Purpose The ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR)γ has been reported to promote the formation of platelets from megakaryocytes and accelerate platelet recovery. The endogenous prostaglandin 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2) is a ligand of PPARγ. Fucoidans are constituents of brown seaweed, and their anticoagulant and antithrombotic actions are recognized. In this study, we analyzed how PPAR ligand and brown seaweed based compound affect megakaryocyte and platelet in vitro. Methods Platelets were isolated from discarded plateletpheresis products by centrifugation. We used DAMI and Meg-01 cells as well. We treated these cells with 15d-PGJ2, GW9662, fucoidan, GW6471, and celecoxib. Then their effects on platelet membrane glycoproteins and PPARγ, PPARα and COX-2 expression, thrombogenicity and genetic regulation were analyzed. Results 15d-PGJ2 induced megakaryocytic differentiation evidence in morphology and flow cytometry analysis. In microarray analysis, fucoidan showed early growth response 1 (EGR1), Fc receptor, IgG, high affinity 1 (FCGR1), YME1 like 1 ATPase (YME1L1), stanniocalcin 1 (STC1) and cytochrome P450 family 11 subfamily B member 2 (CYP11B2) genes upregulation on megakaryocytes. Conclusion These findings support PPAR ligand, and brown seaweed based compound plays a role in megakaryocytic differentiation even in genomic level. Keywords: Megakaryocytes, Platelet, PPAR ligand, Fucoidan

Background: Nuclear receptors aretranscription factors that are activated by ligands and subsequently bind to regulatory regions in target genes. Recently ligand-activated transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) has been reported to promote the formation of platelets from megakaryocytes and accelerate platelet recovery after radiation-induced bone marrow injuries. The endogenous prostaglandin 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2) are ligands of PPARγ. Accumulating evidences also suggest that PPAR activation is involved in inflammation responses and atherosclerosis. In the present study, we analyzed how PPAR ligands and some brown algae-based compounds affect platelet differentiation and functions in vitro.

BACKGROUND:An association has been reported between CYP2C19 polymorphism and the altered antiplatelet activity of clopidogrel. We investigated this association using the newly introduced platelet function analyzer (PFA)-200 (INNOVANCE PFA-200 System; Siemens Healthcare, Germany) P2Y test.METHODS:Polymorphisms of CYP2C19*2, *3, *17 and the degree of inhibition of platelet function were determined in 83 patients. Three different platelet function tests were used to evaluate the degree of platelet inhibition and to check the association with genotype.RESULTS:The post-procedure PFA-200 values of extensive metabolizers (EM) patients (285.3±38.8) were higher than those of intermediate metabolizers (IM) and poor metabolizers (PM) patients (227.7±98.3 and 133.7±99.2, respectively; P=0.024). Light transmittance aggregometry (LTA) and the VerifyNow system showed that the post-procedure values for EM patients were lower than those of IM and PM patients (LTA: 24.4±15.7, 34.1±17.6, and 42.2±16.9, respectively, P<0.001; VerifyNow: 133.2±60.5, 171.5±42.6, and 218.7±59.3, respectively, P<0.001). The high residual platelet reactivity (HPR) rates were significantly different among the EM, IM, and PM groups using PFA-200 (PM:IM:EM=82.4:40.6:11.8, P<0.001).CONCLUSIONS:Approximately, 59.0% of Korean patients with cardiovascular disease receiving clopidogrel had CYP2C19 loss-of-function genotypes classified as IM or PM, and the frequency was similar to the data from Asian people. The PFA-200, LTA, and VerifyNow platelet function tests revealed evidence of a significant association between the efficacy of clopidogrel and CYP2C19 genotypes.

