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Influence of Formalin Fixation Prior to in vitro Ultrasound Examination of PorcineArteries

Wilhjelm, Jens E.; Vogt, Katja; Jespersen, Søren Kragh; Nordmann, Niels; Sillesen, Henrik

Published in:Proceedings of the 10th Nordic-Baltic Conference on Biomedical Engineering

Publication date:1996

Document VersionPublisher's PDF, also known as Version of record

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Citation (APA):Wilhjelm, J. E., Vogt, K., Jespersen, S. K., Nordmann, N., & Sillesen, H. (1996). Influence of Formalin FixationPrior to in vitro Ultrasound Examination of Porcine Arteries. In Proceedings of the 10th Nordic-Baltic Conferenceon Biomedical Engineering (Vol. 34/1, pp. 355-356)

1.7 .1 .03

inInfluence of Formalin Fixation Prior to

vitro Ultrasound Examination of Porcine Arteries

J. E. Wilhjelmt, K. Vogt+, S. K. Jespersent, N., Nordmannt & H. Sillesen+, Center for Arterio-sclerosis detection witliUltrasound (CADUS). tlnst. of Information Tech., Tech. Univ. of Den-mark, Bldg. 349, DK-2800 Lyngby, Denmark. +Dept. of Vasc. Surg., Rigshospitalet, Univ. ofCopenhagen, Blegdamsvej 9, DK-2100 Copenhagen @, Denmark.

Abstract: This paper reports on an investigation of theinfluence on the echo signal from porcine artery wallsdue to two different formalin fixation procedures. Thelumen diameter, the wall thickness and the mean echo-genicity were calculated. In general, the fixation resultedin a more rigid wall. While conventional formalin fixationresulted in shrinking and a slight loss in echogenicity (p< 0.01), fixation of the artery under stretch and pressuredid not. The results of the study suggest that fixation withpressurized formalin is the preparation method of choicefor in vitro investigation of arteries.

INTRODUCTIONFixation with formalin is a widespread technique for

conservation of biological specimens and as a preparationfor histological analysis. Specifically, if biological spec-'imens are to be kept for more than 24 hours (e'9. duringexperimental ultrasound examination), some kind ofpreparation is needed in order to avoid degradation of theiissue. However. the influence of this fixation on theultrasound echoes is only scarcely described in theliterature.

In vitro experiments using ultrasound methods are

being increasingly applied to study the nature of ath-erosclerosis. As such work often requires some fixation,this work is aimed at studying the influence on vesselgeometry and ultrasound reflectivity of two differentpreparation techniques: fixation with formalin with and

without "normal pressure" inside the vessel.

METHODThe thoracic aorta from five pigs (weight 16 - 46 kg)

were excised immediately after death. Branches wereeither ligated or sutured. Each artery was subsequentlycut into two segments corresponding to the two differentpreparations. Each end of an artery segment was fixed tospecially designed acrylic fittings; the fittings remainedon the artery segments during the entire experiment.

The artery segment with fittings and extension tubeswere mounted onto a specially designed vessel holderwhich was placed in a scanning tank. For obtaining twodifferent transmural pressures, the extension tubes wereeither connected to a 0.1 m water column (low presSure)

or a 1.0 m water column (high pressure).Each of two fresh artery segments (from the same pig)

with fittings and mounted to the extension tubes weresequentially placed in the vessel holder. The arterysegment was stretched longitudinally to ll0 vo of itsexcised resting length in order to approximate the typicalin vivo length. The vessel holder was next installed in thescanning tank, the extension tubes were connected to thewater columns and air inside the artery segment wasflushed out. Next, three cross-sectional ultrasound imageswere recorded at low and high transmural pressure. Eachtime the scanning was accomplished, one of the twodifferent preparations, outlined below, was commenced:

Fixation in formalin bath: The artery segment wasdetached from the vessel holder, and placed in 5 Vo

buffered aldehyd (formalin) bath at 21" C for 24hours.Pressurized frxation with formalin: The vesselholder with artery segment was removed from thescanning tank, and still under high pressure, it couldbe inspected for possible leakages. The I m watercolumn was exchanged with a formalin column ofI m and the artery was submerged into formalin for24 hours. After this period of time, the formalin wasremoved.After these preparations, the artery segments were

scanned again with exactly the same longitudinallystretching, as had been used prior to preparation for thatparticular artery segment, and with the same two trans-mural pressures.

As three cross-sectional images were obtained fromeach of the 10 artery segments at high and low pressure,before and after preparation, a total of 120 cross-sectionalimages were obtained.

The ultrasound scanning was performed with anexperimental ultrasound system that consisted of acommercial diagnostic ultrasound scanner (B&K MedicalA/S, type 3535) which had been modified to provide theradio frequency (ö signal from the multi-channelreceiver. The scanner's 7.5 MHz transducer was mountedin a 3D translation system. The rf signal from the scannerwas digitized by a digital storage oscilloscope, DSO, thatin turn was connected via a GPIB interface to a workstation. By use of an RS232 interface, the 3D translationsystem was also connected to this work station.

The digitized data was transferred to the computer,bandpass filtered, envelope detected and finally arrangedas a B-mode image. The diameter and wall thicknessmeasurements were taken at the scanline that coincidedwith the largest distance from the protimal to the distalwall. A peak detection algorithm was applied to thescanline that coincided with the diameter to identify thefour peaks that corresponded to the following fourinterfaces: (1) water to outer wall, (2) inner wall to waterin lumen, (3) water in lumen to inner wall, and (4) outerwall to water. From the location of the peaks, the lumendiameter and the wall thickness of the proximal wallcould be estimated.

