(3) summary of the invention
Headspace solid-phase microextraction (HS-SPME)-cooled injection system-gaschromatographic mass spectrometry (GC/MS) coupling that the object of this invention is to provide a kind of novelty carries out method that is qualitative, semi-quantitative analysis to the volatile organic matter in pleural effusion, whether exist in good malignant pleural effusion each material on this basis and content height compare, thus evaluate their value in good the malignant pleural effusion even diagnosis of pleural fluid of patients with lung cancer, and whether there is the potential as specificity and the higher tumor markers of sensitivity.
For achieving the above object, the present invention adopts following technical scheme:
The analytical approach of the headspace solid-phase microextraction-cooled injection system-gas chromatography combined with mass spectrometry of volatile organic matter in a kind of pleural effusion, described analytical approach is: by pleural effusion sample, solid sodium chloride, stirring magneton adds in the ml headspace bottle with poly tetrafluoroethylene dottle pin, at 40 ~ 60 DEG C, under 500 ~ 900rmp stirs, headspace solid-phase microextraction is carried out by the extracting head of CAR/PDMS fiber, after extraction 15 ~ 30min, rapidly extracting head is inserted gas chromatographic sample introduction mouth to resolve, now gas chromatographic column is immersed in the container filling liquid nitrogen near the part of column cap and forms cooled injection system, after resolving 2 ~ 5min, the part of gas chromatographic column near column cap is taken out from the container filling liquid nitrogen, extracting head is taken out from gas chromatographic sample introduction mouth simultaneously, start gas phase program and carry out GC/MS detection analysis, the condition of work of gas chromatograph-mass spectrometer is:
GC conditions is: chromatographic column is low-pole column, selects UA-5 metal capillary post, specification 30m × 0.25mmi.d. × 0.25 μm thickness, 5% methyl-polysiloxane; Post case: initial temperature 35 DEG C, keeps 3 ~ 10min, is raised to 80 ~ 120 DEG C, then is raised to 220 ~ 250 DEG C with 10 ~ 15 DEG C/min speed with 5 ~ 10 DEG C/min speed; Injector temperature is 230 DEG C ~ 300 DEG C; Input mode adopts front 0.1 ~ 1.0min Splitless injecting samples; Carrier gas is helium, flow velocity 1.0mL/min;
Mass Spectrometry Conditions is: electron impact ion source, and ionizing energy is 70eV, ion source temperature 200 ~ 250 DEG C, transmission line temperature 210 ~ 250 DEG C, ion scan scope 40 ~ 550m/z;
Analytic process obtains total ion current figure and the mass spectrogram of pleural effusion sample, in gained total ion current figure, each peak represents a volatile organic components of pleural effusion sample, and each peak obtains corresponding mass spectrogram separately, the mass spectrogram corresponding by each peak of Nist2.0 mass spectrum standard spectrum library searching carries out qualitative analysis to the volatile organic matter in pleural effusion, according to the peak intensity at each peak in total ion current figure, by calculating peak area, semi-quantitative analysis is carried out to the volatile organic matter in pleural effusion, thus obtain the analysis result of volatile organic matter in pleural effusion.
Pleural effusion of the present invention all adopts thoracocentesis collection, is stored in-20 DEG C of refrigerators with the sealing thermal insulation box of built-in Medical ice bag by the pleural effusion of collection, takes out and thaw before analyzing, centrifugal and pleural effusion sample made by fast fetching supernatant.
In analytical approach of the present invention, the consumption of described solid sodium chloride is the amount reached capacity in pleural effusion sample.
Preferred described GC conditions is: chromatographic column is low-pole column, selects UA-5 metal capillary post, specification 30m × 0.25mmi.d. × 0.25 μm thickness, 5% methyl-polysiloxane; Post case: initial temperature 35 DEG C, keeps 3min, is raised to 80 DEG C, then is raised to 230 DEG C with 10 DEG C/min speed with 5 DEG C/min speed; Injector temperature is 250 DEG C; 0.7min Splitless injecting samples before input mode adopts; Carrier gas is high-purity helium (99.99%), flow velocity 1.0mL/min.
Preferred described Mass Spectrometry Conditions is: electron impact ion source (EI), ionizing energy 70eV, ion source temperature 210 DEG C, transmission line temperature 230 DEG C, ion scan scope 40 ~ 500m/z.
