Detection of Coxiella burnetii in market chicken eggs and mayonnaise.

We tried to detect C. burnetii in market chicken eggs and mayonnaise by nested PCR assay. The PCR target was the com 1 gene of C. burnetii. The positive rate for egg and mayonnaise samples was 4.2% and 17.6%, respectively. Direct sequence of some of the positive egg samples shows mutations whereas no mutation was found in the positive mayonnaise samples. The number of molecules of the Q fever agent is estimated at 10(4) to 10(6) per egg, according to our quantitative PCR test.

Autonomic nervous system activity in the late luteal phase of eumenorrheic women with premenstrual symptomatology.

The majority of women of reproductive age experience a regular recurrence of various symptoms in the premenstrual phase. The etiopathogenesis of premenstrual symptomatology, however, remains inconclusive. The present study was proposed to evaluate whether the activity of the autonomic nervous system (ANS), which largely contributes to the relative stability of a human's internal environment, is altered during the menstrual cycle of women with premenstrual symptomatology. Thirty eumenorrheic young women participated in this study. All subjects were investigated during the follicular and late luteal phases. The ANS activity was assessed by means of heart rate variability power spectral analysis during supine rest. No intramenstrual cycle differences in the ANS activity were found in women experiencing no or small increases in premenstrual symptoms. In contrast, the sympathetic nervous system (SNS) activity significantly increased and the parasympathetic nervous system (PNS) activity apparently decreased in the late luteal phase in subjects whose premenstrual symptomatology was not unbearable, but substantially increased (> 20%) compared to the symptom-free follicular phase. The women with greater degrees of premenstrual distress possessed higher SNS activity and lower PNS activity in the late luteal phase than the women with less symptomatology. The ANS activity in the follicular phase did not differ among the subjects regardless of their premenstrual symptoms. Although causes and consequences continue to elude, the present study provides additional intriguing evidence that the altered functioning of ANS in the late luteal phase could be associated with diverse psychosomatic or behavioral symptoms appearing premenstrually.

Tissue factor pathway inhibitor can interact with platelets.

Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation. However, there is no report on interaction between TFPI and platelets other than that by Tsuji, who found that whole blood anticoagulated with TFPI exhibited remarkable decrease in platelet count. Our study revealed that washed platelets suspended in modified Tyrode's buffer (8 mM CaCl2) containing TFPI exhibit platelet aggregation. However, platelets aggregation was observed without TFPI, but its increase and intensity were slow and weak, compared to that in the presence of TFPI. This aggregation was inhibited by anti-CD41 (anti-GPIIb) antibody. This finding suggested that TFPI promotes platelet aggregation.

Role of mitogen-activated protein kinase family in serum-induced leukaemia inhibitory factor and interleukin-6 secretion by bone marrow stromal cells.

1: In the haematopoietic microenvironment, bone marrow stromal cells play an important role in regulating haematopoiesis by expressing various cytokines, including leukaemia inhibitory factor (LIF) and interleukin-6 (IL-6). However, the intracellular signal that regulates cytokine secretion in bone marrow stromal cells has not been determined. The aim of this study was to evaluate the role of mitogen-activated protein kinase (MAPK) family in serum-induced secretion of LIF and IL-6 by bone marrow stromal cells. 2: Transformed human bone marrow stromal cells (HS-5) were stimulated with foetal calf serum (FCS) to produce LIF and IL-6. FCS also induced activation of extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun NH(2)-terminal kinase (JNK). 3: Both PD98059 (MAPK/ERK kinase inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated FCS-induced LIF protein production and gene expression. SB203580 decreased IL-6 production and gene expression, but PD98059 had no effect on IL-6 production and gene expression. 4: Expression of a dominant-negative mutant form of JNK1 that blocked FCS-induced JNK activity had no effect on protein production and gene expression of these cytokines. 5: These findings demonstrate that both ERK and p38 MAPK are involved in FCS-induced LIF secretion, whereas only p38 MAPK is important for IL-6 secretion, and that FCS-induced activation of JNK has no effect on the production of LIF and IL-6. We conclude that, in spite of their similar biological effects, they are differentially regulated at the level of MAPK activity in bone marrow stromal cells.

