Blood Bank Reagents

What are the 4 basic categories of reagents?

1. RBCs w/ known Ags

2. Antisera w/ known Abs

3. Antiglobulin reagents: anti-IgG (anti-immunoglobulin G)

4. Potentiators (enhance Abs)

Reagent Regulation & QC

• FDA criteria

  • Specificity: recognition of the antigenic determinant and its corresponding antibody

  • Potency: Strength of rxn

• determine the accuracy and precision of equipment, reagents, and procedures

  • statement, documentation, corrective action

Commercial Antibody Reagents

• Polyclonal Antibodies

  • Ex: Antihuman globulin (AHG)

• Monoclonal Antibodies

  • Ex: anti-A, anti-c, & anti-IgG Abs

Polyclonal Antibodies

made from several diff clones of B cells that secret of Abs of DIFF. specificities

Monoclonal Antibodies

Made from single clones of B cells that secrete Abs of SAME specificity, recognizes single epitope

Monoclonal Abs vs Polyclonal Abs

ABO Typing

• Anti-A & Anti B reagents used to determine ABO blood type

• Both Antisera directed toward specifics on the pt’s RBCs

  • Anti-A → A ag

  • Anti-B → B ag

ABO Red Cell Testing (ABO Forward Grouping)

ABO Typing Reagent Testing

• Reagents are formulated to give a strong reaction (3+ to

  4+)

• Testing is performed in the immediate-spin (IS) phase

• Confirmation testing to check expected ABO Abs

D Antigen Typing Importance

• Most important in Rh blood group system due to INC. immunogenicity

• AABB requires all blood samples typed for D antigen

D Antigen Typing Procedure

• commercial anti D + pt & donor RBCs

  • Agglutination = presence of D antigen

  • No agglutination = absence of D antigen

• (-) control reagent to ensure lack of false (+) result

Anti D Reagents

• High-Protein Reagents

  • Contain polyclonal antibodies

  • ~ 20% bovine albumin

  • Promotes false (+) agglutination

• Low-protein reagents

  • monoclonal abs (IgM) or mono/polyclonal blend

  • ~ 6% bovine albumin

  • replaced high-protein reagents

Low-Protein Reagent Contro

Ex: Monoclonal Control

• ensure that typing results are correct, SHOW NO AGGLUTINATION

• False(+) agglutination can result from

  • Strong cold autoantibodies

  • Protein abnormalities

When is a separate control used?

• used if RBCs agglutinate with all ABO antisera

• is not needed if there is a (-) result using any of the ABO low-protein reagents

What are Reagent RBCs and when are they used?

• contain known Ags to confirm the presence of Abs in patient serum or plasma

• Procedures that use commercial RBCs

  • ABO serum testing

  • Screening tests

  • Antibody identification

A1 and B Cells

Commercial A1 and B cells are combined with the patient’s serum or plasma to determine the reverse grouping

• Pts have Abs directed against the Ag of the ABO system that is lacking on their RBCs

• Can be from a single donor or a pool of donors

• Resuspended to a 2% to 5% concentration

• Negative for Rh antigens (D, C, and E)

• Should not be used if red cells darken, agglutinate in the vial, or show hemolysis

ABO Serum Testing (Reverse Grouping)

Screening Cells

• used in Ab screen (detection) tests for unexpected Abs

• Available in sets of 2 or 3 vials

• Each vial may be from a 1 donor or 2 donors (pooled together)

  • Pooled cells can be used for donor testing, but only single-donor vials are used when a person is about to receive a transfusion (e.g., recipient)

  • Single-donor cells tend to give stronger rxn

Screening Cells Antigram

• shows the antigen profile

• cells must express antigens associated w/ the most clinically significant Abs

• screening cells are regulated by the FDA for:

  • D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, and Jkb

Panel Cells

• like screening cells but w/ 10+ vials

• used for identifying Abs in a procedure called an Ab pane

• Each lot of panel cells will have an antigram that shows the antigenic profile of each vial

Antiglobulin Test

• Principle of test

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

• Reagent contains antibodies toward:

  • IgG (anti-IgG) and/or

  • Complement (anti-C3d, anti-C3b)

What are the two types of Antiglobulin test and how do they differ?

• Direct Antiglobulin Test (DAT)

  • IgG or Complement on RBCs in VIVO

• Indirect Antiglobulin Test (IAT)

  • IgG or Complement on RBCs in VITRO

Diret Antiglobulin Test (DAT)

• Detects IgG or complement bound to RBCs in vivo

• In the procedure, AHG reagent is added after the RBCs have been washed

• Agglutination demonstrates whether IgG or complement was attached to the RBCs

Positive DAT

Indirect Antiglobulin Test (IAT)

• Detects IgG or complement bound to RBCs in vitro

• Two-step procedure

  1. Antibodies (in serum) are incubated at 37° C with RBC antigens in vitro

  2. RBC suspension is washed and then combined with AHG reagent to detect agglutination

Applications of IAT

Common Sources of False-Positive Error in Antiglobulin Testing

Common Sources of False-Negative Error in Antiglobulin Testing

Antiglobulin Reagents: Poly vs Monospecific AHG

Containing both anti-IgG and anti- C3d antibodies vs containing either anti-IgG or anti- C3b/C3d, but not both

Polyspecific AHG

Contains both anti-IgG and anti- C3d antibodies

• from polyclonal or monoclonal sources

• Agglutination = that IgG or complement is coating the RBCs

  • If positive, a differential DAT is performed

Monospecific AHG

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

• Anti-IgG combines with human gamma chains

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

  • Intravascular hemolysis

  • Extravascular hemolysis

IgG-Sensitized Cells

• Required by AABB as a control system when AHG results are negative

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

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

What are potentiators? What are the types of potentiators?

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

• Low ionic strength saline - LISS

• Polyethylene glycol - PEG

• Proteolytic enzymes: Papain and Ficin

• Bovine serum albumin - BSA

Potentiators

Bovine Serum Albumin

• Available in 22% or 30% concentration

• Allows antibody-sensitized cells to become closer together than is possible with saline

• Favors direct agglutination with Rh antibodies

• Enhances sensitivity of the IAT

Lectins purpose and common types

• Seed extracts that have specificity toward certain RBC antigens

• Bind to carbohydrate determinants of RBC antigens

Gel Technology uses?

Dextran acrylamide gel traps agglutinated cells.

• Gel particles + reagents predispensed in microtubes.

• Add RBCs/plasma/serum, incubate, centrifuge.

• Agglutinates trapped at top (4+), nonagglutinated cells at bottom (0).

Microplate Testing

• 96-well microtiter plate replaces test

  tubes

  • A concentrated button indicates a

    reaction

  • Dispersed cells indicate no reaction

Solid Phase

• Uses microplate wells with immobilized reagent

  • Cells that adhere to the sides and bottom of the wells are (+)

  • Cells that settle to the bottom of the wells are (-)