Sample collection for pre-transfusion crossmatching: Benefits of using an electronic identification system.

BACKGROUND: Transfusion of ABO blood group-mismatched blood or administration to the wrong recipient may result in fatal adverse events. To prevent these types of errors, various strategies have been employed. Recently, we developed a novel sample collection workflow for the pre-transfusion crossmatching test and patient recognition. This study aimed to analyse the usage of the new workflow and improvements in outcomes. METHODS: We analysed the number of crossmatching and wrong-patient errors among the blood transfusion cases during 3 years of data collection (from August 2018 to July 2021). From May 2021 to July 2021, the new workflow was implemented. Outcomes were calculated according to the department type, patient age and processing time. The sample processing time was defined as the time from placing the order to lab arrival. RESULTS: The new workflow utilisation increased from 50.7% to 80.3% and wrong-patient errors decreased annually. The new workflow was used for more adults (3001/3680 samples, 81.5%) than paediatric cases (345/522 samples, 65.5%; p < 0.001) and in general wards than in the emergency room or intensive care unit. The sample processing time differed according to ward type and timing of the request (day: 28.80, 2.43-3889.43 min, night: 3.36, 2.72-1671.47 min; p < 0.001). CONCLUSION: Wrong-patient errors were reduced without increasing sample-processing time after introducing the new workflow which included using an electronic identification system. The time needed for the blood processing differed according to the ward type, patient age, and timing of the request. Patient safety can be promoted by managing these factors and using an electronic identification system.

Effects of Different Centrifugation Protocols on the Detection of EGFR Mutations in Plasma Cell-Free DNA.

OBJECTIVES: Various preanalytical factors, including the collection tube, storage conditions, and centrifugation, affect the detection results of plasma cell-free DNA (cfDNA). We compared the effect of different centrifugation protocols on the detection of EGFR mutations in cfDNA. METHODS: We analyzed 117 plasma specimens from 110 patients with non-small cell lung cancer using the cobas EGFR Mutation Test v2 (Roche Diagnostics). We compared the identified EGFR mutations and semiquantitative index values from the 1- and 2-step centrifugation groups and confirmed the clinical impact of differences in the results after further high-speed centrifugation. RESULTS: We detected EGFR mutations in 44 (37.6%) and 47 (40.2%) samples that were centrifuged once and twice, respectively; the 2 groups showed an 89.7% (105/117) concordance and a strong correlation in their semiquantitative index values (r = 0.929). Among the 12 inconsistent result pairs, 9 samples of 2-step centrifugation (75%) were consistent with the results of a recent tissue biopsy. CONCLUSIONS: Additional high-speed centrifugation has been shown to increase the sensitivity of EGFR mutation detection in a commercial in vitro diagnostic real-time polymerase chain reaction device and is an optimal preanalytical factor for detecting low-allele frequency gene mutations using low concentrations of cfDNA.

Chimerism Assay Using Single Nucleotide Polymorphisms Adjacent and in Linkage-Disequilibrium Enables Sensitive Disease Relapse Monitoring after Hematopoietic Stem-Cell Transplantation.

BACKGROUND: Short tandem repeat (STR)-based chimerism analysis has been widely used for chimerism monitoring after hematopoietic stem-cell transplantation (HSCT), but technical artifacts can be problematic. We designed a chimerism assay using single nucleotide polymorphisms (SNPs) adjacent and in linkage-disequilibrium (CASAL), which doubly checked for SNP pairs, and thus could reduce background errors and increase analytical sensitivity. METHODS: CASAL targeted 84 SNP pairs within 10 bp distance and in perfect linkage-disequilibrium. Using undiluted and serially diluted samples, baseline error rates, and linearity was calculated. Clinical performance of CASAL was evaluated in comparison with a conventional STR assay, using 191 posttransplant samples from 42 patients with HSCT. RESULTS: CASAL had ∼10 times lower baseline error rates compared to that of ordinary next-generation sequencing. Limit of detection and quantification of CASAL were estimated to be 0.09 and 0.39%, respectively, with a linear range of 0.1-100%. CASAL correlated well with STR assay (r2 = 0.99) and the higher sensitivity enabled detection of low-level recipient chimerism and earlier prediction of relapse. CONCLUSIONS: CASAL is a simple, analytically sensitive and accurate assay that can be used in clinical samples after HSCT with a higher performance compared to that of traditional assays. It should also be useful in other forensic and archeological testing.