Differential effects of fibroblast growth factor-4, epidermal growth factor and transforming growth factor-beta1 on functional development of stromal layers in acute myeloid leukemia.

The hematopoietic supporting abilities are known to be impaired in marrow stromal layers developed from patients with acute myeloid leukemia (AML). In this study, fibroblast growth factor-4 (FGF-4), epidermal growth factor (EGF) or transforming growth factor-beta1 (TGF-beta1) were studied to see whether these growth factors can modify the functional development of leukemic stromal layers. Adherent stromal layers from 13 patients with AML and from six non-leukemic controls were established with 3ng/ml of FGF-4, EGF or TGF-beta1. Established stromal layers were washed three times and irradiated, followed by recharge of allogenic peripheral CD34 positive cells as an indicator of supportive function. Progenitor-outputs into supernatant were evaluated at biweekly interval with colony-forming assay until 6 weeks. The results showed that both leukemic and non-leukemic stromal cells established with FGF-4, but not with EGF, showed significantly higher progenitor cell-outputs compared with control stromal cells. By contrast, stromal cells developed with TGF-beta1 showed significantly lower progenitor cell-outputs compared with control. These differences were significant at later than 4 weeks after the recharge of indicator cells, suggesting that the stromal layer developed with EGF or TGF-beta1 preferentially affected the primitive progenitors rather than committed ones. These results indicate that FGF-4 and TGF-beta1 differentially affect the functional development of leukemic as well as of normal stromal layers.

Simple high-density lipoprotein cholesterol assay based on dry chemistry.

BACKGROUND: Currently, high-density lipoprotein cholesterol (HDL-C), a factor which prevents progression of arteriosclerosis, is measured using laboratory-based chemistry analyzers without a pretreatment step. Because HDL-C is measured with a pretreatment step in many point-of-care testing systems, a direct assay is needed. METHODS: A dry-chemistry-based assay using surfactants has recently been developed in parallel with the development of a dedicated reagent. A simple analyzer that accepts whole blood samples was also developed. RESULTS: The assay demonstrated excellent precision, dilution linearity and intermethod comparison. In an interference test, assay values tended to be lower in the presence of high concentrations of hemoglobin, conjugated or unconjugated bilirubin. Neither ascorbic acid up to 20 mg/dl, nor formazin turbidity up to 2100, had an effect on the assay. CONCLUSIONS: This dry-chemistry assay using only surfactants for specificity in the direct HDL-C method was judged useful for point-of-care instrumentation in terms of equipment compactness, operational simplicity and rapid responsiveness.

Automated image processing. Past, present, and future of blood cell morphology identification.

Automated image processing analysis for leukocyte differential counting started 30 years ago principally as a mimic of the traditional microscopic method. Several types of systems were used in the 1970s and 1980s. In the late 1990s, two new image processing systems were developed with new technology for cell image analysis. They possess an intelligent neural network software and can be connected to an Ethernet for telehematologic diagnosis and consultation.

Changes in hypervariable region 1 in patients with chronic hepatitis C of genotype 1b with biochemical response to interferon.

BACKGROUND/AIMS: The outcome of interferon (IFN) therapy of hepatitis C virus (HCV) infection can be classified as a complete viral response (CR), biochemical response (BR), or no response (NR). Why alanine aminotransferase (ALT) activity decreases in patients with BR despite viral persistence is unknown. METHODS: Of 158 patients infected with HCV genotype 1b, all 20 patients with BR and 20 of the 114 patients with NR, matched for viral load to the BR group, were studied. We sequenced nucleotides in the hypervariable region (HVR) of serum HCV RNA, and analyzed quasispecies of this region by the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP). RESULTS: In HVR 1, SSCP patterns differed after therapy; the major clone before therapy disappeared with therapy in eight of the 20 BR patients, but in none of the 20 NR patients (P=0.0033; Fisher's exact test). PCR products of HVR 1 from six patients were cloned before and after therapy, and 40 clones from each patient were sequenced each time. Results of cloning and sequencing were generally consistent with those of SSCP. For the six patients, a major clone could be identified both before and after therapy. In two patients with BR, there were many changes in the amino acid sequence of the major clone after IFN; in one patient with NR, mutations were not found. CONCLUSION: Changes in the major viral clone with IFN treatment may be related to the decrease in ALT activity in some patients, in spite of the continued presence of HCV RNA.

