Antibody Detection & Identification

What are some tools and tests used for detecting non-ABO antibodies?

• antibody screens

• antibody panels

• DAT

• reagent cell treatments

• phenotyping

• elution

• adsorption

What types of patients must undergo antibody screens?

• patient who need transfusions

• pregnant women

• patients who have had transfusion reactions

• also performed on blood and plasma donations

How do antibody screens work?

• patient plasma/serum is added to reagent red cells (with known phenotypes) to detect antibodies

  • unexpected alloantibodies are the result of RBC stimulation via transfusion or pregnancy

alloantibodies can be:

• clinically significant

  • IgG, reacts best at 37 C and during the AHG phase

  • associated with HTRs and HDFN

• OR clinically insignificant

  • IgM

What cells are used for antibody screening?

• screen cells

• group O (some are Rh+ some are Rh-)

• each screen cell kit comes with an antigram

  • typically, the clinically significant antigens of the Rh, Kell, Duffy, Kidd, and MNS systems will show homozygous expression

• reaction to one or more cells indicates the presence of an unexpected antibody

What are the steps in performing an antibody screen?

1. patient plasma/serum is incubated with screening cells and a potentiator (LISS, PEG, saline, BSA, or the proteolytic enzymes ficin or papain)

2. after incubation, the serum and cell mixture is washed with saline to remove any unbound proteins that could neutralize the AHG reagent

3. AHG is added to the washed cells

   1. reagent binds to any antibody that is bound to red cells

4. agglutination = positive for antibody, no agglutination = negative for antibody

What is done after negative antibody screens (no agglutination)?

• check cells (red cells sensitized with IgG) are added to the mixture to confirm negative results

  • a positive reaction (agglutination) indicates confirmation of negative results

What are some causes of false-positives in antibody screens (4)?

• rouleaux

• antibodies to reagent preservatives

• fibrin contamination of sample

• PEG

  • causes false-positives if the reaction is read right after the 37 C incubation, before the washing step

• also review the patient’s diagnosis, history, and any medications they are taking

Why is are autocontrols performed alongside antibody screens?

• to determine if the patient has an alloantibody or an autoantibody

  • negative autocontrols indicate an alloantibody

  • positive autocontrols indicate an autoantibody OR alloantibody to recently transfused cells

• remember: autocontrols involve the testing of a patient’s serum with their own red cells

What must be done after a positive autocontrol result during antibody screening and identification?

• a DAT has to be performed

  • to accurately identify in vivo coating of RBCs with antibody (autoAB or alloAB from a recent transfusion)

What important information can you obtain from looking at a patient’s history when performing antibody screens and identification?

• if the patient has received a transfusion in the past 3 months

  • may indicate that the patient has a mixed RBC population

• if the patient has come from another hospital with another blood bank

• if the patient has certain diseases associated with antibodies

  • Mononucleosis or Mycoplasma pneumoniae, for example

• the patient’s race

  • some antibodies occur at higher rates in some races

What are some common IgM antibodies that react at room temperature (during the IS phase)?

anti-Le^a, anti-Le^b, anti-M, anti-N, and anti-I

What do reactions of varying strengths indicate during antibody identification?

• multiple antibodies are present

• OR the antibody is affected by dosage

  • a panel cell that has a double dose of an antigen will show stronger reactions with the corresponding antibody

What is the rule of 3 when positively identifying an antibody?

3 antigen positive cells must react and 3 antigen negative cells must not react with the patient’s plasma/serum

• panel 8.2 on page 200 for context

What must be done to confirm the presence of an antibody after antibody identification?

• you have to phenotype the patient

  • patient RBCs can be phenotyped with specific antisera to confirm antibody identification results

When can phenotyping be performed on a patient after transfusion?

• after 3 months

  • phenotype results may not be valid after transfusion because positive results may be due to the transfused cells

What additional techniques are used to help identify multiple antibodies detected in AB ID?

using selected cells, enzymes, and chemicals (like DTT, which denatures Kell antigens)

What are selected cells, and how are they used to resolve multiple antibodies?

• def: reagent red cells chosen from a different panel to confirm or eliminate the possibility of an antibody

• example: You suspect the patient has anti-Jk^a, but you cannot rule out the other antibody, anti-E yet. You should find another panel cell that is negative for Jk^a antigen and positive for E antigen; this will confirm or deny the presence of anti-E.

How can proteolytic enzymes be used to resolve multiple antibodies?

