in vitro comparison of water displacement method and 3 tesla mri for mr-volumetry of the olfactory...

Original Investigation

In Vitro Comparison of WaterDisplacement Method and 3 Tesla

MRI for MR-Volumetryof the Olfactory Bulb:

Which Sequence Is Appropriate?

Hartmut Peter Burmeister, MD, Constanze M€oslein, Thomas Bitter, MD, Rosemarie Fr€ober, MD,Karl-Heinz Herrmann, PhD, Pascal Andreas Thomas Baltzer, MD, Hilmar Gudziol, MD,Matthias Dietzel, MD, Orlando Guntinas-Lichius, MD, Werner Alois Kaiser, MD, MSc

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Rationale and Objectives: Magnetic resonance imaging olfactory bulb (OB) volumetry (OBV) is already used as a complementary prog-nostic tool to assess olfactory disorders. However, a reference standard in imaging forOBVhas not been established. The aimof this in vitro

study was to compare volumetric results of different magnetic resonance sequences for OBV at 3 T to genuine OB volumes measured by

water displacement.

Materials and Methods: The volumes of 15 human cadaveric OBs were measured using the water displacement method in this institu-

tional review board–approved prospective study. The magnetic resonance imaging protocol at 3 T included constructive interference in

steady state (CISS), T2-weighted (T2w) three-dimensional (3D) sampling perfection with application-optimized contrasts using different

flip-angle evolutions (SPACE), T2w two-dimensional (2D) turbo spin-echo (TSE), and T1-weighted (T1w) 3D fast low-angle shot (FLASH)sequences. Two blinded observers independently performed two OB volumetric assessments per bulbus and sequence. Intraobserver

and interobserver reliabilities were assessed by intraclass correlation coefficients. Bland-Altman plots were analyzed to evaluate system-

atic biases and concordance correlation coefficients to assess reproducibility.

Results: For both observers, intraclass correlation coefficient analysis yielded almost perfect results for intraobserver reliability (CISS,

0.94–0.98; T2w 3D SPACE, 0.93–0.98; T2w 2D TSE, 0.98–0.98; T1w 3D FLASH, 0.95–0.99). Interobserver reliability showed almost perfect

agreement for all sequences (CISS, 0.98; T2w 3D SPACE, 0.89; T2w 2D TSE, 0.93; T1w 3D FLASH, 0.97). The CISS sequence yielded the

highest mean concordance correlation coefficient (0.95) and the highest combination of precision (0.97) and accuracy (0.98) values. Incomparison with the water displacement method, Bland-Altman analyses revealed the lowest systematic bias (�0.5%) for the CISS

sequence, followed by T1w 3D FLASH (�1.3%), T2w 3D SPACE (–7.5%), and T2w 2D TSE (�10.9%) sequences.

Conclusions: Compared to the water displacementmethod, the CISS sequence is suited best to validly and reliably measure OB volumesbecause of its highest values for accuracy and precision and lowest systematic bias.

Key Words: Olfactory bulb; volumetry; cadaver study; reproducibility; 3 Tesla MRI.

ªAUR, 2011

Olfactory bulb (OB) volumetry (OBV) has become

a growing topic (1) in the field of understanding

olfactory dysfunction, for which studies have indi-

ad Radiol 2011; -:1–8

om the Institute of Diagnostic and Interventional Radiology (H.P.B., C.M.,A.T.B., M.D., W.A.K.); Medical Physics Group (K.-H.H.), Universityspital - Friedrich Schiller University Jena, Philosophenweg 3, D-07740na, Germany; and the Department of Otorhinolaryngology (T.B., H.G.,.G.-L.), University Hospital - Friedrich Schiller University Jena,ssingstrasse 2, D-07743 Jena, Germany; and the Institute of Anatomy I,iedrich Schiller University Jena, Teichgraben 7, D-07743 Jena, Germany.F.). Received May 16, 2011; accepted June 27, 2011. Address correspon-nce to: H.P.B. e-mail: [email protected]

AUR, 2011i:10.1016/j.acra.2011.06.008

cated a prevalence of about 20% in the Western world (2,3).

OB volume decreases have been identified in all of the five

most frequent etiologies (4) of smelling disorders (ie, sinonasal

disease [5], postinfectious [6], posttraumatic [6], neurodegen-

erative diseases [7], and idiopathic olfactory disorders [8]).

