Antibody Characteristics and Potentiators

Antibody characteristics:
Some antibodies react in colder temperatures, some react in warmer temperatures. Some react better when treated with enzymes, and some not. It is important to know these antibody characteristics in order to be able to think through problems.

Alloantibody versus autoantibody:
This is simple but important vocabulary.

An alloantibody is defined as an antibody produced following introduction of an alloantigen (an antigen present in only some individuals of a species) into the system of an individual lacking that particular antigen.

An autoantibody is defined as an antibody produced by an organism in response to a constituent of its own tissues.

Cold antibodies (4⁰C which is ~39⁰F):
Cold autoantibodies can be benign and clinically insignificant. They tend to be more significant when active at higher temperatures. They can interfere with blood typing results, 1+ or 2+ results seen in reverse typing can be associated with a cold allo/auto antibody.

Study Tip
To remember antibodies that react in the cold (4⁰C), use the phrase “Pinch Mold Lewis.” This phrase needs some slight tweaking, switch the M with the C to get the word cold. This reminds you these are cold reacting antibodies, and the remaining letters are the antibodies that react in the cold, P, I, N, M, H, and Lewis. This phrase can also be used to remember which antibodies are generally IgM.

Warm antibodies (37⁰C which is ~98⁰F):
Warm autoantibodies can also be benign however when encountered they must all be treated as clinically significant because there is no way to tell the difference between the autoantibodies that will cause a problem and the ones that won’t.

Study Tip
To remember antibodies that react at warm temperatures (37⁰C), use the phrase “DuKe the Kidd is Rheal warm.” This tells you that Duffy, Kell, Kidd, and Rh react at warm temperatures. This phrase can also be used to remember which antibodies are generally IgG.

Dosage:
Dosage is an important concept to understand and it’s quite simple it just isn’t named very well. Dosage simply means that an antibody will react at a different strength if the patient is homozygous vs heterozygous for a particular antigen. For example, let’s say one patient is homozygous for Duffy A, Fy(a+b-) and another is heterozygous for Duffy A and B, Fy(a+b+). Since Duffy exhibits dosage, the homozygous reaction will give a stronger reaction than the heterozygous reaction.

Study Tip
To remember which antibodies exhibit dosage use this phrase:
“Duffy and NoD the Rh Kidd had a dose of M+Ns.” This tells you that the groups that exhibit dosage are: Duffy, Rh (except for D, that’s where NoD comes from), Kidd, and M, N, S, s.

Enzyme treatment:
There are four enzymes, bromelin, ficin, papain, and trypsin, that when added to a blood sample will remove sialic acid. The removal of sialic acid will decrease the negative surface charge making cell agglutination easier. This will enhance the reactivity of Rh, I, P, Lewis, and Kidd antibodies. However, these enzymes will also destroy M, N, S, s, and Fy antigens. It is important to know which are enhanced and which are destroyed (Note: S and s are variable with enzyme treatment).

Study Tip
To remember the reactivity that is enhanced by enzymes use the phrase “Rest in peace (RhIP) Lewis the Kidd.” This will tell you that Rh, I, P, Lewis, and Kidd groups are enhanced using enzymes.

Study Tip
To remember the antigens that are destroyed using enzymes use the phrase “Must Not Ssmell Duff’s destroyer.” This tells you M, N, S, s, and Fy antigens are destroyed by enzymes.

Other notes about well-known antigens:

M and N antigens are usually developed at birth. Anti-M and anti-N are usually IgM, naturally occurring, and not clinically significant. The glycophorin molecule of the M, N, S, s system is a receptor for Plasmodium falciparum.

K antigen is the most immunogenic after ABO/Rh. K antigen can be destroyed by sulfhydryl reagents dithiothreitol (DTT) and 2-aminoethylisothiouronium (AET). DTT and AET will both break disulfide bonds which are present on Kell antigens.

Anti-S, anti-s, and anti-U are usually IgG and clinically significant. Anti-U is very rare because 99.9% of the population has the U antigen. However, those lacking U antigen will be lacking S and s also.

i antigen is usually only present on cord blood cells. i antigen will convert to I antigen in most children by the age of 2. Anti-i has an association with mononucleosis. The autoantibody anti-I is associated with cold agglutinin syndrome.

Nerdy Note
An example of molecular adaptation at its finest are the Duffy antigens Fya and Fyb. Duffy null phenotype Fy(a-b-) is relatively rare except in a few places including West Africa where about 95% of the population has this “rare” genotype. What’s the reason for this? The Duffy antigen is a receptor for the malaria species Plasmodium vivax. People without Duffy antigens are immune to this malaria species. The Duffy antigen system is extremely complex as are malaria species, and this story is still being studied, debated, and researched to determine how this genotype evolved. In addition, the malaria is adapting as well. There are reported cases of people with Fy(a-b-) who have been infected with Plasmodium vivax.

Potentiators:

Zeta potential:
Zeta potential is a term used to describe the degree of electrostatic repulsion between adjacent, similarly charged particles. It all has to do with charge chemistry. In terms of RBCs, negatively charged RBCs and clouds of positive ions surrounding them keep adjacent RBCs a certain distance away from each other. This distance is significant because in a typical blood sample, RBCs are not close enough together for IgG antibodies to crosslink them. Crosslinking is needed for RBC agglutination to detect potentially dangerous antibodies.

Enhancement reagents (aka potentiators) decrease the charges surrounding RBCs allowing them to get closer to adjacent RBCs.

Albumin:
The use of bovine albumin to enhance RBC agglutination by IgG antibodies has been around since the 1940’s. Albumin can be added before the 37C incubation to decrease zeta potential and enhance reactivity.

Low ionic strength solution (LISS):
LISS consists of glycine in an albumin solution. LISS also decreases the zeta potential but also shortens the incubation time required to detect most antibodies. Put another way it increases the speed of antibody sensitization which can also increase the possibility of agglutination.

Polyethylene glycol (PEG):
PEG is a water soluble polymer that “eliminates” water allowing RBCs to get closer together. PEG is more effective than LISS or albumin in detecting weak antibodies. PEG is added after the immediate spin (IS) and incubated. There is no 37C interpretation when using PEG because centrifuging RBCs with added PEG can cause false positives. Instead of interpreting, the RBCs are washed to remove PEG and excess antibodies before proceeding to the AHG phase.