ASHI Forums

Single antigen beads antibody analysis

2009-06-24T09:02:26Z

Hey, everyone!
Please help. I’ve been doing antibody analysis with one lambda beads for a few years. Several months ago we had noticed decreased MFI values of pos control. DTT treatment (ASHI procedure manual) significantly improved results with excellent correlation between FC/CDC XM data. The reason was presence of blocking anti-HLA and anti-idiotypic IgM. However, in almost 40% of my tests low bead counts were observed (<50) making the results unreportable to clinicians. Watching the procedure in details I had revealed formation of agglutinates after 1st washing of beads/serum mixture. This resulted in probe clogging and low bead counts consequently unless agglutinates were removed mechanically. IF anybody saw something like this and had an idea(s) how to circumvent this problem I will greatly appreciate,
Thank you very much.
Andrew

Pronase

2009-04-01T05:34:07Z

What is the best pronase (with cat#) to be used for flow cytometry crossmatching?
and what is the best protocol used?

Does psoralen interfere with bead assays?

2009-03-17T12:27:01Z

This is an archive from the old ASHI Forum

-----

William W. Ward

Posted: Wed May 21, 2008 2:03 pm Post subject: Does psoralen interfere with bead assays? Reply with quote
There have been no other posts to this section of the ASHI forum, so as moderator, I’d like to kick things off with a question.

For labs whose heart and/or lung transplant programs use photophoresis on highly sensitized patients, have you seen depressed positive control (PC) bead and elevated negative control (NC) bead responses in the OL LABScreen single antigen assays when testing serum from these patients? We are seeing squirrely results on the first two highly sensitized patients one of our centers is treating with photophoresis. I’m thinking it may have something to do with the drug used (methoxypsoralen) to sensitize cells to UV irradiation. Can anyone tell me if psoralen binds directly to plastic (i.e. the Luminex bead) and/or fluoresces such that it could either increase the MFI signal or quench it? Any insight would be greatly appreciated. We also see paradoxical results when we try AbsorbOut on these patient’s sera.

Thanks - Bill Ward

-----

mpdowns

Posted: Wed May 28, 2008 3:53 pm Post subject: Reply with quote
We’ve seen “plastic” antibodies, as we call them on several patients.
Usually dialysis patients, sometimes pregnant women( non-transplant, non-dialysis).
We don’t do heart patients yet, so I don’t know about that.
Some of the Immunology tests we have non-specific matrix binding only on a very few patients, but haven’t found a pattern yet.
_________________
Marilyn
HLA Lab @SW
Temple,TX

-----

William W. Ward

Posted: Thu May 29, 2008 12:03 pm Post subject: Reply with quote
Marilyn -

This phenomenon in the sensitized heart patients on photophoresis does not appear to be the often-seen anti-albumin or anti-plastic (so-called “antibead") antibodies. That is why I am curious about being related to the presence of psoralen.

Bill

HLA Typing report for Solid Organ TX patient

2009-03-16T15:48:29Z

This is an archive from the old ASHI Forum

-----

alzayerfadi

Posted: Mon Oct 30, 2006 2:22 am
We are reporting HLA typing of Kidney patient as serological level even if we do it by Molecular SSP. My question is, If we have an allele that do not have a serological equivalent, shall we report it as blank or report that particular allele?
_________________
Fadi Al Zayer. CHT.CHS(ABHI)

-----

dannyy

Posted: Thu Nov 02, 2006 12:40 pm

I believe labs should report all alleles they detect. Like you, our lab reports serological equivalents for solid organ transplantation, however, if an allele has no serological equivalent, we then report the low resolution allele. This makes a big difference for HLA-C, where Cw*12-18 have no serological equivalents. Our lab assigns unacceptable antigens for Cw12-18, based on screening with single antigen beads, and we have several times received imported kidneys that were incompatible for patients with Cw12-18 antibodies, but these patients did not get ruled out for these kidneys because the originating lab did not report Cw12-18 even though they detected it, because these alleles have “no serological equivalent”. I believe the original point of serological equivalents was to provide a way for labs to report things like B62, B63, DQ7, etc. that would be more informative than the corresponding low-resolution molecular results of B*15, B*63, DQB1*03. Cw12-18 will never have “serological equivalents”, but we can’t pretend that donors don’t have the corresponding antigens, especially when patients can clearly make antibodies to these antigens.

