Antibody Titration

[hmust1]

Hello all,

I'm reviewing our Antibody Titration procedure and read in the Technical manual that using enhancement media (LISS or albumin) is not recommended. Currently, we treat a titer the same as an antibody screen and read IS, 37 degree LISS, and AHG reactions. It seems simpler to just incubate "plain" for 60 minutes as recommended and only read once.

What is everyone else doing?

Thanks!

Heather

[GilTphoto]

One reading at IAT, 60 minutes, no LISS. The technical manual also states to read the endpoint as the last tube showing a 1+ macroscopic reaction. This is not always the case.

There are several reasons for performing titers.

1. Pregnant mother with significant antibody. Read macro only

2. To tell whether an anti-D is passively acquired due to RhIG or immune. <1:4 is RhIG. read microscopically - last positive. Only done if reaction >2+ or reactive in a phase other than IAT (IS or 37C)

3. To tell whether a high incidence weakly reactive antibody is a HTLA antibody (>64) read microscopically - last positive.

4. Titers used to be performed on cold agglutinins for mycoplasma pneumonia before better tests were available. Titers >1000 usually indicate CAD (Cold Agglutinin Disease or CHD). DAT is normally positive with C3 and it can cause a hemolytic anemia. A blood warmer is only recommended for cold autos when they are this strong and don't prewarm away.

Gil

[Mary]

Sending them out to the Blood Center Reference Lab.

[Mabel Adams]

We got re-educated quite a few years ago and quit doing LISS titers. Now it's saline, 60 min, IgG, endpoint is last tube showing 1+ or greater.

Titers < 4 don't speicifically determine RhIG, but it has been used as a reasonable assumption.

[Cathy]

We do the same as Mabel. After getting too high on a survey, we reviewed the Technical Manual and changed our procedure accordingly.

[David Saikin]

We use 6-8% albumin as diluent (but otherwise no enhancements) with anti-IgG after 60 minutes.

[Malcolm Needs ☆]

One reading at IAT, 60 minutes, no LISS. The technical manual also states to read the endpoint as the last tube showing a 1+ macroscopic reaction. This is not always the case.

There are several reasons for performing titers.

1. Pregnant mother with significant antibody. Read macro only

2. To tell whether an anti-D is passively acquired due to RhIG or immune. <1:4 is RhIG. read microscopically - last positive. Only done if reaction >2+ or reactive in a phase other than IAT (IS or 37C)

3. To tell whether a high incidence weakly reactive antibody is a HTLA antibody (>64) read microscopically - last positive.

4. Titers used to be performed on cold agglutinins for mycoplasma pneumonia before better tests were available. Titers >1000 usually indicate CAD (Cold Agglutinin Disease or CHD). DAT is normally positive with C3 and it can cause a hemolytic anemia. A blood warmer is only recommended for cold autos when they are this strong and don't prewarm away.

Gil

Another reason for performing titrations that is becoming more common is for major side ABO incompatible renal transplants (i.e. A2 donor, O recipient).

[BBK710]

Is anyone using the "standardized uniform procedure"? That incubation is only 30 minutes, no potentiator and w+ read macroscopically as the endpoint.

[L106]

Yes, just this year.

[YorkshireExile]

So is it wrong to use the Diamed cells and reagents for an antibody titration? We do the doubling dilutions in saline, pick the appropriate Diamed Screening cell reagent, add them to an AHG gel card and then incubate for 15 minutes, then spin and read. Just like the procedure for the routine antibody screen. End point is a 1+ reaction. Should we be resuspending our cells in saline and using a longer incubation time with tubes??

[Malcolm Needs ☆]

So is it wrong to use the Diamed cells and reagents for an antibody titration? We do the doubling dilutions in saline, pick the appropriate Diamed Screening cell reagent, add them to an AHG gel card and then incubate for 15 minutes, then spin and read. Just like the procedure for the routine antibody screen. End point is a 1+ reaction. Should we be resuspending our cells in saline and using a longer incubation time with tubes??

No, you are perfectly okay doing your titrations in DiaMed columns BoroCliff.

We have been doing this for at least two years (probably longer) in the NHSBT, following a national study and change control. The correlation between our tube and DiaMed titration end points was almost perfect. Obviously, there was the odd antibody that was a couple of dilutions more or less in either method (sometimes the tubes gave, for example, a titre of 64 and the DiaMed 256, sometimes the tubes gave, for example, a titre of 256 and the DiaMed 64 - 2 dilutions difference, so the actual titre was probably 128; this was using master dilutions), but we already know that certain antibodies react differently by certain techniques (tile antiglobulin, tube antiglobulin, capillary tube antiglobulin, liquid-phase microplates, solid-phase microplates, column agglutination technology - you name it).