However, volume increases have also been shown (eg, after

successful olfactory rehabilitation in sinonasal diseases [9]),

which exemplifies the reversibility of OB volume changes.

These dynamic changes are most probably due to the fact

that the human OB retained the ability of neuroneogenesis

(10) and therefore exhibits high structural plasticity, in which

its volume is correlated to afferent neural activity transmitted

by the olfactory receptor neurons (11). OBV has already been

1

mailto:[email protected]

http://dx.doi.org/10.1016/j.acra.2011.06.008

BURMEISTER ET AL Academic Radiology, Vol -, No -, - 2011

used as a complementary prognostic tool for radiologic diag-

nosis to predict outcomes in olfactory disorders (7).

In 1997, Yousem et al (12) described magnetic resonance

(MR) imaging (MRI) as a feasible method for OBV on the

basis of T1-weighted (T1w) sequences at 1.5 T. Further devel-

opment of MRI techniques and the use of MRI at higher field

strength (ie, 3 T) could facilitate the quantification of volu-

metric results of this small paleocortical structure because

the increased signal-to-noise ratio can be invested in better

spatial resolution (13). Therefore, future studies dealing with

the subject of high-resolution MROBV should be based on

standardized imaging, among other factors, for compatibility

reasons. However, until now, a systematic radiologic compar-

ison of various standardized sequences for OB volumetric

purposes, which logically justifies a reference standard in

imaging for upcoming studies, has not been finalized.

Previous in vivo studies have evaluated image quality (14)

and systematic biases (15) of different MR sequences for

OBVat 3 T. However, these studies were not suited for com-

menting on the true accuracy of volumetric measurements,

because of the lack of a direct anatomic correlation (12). An

additional measurement of genuine cadaveric OB volumes

using the water displacement method (WDM) would be suit-

able (12) to verify the accuracy of MROBVand remains the

missing link in the collection of prerequisites to define a valid

and reliable reference standard. Until now, this type of verifi-

cation procedure had to be precluded because of the paucity of

cadaveric OBs available for research purposes.

Consequently, in this in vitro MRI study, we evaluated the

reliability, reproducibility, and systematic bias of different

MRI sequences used for MROBV at a field strength of 3 T

and correlated with realistic OB volumes measured by water

displacement.

MATERIALS AND METHODS

Subject Recruitment, Sampling, and ExperimentalDesign

In this prospective study, we examined a series of 15 human

cadaveric OBs from 11 human body donors from our institute

of anatomy. Authorized agreement for scientific medical

research and education was provided before death, and insti-

tutional review board approval was obtained.

OBs were included in this study if the following criteria

were fulfilled: donor age $ 18 years at the date of death,

macroscopic normal OB shape, an estimated regular OB

size, and completeness of the OB and the transitional zone

between the OB and the olfactory tract after resection. Exclu-

sion criteria were as follows: anamnestic data (if available)

indicating anosmia, a traumawith fracture of the anterior skull

base, or any known previous surgery of the anterior skull base.

Immediately after transfer to our institute of anatomy,

embalmment was initialized by injecting embalming fluid

into the human cadaver (aqueous ethanol-formaldehyde solu-

tion; application of about 10 L via the right femoral artery).

2

The complete brain (including the OBs) was removed 1 to

2 days after the donor’s death, and all brains were subsequently

stored within aqueous formalin solution (formaldehyde 5%).

Furthermore, the principal prosector (R.F.) carefully resected

all OBs, including the adjacent part of the olfactory tract.

Pipetting tubes were filled with aqueous formalin solution

(formaldehyde 5% stabilized with methanol) for immersion

fixation purposes, and the OBs were placed in the pipetting

tubes with the frontal pole of the OBs pointing downward

(Fig 1). Subsequently, the tubes were stored at room temper-

ature for 2 weeks to complete the chemical reactions of fixa-

tion and to equalize the grade of formalin impregnation of the

bulbs at the best possible rate. Then, the OBs were washed

over 48 hours with four isotonic saline solution (sodium chlo-

ride 0.9%) changes at room temperature. Repetitive T1w and

T2-weighted (T2w)MRI of the OBs was performed between

each of the four saline solution changes to define the end point

of the washout procedure on the basis of a finalization of signal

intensity changes. After the fourth pass, pipetting tubes were

filled with saline solution, the OBs were cautiously inserted

into the tubes, and the saline solution’s supernatant was care-

fully taken up with a pipette for OB volume measurement

analogous to the WDM (T.B.). Consequently, the formalin

solution was completely replaced by isotonic saline solution,

and the tubes were filled bubble-free with saline solution.

