Vito Brusasco

1. Joint course with ERS “How to write an article” Material and methods Vito Brusasco Department of Internal Medicine Medical School University of Genoa, Italy Respiratory Pathophysiology Unit San Martino University Hospital Genoa, Italy

2. Material and methods • It is usually placed right after the Introduction • Be concise but complete • Organized as: • Study subjects or animals • Study design • Procedures • Analysis

3. Material and methods General guidelines

4. Material and methods Style • Always use past tense • Active or passive voice? • Active: clearer and more direct, emphasizes the role of the operator. “We chose method A instead of method B because we found it more reproducible (unpublished observations)”. • Passive: it is often preferred (or mandatory) to indicate objective procedures that can be replicated by others. The individual doing the experiment is therefore relatively unimportant.“Lung function was measured by spirometry” is better than “We measured lung function by spirometry”.

5. Material and methods Style • Active or passive voice? • Active: Sometimes emphasizes the role of the subject under study“To have lung volume measured, the subject took a deep breath……..” . • Passive: Sometimes emphasizes the role of the operator “To have lung volume measured, subjects were asked to take a deep breath……..”. • Either form is acceptable

6. Material and methods Style • Avoid redundant wording such as • a total of n subjects • four different groups • absolutely essential • there were several subjects who completed… • small in size • period of time • also included • except for • Use because instead ofbased on the fact that • Use for or to insteadoffor the purpose of • by means of • during the course of

7. Material and methods Style Avoid ambiguity Varying -- Distinguish from various or differing. Using varying amounts means individually changing amounts rather than a selection of various or different ones. Apparent -- means obvious, clear, plainly evident, but also means seemingly or ostensibly as well as observably. You know the meaning that you intend, but readers may not. Compare with, compare to -- Compare with means to examine differences and similarities; compare to means to represent as similar. “The music of Brahms compares to that of Beethoven”but to say so, one must first “compare the music of Brahmswith that of Beethoven”.

8. Material and methods Style Avoid ambiguity High(er), low(er) – Don’t use for other words such as greater, lesser, larger, smaller, more, fewer. “Occurrences of higher concentrations were lower at higher levels of effluent outflow”. One interpretation is that greater concentrations were less frequent as effluent volume increased, but others also are possible. Hyphening -- Often needed to clarify what is modifying what. “a batch of 3 10-mL vials” is different from a “batch of 10 3-mL vials”, as “a man eating fish”is very differentfrom“a man-eating fish”!

9. Material and methods Study subjects or animals • Describe how they were identified • State the inclusion criteria for case-controls or subjects • Provide anthropometric characteristics of subjects in a table, including physiological and clinical data that are not variables of the study • Provide details of animal species and/or strains • Make statements on study approval from local Ethics Committee (for all human studies) and regulatory authority (for clinical trials) or compliance with the Helsinki convention (for animal studies)

10. Material and methods Study design • Clearly state what are the main objective(s) of the study. • Describe how the study was structured (e.g., randomization, blinding, parallel groups, cross-over) • Describe the animals used in the study by giving: • source (supplier or where and how they were collected) • typicalsize (weight, length) • age (newborn, young, adults) • how they were handled, fed, and housedbefore and during the experiment(s)

11. Material and methods Study design • Always identify treatments by the variable or treatment name, NOT by an ambiguous, generic name or number “Bronchodilator group” and “Placebo group” instead of “Group 1” and “Group 2”. • When your paper includes more than one experiment, use sub-headings to help organize your presentation by experiment. • State how the sample size was calculated in order to have enough subjects (animals) to address the question.

12. Material and methods Study design • Give details on location and time of study only when necessary. • In field-based studies, usually authors describe the study region in general terms in the Introduction and then describe the study site and climate in detail in the Materials and Methods section. • For clinical trials, information on settings (hospital, primary care) and location is essential to judge for applicability and generalizability of results. • For laboratory studies, usually unnecessary unless you have performed experiments at a particular location or lab because it is the only place to do it and you want to give the opportunity to others to use it.

