Tetrodotoxin (TTX) is a neurotoxin mainly found in pufferfish. Although food poisoning caused by TTX has been reported in recent years, TTX measurement in biological samples derived from patients has rarely been performed at medical facilities. Therefore, we attempted to establish a high-performance liquid chromatography (HPLC) analytical method using an anion-exchange column to detect and quantify TTX. First, TTX samples (0.5–50 μg/mL) diluted in citrate buffer solution were analyzed by HPLC. According to this method, TTX chromatograms were detected in the retention time range of 0.1–0.4 min. Furthermore, TTX in citrate buffer could be detected in the concentration range of 1.0–50 μg/mL, and the standard curve was successfully created on the basis of the obtained results. Next, we examined whether TTX diluted in human serum or urine could be detected and quantified. Despite deproteinization, TTX in serum could not be measured. In urine samples, TTX was detected in the concentration range of 5.0–50 μg/mL; however, the calculated value was lower than the actual TTX concentration. Next, the calculated TTX concentration was corrected by applying the recreated standard curve based on the measurement results of TTX samples diluted in urine. In conclusion, this HPLC analytical method may be adapted to urine samples from patients with TTX poisoning.

Hippuric acid (HA) is excreted in urine as a metabolic product of toluene, and HA measurement is the most popular health diagnostic test for special workers using organic solvents. This test is required by Japanese law to be carried out at least once every six months. Currently, HA is measured by high-performance liquid chromatography (HPLC). Measurement by HPLC requires a special instrument, and the test measurement output per unit time is smaller owing to batch processing. For high-speed processing and labor saving, we developed a new enzymatic reagent for HA measurement using an open-type automatic analyzer. The HPLC method was studied using JCA-BM8040G (JEOL, Tokyo, Japan), a clinical biochemistry analyzer, and we found that this method showed good accuracy, linearity, and sufficient LOD. In addition, there is a good correlation between the HPLC method and the absence of influence by methyl hippuric acid, which has a similar structure to HA. These findings suggest the usefulness of this new method for health diagnostics.

In secondary antiphospholipid syndrome (APS) associated with SLE, a wide variety of antiphospholipid antibodies appear in the blood, which cause repeated serious arterial and/or venous thrombotic complications. Our previous study revealed that IgG fractions purified from APS patients promote thrombus formation by increasing the levels of monocyte surface TF expression and inflammatory cytokine production. In this study, we investigated the effects of inflammatory and anti-inflammatory cytokines present in SLE patients’ sera on thrombus formation caused by IgG-aPLs. After pretreatment of healthy human monocytes with various cytokines, we analyzed the changes in monocyte surface TF expression levels caused by IgG-aPLs. Monocyte surface TF expression was assessed by measuring TF on the surface of CD14-positive cells by flow cytometric analysis. We found that the pretreatment of monocytes with TNF-α, IL-1β, and IL-6 caused a positive priming effect (increasing the level of TF expression in monocytes). On the other hand, the pretreatment with IL-10 caused a negative priming effect (suppression of TF expression in monocytes). In SLE patients, many components of the immune system, including monocytes, T cells, autoantibodies, and cytokines secreted by cells, are considered to be involved in the pathologic processes that underlie the development of thrombotic complications. The results of our study suggest that IgG-aPLs cause persistently high TF expression levels on monocyte surfaces by interacting with inflammatory cytokines, which may be an important mechanism in the pathogenesis of recurrent arterial and/or venous thrombotic complications peculiar to SLE patients.