An estimate of the mean echogenicity of the proximalwall was calculated as the rms value over a region aroundthe center scanline. As the echoes from the four interfacesbetween artery wall and water might.be predominant overthe echoes from the wall material itself, the regions inthe signal that originated from these interfaces wereexcluded.

RESULTSThe results of calculating the relative change in

diameters, due to the preparation, are summarized inTable 1. Specifically, the table shows the relative changein diameter from fresh to processed state. p values are

Medical & Biological Engineering & Computing Vol. 34,The 1Oth Nordic-Baltic Conference on Biomedical Engineering,

Supplement 1, Part 1, 1996June 9-13, 1996, Tampere, Finland 355

calculated with Wilcoxon mntched pairs test.For low transmural pressure, moderate positive and

negative changes were observed for the arteiy segmentsthat have been prepared with formalin. For- thJarterysegments prepared with pressurized formalin, consistentfarge positive changes were observed (pindicating that the prepared artery maintains its dimensioneven though the transmural pressure is lowered.

Next consider high transmural pressure. For the arterysegments prepared with formalin, large negative changeswere ob-served (p < 0.001), i.e. the prepared artery loosesits elasticity. Eventually, for the artery segments preparedwith pressurized formalin, small random changres wereobserved, again indicating that the artery maintains itsdimension.

Table I Relative changein lumen diameter from prior to to afier

preparatton gtten as

18

14

Formalin bath

36912t5lmage number

3691215lmage number

)rot1oEö

)rof,(dl

u,E

Transmural pressureLow Hish

Formalin bath 1.6 (-8.0/+11.9) Vo -11.4 (-25.8/0) Eo

Pres. form. 15.2 (8.6/23.4) Eo -0.3 (-4.213.3) 7o

The results of the wall thickness changes are sum-marized in Table 2, which is organized as Table l.

Consider low transmural pressure first. For the arterysegments prepared with formalin and pressurized for-malin, mainly positive (p < 0.02) and negative (p <0.0015) changes were observed, respectively.

Next consider high transmural pressure. For the arterysegments prepared with formalin, large positive changeswere seen, indicating wall thickening (pEventually, for the artery segments prepared with pres-surized formalin, the changes are random and relatively9mall, again indicating that the prepared artery maintainsits dimensions in this case.

Table 2 Relative changein wall thickness from prior to to after

p.epatatton gt"en as

Transmural pressureMethod Low High

Figure I Estimate of mean echogenicity for proximalwall. (-) and (-) indicates low and high trånsmuralpressure, respectively, at fresh state. (..-.) and (' ' ')indicates low and high transmural pressure, respectively,for prepared artery segments.

panied by a decrease in wall thickness and vice versa.However, this effect was not present as clearly for theartery segments prepared with formalin when the mea-surements were done at low transmural pressure.Examination of the corresponding images reveåled thatformalin fixation often gives the wall apeculiar lookingshape. An explanation may be that the artery walundergoes uneven shrinking due to branches, sutures,ligations, etc. Such an effect is not present in the arteryfixed under stretch and high transmural pressure. How-ever, this study was not designed to clarify these matters.

The results for the mean echogenicity shows largevariation from image to image. This is due to the largephase cancellation at the receiving transducer. As sug-gested by_ statistical analysis,

" ,nrun echogeniciiydecreases for the artery prepared in formalin bath.However, due to the large variation in echogenicity, thisobservation should be confirmed by a largei study.

CONCLUSIONSThis study concludes that formalin fixation (with and

without ligh transmural pressure and stretching)decreases the elasticity of the artery wall. However, whileconventional formalin fixation carries an increase in wallthickness, pressurized formalin fixation can secure thatthe artery maintains its dimensions. Furthermore, theechogenicity of the proximal wall was reduced due to thefixation in formalin bath (p < 0.01). In summary, fixationwith pressurized formalin is suggested as the mostappropriate fixation method for intact artery specimens.

ACKNOWLEDGEMENTSCADUS is supported by rhe Danish Technical and

Medical Research Councils. The authors eratefullvacknowledge the contributions to the experi-"nål systeÅby instrument maker K. Martinsen.

REFERENCEStll J. C. Park, R. J. Siegel & L. L. Demer: Effect ofcalcification and formalin fixation on in vitro distensi-bility of human femoral arteries. Am. Heart J. Vol. 125,No. 2, part I , pp. 3M - 349, February 1993.

Method

Form. bath 25.3 (-26.91123.5) Vo

Pres. form. -13.9 (-36.8/11.1) Vo

40.0 (9. t/r29.4) Vo

-6.8 (-25.0/13.6) Vo

Estimates of the mean echogenicity of the proximalwall in each image are provided in Figure 1. For theprepared artery segments, the levels are generally inde-pendent of transmural pressure, as expected. Only-for theartery segments prepared in formalin bath, an almostconsistent fall in echogenicity was found (p < 0.01).

DISCUSSIONIn terms of geometrical distortion, the results in Table

I indicate, in agreement with other resultslrl, that theartery segments prepared by formalin becomes morerigid: While the artery segments prepared by formalin failto expand at high transmural pressure, the artery segmentsprepared by pressurized formalin fail to relax at lowtransmural pressure.

Change in wall thickness follows the observations forthe lumen diameter change, except for one case. Spe-cifically, an increase in lumen diameter should be accom-

Medical& Bio.logical Engineering & computing Vol.34, supplement 1, part 1, 1996The 1oth Nordic-Baltic conference on diomedical eniineering, June g-13, 1996, Tampere, Finland

Pres. Formalin

t-

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