Furthermore, an embedding of the pipetting tubes in an

agar-agar phantom (600 mL water, 7% agar-agar) was per-

formed, with a special focus on avoiding adjacent air bubbles.

The tubes were arranged in circular and clockwise order (Fig

1) according to the body register of the institute of anatomy

(order based on date of death and the OB’s side).

Imaging Procedures

All OBs underwent 3-T MRI examinations (Magnetom Tim

Trio; Siemens Medical Systems, Erlangen, Germany) at our

institute of radiology using a dedicated 12-channel head coil

provided by the manufacturer for image acquisition. The

OBs’ orientations were equivalent to those of patients exam-

ined in the prone position with a coronal slice orientation

tilted by 90% to the horizontal lamella of the cribriform plate.

The imaging protocol included the following sequences:

a coronal constructive interference in steady state (CISS)

three-dimensional (3D) Fourier transformation sequence

(repetition time [TR], 11.68 ms; echo time [TE], 5.84 ms;

matrix size, 512 � 512; field of view, 256 mm; voxel size,

0.5� 0.5� 0.5 mm [0.125 mm3]; flip angle, 70�; bandwidth,130 Hz/pixel; averages, 1; acquisition time [TA], 7 minutes

48 seconds), a coronal T2w 3D sampling perfection with

application-optimized contrasts using different flip-angle

evolutions (SPACE) sequence (TR, 1000 ms; TE, 132 ms;

matrix size, 384 � 384; field of view, 200 mm; voxel size

0.5 � 0.5 � 0.5 mm [0.125 mm3]; flip angle, 120�; band-width, 289 Hz/pixel; averages, 2; TA, 4 minutes 18 seconds),

and a coronal T2w two-dimensional (2D) turbo spin-echo

(TSE) sequence (TR, 4800 ms; TE, 150 ms; matrix, 512 �

Figure 1. Experimental design for volumetricmeasurements to compare magnetic reso-

nance (MR) sequences to the water displace-

ment method (WDM) for olfactory bulb (OB)

volumetry at 3 T. (1) Resection of the OBincluding an adjacent part of the olfactory tract.

(2) Immersion fixation with aqueous formalin

solution (formaldehyde 5% stabilized with

methanol) and storage of the OBs for 2 weeks,followed by a quadruplicated washout proce-

dure with isotonic saline solution (sodium chlo-

ride 0.9%). Taking up saline solution’ssupernatant with a pipette analogous to the

WDM to determine OB volumes (V1). (3) Embed-

ding the pipetting tubes including the OBs

(frontal pole pointing downward) in an agar-agar phantom (4). (5) MR imaging (MRI) and

acquisition of the four sequences (Fig 2) for

comparison to the WDM. (6) Use of multiplanar

reconstructions to perform MR volumetry (7)and to determine OB MRI volume (V2).

Academic Radiology, Vol -, No -, - 2011 IN VITRO 3-T OLFACTORY BULB MR VOLUMETRY

512; field of view, 230 mm; voxel size, 0.4 � 0.4 � 2.0 mm

[0.32 mm3]; no distance factor; flip angle, 150�; bandwidth,130 Hz/pixel; averages, 2; TA, 8 minutes 50 seconds).

Furthermore, the imaging protocol was completed with an

additional coronal T1w 3D fast low-angle shot (FLASH)

sequence (TR, 20.0 ms; TE, 2.46 ms; matrix size, 384 �384; flip angle, 20�; voxel size, 0.6 � 0.6 � 0.6 mm

[0.216 mm3]; averages, 2; TA, 16 minutes; no fat saturation).

Imaging Data Analysis

Acquired images were transferred to two Advantage Worksta-

tion VolumeShare 2 workstations (Advantage Workstation

version 4.4; GE Medical Systems, Milwaukee, WI) for

analysis.

