The influences of sample distribution and age on reference intervals for adult males

This investigation was designed to improve reference information and to evaluate the influences of sample distribution and age on the derived reference intervals. Specimens from 127 men were collected after a 12- to 14-hour fast and analyzed by 60 different laboratory procedures. Differences in the reference intervals derived, using three separate statistical methods, appeared to be unimportant clinically, but the percentile method was preferred because it required no assumptions concerning the frequency distribution. Relationships between age and analyte concentrations were examined by linear regression analysis, and the analytes were placed in one of three groups, according to the significance of this relationship: greatest significance (P less than or equal to 0.01), 18 analytes; intermediate significance (0.01 less than or equal to P less than or equal to 0.05), 12 analytes; and least significance (P greater than 0.05), 30 analytes. The age-related changes for each analyte were evaluated according to analytic variation, population dispersion, and clinical relevance. Age-dependent reference intervals for adult males are recommended for albumin, cholesterol, phosphorus, and sedimentation rate.     

High density lipoprotein cholesterol as a determinant factor in coronary heart disease in Africans

A study of the lipid profile of 200 normal Nigerian subjects (Group A) shows a steady increase in the total cholesterol and triglyceride values with increasing age in both sexes, while the high density lipoprotein (HDL) cholesterol and percent HDL cholesterol values show a steady decrease with increasing age in both sexes. A similar study of 160 patients with high-risk factors (Group B), ie, patients with hypertension, diabetes mellitus, cigarette smokers, and obese patients, shows significantly higher values of mean triglyceride than in the normal subjects (P less than 0.001). The HDL cholesterol and percent HDL cholesterol values are significantly lower in the high risk patients than in the normal subjects (P less than 0.001). A study of the lipid profile of 15 Nigerian patients with coronary heart disease (CHD) (Group C) shows significantly lower in the high-risk patients than in the percent HDL cholesterol than normal subjects (P less than 0.001). These values were also found to be significantly lower in Group C patients than in Group B patients (P less than 0.01). A comparison of the lipid profile of normal Nigerian subjects with those of black Americans shows that the total cholesterol values of normal black Americans are significantly higher than those of normal Nigerians of comparable age and sex (P less than 0.001). Although there is no significant difference in the HDL cholesterol values of both black American and Nigerian males and females, the values of the percent HDL cholesterol of black Americans are significantly lower (P less than 0.01) than those of Nigerians of comparable age and sex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Controlled comparison of the BacT/Alert and BACTEC 660/730 nonradiometric blood culture systems.

In a collaborative study at three university hospitals, the recovery of microorganisms and the speed of detection of microbial growth by the BacT/Alert (Organon Teknika Corporation, Durham, N.C.) and BACTEC 660/730 (Becton-Dickinson Diagnostic Instrument Systems, Sparks, Md.) nonradiometric blood culture systems were compared. A total of 5,918 comparisons were made between BacT/Alert aerobic and BACTEC NR 6A bottles and 5,992 comparisons were made between BacT/Alert anaerobic and BACTEC NR 7A bottles. Each bottle was inoculated with 5 ml of blood. The overall recoveries of microorganisms from the two aerobic bottles were comparable; members of the family Enterobacteriaceae were recovered more often from BacT/Alert aerobic bottles alone (P less than 0.001). The overall recoveries of microorganisms from the anaerobic bottles were not significantly different. Growth of Staphylococcus aureus (P less than 0.001), coagulase-negative staphylococci (P less than 0.01), streptococci (P less than 0.001), Escherichia coli (P less than 0.01), other members of the family Enterobacteriaceae (P less than 0.02), and Pseudomonas aeruginosa (P less than 0.05) was detected earlier in BacT/Alert aerobic bottles. Growth of S. aureus (P less than 0.001), coagulase-negative staphylococci (P less than 0.05), enterococci (P less than 0.01), Streptococcus pneumoniae (P less than 0.02), viridans group streptococci (P less than 0.05), E. coli (P less than 0.001), Klebsiella pneumoniae (P less than 0.01), and other members of the family Enterobacteriaceae (P less than 0.001) was detected earlier in BacT/Alert anaerobic bottles. In a system-versus-system comparison, more gram-positive cocci were recovered from the BACTEC system alone (P < 0.05), and more members or the family Enterobacteriaceae were recovered from the BacT/Alert system alone (P < 0.001). As a system, the BacT/Alert system detected growth of S. aureus (P < 0.001), coagulase-negative staphylococci (P < 0.01), streptococci (P < 0.001), E. coli (P < 0.001), other members of the familyEnterobacteriaceae (P < 0.001), and P. aeruginosa (P < 0.05) earlier than the BACTEC system did.Significantly fewer (40 versus 1,183) false-positive results occurred with the BacT/Alert system. We conclude that the BacT/Alert and BACTEC 660/730 nonradiometric systems are comparable for recovering clinically significant microorganisms form adult patients with bacteremia or fungemia, but that the BacT/Alert system detects microbial growth earlier than the BACTEC system does, with significantly fewer false-positive results.     

