Journal of the Neurological Sciences, 1987, 77:49-57 49

Elsevier

JNS 02748

A simple test for the random arrangement of muscle

fibres
D e n i s e H o w e l and C h r i s t o p h e r B r u n s d o n
Department of Medical Statistics, University of Newcastle upon Tyne, Newcastle upon Tyne (U.K.)
(Received 30 January, 1986)
(Revised, received 3 July, 1986)
(Accepted 3 July, 1986)

SUMMARY

The random arrangement of a given muscle fibre class has been assessed by
estimating 'mean cluster size' in transverse sections of skeletal muscle. The method was
found to be useful when the proportion of the fibre class of interest was low. The
statistical distribution of this measure was investigated by computer simulation using
a hexagonal lattice model of muscle fibre arrangement. An approximate significance test
was developed by considering the extreme points of the distribution. Minor changes to
the hexagonal lattice model were incorporated to give a more realistic representation of
fibre arrangement and these were found to give very similar results to the simpler model.

Key words: Fibre count; Monte Carlo method; Muscle fibres; Spatial distribution

INTRODUCTION

Muscle fibres may be divided into two classes in a variety of ways, e.g. fast/slow
twitch, or normal/necrotic fibres. The presence or absence of grouping of fibres of
uniform histochemical type has been found to be a useful indicator of possible
remodelling of motor units in a variety of neuromuscular disorders. Grouping is usually
defined in terms of a non-random arrangement of either or both histochemical fibre
types as observed in representative samples of a muscle biopsy. Statistical techniques
have been necessary since visual appraisal can be misleading; a random arrangement

Correspondenceaddress: Denise Howel,Department of CommunityMedicine,The University,32,

Hyde Terrace, Leeds LS2 9LN, U.K.

0022-510X/87/$03.50 1987 Elsevier Science Publishers B.V. (BiomedicalDivision)

50

of two fibre types can itself lead to large clusters of uniform type if there is a reasonably
high proportion of these.
Current statistical techniques tend to be of two kinds: approximate tests requiring
little data collection (Johnson et al. 1973a; Lexell et al. 1983) and more sophisticated
approaches better suited to automated data collection (Cliff and Ord 1971 ; James 1971 ;
Lester et al. 1983). The method described here belongs to the first group and aims to
investigate the distribution of easily collected statistics and to construct a test of the
random arrangement of a given fibre class. Existing approximate methods have used
an 'enclosed fibre count' (Jennekens et al. 1971) i.e. the number of fibres of a given class
completely surrounded by fibres of the same class. This works well when the chosen
fibre class is common, when 'enclosed' fibres can be expected. However, the impetus
for this study came from an investigation of the arrangement of necrotic fibres, which
are usually present only in small proportions and for which an enclosed fibre count is
not applicable. A new statistic, the 'mean cluster size' has been investigated for use when
the proportion of fibres of interest is low. The distribution of'mean cluster size' has been
found by computer simulation under the assumption of a random distribution of a
number of fibres of given class in a fascicle where the fibres are packed into a hexagonal
array. Interest is concentrated on the extreme points of this distribution, where there
is strong evidence that the fibre distribution is non-random. These have been
investigated for the ~o incidence of fibre class of interest (hereafter known as Class X)
varying from 2~o to 30~o and for fascicle sizes of between 200 and 600 fibres. This
method was found to be suitable for use as an approximate one-sided test of the random
spatial distribution of Class X fibres.

MATERIALS AND METHODS

Equipment
The computer programs described below were first written in APL for a Sirius
microcomputer and later translated into F O R T R A N for an A M D A H L 5860 mainframe
machine.

Model of fibre arrangement and clustering

The model formulated by Johnson et al. (1973a) assumes that muscle fibres are
arranged in a regular hexagonal lattice, as illustrated in Fig. 1A. There are S fibres in
the lattice, which represents a muscle sample (which may be all or part of a fascicle)
of which a percentage (~oX) are of Class X: these are shaded black in Fig. lB. The
spatial distribution of Class X fibres is determined by the extent to which they group
together. A 'cluster' of these fibres is def'med to be a collection of Class X fibres in which
any fibre is in contact with at least one other fibre in the collection. The 'size' of the
cluster is defined as the number of fibres within it. An isolated Class X fibre will be
considered as a cluster of size one. The object of the study is to use some function of
the size of all Class X clusters in a muscle sample to test whether their spatial
distribution is random. The alternative to the hypothesis of random distribution is that
Class X fibres form groups, leading to large and presumably compact clusters. The
 51

A B
Fig. 1. A: A schematic representation of skeletal muscle as a hexagonal lattice with constant row length.
B: An exampleof the distribution of two fibre classes (Type X shaded black) showing one cluster of size 1
and one of size 3.

