Laboratory Preparation for Digital Medicine in Healthcare 4.0: An Investigation Into the Awareness and Applications of Big Data and Artificial Intelligence.

Background: Healthcare 4.0. refers to the integration of advanced technologies, such as artificial intelligence (AI) and big data analysis, into the healthcare sector. Recognizing the impact of Healthcare 4.0 technologies in laboratory medicine (LM), we seek to assess the overall awareness and implementation of Healthcare 4.0 among members of the Korean Society for Laboratory Medicine (KSLM). Methods: A web-based survey was conducted using an anonymous questionnaire. The survey comprised 36 questions covering demographic information (seven questions), big data (10 questions), and AI (19 questions). Results: In total, 182 (17.9%) of 1,017 KSLM members participated in the survey. Thirty-two percent of respondents considered AI to be the most important technology in LM in the era of Healthcare 4.0, closely followed by 31% who favored big data. Approximately 80% of respondents were familiar with big data but had not conducted research using it, and 71% were willing to participate in future big data research conducted by the KSLM. Respondents viewed AI as the most valuable tool in molecular genetics within various divisions. More than half of the respondents were open to the notion of using AI as assistance rather than a complete replacement for their roles. Conclusions: This survey highlighted KSLM members' awareness of the potential applications and implications of big data and AI. We emphasize the complexity of AI integration in healthcare, citing technical and ethical challenges leading to diverse opinions on its impact on employment and training. This highlights the need for a holistic approach to adopting new technologies.

Respiratory Viruses Co-detection on a Multiplex RT-PCR Panel: a Comparative Clinical Study.

BACKGROUND: The FilmArray Respiratory Panel RP 2.1 plus (FilmArray RP) is a point-of-care syndromic panel for respiratory pathogens. Although highly valuable in the clinical settings, the co-detection of pathogens in FilmArray RP may confound result interpretation. METHODS: Nasopharyngeal swab specimens collected from patients with respiratory symptoms were analyzed by comparing co-detection results from FilmArray RP with those of Allplex Respiratory Panels (Allplex RP: Power-Chek for SARS-CoV-2). RESULTS: Out of 765 FilmArray RP tests, 143 (18.7%) showed co-detections (two: 122 (85.3%), three: 18 (12.6%), four: 2 (1.4%), and five viruses: 1 (0.7%). The most frequent co-detection was human rhinovirus/enterovirus (HRV/HEV) with respiratory syncytial virus (RSV) (22.3%, 32/143). The overall discordance rate between Film-Array RP and other tests was 32.9%. Notably, discordance in detecting adenovirus (AdV) was significant, with cases detected by FilmArray often not appearing in Allplex RP. CONCLUSIONS: Discordances were varied by virus combination. It is advisable to perform additional confirmatory testing based on clinical relevance.

Antibody response to second dose of the BNT162b2 mRNA vaccine in the first 12 weeks in South Korea: A prospective longitudinal study.

OBJECTIVES: To characterise the antibody response for 12 weeks following second dose of the Pfizer/BioNTech BNT162b2 mRNA vaccine in hospital workers of a Korean general hospital. METHODS: We measured the level of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-receptor binding domain (anti-RBD) and neutralising antibodies every week in the first 4 weeks, and at weeks 8 and 12 following the second dose of vaccination in 71 hospital workers. RESULTS: The initial median level of anti-RBD and neutralising antibodies were 3898.0 U/mL (interquartile range [IQR], 2107.5-5478.5) and 97.54 % (IQR, 96.85-97.81), respectively. The levels declined the fastest and the most significantly between weeks 1 and 2 (p < 0.01, both), and continuously decreased thereafter, and were 1163.0 U/mL (683.4-1743.0) and 94.87% (89.24-96.99) at weeks 12. The antibodies levels showed a trend of rapid decrease in the older group over time. The slope of the decrease in the antibodies level was observed for each individual. Within 8 weeks, the anti-RBD antibody levels decreased to less than half of the initial levels in most of the participants (88.7%: 63/71). The SARS-CoV-2 anti-RBD and neutralising antibodies levels showed a strong positive correlation (Spearman's coefficient = 0.7833). CONCLUSIONS: Considerably high levels of SARS-CoV-2 anti-RBD and neutralising antibodies were produced following the second dose of vaccination. The levels decreased continuously, showing a tendency to decline over time; however, reasonable levels persisted up to weeks 12. Moreover, considering individual variations in antibody response following vaccination, a further inter-individual analysis is needed.

Molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae isolates.

BACKGROUND: The emergence and transmission of tigecycline- and carbapenem-resistant Klebsiella pneumoniae (TCRKP) have become a major concern to public health globally. Here, we investigated the molecular epidemiology and mechanisms of tigecycline resistance in carbapenem-resistant K pneumoniae (CRKP) isolates. METHODS: Forty-five non-duplicate CRKP isolates were collected from January 2017 to June 2019. We performed antimicrobial susceptibility tests, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). PCR and DNA sequencing were performed for the detection and mutation analysis of acrR, oqxR, ramR, rpsJ, tet(A), and tet(X) genes, which are related to tigecycline resistance. The expression levels of efflux pump genes acrB and oqxB and their regulator genes rarA, ramA, soxS, and marA were assessed by quantitative real-time PCR. RESULTS: The resistance rate to tigecycline in CRKP isolates was 37.8% (17/45). K pneumoniae ST307 was a predominant clone type (70.6%, 12/17) among the TCRKP isolates. The expression levels of acrB (P < .001) and marA (P = .009) were significantly higher in the tigecycline-resistant group than in the tigecycline-intermediate and tigecycline-susceptible groups. Increased expression of acrB was associated with marA expression (r = 0.59, P = .013). CONCLUSIONS: We found that the activated MarA-induced overexpression of AcrAB efflux pump plays an important role in the emergence of tigecycline resistance in CRKP isolates.

Emergence and transmission of New Delhi metallo-beta-lactamase-5-producing Escherichia coli Sequence Type 361 in a Tertiary Hospital in South Korea.

BACKGROUND: The emergence of carbapenem-resistant Escherichia coli (E coli) is a serious global health threat, but little is known about carbapenemase-producing E coli in Daejeon, South Korea. The aim of this study was to investigate characteristics of thirteen carbapenem-resistant E coli isolates in a tertiary hospital. METHODS: Thirteen non-duplicate carbapenem-resistant E coli strains were collected from October 2017 to January 2018. Antimicrobial susceptibility was determined with the E test or disk diffusion method. The carbapenem minimum inhibitory concentrations (MICs) were determined by the agar dilution method. The colistin and tigecycline MICs were determined by broth microdilution. The resistance genes, including carbapenemase genes, were evaluated by polymerase chain reaction, and DNA sequencing was performed to characterize the genes. Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) were performed to evaluate the clonal relatedness of isolates. The clinical data of patients were retrospectively reviewed. RESULTS: All the E coli isolates harbored blaNDM-5 gene and were resistant to most of the antimicrobial agents, such as carbapenem, cephalosporins, ciprofloxacin, and chloramphenicol, excluding amikacin and colistin. Other resistant genes, such as blaTEM-1 , blaCTX-M-15 , blaCMY-2 , aac(6')-Ib-cr, and qepA, were detected. The E coli isolates harboring blaNDM-5 belonged to ST361 (n = 11), ST12 (n = 1), ST410 (n = 1), and PFGE types A (n = 11), B (n = 1), and C (n = 1). CONCLUSIONS: This study reports on an outbreak of a predominant epidemic clone, the NDM-5 producing, multidrug-resistant E coli ST361 isolate. These findings suggest that we should pay attention to infection control measures to limit the spread of NDM-5-producing pathogens.

Comparison of the Genedia MTB/NTM Detection Kit and Anyplex plus MTB/NTM Detection Kit for detection of Mycobacterium tuberculosis complex and nontuberculous mycobacteria in clinical specimens.

