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Watcharit Anantakal M.D., Somboon amtakerngkit M.D., Vijarn Vachirawongsakorn M.D., Ph.D.

Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ailand.

Comparison of Heart Valve Circumference

Examined Before and After 10% Formalin Fixation

ABSTRACT

Objective: To compare the heart valve circumference before and aer 10% formalin xation.

Materials and Methods: e study analyzed 63 ai human cadaveric hearts. Each heart valve circumference was

separately measured in the fresh state by specically designed equipment. Aer that, the hearts were xed in 10%

formalin for 3 days. en each heart valve circumference was measured by the same equipment and by the thread

and ruler technique. e results were analyzed using SPSS package to nd the association between the heart valve

circumference before and aer formalin xation.

Results: is study showed that the average circumferences of the heart valve measured in the fresh state were 13.329

cm in the tricuspid valve, 10.617 cm in the mitral valve, 8.416 cm in the pulmonic valve, and 7.122 cm in the aortic

valve. e average circumferences of the heart valve measured aer 10% formalin xation were 11.019 cm in the

tricuspid valve, 8.714 cm in the mitral valve, 6.751 cm in the pulmonic valve, and 6.089 cm in the aortic valve. e

average ratios of the heart valve circumference measured fresh and aer 10% formalin xation were 0.8267 in the

tricuspid valve, 0.8235 in the mitral valve, 0.8050 in the pulmonic valve, and 0.8573 in the aortic valve. ere were

signicant dierences in the heart valve circumference between the fresh state and aer formalin xation (p < 0.001).

Conclusion: is study revealed important information on the dimensional changes of all the formalin-xed heart

valves. We found that the heart valve shrank aer formalin xation, with the formalin-xed hearts an estimated 0.8

times smaller than the fresh cadaveric hearts.

Keywords: Heart valve circumference; formalin xation (Siriraj Med J 2021; 73: 478-484)

Corresponding Author: Vijarn Vachirawongsakorn

E-mail: vijarn.vac@mahidol.ac.th

Received 2 March 2021 Revised 22 April 2021 Accepted 26 April 2021

ORCID ID: http://orcid.org/0000-0002-7782-5209

http://dx.doi.org/10.33192/Smj.2021.64

INTRODUCTION

e number of people with cardiovascular disease

is increasing globally and it is now one of the leading

causes of death worldwide, not only in industrial countries

but also in developing countries, such as ailand.

1,2

Valvular heart disease represents an important public

health problem and its rising incidence is leading to an

increased mortality rate among the general population.

3,4

Acute rheumatic fever and chronic rheumatic heart

diseases, for example, were responsible for 115 deaths in

2013, leading to a death rate of 0.2 per 100,000 persons

per year in ailand; while the number of deaths had

increased to as many as 226 persons in 2017, leading to

a death rate of 0.3 per 100,000 persons per year.

e heart valves play a signicant role in controlling

blood circulation between the heart chambers and systemic

circulation. e four heart valves are the tricuspid valve

(TV), pulmonic valve (PV), mitral valve (MV), and aortic

valve (AV). e cardiac valves are aected by various

factors, including genetics, aging, sex, and lifestyle, as

well as by infection. Pathological changes of the heart

valves can aect the circulation, such as regurgitation

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and stenosis. People with severe heart valve disease may

need to undergo a surgical procedure to replace the

damaged valve with an articial heart valve.

Knowledge of the cardiac anatomy plays an

important role in cardiothoracic surgery as well as in

understanding the pathophysiology of various cardiac

diseases. Several studies from a variety of countries and

based on dierent ethnicities have been performed to

nd the average heart valve circumference to aid more

accurately estimating the circumference needed for an

articial heart valve.

5-15

In general, previous studies took

measurements on formalin-xed hearts. However, it

is well known that formalin xation can shrink fresh

tissues in an unpredictable manner. Consequently, in

this study we thought it would be interesting to study

the comparison between the heart valve circumference

before and aer 10% formalin xation. In addition, this

study aimed to compare dierent methods of measuring

the heart valve circumference.

