In recent cancer therapy, the number of requests for diagnosis using not only tissue specimens but also cytological specimens has been increasing. Immunostaining of cytological specimens has attracted attention, and the expansion of its application has been anticipated. In a routine histopathological examination, immunoenzyme staining is the most commonly used diagnostic tool, and remarkable developments and progress have been made in developing automated staining equipment and new staining methods in recent years. Moreover, immunostaining is performed in many facilities. However, the number of staining operators who have never performed manual staining is increasing annually, and some of them often lack understanding of the principles or methods of immunostaining. Therefore, they may send poorly stained samples to a pathologist because they fail to determine by themselves whether the staining is sufficient. The importance of performing staining surveys has been increasing significantly so that those involved may become aware of the differences in the level of staining properties between that observed at their own facility and that observed at other facilities. Staining surveys have been conducted, and the level of awareness regarding the quality control of staining has been changing. We need to perform everyday tasks in collaboration with pathologists by recognizing that a poorly stained sample may lead to an incorrect diagnosis, and the preparation of more precisely stained samples can be of benefit to both society and patients. In this article, I will discuss the quality control of staining in addition to the current situation and new techniques of immunostaining.

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Clinical laboratory tests are essential for diagnosing diseases, monitoring therapeutic regimens, and determining the prognosis of patients in daily clinical practice. However, before interpreting test results for any such purpose, we need to consider the possible sources of variation (SVs) other than those caused by pathological processes. Nonpathological SVs include biological (physiological) sources of variation (BSVs) and analytical sources of variation (ASVs). BSVs are divided into interindividual SVs, such as age, gender, environment, lifestyle, and genetics, and intraindividual SVs, which affect sampling conditions, such as posture, physical activities, and time of day. In this review article, the effects of interindividual SVs, namely, age, gender, obesity, and habits of smoking and drinking, and those of intraindividual SVs, namely, posture, physical exercise, diurnal rhythm, and short-term effects of smoking and drinking, on test results are discussed systematically on the basis of the authors’ experiences. All lines of evidence on BSVs should be of considerable relevance to the proper interpretation of clinical laboratory test results.

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We determined the conditions of preventive measures against Norovirus infection among food handlers in Niigata Prefecture and the infection rate among healthy carriers. A questionnaire with 27 questions was sent to 33 food handlers. Of the 27 questions, 22 were direct yes-or-no questions. The data obtained were compared between two groups (group A: employers with ≤ 10 employees; group B: employers with ≥ 11 employees). Norovirus genotypes I and II were detected in feces samples collected from 377 employees (204 men and 173 women) by real-time reverse transcription-polymerase chain reaction analysis. The numbers of items for which all food handlers in groups A and B acted in accordance with the teaching guidelines were two (2/22, 9.1%) and seven (7/22, 31.8%), respectively (p < 0.05). The rates of compliance with the guidelines, namely, “use of a nail brush in hand washing” and “their own manual preparation regarding the prevention of food poisoning”, were significantly lower in group A than in group B (p < 0.05). Genotype II was detected in the samples from two men (0.98%) in group A. To prevent Norovirus infection, action in accordance with the teaching guidelines provided by healthcare centers is needed, and restrictions (such as leaving the food-handling area or staying at home) should be imposed on the asymptomatic carriers of Norovirus who are still positive for the virus.

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The measurement error generated by non-ionic detergents (Brij 35 and Triton X-100) in nine commercial pH test strips [thymol blue (TB), bromophenol blue (BPB), bromocresol green (BCG), bromocresol purple (BCP), bromothymol blue (BTB), methyl red (MR), phenol red (PR), cresol red (CR), alizarin yellow (AZY)] was investigated. A false negative error was produced by the detergents. The measurement error rates, which were proportional to the detergent concentration, were high in BTB, BCG, BCP and BPB in the presence of Brij 35, and in BTB and BCG in the presence of Triton X-100. The measurement error rates in PR, CR, MR, and AZY were low. The measurement error rate tended to correlate with the molecular weight of the pH indicator.

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Since the first report showing that the level of plasma presepsin [presepsin(p)] is significantly elevated in sepsis patients, presepsin(p) has been widely used as a diagnostic marker to determine the severity of sepsis and follow its course. However, owing to the renal excretion of presepsin(p), it seems that the evaluation of presepsin(p) level needs special attention in the presence of renal dysfunction. The effect of renal function on presepsin(p) level has not been well characterized. Herein, we have studied the level of presepsin(p) in renal dysfunction. (1) Out of 98 hemodialysis patients lacking any evidence of sepsis, 96 show a presepsin(p) level above the normal range, indicating that the presepsin(p) level in these patients is significantly higher than that in healthy controls (p < 0.001). (2) Furthermore, in the patients with chronic renal failure but without a history of hemodialysis, the level of presepsin(p) reversely correlates with the estimated glomerular filtration rate (eGFR) (R² = 0.735). (3) On the other hand, it is found that hemodialysis significantly lowers presepsin(p) level (p < 0.001). Cumulatively, our findings indicate that it is critical to pay careful attention to renal function and hemodialysis in the evaluation of presepsin(p) level in sepsis patients.