Some drugs have severe adverse effects such as impaired consciousness and shock. Drug testing is effective for determining the causes of severe adverse effects, but the analyses of drugs require precision instruments that are complicated and expensive to operate and need time and expertise to achieve proficiency and obtain results. Therefore, analytical methods are not easy to establish in any laboratory. Given this background, we have calculated the factors related to the severity of adverse effects by univariate analysis using the data from 197 drug-addicted patients who were brought to the Kawasaki Medical School Emergency Medical Center, for the purpose of developing a universal method using pathologic parameters for laboratories to test, such as clinical laboratory results, parameters, vital signs, medication, and treatment histories. We have derived the equation that can be used to predict the necessity of treatments of severe cases by multivariate logistic regression analysis using these parameters. Furthermore, we have calculated the validity by ROC analysis with multiple patterns from the obtained factors. For the prediction of severity, CK-MB level, APTT, monocytes %, overdose, and GCS score were the important factors. As suggested by ROC analysis, although 0.701 was only based on patient background and 0.700 on the clinical laboratory results, 0.789 on the combined analyses was considered to indicate a high validity of discrimination level.

Urinary hyaline casts are the most frequently encountered type of casts and are found in small numbers in healthy individuals. Although the detection of hyaline casts in urine has recently been reported to be useful for the estimation of various pathological conditions, the mechanism of formation and components of hyaline casts are not fully understood. In this study, we investigated various in vitro conditions for hyaline cast formation and used laser microdissection to identify the constituents of hyaline casts. The number of hyaline casts was significantly increased under the following conditions: (1) acidic pH, (2) increased protein concentration, (3) concentrated urine, and (4) 24 hours of stagnation. Interestingly, the generated hyaline casts were formed as a mold. Proteins were identified by mass spectrometry in hyaline and waxy casts collected by laser microdissection. Seventy-eight different proteins were detected in both types of cast. The number of proteins significantly detected in the hyaline casts was 18, the same number of proteins detected in both casts was 41, and the number of proteins significantly detected in the waxy casts was 19. Importantly, the most predominant protein in the hyaline casts was uromodulin. On the basis of the identified protein information, the proteins required for hyaline cast composition were examined, and the addition of transferrin enabled the generation of hyaline casts similar to those found in clinical specimens. These results provide the basis for the mechanism of hyaline cast appearance and may be useful in understanding the relationship between hyaline casts and pathophysiology.

Currently, the adequacy and reproducibility of specimen staining is subjectively assessed with microscopic observation by medical technologists or pathologists. However, this method is highly dependent on individual experience, skills, and preference, making standardization extremely difficult. Therefore, it has become necessary to establish an objective method. Accordingly, an analysis application was developed to calculate and compare representative color values from photographed images of stained specimens. This application features automated and easy handling of the analytical processes of stained specimen quantifications, analyses, and plotting. Furthermore, Microsoft Excel is used to simplify the introduction of the application, as it is well known and commonly used. Six different comparative methods are available for analysis, using HSV color space, CIE L*a*b* color space, and the CIE ΔE2000 color-difference formula that take into consideration human perceptual characteristics. The application also includes filters for excluding backgrounds and for choosing colors. By selecting and analyzing these features appropriately, one can easily and consistently compare and evaluate many stained specimens. The use of this application to manage staining conditions is expected to contribute to the promotion of the standardization of stained specimens and internal quality control.

One of the complications of carotid endarterectomy (CEA) is cerebral ischemia caused by the clumping of the internal carotid artery. The incidence of cerebral ischemia depends more on the blood supply from other arteries than on the operated internal carotid artery. We investigated the relationship between the structure of the Willis artery circle on magnetic resonance angiography (MRA) images and the attenuation of the amplitudes of somatosensory (SEP)- and motor (MEP)-evoked potentials during the clumping of the internal carotid artery. The subjects were 205 patients who underwent SEP and MEP intraoperative monitoring during CEA. They were classified into four groups according to the MRA pattern of the anterior part of the Willis artery circle, i.e., the cerebral artery origin (A1) and the anterior communicating artery (A-com). During the clumping of the internal carotid artery, the SEP and MEP amplitudes decreased by 50% or more,in comparison with those before clumping. The incidence of SEP/MEP reduction of more than 50% was 6.1% in the group with bilateral A1 and A-com on MRA, 21.1% in the group with no A1 on the operated side, 50.0% in the group with no A1 on the non-operated side, and 100% in the group with no A-com. The presence or absence of blood supply from the non-operated to the operated side was one of the factors related to cerebral ischemia during the clumping of the internal carotid artery. The absence of A1 or A-com on MRA on the non-operated side indicates a high risk of cerebral ischemia during the clumping of the internal carotid artery in CEA.

