1009-LBP: Non-Invasive Molecular Protocol for Deduction of Mismatched Donor HLA from Urine Samples of Renal Transplant Recipients

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• 作者：Ricky Lau¹ ; Gavin Chan² ; Leo Choi¹ ; M.F. Lam³ ; Maggie Mok³ ; W.L. Chak⁴ ; Au Cheuk⁵ ; K.W. Chan⁶ ; K.F. Chau⁴ ; Matthew Tong⁵ ; T.M. Chan³ ; Janette Kwok¹
• 刊名：Human Immunology
• 出版年：June 2014
• 年：2014
• 卷：75
• 期：6
• 页码：483
• 全文大小：28 K

文摘

Previous studies demonstrated that mismatched donor HLA phenotype could be successfully deduced from either fresh or archival formalin-fixed paraffin-embedded renal allograft tissue by polymerase chain reaction-specific sequence primers (PCR-SSP) genotyping [1] and [2]. In this study, a modified non-invasive urine protocol was further validated.

Methods

Fresh early morning urine was centrifuged and the cell pellets were washed with phosphate buffered saline. DNA was extracted from the urinary cells by TBG EZmag Genomic DNA Whole Blood Kit. The HLA-A, B and DRB1 loci were typed by PCR-SSP. The performance of the PCR reaction was enhanced by using a hot-start Taq polymerase together with tailor-made extended PCR profiles according to the DNA yields.

Results

Urine samples were collected from recipients of renal transplant centers in Hong Kong during May to December 2012. 34.8%, 25.8%, 18.1% and 21.3% yielded DNA amount >5000, 2000–5000, 1000–2000 and <1000 ng respectively. Among which, thirty-five non-duplicate specimens (10, 10, 10 and 5 respectively) were retrospectively selected for testing by the protocol as described previously [3]. The HLA phenotypes of the donor-recipient pairs could be completely determined in all tested samples.

Conclusions

In previous study, it was revealed that the presence of sufficient donor-derived substrate for HLA typing appeared critical [4]. By adopting the modified procedures, mismatched donor HLA phenotypes in urine samples containing various DNA quantities could be successfully deduced. This non-invasive approach is promising and reliable for the identification of mismatched donor HLA in kidney transplant recipients with unknown donor HLA phenotype.