The present invention fills in the stainless steel cup of liquid nitrogen form cooled injection system by being immersed near the part of column cap by gas chromatographic column, thus successfully isolates high-volatile organism, improves sensitivity for analysis, improves widening problem; Concrete, described gas chromatographic column near the part of column cap is: the post scope of one section of 5 ~ 15cm length at distance column cap 10 ~ 30cm length place.
Analytical approach of the present invention detect and qualitative go out the higher volatile organic matter of 65 kinds of matching degrees in pleural effusion, obtain it and detect frequency and other average peak area of each material type, the difference of volatile organic matter in com-parison and analysis lung cancer and benign disease chest hydrops.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: headspace solid-phase microextraction treasury is got, enrichment and sample introduction in one, easy and simple to handle, without the need to the pretreatment process of complexity, eliminate the matrix interference of biological sample complexity, do not cause again the distortion of component information in sample; Cooled injection system technology makes high-volatile organism condensation and secondary parsing at low temperatures in pleural effusion, and is effectively separated under suitable chromatographic condition; The method can detect the volatile organic matter in pleural effusion fast, and carries out quantitative and semi-quantitative analysis, then uses difference analysis to distinguish that the screening of even lung cancer marker has very important meaning to good malignant pleural effusion.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is further described, but protection scope of the present invention is not limited in this.
Analyze sample: pleural effusion sample adopts thoracocentesis collection by Zhejiang Provincial People's Hospital's Respiratory Medicine.Sample is fetched with the sealing thermal insulation box of built-in Medical ice bag by experimenter, is stored in-20 DEG C of refrigerators stand-by.Before analysis, pleural effusion sample is taken out from the refrigerator of-20 DEG C, is placed in thawed at room temperature.Thaw complete, sample hose is placed in the centrifugal 10min of hydro-extractor of 3000rmp, pipette supernatant liquor 1mL place magnetic stir bar in advance in 15mL and have in the ml headspace bottle of poly tetrafluoroethylene dottle pin, and take 0.38gNaCl solid and add in sample, make pleural effusion sample to be measured.All patients with lung cancer are the first visit patient that histopathology confirms.
Analytical equipment: the Gc/ms Analyser (GC-MS) of U.S. ThermoFinniganTraceDSQ; U.S. Supleco company 57330U type hand sampling SPME handle and CAR/PDMS extracting fiber.
Embodiment 1:HS-SPME-cooled injection system-GC/MS method is to the analysis of pleural effusion sample
(1) experimental technique
The above-mentioned ml headspace bottle that pleural effusion sample to be measured is housed is placed on magnetic stirring apparatus; moderate-speed mixer (800rmp) is also heated to 50 DEG C; after balance 3min, the extracting head of 75 μm of CAR/PDMS fibers is stretched out stainless steel protection needle tubing; be exposed to the overhead of pleural effusion; after extraction 20min extracting head to be retracted needle tubing; release extracting head in the vaporizer rapidly needle tubing being inserted completely gas chromatography, utilize the high temperature of vaporizer to resolve.Period, the post scope of one section of 10cm length of gas chromatographic column distance column cap 20cm length immerses to fill in the stainless steel cup of liquid nitrogen and forms cooled injection system, and gas phase injection port diverting valve cuts out.After 3min to be resolved, remove the stainless steel cup filling liquid nitrogen, extracting head is taken out from gas chromatography vaporizer simultaneously, start gas phase program and carry out GC/MS detection analysis.
GC conditions is: injector temperature 250 DEG C, and carrier gas is high-purity He (99.99%), flow velocity 1.0mL/min, front 0.7min Splitless injecting samples.Chromatogram column temperature adopts temperature-programmed mode: initial temperature is 35 DEG C, keeps 3min, is warmed up to 80 DEG C, then is warmed up to 230 DEG C with the speed of 10 DEG C/min with the speed of 5 DEG C/min.
Mass Spectrometry Conditions is: electronics bombardment (EI) ion gun, ionization voltage 70eV, transmission line temperature 230 DEG C, ion source temperature 210 DEG C, full scan mass range 40 ~ 500m/z, and molten Ji time delay is 0min.
