Dukek, Brian A.

Abstract Background Lymphocyte function and immune competence can be measured by proliferative response to stimuli and is useful in several clinical contexts including primary (or genetic) and secondary immunodeficiencies (e.g., immunosuppression following transplantation). Proliferation assays have traditionally been prepared manually due to the difficulty in maintaining the sterile environment required to prevent bacterial and fungal impact on cell growth. On most automated liquid handlers, the sample and reagents are placed inside a cabinet whose inner surfaces include rails, pullies, pumps, and circuitry that face the sample and are not amenable to sterilization. The Andrew+ (Waters, MA) is a small surface cleanable liquid handler with all machinery enclosed. We demonstrate here the use of an Andrew+ placed in a biological safety cabinet (BSC) to perform sterile assay setup. Methods Peripheral blood monocytes (PBMCs) from five individuals were manually generated via buffy coat isolation, then brought to standardized concentration. An Andrew+ was placed in a 4’ BSC and all surfaces disinfected with Oxivir Tb (Diversey, SC). The Andrew+ prepared three serial dilutions of two stimulant reagents (phytohemagglutinin (PHA) and pokeweed (PWM)), then dispensed stimulant and control reagents horizontally and PBMCs vertically to a 48 well plate. This process was duplicated manually from the same specimens to create a reference plate. The plates were incubated at 37°C and 5% CO2 for three days, then pulsed with EdU (Thermo Fisher, MA) and incubated for one additional day. Cells were harvested and counted via flow cytometry. Overall time and hands-on time were measured. Results A total of five samples were tested inside a using both manual and automation setup under eight conditions including with isotype control, media without stimulant, three concentrations of PHA, and three concentrations of PWM. The percent proliferation for various cell types as induced by each stimulant was calculated from the difference of the highest stimulant signal from the background (media-only) signal. Comparing the automated and manual method results showed a slope of 1.0217 and R2 of 0.9966. For a simulated run of 6 samples, technologist time for automated steps was under 8 minutes with a total time of 56 minutes. When these steps were performed manually, the time was 30 minutes. Automation increased total time by 26 minutes but saved 22 minutes of technologist hands-on time. Conclusion The Andrew+ was able to perform sterile setup of a cell culture assay. While slower overall than manual processing, technologist time was reduced. Ergonomic, inter-tech variability, and sample transcription error issues were subjectively observed to be less likely. The Andrew+ can work with up to four “dominos” (reagent or tip blocks) in width and up to three dominos in depth. Non-custom, standard depth BSCs from a variety of manufacturers (Labconco, Nuaire, ThermoFisher) have room for one domino in depth so, except for timings performed in this evaluation, all steps were broken down to be performed with a max of four dominos. Efficient sterile use of the system would require an extended depth BSC.

Abstract Background We evaluated the Tecan Fluent Mix and Pierce Workstation as an automated platform to aliquot directly from whole blood collection tubes. The Fluent Mix and Pierce Workstation uses the Tube Rotator™, an on-deck rotisserie, to keep whole blood in suspension, and 8 Piercing Tips™ to cross the pierceable caps of collection tubes to aliquot samples. Methods We performed accuracy and precision testing by aliquoting residual whole blood specimens into an on-deck gravimetric scale. To test carryover, we aliquoted alternating blanks and high calibrators for several analytes and performed mass spectrometry on the blanks. We performed 168 runs over a 21-day period to test the robustness of the platform. We aliquoted and analyzed 2 batches of samples on both our clinical platform and the Mix and Pierce Workstation to check concordance. Results We observed that the Fluent Mix and Pierce Workstation can aliquot 200 µL with 0.5% inaccuracy and 0.9%CV and can aliquot 300 µL with 0.1% inaccuracy and 0.7%CV. We were unable to detect any analyte carryover using mass spectrometry. We observed no hardware failures during the instrument stress test. Clinical comparison runs indicated that the performance of aliquots made on the Fluent Mix and Pierce Workstation was equivalent to that of aliquots made by our current clinical method. Conclusions Overall, our evaluation showed that the Tecan Fluent Mix and Pierce Workstation is a viable platform for aliquoting whole blood for downstream work flows and can eliminate labor-intensive sample decapping and recapping in high-throughput settings.

