Antibody Titers Gel vs. Tube

[SarahE 0]

We are in the process of validating antibody titers on the Ortho Vision.  We are having trouble doing comparison studies between the gel method and our current tube method, since gel is so much more sensitive.  I'd like to know what other facilities have done when validating the gel process.  Is there an increase in the value that is considered "critical" for physicians?  Do you use the reciprocal of the 1+ reaction, or the W+ reaction? 

[Baby Banker 85]

I think Johns Hopkins does titres in gel.  We looked at it a few years ago for our isohemagglutinin titres, but decided to stay with tubes.

[Townsend 90]

We do our titers in gel (ABO titers only as we are not a delivering hospital; pediatric only).  Any positive reaction seen in gel should be interpreted as 1+ since there is no w+ interp in the MTS gel interp. guide - there is also no w+ on the Vision.  So, our end-point is the dilution with the last 1+ positive. Our results have correlated well with the CAP samples done in tube when looking at all of the methods reported, but we have only done these for about a year now (only about 3 CAP survey's so far).

We haven't looked at bringing this onto the Vision yet..... all though I am aware that it is now able to do the dilutions for both the buffered card and IgG titer reactions; so maybe with the next software upgrade???  Would love to hear back if you successfully implement the titers on the Vision!

Stephanie

[Malcolm Needs ★ 4,910]

IN the UK, the NHSBT (at least) has been performing titrations of all antibodies, of all specificities, in gel, after an extensive amount of work performed by my friend Gordon Burgess showed that there was very good correlation between these titres and those obtained by tube technique.

[TreeMoss 71]

The AABB Technical Manual states that antibody titers should not be performed using gel technology, so we revised our procedure to go back to tube method.

 

[galvania 804]

Does the AABB state a reason, Treemoss?

[SMILLER 870]

On p. 563 of AABB Tech Manual 18th edition, it only mentions that titer methods other than "saline AHG 60 minute incubation" in tube may result in higher titers and "should be validated with clinical findings" (see Malcolm's post, above).  So it does not seem to say one cannot use gel or other methods, just that you need to document validation.

I have always been a bit uncomfortable with identifying an antibody with gel (for a prenatal), then doing the titers in tube.  But then again, I guess it is the comparison of the series of tube titers that they are looking at.

Scott

[LCoronado 50]

We are a small facility which uses manual gel technology. We rarely see titers ordered.  However, when reporting an antibody on a pregnant patient (detected and identified in gel) we faced this dilemma: the antibody was so weak it was not detectable in tube. We then started using gel for titers with the understanding that it is the change in titer over time that is significant.  For proficiency, we use the split sample method every 6 months.  CAP and State (NJ)  inspectors have been OK with this.

[lpregeno 17]

We were doing titers in the gel and kept failing our CAP surveys for titers. They were always one titer too high (CAP gives you a 3 titer range the results can be within to pass). After investigation (I was new at this job at the time) I found where the Technical Manual says that they shouldn't be performed in gel. (Like SMILLER says above). It goes on to state that there is a danger with interpretation by the physician and the higher results of unnecessary invasive procedures performed due to that combination. They, strangely, do not tell what studies they base that comment on. We switched to the tube method and haven't failed a survey since.

We are in the process of deciding on new Blood Bank automation. When we saw the Vision, that seemed to be one of the "hot" selling points - the ability to perform titers. We questioned them about how they felt about it being contrary to AABB recommendations to do titers in the gel. The response was something like, "Do you want it to be faster and more hands-off or more exact? We would rather see it be more hands-off." (Not a direct quote, but you get the picture.) Personally, I would prefer the "exact" results to one that may or may not be too high. I would also prefer to pass my CAP competency surveys!

Just saying.

[SMILLER 870]

 "Do you want it to be faster and more hands-off or more exact?"  They really said that?  Yikes!

Scott

[jalomahe 132]

We do our titers in tube. Years back when a lot of places had switched or were getting ready to switch to gel there was a conversation about the difference in titers due to sensitivity of gel. The basic conclusion at least in our geographical patient care area: we didn't want physicians to be getting different titers from different labs solely due to differing methodologies as it could lead to unnecessary concern/procedures for the patient. So we all stick with tube method.

In those instances where we detect the antibody by a more sensitive method i.e. gel or Capture but the titer is negative then we report the antibody titer as less than 1 (<1).

Jan

 

[Sandy L 113]

I am looking at the Participant Summary for latest CAP proficiency, anti-D titer.  For tube testing using the "uniform procedure tube method" that CAP suggests, they reported the following results: 333 participants, mode 64, consensus range 16 to 256.  For Gel testing "uniform procedure gel method", 138 participants, mode 256, consensus range 64 to 1024.  Per CAP, Consensus is determined by the Mode +/- two of the most frequent titers.