13. Material and methods Procedures • The usual order of presentation of methods is chronological, but related methods may need to be described together and strict chronological order cannot always be followed. • Describe the protocol for your study in sufficient detail that other scientists could repeat your work to verify your findings. Foremost in your description should be the "quantitative" aspects (e.g., masses, amounts, volumes, times, concentrations, temperature, dosage and route of administration). • Always use decimal metric system and degrees Celsius.

14. Material and methods Procedures • Equipment and materials available off the shelf should be described exactly and sources of materials should be given if there is variation in quality among supplies. Modifications to equipment or equipment constructed specifically for the study should be carefully described in detail. • Provide relevant citations for methods previously published, but make sure to describe any modifications you have made for the purpose of your study.

15. Material and methods Procedures • Avoid wordy or overly details. • “The petri dish was placed on the turntable. The lid was then raised slightly. An inoculating loop was used to transfer culture to the agar surface. The turntable was rotated 90 degrees by hand. The loop was moved lightly back and forth over the agar to spread the culture. The bacteria were then incubated at 37 C for 24 h”. Bad • “Each plate was placed on a turntable and streaked at opposing angles with fresh overnight E. coli culture using an inoculating loop. The bacteria were then incubated at 37 C for 24 h”. Better • “Each plate was streaked with fresh overnight E. coli culture and incubated at 37 C for 24 h”. Best

16. Material and methods Procedures • Avoid using ambiguous terms. • “A Spec 20 was used to measure A600 of Tubes 1,2, and 3 immediately after chloroplasts were added (Time 0) and every 2 min. thereafter until the DCIP was completely reduced. Tube 4's A600 was measured only at Time 0 and at the end of the experiment”. • “A Spec 20 was used to measure A600 of the reaction mixtures exposed to light intensities of 1500, 750, and 350 uE/m2/sec immediately after chloroplasts were added (Time 0) and every 2 min. thereafter until the DCIP was completely reduced. The A600 of the no-light control was measured only at Time 0 and at the end of the experiment”.

17. Material and methods Analysis • Clearly state and define the main outcome measure(s). • Indicate what types of descriptive statistics were used and which analyses (usually hypothesis tests) were employed to answer each of the questions or hypotheses tested and determine statistical significance. • Briefly state the statistical methods you used if they are standard. New methods should be described in detail with justification.

18. Material and methods Analysis • Be clear about what the outcome measure involves: • Counting people, animals, or objects (categorical data) • Taking measurements on people, animals, or objects (continuous data) • Time-to event data (e.g., survival data)

19. Material and methods Analysis Use the appropriate test:

20. Material and methods Analysis • Use the appropriate test: • Use ANOVA or Kruskall-Wallis test to compare differences of the means for more than two groups. • Use repeated-measure ANOVA or Friedman test to compare paired data for more than two groups. • Use mixed between-within groups ANOVA for repeated measures in different groups. • Use post-hoc tests for multiple comparisons when ANOVA yields significant effects, or consider adjusting p-values for multiple comparisons, or provide 99% confidence intervals.

21. Material and methods Analysis • Use the appropriate test: • Be clear about repeated measures data. A study of 10 repeated measurements of a variable on 10 subjects is not the same as 1 measurement on 100 subjects; there are still only 10 subjects. • Subgroup analyses tend not to produce reliable results if there are (a) too few patients or (b) too few events.

22. Material and methods Analysis • Use the appropriate test: • For screening or diagnostic studies use "detection rate" (sensitivity) and "false-positive rate" (1-specificity). • Using specificity to compare two tests can mask an important difference. For example, specificities of 96% versus 98% both look high. However, the corresponding false-positive rates are 4% versus 2% (one is twice as large as the other).

23. Material and methods Analysis • Use the appropriate test: • For assessing agreement between methods do not use correlation but Bland and Altman plot. Good correlation r=0.94 (p<0.001) Considerable lack of agreement (±80 L/min)

24. Material and methods Analysis • Mean ± SD or Mean ± SEM? • SEM = SD / sqr(n) • SD represents variation in the values of a variable, it gives an idea of the variability of single observations. • SEM represents the spread that the mean of a sample of the values would have if you kept taking samples, it gives an idea of the accuracy of the mean. • SEM does not convey • statistical significance!