Problems on national and locale external quality assurance schema (NEQAS and LEQAS); based on Japanese experience.

The quality and stability of surveillance materials is a critical issue to perform good external quality assessment schema. In the case of nationwide surveillance, more stable materials are required than those in the case of locale external quality control schema. For locale surveillance, fresh blood, plasma or serum materials are prepared for hematology and chemistry tests. On the other hand, dried materials are used for the chemistry test and semi-fixed blood is used for hematology tests in the national surveillance. However to evaluate automated white cell differential in hematology, there has been no good materials. We therefore prepared a mixture of EDTA and ACD solution for our locale surveillance. It gave us excellent results on the differential when we used it within 3 days after blood collection.

Problems related to rapid methods for erythrocyte sedimentation rate test and their solution.

The erythrocyte sedimentation rate (ESR) test is one of the most common and traditional laboratory tests in the world. It reflects both plasma concentration of acute-phase proteins of large molecular size and anemia (International Council for Standardization in Hematology, 1993). The ESR test method is easy to perform and inexpensive. Therefore, it is used today as a routine test worldwide. However, the ESR has some demerits, in requiring a large volume of sodium citrate or ethylenediamine tetraacetic acid (EDTA) blood and at least 2-hour testing time. When ESR is tested manually, incorrect results are obtained if the ESR test tube is not stood strait-up and vertically (Dobashi et al, 1994). Reading error for the meniscus line and surrounding temperature at the testing site cause inaccuracy (Manley, 1957). The 2-hour testing time is not practical for modernized automated laboratories. Its test procedures present the risk of infection from contact with pathogen-bearing blood (Imafuku and Yoshida, 2001). In this context, several kinds of simple, rapid and safe methods have been developed. Of these new systems, we selected 4 and evaluated their performance. This paper reports critical reviews of such devices.

Hematology tests of blood anticoagulated with magnesium sulphate.

Venous blood treated with magnesium sulphate (MgSO4) at blood collection was used for hematology tests. Complete blood counts and automated leukocyte differentials were obtained using a hematology analyzer, and the results obtained for blood treated with MgSO4 were similar to those for blood treated with dipotassium ethylenediaminetetraacetic acid (EDTA K2). Coagulation tests such as activated partial thromboplastin time, platelet factor 3 availability and prothrombin time were dose-dependently affected by the use of MgSO4. Thrombin time was prolonged by addition of MgSO4, and fibrin concentrations determined by coagulometry tended to decrease with addition of MgSO4, although fibrin concentrations determined by the weighing method were unaffected by MgSO4. MgSO, was thus found to potently inhibit blood coagulation and can be used as an anticoagulant for hematology tests.

Evaluation of hematological values obtained with reference automated hematology analyzers of six manufacturers.

We evaluated assays of the same fresh blood samples with six different types of reference automated hematology analyzers developed by the following manufacturers: Beckman Coulter, Sysmex, Bayer, Abbott, Nihon Kohden and Horiba. Fresh whole blood samples treated with dipotassium ethylenediaminetetraacetic acid (EDTA K2) were collected from three healthy adult volunteers. The complete blood counts (CBC) including red blood cell count (RBC), hemoglobin (Hgb), hematocrit (Hct), mean corpuscular volume (MCV), white blood cell count (WBC), platelet count (Plt), reticulocyte percentage (Ret) and leukocyte differential counts including % neutrophils (Neu), % lymphocytes (Lym) and % monocytes (Mon) were surveyed with a reference automated hematology analyzer from each manufacturer. The process from sampling to analysis was performed according to procedures in hospital clinical laboratories. RBC, Hgb, Hct and MCV exhibited allowable differences within 5% of mean value among all instruments. Large differences greater than 10% of mean value in WBC, Neu and Lym between Horiba and other manufacturers, and in Plt between Nihon Kohden and other manufacturers, were observed. Ret and Mon exhibited large differences over 10% of mean value among almost all of the instruments tested. This survey suggests that all parameters exhibiting differences greater than 10% of mean value among instruments should be improved for clinical use to ensure good external quality control in blood cell counting and leukocyte differential counting using automated instruments.

