NicolePCanada - I agree with both Ward_X and Malcolm's comments. There are definitely situations (patient groups, diagnoses) where a less sensitive methodology like LISS-IAT can be useful to work around "junk" that may be detected in Gel, Solid Phase or PEG test systems. But...they should be employed only by operators who understand the consequences of such actions, AND have the support of their medical staff.

2 hours ago, NicolePCanada said:

Since we opened this topic back up........I was simply going to stop using LISS rather than validating a new type. We never use it anyway. If we can't solve it we send it to Canadian Blood Services. Is there a good reason why we should keep it? Malcolm?

The "Swiss Army Knife" approach to serological problem solving used to be fun. Having the ability to tinker with a variety of test methods and come up with your own conclusions was one of the addictive parts of immunohematology. Alas, that is no longer viable in today's era of validate everything and demonstrate competency several times a year.

 

I used a lot of words to say: "If you don't use it, get rid of it".

14 hours ago, Malcolm Needs said:

exlimey, I would most certainly agree with your first comment.  In the VERY old days, when I was merely middle aged, and we only had access to tube tests and reagents, including AHG, and techniques (such as tile techniques), we didn't kill patients by the thousand, so techniques that are a little less sensitive than are available these days, will not necessarily condemn the patient to a certain and painful death, despite the deafening shouts of those who are gainfully (and, in many cases, VERY gainfully) employed within the neo-science of quality for quality's sake (we needed to pull up our socks in terms of quality, but it has got out of hand).

On the other hand, having seen a fair smattering of Jk(a-b-) patients with anti-Jk3, I am unaware of "sticky" Jk(a-b-) red cells, even when they have been cryopreserved and reconstituted.  I am willing to, and will freely admit to being wrong, if I am proved so.

I like your "neo-science" quote !

As the actual science has progressed, and the sensitivity of assays has increased, workers have encountered "-only" antibodies: albumin-only, enzyme-only, LISS-only, PEG-only, solid-phase-only, Gel-only. As you point out, if the older assays were that bad, we'd have patients dropping all over the place due to the presence of "missed" antibodies and false-negative crossmatches.

My point about the "stickiness" is that the gel system is notoriously unforgiving with anything but completely normal, healthy, well-washed cells, preferably in the manufacturer's diluent. Throw anything at it that's little different and you risk incomplete or unsatisfactory centrifugation of red cells. I don't know if that specifically happens with Jk(a-b-) cells, but I have seen frozen/thawed cells behave badly.

After reviewing the posts here , I think it's perhaps a little more worrying that jojo808's group may consider transfusion of "least-incompatible". I understand that needs must, but that's a dangerous proposition if one doesn't know the specificity of the antibody(ies) causing the incompatibility.

9 hours ago, jojo808 said:

Patient has the following antibodies: (Pt is B+)

Jk3, E,c, He phenotypes K, Fya, S, N negative. Our ref lab found us 2 units that are phenotyped matched, one B+ and one O+ rbc. They are both incompatible, the O+ is 1+ in Gel, the B+ +/-.

Auto control Neg, DAT neg (reference lab results). What's our next step??? BTW hope you all are doing well during this time. 

This may be a little heretical, but I think I would avoid the gel test in this case. Perhaps do the cross-matching in a test system that's not as sensitive ? Local policy permitting, of course. A plain old tube test would probably result in compatible crossmatches.

The gel test system has probably not seen many cells with the Jk(a-b-) phenotype, and I also suspect that they're probably "sticky", especially if the cells have be frozen/deglycerolized.

39 minutes ago, diplomatic_scarf said:

Thank you for replying.  Everything was done by gel card, we don't use the tube method.  Patient was typed A Pos. The antibody screen was 1+ positive on Screening cell 1 and 3, negative on Screening cell 2.  But when we did the Antibody Identification panel and Auto control , they were all negative. We repeated everything and got the same results.  We redrew the patient for a new specimen and got the same results. 

We sent out the specimen for an Antibody Identification at a reference lab. The result we got back was Anti-M. 

I am just wondering, why didn't our panels picked this up? Why only the screening cells were positive? What was the reasoning behind this? The panel and all our reagents have passed QC and are not expired. There was no signs of contamination in our specimen or reagents. Thank you. 

Reactivity with two of three cells effectively rules out the "antibody to a low incidence antigen" argument.

Is the supplier/manufacturer of the Screening Cells the same as the supplier/manufacturer of the panel ? If not, then I suspect formulation differences of the two products may be the answer, specifically pH of testing environment. 

3 hours ago, Malcolm Needs said:

Maybe it is a mixed cold and warm AIHA - rare, but they exist (I co-wrote a paper on a case), in which case the "cold" could be an auto-anti-H, or even just the normal auto-anti-H commonly found in group A plasma.  That would explain the weak reactions with group A red cells.

