Report of the International Council for Standardization in Haematology working group for standardization of reticulocyte parameters.

INTRODUCTION: The International Council for Standardization in Haematology convened a working group to assess and propose improvements upon the state of standardization and harmonization of reticulocyte parameters among commercial hematology analyzers. METHODS: An international group of laboratory hematologists prospectively collected and analyzed clinical samples using locally available IVD commercial hematology analyzers. Eight hundred and fifty-five total samples were collected at 6 sites using 9 distinct analyzer types. Samples were assessed for reticulocyte percent (RET%), immature reticulocyte fraction (IRF), and reticulocyte hemoglobin content (RHC). Method comparison and regression statistics were calculated. These analyses were used to determine whether statistical recalibration offered a potential avenue for increasing comparability between these methods. RESULTS: While methods producing reticulocyte percent were the most comparable in this study, the state of harmonization for the IRF and RHC was reduced with pearson correlation coefficients ranging from 0.955 to 0.77 and 0.927 and 0.680, respectively. Nevertheless, use of parameters from the Passing Bablok regression substantially improved the comparability of the results. In addition, precision data was derived which also demonstrated substantial differences between analyzer systems. CONCLUSION: While reticulocyte counting is correlated between the automated methods evaluated in this study, the current state of harmonization of other reticulocyte parameters is not as strong. A major challenge in moving this field forward is the need for commutable materials to facilitate comparisons between analyzers not co-located. A potential alternate approach to improve the current state would be instrument re-calibration. However, this is challenging both technically and due to national regulatory frameworks.

Current Status of Flow Cytometric Immunophenotyping of Hematolymphoid Neoplasms in Korea.

Background: Flow cytometric immunophenotyping of hematolymphoid neoplasms (FCI-HLN) is essential for diagnosis, classification, and minimal residual disease (MRD) monitoring. FCI-HLN is typically performed using in-house protocols, raising the need for standardization. Therefore, we surveyed the current status of FCI-HLN in Korea to obtain fundamental data for quality improvement and standardization. Methods: Eight university hospitals actively conducting FCI-HLN participated in our survey. We analyzed responses to a questionnaire that included inquiries regarding test items, reagent antibodies (RAs), fluorophores, sample amounts (SAs), reagent antibody amounts (RAAs), acquisition cell number (ACN), isotype control (IC) usage, positive/negative criteria, and reporting. Results: Most hospitals used acute HLN, chronic HLN, plasma cell neoplasm (PCN), and MRD panels. The numbers of RAs were heterogeneous, with a maximum of 32, 26, 12, 14, and 10 antibodies used for acute HLN, chronic HLN, PCN, ALL-MRD, and multiple myeloma-MRD, respectively. The number of fluorophores ranged from 4 to 10. RAs, SAs, RAAs, and ACN were diverse. Most hospitals used a positive criterion of 20%, whereas one used 10% for acute and chronic HLN panels. Five hospitals used ICs for the negative criterion. Positive/negative assignments, percentages, and general opinions were commonly reported. In MRD reporting, the limit of detection and lower limit of quantification were included. Conclusions: This is the first comprehensive study on the current status of FCI-HLN in Korea, confirming the high heterogeneity and complexity of FCI-HLN practices. Standardization of FCI-HLN is urgently needed. The findings provide a reference for establishing standard FCI-HLN guidelines.

COUP-TFII plays a role in cAMP-induced Schwann cell differentiation and in vitro myelination by up-regulating Krox20.

