Reagents and Methods for Testing in the Blood Bank

Major Focus Constructed around detecting Antigens and Antibodies Historically viewed by agglutination: now includes Gel Solid phase Molecular techniques Automation With the exception of molecular, they are still Antibody-Antigen reactions

Routine Testing Typing of the ABO and Rh Antigens Typing for Antigens of Other Blood Group Systems Antibody Screen Antibody Identification Compatibility Test (Crossmatch) Direct Antiglobulin Test

ABO and Rh Antisera Where to the Antigens come from?

Antibody Screen Antigens Where do the Antibodies come from?

Reagent cells for reverse typing Where do the Antibodies come from?

Anti-Human Antiglobulin Our new best friend

Principle: Antihuman globulins (AHG) from immunized animals bind to human globulins whether free in serum or attached to RBC’S IgM is so large it usually reacts at room temperature spin IgG needs a little help- a bridge molecule to agglutinate RBC’s AHG acts as a bridge molecule

Polyspecific vs Monospecific AHG Antibodies to both human IgG and to C3d Advantage is that they may detect complement dependent antibodies on RBC’s (Anti-Jka) Disadvantage is it may cause more nuisance positives Mono Specific Antibodies to Human IgG or C3d only May miss some antibodies but has less nuisance positives

Used with all negative AHG reactions Coombs Check Cells Check cells are added to see if AHG is present and functioning

This methodology is vital to blood bank testing

Antibody Screen (Indirect) Purpose is to detect In Vitro sensitization Detection of unexpected Antibody Uses O cells with antigens represented on 2-3 different cells of DCEce, MNSsP, Lea Leb, Kk Fya, Fyb, Jka, Jkb. Homozygous expression of Antigens is valued over heterozygous (may show dosage effect, greater antigen density per cell the greater the sensitivity Need to be sure to run Auto Control

Antibody Screen Limitations Low frequency antibodies may not be detected Antibodies with low titers may not be detected ABO antibodies will not be detected

Direct Antiglobulin Test (DAT) Detects in Vivo sensitization Examples when this would be used would be HDN, Hemolytic Anemia, Auto Immune Hemolytic Anemia (AIHA) Usually would use polyspecific AHG Need to consider if there is in vivo hemolysis Patient recently transfused Medications Unexpected Allo antibody

Antibody Identification Use multiple cell panels, enhancement processes and varying incubations to help identify the Antibody(ies). What you need to consider in the process is Patient Medical History Antigen profile of Panel cells Results of Auto control What phase, what strength of agglutination Crossing out procedure Does this antibody match the reaction pattern

Example of Antigram

Enhancement Media Enhancement media assists the attachment of an antibody to the specific antigen on the red cell Bovine Albumin ( used during incubation) Low-Ionic Strength Saline (what commercial test cells are in) Polyethylene Glycol (concentrates Antibody) Enzymes (Ficin and Papain) Reduces negative charge. Some Antibodies are enhanced like Rh, Lewis others are destroyed like Duffy and M,N,S

Alternate Test Methods Automation Gel Technology Microplate testing Solid Phase Testing Solid and Microplate have exactly the opposite interpretations of reaction Molecular Biology (above my pay grade) Other than Automation and Gel, these other processes are used in Donor centers or large Reference Labs.

Lab Assignment We will be following Procedure seen on the video We will be using manual wash for the AHG testing, this will be demonstrated for you in class Try to limit the centrifuge spin time to 20 seconds especially on the wash cycle. We need to learn a good technique on manual wash, it will mean the right result, not just any result. This project was funded at $3,000,000 (100% of its total cost) from a grant awarded under the Trade Adjustment Assistance Community College and Career Training Grants, as implemented by the U.S. Department of Labor’s Employment and Training Administration. Rogue Community College is an equal opportunity employer/program. Auxiliary aids and services, alternate form and language services are available to individuals with disabilities and limited English proficiency free of cost upon request.   This work is licensed under a Creative Commons Attribution 4.0 International License.