MATERIALS AND METHODS

is study was approved by the Human Research

Ethics Committee of the Faculty of Medicine Siriraj

Hospital, Mahidol University (Si 483/2020(IRB2) on July

20, 2020). is descriptive study included a total of 63

hearts, which were collected from ai adult male and

female cadavers at the Department of Forensic Medicine,

Faculty of Medicine Siriraj Hospital from 1 August 2020

to 31 December 2020. Hearts with disease, trauma, or

postmortem changes were excluded from this study.

e heart samples were collected from dead bodies

with known age, sex, weight, and height. e heart was

removed from the pericardial cavity. e great vessels

connecting the heart with the other organs were separated

and cut without damage to the valvular structure. en,

the heart chamber was cut in a horizontal line through

the le and right ventricle, and blood clots were removed

to show all the heart valves. Measurement of each valve

circumference was performed using specially designed

equipment (Method 1). is cone-shaped equipment

(Fig 1) was fully accredited and designed to directly

measure the exact size of a heart valve circumference.

Next, each heart was xed in 10% formalin solution

for three days. A thread was used to hold the heart

within the formalin to maintain the normal shape of

the heart. e circumference of all the formalin-xed

heart valves was observed again and measured with the

same equipment (Method 2). Subsequently, the heart

chambers were routinely opened to expose the opened

valve annulus, then the thread was placed along the

boundary of each valvular annulus and measured with

a ruler (Fig 2) (Method 3). All the data were recorded

in millimeters to 2 decimal points.

All the data were collected in Excel 2016 and analyzed

using the SPSS package (PASW 18.0 for Windows).

Quantitative data according to the average and the standard

deviation of the data were calculated. To determine

whether the fresh and formalin-xed heart samples, as

well as the measurements obtained by the direct and

thread and ruler techniques, were statistically dierent

(p < 0.05), and ANOVA or non-parametric Freidman

tests were performed on the data. Lastly, assessment

of the intra-observer and inter-observer variability in

the measurements was also conducted with intra-class

correlation coecients.

Fig 1. Specially designed cone-shaped equipment.

Fig 2. Measuring the heart valve circumference; (A) by specially

designed equipment in the fresh state; (B) by specially designed

equipment aer formalin xation; (C) by the thread and ruler method

aer formalin xation.

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RESULTS

In this study, 63 hearts were examined to observe the

normal values regarding the heart valve circumferences.

e males included in this study were aged between 14

and 64 years old, and the females between 16 and 68 years

old. In the study sample, the mean age of the male and

female cadavers was 34.31 ± 14.2 years old and 41.58 ±

15.85 years old, respectively. A summary of the descriptive

data from the heart valve circumference of the mixed

group (both male and female) is provided in Table 1. All

the measurements were found to be normally distributed.

is study showed that the average circumference of

the tricuspid valve was the highest, while the average

circumference of the aortic valve was the lowest. Among

the measurements of the heart valve circumference, this

study indicated that the heart valves aer 10% formalin

xation were smaller than those before xation.

Dierences between the measurements were also

explored. Comparisons of the measurements taken before

and aer 10% formalin xation with the dierent methods

are shown in Table 2. e circumference ratios of all the

valves aer 10% formalin xation compared with those

before 10% formalin xation were less than 1 throughout

all the valves (p < 0.001 by t-test). e average ratios of

heart valve circumference measured by the specially

designed equipment before and after 10% formalin

xation were 0.8267 in the tricuspid valve, 0.8235 in the

mitral valve, 0.8050 in the pulmonic valve, and 0.8573

in the aortic valve (Table 2).

Different plots were conducted to explore the

agreement between Method 2 and Method 3 (Fig 3). A

Bland-Altman plot was used in this study since neither

of the two methods are reference techniques. Almost all

the measurement values were within a 95% condence

interval of the mean dierence, implying that the variability

of the measurements was low. In addition, intra-class

correlation coecients (ICCs) were determined to identify

the relationship between the direct measurement and

the measurement by the thread and ruler method. is

study showed excellent reliability between the two types

of measurement. e ICC of the tricuspid valve was

0.952, the mitral valve was 0.958, the pulmonic valve

was 0.947, and the aortic valve was 0.956 (Fig 3).

e reliability of this study, particularly the intra- and

inter-observer variations, was also evaluated to exclude

the bias of heart valve circumference measurement by

human error. Measurements were performed on 15

samples using all the measurement methods and the

results compared with previous results. Based on Kappa

statistics, the average values of ICC were more than

0.9 for both the intra- and inter-observer reliabilities.

erefore, this study showed excellent reliability of all

the measurements.

TABLE 1. Descriptive statistics for all the heart valve circumferences; Method 1 = Measurement with specially

designed equipment before 10% formalin xation; Method 2 = Measurement with specially designed equipment aer

10% formalin xation; Method 3 = Measurement with the thread and ruler technique aer 10% formalin xation.