Schwann cells (SCs) are known to produce myelin for saltatory nerve conduction in the peripheral nervous system (PNS). Schwann cell differentiation and myelination processes are controlled by several transcription factors including Sox10, Oct6/Pou3f1, and Krox20/Egr2. Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII/NR2F2) is an orphan receptor that plays a role in the development and differentiation. However, the role of COUP-TFII in the transcriptional regulatory network of SC differentiation has not been fully identified yet. Thus, the objective of this study was to investigate the role and molecular hierarchy of COUP-TFII during cAMP-induced SC differentiation. Our results showed that dibutyryl-cAMP (db-cAMP) increased expression levels of COUP-TFII along with the expressions of Oct6, Krox20, and myelin-related genes known to be related to SC differentiation. Our mechanistic studies showed that COUP-TFII acted downstream of Hsp90/ErbB2/Gab1/ERK-AKT pathway during db-cAMP-induced SC differentiation. In addition, we found that COUP-TFII induced Krox20 expression by directly binding to Krox20-MSE8 as revealed by chromatin immunoprecipitation assay and promoter activity assay. In line with this, the expression of COUP-TFII was increased before up-regulation of Oct6, Krox20, and myelin-related genes in the sciatic nerves during early postnatal myelination period. Finally, COUP-TFII knockdown by COUP-TFII siRNA or via AAV-COUP-TFII shRNA in SCs inhibited db-cAMP-induced SC differentiation and in vitro myelination of sensory axons, respectively. Taken together, these findings indicate that COUP-TFII might be involved in postnatal myelination through induction of Krox20 in SCs. Our results present a new insight into the transcriptional regulatory mechanism in SC differentiation and myelination.

Secretory SERPINE1 Expression Is Increased by Antiplatelet Therapy, Inducing MMP1 Expression and Increasing Colon Cancer Metastasis.

Contrary to many reports that antiplatelet agents inhibit cancer growth and metastasis, new solid tumors have been reported in patients receiving long-term antiplatelet therapy. We investigated the effects of these agents directly on cancer cells in the absence of platelets to mimic the effects of long-term therapy. When four antiplatelet agents (aspirin, clopidogrel, prasugrel, and ticagrelor) were administered to colon cancer cells, cancer cell proliferation was inhibited similarly to a previous study. However, surprisingly, when cells were treated with a purinergic P2Y12 inhibitor (purinergic antiplatelet agent), the motility of the cancer cells was significantly increased. Therefore, gene expression profiles were identified to investigate the effect of P2Y12 inhibitors on cell mobility, and Serpin family 1 (SERPINE1) was identified as a common gene associated with cell migration and cell death in three groups. Antiplatelet treatment increased the level of SERPINE1 in cancer cells and also promoted the secretion of SERPINE1 into the medium. Increased SERPINE1 was found to induce MMP1 and, thus, increase cell motility. In addition, an increase in SERPINE1 was confirmed using the serum of patients who received these antiplatelet drugs. With these results, we propose that SERPINE1 could be used as a new target gene to prevent the onset and metastasis of cancer in patients with long-term antiplatelet therapy.

High anemia prevalence in Korean older adults, an advent healthcare problem: 2007-2016 KNHANES.

BACKGROUND: Anemia is associated with high morbidity and mortality in older people. However, the prevalence and characteristics of anemia in older individuals are not fully understood, and national data on these aspects in older Korean adults are lacking. This study aimed to evaluate the prevalence and characteristics of anemia in older adults using data from the Korea National Health and Nutrition Examination Survey (KNHANES), which is a nationwide cross-sectional epidemiological study conducted by the Korean Ministry of Health and Welfare. METHODS: Data from a total of 62,825 participants of the 2007-2016 KNHANES were compiled and analyzed to investigate differences in participant characteristics and potential risk factors for anemia. Differences in clinical characteristics of participants were compared across subgroups using the chi-square test for categorical variables and independent t-test for continuous variables. Univariate and multivariate analyses using logistic regression were performed to identify related clinical factors. RESULTS: The prevalence of anemia was higher in the population aged ≥65 years than in the younger population. Anemia was also more prevalent among females than among males, but this difference was not significant in people aged > 85 years. Being underweight, receiving a social allowance, living alone, and having comorbidities such as hypertension, rheumatoid arthritis, diabetes mellitus (DM), cancer, and chronic renal failure (CRF) were more common among older adults with anemia than among the population without anemia. In univariate and multivariate analyses, older age, female sex, underweight, and presence of comorbidities including rheumatoid arthritis, DM, cancer, and CRF were associated with an increased risk of anemia. CONCLUSIONS: This study revealed that age, female sex, underweight, and the presence of comorbidities such as rheumatoid arthritis, DM, cancer, and CRF were associated with an increased risk of anemia in older Korean adults. Further study on causal relationships between anemia and other variables in the older population is necessary.

