Assessing anti-SARS-CoV-2 cellular immunity in 571 vaccines by using an IFN-γ release assay.

Memory T cell responses have been analyzed only in small cohorts of COVID-19 vaccines. Herein, we aimed to assess anti-SARS-CoV-2 cellular immunity in a large cohort using QuantiFERON assays, which are IFN-γ release assays (IGRAs) based on short-term whole blood culture. The study included 571 individuals receiving the viral spike (S) protein-expressing BNT162b2 mRNA vaccine. QuantiFERON assays revealed antigen-specific IFN-γ production in most individuals 8 weeks after the second dose. Simultaneous flow cytometric assays to detect T cells expressing activation-induced markers (AIMs) performed for 28 randomly selected individuals provided data correlating with the QuantiFERON data. Simultaneous IFN-γ enzyme-linked immunospot and AIM assays for another subset of 31 individuals, based on short-term peripheral blood mononuclear cell culture, also indicated a correlation between IFN-γ production and AIM positivity. These observations indicated the acquisition of T cell memory responses and supported the usability of IGRAs to assess cellular immunity. The QuantiFERON results were weakly correlated with serum IgG titers against the receptor-binding domain of the S protein and were associated with pre-vaccination infection and adverse reactions after the second dose. The present study revealed cellular immunity after COVID-19 vaccination, providing insights into the effects and adverse reactions of vaccination.

Accuracy of rapid antigen detection test for nasopharyngeal swab specimens and saliva samples in comparison with RT-PCR and viral culture for SARS-CoV-2 detection.

INTRODUCTION: Rapid antigen detection (RAD) tests are convenient tools for detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinics, and testing using saliva samples could decrease the risk of infection during sample collection. This study aimed to assess the accuracy of the SARS-CoV-2 RAD for testing of nasopharyngeal swab specimens and saliva samples in comparison with the RT-PCR tests and viral culture for detecting viable virus. METHODS: One hundred seventeen nasopharyngeal swab specimens and 73 saliva samples with positive results on RT-PCR were used. Residual samples were assayed using a commercially available RAD test immediately, and its positivity was determined at various time points during the clinical course. The concordance between 54 nasopharyngeal swab samples and saliva samples that were collected simultaneously was determined. Viral culture was performed on 117 samples and compared with the results of the RAD test. RESULTS: The positive rate of RAD test using saliva samples was low throughout the clinical course. Poor concordance was observed between nasopharyngeal swab specimens and saliva samples (75.9%, kappa coefficient 0.310). However, a substantially high concordance between the RAD test and viral culture was observed in both nasopharyngeal swab specimens (86.8%, kappa coefficient 0.680) and saliva samples (95.1%, kappa coefficient 0.643). CONCLUSIONS: The sensitivity of the SARS-CoV-2 RAD test was insufficient, particularly for saliva samples. However, a substantially high concordance with viral culture suggests its potential utility as an auxiliary test for estimating SARS-CoV-2 viability.

[Evaluation of Immature Platelet Fraction Measurements Using an Automated Hematology Analyzer, Sysmex XN].

The immature platelet fraction (IPF%) is a parameter for the automatic quantification of reticulated plate- lets and is considered to reflect platelet production. We evaluated IPF% measurements using an automated analyzer, the Sysmex XN. We measured the platelet counts and the IPF% values in 35 healthy subjects and 275 patients with various diseases using both the XN analyzer and a conventional analyzer, the Sysmex XE. Significant correlations in the platelet count and the IPF% were observed between the XN results and the XE results. In the same samples, significant inverse correlations were observed between the platelet count and the IPF% in both the XN and XE results. The coefficient of variation values for the platelet count and the IPF% measurements obtained using the XN analyzer were lower than those obtained using the XE ana- lyzer. In patients who had undergone hematopoietic stem cell transplantation, the IPF% measurements obtained using the XN analyzer increased several days before platelet recovery. IPF% measurements performed using the XN analyzer are adequate for clinical use. This parameter may be a useful marker for the prediction of platelet recovery. [Original].

The shortest isoform of C/EBPß, liver inhibitory protein (LIP), collaborates with Evi1 to induce AML in a mouse BMT model.