25. Material and methods Analysis • P<0.05, is there anything is special about it? • An hangover from the days before computers, when it was difficult to calculate exact p values for the value of a test statistic. • P<0.05 does not mean that there is a difference • P>0.05 does not mean that there is no difference. • P values for these two • tests are p=0.051 • and p=0.049, but nobody • can tell which is which.

26. Material and methods Analysis • P<0.05 is regarded as unlikely enough to reject the null hypothesis. It allows to say“the effect is statistically significant at the 5% level”. But some conclude “there is a real effect”. • P>0.05 means there is not enough evidence to reject the null hypothesis. It allows to say “the effect is statistically non-significant”. But some conclude“there is no effect”. • For between-groups comparison, provide estimates of effect sizes and the corresponding CI or p values. • Report all main effect sizes whether statistically significant or not.

27. Material and methods Analysis • State which is the probability level you accept as significant. • Calculate exact p values with 2 or 3 decimal digits • There is no need to show the value of the test used (t, F, chi-square, U). • For regression and correlation calculate r2 to give an estimate of the variance explained.

28. Material and methods Analysis • We can disprove things only in pure mathematics, not in real life. • Failure to reject the null hypothesis doesn't mean we have to accept it. • In any case, true effects are always "real”. • The null hypothesis is always false! • The P value is not a probability of anything in reality. • Some useful effects aren't statistically significant. • Some statistically significant effects aren't useful. • Non-significant is usually misinterpreted as unpublishable. • So good data don't get published.

29. Material and methods Clinical trials The International Committee of Medical Journals Editors requires that clinical trials are registered before the beginning of patient enrollment. For this purpose,a clinical trial is defined as any research project thatprospectively assigns human subjects to intervention or comparisongroups to study the cause-and-effect relationship between amedical intervention and a health outcome. Information on the registry where the study was registered should be given. Reporting of randomized controlled trials should conform to the CONSORT statement, which provides a set of recommendations comprising a list of items to report and a patient flow diagram (www.consort-statement.org)

30. Joint course with ERS “How to write an article” Discussion Vito Brusasco Department of Internal Medicine Medical School University of Genoa, Italy Respiratory Pathophysiology Unit San Martino University Hospital Genoa, Italy

31. Discussion Function • To interpret your results in light of what was already known about the subject of the investigation, and to explain the new understanding of the problem in the light of the results of the study. • Connects to the Introduction by way of the question(s) or hypotheses posed and the literature cited, but it does not simply repeat or rearrange the Introduction. • It tells how the study has moved us forward from the place you left us at the end of the Introduction.

32. Discussion General guidelines

33. Discussion Style • Past or present tense? • Past tense, use it to summarize your findings or those of previous studies • Present tense, use it to make statements on the interpretation of data and conclusions. • Active or passive voice? • Use the active voice whenever possible. Use of the first person is okay, but too much use of the first person may actually distract the reader from the main points.

34. Discussion Content • Comment on the methodological limitations of your study. Some place this after the comments on results but before is preferable. • Address each of the experiments or studies for which you presented results. Discuss each in the same sequence as presented in the Results. The most important findings should be presented first. • Do not restate your results; if you need to remind the reader of the result to be discussed, use "bridge sentences" that relate the result to the interpretation. "The greater response of the treated group relative to controls suggests that...[interpretation]“.

35. Discussion Content • Comment on others’ studies that helps interpret your own data, or reinterpret others' findings in light of yours. • Consider how the results of other studies may be combined with yours to derive a new or better substantiated understanding of the problem. • Don't ignore or bury the major issue. Did the study achieve the goal presented in the Introduction? • Don't overgeneralize. • Avoid speculation that cannot be tested in the foreseeable future.

36. Discussion Content • In general, new results should not be introduced in the Discussion, but there are exceptions to this rule. • You may include in this section tables and figures which help explain something you are discussing. • You may include flow diagrams, accumulation of data from the literature, or something that shows how one type of data leads to or correlates with another, etc. • You may also include additional data that were not part of the experimental design but may help explain the results.

37. Discussion Conclusions The discussion should end up with one or more conclusions. Many times the authors are very cautious and this results in ending of an manuscript with “Our findings suggest”, or “Our preliminary results indicate”. Many studies also end up with the recommendation that more studies are needed, or higher number of subjects should have been included. These sentences weakens the message. Try to find a very positive finding and state this clearly.