BACKGROUND: Real-time (RT) PCR is a rapid and accurate method that is widely used for the detection of Mycobacterium tuberculosis complex (MTB). The aim of this study was to evaluate and compare the performance of the Genedia MTB/NTM Detection Kit and the Anyplex plus MTB/NTM Detection kit in the detection of MTB and nontuberculous mycobacteria (NTM) in clinical specimens. METHODS: From October 2017 to February 2018, 236 respiratory specimens and 137 non-respiratory specimens, from patients with suspected tuberculosis, were examined. AFB smear, culture, and RT-PCR using the Genedia MTB/NTM Detection kit (Green Cross Medical Science Corp.) and the Anyplex plus MTB/NTM Detection kit (Seegene) were applied. PCR performance in the detection of MTB and NTM was evaluated in relation to culture results and between the two assays. RESULTS: Culture was positive for MTB in 30 (8.0%) of the 373 specimens and for NTM in 23 (6.2%). The sensitivity and specificity of MTB detection with the Genedia kit were 76.7% and 99.7%, respectively, whereas the Anyplex kit sensitivity and specificity for MTB detection were 86.7% and 97.5%, respectively. Both kits exhibited the same sensitivity (73.9%) for NTM detection, and the specificity was 100% and 99.4% for the Genedia and Anyplex kits, respectively. CONCLUSIONS: The Genedia and Anyplex kits demonstrated high sensitivity and specificity for the detection of MTB and NTM. Both kits have a high concordance rate and can be used more widely in clinical laboratories for the early detection of tuberculosis.

Molecular epidemiology and virulence factors of methicillin-resistant Staphylococcus aureus isolated from patients with bacteremia.

BACKGROUND: The various virulence factors of methicillin-resistant Staphylococcus aureus bacteremia (MRSAB) are associated with a high mortality rate worldwide. Further studies are warranted to confirm the significant relationship between the strains and virulence genes. Here, we prospectively investigated the molecular characteristics underlying the genotypes and virulence factors of MRSA isolated from patients with bacteremia. METHODS: We collected 59 MRSA isolates from adult patients with bacteremia. Antimicrobial susceptibility results were obtained with the Vitek2 automated system. Genotypes were identified with multi-locus sequence typing (MLST) and pulse-field gel electrophoresis (PFGE), and 21 virulence genes were detected with polymerase chain reaction (PCR). RESULTS: The 59 MRSA isolates mainly comprised ST5 (n = 31, 52.5%) and ST72 (n = 22, 37.2%). Most ST5 isolates and all ST72 isolates were clustered into one and two PFGE groups, respectively. The mean number of virulence genes was higher in ST5 than in ST72. Sel was more frequently detected in ST5 than in ST72, whereas sec and sed were found only in ST5. ST5 had significantly higher resistance against many antibiotics than ST72. CONCLUSION: Most MRSA isolates causing bacteremia were ST5 (CC5) and ST72 (CC8), and those belonging to the same STs were divided into only a few PFGE groups. ST5 was associated with higher antibiotic resistance and staphylococcal superantigen toxin genes, than ST72, which may be related to its higher virulence.

Cytogenetic evolution in myeloproliferative neoplasms with different molecular abnormalities.

We investigated the changes in chromosomal abnormalities in myeloproliferative neoplasm (MPN) patients during long-term follow-up. In total, 28 MPN patients (22 with primary myelofibrosis and 6 with polycythemia vera) were included. Among them, 25 patients underwent serial bone marrow (BM) biopsies during disease progression, and 3 patients had cytogenetic abnormalities at initial diagnosis but lacked follow-up BM biopsies. JAK2, CALR, and MPL mutation analyses were performed. Targeted sequencing analysis was conducted in 11 patients. Among the 28 patients, 21 (75.0%) had cytogenetic abnormalities either at diagnosis (8/26) or during follow-up. The median time from the initial analysis to the appearance of additional cytogenetic abnormalities was 8.4 years. Among the chromosomal abnormalities at initial diagnosis, trisomy 8 (3/26, 11.5%) was the most frequent, followed by gain of 1q, del(20q), and del(9q) (each in 2/26). Among all chromosomal abnormalities, including those that occurred during follow-up, the most frequent was del(20q) and +1q (8/28, 28.6%), followed by del(6p) (14.3%) and trisomy 8 (10.7%). Del(20q) was more frequent in CALR-mutated patients (4/6, 66.7%) than in JAK2-mutated patients (3/19, 15.8%, P = 0.016). The presence of cytogenetic abnormalities at initial diagnosis was associated with poor prognosis. Cytogenetic evolution may provide interesting insights into the disease course.