Evaluation of neutrophil extracellular traps as the circulating marker for patients with acute coronary syndrome and acute ischemic stroke.

INTRODUCTION: Neutrophil extracellular traps (NETs) are known to be induced by various factors. In this study, we tried to identify circulating levels of NETs in patients with acute coronary syndrome (ACS) and acute ischemic stroke (AIS) and to confirm its suitability as a new circulating marker in their detection. METHODS: We prospectively enrolled 95 patients with a diagnosis of ACS (N = 37) or AIS (N = 58) in Dong-A University Hospital, Busan, Korea. The control group was selected from healthy adults (N = 25) who visited the hospital for health screening. Circulating levels of NETs were evaluated by measuring plasma concentrations of double-stranded DNA (dsDNA) and DNA-histone complex. RESULTS: The concentrations of dsDNA were statistically higher in patients with ACS or AIS than those in the control group (both P < .001). In the univariable and multivariable analyses, statistically significant risk factors were troponin I (TnI) level and dsDNA concentration in the ACS group (P = .046 and P = .015, respectively) and only dsDNA concentration in the AIS group (P = .002). In the receiver operating characteristic curve analyses, the area under the curve values for TnI level and dsDNA concentration in the ACS group were 0.878 and 0.968, respectively, and the value for dsDNA concentration in the AIS group was 0.859. CONCLUSIONS: In this study, it was confirmed that the circulating level of NETs was increased in patients with ACS and AIS at initial presentation. Findings in this study show that NETs could be used as a new circulating marker for the initial diagnosis of ACS or AIS.

Performance evaluation of new Abbott Alinity hq hematology analyzer.

INTRODUCTION: Abbott Alinity hq is a next-generation automated hematology analyzer providing complete blood count (CBC) with 6-part white blood cells (WBC) differential counts. The purpose of this study was to evaluate the performance of the analyzer to verify the diagnostic and clinical utility of the Abbott Alinity hq automated system. METHODS: We evaluated specimen stability, precision, linearity, carry-over, and method comparison to assess the performance of Alinity hq. For comparison of the Alinity hq with Sysmex XN-9000, totally 314 samples from adult and pediatric patients including both normal and abnormal hematology profiles were analyzed in parallel. The Alinity hq was also compared with the manual differential counts for the same 314 samples. RESULTS: At 4°C, the Alinity hq analyzer showed no significant changes in CBC and WBC differential count up to 48 hours. When stored at room temperature (18-25°C), all parameters except the mean platelet volume (MPV) were stable up to 36 hours. The Abbott Alinity hq analyzer demonstrated excellent reproducibility and between-batch precision for all CBC and WBC differential parameters. WBC, red blood cells (RBC), hemoglobin (HGB), and platelets showed good linearity and acceptable carry-over. Comparison with a Sysmex XN-9000 analyzer and manual 400-cell differential showed excellent correlation for CBC and WBC differential count parameters (correlation coefficient = 0.815-0.999) except for mean corpuscular hemoglobin concentration (MCHC) and basophils. CONCLUSION: We performed initial validation studies and confirmed performance specifications on specimen stability, precision, linearity, carry-over, and method comparison. The Abbott Alinity hq analyzer showed good analytical performance for all standard CBC parameters.

Recent progress in laboratory diagnosis of thalassemia and hemoglobinopathy: a study by the Korean Red Blood Cell Disorder Working Party of the Korean Society of Hematology.

