Blood Banking: blood banking reagents; overview and applications

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114 Terms

1

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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proteolytic enzymes

enzymes that denature certain proteins

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Antibody potentiator: Low-ionic-strength saline (LISS)

-Increases rate of antibody uptake

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Antibody Potentiator: Polyethylene glycol (PEG)

-Concentrates the antibody in the test environment in LISS

-removes water molecules

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Antibody potentiator: Proteolytic enzymes (papain, ficin, and bromelin)

-Removes negative charges from the red cell membrane, which reduces the zeta potential; denatures some red cell antigens

-enhance warm and cold autoantibodies

-ficin comes from figs

-bromelin from pineapple

-papain from papaya

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Antibody potentiators: Bovine serum albumin (BSA)

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

-available in either 22% or 30% concentrations

-does NOT enhance warm autoantibodies

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Lectins

plant extracts useful as blood banking reagents; they bind to carbohydrate portions of certain red cell antigens and agglutinate the red cells

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Common lectins in Blood Bank: Dolichos biflorus

-origin: seed extract/plant

-antigen specificity: A1

<p>-origin: seed extract/plant</p><p>-antigen specificity: A1</p>
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Common lectins in Blood Bank: Ulex europaeus

-origin: gorse (flowering plant)

-antigen specificity: H

<p>-origin: gorse (flowering plant)</p><p>-antigen specificity: H</p>
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Common lectins in Blood Bank: Vicia graminea

-origin: seed extract/plant

-antigen specificity: N

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Common lectins in Blood Bank: Iberis amara

-origin: plant

-antigen specificity: M

<p>-origin: plant</p><p>-antigen specificity: M</p>
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Gel test

- ID-MTS Gel card

-uses dextran acrylamide gel particles to trap agglutinated red cells

<p>- ID-MTS Gel card</p><p>-uses dextran acrylamide gel particles to trap agglutinated red cells</p>
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96

Microtiter plate

-96 wells serves as the substituted test tubes

-each well is considered a short test tube

-either U-shaped or V-shaped bottom in the microtiter plate well

-U-bottom well is more used

-small quantities of red cells and antisera are added to the microtiter wells, followed by centrifugation of the microtiter plates

-cell buttoms are resuspended by manually tapping the plate

-concentrated button of red cells is indicative of Ag-Ab reactions, whereas the red cells in a negative result are dispersed throughout the well

<p>-96 wells serves as the substituted test tubes</p><p>-each well is considered a short test tube</p><p>-either U-shaped or V-shaped bottom in the microtiter plate well</p><p>-U-bottom well is more used</p><p>-small quantities of red cells and antisera are added to the microtiter wells, followed by centrifugation of the microtiter plates</p><p>-cell buttoms are resuspended by manually tapping the plate</p><p>-concentrated button of red cells is indicative of Ag-Ab reactions, whereas the red cells in a negative result are dispersed throughout the well</p>
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Solid-phase red cell adherence methods

-uses microplate test wells with immobilized reagent red cells

-for antibody screening, antibody identification, and compatibility testing

-suitable for automation

-antigen or antibody is immobilized to the bottom and sides of the wells

-In direct test: antibody is fixed to the wells. Ag-positive red cells from donor adhere to sides and bottom of well. Ag-negative red cells from donor settle to the bottom of the well and form a button after centrifugation

-In indirect test: red cell membranes are bound to wells. unknown patient serum is added and allowed to react. allows for the capture of IgG antibodies from patient serum to the rbc membrane. a washing step removes unbound IgG antibodies. In a positive indirect test: the indicator cells adhere to sides and bottom of wells. In negative indirect test: indicator cells settle to the bottom of wells and form button after centrifugation.

<p>-uses microplate test wells with immobilized reagent red cells</p><p>-for antibody screening, antibody identification, and compatibility testing</p><p>-suitable for automation</p><p>-antigen or antibody is immobilized to the bottom and sides of the wells</p><p>-In direct test: antibody is fixed to the wells. Ag-positive red cells from donor adhere to sides and bottom of well. Ag-negative red cells from donor settle to the bottom of the well and form a button after centrifugation</p><p>-In indirect test: red cell membranes are bound to wells. unknown patient serum is added and allowed to react. allows for the capture of IgG antibodies from patient serum to the rbc membrane. a washing step removes unbound IgG antibodies. In a positive indirect test: the indicator cells adhere to sides and bottom of wells. In negative indirect test: indicator cells settle to the bottom of wells and form button after centrifugation.</p>
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98

Microplate test method and interpretation: Microtiter Plate Assay

*Method:

-1 drop of anti-A and 1 drop anti-B are placed in separate wells of a U-bottom microplate

-1 drop of a 2-5% saline suspension of red cells is added to each well

-wells are mixed by gently tapping them

-the plate is centrifuged at an appropriate time and speed

-the cell button is resuspended by manually tapping the plate or using a mechanical shaker or placed at an angle for the tilt and stream method

-reactions are read, interpreted, and recorded

*Reactions of Microplate Testing:

-positive reaction: concentrated button of red blood cells

-negative reaction: smooth suspension of red blood cells or a streaming pattern of red blood cells when the plate is placed on an angle

<p>*Method:</p><p>-1 drop of anti-A and 1 drop anti-B are placed in separate wells of a U-bottom microplate</p><p>-1 drop of a 2-5% saline suspension of red cells is added to each well</p><p>-wells are mixed by gently tapping them</p><p>-the plate is centrifuged at an appropriate time and speed</p><p>-the cell button is resuspended by manually tapping the plate or using a mechanical shaker or placed at an angle for the tilt and stream method</p><p>-reactions are read, interpreted, and recorded</p><p>*Reactions of Microplate Testing:</p><p>-positive reaction: concentrated button of red blood cells</p><p>-negative reaction: smooth suspension of red blood cells or a streaming pattern of red blood cells when the plate is placed on an angle</p>
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99

Microplate technique apply the ___ principle of hemagglutination as the tube test. Solid-phase technology reactions are ____. A positive reaction is adherence to the ___; a negative reaction is a ___ ___ ______.

same; opposite; well; red cell button

<p>same; opposite; well; red cell button</p>
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100

What is the purpose of including a reagent control when interpreting group AB, D-positive red cells after testing with a low-protein anti-D reagent?

a. to detect false-positive agglutination reactions

b. to detect false-negative agglutination reactions

c. to identify a mix-up with a patients sample

d. to confirm ABO typing results

A. to detect false-positive agglutination reactions

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