Molecular diagnosis of hereditary spherocytosis by multi-gene target sequencing in Korea: matching with osmotic fragility test and presence of spherocyte.

BACKGROUND: Current diagnostic tests for hereditary spherocytosis (HS) focus on the detection of hemolysis or indirectly assessing defects of membrane protein, whereas direct methods to detect protein defects are complicated and difficult to implement. In the present study, we investigated the patterns of genetic variation associated with HS among patients clinically diagnosed with HS. METHODS: Multi-gene targeted sequencing of 43 genes (17 RBC membrane protein-encoding genes, 20 RBC enzyme-encoding genes, and six additional genes for the differential diagnosis) was performed using the Illumina HiSeq platform. RESULTS: Among 59 patients with HS, 50 (84.7%) had one or more significant variants in a RBC membrane protein-encoding genes. A total of 54 significant variants including 46 novel mutations were detected in six RBC membrane protein-encoding genes, with the highest number of variants found in SPTB (n = 28), and followed by ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations of genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). Positive rate of osmotic fragility test was 86.8% among patients harboring HS-related gene mutations. CONCLUSIONS: This constitutes the first large-scaled genetic study of Korean patients with HS. We demonstrated that multi-gene target sequencing is sensitive and feasible that can be used as a powerful tool for diagnosing HS. Considering the discrepancies of clinical and molecular diagnoses of HS, our findings suggest that molecular genetic analysis is required for accurate diagnosis of HS.

Genetic and phenotypic characterizations of drug-resistant Mycobacterium tuberculosis isolates in Cheonan, Korea.

BACKGROUND: Mycobacterium tuberculosis (MTB) causes tuberculosis (TB), which is a fatal disease. Cases of drug-resistant MTB have increased in recent years. In this study, we analyzed 7 sites of MTB DNA sequences, including the rpoB and inhA gene, to investigate the relationship between gene mutations and drug resistance in MTB. METHODS: Mycobacterium tuberculosis liquid culture samples (197 specimens from 74 cases) were collected between June 2015 and May 2016 and sequenced. The results were compared with those obtained from antibiotic susceptibility tests. RESULTS: In 65 (87.8%) cases, the antibiotic-resistant phenotype was consistent with genotyping results, whereas in 9 (12.2%) cases, there was no match. Eight mutations were detected in the rpoB gene, which showed the highest mutation rate. Sequencing results indicated that these mutations were present in 12 cases. CONCLUSION: Previously published data on antibiotic resistance genes are insufficient for effective prevention of multidrug- or extensive drug-resistant TB. Additional studies are needed to characterize the complement of antibiotic resistance genes in MTB.

Manual versus automated streaking system in clinical microbiology laboratory: Performance evaluation of Previ Isola for blood culture and body fluid samples.

BACKGROUND: The process of plate streaking has been automated to improve routine workflow of clinical microbiology laboratories. Although there were many evaluation reports about the inoculation of various body fluid samples, few evaluations have been reported for blood. In this study, we evaluated the performance of automated inoculating system, Previ Isola for various routine clinical samples including blood. METHODS: Blood culture, body fluid, and urine samples were collected. All samples were inoculated on both sheep blood agar plate (BAP) and MacConkey agar plate (MCK) using Previ Isola and manual method. We compared two methods in aspect of quality and quantity of cultures, and sample processing time. To ensure objective colony counting, an enumeration reading reference was made through a preliminary experiment. RESULTS: A total of 377 nonduplicate samples (102 blood culture, 203 urine, 72 body fluid) were collected and inoculated. The concordance rate of quality was 100%, 97.0%, and 98.6% in blood, urine, and other body fluids, respectively. In quantitative aspect, it was 98.0%, 97.0%, and 95.8%, respectively. The Previ Isola took a little longer to inoculate the specimen than manual method, but the hands-on time decreased dramatically. The shortened hands-on time using Previ Isola was about 6 minutes per 10 samples. CONCLUSION: We demonstrated that the Previ Isola showed high concordance with the manual method in the inoculation of various body fluids, especially in blood culture sample. The use of Previ Isola in clinical microbiology laboratories is expected to save considerable time and human resources.