Ecotropic viral integration site 1 (Evi1) is one of the master regulators in the development of acute myeloid leukemia (AML) and myelodysplastic syndrome. High expression of Evi1 is found in 10% of patients with AML and indicates a poor outcome. Several recent studies have indicated that Evi1 requires collaborative factors to induce AML. Therefore, the search for candidate factors that collaborate with Evi1 in leukemogenesis is one of the key issues in uncovering the mechanism of Evi1-related leukemia. Previously, we succeeded in making a mouse model of Evi1-related leukemia using a bone marrow transplantation (BMT) system. In the Evi1-induced leukemic cells, we identified frequent retroviral integrations near the CCAAT/enhancer-binding protein ß (C/EBPß) gene and overexpression of its protein. These findings imply that C/EBPß is a candidate gene that collaborates with Evi1 in leukemogenesis. Cotransduction of Evi1 and the shortest isoform of C/EBPß, liver inhibitory protein (LIP), induced AML with short latencies in a mouse BMT model. Overexpression of LIP alone also induced AML with longer latencies. However, excision of all 3 isoforms of C/EBPß (LAP*/LAP/LIP) did not inhibit the development of Evi1-induced leukemia. Therefore, isoform-specific intervention that targets LIP is required when we consider C/EBPß as a therapeutic target.

[The current status of genetic testing for leukemia].

The pathogenic chromosome translocations present in various hematological malignancies result in the formation of fusion genes, which are detected by a reverse transcription-polymerase chain reaction method. Furthermore, with this method, it is possible to sensitively detect minimal residual disease (MRD), which is difficult by a morphological testing. It has now been established that the detection of MRD is important for the diagnosis, treatment policy, evaluation of the prognosis, and monitoring of leukemia. In particular, quantitative analysis of MRD is important for evaluation of the curative effect and prediction of recurrence. In addition, mutation analysis is valuable to decide on the therapeutic protocol for imatinib-resistant patients, and the stratification of treatment for acute myeloid leukemia. At present, however, there is no standard laboratory procedure for genetic testing for leukemia. Here, the problems related to external precision management and analytical error are discussed.

[Performance and clinical evaluation of antinuclear antibody test based on fluorescence enzyme immunoassay].

BACKGROUND: The measurement of antinuclear antibodies (ANA) is used for screening of connective tissue diseases (CTD) in the laboratory. ANA detection is performed by indirect immunofluorescence (IF) assay on HEp-2 cells from human larynx carcinoma. However, it lacks specificity for the identification of specific diseases and antigen reactivity. The aim of the present study was to evaluate the EliA CTD Screen (EliA), a new enzyme fluoroimmunoassay (Phadia AB, Uppsala, Sweden) for detection of ANA in human serum. PATIENTS AND METHODS: The study involved a total of 732 serum samples, 200 from healthy donors, 297 from patients with CTD and 235 from patients with rheumatoid arthritis, vasculitis syndrome and relative disease of CTD. For all sera, ANA was measured by IF, commercial assay (MESACUP) and EliA. RESULT: The sensitivity and specificity of EliA were 73.7% and 78.7%, respectively, whereas those of MESACUP were 80.8% and 64.7%, respectively. Area under the receiver operating curves for EliA, MESACUP and IF were 0.821, 0.786 and 0.730, respectively. The concordance rate between EliA and MESACUP was 84.2%. These discrepancies between those 2 assays were found in 84 sera. Further investigation were done by each ANA antigen tests for the discrepant results of EliA in 83 sera. The discrepancies might be occurred by antigen difference or non-specific response. CONCLUSION: AUC results showed that the diagnostic performance of EliA was superior to MESACUP and IF. EliA had a good performance as method for screening of CTD.

[Difference in measurements of erythrocyte sedimentation rate obtained using two devices of the same model in a patient with marked leukocytosis].

