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Off-line exhaled nitric oxide measurements in children.

The concentration of exhaled nitric oxide (eNO) is a useful marker of asthmatic bronchial inflammation. eNO can now be measured away from the laboratory (off-line), even in children. Short exhalation maneuvers (8 sec) and small samples (1 L) of exhaled gas are probably sufficient in children, but more information is needed about the effect of different measurement conditions. As a preliminary step before conducting epidemiological studies in schoolchildren, we investigated the effects of expiratory flow, dead space, and expiratory time on eNO concentrations collected in 1-L mylar collection bags. We studied 101 cooperative subjects (62 males) aged 5-18 years (30 healthy volunteers, 51 asthmatics, and 20 children with various other respiratory diseases) in our pulmonary function laboratory. On-line and off-line eNO were compared in a single session, and analyzed with a Sievers NOA 280 nitric oxide analyzer. For both methods of collecting expired gas, subjects did a single exhalation without breath-holding against an expiratory pressure 10 cm H(2)O. We investigated the effects of expiratory flow, dead space, and exhalation time on eNO; we also compared on-line and off-line eNO measurements, and the repeatability of both techniques at a given flow rate. Expiratory flows of 58 mL/sec provided more reproducible data than lower flows (coefficient of repeatability 1.1 ppb for 58 mL/sec vs. 2.8 for 27 mL/sec vs. 5.7 for 18 mL/sec). eNO concentrations were about 25% higher in off-line than in on-line recordings if the initial 250 mL of exhaled gas were not eliminated, and 37% higher if exhalation lasted longer (16 sec vs. 8 sec). Eliminating 250 mL of dead space and shortening the filling time to 8 sec yielded off-line eNO values close to those on-line (geometric mean off-line eNO 14.4 ppb, 95% confidence interval: 12.2-17.0) vs. on-line eNO 13.8 ppb (95% confidence interval: 11.6-16.5). On-line and off-line results were highly correlated (r = 0.996, P = 0.000) and had similar coefficients of variation (on-line eNO 2.6%, off-line 2.8%). Neither agreement nor repeatability of eNO measurements were affected by disease status or baseline FEV(1) (% predicted values). Once standardized, the off-line eNO technique using 1-L gas collection bags will provide results similar to those recorded on-line.

Adolescent↗

Clinical and technical factors affecting pH and other biomarkers in exhaled breath condensate.

Exhaled breath condensate (EBC) pH appears to be a robust measure of asthma. However, the association between EBC pH and clinical factors and airway inflammatory markers remains unclear. The objectives of this study were to investigate the factors determining EBC pH in asthmatic children, and the reproducibility and effects of collection devices on EBC pH in nine healthy, nonsmoking adults. EBC was collected once from asthmatic children using EcoScreen, and from adults over 3 consecutive days using both RTubes and EcoScreen. EBC pH was measured immediately in non-deaerated samples by microelectrode pH meter. Concentrations of 8-isoprostane, cysteinyl leukotrienes (cys-LT), and leukotriene B4 (LTB4) were measured using enzyme immunoassay. Exhaled nitric oxide concentration (FeNO) was measured by chemiluminescence. Fifty-eight asthmatics (16 intermittent, 12 mild persistent, and 30 moderate-to-severe persistent) were recruited. EBC pH was lower among patients with moderate-to-severe persistent than intermittent asthma (P = 0.046). This marker correlated inversely with disease severity score (rho = -0.276, P = 0.036), but not FeNO or other EBC biomarkers. Bland-Altman analyses found pH but not other EBC biomarkers to be reproducible, which were confirmed by its low coefficient of variation (2.7%; range, 0.4-5.2%). There was poor correlation between pH in EBC collected by RTube and EcoScreen (rho = 0.059, P = 0.784). Factor analysis selected four factors that explained 67.5% of the total variance, and EBC pH clustered with both cys-LT and LTB4. In conclusion, our results suggest that pH in non-deaerated EBC is influenced by asthma severity in children. EBC pH measurement is reproducible, but is dependent on the collection devices used.

Adolescent↗

Biomarkers of neutrophilic inflammation in exhaled air of cystic fibrosis children with bacterial airway infections.

Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) are inflammatory mediators involved in the neutrophil response to pulmonary bacterial colonization in cystic fibrosis (CF). The aim of this study was to investigate whether the LTB(4) and IL-8 levels in exhaled breath condensate (EBC) could be related to the type of bacterial colonization in CF patients. The pH level in EBC was analyzed as an estimate of airway acidification. Forty children were evaluated: 10 CF patients with P. aeruginosa, 10 CF patients with S. aureus, 10 not colonized CF patients, and 10 healthy children. LTB(4) and IL-8 in EBC were analyzed by specific enzyme immunoassay kits (EIA). The pH of EBC was measured with a pH-meter after deareation by bubbling with argon. Exhaled LTB(4) was higher in CF children with P. aeruginosa compared to those with S. aureus (P < 0.01), not colonized (P < 0.001), and healthy children (P < 0.01). Exhaled IL-8 was elevated in CF patients colonized by P. aeruginosa compared with other subgroups (vs. not colonized, P < 0.05; vs. healthy children, P < 0.001). IL-8 levels were higher in CF children with S. aureus than in healthy children (P < 0.05). There was an increase in IL-8 levels in not colonized CF patients compared with healthy children (P < 0.05). EBC pH was higher in healthy children compared to CF patients not colonized (P < 0.05). Our data suggest that EBC is suitable for evaluating neutrophil inflammatory mediators (LTB(4), IL-8, and pH) involved in the response to pulmonary bacterial colonization in CF children.

Biomarkers↗

Markers of airway inflammation in primary ciliary dyskinesia studied using exhaled breath condensate.

Macroscopically, the airways in primary ciliary dyskinesia (PCD) are inflamed and infected, and the eventual result is bronchiectasis. The measurement of noninvasive markers of inflammation in PCD may allow determination of mechanisms of tissue damage, and even allow monitoring of therapy. The aim of this study was to measure in exhaled breath condensate (EBC) of children with PCD the concentrations of the neutrophil chemoattractants leukotriene (LT) B4 and interleukin (IL)-8 and the marker of oxidative stress 8-isoprostane (8-IP), and to try determining whether these markers can be used to assess mechanisms of airway inflammation in these patients. Concentrations of LTB4, IL-8, and 8-IP in the EBC of 23 PCD and 11 age-matched healthy children were measured using an enzyme immunoassay (EIA). The children also performed spirometry and underwent sputum induction, the latter for differential cell count. The concentrations of 8-IP in EBC of children with stable PCD were significantly increased compared to normal controls (median, 7.8 pg/ml vs. 3.1 pg/ml; P = 0.004). There was no difference in the median concentrations of EBC LTB4 between PCD subjects and healthy controls (28 pg/ml vs. 28 pg/ml; P = 0.5). IL-8 levels were below the detection limit of the assay, and were not analyzed further. There was no correlation between concentrations of either 8-IP or LTB(4) in EBC and forced expired volume in 1 sec in PCD children. Sputum induction was successful in 83% of the subjects; the median induced sputum neutrophil count was 69% (interquartile range, 59.3-73.6). No significant correlation was found between sputum neutrophils and either EBC 8-IP or LTB4 concentrations in PCD children. This study showed that oxidative stress, as reflected by increased exhaled 8-IP concentration, is increased in PCD children. The mechanism of airway neutrophilia is unclear, but is unlikely to be related to increased production of LTB4, at least in stable PCD patients.

Adolescent↗

The analysis of 1-propanol and 2-propanol in humid air samples using selected ion flow tube mass spectrometry.

Following the observation that propanol is present in the breath samples of cystic fibrosis (CF) patients infected by Pseudomonas aeruginosa (PA), a study of the reactions of H(3)O(+), NO(+) and O(2) (+.) with 1-propanol and 2-propanol has been conducted using selected ion flow tube mass spectrometry (SIFT-MS). In this study the number and the distribution of the product ions from NO(+) reactions with the two propanol isomers under humid air conditions were able to differentiate between the two isomers. The reaction mechanisms and the structures of the product ions for these reactions, especially those with H(3)O(+) and NO(+), have been proposed. As an example, 2-propanol was shown to be present in a breath sample from one CF patient infected with PA, and also in a PA isolate from another CF patient grown on Pseudomonas-selective media. The results of this study allow an analytical procedure to be advanced for the analysis of the two propanol isomers, which can also be utilised in other applications.