Distribution of Mycobacterium tuberculosis in Korea in the preceding decade.

BACKGROUND: Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis (MTB); it is transmitted among people through air. The aim of this study was to assess the prevalence of TB and its clinical trends by collecting and analyzing data on specimens in Korea. METHODS: All clinical specimens referred to the Dankook University Hospital Laboratory in Cheonan, Korea, from September 2005 to June 2016 were tested to isolate MTB using solid and liquid cultures, acid-fast bacilli (AFB) smears, and polymerase chain reactions (PCR). RESULTS: In total, 146 150 specimens were collected; the mean TB positivity rate was 7.8%. The highest positivity rate was observed among patients 30-39 years of age (12.6%), followed by those 20-29 years of age (12.2%). The mean positivity rate was highest in 2010 and lowest in 2016 (10.7% and 6.7%, respectively). When comparing 2015-2011, we saw a decrease in the number of TB-positive patients of 3.4%; this represented an annual decrease in 0.9%. CONCLUSION: Our data revealed a trend for a decrease in TB prevalence over time. Moreover, TB positivity rates were highest among the younger age groups in our study. Therefore, rapid diagnosis and treatment of TB in younger individuals are crucial.

A challenging diagnosis: crystal-storing histiocytosis in plasma cell myeloma.

OBJECTIVES: Crystal-storing histiocytosis (CSH) is an uncommon finding in plasma cell neoplasms. CSH is thought to be an intralysosomal deposition of secreted paraproteins or immunoglobulins, which usually express κ immunoglobulin light chains that finally aggregate in crystals. Because of its rarity, CSH in bone marrow often makes diagnosis difficult. METHODS: A 57-year-old woman had IgA κ monoclonal proteinemia and monoclonal proteinuria. In the bone marrow aspirate, plasma cells were initially counted less than what would be expected, whereas histiocytes with intracellular crystals were increased. Then, we used α-naphthyl acetate esterase (ANAE) staining to distinguish between true histiocytes and plasma cells. Immunostaining for κ, CD138, CD56, and CD68 was performed on a bone marrow biopsy specimen. RESULTS: True histiocytes containing crystalline inclusions were stained strongly for ANAE, while unstained cells with intracytoplasmic crystals represented plasma cells. The biopsy specimen revealed diffuse infiltration of CD138-positive plasma cells. We also confirmed the presence of plasma cells, histiocytes, and their crystallized inclusions with the immunostaining. The patient was finally diagnosed with plasma cell myeloma. CONCLUSIONS: The diagnosis was challenging; the bone marrow findings resembled features of other histiocytic disorders. The use of immunohistochemistry enabled the diagnosis of CSH in the presence of plasma cell myeloma.

Influence of ABO type on global coagulation assay results: effect of coagulation factor VIII.

BACKGROUND: As ABO blood type influences the plasma level of coagulation factor VIII (FVIII), it likely also affects activated partial thromboplastin time (aPTT) and thrombin generation assay (TGA) values. Here, we aimed to investigate the effect of ABO type on the normal values of three global coagulation assays: prothrombin time (PT), aPTT, and TGA. METHODS: PT, aPTT, TGA [1 or 5 pmol/L tissue factor (TF)], coagulation factors, anticoagulation factors, and ABO type were measured in 200 healthy adults. RESULTS: aPTT was significantly prolonged in those with type O compared with those with type non-O, whereas PT was not significantly different between those with type O and type non-O. The time to peak induced by 5 pmol/L TF was significantly prolonged, and the peak thrombin level was decreased in those with type O compared with those with type non-O. FVIII was a major contributor to the ABO-specific reference range of aPTT, 5 pmol/L TF-induced time to peak, and peak thrombin level. CONCLUSIONS: The reference ranges of aPTT and TGA (time to peak and peak thrombin level) differed by ABO type. FVIII level is considered a major contributor to ABO type-specific differences with respect to aPTT and TGA.

