Blood bank exam 1 (sara)

Antigens

A molecule capable of binding to specific antibody or T cell receptor usually in blood bank they are found on RBC membranes.

What are the 3 types of antigens?

Immunogen, allogenic, autologous

Immunogen

Any substance capable of stimulating a immune response, not always all antigens are immunogens

Allogenic

Present on introduced non self cells (transfused or transplanted cells)

Autologous

Present on individuals own self cells

Antibodies

A protein substance secreted by plasma cells that is developed in response to and reacting specifically with an antigen.

Where are antibodies found in blood bank?

Plasma or commercial antisera

Immune response in antibodies

Reactions of the body to substances that are foreign or are interpreted as being foreign.

Humoral immunity in antibodies

Pertains to cell destruction response during the early period of the reaction.

What is the role of plasma cells with antibodies?

Plasma cells produce majority of antibodies, memory B cells respond rapidly to second exposure and transform into plasma cells.

What are the most important antibodies in blood bank?

IgG and igM

IgM

Doesn’t cross the placenta and very efficient in activation of complement, intravascular clearance of red cells, immediate spin at room temp for detection.

IgG

Crosses placenta, no as efficient in activation of complement, extra vascular clearance of red cells, antiglobulin test which needs incubation test at 37c for detection.

Antigen-antibody reactions

An immune complex occurs when the antigen and antibody combine and held together by non covalent forces.

Strength of binding in antigen-antibody reactions is influence by?

Size, shape, and electrical charge of an antigen. Antigen bound to antibody can activate the classic complement pathway and can result in lysis of cells.

Primary response

First exposure to antigen, lag phase 5-10 days, igM is produced first, less antibody produced.

Secondary response

Second exposure to antibody response, 1-3 days, mostly igG, antibody levels are high and sustained longer.

How do responses and reactions tie to blood bank?

Exposure to foreign antigens during transfusion or pregnancy may cause production of all antibodies which can result in sensitization.

Antibody screen

Detects alloantibodies, further testing is performed to identify the specific antibody, the identity of antibody present in patients plasma MUST be confirmed before RBC units can be crosshatched and transfused unless physician states urgent, never ignore a positive antibody screen.

Detecting alloantibodies before transfusion.

Helps prevent formation of antigen-antibody complexes , formation of these immune complexes would result in decreased RBC survival of transfused cells and may activate complement proteins.

Complement

Classical pathway activated by antibodies and the pathway that is most important to transfusion medicine.

Agglutination

The clumping together of red blood cells or any particulate matter resulting from interaction of antibody and its corresponding antigen.

Visible agglutination

Antigen-antibody reactions and antigen-antibody complex has formed (positive result)

No visible agglutination

Implies lack of antigen-antibody complex formation (negative result)

2 steps to agglutination

Sensitization and lattice formation

Sensitization

Antibody molecules attach to their corresponding antigenic site on the particle, no visible clumping.

Factors that affect sensitization

Temp (igM reacts at < 22c and igG react at 37c), PH (ideal is 6.5 and 7.5), incubation time, ionic strength (can be adjusted with various enhancement media/reagents)

Lattice formation

Antibody molecules cross link the particles forming a lattice that results in visible clumping or agglutination.

Factors affecting lattice formation.

Zeta potential and zone of equivalence

What is zeta potential?

The force of repulsion that naturally exist between red blood cells in a physiologic saline solution; keeps RBC from sticking together while in circulation.

What is Zone of equivalence?

The condition that exist when the number of binding sites of antigen and antibody are almost equal. This favors maximum amount of agglutination.

Factors affecting agglutination

Prozone, postzone, centrifugation

What is prozone?

Incomplete lattice formation caused by an excess of antibody molecules relative to the number of antigen sites, resulting in false negative reactions.

Postzone

Incomplete lattice formation caused by excess of antigen relative to the number of antibodies resulting in false-negative.

Prozone and postzone

Prozone and postzone could cause a false-negative due to formation of small antigen antibody complexes, commercial antibody and red cell preparations are diluted to obtain the optimal antibody and antigen concentrations in order to obtain the zone of equivalence.

