Emergence of Monosomy 7 in Philadelphia-Negative Cells during MDS Development and not CML Diagnosis Proved by Serial Droplet Digital PCR

Emergence of new clonal chromosomal abnormality (CCA) has been reported in Philadelphia-negative cells in patients with chronic myeloid leukemia (CML) undergoing the tyrosine kinase inhibitor (TKI) treatment. However, the time of emergence and clinical significance of CCA remains to be elucidated. In this study, we report a CML patient undergoing TKI treatment who developed myelodysplastic syndrome (MDS) after 206 months since the diagnosis of CML. Results of droplet digital PCR performed with serial bone marrow samples revealed that monosomy 7 in Philadelphia-negative cells appeared at the time of MDS development that did not exist initially at the time of CML diagnosis.

Emergence of Monosomy 7 in Philadelphia-Negative Cells during MDS Development and not CML Diagnosis Proved by Serial Droplet Digital PCR

Emergence of new clonal chromosomal abnormality (CCA) has been reported in Philadelphia-negative cells in patients with chronic myeloid leukemia (CML) undergoing the tyrosine kinase inhibitor (TKI) treatment. However, the time of emergence and clinical significance of CCA remains to be elucidated. In this study, we report a CML patient undergoing TKI treatment who developed myelodysplastic syndrome (MDS) after 206 months since the diagnosis of CML. Results of droplet digital PCR performed with serial bone marrow samples revealed that monosomy 7 in Philadelphia-negative cells appeared at the time of MDS development that did not exist initially at the time of CML diagnosis.

Performance of Xpert Carba-R Assay for Identification of Carbapenemase Gene in the Clinical Microbiology Laboratory / 이화의대지

Objectives@#The Xpert Carba-R Assay is a diagnostic test designed for the rapid detectionand differentiation of the blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP-1 genes. We verifiedthe performance of Xpert Carba-R Assay for identification of carbapenemase genein the clinical microbiology laboratory. @*Methods@#The analytical limit of detection was determined with two suspensions ofcarbapenemase-producing Enterobacteriaceae (CPE) isolates (KPC and NDM). A totalof 52 specimens were evaluated: 21 bacterial isolates from clinical specimens, 21 rectalswabs, and 10 contrived stool specimens. @*Results@#In bacterial isolates, concordant results between the Xpert Carba-R Assayand PCR were found in 20 of 21; 8 KPC, 8 NDM, 1 IMP, and 2 multiple carbapenamasegenes (KPC/NDM, NDM/OXA) were detected both by Xpert Carba-R Assay and PCR.In one GES-positive isolate, Xpert Carba-R Assay showed a negative result as expected.One VIM-positive isolate tested negative by Xpert Carba-R Assay. Complete concordancewas seen in rectal swab specimens: 4 specimens with KPC and 17 specimenswith negative results both by Xpert Carba-R Assay and surveillance culture. Among the10 contrived stool specimens, Xpert Carba-R Assay detected carbapenemase genes in9 specimens as expected according to the CPE strains spiked into the contrived stool; 2KPC, 4 NDM, 1 IMP, and 2 multiple carabapenamase genes (NDM/KPC, NDM/OXA).One VIM-positive specimen tested negative by Xpert Carba-R Assay. @*Conclusion@#In conclusion, the Xpert Carba-R Assay can be used to identify carbapenemasegene in bacterial isolates cultured from clinical specimens and detect CPE carrierusing rectal swab in clinical laboratories.

Application of Six Sigma Metrics to Improve Quality Control for Point-of-care Glucose Testing / 이화의대지

Objectives@#Six sigma is a quality management system for the assessment of precisionand accuracy. We aim to apply the six sigma rule to quality control (QC) of point-of-care(POC) glucose meters in a tertiary hospital. @*Methods@#Thirty POC glucose meters installed at Ewha Womans University MokdongHospital were monitored between January 2013 and March 2014. The QC data fromthe POC glucose meters at low and high levels were collected. The monthly mean, standarddeviation, bias, coefficient of variation, and mean sigma metrics were calculated.The correlation between accuracy and precision was assessed based on the percentagebias and coefficient of variation. Comprehensive instructions on the QC and maintenanceof the devices were provided in the departments with poor sigma scores. Afollow-up assessment was performed after the intervention. @*Results@#The mean sigma values for the low and high controls were 3.29 and 3.71, respectively.At the low and high controls, 36.6% and 10% of the glucose meters showeda sigma value <3. The causes of low sigma values included the use of expired controlmaterials, prolonged air exposure of the sample strip, lack of user training, and errors indevice maintenance. On follow-up monitoring for 3 months following QC intervention,23.3% (low control) and 6.6% (high control) of the glucose meters scored a sigma value<3, indicating improved QC. @*Conclusion@#Sigma metrics-based QC can successfully improve accuracy and precisionof POC glucose meters in an objective and quantitative manner and can be usedfor follow up after QC intervention.

