Recently, a discussion at my facility occurred over QC' ing expired panels used for cell rule-out in antibody work-ups. Tentatively, it's been decided to keep panels for no longer than one month beyond their expiration date, while retaining a few select cells expressing rarer antigens or unusual phenotypes for more complicated work-ups. This gives us six panels to work with at all times (four 0.8% cell panels from Ortho, two 3% cell panels from Immucor), and a handful of vials that we retain for no more than 3 months beyond their expiration.

My supervisor believes that when QC' ing expired cells, these cells should be tested against stockpiled plasma from previous patients having a known antibody (strong enough to detect heterozygous expressions) versus using commercial antisera alone to ensure that the antigens are intact for agglutination and detection.

The reasoning behind this:

Monoclonal antisera is purer, perhaps of a higher titer, and might react even with cells having significant deterioration of antigen expression ...but against patient's plasma, the antigen expressed on the “old†cells might miss detection for a number of reasons all related to the complexities and variabilities of human plasma. In other words, we want to QC with commercial antisera, but also use antisera that is nearly identical to the patient matrix.

Right now, we're freezing aliquots of plasma from our patient population for as many antibodies to single antigens that we can (it's easy and cheap too). We intend then to use this in combo with the commercial antisera when QC' ing these expired cells.

Question: what is everyone else doing and is there a regulation regarding this practice?

:juggle:

The regulation says that you have to have a policy for using expired panels and must run QC when used. To that I would add that the expired panels should be segregated and conspicuously marked.

We use commercial antisera to QC expired panels. You'd have to validate the use of frozen patient samples as QC for expired panels, since their post-thaw performance and frozen outdating is unknown.

You bring up a good point concerning the validation of patient-derived antisera. There are guidelines but nothing is set in stone. For instance:

BCSH guidelines published in Transfusion Medicine 1996;6:273-283;...

6 months (shelf-life) for separated serum or plasma at -30 C.

Currently, our aliquots are stored at -30 C and it would prudent to stipulate that each aliquot can only be thawed and used once, vs. repeated thawing/freezing. From the literature, I believe the concern over lability of IgG antibodies is mostly due to the repeated thawing/freezing and not the actual storage process. Some studies have been done more recently so demonstrate the good stability of IgG antibodies in unpreserved, human plasma but more needs to be done.

(Here's one study: http://cdli.asm.org/cgi/content/full/10/1/19).

In practice we freeze and thaw aliquots for antibody titers in prenatal patients over the course of their pregnancy and yet never read where this is a validated procedure...except that prenatal plasma isn't used to test reagents or used in the identification of antibodies for other patients, so perhaps it's exempt from stringent validation.

As far as validating our plasma aliquots for activity, we could do this by testing an aliquot each month against a panel of cells heterozygous for the specific antigen (let's say 10 cells) to ensure activity and to devise an SOP for this purpose.

Im curious, just what "regulation" requires this QC?

"The regulation says that you have to have a policy for using expired panels and must run QC when used."

Once we are at the point of using expired panel cells, it is usually only for additional rule outs. So we just run the current patient against a cell positive for the antibody we are identifying.

I guess I used the word "regulation" too loosely, as I occasionally do in conersations ...

We based our "Expired Panel" SOP on several sources:

First, CAP's TRM.31250 that says controls should be run if expired panels are used, and a policy is neessary.

Second, some obscure FDA document mentioned on cbbsweb (http://www.cbbsweb.org/enf/2004/reagentcells_exp2.html), saying that a reactivity check was required.

Last, a discussion in Standards Source a year or so ago -- sorry, I don't have that readily available today.

Because monoclonal antisera are usually very strong, it is NOT appropriate to use undiluted monoclonal antisera for QCing cells of any type. Using undiluted monoclonal antisera can give you a false sense of security that the cell possesses the antigen desired at levels that will react with patient serum/plasma.

If you don't want to use stored plasma containing antibodies, one suggestion is to dilute monoclonal antisera to achieve a 2+ or 3+ reaction with in-date panel cells. If the expired panel cells have deteriorated, you should be able to tell that based on a reduction in the reaction grade.

Yes this method has to be validated but the time spent up front could be well worth it if you prevent missing a clinically significant antibody because the cells were not strong enough to react with the patient’s sample.

It is also important to run a NEGATIVE control with expired panel cells to ensure that they have not become contaminated, particularly with microbes.

Sheryl Kochman

Chief, Devices Review Branch

FDA/CBER/OBRR/DBA

The "obscure FDA document" referred to above is The Guide to Inspection of Blood Banks and is one that all blood collection facilities should have available to them. It is available at: http://www.fda.gov/ora/inspect_ref/igs/blood.html

Another useful document that applies to Licensed and Unlicensed Blood Banks, Brokers, Reference Laboratories, and Contractors is the Compliance Program for Blood and Blood Products. It, and other documents of potential interest, are available at: http://www.fda.gov/cber/cpg/cpg.htm

Sheryl Kochman

I appreciate all the responses on my original question, especially John's question and Lcsmrz's answers leading up to Sheryl's links to the FDA documents.

My lab will perform negative controls as well as positive controls for the outdated cells (I forgot to mention this fact previously).

I'm somewhat unclear as to Sheryl's statement for the use of dliuted monoclonal antisera. I'm assuming Sheryl means using diluted antisera only against those cells used as positive and negative controls and NOT against donor and patient cells, where we follow the insert instructions for each antisera we use.

:coffeecup

Actually, you should use diluted monoclonal antisera only on those QC cells that are positive for the desired antigen. Negative QC cells should be tested with undiluted reagents. Patient samples should not be diluted.

Sorry I wasn't clearer.

Sheryl Kochman

A group-wide "Thanks" to Sheryl for watching this forum!!

Sometimes we scientists try too hard to be compliant, yet miss the whole intent ...

Back to the original comment about the use of expired panel cells.

Here is the AABB Standard (IRL Standards 5.1.4.2):

"The criteria for the use of non-FDA-licensed reagents (including expired reagents) shall be defined."

The standard does not mention a specific QC requirement, only that there must be defined criteria for use.

Our lab discards panel cells 2 months after expiration. We do not perform QC on these cells.

Regarding the use of diluted monoclonal antisera: after sitting in many presentations from Immucor/Gamma, I understand that only the original diluent or resuspension media should be used to dilute monoclonal antisera. Rh Control, for example, is the only proper diluent for monoclonal Anti-D. Additionally, I understand that each diluent is different, so using diluted monoclonal antisera turns into a difficult proposition.

Thanks Dawn and Christie...for these additional clarifications. Will be sure to pass them along. I wasn't too keen on the idea of diluting antisera both for the reason you listed Christie (unique diluents for each antisera), and also because of the added burden of establishing the degree of dilution for each antisera.

The information the two of you have given is more along the lines of common sense.