Vito Brusasco Department of Internal Medicine Medical School University of Genoa, Italy Respiratory...

Vito Brusasco

Department of Internal MedicineMedical School

University of Genoa, Italy

Respiratory Pathophysiology UnitSan Martino University Hospital

Genoa, Italy

Joint course with ERS “How to write an article”

Material and methodsMaterial and methods

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

Material and methodsGeneral guidelines

Question to address How to address them

How did you study the problem?

Briefly explain the general type of experimental procedure you used.

What did you use? (Materials) Describe what materials, subjects, and equipment(chemicals, experimental animals, apparatus, etc.) youused.

How did you proceed? (Methods or Procedures)

Explain the steps you took in your experiment.

Material and methodsStyle

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 “We chose method A instead of method B because we found it more reproducible (unpublished observations)”.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”.


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

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)

Material and methodsStudy subjects or animals

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)• typical size (weight, length)• age (newborn, young, adults)• how they were handled, fed, and housed before and

during the experiment(s)

Material and methodsStudy 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.


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.


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.

Material and methodsProcedures

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.


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


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”.


Material and methodsAnalysis

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.


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)

Counting people or objects

Taking measurements on people or objects

Time-to-event data

Effect size

•Relative risk •Risk difference

•Percentage change in risk

•Difference between 2 means •Difference between 2 medians

•Hazard ratio •Risk difference at a specific time point


Use the appropriate test:

Counting peopleor objects

Taking measurements on people or objects

Time-to-event data

Two separate groups, 1 measurement (unpaired data)

Chi-square testUnpaired t-test if the difference between means is normally distributed

Log rank test

Mann-Whitney test if difference distribution is skewed

One group, 2 repeated measurements (paired data)

McNemar’s testPaired t-test if difference is normally distributed

Not applicable

Wilcoxon pairs test if distribution of difference is skewed

Allow for other factors

Multivariate logistic regression

Multivariate linear regression Cox regression



• 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.



• 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.



• 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).



• 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)


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 conveystatistical significance!


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 twotests are p=0.051and p=0.049, but nobodycan tell which is which.


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.


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.


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.

Material and methodsClinical 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 that prospectively assigns human subjects to intervention or comparison

groups to study the cause-and-effect relationship between a

medical 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)

http://www.consort-statement.org/

Vito Brusasco

Department of Internal MedicineMedical School

University of Genoa, Italy

Respiratory Pathophysiology UnitSan Martino University Hospital

Genoa, Italy

Joint course with ERS “How to write an article”

DiscussionDiscussion

DiscussionFunction

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.

DiscussionGeneral guidelines

Question to address How to address them

What do your observations mean?

Summarize the most important findings at the beginning.

What conclusions can you draw?

• Describe the patterns, principles, relationships results show.

• Explain how results relate to expectations and to literature cited in the Introduction.

• Explain plausibly any agreements, contradictions, or exceptions.

• Describe what additional research might resolve contradictions or explain exceptions

How do your results fit in a broader context?

• Suggest their theoretical implications.• Suggest their practical applications• Extend your findings to other situations or species.• Give the big picture: do your findings help us

understand a broader topic?

DiscussionStyle

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.

DiscussionContent

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]“.

DiscussionContent

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.

DiscussionContent

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.

DiscussionConclusions

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.

Vito Brusasco Department of Internal Medicine Medical School University of Genoa, Italy Respiratory...

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