Children's Hospital Colorado Aurora, Colorado, United States
Background/Case Studies: Molecular human erythrocyte antigen (HEA) typing for the provision of phenotypically matched pRBC units during transfusion has become a common and reliable HEA typing method over the last decade. For the FDA approved HEA typing platforms, whole blood is the specimen of choice, which can lead to issues of extracting enough DNA in cases of low WBC count. Especially in patients with treatment related cytopenias (eg. leukemia patients undergoing chemotherapy), WBC counts are often so low that adequate DNA extraction is impossible using the manufacturer recommended techniques. To overcome this limitation, our laboratory developed and validated a buffy coat extraction technique for typing patients when standard extraction methods failed to yield DNA concentrations >15 ng/µl.
Study
Design/Methods: Fourteen samples were selected, including 6 from patients with WBC counts < 1.0 x 103/µl. 11 of 14 were previously tested for HEA using standard extraction methods to ensure concordance of typing between the standard and buffy coat extraction techniques.
EDTA specimens were allowed to come to room temperature prior to processing. Samples were centrifuged for 10 minutes at 3100-3550 RPM with no brake. Once removed from the centrifuge, a sterile transfer pipet was used to draw up 0.25-0.5 mL of specimen from around the buffy coat area and dispensed into a labeled sterile secondary tube. Using a single channel pipette, 200 µl from the buffy coat sample was drawn up to be substituted for whole blood in our DNA extraction protocol.
Following DNA extraction, all samples were tested for DNA concentration. HEA typing was completed with our FDA approved typing platform using standard operating procedures. For the 11 samples with previous HEA typing, test results were compared to determine antigen concordance.
Results/Findings: Despite buffy coat sampling, not all extractions met the 15 ng/µl threshold. Samples from patients with a WBC count >0.2 x 103/µl were more likely to yield >15 ng/µl, and we obtained accurate HEA results from buffy coat derived DNA with concentrations as low as 5.642 ng/µl. All samples from patients or donors with a historical HEA test demonstrated 100% antigen concordance with previous typing, as shown in Table 1.
Conclusions: With buffy coat extraction technique, we were able to obtain accurate HEA typing results on samples from patients with WBC counts < 1.0 x 103/µl. We showed HEA test result concordance in samples even when DNA concentration did not meet the manufacturer-recommended 15 ng/µl, which supported lowering our lab’s DNA concentration threshold to attempt HEA typing on buffy coat extracted samples. Implementation of buffy coat extraction technique has allowed us to expand our ability to provide phenotypically matched units in medically necessary situations.