Two observers, one specialized in skull base imaging and

volumetry (H.P.B., 9 years’ experience) and the other experi-

enced in skull base imaging (C.M., second year of experience)

independently performed volumetry of all 15 OBs separately

using the four sequences applied (Fig 2).

Each observer performed MR volumetry of all OBs twice.

Repeatability conditions in this study included the same

measurement procedure (sequence), the same observers, the

same measuring instrument (used under the same conditions,

ie, 3-T MRI, Advantage Workstation VolumeShare 2 work-

stations), the same anatomic structure (the OB), and repeti-

tion over a short period of time (1 day). The adjacent part

of the olfactory tract was included in the volumetric

assessment because the transitional zone between the OB

and olfactory tract is sometimes more difficult to determine,

as previously stated (16).

The selection of the volume was performed by planimetric

manual contouring of the OB using a circular tool 1 mm in

diameter, which is described in detail in our previous in vivo

study (15). Multiplanar reconstructions in the axial, sagittal,

and coronal planes were used for performing volumetry.

Each slice orientationwas automatically indicated on the other

reconstruction planes simultaneously showing the currently

selected volume. This approach allows for an immediate visual

correlation and corrections. For 3D sequences (CISS, T2w 3D

SPACE, and T1w 3D FLASH), a selection of the volume was

possible using every multiplanar reconstruction plane ad

libitum. However, for volumetry of the T2w 2D TSE

sequence, preconditions were set analogous to the previously

preferred method for volumetry of 2D T1w and T2w

sequences, as described in detail by Rombaux et al (16). The

selection of the volume in the T2w 2D TSE sequence was

exclusively performed in coronal planes. Using the latter

method, OB volumes were calculated by planimetric manual

contouring (surface of coronal planes in square millimeters),

and all surfaces were added and multiplied by 2 because

of the 2-mm slice thickness to obtain a volume in cubic

millimeters (17).

Artifacts should be assessed only with special focus on the

contour sharpness of the OB’s surface. To prevent habituation

effects during MR volumetry, the order of performing MR

volumetry was randomized concerning OBs and sequences

in each of the two passes. During the second pass, the

observers were blinded to the results of the first pass.

Statistical Analysis

All data analyses were performed using SPSS version 15.0

(SPSS, Inc, Chicago, IL) andMedCalc version 11.0 (MedCalc

Software, Mariakerke, Belgium).

Interobserver and intraobserver reliability with corre-

sponding 95% confidence intervals (CIs) for all sequences

was determined by using intraclass correlation coefficients

(ICCs; type 3.k) (18). Results were benchmarked according

to the classification of Landis and Koch (19) for measurements

of observer agreement for categorical data.

Because interobserver reliability results using ICCs do not

address both precision and accuracy, additional concordance

correlation coefficients (CCCs) were calculated (20). The

3

Figure 2. Comparison of sequences appliedfor in vitro olfactory bulb volumetry at 3 T. (a)Constructive interference in steady state

sequence (repetition time [TR], 11.68 ms;

echo time [TE], 5.84 ms; voxel size,0.125mm3); (b) T2-weighted (T2w) three-

dimensional (3D) sampling perfection with

application-optimized contrasts usingdifferent flip-angle evolutions sequence (TR,

1000 ms; TE, 132 ms; voxel size, 0.125mm3);

(c) two-dimensional T2w turbo spin-echo

sequence (TR, 4800 ms; TE, 150 ms; voxelsize, 0.32mm3); (d) T1-weighted 3D fast low-

angle shot sequence (TR, 20.0 ms; TE,

2.46 ms; voxel size, 0.216 mm3). All images

are in the sagittal plane and depict the sameolfactory bulb.


CCC (rc) provides a means for examining the reproducibility

of continuous volumetric measurements made by multiple

observers using a single method (21).

To estimate systematic bias of the four different sequences

compared to theWDM, Bland-Altman plots (22) were gener-

ated by graphing the percentage difference of each volumetric

measurement result (average of two observers and two passes),

the mean percentage difference, and the confidence limits

(95% limits of agreement) on the vertical against the absolute

average volumetric results of the two methods on the

horizontal.

