Clinical evaluation of the QuantiFERON-TB Gold test in patients with non-tuberculous mycobacterial disease.

OBJECTIVE: To evaluate the clinical usefulness of the QuantiFERON TB-2G (QFT-2G) test in patients with non-tuberculous mycobacterial (NTM) disease without a previous history of tuberculosis (TB). METHODS: The study consisted of 214 patients with NTM disease who satisfied the diagnostic guidelines of the American Thoracic Society. RESULTS: The causative microorganism was Mycobacterium avium in 83 patients, M. intracellulare in 80, M. kansasii in 33, M. marinum in 12, M. szulgai in 3, M. abscessus in 2 and M. chelonei in 1. The positive response rate of QFT-2G test result was 2% in 163 patients with M. avium-intracellulare complex (MAIC) disease, 52% in 33 with M. kansasii disease, 58% in 12 with M. marinum disease, 33% in 3 with M. szulgai disease, 0% in two with M. abscessus disease and 0% in one with M. chelonei disease. The positivity of the QFT-2G test was 52% in patients with NTM disease, thought to be because NTM possesses common M. tuberculosis-specific antigens. CONCLUSIONS: Although QFT-2G may be a useful diagnostic method to differentiate TB from MAIC disease, there are several problems to be resolved before it can be used as a diagnostic method for NTM disease (M. kansasii disease), including the determination of the positive cut-off level for QFT-2G test.

[Reticulocyte as indication of the erythroid hematopoiesis: reticulocyte fractions in peripheral blood and bone marrow].

Reticulocyte fractions of peripheral blood and bone marrow were measured for hematological disease in 235 patients using automated fluorescent reticulocyte analysis of Sysmex R-3000. The comparison with R-3000 and the manual method of bone marrow measurement showed an excellent agreement with a correlation coefficient of r = 0.933. In the ratio of reticulocyte fractions of marrow blood and peripheral blood, the marrow blood rate of reticulocytes, HFR, MFR, and LFR was 3.3, 6.2, 1.6, and 0.9 times higher than the peripheral blood rate, respectively. Cases in which the marrow reticulocyte rate was over ten times higher than the peripheral blood rate were observed in MDS and megaloblastic anemia at ratio of 55% and 100%, respectively. These findings suggest ineffective hematopoiesis in bone marrow. Immature reticulocyte fraction(10% or more) showed an excellent agreement with granulocyte(500/microliter or more) as an indication of the engraftment of allotransplantation of bone marrow, in which the analysis of reticulocyte fractions showed a useful indication of engraftment. In cases of death after bone marrow transplantation unlike in survivors, reticulocyte fractions decreased after engraftment.

[What to do as a laboratory physician in the front hospital].

The doctor of clinical laboratory medicine in the core hospital at a local city should grasp the situation of the clinical laboratory under present medical institute and submit measures for renovation of the laboratory. Further, the doctor should take part in medical consultation based on the laboratory data.

Characterization of two types of mitochondrial creatine kinase isolated from normal human cardiac muscle and brain tissue.

Two types of mitochondrial creatine kinase (Mi-CK), sarcomeric (sMi-) and ubiquitous (uMi-)CKs, were isolated from normal human cardiac muscle and brain tissue, respectively, and their heterogeneity was characterized by means of isoelectric focusing (IEF). Octameric sMi-CK and uMi-CK were electrophoresed cathodic to cytoplasmic muscle-type creatine kinase isoenzyme (CK-MM) and dimeric Mi-CKs were found at the position of CK-MM on a cellulose acetate membrane. The electrophoretic mobilities of sMi-CK were similar to those of uMi-CK. Octameric sMi-CK was focused at pI 7.1-8.0 and dimeric forms at pI 6.55, 6.75, 6.85, and 6.95. New bands appearing at pI 6.65 and 6.75 after treatment of sMi-CK with carboxypeptidase B were found to be delysined forms. sMi-CK reacted with anti-sMi-CK antibodies, and the immune complexes were focused at pI 5.8. The Km value of sMi-CK for creatine phosphate (PCr) was 1.19 +/-0.20 mmol/L (mean +/- standard error), the activation energy (Ea) was 108.3+/-1.2 kJ/ mol, and the residual enzyme activity after heating at 45 degrees C for 20 min was 79.6+/-1.9%. On the other hand, octameric uMi-CK was focused at pI 7.1-7.9 and the dimeric forms were focused at pI 6.6, 6.7, 6.8, 6.9, and 7.0. Delysined forms were focused around pI 6.3, 6.4, 6.8, and 6.9. uMi-CK reacted with anti-sMi-CK antibodies, and the immune complexes were focused at pI 5.8. The Km value of uMi-CK for PCr was 1.07+/-0.03 mmol/L, Ea of uMi-CK was 110.0+/-0.9 kJ/mol, and the residual enzyme activity after heating at 45 degrees C for 20 min was 90.3+/-0.4%. The sMi-CK and uMi-CK were hybridized and the hybrid Mi-CK appeared at pI 6.78, 6.98, and 7.1-7.95. The pIs of the hybrid Mi-CK were between those of sMi-CK and uMi-CK. As described above, sMi-CK and uMi-CK were slightly different from each other with respect to the pI and some enzyme characteristics.

