Joanne P. Scannell

Yep ... maybe that donor is not homozygous for M.

I agree ... it's a cold antibody with the specificity Anti-M (all cold antibodies have specificities, most we just report out 'Cold Antibody of Undetermined Specificity' ... really, they are clinically insignficant unless you are going hypothermic during surgery or such). Your reactions (Mixed Cell) in MTS is classic cold agglutinin (refer to the interpretation guide) ... we see Anti-M like this a lot. Also, MTS is more acidic than tube testing. If you are old enough to remember, we used to acidify serum to enhance Anti-M if we suspected that antibody. So, by using this system that is somewhat acidic, we expect (and do!) see a lot more Anti-M than before. Rouleaux is often seen with cold agglutinins ... they are large molecules (IgM) and increased production can skew the Total Protein/Albumin ratio enough to cause rouleaux. So, be not surprised at that observation at 37oC (the proteins are still there even though the antigen-antibody reaction is not occurring at that temperature). Also, be not surprised the patient is typing M-Pos with your 'Anti-M' reagent (and with a negative auto). I venture to say your patient is really Mg-Pos. (There is something about the M antigens that cross react, so antigen typing with a reagent is not as pure as other typings are. This is very common ... so common that I don't recommend my staff type the patient with Anti-M 'to validate the antibody ID' because it's more confusing to a novice tech than it is worth.) Transfusion: Establish if this Anti-M reacts at 37C. Prewarm testing will help determine that. - If reactive only at Room Temp: Our policy is that if the patient has a cold agglutinin, we recommend a blood warmer. This is only precaution and makes everyone feel better about transfusion 'incompatible' RBCs. (I.S. will be incompatible unless you go through absorptions ... but why bother with all that?) With a cold agglutinin that reacts with only some donors (like Anti-M), we just crossmatch (I.S) and issue the compatible units first. Of course, if the OR is planning hypothermia, it becomes a clinically significant antibody ... - If reactive at 37C it is potentially clinically significant ... go by your current policies for giving antigen-negative/extended crossmatched RBCs when the patient is producing a clinically significant antibody. Sorry if I rambled on this ... we see these a lot now that we are using MTS.

Good morning, Malcom! 'You and I' know that (hence I used the word 'exacerbate') ... but try explaining that to a physician.

Ok, a list of 'agree/disagree' items here: I agree with your supervisor on one plane: Don't write things on documents unless you are instructed to do so. It's very important that things don't 'depend on who is working this shift' ... Blood Banking is a team sport and demands consistency. I disagree with the 'use a blood warmer only if the cold agglutinin reacts at 37C'. If that's the case, what are you circumventing if the antibody reacts at 37 and you are transfusing at 37? Nothing. The idea of using a blood warmer is to keep the temperature of the transfusion ABOVE reaction temperature. If you have a cold agglutinin with a thermal amplitude above 30C, a blood warmer is not going to change the reactivity and the MD needs to start thinking about reducing the antibody concentration and/or production (pheresis, medication) rather than a risky transfusion ... the patient will destroy the donor RBCs just as quickly as the auto RBCs and may even exacerbate the problem (introducing new antigens, stimulate the immune system). I agree with your 'caution, best to use a blood warmer': To assure we are all applying the same rules for every patient, I have instructed my staff to issue RBCs with a blood warmer if they see a demonstrable cold agglutinin at the Immediate Spin/Room Temperature phase (we routinely perform this phase with pooled O cells during initial pretransfusion testing to look for 'room temperature/close to infusion temperature' cold agglutinins and rouleux so we have no such surprises later during crossmatching). Is a blood warmer always needed for these cases? Probably not ... but it is the safer side of caution and provides for continuity of a policy. We can do this automatically because it is written into our procedure which is in reality our Medical Director's instructions to us ... yes, she signs all our procedures ... hence, it is a physician's order. Maybe your supervisor will consider putting such a policy/procedure in place for everyone to follow ... rather than you acting on your own.

Ditto ... except using 1 gel pack on bottom and 1 gel pack on top (small cooler, blue and white to signify 'room temperature storage') and no styrofoam.

Nursing has a tough enough time calculating a 2oC rise with the decimals, i.e. 99.2 to 100.8 = ??? [scratch, scratch], so it's just easier to give them whole numbers to work with.

2oF rise from 98.6oF OR 1oC rise from 37oC

We all 'rule out' many antibodies every day with 1 homozygous cell on our Antibody Screens ... and we perform immediate spin crossmatches based on that result ... and nobody flinches. The 3 cell rule is for antibody identification ... you need 3 to be statistically sound. In other words, don't call it an Anti-K unless you can show me 3 K-pos cells that reacted without any other possibilities. And I'll need 3 Jsb-negative cells if you want to call it an Anti-Jsb. And so on ... Gel or not ... heterozygous cells don't count much. We've got a few examples of antibodies that react ONLY with homozygous cells even in gel.

