Dealing With Cold Agglutinins

[dbarding13]

Hello All!

I know this subject has been discussed extensively.  It continues to be a source of confusion in my TS, due mostly in part to the lack of solid Blood Bankers (all generalists who rotate through).

Currently, our SOPs for working up suspected cold agglutinins include a "cold screen" at 4 degrees using Immucor Panoscreen I and II, and patient autocontrol.

If Cold Screen is indicative of a cold Autoantibody, a Tube Pre-warm screen is performed, and subsequent panel, if warranted.

This is so confusing to my techs.  Last night I received a call at home from my 2nd shift tech, saying she had a patient coming in for transfusion for one unit of red cells, with no prior history with us, was found to be A+, Negative antibody screen in solid phase on Echo, BUT positive Immediate spin crossmatches.  She then proceeded to tell me she "prewarmed the crossmatches and then they were fine."  She wanted to know how to result the Cold Auto!!!!!!!!!!!!!!!

After swallowing to remove my heart from my throat, I explained that this was not a conclusive "work-up" for a Cold auto.  I talked her through the possibility of Rouleaux (She said she only saw "clumps" not "coins" microscopically); the possibility of an A2 or subgroup, but she assured me the reverse reactions were consistent with A.  I then told her the series of procedures to follow for a suspected cold agglutinin, all of which are in our SOPs.  I suspected Rouleaux, and asked her to follow the saline replacement procedure, explaining that if it was true agglutination, saline replacement would not "correct" it.  She performed saline replacement and the IS crossmatches were as smooth as silk. 

 

I guess my question is, does anyone have any suggestions for making this more clear to our staff?  Also, what are your procedures for identifying a cold agglutinin, or autoagglutinin, both specific and non-specific?  How about a flow-chart to make it easier for techs to determine what to do next?  Any info, SOP, chart anyone is willing to share will be so appreciated right now, and worth its weight in GOLD!!

Dawn

[SMILLER]

One thing I can say at this point, is that I do not believe many labs still bother doing a "cold screen".  It just is not useful.  If you are having interference in your screen or crossmatch, you simply use whatever techniques are appropriate to try to clear it up to ensure that no significant antibodies are present.

scott

[dbarding13]

Thank you, Scott.  I was afraid of that.  But you wouldn't just pre-warm an immediate spin crossmatch without any other conlusive evidence of a cold auto, would you?  I guess I am just very leery of prewarm in the hands of the inexperienced in an attempt to just get rid of bothersome reactions they aren't expecting.  I can see the logic in the case I sited above, because the screen was negative, but you have to exclude other possibilities for the imcompatible reaction, not just assume it is a cold and try to warm it away, right?  Or am I being too strict and old school here?

 

[SMILLER]

No, I think you are right about not prewarming a crossmatch at IS.  If we used a prewarm technique for a XM or screen or whatever, we would be carrying it through a 37 incubation and AHG.  That would be for those especially pesky cold agglutinins. 

As you already indicated, the most common fix for a snotty IS is a saline replacement.  (Often needed when a XM is done later on, and the specimen is brought out of the fridge).

The "mini cold panel" is uncecessary because in most cases, the first attempt to clear a gel screen is to go to tube anyway, where interference of any kind is much less likely compared to gel.

Regardless of what SOP you want to use.  You may want to review those procedures and update your BB manual as necessary.  You techs should not be working up stuff willy-nilly!

Scott

 

 

[Malcolm Needs ☆]

Cold agglutinins that do not react at 30^oC and above are a very expensive waste of time that cause unwarranted anxiety to everyone who does not realise this fact.

[dbarding13]

40 minutes ago, Malcolm Needs said:

Cold agglutinins that do not react at 30^oC and above are a very expensive waste of time that cause unwarranted anxiety to everyone who does not realise this fact.

