Thanks

Xinjie

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mpdowns

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

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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. 

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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.

Any input would be appreciated.

HLA Center and Affiliation:
Name of respondent or lab director:
• How many sample analysed per year:
• Nature of the sample:
o Blood:
o Buccal swabs:
o FTA papers:
o Other (please precise):
• rSSO (low res) main vendor:
o Failure rate* observed for each locus tested:
 A:
 B:
 C:
 DRB1:
 DQB1:
(*: The failure rate is defined by the impossibility to give a result and the need to repeat the analysis.)
• rSSO (low res) other vendor used:
o Is failure rate lower, similar or higher as compared to your main vendor:
• SSP (low res) main vendor:
o Failure rate observed for each locus tested:
 A:
 B:
 C:
 DRB1:
 DQB1:
• SSP (low res) other vendor:
o Is failure rate lower, similar or higher as compared to your main vendor:
• SSP (high res) main vendor:
o Failure rate observed for each locus tested:
 A:
 B:
 C:
 DRB1:
 DQB1: 

• SSP (high res) other vendor:
o Is failure rate lower, similar or higher as compared to your main vendor:
• SBT double strand main vendor:
o Failure rate observed for each locus tested:
 A:
 B:
 C:
 DRB1:
 DQB1:
• SBT double strand other vendor used:
o Is failure rate lower, similar or higher as compared to your main vendor:
• How many complementary sequences (ex: HARPS) done per locus (average):
• SBT single strand or new generation main vendor:
o Failure rate observed for each locus tested:
 A:
 B:
 C:
 DRB1:
 DQB1:
• Do the observed failure rates obtained from your main vendor’s kits meet your goals:
• Do the failure rates that you observed remain constant from year to year or do they vary:
• Other typing techniques used:

• Type of DNA extraction system and vendor:
• Whole genome amplification:
• Comment:

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.

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.

My question: when would you rule out B*4441 by allele frequency?

1) Both alleles are CWD alleles (Cano et al. 2007), and this is our current standard for HR results.
2) The NMDP allele frequency tables show that 4402 is very common in the US (high frequency and rank), 4441 is not listed.  However, being new to HR typing, we are nervous about using this data over the CWD standards.
3) Allele frequencies.net shows that B*4441 has the allele frequency of 0.000 for the US.
4) Literature searches and HistoCheck show that B*4441 differs from B*4402 by 2 nucleotides and 2 amino acids.

Thank you for any advice or discussion!