(2) foundation of analytical approach
The foundation of a, cooled injection system method
In order to eliminate the widening that Splitless injecting samples causes, improving the separation of low-boiling-point organic compound, usually needing in research to make cold-trap with liquid nitrogen or dry ice, by the aggegation of volatile constituent secondary in the kapillary of front end.Cold-trap cryosorption also to heat up desorb instantaneously, reduces systematic error, improves the sensitivity of analysis, improve widening problem.Based on cooled injection system theory, derived many commercial cold sampling systems at present, wherein foremost is the cold injection port of CIS (Gerstel, Germany).But this type of equipment price is expensive, general many pretreatment technology couplings with carrying.Therefore, for the problem that this experimental study runs into, a kind of homemade liquid nitrogen cold trap focalizer (Self-madeCooledInjectionSystem is have employed in experiment, SCIS), capillary column is immersed liquid nitrogen frozen near one section, column cap, thus target components is concentrated on column cap, substantially increase the separating power of capillary column.This easy cooled injection system operation highly versatile, more flexibly, cost is low, is applicable to the research work in laboratory.
HS-SPME extracts volatile organic matter in the pleural effusion obtained and carry out Thermal desorption under the hot environment of injection port, and under the drive of carrier gas, all materials all enter chromatographic column column cap, then under liquid nitrogen cryogenics effect, carry out time condensation enrichment.Subsequently, remove liquid nitrogen plan, start gas chromatography start program and heat up, the organism of column cap is separated in the chromatography column.In order to investigate the effect that this cooled injection system technology is analyzed actual sample, the pleural effusion sample choosing a tuberculous pleurisy patient (man, 24 years old) is tested, and compares, obtain result shown in Fig. 1 with common HS-SPME-GC/MS method.Can significantly find from figure, the spectrogram that common HS-SPME-GC/MS method records forms two large peaks between 1.5 ~ 6min, and peak shape obtains obvious improvement after combining cooled injection system technology, not only successfully isolate lower boiling organism, and individual components response also significantly improves.
The optimization of b, column temperature
As shown in (b) in Fig. 1, overwhelming majority Concentration of matter is detected at 1 ~ 12min, under original temperature programme condition, (35 DEG C keep 1min, 150 DEG C are warmed up to the speed of 10 DEG C/min, 230 DEG C are raised to again with 15 DEG C/min) though can be separated, but effect is undesirable.Along with deepening continuously of research, the continuous accumulation of sample, under we find this column temperature, the high volatile volatile organism separating effect of some of complex sample particularly in cancer patient's pleural effusion is unsatisfactory, usually there is overlap peak, embedding peak, the situation of tailed peak, brings certain difficulty to data analysis afterwards.Therefore, in order to the organic separation of compartment analysis requirement, particularly high volatile volatile of the different pleural effusion sample of As soon as possible Promising Policy, consider the impact of cooled injection system effect simultaneously, in experiment, temperature programme condition should be relaxed as far as possible.In this research, choosing an adenocarcinoma patients (female, 89 years old) pleural effusion sample is example, and (a. initial temperature 35 DEG C keeps 1min to have investigated two kinds of different temperature programme conditions, be warmed up to 150 DEG C with the speed of 10 DEG C/min, then be raised to 230 DEG C with 15 DEG C/min; B. initial temperature 35 DEG C keeps 3min, is warmed up to 80 DEG C, then is raised to 230 DEG C with 10 DEG C/min with the speed of 5 DEG C/min) impact on experimental result.Fig. 2 is the gaschromatographic mass spectrometry figure of pleural effusion under distinct program Elevated Temperature Conditions, and after slowing down heating rate, the peak situation that goes out of sample obtains obvious improvement generally.Not only several low-boiling-point organic compounds of about 2min obtain good separation, after higher organism (cyclohexanone and isooctyl alcohol) the peak shape hangover of 2 peak intensities also improve.Therefore in subsequent experimental, select the temperature programme pattern in (b).
C, amount of samples and the selection of the time of parsing
Consider that pleural effusion sample source is special, sample size few (≤10mL) and being more difficult to get, originally the distortion that utilization factor easily causes biological specimen finger-print information was increased by diluted sample, and the focussing force of cold-trap improves the sensitivity of method to a certain extent, perhaps more low dose of sampling amount also can meet the requirement of analysis.Therefore, this experiment is with a tuberculous pleurisy patient (man, 87 years old) pleural effusion sample be example, choose 1mL and 2mL two dosage to test, with all organic go out peak situation to consider the impact of sample dose, the chromatographic peak not significantly change of the result overwhelming majority, this test is final using 1mL sampling amount as final experiment condition, for subsequent analysis research.