Agree, Malcolm.  But if the antibody screen is negative through AHG, but IS crossmatches are rolling off questionable, aren't we obligated identify cold agglutinins, if all other possibilities have been excluded?  Or can we just deem a probable cold agglutinin not of clinical significance?  If "everything else" has been excluded, rouleaux, alloantibodies, etc., but the IS is still "chunky" weak macro or microscopically, would you just do a prewarm crossmatch through 37 degree and AHG phases, and call it good if negative?

Thank you, Scott, for your very valuable input, also!!

Edited March 27, 2016 by dbarding13
Correct typo

[Malcolm Needs ☆]

41 minutes ago, dbarding13 said:

If "everything else" has been excluded, rouleaux, alloantibodies, etc., but the IS is still "chunky" weak macro or microscopically, would you just do a prewarm crossmatch through 37 degree and AHG phases, and call it good if negative?

Yes!

[SMILLER]

Just in case what is making you nervous is the missing "ABO Incompatibility" test at IS when doing a pre-warm XM, the 37 incubation covers that as well, if I am not mistaken.

Scott

[dbarding13]

OK-thank you both so much!  Happy Easter!!

[BloodBankBlake]

What about pre-warming a clinically significant antibody away like an Anti-Vel?

[Malcolm Needs ☆]

We have been using pre-warming in the UK since before I started in Blood Transfusion (circa 1973) and we have never had a clinically significant transfusion reaction caused by warming away an antibody in all that time.

Yes, there have been occasions when, for example, an anti-S has disappeared by pre-warming, but, if you look in most text books, and all reliable text books, anti-S is only rarely clinically significant - and certainly none of those that we have "warmed away" have caused any transfusion reactions at all.

There was one case of an anti-Vel causing a fatal transfusion reaction, BUT, that was not missed through pre-warming; that was missed because EDTA plasma was used, and the anti-Vel could only be detected in serum (confirmed by the IBGRL), and so I think that the worries about pre-warming are vastly over estimated.

It is vital to keep up with competency for this technique, as with any other technique, but probably more so with this technique.

Edited March 29, 2016 by Malcolm Needs
Addition of last sentence.

[StevenB]

Question for you dbarding13:  When confronted with an IS incompatible crossmatch and your ECHO screen is negative, are you performing any IS testing in tubes with screen cells and the autocontrol?  

The patient's reverse type will provide you some information as long as the patient is group A, B or AB (you would obviously have a tube or two that are nonreactive).  If the patient is group O however, the reverse type can not be depended on as a "control" for your IS crossmatch.  In the case of a group O patient, an incompatible crossmatch could be due to anything; rouleaux, cold auto, low inc., high inc., newly formed clincially significant antibody and so on.

My point is, testing screen cells and an autocontrol at initial spin can be very helpful in deciding what the issue may be.  Without these, a tech is only blindly guessing at what is causing the incompatibility at IS.  

In the example you presented, I find it interesting that the rouleaux was not seen in the reverse typing.  In my experience, it is not uncommon for rouleaux to demonstrate variable "reactivity" from cell to cell.  Nice call on having the tech do a saline replacement!  It's a simple and quick procedure that will often clear up your IS issues.

In regards to a "cold screen"; all that will tell you is that you have an antibody that reacts at 4C.  It does not tell you that the same antibody is responsible for the room temperature reactivity.

[goodchild]

4 minutes ago, StevenB said:

My point is, testing screen cells and an autocontrol at initial spin can be very helpful in deciding what the issue may be.  Without these, a tech is only blindly guessing at what is causing the incompatibility at IS.  

In the example you presented, I find it interesting that the rouleaux was not seen in the reverse typing.

This is essentially why we still have a procedure for "cold antibody screen." The AABB technical manual also includes this as their procedure for ABO discrepancies.

I also thought it was interesting that the rouleaux wasn't seen in reverse typing but I'm not familiar with the ECHO.