Analytical Performance of INNOVANCE Free Protein S Antigen on Sysmex CS-5100

BACKGROUND: Protein S deficiency is a common cause of thrombophilia. Free protein S has been suggested as one of the best screening tests for this deficiency. We evaluated an immunoturbidimetric free protein S reagent, INNOVANCE Free Protein S Antigen (Free PS Ag; Siemens Healthcare Diagnostics, Germany), using a CS-5100 coagulation analyzer (Sysmex, Japan). METHODS: The performance of INNOVANCE Free PS Ag was evaluated according to the CLSI guidelines. Precision, linearity, and verification of reference intervals were examined. The INNOVANCE Free PS Ag was also compared by the STA-Liatest Free Protein S immunoturbidimetric assay (Diagnostica Stago, France). RESULTS: The repeatability and within-laboratory imprecision of INNOVANCE Free PS Ag were 0.8% CV and 2.0% CV at the normal level, and 1.3% CV and 2.3% CV at the abnormally low level, respectively. This assay showed linearity from 4.0% to 151.9% (correlation coefficient r=1, P < 0.0001). Reference intervals for males and females were verified as acceptable. INNOVANCE Free PS Ag was comparable with STA-Liatest Free Protein S with a very high correlation (r=0.935, P < 0.0001). The results for the INNOVANCE antigen were higher. CONCLUSIONS: The INNOVANCE Free PS Ag on a Sysmex CS-5100 coagulation analyzer has excellent analytical performance and is comparable with the STA-Liatest Free Protein S assay.

Effects of Pre-analytical Variables on Cell-free DNA Extraction for Liquid Biopsy

BACKGROUND: Extraction of cell-free DNA (cfDNA) is a key step for determining the quality of cfDNA-related molecular diagnostics. We evaluated the effect of sample containers and sample storage conditions on cfDNA extraction. METHODS: The cfDNA extraction using the MagMAX Cell-Free DNA Isolation Kit from five healthy controls and five lung cancer patients was evaluated according to the type of sample container and storage conditions: K2-EDTA container, <1, 6, 24, and 48 hr storage at 4℃ after immediate plasma separation; and Cell-Free DNA BCT container, <1, 3, 7, and 14 days stored at room temperature. Mutation analysis of EGFR exons 18–21 was performed. To assess the effect of a delay in centrifugation, EDTA whole blood samples from five healthy individuals were stored at 4℃ for 6, 12, and 24 hr before plasma separation. RESULTS: There was no significant difference in the amount and nucleic acid size of cfDNA in both controls and patients with cancer when EDTA plasma was stored at 4℃ up to 48 hr. The amount and size of cfDNA in the BCT container were not different up to 7 days; however, the 14-day sample showed an increase in cfDNA concentration due to genomic DNA contamination. EGFR mutations were detected on EDTA containers up to 48 hr and with BCT containers up to 14 days. When EDTA whole blood was stored at 4℃ and plasma separation was delayed, the cfDNA concentration increased from 24 hr. CONCLUSIONS: The cfDNA extraction was affected by the sample containers and storage conditions.

A Clue to Discovering Unstable Hemoglobin Variants via Abnormal WBC Differential Scattergrams Using the Sysmex Automated Hematology Analyzer

Incidentally, hemoglobin (Hb) variants can be detected using HbA1c tests in clinical laboratories. We found 38 patients with Hb variants after reviewing a total of 29,398 HbA1c test results from January 2017 to December 2017. While reviewing the complete blood count results of the patients (N=36) using the Sysmex XN-9000 analyzer (Sysmex, Japan), 35 patients were flagged as unremarkable with respect to differential white blood cell (WBC) counts. However, 1 patient with a normal WBC count did not obtain a differential WBC count while being flagged for an abnormal WBC scattergram in the white blood cell differential (WDF) channel. The WBC histogram showed an abnormally low fluorescent signal in the WDF channel; however, the differential WBC count was normal upon microscopic examination. After testing the patient's buffy coat suspended in normal saline and removing red blood cells (RBCs), the WBC scattergram and differential WBC count returned to normal. This finding suggests that the presence of a patient's RBCs may affect WBC scattergrams and Hb variants may interfere with the fluorescent dye in the differential WBC count. Therefore, when an abnormal WBC scattergram with an abnormally low fluorescent signal is encountered on the Sysmex XN-9000 analyzer, the presence of an Hb variant can be suspected.

