exlimey

I'd like to be a fly-on-the-wall when you have that conversation with an Inspector......just kidding. I suspect many folks are doing just as you describe and for the same reason.

As Malcolm points out, washing is not really important for ABO grouping in the modern era of tube testing. The reagents are formulated to tolerate a degree of neutralization. I assume this is true also for the reagents used in gel cards. Washing is important when using the cells in any test using antiglobulin reagents (as LIMPER55 notes). Perhaps more of a concern is that your in-house procedures should not contradict the reagent manufacturers' instructions. One could even argue that an SOP is not really required if you follow the package insert - that's your SOP! Whichever approach you take, you should definitely be following your SOP. I can see and hear the regulatory folks cringing in their seats as they read your post.

A blanket policy like the one gagpinks describes will result in a lot of clinically irrelevant testing (as Malcolm so elegantly stated). Having a policy is a good idea, but it must be applied with precision, on a case-by-case basis. An eluate may be relevant if there's clinical or laboratory evidence of a transfusion reaction. I've heard of policies where a significant change in the strength of a post-transfusion DAT triggers preparation of an eluate. However, as Malcolm also points out, such eluates in persons with "warm-autos" are almost useless, unless one is prepared to perform adsorptions on the eluates to look for alloantibodies behind the auto.

Agreed. One could also add questions related to the anticipated use in a research project - anticoagulant therapy, aspirin use, etc. That kind of information might be pertinent to the research lab. Wouldn't be a lot of use collecting platelets from a donor who has been taking Plavix, for example.

Good point. I know that the Gamma/Immucor PEG reagent available in the US does have low ionic properties. Don't know about other suppliers, but it's common for reagents like these to be very similar.

PEG-only antibodies are well known, but it's common that they can also be detected in one of the other very sensitive methods (Gel or Solid Phase). The LISS method is less sensitive than PEG and it's not surprising that you can't detect it there. It is possible that the PEG reagent contains a chemical that is critical to the reactivity of your patient's particular antibody. There are lots of documented examples of "chemical-X-dependent" antibodies. The absence of the specific chemical from the LISS test and the Gel system would render it nonreactive/undetectable. Trying another manufacturer's PEG reagent (presumably with a different formulation) might clarify the situation.

I agree completely. Hence why I wrote "partly due to their poor expression". And, don't forget, if antibodies to Lutheran system antigens are in part IgG, they are often they wrong subclass to readily cross the placenta (IgG2 and IgG4).

I agree - mixed-up information. If CD38 is only poorly expressed on cord cells, it would make sense that they would be less likely to react with the therapeutic anti-CD38 antibodies. Many workers believe that antibodies to Lutheran system antigens (also carried by CD38) rarely cause HDFN partly due to their poor expression on cord cells. This seems to parallel one of the mixed concepts above - no antigens on cord cells, no reactivity. If this is true, then K+ cord cells could, perhaps, maybe used to exclude anti-K in Dara patients. Not quite sure how you'd put that in a report. However, having access to a large number of cord cells and the ability to mine them on regular basis for K+ examples seems like a pie-in-the-sky situation. It might be possible in a large, competent Reference Laboratory that has a good working relationship with a large maternity hospital. Might be some ticklish Informed Consent issues, but.......at least its feasible. As to typing the patient - monoclonal IgM anti-K reagents are available. This makes the DAT moot.

A few points, but first, a disclaimer: I am not an expert in FDA or CLIA regulations. I suspect that many labs are using some form of a "home-brew" reagents. The number of internal controls required by various regulatory agencies increases by the day. Add the large number of "competencies" necessary for all the staff and one has to use what is available. Personally, I don't think viral testing should really be an issue (although it would be nice to know) - you're not preparing this stuff for distribution/sale to other facilities; the "treat all materials as potentially infectious" mentality and Universal Precautions approach should cover you. The absence of an expiration date is probably better that assigning an arbitrary one (you have no stability data). In your application, you have a in-built control - it either works or it doesn't (I'm presuming you test the untreated [positive result] and the treated cells [negative result]?).

On the surface, "stability" of red cell products appears quite simple. However, there are a huge number of variables to consider, especially the age of the cells when put into use. Chemical- or enzyme-treatment of those cells adds another huge wildcard - it certainly voids the original expiration date assigned by a commercial supplier. One approach may be to treat cells of a known/fixed age, suspend them in a commercially available red cell preservative, and then test the untreated (in the same preservative) and the treated for the presence/absence of a variety of antigens over the course of a few days or weeks. Ideally one would test all the "clinically relevant" antigens. Or, one could choose those antigens that are thought to be less stable over time. The best serological way to detect weakening of antigens would be to prepare serial dilutions of the test reagents and run titirations against the untreated and treated versions of the test cells. It's a lot of work and all the commercial suppliers have done it for all of their current red cell products. However, I doubt that they will ever supply DTT-treated cells since the market is so small and potential profit very limited.