It's tough to see on the scanned panel sheets, but the D- group O panel cells are w+ reactive, equivalent reactivity to the crossmatched group A cells.

Agree with previous - looks like a warm autoantibody and it's important to see what, if anything is underneath. Adsorptions will be necessary. Interestingly, if I can read the results correctly, the antibody is showing a marked preference for D+ cells. It may have LW specificity, although 4+ is unusually strong for anti-LW.

I'm curious as to why there is "w+ incompatibility with all A pos units". I would have expected those to be 4+ reactive, also.

There may be several issues going on here.

6 hours ago, diplomatic_scarf said:

The antibody screen was positive.

All screening cells reactive, or some other pattern ?

1 hour ago, Malcolm Needs said:

I like explanation one very much exlimey.  Very many of the examples of anti-Vel I have encountered (and I have seen many, as I was working at the BGRL in London in the mid to late 1970's when Dr Bertil Cedegren was studying the percentage of Vel Negative donors in the Swedish population) had a high concentration of IgM, and a low concentration of IgG.

Exactly. And very likely to cause in vitro hemolysis in appropriate test systems (not to mention in vivo hemolyis). I remember doing many 2-stage EDTA tests, using fresh complement and poly AHG. Good times.

I think I may have met Dr. Cedergren when I was working at the BGRL in Oxford during the late 1980's.

I can't really explain your serological findings, but I agree with Malcolm's sentiment: "That stuff'll kill ya!" Anti-Vel is notoriously slippery and infamously dangerous.

Without a more detailed look at your results, I see a few remote possibilities:

1. The anti-Vel may have an IgM component that doesn't like the Gel, but does like the PEG test

2. There's possibly something underlying the anti-Vel, hence the "extra" reactivity

3. The unit you crossmatched is not actually Vel-, but another member of the Vel variant club

While the use of EDTA plasma basically eliminates the chance of detecting hemolytic antibodies (as Malcolm says above), some laboratories still use serum. It's possible some hemolysis might been seen in visual check before taking the serum-RBC-PEG reactants to IAT. One may also recognize loss of RBC volume after the washing process, if some of the cells were destroyed during the incubation phase.

On 12/24/2019 at 11:58 AM, Michelle L said:

Does anyone that uses Immucor's Elu-Kit II test the pH of their eluate once it turns blue? Or do you just go by the blue color indicator as being within the appropriate pH range?

1. Personally, I wouldn't do ANYTHING different/extra than the manufacturer recommends. You may inadvertently "modify" the process and find yourself in a corner that requires validation of your local modification. Yikes !

2. Typical eluate volumes are quite small - often too small to be measured with a pH probe. So I suspect, correct me if I'm off-base, that the pH has been checked using pH paper. If so, eyeballing the color of the pH paper is no better than eyeballing the color of the eluate.

And, as AMcCord suggests, while the blue color may vary, the differences in pH values of the different blues is very small. The manufacturer (Gamma/Immucor) has put a lot of effort into making the kit idiot-proof.

6 hours ago, pathlabtech said:

Here's the antibody screen:

Cell	IS	37C	AHG
SC 1	neg	neg	1+
SC 2	neg	neg	1+
Auto	neg	neg	neg

The possible interpretations are:

1. single warm antibody, antigen present on both cells

2. antibody to high prevalence antigen

3. complement binding by a cold antibody not detected at IS

Can someone explain interpretation #3? 

 

 

 

Interpretation #3 should only be considered if a polyspecific antiglobulin reagent was used, i.e., a "complement coat" will only be detected if the antiglobulin reagent used contains an anti-complement component. Even if that is the case, Interpretations #1 and #2 are far more likely.

1 hour ago, Malcolm Needs said:

As said above, we don't have to make this decision, as all of our units are typed for ABO, D, C, E, c, e and K.

What does the Blood Center do with the K+ units ? Throw them out, or only give them to K+ patients ?

16 hours ago, Mabel Adams said:

So the same logic applies as for E & c--avoid stimulating anti-V/VS during more routine transfusions to save yourself the option to have a compatible crossmatch during a crisis when you may be giving V/VS+ blood to save a life.  If the patient has already made anti-V/VS you can still choose to ignore it and give incompatible units because it is a lesser evil but we would mostly feel better if we could avoid giving crossmatch-incompatible blood because it would be hard in the moment to prove that there wasn't an additional antibody directed against a different low incidence antigen.  That's why we do AHG crossmatches I'd say.  Same argument against it of using a precious resource before the patient has made the antibody.

This is a very interesting thread, partly ethics, partly practical use of resources, and a large dose of "what if".  In the legal sense, the concept of "Prior Restraint" comes into play  - doing something to prevent a possible event regardless of probability.

So.....a not-so-unrealistic scenario:

The hospital has a patient with anti-K and is required to screen/type a number of units to fill a transfusion order. During the process some donors/donations are identified as K+. What should the facility do with those, knowing full well that they may stimulate an immune response in recipients ?