Dear Editor, Interferon (IFN)-γ release assay (IGRA) is a functional assay used to measure T cell responses to Mycobacterium tuberculosis-specific antigens in whole blood or blood-derived mononuclear cells [1,2]. The QuantiFERON-TB Gold In-Tube (QFT-GIT; Cellestis, Carnegie, Australia) is a commercially available IGRA. Significant within-subject variability in QFT-GIT results has been reported in tuberculosis (TB) screening [3,4]. Variability in QFT-GIT results for the retesting of the same patient sample has been observed in a low TB-incidence setting. Twenty-eight (8%) of 366 patients showed results different from those of the initial QFT-GIT, and 24 (85.7%) of these 28 patients had an initial TB response between 0.25 and 0.80 IU/mL [5]. To explore the clinical impact of QFT-GIT results with borderline TB response in an intermediate TB-incidence setting, we specifically assessed the repeatability of QFT-GIT near cut-off points in routine laboratory practice. We prospectively included 748 samples referred for TB screening between March, 2013 and February 2014 in the Dong-A University Hospital, Busan, Korea. This study was approved by the Institutional Review Board of Dong-A University Hospital. The QFT-GIT was performed according to the manufacturer's instructions. A TB response between 0.25 and 0.80 IU/mL was adopted as a borderline range [5]. After the initial test, the residual samples with borderline range of TB response, were stored and retested. According to the manufacturer's interpretation criteria, we compared and analyzed the first and second results. Bland-Altman plots were used for quantitative results. Data analysis was performed by using MedCalc Software (ver. 12.6.1, MedCalc Software, Mariakerke, Belgium). Eighty-five of the 748 patients showed a TB response in the range of 0.25-0.80 IU/mL. Among these 85 patients, 25 (29.4%) were diagnosed as having TB, 28 (33%) had suspected symptoms of TB, and 32 (37.6%) were examined by QFT-GIT to screen for latent TB infection. The median age was 56 yr, and 47 (55.3%) were males. The first QFT-GIT result was positive in 49 patients (57.6%), negative in 35 (41.2%), and indeterminate in 1 (1.2%). Seventy-one patients (83.5%) showed concordance between the results of the first and second QFT-GIT. Of the 14 patients with discordant results (14/85, 16.4%), three showed positive conversion, eight showed negative reversion, one with an indeterminate result in the first QFT-GIT showed a negative result in the second QFT-GIT, and two with positive results in the first QFT-GIT showed indeterminate results in the second QFT-GIT. The discordant results were most frequent (5/17, 29.5%) in the group with second-quartile TB response where the values included the cut-off value of 0.35 IU/mL. However, the likelihood ratio for subsequent discordant results was not statistically significant (P=0.48; Table 1). Bland-Altman plots of the differences in the first and second responses for nil, mitogen, and TB responses are shown in Fig. 1; the mean difference in the mitogen response was the highest. Fig. 1 Bland-Altman plots for the first-second response differences for nil (A), mitogen (B), and TB responses (C); the analysis showed mean differences of -0.02 IU/mL, -0.2 IU/mL, and 0.03 IU/mL, respectively. The lower and upper limits of agreement (mean difference±1.96 ... Table 1 Distribution of discordant results based on the initial TB response The repeatability and reproducibility of QFT-GIT have important clinical considerations because the test uses a single cutoff point to distinguish between positive and negative results with one-time testing. The low positive result for TB response (0.35-0.59 IU/mL) could be supposed to revert to negative in the retest on the basis of inherent variability of the QFT-GIT using a linear mixed effects model [5]. The conversion and reversion may have occurred in patients undergoing serial testing, and this could result in unnecessary preventive therapy while screening for latent TB infection [6,7]. In this study, we observed that a considerable portion of the QFT-GIT results near the cut-off points showed discordance on retesting of the same sample. Thus, it might be worth introducing a borderline decision range for quantitative TB antigen response in QFT-GIT. In addition, we found that the mitogen response would be more dynamic than nil and TB responses, consistent with those of a previous study involving serial QFT-GIT assays in 299 patients [8]. Although we could not examine subjects with a TB response 0.8 IU/mL, nearly two-thirds of the discordant results were observed between the cut-off points minus 0.03 IU/mL and plus 0.19 IU/mL for the TB response (0.32-0.54 IU/mL). This range considerably overlaps a low positive zone (0.35-0.59 IU/mL) defined by a previous study on QFT-GIT variability [5]. In conclusion, we suggest that to improve the clinical interpretation of QFT-GIT results, the results near the cut-off points in particular should be interpreted cautiously, and the establishment of a borderline decision range for quantitative TB antigen response in QFT-GIT should also be considered.

