Evaluating the Reliability of Human Brain White Matter Tractometry

Original Research Manuscript

Evaluating the Reliability of Human Brain White Matter Tractometry

Analysis Methods Connectivity Neuroinformatics, Pipelines, Data, or Data Sharing


The validity of research results depends on the reliability of analysis methods. In recent years, there have been concerns about the validity of research that uses diffusion-weighted MRI (dMRI) to understand human brain white matter connections in vivo, in part based on the reliability of analysis methods used in this field. We defined and assessed three dimensions of reliability in dMRI-based tractometry, an analysis technique that assesses the physical properties of white matter pathways: (1) reproducibility, (2) test-retest reliability, and (3) robustness. To facilitate reproducibility, we provide software that automates tractometry (https://yeatmanlab.github.io/pyAFQ). In measurements from the Human Connectome Project, as well as clinical-grade measurements, we find that tractometry has high test-retest reliability that is comparable to most standardized clinical assessment tools. We find that tractometry is also robust: showing high reliability with different choices of analysis algorithms. Taken together, our results suggest that tractometry is a reliable approach to analysis of white matter connections. The overall approach taken here both demonstrates the specific trustworthiness of tractometry analysis and outlines what researchers can do to establish the reliability of computational analysis pipelines in neuroimaging.


Correspondence arokem@uw.edu

DOI: 10.52294/e6198273-b8e3-4b63-babb-6e6b0da10669

Keywords: Diffusion MRI, Brain Connectivity, Tractography, Reproducibility, Robustness


The white matter of the brain contains the long-range connections between distant cortical regions. The integration and coordination of brain activity through the fascicles containing these connections are important for information processing and for brain health (12). Using voxel-specific directional diffusion information from diffusion-weighted MRI (dMRI), computational tractography produces three-dimensional trajectories through the white matter within the MRI volume that are called streamlines (34). Collections of streamlines that match the location and direction of major white matter pathways within an individual can be generated with different strategies: using probabilistic (56) or streamline-based (78) atlases or known anatomical landmarks (912). Because these are models of the anatomy, we refer to these estimates as bundles to distinguish them from the anatomical pathways themselves. The delineation of well-known anatomical pathways overcomes many of the concerns about confounds in dMRI-based tractography (1314), because “brain connections derived from diffusion MRI tractography can be highly anatomically accurate – if we know where white matter pathways start, where they end, and where they do not go” (15).

The physical properties of brain tissue affect the diffusion of water, and the microstructure of tissue within the white matter along the length of computationally generated bundles can be assessed using a variety of models (1617). Taken together, computational tractography, bundle recognition, and diffusion modeling provide so-called tract profiles: estimates of microstructural properties of tissue along the length of major pathways. This is the basis of tractometry: statistical analysis that compares different groups or assesses individual variability in brain connection structure (91821). For the inferences made from tractometry to be valid and useful, tract profiles need to be reliable.

In the present work, we provide an assessment of three different ways in which scientific results can be reliable: reproducibility, test-retest reliability (TRR), and robustness. These terms are often debated, and conflicting definitions for these terms have been proposed (2223). Here, we use the definitions proposed in (24). Reproducibility is defined as the case in which data and methods are fully accessible and usable: running the same code with the same data should produce an identical result. Use of different data (e.g., in a test-retest experiment) resulting in quantitatively comparable results would denote TRR. In clinical science and psychology in general, TRR (e.g., in the form of inter-rater reliability) is considered a key metric of the reliability of a measurement. Use of a different analysis approach or different analysis system (e.g., different software implementation of the same ideas) could result in similar conclusions, denoting their robustness to implementation details. The recent findings of Botvinik-Nezer et al. (25) show that even when full computational reproducibility is achieved, the results of analyzing a single functional MRI (fMRI) dataset can vary significantly between teams and analysis pipelines, demonstrating issues of robustness.