Estimated Glomerular Filtration Rates Show Minor but Significant Differences Between the Single and Subgroup Creatinine-Based Chronic Kidney Disease Epidemiology Collaboration Equations

The creatinine-based Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation can be calculated according to race, sex, and creatinine concentration (subgroup equation) or in the form expressed by one equation (single equation). Minor differences in the constants used in the CKD-EPI equations (subgroup vs single equations) could result in a significant difference in the estimated glomerular filtration rate (eGFR). We evaluated the impact of this difference in 79,709 Korean patients. The eGFR was calculated as an integer using the single and subgroup CKD-EPI equations. The differences in eGFR and GFR categories between the equations were analyzed. eGFR was higher in the subgroup equation than the single equation by 1 mL/min/1.73 m² for 12,476 (27.4%) Korean females. The GFR category based on the subgroup equation was reclassified using the single equation for 352 (0.77%) females. Based on the results, the constant of the single equation was optimized. There was no difference in eGFR values between equations using a multiplier of 1.0213 instead of 1.018 for the “white or other” females constant in the single CKD-EPI equation. Clinicians should carefully apply the CKD-EPI equation because eGFR values may differ by 1 mL/min/1.73 m² depending on the manner of calculation. To minimize these differences, the constants of the single equation should be revised.

Variable Natural Killer Cell Activity in Hematological Malignancies at Diagnosis

BACKGROUND: Natural killer (NK) cells play a key role in innate immune responses and are an important component of anti-cancer defenses. This study aimed to investigate the clinicopathological characteristics of NK cell activity (NKA) among various hematological malignancies at diagnosis and to evaluate their clinical value as a monitoring marker. METHODS: A total of 111 patients that were newly diagnosed with hematological malignancies were recruited, comprising 18 acute myeloid leukemia (AML), 31 multiple myeloma (MM), and 62 lymphoma. Twenty-three normal control subjects from our health examination center were recruited. NKA was measured using a commercially available enzyme-linked immunosorbent assay kit, which measures interferon-gamma secreted by ex vivo-stimulated NK cells in whole blood. RESULTS: The 111 patients had a median NKA of 202.80 pg/mL (range 40–2,000). NKA was significantly decreased in patients with AML (median 47.05 pg/mL, 40–2,000, P<0.0001), MM (275.00, 40–2,000, P<0.0001), and lymphoma (289.49, 40–2,000, P<0.0001) compared with that in normal controls (1,891, 412–2,000). There was a difference in NKA between AML and lymphoma (P=0.0499). Serial changes in NKA correlated with disease progression. NKA did not correlate with the NK cell count in any group of hematological malignancies. CONCLUSIONS: The measurement of NKA could be useful to evaluate the immunological status in hematological malignancies at diagnosis and during follow-up.

Anaplastic Large Cell Lymphoma with Massive Eosinophilia and Complex Karyotype Initially Misdiagnosed as Chronic Eosinophilic Leukemia

We report a patient with massive eosinophilia and a complex karyotype that was initially misdiagnosed as chronic eosinophilic leukemia (CEL), but later diagnosed as anaplastic large cell lymphoma (ALCL) masked by massive eosinophilia. The complex karyotype observed at initial diagnosis remained unchanged later, after the evidence of bone marrow involvement of ALCL was obtained. At diagnosis, genetic aberrations corresponding to metaphase cytogenetics were not identified by interphase fluorescence in situ hybridization, although abnormal results were noted at follow-up. Together, these observations indicate that the complex karyotype at initial work-up has been derived from a low proportion of lymphoma cells with high mitotic ability that were not identified by microscopy, rather than from massive eosinophils. These findings suggest that our patient had ALCL with secondary eosinophilia rather than CEL since initial diagnosis.

