IMMUNOHEMO: CH 2 - BLOOD BANK REAGENTS

Immunohematology reagents are used to detect

antigen and antibody reactions

Reactions in vitro appear as

agglutination or hemolysis

Testing methods include

Tube testing;. Gel technology;. And Solid-phase adherence technology

All routine test methods use a source of antigen and antibody

to detect agglutination or hemolysis

Sources of antigen

Reagent RBCs;. Patient or donor RBCs

Commercially prepared, Known source of antigen

Reagent RBCs

Usually an unknown source of antigen

Patient or donor RBCs

RBCs are tested with commercial antisera to determine

antigen identity

Sources of antibody

Reagent antisera;. And Patient or donor serum or plasma

Commercially prepared; Known source of antibody

Reagent antisera

Usually an unknown source of antibody

Patient or donor serum or plasma

Serum or plasma is tested with commercial RBCs to determine

identity of antibody or antibodies

4 basic categories of reagents of Blood Banking Reagents

RBCs; with known antigens;. Antisera; with known antibodies;. Antiglobulin reagents;: anti-IgG (anti-immunoglobulin G) and or complement (C3b, C3b and C3d);. And Potentiators; to enhance antibodies

Commercial blood banking reagents are licensed by the

Center for Biologics Evaluation and Research (Food and Drug Administration [FDA])

The FDA's criteria can be found in the

Code of Federal Regulations

Before being licensed;. Reagents must meet minimum standards for

specificity and potency

recognition of the antigenic determinant and its corresponding antibody

Specificity:

strength of the reaction

Potency:

testing to determine the accuracy and precision of equipment, reagents, and procedures

Quality Control (QC)

Components of a QC program include

statement of the criteria for acceptable reagent performance;. Documentation of reagent use;. Corrective actions for lack of performance

Commercial Antibody Reagents

Polyclonal antibodies;. Monoclonal antibodies

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

Polyclonal antibodies

Polyclonal antibodies Recognize multiple

epitopes;. An Example of a polyclonal ab is : antihuman globulin (AHG)

Made from single clones of B cells that secrete antibodies of the same specificity

Monoclonal antibodies

Monoclonal antibodies Use hybridoma technology and Recognize

a single epitope;. Examples: anti-A;, anti-c,; and anti-I g G antibodies

Anti-A and anti-B reagents are used to

determine the ABO blood type

Both anti-A & anti-B; antisera are directed toward

specific antigens on the patient's RBCs

Anti-A: is directed toward

A antigen

Anti-B: is directed toward

B antigen

Reagents are formulated to give

a strong reaction (3+ to 4+)

Testing is performed in the

immediate-spin (IS) phase

Confirmation testing should check for expected

ABO antibodies

D Antigen is part of the

Rh blood group system

The Rh blood group system contains several antigens, but the D antigen is

the most important because of increased immunogenicity

The AABB's Standards for Blood Banks and Transfusion Services requires that

all blood samples be typed for the D antigen

D Antigen Typing Procedure

Commercial anti-D is combined with the patient and donor RBCs. Agglutination indicates the presence of the D antigen; no agglutination indicates the absence of the D antigen.

A negative reagent control ensures that a

false-positive result has not occurred.

Anti-D Reagents include

High & Low-Protein REAGENTS

Reagent that Contains polyclonal antibodies;. Is Approximately 20% bovine albumin;. & Promote false-positive agglutination

High-Protein REAGENTS

Reagent that Contains monoclonal antibodies; I g M;. or monoclonal and polyclonal blends;. is Approximately 6% bovine albumin;. & Have replaced high-protein reagents

Low-Protein REAGENTS

Controls ensure that typing results are correct should show

no agglutination

False-positive agglutination can result from

Strong cold autoantibodies & Protein abnormalities

When is a separate control used?

if RBCs agglutinate with all ABO antisera

A separate control is not needed; if there is a negative result; using any of the

