Kester, Sarah

Purpose: molecular testing is often indicated for recently transfused patients. However, there are no guidelines regarding the potential interference from donor DNA or whether it is necessary to wait for a period of time post-transfusion prior to genetic testing. While the majority of patients are transfused in the non-trauma setting using leukoreduced (LR) red blood cell products, the degree of leukoreduction varies among centers and is not universally practiced. Methods: whole blood units collected from anonymous donors were used in an in vitro transfusion model. One unit was split: half being leukoreduced simulating a leukopenic recipient and half left untreated. Donors were simulated by leukoreduced, partially leukoreduced (PLR), or non-leukoreduced units, transfused in 2, 5, or 16 unit equivalents. DNA from the combinations were subjected to short tandem repeat (STR) analysis for chimerism detection. Results: donor DNA was not detectable in any of the LR combinations, but detected in the PLR combinations, ranging from 0.1 to 1.5% donor DNA in the immunocompetent recipient and 6.3–27.8% in the leukopenic recipient. Non-LR donor DNA was also detected (13–95%). Conclusion: donor-derived DNA from leukoreduced blood products is unlikely to interfere with the interpretation of germline genetic testing in immunocompetent recipients but may interfere in immunocompromised recipients.

Abstract Background Deafness and hearing loss are common conditions that can be seen independently or as part of a syndrome and are often mediated by genetic causes. We sought to develop and validate a hereditary hearing loss panel (HHLP) to detect single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs) in 166 genes related to nonsyndromic and syndromic hearing loss. Methods We developed a custom-capture next-generation sequencing (NGS) reagent to detect all coding regions, ±10 flanking bp, for the 166 genes related to nonsyndromic and syndromic hearing loss. Our validation consisted of testing 52 samples to establish accuracy, reproducibility, and analytical sensitivity. In addition to NGS, supplementary methods, including multiplex ligation-dependent probe amplification, long-range PCR, and Sanger sequencing, were used to ensure coverage of regions that had high complexity or homology. Results We observed 100% positive and negative percentage agreement for detection of SNVs (n = 362), small indels (1–22 bp, n = 25), and CNVs (gains, n = 8; losses, n = 17). Finally, we showed that this assay was able to detect variants with a variant allele frequency ≥20% for SNVs and indels and ≥30% to 35% for CNVs. Conclusions We validated an HHLP that detects SNVs, indels, and CNVs in 166 genes related to syndromic and nonsyndromic hearing loss. The results of this assay can be utilized to confirm a diagnosis of hearing loss and related syndromic disorders associated with known causal genes.

Background: Molecular genetic testing is increasingly utilized in clinical care, typically with peripheral blood as the preferred specimen. Despite the prevalence of therapeutic blood transfusions, whether donor DNA is present in a sufficient quantity to interfere with recipient genetic testing has not been systematically studied. Microchimerism secondary to transfusion of blood products with donor leukocytes has been well-documented in trauma patients receiving whole blood; however, most medical centers currently transfuse leukoreduced (LR) red blood cell products for non-trauma patients. The degree of leukoreduction varies among centers, but to meet AABB standards must be <5x106 white blood cells (WBC) in 95% of units tested. We sought to determine what quantity, if any, of infused LR or non-LR (NLR) products leads to detectable microchimerism that may impact genetic test results. Methods: We performed an in vitro spiking study utilizing four whole blood units collected from different anonymous donors. Three were leukoreduced at varying levels in order to establish a LR (per our institutional guidelines and standards), a partially leukoreduced (PLR) and a NLR unit, which were then considered "donors". The 4th unit was divided to generate two separate "recipients". The first half was left untreated simulating an immunocompetent recipient while the other was leukoreduced to mimic a leukopenic recipient. Based on a 70 kg patient, we calculated the volume of blood from each "donor" to mix with aliquots of each "recipient" to represent a transfusion of 2, 5, or 16 units, with 2 units corresponding to a double apheresis red blood cell (RBC) transfusion and 16 units corresponding to a near total volume transfusion. DNA was extracted from each individual unit as well as all "transfused" combinations within 24 hours of mixing. Chimerism analysis was then performed by STR analysis using the GlobalFiler PCR amplification kit followed by ChimerMarker analysis software. Results: None of the LR units, despite volume transfused, revealed any level of microchimerism in either the immunocompetent or leukopenic "recipients". The PLR transfused combinations displayed levels ranging from 0.14% to 1.52% of donor chimerism for the immunocompetent "recipient", which is below the limit of detection for most clinical assays evaluating germline genetic variation, and 6.3% to 27.78% of donor chimerism for the leukopenic "recipient", which would be expected to impact a subset of clinical genetic assays. The NLR transfused combinations displayed levels ranging from 13.28% to 95.66% of donor chimerism. Discussion:In vitro "transfusion" of LR RBCs into simulated immunocompetent or leukopenic samples does not lead to detectable donor DNA. However, "transfusion" of PLR and NLR units in an in vitro model reveals significant levels of microchimerism dependent on volume transfused and immune status of the "recipient" which implies possible risk for impact on clinical genetic tests. The minimum time required for clearance of donor leukocytes in the recipient is unknown; we were unable to fully evaluate this and other variables in our in vitro system, but follow-up in vivo studies addressing this question are planned. Disclosures No relevant conflicts of interest to declare.