exlimey

RESt = Rabbit Erythrocyte Stroma - basically stabilized red cell membranes from rabbits. There is absolute no DTT in RESt.

I just answered this question. My Score PASS  

I read on the Internet that if a person sinks in water and drowns, they're proven to be a witch........

I suspect that routine use of enzyme-treated cells (in IAT) by "Non-reference Laboratory Staff" would cause more confusion than it would solve. Even the largest, most proficient hospital laboratory doesn't have high caliber serologists available on all shifts. I would suggest that tests with enzyme-treated cells be restricted to more difficult serological pictures, e.g., post-transfusion hemolysis without obvious cause (read "anti-Jka or anti-Jkb"), or for investigation of antibodies to high-incidence antigens. I also suspect that many of the "enzyme-only" specificities have a major IgM component - notoriously difficult to detect by CAT (gel). Just my two cents/pennies.☺

Polyagglutination (one L) is a phenomenon demonstrated by some red cells in the presence of most normal human sera. It has little to do with a positive DAT. Perhaps you mean "spontaneous" or "non-specific" agglutination ?

You bring up some interesting points and I agree with your position. Certainly "the literature" regarding the clinical importance of titration results is confusing. Most of the original work was done on anti-D, using an unorthodox test protocol (I believe the titrant was a high concentration of BSA), but there did seem to be some correlation between titration strength and clinical impact. Workers attempted to shoe-horn other specificities into the same program, with mixed success. Now, today, as you point out, the gel test is becoming a routine way to measure antibody strength. I don't think anyone honestly knows what the titration end-points mean, since the modern results are difficult to interpret/compare to the older literature. Time will tell. I think people have been caught in a little trap with regard to the controls needed for titrations. It is very unusual to have two sequential samples in a clinical assay, even rarer that one of said samples has been stored (frozen). Consider a simple CBC.....many patients with extended hospital stays have multiple tests performed. Their last samples are not run in parallel with the current sample, yet the results are considered valid because the instrument's controls performed as expected - this is the equivalent of using material with a known potency as a control for patient sample testing. The art is in selecting your control.

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.

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).

(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.

That conflicts with you original post: "Set up 4 units of A Pos unit and found to be incompatible. Then we set up 3 A Rh D negative units and found to be incompatible." I'm confused.

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.

On another track......why the switch to Rh-negative units ?

This doesn't fit the pattern for an antibody to a low incidence antigen - in this case, all 7 group A units are incompatible and the 4 group O units are compatible.

Here's my 2-cents, 2-pennies, or 2-any other small denomination coins: First, I am NOT a regulatory expert, but I am familiar with assay development and validation. All assays should be controlled in some fashion, to give the practitioners some confidence that the results are valid AND that batch-to-batch variation is limited. In a titration, especially a serological titration, this is a little more difficult than having one or two pass/fail samples that are typically included in many laboratory tests. If a control is needed for a titration, it doesn't need to be the same specificity as the test antibody (as Malcolm highlights). Ideally, yes, but not necessarily. It does need to be reliable/robust and give the same end point each time, even with the acknowledged variations in serological tests (reagents, test cells, techs, etc.). Tube testing is notoriously variable, while gel testing is believed to reduce some of those nuances. As Malcolm suggests, it might be necessary to have a control for an IgM titration (ABO) and/or a different one for an IgG titration. At the very least, the end-points may be different. An IgM control might be as simple your routine anti-A reagent; a simple IgG control might be an IAT-reactive anti-D or other specificity. A clever option might be a control that contains both - an IgM component and an IgG component. If a test system is adequately controlled each time (and passes), in my opinion, there is no reason to routinely perform parallel testing of successive patient samples. Retention samples might be useful for investigatory reasons if/when a patient's antibody titration changes radically from one sample to the next, or if there's some other medical indication. I getting a sense of deja vu, I think I've written this before.

Good thinking. Might be interesting to see if there's anything in an eluate....