Comparison of dysmorphic erythrocytes with other urinary sediment parameters of renal bleeding

Dysmorphic erythrocytes have been described in the urine sediment of patients with renal parenchymal bleeding. This study compares the prevalence of dysmorphic erythrocytes in 5,128 urine specimens with blood, erythrocytic, or fibrin casts, proteinuria, and significant numbers of exfoliated renal tubular cells (RTCs). Of the 510 samples containing pathologic casts, 15% had dysmorphic erythrocytes, 60% had proteinuria, 71% had RTCs, and 12% had no other urinalysis abnormality. Of the 186 samples containing dysmorphic erythrocytes, 55% had pathologic casts, 42% had proteinuria, 71% had RTCs, and 13% had no other abnormality. Renal hematuria can best be evaluated by examination of all four of these urinalysis abnormalities rather than using a single entity for diagnosis. Patients with urinalysis evidence of renal hematuria should be evaluated further for renal disease rather than lower urinary tract disorders.     

Urine sediment analysis by the Yellow IRIS automated urinalysis workstation

The Yellow IRIS automated urinalysis workstation (International Remote Imaging Systems, Chatsworth, CA 91311) performs urine sediment analysis with the use of Automated Intelligent Microscopy (AIM). It ranks and counts particles based on size. A split sample study using 268 individual urine specimens was performed comparing sediment analyte detection by the Yellow IRIS with that achieved by a standard manual method. The detection ratio ("positive" specimens detected by the Yellow IRIS divided by "positive" specimens detected by the manual method) for the presence of various analytes, as well as the presence of abnormal numbers of analytes, was determined. The significance of each detection ratio was tested by calculating the McNemar's statistic, which is useful in the evaluation of nonparametric data collected by two different methods on identical specimens. Results were considered abnormal if the red blood count was greater than or equal to three per high-power field, the white blood cell count was greater than or equal to five per high-power field, or squamous epithelial cells were greater than or equal to five per high-power field. The results indicate that the Yellow IRIS enhances the detection of urinary sediment components.     

Inhibition of lymphocyte cytotoxicity in chronic active hepatitis.

Inhibitory factors were investigated, using 51Cr-labelled Chang cells as targets, based on the inhibition of lymphocyte-mediated cytotoxicity (LMC) in patients with chronic active hepatitis (CAH). A significant reduction of LMC was noted (P less than 0.001) when autologous sera of patients with CAH was added. The addition of HBsAg-positive and -negative homologous sera from CAH, which were as capable of inhibiting LMC as autologous sera, decreased LMC significantly (P less than 0.001). The LMC fell significantly (P less than 0.001) in the presence of sera of patients with rheumatoid arthritis. A significantly higher inhibition (P less than 0.001) by rheumatoid factor (RF) positive autologous sera of patients with CAH was observed when compared to that of RF-negative sera. Such autologous sera inhibiting LMC contained increased IgG levels (P less than 0.01) when compared to those that failed to inhibit LMC. A significant reduction of LMC was also induced with the addition of anti-HBs (P less than 0.05), HBsAg--anti-HBs (P less than 0.001) and IgG--anti-IgG (P less than 0.01) complexes, or aggregated IgG (P less than 0.001). The LMC was reduced significantly (P less than 0.001) after adding a purified liver-specific membrane lipoprotein (LSP) from human livers. There was some evidence for the inhibition of LMC being effected by these additives as a result of their acting on an effector cell level. These findings suggest that humoral factors including immune complexes, aggregated IgG and LSP play an important role for lymphocyte cytotoxicity in the pathogenesis of CAH.     

