Blood Banking: blood banking reagents; overview and applications

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114 Terms
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reagent red cells are:

known red cell antigens

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antisera are:

known red cell antibodies

<p>known red cell antibodies</p>
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Antiglobulin reagents are:

anti-IgG or anti-C3d or a combination of anti-IgG and anti-C3d

<p>anti-IgG or anti-C3d or a combination of anti-IgG and anti-C3d</p>
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potentiators

Reagents added to the serum-cell mixture to enhance antibody uptake during the incubation phase of the indirect antiglobulin test

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potency

strength of an Ag-Ab reaction

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If reagents are produced for IN-HOUSE use (within the facility), a license is ___ required.

NOT

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QC of reagents is performed ___ on commercial reagent red cells and antisera.

daily

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Reagent product insert must include:

-description

-procedure for proper use

-interpretations

-performance characteristics

-limitations

-quality control

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polyclonal antiserum

made from several different clones of B cells that secrete antibodies of different specificities

(AHG reagents)

<p>made from several different clones of B cells that secrete antibodies of different specificities</p><p>(AHG reagents)</p>
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Polyclonal vs. monoclonal antibodies

<p></p>
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monoclonal antibody

made from single clone of B cells that secrete antibodies of the same specificity

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monoclonal antibody production

<p></p>
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hybridomas

hybrid cells formed by the fusion of myeloma cells and antibody-producing cells; used in the production of monoclonal antibodies

<p>hybrid cells formed by the fusion of myeloma cells and antibody-producing cells; used in the production of monoclonal antibodies</p>
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Epstein-Barr virus (EBV)

also called human herpesvirus 4 (HHV-4) and is one of eight viruses in the herpes family

<p>also called human herpesvirus 4 (HHV-4) and is one of eight viruses in the herpes family</p>
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heterohybridomas

hybrid cells formed by the fusion of lymphocyte of one species with the myeloma cell of a different species

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phenotype

observable expression of inherited traits

<p>observable expression of inherited traits</p>
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Four major blood phenotypes in the ABO blood group system:

A, B, AB, and O

<p>A, B, AB, and O</p>
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Summary of monoclonal antibodies:

-secreted by a single clone of antibody-producing B cells

-one immunoglobulin class (IgG or IgM)

-unique specificity for a particular epitope

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Summary of polyclonal antibodies:

-secreted by several different clones of antibody-producing B cells

-Mixture of IgM and IgG antibodies

-mixture of antibodies that may be directed at different epitopes of the same antigen

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ABO Red Cell Testing (ABO forward grouping):

*ABO blood group antigen: A

-reaction with anti-A

*ABO blood group antigen: B

-reaction with anti-B

*ABO blood group reaction: AB

-reaction with anti-A and anti-B

*ABO blood group reaction: O

- no reaction with either

<p>*ABO blood group antigen: A</p><p>-reaction with anti-A</p><p>*ABO blood group antigen: B</p><p>-reaction with anti-B</p><p>*ABO blood group reaction: AB</p><p>-reaction with anti-A and anti-B</p><p>*ABO blood group reaction: O</p><p>- no reaction with either</p>
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Immediate Spin Phase (IS)

source antigen and source antibody used in immunohematologic testing are combined, immediately centrifuged, and observed for agglutination

<p>source antigen and source antibody used in immunohematologic testing are combined, immediately centrifuged, and observed for agglutination</p>
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ABO antibodies

anti-A, anti-B, and anti-A,B; patients possess the ABO antibody to the ABO antigen lacking on their red cells (eg, group A individuals possess anti-B)

<p>anti-A, anti-B, and anti-A,B; patients possess the ABO antibody to the ABO antigen lacking on their red cells (eg, group A individuals possess anti-B)</p>
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anti-A reagent color

always contains a blue dye

<p>always contains a blue dye</p>
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anti-B reagent color

always contains a yellow dye

<p>always contains a yellow dye</p>
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autoantibodies

antibodies to self antigens

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Typing for D antigen with patient or donor red cells

