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Reasons for Inconclusive Antibody Identification

A Blog from Eric Ching:

After the blood group antigens last year, my blogs for 2017 will be focusing on antibodies, techniques and identification.

La SCMT est heureuse d’annoncer que le blogue d'Eric Ching,  "Raisons pour identification d'anticorps non concluante"  est maintenant aussi disponible en français. ​
                                       Reasons for Inconclusive Antibody Identification

I am sure most of us have encountered inconclusive antibody identification results many times. It may happen in the worst circumstances - urgent needs, understaff, inadequate reagents and so on. When I look back from my comfortable retirement, I can categorize reasons why we don’t see straight forward antibody ID results:

1.  Antibody is weak or at borderline detection level; it may not be as reproducible:
  • For IS, RT (20-24 C) and Saline-IAT: increase serum/cell ratio, manipulate incubation temperature (inconclusive anti-M, anti-P1, anti-Lea, etc. can be enhanced using a lower than RT but not at 4oC to avoid interference from auto anti-I)
  • Enhancement media: LISS, PEG
  • Enzyme: papain, ficin
  • Acidify plasma/serum to detect anti-M
  • Let’s not forget some complement fixing weak anti-Kidd antibodies can be detected using fresh serum sample and polyspecific AHG with papain treated reagent red cells
  • Obstetric and transfusion history may prompt you to ask for a new sample in a day or two to see if you can catch it later!  Remember the time sequence of secondary immune response?!
2.  Presence of warm and/or cold reactive autoantibody
  • DAT or Auto Control is usually 2+ or stronger before “free” autoantibody is present in serum/plasma
  • Use of enzyme will reveal their presence (pan agglutination). Use ZZAP or  rabbit red cell stroma to avoid interference. Caution: prewarm decreases sensitivity.
3.  Interference of HLA and the so-called “HTLA” antibody
  •  Mostly weak to 1+, only a few 1-2+; no pattern, poor reproducibility; titrate 4 or more tubes at same strength (Tip: do a 1/10 dilution to see if there is no change -  2 tubes instead of 5 - learned this from colleagues in Ottawa)
  • Adsorption with human platelet concentrate to remove HLA antibodies or use of 6% AET or 20% CDP treated panel to bypass interference. Beware of limitations of each method.
4.  Multiple alloantibodies
  • It is particularly difficult if multiple alloantibodies are reactive at the same phase; titration, selected panel, selected cells, chemical or enzymatic treatment of panels, adsorption and elution are useful tools for their identification. Please read my previous blog (Divide and Conquer) on this subject.
5.  Single or multiple alloantibodies to low incidence antigens
  • For academic interest only, the late John Case once told us that he encountered a patient with more than 10  alloantibodies to low incidence antigens! You can probably experience it when you work in reference labs with panels of antigen positive of many “lows”.
6.  Antibody to antigen not shown on the antigen profile form (panel sheet)
  • An antibody ID reported as possible HTLA because of the titration characteristics turned out to be an anti-Cob; the technologist didn’t notice that the information was under the special typing column. We titrated a few samples of anti-Cob, more than half of them showed HTLA titration characteristics - personal observation, not reported)
 7.  Antibody to a high incidence antigen
  • Pan agglutination with uniform reactivity is always challenging, I am more fearful if the autologous control is negative and it is a STAT! Good communications with your medical director and the attending physician is paramount in this scenario. In most cases, you are looking at an alloantibody to a high incidence antigen. In some cases, there could be combinations of alloantibodies reactive at the same strength. In the worse situation, there is a combination of multiple alloantibodies with one to a high incidence antigen. For example, anti-e + anti-Fya + anti-Kpb.
8.  Antibody to drugs
  • Drug-induced warm-reactive autoantibody, though less frequent in recent years, can confuse investigation when present at low level; enzyme enhancement and drug history (eg. fludarabine and methyldopa) may be helpful.
  • Decreased incidence of drug induced antibody causing inconclusive antibody identification in recent years because:
    • DAT or autologous control is no longer part of routine pre transfusion testing
    • Plasma has replaced serum thus preventing the detection of in-vitro hemolysis caused by the drug-induced immune complex mechanism.
    • Less frequent use of alpha methyldopa to treat hypertension.
9.  Antibody to ingredients of enhancement media/preservative solutions
  • Panagglutination is the scary hallmark resembling a warm-reactive autoantibody or an alloantibody to a high frequency antigen.
  • Autologous control is positive but DAT is negative: antibody to enhancement media
  • In the case of an antibody to an ingredient in the preservative solution, both autologous control and DAT are negative resembling an alloantibody directed against a high frequency antigen! BUT washed panel cells are NON REACTIVE with patient’s plasma!
  • Proof - adding manufacturer’s diluent to the autologous control will become reactive.
10.  Enzyme specific autoantibody
  • Presence of  “auto-pap” or “auto-ficin” will mask the detection of clinically significant alloantibody. Switching to ficin or papain may be helpful.
  • Patients with scoliosis have higher incidence of auto-papain (personal unpublished observation).
11.  Misinterpretation of results
  • Less experienced technologist may not aware that there is a column special antigen typing, e.g. Anti-Cob was missed and was reported as possible anti-Bg!
12.  Technical errors
  • result interpretation, over-read and under-read microscopic results, faulty cell washer, improper manual washing, instrument failure etc.
13.  Clerical errors
  • transcription, manual sampling, etc.
I had my fair share of 12 and 13 :-(  !

Best regards,



In our blood bank, the antibody identification is repeated every two weeks, cold identification is a common practice if there’s an extra weak reaction on the panel results, extra reactions in forward and reverse grouping.
3/13/2019 4:53:20 AM

CSTM Board of Directors
Thanks for your inquiry and blog posting. We would prefer not to discuss individual results through the blog. The laboratory that issued your report will have a medical director. I suggest that you - or your doctor - may wish to speak to the laboratory medical director about your results. You can contact the laboratory physician by calling the laboratory that issued the report.
3/11/2018 4:52:26 PM

Kaitlyn Jackson
I have Anti-fyA antibodies and my antibody screen also had a note attached stating “anti-fyA was demonstrated at the AHG phase of testings additional weaker reactivity is seen only by solid phase testing that cannot be attributed to a specific antibody. All other common clinically significant antibodies have been ruled out with PEG enhancement. The inconclusive reactivity is too weak to titer” what does this mean? I am pregnant and being monitored of HDF/N
2/21/2018 11:58:13 PM

Eric Ching
Hi Heather, I am happy to learn that you do not produce any blood group antibody following multiple transfusions. You have to thank your parents that you have inherited most or almost all of the most immunogenic (potent) blood group antigens that can elicit antibody production. We refer patients like yourself as "non responders". Take care, Eric
4/9/2017 10:52:28 PM

CSTM Webmaster
Hi Heather,

Eric is out of the country at the moment, but he will post a reply to your question upon his return.

Thank you.

CSTM Webmaster
4/8/2017 7:35:02 AM

I have had hundreds of transfusions but have (fortunately) not developed any antibodies. Is there a way to figure out why this is the case and potentially use this information to help other patients?
4/7/2017 1:29:51 PM

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