Status of the clinical laboratory in the mandatory postgraduate medical training system. (1) Report from a laboratory technologist

According to a new system for postgraduate clinical training, 33 medical trainees have been accepted for the past two years at Osaka General Medical Center. Before practicing clinical medicine in each division by a super-rotated table, orientation is scheduled for 5 days to master the basic systems indispensable to the hospital. In this orientation, training in laboratory medicine is performed for 7 hours (3.5 hours for 2 days). Trainees are divided into 4 groups and learn emergency tests of chemistry, hematology and urinalysis, blood transfusion, physiology and microbiology for 60 min each. Laboratory technologists instruct the trainees to gain the basic skills. The main contents are blood gas measuring in chemistry, sample preparation in hematology and urinalysis, taking each other's ECG, ordering blood products for transfusion, serologic study of infectious diseases, and Gram stain in microbiology. Although it is difficult to find time for routine analysis and instructing trainees in the clinical laboratory, it is a suitable opportunity for revision, also for laboratory technologists, and for communication to discuss clinical matters.     

Silicon contents in normal,fatty streaks and atheroma of human aortic intima: its relationship with glycosaminoglycans.

The silicon (Si) content and glycosaminoglycans (GAG) of normal, fatty streaks and atheroma of human aortic intima was measured. The Si content in fatty streaks and/or atheroma was significantly higher than in normal human aortic intima (P less than 0.05). The GAG content in human aortic intima was inversely correlated with the severity of atherosclerosis as described in many reports. However, the percentage composition of dermatan sulphate (DS) in total GAG was found to increase with the advance of atherosclerosis, and was significantly higher than that in normal and fatty streaks (P less than 0.001, P less than 0.01, respectively). In human aortic intima, a significant negative correlation was seen between Si content and the content of each GAG except GlcUA-GalNAc-6-sulphate (CS-6-S), and a significant positive correlation was noted between Si content and the content of each lipid. Interestingly, the percentage composition of DS in total GAG showed a significant positive correlation with Si content in human aortic intima (r = 0.603, P less than 0.005). These results suggest the increase in Si in the aortic intima is related to the occurrences and/or progression of atherosclerosis, whether primary or secondary.     

Grading of superficial antral gastritis: comparison of cell-counting and photographic-based methods

A cell-counting technique and a photographic-based scoring system for assessing inflammatory cell infiltration were compared for agreement in 275 endoscopic gastric antral biopsies from 55 dyspeptic patients. Each biopsy was independently scored 0-5 for polymorphs and mononuclear cells using both methods, and a mean score was derived for each patient. There was a highly significant correlation between the mean patient scores obtained using each method (P less than 0.001). Agreement statistics showed that the mean score difference (cell-counting minus photographic score) was negative for polymorphonuclear and mononuclear cells, indicating that the photographic method yielded higher mean patient scores. Scatterplots of difference versus mean by the two methods showed that only 3.6 per cent of scores fell outside +/- 2 SD of the mean difference. The use of standard photomicrographs to grade superficial antral gastritis therefore provides accurate results, comparable to those obtained by cell counting, yet is a faster and much cheaper technique.     