Consider that cooled injection system effect concentrates the secondary enrichment of sample components, sample needed again to investigate in the best of injection port time of resolving, this experiment is for No. 252 samples, investigate parsing time (1min, 3min, 5min) on the impact that actual chest hydrops detects, find that the peak intensity of 1min parsing time is general less, and 3min and 5min is larger, partial organic substances peak intensity presents downtrending when the parsing time is 5min, therefore finally selects 3min to be the top condition of this experiment.
D, reappearance are investigated
In order to verify the reappearance of the method, in experiment, three parallel samplings being carried out to same pleural effusion sample, carrying out mensuration respectively under HS-SPME-cooled injection system-GC/MS condition after optimization and obtaining its total ion current figure (Fig. 3).As can be seen from Figure 3 three chromatograms go out peak number amount and peak intensity does not have obvious difference substantially, spectrogram has good reappearance.Certainly, wherein also inevitably there is the Interference Peaks of more type siloxane.Therefore, in order to compare more accurately, method according to mass spectrum Selective ion mode carries out integration respectively to detect in this sample 21 organic peak areas, calculate its RSD value between 1.35 ~ 13.39%, result shows, under the operation of specification, this technology for detection to pleural effusion sample in most volatile organic matter reappearance good, this accuracy also compared for revision test and the data of great amount of samples provides guarantee.
Embodiment 2: the detection analysis of volatile organic matter in different pleural effusion sample
28 pleural effusion samples and clinical data take from 11 routine cancer patients (pernicious group) and 17 routine benign disease patients (optimum group) respectively, adopt thoracocentesis to concentrate between year Dec in January, 2014 to 2014 gather by Zhejiang Provincial People's Hospital's Respiratory Medicine.Cancer patient, after confirmation is admitted to hospital, namely extracted pleural effusion sample second day early morning on an empty stomach, and optimum control group patient also extracts pleural effusion sample in the morning on an empty stomach.Sample is fetched with the sealing thermal insulation box of built-in Medical ice bag by experimenter, is stored in-20 DEG C of refrigerators stand-by.Wherein, patients with lung cancer 9 example in 11 routine malignant pleural effusion samples, colon cancer 1 example, liver cancer 1 example, tuberculous pleurisy 11 example in 17 routine Benign pleural effusions samples, pneumonia 2 example, other 4 examples (pyothorax, pulmonary abscess, hypothyroidism, coronary heart disease companion hydropericardium).
Under the method condition that embodiment 1 is optimized, carry out detecting analyzing to above 28 actual samples, obtained the pleural effusion gaschromatographic mass spectrometry spectrogram that peak intensity is moderate, degree of separation is good.Fig. 4 is typical good malignant pleural effusion total ion current figure.As shown in the figure, containing a large amount of volatile organic matters in pleural effusion, organism quantity in malignant pleural effusion is generally many than the quantity in Benign pleural effusions, and in malignant pleural effusion most organic peak intensity also higher than the intensity at peak corresponding in Benign pleural effusions.In order to the difference of VOCs in better malignant pleural effusion, first Structural Identification is carried out to the organism detected in 28 pleural effusions, mass spectrum Nist2.0 library searching combines artificial parsing, obtains the VOCs of 65 matching degrees higher (>=73.4%) altogether.Table 1 lists the retention time of these compounds, title, molecular formula, molecular weight, fragments characteristic, No. CAS, matching degree and they are in the good pernicious group of frequency detected.
Result shows that most of organic recall rate is about 20 ~ 80%, recall rate fluctuation range is larger, simultaneously the frequency that detects of high-volatile organism is generally than the height of low volatility, and the frequency that the material of the overwhelming majority occurs between benign and malignant diseases group is close.This also illustrates whether the existence of certain volatile organic matter can not bring the type directly judging good malignant pleural effusion.But the degree of the metabolic disorder caused by each disease is different, the peak intensity of its small molecule metabolites produced may be different, so need to carry out quantitative test to differentiate whether the volatile small molecule organism in good malignant pleural effusion exists certain difference to sample further.
