What is the best wavelength for the measurement of hemolysis index?

BACKGROUND: Hemolysis is a common problem in the handling of serum specimens. The hemolysis index (HI) provides a warning of hemolysis in auto-analyzers. However, HI has not been standardized, and each laboratory's original method is applied. Especially, the wavelength used for HI measurement is different in each laboratory. Thus, we investigated the warning ability of HI at various wavelengths. METHODS: We selected 4 wavelength types, and each HI was measured and calculated (410 nm/HI-1, 451 nm/HI-2, 545 nm/HI-3, and 571 nm/HI-4). To compare the 4 HI types, we investigated the influence of 3 interference components using artificially hemolyzed specimens (AHSs). We also investigated both the relationship between HI and hemoglobin concentration (Hb) and that between HI and 31 biochemical test values in AHSs. RESULTS: In the interference assessment, only HI-4 showed no influence on the 3 interference components. The correlation between Hb and HI-4 was very strong (rS = 0.9987). A 1-unit increase in HI-4 corresponded to a 14.8-mg/dL increase in Hb. CONCLUSION: We found the best wavelength for HI to be at or near 571 nm.

Establishment of a Screening Method for Epstein-Barr Virus-Associated Gastric Carcinoma by Droplet Digital PCR.

BACKGROUND: Epstein-Barr virus-associated gastric carcinoma (EBVaGC) is classified as one of the molecular subtypes of gastric cancer. We used droplet digital polymerase chain reaction (ddPCR) to enable highly sensitive and quantitative detection of EBV. METHODS: EBV-DNA load was calculated based on the copy number of the BamH1-W fragment of EBV by ddPCR, and the cut-off value of EBV-DNA load was set. We conducted both ddPCR and EBER1 ISH to examine whether their results coincided in 158 gastric cancer specimens of unknown EBV status. We prepared 26 biopsy specimens and 49 serum samples including EBVaGC and assayed them by ddPCR. RESULTS: The median values of EBV-DNA load for EBVaGC and EBV-negative control were 17.0 and 0.00308, respectively. A cut-off value of 0.032 was determined for which the sensitivity was 1. Among the 158 gastric cancer specimens, 14 lesions were judged as EBV-positive by the 0.032 cut-off value determined by ddPCR. The results of ddPCR and EBER1 ISH were in complete agreement. Even when using a biopsy specimen as a sample for ddPCR, the EBV-DNA load of all EBVaGCs was larger than the cut-off value. CONCLUSIONS: We established a new method of diagnosing EBVaGC from tissue samples by ddPCR.

Clinicopathologic Characteristics of Epstein-Barr Virus-Associated Gastric Cancer Over the Past Decade in Japan.

: Epstein-Barr virus (EBV) is a ubiquitous human herpes virus, but related with several types of malignancies. Among EBV-related malignancies, EBV-associated gastric carcinoma (EBVaGC) has the largest patient's number. We screened for EBV infection in 1067 GC lesions of 1132 patients who underwent surgical resection from 2007 to 2017 in Japan and examined clinicopathological features of EBVaGC. EBV infection was detected by in situ hybridization with EBV-encoded small RNA 1(EBER-1 ISH). EBV was infected in 80 GC lesions (7.1%). Mean age was significantly lower in patients with EBVaGC than with EBV-negative GC. EBVaGC was more frequent in men than in women. EBVaGC was found twice as frequent in the upper or middle stomach as in the lower stomach. Early EBVaGC was more frequent, and submucosally invaded cases were dominant. The presence of lymphatic vessel invasion was less in EBVaGC, but frequency of lymph node metastasis was similar. Carcinoma with lymphoid stroma (CLS) was found in 3.8% (43/1132) of all lesions with 60.5% of EBV positivity. The synchronous or metachronous multiple GC was frequent in EBVaGC. We clarified clinicopathologic characteristics of EBVaGC over the past decade in Japan. EBV infection should be examined in gastric cancer cases showing these characteristics.

A new Krüppel-like factor 1 mutation (c.947G > A or p.C316Y) in humans causes ß-thalassemia minor.