The contribution of the present work is three-fold: to support reproducible research using tractometry, we developed an open-source software library called Automated Fiber Quantification in Python (pyAFQ; https://yeatmanlab.github.io/pyAFQ). Given dMRI data that has undergone standard preprocessing (e.g., using QSIprep (26)), pyAFQ automatically performs tractography, classifies streamlines into bundles representing the major tracts, and extracts tract profiles of diffusion properties along those bundles, producing “tidy” CSV output files (27) that are amenable to further statistical analysis (Fig. S1). The library implements the major functionality provided by a previous MATLAB implementation of tractometry analysis (9) and offers a menu of configurable algorithms allowing researchers to tune the pipeline to their specific scientific questions (Fig. S2). Second, we use pyAFQ to assess TRR of tractometry results. Third, we assess robustness of tractometry results to variations across different models of the diffusion in individual voxels, across different bundle recognition approaches, and across different implementations.



We developed an open-source tractometry software library to support computational reproducibility: pyAFQ. The software relies heavily on methods implemented in Diffusion Imaging in Python (DIPY) (28). Our implementation was also guided by a previous MATLAB implementation of tractometry (mAFQ) (9). More details are available in the “Automated Fiber Quantification in Python (pyAFQ)” section of Supplementary Methods.


The pyAFQ software is configurable, allowing users to specify methods and parameters for different stages of the analysis (Fig. S2). Here, we will describe the default setting. In the first step, computational tractography methods, implemented in DIPY (28), are used to generate streamlines throughout the brain white matter (Fig. S1A). Next, the T1-weighted Montreal Neurological Institute (MNI) template (2930) is registered to the anisotropic power map (APM) (3132) computed from the diffusion data that has a T1-like contrast (Fig. S1B) using the symmetric image normalization method (33) implemented in DIPY (28). The next step is to perform bundle recognition, where each tractography streamline is classified as either belonging to a particular bundle or discarded. We use the transformation found during registration to bring canonical anatomical landmarks, such as waypoint regions of interest (ROIs) and probability maps, from template space to the individual subject’s native space. Waypoint ROIs are used to delineate the trajectory of the bundles (34). See Table S1 for the bundle abbreviations we use in this paper. Streamlines that pass through inclusion waypoint ROIs for a particular bundle, and do not pass through exclusion ROI, are selected as candidates to include in the bundle. In addition, a probabilistic atlas (35) is used as a tiebreaker to determine whether a streamline is more likely to belong to one bundle or another (in cases where the streamline matches the criteria for inclusion in either). For example, the corticospinal tract is identified by finding streamlines that pass through an axial waypoint ROI in the brainstem and another ROI axially oriented in the white matter of the corona radiata but that do not pass through the midline (Fig. S1C). The final step is to extract the tract profile: each streamline is resampled to a fixed number of points, and the mean value of a diffusion-derived scalar (e.g., fractional anisotropy (FA) and mean diffusivity (MD)) is found for each one of these nodes. The values are summarized by weighting the contribution of each streamline, based on how concordant the trajectory of this streamline is with respect to the other streamlines in the bundle (Fig. S1D). To make sure that profiles represent properties of the core white matter, we remove the first and last five nodes of the profile, then further remove any nodes where either the FA is less than 0.2 or the MD is greater than 0.002. This removes nodes that contain partial volume artifacts (16).


We used two datasets with test-retest measurements. We used Human Connectome Project test-retest (HCP-TR) measurements of dMRI for 44 neurologically healthy subjects aged 22–35 (36). The other is an experimental dataset, with dMRI from 48 children, aged 5 years old, collected at the University of Washington (UW-PREK). More details about the measurement are available in the “Data” section of Supplementary Methods.

HCP-TR configurations

We processed HCP-TR with three different pyAFQ configurations. In the first configuration, we used the diffusional kurtosis imaging (DKI) model as the orientation distribution function (ODF) model. In the second configuration, we used constrained spherical deconvolution (CSD) as the ODF model. For the final configuration, we used RecoBundles (8) for bundle recognition instead of the default waypoint ROI approach, and DKI as the ODF model. More details are available in the “Configurations” section of Supplementary Methods.