• enzymes used: ficin or papain

• can be used to eliminate or enhance antibody activity

  • Duffy, MNS, and Xg antigens are destroyed

  • Rh, Kidd, and Lewis antigens are enhanced

• One-stage test: enzymes, RBCs, and patient plasma are incubated together

• Two-stage test: panel cells pretreated with enzymes are tested with plasma

antibodies to high incidence antigens are:

• not common

an alloantibody to a high frequency antigen should be suspected if:

most panel cells are positive

What are high-titer, low-avidity antibodies?

• antibodies to high-frequency antigens that react weakly (2+ reaction or less)

• usually inconsistent in their reactivity

• not enhanced with potentiators and are not clinically significant

antibodies to low frequency antigens (when is it suspected?)

• examples: anti-C^w, anti-Kp^a, and anti-Lu^a

• suspected when there is one positive cell on the panel during AB ID

• when blood is needed for the patient: crossmatch during the AHG phase

How can weak IgG antibody reactions be enhanced during AB ID?

• repeat the panel with enzymes

• use cells from a different panel

• incubate longer

• increase the serum to cell ratio

• remember to follow package insert directions when making changes

cold alloantibodies

IgM antibodies that react during IS crossmatching and sometimes at 37 C

What are some causes of positive autocontrols and DATs?

• cold or warm autoantibodies

• certain drugs

• HDFN

• delayed transfusion reactions

autoantibodies usually react with ___ reagents and self and donor RBCs

all

cold autoantibodies

• antibodies that attach to self red cells and react below body temperature

• reacts during the IS phase of crossmatching

• positive autocontrol and DAT

  • C3 binds to RBCs, not IgG

• may occur in those with a history of mild anemia, M. pneumoniae infection, or infectious mono

• anti-I, anti-H, and anti-HI are common

• cold panels help in identification

What are some methods to avoid cold autoantibody reactivity?

• use a monospecific AHG reagent (anti-IgG) instead of a polyspecific AHG reagent for screens and panels

• do not read reactions at the IS phase of testing

• avoid enzymes that enhance autoantibody activity

  • use 22% bovine serum albumin instead of LISS

• warm your tubes

• perform an adsorption technique

How do you prewarm your tubes to prevent cold autoantibody reactivity?

1. incubate serum and red cells separately

2. mix serum and red cells and incubate again

3. wash cells with saline and add anti-IgG and observe agglutination (add check cells to negative reactions)

adsorption

procedure that uses red cells (with known antigens) to remove red cell antibodies from a solution (plasma)

ex: group A red cells can remove anti-A from plasma

adsorption technique for removing cold autoantibodies from plasma

• plasma is added to rabbit erythrocyte stroma (RESt)

• RESt removes the cold AB from the plasma

• testing is done with the “new” plasma (plasma without cold AB)

when should you suspect the presence of warm autoantibodies in a patient’s plasma?

• when most or all screen cells, panel cells, autocontrols, and DAT are positive

  • positive DAT indicates red cells are coated with IgG

the specificity of warm autoantibodies is often directed towards:

• the Rh system, especially e antigen

  • patient has warm anti-e AND e antigen

  • e negative blood should be given

warm autoimmune hemolytic anemia (WAIHA)

• autoantibodies react with red cells at 37 C or higher

• AB is typically directed towards all cells of the Rh phenotype

• RBCs should be transfused with caution

  • an autoantibody with Rh specificity will attack transfused cells

Why might an adsorption need to be done in those with warm autoantibodies?

• warm autoantibodies may need to be removed from plasma to test for clinically significant alloantibodies

elution

• the removal of antigen-antibody complexes off of RBCs

• antibody that’s recovered (eluate) can be used for further testing (like antibody specificity)

• performed after a positive DAT

What are the 3 types of elution methods?

• glycine acid

  • removes antibody by lowering pH, most common

• heat, freeze, thaw

  • physical removal of antibody, effective for ABO antibodies

• ether methylene chloride chloroform

  • organic solvent removes antibody, sensitive but hazardous

What are the two categories of drugs that cause positive DATs?

• drug-independent

  • drug causes autoimmune processes that resolves itself when the drug is discontinued

  • appears as a warm autoantibody

• drug-dependent

  • drug modifies the RBC membrane, causing proteins to attach

  • OR the drug attaches to the red cell membrane and an antibody to the drug causes red cell clearance by macrophages

phenotyping IgG coated red cells causes false-

• positives

  • how to fix it: treat cells with chloroquine diphosphate, this will dissociate the IgG from the RBCs without harming antigens

Which two drugs commonly cause reactions in the blood bank?

• Daratumumab

  • treatment for MM

  • shows panagglutinin in all IATs

• IVIG

  • causes issues with antibody screens and can mimic warm autoantibodies in plasma