Fig. 2. A cluster of 10 class X fibres can be (A) compact or (B) stringlike.

definition of a cluster here refers to the size and not the shape of a cluster. This means
that both compact and stringlike large clusters (see Fig. 2) are treated equally. This work
concentrates on relatively low proportions of Class X fibres where random clusters are
too small for the shape to be critical. A variety of cluster size statistics were considered,
viz., 'maximum cluster size', 'number of clusters', and 'mean cluster size'. All three were
found to be useful but 'maximum cluster size' was less robust to departures from the
main model. 'Mean cluster size' was eventually chosen as the most useful statistic since
its distribution was little affected by the sample size, which simplified presentation of
results.

Simulation
The distribution of 'mean cluster size' was determined by computer simulation
for a lattice of hexagonal fibres of constant row length with values of sample size (S)
being 200, 300, 400, 500 and 600 and proportion of Class X fibres (~oX) varying from
2-30~o in steps of 2 ~ . Simulation was used since the distribution is analytically
intractable. For each combination of S and ~ X the requisite number of fibres was
randomly chosen from S, without replacement, to represent Class X fibres. The size of
all their clusters was found by Depth First Search (Horowitz and Sahni 1978) and
summarised by 'mean cluster size'. The cycle of assigning Class X fibres and calculating
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the test statistic was repeated 999 times for each combination of S and ~oX and the 50 ~,
and upper 5 ~o points of this empirical distribution were determined.

Extension of main model

It has hitherto been assumed that the fibres in a sample are arranged in rows of
the same length leading to an approximately rectangular fascicle shape. This shape will
affect the number of peripheral or edge fibres which have less than 6 neighbours and
therefore possibly the distribution of cluster size statistics. Four alternative muscle
sample shapes have been considered, viz, rectangular, square, triangular and hexagonal
(see Fig. 3), all of which have a different number of edge fibres for a given sample size.
Edge effects are increasingly important as the sample sizes decreases, so attention was
focused on the four different shapes of virtually the same size (170 + 1 fibre) at the
bottom of the range considered for S in the simulations. The exact sizes of the four
sample shapes are described in Table 1. The percentage points of the distribution were
determined as before.
The assumption of a hexagonal lattice of fibres means that all except edge fibres

A B

C D
Fig. 3. The four muscle sample shapes considered. (A) square, (B) rectangle, (C) triangle, (D) hexagon.

TABLE 1
DETAILS OF FOUR SAMPLE SHAPES COMPARED

Shape Dimension Total fibres

Rectangle 17 10 170
Square 13 13 169
Triangle Side = 18 171
Hexagon Side = 8 170
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A B

Fig. 4. A: All fibres in the standard lattice have 6 neighbours. B: The pinching algorithm investigates the
effect of some fibres having only 5 neighbours.

have 6 neighbours. This is unlikely to be true in practice. Lexell et al. (1983) found the
distribution of the number of neighbours (of internal fibres) in one sample to vary
between 4 and 8 with a mean of 5.8. The effect of weakening this assumption has been
investigated by allowing some fibres to have 5 neighbours. This is done by pinching the
boundary between two fibres to a point so that the pair no longer have a common
boundary and therefore only 5 neighbours each (see Fig. 4). The percentage points of
the mean cluster size were determined as before for a square configuration of 400 fibres
with a varying proportion of pentagonal fibres, leading to mean numbers of neighbours
for internal fibres of 6, 5.8, 5.6 and 5.4.
RESULTS

Simulation of main model

A subset of the 5 ~o and 50 ~ points of the distribution of 'mean cluster size' are
given in Table 2. It can be seen that these increase with ~oX and are relatively unaffected
by sample size. This allows spline functions to be fitted to the data for a given sample
size so that typical and extreme points of the 'mean cluster size' distribution can be
estimated for any value of ~oX between 2~o and 30~o. A graph showing the fitted
relationship of the percentage points with proportion of Type X fibres is given in Fig. 5

TABLE 2

MAIN SIMULATION RESULTS

% Class X

2 6 10 16 20 26 30

5 % points of'distribution of "mean cluster size"

200 1.33 1.50 !.67 2.00 2.35 3.06 3.75
Sample 400 1.33 1.41 1.60 2.00 2.29 2.97 3.75
Size S = 600 1.20 1.33 1.54 1.92 2.26 2.94 3.67

50% points of distribution of 'mean cluster size'

200 1.00 1.20 1.33 1.60 1.90 2.36 2.86
Sample 400 1.00 1.20 1.33 1.68 1.95 2.54 3.08
Size S = 600 1.00 1.20 1.36 1.68 1.97 2.56 3.16

These results are based on 999 runs at each combination of s and % X.

