigen? A, B, Rh(D)
What are the 2 types of antibodies that are of concern in blood banking? naturally occuring ab and immune ab
RBC antibodies are considered naturally occurring when... they are found in the serum of individuals who have never been previously exposed to RBC antigens by transfusion, injection, or pregnancy. Most are IgM cold agglutinis, which react best at room temp or lower, activate complement.
How are Natural ab produced? most likely due to a response in the environment like pollen grains and/or bacteria membranes.
what are the naturally occuring antibodies? ABH, Hh, Ii, Lewis, MN and P blood group systems.
When are RBC antibodies considered immune? when they are found in the serum of individuals who have been transfused or pregnant. These antigens are not generally found in nature, and their molecular makeup is unique to human RBCs.
Most immune RBC are IgG antibodies that react best at 37 degrees an require the use of antihuman globulin sera (Coombs sera) for detection.
What are the most common immune antibodies encountered? those that react with Rh, Kell, Duffy, Kidd, and Ss blood group system.
Blood group A has Anti-B
Blood group B has anti-A
Blood group O has both as well as anti-A,B
Blood group AB has neither
Antibodies are either allo- or autoreactive
Alloantibodies mean are produced after exposure to genetically different, or nonself, antigens of the same species, such as a different RBC antigen after transfusion.
Autoantibodies are produced in response to self antigens.
Why are alloreactive antibodies a potentially serious problem? because transfused patients have produced alloreactive antibodies that are no longer detectable in the patients plasma or serum.
Why is plasma preferred for DAT and elution studies? because they lack fibrin strands, which can cause false-positives.
How is Hemagglutination accomplished? By red cell agglutination reactions
What are the two stages of agglutination? Sensitization and Agglutination
What is Sensitization? it's antigen binding to the antibody. Epitopes on the surfaces of RBC membranes combine with the antigen-combining sites (Fab region) on the variable regions of the immunoglobin heavy & light chains. NO VISIBLE AGGLUTINATION SEEN
What is agglutination (second stage) a lattice-type structure composed of multiple ag-ab bridges between RBC antigens and antibodies is formed. VISIBLE AGGLUTINATION SEEN
What is precipitation reaction? the development of an insoluble ag-ab complex, resulting from the mixture of equivalent amounts of soluble ag-ab.
Agglutination inhibition is a method in which a positive reaction is the opposite of what is normally observed in agglutination.
Agglutination is inhibited when... an antigen-antibody reaction has previously occurred in a test system and prevents agglutination. Ag & Ab can't bind b/c another substrate has been added to the reaction mixture & blocks it.
Agglutination reactions are influenced by the... concentrations of the reactants(ag & ab), pH, temperature, and ionic strength.
Centrifugation is an effective way to enhance agglutination reactions because it decreases reaction time by increasing the gravitational forces on the reactants as well as bringing reactants closer together.
Antigen-Antibody Ratio Ag-ab should have equivalent amounts each. Any deviation decreases the effeciency of the reaction.
Excess of unbound ab results in a prozone effect
a surpluss of antigen results in a postzone effect
A weak expression of antigen on RBCs is called dosage effect and it can also alter the ag-ab ratio.
How do weak expression occur? as a result of the inheritance of genotypes that give rise to heterozygous expression of RBC antigens an resultant weaker phenotypes.
Effect of pH 6.5-7.5 is the ideal pH for ag-ab reactions.
Temperature Different isotypes of antibodies may exhibit optimal reactivity at different temperatures. Because clinically significant ab may be present it's important to do testing with a range of temps.
IgM temp at ambient temps or below 22 degrees
IgG usually require 37 degrees
Ionic Strength of a solution will determine how your ab-ag come together, decreased Ionic strength increases the ag-ab reaction by lowering the # of charged particles in solution, you reduce the shielding affect and allow slightly (+) charged ab to be uptake.
Does IgM need any enhancement media to react strongly with ag? No IgM and their corresponding RBC antigens are easily accomplished in saline medium because it's a larger antibody, fixes complement very well.
To discover the presence of IgG ab,what needs to be done? the use of enhancement techniques
One of the key ways to enhance the detection of IgG ab is to increase their reactivity.
This is accomplished by reducing the zeta potential on RBCs
The net negative charge surrounding RBCs in a cationic media is part of the force that repels RBCs from each other and is due to sialic acid molecules on the surface of RBCs.
Most acids have a neg. charge, and the large concentration of these molecules on RBCs creates a "zone" of negative charge around the RBC, which the zone is protective and keeps RBCs from adhering to each other in the peripheral blood.
A potential is created because of the ionic cloud of cation (positively charged ions) that are attracted to the zone of negative charges on the RBC membrane.
What is the potential around the RBC called? the Zeta Potential and is an expression of the difference in electrostatic charges at the RBC surface and the surrounding cations.
Reducing the zeta potential allows the more positively charged ab to get closer to the negatively charged RBCs and therefore increases RBC agglutination by IgG molecules.
Other enhancement methods are by the use of colloids. a type of clear solution that contains particles permanently suspended in solution.
The colloidal solutes can be either charged or neutral and go into solution because of their microscopic size
Types of Colloids include albumin, polyethylene glycol (PEG), polybrene, polyvinylpyrrolidone (PVP), and protamine.
