StevenB

In my experience, the two most common causes of weak reactivity in Gel and negative reactions in tube are either rouleaux (no, not my pet "go to", but still applicable) or reactivity seen only with the manufacturer's prepared 0.8% reagent cells. We encounter this fairly routinely: Hospital gets Gel reactivity, sends it in, we do a selected panel making our own 0.8% suspensions from various manufactures, get a negative Gel test, test with a manufacture's 0.8% panel and get reactivity. Basically, reactivity dependent upon something in the manufacture's product. Kinda similar to a "LISS panagglutin".

It's not uncommon to see rouleaux interference in Gel testing but it's not something we see frequently. The crossmatch question is interesting: Technically, the screen is negative for alloantibody reactivity so I think you'd be able to follow your procedure for that result. However, with the immediate spin crossmatch, you may have to perform a saline replacement to obtain a "compatible" unit. I wouldn't want to assume an incompatible IS crossmatch is due to the rouleaux without confirming that.

Auto directed at senescent red cells can show mixed-field reactivity. With your additional information, kinda seems the most likely cause of your observed reactions. Garrartty and Petz discuss this in Immune Hemolytic Anemias, 2nd ed., pg 243. The issue with PEG adsorptions is they may work too well, potentially resulting in the loss of weak (low titered) antibodies. Of course, that could happen with any adsorption, I guess, but with the samples we see in our ref. lab, it is the last technique we would use to adsorb out an autoantibody. I'm not saying you are wrong to use it. I'm just saying it has it's drawbacks;

Hmmm.... Not too fond of PEG adsorptions; did it truly adsorb the antibody out or was the antibody lost in the precipitate? Pondering if you have two different things happening: Rouleaux in your reverse (I'm assuming the reverse typing with the A1 and A2 cells was not 3+ or 4+....were the majority of the cells at the bottom or top?) and Gel testing with an alloantibody in PEG? With 4 drops of plasma, you could eliminate the rouleaux as an issue. Thinking out loud....negative DAT, negative autocontrol usually does not equal an autoantibody (one of the reasons I'm cautious about the PEG adsorption resullts), although it is well known that "antibodies don't read books". In the tube tests, how strong was the initial spin prior to adding the PEG? I know I'm reaching a bit on the rouleaux, but I have always found it beneficial to not overlook the simplest explanation before moving on to the more obscure. Is there any additional sample in hematology that you could play with?

DAT performed? Neg autocontrol does not always equal a negative DAT. How strong was the mixed-field and was the antibody screen in tube or Gel? PEG prewarm in tube not mf?

Transfusion history?

Ruling out on zygosity....I'm assuming you are referring to "crossing-out" on heterozygous cells, as opposed to homozygous cells, that are nonreactive in Gel testing?

I'm not aware of any quality control requirements for weak D testing. Looking at two different manufacturer's inserts for anti-D, I did not see any requirements for quality control testing for weak D. In addition, our daily quality control of the anti-D reagent does not involve testing a known weak D. It will be interesting to see if anybody else knows of a requirement for weak D testing.

Hmmm... Never have used the prewarm technique to resolve a forward type. Just "thinking out loud": In the scenario of a cold interfering autoantibody, prewarming is typically used to prevent "free" cold autoantibody from binding onto red cells causing unwanted reactivity. In testing a patient's red cells, there should be no "free" autoantibody present if the sample was adequately washed. The issue with the forward type is the antibody that is already bound to the patient's cells. If warm washing did not remove it, then I'm not sure how prewarming the forward type would be more effective. If it works though, great! Whatever it takes to resolve ABO discrepancies due to cold autoantibody interference, I'm for it! We warm wash the patient's cells with 37C saline. If needed, move to a form of heat elution (incubate or even wash with 40-45C saline) and if that's not effective, treat the cells with 0.01M DTT (treated controls must be included). Reverse type resolution: I- cells, prewarm at 37C spin, and just recently, resolved a back type issue with testing at 37C/AHG with heavy chain specific anti-IgG. I'm sure I don't need to say this, but I'm going to anyway: In a situation where you have an unresolved ABO discrepancy and you have to transfuse, don't guess. Give group O red cells.

