Antibody Detection

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Antibody Detection

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

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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

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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)
• Not clinically significant (IgM)

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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)

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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

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Screening Cells

• Screening cells are single or pooled donor group
  O cells, however, single-donor 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, S, s, P1, Lea, Leb,
  K, k, Jka, Jkb, Fya, Fyb

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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

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2-Cell Antigram
Screening Cells I II
AHG
IS
37
CC
Some manufacturers will use instead of 1 to
indicate the presence of the antigen
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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

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Examples
If patients serum contains anti-Fya, there will
be a stronger reaction because SCI is homozygous
for the Duffy antigen
In this case, the person has anti-Fyb. The
antibody reacts weaker with SCI (heterozygous)
and stronger with SCII (homozygous)
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Screening Cells

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

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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
• Open-heart surgery
• hypothermia

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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
  AHG) to detect in vivo coating

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Autocontrol

• The AC and DAT can help in determining whether
  the antibodies are directed against the patients
  cells or transfused cells (allo-or autoantibody)
• In this hospital

Screen
If positive
Antibody Panel (w/AC)
If positive
DAT
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Potentiators

• Used in antibody detection and identification to
  enhance antigen-antibody 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 antigens)

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Potentiators
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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

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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

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Example 1
Not Done

• IgG antibody
• Single specificity

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Example 2

• IgG antibody
• Multiple specificities

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Example 3
Neg AHG, add CC

• IgM antibody
• Single specificity showing dosage

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Example 2

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