The colloids work by increasing the dielectric constant ( a measure of electrical conductivity), which reduces the zeta potential.
Low Ionic Strength solutions are (LISS) low salt media, generally 0.2% NaCl, they work by decreasing the ionic strength and the result is an increased rate of ab uptake during sensitization and a decreased reaction time. can cause false positives
PEG and Polybrene are macromolecules additives used with LISS to increase agglutination they provide the same advantages, in that poly can detect ABO incompatibility and IgG alloantibodies; PEG produces specific reactions with reduction in false-positive
Enzyme Modification is another way to increase agglutination, they're useful when testing multiple antibodies in a sample.
Enzymes used in the detection and identification of blood group ags are ficin(isolated from fig plants) papain (from papaya) trypsin (from pig stomach) bromelin (from pineapple)
The use of enzymes provides enhanced ab reactivity to Rh, Kidd, P1, Lewis, and I antigens and destroys or decreases reactivity to Fya, Fyb, M, N, and S antigens.
The direct anti-human globulin (AHG) test is designed to determine if RBCs are coated with ab or complement or both. In-vivo sensitization
Polyspecific AHG can determine if RBCs have been sensitized with IgG ab or complement (components C3b or C3d) or both
Monospecific AHG reagents react only with RBCs sensitized with IgG or complement.
Anti-human antibodies are produced by injecting animals usually rabbits or goats with human globulin, which in turn makes antihuman antibodies against foreign human protein.
Polyspecific AHG both the gamma (IgG) and beta (C3b and C3d) globulin fractions of plasma are processed.
Monospecific AHG animals are injected only with IgG and produce ab directed against the gamma heavy chain.
If the ab present in serum cannot cause RBC agglutination but only sensitize the RBCs, then the AHG reagents will allow for agglutination to occur by cross-linking the antibodies on the RBCs.
Chemical modification of IgG are used to help identify the different antibodies present in a mixture of alloantibodies or allloantibodoes occurring with autoantibodies.
Chemical modification reagents generally act on covalent sulfhydryl bonds and facilitate ab ID by removal of either IgG or IgM antibodies
Dithiothreitol (DTT) and B-2-mercaptoethanol(2-ME) are thiol reducing agents that break the disulfide bonds of the J(joining) chain of the IgM molecule but leave the IgG intact.
ZZAP reagent consist of a thiol reagent plus a proteolytic enzyme, causes the dissociation of IgG molecules from the surface of sensitized RBCs and alters the surface antigens of the RBCs
Chemical reduction of the disulfide bond of the IgG molecule is also used to produce chemically modified reagents that react with RBCs in saline.
How does the reduction in the disulfide bond of the IgG molecule cause it to react with saline? by reducing the strong but flexible covalent disulfide bonds in the hinge region of the IgG molecule, allowing the Fab portions more flexibility in faciliting agglutination reactions.
Solid-Phase Adherence Test used to ID both antigens and antibodies, and are ab-ag reactions and adherence to a solid phase support system. Using a direct and indirect method
Direct Solid Phase Test consist of reagent antibody of known specificity fixed to the microplate wells with the addition of RBCs with unknown antigen. used for RBC typing for specific antigen
If the ag-ab reaction occurs, the RBC cover the entire well, and the reaction is considered positive
If no adherence occurs, and the RBCs are free to fall to the bottom of the well and form a button then the reaction is negative
The indirect Solid phase test uses RBCs of known antigenic composition bound to pretreated microplate wells. unknown test serum is added, incubated, and washed free of unbound serum proteins, IgG indicated cells are then added.
A positive indirect solid phase test is demonstrated if the IgG-coated cells cover the well in a uniform pattern.
A negative indirect SPT is demonstrated when indicator cells settle to the bottom of the well in a button.
The Gel Test uses a type of gel matrix instead of saline to separate positive and negative reactions.
The gel test consist of a dextran acrylamide gel that can separate particles based on size is contained in a plastic microtube. it has an upper reaction chamber and a gel column with a tapered bottom. Whatever is being tested is added to the upper chamber, including cells, plasma, or both. Reaction is incubated then centrifuged, the gel acts as a filter to trap RBC agglutinates of different size at different levels.
Larger agglutinates of the gel test stay at the top
Smaller agglutinates (gel test) filter out toward the lower part of the column
Unagglutinated cells are centrifuged to the bottom of the microtube
The Gel technique is used for cell antigen detection and identification, serum antibody detection and identification, and crossmatching and is applicable to automated blood bank methods.
The CH3 on the Fc portion of immunoglobulin molecule is the attachment site for Macrophage
The CH2 on the Fc portion of the immunoglobulin is the attachment site for Complement C1q
Complement peptide fragments mediate an immune response
The activation sequence of complement is C1, 4, 2, 3, 5, 6, 7, 8, 9= Lysis
If fragments of complement bind on RBC without lysis what will happen? it can occur without completion of the sequence, making the RBC Complement sensitization which can cause an immune response.
The bound complement fragments are C3b which will turn to C3d if it's there long enough
Hemolysis in vitro is considered a positive reaction
Created by: Twixy565