Measures of reliability

Tract recognition of each bundle was compared across measurements and methods using the Dice coefficient, weighted by streamline count (wDSC) (37). Tract profiles were compared with three measures: (1) profile reliability: mean intraclass correlation coefficient (ICC) across points in different tract profiles for different data, which quantifies the agreement of tract profiles (3839); (2) subject reliability: Spearman’s rank correlation coefficient (Spearman’s ρ) between the means of the tract profiles across individuals, which quantifies the consistency of the mean of tract profiles; and (3) an adjusted contrast index profile (ACIP): to directly compare the values of individual nodes in the tract profiles in different measurements. To estimate TRR, the above measures were calculated for each individual across different measurements, and to estimate robustness, these were calculated for each individual across different analysis methods. For example, if we calculated the subject reliability across measurements, we would call that “subject TRR,” and if we calculated the subject reliability across analysis methods, we would call that “subject robustness.” We explain profile and subject reliability in more detail below; we explain wDSC and ACIP in more detail in equations 1 and 2 in the “Measures of Reliability” section of the Supplementary Methods.

Profile reliability

We use profile reliability to compare the shapes of profiles per bundle and per scalar. Given two sets of data (either from test-retest analysis or from different analyses), we first calculate the ICC between tract profiles for each subject in a given bundle and scalar. Then, we take the mean of those correlations. We do this for every bundle and for every scalar. We call this profile reliability because larger differences in the overall values along the profiles will result in a smaller mean of the ICC. Consistent profile shapes are important for distinguishing bundles. Profile reliability provides an assessment of the overall reliability of the tract profiles, summarizing over the full length of the bundle, for a particular scalar. We calculate the 95% confidence interval on profile reliabilities using the standard error of the measurement.

In some cases, there is low between-subject variance in tract profile shape (e.g., this is often the case in corticospinal tract (CST)). We use ICC to account for this, as ICC will penalize low between-subject variance in addition to rewarding high within-subject variance. Profile reliability is a way of quantifying the agreement between profiles. Qualitatively, we use four descriptions for profile reliability: excellent (ICC > 0.75), good (ICC = 0.60 to 0.74), fair (ICC = 0.40 to 0.59), and poor (ICC < 0.40) (40).

Subject reliability

We calculate subject reliability to compare individual differences in profiles, per bundle and per scalar, following (41). Given two measurements for each subject, we first take the mean of each profile within each individual, measurement and scalar. Then, we calculate Spearman’s ρ from the means from different subjects for a given bundle and scalar across the measurements. High subject reliability means the ordering of an individual’s tract profile mean among other individuals is consistent across measurements or methods. This is akin to test reliability that is computed for any clinical measure.

One downside of subject reliability is that the shape of the extracted profile is not considered. Additionally, if one measurement or method produces higher values for all subjects uniformly, subject reliability would not be affected. Instead, the intent of subject reliability is to well summarize the preservation of relative differences between individuals for mean tract profiles. In other words, subject reliability quantifies the consistency of mean profiles. The 95% confidence interval on subject reliabilities is parametric.


Tractometry using pyAFQ classifies streamlines into bundles that represent major anatomical pathways. The streamlines are used to sample dMRI-derived scalars into bundle profiles that are calculated for every individual and can be summarized for a group of subjects. An example of the process and result of the tract profile extraction process is shown in Fig. S3 together with the results of this process across the 18 major white matter pathways for all subjects in the HCP-TR dataset.

Assessing TRR of tractometry

In datasets with scan-rescan data, we can assess TRR at several different levels of tractometry. For example, the correlation between two profiles provides a measure of the reliability of the overall tract profile in that subject. Analyzing the HCP-TR dataset, we find that for FA calculated using DKI, the values of profile reliability vary across subjects (Fig. 1A), but they overall tend to be rather high, with the average value within each bundle in the range of 0.77 ± 0.05 to 0.92 ± 0.02 and a median across bundles of 0.86 (Fig. 1B). We find similar results for MD (Fig. S4) and replicate similar results in a second dataset (Fig. 3B).