Genetic hemoglobin disorders are caused by mutations and/or deletions in the α-globin or ß-globin genes. Thalassemia is caused by quantitative defects and hemoglobinopathies by structural defect of hemoglobin. The incidence of thalassemia and hemoglobinopathy is increased in Korea with rapid influx of people from endemic areas. Thus, the awareness of the disease is needed. α-thalassemias are caused by deletions in α-globin gene, while ß-thalassemias are associated with decreased synthesis of ß-globin due to ß-globin gene mutations. Hemoglobinopathies involve structural defects in hemoglobin due to altered amino acid sequence in the α- or ß-globin chains. When the patient is suspected with thalassemia/hemoglobinopathy from abnormal complete blood count findings and/or family history, the next step is detecting hemoglobin abnormality using electrophoresis methods including high performance liquid chromatography and mass spectrometry. The development of novel molecular genetic technologies, such as massively parallel sequencing, facilitates a more precise molecular diagnosis of thalassemia/hemoglobinopathy. Moreover, prenatal diagnosis using genetic testing enables the prevention of thalassemia birth and pregnancy complications. We aimed to review the spectrum and classification of thalassemia/hemoglobinopathy diseases and the diagnostic strategies including screening tests, molecular genetic tests, and prenatal diagnosis.

Early prediction of severity in acute ischemic stroke and transient ischemic attack using platelet parameters and neutrophil-to-lymphocyte ratio.

BACKGROUND: It is still not easy to predict severity promptly in patients with acute ischemic stroke (AIS) and transient ischemic attack (TIA). We investigated that platelet parameters or combinations of them could be a useful tool for early prediction of severity of AIS and TIA at admission and after 3 months. METHODS: We prospectively recruited 104 patients newly diagnosed with AIS and TIA. We investigated their neutrophil-to-lymphocyte ratio (NLR) and platelet parameters. According to the Modified Rankin Scale scores, the patients were divided into two groups. RESULTS: In receiver operating characteristic (ROC) curve analyses, mean platelet volume (MPV), NLR/platelet count (PLT), MPV/PLT, MPV*NLR, and MPV*NLR/PLT showed statistically significant results in both at admission and after 3 months. Values of area under ROC curves for those tests at admission were 0.646, 0.697, 0.664, 0.708, and 0.722, respectively. Also, values after 3 months were 0.591, 0.661, 0.638, 0.662, and 0.689, respectively. CONCLUSION: MPV*NLR/PLT could be used as a relatively good tool for predicting severity at the time of admission and after 3 months than other parameters or combinations of them. Further studies have to be carried out to investigate the best parameter for predicting the severity of AIS and TIA.

Recent advances in cytogenetic characterization of multiple myeloma.

The detection of cytogenetic abnormalities in multiple myeloma (MM) has received more importance over last years for risk stratification and the new risk-adapted treatment strategies. Conventional G-banding analysis should be included in a routine procedure for the initial diagnostic workup for patients suspected of MM. However, the detection of chromosomal abnormalities in MM by conventional cytogenetics is limited owing to the low proliferative activity of malignant plasma cells as well as the low number of plasma cells in bone marrow specimens. Fluorescence in situ hybridization (FISH) or microarray-based technologies can overcome some of those drawbacks and detect specific target arrangements as well as chromosomal copy number changes. In this review, we will discuss different cytogenetic approaches and compare their strength and weakness to provide genetic information for risk stratification and prediction of outcome in MM patients.

Platelet Function Analyzer-200 P2Y Results Are Predictive of the Risk of Major Adverse Cardiac Events in Korean Patients Receiving Clopidogrel Therapy Following Acute Coronary Syndrome.