Sensitive enzymatic assay for erythrocyte creatine with production of methylene blue.

We developed a new, highly sensitive enzymatic method for quantifying creatine in erythrocytes, which comprises creatine amidinohydrolase, sarcosine oxidase, and peroxidase. In the present method, an N-methylcarbamoyl derivative of methylene blue, 10-N-methylcarbamoyl-3,7-bis(dimethylamino)phenothiazine (MCDP), was used as a sensitive chromogenic compound. Potassium ferrocyanide was used to prevent nonspecific oxidation of MCDP. The enzymatic method exhibited good analytical performance: precision, within-run CVs <1.0% and between-day CVs <2.0%; average analytical recovery, 99.3% +/- 1.8%; detection limit, 1.0 micromol/L in hemolysate; and linearity, at least up to 500 micromol/L as creatine concentration in hemolysate. Excellent agreement was observed between the present method (y) and HPLC (x), y = 1.029x - 0.002 micromol/g hemoglobin, r = 0.9998, S(y/x) = 0.053 micromol/g hemoglobin (n = 110). No significant interference was produced by various compounds, including guanidino compounds, amino acids, and reducing materials. The reference intervals (mean +/- 2 SD) for erythrocyte creatine obtained from 60 males and 60 females were (in micromol/g hemoglobin) 1.18 +/- 0.52 (0.66-1.70) for males and 1.35 +/- 0.49 (0.86-1.84) for females. Using this method, we documented changes in erythrocyte creatine in patients with various hemolytic conditions, including hemolytic anemia, liver cirrhosis, renal insufficiency, and chronic renal failure treated with hemodialysis with or without the administration of erythropoietin. We conclude that the use of MCDP allows sensitive measurement of erythrocyte creatine and that MCDP with potassium ferrocyanide can improve the sensitivity of assays that use peroxidase for detection of H2O2.

An enzymatic assay for erythrocyte creatine as an index of the erythrocyte life time.

OBJECTIVES: To establish and estimate an enzymatic measurement of creatine in erythrocytes as an index of the erythrocyte life time. DESIGN AND METHOD: The measurement of creatine in erythrocytes was performed using an enzymatic assay kit that was developed for serum and urine creatine. An erythrocyte sample was subjected to creatine measurement after hemolysis and deproteinization. Performance of the method for creatine measurement in erythrocytes was estimated. Effects of age and gender on the creatine content of erythrocytes were also estimated in 305 normal subjects. RESULTS: The method showed within-run CVs varying from 0.7 to 1.0% (n = 20), and between-day CVs from 1.3 to 1.7% (15 days). Good linearity was observed at least up to 1000 mumol/L as creatine value in hemolyzed sample. The analytical recovery was calculated to be 98.1 +/- 1.3% on average. No considerable interference by various substances, including guanidino compounds and amino acids, with the assay was observed. Excellent correlation was observed between the present method and high performance liquid chromatography. With the unit of mumol/g Hb: slope, 1.034 +/- 0.003 (mean +/- SD); intercept, -0.059 +/- 0.012 (mean +/- SD); correlation coefficient, 0.9996; and Sy.x, 0.069. With the unit of mumol/L RBC: slope, 1.033 +/- 0.003 (mean +/- SD); intercept, -18.23 +/- 3.55 (mean +/- SD); correlation coefficient 0.9996; and Sy.x, 20.40. A significant increase in erythrocyte creatine was observed in females aged 11- to 50 years old as compared with males in the corresponding age bracket, however, a gender difference was not observed in other age bracket. This finding suggests the possibility of a slight decrease in the erythrocyte life time due to menstruation in females. CONCLUSION: This study showed that the present method is favorable for quantifying erythrocyte creatine, and has analytical characteristics suitable for routine work in clinical laboratories.

