We perform our antibody screens at 37C and IAT only (since AABB STD's says methods that detect SIGNIFICANT antibodies).

The problem is, that crossmatches are performed at Immediate Spin.

Cold autos are missed at antibody screening, and the screen is reported as negative.

When techs crossmatch units, the I.S. reading is positive due to a weak cold auto, but several techs keep reporting rouleaux when it's clearly not rouleaux. I have told them over and over, that rouleaux is rare and only seen in patient's with protein abnormalities such as Multiple Myeloma.

They are reporting rouleaux at least 3 times a week. A saline replacement CAN disperse weak agglutination from a cold. What can I do to stop this mis-identification? I have tried over and over to explain and showed them the short cold panel results, but the rouleaux reports keep coming.

Today's specimen was on an A patient with a weakly reactive A cells. The tech did not check for A subgroup with anti-A1 which should have been done with an ABO forward/reverse discrepancy. (It wasn't - patient was A1 with cold autoanti-I.) I have also explained that autoanti-H and IH can be seen with A1 or A1B patients.

I always do an immediate spin on antibody screens, even though we don't record results in computer. Significant or not, I want to know what's there. No surprises for later when crossmatches are needed STAT!

We do see a lot of colds, as our room temp is freezing (18-20C) even though we've in Miami!!! Have never been able to get our A/C fixed so we don't freeze!

Gil

With electronic XM, this problem is moot.

In my patient population, we see a fair amount of rouleaux, which we confirm microscopically and attempt to disperse first with albumin. Saline replacement is the second step.

We see quite a bit of rouleaux. EDTA tubes will have more rouleaux because of the higher protein level.

We also get surprizes, because we use gel, so never see tube reactions until we do IS XMs.

For patients with negative antibody screens who show 'stickiness' at IS (we also have a cold BB room), we examine for rouleaux, if not, we do a quick (5 minutes) 37C inubation. This usually takes care of most weak cold agglutinins.

(I would love to do e-XMs, if not for my BB system).

Linda F

We see quite a bit of rouleaux. EDTA tubes will have more rouleaux because of the higher protein level.

I've never heard that. Why would EDTA tubes have higher protein?

That's what we use: pink tops.

Rouleaux should be rare.

Gil

We do not get too many rouleaux problems. Very rare ...I would say 0.16%...4 out od 2400 samples....We use Gel cards

How long do you centrifuge your specimen?

We use EDTA and see a fair amount of rouleaux. Wish we could go to e-xm. Anticoagulated blood has higher protein because it still has all the clotting proteins.

We have a procedure for managing rouleaux. It must appear microscopically like rouleaux; first we try albumin, then incubate with the albumin, then do saline replacement if nothing less intense works. Or we can always just substitute a gel xm.

There is more rouleaux when using EDTA plasma vs serum because of the fibrinogen in the specimen. (At least I think I've got that right.)

Linda F

the problem isn't that a lot of rouleaux is being seen, it's that it's being called rouleaux when it's really a cold auto. Real rouleaux doesn't go away when you warm it!

As one of Gilda's SNL characters used to say... "never mind".

But anyway, we still do a 5 minute 37C incubation if ABS is negative & XMs are positive at IS AND it is not rouleaux. This seems to fix most minor cold agglutinins.

Linda F

We also have noticed with EDTA specimens, that we need to spin for a longer time than we did for Non-anticoagulated Red top specimens. It is imporatant to get platelet-poor plasma for Gel testing and also if the EDTA samples are not well spun the incidence of rouleux is increased.

It is imporatant to get platelet-poor plasma for Gel testing ]

this is news to me. I've been working with gel for 16 years. Where does this info come from?? I'd be VERY interested to know

If an EDTA specimen has been seperated and stored, we respin the plasma when time permits before testing.

It is imporatant to get platelet-poor plasma for Gel testing

What is the theory behind this and is there documentaion?

We find (and were told by our Ortho trainer in 2003) that respinning the sample helps prevent topline. This certainly makes sense with respect to fibrin bits that can precipitate out in a stored specimen. It seems like platelets, being so much smaller than red cells, would spin through the gel fine, but plt clumps could probably stay on the surface and trap some red cells. And we know from Hematology that there are patients whose plts clump in EDTA.

We usually respin a sample that causes topline before repeating the gel test. It would be interesting to repeat the test using an aliquot that was not respun plus one that was. Also, if you ever run a gel test on a sample you thought was spun but had only settled, you will usually get bad topline.