And.....discuss.....

16 hours ago, Malcolm Needs said:

"reagent quality anti-V and/or anti-VS are both like hen's teeth"

Unless you have the right contacts......

9 hours ago, Malcolm Needs said:

Anti-V and anti-VS have only ever caused very mild delayed transfusion reactions, and only ever caused a positive DAT, rather than clinically significant HDFN.

They may indeed "cause very mild delayed transfusion reactions", but to a multi-transfused (Sickle Cell Disease, SCD) patient, with often a multitude of other alloantibodies, what would be a typically mild reaction in a "normal" patient can be serious, even fatal in SCD patients, especially if it induces a hyperhemolysis event. SCD patients understandably have very fragile immune systems. It doesn't take much to upset the apple cart.

36 minutes ago, Mabel Adams said:

But I bet they make the V/VS positive unit crossmatch incompatible so you can't use it, right?  And that from a very limited pool of units.

They sure do, especially since many laboratories routinely use the super-sensitive assays like PEG-IAT or CAT (gel/bead technology) for crossmatches.

Most examples of anti-K are IgG, and therefore need an antiglobulin test to detect them. While enzyme-treatment of cells may or may not enhance reactivity of anti-K, it rarely turns an indirect agglutinin into a direct agglutinin. It does happen, but I've only seen it with Rh specificities where antigen density supports the process.

 

I presume that the "neutral cards" do not contain anti-IgG and therefore your findings are as expected/typical for most examples of anti-K.

2 hours ago, Baby Banker said:

Issitt says that the antigen expression is variable even in the same donor or patient.  I didn't see anything about sample age, but I just glanced over the entry.

I am Bg(a+) and my expression is usually quite strong; it got super strong after a bout of Infectious Mononucleosis.

I have seen my own cells react with examples of anti-Bga when the cells are fresh and then reactivity dwindle to nothing as cells from the very same collection tube age.

Definitely nonsense.

In my experience, antibodies to Bg antigens ONLY react with the freshest of cells, and only with individuals with unusually strong expression of the antigens, e.g., Bg(a+s). Even when the odds are stacked in your favor - fresh cells from an individual with a strong expression, you're only likely to get barely macroscopic results. It can be very frustrating to chase down one of these to identify it.

13 hours ago, fendi98 said:

Saline tube technique at 37, immediate spin = incompatible with 1+ reaction including auto control

I concur with the autoantibody conclusion. However, unless you've neglected to tell the forum important details about this case, this work-up should have stopped at a negative antibody screen (in cards).

I'm more concerned about why are you doing lots of extra work - especially an enzyme panel. And, why, oh, why are you using a "primitive", "insensitive" tube test when the super-sensitive card tests are negative /compatible?

If this is a "normal" work-up, I believe your testing algorithm needs attention.

41 minutes ago, abillarn said:

Thank you all for the responses and input.. The reagent manufacturer does not really specify what reaction grade is acceptable to validate your Coombs reagent really did its job. We were in the process of making our procedure , just not 100 percent sure what grade , we were leaning on 2+.

Do you plan to grade/record your antiglobulin control cell reactions ? If "YES", then you will need to define an acceptable range. Most workers simply use a check mark to signify satisfactory performance (hence "Check Cells"). In the absence of specific instructions and/or ranges from the antiglobulin control cell manufacturers, I favor the position suggested above by AuntiS: Macroscopic agglutination.

25 minutes ago, Malcolm Needs said:

Grrrrrrrrrrrrrrrrrrrrrrr!!!!!!!!!!!!!!!!!!!

Anyone know the name of this man and his relevance to this thread?????

 

What's wrong Malcolm ? You don't like the idea of using a Coombs Test to find Kell- blood ??

14 minutes ago, JPSCANNELL f. CROKE said:

2+ is the only result possible as you have a mixed cell population in the tube now (patient/donor + check cells).

The definition of a 3+ and 4+ is a clear background = not mixed field.

And 1+ is just too weak, something is wrong.

The avidity/reactivity of freshly-prepared antiglobulin control cells may be sufficiently high that the obligatory mixed-field result is not seen. The agglutination matrix of the sensitized cells often incorporates the un-sensitized test cells - a clear background is very possible.

Some workers believe that 2+ is too strong and that 1+ with antiglobulin control cells is the prefect result and allows detection of a decrease in sensitivity of the assay.

4 hours ago, AMcCord said:

Liquid-in-glass, spirit only. We (the entire facility) got rid of mercury thermometers some years ago - safety hazard.

That's interesting. The last time I purchased spirit thermometers, we were unable to certify them (we didn't pony-up the extra $$$ for NIST-traceable versions). We had serious linearity issues between the ice-point and the working temperature. The mercury replacements had perfect linearity, but I understand the concern regarding mercury.