1-Propanol↗

Determination of N epsilon-(carboxymethyl)lysine in exhaled breath condensate using isotope dilution liquid chromatography/electrospray ionization tandem mass spectrometry.

In order to identify new biomarkers for pulmonary diseases in exhaled breath condensate (EBC) it was the aim of this study to develop an analytical method for the identification and quantification of N epsilon-(carboxymethyl)lysine (CML) in EBC. As detection by liquid chromatography with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) offers the advantage of structurally related detection with the necessary specificity required for the identification of a substance, it was the method chosen for the determination of the non-volatile compound. Specific mass transitions and comparison of retention times with standards under given conditions were used for the unequivocal identification of CML in EBC of healthy subjects. Synthesis of isotopically labelled CML was performed and used as an internal standard for an accurate determination. It was possible to identify the advanced glycation end-product CML in 8 out of 10 healthy subjects. The concentration range determined in the quantifiable examined samples ranged between 35 and 110 pg/mL. EBC samples from 11 patients with different diseases such as diabetes and chronic obstructive pulmonary disease were also measured. In one patient with pneumonia a concentration of 1509 pg CML/mL EBC could be detected. This is the first time that CML has been identified and determined in EBC. The developed LC/ESI-MS/MS method could be used to address the utility of CML as a biomarker in pulmonary diseases.

Aged↗

Determination of patterns of biologically relevant aldehydes in exhaled breath condensate of healthy subjects by liquid chromatography/atmospheric chemical ionization tandem mass spectrometry.

A method for the simultaneous determination of several classes of aldehydes in exhaled breath condensate (EBC) was developed using liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS). EBC is a biological matrix obtained by a relatively new, simple and noninvasive technique and provides an indirect assessment of pulmonary status. The measurement of aldehydes in EBC represents a biomarker of the effect of oxidative stress caused by smoke, disease, or strong oxidants like ozone. Malondialdehyde (MDA), acrolein, alpha,beta-unsaturated hydroxylated aldehydes [namely 4-hydroxyhexenal (4-HHE) and 4-hydroxynonenal (4-HNE)], and saturated aldehydes (n-hexanal, n-heptanal and n-nonanal) were measured in EBC after derivatization with 2,4-dinitrophenylhydrazine (DNPH). Atmospheric pressure chemical ionization of the analytes was obtained in positive-ion mode for MDA, and in negative-ion mode for acrolein, 4-HHE, 4-HNE, and saturated aldehydes. DNPH derivatives were separated on a C18 column using variable proportions of 20 mM aqueous acetic acid and methanol. Linearity was established over 4-5 orders of magnitude and limits of detection were in the 0.3-1.0 nM range. Intra-day and inter-day precision were in the 1.3-9.9% range for all the compounds. MDA, acrolein and n-alkanals were detectable in all EBC samples, whereas the highly reactive 4-HHE and 4-HNE were found in only a few samples. Statistically significant higher concentrations of MDA, acrolein and n-hexanal were found in EBC from smokers.

Air Pressure↗

Validation of self-reported smoking status by simultaneous measurement of carbon monoxide and salivary thiocyanate.

OBJECTIVE: The objective of this study was to determine the validity of tobacco questionnaires when using as gold standard either a single biomarker or a combination of two biomarkers. METHODS: The methods were self-reported smoking compared with salivary thiocyanate and expired carbon monoxide in a 1996, population-based, Swiss survey of 552 men and 565 women. RESULTS: Sensitivity of self-reported smoking relative to salivary thiocynate or carbon monoxide alone was low (38.2% for salivary thiocyanate > or = 100 mg/L, 56.4% for salivary thiocyanate > or = 150 mg/L and 62.6% for carbon monoxide > or = 9 ppm). When defining true positive smokers as people with high concentration of both salivary thiocyanate and carbon monoxide, overall, sensitivity was 88.6% and specificity was 87.2%. In women, sensitivity increased from 85 to 89% when removing subjects exposed to passive smoking. When excluding heavy smokers, sensitivity decreased to 63% in men and to 71% in women. Older women had tendency to misreport smoking. CONCLUSIONS: This comparison of questionnaire data with the simultaneous measurement of salivary thiocyanate and expired carbon monoxide indicates that valid responses can be obtained for self-reported, current smoking in population-based surveys. However, the validity of questionnaires can be underestimated if the gold standard (of exposure to tobacco smoke) is either high levels of carbon monoxide or high levels of salivary thiocyanate.