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We investigated the presence of cst-II, cgtA, and cstB associated with ganglioside mimicry in Guillain-Barre syndrome (GBS) associated with Campylobacter jejuni strains from humans and chicken. To examine the relationship of Campylobacter jejuni isolates with the development of GBS, 246 strains isolated from 30 gastroenteritis patients and 28 chicken liver samples were characterized by a serological test and subjected to PCR analysis to detect cstII, cgtA, and cgtB associated with ganglioside-like mimicry of lipooligosaccharide (LOS) and to an antimicrobial susceptibility test. All these three LOS genes were detected in 26.7% of human-derived strains and 43.5% of chicken-derived strains. The serogroups of the strains harboring the three LOS genes were as follows: serogroups B(O:2), D(O:4,13,16,43,50) and O(O:19) in chicken-derived strains and serogroups D(O:4,13,16,43,50) and O(O:19) in human-derived strains. It was considered that there was a sufficient association because similar serotypes were detected in human-derived strains and chicken-derived strains. However, the serotypes of many strains were not clearly identified. Therefore, the method of detecting the presence of cst-II, cgtA, and cgtB associated with ganglioside mimicry in GBS associated with C. jejuni strains was more useful.

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We examined patient characteristics and related test results of 15 urine specimens in which ammonium urate (AU) crystals were observed in our hospital in the past 3 years. In 13 of the 15 specimens, the urine was acidic, which was in contrast to most reports in which AU crystals were detected in alkaline urine. When some time has passed before examination, urine may become alkaline. However, examination for urinary sediments has recently been performed rapidly in Japan, which may have led to our present findings. Patients with AU crystals in their urine were younger than those without the crystals (n = 60; 19 years vs 66 years; p < 0.001) and were mostly male (p < 0.05). These findings of younger age and male predominance are consistent with those observed among patients with postrenal failure due to AU stones, suggesting a correlation between AU stones and AU crystals. In addition, the average urine specific gravity was significantly higher in samples with AU crystals than in those without the crystals (p < 0.001). Diarrhea, which causes dehydration, was seen frequently (p < 0.001, compared with controls). The presence of AU crystals in urinary sediments could therefore be indicative of AU calculus disease or dehydration.

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We experienced treating a rare case of Clostridium haemolyticum infection of the bone marrow. There are very few reports of cases in which bone marrow necrosis is diagnosed during the lifetime. This bacterium has been found to cause bacterial hemoglobinuria in domestic animals such as cows or horses. The patient’s main complaints were fever and lower back pain, and he consulted our hospital. He was diagnosed as having diffuse large B-cell lymphoma (DCBCL) and was also suspected of having Burkitt lymphoma. He was treated by chemotherapy. After partial resection of the small intestine with ulcer and hemorrhage, bone marrow necrosis was confirmed. The samples were negative for pathogenic microorganisms on the third culture and a Clostridium sp. was detected on the fourth culture. Because it was difficult for us to identify the species of this bacterium by the anaerobic bacterium identification method in our laboratories and we doubted the possibility of Clostridium tetani, we requested other facilities to identify the bacterium while we treated the patient with penicillin G (PCG), tetanus toxoid, and tetanobulin. As a result of continuous treatment by chemotherapy and myeloid irrigation, his lower back pain improved and the bone marrow culture became negative. It is effective to use other methods of bacterium identification and improve the biochemical properties of culture media to increase the identification rate of bacteria. By this strategy, doctors will be able examine and treat patients in whom the suspected causal anaerobic bacterium is difficult to identify.

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Vestibular evoked myogenic potential (VEMP) is a short-latency electromyographic response elicited by acoustic stimuli and is recorded from tonically contracted neck muscles, particularly the sternocleidomastoid muscle (SCM). This potential is used to diagnose lesions of the saccule, inferior vestibular nerve, and vestibulospinal tract. Our case involved a 15-year-old girl whose chief complaints were dizziness and an eyeball abnormality. The left side stimulation showed waveform loss in the examination of VEMP. MRI revealed a T2WI hyperintense area in the left ventral and right dorsal parts of the pons, and a lesion was observed in the cerebral white matter during a follow-up examination. The regions examined in the VEMP test included the nuclei vestibulares, which is located in the dorsal part of the pons and medulla oblongata; the MRI findings for the same region did not concur with the results of the VEMP test. This VEMP test detected dysfunction of the vestibular nucleus or the vestibulospinal tract, which was thought to indicate a lesion that was undetectable by MRI.