The erythrocyte sedimentation rate (ESR) has been used as an index for inflammatory conditions, such as infectious diseases, autoimmune diseases, and malignancies. The ESR values of a 37-year-old male with marked leukocytosis due to chronic myeloid leukemia showed remarkable differences between two devices of the same model (Ves-Matic 30, DIESSE Diagnostica Senese). From the appearance of the tested tube after the ESR measurement, the values obtained using one device might have been falsely low, whereas the values obtained using the other device were likely to have been accurate. The difference of the ESR values between the two devices might have occurred by the false detection of transmitted light during the transition from the erythrocyte layer to the leukocyte layer. These findings suggest that in cases with marked leukocytosis the accuracy of ESR should be confirmed with the appearance of the test tube.

Antagonism of sphingosine 1-phosphate receptor 2 causes a selective reduction of portal vein pressure in bile duct-ligated rodents.

UNLABELLED: Sinusoidal vasoconstriction, in which hepatic stellate cells operate as contractile machinery, has been suggested to play a pivotal role in the pathophysiology of portal hypertension. We investigated whether sphingosine 1-phosphate (S1P) stimulates contractility of those cells and enhances portal vein pressure in isolated perfused rat livers with Rho activation by way of S1P receptor 2 (S1P(2) ). Rho and its effector, Rho kinase, reportedly contribute to the pathophysiology of portal hypertension. Thus, a potential effect of S1P(2) antagonism on portal hypertension was examined. Intravenous infusion of the S1P(2) antagonist, JTE-013, at 1 mg/kg body weight reduced portal vein pressure by 24% without affecting mean arterial pressure in cirrhotic rats induced by bile duct ligation at 4 weeks after the operation, whereas the same amount of S1P(2) antagonist did not alter portal vein pressure and mean arterial pressure in control sham-operated rats. Rho kinase activity in the livers was enhanced in bile duct-ligated rats compared to sham-operated rats, and this enhanced Rho kinase activity in bile duct-ligated livers was reduced after infusion of the S1P(2) antagonist. S1P(2) messenger RNA (mRNA) expression, but not S1P(1) or S1P(3) , was increased in bile duct-ligated livers of rats and mice and also in culture-activated rat hepatic stellate cells. S1P(2) expression, determined in S1P 2LacZ/+ mice, was highly increased in hepatic stellate cells of bile duct-ligated livers. Furthermore, the increase of Rho kinase activity in bile duct-ligated livers was observed as early as 7 days after the operation in wildtype mice, but was less in S1P 2-/- mice. CONCLUSION: S1P may play an important role in the pathophysiology of portal hypertension with Rho kinase activation by way of S1P(2) . The S1P(2) antagonist merits consideration as a novel therapeutic agent for portal hypertension.

Perihepatic lymph node enlargement observed at a general health examination: A cross-sectional study.

AIM: Although perihepatic lymph node enlargement (PLNE) is known as a common finding in chronic liver disease, it can be found occasionally at a general health examination. We aimed to clarify the clinical significance of PLNE in general. METHODS: Between January 2008 and December 2011, 4234 subjects were enrolled, who underwent a general health examination at the University of Tokyo Hospital. RESULTS: PLNE was observed in 69 (1.6%) subjects, among whom 17 (0.4%) had liver disorders and 13 (0.3%) had malignancy, one of whom had both. No disorders were determined in the remaining 40 subjects (0.9%). Among 17 subjects with liver disorder-associated PLNE, anti-hepatitis C virus (HCV) antibody was determined in 11 and serum alanine aminotransferase levels were less than 40 U/L in eight. Among 13 subjects with malignancy-associated PLNE, para-aortic lymph nodes were also enlarged in eight. Among 40 subjects with PLNE of unknown etiology, 27 could be followed up for the mean period of 2.08 years, where no underlying disorders were newly determined with largely unaltered size of PLNE. CONCLUSION: The incidence of PLNE in the general population may vary with the prevalence of chronic liver disease, especially HCV infection. When PLNE is observed, liver disorders should be first surveyed including HCV infection even with normal serum alanine aminotransferase levels. PLNE with para-aortic lymph node enlargement may be suggestive of a malignant lesion. The incidence of PLNE of unknown etiology may be approximately 1% in the general population, which may be just followed up without further change.

Perihepatic lymph node enlargement is a negative predictor for sustained responses to pegylated interferon-α and ribavirin therapy for Japanese patients infected with hepatitis C virus genotype 1.