54

I,.0 q

5*/. Point

3.6
S = 200
S = ~00
--- S=600 , Point
.... 3.2

~ , ~ 2-8

~i 2.1,
,, 2.8

to
1.6

~o 1.2

I
0.8

Oq.
o; 5 10 15 20 25 30
% Class x fibres
Fig. 5. Percentage points of 'mean cluster size' distribution to be used in approximate significance tests.

for sample size S = 200, 400 and 600. Values for S in between these values can be found
by interpolation. This gives both typical values of mean cluster size when the spatial
distribution is random (50% point) and extreme values (5 % point). An example of the
use of the graph is given below.
e.g.: A muscle sample from a polymyositis patient had 416 fibres of which 48 were
necrotic (Class X). The necrotic fibres were grouped as 15 isolated fibres, 6 clusters
size 1, 1 cluster size 6, and 1 cluster of size 15.

48
%X = - 100 = 11.5%
416

48
Mean cluster size = = 2.09
15+6+1+1

Reading from Fig. 5, when % X = 11.5 and S = 416, the 50 % point = 1.39 and the 5 %
 55

TABLE 3
EFFECT OF SAMPLE SHAPE
These results are based on 999 runs at each combination of shape and %X.

Class X

10 20 30

5 % point of "mean cluster size" distn'bution

Rectangle 1.70 2.42 3.92
Sample Square 1.70 2.62 4.25
Shape Triangle 1.70 2.42 4.25
Hexagon 1.70 2.62 3.92

TABLE 4
EFFECT OF RELAXING HEXAGONAL FIBRES ASSUMPTION
These results are based on 999 runs at each combination of parameters.

% Class X

5 10 20

5 % point of "mean cluster size' distribution

Average neighbours per internal fibre 6.0 1.33 1.60 2.29
5.8 1.33 1.60 2.22
5.6 1.33 1.54 2.16
5.4 1.33 1.54 2.16

point = 1.65. But the 'mean cluster size' in this sample is greater than the 5~o point,
therefore the hypothesis of random distribution of necrotic fibres is rejected (P < 0.05).

E x t e n s i o n s to m a i n m o d e l
The effect of varying sample shape on the 5% point of the 'mean cluster size'
distribution is shown in Table 3. The values do vary slightly for different shapes but not
in any consistent manner or to any greater extent than would be expected in repeat
simulations of the same shape. This suggests that sample shape is unimportant as long
as it is fairly regular and contains at least 170 fibres.
The effect of introducing a proportion of pentagonal fibres, thus reducing the
average number of neighbours/fibre, on the 5 % point o f ' m e a n cluster size' distribution
is illustrated in Table 4. There is no effect for lower proportions of Class X fibres but
an indication that the 5 ~o point decreases consistently with average neighbours/fibres
at higher proportions. This will be further discussed elsewhere. However, a marked
departure from the hexagonal fibres assumption has no effect when the fibre type of
interest is rare, which was the situation for which this test was specifically developed.
56

DISCUSSION

Fibre type grouping (when the two types are fast and slow twitch) has already
been studied by a number of methods. The simplest approach is to assess the degree
of grouping by eye, however this can be misleading in all except the most extreme cases.
The 'enclosed fibre count' (Jennekens et al. 1971) provided a more objective method and
approximate significance tests were devised by assuming a hexagonal lattice of fibres
and using the Binomial Theorem (Johnson et al. 1973a) or simulation (Lexell et al.
1983). This measure has been used on patients with a variety of neuromuscular
disorders (Johnson et al. 1973b; Fawcett et al. 1985) and healthy subjects (Lexell et al.
1984). An occurrence of fibre type grouping here indicates that remodelling of motor
units is taking place.
More sophisticated techniques are available which dispense with the assumption
that the fibres are regular hexagons. The Join-Count statistic (Cliff and Ord 1971) was
developed with geographical data in mind but could equally well be used on muscle
cross-sections. James (1971) suggested the use of nearest neighbour techniques much
used in botany. Lester et al. (1983) formulated the Co-Dispersion Index which has the
advantage of providing a measure of the degree of grouping rather than a significance
test. However, all these methods require not only the type of each fibre, but also either
the type of all, or the distance between, neighbouring fibres. This makes these methods
more suitable for automatic data collection.
The 'mean cluster size' statistic suggested in this paper is as simple to calculate
as the 'enclosed fibre count' and could be used on similar data on occasions when the
fibre type of interest is not very common. However, the technique was initially devised
to investigate the apparent non-random arrangement of necrotic fibres in some patients
with inflammatory muscle disorders. In this case, necrotic fibres, the fibre class of
interest, make up only a small proportion of the total fibres in a sample and an 'enclosed
fibre count' will not be suitable.
Computer simulation has enabled a test for randomness to be carried out for a
variety of sample sizes and necrotic fibre proportions. In some cases, the presence or
absence of a random distribution of necrotic fibres may provide evidence for the various
hypotheses for the aetiology of muscle fibre degeneration. The full analysis of the data
on inflammatory muscle disorders, is given in Johnson et al. (1986).
The model of muscle fibre organisation chosen is that of a rectangular array of
regular hexagonal fibres, which is only an approximation to the situation occurring in
practice. However, the features of the model which are actually used in the simulations
are that
(i) each internal fibre has six neighbours;
(ii) there will be a f'Lx~xlproportion of peripheral fibres for a given sample size.
Both of these assumptions have been tested by allowing small departures from
them. The results in Tables 4 and 5 suggest that the distribution of 'mean cluster size'
is robust to these changes, at least when the fibre type of interest is rare.
The proportion of Class X fibres was allowed to vary between 2~o and 30~o. In
practice, proportions of necrotic fibres have occasionally been observed outside these
 57