The digital polymerase chain reaction (dPCR) method is used to quantify the absolute numbers of genes by distributing the samples to each small well and performing PCR. To date, although there are several reports showing the usefulness of amplification of genes of pathogens in the diagnosis of pulmonary tuberculosis by dPCR, there have been only a few reports of genetic diagnosis for pulmonary Mycobacterium avium complex (pMAC) disease by the dPCR method. In this study, we compare the usefulness of the dPCR method in pMAC disease diagnosis using bronchial washing samples with the gold standard culture method and the TRC method. From March 2019 to February 2021, we recruited 135 patients in this study (mean age ± SD: 69.8 ± 10.1; males, 29; females, 106). We conducted dPCR, culture, and TRC methods using their bronchial washing samples and compared the concordance rates among the three. We also investigated the correlation between the number of dPCR-positive wells and the amount of acid-fast bacilli present in the sample smear. The concordance rate between the dPCR method and the culture method was 93% (sensitivity, 100%; specificity, 87%) and that between the dPCR method and the TRC method was 96% (sensitivity, 97%; specificity, 96%). The number of dPCR-positive wells was significantly correlated with smear samples from 0 to 3 Gaffky scale groups. The dPCR method for pulmonary MAC diagnosis has high sensitivity and the same detectability as the TRC method, which makes it useful for the diagnosis of pMAC disease.

Mycoplasma pneumoniae pneumonia is one of the most common community-acquired pneumonias and accounts for 10–20% of the pneumonia cases among children. The precise and timely detection of M. pneumoniae is critical because delayed diagnosis will cause disease exacerbation and prolonged treatment partly because of the development of macrolide resistance. We compared the bacteria detection ability of Smart Gene^®, a fully automated genetic analyzer based on the QP method, with that of the conventional QP method used in our laboratory. M. pneumoniae culture strains of known concentrations were used and 104 patients with suspected mycoplasma pneumonia were recruited in this study. ①M. pneumoniae culture strains were serially diluted with physiological saline and analyzed by both methods and the minimum detection limits were compared between these two methods. The minimum detection sensitivities were almost the same for both methods. ②We compared the detection ability and the presence of resistance mutations using pharyngeal swabs. Both methods showed good agreement in terms of sensitivity, specificity, and match rate of detection and resistance mutation. ③When cryopreserved M. pneumoniae DNA specimens extracted from residual specimens after analysis using the mycoplasma antigen rapid diagnostic kit were used, the sensitivity, specificity, and match rate of Smart Gene^® were 75.9%, 100.0%, and 87.3%, respectively. The results of both methods were consistent for the presence of resistance mutations. Smart Gene^® can detect M. pneumoniae infection and the presence of macrolide-resistant mutations in a short time, and it is considered to be a highly useful test method in mycoplasma treatment because it can contribute to the early diagnosis and appropriate use of antimicrobial agents.

T&T olfactometry (T&T) is important for assessing the degree of olfactory impairment and the effect of treatment. Although T&T is a noninvasive test, it is complicated to perform, and the results may vary depending on the technique and judgment of the examiner. In this study, we attempted to standardize the examination according to our original T&T flowchart. Sixty-four patients (41 with chronic sinusitis, two with allergic rhinitis, one had trauma, four with neurodegenerative diseases, and 16 with olfactory impairment of unknown cause) who visited an otolaryngologist between October 2020 and April 2021 were analyzed. Clinical laboratory technologists were asked to evaluate the effectiveness of T&T using a flow chart. As a result, the clinical laboratory technologists and patients were less confused during examinations. The validity of T&T using the flowchart was evaluated by comparing its results with the scores of the self-administered odor questionnaire (SAOQ) and the visual analogue scale (VAS), which are tools for self-evaluation of olfaction. As a result, we found a strong negative correlation between T&T results and SAOQ scores (r = −0.715). There was also a strong negative correlation between T&T results and VAS scores (r = −0.793). The examiners felt that they had less difficulty in making decisions during the examination, and the patients felt that they can easily identify a scent. Flowchart-based T&T olfactometry has potential use in the standardization of the examiners’ procedural steps and better reflect the patients’ subjective complaints.

