Tube Antibody Titers: Yes or No to Enhancement?

[sgoertzen]

We have a busy Fetal/Maternal Center for high risk pregnancies at our hospital.  Our primary method of testing is solid phase (Capture) for antibody detection and identification, and our secondary method is PEG tube testing.  The perinatologists are requesting antibody titers on all pregnant women with clinically significant alloantibodies.  We have repeatedly seen clinically significant antibodies that react 3+ to 4+ with solid phase methodology that end up being "too weak to titer" when we move them to the tube for saline/37C/AHG titering.  Even the non-diluted plasma reacts negatively in the tube at 37C and AHG without any enhancement.  This is confusing to the perinatologists and I understand why.  It doesn't make sense to them that a 4+ strong antibody can be too weak to titer.  Does anyone else frequently experience this?   I'm just curious whether anyone is routinely using any kind of enhancement when performing antibody titers on known clinically significant antibodies (such as CcDEe, K, Fy, Jk, etc).  I know that the CAP ABT Survey choices for titer "diluent" are saline, 0.5% albumin, 6% albumin, 22% albumin and their "technique" choices include various versions of IS, RT, 37C, AHG, DTT (we're using saline w/ 37C incubation and poly AHG).  With the exclusion of a gel titer, if you have a procedure (with criteria) for performing an "enhanced" titer, would you be willing to share it?  For those of you using albumin for diluent, do you find this helps enhance the reaction of the antibody and what strength of albumin do you use? 

[Ensis01]

My experience is that the BB reports out the antibody identification. Never the reactivity! If a titer is ordered the only thing reported is the titer or “too weak to titer”. 
As the rise in titer is the most relevant result, consistency in method and technique is very important, both within your hospital system and the reference lab you use. 
Physicians are interested in your results not the process. Keep that simple. If they have questions your Medical Director can enlighten them. 

[butlermom]

I get what you're saying, but remember, with the antibody screen and panel you are using sensitive methods to detect the presence (and i.d.) of a clinically significant antibody. The titer is merely measuring the "concentration," if you will, of the antibody in solution. They are really two unrelated attributes. We use gel method for screens and i.d., but perform the tube method titer in saline using the CAP recommended Uniform Method. 

[exlimey]

An excellent discussion point. I think many others have similar questions and concerns. The have been several other threads on this forum with similar subject matter.

As an Old Fart, I feel obliged to spout some (un-referenced) history. Most of the original work on clinical significance of antibodies in pregnancies was done in the absence of potentiators and definitely before the use of (semi)automated test systems. I think it was a "saline-IAT" using 22% albumin (BSA) as a diluent. Most of those antibodies were anti-D, for obvious reasons. There's not much out there in the literature in terms of controlled or organized studies regarding other specificities. There are a fair number of one-of-a-kind case studies, but most of the stuff is retrospective analysis of data. Basically, other than anti-D, nobody really knows what an antibody titer means, but as Ensis01 suggests, detecting a change in titer (increase) may be more important.

In an era when basic tube shaking is going away, it only makes sense (we have no other option) to convert to the new techniques and equipment, but I suspect that it has the potential to further confuse an issue which already has enough confusion to (dis)satisfy everyone. I don't envy anyone handling this hairball.

As a last thought...the high-powered potentiators (and techniques) used today don't reflect what's going on in vivo. Arguably, if one ignored the 22% BSA diluent, the saline-IAT is a better mimic of the in vivo scenario.

[David Saikin]

Tubes without enhancement is what I have always used.  I don't care for the CAP method as it is actually a 1:3 serial dilution vs the 1:2 which has classically been used.  (maybe it's ok because the "new" method is read microscopically vs macro read for the classic method).

[exlimey]

3 hours ago, David Saikin said:

(maybe it's ok because the "new" method is read microscopically vs macro read for the classic method).

Hah ! Excellent point, David. I wonder how much emphasis should be put on those "microscopic" reactions, especially when the endpoint a titration is most often defined as the "last MACROscopic (or 1+) reaction"? How do the Powers-That-Be justify that little nugget ?

Can you imagine resulting-out a "change in titer" based on a microscopic reaction ? Talk about piling-on to the already acknowledged confusion level.

Edited July 19, 2021 by exlimey

[carolyn swickard]

To answer your first question - Yes, we have seen several antibodies On ECHO/LUMINA) that we can not see in the titers (saline only / 2 fold dilutions/ 30 min inc).  Especially Anti-E.

I once talked to a reference Lab about titers  (we had an anti-G - such fun) and they felt it was most important to try and replicate the In-Vivo condition in the mother for clinical significance - therefore - no enhancement medias and heterozygous test cells, where possible.   That is what we have done since and we just have the Med Director answer any questions they might have (after a through briefing, of course!).  Hope that helps.  

Titers in gel are always higher than titers in tubes (see CAP results for the various titer methods if you can).  

Consistency in method and full disclosure on method and clinically significant ranges should be the most important part of titers.  We restrict ours to only daycrew techs with proven competency testing (and still hope for the best!).

There was an article:  W. John Judd for the Scientific Section Coordinating Committee of the AABB, Practice Guidelines for Prenatal and Perinatal Immunohematology, revisited Transfusion 2001,41:1445-1452, that answered a lot of my questions - if you can find it.  It was supposed to be revisited each decade, but I never found anything in or around 2011.  May have missed it...

[John C. Staley]

Just a thought, I have not seen anyone mention in this thread, testing the current sample in parallel with the previous sample.  We would start with the very first sample and freeze what was left after the initial titration.  We would then thaw and run it in parallel with the next sample.  This was an attempt to mitigate the, hopefully, minor differences in technique between techs and give us an accurate picture of any increase in antibody levels.

[Malcolm Needs ★]

6 hours ago, John C. Staley said:

Just a thought, I have not seen anyone mention in this thread, testing the current sample in parallel with the previous sample.  We would start with the very first sample and freeze what was left after the initial titration.  We would then thaw and run it in parallel with the next sample.  This was an attempt to mitigate the, hopefully, minor differences in technique between techs and give us an accurate picture of any increase in antibody levels.

Quite right John.  In the UK, we ALWAYS test the previous sample with the latest sample (unless there is a legitimate reason why the previous sample is not available - sample too small, freezer broke down, Malcolm dropped it, etc!).  This test is essential in my book, as antigen expression can vary hugely from one donor to another, resulting in either a falsely high titre or a falsely low titre - and either can be dangerous.

[mrmic]

I agree with everyone!

However, the specific antibody ID methods and/or the titration methods alone do not affect the decision process for the pregnancy. 

A significant antibody identified or a significant change in the titration result does.

But, only does if there have been reported cases of harm to the mother and/or infant based on the specific antibody identification and using the specific method of titration.

A lot of the literature in the (ancient?) past showing correlation with potential harm to the infant were done with saline and/or dilutions of percentages of bovine albumin.  Some of us "old farts" still remember those old reports.

With all the new reagents or methods that are available now, it requires BB technologists to help initiate the investigation and follow up with the physians to report cases of a positive or negative outcomes of pregnancies with the different laboratory methods.  Based on that knowledge, the technologist can select the best antibody identification and titration methods for following pregnancies and the physician can make the best medical plan of action for the pregnancy.

So the best place to start; researching literature then working with obgyn physicians in your area to correlate the best laboratory methods with the best patients' outcomes.

[Malcolm Needs ★]

It was ever thus mrmic, but don't for get the transplant physicians.