Applied Blood Group Serology, 4th Ed. chapter 6, pages 129-132 addresses complement activation by blood group antibodies. It's an interesting read. This is the verbiage we use with a recently transfused patient that has demonstrated a positive DAT, a new antibody in the eluate and perhaps in the plasma/serum: "Serologic results, including a positive DAT due to IgG and complement (C3d), support the occurrence of a delayed serologic transfusion reaction." The DAT portion may be edited as needed. We're not making a diagnosis, but simply altering the physician to its possibility.

Lol...in my opinion, I should look at the screen results more closely. #2 can work...if serum is tested and polyspecific AHG is used, neither of which is a given anymore though.

A few antibodies that can cause the screen results that you showed include; I, M, dosing M, Lea, Leb, P1, IH in a group A individual, newly developing multiple antibodies in a recently transfused patient all come to mind. I'm sure there may be more. In my opinion, coming up with answer #2 with the information provided is a bit of a stretch.

No resources, just experience... we perform what we call "alloadsorptions" on a regular basis.

Any questions Ward_X or are you "good to go"?

Just curious...did you serologically confirm the Rh phenotype of the reagent cell in question?

In Applied Blood Group Serology, 4th edition, pg. 332, Issitt refers to a study by Shirley et al. on this very topic. The study concluded that 5 of 27 patients who had anti-E and only received E- units eventually made anti-c. They suggested this justified the expense and extra time needed to find units that were negative for both E and c. Issitt however, uses the findings to argue just the opposite, "Those of us (PDI...referring to himself) who believe the E- c- blood should be provided only after the patient has made both anti-E and anti-c, use the finding that none of the 5 patients in this study who made anti-c suffered a delayed transfusion reaction, to support their view." While the Technical Manual states the "some experts advocate for avoiding the transfusion of c positive blood in this situation" obviously, some experts do not. In an emergent situation, I don't feel the potential time delay in finding or ordering units that are E- and c- is justified when the patient has not demonstrated they have anti-c. In a routine situation, we recommend E- units, but quote the Technical Manual regarding this situation and let the customer make the decision.

Lol...I like the way you think, Malcom, but I don't have control over those decisions. Being a reference lab though, we push our efforts when testing referred samples. Micro reads are part of our routine if the patient has been transfused or pregnant within the last 3 months. Testing in PEG however is optional and PEG is notorious for revealing micro reactivity which often can not be identified. Having said that, I have on many occasions identified micro only, clinically significant antibodies in PEG. Would I recommend a hospital blood bank read micro? No, I wouldn't.

Thanks yan....the macro negative reaction in PEG at IAT was with the unadsorbed plasma and was tested with Immucor’s murine monoclonal Gamma Clone IgG. As Mabel pointed out, this reagent does not detect IgG4 and the anti-CD47 is....evidently.... an IgG4 antibody. In my hands, it was microscopic reactive so I believe in the future we will drop that read when faced with these patients.

