exlimey

Rhetorical question: Is it a typical request for a "Type & Panel" ? If you are almost certain that the eluate will be reactive, I agree that the screen may be redundant. However, it is still a test against phenoptyped cells and the information is still valuable even if you end up testing a panel.

Preparation and standardization of enzymes is notoriously VERY difficult (making trypsin is a nightmare). Each batch of source material may have a very different activity level than the previous. Stability is also an issue, even when frozen. You can probably get all of the enzymes you list from Sigma, but they will undoubtedly have 15 versions of each and it may be difficult to choose which flavor is most suitable. I would stick with papain and DTT. They'll be most useful, most often. You'll get a lot of information from just those two. Only the very highest-level Reference Labs. should mess with alpha-chymotrypsin, trypsin and pronase.

A good serologist is an efficient serologist. Do the small work first - the screen. Only do the panel if the screen is reactive and you need more information.

Very true and quite possible, especially neutralization by serum antigens. One might even see a positive DAT if the patient's cells sucked-up passive anti-A. On the other hand, a large enough dose of out-of-group platelets might leave some isogglutinins available to mess up the ABO results. Just throwing out ideas.

Passive anti-A from a transfusion ? Possibly from a platelet product ? I agree with testing more group A cells.

This is obviously still a ticklish issue. Several good arguments are presented above, but a couple of things come to mind.... A mis-draw is always a possibility, but some of the algorithms above suggest that it happens on a regular basis. Does anyone here have statistics ? I'd love to see what the data says. Even when drawing a second specimen, based on blood group frequencies, the odds are very favorable that you'll get a corroborative ABO/Rh typing, EVEN IF THE SAMPLE IS COLLECTED FROM A COMPLETELY RANDOM PERSON. Just a couple of thoughts.....

This is a like-for-like replacement using OEM parts, and supposedly, as suggested by the manufacturer (see jmm8427's post above). My opinion: No validation of any kind required.

How does that process "qualify" the Rh- cells ?

Even the very best of managers or management teams cannot work miracles if they lack the appropriate resources.

Well said.

There is a very good reason why "generalists" avoid Blood Bank and transfusion medicine - it's complicated and you need a lot of specific training to do it well. Even today, with a significant level of automation, a warm body is often needed to interpret results and give recommendations. And then add the fact that there is a seemingly endless list of "exceptions", "equivocal", "indeterminate", and other levels of results that confound even a trained (SBB) person, let alone an "every other weekend, third shift" employee. Cross-training is a must for very small, low volume facilities. No question. However, once work gets to a certain level of complexity and volume, institutions should seriously consider having dedicated staff. I don't know how "generalists" manage to maintain their legally-required competency levels.

A sound approach.

I like the approach taken/suggested by Baby Banker and pbaker, but it does need a moderately high skill set to make up the selected panel. Perhaps that's not possible in the "average" blood bank? A follow-up question: Are you performing titrations (potency) of the antibodies that are identified? If so, how often ?

I think I understand what you're saying - we don't want to miss isogglutinins, but why is this practice only applied to cord samples ? If you want to perform a cold autoadsorption, preparing the cells (washing) with WARM saline would be better - this would theoretically eluate off some of the already bound immunoglobulins and free-up antigens for binding of additional (auto)antibodies. You would have to "cool-off" the warm-washed cells before actual use in the adsorption.

The question was somewhat rhetorical: I know why it's done and agree that the approach is quasi-logical (as Malcolm points out). There appears to be more emphasis on detecting isoagglutinins on cord cells than in older patients, perhaps with good reason. I was attempting to make folks think about the logic of some of the tests/processes that have become "normal". One could argue that we should want to detect isoagglutinins on any patient's cells, regardless of age, and therefore we should use "cold" wash solutions universally - what's good for the goose is good for the gander.

In my opinion: Antigens are unlikely to "wash away" or be "altered" by washing with normal saline. [One exception: Lewis antigens may be liberated during washing.] Antibodies, on the other hand, are more likely to be eluted from red cells by excessive washing with acidic saline. I doubt any publications exist that prove excessive washing has the effects you describe, but I would love to be proved wrong.