Minimal residual disease in acute lymphoblastic leukemia: technical aspects and implications for clinical interpretation.

Minimal residual disease (MRD) monitoring has proven to be one of the fundamental independent prognostic factors for patients with acute lymphoblastic leukemia (ALL). Sequential monitoring of MRD using sensitive and specific methods, such as real-time quantitative polymerase chain reaction (qPCR) or flow cytometry (FCM), has improved the assessment of treatment response and is currently used for therapeutic stratification and early detection. Although both FCM and qPCR yield highly consistent results with sensitivities of 10‒4, each method has several limitations. For example, qPCR is time-consuming and laborious: designing primers that correspond to the immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements at diagnosis can take 3‒4 weeks. In addition, the evolution of additional clones beyond the first or index clone during therapy cannot be detected, which might lead to false-negative results. FCM requires experienced technicians and sometimes does not achieve a sensitivity of 10‒4. Accordingly, a next generation sequencing (NGS)-based method has been developed in an attempt to overcome these limitations. With the advent of high-throughput NGS technologies, a more in-depth analysis of IG and/or TCR gene rearrangements is now within reach, which impacts all applications of IG/TR analysis. However, standardization, quality control, and validation of this new technology are warranted prior to its incorporation into routine practice.

Mutation analysis in Korean patients with T-cell acute lymphoblastic leukemia.

Genomic studies have illuminated the alterations in pathways underlying T-cell acute lymphoblastic leukemia (T-ALL) pathogenesis, but detailed mutation data by next-generation sequencing have not been reported in Korean patients. We aimed to investigate mutation frequency, spectrum, and pattern in the Korean patients with T-ALL. We designed a multigene panel targeting 101 genes and validated it using 10 reference materials. The mutation analysis was done in a total of 10 patients with T-ALL. Clinical data and laboratory tests including immunophenotyping, cytogenetics, and molecular genetic tests were also investigated. All of the 10 patients harbored at least one mutation (range 1-6 per patient). A total of 34 clinically significant mutations including 15 novel mutations were identified in 23 genes. The median of variant allelic frequencies (VAFs) and blasts were counted upto 33% (range 5-91%) and 79% (range 38-90%), respectively. Recurrent mutations were involved in epigenetic regulators (60%), NOTCH1 signaling (40%), PI3K-AKT (40%), JAK-STAT (30%), and transcription factors (30%). We found that both NOTCH signaling and JAK-STAT signaling were positively associated with epigenetic regulators, while showed mutually exclusive patterns with PI3K-AKT pathway. This study showed that the frequency of mutations in epigenetic regulators in Korean patients was significantly higher than expected. Distribution of VAF as well as mutation spectrum is considerably heterogeneous in Korean patients with T-ALL. Although from a limited number of patients, this study provides the first detailed mutational portrait of T-ALL of Korean patients, and gives additional insight into molecular pathogenesis of the disease.

T-Cell Receptor Rearrangements Determined Using Fragment Analysis in Patients With T-Acute Lymphoblastic Leukemia.