Adult↗

1,2-Dichlorobenzene-mediated hepatocellular oxidative stress in Fischer-344 and Sprague-Dawley rats.

1,2-Dichlorobenzene (1,2-DCB) is a potent hepatotoxicant in male Fischer 344 (F-344) rats but not in Sprague-Dawley (SD) rats. While Kupffer cell-dependent oxidative stress plays a role in the progression of 1,2-DCB-mediated liver injury, we hypothesize that initiation of liver injury is due to oxidative events within the hepatocyte. This study compared hepatocellular oxidative stress marked by glutathione disulfide (GSSG) and glutathione (GSH) production in either bile, liver, or isolated hepatocytes of F-344 and SD rats following 1,2-DCB administration. Hepatic GSH concentrations were depleted at a greater rate in F-344 than in SD rats within 12 h of 1,2-DCB administration (3.6 mmol/kg ip). In bile, GSSG concentrations were threefold greater in F-344 rats compared to SD rats by 9 h of 1,2-DCB treatment. Moreover, 1-aminobenzotriazole but not gadolinium chloride pretreatment blocked the rise in biliary GSSG concentrations following 1,2-DCB administration. In in vitro studies, isolated hepatocytes of F-344 rats had a 15% increase in cellular GSSG concentrations following 1 h of 1,2-DCB (3.55 nmol) exposure, while GSH decreased 22% by 6.5 h compared to controls. In contrast, isolated SD hepatocytes exposed to 1,2-DCB had no increase in GSSG and only an 8% reduction in GSH. Furthermore, parameters of lipid peroxidation were increased in F-344 rats and not in SD rats. Collectively, these data suggest that hepatocellular oxidative stress is dependent upon bioactivation and the enhanced oxidative stress in the F-344 rat may explain its susceptibility to 1,2-DCB compared to the SD rat.

Animals↗

Clinical usefulness of inflammatory markers in asthma.

Asthma is a significant and increasing health problem. Airway inflammation and hyperresponsiveness are key pathophysiological mechanisms underlying asthma. Currently, effective treatments target these two processes and can lead to clinically important improvements in disease control. At present, decisions to initiate or modify therapy are based on symptoms and measures of airway caliber, with no direct assessment of airway inflammation or hyperresponsiveness. It is now possible to measure airway inflammation using noninvasive markers such as exhaled gases, induced sputum and serum measurements. Exhaled nitric oxide (eNO) and induced sputum eosinophils show the greatest promise as clinically useful markers of airway inflammation in asthma. Induced sputum can now be applied to the diagnosis of airway diseases, based on its ability to detect eosinophilic bronchitis in cough, and to differentiate between eosinophilic and non-eosinophilic asthma. The place of induced sputum and eNO in the ongoing monitoring of patients with asthma are now being investigated in controlled trials.

Asthma↗

Association between exhaled nitric oxide, ambient air pollution and respiratory health in school children.

OBJECTIVES: The aim of the study was to investigate whether the level of exhaled nitric oxide (eNO) provides a more sensitive measure to assess adverse pulmonary effects of air pollution than conventional lung function indices. METHOD: The non-selected cohort studied consisted of 68 children (aged 10-11 years) living in an urban environment. For 7 weeks respiratory complaints were reported daily by these children in a diary, and lung function measures and eNO levels were determined in the children once a week on days with various level of air pollution. RESULTS: During the study the increase in the levels of the various air pollutants was positively associated with eNO (6% to 31% increase; P<0.05) but not with lung function measures. In contrast to the lung function measures, the prevalence of respiratory symptoms such as "sore throat", "runny nose", "having a cold", and "sick at home" were positively associated with the level of eNO measured in the following week. CONCLUSIONS: Though clinically very meaningful, functional pulmonary measures appear to be too course to detect relatively mild clinical symptoms in response to exposure to air pollution. In an epidemiological setting the increase in eNO may represent a more suitable measure to assess such adverse effects.

Air Pollution↗

Comparison of breath, blood and urine concentrations in the biomonitoring of environmental exposure to 1,3-butadiene, 2,5-dimethylfuran, and benzene.