*D-positive: (D-antigen on RBC) reacts with anti-D and

*D-negative: (NO D-antigen on RBC) No reaction with anti-D

<p>*D-positive: (D-antigen on RBC) reacts with anti-D and</p><p>*D-negative: (NO D-antigen on RBC) No reaction with anti-D</p>
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D typing procedure:

Commercial anti-D is combined with patient or donor red cells

-agglutination indicates presence of the D antigen on the red cells tested

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Summary of ABO and D typing reagents: Murine Monoclonal Anti-A and Anti-B

-for slide, tube, and microplate testing

-Anti-A=blue dye

-Anti-B=yellow dye

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Summary of ABO and D typing reagents: Murine monoclonal Anti-A,B

-for slide, tube, and microplate testing

-blend of anti-A and anti-B clones

-Anti-A,B=clear

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Summary of ABO and D typing reagents: Monoclonal Anti-D

-for slide, tube, and microplate testing

-monoclonal-polyclonal blend: IgM anti-D from human-murine heterohybridoma and polyclonal IgG anti-D

-monoclonal blend: IgM and IgG blending of human-murine heterohybridomas

-monoclonal: IgM from single clone

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Examples of Low-protein controls in ABO and D typing:

1) reaction w/ anti-A and reaction w/ anti-D: A and D antigens present: reagent control present? yes; no agglutination w/ anti-B

2) reaction w/ anti-B: B antigens present: reagent control present? yes; no agglutination with anti-A

3) reaction w/ anti-B and anti-D: B and D antigens: reagent control present? yes; no agglutination with anti-A

4) reaction w/ anti-A and anti-B: antigens A and B: control present? yes; no agglutination w/ anti-D

5) reaction w/ anti-A, anti-B and anti-D: antigens: cannot interpret typing: control present? no; reagent control must be tested to determine ABO and D typing results

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Reagent red cells

A1 and group B red cells: testing with patient serum or plasma confirms the ABO type

(known as reverse grouping or ABO serum testing)

<p>A1 and group B red cells: testing with patient serum or plasma confirms the ABO type</p><p>(known as reverse grouping or ABO serum testing)</p>
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ABO Reverse Grouping/Back type

<p></p>
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Reagent red cells for serum testing are obtained from:

selected human donors and are manufactured in several optional packages (most common: two-vial set of A1 and B red cells)

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All reagent red cells are ___ to remove blood group antibodies and are resuspended to a __-__% concentration in a buffered preservative solution to minimize hemolysis and loss of antigenicity during the dating period.

washed; 2-5%

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recipient

patient receiving the transfusion

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Antigram

profile of antigen phenotypes for each donor used in the manufacture of commercially supplied screening and panel cells

<p>profile of antigen phenotypes for each donor used in the manufacture of commercially supplied screening and panel cells</p>
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Screening cells are used in:

antibody screen tests

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Antibody screening test

looks for antibodies with specificity to red cell antigens in patient and donor samples

<p>looks for antibodies with specificity to red cell antigens in patient and donor samples</p>
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An antibody identification panel is performed when the ___ ___ ___ is positive.

antibody screen test

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Reagent red cell antibody identification panels:

are required to determine the specificity of a red cell antibody in a blood banking procedure called antibody identification

(patient or donor serum/plasma is tested with the reagent panel cells to identify an antibody to red cell antigens)

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The antibody identification panel cells are individual group __ donors packaged in sets of __ or more, depending on the manufacturer.