Total laboratory automation in Japan. Past, present, and the future.

The history of systematized automation in clinical laboratories in Japan started in 1981. At that time, about 12 laboratory technicians worked in a typical private University hospital laboratory (average size 1000 beds), whereas in national university hospitals (typical size 600 beds), the number of technicians was as low as 18-25. In 1981, the Kochi Medical School was founded as a new national school, and laboratory staffing was limited by the Ministry of Education to only 19 technicians for the first 3 years. Therefore, we started to develop a fully automated laboratory system by ourselves rather than accepting an intolerable shortage of technicians. The system was based on conveyor and robotic technology, and we called this approach systematization. Ten years later, systematized automation was introduced into the Japanese market. As a result, 72% of the national university hospitals in Japan installed commercial systems for systematization. There is a trend now in hospitals with sufficient numbers of technicians, to introduce fully automated systems in their laboratories as well, and even small hospitals with less than 100 beds are planning to introduce such systems. However, current technology is too expensive and not sufficiently standardized to meet the needs of these market segments in Japan. We recommend that companies agree on common shapes and sizes of racks and include more flexible robotic technology in their sample handling systems, to allow for plug and play systems and to make systematization affordable for every laboratory in the world.

[Simplification of on-line connection of analyzers and standardization problems].

On-line connection of automated analyzers to laboratory information system (LIS) reduces mistakes in inputing data for each samples. It also makes reporting faster in clinical laboratory. Moreover, connection of these instruments with sample transporting system enables analyses without touching samples directly. It, however, costs extremely high to construct such a system. It is because every automated analyzer uses different connecting protocol, so that we have to make a different program for each machine. For solving this problem, we have to make a standard for connecting protocol. It is very difficult to make a standard protocol fitting on all of the analyzers, considering its cost and other things. Furthermore, it must take a few decades to spreading the standard through the end users. ICCLS and NCCLS has been taking a central role for this problem since 1996, with a five-year plan to make an international standard. Until the standard will be laid, each clinical laboratories have to pay high costs to construct their systems, connecting different manufacturers' analyzers each other. Thus, we have developed a novel system which enables us to construct a laboratory automation system in a shorter time. For realizing this new system, we have reduced the number of connecting protocols for analyzers. Moreover, we have corrected the flow of laboratory works in order. Each programs are put together into a system as parts, or modules. This software system is now in operation in the clinical laboratory of Kochi Medical School. In this report, we describe the construction of this novel software system, and the effect obtained by using this system. Furthermore, we would show problems and things to be improved for making international standards for communication protocols between host and analyzing instruments.

[Adherent reaction among activated platelets, polymorphonuclear cells and vascular endothelial cells under thrombin stimulation or shear stress].

Platelet activation in addition to blood coagulation abnormality is regarded as a primary factor of thrombosis by atherosclerotic obstruction. Therefore, the platelet activation on atherosclerosis is considered to correlate with shear stress generated between blood cells and endothelial cells in blood flow. P-selectin on the surface of the platelet membrane is measured by flowcytometry as a marker of platelet activation. Herein, we examined the phenomena of platelet activation and adherent platelets with leukocytes (ad-P.L) on stored platelet concentration (PC) and chronic rheumatoid arthritis (RA), moreover, of the adherent platelets or polymorphonuclear cells (PMN) with endothelial cells (EC) in shear stress by using an apparatus we devised. The rate of platelet activation and ad-P.L increased in PC with storage, and the sensitivity of platelets to thrombin decreased. The rate of platelet activation and ad-P.L increased in RA in vivo. Many adherent platelets with EC were found at a low shear rate on normal EC but at a high shear rate on denatured EC without any specific adherent property. Adherent PMN with EC had several hundred times more denatured EC than normal EC and the relationship with shear rate disappeared on denatured EC. The platelet activation and relationship between platelets or leukocytes and EC as to the cause of thrombosis are important subjects for future studies.

[Detection of antineutrophil cytoplasmic antibodies in sera from patients with rheumatoid arthritis by indirect immunofluorescence technique].