We actually use Diluent2 as our suspension medium, but there is no reason why you should not use the cells in the diluent in which they are supplied (we looked at that too). We just feel, subjectively, that we get crisper results with Diluent2.

:)

[YorkshireExile]

From a first-time poster - thank you Malcolm for your excellent reply. I was beginning to wonder what was going on with people talking about 60 minute incubations and only using tubes!

[David Saikin]

The problem with using gel for titers in the US is that our physicians are not used to the increase in titer seen in gel. It will take a sgnificant education process for them to realize that what was in tube a titer of 16 may be a titer of 32 or 64 in gel. I know some folks here are performing comparison studies, but unless we are all on the same page with these, there is the risk of interventions when they may not be necessary.

[Malcolm Needs ☆]

True, there may be David, but surely they would not intervene physically, unless they had performed other studies such as MCA Doppler/ultrasound?

I know our doctors would only use the titre as a guide, rather than the be all and end all.

[David Saikin]

You are correct . . . but it would avoid the "unnecessary" extra stuff (that is certain to be expensive here in the US of A).

[Malcolm Needs ☆]

Very true. I tend to forget that over this side of the puddle!

[clmergen]

We ran into the problem with a patient having a titer of 1:64 (can't remember which antibody)t at a different hospital. She ws referred to High Risk OB doctor who sends all of his work to us. We got a titer of 1:2. Difference of Gel vs Tube.

[Malcolm Needs ☆]

clmergen, I wouldn't mind betting it was a mixture of an IgG and IgM, and that you were detecting the IgM in the gel (same specificity, but a mixture of immunoglobulins).

[clmergen]

I thought gel was only supposed to detect IgG, much like solid phase. The gel testing was done at another hospital, we do tube titers at my facility. It did cause a lot of concern amongst the staff until I called and asked how the testing was done. Then it was like tring to compare apples and oranges. So I wonder if the patient would have been referred to the specialist if the typing had been done in tube to begin with.

[Malcolm Needs ☆]

No, not necessarily.

We use cassettes that contain anti-IgG and anti-C3d (although cassettes containing anti-IgG only are available). But we often find that when an IgM antibody is present, particularly if it reacts better in the cold (well, cool) as most of them do, the sensitisation occurs before we put the cassettes in to incubate at 37oC, and you then get agglutination when you centrifuge. This is why did so much comparative work and change control.

With experience, you can usually tell (although I'm sure we don't detect every time there is an IgM present).

[clmergen]

I forgot you can get those anti-IgG and anti-C3d cassettes. The only hospital we have in our system using Gel uses IgG only cards. I personally haven't worked with Gel since the 2000, and at that time I remember we did on occasion detect a cold reacting atibody. Of course, I can't remember if we had anti-IgG only or the dual anti-IgG/C3d.

[Malcolm Needs ☆]

In a way though, the anti-C3d is irrelevant. If there is an IgM element to the antibody, as well as an IgG (as can often happen with an antibody produced de novo during a pregnancy) you may still get agglutination prior to 37oC incubation.

[GilTphoto]

Is anyone using the "standardized uniform procedure"? That incubation is only 30 minutes, no potentiator and w+ read macroscopically as the endpoint.

Just saw that in the last CAP Titration Survey instructions.

Of course I had already performed the titer using 60 minutes, titration using a 100 ul pipettor and 1 drop of cells.

THEN I READ THE DIRECTIONS!!!

Repeated using same 100 ul pipettor and a 50 ul pipettor for the cells, incubating 30 minutes

The first titer was one tube higher

BTW we use w+ to denote microscopic readings only. The last macroscopic reading as 1+.

[Mabel Adams]

I wondered how they can call it the "uniform" procedure when it is not even in the Technical Manual. In fact there are 2 different methods for titration in the Tech Manual and they sort of contradict each other as to which is more appropriate for following pregnant women.

To respond to the post further up, I am pretty sure I know of doctors that would order amniocentesis if the titer were 64--trained a long time ago and haven't kept up. I hope I am wrong.

[John C. Staley]

I'm curious, can anyone site a current/recent reference study indicating the titer significance when "enhancement" techniques are used for titration studies of antibodies during pregnancy?

Point #1: The original and only antibody studied for HDN is Anti-D.

Point #2: The technique utilized was saline and 60 minutes incubation.

Point #3: This is the study most OB docs are made aware of during training and they were trained to those numbers. Any numbers provided to them using alternate techniques will be confusing to them and possibly dangerous to the fetus.

A little historic food for thought.