GATA1 Expression in BCR/ABL1-negative Myeloproliferative Neoplasms

BACKGROUND: This study aimed to determine GATA1 expression levels to better characterize subgroups in BCR/ABL1-negative myeloproliferative neoplasms (MPNs). METHODS: This study enrolled 49 patients diagnosed as having BCR/ABL1-negative MPN on the basis of the 2016 World Health Organization classification : nine polycythemia vera (PV), 17 essential thrombocythemia (ET), 12 prefibrotic primary myelofibrosis (prePMF), and 11 overt primary myelofibrosis (PMF). Relevant clinical and laboratory data were retrieved from the medical records. The molecular analysis of CALR and MPL mutations and quantification of JAK2 V617F allele burden were performed. GATA1 expression was assessed by an immunohistochemical assay on bone marrow biopsy. GATA1 expression was analyzed serially in 18 patients. RESULTS: GATA1 expression decreased significantly in PMF compared with that in other subtypes, while no statistical difference was identified between ET and prePMF. GATA1 expression did not differ according to the mutation profiles or the allele burden of JAK2 V617F, but it decreased significantly in patients with overt fibrosis or leukemic transformation. CONCLUSIONS: Our results suggest that GATA1 expression is significantly low in PMF and decreases with progressive fibrosis and possibly with leukemic transformation, although our attempt to accurately distinguish between subgroups using GATA1 immunohistochemical approach did not achieve statistical significance. A large patient cohort with long term follow-up is required to evaluate the prognostic value of GATA1 expression.

Neutrophilic Leukemoid Reaction Associated with Malignancy Initially Suspected as Chronic Neutrophilic Leukemia

Although neutrophilia can manifest from various causes, it is important to be able to distinguish chronic neutrophilic leukemia (CNL) from neutrophilic leukemoid reactions (NLR). In this paper, we describe four cases of leukocytosis with neutrophilia, including one case of CNL with a T618I mutation in colony stimulating factor 3 receptor (CSF3R) and three cases of NLR associated with malignancy or sepsis, which were initially suspected as CNL. Of the three NLR cases, one was associated with ovarian cancer, one with monoclonal gammopathy of undetermined significance and one with multiple myeloma with sepsis. This study demonstrated that confirming the clonality of myeloid cells with CSF3R T618I could contribute to making an accurate differential diagnosis between CNL and NLR in patients with solid cancers or plasma cell neoplasms caused by paraneoplastic syndromes and/or infection.

Two Cases of Medical Device-Related Corynebacterium striatum Infection: A Meningitis and A Sepsis / 대한임상미생물학회지

Corynebacterium striatum is a commonly isolated contaminant in the clinical microbiology. However, it can be an opportunistic pathogen in immunocompromised and even immunocompetent hosts. The increasing prevalence of C. striatum infection has been associated with immunosuppression and prosthetic devices. We report a case of meningitis with cerebrospinal fluid drainage and a case of catheter-related bloodstream infection caused by C. striatum. The isolates were identified as nondiphtherial Corynebacterium species by VITEK 2 (bioMérieux, France) anaerobe and Corynebacterium card. The final identification by 16S rRNA gene sequencing analysis was C. striatum with 99.7% identity and 99.6% identity with C. striatum ATCC 6940, respectively. Both strains were sensitive to vancomycin and gentamicin, but multidrug-resistant to ciprofloxacin, penicillin, erythromycin and imipenem.

Diagnostic Utility of Serum Glycated Albumin for Diabetes Mellitus and Its Correlation With Hyperlipidemia

BACKGROUND: Glycated albumin (GA) is a better marker of short-term glycemic control than glycated hemoglobin (A1c). Dyslipidemia is the main cause of cardiovascular complications in diabetes mellitus (DM). Studies on the correlation of GA with lipid indices are sparse. We investigated the diagnostic utility of GA for DM and its relationship with serum lipid profiles compared with that of A1c. METHODS: The GA enzymatic method was used to determine the diagnostic utility of GA for DM by using samples from 163 normal subjects (group 1) and 102 patients newly diagnosed with type 2 DM (T2DM; group 2). To analyze the lipid profiles, 263 patients with T2DM receiving treatment (group 3) were recruited. RESULTS: GA correlated with A1c (r=0.934, P<0.0001). Linear regression analysis indicated that GA levels were about 2.48 folds those of A1c. In the ROC analysis for GA to diagnose DM, the areas under the curve (0.988, 95% confidence interval 0.972-1.004) was excellent. HDL levels were significantly lower in groups 2 and 3. In group 1, positive correlations were observed between A1c and triglyceride (TG), total cholesterol (TC), LDL, TG/HDL, TC/HDL, and LDL/HDL levels. A negative correlation was observed between HDL and A1c levels. In group 3, HDL levels (P=0.0124 and P=0.0141, respectively) were significantly higher and LDL levels tended to be lower, not statistically significant, in the well-controlled group categorized using the A1c and GA cut-off values. CONCLUSIONS: GA is a potential diagnostic tool for DM. Compared with A1c, GA seems less relevant to dyslipidemia.