So if we pre-warm away those cold agglutinins in IS xms, what are the chances that we are pre-warming away an ABO antibody that is very weak or trying to react with a very weak antigen?

Thinking aloud:

The point of the IS xm is to detect ABO incompatibility. This could be caused by:

1. Mislabeled unit

2. Wrong unit or sample being tested

3. Wrong donor's segments attached to unit (this happened to us once, but it was Rh that differed--it's a good story).

These would usually give the strong incompatible reactions of a reverse type--unless the patient has a weak reverse. Then maybe you could prewarm away the reaction? Especially if it is an add-on xm and you aren't aware that the patient has a weak reverse.

3. Very weak subgroup of donor not detected by donor testing. (extremely rare due to requirements of commercial antisera)

4. Unusual subgroup of donor not detected by donor testing but detected by this patient's sample. (unicorn rare)

These are so rare as to have been deemed inconsequential (i.e. electronic crossmatch)

Anything I missed?

Those are all good points, Mabel.

Yes, ABO incompatibility is a concern, part of our trouble shooting of IS incompatible XMs is to looks at this (is the patient type OK? Unit type OK?), so I am simplifying here.

You had wrong segments attached to a unit? Oy vay!

Linda F

Good point, I forgot to include mistyping the patient. So if we call a patient A when he is really O, then the A unit we try to crossmatch would be strongly incompatible unless the patient had a weak reverse.

We really don't care about rouleaux or cold antibodies because we accept the theory that nothing that we are going to detect in the IS xm (that we don't detect in the Ab screen) is going to be clinically significant except ABO incompatibility. If we were doing IS xms for any reason besides ABO compatibility we would not be able to substitute the e-xm.

I am kind of hammering on this because I have some techs that read that IS xm practically with X-ray vision. I haven't discouraged them, but they spend a lot of time resolving very weak questionable reactions.

I think rouleaux and cold auto( titer below 1000 in 4 drgree C) are all insignificant, the question is correct name it, because they are two different reaction.

Rouleaux is not that rare. If it is presenting with the classical characteristic of "stacking" then it's rouleaux, regardless of the patient's diagnosis. On the other hand the simple act of doing the saline replacement, may warm the tube enough to disperse a weak cold. As far as the staff goes, ask them to check each case under the scope prior to reporting rouleaux.

As long as clinically significant antibodies are ruled out, the immediate spin XM should present no delaying issues, even if you see reactivity. The first thing I would do is a saline replacement, if neg - issue the blood. Does it matter if it's true rouleaux or a weak cold?

Rouleaux is not that rare. If it is presenting with the classical characteristic of "stacking" then it's rouleaux, regardless of the patient's diagnosis. On the other hand the simple act of doing the saline replacement, may warm the tube enough to disperse a weak cold. As far as the staff goes, ask them to check each case under the scope prior to reporting rouleaux.

As long as clinically significant antibodies are ruled out, the immediate spin XM should present no delaying issues, even if you see reactivity. The first thing I would do is a saline replacement, if neg - issue the blood. Does it matter if it's true rouleaux or a weak cold?

Rouleaux can also be caused by medications.

Can saline replacement reduce the strength of significant reactivity, I think this is a question.In AABB Technical Manual suggest to use saline replacement to distinguish the real agglutination from rouleaux, I think spin the tube then spill it gently on a piece of glass( sorry for my poor English), then see it microscopically. The rouleaux is stack of cells ,its edge is smooth seems like been surrounded by fibrin or grume.

Do you have any other words for "grume"? That one doesn't mean anything to me.

Oh, wait, let me go to dictionary.com and look it up.

grume

noun1. a thick viscous liquid 2. a semisolid mass of coagulated red and white blood cells

I hope I am not the only one that had never heard that word.

OK, Yanxia, thanks for teaching me English!

We also have noticed with EDTA specimens, that we need to spin for a longer time than we did for Non-anticoagulated Red top specimens. It is imporatant to get platelet-poor plasma for Gel testing and also if the EDTA samples are not well spun the incidence of rouleux is increased.

Is there a reference you can site for requiring platelet poor plasma for gel testing? I have searched product inserts, the Technical Manual and other sources and cannot find any reference to this. I have a call into technical support about this matter. I am concerned if this is indeed a requirement as we will need to validate/verify our blood bank centrifuges for specimens in providing platelet poor plasma.

Warm at 37C disperses cold anitbodies. Saline replacement resolves rouleaux problem.

Want to know why platelet-poor plasma is required for gel test? Any document support it? Thanks.

CK Cheng, MSc, SBB(ASCP), CQA(ASQ)

July 9, 2008