BACKGROUND:Clopidogrel is a widely used antiplatelet agent for dual antiplatelet therapy and metabolized by CYP2C19. The polymorphism of CYP2C19 is associated with the therapeutic effect of clopidogrel.METHODS:A total of 119 patients diagnosed with acute coronary syndrome (ACS) and underwent percutaneous coronary intervention (PCI) with drug-eluting stents was enrolled. Polymorphisms of CYP2C19 *2,*3,*17 were determined by the Spartan RX CYP2C19 and confirmed by SNP genotyping assay. Genotype was grouped as ultra-rapid metabolizer, extensive metabolizer, intermediate metabolizer, and poor metabolizer. The degree of platelet inhibition was assessed by the VerifyNow P2Y12 system (Accumetrics, USA).RESULTS:The CYP2C19 genotypes were distributed as 4 (3.3%) for UM, 39 (32.8%) for EM, 54 (45.4%) for IM, 22 (18.5%) for PM by evaluation with Spartan RX CYP2C19. The numbers of patients with the *1/*17, *1/*1, *1/*2, *1/*3, *3/*17, *2/*2, *2/*3, and *3/*3 genotype were 4 (3.3%), 39 (32.8%), 40 (33.6%), 13 (10.9%), 1 (0.9%), 11 (9.2%), 10 (8.4%), 1 (0.9%), respectively. The genotyping results between Spartan RX CYP2C19 and SNP genotyping assay showed discrepancy in 2 patients. The discrepancy appeared in *17 allele analysis in both patients as false-positive result.CONCLUSIONS:The false-positive *17 allele couldn't affect IM or PM group associated with thrombotic events, but it could affect UM group associated with bleeding events, which is relatively less investigated. Although the supplement of *17 allele detection should be accomplished, this novel point-of-care CYP2C19 genotyping instrument could determine the response to the clopidogrel and support the appropriate treatment of ACS patients.

Shuhua Li, Jae-Lim Choi, Bo-Ram Kim, Cheol-Soo Kang, RiYoung Goh, KwangSook Woo, and JinYeong Han Department of Laboratory Medicine, Dong-A University Medical Center, Busan, Korea Background: Detection of antiphospholipid antibodies (aPL) can be considered problematic due to assay variability and reagent sensitivity, high false-positive and false-negative rates, and lack of assay standardization. Therefore, utilizing an automated system can improve reproducibility and reduce interlaboratory variation. Here, we evaluated the analytical performance of the new automated ACL AcuStar chemiluminescence assay (Instrumentation Laboratory, USA). This was compared to the results of a panel analyzed with the QUANTA Lite ELISA (INOVA Diagnostics Inc., USA). Methods: We evaluated the inter-assay precision, linearity, and carry-over between the two methods, ACL and ELISA. A reference range study for each of the anticardiolipin (aCL) and anti-b2 glycoprotein-I (ab2GPI) IgG and IgM antibodies were performed using 135 healthy patient samples, which served as controls. We then compared the accuracy among the AcuStar and ELISA systems via four aPL tests. For this comparison, 69 patient samples suspected of an autoimmune disorder were used as the experimental panel. Results: The AcuStar analyzer showed excellent precision, linearity, and carry-over for all four assays. The calculated cutoff values were 20.3 U/mL for aCL IgG, 20.3 U/mL for aCL IgM, 26.3 U/mL for ab2GPI IgG, and 11.9 U/mL for ab2GPI IgM. The consensus between AcuStar and ELISA results were generally comparable. Total agreement varied between 82.6% and 95.7%, and kappa values showed moderate to good agreement. Conclusions: Our study demonstrates that the new AcuStar chemiluminescence assay showed better performance. This automated system leads to improved reproducibility and reduces interlaboratory variability. (J Lab Med Qual Assur 2015;37:134-140)

