ASHI Forums

HD-SSO technology for High-Res DNA typing

2009-08-18T13:30:00-05:00

Hello~
Our lab has adopted the One Lambda HD-SSO system as the first step to reporting high-resolution DNA typing. We are pleased with the results so far, but we are still gathering more information about the strengths, limits, range, and sensitivity of this system. When we find a resulting allele pair that does not resolve to a single Common and Well-Documented allele or allele group, we use the O.L. allele-specific MicroSSP kits. In our hands so far, ~60% of all patient samples require secondary SSP tests to resolve one or more loci; average is 1.1 SSP tests/patient - very manageable so far. While we’ve been having some PCR drama lately getting perfect results (due to DNA conc., well failures, dehydrated wells? etc.), we’re working through these and discussing options with the manufacturer.
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I’m wondering how many other labs are in our same situation. Would anyone would like to communicate and share notes/experiences using the HD-SSO and allele-specific SSP reagents for HR typing??
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Specifically, we’ve detected several allele pairs that just can’t be resolved to single CWD alleles using these reagents, and therefore must be sent out for SBT. It would be great to know about other situations like this, ones that we just haven’t run across yet. We’d be happy to share more about our findings.
I’d appreciate replies to the forum or via personal email.
Thanks!
Carley Shaut

B-cell Antiglobulin Crossmatch

2009-03-18T11:31:17-05:00

This is an archive from the old ASHI Forum

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dannyy

Posted: Thu Feb 14, 2008 3:27 pm Post subject: B-cell Antiglobulin Crossmatch Reply with quote
Many years ago our lab played around with using AHG in the B-Cell cytotoxicity crossmatch, with mixed results. Some cells seemed to be overly susceptible to the reagent and had high cell-death backgrounds in all sera, while others did just fine. We’re planning to evaluate B-cell-AHG again, and would apprecaite hearing others’ experience and advice.

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awhiteley

Posted: Mon Dec 01, 2008 1:46 pm
I think the main issue with B-Cell crossmatching is the CD32 receptors and the non-specific binding of IgG by the FcgR. Does your lab already do a B-cell Flow Crossmatch?

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dannyy

Posted: Wed Jan 21, 2009 1:47 pm
Yes, we perform a B-cell Flow XM, but there are some applications where our customers prefer a cytotoxicity crossmatch.

Luminex PRA Ratio

2009-06-27T14:43:05-05:00

Did any body try to see at what ratio of LABScreen mix beads show MFI of single antigen more than 2000? Any studies?

GTI EZ Type SSP System

2009-03-18T11:27:42-05:00

This is an archive from the old ASHI Forum

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cjordan

Posted: Thu Sep 14, 2006 4:30 pm Post subject: GTI EZ Type SSP System Reply with quote
Is anybody out there in HLA land using the GTI EZ Type SSP System?
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Curt Jordan
Carter BloodCare/Stewart Regional Blood Center
Tyler,TX

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vicki22

Posted: Wed Oct 04, 2006 12:55 pm
We have started to qualify the DRDQ kits but they are not in use yet. We just wanted a backup for our current Pel-Freez SSP trays so we are not in a hurry to implement.

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cjordan

Posted: Wed Oct 04, 2006 3:03 pm
Thanks!

Let me know how you like them. We were having some problems with the ABC trays. getting alot of no amps. I’m just trying to see how many people are using the trays, and if having some of the same problems Smile

The DR/DQ trays seem to be OK
_________________
Curt Jordan
Carter BloodCare/Stewart Regional Blood Center
Tyler,TX

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jtownsend

Posted: Mon Feb 02, 2009 4:14 pm
We use the ABC and DRDQ routinely for deceased donor typings. It is a good system for going molecular for this type of work. Easy to get up and running. A few issues: paperwork a little confusing to start with, and software needed updates but it has been and we will see it in March. Some very weak bands are false positive because they have the correct bp size. Very low resolution, but good for serological equivalence and sure beats serology. Don’t recommend using the heat sealer, can melt trays to the point of not being able to insert pipet tips into wells to extract amplicons, ok just to use adhesive seal.

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cjordan

Posted: Tue Feb 03, 2009 11:56 am
agreed, be careful with the heat sealer, no more than about 5-6 seconds. we have melted trays also. the paperwork and analysis is a little more complicated than other kits.
_________________
Curt Jordan
Carter BloodCare/Stewart Regional Blood Center
Tyler,TX

Amplification problems on Low resolution trays

2009-03-18T10:42:24-05:00

This is an archive from the old ASHI Forum

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Padma

Posted: Fri Feb 06, 2009 2:39 pm
Why would one see false negative amplification while all internal control bands worked? What are possible interferences that would not amplify positive bands.

Marker worked fine. DNA conc 135 ng/ul and ratio of 1.96

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cavergas

Posted: Mon Mar 09, 2009 10:25 am
Were there any positive bands at all? Also, how strong were the internal control bands? Could be a novel allele that the primer(s) are not annealing to. Not sure of answer without having more detail.