Morphometric analysis of the vagus nerve in non-diabetic and ketonuric diabetic Chinese hamsters

Morphometric analysis of axons from the ventral division of the vagus nerve of ketonuric diabetic Chinese hamsters and age-sex matched non-diabetic controls was performed to determine the frequency distribution and numerical and volume density. Myelinated fibres of diabetics displayed a significant reduction in diameter (P less than 0.001) compared with controls, which was correlated inversely with progressive ketonuria (P less than 0.05). The reduced calibre of myelinated fibres was the result of thin myelin sheaths rather than a reduction in axon diameter. A marked decrease in numerical density (P less than 0.05) and volume density (P less than 0.005) was found in the myelinated fibres of diabetics compared with controls. Non-myelinated axons showed a significant shift to smaller diameter (P less than 0.001) in diabetics, which was correlated inversely with duration of ketonuria (P less than 0.05). Numerical density of non-myelinated axons was increased (P less than 0.01) in diabetic hamsters whereas volume density was comparable in diabetic and control animals. These data provide morphological evidence of impairment in the parasympathetic nervous system which may be a major factor underlying previously reported gastrointestinal and pancreatic islet dysfunction in the diabetic Chinese hamster.     

A comparison of six epicutaneous devices in the performance of immediate hypersensitivity skin testing

Six devices commonly used for immediate hypersensitivity epicutaneous skin testing were compared with regard to precision and diagnostic accuracy. Fifteen subjects were tested on the back to 10 mg/ml of histamine phosphate and 50% glycerosaline by prick technique with a smallpox needle (SN), bifurcated needle (BN), Greer "pen" (GP), and blood lancet, and by puncture with the Morrow-Brown needle (MB) and Multi-Test (MT). Five devices were tested in quintuplicate to histamine and once to glycerosaline in each subject; with MT, five histamine and three glycerosaline sites were used. Analysis of the wheal areas obtained with SN, BN, GP, and MB demonstrated comparable degrees of precision (coefficient of variation). The precision of MT was less than the other devices (p less than 0.05). The blood lancet demonstrated intermediate precision. Twenty-two of 45 (49%) of the glycerosaline skin tests performed with MT were falsely positive, significantly more than the other devices (p = 0.0001). We conclude that MB, BN, GP, and SP are comparable devices for use in immediate hypersensitivity skin testing. The low precision and reliability of MT used for testing on the back would appear to make this device less than adequate for diagnostic or research studies. Its high rate of false positive reactions requires caution in interpretation of results when it is used in the clinical diagnosis of allergy.     

Effect of different dialyzer membranes on serum angiotensin-converting enzyme during hemodialysis

The effects of different dialyzer membranes on serum concentration of angiotension-converting enzyme (ACE) and white blood cells during hemodialysis were examined on a cross-over basis in 20 chronically uremic patients. Hemodialysis with cuprophane membranes was associated with a significant (p less than 0.001) fall in the mean leukocyte count during the 1st hour of treatment. The use of polymethylmethacrylate membranes resulted in a more attenuated form of leukopenia and with polyacrylonitrile membranes no change was observed during hemodialysis. Hemodialysis with each membrane caused a comparable, significant (p less than 0.005) increase in serum ACE, independent of the degree of leukopenia but significantly (p less than 0.001) correlated with the increases in serum proteins. We conclude that this increase in serum ACE concentration after hemodialysis does not reflect acute damage of the pulmonary vascular endothelium during treatment and most probably is a result of hemoconcentration. Therefore, serum ACE analysis is not an indicator of dialyzer membrane biocompatibility.     

Saturation-inhibition index as a measure of the risk of calcium oxalate stone formation in the urinary tract.

Studies were carried out on multiple urine samples from eight patients with recurrent idiopathic calcium oxalate stone formation and eight normal persons to define an index of the risk of forming calcium oxalate stones. Under the same conditions of dietary and fluid intake the urine samples of the patients with stone formation were more supersaturated with calcium oxalate (P less than 0.001) and had lower concentrations of protective inhibitors of crystallization (P less than 0.001) than those of the controls. However, the best separation between the groups was defined by a discriminant line relating inhibitory activity and urine saturation. A measure of the risk of forming large crystals, the saturation-inhibition index, was defined as the distance of each urine from the discriminant line. The patients with stone formation had a significantly higher mean saturation-inhibition index than the controls (P less than 0.001). Both the percentage of large calcium oxalate crystals excreted (P less than 0.001) and the stone episode rate (P less than 0.005) were significantly correlated with the saturation-inhibition index.     

