What QC reagent for antibody screen cells-tube?

[suhu]

what do you use for your QC antibody reagent for daily QC of screen cells?

we are automated, but use a 2 cell screen as back-up by manual tube testing.. CAP requires "each cell used for antibody detection must be checked each day of use for reactivity of at least one antigen using antisera of 1+ or greater validity" (TRM. 31400)

We have been diluting commercial antisera as appropriate for each new lot of screen cells, but this is getting rather expensive.

Would inspectors frown on using a diluted anti D for the antibody?

Thanks.

[Malcolm Needs ☆]

what do you use for your QC antibody reagent for daily QC of screen cells?

we are automated, but use a 2 cell screen as back-up by manual tube testing.. CAP requires "each cell used for antibody detection must be checked each day of use for reactivity of at least one antigen using antisera of 1+ or greater validity" (TRM. 31400)

We have been diluting commercial antisera as appropriate for each new lot of screen cells, but this is getting rather expensive.

Would inspectors frown on using a diluted anti D for the antibody?

Thanks.

I'm sorry suhu, but if the inspectors do not frown upon it, they should do (which, folks, is another reason why the inspectors should have a good working knowledge of Blood Transfusion - sorry suhu, another thread!).

Antibody/antigen reactions obey the Law of Mass Action. The association of the antigen with the antibody has its own equilibrium constant (k1), as will the breaking of the association (k2).

K1 and k2 are very different for "strong" and "weak" antibodies. If, therefore, you dilute a strong antibody to the point where it mimics a weak antibody, whilst the strength of the agglutination may look identical to the human eye, at a molecular level it is very different.

Using a diluted strong antibody is, therefore, not the same as using a genuine weak antibody as a control, and using such diluted "controls" would give you a false sense of security. WHilst you may detect this diluted strong antibody, you would not necessarily detect a genuine weak antibody.

Miss a weak antibody, give incompatible blood, and you end up with a strong antibody. If this is in a female of child bearing potential, you are storing up problems for the future (and, quite possibly, a legal case against you).

This is good news for you in one way; you can cut the cost of buying strong antisera and diluting it!

It does, however, leave you with another problem, and that is where you get hold of geuine weak antisera (without using a patient's plasma, which, in the UK anyway, is illegal, unless the patient has agreed to it in writing).

Sorry, this answer has probably left you with more problems than you had before you asked it, but that, nevertheless, is the situation.

[clmergen]

We create a weak Anti-D,Anti-c mixture by diluting it. And haven't had any problems with inspections at all. It isn't that expensive to make and it lasts until expiration of one of the ingrediants (which is the saline which usually expires within 30 days for less). Having the Anti-c lets us test the D negative screen cell.

[Malcolm Needs ☆]

We create a weak Anti-D,Anti-c mixture by diluting it. And haven't had any problems with inspections at all. It isn't that expensive to make and it lasts until expiration of one of the ingrediants (which is the saline which usually expires within 30 days for less). Having the Anti-c lets us test the D negative screen cell.

No, I'm not saying you will have problems with inspections clmergen, I'm saying you should!

[David Saikin]

I use the Immucor CorQC antibody. When I used tubes I used it neat (rx 1-2+); I use it for gel diluted 1:10 (4+) I could never dilute it enough to get weak rxs and CAP has changed its absc qc from 1-2+ to just reactive so I don't bother diluting it too much.

[suhu]

David-

I'd like to use the Immucor corQC reagent but manufacturers directions state it is designed for use with solid phase antibody detection. We need a reagent to test our antibody screen by manual tube testing (our back-up).

I dont know if its okay to use this reagent to test by manual methods, for QC purposes.

[David Saikin]

My package insert only has directions for tube testing. It accompanies my current lot in use.

[AMcCord]

I use corQC for tube also and my directions are tube only, though I do use the undiluted serum for a strong positive control with gel. too.

[adiescast]

I'm sorry suhu, but if the inspectors do not frown upon it, they should do (which, folks, is another reason why the inspectors should have a good working knowledge of Blood Transfusion - sorry suhu, another thread!).

Antibody/antigen reactions obey the Law of Mass Action. The association of the antigen with the antibody has its own equilibrium constant (k1), as will the breaking of the association (k2).

K1 and k2 are very different for "strong" and "weak" antibodies. If, therefore, you dilute a strong antibody to the point where it mimics a weak antibody, whilst the strength of the agglutination may look identical to the human eye, at a molecular level it is very different.

Using a diluted strong antibody is, therefore, not the same as using a genuine weak antibody as a control, and using such diluted "controls" would give you a false sense of security. WHilst you may detect this diluted strong antibody, you would not necessarily detect a genuine weak antibody.

Miss a weak antibody, give incompatible blood, and you end up with a strong antibody. If this is in a female of child bearing potential, you are storing up problems for the future (and, quite possibly, a legal case against you).

This is good news for you in one way; you can cut the cost of buying strong antisera and diluting it!

It does, however, leave you with another problem, and that is where you get hold of geuine weak antisera (without using a patient's plasma, which, in the UK anyway, is illegal, unless the patient has agreed to it in writing).

Sorry, this answer has probably left you with more problems than you had before you asked it, but that, nevertheless, is the situation.

What do you suggect using for reagent QC?

Fortunately for many of us, no one has decided to apply the Law of Mass Action to our QC policies. However, the manufacturers have decided to apply the Law of Supply and Demand instead, which may neatly take care of it. (Human source reagents are mostly not available any more no matter how much you demand them! Monoclonal antibodies do not suffer dilution well.) I don't think the alternative of purchasing QC kits will cut the price much, given the current upwardly mobile pricing we have observed in other threads.