All statistical analyses in this study were based on a signifi-

cance level of a = 5%. To address possible a error accumula-

tion, P values in this paper are given as calculated. For

interpretation of results, classical Bonferroni correction was

applied (23).

RESULTS

Fifteen of 22 OBs (nine right sided, six left sided) met the

inclusion criteria and underwent both WDM and 3-T MRI

volumetry between February 5 and February 15, 2011. Prior

to resection, seven OBs were excluded because of estimated

hypotrophy and/or irregular shape. Regarding our exclusion

criteria, no OB had to be excluded, and consecutively all 15

resected OBs were eligible for volumetry and statistical evalu-

ation. The ages of the body donors ranged from 59 to 90 years

(mean, 76.5� 13.8 years), and the dates of death ranged from

2008 to 2011. Because in five body donors, some of the

demographic data were anonymized before this study, no

gender-specific differences were evaluated.

OB volumes determined by water displacement ranged

from 75 to 155 mm3 (mean, 106.33 � 21.05 mm3). Corre-

sponding MR volumetric measurements ranged from 77.5

to 157.5 mm3 (mean, 105.58 � 20.00 mm3) for the CISS

sequence, 64.5 to 135 mm3 (mean, 98.31 � 16.64 mm3) for

the T2w 3D SPACE sequence, 66 to 131.5 mm3 (mean,

95.11 � 16.89 mm3) for the T2w 2D TSE sequence, and

4

74.5 to 145 mm3 (mean, 104.83 � 19.34 mm3) for the

T1w 3D FLASH sequence. Artifacts did not significantly

restrict the evaluation of the OBs and olfactory tracts con-

cerning the aim of this study.

Intraobserver reliability for the first observer resulted in

ICCs of 0.94 (95% CI, 0.83–0.98) for the CISS sequence,

0.93 (95% CI, 0.80–0.98) for the T2w 3D SPACE sequence,

0.98 (95% CI, 0.94–0.99) for the T2w 2D TSE sequence, and

0.95 (95% CI, 0.86–0.98) for the T1w 3D FLASH sequence.

Intraobserver reliability for the second observer resulted in

ICCs of 0.98 (95% CI, 0.93–0.99) for the CISS sequence,

0.98 (95% CI, 0.95–0.99) for the T2w 3D SPACE sequence,

0.98 (95% CI, 0.96–0.99) for the T2w 2D TSE sequence, and

0.99 (95% CI, 0.95–0.99) for the T1w 3D FLASH sequence.

Interobserver reliability resulted in ICCs of 0.98 (95% CI,

0.90–0.99) for the CISS sequence, 0.89 (95% CI, 0.21–

0.97) for the T2w 3D SPACE sequence, 0.93 (95% CI,

0.78–0.98) for the T2w 2D TSE sequence, and 0.97 (95%

CI, 0.92–0.99) for the T1w 3D FLASH sequence.

The results of reproducibility evaluation using CCCs with

corresponding values for 95% CI, precision, and accuracy are

summarized in Table 1.

The mean biases and 95% limits of agreement for

volumetric measurement results (percentage values in cubic

millimeters) regarding the comparison of the WDM and the

four sequences applied are displayed in the corresponding

Bland-Altman plots in Figure 3.

DISCUSSION

According to our results, for both observers, ICC analyses of

intraobserver reliability yielded almost perfect agreement for

the CISS, T2w 2D TSE, and T1w 3D FLASH sequences.

Additionally, the widths of associated 95% CIs did not extend

beyond the limits of 0.81 to 1.00 for almost perfect agreement

according to the classification of Landis and Koch (19).

Although the T2w 3D SPACE sequence also resulted in

almost perfect agreement for both observers, the range of

TABLE 1. CCCs, Interobserver Reliability, and Reproducibility (n = 15)

Sequence CCC (rc) 95% CI Pearson’s r (Precision)

Bias Correction

Factor cb (Accuracy)

CISS

First evaluation 0.92 0.80–0.97 0.95 0.97

Second evaluation 0.90 0.73–0.96 0.91 0.99

Average 0.95 0.88–0.98 0.97 0.98

T2w 3D SPACE



Average 0.80 0.57–0.91 0.91 0.88

T2w 2D TSE



Average 0.85 0.63–0.95 0.86 0.99

T1w 3D FLASH



Average 0.94 0.84–0.98 0.94 0.99

CCC, concordance correlation coefficient; CI, confidence interval; CISS, constructive interference in steady state; FLASH, fast low-angle shot;

SPACE, sampling perfection with application-optimized contrasts using different flip-angle evolutions; 3D, three-dimensional; T1w,

T1-weighted; TSE, turbo spin-echo; T2w, T2-weighted; 2D, two-dimensional.