BACKGROUND: Clopidogrel is one of the most commonly used anti-platelet agents in cardiovascular diseases. We analyzed the relationship between the platelet function analyzer (PFA)-200 P2Y (INNOVANCE PFA-200 System, Siemens Healthcare, Germany) results and occurrence of major adverse cardiac events (MACEs) in Korean patients with recent-onset acute coronary syndrome (ACS) taking clopidogrel. METHODS: Between August 2013 and June 2016, we prospectively enrolled 106 patients with recent-onset ACS who had been treated with clopidogrel. We obtained blood samples and measured closure time (CT) using the PFA-200 P2Y test. Patients were divided into two groups on the basis of a CT cut-off value of 106 seconds. We compared patient characteristics and various MACEs that occurred during the follow-up period. RESULTS: The CTs for 78 patients exceeded the cut-off value. At the time of these analyses, 11 patients had been diagnosed with MACEs. In the time-to-event analysis, there was a difference between the two groups (P<0.001). After adjusting other variables associated with MACE occurrence, CT value was the strongest predictor of MACEs, with a 7.30-fold occurrence risk (P=0.002). CONCLUSIONS: We found a strong relationship between CT and MACE risk in Korean patients with recent-onset ACS taking clopidogrel. Accordingly, PFA-200 P2Y results could be used as a predictive marker for MACE risk in such patients.

Holotoxin A1 Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells.

Marine triterpene glycosides are attractive candidates for the development of anticancer agents. Holotoxin A1 is a triterpene glycoside found in the edible sea cucumber, Apostichopus (Stichopus) japonicus. We previously showed that cladoloside C2, the 25(26)-dihydro derivative of holotoxin A1 induced apoptosis in human leukemia cells by activating ceramide synthase 6. Thus, we hypothesized that holotoxin A1, which is structurally similar to cladoloside C2, might induce apoptosis in human leukemia cells through the same molecular mechanism. In this paper, we compared holotoxin A1 and cladoloside C2 for killing potency and mechanism of action. We found that holotoxin A1 induced apoptosis more potently than cladoloside C2. Moreover, holotoxin A1-induced apoptosis in K562 cells by activating caspase-8 and caspase-3, but not by activating caspase-9. During holotoxin A1-induced apoptosis, acid sphingomyelinase (SMase) and neutral SMase were activated in both K562 cells and human primary leukemia cells. Specifically inhibiting acid SMase and neutral SMаse with chemical inhibitors or siRNAs significantly inhibited holotoxin A1-induced apoptosis. These results indicated that holotoxin A1 might induce apoptosis by activating acid SMase and neutral SMase. In conclusion, holotoxin A1 represents a potential anticancer agent for treating leukemia. Moreover, the aglycone structure of marine triterpene glycosides might affect the mechanism involved in inducing apoptosis.

In vitro and in vivo anti-leukemic effects of cladoloside C2 are mediated by activation of Fas/ceramide synthase 6/p38 kinase/c-Jun NH2-terminal kinase/caspase-8.

We previously demonstrated that the quinovose-containing hexaoside stichoposide C (STC) is a more potent anti-leukemic agent than the glucose-containing stichoposide D (STD), and that these substances have different molecular mechanisms of action. In the present study, we investigated the novel marine triterpene glycoside cladoloside C2 from Cladolabes schmeltzii, which has the same carbohydrate moiety as STC. We assessed whether cladoloside C2 could induce apoptosis in K562 and HL-60 cells. We also evaluated whether it showed antitumor action in mouse leukemia xenograft models, and its molecular mechanisms of action. We investigated the molecular mechanism behind cladoloside C2-induced apoptosis of human leukemia cells, and examined the antitumor effect of cladoloside C2 in a HL-60 and K562 leukemia xenograft model. Cladoloside C2 dose- and time-dependently induced apoptosis in the analyzed cells, and led to the activation of Fas/ceramide synthase 6 (CerS6)/p38 kinase/JNK/caspase-8. This cladoloside C2-induced apoptosis was partially blocked by specific inhibition by Fas, CerS6, and p38 siRNA transfection, and by specific inhibition of JNK by SP600125 or dominant negative-JNK transfection. Cladoloside C2 exerted antitumor activity through the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 without showing any toxicity in xenograft mouse models. The antitumor effect of cladoloside C2 was reversed in CerS6 shRNA-silenced xenograft models. Our results suggest that cladoloside C2 has in vitro and in vivo anti-leukemic effects due to the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 in lipid rafts. These findings support the therapeutic relevance of cladoloside C2 in the treatment of human leukemia.