Mabel was kind enough to share this sample with our lab as we had not had the pleasure of seeing one of these. The sample was limited and it technically was not a referral, so my work was centered on getting to know anti-CD47. The patient forward typed as an A with no issues (I've heard the forward type can at times be affected), but reverse typed as an O, with 2+ reactivity in the A1 and A2 reverse cells. The patient typed Rh positive; no reactivity noted in the Rh control. The DAT was micro positive with poly specific AHG, heavy-chain IgG (HC) and C3, but was not interpret-able due to a micro positive saline control. Warm washing x4 with 37C saline resolved the positive saline control and still demonstrated micro reactions in the previously mentioned AHG sources. Interestingly, the reactivity was actually slightly stronger than the initial testing. Initial antibody testing was 2+ reactive in saline at initial spin, 3+ in saline at 37C spin (post 30 minute incubation) and 3-4+ reactive in saline at IAT with HC IgG, 3-4+ reactive in PEG at IAT (4+ reactions with the Rh negative cells tested as compared to 3+ reactivity with Rh positive cells; consistent with reports in literature), and 4+ reactive with papain treated cells (carryover reactivity was noted as a "no spin read" was 3+ and agglutination was noted prior to the addition of HC AHG). Testing with Immucor's murine monoclonal IgG Gamma Clone in PEG at IAT was macro negative, micro positive. Alloadsorption x2 (using 2 volumes of cells to one volume of plasma) onto papain-treated R1R1 and rr cells of known phenotypes (we usually do R1, R2 and r cells, but with the limited sample I dropped the R2 adsorption) removed the initial spin reactivity and confirmed the patient was group A. Alloadsorptions x3 were 1+ reactive in saline at 37C, and 3+ reactive at IAT using the HC IgG. x4 adsorptions yielded the same results. Out of curiosity, I did run a D-- cell with the x4 and it was nonreactive at 37C, but 2+ reactive at IAT. For fun, I performed titration studies on both the R1 and rr x4 adsorptions (saline, 60 minutes 37C incubation). Macro reactivity at 37C was noted at 1:32 in both samples, but technically they had a titer of 4 (1st observed 1+ reaction). At IAT, both samples had a titer of 16,384.... AFTER 4 adsorptions! A recent report in IMMUNOHEMATOLOGY stated that using equal volume (cells:plasma) adsorptions onto papain-treated cells x3 and x4 resulted in "the majority" of the samples being nonreactive in saline at initial spin and in PEG at IAT. Lastly, I did perform EGA treatment of the patient's red cells to see its affect on the positive DAT. The micro reactivity was removed following a 1min. 30sec treatment of the patient's cells, but since the sample was way beyond the manufacturer's specimen recommendation, I'm not totally confident in the observed results. Net result: It looks like alloadsorptions will be needed to resolve any observed ABO discrepancies due to reverse cell issues and PEG testing with Immucor's murine monoclonal Gamma Clone, with a macro only read, would resolve any the issue of detecting underlying alloantibodies. Thanks much Mabel for sharing this sample with us!

Antibody reacts consistently with all cells tested, including a phenotypically "matched" cell, but is nonreactive with the patient's autocontrol is indicative of an antibody to a high frequency antigen. Is initial testing in Gel, solid phase, LISS, PEG? The reason I ask is that in my experience, HTLA antibodies tend to show variable reactivity in tube, but can react more uniform in Gel testing (just guessing the same would be true for solid phase). Chido/Rodgers are possibilities; U is not as the patient is s+. Ena is a possibility as are Ge:2 and Ge:4.

For me, it's completely dependent upon the results I obtained in my serum/plasma testing and the amount of eluate I have to test.

We use cold and cold, not that it would have mattered in this case. It would have been interesting to see what a Lui Freeze eluate would have yielded. What did your ARC come-up with if you don't mind my asking?

It is a bit annoying that the manufacturers do not have grades below 1+ as we see these all too often. However, we never interpret these reactions as 1+ as that would not be consistent with the manufacturer's parameters. When faced with this reactivity, we will record +w or w+ and define these reactions as being less than 1+ on the worksheet. For titers in Gel, I would think this would be an important distinction as the end point for a titer is the last 1+ result, not the last result that demonstrates reactivity. That could easily be a 2 to 3 tube difference and may present the physician with information that is not totally accurate.

Just curious...what type of eluate did you perform and if Acid, did you use cold saline and wash solution when prepping the patient's cells?

Interesting topic. In just a few posts, it is easy to see there is a variety of ways labs approach the "how often are elutions performed" question and under what circumstances. I too agree with Malcolm; in the presence of AIHA and a positive DAT, most likely you will get off a panagglutinin every time you perform an elution. Once this occurs, there is no point in performing additional elutions on a routine basis. In the patient though who has a positive DAT and is transfused on a regular basis, and has a negative eluate (yes, this does occur) the elution question is a bit different. Technically, any transfusion can result in the production of an alloantibody that may or may not present itself in a hemolytic fashion. It is possible to have a "delayed serologic transfusion reaction" that shows no signs or symptoms of a hemolytic process. In this scenario, the idea of not doing an elution on a regular basis because it has never revealed anything in the past, may result in missing a newly formed, clinically significant antibody that is only detectable in the eluate. Not performing an eluate in this scenario is not without risk and should not ever become "policy" without proper overview of the clinical situation.