Centrifugtation

Centrifuges are calibrated for optimum lattice formation over centrifugation and or operation over optimal RPM= false positive, under centrifugation and or operation below optimal = false negative.

Routine testing

Commercially prepared blood banking reagents are used to detect antigen and antibody reactions

ABO/D typing (forward testing)

Detects A and B and D antigens, patients red cells (antigen source), commercial anti-A and anti-B and anti-D (antibody)

ABO serum testing (reverse grouping)

Detects ABO antibodies, reverse grouping cells (A and B) antigen source, patients serum or plasma antibody source.

Antibody screen

Detects antibodies with specifically to red cell antigens, screening cells (antigen source), patients serum or plasma (antibody source).

Antibody identification

Identifies specifically of red cell antibodies, panel cells (antigen), patients serum or plasma (antibody)

Cross match

Determines serologic compatibility between donor and patient before transfusion, donors red cells (antigen), patients serum or plasma (antibody)

Testing methods

Tube testing (hemagglutination), gel technology, solid-phase adherence technology, microtiter plates

Grading reactions

Antigen and antibody reactions are measured qualitatively; is an antibody present or not and qualitatively how strong they are (1-4+), conventional grading system, microscopic and macroscopic.

Blood bank reagents

Used in blood bank to detect antigen-antibody reactions.

what are the 4 basic categories of blood bank reagents?

RBC with known antigens, antisera with known antibodies, antiglobulin reagents (anti-igG or anti complement), potentiators to enhance antigen/antibody reactions.

Reagent regulation

All commercial blood bank reagents are licensed by the FDA and have expiration dates.

What must package inserts include?

Detailed description of reagent, procedure for use, interpretation of reactions, performance characteristics, limitations, quality control requirements

What must happen before changing a parameter of a package insert?

Must be changed in the facilities standard operating procedure (SOP) before being put into use.

Quality control

Testing to determine the accuracy and precision of equipment, reagents, and procedures.

First 50

QC programs for institutions

Statement of acceptable criteria, documentation of reagent use, corrective actions, don’t use reagents that have unacceptable results during QC for patient testing.

Testing ABO typing

Anti-A and anti-B reagents, used to determine the ABO blood type, directed toward specific antigens on patients red blood cells.

What can ABO typing be known as?

Grouping, forward grouping, or forward typing

How is ABO typing performed?

Adding patient cells (unknown antigens) to antisera (unknown antibodies), Anti-A is directed towards A antigen and anti-B is directed towards B antigen, no such thing as O because O lack both and is not a antigen.

Reagents in ABO typing

Formulated to give strong reactions (3-4+), test is performed in immediate spin phase.

A1 and B cells

Testing also checks for ABO antibodies in patient plasma or serum, can act as a check.

How is A1 and B cells performed?

Adding patient plasma (unknown antibodies) to commercial A1 and B red cell reagents (known antigens), patients posses the antibody directed against the antigen of the ABO system that is lacking on their RBCs.

What can cause false reactions in A1 and B testing?

2-5% concentration of donor cells that lack D antigen which could cause false reactions if the alloantibody is present in the plasma.

D antigen typing

D antigen is most important in the Rh group.

What is anti-D linked to?

Hemolytic disease of fetus and newborn and hemolytic transfusion reactions meaning all blood samples submitted to the lab must be tested for the d antigen as well as ABO group.

Procedure for D antigen typing

Performed concurrently with ABO typing, commercial anti D antisera is combined with the patient or donor RBCs.

What indicates the presence of D antigen?

Agglutination indicates presence of D antigen.

What indicates absence of D antigen?

No agglutination

Why do we want a negative control in D antigen typing?

Because a negative reagent control ensures that a false positive result hasn’t occurred due to the presence of a panagglutin. If the patient is AB positive a separate control must be used.

Low protein reagent control

Controls ensure that typing results are correct, should show no agglutination (negative), control can detect false positives which can be caused from strong cold autoantibodies.

When must a second control be used in low protein reagent control?

RBCs agglutinate with all ABO/Rh, to ensure accuracy, high protein D reagent.

RBC reagents

Commercial RBCs with known antigens used in testing to confirm the presence of antibodies in the plasma/serum.

