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Cold Agglutinins - Tech Tips

A Blog from Eric Ching:

A case on cold agglutinin was presented at the recent CSTM Prince George education meeting; I was surprised that the incubation time used was 24 hours! 
In the 70’s, working at the Calgary Red Cross, a titration of cold agglutinin was incubated for 30 minutes at 4oC.
When I started working part time at the Foothills Hospital, there was a procedure they used called cold agglutinin screen which called for an incubation time of only 15 minutes. It was set up and had been used since the late 60’s to establish if a cold agglutinin was present to cause non-specific reactions or panagglutination or for the diagnosis of patients  for cold agglutinin disease. Titration of cold agglutinins was also incubated for 15 minutes only. This procedure has been used by local hematologists for decades! Decades of clinical validation certainly justified its continued use. (AABB Technical Manual- 30-60 minute incubation for cold agglutinin specificity determination; 60-120 minutes for titre)
  • 4 labelled tubes: Auto, SI, SII and Cord
  • two drops of patient’s serum/plasma into each tube followed by adding patient’s red cells, screening cells and group O cord cells
  • mix and incubate for 15 minutes at 4oC
  • spin and read. 
A 3-4+ would result in a patient’s cold agglutinin titre. A 1/10 dilution of patient’s sample was made on negative result and repeated to see if prozone had occurred. I believe this is the only incidence in routine blood group serology where prozone could cause a false negative result. I think prozone is different from the “ blocking phenomenon” of reagent anti-D with a heavily maternal IgG anti-D coated baby red cells where steric hindrance and occupied sites are the cause of a false negative result rather than antibody excess in the prozone phenomenon. Please let me know if I misunderstand this part.
Please share your thoughts on this procedure and I am sure there are variations at different sites.
Technical Challenges of a strong cold auto:
  • ABO RH determination: use inert AB serum or clone control to ensure patient’s red cells do not autoagglutinate - if they do, you can wash patient red cells 3-6 x using saline kept at 37oC. My favourite is at 45oC which I found is more effective as it won’t cause significant hemolysis. ZZAP (WARMTM) is always effective as the 0.2M DTT in it destroys the joint chain of the pentameric IgM, thus rendering the cold agglutinins non-agglutinable. If DTT is available in your lab, you can just make a 0.01M solution.
  • Cold autoadsorption: do you have a special collection procedure which calls for taking two beakers, one with ice and the other saline warmed to 40-45oC, keep the drawn EDTA tube in warm water and place the clotted sample in ice?
  • To avoid carry-over causing a falsely high titre, change disposable pipette or pipette tip to transfer from one tube to the next. Use 0.5 ml 2 fold dilutions to have better reproducibility.
  • Always ask for patient’s diagnosis (current or past, if available) which may clue you in on specificity.
    • Anti-I: Cold Agglutinin Disease (aka cold hemagglutinin diseases, cold agglutinin syndrome), Atypical pneumonia due to Mycoplasma pneumonia and Raynaud phenomenon and   lymphoproliferative disorders
    • Anti-i:  Infectious mononucleosis, Wadenstrom’s macroglobulinemia and lymphoma
    • Anti-P: PCH IgG biphasic hemolysin aka Donath-Landsteiner antibody
    • Anti-Pr: rare IgG cold auto that could cause acute intravascular hemolysis
  • For academic interests, Ii are NOT the only antigens that show significant difference in reactivity between adult and cord red cells.
    • Adult>Cord: I, Lud, Fl,Rx
    • Cord>Adult: i, Vo, Li
    • Adult=Cord: Pr, Me, Om, Ju
  • More information can be found in Geoff Daniel’s text book or Roelcke’s review in Trans Med Rev 1989; 3:140-166, if you want to see how sialidase, papain and human milk can differentiate various specificities.
  • Wide thermal range of cold agglutinin is more clinically significant than concentration (titre). Using a prewarm technique, I used to set up 4 temperature points to determine the thermal range: 37oC, 30oC and RT, I would start off with the 37oC and stop at the temperature at which the reaction is seen.
I recall on a few occasions, usually in early winters, my boss, Dr. BA Ruether, came to the blood bank with his residents and medical students.  He would ask a tech to find an EDTA sample from the fridge. He would say "notice the abnormal  plasma colour and no free flowing red cells!"  He would then ask, "What do you expect the hematology results would be?"
I hope a hematopathologist will give our technical colleagues a brief review on the lab findings of cold agglutinin syndrome.
I welcome your input and feedback.


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