Figure 3. Systematic biases of magnetic reso-nance sequences compared to the water

displacement method (WDM) for in vitro olfac-

tory bulb volumetry. Bland-Altman plots of

percentage differences of volumetric measure-ment results (y axis; percentage) against

average volumetric measurement results

(x axis; absolute values), with mean percentagedifference (ie, bias; continuous line) and 95%

limits of agreement (dashed lines) for (a)comparison of the constructive interference in

steady state (CISS) sequence and the WDM,(b) comparison of the T2-weighted (T2w) three-

dimensional (3D) sampling perfection with

application-optimized contrasts using different

flip-angle evolutions sequence and the WDM,(c) comparison of the T2w two-dimensional

turbo spin-echo sequence and the WDM, and

(d) comparison of the T1-weighted 3D fastlow-angle shot sequence and the WDM. The

dash-dotted regression lines proved not to be

significant in (a) (slope = �0.988, P = .332), (b)(slope = �1.111, P = .276), (c) (slope = �0.969,P = .341), and (d) (slope = �0.601, P = .553).

All values are given in cubic millimeters. SD,

standard deviation.


the associated 95% CI for the first observer (0.80–0.98) only

barely missed the limits for almost perfect agreement.

Regarding interobserver reliability, the CISS, T2w 2D

TSE, and T1w 3D FLASH sequences showed almost perfect

agreement, whereas the T2w 3D SPACE sequence resulted in

only substantial agreement. However, apart from the fact that

the width of the associated 95% CI (0.21–0.97) for T2w 3D

SPACE clearly extended beyond the limits for almost perfect

agreement, the associated 95% CI (0.78–0.98) for T2w 2D

TSE narrowly missed those limits.

Nevertheless, intraobserver reliability for both observers

and interobserver reliability using the CISS, T2w 3D SPACE,

and T2w 2D TSE sequences were noticeably higher

compared to the previously reported in vivo results from

our previous study at 3 T (15). Comparing intraobserver reli-

abilities between the two observers, the greater experience of

5


the first observer had no favorable impact; conversely, the less

experienced observer performed slightly better. These latter

two facts are most likely attributable to the peculiarity of

this in vitro study design (eg, no surrounding soft tissue or

osseous structures, no motion artifacts; see below).

On the topic of reproducibility, the CISS sequence yielded

the best results for CCC and the best combination of precision

and accuracy values (Table 1) compared to the T2w 3D

SPACE and T2w 2D TSE sequences. It must be stated that

except for the T2w 3D SPACE sequence, all sequences

showed lower precision than accuracy.

According to Bland-Altman plot analysis, in relation to the

WDM, the mean biases were�7.5% for the T2w 3D SPACE

sequence,�10.9% for T2w 2D TSE, and�1.3% for T1w 3D

FLASH. Additionally, associated limits of agreement of the

latter sequences had (in part) a considerably broader range

compared to the CISS sequence (Fig 3). The CISS sequence,

however, yielded the relatively most accurate mean bias of

�0.5% and the narrowest range for associated limits of agree-

ment (8.7% to �9.8%).

Bland-Altman plot analysis implies prima facie that the

methods (MR sequences) and agreement tended to be related

to the underlying size of the OB being measured. However,

none of the dash-dotted regression lines (Fig 3) is related to

a significant regression (for slope and P values, refer to the

legend of Fig 3). Therefore, it is not mandatory to create

size-related conversion tables to adapt the volume measured

by MROBV to the real individual OB’s volume. However,

we recommend this latter conclusion to be restricted to the

volume bandwidth evaluated in this study. Consequentially,

the necessity to use size-related conversion tables for OBs

with hypoplasia or disease-related atrophy should be evaluated

in a separate study.

