Mahaffey, Victor

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.

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.

Abstract Introduction: Monitoring BCR-ABL fusion transcript levels in peripheral whole blood (WB) of patients on tyrosine kinase inhibitor (TKI) therapy using real-time quantitative PCR (RT-qPCR) is standard of care in the management of Chronic Myeloid Leukemia (CML). GeneXpert® BCR-ABL V2¥¥ or Xpert® BCR-ABL Ultra¥ (Ultra), a cartridge-based assay for use on the GeneXpert Instrument System, automates and standardizes the RT-qPCR process in less than 2 hours, using a lysate prepared from 4mL WB, resulting in an effective WB input volume of 600μL. There are, however, clinical situations where the total RNA isolated from high numbers of white blood cells (WBC) circulating in the patient's blood or in bone marrow (BM) can overload the Ultra cartridge and other quantitative BCR-ABL assays, requiring subsequent dilution of the sample to generate valid assay results. In this set of experiments, we sought to define the WBC input limits for Ultra that would predict cartridge overload, and thereby provide guidance regarding when to dilute the patient's sample in the presence of high WBC. Methods: Serial dilutions of WB specimens were tested in Ultra to determine the upper and lower WBC input limits corresponding to the valid ABL reference gene Ct cutoffs of 10 and 18, respectively. Sample prep procedures were developed to allow using 50μL or lower input volume from WB, BM, or the 1st sample lysate, with or without the WBC count (WBCC) information. BCR-ABL log% IS results from a subset of samples prepared and tested with various specimen input volumes in Ultra were compared in linear regression analyses with results from the Qiagen Ipsogen BCR-ABL1 Mbcr IS-MMR assay (IS-MMR). Results: The WBC input number of ~20 million cells/mL WB corresponded to the upper limit, and ~150K cells/mL WB corresponded to the lower limit, of the valid ABL Ct range for Ultra. For CML specimens with WBCCs <20 million cells/mL WB, the standard 4mL WB input yielded ABL Ct within acceptable limits. For specimens with WBCCs ≥20 million cells/mL, reaching as high as >500 million, sample prep procedures were developed to use 50μL WB, BM, or 1st lysate input volume. When 50μL was used but still yielded ABL Ct <10, a lower input volume (10μL or lower) can be used (Table 1). For situations in which the WBCC information is not available, but there is suspicion of high WBC yielding a very viscous 1st lysate that is hard to pipette, a 1st lysate treatment procedure for dilution was developed to allow for testing with 50μL or lower WB or BM input volume. These procedures were validated in Ultra by testing in CML specimens with various WB or BM input volume and showed high concordance (R2=0.98) when compared to the IS-MMR. Conclusions: In summary, sample prep procedures were developed in cases where high WBCC is known, or is suspected, or when repeat testing is needed for Invalids with ABL Ct <10, allowing the use of Ultra with various input sample volume for WB or BM in a wide variety of clinical situations. ¥ In vitro diagnostic medical device. May not be available in all countries. Not available in the United States. ¥¥ RUO; For research use only. Not for diagnostic use. Not reviewed by any regulatory body. Disclosures Day: Cepheid: Employment.