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Performing titers:

Pregnant Females sensitized with clinically significant antibodies - Does your blood bank follow the AABB procedure recommendation or have you validated using gel?

Other patients:  Do you use tube as well or use gel?

Any input is greatly appreciated! 

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On the same subject, sort of.... What type of QC are you performing? Our facility was recently cited by CLIA "The failed to include a negative and a titered control for antibody titration"...This was specifically referring to Anti-A and Anti-B but we also rarely perform titers for alloantibodies. 

Any advice would be appreciated

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53 minutes ago, Santa Beauchamp said:

On the same subject, sort of.... What type of QC are you performing? Our facility was recently cited by CLIA "The failed to include a negative and a titered control for antibody titration"...This was specifically referring to Anti-A and Anti-B but we also rarely perform titers for alloantibodies. 

Any advice would be appreciated

Interesting!  Thank you for bringing this up.  I wonder if we are meeting that requirement as well?  We do 2 CAP surveys per year for antibody titers. In addition, we freeze an aliquot to test in parallel with the next sample.  We only do the parallel on returning patients - I'm going to have to look into this.  Should we doing a parallel with each titer?

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I find this interesting too - from another point of view.

Do CLIA ask you to perform IgM and IgG antibody titres of ABO, because the two may be quite different, BUT, ABO IgG antibodies are often agglutinating antibodies, even at low temperatures, so, just because an ABO antibody titre may be, say, 64, it is VERY important to know whether this is the IgM or IgG titre?  This is particularly important in cases of solid organ transplant and stem cell transplant, as, not only may the antibodies contribute to red cell destruction through complement activation and/or destruction in the reticulo-endothelium system, but may also result in immune complex destruction.

MANY years ago now, Prof Patrick Mollison wrote about the fact that it is more difficult to inhibit IgG antibodies than IgM antibodies in vivo, so this is really important.

Did your CLIA inspector ask about this, because, if not, their citation was a waste of time?  If they did, good for them.

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No they did not ask these questions. As a matter of fact the standard they sited "Test procedures producing graded or titered results, include a negative control material and a control material with graded or titered reactivity", refers to Control Procedures, not specifically Immunhematology. Their only concern was a negative control and a positive control that titers at least as far as the reportable range, not actual Blood Bank information. We also participate in the 2 CAP surveys per year and were recently inspected by AABB and CAP without this being mentioned. CLIA did not deem this sufficient

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10 hours ago, Santa Beauchamp said:

No they did not ask these questions. As a matter of fact the standard they sited "Test procedures producing graded or titered results, include a negative control material and a control material with graded or titered reactivity", refers to Control Procedures, not specifically Immunhematology. Their only concern was a negative control and a positive control that titers at least as far as the reportable range, not actual Blood Bank information. We also participate in the 2 CAP surveys per year and were recently inspected by AABB and CAP without this being mentioned. CLIA did not deem this sufficient

So, what EXACTLY is this controlling I wonder?  IF the positive control material is an antibody directed against an antigen that is different to the one you are titrating (say, for example, the control material was an anti-D, and your test material was an anti-Bpa - and you are unlikely to have a control anti-Bpa available), then this is not even controlling the antigen expression on the red cells, let alone anything else.  I would be more than a little inclined to challenge (and re-challenge and re-challenge) CLIA, until they come up with a proper answer, which, I would suggest, they would find VERY difficult.

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On ‎03‎/‎16‎/‎2018 at 11:39 AM, TreeMoss said:

We follow the AABB guideline and test only in tube method.

Do you perform any parallel testing/control with every titer?  We only perform if we have the previous titer on the same patient. 

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22 hours ago, BloodBanker80 said:

Do you perform any parallel testing/control with every titer?  We only perform if we have the previous titer on the same patient. 

We do perform in parallel if we have a previous specimen from the same pregnancy.

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Here's my 2-cents, 2-pennies, or 2-any other small denomination coins:

First, I am NOT a regulatory expert, but I am familiar with assay development and validation.

All assays should be controlled in some fashion, to give the practitioners some confidence that the results are valid AND that batch-to-batch variation is limited. In a titration, especially a serological titration, this is a little more difficult than having one or two pass/fail samples that are typically included in many laboratory tests.

If a control is needed for a titration, it doesn't need to be the same specificity as the test antibody (as Malcolm highlights). Ideally, yes, but not necessarily. It does need to be reliable/robust and give the same end point each time, even with the acknowledged variations in serological tests (reagents, test cells, techs, etc.). Tube testing is notoriously variable, while gel testing is believed to reduce some of those nuances. As Malcolm suggests, it might be necessary to have a control for an IgM titration (ABO) and/or a different one for an IgG titration. At the very least, the end-points may be different. An IgM control might be as simple your routine anti-A reagent; a simple IgG control might be an IAT-reactive anti-D or other specificity. A clever option might be a control that contains both - an IgM component and an IgG component.

If a test system is adequately controlled each time (and passes), in my opinion, there is no reason to routinely perform parallel testing of successive patient samples. Retention samples might be useful for investigatory reasons if/when a patient's antibody titration changes radically from one sample to the next, or if there's some other medical indication.

I getting a sense of deja vu, I think I've written this before.

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"Tube testing is notoriously variable, while gel testing is believed to reduce some of those nuances. "

Yes, but it is also - according to years of CAP survey results - routinely 1-2 endpoints higher on every titration run.  So, make sure you are telling your physicians that your facility is doing titers with gel and that the actionable titers for their patients might be at higher endpoints than those historically with tube testing.

"It does need to be reliable/robust and give the same end point each time, even with the acknowledged variations in serological tests (reagents, test cells, techs, etc.)"

I wonder if the inspector would have been OK with procedures with this kind of control if it was used when there was no retained sample (no control avail) and not used when the new specimen was run in parallel with it's retained pair (internal control avail)??  I guess we will have to see, because that is what we are going to try doing.

 

 

 

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19 hours ago, cswickard said:

Yes, but it is also - according to years of CAP survey results - routinely 1-2 endpoints higher on every titration run.  So, make sure you are telling your physicians that your facility is doing titers with gel and that the actionable titers for their patients might be at higher endpoints than those historically with tube testing.

I wonder if the inspector would have been OK with procedures with this kind of control if it was used when there was no retained sample (no control avail) and not used when the new specimen was run in parallel with it's retained pair (internal control avail)??  I guess we will have to see, because that is what we are going to try doing.

You bring up some interesting points and I agree with your position.

Certainly "the literature" regarding the clinical importance of titration results is confusing. Most of the original work was done on anti-D, using an unorthodox test protocol (I believe the titrant was a high concentration of BSA), but there did seem to be some correlation between titration strength and clinical impact. Workers attempted to shoe-horn other specificities into the same program, with mixed success. Now, today, as you point out, the gel test is becoming a routine way to measure antibody strength. I don't think anyone honestly knows what the titration end-points mean, since the modern results are difficult to interpret/compare to the older literature. Time will tell.

I think people have been caught in a little trap with regard to the controls needed for titrations. It is very unusual to have two sequential samples in a clinical assay, even rarer that one of said samples has been stored (frozen). Consider a simple CBC.....many patients with extended hospital stays have multiple tests performed. Their last samples are not run in parallel with the current sample, yet the results are considered valid because the instrument's controls performed as expected - this is the equivalent of using material with a known potency as a control for patient sample testing. The art is in selecting your control.

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