OBJECTIVES: To investigate and compare alveolar, blood, and urine concentrations of 1,3-butadiene, 2,5 dimethylfuran, and benzene, in non-occupational exposure to these products. METHODS: Benzene, 2,5-dimethylfuran and 1,3-butadiene were measured in the breath, blood, and urine samples of 61 subjects living in small mountain villages. All 61 were regularly employed as forestry workers. Sampling was done during the long winter-season non-working period. Samples were collected after overnight rest and analysed by headspace and GC-mass spectrometry methods. RESULTS: The median 1,3-butadiene level was 1.2 ng/l (range: <0.8-13.2 ng/l) in alveolar air, 2.2 ng/l (range: <0.5-50.2 ng/l) in blood, and 1.1 ng/l (range: <1-8.9 ng/l) in urine. The median benzene level was 5.7 ng/l (range: <1-24.9 ng/l) in alveolar air, 62.3 ng/l (range: 33.5-487.2 ng/l) in blood, and 63.4 ng/l (range: 25.8-1099.1 ng/l) in urine. The median 2,5-dimethylfuran level was 0.5 ng/l (range: <1-12.5 ng/l) in alveolar air, 2.5 ng/l (range: <5-372.9 ng/l) in blood, and 51.8 ng/l (range: <5-524.9 ng/l) in urine. In several cases, 2,5-dimethylfuran levels were below the detection limit in alveolar air and blood, especially in non-smokers. 1,3-Butadiene, 2,5-dimethylfuran and benzene levels were significantly higher in smokers than non-smokers in all biological media. CONCLUSIONS: 1,3-Butadiene and benzene, as ubiquitous pollutants, are detectable and quantifiable in human alveolar air, blood and urine. 2,5-Dimethylfuran, which is not a usual environmental pollutant, is almost always detectable in biological media, but only in smokers.

Adult↗

Breath, urine, and blood measurements as biological exposure indices of short-term inhalation exposure to methanol.

Due to their transient nature, short-term exposures can be difficult to detect and quantify using conventional monitoring techniques. Biological monitoring may be capable of registering such exposures and may also be used to estimate important toxicological parameters. This paper investigates relationships between methanol concentrations in the blood, urine, and breath of volunteers exposed to methanol vapor at 800 ppm for periods of 0.5, 1, 2, and 8 h. The results indicate factors that must be considered for interpretation of the results of biological monitoring. For methanol, concentrations are not proportional to the exposure duration due to metabolic and other elimination processes that occur concurrently with the exposure. First-order clearance models can be used with blood, breath, or urine concentrations to estimate exposures if the time that has elapsed since the exposure and the model parameters are known. The 0.5 to 2-h periods of exposure were used to estimate the half-life of methanol. Blood data gave a half-life of 1.44+/-0.33 h. Comparable but slightly more variable results were obtained using urine data corrected for voiding time (1.55+/-0.67h) and breath data corrected for mucous membrane desorption (1.40+/-0.38 h). Methanol concentrations in blood lagged some 15-30 min behind the termination of exposure, and concentrations in urine were further delayed. Although breath sampling may be convenient, breath concentrations reflect end-expired or alveolar air only if subjects are in a methanol-free environment for 30 min or more after the exposure. At earlier times, breath concentrations included contributions from airway desorption or diffusion processes. As based on multicompartmental models, the desorption processes have half-lives ranging between 0.6 and 5 min. Preliminary estimates of the mucous membrane reservoir indicate contributions of under 10% for a 0.5-h exposure and smaller effects for longer periods of exposure.

Adult↗

The balloon technique: a convenient method to measure exhaled NO in epidemiological studies.