Joint fluid is tested to identify the cause of joint effusions, and crystal classification is particularly useful for the diagnosis of crystal-induced arthritis. However, quality control and educational activities on joint fluid analysis are not sufficiently widespread, and the reasons are attributed to the small number of requests for analysis and unstable preservation. In this study, we examined the possibility of long-term preservation of crystals in specimens with calcium pyrophosphate crystals (CPPD crystals) or sodium urate crystals (MSU crystals) in joint fluid, using 10% neutral buffered formalin-fixed or anhydrous ethanol-fixed cell block specimens. CPPD crystals were significantly well preserved by 10% neutral buffered formalin fixation. In contrast, MSU crystals were significantly well preserved by anhydrous ethanol fixation. The crystals detected by polarized light microscopy also showed characteristic birefringence for CPPD and MSU crystals. In the online microscopy training using cell block specimens, the survey results showed that more than 70% of the participants responded ‘good’. These results suggest that the use of cell block specimens prepared by anhydrous ethanol fixation enables long-term preservation of both CPPD and MSU crystals and can be useful for education in joint fluid analysis.

There are many fluorescent substances in urine and urinary sediments. We have confirmed the fluorescence of xanthine and 2,8-dihydroxyadenine (2,8-DHA) crystals, which can be formed by purine catabolism, and tosufloxacin crystals that are derived from the tosufloxacin tosilate antimicrobial drug. A fluorescent pale blue color is obtained from salts/crystals in casts, human hair, nails, and stratified squamous keratinized epithelia. Commercially available uric acid is a colorless and thin crystal and has a faint pale blue fluorescence. Uric acid crystals obtained from the urine of students and kept in a refrigerator were found to be very pure (over 98% purity) by infrared absorption spectroscopy. They also showed different colors, namely, yellow brown, and red brown under visible light and pale blue and yellow green under fluorescent light. Each color may be included owing to the fluorescence of uric acid and the incorporation of urinary colored pigments.

The soluble interleukin-2 receptor (sIL-2R) is considered to be useful as a diagnostic aid for non-Hodgkin’s lymphoma and adult T-cell leukemia, as well as for determining therapeutic efficacy. In this study, Tosoh Corporation has developed a reagent for chemiluminescent enzyme immunoassay (CLEIA) that can be used in 15 min. In this study, we evaluated the basic performance of this reagent using the fully automated CLEIA system AIA^®-CL1200. As a result, the reproducibility, selectivity, and limit of quantitation using this reagent were found to be high, and dilution linearity test was confirmed up to 44,131 U/mL. The correlation with other methods was good, and no extremely deviated sample was found using LUMIPULSE based on the same measurement principle were observed. However, in comparison with Nanopia which uses latex immunoturbidimetry as its principle, two divergent cases were identified in which Nanopia showed higher values. The results of the analysis suggest that IgM in sample 1 was involved in the assay reaction system, and in sample 2, it was possible that Human anti-mouse antibody (HAMA) in this sample caused the nonspecific reaction. The basic performance of this reagent is good, and it is expected to contribute to clinical diagnostics since it has sufficient performance for routine examinations.

The standardization of thyroid-related tests, such as TSH, FT4, and FT3, is clinically important since several guidelines for thyroid diseases recommend the therapeutic targets based on their absolute values. Since the thyroid-related tests are performed with an immunoassay, however, the absolute values are largely influenced by the analytical devices and reagents used for the assays. The International Federation of Clinical Chemistry and Laboratory Medicine Committee for Standardization of Thyroid Function Tests (IFCC C-STFT) promotes the standardization of FT4 and the harmonization of TSH, and the latter is now ongoing in Japan. However, at present, there are few reports comparing the absolute values yielded by multiple thyroid-related tests conducted simultaneously. In this study, we compared the precisions and correlations among four different methods, including those offered by Abbott, Siemens, Tosoh, and Fujirebio. We obtained favorable results on the precisions and correlations; however, we observed substantial discrepancies among the absolute values obtained by using different methods. These results suggest that harmonization especially on the absolute values is further required for thyroid-related tests in Japan.