65 organism identified can be divided into alcohols, aldehydes, ketone, ester class, alkane olefines, benzene and its derivative, heterogeneous ring compound, terpene and other compounds non-classified.As can be seen here, in pleural effusion volatile organic matter mainly based on saturated alkane class, oxygenatedchemicals and benzene series derivant.Wherein, ethanol, acetone, 2-methylpentane, 3-methylpentane, ethyl acetate, tetrahydrofuran, methyl cyclopentane, cyclohexane, benzene, normal butyl alcohol, 2 pentanone, heptane, toluene, hexanal, octane, 2, more than 20 the material quilt such as 4-dimethyl heptane, 4-methyloctane, dimethylbenzene, ethylbenzene, phenol, cyclohexanone, 2-ethyl-1-hexanol (isooctyl alcohol), aldehyde C-9 is successively reported in tidal air, blood, urine and the cell tissue appearing at patients with lung cancer, and is considered to potential lung cancer marker; 1-octene-3 alcohol, n-hexyl aldehyde, octane are considered to hepatic carcinoma mark, this also illustrates that pleural effusion is a good biological specimen of screening tumor markers, and its small-molecule substance relevant to pathology comprised may than abundanter in blood, urine, tissue.
Fig. 5 is the average peak area histogram of each chemical classes material in 28 routine good malignant pleural effusions.As seen from the figure, the organic peak intensity in good malignant pleural effusion is completely different.Except the peak intensity of alkane olefin compound is a little less than except optimum group in malignant pleural effusion group, the organism average peak intensity of other classifications is all higher than optimum group.Wherein, the average peak area comparison in difference of alcohols, ketone, aldehydes and ester class is obvious, and it is visibly different for namely expressing containing the metabolism of oxygen small organic molecule in good malignant pleural effusion, can be used as primary part observation object.Secondly, the main organism classification in good pernicious group is also discrepant.Be mainly ketone, alcohols and ester class in pernicious group, and in optimum group, be mainly ketone, alcohols and benzene and derivant class.
Embodiment 3: the comparative analysis of volatile organic matter in lung cancer and lung's benign disease chest hydrops
18 routine pleural effusion samples and clinical data take from 9 routine patients with lung cancer (cancer group) and 9 routine lung inflammation patients (inflammation group) respectively, wherein, gland cancer 5 example in 9 routine malignant pleural effusion in patients with lung cancer samples, the not clear and definite Lung Cancer Types of other 4 examples, tuberculous pleurisy 7 example in 9 routine Benign pleural effusions samples, pneumonia 1 example, pulmonary abscess 1 example.
(1) each organism average peak area is analyzed
In quantitative test and otherness screening substances process, the material accidentally occurred does not have statistical significance, and can produce certain error to result.So, for the qualitative results of select 18 routine samples, filter out verification and measurement ratio and the higher 21 kinds of organism of matching degree, the comparative analysis of the peak area that is averaged.Fig. 6 is 21 organism at average peak area histogram (the former figure of a of cancer group and inflammation group; B amplifies 60 times).From figure (a), the volatile organic matter that in the pleural effusion that this law detects, peak intensity is larger has 7 kinds, i.e. acetone, methylene chloride, ethyl acetate, toluene, cyclohexanol, cyclohexanone and 2-ethyl-1-hexanol.Wherein, toluene, acetone all have report to think potential lung cancer marker, but both average peak area in the lung cancer chosen and lung inflammation pleural effusion sample relatively, and difference is also not obvious.Then there is larger difference in ethyl acetate, cyclohexanol, cyclohexanone and these four kinds of organism of 2-ethyl-1-hexanol, certainly, whether really the property of the there are differences expression of these four kinds of materials also need the analysis making large sample further.
Shown in figure (b), can find after column being amplified 60 times, except the organism that above-mentioned 7 kinds of peak intensities are larger, although other organism intensity are lower, but also show difference trend, such as cyclopentane, methenyl choloride, normal heptane, octane, ethylbenzene and dimethyl benzene etc. detect higher content in cancer group, and ethanol, acetaldehyde, 2-methylpentane, 3-methylpentane then has higher expression in inflammation group.
(2) the organic box-shaped map analysis of otherness
More tentatively think that 7 kinds of volatile organic matters (acetone, methylene chloride, ethyl acetate, toluene, cyclohexanol, cyclohexanone and the 2-ethyl-1-hexanol) intensity detected in pleural effusion is higher previously by peak area average size, certain value may be there is to the discriminating of lung cancer and lung benign disease patient.In order to compare this 7 kinds of organic content distribution and difference in lung cancer group and inflammation group sample better, be depicted as box-shaped figure respectively, as shown in Fig. 7 a ~ 7g.