Therapeutic Plasma Exchange for a Patient with Severe Cold Agglutinin Disease / 대한수혈학회지

Cold agglutinin disease is a kind of autoimmune hemolytic anemia, caused by cold agglutinin, serum autoantibodies activated at reduced body temperatures to produce red blood cell agglutination and hemolysis. In this paper we described a case of severe hemolytic anemia in a cold agglutinin disease patient treated with therapeutic plasma exchange. Therapeutic plasma exchanges were performed four times every other day. Over the same period, a total of 8 units of washed red blood cells were transfused. Then hemoglobin was increased from 4.0 g/dL to 7.8 g/dL. On the 12th hospital day hemoglobin level was decreased again to 4.2 g/dL and fludarabine chemotherapy was started on the 14th hospital day. The patient's symptoms were relieved and she was discharged on the 30th hospital day. As in this case, therapeutic plasma exchange could be considered as secondary therapy for temporary improvement of acute severe hemolytic anemia in cold agglutinin disease.

Evaluation of the Performance of a New Chromatographic Assay BD Veritor System for Rapid Detection of Influenza A & B / 대한임상미생물학회지

BACKGROUND: For early diagnosis and treatment of influenza, rapid influenza diagnostic tests are commonly used. We evaluated the performance of the BD Veritor System for Rapid Detection of Flu A+B (BD Veritor System; BD Diagnostics, USA) compared to multiplex real-time RT-PCR. METHODS: A total of 3,213 nasal and nasopharyngeal swab specimens in transport media from patients with influenza-like symptoms were tested with the BD Veritor System from December 2013 to April 2014. The sensitivity and specificity of 127 specimens were determined simultaneously using multiplex real-time RT- PCR with the AdvanSure RV real-time PCR (AdvanSure PCR; LG Life Sciences, Korea). RESULTS: Influenza viruses were detected in 41.3% (1,327/3,213) of all specimens tested using the BD Veritor System. Of the 127 specimens, 27 influenza A and 17 influenza B viruses were identified by the AvanSure PCR. The sensitivity and specificity of the BD Veritor System relative to the AdvanSure PCR was 85.2% and 99.0% for influenza A, and 58.8% and 99.1% for influenza B. Of the 190 specimens that tested negative using the BD Veritor System, the AdvanSure PCR detected influenza A and influenza B in 19 and 13 specimens, respectively. The mean threshold cycle (Ct) values of the antigen positive specimens were lower than those of the antigen negative specimens. CONCLUSION: The BD Veritor System showed excellent specificity for both influenza types and good sensitivity for influenza A. However, the system was less sensitive for influenza B compared to multiplex real-time RT-PCR. For accurate diagnosis of false negative specimens, a molecular diagnostic test should be performed. The BD Veritor system could be a useful tool for screening and early diagnosis of influenza.

Clinical and Microbiological Characteristics of Six Staphylococcus pettenkoferi Isolates From Blood Samples

Coagulase-negative staphylococci (CoNS) are reported to be the leading cause of nosocomial bloodstream infections. Staphylococcus pettenkoferi is a novel member of CoNS that was first isolated from the human blood and bursitis wound in 2002. We have reported cases of 6 S. pettenkoferi strains isolated from blood specimens, including one pathogen and 5 contaminants and catheter colonizers. Brucker Biotyper (Brucker Daltonics, Bremen, Germany) and molecular typing with 16S rRNA gene sequencing confirmed the 6 isolates as S. pettenkoferi. The conventional phenotypic identification of these isolates is not reliable owing to their inconsistent biochemical characteristics. Five of the 6 isolates were found to be resistant to oxacillin, and all isolates showed susceptibility to vancomycin and linezolid. For accurate identification of this novel species, advanced methods by using Brucker Biotyper or molecular methods such as 16S rRNA gene sequencing are required.