Objective The neutrophil-to-lymphocyte ratio (NLR) has been shown to predict adverse outcomes in several pathologic conditions. The majority of indeterminate interferon (IFN)-γ release assays were due to inadequate IFN-γ response to the phytohemagglutinin. We sought to study the value of NLR to predict an indeterminate result of QuantiFERON-TB Gold In-Tube (QFT-GIT) performed in routine laboratory practice. Methods Results from 2,773 QFT-GIT assays were analyzed. Data collection included demographic data, the level of IFN-γ to nil, mitogen, and TB antigen of QFT-GIT, total WBC, and a differential count. We calculated the absolute neutrophil count, lymphocyte count, and NLR. Results Of the total, 224 (8.1%) indeterminate results were observed. Twelve (1.8%) showed indeterminate results in the NLR range from 1.71 to 2.84, but 132 (19.2%) had indeterminate results in NLR≥5.18 (p<0.0001). The likelihood ratio for indeterminate results were 2.70 (95% CI, 2.36-3.08) in NLR ≥5.18 and 1.93 (95% CI, 1.64-2.27) in lymphocyte count ≤1050/μL. NLR and neutrophil count were independent predictors for indeterminate QFT-GIT result in multiple regression analysis. The IFN-γ response to PHA was negatively associated with NLR (r=-0.33, p<0.001). Conclusion We showed that the NLR is an independent predictor of indeterminate QFT-GIT result. Low frequency of indeterminate results in group with normal NLR may imply the importance of a balance between two cellular compartments in physiological and pathological conditions.

김보람·최재림·김지은·우광숙·김경희·김정만·김성현·한진영 Bo-Ram Kim, M.D., Jae-Lim Choi, M.D., Ji-Eun Kim, M.D., Kwang-Sook Woo, M.D., Kyeoung-Hee Kim, M.D., Jeong-Man Kim, M.D., Sung-Hyun Kim, M.D., Jin-Yeong Han, M.D. 동아대학교 의과대학 진단검사의학교실, 내과학교실 Departments of Laboratory Medicine, Division of Hematology-Oncology, Department of Internal Medicine, Dong-A University College of Medicine, Busan, Korea 원저 Lab Med Online Vol. 5, No. 3: 133-136, July 2015 http://dx.doi.org/10.3343/lmo.2015.5.3.133 진단혈액학

Background: Clinical data of serial interferon-gamma release assay (IGRA) testing in routine laboratory practice are limited. IFN-gamma response to mitogen is used as a positive control in IGRA. We assessed the association between IFN-gamma response to nil, mitogen, tuberculosis (TB) mycobacterial antigens, and the variations from the results of the serial testing.Method: A total of 299 patients with serial QuantiFERON-TB Gold In-Tube (QFT-GIT) were enrolled. The medical records of patients were reviewed for demographic information, status of Mycobacterium tuberculosis infection, treatment of tuberculosis, and the quantitative response to nil, mitogen, and TB antigen.Results: The initial QFT-GIT result was positive in 142 patients (47.5%), negative in 139 (46.5%), and indeterminate in 18 (6.0%). Of total, 79.6% showed concordant results in serial testing. The discordance in serial tests was significantly high in patients with a low mitogen response (<= 3.93 IU/ml) (p < 0.0001). Quantitative TB responses around the cut-off point in serial QFT-GIT were associated with an increased conversion and reversion rates (p = 0.01, p = 0.0005), respectively.Conclusion: Because IGRAs are dynamic assays, integrated interpretation of quantitative TB response with mitogen and nil response would be helpful in serial QFT-GIT. Recommendations for the interpretation of results of serial testing for active TB will be required. (C) 2013 Published by Elsevier B.V.

The major goal of traditional platelet function tests has been to screen and diagnose patients who present with bleeding problems. However, as the central role of platelets implicated in the etiology of arterial thrombotic diseases such as myocardial infarction and stroke became widely known, platelet function tests are now being promoted to monitor the efficacy of antiplatelet drugs and also to potentially identify patients at increased risk of thrombosis. Beyond hemostasis and thrombosis, an increasing number of studies indicate that platelets play an integral role in intercellular communication, are mediators of inflammation, and have immunomodulatory activity. As new potential biomarkers and technologies arrive at the horizon, platelet functions testing appears to take on a new aspect. This review article discusses currently available clinical application of platelet function tests, placing emphasis on essential characteristics.