Problems in determining immune status in borderline specimens in an enzyme immunoassay for rubella immunoglobulin G antibody

A total of 374 sera, found by enzyme-linked immunosorbent assay (Rubazyme; Abbott Laboratories, North Chicago, Ill.) to have borderline rubella antibody levels, were tested by hemagglutination inhibition. All sera had Rubazyme indexes in the range of 0.500 to 1.499. Rubazyme sensitivity was 59.0%, and specificity was 80.8%. The predictive value of Rubazyme-positive result was 91.4%, and that for a negative result was 36.4%. Immune and nonimmune results were significantly different between the two methods (P less than 0.001). The same sera were retested with the Rubazyme test, with an inter-run agreement of 75.3%. A significant difference in Rubazyme indexes between runs (P less than 0.001) was observed. An alternative method of testing specimens in the range close to the Rubazyme index cutoff value of 1.000 may be indicated.     

Association of HLA-DRw9 with myasthenia gravis in Chinese

HLA phenotypes were studied in 59 Chinese myasthenia gravis (MG) patients. Unfractionated peripheral blood mononuclear cells were used for typing of HLA-A, B and C, and B-enriched lymphocytes for HLA-DR. Seventy-nine healthy subjects were included as controls. When compared with healthy controls, the patients showed significant increases in HLA-Bw46 (47.5% versus 14%, chi 2 = 18.7, P less than 0.001), HLA-DRw9 (59.3% versus 11%, chi 2 = 35.7, P less than 0.001), while HLA-DR3 was decreased (3.4% versus 32.9%, chi 2 = 18.2, P less than 0.001). Further analysis showed that the primary association was with DRw9 and the increase in Bw46 was secondary to it. Both HLA-Bw46 and -DRw9 were increased in all subgroups except where the age of onset was greater than 40 years. Finally, the HLA-A2-Bw46-DRw9 combination was also significantly increased in patients (25.4% versus 5.1%, chi 2 = 11.5, P less than 0.001), especially in the subgroups of MG with age of onset of less than 10 and males with age of onset of less than 40 years.     

A multicenter, controlled trial of ursodiol for the treatment of primary biliary cirrhosis. UDCA-PBC Study Group.

BACKGROUND. In primary biliary cirrhosis the hepatic lesions may result, at least in part, from the intracellular accumulation of potentially toxic endogenous bile acids. Preliminary work suggests that the administration of ursodiol (also called ursodeoxycholic acid), a hydrophilic bile acid without hepatotoxicity, leads to improvement in the condition of patients with primary biliary cirrhosis. METHODS. We conducted a two-year, multicenter, double-blind trial to compare the efficacy of ursodiol with that of placebo. Patients with biopsy-proved primary biliary cirrhosis were randomly assigned to receive either ursodiol (13 to 15 mg per kilogram of body weight per day) (n = 73) or placebo (n = 73). Treatment failure was defined as a doubling of bilirubin levels to more than 70 mumol per liter or the occurrence of a severe complication (ascites or variceal bleeding) or an adverse reaction. RESULTS. Treatment failed in 6 patients in the ursodiol group, as compared with 13 in the placebo group (P less than 0.01 by Cox regression model). A single patient in each group withdrew because of minor adverse effects. After two years of treatment, the proportion of patients with clinically overt disease decreased only in the ursodiol group (P less than 0.02). The patients treated with ursodiol had significant improvements in serum levels of bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, cholesterol, and IgM (all P less than 0.001); the antimitochondrial-antibody titer (P less than 0.01); and the Mayo risk score (P less than 0.001). Follow-up analysis of 95 liver-biopsy specimens showed a significant improvement in the mean histologic score (P less than 0.002) and in all the characteristic histologic features except fibrosis only in the group given ursodiol. CONCLUSIONS. Ursodiol is a safe and effective treatment for primary biliary cirrhosis.     

