Antibody Detection

Renee Wilkins, PhD, MLS(ASCP)cm CLS 325/435 School of Health Related Professions University of Mississippi Medical Center

Antibody Detection

The test used to detect antibodies is called an antibody screen Antibody screens are used for:
Patients needing a transfusion Pregnant women Cases of transfusion reactions Blood and plasma donors

Antibody Screen

Uses patients plasma/serum against reagent red cells to detect unexpected antibodies Unexpected antibodies are found in addition to the expected anti-A and/or anti-B Unexpected antibodies are a result of red cell stimulation (transfusion, HDN) Unexpected antibodies may be:

Clinically significant (IgG)

Clinically significant antibodies

Usually IgG React best at 37 and AHG phase (IAT) Clinically significant antibodies are associated with hemolytic transfusion reactions (HTR) and hemolytic disease of the newborn (HDN)

Performing an antibody screen

Patients plasma/serum is incubated with screening cells After incubation, an IAT is performed (indirect antiglobulin test) using AHG reagent This will detect any IgG antibodies

Screening Cells

Screening cells are single or pooled donor group O cells, however, singledonor vials offer increased sensitivity Why group O? so anti-A and anti-B wont react Screening cells come in sets of 2 or 3 vials each Each vial (donor) has been phenotyped for each antigen 18 antigens are required on at least one of the vials: D, C, E, c, e, M, N,

Screening cells

Screening cells come with a sheet of paper called an antigram Screening cells are an already prepared 2-5% RBC suspension An antigram (2 or 3 cells) will list the antigens present in each vial A reaction to one or more cells indicates the presence of an unexpected antibody

2-Cell Antigram
Screening Cells I & II

IS 37

Some manufacturers will use + instead of 1 to indicate the presence of the antigen

AHG

CC

Screening cells

The technologist should be aware that some antigens demonstrate dosage An attempt should be made to used screening cells that are homozygous for the clinically significant antigens (Rh, Duffy, Kidd). Just be aware that different strengths can occur
Homozygous antigens will react stronger Heterozygous antigens will react weaker

Examples
Fya SCI SCII + 0 Fya SCI SCII + 0 Fyb 0 + Fyb + + 2+ 4+
In this case, the person has anti-Fyb. The antibody reacts weaker with SCI (heterozygous) and stronger with SCII (homozygous)

4+ 0

If patients serum contains anti-Fya, there will be a stronger reaction because SCI is homozygous for the Duffy antigen

Screening Cells

Screening cells may also contain lowincidence antigens like V, Cw, and Kpa The presence of these antigens is not required for screening cells

Pretransfusion Screening

Screening for antibodies is normally performed prior to blood transfusion to detect antibodies that react at body temperature (37) Colder reacting antibodies (RT and below) are therefore considered insignificant and just cause interference when performing lab testing The only important thing to remember concerning cold antibodies is that they may bind complement if a persons body temperature becomes low

Autocontrol

Tests patient serum with their own red cells Some labs may or may not perform an autocontrol (AC) with the screendepends on the hospital However, the AC should be run with the antibody panelwell discuss this later AC is incubated with the antibody screen (or antibody panel) If a lab uses an AC with the screen and it is positive, they may run a DAT (patient cells

Autocontrol

The AC and DAT can help in determining whether the antibodies are directed against the patients cells or transfused cells (allo-or autoantibody)Scree n If In this hospital: positive
Antibody Panel (w/AC) If
positive

DAT

Potentiators

Used in antibody detection and identification to enhance antigenantibody reaction

Saline (may only enhance if incubated long time) Low-ionic strength solution (LISS) common Bovine serum albumin (BSA) Polyethylene glycol (PEG) Proteolytic enzymes (can destroy some

Potentiators
Albumin
Serum/cell mixture should incubate at least 20 minutes, 30 minutes preferred; doesnt enhance warm autoantibodies Incubation time of 10 minutes; lowers ionic strength allowing better reaction; sensitive and quick! Enhances warm autoantibodies; does not react well with insignificant antibodies (IgM)

LISS

PEG

Limitations

Very effective in detecting antibodies If negative, then the crossmatch should be compatible However,
Dosage may weaken a reaction Antigen may not be present on screening cells Patient may have a passive ABO antibody

Patient History

GET THE HISTORY!!

Mixed red cell populations from a previous transfusion can remain for up to 3 months Patient may have come from another hospital Some diseases are associated with antibodies Some antibodies occur at a higher frequency in some races Get diagnosis, age, race, etc

Example 1
Screenin g Cell I II IS 0 0 37C 0 0 AHG 0 2+ CC ND
Not Done

IgG antibody Single specificity

Example 2
Screening Cell I II IS 0 0 37C 0 2+ AHG 3+ 3+ CC

IgG antibody Multiple specificities

Example 3
Screening Cell I II IS 1+ 3+ 37C 0 0 AHG 0 0 CC
Neg AHG, add CC

IgM antibody Single specificity showing dosage

Example 2
Screening Cell I II IS 0 0 37C 0 0 AHG 2+ 2+ CC

IgG antibody Allo- or autoantibody? (dont know without further testing)