BACKGROUND: Chromosomal abnormalities and common genetic rearrangements related to T-acute lymphoblastic leukemia (T-ALL) are not clear. We investigated T-cell receptor (TCR) rearrangement in Korean T-ALL patients by fragment analysis, examining frequency, association between clinicopathologic characteristics and TCR clonality, and feasibility for detecting minimal residual disease (MRD). METHODS: In 51 Korean patients diagnosed as having T-ALL, TCR rearrangement was analyzed using the IdentiClone TCR gene clonality assay (InVivoScribe Technologies, San Diego, CA, USA) from archived bone marrow specimens. Limit of detection (LOD) and clonal stability at relapse were evaluated. The association between clinical prognosis and TCR clonality was examind by age and immunophenotypic classification. RESULTS: Thirty-eight patients (74.5%) had 62 clonal products of TCRß, TCRγ, and/or TCRδ rearrangements at diagnosis. Children with T-ALL (<12 years) showed a higher frequency of clonality (93.8%) than adolescents/adults (65.7%; ≥12 years). Patients with a mature immunophenotype (84.4%) showed a relatively higher frequency of clonality than those with the immature immunophenotype (57.9%). Survival and event-free survival were not influenced by immunophenotype or TCR clonality. The LOD was 1%. Clonal evolution at the relapse period was noted. CONCLUSIONS: The overall detection rate of TCR clonality was 74.5%. Survival did not differ by TCR clonality or immunophenotype and age group. Fragment analysis of TCR rearrangement cannot be used to assess MRD due to low sensitivity. Further research on the relationship between prognosis and frequency of TCR rearrangements is needed, using more sensitive methods to detect clonality and monitor MRD.

MLL leukemia induction by t(9;11) chromosomal translocation in human hematopoietic stem cells using genome editing.

Genome editing provides a potential approach to model de novo leukemogenesis in primary human hematopoietic stem and progenitor cells (HSPCs) through induction of chromosomal translocations by targeted DNA double-strand breaks. However, very low efficiency of translocations and lack of markers for translocated cells serve as barriers to their characterization and model development. Here, we used transcription activator-like effector nucleases to generate t(9;11) chromosomal translocations encoding MLL-AF9 and reciprocal AF9-MLL fusion products in CD34+ human cord blood cells. Selected cytokine combinations enabled monoclonal outgrowth and immortalization of initially rare translocated cells, which were distinguished by elevated MLL target gene expression, high surface CD9 expression, and increased colony-forming ability. Subsequent transplantation into immune-compromised mice induced myeloid leukemias within 48 weeks, whose pathologic and molecular features extensively overlap with de novo patient MLL-rearranged leukemias. No secondary pathogenic mutations were revealed by targeted exome sequencing and whole genome RNA-sequencing analyses, suggesting the genetic sufficiency of t(9;11) translocation for leukemia development from human HSPCs. Thus, genome editing enables modeling of human acute MLL-rearranged leukemia in vivo, reflecting the genetic simplicity of this disease, and provides an experimental platform for biological and disease-modeling applications.

Clinical Pharmacogenetic Testing and Application: Laboratory Medicine Clinical Practice Guidelines.

Pharmacogenetic testing for clinical applications is steadily increasing. Correct and adequate use of pharmacogenetic tests is important to reduce unnecessary medical costs and adverse patient outcomes. This document contains recommended pharmacogenetic testing guidelines for clinical application, interpretation, and result reporting through a literature review and evidence-based expert opinions for the clinical pharmacogenetic testing covered by public medical insurance in Korea. This document aims to improve the utility of pharmacogenetic testing in routine clinical settings.

Correlation of NPM1 Type A Mutation Burden With Clinical Status and Outcomes in Acute Myeloid Leukemia Patients With Mutated NPM1 Type A.