limits, but a handful of necrotic fibres does not provide enough information for a
significance test. At the other end of the scale, values above 30~o were omitted, because,
at these levels a statistic which considers cluster shape as well as size would be
preferred. It has been suggested (Brunsdon 1985) that when ~/oX lies between values
suitable for usage of either 'mean cluster size' or 'enclosed fibre count' a suitable statistic
might be 'number of Class X fibres in largest cluster with 4 neighbours of same class'.
Extreme values of this statistic would indicate large compact clusters.
A more exact significance test could be done by running the simulation program
for the exact size and Class X proportions of a given sample. However, the program
is time-consuming, particularly on a microcomputer, and the combinations of S and
~oX already simulated should give reasonable significance test results for most other
combinations by interpolation between the values in Fig. 5.
In conclusion, the 'mean cluster size' statistic is a useful addition to the tests
available for the random distribution of muscle fibre classes. Its particular value is in
the investigation of possible clustering of fibres of uniform histochemical type when the
particular fibre type comprises less than 30~ of the total population. Thus the method
is especially suitable for studies of the distribution of necrotic fibres in toxic myopathies
or inflammatory muscle disorders.

ACKNOWLEDGEMENTS

We wish to thank Dr. M. Johnson of the Muscular Dystrophy Group

Laboratories and Dr. D. Appleton of the Department of Medical Statistics in the
University of Newcastle upon Tyne for many helpful discussions.

REFERENCES

Brunsdon, C. F.M. (1985) A Study of Necrotic Fibre Clustering in Polymyositis, M.Sc Thesis, University of
Newcastle upon Tyne.
Cliff, A. D. and J.K. Ord (1971) Spatial Processes --Models and Applications, Methuen & Co Ltd., London.
Fawcett, P. R.W., M.A. Johnson and I.S. Schofield (1985) Comparison of electrophysiological and histo-
chemical methods for assessing the spatial distribution of muscle fibres of a motor unit within a muscle,
J. Neurol. Sci., 69: 67-79.
Horowitz, E. and S. Sahni (1978) Fundamentals of Computer Algorothms, Pitman, London.
James, N.T. (1971) A geometrical probability study of Type 1 muscle fibres in the rat and guinea pig, J.
Neurol. Sci., 14: 381-387.
Jennekens, F. G. I., B.E. Tomlinson and J.N. Walton (1971) Data on the distribution of fibre types in five
human limb muscles, 3. Neurol. Sci., 14: 245-257.
Johnson, M.A., J. Polgar, D. Weightman and D. Appleton (1973a) Data on the distribution of fibre types
in thirty-six human muscles - - An autopsy study, J. Neurol. Sci., 18:111-129.
Johnson, M.A., G. Sideri, D. Weightman and D. Appleton (1973b) A comparison of fibre size, fibre type
constitution and spatial fibre type distribution in normal human muscle and in muscle from cases of
spinal muscular atrophy and from other muscular disorders, J. Neurol. Sci., 20: 345-361.
Johnson, M.A., M. Comola, D. Howel and C.M. Brunsdon (1986) Spatial distribution of muscle necrosis
in biopsies from patients with inflammatory muscle disorders, Muscle and Nerve, 9: 216.
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dispersion index for the measurement of fibre type distribution patterns, Muscle andNerve, 6: 581-587.
Lexell, J., D. Downham and M. SjSstrtim (1983) Distribution of different fibre types in human skeletal
muscles, J. Neurol. Sci., 61: 301-314.
Lexell, J., D. Downham and M. SjOstrOm (1984) Distribution of different fibre types in human skeletal
muscle, J. Neurol. Sci., 65: 353-365.