[dbarding13]

Yes, like Quality Guy, this is why we still have the procedure for the cold screen.  We test the patient auto along with screen cells at IS, and 4 degrees.  As far as the rouleaux not being an issue with the reverse typing, the Echo testing platform cannot reliably detect hemagglutination reactions that are graded as 1+ or less in test tube methodology.  The IS XM reactions with the example above were graded as W+ by the tech.  There may or may not have been something "funny" about the reverse on the Echo, but it did not trigger a NTD (no type determined) flag.  If it had, the type would have to be performed in tube.  We do see NTD fairly often when the reaction strengths are very weak in reverse typing.

[SMILLER]

Someone check me on this, but I believe that if you do a "cold screen" on any patient, a large number will come out "positive', even though for almost all of these there will be no interference in usual screening methods.

 

Scott

[Malcolm Needs ☆]

55 minutes ago, SMILLER said:

Someone check me on this, but I believe that if you do a "cold screen" on any patient, a large number will come out "positive', even though for almost all of these there will be no interference in usual screening methods.

 

Scott

Completely and utterly 100% correct Scott.

[StevenB]

Just want to reemphasize this: The reactivity you see at 4C can not be compared to what you see at room temp...regardless of what the Technical Manual says.  It's at least a 16C change in the test parameter...without additional testing, I'm not sure how you can justify saying the reactions at both phases are due to the same antibody.

Thinking on it...the only way I would ever consider reactivity at RT and 4C to be caused by the same antibody would be if I performed a cold autoadsorption and the reactivity disappeared in both test phases.

Needless to say, lol, I'm not too fond of the 4C screen.  The 4C incubation for enhancing weak reverse reactions is a good technique as long as you include your control cells.  The 4C screen....I'm not sure I have used that test in over a couple of decades, if ever.

[Malcolm Needs ☆]

Agreed StevenB, but, unless your laboratories room temperature is particularly warm (30^oC or above), the antibody detected at RT is still not certain to be clinically significant - which begs the question, why perform the screen at that temperature?

Edited March 30, 2016 by Malcolm Needs
I put a full stop instead of a question mark.

[SMILLER]

Why do we make auto-anti-I in the first place?  Sometimes I think God just likes to mess with us...

 

Scott

[StevenB]

1 hour ago, Malcolm Needs said:

Agreed StevenB, but, unless your laboratories room temperature is particularly warm (30^oC or above), the antibody detected at RT is still not certain to be clinically significant - which begs the question, why perform the screen at that temperature?

Lol....great question Malcolm!  I know the MI for PeG specifically states to run an initial spin test prior to adding PeG to the test, so we are stuck.  Immucor's LISS does not, yet we run the first panel at initial spin, prior to adding the LISS.  Habit I guess.  We do consider it to be a "control" for the reverse type results which helps as we see rouleaux all too often with the plasma testing or that pesky marauding anti-M at IS.  I can think of a number of "what ifs", but they happen so rarely it's not worth the conversation.

[Clarest]

As the prewarming technique was also mentioned here, I am wondering if my confusion with the Notes below in the Method 3-6 Prewarming Procedure of AABB Technical Manual (20th ed.) could also be discussed here.

"2.      Cold-reactive antibodies may not be detectable when room temperature saline instead of 37 C saline is used in the wash step.³ The use of room temperature saline may avoid the elution of clinically significant antibody(ies) from reagent red cells that can occur with the use of 37 C saline. Some strong cold-reactive autoantibodies, however, may still react and therefore require the use of 37 C saline to avoid their detection."

Why are the cold-reactive antibodies not be detected when washing with RT saline, but are detected with a warm saline wash? I was thinking the opposite way And the last sentence in the above notes seems to confirm my thoughts 

[Neil Blumberg]

I don't think the AABB comments are evidence based.  Washing with 37 degree saline is extremely unlikely to cause false negatives with clinically significant antibodies,  and I'm unaware of any evidence that this is so.  Any such antibody would be very low affinity to be washed away by saline at any temperature, and unlikely to have in vivo/clinical significance. 

As argued persuasively above by Malcolm Needs, anything that doesn't react at 30 degrees or above in typical serologic testing isn't going to cause clinical problems.  Patients are neither at 30 degrees nor centrifuged :).  Our serologic techniques are overly sensitive,  in general,  for clinically insignificant agglutinins. 