Could be an (auto) anti-LW, not uncommon in older folks. DTT- or AET-treatment of the test cells might give some insight.

Most of the common Rh antibodies have been reported as IgM. A "saline-reactive" anti-E is probably the most often seen. I've personally seen an anti-e that appeared to be IgM. Way back when, in the dawn of time........there was serious concern over examples of IgM anti-D potentially causing ABO reverse typing discrepancies - hence why the commercial reverse calls are all Rh-.

It's a bit unusual for the tube typing to be more "sensitive" than the automated (and I use that term loosely). May I presume that the anti-B used on the Provue and in the tube tests are formulated from different monoclonal cell lines?

Mabel is quite correct - I also remember that there were a number of ticklish "administrative" issues. A more technical note: Since the volume red cells from one cord are small, I suspect the the products that were distributed were POOLS of cord cells. That is the only way that the manufacturers could have enough volume to make a viable product. A pool would mean a mix of phenotypes and the product wouldn't be much for the application suggested by WisKnow.

Pony, I don't think anyone suggested that they get a shipment of reagents (with the same lot number) with varying inserts.

A word of caution: The "old" insert may still be relevant to in-date reagents on your or others shelves. But saying that, it doesn't excuse the absence of the new insert.

I agree, Brenda. Immucor does a relatively good job of indicating changes. It can get a little ugly when the same "new" insert (with highlighted changes) is still being issued many years after the change. Perhaps Immucor buys a minimum number of inserts with highlighted changes and at some point transitions to the "clean" version ? In the recent years, in the wake of mergers and consolidations, I've noted that the changes are often just name, logo, address or telephone numbers - of no real impact to the average user (unless you need to call Customer Service, of course).

The short answer: Money - throwing stuff out is expensive. The long answer: To get a good price for printed materials (package inserts, labels, etc.), one must purchase tens of thousands of copies. If changes are required, but not especially critical, the regulating bodies (FDA) will allow manufacturers a fair amount of leeway to use up current stock. Throwing out anything has an associated cost and since the commercial manufacturers are in the for-profit arena, they will tend to shy away from such actions. I'm sure this contributed to Immucor's tardiness. Another contributor to slow moving changes: In the US, any changes to a package insert (or label) must be approved by the FDA before that new document is distributed. This process can take 6 - 12 months depending upon the complexity of the changes and the number of times the document goes backwards and forwards between the two parties.

Reagent red cells certainly do not "express antigens weakly". There is no way a commercial entity would risk distributing a product that might give rise to customer complaints - the regulating bodies (the FDA in the USA) would be all over those issues. There may be some weakening of antigen strength over the life of commercial red cell products, but this weakening would probably only be noticed with a borderline, wishy-washy antibody - perhaps you have stumbled upon such a beast. Commercial reverse cells (A1, A2, B, O) are washed extensively before preparation of the products. Additional washing will not "uncover more antigen sites". At most, washing (with saline) will remove the commercial red cell diluent/preservative and change the chemical environment of the test system. This change may indirectly enhance or suppress reactivity of some antibodies. If you are using unwashed cells in ABO tests, remember that ABO substance will also be in the plasma/serum and may neutralize an antibody before it has a chance to bind to the red cells. In such a case, washing will definitely enhance reactivity.

Red cells that contain HbSS (or HbSC) are more resistant to hemolysis in hypotonic environments than cells carrying "normal" hemglobin. This makes it a useful technique to recover autologous cells from transfused sickle cell patients (providing the patients have some level of native RBC production). It is not useful for harvesting autologous cells from transfused patients who do not have HbSS. A reference: A Rapid Method for Harvesting Autologous RBCs from Patients with Hemoglobin S Diesease. Brown D., Transfusion 1988; 28:21-3

Sandy, How do you deal with discordant results? There's a great difference in sensitivity of the assays you mentioned. Or do you bias the results beforehand by making sure your test samples perform in each system? Not a criticism, just wondering.

Perception is important, but education can help smooth over the concerns. I think it's important to remember that the outside of the bag "lost it's shine" as soon as it left the shipping box and was used to collect blood. There is nothing sterile about a church hall, a high school gymnasium or any other of the myriad of places blood is collected. I wonder how hospitals reconcile the fact that "dirty" blood bags are transported into and used in their super-clean surgical suites ?

A valid point. It is not uncommon to find a spurious positive DAT in a sample collected in EDTA tube, especially in "older" tubes. My point is that crossmatches use segments (pigtails) and the presence of the sugary-goodness in the collection systems can also cause positive DATs.

I agree with Malcolm's comments and would add that I suspect that many positive DATs in donors occur after collection of the blood, i.e., the donor is not actually DAT+ in vivo, but somehow the red cells pick up globulins during exposure to the chemicals and environment of the collection system.

sbazel - bad idea to use the earliest expiration date of the components of your cocktail. Neither apply to your working reagent. Certainly the anti-D has been grossly modified from its original. To assign a genuine expiration date to your cocktail, you would be obliged to perform a formal stability protocol.