Abstract Background Our institution is facing increasing demand for oncology testing that requires high quality DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The current DNA extraction workflow involves a time and labor-intensive manual process that limits our ability to accommodate increasing downstream clinical testing volumes. We evaluated the KingFisher (KF) Flex utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit as a possible high-throughput, automated DNA isolation workflow option that would decrease hands-on time and be conducive to volume growth without the need for additional staff. Methods DNA from 52 FFPE tissue samples (n=24 unique cases previously extracted by the current “manual method”) were extracted using Autolys M tubes, the MagMAX FFPE DNA/RNA Ultra Kit, and the KF Flex (Thermo Fisher Scientific Waltham, MA) over 4 “automated method” runs. Method modifications to the published procedure were introduced over the runs to determine any effects on DNA quality/quantity. Timings were taken during each run to compare hands-on and total run time against that of the manual method. Precision experiments were performed by extracting 4 replicates of sample on a single run (intra-assay precision) and 2 samples from 2 cases over 2 runs (inter-assay precision). Three cases were processed using varying tissue input levels to assess input-yield correlation. DNA yield was measured using HS dsDNA Qubit (Thermo Fisher Scientific, Waltham, MA). A subset of samples was tested by digital droplet PCR (ddPCR) (n=10), chromosomal microsomal microarray (CMA) (n=1), and next-generation sequencing (NGS) (n=3). Variants and QC metrics of the KF DNA were compared to those of the manual method DNA. Results The average total DNA yield was 3% lower from samples extracted following the manufacturer recommended KF procedure compared to matched samples extracted using the manual method (n=28 samples from 23 unique cases). A CV of 2.8% was observed in an intra-assay experiment (n=4 replicates from 1 case) and 3.0% in an average inter-assay experiment (n=2 replicates from 2 cases). There was a direct correlation between the tissue input amount (4 amounts of varied tissue input) and the total DNA yield (n=3 samples). We observed concordant fractional abundance values for ddPCR samples and concordant variant detection for NGS and CMA samples. We did not see a significant difference in the quality metrics tracked for any of the downstream testing assays. Conclusions This evaluation demonstrated that, as compared to our manual method, the automated method resulted in comparable DNA yields and downstream assay results. The incorporation of this automation would significantly increase our FFPE DNA extraction capacity without requiring additional staff while reducing hands-on time per run by about six hours. We would also be able to reduce ergonomic hazards related to tube cap manipulation, eliminate use of hazardous reagents, and leverage the plate format to better integrate extracted DNA into our clinical sample preparation workflows. These operational benefits highlight the value of automating high-throughput workflows.

Background Daratumumab (DARA), a human IgG1 K monoclonal antibody targeting CD38, is used to treat refractory multiple myeloma patients. CD38 is expressed on many cell types (RBCs, granulocytes, lymphocytes, etc.), and thus, DARA can interfere with serological tests. Information regarding how DARA affects anti‐granulocyte antibody (AGA) testing and optimal neutralization of DARA will help laboratories perform accurate testing. Methods Screening of AGA was performed by the granulocyte agglutination test (GAT) and the flow cytometric granulocyte immunofluorescence test (Flow‐GIFT). Samples were tested from patients on DARA ( n = 7), non‐transfused blood donors (healthy controls, n = 7) and AGA reactive samples (positive controls, n = 5). Two neutralization experiments, CD38 removal with DTT and DARA epitope blockage with mouse anti‐CD38, were evaluated. Results Positive reactivity of human IgG binding was observed in 5/7 DARA cases when tested by Flow‐GIFT; however, all 7 cases had negative GAT agglutination results. Further studies by Flow‐GIFT revealed DARA concentrations >0·63 μg/ml bound to granulocytes. DARA binding was negated by DTT though a reduced Flow‐GIFT sensitivity was observed in positive control samples due to increased background detection of human IgG. Mouse anti‐CD38 neutralized the detection of human IgG observed in DARA‐treated patient serum without effecting controls. Conclusion We established that DARA can interfere with AGA testing, leading to false positive Flow‐GIFT results without causing GAT agglutination. DTT treatment increased background binding of secondary antibodies causing a decrease in Flow‐GIFT sensitivity. In comparison, blockage of the DARA binding epitope using mouse anti‐CD38 antibody was effective in neutralizing DARA interference while maintaining Flow‐GIFT sensitivity.

OBJECTIVES: Pancreatic cystic lesions (PCLs) may be precancerous. Those likely to harbor high-grade dysplasia (HGD) or pancreatic cancer (PC) are targets for surgical resection. Current algorithms to predict advanced neoplasia (HGD/PC) in PCLs lack diagnostic accuracy. In pancreatic tissue and cyst fluid (CF) from PCLs, we sought to identify and validate novel methylated DNA markers (MDMs) that discriminate HGD/PC from low-grade dysplasia (LGD) or no dysplasia (ND). METHODS: From an unbiased whole-methylome discovery approach using predefined selection criteria followed by multistep validation on case (HGD or PC) and control (ND or LGD) tissues, we identified discriminant MDMs. Top candidate MDMs were then assayed by quantitative methylation-specific polymerase chain reaction on archival CF from surgically resected PCLs. RESULTS: Of 25 discriminant MDMs identified in tissue, 13 were selected for validation in 134 CF samples (21 cases [8 HGD, 13 PC], 113 controls [45 ND, 68 LGD]). A tree-based algorithm using 2 CF-MDMs ( TBX15 , BMP3 ) achieved sensitivity and specificity above 90%. Discrimination was significantly better by this CF-MDM panel than by mutant KRAS or carcinoembryonic antigen, with areas under the receiver operating characteristic curve of 0.93 (95% confidence interval: 0.86–0.99), 0.71 (0.57–0.85), and 0.72 (0.60–0.84), respectively. Cutoffs for the MDM panel applied to an independent CF validation set (31 cases, 56 controls) yielded similarly high discrimination, areas under the receiver operating characteristic curve = 0.86 (95% confidence interval: 0.77–0.94, P = 0.2). DISCUSSION: Novel MDMs discovered and validated in tissue accurately identify PCLs harboring HGD/PC. A panel of 2 MDMs assayed in CF yielded results with potential to enhance current risk prediction algorithms. Prospective studies are indicated to optimize and further evaluate CF-MDMs for clinical use.