Subject reliability assesses the reliability of mean tract profiles across individuals. Subject FA TRR in the HCP-TR also tends to be high, but the values vary more across bundles with a range of 0.57 ± 0.24 to 0.85 ± 0.12 and a median across bundles of 0.73. We can see that subject TRR is lower than profile TRR (Fig. 2). This trend is consistent for MD (Fig. S5) as well as for another dataset (Fig. 3C).

Fig. 1. Fractional anisotropy (FA) profile test-retest reliability (TRR). (A) Histograms of individual subject intraclass correlation coefficient (ICC) between the FA tract profiles across sessions for a given bundle. Colors encode the bundles, matching the diagram showing the rough anatomical positions of the bundles for the left side of the brain (center). (B) Mean (± 95% confidence interval) TRR for each bundle, color-coded to match the histograms and the bundles diagram, with median across bundles in red.

TRR of tractometry in different implementations, datasets, and tractography methods

We compared TRR across datasets and implementations. In both datasets, we found high TRR in the results of tractography and bundle recognition: wDSC was larger than 0.7 for all but one bundle (Fig. 3A): the delineation of the anterior forceps (FA bundle) seems relatively unreliable using pyAFQ in the UW-PREK dataset (using the FA scalar, pyAFQ subject TRR is only 0.37 ± 0.28 compared to mAFQ’s 0.84 ± 0. 10). We found overall high-profile TRR that did not always translate to high subject TRR (Fig. 3B–G). For example, for FA in UW-PREK, median profile TRRs are 0.75 for pyAFQ and 0.77 for mAFQ, while median subject TRRs are 0.70 for pyAFQ and 0.75 for mAFQ. Note that profile and subject TRRs have different denominators (e.g., subjects that have similar mean profiles to each other would have low subject TRR, even if the profiles are reliable, because it is harder to distinguish between subjects in this case). mAFQ is one of the most popular software pipelines currently available for tractometry analysis, so it provides an important point for comparison. In comparing different software implementations, we found that mAFQ has higher subject TRR relative to pyAFQ in the UW-PREK dataset, when TRR is relatively low for pyAFQ (see the FA bundle, CST L, and ATR L in Fig. 3C). On the other hand, in the HCP-TR dataset pyAFQ, we used the Reproducible Tract Profile (RTP) pipeline (4243), which is an extension of mAFQ, and found that pyAFQ tends to have slightly higher profile TRR than RTP for MD but slightly lower profile TRR for FA (Fig. 3D). The pyAFQ and RTP subject TRR are highly comparable (Fig. 3E). In FA, the median pyAFQ subject TRR for FA is 0.76, while the median RTP subject TRR is 0.74. Comparing different ODF models in pyAFQ, we found that the DKI and CSD ODF models have highly similar TRR, both at the level of wDSC (Fig. 3A) and at the level of profile and subject TRRs (Fig. 3F, G).

Fig. 2. Subject test-retest reliability. (A) Mean tract profiles for a given bundle and the fractional anisotropy (FA) scalar for each subject using the first and second session of Human Connectome Project test-retest (HCP-TR). Colors encode bundle information, matching the core of the bundles (center). (B) Subject reliability is calculated from the Spearman’s ρ of these distributions, with median across bundles in red (± 95% confidence interval).

Robustness: comparison between distinct tractography models and bundles recognition algorithms

To assess the robustness of tractometry results to different models and algorithms, we used the same measures that were used to calculate TRR.