ABO low-protein reagents

Commercial reagent RBCs contain known antigens to

confirm the presence of antibodies in patient serum or plasma

Procedures that use commercial RBCs

ABO serum testing;. Screening tests;. & Antibody identification

Commercial A1 and B cells are combined with the patient's serum or plasma to

determine the reverse grouping

Patients possess the antibody directed against the antigen of the ABO system that is

lacking in their RBCs

Can be from a single donor or a pool of donors

A1 and B Cells

A1 and B Cells are Resuspended to a

2% to 5% concentration

A1 and B Cells are Negative for

Rh antigens D, C, and E

A1 and B Cells Should not be used if

red cells darken,; agglutinate in the vial,; or show hemolysis

What are used in antibody screen (detection) tests for unexpected antibodies

Screening cells

Screening cells Available in sets of

2 or 3 vials

Each vial may be from a single donor or

two 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 reactions

Each lot of screening cells comes with an antigram that shows

the antigen profile

The cells must express antigens associated with

the most clinically significant antibodies

Screening cells are regulated by the FDA for:

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

Panel cells are similar to screening cells, except they are

obtained in vials of 10 or more

Panel cells are used for identifying antibodies in a procedure called

an antibody panel

Each lot of panel cells will have an antigram that shows the

antigenic profile of each vial

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

Antiglobulin Test Principle

Antiglobulin Test Reagent contains antibodies toward

IgG (anti-IgG) and or Complement (anti-C3d, anti-C3b)

Types of Antiglobulin Tests

DAT and IAT

Detects IgG or complement bound to RBCs in vivo

DAT

AHG reagent is added after the RBCs have been washed

DAT procedure

In the DAT;, Agglutination demonstrates whether IgG or complement was attached to

the RBCs

Conditions that can cause a Positive DAT

Transfusion reaction;. HDFN;. Autoimmune hemolytic anemia;. And drugs

positive Dat; due to a Transfusion reaction; is caused by donor cells coated with I g G antigens;. Which leads to

production of I g G antibodies from the recipient;

Positive Dat; due to HDFN; is caused by the baby's I g G coated cells;. Which leads to

the mother's I g G antibodies pass through the placenta

Positive Dat;. Due to autoimmune hemolytic anemia;. Is Caused by I g G or C3 on patient RBCs;.

Leading to patient autoantibody

Positive Dat;. Due to drugs;. Is caused by I g G drug complex attached to cells;. Leading to an

immune complex formed with the drug

Detects IgG or complement bound to RBCs in vitro

IAT

Antibodies in serum; are incubated at 37° C; with RBC antigens in vitro;.

IAT

RBC suspension is washed; and combined with AHG reagent; to detect agglutination

IAT Two-step procedure

IAT is used for

Antibody screening;. Identification;, crossmatch;, and antigen typing

Detects antibodies with specificity to red cell antigens

antibody screening

Identifies specificity to red cell antibodies

antibody identification

Determines serological compatibility between donor and patient before transfusion

crossmatch

Identifies a specific red cell antigen in a patient or donor

antigen typing

IAT Sources of Error that cause false positives

Cold antibody;. Contaminates;. Cells packed to tight

What is the likely cause if RBCs agglutinate before washing & adding AHG

cold antibody from patient

Using dirty glassware can cause what kind of result with IAT

false positive due to contamination

What is the result of improper centrifugation with IAT

false positive due to super packed RBCs that won't disperse

Types of Antiglobulin Reagents

Polyspecific AHG;.and Monospecific AHG

Contains both anti-IgG and anti-C3d antibodies

Polyspecific AHG

Polyspecific AHG is Derived from

polyclonal or monoclonal sources

Polyspecific AHG Agglutination indicates that IgG or complement is

coating the RBCs

If results are positive with Polyspecific AHG,

a differential DAT is performed

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

Monospecific AHG

With Monospecific AHG ;. Anti-IgG combines with

human gamma chains

With Monospecific AHG ;. Anti-C3b/C3d specifically detects; complement proteins as a result of

activation of the classical pathway

Activation of the classical pathway causes

Intravascular hemolysis and or Extravascular hemolysis

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

I g G-Sensitized Cells

I g G-Sensitized Cells added to a negative AHG test, should cause

agglutination

Commercial reagents are type O RBCs prepared with

I g G antibodies attached

False-negative results are caused by

Failure to add the AHG reagent;. Failure of the AHG reagent to react;. Or Failure to wash the RBCs adequately

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

Potentiators