I hope the US does not decide to make it illegal for us to use patient plasma (already in hand and relabeled for anonymity) in our in-house processes. Our students would truly suffer!

[Malcolm Needs ☆]

What do you suggect using for reagent QC?

Fortunately for many of us, no one has decided to apply the Law of Mass Action to our QC policies. However, the manufacturers have decided to apply the Law of Supply and Demand instead, which may neatly take care of it. (Human source reagents are mostly not available any more no matter how much you demand them! Monoclonal antibodies do not suffer dilution well.) I don't think the alternative of purchasing QC kits will cut the price much, given the current upwardly mobile pricing we have observed in other threads.

I hope the US does not decide to make it illegal for us to use patient plasma (already in hand and relabeled for anonymity) in our in-house processes. Our students would truly suffer!

It rather depends on what you want to do; the full Monty or the minimum.

I would always include a weak anti-Fya, for reasons given above.

I know exactly what you mean about the Law of Supply and Demand. Over this side of the pond we are lucky in that the NHSBT Reagents Department actually produces these weak antibodies for us and, being an NHSBT Reference Laboratory, we are supplied them free and gratis. It is not the same for the hospitals.

While you've got the chance, I would use patients' samples if I were you.

Over here, anyone who has been transfused cannot give blood (at least, for therapeutic use, because of vCJD), so we have to rely on antibodies stimulated by pregnancy, but I don't think the same applies in the USA (does it?). If not, you might be able to get some weak antibodies from your blood supplier, if they take off the plasma from whole units.

[eric1980]

My BB dilutes the Immucor CorQC anti-serum in order to obtain a 2+ or 3+ reaction in tube and gel QC. It has always worked so far, and I wasn't taught the idea of Law of Mass Action. =/

[Malcolm Needs ☆]

My BB dilutes the Immucor CorQC anti-serum in order to obtain a 2+ or 3+ reaction in tube and gel QC. It has always worked so far, and I wasn't taught the idea of Law of Mass Action. =/

Mollison's Blood Transfusion in Clinical Medicine, 11th edition, editors Harvey G Klein and David J Anstee, Blackwell Publishing, Chapter 3, pages 85 and 86.

[TimOz]

I have an interest in QC materials and I find this post interesting. I have 2 comments:

1. I think the often held view that Fy antigens degrade or fall off red cells is a myth. I believe that this has come from some papers on antigen stability (especially in Low Ionic solutions) where Fy antigens were seen to degrade. I think this was due to poorly washed red cells having some remaining white cells that age and lyse, releasing a range of proteases that knock of the Fy antigens - like papain does. I do not believe this happens if the cells are washed well and are clean. I have studied this in detail in a range of solutions and diluents and never seen it happen. The antigens that appear to weaken over time in some of the lower ionic strength solutions are Rh and c and e are the first to go. This is unlikely to be the antigen degrading but due to membrane changes and crenation due to the non-isotonic nature of the diluents. But I could be wrong.

Did you know that Alsevers solution that is used for most 3% red cell reagents is 70 years old this year?

2. The The focus of many of the posts above seems to be on what can be called "reagent QC" or in this case, whether the screening cell antigens are OK. That is fine and important but I always think the main reason to run controls is to detect the dangerous problems that can occur in a test. Statistically, this is errors made by the carbon based lifeforms in a lab rather than a reagent red cell failing (see the SHOT scheme report if you doubt this). I like to think of controls as "process controls". They should look and feel like a patient sample and go through the entire testing process. For this reason, commercial controls that have 3% cell suspensions and a few mLs of plasma as sub-optimal. Instrument controls that have barcodes so the instrument knows that they are controls and treats them differently from patient samples are sub-optimal.

In short, grouping and antibody screening controls should (roughly in order of importance) detect:

1. Human or instrument transposition error.

2. Human or instrument transcription error (interface errors can and do happen).

3. Test performace overall.

4. Individual reagent performance (including red cell antigen sensitivity in an Ab Screen).

I often see labs making their own controls or using a sample from yesterday. They often see "issues" and "drift" in QC scores so they change their QC material to suit. I like to use this analogy. If I was studying and wrote the test paper, filled in the answers and marked it myself I probably will always pass. If I always pass QC, I am suspicious of the QC material and that it is not testing the limitations of the assay. A "slam dunk" control is a waste of time and money. Controls shoudl fail - when something is wrong.

I think using proper QC material from an externally accredited source is going to soon be mandatory and this is already so in the European Union IVD directive. This not cheap and has to be funded. After all, is their a Biochemist who would report a glucose result without running a standard curve then a low, normal and high control a few times before trusting the analyser? Yet we seem happy to stagger into a lab at 2:00AM, knock over a few groups, crossmatch 12 units of blood for 3 patients and the most recent QC was a thermometer in the waterbath 19 hours before.

Sorry about - better have a cup of tea.

Edited July 24, 2009 by TimOz
typo

[Malcolm Needs ☆]

TimOz, I agreed with everything you said until you got to the bit when you said,

"Yet we seem happy to stagger into a lab at 2:00AM, knock over a few groups, crossmatch 12 units of blood for 3 patients and the most recent QC was a thermometer in the waterbath 19 hours before."

I disagree strongly with the use of the word "happy".

Although it is mitigated by the fact that I am being paid for on-call (albeit about an eigth of what I would get if I were a plumber on-call), I have never been happy about being called out at this time; you just ask my wife!!!!!!!!!!

"tolerate" maybe; "happy" NO!

Seriously though, you make some excellent points.

[TimOz]

Mea culpa Malcolm. Change that to "seriously annoyed at being woken up, but not concerned enough to control the riskiest tests in the pathology lab."

And I hear you when it comes to remuneration for long hours.