Heart valve Mean Standard Minimum Maximum

(n = 63) (cm) deviation (cm) (cm) (cm)

Type Method

Tricuspid Valve (TV) 1 13.33 0.92 11.00 15.00

2 11.02 1.10 8.60 13.70

3 10.81 1.02 8.50 13.50

Mitral valve (MV) 1 10.62 1.13 8.20 13.50

2 8.71 0.99 7.00 12.00

3 8.65 0.91 7.20 12.20

Pulmonic Valve (PV) 1 8.42 1.01 6.80 10.70

2 6.75 0.80 5.00 8.70

3 6.66 0.70 5.30 8.10

Aortic Valve (AV) 1 7.12 0.79 5.50 9.00

2 6.09 0.70 4.90 8.30

3 6.00 0.69 5.00 7.80

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TABLE 2. Descriptive data of the proportions among the dierent measurement methods.

Ratio of Mean of 95% Condence

Different Mean Standard Standard Sig. Mean Interval of the

Methods Deviation Error (2-tailed) Difference Difference

Lower Upper

/ TV

0.83 0.06 0.01 0.00 -0.17 -0.19 -0.16

/ TV

0.81 0.06 0.01 0.00 -0.19 -0.20 -0.17

/ MV

0.82 0.07 0.01 0.00 -0.18 -0.20 -0.16

/ MV

0.82 0.06 0.01 0.00 -0.18 -0.20 -0.17

/ PV

0.81 0.07 0.01 0.00 -0.20 -0.21 -0.18

/ PV

0.80 0.07 0.01 0.00 -0.20 -0.22 -0.19

/ AV

0.86 0.07 0.01 0.00 -0.14 -0.16 -0.13

/ AV

0.85 0.07 0.01 0.00 -0.15 -0.17 -0.14

Fig 3. Dierences between Methods 2 and 3: (A) Tricuspid valve; (B) Mitral valve; (C) Pulmonic valve; (D) Aortic valve; ICC: Intra-class

correlation coecient.

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DISCUSSION

It is well known that tissues shrink aer formalin

xation. With reference to previous studies, however, a

variable reduction in the percentage of tissue shrinkage

was observed.

16-20

Most of these works used dierent so

tissues as their samples and they reported dierent results.

Pritt et al. reported that there was no signicant change

in 96% of the breast tumor size between the fresh and

xed states.

Also, Jonmarker et al. found no signicant

decrease in prostate tissue diameter aer formalin xation,

but observed a signicant weight loss.

In contrast, a

dierent result was found by Hsu et al. in their study,

whereby they found that the measured dimensions of the

oral cavity mucosa and tongue muscle shrank by 30.7%

aer formalin xation.

It was also reported that the mean

diameter of breast cancer tissues decreased by 4.5% aer

formalin xation.

Lastly, the average shrinkage of the

maximum diameter of head and neck cancer samples

was 4.4% in the study by Chen et al.

In our study,

the average heart valve circumference aer formalin

xation was 15-20% smaller than that before formalin

xation. Regarding the variable shrinkage reported in

the aforementioned works, it is worth mentioning that

possibly the constitution and type of tissue (e.g., amount

of elastic tissue or fat) may have had an eect on the

overall degree of shrinkage.

Many studies have been carried out in formalin-

xed hearts rather than fresh human hearts.

6,9-11,13,15

Formalin is widely used to preserve tissues for routine

histological examination. e most common formula is

10% formalin, consisting of 3.7% formaldehyde in water

with 1% methanol.

When using to preserve tissues, the

process includes a two-phase xation. e rst phase is

the alcohol xation phase, described as alcohol-induced

dehydration and hardening of the tissue. Shrinkage of

the tissue occurs during this phase. Subsequently, this is

followed by the cross-linking phase formed by a cross-

linking of formaldehyde and peptide. ere are two factors

that inuence formaldehyde xation: penetration and

xation.

e former is the potential of the solution to

penetrate the tissue, while the latter is the ability of the

formaldehyde to form cross-linking. e completion of

formalin xation is dependent on many factors, such

as the temperature, pH, time, type of tissue, and the

concentration of formaldehyde.

21-22

erefore, it can

be assumed that the average heart valve circumference

varies among dierent studies using dierent xative

methods, as we found in this study.