Pseudomonas oryzihabitans is frequently found in various sites within hospital settings, including sink drains and respiratory therapy equipment. Although it rarely causes human infections, P. oryzihabitans has recently been considered a potential nosocomial pathogen, especially in immunocompromised hosts. We report our experience of an outbreak of P. oryzihabitans pseudobacteremia, presumably due to faulty aseptic preparation of a saline gauze canister.

BACKGROUND:Accurate renal function measurements are important in the diagnosis and treatment of kidney diseases. In contrast to creatinine, the production of serum cystatin C has been extensively reported to be unaffected by body muscle mass, age, gender, and nutritional status.METHODS:Our study included 37 samples from diabetic chronic kidney disease (CKD) patients for whom serum creatinine tests had been requested and 40 samples from a healthy populations in Dong-A University Hospital between May 2010 and June 2010. The assay precision (i.e., the coefficient of variation) and the reference range of the serum cystatin C test were evaluated. We compared the estimated glomerular filtration rates (GFRs) based on cystatin C with those based on creatinine. Moreover, we investigated the influences of age, gender, weight, and muscle mass on serum creatinine and serum cystatin C.RESULTS:There was a positive correlation between GFR based on creatinine and that based on cystatin C (r=0.79, P<0.0001) among the diabetic CKD patients. Serum creatinine and cystatin C were significantly correlated with body weight and muscle mass, but the strengths of these correlations were greater for serum creatinine. The precision study revealed excellent results for both the high and low controls. The 95% reference interval of cystatin C in the healthy population was 0.371 to 1.236 mg/L.CONCLUSION:Based on these results, we conclude that, despite the strong correlation between serum creatinine and cystatin C, cystatin C is less affected by weight and muscle mass and might represent a better alternative for the assessment of renal function.

Background: Specific cytogenetic aberrations detected by conventional karyotyping or FISH play a major role in the diagnosis, prognosis, and treatment of patients with acute leukemia. The FISH technique enhances the capacity of conventional karyotyping to detect subtle chromosomal aberrations. Multiprobe FISH assay (Cytocell, UK) can hybridize multiple probes to a single slide, thereby increasing the detection rate of cytogenetic aberrations. This study aimed to evaluate multiprobe FISH in detecting cytogenetic abnormalities in acute leukemia.Methods: Thirty newly diagnosed acute leukemia patients who attended the hematology clinic at Dong-A University Hospital from October 2008 to October 2012 were enrolled in the study. The multiprobe FISH results were compared with those of G-banding.Results: Multiprobe FISH detected the chromosomal aberrations identified by G-banding, as well as additional aberrations in 6 of 30 (20.0%) cases, which included ETV6/RUNX1 translocation, p16 deletion, TP53 deletion, and IGH break-apart.Conclusions: The multiprobe FISH assay was a more sensitive and reliable technique compared with G-banding. It was also more cost-effective and yielded faster results.

Shigella bacteremia is rare, occurring mainly in children. Shigella species often cause diarrhea or gastrointestinal inflammation in humans and are rarely associated with bacteremia. This report describes an unusual case of Shigella boydii bacteremia in an 84-year-old patient visiting our hospital after experiencing nausea, vomiting, and febrile sensation for 2 days. Peripheral blood cultures revealed S. boydii and 16S rDNA sequence analysis produced the same result. However, the organism was not isolated from the patient’s stool. She was started on ciprofloxacin, to which this organism is sensitive, and was subsequently discharged with instructions to complete a 14-day course of ciprofloxacin. Shigellosis is usually a self-limiting enteric disease. However, in contrast to its isolation from both blood and stool, isolation of the organism from blood only is associated with a high mortality rate. As is frequently pointed out, blood cultures should be obtained from elderly or immunocompromised patients with acute febrile gastroenteritis to detect infection caused by enteric pathogens, including Shigella. (Ann Clin Microbiol 2014; 17:20-22)

Clostridium difficile, an anaerobic, spore-forming, gram-positive, rod-shaped bacterium, is the most common nosocomial pathogen causing pseudomembranous colitis. C. difficile is not intrinsically invasive and rarely infects extraintestinal sites. The bacterium, therefore, is not commonly detected in blood cultures. Here, we report a case of C. difficile bacteremia in a patient who had underwent loop ileostomy because of rectal obstruction following metastatic colon cancer originated from prostate cancer.