Antibody Titers

[JoyG]

It's been a long time since I've performed antibody titers for HDN.  In the 17th edition of the Technical Manual and John Judd's Methods states to incubate 37C for 1 hour however the CAP proficiency is for 30 minutes.  What does everyone use; 1 hour or 30 minutes?  Why was it changed to 30 minutes for CAP?

[David Saikin]

I use one hour BUT I do not use the method as defined by both CAP and AABB because with these methods you are making a 1:3 dilution not 1:2. (1 drop of cells and 2 drops of dilution - adds up to 1:3). Who knows why CAP changed it . . . ?

[Malcolm Needs ☆]

We have tried all sorts, but now use the usual 20 minutes with column agglutination technology (Bio-Rad), and this works well, with good validation against the tube technique we used to use.

 

No unexpected cases of HDFN yet, after a good 5 years (or longer).

[Sko681]

We use 1 hr incubation and follow AABB.   IDK the deal with the CAP proficiencies...I think the instructions for use leave a little up to interpretation as well (without further augmentation  )

[Liz0316]

We use the 1 hr incubation described in the AABB procedure.

[Eagle Eye]

use saline as diluents and incubate one hour at 37C.

 

If you use CAP procedure, the grading/end point is also different...

[Mabel Adams]

I believe that the CAP method is based on a study that they did at several sites.  They concluded that they got the most consistent results between sites using that method.  I think the study may still be cited on the CAP website.  I didn't want to change to an endpoint that was less than 1+ so we don't use the CAP method.  1 hr at 37 with no additive, 1+ endpoint.  I believe the Technical Manual has two different titration methods, one in the perinatal section (which I prefer for HDFN assessment) and one in the antibody ID section which includes scoring as I recall.

[JoyG]

Thank you all for your responses!  Much appreciated!

[nmartin]

We use the AABB method for HDFN:  dilute with saline, no enhancement, 1 hour incubation,  and report 1+ as the endpoint.

[Rh-fan]

We have tried all sorts, but now use the usual 20 minutes with column agglutination technology (Bio-Rad), and this works well, with good validation against the tube technique we used to use.

 

No unexpected cases of HDFN yet, after a good 5 years (or longer).

 

Mostly the column technology is much more sensitive than a tube test without aditives. Do you do not have that experiance?

 

Peter

[Malcolm Needs ☆]

Yes, we do, but, strangely, not with titre end points.  There is no doubt, however, that if you scored the reactions, the score in CAT would be much higher than tube technique.

 

I have absolutely NO idea why the titres should be so close to one another; but they are!

[SMILLER]

Re: titration in gel vs. tube. 

 

We just did a titre for a prenatal (don't get too many of these  here).  The AB screen and panel in gel were all 4+ for anti-E.  For titre: did standard 60 min 37 C incubated then AHG in tube (neat, 1:2, 1:4, etc.) was 1+ only up to 1:2.  Was expecting a much higher titre!

 

Scott

[Malcolm Needs ☆]

Re: titration in gel vs. tube. 

 

We just did a titre for a prenatal (don't get too many of these  here).  The AB screen and panel in gel were all 4+ for anti-E.  For titre: did standard 60 min 37 C incubated then AHG in tube (neat, 1:2, 1:4, etc.) was 1+ only up to 1:2.  Was expecting a much higher titre!

 

Scott

 

Hi Scott,

 

Did you try running the titre in gel to see the end point (as opposed to the strength of reaction)?

[SMILLER]

Malcolm

 

Thought about trying gel for the titre, but have not have time.  I am starting to wonder, though, if the standard method of titering is the best way to do this.

 

Thing is, got to compare apples to apples anyway.  If we do another titre later on for this patient, we will be comparing that one to another repeated done on this specimen for the report.  And besides, I am guessing our OB's are used to seeing results from the standard method anyway.  Not sure what they would think of the higher titres reported from gel even for an isolated titre.

 

Scott

[Mabel Adams]

On CAP surveys the titers done via gel always have a higher mean by about 1 dilution as I recall.  Some that have switched to titrating in gel turn them out with different cut offs (i.e. values above which evaluation should be made by doppler ultrasound etc.).  I always worry that the docs will trust the cut offs listed in ACOG guidelines and won't realize that we are using a different method than those are based on.  Still, nowadays they would not be jumping to amniocentesis so the risk is smaller.  Sometimes I wonder if they won't just go to monitoring all sensitized moms with US instead of titers.  Titers are probably still cheaper.