Quantitative analysis of quadriceps muscle biopsy

Journal of the Neurological Sciences, 1984, 66:319-326 Elsevier

319

Quantitative Analysis of Quadriceps Muscle Biopsy Results in 30 Healthy Females Carlo Doriguzzi 1, Tiziana Mongini 1, Laura Palmucci 1, Enrico Gagnor 2 and Davide Schiffer' 'Clinica Neurologica 11 and 21stituto Patologia Chirurgica I, Universitdl di Torino, Turin (Italy) (Received 27 March, 1984) (Accepted 26 July, 1984)

SUMMARY

Open biopsy was performed in fight quadriceps muscle (vastus lateralis) in 30 healthy female volunteers between 20 and 50 years of age. Histometric analysis was carried out on ATPase stained sections preincubated at pH 4.5. Type 1, 2a, 2b, and 2 fibres were quantified taking into account the following parameters: percentage of fibre types, mean diameter, atrophy and hypertrophy factor, variability coefficient of mean diameter and mean diameter ratio of type 1: type 2 fibres. Results showed that there is a large variation in fibre type percentage, mean diameter and hypertrophy factor, and consequently ranges of normal values are wider than those previously reported. Atrophy factor and variability coefficient are the least variable parameters. The importance of normal controls is stressed to avoid false positivities in histometric evaluation of muscle biopsy with minimal changes.

Key words: A TPase reaction - Healthy f e m a l e s - Histometric analysis - Quadriceps muscle biopsy

This research was supported by Grant 500.6/Ric.81/2597 from the Ministero della Sanifft, Dir. Gen. Serv. Med. Soc. Div. Address for correspondence: Carlo Doriguzzi, Clinica Neurologica II, Via Cherasco, 15, 10126 Turin, Italy. ( 22-SI(X'84,'S03.)~ ,'~: 1984 Elsevier Science Publishcr~ B.V.

320 INTRODUCTION Histometric analysis of muscle biopsy has been largely utilized for the quantitative evaluation of pathological c h ~ s in neuromuscular disease (Brooke and Engel 1969a, b, c; Dubowitz and Brooke 1973; Johnson etal. 1973b; Tosi and Jerusalem 1976; Sandstedt et al. 1982). In particular, quantitation of muscle biopsy may be useful when the demonstration~ of minimal changes has diagnostic importance, such as in the detection of Duchenne carriers. However, a correct evaluation of quantitative changes requires reliable normal data, whereas studies in normal subjects differ from one another in sex, the muscle studied, and the paramet~s taken into account (Reske-Nielsen 1970; Johnson et al. 1973a; Meltzer et al. 1976; Mahon et al. 1984). Moreover, no exhaustive morphometric studies considering also subgroups of type 2 fibres (2a and 2b) are av~able in females. Preliminary results of an histometric study on normal muscle carried out in our laboratory (Schiffer et al. 1980) showed values discordant from those currently used (Dubowitz and Brooke 1973) and also partially different from those recently reported in 10 female controls (Maunder-Sewry and Dubowitz 1981). In order to contribute to a better definition of normal histometric values in quadriceps muscle biopsy in females, we have increased the number of controls and we report the results in 30 female volunteers. MATERIALS AND METHODS A callfor healthy fcraalevolunteers yielded 176 w o m e n answering the appeal, of w h o m 30 were selected: 10 between 20 and 30, 10 between 30 and 40 and 10 between 40 and 50 years of age. The selection was made on the basis of the following criteria: (a) negative family history for neuromuscular diseases; (b) absence of any neurological disease in the history; (c) normal clinicalexamination (subjects with varicose veins or excess adipose tissue were excluded); (d) normal neurological examination; (e) normal serum creatine kinase (CK). Volunteers were also asked about their physical activity: none of them participated in sport activity. All volunteers were informed in detailof the surgicaltechnique of the biopsy, time of recovery, aspect and size of the scar and aim of the study. A sample of the lateralportion of the right quadriceps muscle was obtained by open biopsy under local anaesthesia. Th© length of the surgicalincisionwas about 2 cm. The weight of the muscle specimen was 200-300 rag. All specimens were rapidly frozen in isopentane cooled in liquid nitrogen and cryostat sections (8 #m) were stained with routine histological and histochcmical techniques (hematoxylin-eosin, modified Gomori trichrome, oxydative enzymes, ATPase). Quantitative analysis was made on sections stained for myosin ATPase after prcincubation at p H 4.5, which in our experience gives the best distinction in 3 fibre types (i, 2a, 2b). In all cases intermediate fibres were verified as 2b, by comparing identical fieldsof serial sections stained for myofibrillary ATPase after p~eincubation at p H 4.3 and 4.6. Values for type 2 fibres were obtained by considering fibres 2a and 2b together.