[Past, present and future of external quality assessment of medical laboratory testing in Japan].

In Japan, three major bodies have conducted external quality surveillance of medical laboratory performance: the Japan Medical Association, the Japan Association of Medical Technologists, and the Japan Society of Registered Medical Laboratories. Although these systems are working very well to elevate the quality of laboratory tests, each has adopted its own surveillance system with little communication. All of these surveillance systems have problems in the evaluation of laboratory proficiency. Thus, the 3 bodies should be unified first to establish an efficient National External Quality Assurance Scheme. New IT technology should also be introduced into the system in the future.

[Education of medical technologists in North America].

The increasing complexity of diseases and advancing medical technologies have recently raised the number of test items and their use. This has caused each department to ask for clinical support by medical technologists, including consultation services on clinical laboratory tests in Japan. Under these circumstances, we think it is necessary to consider a Japanese education system for medical technologists to foster people with advanced ability capable in providing adequate clinical support. It is important that we study medical technologist systems and education systems superior to than ours. Therefore, to investigate the state of the Japanese education system for medical technologists, we conducted a comparison between the medical technologist systems and education systems in foreign countries and those of Japan. The social background in which medical technologists are positioned, their scope of work, and the education systems overseas are different from Japan on many points and we were given many suggestions for what a system in Japan should be like.

[A case of amelanotic melanoma of the nasal cavity].

A 68-year-old male visited Hospital A for treatment of epistaxis, his chief complaint. He was told that he had an easily-bleeding tumor in the nasal cavity. Based of biopsy, a diagnosis of amelanotic melanoma was made. Operation was performed for removal of the tumor. About 8 months after discharge, he visited Hospital B with complaints of lumbar pain and epistaxis. After biopsy at Hospital B, malignant lymphoma (diffuse large cell) was diagnosed, and the patient was referred to our hospital. On bone marrow puncture and biopsy, tumor cell infiltration was observed. Flow cytometric surface marker analysis revealed that these tumor cells were negative for CD45. Results of HE staining of the nasal cavity tumor were insufficient for diagnosis, and staining by immunohistochemistry was necessary to confirm the diagnosis. On immunohistochemical staining of the nasal cavity tumor tissue and bone marrow biopsy tissue, LCA, L26 and UCHL-1 were negative, and S-100 and HMB-45 positive. Recurrence of amelanotic melanoma accompanied by bone marrow infiltration was therefore diagnosed. The incidence of amelanotic melanoma with primary lesions in the nasal cavity is low. However, in making the diagnosis of a nasal cavity lesion, the possibility of such a melanoma should be kept in mind. In many cases, it is difficult to diagnose amelanotic melanoma with HE staining alone, and immunohistochemistry must be used.

[Chronic myelogenous leukemia treated with non-myeloablative stem cell transplantation after discontinuing myeloablative stem cell transplantation due to mental aberrations].

A 54-year-old woman with chronic myelogeneous leukemia was admitted to our hospital on February 15th, 2001 to undergo allogeneic bone marrow transplantation (BMT). We started the transplantation preconditioning with busulfan and high-dose cyclophosphamide on February 22nd, 2001. However, symptoms of a psychiatric nature, such as hallucination, persecution complex, auditory hallucination and sleeplessness, occurred by the third day of treatment with busulfan. Thus, we decided to discontinue conditioning and stopped the administration of BMT at that point. However, pancytopenia persisted for more than 20 days. She finally underwent BMT followed by reduced-intensity conditioning with fludarabine and ATG from a sex-mismatched, HLA-identical sibling donor on April 19th, 2001. To prevent any exacerbation of the psychotic symptoms, the patient was hospitalized in a laminar flow instead of a bio-free room. Graft-versus-host disease occurred on the 32nd hospital day, and was brought under control by steroid treatment. Achievement of complete chimeras was confirmed on the 54th hospital day. Her mental condition was kept stable with antidepressant drugs and tranquilizers, although minor changes in the combination of drugs were required to treat transient exacerbation of psychosis after a short period at home. She was discharged on September 1st, 2001. We think that non-myeloablative stem cell transplantation is a useful treatment for patients with hematological malignancy complicated with psychiatric disorders.