BACKGROUND: Nucleophosmin gene (NPM1) mutation may be a good molecular marker for assessing the clinical status and predicting the outcomes in AML patients. We evaluated the applicability of NPM1 type A mutation (NPM1-mutA) quantitation for this purpose. METHODS: Twenty-seven AML patients with normal karyotype but bearing the mutated NPM1 were enrolled in the study, and real-time quantitative PCR of NPM1-mutA was performed on 93 bone marrow (BM) samples (27 samples at diagnosis and 56 at follow-up). The NPM1-mutA allele burdens (represented as the NPM1-mutA/Abelson gene (ABL) ratio) at diagnosis and at follow-up were compared. RESULTS: The median NPM1-mutA/ABL ratio was 1.3287 at diagnosis and 0.092 at 28 days after chemotherapy, corresponding to a median log10 reduction of 1.7061. Significant correlations were observed between BM blast counts and NPM1-mutA quantitation results measured at diagnosis (γ=0.5885, P=0.0012) and after chemotherapy (γ=0.5106, P=0.0065). Total 16 patients achieved morphologic complete remission at 28 days after chemotherapy, and 14 (87.5%) patients showed a >3 log10 reduction of the NPM1-mutA/ABL ratio. The NPM1-mutA allele was detected in each of five patients who had relapsed, giving a median increase of 0.91-fold of the NPM1-mutA/ABL ratio at relapse over that at diagnosis. CONCLUSIONS: The NPM1-mutA quantitation results corresponded to BM assessment results with high stability at relapse, and could predict patient outcomes. Quantitation of the NPM1-mutA burden at follow-up would be useful in the management of AML patients harboring this gene mutation.

Bone marrow findings in severe fever with thrombocytopenia syndrome: prominent haemophagocytosis and its implication in haemophagocytic lymphohistiocytosis.

AIMS: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by the SFTS virus; primary manifestations are fever, thrombocytopenia, leukopenia and gastrointestinal symptoms. Before an aetiological diagnosis is made, SFTS patients can undergo bone marrow examination due to cytopenias. Although several studies have reported on bone marrow examination in SFTS patients, most do not provide adequate details. Bone marrow findings in SFTS patients were investigated in this study. METHODS: An observational study was conducted in SFTS patients who were hospitalised between 2013 and 2014 in two university hospitals in South Korea. Patients were included in the study if SFTS was confirmed by real-time PCR for the SFTS virus and a bone marrow examination was conducted. The morphologic findings of the bone marrow samples were reviewed. RESULTS: Three cases met the study inclusion criteria. One patient died of multiple organ failure. Haemophagocytosis was evident in the bone marrow samples of all three patients. Histiocytic hyperplasia and haemophagocytosis were more pronounced in the fatal case. One patient was diagnosed as having haemophagocytic lymphohistiocytosis. CONCLUSIONS: Haemophagocytosis in the bone marrow of SFTS patients may be common. In SFTS endemic areas, SFTS should be one of the differential diagnoses of fever of unknown origin with haemophagocytosis in the bone marrow.

High total metabolic tumor volume in PET/CT predicts worse prognosis in diffuse large B cell lymphoma patients with bone marrow involvement in rituximab era.

Bone marrow involvement (BMI) in diffuse large B cell lymphoma (DLBCL) was naively regarded as an adverse clinical factor. However, it has been unknown which factor would separate clinical outcomes in DLBCL patients with BMI. Recently, metabolic tumor volume (MTV) on positron emission tomography/computed tomography (PET/CT) was suggested to predict prognosis in several lymphoma types. Therefore, we investigated whether MTV would separate the outcomes in DLBCL patients with BMI. MTV on PET/CT was defined as an initial tumor burden as target lesion ≥ standard uptake value, 2.5 in 107 patients with BMI. Intramedullary (IM) MTV was defined as extent of BMI and total MTV was as whole tumor burden. 260.5 cm(3) and 601.2 cm(3) were ideal cut-off values for dividing high and low MTV status in the IM and total lymphoma lesions in Receiver Operating Curve analysis. High risk NCCN-IPI (p<0.001, p<0.001), bulky disease (p=0.011, p=0.005), concordant subtype (p=0.025, p=0.029), high IM MTV status (p<0.001, p<0.001), high total MTV status (p<0.001, p<0.001), and ≥ 2CAs in BM (p=0.037, p=0.033) were significantly associated with progression-free survival (PFS) and overall survival (OS) than other groups. In multivariate analysis, high risk NCCN-IPI (PFS, p=0.006; OS, p=0.013), concordant subtype (PFS, p=0.005; OS, p=0.007), and high total MTV status (PFS, p<0.001; OS, p<0.001) had independent clinical impacts. MTV had prognostic significances for survivals in DLBCL with BMI.