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We studied the diagnostic efficacy a cell block method using disposable swab tubes and compared it with that of a conventional cytologic smear method. Seventy-four patients were recruited in this study. Cell blocks were prepared from pancreatic juice from 11 patients, bile from 28 patients, EUS-FNA specimens from the pancreas from 10 patients, and effusion fluid from 25 patients. Immunohistochemical analysis was performed on 15 patients. Among the 74 patients diagnosed using cytologic smear preparations, 31% (23/74), 53% (39/74), and 16% (12/74) showed positive, negative, and equivocal findings, respectively. As for the diagnosis using cell block sections, 41% (30/74), 53% (39/74), and 6% (5/74) showed positive, negative, and equivocal findings, respectively. The cell block method showed a higher positivity than the cytologic smear method. Cell block sections showed clear histologic constitutions. Our method is easy and useful for preparing cell blocks. Combination of conventional cytologic smear method, cell block method, and immunohistochemical analysis can improve the positivity rate in diagnosis.

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The lumbrical-interossei comparison method (2L-INT method) is recommended for the diagnosis of carpal tunnel syndrome owing to its high sensitivity and simplicity. However, it is not easy to find the second lumbricalis (2L) on the palm surface to obtain the compound muscle action potential (CMAP). In this study, therefore, we explored a new approach to locating the proper electrode position in the 2L-INT method. We drew a line starting from the joint between the second finger and the third finger to the carpal center, and on this line, we determined point “a” at the metacarpal head height, point “d” at the boundary between this line and the thenar muscle, and points “b” and “c” respectively at the first and second one-third points on the line from “a” to “d”. We then examined, at each electrode position (a, b, c, and d) the following: (1) latency, (2) CMAP amplitude, and (3) CMAP rising time slopes of both 2L-CMAP and INT-CMAP. We also examined (4) the 2L-INT latency difference and (5) the different results obtained by different examiners. At points “a” and “d,” low amplitude and positive rising time slopes were observed occasionally. The 2L-INT time was also extended in some cases, possibly causing a false positive result. Thus, points “a” and “d” seemed unsuitable for an active electrode. In contrast, at the midpoint between points “b” and “c,” the amplitude was high, and there were few changes in the 2L-INT time caused by the electrode position differences. Thus, this location seemed suitable for an active electrode.

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The performance of a modified selective solubilization assay for serum low-density lipoprotein cholesterol (MetaboLead^® LDL-C) was compared with that of the currently used assay (Determiner^® L LDL-C). The within-run coefficients of variation (CVs) for the modified LDL-C assay ranged from 0.5 to 1.3% (n = 29) for pooled sera. The range of run-to-run CVs was from 0.4 to 0.6% (n = 10). The assay results for the serially diluted LDL-C standard solution showed excellent linearity up to 677 mg/dl. No interference was observed when bilirubin, hemoglobin, Intralipid^®, rheumatoid factor, heparin, or Intrafat® was included in samples for the measurement of LDL-C with the modified assay. Moreover, there was a good correlation between the results of the modified and the currently used assays (y = 0.95x − 0.9, r = 0.995). In samples with high TG levels, the LDL-C level determined by the currently used assay was higher than that determined by the modified assay. The difference in LDL-C level between the modified assay and the modified BQ method was smaller than that between the modified assay and the currently used assay. This may be due to the presence of remnant-like particle cholesterol in serum. In conclusion, the modified assay can be used to measure serum LDL-C levels, which are close to those measured by the modified BQ method.

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Clostridium difficile infection (CDI) is one of the major causes of nosocomial diarrhea, particularly in patients with microbial substitution on antimicrobial therapy. For proper treatment of the diarrhea and prevention of the spread of C. difficile, high-sensitivity detection of the organism is preferred. To diagnose CDI, a rapid immunoassay kit (Alere Medical Co.) to detect both glutamate dehydrogenase (GDH) and CD toxin A or B in stool specimens has been employed in our hospital. Since false-negative cases existed in tests using a stool-direct assay, we applied a culture test to improve the detection of the CD toxins. GDH-positive and CD-toxin-negative stool specimens were anaerobically cultured, and the productions of the CD toxins by the strains grown were tested using the kit. The CD toxins were positive in 34 (7%) out of 457 cases of suspected CDI from October 2013 to March 2014 by the stool-direct assay alone, and the number of toxin-positive cases increased to 58 (13%) with the additional culture test. For the precise diagnosis of CDI, we recommend that the direct toxin detection in stools should be combined with stool culture in cases in which the stool specimens are positive for GDH.