Asymmetric aneuploidy in mesenchymal stromal cells detected by in situ karyotyping and fluorescence in situ hybridization: suggestions for reference values for stem cells.

Cytogenetic testing is important to ensure patient safety before therapeutic application of mesenchymal stromal cells (MSCs). However, the standardized methods and criteria for the screening of chromosomal abnormalities of MSCs have not yet been determined. We investigated the frequency of cytogenetic aberrations in MSCs using G-banding and fluorescence in situ hybridization (FISH) and suggest reference values for aneuploidy in MSCs. Cytogenetic analysis was performed on 103 consecutive cultures from 68 MSCs (25 adipose-origin, 20 bone marrow-origin, 18 cord blood-origin, and 5 neural stem cells; 8 from adipose tissue of patients with breast cancer and 60 from healthy donors). We compared the MSC aneuploidy patterns with those of hematological malignancies and benign hematological diseases. Interphase FISH showed variable aneuploid clone proportions (1%-20%) in 68 MSCs. The aneuploidy patterns were asymmetric, and aneuploidy of chromosomes 16, 17, 18, and X occurred most frequently. Clones with polysomy were significantly more abundant than those with monosomy. The cutoff value of maximum polysomy rates (upper 95th percentile value) was 13.0%. By G-banding, 5 of the 61 MSCs presented clonal chromosomal aberrations. Aneuploidy was asymmetric in the malignant hematological diseases, while it was symmetric in the benign hematological diseases. We suggest an aneuploidy cutoff value of 13%, and FISH for aneuploidy of chromosomes 16, 17, 18, and X would be informative to evaluate the genetic stability of MSCs. Although it is unclear whether the aneuploid clones might represent the senescent cell population or transformed cells, more attention should be focused on the safety of MSCs, and G-banding combined with FISH should be performed.

MYD88 L265P mutations are correlated with 6q deletion in Korean patients with Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a malignant lymphoplasma-proliferative disorder with IgM monoclonal gammopathy. A recent whole-genome study identified MYD88 L265P as the key mutation in WM. We investigated MYD88 mutations in conjunction with cytogenetic study in 22 consecutive Korean WM patients. Conventional G-banding and interphase fluorescence in situ hybridization (FISH) were performed at regions including 6q21 using bone marrow (BM) aspirates. Sixteen patients were subjected to Sanger sequencing-based MYD88 mutation study. Five patients (28%) showed cytogenetic aberrations in G-banding. The incidence of 6q21 deletion was 17% by conventional G-banding and 37% by FISH. Ten patients (45%) showed cytogenetic aberrations using FISH: 6q deletion in eight (37%) and IGH rearrangement in four (18%). Two patients had both the 6q deletion and IGH rearrangement, and two had only the IGH rearrangement. Eleven patients (69%) presented with the MYD88 L265P mutation. MYD88 mutations were significantly associated with the presence of 6q deletions (P = 0.037). Six patients with the 6q deletion for whom sequencing was possible were found to harbor MYD88 mutations. The MYD88 L265P mutation was also associated with increased lymphocyte burden in BM biopsy. This is the first report of high frequency MYD88 L265P mutations in Korean WM patients.

Changes in plasma levels of natural anticoagulants in disseminated intravascular coagulation: high prognostic value of antithrombin and protein C in patients with underlying sepsis or severe infection.