Because of the issues written in these postings (and others), we try to switch to Rh-Pos as early as possible when we know there will likely be a large amount of blood needed for the case. Yes, we require pathologist's approval (our Med Dir isn't always on call) and we document that approval. We do NOT even think about Rh-Ig ... after a unit or two, it is too risky. If the patient were a woman under 50yrs, we'd consider it. Key word: 'Consider'. Once a patient recieves Rh-Pos

Careful about this ... Can't recall off the top of my head (must be tucked away somewhere) ... but I do remember reading that it's dangerous to give Rh-Ig to a patient who has recieved greater than 20% of their blood volume ... you are inducing a 'passively acquired' extravascular hemolytic reaction.

Correct ... Immunomodulation.

Ditto! Keeps it simple! Plus, we are now using the bands that have ONLY the unique BB# so the 'name/number doesn't match the name/number on the BB Band' questions don't exist anymore. BB Band# is our primary ID#.

I just looked up the actual validation study writeup. We prepared plasma from Autologous units (because they would have been discarded anyway) and folded them over a thermometer (between the two 'sides', not in the crease). Then we placed them in 37oC waterbath until they reached approx. 37oC (current temp of waterbath). Then we placed them in the refrigerator in a holder (to simulate how they'd be situated if stored in the refrigerator). We recorded the temperature every 10min. until they reached 6oC (remember these are folded). Summary: range was 70-120 minutes (note volume is not consistent so this probably affected the cooling rate) for units to reach below 10oC (sorry, I mis-stated that in my earlier post). Therefore, we felt it conservative to consider Thawed Plasma 'Issued Prior to Cooling' for up to 60 min past thaw time (even if it's been placed in the refrigerator prior to issue). It's also an easy time frame for the techs to remember. "A unit of Thawed Plasma will be considered 'Issued Prior to Cooling' for up to 60 minutes past thaw time. After that, it will be considered 'cool' and must remain stored at 1-6oC or maintained under 10oC during transportation."

Transfusion, 2002 Sep;42(9):1180-3 Anti-Vel reactivity diminished by adsorption with rabbit RBC stroma Transfusion, 2010 May:50(5):1139-43 Immunoglobulin M red blood cell alloantibodies are frequently adsorbed by rabbit erythrocyte stroma. To cite a couple ... there are more out there.

Hear, hear! And yes, I believe the 'missed clinically significant antibodies with pre-warm' is more of a function of it being a less sensitive method (it used to be 'no enhancement, 1hr inc, do not read at 37oC) rather than the pre-warming. (Observe, all antibody screens are 'prewarmed' in the ProVue because the cards are set in a 37C incubation rack as they wait to be loaded ... haven't heard any stories of missing antibodies.) Let me add to Mabel's notation ... Recently, we sent a sample that was reacting 'same strength' with all cells in the panel to our local reference lab. nb We test for cold agglutinin = negative at Rt with pooled O cells. Their answer: Cold Agglutinin. Their reasoning: Reactions were seen at 4oC and reactivity diminished/disappeared after treatment with RESt. Ummm ... hmmm. Knowing RESt has been documented to absorb some clinically significant antibodies, well, luckily, the patient went home and didn't need a transfusion. So, not only do we need to be mindful of the capabilities of pre-warming, we need to be fully aware of the capabilities of the 'specialities' we employ.

Recognizing that FFP is often 'issued prior to cooling' along with the focus on the FDA rules about storage/transport temperatures being maintained, here's how we deal with this problem: First, we determined (with appropriate documentation/validation) how long it takes for FFP to cool down to below 6oC using worst case scenerio, i.e. FFP starts at 37oC. We found that it took around 60 min. So, I set up a protocol: - When issuing the FFP, if it has been thawed within the past 60 min, we issue it with the comment 'IPC = Issued prior to cooling' to provide documentation that it went out 'likely warm'. - If it comes back, it may be accepted back into inventory (refrigerator) if it has been less than 6 hours since thaw time or less than 4 hrs from pool time, whichever applies. nb Our protocol is to take the temperature of all units that are returned (or the cooler temperature) to assure they are still within acceptable range, but we do not take the temperature of these IPC units because they are not expected to be 1-6oC.

With gel, the first thing we consider: 'Is there mixed cell reactivity?' Mixed cell reaction is highly suggestive of cold agglutinin reactivity (given the screening cells are single donor) or strong rouleaux. If yes: To confirm that, test pooled O cells vs the patient's plasma at Room Temp. If they react, you likely have a cold agglutinin or strong rouleaux (or both) which you can actually visualize under the microscope if you'd like. Final Interpretation: Cold Antibody of Undetermined Specificity or Cold Auto-Antibody of Undetermined Specificity (depending on if you perform testing to make the differentiation) If no: Run a panel. You stated your panel was inconclusive which is strongly suggestive of being either HLA or HTLA antibody(ies). Gel is very good at picking up these antibodies so the old 'HLA is weak reacting' doesn't apply to gel. To 'confirm' this, we run an antibody screen using an enhancement medium that is not so sensitive to HLA/HTLA, e.g. OES (Ortho Clinical). More often than not, we have found that this test is negative, i.e. 'no clinically significant antibodies detected'. Final Interpretation: = 'Probably HLA/HTLA Antibodies'

12.5mcl of 4% cell suspension 25 mcl of Anti-C3b/d Incubate at 20-22oC for 5 min. Spin, etc.

