ASHI Forums

airfuge input

2011-02-03T21:56:55Z

What do you see as its major value/advantage?
If you use one, do you use it for all testing?...or just selected use?

LSM QC

2011-12-21T13:32:08Z

We currently use a hydrometer and graduated cylinder to QC new shipments of density gradient (we use LSM by MP Biomedicals). Just wanted to see if there was another way to QC the reagent that doesn’t require opening 4 bottles.
I can’t find anything in my internet searches except big instrumentation that would be overkill.

Any input would be appreciated.

instrument coorelation

2011-01-07T14:37:00Z

For those labs that have more than one Luminex or Flow. Do you (and if yes, how often) coorelate results between the instruments?

We just purchased a second Luminex and might be getting a second Flow Cytometer. Of course we are validating the new instrument but, after the initial validation do you do any further routine checks to make sure they are both making the same calls? If yes, how often?

Thanks in advance.

Wanted: Electrophoresis Chambers

2011-05-11T10:41:40Z

Does anyone have the old ABgene Electrofast 96 chamber/comb setup and are willing to part with it?
They are no longer being made.

Isolation DNA from small amount of blood sample with good yield of DNA by EZ1 DNA extraction

2010-09-24T10:23:12Z

Isolation DNA using AS WHOLE BLOOD SAMPLES (1 small yellow top).

a. Centrifuge small yellow top tubes for 10 minutes at 2500RPM. Discard top part of the serum and mixed the rest of the blood three times by inverting to mix the buffy coat with the the rest of the red cells.

b. Per ASHI sample: transfer 350µl of the blood mixture into three 2.0ml sterile sample tubes.

c. Insert the EZ1® Whole Blood Card into the card slot. Turn on the EZ1® BioRobot and open the workstation door.

d. Invert each reagent cartridge twice to mix the magnetic particles. Then tap the reagent cartridges to deposit the reagents at the bottom of their wells. Load the EZ1® cartridge rack with three reagent cartridges per ASHI sample (See procedural notes).

e. Load the EZ1® worktable: the uncapped 1.5 ml elution tubes in the 1st row, the disposable filter tips with disposable tip holders in row 2 (row 3 is empty), and the ASHI sample uncapped 2.0ml sample tubes with 350µl whole blood in the 4th row of the worktable. Double check this order by following the protocol instructions listed on the LCD screen of the instrument. (See procedural notes) Also, when loading the samples onto the instrument, be sure to double check and make sure the sample tube and elution tube labels match for each patient and are lined up on the machine correctly.

f. Select protocol #2 (350µl whole blood). Press enter.

g. Choose 200µl as the final elution volume. Choose NO to pure ethanol wash.

h. Proceed to the direction on the screen to the “Start” button and start.

DNA yield is about 50µg from 2ml of whole blood.

HD-SSO technology for High-Res DNA typing

2009-08-18T13:30:00Z

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.
~
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??
~
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

2009-03-18T11:31:17Z

This is an archive from the old ASHI Forum

-----

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.

-----

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?

-----

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

2009-06-27T14:43:05Z

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

2009-03-18T11:27:42Z

This is an archive from the old ASHI Forum

-----

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?
_________________
Curt Jordan
Carter BloodCare/Stewart Regional Blood Center
Tyler,TX

-----

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.

-----

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

-----

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.

-----

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

2009-03-18T10:42:24Z

This is an archive from the old ASHI Forum

-----

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

-----

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.