Jump to content

Adsorption/elution studies for null phenotypes


Recommended Posts

Hello, fellow blood bankers.

I got a question about adsorption/elution studies for weak antigen detection.

Literature describes in detail how to detect weak A and B antigens or weak D antigens using this method.

However, literature describes little regarding other than these weak antigens.

For example, when the Kell null phenotype is found, we need to confirm the absence of weak K and k antigens using adsorption/elution technique.

Imagine that we have only anti-K monoclonal IgM and anti-k monoclonal IgG. How can I perform adsorption/elution studies for K and k antigens?

My guess is that for anti-K monoclonal IgM, I can use room temperature incubation, heat elution, and immediate spin test with K+reagent RBCs, while

for anti-k monoclonal IgG, I can use 37C incubation, acid elution, and IAT with k+ reagent RBCs. 

Do you have any opinion about this or can share your SOP with me?

I look forward to your help. 

Link to comment
Share on other sites

I can see no reason why this would not work as a method.  We used to use something very similar when testing individuals expressing the "McLeod" phenotype.  However, these days it would be easier (and certainly more accurate) to perform gene sequencing on both the KEL and the XK genes.  Don't forget that a true Duffy null phenotype (FY/FY), as opposed to a "GATA-1" type Fy(a-b-) is, these days, established by molecular techniques.

Link to comment
Share on other sites

Malcolm

Thank you for your clearcut answer.

I have another question for you. 

Is it possible for me to perform adsorption/elution studies using monoclonal anti-K and anti-k, not polyclonal human anti-K and anti-k?

AABB Technical Manual says polyclonal antibodies are preferred.

Link to comment
Share on other sites

I must admit, we always used to use polyclonal antibodies, and there is a reason for this.  Even blended monoclonal antibodies will still only recognise certain epitopes within the antigen, and while the K and k antigens tend to be single amino acid substitutions of methionine at position 193 for the K antigen, and threonine at position 193 for the k antigen, this is by no means universal.  A weak expression of K is seen when either an argenine or a serine residue replace the methionine residue at position 193.  As with amino acid substitutions leading to weakened expression of the K antigen, so the same can happen with the k antigen, but these substitutions may not actually be at position 193.  A recent publication has shown that a substitution of Leu196Val weakens the expression of the k antigen (see Uchikawa M, Onodera T, Tsuneyama H, Enomoto T, Ishijima A, Yuasa S, Murata S, Tadokoro K, Nakajima K, Juji T.  Molecular basis of unusual Kmod phenotype with K+wk-.  Vox Sang 2000; 78 Suppl. 1: Abstract O011, Poole J, Warke N, Hustinx H, Taleghani BM, Martin P, Finning K, Crew VK, Green C, Bromilow I, Daniels G.  A KEL gene encoding serine at position 193 of the Kell glycoprotein results in expression of KEL1 antigen.  Transfusion 2006; 46: 1879-1885 and Millard GM, Lopez GH, Turner EM, Lizarazu ME, Roots NM, Liew Y-W, Flower RL, Hyland CA. Modified expression of the KEL2 (k) blood group antigen attributed to p.Leu196Val amino acid change three residues from the K/k antigen polymorphism site: implications for donor screening.  Transfusion 2019; 59: 1156-1158.).  Therefore, it is more likely that you may get a false negative result using a monoclonal antibody in adsorption/elution tests, than by using a polyclonal antibody, with its wider breadth of specificity to the antigen's epitopes.

Link to comment
Share on other sites

Thank you, Malcolm for your kind explanation.

Now I understand polyclonal is prefered over monoclonal when adsorption/elution studies are used. 
 

My another concern is how I can buy or acquire

polyclonal reagents such as anti-K, anti-k, anti-kpa, anti-kpb? Particularly, all vendors sell monoclonal anti-K, not polycloanl anti-K.

Since our country has no reference lab, I can not access in house polyclonal antibodies.

 

 

Link to comment
Share on other sites

Phew, that's a difficult one!

You could begging some from one of the world's big organisations, such as the International Blood Group Reference Laboratory in the UK, somewhere like the Mayo Clinic in New York, Amsterdam, South Africa, etc, but other than that, I don't know.  Some of the large reagent suppliers, such as Ortho or BioRad may possibly have some in store too.

Link to comment
Share on other sites

Hemo bioscience sells Polyclonal anti K and Anti Cellano, both are FDA approved potency and could probably be diluted in a 6%BSA solution to be used for this purpose.  info@hemobioscience.com for pricing info...  Also your local American Red Cross may have donated units of Anti Kell plasma you might use but obviously watch out for the ABO types as these are not adsorbed..... 

Link to comment
Share on other sites

Create an account or sign in to comment

You need to be a member in order to leave a comment

Create an account

Sign up for a new account in our community. It's easy!

Register a new account

Sign in

Already have an account? Sign in here.

Sign In Now
  • Recently Browsing   0 members

    • No registered users viewing this page.
  • Advertisement

×
×
  • Create New...

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.