IgG Blocking to obtain a phenotype on +DAT patient

[Jyoung]

Does anyone perform what is called IgG Blocking on a patient with a strong +DAT? I just wanted to get some info on this and see if it is something worth trying or implementing.  I think some IRLs  in US may use this technique but not sure how well it is accepted and how well it works.

 

 

Thanks,

Edited June 19, 2013 by Jyoung

[Malcolm Needs ☆]

We certainly don't in my own Reference Laboratory. We send such samples to the IBGRL for genotyping - but I fully realise that, in the UK, we are extremely lucky, because we don't get charged for this.

[Malcolm Needs ☆]

Having said that, we will, on occasion, try treating the red cells with Choroquine.

[David Saikin]

 You could also try W.A.R.M.  I just use that to destroy Kell system ags but it is made to remove autoab so you can type cells.

[Emwilson7]

I've never heard of it. You could treat with EGA or cholorquine, or use monoclonal reagents for typing.

[Rh-fan]

We use EGA or do genotyping.

 

How does these 'blocking' works?

 

I have also heard about IgG monoclonals from mouse, and by the use of anti Mouse-IgG (instead of anti Human-IgG) they gave good results.

 

 

David, W.A.R.M. is the comercial name of ZZAP (or not?), if it is the same as ZZAP then you destroy more than Kell (also Fy, MNS, Lu).

 

Peter

[Jyoung]

I had never heard of it either until after attending a SCAB conference a few months ago.  Apparently you can add Combs AHG (IgG) to bind to the antibodies that are causing a positive DAT.   This will then block those antibodies from reacting when phenotyping a patient through the AHG phase. How or why this doesn't block the antigen when phenotyping I do not know? But I was told it doesn't.  I was also told this technique is mainly used for those patients with a strong postive DAT, where the DAT is still reacting after treatment with EGA.

 

Thanks,

Edited June 20, 2013 by Jyoung

[Malcolm Needs ☆]

I had never heard of it either until after attending a SCAB conference a few months ago.  Apparently you can add Combs AHG (IgG) to bind to the antibodies that are causing a positive DAT.   This will then block those antibodies from reacting when phenotyping a patient through the AHG phase. How or why this doesn't block the antigen when phenotyping I do not know? But I was told it doesn't.  I was also told this technique is mainly used for those patients with a strong postive DAT, where the DAT is still reacting after treatment with EGA.

 

Thanks,

Edited June 21, 2013 by Malcolm Needs

[Malcolm Needs ☆]

Sounds a bit dodgy to me!!!!!!!!!!!!!!!!

Edited June 21, 2013 by Malcolm Needs

[Rh-fan]

Maybe they use anti human IgG Fab fragment that bind to the bound IgG. If you then add anti Fya and after washing complete anti human IgG. That sounds like something that could work.

 

Peter

[mpmiola]

I've used this technique successfully in detales see the article:

 

"A quick and simple method for phenotyping IgG-sensitized red blood cells

T.S. SERERAT, D. VEIDT, AND A. DUTCHED

Positive (IgG) direct antiglobulin test (DAT) reactivity ranging from weakly positive to 2+ can be eliminated using a simple “blocking” technique with anti-IgG. This method can be used for antigen typing DAT-positive red blood cells that require the antiglobulin technique. Immunohematology 2000;16:154–156."

[Malcolm Needs ☆]

I'll give it a try!  Thank you!

[jill]

There is an excellent article in Immunohematology, Vol 16, #4,2000 that decribes

the validation of this procedure by the ARC in Columbus, OH.  It compard 26 samples

using IgG blocking, glycine acid EDTA, and chloroquine diphosphate treated red

blood cells in typing for s,S,Fya,Fyb,Jka, and Jkb.  The procedure works well with

samples that have a w+ to 2+ DAT.  One sample tested Fyb+ with the blocking method

and Fyb- with chloroquine and glycine acid EDTA treated cells.  I remember reading in

the insert that chloroquine diphosphate treatment of red cells can weaken the Fyb antigen.

 

 

 

[jill]

The article I just cited was the same article as above by Sererat, Veidt and Dutched. (sorry)

[dawn w]

IgG blocking study was performed by C.Wong at CarterBloodCare in 2001. It was published as an abstract in Transfusion. The reference is "Evaluation of a Method to Prepare IgG-Sensitized Red Blood Cells for Phenotyping" 2001- Vol.41, Supplimen:110S 

Outline steps are:

1. Wash red cells to be treated 3 times with isotonic saline

2. After the last wash resuspend the red cells to 3-5%

3. In a separate tube add 20 drops of the washed red cells

4. Centrifuge 1 min

5. Remove and discard supernatant, be careful not to disturb the red cells

6. Add 20 drops of IgG to the cells button

7. Incubate at room temperature for 15-30 minutes

8. Centifuge for 30 sec

9. Remove supernatant

10. Wash at least 4 times with isotonic saline

11. Perform IgG DAT on washed,treated cells. Be sure to include a 6% albumin control to verify the immunoglobulin coating has been removed.

If the DAT is negative the cells are ready for typing.

If the DAT is positive repeat the treatment. No more than three times.

*This procedure works best for initial DATs no stronger than 2+

**This procedure can be used on EGA treated cells.

***This method peserves the KELL system antigens if used with non EGA treated cells.

If you have any questions please email me at dwebber@carterbloodcare.org

 

 

[Jyoung]

Thanks you guys for the references!