Anti-neutrophil cytoplasmic antibodies (ANCA) in sera from 52 patients with rheumatoid arthritis (RA), including 9 patients with malignant RA (MRA) and 20 healthy controls were examined by indirect immunofluorescence technique (IIF). Nine out of 52 RA patients showed positive ANCA staining. None of MRA patients had, however, ANCA in sera. The staining pattern for ANCA was either perinuclear for 4 sera or non-specific for 5 sera, but not cytoplasmic. Furthermore, anti-nuclear antibodies (ANA) in 9 ANCA positive RA sera were tested by IIF, using Hep-2 cells. Six sera had positive ANA. Three sera showed as nucleolar and 1 serum as centromere in ANA staining pattern. The incidence of these ANA staining pattern in ANCA positive sera (4 out of 9) was higher than in ANCA negative sera (1 out of 19). The clinical profiles and laboratory findings of 9 RA patients with positive ANCA revealed that they had suffered and treated for more than 10 years and had still active joint inflammation, like intractable RA. These results indicate that ANCA in RA are not associated with vasculitis.

[Study on the relation of human red cell membrane protein band 4.2 to red cell aging--in cases of obstructive jaundice].

The relation of human red cell membrane protein band 4.2 to red cell aging both in normal controls and in cases of obstructive jaundice was studied. The studied subjects were 9 people aged 20 to 73 years, the serum bilirubin levels of whom ranged from 4.8 to 26.7mg/dl. And normal controls were 9 people aged 20 to 30 years. First, we separated red cells into several fractions depending on red cell aging by the method of Vettore et al. The analyses of polyacrylamide gel electrophoresis (PAGE) of each RBC membrane sample by Fairbanks' system revealed the results as follows. Primarily, it was observed that in comparison with band 4.1, band 4.2 both in normal controls and in cases of obstructive jaundice increased depending on red cell aging. Secondly, it was suggested that the decrease of band 4.2 protein in cases of obstructive jaundice should not be due to the higher level of serum bilirubin but to the lower level of red cell aging. Because the obstructive jaundice could shorten the life span of red cells and make the average aging level of red cells younger.

[The correlation between hematopoietic status of the bone marrow and peripheral or marrow reticulocyte classification by using the automated reticulocyte analyzer Sysmex R-3000].

In this study peripheral and marrow reticulocytes were counted by using the automated reticulocyte analyzer Sysmex R-3000 with the quick and accurate function of reticulocyte classification dependent on reticulocyte maturation. The correlation of reticulocyte count of the R-3000 to that of visual reticulocyte counting method was r = 0.926, y = 0.88x + 0.305 and r = 0.933, y = 1.32x + 0.328 for peripheral blood and bone marrow, respectively. The hematopoietic status of the bone marrow was reflected in peripheral reticulocytes better than in leukocytes or platelets. Moreover, at recovery stage from bone marrow suppression after chemotherapy, the ratio of immature type (HFR fraction) in peripheral reticulocytes increased to precede in several days increasing total reticulocytes. The relative reticulocyte counts in bone marrow was about 3 times higher than peripheral blood to be drawn at the same time higher and the patients suffering from megaloblastic anemia, MDS or DIC tended to have a much higher reticulocyte count in bone marrow than in peripheral blood.

[A study on creatine uptake into human erythrocytes: relation to erythrocyte aging].

In order to assess the relation between creatine uptake into human erythrocytes and erythrocyte aging, actual influx of creatine into erythrocytes and creatine contents in erythrocytes were measured by using method of high performance liquid chromatography and isotopic estimation of 14C-creatine concentration. Actual influx of creatine into erythrocytes was showed rapidly, in spite of constant contents of erythrocyte creatine under the condition to be incubated at 37 degrees C for approximate 4 hours in isotonic saline containing high concentration of creatine. Km values of the creatine uptake into young erythrocytes were evidently smaller than that into old erythrocytes. On the other hand, no significant difference of Vmax values was observed to be dependent on erythrocyte aging.

[Clinical usefulness of measurement of creatine contents in human erythrocytes as an index of erythropoiesis].