BACKGROUND: Dysfunctional natural anticoagulant systems enhance intravascular fibrin formation in disseminated intravascular coagulation (DIC), and plasma levels of natural anti coagulants can be used in the diagnosis and prognosis of DIC. Herein, the diagnostic value of 4 natural anticoagulants was assessed, and the prognostic value of antithrombin and protein C were validated in a large population. METHODS: Part 1 study included 126 patients with clinically suspected DIC and estimated plasma levels of 4 candidate anticoagulant proteins: antithrombin, protein C, protein S, and protein Z. Part 2 comprised 1,846 patients, in whom plasma antithrombin and protein C levels were compared with other well-known DIC markers according to the underlying dis eases. The 28-day mortality rate was used to assess prognostic outcome. RESULTS: Antithrombin and protein C showed higher areas under the ROC curve than protein S and protein Z. In part 2 of the study, antithrombin and protein C levels significantly correlated with DIC score, suggesting that these factors are good indicators of DIC severity. Antithrombin and protein C showed significant prognostic power in Kaplan-Meier analyses. In patients with sepsis/severe infection, antithrombin and protein C showed higher hazard ratios than D-dimer. Platelet count showed the highest hazard ratio in patients with hemato logic malignancy. In patients with liver disease, the hazard ratio for antithrombin levels was significantly high. CONCLUSIONS: Decreased plasma anticoagulant levels reflect florid consumption of the physiologic defense system against DIC-induced hypercoagulation. Plasma antithrombin and protein C levels are powerful prognostic markers of DIC, especially in patients with sepsis/severe infection.

The application of an in situ karyotyping technique for mesenchymal stromal cells: a validation and comparison study with classical G-banding.

The cytogenetic analysis of mesenchymal stromal cells (MSCs) is essential for verifying the safety and stability of MSCs. An in situ technique, which uses cells grown on coverslips for karyotyping and minimizes cell manipulation, is the standard protocol for the chromosome analysis of amniotic fluids. Therefore, we applied the in situ karyotyping technique in MSCs and compared the quality of metaphases and karyotyping results with classical G-banding and chromosomal abnormalities with fluorescence in situ hybridization (FISH). Human adipose- and umbilical cord-derived MSC cell lines (American Type Culture Collection PCS-500-011, PCS-500-010) were used for evaluation. The quality of metaphases was assessed by analyzing the chromosome numbers in each metaphase, the overlaps of chromosomes and the mean length of chromosome 1. FISH was performed in the interphase nuclei of MSCs for 6q, 7q and 17q abnormalities and for the enumeration of chromosomes via oligo-FISH in adipose-derived MSCs. The number of chromosomes in each metaphase was more variable in classical G-banding. The overlap of chromosomes and the mean length of chromosome 1 as observed via in situ karyotyping were comparable to those of classical G-banding (P=0.218 and 0.674, respectively). Classical G-banding and in situ karyotyping by two personnel showed normal karyotypes for both cell lines in five passages. No numerical or structural chromosomal abnormalities were found by the interphase-FISH. In situ karyotyping showed equivalent karyotype results, and the quality of the metaphases was not inferior to classical G-banding. Thus, in situ karyotyping with minimized cell manipulation and the use of less cells would be useful for karyotyping MSCs.

Contributions of procoagulants and anticoagulants to the international normalized ratio and thrombin generation assay in patients treated with warfarin: potential role of protein Z as a powerful determinant of coagulation assays.

BACKGROUND: The effects of warfarin are measured with the international normalized ratio (INR). However, the thrombin generation assay (TGA) may offer more information about global coagulation. We analyzed the monitoring performance of the TGA and INR and investigated the impact of procoagulants (fibrinogen, factor (F)II, FVII, FIX, and FX) and anticoagulants (proteins C, S, and Z) on them. METHODS: The TGA was performed on a calibrated automated thrombogram, producing lag time, endogenous thrombin potential (ETP), and peak thrombin in 239 patients treated with warfarin. Pro- and anticoagulant levels were also measured. RESULTS: The INR was significantly and inversely correlated with ETP. The therapeutic range of ETP comparable to an INR range of 2.0-3.0 was 290.1-494.6. ETP showed comparable performance to the INR as a warfarin-monitoring parameter with respect to clinical complication rate. The median levels of FII, FVII, FIX, and FX and proteins C and Z tended to decrease gradually with increasing anticoagulation intensity according to the INR or ETP. Of note, protein Z levels decreased dramatically with increasing anticoagulation status. INRs were significantly determined by FII, FVII, and protein Z. ETP was significantly dependent on FVII, and proteins C and Z concentration. Protein Z significantly reduced the total amount of thrombin generation and prolonged PT value in vitro. CONCLUSIONS: The INR and ETP exhibit similar efficacy for warfarin monitoring according to the clinical complication rate. Protein Z is considered to be a significant determinant of INR and ETP in patients on warfarin therapy.