Tractometry results can be robust to differences in ODF models used in tractography

We compared two algorithms: tractography using DKI- and CSD-derived ODFs. The weighted Dice similarity coefficient (wDSC) for this comparison can be rather high in some cases (e.g., the uncinate and corticospinal tracts, Fig. 4A) but produce results that appear very different for some bundles, such as the arcuate and superior longitudinal fasciculi (ARC and SLF) (see also Fig. 4D). Despite these discrepancies, profile and subject robustness are high for most bundles (median FA of 0.77 and 0.75, respectively) (Fig. 4B, C). In contrast to the results found in TRR, MD subject robustness is consistently higher than FA subject robustness. The two bundles with the most marked differences between the two ODF models are the SLF and ARC (Fig. 4D). These bundles have low wDSC and profile robustness, yet their subject robustness remains remarkably high (in FA, 0.75 ± 0.17 for ARC R and 0.88 ± 0.09 for SLF R) (Fig. 4C). These differences are partially explained due to the fact that there are systematic biases in the sampling of white matter by bundles generated with these two ODF models, as demonstrated by the non-zero ACIP between the two models (Fig. 4E).

Most white matter bundles are highly robust across bundle recognition methods

We compared bundle recognition with the same tractography results using two different approaches: the default waypoint ROI approach (9) and an alternative approach (RecoBundles) that uses atlas templates in the space of the streamlines (44). Between these algorithms, wDSC is around or above 0.6 for all but one bundle, Right Inferior Longitudinal Fasciculus (ILF R) (Fig. 5). There is an asymmetry in the ILF atlas bundle (7), which results in discrepancies between ILF R recognized with waypoint ROIs and with RecoBundles. Despite this bundle, we find high robustness overall. For MD, the first quartile subject robustness is 0.82 (Fig. 5C, D).

Tractometry results are robust to differences in software implementation

Overall, we found that robustness of tractometry across these different software implementations is high in most white matter bundles. In the mAFQ/pyAFQ comparison, most bundles have a wDSC around or above 0.8, except the two callosal bundles (FA bundle and forceps posterior (FP)), which have a much lower overlap (Fig. 6A). Consistent with this pattern, profile and subject robustness are also overall rather high (Fig. 6B, C). The median values across bundles are 0.71 and 0.77 for FA profile and subject robustness, respectively.

For some bundles, like the right and left uncinate (UNC R and UNC L), there is large agreement between pyAFQ and mAFQ (for subject FA: UNC L ρ = 0.90 ± 0.07, UNC R ρ = 0.89 ± 0.08). However, the callosal bundles have particularly low MD profile robustness (0.07 ± 0.09 for FP, 0.18 ± 0.09 for FA) (Fig. 6B).

The robustness of tractometry to the differences between the pyAFQ and mAFQ implementation depends on the bundle, scalar, and reliability metric. In addition, for many bundles, the ACIP between mAFQ and pyAFQ results is very close to 0, indicating no systematic differences (Fig. 6D). In some bundles – the CST and the anterior thalamic radiations (ATR) – there are small systematic differences between mAFQ and pyAFQ. In the forceps posterior (FP), pyAFQ consistently finds smaller FA values than mAFQ in a section on the left side. Notice that the forceps anterior has an ACIP that deviates only slightly from 0, even though the forceps recognitions did not have as much overlap as other bundle recognitions (see Fig. 6A).


Previous work has called into question the reliability of neuroimaging analysis (e.g., (254546)). We assessed the reliability of a specific approach, tractometry, which is grounded in decades of anatomical knowledge, and we demonstrated that this approach is reproducible, reliable, and robust. A tractometry analysis typically combines the outputs of tractography with diffusion reconstruction at the level of the individual voxels within each bundle. One of the major challenges facing researchers who use tractometry is that there are many ways to analyze diffusion data, including different models of diffusion at the level of individual voxels; techniques to connect voxels through tractography; and approaches to classify tractography results into major white matter bundles. Here, we analyzed the reliability of tractometry analysis at several different levels. We analyzed both TRR of tractometry results and their robustness to changes in analytic details, such as choice of tractography method, bundle recognition algorithm, and software implementation (Fig. 6).