What is the reasoning behind using "cold saline" for cords and not other samples?

Based on your initial screen, it appears that the patient's cells are probably already coated with antibody (DAT-positive). Unless you are DTT-treating the patient's cells - not typically part of the testing protocol - they will remain DAT-positive and therefore reactive in the second series of tests using the DTT-treated screening cells.

Please explain your logic.

Questions: Is the saline used for tube washing the same as that used in the automated cellwasher ? What is the pH of your saline(s) ? Why was testing repeated ? What was "wrong" with the original results ? If the pH of the saline used is low (acidic), it can cause elution of bound antibodies. A total of eight washes in mildly acidic saline in the first case may have resulted in a negative DAT. A weakly positive result in the second case may be because the cells were not exposed to the same degree of acidity (fewer washes, less time exposure).

Don't forget....in this case......Rh-negative units are required. Multiply by 0.15 !!!

As I understand it, IgG2 and IgG4 do not cross the placenta easily, whereas IgG1 and IgG3 are "actively" transported from Mother to baby. Obviously the baby benefits from this passive immunity. Since most examples of anti-Yta are predominantly IgG4, that's why they don't cause HFDN, especially since, as you mentioned, the Yta antigens are poorly expressed in utero. Never say never, but I doubt that anti-Yta occurs naturally. Cartwright system antigens are poor immunogens, so from my point of view, the antibody's existence implies repeated stimulation. That being said, your patient may be a "super-responder". I don't if anyone really understands why immune responses vary or how some result in different IgG subclasses.

I think matching Rh and K is a good idea. My foggier-by-the-day memory leads me to believe that most of the examples of anti-Yta that I've seen were single specificity. I do remember a couple with anti-D and at least one with anti-c. As to longevity......anti-Yta tends to fade away over time in the absence of additional stimulation. In your patient, additional pregnancies may be a source of re-stimulation, so her antibody might be more persistent. As usual, these are generalized statements and opinions. There are always antibodies that don't read the literature.

I echo Malcolm's sentiments - you appear to in control of the situation. One wrinkle, perhaps: Are you able to "sell it" to the physicians ? It has been many moons, but I have performed dozens of MMAs on examples of anti-Yta. Almost all were considered insignificant and even those that were over the threshold were barely above. Add the fact that they are almost universally IgG4 - no harm to the baby and minimal risk of a transfusion reaction. The examples that did just make it into the "significant" interpretation usually had a touch of IgG1 and/or IgG3. I have no hard data to support it, but I'm quite sure that many of the patients (even those with "significant" results) received Yt(a+) blood without consequence. If the case arises and in my opinion, giving Rh-negative blood is way more important than fussing about anti-Yta. Given your geography, it might wise, albeit extremely cautious, to suggest the patient move to the "big city" (Portland) for Christmas, but that would only hold true if the physicians want easier access to Yt(a-) blood.

Bottom line: If a facility "manufactures" a reagent, it is obliged to prove that it functions as designed and can be made repeatedly/reliably. However, the rules and regulations that apply to licensed and registered reagents do not necessarily apply to "home-brew" materials. Providing the material will only be used in-house and not distributed, there is no need for "parallel testing" of any other formulation, no matter how crude the manufacturing process appears to be. Such testing is only required for licensed or registered reagents and devices (under the umbrella of Design Control). I'm assuming (correct me if I'm wrong) that the PBS used in preparation of the DTT is made immediately before use, and therefore the stability of it is irrelevant. Stability of the finished reagent may be important if the "manufacturers" wish to assign an expiration date. This may not be necessary, since in the case of DTT, controls are included EVERY time it is used - the operator has a current and immediate indication of whether the reagent worked or not. Validation is essential to provide confidence when it is impossible to directly prove that a process has worked. Think of vaccine manufacture: One cannot test every vial/dose to see if it meets specifications - all of the product would be compromised. When integral testing proves that a process works each time, validation requirements are minimized.