321 Two cases, in which we could not distinguish the three fibre types, were excluded from the study and substituted by two other volunteers of the same age. A total of 250-300 fibres were counted in each biopsy. Histometric evaluation was made according to the method of Dubowitz and Brooke (1973) and the following parameters were considered: percentage of fibre types, mean diameter, variability coefficient, atrophy and hypertrophy factor and the ratio of type 1 : type 2 mean fibre diameter. The variability coefficient for each fibre type was calculated as follows: SD/mean diameter × 1000 (Dubowitz and Brooke 1973). Atrophy factor and hypertrophy factor were calculated by giving a score to the fibres outside the range 30-70 #m, (Dubowitz and Brooke 1973). Fibres with 20-30 #m diameter were given a score of 1, those 10-20 #m were given score 2 and those less than 10 #m score 3. The products were summed up, divided by the total number of fibres of the type measured and the result was multiplied by 1000 to obtain the atrophy factor. A similar method was used to obtain hypertrophy factor by giving a score to fibres above 70 #m (e.g. a score of 1 to fibres 70-80 #m, of 2 to fibres 80-90/~m etc.). In cases 13, 19 and 26 (Table 1) histometric evaluation was repeated twice by different operators; the results did not show significant differences. In 6 cases (2, 9, 13, 19, 24, 26; Table l)histograms were also constructed on ATPase stained sections without acid preincubation. The results were the same as those derived from sections preincubated at pH 4.5. A chi-square test for goodness of fit to normal distribution has been calculated for: (a) the diameter values of all fibre types in each subject; (b) percentage of fibre types, mean diameter, atrophy and hypertrophy factors of each fibre type of all 30 controls together. RESULTS Results are shown in Table 1. Table 2 shows ranges (the lowest and the highest values) of single parameters in each age group and in the total sample of females. In Table 3 mean values + SD of mean diameter of each fibre type in all 30 volunteers are reported. The chi-square test showed that: (a) in each subject the values of the diameter of all fibre types were normally distributed; (b) in all 30 controls the values of the mean diameter of all fibre types were normally distributed, whereas frequency distributions of percentage, atrophy and hypertrophy factor of all fibre types were non-gaussian. The percentage of fibre was very variable; however, type 2 fibres were prevalent in all individuals but one (see Table 1, case 26). The mean diameter varied in all fibre types without correlation with age. Type 1 fibres were larger than type 2 fibres in 20 cases, as shown by the mean diameter ratio of type 1 : type 2 fibres (Table 1), but when considering type 2a and 2b fibres separately, type 2a fibres were larger than type I fibres in 15 cases and the mean value of type 2a fibre mean diameter of all 30 controls was higher than that of type 1 fibres (Table 3). Type 2b fibres were the smallest fibres in 23 cases.

20 23 23 24 24 24 28 28 29 29

32 32 33 34 35 36 36 36 38 39

41 42 42 42 43 43 45 48 49 50

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 20

21 22 23 24 25 26 27 28 29 30

34 35 30 39 48 68 40 37 3b 47

30 35 29 25 32 28 48 23 46 32

35 42 30 41 41 35 45 37 36 41

1

y,;

31 29 26 47 31 18 27 29 34 29

33 32 51 39 35 45 21 45 38 33

38 30 27 22 30 31 27 35 36 30

2a

35 36 44 14 21 14 33 34 30 24

37 33 20 36 33 27 3t 32 16 35

27 28 43 37 29 34 28 28 28 29

2b

66 65 70 61 52 32 60 63 64 53

70 65 71 75 68 72 52 77 54 68

65 58 70 59 59 65 55 63 64 59

2

+ 8.8 ± 10.6 _+ 11.2 + 12.3 ± 9.1 ± 10.5 +_ 8,1 + 9.2 ± 10.2 ± 8.5

57 -+ 9.4 50 + 10.8 52 +_ 12.4 63 _+ 12.9 52 ± 9.6 76 ± 13.7 50 _+ 10.5 47_+ 7.8 5 0 4 10.3 46 ± 911

