Antibody Titer methodology

[Sue Lawry]

I am interested in finding out if anyone is using gel technology to do antibody titers  on pregnant women?  If so, did you validate it against tube saline/AGT titers and were the results comparable?

[Malcolm Needs ☆]

Yes, and yes...............and now it is done nationally.

[YorkshireExile]

Malcolm - For the antibody titre methodology in the UK when using the gel cards, do you dilute the plasma in phosphate-buffered saline or in 0.9% Normal Saline? Does it actually matter?

[Malcolm Needs ☆]

We use PBS and always have done.  I can't see that 0.9% normal saline would make that much difference, BUT, I suspect that rather depends on the original pH of the distilled water (whether it is really pure distilled water) and the pH of the final 0.9% normal saline.

[carolyn swickard]

The CAP survey for Antibody Titers has been following the different methodologies for some time.  The result bell curves for gel is always higher than any tube methodology using the Uniform Procedure CAP recommends (The titer is run in saline with no enhancement medias).   If you switch to gel you will need to let your physicians know why the results are higher.

[David Saikin]

I don't like the "uniform procedure"  it is actually not a x2 dilution but a times 3, 1 volume cells and 2 volumes diluent.

[galvania]

You do have to be a bit careful when using either saline or PBS in gel cards as it can mess up the buffer balances and cause cells to 'hang' in the gel.  But the really important thing is that whatever you do you always do the same thing, and preferably re-test the previous sample in parallel (unless you have standardised frozen cells) to compensate for cell-to-cell variation in antigen strength.  That way you know whether an apparant increase really is an increase and not just due to technical factors.

[Winter]

When using gel antibody titers, what endpoint do you use? We report the reciprocal of the highest  titer showing a 1+ in tube. Currently, we are doing our screens in gel and our titers in tube. Yesterday, I had one that is barely positive in gel so guess what the tube in titer looked like!

[Maureen]

When using gel antibody titers, what endpoint do you use? We report the reciprocal of the highest  titer showing a 1+ in tube. Currently, we are doing our screens in gel and our titers in tube. Yesterday, I had one that is barely positive in gel so guess what the tube in titer looked like!

 

We see this situation often, performing the antibody screen/ID with solid phase (Immucor Echo Capture) obtaining signficant reactivity, but finding the antibody is 'below titerable levels' performing the CAP Uniform Procedure.

In the lab I worked previously the Antibody titers were done using Ortho gel - there were no issues.  I expect the change from tube to gel was validated, but before I worked there.  I also expect that the validation results required some explanation in the documentation - as gel (like solid phase) can detect IgG antibodies at lower levels.

[John C. Staley]

Just a thought and I'm afraid I'm not up to date with the current literature, has there been correlation studies done on the clinical picture of newborns in relation to the titre level produced by enhancement methods?  I would think that is the info that really matters.     I suspect that if anyone has this data it will probably be Malcolm since it appears to be a national standard in the UK.

[Malcolm Needs ☆]

I haven't got the data myself John, but I think I know someone who will - sorry, I mean I know someone who I think will - of course, I know I know them!!!!!!!!!!!!!!!!

[Abdulhameed Al-Attas]

Titre: The reciprocal of the highest serum dilution that causes macroscopic agglutination when serial dilutions of an antibody are tested against selected red cells.

Applications:

Prenatal testing 

Identification of HTLA

Comlex Antibody Identification 

Differentiation of pathological and harmless autoanti- I  and  Procurement of antisera Quality assurance of reagents.

 

Limitations:

Titrations are only semiquantitative estimates of antibody reactivity due to several variables that affect their performance. Three main variables are the technologist, the red cells, and the method.

Ways to Minimize Variables:

1. technologist: experienced with proven technique
   
2. red cells: ideally when titres of samples are to be compared, use fresh red cells (antigens deteriorate on storage) from the same donor (same number of antigenic sites present). If this is not possible, use commercial red cells of the same apparent genotype.
   
3. method: when sequential samples are examined for change in titre, store samples frozen and run new samples in parallel with the immediately preceding sample. This is the most practical way to control that an increase in titre is real.
   

Prozone Phenomenon:

This may cause reactions to be weaker in the first tubes than in higher dilutions and is believed to be caused by an antibody excess in which all antigenic sites are sensitized with antibody leaving none free to form cross-links

Significant Difference in Titres:

When comparative studies are done, such as in prenatal testing, a difference in titre of at least 2 tubes is required to be considered a significant difference. For example, if the titre changes from 32 to 64, this is not considered to be significant (difference of only 1 tube); however, a change from 32 to 128 would be significant (2 tube difference).

These are the important points to know about AntibodyTitration.

Edited July 5, 2014 by Abdulhameed Al-Attas

[DPruden]

What is the advantage of doing a titer in gel? I would think that it would be confusing to a clinician, given the increase in sensitivity.  It is certainly cheaper to use tubes and saline.

[Yanxia]

What is the advantage of doing a titer in gel? I would think that it would be confusing to a clinician, given the increase in sensitivity.  It is certainly cheaper to use tubes and saline.

I think the advantage is convenient and easy to read ,easy to control.