Antiplatelet effect of ticagrelor compared to tirofiban in non-ST-segment elevation ACS patients undergoing PCI. The result of the TE-CLOT trial.

Addition of a potent P2Y12 inhibitor to aspirin is the standard therapy for non-ST-segment elevation acute coronary syndrome (NSTE-ACS) patients undergoing percutaneous coronary intervention (PCI). Glycoprotein IIb/IIIa inhibitor, together with antiplatelet therapy, may be considered as part of initial therapy in NSTE-ACS patients with high-risk features. This study investigated the antiplatelet effect of ticagrelor loading dose (LD) versus tirofiban bolus injection with a post-bolus infusion on top of aspirin among NSTE-ACS patients planned to PCI. NSTE-ACS patients were randomised to receive either ticagrelor (n = 47) or tirofiban (n = 48). Platelet reactivity was assessed by light transmittance aggregometry at 0, 2, 8, and 24 hours (h) after treatment initiation. Primary endpoint was inhibition of platelet aggregation (IPA, 20 µM ADP, final extent) at 2 h after LD therapy, with a non-inferiority margin of 10%. The prevalence of high on-treatment platelet reactivity (HPR) was also compared at 0, 2, 8, and 24 h. The mean difference in IPA between ticagrelor and tirofiban was -9.9% (95% confidence interval: -25.7% to 5.9%) at 2 h, -1.6% (-8.0% to 4.8%) at 8 h, and -3.3% (-18.4% to 12.0%) at 24 h. The prevalence of HPR did not differ between the two groups at any time point (all p values ≥ 0.059), which was almost abolished by 8 h post-LD (< 5%). In conclusion, the antiplatelet effect during the early phase (~2 h) after ticagrelor LD appeared to be relatively strong, but it did not reach that of tirofiban in NSTE-ACS patients.

Incidences and Prognostic Impact of c-KIT, WT1, CEBPA, and CBL Mutations, and Mutations Associated With Epigenetic Modification in Core Binding Factor Acute Myeloid Leukemia: A Multicenter Study in a Korean Population.

BACKGROUND: To identify potential molecular prognostic markers in core binding factor (CBF) AML, we analyzed incidences and prognostic impacts of mutations in c-KIT, WT1, CEBPA, CBL, and a number of epigenetic genes in CBF AML. METHODS: Seventy one and 21 AML patients with t(8;21) and inv(16) were enrolled in this study, respectively. NPM1, CEBPA, c-KIT, IDH1/2, DNMT3A, EZH2, WT1, and CBL mutations were analyzed by direct sequencing. Patients were categorized with respect to c-KIT and WT1 mutation status, and both clinical features and prognoses were compared. RESULTS: The incidences of FLT3 internal tandem duplication (ITD), NPM1, CEBPA, IDH1/2, DNMT3A, EZH2, and CBL mutations were low (≤5%) in CBF AML patients. However, c-KIT and WT1 mutations occurred frequently (10.9% and 13.8%, respectively). t(8;21) patients with c-KIT mutations showed significantly shorter overall survival (OS) and disease free survival (DFS) periods than those without mutations (P<0.001, for both); however, although the limited number of t(8;21) patients were analyzed, WT1 mutation status did not affect prognosis significantly. Relapse or death during follow-up occurred more frequently in t(8;21) patients carrying c-KIT mutations than in those without the mutation, although the difference was significant only in a specific patient subgroup with no WT1 mutations (P=0.014). CONCLUSIONS: The incidences of mutations in epigenetic genes are very low in CBF AML; however, c-KIT and WT1 mutations occur more frequently than others. The poor prognostic impact of c-KIT mutation in t(8;21) AML patients only applies in a specific patient subgroup without WT1 mutations. The prognostic impact of WT1 mutation in CBF AML is not evident and further investigation is required.