It is important to report the serum information of a specimen in a clinic for the hemolysis of the specimen, which affects the examination measurements and the interpretation of test results. We investigated the effect of hemolysis on biochemical laboratory data, set a hemolysis comment addition origin, and examined the setting of the grades of hemolysis judgment of an autoanalyzer. We collected blood and serum samples into sodium heparin tubes from five people who agreed to the use of their blood and to participate in this study, and then we made 10 phases of hemolysate and examined the effect of hemolysis on 28 biochemical test items. As a result, the mean rate of change of 400 mg/dL was in the order of LD, Fe, AST, D-BIL, T-BIL, and K when we compared it with the Hb concentration of 0 mg/dL. We considered that a change in K level had the biggest effect on the data reading and that half (0.12 mEq/L) of the standard deviation of the physiological band for K in individuals is the change limit of tolerance. A hemolysis grade of more than this indicates a Hb concentration of 50 mg/dL, and the grade of hemolysis judgment at that time was 1.41. Because increases in LD, Fe, AST, and K levels at the Hb concentration of 50 mg/dL were beyond the change limit of tolerance, we considered it significant to the hemolysis comment addition to report a reference level.

The latex immunoassay using the Helicobacter pylori antibody test reagent “H. pylori-latex Seiken” (Denka Co., Ltd.) has been considered to be sufficiently accurate with a cut-off value of 10 U/mL. However, there have been reports indicating the presence of H. pylori infection among antibody-negative patients. We aimed to re-evaluate H. pylori infection among antibody-negative patients with high (3.0–9.9 U/mL) and low (< 3.0 U/mL) negative antibody levels with results of endoscopies conducted alongside antibody tests. From our hospital, we enrolled 53 patients who were endoscopically diagnosed as having chronic atrophic gastritis from 214 patients negative for H. pylori antibodies and no clinical records of H. pylori eradication treatment or histories of gastric cancer. Of these 53 patients, 31 (58%) had low negative antibody levels and 22 (42%) had high negative antibody levels. Re-evaluation of endoscopic findings revealed that 23 (43.4%) of these 53 patients were possibly currently infected with H. pylori or needed further examination to verify infection. Of these 23 patients, 8 had low negative antibody levels (8/31, 25.8%) and 15 had high negative antibody levels (15/22, 68.2%). We discovered false-negative results of the H. pylori antibody test, with a higher ratio in the patients with high negative antibody levels than in those with low negative antibody levels. If these patients were not examined by endoscopy in addition to antibody tests, the infection might have been missed. From our results, it appears necessary to use additional H. pylori tests such as urea breath tests and/or endoscopy for patients with high negative antibody levels.

Adverse reactions such as fever, headache, and fatigue after vaccination are one of the major anxiety factors for the general public in preventing the spread of Covid-19 infection. The subjects were 269 staff members (M:F = 60%:40%) who received the second dose of Pfizer’s mRNA vaccine at our hospital. The association of adverse reactions with the IgG antibody titer for the binding domain was examined considering factors such as age and gender. The median sIgG antibody titer was 1,299.5 AU/mL (800.2 AU/mL to 1,947.3 AU/mL), and all the subjects were antibody-positive (50 AU/mL or higher), with an extensive range. There was no significant difference between men and women. When the subjects were divided into four age groups from the 20s to 50s and the antibody titers were compared, a statistically significant difference was observed among the age groups. Drinking habits and taking anti-allergic drugs did not affect the sIgG antibody titers. The criteria for “with adverse reactions” in this study were having a fever of 37.5°C or higher, and one or more systemic symptoms of headache, malaise, and arthralgia whose severity was “disturbing life”. Overall, males and females in the group with adverse reactions all tended to have significantly higher sIgG antibody titers. Any systemic symptoms other than fever as an adverse reaction could be a predictive factor for the acquisition of neutralizing antibodies.

