C3b,-d Homemade check cells

[Lindz82]

Bear with me as I am new not only to bloodbanktalk but Blood bank in general.

I am trying to decide if it is worth the trouble to make our own complement check cells. Currently we use Gel for Anti-C3b,-d testing and the reactivity of check cells is much improved from tubes.

My main curiosity is are homemade check cells worth the trouble? The money it will save. That's what it is all about... right?

Please share your adventures in cooking up reagents!

[David Saikin]

I think you will find that you will make them fairly often. Whenever we did this (in the past) we found that they hemolyzed easily and if they lasted for a week that was a good thing. Personally, I don't think it is worth the time UNLESS you have a student (or lab aide) that can do it for most of the year.

[NedB]

Boy, David, that takes me back. We were lucky when we could make them last from Fiday to Monday and didn't have to do it on the weekend. I was amazed when Immucor offered their product with a 28 day expiration, and it works. But to the original questin, if you are using the gel, why do you want check cells? I don't use that card, but the other gel cards state that use of check cells is not required.

[Malcolm Needs ☆]

Like David and NedB, we used to make them up assiduouisly, and then someone asked "Why?" (classic question, of course).

We no longer do so.

The only time they were ever of any real use was when, back in the day, I used to have to produce AHG, and they were used for standardisation (which, considering there was absolutely no standardisation of the C3d-coated red cells, was a bit of a joke in itself)!

Good luck in Blood Banking; it's a fascinating subject that many of us have learned quickly to love.

[TimOz]

Hi all. I have been away but now I am back.

We have quite a bit of experience with QCing Anti-Complement. The often used method is the cold sucrose method that is also called the Fruitstone coat method. The problem with this method is that it uses Trypsin to convert C4b to C3d and C4d - in theory. Trypsin is not complement convertase and it cleaves differently. Our experience using this method is that it appears to work OK with polyclonal Anti-C reagents but is very variable and unreliable with monoclonal Anti-C reagents. Sometimes it will apear to work and you can repeat it at the same time with the same reagents and it fails. This makes for a very poor QC method as you don't know if the reagent is the problem, or the QC cells. We have tested commercially available C' coated cells and they seem to suffer from the same problem.

I think that the monoclonal Anti-C reagents are excellent and the clones I have seen are very stable. More stable thean the polyclonal Anti-IgG component. Defacto QC testing of the AHG by minitoring the Anti-IgG sensitivity is one way to go or the other way is to get your hands on real complement coated cells and freeze them. This of course takes liquid nitrogen and a good freeze thaw procedure.

Below is an exchange on the subject from the CBBS forum:

John Case wrote that in the case of polyspecific Anti-Human Globulin, it is generally argued that to demonstrate continuing anti-IgG reactivity is to cover the anti-C3d component. If the anti-IgG component is working, then the anti-C3 must be working also. This argument has many convinced adherents, but is not very rational. The anti-C3 component has a significantly lower titer than the anti-IgG; hence, you could have your reagent so diluted (through leaving too much saline on the cells after the last wash), or so impaired by partial neutralization, as to diminish the anti-C3 reactivity while leaving the anti-IgG ostensibly intact. However valid that reasoning may be for polyspecific AHG, it does not legitimately apply to anti-C3b,-C3d, which was the subject of the question. One feels that one undoubtedly ought to be doing regular quality control, if for no other reason than that this is supposedly required by Standards. Speaking personally, I'd say it is especially important in the case of this specificity, because anti-complement tests are not, generally speaking, carried out well. Agglutination due to anti-C3b and anti-C3d develops slowly; yet there is still an expectation that it ought to come up at immediate spin. Even when the manufacturers' recommendation to wait for five minutes between adding the reagent to the washed cells and centrifugation is faithfully carried out, the resulting agglutinates are fragile, and are easily dispersed by the lack of gentle shaking. A positive control is a useful means of reminding the user what to expect. This reason could apply equally to Polyspecific AHG, needless to say. Where to get the necessary control cells? Well, you can make your own. There are methods in the literature, but they are admittedly rather tedious, and applying them may entail complications arising from the observance of good manufacturing practices, which most laboratories would (understandably) prefer to avoid. It would be hard to justify the hassle, particularly as your home-made control cells would have a very short storage life (due to being made without aseptic precautions). The alternative is to buy them.

To which John Judd commented that just because Standards says we should or must, does not mean it is relevant to patient care! If we need to perform daily QC on our anti-C3 reagents, then surely - by the same token - we need to perform daily QC studies on each antigen claimed to be present on reagent cells used for antibody detection and identification. How do we know that we haven't denatured S antigen on these cells by too liberal use of NaClO? We also need - by the same token - to show that our acid elution kits are still active (eg, we haven't inadvertently contaminated the product with NaOH). When can we stop this nonsense?

We rarely use anti-C3 reagents alone in antibody detection/identification tests. When we do, it is purely for academic reasons. We do use anti-C3 in the workup of a patient with autoantibodies or when immune-mediated hemolysis is suspected. The test with "monospecific" anti-C3 is invariably done in parallel with or after a positive test with polyspecific AHG. In our institution we use reagents from two different manufacturers for this testing. I doubt if there will ever be a situation in which 2 different polyspecific AHGs and two different anti-C3 will be inactive, for whatever reason, unless the whole rack of AHG reagents got fried or was left at RT for weeks on end (which it never is). Regardless of laboratory findings, the diagnosis of immune hemolysis is based on clinical observations. I say that this sort of QC testing - although claimed to be essential by those who have nothing better to do but conjure "quality processes" - is sheer and utter twaddle!

To which John Case commented that in his view no daily quality control should be done that does not provide truly meaningful information. Applying that principle unquestionably precludes testing the integrity of antigens on Reagent Red Blood Cells, which offers nothing useful besides confirming the reactivity of that antigen with that example of its corresponding antibody in the hands of that particular bench worker.

One could argue, however, that to run control tests on day of use with Anti-C3 reagents does at least (as I argued) give the user a hint of what is to be expected in terms of a positive result. Beyond that, I don't suppose it tells you much. For instance, "day of use" testing doesn't allow for differences of manipulative skill in resuspending the cells, which is of particular relevance when agglutination is as fragile as that produced by anti-complement components. I am at something of a loss to know why testing any sample needing to be tested for a coating of complement with two examples of polyspecific and two examples of anti-C3 would be easier, cheaper and more informative than confirming the reactivity of a licensed anti-C3 with a positive control.

To which Mr. Judd admitted that Mr. Case was absolutely right that our duplicate testing seems excessive, but we have had trouble with these products in the past, ever since one manufacturer ceased marketing blood bank reagents. We have seen discrepancies between manufacturers, that are not always consistent, and I don't quite know what to make of it. In any event, when I have nothing better to do I need to review our most recent data. As to what we do when the test with one reagent is positive and the other negative, we report the results as equivocal. I have never been convinced that such discrepancies are due to reagent inactivation or deterioration, the reagent that is non-reactive with the sample in question still reacts with other complement-coated RBCs. We would, of course, review the clinical findings on the patient and discuss our results with the attending physician.

For the full information, go here: http://www.cbbsweb.org/enf/1999_2000/antic3qc.html