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An immunochromatographic assay kit is frequently used in the diagnosis of legionellosis. We evaluated the usefulness of Immunocatch^®-Legionella, a newly developed immunochromatographic assay kit for the detection of the Legionella antigen. The kit showed a higher detection sensitivity than BinaxNOW^® Legionella and Check-Legionella, and showed an equivalent detection sensitivity to Q-line Kyokuto Legionella. In addition, Immunocatch^®-Legionella as well as Q-line Kyokuto Legionella showed the most rapid determination. A total of 100 specimens from patients with pneumonia symptoms were tested using these immunochromatographic assay kits. The positive agreement, negative agreement, and overall agreement rates of Immunocatch^®-Legionella and BinaxNOW^® Legionella were 100% (1/1), 99.0% (98/99), and 99.0% (99/100), respectively. One case was negative as determined using BinaxNOW^® Legionella, but was positive as determined using Immunocatch^®-Legionella. The use of a concentrated specimen of this case resulted in a positive result using BinaxNOW^® Legionella. These results suggest that BinaxNOW^® Legionella has an insufficient sensitivity, and the determination using it may lead to a false-negative result. Immunocatch^®-Legionella has a simpler procedure and higher sensitivity than the other kits. It also showed good performance with clinical specimens. In conclusion, Immunocatch^®-Legionella is a clinically useful rapid diagnostic kit for the detection of Legionella antigen.

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We conducted cerebrospinal fluid analysis to examine the incidences of meningitis and suspected meningitis and compared its findings with cerebrospinal fluid culture results. We also investigated the rate of bacteria detection and its annual changes. These examinations were conducted over 5 years from 2008 to 2012 in three hospitals with different numbers of beds (Fukuoka, Nagano and Okinawa). The examination results from the three hospitals showed that the most commonly detected bacteria were Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli, and methicillin-sensitive Staphylococcus aureus (MSSA) in this order. These investigations revealed that the rate of uncultivable bacteria is as high as 41.1% and the rate of detection of other microorganisms is as low as 3.6%. Laboratory findings showed a lower ratio of the level of serum glucose to that of cerebrospinal fluid glucose in the culture-positive group than in the culture-negative group. From the annual changes in the rate of detection of pathogenic bacteria causing meningitis, it was shown that H. influenzae has not been detected since 2011 and the rate of S. pneumoniae detection transiently decreased in 2010, but continued to increase thereafter. It is considered that medication can be administered confidently when genetic testing shows positivity for microorganisms even in cases in which a pathogenic bacterium is not detected, which can lead to the treatment of meningitis. Therefore, it is also suggested that genetic testing is required in future examinations.

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A rapid-clotting blood collection tube, which is coated with thrombin on the inner wall of the orifice, is suitable for the rapid reporting of test results. However, the effects of thrombin coating on test results have been evaluated only for limited test items. Therefore, we examined the test results of blood collected from healthy volunteers using three types of blood collection tube, namely, a glass blood collection tube, a conventional blood collection tube, and a rapid-clotting blood collection tube, for 146 test items, which included 59 biochemical test items, 38 tumor marker/hormone test items, 24 infection test items, and 25 autoimmunity test items. Although statistically significant differences (p < 0.05) between the rapid-clotting blood collection tube and the other types of collection tube were observed in some test items, the differences appeared to be negligibly small in daily clinical practice. In healthy volunteers, the rapid-clotting blood collection tube, in comparison with the conventional blood collection tube, did not show any clinically detrimental effect on test results.

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The purpose of this study is to determine the Clostridium difficile toxin genotypes and the prevalence of binary toxin genes. We investigated 587 stool specimens with suspected CDAD submitted between April 2010 and March 2011 and between July 2012 and December 2012. As a result, of the 587 stool specimens examined by culture, 18.9% were positive and 81.1% were negative for the toxin genes. We examined the toxin type of 107 toxin-gene-positive specimens. The breakdown of toxin genotypes was as follows: 60.7%, toxin A⁺B⁺; 15.0%, toxin A⁻B⁺; 24.3%, toxin A‍⁻B‍⁻. In addition, binary-toxin-gene-positive strains were found in one specimen. The binary-toxin-gene-positive strains analyzed by PCR ribotyping and slpA sequence typing were identified as having the slpA sequence type y05-02/PCR ribotype hu13027. In conclusion, epidemiological studies of C. difficile using methods such as culture examination, toxin genotyping, and binary toxin gene analysis are considered to be very important for understanding the current status of the epidemic strains of C. difficile in a region and in individual hospitals.

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