Various techniques have been conducted using a

variety of methods, such as the thread and ruler method,

the direct measurement, echocardiography, and computer

soware. Currently, there is no standard method to

measure heart valve circumference. With reference to

previous studies

5-8,10,12,13

, the thread and ruler technique

was the most popular practice to evaluate the heart valve

circumference. Because none of the above-mentioned

studies provided data on direct measurement, no

comparisons can be made. In this study, we measured

the heart valve circumferences by the direct measurement

using a specially designed instrument and by the thread

and ruler technique. e results showed that there was

excellent reliability between the two types of measurement.

us, we recommend the direct measurement method

for assessing the heart valve circumference because of

its easy manipulation.

e next issue we considered was that of the variation

in heart valve circumference. Westaby et al.

reported a

wide individual variation in heart valve circumference and

identied that the size of the heart valve circumference

was unrelated to the body habitus. Many studies have

performed detailed analyses of the heart valve circumferences

in various populations (Table 3). However, the data

in the previous studies showed some dierent results

compared with this study. Before formalin xation,

the average heart valve circumference in this study was

slightly higher than in almost all the previous studies

5-14

but lower than that reported by Tei et al.

and coincided

with those reported by Jatene et al.

and Alison et al.

Nevertheless, the average heart valve circumference

aer formalin xation was slightly lower or similar to

the above-mentioned studies. Why there was such a

dierence between our study and previous studies might

be explained by the dierent socio-economic status and

population groups from which the hearts were obtained

as well as by the factors aecting formalin xation, as

mentioned above.

To the best of our knowledge, this study is the rst

study to investigate the eects of formalin xation on

heart valve samples. Previous studies were conducted

using formalin-xed hearts and these may be dierent

from the values from fresh hearts. In this study, an eort

was made to describe this nding, which might be used

to compare the valve orice size before and aer formalin

xation. us the data from this study can be taken as

a useful guide in medical practice, especially being a

reference in the diagnosis and treatment of valvular

heart disease. is study should help in the choice of

prosthetic replacement as well. It is realistic to use a

mean circumference of each heart valve as a guide for

valve surgery. e measurements that we have taken will

help a surgeon to estimate the correct size of prosthesis

to t accurately in the valve orice in a person. Further

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TABLE 3. Comparison with the previous studies’ heart valve measurements.

Nationality Heart status Measurement Heart Circumference

Study Method valve (cm)

Mean S.D.

Ilankathir

India Fresh Thread TV 10.37 -

PV 6.82 -

MV 8.29 -

AV 7.54 -

Deopujari et al.

India Fresh Thread MV 8.27 1.25

Jatene et al.

Brazil Fresh Thread AV 7.38 1.01

Udhayakumar and Sri Lanka Fresh Thread AV 6.47 0.70

Yasawardene

Alison et al.

U.S. Living Echocardiograph MV 10.70 1.46

Tei et al.

U.S. Fresh Ruler TV 13.50 0.80

MV 11.40 0.70

  Formalin-xed Ruler TV 12.20 0.80

MV 10.70 0.50

Gupta et al.

 India Formalin-xed Imageanalysis MV 9.11 0.44

Nayak et al.

 India Formalin-xed Thread MV 7.92 0.50

Westaby et al.

 U.S. Formalin-xed Ruler TV 11.63 1.39

PV 7.63 0.93

MV 9.79 1.23

AV 7.28 0.92

Garg et al.

 India Formalin-xed Thread PV 6.50 0.59

Lama et al.

 Nepal Formalin-xed Thread TV 11.22 0.20

MV 9.22 1.49

The present study Thailand Fresh Equipment TV 13.33 0.92

PV 8.42 1.01

MV 10.62 1.13

AV 7.12 0.79

  Formalin-xed Equipment TV 11.02 1.10

PV 6.75 0.80

MV 8.71 0.99

AV 6.09 0.70

Thread TV 10.81 1.02

PV 6.66 0.70

MV 8.65 0.91

AV 6.01 0.69

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studies concerning a survey of a larger sample size and

comparisons with radiological and echocardiographic

examinations should be conducted.

ACKNOWLEDGMENTS

We would like to thank the sta of the Forensic

Pathology Unit, Department of Forensic Medicine, Faculty

of Medicine Siriraj Hospital for supporting this project.

Also, we greatly appreciate the help of Mr. Suthipol

Udompunthurak for his support with the statistical

analyses.

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