AIM: Although perihepatic lymph node enlargement (PLNE) is reportedly associated with the negative outcome of interferon therapy for chronic hepatitis C, there were limitations in that the results were obtained in patients with various genotypes, viral loads and treatment regimens. We aimed to precisely clarify the significance of PLNE in interferon therapy for chronic hepatitis C. METHODS: Between December 2004 and June 2005, 112 patients with hepatitis C virus (HCV) genotype 1 and HCV RNA of more than 100 KIU/mL were enrolled, who underwent pegylated interferon-α plus ribavirin therapy thereafter. PLNE was defined as a perihepatic lymph node of more than 1 cm in the longest axis by ultrasonography. RESULTS: The sustained virological response (SVR) rate was lower in patients with PLNE (4/22, 18.2%) than in those without (37/90, 41.1%; P = 0.045) and viral load decline was smaller in patients with PLNE than in those without (P = 0.028). The proportion of PLNE positive patients was the smallest in the SVR group (P = 0.033) among the patient groups divided by the treatment outcome. PLNE was retained as a negative predictor for SVR by multivariate logistic regression analysis (P = 0.012). Furthermore, PLNE was not significantly associated with the mutations at HCV core protein and at interferon sensitivity-determining region, or interleukin-28B polymorphism in 45 patients with HCV genotype 1, enrolled between December 2011 and March 2012. CONCLUSION: PLNE is a negative predictor for SVR in patients with HCV genotype 1 and HCV RNA of more than 100 KIU/mL treated with pegylated interferon-α plus ribavirin, independent of other known predictors for SVR.

Sphingosine 1-phosphate release from platelets during clot formation: close correlation between platelet count and serum sphingosine 1-phosphate concentration.

BACKGROUND: Sphingosine 1-phosphate (Sph-1-P), abundantly stored in platelets and released extracellularly upon activation, plays important roles as an extracellular mediator by interacting with specific cell surface receptors, especially in the area of vascular biology and immunology/hematology. Although the plasma Sph-1-P level is reportedly determined by red blood cells (RBCs), but not platelets, this may not be true in cases where the platelets have been substantially activated. METHODS AND RESULTS: We measured the Sph-1-P and dihydrosphingosine 1-phosphate (DHSph-1-P) levels in serum samples (in which the platelets had been fully activated) from subjects with (n = 21) and without (n = 33) hematological disorders. We found that patients with essential thrombocythemia exhibited higher serum Sph-1-P and DHSph-1-P concentrations. The serum Sph-1-P concentration was closely correlated with the platelet count but was very weakly correlated with the RBC count. Similar results were obtained for DHSph-1-P. The serum Sph-1-P and DHSph-1-P levels were inversely correlated with the level of autotaxin (ATX), a lysophosphatidic acid-producing enzyme. A multiple regression analysis also revealed that the platelet count had the greatest explanatory impact on the serum Sph-1-P level. CONCLUSIONS: Our present results showed close correlations between both the serum Sph-1-P and DHSph-1-P levels and the platelet count (but not the RBC count); these results suggest that high concentrations of these sphingoid base phosphates may be released from platelets and may mediate cross talk between platelet activation and the formation of atherosclerotic lesions.

[Future roles of clinical laboratories and clinical laboratory technologists in university hospitals].

Clinical laboratories in university hospitals should be operated with a good balance of medical practice, education, research, and management. The role of a clinical laboratory is to promptly provide highly reliable laboratory data to satisfy the needs of clinicians involved in medical practice and health maintenance of patients. Improvement and maintenance of the quality of the laboratory staff and environment are essential to achieve this goal. In order to implement these requirements efficiently, an appropriate quality management system should be introduced and established, and evaluated objectively by a third party (e.g. by obtaining ISO 15189 certification). ISO 15189 is an international standard regarding the quality and competence of clinical laboratories, and specifies a review of the efficient operational system and technical requirements such as competence in implementing practical tests and calibration. This means the results of laboratory tests reported by accredited laboratories withstand any international evaluation, which is very important to assure the future importance of the existence and management of clinical laboratories as well as internationalization of medical practice. "Education" and "research" have important implications in addition to "medical practice" and "management", as the roles that clinical laboratories should play in university hospitals. University hospital laboratories should be operated by keeping these four factors in good balance. Why are "education" and "research" required in addition to "medical practice" services? If individual clinical laboratory technologists can provide an appropriate response to this question, the importance of the existence of clinical laboratories would be reinforced, without being compromised.