Here we describe a Japanese patient with mild ß-thalassemia (ß-thal) with an intact ß-globin gene but a new missense mutation of c.947G > A or p.C316Y in the erythroid Krüppel-Like Factor (KLF1) gene which is strongly associated with the expression of the ß-globin gene. The association of the KLF1 mutation with ß-thal, is here described. The p.C316Y mutation occurred at one of the cysteines that constitute the second zinc finger motif of KLF1, and would have changed the zinc finger conformation to impair the DNA binding properties or the promoter function of the ß-globin gene. Our expression study found that the mutant KLF1 gene had a markedly negative effect on the ß-globin gene expression, or 7.0% of that of its normal counterpart. A presumed heterozygous state, or equimolar presence of the mutant and normal KLF1s reduced the expression rate to 70.0% of the normal alone. This degree of the decrease may explain the very mild phenotype of the patient's ß-thal. Furthermore, the patient's whole-exome analysis using next-generation sequencing revealed that the ß-thal defect is caused by only this KLF1 gene mutation. The Hb A2 and Hb F levels that are frequently elevated in KLF1 mutations were elevated by 4.1 and 1.3%, respectively, in this case. The contribution to their elevation by KLF1: p.C316Y is uncertain.

Oxidation status of ß-thalassemia minor and Hb H disease, and its association with glycerol lysis time (GLT50).

ß-Thalassemia (ß-thal), especially ß-thalassemia major (ß-TM), is reported to be related to reactive oxygen species (ROS) and enhanced oxidation status. It is reflected by increased malondialdehyde (MDA), by membrane lipid peroxidation and decreased by the newly developed total antioxidant capacity (TAC). However, there is less evidence for ß-thal minor and Hb H (ß4) disease on its association with oxidation status. On the other hand, hemolysis by glycerol lysis time (GLT50) is invariably prolonged in thalassemia. The reason for the prolongation of GLT50 is not well understood. The aim of this study was to investigate the oxidation state in ß-thal minor and Hb H disease and to find out the association of the oxidation with the prolongation of GLT50. Blood samples from 39 subjects (33 with ß-thal minor, six with Hb H disease) were collected from individuals living in Japan. The clinical screening tests and molecular identification of the thalassemias were performed. Malondialdehyde and TAC were measured using spectrophotometric analyses. In ß-thal minor and Hb H disease, the plasma MDA level was significantly elevated and the TAC reduced. A highly reversed correlation between MDA and TAC was noted. Their GLT50 levels were evidently prolonged, and the GLT50 has significant correlations with MDA and TAC. ß-Thalassemia minor and mild Hb H disease are evidently in a milieu of reduced redox state, and GLT50 prolongation in ß-thal minor and Hb H disease has a close correlation with the oxidation state, possibly by oxidative impairment of the membrane protein of the red cell.

A new ß(0)-thalassemia mutation (codon 102, AAC>ATCAC) in coexistence with a heterozygous P4.2 Nippon gene.

A new ß-thalassemia (ß-thal) frameshift mutation was found at codon 102 (AAC>ATCAC) in a 17-year-old Japanese male and his 14-year-old sister. Both demonstrated a more severe phenotype than the usual ß-thal minor with mild hemolytic involvement. No mRNA derived from the thalassemic allele, or ß(T)mRNA, was detected in the sequencing analysis of the whole mRNA (cDNA). However, the ß(T)mRNA from the whole ßmRNA was specifically amplified by amplification refractory mutation system (ARMS), and was actually found to be present. Furthermore, quantitative polymerase chain reaction (q-PCR) demonstrated a negligible amount of ß(T)mRNA. Thus, their more severe phenotype was not caused by the "dominant type" ß-thal in which a considerable amount of the ß(T)mRNA would be expected. In fact, our proband had a total ßmRNA level that was mostly normal. Thus, the cause of a ß-thal phenotype by the frameshift mutation was ascribed to the reduced amount of mRNA. We further searched for the cause of their severe phenotype. However, factors that exacerbated the phenotype of ß-thal, such as α-globin gene triplication, coexisting iron deficiency and infection were not found. Finally, we noticed that the red cell morphology revealed ovalocytosis and small numbers of stomatocytes that were seen in the hereditary spherocytosis (HS), especially by P4.2 mutations. The sequence of the P4.2 gene disclosed heterozygous P4.2 Nippon, or missense mutation at codon 142 (GCT>ACT) on exon 3, the most common mutation of Japanese HS. Frequent mutations of other membrane proteins, Band 3 and ankyrin that are common cause of HS in the Japanese population, other than P4.2, were not detected. When HS by P4.2 Nippon develops it is homozygous, and no P4.2 protein is observed in sodium dodecilsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), while in our case the amount of the P4.2 was almost normal in the SDS-PAGE. However, there are several reports that revealed more severe phenotypes of ß-thal by the coexisting abnormality of membrane protein. It is uncertain, but the presence of heterozygous P4.2 Nippon may be associated with the exacerbation of the phenotype of ß-thal minor.