-----

alzayerfadi

Posted: Fri Nov 03, 2006 2:26 am

What going to be the case for the alleles that the serological specificity is different than the first 2 digits (eg. B*4026=B21).
If we are going to report the B*40, can we accept patient with Anti B21(which is B50)?!
_________________
Fadi Al Zayer. CHT.CHS(ABHI)

-----

dannyy

Posted: Mon Nov 06, 2006 11:19 am
That’s a more difficult question. Just because your patient has anti-B49, B50, doesn’t mean she also has anti-B*4026. Donors with unusual antigens not represented on a screening panel will always be problematic. These rare antigens could cause a crossmatch to be unexpectedly positive or unexpectedly negative. In the end, I suppose your approach should depend on whether your center thinks it better to potentially and unnecessarily exclude a patient from receiving that donor’s kidney, or whether it’s better to bring the patient in and potentially have a positive crossmatch. My inclination would have been to run the crossmatch. If it’s negative, good. If it’s positive, now you know to avoid B*4026 the next time.

-----

mpdowns

Posted: Tue Nov 07, 2006 1:51 pm
As suggested, single antigen beads are very helpful. There are articles regarding the use of single antigen beads vs a crossmatch. A positive crossmatch, depending on methods, etc should be interpreted with the patient’s sensitization and antibody characterization history. Of course, your patient/donor population comes into this also. As the commercial beads don’t have the rarer antigens.
_________________
Marilyn
HLA Lab @SW
Temple,TX

AHG-CONTROL

2009-03-16T15:41:41Z

This is an archive from the old ASHI Forum

-----

alzayerfadi

Posted: Sat Nov 25, 2006 8:17 am Post subject: AHG-CONTROL Reply with quote
I would like to know, how you QC the AHG for the CDC crossmatch?
Any ideas..
_________________
Fadi Al Zayer. CHT.CHS(ABHI)

-----

mpdowns

Posted: Thu Nov 30, 2006 11:42 am
Some ideas include:
Titer the control against the current AHG dilution, as well as using some wells for no complement, and/or inactivated complement , and parallel this with same set-up and no-AHG. This way you know if it is toxic on its own, what the endpoint is, and if it actually works. We have found that it is not consistent with all cells/targets depending on where you get your AHG control.
You could also run it on a cell panel tray at different dilutions with and without AHG.

Technical Considerations (Tips and Tricks) for Flow/Luminex/

2009-03-16T15:34:31Z

This is an archive from the old ASHI Forum

-----

William W. Ward

Posted: Thu Aug 17, 2006 4:33 pm
I am going to be drafting a section for a new CLSI (formerly NCCLS) document on “HLA Detection by Flow Cytometry” dealing with special considerations for antibody testing using the microparticle solid phase assays on the flow and Luminex platforms.

I would like to hear from anyone with technical pointers to share on the interpretation of these assays, especially how to identify and avoid “pitfalls”. Some considerations along these lines might include: Proper selection and use of negative controls; How to handle “Left-Shift” results; What to do about “Anti-bead” antibodies and other causes of high background; Just How Do You Define a Positive result? Is a weak result relevant, and if so, how weak? etc.

Any and all suggestions for topics to include in this section are welcome. Please email me directly if you don’t wish to respond here.

Thanks - Bill Ward

-----

shelman

Posted: Mon Jun 09, 2008 10:18 am
Was this ever completed?

-----

William W. Ward

Posted: Fri Jul 11, 2008 1:14 pm
Yes. The project was completed and published as a proposed guideline. It has been through the review process and I believe it is now an approved guideline. I haven’t seen it advertised for sale yet though.

DP antigen/antibody

2009-03-16T15:22:11Z

This is an archive from the old ASHI Forum

-----

mpdowns

Posted: Thu Jun 05, 2008 10:04 am
Anyone routinely testing donors and recipients for DP?
_________________
Marilyn
HLA Lab @SW
Temple,TX

-----

William W. Ward

Posted: Thu Jun 05, 2008 10:26 am
Not yet, but plan to campaign with the OPO for routine DP on all deceased donors, and with tplt progs for LD’s where the patient has DP antibodies which we now routinely identify with S.A. testing.

I would also be very interested in knowing if others are typing now, or plan to in the near future.

-----

dannyy

Posted: Mon Sep 29, 2008 12:45 pm
Our lab has been screening transplant candidates for DP antibodies for several years, and when a candidate with DP antibodies is offered a kidney, we DP type the deceased donor. The results are available about the same time as the final crossmatch.