Blood Bank Reagents

Polyclonal antibodies

• made from several different clones of B cells that secrete antibodies of different specificity

• recognize multiple epitopes

• mixture of IgM and IgG antibodies

Monoclonal antibodies

• made from single clones of B cells that secrete antibodies of same specificity

• recognize single epitope

• one immunoglobulin class (IgG or IgM)

ABO typing

• reagents are formulated to give strong reaction

• testing performed in the immediate-spin (IS) phase

• confirmation testing should check for expected ABO antibodies

D antigen typing

• negative control ensures that false-positive result has not occurred (low protein reagent control)

• false-positive agglutination can result from strong cold autoantibodies or protein abnormalities

Reagent RBCs

• contain known antigens to confirm the presence of antibodies in pt serum or plasma

• procedures: ABO serum testing, screening test, antibody ID

A1 and B cells

• resuspended to 2-5% concentration

• negative for Rh antigen

• should not be used if red cells darken, agglutinated in vial, or show hemolysis

Screening cells

• used in antibody screen (detection) tests for unexpected antibodies

• each vial may be from single donor or two donors

  • polled cells can be used for donor testing, but only single donor vials are used in transfusion

Panel cells

used for identifying antibodies in a procedure called an antibody panel

Antiglobulin Test

Commercial antibody with a specificity toward human globulins is used to agglutinate antibody-coated RBCs

Direct Antiglobulin Test (DAT)

• detects IgG or complement bound to RBCs in vivo

• AHG reagent added after RBCs have been washed

Positive DAT

Indirect Antiglobulin Test (IAT)

• detects IgG or complement bound to RBCs in vitro

• antibodies are incubated at 37C with RBC antigens in vitro then washed and combined with AHG reagent to detect agglutination

Application of IAT

Polyspecific AHG

• contains both anti-IgG and anti-C3d antibodies

• agglutination indicates that IgG or complement is coating RBCs

• different DAT performed if positive

Monospecific AHG

• Contains either anti-IgG or anti-C3b/C3d, but not both

• Anti-C3b/C3d specifically detects complement proteins as a result of activation of the classical pathway

IgG-Sensitized Cells

• when added to a negative AHG test, reagent should cause agglutination

• Commercial reagents are type O RBCs prepared with IgG antibodies attached

• False-negative results are caused by

  • failure to add AHG reagent

  • failure of AHG reagent to react

  • failure to wash RBCs adequately

Potentiators

Reagents that enhance detection of IgG antibodies by increasing reactivity

Gel Technology

Uses dextran acrylamide gel particles to trap agglutinated cells

Microplate

microliter plate replaces test tubes, same principles

Solid Phase

Uses microplate wells with immobilized reagent

• cells adhere to sides and bottom = positive

• cells that settle to bottom = negative