O; 10

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Three types of reagent red cells for routine testing include:

-A1 and B cells in ABO serum testing

-Screening cells to detect red cell antibodies

-panel cells to identify red cell antibodies

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neutralization

blocking antibody sites, causing a negative reaction

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antiglobulin test (Coombs test)

detects IgG antibodies and complement proteins that have attached to red cells either in vitro or in vivo but do not produce visible agglutination

<p>detects IgG antibodies and complement proteins that have attached to red cells either in vitro or in vivo but do not produce visible agglutination</p>
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Polyspecific AHG reagent contains:

antibodies to IgG molecules (anti-IgG) and complement proteins (anti-C3d, anti-C3b)

<p>antibodies to IgG molecules (anti-IgG) and complement proteins (anti-C3d, anti-C3b)</p>
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Essential that red cells be washed with ___ ___ to remove any unbound molecules before the addition of the ___ reagent.

physiologic saline; AHG

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AHG test washing steps:

-filling test tube with saline to mix with red cells already present in the tube

-saline-suspended red cells are centrifuged

-saline wash is decanted

-repeat for 2-3 cycles

-saline is removed, and the tube is blotted dry to remove most traces of the saline

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to detect potential neutralization:

IgG-sensitized cells are added to tubes w/ negative reactions

(after centrifugation, a positive reaction should be observed to confirm that washing was adequate)

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sensitized

Immunoglobulin or complement attached to the cells from the immune system (in vivo) or from a test procedure (in vitro).

<p>Immunoglobulin or complement attached to the cells from the immune system (in vivo) or from a test procedure (in vitro).</p>
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Direct Antiglobulin Test (DAT)

test used to detect antibody bound to red cells in vivo

<p>test used to detect antibody bound to red cells in vivo</p>
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Indirect Antiglobulin Test (IAT)

test used to detect antibody bound to red cells in vitro

<p>test used to detect antibody bound to red cells in vitro</p>
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Autoimmune Hemolytic Anemia

immune destruction of autologous (self) red cells

<p>immune destruction of autologous (self) red cells</p>
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DAT

-ordered to detect IgG or complement proteins bound to patient cells

-positive DAT is an important indicator of potential immune-mediated red cell destruction in the body

-b/c of IgG or complement attachment to red cells, macrophages are signaled to clear them

-this signals immune destruction of red cells and often leads to anemia

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DAT procedure:

-A patient's RBCs are obtained and washed (3-4x) with physiologic saline

-Coomb's Reagent is added which will bind to any antibodies present on the RBCs

-Agglutination will occur if there are antibodies on the RBCs

<p>-A patient's RBCs are obtained and washed (3-4x) with physiologic saline</p><p>-Coomb's Reagent is added which will bind to any antibodies present on the RBCs</p><p>-Agglutination will occur if there are antibodies on the RBCs</p>
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polyspecific AHG reagent

contains both anti-IgG and anti-C3d antibodies and detects both IgG and C3d molecules on red cells

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monospecific AHG reagent

reagents prepared by separating the specificities of the polyspecific AHG reagents into individual sources of anti-IgG and anti-C3d/anti-C3b

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sample of choice for a DAT is collected in a:

EDTA tube

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Clinical examples causing a positive DAT: Transfusion reaction

-caused by: donor cells coated with IgG

-source of IgG: recipient (patient) antibody

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Clinical examples causing a positive DAT: Hemolytic disease of the fetus and newborn

-caused by: fetal red cells coated with IgG

-source of IgG: maternal antibody crossing the placenta

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Clinical examples causing a positive DAT: Autoimmune hemolytic anemia

-caused by: IgG or C3 on patient red cells

-source of IgG: patient autoantibody

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Clinical examples causing a positive DAT: Drug-related mechanism

-caused by: IgG-drug complex attached to cells

-source of IgG: immune complex formed with drug

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IAT:

-detects in vitro sensitization of RBCs

-two stage procedure

1) antibodies first combine with red cell antigens in vitro during an incubation step

(plasma source is incubated at body temperature with a red cell source to allow the attachment of IgG antibodies to specific red cell antigens)

-red cell suspension is washed with physiologic saline to remove unbound antibody/complement

-after washing, AHG reagent is added to the test and centrifuged

<p>-detects in vitro sensitization of RBCs</p><p>-two stage procedure</p><p>1) antibodies first combine with red cell antigens in vitro during an incubation step</p><p>(plasma source is incubated at body temperature with a red cell source to allow the attachment of IgG antibodies to specific red cell antigens)</p><p>-red cell suspension is washed with physiologic saline to remove unbound antibody/complement</p><p>-after washing, AHG reagent is added to the test and centrifuged</p>
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reaction phase

observation of agglutination at certain temperatures, after incubation, or after addition of AHG

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Polyspecific AHG reagents are used primarily in the ___ to determine that either IgG or complement molecules have attached to the red cells in vivo.