Continuous subcutaneous insulin infusion (CSII) and sensor-augmented pump (SAP) therapy in patients with type 1 diabetes are close to physiological insulin secretion because they can regulate basal insulin according to the amount of individual insulin. In January 2022, insulin pumps equipped with a hybrid closed-looped (HCL) system were made available in Japan. HCL therapy is a technology with which the amount of basal insulin can be automatically adjusted on the basis of glucose levels obtained from continuous glucose monitoring (CGM) using a glucose sensor. At our hospital, a clinical laboratory technician is in charge of the introduction and guidance on the use of continuous insulin pumps. We report a case of type 1 diabetes in which blood glucose level was controlled using HCL therapy. Glycemic control with SAP therapy resulted in HbA1c levels of 7.7–8.2%. After the introduction of HCL therapy, HbA1c levels decreased to 7.0–7.2%, and blood glucose levels tended to decrease. Furthermore, time in range (TIR) increased from 51–56% with SAP therapy to 64–69% with HCL therapy. HCL therapy can increase TIR without promoting hypoglycemic events and is effective for improving patient quality of life and preventing diabetic complications.

We report a case of IgG4-related disease (IgG4-RD) with severe hypereosinophilia. A woman in her 70s with suspected IgG4-RD noticed swellings on both submandibular regions. She was referred to our hospital for further examination. Laboratory data revealed a high level of serum IgG4 (3,420 mg/dL) and leukocytosis (115.2 × 10⁹/L), but neutrophils and eosinophils could not be calculated on an automated hematology analyzer. By visual inspection, we found that 96.9% of the total leukocytes were eosinophils, many of which showed abnormal nuclear lobulations and decreased counts of intracytoplasmic granules. She underwent a surgical biopsy of the submandibular gland and supraclavicular lymph nodes, and was eventually diagnosed as having IgG4-RD (definite) according to the comprehensive diagnostic criteria of IgG4-RD. In the imprinted smears of lymph node specimens subjected to May–Giemsa staining, eosinophils were easily recognized together with numerous plasma cells and lymphocytes. However, in the imprinted smears of lymph node subjected to Papanicolaou staining, eosinophils were not distinguishable from neutrophils because the acidophilic granules were not stained. Every additional test for determining the cause of hypereosinophilia showed negative results. Thereafter, steroid administration for IgG4-RD treatment improved her symptoms and laboratory data, including hypereosinophilia. On the basis of these findings, hypereosinophilia was judged to be a secondary response associated with IgG4-RD. On a practical level of medical technologists, it should be recognized that automated blood cell analyzers may not distinguish between neutrophils and eosinophils in patients with severe hypereosinophilia. Thus, for observing cytological smears with suspected eosinophilic infiltration, May–Giemsa staining, together with Papanicolaou staining, is advisable.

Malignant melanoma frequently occurs on the skin, head, and neck, and is known to be one of the tumors with the worst prognosis. It is rare for this cancer to metastasize to the urinary tract and to be detected while the patient is still alive. In this report, we show that the detection of melanoma cells and melanophages in urinary sediment can help detect multiple metastases of malignant melanoma. The patient was a male in his 70s, and a urinalysis during an outpatient visit revealed characteristic atypical cells in the urinary sediment. The atypical cells with a high nuclear-to-cytoplasm ratio, hyperchromasia, enlarged nucleoli, and dark brown melanin granules in the cytoplasm were identified as melanoma cells by various staining methods and immunohistochemical staining. Furthermore, we mentioned the characteristics of melanophages with small and unevenly distributed nuclei and the difference of melanin granules from similar components. Importantly, the number of melanophages in urinary sediment correlated with serum LD and UA levels. In conclusion, melanoma cells and melanophages detected in urinary sediment could be novel markers for malignant melanoma, prognosis prediction, and understanding of the melanoma pathogenesis.