OBJECTIVES: The aim of the present study was to evaluate the balloon procedure to measure exhaled nitric oxide (NO). METHODS: This was performed by comparing the procedure with the well-established on-line measurement of NO (direct exhalation into the NO module). Using both procedures exhaled NO was measured in 16 healthy subjects on two days with different level of air pollution. RESULTS: Exhaled NO measured on-line was 3.8-4.5 times lower than exhaled NO obtained using the balloon technique but the two sets of values correlated linearly (r 0.93-0.97). Mean NO level on day 1 with low air pollution and day 2 with high air pollution was 6.6 and 8.1 parts per billion (ppb; on-line measurement) and 25.2 and 36.9 ppb (balloon method), respectively. The day 1 to day 2 ratio differed per subject but was independent of the technique of measurement. Mean day-to-day ratio of exhaled NO using the balloon technique (1. 65 +/- 0.13) was not different (P < 0.05) from the ratio of NO levels measured on-line (1.49 +/- 0.13). Based on these ratios the increase in level of outdoor air pollution appears to be associated with a 49-65% increase in exhaled NO. CONCLUSION: Exhaled NO is proposed as a de novo individual biomarker to monitor the adverse effects of air pollution. The balloon procedure offers a sound and convenient alternative for the on-line procedure to measure exhaled NO in large populations as required in epidemiological studies.

Adult↗

Human glomerular mesangial IP15 cell line as a suitable model for in vitro cadmium cytotoxicity studies.

Cadmium represents a major environmental pollutant that may induce severe damage, especially in the kidney where cadmium accumulates. While cadmium is known to severely impair renal tubular functions, glomerular structures are also potential targets. Owing to their contractile properties, glomerular mesangial cells play a major role in the control of glomerular hemodynamics and influence the ultrafiltration coefficient. Cell cultures provide alternative and fruitful models for study of in vitro toxicology. However, the use of primary human mesangial cell cultures is hampered by their limited survival span and their rapid dedifferentiation during passages. This study presents a human stable immortalized mesangial cell line, designated IP15. Cell characteristics were investigated by the detection of known mesangial markers, as well as their ability to contract in response to angiotensin II. IP15 cells were used to investigate cadmium uptake and morphological changes such as cell contraction and cytoskeleton protein expression. The IC(50) cytotoxicity index was obtained with 3.55 micromol/L using neutral red assay for 24 h. After cadmium exposure (1 micromol/L, determined as nonlethal concentration), 0.38 microg Cd/mg protein was internalized by the cells as evaluated by inductively coupled plasma optical emission spectrometry (ICP/OES). Cadmium induced a significant cell surface reduction that correlated with smooth-muscle alpha-actin disorganization. Thus, the IP15 cell line is a suitable model for study of in vitro cadmium cytotoxicity in mesangial cells and allows sufficient material to be obtained for future studies of the intracellular effects of cadmium exposure.

Animal Testing Alternatives↗

Chemokine receptor CXCR4 expression in breast cancer as a potential predictive marker of isolated tumor cells in bone marrow.

Interactions between the CXCR4 chemokine receptor in breast cancer cells and the ligand CXCL12/SDF-1alpha are thought to play an important role in breast cancer metastases. In this pilot study, CXCR4 expression along with other biomarkers including HER2-neu and EGFR, were measured in primary tumor samples of patients with operable breast cancer to test whether any of these biomarkers alone and in combination could indicate breast cancer with high likelihood of metastasizing to bone marrow. Cytokeratin (CK) positive cells in bone marrow were identified by flow-cytometry following enrichment with CK 7/8 antibody-coupled magnetic beads. Primary tumors (n = 18) were stained with specific antibodies for CXCR4, HER2-neu, EGFR, and PCNA using an indirect avidin-biotin horseradish peroxidase method. The majority of the patients had T2/T3 tumors (72%), or lymph node involvement (67%) as pathologic characteristics that were more indicative of high-risk breast cancer. High CXCR4 cytoplasmic expression was found in 7 of 18 patients (39%), whereas 6 of 18 patients (33%) were found to have CK positivity in bone marrow. The median number of CK(+) cells was 236 (range, 20-847) per 5 x 10(4) enriched BM cells. The presence of CK(+) cells in bone marrow was found to be associated with increased expression of CXCR4 alone or in addition to EGFR and/or HER2-neu expression (P = 0.013, P = 0.005, and P = 0.025, respectively) in primary tumors. Furthermore, three patients with high CK positivity (>236 CK(+) per 5 x 10(4) enriched bone marrow cells) in bone marrow exclusively expressed high levels of CXCR4 with EGFR/HER2-neu (P = 0.001). Our data suggest that high CXCR4 expression in breast cancer may be a potential marker in predicting isolated tumor cells in bone marrow. CXCR4 coexpression with EGFR/HER2-neu might further predict a particular subset of patients with high CK positivity in bone marrow.

Adult↗