exlimey

Perhaps I'm a little naive, but I find some of the "old time" logic somewhat illogical. I appreciate that a unit of red cells being transfused would potentially be "cold" - 1 - 6 C at the start of infusion, i.e., might cause a cold-agglutinin issue, but almost immediately, the infused portion would equilibrate to the temperature of the circulating blood. Additionally, the unit itself would start to warm-up to room temperature. Certainly additional problems could arise from "by-pass" procedures, but are the devices\pumps "cold" - 1 - 6 C ?? I suspect they operate at room temperature, nowhere close to refrigerator temperatures. After all that rambling, I meant to say that I don't why anyone would test "cold autoantibodies" at temperatures below that of typical (surgical) rooms. However, I'm sure there is a a whole library of circumstantial, anecdotal evidence supporting such extreme testing protocols.

Does the MTS gel card you typically use contain polyspecific antiglobulin reagent (anti-IgG + anti-complement) or does it just contain anti-IgG ? I think most users are using anti-IgG cards, and if that is the case, they're already dealing with the "Is it possible to miss a complement binding IgM antibody early on by using IgG only." issue.

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....

Might be a rare IgG anti-A1 - you may not see in in the Reverse (presumably IS or buffer-only gel card), but is detected when you do anything with an antiglogulin reagent. You could try doing the Reverse by IAT. The DAT may just be a red herring, but it might represent a weird autoantibody that favors group A cells.

Please excuse my ignorance, but how can a "DAT Control" not have an antiglobulin reagent?

CD = chloroquine diphosphate. A chemical treatment to remove immunoglobulins from red cells, in the hope of getting a negative DAT, thereby allowing the use of antiglobulin-reactive antisera without interference from a positive DAT.

Please supply the reference for this absolute nonsense. It sounds like I would enjoy reading such fiction.

I agree with Malcolm - all the best people are DCeDCe.☺ I also agree with his analysis. The differences you are seeing are probably because you are not doing as complete an adsorption as the IRL. Indeed, this partial and sequential adsorption process was the heart of the early work by Dr. Issitt (and others) when they were trying to determine the specificities of autos. Most warm autoantibodies mimic Rh specificities (there are exceptions). The older literature is full of anti-e-like autos and antibodies to compound antigens (Ce, cE, etc.). There are also a smattering of anti-D-like autos. Not uncommon, by any means. Some labs used to make an effort to ID autos, but in recent years, time and money have have almost eliminated this potentially interesting technical challenge.

A very good point, Anna. The same applies to anything done "off-label", regardless of how much validation has been done.

Scandalous, Scott !!!!!

I think you're joking, but just in case...... It's a simple C1 x V1 = C2 x V2 calculation. One of the few times when algebra gets applied outside of high school. To do this accurately, you must first know the hematocrit (concentration) of your "packed cells" - this will be used as C2. For this example, let's say the hct is 75%. C1 = desired hematocrit (concentration) - 3%; V1 = desired volume - for this example, let's say we want 100 mL of 3% cells. Using the formula and information above.... 3 x 100 = 75 x V2, which resolves to 3 x 100 ÷ 75 = 4 mL. Ergo: 4 mL of "packed cells" in 100 ml pf PBS will yield ~3% suspension. This process works for low cell concentrations where the added RBC volume is only a small portion of the whole (4 mL added to 100 mL). If you try to make higher concentrations, you have to take the RBC volume into consideration as part of the whole volume.☺