[Sphingolipids, possible biomarkers for atherosclerotic disorders].

Sphingolipids have been recently elucidated to be not only mere components of the plasma membrane but also bioactive mediators which can induce various biological responses. Among these lipids, sphingomyelin(SM) and sphingosine 1-phosphate (Sph-1-P) are proposed to be involved in the pathogenesis of atherosclerosis: SM is abundant in atherosclerotic lesions and Sph-1-P is bound to HDL and attributes to the anti-atherosclerotic properties of HDL at least partly. Therefore, Sph-1-P and SM can be useful biomarkers for atherosclerotic disorders. However, at present, the measurement of Sph-1-P and SM levels has not been brought into clinical practice, yet. The main obstacle is the difficulty in measuring these sphingolipids precisely, rapidly, and conveniently. Recently, we have developed new methods for measuring Sph-1-P (HPLC method) and SM (enzymatic method). These methods are easy to be introduced into clinical laboratory testing because they do not need any special techniques and equipment. With this method for SM, we have demonstrated that the SM concentration was significantly higher in subjects with acute coronary syndrome. In this paper, we reviewed the possibility of sphingolipids as biomarkers for atherosclerotic disorders.

Humoral and cellular immune response dynamics in Japanese healthcare workers up to six months after receiving a third dose of BNT162b2 monovalent vaccine.

Longitudinal data on the immune response from the first dose to several months after the third dose of COVID-19 vaccine are limited. We analyzed the immune response in 406 Japanese healthcare workers who received at least three doses of vaccine. The geometric mean anti-receptor binding domain IgG antibody titers and antigen-stimulated T-cell interferon-gamma levels after 6 months after receiving a third dose were similar to those 8 weeks after receiving a second dose. Humoral and cellular immunity induced by the third dose was more durable than that induced by the second dose. UMIN Clinical Trials Registry ID: UMIN000043340.

Increased activity of serum mitochondrial isoenzyme of creatine kinase in hepatocellular carcinoma patients predominantly with recurrence.

BACKGROUND & AIMS: Mitochondrial isoenzyme of creatine kinase (MtCK) is reportedly highly expressed in hepatocellular carcinoma (HCC). Clinical relevance of serum MtCK activity in patients with HCC was assessed using a novel immuno-inhibition method. METHODS: Among patients with cirrhosis caused by hepatitis B or C virus, 147 patients with HCC (12 with the first occurrence and 135 with recurrence) and 92 patients without HCC were enrolled. RESULTS: Serum MtCK activity was higher in cirrhotic patients with HCC than in those without HCC or healthy subjects. Elevated serum MtCK activity in HCC patients decreased after radiofrequency ablation. In case of prediction of HCC, MtCK had a sensitivity of 62.6% and a specificity of 70.7% at a cut-off point of 8.0 U/L, with an area under the receiver operating curve of 0.722 vs. 0.713 for alpha-fetoprotein (AFP) and 0.764 for des-gamma-carboxy prothrombin (DCP). Among the HCC patients, serum MtCK activity was elevated in 52.9% individuals with serum AFP level < 20 ng/ml and 63.2% individuals with serum DCP level < 40 mAu/ml. Even in patients with a single HCC ≤ 2 cm, the sensitivity of serum MtCK activity for the prediction of HCC was 64.4%, which was comparable to the overall sensitivity. This increased activity was due to an increase in ubiquitous MtCK, not sarcomeric MtCK, and the enhanced mRNA expression of ubiquitous MtCK was observed in cell lines originating from HCCs in contrast to healthy liver tissues. CONCLUSIONS: Serum MtCK activity merits consideration as a novel marker for HCC to be further tested as for its diagnostic and prognostic power.