It looks like in the previous 2 surveys for the gel anti-D titers, the mode for gel was 1 to 2 dilutions higher than tube, but so was the consensus range.  It seems like there are a fair number of labs reporting gel and if you report that as your method you should be compared to other gel titer users.  I would also think as more instruments are implemented that can do gel titers, the number reporting gel will go up. 

And of course what ever you do, perform method correlation and communicate with the obstetricians any changes they may see in titer results.  We are contemplating this also. 

Also I am little confused by ""Do you want it to be faster and more hands-off or more exact?"  It seems to me that automated titers in gel should be much more reproducible. 

We are just starting to look in to performing titers on Vision.

Edited August 5, 2017 by Sandy L
added some additional thoughts

[SMILLER 870]

The only thing I would worry about with pre-natals, where titers from one month are compared to the next, concerns things that may intermittently cause gel interference.  For example, if I am not mistaken, CRP goes up and down with any pregnancy, and could cause gel interference (potentially upping the titer I would think) for one month but not necessarily the next.  What would one do with gel titers were interference is noted in one specimen but not another?  Or maybe this would never happen?

Scott

 

[Malcolm Needs ★ 4,910]

1 hour ago, SMILLER said:

The only thing I would worry about with pre-natals, where titers from one month are compared to the next, concerns things that may intermittently cause gel interference.  For example, if I am not mistaken, CRP goes up and down with any pregnancy, and could cause gel interference (potentially upping the titer I would think) for one month but not necessarily the next.  What would one do with gel titers were interference is noted in one specimen but not another?  Or maybe this would never happen?

Scott

 

I have never seen this phenomenon interfering with titre results.  That is not to say it couldn't happen - it just means that I have never seen it.

[galvania 804]

For tube testing using the "uniform procedure tube method" that CAP suggests, they reported the following results: 333 participants, mode 64, consensus range 16 to 256.  For Gel testing "uniform procedure gel method", 138 participants, mode 256, consensus range 64 to 1024. 

Sorry but that range of results is just HUGE regardless of method. FIVE dilutions difference.   Something tells me there is no standardisation in either method.  Things like dilution medium can make a big difference as can the choice of cell phenotype used to carry out the titre.  I hope individual labs are not getting that level of range when they repeat a titre!

I also have never heard of CRP affecting results in gel; and I fail to see by what mechanism it could.  I have, on the other hand, heard that transfusion during a period when the patient has a high CRP can be more likely to induce de novo antibody formation

[SMILLER 870]

Anna, in the bit about gel interference I posted above, CRP was just mentioned as an example of something that can cause gel interference (along with cold agglutinins, high levels of immunoglobulins, etc..).  CRP is an acute phase protein where huge amounts of it could conceivably sticky-up RBCs in gel. 

The main question I had was wondering if something that causes gel interference could result in an over-reading of titres when comparing results from serial pre-natal specimens.  But as Malcolm has pointed out above, it does not seem to be an issue.

Scott

Edited August 8, 2017 by SMILLER
clarification

[Mabel Adams 1,068]

Another caveat about doing titrations on the Vision is that it always runs all 10 (or 12?) dilutions.  That will burn through a lot of reagent cells unnecessarily on a titer of 4!  I agree with those above that it is critical that the OB/GYNs know that you are using a method that gives different results than their textbooks are based on.  Every gel titer result should go out with a comment explaining how its results correlate to the literature for further evaluation of the pregnant person.  At least nowadays they are likely to follow with Doppler ultrasounds rather than riskier, invasive amniocentesis.  I think a review of the CAP survey results is very enlightening.

[TreeMoss 71]

On ‎7‎/‎27‎/‎2017 at 5:38 AM, galvania said:

Does the AABB state a reason, Treemoss?

I looked up Method 5-3 "Using Antibody Titration Studies to Assist in Early Detection of Hemolytic Disease of the Fetus and Newborn" in the AABB Technical Manual, 18 edition -- One of the NOTES states: "Do not use enhancement techniques [albumin, polyethylene glycol, low-ionic-strength saline (LISS)] or enzyme-treated red cells because falsely elevated titers may be obtained.  Gel testing is not recommended."

The next note states: "LISS should not be used as a diluent in titration studies; nonspecific uptake of globulins may occur in serum-LISS dilutions."

[mrmic 55]

Gel testing is just a "miniature" tube system using a "controlled" % cell suspension and a Low ionic environment.   Although the tube test can be similar, unless you are using the same %, concentrations and ratio, you might find it difficult to compare in-vitro results.  Although the sensitivity may be slightly increased for some antigen-antibody reactivity, maybe those we don't want to see, it also has similar problems as other low-ionic methods with the Kell system.