The apparent half-life of erythrocytes in human blood have usually been measured using 51Cr or 32P-labeled erythrocytes. However, these methods are not suitable for routine methods for the determination of the half-life of erythrocytes because of requirement for both patient admission and complicated procedure. In 1967 Griffiths and Fitzpatrick suggested that creatine level in human packed erythrocytes reflected the mean-age of erythrocyte population. Fehr and Knob reported that erythrocyte creatine levels correlated closely with half-life of erythrocytes by means of 51Cr in severe and milder hemolytic anemias. In order to verify the clinical usefulness of measurement of creatine contents in human erythrocytes as an index of erythropoiesis, the number of reticulocytes counted by an automated reticulocyte counter (R-3000, Toa medical instruments) and creatine contents in erythrocytes measured by high performance liquid chromatography were compared. To evaluate the erythropoietic dynamics, we tried to determine the frequency distribution for individual erythrocyte cell age using erythrocyte creatine. The effect of erythrocyte life-span on hemoglobin A1c(HbA1c) was also appraised in normal blood sugar subjects. A significant increase (p less than 0.005) of erythrocyte creatine was found both in inactive autoimmune hemolytic anemia (AIHA) and in active AIHA. In severe AIHA, the frequency distribution of erythrocyte creatine shifted to the area of high level erythrocyte creatine, and showed a broad-pattern form corresponding to erythropoiesis. Negative correlation (r = -0.707, p less than 0.005) was revealed between erythrocyte creatine and HbA1c in normal blood sugar subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein variants in Hiroshima and Nagasaki: tales of two cities.

The results of 1,465,423 allele product determinations based on blood samples from Hiroshima and Nagasaki, involving 30 different proteins representing 32 different gene products, are analyzed in a variety of ways, with the following conclusions: (1) Sibships and their parents are included in the sample. Our analysis reveals that statistical procedures designed to reduce the sample to equivalent independent genomes do not in population comparisons compensate for the familial cluster effect of rare variants. Accordingly, the data set was reduced to one representative of each sibship (937,427 allele products). (2) Both chi 2-type contrasts and a genetic distance measure (delta) reveal that rare variants (P less than .01) are collectively as effective as polymorphisms in establishing genetic differences between the two cities. (3) We suggest that rare variants that individually exhibit significant intercity differences are probably the legacy of tribal private polymorphisms that occurred during prehistoric times. (4) Despite the great differences in the known histories of the two cities, both the overall frequency of rare variants and the number of different rare variants are essentially identical in the two cities. (5) The well-known differences in locus variability are confirmed, now after adjustment for sample size differences for the various locus products; in this large series we failed to detect variants at only three of 29 loci for which sample size exceeded 23,000. (6) The number of alleles identified per locus correlates positively with subunit molecular weight. (7) Loci supporting genetic polymorphisms are characterized by more rare variants than are loci at which polymorphisms were not encountered. (8) Loci whose products do not appear to be essential for health support more variants than do loci the absence of whose product is detrimental to health. (9) There is a striking excess of rare variants over the expectation under the neutral mutation/drift/equilibrium theory. We suggest that this finding is primarily due to the relatively recent (in genetic time) agglomeration of previously separated tribal populations; efforts to test for agreement with the expectations of this theory by using data from modern cosmopolitan populations are exercises in futility. (10) All of these findings should characterize DNA variants in exons as more data become available, since the finding are the protein expression of such variants.

Search for mutations altering protein charge and/or function in children of atomic bomb survivors: final report.

A sample of (1) children whose parents had been proximally exposed (i.e., less than 2,000 m from the hypocenter) at the time of the atomic bombings of Hiroshima and Nagasaki and (2) a suitable comparison group have been examined for the occurrence of mutations altering the electrophoretic mobility or activity of a series of 30 proteins. The examination of the equivalent of 667,404 locus products in the children of proximally exposed persons yielded three mutations altering electrophoretic mobility; the corresponding figure for the comparison group was three mutations in 466,881 tests. The examination of a subset of 60,529 locus products for loss of enzyme activity in the children of proximally exposed persons yielded one mutation; no mutations were encountered in 61,741 determinations on the children of the comparison group. When these two series are compared, the mutation rate observed in the children of proximally exposed persons is thus 0.60 x 10(-5)/locus/generation, with 95% confidence intervals between 0.2 and 1.5 x 10(-5), and that in the comparison children is 0.64 x 10(-5)/locus/generation, with 95% intervals between 0.1 and 1.9 x 10(-5). The average conjoint gonad doses for the proximally exposed parents are estimated to be 0.437 Gy of gamma radiation and 0.002 Gy of neutron radiation. If a relative biological effectiveness of 20 is assigned to the neutron radiation, the combined total gonad dose for the parents becomes 0.477 Sv. (Organ absorbed doses are expressed in gray [1 Gy = 100 rad]; where dose is a mixture of gamma and neutron radiation, it is necessary because of the differing relative biological effectiveness of gamma and neutron radiation to express the combined gamma-neutron gonad exposures in sieverts [1 Sv = 100 rem]).