In terms of intraobserver reliability, interobserver reli-

ability, reproducibility, and systematic bias analyses, we inter-

pret the results as indicating a clear tendency in favor of the

CISS sequence, in particular in consideration of the corre-

sponding results of our previous in vivo studies (14,15). For

the reasons we focused exclusively on T2w imaging in

these two preceding studies, please refer to Burmeister et al

(14,15). However, under the condition of an in vitro

evaluation, the idea was taken up anew to evaluate a high-

resolution T1w 3D FLASH sequence at 3 T for comparison

purposes. It must be stated that although T1w 3D FLASH

imaging did not reach the resolution of the CISS and T2w

3D SPACE sequences, in this study, T1w 3D FLASH

imaging performed second best, and the results were sur-

passed only by the CISS sequence. However, the T1w 3D

FLASH sequence is an exception, because an acquisition

time of 16 minutes was necessary to obtain approximately

comparable resolution. The latter issue and the consecutive

aggravated risk for developing motion artifacts make it diffi-

cult to include such a sequence in clinical routine. For these

reasons, T1w imaging should not yet be favored for OBV

purposes, but the possibility should be kept in mind for future

research.

6

Although this systematic evaluation of sequences regarding

their suitability for OBV purposes was performed under opti-

mized conditions as much as possible, this study was not

immune to limitations. Formaldehyde-fixed nervous tissues

are well suited for high-resolution, time-intensive MRI

acquisitions without motion artifacts (24). However, it is

well known that the use of aldehyde fixatives may significantly

alter tissue MRI properties. For this very reason, we tried to

equalize the grade of formalin impregnation of the bulbs at

the best possible rate, as described under ‘‘Materials and

Methods.’’ Nevertheless, after the end point of the washout

procedure and a finalization of signal intensity changes, we still

had to accept minimal contrast differences between the OBs

of body donors with dates of death after (Fig 1, specimens

1–9) and before (Fig 1, specimens 10–15) 2010. We cannot

rule out that minimal contrast differences between the OBs

and surrounding saline solution in these two subgroups led

to a systematic error. However, we were aware of the fact

that this systematic error could have been a constant factor,

possibly slightly skewing results in general but not regarding

the relation among the sequences. Fortunately, however, it

was possible to adjust these residual contrast differences by

fine-tuning the window width and level, and therefore we

do not expect the results of MROBV to be significantly influ-

enced. Furthermore, adhesive forces at the pipetting tubes’

lateral walls may have led to minimal impreciseness when

assessing volumes using the WDM. Therefore, the WDM

was performed twice, and the second pass confirmed all

first-pass WDM results. In this context, we should point out

that because of enhanced susceptibility at 3 T, the CISS

in vivo tends to underestimate the cerebrospinal fluid, whereas

the OB soft tissue consequently might be overestimated. We

hypothesize that the minimal imprecision using the WDM

(presumably a systematic overestimation of OB volume)

might have been the reason the mean volumetric results using

the WDM surpassed those using the CISS sequence by 0.5%,

although this relation should in fact have been the reverse.

However, because of the negligible difference of 0.5%, we

think that these overestimation effects of both the CISS

sequence and the WDM cannot be as pronounced as previ-

ously assumed.

Another limitation was that the occurrence of artifacts

(eg, Gibbs ringing artifacts due to sharp changes in intensity

at the pipetting tubes’ surfaces). These artifacts cannot be

avoided completely, and it was not possible to eliminate these

artifacts from the region of interest (25).

Unfortunately, no studies dealing with the subject of MR

volumetry of comparably small nervous structures of the

head exist using 1.5-T or 3-T MRI. The only studies at

1.5 T that seem most likely comparable to this study evaluated

the volume of the vestibulocochlear organ (26,27), whose

volume is at least twice as large (230 mm3) as that of the

OB. The only phantom study evaluating the reproducibility

and reliability of volumetric measurements of olfactory

eloquent structures (including the OB and olfactory tract)

was performed in 1997 by Yousem et al (12). The smallest

Figure 4. Comparison of the configuration of two left olfactory

bulbs, the posterior transitional zone, and the small adjacent part

of the olfactory tract in vitro (top view, scale bar on the left sideamounts to 1.2 cm, edge length of the grid 1 mm). (a) Olfactory

bulb diameter of the equatorial section approximately 4.5 mm (white

arrowhead), transitional zone (expected to be between millimeters 8

and 10; dashed white lines, 2-mm range) continuously merging intoan also conically tapering distal olfactory tract (red arrowheads)

with a diameter of the equatorial section of approximately 2.5 mm

(measured 11-mm distance from anterior end; bulb-to-tract ratio,1.8). (b) Olfactory bulb diameter of the equatorial section approxi-

mately 5 mm (white arrowhead), slightly tapering transitional zone

(expected to be between millimeters 8 and 10; dashed white lines,

2-mm range) merging into a constantly broad (green arrowheads)distal olfactory tract (diameter of the equatorial section approxi-

mately 3.5 mm, bulb-to-tract ratio, 1.4).