47 + 8.2 58 ± 14.3 70 +_ 2k2 59 + 9:6 43 ± 6.4 54 ± 12.8 4 3 ± 9.7 59 ± 12.7 63 ± 15.7 51 ± 10.0

53 54 52 58 51 49 46 55 51 54

1

_+ 8,5 +_ 14,8 ± 8.5 ± 8.6 _+ 9.4 -+ 9.0 + 8.6 + 10.7 +_ 10.5 +_ 10.7

59 ± 10.9 60 + 8,3 50 ± 9.5 49 + 11.5 52 _+ 9.3 69 ± 14.3 49 ± 10.1 4 6 ± 9.0 45 -+ 9.7 48 ± 10.9

60 _+ 9.2 62 _+ 9.5 75 + 14.6 54 +_ 10.4 45 + 7.2 47 ± 10.3 4 4 + 6.5 6 2 ± 10.3 46 ± 12.9 51 ± 11.1

52 66 57 60 50 55 57 51 48 57

2a

+ ± ± ± + + + ± ± ±

9.5 9.6 17.8 8.6 7.2 9.3 7.2 10.0 9,3 12.2

_+ 8.4 + 16.8 _+ 8.9 + 8.7 + 8.4 + 9.7 + 12.0 _+ 9.5 + 12.1 + 10.7

49 ± 13.4 45 + 8.8 3 6 _ + 7.7 39 ± 9.1 43 ± 9.8 60 ± 15.3 42 ± 10.7 4 7 _ + 8.9 39 ± 9~5 47 _+ 9.9

66 51 72 39 45 40 41 49 40 46

39 61 39 50 42 45 61 45 37 51

2b

Mean diameter ( # m ) ( ± SD)

ANALYSIS OF QUADRICEPS

± 10.5 _+ 15.9 + 12.4 ± 9.8 + 10.1 +_ 10.6 + 10.6 ± 10.0 +_ 12.6 ± 11.1

54 ± 13.2 51 -+ 11.4 41 ± 10.9 47 -+ 11.8 48 ± 10.5 66 ± 15,5 45 _+ 10.9 47-+ 8.9 42 ± 9.8 47 + 10.4

63 ± 9.8 56 _+ 11.0 74 _+ 15.5 47 ± 12.1 45 + 7.3 45 _+ 10.5 42_+ 7.1 56 ± 12,1 44 ± 12.3 49 -+ 11.9

47 63 46 54 46 50 59 48 43 54

2

0 12 65 0 8 0 29 11 18 48

16 0 0 0 9 12 6 0 0 11

18 22 34 24 0 18 12 0 9 0

1

AF

0 0 21 48 13 0 42 41 86 66

0 0 0 18 9 38 14 0 131 10

0 30 13 0 0 10 0 14 69 0

2a

95 57 250 ~ 167 98 42 116 12 183 16

0 12 0 149 9 141 47 14 152 54

80 0 164 0 62 54 20 53 382" 14

2b

49 32 166 74 47 18 82 25 131 43

0 6 0 80 9 77 28 6 137 32

33 8 107 0 30 28 10 19 205 a 7

2

117 12 56 375 34 1262 ~ 28 0 9 0

0 310 1197 a 111 0 72 31 274 523 ~ tI

0 66 22 180 18 18 0 14 26 20

1

HF

M U S C L E B I O P S Y I N 30 C O N T R O L S

~ Values exceeding ranges of Dubowitz and Brooke (1973). AF = atrophy factor; HF = hypertrophy factor; VC = variability coefficient of mean diameter.

Age (yr)

No.

