exlimey

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.

What do you mean by "extensive in-house validation" ? What is "in-use testing" ?

In my opinion: Yes, you can. That statement on chemicals is meant to tell you that you can't use it in a medical or nutritional fashion. You will not find a package insert for any raw chemical - the supplier has absolutely no idea what the buyers are going to do with the materials. If you buy plain old sodium chloride (NaCl), it doesn't have a package insert, exactly for the reasons stated above. Anything anyone is doing with DTT and/or other exotic chemicals in the Blood Bank realm is completely out of the typical regulated environment. These chemicals assist in a complex investigation, they are not making a diagnosis. Sometimes it is necessary to go beyond the use of licensed, registered, validated reagents to best serve a patient. That being said, the DARA issue has brought DTT use into some routine labs. Complex serological investigations should be left to experts - the high level Reference Laboratories, who understand the pros, cons and limitations of the specialized reagents they use. End of rant.☺

You may be referring to trypsin-treatment of the red cells (screening cells). Apparently CD38 is destroyed/inactivated (along with Lutheran system determinants) but Kell system antigens remain intact. Other blood group antigens are also affected by trypsin, so I think the modified approach involves testing the patients' samples against both DTT-treated and trypsin-treated cells. To further complicate matters.....manufacturing a reliable, consistent trypsin reagent is VERY difficult. The enzyme activity of source material varies immensely and, as with other enzymes, stability is a problem.

You performed stability testing on your home-made material ?

Feisty rainbows? Some grayling? I'm very jealous - haven't made it to Alaska yet.......

I concur. A good writer should be able to finagle that logic into a validation plan. Getting hold of said cells may still be problematic, but at least it might be simpler than an expedition to catch the Loch Ness Monster.

First, a disclaimer: I am not a Regulatory expert. Perhaps you're over-stretching? I don't think you're required to validate the new system in the sense that the manufacturer has to do for licensing - that's a LOT of work and an attempt to cover every variable imaginable. In your case, perhaps just proving the system works in your facility is enough. That would probably only need to include a couple of examples of weak-D, rather than the whole gamut.

Well said. The world is so "dangerous" these days, it's a wonder we're still around.

Contrary to the manufacturer's instructions ? That's really living on the edge. ☺

I must confess.....I was poking the bear. I have worked with LN2 for 25+ years and personally believe the risk of asphyxiation is highly over exaggerated. Any room with adequate ventilation (and that's a very loose term) is safe. Most interactions with an LN2 environment are short term, except perhaps for the worker(s) at the NHSBT National Frozen Blood Bank where special ventilation systems should have been designed into their operation. I was expecting answers involving cryogenic burns, too.

What, precisely, are the "H&S" risks?

Anything is possible, but the screening cells are designed with the intent to detect all (common) antibodies, including the antibodies traditionally considered "colds" - MN, Lea/b, P1, etc. It is remotely possible that the frequencies/incidences of an antigen have come together perfectly to yield these results, but you'd have to be very lucky or unlucky, depending upon your point of view.

If the screen is negative by IS, it rules out the presence of a generic "cold". Are you only seeing this phenomenon during an IS crossmatch ? Are you only crossmatching group A units ? Does the same "incompatibility" happen if you crossmatch group O units ? Typically antibodies detected prior to an antiglobulin phase are IgM. These do not necessarily "carry through" to the antiglobulin phase, especially if they have limited thermal amplitude.

I don't know why the Control is positive, but the actual test is negative. In theory, the anti-D and control should be a matched set - they should be formulated exactly the same, except for the presence of the anti-D. Every manufacturer has "secret ingredients" - potentiators and other chemicals that stabilize and enhance the antibody reactivity. The controls, if supplied, should include the same ingredients. If you are using a control from a different manufacturer or product line, it might explain your findings. Are you saying that a monospecific anti-complement reagent is reactive with the patient's cells? It's not unheard of for warm-autos to bind complement, but since this looks like an autoanti-e, it would be highly unusual.

Nicely done. Often, a little strategic word-smithing will save the time and effort of revising a procedure or policy.