phantom measured had a volume of 1.5 cm3. Repeatedly per-

formed MR volumetric measurements yielded a mean varia-

tion (12%, or about 180 mm3), which, compared to the true

phantom’s volume, clearly surpasses the volume of a normal

genuine OB. However, those three studies neither performed

a systematic comparison of sequences for MR volumetry nor

compared to volumetric results of cadaveric organs.

The potential for inaccuracy or imprecision is large when

one is dealing with volumes as low as that of the OB. The

smaller the anatomic structure becomes, particularly if the

OB volume is reduced because of illness, the more important

it is to minimize the potential error using smaller voxel sizes

(27,28). Additionally, high accuracy and precision play

important roles when comparing sequences for OBV.

Furthermore, the smaller the structure measured with MRI,

the more the range for intraobserver and interobserver

reliability is expected to increase (12). Therefore, to facilitate

OBV for future research purposes, it might be useful to apply

standardized modern automated volumetric methods.

However, until now, it has not been possible to replace the

manual segmentation procedure of this primary olfactory

paleocortical structure by modern automated volumetric

methods such as voxel-based morphometry (29,30), which

is used for assessing secondary olfactory structures.

Conversely, a reason why manual segmentation should still

be used is the problem of exactly defining the OB’s

posterior end (7) using high-resolution 3-T imaging. The

posterior part of the OB does not abruptly end with a sudden

change in diameter between the OB and the olfactory tract, as

was pretended using 2-mm slice thicknesses in coronal planes.

However, OBs’ posterior ends form intraindividual and inter-

individual variably shaped transitional zones (Figs 1a–h in

Bhatnagar et al [31]) showing a smooth transition (Fig 4)

where the OB intermingles with and merges into the olfac-

tory tract (which only might be exactly seen on histologic

sections). Our opinion is that defining the posterior end of

an individual OB cannot be automated or standardized (eg,

in particular in atrophic or hypoplastic OBs) and remains

a task for the assessing radiologist. Therefore, to define the

OB’s posterior end to the best possible extent for MROBV

purposes, we propose that this task preferably be performed

by manual segmentation in multiplanar reconstructions (eg,

Figs 1a–c in Burmeister et al [15]).

Considering our previous results of image quality ratings

(14) and in vivo OBV evaluation (15), we recommend using

a high-resolution CISS sequence at 3 T as the reference

standard sequence in future imaging studies to gain precise

OB volumetric results. Thus, future studies should gain

the following advantages: a precisely rendered correlation

curve of OB volume with age-related olfactory function

(32), an easier categorization of pathologic findings, compa-

rability to other volumetric studies (eg, in case of neurologic

and/or psychological disorders) (33), and the possibility for

OBV to be used as a valid and reliable complementary prog-

nostic tool for the prediction of outcomes in olfactory

disorders (6).

CONCLUSIONS

The in vitro comparison of T1w and T2w sequences for MRI

OBVat 3 Twith WDM confirms previous in vivo results for

the CISS sequence to yield the best results for intraobserver

and interobserver reliability and reproducibility and the high-

est combination of accuracy and precision. On the basis of

direct anatomic correlation, the CISS sequence allows MRI

OBV to gain exact volumetric results. Therefore, OBV can

be implemented in routine clinical practice as a valid and reli-

able method for the evaluation of olfactory disorders.

ACKNOWLEDGMENTS

In particular, our special thanks go to all body donors who

bequeathed their bodies for scientific medical research and

education; without their generous donations, it would not

have been possible for us to do this research. Furthermore,

we would like to thank our photographer, Mrs Astrid Wetzel,

for her excellent cooperation.

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