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Molecular Test Identifies Acute Transplant Rejection

By LabMedica International staff writers
Posted on 25 Nov 2014
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Fluidigm\'s BioMark HD and EP1 platform with Digital Array Integrated Fluidic Circuits for quantitative real-time polymerase chain reaction
The BioMark HD and EP1 platform with Digital Array Integrated Fluidic Circuits for quantitative real-time polymerase chain reaction (Photo courtesy of Fluidigm)
The development of noninvasive molecular assays to improve disease diagnosis and patient monitoring is a critical need in renal transplantation, as acute rejection (AR) increases the risk for chronic graft injury and failure.

Acute rejection after kidney transplantation occurs in about 15% to 20% of patients despite immunosuppressive therapy and rejection is usually heralded by an increase in the patient's serum creatinine which is a marker of kidney function, and a kidney biopsy is then performed to confirm whether rejection is taking place.

Scientists at the University of California (San Francisco, CA, USA) working with an international team used 558 peripheral blood (PB) samples from 438 adult and pediatric renal transplant patients from eight renal transplant centers in the United States, Mexico, and Spain and enrolled between 2005 and 2012 to develop a quantitative real-time polymerase chain reaction (qPCR) test. They developed the test in 143 samples from adults with and without acute rejection as determined by kidney biopsy, and then finalized and validated the test in three cohorts of patients.

Total ribonucleic acid (RNA) was extracted using the column-based method kits and was measured for RNA integrity using the RNA 6000 Nano LabChip Kit on a 2100 Bioanalyzer (Agilent Technologies; Santa Clara, CA, USA). Microfluidic qPCR was performed on the 96.96 dynamic array and the chip loaded via the HX IFC Controller and performing the qPCR in the BioMark qPCR platform (Fluidigm; South San Francisco, CA, USA).

The team evaluated the differential expression of 10 out of 43 genes, selecting the same ten genes that have been validated as diagnostic for AR in PB using standardized clinical trial sample collection and processing protocols. To improve the diagnostic accuracy for AR in the 143 adult samples, various statistical modeling methods were used to include an additional seven genes which improved the accuracy of the gene panel for discriminating AR from No-AR samples, for a final selection of 17 genes. The assay was called kSORT and was able to classify patients as high risk versus low risk for AR. The kSORT assay was able to detect AR in blood independent of age, time post-transplantation, and sample source without additional data normalization.

The authors concluded that the kSORT assay is a simple, robust, and clinically applicable blood test. They are using kSORT in a prospective observational trial as well as a second prospective, randomized, double-blinded clinical interventional trial, which will establish how kSORT can be used serially post-transplant to complement current clinical practice guidelines for stratifying patient immune risk, medication load, and requirement for biopsy. The study was published on November 11, 2014, in the journal Public Library of Science Medicine.

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