Box-shaped figure (Box-plot) is a kind of position distribution of expression data set and statistical graph of variation situation of being used for, especially for the position and the change that find and represent different pieces of information, and the exceptional value that simple and clear ground identification data is concentrated.Although our test of hypothesis is based on average, what box-shaped figure provided is not average, but median information.Box-shaped figure 6 back end have done simple summary to data set, and these 6 points comprise first quartile (Q1), the second quartile (Q2), the 3rd quartile (Q3), coboundary, lower limb and exceptional value.Q1 refers to that in single group sample, all numerical value is positioned at the numerical value of 25% after arranging from small to large, Q2 and median, refer to that in single group sample, all numerical value is positioned at the numerical value of 50% after arranging from small to large, Q3 refers to that in single group sample, all numerical value is positioned at the numerical value of 75% after arranging from small to large.The length of Q1 to Q3 is four points of spacing.Coboundary refers to that Q3 arrives distance Q31.5 doubly four points of interior maximal values occurred of spacing, and lower limb refers to that Q1 arrives distance Q11.5 doubly four points of interior minimum value occurred of spacing.Exceed case edge but the point that distance edge is less than 3 four points of spacing is called exceptional value, the point that distance case edge is greater than 3 four points of spacing is called extreme exceptional value.Box-shaped map analysis is not to the requirement of imposing any restrictions property of data, and it is the style of true representation of data shape intuitively.
Fig. 7 a ~ 7g is these 7 kinds organic average peak area distribution box-shaped figure [(a) acetone, (b) toluene, (c) methylene chloride, (d) ethyl acetate, (e) cyclohexanol, (f) 2-ethyl-1-hexanol, (g) cyclohexanone].Although with regard to average peak area, slightly high than inflammation group of the acetone peak intensity of lung cancer group, bigger but than lung cancer group in the acetone population distribution that box-shaped figure shows inflammation group.Main cause is that the content distribution of acetone in lung cancer group is relatively even, although and low than lung cancer group of content of acetone in inflammation group in most of sample, also there is the sample of fraction high-load, dispersion is compared in tolerance distribution.In the same manner, although large than in inflammation group of the toluene peak area population distribution of lung cancer group, dispersion is compared in its distribution, much little than in inflammation group of the value of median.So acetone and the difference of toluene between lung cancer and inflammation pleural effusion are not remarkable, its content size in two groups of pleural effusions can not be pointed out clearly, need larger sample size to differentiate its distribution trend and diversity factor.The otherness of methylene chloride and ethyl acetate is then relatively more remarkable, and the content of inflammation group all remains under the Median levels of lung cancer group, and population distribution is starkly lower than lung cancer group.The recall rate of cyclohexanol in inflammation group is, in 22.2%, box-shaped figure distribution, these two numerical value are made extreme outlier processing, therefore cannot form box-shaped.Therefore, the cyclohexanol of lung cancer group has the organic value of otherness more.2-ethyl-1-hexanol is one of potential lung cancer marker thought in HS-SPME-GC/MS research early stage, what in the sample that this research is chosen, there is some difference equally, but otherness alleviates and the serious skewness that distributes to some extent, this shows that the difference of sample may affect the analysis of otherness result, so whether 2-ethyl-1-hexanol still can need the tracking verification of more longer-terms as the research of lung cancer marker and sensitivity thereof.Cyclohexanone is the most obvious material of otherness in 7 organism, and the peak area of inflammation group cyclohexanone will be far smaller than the peak area of the cyclohexanone detected in lung cancer group, and this result is consistent with the conclusion of seminar early-stage Study.It should be noted that, the extreme exceptional value that this box-shaped map analysis differentiates, be prevalent in certain two sample of lung cancer group, inflammation group then relates to more than 5 samples, this also illustrates that the interference ratio of inflammation group group difference is comparatively large, and this may be relevant with the diversity of the cause of disease.
Be different from mean analysis, box-shaped map analysis more tool has showed this 7 kinds of organism Data distribution8 situations with resembling, and eliminates the interference of exceptional value.On this basis, tentatively think that methylene chloride, ethyl acetate, cyclohexanol, cyclohexanone and these 5 kinds of organism of 2-ethyl-1-hexanol have larger otherness between two groups, have the potential differentiating lung cancer and struvite pleural effusion.