Discovery of a novel HLA‐B*45:22 allele in a Middle Eastern heart and lung transplant candidate.

Early detection improves hepatocellular carcinoma (HCC) outcomes, but better noninvasive surveillance tools are needed. We aimed to identify and validate methylated DNA markers (MDMs) for HCC detection. Reduced representation bisulfite sequencing was performed on DNA extracted from 18 HCC and 35 control tissues. Candidate MDMs were confirmed by quantitative methylation‐specific PCR in DNA from independent tissues (74 HCC, 29 controls). A phase I plasma pilot incorporated quantitative allele‐specific real‐time target and signal amplification assays on independent plasma‐extracted DNA from 21 HCC cases and 30 controls with cirrhosis. A phase II plasma study was then performed in 95 HCC cases, 51 controls with cirrhosis, and 98 healthy controls using target enrichment long‐probe quantitative amplified signal (TELQAS) assays. Recursive partitioning identified best MDM combinations. The entire MDM panel was statistically cross‐validated by randomly splitting the data 2:1 for training and testing. Random forest (rForest) regression models performed on the training set predicted disease status in the testing set; median areas under the receiver operating characteristics curve (AUCs; and 95% confidence interval [CI]) were reported after 500 iterations. In phase II, a six‐marker MDM panel (homeobox A1 [HOXA1], empty spiracles homeobox 1 [EMX1], AK055957 , endothelin‐converting enzyme 1 [ECE1], phosphofructokinase [PFKP], and C‐type lectin domain containing 11A [CLEC11A]) normalized by beta‐1,3‐galactosyltransferase 6 (B3GALT6) level yielded a best‐fit AUC of 0.96 (95% CI, 0.93‐0.99) with HCC sensitivity of 95% (88%‐98%) at specificity of 92% (86%‐96%). The panel detected 3 of 4 (75%) stage 0, 39 of 42 (93%) stage A, 13 of 14 (93%) stage B, 28 of 28 (100%) stage C, and 7 of 7 (100%) stage D HCCs. The AUC value for alpha‐fetoprotein (AFP) was 0.80 (0.74‐0.87) compared to 0.94 (0.9‐0.97) for the cross‐validated MDM panel (P < 0.0001). Conclusion : MDMs identified in this study proved to accurately detect HCC by plasma testing. Further optimization and clinical testing of this promising approach are indicated.

A new allele, now named HLA‐C*02:138, was discovered during testing of a registry donor for possible stem cell transplantation.

Abstract Purpose: Gastric adenocarcinoma is the third most common cause of cancer mortality worldwide. Accurate and affordable noninvasive detection methods have potential value for screening and surveillance. Herein, we identify novel methylated DNA markers (MDM) for gastric adenocarcinoma, validate their discrimination for gastric adenocarcinoma in tissues from geographically separate cohorts, explore marker acquisition through the oncogenic cascade, and describe distributions of candidate MDMs in plasma from gastric adenocarcinoma cases and normal controls. Experimental Design: Following discovery by unbiased whole-methylome sequencing, candidate MDMs were validated by blinded methylation-specific PCR in archival case–control tissues from U.S. and South Korean patients. Top MDMs were then assayed by an analytically sensitive method (quantitative real-time allele-specific target and signal amplification) in a blinded pilot study on archival plasma from gastric adenocarcinoma cases and normal controls. Results: Whole-methylome discovery yielded novel and highly discriminant candidate MDMs. In tissue, a panel of candidate MDMs detected gastric adenocarcinoma in 92% to 100% of U.S. and South Korean cohorts at 100% specificity. Levels of most MDMs increased progressively from normal mucosa through metaplasia, adenoma, and gastric adenocarcinoma with variation in points of greatest marker acquisition. In plasma, a 3-marker panel (ELMO1, ZNF569, C13orf18) detected 86% (95% CI, 71–95) of gastric adenocarcinomas at 95% specificity. Conclusions: Novel MDMs appear to accurately discriminate gastric adenocarcinoma from normal controls in both tissue and plasma. The point of aberrant methylation during oncogenesis varies by MDM, which may have relevance to marker selection in clinical applications. Further exploration of these MDMs for gastric adenocarcinoma screening and surveillance is warranted. Clin Cancer Res; 24(22); 5724–34. ©2018 AACR.