DAT

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Comparison of DAT and IAT procedures: (DAT)

-detects IgG / complement-coated red cells

-IgG attachment to red cells has occurred within the PATIENTS BODY

-one-stage procedure

-patients red cells are tested with antiglobulin reagent withOUT an incubation step

-test for certain clinical conditions: HDFN, hemolytic transfusion reaction, and autoimmune hemolytic anemia

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Comparison of DAT and IAT procedures: (IAT)

-detects IgG and complement-coated red cells

-IgG attchment to red cells occurred during the incubation step

-two-stage procedure

-test requires an incubation step before the addition of AHG reagent

-used as a reaction phase of several tests in immunohematology: antibody screen and antibody identification panel

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Common sources of false-positive error in antiglobulin testing:

-Red cells are agglutinated before washing step and addition of antihuman globulin reagent

Possible explanation: potent cold reactive antibody of patient origin

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Common sources of false-positive error in antiglobulin testing:

-Use of dirty glassware

Possible explanation:

particles or contaminants

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Common sources of false-positive error in antiglobulin testing:

improper centrifugation-overcentrifugation

possible explanation: red cell button packed so tightly on centrifugation that nonspecific clumping cannot be dispersed

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Common sources of false-negative error in antiglobulin testing:

Failure to wash cells adequately during the test procedure before the addition of AHG reagent

possible explanation: unbound human serum globulins neutralize AHG reagent

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Common sources of false-negative error in antiglobulin testing:

testing is interrupted or delayed; AHG reagent is not added immediately after washing

possible explanation: Bound IgG or complement may detach from the coated red cells

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Common sources of false-negative error in antiglobulin testing:

failure to identify weak positive reactions

possible explanation: technical error in testing

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Common sources of false-negative error in antiglobulin testing:

loss of reagent activity

possible explanation: improper reagent storage, bacterial contamination, or contamination with human serum

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Common sources of false-negative error in antiglobulin testing:

Failure to add AHG reagent

possible explanation:

technical error in testing

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Common sources of false-negative error in antiglobulin testing:

improper centrifugation: undercentrifugation

possible explanations: conditions for promoting agglutination are not optimal

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Common sources of false-negative error in antiglobulin testing:

inappropriate red cell concentrations-red cell suspensions fall outside the optimal 2%-5%

possible explanation: concentration of red cells influences the agglutination reaction

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Differential DAT

test that uses monospecific anti-IgG and monospecific anti-C3d/anti-C3b reagents to determine the cause of a positive DAT with polyspecific antiglobulin reagents

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IgG sensitized cells

-control system for antiglobulin tests interpreted as negative

-referred to as check cells or Coombs control cells

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Potential reasons for a false-negative result detected by the use of IgG-sensitized red cells in an antiglobulin test:

-failure to add the antiglobulin reagent to the test

-failure of the added antiglobulin reagent to react

-failure to wash red cells adequately

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Antihuman globulin reagents:

-Polyspecific: anti-IgG and anti-C3d; use in DAT

-Monospecific: either anti-IgG or anti-C3d/C3b; use in antibody screen/ identification, differential DAT

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IgG sensitized red cells:

-added to negative AHG reactions

-should agglutinate after addition

-checks: sufficient washing, addition of AHG, AHG reagent was potent

-does NOT ensure that the AHG reaction is negative

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antibody potentiators

(aka: enhancement media)

Reagents or methods that enhance or speed up the antibody-antigen reaction

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enhancement media

reagents that enhance or speed up the antibody-antigen reaction

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