Home/Resources / CSTM Blog / Tech Tips: Divide and Conquer - Separating Multiple Antibodies

Tech Tips: Divide and Conquer - Separating Multiple Antibodies

A Blog from Eric Ching:
Although blood group genotyping has helped us to assume possible combinations of alloantibodies, identification of multiple alloantibodies is still a challenge to even seasoned blood bankers occasionally.
In this entry, I’d like to classify different ways to separate antibodies:

Physical separation
Adsorption and Elution
Known Antibody

Warm and cold autoantibodies can be adsorbed by autologous or allogeneic red cells in differential adsorption or by the use of reagent rabbit red cell stroma.
Adsorption of isohemagglutinins using “in house” rare reagent antiserum taken from a non-AB patient has been performed in a few occasions. For example, if a group A patient has anti-k, ideally, k negative, group B red cells should be used to adsorb patient’s anti-B. If it is not available and if the anti-k is more than 2+, adsorption can be done at 4oC with minimal loss of reactivity. If it is less than 2+, DTT treated cells can be used.           
Unknown antibodies (blind adsorption)
Let say that there are six alloantibodies in the sample: you just pick a random donor that may be positive for two of the six antigens; your eluate and the adsorbed plasma can be run against a regular panel and an enzyme treated panel. For example, anti-D,-C,-E,-K,-Fya and Jka are present, knowing the patient is D negative, a D- donor is used to adsorb patient’s plasma. It is likely that the donor is positive for at least Jka, if not also for Fya and likely K-. The enzyme panel on the eluate will show anti-Jka, and Anti-Fycan be revealed along with the anti-Jka in the untreated panel using with PEG or LISS. The panels done on the adsorbed serum will show anti-D,-C,-E and –K.  
Serological Differentiation
Variation in reactivity
See if there is a pattern that you recognize – for example, the two or three E+ panel cells are 4+ while others are less reactive. Strong/weak - consistent grading is key to detect combinations.

Beware of:
  • Uniform or variable - is it due to dosage? E.g., MNS, K, Lu, Duffy, Kidd, Do, Co.
  • Antigen expression varies among normal individuals.
  • Blood Group Systems and Collections showing variation in antigen expression: P1, Ii, Sid, Vel and HTLA including Ch/Rg, Gy/Hy, Cromer, Knops, Cartwright and Cost and York.
Avoiding known antibody(ies)
Knowing anti-c frequently accompanies anti-E, I would then compile a c- panel to reveal other antibody combinations.
1.     Commercially available soluble blood group substance: P1, Lea, Leb etc
2.     Known Ch- and Rg- plasma
3.     Dialyzed, pooled and pH adjusted urine as a source of Sda.
Membrane Modification (please look up abbreviations from AABB’s Technical Manual)
Membrane modification can be used to weaken or destroy selected red cell antigens
  1. Enzyme sensitive antigens : Fya, Fyb, MNS, EnaFS/PS(ficin or papain sensitive), Lu8,Ch, Rg, Pr, Yta, JMH, Ge2,Ge4,Tn, Mg, Mia/Vw, Cla, Jea, Ina, Inb and Nya. Of all these antigens,  EnaFS/PS, Ch, Rg, Pr, Yta, JMH, Lu8 and Inb are of high incidence.
  2. DTT sensitive antigens: Kell, Dombrock, Cartwright, LW, Indian and Lutheran system antigens. Note that Kel: 2,4,5,7,11,12,13,14,18,22 ;Yta; Lwa, Inb; Lu: Lub, 4,5,6,7,8,12,13,16 and 17 are of high incidence.
  3. ZZAP sensitive antigens: All of the DTT sensitive antigens as well as the following high incidence antigens Ge2,3,4,Hy, Gy, Yka,McCa/Kna
  4. EDTA-Glycine Acid EGA sensitive antigens: Kell system antigens, Erand Bg’s.
  5. AET sensitive antigens: Yta, Gy, Hy, Kna, McCa, Yka, LWa and  antigens in the Kell, Lutheran, and Cromer Blood Group Systems
  1. Distinguish the strongest antibody from a mixture (circumvent activity by using a selected antigen negative  panel or modify panel red cells with enzyme and/or chemicals (see below))
  2. Stronger reacting panagglutinin may mask weaker alloantibody (avoid interference by  modifying panel red cells) e.g., Anti-Kpb and anti-E can be identified by the use of ZZAP.
  3. Reveal HTLA-like reactivity (modify panel red cells to prevent interference). For example, AET treated panel may reveal an anti-Fya in the presence of anti-Kna.
Blood Banker’s sixth sense
I guess it comes with experience when antibody ID is your daily routine. Below are some of my “not-so-ancient” Chinese secrets.

My usual approach to panagglutination  is to see if there is variation in reactivity; that is, if the panagglutination is uniform, I would titrate the patient’s plasma to reveal the most reactive antibody(ies)  and at the same time, see if there is any “HTLA” behavior.
When I see increased reactivity with enzyme treated panel and there is no discernable pattern, I might think of a weak autoantibody being enhanced.
Sometimes a seemingly complicated case of weak multiple alloantibodies became a lot more straight forward when you work on a new sample collected 2-3 days later- remember the anamnestic immune response?
I always use RZR1(E+c-) to rule out anti-E in the presence of anti-c and RZR2 (C+e-) to rule out anti-C in the presence of anti-e.
I know there is/are more unidentified antibody(ies) when your phenotyped donor is incompatible.
I always ask if the patient has an antibody card when I encountered antibody ID difficulty. I remember a patient moved from Prince George, BC to Calgary with pure red cell aplasia; his wife told the nurse that they had to get “special blood” from Vancouver. Instead of doing our own extensive investigation, we phoned CBS Vancouver and learned that he had six alloantibodies; we continued to support him with monthly two-unit transfusions for the near two years before he passed away; he developed two more alloantibodies and a warm auto!
My dear fellow blood bankers, please share your techniques with us.


Blog post currently doesn't have any comments.
 Security code