No need for cold panels ever, with rare exception, and more for intellectual curiosity than clinical decision making.  Perhaps a mini-cold screen someetimes just to confirm you are indeed detecting a weak cold agglutinin in 37 degree testing, which disappears with prewarm technique. 

Like Malcolm, I've never seen a patient with an hemolytic reaction due to an antibody that disappears with prewarming, in close to 50 years of clinical practice.  I know there are in vitro examples of clinically significant antibodies that weaken or disappear with prewarm, but I've never seen any clinical consequences.

Edited January 29 by Neil Blumberg

[snance]

The following references may be of interest:

Leger RM, Garratty G. Weakening or loss of antibody reactivity after prewarm technique. Transfusion. 2003 Nov;43(11):1611-4. doi: 10.1046/j.1537-2995.2003.00563.x. PMID: 14617322

From the abstract of the above publication:

 

"Results: PW PBS-IAT and PW LISS-IAT showed that 40 and 47 percent of antibodies were weakened, respectively, compared to LISS-IAT; reactivity for 14 percent of antibodies was completely lost by each PW method. By PW PBS-IAT, 34 percent of antibodies were weakened compared to PBS-IAT. PW PEG-IAT showed weakened reactivity by 56 percent of antibodies compared to PEG-IAT; reactivity of seven out of seven PEG-dependent antibodies was completely lost. Of 67 antibodies, 19 percent were defined as low affinity. Of 64 samples tested by the PW method and for low-affinity antibodies, only 6 of 30 that showed decreased reactivity by the PW method appeared to be due to low-affinity antibodies; only 6 of 12 samples that appeared to contain low-affinity antibodies also showed decreased reactivity by the PW method.

Conclusion: Antibody reactivity of potentially clinically significant antibodies can be decreased or missed by PW methods. Antibody enhancement media does not ensure antibody detection by PW methods."

Other publications of possible interest:

Storry JR, Mallory D. Misidentification of anti-Vel due to inappropriate use of prewarming and adsorption techniques. Immunohematology. 1994;10(3):83-6. PMID: 15945800.

Hopkins C, Walters TK. Thermal amplitude test. Immunohematology. 2013;29(2):49-50. PMID: 24094235.

Dupuis S. Use of the prewarm method for detecting clinically significant alloantibodies in the presence of cold autoantibodies. Immunohematology. 2018 Dec;34(4):148-150. PMID: 30624948.

[Jessica Reed]

On 1/28/2024 at 11:15 AM, Clarest said:

As the prewarming technique was also mentioned here, I am wondering if my confusion with the Notes below in the Method 3-6 Prewarming Procedure of AABB Technical Manual (20th ed.) could also be discussed here.

"2.      Cold-reactive antibodies may not be detectable when room temperature saline instead of 37 C saline is used in the wash step.³ The use of room temperature saline may avoid the elution of clinically significant antibody(ies) from reagent red cells that can occur with the use of 37 C saline. Some strong cold-reactive autoantibodies, however, may still react and therefore require the use of 37 C saline to avoid their detection."

Why are the cold-reactive antibodies not be detected when washing with RT saline, but are detected with a warm saline wash? I was thinking the opposite way And the last sentence in the above notes seems to confirm my thoughts 

I read this as washing with room temp saline is sufficient to remove reactivity from cold-reactive antibodies. Some strong ones may require washing with warmed saline, but you risk losing some clinically significant antibodies. In practice, most of our cold antibodies picked up in gel do not require pre-warm and it's unnecessary extra time. Our current procedure is to go straight from our most sensitive method (gel) to our least sensitive method (PW PBS-IAT) and I'd like to change that! Thank you @snance for the references!

[REN_NH]

Does anyone have a flowchart for the workup of cold-reactive antibodies? I'm a Generalist using GEL BT, and GEL anti-IgG with tube IS XM and feedback on this is much appreciated as I am also working on updating policies.