We test both Anti-IgG and Anti-C3bd Direct Coombs Tests in gel. Very clear results. Validated this and found sometimes the reactions are stronger in gel (sometimes not), so we did notify the physicians about the change.

Ditto!

We are using gel which picks up warm-autoantibodies 'wonderfully'. When this is suspected, we run an OES (Ortho Enhancement) Antibody Screen. That usually effectively clears the warm-auto and we go from there. If it doesn't clear it, we try Albumin and go from there. If that doesn't clear it, we send samples to our local reference lab where they perform auto or differential absorptions as indicated. We revert to antigen-negative units if this fails.

Ditto! Only we don't use the '30 minute rule' (it cannot be validated), we take the temperature of all returned units to assure they are still within required temperature range. n.b. They don't stay below 10oC (nevermind 6C!) after about 10 min. I'm thinking that 'special units' should be issued in a small cooler or thermal That way, if the initiation of the transfusion IS delayed, at least that precious blood would be protected. Hmmm ... I think I will put this in our policy. Re: Nursing not having their act together BEFORE they call for the units: We will not issue a unit of blood without a 'Blood Bank Release Request' form FILLED OUT AND SIGNED by the infusionist. This form bears a checklist of items that both nursing and the BB decided must be done BEFORE requesting blood issue, e.g. 'Consent Form Signed'. It also bears patient information (name, Hospital #) and what product they are requesting, e.g. Thawed Plasma. So, in theory, we are assured that they are 'ready to go' when we issue the unit.

The FIRST thing you need to do is check out the capabilities of your Anti-D reagent(s)! Some detect 'weak D' by I.S. while others need 37oC incubation, while others need AHG Phase. Furthermore, some detect 'weak D' but not DVI (D Category VI) in the same. Read your package inserts, that info is there. DVI, if I am correctly informed, is the 'weak D' that will develop Anti-D. The others don't. Anti-D with MTS (ABD Card, Anti-D Card) will not detect DVI, therefore correctly categorizing the patient as D neg. 'All' other 'weak D' are positive, therefore correctly classified as D-pos. btw: I do believe the reason we stopped using the term 'Du variant' or 'Du Pos' was because, as we got to understand the D antigen better, these cells really are D-pos; they just bear either smaller amounts of D genetically or inhibited by C (and some claim E as well) in cis or trans position ... so different from what I was taught as a student! Bottom line: Know the reagents you are using and be sure you are interpreting the results using the specific reagents limitations/capabilities. And realize that reagents are different and that they have changed over the years ... even within the same manufacturer.

I think the big question is: Do you really need to do this in your lab? The concern is mainly about Rh-immune globulin candidacy. (It's easy enough to find D-neg,C-neg RBCs for transfusion, so pretransfusion candidates are not an issue here.) How often will a pregnant woman present in your lab with "Anti-D,-C" ... possible ONLY Anti-G or a combo of Anti-D,-G? If that number is low/rare, is it worth it to find all these cells ... and to actually find enough to accomplish these elutions (and don't forget controls and competency!) for those few times? Or, is it better to send the sample to a reference lab? OR is it better to just make a policy to classify patients producing Anti-D,-C as candidates and give them the Rh-Ig and not be concerned if it's ONLY Anti-G? (Which in itself, I hear is unlikely.)

Ortho had addressed this problem a while ago, suggesting we keep our reagent red cells in the dark. It sort of makes sense ... the screening cells are out in the lights most of the time and the panel cells are not ... same diluent, different reactions. Anyway, we got some plastic 'sleeves' from a local vendor for the bottles (Wescott Laboratory Solutions) and we don't seem to be plagued by 'Antibody Screen Positive/ Panel negative' issues. Yes, the sleeve covers the outside of the bottle, but we put the reagent number on the cap so we can see which cell it is. You can take the bottle out and look at it very easily when you need to. As far as 'random' positives on screening cells AND panel cells ... MTS is more sensitive to HLA Antibodies than tube testing. If MTS Antibody Screen results are less than 2+, we run the MTS panel 'maxtime'. If we get extraneous similarly weak random results and no possible antibody ID, it's an HLA or HTLA antibody. If you are nervous about that, repeat the screen using tube testing ... it will be gone and real clinically significant antibodies should still be there.