Types of testing that use RBC reagents

ABO serum/plasma testing (A1and B cells), screening test (screening cells), antibody identification (panel cells).

Landsteiners law

And individual naturally possess the antibody directed against the antigen of the ABO system that is absent from their red cells.

Screening cells

Used in antibody screen test for detection of unexpected antibodies, available in 2,3,4 vial sets, group O RBC no ABO antibodies react with them.

Screening cells antigram

Shows the antigen profile, += antigen present, o= antigen absent, screening cells must express antigens associated with the most clinically significant antibodies.

Panel cells

Like screening cells, has sets of 10 vitals or more, used for determining antibody specificity in and antibody identification procedure (ABID), each lot of panel cells has a antigram that shows to antigenic profile of each vial.

Antiglobulin test

Commercial antibody with a specifically toward human globulins (anti human globulin or AHG) is used to agglutinate antibody coated RBC, AHG test detects igG and or complement molecules that have attached to red cells but have no caused visable agglutination through cross linkage with sensitized cells.

Polyspecific

Reagent contains antibody toward anti-IgG + anti-complement

Monospecific

Reagent contains antibodies toward anti-IgG

? Specific

Reagent contains antibodies towards anti-complement

Direct antiglobulin test (DAT)

Detects IgG or complement bound to RBC in vivo, no incubation phase requires, agglutination determines whether IgG or complement was attached to the RBC.

Clinical conditions of direct antiglobulin test (DAT)

Transfusion reaction (donor cells coated with IgG), hemolytic disease of fetus (fetal red cells coated with IgG), autoimmune hemolytic anemia (IgG or C3 on red cells), drug related (IgG drug complex attached to cell).

Indirect antiglobulin test (IAT)

Detects IgG that bind to RBCs in vitro

2 step procedure of indirect antiglobulin test.

Antibodies (in patient plasma) are incubated at 37c with RBC antigens and with enhancement media, the RBC suspension is washed and then combined with AHG reagent to detect agglutination.

Applications of indirect antiglobulin test.

Multiple blood bank test incorporate as part of procedure, antibody screening, antibody identification, cross match, antigen typing

False positive errors

Failure to wash cells adequately which causes unbound human serum globulins neutralize AHG reagent, red cells agglutinated before washing step, improper centrifugal ion, dirty glassware.

Polyspecific AHG

Contains both anti IgG and anti-C3b/C3d antibodies, agglutination indicates that IgG or complement is coating the RBCs.

What happens if direct antiglobulin test is positive in Polyspecific AHG?

A perform DAT with anti-IgG and perform DAT with anti-C3b/C3d.

Monospecific AHG

Contains either ant-IgG or anti-C3b/C3d but not both, anti IgG combines with human gamma chains, anti-C3b/C3d specifically detects complement proteins.

IgG sensitized cells

Referred to as chech cells; commercial reagent that is type O RBCs prepared with IgG antibodies attached, required by AABB as control when AHG results are negative to verify the validity of the negative results.

What should happen when IgG sensitized cells are added to a negative AHG test?

Should cause agglutination, no agglutination= invalid

What are false negative results caused by I’m IgG sensitized cells?

Failure to add AHG reagent, failure to wash adequately, failure of reagent to react.

Potentiators

Reagents that enhance the detection of IgG antibodies by increasing their reactivity.

Potentiator low ionic strength saline (LISS)

Increases rate of antibody uptake

Polythylene glycol (PEG) potentiator

Concentrates the antibody in the test environment in LISS

Proteolytic enzymes potentiator

Papain or ficin removes negative charges from the red cell membrane

Bovine serum albumin (BSA)

Reduces the repulsion between cells but does not shorten the incubation time.

Lectins

Seed extracts with specificity toward certain RBC antigens and bind to the carbohydrate determinants of RBC antigens, anti-A, (dolichos biflorus) is most used and often found in hospital BB.

Dolichos biflorus (lectin)

Uses A1 phenotype antigen

Ulex europaeus (lectin)

Uses H antigen

Vicia graminea (lectin)

Uses N antigen specificity

Iberis amara (lectin)

Uses M antigen specificity

A 96 indicates a well microplate replaces test tubes

A concentrated button indicates a reaction, dispersed cells indicate no reaction