Comparison of costs of endovascular repair versus open surgical repair for abdominal aortic aneurysm in Korea

This study was designed to compare the hospital-related costs of elective abdominal aortic aneurysm (AAA) treatment and cost structure between endovascular aneurysm repair (EVAR) and open surgical repair (OSR) in Korean health care system. One hundred five primary elective AAA repairs (79 OSRs and 26 EVARs) performed in the Seoul National University Hospital from 2005 to 2009 were included. Patient characteristics were similar between two groups except for older age (P = 0.004) and more frequent history of malignancy (P = 0.031) in EVAR group. Thirty-day mortality rate was similar between two groups and there was no AAA-related mortality in both groups for 5 yr after repair. The total in-hospital costs for the index admission were significantly higher in EVAR patients (mean, KRW19,857,119) than OSR patients (mean KRW12,395,507) (P < 0.001). The reimbursement was also significantly higher in EVAR patients than OSR patients (mean, KRW14,071,081 vs KRW6,238,895, P < 0.001) while patients payments was comparable between two groups. EVAR patients showed higher follow-up cost up to 2 yr due to more frequent imaging studies and reinterventions for type II endoleaks (15.4%). In the perspective of cost-effectiveness, this study suggests that the determination of which method to be used in AAA treatment be more finely trimmed and be individualized.     

Different response of body weight change according to ketonuria after fasting in the healthy obese

The relationship between obesity and ketonuria is not well-established. We conducted a retrospective observational study to evaluate whether their body weight reduction response differed by the presence of ketonuria after fasting in the healthy obese. We used the data of 42 subjects, who had medical records of initial urinalysis at routine health check-up and follow-up urinalysis in the out-patient clinic, one week later. All subjects in the initial urinalysis showed no ketonuria. However, according to the follow-up urinalysis after three subsequent meals fasts, the patients were divided into a non-ketonuria group and ketonuria group. We compared the data of conventional low-calorie diet programs for 3 months for both groups. Significantly greater reduction of body weight (-8.6 ± 3.6 kg vs -1.1 ± 2.2 kg, P < 0.001), body mass index (-3.16 ± 1.25 kg/m(2) vs -0.43 ± 0.86 kg/m(2), P < 0.001) and waist circumference (-6.92 ± 1.22 vs -2.32 ± 1.01, P < 0.001) was observed in the ketonuria group compared to the non-ketonuria group. Fat mass and lean body mass were also more reduced in the ketonuria group. In addition, serum free fatty acid concentration after intervention in the ketonuria group showed significant more increment than in the non-ketonuria group. The presence of ketonuria after fasting may be a predicting factor of further body weight reduction.     

Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity

Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution.     

Thymidine labeling index, flow cytometric S-phase measurement, and DNA index in human tumors. Comparisons and correlations

The results of flow cytometry were compared with the tritiated thymidine labeling index (TLI) in 314 tumors studied in one laboratory and 163 tumors from a second laboratory, a total of 477, including 306 breast carcinomas. Flow cytometric DNA measurements were obtained on mechanically dissociated fresh tissue stained with propidium iodide or DAPI. Spearman's rank order correlation coefficients (r) for TLI versus flow cytometric S-phase measurement (%S) were 0.38 to 0.55 (P less than 0.001) for diploid DNA index (DNAI) and 0.59 and 0.67 (P less than 0.001) for aneuploid DNAI. The correlations were similar for breast and nonmammary tumors. No correlation of DNAI with TLI or %S was observed for aneuploid tumors, although the aneuploid tumors had significantly higher TLI and %S than diploid tumors (P less than 0.001). Mean TLI for the breast carcinomas was 4.6% and 3.6% for diploid tumors in Series 1 and 2, respectively; for aneuploid tumors, 9.9% and 9.3%. Respective mean %S for diploid breast carcinomas was 6.8% and 7.4%; for aneuploid breast carcinomas, it was 13.6% and 14.5%. The excess of %S over TLI was greatest for tumors with low TLI and appeared to result largely from debris in DNA histograms. TLI, %S and DNAI each predicted both absolute survival and relapse-free survival of breast carcinoma patients.