RESULTS OF HISTOMETRIC

TABLE 1

206 a 130 21 24 0 848" 0 0 0 13

118 187 a 1164 a 35 0 23 0 260 a 28 42

8 591 a 38 89 24 29 83 29 17 107

2a

68 0 0 0 0 375 ~ 12 12 21 16

382 a 0 1070 ~ 0 0 0 0 14 0 54

0 443 a 0 0 0 0 240 a 0 0 42

2b

134 57 8 21 0 649" 6 6 10 14

257 ~ 92 1137 ~ 19 0 14 0 159 a 20 48

5 520" 15 33 12 14 163 a 5 10 75

2

166 217 239 206 186 179 209 165 208 198

175 248 303 a 165 150 236 183 213 249 197

167 198 215 213 179 215 178 167 202 159

1

VC

186 139 190 234 .181 203 207 195 219 228

154 154 195 193 168 219 148 167 281 ~ 218

I65 226 149 146 188 164 150 210 218 191

2a

274 a 197 219 234 230 252 253 188 247 212

145 189 248 221 161 232 176 207 235 264 ~

214 274" 228 175 201 215 197 213 332 a 212

2b

246 223 268 a 252 218 235 241 191 234 221

156 196 211 265 ~ 164 236 I68 216 279 ~ 244

225 250 272 a 185 220 212 179 208 291 ~ 208

2

1.05 0.97 1.27 1.34 1.07 1,16 1.11 1.00 1.18 0~98

0.74 1.02 0.94 1.24 0;96 1.22 1.25 1.05 1.43 1.04

1.13 0.84 1.13 1,07 1.09 0.97 0.77 1.15 1.16 1.00

Ratio 1:2

23 . 48

30 . 68

23 . 68

30-40 (10 cases)

40-50 (10 cases)

20-50 (30 cases)

.

18 . 51

18 . 47

21 . 51

22 . 38

2a

.

14 . 44

16 . 37

43

27

2b

14 . 44

.

70

55

2

32 . 77

32 . 70

52 . 77

.

.

43 . 76

46 . 76

43 . 70

58

46 .

44 . 75

45 . 69

44 . 75

66

48 .

36 . 72

60

36

39 . 72

61

37

.

.

41 . 74

66

41

42 . 74

63

43

Mean diameter ( # m ) . . . . 1 2a 2b 2

.

.

0 . 65

65

0

0 . 32

34

0

.

AF . 1

.

0

0 . 131

0 . 86

0 . 131

69

. 2a

0 . 382

12 . 250

0 . 152

0 . 382

2b

0 . 205

18 . 166

0 . 137

0 . 205

2

AF = atrophy factor: HF = hypertrophy factor; VC = variability coefficient of mean diameter.

30 . 45

1

°,,

20-30 (10 cases)

Age (yr)

180

0 .

8 591

2a

0 . 848

0 0 . . . 1 2 6 2 1164

0 . 1262

0 0 . . . 1 1 7 7 1164

.

1

HF

RANGES OF SINGLE PARAMETERS 1N EACH AGE G R O U P AND IN ALL HEALTHY FEMALES

TABLE 2

.

0

375

0

0 . 1070

.

0 . 1070

443

2b

.

.

5 . 520

0 . 1137

649

0

0 . 1137

2

.

.

.

303

149

238

165

303

149

159 . 214

1

VC

.

.

.

281

139

234

139

281

139

146 . 226

2a

.

.

.

330

145

274

188

264

145

175 . 330

2b

.

.

.

291

156

268

191

279

156

291

179

2

1.43

0.74

1.34

0.97

1.43

0.74

1.16

0.77

Ratio 1: 2

b~

324 TABLE 3 MEAN VALUES ( + SD) OF MEAN DIAMETER (/am) IN 30 HEALTHY FEMALES I

2a

2b

2

53.7 + 7.3

54.2 +_ 7.6

46.8 + 9.0

50.6 + 7.9

In all age groups we found a narrow range of atrophy factor in all fibre types, cxcept in type 2b, which showed the highest values of atrophy factor. The hypertrophy factor was the most variable of all considered paranaeters;it increased in people over 30 years of age, especiallyin type I fibres,but the difference among the 3 age groups was not statisticallysignificant,as demonstrated by Kendalrs S-test (Kendall 1955). DISCUSSION