I agree with Ms. Adams comments.  It is important that the OB/GYNs know how you are performing the titrations and if the method(s) correlates with clinical outcomes.  This has been established with the tube method, maybe the tile as well, but I am not aware of references for the "gel" method.   I believe, as long as the OB/GYN is aware that the "trigger" titer number for clinical intervention may be different for the gel method, however, the monitoring during pregnancy looking for a significant change in titer may be consistent with both methods.  I also agree that use of other clinical data (Doppler etc.) is becoming a better standard of practice than amniocentesis and it is less risky for the  mother and child.   Although in suspected severe cases of HDN, amniocentesis may still be warranted.

Better have your validations and IQCP ready...

[SarahE 0]

Thank you for all your responses!  I will be doing more research, and discussing the topic with our pathologists.  Since our current method does not use any enhancement techniques, and "gel" does, the correlations are poor.  There difference is at very least 2-fold.  I agree that reproducibility and tech interpretation are two major factors with the tube technique, and I believe that "gel" would help with both of those.  What do your physicians use as a "critical" number as far as titers go?  I've read that using tube, some physicians consider a titer of 32 as "critical", where this would have to be modified for "gel."

We have not gone "live" with our Ortho Vision testing yet, but I will keep you informed with our decisions. 

[Malcolm Needs ★ 4,910]

We still use 32 for our gel titres in the UK (except for anti-K and Kell-related antibodies, but even 32 seems to be "belt and braces" for most antibodies outside of the Rh and Kell Blood Group Systems - although not all).

[galvania 804]

Sorry mrmic, but I have to disagree with you.  Gel testing is not just a miniature tube method.  Especially when you are talking about any reactions relating to AHG.  The principles behind testing are really quite different. 

[StevenB 118]

On ‎7‎/‎26‎/‎2017 at 5:33 AM, Townsend said:

We do our titers in gel (ABO titers only as we are not a delivering hospital; pediatric only).  Any positive reaction seen in gel should be interpreted as 1+ since there is no w+ interp in the MTS gel interp. guide - there is also no w+ on the Vision.  So, our end-point is the dilution with the last 1+ positive. Our results have correlated well with the CAP samples done in tube when looking at all of the methods reported, but we have only done these for about a year now (only about 3 CAP survey's so far).

We haven't looked at bringing this onto the Vision yet..... all though I am aware that it is now able to do the dilutions for both the buffered card and IgG titer reactions; so maybe with the next software upgrade???  Would love to hear back if you successfully implement the titers on the Vision!

Stephanie

It is a bit annoying that the manufacturers do not have grades below 1+ as we see these all too often.  However, we never interpret these reactions as 1+ as that would not be consistent with the manufacturer's parameters.  When faced with this reactivity, we will record +w or w+ and define these reactions as being less than 1+ on the worksheet.  For titers in Gel, I would think this would be an important distinction as the end point for a titer is the last 1+ result, not the last result that demonstrates reactivity.  That could easily be a 2 to 3 tube difference and may present the physician with information that is not totally accurate.

[Transfusion98 0]

Hi Sarah,  

Do you have a validation protocol you would be willing to share for tube vs gel titers?

[mrmic 55]

Wow, since retirement and having time to review past posts, I found a missed opportunity to respond to Galvania's comment on my LISS/gel column comparison.   I agree they are different "methods" however, atigen- antibody reactivity does take place in an ionic environment. That being said, the LISS tube test requires manually added LISS, while the column technologies the gel or bead matrix is prepared by the manufacturer for you.  I haven't seen the actual composition of the different column matrixes and their ionic strength, but they must have one. Although, in the late 80's we could actually make our own columns, as originally described by LAPIERRE, and then by Luc Noel in " Micromethods en Immuno-Hematologie", Societe Nationals De Transfusion Sanguine, 1989.  And even the tube LISS test can be improved by using a lighter red cell suspension and having a better serum to red cell ratio.  If only our eyes were better!  The standardized micro column matrixes have made this easier to read and sustain the agglutinates for longer readings. Oh, and no more subjective shaking the tube!  It is nice that standardized pre-prepared tests cards/strips, cell suspensions are available to provide better reproducible tests among staff.  So back to my comment and keep in mind that I'm just old and opinionated these days, I still look at the gel/bead columns as modified miniature LISS tube tests with basically the same principles as the standard LISS tube test when antigen-antibody reactions are the subject. At least that's what we thought when we made our own columns.  So I hope we can respectfully agree to disagree.