Ror Phenotype

[SMILLER]

Is there anything special about the Ror phenotype?  On a recent prenatal antibody work-up, we noticed that along with all the weak reactions for the D pos panel cells (gel), the cells with the Ror phenotype were completely negative.  (The patient was given a dose of anti-Rh two months ago.)  Is the D antigen more weakly expressed on these cells for some reason?

Thanks, Scott

[exlimey]

Two thoughts:

1. The other D+ cells on the panel probably have "homozygous expression", whereas the Ro donor may be a single dose. You may just be seeing a dosage effect.

2. The Ro cell may be DcatIV, which is missing a few epitopes found on normal expressions of D antigen (I don't remember the details). If the donor is heterozygous (single dose), expressing only the DcatIV gene, the anti-D in the patient may not have the ability to react with the modified expression.

If Malcolm Needs reads this, I'm sure he'll correct my terminology deficits to the more modern versions.

[Malcolm Needs ☆]

1 hour ago, SMILLER said:

Is there anything special about the Ror phenotype?  On a recent prenatal antibody work-up, we noticed that along with all the weak reactions for the D pos panel cells (gel), the cells with the Ror phenotype were completely negative.  (The patient was given a dose of anti-Rh two months ago.)  Is the D antigen more weakly expressed on these cells for some reason?

Thanks, Scott

No Scott.  According to the table in Reid ME, Lomas-Francis C, Olsson ML.  The Blood Group Antigen FactsBook.  3rd edition, 2012, Academic Press, the number of D antigen sites on such red cells varies between 12, 000 and 20, 000, but this is more than for the R₁r (R₁R_o or R_or') types, which vary between 9, 900 and 14, 600.  Mind you, there may not have been an R₁r phenotype on your panel.  If not, then the chances are that your panel cells expressing the fewest number of D antigen sites would be your R₁R₁ (R₁r') red cells, with a range of 14, 500 to 19, 300, and if this was true, it could well be that it is the number of D antigens expressed that is causing the phenomenon you are seeing, if all of the other D Positive red cells are giving weak reactions.

My own question would be, "How do you know your R_o red cells are R_or?"  If the donors were from the White ethnicities, then the chances are that they are R_or (as only 2.1% of this population have the R_o phenotype, so they are unlikely to ALL come from this population).  If the donors were from the Black ethnicities, then the chances are that they are R_oR_o (as about 46% of this population have the R_o phenotype, so it is likely that ALL of the donors used for the panel come from this population).  Either way though, unless you have access to some VERY rare examples of anti-D, anti-D does not show dosage in the way we know it (for example, with anti-M or anti-Jk^a), and so, unless molecular studies are undertaken, it is impossible to decide, serologically, if the RHD gene is present in the homozygous form or the hemizygous form (as there is no such gene as RHd, there cannot be a heterozygous form - exlimey!).

Lastly, one would hope that, if the panel you are using is a commercial panel, they would have done something (perhaps a fluorescence-labelled anti-D and a FACS) to ensure that no red cells are chosen with a particularly low number D antigens expressed.

The exons involved in the RHD gene leading to Partial D Category IV, are 2, 3 and 7 (exlimey), but even these express around 9, 000 D antigens.

[exlimey]

16 minutes ago, Malcolm Needs said:

 

(as there is no such gene as RHd, there cannot be a heterozygous form - exlimey!) - I stand corrected ! Thank you.

Lastly, one would hope that, if the panel you are using is a commercial panel, they would have done something (perhaps a fluorescence-labelled anti-D and a FACS) to ensure that no red cells are chosen with a particularly low number D antigens expressed. - Not a chance of that happening.☺

The exons involved in the RHD gene leading to Partial D Category IV, are 2, 3 and 7 (exlimey), but even these express around 9, 000 D antigens. I was on the right track.

[Malcolm Needs ☆]

21 minutes ago, exlimey said:

(as there is no such gene as RHd, there cannot be a heterozygous form - exlimey!) - I stand corrected ! Thank you.

Lastly, one would hope that, if the panel you are using is a commercial panel, they would have done something (perhaps a fluorescence-labelled anti-D and a FACS) to ensure that no red cells are chosen with a particularly low number D antigens expressed. - Not a chance of that happening.☺

The exons involved in the RHD gene leading to Partial D Category IV, are 2, 3 and 7 (exlimey), but even these express around 9, 000 D antigens. I was on the right track.

You most certainly were!

[SMILLER]

Yeah, I wondered about that hetero/homozygous D thing as well...   I will also mention that according to Ortho, Panel cells are all tested with specific antisera for each antigen for appropriate reaction strength before being released for shipping.

Scott

[exlimey]

38 minutes ago, SMILLER said:

Yeah, I wondered about that hetero/homozygous D thing as well...   I will also mention that according to Ortho, Panel cells are all tested with specific antisera for each antigen for appropriate reaction strength before being released for shipping.

Scott

Yeah, that's one of the many fuzzy statements found in package inserts. I suspect that it means they perform a test to detect antigens and the reaction has to meet a certain grade. I doubt that they test the strength of antigens by titration or by another other more exotic means (FACS).

[SMILLER]

On 3/23/2018 at 3:03 PM, exlimey said:

Yeah, that's one of the many fuzzy statements found in package inserts. I suspect that it means they perform a test to detect antigens and the reaction has to meet a certain grade. I doubt that they test the strength of antigens by titration or by another other more exotic means (FACS).

Actually, I was talking to an Ortho tech the other day in regards to another issue, and asked them about their QC for gel panels.  They said they test each antigen on each cell to make sure that they get a decent reaction (pos or neg depending).  Did not ask what they used for QC anti-sera though.

sCOTT  

[Malcolm Needs ☆]

19 minutes ago, SMILLER said:

They said they test each antigen on each cell to make sure that they get a decent reaction.

Did "they" expand on what they think of as "a decent reaction"?  This sounds very subjective to me.

[SMILLER]

1 hour ago, Malcolm Needs said:

Did "they" expand on what they think of as "a decent reaction"?  This sounds very subjective to me.

Well, it may seem subjective to YOU...

The particular problem we had was with a few panel cells on a new panel coming up only slightly hazy--not even a weak 1+.  (These are R1R1 cells that should be clearly positive with our usual QC anti-sera which contains anti-D and ant-e.  So that is what is under investigation.)  Ortho said that we should be seeing a definite 1+ at the least.  We are used to seeing 2+ or 3+ with these gel panel cells.

Scott

[exlimey]

21 hours ago, SMILLER said:

Well, it may seem subjective to YOU...

The particular problem we had was with a few panel cells on a new panel coming up only slightly hazy--not even a weak 1+.  (These are R1R1 cells that should be clearly positive with our usual QC anti-sera which contains anti-D and ant-e.  So that is what is under investigation.)  Ortho said that we should be seeing a definite 1+ at the least.  We are used to seeing 2+ or 3+ with these gel panel cells.

Scott

Your results suggest to me that your QC reagent is deteriorating, maybe, perhaps, subjectively speaking, of course.☺

I am well aware that manufacturers test every cell for every antigen and that the results must meet a certain threshold. However, as Malcolm suggests, "decent strength" can be a very elusive target. One direct test with undiluted antisera (or a strategically-diluted sample) does not always give an honest indication of antigen strength.