Our resultsdifferpartiallyfrom those reported by Dubowitz and Brooke (1973) and by Maunder-Sewry and Dubowitz (1981).In factin 14 out of our 30 controls,one or more parameters were out of the conventional range (see Table l) and in 8 of them the differencewas so marked that they might be considered "'pathological"ifmatched with the values usuallyemployed in quantitativeevaluationof muscle biopsy (Dubowitz and Brooke 1973). In particular7 of them presented high hypertrophy factor,which indicates that "hypertrophy" of muscle fibres may be a c o m m o n finding in normal population. In thisregard itisworth mentioning that severalstudiesin humans (Gollnick ct al. 1972; Prince et al. 1976) and in animals (Silberman ct al. 1973) demonstrated that both trainingand aging can determine hypertrophy of muscle fibres,suggestingthatfibre sizecan increasein differentphysiologicalconditions.A hypertrophy factorhigherthan thatpreviouslyreported (Dubowitz and Brooke 1973) has alsobeen found by MaunderSewry and Dubowitz (1981) in 3 out of 10 controls. W e have found a wide range of mean diameter in allfibretypes (Table 2); this agrees with the result of other studies in normal human muscle (see Mahon et al. 1984 for review) and it is basically due in our controls to the presence of some subjects with high values of mean diameter such as cases 13 and 26 (Table 1), which showed the highest values of mean diameter and hypertrophy factor. Wc could not relate this finding to any particular anthropometric or physiological characteristic. We conftrm that the sizes of type 1 and type 2 fibres are interrelated (Maunder-Sewry and Dubowitz 1981) and that type 1 fibres are generaUy larger than type2 fibres (Jennekens etal. 1971; Maunder-Sewry and Dubowitz 1981). In our controls this is due to the small size of type 2b fibres. The percentage of muscle fibre types was also very variable; this agrees with the results of most studies in human normal muscle (see Mahon et al. 1984)and with the

325 demonstration that the proportion of fibre types may be influenced by several factors such as the depth of the biopsy (Johnson et al. 1973a; Henriksson-Larsen et al. 1983) and training (Gollnick et al. 1972; Prince et al. 1976; Andersen and Henriksson 1977; Schantz et al. 1982; Ball et al. 1983). Atrophy factor and variability coefficient were the least variable parameters. In type 2b fibres we have found values of atrophy factor higher than those reported by Dubowitz and Brooke (1973); in type 1, 2a and 2 fibres values of atrophy factor were comparable to the results of Dubowitz and Brooke (1973) and of Maunder-Sewry and Dubowitz (1981). Values of mean diameter variability coefficient were higher in all fibre types than those reported by Dubowitz and Brooke (1973) and by Maunder-Sewry and Dubowitz (1981), but none of them exceeded the limit used by Cornelisse et al. (1980) to define normal muscle. In conclusion, our quantitative analysis of normal female muscle samples shows a large variability of results. This observation is comparable to the results in normal males reported by Mahon et al. (1984) who not only observed a significant difference of fibre sizes in muscle sample from different subjects, but also within different muscle samples of the same individual. These data demonstrate the importance of studying normal subjects to obtain more reliable histometric normal values from muscle samples and to avoid false positives in histometric evaluation of muscle biopsy. Comparison of the parameters proposed (Dubowitz and Brooke 1973) for the quantitative evaluation of muscle samples shows that the variability coefficient of the mean diameter and the atrophy factor may be the most useful ones because of their smaller variability in normal subjects. ACKNOWLEDGEMENTS

We gratefully thank all the volunteers, who provided muscle samples, and the newspaper Stampa Sera, Dr. L. Bussi and Cav. C. Meliga (local section of UILDM) for their collaboration. We also thank Dr. Guelfa Corbascio and Dr. Emanuela Terazzi for technical assistance and Prof. Alberto Piazza for helpful advice in the statistical evaluation of our data. REFERENCES Andersen, P. and J. Henriksson (1977) Training induced changes in the subgroups of human type II skeletal muscle fibres, Acta Physiol. Scand., 99: 123-125. Ball, M.E., H.E. Green and M.E. Houston (1983) Alterations in human muscle fibre type distribution induced by acute exercise, Histochemistry, 79: 53-57. Brooke, M.H. and W.K. Engel (1969a) The histographic analysis of human muscle biopsies with regard to fiber types, Part 1 (Adult male and female), Neurology (Minneap.), 19: 221-233. Brooke, M.H. and W.K. Engel (1969b) The histographic analysis of human muscle biopsies with regard to fiber types, Part 2 (Diseases of the upper and lower motor neuron), Neurology (Minneap.), 19: 378-393. Brooke, M. H. and W. K. Engel (1969c) The histographic analysis of human muscle biopsies with regard to fiber types, Part 3 (Myotonias, myasthenia gravis and hypokalemie periodic paralysis), Neurology (Minneap./, 19: 469-477. Cornelisse, C.J., G. Th. A.M. Bots, A.R. Wintzen, J.S. Ploem and K. Van den Broek (1980) Realtime

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