Analysis for the combination expression of CK20, FABP1 and MUC2 is sensitive for the prediction of peritoneal recurrence in gastric cancer.

Prediction of peritoneal recurrence in gastric cancer patients is important for application of adjuvant chemotherapy. After surgery, occasional patients have peritoneal recurrence despite negative cytology of the peritoneal washings. Thus, molecular detection of a subliminal number of cancer cells in peritoneal washings may overcome the sensitivity limitation of conventional cytology. In this study, expressions of five specific marker genes, namely, TFF1, TFF2, CK20, FABP1 and MUC2, were evaluated for their usefulness as markers of micro-dissemination. It was found that reverse transcriptase-polymerase chain reaction for these five genes yielded results highly specific for the depth of invasion and disease stage. Furthermore, the expression of CK20, FABP1 and MUC2 was a reliable prognostic indicator of peritoneal metastasis. Our results suggest that evaluation of the expression of CK20, FABP1 and MUC2 in peritoneal washings is a useful tool for identifying patients at high risk of peritoneal recurrence who may need adjuvant chemotherapy.

[Study on the analytical error factors and evaluation of an internal control gene for leukemia gene expression analysis].

Quantitative analysis of the leukemia fusion gene by real-time PCR is a sensitive method to monitor minimal residual disease; the data obtained are very useful to evaluate the disease stage and prognosis, contributing to the clinical practice of hematology. However, there is no standard laboratory procedure for leukemia genetic testing. Therefore, this genetic testing has some problems related to precision management. To minimize analytical error factors, normalization by an internal control gene is necessary. Additionally, it is important to choose a gene suitable for leukemia gene expression analysis because the expression of an internal standard gene changes due to various factors. In this study, we examined analytical error factors (RNA extraction efficiency, reverse transcription reaction efficiency) and evaluated an internal control gene. As a result, in RNA extraction, the extraction efficiency of the acid-guanidium-phenol-chloroform (AGPC) method was high compared to the silica method. The reverse transcription reaction efficiency was significantly different with each reaction reagent. Furthermore, since three kinds of gene (18s rRNA, GUS, beta-actin) had few differences between samples, they were considered to be suitable as internal standards.

[Condition setting for the measurement of blood coagulation factor XIII activity using a fully automated blood coagulation analyzer, COAGTRON-350].

The automated laboratory analyzer COAGTRON-350 (Trinity Biotech) is used for routine and specific coagulation testing for the detection of fibrin formation utilizing either mechanical principles (ball method) or photo-optical principles, chromogenic kinetic enzyme analysis, and immune-turbidimetric detection systems in one benchtop unit. In this study, we demonstrated and established a parameter for the measurement of factor XIII (FXIII) activity using Berichrom FXIII reagent and the COAGTRON-350 analyzer. The usual protocol used for this reagent, based on the handling method, was slightly modified for this device. The analysis showed that fundamental study for the measurement of FXIII activity under our condition setting was favorable in terms of reproducibility, linearity, and correlation with another assays. Since FXIII is the key enzyme that plays important roles in hemostasis by stabilizing fibrin formation, the measurement of FXIII is essential for the diagnosis of bleeding disorders. Therefore, FXIII activity assessment as well as a routine coagulation testing can be conducted simultaneously with one instrument, which is useful in coagulopathy assessment.

[Usefulness of hepatitis C virus (HCV) genotype test using direct sequencing of 5' untranslated region--application from 2003 to 2011].

Hepatitis C virus (HCV) genotype is the key to prediction of the therapeutic effect of combination therapy of pegylated interferon and ribavirin. HCV grouping test, which is used as a routine testing method, is simple and practical, but the results are sometimes not conclusive. In order to cope with such cases, we have adopted a direct sequencing method since 2003. This paper summarizes the HCV genotype test results obtained between 2003 and 2011. The results showed that the genotypes of 129 of 255 specimens which were not determined by the HCV grouping test were all successfully determined by the direct sequencing method. In the serotype determined samples the concordance rate with direct sequencing was 99.0% in HCV serotype 1 and 88.0% in HCV serotype 2. These findings suggest that the direct sequencing method is useful to determine the genotypes of specimens when untypeable with the HCV grouping test.